Basic Aspects of Receptor Biochemistry: Proceedings of the International Symposium, Vienna, Austria September 10–12, 1982 [1st ed.] 978-3-211-81733-9;978-3-7091-4408-4

Table of contents :
Front Matter ....Pages I-IX
The Normal and Aberrant Development of Synaptic Structures Between Parallel Fibers and Purkinje Cell Dendritic Spines (A. Hirano)....Pages 1-8
High-Resolution Radioautographic Study of Dopamine Binding Sites in the Rat Neostriatum Using 3H-Domperidone (M. Arluison, Marie-Pascale Martres, P. Sokoloff)....Pages 9-24
Neurotransmitter Related Immunocytochemistry of the Human Central Nervous System (J. Pearson, L. Brandeis, M. Goldstein, A. Claudio Cuello)....Pages 25-31
Studies on the Neurotransmitter Binding to Pig Brain Microvessels (W.-D. Rausch, W. Rossmanith, J. Gruber, P. Riederer, K. Jellinger, M. Weiser)....Pages 33-44
Coordination Chemical Aspects of Receptor Biochemistry (H. Noller, Elisabeth Kienzl, P. Riederer)....Pages 45-54
Energetics of Ligand Binding to Receptors (T. J. Franklin)....Pages 55-60
Receptor Blockade and Synaptic Function (D. M. J. Quastel, P. Pennefather)....Pages 61-81
Patch-Clamp Measurements of Elementary Chloride Currents Activated by the Putative Inhibitory Transmitters GABA and Glycine in Mammalian Spinal Neurons (B. Sakmann, O. P. Hamill, J. Bormann)....Pages 83-95
Calmodulin, Ca2+-Antagonists and Ca2+-Transporters in Nerve and Muscle (J. D. Johnson, Laura A. Wittenauer, R. D. Nathan)....Pages 97-111
Adenosine: Transport, Function and Interaction with Receptors in the CNS (G. W. Kreutzberg, M. Reddington, K. S. Lee, P. Schubert)....Pages 113-119
Regulation of Noradrenergic Receptor Systems in Brain that Are Coupled to Adenylate Cyclase (F. Sulser, D. H. Manier, A. J. Janowsky, F. Okada)....Pages 121-130
The Search for Selective Dopaminergic Autoreceptor Agonists (S. Hjorth, A. Carlsson, D. Clark, K. Svensson, H. Wikström, D. Sanchez et al.)....Pages 131-137
Evidence That the D-2 Dopamine Receptor in the Intermediate Lobe of the Rat Pituitary Gland Is Associated with an Inhibitory Guanyl Nucleotide Component (T. E. Cote, E. A. Frey, C. W. Grewe, J. W. Kebabian)....Pages 139-147
Dopamine Receptor Sites and States in Human Brain (Susan George, Karen Binkley, P. Seeman)....Pages 149-156
Biochemical and Pharmacological Differentiation of Neuroleptic Effect on Dopamine D-1 and D-2 Receptors (J. Hyttel, Anne Vibeke Christensen)....Pages 157-164
Evidence for the Existence of Receptor—Receptor Interactions in the Central Nervous System. Studies on the Regulation of Monoamine Receptors by Neuropeptides (K. Fuxe, L. F. Agnati, F. Benfenati, M. Celani, I. Zini, M. Zoli et al.)....Pages 165-179
(3,4-Dihydroxyphenylimino)-2-Imidazoline (DPI) and Its Action at Noradrenergic and Dopaminergic Receptors in the Nucleus Accumbens of Rats: Mesolimbic Catecholamine Receptors and Hyperactivity (A. R. Cools, S. K. Oosterloo)....Pages 181-188
Interactions of Ergot Compounds with Dopamine Receptors and Endocrine Functions (E. Flückiger)....Pages 189-204
Long-Term Adaptive Changes in Striatal Dopamine Function in Response to Chronic Neuroleptic Intake in Rats (P. Jenner, R. Kerwin, N. M. J. Rupniak, K. Murugaiah, M. D. Hall, S. Fleminger et al.)....Pages 205-212
Cotransmitters: Pharmacological Implications (A. Guidotti, L. Saiani, B. C. Wise, E. Costa)....Pages 213-225
Multiple Opiate Receptors and Their Functional Significance (A. Herz)....Pages 227-233
Peptidases Involved in the Inactivation of Exogenous and Endogenous Enkephalins (J. C. Schwartz, Sophie de la Baume, C. C. Yi, P. Chaillet, Hélène Marcais-Collado, J. Costentin)....Pages 235-243
D2-Protein and D3-Protein as Markers for Synaptic Turnover and Concentration (O. S. Jørgensen)....Pages 245-255
Therapeutic Potentials of Centrally Acting Dopamine and α2-Adrenoreceptor Agonists (M. Goldstein, J. Engel, A. Lieberman, I. Regev, A. Bystritsky, S. Mino)....Pages 257-263
Dopamine Receptor Changes in Schizophrenia in Relation to the Disease Process and Movement Disorder (A. J. Cross, T. J. Crow, I. N. Ferrier, E. C. Johnstone, R. M. Mccreadie, F. Owen et al.)....Pages 265-272
Preliminary Studies of Human Cortical 5-HT2 Receptors and Their Involvement in Schizophrenia and Neuroleptic Drug Action (G. P. Reynolds, M. N. Rossor, L. L. Iversen)....Pages 273-277
Brain Receptor Changes in Parkinson’s Disease in Relation to the Disease Process and Treatment (U. K. Rinne, J. O. Rinne, J. K. Rinne, K. Laakso, A. Laihinen, P. Lönnberg)....Pages 279-286
Serotonin Binding in Rat Brain: Circadian Rhythm and Effect of Sleep Deprivation (W. Wesemann, N. Weiner, M. Rotsch, E. Schulz)....Pages 287-294
General Properties of 14C-L-Valine-Binding to Human Brain Tissue (P. Riederer, E. Kienzl, K. Jellinger, H. Noller, G. Kleinberger)....Pages 295-306
5-Hydroxytryptamine Receptors in Neurones and Glia (G. Fillion, Dominique Beaudoin, Maria-Paule Fillion, J.-C. Rousselle, Christine Robaut, Y. Netter)....Pages 307-317
Pharmacological and Therapeutic Actions of GABA Receptor Agonists (B. Zivkovic, B. Scatton, P. Worms, K. G. Lloyd, G. Bartholini)....Pages 319-326
A Possible Relationship Between Folic Acid Neurotoxicity and Cholinergic Receptors in the Pyriform Cortex and Amygdala (P. L. McGeer, Edith G. McGeer, T. Nagai, P.-T. Wong)....Pages 327-344
Association of Proteins Irreversibly Labeled by 3H-Flunitrazepam with Different Benzodiazepine Receptors (W. Sieghart)....Pages 345-352
Tubocurarine, a Partial Agonist for Cholinergic Receptors (A. Trautmann)....Pages 353-361
Differentiation—Dependent Changes of the Nicotinic Acetylcholine Receptor and Other Synapse-Associated Proteins (V. Witzemann)....Pages 363-368
Cellular Mechanism Involved in the Synthesis of Cyclic GMP in Nervous Tissues (T. Deguchi, S. Ohsako, M. Nakane, M. Ichikawa, M. Yoshioka)....Pages 369-378
Closing Remarks (M. Goldstein)....Pages 379-380
Back Matter ....Pages 381-392

Citation preview

Journal of Neural Transmission Supplementum 18

Basic Aspects of Receptor Biocheltlistry

Springer- Verlag Wien GlDhH

Journal of Neural Transmission Supplementum 18

Basic Aspects of Receptor Biochentistry Proceedings of the International Symposium,Vienna, September 10-12, 1982 Edited by M.Goldstein, K. Jellinger, and P. Riederer

Springer-Verlag Wi en GmbH

Prof Dr. Menek Goldstein

Department' of Psychiatry, New York University Medical Center, New York, N.Y., U.S.A.

