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Studies in neurofibrillar development in the bird and mammal

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U n p u b lis h e d t h e s e s s u b m i t t e d f o r th e M a s t e r ’ s and D o c t o r ’ s d e g r e e s and d e p o s i t e d in th e N o r t h w e s t e r n U n i v e r s i t y L i b r a r y a r e op en f o r i n s p e c t i o n , b u t a r e to be u s e d o n l y w i t h due r e g a r d to t h e r i g h t s o f t h e a u t h o r s . Bibliographical r e f e r e n c e s may b e n o t e d , b u t p a s s a g e s ma y b e c o p i e d o n l y w i t h t h e p e r m i s s i o n o f t h e a u t h o r , a nd p r o p e r c r e d i t m u s t be g i v e n in su bsequ ent w r i t t e n or p u b lis h e d work. E xtensive copying or p u b l i c a t i o n of th e th e s e s in whole or in p a r t r e q u i r e s a lso t h e c o n s e n t o f t h e Dean o f t h e G r a d u a t e S c h o o l o f N o r t h w e s t e r n U niversity. T h i s t h e s i s by . h a s b e e n u s e d by t h e f o l l o w i n g a t t e s t t h e i r acceptance of the patrons

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DATE

NORTHWESTERN UNIVERSITY

STUDIES IN NEUROFIBRILLAR DEVELOPMENT IN THE BIRD AND MAMMAL

A DISSERTATION SUBMITTED TO THE GRADUATE SCHOOL IN PARTIAL FULFILLMENT OF THE REQUIREMENTS f o r th© d e g r e e DOCTOR OF PHILOSOPHY

DEPARTMENT OF ANATOMY

BY R u th R h in e s

C h io a g o , I l l i n o i s J u l y , 1942

P ro Q uest Number: 10101882

All rights re se rv e d INFORMATION TO ALL USERS The quality o f this re p ro d u c tio n is d e p e n d e n t u p o n t h e quality o f t h e c o p y su b m itte d . In th e unlikely e v e n t t h a t th e a u th o r did n o t s e n d a c o m p l e t e m an u sc rip t a n d th e re a r e missing p a g e s , t h e s e will b e n o te d . Also, if m aterial h a d to b e r e m o v e d , a n o te will in d ic a te t h e d eletio n .

uest. P ro Q uest 10101882 Published by P ro Q u est LLC (2016). C opyright o f t h e Dissertation is h e ld by t h e Author. All rights reserved. This work is p r o t e c t e d a g a in s t u n au tho rized c o p y in g u n d e r Title 17, United States C o d e Microform Edition © P ro Q u est LLC. ProQ uest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106 - 1346

TABLE OF CONTENTS In tr o d u ctio n

2

Part I .• • • T h e E a rly Development o f th e F a s e ic u lu s L o n g itu d i­ n a l i s M ed ia lis and A sso c ia te d Secondary Neurons in th e R at, Cat and Man M a teria ls and Methods O bservations D is c u ss io n C on clu sion s

8 10 11 19 22

P art II* ••O b se r v a tio n s on th e Growth o f lo n g itu d in a l T racts in th e B rain o f th e Chick Embryo up to 6 days* Four and one h a l f Days F iv e Days S ix Day

24 27 28 31

P art I I I . . E f f e c t s o f S u r g ic a l Removal o f P a rts o f th e B rain upon Development o f th e Brain Method and M a teria l E f f e c t s o f o p era tio n s upon th e shape o f th e b ra in and o r ie n t a tio n o f f ib e r s D is c u ss io n A lte r a tio n s i n th e M edial L on gitu d in al F a sc ic u lu s and o th er L o n g itu d in a l T r a c ts, caused by Absence o f Various N u clei o f th e B rain Embryos w ith o u t a F oreb rain E x c is io n o f P a rts o f th e Tectum Embryos Deprived o f F orebrain and Midbrain Embryos Deprived o f F oreb rain, Midbrain and P art o f th e Metane ephalon S ep a ra tio n o f th e Neural Tube a t th e L evel o f th e © to c y sts D is c u ss io n C onclusions Abnormal Nerves P e rip h e r a l Course o f Nerves Deprived o f t h e ir Normal D e s tin a tio n A lte r a tio n s in th e Oculomotor and T rochlear Nerves D isc u ssio n P art I V ...I n c id e n c e o f Spontaneous M o t ilit y in Operated Embryos D isc u ssio n C onclusions Summary B ib lio g ra p h y I ll u s t r a t i o n s Tables V ita

32 32 35 39 41 41 44 46 48 49 51 57 58 58 65 68

74 75 76 77 90 94 1°7 HI

INTRODUCTION Although i t has lo n g been known th a t th e m edial lo n g itu d in a l f a s c i ­ c u lu s c o n ta in s a component d escen d in g from th e diencephalon* th e e x te n t and c o n n e ctio n s o f t h a t f ib e r bundle are not known* in s p it e o f th e ex ten ­ s iv e resea rch th a t has been devoted t o th e m ed ial lo n g itu d in a l f a s c ic u ­ lu s o f a d u lt a n im a ls.

S in ce understanding o f th e n ature and r e la t io n ­

s h ip s o f a d u lt nervous s tr u c tu r e s can be fu rth ered by o b se rv a tio n o f th e d ev e lo p in g nervous system w ith s i l v e r red u ctio n tech n iq u es* i t i s t o be ex p ected th a t th e s e methods should p rovid e much in form ation about th e d ie n c e p h a lic p a rt o f th e m edial lo n g itu d in a l f a s c ic u lu s * because th a t f a s c i c l e i s th e f i r s t lo n g itu d in a l t r a c t t o appear in th e embryonic b r a in . D e s c r ip tio n s o f th e m edial lo n g itu d in a l f a s c ic u lu s have been g iv en in s tu d ie s o f n e u r o f ib r illa r d if f e r e n t ia t io n in v a rio u s s p e c ie s* in c lu ­ d in g th e ch ick (Mesdag* 1909) (Bok* 1915) (T e llo * 1923) (W indle and Aus­ t in * 1936) ( V i s i n t i n i and M on tcalcin i* 1939); th e mouse (T e llo * 1934); th e r a t (W indle and Baxter* 1936) (A ngulo, 1939); th e ca t (Windle* 1932 a* 1932 b* 1933* 1 9 3 5 ); th e rat* c a t and man (R hines and 7/indle* 1 9 4 1 ). N ev e rth ele ss* th e r e has been c o n sid e r a b le disagreem ent as t o th e n a tu re o f t h i s f a s c i c l e .

Some workers (Mesdag* Bok* Angulo) b e lie v e

th a t in e a r ly s ta g e s i t i s composed e n t ir e ly o f descen d in g f i b e r s .

Others

(W indle* T ello * Windle and A u stin , Windle and Baxter* Rhines and W indle) concluded th a t ascen d in g f ib e r s me,t and overlapped th o se o f d ie n c e p h a lic o r ig in * ob scurin g th e complex str u c tu r e o f th e t r a c t .

Indeed* th e l a s t

stu d y u sin g mammalian embryos from th e sta g e of e a r l i e s t n e u r o f ib r illa r d if f e r e n t ia t io n * was undertaken t o observe th e m edial lo n g itu d in a l f a s c i ­ c u lu s b efo re o th er lo n g itu d in a l f ib e r s could com p licate th e s it u a t io n .

The o r i g i n an d e a r l y r e l a t i o n s h i p s o f t h e t r a c t c o u l d be c l e a r l y f o l l o w e d , and t h e r e s u l t s

o f t h a t w o rk c o n s t i t u t e t h e f i r s t

s e c ti o n o f t h i s p a p e r*

S h o r tly a f t e r t h e a p p e a ra n c e o f t h e d ie n c e p h a lic b u n d le , o th e r f o c i of d i f f e r e n t i a t i o n b e g in t o p a r t o f th e b ra in .

c o n tr ib u te lo n g itu d in a l f i b e r s t o th e v e n t r a l

T h e s e f i b e r s a r e b o th a s c e n d i n g an d d e s c e n d i n g , and

v e r y s o o n , e l e m e n t s o f d i v e r s e o r i g i n e n t e r t h e p a th w a y i n c o n s i d e r a b l e n u m b e rs , m a k in g i t

im p o s s ib le t o

a s c e r ta in th e te r m in a tio n o f th e d ie n ­

c e p h a lic f i b e r s . T h e re fo re , to

f a c i l i t a t e t h e s tu d y o f t h e v a r i o u s c o m p o n e n ts o f t h e

m e d i a l l o n g i t u d i n a l f a s c i c u l u s , e x p e r i m e n t a l m e th o d s w e re em p lo y ed i n c o n j u n c t i o n w i t h t h e p y r i d i n e - s i l v e r m eth o d *

A f t e r re m o v in g t h e f o r e ­

b r a i n o f a 3 8 -4 0 h o u r c h i c k em b ry o , t o e x c l u d e t h e n u c l e u s o f t h e d i e n c e ­ p h a l i c c o m p o n e n t, an d p e r m i t t i n g s u b s e q u e n t i n c u b a t i o n f o r v a r y i n g p e r i o d s of tim e , th e e f f e c t o f i t s

a b s e n c e u p o n t h e m e d ia l l o n g i t u d i n a l f a s c i c u l u s

o f m ore c a u d a l r e g i o n s w as s t u d i e d .

To e x c l u d e t h e c r o s s e d t e c t a l t r a c t ,

a n d o t h e r f i b e r s o f m i d b r a in o r i g i n , tw o t y p e s o f o p e r a t i o n w e re u s e d . I n o n e , b o th t h e f o r e b r a i n and m i d b r a in w e re re m o v e d ; t h e o t h e r a p p r o a c h t o t h i s p r o b le m c o n s i s t e d o f e x c i s i o n o f t h e t e c t u m .

A n o th e r t y p e o f

o p e r a t i o n i n v o l v e d re m o v a l o f a l l o f t h e b r a i n r o s t r a l t o t h e lo w e r m et e n ­ c e p h a lo n .

A tte m p ts w e re a l s o made t o e x c i s e s e c t i o n s o f t h e b r a i n , l e a v ­

i n g m ore r o s t r a l an d c a u d a l p a r t s .

A s e r i e s o f em b ry o s i n w h ic h t h e b r a i n

w as i n t e r r u p t e d a t t h e l e v e l o f t h e o t o c y s t s w as o b t a i n e d , b u t t h o s e w i t h th e h i a t u s p la c e d a t

ohe t r i g e m i n a l r e g i o n w e re n o t s u i t a b l e f o r s t u d y .

A lso u n s u c c e s s f u l , w e re a t t e m p t s t o e x c i s e a l a t e r a l h a l f o f a n y r e g i o n of th e b ra in . E m bry o s o f e a c h t y p e o f o p e r a t i o n w h ic h p r o v e d p r a c t i c a b l e w e re o b t a i n e d a t 1 / 2 d ay i n t e r v a l s fro m 4 t o 6 d a y s i n c u b a t i o n a g e .

A com -

p a ra b le normal s e r i e s was p rep ared. The work e n t a ile d a c o n sid e r a b le amount o f e x p lo r a tio n , because s im ila r methods f o r th e study o f th e brain have not been used by oth er w ork ers.

S u r g ic a l tec h n iq u es have been employed in em bryologic r e se a r c h ,

but n o t f o r th e purpose o f stu d yin g development o f t r a c t s in th e b ra in . Fugo (1940) removed th e fo r e b r a in in order t o observe th e development of hypophysectom ized chick embryos; Jones (1 9 3 7 , 1939, 1941, 1942) produced h ia tu s e s in th e c e n tr a l nervous system in s tu d ie s on p e r ip h e r a l n e r v e s , and a ls o performed o p e ra tio n s t o remove th e c e p h a lic c r e s t ; Hamburger (1934) noted th e e f f e c t o f l o s s o f a lim b bud on th e d ev elo p in g s p in a l co rd .

W eiss (1 9 3 3 , 1934 a , b , c) has made e x te n s iv e u se o f exp erim en tal

methods on th e ch ick embryo, but h is work has been la r g e ly d ir e c te d toward th e p e r ip h e r a l nervous system , and h is tech n iq u es have c o n s is te d m ainly o f e x p la n ta tio n in to t i s s u e c u lt u r e s .

By f a r th e g r e a te r p art of th e

exp erim en tal work in th e f i e l d o f embryology has been done on amphibian embryos.

Much o f th a t work which has been s p e c i f i c a l l y concerned w ith

n e u r o lo g ic a l in v e s t ig a t io n has come from th e la b o r a to r ie s o f H arrison (1 9 0 4 , 1907 a , 1907 b, 1910 a , 1910 b, 1924) and B e tw ile r (1 9 3 4 , 1936, 1 9 3 7 , 1 9 24, 1928) (B e tw ile r and Van Dyke, 1 9 3 4 ). Furtherm ore, s u r g ic a l methods have not been combined w ith prepara­ t i o n o f m a te r ia l by th e p y r id in e - s ilv e r te c h n iq u e .

Since th e s i l v e r -

r e d u c tio n method fo r dem onstrating n e u r o f ib r ils was introdu ced by C a ja l, v a rio u s improvements have been made in th e tech n iq u e by l a t e r w ork ers. The p y r id in e - s ilv e r method (D avenport, V in dle and Beech, 1934) has proved p a r t ic u la r ly u s e fu l because in a good p r e p a r a tio n , th e im pregnation o f th e n e u r o f ib r ils ca u ses botn c e l l b od ies and f ib e r s t o appear in sharp c o n tr a st t o th e lig h t - c o lo r e d background.

That th e com bination o f exp erim en tal methods w ith th e p y r id in e s i l v e r tech n iq u e has g rea t p o s s i b i l i t i e s , has been dem onstrated by th e r e s u l t s obtained so f a r , and many new avenues o f resea rch have been suggested#

I t must be empnasized th a t th e r e s u lt s rep orted here rep re­

se n t only a b egin n in g o f the work, and th a t much uf th e resea rch has in v o lv ed a working out o f o p e r a tiv e p roced u res.

While new f in d in g s con­

cern in g th e n ature o f one components o f th e m edial lo n g itu d in a l f a s c ic u lu s have been added t o th o se ob tain ed from ob servin g normal embryos, a com plete un d erstan d in g o f t h i s f a s c i c l e has not y e t been a c h iev ed .

By in c r e a s in g

th e range o f th e p re se n t s e r i e s , and im proving th e te c h n iq u e , i t i s pro­ b a b le t h a t th e r e la t io n s m p s o f th e embryonic m edial lo n g itu d in a l f a s c i ­ cu lu s w i l l be thorougniy worked out# The s e r i e s o f exp erim en tal embryos was u s e fu l f o r s tu d ie s n ot p er­ t a in in g t o th e developm ent o f lo n g itu d in a l t r a c t s .

The m a te r ia l was e s ­

p e c i a l l y va lu a b le f o r o b servin g th e f a t e o f th e oculom otor, t r o c h le a r , and ophthalm ic n e r v e s , deprived o f t h e ir normal d e s t in a t io n , and t h i s method should be a p r o f it a b le m^ans of in v e s t ig a t in g th e f a c to r s d ir e c t in g th e growth o f p e r ip h e r a l n e r v e s . Inform ation concerning th e in flu e n c e s d eterm ining th e in tram ed u llary co u rse o f nerve p r o c e s s e s may be o b ta in a b le by t h i s method.

The p o s s ib i­

l i t y o f p u rsuing such an in v e s t ig a t io n i s su g g ested by th e f a c t t h a t , in t h e m a te r ia l u sed , a lt e r a t io n s of th e course o f axons near th e s i t e o f o p er a tio n were q u ite c o n s is t e n t ly n o ted , and th a t th e s e changes fo llo w ed a t y p ic a l p a tx ern , and were not m erely random resp on ses t o abnormal con­ d itio n s *

Furtherm ore, in a few in s ta n c e s , d eparture o f th e tr o c h le a r

n erve from i t s normal course was ob served .

To some d e g r e e , th e methods alread y o u tlin e d are ra th e r n eg a tiv e * I t would be d e s ir a b le , i f p o s s ib le , t o stren g th en th e c o n c lu sio n s reach ed , w ith a more p o s i t i v e approach.

S tu d ies on d eg en era tio n and r eg en era tio n

o f axons in th e embryonic c e n tr a l nervous system might p rovid e a means o f ch eck in g upon r e s u lt s o f " elim in ation " te c h n iq u e s , and th ey would a ls o c o n s t it u t e an in t e r e s t in g problem .

The e x e c u tio n o f such an in v e s t ig a t io n

on th e c e n tr a l nervous system should not be b e se t w ith any g rea t d i f f i ­ c u l t i e s , and i t i s p o s s ib le t h a t , by e x c is in g th e n eu ral c r e s t and d o rsa l p art o f th e n eu ra l tu b e , as has been done by D e tw ile r (1937) and H arrison (1924) in am phibia, some f a c t s about th e r o le p layed by th e neurilemma in p e r ip h e r a l r e g e n e r a tio n may be le a r n e d .

T h is would in v o lv e h atch in g

th e c h ic k , and s e c tio n in g p e r ip h e r a l n e r v e s , and i t msy be th a t oth er so u r ces o f neurilemma would in t e r f e r e w ith r e s u l t s . In th e absence o f a la r g e p art o f th e b r a in , spontaneous m o t il it y was ob served .

By ex ten d in g th e in cu b a tio n p erio d o f embryos o f t h i s typ e

a n d o f some w i t h s p e c i f i c a l l y l o c a l i z e d e x c i s i o n s , s o m e th in g a b o u t t h e p a th w a y s in v o l v e d i n e a r l y m ovem ents i n r e s p o n s e t o e x t e r n a l s t i m u l a t i o n s h o u ld b e r e v e a l e d . The t e r m i n o l o g y u s e d i n f o l l o w i n g s e c t i o n s o f t h i s p a p e r n e e d s d e f i ­ n i t i o n , b e c a u s e t h e nam es a p p l i e d t o d i f f e r e n t m e a n in g by v a r i o u s a u t h o r s .

some t r a c t s h a v e b e e n u s e d 7 /i th

A r a t h e r d i f f u s e , p o o r l y c ir c u m ­

s c rib e d t r a c t a t th e p e rip h e ry , j u s t l a t e r a l to th e f lo o r p la te

(d e s ig ­

n a t e d h e r e a s t h e v e n t r a l l o n g i t u d i n a l f a s c i c u l u s ) h a s b e e n i n c l u d e d by some a u t h o r s u n d e r t h e t e r m , m e d ia l l o n g i t u d i n a l f a s c i c u l u s ;

an d t h e

e x t e n s i o n o f t h i s t r a c t i n t o t h e m y e le n c e p h a lo n an d s p i n a l c o r d , h a s b e e n c o n s i d e r e d t o be a d i r e c t c o n t i n u a t i o n o f t h e m e d i a l l o n g i t u d i n a l f a s c i c u -

l« * In e a r ly mammalian embryos t h i s term in c lu d e s f ib e r s o f th e M edial L o n g itu d in a l F a sc ic u lu s which cannot be d is tin g u is h e d from th o se o f th e V en tral L ongitu d inal F a s c ic u lu s .

7

l u s (A ngulo, 1939)« erroneous*

.

I t should he emphasized th a t such assum ptions are

The m ed ial lo n g itu d in a l f a s c ic u lu s i s th e d o r s a l, compact

p a r t o f the t r a c t b ordering th e f lo o r p l a t e , and t h i s usage i s compara­ b le t o what o f a d u lt neurology*

In th e e a r ly embryo, t h i s f a s c i c l e i s

co n fin e d t o th e m esencephalon and m etencephalon; a t 6 days in th e c h ic k , end comparable s ta g e s in mammals, i t exten d s only t o th e upper vagus r e g io n . F ib e rs in th e r e s t o f th e b a sa l p l a t e , excep t th e la te r a lm o s t p a r t, com prise th e r e t ic u la r fo rm a tion , and th e t r a c t im m ediately ven tro-m ed ial t o th e su lc u s lim ita n s i s c a lle d th e l a t e r a l lo n g itu d in a l f a s c i c u lu s . Secondary r e f e r s t o rhom bencephalic in te m e u r o n s .

L o n g itu d in a l f ib e r s

a re th o se p a r a l l e l t o th e lo n g a x is o f th e n eu ral tu b e .

The terms d o r sa l

v e n tr a l are used w ith r e fe r e n c e t o th e tr a n sv e r s e p lan e o f th e n eural tu b e a t any g iv en p o in t , r e g a r d le s s o f th e curvature of th e n eu ral tu b e . T h er e fo re, th e f lo o r p la te i s always v e n t r a lly s it u a t e d .

In exp erim en tal

m a te r ia l, th e ty p e o f o p era tio n i s s ig n if ie d by th e h ig h e s t e x is t in g l e v e l in th e b r a in .

In "m idbrain** specim ens, th e oculom otor reg io n i s most

r o s t r a l ; in "tr ig e m in a l ** embryos, both fo r e b rain and m idbrain have been removed; "f a c i a l " embryos have no brain r o s t r a l t o th e f a c ia l- a c o u s t i c l e v e l ; "su b th alam ic" embryos are th o se in which most o f th e fo r e b r a in i s gone.

a

THE EARLY DEVELOPMENT OF THE FASCICULUS LONGITUDINAL1S MEDIALIS AND ASSOCIATED SECONDARY NEURONS IN THE RAT, CAT, AND MAN An a t t e m p t h a s b e e n m ade h e r e t o d e t e r m i n e , a s f a r a s m i c r o s c o p i c o b s e r v a t i o n 1 0 .1 1 p e r m i t , t h e o r i g i n an d c o u r s e o f t h e d i e n c e p h a l i c com­ p o n e n t o f t h e m e d ia l l o n g i t u d i n a l f a s c i c u l u s i n e a r l y c a t , r a t an d hum an em bryos* S i n c e o t h e r l o n g i t u d i n a l n e r v e f i b e r s i n t h e a r e a a d j a c e n t t o th e f l o o r p l a t e a re c l o s e l y r e l a t e d to th e d ie n c e p h a lic p o r ti o n o f th e m e d ia l l o n g i t u d i n a l f a s c i c u l u s , t h e p r e s e n t s t u d y i n c l u d e s a l l l o n g i t u d ­ i n a l n e r v e ‘f i b e r s i n t h e v e n t r a l p a r t o f t h e e m b ry o n ic b a s a l p l a t e * I t i s p o s s i b le t h a t v a r i a t i o n s i n th e te rm in o lo g y u s e d i n d e s c r ib in g n e u r a l s t r u c t u r e s a r e p a r t l y r e s p o n s i b l e f o r l a c k o f a g re e m e n t c o n c e r n i n g th e n a tu r e o f th e e a r l y lo n g i tu d in a l t r a c t s *

The te r m s u s e d i n t h i s r e p o r t

a r e d e f i n e d a s f o l l o w s : m e d ia l l o n g i t u d i n a l f a s c i c u l u s d e s i g n a t e s o n l y t h e p rim o rd iu m o f t h e d e s c e n d i n g , d i e n c e p h a l i c co m p o n en t o f t h e a d u l t m e d ia l l o n g i t u d i n a l f a s c i c u l u s ( v e s t i b u l a r c o m p o n e n ts a r e n o t y e t r e p r e s e n t e d ) ; v e n t r a l l o n g i t u d i n a l f a s c ic u lu s i n d i c a t e s I n c o m p le te ly i d e n t i f i a b l e f a s ­ c i c l e s o f l o n g i t u d i n a l f i b e r s o c c u p y in g t h e a r e a j u s t l a t e r a l t o t h e f l o o r p l a t e ( i n c l u d i n g t h e m e d ia l l o n g i t u d i n a l f a s c i c u l u s w hen i t b y o t h e r e l e m e i t s an d i s

i n d i s t i n g u i s h a b l e fro m th e m ) ;

i s a c c o m p a n ie d

sec o n d a ry r e f e r s to

r h a m b e n c e p h a lic i n t e r n e u r o n s - b o t h a s s o c i a t i o n a l an d c o m m is s u ra l* The te r m s d o r s a l an d v e n t r a l a r e u s e d i n r e f e r e n c e t o p l a n e s d raw n a t r i g h t a n g l e s t o t h e lo n g a x i s o f t h e n e u r a l t u b e , r e g a r d l e s s o f a c t u a l c u r v a t u r e o f t h e n e u r a l tu b e * L o n g i t u d i n a l f i b e r s a r e t h o s e c o u r s i n g p a r a l l e l t o th e f lo o r p la te * T h e d i e n c e p h a l i c p a r t o f t h e m e d ia l l o n g i t u d i n a l f a s c i c u l u s h a s b e e n d e s c r i b e d i n t h e c a t em bryo i n t h e c o u r s e o f m o re g e n e r a l s t u d i e s o f n e u r o ­ f i b r i l l a r d e v e lo p m e n t ( W in d le , *32 a , ’ 32 b ,

f33,

*3 5 )* I n t h e 5*5 mm*

9

c a t e m b ry o , i t w as s e e n a r i s i n g fro m a n u c l e u s a t t h e j u n c t i o n o f t h e m e s­ e n c e p h a lo n an d d ie n c e p h a lo n * I t c e p h a lic f le x u r e to

d e c r e a s e d fro m e i g h t y - f i v e f i b e r s a t t h e

s ix ty -fiv e f ib e r s ju s t r o s tr a l to

t h e t r i g e m i n a l m o to r

n u c l e u s , a n d w as p r a c t i c a l l y a b s e n t i n t h e f a c i a l r e g i o n . B elo w t h e f a c i a l le v e l,

a v e n tra l lo n g itu d in a l t r a c t ,

a t t h a t tim e t h o u g h t t o b e p a r t o f t h e

m e d ia l l o n g i t u d i n a l f a s c i c u l u s , r e a p p e a r e d an d a g a in b ecam e p r o m in e n t* I t was o b s e r v e d t h a t :

lfThe s i z e o f t h e m e d ia l l o n g i t u d i n a l f a s c i c u l u s seem s

t o v a r y d i r e c t l y w i t h t h e num ber o f s e c o n d a r y n e u r o b l a s t s i n t h e m a n tle l a y e r a n d t h e num ber o f c o m m is s u ra l f i b e r s i n t h e lo w e r rh o m b e n c e p h a lo n and s p i n a l c o r d ” ( W in d le , *32 b )* I n t h e p r e s e n t s t u d y t h i s

a n d a n o t h e r em bryo

h a v e b e e n r e v i e w e d . L i t t l e c a n b e ad d e d t o t h e e a r l i e r d e s c r i p t i o n o f t h e s e tw o s p e c im e n s . The m o s t r e c e n t s tu d y o f t h e s t r u c t u r e s i n q u e s t i o n h a s b e e n r e o o r t e d

(%)

b y A n g u lo ( f 39) i n r a t em b ry o s 2 .0 mm. t o 5 . 5 mm. g r e a t e s t l e n g t h . ~ w rite r e x p ressed th e b e l i e f t h a t;

”. . .

T h is

. i n th e s ta g e s u n d er c o n s id e r a tio n

t h e f a s c i c u l u s l o n g i t u d i n a l ! s m e d i a l i s an d o t h e r c o m p o n e n ts t h a t e n t e r i n t o t h e f o r m a t i o n o f t h e v e n t r a l l o n g i t u d i n a l p a t h a r e com posed e n t i r e l y o f d e s ­ c e n d in g f i b e r s . ” A n g u lo saw t h e f i r s t d e v e lo p m e n t o f t h e n u c l e u s o f t h e med­ i a l l o n g i t u d i n a l f a s c i c u l u s i n h i s 2 .0 mm. em b ry o , t h e v e n t r a l l o n g i t u d i n a l f a s c i c u l u s w as s a i d t o h a v e r e a c h e d t h e lo w e r l e v e l o f t h e m e d u lla o b l o n g a t a a t 4 . 0 ram., a t 4 . 5 mm. i t in v a d e d t h e c e r v i c a l c o r d , a n d a t 5 .5 mm. i t b e ­ came a w e l l e s t a b l i s h e d p a t h th r o u g h o u t t h i s r e g i o n . O th e r s who s t u d i e d t h e r a t h a d p r e v i o u s l y s t a t e d t h a t t h e m e d i a l lo n g ­ i t u d i n a l f a s c i c u l u s w as a b s e n t i n one o f a l i t t e r o f s e v e n r a t em b ry o s 5 t o 4 mm. lo n g ( f r e s h m e a s u r e m e n t) , and t h a t i t s m o s t c a u d a l e x t e n t i n a n y s p e c -

The a u t h o r d o e s n o t s a y w h e th e r t h e em b ry o s w e re m e a s u re d b e f o r e o r a f t e r f i x a t i o n . I f h i s a r e p o s t f i x a t i o n m e a s u r e m e n ts , t h e em b ry o s c o r r e s p o n d t o o u r s p e c im e n s o f a p p r o x im a te ly 3 mm. t o 7 .5 m m ., f r e s h m e a s u r e m e n t.

10

im en w as t h e m i d d le o f t h e t r i g e m i n a l r e g i o n (W in d le and B a x t e r , E ven b e f o r e t h i s ,

* 3 6 )•

o n e i n v e s t i g a t o r s u g g e s te d t h a t t h e f i b e r s o f t h e

r a t e m b ry o ’ s m e d ia l l o n g i t u d i n a l f a s c i c u l u s c r o s s i n t h e f l o o r p l a t e o f t h e p o n s and s y n a p s e f r it h f l o o r p l a t e n e u r o n s w h ic h c a r r y t h e im p u ls e on c a u d a lw a r d (H o g g ,

* 3 0 ).

