Stress: Physiology, Biochemistry, and Pathology: Handbook of Stress Series, Volume 3 [3, 1 ed.] 0128131462, 9780128131466

Table of contents :
Cover
STRESS: PHYSIOLOGY, BIOCHEMISTRY, AND PATHOLOGY
Copyright
Contributors
Preface
1. Arousal
Evidence for the Existence of GA
Physical and Quantitative Properties of GA
Neurologic Maladies and Public Health Problems From Dysregulation of GA
Neurons Critical for GA
Arousal and Psychiatric Disorders
Psychiatric Disorders Associated With Hyperarousal
Conditions Featuring Arousal Dysregulation
Psychiatric Disorders Associated With Hypoarousal
Summary
References
2. Resilience of the Brain and Body
Introduction
Brain-Body Basics
Central Role of the Brain
Plasticity of the Adult and Developing Brain
Stress-Induced Structural Plasticity
Epigenetics
Brain Gene Expression Is Continually Changing
Development of the Capacity for Resilience
How the Brain Gets “Stuck”
Prevention
Neurobiological Mechanisms of Overcoming Loss of Resilience
Some Examples of Opening Windows to Promote Resilience
Other Top-Down Therapies That Change the Brain
Conclusion
References
3. Cerebral Metabolism, Brain Imaging and the Stress Response
Introduction
Imaging the Stress Response Using the Example of Posttraumatic Stress Disorder
Resting State Brain Function in Humans
Brain Function During Flashbacks, After Traumatic Reminders and Alleviating Interventions
Effects of Mindfulness
Pharmacological Interventions
Limbic-Hypothalamic-Pituitary-Adrenal Axis
Road Traffic Accidents
Alternative Activation Strategies
Pharmacological Imaging
Dopamine
Serotonin
Cannabinoid Receptor
Glutamate Receptor
Stress Mechanisms in the Etiology of Other Psychiatric Disorders
Chronic Stress and Its Effect on Structural In Vivo Brain Imaging
Early Life Stress
Everyday Stress
Future Developments
References
4. Stress-Hyporesponsive Period
Introduction
Stress-Hyporesponsive Period
SHRP, the Adrenal and Corticosterone
SHRP and the Pituitary
SHRP and the Brain
Corticosteroid Feedback
Conclusion
References
5. Hippocampus and Hippocampal Neurons∗
Overview
Hippocampal Formation
Laminar Organization
Cross-Sectional Organization—Trisynaptic Circuit
Principal Neurons
Interneurons
Intrinsic and Extrinsic Neural Connections
Neurochemistry
Amino Acid Neurotransmitters
Biogenic Amines
Other Neuromodulators
Neuroplasticity
Hippocampal Function
References
6. Memory and Stress
STRESS
IMPORTANT FACTORS TO CONSIDER WITH REGARDS TO METHODOLOGY
Modulation of Stress Hormones
Valence of the To-Be-Remembered Material
IMPACT OF STRESS ON MEMORY
Impact of Stress on Memory Encoding and Memory Consolidation
Impact of Stress on Memory Retrieval
Impact of Stress on Reactivated Memories and Memory Reconsolidation
IMPLICATIONS FOR TRAUMATIC MEMORIES
INSIGHTS FOR FUTURE STUDIES
References
7. Adult Neurogenesis and Stress
Introduction
Species Differences in Adult Hippocampal Neurogenesis
Stress Effects on Adult Neurogenesis
A Role for Stress Hormones in Adult Neurogenesis
Sex Differences in Stress-Induced Changes in Adult Neurogenesis
Function of Stress-Induced Changes in Adult Neurogenesis
Conclusions
References
8. Stress, Corticosterone, and Hippocampal Plasticity
Introduction
Induction of Stress
Molecular Mechanisms
Network Mechanisms
Conclusions
References
9. Dopamine and Stress
Introduction
Nerve Cells, Synaptic Transmission, and DA Pathways in the CNS
Responses of Dopaminergic Systems to Acute Stressful Stimuli
Responses of Dopaminergic Systems to Chronic Stress
Interactions Between DA and Other Neurochemical Systems Altered by Stress
DA and the HPA Axis
DA and Excitatory Amino Acids
DA and NE
DA and Serotonin
DA and DA
Developmental Modulation
References
10. Serotonin in Stress
Introduction: Stress, Serotonin, and Human Psychopathology
Effect of Stress on Serotonin Parameters in the Brain
Animal Models
Human Imaging
Effect of Serotonergic Drugs on Stress Responses: Serotonin and HPA Axis Activity
Animal Models
Human Studies
Stress, Serotonin, and Human Psychopathology
Anxiety, PTSD, and Depression
Psychoses
Conclusions
References
11. Excitotoxicity
Introduction
Excitotoxic Mechanisms
Evidence That Physiological and Psychological Stress Can Endanger Neurons
Stress Hormones and Excitotoxicity
Ketone Bodies and Resistance to Excitotoxicity
Excitoprotective Effects of Mild Neuronal Stress
Environmental and Genetic Risk Factors for Stress-Mediated Excitotoxic Neuronal Degeneration
References
12. Chaperone Proteins and Chaperonopathies
Objectives and Scope
Chaperones and the Chaperoning System
Chaperonopathies
Structural Hereditary Chaperonopathies
sHsp Chaperonopathies
Hsp60 and CCT Chaperonopathies
Hsp40(DnaJ), Hsp70(DnaK), and Super Heavy Chaperones
Chaperonopathies Associated With Abnormal Organelle Chaperones
Chaperonopathies Associated With Dedicated Chaperones
Gene Polymorphisms and Chaperonopathies
Chaperonopathies Attributable to Chaperone-Gene Dysregulation
Other Types of Chaperonopathies
Chaperones and Metabolic Pathways
Acquired Chaperonopathies
Autoimmunity and Chronic Inflammation
Carcinogenesis
Indeterminate Clinical Pictures Which Could Implicate Chaperonopathies
Chaperonotherapy
Conclusions and Perspectives
Acknowledgments
References
13. Oxidative Stress: Eustress and Distress in Redox Homeostasis
Introduction
Concept of Oxidative Stress
Brief Historical Remarks
Oxidative Eustress and Oxidative Distress
Adaptive Oxidative Stress Responses
Hormesis
Kinds of Oxidative Stress
Nutritional Oxidative Stress
Postprandial Oxidative Stress
Photooxidative Stress
Radiation-Induced Oxidative Stress
Reductive Stress
Nitroxidative, Nitrosative, Nitrative Stress
Oxidant Sources
Endogenous (Cellular) Oxidant Sources
Exogenous Oxidant Sources: Exposome
Consequences in Health and Disease
Some Current Lines of Development
Relation to Calcium Signaling
Toxicology
Stress Response
Circadian Rhythm
Aging
Plant Research
Medical (Small Sample of Extensive Current Activity)
Environment
Concluding Remarks
Acknowledgments
References
14. Gender and Stress
Evidence Supporting Sex Differences in Stress Responses
Sex Differences in Adrenal Function
Sex Differences in Neuroendocrine Function
Pituitary Hormone Secretion
Hypothalamic Function
Negative Feedback Regulation
Sex Differences in Behavioral Responses to Stress
Basic Mechanisms of Sexual Differentiation of Neural Function
Organizational Effects of Gonadal Steroid Hormones on Stress Responses
Activational Effects of Gonadal Steroid Hormones on Stress Responses
Clinical Implications for Gender Differences in Stress Responses
References
15. Atrial Natriuretic Peptide, the Hypothalamic–Pituitary–Adrenal Axis, and Panic Attacks
Introduction
The NPs System
NPs and the HPA System
Effects on Anxiety and Panic Attacks
Outlook
References
16. Stress, Reward, and Cognition in the Obese Brain
Introduction: Stress, Appetite, and Control
Stress, Craving, and Motivational/Affective Biases in Obesity
Stress and Cognition in Obesity
Stress and Brain Function and Structure in Obesity
Conclusions
References
17. The Innate Alarm System: A Translational Approach
Introduction
Innate Defense Responses in Animals
The Defense Cascade Model
Innate Defense Responses in Humans
The Innate Alarm System
Conscious and Subconscious Processing of Threat in PTSD
Conscious Threat Processing in PTSD
Subconscious Threat Processing in PTSD
PTSD Symptomatology and the IAS During Threat Processing
Functional Connectivity of Brain Regions Associated With the IAS in PTSD
The Role of the Amygdala in Innate Defensive Responding in PTSD
Brainstem Regions and Innate Defensive Responding in PTSD
Clinical and Research Implications
Acknowledgments
References
18. Stress-Induced Anovulation
Definitions
Introduction
Neuroendocrine Mechanisms Linking Cognition, Mood, Behavior, and GnRH Drive
Pathogenesis of Stress-Induced Anovulation
Behavioral, Nutritional, and Metabolic Influences on the Reproductive Axis
Behavioral Influences
Nutritional and Metabolic Influences
Synergism Among Stressors
Treatment Considerations
Acknowledgments
References
19. Multidrug Resistance P-Glycoprotein (P-gb), Glucocorticoids, and the Stress Response
Introduction
P-glycoprotein: An Overview
P-gp Substrates
P-gp Localization and Regulation
Blood–Brain Barrier
Other Brain Regions
Pituitary and Adrenal Gland
Glucocorticoid Excretion
Stress-Related Substrates and HPA Function
P-gp and Development
Placenta
Fetal Blood-Brain Barrier
Concluding Remarks
Disclosure
Acknowledgments
References
20. Stress and Glucocorticoids as Experience-Dependent Modulators of Huntington's Disease
Introduction
Modeling Huntington's Disease in Mice
Glucocorticoids and the Stress Response
Mechanisms of Stress-Induced Changes
The Effects of Stress Depend on Many Variables
Stress Paradigms Used in Rodents
Corticosterone in Drinking Water
Restraint Stress
Chronic Unpredictable Stress
Social Defeat and Predator Exposure
Stress in Neurological and Psychiatric Disorders
Stress in Huntington's Disease
Psychological Stress
Abnormal Stress Response
The Effects of Stress and Stress Hormone Inventions in HD Mice
Oral CORT Treatment
Corticosterone Treatment Accelerated the Onset of Y-Maze Memory Deficits in Male HD Mice
Prolonged CORT Treatment–Induced Anhedonia Only in Female Mice
Motor Coordination Was Unaffected by CORT Treatment
Reduced Hippocampal MR Levels in R6/1 Mice at a Young Age
The Effects of Elevated Corticosterone Treatment on Novel Behavioral Phenotypes in HD Mice
Two Weeks of CORT Treatment Impaired Olfactory Sensitivity in Female Mice
CORT Treatment–Enhanced Female Social Interaction in Male R6/1 and WT Mice
The Effects of Chronic Restraint Stress on the HD Phenotype
Chronic Restraint After 9 weeks Is Still “Stressful”
Response to Chronic Stress Between the Genotypes
Restraint Enhanced Rotarod Performance and Induced HyperLocomotion in Male Mice
Sex Difference in Restraint-Induced Rotarod Effect
Restraint Transiently Reduced Saccharin Preference and Nest Quality in Female WT Mice
Olfactory Sensitivity Is Modulated by Restraint and the HD Mutation
Olfactory Deficits in Female R6/1 Mice Were More Vulnerable to 2 Weeks of Restraint Stress Compared to WT Littermates
Olfactory Sensitivity Deficits in Male Mice Were Impaired by Restraint Stress
Stress as a Novel Environmental Modulator of HD
Future Directions
Conclusions
References
Further Reading
21. PACAP: Regulator of the Stress Response
Introduction to Pituitary Adenylate Cyclase–Activating Polypeptide
Discovery, Characterization, and Evolution
General Functions
Distribution
PACAP Receptors
Receptor Characterization
Associated Pathways
Receptor Agonists and Antagonists
Uncovering PACAP as a Stress Peptide: The Role of Functional Genomics
PACAP Regulation of the Autonomic Nervous System
The Primary Neurotransmitter at the Sympathetic Adrenomedullary Synapse
PACAP's Role in the SNS Outside of the Sympathetic Adrenomedullary Axis
Regulation of Pre- and Post-Ganglionic Sympathetic Nerve Activity
Central Regulation of the SNS
PACAP and the Hypothalamic–Pituitary–Adrenal Axis
Regulation of the HPA Axis
Extrahypothalamic Regulation of the HPA Axis
PACAP in the Pathophysiology of Stress Disorders: A Maladaptive Response to Stress
PACAP's Sex-Specific Association With PTSD Risk: Clinical Association and Mechanistic Evidence
Summary
Conflicts of Interest
References
22. Glucose Transport
Introduction
Glucose Transporter Proteins
Glucose Transport
Overview of Glucose Transport Regulation
Stress Hormones and Glucose Transport
GLUTs as Stress-Responsive Proteins
Metabolic Stresses and Glucose Transport
Hypermetabolism
Mitochondrial Inhibitors
Glucose Deprivation
Obesity, Type 2 Diabetes Mellitus, and Cardiovascular Disease
Overall Effects of Transport Regulation
Signaling Cascades and Glucose Transport
GLUTs, Glucose Transport, and Metabolism in Chronic Disease States—Cancer
Summary
Acknowledgments
References
23. Links Between Glucocorticoid Responsiveness and Obesity: Involvement of Food Intake and Energy Expenditure
Introduction
Nexus Between Body Weight, Obesity and Activation of the HPA Axis
Physiological Determinants of Glucocorticoid Responsiveness: Selection of LR and HR Individuals
Cortisol Responsiveness and Innate Predisposition to Weight Gain
Cortisol Responsiveness and the Neural Control of Food Intake
Cortisol Responsiveness and Thermogenesis
Neuroendocrine Determinants of Altered Thermogenesis in LR and HR
Cortisol Responsiveness, Coping Strategies, and Physical Activity
Future Perspective
References
24. Blood–Brain Barrier: Effects of Inflammatory Stress
Introduction
Structure and Function of the Blood–Brain Barrier
Physical Barrier
Functional Barrier
The Metabolic and Enzymatic Barrier
The Neurovascular Unit
Neuroinflammation and BBB Physiology
BBB Integrity Impairment and Immune Cell Trafficking
Regulation of Transport Activities
Inflammatory Modulation of Metabolism
Inflammatory Stress at the BBB in Pathological Contexts
Neurodegenerative Diseases
Hypoxia-Ischemia Brain Injury and Stroke
Epilepsy
Infectious Diseases
Diabetes
Conclusions
Acknowledgments
References
25. Blood–Brain Barrier in Alzheimer's Disease
Introduction
Blood–Brain Barrier
BBB Characteristics
BBB Constitution
Neurovascular Unit
BBR Dysfunction in AD
AD Pathogenesis
Disruption of Barrier Properties in AD
Dysregulation of Transport Systems in AD
Concluding Remarks
Acknowledgments
References
26. Thermal Stress and Its Physiological Implications
Introduction
Exogenous and Endogenous Sources of Thermal Stress
Climate Change and the Speciation of Homo Sapiens
Generalisations Concerning Thermal Stress
Our Thermal Environment
First Principles
Stress and Strain in the Human Thermal Context
The Thermal and Water Vapour Pressure Continua
Thermodynamics
The Impact of Composition and Shape on Heat Exchanges
Quantification of the Thermal Environment
Indices of Stress and Strain
Concepts of Mammalian Homoeothermy
Morphological Considerations
The Cutaneous Vascular Network
Eccrine Sweat Gland Distributions
Skeletal Muscles
Principles of Physiological Control and Regulation
Passive and Active Systems
Homoeostasis
Normothermia, Physiological Accommodation, and Zones of Thermoregulation
Thermal Adaptation
Thermally Mediated Cutaneous Vasomotor Responses
Thermally Mediated Sudomotor Responses
Morphological Determinants of Cutaneous Blood Flow and Sweating
Predicting Scenarios of Adverse Strain
Interactive Influences
Interactions With Other Homoeostatic Mechanisms
Nonthermal Sudomotor Responses: Psychological Stress
The Interactive Impact of Clothing
Conclusion
Acknowledgments
References
27. Stress and Salt Appetite
Stress-Induced Salt Appetite in Animals
ACTH-Induced Salt Appetite
Hormones Influencing Salt Appetite in Stressed Animals
Adrenocorticotropic Hormone
Renin, Angiotensin, Aldosterone
The Effects of Increased Sodium Levels on Stress and Anxiety
Stress and Human Salt Appetite
References
28. Central Mechanisms Generating Cardiovascular and Respiratory Responses to Emotional Stress
Introduction
Pattern of Cardiovascular and Respiratory Responses Associated With Emotional Stress
Key Brain Regions Activated by Emotional Stress
DMH/PeF Region
Amygdaloid Complex
Midbrain PAG
Medial Prefrontal Cortex
Summary and Conclusions
References
29. Febrile Response and Seizures
Fever and Seizures
Causative Factors Mediating Seizures Caused by Fever
Genetic Susceptibility
Increased Brain Temperature Affects Permeability of the Ion Channels
The Role of the Innate Immune System
Clinical Studies
Experimental Studies
Alkalosis and FS
Potassium Chloride Cotransporter
FSs and Epilepsy: Human and Animal Studies
FSs and Cognitive Dysfunction
Implications for Therapy
References
Index
A
B
C
D
E
F
G
H
I
J
K
L
M
N
O
P
R
S
T
U
V
W
X
Y
Z
Back Cover

Citation preview

STRESS: PHYSIOLOGY, BIOCHEMISTRY, AND PATHOLOGY HANDBOOK OF STRESS VOLUME 3 Edited by

GEORGE FINK Florey Institute of Neuroscience and Mental Health University of Melbourne Parkville, Victoria, Australia

Academic Press is an imprint of Elsevier 125 London Wall, London EC2Y 5AS, United Kingdom 525 B Street, Suite 1650, San Diego, CA 92101, United States 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom Copyright © 2019 Elsevier Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library ISBN: 978-0-12-813146-6 For information on all Academic Press publications visit our website at https://www.elsevier.com/books-and-journals

Publisher: Nikki Levy Acquisition Editor: Natalie Farra Editorial Project Manager: Pat Gonzalez Production Project Manager: Paul Prasad Chandramohan Cover Designer: Mark Rogers Typeset by TNQ Technologies

Contributors Tamas Bartfai Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden

Matthew W. Hale School of Psychology and Public Health, La Trobe University, Melbourne, VIC, Australia

Sarah L. Berga Division of Reproductive Endocrinology and Infertility, Department of Gynecology and Obstetrics, University of Utah School of Medicine, Salt Lake City, UT, United States

Robert J. Handa Department of Biomedical Sciences, Colorado State University, Fort Collins, CO, United States Anthony J. Hannan Florey Institute of Neuroscience and Mental Health, Melbourne Brain Centre, University of Melbourne, Parkville, VIC, Australia; Department of Anatomy and Neuroscience, University of Melbourne, Parkville, VIC, Australia

Sondra T. Bland Department of Psychology, University of Colorado Denver, Denver, CO, United States Enrrico Bloise Department of Morphology, Federal University of Minas Gerais, Belo Horizonte, Brazil

Belinda A. Henry Metabolism, Diabetes and Obesity Program, Monash Biomedical Discovery Institute, Department of Physiology, Monash University, Clayton, VIC, Australia

Jenna E. Boyd Department of Psychology, Neuroscience, and Behaviour, McMaster University, Hamilton, ON, Canada; Mood Disorders Program, St. Joseph’s Healthcare Hamilton, Hamilton, ON, Canada; Homewood Research Institute, Guelph, ON, Canada

Holger Jahn Department of Psychiatry and Psychotherapy, University Medical Center Hamburg-Eppendorf, Heiligenhafen, Germany

Brandy A. Briones Princeton Neuroscience Institute, Princeton University, Princeton, NJ, United States

Naomi Kakoschke Monash Institute of Cognitive and Clinical Neurosciences, Monash University, Melbourne, VIC, Australia

Maria Alexandra Brito Research Institute for Medicines, Faculty of Pharmacy, Universidade de Lisboa, Lisboa, Portugal

Hagar Kandel Laboratory of Neurobiology and Behavior, The Rockefeller University, New York, NY, United States

Wilson C.J. Chung Department of Biological Sciences, School of Biomedical Sciences, Kent State University, Kent, OH, United States

Ruth A. Lanius Department of Psychiatry, University of Western Ontario, London, ON, Canada; Department of Neuroscience, University of Western Ontario, London, ON, Canada; Imaging Division, Lawson Health Research Institute, London, ON, Canada

Iain J. Clarke Neuroscience Program, Monash Biomedical Discovery Institute, Department of Physiology, Monash University, Clayton, VIC, Australia

Sonia J. Lupien Center for Studies on Human Stress, Research Center of the Montreal Mental Health University Institute, Montreal, Canada; Department of Psychiatry, Faculty of Medicine, University of Montreal, Montreal, Canada

Daemon L. Cline Northern Medical Program, University of Northern British Columbia, Prince George, BC, Canada Everly Conway de Macario Department of Microbiology and Immunology, School of Medicine, University of Maryland at Baltimore-Institute of Marine and Environmental Technology (IMET), Columbus Center, Baltimore, MD, United States; Euro-Mediterranean Institute of Science and Technology (IEMEST), Palermo, Italy

Alberto J.L. Macario Department of Microbiology and Immunology, School of Medicine, University of Maryland at Baltimore-Institute of Marine and Environmental Technology (IMET), Columbus Center, Baltimore, MD, United States; Euro-Mediterranean Institute of Science and Technology (IEMEST), Palermo, Italy

R.A.L. Dampney School of Medical Sciences (Physiology) and Bosch Institute, University of Sydney, Camperdown, NSW, Australia

Nicola Maggio Department of Neurology and Neurosurgery, The Sackler Faculty of Medicine, Tel Aviv University, Israel

Cle´mence Disdier The Alpert Medical School of Brown University, Department of Pediatrics, Women & Infants Hospital, Providence, RI, United States

Elizabeth Gould Princeton Neuroscience Institute, Princeton University, Princeton, NJ, United States

Marie-France Marin Center for Studies on Human Stress, Research Center of the Montreal Mental Health University Institute, Montreal, Canada; Department of Psychology, Faculty of Social Sciences, Universite´ du Que´bec a` Montre´al, Montreal, Canada; Department of Neurosciences, Faculty of Medicine, University of Montreal, Montreal, Canada

Sarah L. Gray Northern Medical Program, University of Northern British Columbia, Prince George, BC, Canada

Cristina Martin-Perez Mind, Brain and Behavior Centre, Universidad de Granada, Granada, Spain

Klaus P. Ebmeier Department of Psychiatry, University of Oxford, Warneford Hospital, Oxford, United Kingdom

ix

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CONTRIBUTORS

Stephen G. Matthews Department of Physiology, Obstetrics & Gynaecology and Medicine, University of Toronto, Toronto, ON, Canada M.P. Mattson Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD, United States Anthony L. McCall Division of Endocrinology & Medicine, University of Virginia (Emeritus), Charlottesville, VA, United States; Division of Nutritional Sciences, Cornell University, Ithaca, NY, United States Bruce S. McEwen Laboratory of Neuroendocrinology, The Rockefeller University, New York, NY, United States Michael J. McKinley Florey Institute of Neuroscience and Mental Health, University of Melbourne, VIC, Australia Margaret C. McKinnon Mood Disorders Program, St. Joseph’s Healthcare Hamilton, Hamilton, ON, Canada; Homewood Research Institute, Guelph, ON, Canada; Department of Psychiatry and Behavioural Neurosciences, McMaster University, Hamilton, ON, Canada Christina Mo Florey Institute of Neuroscience and Mental Health, Melbourne Brain Centre, University of Melbourne, Parkville, VIC, Australia; Department of Neurobiology, University of Chicago, Chicago, IL, United States Donald W. Pfaff Laboratory of Neurobiology and Behavior, The Rockefeller University, New York, NY, United States Daniela Rabellino ON, Canada

Homewood Research Institute, Guelph,

Catherine Raymond Center for Studies on Human Stress, Research Center of the Montreal Mental Health University Institute, Montreal, Canada; Department of Psychology, Faculty of Social Sciences, Universite´ du Que´bec a` Montre´al, Montreal, Canada; Department of Neurosciences, Faculty of Medicine, University of Montreal, Montreal, Canada Thibault Renoir Florey Institute of Neuroscience and Mental Health, Melbourne Brain Centre, University of Melbourne, Parkville, VIC, Australia

Philip J. Ryan Florey Institute of Neuroscience and Mental Health, University of Melbourne, VIC, Australia Mathias V. Schmidt Max Planck Institute of Psychiatry, Munich, Germany Menahem Segal Department of Neurobiology, The Weizmann Institute, Rehovot, Israel Helmut Sies Institute of Biochemistry and Molecular Biology I, Heinrich-Heine-University Du¨sseldorf, Du¨sseldorf, Germany; Leibniz Research Institute for Environmental Medicine, Heinrich-Heine-University Du¨sseldorf, Du¨sseldorf, Germany Robert L. Spencer Department of Psychology and Neuroscience, University of Colorado Boulder, Boulder, CO, United States Gregg D. Stanwood Department of Biomedical Sciences, Florida State University College of Medicine, Tallahassee, FL, United States Barbara S. Stonestreet The Alpert Medical School of Brown University, Department of Pediatrics, Women & Infants Hospital, Providence, RI, United States Nigel A.S. Taylor Centre for Human and Applied Physiology, School of Medicine, University of Wollongong, Wollongong, Australia Maarten van den Buuse School of Psychology and Public Health, La Trobe University, Melbourne, VIC, Australia Antonio Verdejo-Garcia Monash Institute of Cognitive and Clinical Neurosciences, Monash University, Melbourne, VIC, Australia Annamaria Vezzani Department of Neuroscience, Mario Negri Institute for Pharmacological Research IRCCS, Milano, Italy EnikT Zsoldos Department of Psychiatry, University of Oxford, Warneford Hospital, Oxford, United Kingdom

Preface

physiology, biochemistry, and pathology of stress has increased exponentially since 1932 due in large part to new stress concepts, the discipline of neuroendocrinology which first matured in the 1950s (covered in Volume 2 of the Handbook of Stress) and astonishing new technologies such as human brain imaging, neurochemistry, genetics, optogenetics, genomics, and studies of behavior. Many of the quantum advances in stress knowledge are the subject of this volume. I am grateful to our distinguished authors who have given so generously of their time and knowledge, Pat Gonzales for her excellent assistance in collating and preparing the chapters for Production and Natalie Farra for her encouragement, oversight, support, and wise guidance. Finally, as always, I thank Ann Elizabeth Fink for her steadfast forbearance and support and my children Naomi and Jerome who forever cheer from the sidelines.

It isn’t the mountain ahead that wears you out; it’s the grain of sand in your shoe. Robert W. Service (Bard of the Yukon) “One of the most striking features of our bodily structure and chemical composition that may reasonably be emphasized, it will be recalled, is extreme natural instability. Only a brief lapse in the coordinating functions of the circulatory apparatus, and a part of the organic fabric may break down so completely as to endanger the existence of the entire bodily edifice. In many illustrations we have noted also how infrequently they bring on the possible dire results. As a rule, whenever conditions are such as to affect the organism harmfully, factors appear within the organism itself that protect it or restore its disturbed balance.”

So wrote the great Harvard physiologist, Walter Bradford Cannon in his landmark Wisdom of the Body in which he coined the term “homeostasis” and described the “fight-or-flight” response. Cannon continues: “A noteworthy prime assurance against extensive shifts in the status of the fluid matrix is the provision of sensitive automatic indicators or sentinels, the function of which is to set corrective processes in motion at the very beginning of the disturbance.” Cannon’s prescience is underscored by the fact that the epilogue of his book is focused on the “relations of biological and social homeostasis,” relations which are now the subject of intense investigation. Notwithstanding the important principles established in the Wisdom of the Body, our knowledge of the

Reference: Cannon WB 1932. The Wisdom of the Body. WW Norton &Co Inc, New York Pp. 1e312 (quotations from pages 268e270). George Fink Florey Institute of Neuroscience and Mental Health University of Melbourne Parkville, VIC, Australia 2018

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C H A P T E R

1 Arousal Hagar Kandel, Donald W. Pfaff Laboratory of Neurobiology and Behavior, The Rockefeller University, New York, NY, United States O U T L I N E Evidence for the Existence of GA

2

Physical and Quantitative Properties of GA

3

Neurologic Maladies and Public Health Problems From Dysregulation of GA

Psychiatric Disorders Associated With Hyperarousal

7

Conditions Featuring Arousal Dysregulation

12

5

Psychiatric Disorders Associated With Hypoarousal

13

Neurons Critical for GA

6

Summary

14

Arousal and Psychiatric Disorders

6

References

14

• Several components of the nervous system such as the medullary reticular formation, thalamus, and cortex contribute to GA and have been analyzed with respect to neuroanatomical pathways, electrophysiological features, and some of the most important genes involved in the generation of GA. • GA has been given an operational definition and criteria for successful operation. • GA is proven to exist by psychological, genetic, statistical, and mechanistic findings. • Surprisingly, GA can be abnormally high during melancholic depression. • GA is out of control, in association with bipolar disorders.

Several years ago, we proposed the concept of “generalized central nervous system (CNS) arousal (GA),”3 now updated and extended.4 There is a marked asymmetry between the concept of GA and the concept of stress: You can be aroused without stress, but you cannot be stressed without arousal. This chapter represents an update of Pfaff, Martin, and Ribeiro.5 KEY POINTS • Generalized CNS arousal (GA) is the most elementary function of vertebrate nervous systems. It is a nonspecific neuronal “force” that activates ascending and descending systems, facilitating the initiation of any behavior responding to external stimulation and emotional expression

Stress: Physiology, Biochemistry, and Pathology https://doi.org/10.1016/B978-0-12-813146-6.00001-1

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Copyright © 2019 Elsevier Inc. All rights reserved.

2

1. AROUSAL

Constant throughout has been the definition of GA: Operational definition: A more aroused animal or human, with higher GA, is more alert to sensory stimuli in many sensory modalities (S), more active motorically (M) and more reactive emotionally (E). One can also consider operating requirements: Four which can be justified on a theoretical basis: (1) GA mechanisms must work fast enough to allow the individual to escape danger, (2) there must be great convergence of inputs onto GA mechanisms so that a wide variety of incoming signals can trigger adequate behavioral responses, (3) there must be great divergence of signals emanating from GA mechanisms so that a wide variety of behavioral responses can be initiated, and (4) GA mechanisms must be robust enough so that they will not fail. We propose that GA mechanisms work in all vertebrate brains including, of course, the human brain. GA is a primitive, undifferentiated force deeper than and additive to the usual motivational states (sex, hunger, etc.). GA contributes to many different types of behaviors, normal and abnormal. GA is the ur-arousal*, the force for the initiation of behavior conceptually and mechanistically deep to all of the more superficial and individuated forces. For example, fight or flight depends on GA but is more situationally dependent than GA.

EVIDENCE FOR THE EXISTENCE OF GA We cite four lines of evidence: 1. Psychology. The oldest line of evidence for the existence of a brain function called “generalized arousal” came from psychologists who study normal human behavior and personality. Virtually, all personality theorists included a dimension called “arousal” or a similar term in their description of the fundamental axes of personality. 2. Genetics. The newest evidence for GA comes from genetics; modification of GA, and

therefore to its existence (reviewed in the study by Pfaff4). This genetic evidence comes from three approaches. A perfect example of the first approach is the gene for hypocretin (reviewed in the study by Li et al.6,7) expressed in about 3000 neurons in a very restricted portion of the lateral hypothalamus. Optogenetic activation of hypocretin neurons can wake up mice from sleep. Conversely, optogenetic silencing of hypocretin neurons can induce sleep during the light phase of the daily light cycle. Part of the power of the hypocretins in preserving wakefulness seems to lie in their ability to work through classical monoaminergic systems that serve arousal. These include noradrenergic neurons since hypocretin axons project to the noradrenergic source in the hindbrain the locus coeruleus. Another monoamine systems affected are dopamine (emanating from the ventral tegmental area), histamine (produced in the tuberomammillary nucleus in the medial hypothalamus) and serotonin (produced in neurons in the raphe neurons on the midline of the midbrain). Importantly, hypocretin neurons project to the large cholinergic neurons of the basal forebrain, neurons which are important for waking activity in the cerebral cortex. A second approach to the genetics of arousal follows a different route to discovery. In the field of work just reviewed, the scientists cloned a gene involved in GA and thus stimulated an entirely new field of work: describing neuroanatomical projections, discovering cognate receptors, analyzing mechanisms of action, and so forth. This second approach comes from gene knockout studies that were initiated for a different reason, and the arousal data were eventually discovered in later, follow-up studies. For example, the gene for estrogen receptor a (ER-a) first garnered great interest because of its involvement in sexual behaviors.8,9 Later, Joan Garey10 extended our behavioral analyses to include arousal measurements.

* Ur-arousal means the oldest, most primitive and most fundamental form of CNS arousal

PHYSICAL AND QUANTITATIVE PROPERTIES OF GA

Other studies measuring motoric activity gave similar results. For example, we know that reduction of the ER-a gene product specifically in the neurons of the medial preoptic area reduced movement. This finding replicated early work from our lab which had also reported that gonadectomized a-ERKO (estrogen receptor knockout) females were significantly less active than a wild-type (WT) mice in open field tests, whereas beta-ERKO females tended to be more active than beta-WT mice. A third line of genetic evidence for generalized arousal is that you can breed for this function. You can’t breed for a brain function that does not exist. That is, if the highest and lowest males and females are selected generation after generation according to the operational definition of arousal (mentioned previously), then successful creation of a high and a low line proves that a concept matching that operational definition must exist. Since we knew that we would be breeding mice for a multigenic function, we went for advice to behavior geneticist David Blizard of Pennsylvania State University. He advised us to start with a strain of mice that had a high-degree genetic heterogeneity. This type of strain had indeed been achieved by Gardner Lindzey and Donald Thiessen many years before. The high genetic heterogeneity had resulted from an extensive intercross of more than eight outbred strains (and was called Het-8). The generalized arousal assay in our lab featured mice housed singly and cut off absolutely from the outside world. No sound, no vibration, no odors. Using this assay and, over 10 generations, mating high arousal males with high arousal females (and low with low), it was possible to achieve a high arousal line and a low arousal line of mice.11 3. A third line of evidence showing the existence of GA comes from mathematical statistics. As summarized by Calderon et al.12 principal components analyses of mouse behavioral data related to arousal reveal a large GA component which accounts, in different experiments, for between 29% and 45% of the data. That means in a differential equation which mathematically describes changes in arousal, on the right side

3

of the equal sign, there would be one term representing GA, and many other terms representing specific forms of arousal, such as sex, hunger, thirst, fear, anger, and so on. Thus, the statistics of principal components analysis support the conclusion that GA exists but also indicates the importance of other, specific forms of arousal. 4. Brain mechanisms. You cannot have mechanisms for a function that does not exist. During the last 30 years or so, the neurobiological mechanisms for changes of state of the entire CNSdthe exact opposite of specific sensory systemsdhas “caught up” enough to merit a book-length treatment. An example would be the neuroanatomical delineation of reticular formation neuroanatomy by the McGill University neurobiologist Barbara Jones. Brain mechanisms have spelled out and reviewed.4 With four independent lines of evidence for the existence of GAdpsychological, genetic, statistical, and mechanisticdit is timely to theorize about GA as a physical process. It turns out that flipping from a not-aroused state appears to have the property of a physical phase transition and should demonstrate the “scaling” and accompanied “power law behavior”dbehavior governed by a simple exponential equation that produces the same dynamics from tiny scales to huge scales. In laboratory mice, this prediction proves true, and this type of transition likely is universal among vertebrates.

PHYSICAL AND QUANTITATIVE PROPERTIES OF GA Pursuing the conviction to think about arousal systems with a precision typical of the physical sciences, we turned to Penn State Professor of Physics, Jayanth Banavar. We knew we needed to generate a systematic set of hypotheses about the regulation of GA as a function that bears on virtually all aspects of human and animal behavior. These ideas were expected to apply universally among vertebrates. We started with the idea that when rapid changes of state of the CNS would be requireddfor example, when a rapid response to a stimulus would be important

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to achievedthat linear dynamics in generalized arousal mechanisms would not be sufficient. Nonlinear dynamics, as found in chaotic systems, could provide tremendous amplification of CNS arousal signals and would also confer exquisite sensitivity to the initial state of the system. The hypothesis, therefore, that in the notaroused state chaotic dynamics prevail, is very attractive because they are deterministic and because they link the elegant mathematics of chaos to the concept of a fundamental property of the vertebrate CNS. But for coordinated movements as part of the behaviors thus initiated, the system will have to emerge from chaos. Thus, the second idea was that as neural systems pass from the chaotic nonaroused state to aroused states, they pass through a classically defined phase transition. With the behavioral response activated, orderly movement control neurophysiology takes over.13 To understand this theory clearly, consider the analogy to a classical physical example of a phase transition, the liquid crystal. Arousal systems in the not-aroused or low-aroused animal are in a chaotic state. The controlled-chaotic state of Ott et al. would be perfectly appropriate. When the animal is sufficiently stimulated, the nonlinear dynamics of deterministic chaos provide exponential amplification so that CNS systems can initiate orderly movement in response. By analogy to the liquid crystal, the disordered molecules at a higher temperature go through a phase transition to the ordered, crystalline state. Experimental scientists are beginning to think along these lines, and some evidence for our theory has accrued. For example, with magnetoencephalographic data from human subjects who were performing a finger-tapping task, a variety of mathematical approaches were used to analyze several spectral domains in the subjects’ cerebral cortical activity. The results showing the degree of synchronizability of this activity demonstrated; in their words, the brain networks are located dynamically on a critical point of the order/disorder transition. That is, their networks were close to the threshold of order/disorder transformation in all frequency bands, just like our theoretical liquid crystal analogy. Another example of the importance of thinking about chaotic dynamics in relation to

neural activity comes from findings in auditory neurophysiology. Certain nonlinear equations yielding chaotic dynamics demonstrate instabilities at fixed, special values of some given system parameter called “Hopf bifurcations.” Marcelo Magnasco and his colleagues have presented evidence that the tuning curves of the cochlea in the auditory system are partly shaped by a set of mechanosensors poised precisely at the threshold of a Hopf instability. This application of nonlinearity in hearing achieves the advantages of a high degree of amplification and a sharp tuning curve even at low input intensity. Magnasco and his colleagues have extended their evidence for “dynamic criticality” to the electrical activity of the human cerebral cortex. Dynamic criticality refers to “systems that persist at the boundary between stability and instability” and is typified by “systems highly susceptible to small external perturbations.” It can be argued that we need neural mechanisms to constitute an extended dynamical system that is close to a critical point and that will neither decay nor explode, thus allowing for longrange communication across the entire system. This type of system is just what is needed, theoretically, to create a GA system that protects us from dangers in the external world. Alex Proekt (2012), now at the University of Pennsylvania Medical School, noticed that the timing of many diverse behaviors from human communication to animal foraging form complex self-similar temporal patterns reproduced on multiple time scales. We envisioned a general framework for understanding how such scale invariance may arise in nonequilibrium systems, including those that regulate mammalian behaviors. Below is described how we demonstrated that the predictions of this framework are in agreement with detailed analysis of spontaneous mouse behavior observed in a simple unchanging environment. Neural systems operate on a broad range of time scales, from milliseconds to hours. Analyses revealed that the specifics of the distribution of resources or competition among several tasks are not essential for the expression of scale-free dynamics. Importantly, we showed that scale invariance observed in the dynamics of behavior can arise from the dynamics intrinsic to the brain.

NEUROLOGIC MALADIES AND PUBLIC HEALTH PROBLEMS FROM DYSREGULATION OF GA

In physical systems, one observes scale invarianceda repetition of shape and dynamics from tiny physical scales through huge physical scalesdnear a critical point, for example, where water turns into steam or where the unaroused animal can become aroused. It has been suggested that the presence of power laws in diverse living systems might imply that biological systems are poised in the vicinity of phase transitions. There are, however, fundamental differences between scale invariance exhibited by biological and physical systems. Criticality, the supersensitive responses to small stimuli are is confined to a small region in parameter space, and it is not clear how diverse biological systems are fine-tuned to exhibit criticality. But, in physics, critical systems are at equilibrium, whereas most processes occurring in living systems including animal behavior are not in equilibrium. Behavior is often conceived as serving a particular purpose or as a response to a specific stimulus. However, even in the relative absence of these phenomena, all animals including humans readily exhibit spontaneous behavior. Spontaneous activation of behavior is the simplest case of animal behavior because it avoids the complexities added by specific behavioral tasks, interactions among individuals, and the specifics of the structure of the environment. Understanding the dynamics of spontaneous behavior therefore is a prerequisite for understanding behavioral dynamics in more complex settings. This was the focus of Proekt’s analysis.14 Proekt worked with Professor Banavar and mathematician Amos Maritan to analyze the fine structure of the movements of mice in the GA assay described previously. Importantly, systems going through a phase transition behave according to simple exponential equations called “power laws.” Plotted on log: log coordinates, both the X-axis of the graph and the Y-axis of the graphs are scaled logarithmically rather than linearly, such systems yield straight lines. In accordance with theory, mice, during the dark phase of the daily light cycle, demonstrated straight lines over three orders of magnitude. These results14 are consistent with the phase transition theory summarized previously.

5

Still working with the physicist Jayanth Banavar, we are now asking mathematical questions about the performance of laboratory mice as they go through the hypothesized phase transitions from the light part of the day (low CNS arousal) to the dark part (high CNS arousal) in a 12-h light 12-h dark daily cycle. Working with equipment that provides temporal resolution of 20 ms, we ask, with the data from individual mice on individual days, what mathematical curves fit their activity change and what do whose equations suggest? How can we describe individual differences? Is the phase transition from high to low arousal the mirror image of the transition from low to high? Within a few months, these new studies may offer answers to these questions.

NEUROLOGIC MALADIES AND PUBLIC HEALTH PROBLEMS FROM DYSREGULATION OF GA There are many serious medical and public health problems resulting from failures of GA. One obvious category is disorders of consciousness. Coma is, by definition, a temporary condition. Either the patient escapes from coma and enters a vegetative state or he dies. Vegetative state patients are not uniform in their range or severity of symptoms. Recently, there has been special attention given to “high-end” vegetative state patientsdthose who sporadically have shown some communicationdbecause such patients may be responsive to treatments such as deep brain stimulation. Stupor, as well, certainly involves arousal problems. In the working world, GA plays especially important roles in certain jobs that require high and sustained vigilance. The military is one example. It is said that even a trained sniper cannot maintain the necessary level of attention for longer than about 30 min. Shift work in which an individual rotates through two or three daily shifts takes its toll because of the challenge to the individual’s circadian rhythms. Dangerous occupations like slicing meat or fish explode the size of the potential lossesdfor example, decreased

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arousal resulting in even a moment of lapsed attention can cause the loss of a limb. Public health failures like elevated lead concentration in drinking water can reduce cognitive performance through routes that included decreased GA. Perhaps, the mysterious “fatigue states” are related. Thus, some scientists think that they can follow certain environmental exposures that lead to chronic fatigue syndrome, fibromyalgia syndrome, and Gulf War Syndrome. The first two are much more common among women and the third in men, but they all share many symptoms, one of which is decreased GA. Almost all the foregoing conditions depend on underperformance of GA mechanisms. But for some patients, reducing arousal level is necessary. Here are two examples. First, it is estimated that about 15%e20% of American adults have sleep problems: some cannot get to sleep, while others have badly fragmented sleep or wake up too early. Second, anesthesia, as for surgery, is a highly sophisticated branch of medicine. The regulated reduction in arousal level was mentioned earlier in this chapter.

NEURONS CRITICAL FOR GA Evidence has piled up that large medullary reticular neurons in a group called nucleus gigantocellularis (NGC; also called reticularis gigantocellularis) are crucial for maintaining CNS arousal levels and for the initiation of a wide variety of behaviors. Elevating electrical activity in these glutamatergic neurons is associated with the activation of behavior15 and with an aroused electrical pattern in the cerebral cortex (the electroencephalogram [EEG]). Decreased activity has the opposite effect. Large medullary reticular neurons express genes for arousalrelated neuropeptide receptors.16 We now have the entire transcriptome expressed by a subset of NGC neurons, demonstrating a unique expression of one gene and an unusually intimate relation to the nearby vasculature. The hypothesis has been put forth4 that these NGC neurons function in a large anterior/posterior integrated network. In terms of the

connectivity of individual neurons within the network, they may have a “scale-free” property; in that many neurons have few connections, while only a few, like NGC, have a large number of connections.

AROUSAL AND PSYCHIATRIC DISORDERS Arousal regulation in the human brain is a complex phenomenon; it describes a dynamic process of cortical and behavioral activation in response to varying degrees of stimulation; and accordingly, the relationship between stress, cortical activity, and performance. Its dysregulation has been implicated in different psychiatric disorders.17,18 Classically, beginning with the pioneering work of Professors Tarchanoff, Peterson, and Jung, researchers started to study dysregulation of arousal in different psychiatric disorders.19 The Research Domain Criteria (RDoC) project of the National Institute of Mental Health develop new ways of classifying mental disorders for research purposes; they shift away from symptom-based diagnoses toward a transdiagnostic neurobiological focus in the study of mental disorders. The major RDoC framework consists of Matrices; there are five domains in it: Negative Valence Systems, Positive Valence Systems, Cognitive Systems, Systems for Social Processes, and Arousal/ Regulatory Systems.20e22 The Arousal construct group defines arousal as a continuum of sensitivity of the organism to stimuli both external and internal; it facilitates interaction with the environment; it can be evoked by either external/environmental stimuli or internal stimuli; it can be modulated by the physical characteristics and motivational significance of stimuli; it varies along a continuum that can be quantified in any behavioral state; it is distinct from motivation and valence; it may be associated with increased or decreased locomotor activity; and it can be regulated by homeostatic drives. The group identified a number of genes, molecules, circuits, and neurotransmitter

PSYCHIATRIC DISORDERS ASSOCIATED WITH HYPERAROUSAL

systems that were relevant to arousal, which are included in the arousal matrix.23 Arousal can be assessed by • Autonomic measures: heart rate variability (HRV; Beauchaine and Thayer24), electrodermal responding.25 • Cognitive measures: psychomotor vigilance task26 and the Vigilance Algorithm Leipzig (VIGALL); where different EEG vigilance stages from full alertness to sleep onset can be separated during rest.27,28,29,30 • Psychological measures: the Arousal Predisposition Scale31 and the Scale of Trait Arousability.32 This part of the chapter presents examples of different psychoepathoephysiological proposed mechanisms that potentially can contribute to discoordination of arousal regulatory systems, leading to hyper or hypo arousal in different psychiatric disorders.

PSYCHIATRIC DISORDERS ASSOCIATED WITH HYPERAROUSAL Major depressive disorder (MDD) is a mental disorder characterized by at least 2 weeks of pervasive depressed mood, loss of pleasure in daily activities, weight loss, insomnia, agitation, fatigue, feelings of worthlessness or guilt, attentional problems, thoughts of death, and suicidal ideation.33 The arousal regulation model of affective disorders denotes that the upregulation of arousaldnegative emotional arousaldis a central pathogenic factor in MDD. This is paradoxical at first glance, but this model provides a simple explanation, that withdrawal and sensation avoidance in depression are proposed to be a reaction to the tonically high brain arousal. It explains several clinical phenomena typically seen in MDD such as prolonged sleep onset latencies, avoidance of arousal-increasing external stimulation and the response to therapeutic sleep deprivation.34 In line with this model, wakefulness-promoting cytokines, “especially IL-13,” were found to be significantly associated

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with hyper-stable EEG vigilance recording in MDD patients.29 Severe depression drastically reduces the amount of time spent in Stage 4 (delta) sleep. Furthermore, depressed patients have more reduced rapid eye movements (REM) sleep, and REM sleep occurs earlier in the night (reduced REM latency), indicating increased arousal.35 This can open the door for future evaluation of vigilance measures as a biomarker in MDD. Zobel and colleagues found that genetic factors, elevated neuroticism, and HPA dysregulation moderate as risk factors for depressive disorders and also reflect a predisposition toward coping less effectively with stress and its related challenges.36 Animal evidence indicates that stress exacerbates the effects of reduced brain-derived neurotrophic factor (BDNF) on both hippocampal networks and autonomic arousal.37 The effects of the interaction of the BDNF Val66Met polymorphism and exposure to early life stressors (ELS) on neural circuitry and autonomic arousal pathways that in turn predict syndromal depression and anxiety have been identified by Gatt et al. They found that BDNF Met carriers exposed to greater ELS have smaller hippocampal and amygdala volumes (P ¼ .013), heart rate (HR) elevations (P ¼ .0002), and a decline in working memory (P ¼ .022), also the combination of Met carrier status and exposure to ELS predicted reduced gray matter in hippocampus (P < .001), and associated lateral prefrontal cortex (P < .001) and, in turn, higher depression (P ¼ .005). Higher depression was associated with poorer working memory (P ¼ .005) and slowed response speed. The BDNF MeteELS interaction also predicted elevated neuroticism and higher depression and anxiety by elevations in body arousal (P < .001).38 Schmidt et al. studied arousal regulation between depressed patients and healthy controls and also responders and nonresponders to antidepressant “Escitalopram” using the VIGALL 2.1. In 65 unmedicated depressed patients; 15min resting-state EEGs was recorded. In 57 patients, an additional EEG was recorded 14  1 days following onset of escitalopram.

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There were 29 responders and 36 nonresponders. They found that responders and nonresponders differed in distribution of overall EEG vigilance stages (P ¼ .009), with responders showing significantly more high vigilance stage A and less low vigilance stage B. They concluded that responders to antidepressants show a higher brain arousal level compared to nonresponders and that could confirm the hypothesis of a higher brain arousal level in responders compared to nonresponders to antidepressant treatment.39 Olbrich et al. investigated the hypothesis of a decline in CNS and autonomic nervous system (ANS) arousal by treating depressed patients with selective serotonin reuptake inhibitor (SSRI). The data were derived from a small, independent exploratory dataset (N ¼ 25) and replicated using data from the randomized international Study to Predict Optimized Treatment Response in Depression (iSPOT-D; N ¼ 1008). CNS arousal was assessed using VIGALL (see previously). Analysis of the exploratory dataset revealed a significantly more negative CNS arousal slope (P < .03; Cohen’s d ¼ 0.84) and a trend for a faster declining ANS arousal (P < .06; Cohen’s d ¼ 0.94) in responders compared with nonresponders to SSRI treatment after 2 weeks. Analysis of iSPOT dataset results were not significant for CNS arousal slope (P ¼ .57; Cohen’s d ¼ 0.34) but were for ANS arousal slope (P < .04; Cohen’s d ¼ 0.86).40 Taken exploratory dataset and iSPOT dataset together, when the means of ANS and CNS arousal parameters were used to assign subjects to SSRI or SNRI treatment retrospectively, response rates for SSRI treatment increased from 63.5% to 73.3%, and remission rate increased from 47.1% to 58.4%. For treatment with the SNRI, response rates increased from 64.7% to 72.3%, and remission rates increased from 43.2% to 46.5%. These findings underline the importance of the RDoC announced by the National Institutes of Health and validate CNS and ANS arousal systems as future potential predictive biomarkers to guide positive treatment outcome in MDD patients.40 Acute stress disorder (ASD) characterized by presence of nine (or more) of symptoms from any of the five categories of intrusion, negative

mood, dissociation, avoidance, and arousal, beginning or worsening after the traumatic event(s), experienced during the first month of the trauma. Arousal symptoms include sleep disturbance, irritable behavior, and angry outbursts (with little or no provocation), typically expressed as verbal or physical aggression toward people or objects, hypervigilance, problems with concentration, and/or exaggerated startle response.33 Recent evidence points to hyperarousal being a critical component of the acute trauma response, and that hyperarousal is associated with acute psychopathology levels. Nixon and Bryant provide evidence that re-experiencing is directly associated with elevated states of arousal, by investigating Civilian trauma survivors with (n ¼ 18) and without ASD (n ¼ 14), using hyperventilation provocation test (HVPT) and Physical Reactions Scale (PRS). They found that significantly more ASD participants described flashback experiences (72%) than non-ASD participants (29%), c2(1, n ¼ 32) ¼ 4.40, P < .05. Similarly, ASD participants were more distressed as a result of the HVPT procedure (72%) than non-ASD participants (7%), c2(1, n ¼ 32) ¼ 11.04, P < .001. ASD participants had more intense flashback-type experiences (M ¼ 2.23  0.73) than non-ASD participants (M ¼ 1.25  0.50, t(15) ¼ 2.50, P < .05).41 ASD participants reported higher arousal as a result of the HVPT on the PRS (M ¼ 17.28  11.91) than non-ASD participants (M ¼ 9.36  9.24, t(30) ¼ 2.05, P ¼ .05). ASD participants reported greater avoidance of traumarelated thoughts during the experiment (M ¼ 6.04  2.33) than non-ASD participants (M ¼ 3.67  1.99, t(30) ¼ 3.05, P < .005). Pearson correlations indicated that the number of intrusions was positively correlated with PRS scores (r ¼ 0.42, P < .05). The findings provide evidence that re-experiencing of the trauma is directly associated with elevated states of arousal.41 Stress Trauma Symptoms Arousal Regulation Treatment (START) is a short manualized structured intervention to stabilize and modulate arousal for highly stressed minor refugees through working with them immediately on arrival to Germany. It is used for children and

PSYCHIATRIC DISORDERS ASSOCIATED WITH HYPERAROUSAL

adolescents suffering from intense stress and acute tension or desperation. START was accepted by the refugee children and adolescents and observed to reduce stress in children and supervising professionals. Its efficacy and effectiveness are currently targets of a standardized pre- and post-test evaluation.42 Posttraumatic stress disorder (PTSD) criteria include exposure to a stressor beyond the normal range of human experience, symptoms clustering in three areas that interfere with daily functioning: re-experiencing the trauma, avoiding the stimuli associated with the trauma, and experiencing increased arousal levels. Those symptoms typically remain more than 4 weeks.33 Hyperarousal can manifest itself as sleep difficulties, hypervigilance, startle response, and intrusive thoughts.43 PTSD brain dysfunction has been presented under a frontolimbic model that includes the amygdala, medial prefrontal cortex (mPFC), and hippocampus as core-implicated structures. This model explained that an overactive amygdala is responsible for heightened arousal and exaggerated fear, aggravated by loss of topdown inhibition due to a dysfunctional mPFC; the hippocampus fails to identify safe or otherwise nonthreatening situations thereby contributing to avoidance and re-experiencing.44 Akiki et al.45 tried to present the evidence of functional alterations in the broader framework of large-scale network dysfunctiondthe salience network (SN), which is involved in the detection of salient internal and external stimuli. Core structures that are part of the SN are the amygdala, insula, and dorsal anterior cingulate cortex. Within the SN, based on the perceived threat level, the anterior insula is thought to modulate the dynamics between central executive network (the middle frontal gyrus, precuneus, and parts of the premotor cortex) and default mode network (posterior cingulate cortex, ventromedial prefrontal cortex, and medial temporal lobe, including the hippocampus). Consequently, this dysfunction in the SN may alter the threat detection functions and could underlie behaviors such as hyperarousal.45 BDNF, which is known to regulate neuronal survival, growth, differentiation, and synapse

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formation hence the plasticity of the brain, can also regulate the stress response. It has been implicated in a number of psychiatric disorders, such as MDD and PTSD. A common singlenucleotide polymorphism in the BDNF gene leading a valine to methionine substitution at position 66 (Val66Met) influences human hippocampal volume, memory, and susceptibility to PTSD.46,47 Startle is a core symptom of hyperarousal in PTSD observed to be associated with polymorphism. The association between BDNF Val66Met and the startle score of PTSD Checklist has been studied by Zhang et al. Met/Met frequency distribution was significantly different between subjects with and without exaggerated startle. The frequency of the Met/Met genotype was almost fourfold (12.2% vs. 3.3%) higher in subjects with exaggerated startle than in those without exaggerated startle. In addition, the frequency of the Met allele was higher in subjects with exaggerated startle than in those without exaggerated startle (24.4% vs. 15.3%), indicating that Met/Met is associated with hyperarousal vulnerability.48 Chronic hyperarousal can lead to abnormal levels of stress-related hormones such as norepinephrine and cortisol or change in the number or sensitivity of receptors to these substances as it makes victims of past traumatic events more vulnerable to current life stressors through a process of sensitization or it may alter certain brain structures, such as the hippocampus.49 There is evidence that corticotropin releasing factor (CRF) and norepinephrine (NE) interact to increase fear conditioning and encoding of emotional memories, through a feedforward circuit connecting the amygdala and the hypothalamus with the LC, to enhance arousal and vigilance and integrate endocrine and autonomic responses to stress.50 Baker et al. measured CRF in CSF using serial cerebrospinal fluid (CSF) sampling in a group of 11 combat veterans with PTSD and 12 matched normal volunteers, they found high basal CSF CRH concentrations in veterans than in normal subjects (55.2 pg/mL  16.4 vs. 42.3 pg/ mL  15.6); no correlation was found between CSF CRH concentrations and PTSD symptoms,

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while there was no significant difference between groups in 24-h urinary-free cortisol excretion, the correlation between 24-h urinary-free cortisol excretion, and PTSD symptoms was negative and significant (Baker et al., 1999). In various animal models, increased CNS CRF activity may promote certain cardinal features of PTSD, as conditioned fear responses, increased startle reactivity, sensitization to exposure to stressors, and hyperarousal.50 Several studies have demonstrated the efficacy of alpha-1 adrenergic blocker in reducing nightmares and hyperarousal related to PTSD. Raskind and colleagues tested 10 Vietnam combat veterans with chronic PTSD and severe trauma-related nightmares, using prazosin and placebo in a 20week double-blind crossover protocol. They found that subjects were more improved when they were taking prazosin (M ¼ 9.5 mg/day at bedtime 0.5) than when they were taking placebo on the primary outcome measures of nightmares, sleep disturbance, and global change in PTSD severity and functional status. Moreover, prazosin was more effective for re-experiencing, avoidance, and hyperarousal symptom cluster scores as well as total scores on the ClinicianAdministered PTSD Scale. Effect size analyses for dependent variables showed robust and clinically meaningful reductions in symptoms across all outcomes measured.51 It is hypothesized that yohimbine (an alpha-2 adrenergic receptor antagonist) increases noradrenergic activity and so emotional distress during prolonged exposure therapy (PE), which is considered a gold-standard treatment for PTSD. Yohimbine facilitates enhanced emotional engagement with trauma memories in PTSD so that PE “can correct information by pairing them with distress for new learning to occur”.52 Tuerk and colleagues52 conducted a randomized placebo-controlled double-blind clinical trial for 5 years. The trial investigated the effects of pairing one 21.6 mg oral dose of yohimbine with the first imaginal exposure in PE on trauma-related HR reactivity (primary outcome) and on the slope of patient-rated PTSD, depression, and exposure-related distress throughout the remaining course of treatment (secondary outcomes) in the intention-to-treat sample.

The sample consisted of 26 male combat veterans of Operations Enduring Freedom and Iraqi Freedom, they found that participants randomized to yohimbine were more likely to experience an increase in HR from the time of drug administration to 1 h later, compared with placebo (c2 ¼ 3.91, N ¼ 26, P < .04, adj. P ¼ .09), with 43% of the yohimbine group, and only 8% of the placebo group experiencing an increase of at least five beats per minute. Participants randomized to yohimbine also evidenced increased systolic BP 1 h after drug administration compared with placebo (t ¼ 2.17, df ¼ 23, P ¼ .02, adj. P ¼ .04, d ¼ 0.66), with an average increase of 7.5 mm Hg (8.06, 95% confidence interval [CI]: 2.85e12.15) and no increase for placebo, 0.58 mm Hg (7.23, 95% CI: 4.01 to 5.18). Yohimbine resulted in increased physiological arousal and subjective distress during the drug/exposure visit compared with placebo led to significantly lower trauma-cued HR reactivity 1 week after administration and greater between- and within-session declines in distress.52 Further studies are needed to replicate the findings. Generalized anxiety disorder (GAD) is characterized by excessive anxiety or worry over more than 6 months. That is present most of the time regarding many activities with inability to manage these symptoms and at least three of the following: restlessness, fatigue, problems concentrating, irritability, muscle tension, and problems with sleep. These symptoms result in problems with functioning.33 To diagnose GAD using ICD-10, at least one from autonomic arousal symptoms must be preset (palpitations or pounding heart or accelerated HR, sweating, trembling or shaking, dry mouth [not due to medication or dehydration]). Barlow has termed the fundamental process to conceptually understand anxiety disorders as “anxious apprehension.” Anxious apprehension refers to a future-oriented mood state in which one becomes ready or prepared in an attempt to cope with upcoming negative events. This mood state is associated with a state of high negative affect and chronic overarousal, a sense of uncontrollability, and an attentional focus on threat-related stimuli. The content of anxious

PSYCHIATRIC DISORDERS ASSOCIATED WITH HYPERAROUSAL

apprehension varies from disorder to disorder (e.g., anxiety over future panic attacks in panic disorder, anxiety over possible negative social evaluation in social phobia).53 Pathological worry shifts the nature of the cognition toward negative verbal thoughts as denoted by “the Cognitive Avoidance Theory,” which proposes that worry is implemented by patients as an avoidance strategy, aimed at controlling physiological arousal engendered by anxiety.54,55 Makovac and colleagues used resting-state functional magnetic resonance imaging and measure HR variability (HRV) in 19 patients with GAD and 21 control subjects to define neural correlates of autonomic and cognitive responses before and after induction of perseverative cognition. They found that patients with GAD have higher HR compared with the healthy control (67.35  8.83 vs. 61.65  7.63, P < .001), with baseline HR being lower compared with HR after the induction (63.84  9.3 vs. 65.37  8.63, P < .05). Compared with HC subjects, patients with GAD reported lower connectivity between the right amygdala and right superior frontal gyrus, right paracingulate/anterior cingulate cortex, and right supramarginal gyrus. They link functional brain mechanisms to parasympathetic autonomic dyscontrol, highlighting overlap between cognitive and autonomic responses in patients with GAD.56 One of the two principal components that should form the targets of a treatment intervention for GAD is the persistent overarousal accompanying the uncontrollable worry. Brown and colleagues’ relaxation training in their treatment protocol for GAD taught patients the rationale that relaxation is aimed at alleviating the symptoms associated with the physiological component of anxiety, partly via the interruption of the learned association between autonomic overarousal and worry.57 Obsessiveecompulsive disorder (OCD) is characterized by presence of obsessions, compulsions, or both. Obsessions are defined by recurrent, persistent, intrusive and unwanted thoughts, urges, or impulses, causing marked anxiety or distress, and the individual attempts to ignore, neutralize, or suppress such thoughts, urges, or images, with some other thought or

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action. Compulsions are defined by repetitive behaviors or mental acts that the individual feels driven to perform in response to an obsession or according to rules that must be applied rigidly; they are aimed at preventing or reducing anxiety or distress.33 The neurobiological basis of emotional experience is based on the interaction between the limbic brain areas and prefrontal control mechanisms to appraise salient stimuli and adequately regulate emotional responses; exaggerated anxiety in OCD has been linked to functional changes in these brain areas.58,59 Besides abnormal neural activity in those brain areas during threat processing, clinical anxiety is also characterized by excess attention to threatening stimuli; enhanced processing of phobic stimuli is reflected in the late positive potential (LPP) in the event-related potential (ERP). The LPP shows its maximum effect over centro-parietal scalp sites and is enhanced by emotional compared with neutral pictures.60 This was investigated by Paul et al.61 24 patients with OCD and 24 HC were studied using ERPs to disorder-relevant, to record aversive and neutral pictures while participants were instructed to either maintain or reduce emotional responding using cognitive distraction or cognitive reappraisal. They found that relative to OCD patients, HCs showed greater regulation effects in the LPP for both distraction (t(46) ¼ 2.25, P ¼ .03) and reappraisal (t(46) ¼ 1.81, P ¼ .08). OCD patients rated aversive pictures as less arousing when using reappraisal compared with distraction (t(23) ¼ 3.18, P ¼ .01), which was absent in HCs (P ¼ .99), and only distraction reduced arousal in response to neutral pictures in HC (t(23) ¼ 2.53, P ¼ .06), while reappraisal failed to reach significance (P ¼ .17). This should draw more attention for further investigation because quickly responding to aversive stimuli has proven to be critical for survival.61 Further studies showed morphometric gray matter abnormalities in regions associated with the frontal-subcortical loops, and functional neuroimaging studies demonstrated activation of the orbitofrontal and anterior cingulated loops that are associated with provocation of OCD symptoms.62 Gonc¸alves et al. investigated 15 patients with OCD, and 12 healthy controls

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underwent functional magnetic resonance imaging acquisition while being exposed to emotional pictures, with different levels of arousal. They found that patients with OCD when compared with healthy controls showed significantly less activation in the superior occipital gyrus, the right precentral gyrus, left paracentral lobule, left superior occipital gyrus, and left fusiform gyrus. That means OCD patients show evidence of altered basic survival circuits, particularly those associated with the visual processing of the physical characteristics of emotional stimuli.63 Olbrich et al.64 studied unmedicated OCD patients altered vigilance regulation during a 15min resting-state EEG recording in comparison to healthy controls. Thirty OCD patients and 33 HC enrolled. The post-hoc Scheffe´ test revealed a significantly higher EEG vigilance for OCD patients in comparison to HCs for minutes 9e12 (P < .003).64 This finding may be in line with study showed increased cortisol and adrenocorticotropic hormone,65 which represents the neuro-endocrinological analogy of altered EEG vigilance regulation in OCD. Differences in sleep behavior have been documented in patients with OCD, especially delayed sleep phase disorder (DSPD) in patients with severe OCD.66 Nota et al. examined quantitative information about the sleep of patients with OCD in comparison to healthy individuals by doing a meta-analysis including 12 articles. They found that sleep duration was shorter in individuals with OCD compared with healthy individuals. The magnitude of this difference is in the medium range (g ¼ 0.60; 95% CI: 0.90 to 0.31); heterogeneity among studies was low and not statistically significant (Q ¼ 11.81, P ¼ .22; I2 ¼ 23.81%), and the prevalence of DSPD in the individuals with OCD was also significantly greater than healthy individuals. The magnitude of this difference is large (g ¼ 2.28; 95% CI: 1.28, 3.27); heterogeneity among studies was moderate but not statistically significant (Q ¼ 4.72, P ¼ .09; I2 ¼ 57.61%). 67 Further studies are needed to clarify these findings. A retrospective study was performed by Dohrmann et al. to examine whether EEGbased CNS arousal markers differ for patients

suffering from OCD that either respond or do not respond to cognitive behavioral therapy (CBT), SSRIs, or their combination using VIGALL, and to identify specific responsepredictors for the different therapy approaches, Clinical Global Impression scores were used to assess response or nonresponse after 3e 6 months following therapy (CBT, n ¼ 18; SSRI, n ¼ 11; or combination, n ¼ 22). Fifty-one patients enrolled. These results revealed that there is a significant difference between responders and nonresponders only for stage 0 with F(1, 49) ¼ 5.76, P < .02, but for no other stage, responders spent significant less time at highest CNS-arousal stage 0. Comparisons between the wakefulness profiles of responders of the three treatment groups revealed that subjects with lowest wakefulness profiles (i.e., lowest amounts of high arousal stages) were more likely to respond to a combined treatment approach than to SSRI or CBT treatment alone.68

CONDITIONS FEATURING AROUSAL DYSREGULATION Bipolar affective disorder involves the alternation between manic, hypomanic, and depressive episodes. Manic episodes are characterized by abnormally elevated mood lasting at least 1 week, together with inflated self-esteem, talkativeness, and flight of ideas, distractibility, increased goal-directed activity, decreased need for sleep, and excessive involvement in pleasurable activities. Hypomania is distinct from mania in that there is no significant functional impairment and lasting at least four consecutive days.33 The vigilance regulation model denotes that in vulnerable subjects, “genetically” an unstable vigilancedthe term vigilance denotes tonic neurophysiologic arousaldinduces exaggerated autoregulatory behavior “sensation and novelty seeking, hyperactivity, talkativeness, distractibility, and impulsivity.” This behavior overrides the physiological tendency to seek sleep, thus aggravating the sleep deficits and therefore the instability of vigilance. A pathogenic vicious circle is started, which then contributes to fullblown mania.34

PSYCHIATRIC DISORDERS ASSOCIATED WITH HYPOAROUSAL

HRV as one of arousal regulation measures has been reviewed by Faurholt-Jepsen et al. in a meta-analysis included 15 articles, for it being an important risk factor for coronary heart disease, atherosclerosis, heart failure, and arrhythmias when it is reduced. Reduced HRV (g ¼ 1.77, 95% CI: 2.46 to 1.09, P < .001, 10 comparisons, n ¼ 1581) was observed in patients with bipolar disorder compared with healthy control individuals.69 It is possible that a reduced HRV in bipolar disorder could predict sudden cardiac death in this population, further prospective and retrospective studies are needed to further investigate it. Wittekind and colleagues investigated brain arousal regulation in different affective episodes in patients with bipolar disorder using VIGALL (see previously). Twenty-eight patients with bipolar disorder received a 15-min resting EEG during a depressive episode, 19 patients received the same during a manic/hypomanic episode and 28 healthy control subjects. When comparing patients and controls, unstable arousal regulation was highest in patients with manic episodes, who showed significantly less stable arousal regulation than controls (P ¼ .004, h2 ¼ 0.168); however, patients with depressive episodes showed significantly more stable arousal regulation than patients with manic episodes (P  .001, h2 ¼ 0.257). By comparing the groups, they revealed that manic patients had the lowest vigilance level, with the mean vigilance values being significantly lower than that in the control sample (F(1, 45) ¼ 4.981, P ¼ .031, h2 ¼ 0.100), while depressive patients showed the highest vigilance level with a significantly higher mean vigilance than both manic patients (F(1, 45) ¼ 19.246, P  .001, h2 ¼ 0.300) and healthy controls (F(1, 54) ¼ 4.213, P ¼ .045, h2 ¼ 0.072).70 Brain arousal-stabilizing drugs have been studied in the last decade for their potential to stabilize vigilance dysregulation in manic and attention deficit hyperactivity disorder (ADHD) patients.71e77 Based on these findings, an international randomized placebo-controlled clinical trial was started to assess efficacy and safety of treatment with methylphenidate in mania.78 Forty-two patients were randomly assigned to

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receive 20e40 mg per day of methylphenidate or placebo. Futility was declared for methylphenidate, as there was no significant difference between both groups F(1, 37) ¼ 0.23; P ¼ .64; difference from placebo ¼ 4.50 points; effect size (Cohen’s d) ¼ 0.48; 95% CI: 1.08 to 0.14. Given this result, the randomized controlled trial was stopped.79 Those results are in line with the cohort done using linked Swedish national registries on 2307 adults with bipolar disorder who initiated therapy with methylphenidate between 2006 and 2014. They found that manic patients on methylphenidate monotherapy displayed an increased rate of manic episodes within 3 months of medication initiation (hazard ratio ¼ 6.7, 95% CI: 2.0e22.4), while patients taking mood stabilizers, the risk of mania was lower after starting methylphenidate (hazard ratio ¼ 0.6, 95% CI: 0.4e0.9).80

PSYCHIATRIC DISORDERS ASSOCIATED WITH HYPOAROUSAL ADHD is childhood-onset psychiatric disorder, which is characterized by ageinappropriate levels of the core symptoms inattention, hyperactivity, and impulsivity.33 The presentation specifiers for ADHD are predominantly inattentive subtype and predominantly hyperactiveeimpulsive subtype. The inattentive subtype can be explained by unstable arousal regulation. In the combined presentation, additional autoregulatory aspects supervene with sensation seeking and hyperactivity as an attempt to stabilize arousal, according to vigilance regulation model “which denotes that in vulnerable subjects an unstable vigilance induces exaggerated autoregulatory behavior as hyperactivity, talkativeness, and distractibility,” so we can explain hyperactivity not as a primary disorder per se, but as an auto-regulatory response, which may or may not be present.81e83 Hypoarousal in ADHD has been documented for many years started by the work of Satterfield and Dawson; they illustrated a lower general skin conductance leveldan established indicator of autonomic arousaldin ADHD patients at

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rest.84 Several resting EEG studies have also suggested ADHD-associated hypoarousal in children and adults by demonstrating excess of slow frequencies, especially increased theta and reduced faster alpha and beta activities.85e87 The increased ratio of theta-to-beta activity (TBA) during rest has perceived as a marker of the disorder indicating hypo arousal.88e90 Further studies are needed to examine TBA and its correlation to executive functions.91e93 Unstable vigilance and behavioral variability in ADHD might be due to altered connectivity between regions of the default mode network, which is a distributed set of brain regions in frontal (inferior frontal cortex), parietal (precuneus, inferior parietal cortex), temporal (inferior temporal gyrus, amygdala), and medial regions.94e96 When arousal mechanisms are performing at a low level, it is claimed that arousal-related neurons utilizing NE and DA and pyramidal neurons in the prefrontal cortex are unable to distinguish important neuronal signals from unimportant signals. These patients cannot focus on one thing and cannot sustain attention because it is easy to be distracted from one signal to another, as if all signals are the same.97 Psychostimulants therapeutic effects are well established in treatment of ADHD. The rapid effects of stimulants could be explained by their arousal-stabilizing properties, which could interrupt the autoregulatory hyperactivity and sensation-seeking behavior.98e100This effect was documented by reduction of EEG slow-wave activity under treatment.101 Children having less beta activity show a good response to treatment with methylphenidate.102 Ludyga and colleagues103 examined the effect of acute moderately intense aerobic exercise on cognitive flexibility and task-related HRV in children with ADHD (n ¼ 18) and healthy controls (n ¼ 18) in a cross-over design. The analysis indicated a lower HR during cognitive testing following the control condition (78.8  10.3 bpm) compared with aerobic exercise (85.8  10.2 bpm); F(1, 32) ¼ 26.5, P < .001, h2 ¼ 0.45. Regarding the acute effects of exercise on task performance, the results revealed a significant multivariate main effect for condition, Wilks’s l ¼ 0.725, F(4, 29) ¼ 2.8, P ¼ .047,

h2 ¼ 0.28, and power to detect the effect was 0.68, these effects indicated higher scores following aerobic exercise compared with the control condition. In this respect, acute intense aerobic exercise might be seen as a complementary treatment, which allows a temporary enhancement of executive function beyond the normal range.103 Further studies are needed to replicate the finding.

SUMMARY Changes in arousal are associated with many neuropsychiatric disorders. For example, hyperarousal is associated with major depression, GAD, and PTSD; while hypoarousal correlates with ADHD. Arousal can be assessed by cognitive, psychological, or autonomic measures. Shedding the light on the brain mechanisms for fine tuning of arousal levels potentially can help in the understanding and treatment of certain psychiatric disorders.

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72. Waxmonsky J, Pelham WE, Gnagy E, et al. The efficacy and tolerability of methylphenidate and behavior modification in children with attention-deficit/hyperactivity disorder and severe mood dysregulation. J Child Adolesc Psychopharmacol. 2008;18(6):573e588. 73. Carlson PJ, Merlock MC, Suppes T. Adjunctive stimulant use in patients with bipolar disorder: treatment of residual depression and sedation. Bipolar Disord. 2004;6(5):416e420. 74. El-Mallakh RS. An open study of methylphenidate in bipolar depression. Bipolar Disord. 2000;2(1):56e59. 75. Fernandes PP, Petty F. Modafinil for remitted bipolar depression with hypersomnia. Ann Pharmacother. 2003;37(12):1807e1809. 76. Lydon E, El-Mallakh RS. Naturalistic long-term use of methylphenidate in bipolar disorder. J Clin Psychopharmacol. 2006;26(5):516e518. 77. Nasr S, Wendt B, Steiner K. Absence of mood switch with and tolerance to modafinil: a replication study from a large private practice. J Affect Disord. 2006; 95(1e3):111e114. 78. Kluge M, Hegerl U, Sander C, et al. Methylphenidate in mania project (MEMAP): study protocol of an international randomized double-blind placebo-controlled study on the initial treatment of acute mania with methylphenidate. BMC Psychiatry. 2013;13:71. 79. Hegerl U, Mergl R, Sander C, et al. A multi-centre, randomised, double-blind, placebo-controlled clinical trial of methylphenidate in the initial treatment of acute mania (MEMAP study). Eur Neuropsychopharmacol. 2018;28(1):185e194. 80. Viktorin A, Ryde´n E, Thase ME, et al. The risk of treatment-emergent mania with methylphenidate in bipolar disorder. Am J Psychiatry. 2017;174(4):341e348. 81. Hurtig T, Ebeling H, Taanila A, et al. ADHD symptoms and subtypes: relationship between childhood and adolescent symptoms. J Am Acad Child Adolesc Psychiatry. 2007;46:1605e1613. 82. Willcutt EG, Nigg JT, Pennington BF, et al. Validity of DSM-IV attention deficit/hyperactivity disorder symptom dimensions and subtypes. J Abnorm Psychol. 2012; 121:991e1010. 83. Geissler J, Romanos M, Hegerl U, et al. Hyperactivity and sensation seeking as autoregulatory attempts to stabilize brain arousal in ADHD and mania? ADHD Atten Def Hyp Disord. 2014;6:159e173. 84. Satterfield JH, Dawson ME. Electrodermal correlates of hyperactivity in children. Psychophysiology. 1971;8(2): 191e197. 85. Barry RJ, Clarke AR, Johnstone SJ. A review of electrophysiology in attention-deficit/hyperactivity disorder: I. Qualitative and quantitative electroencephalography. Clin Neurophysiol. 2003;114(2):171e183. 86. Paucke M, Sander C, Hegerl U, et al. P 150 the correlation of attention and neurophysiological characteristics in attention deficit hyperactivity disorder (ADHD). Clin Neurophysiol. 2017;128(10):401.

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87. Strauß M, Ulke C, Paucke M, et al. Brain arousal regulation in adults with attention-deficit/hyperactivity disorder (ADHD). Psychiatry Res. 2018;261:102e108. 88. Monastra VJ, Lubar JF, Linden M, et al. Assessing attention deficit hyperactivity disorder via quantitative electroencephalography: an initial validation study. Neuropsychology. 1999;13(3):424e433. 89. Clarke AR, Barry RJ, Dupuy FE, et al. Excess beta activity in the EEG of children with attention-deficit/hyperactivity disorder: a disorder of arousal? Int J Psychophysiol. 2013;89(3):314e319. 90. Bresnahan SM, Barry RJ. Specificity of quantitative EEG analysis in adults with attention deficit hyperactivity disorder. Psychiatry Res. 2002;112(2):133e144. 91. Barry RJ, Clarke AR, Johnstone SJ, et al. Electroencephalogram theta/beta ratio and arousal in attention-deficit/hyperactivity disorder: evidence of independent processes. Biol Psychiatry. 2009;66(4): 398e401. 92. Hsu CF, Broyd SJ, Helps SK, et al. “Can waiting awaken the resting brain?” A comparison of waitingand cognitive task-induced attenuation of very low frequency neural oscillations. Brain Res. 2013;1524: 34e43. 93. Zhang DW, Li H, Wu Z, et al. Electroencephalogram theta/beta ratio and spectral power correlates of executive functions in children and adolescents with AD/ HD. J Atten Disord. 2017, 1087054717718263. 94. Fair DA, Posner J, Nagel BJ, et al. Atypical default network connectivity in youth with attention-deficit/ hyperactivity disorder. Biol Psychiatry. 2010;68(12): 1084e1091. 95. Tian L, Jiang T, Liang M, et al. Enhanced resting-state brain activities in ADHD patients: a fMRI study. Brain Dev. 2008;30(5):342e348. 96. Raichle ME, MacLeod AM, Snyder AZ, et al. .A default mode of brain function. Proc Natl Acad Sci USA. 2001; 98(2):676e682. 97. Petrescu-Ghenea C, Trutescu C, Mihailescu I, et al. Arousal modulation in ADHD. Rom J Child Adolesc Psychiatry. 2013;1(1). 98. Pietrzak RH, Mollica CM, Maruff P, et al. Cognitive effects of immediate-release methylphenidate in children with attention-deficit/hyperactivity disorder. Neurosci Biobehav Rev. 2006;30(8):1225e1245. 99. Riccio CA, Waldrop JJ, Reynolds CR, et al. Effects of stimulants on the continuous performance test (CPT): implications for CPT use and interpretation. J Neuropsychiatry Clin Neurosci. 2001;13(3):326e335. 100. Spencer T, Biederman J, Wilens T, et al. A large, double blind, randomized clinical trial of methylphenidate in the treatment of adults with attention-deficit/hyperactivity disorder. Biol Psychiatry. 2005;57(5):456e463. 101. Bresnahan SM, Barry RJ, Clarke AR, et al. Quantitative EEG analysis in dexamphetamine-responsive adults with attention-deficit/hyperactivity disorder. Psychiatry Res. 2006;141(2):151e159.