Prof Dr. Kurt Jellinger

Ludwig Boltzmann-Institut flir Klinische Neurobiologie und Neurologische Abteilung, Krankenhaus der Stadt Wien-Lainz, Wien, Austria

Prof. Dr. Peter Riederer

Ludwig Boltzmann-Institut fiir Klinische Neurobiologie Krankenhaus der Stadt Wien-Lainz, Wien, Austria

This work is subject to copyright. All rights are reserved, whether the whole or part of the material is concerned, specifically those of translation, reprinting, re-use of illustrations, broadcasting, reproduction by photocopying machine or similar means, and storage in data banks. © 1983 by Springer-Verlag Wien Originally published by Springer-Verlag Wien-New York in 1983 Softcover reprint of the hardcover I st edition 1983

With 100 Figures

Library of Congress Cataloging in Publication Data. Main entry under title: Basic aspects of receptor biochemistry. Oournal of neural transmission. Supplementum, ISSN 0303-6995-; 18) "Symposium on 'Basic Aspects in Brain Receptor Biochemistry', organized by the Ludwig Boltzmann-Institute of Clinical Neurobiology, Vienna"-P. 1. Neurotransmitter receptorsCongresses. I. Goldstein, Menek. II. ]ellinger, K (Kurt), 1931- . III. Riederer, P., 1942- . IV. Ludwig Boltzmann-Institut ftir Klinische Neurobiologie. V. Symposium on "Basic Aspects in Brain Receptor Biochemistry" (1982 : Vienna, Austria). VI. Series. [DNLM: 1. Receptors, Sensory-Congresses. WI ]0781A no. 18 ! WL 102.9 B3111982]. QP364.7.B37. 1983. 599.01'88. 83-4762

ISBN 978-3-7091-4410-7

ISSN 0303-6995

ISBN 978-3-7091-4408-4 (eBook)

DOl 10.1007/978-3-7091-4408-4

Preface In recent years a number of research fields in biology have focussed on the concept of receptors. The selective binding of small molecules to specific sites of the cell membrane is being increasingly recognized as the basic mechanism by which cells communicate with each other. It has been shown that neurotransmitters, hormones and drugs must first interact with specific receptors in order to elicit their biochemical, physiological, and pharmacological effects. The basic field of "cellular receptors" and the fundamental concepts of receptor activity embrace a broad spectrum of biomolecular interactions which have been the subject of intense investigations. Although considerable progress has been made in the understanding of the receptor-related phenomena, e.g. the location, sensitivity, and kinetics of neurotransmitter receptors, little progress has been made in elucidating the structures of receptors and the biochemical and biophysical components coupled to them. Many basic problems in the function and dysfunction of receptors and their importance in neurophysiology, neurochemistry, endocrinology, pharmacology, and in clinical neurosciences are not yet fully understood. It was, therefore, the object of a Symposium on "Basic Aspects in Brain Receptor Biochemistry", organized by the Ludwig BoltzmannInstitute of Clinical Neurobiology, Vienna, as a Satellite Symposium to the IXth International Congress of Neuropathology in Vienna, September 1982, to provide an up-to-date overall view of the present state of knowledge of "receptorology" with particular emphasis on the nervous system. Studies of different receptors have progressed at different rates and along different lines. Some receptor-related mechanisms have been found to play an important role in behavioural regulation, aging, and in the pathophysiology and pathogenesis of nervous and psychiatric disorders, e.g. of extrapyramidal diseases or schizophrenia, and in the effects and side-effects of drug treatment. However, since many of the fundamental mechanisms of action of receptors in the normal and diseased nervous system are still poorly understood, it may well be an appropriate time for compiling informations on this special field in order to give some "crossfertilization" of ideas

VI

Preface

and approaches from and to neuroanatomy, physiology, biochemistry, molecular biology, pharmacology, behavioral and clinical neurosciences. Thus, it was also the aim of the Symposium to seed such an interdisciplinary interaction and to give new impacts to both basic research and applied neurosciences. We would like to express our gratitude to all participants of this Symposium for contributing their efforts and for presenting the manuscripts so quickly. We are indebted to Hoffmann-La Roche, Vienna, for supporting the organization of the meeting, and to Springer-Verlag, Vienna, for providing the rapid publication of this volume and for their appreciation of the editorial concerns. M. Goldstein

K.}ellinger

P. Riederer

Contents Hirano, A.: The Normal and Aberrant Development of Synaptic Structures Between Parallel Fibers and Purkinje Cell Dendritic Spines . . . . . . . . . . . . . . . . . . . . . .

1

Arluison, A., Martres, Marie-Pascale, Sokoloff, P.: High-Resolution Radioautographic Study of Dopamine Binding Sites in the Rat Neostriatum Using 3H-Domperidone. . . . . . . . .

9

Pearson, J., Brandeis, L., Goldstein, M., Claudio Cuello, A.: Neurotransmitter Related Immunocytochemistry of the Human Central Nervous System . . . . . . . . . . . . . . .

25

Rausch, W.-D., Rossmanith, W., Gruber, J., Riederer, P., Jellinger, K., Weiser, M.: Studies on Neurotransmitter Binding to Pig Brain Microvessels. . . . . . . . . . . . . . . .

33

Noller, H., Kienzl, Elisabeth, Riederer, P.: Coordination Chemical Aspects of Receptor Biochemistry. . . . . . . . . . . .

45

Franklin, T.J.: Energetics of Ligand Binding to Receptors

55

Q!lastel, D. M.J., Pennefather, P.: Receptor Blockade and Synaptic Function . . . . . . . . . . . . . . . . . . . . .

61

Sakmann, B., Hamill, O. P., Bormann, J.: Patch-Clamp Measurements of Elementary Chloride Currents Activated by the Putative Inhibitory Transmitters GABA and Glycine in Mammalian Spinal Neurons. . . . . . . . . . . . . . . .

83

Johnson, D.J., Wittenauer, Laura A., Nathan, R. D.: Calmodulin, Ca2+-Antagonists and Ca2+-Transporters in Nerve and Muscle

97

Kreutzberg, G. W., Reddington, M., Lee, K. S., Schubert, P.: Adenosine: Transport Function and Interaction with Receptors in the CNS . . . . . . . . . . . . . . . . . . . .

