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Histochemical localization of choline acetyltransferase in the deep cerebellar nuclei of the rat Petrali, Elena Harriet 1972

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The  Histochemical Localization  Choline Acetyltransferase Deep C e r e b e l l a r  Nuclei  in  of  of  the  the  Rat  by  Elena Harriet B.Sc.  A  (Hon.),  University  Thesis submitted  Petrali  of B r i t i s h  in partial  the requirements Master  of  in  Columbia,  fulfillment  f o r the degree  of  Science  the  ;  Department of P h y s i o l o g y  We  accept this the  The  thesis  required  University  as  conforming  standard  of B r i t i s h  September,  to  1972  Columbia  of  1969  In p r e s e n t i n g t h i s  thesis  an advanced degree at the L i b r a r y I  the U n i v e r s i t y  s h a l l make i t  f u r t h e r agree  in p a r t i a l  freely  f u l f i l m e n t o f the of B r i t i s h  available  for  requirements  Columbia, I agree  for  that  r e f e r e n c e and s t u d y .  that permission for extensive copying of t h i s  thesis  f o r s c h o l a r l y purposes may be g r a n t e d by the Head o f my Department o r by h i s of  this  representatives.  It  thesis for financial  i s understood that gain s h a l l  written permission.  Department o f  1*H VX/aLoCY  The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8, Canada  copying o r p u b l i c a t i o n  not be a l l o w e d without my  ABSTRACT A histochemical  method f o r t h e l o c a l i z a t i o n o f  choline a c e t y l t r a n s f e r a s e , the synthesizing of a c e t y l c h o l i n e has been r e c e n t l y Conditions and  o f t h e method  optimized  developed.  were f i r s t  i n the s p i n a l cord  investigated  o f t h e r a t where  c h o l i n e r g i c n e u r o n e s a r e known t o be Following  enzyme  present.  s t a n d a r d i z a t i o n o f t h e method t h e  l o c a l i z a t i o n o f t h e enzyme was s t u d i e d i n t h e deep c e r e b e l l a r n u c l e i .  A series of  transverse  s e c t i o n s were s t a i n e d t o f a c i l i t a t e , a l l o c a t i o n o f c e l l s d e m o n s t r a t i n g enzyme a c t i v i t y r e s p e c t i v e n u c l e i as d e s c r i b e d  to their  i n the l i t e r a t u r e .  The p r e s e n c e o f c h o l i n e a c e t y l t r a n s f e r a s e  was  used as a d i r e c t i n d i c a t i o n o f a c h o l i n e r g i c n e u r o n e , and  was f o u n d p r e d o m i n a n t l y i n t h e c e l l s  v e n t r a l p o r t i o n of the l a t e r a l nucleus,  i n the on t h e  b o r d e r o f t h e i n t e r p o s i t u s and l a t e r a l n u c l e i and i n the caudal  p o r t i o n of the medial nucleus.  results obtained  are consistent with  i n d i c a t i n g t h a t a p o r t i o n of: t h e pathway i s c h o l i n e r g i c .  other  The studies  cerebello-thalamic  tm  T A B L E OP  CONTENTS  Abstract  ii  List  of Figures  v  List  of Abbreviations  vii  Acknowledgements  •  ix  Introduction A.  G e n e r a l Comment  B.  Morphology  C.  1  of the Cerebellar  Nuclei  6  I.  Nucleus  medialis  7  II.  Nucleus  interpositus  9  III.  Nucleus  lateralis  Fibre  Connections  12  General  12  I. II. III.  IV.  comment  Cortico-nuclear Brachium  projections  conjunctivum  10  13 16  1.  Nucleus  medialis  17  2.  Nucleus  i n t e r p o s i t u s and l a t e r a l i s  21  Summary  23  Methods A.  Preparation  25  B.  Cholineacetyltransferase  25  C.  Acetylcholinesterase  27  D.  Histological  28  Staining  (IV)  /  Table  o f Contents. -  continued  Results A.  29 S p i n a l Cord I.  Sectioning  30  II.  Incubation  30  1.  Precipitation  2. III. IV. B.  30  o f CoA-SH and  acetyl^  CoA w i t h  lead  31  Controls  and i n h i b i t o r s  35  Sodium  Sulphide  Histochemical  38  localization  o f AChE  Cerebellum I. II.  40 43  Nucleus medialis  43  Nuclei  49  i n t e r p o s i t u s and l a t e r a l i s  Discussion A.  Evaluation I. II. III. IV.  o f t h e Method  Histochemical  55  localization  55  Substrates Mechanism  56 of histochemical  localization  The enzyme-ChAc  B.  The C e r e b e l l o - T h a l a m i c  C.  Summary  60 Pathway  64  and Conclusions  71  Bibliography Appendix  I II III IV V  58  72 Choline  acetyltransferase  80  Acetylcholinesterase  82  Paraffin  84  Sections  Cresyl Violet Haematoxylin-Darrow  86 Red  88  (V)  LIST  OF  Figure  1  Subdivision  Figure  2  Relative  FIGURES  of the c e r e b e l l a r  distribution  cerebellar  of c e l l  3  Phylogenetic  Figure  4  Comparison  of c e l l  Figure  5  Morphology  of r a t c e r e b e l l a r  Figure  6  Longitudinal  Figure  7  development  nuclear  anatomical  zones  nuclei. of  cortico-  of Brodal's  longitudinal cortico of the c a t  according  Voogd.  8  Fibre  Figure  9  Pathological  composition  of brachium  changes  conjunctivum.  i n the c e r e b e l l a r n u c l e i  cat following unilateral  brachium  transection of  conjunctivum.  Figure  10  Effect  of  Figure  11  Effect  of doubling  Figure  12  Effect  of o l d coenzyme.  Figure  13  a) E f f e c t o f b)  of c e r e b e l l a r n u c l e i  s i z e s of c e r e b e l l a r n u c l e i  p r o j e c t i o n zones  Figure  the  found i n  p r o j e c t i o n s of the c a t .  Modification  to  types  nuclei.  Figure  nuclear  nuclei.  Control  time. t h e coenzyme  pre-incubation.  result.  Figure  14  Effect  Figure  15  Typical  Figure  16  Acetylcholinesterase  of formalin  fixation.  result. result.  concentration  Figure  17  Middle the  Figure  18  1/3 o f r o s t r o - c a u d a l  cerebellar Violet.  Caudal  2/3 o f r o s t r o - c a u d a l  Figure  19  20  21  i n vagal  22  of the r a t .  staining f o r  and m e d i a l  nuclei.  46  V i o l e t and AChE s t a i n i n g i n  caudal  medial  nucleus.  47  staining  i t s corresponding  medial Figure  of  45  C o m p a r i s o n o f ChAc and  extent  Violet.  Cresyl the  Figure  nuclei  Comparison o f c e l l s ChAc  Figure  44  cerebellar  Cresyl  of  nuclei of the r a t .  Cresyl  the  extent  section  control  i n the  nucleus.  Representative bution  48 section  of cells  showing  exhibiting  distri-  ChAc  activity. Figure  23  Cresyl small  V i o l e t a n d AChE s t a i n i n g i n celled ventral  lateral Figure  24  ChAc  50  portion  of the  nucleus.  51  and AChE s t a i n i n g i n s m a l l  ventrolateral  portion  of the  celled  lateral  nucleus. Figure  25  Figure  26  Figure  27  Interpositus  52 cells  s t a i n i n g f o r ChAc  and AChE  53  ChAc  i n the cerebellum.  66  AChE i n t h e c e r e b e l l u m .  67  tv\\)  LIST  Acetyl-CoA  OJF A B B R E V I A T I O N S  acetyl-Coenzyme  A  ACh .  acetylcholine  AChE  acetylcholinesterase  ANS  ansiform  1  ANSU  ;  ansula  lobe (Bolk)  ANT  anterior  lobe  BC  brachium  conjunctivum  C  cytoplasm A  ChAc  v., v  CV  cholineacetyltransferase cresyl  DFP  »  di-isopropylfluorophosphate  F  Nucleus  FLO  violet  '  fastigius  ..'. ' f l o c c u l u s  Fr  fragmentation of tissue  HEPES  N-2-Hydroxyethylpiperazine-N-2ethanesulfonic acid  HDR  Haematoxylin-Darrow  Red  A  HSR  ,.  ,  Harleco Synthetic  Resin  I  Nucleus  IA  Nucleus  interpositus  anterior  IP  Nucleus  interpositus  posterior  L  :  L  c  L  r  M  Nucleus pars  interpositus  lateralis  convexa  . ' • •" p a r s Nucleus  of Nucleus  rotunda  of Nucleus  medialis  lateral lateral  (viii) List  of Abbreviations - continued  mis  millilitres  Mo  motoneurone  N  neurone  Nc  nucleus  NP  non-specific  PFL  paraflocculus v  PMD RN  paramediam  lobe  r e d nucleus  SIM  . lobulus simplex  SLP  subnucleus  SMP  V  ul  •  •;  lateralis parvicellularis  microlitre -  VL < ..  (Bolk)  subnucleus m e d i a l i s p a r v i c e l l u l a r i s  V  WM  precipitation  ,  •'."•:  vestibular  nuclear  nucleus v e n t r a l i s •, .. t h a l a m u s white  matter  complex l a t e r a l i s of the  . . (ix) ACKNOWLEDGEMENTS  The a u t h o r w i s h e s the  project  V '  t o t h a n k D r . H. M c L e n n a n f o r s u g g e s t i n g  and f o r h i s c o n s t r u c t i v e  throughbut! t h e development Many t h a n k s a l s o  of this  a d v i c e and c r i t i c i s m thesis.  t o Mr. Ron Walker  technical  assistance  practical  histological  forh i s excellent  and v e r y h e l p f u l i n s t r u c t i o n i n technique.  The c o n t r i b u t i o n s o f  Mr. K u r t Henze f o r p r i n t i n g t h e photographs  and M r s . Yvonne  Heap f o r h e r c o n t i n u e d i n t e r e s t i n a l lp h a s e s are  gratefully  of the project  acknowledged.  A s p e c i a l thanks t o Diane copy  .  the  rough  for  her conscientious  Cumming f o r c h e e r f u l l y t y p i n g .  a n d d r a w i n g F i g . 22 a n d t o M i s s P a t t i typing  Miller  of the f i n a l manuscript..  INTRODUCTION  A.  General It  (cytoand in  Comment  i s now g e n e r a l l y  and m y e l o a r c h i t e c t u r e ) ,  ontogenetic), both  that  rostro-lateral medial pair  (n.  man) m e d i a l l y , globosus  lateral  pair  laterally, of  In  medially  (1961;  addition  nucleus;  does s u b d i v i d e caudal pars and  The  nucleus  nucleus  caudo-  (n. f a s t i g i u s  posterior  i t .  a  The  .,  rostro-  (n. dentatus  anterior  o f man)  (n. emboliformus  i t (FLOOD a n d J A N S E N , 1 9 6 1 ; M A S S I O N ,  1967). t o these  and a subnucleus  does n o t mention  medio-lateral  (VOOGD, 1 9 6 4 ) ;  with  interpositus  subdivided  distinguishable.in  interpositus  four  main d i v i s i o n s  portion  lateralis  convexa  a ventro-lateral  medialis  nucleus  parvicellularis  c  rotunda  into  with  parvi-  of the medial  nucleus.  (SLP) VOOGD  of the medial nucleus  ( L ) merging pars  portion  of the l a t e r a l  a subdivision  the lateral  FLOOD a n d J A N S E N  i n the c a t a subnucleus  (SMP) i n t h e c a u d o - m e d i a l  the v e n t r o l a t e r a l  and  (Fig. 1).  laterally  with  Fig. 2 ) ,described  cellularis  pair  of the lateral  and the nucleus  NIEWENHUYS,  of.two  morphology  (phylogenetic  c a n be  nuclei  of the medial or roof  o f man) m e r g i n g  man) m e r g i n g  1967;  i n two groups  and t h e nucleus  consists  nuclei  The f o u r  and a caudo-medial  consists  of their  and development  (WEIDENREICH, 1 8 9 9 ) ,  (BRUNNER, 1 9 1 9 ) .  mammals c a n b e f o u n d  on t h e b a s i s  the cerebellar  rostro-caudal  directions  of  agreed  two p a r t s ;  interpositus  (L ) r o s t r a l l y  a  i n (1964)  a l t h o u g h he dorso-  a n t e r i o r (IA) ( F i g .1&2).  -2-  BRUNNER  MED  WEIDENREICH OGAWA  t i g . 23. H o r i z o n t a l section o f the cerebellum o f the cat. illustrating the mediolatcral s u b d i v i s i o n o f Brunner a n d the rostrocaudal  subdivision o f the central cerebellar nuclei o f W e i d e n r e i c h - O g a w a .  H a t c h e d : fastigial nucleus ( F ) . S m a l l dots: posterior interposed interposed  n u c l e u s (IA) p a r s c o n v e x a o f t h e l a t e r a l  nucleus (Lr). F o r abbreviations  F I G U R E 1. S u b d i v i s i o n  - n. i n t e r p o s i t u s  LAT - n .  see p. 131.  of thecerebellar nuclei  MED - n . m e d i a l i s INT  n u c l e u s (IP). F i l l e d c i r c l e s : a n t e r i o r  n u c l e u s (Ix) a n d p a r s r o t u n d a o f t h e l a t e r a l  lateralis  (VOOGD, 1 9 6 7 )  -3-  <Z2&>  i.,  i  12  ;'..;<'.7  -v-  20 nuc. rn , nuc.  -f  .  V^V  •• . —<&r— sup  r  28  Fig. 1 A series of sections through the cerebellar nuclei of a kitten. The numbers (4, 12, 20 etc.) refer to the number of the section in the series used for the drawings, number 1 being the section through the rostral bonier of the nuclear complex. On the right side the relative size of the nerve cells and their distribution within the nuclear subdivisions are indicated. The left half shows the boundaries of the nuclei as defined in the present investigation. Nuc. i., nucleus interpositus; nuc. 1., nucleus lateralis; nuc. m., nucleus niedialis or faatigii. This diagram is used in figures 3 to 7 to illustrate the pathological changes observed in tho experiments.  C> 11  F I G U R E 2.  R e l a t i v e d i s t r i b u t i o n o f c e l l types found i n c e r e b e l l a r n u c l e i ( M o d i f i c a t i o n o f Jansen and Jansen. (1955); to i n c l u d e i n f o r m a t i o n from F l o o d and Jansen (1961). DP  - dorsolateral situated  p r o t u b e r a n c e o f Goodman. L a r g e  between f a s t i g i a l  and i n t e r p o s i t u s  SLP  - subnucleus  parvicellularis  lateralis  SMP  - subnucleus  parvicellularis  medialis  <  cells nuclei.  H o w e v e r , FLOOD a n d J A N S E N lateral  small  equivalent In  celled  portion of the lateral  t o VOOGD's  comparative  (1961) do n o t c o n s i d e r  their  nucleus  ventro-  t o be  h^.  studies  of the cerebellum  i t has been  found  (DOW, 1 9 4 2 ; A R I E N S e t a l . , 1 9 3 6 ; C R O S B Y e t a l . , 1 9 6 2 ) t h a t portions i.e.,  o f i t s hemispheres  the vermal  medially  placed  zone  c e r e b e l l a r nucleus.  of the lateral  from  l o w e r mammals t h r o u g h  3).  Similarly  variation  throughout  nucleus.  DOW  of  man, s t a t i n g t h a t  forms bulk  increase lateral  and  part  increase  by an i n c r e a s e  nucleus  shows much  r e f e r s t o t h e two p a r t s  nucleus  phylogenetic  the medial  o f the dentate  appears  t o be  of phylogenetically  e x p a n d e d new p a r t , w h i c h  older  comprises the  i n man, i s s i t u a t e d v e n t r o l a t e r a l l y . T h e  i n the ascending phylogenetic  nucleus(ventro-  scale  i s parallel-  i n the s i z e of the parvocellular red nucleus s y s t e m i n man.  Homology o f t h e c e r e b e l l a r n u c l e i i n t h e p a s t based  nucleus  greater  than does  the dorso-medial portion  the cerebello-thalamic  principally  i n the  reflectedi n  i n the size of the parvocellular lateral portion)  t h e more  of the lateral  t h e mammalian s p e c i e s  the lateral  of the nucleus  The g r a d u a l  t o man i s p a r t i c u l a r l y  the lateral  and t h e l a t e r a l l y  with  older,  ( F i g . 5) o f t h e c e r e b e l l a r h e m i s p h e r e s  (1942) a l s o  homologous w i t h  ed  zone  increasing size of the lateral  (Fig.  phylogenetically  (Fig. 5 ) ,are associated  size  the  considered  those  on t h e i r  development.  comparison o f t h e i r  fibre  morphology  and t h e i r  has been  ontogenetic  and  Few s t u d i e s w e r e a v a i l a b l e t o a l l o w connections  More r e c e n t l y however, v a r i o u s  and p h y s i o l o g i c a l f u n c t i o n s .  