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102. Clarke AR, Barry RJ, McCarthy R, et al. EEG differences between good and poor responders to methylphenidate and dexamphetamine in children with attention-deficit/hyperactivity disorder. Clin Neurophysiol. 2002;113(2):194e205. 103. Ludyga S, Gerber M, Mu¨cke M, et al. The acute effects of aerobic exercise on cognitive flexibility and taskrelated heart rate variability in children with ADHD and healthy controls. J Atten Disord. 2018, 1087054718757647.

104. Rotge JY, Langbour N, Guehl D, et al. Gray matter alterations in obsessive-compulsive disorder: an anatomic likelihood estimation meta-analysis. Neuropsychopharmacology. 2010;35(3):686e691. 105. World Health Organization. International Statistical Classification of Diseases and Related Health Problems, 10th Revision (ICD-10). Geneva: WHO; 1992.

C H A P T E R

2 Resilience of the Brain and Body Bruce S. McEwen Laboratory of Neuroendocrinology, The Rockefeller University, New York, NY, United States O U T L I N E Introduction Brain-Body Basics Central Role of the Brain Plasticity of the Adult and Developing Brain Stress-Induced Structural Plasticity Epigenetics Brain Gene Expression Is Continually Changing Development of the Capacity for Resilience How the Brain Gets “Stuck”

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Prevention Neurobiological Mechanisms of Overcoming Loss of Resilience Some Examples of Opening Windows to Promote Resilience Other Top-Down Therapies That Change the Brain

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INTRODUCTION

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Conclusion

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References

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development over the life course is a “one-way street,” we need to understand that “reversal” is not possible and appreciate how resilience, recovery, and redirection are key to promoting a favorable trajectory over lifespan. There are a growing number of interventions that appear to promote beneficial changes in trajectory.

“Resilience” can be defined as the ability to achieve a successful outcome in the face of adversity. The purpose of this chapter is to put the concept of resilience into the context of the reciprocal communication between the brain and the body via neuroendocrine, autonomic, immune, and metabolic mechanisms viewed over the life course. What do we need to know? First, we need to understand the nonlinear, reciprocal communication between brain and body that promotes adaptation to a changing physical and social environment, often called “stress,” but which also can lead to pathophysiology when over used and/or dysregulated. Second, it is important to understand the epigenetic plasticity and vulnerability of the brain. Finally, since

Stress: Physiology, Biochemistry, and Pathology https://doi.org/10.1016/B978-0-12-813146-6.00002-3

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KEY POINTS • “Resilience” can be defined as the ability to achieve a successful outcome in the face of adversity. • This is particularly relevant to the brain, which is the central organ of stress and adaptation to stress because it perceives threatening challenges and determines physiological and behavioral responses.

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Copyright © 2019 Elsevier Inc. All rights reserved.

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• The brain together with the neuroendocrine, immune, autonomic, and metabolic systems controls “allostasis” (stability through adaptive physiological change) that maintains “homeostasis” (physiological equilibrium through stability). • Chronic stress and resulting changes in health-related behaviors such as diet, physical activity, and sleep contributes to pathophysiology (“allostatic load/ overload”). • The mature, as well as developing brain, possess a remarkable ability to show structural and functional plasticity and resilience in response to stressful experiences, by way of neuronal replacement, dendritic remodeling, and synapse turnover. • However, the life course is a “one-way street” and gene expression is continually changing via epigenetic mechanisms; one cannot “roll back the clock” and “reverse” change but rather promote “recovery” and “resilience.” • The purpose of this chapter is to put the concept of resilience into the context of the reciprocal communication between the brain and the body via neuroendocrine, autonomic, immune, and metabolic mechanisms and to discuss intervention strategies to promote brain and body health.

Brain-Body Basics In talking about resilience we often refer to the response to stress, which, however, is an ambiguous word. A more biological way of looking at stress is embodied in the concepts of “allostasis” and “allostatic load/overload” that are described later after distinguishing “good stress” from “tolerable stress” and from “toxic stress” based on definitions from the National Scientific Council for the Developing Child (http:// developingchild.harvard.edu/science/nationalscientific-council-on-the-developing-child/).

Good stress involves our taking a chance on something one wants, like interviewing for a job or school, or giving a talk before strangers, and feeling rewarded when we are successful. Tolerable stress means that something bad happens, like losing a job or death of a loved one, but we have the personal resources and support systems to weather the storm. Toxic stress refers to the response to a major life event where the individual does not have the personal resources or support systems, and, as a result, lacks a sense of control, leading to mental and physical health problems over time, particularly if the situation is not resolved. Now let us put these three forms of stress into a biological and behavioral context. We know that “homeostasis” means the physiological state that the body maintains to keep us alived that is, body temperature and pH within a narrow range and adequate oxygen supply. In order to maintain homeostasis, our body activates hormone secretion and turns on our autonomic and central nervous system (we call these “mediators” like cortisol, adrenalin, the immune system and metabolism) to help us adapt, for example, when we get out of bed in the morning, walk up a flight of stairs, or have a conversation. These systems are also turned on when we are surprised by something unexpected, or get into an argument, or run to catch a train. Some of these experiences we may refer to as “stressful” but others we do not. So using the word stress does not really recognize all of the underlying biology. The mediators help us adapt as long as they are turned on in a balanced way when we need them and then turned off again when the challenge is over. When that does not happen, they can cause unhealthy changes in brain and body. This is also the case when the mediators are not produced in an orchestrated and balanced manner; for example, too much or too little cortisol or an elevated or too low blood pressure. When this happens and continues over weeks and months, we call it allostatic load to refer to the wear and tear on the body that results from the chronic overuse and imbalance of the mediators. Accumulation of belly fat is an example, as is the development of chronic hypertension.

INTRODUCTION

When the wear and tear is strongest we call it allostatic overload, and this is what is occurring in toxic stress. An example is when hypertension leads to coronary artery blockade. In fact, belly fat contributes chemicals that accelerate that coronary artery blockade. Note, however, that we are talking not about one mediator, like cortisol, but a host of mediators that are all released in allostasis in a coordinated manner to help us adapt but which can also cause damage when overused and dysregulated as described earlier. Because cortisol is well known in relation to stress, its role is often misunderstood. We must go one step further and recognize that bad health behaviors, often the result of a stressful life style, like eating too much of the wrong things, smoking, drinking, loneliness, poor sleep, and lack of exercise, all contribute to that hypertension, belly fat, and blockade of coronary arteries. And they do so through the same mediators that are activated to help us adapt but that also, when overused and dysregulated, cause allostatic load and overload.1,2 So the mediators that help us adapt and enable us to maintain our homeostasis and survive can also contribute to well-known diseases of modern life. These ideas are the basis of the concepts of allostasis and allostatic load/overload, whereas they are not so obvious from the word stress, which is usually explained as the “fight or flight response” when we are, for example, threatened by a mugger and run away. What really affects our health and well-being are the more subtle, gradual, and long-term influences from our social and physical environment like family and neighborhood chaos and conflict, demands of a job, shift work and jet lag, sleeping badly, living in an ugly, noisy, and polluted environment, being lonely, not getting enough physical activity, eating too much of the wrong foods, smoking, and drinking too much alcohol. All these contribute to allostatic load and overload through the same biological mediators that help us adapt and keep us alive, and they shape our brains.

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Central Role of the Brain The brain is the central organ of stress and adaptation to stress because it perceives and determines what is threatening, as well as the behavioral and physiological responses to the stressor, which promote adaptation (allostasis) but also contribute to pathophysiology (allostatic load/overload) when overused and dysregulated. The brain “keeps the score” by storing memories from bad as well as good experiences, and yet the brain, working with the body, also “knows what to do” to keep us alive if we give it a chance by minimizing those subtle and long-term influences that cause allostatic load and overload.3 We call that the “wisdom of the body,” and it refers back to allostasis, the active process of biological adaptation, and its role in maintaining homeostasis. Indeed the brain is a plastic and vulnerable organ of the body and is continually sculpted by experiences. The brain is also a vulnerable organ, along with our heart, liver, and kidneys and other organs. The brain changes its architecture and function as part of allostasis, and the development of the notion of structural plasticity of the brain has come about over the last half century with an accelerating pace as will be described later! We also need to consider where our genes fit in and understand that they do not rigidly determine our destiny but, rather, provide the foundation on which our experiences shape our brains and bodies over the life course via “epigenetic” mechanisms that promote resilience. Finally, what does this tell us about interventions to either prevent adverse outcomes or redirect the brain and body in a healthier direction?

Plasticity of the Adult and Developing Brain Long regarded as a rather static and unchanging organ, except for electrophysiological responsivity, such as long-term potentiation,4 the brain has gradually been recognized as capable of undergoing rewiring after brain

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damage,5 and also able to grow and change as seen by dendritic branching, angiogenesis, and glial cell proliferation during cumulated experience.6,7 More specific physiological changes in synaptic connectivity were also recognized in relation to hormone action in the spinal cord,8 and in environmentally directed plasticity of the adult songbird brain.9 Seasonally varying neurogenesis in restricted areas of the adult songbird brain is recognized as part of this plasticity.10 Indeed, adult neurogenesis in the adult mammalian brain was initially described11,12 and then suppressed,13 only to be rediscovered in the dentate gyrus of the hippocampus14,15 in the context of studies of neuron cell death and actions of adrenal steroids and excitatory amino acids in relation to stress. Neurogenesis in the dentate gyrus has gone on to become a huge topic related to effects of stress,16 exercise,17 enriched environment,18 antidepressants,19 and learning and memory.20 More than neurogenesis, structural plasticity includes dendrite remodeling, synapse formation, and synaptic pruning. For example, a recent study shows how the brain architecture of a mother is sculpted during pregnancy as part of the formation of attachment to the child.21 Moreover, a musician’s brain develops with enhanced size and connections of sensory and motor control regions of the cerebral cortex!22

Hebb.30 For stress-induced remodeling, which can be mimicked by chronic glucocorticoid treatment, excitatory amino acids, and other cellular mediators are involved.25,31 What emerged from this as well as the work of Robert Sapolsky that emphasized damaging aspects of glucocorticoid action on hippocampus mediated also by excitatory amino acids32e34 is an inverted U-shaped dose-response curve (see Fig. 2.1) in which physiological levels of glucocorticoids and excitatory amino acids operate synergistically and beneficially to facilitate long-term potentiation (LTP) and memory, as shown by Constantine Pavlides at Rockefeller University and also by David Diamond35,36 over a short time frame of minutes to hours and promote dendritic remodeling over a time frame of weeks that shows resilience,25 but acute traumatic events such as stroke, seizures, and head trauma cause permanent damage and neuron loss via synergy between glutamate and glucocorticoids.33,37 The prefrontal cortex also responds to what we can call “tolerable stress.” As an example, in a group of medical student volunteers, perceived stress, i.e., how much or little they felt in control of their daily lives, revealed that those with the highest perceived stress were slower in doing a cognitive-flexibility test and also had slower functional connectivity in a brain circuit involving the prefrontal cortex when tested in a functional magnetic resonance

Stress-Induced Structural Plasticity Our demonstration of stress-induced remodeling of dendrites in hippocampal CA3 neurons provided a neuroanatomical mechanism that helped to explain behavioral effects of stress on memory and related processes.23e28 These discoveries have led to a cascade of investigations in stress neurobiology that have increasing relevance to human mental and physical health. What these findings helped to demonstrate is the remarkable feature of the adult as well as developing brain, namely, recognition of its capacity for remodeling of dendrites, turnover of synapses, and neurogenesis that began with the enriched environment studies on brain cortex thickness6,29 based on the work of Donald

FIGURE 2.1 Inverted U-shaped dose-response curve.

INTRODUCTION

imaging (MRI) machine.38 The reason we can call this tolerable stress is that, after a vacation, these impairments disappeared, showing the resilience, at least of the young adult brain! Parallel studies on an animal model of perceived stress revealed shrinkage of neuronal dendrites and reduction of synapses in the prefrontal cortex that explained the deficits in cognitive flexibility.39,40 To complete the present story of brain plasticity we need to describe what happens in the amygdala under the same stressors that cause dendrites to shrink and synapses to be lost in the prefrontal cortex and hippocampus, namely, that dendrites in the basolateral amygdala grow and become more branched and, as a result, there is increased anxiety. This was discovered in the laboratory of Sumantra Chattarji at the National Centre for Biological Sciences in Bangalore, India.41 Of note, dendrites in the orbitofrontal part of the prefrontal cortex also expand with chronic stress, and there is increased vigilance.39 In the short term, these changes may be adaptive, as anxiety and vigilance are adaptive in a dangerous or uncertain environment; but, if the threat passes and the behavioral state “gets stuck” and persists along with the changes in neural circuitry, such maladaptation requires intervention to open “windows of plasticity” with a combination of pharmacological and behavioral therapies. A special example of plasticity relates to posttraumatic stress disorder (PTSD). With Chattarji, we found that a single, traumatic stressor causes new synapses to form in basolateral amygdala with a delay of a week or so that is accompanied by a gradual increase in anxiety.42 This type of delay is a feature of human PTSD. What we have further shown with the Chattarji group is that a timed elevation of cortisol at the time or, or shortly after, a traumatic stressor actually prevents the delayed increase in amygdala synapses and anxiety-like behavior.43 Now there is evidence for human PTSD that low cortisol at the time of trauma, e.g., during open heart surgery or after a traffic accident, is a risk factor and that elevating cortisol during or right after trauma can reduce later PTSD symptoms.44,45

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Epigenetics What are the mechanisms for these changes in neuron structure and function? Indeed, they involve changes in gene expression caused by the stressful experience, and now the term epigenetics is used to refer to how experiences affect the brain and body to promote adaptation or maladaptation. Epigenetics originally meant something quite different, namely, the emergence of characteristics as a fertilized egg develops into a living organism characteristic of that species.46 This is programmed into each species, but the individual characteristics are influenced by experiences, and that is where the modern use of epigenetics comes from. An example of this is a pair of identical twins with genes that predispose them to schizophrenia or bipolar illness. Even with the same DNA, the probability that one twin will develop the disease when the other twin gets it is only in the range of 40%e60%, which leaves plenty of room for experiences and other environmental factors to either prevent or precipitate the disorder. As an indicator of this, the methylation patterns of DNA diverge as identical twins grow older.47 Thus, epigenetics now meaning “above the genome,” that is, not changing the genetic code, replaces and makes unnecessary the old question: “which is more important, genes or environment?” The CpG methylation of DNA is now a well-known form of epigenetic modification.48 Evidence from CpG methylation of DNA indicates the embedded influence of early adversity49 and this will be discussed further later. But there are other mechanisms that include histone modifications that repress or activate chromatin unfolding50 and the actions of noncoding RNAs,51 as well as transposons and retrotransposons52 and RNA editing.53 We shall see how this plays out for one brain region, the hippocampus.

Brain Gene Expression Is Continually Changing As the first extra-hypothalamic brain structure recognized to have receptors for adrenal steroids,54 the hippocampus is an important

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gateway for understanding the effects of glucocorticoids and stress on gene expression in the brain. Recent technological advances have allowed high-throughput analysis of gene expression changes in response to stress.55 For example, work by current postdoctoral fellow Jason Gray, using a microarray analysis of whole hippocampus after acute stress, chronic stress, and stress recovery in mice, revealed that acute and chronic stress modulate a core set of genes, but that numerous changes are exclusive to each condition, highlighting how duration and intensity of stress alters reactivity.56 Furthermore, corticosterone injections do not yield the same expression profile as acute stress, suggesting that in vivo stressors activate a diverse set of pathways independent of glucocorticoid receptor (GR) activation.56 Finally, characterization of expression profiles after extended recovery from 21 days of chronic stress showed that, despite a normalization of anxiety-related behaviors, recovery did not represent a return to the stress-naı¨ve baseline, but rather represents a new state in which reactivity to a novel stressor produces a unique expression profile.56 Studies in rats confirm that gene expression profiles can vary significantly from the immediate end of stress to 24 h later57 and that chronic stress can alter the transcriptional response to an acute corticosterone injection in dentate gyrus, as shown in a collaboration with Dutch scientists Nicole Datson and Ron de Kloet.58 Together, these studies demonstrate that a history of stress exposure can have a lasting impact on future stress reactivity and hippocampal function. It seems logical to assume that this generalizes to experiences that we have, whether or not we call them stress. Histone modifications are keys to epigenetic regulation of gene expression. Besides the acetylation of histones involved in the upregulation of mGlu2 gene expression described before, repressive epigenetic modifications of histones are also evident after acute and chronic stress, as shown by Richard Hunter, now a faculty member at UMass, Boston. Acute stress dramatically increased the levels of H3K9 trimethylation (H3K9me3) in the dentate gyrus (DG) and CA1,

while chronic restraint stress (CRS) for 21 days abolished this effect. Treatment with fluoxetine during CRS reversed the decrease in DG H3K9me3.59 To dig deeply into the substantial, regionally specific, increase in hippocampal levels of the repressive histoneH3 lysine 9 trimethylation (H3K9me3), Hunter used ChIP coupled with next-generation sequencing (ChIP-Seq) to determine the genomic localization of the H3K9me3 response. We found that acute stress increases in H3K9me3 trapped and therefore repressed expression enrichment at transposable element loci and, using RT-PCR, we demonstrated that this effect represses expression of intracisternal-A particle endogenous retrovirus elements and B2 short-interspersed elements, but it does not appear to have a repressive effect on long-interspersed element RNA. In addition, the histone H3K9-specific methyltransferases Suv39h2 is upregulated by acute stress in the hippocampus, and this may explain the hippocampal specificity.60 This response may represent a genomic stress response aimed at maintaining genomic and transcriptional stability in vulnerable brain regions such as the hippocampus, although the transposome might have adaptive functions at the level of both evolution and the individual organism.61

Development of the Capacity for Resilience Another important element so far in this discussion of stress is the influence of events early in life and their epigenetic effects on brain and body development in developing the capacity for resilience. Michael Meaney has led the way in demonstrating the important role of postnatal maternal care in emotional and cognitive development. Meaney and Robert Sapolsky investigated ontogeny of glucocorticoid receptors in the neonatal rat brain,62,63 which led them to later investigate the ability of neonatal “handling” of infant rats to slow down brain aging.64 Meaney went on to show that handling,65 i.e., separating pups from the mother for 10e20 min, increases maternal care when the pups are returned. Going on to explore the role

INTRODUCTION

of maternal care, Meaney and Darlene Francis showed that infant rats raised with a nurturing mom develop less emotionality and great ability to explore novel places and things, while pups raised with an anxious mom that provides inconsistent care shows the opposite outcome.66 This was done by Francis switching embryos in the womb67 and also by cross-fostering pups postnatally between mothers of two mouse strains.68 Subsequently, it was shown that the consistency of maternal care and not the amount has the most positive effect.69 Indeed, chaos in the nest has negative effects on the offspring as it does in humans.70,71 Cross-fostering of infants between good and bad moms alters the outcome, pointing to what is now referred to as epigenetic transgenerational behavioral transmission.66 John Kral at Downstate Medical Center, Brooklyn, New York, introduced other forms of transgenerational transmission of traitsdeven before conception and during life in the womb, paternal and maternal obesity can affect the child.72,73 These may involve epigenetic changes of the DNA of the sperm and egg that do not alter the genetic code per se but, rather, how it is read. In the case of parental obesity, this increases the risk that the child will also become obese. Indeed, as noted, we cannot “roll back the clock” and “reverse” the effects of experiences, positive or negative. Rather, we must think of “recovery” and “redirection” and “resilience,” rather than “reversal”56 (see Fig. 2.2). We therefore think about “changing trajectories” of function resulting in compensatory changes in the brain and body over the life course. A pediatrician researcher at UCLA, Neal Halfon, has written and spoken about “life course health development,” or LCHD, as the most up-todate overview of medicine, contrasting that with the view of “magic bullets” like penicillin that revolutionized treatment of infectious disease but does not apply to antidepressants or drugs, like statins, that help but do not “cure” the in-principle preventable diseases of modern life.74 Going beyond the psychosocial model of how health behaviors and toxic stress

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FIGURE 2.2 Gene expression hippocampus after a bolus of corticosterone compared to the effects of a novel acute forced swim (FST) in naı¨ve, chronically restrained (CRS) and recovered (Rec after CRS) showing largely unique gene expression responses, indicating that the brain is continually changing with experience. There is a set of genes always activated by FST (e.g., immediately early genes).56

cause those diseases,75 LCHD notes the importance of events preconception, prenatally, and throughout the life course in which income and education have a huge influence. The determinants of diabetes, depression, and dementia are a good example of this.76

How the Brain Gets “Stuck” Resilience is decreased and vulnerability is increased by adverse childhood experiences (ACE) and poverty that lead to “biological embedding” of trajectories of response to stressful life events77 throughout the life course,74 which contribute disproportionately to allostatic overload in the form of physical and mental health disorders over the life course and impaired brain development.78,79 Depression and anxiety disorders, often exacerbated by early life adversity, illustrate loss of resilience. This means that changes in brain circuitry and function, caused by the stressors that precipitate the disorder, become “locked” in a particular state and thus need external intervention. Indeed, prolonged depression is associated with shrinkage of the hippocampus80,81 and prefrontal cortex.82 While there appears to be no neuronal loss, there is evidence for glial cell loss and smaller neuronal cell nuclei,83,84 which is consistent with a shrinking of the dendritic tree

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described previously after chronic stress. As far as reversal of these changes, there are a few studies that indicate that pharmacological treatment reverses the decreased hippocampal volume in unipolar85 and bipolar86 depression, but the possible role of any concurrent cognitive-behavioral therapy in these studies is unclear. Aging is also an example of loss of resilience to the effects of chronic stress, based on studies of the rodent prefrontal cortex.87 What is not clear yet is whether this loss of resilience can be reversed or prevented, although pharmacological studies do indicate some retardation of age-related changes in morphology, neurochemical markers, and cognitive function.88,89 Although not directly addressing recovery of resilience, studies of the beneficial effects of physical activity on the aging brain are revealing the retention with age of the capacity for structural plasticity. There is loss of resilience with aging87 that can be redirected by exercise90 and possibly by pharmacological intervention.89 Next we consider more examples of this, after first considering interventions that prevent adverse outcomes before they can occur.

Prevention Adverse early life experiences for a child can be prevented by interventions with the family. One example is visitation by a skilled nursesocial worker who provides support and information to an expectant mother and her partner, if there is one. The “Nurse-family Partnership” (https://www.nursefamilypartnership.org/) has a documented success in reducing abuse and neglect.91 Moreover, it is possible to intervene later in a child’s life to reduce the progression of effects of poverty and discrimination, as shown in a recent report on a 7-week intervention with 11-years-old African American youth and their caregivers; there were reductions in depression and prevention of brain atrophy by 18 and 25 years of age, respectively.92 But when there is substantial loss of resilience, other strategies must be employed.

Neurobiological Mechanisms of Overcoming Loss of Resilience A totally different domain of neuroscience has brought some new thinking about what might be possible when the brain is “stuck”dnamely, the reversal of amblyopia and other conditions by “releasing the brakes” that retard structural and functional plasticity.93 Brain-derived neurotrophic factor (BDNF) may be a key feature of the depressive state, and elevation of BDNF by diverse treatments ranging from antidepressant drugs to regular physical activity may be a key feature of successful treatment.94 Yet, there are other potential applications, such as the recently reported ability of fluoxetine to enhance recovery from stroke.95 However, a key aspect of this new view96 is that the drug is opening a “window of opportunity” that may be capitalized on by a positive behavioral intervention, e.g., behavioral therapy in the case of depression or intensive physiotherapy to promote neuroplasticity to counteract the effects of a stroke. “Opening a window of plasticity” is consistent with studies in animal models that show that ocular dominance imbalance from early monocular deprivation can be reversed by patterned light exposure in adulthood that can be facilitated by fluoxetine, on the one hand,97 and caloric restriction, on the other hand,98 in which reducing inhibitory neuronal activity appears to play a key role. Investigations of underlying mechanisms for the reestablishment of a new window of plasticity are focusing on the balance between excitatory and inhibitory transmission and removing molecules that put the “brakes” on such plasticity.93 The caloric restriction study also showed that putting cortisol in the drinking water instead of caloric restriction98 was able to open a window of plasticity and enable binocular visual stimulation to correct amblyopia. This may be explained, at least in part, by the key role of physiologic levels of cortisol in promoting turnover of spine synapses and the important of circadian patterns of glucocorticoid elevation in spine formation and elimination in relation to

INTRODUCTION

motor learning and possibly other forms of learning.99,100 We shall now summarize some examples of interventions that open windows of plasticity and use them to promote resilience from adverse experiences or the aging process that causes the brain to “get stuck.”

Some Examples of Opening Windows to Promote Resilience Regular physical activity: Regular physical activity has effects not only on the cardiovascular and metabolic systems but also on the brain. It improves prefrontal and parietal cortex blood flow and enhances executive function.101 Moreover, regular physical activity, consisting of walking an hour a day, 5 out of 7 days a week, increases hippocampal volume in previously sedentary elderly adults,90 and this complements another study showing that fit individuals have larger hippocampal volumes than sedentary adults of the same age range.102 Regular physical activity is an effective antidepressant and protects against cardiovascular disease, diabetes, and dementia.103,104 Moreover, intensive learning has also been shown to increase the volume of the human hippocampus, based on a study on medical students.105 Perception based therapy: A new therapeutic approach106 is based upon training older adults in visual perceptual discrimination using Gabor patches that have built-in animation for directed motion.107 Ten hours of training were found to improve on-task perception, and the training also benefitted working memory for a delayedrecognition motion direction task. Moreover, electroencephalography (EEG) showed that training produced more efficient sensory encoding of the stimuli, which correlated with gains in working memory performance. This finding fits with other evidence that perceptual training improves the ability to detect signal over noise and thus produces some generalized cognitive benefits. The authors suggest that there are two fundamental design elements that drive neuroplasticity in this type of intervention, because they personalize training to the capacity of each person and allow abilities to improve over time. To do so, the training incorporates

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continuous performance feedback to provide repeated cycles of reward to the subject. Moreover, training is designed to adapt to the trainee’s on-going performance using psychophysical staircase functions that enhance the challenge in response to accurate performance and reduce it for inaccurate performance. Mindfulness and meditation: Therapies addressing functional links between brain and body may be particularly effective in treating the range of symptoms associated with many chronic diseases.108 Successful cognitive behavioral therapies, which are tailored to individual needs, can produce volumetric changes in both prefrontal cortex in the case of chronic fatigue,109 and in amygdala in the case of chronic anxiety,110 and in brainstem area associated with wellbeing.111 Mindfulness-based stress-reduction (MBSR) has been shown to increase regional brain grey matter density in hippocampus, cerebellum, and prefrontal cortex, which are brain regions involved in learning and memory processes, emotion regulation, self-referential processing, and perspective taking.112 Indeed, enhancing self-regulation of mood and emotion appears to be an important outcome.113 More studies showing brain changes after MBSR have been reviewed very recently.114 In relation to MBSR effects on amygdala volume that accompany anxiety reduction in generalized anxiety disorder (GAD),110 a follow-up study of symptom improvements followed GAD patients who were randomized to an 8week MBSR or a stress management education (SME) active control program. In GAD patients, amygdala activation in response to neutral faces decreased following both interventions, whereas BOLD responses in ventrolateral prefrontal regions (VLPFC) showed greater increases in MBSR than in SME participants. Furthermore, functional connectivity between amygdala and PFC increased significantly pre-to postintervention within the MBSR subjects, but did not do so in the SME group, at least not to a level that has clinical relevance, based on changes in Beck Anxiety Inventory scores. Amygdalaeprefrontal connectivity turned from negative coupling, as typically seen in downregulation of emotions, to positive coupling suggesting a unique

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2. EPIGENETICS OF ALLOSTASIS AND ALLOSTATIC LOAD OVER THE LIFE COURSE

mechanism of mindfulness involving other components of the complex prefrontal cortex. These findings suggest that, in GAD, MBSR training leads to changes in frontolimbic areas crucial for the regulation of emotion and may do so in ways unique to MBSR.115 Meditation is reported to enlarge volume of the hippocampus and to do so differently in men and women, suggesting to the authors that meditation practices and, most likely, MBSR, operate differently in males and females.116 This suggestion is reminiscent of very recent work showing sex differences in rats differing in fear responses. During fear conditioning and extinction, the work revealed that, despite no overall sex differences in freezing behavior, the neural processes underlying successful or failed extinction maintenance are sex specific.117 Given other work showing sex differences in stressinduced structural plasticity in prefrontal cortex projections to amygdala and other cortical areas,118 these findings are relevant not only to sex differences in fear conditioning and extinction but “also to exposure-based clinical therapies, which are similar in their premises to those of fear extinction and which are primarily used to treat disorders that are more common in women than in men.”117 Another domain where MBSR and meditation practices are reported to have positive effects on brain function is in age-related cognitive decline.119 Fluid intelligence declined slower in aging yoga practitioners and in aging MBSR practitioners then in controls.113 Resting state functional networks of yoga practitioners and meditators were more integrated and more resilient to simulated damage than those of controls. Furthermore, the practice of meditation was found to be positively correlated with fluid intelligence, resilience, and global network efficiency.113 Moreover, grey matter volume is reported to be preserved in meditators versus age-matched controls.120 Building upon these findings, a recent study121 investigated whether targeted mental training of different cognitive and social skills over 9 months would induce specific changes in brain morphology using MRI and improve functional performance. Using daily mental

exercises and weekly group session on adults between 20 and 55 years of age, training protocols specifically addressed three functional domains: (1) mindfulness-based attention and interoception, (2) socioaffective skills (compassion, dealing with difficult emotions, and prosocial motivation), and (3) sociocognitive skills (cognitive perspective-taking on self and others and metacognition). MRI-based cortical thickness analyses revealed that different cortical areas responded to the different training modules producing divergent changes in cortical morphology. For example, training of presentmoment focused attention led to increases in cortical thickness in prefrontal regions, whereas socioaffective training induced plasticity in frontoinsular regions, while sociocognitive training included changes in inferior frontal and lateral temporal cortices. These specific patterns of structural change correlated with training-induced behavioral improvements in the same individuals in domain-specific measures of attention, compassion, and cognitive perspective-taking, respectively, that overlapped with task-relevant well-known socioaffective and sociocognitive functional networks. According to the authors, “these findings could promote the development of evidence-based mental training interventions in clinical, educational, and corporate settings aimed at cultivating social intelligence, prosocial motivation, and cooperation.”121

Other Top-Down Therapies That Change the Brain Social integration and support, and finding meaning and purpose in life, are known to be protective against allostatic load122 and dementia,123 and programs such as the Experience Corps that promote these along with increased physical activity have been shown to slow the decline of physical and mental health and to improve prefrontal cortical blood flow in a similar manner to regular physical activity.124,125 It should be noted that many of these interventions that are intended to promote plasticity and slow decline with age, such as physical activity and positive social interactions that give

REFERENCES

meaning and purpose are also useful for promoting “positive health” and “eudemonia,”126,127 independently of any notable disorder and within the range of normal behavior and physiology.

CONCLUSION The adult as well as developing brain is thus capable of considerable structural plasticity, and resilience refers to this ability to change and not only adjust but also to benefit in the aftermath of adversity through a learning process. Moreover, in the spirit of integrative medicine,128 it is important to focus upon strategies that center around the use of targeted behavioral therapies along with treatments, including pharmaceutical agents, that “open up windows of plasticity” in the brain and facilitate the efficacy of the behavioral interventions to promote resilience.129 This is because a major challenge throughout the life course is to find ways of redirecting future behavior and physiology in more positive and healthy directions.74 Again, to emphasize, by resilience, we do not mean “reversibility” as in “rolling back the developmental clock” but rather “redirection” of those features of a species that can be modified by experiences, since, as noted, gene expression continually changes with experience. As an example, the response of the brain to stressors is a complex process involving multiple interacting mediators that utilizes both genomic and nongenomic mechanisms from the cell surface to the cytoskeleton to epigenetic regulation via the cell nucleus. Resilience in the face of stress is a key aspect of a healthy brain, even though gene expression shows a brain that continually changes with experience.130 Indeed, resilience may be thought of as an active process that implies ongoing adaptive plasticity without external intervention.131 Therefore, recovery of stress-induced changes in neural architecture after stress is not a reversal but a form of neuroplastic adaptation and resilience that may be impaired in mood disorders, when the brain gets stuck and needs external intervention. Loss of resilience may also occur with aging

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and need external intervention such as exercise.87 As we have seen, the brain is continually changing with experiences, creating memories and alerting brain architecture via mechanisms that are facilitated in part by circulating sex, stress, and metabolic hormones and chemicals produced by the immune system. This has led to a new view of the epigenetic changes over the life course that determine trajectories of health and disease, and the plasticity of the brain offers opportunities for changing the trajectory toward an improved “healthspan.”74

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120. Luders E, Cherbuin N, Kurth F. Forever Young(er): potential age-defying effects of long-term meditation on gray matter atrophy. Front Psychol. 2014;5:1551. 121. Valk SL, Bernhardt BC, Trautwein FM, Bockler A, Kanske P, et al. Structural plasticity of the social brain: differential change after socio-affective and cognitive mental training. Sci Adv. 2017;3:e1700489. 122. Seeman TE, Singer BH, Ryff CD, Dienberg G, LevyStorms L. Social relationships, gender, and allostatic load across two age cohorts. Psychosom Med. 2002;64: 395e406. 123. Boyle PA, Buchman AS, Barnes LL, Bennett DA. Effect of a purpose in life on risk of incident Alzheimer disease and mild cognitive impairment in communitydwelling older persons. Arch Gen Psychiatry. 2010;67: 304e310. 124. Carlson MC, Erickson KI, Kramer AF, Voss MW, Bolea N, et al. Evidence for neurocognitive plasticity in at-risk older adults: the experience corps program. J Gerontol A Biol Sci Med Sci. 2009;64:1275e1282. 125. Fried LP, Carlson MC, Freedman M, Frick KD, Glass TA, et al. A social model for health promotion for an aging population: initial evidence on the experience corps model. J Urban Health: Bull NY Acad Med. 2004;81:64e78. 126. Ryff CD, Singer B. The contours of positive human health. Psychol Inq. 1998;9:1e28. 127. Singer B, Friedman E, Seeman T, Fava GA, Ryff CD. Protective environments and health status: cross-talk between human and animal studies. Neurobiol Aging. 2005;26S:S113eS118. 128. Bell IR, Caspi O, Schwartz GE, Grant KL, Gaudet TW, et al. Integrative medicine and systemic outcomes research: issues in the emergence of a new model for primary health care. Arch Intern Med. 2002;162: 133e140. 129. McEwen BS. Brain on stress: how the social environment gets under the skin. Proc Natl Acad Sci USA. 2012;109(Suppl. 2):17180e17185. 130. McEwen BS, Gray J, Nasca C. Recognizing resilience: learning from the effects of stress on the brain. Neurobiol Stress. 2015;1:1e11. 131. Russo SJ, Murrough JW, Han MH, Charney DS, Nestler EJ. Neurobiology of resilience. Nat Neurosci. 2012;15:1475e1484.

C H A P T E R

3 Cerebral Metabolism, Brain Imaging and the Stress Response Klaus P. Ebmeier, EnikT Zsoldos Department of Psychiatry, University of Oxford, Warneford Hospital, Oxford, United Kingdom O U T L I N E Introduction

35

Dopamine Serotonin Cannabinoid Receptor Glutamate Receptor

Imaging the Stress Response Using the Example of Posttraumatic Stress Disorder 36 Resting State Brain Function in Humans 36 Brain Function During Flashbacks, After Traumatic Reminders and Alleviating Interventions 38 Effects of Mindfulness Pharmacological Interventions Limbic-Hypothalamic-Pituitary-Adrenal Axis Road Traffic Accidents Alternative Activation Strategies

Pharmacological Imaging

38 39 39 39 40

40

INTRODUCTION

40 41 41

Stress Mechanisms in the Etiology of Other Psychiatric Disorders

41

Chronic Stress and Its Effect on Structural In Vivo Brain Imaging Early Life Stress Everyday Stress

42 42 43

Future Developments

44

References

45

modern in vivo imaging, in particular positron emission tomography (PET), is able to detect very small (nano- to picomolar) signals in brain metabolism and pharmacology, and can localize such signals anatomically within the mm-range.1 Assuming that a pharmacokinetic model of radiotracer brain uptake exists, and an input curve can be measured, such signals can be quantified.

While neuroendocrinology has afforded us a “window into brain metabolism,” which has been exploited extensively in stress research (reviewed in several chapters of this volume, as well as volume 2 of the Handbook of Stress, series: “Stress: Neuroendocrinology and Neurobiology”),

Stress: Physiology, Biochemistry, and Pathology https://doi.org/10.1016/B978-0-12-813146-6.00003-5

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35

Copyright © 2019 Elsevier Inc. All rights reserved.

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3. CEREBRAL METABOLISM, BRAIN IMAGING AND THE STRESS RESPONSE

KEY POINTS • Advanced imaging techniques have been used to examine mental states and brain changes associated with acute and chronic stress responses • Certain brain structures are both part of the brain networks underlying the stress response and the end organ of potential damage caused by stress-induced metabolic changes • There is a complex relationship between acute and chronic stressors, involvement of frontolimbic networks and behavioral syndromes While X-ray computed tomography (CT) and magnetic resonance imaging (MRI) provide structural information, functional MRI (fMRI), H2O-PET, and FDG-PET provide information about brain activity, blood flow, and metabolism, respectively, and specific ligands are available for PET to label a number of diverse molecular targets in the brain (see Table 3.1).2e5

IMAGING THE STRESS RESPONSE USING THE EXAMPLE OF POSTTRAUMATIC STRESS DISORDER From a scientific point of view, posttraumatic stress disorder (PTSD) can be seen as an experiment of nature that allows us to isolate the effects of more extreme stressors on the brain, given that confounding factors have been controlled for in a case-control design. Furthermore, rodent models are available that use control (tone presentation), fear conditioning, and extinction retrieval phases to map brain activity.6 The amygdala appears to play a key role in fear memory formation, and the insular cortex is related to the retrieval of extinction memory.6 Medial prefrontal cortex (PFC) dysfunction has been highlighted as the cause of abnormal contextualization of traumatic stimuli and therefore one of the central mechanisms for PTSD.7 A conceptual framework suggesting specific abnormalities and associations of clinical syndromes in PTSD with established

human connectivity networks has been proposed, i.e., the central executive network in relation to cognitive dysfunction, the salience network to increased and decreased arousal and interoception, and the default mode network to an altered sense of self.8 The salience network consists of the dorsal anterior cingulate cortex (ACC) and the frontoinsular cortex and plays a central role in directing behavior to the most pertinent actions.8 The default mode network includes anterior and posterior medial cortices and lateral parietal lobes, and is activated during disengagement from tasks, i.e., it is interpreted as being linked to self-referential processing.8 These networks interact via the anterior insula (salience network), which mediates the engagement of the central executive network and disengagement of the default mode network.8

Resting State Brain Function in Humans Resting state brain metabolism images depend on the product of neuronal loss and the metabolic activity of the remaining cells. They therefore represent both structural and functional brain changes. At one extreme, virtual reality simulation of warfare scenarios over 10 minutes does not generate any effects on brain metabolism in healthy soldiers.9 On the other hand, combat-related PTSD frequently co-occurs with mild traumatic brain injury (TBI), for example, blast injury.10 By selection of patient groups, it is possible to try and tease apart such differences. It appears that PTSD in addition to TBI is associated with larger volumes of hypometabolism, which are moreover associated with impaired neuropsychological function.10 Similarly, PTSD after psychological and physical torture, but without obvious brain injury, is found to be associated with hypometabolism in occipital lobes, caudate nuclei, and less frequently temporal lobe, frontal lobe, and posterior cingulate.11 PTSD after sexual assault was associated with left hippocampal and basal ganglia hypometabolism.12 Apart from the relatively small size of such imaging studies, the association of PTSD with varying degrees of physical (as well as psychological) trauma makes the interpretation of such post hoc brain metabolism studies difficult.

IMAGING THE STRESS RESPONSE USING THE EXAMPLE OF POSTTRAUMATIC STRESS DISORDER

TABLE 3.1

37

Some Imaging Modalities Used in Examining Stress Disorders

Modality

Method

Paradigm

Results

CT

X-ray CT

Brain structure

Widely available

Structural MRI

T1-weighted MRI

Brain structure

Widely available

Structural MRI

FLAIR/T2-weighted MRI

White matter changes

Widely available

Functional MRI

BOLD fMRI

Regional brain activation

Mainly research use

DTI

MRI sequence

White matter integrity

Mainly research use

PET

18

Brain metabolism

Routine use

PET

15

OeH2O

Regional cerebral blood flow

Research use

PET

11

C-DASB

5HT transporter (SERT) ligand

Research use

11

5HT transporter (SERT) ligand

Research use

11

5-HT(1A) receptor ligand

Research use

PET

11

NK1 receptor

Research use

PET

11

Benzodiazepine-GABA(A) receptor

Research use

PET

18

F-fallypride

Dopamine D2/D3 receptor ligand

Research use

11

C-(þ)-PHNO

Dopamine D3/D2 receptor ligand

Research use

mGluR5 ligand

Research use

CB1-selective ligand

Research use

Routine protocol perfusion scan

Widely available

Dopamine transporter ligand

Widely available

F-FDG PET

C-McN 5652 C-WAY100635

PET

C-GR205171 C-flumazenil

18

F-FPEB

11

C-OMAR

SPECT SPECT

ECD, HMPAO 123

FP-CIT

99m

Tc-SPECT

I-SPECT

11 C-McN 5652, 6-[4-(fluoranylmethylsulfanyl)phenyl]-1,2,3,5,6,10b-hexahydropyrrolo[2,1-a]isoquinoline; 11C-OMAR, 4-cyano-1-(2,4-dichlorophenyl)-5-(4-[11C] methoxyphenyl-N-(pirrolodin-1-yl)-1H-pyrazole-3-carboxamide; 123I-IMP, n-isopropyl-123IePiodoamphetamine; 18F-FDG, 2-deoxy-2-[18F] fluoro-D-glucose; 18F-FPEB, 18F-3-fluoro-5-[(pyridin-3-yl)ethynyl] benzonitrile; 99mTc-ECD, 99m Tc-ethylcysteinate dimer; 99mTc-HMPAO, 99mTc-hexamethylpropyleneamine oxime; BOLD, Blood oxygen level dependent; CB1, Cannabinoid Type 1; CT, computed tomography; DASB, 3-amino-4-(2-dimethyl-aminomethylphenylsulfanyl)-benzonitrile (higher specific binding than 11C-McN 5652); DTI, Diffusion Tensor Imaging; FLAIR, Fluid attenuated inversion recovery; FP-CIT, 123I-labeled N-(3fluoropropyl)-2b-carbomethoxy-3b(4-iodophenyl) nortropane; GR205171, N-((2-methoxy-5-(5-(trifluoromethyl)-1H-tetrazol-1-yl)phenyl) methyl)-2-phenyl-3-piperidinamine; mGluR5, metabotropic glutamate receptor type 5; MRI, Magnetic resonance imaging; NK1, Substance P/neurokinin-1 (SP/NK1) system; PET, Positron emission tomography; PHNO, (þ)-4-propyl-9-hydro naphthoxazine; SPECT, Single photon emission computed tomography; WAY100635, N-[2-[4-(2-methoxy phenyl) piperazin-1-yl]ethyl]-N-pyridin-2-yl cyclohexane carboxamide.

The specific link between mechanisms of the stress response and brain changes is likely to be confounded by the severity of direct traumatic effects on the brain, or acute vascular damage with systemic physical nonbrain trauma. First attempts have been made to differentiate PTSD into danger-related (threat to own and others’ lives) and not-danger-related PTSD (traumatic aftermath of violence, loss, and moral injury), based on detailed self-reports of each combat participant’s “worst” and most currently distressing trauma.13 The authors hypothesized from previous reports that fear-related scripts

would be linked to amygdala and right rostral ACC dysfunction, with predicted trauma script related changes in right dorsal ACC and left precuneus/posterior cingulate cortex (PCC). In the former group of patients, PTSD symptom severity was in fact associated with higher metabolism in the precuneus and dorsal anterior cingulate and lower metabolism in the left amygdala. In the latter group, symptom severity was associated with higher precuneus metabolism and lower right amygdala metabolism. The authors conclude that elevated amygdala metabolism may represent a trait marker of

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3. CEREBRAL METABOLISM, BRAIN IMAGING AND THE STRESS RESPONSE

susceptibility to develop PTSD,14 “or it may be a state marker resulting from danger-based trauma, stressful life events, current life stressors, or it may reflect a pre-potent vigilance.”13 PTSD patients have been further divided into a “dissociative” versus a “nondissociative” subgroup.15 Heightened detection of bodily arousal is a feature of the latter, and from script-driven activation studies (see below), such symptoms have been associated with amygdala activation. While both PTSD groups showed increased insula-amygdala connectivity at rest, the dissociative as opposed to the nondissociative group showed even greater functional connectivity between various insula nuclei and left basolateral amygdala, which was also associated with measures of depersonalization and derealization.15 Finally, in a study of the interaction between preferred sleep-wake timing (chronotype or morningness-eveningness) and PTSD in soldiers, “eveningness was associated with greater lifetime PTSD symptoms, more disturbed sleep, and more frequent and intense nightmares. It was also associated with greater brain activity in posterior cingulate/precuneus and brainstem regions across wakefulness and REM sleep, overlapping with regions related to arousal and REM sleep generation.”16 In general, the limitations of cross-sectional functional activity studies are thus: the confounding with anatomy, the lack of causal clarity because the relative time course of correlates cannot be established, and the absence of evidence of reversibility of changes, which would distinguish state from trait-related imaging features.

Brain Function During Flashbacks, After Traumatic Reminders and Alleviating Interventions To examine such aspects, repeat measurements of brain variables are required. For practical and reliability reasons, such repeat examinations tend to take place in short order, often during the same research visit. Longitudinal follow-up studies are rarer; dropout rates are higher, confounding changes related to aging

and intervening events influence the results, and technology and methods tend to change fast over the period of years and months. To identify functional changes over short intervals in an experimental fashion, the provocation of symptoms by traumatic scripts17e20 can be balanced by scanning before and after treatment21e24 or pharmacological intervention.25e28 In addition, covariance with other measures of stress29 and interaction with unrelated stimulation procedures18,30 have been attempted. Effects of Mindfulness PTSD has been reported to involve the brain circuitry, including medial prefrontal-, parietal-, and insular cortex, amygdala, and hippocampus.21 Mindfulness-based stress reduction (MBSR) technique appears to reduce blood pressure and blood pressure reactivity to acute stress.24 A recent systematic review of the effects of MBSR on brain function reported increased activity, volume, and connectivity in the prefrontal cortex, cingulate cortex, insula, and hippocampus in stressed, anxious, and healthy participants. The amygdala showed decreased functional activity, improved functional connectivity with prefrontal cortex, and earlier deactivation after exposure to emotional stimuli.23 Specifically applied to PTSD after combat, patients who have undergone MBSR training had increased functional activation in the anterior cingulate and inferior parietal lobule and decreased activation in the insula and precuneus, in response to traumatic reminders (Iraq combat-related slides) compared with the control (present-centered group therapy) group.21 Moreover, in a study of PTSD with combatrelated symptoms treated with mindfulnessbased exposure therapy (MBET), treatment specifically resulted in increased resting-state functional connectivity of the default mode and executive networks (regions of interest [seed] in PCC connected with dorsolateral prefrontal cortex [DLPFC] and dorsal ACC). In comparison with present-centered group therapy, there was a small, not significant, difference in treatment efficacy, but a group by time interaction. MBET was associated with greater connectivity between PCC DLPFC, PCC dorsal

IMAGING THE STRESS RESPONSE USING THE EXAMPLE OF POSTTRAUMATIC STRESS DISORDER

ACC than present-centered group therapy, while PCC DLPFC connectivity correlated with improvement in avoidant and hyperarousal PTSD symptoms.22 Pharmacological Interventions Selective serotonin reuptake inhibitors (SSRIs) provide successful pharmacological treatment for PTSD. The SSRI paroxetine was compared with placebo after 12 weeks’ treatment, examining the contrast between exposure to neutral scripts and personalized trauma scripts with fluorodeoxyglucose FDG-PET.25 While there was an increase in glucose uptake in the ACC in both paroxetine and placebo groups, an increase from neutral to trauma scripts was only present in the paroxetine group only in the orbitofrontal cortex. Both groups showed decreases in PTSD symptoms following “treatment”; paroxetinetreated participants showed a slightly greater percentage decrease in symptoms.25 Limbic-Hypothalamic-Pituitary-Adrenal Axis Neural substrates underlying activation of the limbic-hypothalamic-pituitary-adrenal (LHPA) axis during PTSD are not well understood in humans.27 Comparing Vietnam combat veterans with and without PTDS during autobiographic script-driven imagery, acute adrenocorticotropic hormone (ACTH) responses occurred in both groups equally frequently.27 Both veterans with and without ACTH response had increased regional cerebral blood flow (rCBF) to the right insula. However, veterans with ACTH responders had deactivated rostral medial prefrontal cortex (mPFC) and rostral ACC, whereas nonresponders activated this same mPFC region, and deactivated the amygdala, hippocampus, and temporal poles. Directly comparing ACTH responders and nonresponders, the responders had significantly higher rCBF in the right insula and right temporal pole, whereas nonresponders had higher rCBF in rostral mPFC and dorsal ACC.27 While the right insula may therefore be involved in the activation of the LHPA axis by relevant psychological stimuli, rostral mPFC may negatively modulate LHPA axis responses, independently from diagnosis of PTSD.27 In a

39

study involving the use of aversive pictures and autobiographic narratives, ACTH responses covaried with rCBF in the dorsal mPFC, rostral ACC, and right insula, with some differences between PTSD patients and controls.29 In combatPTSD patients only, prestimulus cortisol levels covaried with rCBF in the subgenual ACC.29 In pharmacological stimulation experiments, veterans with and without PTSD were injected with 17.5 mg hydrocortisone sodium succinate or placebo followed by a 2-deoxy-2-[fluorine18]fluoro-D-glucose (18F-FDG) injection in preparation for glucose uptake measurement by PET. In a first study, hydrocortisone restored a normal inverse association between the ACC and amygdala in the PTSD group, but disrupted this neural network in veterans without PTSD.26 Working memory performance was associated with greater hemispheric laterality in the dorsal amygdala after hydrocortisone injection in both groups. In a second study, veterans of the Gulf wars with PTSD showed greater cortisol and ACTH suppression after dexamethasone, but only veterans without PTSD showed a reduction in hippocampal metabolism in response to hydrocortisone injection. The authors concluded that differences in peripheral and central glucocorticoid receptor responsiveness may account for differences in brain metabolic responses between Gulf war veterans with and without PTSD.28 Road Traffic Accidents While less newsworthy, PTSD after road traffic trauma is probably more common than battleinduced trauma. Some studies have examined the effects of such PTSD on brain activity. Subjects with PTSD exhibit a significant increase in heart rate after traumatic scripts, while trauma survivors without PTSD do not.19 Correlations between regional cerebral blood flow and heart rate were found only in patients with PTSD, in orbitofrontal, precentral, and occipital regions, but not in the amygdala or hippocampus. The authors conclude that “top-down” central nervous system regulation of autonomic stress response in PTSD may involve associative, sensory, and motor areas in addition to regions commonly implicated in fear conditioning.19 In

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3. CEREBRAL METABOLISM, BRAIN IMAGING AND THE STRESS RESPONSE

a separate approach that compared individuals with acute stress disorder after serious motor collisions with a healthy control group, subjects postaccident showed increased Beck depression scores, hyperperfusion in the right medial PFC/ ACC and hypoperfusion in the right amygdala at rest.20 In postaccident participants, trauma scripts were associated with relatively decreased rCBF in both amygdalae, while symptom scores correlated negatively with right hippocampal rCBF.20 Alternative Activation Strategies While the most obvious way to induce stressrelated brain activity patterns is by traumarelated imagery and scripts, some authors have used the recall of stressful, but traumaunrelated, personal events18 or have driven cognitive networks to highlight the effects of PTSD on the brain.30 Trauma-unrelated stressful imagery resulted in a significant interaction between group (male and female veterans with and without PTSD) and imagery content (trauma-unrelated stressful vs. neutral) in the mPFC, which represented greater rCBF decreases in the PTSD group. There was no such effect in the amygdala.18 To test for top-down versus bottom-up dysfunction in the interaction of cognitivecontrol circuitry and emotion-processing circuitry in women with interpersonal trauma, an attentional interference task with emotional distractors was used. Compared to controls, patients with PTSD showed hyperactivity in several brain regions, including the amygdala, insula, as well as dorsolateral PFC and ventral PFC.30 These findings suggest that the specific emotional conflict task used implicit or automatic emotional regulation instead of explicit or effortful emotional regulation.30

Pharmacological Imaging Dopamine Repeat PET examinations with the dopamine D(2/3) agonist, 11C-(þ)-PHNO, examined under an arithmetic control task (without time pressure and negative feedback) and the Montreal Imaging Stress Task in 11 healthy young volunteers,

who were previously assessed with the neuroticism-extraversion-openness personality inventory, showed a reduced response to stress in proportion to the “Angry-Hostility,” “Vulnerability,” and “Depression” traits in the associate striatum (D2), limbic striatum, and globus pallidus (mixed D2/3). “Openness to values” was associated with reduced dopamine release in the substantia nigra (D3).31 The authors interpret these findings as personality traits variably affecting dopamine response to performance stress. Disturbance of dopamine transmission has been found in association with schizophrenia and psychosis (see below). Serotonin Stress has been identified as an important etiological factor in major depressive disorder. This is commonly linked to serotonin function.32e34 5-HT1A receptor binding has also been described in patients with PTSD.35,36 Higher 5-HT1A binding was shown in 20 PTSD patients in all cortical regions, in the amygdala, and the raphe nuclei in the brainstem, but not in the hippocampus.35 While there was comorbidity with major depression in the majority of patients, the same significant effect was observed in patients with PTSD only, compared with 39 healthy volunteers.35 The authors interpret this as evidence of receptor upregulation with presumably reduced serotoninergic transmission in patients. Sixteen patients with “reaction to severe stress, and adjustment disorder” (F43) according to the International Statistical Classification of Diseases and Related Health Problems (ICD10), who attributed their illness to prolonged work-related stress, after 60e70 working hours/week continuously over several years before symptom onset, were compared with 16 nonstressed healthy volunteers.36 Patients showed a functional disconnection between the amygdala, the ACC, and the medial prefrontal cortex, and a smaller activation of the ACC to neutral odors (10% butanol, or undiluted cedar oil, lavender oil, and eugenol). 5-HT1A receptor binding was lower in the ACC, the insular cortex, and the hippocampus. Performance in attention-, odor discrimination-, and semantic memory tasks was impaired, and was correlated

IMAGING THE STRESS RESPONSE USING THE EXAMPLE OF POSTTRAUMATIC STRESS DISORDER

with 5-HT1A receptor binding. Finally, the degree of perceived stress was inversely correlated with odor activation in the ACC, and 5-HT1A receptor binding in the amygdala and hippocampus.36 Although this was a post hoc cross-sectional study, findings suggest that years of stressful work conditions are associated with a limbic reduction of 5-HT1A receptor binding and “functional disintegration” of the ACC and mPFC.36 The authors speculate that chronic stress with high cortisol levels may have been causal to the downregulation of 5-HT1A receptors.36 In a further study of 18 PTSD patients and 18 healthy controls, all were examined using PET for 5HT-transporter (11C-DASB) and substance P/neurokinin-1 (NK1) receptor (11C-GR205171) binding.37 Symptoms of PTSD were negatively correlated with 5HT-transporter binding in the amygdala, and NK1 receptor levels moderated this relationship. Higher 5HT-transporter binding was also found in the precentral gyrus and PCC of PTSD patients. Comparing expression of 5HT-transporter and NK1 binding, patients showed less overlap in putamen, thalamus, insula, and lateral orbitofrontal gyrus. Smaller overlap was associated with more PTSD symptoms. The authors speculate that “aberrant serotonergic-SP/NK1 couplings contribute to the pathophysiology of PTSD.”37 Cannabinoid Receptor In a study of 35 untreated individuals with noncombat PTSD, 12 healthy controls with lifetime histories of trauma and 24 controls without such histories, participants were examined with MRI, PET with the CB1 receptor antagonist radiotracer 11C-OMAR, and peripheral measures of the endocannabinoid anandamide and cortisol.38 While cortisol levels were lower in patients and controls previously exposed to trauma, anandamide levels were lower and 11C-OMAR binding higher in PTSD patients compared to the two control groups. Results indicate that “abnormal CB1 receptor-mediated anandamide signaling is implicated in the etiology of PTSD.”38 Glutamate Receptor The possible involvement of the metabotropic glutamate receptor mGluR5 was examined using

41

16 PTSD patients (nine comorbid with major depression) and 16 controls in a PET study of the mGluR5-ligand 18F-FPEB, and 19 brains of PTSD patients (19 controls) in a postmortem study. The PET study demonstrated higher cortical mGluR5 availability in PTSD compared with controls and a positive correlation between mGluR5 availability and avoidance symptoms.39 In the postmortem brains, SHANK1, a scaffolding protein that anchors mGluR5 to the cell surface, was upregulated, and FKBP5, a cochaperone for the glucocorticoid receptor, was decreased, suggesting aberrant glucocorticoid functioning in PTSD.39 While the authors discuss the implications of the findings in detail, they acknowledge the possible lack of specificity for PTSD of their findings.39

Stress Mechanisms in the Etiology of Other Psychiatric Disorders Stress is an important etiological factor in many psychiatric disorders, including major depressive disorder (MDD),32e34 psychosis and schizophrenia,34,40e43 substance dependence,44,45 and dementia.46 For example, childhood sexual and physical abuse is associated with PTSD, but also depression in later life, and both may demonstrate reduced serotonin transporter binding capacities.33 Rapid eye movement (REM) sleep disturbances are associated with PTSD and MDD, and may predict poor clinical outcomes.32 PTSD is associated with increased REM sleep limbic and paralimbic glucose uptake, while MDD may be associated with similar wake and REM hypermetabolism.32 The etiology of schizophrenia has been linked to chronic psychosocial stressors, such as childhood adversity, migration background, belonging to an ethnic minority, and living in cities.40 Acute stress also plays a role in triggering psychotic symptoms.40 There is good evidence to suggest that stressful stimuli are associated with increased striatal dopamine release in patients with schizophrenia, those at high risk of psychosis, and following childhood adversity.40 While cannabis use is an independent predictor of psychosis in high risk subjects, its mechanism is also likely to be independent of stress-induced

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3. CEREBRAL METABOLISM, BRAIN IMAGING AND THE STRESS RESPONSE

dopamine hyperactivity.42 Individuals at high risk of schizophrenia show increased endogenous dopamine release in most striatal regions, as evidenced by reduced binding potential for a displaceable D2/3 receptor ligand during PET, during the Montreal Imaging Stress task as compared with a sensory-motor control task. In contrast, those high-risk participants who also used cannabis showed reduced endogenous dopamine release in the same regions.42 Psychosocial stress in healthy volunteers induces detectable amounts of dopamine release throughout the prefrontal cortex, with dopaminergic activity in bilateral ventromedial prefrontal cortex associated with subjectively rated experiences of psychosocial stress.17 In contrast to striatal dopamine measures, first-degree relatives of patients with a psychotic disorder showed an attenuated response that was associated with greater subjectively rated stress and psychotic experiences.43 Endogenous dopamine release in ventromedial prefrontal cortex was further associated with psychotic reactivity to daily life stress in healthy first-degree relatives of individuals with a psychotic disorder and in healthy controls.41 Lower endogenous dopamine release to stress predicted increased prepsychotic reactivity to daily life stress.41 Alcohol dependence and PTSD are often comorbid, and both have been linked to the neurokinin 1 (NK1) receptor for substance P.45 Patients with PTSD have elevated basal levels of substance P in cerebrospinal fluid, which are further increased by presentation of trauma-associated cues.45 Aprepitant is a selective and highaffinity NK1 antagonist, and therefore a potential treatment for PTSD. It increases ventromedial PFC blood oxygen leveledependent MRI responses to aversive visual stimuli, but does not affect PTSD symptoms.45 The central role of the insular cortex in PTSD and in the interplay between crucial brain networks8 makes it an important structure affected in mood, panic, obsessive-compulsive, eating disorders, and schizophrenia.34 The effect of chronic stress on the hippocampus suggests that PTSD may represent a risk factor for the condition with hippocampal atrophy as a specific marker, i.e., Alzheimer disease. Evidence from

the US Department of Defense Alzheimer’s Disease Neuroimaging Initiative grant, which enrolled Vietnam veterans with PTSD or traumatic brain injury, so far suggests that this is not the case, but is awaiting further results.46

CHRONIC STRESS AND ITS EFFECT ON STRUCTURAL IN VIVO BRAIN IMAGING We have previously provided a detailed description of the link between neuroendocrine stress response and brain structure (Handbook of Stress, Vol. 1, Ch. 38). Primary and secondary markers of allostatic load and the chronic stress response are associated with brain mechanisms that regulate the stress response, but also with damage to certain brain structures, if the stress response persists for too long. Brain regions, such as the prefrontal cortex, hippocampal formation, and the amygdala, are thus not only instrumental in regulating the stress response but are also its targets. We will provide examples here of chronic every-day or traumatic stressors that result in lasting physiological and psychological effects, and their relationship with structural brain measures.

Early Life Stress Adversity in early childhood activates the stress response system excessively. This can have lasting consequences. Early psychosocial adversity takes many forms: neglect (the absence of sensitive and responsive caregiving), disrupted parent-child interaction (e.g., due to interpersonal loss or family instability), physical, sexual and emotional abuse, and violence. It increases the risk of adverse mental, cognitive, physical, and social outcomes in childhood, adolescence, and adulthood, as well as poor academic achievement, lifetime productivity, and impairment in multiple brain structures.47,48 A prospective community-based longitudinal birth cohort found evidence for independent effects of stress measured during three distinct neurodevelopmental periods (prenatal maternal stress, early childhood, and adolescence) on

CHRONIC STRESS AND ITS EFFECT ON STRUCTURAL IN VIVO BRAIN IMAGING

structural properties of the white matter in 393 young adult men.49 Prenatal stress was associated with a lower magnetization transfer ratio (MTR) in lobar white matter (a sensitive and sometimes more specific MRI measure of tissue change) and in the corpus callosum (with lower myelin water fraction in the corpus callosum as well). Stress during early childhood was associated with higher MTR in the splenium, and during adolescence with MTR in the genu and lower mean diffusivity in the splenium of the corpus callosum. A possible explanation for the specific effects of stress is due to the different developmental processes operating at critical time periods, which may be differently affected by glucocorticoid exposure. The authors speculate that associations with prenatal stress may, for example, relate to abnormalities in neurogenesis, affecting the number and density of axons. On the other hand, postnatal stress may interfere with processes related to myelination or radial axon growth. In the same cohort, total number of family adversities (such as interpersonal loss, family instability, and abuse toward the child or his mother) between the ages of 8 months and 6 years was associated with more depressive and anxiety symptoms (“internalizing disorder”) in childhood and lower grey matter volume in the anterior cingulate cortex and the precuneus in early adulthood (N ¼ 494 young men).50 Symptoms of depression and anxiety in childhood were also associated with lower grey matter volume in the right superior frontal gyrus. This association was mediated by a greater internalizing disorder. A recent meta-analysis of magnetic resonance imaging studies found only moderate evidence of negative impact of childhood adversity on brain structures in healthy adults without significant psychopathology.51 Those with a history of childhood adversity had slightly smaller hippocampal volumes than those without childhood adversity (hedges g ¼ 0.15, P ¼ .015), however, this difference was greater in studies where participants with psychopathology were included in the childhood adversity group (hedges g ¼ 0.66, P ¼ .007). These findings provide little evidence for the chronic hypothalamicpituitary-adrenal (HPA)-axis activation and

43

secretion of stress hormones, and cognitive dysregulations as potential mechanism of stress-related brain changes in adults without considerable psychopathology.52 According to this model, the chronic secretion of stress hormones affects the development of the prefrontal cortex, hippocampus, and amygdala, and consequently the underlying mechanisms that serve decision-making, memory formation and retrieval, and emotion processing, respectively, in turn, increasing vulnerability to specific mental disorders in adulthood. The Bucharest Early Intervention Project is the only randomized controlled trial of a foster care intervention (vs. institutional care as usual) for infants and young children who were exposed to early adversity.53 Besides exhibiting aggressive behavior,54 abnormal patterns of attachment,55 and lower IQ,56 children who were randomized to institutional care as usual also displayed significantly lower EEG alpha power.57 Lower alpha power reflects dampened brain metabolism,58 underlying a disruption of normal developmental increases in information processing in frontal and central cortical regions, visual attention, and alertness in occipital regions.59 These effects were largely compensated for, only if foster care began before the age of 2 years. Although advances have been made to better understand the effects of chronic stress on the developing and adult brain, results from crosssectional and longitudinal studies are mostly correlational, often including retrospective reporting of early adversity and life events.60 In addition, study designs often fail to appropriately address history of psychopathology and Complementary internalizing disorders.51 assessment of internalizing disorders and brain imaging in children and adolescents can shed light on the development of key regions and emotional vulnerability at critical time periods, as well as in early adulthood.61

Everyday Stress There is limited evidence that chronic perceived stress and the number of stressful life events is associated with decreased grey matter

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3. CEREBRAL METABOLISM, BRAIN IMAGING AND THE STRESS RESPONSE

volumes in the limbic and prefrontal cortex. For example, average chronic perceived stress predicted lower grey matter volume in the right orbitofrontal cortex and right hippocampus in healthy postmenopausal women, even after controlling for standard demographic variables.62 Decreased grey matter volume in the anterior cingulate gyrus, the left parahippocampal gyrus, and the right hippocampus was correlated with the number of stressful life events.63 Cumulative exposure to adverse life events was also associated with lower grey matter density of the prefrontal and limbic brain regions in a community sample of middle-aged adults.64 In an MRI study of a group of seniors (N ¼ 240; age >60 years; 54% depressed), crosssectional and longitudinal relationships between stress measures and white matter hyperintensity volumes were assessed.65 The self-administered 20-item questionnaire assessed stressor exposure at baseline and over a year-long period, including a variety of positive and negative stressful events common with aging, for example, the development of a physical illness, separation from a loved one, marriage or divorce, addition or loss of a family member, work-related difficulties, financial struggles, and retirement. There was no association between cross-sectional baseline stress measures and baseline hyperintensity volume or 2-year change in white matter hyperintensity volume. After controlling for demographic variables and baseline measures, increased stressor exposure was associated with greater increases in white matter hyperintensity volume, while reductions in stressor exposure were associated with a smaller increase in hyperintensity volume. Depression and medical comorbidities did not have a significant effect on the results. Little is known about the association between socioeconomic position and morphologic brain changes in older age, despite low socioeconomic position being a risk factor for cognitive decline. In a population-based cohort of 1328 older adults aged 65e80 years, participants with lower midlife socioeconomic position had smaller hippocampal volume (0.08 cm3; 95% confidence interval, 0.15 to 0.01) and 0.17% (95% confidence interval, 0.04%e0.30%) greater

hippocampal atrophy than participants with higher midlife status. Childhood and early adulthood socioeconomic status were not predictors of hippocampal volume in older age. The accumulation of socioeconomic disadvantage and declining socioeconomic trajectories were related to faster hippocampal atrophy, a cerebral change linked to cognitive disorders.66 Chronic occupational stress has also been associated with partially reversible structural abnormalities in brain regions considered key for the stress response, i.e., reduced cortical thickness in the right prefrontal cortex and left superior temporal gyrus, reduced caudate volumes, and enlarged amygdala volumes. These were correlated with perceived stress levels and were more pronounced in women, perhaps highlighting an increased cerebral vulnerability to stress-related psychopathology.67 The functional correlates of perceived occupational stress are not clear. Perceived stress reflects the extent to which situations are appraised as stressful at a given point in one’s life. The association between perceived occupational stress and cortical activity was investigated over the bilateral frontotemporal regions during a verbal fluency test in 68 middle-aged Chinese adults (51 women, 20e 62 years), using near-infrared spectroscopy. Significant negative associations were found between occupational stress and brain cortical activity over the frontopolar cortex and DLPFC during verbal fluency test performance.68

FUTURE DEVELOPMENTS A striking observation in stress-related research is the increasing importance of longitudinal studies that allow for the collection of prospective unbiased data related to stress that can then be related to brain outcomes and clinical consequences. While cross-sectional studies and experiments will remain useful in elucidating mechanisms, particularly with respect to newly discovered mechanisms explored with novel ligands and imaging techniques, a causal analysis relies on the painstaking and patient follow-up of participants over years, if not decades, using replicable and stable methods. The inherent

REFERENCES

contradiction with a trade-off between generating reliable change measures while forgoing technical innovation with promising novel and exciting results will continue to exercise researchers. We can look forward, therefore, to a steady but gradual increase in knowledge about the effects of stress on the brain.

References 1. Glickson JS. Molecular imaging. In: Bryan RN, ed. Introduction to the Science of Medical Imaging. New York: Cambridge University Press; 2010:275e291. 2. Im JJ, Namgung E, Choi Y, Kim JY, Rhie SJ, Yoon S. Molecular neuroimaging in posttraumatic stress disorder. Exp Neurobiol. 2016;25(6):277e295. 3. Fredrikson M, Faria V. Neuroimaging in anxiety disorders. Mod Trends Pharmacopsychiatry. 2013;29: 47e66. 4. Nikolaus S, Antke C, Beu M, Muller HW. Cortical GABA, striatal dopamine and midbrain serotonin as the key players in compulsive and anxiety disorderse results from in vivo imaging studies. Rev Neurosci. 2010;21(2):119e139. 5. Francati V, Vermetten E, Bremner JD. Functional neuroimaging studies in posttraumatic stress disorder: review of current methods and findings. Depress Anxiety. 2007; 24(3):202e218. 6. Zhu Y, Du R, Zhu Y, et al. PET mapping of neurofunctional changes in a posttraumatic stress disorder model. J Nucl Med. 2016;57(9):1474e1477. 7. Liberzon I, Sripada CS. The functional neuroanatomy of PTSD: a critical review. Prog Brain Res. 2008;167: 151e169. 8. Lanius RA, Frewen PA, Tursich M, Jetly R, McKinnon MC. Restoring large-scale brain networks in PTSD and related disorders: a proposal for neuroscientifically-informed treatment interventions. Eur J Psychotraumatol. 2015;6:27313. 9. Wojtlowska-Wiechetek D, Tworus R, Dziuk M, et al. Estimation of usefulness of positron emission tomography (PET) in the diagnosis of post-traumatic stress disordersepreliminary report. Stud Health Technol Inform. 2013;191:178e180. 10. Buchsbaum MS, Simmons AN, DeCastro A, Farid N, Matthews SC. Clusters of low (18)Ffluorodeoxyglucose uptake voxels in combat veterans with traumatic brain injury and post-traumatic stress disorder. J Neurotrauma. 2015;32(22):1736e1750. 11. Zandieh S, Bernt R, Knoll P, et al. Analysis of the metabolic and structural brain changes in patients with torture-related post-traumatic stress disorder (TRPTSD) using (1)(8)F-FDG PET and MRI. Medicine (Baltimore). 2016;95(15):e3387.

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12. Kim SY, Chung YK, Kim BS, Lee SJ, Yoon JK, An YS. Resting cerebral glucose metabolism and perfusion patterns in women with posttraumatic stress disorder related to sexual assault. Psychiatry Res. 2012;201(3): 214e217. 13. Ramage AE, Litz BT, Resick PA, et al. Regional cerebral glucose metabolism differentiates danger- and nondanger-based traumas in post-traumatic stress disorder. Soc Cogn Affect Neurosci. 2016;11(2):234e242. 14. Admon R, Milad MR, Hendler T. A causal model of post-traumatic stress disorder: disentangling predisposed from acquired neural abnormalities. Trends Cogn Sci. 2013;17(7):337e347. 15. Nicholson AA, Sapru I, Densmore M, et al. Unique insula subregion resting-state functional connectivity with amygdala complexes in posttraumatic stress disorder and its dissociative subtype. Psychiatry Res. 2016; 250:61e72. 16. Hasler BP, Insana SP, James JA, Germain A. Eveningtype military veterans report worse lifetime posttraumatic stress symptoms and greater brainstem activity across wakefulness and REM sleep. Biol Psychol. 2013; 94(2):255e262. 17. Lataster J, Collip D, Ceccarini J, et al. Psychosocial stress is associated with in vivo dopamine release in human ventromedial prefrontal cortex: a positron emission tomography study using [(1)(8)F]fallypride. Neuroimage. 2011;58(4):1081e1089. 18. Gold AL, Shin LM, Orr SP, et al. Decreased regional cerebral blood flow in medial prefrontal cortex during trauma-unrelated stressful imagery in Vietnam veterans with post-traumatic stress disorder. Psychol Med. 2011; 41(12):2563e2572. 19. Barkay G, Freedman N, Lester H, et al. Brain activation and heart rate during script-driven traumatic imagery in PTSD: preliminary findings. Psychiatry Res. 2012; 204(2e3):155e160. 20. Osuch EA, Willis MW, Bluhm R, Group CNS, Ursano RJ, Drevets WC. Neurophysiological responses to traumatic reminders in the acute aftermath of serious motor vehicle collisions using [15O]-H2O positron emission tomography. Biol Psychiatry. 2008;64(4):327e335. 21. Bremner JD, Mishra S, Campanella C, et al. A pilot study of the effects of mindfulness-based stress reduction on post-traumatic stress disorder symptoms and brain response to traumatic reminders of combat in Operation Enduring Freedom/Operation Iraqi Freedom combat veterans with post-traumatic stress disorder. Front Psychiatry. 2017;8:157. 22. King AP, Block SR, Sripada RK, et al. Altered default mode network (DMN) resting state functional connectivity following a mindfulness-based exposure therapy for posttraumatic stress disorder (PTSD) in combat veterans of Afghanistan and Iraq. Depress Anxiety. 2016; 33(4):289e299.

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23. Gotink RA, Meijboom R, Vernooij MW, Smits M, Hunink MG. 8-week mindfulness based stress reduction induces brain changes similar to traditional longterm meditation practice e a systematic review. Brain Cogn. 2016;108:32e41. 24. Nyklicek I, Mommersteeg PM, Van Beugen S, Ramakers C, Van Boxtel GJ. Mindfulness-based stress reduction and physiological activity during acute stress: a randomized controlled trial. Health Psychol. 2013; 32(10):1110e1113. 25. Fani N, Ashraf A, Afzal N, et al. Increased neural response to trauma scripts in posttraumatic stress disorder following paroxetine treatment: a pilot study. Neurosci Lett. 2011;491(3):196e201. 26. Yehuda R, Harvey PD, Golier JA, et al. Changes in relative glucose metabolic rate following cortisol administration in aging veterans with posttraumatic stress disorder: an FDG-PET neuroimaging study. J Neuropsychiatry Clin Neurosci. 2009;21(2):132e143. 27. King AP, Abelson JL, Britton JC, Phan KL, Taylor SF, Liberzon I. Medial prefrontal cortex and right insula activity predict plasma ACTH response to trauma recall. Neuroimage. 2009;47(3):872e880. 28. Yehuda R, Golier JA, Bierer LM, et al. Hydrocortisone responsiveness in Gulf War veterans with PTSD: effects on ACTH, declarative memory hippocampal [(18)F] FDG uptake on PET. Psychiatry Res. 2010;184(2): 117e127. 29. Liberzon I, King AP, Britton JC, Phan KL, Abelson JL, Taylor SF. Paralimbic and medial prefrontal cortical involvement in neuroendocrine responses to traumatic stimuli. Am J Psychiatry. 2007;164(8):1250e1258. 30. Bruce SE, Buchholz KR, Brown WJ, Yan L, Durbin A, Sheline YI. Altered emotional interference processing in the amygdala and insula in women with PostTraumatic Stress Disorder. Neuroimage Clin. 2012;2:43e49. 31. Suridjan I, Boileau I, Bagby M, et al. Dopamine response to psychosocial stress in humans and its relationship to individual differences in personality traits. J Psychiatr Res. 2012;46(7):890e897. 32. Ebdlahad S, Nofzinger EA, James JA, Buysse DJ, Price JC, Germain A. Comparing neural correlates of REM sleep in posttraumatic stress disorder and depression: a neuroimaging study. Psychiatry Res. 2013;214(3): 422e428. 33. Miller JM, Kinnally EL, Ogden RT, Oquendo MA, Mann JJ, Parsey RV. Reported childhood abuse is associated with low serotonin transporter binding in vivo in major depressive disorder. Synapse. 2009;63(7): 565e573. 34. Nagai M, Kishi K, Kato S. Insular cortex and neuropsychiatric disorders: a review of recent literature. Eur Psychiatry. 2007;22(6):387e394. 35. Sullivan GM, Ogden RT, Huang YY, Oquendo MA, Mann JJ, Parsey RV. Higher in vivo serotonin-1a binding in posttraumatic stress disorder: a PET study with [11C] WAY-100635. Depress Anxiety. 2013;30(3):197e206.