113

Sulser, F., Manier, D. H., Janowsky, A.J., Okada, F.: Regulation of Noradrenergic Receptor Systems in Brain that Are Coupled to Adenylate Cyclase. . . . . . . . . . . . . . . . .

121

Hjorth, A., Carlsson, A., Clark, D., Svensson, K., Wikstrom, H., Sanchez, D., Lindberg, P., Hacksell, U., Arvidsson, L.-E., Johansson, A., Nilsson, J. L. G.: The Search for Selective Dopaminergic Autoreceptor Agonists . . . . . . . . . .

131

Contents

VIII

Cote, T. E., Frey, E. A., Grewe, C. W., Kebabian, J. W.: Evidence That the D-2 Dopamine Receptor in the Intermediate Lobe of the Rat Pituitary Gland Is Associated with an Inhibitory Cuanyl Nucleotide Component. . . . . . . . . . . . . . . .

139

George, Susan, Binkley, Karen, Seeman, P.: Dopamine Receptor Sites and States in Human Brain. . . . . . . . . . . .

149

Hyttel, J. Christensen, Anne Vibeke: Biochemical and Pharmacological Differentiation of Neuroleptic Effect on Dopamine D-1 and D-2 Receptors. . . . . . . . . . . . . . . . . .

157

Fuxe, K., Agnati, L. F., Benfenati, F., Celani, M., Zini, I., Zoli, M., Mutt, V.: Evidence for the Existence of Receptor-Receptor Interactions in the Central Nervous System. Studies on the Regulation of Monoamine Receptors by Neuropeptides. . .

165

Cools, A. R., Oosterloo, S. K.: (3,4-Dihydroxyphenylimino)-2-Imidazoline (DPI) and Its Action at Noradrenergic and Dopaminergic Receptors in the Nucleus Accumbens of Rats: Mesolimbic Catecholamine Receptors and Hyperactivity. . . . .

181

Fluckiger, E.: Interactions of Ergot Compounds with Dopamine Receptors and Endocrine Functions. . . . . . . . . . .

189

Jenner, P., Kerwin, R., Rupniak, N. M. J., Murugaiah, K., Hall, M. D., Fleminger, S., Marsden, C. D.: Long-Term Adaptive Changes in Striatal Dopamine Function in Response to Chronic Neuroleptic Intake in Rats. . . . ... . . . .

205

Guidotti, A., Saiani, L., Wise, B. C., Costa, A.: Cotransmitters: Pharmacological Implications . . . . . . . . . . . . .

213

Herz, A.: Multiple Opiate Receptors and Their Functional Signiflcance

227

J. C.,

de la Baume, Sophie, Vi, C. C., Chaillet, P., Schwartz, Marcais-Collado, Helene, Costentin, J.: Peptidases Involved in the Inactivation of Exogenous and Endogenous Enkephalins

235

J0rgensen, O. S.: D2-Protein and D3-Protein as Markers for Synaptic Turnover and Concentration. . . . . . . . . . . . . .

245

Goldstein, M., Engel, J., Lieberman, A., Regev, I., Bystritsky, A., Mino, S.: Therapeutic Potentials of Centrally Acting Dopamine and az-Adrenoreceptor Agonists . . . . . . . . . . . .

257

Cross, A.J., Crow, T.J., Ferrier, I. N.,Johnstone, E. C., McCreadie, R. M., Owen, F., Owens, D. G. C., Poulter, M.: Dopamine Receptor Changes in Schizophrenia in Relation to the Disease Process and Movement Disorder. . . . . . . . . . . .

265

Reynolds, G. P., Rossor, M. N., Iversen, L. L.: Preliminary Studies of Human Cortical 5-HT z Receptors and Their Involvement in Schizophrenia and Neuroleptic Drug Action . . . . . . .

273

Contents

IX

Rinne, U. K., Rinne, J. 0., Rinne, J. K., Laakso, K., Laihinen, A., Lonnberg, P.: Brain Receptor Changes in Parkinson's Disease in Relation to the Disease Process and Treatment . . . . .

279

Wesemann, W., Weiner, N., Rotsch, M., Schulz, E.: Serotonin Binding in Rat Brain: Circadian Rhythm and Effect of Sleep Deprivation . . . . . . . . . . . . . . . . . . . .

287

Riederer, P., Kienzl, E., Jellinger, K., Noller, H., Kleinberger, G.: General Properties of 14C-L-Valine-Binding to Human Brain Tissue . . . . . . . . . . . . . . . . . . . . . .

295

Fillion, G., Beaudoin, Dominique, Fillion, Maria-Paule, Rousselle,J.-C., Robaut, Christine, Netter, Y.: 5-Hydroxytryptamine Receptors in N eurones and Glia . . . . . . . . . . . .

307

Zivkovic, B., Scatton, B., Worms, P., Lloyd, K. G., Bartholini, G.: Pharmacological and Therapeutic Actions of GABA Receptor Agonists. . . . . . . . . . . . . . . . . . . . . .

319

McGeer, P. L., McGeer, Edith G., Nagai, T., Wong, P.-T.: A Possible Relationship Between Folic Acid Neurotoxicity and Cholinergic Receptors in the Pyriform Cortex and Amygdala. . . . . .

327

Sieghart, W.: Association of Proteins Irreversibly Labeled by 3H-Flunitrazepam with Different Benzodiazepine Receptors .

345

Trautmann, A.: Tubocurarine, a Partial Agonist for Cholinergic Receptors . . . . . . . . . . . . . . . . . . . . .

353

Witzemann, V.: Differentiation-Dependent Changes of the Nicotinic Acetylcholine Receptor and Other Synapse-Associated Proteins. . . . . . . . . . . . . . . . . . . . . .

363

Deguchi, T., Ohsako, S., Nakane, M., Ichikawa, M., Yoshioka, M.: Cellular Mechanism Involved in the Synthesis of Cyclic GMP in Nervous Tissues . . . .

369

Goldstein, M.: Closing Remarks.

379

Subject Index. . . . . . . .

381

J.

Neural Transmission, SUpp!. 18, 1-8 (1983)

The Normal and Aberrant Development of Synaptic Structures Between Parallel Fibers and Purkinje Cell Dendritic Spines A.Hirano Division of Neuropathology, Department of Pathology, Montefiore Hospital and Medical Center, Albert Einstein College of Medicine, Bronx, N. Y., U.S.A. With 4 Figures

Summary Under normal circumstances the dendritic spines of the cerebellar Purkinje cell differentiate in association with the parallel fibers formed by the descending granule cells. In the adult these elements form the most frequent synapse of the cerebellar cortex. In certain conditions, however, the granule cells are destroyed before they form the parallel fibers. Nevertheless, unattached dendritic spines are found which are complete with submembranous densities. Their cytochemical reactions and their morphology in both thin section and after freeze fracture are indistinguishable from normal spines except for the fact that they are unattached to any presynaptic elements. Examples of the formation of unattached presynaptic endings have also been observed. We conclude, therefore, that at least in some instances, pre- and postsynaptic terminals may form without benefit of the direct oneto-one influence of their synaptic mates.