T e l l o ( * 3 4 ) m e n tio n s t h e m e d ia l l o n g i t u d i n a l f a s c i c u l u s i n a n i n t r o ­ d u c t o r y s u r v e y o f t h e b r a i n s t e m o f t h e m o u se em b ry o . H is o b s e r v a t i o n s o n s e c o n d a r y n e u r o b l a s t s an d t h e v e n t r a l c o m m issu re a r e i n a g re e m e n t w i t h t h o s e on t h e c a t ( W in d le , *32 a , * 3 3 ) . H is s t u d y i s s c a r c e l y a p p l i c a b l e t o t h e p r e s e n t r e p o r t b e c a u s e t h e 4 .0 mm. m o u se em b ry o , h i s s m a l l e s t s p e c im e n , i s c o m p a ra b le w i t h t h e 7 - 8 mm. c a t a n d a p p e a r s t o b e m ore a d v a n c e d i n d e ­ v e lo p m e n t t h a n t h o s e we h a v e u s e d * o t h e r t h a n t h e s t u d i e s o n mammals n o te d a b o v e , M esdag ( ’0 9 ) , Bok ( * 1 5 ) , T e llo (*23)

an d W in d le a n d A u s t i n ( * 3 6 ) h a v e p r e s e n t e d o b s e r v a t i o n s o n

c h i c k e m b ry o s . The d e v e lo p m e n t o f t h e n e r v o u s s y s te m o f t h e c h i c k i s

som e­

w h a t d i f f e r e n t fro m t h a t i n mammals a n d c o m p a r is o n s s h o u ld b e d raw n c a u ­ tio u s ly . MATERIALS M D METHODS I n t h e p r e s e n t w o rk , m e th o d s c o n s i s t e d e n t i r e l y o f d i r e c t m i c r o s c o p i c o b s e r v a t i o n , s u p p le m e n te d b y t h e c o n s t r u c t i o n o f a c a r d b o a r d m o d e l o f o n e em bryo and n e r v e f i b e r c o u n t s o f d e v e l o p i n g t r a c t s i n n e a r l y a l l e m b ry o s . A l l s p e c im e n s w ere p r e p a r e d b y t h e R an so n p y r i d i n e - s i l v e r te c h n i q u e ( D a v e n p o r t, W in d le and B e e c h , * 3 4 ) . The c a t an d r a t em b ry o s w e re o b t a i n e d fro m a n i m a ls o f known m a tin g d a t e s , im m e d ia te ly u p o n re m o v a l fro m t h e u t e r u s , t h e y w e re p l a c e d i n t h e f i x i n g f l u i d . R e s u l t s o f p r e v i o u s w o rk i n t h i s l a b ­ o r a t o r y i n d i c a t e t h a t a minimum am ount o f d e l a y i n f i x a t i o n

and a minimum

o f h a n d lin g p ro d u c e th e b e s t h i s t o l o g i c p i c t u r e w ith th e p y r i d i n e - s i l v e r s t a i n i n g m e th o d . K or t h i s

r e a s o n , ti m e was n o t t a k e n t o m e a s u r e t h e em b ry o s

11

e x a c t l y , b u t t h e i r g r e a t e s t l e n g t h s w e re e s t i m a t e d w h ile i n t h e am n io n an d b e f o r e f i x a t i o n . Two hum an s p e c im e n s w ere o b t a i n e d a t o p e r a t i o n s an d p r e s e r v e d i m m e d i a te l y . D e t a i l s c o n c e r n in g t h e m a t e r i a l u s e d a r e su m m a riz e d i n t a b l e 1* OBSERVATIONS The 1 6 - d a y c a t em b ry o s o f l i t t e r

846 a r e t h e s m a l l e s t s p e c im e n s s t u d ­

i e d , T hey h a d b e tw e e n t e n an d t h i r t e e n p a i r s o f s o m i t e s . The b r a i n p r e s e n t s v e r y n e a r ly th e b e g in n in g o f n e u r o f i b r i l l a r d i f f e r e n t i a t i o n . E m bryos 1 an d 2 a r e a b o u t a l i k e i n a p p e a r a n c e end d e g r e e o f d e v e lo p m e n t. T h e re i s no n e u r o f i b r i l l a r d i f f e r e n t i a t i o n r o s t r a l t o t h e o t o c y s t s , an d t h e r e f o r e no m e d ia l l o n g i t u d i n a l f a s c i c u l u s i s p r e s e n t . I n f a c t t h e r e a r e n o l o n g i t u d i n a l f i b e r s p r e s e n t an y w h ere i n t h e n e r v o u s s y s te m o f t h e s e em­ b r y o s , P r im a r y m o to r n e u r o b l a s t s o f t h e rh o m b e n c e p h a lo n a r e c l e a r l y d i s t i n (3 ) g u is h a b le , A t t h e sam e l e v e l s , an d a lm o s t a s c o n s p ic u o u s a s t h e s e m o to r e l e m e n t s , s e c o n d a r y n e u r o b l a s t s a r e fo u n d n e a r t h e s u l c u s l i m i t a n s . T h ey s e n d t h e i r a x o n s v e n t r a d , A fe w r e a c h t h e f l o o r p l a t e b u t no v e n t r a l com­ m i s s u r e i s f o r m e d , f o r n o n e o f them h a s c r o s s e d y e t . The g l o s s o p h a r y n g e a l v a g u s r e g i o n o f em bryo num ber 2 i s i l l u s t r a t e d i n f i g u r e 5 , Em bryo nu m b er 3 w as s e o t i o n e d i n t h e

s a g it ta l p la n e . D if f e r e n tia tio n

i s f a r t h e r a d v a n c e d t h a n i n 1 a n d 2 , The m e d ia l l o n g i t u d i n a l , f a s c i c u l u s a s w e l l a s t h e o c u lo m o to r a n d t r i g e m i n a l m o to r n u c l e i a r e b e g i n n i n g t o T he m e d ia l l o n g i t u d i n a l f a s c i c u l u s o r i g i n a t e s j u s t r o s t r a l t o ,

fo ra .

and e x te n d s

o n l y a s f a r c a u d a l a s t h e o c u lo m o to r n u c l e u s * I t c o n s i s t s o f f i v e o r s i x f i b e r s on e i t h e r s i d e . No v e n t r a l l o n g i t u d i n a l t r a c t h a s b e e n fo rm e d b y t h e r h o m b e n c e p h a lic s e c o n d a r y n e u r o b l a s t s . One em bryo o b t a i n e d fro m t h e c a t ( n o , 841) o p e r a t e d u p o n 17 d a y s a f t e r ( s i M o to r n u c l e i o f a l l em b ry o s u s e d i n t h e p r e s e n t s t u d y o c c u p y m e d ia l p o s i t i o n s a d j a c e n t t o t h e f l o o r p l a t e ( W in d le , * 3 3 ) ,

12

i n s e m i n a t i o n h a d tw e n ty - tw o p a i r s o f s o m ite s # C e r t a i n a d v a n c e s i n d e v e lo p ­ m e n t c a n h e c l e a r l y d i s t i n g u i s h e d # A l l p r i m a r y m o to r n u c l e i e x c e p t t h e a b d u c en s a re p r e s e n t , a lth o u g h th e t r o c h l e a r i s is

fo u n d on one s i d e o n l y an d

com posed o f b u t f o u r n e u r o b l a s t s # T he o n ly l o n g i t u d i n a l t r a c t i n t h e

n e r v o u s s y s te m i s t h e m e d ia l l o n g i t u d i n a l f a s c i c u l u s , i t a r i s e s fro m a fe w l i g h t l y

s t a i n e d , b i p o l a r n e u r o b l a s t s a t t h e j u n c t i o n o f t h e m e se n c e p h ­

a l o n and d ie n o e p h a lo n # A t i t s

o r ig i n i t c o n s is ts o f a b o u t te n axons c o u rs in g

c a u d a d i n s i d e t h e e x t e r n a l l i m i t i n g m em b ran e; o n ly tw o c o n t i n u e f o r a n y d i s t a n c e . T h ey r e a c h t h e t r o c h l e a r n u c l e u s , a d i s t a n c e o f 168

fro m t h e

n u c le u s o f o rig in # S e c o n d a r y n e u r o b l a s t s a r e m ore n u m e ro u s a t t h e l e v e l o f t h e v a g u s a n d a c c e s s o r y n e r v e s t h a n e l s e w h e r e , b u t a fe w h a v e a p p e a r e d i n t h e t r i g e m i n a l r e g io n # A xons o f s e c o n d a r y n e u r o b l a s t s i n t h e lo w e r rh o m b e n c e p h a lo n h a v e p e n e t r a t e d t h e f l o o r p l a t e # P o u r s e c t i o n s fro m t h i s em bryo a r e i l l u s t r a t e d i n f i g u r e s 6 , 7 , 1 4 an d 15# The f o u r em b ry o s o f l i t t e r 8 4 5 , o b t a i n e d a b o u t 17 d a y s a n d 4 h o u r s a f ­ t e r in s e m in a tio n , a re c o n s id e r a b ly f a r t h e r advanced i n n e u r o f i b r i l l a r d e­ v e lo p m e n t t h a n num ber 841# S o m ite s v a r y b e tw e e n t w e n t y - s e v e n a n d t h i r t y f o u r # A l l p r i m a r y m o to r n u c l e i a r e p r e s e n t an d m o s t o f them h a v e w e l l - o r g a n ­ i z e d n e r v e r o o t s e x t e n d i n g i n t o t h e m esenchym e# s e c o n d a r y n e u r o b l a s t s a r e n u m e ro u s ; t h e i r ax o n s fo rm a p r o m in e n t v e n t r a l c o m m issu re and a v e n t r a l l o n g ­ i t u d i n a l f a s c i c u l u s i n m o st r e g io n s . S e c o n d a ry n e u r o b la s t s a re a b s e n t a t t h e l e v e l o f c r a n i a l m o to r n u c l e i I I I ,

IV a nd V I I # The m e d ia l l o n g i t u d i n a l

f a s c i c u l u s a r i s e s fro m a s i n g l e g ro u p o f c e l l s . T h i s l i e s j u s t r o s t r a l t o t h e o c u lo m o to r n u c l e u s a n d c o n t a i n s b i p o l a r an d p i r i f o r m n e u r o b l a s t s ( f i g s . 1 1 , 1 9 ;# The a x o n s o f t h e s e n e u r o b l a s t s c o u r s e c a u d a d i n s i d e o f t h e e x t e r n a l l i m i t i n g m em brane# A t t h e o c u lo m o to r l e v e l t h e y sw eep m e sa d , s p r e a d i n g o u t a s t h e y p a s s t h r o u g h t h e o c u lo m o to r n u c le u s # A t t h i s p o i n t t h e t r a c t i s f u l l o f t e r m i n a l g ro w th c o n e s , f o r m o s t o f i t s

f i b e r s h a v e p r o g r e s s e d no f a r t h e r

13

c a u d a d ( f i g * 1 1 )* B e lo w t h e o c u lo m o to r n u c l e u s , t h e f e w r e m a in in g f i b e r s o f t h e m e d ia l l o n g i t u d i n a l f a s c i c u l u s c o n v e r g e an d t a k e u p a p o s i t i o n a d ­ ja c e n t to th e f lo o r p l a t e . M o re d e t a i l e d d e s c r i p t i o n s o f t h e b r a i n s o f i n d i v i d u a l em b ry o s o f t h i s s e r i e s a r e n e c e s s a r y b e c a u s e n e u r o f i b r i l l a r d e v e lo p m e n t i n t h e m e s e n c e p h a lo n an d u p p e r rh o m b e n c e p h a lo n h a s p r o c e e d e d a t d i f f e r e n t s p e e d s i n t h e s e v e r a l s p e c im e n s * I n t h e r e g i o n b e tw e e n n u c l e i o f t h e o c u lo m o to r a n d t r o c h l e a r n e r v e s o f em bryo num ber 4 , t h e m e d ia l l o n g i t u d i n a l f a s c i c u l u s c o n s i s t s o f t e n s m a ll d a rk f i b e r b u n d le s s e c tio n e d t r a n s v e r s e l y . J u s t l a t e r a l t o i t

are f iv e

l i g h t l y s t a i n e d f i b e r s w h ic h h a v e b e e n s e c t i o n e d m ore d i a g o n a l l y ( t h e y a r e c o u rs in g i n

a s l i g h t l y d i f f e r e n t p l a n e ; . T h e se fo rm t h e r o s t r a l en d o f t h e

v e n t r a l l o n g i t u d i n a l f a s c i c u l u s . F a r t h e r c a u d a l l y , t h e m e d ia l l o n g i t u d i n a l f a s c i c u l u s d e c r e a s e s i n s i z e , and j u s t b e f o r e i t d i s a p p e a r s i n t h e m id d le t r o c h l e a r r e g i o n , i t s b u n d l e s s p l i t u p i n t o s e v e n t e e n f i n e f i b e r s . The m o re l a t e r a l g ro u p o f f i b e r s ( v e n t r a l l o n g i t u d i n a l f a s c i c u l u s ) i n c r e a s e s i n s i z e ( t h i r t y f i b e r s a t t h e l e v e l o f IV ) a s i t i s

f o ll o w e d c a u d a d , b u t t h e r e i s

c o n s i d e r a b l e f l u c t u a t i o n i n t h e n um ber o f i t s

f i b e r s th ro u g h o u t th e tr ig e m ­

i n a l r e g i o n . I n t h e c a u d a l p a r t o f t h e t r o c h l e a r r e g i o n (2 4 0

cau d al to

th e f i r s t a p p e a ra n c e o f th e v e n tr a l lo n g itu d in a l f a s c ic u lu s ) th e f i r s t s e c ­ o n d a r y n e u r o b l a s t s a r e o b s e rv e d *

s i x s e c t i o n s (721* ) m ore c a u d a l l y t h e f i r s t

v e n t r a l c o m m is s u re I s f o u n d , i i e r e o n e f i b e r c r o s s e s t h e f l o o r p l a t e . I n s u c c e e d in g s e c t i o n s t h e v e n t r a l c o m m issu re e n l a r g e s , t h e a v e r a g e num ber o f f i b e r s p e r s e c ti o n i n th e f l o o r p l a t e o f th e tr ig e m in a l r e g io n b e in g f o u r V.f i g * 1 0 ) . I n em bryo num ber 2 t h e m e d ia l l o n g i t u d i n a l f a s c i c u l u s i s co m p o sed o f tw e n t y f i n e f i b e r s l o c a t e d i n t h e a r e a j u s t l a t e r a l t o t h e f l o o r p l a t e a n d j u s t c a u d a l t o t h e o c u lo m o to r n u c l e u s . B e tw e e n t h e t r o c h l e a r an d t r i g e m i n a l m o to r n u c l e i , a few l o n g i t u d i n a l f i b e r s a p p e a r l a t e r a l t o t h e m e d ia l l o n g -

14

itu d in a l fa s c ic u lu s * fib e rs ,

r h e y are n o t o rg a n iz e d i n t o

a d e f in ite tra c t*

T hese

s e e n i n f i g u r e 8 , a r e c u t s l i g h t l y d ia g o n a lly w h ile th o s e o f t h e

m e d ia l l o n g i t u d i n a l f a s c i c u l u s a r e s e c t i o n e d m o re t r a n s v e r s e l y a t t h i s p o i n t * A p p a r e n t l y , t h e y m e rg e w i t h t h e l a t e r a l b o r d e r o f t h e m e d ia l lo n g ­ i t u d i n a l f a s c i c u l u s m o re c a u d a l l y ,

f o r no s e p a r a t e l a t e r a l t r a c t i s s e e n

t h e r e * The l o n g i t u d i n a l l y c o u r s i n g f i b e r s i n c r e a s e i n n u m b er f a r t h e r c a u ­ d a l l y , t h e m o re m e d ia l f i b e r s b e c o m in g d i a g o n a l , w h ile t h e l a t e r a l o n e s a re c u t tr a n s v e r s e ly * A t t h i s p o in t , th e r o s t r a l tr ig e m in a l l e v e l , n e u ro ­ b l a s t s o f t h e s e c o n d o r d e r h a v e b e g u n t o d i f f e r e n t i a t e a n d a few s e c t i o n s c a u d a l t o t h i s a v e n t r a l c o m m issu re i s f i r s t s e e n * As t h e y c o u r s e f a r t h e r c a u d a d , t h e m e d ia l l o n g i t u d i n a l f a s c i c u l u s f i b e r s t e n d t o fo rm b u n d l e s w h ic h h a v e t h e a p p e a r a n c e o f l a r g e d a r k l y s t a i n e d f i b e r s * T h e s e b u n d le s s e p a r a t e i n t o

fin e f ib e r s j u s t b e fo re th e

m e d ia l l o n g i t u d i n a l f a s c i c u l u s e n d s i n t h e t r i g e m i n a l r e g io n * f i b e r s

fro m

a e c o n d a r y n e u r o b l a s t s i n t h i s r e g i o n fo rm a h o m o l a t e r a l co m p o n en t o f t h e v e n t r a l lo n g itu d in a l f a s c ic u lu s ; th e y can b e c l e a r l y se e n to e n te r th e tra c t*

H ere a l s o a few f i b e r s fro m t h e r e t i c u l a r f o r m a t i o n a p p e a r t o

s h ift

i n t o t h e v e n t r a l l o n g i t u d i n a l f a s c i c u l u s ( f i g * 1 2 ;* S e c o n d a r y f i b e r s p a s s t h r o u g h t h e u r i g e m i n a l m o to r n u c l e u s an d t h e " v e n tra l l o n g i t u d i n a l f a s c i c u l u s o f t h e sam e s i d e t o

fo rm t h e v e n t r a l c o m m issu re an d o p p o s i t e v e n t r a l lo n g ­

i t u d i n a l f a s c i c u l u s * A fe w r o s t r a l l y d i r e c t e d g ro w th c o n e s w ere o b s e r v e d in th e fa s c ic u lu s * The m e d ia l l o n g i t u d i n a l f a s c i c u l u s o f em bryo n u m b er 3 c o n t a i n s s is c ty f i b e r s c a u d a l to th e n u c le u s o f o r i g i n and tw e n ty - f iv e f i b e r s j u s t c a u d a l t o t h e o c u lo m o to r n u c l e u s . No s e p a r a t e l a t e r a l g ro u p o f f i b e r s was o b s e r v e d i n t h i s em bryo* T he lo w e r p a r t o f t h e m e d ia l l o n g i t u d i n a l f a s c i c u l u s r e ­ t a i n s a c o n s ta n t s i z e u n t i l a f t e r sec o n d a ry n e u r o b la s ts have a p p e a re d i n t h e t r i g e m i n a l r e g io n * A t t h i s p o i n t t h e t r a c t h a s d e c r e a s e d t o t w e n t y tw o f i b e r s , an d t h e n im m e d ia te ly i n c r e a s e s t o t w e n t y - s e v e n f i b e r s . T h is

15

in c re a s e i s

due t o t h e a d d i t i o n o f s e c o n d a r y n e u r o n s . H e a r t h e m i d d le o f

th e tr ig e m in a l re g io n th e v e n tr a l lo n g itu d in a l f a s c ic u lu s c o n ta in s f o r ty f i b e r s ( f i g * 2 0 ) • T h e r e a f t e r , i t b eco m es s m a l l e r an d d i s a p p e a r s i n t h e f a c i a l re g io n ( f i g *

2 1 )* H o w e v er, t h r o u g h o u t t h e f a c i a l r e g i o n a fe w f i b e r s a p p e a r

h e r e an d t h e r e i n t h e p o s i t i o n o f t h e v e n t r a l l o n g i t u d i n a l f a s c i c l e - t h e y do n o t c o u r s e f o r m o re t h a n tw o o r t h r e e s e c t i o n s . I f t h e s e a r e

se c o n d ary

n e u r o n s , t h e y a r i s e o n t h e h o m o l a t e r a l s i d e . As i n em bryo 2 , a v e n t r a l com­ m i s s u r e b e g i n s a fe w s e c t i o n s c a u d a l t o t h e a p p e a r a n c e o f s e c o n d a r y n e u r o ­ b l a s t s * A g r a p h i c r e p r e s e n t a t i o n o f num ber o f f i b e r s i n t h e l o n g i t u d i n a l t r a c t s a n d t h e c o m m is s u re s a t v a r i o u s l e v e l s w i l l b e s e e n i n f i g u r e 1* Em bryo num ber 1 i s v e r y l i g h t l y s t a i n e d and i t i s t i n g u i s h s e c o n d a r y n e u r o b l a s t s * H e rv e f i b e r fib e rs

d i f f i c u l t to d is ­

c o u n ts ( t w e n t y l o n g i t u d i n a l

c a u d a l t o t h e o c u lo m o to r n u c l e u s ) c a n n o t b e c o n s i d e r e d as r e l i a b l e

as i n th e b e t t e r

s t a i n e d e m b ry o s . H o w ev er, a g r a p h c o n s t r u c t e d o n t h e b a s i s

o f t h e s e c o u n t s , f o l l o w s t h e same p a t t e r n a s i n t h e o t h e r em b ry o s o f t h i s s e r i e s * A m e d ia l a n d l a t e r a l g r o u p in g o f f i b e r s i s a p p a r e n t a s f a r

as th e

m id d le t r i g e m i n a l r e g i o n . The v e n t r a l l o n g i t u d i n a l f a s c i c u l u s d i s a p p e a r s c a u d a l t o t h e e m e rg in g t r i g e m i n a l m o to r r o o t an d r o s t r a l t o t h e o t o c y s t * The lo w e r rh o m b e n c e p h a lo n c a u d a l t o t h e t r i g e m i n a l r e g i o n i s i n a l l f o u r em b ry o s o f l i t t e r

s im ila r

845* The v e n t r a l l o n g i t u d i n a l f a s c i c u l u s d i s ­

a p p e a r s i n t h e f a c i a l r e g i o n . Ah e v e n t r a l co m m issu re i s

com posed e n t i r e l y o f

c r o s s i n g f a c i a l m o to r ax o n s a t t h i s l e v e l ( .f ig * 2 1 ; . T h e s e a r e t h i c k , d a r k f i b e r s w i t h l a r g e c l u b - s h a p e d g ro w th c o n e s . M any o f th e m c a n b e t r a c e d d i r e c t l y fro m m o to r n e u r o b l a s t s . A t t h i s l e v e l ,

sec o n d a ry n e u r o b la s ts a re

v e r y s c a r c e a n d im m a tu r e . I n m o s t i n s t a n c e s t h e y h a v e s h o r t , v e n t r a l l y d i r e c t e d a x o n s e n d in g i n g ro w th c o n e s a fe w m i c r a fro m t h e i r o r i g i n s * A t th e c a u d a l end o f th e f a c i a l r e g io n , th e v e n t r a l lo n g i tu d in a l f a s ­ c i c u l u s r e a p p e a r s o r a g a i n b eco m es a p r o m in e n t t r a c t * A few s e c t i o n s l a t e r , m o re m a tu r e s e c o n d a r y n e u r o b l a s t s , w hose a x o n s c o u r s e v e n t r a d an d m in g le

16

w i t h t h e d o r s a l l y r u n n i n g i n t r a - m e d u l l a r y m o to r r o o t f i b e r s , a p p e a r # A t f i r s t th e re

a r e o n l y o n e o r tw o p e r s e c t i o n , b u t t h i s n um ber r a p i d l y i n ­

c r e a s e s t o t e n o r m o r e . F i b e r s c a n b e s e e n c o u r s i n g fro m t h e i n t r a - m e d u l l a r y m o to r b u n d le t h r o u g h t h e m o to r n u c l e u s a n d i n t o t h e v e n t r a l c o m m is s u re . T h e re i s a c o r r e l a t i o n b e tw e e n t h e s i z e o f t h e v e n t r a l c o m m issu re a n d t h e num ber o f f i b e r s i n t h e v e n t r a l l o n g i t u d i n a l f a s c i c u l u s #

T h is i s show n i n

f i g u r e 1 . Ho h o m o l a t e r a l c o n t r i b u t i o n s t o t h e v e n t r a l l o n g i t u d i n a l f a s c i c u l u s w e re o b s e r v e d i n t h e lo w e r r h o m b e n c e p h a lo n . The i n c r e a s e i n s i z e o f v e n t r a l c o m m issu re an d v e n t r a l l o n g i t u d i n a l f a s c i c l e i n t h e v a g u s r e g i o n i s i l l u s ­ t r a t e d i n f i g u r e s I S an d 2 2 . S e v e n r a t em b ry o s o f 19 t o

25 s a m i t e s , o b t a i n e d 272 h o u r s a f t e r in s e m ­

i n a t i o n , w e re re e x a m in e d # Ho m e d ia l l o n g i t u d i n a l f a s c i c u l u s i s p r e s e n t i n t h e s m a l l e s t s p e c im e n ( n o . 5) n o r a r e t h e r e

an y s e c o n d a r y e l e m e n ts i n t h e

rh o m b e n c e p h a lo n . I n em bryo num ber 2 , s l i g h t l y l a r g e r , t h e m e d ia l l o n g i t u d i n a l f a s c i c u l u s h a s j u s t b e g u n t o fo rm an d e x te n d s o n l y a s f a r

as th e t r o c h l e a r

l e v e l ( s e e f i g . 5 , W in d le an d B a x t e r , * 3 6 ) . The p r e s e n c e o f s e c o n d a r y n e u r o n s i s i n d e t e r m i n a b l e b e c a u s e o f a d a r k l y s t a i n e d b a c k g r o u n d . I n em b ry o s num­ b e r s 6 and 7 , th e m e d ia l l o n g i tu d in a l f a s c i c u l u s re a c h e s t h e r o s t r a l t r i ­ g e m in a l l e v e l a n d e n d s t h e r e w i t h o u t d e c u s s a t i n g . S e c o n d a r y n e u r o b l a s t s a r e b e g i n n i n g t o d e v e lo p i n t h e t r i g e m i n a l r e g i o n , b u t no v e n t r a l com m is­ s u r e and no v e n t r a l l o n g i t u d i n a l f a s c i c u l u s h a v e b e e n fo rm e d t h e r e . Ho lo n g ­ i t u d i n a l t r a c t i s p r e s e n t i n th e v e n tr a l p a r t o f th e b a s a l p l a t e a t o th e r l e v e l s . H o w ev er, a f e w n e u r o b l a s t s o f t h e

second o rd e r c a n b e re c o g n iz e d in

t h e lo w e r rh o m b e n c e p h a lo n . T h e s e a p p e a r t o b e l e s s m a tu r e t h a n t h o s e a t t h e t r i g e m i n a l l e v e l . T he m e d ia l l o n g i t u d i n a l f a s c i c u l u s o f em b ry o s 1 , 3 an d 4 e x t e n d s t o t h e c a u d a l t r i g e m i n a l r e g i o n . Em bryo 3 i s

s e c tio n e d i n a s a g i t t a l

p l a n e , w hich m ak es r e l a t i o n s h i p s b e tw e e n t h e l o n g i t u d i n a l t r a c t , v e n t r a l c o m m issu re an d n e u r o b l a s t s o f t h e se c o n d o r d e r d i f f i c u l t t o d e t e r m i n e . E m bryos 1 and 4 r e p r e s e n t s t a g e s o f d e v e lo p m e n t s i m i l a r i n some r e s -

17

p e c t s t o t h o s e i n 4 mm# t o 5 mm* c a t em b ry o s# B etw een t h e t r o c h l e a r an d trig e m in a l re g io n s , th e lo n g itu d in a l t r a c t d iv id e s in to l i t t l e

b u n d le s #

A t some l e v e l s t h e s e b u n d le s a r e d e f i n i t e l y a r r a n g e d m e d i a l l y an d l a t e r a l l y . A m e d ia l g ro u p o f t h e s e i s m o re c o m p a c t and d a r k e r t h a n t h e m o re l a t e r a l b u n d le s a n d c o m p r is e s t h e d e s c e n d in g d i e n c e p h a l i c co m p o n en t o f t h e m e d ia l lo n g itu d in a l fa s c ic u lu s #

I n o t h e r r e g i o n s , h o w e v e r, t h e f i b e r s a r e m o re

e v e n ly d i s t r i b u t e d a t th e p e r ip h e r y o f th e an d i t i s c ic u lu s #

a re a l a t e r a l to

th e f lo o r p l a t e ,

n o t p o s s i b l e t o s a y w h ic h b e lo n g t o t h e m e d ia l l o n g i t u d i n a l f a s ­ I n some s e c t i o n s , p a r t i c u l a r l y i n t h e c a u d a l t r i g e m i n a l r e g i o n ,

f i b e r s c a n b e s e e n r u n n in g fro m o n e b u n d le t o

a n o th e r, s u g g e s tin g t h a t an

in te r m in g lin g o f f i b e r s ta k e s p la c e . T he t o t a l num ber o f f i b e r s i n b o t h m e d i a l and l a t e r a l b u n d le s j u s t c a u d a l t o t h e t r o c h l e a r l e v e l d r o p s b e lo w t h e c o u n t o f f i b e r s i n t h e m e d ia l lo n g itu d in a l fa s c ic u lu s

a t t h e t r o c h l e a r l e v e l , -where o n ly one t r a c t i s

a p p a r e n t* A few s e c t i o n s f a r t h e r c a u d a l l y , h o w e v e r, t h e t o t a l c o u n t o f t h e m e d ia l an d l a t e r a l f i b e r s o f t h e v e n t r a l l o n g i t u d i n a l t r a c t i n c r e a s e s an d s u r p a s s e s t h a t o f t h e m e d ia l l o n g i t u d i n a l f a s c i c u l u s f i b e r s

stb t h e t r o c h l e a r

le v e l• S e c o n d a r y n e u r o b l a s t s i n t h e t r i g e m i n a l r e g i o n s e n d axons v e n t r a d t o accom pany t h e i n t r a m e d u l l a r y p r im a r y m o to r r o o t s o f t h e t r i g e m i n a l n e r v e # Some o f t h e s e a x o n s r e a c h t h e v e n t r a l p a r t o f t h e n e u r a l t u b e ; a fe w l i g h t f i b e r s c a n be se e n p a s s i n g v e n tro m e s a d th r o u g h t h e m o to r n u c l e u s . I n em bryo num ber 1 , some o f t h e s e f i b e r s

c o u r s e v e n t r a d fro m t h e i n t r a - m e d u l l a r y mo­

t o r b u n d le an d e n d a t t h e l a t e r a l b o r d e r o f t h e v e n t r a l l o n g i t u d i n a l f a s ­ c i c u l u s o f t h e same s i d e ( f i g * 9 ) . They e n t e r t h i s t r a c t # T he v e n t r a l l o n g i t u d i n a l t r a c t e n d s a t t h e f a c i a l r e g i o n ; no l o n g i t u d ­ i n a l f i b e r s a r e fo u n d n e a r t h e f l o o r p l a t e c a u d a l t o t h i s i n em b ry o s 1 and 4 . I n b o t h o f t h e s e e m b ry o s, t h e f o r m a t i o n o f a v e n t r a l co m m issu re i s j u s t b e g i n n i n g i n t h e t r i g e m i n a l r e g i o n . T h re e o r f o u r f i b e r s i n a s m any s e c t i o n s