36. Jovanovic H, Perski A, Berglund H, Savic I. Chronic stress is linked to 5-HT(1A) receptor changes and functional disintegration of the limbic networks. Neuroimage. 2011;55(3):1178e1188. 37. Frick A, Ahs F, Palmquist AM, et al. Overlapping expression of serotonin transporters and neurokinin-1 receptors in posttraumatic stress disorder: a multitracer PET study. Mol Psychiatry. 2016;21(10):1400e1407. 38. Neumeister A, Normandin MD, Pietrzak RH, et al. Elevated brain cannabinoid CB1 receptor availability in post-traumatic stress disorder: a positron emission tomography study. Mol Psychiatry. 2013;18(9):1034e1040. 39. Holmes SE, Girgenti MJ, Davis MT, et al. Altered metabotropic glutamate receptor 5 markers in PTSD: in vivo and postmortem evidence. Proc Natl Acad Sci U S A. 2017;114(31):8390e8395. 40. Howes OD, McCutcheon R, Owen MJ, Murray RM. The role of genes, stress, and dopamine in the development of schizophrenia. Biol Psychiatry. 2017;81(1):9e20. 41. Hernaus D, Collip D, Lataster J, et al. Psychotic reactivity to daily life stress and the dopamine system: a study combining experience sampling and [18F]fallypride positron emission tomography. J Abnorm Psychol. 2015;124(1):27e37. 42. Mizrahi R, Kenk M, Suridjan I, et al. Stress-induced dopamine response in subjects at clinical high risk for schizophrenia with and without concurrent cannabis use. Neuropsychopharmacology. 2014;39(6):1479e1489. 43. Lataster J, Collip D, Ceccarini J, et al. Familial liability to psychosis is associated with attenuated dopamine stress signaling in ventromedial prefrontal cortex. Schizophr Bull. 2014;40(1):66e77. 44. Mizrahi R, Suridjan I, Kenk M, et al. Dopamine response to psychosocial stress in chronic cannabis users: a PET study with [11C]-þ-PHNO. Neuropsychopharmacology. 2013;38(4):673e682. 45. Kwako LE, George DT, Schwandt ML, et al. The neurokinin-1 receptor antagonist aprepitant in comorbid alcohol dependence and posttraumatic stress disorder: a human experimental study. Psychopharmacology (Berlin). 2015;232(1):295e304. 46. Weiner MW, Harvey D, Hayes J, et al. Effects of traumatic brain injury and posttraumatic stress disorder on development of Alzheimer’s disease in Vietnam Veterans using the Alzheimer’s Disease Neuroimaging Initiative: preliminary report. Alzheimers Dement (N Y). 2017;3(2):177e188. 47. Anda RF, Felitti VJ, Bremner JD, et al. The enduring effects of abuse and related adverse experiences in childhood. A convergence of evidence from neurobiology and epidemiology. Eur Arch Psychiatry Clin Neurosci. 2006;256(3):174e186. 48. Edwards VJ, Holden GW, Felitti VJ, Anda RF. Relationship between multiple forms of childhood maltreatment and adult mental health in community respondents: results from the adverse childhood experiences study. Am J Psychiatry. 2003;160(8):1453e1460.

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49. Jensen SKG, Pangelinan M, Bjornholm L, et al. Associations between prenatal, childhood, and adolescent stress and variations in white-matter properties in young men. Neuroimage. 2017, Oct 21; https:// doi.org/10.1016/j.neuroimage.2017.10.033. 50. Jensen SK, Dickie EW, Schwartz DH, et al. Effect of early adversity and childhood internalizing symptoms on brain structure in young men. JAMA Pediatr. 2015; 169(10):938e946. 51. Calem M, Bromis K, McGuire P, Morgan C, Kempton MJ. Meta-analysis of associations between childhood adversity and hippocampus and amygdala volume in non-clinical and general population samples. Neuroimage Clin. 2017;14:471e479. 52. Raymond C, Marin MF, Majeur D, Lupien S. Early child adversity and psychopathology in adulthood: HPA axis and cognitive dysregulations as potential mechanisms. Prog Neuro-Psychopharmacol Biol Psychiatry. 2018;85: 152e160. 53. Zeanah CH, Nelson CA, Fox NA, et al. Designing research to study the effects of institutionalization on brain and behavioral development: the Bucharest Early Intervention Project. Dev Psychopathol. 2003;15(4): 885e907. 54. Zeanah CH, Egger HL, Smyke AT, et al. Institutional rearing and psychiatric disorders in Romanian preschool children. Am J Psychiatry. 2009;166(7):777e785. 55. Zeanah CH, Smyke AT, Koga SF, Carlson E, Bucharest Early Intervention Project Core G. Attachment in institutionalized and community children in Romania. Child Dev. 2005;76(5):1015e1028. 56. Smyke AT, Koga SF, Johnson DE, et al. The caregiving context in institution-reared and family-reared infants and toddlers in Romania. J Child Psychol Psychiatry. 2007;48(2):210e218. 57. Marshall PJ, Fox NA, Bucharest Early Intervention Project Core G. A comparison of the electroencephalogram between institutionalized and community children in Romania. J Cogn Neurosci. 2004;16(8):1327e1338. 58. Buzsaki G, Kaila K, Raichle M. Inhibition and brain work. Neuron. 2007;56(5):771e783.

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59. Vanderwert RE, Marshall PJ, Nelson 3rd CA, Zeanah CH, Fox NA. Timing of intervention affects brain electrical activity in children exposed to severe psychosocial neglect. PLoS One. 2010;5(7):e11415. 60. McEwen CA, McEwen BS. Social structure, adversity, toxic stress, and intergenerational poverty: an early childhood model. Annu Rev Sociol. 2017;43:445e472. 61. Cameron JL, Eagleson KL, Fox NA, Hensch TK, Levitt P. Social origins of developmental risk for mental and physical illness. J Neurosci. 2017;37(45):10783e10791. 62. Gianaros PJ, Jennings JR, Sheu LK, Greer PJ, Kuller LH, Matthews KA. Prospective reports of chronic life stress predict decreased grey matter volume in the hippocampus. Neuroimage. 2007;35(2):795e803. 63. Papagni SA, Benetti S, Arulanantham S, McCrory E, McGuire P, Mechelli A. Effects of stressful life events on human brain structure: a longitudinal voxel-based morphometry study. Stress. 2011;14(2):227e232. 64. Ansell EB, Rando K, Tuit K, Guarnaccia J, Sinha R. Cumulative adversity and smaller gray matter volume in medial prefrontal, anterior cingulate, and insula regions. Biol Psychiatry. 2012;72(1):57e64. 65. Johnson AD, McQuoid DR, Steffens DC, Payne ME, Beyer JL, Taylor WD. Effects of stressful life events on cerebral white matter hyperintensity progression. Int J Geriatr Psychiatry. 2017;32(12):e10ee17. 66. Elbejjani M, Fuhrer R, Abrahamowicz M, et al. lifecourse socioeconomic position and hippocampal atrophy in a prospective cohort of older adults. Psychosom Med. 2017;79(1):14e23. 67. Savic I, Perski A, Osika W. MRI shows that exhaustion syndrome due to chronic occupational stress is associated with partially reversible cerebral changes. Cereb Cortex. 2018;28(3):894e906. 68. Chou PH, Lin WH, Hung CA, et al. Perceived occupational stress is associated with decreased cortical activity of the prefrontal cortex: a multichannel nearinfrared spectroscopy study. Sci Rep. 2016;6:39089.

C H A P T E R

4 Stress-Hyporesponsive Period Mathias V. Schmidt Max Planck Institute of Psychiatry, Munich, Germany O U T L I N E Introduction

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SHRP and the Brain

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Stress-Hyporesponsive Period

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Corticosteroid Feedback

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SHRP, the Adrenal and Corticosterone

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Conclusion

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SHRP and the Pituitary

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References

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INTRODUCTION

the developing organism. Abundant evidence indicates that the rules that govern the activity of the hypothalamic-pituitary-adrenal (HPA) axis in the adult are very different in the neonate.

Based primarily on the pioneering work of the late Hans Selye, the stress response has become somewhat synonymous with the release of hormones from the pituitary and the adrenal glands.1 Thus, in most adult mammals stimuli presumed to be stressful result in a systematic release of adrenocorticotropic hormone (ACTH) and the subsequent secretion of glucocorticoids from the adrenal. This simplistic view of the pituitary-adrenal axis as first described by Selye has been elaborated on extensively. The regulation of the so-called stress hormones clearly involves specific peptides synthesized and stored in the brain (i.e., corticotropin-releasing factor (CRF) and arginine vasopressin) and brainderived neurotransmitters (i.e., norepinephrine). Thus the brain must be included as a critical stress-responsive system. However, the sequences of responses observed consistently in the adult are in many ways very different in

Stress: Physiology, Biochemistry, and Pathology https://doi.org/10.1016/B978-0-12-813146-6.00004-7

KEY POINTS • The stress-hyporesponsive period (SHRP) marks a developmental period of relative quiescence of the adrenal cortex to secrete glucocorticoids following stress exposure • The SHRP is kept in check via a highly sensitive glucocorticoid receptor feedback signal at the level of the pituitary • Other stress system organs, especially the brain, are highly responsive to stressors throughout the SHRP • Under specific, mostly life-threatening, circumstances the suppression of the hypothalamic-pituitary-adrenal axis can be overcome

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Copyright © 2019 Elsevier Inc. All rights reserved.

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• Disruption of the SHRP, a process that requires corticotropin-releasing factor signaling in the brain, is a key process for shaping long-term physiology and behavior of an individual.

STRESS-HYPORESPONSIVE PERIOD In 1950, a report appeared that first indicated that the neonatal response to stress deviated markedly from that observed in the adult and thus created a field of inquiry that has persisted for over four decades.2 Using changes in adrenal ascorbic acid as the response to stress, Jailer reported that the neonate did not show any response to stress. By the early 1960s, Shapiro placed a formal label on this phenomenon and designated it as the ‘‘stress nonresponsive period’’ (SNRP).3 It is important to note that for the most part the database for this description was the inability of the rat pup to show significant elevations of corticosterone following stress. There was one study that received little attention at the time but did raise important questions concerning the validity of the notion of an SNRP. In that study,4 in addition to exposing the pup to stress and demonstrating a lack of a corticosterone response, another group was injected with ACTH. These pups also failed to elicit a corticosterone response, which indicated that one of the factors contributing to the SNRP could be a decreased sensitivity of the adrenal to ACTH. Therefore, it was conceivable that other components of the HPA axis might be responsive to stress. The resolution of this question was dependent on the availability of relatively easy and inexpensive procedures for examining other components of the HPA axis. The methodological breakthrough that altered most of endocrinology and had a major impact on our understanding the ontogeny of the stress response was the development of radioimmune assay (RIA) procedures. The initial impact of the RIA was to change the designation of this developmental period for the SNRP to the ‘‘stress-hyporesponsive period’’ (SHRP).5,6 This change was a result of studies that showed a small but significant rise in

corticosterone when measured by the RIA. Thus, although the response of the adrenal was reduced markedly during the SHRP, the adrenal was capable of releasing low levels of corticosterone. While mostly studied in rodents, equivalent periods of relative glucocorticoid quiescence have also been observed in other species, including humans.7e9 It has been argued, that the biological function of the SHRP is to protect the developing organism, specifically the brain, from an excess of circulating glucocorticoids. There is abundant evidence that high levels of glucocorticoids affect neurogenesis and neural growth.10,11 Thus, under basal circumstances it seems beneficial for the neonate to suppress the activity of the stress system. However, under situations that threaten health or even survival, an activation of the HPA system might be crucial. The HPA axis during development is unique in that it maintains a delicate balancing act between the regulation of stable levels of corticosterone and the ability to respond to systemic stimuli that may be life threatening. Thus the neonate can more precisely discriminate between stressors that require higher-level cognitive processing and life-threatening events. Furthermore, the stress response of the neonate shapes HPA axis development, which can also be beneficial for the adaptation to adverse environments later in life.12 Indeed, when investigators began to examine other components of the neonates’ HPA axis, it became apparent that the SHRP was by no means absolute and that the different components of the axis responded differentially and were stimulus specific. The question addressed in this chapter is whether the concept of the SHRP is still valid. In order to confront this question, the development of several components of the HPA axis is examined, specifically, the adrenal, the pituitary, and the brain.

SHRP, THE ADRENAL AND CORTICOSTERONE This chapter focuses on how the development of the different components of the HPA axis bears on the issue of the SHRP. It is generally agreed that in response to most stressors the

SHRP, THE ADRENAL AND CORTICOSTERONE

neonate fails to elicit adrenocortical response, or does so minimally. There are several features that characterize the function of the pup’s adrenal. The first and most obvious characteristic of the adrenal is that basal levels of corticosterone during the SHRP are considerably lower than observed immediately following parturition, and after the pup emerges from the period of adrenal quiescence, which is normally between postnatal days 4e14 in the rat (postnatal days 1e12 in the mouse).13 Further, numerous investigators have reported that the neonate can elicit a significant increase in plasma levels of corticosterone following a variety of challenges.14 However, invariably the magnitude of the response is small compared to older pups that are outside the SHRP and of course to the adult. Thus, whereas the reported changes in corticosterone levels following stress in the adult can at times exceed 500 ng/mL, rarely does the infant during the SHRP reach levels that exceed 100 ng/mL. These levels are reached only under special circumstances, which shall be described later. Thus the ability of the neonatal adrenal to secrete corticosterone seems to be impaired markedly. There is an important caveat in making the assumption that the reduced level of corticosterone following stress indicates a reduction in biological activity. Corticosterone exists in the circulation in two forms, bound and unbound. The large majority of corticosterone in the adult is bound to corticosteroid-binding protein (CBG) and other binding proteins. Only a small fraction exists in the free form, which is considered to be the biologically active form. Another aspect of the SHRP in rodents is the relative absence of CBG during the SHRP.15 Thus, although absolute values of corticosterone, which normally include both bound and unbound hormone, are very low in the absence of CBG, the actual fraction of corticosterone that is available in the free form for binding to corticosteroid receptors may actually be higher than is observed in the adult. Further, the clearance of corticosterone from the circulation is significantly slower in the pup, and, as a consequence, corticosterone is available for a more prolonged period. Thus, the biologically active corticosterone does have a more prolonged period of

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time to exert its effects in the periphery and the brain. There is also the possibility that corticosterone is replaced by aldosterone as the main adrenocortical stress hormone during the SHRP. Several reports indicated that while the corticosterone response to a challenge during the SHRP is minimal, the response of aldosterone is significant and also much stronger compared to adults.16 Further, the levels of 11beta-hydroxysteroid dehydrogenase 2 (11bHSD2), an enzyme enabling preferential effects of aldosterone on mineralocorticoid receptors, were found to be increased in pups. Although there appear to be rate-limiting factors that act developmentally to limit the secretion of corticosterone in the neonate, evidence indicates that the adrenal is actively suppressed during the SHRP, and may thus be viewed as the most proximal cause of the SHRP.17 It has been extensively documented that certain aspects of the rodent maternal behavior play an important role in regulating the neonate HPA axis. In particular, two specific components of the dam’s care-giving activities seem to be critical; licking/stroking and feeding. Numerous studies have demonstrated that feeding is in part responsible for the suppression of the pups’ capacity to both secrete and clear corticosterone from the circulation.18e20 Thus, removing the mother from the litter for 24 h (and thus fasting the pup) results in a significantly higher basal level and a further increase in the secretion of corticosterone following stress or administration of ACTH. The initial activation of the HPA axis occurs between 4 and 8 h of maternal absence.21 This process is likely mediated by metabolic signals, since replacement with glucose or blockade of ghrelin receptors during deprivation prevent HPA axis activation.20 In fact, rat pups that were artificially reared from postnatal day 5 onwards also demonstrate a stress hyporesponsiveness similar to dam-reared pups, as long as they were adequately fed.22 It has been postulated that one of the consequences of maternal deprivation is to increase the sensitivity of the adrenal to ACTH. This has been demonstrated in several ways; (1) following deprivation, significantly lower doses of ACTH are required to induce the

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adrenal to secrete corticosterone; (2) although the levels of ACTH are equivalent between deprived and nondeprived pups under certain experimental conditions, the levels of corticosterone are greater in deprived pups; and (3) studies indicate that following a mild stress (injection of isotonic saline) there is an increase in c-fos gene expression in the adrenal cortex of the deprived neonate, whereas the nondeprived pup exhibited almost no detectable levels of c-fos mRNA.23 If maternally deprived pups are provided with food during the period of maternal deprivation, both basal and stress levels of corticosterone no longer differ from mother-reared pups.

SHRP AND THE PITUITARY The concept of an absolute SHRP regarding the response of the pituitary following stress in the neonate is much more problematic. Whether the pituitary can show an increase in ACTH in response to stress is dependent on numerous factors. Among these are the age of the neonate, the type of stress imposed, and, once again, maternal factors. The early findings concerning the stress response of the pituitary suggested that there was a deficiency in the neonates’ capacity to synthesize ACTH. Thus, as a result, the pup should exhibit a reduction in the magnitude of the ACTH stress response. However, sufficient data now indicate that the pituitary of the neonate does have the capacity to synthesize and release ACTH that closely resembles the adult response. What seems to discriminate the neonate from the adult is that for the pup the response of the pituitary is much more stimulus dependent. While neonates can indeed mount an ACTH response to challenges as endotoxin injection, ether vapor or cold, milder stressors as novelty or a saline injection have little or no effect.14,24 The capacity of the pituitary to release ACTH seems thereby not different from the situation in adult animals. Three hours following adrenalectomy (ADX), a robust increase in ACTH occurs as early as postnatal day 5.25 This magnitude of the ACTH response is as great as that seen in older neonates at postnatal day 18, which are well out of the

SHRP. Under some circumstances the pup can show an even greater ACTH response early in development than later. In contrast, brief periods of maternal separation, exposure to novelty, injections of isotonic saline, and restraint for 30 min all failed to elicit an ACTH response in normally reared pups until they escaped from the SHRP. Recent studies in the mouse have revealed that the low and suppressed ACTH response during the SHRP is indeed largely mediated by an enhanced glucocorticoid receptor (GR)-mediated negative feedback.26e28 Treatment with the GR antagonist RU486 resulted in a pronounced increase of basal ACTH and corticosterone values in 9-day-old pups to levels greater than in the adult (see Fig. 4.1). As a different study observed only a small effect of RU486 when applied locally at the paraventricular nucleus of the hypothalamus (PVN), a direct GRmediated suppression of ACTH production and release at the pituitary level seems likely, at least towards the end of the SHRP. The data also indicate that the hypoactivity of the neonatal stress system may be restricted to the pituitaryadrenal axis. These data were confirmed in mice with a genetic deletion of the glucocorticoid receptor in the pituitary, which show an excessive oversecretion of corticosterone during the SHRP already under basal conditions, which normalizes again in adult animals. The high sensitivity of the pituitary GRs during the SHRP might be a consequence of the low levels of circulating CBG or other factors that affect GR sensitivity, such as the expression of cochaperones. However, so far there is little known of why and how neonates discriminate between different classes of stimuli and how the GR suppression of the peripheral HPA axis response is overwritten under such circumstances.

SHRP AND THE BRAIN The brain is clearly a stress-sensitive organ. It is not the purpose of this chapter to review all of the changes in the brain that have been shown to occur in response to stress in the neonate. For this chapter the focus will be on the specific

SHRP AND THE BRAIN

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FIGURE 4.1

Plasma corticosterone (A) and ACTH (B) in 9-day-old mouse pups during the SHRP. Animals were injected i.p. 16 and 8 h before testing with either vehicle (polyethylene glycol, n ¼ 10), the MR antagonist RU28318 (n ¼ 15) or the GR antagonist RU38486 (n ¼ 15). Data represent mean  SEM. *Significant from vehicle-treated animals, P NAC > striatum). Behavioral stress also induces complex neuroadaptations in dopaminergic neurons that are very similar to those induced by drugs of abuse.8,12e14 Collectively, these data demonstrate that increases in DA neurotransmission occur during exposure to acute stress. But what functional significance do these changes in DA activity represent? There are several lines of evidence suggesting that DA systems play critical roles in stress responses. First, antianxiety drugs such as benzodiazepines can reduce stress-induced increases in DA release, suggesting that the therapeutic actions of these compounds may be due at least in part to reversing increased dopaminergic activation. Second, lesions of midbrain DA neurons lead to a partial suppression of endocrine stress responses (see Section “DA and the HPA Axis”in the following for a more complete discussion). Third, enhancement of DA signaling in the amygdala attenuates stressinduced ulcer formation, and stress-induced increases in dopaminergic activity in the prefrontal cortex have been shown to be associated with

NERVE CELLS, SYNAPTIC TRANSMISSION, AND DA PATHWAYS IN THE CNS

behavioral coping responses. These and other data have been interpreted as indicating that stress-induced increases in dopaminergic activity are important components of coping responses to environmental stressors rather than being a reflection of anxiety.7 Social context and biological sex-dependent modulation also affect the nuances of DA responses to stress.15 Yet, in trying to understand the functional significance of the activation of brain DA systems, it must be kept in mind that DA neurotransmission can be increased by a variety of environmental and behavioral challenges that are nonaversive. Such challenges include feeding, sexual activity, and exposure to drugs of abuse. Indeed, the dopaminergic pathway from the VTA to the NAC is critical for the reinforcing effects of many addictive drugs, including cocaine, morphine, and nicotine, as well as direct brain stimulation. These two seemingly contradictory observationsdDA release is increased by stress and by positively reinforcing stimulidcan be reconciled if one assumes that any stimulus that causes behavioral arousal leads to an increase in DA neurotransmission. Dopaminergic activation is probably essential for attention and survival. However, at some point, the amount of activation becomes detrimental. For example, a rat trained to lever press in order to receive injections of cocaine will respond at a very steady rate. Such an animal will exhibit elevated rates of DA release in the NAC and other brain regions. If the dose of cocaine per injection is suddenly increased, however, the animal will reduce its rate of lever pressing such that only enough cocaine is present to maintain the previous level of DA release. In other words, if given the choice, the animal will not allow the extent of DA activation to exceed some optimal level.16 It is likely that this optimum varies both from individual to individual and from physiological state to state. A similar “inverted U” relationship between stress-induced DA activation and working memory has been well-documented within the frontal cortex,17e19 and an illustration of this concept is displayed as Fig. 9.3. Recent human imaging studies have similarly supported distinct roles for dopaminergic neurotransmission in

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mediating both expectations regarding the stressor(s) and threat evaluation.20

Responses of Dopaminergic Systems to Chronic Stress Animals are often confronted with repeated or prolonged exposure to stressors, and this can lead to additional neurobiological and behavioral changes. Under certain conditions, habituation can resultdthe animals cease responding to the stimulus. But under other conditions, sensitization can occur. In this case, the animal’s biological and/or behavioral responses increase to the next presentation of the same stimulus or to a different stimulus. Whether or not a given paradigm leads to habituation or sensitization is likely determined by the exact nature and duration of the stressors used, the species, and/or the specific neuronal subpopulation examined. These processes likely contribute to stressrelated disorders such as anxiety, depression, and fibromyalgia. Chronic stress can also lead to “crosssensitization” to drugs of abuse such as cocaine, amphetamine, and opiates at both the behavioral and cellular level. It is possible that sensitization plays a role in the development of posttraumatic stress disorder, panic attacks, and psychosis. Furthermore, as DA can oxidize to form potentially toxic metabolites, it has been hypothesized that the prolonged elevations in DA observed with repeated stress may lead to impairments of cellular functioning,8,21,22 although this possibility has not yet been rigorously examined. Many cellular and molecular changes have been described in the development of neurochemical and behavioral sensitization following chronic stress.7,23e27 For example, chronic intermittent or sustained stress has been shown to induce changes in the number of DA receptors in several brain regions, including the prefrontal cortex and NAC. Chronic stress also leads to changes in TH expression in the VTA and in the activity of several components of DA receptor signaling in the NAC, dorsal striatum, and amygdala. Recent studies have demonstrated great heterogeneity within subsets of DA neurons, even within constrained anatomical regions.13,14

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FIGURE 9.3 The “inverted U” model for dopaminergic regulation of prefrontal function which was originally proposed by Patricia Goldman-Rakic and colleagues.17e19 Top: A generalized model for DA function is depicted which assumes that, under normal conditions, there is a range of DA concentration over which signal processing in prefrontal neurons operates efficiently and working memory performance is sufficient to the task. At the center of this range is an optimum for cortical DA level, and D1 receptor stimulation in particular, at which performance is maximal. The exact shape and slope of this curve are likely task-specific, such that complex tasks may be more sensitive to a narrower range of DA concentrations. Bottom: The curve may be altered in shape, slope (blue and yellow gradients), or be shifted to the left or right (green and brown gradients, respectively) depending on genotype, life history, and/or conditions. Individuals that suffer from deficiency of prefrontal DA transmission may benefit from DA receptor stimulation by agonists, whereas those that suffer from excessive endogenous transmission may benefit from treatment with DA receptor antagonists.

INTERACTIONS BETWEEN DA AND OTHER NEUROCHEMICAL SYSTEMS ALTERED BY STRESS DA and the HPA Axis Many neurochemical and hormonal systems are activated by stress. One of the bestcharacterized physiological responses to stress is the activation of the hypothalamicepituitarye adrenal (HPA) axis. The HPA system is a principal component of the stress response that regulates the secretion of glucocorticoid hormones from the adrenal gland. Interestingly, several dopaminergic regions in the brain express glucocorticoid receptors, and the density

of the receptors in these regions parallels the ability of stress to increase the activity of DA neurons: receptor density is higher in VTA than SN. There is reason to believe that the correlation between glucocorticoid receptors and stressinduced changes in DA release has functional significance.25 Administration of glucocorticoids can increase extracellular DA. Moreover, the suppression of stress-induced corticosterone secretion causes a reduction in the dopaminergic response to stress. Glucocorticoid regulation of midbrain DA neurons also appears to be a biological substrate of the effects of stress on the propensity to develop drug abuse.25 Conversely, midbrain

INTERACTIONS BETWEEN DA AND OTHER NEUROCHEMICAL SYSTEMS ALTERED BY STRESS

DA neurons also seem capable of regulating the HPA axis since loss of DA decreases the basal and stress-induced secretion of corticosterone.

DA and Excitatory Amino Acids Stress has been shown to increase the release of glutamate and other excitatory amino acids in many brain regions, including the prefrontal cortex, striatum, NAC, VTA, and hippocampus.28 The presence of glucocorticoids appears to be necessary for this response as adrenalectomy abolishes the stress-induced increase of extracellular glutamate, and corticosterone replacement restores this response. Glutamatergic neurons in the cortex project to the striatum and NAC as well as SN and VTA. Furthermore, glutamate agonists appear to increase the synthesis and release of DA in each of these regions, and it has been hypothesized that stress-induced activation of glutamate may contribute to stressinduced increases in DA. The local administration of glutamate antagonists into the region of the midbrain containing DA cell bodies can block stress-induced increases in extracellular DA in the striatum. Additionally, stressinduced increases in DA synthesis can be blocked by local administration of a glutamate antagonist directly into the striatum itself. Glutamate antagonists can also block stressinduced increases in extracellular DA in the prefrontal cortex. Intracellularly, glutamatergic and dopaminergic signals can converge on a single protein, the DA- and 3’,5’-cyclic adenosine monophosphate ( cAMP-) regulated phosphoprotein (DARPP-32), a protein whose activation is a crucial component of neural signaling responses.29,30

DA and NE NE is formed from DA and shares many of its basic characteristics. Acute exposure to stress causes an activation of NE neurons in the locus coeruleus and an increase in both the synthesis and release of NE. The exact relationship between these events and stress-induced changes in DA are unclear. NE terminals in the prefrontal cortex modulate the dopaminergic responses to stress and interactions between NE and DA in

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the prefrontal cortex modulate synaptic function interactively.31 In fact, the NE transporter, rather than the DA transporter, is responsible for the reuptake of DA within the prefrontal cortex.32,33 The NAC and striatum, on the other hand, exhibit increases in dopaminergic activity with stress but are NE-poor regions; presumably, NEeDA interactions have little significance in these regions. However, novel roles for the NErich bed nucleus of the stria terminalis in mediating polysynaptic responses to stress and drugs of abuse have recently been implicated and are a current area of intense scrutiny.34

DA and Serotonin The involvement of 5-HT in animal models of stress and depression has received great attention due to the antidepressant effects of selective 5-HT selective serotonin reuptake inhibitors (SSRIs) such as fluoxetine (Prozac). Acute exposure to stress leads to dramatic increases in 5-HT release in the striatum and other brain regions. Local administration of 5-HT agonists into the striatum can increase DA release, suggesting it is possible that stress-induced increases in DA release are at least partially mediated by increases in 5-HT. Anatomical and electrophysiological evidence also suggests prominent interactions between 5-HT and DA in both cell body and terminal regions.35 These effects appear to be mediated via multiple families of 5-HT receptors, including the 5-HT1A, 5HT2C, and 5-HT4 subtypes.

DA and DA In developing an understanding of the role of DA neurotransmission in stress, one must take into consideration the functional differences among distinct brain DA systems. In particular, it must be remembered that mesocortical DA neurons inhibit glutamatergic cortical pyramidal neurons which in turn can stimulate dopaminergic activity in nigrostriatal and mesoaccumbens neurons (see Section “Dopamine and Glutamate”). Therefore, the less pronounced effects of acute stress on subcortical DA systems may be due in part to the suppression of subcortical DA systems by activated mesocortical DA

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neurons. Consistent with this hypothesis, depletion of DA in the prefrontal cortex enhances the stress-induced increase in dopaminergic transmission in the NACshell. Furthermore, injection of a DA receptor antagonist into the prefrontal cortex has been shown to block the DA stress response in the NAC. Lastly, under certain conditions, chronic stress produces neurochemical tolerance in the prefrontal cortex but sensitization in subcortical areas. Interactions between cortical and subcortical DA systems have also been proposed in the pathophysiology of schizophrenia. A dysfunction of dopaminergic signaling in the prefrontal cortex can produce secondary disruptions in subcortical DA systems and vice-versa. In fact, decreases in the expression of TH and DA receptors in the prefrontal cortex have recently been described. The effects of cortical DA depletion on subcortical DA stress responses may thus be of particular clinical importance, given that stress is known to exacerbate psychotic symptoms in schizophrenic patients. Individual variability in certain traits, such as impulsivity, also associates with complex brainregion and molecular differences in DA system functioning.36

Developmental Modulation Finally, recent studies have demonstrated major effects of altering neurodevelopmental trajectory on subsequent functioning of brain DA systems. For example, prenatal exposure to stress or drugs of abuse can permanently alter the anatomical organization and cellular responses of both midbrain DA neurons and their forebrain targets.37,38 The quality of parental care has also been observed to alter these relationships in both animal models and in humans. For example, repeated periods of maternal separation during early life decreases DA transport and increases behavioral and brain DA responses to stress.39e41 DA systems are also uniquely sensitive to environmental, genetic, and/or pharmacological modifications during adolescence.42,43 Responses of brain DA systems

to stress, therefore, are critically impacted by genetic and environmental risk factors. Elucidation of these risk factors, their specific effects on brain maturation, and the development of therapeutic methods to normalize developmental trajectory in their presence are all important areas of active research.

References 1. Purves D, et al. Neuroscience. 6th ed. Oxford University Press; 2018. 2. Beaulieu JM, Gainetdinov RR. The physiology, signaling, and pharmacology of dopamine receptors. Pharmacol Rev. 2011;63:182e217. https://doi.org/ 10.1124/pr.110.002642. 3. Deutch AY, Roth RH. The determinants of stressinduced activation of the prefrontal cortical dopamine system. Prog Brain Res. 1990;85:367e402. discussion 402-363. 4. Horger BA, Roth RH. The role of mesoprefrontal dopamine neurons in stress. Crit Rev Neurobiol. 1996;10: 395e418. 5. Finlay JM, Zigmond MJ. The effects of stress on central dopaminergic neurons: possible clinical implications. Neurochem Res. 1997;22:1387e1394. 6. Moghaddam B, Jackson M. Effect of stress on prefrontal cortex function. Neurotox Res. 2004;6:73e78. 7. Cabib S, Puglisi-Allegra S. The mesoaccumbens dopamine in coping with stress. Neurosci Biobehav Rev. 2012;36:79e89. https://doi.org/10.1016/j.neubiorev.2011.04.012. 8. Belujon P, Grace AA. Regulation of dopamine system responsivity and its adaptive and pathological response to stress. Pro Biol Sci. 2015;282. https://doi.org/ 10.1098/rspb.2014.2516. 9. Abercrombie ED, Keefe KA, DiFrischia DS, Zigmond MJ. Differential effect of stress on in vivo dopamine release in the striatum, nucleus accumbens, and medial prefrontal cortex. J Neurochem. 1989;52: 1655e1658. 10. Pezze MA, Feldon J. Mesolimbic dopaminergic pathways in fear conditioning. Prog Neurobiol. 2004;74: 301e320. 11. Francis TC, Lobo MK. Emerging role for nucleus accumbens medium spiny neuron subtypes in depression. Biol Psychiatr. 2017;81:645e653. https://doi.org/10.1016/ j.biopsych.2016.09.007. 12. Piazza PV, Le Moal M. The role of stress in drug selfadministration. Tr Pharmacol Sci. 1998;19:67e74. 13. Holly EN, Miczek KA. Ventral tegmental area dopamine revisited: effects of acute and repeated stress. Psychopharmacology. 2016;233:163e186. https://doi.org/ 10.1007/s00213-015-4151-3.

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14. Lammel S, Lim BK, Malenka RC. Reward and aversion in a heterogeneous midbrain dopamine system. Neuropharmacology. 2014;76:351e359. https://doi.org/10.1016/ j.neuropharm.2013.03.019. Pt B. 15. Trainor BC. Stress responses and the mesolimbic dopamine system: social contexts and sex differences. Horm Behav. 2011;60:457e469. https://doi.org/10.1016/ j.yhbeh.2011.08.013. 16. Ator NA, Griffiths RR. Principles of drug abuse liability assessment in laboratory animals. Drug Alcohol Depend. 2003;70:S55eS72. 17. Arnsten AF, Wang M. Targeting prefrontal cortical systems for drug development: potential therapies for cognitive disorders. Annu Rev Pharmacol Toxicol. 2016;56: 339e360. https://doi.org/10.1146/annurev-pharmtox010715-103617. 18. Zahrt J, Taylor JR, Mathew RG, Arnsten AF. Supranormal stimulation of D1 dopamine receptors in the rodent prefrontal cortex impairs spatial working memory performance. J Neurosci. 1997;17:8528e8535. 19. Goldman-Rakic PS, Castner SA, Svensson TH, Siever LJ, Williams GV. Targeting the dopamine D1 receptor in schizophrenia: insights for cognitive dysfunction. Psychopharmacology. 2004;174:3e16. https://doi.org/ 10.1007/s00213-004-1793-y. 20. Vaessen T, Hernaus D, Myin-Germeys I, van Amelsvoort T. The dopaminergic response to acute stress in health and psychopathology: a systematic review. Neurosci Biobehav Rev. 2015;56:241e251. https://doi.org/10.1016/j.neubiorev.2015.07.008. 21. Kulak A, et al. Redox dysregulation in the pathophysiology of schizophrenia and bipolar disorder: insights from animal models. Antioxidants Redox Signal. 2013;18:1428e1443. https://doi.org/10.1089/ ars.2012.4858. 22. Alghasham A, Rasheed N. Stress-mediated modulations in dopaminergic system and their subsequent impact on behavioral and oxidative alterations: an update. Pharmaceut Biol. 2014;52:368e377. https:// doi.org/10.3109/13880209.2013.837492. 23. Lucas LR, et al. Repeated exposure to social stress has long-term effects on indirect markers of dopaminergic activity in brain regions associated with motivated behavior. Neuroscience. 2004;124:449e457. 24. Arnsten AF. Prefrontal cortical network connections: key site of vulnerability in stress and schizophrenia. Int J Dev Neurosci. 2011;29:215e223. https://doi.org/ 10.1016/j.ijdevneu.2011.02.006. 25. Marinelli M, Piazza PV. Interaction between glucocorticoid hormones, stress and psychostimulant drugs. Eur J Neurosci. 2002;16:387e394. 26. Muir J, et al. In Vivo fiber photometry reveals signature of future stress susceptibility in nucleus accumbens. Neuropsychopharmacol. 2017. https://doi.org/10.1038/ npp.2017.122.

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27. Tye KM, et al. Dopamine neurons modulate neural encoding and expression of depression-related behaviour. Nature. 2013;493:537e541. https://doi.org/ 10.1038/nature11740. 28. Moghaddam B. Stress activation of glutamate neurotransmission in the prefrontal cortex: implications for dopamine-associated psychiatric disorders. BiolPsychiatr. 2002;51:775e787. 29. Fienberg AA, et al. DARPP-32: regulator of the efficacy of dopaminergic neurotransmission. Science. 1998;281: 838e842. 30. Svenningsson P, et al. DARPP-32: an integrator of neurotransmission. Ann Rev Pharmacol Toxicol. 2004;44: 269e296. 31. Xing B, Li YC, Gao WJ. Norepinephrine versus dopamine and their interaction in modulating synaptic function in the prefrontal cortex. Brain Res. 2016;1641:217e233. https://doi.org/10.1016/j.brainres.2016.01.005. 32. Mazei MS, Pluto CP, Kirkbride B, Pehek EA. Effects of catecholamine uptake blockers in the caudateputamen and subregions of the medial prefrontal cortex of the rat. Brain Res. 2002;936:58e67. 33. Yamamoto BK, Novotney S. Regulation of extracellular dopamine by the norepinephrine transporter. J Neurochem. 1998;71:274e280. 34. Kash TL. The role of biogenic amine signaling in the bed nucleus of the stria terminals in alcohol abuse. Alcohol. 2012;46:303e308. https://doi.org/10.1016/ j.alcohol.2011.12.004. 35. Hensler JG, et al. Catecholamine/Serotonin interactions: systems thinking for brain function and disease. Adv Pharmacol. 2013;68:167e197. https://doi.org/ 10.1016/b978-0-12-411512-5.00009-9. 36. Bosker WM, Neuner I, Shah NJ. The role of impulsivity in psychostimulant- and stress-induced dopamine release: review of human imaging studies. Neurosci Biobehav Rev. 2017;78:82e90. https://doi.org/10.1016/ j.neubiorev.2017.04.008. 37. Frederick AL, Stanwood GD. Drugs, biogenic amine targets and the developing brain. Dev Neurosci. 2009;31: 7e22. https://doi.org/10.1159/000207490. 38. Thompson BL, Levitt P, Stanwood GD. Prenatal exposure to drugs: effects on brain development and implications for policy and education. Nature reviews. Neuroscience. 2009;10:303e312. https:// doi.org/10.1038/nrn2598. 39. Pruessner JC, Champagne F, Meaney MJ, Dagher A. Dopamine release in response to a psychological stress in humans and its relationship to early life maternal care: a positron emission tomography study using [11C]raclopride. J Neurosci. 2004;24:2825e2831. https:// doi.org/10.1523/jneurosci.3422-03.2004. 40. Pruessner JC, et al. Stress regulation in the central nervous system: evidence from structural and functional neuroimaging studies in human populations - 2008

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Curt Richter Award Winner. Psychoneuroendocrinology. 2010;35:179e191. https://doi.org/10.1016/j.psyneuen. 2009.02.016. 41. Teicher MH, et al. The neurobiological consequences of early stress and childhood maltreatment. Neurosci Biobehav Rev. 2003;27:33e44. 42. Burke AR, Miczek KA. Stress in adolescence and drugs of abuse in rodent models: role of dopamine, CRF, and

HPA axis. Psychopharmacology. 2014;231:1557e1580. https://doi.org/10.1007/s00213-013-3369-1. 43. Ernst M, Romeo RD, Andersen SL. Neurobiology of the development of motivated behaviors in adolescence: a window into a neural systems model. Pharmacol Biochem Behav. 2009;93:199e211. https://doi.org/10.1016/ j.pbb.2008.12.013.

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10 Serotonin in Stress Maarten van den Buuse, Matthew W. Hale School of Psychology and Public Health, La Trobe University, Melbourne, VIC, Australia O U T L I N E Introduction: Stress, Serotonin, and Human Psychopathology 115

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Stress, Serotonin, and Human Psychopathology Anxiety, PTSD, and Depression Psychoses

Effect of Serotonergic Drugs on Stress Responses: Serotonin and HPA Axis Activity

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INTRODUCTION: STRESS, SEROTONIN, AND HUMAN PSYCHOPATHOLOGY It is widely accepted that stress plays a role in the pathology of several psychiatric disorders. This is most notable in mood and anxiety disorders such as major depression, general anxiety disorder, and posttraumatic stress disorder (PTSD) but, similarly, stress has been shown to play a role or exacerbate symptoms in psychotic illnesses, such as schizophrenia (Fig. 10.1, ➀). Serotonin systems in the brain are implicated in all

Stress: Physiology, Biochemistry, and Pathology https://doi.org/10.1016/B978-0-12-813146-6.00010-2

these disorders (Fig. 10.1, ➁). There is a wealth of literature showing that stress affects serotonergic signaling in the brain (Fig. 10.1, ➂) and thereby influences disease development. Conversely, serotonergic drugs can modulate or mitigate the effects of stress in psychopathology (Fig. 10.1, ➃). The role of stress in specific psychiatric illnesses is described in other chapters in this handbook. This chapter will summarize the complex and reciprocal interrelationship between stress and brain serotonin signaling in the development and symptomatology of psychiatric disorder.

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Copyright © 2019 Elsevier Inc. All rights reserved.

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KEY POINTS • Both serotonin and stress are implicated in a range of psychiatric disorders. • Stress affects several aspects of serotonergic signalling in the brain and serotonergic drugs can modulate the effects of stress. • Stress effects on the serotonin system are highly complex, regionally different throughout the brain, and dependent on the type of stress, serotonergic marker, and individual differences in resilience and genetic background. • Further pre-clinical as well as clinical studies are needed to increase our understanding of the interaction of stress, serotonin and human psychopathology, ultimately leading to more effective and selective treatments for psychiatric disorders.

EFFECT OF STRESS ON SEROTONIN PARAMETERS IN THE BRAIN Animal Models Already several decades ago, early animal model studies have addressed the effects of single and chronic stress on serotonin

FIGURE 10.1 Stress and serotonin systems in the brain interact to affect psychiatric disorder development. Stress plays a role in the pathology of several psychiatric disorders, including mood and anxiety disorders and psychosis (➀). Serotonin systems in the brain are implicated in these disorders (➁). Stress affects serotonergic signaling in the brain (➂) and, conversely, serotonergic drugs can modulate the effects of stress in psychopathology (➃).

concentrations in the brain and on the activity of tryptophan hydroxylase, the rate-limiting enzyme in the biosynthesis of serotonin.1 This has been followed by a large animal model literature showing that stress effects on the serotonergic system are strongly regional in the brain and depend on the type of stress, its duration, the serotonergic marker measured, and individual difference in stress resilience of the tested animals. For example, when cynomolgus monkeys were characterized as high stress resilient, medium stress resilient, or low stress resilient, the stress-sensitive animals had decreased expression of a number of genes that are essential for serotonergic system development, including Plasmacytoma expressed transcript 1 (PET-1), which codes for a transcription factor that is critical for differentiation and development of brain serotonergic systems, TPH2, which codes for tryptophan hydroxylase, SLC6A4, which codes for the serotonin transporter (SERT), and HTR1A, which codes for the serotonin 1A receptor, but increased expression of corticotropinreleasing hormone (CRH) in the dorsal raphe nucleus (DRN) than stress-resilient animals,2 suggesting that the serotonin system is involved in determining stress sensitivity and resilience. Because stress elicits a large number of neuroendocrine and neurochemical responses in the brain, all of which could directly or indirectly affect serotonin function, several studies have focused on individual components of the stress response. In particular, the effect of cortisol (corticosterone in rodents) on serotonergic function has been widely characterized (for references, see studies by Chaouloff3 and Meijer and de Kloet4). In this context it is important to note that there are two intracellular receptors for cortisol in neurons, mineralocorticoid, and glucocorticoid receptors, with high and low affinity for cortisol, respectively.5 At a cellular level, occupation of mineralocorticoid receptors reduced electrophysiological effects of serotonin in the hippocampus in vitro, an effect mediated by serotonin 1A receptors but not seen with selective glucocorticoid receptor ligands.6 Later experiments showed that activation of glucocorticoid receptors can enhance raphee hippocampus serotonin transmission.4 However, chronic treatment with corticosterone7 or chronic

EFFECT OF STRESS ON SEROTONIN PARAMETERS IN THE BRAIN

unpredictable stress8 induced a similar reduction of serotonin 1A receptoremediated effects.9 Thus, the effects of glucocorticoids on the brain in general and on serotonergic transmission in particular greatly depend on a number of factors. Rapid in vivo onset and short duration effects are mediated by a membrane-bound, nongenomic receptor mechanism, whereas sloweronset, longer-duration effects are mediated by nuclear receptors and a genomic action. Chronically increased levels of corticosteroids, as in chronic stress, reduce serotonin signaling in the hippocampus.4 Consistent with a role for serotonin in the central response to stress, exposure to stress-related stimuli activates serotonergic neurons in the

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DRN, the brain region that contains the majority of forebrain-projecting serotonergic neurons. Serotonergic neurons in the DRN are highly topographically organized and can be subdivided into anatomically and functionally distinct subpopulations of neurons.10 Exposure of the rats to the mild stress of being placed on an elevated plus maze elicited activation of serotonergic cells in the dorsal and the ventrolateral parts of the DRN (DRD and DRVL, respectively) versus the ventral and interfascicular parts of the DRN (DRV and DRI, respectively; Fig. 10.2, for more details, see the study by Lawther et al.11). Physiological stressors (such as hypercapnia) or intense physiological/psychological stressors such as cold water swim stress or social defeat, increase

FIGURE 10.2 Exposure to the elevated plus maze (EPM) activates subpopulations of serotonergic neurons in the dorsal raphe nucleus (DRN). Photomicrographs show immunohistochemical staining for c-Fos, the protein product of the immediate early gene, c-fos (used as a marker of neuronal activation, which appears as a blue-black stain localized to the nuclei) and TPH-2 (used as a marker of serotonergic neurons, which appears as a brown/orange stain localized to the cytoplasm). Compared with rats exposed to home cage control conditions, rats exposed to the EPM for 5 min in low-light conditions (a relatively mild stress-related stimulus) show increased c-Fos expression within serotonergic neurons in the dorsal and ventrolateral parts of the DRN (DRD and DRVL, respectively) but not the ventral and interfascicular parts of the DRN (DRV and DRI, respectively). Serotonergic neurons in the DRD and DRVL are associated with anxiety-like and antipanic responses respectively.10,12 Boxes in the low magnification images in the top row are shown at higher magnification in the bottom row. Black boxes in the images in the bottom row are shown at higher magnification in the insets. Black arrowheads show c-Fosimmunopositive/TPH-immunopositive (double labeled, i.e., activated) serotonergic neurons, while white arrowheads show c-Fos-immunonegative/TPH-immunopositive (i.e., nonactivated) serotonergic neurons. Scale bar, 200 mm (top row), 100 mm (bottom row), 50 mm (insets). For full experimental details, see the study by Lawther et al.11

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expression of the protein product of the immediate early gene, c-fos (c-Fos) in all subpopulations of serotonergic neurons.12 Taken together, these data suggest that serotonergic systems are sensitive to stress-related stimuli, but the extent of serotonergic activation depends on the nature, duration, and intensity of the stressor (Fig. 10.2).

Human Imaging While several studies have investigated changes in serotonergic parameters in human psychopathology associated with stress (see Section Stress, Serotonin, and Human Psychopathology), few studies have actually directly assessed the effect of stress or components of the stress response on serotonergic factors in healthy humans. The cortisol awakening response, a rise in circulating cortisol levels around 30 min after awakening, has been proposed as an index of HPA axis dynamics.13 This response was negatively associated with binding to the serotonin 4 receptor in the prefrontal cortex, anterior cingulate cortex, and striatum, but not hippocampus. The authors interpreted these findings as indicating higher state level of serotonin release in the brain induced by the cortisol awakening response. This was in contrast to earlier studies which established a positive relationship between the cortisol awakening response and SERT binding which was interpreted as a trait phenomenon.14

EFFECT OF SEROTONERGIC DRUGS ON STRESS RESPONSES: SEROTONIN AND HPA AXIS ACTIVITY Animal Models The relationship between stress and serotonin is clearly reciprocal, with several studies showing that serotonin and serotonergic drugs influence activity of several components of the HPA axis and stress response more general. Anatomical evidence indicates serotonin-immunoreactive terminals throughout the hypothalamus, including

the parvocellular and magnocellular divisions of the paraventricular nucleus (PVN) of the hypothalamus.15 These serotonergic terminals form axodendritic and axosomatic contacts with CRH-containing neurons in the PVN, and activation of serotonergic receptors in this region increases HPA axis activation. Consistent with this observation, early studies (reviewed by Holsboer and Barden16) showed that administration of the serotonin releaser, fenfluramine, caused increased release of CRH, ACTH (adrenocorticotropic hormone), as well as corticosterone.17,18 This conclusion was based on evidence obtained both in vitro and in vivo and implicated both serotonin 1A receptors and serotonin 2A receptors in these effects, but an involvement of other serotonin receptor subtypes was not excluded.19,20 Stress-induced changes in blood pressure and heart rate were also affected by 5-HT1A receptor agonists; however, these effects depended on the rat strain tested.21 A large number of studies have focused on the effect of serotonin reuptake inhibitors on components of the HPA axis.20 These drugs, which are used clinically to treat anxiety and affective disorders, inhibit the reuptake of serotonin into the presynaptic terminal, thereby acutely increasing extracellular concentrations of serotonin. Preclinical evidence suggests that acute administration of fluoxetine (Prozac, Sarafem), which is one of the most commonly used and best-tolerated serotonin reuptake inhibitor drugs,22 potently increases extracellular concentration of serotonin in the hypothalamus, including within the PVN, which contains the CRH-containing neurons. Consistent with a role for serotonin in the control of the HPA axis, acute administration of serotonin reuptake inhibitors increases indices of HPA axis activity, including increased CRH mRNA in the PVN, increased c-Fos expression in CRH-containing neurons in the PVN, increased plasma ACTH, and increased plasma corticosterone. Serotonin acts at several different receptors in the PVN to alter HPA axis activity.23 Microinjection studies using selective agonists, or the amine itself, suggest that serotonin can activate or inhibit the HPA axis depending on the dose and receptor subtype involved.24 For example, intra-PVN

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administration of serotonin 1A receptor agonists inhibits HPA axis activity at low doses but increases HPA axis activity at higher doses. Administration of serotonin 2A receptor agonists increases plasma ACTH and corticosterone concentrations.24 While the acute effects of serotonin reuptake inhibitor drugs on the HPA axis are relatively well characterized, it is less clear how these acute effects relate to the antidepressant properties of these drugs, which take weeks to become evident. Pharmacological evidence suggests that chronic administration of serotonin reuptake inhibitor drugs reduces HPA axis responsiveness. For example, chronic administration of citalopram decreases plasma ACTH and CRH mRNA in the PVN.25 However, it is unclear how these effects specifically relate to changes in serotonergic activity, as similar chronic effects have been reported with antidepressant drugs with a predominantly noradrenergic mechanism of action.16 As concluded by Holsboer et al., “Apparently, quite different pharmacological effects can act clinically to improve depression, suggesting that depression can be caused by different aetiologies that all ultimately produce impaired HPA function.”16 This view is consistent with the previously mentioned multifactorial model of regulation of stress and the HPA axis, including a large number of nuclei involved in responses to systemic stressors. These pathways include brainstem nuclei, for example, the nucleus tractus solitarius, raphe nuclei, and locus coeruleus, all of which directly project to the PVN to influence CRH secretion.19

Human Studies Acute manipulation of serotonergic activity or administration of serotonergic drugs has been used in healthy humans as a tool to probe the interaction of brain serotonin, stress reactivity, and HPA axis activity. Several of these studies investigated the effect of a variety of serotonergic drugs on circulating levels of ACTH or cortisol, as well as other hormones indicative of stress responses.20 More indirectly, the effect of serotonergic drugs on stress responses in humans has

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been assessed. For example, reduction of brain serotonin levels by dietary tryptophan depletion caused attenuation of fear responses and was accompanied by lower fear signals in the amygdala and orbitofrontal cortex.26 On the other hand, in PET imaging studies, lower SERT availability in the amygdala and cortical regions was shown to predict enhanced fear responses in a conditioning paradigm.27 Public speaking and its associated activation of the HPA axis have been used in experimental studies in humans to investigate the role of serotonin in stress responses.28 Several serotonergic drugs have been tested in human paradigms of public speaking stress, including serotonin 1A receptor agonists such as buspirone and ipsapirone, serotonin 2A receptor antagonists such as ritanserin, serotonin releasers such as fenfluramine, and reuptake inhibitors such as escitalopram (for references, see Garcia-Leal et al.28). The overarching conclusion of these studies was that serotonin inhibits fear as probed in a public speaking task. This was in line with findings that tryptophan depletion, which reduces serotonin levels, enhanced anxiety responses in this task, at least in women. Such a result would seem inconsistent with fear conditioning studies mentioned previously; however, this could be due to serotonin exerting opposite effects on different brain substrates involved in fear and anxiety. While conditioned fear is consistently associated with the amygdala, public speaking stress may be associated with the periaqueductal gray.28 These regional differences in the involvement of serotonin in stress responses are in line with animal data for differential involvement of subregions of the DRN in selected components of the HPA axis.10,12

STRESS, SEROTONIN, AND HUMAN PSYCHOPATHOLOGY Given the widely accepted role of stress as a risk factor for a number of psychiatric illnesses, such as major depression, PTSD, or schizophrenia, several studies have investigated

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postmortem and genetic markers of serotonergic dysfunction in these illnesses.

Anxiety, PTSD, and Depression Several reviews have addressed the changes in serotonin system activity seen in mood and anxiety disorders associated with stress reactivity.29 For example, in patients with panic disorder or with major depressive disorder, a marked increase in brain serotonin turnover was observed. These disease states were furthermore associated with reduced serotonin 1A receptor density (for references, see Hale et al.12). Recently, Frick et al. used PET to assess SERT density in patients with PTSD compared with controls.30 These authors observed increased SERT availability in the precentral gyrus and posterior cingulate cortex in PTSD, with no changes in the amygdala. There was a negative correlation between SERT availability in the amygdala and PTSD symptoms.30 These results were in agreement with previous observations of a negative correlation between amygdala SERT availability and anxiety scores in PTSD but were in contrast to reduced SERT binding in the amygdala in that study.31 Other changes in serotonergic markers in PTSD include reduced binding of the serotonin-1B receptor in the amygdala, caudate nucleus, and anterior cingulate cortex.32 This reduction was particularly prevalent at an earlier age of first trauma exposure; however, it was also seen in participants who had undergone trauma but were not suffering from PTSD.32 The authors concluded that, in addition to the longterm effects of trauma, individuals who proceed to develop PTSD will have additional risk factors that play a role. Similar studies in major depression have been the subject of several meta-analyses.29 Kambeitz and Howes concluded that in major depression, imaging studies showed reduced SERT binding in the striatum, amygdala, and brainstem but not the thalamus or hippocampus.33 This was in contrast to postmortem studies, which did not reveal such differences.33 Overall, however, there is great variability in the results of available SERT imaging studies34 preventing clear conclusions about the role of the SERT in major depression. This lack of consensus prevents a clear

understanding of how selective serotonin reuptake inhibitors, the most successful class of antidepressant drugs, are efficacious in major depression and, for example, why it takes weeks for these drugs to show clinical effects despite having acute pharmacological effects and novel treatment, such as ketamine, having much faster onset of action. PET-1 is a transcription factor for the development of serotonin neurons, and PET-1 knockout mice are known to show loss of serotonin projections in the brain and behavioral deficits. Using functional MRI analysis, it was shown that a polymorphism in the PET-1 gene (rs860573) was associated with threat-induced amygdala reactivity and risk for psychopathology,35 illustrating that developmental abnormalities in the serotonergic system are associated with altered stress effects. Genetic variations in the SERT and in serotonin receptors have also been extensively studied for their association with anxiety, depression, PTSD, and other psychiatric illnesses.29 For example, a common polymorphism in the promoter region of the SERT gene (5-HTTLPR) results in a “short” allelic form (S), which results in lower levels of SERT binding in the brain as well as reduced serotonin uptake compared with the “long” allelic variant (L).36 Among the reported effects of this polymorphism is increased risk of anxiety, heightened fear responses, and increased neuroticism (for references, see the studies by Hariri and Holmes36 and Canli and Lesch37). Individuals with one or two alleles furthermore showed higher rates of depressive symptoms and suicidality in some studies,38,39 although other studies, including meta-analyses, have not been able to confirm this.40,41 A common polymorphism in the gene that codes for the serotonin 1A receptor (HTR1A; RS6295) has been associated with major depression and bipolar disorder although this may be dependent on the ethnic background of the subjects.42,43 Finally, the association of two common polymorphisms in the gene that codes for the serotonin 2A receptor (HTR2A; T102C and A-1438G) with major depression has been the subject of several meta-analyses. One such review found no association of major depression susceptibility with the T102C polymorphism in allelic analysis and genotypic analysis, but it

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was concluded that the A allele of A-1438G polymorphism might play a role in major depression susceptibility.44 Rather than gene expression, polymorphisms, or density levels of serotonin receptors and transporters, increasingly it is being recognized that the chronic effects of stress in mood disorders are likely related to epigenetic changes in the genes of these factors. For example, a number of prenatal and postnatal adverse events associated with methylation status of the SERT gene45 although the relationship of this association with psychopathology remains to be determined. However, a preliminary consensus view is that of increased vulnerability to psychopathology in carriers of the short allele of SERT gene as a result of methylation-driven effects of developmental stress.46 Such epigenetically-mediated alterations in gene expression may also be important for the efficacy of serotonergic antidepressant drugs.29

Psychoses The role of stress as a risk factor in schizophrenia and other psychotic illnesses is supported by epidemiological evidence of higher incidence of these illnesses after childhood trauma or abuse47 or in the offspring of people who have been subjected to traumatic life events.48 Studies over several decades have shown changes in serotonergic parameters in the brain in psychosis, and these findings have been the subject of meta-analyses (for example, the study by Selvaraj et al.49). Here, combining data from 50 postmortem studies, the authors found serotonin 1A and 2A receptor density in the prefrontal cortex to be significantly increased and decreased, respectively, but no firm conclusions could be drawn about alterations in the levels of other serotonin receptors or the SERT.49 The longstanding interest in serotonergic involvement in psychosis has led to attempts to develop novel antipsychotic drugs with a pharmacology that combined dopamine receptor antagonism/partial agonism and serotonin 1A or 2A receptor binding.50 However, fewer studies have attempted to directly link stressful life events with serotonergic function in psychosis.

Aas et al. noted that the effect of childhood abuse on cognition in psychotic patients was dependent on their SERT genotype, with s-carriers being more affected by abuse than any of the other groups.51 These results were opposite of those Goldberg et al. who found that psychotic symptoms were more prominently associated with the l-allele and that the relationship between SERT genotype and depressive and negative symptoms also was reversed in psychotic illness compared with healthy controls.40 The authors emphasized the importance of geneegene interactions (epistasis) meaning the effects of specific serotonergic genes could strongly depend on polymorphisms in other genes. Therefore, more recently, others have investigated polymorphisms in serotonin genes in the context of comprehensive genetic analysis, that is, socalled convergent functional genomics.52 Such studies view the resulting genetic risk prediction score, rather than individual gene alterations, in combination with neurodevelopmental stress, as predictive of disease risk. It should be noted that in this comprehensive analysis, so far only the serotonin 2A receptor gene was included.52 Others have postulated that receptor density or polymorphisms are not necessarily a reliable indicator of the effect of chronic stress on the serotonin system in psychosis but that, instead, stress-induced epigenetic changes such as methylation of serotonin receptor genes should be considered, similar to studies on mood disorders. For example, Parade et al. observed that moderate to severe childhood maltreatment was associated with altered 5-HT2A receptor gene methylation, and that the association with psychopathology and direction of the effect was dependent on the CpG site and HTR2A polymorphism genotype investigated.53

CONCLUSIONS There is no doubt that stress has significant effects on the serotonergic system in the brain, both directly at the level of serotonergic neurons in the raphe and indirectly on serotonin release, and receptor and transporter levels. These effects

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can be chronic and involve altered gene expression and epigenetic control. As yet, this complex array of effects of stress on the serotonin system does not allow clear understanding as to how serotonin is involved in the effects of stress on psychopathology. For example, it is essentially unknown how serotonergic drugs are beneficial in major depression, despite their widespread clinical use. This lack of insight has hampered the development of novel classes of serotonergic antidepressants, as well as novel treatments targeted at the serotonin system in anxiety and psychotic disorders. Recent developments in comprehensive functional genomics, pharmacogenomics, and human imaging are expected to increase our understanding of the complex interaction of stress, serotonin, and human psychopathology, ultimately leading to more effective and selective treatments for psychiatric disorders.

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10. Hale MW, Lowry CA. Functional topography of midbrain and pontine serotonergic systems: implications for synaptic regulation of serotonergic circuits. Psychopharmacology. 2011;213(2e3):243e264. 11. Lawther AJ, Clissold ML, Ma S, et al. Anxiogenic drug administration and elevated plus-maze exposure in rats activate populations of relaxin-3 neurons in the nucleus incertus and serotonergic neurons in the dorsal raphe nucleus. Neuroscience. 2015;303:270e284. 12. Hale MW, Shekhar A, Lowry CA. Stress-related serotonergic systems: implications for symptomatology of anxiety and affective disorders. Cell Mol Neurobiol. 2012;32(5):695e708. 13. Jakobsen GR, Fisher PM, Dyssegaard A, et al. Brain serotonin 4 receptor binding is associated with the cortisol awakening response. Psychoneuroendocrinology. 2016;67:124e132. 14. Frokjaer VG, Erritzoe D, Holst KK, et al. Prefrontal serotonin transporter availability is positively associated with the cortisol awakening response. Eur Neuropsychopharmacol. 2013;23(4):285e294. 15. Sawchenko PE, Swanson LW, Steinbusch HW, Verhofstad AA. The distribution and cells of origin of serotonergic inputs to the paraventricular and supraoptic nuclei of the rat. Brain Res. 1983;277(2):355e360. 16. Holsboer F, Barden N. Antidepressants and hypothalamicpituitary-adrenocortical regulation. Endocr Rev. 1996;17(2): 187e205. 17. Fuller RW. The involvement of serotonin in regulation of pituitary-adrenocortical function. Front Neuroendocrinol. 1992;13(3):250e270. 18. Fuller RW. Serotonin receptors involved in regulation of pituitary-adrenocortical function in rats. Behav Brain Res. 1996;73(1e2):215e219. 19. Myers B, Scheimann JR, Franco-Villanueva A, Herman JP. Ascending mechanisms of stress integration: implications for brainstem regulation of neuroendocrine and behavioral stress responses. Neurosci Biobehav Rev. 2017;74(Pt B):366e375. 20. Raap DK, Van de Kar LD. Selective serotonin reuptake inhibitors and neuroendocrine function. Life Sci. 1999; 65(12):1217e1235. 21. van den Buuse M, Wegener N. Involvement of serotonin1A receptors in cardiovascular responses to stress: a radio-telemetry study in four rat strains. Eur J Pharmacol. 2005;507(1e3):187e198. 22. Jakobsen JC, Katakam KK, Schou A, et al. Selective serotonin reuptake inhibitors versus placebo in patients with major depressive disorder. A systematic review with meta-analysis and trial sequential analysis. BMC Psychiatry. 2017;17(1):58. 23. Jensen JB, Jessop DS, Harbuz MS, Mork A, Sanchez C, Mikkelsen JD. Acute and long-term treatments with the selective serotonin reuptake inhibitor citalopram modulate the HPA axis activity at different levels in male rats. J Neuroendocrinol. 1999;11(6):465e471. 24. Mikkelsen JD, Hay-Schmidt A, Kiss A. Serotonergic stimulation of the rat hypothalamo-pituitary-adrenal

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11 Excitotoxicity M.P. Mattson Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD, United States O U T L I N E Introduction

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Glossary Excitotoxicity Cell injury or death caused by the excessive activation of excitatory transmitter receptors, particularly glutamate receptors. Neurons become vulnerable to excitotoxicity under conditions of reduced energy (glucose) availability and increased oxidative stress. Hippocampus A structure in the temporal lobe of the brain that plays a major role in learning and memory processes and that is a focus of nerve cell degeneration in several prominent disorders including stroke, epilepsy, and Alzheimer’s disease. Ketone Bodies The neuroactive fatty acid metabolites b-hydroxybutyrate and acetoacetate

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which are produced during fasting, exercise, and ketogenic diets. Neurodegenerative Disorders Disorders characterized by progressive nerve cell degeneration in the brain or spinal cord; examples include Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis. Neuronal Calcium Homeostasis The ability of the nerve cell to regulate intracellular free calcium levels during physiological conditions and in response to stress. Membrane-associated ion channels, calcium transporters, and cytosolic calcium-binding proteins participate in this process. Reactive Oxygen Species One of several oxygen-derived molecules that have the potential to damage cellular proteins, nucleic acids, and lipids; examples include superoxide anion radical, hydroxyl radical, peroxynitrite, and hydrogen peroxide.

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Copyright © 2019 Elsevier Inc. All rights reserved.

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INTRODUCTION The major excitatory neurotransmitter in the nervous system is glutamate. Most, if not all, neurons in the brain and spinal cord express one or more types of glutamate receptors. Glutamate receptors are classified as ionotropic (ion channels) and metabotropic (linked to GTP-binding protein signaling pathways). Ionotropic glutamate receptors include N-methyl-D-aspartate (NMDA) receptors (NMDAR), which flux high levels of Ca2þ, and kainate and a-amino-3-hydroxy-5methylisoxazole-4-propionate (AMPA) receptors, which flux mainly Naþ. Glutamatergic synapses play major roles in most functions of the nervous system, including learning and memory and motor and sensory processing. However, the overactivation of glutamate receptors, particularly under conditions of reduced energy availability and increased oxidative stress, can damage and kill neurons.1 The excitotoxic mechanism, which is reviewed here, involves the massive influx of Ca2þ through NMDAR and voltage-dependent Ca2þ channels and the subsequent generation of reactive oxygen species (ROS) and mitochondrial dysfunction.1 Classic examples of excitotoxic neuronal degeneration in humans include severe epileptic seizures and stroke. There is also considerable evidence that excitotoxicity contributes to the neurodegenerative process in chronic neurodegenerative conditions, including Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, and amyotrophic lateral sclerosis (ALS).2 This article considers the emerging evidence that the neuroendocrine stress response and cellular stress responses in the nervous system can modify excitotoxic neurodegenerative processes in neurodegenerative disorders.

KEY POINTS • Glutamate is the major excitatory neurotransmitter in the central nervous system (CNS). • Unconstrained activation glutamate receptors can result in excitotoxic neuronal death.

• Chronic stress and hypothalamice pituitaryeadrenal axis activation render neurons vulnerable to excitotoxicity. • Excitotoxicity is involved in epilepsy, acute CNS injury, and neurodegenerative disorders. • Fasting and exercise can protect neurons against excitotoxicity. • Drugs that reduce neuronal excitability or bolster cellular stress resistance can prevent excitotoxicity.

EXCITOTOXIC MECHANISMS Excitotoxicity is a complex process triggered by glutamate receptor activation that results in the degeneration of dendrites and cell death. All subcellular compartments are affected by the excitotoxic process, with changes in the cytosol, mitochondria, endoplasmic reticulum (ER), and nucleus being pivotal. Normal amounts of glutamate receptor activation can damage neurons under conditions of metabolic and oxidative stress, which occur after a stroke or traumatic brain injury or in age-related neurodegenerative disorders. Excitotoxicity is triggered by the overactivation of glutamate receptors, resulting in Naþ and Ca2þ influx across through the plasma membrane as the result of opening of glutamate receptor (AMPAekainate and NMDA) channels and voltage-dependent Ca2þ channels (Fig. 11.1). In addition, the activation of GTP-binding protein-coupled metabotropic glutamate receptors stimulates inositol trisphosphate (IP3) production and the release of Ca2þ from the ER. Depending on the particular type of neuron, its developmental stage and various environmental factors, the relative contributions of AMPAe kainate, NMDA, and metabotropic receptors and voltage-dependent Ca2þ channels to excitotoxicity may differ. An antagonist that selectively blocks one of the different glutamate receptors or Ca2þ channels may therefore exhibit differential effectiveness in protecting different populations of neurons against excitotoxicity. A neuron’s abilities to remove and buffer Ca2þ are also

EXCITOTOXIC MECHANISMS

FIGURE 11.1 Excitotoxic mechanisms. The binding of glutamate to AMPA receptors and kainate receptors (KAR) opens the receptor channels, resulting in Naþ influx and consequent membrane depolarization and opening of voltage-dependent Ca2þ channels (VDCC). Some forms of AMPA receptor are also permeable to Ca2þ. The binding of glutamate to NMDAR under depolarizing conditions opens the NMDAR channel, resulting in large amounts of Ca2þ influx. The activation of metabotropic glutamate receptors (MetR) induces IP3 production and the activation of IP3 receptor and ryanodine receptor channels in the endoplasmic reticulum (ER) membrane, resulting in the release of Ca2þ from the ER into the cytoplasm. The increases in cytoplasmic Ca2þ levels in response to glutamate receptor activation can induce Ca2þ uptake into the mitochondria which, if excessive, can induce the production of reactive oxygen species (ROS) and inhibit adenosine triphosphate (ATP) production. By activating proteases and inducing oxidative stress, Ca2þ is a key mediator of excitotoxic cell death. AMPA, a-amino-3-hydroxy-5-methylisoxazole-4propionate; NMDAR, N-methyl-D-aspartate receptors; IP3, inositol trisphosphate. Modified from Mattson MP. Excitotoxic and excitoprotective mechanisms: abundant targets for the prevention and treatment of neurodegenerative disorders. Neuromol Med. 2003;3:65e94.

important determinants of its susceptibility to excitotoxicity. Examples of such excitoprotective mechanisms are Ca2þ-ATPases in the plasma membrane and ER, Naþ/Ca2þ exchangers, the mitochondrial Ca2þ uniporter, and various Ca2þ-binding proteins.1 Both the magnitude and the duration of the increase of the intracellular Ca2þ concentration after glutamate receptor activation are important determinants of whether the neuron degenerates. Under resting conditions, the cytoplasmic Ca2þ concentration in neurons is typically

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approximately 100 nM. Large elevations of intracellular Ca2þ (low micromolar concentrations) can be tolerated as long as they are transient (recovering within seconds to minutes). On the other hand, even a lesser increase of the cytoplasmic Ca2þ concentration (e.g., to 500 nM) that is sustained for more than 20e30 min can kill the neuron. Ca2þ damages dendrites and kills neurons, in part, by activating cysteine proteases called calpains that degrade a variety of substrates, including cytoskeletal proteins, membrane receptors, and metabolic enzymes. Oxidative stress involves the production of ROS such as superoxide anion radical, hydrogen peroxide, and hydroxyl radical. ROS-mediated damage to proteins, membranes, and DNA plays a key role in excitotoxic damage to neurons. Glutamate-induced Ca2þ influx can cause ROS production by activating cyclooxygenases and lipoxygenases, perturbing mitochondrial metabolism and inducing membrane lipid peroxidation. Membrane lipid peroxidation may render neurons susceptible to excitotoxicity by impairing the function of membrane ion-motive ATPases and of glucose and glutamate transporters. The importance of oxidative stress in excitotoxicity has been demonstrated in cell culture and animal models in which antioxidants such as vitamin E, lipoic acid, and glutathione protect neurons. Because of the high amounts of energy (ATP) required to maintain and restore ion gradients, neurons are particularly vulnerable to excitotoxicity when under conditions of reduced energy availability such as hypoglycemia and ischemia. Glutamate receptor antagonists can prevent the death of neurons under such conditions, as can agents such as creatine that promote the maintenance of ATP levels. Damage to the DNA of neurons, such as occurs under conditions of oxidative stress or exposure to certain toxins, can render neurons vulnerable to excitotoxicity. DNA damage activates the enzyme poly(ADP-ribose) polymerase, a nicotine adenine dinucleotideedependent enzyme that can consume large amounts of ATP. DNA damage can also induce the production and activation of a protein called p53, which can trigger apoptosis. Excitotoxic neuronal death can occur rapidly as the result of massive influx of Naþ through

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AMPAekainate receptors and voltage-dependent Naþ channels, resulting in cell swelling and lysis, a process called necrosis. Another subtler form of neuronal death called apoptosis is characterized by cell body shrinkage, the formation of cell surface membrane blebs, and nuclear chromatin condensation and fragmentation. Excitotoxic apoptosis involves pivotal mitochondrial changes including increased membrane permeability and the release of cytochrome c. Cytochrome c then binds to a protein called Apaf-1, which mediates the activation of caspases 9 and 3. Drugs that stabilize mitochondrial membrane, as well as caspase inhibitors, can protect neurons against excitotoxic death. Many different signaling mechanisms can protect neurons against excitotoxicity. These include those activated by neurotrophic factors such as brain-derived neurotrophic factor (BDNF), basic fibroblast growth factor, and insulin-like growth

factors. Two transcription factors that are known to induce the expression of excitoprotective genes are nuclear factor (NF)-kB and cAMP response element binding protein (CREB). Examples of such protective genes are those encoding the antioxidant enzyme manganese superoxide dismutase and the antiapoptotic protein Bcl-2. These endogenous protective pathways can be activated by certain drugs. For example, bacterial alkaloids that activate neurotrophic factor signaling pathways can protect hippocampal neurons against seizure-induced excitotoxic damage (Fig. 11.2).

EVIDENCE THAT PHYSIOLOGICAL AND PSYCHOLOGICAL STRESS CAN ENDANGER NEURONS Early studies of the impact of stress on the brain focused on the action of sustained

FIGURE 11.2 Cresyl violet-stained coronal sections of hippocampus showing that bacterial alkaloids that activate neurotrophic factor signaling pathways protect hippocampal neurons against excitotoxic death. (A) Section from a shamoperated rat not receiving the excitotoxin kainic acid; (B) Section from a rat administered saline for 7 days and then injected with kainic acid; (C) Section from a rat administered K252a for 7 days and then injected with kainic acid; (D) Section from a rat administered K252b for 7 days and then injected with kainic acid. Note the extensive loss of neurons in the saline-treated mouse compared to the mice treated with K252a and K252b. Modified from Smith-Swintosky VL, Kraemer PJ, Bruce AJ, et al. Bacterial alkaloids mitigate seizure-induced hippocampal damage and spatial memory deficits. Exp Neurol. 1996;141:287e296.

STRESS HORMONES AND EXCITOTOXICITY

elevations of glucocorticoids.3 It was shown that damage to hippocampal neurons induced by the excitotoxin kainic acid was reduced in adrenalectomized rats and exacerbated in rats administered corticosterone. The suppression of endogenous glucocorticoid production, effected by administration of the 11-b-hydroxylase inhibitor metyrapone, significantly reduced kainic acideinduced damage to hippocampal neurons. Metyrapone administration also significantly reduced brain damage in two different rat models of stroke, a middle cerebral artery occlusion model of focal cerebral ischemia and a transient global forebrain ischemia paradigm that results in the selective loss of CA1 hippocampal neurons. The latter studies showed that both seizures and ischemia resulted in a massive stress response (i.e., large increases in plasma corticosterone levels) that was effectively suppressed by metyrapone.4 Consistent with a role for the elevation of glucocorticoids in the endangerment of neurons in human neurodegenerative conditions are data showing that plasma glucocorticoid levels are increased in patients with temporal lobe epilepsy, stroke, and Alzheimer’s, Parkinson’s, and Huntington’s diseases. Not only were levels of circulating glucocorticoids shown to be elevated in stroke patients and patients experiencing severe epileptic seizures but increased levels and duration of elevation were correlated with worse outcome.5 Despite these and other compelling data, clinical trials of agents that suppress glucocorticoid production in these disorders have not yet been pursued. Recent animal studies have shown that physiological stress (physical stressors such as cold temperature or pain and psychological stressors such as crowding or exposure to dominant males) can endanger neurons in the brain. The subjection of adult rats to chronic stress resulted in the atrophy of apical dendrites of hippocampal CA3 pyramidal neurons. The subjection of adult rats to an intermittent stress regimen resulted in an increase in vulnerability of their hippocampal neurons to seizureinduced injury.

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STRESS HORMONES AND EXCITOTOXICITY Studies of primary hippocampal and cortical neurons in cell culture have provided direct evidence that glucocorticoids can increase neuronal vulnerability to excitotoxicity. Based on analyses of the effects of glucocorticoids on glucose uptake and metabolic parameters (e.g., ATP levels) in cultured neurons, Sapolsky and coworkers proposed that a primary mechanism underlying the endangering effects of glucocorticoids involves impaired energy availability.6 This hypothesis is consistent with many studies showing that neuronal vulnerability to excitotoxicity is increased under conditions of reduced energy availability. Further supporting the metabolic compromise hypothesis are data showing reduced energy availability to neurons in Alzheimer’s disease.7 The exposure of cultured hippocampal neurons to corticosterone resulted in a reduction in the level of glucose transport. It has not been established whether the adverse effect of glucocorticoids on glucose transport is a transcription-dependent process mediated by classic steroid receptors. Indeed, an alternative mechanism of action is suggested by data showing that the inhibition of glucose transport occurs rapidly and requires higher concentrations of corticosterone than would be expected for a receptor-mediated process. Measurements of intracellular calcium levels in hippocampal neurons exposed to glutamate and other excitatory amino acids have shown that glucocorticoids disrupt cellular calcium homeostasis and promote calcium overload. It is not known whether this calcium-destabilizing action of glucocorticoids can be explained solely on the basis of impaired glucose uptake and ATP depletion or whether it involves one or more of the recently described actions of glucocorticoids on ion channel function. Interestingly, estrogen protects cultured hippocampal neurons against excitotoxic, metabolic, and oxidative insults by a mechanism involving the suppression of oxidative stress. In particular, estrogen suppresses membrane lipid peroxidation,

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apparently via the direct inherent antioxidant activity of this steroid. Indeed, estrogen can prevent the impairment of ion-motive ATPases and glucose and glutamate transporters in cortical synaptosomes, a preparation lacking nuclei. Other possible endangering mechanisms of glucocorticoids include the suppression of the production of neurotrophic factors and the suppression of antioxidant defense mechanisms. Indeed, glucocorticoids increase the vulnerability of cultured hippocampal neurons to oxidative insults, whereas neurotrophic factors protect neurons against such insults. Amyloid b-peptide is a 40- to 42-amino acid peptide that forms insoluble aggregates (plaques) in the brain in Alzheimer’s disease; considerable evidence indicates that amyloid b-peptide may directly damage neurons and/or increase their vulnerability to excitotoxicity. Glucocorticoids significantly increase the vulnerability of hippocampal neurons to cell death induced by amyloid b-peptide. Measurements of levels of ROS in hippocampal neurons have shown that amyloid b-peptide increases peroxide accumulation and membrane lipid peroxidation and that corticosterone exacerbates such oxidative stress. Membrane lipid peroxidation is believed to play a major role in the neurodegenerative process in Alzheimer’s disease by impairing the function of membrane ion-motive ATPases (Naþ/Kþ-ATPase and Ca2þ-ATPase) and glucose transporters. Collectively, the available data suggest that excessive physiological and psychological stress can increase the vulnerability of at least some populations of neurons in the brain to excitotoxicity. The mechanism may involve metabolic compromise secondary to the suppression of glucose transport and/or alterations in cellular calcium homeostasis. Excitotoxic neurodegeneration involves oxidative stress and mitochondrial dysfunction, and glucocorticoids can exacerbate such oxidative stress either by directly affecting antioxidant pathways or by indirectly enhancing oxyradical production by disturbing calcium and energy homeostasis. Although much attention has been given to the roles of glucocorticoids in exacerbating

excitotoxicity, there are many other stressinduced hormones that may also influence a neuron’s vulnerability to excitotoxicity. For example, corticotropin-releasing hormone (CRH) and a related neuropeptide called urocortin have been shown to affect the vulnerability of neurons to oxidative, metabolic, and excitotoxic injuries.8 CRH and urocortin act on receptors coupled to cAMP production. The activation of these receptors can protect cultured neurons against oxidative stress and excitotoxicity. Opioid peptides are another class of stress-related hormones that can affect the excitotoxic process. For example, dynorphin and nociceptin can exacerbate excitotoxicity, whereas s receptor ligands can have excitoprotective actions.