extensive  studies  have been  done  -sI  I  '  med.va.1  "•  CAT  PHAL ANGER  rostral<—  MONKEY  -» caudal  F I G U R E 3. P h y l o g e n e t i c d e v e l o p m e n t o f c e r e b e l l a r n u c l e i (Voogd, 1967) T r e n d shows d i m i n i t i o n i n t h e r e l a t i v e s i z e o f t h e medial n u c l e u s , and t h e i n c r e a s e d s e p a r a t i o n between IA + I P and L c + I P . A l s o t h e development and differentiation of Lc into a rostral magnocellular and c a u d a l p a r v o c e l l u l a r p a r t c o r r e s p o n d i n g t o t h e human d e n t a t e n u c l e u s . :  :  F - f a s t i g i a l (medial nucleus). IA + IP a n t e r i o r + p o s t e r i o r i n t e r p o s i t u s , L c + L r p a r s convexa and r o t u n d a o f l a t e r a l n.  in  t h e c a t (FLOOD a n d JANSEN., 1 9 6 6 ) , a n d m o n k e y  on  the efferent  connections;  EAGER, 1963) , a n d monkey projections  which  establishment of true  a n d i n t h e c a t (VOOGD, 1 9 6 4 ;  (EAGER, 1966) o n  thereby  provide  been done on t h e m o r p h o g e n e s i s cerebellar  and  on t h e c o r t i c o - n u c l e a r  nuclei  this  the  available  study  cerebellar  their  hemispheres  nuclei  morphology  projections  connections In  outlining  attention  will  to the  (BC),  here  lateral  cerebellar  fastigial uncinate B.  scanty  of the nuclei.  projections  of on  as d e s c r i b e d  (1968a) and w i t h  by  refer-  efferent  projection  on t h e p r o p o r t i o n a l  nuclei  particular  contribution  makes t o t h e b r a c h i u m  as t h e a s c e n d i n g nuclei  systems  conjunctivum  t r a c t a r i s i n g from  and i n c l u d i n g  that  the  ipsi-  the contralateral branch of the  fasciculus. of the Cerebellar  cerebellar  horizontal in  only  be b a s e d p r i m a r i l y  component known as t h e a s c e n d i n g  Morphology The  i s s t i l l  established.  be f o c u s e d  defined  cerebellar  done on t h e c a t where t h e  the efferent  each of the c e r e b e l l a r  have  of  the nomenclature of the d i v i s i o n s  and c o r t i c o - n u c l e a r  have been  and f i s s u r e s  connections  of the r a tw i l l  to similar studies  f o rthe  (LARSELL, 1952; KORNELIUSSEN,1968b)  GOODMAN e t , a l . | 1 9 6 3 ) a n d K O R N E L I U S S E N ence  criteria  of the lobes  on t h e e f f e r e n t  therefore  cortico-nuclear  I n t h e r a t , d e t a i l e d studies  (GOODMAN e t a l . , 1 9 6 3 ) b u t t h e r e  information In  further  homologies.  the  (RAND, 1 9 5 4 )  nuclei  and s a g g i t a l  Nuclei  have been c l e a r l y d e f i n e d  sections  with  i n  N i s s l and m y e l i n  transverse, stains  t h e c a t (VOOGD, 1 9 6 4 ; FLOOD a n d J A N S E N , 1 9 6 1 , 1 9 6 6 ) a n d i n t h e  -7-  white  r a t (GOODMAN e t a l . , 1 9 6 3 ; K O R N E L I U S S E N ,  general of  cyto-architecture  cells  similar  found  dorsal  part  t o the large  flattened.  portions  portions,  The l a r g e  and t h e small  portions  Figure cerebellar  cells  nucleus  polygonal  cells  t h e medium  cells  cells  of the kitten. nucleus  also  found  cells  (MATSUSHITA  cat  a n d r a t showed i t t o be composed o f c e l l s  i n the  i nthe central ventro-lateral  throughout the  silver  the  cells  sometimes  of the cells  Golgi  types  medium  (FLOOD a n d J A N S E N ,  4 shows t h e r e l a t i v e s i z e s  i n the medial  cells,  p a r t i c u l a r l y in.the  nuclei  The  are three  a r e commonly  but scattered  o f the other  nuclei  there  cells, and small  of the nuclei,  of the lateral  ventral  that  i n a l l nuclei,.large  i n shape  appearing  indicates  1968a).  1961).  found  studies  i n the  done on  e t a l . , 1971) o f t h e of the following  dimensions: large medium small Cells the  cells cells  X 40^  20-30 .  X 30^  A  cells  o f s i m i l a r dimensions  35-40^  12.5-20 X 2 0 A  have a l s o  been found  4  i na l l nuclei of  c e r e b e l l u m o f t h e c a t (EAGER, 1 9 6 8 ) a n d m o n k e y ( C O U R V I L L E e t a l . ,  1970).  .  I.  .  Medial This  fourth  nucleus  nucleus  i s situated  '  o f middle  sized  j u s t above t h e r o o f  o f f caudally. cells.  .  of cells  tending  i s no s p e c i f i c r e f e r e n c e  cells  i n the c a tb u t these  I t i s composed  protuberance  t o be. l a r g e r  There  ofthe pre-  The r o s t r a l t h i r d o f t h e  i n the r a t has a d o r s o l a t e r a l  consisting  '  nucleus  v e n t r i c l e and tapers  dominantly  \  ( F i g . 5)  (GOODMAN e t a l . , 1 9 6 3 ) .  t o t h e above mentioned  seem t o c o r r e s p o n d  group o f  t o t h e group o f  r.-i Nuc. f  Nuc.  i  Nuc.  1  4  V  Figure 2 Fig. 2 Types of normnl nerve cells from the cerebellar nuclei (to the left) and cells displaying retrograde reactions (to the right). Nuc. f., nucleus fastigii; nuc. i., nucleus interpositus; nuc. 1., nucleus lateralis, Camera lueida drawing. Leitz oil imm. 1/12. Oc. 5, reduced to X 530.  F I G U R E 4. C o m p a r i s o n o f c e l l s i z e s o f c e r e b e l l a r (Jansen and J a n s e n , 1955)  nuclei  7neurones  lying  between  the medial  t h a t JANSEN-and JANSEN J R . be r e f e r r e d In  the caudal  scattering sized is  to later  two-thirds of the medial  become  more  length  approximately  will  connections.  there  i s a and s m a l l  and JANSEN s 1  1  there  SMP', 1966)  to one-sixth of the  total  done by MATSUSHITA e t a l . (1971) ,  justification  rostral  portions.  II.  uncinate  cylindrical  a difference  portion  The d i v i s i o n sections  difficult  between  and  IA and IP i s b e s t  complex  (IA) i s  two-thirds, while the caudal  visible  one-  i n  (VOOGD, 1 9 6 4 ) b u t i n t r a n s v e r s e s e c t i o n s i t  will  The r o s t r a l  up t h e r o s t r a l  (IP) i s o v o i d and o c c u p i e s  to define.  interpositus  the caudal  portion of the interpositus  i n shape and takes  the p o s t e r i o r  between  but the axonal  interpositus  The a n t e r i o r  For the purposes of this  be c o n s i d e r e d pole  medium  and s m a l l s i z e d  middle  third,  Ventrally  medial  the contralateral  f o r s u b d i v i d i n g the nucleus  to indicate  Nucleus  horizontal  to join  of the caudal  Thus on t h e b a s i s o f c y t o - a r c h i t e c t u r e t h e r e i s  seems  the  study  cross i n the cerebellum  pattern  is  (FLOOD  found t h a t t h e axons o f t h e c e l l s  fasciculus.  third.  nucleus  group  packed, and v e n t o - m e d i a l l y  one-fifth  the Golgi s i l v e r  nucleus  little  This  (Fig.2)  of the nucleus.  In it.was  noted.  nuclei  d o r s o - m e d i a l l y , t h e medium  closely  a group o f s m a l l c e l l s  extending  (1955) have  i n considering the efferent  of large cells  cells  and i n t e r p o s i t u s  cells.  Medium c e l l s cells  then  unit.  of the interpositus  and l a r g e s i z e d  i n the caudal  as a  investigation  complex c o n s i s t s  predominate i n the  i n the caudal  one-third small cells  third.  are found  (FLOOD  of  -10-  and  JANSEN, In  t h e r a tand c a t t h ed i v i s i o n  lateralis et  nuclei  by  a dorsolateral  on t h e border,  that  has c e l l s  theadjacent  toward  appear  nucleus  this  (1968a)  suggests  portion with that  thedivision  I l l .  and taken Nucleus In  of large  medium a n d s m a l l  belonged  t o the  on t h e c a t,  a n d KORNELIUSSEN as an e n t i t y i n  o f GOODMAN e t a l . (196 3)  will  t o the'hump' a s i n d i c a t e d i n F i g . 5 .  lateralis  cells  two-thirds  dorsally with  The v e n t r a l h a l f  sized cells  medium a n d l a r g e axes p o i n t i n g  nucleus  be c o n s i d e r e d  o f medium s i z e d c e l l s .  nucleus  i s composed  medium a n d s m a l l  I n t h e caudal  predominate with  sized cells  toward  this  cells  i s composed c h i e f l y  areoften  theorigin  t h e most ventrally,. s i t u a t e d ' c e l l s  appearance.  portion  projections  t o either of theadjacent nuclei.  lateral  i t s rostral  found c e n t r a l l y .  