18

a r e s e e n e n t e r i n g t h e f l o o r p l a t e an d e n d i n g t h e r e * No f i b e r c r o s s e s t o t h e o t h e r s i d e * A fe w f l o o r p l a t e f i b e r s

a re e n c o u n te re d i n th e f a c i a l

r e g i o n . T h e s e a r e l a r g e , d a r k l y s t a i n e d a x o n s i n c o n t r a s t w ith t h e d e l i ­ c a t e p r o c e s s e s o f s e c o n d a r y n e u r o b l a s t s an d a r e u n d o u b te d ly p r im a r y m o to r f i b e r s fo r m in g t h e c r o s s e d fo m p o n e n t o f t h e f a c i a l n e r v e * T he s m a l l e s t human em bryo (n o * 8 2 1 ) , 26 d a y s 1 f e r t i l i z a t i o n d e a d b e f o r e a b o r t i o n . An a c c u r a t e s t u d y o f t h e n e r v o u s s y s te m i s

a g e , w as im p o s s ib le

b u t a fe w s i g n i f i c a n t n e u r o f i b r i l l a r s t r u c t u r e s a r e v i s i b l e . Some o f t h e p r im a r y m o to r n u c l e i h a v e a p p e a r e d , and s e c o n d a r y n e u r o n s a r e f o r m in g i n t h e rh o m b e n c e p h a lo n * T h e re i s no m e d ia l l o n g i t u d i n a l f a s c i c u l u s a s y e t ; i n f a c t no n e u r o b l a s t s c a n b e s e e n an y w h ere i n t h e d ie n c e p h a l o n a n d t e l e n c e p h ­ a lo n * The s e c o n d a r y n e u r o n s o f t h e b r a i n a r e c o n f in e d t o t h e l e v e l s o f t h e g lo s s o p h a r y n g e a l an d v a g u s n e r v e s . A few t h i n f i b e r s c r o s s t h e f l o o r p l a t e i n t h e lo w e r rh o m b e n c e p h a lo n a n d a v e n t r a l l o n g i t u d i n a l f a s c i c u l u s h a s fo rm e d i n t h i s r e g i o n o n l y . I t s

co u rse i s

s h o rt, fo r i t

is

seen n e ith e r in

th e tr ig e m in a l re g io n n o r in th e u p o er c e r v i c a l s p in a l c o rd . One em bryo o f 30 d a y s 1 f e r t i l i z a t i o n

ag e ( n o . 8 2 8 ) i s o u r s m a l l e s t

hum an s p e c im e n i n w h ic h a m e d ia l l o n g i t u d i n a l f a s c i c u l u s a p p e a r s . I t i s m ore a d v a n c e d t h a n t h e l a r g e s t r a t and c a t e m b ry o s . The r o s t r a l o r i g i n o f th e m e d ia l lo n g i t u d i n a l f a s c ic u lu s i s o b s e rv e d i n a g ro u p o f n e u r o b la s t s l o c a t e d i n f r o n t o f and s l i g h t l y d o r s a l t o t h e o c u lo m o to r n u c l e u s . T h i s c e l l g ro u p i s t h e m o s t h i g h l y d e v e lo p e d s t r u c t u r e r o s t r a l t o t h e m e s e n c e p h ­ a lo n . I t g iv e s r i s e

to s e v e r a l c l o s e l y a s s o c ia te d b u n d le s o f f in e f i b e r s

w h ic h c o u r s e c a u d a d o n t h e samd s i d e o f t h e b r a i n . T h ey come t o l i e

ju s t

b e n e a t h t h e e x t e r n a l l i m i t i n g m em brane a t t h e j u n c t i o n o f f l o o r p l a t e

an d

b a s a l p l a t e i n t h e r e g i o n o f t h e o c u lo m o to r n e r v e ( f i g . 1 6 ) . Some o f t h e b u n d le s o f t h i s

f i b e r t r a c t a r e a c t u a l l y i n t h e f l o o r p l a t e . N ear i t s

o rig in

i t c o n t a i n s m o re t h a n 450 f i b e r s , b u t m any o f t h e s e e n d b e f o r e t h e l e v e l o f

19

t h e t r o c h l e a r n e r v e i s r e a c h e d ; l e s s t h a n 300 f i b e r s c a n b e c o u n t e d a t t h a t l e v e l . A ll o f th e l i t t l e

b u n d le s o f f i n e f i b e r s e n d b y t h e t i m e t h e

t r a c t r e a c h e s t h e l e v e l o f t h e t r i g e m i n a l n e r v e r o o ts *

a t t h i s p o in t th e y

a r e b e i n g r e p l a c e d b y m ore l o o s e l y a r r a n g e d a n d c o a r s e r f i b e r s - s e c o n d a r y n e u r o n s o f l o c a l o r i g i n . T hus t h e m e d ia l l o n g i t u d i n a l f a s c i c u l u s g i v e s w ay t o t h e v e n t r a l l o n g i t u d i n a l f a s c i c u l u s . The t o t a l num ber o f f i b e r s i n t h i s t r a c t a t t h e l e v e l o f t h e t r i g e m i n a l m o to r n u c l e u s i s l e s s t h a n 1 5 0 , T h e re i s n o t h i n g e x c e p t i o n a l i n t h e mode o f t e r m i n a t i o n o f t h e f i n e

fib e rs

fro m t h e n u c l e u s o f t h e m e d ia l l o n g i t u d i n a l f a s c i c u l u s . T hey do n o t c r o s s t o t h e o p p o s i t e s i d e f o r n o v e n t r a l c o m m issu re i s p r e s e n t i n t h e p la te

flo o r

ab o v e t h e t r i g e m i n a l l e v e l . As t h e b u n d l e s b r e a k u p t h e y s im p ly e n d

on t h e

same s i d e . B elov/ t h e t r i g e m i n a l r e g i o n t h e v e n t r a l l o n g i t u d i n a l

f a s c i c u l u s im m e d ia te ly i n c r e a s e s i n num ber o f f i b e r s ( f i g s . 1 7 , 1 8 ) * C or­ r e l a t e d w ith t h i s

i n c r e a s e , t h e v e n t r a l co m m issu re e n l a r g e s . DISCUSSION

W ith m in o r s p e c i e s an d v e r y s l i g h t i n d i v i d u a l v a r i a t i o n , we f i n d t h a t t h e m e d i a l l o n g i t u d i n a l f a s c i c u l u s an d i t s ila rly

a s s o c i a t e d t r a c t s d e v e lo p sim ­

i n t h e c a t , r a t an d m an . We f i n d t h a t t h e t r a c t w h ic h h a s b e e n assu m ed

t o b e t h e p rim o rd iu m o f t h e a d u l t f a s c i c u l u s l o n g i t u d i n a l i s m e d i a l i s I s t h a t o n ly i n p a r t . T h e r e c a n b e no r e a s o n a b l e d o u b t t h a t t h e f i b e r s d e s c e n d i n g fro m a g ro u p o f c e l l s a t t h e j u n c t i o n o f e m b ry o n ic m e s e n c e p h a lo n an d d i e n ­ c e p h a lo n c o n s t i t u t e t h e o l d e s t p a r t o f t h e m e d ia l l o n g i t u d i n a l f a s c i c u l u s . T h i s i s t h e o n ly p a r t o f t h e m e d ia l l o n g i t u d i n a l f a s c i c u l u s p r e s e n t i n em­ b ry o s o f th e s iz e s

s t u d i e d h e r e . T h is g ro u p o f f i b e r s fo rm s a h o m o l a t e r a l

d e s c e n d i n g p a th w a y w h ic h t a k e s no p a r t i n c o n s t r u c t i o n o f t h e v e n t r a l com­ m i s s u r e i n b r a i n s o f em b ry o s o f t h e s i z e s t u d i e d h e r e . A v e n t r a l l o n g i t u d i n a l f a s c i c u l u s m ore c a u d a l l y o c c u p i e s a p o s i t i o n c o m p a ra b le w i t h t h a t o f t h e m e d ia l l o n g i t u d i n a l f a s c i c u l u s . I n t h e t r i g e m -

20

in & l r e g i o n t h i s t r a c t i s s m a l l . I n a l l t h r e e s p e c i e s , t h e m e d i a l lo n g ­ i t u d i n a l f a s c i c u l u s i s e v e n t u a ll y o v e rla p p e d b y th e o th e r lo n g i t u d i n a l f i b e r s . I n m o s t c a t e m b ry o s , t h e tw o t r a c t s

are d i s t i n g u i s h a b l e

fro m o n e

a n o t h e r b e c a u s e t h e y a r e s e p a r a t e an d b e c a u s e t h e i r f i b e r s t a k e a s l i g h t l y d i f f e r e n t d i r e c t i o n . I n t h e hu#ian, t h e f i b e r s o f t h e m e d ia l l o n g i t u d i n a l f a s c i c u l u s a r e f i n e r t h a n t h o s e o f t h e v e n t r a l l o n g i t u d i n a l f a s c i c u l u s * The d e v e lo p m e n t o f t h e r a t r o s t r a l t o t h e f a c i a l r e g i o n r e s e m b le s t h a t i n t h e c a t . N e u r o b l a s t s o f t h e s e c o n d o r d e r s t a r t t o fo rm a v e n t r a l l o n g i t u d i n a l f a s c i c u l u s b y t h e tim e t h e m e d ia l l o n g i t u d i n a l f a s c i c u l u s r e a c h e s t h e t r i ­ g e m in a l r e g i o n . I f f o r no o t h e r r e a s o n , t h e p r e s e n c e o f a v e n t r a l l o n g i t u d ­ i n a l f a s c i c u l u s s h o u ld b e I n d i c a t e d i n t h e r a t b y t h e

f a c t t h a t t h e num ber

o f l o n g i t u d i n a l f i b e r s i n t h e r o s t r a l t r i g e m i n a l r e g i o n becom es g r e a t e r t h a n a t th e tro c h le a r le v e l;

f i b e r s a r e b e i n g ad d ed fro m a s o u r c e b e lo w t h e

t r o c h l e a r l e v e l . The v e n t r a l l o n g i t u d i n a l f a s c i c u l u s i s b o th c r o s s e d a n d u n c ro s s e d in th e trig e m in a l re g io n . I n t h e y o u n g e r e m b ry o s , b o t h t r a c t s

are a b s e n t i n th e f a c i a l r e g io n ,

b u t a t t h e l e v e l o f t h e g lo s s o p h a r y n g e a l an d v a g u s n u c l e i t h e v e n t r a l l o n g ­ i t u d i n a l f a s c i c u l u s r e a p p e a r s . T h is t r a c t a p p e a r s t o b e fo rm ed e n t i r e l y b y s e c o n d a r y n e u r o n s w hose c e l l b o d ie s a r e l o c a t e d a t t h e j u n c t i o n o f t h e a l a r an d b a s a l p l a t e s i n t h e lo \v e r r h o m b e n c e p h a lo n . I t i s p r e d o m i n a n t l y c r o s s e d an d a s c e n d i n g a t t h i s l e v e l * We c a n b e r e a s o n a b l y c e r t a i n t h a t t h e v e n t r a l l o n g i t u d i n a l f a s c i c u l u s d oes n o t r e p r e s e n t th e v e s t i b u l a r p a r t o f th e f a s c ic u lu s l o n g i t u d i n a l i s m e d i a l i s , b e c a u s e b o t h p r i m a r y and s e c o n d a r y v e s t i b u l a r n e u r o n s a r e u n ­ d e v e lo p e d i n em b ry o s w i t h w e l l fo rm ed v e n t r a l l o n g i t u d i n a l f a s c i c u l i . We suggest th a t th is

tra c t is

a compound s e c o n d a r y a f f e r e n t p a th w a y f o r t r i ­

g e m in a l, g l o s s o p h a r y n g e a l an d v a g u s n e r v e s (a n d l a t e r ,

the f a c i a l ) .

We c a n n o t b e c e r t a i n t h a t t h e v e n t r a l l o n g i t u d i n a l f a s c i c u l u s c o n t a i n s no c a u d a l l y d i r e c t e d f i b e r s . O ur c o n c l u s i o n t h a t i t i s p r e d o m i n a n tl y a s c e n d ­

21

i n g i s b a s e d p a r t l y on r e l a t i o n s h i p s l i k e i.e.,

th o s e i l l u s t r a t e d i n f i g u r e 1 ,

o n e n u m e r a ti o n o f co m p o n en t f i b e r s i n t h e t r a c t

and i n t h e c o m m issu re

a t d i f f e r e n t l e v e l s . F u r th e r m o n e , n o c a u d a l l y d i r e c t e d g ro w th c o n e s w e re o b s e r v e d i n t h e v e n t r a l l o n g i t u d i n a l f a s c i c u l u s o f t h e lo w e r rh o m b e n c e p h a lo n a l t h o u g h t h e y w e re p r e s e n t i n t h e m e d ia l l o n g i t u d i n a l f a s c i c u l u s a t h i g h e r l e v e l s , n o s t r a l l y d i r e c t e d g ro w th c o n e s w e re s e e n i n t h e v e n t r a l l o n g i t u d i n a l f a s c i c u l u s . I n a n e a r l i e r s t u d y i t was r e p o r t e d t h a t t h e f i r s t a s c e n d i n g f i b e r s r e a c h t h e a n l a g e o f t h e th a la m u s i n t h e 8 .0 mm. c a t em bryo ( W in d le , * 3 5 ) . The v e n t r a l l o n g i t u d i n a l f a s c i c u l u s a p p e a r s t o b e t h e s o u r c e o f m any o f th e s e f ib e r s . T h e s e o b s e r v a t i o n s do n o t s u p p o r t t h e c la im t h a t i n t h e r a t t h e me d i a l l o n g i t u d i n a l f a s c i c u l u s and t h e v e n t r a l l o n g i t u d i n a l f a s c i c u l u s d e s c e n d t h r o u g h o u t t h e n e r v o u s s y s te m b e f o r e an y a s c e n d i n g s e c o n d a r y e l e m e n t s a r e c o n t r a i b u t e d ^ A n g u lo , * 3 9 ) . S u c h a c o n c l u s i o n c a n n o t b e r e a c h e d i n o b j e c t i v e a n a l y s i s o f a d e q u a t e l y s t a i n e d h i s o l o g i c p r e p a r a t i o n s o f n o rm a l e m b ry o n ic m a t e r i a l . A n g u lo ( * 3 9 ) h a s a s s i g n e d t o t h e l o n g i t u d i n a l f a s c i c u l i a f u n c t i o n o f d e s c e n d in g i n t e g r a t i o n , p r o v i d i n g an a n a t o m ic a l b a s i s f o r t h e t h e o r y t h a t m am m alian em bry o s e x h i b i t a " t o t a l r e a c t i o n ” m o to r p a t t e r n c o m p a r a b le w i t h t h a t fo u n d b y C o g h i l l ( * 2 9 ) i n A m b ly sto m a. O ur p r e s e n t s t u d y d e m o n s t r a t e s t h a t a s c e n d i n g s e c o n d a r y n e u r o n s p r e c e d e t h e d e v e lo p m e n t o f a d e s c e n d i n g i n t e g r a t i n g m e c h a n ism i n c a t an d human e m b ry o s . D i f f e r e n t i a t i o n o f s e c o n ­ d a r y e l e m e n ts o f t h e lo w e r rh o m b e n c e p h a lo n t a k e s p l a c e r e l a t i v e l y e a r l i e r i n t h e c a t t h a n i n t h e r a t , b u t i n n e i t h e r s p e c i e s i s t h e r e an y d o u b t r e ­ g a r d in g th e g r e a t p re p o n d e ra n c e o f a s c e n d in g o v er d e s c e n d in g n e u ro n s i n th e e m b ry o n ic b r a i n s t e m . We c o n c e d e t h a t t h e m e d ia l l o n g i t u d i n a l f a s c i c u l u s may u l t i m a t e l y f u n c t i o n i n m o to r i n t e g r a t i o n , b u t i t i s th e e n tir e

f a l l a c i o u s to h o ld t h a t

s y s te m o f v e n t r a l l o n g i t u d i n a l n e u r o n s c o n s t i t u t e

a g r e a t de­

s c e n d in g p a th w a y i n y o u n g m am m alian e m b ry o s . O ur o b s e r v a t i o n s do n o t s u b s t a n t i a t e t h e c la im s t h a t :

" D u r in g t h e

22

e a r l y s ta g e s o f g e n e s is th e f i b e r s t h a t e n te r t h i s h a v e b u t one s o u r c e o f o r i g i n :

( v e n t r a l ) c o m m is s u re

t h e v e n t r a l l o n g i t u d i n a l p a t h . M o re o v e r t h i s

c o m m is s u re h a s a s e q u e n t i a l o r d e r o f a p p e a r a n c e , b e g i n n i n g i n t h e m i d b r a i n an d p r o g r e s s i v e l y a p p e a r i n g a t t h e m ore c a u d a l l e v e l s w i t h t h e i n c r e a s e i n ag e o f t h e f e t u s " (.A n g u lo , * 3 9 ) . We f i n d t h a t i n t h e r a t t h e v e n t r a l c o m m issu re b e g i n s t o b la s ts

fo rm i n t h e t r i g e m i n a l r e g i o n ( w h e re s e c o n d a r y n e u r o ­

a r e a l r e a d y p r e s e n t a n d q u i t e w e l l a d v a n c e d ; b e f o r e a v e n t r a l com­

m i s s u r e a p p e a r s a t t h e o c u lo m o to r an d t r o c h l e a r l e v e l s (w h e re t h e m e d ia l l o n g i t u d i n a l f a s c i c u l u s i s t h e o n ly t r a c t p r e s e n t ) . T h is i s

ev en m o re s t r i k ­

in g i n t h e c a t w h e re f o r m a t i o n o f t h e v e n t r a l co m m issu re t a k e s p l a c e i n a r e a s f a r rem o v ed fro m t h e c a u d a l l i m i t o f t h e m e d ia l l o n g i t u d i n a l f a s c i c u l u s * i n t h e s e r e g i o n s , t h e co m m issu re d e v e lo p s b y g ro w th o f s e c o n d a r y a x o n s a c ro s s th e f lo o r p l a t e i n th e ab sen ce o f any lo n g itu d in a l t r a c t . T he d i e n c e p h a l i c co m p o n en t o f t h e m e d ia l l o n g i t u d i n a l f a s c i c u l u s c a n b e s a i d t o b e t h e f i r s t t r a c t o f n e r v e f i b e r s i n t h e e m b ry o n ic b r a i n . How­ ev e r, i t

d o e s n o t s p r i n g fro m t h e f i r s t g ro u p o f i n t e r n e u r o n s t o d e v e l o p .

B e f o r e t h e r e i s a n y d i f f e r e n t i a t i o n i n t h e n u c l e u s o f t h e m e d ia l l o n g i t u d i n a l fa s c ic u lu s ,

s e c o n d a r y n e u r o b l a s t s o f t h e lo w e r rh o m b e n c e p h a lo n ( g l o s s o ­

p h a r y n g e a l , v a g u s an d a c c e s s o r y r e g i o n ) a r e s e n d in g t h e i r ax o n s to w a r d t h e f l o o d p l a t e . T h e s e ax o n s fo rm t h e v e n t r a l l o n g i t u d i n a l f a s c i c u l u s i n th is firs t

r e g i o n b y c r o s s i n g t h e f l o o r p l a t e a n d t u r n i n g r o s t r a d o n ly a f t e r t h e fe w d e s c e n d i n g f i b e r s o f t h e m e d ia l l o n g i t u d i n a l f a s c i c u l u s a p p e a r . CQUCLUSIONS The e a r l y d e v e lo p m e n t o f t h e m e d ia l l o n g i t u d i n a l f a s c i c u l u s an d a s s o ­

c i a t e d n e u r o n s w as s t u d i e d i n p y r i d i n e s i l v e r s t a i n e d r a t ,

c a t a n d human

e m b ry o s . The f i r s t co m p o n en t o f th e m e d ia l l o n g i t u d i n a l f a s c i c u l u s

a ris e s

fro m a n u c l e u s a t t h e r e g i o n o f j u n c t i o n o f m e s e n c e p h a lo n an d d i e n c e p h a l o n a t a p p r o x im a te ly 272 h o u r s a f t e r i n s e m i n a t i o n i n t h e r a t ( a b o u t 24 s o m i t e s ) , 16 d a y s a f t e r i n s e m i n a t i o n i n t h e c a t ^ a b o u t 1 0 -1 3 s o m i t e s ; , an d b e tw e e n

23

26 a n d 30 d a y s ( f e r t i l i z a t i o n

a g e ) i n man* T h is i s t h e o n ly c o m p o n e n t o f

t h e m e d i a l l o n g i t u d i n a l f a s c i c u l u s p r e s e n t i n t h e b r a i n s o f em b ry o s l e s s t h a n 5 mm. o r 6 mm. t o t a l l e n g t h ( m e a s u r e d b e f o r e f i x a t i o n ) . I t d e s c e n d s h o m o l a t e r a l l y a s a f a r a s t h e t r i g e m i n a l r e g i o n -w ith o u t f o r m in g a c o m m is s u re in th e

s p e c im e n s s t u d i e d .

A v e n t r a l l o n g i t u d i n a l f a s c i c u l u s , p r e d o m i n a n tl y a s c e n d i n g ,

d e v e lo p s

i n t h e rh o m b e n c e p h a lo n an d com es t o o v e r l a p ( i t s f i b e r s o f t e n m i n g l i n g w i t h ) t h e d i e n c e p h a l i c c o m p o n e n t o f t h e m e d ia l l o n g i t u d i n a l f a s c i c u l u s . T h is t r a c t a r i s e s fro m s e c o n d a r y n e u r o b l a s t s a t t h e in u n c tio n o f a l a r an d b a s a l p l a t e s i n t h e r e g i o n o f t h e t r i g e m i n a l , g l o s s o p h a r y n g e a l , v a g u s and a c c e s s o r y n e rv e s . I t i s d e f ic ie n t in th e

f a c i a l an d v e s t i b u l a r r e g i o n s a t f i r s t ,

rh e

p o r t i o n a r i s i n g fro m t h e lo w e r rh o m b e n c e p h a lo n i s o l d e r th em t h a t a r i s i n g i n th e tr ig e m in a l re g io n o f t h e

c a t e m b ry o . The c o n v e r s e a p p e a r s t o b e

t r u e o f t h e r a t e m b ry o , i h e rhom b e n c e p h a l i c co m p o n en t i s p r e d o m i n a n t l y a c r o s s e d a s c e d d in g s e c o n d a r y p a th w a y . The p o r t i o n a r i s i n g i n t h e t r i g e m i n a l r e g i o n h a s b o t h h o m o l a t e r a l a n d c o n t r a l a t e r a l o r i g i n s an d i t c o u r s e s r o s t r a d . T he v e n t r a l co m m issu re i s

fo rm ed m a i n l y b y s e c o n d a r y n e u r o n s o f t h e

rh o m b e n c e p h a lo n . A fe w p r im a r y m o to r n e u r o n s o f t h e f a c i a l n e r v e c r o s s , b u t no f i b e r s o f t h e d i e n c e p h a l i c com p o n en t o f t h e m e d ia l l o n g i t u d i n a l f a s c ic u lu s e n te r th e flo o r p l a t e . T he v e n t r a l l o n g i t u d i n a l f a s c i c u l u s o f em bryos l e s s t h a n 5 mm. o r 6 mm. lo n g i s n o t t h e p rim o rd iu m o f t h e v e s t i b u l a r p o r t i o n o f t h e m e d ia l l o n g i t u d i n a l f a s c i c u l u s . I t m ay b e a com pound s e c o n d a r y a f f e r e n t p a th w a y ( p a r t l y v i s c e r a l le m n i s c u s ? ) w h ic h w i l l l i n k a f f e r e n t n e u r o n s o f t h e t r i ­ g e m in a l, g l o s s o p h a r y n g e a l a n d v a g u s n e r v e s ( l a t e r a l s o t h e f a c i a l ) w ith d ie n c e p h a lic c e n t e r s .

OBSERVATIONS ON THE GROWTH OF THE LONGITUDINAL TRACTS

IN

THE BRAIN OF THE CHICK EMBRYO« UP TO 6 Davs T h is d e s c r ip tio n o f th e b rain o f th e normal ch ick embryo i s lim it e d t o th e lo n g itu d in a l t r a c t s * and r e la te d n u c le i .

I t i s based on d escrip *

t i o n s by Mesdag ( 1 9 0 9 ), T e llo (1 9 2 3 ), and Windle and A ustin (1936) and supplem ented, e s p e c i a l l y f o r s ta g e s a f t e r 72 h ou rs, by o b se r v a tio n s on th e normal c o n t r o ls o f th e p r e se n t s e r ie s * The f i r s t n e u r o f ib r illa r d if f e r e n t ia t i o n in th e b rain o f th e normal ch ick embryo ta k e s p la c e a t about 16 so m ites (3 8 hours) in th e l a t e r a l w a ll o f th e rhombencephalon near th e l e v e l o f th e o t o c y s t s .

By 40-42

h o u r s, a second fo c u s o f development appears a t th e ju n c tio n o f m esence­ phalon and diencephalon* In th e 20 som ite embryo th e more r o s t r a l group o f n e u r o b la sts sends a few sh ort axons caudad in to th e upper m idbrain, and th e c e l l s o f th e rhombencephalon have begun t o form a v e n tr a l com m issure.

Thus, th e axons

o f th e d ie n c e p h a lic n u c le u s, though sh o r te r than th o se o f th e rhombence­ p h a lo n , form th e f i r s t lo n g itu d in a l f a s c i c l e in th e embryonic b r a in .

At

22 so m ites th e m edial lo n g itu d in a l f a s c ic u lu s has reached th e caudal end o f th e c e p h a lic f le x u r e , from th e commissure, and perhaps from d ir e c t f i b e r s a l s o , a lo n g itu d in a l t r a c t i s b eing formed in th e hindbrain* During th e n ex t few h o u rs, th e m edial lo n g itu d in a l f a s c ic u lu s le n g ­ t h e n s , and g rea t numbers o f growth cones are p resen t in th e t r a c t a t th e l e v e l o f th e upper m idbrain.

The fo c u s o f d i f f e r e n t i a t i o n in th e hind brain

h a s spread r o s t r a l l y and c a u d a lly , c o n tr ib u tin g sh ort axons t o a con tinu ous lo n g it u d in a l psbfe which merges w ith d escen d in g d ir e c t t e c t a l and d ien ce­ p h a lic f ib e r s in th e m idbrain and w ith lo n g itu d in a l in tern eu ro n s o f th e

25 c e r v ic a l co rd .

.

At th e oculom otor l e v e l , t e c t a l n e u r o b la sts have begun

t o form a sm all com missure. At 60 hours (37 so m ite s) an in c r e a s e in th e s iz e o f th e v e n tr a l lo n g itu d in a l f a s c ic u lu s o c c u r s.

Because o f overlap p in g f ib e r s at most

l e v e l s , th e ex a c t te rm in a tio n o f th e v a rio u s components can not be a sc e r ­ ta in ed *

In th e b a sa l p la te o f th e h in d b ra in , near th e s u lc u s lim it a n s ,

a l a t e r a l lo n g itu d in a l f a s c ic u lu s ap p ears. No r e c o g n iz a b le r e t ic u la r form ation becomes e v id e n t u n t i l th e 72 hour (37 so m ite) s ta g e .

T his i s formed in th e rhombencephalon by f in e

f i b e r s from l i g h t l y s ta in e d b a sa l p la te c e l l s .

Probably most o f th e s e

f i b e r s are h o m o la ter a l, but some c r o s s in th e commissure t o th e o p p o site r e t i c u l a r fo rm a tio n .

At t h i s s ta g e , more darkly sta in e d c e l l s near th e

s u lc u s lim it a n s a t th e tr ig e m in a l and f a c ia l* a c o u s t ic l e v e l s send t h e ir axons toward th e m edial lo n g itu d in a l f a s c ic u lu s and some of them e n te r th e d o r sa l p a rt o f th e com missure.

They form prominent bundles which run

p a r a l l e l t o th e tr ig e m in a l and f a c i a l motor axons co u rsin g from th e m edial motor n u c le i t o th e s u lc u s lim it a n s t o em erge.

E n terin g th e hom olateral

and c o n t r a la te r a l m edial lo n g itu d in a l f a s c i c l e s , th e s e axons of c e l l s form ing th e prim ordia o f tr ig e m in a l and v e s tib u la r secondary sensory n u c l e i are predom inantly a scen d in g .

Mesdag, however, b e lie v e s th a t a l l

components o f th e m edial lo n g itu d in a l f a s c ic u lu s in e a r ly s ta g e s are d esc en d in g . The upper p a rt o f th e lo n g itu d in a l path r e c e iv e s th alam o-tegm ental f i b e r s , e n te r in g th e m idbrain l a t e r a l t o th e d escen d in g m edial lo n g itu d i­ n a l fa s c ic u lu s .

These d ie n c e p h a lic f ib e r s become in d is tin g u is h a b le from

d ir e c t t e c t a l f i b e r s . The normal 4 day, 40 so m ite, embryo has a w e ll-d e v e lo p e d m edial lo n g i*

t u d in a l f a s c ic u lu s d escen d in g from th e d ien cep h a lo n . and t r o c h le a r n u c le i i t c o n s is t s of 180 f i b e r s .

At th e oculom otor

In t h i s r e g io n none of

th e f ib e r s are d eriv ed from th e sm all commissure formed by cro ssed t e c t a l a x o n s.

Caudal t o th e tr o c h le a r l e v e l th e t r a c t d im in ish es t o 120 f ib e r s

(T ab le 3 ) .