The architecture of the cerebellum is better understood than most other areas of the central nervous system (Palay and ChanPalay, 1974). It therefore seemed a good choice for the study of synaptic development, especially for the interaction of granule cells and Purkinje cells which are the major neurons of the cerebellar cortex. Early in development, the immature granule cells occupy the external germinal layer while the Purkinje cells are confined to a deeper level. As the granule cells descend into the molecular layer the 1 Journal of Neural Transmission, Suppl.18

2

A.Hirano

Purkinje cell dendrites arborize and grow upwards as though to meet them. During their descent the axons of the granule cells, the parallel fibers, are formed and remain behind to become the afferent connection to the Purkinje cell dendrites. The fine structure of this process has been documented (Larramendi, 1969). If one examines the cerebellum during development a broad spectrum of the developmental sequence can be seen even at a single age; beginning at the more superficial layers and extending to the deeper areas. At 14 days after birth the more superficial portions of the molecular layer in the rat (Fig. 1) show many examples of young parallel fibers, dendritic spines and even some synapses as well as astrocytic

Fig.l. A relatively superficial stratum of the molecular layer of the cerebellum of a 14 day old rat. X 36,000

Development of Normal and Aberrant Synaptic Structures

3

Fig.2. A deeper stratum of the molecular layer of the cerebellum of a 14 day old rat. X 36,000

processes. The parallel fibers are relatively wide at this time and contain several microtubules. The spines, on the other hand, are relatively small. The synapses consist of apposed pre- and postsynaptic endings. The former contain a small number of synaptic vesicles and a submembranous density and are attached to a dendritic spine which contains a clear but diminutive submembranous density. Some extracellular dense material is present between the apposing submembranous densities. Astrocytic processes are often present nearby but do not yet invest the synapse.

4

A.Hirano

Somewhat deeper in the molecular layer the 14 day old rat cerebellar cortex (Fig. 2) shows more advanced shapes of development. The parallel fibers are distinctly narrow with fewer microtubules. The dendritic spines are substantially larger. Complete synapses are more frequent and more prominent. The presynaptic terminal is larger and contains abundant synaptic vesicles as well as a mitochondrion or two. The submembranous densities of both elements are wider and denser and the cleft material is considerably more distinct. Astrocytic processes are more intricately inserted among the synaptic com-

Fig. 3. A fully developed synapsis between a parallel fiber and a Purkinje cell dendritic spine in the molecular layer of the cerebellum of an adult rat. X l30,000. Reproduced by permission [Hirano, A., et at.: An electron microscopic study of cycasin-induced cerebellar alterations. ]. N europathol. Exp. N eurol. 31, 113-125 (1972)]

Development of Normal and Aberrant Synaptic Structures

5

plexes and often seem to invest and separate single synapses. In many ways the synapses of the deeper portions of the molecular layer of the 14 day old rat are very similar to those seen in the adult (Fig. 3). The most common speculation concerning the machanisms of this apparently straightforward process is that specific receptors in each synaptic terminal respond to the presence of the synaptic mate and development proceeds from that point on. Several observations, however, indicate that this may not be entirely true. First of all, even in some normal animals dendritic spines are sometimes seen which are complete with submembranous densities but are unattached to any presynaptic element (Hirano et al., 1977). One might argue that these rare observations are the result of a loss of a presynaptic mate after development is complete. This possibility cannot be ruled out. Other observations of unattached dendritic spines are not as easily explained. Several conditions result in the loss of granule cells before parallel fibers are formed in large numbers. Virus infection (Herndon et al., 1971), cycasin intoxication (Hirano et al., 1972), certain genetic deficiencies (Hirano and Dembitzer, 1973) among other conditions (Hirano, 1979), all result in this condition. In each of these, unattached dendritic spines (Fig. 4 A) are found in abundance. So far as we can tell, except for their lack of a presynaptic mate, these spines are identical to those in the normal animal. Their staining reactions to phosphotungstic acid, bismuth iodide and uranyl acetate are indistinguishable from those of dendritic spines in the normal animal (Hirano and Dembitzer, 1973). Freeze fracture studies can show no differences (Hanna et al., 1976). It is difficult to believe that these unattached spines are the result of the loss of presynaptic elements after the initiation or completion of development. There simply are not enough parallel fibers for each spine to have been contacted. Furthermore, if one follows the development of these spines one cannot detect a stage at which a spine is attached to a degenerating parallel fiber ending (Hirano et al., 1977). It seems, therefore, that a presynaptic process or even a presumptive presynaptic process is not required for the development of what seems to be a fully differentiated dendritic spine. We cannot rule out the possibility that some point on the surface of the Purkinje cell was, indeed, in contact with a parallel fiber since a few parallel fibers are present under any of the conditions referred to above. It seems clear, however, that a Purkinje cell dendritic spine can differentiate without benefit of the immediate influence of a presynaptic ending on a oneto-one basis.

A Fig.4A. Two unattached dendritic spines surrounded by an astrocyte in the cerebellar cortex of a cycasin-treated mouse. X 130,000. Reproduced by permission [Hirano, A., et at.: An electron microscopic study of cycasin-induced cerebellar alterations. J.Neuropathol. Exp. Neurol. 31, 113-125 (1972)]

Fig.4B. Unattached presynaptic terminals in a cerebellar tumor of an 18 month old boy. X 133,000. Reproduced by permission [Hirano, A., Shin, W.-Y.: Unattached presynaptic terminals in a cerebellar neuroblastoma in the human. J. N europathol. A ppl. Neurobiol. 5, 63-70 (1979)]

A. Hirano: Development of Normal and Aberrant Synaptic Structures

7

The opposite phenomenon, i.e. the development of presynaptic elements, unattached to any target cell has also been observed during normal development of the spinal cord of an amphibian embryo (Hayes and Roberts, 1974). In addition, we have observed numerous presynaptic elements, complete with synaptic vesicles and submembranous densities, in a cerebellar tumor removed from an 18 month old infant (Hirano and Shin, 1979) (Fig. 4 B). The significance of these findings is not clear. It is impossible to suggest that the cellular environment of a developing system is not crucial. On the other hand, the possibility of a preprogrammed mechanism that may be relatively independent of direct interaction with other cells, cannot be ruled out at least in regard to the development of the synapse described above.