and  i tcould  t h e study  observed  long  thelateral  n o tbelong  be  this  on e f f e r e n t  however, i n s t u d i e s  and thereby  this  The  f l a t t e n e d and oriented  complex.  include  chiefly  (i.e.,  indicated that  FLOOD a n d , J A N S E N ( l 9 6 6 )  In  ( F i g . 5)  t o be s i m i l a r t o t h e c e l l s  t h e h i l u s o f BC) h o w e v e r , s t u d i e s  interpositus  chiefly  'hump' a r e a  GOODMAN  o f t h e i n t e r p o s i t u s and l a t e r a l  that  lateral  t h e same a u t h o r  itself  between t h e nucleus  and i n t e r p o s i t u s has been v a r i o u s l y placed.  a l . (1963), d e s c r i b e s  situated  of  1961).  o f medium  third the few l a r g e  oriented  o f thebrachium  with  cells. their  conjunctivum  areflattened i n  -n-  NUCLEUS VERMAL PARAVERMAL! |  ZONE  NUCLEUS DENTATUS  FASTIGII  ZONE  I  ,,_J  |,C[Nllf»T, /^•CtNUAL'l LATERAL ZONE  '  CULMENI  r  9  PARAROCCULUS  h FLOCCULONODUIAR'LOBE NUCLEUS INTERPOSITUS  B Fig. 1 A , Schematic representation of surface topography o f rat cerebellum (modified after L a r s e l l , "52). F u n c t i o n a l cortical zones are indicated. B , Serial diagrams o f every eighth transverse section through the ipsilateral cerebellar nuclear complex. Oblique lines i n nucleus fastigii, levels c through e, indicate region o f dorsolateral protuberance; s t i p p l i n g i n m e d i a l aspect of nucleus interpositus, levels b through e, identify the crest region; a n d vertical lines i n lateral aspect o f nucleus interpositus indicate the dorsolateral " h u m p " region.  F I G U R E 5. M o r p h o l o g y o f R a t c e r e b e l l a r : (Goodman e t a l y  nuclei :  -\%-  pars on  VOOGD's  (1964) d i v i s i o n o f t h e l a t e r a l  convexa  (L ) a n d p a r s  cytoarchitectonic  connections. cellularis of  lateralis  The F i b r e I.  that  carry  peduncles. peduncle  of  small  afferent  and/or e f f e r e n t  The p r i m a r y  fibres to structures  primarily  terminating  reticularis  VOOGD, The  fibres  cells.  root  cerebellar  branch  i t (VOOGD, 1 9 6 4 ,  1967;  component,  and i n t e r p o s i t u s  nuclei  nucleus The  c e r e b e l l a r peduncle  tract,  consists of  tectocerebellar and  (BRODAL e t a l . , 1 9 5 4 ) ,  of the fifth  efferent  theascending  (BRODAL e t a l . , 1 9 7 2 ) .  thesuperior  bundles  the cerebellar  contains  i nthecontralateral  pontis  fibres  of fibre  has a descending  thelateral  spinocerebellar  rubrocerebellar  joins  I talso  from  component-of  mesencephalic  c  i n t h e mes- a n d d i e n c e p h a l o n  together with  principally  tegmenti  the p o s t e r i o r  staining  nerves./ termed  conjunctivum, which  t h e uncinate f a s c i c u l u s which  1966;  darkly  thecaudal L  component o f t h e s u p e r i o r  i t s ascending.branch,  afferent  with  parvi-  comment  i s thebrachium  originating  fibre  subnucleus  t o coincide  as c o n t a i n i n g  FLOOD a n d J A N S E N , 1 9 6 6 ) .  and  (1961)  on  c e r e b e l l u m has three main p a i r s  cerebellofugal via  butalso  solely  Connections  General The  appears  into a  (L ) i s n o t based  considerations  FLOOD a n d J A N S E N ' s  VOOGD d e s c r i b e d  C.  rotunda  nucleus  c r a n i a l nerve  and f i b r e s o f t h e (FLOOD a n d J A N S E N ,  1964).  middle  c e r e b e l l a r peduncle  containing  probably purely  i sa well  defined  cerebellopetal  bundle o f  afferent  fibres.  The  inferior  restiform the  and  inferior  direct  and  cerebellar  juxtarestiform peduncle  that  uncinate fasciculus.  the  descending  the  juxta-restiform  Marchi by  branch  fibres  direct  (VOOGD,  of  investigation  c a t and  corpus  this  cerebelli  i s arranged  zones;  a vermal  vermal  zone c o n n e c t e d w i t h  lateral  zone r e l a t e d  zone p r o j e c t i n g  were proposed  t o be  (2) r o s t r o - c a u d a l l y cortical  found  i t .  and  The  (Fig. 6):  using  and  lateral  t h e r e was the  (1) e a c h  principles  half  of  cortical para-  n u c l e u s and  s y m m e t r i c a l and  an  deep  f o l l o w i n g two  nucleus.  the  undertaken  i n three longitudinal  to the c e r e b e l l a r  localization  based  on  These  a divisions  non-overlapping;  nuclei.  Functional  stimulation  experiments  of  was  to correspond to these anatomical observations of  JANSEN e t a l . (1940),(CHAMBERS and propositions  whether  the i n t e r p o s i t u s  bilaterally  and  through  to the medial nucleus, a  to the  form  there i s a point-to-point.localization  projections  longitudinal  cortex  betweeen the c o r t e x  investigation  the  tract  pass  monkey was  to determine  i n - the white matter below from  are  together  fastigio-bulbar  the c e r e b e l l a r  rabbit,  (1940)  relationship  that  of  1964).  An  BRODAL  main components  the uncinate f a s c i c u l u s  body  the  concern:-this i n v e s t i g a t i o n  The  of  technique i n the  resulted  also  The  projections  anatomical  the  bodies.  Cortico-nuclear  JANSEN and  nuclei  i s composed o f  crossed fastigio-bulbar  the  II.  peduncle  were q u e s t i o n e d by  monkey and  by  SPRAGUE, 1 9 5 5 ) .  EAGER  GOODMAN e t a l . ( 1 9 6 3 )  (1963,  1966)  These i n the  i n the r a t .  These  cat  -m-  N. ve*tib.  Fig. 4. Diagram of the main principles in the corticonuclear projection in the cerebellum of the cat as established in Marchi studies. The mutually interconnected divisions of the cerebellar cortex and the intracerebellar nuclei are marked with identical symbols. The drawings of the transversely sectioned nuclei are arranged from rostral (above) to caudal (below). Note longitudinal zonal pattern. Cf. text. From Jansen and Brodal (1940).  F I G U R E 6. L o n g i t u d i n a l a n a t o m i c a l z o n e s o f c o r t i c o - n u c l e a r projections of the cat. ( B r o d a l , 1967) ' !  FLO  j|| zone A and caudal  vermis  zoneB zoneB' zone C and C' O, zone D and Cf  F I G U R E 7. M o d i f i c a t i o n o f B r o d a l ' s l o n g i t u d i n a l c o r t i c o n u c l e a r p r o j e c t i o n z o n e s o f t h e c a t a c c o r d i n g t o VOOGD. (Voogd, 1964) ~~ ' ' ~ Only narrow d i v i s i o n s of c o r t e x p r o j e c t s p e c i f i c a l l y t o one n u c l e u s as i n d i c a t e d above. Division of c e r e b e l l a r n u c l e i a s i n F i g . 1. ANT - a n t e r i o r SIM  - lobulus  lobe simplex  PMD - p a r a m e d i a n PFL  lobe  - paraflocculus  ANS - a n s i f o r m ANSU -  ansula  lobe (Bolk)  (Bolk)  authors exist had  concluded  they  to  a r e n o t as s t r i c t l y  claimed.  distinct,  that although  VOOGD  (1964) a l s o  observation  into  pattern  Suffice  i t here  Brachium  tion will  and areas  the  be  mentioned.  The  fibres  and compact.  latter  about  nuclei. with  fibres  Only  raphes.  BRODAL projection  b y VERHAART into  conjunctivum (1970):  surrounded  In the lateral  spread  except  parts  laterally  , coarse  and l a t e r a l  fine  part,  already i n the  from  the central  i n which  fibres  investiga-  i n the r a t  of a centre richly  by an area  where  areas o f  ' BC i s m o d e r a t e l y  a medial  i sdistinct  part consists  major  and term-  pertinent to this  t h e two p a r t s o r i g i n a t e  less.  very  reviewed.-by  zones  conception of the  BC.  o f the brachium  I t sd i v i s i o n  The m e d i a l  coarse  termed  t o be c o n s i d e r e d t h e o r i g i n  twice as l a r g e  c e r e b e l l u m , where  on t h e  nuclei.  