R o stra l t o th e tr ig e m in a l reg io n i t e n la r g e s t o 150 f ib e r s

( in d ic a t in g th a t i t co n ta in s components not d eriv ed from th e d ien c e p h a lo n ). At th e l e v e l o f th e tr ig e m in a l r o o t , a r e l a t i v e l y la r g e , d if f u s e t r a c t appears v e n tr a l t o th e compact m edial lo n g itu d in a l f a s c ic u lu s ( f i g . 2 5

).

The v e n tr a l commissure shows a g rea t in c r e a s e in s i z e , due p a r tly t o con­ t r ib u t io n s by a rcu a te f ib e r s from th e sen sory tr ig e m in a l n u c l e i .

At t h i s

s t a g e , many o f th e s e f ib e r s have not y e t reached th e com missure.

J u st

below th e tr ig e m in a l l e v e l , th e m edial lo n g itu d in a l f a s c ic u lu s i s la r g e but i t d oes n o t extend in to th e m yelencephalon, fo r in th e low er f a c i a l r e g io n , no compact d o r sa l t r a c t i s p r e s e n t.

The v e n tr a l lo n g itu d in a l

f a s c ic u lu s i s sm a lle r h e r e , as i s th e com missure. A s m a ll, d i f f u s e , lo n g itu d in a l t r a c t of th e r e t ic u la r form ation b e g in s t o appear at th e tr o c h le a r l e v e l , and i s composed o f uncrossed t e c ­ t a l f ib e r s sw inging mesad from th e l a t e r a l w a ll in to th e b a sa l p l a t e . R o«trn l t o th e tr ig e m in a l ro o t s e v e r a l sm all bundles com prise th e r e t ic u ­ l a r form ation and a few b a sa l p la te n eu ro b la sts appear.

A c e l l column

in th e a la r p la t e r e p r e s e n ts th e primordium o f th e cereb ellu m .

At th e

tr ig e m in a l l e v e l th e l a t e r a l part o f th e r e t ic u la r form ation , adjacen t t o th e l a t e r a l lo n g itu d in a l f a s c ic u lu s , i s formed by a f a i r l y compact t r a c t o f lig h t fib e r s .

The more m edial part o f th e r e t ic u la r form ation c o n ta in s

v e ry few lo n g itu d in a l f i b e r s , and seems alm ost blank as compared w ith the v e n tr a l and l a t e r a l lo n g itu d in a l f a s c i c l e s .

Even s o , th e f ib e r s in th e

r e t i c u l a r form ation a t t h i s l e v e l are more numerous than in th e f a c i a l

r e g io n and b elow .

At th e f a c i a l l e v e l , th e l a t e r a l t r a c t o f th e r e t ic u la r

fo rm a tio n has alm ost d isa p p ea red . 4 1 /2 DAYS In th e 4 1 /2 day embryo, a la r g e m edial lo n g itu d in a l f a s c ic u lu s i s p r e se n t a t th e oculom otor l e v e l .

Here th e d ie n c e p h a lic component i s d i s ­

t i n c t from th e cr o sse d t e c t a l t r a c t which i s form ing v e n tr a l t o it * growth cones can be observed in both t r a c t s .

Many

Im m ediately l a t e r a l t o th e

m ed ial lo n g itu d in a l f a s c ic u lu s i s a sm all group of dark f ib e r s which seem t o come from th e l a t e r a l d ie n c e p h a lic w a ll.

In i t a few c a u d a lly d ir e c te d

growth cones appear. In th e tr o c h le a r reg io n th e m edial lo n g itu d in a l f a s c ic u lu s c o n s is t s o f se v e r a l bundles spread l a t e r a l l y .

The r e t ic u la r form ation i s composed

o f many d iag o n a l b u n d les, formed by horaolateral t e c t a l f i b e r s . Below th e tr o c h le a r l e v e l th e m edial lo n g itu d in a l f a s c ic u lu s i s s t i l l spread out but th e r e are few er sm all l a t e r a l b u n d les.

Most o f th e f ib e r s

a re lo c a te d in narrow, p l a t e - l i k e bundles o f th e m edial p art of th e t r a c t . The commissure i s made up o f s e v e r a l f ib e r s which seem t o go t o th e m edial p a rt o f th e r e t ic u la r fo rm ation .

F ib ers form ing th e commissure ap p aren tly

a r e d eriv ed from th e an lage o f th e cereb ellu m .

J u st caudal t o t h i s l e v e l ,

a few f ib e r s o f th e v e n tr a l lo n g itu d in a l f a s c ic u lu s appear.

Many dark

f ib e r s are p resen t on th e p erip h ery o f th e bundles o f th e m edial lo n g i­ t u d in a l f a s c i c u l u s . R o stra l t o th e tr ig e m in a l r e g io n , th e sm all l a t e r a l bundles o f th e m ed ia l lo n g itu d in a l f a s c ic u lu s have jo in e d th e m edial group o f f i b e r s . The v e n tr a l lo n g itu d in a l f a s c ic u lu s has in crea sed in s iz e but i s much sm all than th e m edial lo n g itu d in a l f a s c ic u lu s .

F ib ers o f th e r e t ic u la r form ation

h e r e are much few er th an in th e m idbrain.

The l a t e r a l lo n g itu d in a l f a s ­

c ic u lu s i s form ing from f ib e r s which course a cr o ss th e reg io n o f th e s u l­ cu s lim ita n s t o ta k e a p o s it io n in th e l a t e r a l p art o f th e b a sa l p l a t e . At th e tr ig e m in a l l e v e l , th e m edial lo n g itu d in a l f a s c ic u lu s a t t a in s i t s g r e a t e s t s i z e and i s a w e ll-c ir c u m sc r ib e d bundle ( f i g . 23 ) .

F ib ers

from th e secondary sen so ry n u c le i e n te r th e t r a c t ; many o f them form a d a rk , d o r sa l p art o f th e com missure.

The v e n tr a l lo n g itu d in a l f a s c ic u lu s

i s d i f f u s e , but much la r g e r than a t more r o s t r a l l e v e l s .

Many n e u r o b la sts

have d i f f e r e n t ia t e d in th e b a sa l p la t e , and c o n tr ib u te t o th e r e t ic u la r form ation which i s very d if f u s e m e d ia lly , but more compact l a t e r a l l y . In th e a n g le formed by th e motor axons as th e y tu rn ventrad t o em erge, l i e s a prominent bundle, th e l a t e r a l lo n g itu d in a l f a s c i c u lu s . From t h i s p o in t t o th e f a c i a l- a c e u s t i c r e g io n , th e m edial lo n g it u d i­ n a l f a s c ic u lu s m ain tain s i t s s i z e , but fa r th e r c a u d a lly i t d ecrea ses r a p id ly , and a t th e g lo sso p h a ry n g ea l l e v e l i t i s n ot p r e s e n t.

The v e n tr a l lo n g it u ­

d in a l f a s c ic u lu s in c r e a s e s g r e a t ly through th e m etencephalon and becomes a l a r g e , t r ia n g u la r t r a c t in th e upper m yelencephalon.

The r e t ic u la r

form ation has d im in ish ed a t th e f a c i a l l e v e l , and ex cep t f o r th e l a t e r a l compact t r a c t , i t rem ains sm all through th e m yelencephalon.

The l a t e r a l

t r a c t o f th e r e t ic u la r form ation and th e s p in a l Vth t r a c t are c lo s e ly r e la t e d t o th e l a t e r a l lo n g itu d in a l f a s c ic u lu s , and t h e ir boundaries are n o t d i s t i n c t , in d ic a tin g th a t th e r e may be some exchange o f f ib e r s .

The

two secondary t r a c t s show a v a r ia b le s iz e in th e h in d b ra in , becoming q u ite d i f f u s e in th e vagus r e g io n . 5 DAYS At f i v e days th e cro ssed t e c t a l t r a c t appears v e n tr a l t o th e d ie n -

c e p h a lic m edial lo n g itu d in a l f a s c ic u lu s a t th e oculom otor l e v e l .

The

u n cro ssed t e c t a l t r a c t b eg in s i t s form ation t h e r e , but c o n tin u es t o e n te r t h e tegmentum u n t i l th e isthm us sep a ra tes th e tectum from th e b a sa l p art o f th e b r a in .

Through th e m idbrain, th e h om olateral t e c t a l t r s c t s h i f t s

ven trad in to th e b a sa l p l a t e .

The m edial lo n g itu d in a l f a s c ic u lu s , la r g e r

th an in th e p reced in g s t a g e , s t i l l exten d s from th e d ien cep h alon on ly t o th e g lo sso p h a ry n g ea l l e v e l . Throughout th e h in d b rain th e m edial lo n g itu d in a l f a s c ic u lu s i s a m e d ia lly lo c a t e d , compact t r a c t . more r o s t r a l l y .

I t i s la r g e r a t th e f a c i a l l e v e l than

In th e tr ig e m in a l and f a c i a l r e g io n s , sen so ry n u c le i ,

a r c u a te f ib e r s and th e dark, d o r sa l part o f th e commissure have in crea sed ( f i g . 28 and 35 ) .

R o str a l t o th e tr ig e m in a l reg io n th e t r a c t becomes

spread out l a t e r a l l y in s e v e r a l b u n d les.

At th e upper tr o c h le a r l e v e l

th e l a t e r a l groups o f f ib e r s have d isap p eared , but j u s t r o s t r a l t o t h i s , f i b e r s branch o f f th e t r a c t l a t e r a l l y and form a t h in row of f ib e r s con­ t a in in g r o s t r a l l y d ir e c te d growth c o n e s.

A few c a u d a lly growing f ib e r s

from a t r a c t r o s t r a l t o th e n u cleu s o f m edial lo n g itu d in a l f a s c ic u lu s m ingle w ith t h e ascen d in g axon s. A la r g e but d if f u s e v e n tr a l lo n g itu d in a l f a s c ic u lu s i s encountered in th e m idbrain and h in d b ra in .

The r e t ic u la r form ation has in crea sed

g r e a t ly d uring th e 1 /2 day sin c e th e p reviou s s t a g e .

In th e m etencephalon,

r o s t r a l t o th e tr ig e m in a l r e g io n , b a sa l p la te c e l l s are numerous, and c o n tr ib u te t o th e r e t ic u la r form ation and perhaps t o th e commissure.

Axons

from th e c e r e b e lla r an lage are d ir e c te d vent r a l ly and many o f them probably e n ter

th e commissure at t h i s l e v e l .

At th e tr ig e m in a l l e v e l n e u r o b la sts

o f th e r e t ic u la r form ation are e v e n ly d is t r ib u te d in th e b a sa l p la t e , where g r ea t numbers o f them appear in th e m antle la y e r .

T heir axons are

d ir e c te d v e n t r a lly in to th e h om olateral r e t ic u la r form ation , or d ia g o ­ n a l l y through th e commissure t o th e r e t ic u la r form ation o f th e o p p o site s id e .

They f i l l th e m arginal la y e r o f th e b a sa l p la t e w ith d if f u s e f ib e r s

m e d ia lly and a r a th e r compact t r a c t l a t e r a l l y .

At th e sev en th l e v e l ,

f i b e r s o f th e r e t i c u l a r form ation are much l e s s numerous, e s p e c ia lly in th e m ed ial p a r t.

Through th e m yelencephalon th e y g ra d u a lly d im in ish .

J u s t v e n tr a l t o th e s u lc u s lim it a n s , th e l a t e r a l lo n g itu d in a l f a s c ic u lu s i s la r g e and com pact.

T his t r a c t i s very prominent throughout th e hind­

b r a in , alth ou gh i t v a r ie s somewhat at d if f e r e n t l e v e l s , and in th e g lo s s o ­ pharyngeal r e g io n , th e f ib e r s o f th e s p in a l Vth t r a c t m ingle w ith i t , and i t i s im p o ssib le t o say how g rea t a p rop ortion o f th e f ib e r s h e r e , are o f g a n g lio n ic o r ig in .

The v e n tr a l commissure at a l l l e v e l s shows a g rea t

in c r e a s e , corresp on d in g t o th a t o f lo n g itu d in a l t r a c t s end to th e rapid d i f f e r e n t i a t i o n o f c e l l u l a r elem en ts. In th e 5 1 /2 day norm al, f ib e r s appear th in n e r , and t r a c t s ta k e up a r e l a t i v e l y sm a ller amount o f space in th e b r a in .

The row o f ascending

f ib e r s noted in th e upper m idbrain at 5 days has reached th e l e v e l o f th e i n t e r s t i t i a l n u c le u s.

The m edial lo n g itu d in a l f a s c ic u lu s i s la r g e s t at

th e tr ig e m in a l and f e c i a l l e v e l s ( f i g . 34 ) , g rad u ally d im in ish in g below t h a t , but a sm all compact t r a c t , d o r sa l t o th e v e n tr a l lo n g itu d in a l f a s c i ­ c u lu s , i s p r esen t in th e upper vagus r e g io n . The cr o ssed t e c t a l t r a c t i s la r g e r , and at l e a s t some of i t s f ib e r s occupy th e v e n tr a l lo n g itu d in a l f a s c ic u lu s which now exten d s from th e oculom otor l e v e l cau d ally*

J u st above th e tr ig e m in a l r o o ts th e m edial

lo n g itu d in a l f a s c ic u lu s becomes reduced in s i z e .

At th e caudal tr ig e m in a l

l e v e l i t in c r e a s e s but i s co n cen trated near th e commissure, in c o n tr a st t o th e l a t e r a l l y d is t r ib u te d bundles in th e m idbrain.

The r e t i c u l a r form ation hag in creased at a l l l e v e l s but i s d if f u s e and e v e n ly d is t r ib u te d through th e m idbrain.

At th e l e v e l o f th e c e r e ­

b e l l a r a n la g e , a row o f f ib e r bundles d iv id e s th e r e t ic u la r form ation in to m ed ia l and l a t e r a l h a lv e s .

A l i t t l e more c a u d a lly , another row o f sim i­

l a r appearing bundles appears l a t e r a l t o th e f i r s t .

The l a t t e r group

has moved mesad from th e re g io n o f th e m esen cep halic Vth t r a c t .

The

r o s t r s l e x te n t o f th e compact t r a c t o f th e r e t ic u la r form ation i s a t th e tr ig e m in a l l e v e l . p e r ip h e r y .

Here th e l a t e r a l t r a c t l i e s at some d ista n c e from the

The r e t ic u la r fo rm ation , s t i l l th e most d if f u s e o f th e lo n ­

g it u d in a l t r a c t s , has in crea sed g r e a t ly , as have b a sa l p la te c e l l s . The l a t e r a l lo n g itu d in a l f a s c ic u lu s i s more d i f f i c u l t t o f o llo w at t h i s s t a g e , f o r i t i s in term in gled w ith th e s p in a l Vth t r a c t .

Between

t h e tr ig e m in a l and f a c i a l l e v e l s i t l i e s deep t o th e primary t r a c t .

In

th e m yelencephalon i t appears on th e periphery* 6 DAY NORMAL With some in c r e a se in s i z e , th e 6 day embryo has e s s e n t i a l l y th e same appearance as th a t o f 5 1 /2 d a y s.

In th e ascend in g t r a c t near th e

oculom otor n u cleu s many growth con es appear.

The r e t ic u la r form ation

s t i l l i s l e a s t w e ll developed a t th e f a c i a l l e v e l .

In th e glossoph aryn ­

g e a l reg io n i t appears blank excep t fo r th e deep l a t e r a l t r a c t . vagus l e v e l , a p e r ip h e r a l l a t e r a l t r a c t appears a ls o .

At th e

32

EFFECTS OF SURGICAL REMOVAL OF PARTS OF THE BRAIN OF THE CHICK EMBRYO UPON DEVELOPMENT OF THE BRAIN Method and M a teria l S im ila r methods o f o p er a tio n have been used by v a rio u s authors* The w r itin g s o f Jones (1 9 3 7 ) and Hamburger (1934* 1938) have been very u s e f u l as a b a s is upon which t o develop an o p e r a tiv e p rocedu re.

The

method used by Fugo (1940) t o remove th e fo r e b rain served as a very h e lp fu l guide in perform ing s im ila r o p e r a tio n s fo r th e p resen t work* The method o f o p era tio n as used in th e p resen t experim ents i s as fo llo w s s

Eggs were incubated in a h o r iz o n ta l p o s it io n a t 38-39° C.

f o r 38-40 hours*

They were th en candled and marked, a f t e r which th e y

were returned t o th e incubator*

Through a h o le made in th e sm all end

o f th e egg» about 2 cc* o f albumin was drawn o f f w ith a s y r in g e . was d isca rd ed and th e h o le plugged w ith wax*

T h is

The marked area was wiped

o f f w ith a lc o h o l, and a window 1 cm* or more in diam eter was made in th e s h e l l 9 u sin g a d e n ta l d r i l l equipped w ith an emery d i s c .

The s h e ll

membrane was cu t through and th e sawed-out s h e l l l i f t e d o f f and d is c a r ­ ded*

The o p er a tio n was performed on a warming t a b le w ith th e a id o f

a d i s s e c t in g m icroscope and a lamp provided w ith a g la s s which prevented t h e beam o f l i g h t from becoming hot*

In most c a s e s a f i l t e r o f b lu e

o r green g la s s was p la ced in fr o n t o f th e l i g h t , t o aid in v i s u a liz in g t h e o u t lin e o f th e embryo*

U su ally no s o lu t io n was p laced on th e embryo,

a lth o u g h in a few c a s e s in which th e membrane seemed t o be drying out L o c k 's, or R in g e r 's , s o lu t io n was u sed . The v i t e l l i n e membrane was picked up w ith a n eed le and s p l i t over

“th e area o f op eration *

In c a s e s where " d eca p ita tio n " was th e o b jec­

t i v e , a h a ir loop run through a blu nted hypodermic n ee d le so th a t i t cou ld be drawn up in to th e n e e d le , was employed*

A fte r th e loop was

slip p e d around th e f r e e end o f th e head, i t was p laced at th e d e s ir e d l e v e l and c lo s e d , th u s c o n s t r ic t in g o f f th a t p art of th e head ly in g r o s tr a l to it*

In t h i s manner th e fo r e b r a in , fo re-a n d m idbrain, or f o r e -

mid-and upper h in d b rain were removed*

T h is ty p e o f o p era tio n was most

s u c c e s s f u l when performed w h ile th e embryo was s t i l l s tr a ig h t but had a c o n sid e r a b le p art o f i t s head fr e e *

At t h i s sta g e th e th r e e primary

brain v e s i c l e s are c le a r ly marked, and serv e as landmarks f o r l o c a l i z i n g th e op eration*

The removed p art was l i f t e d out and discard ed *

In oth er

c a s e s , s e c tio n s o f th e n eu ral tube were removed, le a v in g more r o s t r a l and caudal p a rts*

L ev els o f o p era tio n were a t th e o t o c y s t , th e t r i g e ­

m inal r e g io n , and th e midbrain*

Attem pts were a ls o made t o e x c is e th e

c e p h a lic n eu ra l c r e s t c o n ta in in g th e primordium o f th e sem ilunar gang­ l i o n , but t h e s e were not w h olly s u c c e ss fu l*

E f f o r ts t o e lim in a te one

l a t e r a l h a l f o f th e n eu ral tube r e s u lte d in such d is t o r t io n th a t th e specim en was n ot s u it a b le fo r study* A fte r th e o p era tio n th e opening in th e on

a p ie c e o f g la s s w ith beeswax*

s h e l l was c lo s e d by s e a lin g

In cub ation was con tin u ed u n t i l th e

d e s ir e d age f o r f ix a t io n was reach ed .

A ll m a te r ia l was f ix e d in ammo-

n ia te d a lc o h o l and sta in e d by th e p y rid in e s i l v e r method (D avenport, W indle, Beech, 1934) and se c tio n e d a t 12 m icra. By t h i s method a s e r i e s o f embryos was obtained which rep resen ted 4 to

5 1 /2 or 6 day embryos of each ty p e o f operation*

th e r e were d u p lic a t e s .

In many c a se s

A s e r ie s o f normal ch ick embryos of correspond­

in g ages was a lso prepared.

The m a te r ia l used i s shown in Table 2 .

The operated embryos were compared w ith normal c o n t r o ls t o e s t i ­ mate f ib e r r ed u c tio n in lo n g itu d in a l t r a c t s *

The f ib e r s o f th e m edial

lo n g itu d in a l f a s c ic u lu s were counted in 4 day specim en s, but in o ld e r s t a g e s , a ccu ra te enum eration o f f ib e r s was d ou btfu l*

Comparisons were

checked upon by m easuring th e diam eter o f th e m edial lo n g itu d in a l f a s ­ c ic u lu s w ith an o cu la r micrometer*

In embryos s e c tio n e d f r o n t a l l y , th e

w idth o f th e t r a c t was m easured, and th e number o f s e c t io n s in which i t appeared, was counted*

In t h i s manner, a rough estim a te o f th e s iz e

o f th e m ed ial lo n g itu d in a l f a s c ic u lu s was made* en ces in s iz e can n ot be determ ined in t h i s way*

However, sm all d i f f e r ­

35.

THE EFFECT OF OPERATIONS UPON THE SHAPE OF THE BRAIN AND ORIENTATION OF FIBERS A fte r removal o f a p a rt o f th e b r a in , th e exp erim en tally-p rod u ced te r m in a tio n o f th e n eu ra l tu be h ealed over*

No scar* or oth er evid en ce

o f trauma* t o mark th e p o in t o f tr a n a e c tio n o f th e n eu ral tu be was seen* a lth o u g h th e surrounding mesenchyme o fte n showed s ig n s o f hemorr­ hage*

In many c a s e s th e t i p o f th e b rain bent vent r a lly *

In "midbrain "

specim ens th e oculom otor n u cleu s o fte n appeared t o l i e in a pocket of t h e f lo o r p la te *

The ty p e o f c lo su r e v a ried somewhat* accord in g t o th e

l e v e l o f operation*

I f th e b rain ended in th e p r e t e c t a l r e g io n , or th e

r o s t r a l end o f th e midbrain* th e edge o f th e tectum merged w ith th e b a sa l p a rt o f th e n eu ra l tube* and l i t t l e departure from th e normal shape o f th e b r a in , or u su a l arrangement o f f ib e r s was noted*

In one

e x c e p tio n a l c a s e , developm ent o f th e tectum was suppressed and th e midbrain* th ic k w a lle d and sm all in diam eter* ta p ered and formed a rounded en d in g tr a v e r se d by ascen d in g fib e r s *

This brain ap p aren tly had been

t w is t e d during o p e r a tio n , so th a t i t s l a t e r a l w a lls were lo c a te d dors a l l y and vent r a lly * A rounded ending was t y p ic a l o f th e rhombencephalon*

In capping

over th e ending o f th e b r a in , th e th r e e la y e r s r e ta in e d t h e ir normal r e la tio n s h ip s *

The ependyma lin e d th e v e n t r ic le * and th e m arginal la y e r

was outerm ost*

The term in a tio n caused th e f lo o r p la te * ro o f p la te and

su lc u s lim ita n s t o be a b olish ed * r e s u lt in g in a h o llo w , dome-shaped s tr u c t u r e .

However* a short d ista n c e from th e end, th e p la t e s form ing

th e n eu ra l tube became apparent, and th e b rain assumed i t s normal shape* but i t u s u a lly was d im inished in s i z e .

Near th e end o f th e b rain th e

v e n t r i c l e u s u a lly was lo c a te d d o r s a lly as in th e norm al, but was covered by a th ic k roof# At th e r o s t r a l end o f th e b r a in , a lt e r a t io n in shape was accom­ p an ied by abnormal o r ie n t a tio n o f fib e r s #

N eu ro b la sts o f th a t part o f

th e m antle la y e r p a r t ic ip a t in g in capping over th e n eu ral tube sen t t h e i r ax o n s, ventrad and caudad#

F ib e r s o r ig in a tin g near th e m id lin e

cou rsed d i r e c t l y caudad u n t i l th e brain became normal in shape and a t y p ic a l commissure appeared, and th en en tered th e b a sa l p la te w ith ­ out c r o s s in g each other#

P r o c e sse s a r is in g more l a t e r a l l y became

c o n t r a la t e r a l and to o k a d ir e c t cou rse t o e n te r th e v e n tr a l lo n g itu ­ d in a l f a s c ic u lu s or r e t ic u la r form ation o f th e o p p o site s id e , w ith out cu rv in g t o become t r a n s v e r s e , as do f ib e r s in a normal commissure. In th e ventrom edian p art o f th e n eu ral tu b e , t h i s r e s u lte d in a r a d ia l arrangement o f f i b e r s , resem b ling th e spokes o f a w heel ( f i g 47 )• Caudal t o th e l e v e l at which a normal f lo o r p la te appeared, th e commis­ sure g r a d u a lly took on a normal appearance# A scending f ib e r s fo llo w ed th e contour o f th e n eu ral tube end t r a c t s o f each s id e became tr a n sv e r s e a t th e en d in g, sometimes c r o s s ­ in g each o th e r .

In th e o ld er s ta g e s stu d ied here (5 1 /2 days end

more) many o f th e s e f ib e r s emerged from th e end o f th e b ra in end wandered in th e mesenchyme or went t o th e ectoderm near by.

Often

th e y term in ated alon g th e in s id e o f th e ectoderm , but in one embryo o f 6 days a la r g e bundle composed o f f ib e r s from ascen ding sensory t r a c t s and th e m ed ial lo n g itu d in a l f a s c i c l e s p en etra ted th e ectoderm and formed a ta n g le d mass on i t s o u ter su rfa ce ( f i g . 38 ) .

in fron ­

t a l l y s e c tio n e d embryos o f t h i s ty p e , th e m edial lo n g itu d in a l f a s c i -

37.

c u iu s cou ld be e a s i l y d is tin g u is h e d from th e v e n tr a l lo n g itu d in a l f a s c i c u l u s and th e r e t ic u la r form ation*

Many f ib e r s o f th e r e t ic u l a r

form ation were a sce n d in g , but ap p aren tly were sh o r te r than th o se o f t h e m edial lo n g itu d in a l f a s c ic u lu s or sen sory t r a c t s *

No ascen ding

f ib e r s were found in th e l a t e r a l lo n g itu d in a l f a s c ic u lu s but t h i s t r a c t was somewhat obscured by th e compact sen sory t r a c t s .

i n tr a n s ­

v e r s e s e c t io n th e d is p o s it io n o f f ib e r s was more d i f f i c u l t t o d eterm in e, f o r th e y were cu t d ia g o n a lly as th e y curved t o c r o s s th e end o f th e b rain ( f i g . 46 ) . When a gap e x is t e d in th e h in d b rain , th e caudal end o f th e upper p a r t o f th e brain was arranged in a s im ila r manner t o th e one j u s t d escrib ed *

In t h e s e c a s e s , c a u d a lly co u rsin g f ib e r s cro ssed th e roun­

ded te r m in a tio n and in o ld e r sta g e s some f ib e r s emerged from th e neu­ r a l tube*

The low er s e c t io n , however, tended t o resem ble th e normal

i n sh ap e, but was elo n g a ted d o rso -v eirtra lly *

Near th e s i t e o f opera­

t i o n , n e ith e r lo n g itu d in a l t r a c t s , nor motor n u c le i, cou ld be id e n t i­ fie d * Endings r e s u lt in g from d is c o n t in u ity a t or near th e m idbrain were l e s s c o n s is te n t*

S ep aration o f th e m esencephalon from th e meten-

cep h alon had l i t t l e e f f e c t on th e h in d b ra in , but th e m esencephalon was very sm all and ra th er f la t t e n e d , w ithout any d if f e r e n t ia t io n o f p l a t e s or motor n u c le i*

No t y p ic a l tectum was p resen t*

L on gitu d in al

f i b e r s seemed t o s p ir a l around th e n eu ral tube and no s p e c if i c t r a c t s c o u ld be id e n t if ie d * In one ca se o f se p a r a tio n o f th e oculom otor and tr o c h le a r r e g io n s , t h e d ien cep h a lo n was n e a r ly normal in shape but near th e ending i t was d is to r te d *

The n u c le i o f th e m edial lo n g itu d in a l f a s c i c l e s appeared

t o be pushed t o g e t h e r , but a r ig h t and l e f t t r a c t d ev elo p ed , and emerged in to th e mesenchyme*

The f a s c i c l e on th e r ig h t turned r o s­

t r a l l y and fo llo w e d th e l a t e r a l w a ll o f th e d ien cep h a lo n , ending a f t e r a sh o r t d is t a n c e , whereas th a t o f th e l e f t s id e coursed caudad in a d i r e c t l i n e t o th e low er p o r tio n o f th e b r a in , which i t en tered on th e r ig h t s id e j u s t caudal t o th e tr o c h le a r n ucleus*

(The tr o c h le a r nerve

emerged v e n t r a l ly , and w i l l be d isc u s se d in a l a t e r s e c tio n o f t h i s p aper*)

The shape o f th e brain a t th e e x trem ity o f th e low er p art

was f a i r l y norm al, excep t th a t th e development of one s id e was some­ what suppressed*

T h is probably was due t o l o s s o f t is s u e *

Another

embryo w ith a gap in th e m idbrain was very abnormal on both s id e s o f th e o p e r a tio n , but th e r e was evid en ce o f some m aceration , e s p e c ia lly in th e upper part*

H ere, only one oculom otor n u cleu s was p r e s e n t, and

a sm all lo n g itu d in a l t r a c t which went through th e mesenchyme t o reach th e low er p a rt was not as c l e a r ly i d e n t if i a b le as t h a t in th e preced­ in g d e s c r ip tio n * and amorphous*

The low er dienceph aloh and upper midbrain were to rn The caudal p art o f th e mid brain was sm all and rounded,

but th e m etencephalon appeared normal in shape. O ften , abnormal emergence o f f ib e r s took p la c e , .some d is ta n c e ca u d a l t o th e end o f th e b r a in .

There seemed to be no p a r tic u la r

s i t e o f e x i t , but th e f ib e r s alumys emerged v e n t r a lly .

In a 5 day

" fa c ia l" embryo a la r g e trunk formed at th e m iddle of th e f lo o r p la te in th e Xth r e g io n (400 m icra from th e end o f th e b r a in ).

I t was com­

posed o f eoramissural f ib e r s which reached th e f lo o r p la te and th en tu rn ed v e n t r a lly , form ing a fu n n el shaped "commissure".