References Hanna, R. B., Hirano, A., Pappas, G. D.: Membrane specializations of dendritic spines and glia in the weaver mouse cerebellum. A freeze-fracture study. J. Cell BioI. 68, 403-410 (1976). Hayes, B. P., Roberts, A.: The distribution of synapses along the spinal cord of an amphibian embryo: An electronmicroscopic study of junctional development. Cell Tiss. Res. 153, 227-244 (1974). Herndon, R. M., Margolis, G., Kilham, L.: The synaptic organization of the malformed cerebellum induced by perinatal infection with the feline panleukopenia virus (PLV) in the Purkinje cell and its afferents. J. Neuropathol. Exp. Neurol. 30, 557-570 (1971). Hirano, A.: On the independent development of the pre- and postsynaptic terminals. In: Progress in Neuropathology, pp. 79-99. New York: Raven Press. 1979. Hirano, A., Dembitzer, H. M.: Cerebellar alterations in the weaver mouse. J. Cell BioI. 56, 478-486 (1973). Hirano, A., Dembitzer, H. M., Jones, M.: An electron microscopic study of cycasin-induced cerebellar alterations. J. N europathol. Exp. N eurol. 31, 113-125 (1972). Hirano, A., Dembitzer, H. M., Yoon, C. H.: Development of Purkinje cell somatic spines in the weaver mouse. Acta Neuropathol. (Bed.) 40, 85-90 (1977). Hirano, A., Shin, W-Y.: Unattached presynaptic terminals in a cerebellar neuroblastoma in the human. J.Neuropathol. Appl. Neurobiol. 5, 63-70 (1979). Larramendi, L. M. H.: Analysis of synaptogenesis in cerebellum of the mouse. In: Neurobiology of Cerebellar Evolution and Development,

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A. Hirano: Development of Normal and Aberrant Synaptic Structures

pp.803-843. Chicago: American Medical Association/Education and Research Foundation. 1969. Palay, S. L., Chan-Palay, v.: Cerebellar Cortex. Berlin-Heidelberg-N ew York: Springer. 1974. A uthors' address: Dr. A. Hirano, Division of Neuropathology, Department of Pathology, Montefiore Hospital and Medical Center, Albert Einstein College of Medicine, 111 East 210th Street, Bronx, NY 10467, U.S.A.

J.

Neural Transmission, Supp!. 18, 9-24 (1983)

High-Resolution Radioautographic Study of Dopamine Binding Sites in the Rat Neostriatum Using 3H-Domperidone M. Arluison 1, Marie-Pascale Martres 2, and P. Sokoloff lCNRS ERA 884, Laboratoire de Cytologie, Universite P. et M.Curie, Paris, France 2INSERM U 109, Paris, France With 19 Figures

Summary The possibility to use the new ligand 3H-domperidone to identify some dopamine binding sites at the ultrastructural level was assessed in the neostriatum after in vivo administration and high-resolution radioautography. Since this ligand does not cross the blood-brain barrier, intracerebral injections were performed, which resulted in a gradient of diffusion of the tracer. According to increasing distances to the injection site, a quantitative study of the radioautographic reaction was realized. An intense and diffuse reaction took place in the vicinity of the injection site in control rats. On the contrary, numerous accumulations of silver grains were observed in the peripheral zone. The statistical analysis of the distribution of the clusters showed that they were more numerous over the contacts between nerve terminals and dendritic spines than expected from a distribution at random; moreover half of these labelled contacts were differentiated in synapses of the asymmetric type. When the animals were pretreated with haloperidol in order to block the dopaminergic binding sites, we found a decrease in the total number of the number of silver grains. A decrease in the number of clusters of silver grains was noted over nerve terminals and synaptic contacts in both peripheral zones while the nonspecific labelling was increased over other structures. We conclude to the possibility of the detection of the dopaminergic binding sites by electron microscopic radioautography. Moreover we confirm the existence of dopaminergic synapses in the neostriatum with this new technique.

10

M. Arluison, Marie-Pascale Martres, and P. Sokoloff

Introduction In the last few years the pharmacological characterization of the dopaminergic binding sites was developed extensively by studying in vitro the binding of radiolabelled ligands to membrane fractions of the brain (Seeman, 1980). Although dopaminergic agonists were also used in theses studies, the dopaminergic antagonists (neuroleptics) were particularly useful. Thus, the regional distribution of dopaminergic binding sites was established using 3H-haloperidol, 3H-doperidone or 3H-spiperone binding (Seeman, 1980). Due to its very high affinity for the dopaminergic binding sites, to the slow rate of dissociation and to a high specific activity (Laduron et al., 1978), this last compound was the first tracer to be used in vivo for light microscopic radioautography. Hollt and Schubert (1978), Kuhar et al. (1978) and Murin and Kuhar (1979) have demonstrated with this technique that preferential accumulation of silver grains occurred in dopaminergic areas of the brain after intravenous injections. The labelling observed, which was displace able by other dopaminergic blockers, was considered by these authors as specific of the dopaminergic receptors. However, Leysen et al. (1978) have shown that spiroperidone binds also with high affinity to the serotoninergic receptors of the brain. So, we have preferred to use the new ligand 3H-domperidone for electron microscopic radioautography (EMRA) owing to its excellent binding characteristics, its better selectivity and its lower solubility in fats (Baudry et al., 1979; Laduron et al., 1979). Using intrastriatal injections, since domperidone does not cross the bloodbrain barrier, we have tried to localize some of the dopaminergic binding sites of the rat striatum at the ultrastructural level. We expected that (1) the perfusion of aldehydes is able to retain large amounts of the tracer closely to the binding sites and (2) the dopaminergic sites which are labelled, are not too diffusely distributed to permit the radioautographic detection owing to the low yield of the reaction. Multiple dopamine sites have been postulated from binding studies (Titeler et al., 1978; Kababian and CaIne, 1979). Among the four classes of dopaminergic sites which are currently described now (Seeman, 1980; Sokoloff et al., 1980a, b), the classes D z and D4 were shown to bind more efficiently the antagonists. The striatal sites D4 are probably localized on cortico-striatal fibres (Schwartz et al., 1978), while the striatal sites D z are likely localized post-synaptically. The aim of this work was to identify these sites and to confirm the morphological data on the existence of dopaminergic synapses which were obtained in the rat neostriatum by other techniques (Arluison et al., 1978a, b; Descarries et al., 1980; Hassler et al., 1978; Specht et al., 1981).

Dopamine Binding Sites in the Rat

11

Materials and Methods Three couples of rats weighing 250-300 g. were used. Half of the animals were injected intraperitoneally with 5 mg/kg of haloperidol two hours before the intracerebral injection of the tracer, in order to block the dopaminergic receptors. All rats were anaesthetized with chloral hydrate (350 mg/kg, i.p.) and fixed in a stereotaxic apparatus (Horsley-Clarke). After trepanation, the animals were bilaterally injected in the head of the caudateputamen at coordinates: A = 70, L = 30, H = 65 (according to the atlas of Albe-Fessard et a!., 1965), using a stainless steel canula of 300 j.lm diameter connected to a 5 j.ll Hamilton seringe by mean of a fine catheter. Four ml of 4.1O- 7M 3H-domperidone (specific activity 10 or 40 Ci/mM; I.R.E., Belgium), were slowly injected in each striatum. The survival time was 15 min. Then, the animals were perfused via the ascending aorta with 400 ml of a fixative composed of 1% glutaraldehyde2%formaldehyde in a 0.15 M, pH 7.4 phosphate buffer. After post-fixation, the injected striata were cut in horizontal slices perpendicular to the needle track. At the level of the needle tip, several series of three blocks of tissue were taken off at increasing distances from the injection site 0-0.7 mm (level A), 0.7-1.4 mm (level B) and 1.4-2.1 mm (level C). Blocks of tissue were dehydrated in alcohol and embedded in Araldite. Ultrathin sections were contrasted in uranyle acetate and lead citrate. They were processed according to the method of Larra and Droz (1970), using an Ilford L4 emulsion for high-resolution radioautography. The exposure time was either 3 or 5 months according to the specific activity of the 3H-ligand. Due to the limited resolution of the radioautographic technique, numerous silver grains can be observed on structures out of the presumed specific localization of the tracer. So, the circle method (Halasz et al., 1981; Kent et a!., 1974; Kramer and Ceuze, 1980) was used to assess the existence of a linkage between silver grains and some components of the striatum. The principe of the technique is to compare the actual distribution of silver grains observed above the different nervous structures (primary items) or membrane appositions (secondary items) with a distribution at random established using a predetermined squaring of the photographs (magnification 25.000). All the clusters of silver grains (and neighbouring single silver grains) were photographed. The exact localization of the observed or putative silver grains were determined using circles which were centered on the silver grains the best as possible and whose diameter correspond to 0.4 j.lm. When circles covered two types of membrane appositions, half units were used and when silver grains were more or less dispersed, intersecting circles were considered to correspond to only one cluster. For each item, the observed and theoretical frequences were compared using the t-test for percentages. A quantitative analysis of the striatal binding of 3H-domperidone was also performed in both groups of rats. The intensity of the radioautographic reaction in homologous regions was compared in normal and haloperidoltreated rats in order to determine the relative importance of specific and