KETONE BODIES AND RESISTANCE TO EXCITOTOXICITY During fasting and/or extended periods of exercise, liver glycogen stores are depleted, and fatty acids released from adipose cells are metabolized in hepatocytes and astrocytes to generate ketone bodies. While in the fed state, neurons utilize primarily glucose as their energy source, during fasting they use the ketone bodies acetoacetate and b-hydroxybutyrate (BHB) for ATP generation. By sustaining cellular ATP levels, ketones enable neuronal ion-motive ATPase to operate normally, thereby guarding against excitotoxicity.9 Fasting and the ketone BHB can suppress excitotoxic epileptic seizures, and fatty aciderich ketogenic diets are prescribed to epilepsy patients who do not respond to anticonvulsant drugs. Intermittent fasting has been shown to protect neurons in the brain against degeneration and thereby improve functional outcomes in animal models of epilepsy, stroke, and Alzheimer’s and Parkinson’s diseases. BHB may play a role in neuroprotection by intermittent fasting by enhancing GABAergic tone and by inducing the expression of BDNF.10 By stimulating mitochondrial biogenesis, BDNF may further bolster neuronal resistance to excitotoxicity.

EXCITOPROTECTIVE EFFECTS OF MILD NEURONAL STRESS

EXCITOPROTECTIVE EFFECTS OF MILD NEURONAL STRESS The kinds of data described so far provide strong evidence that excessive activation of the neuroendocrine stress axis is detrimental to neurons in certain regions of the brain. However, emerging findings suggest that moderate levels of stress may enhance neuronal plasticity and increase the resistance of neurons to various insults, including excitotoxic conditions.11 It has been repeatedly demonstrated that exposure of neurons, in vivo and in cell culture, to a moderate (subtoxic) level of stress can protect those neurons against a subsequent intense insult that would otherwise be neurotoxic. For example, the exposure of cultured hippocampal or cortical neurons to subtoxic levels of heat shock or excitotoxins renders them resistant to excitotoxicity. Moreover, the extent of the brain damage caused by cerebral ischemia in adult rodents is greatly reduced when the animals are subjected to a mild alchemic episode prior to exposure to severe ischemia. Such preconditioning hormesis may be mediated by the upregulation of heat shock proteins and antiapoptotic genes. The increased expression of heat shock proteins occurs in both acute neurodegenerative conditions, such as stroke and severe epileptic seizures, and in chronic neurodegenerative disorders, such as Alzheimer’s disease. The available data suggest that these stress-responsive gene products serve neuroprotective functions. In acute neurodegenerative conditions such as stroke, epileptic seizures, and traumatic brain injury, there is a robust and quite rapid increase in the expression of several different cytokines and neurotrophic factors. Considerable data suggest that this particular aspect of the stress response represents an attempt of brain cells to prevent neuronal death and promote recovery. Indeed, cell culture and in vivo studies have shown that the administration of some neurotrophic factors (e.g., basic fibroblast growth factor, nerve growth factor, BDNF, and insulin-like growth factors) and cytokines (e.g., tumor

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necrosis factor [TNF]) can protect neurons against excitotoxic, metabolic, and oxidative insults. An example of a stress-responsive signaling pathway that increases neuronal resistance to excitotoxicity involves the activation of the transcription factor NF-kB by intercellular signals such as TNF and secreted b-amyloid precursor protein (APP), as well as by increased levels of oxidative stress. The NF-kB induces the expression of several cytoprotective gene products including Mn-superoxide dismutase. The mitochondrial enzyme called SIRT3 mediates adaptive excitoprotective responses to excitatory and bioenergetic challenges including exercise.13 SIRT3 is a protein deacetylase located in mitochondria where it targets multiple proteins involved in energy metabolism and resistance to oxidative stress and apoptosis. SIRT3 expression is induced by physiological levels of glutamate receptor activation, and SIRT3 protects neurons against excitotoxicity by deacetylating superoxide dismutase 2 and cyclophilin D to reduce mitochondrial free radical levels and prevent opening of membrane permeability transition pores. A final example of a scenario in which moderate stress may be beneficial for the brain comes from recent studies of the impact of food restriction on aging of the brain and on the brain’s response to metabolic and excitotoxic insults. Well-established method for increasing the lifespan of laboratory rodents and reducing the incidence of age-related cancers and immune alterations is to reduce their caloric intake. A decrease in levels of cellular oxidative stress appears to play an important role in the beneficial effects of food restriction. Recent studies have provided evidence that food restriction retards age-related alterations in the brain such as increases in glial reactivity and impaired performance on learning and memory tasks. With respect to interactions between stress and excitotoxicity, it was recently reported that food restriction in adult rat (alternate day feeding regimen for 2e4 months) results in resistance of hippocampal neurons to excitotoxin-induced

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degeneration and of striatal neurons to degeneration induced by the mitochondrial toxins 3-nitropropionic acid and malonate.14 Food restriction dramatically reduced excitotoxininduced deficits in learning and memory and also prevented the mitochondrial toxin-induced impairment of motor function. Although the mechanism underlying the beneficial effect of chronic food restriction in these rodent models of excitotoxic brain injury has not been established, it is possible that the food-restriction regimen subjects the body and brain to a moderate level of stress, resulting in the upregulation of cellular defense mechanisms.15 In support of this scenario, data show that levels of activation of the hypothalamicepituitaryeadrenal axis and levels of heat shock proteins in some tissues are increased in food-restricted rodents.

ENVIRONMENTAL AND GENETIC RISK FACTORS FOR STRESSMEDIATED EXCITOTOXIC NEURONAL DEGENERATION An increasing number of environmental factors (e.g., diet, exercise, and exposure to toxins) and genetic factors are being identified that can affect the vulnerability of neurons to excitotoxicity (Fig. 11.3). Several different environmental toxins have been identified that can induce nervous system damage and behavioral deficits that are remarkably similar to those seen in human neurodegenerative conditions. As already described, kainic acid (produced by seaweed) can induce seizures and hippocampal damage reminiscent of human temporal lobe epilepsy. Similarly, domoic acid (produced by algae and

FIGURE 11.3 Excitotoxicity is a convergence point in the neurodegenerative cascades of each of the major acute and chronic neurodegenerative disorders. The genetic and environmental factors that initiate the neurodegenerative process may differ among disorders. For example, stroke is caused by the atherosclerotic occlusion of a cerebral blood vessel; Alzheimer’s disease can result from mutations in the b-amyloid precursor protein or presenilins or by age-related increases in oxidative and metabolic stress, resulting in increased production of neurotoxic forms of amyloid b-peptide; and Huntington’s disease is caused by polyglutamine expansions in the huntingtin gene. Despite such differences in initiating factors, each disorder results in similar neurodegenerative cascades that involve increased oxidative stress, metabolic impairment, and overactivation of glutamate receptors, resulting in excessive Ca2þ influx and excitotoxic cell death. Stress, dietary factors, and certain aspects of lifestyle can influence the risk of both the acute neurodegenerative conditions listed on the left and the chronic neurodegenerative disorders listed on the right. Modified from Mattson MP. Excitotoxic and excitoprotective mechanisms: abundant targets for the prevention and treatment of neurodegenerative disorders. Neuromol Med. 2003;3:65e94.

ENVIRONMENTAL AND GENETIC RISK FACTORS FOR STRESS-MEDIATED EXCITOTOXIC NEURONAL DEGENERATION

concentrated in shellfish) can cause hippocampal neuron degeneration and severe memory loss. IMethyl-4-phenyl-1,2,3,6-tetrahydropyridine damages dopaminergic neurons by a mechanism involving, in part, the excessive activation of glutamate receptors under conditions of increased oxidative stress. The ingestion of a mitochondrial toxin (3-nitropropionic acid) produced by a mold results in selective damage to striatal neurons and motor deficits similar to those seen in Huntington’s disease. An excitotoxin in the cycad seed may play a role in the widespread neurodegeneration in brains and spinal cords of individuals suffering from ALSe Parkinsonismedementia complex of Guam. There is evidence that both the neuroendocrine stress response and neuronal stress responses can modulate the adverse effects of such excitotoxins (Fig. 11.4). Genetic defects have been identified that cause several major neurodegenerative disorders in which excitotoxicity is believed to play a role. For example, mutations in APP and the presenilins (PS-1 and PS-2) are responsible for some cases of early-onset autosomal-dominant familial Alzheimer’s disease. Mutations in APP may render neurons vulnerable to age-related metabolic and oxidative stress by altering the proteolytic processing of APP in a manner that increases the production of neurotoxic amyloid b-peptide and decreases the levels of a neuroprotective secreted form of APP. Presenilin mutations appear to place neurons at risk by perturbing calcium regulation in the R, which, in turn, leads to enhanced oxidative stress and mitochondrial dysfunction when neurons are subjected to metabolic and excitotoxic insus. Glucocorticoids have been shown to exacerbate the neurodegenerative process initiated by amyloid b-peptide and may also interact with presenilin mutations to disturb neuronal calcium homeostasis. Conversely, estrogen protects neurons against the death-enhancing effects of PS-1 mutations. ALS, a disease in which the lower motor neurons degenerate, is believed to involve increased oxidative stress and excitotoxicity. Mutations in the antioxidant enzyme Cu/Zn-superoxide dismutase are responsible for some cases of

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INTERMITTENT BIOENERGETIC CHALLENGES Exercise; Energy restriction/Fasting; Pharmacological agents

Adaptive Responses

Cellular Stress Reduction

3-hydroxybutyrate (ketone) Neurotrophic factors (BDNF) Deacetylases (SIRT1 and SIRT3) DNA repair enzymes Protein chaperones Autophagy Mitochondrial biogenesis GABAergic tone

Bolstered Bioenergetics Improved Calcium Handling Reduced Oxidative Damage Enhanced Autophagy Reduced inflammation

ENHANCED NEUROPLASTICITY RESISTANCE TO EXCITOTOXICITY AND DISEASE

FIGURE 11.4 Mechanisms by which intermittent bioenergetic challenges can protect neurons against excitotoxicity. Neurons respond adaptively to physiological bioenergetic challenges (exercise and dietary energy restriction/fasting) by shifting cellular fuel from glucose to ketones and by upregulating neurotrophic factors such as BDNF, protein deacetylases such as SIRT1 and SIRT3, DNA repair enzymes, and protein chaperones. Three prominent adaptive responses of neurons to intermittent bioenergetic challenges that likely contribute to resistance to excitotoxicity are enhancement of autophagy, increased mitochondrial biogenesis, and enhanced GABAergic tone. Via such mechanisms, levels of cellular stress are reduced, neuroplasticity (synaptic plasticity, neurogenesis, and learning and memory) is enhanced, and neurons are better able to resist excitotoxicity. Many of the same adaptive excitoprotective signaling pathways engaged by exercise and fasting can also be activated by pharmacological agents that impose a mild bioenergetic stress including 2-deoxyglucose and mitochondrial uncouplers.12

inherited autosomal-dominant ALS. Huntington’s disease is a genetic disorder in which the aberrant gene (called huntingtin) exhibits expansions of a trinucleotide repeat encoding the amino acid glutamine. Although it has not been established how the trinucleotide repeats promote the degeneration of striatal neurons, available data suggest an alteration that increases neuronal vulnerability to excitotoxicity. In each of these genetic neurodegenerative disorders, the defective gene is widely expressed in various cell types throughout the body and nervous system. The available data suggest that a major reason neurons are selectively vulnerable in these disorders is because of their high metabolic demands and unique vulnerability to excitotoxicity. For the same reason, various extrinsic and endogenous stressors (including the aging

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process itself) can have profound effects in promoting excitotoxic neuronal degeneration.

References 1. Mattson MP. Excitotoxic and excitoprotective mechanisms: abundant targets for the prevention and treatment of neurodegenerative disorders. Neuromol Med. 2003;3:65e94. 2. Beal MF. Does impairment of energy metabolism result in excitotoxic neuronal death in neurodegenerative illnesses? Ann Neurol. 1992;31:119e130. 3. McEwen BS. Protection and damage from acute and chronic stress: allostasis and allostatic overload and relevance to the pathophysiology of psychiatric disorders. Ann N Y Acad Sci. 2004;1032:1e7. 4. Smith-Swintosky VL, Pettigrew LC, Sapolsky RM, et al. Metyrapone, an inhibitor of glucocorticoid production, reduces brain injury induced by focal and global ischemia and seizures. J Cereb Blood Flow Metab. 1996;16:585e598. 5. Fassbender K, Schmidt R, Mossner R, et al. Pattern of activation of the hypothalamic-pituitary-adrenal axis in acute stroke: relation to acute confusional state, extent of brain damage, and clinical outcome. Stroke. 1994;25: 1105e1108. 6. Sapolsky RM. Stress, glucocorticoids, and damage to the nervous system: the current state of confusion. Stress. 1996;1:1e19. 7. Pomara N, Greenberg WM, Branford MD, et al. Therapeutic implications of HPA axis abnormalities in Alzheimer’s disease: review and update. Psychopharmacol Bull. 2003;37:120e134.

8. Pedersen WA, Wan R, Zhang P, et al. Urocortin, but not urocortin II, protects cultured hippocampal neurons from oxidative and excitotoxic cell death via corticotropin-releasing hormone receptor type I. J Neurosci. 2002;22:404e412. 9. Camandola S, Mattson MP. Brain metabolism in health, aging, and neurodegeneration. EMBO J. 2017;36: 1474e1492. 10. Marosi K, Kim SW, Moehl K, et al. 3-hydroxybutyrate regulates energy metabolism and induces BDNF expression in cerebral cortical neurons. J Neurochem. 2016;139:769e781. 11. Raefsky SM, Mattson MP. Adaptive responses of neuronal mitochondria to bioenergetic challenges: roles in neuroplasticity and disease resistance. Free Radic Biol Med. 2017;102:203e216. 12. Geisler JG, Marosi K, Halpern J, et al. DNP, mitochondrial uncoupling, and neuroprotection: a little dab’ll do ya. Alzheimers Dement. 2017;13:582e591. 13. Cheng A, Yang Y, Zhou Y, et al. Mitochondrial SIRT3 mediates adaptive responses of neurons to exercise and metabolic and excitatory challenges. Cell Metab. 2016;23:128e142. 14. Bruce-Keller AJ, Umberger G, McFall R, et al. Food restriction reduces brain damage and improves behavioral outcome following excitotoxic and metabolic insults. Ann Neurol. 1998;45:8e15. 15. Mattson MP. Energy intake and exercise as determinants of brain health and vulnerability to injury and disease. Cell Metab. 2012;16:706e722.

C H A P T E R

12 Chaperone Proteins and Chaperonopathies 1

Alberto J.L. Macario1,2, Everly Conway de Macario1,2

Department of Microbiology and Immunology, School of Medicine, University of Maryland at BaltimoreInstitute of Marine and Environmental Technology (IMET), Columbus Center, Baltimore, MD, United States; 2Euro-Mediterranean Institute of Science and Technology (IEMEST), Palermo, Italy O U T L I N E Objectives and Scope

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Chaperones and the Chaperoning System

137

Chaperonopathies

139

Structural Hereditary Chaperonopathies sHsp Chaperonopathies Hsp60 and CCT Chaperonopathies Hsp40(DnaJ), Hsp70(DnaK), and Super Heavy Chaperones Chaperonopathies Associated With Abnormal Organelle Chaperones Chaperonopathies Associated With Dedicated Chaperones

139 139 139 139 139 140

Gene Polymorphisms and Chaperonopathies 140

Chaperonopathies Attributable to Chaperone-Gene Dysregulation

140

Other Types of Chaperonopathies

144

Chaperones and Metabolic Pathways

144

Acquired Chaperonopathies Autoimmunity and Chronic Inflammation Carcinogenesis Indeterminate Clinical Pictures Which Could Implicate Chaperonopathies

144 144 145

Chaperonotherapy

145

Conclusions and Perspectives

147

Acknowledgments

148

References

148

OBJECTIVES AND SCOPE

KEY POINTS

Molecular chaperones are typically considered as key players in antistress mechanisms; they are cytoprotective. However, abnormal chaperones can be pathogenic.

Stress: Physiology, Biochemistry, and Pathology https://doi.org/10.1016/B978-0-12-813146-6.00012-6

145

135

• Molecular chaperones, also called heat shock proteins although many of them are not coded by genes inducible by heat stress, are crucial in antistress mechanisms.

Copyright © 2019 Elsevier Inc. All rights reserved.

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• The functions of molecular chaperones are canonical and noncanonical. The former pertain to maintenance of protein homeostasis in normal and stress conditions, whereas the noncanonical (moonlighting) functions pertain to other cellular and organismal processes. • Molecular chaperones are proteins of the chaperoning system, which includes also co-chaperones, chaperone co-factors, and chaperone receptors and interactors. • The chaperoning system’s major partners in complex organisms, like humans, are the ubiquitineproteasome system and the chaperone-mediated autophagy machinery, mostly for protein quality control (canonical functions). For noncanonical functions, one of the main partners is the immune system. • Chaperones are typically cytoprotective, for example, against stressors, but if abnormal, they can be pathogenic. • Diseases in which chaperones play an etiopathogenic role are the chaperonopathies. • Chaperonopathies can be caused by changes (e.g., mutation) in a chaperone gene or by alterations (e.g., aberrant posttranslational modification) in the protein. The former are the genetic, and the latter are the acquired chaperonopathies. • Genetic chaperonopathies show complex phenotypes, involving diverse tissues, frequently more noticeably nerves and muscles. • The most important and widespread acquired chaperonopathies include numerous chronic inflammatory and autoimmune conditions and various types of cancer. • Chaperonopathies can be by defect (qualitatively or quantitatively), excess (qualitatively or quantitatively), or mistake (an apparently normal chaperone participates in a pathogenic pathway or favors a malignant cell).

• Since chaperonopathies, genetic and acquired, can affect any tissue and organ, they are of interest over a wide range of medical specialties. • Chaperonotherapy uses chaperones as targets or therapeutic agents, depending on whether the chaperone has to be eliminated/blocked or functionally boosted/replaced (e.g., gene therapy or administration of normal chaperone molecules). Many diseases, the chaperonopathies, have abnormal chaperones as etiopathogenic factors, primary or auxiliary. In the former case, abnormal chaperones are the main cause, that is, the etiology, of the disease. However, there may be other factors in addition to the abnormal chaperone also contributing to the pathologic process. Instead, when an abnormal chaperone is an auxiliary etiopathogenic factor, the chaperone contributes to the pathogenesis of diseases with other etiologies. Whatever the role of the abnormal chaperone, primary or auxiliary etiopathogenic factor, these pathologic conditions bring chaperones to the main stage, and they have to become the focus of diagnosis and treatment. This article aims at presenting an overview of the field of chaperonopathies as an introduction to it for scientists and physicians in practice and clinical research. It summarizes information in the literature and databases to serve as a starting point for those still unfamiliar with the field. Detailed discussion of each disease, or even of a few paradigms, is not intended. Rather, the purpose is to provide an aerial view, so to speak, of this new area of Medicine for those interested in the biomedical sciences, so they can appreciate its full dimension and become aware of its multiple possibilities in practice and research. Consequently, it is hoped that this article will not only inform but also alert physicians to look for chaperonopathies in their patients. This attitude will open new roads toward

CHAPERONES AND THE CHAPERONING SYSTEM

developing chaperonotherapy, namely the treatment of diseases using chaperones as targets or agents. We have attempted to present in Tables a representative list of chaperonopathies to provide an overview of the field in virtually all its dimension across various medical specialties: internal medicine, neurology, hematology, immunology, rheumatology, cardiology, gastroenterology, endocrinology, gynecology, and so on. It is easily seen that the field is quite large and encompasses many pathologic conditions, many very serious, and many very prevalent (particularly within the group of acquired chaperonopathies). This work is an extension and update of previous publications cited as needed, including a Website (http://www.chaperones-pathology. org), so it should be easy for the interested reader to visit those publications and Website in search of in-depth information and extended bibliography. Here, we have limited the references to include those earlier publications in which this one is based so that they can be consulted for detailed descriptions, plus a selected few updates. The latter should help in assessing the current importance of the field and in visualizing

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its direction: what can be done now and where to go in research and clinical practice in the near future.

CHAPERONES AND THE CHAPERONING SYSTEM It is convenient for studious of Medicine, including practicing physicians to consider the entire set of chaperones of an organism as a physiological system, the chaperoning system.1,2 It is constituted of chaperones, co-chaperones, chaperone co-factors, and chaperone receptors and interactors (Fig. 12.1). A list of chaperones is displayed in Table 12.1.2e6 Perhaps, the most basic and ancient function of chaperones was protection against stressors and reconstitution of molecules damaged by stress. Consequently, it is important to bear in mind that many chaperones are heat shock proteins (Hsp), namely their genes are inducible by heat shock and other stressors. On the other hand, many chaperones are not Hsp and vice versa, many Hsp are not chaperones. However, for years, the terms chaperone and Hsp have been used interchangeably, as if they were true

FIGURE 12.1 The chaperoning system (represented by a 5-pointed star) of an organism consists of its entire set of molecular chaperones, co-chaperones, and chaperone co-factors, plus the chaperone receptors on the structures (e.g., cells of the innate immune system) that interact with chaperones and other molecules, the chaperone interactors. The chaperones discussed in this article are proteins and have canonical and noncanonical functions, the former pertaining to protein homeostasis under normal conditions and in response to stress and the latter related to other cellular processes. The chaperone system has as important partners in the maintenance of protein homeostasis the ubiquitin-proteasome system (UPS) and the chaperone-mediated autophagy (CMA) machinery. Many noncanonical functions involve interaction with the immune system (IS), which is a major partner of the chaperoning system in health and particularly in disease (e.g., in chronic inflammatory and autoimmune disorders). Also, the chaperoning system plays key roles in carcinogenesis, by itself or through its interactions with the immune system.

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TABLE 12.1 Subpopulations of Hsp-Chaperones by Molecular Weight kDa

Classical Family

Others Causing Chaperonopathies

>200

None

Sacsin

100e199

Hsp100e110

81e99

Hsp90

Paraplegin (SPG7); UNC45B; p97 (VCP)

65e80

Hsp70(DnaK)

Spastin (SPG4)

55e64

Hsp60 (chaperonins Groups I and II, e.g., Cpn60 and CCT-BBS)

BAG3; immunophilins FKB-type: peptidyl-prolyl cis-trans isomerases (PPI; FKBP5; FKBP10); myocilin; protein disulfide isomerases (PDI; ER); SERPINH1 (Hsp47)

35e54

Hsp40(DnaJ)

AIP; AIPL1; BCS1L; CALR; clusterin; DNAJC19; FOXRED1; melusin; morgana; SIL1; TBCE; torsin A

70% of the money), which tends to generate a strong negative affective reaction in the participant, reflected in activation of brain’s emotional regions such as the amygdala, the insula, and the anterior cingulate cortex.52 We reasoned that overweight and obese participants

would be more sensitive to unfair offers, and hence we could detect brain activation differences with healthy-weight controls. Indeed we found that participants who were overweight or obese showed abnormally decreased activation than controls in the anterior cingulate and the insula, as well as midbrain regions associated with reward prediction.53 Although the psychological interpretation of these brain findings is uncertain, we speculated that the high levels of interpersonal stress suffered by overweight and obese individuals had made them “give up” on fairness (akin to a sense of hopelessness), as reflected in their blunted brain’s emotional response. Fewer studies have examined the impact of chronic stress on brain function or structure, but importantly, their results are consistent with those from acute stress studies. By grouping participants into high and low chronic stress groups according to their scores in a self-report questionnaire (The Wheaton Chronic Stress Inventory), a study has shown that chronic stress

REFERENCES

is associated with hyperactivation of regions of the brain’s reward and emotional systems in response to high-energy food images.54 Specifically, participants with high chronic stress, compared with low-stress controls, showed higher activation in the striatum, the orbitofrontal cortex, the amygdala, and the anterior cingulate cortex, as well as higher connectivity between the striatum and the amygdala (i.e., reward and emotional regions). Interestingly, they also showed lower activation in brain regions implicated in cognitive control, such as the dorsolateral and ventrolateral prefrontal cortices. In another study, in which chronic stress was operationalized as low perceived maternal care during childhood, higher levels of chronic stress were also associated with less activation in superior frontal, parietal, and temporal cortex regions implicated in cognitive control.46 Studies examining the impact of chronic stress on brain structure have primarily focused on gross markers of brain atrophy. For example, a recent study has shown that excessive adiposity and higher levels of C-reactive protein are significantly associated with ventricular enlargement.55 Importantly, using brain perfusion measures, it has also been established that fat accumulation is associated with reduced myocardial perfusion after a pharmacologically induced (dobutamine) stress challenge.56 Longitudinal research has also shown that increases in adiposity are significantly associated with progressive atrophy in the hippocampus, which is one of the key regions for stress regulation.21 These findings indicate that, although chronic stress is a risk factor for obesity, excessive adiposity can also aggravate the impact of acute and chronic stressors on brain structure and function.

CONCLUSIONS Stress has a significant impact on consumption of highly palatable foods, via its impact on motivational, affective, learning, and higher-order cognitive processes, underpinned by the crosstalk between the hypothalamus and other

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homeostatic regulation regions and the brain’s reward and emotional systems. People who are overweight and obese experience high levels of stress and are particularly sensitive to the effects of stress on appetite and cognition. This “double vulnerability” is important to understanding the problems that overweight and obese individuals experience when trying to control food intake and manage weight. In the long-term, both stress and adiposity have neuroadaptive effects on key regions for cognitive control and emotion regulation, including the prefrontal cortex and the hippocampus. Therefore, the cognitive, affective, and brain structural and functional characteristics of overweight and obese individuals need to be factored in during treatment. Although the causal and temporal pathway of the interactions between stress, cognitive and affective phenotypes, and brain alterations is not yet fully understood, current evidence supports the view that early stress can generate trait- and state-related cognitive-affective vulnerabilities, which result in unhealthy eating, weight gain, and body fat accumulation, and ultimately brain deterioration. Therefore, preventative strategies directed to reduce or regulate stress among children and adolescents can also be beneficial to consolidate healthy eating habits and prevent obesity.

References 1. Adam TC, Epel ES. Stress, eating and the reward system. Physiol Behav. 2007;91(4):449e458. 2. Sinha R, Jastreboff AM. Stress as a common risk factor for obesity and addiction. Biol Psychiatr. 2013;73(9): 827e835. 3. Yau YH, Potenza MN. Stress and eating behaviors. Minerva Endocrinol. 2013;38(3):255e267. 4. Burghardt PR, Love TM, Stohler CS, et al. Leptin regulates dopamine responses to sustained stress in humans. J Neurosci. 2012;32(44):15369e15376. 5. Maniam J, Morris MJ. The link between stress and feeding behaviour. Neuropharmacology. 2012;63(1): 97e110. 6. Sominsky L, Spencer SJ. Eating behavior and stress: a pathway to obesity. Front Psychol. 2014;5:434. 7. Dallman MF. Stress-induced obesity and the emotional nervous system. Trends Endocrinol Metab. 2010;21(3): 159e165.

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8. Girotti M, Adler SM, Bulin SE, Fucich EA, Paredes D, Morilak DA. Prefrontal cortex executive processes affected by stress in health and disease. Prog Neuro Psychopharmacol Biol Psychiatry. 2017;85:161e179. 9. McEwen BS, Eiland L, Hunter RG, Miller MM. Stress and anxiety:Structural plasticity and epigenetic regulation as a consequence of stress. Neuropharmacology. 2012;62(1):3e12. 10. Pruessner JC, Dedovic K, Pruessner M, et al. Stress regulation in the central nervous system: evidence from structural and functional neuroimaging studies in human populationsd2008 Curt Richter Award Winner. Psychoneuroendocrinology. 2010;35(1):179e191. 11. Roberts CJ, Campbell IC, Troop N. Increases in weight during chronic stress are partially associated with a switch in food choice towards increased carbohydrate and saturated fat intake. Eur Eat Disord Rev. 2014;22(1): 77e82. 12. Hemmingsson E. A new model of the role of psychological and emotional distress in promoting obesity: conceptual review with implications for treatment and prevention. Obes Rev. 2014;15(9):769e779. 13. Agerstro¨m J, Rooth DO. The role of automatic obesity stereotypes in real hiring discrimination. J Appl Psychol. 2011;96(4):790e805. 14. Strauss RS, Pollack HA. Social marginalization of overweight children. Arch Pediatr Adolesc Med. 2003;157(8): 746e752. 15. Dulloo AG, Jacquet J, Montani JP, Schutz Y. How dieting makes the lean fatter: from a perspective of body composition autoregulation through adipostats and proteinstats awaiting discovery. Obes Rev. 2015;16(S1): 25e35. 16. Morris MJ, Beilharz JE, Maniam J, Reichelt AC, Westbrook RF. Why is obesity such a problem in the 21st century? The intersection of palatable food, cues and reward pathways, stress, and cognition. Neurosci Biobehav Rev. 2015;58:36e45. 17. Lasikiewicz N, Hendrickx H, Talbot D, Dye L. Stress, cortisol and central obesity in middle aged adults. Obes Facts. 2013;6:44. 18. Verdejo-Garcia A, Moreno-Padilla M, Garcia-Rios MC, et al. Social stress increases cortisol and hampers attention in adolescents with excess weight. PLoS One. 2015a; 10(4):e0123565. 19. Yang Y, Shields GS, Guo C, Liu Y. Executive function performance in obesity and overweight individuals: a meta-analysis and review. Neurosci Biobehav Rev. 2017; 84:225e244. 20. Brook JS, Zhang C, Saar NS, Brook DW. Psychosocial predictors, higher body mass index, and aspects of neurocognitive dysfunction. Percept Mot Skills. 2009;108(1): 181e195. 21. Cherbuin N, Sargent-Cox K, Fraser M, Sachdev P, Anstey KJ. Being overweight is associated with hippocampal atrophy: the PATH through Life Study. Int J Obes. 2015;39(10):1509e1514.

22. Rivera M, Rovira P, Cervilla J, et al. The VAL66MET BDNF genetic polymorphism does not modify the association between major depression and body mass index (BMI). Eur Neuropsychopharmacol. 2017;27:S447. 23. Sevgi M, Rigoux L, Ku¨hn AB, et al. An obesitypredisposing variant of the FTO gene regulates D2Rdependent reward learning. J Neurosci. 2015;35(36): 12584e12592. 24. Waters A, Hill A, Waller G. Bulimics’ responses to food cravings: is binge-eating a product of hunger or emotional state? Behav Res Ther. 2001;39(8):877e886. 25. Weingarten HP, Elston D. The phenomenology of food cravings. Appetite. 1990;15(3):231e246. 26. White MA, Whisenhunt BL, Williamson DA, Greenway FL, Netemeyer RG. Development and validation of the food-craving inventory. Obesity. 2002; 10(2):107e114. 27. Potenza MN, Grilo CM. How relevant is food craving to obesity and its treatment? Front Psychiatr. 2014;5:164. 28. Lemmens SG, Rutters F, Born JM, WesterterpPlantenga MS. Stress augments food ‘wanting’and energy intake in visceral overweight subjects in the absence of hunger. Physiol Behav. 2011;103(2):157e163. 29. Rosenberg N, Bloch M, Avi IB, et al. Cortisol response and desire to binge following psychological stress: comparison between obese subjects with and without binge eating disorder. Psychiatr Res. 2013;208(2):156e161. 30. Kirschbaum C, Pirke KM, Hellhammer DH. The ‘trier social stress test’ea tool for investigating psychobiological stress responses in a laboratory setting. Neuropsychobiology. 1993;28(1e2):76e81. 31. Lim SS, Norman RJ, Clifton PM, Noakes M. Hyperandrogenemia, psychological distress, and food cravings in young women. Physiol Behav. 2009;98(3):276e280. 32. Tryon MS, DeCant R, Laugero KD. Having your cake and eating it too: a habit of comfort food may link chronic social stress exposure and acute stressinduced cortisol hyporesponsiveness. Physiol Behav. 2013b;114:32e37. 33. Macedo DM, Diez-Garcia RW. Sweet craving and ghrelin and leptin levels in women during stress. Appetite. 2014;80:264e270. 34. Chao A, Grilo CM, White MA, Sinha R. Food cravings mediate the relationship between chronic stress and body mass index. J Health Psychol. 2015;20(6):721e729. 35. Chao AM, Jastreboff AM, White MA, Grilo CM, Sinha R. Stress, cortisol, and other appetite-related hormones: prospective prediction of 6-month changes in food cravings and weight. Obesity. 2017;25(4):713e720. 36. Jansen A, Houben K, Roefs A. A cognitive profile of obesity and its translation into new interventions. Front Psychol. 2015;6:1807. 37. Werthmann J, Jansen A, Roefs A. Worry or craving? A selective review of evidence for food-related attention biases in obese individuals, eating-disorder patients, restrained eaters and healthy samples. Proc Nutr Soc. 2015;74(2):99e114.

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38. Newman E, O’Connor DB, Conner M. Attentional biases for food stimuli in external eaters: possible mechanism for stress-induced eating? Appetite. 2008;51(2): 339e342. 39. Hepworth R, Mogg K, Brignell C, Bradley BP. Negative mood increases selective attention to food cues and subjective appetite. Appetite. 2010;54(1):134e142. 40. Davis C, Levitan RD, Muglia P, Bewell C, Kennedy JL. Decision-making deficits and overeating: a risk model for obesity. Obesity. 2004;12(6):929e935. 41. van Strien T, Ouwens MA, Engel C, de Weerth C. Hunger, inhibitory control and distress-induced emotional eating. Appetite. 2014;79:124e133. 42. Delgado-Rico E, Rı´o-Valle JS, Gonza´lez-Jime´nez E, Campoy C, Verdejo-Garcı´a A. BMI predicts emotiondriven impulsivity and cognitive inflexibility in adolescents with excess weight. Obesity. 2012;20(8): 1604e1610. 43. Mobbs O, Cre´pin C, Thie´ry C, Golay A, Van der Linden M. Obesity and the four facets of impulsivity. Patient Educ Couns. 2010;79(3):372e377. 44. Stinson EJ, Krakoff J, Gluck ME. Depressive symptoms and poorer performance on the stroop task are associated with weight gain. Physiol Behav. 2018;186:25e30. 45. Lasselin J, Magne E, Beau C, et al. Low-grade inflammation is a major contributor of impaired attentional set shifting in obese subjects. Brain Behav Immun. 2016;58: 63e68. 46. Machado TD, Dalle Molle R, Reis RS, et al. Interaction between perceived maternal care, anxiety symptoms, and the neurobehavioral response to palatable foods in adolescents. Stress. 2016;19(3):287e294. 47. Jastreboff AM, Potenza MN, Lacadie C, Hong KA, Sherwin RS, Sinha R. Body mass index, metabolic factors, and striatal activation during stressful and neutral-relaxing states: an FMRI study. Neuropsychopharmacology. 2011;36(3):627e637.

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48. Jastreboff AM, Sinha R, Lacadie C, Small DM, Sherwin RS, Potenza MN. Neural correlates of stressand food cueeinduced food craving in obesity: association with insulin levels. Diabetes Care. 2013;36(2):394e402. 49. Rudenga KJ, Sinha R, Small DM. Acute stress potentiates brain response to milkshake as a function of body weight and chronic stress. Int J Obes. 2013;37(2):309e316. 50. Harding IH, Andrews ZB, Mata F, et al. Brain substrates of unhealthy versus healthy food choices: influence of homeostatic status and body mass index. Int J Obes. 2018;42(3):448e454. 51. Contreras-Rodrı´guez O, Vilar-Lo´pez R, Andrews ZB, Navas JF, Soriano-Mas C, Verdejo-Garcı´a A. Altered cross-talk between the hypothalamus and nonhomeostatic regions linked to obesity and difficulty to lose weight. Sci Rep. 2017;7(1):9951. 52. Sanfey AG, Rilling JK, Aronson JA, Nystrom LE, Cohen JD. The neural basis of economic decisionmaking in the ultimatum game. Science. 2003; 300(5626):1755e1758. 53. Verdejo-Garcı´a A, Verdejo-Roma´n J, Rio-Valle JS, Lacomba JA, Lagos FM, Soriano-Mas C. Dysfunctional involvement of emotion and reward brain regions on social decision making in excess weight adolescents. Hum Brain Mapp. 2015b;36(1):226e237. 54. Tryon MS, Carter CS, DeCant R, Laugero KD. Chronic stress exposure may affect the brain’s response to high calorie food cues and predispose to obesogenic eating habits. Physiol Behav. 2013a;120:233e242. 55. Lai YH, Liu CC, Kuo JY, et al. Independent effects of body fat and inflammatory markers on ventricular geometry, midwall function, and atrial remodeling. Clin Cardiol. 2014;37(3):172e177. 56. Hall ME, Brinkley TE, Chughtai H, et al. Adiposity is associated with gender-specific reductions in left ventricular myocardial perfusion during dobutamine stress. PLoS One. 2016;11(1):e0146519.

C H A P T E R

17 The Innate Alarm System: A Translational Approach Daniela Rabellino4, Jenna E. Boyd2,3,4, Margaret C. McKinnon3,4,5, Ruth A. Lanius1,6,7

1

Department of Psychiatry, University of Western Ontario, London, ON, Canada; 2Department of Psychology, Neuroscience, and Behaviour, McMaster University, Hamilton, ON, Canada; 3Mood Disorders Program, St. Joseph’s Healthcare Hamilton, Hamilton, ON, Canada; 4Homewood Research Institute, Guelph, ON, Canada; 5Department of Psychiatry and Behavioural Neurosciences, McMaster University, Hamilton, ON, Canada; 6Department of Neuroscience, University of Western Ontario, London, ON, Canada; 7Imaging Division, Lawson Health Research Institute, London, ON, Canada O U T L I N E Introduction

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Innate Defense Responses in Animals The Defense Cascade Model

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Innate Defense Responses in Humans

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The Innate Alarm System

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Conscious and Subconscious Processing of Threat in PTSD Conscious Threat Processing in PTSD Subconscious Threat Processing in PTSD PTSD Symptomatology and the IAS During Threat Processing

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The Role of the Amygdala in Innate Defensive Responding in PTSD 206 Brainstem Regions and Innate Defensive Responding in PTSD 206

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Clinical and Research Implications

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Acknowledgments

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References

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INTRODUCTION Innate defense responses are evolutionarily adaptive mechanisms that increase the chance of survival in situations involving imminent

Stress: Physiology, Biochemistry, and Pathology https://doi.org/10.1016/B978-0-12-813146-6.00017-5

Functional Connectivity of Brain Regions Associated With the IAS in PTSD

danger. Here, threat processing occurs at a subconscious level (below the threshold of conscious awareness), without the need for conscious appraisal of the threat, thus allowing for fast, reflexive physiological and behavioral

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Copyright © 2019 Elsevier Inc. All rights reserved.

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responses.1,2 Fight-or-flight reactions provide a key behavioral example of response to imminent threat, a pattern that is common across humans and several other species, especially mammals.3 Indeed, studies examining preclinical, animal models may help to elucidate innate, reflexive responses in humans, following a phylogenetic continuity that has ensured survival across species.4 KEY POINTS (HIGHLIGHTS) • The innate alarm system refers to the neurocircuitry underlying reflexive innate defense responding necessary to adaptively survive imminent danger. • Preclinical, animal models provide useful insights into the neural correlates underlying innate defense responses common across mammals and conserved in humans. • Subconscious processing of threatening cues allows for investigation of the innate alarm system in humans. • Posttraumatic stress disorder provides a disease model to increase the understanding of rapid innate threat processing; individuals with this disorder display aberrant neural activity and functional connectivity in critical regions of the innate alarm system. • Key regions of the innate alarm system include the amygdala, the prefrontal cortex, the pulvinar, the hypothalamus, brainstem regions (superior colliculus, locus coeruleus, periaqueductal gray, vestibular nuclei), and the cerebellum. In this chapter, we review the innate behavioral reactions that may occur in response to threat and their underlying neural substrates in preclinical animal models and in healthy humans. Specifically, research in humans that has focused on subconscious processing of threat-related stimuli in an attempt to investigate immediate reactions that occur below the consciousness threshold of the individual is reviewed.5 The innate alarm system (IAS) is

introduced as a neural network underlying fast, reflexive innate defense reactions in organisms facing imminent threat. The IAS encompasses brainstem regions, subcortical regions such as the amygdala, the hypothalamus, and the pulvinar, as well as prefrontal cortical areas.6e11 Conscious and subconscious threat processing in relation to IAS brain regions in posttraumatic stress disorder (PTSD), a psychiatric syndrome that may ensue after experiencing or witnessing traumatic events, is then reviewed. Critically, innate defense mechanisms appear to play a prominent role in the maintenance of the disorder.12,13 Potential clinical and research implications of the IAS are discussed.

INNATE DEFENSE RESPONSES IN ANIMALS Given the conserved nature of key defensive response circuits from animals to humans, research on animal models provides useful insights into the behavioral responses and neural circuits underlying innate defense responses in humans. Moreover, phylogenetic continuity that allows for comparisons across mammals is informative in highlighting potentially shared defensive mechanisms across species.4 Innate fear represents a basic emotional state elicited by stimuli that intrinsically implicate a threat, such as the fear of predators in animals. By contrast, learned fear refers to a neutral stimulus that has been previously associated with an innate threat, leading to association of this previously neutral stimulus with threat and thus future perception of the stimulus as threatening.3 Different neural circuits have been identified in animals depending on the type of threat that elicits a response, including fear of predators, fear of aggressive conspecifics, or fear of pain. Our more recent understanding of innate fear circuits arises from research in rodents and is based mainly on the fear of predators. Here, Silva and colleagues11 delineate a neurocircuitry of fear organized as interconnected functional subunits, including (1) a detection unit that receives sensory inputs and includes association cortices (auditory, visual, olfactory systems), brainstem

INNATE DEFENSE RESPONSES IN ANIMALS

structures for pain detection (periaqueductal gray [PAG]), and the amygdala for polymodal sensory information; (2) an integration unit that integrates sensory inputs and downstream outputs (from the central to the peripheral nervous system) to generate an adaptive response to the perceived threat; this unit encompasses the hypothalamus, the amygdala, the superior and inferior colliculi, the bed nucleus of the stria terminalis, the lateral septum, and the solitary tract nucleus; and (3) an output unit comprising midbrain structures that initiate responses to threatening stimuli, including the ventrolateral PAG (vlPAG) and dorsolateral PAG (dlPAG). The role of the amygdala, in particular, has been compared with that of a switchboard in that this structure converges distinct afferent inputs and channels them through different downstream pathways depending on the type of threatening stimulus. Each amygdala subdivision appears to play a specific role in this task. As an example, in an experiment where rats were exposed to cat odors (a predator cue), lesions to the medial nucleus of the amygdala resulted in almost complete loss of defensive responses. By contrast, lesions to the posterior basomedial amygdala or to the lateral amygdala produced alterations in defensive responding, including reduced freezing and increased risk assessment behavior.14 The central nucleus of the amygdala appears further capable of switching between outputs that induce freezing responses (via the vlPAG) and responses involved in arousal and risk assessment (via the substantia innominata in the basal forebrain).15 Finally, the basolateral amygdala (BLA) and centromedial amygdala (CMA) play important and differential roles in threat response. Here, the BLA receives and integrates sensory information while communicating with the CMA, thus orchestrating the fear response via connections with the hypothalamus and the PAG.16,17 The medial hypothalamus is also an important player in the integration unit and is a crucial relay station receiving afferents from the amygdala, the parabrachial nucleus, and the medial prefrontal cortex (mPFC), and sending efferents to the PAG, the superior colliculus (SC), and the vagal motor nerve.11,18 In rodents,

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the integration unit has shown to be driven by a defensive system mediated by the hypothalamus, referred to as the medial hypothalamic defensive system. This system is thought to organize innate defensive behaviors19 and to mediate states of basic emotions in animals (e.g., fear; for complete references, see the review by Silva et al.11). The medial hypothalamic defensive system drives differential responses to threat according to the context and imminence of a threatening cue (freezing, avoidance, escape, risk assessment, anxiety, autonomic response; Silva et al.,11 Wang et al.20) and mediates PAG activity in response to the imminence of threat and contextual factors (presence of escape routes). On balance, however, the PAG is likely the most important structure in producing active versus passive defensive responses serving as a common pathway for all types of defensive responses.22,23 The PAG receives inputs from the central amygdala, the hypothalamus, and the mPFC to regulate defensive behaviors.24 Critically, the PAG can be functionally subdivided in the ventrolateral portion, which is involved in passive defensive responses (e.g., freezing) and a dorsolateral section involved in active defensive strategies (e.g., fight-orflight24). In mice, disinhibition of vlPAG excitatory outputs to premotor medullary regions has been associated with freezing states, a behavior manifested across species and characterized by restricted movement and information gathering for environmental threat cues. This disinhibition has been linked to firing of glutamatergic neurons in the vlPAG.24 By contrast, electrical stimulation of the dorsal PAG in rats elicits freezing or flight and visceral responses.25 In addition, combined inhibition of passive and active defensive responses has been recorded in rats during inescapable shock and has been attributed to inhibition of the dorsal PAG and a decrease in resilience to stress.25 Here, it is important to note that the circuits involved in the opposite defensive responses need to be interacting with each other in order to allow rapid adaptive switching between alternative defensive behavioral expressions.

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Thus far, we have identified three main structures (in addition to others) that play a crucial role in the innate defense response circuitry in animals: the amygdala, the medial hypothalamus, and the PAG. Notably, however, the cerebellum is often overlooked in models outlining the neural circuitry underlying fear-based responses to predators. Studies on rats with vermal cerebellar lesions reveal reduced unconditioned freezing responses in the presence of a cat,3,26 where the vermis is thought to connect to the dlPAG via the fastigial nucleus and the SC, perhaps modulating activation of the dlPAG.

The Defense Cascade Model In a pattern complementary to the neurocircuitry model of fear outlined previously, the defense cascade model proposed by Schauer and Elbert27 is essential to understanding innate behavioral defensive responses and their corresponding neural circuits. Specifically, the defense cascade model identifies distinct states of defensive responding including freezing, fight-orflight, and tonic or collapsed immobility. This model is critical to a translational approach that aims to understand human expressions of defensive posturing including fight or flight (hyperaroused states) and reduced responding to threat (dissociative states or dissociation). Here, dissociation refers to alterations in consciousness that disrupt the normal integration of thought, memory, emotions, sense of self, body awareness, and perception of the external environment.12 Among humans, common symptoms of dissociation include depersonalization (feeling outside of or as if one does not belong to one’s own body), derealization (feeling as though things around you are strange or unfamiliar), disengagement (feeling spaced out or disconnected), emotional constriction (reduced range of affect and experience of emotions), memory

1

disturbance, and identity dissociation (feeling as though there is more than one identity inside you).28 The autonomic and behavioral responses associated with the defense cascade have been well documented in Schauer and Elbert’s seminal article describing the cascade “FreezeFlight-Fight-Fright-Flag-Faint” as a continuum of escalating fear responses depending on the imminence of threat and situational variables (escapability). Kozlowska and colleagues29 expand this model, describing in detail the neural circuitry underlying the defense cascade model. Although the defense cascade model arises from the animal literature, it has been used subsequently to understand the fear cascade in humans. The neural circuitry underlying the defense cascade model includes key regions including the amygdala, hypothalamus, PAG, ventral and dorsal medulla (the sympathetic and vagal nuclei), and the spinal cord. Briefly, the first step in the defense cascade is the arousal state,1 where the hypothalamus plays a critical role in activating the sympathetic nervous system, resulting in increased muscle tone, heart rate, and respiration necessary to prepare the body for action. This stage is followed by active defensive responsesdfight-or-flightdarising when threat becomes imminent (e.g., predator’s attack) and associated with activation of somatomotor (e.g., motor patterns of fight-or-flight), visceromotor (e.g., increased blood pressure), and pain (e.g., endocannabinoid-mediated analgesia) components of the defense response. Notably, the dlPAG mediates this type of response, activating the pons and medullar centers to promote a motor response via the spinal cord.30 The cerebellum and basal ganglia are also thought to contribute at this stage. Among rats, the dorsal hypothalamus produces widespread sympathetic autonomic activation and stimulates midbrain structures (the cuneiform nucleus

Compared with Kozlowska’s description of the defense cascade model,29 the original Schauer and Elbert’s model27 follows a different order along the progress of the defense states: freeze (attentive immobility), fight/flight, fright (unresponsive immobility), flag (shutdown of activities), faint. In this chapter, we summarize Kozlowska’s updates to the defense cascade, thus reporting the following order of defensive response states: arousal, fight/flight, freezing, tonic immobility, collapsed immobility, quiescent immobility (return to rest and healing, not described in this chapter).

INNATE DEFENSE RESPONSES IN HUMANS

and the dlPAG) to inhibit the baroreflex bradycardia reducing parasympathetic responding.31 The next step in the defense cascade is a state of attentive immobility,32,33 where switching back to the fight-or-flight stage is still possible. Here, the central nucleus of the amygdala activates the vlPAG, which acts as a brake for the dlPAG and prevents active responses. The hypothalamic pathway increases sympathetic tone (muscle tone, respiration rate34), and an opioidmediated analgesic state is induced. Critically, when all previous strategies have failed (e.g., the animal is captured by the predator), a state of tonic immobility can occur. This extreme defense behavior is found across species and is characterized by a shutdown of the active responses driven by vlPAG output and activation of phylogenetically older structures, such as the spinal cord, the solitary tract nucleus, and the parabrachial nucleus in the brainstem. The dorsal motor nucleus of the vagus promotes parasympathetic activity mediated by the vlPAG, with consequent bradycardia, decreased temperature and respiration rate, and opioid-mediated analgesia.35e37 Importantly, tonic immobility has been recording in humans, particularly among individuals with PTSD.38,39 Finally, in some animal species (e.g., opossums) a subsequent stage, collapsed immobility, is reached.29 This state is similar to tonic immobility, with the exception that this state includes hypoxia and consequent loss of muscle tone (collapse), usually referred to as “death feigning” in animals. Critically, this state can have potential lethal risks. On balance, the defense cascade model is well supported by literature on innate defensive responses in animals and provides an eloquent description of innate human defensive behaviors during threat processing (as an example, tonic immobility in PTSD38e40). Importantly, the neurocircuitry underlying this model overlaps significantly with the neurocircuitry underlying the innate alarm system (IAS), described in detail below, pointing toward key future research directions surrounding stress response in humans.

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INNATE DEFENSE RESPONSES IN HUMANS Innate defense responses are vital for survival, as they allow rapid and automatic responding to potential danger without the engagement of higher-order cognitive functions.35,41 Such strategies rely on phylogenetically ancient innate mechanisms that have been shown to be adaptive from an evolutionary perspective.41 To investigate innate defense responses in humans, researchers have focused on subconscious processing of threat, utilizing masked subliminal stimuli that are perceived under the threshold of conscious awareness (e.g., due to rapid display time).5 A subcortical pathway, also known as a “lowroad” model (as opposed to a “high-road” model employing processing via cortical structures),42 appears the most efficient route for rapid reactions to imminent danger.6,7,9,43 Here, fundamental work by Liddell and colleagues6 using masked fearful stimuli with healthy participants identifies a brainstem-amygdala-cortical alarm system. This alarm system represents the designated route for processing subconsciously perceived threat cues. Brainstem regions closely associated with this system include the SC and locus coeruleus (LC), critical for integrating salient cues and orienting the response, respectively. In addition, the amygdala provides alerting signals to threat, and the pulvinar and prefrontal regions are, in turn, activated by subcortical structures (LC and SC) to process incoming threat. In line with Liddell and colleagues’ findings, increased functional connectivity was reported between the right amygdala, pulvinar, and SC among healthy volunteers in response to processing of masked fearful faces (i.e., subconscious processing) as opposed to processing of consciously processed (unmasked) fearful faces.44 Similar findings emerged in a more recent study on functional connectivity during subconscious perception of fearful faces, where the authors reported positive connectivity between the right amygdala,

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pulvinar, and SC.8 Of note, despite the visual nature of the stimuli being processed, the visual cortex is not involved in this rapid threatdetection route. In keeping with this conclusion, a study using intracranial electrophysiological methods identified early activity in the amygdala preceding activation of the visual cortex in epileptic patients during processing of low spatial frequency components of fearful faces,45 suggesting that processing via the visual cortex is not necessary in order to detect threat. Likewise, a magnetoencephalographic approach identified an amygdalaepulvinar route specific to early visual processing of fearful (but also happy and neutral) faces in healthy participants,46 supporting the notion of a subcortical

amygdalaepulvinar pathway for early threat detection (though bearing in mind that no differences in responding were found for different emotional valences). Taken together, these findings point toward a neural circuit, including the SC, LC, amygdala, and pulvinar that together serve as a preferential subcortical route for fast defensive response to threat in humans.

THE INNATE ALARM SYSTEM The human IAS (Fig. 17.1) is a neural circuitry system responsible for fast defensive responses to imminent threat that, when viewed collectively, demonstrates the conserved nature of the

Sensory Input

Prefrontal Cortex/ACC VL Pulv

HP SC

PAG Amg

Cerebellum

LC

VN Imminent Threat

Spinal cord

Visceral Organs FIGURE 17.1 The innate alarm system. In the figure, red arrows represent unidirectional interactions, whereas black arrows depict bidirectional interactions. ACC, anterior cingulum; Amg, amygdala; HP, hypothalamus; LC, locus coeruleus; PAG, periaqueductal gray; Pulv, thalamic pulvinar; VL, ventrolateral n thalamic nucleus; VN, vestibular nuclei. Adapted from Lanius RA, Rabellino D, Boyd JE, Harricharan S, Frewen PA, Mckinnon MC. The innate alarm system in PTSD: conscious and subconscious processing of threat. Curr Opin Psychol. 2017;14:109e115, Copyright (2017), with permission from Elsevier.

CONSCIOUS AND SUBCONSCIOUS PROCESSING OF THREAT IN PTSD

neurocircuitry of innate defensive responses in animals. Specifically, the IAS relies upon evolutionarily adaptive mechanisms, conserved across species, and anchored in a subcortical pathway to produce an immediate response to threat, thus increasing the chance of survival5e7,9 (also see Liddell and colleagues’ alarm system6; Schutter and colleagues’ cerebello-limbicthalamo-cortical network for detection and evaluation of unsafe environments47; Porges’ neural model to detect internal and external threats to safety, namely “neuroception” model35,48). The IAS is comprised of a network of brain regions, many of which play a prominent role in the neurocircuitry of fear proposed by Silva and colleagues11 and in the defense cascade model of threat responding.27,29 In particular, the IAS is comprised of midbrain structures including the SC, PAG, LC, amygdala, vestibular nuclei, cerebellum, and cortical regions.6,8,42,43,49 In particular, when an individual faces an imminent threat, the SC receives and integrates afferent visual, auditory, and somatosensory inputs to orient the response and communicates with the PAG to activate either the vlPAG or dlPAG to enact passive or active defense reactions, respectively, influencing autonomic (e.g., activation of the sympathetic nervous system) and behavioral outcomes (e.g., defensive responding).6,50e52 SC and PAG projections to the pulvinar within the thalamus allow for coordination of orienting and alerting mechanisms involving further the amygdala in producing an immediate response to threat.15,24,48,51,53,54 The amygdala, in turn, can communicate with prefrontal cortical regions for appraisal of incoming danger.6,55 The amygdala, however, is also able to drive independently an immediate response to threat. Here, the amygdala innervates both the LC and PAG, where the LC is a noradrenergic structure in the pons responsible for driving the physiological response to stress,56 and the PAG plays a central role in autonomic regulation and initiating a defense cascade from hyper-arousal to fight-or-flight to freezing/immobility responses (as in the defense cascade model described previously).29,57 In order to carry out defensive responses, the LC and PAG project downward to the spinal cord

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and visceral organs. Amygdala efferents also project to the hypothalamus, which, in turn, influences the PAG to coordinate physiological and neuroendocrine outputs to sustain the defense response.3,11 The cerebellum plays a homeostatic role within the IAS, contributing with projections to the entire process through the thalamus (ventrolateral nucleus), as well as through several brainstem structures, including for the SC, LC, PAG, and the vestibular nuclei.47,58,59 Finally, the vestibular nuclei are considered crucial for several automatic functions, such as the vestibulo-ocular reflex to orient the oculomotor movements, postural movements, and balance60,61 (see Fig. 17.1 for an illustration of the IAS).

CONSCIOUS AND SUBCONSCIOUS PROCESSING OF THREAT IN PTSD Conscious Threat Processing in PTSD Individuals with PTSD display increased attentiveness to salient threatening stimuli in the environment, providing an ideal study sample for the investigation of threat processing. Extant knowledge concerning threat responding in PTSD stems largely from studies examining conscious threat processing in humans, where participants are presented with threat-related stimuli above the threshold of consciousness (e.g., script-driven imagery, trauma-related stimuli). This research has focused mainly on the neural correlates of hyper-reactivity symptoms (e.g., hyperarousal and re-experiencing symptoms) elicited by conscious processing of trauma-related or fearful stimuli. Here, decreased neural activity in key cortical regions associated with emotion regulation (such as the mPFC) and concomitant increased neural activity in limbic regions involved in emotional processing (amygdala, perigenual anterior cingulum), in interoceptive and emotional awareness (anterior insula), and in selfreflective processing and memory recall (precuneus, hippocampus, and midline retrosplenial cortex) have been identified as neural markers of conscious threat processing.55,62e68 This

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neural pattern, described as impaired topdown inhibition of limbic regions and referred to as emotional undermodulation, is representative of emotion dysregulation in PTSD, particularly characterizing individuals with predominant hyperemotionality and hyperarousal symptomatology.69e71 Conversely, the recently identified dissociative subtype of PTSD (PTSD þ DS)12,72,73 displays an opposite response to consciously processed threat, favoring instead detachment from the environment and from one’s own body and mind. This dissociative state is characterized by increased neural activity of emotion regulation regions (mPFC, dorsal anterior cingulum, dACC) and reduced activation of limbic regions involved in emotion processing (e.g., amygdala and anterior insula), referred to as emotion overmodulation.69,74 Here, complementary results have emerged from volumetric studies in PTSD, where gray matter reduction was found in the mPFC, ACC, and hippocampus, key regions associated with threat processing.75 Notably, the majority of studies of participants with PTSD do not distinguish between patients with or without dissociative symptoms, instead considering them a homogeneous sample. This approach likely contributes to the mixed pattern of findings observed in relation to conscious threat processing in PTSD, with some studies reporting findings of neural patterns consistent with overmodulation and others reporting neural patterns consistent with undermodulation.76,77

appears to play a prominent role in subconscious threat processing among individuals with PTSD. For example, a recent study investigating subconscious processing in response to individualized trauma-related stimuli in PTSD13 revealed increased neural activity in the right cerebellar lobule VI and the posterior cingulum among individuals with PTSD compared with healthy controls. Importantly, cerebellar lobule VI is part of the most phylogenetically recent region of the cerebellum, the posterior cerebellum, also known as the “cognitive cerebellum,” which is involved in integration of perceptions, emotions, and behaviors.85,86 Increased recruitment of this region in PTSD during subconscious defense responses to threat could indicate an attempt to integrate subconscious processing into conscious experiences. Interestingly, some work has elucidated differential subconscious threat processing among individuals with PTSD who experience dissociative symptoms and those with PTSD who do not experience prominent dissociative symptoms. For example, a study carried out by Felmingham and colleagues74 reported heightened neural responding within the amygdala and the parahippocampus during subconscious processing of fear in PTSD þ DS compared with PTSD without dissociative symptoms. These results suggest hyperactivity of limbic regions involved in innate alarm responses in PTSD þ DS as well as emotional overmodulation driven by prefrontal regions seen during conscious threat processing.

Subconscious Threat Processing in PTSD

PTSD Symptomatology and the IAS During Threat Processing

Studies of subconscious threat processing in PTSD have revealed key findings surrounding innate defensive responses in this disorder. For example, numerous studies report increased neural activity among key regions implicated in the IAS, including the amygdala,78e82 brainstem regions,78 mPFC,78,79 thalamus,78 parahippocampus,83 and visual cortex in response to masked fearful stimuli.84 In addition to those regions more commonly associated with threat responding (e.g., amygdala, brainstem regions, mPFC), the cerebellum

Correlations between neural activity during subconscious threat processing and PTSD symptoms support the model of altered emotional modulation in PTSD, where emotion undermodulation is associated with dissociative symptoms and emotion overmodulation is associated with hyperarousal symptoms.13,69 Specifically, hyperarousal and re-experiencing symptoms were associated with hyperactivity in the amygdala during subconscious threat processing and in brainstem regions (PAG and SC) during

CONSCIOUS AND SUBCONSCIOUS PROCESSING OF THREAT IN PTSD

conscious threat processing among individuals with PTSD, suggesting impaired regulation of limbic regions.7 By contrast, during subconscious fear processing in PTSD, avoidance symptoms were positively correlated with neural activity in the inferior frontal gyrus, a brain region implicated in emotion regulation, suggesting overregulation of limbic regions.13 In addition, reduced activation of the left anterior lobule IV-V of the cerebellum was associated with avoidance symptoms in PTSD during subconscious threat processing.13 As the anterior cerebellum has been functionally identified as the sensorimotor cerebellum,87 these results may indicate an impaired integration of sensorimotor perception in PTSD during subconscious threat processing. Moreover, a recent study on the functional connectivity of the cerebellum at rest in PTSD has shown decreased functional connectivity of the posterior cerebellum (involved in cognitive processing) with prefrontal regions involved in emotion regulation in PTSD compared with healthy controls, suggesting a reduced ability to engage brain regions involved in top-down regulation of threat, and potential overreactivity in regions implicated in the IAS in PTSD.88 By contrast, PTSD þ DS was associated with increased functional connectivity with prefrontal regions at rest compared with PTSD, suggesting excessive prefrontal inhibition of limbic and IAS regions and associated emotional detachment. Taken together, these findings support the hypothesis of aberrant responding by cerebellar regions in PTSD compared with controls and are in keeping with previous findings pointing toward differential alterations in functional connectivity between individuals with PTSD and with PTSD þ DS. On balance, the findings reviewed previously suggest strongly that the cerebellum is a key structure in the IAS and plays a central role in mediating subconscious threat responses in individuals with PTSD. Indeed, the IAS may be best characterized as a cerebellarelimbicethalamoe cortical network47 involved in innate human defense responses to threat. In comparison to the more widely accepted limbicethalamoe cortical network,6 inclusion of the cerebellum in

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this network is novel and potentially crucial as the cerebellum has emerged as an essential brain region underlying emotional processing and modulation.85 Indeed, we hypothesize that the cerebellum may be included in the human IAS (Fig. 17.1). Critically, all nodes of the suggested cerebellarelimbicethalamoecortical network have been shown to be altered in PTSD.13,78,79

Functional Connectivity of Brain Regions Associated With the IAS in PTSD In addition to studying neural responding in single brain regions, it is necessary to study synchronicity between key brain regions involved in innate defense responding and the IAS during subconscious processing of threat. Studies reporting functional connectivity patterns during subconscious processing in PTSD can elucidate how different and sometimes distant brain regions work together to affect the stress response. For example, Bryant and colleagues79 investigated functional connectivity between the mPFC and the amygdala in PTSD while participants viewed masked fearful stimuli. Here, enhanced connectivity between the right amygdala and the left dorsomedial PFC was found in PTSD compared with controls.79 Similarly, in another study investigating subconscious processing of fearful faces in soldiers with PTSD,89 hypersynchrony was found among a neural network encompassing IAS regions, including the amygdala, the ventral mPFC, and the posterior cingulum. In a recent study from our group comparing PTSD to controls during subconscious processing of threat, we observed aberrant coupling of key brain regions implicated in innate defensive responding within intrinsic connectivity networks90: the default mode network (associated with rest and dedicated to self-referential processing, emotion regulation, and memory retrieval91,92), the salience network (involved in filtering salient stimuli to guide behavior91,93), and the central executive network (involved in cognitive and executive control for goal-directed behavior94). Specifically, key nodes of each network that are also implicated in the IAS (e.g., amygdala, mPFC, and dACC) were functionally connected

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with different networks in PTSD when compared with controls. Here, the amygdala was connected with the default mode network (as opposed to the salience network), which includes the mPFC as a key region, mirroring previous findings of increased connectivity between IAS regions (the amygdala and mPFC) during subconscious threat processing in PTSD.79 These findings indicate a reorganization of the networks involved in rest and selfreferential processing and those involved in salience detection, including detection of threat as well as increased connectivity between IAS regions among individuals with PTSD during subconscious processing of threat. In addition, brain areas associated with consciousness and self-awareness (precuneus and claustrum) were decoupled across networks (precuneus within the DMN and CEN, claustrum within the SN) when comparing PTSD to controls during subconscious threat detection, suggesting reduced self-awareness. These results illustrate key alterations in the defense response system during threatening situations in PTSD.90

The Role of the Amygdala in Innate Defensive Responding in PTSD Across the studies reviewed thus far, the amygdala has emerged as a key region for processing emotional responses at the subconscious level and also features as a key region in the IAS.5e7,9 In addition to studying the amygdala as a whole, several preclinical animal studies point toward identification of the differential roles of the various amygdala subdivisions as critical to the understanding of innate defense mechanisms.95 Patterns of distinct connectivity of amygdala subdivisions have also been found among individuals with PTSD during conscious and subconscious processing of threat.96 Here, conscious processing of threat has been associated with increased connectivity of the left CMA (involved in behavioral responding to emotion) with the left pulvinar, a thalamic subregion crucial for quick defensive responses and salience detection, suggesting increased activation of behavioral responses to threat in PTSD.97,98 By contrast, increased connectivity between the right

CMA and the superior frontal gyrus and decreased connectivity between the right BLA (involved in integration of sensory information associated with threat) and the SC emerged during subconscious processing of trauma-related cues among individuals with PTSD.96 Our findings reveal altered orchestration of the emotional response in PTSD, featuring undermodulation of prefrontal regions receiving afferents from the CMA (where the CMA would exert an excitatory influence on the prefrontal regions79) leading increased reactivity to threat cues, together with disrupted sensory integration and consequent impairment in evaluating incoming information due to decreased connectivity between the BLA and the SC. Together, the CMA, BLA, and SC play crucial roles in the IAS.

Brainstem Regions and Innate Defensive Responding in PTSD The PAG plays an essential role in driving innate defense responses via the IAS (see also section “Innate Defense Responses in Animals”), mediating both active and passive physiological and behavioral responses to threat.25 Among individuals with PTSD, increased functional connectivity between the dlPAG (involved in enacting motor patterns of fight-or-flight) and brain regions involved in emotion regulation (dorsal anterior cingulum and anterior insula) is reported during rest.53 In addition, increased connectivity of both the vlPAG (a region that acts as a brake for the dlPAG) and dlPAG with the fusiform gyrus (an occipital region dedicated to face, voice, and movement recognition) has been observed among individuals with PTSD and may be related to search for threatening faces in the environment. In another recent resting-state PTSD study, the PAG showed widespread connectivity with cortical regions involved in central autonomic functioning, suggesting hyperarousal and maladaptive physiological regulation driven by the PAG even at rest in PTSD.57 In keeping with these findings, an additional study on the neural activity associated with autonomic regulation during subconscious processing of threat in PTSD99 found decreased activity in the dorsal anterior insula,

CLINICAL AND RESEARCH IMPLICATIONS

an area involved in the autonomic adaptation to internal and external stress,100 in PTSD compared with controls. These results suggest a disruption in the central autonomic circuitry in PTSD, which appears evident even without conscious awareness of the perceived threat.101 Indeed, even at rest, individuals with PTSD show increased functional connectivity between insular subregions and the BLA.102 Taken together, these findings point toward the urgent need for research investigating the role of the PAG in subconscious threat processing in PTSD using masked threatening stimuli. The SC is also central to innate defense responses and the IAS where this region is responsible for fast, lower-order multisensory integration and threat-detection mechanisms.50,54 A recent investigation of SC functional connectivity in PTSD at rest revealed hyperconnectivity between the left SC and the right dorsolateral PFC in participants with PTSD, as well as increased connectivity between the right SC and the left temporoparietal junction (TPJ) in individuals with PTSD þ DS.52 These results have been linked to overmodulation of emotional responses in PTSD (top-down prefrontal modulation) and to passive defensive reaction involving depersonalization symptoms in PTSD þ DS due to increased TPJ connectivity with the SC, where the TPJ plays a key role in bodily self-consciousness and depersonalization symptomatology.52,103 One final, unique method of studying subconscious threat processing involves investigation of neural responses to direct eye contact. Direct eye contact may be perceived as an imminent threat in PTSD, thus generating subconscious processes necessary for quick defensive responses.104,105 Notably, processing of direct eye contact also engages neural regions identified as key to the innate defense response, including the SC, PAG, pulvinar, and amygdala.105,106 Notably, during direct versus averted eye gaze processing, in comparison to healthy controls, individuals with PTSD showed increased neural activity within the SC, LC, and PAG, along with decreased activity in cortical regions associated with social cognition (e.g., the TPJ and dorsomedial PFC).51 Moreover, increased functional

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connectivity of the SC and LC with the insula, the inferior frontal cortex, and the anterior cingulum has been observed during processing of direct eye gaze in PTSD.54 Taken together, these findings point toward a hyperactive subcortical pathway in PTSD that is engaged in response to direct eye contact and appears to trigger a subconscious defense response operating independently from regulatory cortical structures. On balance, the findings reviewed here suggest that innate defensive responding in PTSD can be understood through the IAS framework. In particular, our review of studies in PTSD during conscious and subconscious threat processing highlights aberrant connectivity and reactivity of critical IAS brain regions, including the amygdala, PAG, SC, LC, and mPFC, as well as the cerebellum (involved in sensorimotor perception and cognitive processing).13,51e54,57,74,78,79,89,90,99 These alterations may be associated with exaggerated threat responding in individuals with PTSD.

CLINICAL AND RESEARCH IMPLICATIONS Alterations in activity and connectivity of IAS brain regions in individuals with PTSD may underlie the enhanced reactivity to threat observed in this disorder, resulting in hyperarousal symptoms (Fig. 17.1). The findings reviewed here suggest further that chronic stress often underlying the etiology of PTSD (e.g., childhood abuse) may lead to alterations in defensive response circuits, resulting in hyperreactivity and a loss of adaptive mechanisms (e.g., inability to accurately perceive and react to threatening stimuli). Although the majority of treatments for PTSD focus on symptoms that occur in response to conscious threat (e.g., exposure therapy), our review suggests strongly that individuals with PTSD are susceptible to subconscious threat processing. Thus, a better understanding of subconscious threat processing in PTSD may lead to alternative treatment modalities.

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Future work should delineate further the involvement of subcortical brain regions, including the PAG and SC, in subconscious threat processing in order to better understand the functioning of the IAS in individuals with PTSD. Future work may also evaluate differences in IAS neurocircuitry among individuals with PTSD and PTSD þ DS, given the differential responses to threatening stimuli observed in these populations and characterized as emotion undermodulation (hyperemotionality) and emotion overmodulation (hypoemotionality), respectively. Future research may also aim to understand changes in IAS neurocircuitry in other anxiety- or fear-based disorders, including anxiety disorders and dissociative disorders.

Acknowledgments Daniela Rabellino was supported by fellowship from MITACS and Homewood Research Institute.

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amygdala. Nature. 1998;393(6684):467e470. https:// doi.org/10.1038/30976. Merker B. The efference cascade, consciousness, and its self: naturalizing the first person pivot of action control. Front Psychol. 2013;4:501. https://doi.org/ 10.3389/fpsyg.2013.00501. Steuwe C, Daniels JK, Frewen PA, et al. Effect of direct eye contact in PTSD related to interpersonal trauma: an fMRI study of activation of an innate alarm system. Soc Cogn Affect Neurosci. 2014;9:88e97. https://doi.org/ 10.1093/scan/nss105. Olive´ I, Densmore M, Harricharan S, The´berge J, McKinnon MC, Lanius R. Superior colliculus resting state networks in post-traumatic stress disorder and its dissociative subtype. Hum Brain Mapp. 2018;39(1):563e574. https://doi.org/10.1002/ hbm.23865. Harricharan S, Rabellino D, Frewen P, et al. fMRI functional connectivity of the periaqueductal gray in PTSD and its dissociative subtype. Hum Brain Mapp. 2016;6(12):e00579. https://doi.org/10.1002/ brb3.579. Steuwe C, Daniels JK, Frewen PA, Densmore M, Theberge J, Lanius RA. Effect of direct eye contact in women with PTSD related to interpersonal trauma: psychophysiological interaction analysis of connectivity of an innate alarm system. Psychiatry Res Neuroimaging. 2015;232(2):162e167. https://doi.org/10.1016/ j.pscychresns.2015.02.010. Williams LM, Kemp AH, Felmingham K, et al. Trauma modulates amygdala and medial prefrontal responses to consciously attended fear. Neuroimage. 2006;29(2):347e357. https://doi.org/10.1016/ j.neuroimage.2005.03.047. Ziegler DR, Cass WA, Herman JP. Excitatory influence of the locus coeruleus in hypothalamic-pituitary- adrenocortical axis responses to stress. J Neuroendocrinol. 1999;11(5):361e369. Thome J, Densmore M, Frewen PA, et al. Desynchronization of autonomic response and central autonomic network connectivity in posttraumatic stress disorder. Hum Brain Mapp. 2017;38(1):27e40. https://doi.org/ 10.1002/hbm.23340. Strata P, Scelfo B, Sacchetti B. Involvement of cerebellum in emotional behavior. Physiol Res. 2011; 60(suppl 1):S39eS48. Bagnall M, du Lac S, Mauk M. Cerebellum. In: Fundamental Neuroscience. 4th ed. Academic Press BooksElsevier; 2013:677e696. https://doi.org/10.1016/ B978-0-12-385870-2.00031-7. Lopez C, Blanke O. The thalamocortical vestibular system in animals and humans. Brain Res Rev. 2011; 67(1e2):119e146. https://doi.org/10.1016/j.brain resrev.2010.12.002. Lopez C. The vestibular system: balancing more than just the body. Curr Opin Neurol. 2016;29:74e83. https://doi.org/10.1016/j.cub.2005.07.053.

62. Patel R, Spreng RN, Shin LM, Girard TA. Neurocircuitry models of posttraumatic stress disorder and beyond: a meta-analysis of functional neuroimaging studies. Neurosci Biobehav Rev. 2012;36(9):2130e2142. https://doi.org/10.1016/j.neubiorev.2012.06.003. 63. Koch SBJ, van Zuiden M, Nawijn L, Frijling JL, Veltman DJ, Olff M. Aberrant resting-state brain activity in posttraumatic stress disorder: a meta-analysis and systematic review. Depress Anxiety. 2016;33: 592e605. https://doi.org/10.1002/da.22478. 64. Sartory G, Cwik J, Knuppertz H, et al. In search of the trauma memory: a meta-analysis of functional neuroimaging studies of symptom provocation in posttraumatic stress disorder (PTSD). PLoS One. 2013;8(3): e58150. https://doi.org/10.1371/journal.pone.0058150. 65. Hayes JP, Hayes SM, Mikedis AM. Quantitative metaanalysis of neural activity in posttraumatic stress disorder. Biol Mood Anxiety Disord. 2012;2(1):9. https://doi.org/10.1186/2045-5380-2-9. 66. Shin LM, Wright CI, Cannistraro PA, et al. A functional magnetic resonance imaging study of amygdala and medial prefrontal cortex responses to overtly presented fearful faces in posttraumatic stress disorder. Arch Gen Psychiatry. 2005;62(3):273e281. https:// doi.org/10.1001/archpsyc.62.3.273. 67. Shin LM, Liberzon I. The neurocircuitry of fear, stress, and anxiety disorders. Neuropsychopharmacology. 2010; 35(1):169e191. https://doi.org/10.1038/npp.2009.83. 68. Duval ER, Javanbakht A, Liberzon I. Neural circuits in anxiety and stress disorders: a focused review. Ther Clin Risk Manag. 2015;11:115e126. https://doi.org/ 10.2147/TCRM.S48528. 69. Lanius RA, Vermetten E, Loewenstein RJ, et al. Emotion modulation in PTSD: clinical and neurobiological evidence for a dissociative subtype. Am J Psychiatry. 2010;167(6):640e647. https://doi.org/10.1176/ appi.ajp.2009.09081168. 70. Hopper JW, Frewen PA, van der Kolk BA, Lanius RA. Neural correlates of reexperiencing, avoidance, and dissociation in PTSD: symptom dimensions and emotion dysregulation in responses to script-driven trauma imagery. J Trauma Stress. 2007;20(5):713e725. https://doi.org/10.1002/jts. 71. Rauch SL, Shin LM, Phelps EA. Neurocircuitry models of posttraumatic stress disorder and extinction: human neuroimaging research-past, present, and future. Biol Psychiatry. 2006;60(4):376e382. https://doi.org/ 10.1016/j.biopsych.2006.06.004. 72. Steuwe C, Lanius RA, Frewen PA. Evidence for a dissociative subtype of PTSD by latent profile and confirmatory factor analyses in a civilian sample. Depress Anxiety. 2012;29(8):689e700. 73. Lanius RA, Brand B, Vermetten E, Frewen PA, Spiegel D. The dissociative subtype of posttraumatic stress disorder: rationale, clinical and neurobiological evidence, and implications. Depress Anxiety. 2012; 29(8):701e708. https://doi.org/10.1002/da.21889.

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C H A P T E R

18 Stress-Induced Anovulation Sarah L. Berga Division of Reproductive Endocrinology and Infertility, Department of Gynecology and Obstetrics, University of Utah School of Medicine, Salt Lake City, UT, United States O U T L I N E Definitions

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Introduction Neuroendocrine Mechanisms Linking Cognition, Mood, Behavior, and GnRH Drive Pathogenesis of Stress-Induced Anovulation Behavioral, Nutritional, and Metabolic Influences on the Reproductive Axis

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DEFINITIONS Allostasis A sustained neuroendocrine and metabolic adjustment that promotes survival in response to chronic challenge and if sustained may increase acute and chronic health burden. Amenorrhea The absence or cessation of uterine bleeding. Anovulation The absence or cessation of ovulation. Cognitive behavioral therapy A form of psychoeducation or talk therapy that addresses problematic thoughts, cognitions, attitudes, and conceptualizations. Eumenorrhea Regular and predictable menstrual cycles; the presence of eumenorrhea cannot be taken as evidence as ovulation. Gonadotropin-releasing hormone (GnRH) A decapeptide produced and released by hypothalamic neurons that stimulates the release of pituitary luteinizing hormone (LH) and follicle-stimulating hormone (FSH) to drive gonadal function. Hypothalamic hypogonadism A condition characterized by suppression of GnRH drive that manifests as

Stress: Physiology, Biochemistry, and Pathology https://doi.org/10.1016/B978-0-12-813146-6.00018-7

reduced pituitary secretion of LH and FSH and, in turn, reduced or absent gonadal steroidogenesis and gametogenesis; causes may be functional (behavioral) or organic. Hypothalamic hypothyroidism An allostatic adjustment in the feedback sensitivity of the hypothalamice pituitaryethyroidal axis that reduces metabolism and manifests in the circulation as normal levels of pituitary thyroid-stimulating hormone in the presence of reduced levels of the thyroid hormones triiodothyronine (T3) and thyroxine (T4). Limbicehypothalamicepituitaryeadrenal axis Neural circuitry located in the limbic lobe transduces perceived stress into increased release into the portal circulation of hypothalamic corticotropin-releasing hormone, which then elevates pituitary release of ACTH and adrenal secretion of cortisol. Metabolism Biochemical processes that regulate energy expenditure and storage. Secondary amenorrhea Cessation of menses in a woman of reproductive age who was previously eumenorrheic.

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Copyright © 2019 Elsevier Inc. All rights reserved.

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Stress Physical and psychological stimuli that challenge the status quo and elicit homeostatic or allostatic biobehavorial responses with associated neuroendocrine and metabolic concomitants. Stress-induced anovulation Often termed functional hypothalamic amenorrhea to indicate a potentially reversible suppression of hypothalamic GnRH drive due to behavioral causes.