Many of th e s e

f i b e r s emerged, and th e trunk so formed coursed ven tra d , c lo s e t o th e b a s i l a r a r te r y and on t o th e notochord which was very sm all here*

From t h i s p o in t , a few f ib e r s t r a v e lle d toward th e fo reg u t and ended near i t s w a ll.

A common abducens tru n k , formed from s e v e r a l b i l a t e r a l

r o o t l e t s r o s t r a l t o th e emergence o f th e commissural f i b e r s , fo llo w ed a s im ila r c o u r se , growing ro stra d and ventrad alm ost t o th e gut*

These

tru n k s go in an o b liq u e d ir e c t io n , and reach r o s t r a l ly about 300 m icra from t h e i r p o in t o f emergence* In a 4 day "rhombencephalic" embryo a few f ib e r s emerged in th e r e g io n between th e tr ig e m in a l and f a c i a l roots* toward th e notochord and r o s t r a l l y .

These a ls o coursed

The f lo o r p la t e at and near th e

p o in t o f e x i t was drawn out in t o a p o in t toward th e n otoch ord . In a 5 day embryo in which th e n eu ral tu be was sep arated a t th e o to c y s t l e v e l , two bundles l e f t th e la t e r o - v e n t r a l a sp ect o f th e neu­ r a l tube and t r a v e lle d ro stra d in th e shrinkage space along th e e x te r ­ n a l lim it in g membrane*

The la r g e r o f th e s e t r a c t s reached r o s t r a l ly

f o r about 400 m ic ra , g ra d u a lly ending in growth cones*

The sm a ller

t r a c t ends s im ila r ly but i s much shorter* DISCUSSION F req u en tly th e notochord was absent near th e a r t i f i c i a l end o f th e n e u r a l tu b e in operated embryos. t o th e shape o f th e b r a in .

This seemed t o have no r e la t io n s h ip

Some b rain s recovered t h e ir normal shape

b e fo r e th e notochord appeared, whereas o th e r s , having a notochord ven­ t r a l t o them , were very d is t o r t e d .

In some specim ens th e p resence of

th e b a s ila r a r te r y seemed to be c o r r e la te d to th e form ation o f th e f lo o r p l a t e , but t h i s was not c o n s is te n t*

Lehmann ( 1926) and H o ltf r e t e r ( 1932$ >

by producing a d e f e c t , or p rev en tin g th e development of th e notochord in amphibian embryos, found a c o r r e la t io n between th e p resen ce o f th a t

s tr u c t u r e and a norm ally shaped s p in a l cord*

I t was observed th a t i f

m uscle rep la ced th e n otoch ord , v e n tr a l t o th e n eu ra l tu b e , th e c e n tr a l ca n a l became s it u a t e d d o r s& lly , and th e sp in a l cord was round*

Some o f

t h e n eu ra l tu b e s in operated embryos d escrib ed here had a very s im ila r appearance, but s in c e th e r eg io n under c o n s id e r a tio n i s b ra in , ra th er than s p in a l co rd , a d o r s a lly p la ced v e n t r ic le was probably due t o th e normal ten d en cy t o form a t h in roof*

The f lo o r p la t e and b a sa l p la te were ab­

n orm ally t h ic k , but no m uscle t i s s u e was p resen t v e n tr a l t o th e brain in th e median lin e * The arrangement o f f ib e r s at th e end o f th e brain conformed t o a c e r t a in p a ttern *

At th e r o s t r a l m idbrain, in most c a s e s , th ere was l i t t l e

d ep artu re from th e normal*

In th e h in d -b r a in , lo n g itu d in a l t r a c t s curved

m e d ia lly , fo llo w in g th e contour o f th e b ra in , and in o ld er embryos, a f t e r reach in g th e m id lan e, th e f ib e r s continued r o s tr a d , emerging from th e n eu ra l tu b e .

These o b se rv a tio n s su g g est th a t f ib e r s f o llo w th e alignm ent

o f th e substratum , but th a t oth er f a c to r s a lso in flu e n c e t h e ir d ir e c t io n . Tn th e s e ex p erim en ts, i t i s n ot th e a t t r a c t io n o f two d evelop in g c e n te r s fo r one another th a t cau ses ascen ding f ib e r s t o grow r o s tr a d , as su ggested by W eiss ( 193-$, fo r in th e absence o f any more r o s t r a lly lo c a te d fo cu s o f d i f f e r e n t i a t i o n , th ey cou rse t o th e end of th e b ra in , le a v e i t and go t o th e ectoderm . A commissure resem b lin g an a s t r a l fig u r e was q u ite r e g u la r ly found in th e hind b r a in , at the en ding.

The very d ir e c t , ob liq u e course taken

by f ib e r s o f l o c a l o r ig in was in s t r ik in g c o n tr a st t o th e curved course o f commissural f ib e r s an normal embryos.

The very th ic k f lo o r p la te and

b a sa l p la te may have had some in flu e n c e , p erm ittin g f ib e r s t o tak e a more d i r e c t c o u r se .

The p o s s i b i l i t y th a t th e rounding up o f th e end o f th e

b ra in may have s e t up new l i n e s o f s t r e s s should be co n sid e r e d .

ALTERATIONS IN THE MEDIAL LONGITUDINAL FASCICULUS

AND OTHER LONGITUDINAL TRACTS. CAUSED BY ABSENCE OF VARIOUS NUCLEI OF THE BRAIN When compared w ith normal c o n t r o ls , operated embryos sometimes showed a s l i g h t r e ta r d a tio n in gen eral development and n e u r o f ib r illa r d i f f e r e n t i a t i o n , but th e y u s u a lly overcame t h i s la g a f t e r f i v e d ays. R eta rd a tio n was not c o n s is t e n t ly found, however.

When p r e s e n t, i t

occurred in embryos w ith a la r g e amount o f b rain m is s in g . EMBRYOS WITHOUT A FOREBRAIN In one 4-day operated embryo, th e oculom otor reg io n marked th e r o s tr a lm o st l i m i t o f th e b r a in . behind th a t o f th e co n tro l*

N e u r o f ib r illa r d if f e r e n t ia t io n was a l i t t l e N e v e r th e le s s , th e only s t r ik in g d iffe r e n c e

between i t and th e c o n tr o l was found in th e mid b r a in .

At th e oculom otor

l e v e l th e r e was a sm all commissure d erived from t e c t a l n e u r o b la s ts , but th e v e n tr a l lo n g itu d in a l lo n g itu d in a l f a s c ic u lu s was e n t ir e ly a b se n t. At th e l e v e l o f th e tr o c h le a r n u c le u s, a t r a c t of 5 f ib e r s appeared j u s t l a t e r a l t o th e f lo o r p l a t e .

I t in crea sed r a p id ly as i t was tra ced

caudalw ard, c o n s is t in g o f 30 f ib e r s r o s t r a l t o th e tr ig e m in a l r e g io n , and a t th e l e v e l o f th e tr ig e m in a l motor ro o t ( f i g .24 ) , i t t o t a l l e d 125 f ib e r s (T ab le 3 ) .

At th e f a c i a l l e v e l , no d iff e r e n c e between th e

normal and operated embryos was d e te c te d .

The r e t ic u la r form ation and

l a t e r a l lo n g itu d in a l f a s c is u lu s were s im ila r t o th e normal. Another 4-day "midbrain" embryo, s l i g h t l y more advanced in d evelop ­ ment than th e one j u s t d e sc r ib e d , had la r g e r t e c t a l t r a c t s at th e l e v e l o f th e oculom otor and tr o c h le a r n u c le i.

The cro ssed t e c t a l t r a c t was

a s m a ll, sh o rt bundle lim ite d t o th e oculom otor l e v e l .

The uncrossed

t e c t a l t r a c t was more prom inent, and th e m edial p art o f i t coursed mesad and caudad and ended a t th e r o s t r a l end o f th e tr o c h le a r nucleus*

At

th e caudal end o f th a t b u n d le, th e most r o s t r a l f ib e r s o f th e ascending m ed ial lo n g itu d in a l f a s c ic u lu s were ob served. and th e t r a c t r a p id ly in c r e a s e d . l e v e l 70 f ib e r s were p r e s e n t.

Four f ib e r s appeared here

Some d ista n c e above th e tr ig e m in a l

Below t h i s p o in t , th e s e c tio n s became

o b liq u e and no accu ra te cou n ts could be made, but i t was ev id e n t th a t th e in c r e a s e co n tin u ed .

The o b liq u it y of th e s e c tio n s afford ed an

o p p o rtu n ity t o observe r o s t r a ll y d ir e c te d growth cones in th e m edial lo n g it u d in a l f a s c i c u l u s . In a 4 1 /2 day embryo in which th e d ie n c e p h a lic m edial lo n g itu d in a l f a s i c u l u s was a b se n t, th e cro ssed t e c t a l t r a c t formed a la r g e bundle l a t e r a l t o th e oculom otor n u c le u s.

The m edial lo n g itu d in a l f a s c ic u lu s

was la r g e r in th e tr o c h le a r reg io n than above or below . tr ig e m in a l r e g io n , la r g e dark f ib e r s appeared on l a t e r a l s u r fa c e s .

R o str a l t o th e

i t s d o r sa l and

At t h i s l e v e l a few sm all bundles appeared l a t e r a l

t o th e main group o f f i b e r s .

At th e tr ig e m in a l reg io n and more ca u d a lly

th e p lan e o f s e c tio n o f t h i s embryo was o b liq u e , but a la r g e m edial lo n g itu d in a l f a s c ic u lu s was p resen t as f a r as th e f a c i a l le v e l*

There

was no v e n tr a l lo n g itu d in a l f a s c ic u lu s in th e tr o c h le a r r e g io n , but j u s t caudal t o t h i s l e v e l , s e v e r a l growth cones appeared on th e p e r i­ p h ery, l a t e r a l t o th e f lo o r p la t e .

C au d ally, th e r e was an in c r e a se in

t h i s t r a c t , due p a r t ly t o an in crea se in ascend ing f i b e r s , and p a r tly t o m edial s h i f t i n g o f h om olateral t e c t a l f i b e r s .

A more l a t e r a l d i v i ­

s io n o f th e u n crossed t e c t a l t r a c t occupied th e r e t ic u la r form ation .

In th e absence o f d ie n c e p h a lic f i b e r s , th e t e c t a l t r a c t s a t 5 d a y s, co u ld be e a s i l y i d e n t i f i e d in th e m edial lo n g itu d in a l f a s c ic u lu s and th e v e n tr a l lo n g itu d in a l f a s c ic u lu s o f th e m esencephalon.

The cro ssed

t e c t a l f ib e r s ended j u s t caudal t o th e tr o c h le a r n u c le u s.

In t h i s reg io n

th e m ed ial lo n g itu d in a l f a s c ic u lu s in c r e a s e d .

The h om olateral t e c t a l

f i b e r s extended fa r th e r c a u d a lly and became in d is tin g u is h a b le from oth er f ib e r s o f th e v e n tr a l lo n g itu d in a l f a s c ic u lu s and th e r e t ic u la r form ation . Ascending f ib e r s form ing th e l a t e r a l bundles o f th e m edial lo n g i­ tu d in a l f a s c ic u lu s en tered th e m idbrain at 5 d a y s.

They were r e a d ily

t r a c e d , f o r th e y veered la te r a d v e n tr a l t o th e tr o c h le a r n ucleus and ended in th e l a t e r a l p art o f th e b a sa l p la t e . At th e tr ig e m in a l l e v e l , th e m edial lo n g itu d in a l f a s c ic u lu s formed a m e d ia lly lo c a t e d , compact bundle ( f i g . 2 9 ) , which did n ot vary s ig n i­ f i c a n t l y in s iz e from th e norm al, f o r i t was a t i g h t l y packed group o f f i b e r s , whereas in th e normal i t was a l i t t l e more d i f f u s e , though w e llcircu m scrib ed ( f i g . 28 ) .

As in th e c o n t r o l, th e m edial lo n g itu d in a l

f a s c ic u lu s in crea sed toward th e f a c i a l l e v e l , and g ra d u a lly disappeared in th e m yelencephalon. There seemed t o be no red u ctio n o f f ib e r s in th e r e t ic u la r forma­ t i o n or th e l a t e r a l lo n g itu d in a l f a s c ic u l u s , but th e f ib e r s in th e op erated embryo ten d ed t o clump to g e th e r and p resen ted a l e s s o rd erly appearance th a n in th e norm al. In a “midbrain" specim en alm ost 5 1 /2 days o ld , no t y p ic a l tectum was p r e s e n t.

The w a lls o f th e m idbrain were t h ic k , and a t th e ocu lo­

m otor l e v e l th ey formed a rounded ending a cr o ss which many f ib e r s cou rsed . T h ese, and o th ers which l e f t th e m idbrain in la r g e bundles below t h is p o i n t , were a scen d in g .

They formed conspicuous lo n g itu d in a l t r a c t s ,

re sem b lin g th e r e t i c u l a r fo rm a tio n .

Probably d escen d in g f ib e r s o rig i-v

n a tin g in th e tegmentum were a ls o p r e se n t in t h i s a rea .

The oculom otor

n u c l e i were very a sym m etrical, th e one on th e l e f t bein g much la r g e r b ecau se th a t s id e o f th e b rain was turned v e n t r a lly .

Between i t and th e

s m a lle r , d is t o r t e d , n u cleu s o f th e r ig h t s id e , many tr a n sv e r s e f ib e r s co u rsed , but th ey d id not have th e appearance o f a tr u e commissure, and probably were some o f th e ascen d in g f i b e r s .

The only m edial lo n g i­

tu d in a l f a s c ic u lu s in th e m ldbrain was com prised o f s e v e r a l sm all dark b u n d le s, th e f ib e r s o f which sep arated near th e end o f th e brain and co u rsed t o th e p erip h ery t o j o in th e more d if f u s e f i b e r s .

Above th e

tr ig e m in a l r e g io n , where th e s e c t io n s have become o b liq u e , th e m edial lo n g it u d in a l f a s c ic u lu s was spread l a t e r a l l y .

At th e l e v e l o f th e t r i ­

gem inal r o o t , th e s c a tte r e d bundles j o in to g e th e r t o form a t y p ic a l m ed ia l lo n g itu d in a l f a s c ic u lu s (fig J 3 9

).

Here hom olateral and commi-

s u r a l ascen d in g f ib e r s c o n tr ib u te t o th e m edial lo n g itu d in a l f a s c ic u l u s . No specim ens o ld e r th an 5 1 /2 days had th e oculom otor n u cleu s as t h e ir r o str a l le v e l.

S ev era l embryos, 5 1 /2 and 6 days o ld , which had

been operated upon a t th e ju n c tio n o f th e m idbrain and fo r e b r a in , had d ie n c e p h a lic m edial lo n g itu d in a l f a s c i c l e s and could not be used fo r a stu d y

o f lo n g itu d in a l t r a c t s a t low er l e v e l s .

in g f ib e r s

However, some ascend­

were apparent at th e l e v e l o f th e i n t e r s t i t i a l n u c le u s. EXCISION OF PARTS OF THE TECTUM

In an embryo f ix e d a t 4 1 /2 d ays, p art o f th e tectum o f one sid e had been removed.

The r o s t r a l part o f th e tectum was i n t a c t , and th e

c r o sse d t e c t a l t r a c t a p p aren tly was not a f f e c t e d , f o r th e commissure a t th e oculom otor l e v e l seemed norm al.

In th e m idbrain, th e r e t ic u la r

45«

fo rm a tio n o f th e operated s id e was dim inished and d is t o r t e d , hut in th e h in d b ra in t h i s a rea had a normal appearance*

The m edial and v e n tr a l

lo n g it u d in a l f a s c i c l e s , and l a t e r a l t r a c t s were not a f f e c t e d . In a 5 day embryo most o f one sid e o f th e tectum and tegmentum between th e oculom otor and tr o c h le a r n u c le i was a b se n t.

The tr o c h le a r

n u c le u s seems t o have been d isp la c e d caudad in to th e upper m etencephalon on th a t s id e , and i t i s probable th a t th e a la r p i s t e co n ta in in g th e c e r e b e lla r primordium was a ls o d e f i c i e n t .

The w a ll o f th e brain on th e

a f f e c t e d s id e was extrem ely t h in , so th a t i t was im p o ssib le fo r lo n g i*

tu d in a l t r a c t s o f th a t s id e t o grow in i t .

At th e oculom otor l e v e l ,

th e d ie n c e p h a lic m edial lo n g itu d in a l f a s c ic u lu s and th e t e c t a l t r a c t c r o s s in g from th e oth er sid e l e f t th e b rain l a t e r a l l y and fo llo w ed th e o u ts id e o f th e b ra in w a ll caudad.

Some o f th e f ib e r s coursed dorsad

a lon g t h i s t h in membrane and ended, but th e g r e a te r p art o f th e t r a c t reached th e tr o c h le a r n u c le u s .

The tr o c h le a r f ib e r s on t h i s sid e emer­

ged v e n t r o la t e r a lly and a t t h i s p o in t, th e descen d in g t r a c t reen tered th e b rain ( f i g . 42 ) , coursed mesad and caudad, and b efore a r r iv in g at th e tr ig e m in a l l e v e l , had assumed i t s normal p o s it io n l a t e r a l t o th e f lo o r p la t e ( f i g . 32 ) .

I t i s probably th a t ascen d in g f ib e r s of th e

m ed ia l lo n g itu d in a l f a s c ic u lu s m ingled w ith th e d escen d in g f a s c i c l e as i t reen tered th e brain* but none could be d is t in g u is h e d .

The v e n tr a l

lo n g itu d in a l f a s c ic u lu s and r e t ic u la r form ation were dim inished on th e op erated s id e ( f i g . 32 ) .

T his was most s t r ik in g in th e m etencephalon,

but was s t i l l e v id e n t in th e vagus r e g io n .

The m ed ial lo n g itu d in a l

f a s c ic u lu s was not a lt e r e d . The b rain o f an embryo incubated f o r 5 1 /2 days was found t o be c o m p le tely d isc o n tin u o u s between th e oculom otor and tr o c h le a r n u c le i.

O verlapping o f th e r o s t r a l and caud al p o r tio n s caused th e oculom otor r e g io n t o appear in th e same s e c t io n w ith th e tr o c h le a r n u c le u s, even though th e b rain a t th e l a t t e r l e v e l was in tr a n sv e r s e sectio n #

From

th e n u cleu s o f th e m edial lo n g itu d in a l f a s c i c u lu s , a th ic k bundle d es­ cen d ed , p a ss in g th e oculom otor n u cleu s and emerging in to th e mesenchyme# The f a s c i c l e coursed d i r e c t l y to th e m etencephalon (fig * 5 1 i t j u s t caudal t o th e tr o c h le a r n u cleu s o f th e r ig h t sid e#

) and en tered As i t assumed

a d escen d in g cou rse l a t e r a l t o th e f lo o r p l a t e , i t was jo in e d by oth er lo n g itu d in a l f ib e r s ( f ig # 3 3 ) .

P r o g ressin g caudad, th e m edial lo n g itu ­

d in a l f a s c ic u lu s r a p id ly d ecreased as f ib e r s o f rhom bencephalic o r ig in in c r e a s e d , and soon became in d is tin g u is h a b le from them# f ib e r s reached th e l e f t s id e o f th e metencephalon#

No d ien c e p h a lic

The hindbrain was

asym m etrical; a t th e r o s t r a l term in a tio n th e r ig h t tr o c h le a r and l e f t tr ig e m in a l r e g io n s occupied th e r e s p e c tiv e h a lv es o f th e n eu ral tube# However, th e m edial lo n g itu d in a l f a s c ic u lu s o f s im ila r l e v e l s was mea­ su r ed , and in th e tr ig e m in a l reg io n i t was la r g e r on th e r ig h t s id e , co n ta in in g th e d ie n c e p h a lic component, but a t th e f a c i a l l e v e l th e f a s c i c l e s were about equal#

AH attem pt was made t o determ ine th e

e x te n t o f th e d ie n c e p h a lic t r a c t , but i t became l o s t among other f ib e r s r o s t r a l t o th e tr ig e m in a l roots#

The tectum was co m p letely

m is s in g and th e r e was a c o n sid era b le tegm en tal l o s s , but d iscrep an cy in l e v e l s and d i s t o r t io n o f th e n eu ral tu be precluded accu rate com­ p a r iso n s o f d if f u s e t r a c ts # TEMRRYOS DEPRIVED OF FOREBRAIN AND MIDBRAIN A fou r-d a v “tr ig e m in a l” embryo was retard ed in growth#

Though th e

amount o f f ib e r s in th e h indbrain was l e s s than in th e c o n t r o l, a t y p ic a l

v e n tr a l lo n g itu d in a l f a s c ic u lu s o f 35 f ib e r s was p resen t* In two 4 l / 2 day embryos o f t h i s s e r i e s , th e lo n g itu d in a l t r a c t s were comparable w ith th e normal ex cep t fo r a short d is ta n c e caudal t o th e opera­ tio n *

The m edial lo n g itu d in a l f a s c ic u lu s appeared alm ost id e n t ic a l w ith

th a t o f th e c o n tr o l ( f i g . 26 ) .

In a f iv e -d a y embryo o f t h i s ty p e , th e

s iz e o f i t s s p in a l cord in d ic a te d th a t i t belonged t o th e 4 1 /2 day s e r ie s * Development o f lo n g itu d in a l t r a c t s was comparable to th e 4 1 /2 day c o n tr o l. The m edial lo n g itu d in a l f a s c ic u lu s o f a fiv e -d a y specimen was s im ila r t o th e normal ( f i g . 30 ) #

The v e n tr a l lo n g itu d in a l f a s c ic u lu s appeared t o

be somewhat d im in ish ed , but disarrangem ent of f ib e r s near th e end o f th e b ra in made i t d i f f i c u l t to be c e r ta in o f t h i s .

Other t r a c t s ap p aren tly

were n ot a f f e c t e d . The 5 1 /2 day specimen had a sm a ll, d is to r te d neural tube in th e upper rhombencephalon.

In th e tr ig e m in a l reg io n th e m edial lo n g itu d i­

n a l f a s c ic u lu s was co n sid era b ly sm a ller than in th e norm al.

At th e f a c i a l

l e v e l th e m edial lo n g itu d in a l f a s c ic u lu s i s n ea r ly comparable t o th e normal ( f i g .^ 7 ) t>ut t,he v e n tr a l lo n g itu d in a l f a s c ic u lu s and r e t ic u la r form ation show a r e d u c tio n .

Even at th e vagus l e v e l , th e b rain appeared

t o d i f f e r from th e norm al, though f ib e r red u ctio n was not so g r e a t. In an another embryo, th e s i t e o f op eration was a l i t t l e low er and d is ­ o r ie n t a tio n o f f ib e r s at th e tr ig e m in a l l e v e l made com parisons impos­ s ib le .

Even h e r e , nowever, th e s iz e o f th e m edial lo n g itu d in a l f a s c ic u ­

lu s was g r e a te r than in th e p reced in g embryo. At 6 days th e on ly tr a n s v e r s e ly s e c tio n e d " trigem in al" specimen was very d is o r ie n te d at th e tr ig e m in a l l e v e l .

Though th ere was a g rea t

r e d u c tio n in lo n g itu d in a l f i b e r s , a m edial lo n g itu d in a l f a s c ic u lu s , sm a lle r than in th e norm al, was p resen t*

That some e x te r n a l in flu e n c e

was r e ta r d in g th e h in d b rain o f t h i s embryo, became ev id e n t from study o f an other embryo o f id e n t ic a l age and l e v e l o f o p e r a tio n .

The t r i g e ­

m in al r e g io n o f th e l a t t e r embryo, in f r o n ta l s e c t io n , con tain ed a l* r g e a scen d in g m edial lo n g itu d in a l f a s c ic u lu s and r e t ic u la r form ation . Another 6 day embryo had a m edial lo n g itu d in a l f a s c ic u lu s which was prob ab ly a l i t t l e sm a ller than th e normal at th e tr ig e m in a l l e v e l . T h is appeared t o be e n t ir e ly a scen d in g , and w ith th e sen sory t r a c t s and f ib e r s o f th e r e t ic u la r fo rm a tio n , i t l e f t th e end o f th e brain t o form a ta n g le d mass under th e ectoderm ( f i g . 38 ) . EMBRYOS DEPRIVED OF FOREBRAIN, MIDBRAIN AND PART OF THE METENCEPHAXiQN

A 4 1 /2 day embryo in which th e tr ig e m in a l r o o ts e n te r th e upper t i p o f th e n eu ral tu b e and th e sem ilunar g a n g lia l i e r o s t r a l t o th e b r a in , was se c tio n e d in th e f r o n ta l p la n e .

The s i z e o f th e m edial lo n ­

g it u d in a l f a s c ic u lu s appears equal t o th a t o f th e norm al.

That most

o f i t s f ib e r s were a scen d in g , was in d ic a te d by t h e i r curved course a c r o s s th e end o f th e n eu ra l tu b e .

Many ascen din g f ib e r s o f th e r e t i ­

c u la r form ation curved toward th e m id lin e but did not meet ( f i g . 47 ) . An embryo in which th e n eu ral tube ends ab ru p tly a t th e low er f a c i a l l e v e l , so th a t th e a c o u s tic g a n g lia l i e r o s t r a l t o th e end o f th e b r a in , was in cu b ated f o r 5 d a y s, but th e s iz e o f i t s s p in a l cord is a lit t le

sm a ller than th o se o f th e embryos in th e 4 1 /2 day s e r i e s .

C o n sid erin g th e sm a ller s iz e o f t h i s embryo, i t s lo n g itu d in a l t r a c t s

compare fa v o r a b ly w ith th e 4 1 /2 day normal embryo at th e vagus l e v e l . However, f ib e r s are not q u ite norm ally o r ie n te d here and i t i s d i f f i ­ c u l t t o make com parisons. A 5 day embryo w ith no brain r o s t r a l t o th e f a c i a l l e v e l was s e c ­ t io n e d f r o n t a l l y so th a t no d ir e c t com parisons cou ld be made w ith th e t r a n s v e r s e ly s e c tio n e d c o n t r o l.

However, a la r g e ascen din g m edial lo n ­

g itu d in a l f a s c ic u lu s was p r e s e n t.

The r e t ic u la r form ation and l a t e r a l

lo n g it u d in a l f a s c ic u lu s were w e ll rep r e se n te d , but e s tim a tio n s o f t h e ir s i z e s in r e la t io n t o th e normal were im p o ssib le .

Many o f th e f ib e r s of

th e r e t i c u l a r form ation were a scen d in g . SEPARATION

OFTHE NEURAL TUBE AT THE LEVEL OF THE OTOCYSTS

With a h ia tu s

in th e n eu ral tube at th e f a c i a l l e v e l , a 4 1 /2 day

embryo had a sm a ller m edial lo n g itu d in a l f a s c ic u lu s than th e c o n tr o l a t th e tr ig e m in a l l e v e l ( f i g . 27 ) , even though th e operated embryo had a s l i g h t l y la r g e r b r a in , and in g en era l appearance seemed t o be a little

o ld e r .

R o str a l t o th e tr ig e m in a l r e g io n , th e m edial lo n g itu d in a l

f a s c ic u lu s o f th e exp erim en tal embryo in crea sed in s iz e and at th e tr o c h ­ l e a r and oculom otor l e v e l s i t was la r g e r th an in th e norm al. In a 5 day embryo o f t h i s t y p e , th e upper p o r tio n o f th e brain ended below th e tr ig e m in a l l e v e l on th e l e f t s id e , and in th e tr ig e m i­ n a l r e g io n on th e r ig h t s id e .

At th e very sm all en d in g, a lo n g , t h in

trunk from th e a c o u s tic g a n g lio n e n te r e d , c o n tr ib u tin g some sensory f i b e r s t o th e d is o r ie n te d t r a c t s .

Here th e n eu ral tube was tr a v e r se d

by f ib e r s but no commissure or lo n g itu d in a l t r a c t s were e v id e n t. r o s t r a l t o th e end ed on th e r ig h t s id e .

J u st

o f th e brain a m edial lo n g itu d in a l f a s c ic u lu s appear­ I t was con tin u ou s w ith a bundle o f f ib e r s arch­

in g a cr o ss th e n eu ral tube and sen din g some f ib e r s out o f th e brain t o

50.

t r a v e l r o str a d a lo n g th e e x te r n a l lim it in g membrane fo r 400 m icra. Other f ib e r s jo in e d t h i s extram ed u llary ascen d in g f a s c i c l e .

The m edial

lo n g it u d in a l f a s c ic u lu s in crea sed r a p id ly when fo llo w e d r o s tr a d , and at th e tr o c h le a r l e v e l i t appeared norm al.

On th e l e f t s id e , no m edial

lo n g it u d in a l f a s c ic u lu s was observed at th e en d in g .

A commissure and

v e n tr a l lo n g itu d in a l f a s c ic u lu s appeared, and below th e tr ig e m in a l r e g io n th e t r a c t in c r e a se d g r e a t ly and a p oorly d e lim ite d m edial lo n g i­ t u d in a l f a s c ic u lu s became e v id e n t.

O pposite th e tr ig e m in a l ro o t th e

f a s c i c l e seemed la r g e r th an in th e c o n t r o l, but i t was more d if f u s e and d ia g o n a l. At 5 1 /2 d a y s, sep a ra tio n o f th e n eu ral tu be at th e f a c i a l l e v e l caused th e low er end o f th e r o s t r a l p ie c e o f th e b rain t o be very sm all and rounded.

N e v e r th e le s s , lo n g itu d in a l t r a c t s and a commissure were

p r e se n t a t th e te r m in a tio n .

T h is probably rep resen ted th e low er f a c i a l

or upper g lo sso p h a ry n g ea l r e g io n , s in c e an abducens nerve l e f t th e brain and went t o th e r e g io n o f th e e y e .

T h erefo re, though th e abnormal shape

o f th e b rain made th e i d e n t i f ic a t io n o f n u c le i im p o ssib le , i t i s l i k e l y th a t th e v e s t ib u la r n u c le i were r e p resen ted .

The d o r sa l p art o f th e

v e n tr a l lo n g itu d in a l f a s c ic u lu s was com pact.