12

M. Arluison, Marie-Pascale Martres, and P. Sokoloff

Figs. 1-3 . Local injection of3H-domperidone in the caudate-putamen of the rat; electron microscopic radioautography after 4 months exposure. Three dendritic spines (5) which participate to synaptic contacts (small arrows) are significantly by several silver grains. D dendrite; MAx. myelinated axon. T nerve terminal. Fig.! X 32,000; Fig. 2x27,SOO; Fig.3x2S,000

Dopamine Binding Sites in the Rat

13

Figs.4-11. Examples of reactive synaptic contacts of the asymmetric type between a nerve terminal and a dendritic spine (S). Several silver grains are often seen just above the active zone which is unique and exhibits a prominent post-synaptic thickening (small arrows). In the nerve terminal, the synaptic vesicles of 40 nm in diameter are numerous and crowded (type I of Hassler et al., 1978). Some neighbouring mitochondria are equally labelled by one or more silver grains (large arrow). Fig.4 X 36,000; Fig.5 X 30,000; Fig.6 and 8 X 27,000; Figs. 7, 10,11 X 25,000; Fig. 9X 31,250

14

M.Arluison, Marie-Pascale Martres, and P. Sokoloff

aspecific labelling with the decreasing concentration of the tracer due to the gradient of diffusion. In this case, a total surface area of 50.000 /-lm2 was examined in the zone A, as well as 0.01 mm 2 in the peripheral zones Band C. Results

of 3H-Domperidone Binding Sites Within the Striatum of Control Rats

I. Morphological Identification

The intensity of the radioautographic reaction according to the distance to the injection site appears proportional to the decreasing gradient of concentration due to the diffusion of the tracer. In the central zone A, we observe a high density of silver grains, but this reaction is essentially diffuse with numerous single silver grains over all the nervous elements. In the zone C, on the contrary, the diffuse reaction is low and silver grains are often grouped in clusters, which are scattered over the tissue, but which exhibit some electivity for various elements of the caudate-putamen. We have studied more particularly this peripheral region since clusters of silver grains correspond probably to local retentions of 3H-domperidone, and perhaps to dopaminergic sites when they are repeatedly observed over identical structures; moreover this zone exhibits the best ultrastructural preservation since local injection results often in bad perfusion of the tissue (due to the destruction of blood vessels). Because of the limited resolution of the radioautographic technique, clusters of silver grains can be observed over all kinds of structures within the striatum. Such scattered but heavy labellings are encountered over nerve terminals, unmyelinated axons, perikarya, dendrites and dendritic spines. In addition, clusters as well as single silver grains are also relatively numerous over glial processes, myelinated axons and mitochondria. There is strong evidence that a significant part of the clusters of silver grains is all the same related to nerve terminals and dendritic spines and more particularly to their membrane appositions; moreover, a number of these contacts appears differentiated in synapses of the asymmetric type. The density of the reactive synaptic contacts in the peripheral zone is low as compared to the large number of dopamine nerve terminals evidenced with the histofluorescence method; however, they may appear grouped in some regions of the tissue sections and rare elsewhere. Figs. 1-3 illustrate the clusters of silver grains which were found over some dendritic spines of the caudate-putamen, and equally, the strong electivity of this

Dopamine Binding Sites in the Rat

15

labelling. The reactive synaptic contacts between dendritic spines and nerve terminals are often labelled just above the active zone (Fig. 4-8) but in some cases, the accumulation of silver grains can be located over the synaptic vesicles within the nerve terminal (Fig. 9), or laterally on the edge of the active zone (Fig. 10) owing to the low resolution of the technique. The nerve terminals which form labelled asymmetric contacts with dendritic spines are of small to medium size and contain only occasionally large granular vesicles (90-100 nm in diameter), they belong to two morphological types according to Hassler et al. (1978). The reactive type III is the most numerous (Figs.4-1O) and is characterized by its medium size, numerous (crowded) small synaptic vesicles of about 40 nm in diameter and a single active zone with prominent post-synaptic thickening. The labelled type I is less numerous (Figs. 2 and 12) and characterized both by a small size and by loosely arranged synaptic vesicles of the same diameter. Labelled nerve terminals filled with synaptic vesicles of 40 nm in diameter are also seen in synaptic contact with either dendritic trunks (Fig. 7-9) or perikarya (Fig. 10); in this case, the active zone is slightly asymmetric or symmetric. These synapses correspond probably to the type VII of Hassler et al. (1978). II. Statistical Analysis of the Distribution of Radioautographic Silver Grains in Control Rats by the Circle Method (Table 1) When silver grains are distributed at random over the tissue section the number of silver grains encountered over each type of structure (primary items) is directly proportional to the surface occupied by the different structures in the section. We have been interested to analyze more particularly the distribution of the clusters of silver grains in the peripheral zone C: in the case of primary items, we have found that their distribution is closely similar to a distribution at random (Table 1); only the number of clusters on nerve terminals and dendritic spines was slightly higher (over nerve terminals only the percentage of single silver grains is significantly higher than the frequence at random). Moreover, contrasting with an idea issued from our qualitative observations, we have found no significant difference in the observed and theoretical frequences of silver grains (clusters or neighbouring single silver grains) over myelinated axons and over mitochondria. The percentages for the clusters of silver grains observed above the membrane appositions of the terminal-dendrite and terminalterminal type are not different from those corresponding to a distribution at random too. However, we observe a highly significant dif-

Fig.12. A small nerve terminal with loosely arranged synaptic vesicles torms an axospinous synaptic contact which is labelled and which belong probably to the type I of Hassler et al., 1978; X 27,500 Figs. 13-15. In these labelled synaptic junctions (probably of the type VII) three small nerve terminals are filled with synaptic vesicles of 40 nm in diameter and form symmetric, or slightly asymmetric, synaptic contacts (bent arrows) with dendritic profiles (D). Fig. 13 X 27,500; Fig. 14 X 32,000; Fig.15 X 25,000 Fig.16. Axo-somatic synapses are scarce in the neostriatum but one of them was labelled in our material. The active zone appeared equally symmetric (bent arrow). N nucleus of the perikaryon. X 30,000 .