KEY POINTS • Stress is one of the most common and most commonly underappreciated causes of infertility and reproductive compromise in men and women. • Stress-induced hypothalamic hypogonadism increases the acute and chronic health burden for individuals and their offspring. • Our findings regarding the pathogenesis stress-induced anovulation/functional hypothalamic amenorrhea (SIA/FHA) reveal a powerful synergism between energetic imbalance and psychogenic challenge and underscore why seemingly mundane psychological states evoke not only reproductive compromise but also concomitant neuroendocrine and metabolic allostasis. • These mechanistic pathobiological insights have been harnessed to develop therapeutic options aimed at ameliorating sustaining cognitive and behavioral variables. • By showing that cognitive behavior therapy (CBT) reverses SIA/FHA, we can appreciate the inherent neuroplasticity that forms the rationale for coupling mindfulness approaches with the judicious use of psychopharmacologic and technical interventions to yield improved reproductive and overall health.

INTRODUCTION A wealth of scientific and clinical evidence supports the notion that stress causes reproductive compromise and increases health burden. While acute stress elicits transient neuroendocrine, metabolic, and behavioral responses that promote survival in the face of perceived challenge, chronic stress provokes allostatic (sustained) neuroendocrine and metabolic adjustments that also promote survival, but at the expense acute and chronic health (Fig. 18.1). Reproductive compromise impedes population replenishment and increases health burden in women, men, and children due to metabolic dysfunction, obesity, diseases of aging, preterm delivery, birth defects, costly and risky infertility therapies, and, through epigenetic mechanisms, imprints the next generation. Stress is the most common and most commonly underappreciated cause of reproductive dysfunction (Table 18.1,1). Stress-induced anovulation (SIA), often termed functional hypothalamic amenorrhea (FHA), causes infertility and increases acute and chronic health burden. Behaviors that chronically activate the limbice hypothalamicepituitaryeadrenal (LHPA) axis elicit a constellation of neuroendocrine adjustments including suppression of the hypothalamicepituitaryegonadal (HPG) axis and the hypothalamicepituitaryethyroidal (HPT) axis in both women2 and men by reducing hypothalamic gonadotropin-releasing hormone (GnRH) drive. Individuals with functional (not due to organic conditions) hypothalamic hypogonadism typically engage in a combination of behaviors in response to perceived endogenous and exogenous psychosocial stressors that concomitantly induce intermittent or chronic energy imbalance. Chronic adrenal activation may increase the energetic cost of common activities undertaken to manage stress such as exercise. Not only did women with FHA show an exaggerated rise in cortisol during graded exercise compared with eumenorrheic, ovulatory

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INTRODUCTION

Homeostasis

Chronic Stress Cognitive Behavior Therapy

Metabolic Challenge -excessive exercise -undernutrition -nutrient deficiency

Allostasis

Psychogenic Challenge - performance pressure - unrealistic expectations - poor coping strategies

Central Neuromodulation

Hypothalamic adjustment

Pituitary

Glands Thyroid, Parathyroid, Gonads, Adipocytes, Pancreas, Adrenal

FIGURE 18.1 Conceptualization of synergism between metabolic and psychogenic stress in the pathogenesis of stressinduced anovulation.

women, but they also showed a drop in glucose.3 In contrast, exercise elicited a rise in circulating glucose in eumenorrheic, ovulatory women. We posited that the drop in circulating glucose levels in women with FHA elicited the greater rise in cortisol during exercise and that the drop in glucose reflected hepatic depletion of glycogen due to chronic LHPA activation. In women, functional hypothalamic hypogonadism exists on a spectrum that includes polymenorrhea, eumenorrhea with reduced luteal progesterone secretion, and anovulatory TABLE 18.1

Common Causes of Anovulation/ Amenorrhea

Functional hypothalamic amenorrhea

34%

Hyperandrogenism/Polycystic ovary syndrome (PCOS)

29%

Hyperprolactinemia

13%

Premature menopause

12%

Asherman’s syndrome

5%

Other

7%

Adapted from Reindollar RH, Novak M, Tho SP, McDonough PG. Adult onset amenorrhea: a study of 262 patients. Am J Obstet Gynecol. 1986; 155:531e543.

eumenorrhea, oligomenorrhea, or amenorrhea. In men, oligoasthenospermia may result, but this is typically not clinically evident unless infertility results or significant testosterone deficiency causes phenotypic changes such as muscle wasting. This chapter focuses on SIA in women, a condition that is often termed FHA or functional hypothalamic chronic anovulation. SIA/FHA typically results from psychogenic stress coupled with mild energy imbalance and represents an allostatic adaptation, that is, a stable change in behaviors, neuroendocrine secretory patterns, and metabolism that promotes acute survival but at some health cost. While SIA/FHA affects roughly 5% of women of reproductive age, less severe forms of hypothalamic hypogonadism are more common and harder to detect clinically (Fig. 18.2). SIA is theoretically reversible, but reproductive recovery appears to depend upon restoration of eucortisolemia and at least partial recovery from functional hypothalamic hypothyroidism.4,5 Hormone replacement regimens have limited benefit for women with SIA/FHA because their use does not promote recovery from allostatic endocrine adjustments. Indeed, the rationale for the use of sex steroid replacement is based on the erroneous assumption

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FIGURE 18.2 The continuum of ovarian function in women with preserved menstrual cyclicity. Pituitary gonadotropin (LH and FSH ) and ovarian sex steroid (estradiol and progesterone ) levels were obtained daily for one cycle. Examples include ovulatory eumenorrheia (panel A), luteal insufficiency (panel B), and eumenorrheic anovulation (panels C and D).

that functional hypothalamic hypogonadism represents only or primarily a loss of sex steroid exposure due to isolated anovulation. Furthermore, replacement of sex hormones masks deficits that accrue from chronically altered adrenal and thyroidal function. Long-term deleterious

consequences of SIA likely include an increased risk of cardiovascular disease, osteoporosis, depression, other psychiatric conditions, dementia, and neurodevelopmental compromise in offspring. While fertility can be restored with exogenous administration of gonadotropins or

INTRODUCTION

pulsatile GnRH, fertility management alone does not promote neuroendocrine and metabolic recovery. Pregnancy in the face of ongoing psychogenic stress and metabolic imbalance may increase the likelihood of poor obstetrical, fetal, or neonatal outcomes. In contrast, behavioral and psychological interventions that address problematic behaviors and attitudes have the potential to facilitate reproductive, neuroendocrine, and metabolic recovery. In short, full endocrine recovery offers better individual, maternal, and child health. The recent Endocrine Society Clinical Practice Guideline offers a full consideration of the pros and cons of treatment options.6

Neuroendocrine Mechanisms Linking Cognition, Mood, Behavior, and GnRH Drive Scientific delineation of the neuroendocrine and metabolic responses to thoughts, feelings, and behaviors has refined our conceptualization of the bidirectional interaction between behavior and gonadal function. The proximate cause of hypothalamic forms of hypogonadism, including SIA, is reduced hypothalamic GnRH input. Gonadal function depends directly upon secretion from the hypothalamus of GnRH as pulses occurring at the frequency of once per 90 min. Declines in pulsatile GnRH secretion reduce pituitary secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) and wholly or partially compromise folliculogenesis. Thus decreased GnRH drive is a common cause of anovulation and amenorrhea. Decrements in central GnRH-LH/FSH drive exist on a continuum, however, and may vary from day-to-day,7 leading to variable folliculogenesis and a range of menstrual patterns including polymenorrhea (menstrual interval 35 days), or amenorrhea (no cycles for >3 months). Clinically, the decreased ovarian function can be clinically occult or obvious. In men, decreased central GnRH drive may cause oligoasthenozoospermia (very low sperm count with poor motility and morphology). Typically, hypothalamic hypogonadism gonadal

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compromise in men is clinically occult unless it results in “low testosterone (T) syndrome” and/ or infertility, decreased libido, diminished muscle mass, or altered hair (beard) growth. The most common cause of reduced GnRH drive is functional; that is, it is not due to identifiable organic causes such as hypothalamic tumors or pituitary adenomas. Genetic causes are more difficult to detect and may be one factor that sensitizes women to stressors and the development of SIA/FHA.8 Functional hypothalamic hypogonadism/SIA/FHA is theoretically reversible. The mechanisms by which stress disrupts GnRH drive are poorly understood and complex. GnRH neurons are diffusely distributed in the medial basal hypothalamus, and most of the axons project to the median eminence, allowing pulses of GnRH to be released into the portal vasculature. Although GnRH neurons are endogenously pulsatile, their activity must be synchronized by GnRH-to-GnRH synapses for the GnRH bolus released into the portal vasculature to be of sufficient magnitude to release of LH and FSH. Peripheral substances communicate to the GnRH neuronal network via specialized neurovascular cells that line the fenestrated bloodebrain barrier at the level of the hypothalamus and median eminence, so modulation of GnRH secretion is not limited to central factors.9 The brain-gut axis appears to communicate metabolic status of an individual to the hypothalamic GnRH neurons through multiple mechanisms.10 In humans, GnRH pulsatile secretion can be inferred from the pattern of LH secretion in the circulation. However, to define LH pulse patterns requires that blood samples be obtained via an indwelling intravenous catheter at intervals of 10e15 min for durations of 12e24 h. The tedious nature of quantifying central GnRH drive explains why quantifying LH pulse patterns is not routinely done for clinical evaluation. Technical limitations also plague recognition and quantification of stress and metabolic status. The accuracy of psychometric inventories for assessing and quantifying stress, mood, and cognitive patterns is inherently constrained by reporting biases, while endocrine or biophysical indices

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of stress and metabolic status are also technically cumbersome and expensive to collect. Neuroimaging techniques have afforded a new window into the neurochemistry and neuroanatomy of behaviors and cognitions, but these techniques have not yet been utilized to understand the pathogenesis of stress-induced reproductive compromise in humans. However, we recently found that subordinated monkeys, previously shown to have hypothalamic hypogonadism and its associated concomitants,11 have increased prefrontal GABAergic tone as detected by neuroimaging that is reversed by the CRH antagonist astressin B.12 Furthermore, over the last decade, it has been established that the neuropeptide, kisspeptin, regulates GnRH neurons via its receptor, and that the signaling systems that communicate metabolic state to GnRH neurons involve kisspeptin-neurotrophin-dynorFurthermore, phin (KNDy) neurons.13 metabolic signals such as leptin and ghrelin appear to regulate KNDy neurons via a GABAergic mechanism.10,14

Pathogenesis of Stress-Induced Anovulation The best biochemical evidence supporting the concept that stress impairs ovarian function in women is the consistent demonstration that women with hypogonadotropic hypogonadism not due to defined organic conditions have higher cortisol levels than eumenorrheic, ovulatory women.2 Women athletes show an inverse relationship between degree of ovarian compromise and cortisol levels.15 When compared with eumenorrheic but sedentary women, eumenorrheic athletes had lower luteal progesterone secretion as evidenced by lower urinary levels of the metabolite pregnanediol glucuronide, fewer LH pulses in a day, and higher cortisol levels. Amenorrheic anovulatory athletes had the fewest LH pulses in a day and the highest cortisol levels despite comparable levels of exertion and fitness. Women with SIA/FHA display a constellation of neuroendocrine and metabolic alterations in addition to activation of the hypothalamice pituitaryeadrenal (HPA) axis and suppression

of the HPG2,16 that reflect the neuroanatomical and neurophysiological integration of the neural networks that synchronize hypothalamic function. The goal of the resulting “endocrine action plan” is to preserve the organism in the face of challenge and includes metabolic mobilization and energy conservation. In addition to heightened cortisol secretion, women with SIA/FHA also displayed hypothalamic hypothyroidism with preservation of thyroid-stimulating hormone (TSH) secretion despite decreased levels of thyronine (T3) and thyroxine (T4) and decreased GnRH-LH/FSH drive despite decreased ovarian secretion of estradiol and progesterone. The secretory patterns of growth hormone, prolactin, and melatonin also differed from those of eumenorrheic women (EW). At a minimum, these neurosecretory alterations reflect altered hypothalamic feedback sensitivity to estradiol, cortisol, and thyroxine. Other feedback sensitivities that are likely to be altered include leptin and ghrelin signaling. Vulliemoz et al.17 showed that administration of a CRH antagonist reversed the inhibition of LH pulsatility induced by the administration of a potent orexigenic signal, ghrelin. Women recovering from FHA showed a decrease in cortisol and an increase in leptin independent of weight gain.5 We demonstrated that increased circulating and cerebrospinal fluid (CSF) cortisol levels are specific to FHA and were not found in women with anovulation due to other causes.18,19 In short, SIA/FHA is more than an isolated suppression of GnRH leading anovulation and amenorrhea. The constellation of neuroendocrine aberrations that accompany SIA/FHA supports the notion that central neurotransmission has been chronically altered to forge an allostatic state.2 The aim of allostatic adjustments is to allow the individual to cope with chronic as opposed to acute challenge. In this context, then, the hypothalamus links the external environment, the internal milieu, and gonadal function. Recovery from SIA/FHA involves amelioration of HPA activation.5 We reported that most women with SIA/FHA treated with cognitive behavioral therapy (CBT) displayed return of ovarian function and ovulation and reduced cortisol levels, but only partial recovery from

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hypothalamic hypothyroidism and no weight gain.4 These data, coupled with our psychometric data showing that women with SIA/FHA differed from EW in their attitudes toward eating and desire for thinness, raised the possibility that intermittent or mild energy imbalance may persist after CBT-induced reproductive recovery. We posit that attitudes and behaviors that initiate and sustain SIA/FHA must be ameliorated to foster full neuroendocrine and metabolic recovery. The characteristic hypothalamic alterations associated with SIA/FHA only become problematic when ongoing challenges elicit a chronic rather than acute response. Stress appears to increase the risk of dementia and other neurodegenerative disorders. Exogenous steroids fail to stimulate appropriate bone accretion in women with SIA/FHA due to persistent catabolism.6 Because of the concomitant endocrine and metabolic disturbances, hypothalamic hypogonadism deserves clinical attention even when fertility is not an immediate goal.6 The central neuromodulators responsible for the initiation and maintenance of the disruption of GnRH are difficult to identify in humans and may be multiple but most certainly involve a cascade that includes kisspeptin.13 To date, efforts to identify these neuromodulators in humans have yielded inconsistent results. Thus naloxone, an opioidergic blocker, increased LH pulse frequency or levels in some, but not all, women with SIA/FHA.20 An infusion of metoclopramide, a dopamine receptor blocker, to women with FHA accelerated LH pulse frequency, while that of EW remained constant.21 These data suggest that there may be dopaminergic as well as opioidergic inhibition of GnRH drive. We performed lumbar punctures to obtain rostral cerebrospinal fluid in women with SIA/FHA and those with eumenorrhea.22 CRH levels were identical in women with SIA/ FHA and eumenorrhea, vasopressin levels were similar, but surprisingly, b-endorphin levels were lower in SIA/FHA. Furthermore, free cortisol in the CSF was approximately 30% higher in those women with SIA/FHA.19 In aggregate, these findings (Fig. 18.3) support the notion that preservation of CSF CRH levels in

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FIGURE 18.3 Mean  standard error CSF cortisol and CRH in eumenorrheic women (EW) and those with SIA/ FHA demonstrating allostatic resistance to cortisol feedback suppression of CRH.CRH, corticotropin-releasing hormone; FHA, functional hypothalamic amenorrhea; SIA, stress-induced anovulation. Adapted from Brundu B, Loucks TL, Adler LJ, Cameron JL, Berga SL. Increased cortisol in the cerebrospinal fluid of women with functional hypothalamic amenorrhea. J Clin Endocrinol Metab. 2006;91:1561e1565.

the presence of elevated CSF cortisol reflects chronic stress-induced (allostatic) resistance to negative feedback suppression by cortisol. Other putative central neurotransmitters that may contribute to the initiation and maintenance of SIA/FHA are central serotonergic and GABAergic systems. Several lines of evidence suggest that serotonergic function gates stress reactivity and metabolism. First, stress-sensitive monkeys show larger cortisol elevations and reduced prolactin responses to the serotonergic agonist fenfluramine, indicating reduced central serotonergic tone in stress-sensitive as contrasted with stress resilient monkeys.23 Second, serotonergic neurons, which express leptin receptors, terminate on GnRH neurons. Serotonin has satiety effects similar to those of leptin and may modulate the response to orexigenic and anorectic signals. When fasting female mice were exposed to saline, leptin, or fluoxetine, a selective serotonin reuptake inhibitor antidepressant, leptin, and fluoxetine prevented

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fasting-induced estrous cycle lengthening, while a serotonin receptor antagonist metergoline blocked estrous cycle rescue by fluoxetine.24 Furthermore, there is abundant evidence of a role for the serotonergic system in the regulation of stress reactivity and feedback inhibition of the HPA axis by cortisol. In addition, variation in transporter-facilitated uptake of serotonin has been implicated as a contributor to stress sensitivity and anxiety and depression in animal models and humans. Human serotonin reuptake transporter (SERT) gene transcription is modulated by a common polymorphism in its upstream regulatory region. The short variant reduces the transcriptional efficiency of the SERT gene promoter, resulting in decreased SERT expression and serotonin reuptake. In humans, the SERT polymorphisms moderated the influence of stressful life events on depression in a representative birth cohort.25 Stresssensitive monkeys had lower expression of SERT mRNA and monoamine oxidase A (MAO-A) in the caudal region of the dorsal raphe nucleus, while no differences were detected between stress-sensitive and stressresilient monkeys in the mRNA for the serotonin 1A autoreceptor or MAO-B.26 Alprazolam, a gamma-Aminobutyric Acid (GABA) receptor agonist, decreased cortisol levels and increased LH pulse frequency from 0.8 to 2.0 pulses/8 h in women with stressrelated anovulation, while its administration decreased LH pulse frequency in EW in the follicular phase.27 These data suggest a role for GABA neurons in the stress-induced neuromodulation of GnRH pulsatility. As noted earlier, we found increased GABAergic binding in the frontal cortex of subordinated monkey and showed that the increase was reversed by the CRH antagonist astressin B.12 Not surprisingly, the neurochemistry of stress and SIA/FHA is far from simple, and firm mechanistic conclusions are not possible at present.

Behavioral, Nutritional, and Metabolic Influences on the Reproductive Axis Identification of the initiating and sustaining behavioral factors that activate the adrenal axis

and suppress the thyroidal and ovarian axes can be challenging. Psychogenic stressors have metabolic costs and metabolic stressors, such as food restriction and excessive exercise, often initiated to cope with psychogenic stress.28 Furthermore, independent of the stressor type, some individuals are more stress-sensitive or -resilient than others.

BEHAVIORAL INFLUENCES A number of behavioral and other psychogenic factors including exercise, low weight and weight loss, affective and eating disorders, various personality characteristics, drug use, and external and intrapsychic stresses have been associated with functional hypothalamic hypogonadism. Likely, any given stressor, when the “dose” is large enough, can activate the central neural pathways leading to the disruption of GnRH. In clinical research, the trend has been to study single stressors and to partition as separate populations women with “exercise amenorrhea” anorexia nervosa, and “idiopathic amenorrhea.” Populations studied in clinical research settings may not be entirely representative of all women with SIA because research subjects must meet relatively strict inclusion and exclusion criteria. In general, women with SIA/FHA do not report or do not have an easily identified solitary stressor. Typically, there are multiple, seemingly minor stressors such as a combination of job or school pressures, poor eating habits, and increased energy expenditure through activity or exercise.29e31 To understand the role of psychological variables, such as attitudes and expectations, in the pathogenesis of SIA/FHA, we compared three groups of women: those with eumenorrhea and demonstrable luteal adequacy; those with SIA/FHA unrelated to excessive exercise, weight loss, an eating disorder, drug use, or an affective disorder; and those with anovulation due to an identifiable organic cause. Being amenorrheic, regardless of cause, was associated with a compromised sense of psychological equilibrium as reported on psychometric inventories, but, as a group, only women with SIA/ FHA differed from the other groups on scales

BEHAVIORAL INFLUENCES

that measured unrealistic expectations and dysfunctional attitudes (defined as those attitudes likely to impair coping responses).32 For instance, women with SIA/FHA were both highly perfectionistic and sociotrophic (high need for social approval).29 In that perfectionism interferes with social approval or acceptance, one interpretation is that the concomitant high drive for perfectionism and sociotrophy creates an intrapsychic conflict that women with SIA/FHA may not possess the appropriate coping skills to resolve. Another interpretation is that the expectation of simultaneously being perfect and garnering social approval is an unrealistic expectation of self and others. Our earlier study suggested that women with SIA/ FHA had trouble relaxing and having fun, attributes that may further predispose them to value performance at the expense of psychological needs. Although women with SIA/FHA do not typically meet criteria for an eating disorder, they display many attitudes and behaviors similar to women with eating disorders. One is a drive for thinness and disordered eating. What appears to discriminate women with undifferentiated SIA/FHA from those with an eating disorder is the degree of disturbance, including the degree of food restriction, weight loss, bingeing, and perfectionism. The key to recovery is to change both the behaviors and attitudes that have initiated and now sustain that allostatic adjustments including reduced GnRH drive. Generally, a mix of multiple, seemingly minor, psychogenic and metabolic stressors appears to be more deleterious to reproductive function than a solitary stressor.33 Our studies suggest that it may take a prolonged duration of energy balance and improved psychological equilibrium for the hypothalamic allostasis to reverse and for ovulatory function to return.4,5

Nutritional and Metabolic Influences Nutritional and metabolic signals play critical roles in the elaboration of homeostatic and allostatic responses to ongoing challenges and can influence the reproductive system at many levels. Few studies have characterized the role

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of nutritional signals in the modulation of reproductive function in men, but available evidence suggests that undernutrition is as deleterious to reproductive competency in men as it is in women. Metabolic imbalance occurs when energy expenditure exceeds energy intake (negative energy balance). The brain is the most metabolically active tissue in the body, requiring 16 times more energy per unit mass than muscle tissue. Because humans have much bigger brains relative to body size than do other primates or other species, they utilize more of their daily energy intake than any other species to supply their brain; humans are estimated to use 25%, monkeys 8%, and rodents 5%. Indeed, as a species, humans may be uniquely sensitive to energy imbalance. Chronic energy deficiency alters thyroidal function to slow metabolism and correct negative energy balance. Food intake is influenced by availability, emotional state, social cues, and learned behaviors. Peripheral signals convey information about energy stores and immediate energy availability to the hypothalamus and the associated neural cascade that modulates hypothalamic function; leptin and insulin, which are actively transported across the bloodebrain barrier, potentiate satiety signals. Thus the level of any one signal per se may be less important than the action of that hormone, and action is likely to be gated by the constellation of other metabolic signals. Putative orexigenic signals include ghrelin, neuropeptide-Y (NPY), orexins A and B, melanin-concentrating hormone, and agouti-related peptide. Putative anorectic and satiety signals include (but are not limited to) cortisol, CRH, insulin, glucose, resistin, leptin, POMC, CART (cocaine- and amphetamineregulated transcript) peptide, PYY (peptide YY), and glucagon-like peptide 1. Adipokines (adipocyte-derived hormones) implicated in energy regulation include leptin, adiponectin, and resistin. Leptin is the dominant long-term energy signal informing the brain of adipose energy reserves; it also functions as a satiety signal. Ghrelin, a 28 amino acid acylated hormone that is also a growth hormone secretagogue, is produced by the gastrointestinal tract, especially the fundus of the stomach. Plasma ghrelin levels rise during

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fasting and immediately before anticipated mealtime and then fall within an hour of food intake, suggesting that ghrelin is important for meal initiation. Women with anorexia nervosa and exercise amenorrhea showed higher ghrelin levels than control women.34 Miljec et al.35 reported “ghrelin insensitivity” in response to an infusion of ghrelin in women with anorexia nervosa compared with EW, but appetite was measured only by self-report and not by food intake. Leptin levels rose in women with FHA treated with CBT concomitant with reduced cortisol levels. In summary, a plethora of metabolic signals reflect and convey the acute and chronic nutritional state of a given individual and thereby engender homeostatic and allostatic responses. Given the energetic demands of reproductive processes, however, undernutrition has the potential to induce more significant reproductive compromise than overnutrition.

SYNERGISM AMONG STRESSORS Stressors are often viewed as categorical, that is, either psychosocial (cognitive and emotional) or energetic (metabolic). In that the definition of a stressor is an experience that increases cortisol and other concomitants, it may be a false dichotomy to view stressors as categorical.28 Additionally, experiences have valences, and not all individuals have the same fitness or habitus, thus what is stressful to one may be more or less so to another. For instance, neuroendocrine and metabolic responses to acute exercise were greater in men whose HPA axis did not suppress in response to dexamethasone before the exercise challenge.36 Conversely, lactating women were hyporesponsive to exercise challenge.37 We recently showed that a graded exercise challenge elicited a greater cortisol response in women with SIA/FHA than those with ovulatory eumenorrhea.3 Furthermore, glucose responses diverged with women with SIA/FHA showing a 10% decrease in glucose while EW had a modest 3% increase in glucose, presumably to cope with the energetic demand of exercise. Interestingly, these two groups did not differ at

baseline with regard to cortisol or glucose levels. The decrement in glucose seen in SIA/FHA but not EW suggests latent metabolic compromise and indicates that SIA/FHA are unable to meet energetic demands of ongoing activities presumably due to chronic mild undernutrition or overexertion. Taken together, these data indicate that prior HPA activation potentiates the neuroendocrine and metabolic responses to subsequent challenge and buttress the notion that responses are gated not only by the stressor type but also by host factors. Women who developed SIA/FHA unrelated to weight loss, exercise, and definable psychiatric disorders held negative attributions about recent life events and displayed more unrealistic expectations of self and others than eumenorrheic, ovulatory women.29 Men who did not habituate when exposed to repeated psychogenic challenge viewed themselves as less attractive, had lower self-esteem, and reported being in a depressed mood more often. Apparently, unachievable ambitions or other cognitions sensitize individuals to life’s inevitable challenges and likely heighten responsivity to metabolic stressors such as exercise or food restriction. In our monkey model, the converse was true and energetic imbalance augmented reactivity to psychogenic stressors.33 Loucks and Thurma38 applied a graded energy restriction for 5 days to EW in the follicular phase and measured cortisol, glucose, and GnRH-LH drive. An energy deficit of 33% had no impact on LH pulse frequency, whereas an energy deficit of about 75% induced a decline in LH pulse frequency of about 40%. The induction of an energy deficit resulted in a graded increase in cortisol levels. At an energy availability of 10 kcal/kg of LBM (75% deficit), cortisol was increased by about 30%, which is the amount of increase typically seen in women with SIA/ FHA. Much like stress-sensitive monkeys, women whose luteal phase progesterone levels were lowest at the initiation of the energy restriction showed the greatest response to the imposed metabolic challenge. Most women with SIA/FHA, when carefully evaluated, display more than one trait, state, or behavior capable of activating stress response cascades or inducing a mild metabolic deficit, but most

TREATMENT CONSIDERATIONS

do not have a profound metabolic deficit that alone would explain the reduction in central GnRH drive. Because of the synergism between metabolic and psychogenic stressors, a combination of multiple, small magnitude, mixed stressors may be potentially more disruptive of reproductive function than a single large stressor limited to one category.

TREATMENT CONSIDERATIONS SIA/FHA is difficult to recognize clinically in the absence of altered menstrual cyclicity. Before rendering a diagnosis of stress as the cause, it is important to exclude all other causes and contributors including low ovarian reserve (low oocyte count). Infertility is a common clinical presentation of SIA/FHA. In the absence of other causes, the more clinically evident the ovarian compromise, the greater is the neuroendocrine allostasis and energetic imbalance. The Endocrine Society recently published guidance on the treatment of women with FHA.6 If a woman with functional hypothalamic hypogonadism is seeking to become pregnant, ovulation induction can be initiated with exogenous administration of pulsatile GnRH therapy or gonadotropins. Administration of exogenous GnRH therapy is advantageous insofar as it minimizes the risk of ovarian hyperstimulation and multiple gestation associated with gonadotropins. Clomiphene citrate is an option, but it may be ineffective because of its hypothalamic site of action and the altered feedback sensitivity in SIA/FHA. Ovulation induction, particularly in women with a low body mass index, may increase risk for premature labor and intrauterine growth restriction. The parenting skills of women with SIA/FHA may be impaired because they are already overwhelmed and stressed prior to pregnancy and delivery, and their psychological precariousness may place their children at risk for poor psychosocial development. Furthermore, a recent study showed that children born to mothers with clinically occult hypothyroidism due to autoimmune thyroiditis had a mean fullscale intelligence quotient that was seven points lower than the control population.39 Women

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with clinically silent hypothyroidism had a 30% reduction in thyroxine, which is similar to what is observed in women with SIA/FHA.2 Of critical importance is the fact that maternal thyroxine is the only source of fetal thyroxine in the first trimester, and the predominant fetal source in the second and third trimesters. Because the fetal brain requires an appropriate amount of thyroxine for neurogenesis, even small deficits in thyroxine may compromise fetal neurodevelopment. Increased maternal cortisol may also have independent and/or synergistic effects upon fetal neurodevelopment and organogenesis. Recent evidence showed that severe stress such as that associated with the unexpected death of a child increased the risk of congenital anomalies of the cranial neural crest eightfold.40 A popular approach to a woman with SIA/ FHA who is not seeking immediately to become pregnant is to offer her hormone replacement. This approach assumes that sex steroid deprivation is the primary therapeutic issue. This approach has inherent limitations. First, exogenous sex steroid exposure does not fully promote bone accretion or cardioprotection in the presence of ongoing metabolic derangements.6 Second, ongoing insults to the brain from chronic amplification of stress cascades go unchecked. Furthermore, estrogen therapy does not correct hypothalamic hypothyroidism. In short, hormone therapy may mask potentially deleterious processes that are unlikely to be ameliorated by hormone exposure alone. It is critical to remember that SIA/FHA is more than an isolated disorder of reduced GnRH secretion. Hormone therapy per se is unlikely to be harmful, but more than hormone administration is needed. The optimal intervention is to ameliorate the stress process and thereby facilitate neuroendocrine and metabolic recovery. An integral goal of the treatment plan for women with SIA/FHA is to help them identify and address stressor and provide emotional support while coping mechanisms other than dieting or exercising are learned. Nonpharmacologic interventions such as stress management, relaxation training, or psychoeducation empower

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FIGURE 18.4 Serum estradiol and progesterone levels and menstrual bleeding in a woman with SIA/FHA who was observed and did not recover (top panel) compared with a woman with SIA/FHA who was treated with cognitive behavioral therapy (CBT) and recovered (lower panel). Adapted from Berga SL, Marcus MD, Loucks TL, Hlastala MS, Ringham R, Krohn MJ. Recovery of ovarian activity in women with functional hypothalamic amenorrhea who were treated with cognitive behavior therapy. Fertil Steril 2003;80:976e981.

REFERENCES

individuals by fostering self-care and competency. In this regard, nonpharmacologic therapies have the potential to produce long-term benefits upon psychological and physical health. Given these considerations, we recently studied whether CBT aimed at ameliorating problematic attitudes and behaviors would permit ovarian recovery in normal-weight women with SIA/FHA.4,5 Women with SIA/FHA were randomized to observation versus CBT. CBT consisted of 16 visits over 20 weeks with a team comprised of a physician, therapist, and nutritionist. The two groups were followed for return of menses for up to 8 weeks following the intervention. Estradiol and progesterone levels were monitored at weekly intervals for 4 weeks before and after observation versus CBT. About 88% of those who underwent CBT had return of ovarian function, whereas only 25% of those in the observation arm did.4 Fig. 18.4 illustrates the recovery of sex steroid secretion in a woman treated with CBT who showed ovarian recovery and in a woman randomized to observation who did not. Interestingly, ovarian recovery was not associated with significant weight gain. This does not mean that subjects did not alter food intake or energy expenditure. Improved nutrition generally restores the thyroidal axis, thereby leading to an increased basal metabolic rate. We recently compared metabolic variables in women who did and those who did not recover from SIA/ FHA after CBT intervention and observed that ovarian recovery was associated with a decline in cortisol and an increase in TSH and leptin.5 Ultimately, behavioral and psychological interventions that address problematic behaviors and attitudes permit resumption of ovarian function along with recovery of the LHPA and HPT axes. In short, full endocrine recovery offers better individual, maternal, and child health.

Acknowledgments Some of the research presented in this chapter was funded by grants from the National Institutes of Health (R01MH50748 to SLB and RR-00046 to the General Clinical Research Center at the University of Pittsburgh).

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transporter gene modify the consequences of social status on metabolic health in female rhesus monkeys. Physiol Behav. 2008;93:807e819. Judd SJ, Wong J, Saloniklis S, et al. The effect of alprazolam on serum cortisol and luteinizing hormone pulsatility in normal women and in women with stress-related anovulation. J Clin Endocrinol Metab. 1995;80:818e823. Berga SL. Stress and reproduction: a tale of false dichotomy? Endocrinology. 2008;149:867e868. Giles DE, Berga SL. Cognitive and psychiatric correlates of functional hypothalamic amenorrhea: a controlled comparison. Fertil Steril. 1993;60:486e492. Marcus MD, Loucks TL, Berga SL. Psychological correlates of functional hypothalamic amenorrhea. Fertil Steril. 2001;76:310e316. Berga SL, Girton LG. The psychoneuroendocrinology of functional hypothalamic amenorrhea. Psychiatr Clin North Am. 1989;12:105e116. Berga SL. Behaviorally induced reproductive compromise in women and men. Semin Reprod Endocrinol. 1997;15:47e53. Williams NI, Berga SL, Cameron JL. Synergism between psychosocial and metabolic stressors: impact on reproductive function in cynomolgus monkeys. Am J Physiol Endocrinol Metab. 2007;293:E270eE276. Tanaka M, Naruo T, Yasuhara D, et al. Fasting plasma ghrelin levels in subtypes of anorexia nervosa. Psychoneuroendocrinology. 2003;28:829e835. Miljic D, Pekic S, Djurovic M, et al. Ghrelin has partial or no effect on appetite, growth hormone, prolactin, and cortisol release in patients with anorexia nervosa. J Clin Endocrinol Metab. 2006;91:1491e1495. Petrides JS, Mueller GP, Kalogeras KT, Chrousos GP, Gold PW, Deuster PA. Exercise induced activation of the hypothalamic-pituitary-adrenal axis: marked differences in the sensitivity to glucocorticoid suppression. J Clin Endocrinol Metab. 1994;79:377e383. Altemus M, Deuster PA, Galliven E, Carter CS, Gold PW. Suppression of hypothalamic pituitaryadrenal axis responses to stress in lactating women. J Clin Endocrinol Metab. 1995;80:2954e2959. Loucks AB, Thurma JR. Luteinizing hormone pulsatility is disrupted at a threshold of energy availability in regularly menstruating women. J Clin Endocrinol Metab. 2003;88:297e311. Haddow JE, Palomaki GE, Allan WC, et al. Maternal thyroid deficiency during pregnancy and subsequent neuropsychological development of the child. New Engl J Med. 1999;341:549e555. Hansen D, Lou HC, Olsen J. Serious life events and congenital malformations: a national study with complete follow-up. Lancet. 2000;356:875e880.

C H A P T E R

19 Multidrug Resistance P-Glycoprotein (P-gb), Glucocorticoids, and the Stress Response Enrrico Bloise1, Stephen G. Matthews2

1

Department of Morphology, Federal University of Minas Gerais, Belo Horizonte, Brazil; 2Department of Physiology, Obstetrics & Gynaecology and Medicine, University of Toronto, Toronto, ON, Canada O U T L I N E Introduction

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P-glycoprotein: An Overview P-gp Substrates

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P-gp Localization and Regulation BloodeBrain Barrier Other Brain Regions Pituitary and Adrenal Gland Glucocorticoid Excretion

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Stress-Related Substrates and HPA Function 234

INTRODUCTION The steroid properties of endogenous (i.e., physiologic) and synthetic (i.e., therapeutic) glucocorticoids allow for a widespread biodistribution throughout the body. While naturally occurring glucocorticoids impact nearly every aspect of the mammalian physiology, synthetic glucocorticoids (sGC) aid in the treatment of a number of conditions, including

Stress: Physiology, Biochemistry, and Pathology https://doi.org/10.1016/B978-0-12-813146-6.00019-9

P-gp and Development Placenta Fetal Blood-Brain Barrier

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Concluding Remarks

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Disclosure

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Acknowledgments

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References

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those involving inflammation, immunological disorders, adrenal insufficiency, and developmental disorders such as congenital adrenal hyperplasia and preterm labor (PTL). Actions of distinct glucocorticoids are dependent upon their intracellular availability, glucocorticoid versus mineralocorticoid biological potency, glucocorticoid receptor (GR), and mineralocorticoid receptor (MR) density and affinity, as well as dosage and duration of action (i.e., biological

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Copyright © 2019 Elsevier Inc. All rights reserved.

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stability). Importantly, intracellular glucocorticoid availability is controlled by interconversion of cortisol and cortisone (corticosterone and 11dehydrocorticosterone in rodents) by specific intracellular enzymes and transmembrane efflux transporters.

KEY POINTS • P-glycoprotein (P-gp) is produced in many cells including those that form tissue barriers (bloodebrain barrier, bloode testes barrier) and those with important transport functions (hepatocytes, gastrointestinal epithelial cells). • P-gp can transport a very broad spectrum of endogenous and exogenous substrates, including synthetic and endogenous glucocorticoids (GCs), cytokines, drugs, and environmental toxins • P-gp modulates transfer of GCs into the brain and at other sites regulates release, metabolism, and excretion of GCs; profoundly influencing hypothalamice pituitaryeadrenal function. • P-gp plays an important role in maintaining low levels of GC in the fetus through actions at the placenta and in the developing bloodebrain barrier. • GC, inflammation, and therapeutic drugs modulate P-gp activity in different tissues, leading to altered transport of stressrelated factors and modified responsiveness to stress.

The intracellular 11b-hydroxysteroid dehydrogenase (11b-HSD) isozymes 1 and 2 interconvert cortisol and cortisone.1 Expression of 11b-HSD1 and 11b-HSD2 is highly tissue-specific and is developmentally regulated. 11b-HSD1 has reductase activity and reactivates inactive cortisone into active cortisol, whereas 11b-HSD2 displays dehydrogenase activity and converts cortisol into cortisone. Specificity and affinity of these enzymes are major determinants of

intracellular glucocorticoid levels, and thus GR and MR activation.1,2 However, emerging evidence, primarily extracted from studies investigating transfer of glucocorticoids in biological barriers,3 have demonstrated another important mechanism capable of limiting (or regulating) intracellular glucocorticoid levels. This involves cell membrane activity of the multidrug resistance transporter P-glycoprotein (P-gp; Fig. 19.1), a member of the ABC efflux transporter superfamily.4 These transmembrane active transporters hydrolyze Adenosine triphosphate (ATP) to translocate a number of substrates across cellular membranes.2 In this chapter, we review and discuss the most important biological aspects of P-gp expression and function in the context of the hypothalamice pituitaryeadrenal axis (HPA) axis and local regulation of glucocorticoid levels during development and in adulthood, in both physiological and some pathological conditions.

P-GLYCOPROTEIN: AN OVERVIEW P-gp was discovered as a surface glycoprotein on a drug-resistant Chinese hamster ovary cell line over 40 years ago. This represented a turning point in the understanding of acquired resistance to chemotherapeutic agents. P-gp was then identified in various cancers, with particularly high levels in cells with high resistance to antineoplastic drugs.5 Subsequent studies identified that P-gp was also produced and functional in many normal cells including those that form tissue barriers (i.e., bloodebrain barrier [BBB], bloodetestes barrier) and those with important transport functions (liver hepatocytes, mammary, and gastrointestinal epithelial cells). At a cellular level, it is now well-established that Pgp limits the accumulation of its substrates within the cell but also facilitates the transport of specific substrates produced by a cell and waste metabolites, out of the cell. With respect to its role at barrier sites, P-gp acts to reduce/ prevent substrate transfer, thus conferring protection. Dysregulation of P-gp can lead to altered tissue sensitivity, organ protection, and ultimately toxicity by noxious substrates. P-gp has

P-GLYCOPROTEIN: AN OVERVIEW

229

FIGURE 19.1 Control of intracellular availability of glucocorticoids (GC): GCs enter the cell by passive diffusion. Cortisol may then be inactivated into cortisone by the 11b-HSD2 enzyme or be mutually extruded into the extracellular space with other GC, through the actions of the transmembrane transporter P-glycoprotein (P-gp). Both mechanisms decrease intracellular levels of active GC and their availability to bind glucocorticoid receptors (GR) and mineralocorticoid receptors (MR). Once GC bind the GR and MR, complexes transfer into the nucleus to control target gene transcription.

a very broad spectrum of substrates that include steroid hormones such as endogenous and sGC. Thus, P-gp plays an important role in regulating glucocorticoid biodisposition and diverse biological responses in different stress-related tissuesda focus of this chapter. In the human and 58 other species, P-gp is encoded by a single gene, ABCB1. In contrast, in the rat, mouse, and hamster, two genes, Abcb1a and Abcb1b, encode distinct P-gp isoforms.6 P-gp is a 170 kDa glycoprotein, primarily localized on the surface of cell membranes. This particular cellular localization determines the efflux direction of substrates, that is, from within the cell to the extracellular space. Moreover, in most cases, P-gp is localized at the apical membrane of tissue barriers. Structurally, P-gp is formed by two transmembrane domains (TMDs) and two cytosolic nucleotide-binding domains (NBDs). When ATP binds to NBDs, it induces conformational changes in NBD and

TMD structure and triggers a transport cycle. P-gp captures substrates as they enter into the inner leaflet of the lipid bilayer of cell membranes. P-gp then extrudes the “captured” substrates back to the outer cell space, preventing their accumulation inside cells.4 In the following section, we will describe the most important physiological and pharmacological substrates of P-gp, with focus on those related to stress.

P-gp Substrates P-gp can transport a very broad spectrum of endogenous and exogenous substrates including hydrophilic and hydrophobic compounds. This is likely attributable to the numerous binding sites specific for different substrate classes, as well as a large flexible binding pocket present in its TMDs, capable of binding diverse compounds.7,8 As a result, P-gp is involved in the absorbance, distribution, metabolism, and/or

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excretion of over 300 pharmacological substrates.7 Endogenous P-gp substrates that are relevant to stress include hormones: glucocorticoids, aldosterone, estriol, estrone, pregnenolone, 17-hydroxyprogesterone, and testosterone; antiand pro-inflammatory cytokines: interleukins (IL)4, IL-1b, IL-2, IL-6, interferon (IFN)-g, and tumor necrosis factor (TNF)-a; chemokines: chemokine CeC motif ligand 2; and nutrients: folate. P-gp also extrudes a very broad spectrum of xenobiotics, including environmental toxins (pesticides and herbicides) and many classes of commonly administered drugs such as sGC, antibiotics, antiretrovirals, and chemotherapeutic agents.9 The C6 alpha, C11, and C16 positions in P-gp structure are key to its affinity for, and transport of, glucocorticoids. 10 A range of glucocorticoids can be transported by P-gp (Table 19.1). However, P-gp transepithelial transport efficiency varies for different glucocorticoids with P-gp exhibiting greater transport efficiency for sGC (methylprednisolone > prednisolone > betamethasone > dexamethasone/prednisone) over cortisol, which in turn, exhibits a greater transport efficiency over cortisone.10 Other P-gp substrates related to stress include antidepressant drugs, which act by increasing central GR messenger RNA (mRNA) and protein levels, as well as GR function.11,12 Antidepressant drugs that are P-gp substrates include the specific selective serotonin reuptake inhibitors (SSRIs) and tricyclic antidepressants13 (Table 19.1). Thus, P-gpemodulated brain entry of these and other compounds may impact HPA function under normal and pathological conditions.

P-GP LOCALIZATION AND REGULATION BloodeBrain Barrier P-gp is highly expressed at the BBB. It is localized at the luminal (apical) and abluminal membranes of brain endothelial cells (BECs) and in the plasma membranes of adjacent pericytes and astrocytes, suggesting that P-gpemediated efflux takes place in different cellular components of the BBB (Fig. 19.2). Of note, subcellular localization of P-gp has also been described in BECs, that is, alongside the nuclear envelope, in caveolae, cytoplasmic vesicles, Golgi complex, and rough endoplasmic reticulum; however, there has been little consideration of the potential role of P-gp at these sites. Astrocytes and pericytes are also key components of the BBB. Astrocytes extend foot processes that ensheathe the outer surface of brain microvessels. Coculture of astrocytes with BECs increases P-gp transport efficiency and decreases paracellular transport in the brain endothelium. P-gp has also been localized to the plasma membrane of astrocytes and at astrocyte foot processesdin close contact to the abluminal membrane of BECs. It is likely that P-gp mediates transport of trophic factors from astrocytes to maintain BBB integrity. Similarly, pericytes are critical for maintaining endothelial cell integrity at the BBB. P-gp expression has been detected in the plasma membrane of pericytes,14 but its functional relevance to pericyte biology and to the integrity of the BBB is not known. Localization of P-gp at the luminal and abluminal membranes of BECs denotes that the efflux direction of its substrates occurs from

TABLE 19.1 Examples of P-gp Substrates Related to Stress and HPA Function Nature

P-gp Substrate

Reference

Endogenous glucocorticoids

Cortisol, corticosterone, cortisone, deoxycorticosterone

44,83,84

Synthetic glucocorticoids

Beclomethasone, betamethasone, budesonide, dexamethasone, hydrocortisone, methylprednisolone, prednisone, prednisolone

84e86

Antidepressants

Amitriptyline, citalopram, doxepin, escitalopram, fluvoxamine, imipramine, levomilnacipran, nortriptyline, paroxetine, trimipramine, vilazodone, venlafaxine

13,87

P-GP LOCALIZATION AND REGULATION

231

FIGURE 19.2 P-glycoprotein (P-gp) localization at the blood brain barrier: (A) P-gp is localized at the luminal and abluminal membranes of brain endothelial cells, pericytes, and astrocyte foot processes. P-gp limits the transfer of glucocorticoids (GC) and other substrates into the central nervous system. Localization of P-gp in astrocyte foot processes and pericytes suggests that P-gpemediated efflux takes place in different cellular components of the BBB.

within the BECs toward the peripheral blood and is consistent with the notion that P-gp acts as an important protective component of the BBB, limiting entry of its substrates into the brain. Schinkel and co-workers in 1995 provided the first evidence that P-gp regulates the entry of many drugs, including glucocorticoid, into the brain. Abcb1a (Mdr1a) knockout (KO) mice injected with [3H]-dexamethasone ([3H]-DEX) exhibited a 2.5-fold increase of levels in the brain, while exhibiting similar [3H]-DEX levels in the peripheral circulation, compared with wildtype animals.15 Subsequently, it was elegantly demonstrated that bi-laterally adrenalectomized Abcb1a KO mice, injected with [3H]-DEX or [3H]prednisolone, exhibited a fivefold and threefold increase in radioactivity levels in cerebellum homogenates, respectively.16,17 Moreover, using autoradiography, these authors demonstrated a 10-fold increase in [3H]-DEX-GR binding in the hippocampus and the hypothalamic paraventricular nuclei (PVN) compared with wild-type animals.17 Similarly, brain uptake of systemically administered [3H]-DEX, [3H]cortisol, and [3H] corticosterone was increased in Abcb1a and Abcb1b (Abcb1a/1b) double KO mice.18 Together, these studies conclusively demonstrated that Pgp plays an important role in regulating BBB transfer of glucocorticoids into the brain. However, it is important to note that one study using the Abcb1a KO mouse demonstrated that, while peripherally administered, [3H]cortisol was

excluded from the brain by P-gp, encoded by Abcb1a in the BBB, [3H]corticosterone was not.19 The reasons for the disparity between studies are not clear. Since intracellular availability and barrier transfer of steroid hormones are controlled by P-gp, it is perhaps not surprising that glucocorticoids regulate P-gp expression and function at the BBB. Primary cultures of adult rat BECs exposed to DEX exhibited a GR-dependent increase in P-gp function and elevated expression of P-gp protein and Abcb1a/1b mRNA levels compared with controls.20 DEX treatment of BECs isolated from adult male rats exhibited increased P-gp protein expression and activity,21,22 and this was associated with a concomitant decreased quinidine accumulation in the brain (a well-described P-gp substrate).23 These data provide direct biological evidence that glucocorticoids increase P-gp expression and activity in the adult BBB. A number of pro- and anti-inflammatory cytokines and chemokines are selectively transported by P-gp. In addition, some of these cytokines have been shown to regulate P-gp activity. In vivo and in vitro experiments have demonstrated that exposure to TNF-a or IFN-g can inhibit P-gp activity in BBB of rodents and human BECs.24-26 Furthermore, lipopolysaccharide (LPS), which mimics gram-negative bacterial infection and acts through toll-like receptor 4, rapidly inhibited P-gp activity in brain

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capillaries isolated from adult rats.25 A similar reduction in P-gp activity was reported when LPS was injected (i.p.) in adult mice, in vivo; and this persisted 18e36 h following LPS insult.27 This effect was mediated via nuclear factor kB (NF-kB), a transcription factor that regulates cellular inflammatory responses to infection.28 Inhibition of P-gp activity at the BBB, as a result of cytokine exposure or infection, will increase the transfer of P-gp substrates into the brain. As such, proinflammatory states and infection have the potential to result in an increased transfer of glucocorticoids and other stress-related substrates into the brain, leading to a change in HPA function. Further studies are required to investigate this relationship. Major depressive disorder (MDD) has been associated with dysregulation of the HPA axis, including increased cortisol concentrations and impaired glucocorticoid negative feedback, through blunted central GR sensitivity. In addition, an increasing number of studies have reported elevated cytokine levels (i.e., IL-1b, TNF-a, and IL-6) and some ABCB1 genetic variants in MDD.13,29 Thus, altered P-gp activity in the BBB of MDD patients may result from an interplay of glucocorticoids, proinflammatory cytokines, and ABCB1 genetic variants. It is important to note that a number of commonly prescribed antidepressants are P-gp substrates (see Table 19.1) that can themselves modulate P-gp activity. The SSRI, sertraline, can potently inhibit P-gp function in BECs isolated from adult male guinea pigs. A timedependent in vivo effect of sertraline, increasing brain levels of the P-gp substrate [3H] digoxin has also been reported. However, the same effect was not evident following fluoxetine treatment (another SSRI).30 In contrast, in rats, venlafaxine decreased transport of [11C]verapamil (a P-gp substrate) into the brain.31 Furthermore, a combination of different antidepressants (see the study by de Klerk et al.32) in patients with MDD was associated with decreased penetration of [11C] verapamil in the brain, effects that were more evident in frontal and temporal brain regions.32 Together, these studies suggest that specific antidepressant drugs may differentially affect P-gp activity at the BBB. Further studies investigating

the individual or combined effects of clinically prescribed antidepressants on P-gp activity at the BBB are warranted and should clarify how and which drugs normalize HPA axis through modification of P-gp function at the BBB. The pathogenesis of Alzheimer’s disease involves excessive accumulation of the amyloid b-peptide in the brain, through different mechanisms, including decreased clearance of amyloid b-peptide from the central nervous system. The amyloid b-peptide is a P-g substrate33 and is increased in the brain of adult Abcb1a/1b KO mice compared with controls.34 Moreover, pharmacological induction of P-gp in the BBB resulted in decreased accumulation of amyloid b-peptides in the adult mouse brain parenchyma.35 In humans, P-gp expression in the brain vasculature was inversely correlated with amyloid b40 and amyloid b42 deposition in the medial temporal lobe of elderly nondemented individuals. 36 As such, it has been proposed that P-gp in the BBB plays a role in Alzheimer’s disease progression. It is yet to be determined how dysfunction of P-gp in the BBB, resulting from chronic or acute stress, depression, ABCB1 genetic variants, and or infection/inflammation during the lifespan, could increase the risk of Alzheimer’s disease. This possibility clearly warrants further investigation.

Other Brain Regions In addition to P-gp expression in microvessels (BECs, pericytes, and astrocytes), P-gp is present in other brain structures, including fenestrated capillaries in the circumventricular organs (CVOs), epithelial cells of the choroid plexus,14 ependymal cells lining the ventricles, and in neurons. CVOs are considered “leaky regions” that enable transfer of specific factors from the peripheral blood to the brain and contribute to maintaining thermal, fluid, and energy homeostasis, as well as regulating neuronal immune/ inflammatory processes.37,38 P-gp has been identified in a number of the CVOs including the subfornical organ, neurohypophysis, area postrema, and pineal organ.14 The role of P-gp in CVOs is not known, and whether P-gp impacts

P-GP LOCALIZATION AND REGULATION

glucocorticoid transfer at these sites remains to be determined. P-gp has been detected at the apical membrane of the epithelial cells of the choroid plexus where it transports brain-derived substrates into the Cerebrospinal fluid (CSF).39 However, P-gp immunostaining was detected in less than 5% of the adult human choroid plexus.14 In situ brain/choroid plexus perfusion analysis did not find evidence that P-gp regulates [3H]cortisol and [3H]corticosterone transfer in the rodent choroid plexus.40 Thus, a potential role for P-gp at this site, in regulating HPA function, is unlikely. Ependymal cells lining the third and the lateral ventricles express P-gp in the adult human brain.14 However, function of P-gp in this site has yet to be determined. P-gp has been shown to be expressed in subpopulations of neurons in the cingulate cortex, hippocampus (especially interneurons), the habenula, and the lateral geniculate of the human brain. P-gp has also been identified in the hypothalamic supraoptic, PVN, and suprachiasmatic nuclei, as well as the substantia nigra and the locus coeruleus.14 In the cerebellum, P-gp is present in Purkinje cells, Golgi 1 and Golgi 2 cells, and in a few granular cells.14 P-gp function in neurons is not well understood; however, it has been postulated that P-gp is involved in the purinergic neuromodulation of the medial habenula.14 Since P-gp can transport a myriad of factors that have the potential to modulate neuronal survival, growth, and neurotransmission, further studies of P-gp function at these sites are warranted. Given the role of the hypothalamic and midbrain sites (where neuronal P-gp has been reported) in regulation of HPA function and stress responses, it is quite possible that P-gp at these sites can influence stress biology.

Pituitary and Adrenal Gland P-gp has been identified in the human pituitary, localized to capillaries, pituicytes, and anterior pituitary cells. 14 However, localization of P-gp to specific subpopulations of anterior pituitary cells has not been investigated. Studies have shown P-gp expression and/or activity in

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prolactin-releasing and growth hormonee releasing pituitary tumor cell lines.41-43 However, the role of P-gp in anterior pituitary cells on HPA function is less clear as pituitary uptake of [3H]-DEX, [3H]cortisol, and [3H]corticosterone was unaffected in Abcb1a and Abcb1a/1b KO mice compared with controls.17,18,44 P-gp is present at the cellular membrane of adrenocortical cells as well as in clusters of cells in the adrenal medulla.45,46 Studies have not been undertaken to localize P-gp to any particular region of the adrenal cortex. P-gp has been shown to be involved in the regulation of aldosterone and cortisol secretion in adrenal cell lines. 47,48 As such, P-gp may be involved in the adrenal corticosteroid release. It has been suggested that P-gp protects the adrenal cortex from exposure to xenobiotics and environmental toxicants.49 Another attractive hypothesis is that P-gp acts to protect adrenocortical and adrenomedullary cells from the very high levels of glucocorticoids present in the adrenal gland, although this remains to be tested. No studies have considered the potential role of P-gp in chromaffin cells. However, P-gp has been shown to modulate uptake of [3H]-adrenaline into hepatic cells,50 suggesting that P-gp may be involved in the biodistribution of catecholamines.

Glucocorticoid Excretion Metabolism and excretion represent important determinants of glucocorticoid action. Although different cells are capable of metabolizing glucocorticoids, the liver is the primary site of glucocorticoid degradation, whereas the kidney is the primary site of glucocorticoid metabolite excretion. Hepatic enzymatic conversion of glucocorticoids to less biologically active metabolites (sulfates and glucuronides), with increased water solubility, facilitates urinary excretion (w90% cortisol). The remainder is excreted by the biliary system, even though metabolized products are likely to be reabsorbed by the enteric system.51 P-gp is expressed at high levels on the biliary canalicular surface of hepatocytes and the apical surface of small biliary ductules,52 facing the

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liver excretory compartment. This hepatic localization suggests that P-gp extrudes its substrates from the liver toward the biliary tract, thus participating in the excretion of cortisol and possibly specific glucocorticoid metabolites into the intestines. Likewise, in the kidney, P-gp is abundantly expressed in the apical surface of proximal tubular epithelium, indicating an important excretory transport function into the glomerular filtrate.52 Due to its important role in extruding glucocorticoids, a potential role of liver P-gp is to deliver endogenously secreted cortisol to the enteric system through the bile, where cortisol acts to modulate the gut barrier functional characteristics and impact the gut microbiota.53,54 It remains unclear as to whether sulfate and glucuronide conjugates derived from cortisol metabolism are P-gp substrates. However, the water-soluble estradiol metabolite, estradiol-17b-glucuronide, is a P-gp substrate9 and is secreted into the urine, indicating a possible involvement of P-gp in extruding cortisol metabolites via the kidney and the hepatobiliary system.

STRESS-RELATED SUBSTRATES AND HPA FUNCTION Glucocorticoids tightly regulate HPA basal activity and responsiveness to stress. Stress activates the HPA axis resulting in the release of cortisol in humans and corticosterone in rodents. These glucocorticoids, which gain access to the brain, bind to GR in most brain regions and MR in the hippocampus and central amygdala to induce physiological and behavioral responses, through a rapid nongenomic phase and a second slow, genomic phase.55 Due to the important role that P-gp plays in limiting glucocorticoid transfer from the peripheral circulation to the brain, it has been suggested to be an important regulator of HPA function and the stress response. Indeed, Abcb1a/1b KO mice exhibited reduced adrenocorticotropic hormone (ACTH) levels under basal and stressful conditions, which were paralleled with decreased stress-induced corticosterone levels (compared with controls).56 This would be

consistent with increased corticosterone transfer into the brain resulting in increased glucocorticoid negative feedback and a blunted HPA axis response to stress. Other studies have demonstrated a reduction in HPA axis activation following stress combined with alterations of stress-coping behaviors in Abcb1a/1b KO mice and following pharmacological inhibition of P-gp. Reduced P-gp function prevented stress-induced increases in plasma and brain corticosterone levels, impaired behavioral response to novelty, and attenuated corticosterone-induced antianxiety effects.57,58 Most recently, a study in Abcb1a/1b KO mice identified an increase in microglia density in the CA3 region of the hippocampus, associated with altered behavioral responses to stress, suggesting that the role of P-gp might not be limited to the BBB. Interestingly, baseline concentrations of corticosterone were reduced in the brains of adult Abcb1a/1b KO mice.58 This paradoxical decrease has been attributed to increased life-long central glucocorticoid negative feedback activity in these animals, characterized by reduced corticotropin-releasing hormone mRNA levels in the PVN, reduced plasma ACTH levels, and by an overall increase of GR mRNA levels in the brain. Decreased HPA tone was likely elicited by the excessive entry of corticosterone into the brain.56,58 As such, the adult phenotype probably represents a life-long adaptation to the chronic overload of corticosterone in the brain caused by the lack of P-gp at the BBB. The HPA axis integrates complex regulatory responses of the neuroendocrine, metabolic, and immunological systems to restore homeostasis following stress exposure. Infective and or inflammatory challenges are common and lead to the release of proinflammatory cytokines including TNF-a, IL-1, IL-6, and IFN-a (innate response) and IL-2 and IFN-g (adaptive response). These cytokines stimulate the HPA axis, and glucocorticoids exhibit potent antiinflammatory actions to suppress further release of cytokines and prevent immune hyperactivation.59 Given that P-gp can transport both glucocorticoids and selective cytokines, together with the fact that glucocorticoids increase, while

P-GP AND DEVELOPMENT

cytokines inhibit P-gp activity in BECs, suggests that P-gp may play a central role in modulating the interplay of stress and immune function. Clearly, further work is required to further investigate this complex relationship.

P-GP AND DEVELOPMENT Development of P-gp expression and function has been extensively characterized in the placenta and the developing BBB by our group and others. P-gp is expressed in the placenta with the highest levels at the apical surface of the microvillus membrane of the syncytiotrophoblast. This pattern of placental localization implicates P-gp as an important efflux component of the syncytiotrophoblast barrier and is consistent with its role in fetal protection, preventing excessive transfer of maternal glucocorticoids and other steroids,

FIGURE 19.3

235

cytokines, xenobiotics, and environmental toxins into the fetal circulation.9

Placenta Placental P-gp localization is illustrated in Fig. 19.3. Placental levels of ABCB1 mRNA and P-gp protein change as a function of gestational age in a number of species. In the human placenta, ABCB1 mRNA and P-gp protein are highest in the first trimester and lowest toward the end of the third trimester,9,60,61 suggesting that placental P-gpemediated fetal protection decreases in late gestation. In this regard, in vivo transport studies in the mouse have shown that fetal accumulation of [3H]digoxin (a P-gp substrate) increases dramatically in late gestation, coincident with the decrease in placental P-gp.62 In the late gestation fetus, there are rapid increases in the expression of ABCB1 mRNA and

Placental P-glycoprotein (P-gp) localization: (A) P-gp is localized at the brusheborder apical membrane of the syncytiotrophoblast, where it acts to efflux glucocorticoids, steroid hormones, cytokines, xenobiotics, and environmental toxins back into the maternal blood in the placental intervillous space. This decreases transfer from the maternal circulation to the fetus. P-gp is also expressed in the endothelial cells of fetal capillaries, where its activity has been recently described in isolated human placental microvascular endothelial cells; but its efflux direction is yet to be determined.88 (B) P-gp immunostaining in the brusheborder apical membrane of the syncytiotrophoblast in the first trimester human placenta and negative control (IgG1). CT, cytotrophoblast; P-gp, P-glycoprotein; ST, syncitiotrophoblast.

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19. MULTIDRUG RESISTANCE P-GLYCOPROTEIN (P-GB), GLUCOCORTICOIDS, AND THE STRESS RESPONSE

P-gp protein in the BBB and at other barrier sites including the oral and gastrointestinal epithelium and in skin (personal observation).63 These developmental changes presumably relate to the fetal to neonatal transition (i.e., loss of the protective placenta).2,64 The developing fetus is extremely sensitive to glucocorticoids, which if present in excess, can decrease proliferation and reduce fetal growth. As such, there is a 10- to 20-fold gradient in glucocorticoid concentrations between mother and fetus. A series of landmark studies from Seckl and others have clearly demonstrated an important role of placental 11b-HSD2 in protecting the fetus from the high levels of glucocorticoids in the maternal circulation. In the human, 11b-HSD2 converts cortisol into inactive cortisone, whereas in rats, corticosterone is converted to 11-dehydrocorticosterone, thus decreasing transfer of endogenous active glucocorticoids into the fetus.1 In humans, placental 11b-HSD2 levels increase as gestation proceeds,65 but decrease between the 38th and 40th week of pregnancy.66 The decrease in placental 11b-HSD2 activity in late gestation allows increased transfer of maternal cortisol to the fetus facilitating a surge of fetal cortisol,66 which is essential for the maturation of several fetal organs including the lungs, brain, and kidney.2 The placental profiles of P-gp and 11b-HSD2 across gestation in several species, including humans, would indicate that active glucocorticoid transport (P-gp) and metabolism (11b-HSD2) are important in protecting the fetus from the high levels of glucocorticoid present in the maternal circulation. At term, both placental P-gp and 11b-HSD2 decrease allowing an increase in transfer of maternal glucocorticoid to the fetus and facilitating the natural fetal glucocorticoid surge. Further studies are required to understand the relative importance of P-gp and 11b-HSD2 in protection of the fetus from high levels of glucocorticoid in the maternal circulation, both under basal conditions and during periods of maternal stress, when endogenous glucocorticoid levels are elevated. In this connection, maternal distress has been directly correlated with reduced expression of human placental ABCB1 mRNA levels at termdan effect that was highly sexually

dimorphic.67 This would suggest that under stressful circumstances, the fetus may become more vulnerable to the increase in maternal glucocorticoids, as well as other xenobiotics present in the maternal circulation. There are a number of circumstances in human pregnancy when glucocorticoids are administered to the mother. Synthetic GC are administered to improve success of in vitro fertilization protocols (hydrocortisone, dexamethasone, prednisolone, and methylprednisolone)68 to prevent virilization of female fetuses at risk of congenital adrenal hyperplasia (dexamethasone)69 or to improve newborn survival rates in pregnancies threatened by PTL (dexamethasone and betamethasone).64 In contrast to endogenous glucocorticoids, 11b-HSD2 has a low affinity for sGC,2 restricting the ability of placental 11b-HSD2 to limit transfer of active glucocorticoid to the fetus. In contrast, and as discussed previously, P-gp has a high affinity for sGC, and as such placental P-gp likely acts to decrease transfer of sGC to the fetus. This clearly has implications if the fetus is the intended target. In addition, given the decreases that occur in placental P-gp in late gestation, fetal exposure to maternally administered sGC will likely increase in late gestation, having implications for dose requirements. Virtually, no studies have considered the role of placental P-gp in modulating transfer of sGC from the mother to fetus. However, some studies have considered the impact of sGC exposure on the expression of placental P-gp. Antenatal betamethasone therapy in cases of threatened human pregnancies did not alter placental ABCB1 and P-expression.70 However, in the mouse, sGC treatments upregulate placental Abcb1a and P-gp protein,71 while sGC decrease placental Abcb1 mRNA and P-gp protein levels in guinea pigs,72 suggesting species-specific regulation. Infection can occur through multiple routes in human pregnancy, and 40% of preterm birth is associated with infection.73 In human pregnancy, infection has been associated with a reduction in placental P-gp in cases of preterm birth.73 Interestingly, in vivo studies in the mouse have demonstrated LPS exposure inhibits placental P-gp activity and increases fetal accumulation

P-GP AND DEVELOPMENT

of P-gp substrates.74 Viral infections have also been shown to affect placental ABCB1.75 Changes in placental P-gp resulting from infection will likely alter transfer of sGC (administered in cases of threatened preterm labor) from the maternal to fetal compartment.

Fetal Blood-Brain Barrier P-gp expression in the fetal BBB provides the developing brain with an additional level of protection against maternally derived P-gp substrates including glucocorticoids. In the human, P-gp immunoreactivity was detected in fetal brain microvessels by the 20th week of gestation. Levels then progressively increased up to 3e6 months postnatally, when P-gp reached the same levels similar to those in the adult brain.76 Similar expression patterns have been described in the developing BBB of the mouse and guinea pig,77,78 with dramatic increases in P-gp expression and activity in late gestation.78 As such, increases in P-gpemediated protection of the fetal brain coincide with the reduction in P-gp in the placenta. The former is critical as the fetus transitions to neonatal life. These developmental changes in P-gp expression in the BBB will clearly have implications for transfer of glucocorticoids and other P-gp substrates into the fetal brain. Interestingly, the increase in P-gp at the fetal BBB in late gestation coincides with the fetal glucocorticoid surge. Recent studies have shown that maternal treatment with sGC can lead to increases in P-gp in brain microvessels in guinea pig fetuses at gestational day (GD) 50 (75% of gestation), which is prior to the natural glucocorticoid surge.79 In vitro, cortisol and dexamethasone did not alter P-gp activity of BECs derived from guinea pig fetuses at GD 40, but profoundly increased P-gp activity in BECs derived at GD 50, 60, 65 (term w68 days) and postnatal day (PND) 14.77 As such, it appears that while P-gp can actively reduce transfer of glucocorticoids into the fetal brain in late gestation, glucocorticoids themselves represent an important trigger for late gestation development of the BBB. The fact that sGC exposure can prematurely mature the P-gp system in the BBB clearly has implications for the therapeutic use of sGC in the management

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of threatened preterm labor. It is also important to note that exposure to sGC or maternal stress in pregnancy leads to long-term programing of cardiometabolic, endocrine, and behavioral function in offspringdeffects that can last across multiple generations.2,64,80 Mechanistically, glucocorticoid actions in the fetal brain are, at least in part, responsible for such programing, further implicating the importance of understanding Pgpemediated brain protection. Infection and related proinflammatory cytokine exposure have been shown to impact P-gp expression and function in the developing BBB. Maternal treatment with Poly:IC (a viral mimic) decreased P-gp activity in the fetal mouse BBB, resulting in increased accumulation of [3H] digoxin in the fetal brain.81 Consistent with the effects of Poly:IC, proinflammatory cytokines inhibit P-gp expression and activity in the developing BBB, though the effects were dependent on the stage of development when exposure occurred. Exposure of BECs derived from guinea pig fetuses on GD65 and PND 14 to IL-1b, IL-6, or TNF-a, in vitro, resulted in a reduction in Pgp expression and activity, with greatest effect at PND14.82 Interestingly, no change in P-gp activity was observed in BECs derived on GD50, which is prior to developmental increase in P-gp in the BBB, and indicates developmental differences in sensitivity.82 Together, these studies suggest that infection may result in an increase in P-gp substrate transfer, including glucocorticoids into the developing brain. It is clear that there is a complex interplay of factors in the regulation of P-gp in the developing BBB, and further studies are required to understand the implication of these regulatory mechanisms in modulating exposure of the developing brain to P-gp substrates, particularly in the context of pathophysiology. For example, preterm labor is often associated with infection, which will result in release of proinflammatory cytokines and inhibition of P-gp at the developing BBB. However, sGC are administered in cases of threatened preterm labor, and sGC have been shown to mature the BBB and increase the inhibitory actions of cytokines on P-gp. This interplay will have significant implications for exposure of the fetal brain to P-gp substrates, including glucocorticoids.

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CONCLUDING REMARKS P-gp efflux activity is a central component of glucocorticoid biodisposition and excretion. P-gp at the BBB regulates access of glucocorticoids to the brain and by extension modulates HPA function in both basal and challenged states. Localization of P-gp in the adrenal cortex, liver, and kidney indicate that P-gp likely plays an important role in glucocorticoid release, metabolism, and excretion as well as possibly cellular protection in these peripheral tissues. P-gp is also enriched in developmental barriers and functions to protect the fetus from maternally derived glucocorticoids, xenobiotics, and environmental toxins. The fact that sGC, inflammation, and drugs (e.g., SSRIs) can modulate P-gp expression and activity in different tissues, suggests that these factors likely indirectly regulate access of endogenous glucocorticoid to the brain and therefore HPA axis function and stress responsiveness. Clearly, further work is required to investigate the role and regulation of P-gp in normal tissues and pathophysiolgy. Such knowledge will improve understanding of glucocorticoid physiology, as well as facilitate development of novel therapeutic strategies.

Disclosure The authors report no conflicts of interest in this work.

Acknowledgments This work was supported by CNPq (Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico, Brasilia, Brazil), grant number: 422410/2016-0 to E.B. This work was also supported by Canadian Institutes for Health Research (FDN-148368) to S.G.M. The authors thank Ms Phetcharawan Lye for the immunohistochemistry images displayed on Fig. 19.3.

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C H A P T E R

20 Stress and Glucocorticoids as Experience-Dependent Modulators of Huntington’s Disease 1

Christina Mo1,2, Thibault Renoir1, Anthony J. Hannan1,3

Florey Institute of Neuroscience and Mental Health, Melbourne Brain Centre, University of Melbourne, Parkville, VIC, Australia; 2Department of Neurobiology, University of Chicago, Chicago, IL, United States; 3Department of Anatomy and Neuroscience, University of Melbourne, Parkville, VIC, Australia O U T L I N E Introduction

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Modeling Huntington’s Disease in Mice Glucocorticoids and the Stress Response Mechanisms of Stress-Induced Changes The Effects of Stress Depend on Many Variables Stress Paradigms Used in Rodents

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Corticosterone in Drinking Water Restraint Stress Chronic Unpredictable Stress Social Defeat and Predator Exposure

Stress in Neurological and Psychiatric Disorders Stress in Huntington’s Disease Psychological Stress Abnormal Stress Response

The Effects of Stress and Stress Hormone Inventions in HD Mice Oral CORT Treatment Corticosterone Treatment Accelerated the Onset of Y-Maze Memory Deficits in Male HD Mice

Stress: Physiology, Biochemistry, and Pathology https://doi.org/10.1016/B978-0-12-813146-6.00020-5

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Prolonged CORT TreatmenteInduced Anhedonia Only in Female Mice Motor Coordination Was Unaffected by CORT Treatment Reduced Hippocampal MR Levels in R6/1 Mice at a Young Age

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The Effects of Elevated Corticosterone Treatment on Novel Behavioral Phenotypes in HD Mice 253 Two Weeks of CORT Treatment Impaired Olfactory Sensitivity in Female Mice 254 CORT TreatmenteEnhanced Female Social Interaction in Male R6/1 and WT Mice 254 The Effects of Chronic Restraint Stress on the HD Phenotype Chronic Restraint After 9 weeks Is Still “Stressful” Response to Chronic Stress Between the Genotypes Restraint Enhanced Rotarod Performance and Induced HyperLocomotion in Male Mice Sex Difference in Restraint-Induced Rotarod Effect

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Restraint Transiently Reduced Saccharin Preference and Nest Quality in Female WT Mice Olfactory Sensitivity Is Modulated by Restraint and the HD Mutation

Stress as a Novel Environmental Modulator of HD 256 256

Olfactory Deficits in Female R6/1 Mice Were More Vulnerable to 2 Weeks of Restraint 257 Stress Compared to WT Littermates Olfactory Sensitivity Deficits in Male Mice Were Impaired by Restraint Stress 257

INTRODUCTION Huntington’s disease (HD) is a fatal neurodegenerative disease caused by a gene mutationd an unstable CAG trinucleotide expansion in exon 1 of the HD gene, which is situated on chromosome 4.1 In healthy individuals, this gene has 9e35 repeats of the trinucleotide tract, but in HD this has expanded to >35 repeats,1 although incomplete penetrance has been observed in the range of w36e39 repeats, presumably due to genetic and environmental modifiers. KEY POINTS • Although Huntington’s disease (HD) is an inherited neurodegenerative disorder, environmental factors can alter symptomatology. • HD patients show physiological abnormalities in the stress response. • Transgenic mouse models have been instrumental in advancing the understanding of HD pathology. • We were one of the first teams to investigate the effects of chronic stress (i.e., long-term treatment with corticosterone) on the development of the HD phenotype in both female and male R6/1 HD mice.

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• Stress accelerated onset of specific deficits in HD mice (potentially expressed in a sexually dimorphic manner). • The HD gene mutation may confer a susceptibility to the negative effects of stress. • Levels of hippocampal mineralocorticoid receptor (MR) gene expression were reduced in R6/1 mice of both sexes at early stage (i.e., 7 weeks of age). The disease phenotype is expressed as a triad of cognitive, psychiatric, and motor symptoms.2 The majority of cases are adult-onset, manifesting in the third to fourth decade of life, but 5% of cases are the aggressive juvenile form, which can manifest as early as 2 years of age.3,4 The specific age of disease onset is highly inversely correlated with the length of the CAG expansion.5,6 The HD mutation shows full penetrance in individuals with more than 40 repeats given a normal lifespan.7 Transmission follows autosomal dominant inheritance, conferring a 50% probability that the gene will be passed onto offspring. Due to its high genetic load, HD has been regarded as the epitome of genetic determinism, reflecting the long-held view that the genetics alone was responsible for phenotype and behavior.8

MODELING HUNTINGTON’S DISEASE IN MICE

HD is the most commonly inherited neurodegenerative disorder.9 HD follows an autosomal dominant inheritance pattern. Status for the HD mutation can be determined by a genetic test for the length of the CAG repeat. Currently, effective treatments to slow the disease progression remain elusive. HD encompasses a triad of symptoms consisting of cognitive decline, psychiatric abnormalities, and motor dysfunctions. The hallmark motor feature is Huntington’s chorea, the involuntary, jerky movements of the head, trunk, and limbs.10 The onset of motor symptoms is used for diagnosis along with a family history of the HD mutation.11 Cognitive symptoms present up to 20 years prior to the onset of motor symptoms in HD.12e16 This interferes with activities of daily living; the ability to work, manage finances, and perform domestic chores.17 Manifest HD patients eventually progress to global dementia.18 Psychiatric symptoms are also present early in premanifest HD individuals.19 The symptoms include depression, anxiety, apathy, irritability, impulsiveness, and extroverted hostility.20,21 Suspiciousness and paranoia can also present close to the age of diagnosis.22 Depression is the most common psychiatric disturbance with a prevalence in HD sufferers twice that of the general population.23e25 The symptoms of HD extend outside the characteristic triad of motor, cognitive, and psychiatric dysfunctions to include hypothalamic, neuroendocrine, metabolic, immune, and peripheral abnormalities.26,27 The HD mutation encodes a dysfunctional huntingtin protein; however, the cascades that lead to cellular dysfunctions and the pathologies that underlie symptoms remain elusive. The huntingtin protein forms insoluble highmolecular-weight protein aggregates only when the number of CAG repeats is within the pathogenic range.28 A hypothesis for the pathological process in HD is that mutant huntingtin causes cellular dysfunctions that accumulate over time, reaching a critical threshold which then manifests in clinical features.

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MODELING HUNTINGTON’S DISEASE IN MICE The identification of the causative HD mutation1 has allowed the development of animal models with excellent construct validity. Animal models of HD include worms, flies, sheep, monkeys, rats, and mice. Many HD models also have good face validity, recapitulating well the progressive behavioral, neuroanatomical, and histological abnormalities seen in human patients.29 In particular, transgenic mouse models have been instrumental in advancing the understanding of HD pathology30 and the screening of potential therapeutic agents.31 The full-length and fragment HD models show good face and construct validity. They develop the motor, cognitive, and affective phenotypes, as well as the peripheral abnormalities such as muscle wasting and circadian disruptions seen in patients. They also generally reflect cortical and striatal atrophy, aggregates, and histopathologies of HD.29 We performed the first preclinical stress studies, using the R6/1 HD mouse model, which is ideal to study the early cognitive and affective changes in HD,32e35 which precede motor deficits. Learning and memory problems are present and progressive from an adult age,36,37 but motor symptoms, which may confound cognitive tests, are not apparent until 14e16 weeks of age.38,39 Studies report dysfunctions in various tests of spatial memory: T-maze (working memory), Y-maze (short-term memory), Morris water maze, and Barnes circular maze (long-term memory).36e41 Hippocampal function in R6/1 mice is impaired by at least 8 weeks of age.36 Affective abnormalities in R6/1 mice also develop prior to the onset of motor phenotype, reflective of the human condition. In the R6/1 model, female (but not male) mice show increased immobility in the forced-swim test, suggestive of a depressive-like behavior. They also show a longer latency to approach food in the novelty-suppressed feeding test, indicating enhanced anxiety. An anhedonic phenotype is suggested by a reduced preference for a sweet

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saccharin solution.34,35,42e44 The reason for the sex difference is not fully understood, but a dysregulated serotonin system and adrenal pathophysiology are implicated.34,43,44 Other transgenic models also show sex differences in the HD phenotype.45,46 Ultrastructural neuropathological features of R6/1 mice include cortical, cerebellar, and striatal cell loss with widespread inclusions.47e49 R6/1 animals also show weight loss, premature death,50 increased thirst,43,44,51 testicular degeneration,52 and sleep disturbances.53,54 The attraction of the R6/1 model is its adult onset with progressive development, making it a clinically relevant model of symptom development. To date, there is evidence from both clinical and rodent studies that nongenetic factors (diet, physical activity, and cognitive activity) can affect the progression of HD, which has been most clearly demonstrated in the preclinical models (reviewed by Mo et al.55). Such factors are feasible as interventions, and their identification may help delay the onset of HD. One major environmental factor that has only recently been investigated is stress.