I t did not have th e ap­

p earance o f a t y p ic a l m edial lo n g itu d in a l f a s c ic u lu s and i t was sm a ll, but i t probably co n ta in ed some v e s tib u la r f i b e r s , and perhaps a few d ie n c e p h a lic axon s.

From th e end o f th e brain a bundle o f f ib e r s d es­

cended in th e mesenchyme.

The m edial lo n g itu d in a l f a s c ic u lu s in creased

and became more normal r o s t r a l l y , but th e b rain remained sm all and rounded in th e tr ig e m in a l r e g io n . In a l l th r e e specim ens th e end o f th e low er part o f th e brain was v ery abnormal.

Motor n u c le i were su p p ressed , and lo n g itu d in a l t r a c t s

w ere ab sen t ex cep t f o r a few sm all bundles s c a tte r e d in th e b asal p l a t e . An a t y p ic a l commisBure appeared, and a l i t t l e more c a u d a lly , th e motor r o o t s , lo n g itu d in a l t r a c t s and commissure were more norm al, but th e b ra in remained d is t o r t e d in shape. DISCUSSION

The n u c le i o f th e embryonic nervous system develop in a p r o g r e ssiv e manner, s t a r t in g w ith v ery few n e u r o b la sts and g ra d u a lly en la rg in g by con­ t in u a l d i f f e r e n t i a t i o n o f n e u r o b la sts over a c o n sid era b le p erio d of tim e . T h e r e fo r e , th e o ld e s t f ib e r s in a t r a c t extend a g r e a te r d ista n c e than t h o s e form ing th e bulk o f th e t r a c t .

C on seq uently, when a t r a c t i s ex­

clu d ed from th e b r a in , th e absence o f i t s lo n g e s t f ib e r s i s not d e t e c ta b le , e s p e c i a l l y a f t e r many lo n g itu d in a l f ib e r s appear.

In c o n c lu sio n s drawn from

t h i s m a te r ia l, th e estim a ted term in a tio n o f any t r a c t probably f a l l s sh o r t o f th e l e v e l reached by f ib e r s form ing i t s t i p .

However, th e method

would show th e absence o f any s u b s ta n tia l p art o f a t r a c t . S in ce absence o f th e d ie n c e p h a lic component o f th e m edial lo n g itu d in a l f a s c ic u lu s a f f e c t s th e s iz e o f t h i s f a s c i c l e in th e m idbrain in th e 4 -5 day s ta g e , but has l i t t l e e f f e c t on th e t r a c t at th e tr ig e m in a l l e v e l a t 5 1 /2 d a y s, i t i s assumed th a t s ig n if ic a n t numbers o f th e d ie n c e p h a lic f ib e r s do n ot e n te r th e tr ig e m in a l reg io n b efo re 5 1 /2 d a y s.

That th ey

a r e p r e se n t in th e upper met encephalon was dem onstrated in an embryo in w hich o n ly one d ie n c e p h a lic t r a c t descended below th e oculom otor l e v e l . The m edial lo n g itu d in a l f a s c ic u lu s , as e a r ly as 72 h ou rs, i s o f d iv e r s e o r ig in , being d eriv ed from th e i n t e r s t i t i a l n u c le u s, a secondary sen­ so ry tr ig e m in a l n u cleu s and secondary v e s tib u la r n u c le i. G rad u ally th e r e i s overlap pin g o f th e d escen din g d ie n c e p h a lic w ith

a sce n d in g tr ig e m in a l com ponents, and ascending v e s tib u la r f ib e r s in c r e a s e t h e s i z e o f th e t r a c t in th e m etencephalon w h ile descending v e s tib u la r f i b e r s exten d th e cau d al l i m i t o f th e t r a c t in to th e m yelencephalon# The d ie n c e p h a lic elem ent o f th e m © dial lo n g itu d in a l f a s c ic u lu s h as been r ec o g n ized in th e a d u lt , and v e s tib u la r components have been q u ite th o ro u g h ly traced #

A tr ig e m in a l c o n tr ib u tio n , however, has not

been rep orted from re sea r c h e s on a d u lt m a te r ia l,

E xperim ental m a te r ia l

in d ic a t e s th a t many o f th e tr ig e m in a l f ib e r s le a v e th e main p art o f th e t r a c t in th e upper m etencephalon and go la te r a d in sm all bundles#

They

approach th e m esen cep h alic Vth t r a c t where i t l i e s a t th e edge o f th e m antle la y e r o p p o site th e su lc u s lim it a n s , and at 5 days th ey do not seem t o ex ten d f a r t e r r o s t r a l l y .

In p o s it io n , th e s e bundles are ana­

lo g o u s t o th e d o r s a l secondary tr ig e m in a l t r a c t o f th e a d u lt.

In th e

b ir d a homologue o f t h i s t r a c t has been tr a ced t o the m esencephalon but n o t to th e th alam u s.

The q u in to -fr o n ta l t r a c t o f th e ad u lt b ird occu p ies

t h e c e n t e r - o f th e tegmentum o f th e m idbrain, a co n sid era b le d ista n c e from th e m edial lo n g itu d in a l f a s c ic u lu s (W allenberg, 1 9 0 3 ).

In ** sub­

th alam ic" specim ens o f t h i s s e r i e s , ascend in g f ib e r s le a v in g the m edial lo n g itu d in a l f a s c ic u lu s l a t e r a l l y , caudal t o th e oculom otor n u c le u s, w ere ob served .

I t i s not l i k e l y t h a t , in th e sta g e s s tu d ie d , a focu s

f o r a scen d in g f ib e r s o f th e mBdial lo n g itu d in a l f a s c ic u lu s i s p resen t a b ove th e tr ig e m in a l r e g io n .

Furtherm ore, m edial ascen d in g f ib e r s alm ost

rea ch th e tr o c h le a r l e v e l at 4 d ays.

T h erefo re, i t i s most probable th a t

t h e s e f ib e r s are o f tr ig e m in a l o r ig in . In some w ays, th e nervous system o f th e bird d i f f e r s g r e a t ly from th a t o f mammals, but in e a r ly development th e form ation o f th e m edial lo n g it u d in a l f a s c ic u lu s i s very s im ila r in th e b ird and mammalian

embryos rep o rted on in t h i s p ap er.

T his m a teria l a ffo r d s no b a s is fo r

agreement w ith th e cla im s o f Mesdag (1909)^ Bok (1 9 1 5 ), and V is in t i n i and Montjfcalcini (1939) th a t a l l v e n tr a l lo n g itu d in a l f ib e r s in th e e a r ly embryo are d esc en d in g .

R e su lts obtained in t h i s work dem onstrate t h a t ,

v ery soon a f t e r n e u r o f ib r illa r d if f e r e n t i a t io n commences, th e c e n tr a l nervous system becomes q u ite com plex.

Much overlap p in g of t r a c t s occurs

an'* i t i s im p o ssib le s p e c i f i c a l l y t o d esig n a te term in a tio n s of t r a c t s a s a w h ole.

Even in very young embroys, growth cones of r e l a t i v e l y few

f ib e r s are s e e n , and th ey become p r o g r e s s iv e ly l e s s e v id en t as f ib e r s become more numerous. Becaus o f d istu rb e d r e la t io n s h ip s near th e s i t e of o p era tio n , and th e im p o s s ib ilit y of id e n t if y in g t r a c t s at th e ending of th e n eu ral tu b e , e s p e c i a ll y in tr a n s v e r s e s e c t io n s , th e r e s u lt s o f t h i s p a r tic u la r problem are n ot as s p e c i f i c a s i t was hoped th ey would b e.

However, fr o n ta l

s e c t io n s o f s im ila r embryos dem onstrated th a t a m edial lo n g itu d in a l f a s c i c u l u s , a p p a ren tly comparable t o th e normal in s i z e , i s p resen t in th e tr ig e m in a l r e g io n , and i t s emergence from th e n eu ra l tube ( f i g . 38 ) . shows th a t i t i s ascen d in g to h ig h er l e v e l s .

At 5 1 .2 and 6 d ays, th e r e

seems t o be a s l i g h t red u ctio n in th e m edial lo n g itu d in a l f a s c ic u lu s of th e m etencephalon in embryos deprived o f more r o s tr a l p a r ts o f th e b ra in . T his s e r i e s should be extended s e v e r a l days and th e embryos should be compared w ith " m idbrain" embryos o f s im ila r a g e. To f o llo w c l o s e ly th e caudal growth of th e d ie n c e p h a lic component, i t should be i n s t r u c t iv e to remove th e a la r p la t e , and perhaps a sm all amount o f th e b a sa l p la te o f the tr ig e m in a l region ; and in some embryos t h a t o f th e f a c i a l r e g io n , a l s o . th u s be e lim in a te d .

Most o f th e ascen din g components could

I t i s probable th a t a u n ila t e r a l o p era tio n o f t h is

ty p e would be s u c c e s s f u l f o r , u n lik e u n su c c e ss fu l u n ila t e r a l o p era tio n s th a t have been attem p ted , th e d e fe c t would be sm all in comparison to th e amount o f n e u r a l t i s s u e l e f t t o d e v e lo p . would depend upon th e r e s u lt s o b ta in ed ,

The age li m it o f such a s e r ie s The cro ssed t e c t a l t r a c t , la g g in g

behind th e d ie n c e p h a lic b in d le , probaHy would n ot in t e r f e r e w ith obser­ v a t io n s , but a s a check upon r e s u l t s , fu r th e r attem pts to p revent i t s growth shou ld be made* Very l i t t l e l o s s was d e te c te d in th e more d if f u s e lo n g itu d in a l t r a c t s U n til o ld er s t a g e s , and embryos w ith u n ila t e r a l o p e r a tio n s, have been o b ta in e d , few c o n c lu sio n s regarding th e r e t ic u la r form ation and v e n tr a l end l a t e r a l lo n g itu d in a l f a s c i c l e s can be drawn.

The most s ig n if i c a n t

f in d in g was t h a t re d u c tio n o f f ib e r s was s l i g h t . The f i r s t f ib e r s o f th e cro ssed t e c t a l t r a c t appear t o j o in th e d ie n c e p h a lic m ed ial lo n g itu d in a l f a s c ic u l u s .

L ater, f ib e r s from th e ro s­

t r a l m esen cep h alic commissure are more d i s t i n c t v e n tr a l to th e m edial lo n g itu d in a l f a s c i c u l u s , and probably some o f th e t r a c t l i e s in th e v e n tr a l lo n g itu d in a l f a s c i c u l u s .

T his part o f th e d escen din g t e c t a l s y s ­

tem d ev e lo p s r e l a t i v e l y l a t e , and at th e o ld e s t sta g e stu d ie d h e r e , i t i s not l i k e l y t h a t th e t r a c t exten d s beyond th e m idbrain. In some o f th e operated m a te r ia l, th e m edial p art o f th e uncrossed t e c t a l t r a c t cou ld be fo llo w e d in to th e v e n tr a l lo n g itu d in a l f a s c ic u lu s . S in ce t h e s e f ib e r s had n ot l e f t th e m idbrain at 4 1 /2 d ays, i t i s un­ l i k e l y th a t th ey e n te r th e m yelenceohalon by 5 d a y s.

However, th e r e ­

d u c tio n o f f ib e r s in th e v e n tr a l lo n g itu d in a l f a s c ic u lu s at th e vagus l e v e l o f a 5 day embyyo w ith a u n ila t e r a l d e fe c t in th e mid brain i s d i f ­ f i c u l t t o e x p la in .

Other p o s s ib le sou rces of th e m issin g f ib e r s are th e

tegmentum o f th e mid b r a in , and th e c e r e b e lla r a n la g e .

Great numbers of

in te r n e u r o n s o f th e b a sa i p la te o f the m idbrain appear ra th er l a t e (5 -5 1 /2 d a y s ) , so th a t t h e ir axons could hard ly reach th e m yelencephalon u n t i l a much l a t e r stage*

N eu ro b la sts in th e c e r e b e lla r anlage are f i r s t obser­

ved a t about 3 1 /2 days*

Exam ination o f normal m a te r ia l in d ic a te s th a t

many o f th e s e f ib e r s probably e n te r the commissure a t th e tr o c h le a r le v e l* I t i s q u ite p ro b a b le, however, th a t some of th e s e axons are hom olateral* I t i s im p o s s ib le , in normal m a te r ia l, to d e f i n i t e l y s t a t e t h e ir p o s it io n in th e b a sa l p l a t e .

To account fo r th e l o s s in th e v e n tr a l lo n g itu d in a l

f a s c ic u lu s o f th e m yelencephalon, i t seems most reason able to assume th a t i p s i l a t e r a l c e r e b e lla r f ib e r s occupy th a t f a s c i c l e , but d e f in it e c o n c lu sio n s in regard t o t h i s must w ait fu r th e r exp erim en tation .

Remov­

a l o f one a la r p la te of th e r o s t r a l m etencephalon and a d d itio n a l u n ila ­ t e r a l t e c t a l e x c i s io n s , w ith and w ith ou t tegm en tal d e f i c ie n c ie s , w i l l be u s e fu l.

Other components o f th e v e n tr a l lo n g itu d in a l f a s c ic u lu s have not

been d eterm in ed , but th e p resen ce o f ascend ing f ib e r s has been observed in th e mid b r a in .

T h eir e x a c t o r ig in i s unknown*

The r e t ic u la r form ation i s a la r g e , complex t r a c t .

The la ck of any

s t r ik in g l o s s in i t s i g n i f i e s th a t i t i s composed m ainly o f sh ort f ib e r s a t e a r ly s t a g e s .

In th e 5 day embryo w ith lo s s in th e m idbrain, a d e f i ­

n i t e r e d u ctio n o f t h i s area was noted at th e tr ig e m in a l l e v e l .

It is

probable th a t some o f th e f ib e r s from th e l a t e r a l p art o f th e uncrossed t e c t a l t r a c t invade th e m etencephalon by 5 d a y s.

D im inution of tegm en tal

c e l l s may a ls o be r e s p o n s ib le f o r th e l o s s . The compact l a t e r a l t r a c t of th e r e t ic u la r form ation was u n a ffected in th e operated m a te r ia l.

This pathway and th e la t e r a l lo n g itu d in a l f a s ­

c i c u l u s , t o which i t i s c lo s e ly r e la t e d , are app arently composed of sh ort

c h a in s o f f i b e r s , and c a r e f u lly lo c a liz e d u n ila t e r a l o p e r a tio n s, carryin g th e embryos t o a c o n sid e r a b ly la t e r sta g e w i l l be n ecessary fo r an an aly­ s is of th e se tr tc ts *

Removal of th e sem ilun& r, and p o s s ib ly th e f a c i a l

and a c o u s tic g a n g lia , alon g w ith th e o t o c y s t s , t o exclu de primary a ffe r e n t t r a c t s would be a d v is a b le .

Almost t o t a l e lim in a tio n o f th e tr ig e m in a l

t r a c t s was accom plished in one embryo in which only a sm all group of c e l l s o f th e sem ilunar g a n g lio n rem ained. S p e c if ic t r a c t s of th e r e t ic u la r form ation noted in the upper meten­ cep h alon at 5 l / 2 and 6 days were n ot a f f e c t e d .

Being d e f i n i t e , ra th er

compact t r a c t s , i t should be p o s s ib le t o le a r n som ething about them from ex p erim en ta l m a te r ia l ca r r ie d t o l a t e r s t a g e s .

5 .? u

CONCLUSIONS 1.

The components of th e m e d ia llo n g itu d in a l f a s c ic u lu s in

th e e a r ly ch ick embryo are of d iv e r se o r ig in :

th e i n t e r s t i t i a l n u cleu s;

a tr ig e m in a l secondary sen sory n u cleu s; and secondary v e s tib u la r n u c le i. A fte r 4 days some cro sse d t e c t a l f ib e r s probably c o n trib u te to th e t r a c t . 2.

In th e 6 day c h ic k , th e m edial lo n g itu d in a l f a s c ic u lu s does

n o t extend fa r th e r caud& lly than th e upper vagus r e g io n .

The caudal part

o f th e t r a c t i s formed by descending v e s tib u la r f i b e r s . 3.

At 5 l / 2 d a y s, th e d ie n c e p h a lic component of th e m edial

lo n g itu d in a l f a s c ic u lu s exten d s alm ost t o th e tr ig e m in a l le v e l a scen d in g f ib e r s reach th e oculom otor l e v e l .

A few

Other ascending f ib e r s le a v e

th e m edial lo n g itu d in a l f a s c ic u lu s in th e upper m etencephalon and c o n s t i­ t u t e « ev era l l a t e r a l bundles exten d in g t o th e tr o c h le a r le v e l* 4.

The uncrossed t e c t a l t r a c t extend s through th e midbrain at

4 1 /2 d a y s, and e n te r s th e m etencephalon at 5 d ays. 5.

The cro ssed t e c t a l f ib e r s begin t o form a lo n g itu d in a l

t r a c t at 4 days; and reach th e low er tr o c h le a r l e v e l at 5 d a y s.

ABNORMAL NERVES P e r ip h e r a l Course o f N erves Deprived o f t h e ir Normal D e s tin a tio n The ex p erim en ta l m a te r ia l used here provided an op p ortun ity t o o b serv e th e cou rse o f c r a n ia l n e r v e s , deprived o f t h e ir normal d e s t i ­ n a tio n *

In o p e r a tio n s e lim in a tin g th e fo r e b ra in , th e eye and much o f

th e surrounding re g io n was found t o be a b se n t, but th e oculom otor, t r o c h le a r and ophthalm ic n erv es were p resen t* Da « fo u r day embryo o f t h i s ty p e th e oculom otor nerve proceeded t o th e ectoderm , and spread out upon i t s in n er surface*

These f ib e r s

descended caudad, and a t th e tr ig e m in a l l e v e l , much dim inished in number, th e y were jo in e d by s e v e r a l f ib e r s from th e G asserian g a n g lia , a f t e r which th e f ib e r groups o f both s id e s in term in gled and disappeared* t r o c h le a r nerve was s t i l l in tram ed u llary at t h i s s ta g e .

The

Very sm all

abducens trunks ended in th e mesenchyme a t th e tr ig e m in a l le v e l* The oculom otor n erves o f a s im ila r embryo o f th e same a g e , formed tr u n k s, each o f which jo in e d th e ophthalm ic nerve o f th e same side* Some f ib e r s coursed between th e r ig h t and l e f t tr ig e m in a l n erves a t t h i s le v e l*

The tr o c h le a r nerve emerged, but ended in th e mesenchyme

l a t e r a l t o th e n eu ra l tube*

The abducems nerve ended near th e tr ig e m i­

n a l le v e l* A 4 1 /2 day embryo had many aberrant oculom otor r o o t l e t s r o s t r a l t o th e r e g u la r oculom otor n u c le u s. t h e i r o r ig in .

They ended in th e mesenchyme near

The main oculom otor root approached and jo in e d th e Gas-

e e r ia n g a n g lio n , n ear which i t gave o f f a nerve t h a t could be fo llo w ed t o th e m andible.

The tr o c h le a r nerve wae in th e mesenchyme but ended

B9. l a t e r a l t o th e n eu ra l tube*

The abducens nerve was sm all but had a

few f ib e r s communicating w ith th e oculom otor trunk* In a 5 day "midbrain” embryo, th e oculom otor n u c le i and surround­ in g b ra in were drawn out in to two prom inences, from which th e n e r v e s emerged*

T h is p a ir was very la r g e and was jo in e d by th e tr o c h ­

l e a r p a ir , form ing a c e n tr a l trunk*

A few f ib e r s o f th e tr o c h le a r

n er v es d id not e n te r in to t h i s , but jo in e d each oth er v e n tr a l t o th e main trunk*

The s in g le oculom otor—tr o c h le a r bundle coursed caudad a

l i t t l e t o th e l e f t @f th e m id lin e , and sen t a few communicating f ib e r s t o a combined ophthalm ic-m andibular nerve o f th e r ig h t s id e . r e c e iv e d a bundle o f f ib e r s from th e l a t t e r n e rv e.

I t a ls o

The oculom otor nerve

ended a sh ort d ista n c e fa r th e r c a u d a lly , w ith out any oth er con n ections* The ophthalm ic-m andibular branch ended by sendin g a la r g e part o f i t s f i b e r s a c r o ss th e m id lin e .

C lo ser t o th e g a n g lio n , th e ophthalm ic and

m andibular components became sep arate and could be observed t o e n te r th e G a sseria n g a n g lio n by in d iv id u a l p o le s .

Near th e m id lin e both ophthal­

m ic and m andibular n erves gave o f f a branch which en tered a la r g e c e n tr a l g a n g lio n a l i t t l e more cau d ally*

The c e l l mass became lo c a te d at th e t ip

o f a V formed by th e l a t e r a l r e c tu s m uscle m asses.

The r o s t r a l ends o f

t h e abducens n erves appeared a t t h i s l e v e l and s e v e r a l f ib e r s passed between them and th e g a n g lio n .

The m uscle m asses th en d iv id ed and th e

g a n g lio n la y between them, bounded d o r s a lly and v e n t r a lly by two sm all blood v e s s e l s , th e in te r n a l c a r o tid a r t e r i e s .

Near i t s caudal t i p , th e

c e l l group s e n t a loop to surround th e d o rsa l a r te r y . The l e f t tr ig e m in a l nerve did not e n te r th e com plex.

I t s mandibular

branch went ro str a d a short d ista n c e along th e m edial s id e of th e G asse­ r ia n g a n g lio n t o end in a m uscle mass more s u p e r f ic ia l ly p laced than th e

l a t e r a l rectu s*

The ophthalm ic and m andibular n erves j o in it *

A sm all

branch from t h i s com bination went la te r a d and became spread out under th e ectoderm .

There were a few n e u r o b la sts in th e network o f n erves

formed by th e tr ig e m in a l b ran ches, but th e y la y very c lo s e t o th e sem ilu n a r g a n g lio n , and may have been part of th a t c e l l group* At 5 1 /2 d a y s, th e cou rse o f th e s e n erv es was s im ila r in th r e e embryos*

F o llo w in g i s a d e s c r ip tio n o f one o f them.

The tr o c h le a r

n er v es coursed ventrad in th e mesenchyme, and, a f t e r p a ssin g o u tsid e of t h e oculom otor p a ir , swung m ed ia lly t o meet and exchange f ib e r s (fig * 4 1 )• The oculom otor n erv es took no part in t h i s common tru n k , but continued caudad, p a ss in g through th e tr o c h le a r lo o p , undergoing a p ro g r e ssiv e and v er y c o n sid e r a b le enlargem ent.

These la r g e tru n ks became ap p lied t o th e

m ed ia l su r fa c e o f each l a t e r a l r e c tu s m uscle mass*

Caudal t o t h i s , th e

**

m uscle m asses fu sed w ith one another alm ost surrounding *the in te r n a l caro­ t i d a r te r y which was s in g le *

The ends o f th e abducens n erves m et, a ls o

lo o p in g v e n tr a l t o th e oculom otor.

A la r g e branch from each sem ilunar

g a n g lio n en tered th e m uscle m ass, l a t e r a l t o th e abducens and s p l i t alm ost a t once in t o a l a t e r a l branch which went p e r ip h e r a lly and presumably repre­ sen te d th e m a x illa r y nerve*

The remainder o f th e tr ig e m in a l f ib e r s (^oph­

th a lm ic ? ) arched v e n tr a l t o th e oculom otor p a ir and met tr ig e m in a l f ib e r s o f th e o p p o site s id e , forming a loop in co n ta ct w ith th e oculomotor nerves and c o n tr ib u tin g t o a c e n tr a l mass o f f ib e r s surrounding a la r g e median g a n g lio n ( f i g . 40 ) .

I t i s probable t o o , th a t some abducens axons en tered

t h i s t a n g le o f f i b e r s .

A fter th e tr ig e m in a l loop formed, a s in g le la r g e

trunk o f tr ig e m in a l f ib e r s l e f t th e ventromedian p art o f th e m uscle mass t o cou rse t o th e p erip h ery o f th e mandible*

In con n ection w ith th e t r i ­

gem inal f ib e r s in th e m uscle m ass, a sm all group o f c e l l s , n ot connected

^ t t h th e s in g le c e n t r a l g a n g lio n , was observed on each sid e* probably were th e sp h en o p a la tin e g a n g lia .

They

Below th e tr ig e m in a l lo o p ,

th e f ib e r s o f th e g r e a t ly en larged oculom otor p a ir jo in e d in th e m iddle, v e n tr a l t o th e s in g le g a n g lio n .

The m uscle mass a t t h i s l e v e l had alm ost

sep a ra ted in to two p a r t s , each c o n ta in in g an abducens nerve*

Below t h i s ,

t h e p a ir o f m uscle m asses disappeared and th e abducens nerves approached th e b r a in .

The c e n tr a l mass o f n erves sep arated in to two la r g e trunks

w hich cou ld be fo llo w e d a l i t t l e fa r th e r c a u d a lly where th ey con trib u ted t o , or r e c e iv e d f ib e r s from, th e sen sory branch o f th e mandibular nerve* The g r e a t s u p e r f i c i a l p e tr o s a l nerve l e f t t h i s trunk and went t o th e g en i­ c u la t e gan glion * V a r ia tio n s o f t h i s p a tte r n were found in oth er 5 1 /2 day embryos, and a t 6 and 6 1 /2 d a y s, but c e r ta in common f a c to r s were found.

The charac­

t e r i s t i c lo o p s formed by th e t r o c h le a r , abducens, ophthalm ic and ocu lo­ m otor p a ir s o f n erv es were u s u a lly observed in th a t r o str o -c a u d a l order* In one c a s e , th e abducens d id n ot form a lo o p .

The s in g le (som etim es

p a r t i a l l y b i l a t e r a l ) g a n g lio n was alw ays a s s o c ia te d w ith th e oculom otor p a ir and th e whole complex was formed in a s in g le m uscle mass which o fte n became b i l a t e r a l a t i t s r o s t r a l and caudal ends* o f sm all g a n g lia were found.

U su a lly a sep arate p a ir

The oculom otor n erves always showed a great

in c r e a s e in s iz e as th e y approached th e com plex. The m andibular nerve did not always e n te r th e com plex.

Sometimes

t h e o r ig in o f th e ophthalm ic branch was q u ite se p a r a te , but i t u s u a lly communicated w ith e it h e r th e m andibular, or th e m a x illa ry nerve* In some embryos th e oculom otor nerve on one or both s id e s , was formed by two tru n k s and in o th er specim ens, groups of f ib e r s l e f t one o f th e oculom otor n erves and coursed ro stra d alon g th e s in g le c a r o tid a r te r y .

62 A "midbrain" embryo, s l i g h t l y under 5 1 /2 d a y s, d iff e r e d c o n sid era ­ b ly from o th e r s in th e group*

I t s m idbrain was t w is t e d , and th e ocu lo­

m otor n u c le i were asym m etrical*

The oculomotor tr o c h le a r n erves o f both

s id e s were d ir e c te d toward th e r ig h t G asserian g a n g lio n .

The l e f t tro ch ­

l e a r n e r v e , p art o f which t r a v e lle d ro stra d in co n ta ct w ith th e l a t e r a l w a ll o f th e b r a in , headed t o th e r ig h t but ended w ith out c r o s s in g th e mid­ lin e *

The r ig h t tr o c h le a r nerve went t o th e h om olateral G asserian gang­

lio n *

Some o f th e f ib e r s p assed through th e g a n g lio n and Joined th e r ig h t

oculom otor nerve which had gone through th e g a n g lio n a l i t t l e more r o str a lly . A v ery la r g e oculom otor nerve on th e l e f t , emerged a t th e median lin e ? because th e f l o o r p la t e was r o ta te d t o th e r ig h t .

(C on sid erab le d is p la c e ­

ment o f th e m idbraln had taken p la c e , and due t o an abnormal fle x u r e in th e tr ig e m in a l r e g io n , th e r o s t r a l end o f th e b rain la y in co n ta ct w ith t h e m andible*) l a r nerve*

The l e f t oculom otor nerve merged w ith th e r ig h t mandibu­

A fte r p a ss in g through th e G asserian g a n g lio n th e l e f t and

r ig h t oculom otor and some o f th e tr ig e m in a l f ib e r s , communicated w ith th e abducens nerve*

At t h i s p o in t a con spicuou s g a n g lio n was observed, in th e

l a t e r a l r e c tu s m uscle m ass, alon g th e communicating f i b e r s .

The la r g e

o cu lo m o to r-trig em in a l trunk continu ed caudad, and o p p o site th e gen icu ­ l a t e g a n g lio n , th e f a c i a l nerve cro ssed through it *

A prominent g a n g lio n

appeared a t t h i s p o in t , end was p resen t a f t e r th e f a c i a l f ib e r s were no lo n g e r in c o n ta c t w ith th e trunk*

A few stran d s o f f ib e r s connect th e

o c u lo m o to r-trig em in a l trunk w ith th e p e tr o s a l g a n g lio n . The tr ig e m in a l nerve o f th e r ig h t s id e remains is o la t e d from co n tra t e r a l , and o th er h o m o la tera l, n e r v e s.

The ophthalm ic branch e n te r s th e

l a t e r a l r e c tu s m uscle m ass, and shares a g an glion w ith th e mandibular

branch*

The l a t t e r n e r v e , combines w ith th e abducens*

The abducens

n e r v e s are very la r g e a t th e l e v e l o f th e G asserian g a n g lia , d ecrea sin g c o n s id e r a b ly toward t h e i r o r ig in . 3h th e absence o f fo r e b r a in and m idbrain, th e sem ilunar gan glion u s u a lly underwent a t o r s i o n , ca u sin g th e ophthalm ic p o le s , when id e n t i­ f i a b l e , t o be d ir e c te d m ed ially*

The mandibular and m a x illa r y nerves

were d is t in g u is h a b le and more or l e s s norm ally o r ie n te d , but one or th e o th er communicated w ith th e ophthalm ic branch* In a 4 1 /2 day embryo, th e ophthalm ic and mandibular branches and th e abducens nerve met in th e m id line*

The mandibular branches th en

l e f t th e p le x u s and went in to th e mandible* Three 5 day embryos were q u ite v a r ia b le as t o th e cou rse and con­ n e c t io n s o f th e ophthalm ic n e rv e.