M. Arluison et al.: Dopamine Binding Sites in the Rat

17

ference in the frequences for the contacts between nerve terminals and dendritic spines. Moreover, we have found that the labelled asymmetric synaptic contacts were significantly more numerous (two times) than in the distribution at random. Symmetrical synapses are not shown in Table 1 because their number was too limited in our material. Table 1. Statistical analysis of the distribution of the clusters of silver grains (and neighbouring single silver grains) in the peripheral zone C with the circle method (see text). The observed distribution is compared to a distribution at random determined by a squaring of the photographs. There are no significant differences in the frequences, excepted for the contacts between nerve terminals and dendritic spines. In this case, the membrane appositions are three times more labelled, and the asymmetric synaptic contacts two times more labelled than at random PUTATIVE SOURCES EXPECTED Nb ACTUAL NUMBER OF OF SILVER GRAINS SILVER GRAINS OF RADIATION (SELECTION) IF RANDOM SINGLE CLUSTERS NEURONAL

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186±9 339±9 1.13±0.02 (119) 4.17±0.17 (24)

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MEPC Amplitude (nA) Control After paraoxon Percent increase

Untreated

0.85 ± 0.03 (11) 3.31±0.17 (13)

109±4

2.36±0.13 (11) 3.12±0.12 (13) 32±9

a-BuTX (5 f.lg Lv.)

1.02±0.03 (11) 3.86±0.25 (7)

107±32 186± 7

3.11 ± 0.14 (11) 3.90±0.13 (7) 25±6

MIgG 2 days

0.96 ± 0.04 (8) 3.71±0.41 (9)

73± 17 109± 9

2.17 ± 0.08 (8) 3.06±0.19 (9) 43± 10

4 days

Table 2. Modification of MEPC amplitude, "time constant", and sensitivity to dTC by AChE poisoning, MlgG, and a-BuTX

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D. M. J. Quastel and P. Pennefather

Results and Discussion

Table 2 lists the characteristics of MEPCs recorded at a holding potential of -80 mV, at end-plates of untreated animals, and after irreversible partial blockade of receptors, in each case before and after poisoning of AChE by paraoxon. Also listed are the IC 50s for (+)-tubocurarine (dTC), i.e. the concentrations of dTC (determined by interpolation) that caused a 50 percent reduction of ME PC height from the value in the absence of dTe. In agreement with the predictions made on the basis of the above models the IC 50 for dTC was substantially increased by AChE poisoning by paraoxon, and reduced by irreversible blockade of receptors. It is notable that for MEPCs of the same amplitude obtained in three different ways-2 days MIgG before paraoxon, and either a-BuTX or 4 days MIgG after paraoxon, the IC 50 for dTC was the same, in accord with equation (5). With regard to the effects of irreversible receptor blockade on the time constant of MEPC decay it is notable that MIgG was relatively less effective than a-BuTX in reducing the time constant (,). The reduction of, by MIgG was also consistently less than that produced by concentrations of dTC producing equivalent depression of MEPC height (Pennifather and Quastel 1980 a), a result we interpret as indicating that with MIgG some receptors that are incapable of opening post-synaptic channels may be capable of transiently binding ACh, thereby buffering diffusion of ACh in the cleft (Katz and Miledi, 1973). The data shown in Fig. 3 also agree well with the theoretical model and provide a quantitative estimate of the normal capture of ACh in a quantum by receptor. With MEPCs made small by dTC (and/or by a-BuTX or MIgG) the effectiveness of AChE poisoning to increase MEPC height was continuously graded with MEPC height before AChE poisoning (ga). As predicted by equation (6), the graph of g/gd vs. ga is linear. Extrapolation of the line, to g/gd = 1, gives a value of 5.1 ± 0.6 nA for gm' the height of the MEPC that would occur if all the ACh in the quantal package were captured by receptor. Since the normal MEPC had a height of 4.03 nA, this result implies that normally 74% of the ACh discharged in a quantum is captured by receptor, while after poisoning of AChE 92% is captured. In Fig. 4 are shown graphs of the inverse of ME PC height vs. the concentration of dTe. Here a linear plot was to be expected only if dTC combines with receptor on a 1 : 1 basis, which is unlikely in view of a variety of evidence (e.g. Colquhoun et al., 1979) that dTC competes with ACh on a 1 : 1 basis, while two molecules of ACh must combine with receptor in order to open a subsynaptic ionic channel (Drryer et

69

Receptor Blockade and Synaptic Function

al., 1978). However, the present data is compatible with the existence of two distinct dTC binding sites on the receptor, with different affinities, so that in the range of concentration used here there is essentially no more than one dTC molecule per receptor. These results fit very well the prediction of equation (4). There is a linear A Can

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B. Sakmann, O. P. Hamill, and

92

J. Bormann

application of 50 jiM glycine to the "whole-cell" membrane, the current rises smoothly reflecting the nearly simultaneous activation of around 80 channels. The steady state current shows characteristic "noise", i.e. smooth fluctuations representing statistical variations in the average number of open channels. Following isolation of a membrane patch the background noise drops to 0.2-0.5 pA. After addition of 50 jiM glycine the current increases in a stepwise fashion and then fluctuates between discrete, evenly spaced levels representing the opening and closing of individual Cl- channels (Fig. 5 d). In the "cell-free" configuration Na+ and K+ can be replaced on either membrane face by impermeant cations like Tris. The conductance and gating properties of GABAR- and GlycineR-channels can be studied on a much larger voltage range than in the "cell-attached"

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Patch-Clamp Measurements of Elementary Chloride Currents

93

configuration. In symmetrical CI- transmembrane concentrations the conductances of GABAR- and GlycineR-channels are 29 pS and 46 pS. This value is larger than the values obtained in the cellattached recording configuration.

Conclusion The different configurations of the patch-clamp technique can be used to separate the GABA - and glycine-dependent Cl- currents in the somal membrane of spinal cord cells from other membrane currents. For both GABA and glycine at least two molecules must bind to their receptors to open the associated ion channel. The conductance of GABA-activated ion channels is 29 pS whereas of glycine-activated channels of 46 pS in symmetrical, 150 mM transmembrane CI- concentrations. The elementary CI- current shows brief interruptions. If channel activation and desensitization is represented according to the reaction scheme suggested by del Castillo and Katz (1957) and Katz and Thesliff(1957) for end-plate channels these rapid interruptions presumably reflect transitions of the liganded receptor-channel complex between the open (AzR ,C) and an intermediate closed state (AzR). k~l fJ 2A+R ~AzR~AzR*

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k~l

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The average duration of the elementary current is determined by both the occupation time (lIk~l) of the receptor-transmitter complex and the channel opening and closing rates fJ and a (Colquhoun and Hawkes, 1977). In the presence of GABA concentrations exceeding· 5 f.lM elementary currents occur in bursts separated by long intervals. Presumably, this bursting reflects predominantly fluctuations of the receptor-channel complex between "activatable" states (R, AzR, AzR"), where the channel can be either open (AzR"') or closed (R, AzR), and desensitized states (D, AzD). The gating behavior described here for Cl- channels gated by putative inhibitory transmitters in neurons derived from the CN.S.