particularly  are d e s c r i b e d as f o l l o w s large  i s based  of the longitudinal  t o VOOGD"s  o f t h e component p a r t s o f  termination  on t h a t o f  conjunctivum  There remains ination  fibres  (1940)  relate  to say that the cortico-nuclear  of the cerebellar  III.  based  The d i a g r a m  have been c r i t i c a l l y  l e n d s more s u p p o r t  divisions  nuclei  as they  sharply,circumscribed bundles,  These i n v e s t i g a t i o n s  do  t h a t - t h e zones were n o t  h i sresults  (Fig. 1).  that the efferent  collected  (1967).  found  of the cerebellar  W E I D E N R E I C H a n d OGAWA  zones  d e f i n e d a s J A N S E N a n d BRODAL  and F i g . 7 i l l u s t r a t e s  the subdivision  are  the longitudinal  they  a r e more  fibres  provided dominate  evenly  a r e numerous.'  These d i v i s i o n s well  (Fig. 8).  indicated  and  followed  by  In the  l a t e r a l i s of  Nucleus  tracts;  i n the  the main area of the  of  t h e BC  thalamus  i s  red nucleus.  (VL)  METTLER, 1954  as  mesencephalon  t e r m i n a t i o n i s the (ALLEN,  Beyond  nucleus  1924;  ).  medialis  The  medial nucleus  gives rise  the  first,  from  nucleus,  i n the c a t  course  a p a r t i a l ' t e r m i n a t i o n i n the  VOOGD,1964; CARREA a n d 1.  obvious  same F i g u r e ' t h e  shown t o d e c u s s a t e  the red nucleus ventralis  are d i s t i n c t - a n d  arising  i s composed o f  the  s m a l l and  t o two  fastigio-bulbar  caudal two-thirds of  medium s i z e d  fibres  r o s t r a l l y i n the a n t e r i o r  to  the uncinate f a s c i c u l u s ;  the  one-third  the n u c l e u s , i s composed  rostral coarse  f i b r e s , and  fastigio-bulbar enter  the  1964). t h e BC  does not  fibres  The and  at and  positions  the  thin  and  body  gives rise  (FLOOD a n d  a descending  hooks over  branch.  i n the brachium, medium f i b r e s  the coarse  fibres  medial,(VOOGD, 1964).. i n which  a  from  The  to the  and  from  i n the extreme  arising  from  the  direct  and VOOGD,  an  branch pole  takes  course  caudal two-thirds  ventromedial corner rostral half  JANSENand JANSEN JR. lesion  into  ascending  the  of  traverses through  i n the dorsomedial  arising  the  JANSEN, 1966;  t h e i r p o i n t of separation s p l i t s  the m e d i a l n u c l e u s , and  experiment  arises  cerebellum  j o i n the u n c i n a t e f a s c i c l e  uncinate fasciculus  two  found  cross but  that  juxta-restiform  ascending  of  or one-half of  second  the  and  crosses form  commissure of  the  i n the brachium  (1955) was  in  made  of the an which  are n.  Fig. 6. Diagram of the fibre composition of the brachium conjunctivum and its termination in the contralateral red nucleus. A. origin, localization in the brachium conjunctivum at the level of the isthmus and termination in the contralateral red nucleus of the fibres arising from the subdivisions of the central cerebellar nuclei (Voogd, 1964). B, somatotopically arranged origin of the fibres of the rubrospinal tract from the red nucleus (Pompeiano and Brodal, 1957b).  L F I G U R E 8. F i b r e c o m p o s i t i o n o f B r a c h i u m (Pompeiaho, 1967)  Conjunctivum  S h o w s d i v i s i o n o f BC i n t o m e d i a l 1/3 a n d 2/3 c e r e b e l l a r n u c l e i a s i n F i g . 1.  lateral  spared  t h e ventromedial corner, found  cells  remaining between t h e medial  VOOGD  (1964)  coarse  fibres  Perhaps  this  suggested found also  that  this  The  corresponds  fasciculus parallel  previously.  crossed portion  l e a v e s t h e BC j u s t  BC i n t h a t  scarce  i n the. nucleus many  they  fibres  theintralaminar  of  thenuclei  ventralis  latter  (1954).  rostral  uncinate  from  BC.  The t r a c t  thalamic nuclei,  itself  lateralis  originating  and a r e which  crosses a t  a l l  t e r m i n a t i o n s appear  , ventralis posteromedialis,  and v e n t r a l i s  i s that  those  t h e most v e n t r o m e d i a l p a r t s  between t h e ascending  projects  from  o f Darkschewiisch and other r e g i o n s  posterolateralis  brachium  The t e r m i n a t i o n s  i n the r e dnucleus  t h e former  anterior.  Another  uncinate and t h e projects  bilaterally  while  contralaterally.  T h e r e h a s b e e n some c o n t r o v e r s y o v e r fibres  b u t more  aredistinguishable  a r e n o t found  vehtralis  difference  ascending the  protuberance  caudal t o i t s decussation, passes  o f t h e m e s e n c e p h a l o n b e f o r e many  in  major  of the  The c o a r s e  o f the ascending  uncinate tract  of"the  levels  (Fig.9),  t o i t a n d may b e t h e e q u i v a l e n t o f t h e u n c r o s s e d  the ascending  receive  nuclei  identified.  c o m p o n e n t o f BC o f C A R R E A a n d M E T T L E R of  intact  conjunctivum.  to thedorsolateral  i n t h e r e dnucleus  have n o t been  second  i stheorigin  i n the ventromedial brachium  terminate p a r t i a l l y  terminations  and i n t e r p o s i t u s  group  o f GOODMAN e t a l . ( 1 9 6 3 ) m e n t i o n e d fibres  a group o f large  i n themedial nucleus  thalamus.  APPELBERG (1961)  stimulated  the-cerebellar  theexistence of and ending  i n the  i n an e l e c t r o p h y s i o l o g i c a l  nuclei  o f a cat~under  study  Nembutal  -10-  L  Fig.  5  .1 J N .  R  Transsection  of  brachium  conjunctivum  l o g i c a l changes i n the cerebellar nuclei,  iloxtruni. PathoWhere no symbols  no pathological cells observe.!.  F I G U R E 9. P a t h o l o g i c a l c h a n g e s i n c e r e b e l l a r n u c l e i f o l l o w i n g t r a n s a c t i o n of. BC. (Jansen and J a n s e n , 1955) IC  - l a r g e c e l l s b e t w e e n "•• i n t e r p o s i t u s + nucleus l e f t i n t a c t  + represents normal  cells  • represents pathological  cells  o f cat-  medial  -Zlanaesthesia  and  failed  to  find' a  t h a l a m u s when s t i m u l a t i o n ANGAUT e t was  due  a l .  to  thesia.  (1968)  the  nucleus  bilaterally  the  pathway  technique  the  m a k e up as  for  are  n u c l e u s was  failure  found  that  a  aenes-  made  of  a In  terminal  fine  diameter  an  anatomical  degeneration,  demonstrate  a high  ratio  or  J  response  stimulation  s u g g e s t i o n was  Fibres  emerging  the  of  the  '  long either  that  study  the  \  using  COHEN e t a l .  of  thin  fibres  is a  VERHAART strong  lateralis  from the  major p o r t i o n by  and  of  interpositus  the  lateral  (1970).  Reference  projection  of  i s located  magnocellular part strong  the  rostrally  from  of  RN.  a l . , 1954;  interpositus the  and  JANSEN, 1966).  the  termination thalamus  of  red  COURVILLE  f i b r e s are  nucleus  interpositus  of  Figure  (1967)  chiefly  back e  t  (JANSEN and  to  to  BC  shows the  red  that  i n the  the  !  the;  posterior  that  interpositus  a l . , 1966).. A p p r o x i m a t e l y continue  JANSEN  studies  f i b r e s i n the  a l . , 1968;  8  b e e n shown  nuclei  the  states  thought to  Electrophysiological  (ANGAUT e t  to  S i m i l a r l y i t has  reciprocal projection  (BRODAL e t  10-15% of  interpositus  lateral  from i n t e r p o s i t u s  termination  the  and  two-thirds  POMPEIANO  the  find  the  (COURVILLE, 1966).  of  performed.  a  nucleus(RN)  f r o m RN  to  the  i n a r e s p o n s e -with  one.  interpositus  is a  of  system under b a r b i t u r a t e  and  Nuclei  there  there  VL  medial-thalamic, projection.  described  that  to  that  resulted  i n VL,  i n the  medial  i t was  i s a polysynaptic  (1958), w e r e a b l e  2.  