In on e, th e ophthalmic branch o f one

s id e Join ed th e m andibular nerve o f th e o p p o site s id e .

The oth er op hth al­

m ic n erve en tered th e l a t e r a l r e c tu s m uscle m ass, but did not Join th e abducens nerves*

In a second specim en, one ophthalm ic nerve Joined th e

h om olateral m a x illa r y branch, and th en l e f t i t t o c r o ss t o th e o p p o site sid e*

Both ophthalm ic branches o f th e th ir d 5 day embryo coursed rostrad

and formed a lo o p j u s t beyond th e l e v e l a t which th e b rain ended* m a x illa r y nerve jo in e d t h i s com plex.

One

However, another p a ir o f tr ig e m in a l

b ra n ch es, o th er th an th e mandibular and m a x illa ry was p r e s e n t, and th ey probably rep resen ted p a rt o f th e ophthalm ic n erv e.

They l e f t th e gang­

l i o n m e d ia lly and on one o f them e s p e c i a ll y , a prom inent, l i g h t l y sta in ed g a n g lio n appeared*

These n erves ended in th e l a t e r a l r e c tu s m uscle m ass.

The m andible o f one 5 1 /2 day embryo was d isp la c e d fa r t o one s id e , and th e G asserian g a n g lia were asym m etrical in p o s it io n .

The m a x illa ry

n e r v e s formed a common trunk which en tered th e m andible, but f i r s t com-

6 4 .-

mur-icaie'’ w ith th e m andibular n e r v e s .

These f ib e r s probably rep resen ted

th e ophthalm ic branch. In an other 5 1 /2 day embryo , th e l a t e r a l r e c tu s m uscle m asses were fu sed and th e abducens n erves formed a loop*

A tr ig e m in a l branch from

one s id e en tered th e m uscle m ass. At ^ days a s in g le l a t e r a l r e c tu s m uscle mass appeared in th e mid­ lin e .

T rigem inal branches en tered t h is * but communicated w ith each oth er

a ls o .

The abducens n erves were q u ite sm all when th ey l e f t th e neural

tu b e , bu t had in c re a sed g r e a t ly b efore e n te r in g th e m uscle m ass. In a A 1 / 2 day embryo, in which th e brain ended j u s t r o s t r a l t o th e f a c i a l r e g io n , th e G asserian g a n g lia la y r o s t r a l and v e n tr a l t o th e b ra in . No motor r o o ts o f th e tr ig e m in a l nerve formed. cou rsed r o str a d and en tered t h e ir m uscle m asses.

The abducens n erves M edial branches o f th e

G asserian g a n g lia approached each oth er and merged.

Trunks from t h i s com­

bined nerve en tered th e l a t e r a l r e c tu s m uscle m ass, but th ey did n ot j o in th e abducens n e r v e s . p o in t.

The l a t t e r nerves disappeared j u s t beyond t h i s

S u b seq u en tly, th e m uscle m asses fu se d , and th e tr ig e m in a l f ib e r s

formed a netw ork, but fa r th e r c a u d a lly th e y became a trunk o f compact f ib e r s and en tered th e m andible.

At th e l e v e l of th e p le x u s, a l a t e r a l

branch l e f t one G asserian g a n g lio n and communicated w ith th e f a c i a l n erv e. A 5 day embryo had one G asserian g a n g lio n r o s t r a l t o th e b ra in . o th er g a n g lio n was a b se n t. se n t.

The

Only one l a t e r a l r e c tu s m uscle mass was pre­

The abducens n e r v e s , in c r e a s in g in s iz e as th e y went r o str a d ,

en tered th e m uscle mass and formed a lo o p .

F ib e r s from t h i s sprayed out

and were jo in e d by f ib e r s from a branch which l e f t th e caudal end o f th e G a sseria n g a n g lio n , and c o n s titu te d th e o n ly branch o f th e tr ig e m in a l n erv e in t h i s embryo.

From t h i s netw ork, a la r g e tru n k , a f t e r g iv in g

£St

o f f a branch which spread out under th e ectoderm , went t o a m uscle mass in th e m andible* With th e tr ig e m in a l nerve e n t ir e l y a b se n t, th e a c o u s tic g a n g lia o f a 5 day embryo fu sed in th e m id lin e r o s t r a l t o th e b ra in , but sen t p e r i­ p h era l f ib e r s t o th e o to c y sts*

The abducens n erves formed a loop at th e

l e v e l o f t h e i r em ergence, and d id not communicate w ith oth er c r a n ia l n erves* In a 5 1 /2 day embryo w ith th e g e n ic u la te g a n g lia r o s t r a l t o th e end o f th e b r a in , th e abducens n erv es emerged and enlarged as th e y coursed rostra d *

V en tra l t o th e end o f th e b r a in , and caudal t o th e g e n ic u la te

g a n g lia , th e y formed a loop which was en tered by some f ib e r s th a t had l e f t th e end o f th e brain*

Sm all trunks le a v e t h i s loop and go caudad,

v e n tr a l t o th e abducens nerves*

The bundle on one sid e ends soon, but a

few f ib e r s o f th e o th er s id e reach th e l e v e l o f th e vagus nerve and com­ m unicate w ith it * ALTERATIONS IN THE OCULOMOTOR AND TROCHLEAR NERVES In 5 1 /2 day embryo in which th e mid brain was amorphic and th e oculom otor and t r o c h le a r n u c le i and n erves were not r e c o g n iz a b le , apparent­ l y normal c i l i a r y g a n g lia were p r e s e n t, co n n ectin g only w ith th e ophthal­ m ic n erves*

In t h i s embryo th e sem ilunar g a n g lio n o f one s id e was sm all

and had no co n n ectio n w ith th e n eu ral tube* Two embryos in which th e n eu ra l tu be was separated between th e oculom otor and tr o c h le a r n u c le i showed ab n o rm a lities o f th e s e nerves* In one o f them th e r e was some overlap o f th e separated midbrain*

A

r ig h t oculom otor nerve could be tr a c e d from th e is o la t e d p ert o f th e

n e u ra l tu b e t o th e eye region*

I t p assed through th e c i l i a r y ganglion*

No oculom otor n erve cou ld be found on th e l e f t s id e , but a c i l i a r y gang­ l i o n was p r e s e n t, c o n n ectin g only w ith th e ophthalm ic nerve*

The l e f t

t r o c h le a r nerve c r o sse d t o th e o p p o site s id e in th e d o r sa l p art o f th e n eu ra l t u b e , emerged in t o th e mesenchyme ( f i g * 45 ) , and Joined a trunk (m ed ia l lo n g itu d in a l f a s c ic u lu s ? ) which descended from th e upper is o la t e d p ie c e o f th e n eu ra l tube*

More r o s t r a l l y , th e tr o c h le a r nerve o f th e

o th e r s id e emerged l a t e r a l l y w ith ou t c r o s sin g and descended caudad and ventrad in th e mesenchyme t o j o in th e trunk formed by th e l e f t IVth n erve and th e m edial lo n g itu d in a l f a s c ic u lu s *

F arther r o s t r a lly t h i s

bundle o f f ib e r s en tered th e v e n tr a l p art o f th e n eural tube*

The tro ch ­

l e a r n erv es are v ery sm all and a t y p ic a l, but t h e ir r e l a t i v e l y la rg e c e l l s , t h e i r c o u r s e , and p o s it io n id e n t if y them ra th er d e f in it e ly * The o th er embryo, 5 1 /2 days o ld , p o ssessed oculom otor nerves which made normal c o n n e ctio n s w ith th e eye region*

T heir manner of e x it was

somewhat abnormal due t o d is t o r t io n o f th e n eu ral tube*

The r ig h t nerve

emerged t o th e l e f t but descended vent r a l l y and turned t o th e r ig h t below th e n eu ra l tube*

The ending o f th e low er p art o f th e brain was

c h a r a c t e r is t ic o f th e upper m etencephalon on one s id e and no tr o c h le a r n erve was p resen t* The oth er s id e con tain ed an abnormally la r g e tr o c h le a r n u c le u s and some o f i t s n e u r o b la sts had been p u lle d out o f th e brain and la y in th e m ed ial lo n g itu d in a l f a s c ic u lu s , which was e n te r in g from th e mesenchyme.

The tr o c h le a r nerve emerged v e n t r o - la t e r a lly ( f i g . 4 4 ) and

cou rsed r o s tr a d , r a p id ly d im in ish in g in s i z e .

The l a s t few f ib e r s o f i t

ended l a t e r a l t o th e oculom otor n e r v e , alm ost at th e top of th e e y e .

The

t r ig s s in & l r e g io n on th a t s id e had two aberrant motor r o o ts which emerged r o s t r a l t o th e re g u la r tr ig e m in a l motor r o o t, and coursed ro stra d t o j o in

t h e oculom otor nerve near th e ophthalm ic branch o f th e tr ig e m in a l. In a 5 day embryo w ith an abnormal m idbrain» a sm all part o f th e r ig h t t r o c h le a r n erve took an in tram ed u llary course dorsed# but most o f i t emerged l a t e r a l l y (fig * 4 2 )* as th e exfcramedullary m edial lo n g itu d in a l f a s c ic u lu s e n te r e d . la te r a lly .

The tr o c h le a r n u cleu s o f t h i s sid e was s itu a te d

The t r o c h le a r nerve went dorsad and ro stra d in th e mesenchyme *

and f i n a l l y m ingled w ith th e m edial lo n g itu d in a l f a s c ic u lu s .

A bundle

s p l i t o f f from t h i s trunk and coursed vent rad and caudad where i t en tered th e n e u r a l tu b e . On th e o th er s id e th e tr o c h le a r nerve had a normal in tram ed u llary co u rse t o th e d o r s a l p a rt o f th e n eu ral tu b e .

Here* owing t o th e extreme­

l y t h in w a ll o f th e brain* i t could not cross* but continued dorsad along th e tectum o f th e same s id e .

A few o f th e f ib e r s en tered th e tectu m .

The r e s t went caudad t o spread out on th e d o r sa l su r fa ce o f th e isthmus* an* end. In a A day embryo* a d e f e c t in th e r o o f o f th e mid brain r e s u lte d in a g r e a t d im in u tio n o f u n crossed t e c t a l f ib e r s on one s id e .

At th e ro s­

t r a l tr o c h le a r l e v e l o f th a t sid e* no lo n g itu d in a l f ib e r s occupied th e l a t e r a l w a ll o f th e b ra in .

Here th e tr o c h le a r nerve made a l a t e r a l e x it*

and formed a la r g e bundle which ended in th e mesenchyme b esid e th e b ra in . A v e ry sm a ll bundle from th e r o s t r a l end o f th e n u cleu s took a d o rsa l in tr a m ed u lla ry course* but ended b efore reach in g th e r o o f.

On th e other

sid e * th e t r o c h le a r nerve went dorsad as fa r as th e roof* emerged homo1 a te r a lly * and ended im m ediately.

Except fo r th e u n ila t e r a l red u ction

in t e c t a l fib e r s * t h i s embryo appeared norm al.

d isc u ssio n

X" th e form ation o f th e IH-V-VX com plex, th e d ir e c t io n o f f ib e r s and r e la t io n s h ip s o f s tr u c tu r e s w ere, in some ways, very s im ila r t o th e norm al.

The c e p h a lic f le x u r e b rin g s th e eye in to such c lo s e r e la t io n t o

th e G asserian g a n g lio n th a t th e m uscle mass fo r th e oculom otor nerve n orm a lly l i e s d i r e c t l y v e n tr a l t o th e g a n g lio n ( i . e . , between th e gang­ l i o n and th e e y e ) , and caudal t o th e ophthalm ic n erv e.

T h erefore, th e

l a r g e , p a r t i a l l y fu s e d , m uscle mass o f midbrain embryos might reason ab ly be su sp ected o f r e p r e se n tin g th e m uscle prim ordia norm ally inn ervated by th e oculom otor and abducens n e r v e s .

In i t s caudalward co u rse, th e

oculom otor n erve o f operated embryos passed m edial t o th e tr o c h le a r n erve, a s i t does in th e normal embryo.

The d ir e c t io n tak en by th e abducens

was n ot changed, but t h i s i s not rem arkable, s in c e th e p o s it io n o f i t s m u scle mass was r e l a t i v e l y unchanged.

The cou rse o f th e ophthalm ic nerve

was g r e a tly a lt e r e d , and was q u ite v a r ia b le in d if f e r e n t embryos.

Nor­

m a lly , th e ophthalm ic branch p a sse s r o s t r a l t o th e m uscle mass of th e oculom otor nerve b efo re i t c r o s s e s over th e o p tic s t a lk .

In th e e x p e r i­

m ental embryos, i t was u s u a lly d isp la c e d c a u d a lly , and became c lo s e ly r e la te d t o e it h e r th e m andibular, or th e m a x illa ry branch. m ic f i b e r s , alm ost in v a r ia b ly en tered th e m uscle m ass.

The ophthal­

The m a x illa ry nerve

was l e a s t a f f e c t e d , whereas th e mandibular nerve o fte n was d iv erted from i t s cou rse t o e n te r th e c e n tr a l com plex.

However, f ib e r s t e a c h in g th e

m andible; from th e complex most probably were o f mandibular o r ig in .

The

freq u en t p resen ce o f a s i n g l e , m edian, nerve su p p lyin g th e mandibular r e g io n was an abnorm ality probably r e la te d t o th e f a c t th a t th e mandibu­ l a r branches had combined in th e m id lin e .

* 9.

C o n c lu sio n s, con cern in g th e f a c t o r s d eterm in in g th e course taken by a n e r v e , are n ot warranted by such a lim ite d amount o f m a te r ia l, nor w ith o u t a c a r e f u l check on a l l p o s s ib le f a c t o r s .

However, c e r ta in p o s s i­

b i l i t i e s are su g g e sted by th e f a i r l y c o n s is te n t p a tte r n formed by th e n er v es under c o n s id e r a tio n . The reason f o r th e tendency t o form m id lin e s tr u c tu r e s i s not c le a r , Whether th e p resen ce o f a median in te r n a l c a r o tid a r te r y caused th e com plex, composed o f m u scle, n erves and g an glion c e l l s , t o organ ize in c lo s e r e la t io n t o i t , can not be s a id .

The con verse might be t r u e .

The

p resen ce o f lo o p s formed by p a ir s o f n erves was another m a n ife sta tio n o f developm ent toward th e m id lin e .

With th e e y e s a b se n t, much o f th e b ila ­

t e r a l i t y o f th e head was l o s t , and i f t h i s a f f e c t s th e " u ltr a s tr u c tu r a l o r g a n iz a tio n " o f th e substratum (W eiss, 1934 c ) th e c o n d itio n s in th e s e embryos would support th e m echanical th eory o f nerve o r ie n t a tio n .

But

t h e r e are so many p o s s ib le in t e r a c tin g f a c to r s th a t no s in g le one can be g iv en prominence on th e b a s is o f th e evid en ce a t hand. The form ation o f lo o p s by th e tr o c h le a r and ophthalm ic p a ir s o f n erv es m ight seem t o in d ic a te t h a t , in th e absence o f th e normal s i t e o f in n e r v a tio n , a nerve i s more s tr o n g ly a ttr a c te d by i t s p a rtn er than by any o th er s tr u c tu r e , but th e abducens (and oculom otor) n erves o fte n formed lo o p s a f t e r e n te r in g t h e ir r e s p e c tiv e m uscle m asses,

Too, great

numbers o f f ib e r s from th e median p lex u s (p robably ophthalm ic and p o st­ g a n g lio n ic ) coursed r o s tr a d /th e oculom otor n e r v e .

Sometimes part o f th e se

f i b e r s were sep a ra te from th e oculom otor n erv e, and th en la y c lo s e t o th e in te r n a l c a r o tid a r t e r y .

Because ophthalm ic f ib e r s probably coursed

caudad in th e abducens n e rv e , when th e oculom otor was a b se n t, i t might be su g g ested th a t nerve f ib e r s f o llo w th e most a v a ila b le tr a c k .

But t h i s

would n ot e x p la in what happens under normal c o n d itio n s .

Furtherm ore, th e

t r o c h le a r n erv es p assed very c lo s e t o th e oculom otor p a ir , y e t communica­ t i o n s between th e s e n erv es were r a r e .

The f a c t th a t th e tr o c h le a r n erves

remained is o la t e d from th e median p le x u s , str o n g ly su g g e sts th a t a m uscle mass has a very s p e c i f i c a t t r a c t io n fo r i t s n erv e. s im ila r a c tio n o f th e abducens nerve in one c a s e .

This i s supported by The entrance of th e

e x te r o c e p tiv e ophthalm ic nerve in to a m uscle mass may be q u ite in c id e n ta l t o a resp on se t o o th er in f lu e n c e s .

The proxim ity o f th e ophthalmic and

oculom otor n erv es t o th e c i l i a r y g an glion in th e norm al, su g g e sts th a t th e same c a u s a tiv e f a c t o r may be o p era tin g in th e exp erim ental embryos.

Such

a c o n s id e r a tio n , however, c r e a te s some d i f f i c u l t y in e x p la in in g th e f r e ­ quent p a r t ic ip a t io n o f th e mandibular nerve in th e median plexus*

Medial

t o r s io n o f th e ophthalm ic p o le of th e G asserian gan glion may be a fa c to r ca u sin g th e v aryin g d eg rees o f com bination observed between th e ophthal­ m ic and m andibular b ran ch es. Hamburger (1 9 3 4 ), by wing bud e x tir p a tio n in th e ch ick embryo, showed t h a t removal o f m uscle prim ordia r e s u lt s in h y p o p la sia o f th e motor column in corresp on d in g segm ents o f th e s p in a l cord .

By means o f wing and hind-

lim b tr a n s p la n ts (1 9 3 9 ) he produced h y p erp la sia in th e motor column and s p in a l g a n g lia , in th e segments corresponding t o th e supernumerary lim b. He concluded t h a t p e r ip h e r a l f i e l d s e x e r t an in flu e n c e on c e n tr a l nervous system developm ent. No h y p o p la sia o f th e tr o c h le a r n u cleu s of th e operated embryos o f th e p r e se n t s e r i e s was n o ted , but th ey are much younger th an th o se o f Hamburger (1 1 -1 2 d a y s). F ia t t ( 1 9 4 0 ), experim enting on th e motor n erves o f th e hypobranchial and fo r e lim b m usculature o f amblystoma, concluded th a t th e r e i s no n erve-

m u scle s p e c i f i c i t y , and t h a t a l l motor neurons are p o t e n t ia lly a li k e . A ls o , t h e h y p o g lo ss a l nerve p e r s is t e d when i t s m uscles were ab sen t. No check on nerve m uscle s p e c i f i c i t y can be made in th e embryos used h e r e .

The p a ssa g e o f th e mandibular nerve through a "foreign"

m uscle mass prob ab ly had l i t t l e s ig n if ic a n c e , s in c e th e nerve continued on t o end in i t s own m uscle primordium. The f a t e o f th e tr o c h le a r nerves in th e absence o f th e oculomotor p a ir would probably have some bearing on th e problem, fo r i t i s l i k e l y t h a t th e oculom otor m uscle m asses could be spared.

However, o p eration s

in ten d ed t o produce such specim ens have been made, but have not been s u c c e s s f u lly l o c a l i z e d .

The m idbrain o fte n responds t o s u r g ic a l in t e r ­

fe r e n c e by d ev e lo p in g pronounced a b n o r m a litie s. The f a t e o f n erv es deprived o f t h e ir normal organ o f in n e r v a tio n , as observed h e r e , d oes n o t f a l l in lin e w ith th e th eo ry o f n eu ro b io ta x is (K appers, 1 9 1 7 ).

A pparently, th e v a r i a b i l i t y in th e course o f th e den-

drons o f th e ophthalm ic n er v e, i s la r g e ly due t o th e presence or absence o f th e oculom otor n er v e.

I t does not seem probable th a t th e s h if t in g

o f th e cou rse o f t h i s nerve from a r o s t r a l t o a caudal d ir e c t io n , i s at a l l concerned w ith th e lo c a t io n o f s t im u li.

In f a c t , th e str u c tu r e s

norm ally b earin g end organs o f t h i s n erv e, have been removed in th e s e em bryos, y e t , when th e oculom otor nerve i s p r e s e n t, many ophthalmic f ib e r s grow r o s tr a d .

There seems no oth er p o s s ib le source fo r so many f ib e r s

a s are added t o th e oculom otor tru n k , r o s t r a l to th e median p le x u s. That th e la r g e g a n g lio n which occu p ies th e median p lex u s i s th e c i l i a r y , seems v ery p ro b a b le.

I t s prom inent, lig h t - s t a i n in g n eu ro b la sts

g iv e i t th e same appearance as th e normal c i l i a r y g a n g lio n , and i t i s in t im a t e ly r e la te d t o th e oculom otor and ophthalmic n e r v e s, and th e in t e r -

?2.n a l c a r o tid a rtery * The occu rren ce o f a s im ila r ga n g lio n in th e p lex u s formed by th e ophthalm ic and m andibular branches in an embryo in which th e tr ig em in a l n erv e o f t h a t s id e was is o la t e d from any o th er nerve len d s support t o t h e cla im ( C o w g ill* 1942) th a t th e c i l i a r y g a n g lio n i s a d e r iv a tiv e o f th e c e p h a lic n eu ra l c r e s t * r a th er than a c e l l group which has m igrated a lo n g th e oculom otor nerve from th e som atic motor n u cleu s (Jones* 1942)* In one ‘tr ig e m in a l" embryo* a comparable c e l l group was observed on th e ophthalm ic nerve*

However* in o th er "trigem in al" embryos* a c le a r ly

i d e n t i f i a b l e g a n g lio n was n ot seen* although o c c a sio n a l n eu ro b la sts were found*

In two embryos w ith d isco n tin u o u s m idbrains* th e ey es and surroun*

d in g r e g io n s were normal ex cep t th a t only one oculomotor nerve was p resen t i n one c a s e ; and in th e o th er specimen* a l l n erves from th e midbrain were absent*

In both embryos* a normal appearing c i l i a r y gan glion was observed

in c o n ta c t w ith each ophthalm ic nerve*

To fu r n is h p roof fo r neural c r e s t

o r ig in o f th e c i l i a r y gan glion* th e ce p h a lic n eural c r e s t co n ta in in g th e sem ilu n ar primordium should be com p letely removed*

T h is has been accom­

p lis h e d in amphibian embryos by H arrison (1924) and D etw iler (1937)* and a neatfly s u c c e s s fu l attem pt in th e chick by th e author p o in ts t o th e f e a s i b i l i t y o f th e exp erim en t. In specim ens w ith a l a t e r a l l y emerging tr o c h le a r nerve* i t was d i f f i ­ c u l t t o fin d a common f a c to r as a p o s s ib le cause fo r th e abnorm ality. An extram ed u llary m edial lo n g itu d in a l f a s c ic u lu s en tered th e b rain a t , or very near th e s i t e o f emergence o f th e tr o c h le a r nerve in two c a s e s , a«d in one embryo a d ie n c e p h a lic t r a c t occupied th e mesenchyme c lo s e to th e e x i t o f th e IVth n er v e, but en tered th e n eural tube at a d iff e r e n t le v e l*

However* in one in sta n ce* both m edial lo n g itu d in a l f a s c i c l e s

a p p e a r e d e n t i r e l y n o r m a l , y e t one t r o c h l e a r n e r v e e n t e r e d t h e m e sen ­ chym e fro m t h e b a s a l p l a t e * th in ,

I n t h r e e e m b ry o s , t h e r o o f p l a t e w as v e r y

b u t t h e a b n o rm a l t r o c h l e a r n e r v e i n e a c h o f t h e s e s p e c im e n s d i d n o t

c o u r s e a s f a r d o r s a l l y a s w as p o s s i b l e .

T h a t a d e f e c t i n t h e r o o f was

n o t r e s p o n s i b l e f o r t h e l a t e r a l e m e rg e n c e w as a p p a r e n t i n one o f t h e s e e m b ry o s , i n Tidiich t h e t r o c h l e a r n e r v e o f t h e m o re n o rm a l s i d e g rew d o r s a d , b u t l e f t th e b r a in w ith o u t c r o s s in g .

I n a n o t h e r em bryo w ith a t h i c k

r o o f a t t h e i s t h m u s , one t r o c h l e a r n e r v e c r o s s e d , b u t part* o f t h e o t h e r e m erg ed l a t e r a l l y , t i o n fo rm e d .

The b r a i n w as v e r y a b n o r m a l, and no t y p i c a l d e c u s s a ­

I n an em bryo w h ic h h a s n o t b e e n d e s c r i b e d , b o th IV th n e r v e s

e m erg ed h o m o l a t e r a l l y b u t fro m t h e d o r s a l p a r t o f t h e i s t h m u s .

T h e re

h ad b e e n some dam age t o t h e t e c t u m . The o n ly f a c t o r , common t o tra lly

a l l f o u r em b ry o s h a v in g l a t e r a l l y

o r ven-

e m e r g in g t r o c h l e a r n e r v e s , w as a b s e n c e , o r g r e a t r e d u c t i o n , o f t h e

u n c ro sse d t e c t a l t r a c t .

Y e t i n tw o c a s e s , a v e r y s m a l l p a r t o f t h e t r o c h ­

l e a r n e r v e c o u r s e d d o r s a d w i t h i n t h e b r a i n f o r a s h o r t d i s t a n c e , and e n d e d w i t h o u t e m e r g in g .

S in c e t h e s h a p e o f t h e b r a i n a t t h e t r o c h l e a r

l e v e l d i f f e r s fro m t h a t m ore r o s t r a l l y an d c a u d a l l y , one w o u ld e x p e c t a m ore c o m p le x c a u s a t i v e f a c t o r t h a n i s

s u g g e s te d by t h e s e o b s e r v a t i o n s .

H o w e v er, re m o v a l o f t h e t e c t u m , w i t h b e t t e r l o c a l i z a t i o n o f t h e o p e r a t i o n s , t o p r e v e n t dam age t o

s u rro u n d in g r e g io n s , i s

in d ic a te d .

INCIDENCE OF SPONTANEOUS MOTILITY IN OPERATED EMBRYOS In a l l op erated specim ens o f f iv e days and more* spontaneous m o t i l i t y was ob served .

In experim ental* as w e ll as normal* embryos

t h e ty p e o f movement was c a r e f u lly n oted .

O bservations agreed w ith

d e s c r ip t io n s o f a c t i v i t y in t h i s phase o f development* given by oth er a u th o rs (C lark and C lark , 1914) (Orr and W indle, 1934) (P rey er, 1 8 8 5 ). Spontaneous movements were p e r io d ic , as observed by th e Clarks* A b r i e f p erio d o f m o t i l i t y was fo llo w ed by a lo n g er phase o f q u iescen ce. A c t iv it y occurred about every m inute or h a lf-m in u te .

There seemed t o

be no p a r t ic u la r r e la t io n s h ip between am niotic c o n tr a c tio n s and spon­ ta n eo u s movements o f th e embryo.

F requently th e amnion was in e r t* and

a c t iv e movements o f th e embryo o fte n took p la ce at th a t tim e; but th ey d id occur a ls o during am niotic a c t i v i t y . Spontaneous a c t i v i t y fo llo w ed no p a tte r n , but th e random movements were s im ila r in normal and operated specim ens.

In th e p eriod between

4 and 4 1 /2 d a y s, very few movements o f th e trunk were se e n .

Most

movements in v o lv ed th e head or th e t a i l a lo n e , or both moved sim u ltane­ ou sly*

E a r l i e s t movements o f th e head were sim ple v e n tr a l f le x io n and

e x te n sio n or l a t e r a l f l e x i o n s .

U su ally th e movements were quick and

Jerky in ch aracter* and th e r e was no smooth t r a n s it io n from one movement t o a n o th er.

At t h i s stage* independent movements of th e t a i l were

in freq u en t*

An e x c e p tio n t o t h i s was an embryo from which a s e c tio n

o f th e b ra in a t th e o to c y s ts had been removed.

From e a r l i e s t observa­

t i o n s ( 4 - d ays) t a i l movements were predom inant, although one sim ul­ ta n eo u s f le x io n o f th e head was observed .

In la t e r sta g es* t h i s embryo

e x h ib ite d t w i s t i n g and r o l l i n g movements of th e tru n k .

Another embryo

w ith th e raetencephalon sep arated from th e m esencephalon, at 5 l / 2 d ays, showed a s im ila r ty p e o f movement.

No ex cu rsio n s o f th e head were seen*

In some o f th e exp erim en tal embryos, e it h e r head or t a i l movements appeared t o be predom inant, but t h i s seemed t o have no r e la tio n s h ip t o t h e l e v e l o f o p e r a tio n .

Some o f th o se w ith th e fa c ia l- l e v e l at th e end

o f th e b ra in had head movements alm ost e x c lu s iv e ly , w h ile oth ers p o s s e s s ­ in g a tr ig e m in a l r e g io n showed more tru n k , or t a i l , than head movements.

DISCUSSION The appearance o f th e s e movements in embryos la c k in g a l l o f th e brain r o s t r a l t o th e low er m etencephalon i s c o n tr a d ic to r y t o th e claim s o f Angulo (1 93 9 ) th a t th e m edial lo n g itu d in a l f a s c ic u lu s i s th e e s s e n t ia l in t e g r a t in g pathway fo r e a r ly spontaneous movements.

Indeed, in some

m o tile embryos, th e b rain r o s t r a l t o th e m yelencephalon was com p letely sep a ra ted from th e m edulla and s p in a l cord .

(The f a c t t h a t , in some of

t h e s e specim ens movements were con fin ed t o th e low er tru n k , was probably due t o in ju r y t o neck m u scles, fo r s e v e r a l oth er specim ens of s im ila r o p e r a tio n did e x h ib it movements o f th e head, w h ile one separated at th e j u n c tio n o f th e m etencephalon and m esencephalon a ls o had i t s movements lim it e d t o th e tru n k .

The l e v e l of th e low er p art o f th e nervous system

in th e l a t t e r embryo was t r ig e m in a l.) V i s i n t i n i and M o n tca lcin i (1939) b e lie v e th a t endogenous stim u la tio n o f c e r e b r a l c e n te r s i s r e q u is it e f o r normal spontaneous movements, and th e y cla im t o have proved t h i s by p h y s io lo g ic a l experim ents.