Glucocorticoids and the Stress Response Stress is a state of threatened homeostasis by intrinsic or extrinsic factors.56 The detection and activation of biological processes to deal with stressors is essential for everyday function and survival. However, severe or prolonged stress can cause molecular and cellular changes that can contribute to pathophysiology. Upon detection of a stressor, two major systems are activated to induce coping adaptations. The sympathetic-adrenomedullary system rapidly acts to suppress nonessential functions and mobilize those required for quick physiological action. The other major response is the activation of the hypothalamicepituitaryeadrenal (HPA) axis (Fig. 20.1). The hypothalamus synthesizes peptides and catecholamines which are released into the circulatory system to act as hormones. Corticotrophin-releasing hormone (CRH) is synthesized in the paraventricular nucleus of the hypothalamus and secreted into the

hypophysial portal vessel system. CRH in synergy with arginine vasopressin stimulates the release of adrenocorticotropin (ACTH) from the anterior pituitary gland into the systemic circulation. ACTH stimulates the synthesis and release of glucocorticoids from the adrenal cortex57: cortisol in humans or corticosterone in rodents (CORT). Under nonstressful conditions, pulsatile secretion of CRH results in diurnal fluctuations of glucocorticoids, which peak in the morning.58,59 In response to stress, glucocorticoid (GR) synthesis is increased to modulate a wide variety of functions including cognition, sexual behavior, energy metabolism, inflammation, and the stress response itself.56,60 The receptors for CORT are the GRs and MRs. Activated MRs and GRs lead to cellular and functional effects through genomic regulation61 (Fig. 20.1). There are also rapid nongenomic pathways which involve the binding of specific ion channels and receptors.62 For example, CORT binding to MR rapidly increases the frequency of excitatory postsynaptic currents in the hippocampus.63,64 This faster signaling (secondseminutes) may support or act in opposition to slower genomic changes.65 MR is highly expressed in the hippocampus and lateral septum. It binds CORT with high affinity such that a large proportion of MR is occupied at any given time.66 This results in a stable excitatory tone and allows the regulation of novelty appraisal.67e70 MR in balance with GR controls the sensitivity of the stress response system.70,71 GR is highly expressed in limbic regions such as the lateral septum, hippocampus, nucleus of the solitary tract, and central amygdala. The affinity for CORT is one-tenth than that of MR.60,66 As a result, GR is generally unoccupied except when CORT levels are highdduring the diurnal peak of circulating CORT or during stress.66 GR binding and activation triggers translocation to the nucleus to regulate target gene expression, including its own downregulation. This genomic pathway triggers the negative feedback in the HPA axis and return to homeostasis.72 However, prolonged activation of GR can have detrimental effects on synaptic plasticity and

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FIGURE 20.1 Stress-induced activation of the hypothalamicepituitaryeadrenal (HPA) axis results in the release of the glucocorticoids (herein referred to as CORT), corticosterone in rodents and cortisol in the human. Notwithstanding the importance of the sympatheticeadrenalemedullary (SAM) system in the immediate acute response to stress,241 the HPA axis is the major endocrine system responsible for the regulation of stress adaptation. A stressor activates the paraventricular nucleus of the hypothalamus and the secretion of CRH to stimulate the anterior pituitary which triggers the release of adrenocorticotrophic hormone (ACTH), which is subsequently released into the bloodstream. ACTH stimulates the secretion of glucocorticoids from the adrenal cortex. The major glucocorticoid released from the adrenal cortex has peripheral effects and also crosses the blood-brain barrier to bind to receptors in various parts of the brain including the cortex and hippocampus. CORT binds to either of its two intracellular receptors, GR and MR, which then translocate into the nucleus and bind genomic recognition sites to enhance or inhibit target genes. GR and MR have differing affinities for CORT and exert different downstream effects through this regulation of gene expression.242 There is negative feedback effect of CORT release through GR activation in the pituitary, hypothalamus, and hippocampus. The levels of CORT within a particular tissue/cell is also regulated by the enzyme 11b-HSD1, which regenerates the inactive form of CORT (cortisone) to its active form, cortisol or corticosterone.243 11b-HSD1, 11b-hydroxysteroid dehydrogenase type 1; ACTH, adrenocorticotropic hormone; CORT, corticosterone/cortisol; CRH, corticotrophin-releasing hormone; GR, glucocorticoid receptor; MR, mineralocorticoid receptor.

neuronal morphology.73,74 Brain regions which show high expression of GR are particularly prone to stress-induced structural and functional changes.60

Mechanisms of Stress-Induced Changes There is a robust relationship between prolonged glucocorticoid exposure, cognitive impairment, and neuronal dysfunction.60,75,76 Stress-induced GR and MR activation differentially

affect synaptic events such as miniature postsynaptic currents, calcium channel expression, and long-term potentiation (LTP) and depression (LTD).77,78 In rodents, chronic exposure to glucocorticoids can cause dendritic remodeling in the hippocampus,79e82 prefrontal cortex,83e85 and amygdala.86 This remodeling is dependent on activation of GR and N-methyl-d-aspartate receptors.87,88 The persistence of morphological changes depends on the period of stress or stress hormone

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exposure. For example, in the rat CA3 hippocampal region, 4 weeks of daily CORT injections induced changes in mossy fiber morphology, atrophy of apical dendrites, and reduced the number of synapses.89 However, after a 4-week washout (period when stress is removed), both the stress-induced morphological abnormalities and associated deficits in water maze performance were abolished. In contrast, 20 days of corticosterone treatment modified dendritic morphology in the mouse hippocampal CA1 and orbitofrontal cortex, which persisted after a washout period of 1 week. CORT-induced anhedonic behavior also persisted after this shortterm recovery period.81 These studies suggest that prolonged corticosteroid exposure can induce both long-term and reversible behavioral deficits, associated with structural alterations in the hippocampus and frontal cortex. The morphological changes associated with stress may be mediated in part by stress-induced reductions in brain-derived neurotrophic factor (BDNF).90,91 BDNF is a key neurotrophin which supports development, synaptic plasticity, and neuronal survival.92 Glucocorticoids control BDNF gene expression through MR and GR.93,94 In most brain regions, GR activation reduces, whereas MR activation increases BDNF levels.95 In mice, 2 weeks of glucocorticoid treatment reduced hippocampal BDNF and induced depressive-like behaviors. Restoration of hippocampal BDNF levels reversed CORT-induced depressive-like behavior.96 Moreover, decreased BDNF signaling has been associated with impaired stress resilience to depressive-like behavior, supporting that BDNF is a key factor in vulnerability to stress.97,98

The Effects of Stress Depend on Many Variables At the behavioral level, the effects of a stressor depend on the stressor (type, duration) assessment (cognitive, affective), timing (between stress and assessment), and the subject (age, sex, and species).99,100 Acute stress can be facilitative101 or detrimental.102 In learning and memory tasks, this is dependent on the timing

between stress and the task.99,103 In contrast, chronic or severe stress generally shows negative effects.104e106 For example, restraint stress lasting 3 or more weeks impaired performance on spatial memory tests, such as the Y-maze test for short-term memory.107e110 An inverted Ushaped curve for stress and performance has been described where a small amount of stress is facilitative to performance up to an optimal level, but further stress (severity or length) impairs performance.111,112 Stress effects can be sexually dimorphic. Stress can impair male cognition with no effect or enhancement in females,113,114 show benefits only in females,115 or have a positive effect in males but impair learning in females.116,117 Sex hormones have been proposed to explain some of these results.116 For male rats, the effects on spatial memory are also task-specific. In appetite-motivated tasks, chronic stress impairs spatial learning. In fear-motivated tasks, such as a water maze, there is minimal impairment or a facilitation in spatial learning (reviewed in ref. 106).

Stress Paradigms Used in Rodents There are different types of paradigms used to model stress in rodents. They can be broadly classified as psychological, physical, and pharmacological agents. These classifications are arbitrary since many stressors (e.g., social defeat, restraint) have both a psychological and physical component. The stress protocol may also encompass a variety of stressor types presented at differing times of the day to produce an unpredictable stress protocol.87 The literature also classifies stressors by their duration. An acute stressor is one of single administration and time-limited exposure (generally minutes to hours). A chronic stressor is one that is administered repeatedly over many days or months. Due to the variety of stress paradigms and the heterogeneity of the stress response, there are stressor-specific neural responses.56 This makes it difficult to model stress as a general environmental factor. Pacak and Palkovits compared five different types of stressors on their neuroendocrine and regional activation profile.

MODELING HUNTINGTON’S DISEASE IN MICE

The expression of c-Fos, a marker of cell activation, showed that immobilization, hemorrhage, cold exposure, pain, and hypoglycemia stressors differed markedly in areas such as the cingulate cortex, piriform cortex, lateral septum, and central amygdala. As discussed previously, a major stress response is HPA axis activation and subsequent release of CORT. Elevation of serum CORT levels was a common response among these 5 stressors.56 Corticosterone in Drinking Water Supplementation of drinking water with CORT is a noninvasive way to mimic elevated circulating levels during stress.96,118 Chronic oral CORT treatment is used in a mouse model of chronic stress,119,120 hypercortisolemia,121 depression,96,122e124 and to understand stress effects on cognition.125,126 Much of the detrimental effects of chronic stress can be attributed to excess glucocorticoid signaling.79,96,127e131 Chronic CORT treatment produces dendritic atrophy in the medial prefrontal cortex132 and the hippocampus81,82,132 as well as overall reductions in the hippocampal volume.89,133,134 Limbic structures such as the hippocampus, amygdala, and medial prefrontal cortex have high concentrations of glucocorticoid receptors.85,135,136 These regions, which support cognitive and affective functions, may be more vulnerable to CORT effects. Restraint Stress Restraint stress is the most widely used stressor in rodents.137 The animal is placed in a ventilated tube or wire mesh for a period (from 30 min to 6 h) for one session (acute) or over many weeks (chronic).138 Generally the source of stress is not physical pain but the psychologically aversive nature of inescapable, restricted movement.56 A more severe version of restraint is complete immobilization, which involves taping the limbs and body to a frame. A less severe version of restraint is confinement stress in a box or cage.56,139 The effects of restraint stress are well documented with impairments in cognitive, affective, neuroendocrine, and immune functions.106,137 Predictability of the repeated restraint sessions can lead to physiological adaptation of the stress response.140

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Chronic Unpredictable Stress Chronic unpredictable or chronic mild stress is also a widely used protocol to induce persistent behavioral changes in rodents.141 Typically, the protocol involves a set combination of daily stressors, exposed in a pseudorandomized order over 2e5 weeks. Such stressors include restraint stress, wet bedding, cage tilt, forced swimming, and lightedark cycle disruptions.142 This paradigm is clinically relevant and reduces stress adaptation compared to other protocols. However, there is more variability compared to other stress paradigms, and it is more suitable to assess long-term behavioral effects of stress after a washout period.143 Social Defeat and Predator Exposure Social defeat is a relatively severe stressor in rodents based on social hierarchy and dominance. In the resident-intruder model, a male mouse is introduced to the cage of a dominant, aggressive male mouse in repeated bouts over days or weeks.144,145 Long-term defeat is associated with cognitive impairment, social deficits, anxiety, and depressive-like behavior.146,147 This stress protocol is also more potent in males than females, although paradigms using lactating females have also been described.148,149 Rodents can be exposed to a predator such as a cat or fox in its whole form (anesthetized or active) or in the form of its odor. Exposure induces powerful reproductive, anxiogenic, and neurochemical consequences.150e152 Predator stress has been regarded as an emotional stress relevant to posttraumatic stress disorder.153,154 Both social defeat and predator exposure are ethologically relevant stressors. However, individual responses to the dominant mouse or odor can be sources of high variability.

Stress in Neurological and Psychiatric Disorders Prolonged exposure to stress can have negative impacts on the susceptibility and progression of neurological disorders.155 In humans, stress is implicated in the onset or exacerbation of depression,156,157 Alzheimer’s disease (AD),158 multiple sclerosis,159 posttraumatic stress disorder,160

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schizophrenia,161 and epilepsy.162 Even in clinically healthy individuals, high levels of lifetime adversity positively correlated with acute stressinduced neural activity in limbic regions and the prefrontal cortex.163 The negative impact of stress on disease is supported by animal models and is most studied in AD and depression. For example, in various transgenic models of AD, chronic stress accelerated the development of cognitive deficits and increased amyloid or tau pathology.139,164e166 This stress-induced acceleration of cognitive decline is related to HPA axis dysregulation165 and reduced levels of the neurotrophin and BDNF.166 In general, mice subjected to chronic stress show abnormal activity and atrophy in prefrontalelimbicestriatal regions.167,168

Stress in Huntington’s Disease Psychological Stress There is evidence that HD patients show higher stress levels than the general population. This is unsurprising given the stigma and absence of effective treatments or cure.169e171 Diagnosed patients show the highest stress scores172; however, predictive genetic testing itself is associated with increased anxiety and hopelessness 3e4 years posttest, regardless of a positive or negative result.173 Those that test positive also become more pessimistic as they approach the age of disease onset.174 HD affects families, and prediagnosed individuals may also experience emotional distress when care giving for a family member with HD.170,175 Abnormal Stress Response Not only is there substantial psychological stress but increasing evidence supports that HD patients show physiological abnormalities in the stress response. Aziz et al. reported an exaggerated cortisol diurnal rhythm in HD patients compared to age- and sex-matched controls. The peak diurnal secretion of cortisol in the morning (w8.30 a.m.) did not differ between HD patients and controls, but the levels in blood were prolonged in the proceeding 4e5 h. Cortisol levels in the middle of the night also showed an abnormal elevation in HD

patients compared to control subjects. These data suggest that HPA axis regulation of cortisol or MReGR balance is dysfunctional in HD patients.176 In support of a hyperactive stress axis in HD, Bjorkqvist et al. reported increased urinary cortisol levels in HD patients, which correlated with disease progression.177 Furthermore, R6/2 mice showed a progressive increase in serum and urine CORT levels from 5.5 weeks of age and hypertrophy of the adrenal cortex by 12 weeks of age.177 In the less aggressive R6/1 mouse model, there were no differences in serum CORT levels at baseline.42 However, after an acute forced swim stress, female R6/1 mice showed a prolonged elevation of CORT.42 Probing of HPA axis activation revealed a hyperactive adrenal gland response from ACTH stimulation, implicating persistent production or impaired clearance of CORT.34 The extended period of circulating CORT levels may be related to the increased immobility during the forced swim test (representing helplessness) in female R6/1 mice compared to wild-type (WT) mice.34,42 Taken together, there is a hyperactive stress system in patients and mouse models of HD, potentially expressed in a sexually dimorphic manner. Patients also experience more psychological stress compared to the general population. In neurodegenerative diseases, stress can exacerbate symptomatology,178 but the disease state can also confer a vulnerability to stress.166 Despite these lines of evidence, the effect of stress on HD pathogenesis has received little attention. It is now known that the onset of HD has nongenetic components such as controllable lifestyle factors. This presents promising avenues to slow the progression of the disease. However, environmental modulators remain to be identified. Prolonged stress can exacerbate pathogenesis. The ability to cope with stress may be impaired in HD.

THE EFFECTS OF STRESS AND STRESS HORMONE INVENTIONS IN HD MICE The triad of characteristic cognitive, psychiatric, and motor symptoms in HD generally

CORTICOSTERONE TREATMENT ACCELERATED THE ONSET OF Y-MAZE MEMORY DEFICITS IN MALE HD MICE

appear at an adult age. The onset of these symptoms may be able to be modulated by environmental factors.179e181 The stress response is dysregulated by the HD mutation in patients and mouse models.34,176,177 We have demonstrated that an acute confinement stress impaired the acquisition of short-term memory in female R6/1 HD mice, while WT mice and male R6/1 mice were unaffected.181a Female R6/1 mice were also unable to cope physiologically or behaviorally with an acute forced swim stress, compared to males and WT littermates.34 These data suggest that R6/1 HD female mice may be more vulnerable to the impact of acute stress. The effect of chronic stress on the development of HD symptoms has yet to be investigated.

Oral CORT Treatment The stress hormone, cortisol or corticosterone (CORT) in rodents, is a major effector of HPA axis activation and elevates during chronic stress.182,183 Exogenous administration of CORT is a model for enhanced glucocorticoid signaling and produces low experimental variability compared to psychological or social stressors.184 In particular, administration through the dissolution of CORT in the drinking water exposes the animal to “stress” levels of circulating CORT.96,118,121 Delivery by passive drinking also avoids injection stress in control animals and induces elevations of CORT relevant to the circadian rhythm.185 The use of the synthetic CORT hemisuccinate is recommended over corticosterone because hemisuccinate can dissolve directly in drinking water, avoiding the need for vehicles such as ethanol.186 Treatment with oral CORT over 2e4 weeks generally alters body weight gain and affective behaviors but such effects are dosedependent.121,187 For example, a 20 mg/L dose in male mice improved depressive-like behaviors and increased cell proliferation in the hippocampus compared to ethanol-treated vehicle controls.188 At moderate doses for mice (25 mg/L), chronic exposure to CORT has been shown to reduce hippocampal BDNF signaling and spine proliferation.96,122 Even higher doses of 100 mg/L decreased hippocampal

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endocannabinoid signaling119 and caused dramatic metabolic abnormalities in mice.121 High doses in rats impaired hippocampal-dependent spatial memory.125,128 In our studies, we assessed the effects of longterm oral CORT treatment on the development of the HD phenotype in female and male R6/1 HD mice, with WT littermates as controls.188a Our focus was on the characteristic triad of cognitive (Y-maze), affective (saccharin preference test), and motor (rotarod) deficits in HD. We used a moderate dose of CORT which robustly elevates serum levels96,118 but did not induce dramatic perturbations in WT mice.121 We found that the onset of Y-maze memory impairment was brought forward by CORT treatment, but only in male R6/1 mice. We therefore investigated candidates of this susceptibility to CORT in the hippocampus because this region is strongly influenced by stress, supports memory function,189 and is dysfunctional in R6/1 mice.190 To extend the testing to chronic stress, we administered CORT from 6 weeks of age and assessed the impact on cognitive, affective, and motor symptoms. Treatment with the stress hormone (CORT) accelerated the onset of male short-term memory impairment in a mouse model of HD.188a Notably, this stress hormone treatment did not impair memory in WT controls or female R6/1 HD mice, suggesting a vulnerability to the negative effects of stress in male R6/1 mice.188a Chronic CORT treatment induced anhedonia, the reduced experience of pleasure, in female mice regardless of genotype, but motor function was unaffected in any group (see Fig. 20.2 for summary). High levels of stress hormone may preferentially impair cognitive and affective function over motor deficits in HD mice and in a sex-specific manner.

CORTICOSTERONE TREATMENT ACCELERATED THE ONSET OF YMAZE MEMORY DEFICITS IN MALE HD MICE Short-term memory deficits in male R6/1 HD mice develop at 8 weeks of age.188a We report

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FIGURE 20.2 Summary of oral CORT effects in R6/1 HD mice.188a CORT treatment (25 mg/L) from 6 to 14 weeks of age had no effect on the onset or progression of rotarod deficits in male ( ) or female ( ) R6/1 mice. Weight gain was reduced in male R6/1 mice compared to WT controls at 7 weeks of age, and this was further suppressed by CORT treatment in the initial few weeks. The onset of a Y-maze memory deficit in male R6/1 mice was accelerated to 7 weeks of age by 5 days of CORT treatment. Y-maze performance in female R6/1 and WT mice was unaffected. At 7 weeks of age, 5 days of CORT treatment reduced adrenal gland weight and levels of hippocampal cell proliferation in male WT and R6/1 mice, but not female mice. indicates the age of onset of male R6/1 deficits; indicates onset of female R6/1 mice deficits. Dotted blue lines for saccharin preference phenotype indicates that it was not found in the relevant study.188a BDNF, brain-derived neurotrophic factor; GR, glucocorticoid receptor; HD, Huntington’s disease; MR, mineralocorticoid receptor; WT, wild type.

that a 5-day oral CORT treatment accelerated the onset of Y-maze deficits in male R6/1 mice but had no effect on performance in female R6/1 mice or WT mice of either sex.188a These data corroborated with the disruption of Ymaze performance in female R6/1 mice by acute confinement.181a It was interesting that the memory deficit was accelerated in male but not female R6/1 mice despite equal consumption of CORT between the sexes.188a Sex differences in response to stress are often reported in the literature and evidence for a protective effect of estrogens is discussed when the stressor only impairs male animals.113,191e193 In the context of HD, perhaps the earlier disease progression in male compared to female R6/1 mice rendered this group more susceptible to perturbations by CORT treatment. Declining sex hormones in HD may

offer an explanation for the male HD-specific Y-maze results seen in the study.188a Testicular atrophy and low serum testosterone concentrations have been reported in R6/1 HD mice52 and HD patients.194,195 In contrast, female R6/1 mice showed no change in circulating estradiol concentrations compared to WT controls.34 Estradiol levels in female HD patients are not yet available. The decline in serum testosterone in male HD patients was associated with dementia and correlated with HD functional rating scores.194 In general, declining androgen levels have been linked to impaired memory function.196,197 Male HD mice may receive a “double hit” to cognitive processes. The first is the decrease in testosterone conferred directly or indirectly by the HD mutation. This may cause the second hit; that is, subsequent vulnerability to hippocampal damage by CORT treatment.

THE EFFECTS OF ELEVATED CORTICOSTERONE TREATMENT ON NOVEL BEHAVIORAL PHENOTYPES IN HD MICE

Prolonged CORT TreatmenteInduced Anhedonia Only in Female Mice Chronic CORT treatment has been shown to induce anhedonia in mice.96,198,199 We also found this effect, but only in female mice. Furthermore, 4 weeks of CORT treatment was required, whereas 2 weeks of treatment was insufficient.188a In a similar study, oral CORT treatment at a slightly higher dose reduced saccharin preference after 3 weeks.96 Our sex-specific results support the notion that female mice are more sensitive in emotional measures to stress.200

Motor Coordination Was Unaffected by CORT Treatment Chronic CORT treatment beginning from 6 weeks of age did not alter rotarod impairments in R6/1 mice, which started to decline at 12 weeks of age in females and 10 weeks in males.188a Motor performance in WT animals were also unaffected by chronic CORT. Motor function in general could be less sensitive to the effects of chronic elevations of stress hormone. Support for this comes from the distribution of stress receptors, which are more concentrated in regions related to learning, memory, and emotional processing.66 In addition, the dose of CORT in the present study was moderate in its physiological effects compared to other stressors used in the literature. For example, chronic restraint water immersion, a severe stressor, was able to impair rotarod performance in WT mice.201 Therefore, we cannot rule out that more severe stressors may be able to accelerate or exacerbate motor symptoms. Nevertheless, motor deficits appear less perturbed by high levels of stress hormone compared to affective and memory dysfunctions in R6/1 HD mice.

Reduced Hippocampal MR Levels in R6/1 Mice at a Young Age Regardless of CORT treatment, levels of hippocampal MR gene expression were reduced in R6/1 mice of both sexes at 7 weeks of age.188a MR and GR are important mediators of the stress response. We reported an w20% reduction in the HD hippocampus at 7 weeks of age.188a

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The majority of MRs are occupied under baseline conditions and regulate the circadian rhythm of plasma CORT secretion.66 This abnormal reduction in MR gene expression may contribute to the exaggerated diurnal CORT levels in the blood and urine of HD patients and mice.176,177 However, our data only pertain to the hippocampus and deficits in the hypothalamus and/or adrenal glands would also be hypothesized. Inefficient clearance of serum CORT and disrupted circadian rhythm activity could also help explain abnormal CORT regulation in HD. GR mRNA expression levels and function in R6/1 mice do not differ from WT levels at 12 weeks of age,34 corroborating with the present findings at 7 weeks of age. We have shown that repeated elevations of stress hormone accelerated the onset of shortterm memory decline in HD mice. By using a moderate dose of a pharmacological stressor, we were able to highlight the behavioral and cellular vulnerability of the HD hippocampus to stress-induced impairments.188a Future studies investigating the impact of CORT on other symptoms of HD and the use of other stress paradigms would further reveal the extent to which stress contributes to HD onset and progression.

THE EFFECTS OF ELEVATED CORTICOSTERONE TREATMENT ON NOVEL BEHAVIORAL PHENOTYPES IN HD MICE HD encompasses a characteristic triad of cognitive, affective, and motor symptoms, which are recapitulated well in transgenic mouse models using the standard behavioral test battery.29,202 However, the ubiquitous expression of the HD mutation produces many other symptoms including peripheral,27,203 circadian rhythm,204,205 sexual,206,207 and olfactory208,209 disturbances. The combination of these dysfunctions results in the impairments of general daily functioning.210 We extended our work to assess the effects of CORT treatment on symptoms that have received relatively less attentiondolfactory

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impairments and sexual abnormalities in HD. In addition, we assessed nest building as a speciesspecific behavior important for daily function in the mouse. Paradigms with ethological context are advantageous for understanding the functional impact of an experimental intervention. Chronic CORT treatment did not affect nest building or sexual vocalization response in R6/ 1 or WT mice. However, olfactory sensitivity was impaired by CORT treatment in female WT and HD mice. Socially motivated vocalizations were also transiently enhanced by CORT treatment. These results210a show that even primitive behaviors are dysfunctional in HD mice and olfaction in particular is sensitive to stress hormone treatment.

Two Weeks of CORT Treatment Impaired Olfactory Sensitivity in Female Mice Oral CORT treatment from 6 to 8 weeks of age impaired olfactory sensitivity in both genotypes.210a Olfaction is an important sense in mice as it supports navigating, foraging, avoiding predators, social recognition, sexual selection, and parental behaviors.211 Ethological evidence for this comes from the continued migration of neuroblasts along the rostral migratory stream to the olfactory bulb throughout adulthood.212

CORT TreatmenteEnhanced Female Social Interaction in Male R6/1 and WT Mice Six weeks of CORT treatment enhanced the number of ultrasonic vocalizations (USVs) emitted by male mice (WT and R6/1) toward a female mouse.210a The effect was timedependent since it was not apparent after 2 weeks of CORT or after 8 weeks of CORT. Stress by social defeat also showed transient effects on female-induced vocalizations in male mice, but in the opposite direction (reduced by stress).213 The same CORT treatment had no effect on urine-induced vocalizations at 12 weeks of age so the CORT effect seems specific to

socially motivated vocalizations. We thus used an ethological approach and found that olfactory sensitivity was impaired by corticosterone treatment, providing further evidence that stress is an environmental modulator of specific symptoms in HD.

THE EFFECTS OF CHRONIC RESTRAINT STRESS ON THE HD PHENOTYPE We have shown that an acute confinement stress181a and a moderate dose of stress hormone treatment188a impaired memory and olfactory sensitivity in R6/1 HD mice. These stressors were chosen for their mild effects on WT animals, allowing us to test the hypothesis that HD mice were more vulnerable to the effects of stress. Indeed, memory in WT mice was not impacted by 1-h of confinement or 5days of CORT treatment.181a,188a HD patients experience higher levels of stress than the general population.171,172 It is therefore relevant to study the effect of more severe stressors that robustly shift behaviors in WT rodents. In this chapter we used chronic restraint stress, which has more impact on affective and cognitive functions compared to corticosterone administration.214 Chronic restraint stress has been shown to perturb motor, cognitive, and affective functions in WT mice. We expected restraint-induced impacts to be of larger magnitude in R6/1 HD mice compared to WT littermates. We also expected some restraint effects to be sexually dimorphic. The main findings of the study were: (1) Motor coordination and locomotor activity were enhanced by chronic restraint in males regardless of genotype and (2) Olfactory sensitivity was impaired by restraint in R6/1 HD mice, and male WT mice, but not female WT mice.231a (see Fig. 20.3 for summary). Together, these data provide further evidence that specific functions in HD mice are more susceptible to stress compared to that of WT mice. We also contribute data on general sex differences in response to restraint and that this paradigm can have both facilitative and detrimental effects on certain behaviors.

THE EFFECTS OF CHRONIC RESTRAINT STRESS ON THE HD PHENOTYPE

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FIGURE 20.3 The effects of chronic restraint stress on the onset and progression of phenotypes R6/1 HD mice. Restraint stress impaired male and female olfactory sensitivity, and this was specific to R6/1 mice in female animals (WT females not affected). Restraint stress also enhanced performance on the rotarod in male R6/1 and WT mice but had no effect on female animals. Weight gain was suppressed in all restrained animals throughout treatment. Anxiety is not a phenotype of R6/1 mice but lightedark box and elevated-plus maze testing showed restraint-induced hyperlocomotion, regardless of genotype (not shown). No other effects of restraint stress were seen in R6/1 mice. The onset of each behavioral phenotype is presented by a colored bar. Restraint stress was administered daily from 6 to 7 p.m., starting from 6 weeks of age. , and refer to mice of both sexes, male mice and female mice, respectively.

Chronic Restraint After 9 weeks Is Still “Stressful” In our study, restrained mice showed constant suppression of weight gain from the first week to the end of treatment.231a This occurred regardless of genotype or sex; the magnitude of effect was higher in female R6/1 mice compared to WT mice after 5 weeks of treatment. This suggests that restraint exerted continual physiological effects. Furthermore, the last restraint session (56th session) induced robust elevations in serum corticosterone levels compared to nonstressed controls.231a These data suggest that the paradigm remains stress-inducing until the endpoint of treatment.

Response to Chronic Stress Between the Genotypes Struggling activity during restraint has been used as a measure of stress reactivity.215 There was no difference in the presence of struggling during the restraint sessions between the genotypes.231a Restraint also suppressed body weight, regardless of genotype in male animals. However, weight gain in female R6/1 mice was suppressed significantly more than WT mice after

5e8 weeks of restraint.231a A full profile of serum CORT release after a 1-h restraint session may reveal a female R6/1-specific prolonged elevation of CORT after stress.34 However, at least immediately after the last restraint session, elevations of serum CORT were similar across all groups. Taken together, R6/1 and WT mice showed similar physiological responses to chronic restraint stress.

Restraint Enhanced Rotarod Performance and Induced HyperLocomotion in Male Mice Daily restraint increased the time spent on the accelerating rotarod in male WT and R6/1 mice.231a This could be interpreted as improved motor coordination, enhanced alertness, and motivation to remain on the apparatus, generalized hyperlocomotion, or a combination of all three. Our restraint paradigm could have increased motivation to perform on the rotarod in male mice. A general increase in motor output may also contribute to improved performance in the rotarod. Locomotor activity during testing in the lightedark box and elevated-plus maze was

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enhanced by chronic restraint in male animals.231a Similar to another study,201 the restraint effect was cumulative since 4e5 weeks of restraint increased locomotion but 1 week had no effect.231a Other studies also report enhanced locomotor activity after chronic restraint.216e218 The restraint-induced increase in time spent in the light arena and open arms231a may also simply reflect hyperlocomotion, confounding interpretations of anxiety behavior.217,219 Considering the hyperlocomotor behavior, it is not surprising that restrained male mice remained on the rotarod for longer than nonstressed mice. Another hypothesis to explain the enhanced rotarod performance in restrained mice is a shift in the circadian rhythm and therefore, alertness during rotarod testing. Restraint occurred for 1h prior to lights-off (6e7 p.m.). Rotarod performance was tested during the late phase of the light cycle (2e5 p.m.) since mice are more active during this period compared to the morning.220 In fact, the circadian peak of serum CORT levels is just prior to lights on (active period).220 Serum CORT levels can be entrained to elevate during periods of increased activity, such as wheel running.221 Stress can also interrupt circadian activity.222,223 Therefore, in order to deal with daily restraint, the rise in serum CORT may have anticipated the restraint session at 6 p.m. During rotarod testing in the few hours prior to lightsoff, restrained mice may be more alert due to stress-entrained circadian rhythms. Taking a full 24-h CORT profile or monitoring sleepewake activity may be a way to test this hypothesis. Taken together, we suggest that improved rotarod performance in restrained males231a was due to a stress-induced alert state and stress-induced general increase in locomotor activity during rotarod testing.

Sex Difference in Restraint-Induced Rotarod Effect In contrast to males, female mice were not affected by restraint in the rotarod test.231a Time spent in the aversive areas of the lightedark box and locomotion were also no different between restrained and nonstressed females. However, locomotor activity in the elevated-plus maze was

slightly increased by chronic restraint.231a The corresponding absence of effects in anxiety tests in female mice further supports the link between an enhanced hyperlocomotive state and improved rotarod performance in restrained males. Perhaps the male-specific effect of restraint was due to a difference in the perception of restraint between the sexes. However, struggling and serum CORT elevations in response to restraint were similar between females and males.231a Both sexes also showed restraintinduced weight suppression. Rather, the malespecific effects of restraint may reflect a general sex difference in anxiety and motor response to restraint. Other studies showed restraintinduced hyperlocomotion in male rodents, but did not test females.216e219

Restraint Transiently Reduced Saccharin Preference and Nest Quality in Female WT Mice Two weeks of restraint stress reduced preference for a saccharin solution in only female WT mice.231a This anhedonic effect of restraint was abolished after a further 2 weeks of restraint, suggesting adaptation to stress in this measure. Female-specific effects on depressive-like behavior have been reported by others224,225 but so too has female resilience to stress compared to males.226 Female R6/1 HD mice show anhedonia and depressive-like behaviors from 8 weeks of age.42e44 We found that 2 weeks of restraint stress impaired behavior (hedonia) in WT female mice but not R6/1 HD mice.231a A similar WTspecific effect of restraint was found in the nest-building test.231a

Olfactory Sensitivity Is Modulated by Restraint and the HD Mutation HD patients experience deficits in olfactory function prior to motor symptom onset and even prior to diagnosis.208,227e229 We identified an olfactory behavioral test which was sensitive to a moderate olfactory impairment in female R6/1 mice at 8 weeks of age.231a Chronic restraint stress had HD-specific effects.

THE EFFECTS OF CHRONIC RESTRAINT STRESS ON THE HD PHENOTYPE

Olfactory Deficits in Female R6/1 Mice Were More Vulnerable to 2 Weeks of Restraint Stress Compared to WT Littermates Two weeks of restraint stress (from 6 to 8 weeks of age) further impaired olfactory sensitivity in R6/1 mice but did not affect performance in WT mice.231a This HD-specific stress impairment suggests that olfactory function in HD mice is more vulnerable to the negative effects of chronic restraint stress compared to WT controls. The effects of chronic restraint were unlikely to be due to anhedonia since restrained female R6/1 mice showed the same level of saccharin preference as WT mice at 8 weeks of age.231a Two weeks of restraint-induced anhedonia in female WT mice but the same stress treatment did not impair detection of a peanut butter odor. This further supports a dissociation between the hedonic response to a sweet solution and detection of peanut butter olfactant. The molecular mechanism for this olfactory insensitivity in R6/1 mice may be due to reduced subventricular neurogenesis and/or reduced plasticity in the piriform cortex.230,231 It would therefore be of interest to further investigate this with the hypothesis that restrained R6/1 mice would show the lowest levels of plasticity markers such as NCAM and/or neurogenesis. Deficits in subventricular neurogenesis specific to restrained R6/1 mice would align well with the deficits in hippocampal proliferation in CORT-treated male mice.188a Olfactory Sensitivity Deficits in Male Mice Were Impaired by Restraint Stress Nonstressed male R6/1 mice showed difficulty detecting an odor at 8 weeks and 12 weeks of age.231a This was the first report of an olfactory deficit in male R6/1 mice, corroborating with the deficits found in female R6/1 mice.210a Together they demonstrate that R6/1 olfactory impairment presents early prior to the manifestation of motor symptoms. Olfactory function in male WT mice was also impaired by 2 weeks of restraint stress.231a This was the first evidence of a chronic restraintinduced impairment the ability to detect an

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odor in WT mice. Thus the olfactory sensitivity results follow our hypothesis that chronic restraint would impair functions in both genotypes but impact on R6/1 HD function to a larger extent. Chronic restraint impaired olfactory function in both sexes but acted as a locomotor stimulant in male mice. This demonstrates that stress can have positive or negative effects, dependent on the sex of the subject, the behavioral measure, and the duration of stress treatment. Restrained WT and R6/1 mice performed well in tests for Y-maze memory and sexual vocalizations,231a indicating behaviorally specific effects of restraint stress. The results also demonstrate that the progression of the HD phenotype can be modulated by chronic restraint stress. Olfactory sensitivity in female R6/1 mice was impaired by restraint while WT mice function was unaffected. This provides further evidence for the notion that the HD brain is more vulnerable to stress. This body of research provided the first evidence that stress can alter the onset and progression of HD pathogenesis.181a,188a,210a,231a The main findings our work in the R6/1 mouse model of HD can be summarized as follows: 1. Short-term memory in female R6/1 HD mice, but not WT mice, was impaired by an acute stress.181a The same memory function in male R6/1 mice was impaired by stress hormone (CORT) treatment but again, memory in WT mice was unaffected.188a 2. Reduced hippocampal proliferation levels may contribute to the susceptibility to CORT in HD male mice.188a 3. Olfactory impairment in R6/1 mice was worsened by chronic stress treatment (restraint and oral CORT).210a These stressors also generally impaired olfaction in WT mice. Sexual vocalization and nest-building behaviors were generally unperturbed by stress.210a,231a 4. A hyperlocomotive effect was seen after chronic restraint in male mice, regardless of genotype.231a Overall, Fig. 20.4 exemplifies the heterogeneous effects seen in the stress literature. The

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FIGURE 20.4 Summary of chronic CORT and restraint effects in R6/1 HD and WT mice. (Gray boxes indicate the assessment was not conducted). BDNF, brain-derived neurotrophic factor; HD, Huntington’s disease; MR, mineralocorticoid receptor; WT, wild type.

impact of stress depends on factors such as the type of stressor, the type of measure, the timing of the stress with the measure, and the sex of the animal.99,100 Our results in both WT and R6/1 mice can provide an example for each case. For example, the time dependency of stress was shown by the confinement-induced disruption of memory acquisition, but not retention during Y-maze testing.181a The sex dependency of stress was shown by the CORT-induced anhedonia in female mice, but not male mice.188a The effects of stress also depend on the stress paradigm. Different stressors elicit a unique profile of neural adaptations.56 Restraint, which is a psychological and physical stressor, increases metabolic and sympathetic activity evident in the dramatic suppression of weight gain.231a,232,233 Its psychological effects were also evident in the motivation to explore in the anxiety tests and remain on the accelerating rotarod.231a In contrast, CORT treatment is a pharmacological agonist of the stress hormone receptors. Chronic CORT impaired the hedonic response but had subtle effects on body

weight.188a Stress is a heterogeneous and complex response, which makes it difficult to model using one stress paradigm. Comparing the effects of multiple stressors strengthens the validity of interpretations to the clinic. The type of behavioral measure also influenced the effect of stress. For example, nest quality was not perturbed by chronic CORT or restraint.210a,231a However, olfactory sensitivity was impaired by both restraint and CORT treatment.210a,231a This suggests that olfactory function may be more prone to chronic elevations of glucocorticoid levels. It is surprising that there is very little literature on this important sensory modality in rodents and the impact of stress.

STRESS AS A NOVEL ENVIRONMENTAL MODULATOR OF HD Stress is influenced by a multitude of factors. We have demonstrated using three stressors (acute confinement, chronic CORT and chronic

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REFERENCES

restraint) and two mouse-relevant behavioral measures (memory and olfactory functions) that HD mice are impaired by stress more than WT mice.181a,188a,210a,231a The HD mutation confers a vulnerability to stress. The clinical interpretation is that stress may be more detrimental when experienced by HD patients, compared to healthy individuals. This has yet to be investigated in patients. This investigation is of profound relevance to HD gene carriers who experience psychological burden, distress,171,172 and higher rates of depression and suicide compared to the general population.24,25,234 This body of work provides a substantial contribution to the HD stress literature in which there has only been physiological evidence of a dysregulated HPA axis34,176,177 and a depressive-like response during an acute stress.34 Retrospective questionnaires on lifetime stress and traumatic events may reveal that stress also predicts an earlier onset in HD patients, along with high caffeine intake,235 a passive lifestyle,179 and a higher educational level.236

Future Directions Given the vast literature of chronic restraint effects on affective, cognitive, and motor functions, we expected larger effects of our restraint stress paradigm on WT mice. We reported effects in 2 behaviors (olfactory, rotarod) out of the 7 behaviors tested (rotarod, Y-maze, saccharin preference, olfactory sensitivity, vocalizations, nest building, and anxiety). Future studies incorporating a paradigm such as predator stress,150 social defeat,144 or chronic unpredictable stress216 would inform us of how the HD brain copes with severe stress. The underlying mechanism of susceptibility to stress in HD has yet to be found. We identified a potential proliferative impairment in hippocampal cells that may contribute to stress-induced memory decline.188a Other candidates of interest are markers of synaptic plasticity and dendritic morphology. Epigenetic mechanisms may also support interactions between stress237 and HD.238 If stress is confirmed as a modulator of HD development, therapies to promote

psychological resilience may help prevent an accelerated decline, particularly in memory. Promotion of neurophysiological resilience awaits an understanding of the underlying mechanisms of stress susceptibility. While it is almost impossible to avoid life stress, particularly in HD patients, cognitive-behavioral stress management has been shown to improve affective and immune function in diseases such as breast cancer.239,240

CONCLUSIONS The various findings discussed above demonstrate that HD, a genetically determined disorder, can be modified by interventions such as stress. The preclinical discoveries are paving the way for clinical interventions. A delay in clinical onset, induced by an intervention based on preclinical studies, could reduce financial burden on society and disease burden on afflicted individuals and their families. Effective treatments for HD will come from understanding not only the contribution of stress to the disease process, but of other lifestyle and genetic factors.

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254. Guo Z, Rudow G, Pletnikova O, et al. Striatal neuronal loss correlates with clinical motor impairment in Huntington’s disease. Mov Disord. 2012;27(11):1379e1386. 255. Gurpegui M, Jurado D, Luna JD, Ferna´ndez-Molina C, Moreno-Abril O, Ga´lvez R. Personality traits associated with caffeine intake and smoking. Prog Neuropsychopharmacol Biol Psychiatr. 2007;31(5):997e1005. 256. Hall S, Bigler ED, Rutledge JN. Depression preceding choreiform movements in Huntington’s disease: a case study. Arch Clin Neuropsychol. 1989;4(1):79e92. 257. Harper SQ, Staber PD, He X, et al. RNA interference improves motor and neuropathological abnormalities in a Huntington’s disease mouse model. Proc Natl Acad Sci U S A. 2005;102(16):5820e5825. 258. Harrison DJ, Busse M, Openshaw R, Rosser AE, Dunnett SB, Brooks SP. Exercise attenuates neuropathology and has greater benefit on cognitive than motor deficits in the R6/1 Huntington’s disease mouse model. Exp Neurol. 2013;248:457e469. 259. Hart EP, Dumas EM, Giltay EJ, Middelkoop HAM, Roos RAC. Cognition in Huntington’s disease in manifest, premanifest and converting gene carriers over ten years. J Huntingt Dis. 2013;2(2):137e147. 260. Hellsten J, Wennstro¨m M, Mohapel P, Ekdahl CT, Bengzon J, Tingstro¨m A. Electroconvulsive seizures increase hippocampal neurogenesis after chronic corticosterone treatment. Eur J Neurosci. 2002;16(2):283e290. 261. Hermel E, Gafni J, Propp SS, et al. Specific caspase interactions and amplification are involved in selective neuronal vulnerability in Huntington’s disease. Cell Death Differ. 2004;11(4):424e438. 262. Hersch SM, Ferrante RJ. Translating therapies for Huntington’s disease from genetic animal models to clinical trials. NeuroRx. 2004;1(3):298e306. 263. Hess S, Rohr S, Dufour B, Gaskill B, Pajor E, Garner J. Home improvement: C57BL/6J mice given more naturalistic nesting materials build better nests. J Am Assoc Lab Anim Sci. 2008;47(6):25e31. 264. Hickey MA, Kosmalska A, Enayati J, et al. Extensive early motor and non-motor behavioral deficits are followed by striatal neuronal loss in knock-in Huntington’s disease mice. Neuroscience. 2008;157(1):280e295. 265. Hillerer KM, Neumann ID, Couillard-Despres S, Aigner L, Slattery DA. Sex-dependent regulation of hippocampal neurogenesis under basal and chronic stress conditions in rats. Hippocampus. 2013;23(6): 476e487. 266. Ho AK, Sahakian BJ, Brown RG, et al. Profile of cognitive progression in early Huntington’s disease. Neurology. 2003;61(12):1702e1706. 267. Hockly E, Cordery PM, Woodman B, et al. Environmental enrichment slows disease progression in R6/2 Huntington’s disease mice. Ann Neurol. 2002;51(2): 235e242. 268. Hodgson JG, Agopyan N, Gutekunst C-A, et al. A YAC mouse model for Huntington’s disease with fulllength mutant huntingtin, cytoplasmic toxicity, and

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selective striatal neurodegeneration. Neuron. 1999; 23(1):181e192. Ho¨lter SM, Stromberg M, Kovalenko M, et al. A broad phenotypic screen identifies novel phenotypes driven by a single mutant allele in Huntington’s disease CAG knock-in mice. PLoS One. 2013;8(11):e80923. Hotchkiss AK, Pyter LM, Neigh GN, Nelson RJ. Nycthemeral differences in response to restraint stress in CD-1 and C57BL/6 mice. Physiol Behav. 2004;80(4): 441e447. Hull EM, Dominguez JM. Getting his act together: roles of glutamate, nitric oxide, and dopamine in the medial preoptic area. Brain Res. 2006;1126(1):66e75. ˚. Hult Lundh S, Nilsson N, Soylu R, Kirik D, Peterse´n A Hypothalamic expression of mutant huntingtin contributes to the development of depressive-like behavior in the BAC transgenic mouse model of Huntington’s disease. Hum Mol Genet. 2013;22(17): 3485e3497. Imarisio S, Carmichael J, Korolchuk V, et al. Huntington’s disease: from pathology and genetics to potential therapies. Biochem J. 2008;412(2):191e209. Ingersoll DW, Weinhold LL. Modulation of male mouse sniff, attack, and mount behaviors by estrous cycle-dependent urinary cues. Behav Neural Biol. 1987;48(1):24e42. Jacobsen JPR, Mørk A. Chronic corticosterone decreases brain-derived neurotrophic factor (BDNF) mRNA and protein in the hippocampus, but not in the frontal cortex, of the rat. Brain Res. 2006;1110(1): 221e225. Jhanjee A, Anand K, Bajaj B. Hypersexual features in Huntington’s disease. Singap Med J. 2011;25(6): e131ee133. Johnson SA, Fournier NM, Kalynchuk LE. Effect of different doses of corticosterone on depression-like behavior and HPA axis responses to a novel stressor. Behav Brain Res. 2006;168(2):280e288. Jones MB, Phillips CR. Affected parent and age of onset in Huntington’s chorea. J Med Genet. 1970;7(1): 20e21. Kant GJ, Eggleston T, Landman-Roberts L, Kenion CC, Driver GC, Meyerhoff JL. Habituation to repeated stress is stressor specific. Pharmacol Biochem Behav. 1985;22(4):631e634. Karishma KK, Herbert J. Dehydroepiandrosterone (DHEA) stimulates neurogenesis in the hippocampus of the rat, promotes survival of newly formed neurons and prevents corticosterone-induced suppression. Eur J Neurosci. 2002;16(3):445e453. Katz S. Assessing self-maintenance: activities of daily living, mobility, and instrumental activities of daily living. J Am Geriatr Soc. 1983;31(12):721e727. Kazantsev A, Preisinger E, Dranovsky A, Goldgaber D, Housman D. Insoluble detergent-resistant aggregates form between pathological and nonpathological

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lengths of polyglutamine in mammalian cells. Proc Natl Acad Sci U S A. 1999;96(20):11404e11409. Kee N, Sivalingam S, Boonstra R, Wojtowicz JM. The utility of Ki-67 and BrdU as proliferative markers of adult neurogenesis. J Neurosci Methods. 2002;115(1): 97e105. Khalil H, Quinn L, van Deursen R, et al. What effect does a structured home-based exercise programme have on people with Huntington’s disease? A randomized, controlled pilot study. Clin Rehabil. 2013;27(7): 646e658. Kinnally K, Antonsson B. A tale of two mitochondrial channels, MAC and PTP, in apoptosis. Apoptosis. 2007; 12(5):857e868. Kirkwood SC, Eric S, Richard JV, et al. Evaluation of psychological symptoms among presymptomatic HD gene carriers as measured by selected MMPI scales. J Psychiatr Res. 2002;36(6):377e382. Kirkwood SC, Siemers E, Hodes ME, Conneally PM, Christian JC, Foroud T. Subtle changes among presymptomatic carriers of the Huntington’s disease gene. J Neurol Neurosurg Psychiatr. 2000;69(6):773e779. Kitraki E, Kremmyda O, Youlatos D, Alexis MN, Kittas C. Gender-dependent alterations in corticosteroid receptor status and spatial performance following 21 days of restraint stress. Neuroscience. 2004;125(1): 47e55. Kobal J, Meglic B, Mesec A, Peterlin B. Early sympathetic hyperactivity in Huntington’s disease. Eur J Neurol. 2004;11(12):842e848. Kohl Z, Regensburger M, Aigner R, et al. Impaired adult olfactory bulb neurogenesis in the R6/2 mouse model of Huntington’s disease. BMC Neurosci. 2010; 11(1):114. Konkle ATM, Baker SL, Kentner AC, Barbagallo LS-M, Merali Z, Bielajew C. Evaluation of the effects of chronic mild stressors on hedonic and physiological responses: sex and strain compared. Brain Res. 2003; 992(2):227e238. Kovtun IV, Therneau TM, McMurray CT. Gender of the embryo contributes to CAG instability in transgenic mice containing a Huntington’s disease gene. Hum Mol Genet. 2000;9(18):2767e2775. Koyama S, Soini HA, Foley J, Novotny MV, Lai C. Stimulation of cell proliferation in the subventricular zone by synthetic murine pheromones. Front Behav Neurosci. 2013;7:101. Kutiyanawalla A, Terry AV, Pillai A. Cysteamine attenuates the decreases in TrkB protein levels and the anxiety/depression-like behaviors in mice induced by corticosterone treatment. PLoS One. 2011;6(10):e26153. Lanska DJ, Lavine L, Lanska MJ, Schoenberg BS. Huntington’s disease mortality in the United States. Neurology. 1988;38(5):769e772. Latham N, Mason G. From house mouse to mouse house: the behavioural biology of free-living Mus

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musculus and its implications in the laboratory. Appl Anim Behav Sci. 2004;86(3e4):261e289. 297. Leeflang EP, Zhang L, Tavare S, et al. Single sperm analysis of the trinucleotide repeats in the Huntington’s disease gene: quantification of the mutation frequency spectrum. Hum Mol Genet. 1995;4(9): 1519e1526. 298. Li J-Y, Conforti L. Axonopathy in Huntington’s disease. Exp Neurol. 2013;246(0):62e71. 299. Li M, Dai F-R, Du X-P, Yang Q-D, Zhang X, Chen Y. Infusion of BDNF into the nucleus accumbens of aged rats improves cognition and structural synaptic plasticity through PI3K-ILK-Akt signaling. Behav Brain Res. 2012;231(1):146e153.

300. Li SH, Schilling G, Young III WS, et al. Huntington’s disease gene (IT15) is widely expressed in human and rat tissues. Neuron. 1993;11(5):985e993. 301. Li S-H, Li X-J. Huntingtineprotein interactions and the pathogenesis of Huntington’s disease. Trends Genet. 2004;20(3):146e154. 302. Li X, Morrow D, Witkin JM. Decreases in nestlet shredding of mice by serotonin uptake inhibitors: comparison with marble burying. Life Sci. 2006;78(17): 1933e1939. 303. Filali M, Lalonde R, Rivest S. Cognitive and noncognitive behaviors in an APPswe/PS1 bigenic model of Alzheimer’s disease. Gene Brain Behav. 2009;8(2): 143e148.

C H A P T E R

21 PACAP: Regulator of the Stress Response Sarah L. Gray, Daemon L. Cline Northern Medical Program, University of Northern British Columbia, Prince George, BC, Canada O U T L I N E Introduction to Pituitary Adenylate Cyclasee Activating Polypeptide 280 Discovery, Characterization, and Evolution 280 General Functions 281 Distribution 281 PACAP Receptors Receptor Characterization Associated Pathways Receptor Agonists and Antagonists

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Uncovering PACAP as a Stress Peptide: The Role of Functional Genomics 283 PACAP Regulation of the Autonomic Nervous System The Primary Neurotransmitter at the Sympathetic Adrenomedullary Synapse PACAP’s Role in the SNS Outside of the Sympathetic Adrenomedullary Axis

Abbreviations aa Amino acid(s) ACTH Adrenocorticotropic hormone ANS Autonomic nervous system BNST bed nucleus of the stria terminalis cAMP cyclic adenosine monophosphate CNS Central nervous system CREB cAMP response element binding protein CRH Corticotropin-releasing hormone DBH Dopamine b-hydroxylase

Stress: Physiology, Biochemistry, and Pathology https://doi.org/10.1016/B978-0-12-813146-6.00021-7

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Regulation of Pre- and Post-Ganglionic Sympathetic Nerve Activity Central Regulation of the SNS

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PACAP and the HypothalamicePituitarye Adrenal Axis 286 Regulation of the HPA Axis 286 Extrahypothalamic Regulation of the HPA Axis 287 PACAP in the Pathophysiology of Stress Disorders: A Maladaptive Response to Stress 288 PACAP’s Sex-Specific Association With PTSD Risk: Clinical Association and Mechanistic Evidence 288 Summary

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Conflicts of Interest

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References

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ER a Estrogen receptor a ER b Estrogen receptor b ERK Extracellular signal-regulated kinases GPCR G-protein coupled receptor HPA Hypothalamic pituitary adrenal axis ICV Intracerebroventricular IML Intermediolateral IP3 Inositol triphosphate MEK Mitogen-activated protein kinase kinase PACAP Pituitary adenlylate cyclase-activating polypeptide

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Copyright © 2019 Elsevier Inc. All rights reserved.

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PKA Protein kinase A PKC Protein kinase C PLC Phospholipase C PNMT Phenylethanolamine N-methyltransferase PSNS Parasympathetic nervous system PTSD Post-traumatic stress disorder PVN Paraventricular nucleus Rapgef4/2 Rap guanine nucleotide exchange factor 4/2 SCG Superior cervical ganglion SNA Sympathetic nerve activity SNP Single nucleotide polymorphism SNS Sympathetic nervous system TH Tyrosine hydroxylase VMN Ventromedial nucleus of the hypothalamus

INTRODUCTION TO PITUITARY ADENYLATE CYCLASEeACTIVATING POLYPEPTIDE Discovery, Characterization, and Evolution Pituitary adenylate cyclaseeactivating polypeptide (PACAP) was discovered in 1989 by Akira Arimura.1 Literature around this pleiotropic peptide has since exploded, gaining considerable attention for its pleiotropic regulatory activity and therapeutic potential. Although scientific progress has been made in a number of organ systems and cell types involving PACAP, the current chapter will focus on PACAP’s contributions to the stress response or regulation thereof. For an extensive description of PACAP in other roles, see Ref. 2 KEY POINTS 1. Pituitary adenylate cyclaseeactivating polypeptide (PACAP) is a highly conserved neuropeptide now considered a “master regulator” of the adaptation to stress. 2. PACAP induces the hypothalamice pituitaryeadrenal axis via the paraventricular nucleus in response to chronic psychogenic stress. 3. Across the branches of the sympathetic nervous system, PACAP promotes

sympathetic nerve activity in response to physiological and psychogenic stresses. 4. At the sympathetic adrenomedullary synapse, PACAP is the primary neurotransmitter for sustained catecholamine release in response to physiological and psychogenic stress. 5. Recent advancements in the structural characterization and conformational modeling of the PAC1R will aid in the quest for small molecule regulators of the PAC1R that may have therapeutic potential.

PACAP belongs to the glucagon/secretin superfamily of proteins and is closely related to vasoactive intestinal peptide (VIP), sharing 68% amino acid (aa) sequence similarity. Both VIP and PACAP share traits common to their superfamily, such as C-terminal amidation, random N-terminal coils, and C-terminal alpha-helices. Encoded by the ADCYAP1 gene, the peptide is first translated as a 176-aa preproPACAP, which is then spliced into the mature PACAP peptide which exists as two forms: PACAP38 and PACAP27. A suite of enzymes are responsible for the variable processing of preproPACAP, contributing to the diverse actions of PACAP (process illustrated in the study by Vaudry et al.2). PACAP27 is composed of the 27N-terminal residues of PACAP38, which comprise the highly conserved, biologically active region of PACAP. The C-terminal residues are more variable because they contribute to binding of the receptors as opposed to mediating activation of receptors. Investigations of PACAP’s evolutionary history have shown conservation in amino acid sequence among taxa that diverged over 700 million years ago.3 PACAP27 was even discovered in tunicates, an ancient lineage of protochordates, showing only a single amino acid difference from the human form. This strict conservation across diverse evolutionary groups aligns with a fundamental role in the maintenance of homeostasis.

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PACAP RECEPTORS

General Functions PACAP receptors are expressed in all major organ systems in the body, and thus, PACAP regulates diverse physiological functions. In the nervous system, PACAP acts as a nonnoradrenergic, noncholinergic, mostly preganglionic neurotransmitter. In the central nervous system (CNS), PACAP acts as a neurotransmitter, neuromodulator, and neurotrophic factor, and in the peripheral nervous system, as a neurotransmitter or neuromodulator regulating sensory stimuli and endocrine secretion.4 In the circulatory system, PACAP is a potent vasodilator. In the endocrine system, it is an essential hormone with roles in regulation and production of other endocrine factors, such as catecholamines from chromaffin cells of the adrenal medulla and glucagon from pancreatic a-cells in response to hypoglycemia.5,6 Coordination of these organ systems allows PACAP to contribute to neuroprotection, energy metabolism, catecholamine synthesis and release, nociception, circadian rhythms, headache pathology, stressrelated pathologies, and neurological disorders.

Distribution PACAP is expressed widely within the central and peripheral nervous systems, as well as in many peripheral organs. In the CNS, PACAP is expressed in the brainstem nuclei, hypothalamic nuclei, amygdala, thalamic nuclei, cerebral cortex, medulla oblongata, posterior pituitary, thalamus, and nerves in cerebral blood vessels. Highest concentrations of PACAP occur in the hypothalamus, specifically in the paraventricular, periventricular, ventromedial, suprachiasmatic, and supraoptic nuclei.7 PACAP behaves as a hypothalamic-releasing hormone and is actively transported to the anterior pituitary via the hypothalamic-hypophyseal portal system.8 In the autonomic nervous system (ANS), PACAP is most well-known for its expression in preganglionic neurons of the sympathetic and parasympathetic nervous systems (SNS and PSNS, respectively). Indeed, PACAP is the primary neurotransmitter released from neurons at the sympathetic adrenomedullary synapse. PACAP

is widely distributed in the peripheral organs, occurring in (but not limited to) exocrine and endocrine glands, immune cells, gonads, pancreas, and the genitourinary tract.

PACAP RECEPTORS Receptor Characterization Distribution and composition of PACAP receptors are functionally important to the target cell response. Different tissues and cell types exhibit specific and differing PACAP receptor populations. Scientific literature was originally confounded with different published names of the three PACAP receptors until the International Union of Pharmacology established the names PAC1R, VPAC1, and VPAC2,9 here collectively referred to as PACAP receptors. The PACAP receptors have been cloned and correspond to a 495aa PAC1R,10 457aa VPAC1,11 and 438aa VPAC2.12 All three are Class B Gproteinecoupled receptors (GPCRs). Since many clinically utilized pharmaceuticals target GPCRs, the PACAP receptors have received attention for their therapeutic potential. The PAC1R receptor is highly spliced, having at least 11 known isoforms in rodents and 9 in humans. All PAC1R isoforms are selective for PACAP; yet, the affinity of PACAP27 and 38 differs based on receptor splice variants. Splice sites occur at several points within the receptor structure, which govern the presence or absence of a 21aa segment in the extracellular domain and/ or a set of “cassettes” termed HIP (28aa), HOP1 (28aa), and HOP2 (27aa) that can be inserted (PAC1Rnull lacks all three) in the third intracellular loop to create a variety of splice variants (Fig. 21.1). Each splice variant is functionally distinct, having a specific distribution inducing different downstream signal transduction cascades. For a list of the variants and their downstream signaling, see Ref. 3 Crystal structures for other Class B GPCRs receptors, such as glucagon receptor and corticotropin-releasing hormone (CRH) 1 receptor, and the extracellular domain of the PAC1R13 have contributed to recent structural14 and

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FIGURE 21.1 Schematic representation of the three G-proteinecoupled PACAP receptors; VPAC1, VPAC2, and PAC1R. Sites within the PAC1R receptor where splice variants occur are labelled with an *. The blue and orange boxes represent the HIP (28aa) and HOP1 (28aa)/HOP2 (27aa) “cassettes” that can be selectively expressed in the third intracellular loop of the PAC1R (Dickson and Finlayson, 2009). PACAP, Pituitary adenylate cyclaseeactivating polypeptide.

conformational15 modeling of the PAC1R. This work has provided insights into the contribution of certain features of PAC1R to the structural conformation of the receptor during ligandreceptor interaction. When the extracellular domain includes the 21aa insert, the PAC1Rnull isoform retains a stable, open conformation, thus far unique among Class B GPCRs. This ligand-independent stability in the open conformation is maintained by two factors: a flexible linking region between the seven transmembrane domains and the extracellular domain and the formation of a zipper structure between the 21aa insert in the extracellular domain and the third extracellular loop. The work also details conformational changes within the transmembrane domains from the open to closed state that facilitate activation through association with G proteins.15 Understanding the unique PAC1R structural features and allosteric interactions that occur during conformation changes from the unbound to bound state will greatly facilitate the search for therapeutically relevant small molecules that target PAC1R.

Associated Pathways The PACAP receptors are associated with stimulatory G-proteins (Gs and Gq) as well as inhibitory G-proteins (Gi). Gq leads to increased cytosolic Ca2þ through the activation of

phospholipase C (PLC) and, subsequently, inositol triphosphate. Gs causes the activation of adenylate cyclase, PACAP’s namesake, which has a number of downstream effects. This includes activation of protein kinase A (PKA), Rap guanine nucleotide exchange factor 4 (Rapgef4), and Rapgef2, which contribute to inhibition of the apoptotic pathway involving caspase 9 and 3, p38-mediated cell arrest, and neuritogenesis.16 Rapgef4 also regulates cyclic adenosine monophosphate (cAMP) response elements via cAMP-response element-binding protein (CREBP).2 Gi has an inhibitory effect on adenylate cyclase and therefore decreases production of cAMP. Thus differential activation of coupled G-proteins adds yet another level of control for downstream response to activation of PAC1R. The diversity of functions associated with PACAP mirrors the variety of locations in which its receptors are expressed. PAC1R messenger RNA (mRNA) is most abundant in the brain, pituitary, and adrenal glands, and tissues expressing PAC1R have distinct profiles of PAC1R splice variants. VPAC1 and 2 are most abundant in the lung, liver, and testis.2 PACAP action at the target tissue is thus modulated by the type and density of PACAP receptors, PAC1R isoform expression, activation of the associated Gprotein, and the concentration of PACAP available for receptor binding.

PACAP REGULATION OF THE AUTONOMIC NERVOUS SYSTEM

Receptor Agonists and Antagonists Robust and selective agonists and antagonists for these receptors are highly desirable due to the very short half-life of PACAP in the blood (25 min. Maxadilan is an unrelated peptide isolated from the sandfly, Lutzomyia longipalpis, that has been characterized as a potent, specific PAC1R agonist,18 while a shortened version of Maxadilan, called M65, offers a selective antagonist for PAC1R.19 In the field of small molecule ligands, progress has been made with Src kinase inhibitors, and recent advances in PAC1 receptor modeling (described previously) will help in the hunt for small molecule agonists and antagonists with therapeutic potential. Developing selective agonists and antagonists for the VPAC1 and VPAC2 receptors has proven more difficult with few selective molecules identified.

UNCOVERING PACAP AS A STRESS PEPTIDE: THE ROLE OF FUNCTIONAL GENOMICS At the start of the century, several groups generated PACAP-deficient5,20e22 or PAC1Rdeficient mouse lines23 to better understand PACAP function. It was speculated that knockout of such a highly conserved neuropeptide may produce nonviable embryos, inducing devastating effects on neuronal development or severe impairments in neuronal function that would compromise pre- or post-natal survival. Although postnatal survival of the PACAP- and PAC1R-deficient mice were indeed compromised, gross neuronal development was seemingly intact at birth, and many were able to reach adulthood with surprisingly few neurological abnormalities. Perhaps, the most striking finding from characterization of PACAP-null mice was their inability to survive insulininduced hypoglycemia as a result of impaired catecholamine-induced glycogenolysis and gluconeogenesis.5 This work changed the way the PACAP literature was viewed, seeing

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commonality in seemingly diverse functions attributed to PACAP when framed in the context of the stress response. Almost two decades since the first publications characterizing the PACAPdeficient phenotypes,5,20e22 it is now understood that life without normal PACAP function is inextricably linked with an impaired stress response.

PACAP REGULATION OF THE AUTONOMIC NERVOUS SYSTEM The Primary Neurotransmitter at the Sympathetic Adrenomedullary Synapse PACAP is present along with acetylcholine in the terminals of the splanchnic nerve that innervate chromaffin cells of the adrenal medulla.5 In the past 20 years, Eiden and colleagues have convincingly demonstrated a requirement for PACAP at the sympathoadrenomedullary synapse for sustained catecholamine release in response to stress. While acetylcholine triggers release of epinephrine from the chromaffin cell in response to low-intensity firing or short-term high-intensity firing of the splanchnic nerve, PACAP signaling via the PAC1R is required to maintain catecholamine release and replenish catecholamine stores in the chromaffin cell during maintained stress-induced, high-intensity, splanchnic nerve firing.24 Acetylcholine binding to nicotinic receptors results in cation influx and cellular depolarization via the nicotinic receptor and voltagegated sodium channels and subsequent opening of voltage-gated calcium channels for Ca2þinduced exocytosis of catecholamines.25 Like acetylcholine, PACAP-induced secretion of catecholamines also triggers opening of voltagegated calcium channels to drive Ca2þ-mediated exocytosis, rather than the mobilization of intracellular calcium26; however, the mechanism of PACAP-induced cellular depolarization has not been confirmed. It is known to occur independently of voltage-gated sodium channels, but the identity of the channel responsible for cellular depolarization in response to

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PACAP/PAC1R-induced protein kinase C (PKC) and cAMP activation is not known (this topic has recently been reviewed and a model proposed by Eiden et al.24). As alluded previously, a critical role for PACAP in catecholamine secretion from the chromaffin cells was demonstrated in mice lacking PACAP. These mice could induce release of epinephrine acutely in response to hypoglycemia, but its release could not be sustained, demonstrating that PACAP was required for sustained catecholamine biosynthesis and secretion.5 This was confirmed in an independently generated line of PACAPnull mice when multiple stressors, including ether exposure and immobilization, failed to increase blood glucose in mice lacking PACAP.27 Ex vivo work in adrenal slices demonstrated the requirement of PACAP for catecholamine secretion in response to highfrequency stimulation of the splanchnic nerve, mimicking stress-induced nerve transduction, and showed PACAP’s ability to induce catecholamine secretion in sections desensitized to acetylcholine.28 PACAP’s ability to maintain epinephrine secretion under stress is due to its effects on gene expression of three catecholamine biosynthetic enzymes, tyrosine hydroxylase (TH), dopamine b-hydroxylase (DBH), and phenylethanolamine N-methyltransferase (PNMT).5,28,29 Direct effects of PACAP on gene expression of these enzymes were confirmed in primary bovine and porcine chromaffin cells, where PACAP38-induced expression of TH, DBH, and PNMT mRNA,28 which is thought to occur via PKA-CREBP30 and/or ERK-MEK (extracellular signal-regulated kinases and mitogen-activated protein kinase kinase, respectively), induced regulation of TH and PNMT gene transcription.24 In summary, PACAP is the primary neurotransmitter at the sympathoadrenomedullary synapse for epinephrine synthesis and release from the chromaffin cell in response to psychogenic stress (a stressor requiring cognitive processing via the limbic system) and physiological stress (a stressor imposing immediate threat on homeostasis that does not require cognitive processing; Fig. 21.2).

PACAP’s Role in the SNS Outside of the Sympathetic Adrenomedullary Axis Regulation of Pre- and Post-Ganglionic Sympathetic Nerve Activity In addition to being a noncholinergic transmitter at the sympathoadrenomedullary synapse, PACAP acts elsewhere in the ANS predominantly as an SNS stimulant and a PSNS repressor. In this chapter, we will focus on PACAP’s role in the SNS. While it is well accepted that PACAP’s effect in the SNS is that of activation, unlike the sympathoadrenomedullary synapse where PACAP has been defined as the primary neurotransmitter for catecholamine release and production in the chromaffin cell, the same has not be proven for norepinephrine production and secretion from the postganglionic nerves across the branches of the SNS. Additionally, the mechanisms by which PACAP promotes sympathetic nerve activity (SNA) and norepinephrine production and secretion in postganglionic nerves have not been extensively characterized. What is known about PACAP in the SNS is mostly gleaned from work in the superior cervical ganglion (SCG). For example, PACAP is expressed in >95% of the preganglionic nerves originating in the intermediolateral cell column of the spinal cord and acts on neurons of the SCG, which express PAC1R HOP1 variants to enhance production and release of catecholamines via PLC induction.31e34 In addition to preganglionic nerves innervating the SCG, PACAP is also expressed in preganglionic nerves innervating other ganglia of the SNS. In the porcine celiac/cranial mesenteric complex this was inferred by detection of PACAP-immunoreactive fibers within the ganglion,35 and in the stellate ganglion was more definitively determined through detection of PACAP in neurons expressing acetylcholine.36 While there is some evidence that PACAP mRNA is expressed in postganglionic neurons of the SCG,37 other co-localization studies show an absence of PACAP in catecholamine-positive cells in several sympathetic ganglia including the major pelvic ganglia,38 the inferior mesenteric ganglia,39 and the SCG.40 A recent paper

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FIGURE 21.2 PACAP (P) acts centrally and peripherally to regulate stress axes, including the hypothalamice pituitaryeadrenal (HPA) axis, sympathetic adrenomedullary system (SAS), and noradrenergic sympathetic response (NSNS). Evidence suggests involvement of PACAP in the bed nucleus of the stria terminalis (BNST) and amygdala for upstream regulation of corticotropin-releasing hormone (CRH) in the hypothalamus (paraventricular nucleus [PVN]) in response to psychogenic stress. The PVN and ventromedial nucleus (VMN) are suspected sites of hypothalamic regulation of thermogenesis via the sympathetic nervous system. Peripherally, PACAP (P) is released from preganglionic nerves of the sympathetic nervous system regulating downstream catecholamine production and secretion. PACAP, Pituitary adenylate cyclaseeactivating polypeptide.

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detected PACAP immunoreactivity in DBHþ neurons originating in the sympathetic chain ganglia that innervate skin,41 and a small number of PACAP-expressing, DBHþ neurons were detected in the inferior thoracic ganglia.42 In summary, within several branches of the SNS, PACAP colocalizes with acetylcholine in sympathetic preganglionic neurons, similar to the splanchnic-adrenal system. Whether PACAP is required for the maintenance of catecholamine secretion from postganglionic nerves of the SNS has not been established and warrants further investigation (Fig. 21.2). Central Regulation of the SNS Upstream of the ganglia, PACAP has been shown to regulate SNA at the level of the spinal cord where injection of PACAP38 at the T5/T6 level of the spinal cord induces widespread SNA.43 Additionally, pharmacological administration of PACAP to the hypothalamus via intracerebroventricular (ICV) injection into the third ventricle of the brain increases SNA to several organs, including brown adipose tissue, white adipose tissue, kidney, heart, liver, and the adrenal gland.27,44 Mouse models of PACAP deficiency demonstrate an impaired sympathetic response to stress, the example mentioned previously was the impaired epinephrine response to hypoglycemia in PACAP-null mice. In another PACAPnull mouse line, maladaptation to cold stress (another metabolic stress) is reflected by reduced postnatal survival rates of PACAP-null mice that increase with increased housing temperature.21 This has been shown to be associated with impaired catecholamine-induced adaptive thermogenesis in brown adipose tissue.45 These findings are supported by pharmacological gain-of-function experiments that show increased SNA and increased body temperature in response to ICV or VMNespecific injection of PACAP.46e48 Further work to understand the level at which PACAP is acting within the CNS and SNS to regulate adaptive thermogenesis is underway. Taken together, these studies provide evidence that PACAP acts within the hypothalamus,

spinal cord, and at the preganglionic/postganglionic synapses of several sympathetic ganglia to regulate the sympathetic response to stress. Further work examining PACAP’s role in the various sympathetic target organs will determine if PACAP induces common or diverse mechanisms throughout the branches of the SNS to regulate this stress axis in response to threats on systemic and psychogenic homeostasis (Fig. 21.2).

PACAP AND THE HYPOTHALAMICePITUITARYe ADRENAL AXIS Regulation of the HPA Axis PACAP also regulates the other major stress axis, the HypothalamicePituitaryeAdrenal axis (HPA). This axis facilitates glucocorticoid secretion from cells of the zona fasciculata of the adrenal cortex. This signal originates in CRHergic fibers of the hypothalamic paraventricular nucleus (PVN) that receives inputs from a variety of higher brain regions, produced in response to detection of both psychogenic and physiological stressors of both acute and chronic nature. CRH interacts with corticotrophs of the anterior pituitary via the hypophyseal portal system initiating secretion of adrenocorticotrophic hormone (ACTH). While PACAP plays a regulatory role of the HPA axis, PACAP is not required for CRH- or ACTH-induced glucocorticoid secretion.49 Instead, data suggest PACAP regulates activation of the HPA axis at or above the level of the hypothalamus in response to certain types of stress, specifically psychogenic stress and thus is an example of a mechanism by which the CNS imposes temporal and situational regulation of the HPA axis. For example, studies using PACAP-deficient mice have demonstrated PACAP is required for full activation of the HPA axis in response to psychogenic stress but not physiological stress, as PACAP-null mice have an attenuated corticosterone response to acute (single bout restraint stress) and chronic

PACAP AND THE HYPOTHALAMICePITUITARYeADRENAL AXIS

psychogenic stressors (repeated daily restraint stress, social defeat stress, open-field exposure) but not in response to physiological stressors (sepsis, hypoglycemia, acute pain, short-term cold exposure, ether exposure).49e51 The most likely place where PACAP is exerting its effects on the axis is in the regulation of CRH neurons in the PVN, where the interaction and influence of PACAP on these neurons are well established. PACAPergic fibers innervate the PVN, forming synapses with CRH neurons.52 ICV injection of PACAP induces CRH mRNA expression53 and CREB phosphorylation54 in CRH-positive neurons of the PVN.54 In vitro studies in a pituitary cell line (aT3-1) showed PACAP-induced upregulation of CRH to be mediated by the cAMP/PKA signal transduction pathway, but not by PKC.54 Finally, in response to restraint stress, CRH mRNA expression and neuronal activity in CRH neurons of the PVN increased in a PACAP-dependent manner.49,51,55 Collectively, these results provided compelling evidence that PACAP acts directly at the PVN to regulate CRH release in response to psychogenic stress, with particular importance for sustained activation of the HPA axis in response to chronic, psychogenic stress (Fig. 21.2). In addition to PACAP’s influence at the hypothalamus, there is evidence that PACAP may also act at other levels of the HPA axis, including directly on corticotrophs of the anterior pituitary. PACAP was first isolated from the ovine hypothalamus and named for its ability to potently induce adenylate cyclase activity.1 Like other endocrine cells of the anterior pituitary, normal corticotrophs are PACAP-responsive,56 yet in vivo, it has not yet been shown that PACAP is required for ACTH-induced corticosterone secretion.49 At the adrenal cortex, PACAP receptors are not expressed on cells of the zona fasciculata,57 and thus a significant role for PACAP in glucocorticoid synthesis and release has not been identified.

Extrahypothalamic Regulation of the HPA Axis If the major site of regulation by PACAP is via PAC1R activation on CRH neurons of the PVN,

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this raises the question of where do PACAPergic fibers originate from and which extrahypothalamic sites are important for this regulation. One potential site for PACAP-mediated regulation of the HPA axis is the bed nucleus of the stria terminalis (BNST). The BNST is a brain region known to be involved in anxiety-related behaviors and thus the adaptive/maladaptive response to psychogenic stress. PACAP and PAC1R mRNA expression are increased in the BNST with chronic stress (repeated daily restraint stress) but not acute stress (after a single exposure of restraint stress) and associated with increased circulating glucocorticoid levels. Interestingly, glucocorticoid treatment without the stressor did not reproduce these results suggesting the stressor, and not the endocrine response to the stress, is the trigger for PACAP release in the BNST.58 PACAP38 infusion into the BNST transiently enhanced circulating corticosterone, but not if infused into the lateral ventricles.59 Complementary experiments showed that anxiety-like responses and increased circulating corticosterone levels in response to stress could be blocked with chronic PAC1R antagonist infusion (PACAP6-38).60 Genetic ablation of PACAP attenuates peak circulating corticosterone levels in response to repeated daily restraint stress but has no effect on glucocorticoid levels after a single restraint stress exposure compared with wild-type control mice.61 These studies suggest the BNST may be an important site whereby PACAP acts upstream of the hypothalamus to induce HPA activity in response to chronic psychogenic stress (Fig. 21.2). Other regions of the limbic system known to be involved in integrating behavioral responses to external stressors such as pain and fear and associated with PACAP is the central and medial amygdala. While this chapter will not address the large body of literature describing an important role for PACAP in fear learning and nociception, recent literature suggest these regions could be higher order brain regions specifically involved in PACAP’s control of the HPA axis.49,62,63 A functional link between PACAP, fear, and fear learning is a growing area of interest with clinical implications.

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PACAP IN THE PATHOPHYSIOLOGY OF STRESS DISORDERS: A MALADAPTIVE RESPONSE TO STRESS Throughout this chapter, we have highlighted PACAP as a critical, high-order mediator of the response to psychogenic and physiological stress, orchestrating the adaptive response to acute and chronic stress by regulating the SNS or the HPA axis. As such, it is foreseeable that fundamental changes in the regulatory or coding sequences within the PACAP or PACAP receptor genes may impair the coordination of the adaptive response to stress resulting in maladaptive pathology. Clinically, PACAP has been studied in the context of several stress-associated psychopathologies including anxiety, depression, and PTSD. We will use PTSD as an example of a stress-related disorder for which integration of data from clinical and lab-based molecular studies is mounting evidence for a mechanistic role for PACAP in disease pathology.