the  fibres involved  Nauta  within  of  Under c h l o r a l o s e  latency  the  suggested  f  depression  caudal medial  that  of  response  JR.,1955;FLOOD' also  confirm  contralateral  TOYAMA e t  a l . , 1970).  VL  -22In  t h e r a t (GOODMAN e t a l . , 1 9 6 3 ) t h e p r e v i o u s l y  'hump' a r e a - a t t h e b o r d e r nuclei found send  i n this  area  efferents  occupy  tion  into The  splits It  send  fibres  the adjacent dorsal  the l a t e r a l  third  the i p s i l a t e r a l brachium  o f f from  from both  which  that  increases  i n size  tract  studies  sends  branch  fibres  which  to the decussation. originating  (VOOGD, 19 6 4 ) .  i n this  descending  reticularis  The BC,  tegmenti  ( 1 9 6 7 ) , i t was  separate  from  the small celled lower  noted  the posterior anterior  RN  s p e c i e s t o man w h i l e t h e  disappears.  originating  I t i s o f note  that  i n the magnocellular  n o n - e x i s t e n t i n man. showed  one-third  degeneration  studies  changes  that  virtually  o f RN i s a l s o  pons and  1972).  c o n s i d e r a b l y from  the a n t e r i o r  ogical  nuclei  i n the nucleus  noted  magnocellular portion  Further  nucleus  o f medium and s m a l l f i b r e s  RN i s f u n c t i o n a l l y  I t was a l s o  the r u b r o s p i n a l  o f BC a n d  only a small projec-  rostral  a n e x t e n s i v e r e v i e w b y MASSION  portion.  portion  just  sends a s t r o n g p r o j e c t i o n  the anterior  cells  pons.  and l a t e r a l  (BRODAL e t a l . ,  In  third  lateral  o f BC a n d s e n d  t h e main t r a c t  terminates largely  pontis  because  conjunctivum has a descending  interpositus  rotunda  i n the middle  lateral  t o t h e same r e g i o n o f t h e i p s i l a t e r a l  i s made u p p r i m a r i l y  pars  and  i s considered part of the interpositus  medulla, whereas to  of the interpositus  mentioned  that  o f t h e RN  showed t h a t  to occur  RN a n d t h e c o n t r a l a t e r a l  the l a t e r a l  (COURVILLE,1966). lesions  nucleus.  projects Retrograde  i n ' VL c a u s e d  i n the small cells lateral  nucleus  pathol-  of the i p s i l a t e r a l Large  cells  i n the  to  -23-  lateral  nucleus  cerebellar large that the an the  remained  peduncle  cells there  intact.  caused  degeneration  of the lateral i s a direct  small"and: middle 5  i n d i r e c t pathway parvocellular  nucleus.  sized  cells  that-arises  portion  terminate  that  there.  demonstrated to  seem f r o m  this  pathway a r i s i n g  from  of: t h e l a t e r a l i n large  nucleus and  cells  and r e l a y s i n  t h e e x p e r i m e n t does n o t e x c l u d e t h e cells  do: n o t r e l a y  v i a RN b u t s i m p l y  However, e l e c t r o p h y s i o l o g i c a l and s h o r t  cerebellar^stimulation  latency  responses  indicating  as w e l l  as  that  studies  there  probably  pathway  1970,-CONDE  e t a l . , 1 9 7 0 ; . YOSHIDA e t a l . , 1 9 6 6 ) .  cerebellum  can conclude  A l l cerebellar  nuclei  send  b)  A l l cerebellar  nuclei  contribute  c)  Fibres  descending  nucleus  Fibres cross  structures e)  from a l l parts  (O'BRIEN e t a l . ,  the following  fibres  about t h e 1956):  t o VL. t o BC.  o f BC c o n t r i b u t e  to i t s  ascending  branches. originating  i n the caudal portion  i n the rostral  cerebellum  of the medial  and p r o j e c t  bilaterally  i n t h e mes- a n d d i e n c e p h a l o n .  F i b r e s . o r i g i n a t i n g .mainly  the  medial  and  b r a i n s t e m b y way o f c r o s s e d  tracts.  one  i n t h e c a t (COHEN e t a l . , 1 9 5 8 ) a n d r a t ( J A N S E N ,  a)  d)  to  a., m o n o s y n a p t i c  i s a  Summary T h u s i n s u m m a r y we  and  have  i n VL i n r e s p o n s e  di-synaptie  IV.  and  o f RN o n i t s w a y t o V L o f t h e t h a l a m u s  the large  long  superior  i n the small  I t would  lateral-thalamic  (GEREBTZOFF, 1941) a l t h o u g h possibility  Lesion of the  nucleus  project  i n the-.rostral p o r t i o n  to the vestibular and uncrossed  nuclear  of  complex  fastigio-bulbar  f) Fibres  from t h e interposed  the  BC d e c u s s a t i o n  and  diencephalon.  and p r o j e c t  g) I n t e r p o s i t u s reticularis  nuclei  i) Fibres of  L of  VOOGD  c  lateral and  many o f t h e s e  j)Half  The the  1  SLP  anatomical  will  efferent  be f u r t h e r  conditions  t o determine  neurones  o f t h e BC  t o RN b u t  to confirm  terminate  this.'  RN a n d t e r m i n a t e  largely  present.  i s t o determine  i nthe  projection  nuclei  (ChAc).  i fa portion  system  were examined  cord  a  recently  f o rthe localization of The p r e s e n c e o f t h e enzyme  The h i s t o c h e m i c a l  and subsequently  neurone as  method was  on known c h o l i n e r g i c  the proportion  of  i s cholinergic.  using  indication of a cholinergic  discussed.  i n the spinal  optimize  rostral  f i b r e s by-pass  half  technique  acetyltransferase  studied  (1961), and  two-thirds  are required  nucleus  study  end the c e r e b e l l a r  taken as a d i r e c t  nuclei  studies  portions  RN.  developed histochemical  was  o f BC.  and caudal  o f FLOOD a n d J A N S E N  and t h e o t h e r  cerebello-thalamic  choline  1  travel i n the lateral  purpose o f t h i s  this  limb  f i b r e s end i n the magnocellular  are thought t o continue  VL o f t h a l a m u s  t o the nucleus  i n the ventrolateral  of the l a t e r a l  parvocellular  To  (1964),  project  on t o t h e VL o f t h e t h a l a m u s .  originating  interpositus  more d e t a i l e d  in  continue  cross i n  b y way o f t h e d e s c e n d i n g  h) M o s t o f t h e i n t e r p o s i t u s RN a n d 1 0 - 1 5 %  nuclei  c o n t r a l a t e r a l l y t o t h e mes-  and l a t e r a l  tegmenti pontis  and l a t e r a l  i n t h e deep  and l o c a t i o n  first  neurones t o cerebellar of  cholinergic  -25-  METHODS  A .  Preparation The  spinal  male r a t s  this  c e r e b e l l a were  Ti0  excised segment  second was  and c e r e b e l l a  of the Long-Evans  throughout  were  cords  study. removed  last  and W i s t a r  Under  at the levels of  r i b , as-described  (AChE)  and dye  staining  M N a C l a t 0°C, away.  containing  +  of  f o r the  cord  segments  position of the l e v e l of the  (19 3 5 ) .  Fresh  tissue  cholineacetyltransacetylcholinesterase  The  blocks  Fresh  were  i m m e d i a t e l y washed  a n d t h e d u r a m a t e r was  10% s u c r o s e  a t 0°C.  cryostat, and  b y GREENE  the  Cholineacetyltransf erase  sected  also  The  T10-L2.  used  techniques.  Upon r e m o v a l t h e t i s s u e s 0.15  anaesthesia,  the spinal  employed f o r the demonstration (ChAc) and f i x e d t i s s u e  (150-200g)  s t r a i n s were  to the vertebral  ferase  ~B  ether  i n t a c t and  corresponded  of adult  were  and  frozen  carefully dis-  then transferred  25 mM  to a  HEPES b u f f e r e d  sections  (  solution  t o pH  7.0  u) were cut i n a +  ( I n t e r n a t i o n a l E q u i p m e n t Company M o d e l  thawed on c o v e r s l i p s .  i n  Optimum c u t t i n g  CTD),  temperatures  A l lv a r i a b l e s l e f t b l a n k i n the f o l l o w i n g d e s c r i p t i o n were a d j u s t e d i n the course of t h i s study i n order t o a r r i v e a t optimum c o n d i t i o n s f o r t h e d e m o n s t r a t i o n o f the enzyme ChAc, as d e s c r i b e d u n d e r R e s u l t s . The composition of the incubation mixture f i n a l l y determined i s given i n Appendix I .  -1L-  w e r e -16°C  and  -11°C  respectively. covers l i p s in  were p l a c e d i n Columbia  a Dewar f l a s k  pairs  (1970).  