In th e

p r e s e n t m a te r ia l, absence o f th e i n t e r s t i t i a l n u c le u s, or of t e c t a l c e n te r s ,

had no e f f e c t on m o t ilit y *

In some specim ens, even th e la r g e - c e lle d

n u c le i o f th e r e t i c u l a r form ation a t th e tr ig e m in a l and f a c i a l l e v e l s w ere a b sen t or sep a ra ted from th e a ccesso ry and s p in a l n u clei* I n te r r u p tio n s o f th e c e n tr a l nervous system in th e low er m yelencep h a lo n and v a r io u s l e v e l s o f th e s p in a l cord should provide fu rth er in fo rm a tio n about th e n ature o f spontaneous m o tility * The o b se r v a tio n s rep orted h ere are con fin ed t o th e p eriod o f d e v e l­ opment, during which no resp on se t o e x te r n a l s tim u li can be e lic it e d * F u rth er work on t h i s problem , ex ten d in g in cu b a tio n p erio d s fo r s e v e r a l d a y s, and perform ing o p e r a tio n s a t many d if f e r e n t l e v e l s should provide some i n t e r e s t i n g r e s u lts * CONCLUSIONS 1*

Spontaneous m o t ilit y in "m yelencephalic" ch ick embryos

i s comparable t o t h a t in th e c o n tro ls* 2.

^ ie n c e p h a lic , t e c t a l , v e s tib u la r c e n te r s and th e magno-

c e l l u l a r n u cleu s o f th e r e t ic u la r form ation o f th e m etencephalon are n o t n e c e ssa r y fo r spontaneous m o tility *

SUMMARY

The o r i g i n an d e a r l y d e v e lo p m e n t o f t h e m e d ia l l o n g i t u d i n a l f a s c i c u l u s w a s s t u d i e d i n r a t , c a t an d human em bryos* fe re n tia tio n s ta r ts

a t tw o f o c i :

d i e n c e p h a l o n an d m e s e n c e p h a lo n *

The f i r s t n e u r o f i b r i l l a r * d i f ­

t h e h i n d b r a i n and t h e j u n c t i o n o f t h e From t h e l a t t e r f o c u s , p r o c e s s e s o f

n e u r o b l a s t s g ro w c a u d a d w i t h o u t c r o s s i n g , a n d fo rm t h e f i r s t l o n g i t u d i ­ n a l t r a c t I n t h e b r a i n , t h e d i e n c e p h a l i c co m p o n en t o f t h e m e d ia l l o n g i t u ­ d in a l fa s c ic u lu s *

S oon f i b e r s o f t h e r h o m b e n c e p h a lic r e g i o n , h a v in g

c r o s s e d i n t h e f l o o r p l a t e , becom e l o n g i t u d i n a l i n t h e m e d ia l p a r t o f th e b a s a l p la te *

Many o f t h o s e i n t h e m e te n c e p h a lo n a s c e n d , and a s d e v e lo p ­

m e n t p r o c e e d s , tn e y m e e t an d o v e r l a p w i t h t h e d e s c e n d in g d i e n c e p h a l i c fib e rs . I n t h e c h i c k e m b ry o , t h e m e d ia l l o n g i t u d i n a l f a s c i c u l u s d e v e l o p s s im ila rly .

A f te i

o v e rla p p in g o f t r a c t s ta k e s p la c e , i t

d e t e r m i n e t h e t e r m i n a t i o n o f i n d i v i d u a l c o m p o n e n ts . d iffic u lty ,

e x p e r i m e n t a l m e th o d s w e re u s e d .

i s im p o s s ib l e t o

To ov erco m e t h i s

R em oval o f t h e f o r e b r a i n o f

t h e 3 8 - h o u r c h i c k em bryo p r e v e n t e d d e v e lo p m e n t o f t h e d i e n c e p h a l i c compo­ n e n t*

O th e r e l e m e n t s w e re e l i m i n a t e d by e x c i s i n g f o r e - a n d m i d b r a i n , and

f o r e - , m id - an d a s m a l l p a r t o f t h e h i n d b r a i n .

H i a t u s e s w ere made i n t h e

n e u r a l t u b e a t one l e v e l o f t h e o t o c y s t , and i n t h e m id b r a in * g ro w th o f t e c t a l t r a c e s , t h e r o o f o f t h e m i d b r a in w as e x c i s e d .

To p r e v e n t Em bryos

w e re f i x e d a t 1 / 2 d a y i n t e r v a l s fro m 4 t o 6 d a y s , i n c u b a t i o n a g e .

A ll

m a t e r i a l w as p r e p a r e d by t h e p y r i d i n e - s i l v e r m e th o d . E xcept n e a r tn e s i t e s u rp ris in g ly l i t t i e tio n

o f o p e r a t i o n , a b s e n c e o f p a r t s o f t h e b r a i n had

e f f e c t on m ost e m b ry o s .

At t h e a r t i f i c i a l t e r m i n a ­

o f t h e n e u r a l t u b e i n t h e h i n d b r a i n , t h e b r a i n was a l t e r e d in

a p p e a r a n c e , d u e t o a r o u n d in g up o f t h e b r a i n w a l l s t o c l o s e t h e e n d i n g . F i b e r t r a c t s becam e x e a r r a n g e d t o b u t, a t 5 l/2

c o n fo rm t o t h e s h a p e o f t h e n e u r a l tu b e

an d 6 d a y s , a s c e n d i n g f i b e r s c o n t in u e d b ey o n d t h e b r a i n ,

a n d c r o s s e d t h e m esencnym e t o t h e e c to d e r m n e a r b y .

L o c a l, c o n t r a l a t e r a l

f i b e r s fo rm e d a r a d i a l c o m m issu re t o r e a c h t h e r e t i c u l a r f o r m a t io n o f t h e o p p o s ite

s id e ,

in

o p e r a t e d e m b ry o s , f i b e r t r a c t s f r e q u e n t l y em erg ed fro m

th e n e u ra l tu b e . A b se n c e lo n g itu d in a l

0!

£ i n t e r s t i t i a l n u c le u s had no

f a s c i Cl*i u s o f t h e m e te n c e p h a lo n a t

e f f e c t on t h e m e d ia l 5 d a y s , and l i t t l e ,

i f any

l o s s w as n o t e d a t 6 d a y s e v e n w i t h b o th f o r e b r a i n an d r a id b r a in a b s e n t . L oss o f p a r t

o f t h e t e c t u m a n d p o s s i b l y some o f

t h e c e r e b e l l a r a n la g e

r e s u l t e d i n r e d u c tio n o f t h e r e t i c u l a r fo rm a tio n i n th e tr ig e m in a l r e g io n ; and i n t h e v e n t r a l l o n g i t u d i n a l f a s c i c u l u s o f t h e m e te n c e p h a lo n and u p p e r m y e le n c e p h a lo n .

A l t e r a t i o n o f t h e v e n t r a l l o n g i t u d i n a l f a s c i c u l u s w as

p ro b a b ly due t o te g m e n ta l o r c e r e b e l l a r d e f e c t s . l o n g i t u d i n a l f a s c i c u l u s w as a p p a r e n t . c y st re s u lte d in d is to r tio n

No c h an g e i n t h e m e d ia l

A h i a t u s a t th e l e v e l o f th e o to -

o f t h e b r a i n n e a r t h e o p e r a t i o n , an d p r o b a b ly

som e r e d u c t i o n o f t h e m e d ia l l o n g i t u d i n a l f a s c i c u l u s a t t h e t r i g e m i n a l re g io n .

More r o s t r a l l y , t h e b r a i n w as n o r m a l.

The h i n d b r a i n was v e r y

a b n o rm a l n e a r t h e o p e r a t i o n , b u t m ore c a u d a l l y , i t w as c o m p a ra b le t o t h e n o rm a l • On t h e b a s i s o f t h e s e r e s u l t s , i t w as c o n c lu d e d ; lic

t h a t t h e d ie n c e p h a ­

co m p o n en t o f t h e m e d ia l l o n g i t u d i n a l f a s c i c u l u s d o e s n o t e n t e r t h e

t r i g e m i n a l r e g io n b e f o r e 5 1 /2 d a y s .

The a s c e n d in g t r i g e m i n a l com ponent

s e n d s a few f i b e r s a s f a r a s t h e d i e n c e p h a l o n a t 5 1 / 2 - 5 d a y s , b u t m o st of it s

f i b e r s go l a t e r a d

i n b u n d le s c a u d a l t o t h e t r o c h l e a r l e v e l , and

do n o t g e t b ey o n d t h e m i d b r a in in t h e s t a g e s s t u d i e d .

The v e s t i b u l a r

c o m p o n e n ts d e s c e n d t o t h e u p p e r v a g u s l e v e l , an d a s c e n d i n t o t h e u p p e r m e te n c e p h a lo n by 6 d a y s . I n em b ry o s h a v in g no e y e s , t h e o c u lo m o to r , o p h th a lm ic (a n d so m e tim e s t h e a b d u c e n s ) n e r v e s fo rm e d a p l e x u s l o c a t e d i n a f u s e d m u s c le m ass b e tw e e n th e

s e m ilu n a r g a n g l i a .

p a rtia lly

su rro u n d e d th e

A l a r g e m e d ia n g a n g l i o n o c c u p ie d t h e m a s s , and s i n g l e m e d ia n , i n t e r n a l c a r o t i d a r t e r y *

The oph­

t h a l m i c n e r v e u s u a l l y becam e p a r t i a l l y m e rg e d w i t h m a n d ib u la r o r m a x i l l a r y f i b e r s , b u t s e p a r a t e d fro m th e m i n t h e p l e x u s . m ic ) a s c e n d e d i n t h e ab d u cen s.

F i b e r s ( p r o b a b ly o p h t h a l ­

o c u lo m o to r n e r v e , an d so m e tim e s d e s c e n d e d i n t h e

The t r o c h l e a r n e r v e s r a r e l y c o m m u n ica ted w i t h o t h e r s , an d u s u a l l y

fo rm e d a l o o p , t h r o u g h w h ic h t h e o c u lo m o to r n e r v e s d e s c e n d e d .

In th e p le x ­

u s , t h e o c u l o m o to r , o p h th a lm ic ( a n d o f t e n t h e a b d u c e n s ) fo rm e d s i m i l a r lo o p s . I n f o u r e m b ry o s , t h e t r o c h l e a r n e r v e o f one s i d e em erg ed l a t e r a l l y . T h i s o c c u r r e d i n tw o c a s e s , n e a r t h e p o i n t a t w h ic h an e x t r a m e d u l l a r y m e d i a l l o n g i t u d i n a l f a s c i c u l u s w as r e - e n t e r i n g t h e b r a i n , b u t t h e o n ly f a c t o r common t o a l l f o u r s p e c im e n s was a b s e n c e , o r g r e a t r e d u c t i o n , o f u n c ro sse d t e c t a l t r a c t s . A l l o p e r a t e d em b ry o s o f f i v e d a y s o r m o re , show ed s p o n ta n e o u s m o t i l ­ ity

r e s e m b l i n g t h a t s e e n i n t h e n o r m a l.

I n some c a s e s , m o t i l i t y o c c u r r e d

e v e n th o u g h t h e m e d u lla an d s p i n a l c o r d w e re s e p a r a t e d fro m t h a t p a r t o f t h e b r a i n c o n t a i n i n g t h e i n t e r s t i t i a l n u c l e u s , t h e t e c t u m , and t h e l a r g e c e l l e d n u c l e u s o f t h e r e t i c u l a r f o r m a t i o n o f t h e m e te n c e p h a lo n .

T h ere­

f o r e , d e s c e n d i n g t r a c t s f o r t h e s e c o l t e r s a r e n o t e s s e n t i a l f o r sp o n ­ ta n e o u s m o t ili ty .

BIBLIOGRAPHY

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th e C ranial Sympat­ ( in p r e s s)

Davenport, H. A ., W. F . W indle and R. H. Beech 1934. Block s ta in in g o f nervous t i s s u e w ith s i l v e r IV. Embryos. S ta in T ech., v o l . 9, p p . 5 -1 9 . D etw iler , S . R. 1924 E f f e c ts o f b i l a t e r a l e x tir p a tio n o f a n te r io r limb rudim ents in amblystoma embryos. J . Comp. N eu r., v o l 37, pp. 1 -1 4 . _______________ 1928 Further experim ents upon a lt e r a t io n o f th e d ir e c t io n o f growth in amphibian s p in a l nerves* Exp. Zool* v o l . 51, pp. 1-! 1934 An exp erim ental stu d y o f s p in a l nerve segm entation i n Amblystoma, w ith r e fe r e n c e t o th e p lu riseg m en ta l c o n trib u tio n s t o th e b r a c h ia l p le x u s . Exp. Z o o l., v o l . 67, pp. 395-441 _______________

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Jour. Exp.

Hamburger, V ik to r 1934. The e f f e c t s o f wing bud e s tir p a t io n on th e develop­ ment o f th e c e n t r a l nervous system in ch ick embryos* J . Exp. Z o o l., v o l . 68, pp. 449-494.

__ “

1938 M orphogenetic and a x ia l s e l f - d i f f e r e n t i a t i o n o f tr a n s "“p la n te d lim b pr&mordia o f 2-day c h ic k embryos. A. Exp. Z o o l., v o l . 68, pp. 4 4 9 -4 9 4 . ______ 1939 Motor and se n so ry h y p e r p la sia fo llo w in g limb-bud tr a n s ­ p la n ta tio n s i n c h ic k embryos. P h y s io l. Z o o l., v o l . 12, pp. 268-284. 1939 The developm ent and in n er v a tio n o f tra n sp la n te d limb primo rd ia o f c h ic k embryos. J . Exp. Z o o l., v o l . 80, pp. 347-389

H arrison, R. G. 1904 Experiment e l l e Untersuchungen uber d ie Entwiok-1 ungen der S invesorganve der S e i t l i n i e den am phibien. Arch. Entwicklungsm eoh. a r g . V o l. 63, p p. 35-149. ____________ 1907 a . Experim ents in tr a n sp la n tin g lim bs and t h e ir bearing up­ on th e problems o f th e development o f n e r v e s . J . Exp. Z o o l., v o l . 4 , p p . 2 3 9 -2 8 1 . ____________

1907 b . O bservations on th e l i v i n g , d evelop in g nerve f i b e r . Exp. Z o o l., v o l . 4 , pp. 116-118.

J.

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1910 b . The development o f p erip h era l nerve f ib e r s in a lte r e d su rro u n d in g s. Arch. f . m ikr. A n a t., v o l . 3 0 , pp. 1 5 -3 3 .

_________

1924 N eu rob last v s . sheath c e l l i n th e development o f p e r ip ­ h e r a l n e r v e s . J . Comp. N eu r., v o l . 37, pp. 123-205.

Hogg, I . D. 1930 Form ation o f th e f a s c ic u lu s lo n g itu d in a l i s m e d ia lis in embryos o f th e a lb in o r a t . A nat. R eo ., v o l . 45, pp. 222. H o ltfr e te r , Joh . 1933 Nachsreis der in d u k tio n sfu h ig k e it a b z e to te te r Keimfceile* Arch. f . E ntw icklungs- mech. Organ*, v o l. 128, pp. 584633. Jones, D. S . 1937 O rigin o f th e sym pathetic trunks in th e c h ick embryo. A nat. R e o ., v o l 7 0 , pp. 4 5 -6 5 . '

1939 S tu d ie s on th e o r ig in o f sh ea th c e l l s and sym pathetic g a n g lia in th e c h ic k . A nat. R e o ., v o l . 73, pp. 343-357.



1941 Further s tu d ie s on th e o r ig in o f sym pathetic g a n g lia in th e c h ic k embryo. A nat. R eo ., v o l . 1 7 , pp. 7-15*

~11

1942 The o r ig in o f th e v a g i and th e parasym pathetic g a n g lio n c e l l s ' o f th e v is c e r a o f th e c h ic k . Anat. Rec*, v o l . 82, pp. 185—197.

1942 The 'o r ig in o f th e c i l i a r y g a n g lia in th e ch ick embryo. A nat. R e c ., v o l . 8 2 , 0 . 3 2 -3 3 . Kappers, C. U. A riens 1917. Further c o n tr ib u tio n s on n e u r o b io ta x is . Comp. N eu r., v o l . 2 7 , pp. 261-298. Kuntz, A.

1920 The development o f th e sym pathetic nervous system in man. J . Comp* N eu r., v o l . 32, pp. 173-229.

Kuntz, A,

1934 The Autonomic Nervous system , P h ila d e lp h ia , Lea and F e b ig e r , pp. 117-127.

J.

Lehmann, F . E. 1926. Entw icklungs storungen in der m edullaranlage von t r it o n , erzen g t durch u n te r la g e r u n g d e fe ls te . Arch. Enturick-lungsm eoh. O rgan., v o l . 1§>8 p p . 243-282. Mesdaj, T. M. 1909 B ijd rage t o t de ont-w ikkel& ngsgeschiedens van der stru ctu u r der h ersen en b i j hetkepembryo. M. De Waal, Gronigen. Orr &W indle

1934 The Development o f Behavior in ch ick embryos; th e appearance o f som atic movements. J . Comp. N eu r., v o l . 60, pp. 271-284.

P ia t t , Jean . 1940 Nerve m uscle s p e c i f i c i t y in Amblystoma stu d ied by means o f cord g r a f t s . J . exp . Z o o l., v o l . 85, pp. 211. Freyer, W* 1885

S p e c ie lle p h y s io lo g ie des embryo.

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R hines, Ruth and W illiam E. W indle, 1941. The e a r ly development o f th e f a s c ic u lu s lo n g itu d in a l i s m e d ia lis and a s s o c ia te d secondary neurons in th e r a t , 6 a t and man. J . Comp. N eu r., v o l . 75, pp. 165-189. T e llo , J . F . 1923 Les d if f e r e n c ia t io n s neuronales dans l ’ embryon du p o n le t, pend ant l e s prem iers jo u rs de 1*in cub ation* Trav. Lab. Rech. B i o l . , U niv. Madrid, T. 2 1 , pp. 1 -9 3 . "

1934 Les d if f e r e n c ia t io n s n e u r o f ib n illa ir e s dans l e prosencephale de l a s o u r is de 4 a 15 mm. Trav. Lab. Rech. B i o l . , Univ. Madrid, T. 1 9 , p p . 3 3 9 - 3 9 5 .

V is in tin a , Fabio and R ita L evi-M ontale i n i 1939. R elazion e tr a d iffe r e n z ia z o n e s t r u t t u r a le e fu n g io n a le d e i c e n t r i e d e lle v ie nervose n e l l ’ embrione d i p o l i o . A rchives S u is s e s de N eurologie e t de P s y c h ia tr ic W allenberg, A d o lf, 1903 Eine z e n tr ifu g a l le it e n d e d ir e k te Verbindung der fr o n ta le n V orderhiribasis m it der oblongata (+ Ruckenmark?) b e i der E n te. Anatomischer A nzeiger Bd. 22, pp. 289-292. W eiss, Paul

1933 F u n ctio n a l a d a p ta tio n and th e r o le o f groundsu b stan ces in developm ent. Am. N a t., v o l . 67, pp. 322—340.

1934a A nat. Rec* S ecre ta r y a c t i v i t y o f th e inner la y e r o f th e c h ic k as r e v e a le d by th e t i s s u e c u ltu r e , v o l . 58, pp. 299-302. _ _ _ _ _ _ _ _ _ 1934b Motor e f f e c t s o f sen so ry nerves ex p erim en ta lly con n ected w ith m u sc le s. Anat* R ec*, v o l . 60, pp. 437-448 _____________ 1934c In v i t r o experim ents on th e fa c to r s determ ining th e co u rse o f th e outgrowing nerve f i b e r s . J . Exp. Z o o l., v o l . 68, pp. 3 9 3 -4 4 8 . W indle, W* F* 1932a The n e u r o f ib r illa r s tr u c tu r e o f th e 7 mm. c a t embryo. J* Comp. Neur*, v o l . 55, pp. 99-138. 1932b The n e u r o f ib r illa r stru o tu re o f th e 5.5mm. c a t embryo J . Comp. N eu r., v o l . 5 6 , pp. 315-331. ____________

1933 N e u r o fib r illa r defelopm ent in th e c e n tr a l nervous system o f e a t embryos betw een 8 and 12 mm. lo n g . J . Comp. N eur., v o l. 5 8 , pp. 643-723* 1935 N e u r o fib r illa r development o f c a t embryos; e x te n t o f de­ velopm ent in th e telen c e p h a lo n and diencephalon up t o 15 mm. J . Comp. N eu r., v o l . 6 3, pp. 139-171.

W indle, W* F* and M. F . A u stin 1936 N e u r o fib r illa r development in th e c e n tr a l nervous system o f ch ick embryos up to 5 days in cu b ation s J . Comp. Neur*, v o l . 63, pp. 431-463* W indle, W. F* and R* E* Baxter 1936 The f i r s t n e u r o f ib r illa r d iff e r e n t a tio n in a lb in o r a t embryos* J . Comp. N eu r., v o l . 63, pp. 173-187.

ILLUSTRATIONS

F ig . 1 Graph based on th e enum eration o f lo n g itu d in a l f ib e r s in t h e area a d jacen t t o th e f lo o r p la te on one sid e and th e number o f f ib e r s in th e v e n tr a l commissure o f c a t embryo 845s 3 b eginning at th e low er end o f th e n u cleu s o f th e m edial lo n g itu d in a l f a s c ic u lu s ( zero p o i n t ) . The s o li d l i n e r e p r esen ts th e m edial and v e n tr a l lo n g itu d in a l f a s c i c u l i ; th e secondary f ib e r s o f th e v e n tr a l commissure are d esig n a ted by a bro­ ken l i n e ; th e number o f motor f ib e r s in th e v e n tr a l commissure i s shown by a d o tted lin e * L evels o f motor n u c le i are Roman num erals. The sharp d ecrea se in th e number o f lo n g itu d in a l f ib e r s in th e ro s­ t r a l p a rt o f th e brain stem i s due t o th e f a c t th a t th e m edial lo n g itu ­ d in a l f a s c ic u lu s ends a t th e tr ig e m in a l l e v e l at t h i s s t a g e . Caudal t o th e low er end o f th e m edial lo n g itu d in a l f a s c ic u lu s th e r e i s a c o r r e la ­ t i o n between the appearance o f tn e secondary commissure and th e in c r e a s e i n number o f f ib e r s in th e v e n tr a l lo n g itu d in a l fa s c ic u lu s * In th e t r i ­ gem inal reg io n th e peak o f th e curve o f lo n g itu d in a l f ib e r s i s h ig h in p ro p o rtio n t o th e s iz e o f th e commissure because o f a la r g e h o m o la tera l c o n tr ib u tio n t o th e v e n tr a l lo n g itu d in a l tra ct* In th e glossop h aryn ­ g e a l - vagus reg io n th e v e n tr a l lo n g itu d in a l f a s c ic u lu s i s o ld er and lo n g e r , and more f ib e r s per s e c tio n have accum ulated; t h i s i s r e f le c t e d in th e h e ig h t o f th e peaks o f th e upper cu rve. The more caudal p o s it io n of th e peaks o f th e secondary commissure as compared w ith th e peaks formed by p lo t t in g th e number o f lo n g itu d in a l f ib e r s su g g e sts th a t th e lo n g itu ­ d in a l t r a c t i s predom inantly ascending* Figs* 2, 3, 4 Diagrams o f brainstem s of ca t embryos 846, 841 and 8 4 5 , showing th e degree o f development o f th e m edial lo n g itu d in a l f a s ­ c ic u lu s and th e v e n tr a l lo n g itu d in a l f a s c ic u l u s . Roman numerals d e s ig ­ n a te th e l e v e l s o f th e motor n u clei* In fig u r e 2 , th e m edial lo n g itu d in a l f a s c ic u lu s i s a b se n t. Secon­ dary n e u r o b la sts have begun t o d i f f e r e n t ia t e in th e g lo sso p h a ry n g ea lv a g u s-a cc esso r y r e g io n , but no v e n tr a l commissure has been formed. In fig u r e 3 , th e m edial lo n g itu d in a l f a s c ic u lu s , exten d s only t o t h e t r o c h le a r l e v e l . A few secondary n eu ro b la sts have appeared in th e tr ig e m in a l r e g io n . Secondary neurons are most numerous at th e g lo s s o ­ p h aryn geal- vagus l e v e l . Some secondary axons in th e low er rhombence­ phalon reach th e f lo o r p l a t e , but no v e n tr a l lo n g itu d in a l t r a c t has form ed. No secondary n eu ro b la sts are p resen t a t th e f a c i a l l e v e l . In fig u r e 4 , th e m edial lo n g itu d in a l f a s c ic u lu s has en tered th e tr ig e m in a l r e g io n . I t overlap s th e v e n tr a l lo n g itu d in a l f a s c ic u lu s w hich i s formed h o m o la tera lly and c o n t r a la t e r a lly by th e ascen d in g axons o f secondary n e u r o b la sts. In th e rhombencephalon, th e secondary axons have formed a v e n tr a l commissure and a v e n tr a l lo n g itu d in a l f a s c i c u l u s . At th e f a c i a l l e v e l , th e v e n tr a l lo n g itu d in a l f a s c ic u lu s i s in ter ru p ted ( th e commissure i s e n t ir e ly motor) and secondary n e u r o b la sts are j u s t b eg in n in g t o d i f f e r e n t i a t e .

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Camera lu c id a drawings o f d e t a il s o f brain s tr u c tu r e s in p y rid in e s i l v e r sta in e d ca t and r a t embryos. The r e c ta n g le s in th e sm all f i ­ gu res (a ) in d ic a te th e areas drawn in th e la r g e f ig u r e s . M agn ifica­ t i o n f o r f ig u r e s 5 , 6 , 8 , 9 , 1 0 , 11, 12: X 290; fo r fig u r e s 7 , 13: X 515. 5 Cat 846: 2; g lo ssop h aryn geal l e v e l . On th e r ig h t , th e axons of two l i g h t l y sta in e d in tern eu ron s are d ir e c te d v e n t r a lly through th e m otor n u c le u s. A ll oth er s tr u c tu r e s are p a j ts of motor neurons. No v e n tr a l t r a c t and no v e n tr a l commissure are presen t* 6 Cat 841; s p in a l cord . Two sp in a l motor n e u r o b la sts are p r e s e n t. Due t o th e absence o f f ib e r s of th e a ccesso ry nerve at t h i s l e v e l th e course o f secondary neurons i s unobscured. The one f ib e r seen in th e f l o o r p la t e i s a commissural secondary axon. No v e n tr a l lo n g itu d in a l f a s c ic u lu s i s p r e s e n t.

7 Cat 841; v a g u s-a ccesso ry l e v e l . The f ib e r s in th e f lo o r p la t e are axons o f secondary n e u r o b la sts. F ib ers seen in c r o s s s e c tio n l a t e r a l t o t h e th r e e motor n e u r o b la sts are not forming a v e n tr a l lo n g itu d in a l t r a c t but are d ia g o n a lly cut motor axons which were tr a ced to th e intram edul l a r y motor ro o t o f th e vagus in oth er s e c t io n s . 8 Cat 845: 2; r o s t r a l tr ig e m in a l l e v e l . No v e n tr a l commissure i s p r e s e n t. The darker m edial bundle o f f ib e r s (upper) i s th e m edial lo n g i­ tu d in a l f a s c i c u l u s . L ateral t o and below i t , lig h t e r lo n g itu d in a l f i ­ b ers rep resen t th e r o s tr a l p art o f th e v e n tr a l lo n g itu d in a l f a s c ic u lu s . One tr ig e m in a l motor n eu ro b last i s p resen t and p a r ts of another motor axon appear a t r ig h t a n g les t o th e lo n g itu d in a l t r a c t f ib e r s .

9 Tat 633: 1; tr ig e m in a l r e g io n . A shrinkage f o ld in th e b a sa l p l a t e has sc a tte r e d th e elem ents in such a way th a t hom olateral secon ­ dary f ib e r s can be seen en ter in g th e v e n tr a l lo n g itu d in a l f a s c ic u lu s from th e reg io n o f th e motor n u c leu s. The v e n tr a l commissure i s j u s t b eg in n in g t o form. Two r o s t r a lly d ir e c te d growth cones appear in th e v e n t r a l lo n g itu d in a l f a s c ic u lu s . 10 Cat 845: 4; tr ig e m in a l l e v e l . The v e n tr a l lo n g itu d in a l f a s c ic u ­ l u s i s se c tio n e d d ia g o n a lly and co n ta in s one r o s t r a l l y d ir e c te d growth co n e . L i t t l e o f th e motor n ucleus appears. 11 Cat 845: 2 ; s e c tio n through th e oculomotor reg io n a t th e cepha­ l i c f le x u r e . The la r g e n e u r o b la sts at th e upper r ig h t rep resen t th e cau d al p a rt o f th e n u cleu s o f th e m edial lo n g itu d in a l f a s c i c u l u s . The m ed ia l lo n g itu d in a l f a s c ic u lu s cou rses caudad near th e e x te r n a l l i m i t ­ in g membrane. Many o f i t s f ib e r s term in ate in growth cones a t t h i s l e v e l * Figure 19 i s a photomicrograph o f th e same r e g io n . 12 Cat 845: 2; m iddle tr ig e m in a l l e v e l . F ib ers from th e sm all ven­ t r a l commissure are e n te r in g th e v e n tr a l lo n g itu d in a l f a s c ic u l u s . The m ed ial lo n g itu d in a l f a s c ic u lu s has ended above t h i s l e v e l . Compare w ith fig u r e 8 . 13 Cat 845: 3; vagus l e v e l . V e n tr a lly d ir e c te d growth cones o f secon ­ dary neurons appear d o r sa l and v e n tr a l t o , and w ith in th e motor n u c le u s . One d o r s a lly d ir e c te d growth cone can be seen on a f ib e r approaching th e v e n tr a l lo n g itu d in a l f a s c ic u lu s o f t h i s s id e .

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