PACAP’s Sex-Specific Association With PTSD Risk: Clinical Association and Mechanistic Evidence In 2011, Ressler et al.64 reported a singlenucleotide polymorphism (SNP) in the promoter region of the PAC1 receptor gene (ADCYAP1R1) to be associated with PTSD in adult females. This study provided clinical evidence for PACAP as a neuroendocrine regulator of the adaptive response to chronic stress, and additionally, suggested a mechanism for sex-dependent risk associated with PTSD. This SNP (rs2267735) resides in an estrogen response element (ERE) in the ADCYAP1R1 promoter. Estrogen regulates gene expression via estrogen receptor a (ERa) and estrogen receptor b, which subsequently interact with EREs within the promoter of target genes. As predicted, in vitro studies revealed the functional implications of the PAC1R mutant to be impaired estradiol/ERa binding to an ERE located in the PAC1R promoter, which resulted in impaired PAC1R expression.65 In vivo experiments in rodents further supported estrogen-

mediated regulation of PAC1R expression as ICV infusion of estradiol-enhanced PAC1 receptor gene expression in regions of the brain known to respond to psychological stress, such as the BNST and prefrontal cortex.64,65 Detailed reviews of PACAP/estradiol interactions in chronic stress in the context of sex-dependent risk for psychopathologies have recently been published and expand considerably on this topic.66,67 In humans, neurological imaging of female brains showed increased neurological activity and decreased connectivity between the hippocampus and amygdala in subjects carrying two copies of the mutant C allele, compared with the GC or GG genotype, indicating that the PAC1R CC genotype results in altered plasticity in brain regions known to be important for the adaptation to chronic stress.68 Taken together, these data suggest PACAP mediates the adaptive response of fear learning in response to traumatic stress in females by responding to estrogen-mediated upregulation of the PAC1 receptor system. Such dysregulation of PACAPergic signaling may exacerbate anxiety-like behavioral responses after exposure to traumatic stressors resulting in maladaptation. This work may support PACAP as a biomarker for PTSD risk or as a molecular target for therapeutic intervention in individuals who suffer from this debilitating illness.

SUMMARY Given the multitude of varied physiological and psychogenic stressors that can disrupt homeostasis within an organism, regulation of the key stress axes requires complex neural and endocrine control at multiple levels of the axes. It is becoming clear that PACAP is a key neuropeptide required for integration of stress signals at the hypothalamus, initiating the HPA axis and/or the SNS selectively in response to specific stressors and farther down the axes as a primary stimulant for catecholamine production and release in the SNS. The intriguing nature by which PACAP-induced activation of the HPA axis occurs in response to chronic

REFERENCES

psychogenic stress, whereas PACAP-induced SNA occurs in response to both psychogenic and physiological stressors, highlights the role of PACAP as a high order regulator of the stress response. Expanding clinical data supporting a role for PACAP in the pathology of maladaptive disorders to stress supports the extensive foundational work that has established PACAP as a key molecule integrating the stress response.

Conflicts of Interest The authors have no conflicts of interest to declare.

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C H A P T E R

22 Glucose Transport Anthony L. McCall1,2

1

Division of Endocrinology & Medicine, University of Virginia (Emeritus), Charlottesville, VA, United States; 2Division of Nutritional Sciences, Cornell University, Ithaca, NY, United States O U T L I N E

Introduction Glucose Transporter Proteins Glucose Transport Overview of Glucose Transport Regulation Stress Hormones and Glucose Transport GLUTs as Stress-Responsive Proteins Metabolic Stresses and Glucose Transport Hypermetabolism Mitochondrial Inhibitors Glucose Deprivation

Obesity, Type 2 Diabetes Mellitus, and Cardiovascular Disease

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INTRODUCTION Glucose transport supplies fuel that is needed for energy metabolism by most mammalian cells. Glucose is a very common metabolic substrate that is used both as a fuel and a signaling molecule. The supply of glucose is especially important for certain cells, such as brain neurons, which have a high metabolic rate supported by an obligate consumption of glucose as fuel in most circumstances. Transport of glucose is regulated by a variety of factors including those associated with several aspects of cellular stress. Transport proteins that accomplish glucose

Stress: Physiology, Biochemistry, and Pathology https://doi.org/10.1016/B978-0-12-813146-6.00022-9

Overall Effects of Transport Regulation

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Signaling Cascades and Glucose Transport

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GLUTs, Glucose Transport, and Metabolism in Chronic Disease StatesdCancer

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Summary

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transport are modulated in their expression, cellular distribution, synthesis, and half-lives by stress-related factors. Such factors include stress hormones, a variety of metabolic stresses, such as cellular energy demand, metabolic poisons, inflammation, and stress-related kinase signaling, endoplasmic reticulum (ER) stress, and chronic diseases. The net effect of such regulation may be favorable and serve to ensure appropriate distribution of glucose fuel to tissues during stress that most require this particular fuel. However, some adaptations of glucose transport promote or worsen diseases such as cancers.

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Copyright © 2019 Elsevier Inc. All rights reserved.

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KEY POINTS • Glucose transport is a highly regulated process accomplished mostly by facilitated diffusion using carrier proteins to cross cell membranes. • There are many glucose transporters, but the most important are known as GLUTs and five of them (GLUT1-5) that have been most fully characterized, are the primary focus of this chapter. • Stress in a variety of acute and chronic forms affects the activity of glucose transporters. • The signal transduction mechanisms by which glucose transporters are regulated are increasingly being identified and differ depending on the metabolic stress and the tissues affected. • Numerous signaling pathways appear to be involved in transporter regulation. • The stresses that regulate GLUTs are not only acute biological stresses but also chronic low-grade inflammation, and associated chronic diseases such as obesity, type 2 diabetes, cardiovascular disease and the growth and spread of many tumors. • Some of these glucose transport effects are compensatory while others are pathogenic. • Ultimately, manipulation of GLUTs could be used as treatment for some of these chronic diseases.

Glucose Transporter Proteins The nomenclature for glucose transport proteins (commonly GLUTs) has changed over the years since their original cloning1 in HepG2 cells. GLUT proteins (see reviews by Mueckler and Thorens)2,3 are currently 14 in number in humans although GLUT1-5 are the most studied of the members of this superfamily of membrane transport proteins. The SLC2 (SoLute Carrier) family of genes encode these GLUTs. Not all the GLUTs are transporters solely for glucose or even other sugars. Individual members of the GLUT proteins are distinguished by their kinetics and

substrates. They primarily carry sugars, sugar alcohols, and urate. These are among the substrates transported by GLUT proteins (Table 22.1). For some, it is less clear what their physiological roles are. The structure of GLUTs is generally about 500 amino acids with intracellular amino and carboxyl terminals with 12 membrane-spanning domains and an exofacial loop with a carbohydrate moiety (Fig. 22.1). Glucose transport proteins are divided into three general groups based on their sequence homology (Fig. 22.2). These are Class 1 (GLUTs 1e4, 14), Class 2 (GLUTs 5, 7, 9, and 11), and Class 3 (GLUTs 6, 8, 10, 12, and HMIT). See Table 22.1 for further details of the family of GLUT proteins. GLUT proteins have 12 transmembrane segments, a single N-linked glycosylation site, a relatively large, central, cytoplasmic linker domain, and exhibit topologies with both their N and C termini positioned in the cytoplasm.1 The Classes 1 and 2 GLUT proteins are structurally distinguishable from the Class 3 proteins by virtue of the location of their sites of N-linked glycosylation, which reside in the first exofacial linker domains of the Classes 1 and 2 GLUTs and in the fifth exofacial linker domains of the Class 3 proteins. It should be noted that sodiumeglucose cotransporters (SGLT),4,5 usually abbreviated as SGLT, are also present in some tissues. These differ from GLUT protein in that the SGLTs are active energy dependent, whereas GLUT transporters are facilitated diffusion carriers. The GLUT transporter proteins are the primary focus of this review although there are studies indicating compensatory adjustments of SGLTs in response to altered GLUTs due to stress. The following is an abbreviated characterization of the GLUT1-5 proteins (also see Table 22.1). GLUT1 is essentially everywhere within the mammalian body and provides for basal transport of glucose. It has been extensively studied and characterized, and it is considered to be the prototype of GLUT stress responders. Its ability to be regulated in normal healthy circumstances is moderate (often twofold), but under certain conditions of cellular stress or in cancerous cells, it can be increased up to 30-fold. It is highly concentrated in the red blood

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INTRODUCTION

TABLE 22.1

Transporter

Transporter

Primary Isoform (aa)/Molecular Weight (kDa)

GLUT1 (SLC2A1)

Substrates (Affinity-Km)

Major Tissue Expression

492/54

• Glucose (3 mmol/L) Galactose (17 mmol/L) • Mannose (20 mmol/L) Glucosamine (2.5 mmol/L) • DHA (1.1 mmol/L)

Ubiquitous

GLUT2 (SLC2A2)

496/45

• Glucose (14 mmol/L) • Galactose (92 mmol/L) • Mannose (125 mmol/L) Glucosamine (0.8 mmol/L) • Fructose (76 mmol/L)

Pancreas, liver, kidney, small intestine

GLUT3 (SLC2A3)

496/45

• • • • •

Brain, testis

GLUT4 (SLC2A4)

509/55

• Glucose (5.0 mmol/L) • DHA (0.98 mmol/L) • Glucosamine (3.9 mmol/L)

Muscle, fat, heart

GLUT5 (SLC2A5)

501/55

Fructose (6 mmol/L)

Intestine, kidney, testis

GLUT7 (SLC2A6)

507/46

Glucose

Spleen, brain, leucocytes

GLUT7 (SLC2A7)

524/53

• Glucose (0.3 mmol/L) • Fructose(0.2 mmol/L)

Small intestine, colon, testis, prostate

GLUT8 (SLC2A8)

477/51.5

Glucose (2.0 mmol/L)

Testis, brain, adrenal gland, liver, spleen, fat, lung

GLUT9 (SLC2A9)

9a 511/66/ 9b 540/46

• Glucose (0.61 mmol/L) • Fructose (0.42 mmol/L) • Urate (0.9 mmol/L)

Liver, kidney, placenta, pancreas

GLUT10 (SLC2A10)

541/57

• 2-Deoxy-glucose (0.3 mmol/L) • Galactose

Heart, lung, brain, liver, muscle, pancreas, placenta, kidney

GLUT11 (SLC2A11)

• • • •

• Glucose (0.16 mmol/L) • Fructose (0.16 mmol/L

• 11a Heart, muscle, kidney • 11b Placenta, fat, kidney • 11c Heart, muscle, fat, pancreas

GLUT12 (SLC2A12)

617/67

Glucose

Muscle, fat, heart, small intestine, prostate, placenta

HMIT (SLC2A13)

618, 629/69

Myoinositol (0.1 mmol/L)

Brain

GLUT14 (SLC2A14

497/520/N/A

N/A

Testis

496/54 11a 11b 11c

Glucose (1.4 mmol/L) Galactose (8.5 mmol/L) Mannose Xylose DHA

DHA, Dehydroascorbic acid.

cells and the endothelial cells that comprise the bloodebrain barrier.6 There is a haplotype deficiency syndrome of GLUT1 that causes a chronic seizure disorder.7

GLUT2 is characterized by a very high Km for glucose, which is about 17 mM. Originally it was cloned from liver DNA libraries by Thorens et al.8 Its expression is most notably in pancreatic

296

22. GLUCOSE TRANSPORT

FIGURE 22.1

Model of glucose transporter protein.

beta-cells and intestinal and kidney epithelial cells and hepatocytes.9 The high Km effectively allows rapid equilibration of glucose between extracellular and intracellular compartments. GLUT2 is an important part of the glucose stimulation of insulin secretion, but it is not a ratelimiting step (glucokinase is). GLUT2 is also important for portal venous glucose sensing and may perform a sensor like function in other areas including the brain. GLUT3 was originally cloned from fetal skeletal muscle cells by Kayano et al.10, but it is primarily expressed within the brain neurons and testis. It is also expressed in placenta, fetal skeletal muscle, platelets, and leukocytes. See the

FIGURE 22.2

excellent review by Simpson et al.11 that summarizes research over 20 years, much of which his group has contributed. GLUT3 is a stressresponsive glucose transport protein in brain and other tissues. Its presence in brain is related to the high need of neurons for glucose as a fuel and the obligate requirement of this fuel for normal brain function. GLUT4 is best recognized and studied as the insulin-responsive glucose transporter cloned by James and around the same time by Birnbaum.12,13 It is present in adipose tissue, skeletal muscle, heart, and in several important areas within the brain. The function of GLUT4 as insulin-responsive has made it studied

The structure of Classes 1 and 2 GLUT proteins versus Class 3 GLUT proteins.

INTRODUCTION

particularly as a factor in the pathophysiology of diabetes mellitus.14 Its ability to move from intracellular locations to the cell plasma membrane ensures its proper response to insulin binding to its receptor to allow transfer of glucose into insulinsensitive tissues.15,16 Like most of the wellstudied GLUTs, it is altered in response to stresses of several types. GLUT5 is not a glucose transport protein, but rather it is a low-affinity fructose transporter that is highly expressed in the intestine, the testis, the kidney, fat tissue, skeletal muscle, and sperm of some species. It was initially identified by Kayano et al.17 Its expression is increased in type 2 diabetes mellitus in skeletal muscle and intestine. It can increase in response to metabolic stress also.

Glucose Transport Passive diffusion of glucose across cellular phospholipid membranes is limited by its modest hydrophobicity. Transfer of glucose across cell membranes uses facilitated diffusion, which is a carrier protein-mediated process. Two types of facilitated diffusion existdenergydependent and energy-independent. A few tissues, such as the gut and kidney epithelium, transport glucose with an SGLT. For most cells however, energy-independent facilitated diffusion is the main mechanism. Facilitated diffusion via carrier proteins transports glucose down a concentration gradient in a saturable manner with isoform-specific kinetic differences. There are different isoforms of a superfamily of hydrophobic, integral membrane proteins (see Fig. 22.1). The cloning by Mueckler and colleagues1 of the first member of this family, GLUT1, has enabled much of the physiological investigation of glucose transport and these proteins. Table 22.1 lists all members of the GLUT family of proteins and partially characterizes them giving a brief synopsis of the size and molecular weight of the remaining isoforms of GLUT1 through GLUT14. It also indicates their substrate specificity and the major tissue expression that has so far been identified.

297

Overview of Glucose Transport Regulation Glucose transport is highly regulated and varies in kinetics, tissue distribution, and isoforms from one cell type to another.2,3 Some cells, such as red blood cells and brain neurons, have an obligate consumption of glucose. For most cells, facultative use of glucose exists, permitting other metabolic fuels, such as fatty acids, to supply the bulk of local energy requirements. Regulation of transport occurs in response to altered energy requirements of tissues, so it is not surprising that one form of cellular stress, energy lack, or metabolic poisons that alter fuel availability are potent regulators of glucose transport and transporter expression by different tissues. Regulation of GLUT proteins may occur by variation of the amounts of synthesis or degradation of the GLUT protein or messenger RNA (mRNA). An increased transcription of GLUT mRNA or other regulatory effects on GLUTs may occur as a result of growth factors and stress hormones, such as glucocorticoids and epinephrine. In some tissues, these hormones have different effects on GLUTs or transport, emphasizing their tissue-specific control. The effects of growth factors and other physiological factors often involved in stress responses, increase GLUT1 transcription, as first shown by Birnbaum and colleagues.18

Stress Hormones and Glucose Transport Stress hormone responses influence glucose transport in a manner that is specifically based on tissue type and isoform. Among the important influences on glucose transport are the stress hormones, particularly the adrenal glucocorticoids. Another common stress hormone response that regulates glucose transport is the secretion of catecholamines, epinephrine, and norepinephrine, by the adrenal medulla and the sympathetic nervous system. Glucocorticoid stress responses or exposure to synthetic glucocorticoid drugs, such as dexamethasone, regulate transport of glucose in several tissues and also regulate the expression of several

298

22. GLUCOSE TRANSPORT

glucose transporter (GLUT) isoforms. In cultured adipocytes and in skeletal muscle which are both insulin-sensitive tissues, glucocorticoids downregulate glucose transport by decreasing the plasma membrane concentration of glucose transporters, particularly GLUT4, the insulin-sensitive glucose transporter. This was shown by Carter-Su19 and confirmed by several groups. There is a cellular redistribution of GLUT4 that is opposite to the effects of insulin. In adipocytes, glucocorticoids may stimulate the synthesis of GLUT1 however again indicating isoform specificity of responses. Chipkin et al. have described that GLUT1 is also overexpressed in the small blood vessels of the brain (which comprise the bloodebrain barrier) after in vivo dexamethasone treatment.20 b-Adrenergic drugs and the stress hormone catecholamines may also reduce insulin-sensitive glucose transport via action of GLUT4 in tissues such as muscle and fat. Precise mechanisms, were incompletely defined,12 but probably include altered cellular distribution, which initially was incompletely defined.21 Studies subsequently have shown indeed that the effects of epinephrine to create insulin resistance is at least in part due to altered transport activity of GLUT4 that is linked specifically to the inability of GLUT4 to properly translocate to the cell surface in adipocytes.22 These examples illustrate tissue- and isoform-specificity effects of stress on glucose transport. The net effect of such regulation may be to redistribute glucose to tissues that need it as a fuel. They preserve the glucose available to tissues such as the brain, which slowly and incompletely adapt to glucose deprivation. Tissues such as muscle during stress may use other fuels, for example, fatty acids.

GLUTs as Stress-Responsive Proteins Wertheimer and colleagues identified GLUT1, the ubiquitously expressed glucose transporter, which is constitutively expressed at low levels by most cells, as being regulated by cellular stress.23 They found that many cell stressors including glucose starvation, mercaptoethanol, calcium ionophores, tunicamycin, heat shock, and so on upregulated glucose transport and

GLUT1 expression in L8 muscle cells and 3T3L1 fibroblasts in an isoform-specific fashion. By contrast, another glucose transporter, GLUT4, did not respond similarly. The GLUT1 stress responses were regulated in a manner very similar to that seen for another cell stress protein, glucose-regulated protein 78 (GRP78), in these studies. Subsequently, other studies confirmed the stress-related increase in GLUT1 expression in a variety of tissues and cell types. GLUT1 is thus the prototypical stressresponsive GLUT isoform. Nonetheless, many GLUT isoforms can be regulated by factors associated with stress.

Metabolic Stresses and Glucose Transport Hypermetabolism One aspect frequently seen with cellular stress or tissue injury is a transient hypermetabolism. Examples include trauma or ischemia. This hypermetabolism is likely used to provide biochemical energy in the form of adenosine triphosphate (ATP) or its high-energy phosphate-bond equivalents for restoration of ionic cellular equilibrium and energy-dependent repair processes. The increased energy requirements after metabolic stress or cellular injury might exert regulatory effects on glucose transport and transporters through an energy intermediate in the metabolic pathways of glucose. Adenosine monophosphate signaling to AMP kinase (AMPK), which is a fuel sensor enzyme, is an example. Mitochondrial Inhibitors Metabolic stress may regulate glucose transport and transporters in vivo and in vitro. Inhibitors of mitochondrial oxidative phosphorylation have long been known to increase glucose transport in vitro, although the precise metabolic pathways and mechanisms are complex and only partly elucidated. Kalckar and colleagues first described this phenomenon in cultured fibroblasts.24 Mitochondrial inhibitors may similarly affect GLUT proteins. Hypoxia and mitochondrial inhibitors or uncouplers of oxidative phosphorylation (dinitrophenol (DNP),

INTRODUCTION

antimycin, and azide) act similarly to hypoxia25 and increase both GLUT1 mRNA and protein expression in vitro.26 Increasing evidence supports the idea that GLUT327 may also respond to these same stimuli, albeit through different mechanisms than GLUT1 or GLUT4.28 An interesting study by Quintanilla and colleagues29 found evidence to support the importance of cellular Caþ2 in the modulation of GLUT1 responses to metabolic stress in cultured epithelial cells. They found that within normal and slightly low range, cytosolic Caþ2 prevents the upregulation of GLUT1 by metabolic stress in rat liver epithelial cells. It decreases the rate of 2deoxyglucose uptake in a dose-dependent fashion. It appears that intracellular calcium levels are permissive in the activation of the GLUT1 by metabolic stress and insulin under these circumstances. A number of other pathways are used to augment GLUT expression as discussed in the following. Glucose Deprivation In like manner, glucose transport is upregulated by glucose deprivation in vitro and in vivo. This has been shown in fibroblasts, muscle cells, and in many other cell types. Some forms of feedback probably occur either by metabolic intermediates in the metabolism of glucose, signaling cascades, and/or feedback directly from the energy state of the cell. Perhaps, ATP levels or ATP/ADP (adenosine diphosphate) ratios may be involved in mediating this increased glucose transport. Indeed, there is a substantial body of evidence that suggests regulation of glucose transport in many tissues is influenced by the availability of the transport substrate itself, that is, glucose.30 This emphasizes glucose itself as a signaling molecule. Although there has been some controversy about this point, in some tissues,31e33 there is evidence to suggest that local energy metabolism may be affected by such stress-mediated regulation of glucose transporters and glucose transport. For example, with in vivo hypoglycemia, an upregulation of brain endothelial transport of glucose occurs.34,35 At blood glucose levels of about 3 mM (equal to 56 mg/dL), normally subtle brain dysfunction occurs related to lack

299

of adequate glucose entry into the brain to support the rather rigid requirement for glucose by brain metabolism. With an adaptation of glucose transport that occurs within several days after repeated hypoglycemia (in this case produced in vivo by overdoses of exogenous insulin), there is maintenance of glucose-6-phosphate and brain creatine phosphate and ATP levels. Presumably, the increased transport efficiency, which appears to involve both brain vascular (GLUT 1) and the primary neuronal glucose transporters (GLUT3),36,37 effectively operates to shuttle a greater amount of glucose across the brain endothelial and neuronal cellular barriers. This helps to feed starving neurons. Interestingly, although such adaptation would seem an unalloyed benefit, there has been suggestion that a reduced awareness of hypoglycemia occurs also as a result. This reduced awareness may actually increase the risk of subsequent hypoglycemia in those with insulin-treated diabetes, which in turn might risk brain injury from fuel deprivation. A recent study has38 identified a role for brain GLUT4 in the stress responsiveness to hypoglycemia. Using brain selective GLUT4 knockout mice, it was found that these animals have both glucose intolerance and, of considerable interest also have poor counterregulatory responses to induced insulin hypoglycemia. Areas affected in the brain include the ventromedial hypothalamus that has been identified by39e43 Sherwin and colleagues as critical to hypoglycemia counterregulation. Ischemia, like hypoxia and hypoglycemia, is capable of altering glucose transport in several tissues with an upregulation that attempts to repair and preserve viable but injured tissues. The heart consumes more energy than any other organ. The primary substrates used by the heart for fuel are long-chain fatty acids and glucose. The glucose transporters GLUT3, GLUT4, GLUT8, GLUT10, GLUT11, and GLUT12 are known to be expressed in the heart, and SGLT1 also has been found.44 Glucose is a preferred fuel in ischemia, and upregulation of GLUT4 has been observed with acute ischemia with activation of AMPK pathway with activated PKC signaling.45 Downregulation of GLUT1 and GLUT8 in the atria have been

300

22. GLUCOSE TRANSPORT

observed in humans with type 1 diabetes. In diabetes, SGLT1 appears to a degree compensate as for the reduction in other glucose transporters. Ischemia and chronic congestive heart failure alter GLUTs. In the heart, GLUT4 normally makes up about 70% of cardiac glucose transporters. Reduced amounts of GLUT1 and GLUT4 have been observed in chronic heart failure also due to diabetic cardiomyopathy, and in both animal models and humans compensatory, increases in SGLT1 have been observed. In brain ischemia and stroke, the occurrence of hyperglycemia predicts poor recovery and larger injury. It has been speculated that therapeutic upregulation of GLUTs in the brain be considered as a possible therapy for cerebral ischemia associated with poor glycemic control.46 It should be noted that in severe brain or other ischemia, GLUTs are reduced due to reduction in viable tissue. As reviewed by Dandekar,47 ER stress, along with oxidative stress and inflammatory responses together in tissues, comprises several “major defense networks” that aid cells in adapting to survive stress caused by noxious biochemical and pathological stimuli. They are also related to a number of chronic diseases. Cellular stress can be characterized by specific processes such as ER stress, which is also related to GLUT expression.48,49 Chronic low-grade inflammation as a metabolic stress related can in many cases be related to glucose transport. As an example (see in the following), GLUT1 is implicated in the chronic inflammation of obesity, type 2 diabetes, and potentially a number of cancers. A stress term used in some studies is “nutrient stress” that is basically indicative of overfeeding with high-fat diets, which is related to insulin resistance,50 and this is an important link to obesity, cardiovascular disease, and cancer risk. Upregulation of GLUT1 (and other GLUT proteins in specific circumstances) may provide energy for adipose tissue macrophage inflammation, leading to oncogenesis and the chronic inflammation associated with cardiovascular risk and the metabolic syndrome. These processes are not always labeled “stresses” as such, but appropriately should be considered stresses (not acute but chronic), however, and

they are characteristic of many metabolically linked chronic diseases. Other pathways that are involved in acute stress-related modulation of glucose transport include the unfolding protein or ER stress pathways.48 In a rat model of Alzheimer’s disease induced by intracerebral streptozotocin, decreased glucose and glucose transporter levels occurred along with ER stress markers like GRP78, GADD, and caspase. In addition,51 Cura and Carruthers have shown that GLUT1 levels are markedly (33-fold increased GLUT1 mRNA) increased by metabolic intracellular ATP depletion in murine brain, and subsequently, these authors also identified AMP kinase signaling as mechanistically involved52 in such upregulation in mouse brain endothelial cells.

Obesity, Type 2 Diabetes Mellitus, and Cardiovascular Disease Obesity, type 2 diabetes mellitus, atherosclerotic cardiovascular disease, and a number of cancers are thought increasingly to be the product in part of the moderate, but persistent inflammation, which is a form of metabolic stress.53 These four chronic diseases also have common links in that they have overlapping aspects of their pathophysiology. With obesity, which is also observed in type 2 diabetes, macrophages in fat tissues are a source of the low-grade inflammatory response and the link to cardiovascular disease and an increased risk of a number of types of cancer. The fuel for the proinflammatory response of macrophages is glucose. The overexpression of GLUT1 in macrophages50 leads to an increase in inflammatory response and increased secretion of inflammatory mediators that can be blunted by glycolytic inhibition. Oxidative stress and reactive oxygen species are increased by increased GLUT1 and can be lessened pharmacologically by antioxidant treatment. It remains to be seen whether reversal of these chronic inflammatory responses may be possible through manipulation of GLUTs that are affected or by altering abnormal active signaling that typically accompanies. Both may be possible and even complementary.54

INTRODUCTION

Overall Effects of Transport Regulation The net effect of GLUT regulation in stress is to redistribute glucose fuel to tissues that have an obligate or increased requirement for this energy substrate. Glucocorticoids may upregulate GLUT1 in tissues such as the brain, resulting in increased availability of fuel for that tissue. By contrast, in insulin-dependent tissues, which employ GLUT4 as their primary glucose transporter, an intracellular sequestration of this transporter may prevent draining of glucose fuel from muscle during stress responses. There are however several situations increased glucose transporter number or activity, instead of salutary effects, have potentially harmful effects, and may exacerbate chronic disease. Perhaps, the most compelling data exist for cancer, where altered glucose transport is usually increased and with expression of unusual transporters not typically found in such tissue. In most cases there are tissue-specific links to a variety of signaling pathways activating overexpression of transporters.55,56

Signaling Cascades and Glucose Transport Stress in cells and whole organisms is associated with a wide variety of hormonal and biochemical changes that induce cellular responses via many mechanisms. One mechanism for signal transduction in stress occurs via certain types of mitogen-activated protein kinase (also known as MAP kinase) pathways, particularly the p38 and jun-kinase subtypes. The p38 kinases, mammalian homologs of osmotic stress kinases found in yeast, may help to regulate both basal and stress-activated expression of glucose transporters, including GLUT1 and GLUT3, according to work performed in cultured muscle cells.57 One of the most common pathways related to cancer signaling that influences glucose metabolism and expression of GLUT proteins is phosphatidylinositol 3-kinase (PI3K), which has been extensively studied by Cantley and his colleagues (see Fruman et al. for a comprehensive review).54 PI3K-targeted therapies can be used to treat cancers. Other pathways influencing GLUT regulation include hypoxia-inducible

301

factor 1 Ras, c-Myc, and p53 signaling (see Fig. 22.3). There is strong evidence to suggest that altering signaling for these pathways may favorably affect cancer remission, reducing hyperglycemia and hyperinsulinemia (insulin resistance) may also be critical to reverse growth and spread of a number of cancers. Specific therapies directed against GLUTs that are often overexpressed in certain tumors may themselves (see Fig. 22.4) be drug targets that also may be effective in cancer chemotherapy.58,59

GLUTs, Glucose Transport, and Metabolism in Chronic Disease StatesdCancer The high need for anaerobic metabolism via glycolysis in cancer cells has several important aspects in diagnosis, prognosis, and potential treatment of a number of cancers.60 From the standpoint of diagnosis, glucose transporter proliferation in many cancers can be readily detected with 18F-2-fluoro-2-deoxy-D-glucose, which is avidly taken up by cancer cells.61 The uptake is best seen by noninvasive PET scanning. FDG uptake is also a prognostic factor with the more intense uptake indicating generally a poorer prognosis. With some lung cancers, although uptake is not noted by the primary tumor, it is often high in metastatic disease. The prognosis is also worse when tumors stain heavily for GLUT transporters. In preclinical studies,

FIGURE 22.3 Signaling pathways that alter GLUT expression in cancersdmultiple pathways are involved.

302

FIGURE 22.4

22. GLUCOSE TRANSPORT

Ectopic and overexpression of GLUTs in cancer.

using siRNA or other inhibitors of GLUT transporter proteins have shown some promise as potential targeted therapy in several different types of tumors. Table 22.3 lists tumor associated with increase in GLUTs. Cancer cells are in one sense metabolically stressed as they have metabolic abnormalities (some metabolic pathways are limited) and are known to require a high rate of glucose utilization via glycolysis to support their metabolism and spread due to a dependence upon anaerobic glucose metabolism (also known as the Warburg effect).62 As a result, a number of studies in several types of tumors have shown an increase in GLUTs, mostly GLUT1 to support their growth. Some tumor types show alteration in GLUT3 or GLUT4. In some but not all studies, use of agents to decrease GLUT1 or to limit anaerobic glucose metabolism (primarily glycolysis) has shown some promise in tumor treatment by effectively starving tumor cells. For example, in prostate cancer,63 researchers reviewed data and suggest that proliferating cancer cells need

nutrients (mostly glucose and glutamine), and that GLUT1 and GLUT3 appear to be important predictors of poor outcome. As reviewed by Barron et al.64, there are numerous examples; Fig. 22.4 shows changes in GLUTs from normal and cancerous epithelial cells, and Table 22.3 documents alterations in cancers, and that the majority of GLUTs have been found to support the metabolism, growth, and aggressiveness of a number of cancers. Deliberate targeting of GLUT proteins as a therapeutic approach for treating cancers has been reported in a number of studies. Table 22.3 lists examples of this.65e77 Approaches range from use of traditional chemotherapies that affect GLUT proteins to testing of antisense oligonucleotides, shRNA, use of anti-GLUT antibodies, and others. Several therapies have provided improvement with mostly preclinical studies. Clinical trials are in progress to use strategies to alter glucose metabolism and/or GLUT function or amount to assess the viability of these treatments to aid more traditional cancer therapies.

SUMMARY

TABLE 22.2

303

GLUT Isoform Transport Tissue Expression in Tumors

GLUT Isoform Transporter

Tissue GLUT Expression in Different Tumors

GLUT1

Various tumors

GLUT2

Liver, breast, pancreatic, colon, and gastric carcinoma

GLUT3

GLUT3 Lung, brain, breast, bladder, laryngeal, prostate, gastric, head and neck, ovarian, and oral squamous carcinoma

GLUT4

Colon, lymphoid, breast, thyroid, pancreatic, and gastric carcinoma

GLUT5

GLUT3 Breast, renal, colon, liver, testicular, and lymphoid carcinoma

GLUT6

Breast, pancreatic, and endometrial carcinoma, uterine leiomyoma

GLUT8

Endometrial and lymphoid carcinoma, multiple myeloma

GLUT9

Liver, lung, skin, thyroid, kidney, adrenal, testicular, and prostate carcinoma

GLUT10

No association proven

GLUT11

Multiple myeloma, prostate carcinoma

GLUT12

Breast, prostate, lung and colorectal carcinoma, rhabdomyosarcoma, oligodendroglioma, oligoastrocytoma, astrocytoma

HMIT

No association proven

GLUT14

No association proven

SUMMARY Biological stresses, both acute and chronic, are inevitable in life. Clearly, a variety of types of stresses activate glucose transport in some, but not all, tissues in a stimulus and tissue-specific manner. Stresses include many cellular metabolic stresses such as ischemia, hypoxia, nutrient stress, hypoglycemia, and cellular toxins to mention a few. Realization that more subtle stresses such as low-grade but chronic inflammation can also modify glucose transport and contribute to chronic disease has been more recent. GLUT1, the most widely expressed isoform, appears to be a prototype of the stressactivated glucose transporter isoform, but GLUT3 and GLUT4 may increase or decrease in response to certain kinds of cellular stress also. Less is known about regulation of other GLUT isoforms. Hormones associated with stress responses may be important for increasing or, in some instances, decreasing cellular glucose transport (as with GLUT4 and glucocorticoids).

The preservation of adequate supply of glucose fuel to tissues is believed to be adaptive, but as has been delineated previously, occasionally, it may be that redirection of glucose to certain tissues could also have adverse consequences as well. Adaptations of glucose transport and GLUT transporters are responsive to many signaling pathways activated by stress, including hormones, kinase signaling, energy lack, ER stress, and other pathways as yet incompletely delineated. Knowledge of the signaling pathways may help in developing therapies to combat stress-related illness. Stress-mediated changes in glucose transporter amount or function probably act to redistribute this fuel to tissues such as the brain that have an obligate demand for it. Chronic low-grade inflammation also may be related to metabolic regulation of GLUT proteins and may fuel and be at least in part mechanistically linked to cardiovascular disease, type 2 diabetes, the metabolic syndrome, and even growth and spread of certain types of cancer. Work in progress is attempting to

304

TABLE 22.3 Using Targeted Facilitated Diffusion Glucose Transporters for Cancer Therapy Inhibitor/Modulator

Cells/Tissues

Effect

Outcome

GLUT1

Apigenin

Pancreatic cancer cells

Decreases mRNA and protein

Inhibition of proliferation

GLUT1

Fasentin

Prostate cancer and leukemia cells

Inhibits transporter function

Induction of cell death

GLUT1

Oxime derivatives

Lung cancer cells

Inhibits transporter function

Inhibition of proliferation

GLUT1

EF24

Ovarian cancer cells

Decreases protein

Inhibition of proliferation

GLUT1

WZB27, WBZ115

Lung, breast, colon, and cervical cancer cells

Inhibits transporter function

Inhibition of proliferation, induction of apoptosis

GLUT1

shRKA, Cre/Lox

Breast cancer cells and tumors

Decreases mRNA and protein

Inhibition of proliferation and tumor growth

GLUT1

Anti-GLUT1 antibody

Breast and nonesmall cell lung cancer cells

Alters transporter function

Sensitization of cells to cisplatin, inhibition of proliferation, induction of apoptosis

GLUT1

Anti-GLUT1 antibody, shRNA

Head and neck cancer cells

Alters transporter function, decreases mRNA and protein

Inhibition of proliferation, induction of apoptosis

GLUT3

siRNA, miR-195-5p

Bladder cancer cells

Decreases protein

Inhibition of proliferation, apoptosis induction

GLUT3

Glycogen synthase kinase-3b inhibitors

Tumorogenic HeLa cell hybrids

Decreases mRNA

Induction of apoptosis

GLUT3

Adriamycin, Camptothecin

Tumorogenic HeLa cell hybrids

Decreases mRNA

Induction of cell death/ apoptosis

GLUT5

Antisense oligonucleotide

Breast cancer cells

Decreases mRNA and protein

Inhibition of proliferation

GLUT4

shRNA

Multiple myeloma cells

Decrease mRNA and protein

Induction of cell death

Adapted from Barron et al. (2016).

22. GLUCOSE TRANSPORT

GLUT Isoform

REFERENCES

deliberately target glucose transport and metabolism to design new treatments for a number of diseases, especially several types of cancer.

Acknowledgments The author wishes to acknowledge support for laboratory research reported in part in this manuscript from the Department of Veterans Affairs, the National Institutes of Health (NINDS; NIDDK), and the Juvenile Diabetes Foundation International.

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12. James DE, Strube M, Mueckler M. Molecular cloning and characterization of an insulin-regulatable glucose transporter. Nature. 1989;338(6210):83e87. 13. Birnbaum MJ. Identification of a novel gene encoding an insulin-responsive glucose transporter protein. Cell. 1989;57(2):305e315. 14. Klip A, Sun Y, Chiu TT, Foley KP. Signal transduction meets vesicle traffic: the software and hardware of GLUT4 translocation. Am J Physiol Cell Physiol. 2014; 306(10):C879eC886. 15. Czech MP. Molecular actions of insulin on glucose transport. Annu Rev Nutr. 1995;15:441e471. 16. Wu PF, Luo SC, Chang LC. Heat-shock-induced glucose transporter 4 in the slow-twitch muscle of rats. Physiol Res. 2015;64(4):523e530. 17. Kayano T, Burant CF, Fukumoto H, et al. Human facilitative glucose transporters. Isolation, functional characterization, and gene localization of cDNAs encoding an isoform (GLUT5) expressed in small intestine, kidney, muscle, and adipose tissue and an unusual glucose transporter pseudogene-like sequence (GLUT6). J Biol Chem. 1990;265(22):13276e13282. 18. Hiraki Y, Rosen OM, Birnbaum MJ. Growth factors rapidly induce expression of the glucose transporter gene. J Biol Chem. 1988;263(27):13655e13662. 19. Carter-Su C, Okamoto K. Effect of insulin and glucocorticoids on glucose transporters in rat adipocytes. Am J Physiol. 1987;252(4 Pt 1):E441eE453. 20. Chipkin SR, van Bueren A, Bercel E, Garrison CR, McCall AL. Effects of dexamethasone in vivo and in vitro on hexose transport in brain microvasculature. Neurochem Res. 1998;23(5):645e652. 21. Weber TM, Joost HG, Kuroda M, Cushman SW, Simpson IA. Subcellular distribution and phosphorylation state of insulin receptors from insulin- and isoproterenol-treated rat adipose cells. Cell Signal. 1991;3(1):51e58. 22. Mulder AH, Tack CJ, Olthaar AJ, Smits P, Sweep FC, Bosch RR. Adrenergic receptor stimulation attenuates insulin-stimulated glucose uptake in 3T3-L1 adipocytes by inhibiting GLUT4 translocation. Am J Physiol Endocrinol Metab. 2005;289(4):E627eE633. 23. Wertheimer E, Sasson S, Cerasi E, Ben-Neriah Y. The ubiquitous glucose transporter GLUT-1 belongs to the glucose-regulated protein family of stress-inducible proteins. Proc Natl Acad Sci U S A. 1991;88(6):2525e2529. 24. Kalckar HM, Christopher CW, Ullrey D. Uncouplers of oxidative phosphorylation promote derepression of the hexose transport system in cultures of hamster cells. Proc Natl Acad Sci U S A. 1979;76(12):6453e6455. 25. Lokmic Z, Musyoka J, Hewitson TD, Darby IA. Hypoxia and hypoxia signaling in tissue repair and fibrosis. Int Rev Cell Mol Biol. 2012;296:139e185. 26. Loike JD, Cao L, Brett J, Ogawa S, Silverstein SC, Stern D. Hypoxia induces glucose transporter

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expression in endothelial cells. Am J Physiol. 1992;263(2 Pt 1):C326eC333. Christensen DR, Calder PC, Houghton FD. GLUT3 and PKM2 regulate OCT4 expression and support the hypoxic culture of human embryonic stem cells. Sci Rep. 2015;5: 17500. Chen YP, Kuo WW, Baskaran R, et al. Acute hypoxic preconditioning prevents palmitic acid-induced cardiomyocyte apoptosis via switching metabolic GLUT4glucose pathway back to CD36-fatty acid dependent. J Cell Biochem. 2018;119(4):3363e3372. Quintanilla RA, Porras OH, Castro J, Barros LF. Cytosolic [Ca(2þ)] modulates basal GLUT1 activity and plays a permissive role in its activation by metabolic stress and insulin in rat epithelial cells. Cell Calcium. 2000;28(2):97e106. Klip A, Tsakiridis T, Marette A, Ortiz PA. Regulation of expression of glucose transporters by glucose: a review of studies in vivo and in cell cultures. FASEB J. 1994;8(1): 43e53. Pardridge WM. Glucose transport and phosphorylation: which is rate limiting for brain glucose utilization? Ann Neurol. 1994;35(5):511e512. Simpson IA, Chundu KR, Davies-Hill T, Honer WG, Davies P. Decreased concentrations of GLUT1 and GLUT3 glucose transporters in the brains of patients with Alzheimer’s disease. Ann Neurol. 1994;35(5): 546e551. Simpson IA, Davies P. Reduced glucose transporter concentrations in brains of patients with Alzheimer’s disease. Ann Neurol. 1994;36(5):800e801. McCall AL, Fixman LB, Fleming N, Tornheim K, Chick W, Ruderman NB. Chronic hypoglycemia increases brain glucose transport. Am J Physiol. 1986; 251(4 Pt 1):E442eE447. Pelligrino DA, Segil LJ, Albrecht RF. Brain glucose utilization and transport and cortical function in chronic vs. acute hypoglycemia. Am J Physiol. 1990;259(5 Pt 1): E729eE735. Kumagai AK, Kang YS, Boado RJ, Pardridge WM. Upregulation of blood-brain barrier GLUT1 glucose transporter protein and mRNA in experimental chronic hypoglycemia. Diabetes. 1995;44(12):1399e1404. Uehara Y, Nipper V, McCall AL. Chronic insulin hypoglycemia induces GLUT-3 protein in rat brain neurons. Am J Physiol. 1997;272(4 Pt 1):E716eE719. Reno CM, Puente EC, Sheng Z, et al. Brain GLUT4 knockout mice have impaired glucose tolerance, decreased insulin sensitivity, and impaired hypoglycemic counterregulation. Diabetes. 2017;66(3): 587e597. Paranjape SA, Chan O, Zhu W, et al. Influence of insulin in the ventromedial hypothalamus on pancreatic glucagon secretion in vivo. Diabetes. 2010;59(6): 1521e1527. Tong Q, Ye C, McCrimmon RJ, et al. Synaptic glutamate release by ventromedial hypothalamic neurons is part

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56. Venmar KT, Kimmel DW, Cliffel DE, Fingleton B. IL4 receptor alpha mediates enhanced glucose and glutamine metabolism to support breast cancer growth. Biochim Biophys Acta. 2015;1853(5):1219e1228. 57. Taha C, Tsakiridis T, McCall A, Klip A. Glucose transporter expression in L6 muscle cells: regulation through insulin- and stress-activated pathways. Am J Physiol. 1997;273(1 Pt 1):E68eE76. 58. Ganapathy V, Thangaraju M, Prasad PD. Nutrient transporters in cancer: relevance to Warburg hypothesis and beyond. Pharmacol Ther. 2009;121(1):29e40. 59. Pizzuti L, Sergi D, Mandoj C, et al. GLUT 1 receptor expression and circulating levels of fasting glucose in high grade serous ovarian cancer. J Cell Physiol. 2018; 233(2):1396e1401. 60. Airley RE, McHugh P, Evans AR, et al. Role of carbohydrate response element-binding protein (ChREBP) in generating an aerobic metabolic phenotype and in breast cancer progression. Br J Cancer. 2014;110(3):715e723. 61. Challapalli A, Aboagye EO. Positron emission tomography imaging of tumor cell metabolism and application to therapy response monitoring. Front Oncol. 2016;6:44. 62. Nam SO, Yotsumoto F, Miyata K, et al. Warburg effect regulated by amphiregulin in the development of colorectal cancer. Cancer Med. 2015;4(4):575e587. 63. Gonzalez-Menendez P, Hevia D, Mayo JC, Sainz RM. The dark side of glucose transporters in prostate cancer: are they a new feature to characterize carcinomas? Int J Cancer. 2017. 64. Barron CC, Bilan PJ, Tsakiridis T, Tsiani E. Facilitative glucose transporters: implications for cancer detection, prognosis and treatment. Metabolism. 2016;65(2):124e139. 65. Wood TE, Dalili S, Simpson CD, et al. A novel inhibitor of glucose uptake sensitizes cells to FAS-induced cell death. Mol Cancer Ther. 2008;7(11):3546e3555. 66. Tuccinardi T, Granchi C, Iegre J, et al. Oxime-based inhibitors of glucose transporter 1 displaying antiproliferative effects in cancer cells. Bioorg Med Chem Lett. 2013; 23(24):6923e6927. 67. Zhang D, Wang Y, Dong L, et al. Therapeutic role of EF24 targeting glucose transporter 1-mediated metabolism and metastasis in ovarian cancer cells. Cancer Sci. 2013;104(12):1690e1696.

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C H A P T E R

23 Links Between Glucocorticoid Responsiveness and Obesity: Involvement of Food Intake and Energy Expenditure 1

Belinda A. Henry1, Iain J. Clarke2

Metabolism, Diabetes and Obesity Program, Monash Biomedical Discovery Institute, Department of Physiology, Monash University, Clayton, VIC, Australia; 2Neuroscience Program, Monash Biomedical Discovery Institute, Department of Physiology, Monash University, Clayton, VIC, Australia O U T L I N E Introduction

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Activation of the hypothalamoepituitarye adrenal (HPA) axis and the secretion of glucocorticoids influence energy homeostasis via effects on food intake and energy expenditure. Furthermore, abdominal obesity is associated with hyperactivity of the HPA axis, which is driven by impaired glucocorticoid negative feedback. Recent data, however, suggest that altered glucocorticoid secretion may precede the onset of obesity. In any given population, there is great

Stress: Physiology, Biochemistry, and Pathology https://doi.org/10.1016/B978-0-12-813146-6.00023-0

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Cortisol Responsiveness and Thermogenesis 315 Neuroendocrine Determinants of Altered Thermogenesis in LR and HR

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Cortisol Responsiveness, Coping Strategies, and Physical Activity 317 Future Perspective 319 References

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variation in the glucocorticoid secretory response to stress, with individuals identified as either high responders (HR) or low responders (LR). Sheep characterized as HR have relatively increased food intake in response to stress, impaired satiety in response to melanocortin treatment, and reduced energy expenditure. The reduction in energy expenditure in HR is due to diminished thermogenesis in skeletal muscle and decreased physical activity.

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Copyright © 2019 Elsevier Inc. All rights reserved.

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This complex physiological phenotype is associated with increased propensity to become obese in HR animals. This chapter describes the metabolic, neuroendocrine, and behavioral phenotype in ewes characterized as LR and HR. We propose that altered expression of key hypothalamic genes underpin the metabolic, thermogenic, and behavioral sequelae associated with increased weight gain and increased susceptibility to become obese in HR compared with LR animals. The work described herein suggests that innate variation in cortisol responsiveness precedes weight gain and may be a marker to identification individuals at increased risk of becoming obese.

KEY POINTS • Glucocorticoid secretory responses to stress vary greatly. • Animals can be selected for high and low cortisol responsiveness using a simple adrenocorticotropic hormone challenge. • Animals characterized as high cortisol responders have increased propensity to become obese when fed a high-energy diet compared with low cortisol responders. • Increased propensity to develop obesity in high responders is associated with a distinct metabolic, neuroendocrine, and behavioral phenotype. Including: • reduced postprandial thermogenesis in skeletal muscle. • relatively increased food intake in response to stress. • impaired melanocortin signaling and resistance to the satiety effect of a-melanocyteestimulating hormone. • reduced physical activity. • Characterization of cortisol responses in humans may be a marker for identifying individuals with increased susceptibility to gain weight and to strategize weight loss therapies.

INTRODUCTION It is well recognized that, once obese, it is extremely difficult to lose weight and maintain weight loss, due to homeostatic defense mechanisms that reset hunger and energy expenditure. Body weight is determined by the balance between energy intake and the rate of energy expenditure. The latter is comprised of three major facets, including basal metabolic rate, physical activity, and adaptive thermogenesis. Reduced energy expenditure and in particular reduced thermogenesis is a key homeostatic component that confounds weight loss and long-term weight maintenance. The recent identification of functional brown adipose tissue (BAT) in adults has created a surge of interest in harnessing thermogenesis to develop novel weight loss strategies. A common practice is to use a combination of calorie restriction, exercise, and pharmacotherapies to achieve weight loss, but large cohort studies show that only 2% of subjects maintain weight loss at 2 years postintervention.1,2 A “one-size-fits-all” approach is ineffective because people vary greatly in their weight loss response to lifestyle and/or pharmacotherapy interventions. It is hypothesized that formulation of personalized weight loss strategies is the key to long-term weight loss in obese individuals. This chapter addresses the use of cortisol responsiveness to personalize weight loss. In response to any stressor, the amount of cortisol secreted is highly variable, and a proportion of individuals can be identified as either high (HR) or low (LR) cortisol responders. Animal studies have demonstrated that in response to a single injection of adrenocorticotropic hormone (ACTH), which can be performed with ease in large animals, where repeated blood sampling is simple and relatively non-invasive, approximately 10% of individuals can be characterised as LR or HR. Sheep selected as HR have greater propensity to become obese than those characterized as LR, which is associated with innate differences in the “set-point” of genes in the hypothalamus. Studies have demonstrated that high cortisol responsiveness is associated with

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a distinct neuroendocrine, metabolic and behavioral phenotype that ultimately leads to increased food intake (in response to stress) and reduced energy expenditure. This review will detail the relationship between cortisol responses and weight control, highlighting the possible use of cortisol responsiveness as a marker to identify individuals that are highly susceptible to become obese and personalized weight loss strategies.

mammals, including sheep and humans, it is unclear as to whether obesity precedes the dysregulation of cortisol secretion and thus the causative relationship between HPA axis function and obesity remains to be elucidated. Recent work suggests that altered glucocorticoid responsiveness actually precedes the development of obesity and thus may be a marker to identify individuals with increased susceptibility to weight gain and greater propensity to become obese.

NEXUS BETWEEN BODY WEIGHT, OBESITY AND ACTIVATION OF THE HPA AXIS

PHYSIOLOGICAL DETERMINANTS OF GLUCOCORTICOID RESPONSIVENESS: SELECTION OF LR AND HR INDIVIDUALS

Various animal models show increased glucocorticoid levels in obesity. For example, in mice naturally occurring mutations in the gene encoding leptin (ob/ob) results in morbid obesity and elevated corticosterone levels.3e5 Furthermore, adrenalectomy reverses the obese phenotype of ob/ob mice, and glucocorticoid replacement therapy reinstates this.5 In sheep, hypothalamopituitary disconnection (HPD) increases basal secretion of cortisol,6 and this is associated with increased body weight and adiposity.7 It is important to note, that HPD disrupts the entire hypothalamo-pituitary axis and thus dysregulation of cortisol secretion is not the sole endocrine feature of this model. Nonetheless, diet-induced obesity in female sheep leads to hyperactivation of the HPA axis and greater cortisol secretion in response to isolation restraint stress compared to lean animals.8 Thus suggesting, an interconnection between enhanced glucocorticoid secretion and obesity. Similarly, in humans, it is generally thought that obesity leads to hyperactivity of the HPA axis,9,10 but this is somewhat controversial since various studies have produced contradictory data.11 Cortisol secretion follows a diurnal pattern, and obesity may cause a subtle shift to increase overnight secretion.12,13 Abdominal or visceral obesity, however, is associated with impaired glucocorticoid negative feedback as demonstrated by the dexamethasone suppression test14,15; this leads to enhanced cortisol secretion in response to stress.16 In large

Increased glucocorticoid secretion is fundamental to the physiological and behavioral responses to stress. In any given population, however, the glucocorticoid secretory response to stress varies between individuals. Rats exhibit strain differences in the corticosterone response to stress with the Fisher 344 strain displaying relatively higher corticosterone levels across the circadian period and in response to various stressors compared to the LOU/C strain.17 Furthermore, in outbred animals such as sheep, individuals can be identified as low (LR) or high (HR) glucocorticoid responders18 in response to ACTH treatment. Indeed, selection for LR and HR in response to stress or ACTH challenge has been demonstrated in humans,19 sheep,20,21 fish,22 mice,23 and pigs.24 A key feature of LR and HR individuals, irrespective of species, is that basal glucocorticoid levels are similar, and divergence in cortisol/corticosterone concentration is only evident in response to stress-related stimuli. Various physiological factors impact on glucocorticoid responses to stress, including sex,25 seasonality,26 lactation,27,28 and pregnancy.29 Innate variation in glucocorticoid responses, however, can manifest independent of the aforementioned factors and are determined by both genetic and environmental factors.30,31 Inherent differences in HPA axis responsiveness can manifest at various levels including impaired negative feedback at the level of the

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brain and anterior pituitary gland or altered steroidogenesis at the level of the adrenal gland. Characterization of the HPA axis in LR and HR ewes demonstrate innate differences in the “setpoint” of the HPA axis (Fig. 23.1).32 HR ewes have elevated expression of CRF and AVP, as

well as reduced expression of oxytocin in the paraventricular nucleus of the hypothalamus.32 Furthermore, the expression of proopiomelanocortin (POMC), the precursor for ACTH, is also greater in the anterior pituitary gland of HR than LR ewes.32 Thus HR have increased

FIGURE 23.1 Cortisol responsiveness is measured by an adrenocorticotropin (ACTH) challenge. Serial blood samples

are collected before and after the administration of a standardized dose of ACTH (0.2 mg/kg body weight). This allows for identification and selection of high and low cortisol responding animals. Representative profiles of high responders (HR) are shown in green and low responders (LR) in purple. HR and LR animals exhibit differences in the setpoint of the hypothalamoepituitaryeadrenal axis. Expression of corticotropin-releasing factor (CRF) and arginine vasopressin (AVP) is increased in the paraventricular nucleus (PVN) of HR compared with LR. While the expression of oxytocin in the PVN is reduced in HR compared with LR. Furthermore, expression of proopiomelanocortin (POMC) is increased in the anterior pituitary (AP) of HR. This shows that selection for high cortisol responses identifies animals with an innate upregulation of key genes within the hypothalamoepituitaryeadrenal axis.

CORTISOL RESPONSIVENESS AND INNATE PREDISPOSITION TO WEIGHT GAIN

expression of key neuroendocrine factors that act to stimulate the HPA axis, including CRF, AVP, and ACTH. Reduced expression of oxytocin in HR would promote increased stress responsiveness since oxytocin is known to dampen the HPA axis and exert beneficial effects on anxiety and social bonding behaviors.33e36 It is of importance to highlight that these differences in gene expression are observed under basal, nonstressed conditions, emphasizing the fact that HR ewes have an innate upregulation in the “setpoint” of the HPA axis. It has been postulated that individual differences in glucocorticoid responses may be driven by impaired negative feedback through altered expression and/or function of the glucocorticoid receptor (GR) and mineralocorticoid receptor (MR). Glucocorticoids bind to the MR with high affinity, and thus this receptor subtype is important in regulating basal glucocorticoid secretion.37 In contrast, glucocorticoids bind GR with low affinity, but the receptor is engaged when glucocorticoid levels are high, during stress. As such, GR is ubiquitous and is considered to be the primary receptor mediating glucocorticoid negative feedback in the brain and anterior pituitary gland. Indeed, GR in the paraventricular nucleus mediates fast, nongenomic negative feedback on CRF neurons and thus controls stress-induced activation of the HPA axis.38 Despite this, expression of GR and MR in the paraventricular nucleus of the hypothalamus was similar in LR and HR animals.32 The neural pathways that relay glucocorticoid negative feedback have been extensively characterized in rodents,37 showing that the parvocellular neurons of the PVN receive input from a number of brain structures including the nucleus of the solitary tract, raphe nucleus, bed nucleus of the stria terminalis, prefrontal cortex, amygdala, and the hippocampus.39 Indeed, long-term negative feedback effects of corticosterone are thought to be relayed to the CRF neurons in the paraventricular nucleus of the hypothalamus via the medial amygdala and hippocampus.40,41 It remains possible that impaired negative glucocorticoid negative feedback in HR manifests upstream to the paraventricular nucleus, resulting in an upregulation in the steady-state expression of key genes involved in activation of the HPA axis.

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Irrespective of altered gene expression within the hypothalamus and the anterior pituitary gland, HR animals also display greater adrenal responsiveness to ACTH than LR ewes. LR and HR ewes can be identified or selected using a low-dose ACTH challenge (Fig. 23.1). Prior to screening, estrous cycles are synchronized to control for fluctuating levels of ovarian steroids. Studies show that 10% of animals are consistently identified as either low or high cortisol responders (Fig. 23.1). In this model, animal selection is based on innate differences in adrenal responsiveness to ACTH. Despite this, the molecular or cellular mechanisms underpinning altered adrenal responses are unknown. Gene analyses have shown that expression of key steroidogenic enzymes (StAR, 11bHSD1, CYP11A, and CYP17) as well as the melanocortin 2 receptor (MC2R) is similar in the adrenal cortex of LR and HR animals.32 Furthermore, the basal concentration of cortisol in the adrenal gland is similar in LR and HR as are adrenal gland weights and the cortex:medulla ratio.32 It is possible that differences in adrenal gland function will only be evident in response to stress or ACTH challenge, as exemplified by similar basal secretion of cortisol in LR and HR ewes.42 Further work is required to characterize the role of the adrenal cortex in determining innate differences in cortisol responsiveness in LR and HR. Irrespective of the mechanisms that underpin differences in cortisol responsiveness, strong evidence suggests that the glucocorticoid responses are important in determining subsequent metabolic sequelae and may be useful to predict propensity to become obese.

CORTISOL RESPONSIVENESS AND INNATE PREDISPOSITION TO WEIGHT GAIN As outlined previously, in humans, abdominal obesity is associated with increased cortisol secretion in response to stress or stress-related stimuli (CRF/ACTH).10,14,16 Furthermore, high cortisol responsiveness to ACTH in rams is correlated to reduced feed efficiency and increased levels of adiposity.20 This demonstrates an association between obesity and

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dysregulation of the HPA axis. The aforementioned studies, however, do not establish causal links between weight gain and cortisol responsiveness and, importantly, these studies do not address whether differences in cortisol responses precede the onset of obesity. In sheep of normal body weight, chronic elevation in cortisol via daily injection of Synacthen Depot (long-acting synthetic ACTH) causes weight gain.43 Furthermore, the degree of weight gain is correlated to the cortisol response as determined by measuring the area under the curve,43 suggesting that differences in adrenal gland response precede obesity. Indeed, HR and LR ewes have similar body weights at baseline, but HR gain greater adiposity in response to feeding a high energy diet than LR.21 This clearly supports the notion that innate differences in HPA axis function precede the onset of obesity and in fact can be a physiological marker to identify individuals that have greater propensity to gain weight. The inherent differences in predisposition to become obese is associated with a suite of neuroendocrine, metabolic, and behavioral differences that ultimately lead to increased food intake and reduced energy expenditure in HR animals. These mechanisms will be the focus of the second half of this chapter.

that HR individuals eat relatively more in response to stressful stimuli. Indeed, this is further supported by altered expression of key appetite-regulating genes in the hypothalamus of LR and HR ewes46 (Fig. 23.2). Food intake is tightly controlled by the hypothalamus. Endocrine factors including leptin, insulin, and ghrelin modulate food intake via hypothalamic appetite-regulating peptides. These blood-borne factors act primarily at the arcuate nucleus of the hypothalamus to regulate orexigenic and satiety neurons. POMC neurons are activated by leptin47,48 and insulin,49 causing release of a-melanocyteestimulating hormone (aMSH), which elicits satiety via the MC4R in the paraventricular nucleus.50 A second population of neurons contain neuropeptide Y (NPY) and agouti-related protein (AgRP), which are activated by ghrelin but inhibited by leptin and insulin; NPY/AgRP neurons are orexigenic and

CORTISOL RESPONSIVENESS AND THE NEURAL CONTROL OF FOOD INTAKE At baseline, LR and HR individuals eat similar amounts,21 but differences in food intake are unmasked in response to stressful stimuli. In women, stress typically increases food intake, and only around 10% of individuals show a reduced appetite.44,45 Interestingly, in response to psychosocial stress, women characterized as HR show greater preference for “comfort” foods high in fat and sugar.19 Likewise, LR ewes reduce food intake in response to a barking dog (psychosocial/predator stress) with no effect in HR animals.42 Immune challenge caused by lipopolysaccharide administration decreases food intake in both LR and HR ewes, but this effect is amplified in the former.42 This demonstrates

FIGURE 23.2 High (HR) and low (LR) cortisol responders exhibit differential neuroendocrine control of food intake. At baseline, food intake is similar between the two groups; however, HR eat relatively more than LR in response to stressful stimuli. In addition, HR animals are resistant to the satiety effect of a-melanocyte stimulating hormone (aMSH). Despite differences in feeding behavior, expression of genes encoding key appetite-regulating peptides including neuropeptide Y (NPY), agouti-related protein (AgRP), and proopiomelanocortin (POMC) are similar in LR and HR. Thus differences in the neuroendocrine control of food intake manifest downstream to the arcuate nucleus (ARC) at the level of the receptors; expression of the melanocortin 4 receptor (MC4R) in the paraventricular nucleus (PVN) of the hypothalamus is lower in HR than LR.

CORTISOL RESPONSIVENESS AND THERMOGENESIS

increase food intake.51 The primary role of NPY/ AgRP neurons is to protect against starvation since targeted ablation of these neurons reduces food intake and causes wasting, eventually leading to starvation and death.52 Innate susceptibility to become obese in HR animals is not associated with early onset leptin resistance,21 and the expression of NPY, AgRP, and POMC is similar in LR and HR animals46 (Fig. 23.2). Despite this, HR animals exhibit a marked reduction in the expression of the MC4R in the paraventricular nucleus, and this coincides with impaired satiety in response to intracerebroventricular infusion of aMSH.46 Previous studies in sheep demonstrate that decreased food intake in response to immune stress is relayed via the central melanocortin pathway.53 It is therefore hypothesized that reduced expression of MC4R in HR animals is a fundamental neuroendocrine determinant of altered food intake in response to stressful stimuli, and this directly relates to increased susceptibility to become obese in HR individuals (Fig. 23.2).

CORTISOL RESPONSIVENESS AND THERMOGENESIS Body weight is not only determined by food intake but also the rate at which energy is expended. Energy expenditure is comprised of three major facets including basal metabolic rate, physical activity, and adaptive thermogenesis. The latter is defined as the dissipation of energy through specialized production of heat and is well characterized in BAT of rodents. Brown adipocytes are rich in mitochondria and express uncoupling protein 1 (UCP1),54,55 which when activated creates a proton leak across the inner mitochondrial membrane, directing protons away from ATP synthesis and resulting in futile heat production. Furthermore, BAT is highly vascularized and receives profuse innervation from the sympathetic nervous system (SNS),54,55 which is essential to the activation of thermogenesis. Earlier dogma stipulated that BAT was exclusively found in neonates, where it was essential for the maintenance of core body temperature, but recent advances demonstrate that

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BAT is retained in adults. Landmark imaging studies in humans revealed the presence of BAT in the clavicular, neck, and sternal regions of adults,56e58 and as such this has become a significant therapeutic target for the development of weight loss pharmacotherapies. In large mammals, including sheep and humans, BAT does not account for total thermogenic capacity, and additional tissues contribute to thermogenesis. In this regard, skeletal muscle may play an important role. Prior to the identification of functional BAT by positron emission tomography-computed tomography (PETeCT) imaging in adult humans, skeletal muscle was thought to be the primary thermogenic tissue. Initial work demonstrated that muscle accounts for up to 50% of ephedrine-induced thermogenesis, whereas adipose tissue accounts for approximately 5%.59 It is important to emphasize that this earlier work did not study adipose tissue in the neck and supra-clavicular regions, sites where most brown/beige adipocytes are found. More recent work using PETeCT scanning has shown that acute low doses of ephedrine have no effect on BAT activity, but high doses increase BAT activity in lean humans. Furthermore, chronic low-dose ephedrine treatment actually reduces BAT activity,60 and isoprenaline (a nonspecific bAR) treatment increases energy expenditure without an associated activation of BAT.61 Similarly, blockade of the bAR with propranolol had no effect on cold-induced BAT thermogenesis in humans.62 This lack of effect, however, is likely due to receptor specificity, as both isoprenaline and propranolol show preferential agonistic and antagonistic affinity to the b1AR and b2AR, respectively. A64 Trp/Arg genetic polymorphism in the b3AR is linked to the decline in BAT function with ageing in men,63 suggesting a predominant role for this receptor subtype in controlling BAT function in humans. Indeed, in healthy lean men, administration of the b3AR-specific agonist, mirabegron, activates BAT and causes a concurrent increase in resting metabolic rate.64 These studies highlight that, in humans, the b3AR is essential to the SNS-driven activation of BAT thermogenesis. It remains possible, however, that the effects of isoprenaline to increase energy expenditure via

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the b1/b2 AR61 are mediated via skeletal muscle thermogenesis.65 Skeletal muscle thermogenesis occurs via two distinct cellular pathways, including UCP3mediated mitochondrial uncoupling and futile calcium cycling. Myocytes express UCP3, which is capable of uncoupling oxidative phosphorylation in isolated yeast mitochondria.66 Individual variation in UCP3 expression and mitochondrial uncoupling in skeletal muscle is linked to the ability to successfully lose weight and maintain weight loss. In obese women, reduced expression of UCP3 in skeletal muscle causes a weakened proton leak in mitochondria and impaired ability to lose weight.67 Furthermore, innate variation in basal mitochondrial uncoupling in skeletal muscle accounts for 20%e50% of the variation in basal metabolic rate. Thus skeletal muscle appears to be an important site of thermogenesis in humans, contributing to total energy expenditure and long-term regulation of body weight. In addition to mitochondrial uncoupling, futile calcium cycling can drive adaptive thermogenesis in skeletal muscle. In this regard, calcium exits the sarcoendoplasmic reticulum (SR) via the ryanodine receptor (RyR). To maintain cytosolic calcium levels, activation of the sarcoendoplasmic reticulum ATPases (SERCA) propel calcium back into the SR; this effect is driven by the hydrolysis of ATP and results in heat production.68,69 In rodents, sarcolipin is an endogenous activator of SERCA, which uncouples calcium transport from the hydrolysis of ATP, leading to an increase in the futile cycling of calcium and heat production. In the absence of BAT (surgical removal) or UCP1 gene knockout animals, sarcolipin increases muscle thermogenesis and is essential for cold adaptation.68,70 Furthermore, overexpression of sarcolipin in skeletal muscle increases oxygen consumption and fatty acid oxidation, which is associated with resistance to weight gain in mice fed a high-fat diet.71 The role of sarcolipin in thermogenesis in larger mammals, however, is relatively unexplored and requires closer investigation. Nonetheless, in sheep, postprandial thermogenesis is associated with increased expression of RyR1 and SERCA,72 indicative of a role of futile calcium cycling in adaptive thermogenesis in skeletal muscle.

Skeletal muscle accounts for approximately 40% of total body mass, so in large mammals at least, even small differences in muscle thermogenesis could contribute substantially to heat production and total energy expenditure. Consistent with this notion, at normal body weight, HR ewes have reduced postprandial thermogenesis in skeletal muscle, which is likely to significantly impact on energy expenditure. Sheep are a grazing species, so they do not typically display any meal-associated changes in various endocrine factors (e.g., ghrelin) or peripheral heat production. Despite this, if animals are entrained to a fixed feeding regime, whereby food is provided at set “meal times” across a number of days, excursions in both ghrelin73 and thermogenesis can be engendered (Fig. 23.3). In ewes, postprandial heat production in skeletal muscle is not related to any change in femoral artery blood flow but is associated with increased uncoupled or state 4 respiration in mitochondria isolated from skeletal muscle as well as increased expression of UCP3 and key markers of the futile calcium cycling pathway (Fig. 23.3). Postprandial skeletal muscle heat production is greater in HR than LR, and this effect is enhanced with central administration of leptin21 (Fig. 23.3). Thus not only do LR and HR animals display innate differences in the neuroendocrine control of food intake there are clear differences in skeletal muscle thermogenesis. It is proposed that reduced postprandial thermogenesis in skeletal muscle leads to a reduction in energy expenditure and thus contributes to the increased susceptibility of HR to become obese. It is important to note that inherent variation in adaptive thermogenesis manifests specifically in skeletal muscle of HR and LR animals. Sheep are unlike rodents in that they do not have a defined or demarcated brown fat depot, but brown adipocytes are interspersed throughout typically white fat depots.74e76 Consistent with this, discrete adipose tissues display marked variation in the expression of UCP177 and thermogenic potential.74,75 The primary sites of adipose thermogenesis in sheep are sternal and retroperitoneal tissues, both of which show abundant expression of UCP1 and pronounced

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muscle accounts for approximately 40% of total body mass, differences in thermogenesis in this tissue are likely to impact on total energy expenditure and long-term regulation of body weight.

NEUROENDOCRINE DETERMINANTS OF ALTERED THERMOGENESIS IN LR AND HR

FIGURE 23.3 Skeletal muscle thermogenesis is lower in high cortisol responders (HR) than low cortisol responders. Skeletal muscle thermogenesis occurs via two distinct cellular mechanisms, including uncoupling protein 3 (UCP3)-driven mitochondrial uncoupled respiration and futile calcium cycling. Futile calcium cycling occurs across the sarcoendoplasmic reticulum (SR), where calcium (Ca2þ) is extricated from the SR via the ryanodine 1 receptor (RyR1). To maintain cytosolic Ca2þ levels, the sarcoendoplasmic reticulum ATP-dependent ATPases (SERCA) are activated, pumping Ca2þ back into the SR. The activation of SERCA is dependent on the hydrolysis of the ATP, which is a thermogenic process. Reduced skeletal muscle thermogenesis in HR is associated with altered expression of key genes in the hypothalamus including decreased melanocortin 4 receptor (MC4R) and prepro-orexin expression. Both the melanocortin pathway and orexin act within the brain to increase thermogenesis, and thus lowered expression is associated with attenuated postprandial thermogenesis in skeletal muscle of HR compared with LR.

thermogenic responses.74e76 Temperature recordings in retroperitoneal adipose tissue show that postprandial thermogenesis is similar in BAT of LR and HR sheep.21 Thus, in conclusion, reduced thermogenesis is a key component of altered propensity to gain weight in LR and HR sheep; however, this innate divergence manifests primarily in skeletal muscle. Given that skeletal

In addition to controlling food intake, the hypothalamus exerts reciprocal control on energy expenditure and in particular acts to control adaptive thermogenesis. Neuropeptides that regulate food intake have dual effect to regulate thermogenesis, whereby factors that increase food intake typically reduce thermogenesis and vice versa77a. For example, aMSH acts to reduce food intake and increases thermogenesis in both BAT77b and skeletal muscle77c. In contrast, orexin is produced in the lateral hypothalamus (LH) and increases both food intake and energyexpenditure via physical activity and adaptive thermogenesis.78,79 A subpopulation of orexin neurons in the LH project to the raphe pallidus and microinjection of orexin into the latter increased BAT thermogenesis.80 Furthermore, ablation of the orexin neurons in the LHA eliminates cold-, stress- and immune-induced BAT thermogenesis.81,82 A recent study demonstrated that obesity in orexin-knockout mice is associated with reduced adaptive thermogenesis in BAT due to an inability of brown preadipocytes to differentiate into mature brown adipocytes.83 In addition to reduced MC4R expression in the PVN, expression of prepro-orexin is lower in the LH of HR than LR46. Thus increased expression of orexin in the LH of LR animals may be important in mediating the enhanced skeletal muscle thermogenesis.

CORTISOL RESPONSIVENESS, COPING STRATEGIES, AND PHYSICAL ACTIVITY A number of animal models have shown that low cortisol responsiveness is associated with enhanced aggressive behaviors.23,24,84 Similarly,

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in humans, cortisol responses and obesity are associated with distinct behavioral traits. In obese subjects, emotional eating is correlated to impulsiveness and depression, whereas restrained eating is correlated with openness, conscientiousness, and extraverted personality traits.85 Furthermore, low cortisol responses are associated with increased incidence of

neuroticism in women, low extraversion in males, and reduced openness in both sexes.86 Cortisol responsiveness is also associated with differences in coping strategies. In LR and HR ewes, behavioral responses and coping strategies have been assessed using the open field test, arena test, and food competition test (Fig. 23.4). Low cortisol responding ewes have enhanced

FIGURE 23.4 Behavioral differences in high and low cortisol responders (HR and LR) have been assessed by the open field, arena, and food competition tests. The open field test measures the degree of locomotion an animal exhibits in response to isolation in an enclosed field. The arena test measures fear and avoidance behaviors where an animals desire to be with their flockmates is assessed in relation to their fear of approaching a human. Finally, the food competition test measures initiative behaviors by assessing the latency for the test animal (LR or HR) to reach a food reward when in competition with a control flockmate. HR animals show reduced physical activity and initiative, but increased fear that is indicative of a reactive behavioral coping style. In contrast, LR animals exhibit increased physical activity and initiative, but reduced fear, and this is indicative of a proactive coping style. Increased physical activity in the LR animals coincides with greater expression of prepro-orexin in LR than HR. Thus innate differences in the orexin system may also underpin the behavioral phenotypes of LR and HR animals.

REFERENCES

sympathoadrenal activation in response to stress and exhibit proactive coping strategies.87 Ewes selected for low cortisol responsiveness show greater physical activity in response to isolation stress (open field), reduced fear toward humans (arena test), reduced freezing, and increased initiative to reach a food reward (food competition test)42 (Fig. 23.4). It is hypothesized that a proactive coping style rather than a reactive style may be more likely to expend energy. A proactive coping strategy is typically associated with increased aggression and physical activity, which are behaviors that are consistent with increased energy expenditure. Importantly, mutations in the orexin system result in narcolepsy, and orexin is known to increase physical activity.78,88e90 Thus increased expression of prepro-orexin in LR does not only align with increased thermogenesis but also the relative increase in physical activity in LR compared with HR (Fig. 23.4). It is therefore proposed that altered expression of both MC4R and preproorexin are key neuroendocrine determinants of the increased propensity to become obese in HR animals.

Future Perspective There is variation in the susceptibility to gain weight and become obese. In addition, weight loss is also vastly variable across populations. Despite our understanding of the physiological control of body weight, weight loss remains elusive to the vast majority. Long-term studies have shown that approximately 2% of subjects maintain weight loss 5 years after weight loss intervention.1,2 A key component that determines individual differences in either weight gain or the ability to successfully lose weight is influenced by inherent variation in energy expenditure. Collectively, a net increase in food intake and reduced energy expenditure culminate in an obesity prone phenotype. One means in which we may improve weight loss is to strategize treatment through a personalized medicine approach. It may be proposed that characterization of cortisol responsiveness may be a means to identify individuals with increased susceptibility to become obese. Gene

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expression analyses within the hypothalamus indicate that reduced expression of MC4R in the PVN and/or reduced expression of orexin in the LH underpin the physiological and metabolic phenotype in obesity-prone HR animals. A number of the new generation of antiobesity drugs are known to target the melanocortin pathway, including Contrave, Lorcaserin, and Liraglutide.91e93 Our experimental data suggest that HR individuals may be less responsive to these pharmacotherapies and thus would greatly benefit from alternative weight loss strategies and therapies. Future studies need to address whether cortisol responsiveness may be a beneficial clinical marker for improving and maintaining long-term weight loss.

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induced by ephedrine in man. Am J Physiol. 1985;248(5 Pt 1):E507eE515. Carey AL, Pajtak R, Formosa MF, et al. Chronic ephedrine administration decreases brown adipose tissue activity in a randomised controlled human trial: implications for obesity. Diabetologia. 2015;58(5):1045e1054. https:// doi.org/10.1007/s00125-015-3543-6. Vosselman MJ, van der Lans AA, Brans B, et al. Systemic beta-adrenergic stimulation of thermogenesis is not accompanied by brown adipose tissue activity in humans. Diabetes. 2012;61(12):3106e3113. https:// doi.org/10.2337/db12-0288. Wijers SL, Schrauwen P, van Baak MA, Saris WH, van Marken Lichtenbelt WD. Beta-adrenergic receptor blockade does not inhibit cold-induced thermogenesis in humans: possible involvement of brown adipose tissue. J Clin Endocrinol Metab. 2011;96(4):E598e605. https://doi.org/10.1210/jc.2010-1957. Yoneshiro T, Ogawa T, Okamoto N, et al. Impact of UCP1 and beta3AR gene polymorphisms on age-related changes in brown adipose tissue and adiposity in humans. Int J Obes (Lond). 2013;37(7):993e998. https://doi.org/10.1038/ijo.2012.161. Cypess AM, Weiner LS, Roberts-Toler C, et al. Activation of human brown adipose tissue by a beta3-adrenergic receptor agonist. Cell Metab. 2015;21(1):33e38. https:// doi.org/10.1016/j.cmet.2014.12.009. Blaak EE, van Baak MA, Kempen KP, Saris WH. Role of alpha- and beta-adrenoceptors in sympathetically mediated thermogenesis. Am J Physiol. 1993;264(1 Pt 1): E11eE17. Gong DW, He Y, Karas M, Reitman M. Uncoupling protein-3 is a mediator of thermogenesis regulated by thyroid hormone, beta3-adrenergic agonists, and leptin. Journal of Biological Chemistry. 1997;272(39): 24129e24132. Harper ME, Dent R, Monemdjou S, et al. Decreased mitochondrial proton leak and reduced expression of uncoupling protein 3 in skeletal muscle of obese diet-resistant women. Diabetes. 2002;51(8): 2459e2466. Bal NC, Maurya SK, Sopariwala DH, et al. Sarcolipin is a newly identified regulator of muscle-based thermogenesis in mammals. Nature Medicine. 2012;18(10): 1575e1579. https://doi.org/10.1038/nm.2897. Arruda AP, Nigro M, Oliveira GM, de Meis L. Thermogenic activity of Ca2+-ATPase from skeletal muscle heavy sarcoplasmic reticulum: the role of ryanodine Ca2+ channel. Biochimica et Biophysica Acta. 2007; 1768(6):1498e1505. https://doi.org/10.1016/j.bbamem. 2007.03.016. Bal NC, Maurya SK, Singh S, Wehrens XH, Periasamy M. Increased reliance on muscle-based thermogenesis upon acute minimization of brown adipose tissue function. Journal of Biological Chemistry. 2016;291(33):17247e17257. https://doi.org/10.1074/jbc.M116.728188.