of  of  25  from  those  C o n t r o l and  The  The  mis.  complete  and  mM  mM mM  HEPES*  deposited  gently  BURT  accurate freshly  concomitantly (1970)  and  corresponding  comparison prepared  of  in  batches  use.  following  order  (from N u t r i t i o n a l  to  minimize  Acetyl-CoA*  (from  Canlab)  and  Co.)  Biochemicals) replaced  weeks.  hours.  i n a medium l a c k i n g -1  Calbiochem)  w e r e k:ept u n d e r r e f r i g e r a t i o n  at  10 *M  (from  (from N u t r i t i o n a l  performed  37°C'for  Biochemicals)  (from F i s h e r S c i e n t i f i c  I n c u b a t i o n was  containing  #  6.0  Lead N i t r a t e  three to four  +  i n the  +  every  either  to  Choline Chloride  solutions  *  prior  a t pH  Stock  bath  and  the  (final concentrations):  10"^M. D F P 1.8  cerebellum  s e c t i o n s from  m e d i u m was  filtered  precipitation  jars  employed by  test  i n g r e d i e n t s were added  25 mM  and  s e c t i o n s were incubated  l e v e l s were p a i r e d t o f a c i l i t a t e results.  cord  containing dry i c e .  media m o d i f i e d  HEBB  spinal  F o l l o w i n g a i r d r y i n g a t room t e m p e r a t u r e ,  Selected in  f o r the  CUSCM  as  i n a constant  temperature  C o n t r o l s e c t i o n s were c h o l i n e and an  inhibitor  acetyl-CoA o f ChAc  water  incubated or  (BURT,  in  one  1970).  N - 2 - H y d r o x y e t h y l p i p e r a z i n e - N - 2 - e t h a n e s u l f o n i c a c i d , an organic buffer. D i - i s o p r o p y l f l u o r o p h o s p h a t e , an i r r e v e r s i b l e a c e t y l c h o l i n esterase inhibitor. A c e t y l - C o e n z y m e A, w e i g h e d o u t d r y . A l l other chemicals made up  from  stock  solutions.  - 2 ? -  The  c o v e r s l i p s were  water,  treated with  Na2S  acetate-buffered  (pH  ) for  i n distilled  glycerol  d r i e d , and examined under t h e l i g h t  jelly, I n some  solution  instances  containing,  1% f o r m a l d e h y d e sucrose as  and mounted  the sections  25 mM  HEPES  i n Kaiser micro-  were placed  i n a  (pH 7 . 0 ) , 1 0 % s u c r o s e  f o r 5 m i n u t e s , washed w e l l  s o l u t i o n ; then  described  water  i n distilled  seconds, r i n s e d  scope.  C.  then washed r e p e a t e d l y  transferred  and  i n buffered  to the incubation  medium -  above.  Acetylcholines teras e To p r e p a r e cholinesterase  tissues activity,  exsanguination. c a n n u l a was the  f o r the demonstration r a t s were k i l l e d  When t h e h e a r t  inserted  ceased  i n t o the arch  by c o l d  10% f o r m a l i n  t i s s u e was d i s s e c t e d calcium  to beat,  of the aorta  (HOLT e t . a l . ,  1960) u n t i l  blocks  o f t i s s u e were then washed  w a t e r , b l o t t e d d r y , and f r o z e n  II).  on s l i d e s ,  the sections  0.9% s a l i n e ,  formol-  0.88 M s u c r o s e  saturated.  for five  minutes  floated i n the  t i m e was  were washed  r a p i d l y dehydrated  The  i n t h e same  t o K A R N O V S K Y a n d ROOTS  Average incubation  incubation,  b y way o f  -  The i n distilled  o n a CO 2 f r e e z i n g m i c r o t o m e .  w e r e c u t a t 20u a n d f r e e  m e d i u m made u p a c c o r d i n g  glass  2  immersed i n c o l d  arabic  a  by  1.0% C a C l .  out, f i x e d overnight  s o l u t i o n , and then  Sections  with  containing  1% gum  dix  under ether  v e n t r i c l e a n d t h e a n i m a l was p e r f u s e d  followed  of acetyl-  incubation (1964) (Appen-  2 1/4 h o u r s . i n distilled  i n alcohol, cleared  Following water,  mounted  i n xylene,  -2f-  mounted the D .  i n Harleco  Synthetic Resin  (H.S.R.)*  and examined under  light'microscope.  Histologic al  Staining  Two h i s t o l o g i c a l purposes.  Sections  4 -  s t a i n s were used  adjacent  t o those  for orientation i n w h i c h C h A c was.  demonstrated were s t a i n e d by t h e C r e s y l V i o l e t The  s e c t i o n s were c u t from  stained  and mounted  The tissue  formalin bedding 10y  20th  t o AUGULIS e t a l . ,  +  saline  dehydrating,  serial  frontal  was u s e d  clearing  a n d em-  s e c t i o n s were c u t a t  s e c t i o n was s t a i n e d a n d mounted (1969).  This  nuclei  on  followed by  provided  according  a means t o  t h e c o n f i g u r a t i o n and t h e c e l l u l a r  the deep c e r e b e l l a r *  After  i nparaffin,  visualize  - Darrow Red procedure  with physiological  fixation.  and every  fixed,  i n H.S.R.  Haemotoxylin  perfused  fresh frozen tissue,  technique.  composition of  of the r a t .  H.S.R. f r o m t h e H a r t m a n - L e d d o n C o . , P h i l a d e p h i a . Contains 60% d r i e d (H.S.R.) r e s i n i n x y l e n e . Used t o mount f r o m xylene media. P r o c e d u r e s w i t h r e f e r e n c e t o L I L L I E ( 1 9 6 5 ) a n d R. W a l k e r (personal communication). Step-by-step d e t a i l s a r e given i n t h e A p p e n d i c e s I I I , I V , a n d V.  RESULTS The  histochemical  t e c h n i q u e was  the  localization  of  aim  of  questions  and  anatomical  to the the of  answering  of  raised  the  from  e n z y m e was of  presence  of  that  the  end  the  where  taken  enzyme r a t h e r  conditions possible  first  of  INNES, 1963)  and  the  cholinergic,  i e . contain  as  are  ChAc  in  localization importance.  of  the  activity.  were adjusted  to  To  obtain  suitable for observation. lumbar s p i n a l cord present  i s abundant t h a t  ACh,  of  given  i n d i c a t i v e of  known t o be  evidence  prime  i t s level  incubation  i n the  l a r g e motoneurones  only  than  contrast  tested  i m p o r t a n c e was  of  the  physiological  cytological  t o be  study  (ChAc) w i t h  regional distribution  considered  s t a i n i n g was  sharpest  m e t h o d was  not  a t o o l to  earlier  Because  deep c e r e b e l l a r n u c l e i , p r e c i s e the  as  acetyltransferase  investigations.  determination  Intensity  the  choline  used  ChAc and  AChE  of  the  (ZEMAN  these  The rat and  cells  (FELDBERG,  are  1945,  review). The  e f f e c t of  r e a c t i o n was obtained Following was  varying  thus based  with  on  to  conditions  the  maximum i n t e r c i t y ' o f  minimal non-specific  standardization  applied  the  the  of  the  of  the  histochemical staining  precipitation occurring.  procedure  cerebellar tissue.  the  same m e t h o d  -30-  A.  Spinal I.  Cord  Sectioning Difficulty  from  fresh  BURT  (1970).  tease  the  frozen  section the  Sectioning  at  considerably  found  off  the  t i s s u e on a higher  and  also  thicker sections  easily  encountered  t i s s u e at  I t was  coverslip,  but  was  that  the  took  of  the  had  (20u)  8u  time  to  advantages  incurred  by  intermediate  Thus  of  by  to a  receded.  conditions  and  staining,  were  i t was  more  found  that  p r a c t i c a b l e the  dis-  sections  thickness  required  cellular  t h i n t o be  c u t t i n g 20y  recommended  t h a w e d and  a i r dry  sections  mount i t on  improved  incubation.  were too  be  block  longer  sections  so  during  resulted i n darker  8u  advantages,  thickness  m i c r o t o m e k n i f e and  setting  fragmented during  whereas  the  in cutting serial  outweighed  1 4 - 1 6 u was  the  found  to  suitable.  II.  