94

B. Sakmann, O. P. Hamill, and

J. Bormann

is qualitatively similar to that reported for cation-selective end-plate channels. This indicates that the coupling between postsynaptic receptor and ion channel follows the same general rules independent of the particular nature of receptor and channel. The temporal pattern of elementary currents suggests that the receptor-channel complex can adopt two different open states and at least three distinct shut states. For the understanding of neurohormonal and pharmacological regulation of inhibitory synaptic transmission in the C.N.S., a detailed description of these various functionally occurring receptor-channel states and their interconversions is essential. Acknowledgements We thank Drs. F. Sigwarth and E. Neher for computer programs.

References Barker, J L., McBurney, R. N.: GABA and glycine may share the same

conductance channel on cultured mammalian neurones. Nature 277, 234-236 (1979). BarkerJ L., Ransom, B. R.: Amino acid pharmacology of mammalian central neurones grown in tissue culture.]. Physiol. (Lond.) 280,331-354 (1978). Bormann, J: Single channel currents activated by glycine and GABA in spinal cord neurons. Biophys. Struct. Mech. 7, 290 (1981). Castillo, J de!, Katz, B.: Interaction at end-plate receptors between different choline derivatives. Proc. Roy. Soc. (Lond.) B 146, 369-381 (1957). Colquhoun, D., Hawkes, A. G.: Relaxation and fluctuations of membrane currents that flow through drug-operated channels. Proc. R. Soc. (Lond.) B 199, 231-262 (1977). Colquhoun, D., Sakmann, B.: Fluctuations in the microsecond time range of the current through single acetylcholine receptor ion channels. Nature 294, 464-466 (1981). CoombsJ S., EcclesJ c., Fatt, P.: The specific ionic conductances and ionic movements across the motoneuronal membrane that produce the . inhibitory post-synaptic potential.]. Physiol. (Lond.) 130, 326-373 (1955). Dude!,J, Finger, Stettmeier, H.: GABA-induced membrane current noise and the time course of the inhibitory synaptic current in crayfish muscle. Neurosci. Lett. 6, 203-208 (1977). Hamill, 0. P., Marty, A., Neher, K Sakmann, B., Sigworth, F.J: Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. pfliigers Arch. 85-100 (1981). Katz, B.: The Release of Neural Transmitter Substances. Liverpool: University Press. 1969.

w.,

3n

Patch-Clamp Measurements of Elementary Chloride Currents

95

Katz, B., Miledi, R.: The statistical nature of the acetylcholine potential and

its molecular components. J. Physio!. (Lond.) 224, 665-699 (1972).

Katz, B., Thesliff, S.: A study of the "desensitization" produced by acetylcho-

line at the motor end-plate.]. Physio!. (Lond.) 138, 63-80 (1957).

McBurney, R. N., Barker, j. L.: GABA -induced conductance fluctuations in

cultured spinal neurones. Nature 274, 596-597 (1978).

Marty, A., Neher, E.: Tight-seal whole cell recording. In: Single Channel Recording (Sakmann, B., Neher, E., eds.). N ew York: Plenum Press. 1983. Neher, E., Sakmann, B.: Single channel currents recorded from membrane of denervated frog muscle fibres. Nature 260, 799-802 (1976). Peck, E.j.: Receptors for amino acids. Ann. Rev. Physio!. 42, 615-627 (1980). Ransom, B. R., Neale, E., Henkart, M., Bullock, P. N., Nelson, P. G.: Mouse spinal cord in cell culture. 1. Morphology and intrinsic neuronal electrophysiologic properties. ]. N europhysio!. 40, 1132-1150 (1977). Sakmann, B., Hamill, O. P., Bormann,}.: Activation of chloride channels by

putative inhibitory transmitters in spinal cord neurons. Pflugers Arch. 392, Rl9 (1982). Sakmann, B., Patlak, j., Neher, E.: Single acetylcholine-activated channels show burst-kinetics in presence of desensitizing concentrations of agonist. Nature 286, 71-73 (1980). Sigworth, F.}., Neher, E.: Single N a+ channel currents observed in cultured rat muscle cells. Nature 286, 447-449 (1980).

Authors' address: Dr. B. Sakmann, Max-Planck-Institut fur biophysikalische Chemie, Am Fassberg, D-3400 Gottingen, Federal Republic of Germany.

J.

Neural Transmission, Supp\. 18, 97-111 (1983)

Calmodulin, Caz+-Antagonists and Ca2+-Transporters in Nerve and Muscle J. D.Johnson', Laura A. Wittenauer', and R. D. Nathan z Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, Cincinnati, Ohio, U.S.A. 2 Department of Physiology, Texas Tech University Health Sciences Center, Lubbock, Texas, U.S.A.

1

With 4 Figures

Summary Calcium is of fundamental importance in the regulation of both muscle contraction and neurosecretion. Its control of these processes is achieved by its binding and activation of various Ca2+-binding proteins (CBP), including those in the Ca2+ channel, the Na+-Ca z+ antiporter, and intracellular calmodulin (CDR). Generally, Ca2+-binding to regulatory CBP exposes hydrophobic sites on their surface at which the CBP interfaces with its receptor or binds inhibitory hydrophobic ligands. We find that some Ca2+antagonist drugs (Ca-ANT) bind to and inhibit calmodulin and that some calmodulin antagonists (CDR-ANT) block Ca2+ channels. This suggests that CDR and the CBP that regulate the Ca z+ channel may be quite homologous proteins. Ca-ANT and CDR-ANT are not effective inhibitors of the Na+-Ca2+ antiporters of heart sarcolemma and brain synaptosomes, suggesting that these antiporters are fundamentally different from the antiforter of heart mitochondria. These results are discussed in terms of ci -binding proteins being potential targets for pharmacological interventions designed to block specific aspects of the action of calcium.

Introduction

Ca2+ ions are of central importance in the regulation of biological processes as seemingly diverse as muscle contraction, neurosecretion, glandular secretion, and cellular reproduction. Ca2+ does not work 7 Journal of Neural Transmission, Suppl.18

98

J. D.Johnson, Laura A. Wittenauer, and R. D. Nathan

alone, however, to orchestrate this wide variety of biological phenomena with such perfect harmony. Nature has provided Ca2+binding proteins which serve as intracellular messengers to carry the "Ca2+ signal" and to regulate [Ca 2+]. Calmodulin is a Ca2+ receptor of particular importance because it is present in all eukaryotic cells; its structure has been highly conserved throughout evolution, and it has been implicated in the regulation of many diverse Ca2+ dependent processes (see Cheung, 1980,1982, and Klee et aI., 1980 for review). The Ca2+ dependent regulation of neurosecretion and smooth muscle contraction share many common features and provide good examples of the role of calmodulin (CDR) and Ca2+-binding proteins (CBP).

~~C;+~