71. Maurya SK, Bal NC, Sopariwala DH, et al. Sarcolipin is a key determinant of the basal metabolic rate, and its overexpression enhances energy expenditure and resistance against diet-induced obesity. Journal of Biological Chemistry. 2015;290(17):10840e10849. https://doi.org/ 10.1074/jbc.M115.636878. 72. Clarke SD, Lee K, Andrews ZB, et al. Postprandial heat production in skeletal muscle is associated with altered mitochondrial function and altered futile calcium cycling. Am J Physiol Regul Integr Comp Physiol. 2012; 303(10):R1071eR1079. https://doi.org/10.1152/ajpregu. 00036.2012. 73. Takahashi H, Kurose Y, Suzuki Y, et al. Changes in blood pancreatic polypeptide and ghrelin concentrations in response to feeding in sheep. J Anim Sci. 2010;88(6): 2103e2107. https://doi.org/10.2527/jas.2009-1920. 74. Henry BA, Pope M, Birtwistle M, et al. Ontogeny and thermogenic role for sternal fat in female sheep. Endocrinology. 2017;158:2212e2225. https://doi.org/10.1210/ en.2017-00081. 75. Henry BA, Dunshea FR, Gould M, Clarke IJ. Profiling postprandial thermogenesis in muscle and fat of sheep and the central effect of leptin administration. Endocrinology. 2008;149(4):2019e2026. https://doi.org/ 10.1210/en.2007-1311. 76. Henry BA, Blache D, Rao A, Clarke IJ, Maloney SK. Disparate effects of feeding on core body and adipose tissue temperatures in animals selectively bred for Nervous or Calm temperament. Am J Physiol Regul Integr Comp Physiol. 2010;299(3):R907eR917. https://doi.org/10.1152/ ajpregu.00809.2009. 77. Symonds ME. Brown adipose tissue growth and development. Scientifica (Cairo). 2013:305763, 2013. 77a. Verty AN, Allen AM, Oldfield BJ. The endogenous actions of hypothalamic peptides on brown adipose tissue thermogenesis in the rat. Endocrinology. 2010;151(9): 4236e4246. 77b. Song CK, Vaughan CH, Keen-Rhinehart E, Harris RB, Richard D, Bartness TJ. Melanocortin-4 receptor mRNA expressed in sympathetic outflow neurons to brown adipose tissue: neuroanatomical and functional evidence. Am J Physiol Regul Integr Comp Physiol. 2008; 295(2):R417eR428. 77c. Gavini CK, Jones WC 2nd, Novak CM. Ventromedial hypothalamic melanocortin receptor activation: regulation of activity energy expenditure and skeletal muscle thermogenesis. J Physiol. 2016;594(18):5285e5301. https://doi.org/10.1155/2013/305763. 78. Teske JA, Billington CJ, Kotz CM. Neuropeptidergic mediators of spontaneous physical activity and non-exercise activity thermogenesis. Neuroendocrinology. 2008; 87(2):71e90. 79. Madden CJ, Tupone D, Morrison SF. Orexin modulates brown adipose tissue thermogenesis. Biomol Concepts. 2012;3(4):381e386. https://doi.org/10.1515/bmc-20110066.

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80. Tupone D, Madden CJ, Cano G, Morrison SF. An orexinergic projection from perifornical hypothalamus to raphe pallidus increases rat brown adipose tissue thermogenesis. J Neurosci. 2011;31(44):15944e15955. https://doi.org/10.1523/JNEUROSCI.3909-11.2011. 81. Zhang W, Sunanaga J, Takahashi Y, et al. Orexin neurons are indispensable for stress-induced thermogenesis in mice. J Physiol. 2010;588(Pt 21):4117e4129. https://doi.org/10.1113/jphysiol.2010.195099. 82. Takahashi Y, Zhang W, Sameshima K, et al. Orexin neurons are indispensable for prostaglandin E2-induced fever and defence against environmental cooling in mice. J Physiol. 2013;591(22):5623e5643. https://doi.org/ 10.1113/jphysiol.2013.261271. 83. Sellayah D, Bharaj P, Sikder D. Orexin is required for brown adipose tissue development, differentiation, and function. Cell Metab. 2011;14(4):478e490. https:// doi.org/10.1016/j.cmet.2011.08.010. 84. Terenina E, Babigumira BM, Le Mignon G, et al. Association study of molecular polymorphisms in candidate genes related to stress responses with production and meat quality traits in pigs. Domest Anim Endocrinol. 2013;44(2):81e97. https://doi.org/10.1016/j.domaniend. 2012.09.004. 85. Elfhag K, Morey LC. Personality traits and eating behavior in the obese: poor self-control in emotional and external eating but personality assets in restrained eating. Eat Behav. 2008;9(3):285e293. https://doi.org/ 10.1016/j.eatbeh.2007.10.003.

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86. Oswald LM, Zandi P, Nestadt G, et al. Relationship between cortisol responses to stress and personality. Neuropsychopharmacology. 2006;31(7):1583e1591. https:// doi.org/10.1038/sj.npp.1301012. 87. Koolhaas JM, Korte SM, De Boer SF, et al. Coping styles in animals: current status in behavior and stressphysiology. Neurosci Biobehav Rev. 1999;23(7):925e935. 88. Gencik M, Dahmen N, Wieczorek S, et al. A preproorexin gene polymorphism is associated with narcolepsy. Neurology. 2001;56(1):115e117. 89. Lin L, Faraco J, Li R, et al. The sleep disorder canine narcolepsy is caused by a mutation in the hypocretin (orexin) receptor 2 gene. Cell. 1999;98(3):365e376. 90. Nishino S, Ripley B, Overeem S, Lammers GJ, Mignot E. Hypocretin (orexin) deficiency in human narcolepsy. Lancet. 2000;355(9197):39e40. https://doi.org/10.1016/ S0140-6736(99)05582-8. 91. Secher A, Jelsing J, Baquero AF, et al. The arcuate nucleus mediates GLP-1 receptor agonist liraglutide-dependent weight loss. J Clin Invest. 2014;124(10):4473e4488. https://doi.org/10.1172/JCI75276. 92. Billes SK, Sinnayah P, Cowley MA. Naltrexone/bupropion for obesity: an investigational combination pharmacotherapy for weight loss. Pharmacol Res. 2014;84:1e11. https://doi.org/10.1016/j.phrs.2014.04.004. 93. Burke LK, Doslikova B, D’Agostino G, et al. 5-HT obesity medication efficacy via POMC activation is maintained during aging. Endocrinology. 2014;155(10): 3732e3738. https://doi.org/10.1210/en.2014-1223.

C H A P T E R

24 BloodeBrain Barrier: Effects of Inflammatory Stress Cle´mence Disdier, Barbara S. Stonestreet The Alpert Medical School of Brown University, Department of Pediatrics, Women & Infants Hospital, Providence, RI, United States O U T L I N E Introduction

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Structure and Function of the BloodeBrain Barrier Physical Barrier Functional Barrier The Metabolic and Enzymatic Barrier The Neurovascular Unit

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Neuroinflammation and BBB Physiology BBB Integrity Impairment and Immune Cell Trafficking Regulation of Transport Activities Inflammatory Modulation of Metabolism

Abbreviations ABC transporters [ ATP-Binding Cassette transporters AD [ Alzheimer disease BBB [ BloodeBrain Barrier BCRP [ Breast Cancer Resistance Protein CNS [ Central Nervous System CYP450 [ Cytochrome P450 ICAM [ IntraCellular Adhesion Molecule IL-1b [ Interleukine-1b IL-6 [ Interleukine-6J

Stress: Physiology, Biochemistry, and Pathology https://doi.org/10.1016/B978-0-12-813146-6.00024-2

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Inflammatory Stress at the BBB in Pathological Contexts 332 Neurodegenerative Diseases 332 Hypoxia-Ischemia Brain Injury and Stroke 333 Epilepsy 333 Infectious Diseases 333 Diabetes 334 Conclusions

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Acknowledgments

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References

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AMs [ Junctional Adhesion Molecules MCT [ MonoCarboxylate Transporter MRP [ Multi-drug Resistance-associated Protein NF-kB [ Nuclear Factor kB NVU [ Neurovascular Unit P-gp [ P Glycoprotein ROS [ Reactive Oxygen Species TJ [ Tight Junction TLR [ Toll-Like Receptor TNF-a [ Tumor Necrosis Factor a VCAM [ Vascular Cell Adhesion Molecule ZO [ Zonula Occludens

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Copyright © 2019 Elsevier Inc. All rights reserved.

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INTRODUCTION Two biological systems maintain an optimal environment for nervous tissue: the bloode cerebrospinal fluid barrier (CSFB) and the bloodebrain barrier (BBB). The first regulates the exchange between blood and cerebrospinal fluid. The BBB has a 5000 times greater surface area compared with the CSFB, is located in the capillaries of the brain, and regulates exchange between the cerebral parenchyma and the blood compartments. Emerging evidence on the sustained inflammatory response associated with BBB dysregulation in many neurological diseases suggests contributory important roles of both mechanisms in neuronal dysfunction. Consequently, neuroinflammation-targeted therapeutics has gained interest because of the increased appreciation of the importance of neurovascular inflammation in the pathophysiological progression of a variety of neurological diseases. Considering the sensitivity and the key functions of BBB, it appears essential to understand the response to the central nervous system (CNS) gatekeeper to inflammatory stressors. Here, we describe the structure and key functions of the BBB and then summarize information about regulation of BBB physiology in the context of inflammation with reference to the inflammatory effects on major neurological pathologies.

KEY POINTS • The bloodebrain barrier (BBB) and the neurovascular unit (NVU) are fundamental to maintain central nervous system homeostasis. • BBB features include the physical barrier and transport and metabolic functions and result from cooperation with other components of the NVU. • In response to inflammatory stress, BBB functions are impaired, and NVU cells are activated to contribute to signal transduction.

• BBB dysfunction and neurovascular inflammation contribute to the pathogenesis of many neurological disorders. • Anti-inflammatory therapeutic strategies could protect the BBB and NVU resulting in neuroprotective effects.

STRUCTURE AND FUNCTION OF THE BLOODeBRAIN BARRIER The endothelium of the cerebral capillaries constitutes the anatomical basis of the BBB: it has restrictive properties distinguishing it from the peripheral endothelium. These properties result from a combination of physical features including the presence of tight junctions (TJs), scarcity of vesicular transport, and absence of fenestrations as well as transport properties including efflux pumps and metabolic processes such as enzymes that limit paracellular and transcellular passage of many molecules.

Physical Barrier The physical barrier results in very limited paracellular passage between adjacent endothelial cells. This limitation is a result of the presence of junctions between neighboring brain endothelial cells (Fig. 24.1). The protein junctions are connected to the actin cytoskeletal proteins to form a continuous membrane with very high electrical resistance (approximately 2000 U/cm2). These junctions consist of three types: TJs, adherens junctions, and gap junctions (Fig. 24.1).1e3 The attachment between endothelial cells and the extracellular matrix is provided by junctional adhesion molecules (JAMs). These junctions are mediated by homophilic interactions between membrane proteins: cadherin proteins. The cadherin proteins are expressed on the surface of the adjacent cells and have extracellular domains that interact to form a homodimer. Cadherins are linked to the actin cytoskeleton on the cytosolic side via intermediate proteins: catenins and form the primary seal of junctions. Adherens

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FIGURE 24.1 Basic structure of junctions between the BBB endothelial cells.

junctions form a continuous belt around the cells, thereby maintaining contiguous cells and strengthening the TJs. The TJs form a complex protein network at the luminal side of the brain endothelial cells. The latter is composed of three major proteins: claudins, occludins, and molecules of adherens junctions, which interact with accessory proteins including zona occludens (ZO) and cingulins. The accessory proteins are also connected to the actin cytoskeleton. TJs are critical in limiting the paracellular passage of molecules. This organization of the junctions maintains cell polarity by preventing the migration of molecules within the membrane and limits the penetration of xenobiotic molecules into the brain parenchyma. Consequently, most of the molecular traffic across the cerebral vascular endothelium is forced to take a transcellular route. Finally, gap junctions facilitate the exchange of ions and other small molecules between adjacent cellular partners. Gap junctions are particularly important for communication between adjacent cells in tissues with extensive TJ complexes. The physical BBB also limits the extravasation of immune cells into the brain parenchyma under

healthy conditions. The recruitment and infiltration of immune cells termed “diapedesis” depends on the expression of different adhesion molecules by brain endothelial cells and immune cells. The adhesion molecules expressed by endothelial cells include selectins, vascular cell adhesion molecule-1 (VCAM-1), and intracellular adhesion molecule-1 (ICAM-1). These molecules facilitate interactions between brain endothelial cells and immune cells rolling at the endothelial surface.

Functional Barrier The BBB facilitates the passage of oxygen, carbon dioxide, as well as some small hydrophilic and lipophilic molecules but is impermeable to hydrophilic molecules such as glucose or amino acids, which are nevertheless critical for the normal function of brain cells.4 A schematic representation of the different transport mechanisms at the BBB are summarized in Fig. 24.2. First, the paracellular aqueous pathway permits the passage of small water-soluble agents through the TJs between adjacent endothelial cells. Second, transcellular passage or passive diffusion depends on the physicochemical

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FIGURE 24.2 Schematic representation of the different transport mechanisms at the BBB. Adapted from Abbott NJ, Ronnback L, Hansson E. Astrocyte-endothelial interactions at the blood-brain barrier. Nat Rev Neurosci. 2006;7:41e53. doi:10.1038/ nrn1824.

characteristics of the molecules. In this situation, the molecular weight, lipophilicity, pKa, and capacity of the molecules to form hydrogen bonds determine the ability of a molecule to cross the endothelial monolayer. Third, crossing the BBB can occur by endocytosis/transcytosis mediated by a receptor recognizing a specific ligand or by a so-called adsorptive mechanism. Finally, endothelial cells can express a variety of specific carriers that facilitate transport of molecules. These transporters can be expressed on the luminal and/or abluminal endothelial cells. They can be responsible for unidirectional or bidirectional transport and therefore allow the influx (blood to brain) and/or the efflux (brain to blood). There are a very large number of transporters with more or less high substrate specificity. Some

of these carriers are responsible for transporting essential nutrients into the brain, whereas others are responsible for the efflux of toxins and metabolic waste products. These efflux transporters play a major role in detoxification functions of the BBB. Two key families of efflux transporters have been described: Solute carriers (SLC) and ATP-binding cassette (ABC) carriers. The family of SLC carriers is a very diverse family whose main representatives are the GLUT1 or SLC2A1 (glucose transporter), the cationic amino acid transporters, and large amino acid transporters (LATs) that belong to SLC7 family, amino acid transporters in the SLC38 and SLC1 family, and organic anion transporters (OAT and OATP). The ABC family (ABC transporters) is mainly responsible for efflux of substances out of the brain. The main proteins are P-glycoprotein

NEUROINFLAMMATION AND BBB PHYSIOLOGY

(P-gp or ABCB1), Breast Cancer Resistance Protein (BCRP or ABCG2) as well as multidrug resistance-associated proteins (MRPs). The P-gp is located at the luminal side of the endothelium, but it is also expressed by astrocytes end-feet and microglia. P-gp is commonly considered to be the primary transporter in the ABC family because of its large variety of substrates.

The Metabolic and Enzymatic Barrier Brain endothelial cells express several enzymes responsible for the metabolism of neurotransmitters. For example, they express monoamine esterases, cholinesterases, GABA transaminases, aminopeptidases, and endopeptidases. Enzymatic barrier function can be illustrated by 3,4dihydroxyphe´nylalanine (DOPA) carboxylase. Dopamine in the blood cannot cross the BBB because it has lipophilic properties and does not have a specific transport carrier. Therefore, patients with Parkinson disease are treated with L-DOPA, the dopamine precursor that can cross the BBB using the large neutral amino acid transport system. However, the brain distribution of LDOPA is limited by the DOPA decarboxylase and monoamine oxidase within the brain endothelial cells.5 This explains the requirement for large doses of L-DOPA to treat Parkinson disease. Brain endothelial cells also express different systems responsible for the metabolism of drugs typically found in the liver, such as the cytochromes P450 (CYP450 or phase I enzymes) and phase II enzymes of metabolism.6,7 These toxin and drug-metabolizing enzymes contribute along with the efflux transporters to the detoxification function of the BBB. In addition, enzymes are important for the metabolism of endogenous substrates such as fatty acids, hormones, steroids, and vitamins and are implicated in the regulation of the concentration of signaling pathway molecules.

The Neurovascular Unit The specialized characteristics of the cerebral vascular endothelium are enhanced by interactions with related cells and particularly by the physical and paracrine interactions with

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astrocytes and pericytes (Fig. 24.3). The endothelium is surrounded by the cerebral vascular pericytes embedded within the basal laminal membrane that they share with the endothelial cells within the vascular wall. The pericytes contribute to the endothelial barrier properties and the development of the basal lamina by providing structural and metabolic support. The basal lamina is composed of various types of laminin, collagen, glycoproteins, and proteoglycans and provides a structural anchor for the endothelial cells. The capillaries are sheathed by numerous astrocytic extensions that form the glia limitans and have been shown to induce many of the BBB properties of the brain endothelial cells. Astrocytes also provide a cellular link to neurons and express many transporters that participate in the regulation of brain homeostasis. Microglia are resident immune cells in the CNS and can be found in the perivascular area. This multicellular structure facilitates close communication between parenchymal neurons and the endothelium has been termed the neurovascular unit (NVU). The cellular and noncellular elements of the NVU are particularly important to consider in relationship to inflammatory conditions because they are associated with proinflammatory mediator production and activation of proteases. However, in this review, we will mainly focus on the brain capillary endothelium and its response to inflammatory stress.

NEUROINFLAMMATION AND BBB PHYSIOLOGY BBB physiology is influenced both by systemic and locally produced inflammatory mediators and plays an important role in the CNS inflammatory response. Activation of inflammatory pathways such as nuclear factor-kB (NF-kB) in brain endothelial cells enhances endothelial apoptosis, membrane abnormalities, and mitochondrial damage and results in alterations in the physical and functional barriers, which in turn affect CNS function in neuroinflammatory disorders. In addition, the BBB itself plays an active role in modulation of the neuroimmune

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FIGURE 24.3

Schematic representation of the different components of the NVU.

response either by production and transport of inflammatory mediators or by the expression of adhesion molecules. Other constituents of the NVU such as microglia and astrocytes are activated by the inflammatory cascade and act in concert to further propagate the inflammatory wave across the BBB and into the brain parenchyma. Key events occurring at the BBB level in inflammatory state are summarized in Fig. 24.4.

BBB Integrity Impairment and Immune Cell Trafficking The first physiological alteration of the BBB observed after inflammatory stress is impairment of the BBB integrity. The response of the BBB to inflammation is multifactorial. Neuroinflammation is characterized by activation of multiple components of the NVU, including brain

endothelial cells, which can produce proinflammatory cytokines and chemokines. Circulating or locally produced cytokines, such as tumor necrosis factor-a (TNF-a), interleukin-1b (IL-1b), or interleukin 6 (IL-6), induce the expression of endothelial adhesion molecules including selectins, VCAM-1, and ICAM-1, that allow the immune cells (neutrophils, monocytes, and lymphocytes) to infiltrate across the BBB.8 Moreover, systemic as well as neuroinflammatory disorders that are characterized by high concentrations of circulating proinflammatory mediators are often associated with alterations in the molecular composition or organization of TJ proteins.9 Proinflammatory cytokines also activate the expression of matrix metalloproteinases (MMPs), which participate in degradation of the extracellular matrix components in the basement membrane and TJs.10,11 Overall, these events result in impaired BBB structure and

NEUROINFLAMMATION AND BBB PHYSIOLOGY

FIGURE 24.4

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Schematic illustration of the key events at the BBB in inflammatory states.

function, which further facilitates the paracellular infiltration of immune cells and aggravates neuroinflammation. TNF-a has been shown to increase BBB permeability by modulation of TJ proteins.12e15 Restoration of TJ proteins expression and normalization of BBB integrity by inhibition of TNF-a or administration of antieTNF-a antibodies further supports the adverse effects of TNF-a on BBB integrity.16 In vitro studies also suggest that TNF-a increases the expression of adhesion molecules by brain endothelial cells, thereby promoting adhesion and migration of leukocytes and further aggravating alterations in BBB permeability.17 Similarly, IL-1b exposure increases BBB permeability in vitro18 and systemic injections of IL-1b in mice reduces the TJ protein, occludin, by proteolysis resulting in a subsequent injury to the BBB.19 Incubation of microvessels isolated from sheep brain with IL6 protein also results in decreased expression of the TJ proteins, occludin and claudin-5.20 Other features such as endothelial apoptosis and mitochondrial damage are also implicated in BBB integrity loss during inflammatory stress.

Regulation of Transport Activities The transporters of the BBB actively participate in the CNS response to inflammatory mediators. Many cytokines and chemokines including TNF-a and IL-1b are able to penetrate the BBB and are selectively transported across the BBB by the ABC transporters.21 Thus, the transporters are able to regulate the distribution of inflammatory mediators within the CNS. On the other hand, neurovascular inflammation has also been linked to changes in carrier activity and expression. The effect of TNF-a on P-gp is probably one of the best examples. This effect has been shown to be biphasic in isolated microvessels from rats.22,23 After a short exposure to low-dose TNF-a, P-gp expression decreased. In contrast, P-gp expression was upregulated after TNF-a exposure for 6 h. The NF-kB pathway has been demonstrated to be involved in this regulation.24 Multiple other in vitro studies have demonstrated the effects of TNF-a and other cytokines such as IL1-b and IL-6 with variable effects on the BBB.25e28 These discrepancies could be explained by the

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differences in the models used (primary cells versus cell lines). TNF-a has also been shown to enhance its own saturable transport across the BBB.29 Modification in activity and/or the expression of P-gp, BCRP, and other ABC transporters have also been demonstrated in vivo under inflammatory conditions in several animal models.30e32 Modifications in the ABC transporter expression and/or activity have great implications because they alter the detoxification function of the BBB. Certainly, the ABC transporters have a wide variety of substrates, and even small modulations of their efflux capacity could potentially result in accumulation of endogenous and exogenous neurotoxins within the brain parenchyma. In addition, the CNS relies on a continuous supply of nutrients and ions carried mainly by the transporters of the BBB. Therefore, changes in transport function could dramatically affect BBB permeability to nutrients, parenchyma homeostasis and, consequently, neuronal function. Finally, information is not available regarding other mechanisms of transport, for example, involving vesicular transcytosis.

Inflammatory Modulation of Metabolism The effects of inflammation on the metabolic enzymes and their regulation at the BBB are not well understood in comparison with the current knowledge of drug transporters across the BBB. Inflammation in vivo results in CYP450 downregulation at the level of gene transcription with a consequent decrease in messenger RNA, protein, and enzyme/transporter activity in the liver.33 Infection and inflammation in patients are also associated with modulation of the hepatic cytochrome P450 enzymes. In addition, cerebral inflammation has been demonstrated to decrease CYP450 expression in the brain34 and particularly in astrocytes.35 Intermediary constituents including cytokines, proteases, free radicals, and prostaglandins have been implicated in signaling pathways that result in the CYP450 changes in brain. Astrocytes play crucial roles in BBB function. Therefore, it is likely that

changes in their metabolism could result in altered BBB function, particularly in the context of chronic inflammation. Long-term alterations in metabolic activity could have therapeutic implications for chronic neuroinflammatory disorders. However, studies have not examined the effects of metabolic activity at the BBB after inflammatory stress.

INFLAMMATORY STRESS AT THE BBB IN PATHOLOGICAL CONTEXTS Neurodegenerative Diseases Alzheimer disease (AD) is an illustration of a neurodegenerative disease for which BBB dysfunction and neuroinflammation are critical contributors to its pathogenesis. AD is characterized by microglial activation and infiltration of circulating immune cells into the cerebral parenchyma, which are associated with the accumulation of amyloid beta peptides and phosphorylated tau protein. The BBB plays a major role in the pathophysiology of AD.36 A leading mechanism by which the BBB contributes to the progression of AD appears to be accumulation of amyloid beta peptides as a result of impaired brain-to-blood efflux mechanisms that allow accumulation of A-beta in brain. Additional alterations in the BBB have been documented in AD including the loss of TJs, changes in other carrier activities such as the glucose transporter GLUT1, and abnormalities in the basal lamina and pericytes. The brain endothelial cells also actively participate in the inflammatory cascade by releasing various bioactive molecules that activate the inflammatory cascade and serve to further impair the BBB in the AD brain.37 These abnormalities in the BBB accentuate cellular toxicity and neurodegeneration. Therefore, it appears that neuroinflammation and dysfunction of multiple components of the BBB function are two important mechanisms that serve to contribute to AD progression. These abnormalities represent potential avenues for therapeutic targets. However, the interactions between the BBB and

INFLAMMATORY STRESS AT THE BBB IN PATHOLOGICAL CONTEXTS

inflammation in AD require further investigation regarding their roles as initiating or secondary factors of the disease. Similarly, these two factors appear to contribute to Parkinson disease and multiple sclerosis.38 However, there is a still debate regarding their roles in the sequence and progression of these disorders.

Hypoxia-Ischemia Brain Injury and Stroke Hypoxia-ischemia (HI) is a defined as severe reduction in blood flow with subsequent deprivation of oxygen and nutrient supply to the tissues. HI brain injury is characterized by a pronounced inflammatory response along with early structural alterations in the BBB. Most alterations in the BBB are observed early (hours to days) after the insult. A large number of in vitro and in vivo studies have documented increased BBB permeability with alterations in the expression and localization of key TJ components and transporters in the context of HI. Abnormalities in BBB permeability after HI are multifactorial and involve factors such as energy deprivation, release of reactive oxygen species (ROS), and local inflammation.39,40 HI triggers the expression and release of proinflammatory mediators, immune cell chemoattractants, and activates proteases. The microvascular inflammatory environment potentiates TJ disassembly in brain endothelial cells, increases the expression of adhesion molecules, and also damages the extracellular matrix that together result in BBB dysfunction.41 Previous work has shown that HI-related BBB dysfunction can be attenuated by targeting inflammatory processes. This strongly suggests that inflammation is an important contributor to HI-related BBB dysfunction and, consequently, potentiates parenchymal brain injury. We have recently shown that administration of antieIL1b neutralizing antibodies attenuates BBB dysfunction in the brain of the ovine fetus after ischemia42 and also attenuate parenchymal brain injury.43 Although abnormalities in BBB permeability along with alterations in TJ proteins and immune cell infiltration represent the key features of HI-related brain injury, other abnormalities also include changes in the ABC transporters.44e46

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Epilepsy Inflammatory processes are associated with alterations in BBB permeability and abnormalities in transport activities in humans who have epilepsy.47 Changes in the physiology of the BBB have been extensively studied as a potential cause of pharmacological resistance to antiepileptic drugs. However, it remains to be determined whether changes in the BBB phenotype are related to the physiopathology of seizures and/or exposure to antiepileptic drugs, which also are substrates for many transporters. Brain tissue obtained from patients operated for pharmacoresistant epilepsy exhibit substantial modifications in the BBB with changes in TJ proteins levels and modifications in the expression of several transporters including P-gp, MRP1, and the monocarboxylate transporter 1. BBB leakage has been directly implicated in the epileptogenic process as the BBB maintains ionic homeostasis and therefore acts as a regulator of neuronal excitability. Brain inflammation is rapidly activated in many different animal models with upregulation of proinflammatory mediators.48 The proinflammatory pathway such as IL1-b receptor/toll-like receptor (TLR) signaling is activated in many brain cells. This activation affects endothelial TJs, transporters, and the basal lamina. Proinflammatory mediators play a role in seizures by modifying membrane excitability and seizure threshold. Chemokine upregulation also attracts leukocytes and adhesion molecules at brain endothelial cell surfaces and induces BBB dysfunction via extravagation of leukocytes. Therapeutic targeting the IL-1b pathway has been shown to reduce BBB leakage and exhibit anticonvulsive efficacy in a rodent model of epilepsy.49 These findings suggest strong associations between BBB impairment and inflammation as contributing factors to seizures and epilepsy.

Infectious Diseases BBB physiology is critically influenced by proinflammatory mediators such a cytokines and chemokines that are circulating in blood of

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infected patients and act on the various elements of the NVU. Neurovascular inflammation resulting in BBB leakage also enhances the invasion of pathogens by direct entry, paracellular translocation, or by increasing host receptor expression. Some bacterial (e.g., lipopolysaccharide) and viral elements (e.g., virion envelope protein of HIV) interact with brain endothelial cells and directly alter BBB function. Thus, proinflammatory mediators and pathogens act in concert to breach the CNS endothelium. HIV infection has been shown to result in alterations in the function of the BBB in patients and in vitro models. When brains from HIV positive patients with encephalitis or HIV-associated dementia were examined postmortem, monocyte infiltration was observed which was associated with decreased claudin 5, occludin, and ZO-1 expression.50 In addition, three HIV proteins (Tat, gp 120, and Nef) have been implicated in direct alterations in BBB integrity. Similarly, bacterial-related meningeal inflammation also affects BBB integrity by interfering with the TJ complex.51

Diabetes Diabetes mellitus can result in neurocognitive and neurologic complications including vascular dementia and stroke. These clinical outcomes are multifactorial, but the role of the BBB in the pathogenesis of diabetic encephalopathy and other diabetic-related complications in the CNS has been suggested. In vivo and in vitro studies have documented altered BBB integrity in patients with diabetes and in animal models.52 Diabetic states have been shown to be associated with alterations in brain endothelial cells, pericyte degeneration, decreases in TJs proteins, increases in the expression of adhesion molecules, and activation of proteolytic enzymes resulting in the basal lamina abnormalities. BBB dysfunction as a result of hyperglycemic states is multifactorial and includes potential hyperosmolality, overproduction of ROS, and associated inflammation. Hyperglycemia also results in accelerated glucose metabolism in brain endothelial cells and excessive production of ROS. Other mechanisms of hyperglycemia-induced

neuroinflammation include NF-kBedependent production of proinflammatory cytokines, TLR expression, and inflammasome activation. In addition, vascular alterations in hypoglycemic states are also considered to be one of the causes of the comorbidities in diabetic patients including stroke and epilepsy.

CONCLUSIONS Dysfunction of the BBB and neurovascular inflammation are associated with brain pathophysiology in many neurologic disorders including stroke, epilepsy, and neurodegenerative disorders such as Parkinson disease and AD. BBB dysfunction and inflammatory stress represent underlying mechanisms for the manifestation of these neurological diseases and, in turn, further exacerbate the pathological brain lesions. It is important to consider both BBB dysfunction and inflammatory stressors in the development of targeted drug strategies for many neurological disorders. The most common strategy to achieve neuroprotection is to target inflammatory processes in order to reduce the extent of the brain lesions and attenuate BBB dysfunction. Anti-inflammatory drugs may act by several mechanisms. However, they may target the BBB and NVU in addition to their effects on the cerebral parenchyma.

Acknowledgments This chapter was supported in part by National Institutes of Health (NIH) 1R01-HD-057100. Elements of the illustrations were provided by Servier Medical Art by Servier (http:// smart.servier.com) licensed under a Creative Commons Attribution 3.0 Unported license. Some of the elements were adapted.

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25 BloodeBrain Barrier in Alzheimer’s Disease Maria Alexandra Brito Research Institute for Medicines, Faculty of Pharmacy, Universidade de Lisboa, Lisboa, Portugal O U T L I N E Introduction

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INTRODUCTION

KEY POINTS

The relevance of the bloodebrain barrier (BBB) in Alzheimer’s disease (AD) has been progressively recognized and has deserved increasing attention, which is attested by the growing number of publications retrieved in a PubMed search for “Alzheimer’s disease” and “Bloodebrain barrier” as title/abstract words: 34, 147, and 434 results in the 80s, 90, and 00s, respectively, and already 1201 in the current decade. This motivated the construction of the present chapter that addresses the BBB and its structural and functional alterations in AD. Emphasis is put on brain microvascular endothelial cells (BMEC) since they are considered the anatomic basis of the BBB. Whenever relevant, the contribution of other neurovascular unit (NVU) components is also addressed.

Stress: Physiology, Biochemistry, and Pathology https://doi.org/10.1016/B978-0-12-813146-6.00025-4

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• Alzheimer’s disease (AD) is a neurodegenerative disease, characterized by amyloid-b peptide (Ab) deposition in the brain. • Vascular risk factors (e.g., hypertension and diabetes) predispose to AD development, giving raise to the neurovascular hypothesis of AD. • Vascular dysfunction precedes Ab accumulation in the brain parenchyma and involves bloodebrain barrier (BBB) disruption. • BBB disruption is characterized by: • tight junctions’ disruption and hyperpermeability that lead to entrance of blood-borne molecules into the

Copyright © 2019 Elsevier Inc. All rights reserved.

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parenchyma, which elicit toxicity to brain cells. • enhanced receptor for advanced glycation end products, which mediates Ab influx into the parenchyma, as well as by decreased low-density lipoprotein receptor-related protein 1 and Pglycoprotein, which export Ab out of the brain, collectively leading to Ab accumulation in the brain. • Ab accumulation in the brain amplifies the neurodegeneration that ends up with cognitive decline and dementia, characteristic of AD. • Collectively, cerebral blood vessels are the converging point of pathogenic events leading to dementia.

BLOODeBRAIN BARRIER The BBB plays a key role in brain homeostasis by strictly regulating the exchanges between the periphery and the brain, preventing the access of toxic molecules to the brain, while assuring the delivery of essential molecules and the elimination of waste products toward the circulation. Here, it will be presented a brief description of the BBB, as well as of its insertion in the brain parenchyma, within the NVU.

BBB CHARACTERISTICS Endothelial cells lining brain microvasculature constitute the basis of the BBB since they are at the interface between the brain and the periphery and have unique characteristics that account for a restricted permeability. The major feature of BMEC is the elaborate junctional complexes mainly formed by tight junctions (TJ) and adherens junctions (AJ). Although TJ are the main structures responsible for sealing the intercellular space, in the brain, TJ and AJ are intermingled, and AJ also contribute to the restricted permeability.1 Due to such restricted

permeability, the passage of molecules depends on their physic-chemical characteristics and on the existence of transport proteins. Thus, ions and solutes diffuse between adjacent cells (paracellular pathway), small lipophilic molecules, such as oxygen, CO2, and ethanol, can pass the BBB freely by diffusion, and hydrophilic molecules, such as peptides and proteins, may enter the brain through vesicular transport mechanisms mediated by caveolae (transcellular pathway), while polar and lipid-insoluble molecules do not cross the BBB.1 Importantly, rather than a static barrier, BMEC have been recognized as key players in the bidirectional communication between the brain and the periphery and viewed as a relay station. In fact, vascular signals cause the release of cytokines by BBB endothelial cells into the brain parenchyma, which determine brain cells’ action, whereas signaling from brain parenchyma to the periphery via BMEC also occurs.1

BBB CONSTITUTION TJ are elaborate structures located at the apical region of BMEC, constituted by networks of transmembrane proteins (e.g., claudin-5, occludin) that associate laterally with the proteins of apposing membrane of an adjacent cell to form paired TJ strands responsible for the occlusion of the intercellular space. Transmembrane proteins are linked to cytosolic proteins (e.g., zonula occludens-1 [ZO-1]) that connect the transmembrane proteins to the actin cytoskeleton, therefore playing a central role in the macromolecular assembly of TJ.2 Below TJ, there are AJ that give place to a continuous belt, the adhesion belt, which is responsible for the endothelium integrity. AJ are also formed by transmembrane and cytosolic proteins, like vascular endothelial (VE)-cadherin and b-catenin, respectively, linked to the actin cytoskeleton by the peripheral protein.3 Caveolae are small membrane microdomains considered a specific form of lipid rafts.4 They are enriched in cholesterol, sphingolipids, and

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caveolin-1, the major structural protein of caveolae.1 In turn, caveolin-1 levels are regulated by cholesterol.4 Caveolae are flask-shaped invaginations of the plasma membrane that form vesicular structures responsible for the vesicular trafficking of molecules such as albumin and cholesterol, the latest being involved in AD pathogenesis.4 Moreover, caveolae play important roles in signaling pathways and regulation of several aspects of endothelial cell function, including intercellular junctions’ assembly.1 In physiological conditions the number of caveolae at the BBB is usually small, whereas its increase is usually associated with BBB hyperpermeability by the transcellular route in pathological conditions.3 BMEC are additionally characterized by a number of receptors and the presence of influx and efflux transporters that assure the passage of substances into and out of the brain parenchyma in order to guarantee the entrance of molecules required for proper brain function, as well as the elimination of catabolites and potentially harmful molecules.1 Among the several influx transporters normally expressed by BMEC is the receptor for advanced glycation end products (RAGE).5 RAGE is a member of the Ig superfamily of cell surface molecules, with a versatile structure and ability to bind multiple ligands.6 Among RAGE, ligands are included the amyloid-b peptide (Ab), a key intervenient in AD pathogenesis,6 as will be addressed in the following. Upon interaction with ligands, RAGE activates signal transduction pathways that lead to sustained cellular stress as shown in inflammation and AD, among other pathological conditions.6 Among the several efflux transporters at the BBB, there is the low-density lipoprotein (LDL) receptor-related protein 1 (LRP-1), a member of the LDL receptor gene family. LRP-1 interacts with a variety of ligands, mediating the transcytosis of molecules such as Ab across the BBB, as well as the transport of cholesterol associated with ApoE-containing lipoproteins.7 In addition, it plays a role as a signaling receptor, regulates BBB integrity by modulating TJ proteins, and mediates the clearance of major extracellular

matrix (ECM)-degrading proteinases,8 and alterations in this transporter have been associated with pathological conditions such as AD.9 Other important efflux transporters at the BBB are the members of the ATP-binding cassette (ABC) family,1 which mediate the export of substances coupled with the hydrolysis of ATP.10 Among them are the P-glycoprotein (P-gp), also known as multidrug resistance (MDR) 1, or ABCB1, and the breast cancer resistance protein (BCRP), or ABCG2, which play an important role in organ protection by pumping out from the cell a variety of structurally diverse molecules,11 among which is included Ab.9

NEUROVASCULAR UNIT BMEC establish important interactions with surrounding cells, as well as with the basement membrane (BM) and ECM components, within the NVU.3 In fact, BMEC are surrounded by a BM, which in turn is continuous with the ECM. Proteins found on the matrix-proximate faces of ECs like integrins establish the anchorage of the cell cytoskeleton to the BM. The anchorage function of the BM plays an important role in the integrity of cerebral microvasculature and, thus, on BBB stability and properties. BMEC are also ensheathed by pericytes, mural cells that are highly present in brain microvasculature. Pericytes play a key role in the maintenance of the BBB properties, namely through plateletderived growth factor (PDGF)-B signaling in endothelial cells to the corresponding receptor in pericytes, PDGFR-b. BMEC are also extensively covered by astrocytes, the most abundant glial cells, which processes covering the microvasculature play a key role in the restricted permeability. More recently, the contribution of neurons to the enhanced expression of TJ proteins and the barrier properties was also demonstrated. Although the role of microglia in the BBB in physiological conditions is less clear, it has been considered a member of the NVU. Dysregulation of the NVU homeostasis and the cross-talk among its components have been increasingly implicated in brain pathology. For instances, in

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inflammatory conditions, astrocytes and microglia are activated, leading to the release of cytokines, BBB hyperpermeability, and recruitment of leukocytes into the brain. Moreover, matrix metalloproteinases (MMPs) are activated, which leads to the degradation of BM, affecting TJ integrity and leading to alterations in BBB properties, as well as of ECM components that further aggravate the BBB impairment.3,12 The loss of pericytes, leading to a poorer vascular coverage, microvessels fragility, and BBB disruption, has also been established. Therefore, homeostasis within the NVU and proper interplay among its components are pivotal for the maintenance of BBB properties. Despite the relevance of each of the NVU components for brain homeostasis

and disease, the focus of this chapter will be on the central cells of the NVU, BMEC, which main features in healthy conditions, as well as in AD, are illustrated in Fig. 25.1.

BBR DYSFUNCTION IN AD There is accumulating evidence about the involvement of BBB dysfunction in the pathogenesis of a wide range of brain disorders, including neurodegenerative diseases. This chapter illustrates key aspects of BBB impairment, including loss of barrier properties and alterations in BBB transporters, in the most common neurodegenerative pathology, AD.

FIGURE 25.1 Schematic representation of the main features of endothelial cells in the brain microvasculature that forms the bloodebrain barrier in healthy conditions and AD. In healthy conditions (left), well-organized TJ between apposing BMECs obliterate the intercellular space and restrict the paracellular permeability. TJ are formed by transmembrane proteins that are linked to cytosolic scaffold ones, of which are represented the transmembrane claudin-5 and occludin and the cytosolic ZO-1. Among the several transporters of BMEC, there is the receptor for RAGE, P-gp, and BCRP, which are mainly expressed in the luminal surface, and the LRP-1, present in the abluminal surface. In AD (right), there is a disorganization of TJ proteins within the junctional complexes, with increased paracellular permeability. The increased endothelial permeability allows entrance into the brain parenchyma of blood-borne molecules, such as albumin, thrombin, and fibrinogen, which are known to elicit neurotoxicity. The expression of RAGE that promotes the entrance of Ab into the brain parenchyma is increased, while the expression of the efflux transporters LRP-1 and RAGE, which are known to transport Ab out of the brain are decreased, which collectively contribute to Ab accumulation in the brain parenchyma. BCRP is also enhanced and suggested as an attempt to prevent Ab peptide accumulation in the brain.

DISRUPTION OF BARRIER PROPERTIES IN AD

AD PATHOGENESIS AD is a progressive, irreversible, neurodegenerative disease, characterized by cognitive decline and memory loss. It represents the most common form of dementia, afflicting as many as 45% of individuals who survive past the age of 85 years, with estimates of 34 million AD patients in 2025.9 AD is a multifactorial pathology, which pathogenesis is still not fully understood. Hallmarks of the disease are hyperphosphorylated tau in neurofibrillary tangles and the presence of senile plaques containing Ab peptide, which is produced by cleavage of Ab-precursor protein (APP). Deposition in the brain parenchyma of the neurotoxic Ab oligomers triggers a sequence of events that result in neurodegeneration and cognitive impairment, as proposed by the amyloid hypothesis.9,13 However, there is a growing body of evidence suggesting its vascular origin, as vascular risk factors have been implicated in AD onset and progression, and it was suggested that the vascular damage in these diseases would affect the cognitive function.14 These observations led to the neurovascular hypothesis of AD, which proposes that cerebrovascular dysfunction and disruption in the neurovascular integrity contribute to the onset and progression of cognitive decline and that cerebral blood vessels are the converging point of pathogenic events leading to dementia.13 According to this two-hit hypothesis, vascular risk factors in the midlife (hit one) lead to both BBB disruption and reduction in cerebral blood flow (oligemia). These lead to accumulation of toxic molecules and hypoperfusion, respectively, which contribute to the neuronal dysfunction that precedes cognitive impairment and dementia (non-Ab pathway). Dysregulation of BBB transport systems and oligemia, which stimulate APP processing and Ab production, also occur, leading to brain accumulation of the toxic peptide. The increase in Ab (hit two) amplifies neuronal dysfunction and accelerates neurodegeneration, cognitive impairment, and dementia, thus contributing to disease self-propagation.13

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To the neurodegeneration, ending up in the collapse of the NVU further contributes the impaired cross-talk between endothelial cells and pericytes, accounting for the poor pericyte vascular coverage and BBB hyperpermeability, as well as the aberrant astrocyteepericyte signaling, and microglia activation.15

DISRUPTION OF BARRIER PROPERTIES IN AD Accumulation of Ab along brain blood vessels (cerebral amyloid angiopathy) and associated vascular inflammation leads to BBB hyperpermeability and altered communication between the brain and the peripheral immune system that favor the entrance of activated peripheral immune cells into the brain. This may directly or indirectly affect the central nervous immune system and modulate the pathogenesis of AD. Moreover, it provides a possible explanation for the link between chronic diseases like hypertension and diabetes, which are characterized by persistent inflammation, with the increased risk of dementia.14 There are several lines of evidence supporting this assumption, such as hypertension favors Ab deposition in the brain and BBB hyperpermeability,16 antihypertensive drugs restore cerebrovascular dysfunction and improve cognitive function in an AD mouse model,17 and coexistence of diabetes accelerates Ab-related vascular alterations.18 Among the players involved in the cerebrovascular dysfunction in these conditions are oxidative stress and mitochondrial damage, as well as increased expression of inflammatory cytokines and induction of RAGE in the cerebral vasculature.14,19 Besides the previously mentioned nongenetic risk factors, genetic factors also contribute to AD development. APOE, encoding apolipoprotein E, appears as the major contributor since as many as 65%e80% of all AD patients are carriers of the APOEε4 allele (APOE4),9 and this allele was shown to increase the accumulation of senile plaques in AD patients and in cognitively normal people.14

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Several studies performed in AD mice models have demonstrated the compromise of BBB, early in the onset of the disease or even prior to the development of amyloid plaques and cognitive impairment. In fact, studies in the Tg 2576 AD mouse model and analysis of dye leakage showed the BBB breakdown at 4 months of age, a time point that precedes amyloid plaque deposition and cognitive dysfunction, suggesting that functional alteration of the BBB constitute one of the earliest signs of AD and may favor Ab entry into the brain.20 Our own studies, performed by immunohistochemical analysis of cortex and hippocampus of the APP/PS1 Tg AD mouse model along disease progression revealed that BBB permeability to the blood molecules thrombin and albumin increases, whereas the number of cells labeled with the pericyte marker PDGFR-b and the pericyte vascular coverage decrease, pointing to microvessels fragility. These studies further showed that the immunolabeling to the astrocytic and microglial markers, glial fibrillary acidic protein, and ionized calcium-binding adapter molecule 1, respectively, is enhanced in the disease condition, compared with wild-type controls.5 Moreover, they revealed that senile plaques precede vascular and glial alterations in hippocampus, whereas in cortex vascular and glial alterations accompanied the first senile plaques, thus corroborating the contribution of vascular alterations in the disease pathogenesis.5 Another study in the APP transgenic mouse subjected to peripheral inflammation induced by peripheral injection of lipopolysaccharide showed a greater increase in inflammatory cytokines in brain interstitial fluid but not in plasma or peripheral organs and more severe noncognitive symptoms (e.g., attenuation of social interaction behavior).21 Moreover, BBB hyperpermeability was observed, which might constitute a mechanism for peripheral inflammation spreading into the brain. This observation illustrates the potential vulnerability of the BBB to inflammation that may underline the increased severity of behavioral impairment in AD. It was also shown that expression of APOE4 (but not of other APOE isoforms in mice lacking ApoE) leads to BBB breakdown by activating a proinflammatory CypA-nuclear factor-kB-MMP-9 pathway in pericytes, and that

these events occur prior to neuronal dysfunction and neurodegenerative changes.22 As far as AD patients are concerned, BBB disruption was present in 22% of patients with mild-to-moderate probable AD, and in up to 42% of AD patients, as established based on cerebrospinal fluid albumin levels or ratio to plasma albumin.23,24 Postmortem analysis of the prefrontal cortex of AD patients by immunohistochemistry and enzyme-linked immunosorbent assay (ELISA) revealed the presence of plasma proteins like prothrombin in advanced AD (Braak stage V-VI), particularly within the microvessel wall and surrounding neuropil, and that leakage of the BBB was more common in patients with at least one APOE4 allele.25 Immunohistochemistry analysis of BBB disruption indicators, albumin and fibrinogen, and of the TJ proteins claudin-5, ZO-1, and occludin in temporal cortex sections of a population-representative sample also showed BBB leakage, although a wide population variation and considerable overlap between different levels of Alzheimer-type pathology were observed.26 More recently, dynamic contrast material-enhanced magnetic resonance imaging (MRI) of early AD patients and age-matched control subjects showed global BBB leakage and a significantly higher volume fraction of the leaking brain tissue in patients with early AD associated with cognitive decline, suggesting that a compromised BBB may be part of a cascade of pathologic events that eventually lead to cognitive decline and dementia.27 Appearance of pericortical enhancement after contrast administration was observed in older individuals with normal cognition, mild cognitive impairment, and AD patients, illustrating chronic focal superficial BBB leakage in normal aging and dementia.28 Chiaravalloti et al.29 found a significant negative correlation between albumin concentration quotient (Qalb) and 2-deoxy2-(18F) fluoro-D-glucose uptake, markers of BBB permeability and metabolic activity, respectively, with higher Qalb values being related to a reduced glucose consumption in AD patients. Interestingly, this was observed in the temporal lobe, a preferential vulnerable brain region in AD patients in which degeneration is responsible for the memory loss.

DISRUPTION OF BARRIER PROPERTIES IN AD

The BBB disruption associated with abnormal deposition of Ab peptide in the brain has increasingly been related with endothelial cell alterations and referred as endotheliopathy.30 The barrier properties of BMEC exposed to Ab and the underlying mechanisms have been particularly studied in in vitro models of the BBB, relying on the use of two-chamber systems where BMEC are plated onto a semipermeable membrane with the luminal surface facing the upper chamber and the abluminal surface facing the lower one, mimicking the blood and brain compartments, respectively.1 Studies performed by Kook et al.31 showed that exposure of monolayers of the endothelial cell line bEnd.3 to Ab induced structural alterations characterized by reduced levels of claudin-5, occludin, and ZO-1 and increased the permeability to fluorescein isothiocyanate (FITC)-dextran, suggesting the opening of the paracellular pathway due to disrupted TJ integrity. Ab-induced downregulation of ZO-1, occludin, and claudin-5 appears to be mediated by endophilin-1, a target of miR-107, which overexpression largely abrogated Ab-induced disruption of BBB.32 In another study, using the immortalized human BMEC line hCMEC/D3, widely used as a simplified in vitro model of the BBB, Ab induced a marked increase in permeability to the paracellular tracer 70 kD FITC-Dextran associated with a decrease in the TJ protein occludin, but not of claudin-5 and ZO-1. In this study, the decrease in occludin was prevented by JNK and p38 MAPK inhibition, implying these signaling pathways in BBB dysfunction associated with AD.33 It was recently demonstrated that astrocytes contribute to Ab-induced disruption of barrier properties by secreting vascular endothelial growth factor and MMP-9, which is known to mediate claudin-5 disruption, thus pointing out the importance of astrocytes’ mediation in inducing endothelial sensitivity to Ab.34 Importantly, a disrupted BBB will not prevent the entrance into the brain parenchyma of otherwise restricted molecules, such as Ab, which will further amplify the cascade of deleterious events. Interestingly, using an in vitro BBB model consisting of BMEC and pericytes prepared from

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wild-type mice and primary astrocytes prepared from human ApoE3- and ApoE4-knock-in mice revealed a disruption of the barrier function of TJ in the BBB model with astrocytes from apoE4-knock-in mice, providing evidence that TJ integrity in BBB is specifically impaired by ApoE4.35 Despite the recognized influence of AJ on TJ function and integrity and the established disruption of TJ in AD, direct evidence of AJ impairment in AD are missing. In fact, AJ compromise in AD has been suggested based on indirect observations, rather than by analysis of AJ expression in AD experimental models or brain analysis of autopsy material from AD patients. Among such observations is the downregulation of VE-cadherin in a mouse model of hyperhomocysteinemia, a condition that accompanies many cognitive disorders including AD.36 The decreased expression of VE-cadherin was accompanied by Ab and fibrinogen accumulation in the subendothelial matrix, as well as by loss of short-term memory, pointing to a link between disruption of AJ, paracellular hyperpermeability and cognitive impairment. It was also observed that neutrophils transmigration across endothelial cells monolayers occurs at regions of low VE-cadherin expression and that block of the protein adhesive function accelerates recruitment of neutrophils to inflammation sites, raising the hypothesis that the neutrophils transmigration observed in mouse models of AD takes place in brain microvessels with low VEcadherin expression.37 Moreover, loss of VEcadherin was observed in pericyte-deficient mice,38 and loss of pericyte vascular coverage has been reported in different AD mouse models5,39 as well as in AD patients’ autopsy material,40 raising a further connection between impairment of AJ and the paracellular hyperpermeability in AD. Therefore, establishment of VEcadherin and/or other AJ proteins alterations in AD, as well as the underlying signaling pathways and associated impairment of the NVU, is required for a better understanding of BBB dysfunction in AD pathogenesis. Contrasting with the well-characterized disruption of TJ and paracellular hyperpermeability of the BBB summarized previously, the

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eventual alterations in caveolin-1 expression and transcellular permeability remain unknown. In fact, it was demonstrated that brain homogenates of AD patients’ autopsy material and ApoE-deficient mice present increased caveolin-1 levels, which could be related with alterations of cholesterol distribution in the plasma membrane of brain cells in AD.41 Therefore, it would be important to determine if caveolin-1 is also elevated in BMEC and if such increase is associated with a disruption of the BBB by the transcellular pathway.

DYSREGULATION OF TRANSPORT SYSTEMS IN AD Since accumulation of Ab within extracellular spaces of the brain is a hallmark of AD, considerable attention has been given to the mechanisms by which Ab might be transported between the brain and blood. Of particular interest are the receptor and transporter proteins RAGE, LRP-1, and P-gp, which altered expression in BMEC has been associated to Ab accumulation in the brain parenchyma and AD development. RAGE is the main receptor responsible for Ab influx into the brain parenchyma, considered as an entrance gate for peripheral Ab, with several lines of evidence indicating that RAGE mediates Ab-induced perturbations in cerebral vessels in AD.9 In fact, it was shown that RAGE expression in BMEC of the human hippocampus is increased in advanced AD compared with early stage AD and/or individuals with mild cognitive impairment, meaning that microvascular RAGE increases with the onset and severity of AD,42 which may further contribute to Ab accumulation in brain via accelerated Ab influx from blood. Our own studies also revealed a progressive increase in the expression of RAGE in hippocampal microvessels along aging of APP/PS1 transgenic mice and associated disease progression and Ab accumulation.5 Curiously, RAGE expression also increased in wild-type mice, and no significant difference between the AD mouse model and wild-type control was detected,5 suggesting that the enhanced RAGE expression is mostly associated with the aging-associated disease progression. Deane

et al.43 demonstrated that RAGE blockage by its specific inhibitor, FPS-ZM1, inhibited RAGEmediated influx of circulating Ab into the brain, thus reducing Ab levels in the brain in aged APPsw/0 mice overexpressing human APP, a transgenic mouse model of AD with established Ab pathology. FPS-ZM1 binding to RAGE in neural cells further inhibited Ab production and suppressed microglia activation and the neuroinflammatory response, normalizing cognitive performance in the aged APPsw/0 mice. In vitro studies performed by Wan et al.44 showed that incubation of bEnd.3 cells with Ab upregulated RAGE, whereas coincubation with an anti-RAGE antibody or RAGE knockdown by small interfering RNA (siRNA) reversed the upregulation of RAGE and MMP-2 and MMP9, as well as the alterations in TJ scaffold proteins. These observations suggest an important role of RAGE in Ab-induced BBB leakage and point to MMP-2 and MMP-9 as key mediators of TJ disruption. It was further showed that interaction of Ab with RAGE on BMEC induces calcium influx and calcineurin signaling, which leads to MMP secretion, TJ breakdown and increased BBB permeability, effects that were attenuated by neutralizing antibodies against RAGE and inhibitors of calcium signaling and MMPs. These findings, obtained based on in vitro and in vivo studies, corroborate the role of RAGE as a mediator of Ab-induced changes in TJ and breakage of BBB integrity.31 It was also shown that exposure of human BMEC to Ab triggers the activation of the mitogen-activated protein kinases ERK, JNK, and PI3K and promotes endothelial CeC Chemokine Receptor Type 5 (CCR5) expression and transendothelial migration of T cells expressing a CCR5 ligand. These effects were abrogated by antibodies against RAGE and in endothelial cells expressing truncated RAGE, pointing to an Ab-mediated signaling to systemic immune T cells via RAGE at the BBB.45 LRP-1 is the major efflux transporter for Ab across the BBB, promoting Ab clearance from brain to blood through transcytosis. On the other hand, soluble LRP-1 (sLRP-1) is released into the plasma, where it binds to 70%e90% of plasma Ab, preventing free Ab from entering into the

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CONCLUDING REMARKS

brain,9 thus providing a peripheral “sink” for Ab.6 Several studies have indicated that LRP-1 expression in brain endothelium decreases in AD models and AD patients, and that reduced LRP-1 levels in brain microvessels were correlated with Ab cerebrovascular and brain accumulation in AD patients. These findings suggest that LRP-1 downregulation at the BBB may contribute to cerebrovascular and focal parenchymal Ab accumulations by reducing Ab elimination.6,7 LRP-1 expression appears to be regulated by mesenchyme homeobox gene 2, which is greatly reduced at the BBB in AD compared with agematched controls, and its downregulation was associated with Ab accumulation in the brain.7 On the other hand, Ab binding to sLRP-1 in AD patients and AD transgenic mice was shown to be compromised by oxidation of the soluble receptor, which does not bind Ab. This is associated with elevated levels of free Ab peptides in plasma that can reenter the brain via RAGE-mediated transport across the BBB.6 Peripheral clearance of Ab may also be mediated by hepatic LRP-1. Reduced hepatic LRP-1 levels are associated with decreased peripheral clearance of Ab in aged rats, suggesting that faulty LRP-1e mediated Ab clearance contribute to Ab accumulation in the brain.7 Evidence suggests that P-gp plays an important role in Ab transport across the BBB, from brain into the vascular compartment, thus contributing to Ab removal from the brain.9 In fact, it was shown that Ab microinjected into the central nervous system of P-gp null mice was cleared at half the rate of that in wild-type, whereas administration of a P-gp inhibitor to APP transgenic mice led to significantly increased Ab levels within the brain interstitial fluid.46 Moreover, BBB P-gp activity in brain regions affected by AD, namely parietotemporal, frontal, and posterior cingulate cortices, as well as hippocampus, is reduced in patients with mild AD.47 These observations link P-gp to Ab clearance across the BBB. Moreover, they suggest that impaired P-gp activity may contribute to cerebral Ab accumulation in AD and, thus, that Pgp activity at the BBB could affect the risk for developing AD, as well as provide a novel diagnostic and therapeutic target.

In contrast to P-gp, the BBB efflux transporter BCRP was shown to be upregulated in cerebral vessels of AD patients and in AD mouse models.48 The same study also demonstrated an increased transport and accumulation of Ab peptides in the brain of Abcg2 knockout animals, suggesting that this Abcg2 may play a role of gatekeeper at the BBB to prevent circulatory Ab peptides from entering into the brain. Moreover, ABCG2 expression in cultured BMEC was upregulated by paracrine factor(s) released from Ab-activated microglia,48 reflecting the importance of NVU cross-talk probably as an attempt to maintain brain homeostasis. Interestingly, interaction of Ab with its receptor RAGE in BMEC initiates cellular signaling leading to the transendothelial migration of monocytes, which may contribute to the increased presence of inflammatory cells (monocytes/macrophages) and activated microglial cells in Ab-related vascular disorders, like AD,49 thus amplifying the BBB expression of the transporter. Also interesting is the observation that oligomers and monomeric Ab peptides simultaneously induced LRP-1 downregulation and RAGE upregulation in the BBB cell line hCMEC/D3. This was accompanied by disruption of endothelial cells properties and altered transport of Ab forms, showing that in conditions that better resemble the in vivo condition, where several forms of Ab are present, the expression of both influx and efflux transporters is altered.50

CONCLUDING REMARKS In this chapter, it is pointed out the current knowledge about BBB disruption and its relationship with alterations in surrounding NVU components in AD, based on clinical and experimental evidence and on a plethora of methodologies ranging from immunohistochemistry analysis of the brain parenchyma to sophisticated MRI imaging. Available evidence points to BBB breakdown and BBB dysregulated transport as early events in AD pathogenesis, occurring prior to dementia, neurodegeneration, and/or brain atrophy. However, there is some heterogeneity in the reports, particularly in what concerns to BBB alterations

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in patients. This is not surprising considering the variability in age and disease states and the existence of comorbidities contributing to the overall state of the individual, besides the different methodologies with variable sensibilities used in different laboratories. Therefore, further studies in larger cohorts, methodological approaches standardization, and patients’ stratification appear as key aspects to consider in order to achieve a robust categorization of BBB disruption along AD onset and progression and hopefully establishment of BBB indicators as diagnostic and prognostic biomarkers. The findings about Ab and its transporters at the BBB endothelium reveal both influx and efflux transporters as key players in the brain accumulation of the peptide, and that Ab either in the periphery or within the brain parenchyma contribute to their alterations. However, it remains to be established the contribution of such transporters-mediated accumulation of Ab, in face of the BBB hyperpermeability resulting from the structural alterations. Thus, clarification of each player’s contribution to the overall Ab load would be important for the development of targeted therapies. Collectively, the current knowledge establishes BBB disruption as a key player in AD pathogenesis, and that Ab accumulation in the brain parenchyma amplifies the magnitude of the microvascular impairment. Thus, it is conceivable that a vicious circle in which BBB disruption and Ab accumulation participate contribute to the escalating progression of the disease. Not least important is the activation of signaling pathways in BBB endothelial cells and the interplay with surrounding mural and glial cells that contribute to the magnification of the cascade of events that end up with the collapse of the NVU, which contribute to the cognitive impairment in dementia that are characteristics of AD.

Acknowledgments The support of the Portuguese Foundation for Science and Technology, Portugal, through the award of the strategic Project to iMed.ULisboa (UID/DTP/04138/2013) is greatly acknowledged.

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44. Wan W, Cao L, Liu L, et al. Abeta(1-42) oligomer-induced leakage in an in vitro blood-brain barrier model is associated with up-regulation of RAGE and metalloproteinases, and down-regulation of tight junction scaffold proteins. J Neurochem. July 2015;134(2):382e393. 45. Li M, Shang DS, Zhao WD, et al. Amyloid beta interaction with receptor for advanced glycation end products up-regulates brain endothelial CCR5 expression and promotes T cells crossing the blood-brain barrier. J Immunol. May 1, 2009;182(9):5778e5788. 46. Cirrito JR, Deane R, Fagan AM, et al. P-glycoprotein deficiency at the blood-brain barrier increases amyloid-beta deposition in an Alzheimer disease mouse model. J Clin Invest. November 2005;115(11):3285e3290. 47. Deo AK, Borson S, Link JM, et al. Activity of P-glycoprotein, a beta-amyloid transporter at the blood-brain barrier, is compromised in patients with mild Alzheimer disease. J Nucl Med. July 2014;55(7):1106e1111.

48. Xiong H, Callaghan D, Jones A, et al. ABCG2 is upregulated in Alzheimer’s brain with cerebral amyloid angiopathy and may act as a gatekeeper at the blood-brain barrier for Abeta(1-40) peptides. J Neurosci. April 29, 2009;29(17):5463e5475. 49. Giri R, Shen Y, Stins M, et al. beta-amyloid-induced migration of monocytes across human brain endothelial cells involves RAGE and PECAM-1. Am J Physiol Cell Physiol. December 2000;279(6):C1772eC1781. 50. Qosa H, LeVine 3rd H, Keller JN, Kaddoumi A. Mixed oligomers and monomeric amyloid-beta disrupts endothelial cells integrity and reduces monomeric amyloid-beta transport across hCMEC/D3 cell line as an in vitro blood-brain barrier model. Biochim Biophys Acta. September 2014;1842(9): 1806e1815.

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26 Thermal Stress and Its Physiological Implications Nigel A.S. Taylor Centre for Human and Applied Physiology, School of Medicine, University of Wollongong, Wollongong, Australia O U T L I N E Introduction Exogenous and Endogenous Sources of Thermal Stress Climate Change and the Speciation of Homo Sapiens Generalisations Concerning Thermal Stress

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Our Thermal Environment First Principles

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Stress and Strain in the Human Thermal Context The Thermal and Water Vapour Pressure Continua Thermodynamics The Impact of Composition and Shape on Heat Exchanges

Quantification of the Thermal Environment Indices of Stress and Strain

Concepts of Mammalian Homoeothermy Morphological Considerations The Cutaneous Vascular Network Eccrine Sweat Gland Distributions Skeletal Muscles

Principles of Physiological Control and Regulation

Stress: Physiology, Biochemistry, and Pathology https://doi.org/10.1016/B978-0-12-813146-6.00026-6

Passive and Active Systems Homoeostasis Normothermia, Physiological Accommodation, and Zones of Thermoregulation Thermal Adaptation Thermally Mediated Cutaneous Vasomotor Responses Thermally Mediated Sudomotor Responses Morphological Determinants of Cutaneous Blood Flow and Sweating Predicting Scenarios of Adverse Strain

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Interactive Influences Interactions With Other Homoeostatic Mechanisms Nonthermal Sudomotor Responses: Psychological Stress The Interactive Impact of Clothing Conclusion

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Copyright © 2019 Elsevier Inc. All rights reserved.

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INTRODUCTION Changes in one’s internal and immediate external environments elicit feedback from sensory organs and receptors distributed throughout the body. That feedback is relayed to the somatosensory cortex, with parallel signals passing to other central nervous structures. The processing of those neural messages permits the interpretation and understanding of sensory information, leading to both conscious and subconscious responses. The former arise following deviations away from states associated with comfort and pleasure. The latter occurs when challenges, or threats, to the stability of the internal environment occur (milieu inte´rieur1), which we seek to defend between somewhat rigid boundaries.2,3 Those demarcation points (effector activation thresholds) define the characteristics of the fluid in which our cells are bathed (the extracellular fluid), and which are most conducive to life.4 Chemical and physical changes that disturb, or threaten to disturb, the equilibrium of that state are collectively classified as physical stresses,5 and they occur in many forms. Selye called those stimuli “stressors,”6,7 and that term remains in popular use. KEY POINTS 1. Thermodynamic concepts are used to identify and explain the sources of thermal stress and the resulting physiological strain. 2. The principal physical and physiological avenues of heat exchange are identified, along with their likely evolutionary development. 3. Homoeostasis is introduced, with human thermoregulation then described, supported by the anatomical and physiological characteristics of two heatloss pathways. 4. The impact of multiple interacting regulatory systems, as well as those of nonthermal stress and clothing, is considered, resulting in a detailed control model for the physiological impact of thermal stress.

In this chapter, the emphasis is upon factors that alter the thermal energy (heat) content of the body, with a focus primarily on healthy adults, although it is well recognized that sedentary ageing is associated with an unrelenting decline in tolerance across a broad spectrum of stresses.8,9 However, stress is only a starting point. From a physiological perspective, our interests lie in the subconscious (autonomic) responses to stress, and how they work in concert to defend the milieu inte´rieur (homoeostatic regulation), and thereby permit us to accommodate such disturbances.10 Accordingly, we must emphasise the acute physiological responses, which Adolph called “adaptates,”11 that support homoeostasis following stress exposure. Those responses quantify the impact or physiological strain associated with stress, and it will become evident that some variables remain within very narrow bands (e.g., normal deep-body temperature range: 35e39
C; 11.4% variation), while others are much more variable (e.g., normal heart rate range: 50e200 beats/ min; 300% variation). Those differences tell us that some variables are homoeostatically regulated, while others are subservient to one or more of our integrated regulatory processes, and are being controlled primarily to maintain the constancy of the internal environment (regulated variables); these topics will be dealt with in detail in Concepts of Mammalian Homoeothermy. Thus, it is not unusual, particularly during mild stress exposures, to see the status of a regulated variable remaining constant while that of a controlled variable is appreciably modified to sustain the status quo.

Exogenous and Endogenous Sources of Thermal Stress It is commonly considered that changes in the thermal energy content of the body are brought about primarily by external (exogenous) influences that modify the radiative, convective and conductive (dry) heat exchanges, as well as the evaporative heat losses, between the body and its surroundings, resulting in the rise and fall of tissue temperatures. In this regard, we might consider sources of external stress that arise from variations in ambient temperature and water vapour pressure, and from exposure to

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INTRODUCTION

nonionizing radiation (radio waves, microwaves, infrared, and near ultraviolet exposures). Since thermal energy moves inexorably from locations of higher to lower thermal energy (Second Law of Thermodynamics),12 and at a rate that is proportional to the size of the thermal gradient (Newton’s Cooling Law),13,14 then our capacity to either lose or conserve body heat is intimately affected by temperature changes in our immediate surroundings. When that temperature exceeds that of the skin, then it is only possible to dissipate heat through evaporation, and since humans are sweating specialists, then increases in the water vapour content of the air will impede the evaporation of sweat and the associated heat loss. Thus, heat exposures in drier climates are better tolerated than those in the tropics. While these examples represent the most familiar sources of thermal stress, we must also appreciate that we are living engines that generate (endogenous) heat from metabolic processes that convert food (chemical potential

energy) into useful kinetic, mechanical, electrical and radiation energy. Like motor vehicles, we are inefficient energy convertors, with about 80% of chemical energy being converted into thermal energy. Indeed, in a well-rested (basal) state, the unisexual, morphological reference adult (70.0 kg, 1.70 m)15 produces about the same thermal energy as a 100-W light bulb. Since the surface area of that individual is 1.8 m2, then, when resting in an unclothed state at 25
C (water vapour pressure 1.58 kPa [50% relative humidity] and average skin temperature 33
C), all of that heat is readily dissipated through the skin (convective and radiative losses) to the surrounding environment (Fig. 26.1A), so the relationship between the mass and surface area is very important for heat exchange (Section The Impact of Composition and Shape on Heat Exchanges). In this instance, dry heat loss will exceed the resting metabolic heat production, leading to an obligatory and involuntary elevation in heat production (shivering thermogenesis). The summation

FIGURE 26.1

First-principles calculations of the thermal energy exchanges experienced by a unisexual reference adult (70.0 kg, 1.70 m: Miller et al., 1980) during nude, resting air exposures to three ambient conditions, all with an initial deep-body temperature of (36.8
C). Condition A: 25
C, water vapour pressure 1.58 kPa (50% relative humidity), average skin temperature 33
C. Condition B: 33
C, water vapour pressure 2.52 kPa (50% relative humidity), skin temperature 34
C. Condition C: 41
C, water vapour pressure 3.89 kPa (50% relative humidity), skin temperature 35
C.

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26. THERMAL STRESS AND ITS PHYSIOLOGICAL IMPLICATIONS

of those components should approach zero within a thermal steady state, as dictated by the First Law of Thermodynamics.16 If we transfer our person into a 33
C room (50% relative humidity and skin temperature now 34
C), those convective and radiative losses are reduced by 85%, the requirement to shiver is removed and is replaced by a small increase in the required evaporation (sweating: Fig. 26.1B). At 41
C (50% relative humidity and skin temperature now 35
C), heat is gained from the environment through convection and radiation, and is accompanied by a pronounced elevation in evaporative cooling (Fig. 26.1C). In that case, the complete evaporation of 0.6 L of sweat would be sufficient to prevent a rise in deep-body temperature. In 1775, Charles Blagden demonstrated the extraordinary power of evaporative cooling in air exposures of 104e115
C, during which accompanying meat and eggs were cooked, while he (an eccrine sweater) and a companion dog (a salivator) remained unharmed.17 Our sweating (sudomotor) response is both unique and powerful, and there is evidence to suggest that its evolutionary appearance enabled homo sapiens to survive a previously catastrophic climate change.

Climate Change and the Speciation of Homo Sapiens The World Meteorological Organization recently reported that, since the 1940s, the rate of atmospheric carbon dioxide elevation has become almost 100-fold faster than at the end of the most recent ice age.18 That change, in combination with other rapidly rising greenhouse gases, is producing significant climatic disturbances, including, but not limited to, more dramatic and more frequently encountered extremes of temperature. Uninformed skeptics, disinterested capitalists, and opportunistic politicians would have us believe that these tightly linked events are not the result of human activities. While climate change is not a contemporary phenomenon, it does represent a significant stress, with sudden climatic disturbances sometimes leading to mass extinctions, but also the appearance of new species.

In the case of homo sapiens, who evolved in Africa, unusually rapid and large temperature changes (250,000 years ago) converted that continent from a very cold to a tropical habitat within one century.19 With such a rapid transition, less thermally tolerant species became extinct, while homo sapiens arose.20,21 Presumably as a consequence of the previously cold climate and the associated pressures of natural selection, those humans were endothermic. As such, and in common with all mammals and birds,21,22 they possessed a high metabolic rate (tachymetabolism), in combination with behaviourally and autonomically mediated control mechanisms that regulated their body temperatures by controlling the rates of heat production and heat loss (Section Concepts of Mammalian Homoeothermy). That is, they did not permit their body temperature to conform with changes in the thermal environment (ectothermy or poikilothermy: i.e., animals other than mammals and birds), but instead defended a relatively constant deep-body temperature (endothermy or homoeothermy). In Fig. 26.1, we saw that the required heat production (shivering thermogenesis) to stabilise body temperature, when confronted with continued respiratory, convective and radiative heat losses, was almost three times the resting metabolic heat production. That mechanism provided early man with a level of cold defence that could be reinforced by learned behavioural strategies (clothing and shelter), as evident in all sentient creatures,23,24 and even unicellular organisms.25 Indeed, the ubiquitous nature of behavioural thermoregulation across ecto- and endo-thermic species establishes it as the most primitive form of temperature regulation,24 over which was firstly laid the capacity to use existing cutaneous vascular networks for either the conservation or dissipation of heat26; a selected coadaptation present even in ectotherms. “It would be unnecessarily burdensome to require the evolutionary process to create a new system to solve a problem already solved by an existing system”.27 (P21) That vasomotor regulatory mechanism was perhaps followed by nonshivering thermogenesis,28 then by shivering, with sweating probably added last, as it occurs only in

INTRODUCTION

mammals.29 Moreover, eccrine sweat glands are present only in the higher order, bare-skin primates,30,31 with some considering that they possibly appeared contemporaneously with bipedalism.32 It seems that natural selection also favoured heat tolerance that could support endurancebased hunting and escape strategies,33,34 as well as persistence hunting,35 which occurs over several hours and frequently during the heat of the day. Before the African climatic change, the selective requirement for heat dissipation was less powerful. However, the coming of savanna and tropical habitats changed the playing field dramatically. Since most mammals are panters, then heat dissipation during periods of rest was both feasible and efficient, but not when running. Instead, they chose to transiently tolerate the thermal load accompanying heavy exercise, delaying heat dissipation to times of rest. This worked well when everybody operated by the same rules, but the arrival of homo sapiens further complicated survival. They had acquired eccrine sweat glands, which are assumed to have been coselected with bipedal endurance and tools use.32,36

Generalisations Concerning Thermal Stress For thermoconformers, the temperature of the ambient medium directly influences body temperature, and thereby modifies the rate of metabolic reactions and many other biological functions. That effect is reflected in the Arrhenius equation, which demonstrates the thermal dependence of most reactions, and the Q10 temperature coefficient, which provides a numerical solution for the impact of a given temperature change. For most animals, one finds a two- to three-fold elevation in biological reactions for a temperature change of 10
C. Mammals and birds also adhere to those principles, but as obligate thermal regulators, they have autonomically controlled organs that resist forced body temperature changes (Section Concepts of Mammalian Homeothermy). Instead, those species endeavor to defend body temperatures at levels that are generally higher than ambient temperature; for

353

comfortably resting humans, the deep-body temperature will vary around 36.5e37.0
C (depending upon the site of measurement), and the average skin temperature will be about 33
C.37 While that thermoregulatory strategy is energetically expensive, it is advantageous due to the thermal dependence of most biological functions, which are held in a state of readiness for immediate activation. Nevertheless, circumstances exist during which those mechanisms can become overwhelmed or disrupted (excessive heat storage, excessive heat loss and chemical disturbances), forcing body temperatures to move from a regulated to an apparently unregulated state.38 In other cases, temperature variations can be observed, but without regulation being lost. There are two general examples of this permissible or regulated variance. Firstly, when we are resting comfortably, body temperature changes over a range of about 0.6
C can be seen before either shivering (at one end) or sweating are activated. Secondly, during steady-state thermal exposures and exercise, body temperatures may rise (or fall) by as much a 1.5
C, resulting in the recruitment of sweating (or shivering). However, rather than that temperature change continuing unchecked, autonomically controlled structures are able to compensate for the elevated heat storage (or heat loss), resulting in the attainment of a stable body temperature, albeit above (or below) that which obtained when resting comfortably (the basal state). These regulated hyper- and hypothermic states are often misinterpreted as instances of regulatory failure. Instead, they represent efficient accommodations that permit mild-moderate thermal stresses to be physiologically compensated (in the short term) without undue strain, and it is upon those conditions that the greatest emphasis of this contribution will be placed. In the first instance, thermal stresses are detected by sensors located just below the skin surface (thermoreceptors), giving rise to sensations we associate with high and low thermal energy states.39,40 Eventually, the deeper tissues may experience heating or cooling, the combined effect of that thermal feedback allows us to form an opinion concerning how comfortable we are with those thermal stresses,

354

26. THERMAL STRESS AND ITS PHYSIOLOGICAL IMPLICATIONS

and in which set of conditions we prefer to reside (thermal preferendum); some prefer warmer, others prefer cooler temperatures. Nevertheless, depending upon one’s place of residence and the use of air conditioning, thermal comfort is heavily influenced by past and concurrent experiences, and will vary seasonally,41 resulting in people reporting greater comfort with heat in the summer than during the winter months.

OUR THERMAL ENVIRONMENT In January 2017, the World Meteorological Organization declared 2016 the hottest year on record,42 although local hot spots have always existed, with considerable variation across the continents (Table 26.1). For instance, the highest naturally occurring local air temperature on earth is 56.7
C (Furnace Creek, USA: World Meteorological Organization, 2018b),43 with the lowest being 89.2
C (Vostok Station, Antarctica). Survival of unprotected humans is seriously challenged at those extremes. However, technological (behavioural) advances in protective clothing and equipment have not only negated those thermal stresses, but they have enabled exploration of our deepest oceans (Challenger Deep: 10,900 m) and extended residence within the thermosphere (International Space Station: 400 km above earth). Explorers and adventurers must also overcome extremes of physical and psychological stress, often with minimal technical support. Moreover, workers, particularly within less

developed regions, are exposed to extremes of exogenous heat, while themselves generating significant endogenous heat. For instance, within the deep South African mines (3.6 km), rock surface temperatures >60
C are routinely observed.44 In the steel industry, blast furnaces, which typically operate around 1500
C, and molten steel, which is tempered at temperatures between 200 and 600
C, provide significant source of radiant heat. Clearly, some conditions are not conducive to life, with cell walls rupturing at tissues temperatures of about 0.6
C,45 and proteins start to lose the integrity of their molecular structures (denature) at temperatures above 43e45
C,46 with those changes becoming irreversible beyond 57
C.47 Largely as a consequence of stress avoidance and minimisation strategies, most thermal experiences are much less grueling, and depend upon the nature of the local climate and level of physical activity. However, it was once thought that Europeans were ill-suited to life in the tropics,48 with fevers and heat illnesses rendering “. some employments, which are of such a nature, as cannot well be performed in hot and unhealthy countries, by such as are lately arrived, without imminent danger of their health and lives .”.49 (P 104) Not surprisingly, Noe¨l Coward suggested that only “mad dogs and Englishmen go out in the midday sun.”50 We now know those interpretations lacked an understanding of physiological adaptation, with abundant contemporary evidence supporting the absence of ethnic differences in heat tolerance, providing opportunities to adapt were equally available.51e53

TABLE 26.1 Continental Temperature Extremes (World Meteorological Organization, 2018b)43 Continent

Hottest Temperature

Coldest Temperature

Africa

55.0
C (Kebili, Tunisia)

23.9
C (Ifrane, Morocco)

Antarctica

17.5
C (Esperanza Research Station)

89.2
C (Vostok Station)

Asia

Currently under investigation (World Meteorological Organization)

67.8
C (Verkhoyansk, Russia)

Australia

50.7
C (Oodnadatta)

23.0
C (Charlotte Pass)

Europe

48.0
C (Athens, Greece)

58.1
C (Ust ‘Shchugor, Russia)

North America

56.7
C (Furnace Creek, California)

63.0
C (Snag, Yukon Territory)

South America

48.9
C (Rivadavia, Argentina)

32.8
C (Sarmiento, Argentina)

OUR THERMAL ENVIRONMENT

First Principles Stress and Strain in the Human Thermal Context The convention adopted herein will be to restrict the use of some words, which are frequently used synonymously, to their scientific denotations. Accordingly, as noted previously, physical and chemical stimuli will be classified as stresses.5,54 The physiological impact of those stimuli is quantified using indices of strain, and while understanding those stresses is important, they have no physiological significance unless they disturb homoeostasis, or have the potential to do so. To help set the scene for the discussion that follows, the clinical significance of variations in deep-body temperature between the survivable extremes is presented (Table 26.2). What becomes immediately, and perhaps also paradoxically, evident is that humans, who evolved in the tropics, seem to have the capacity to survive body temperature reductions that are about twice the tolerable temperature elevation.55e57 Perhaps, this was because our evolutionary forebears appeared when the earth was much cooler. TABLE 26.2

Clinically Significant Deep-Body Temperatures

Deep-Body Temperature (
C)

General Classification

>45

Probable death unless aggressively cooled

>40

Profound clinical hyperthermia