Incubation The  in  this  choline mM  acetyl-Coenzyme  1.8  mM  lead n i t r a t e  mM  used  initially  N-2-Hydroxyethylpiperazine-N-2-ethanesulfonic  (HEPES.) b u f f e r  adjusted  b y K A S A e t a l . (1970aX I n r  bution  was  chloride  0.2  25  (BURT, 1970)  study:  4 mM  in  f o l l o w i n g medium  of  the  t o pH  6.0,  plus  10 *  following report  each component of  the  M  -1  the  incubation  DFP  as  acid  suggested  individual contri-  medium t o  the  -31-  precipitation the  reaction w i l l  concentrations  non-specific BURT  b y BURT  at  room t e m p e r a t u r e .  at  37°C, a s l i g h t  in  general  incubation slight  of  that  result section of  that  acetyl-CoA  However a f t e r 60-90 m i n u t e s  incubation  i n this  study,  was s o m e t i m e s reacts  I t follows  precipi-  n i t r a t e a n d 0.2 mM  p r e c i p i t a t i o n was o b s e r v e d .  lead  was- n o  lead  m e d i u m w a s made u p e m p l o y i n g  then  with that  i n a non-specific of the tissue.  These  t h e above  the free  observations  concentrations  As o u t l i n e d i n  SH g r o u p o f t h e C o A  any lead-CoA  formed  independently  hydrolytic activity  brown p r e c i p i t a t e appearing Regardless  HEPES,  a l t h o u g h when t h e  observed.  a t t r i b u t a b l e t o ChAc o r o t h e r  acetyl-CoA  there  i n a s o l u t i o n o f 2 5 mM  were confirmed  discussion,  molecule.  showed  lead  lead  1.8 mM  cloudiness  t o cause  optimal.  (1970)  of acetyl-CoA with  6.0, c o n t a i n i n g  the  t o be  increasing  employed by  P r e c i p i t a t i o n o f CoA-SH a c e t y l - C o A w i t h  pH  a  tended  p r e c i p i t a t i o n and t h e concentrations  Studies tation  In general  of the individual solutions  (197fij) w e r e f o u n d 1.  be c o n s i d e r e d .  will  on t h e  o f t h e cause o f h y d r o l y s i s  t h e degree o f p r e c i p i t a t i o n present w i l l  be  d e p e n d e n t on t h e amount o f f r e e CoA-SH a v a i l a b l e i n t h e medium. A stronger  initial  p r e c i p i t a t i o n occurred  the  medium b o t h when t h e coenzyme c o n c e n t r a t i o n  and  when o l d e r  m a t e r i a l was u s e d .  was  I t i s suggested  above mentioned p r e c i p i t a t i o n o c c u r r i n g  increased, that the  on a d d i t i o n o f a c e t y l -  CoA  to the solution containing  the  amount o f f r e e CoA-SH p r e s e n t d u e t o t h e i n s t a b i l i t y o f  acetyl-CoA.  lead  when m a k i n g up  a n d HEPES i s a t t r i b u t a b l e t o  -32-  The lead, BURT the  increase  acetyl-CoA  i n precipitation a n d HEPES o v e r  (1970) , s u g g e s t s greater  neuropil,  will  while  precipitate This  was  time  noted  should  remain r e l a t i v e l y  after  develop  intensity  enzyme was degrees  doubled  tration  effective  I t can be seen t h a t t h e should  be  long  mM  t o 0.4  staining intensity  the acetyl-CoA time.  Filtering  In the later  mM)  enough t o and  due t o  of the co-  showed  similar  b u t darker- and  more  the higher  caused  i t t o become  experiments  greater  concen-  the absolute  quantity  intensity  done on t h e s p i n a l  cord  o f t h e enzyme.  of cellular  that  This  s t a i n i n g and  i n the spinal  showed  precipi-  to incubation  i n t h e c e r e b e l l a r s t u d i e s w h i c h were done  i n v e s t i g a t i o n of the technique  less  added t o t h e i n c u b a t i o n  removed t h e p r e c i p i t a t e p r i o r  i n a decreased  evident  a t 4°C  n o t e d when t h e c o e n z y m e was  also reduced  resulted  the concentration  non-specific precipitation with  over  t a t i o n was medium.  0.2  incu-  (Fig. 11).  Storing  study  a 3 hour  mixture.  i n which  (from  of cellular  concentrated  to  after  o f non-  f o r adequate v i s u a l i z a t i o n  of the incubation  E x p e r i m e n t s done  was  constant.  enough t o m i n i m i z e n o n - s p e c i f i c p r e c i p i t a t i o n  instability  but  greater  lh h o u r s .  time determined  sufficient  period  i n the  i n F i g . 10, where the e x t e n t  staining i s considerably than  by  o f s t a i n i n g due t o t h e i n s o l u b l e  optimum i n c u b a t i o n  short  the incubation  be t h e n o n - s p e c i f i c p r e c i p i t a t i o n  i n the c e l l  bation period  a t 37°C a s n o t e d  the longer  the intensity  i n fact  specific  that  i n the solution containing  cord.  indentical  subsequent A  further  conditions  -33  b) FIGURE 10.  3 hour  incubation.  Not  filtered,  high  Na2S  pH.  E f f e c t of time. Note t h a t i n b n o n - s p e c i f i c p r e c i p i t a t e i s s u p e r i m p o s e d on s e c t i o n . Mo- M o t o n e u r o n e ,  NP  - Non-specific precipitation  -34-  b) FIGURE 11.  0.4 not  mM a c e t y l - C o A , 1*2 h o u r i n c u b a t i o n , f i l t e r e d , h i g h N a 2 S pH.  E f f e c t of d o u b l i n g the coenzyme c o n c e n t r a t i o n . t h e n o n - s p e c i f i c p r e c i p i t a t e i n b i s d a r k e r and p r e v a l e n t t h a n i n a. H o l e s i n t h e t i s s u e c a n be c a u s e d by f r a g m e n t a t i o n o f t i s s u e a t h i g h pH o f Mo- m o t o n e u r o n e , NP WM - w h i t e m a t t e r  Note t h a t more seen Na2S.  - non-specific precipitation  to  those  employed p r e v i o u s l y  The  above r e s u l t s can  amount o f extent,  non-specific the  amount o f  acetyl-CoA  and  the  the  with and  amount o f  increasing with  fresh  and  optimal  an  concentration of  and  a  incubation  of  certain  unstable  staining is  dependent  a v a i l a b l e t o ChAc. age  Thus  the  coenzyme  incubation,  the  non-specific  For the  period  the  12)  the  of  becomes, g r e a t e r .  employed  above r e a s o n s  0.2  mM  of  c o m p l e t e medium j u s t p r i o r  lh  of  hours were  considered  conditions.  Preincubation improvement i n the Fig.  10b)  also  no  media  cellular  i n t a c t acetyl-CoA  coenzyme, f i l t r a t i o n  t o use  to  f r e e CoA-SH r e s u l t i n g f r o m  times  (Fig.  i n t e r p r e t e d t o mean t h a t  i n t e n s i t y of  increasing  precipitation  be  result.  p r e c i p i t a t i o n i s dependent,  on  on  g a v e a much p a l e r  and  of  therefore  contained  i n the  and  expected amount o f  choline  result  r e s u l t s obtained than  10" M  only  that  the  cell  the  two  substrates the  appreciable  (compare F i g .  done r o u t i n e l y .  There  when t h e  13a  with  was  incubation  DFP.  4  from  a c e t y l - C o A were o m i t t e d should  of  omission  from  the  that  control sections  reaction.  not  s h o w e d no  the  medium  a s i m i l a r r e s u l t was  precipitation  the  the  DFP  inhibitors  s t a i n i n g , and and  was  - 3  Omission of  choline  of  10 M. r a t h e r  Controls  cellular  tissue with  clarity  difference  2.  the  which  r e a c t i o n end  incubation  obtained  ( F i g . 13b).  contain  a certain  are  necessary  product  medium.  were never  eliminated when  I t might  be  endogenous to  result in  i n s p i t e of  H o w e v e r , i t was seen to  both  exhibit a  their found cellular  -36-  b) O l d e n z y m e , F I G U R E 12.  filtered,  low Na S 2  pH.  E f f e c t o f o l d coenzyme. I n b: n o t e t h a t o l d enzyme r e s u l t s i n paler c e l l u l a r s t a i n i n g ; f i l t r a t i o n c o n s i d e r a b l y reduces n o n - s p e c i f i c p r e c i p i t a t i o n a n d l o w e r pH o f N a S r e d u c e s f r a g mentation of t i s s u e . A l s o note p r e c i p i t a t i o n pattern i n white matter. 2  M«- motoneurone E> - f r a g m e n t a t i o n  NP - n o n - s p e c i f i c WM  - white  precipitation  matter  b) C o n t r o l - o m i s s i o n o f c h o l i n e 1*5 h o u r i n c u b a t i o n , f i l t e r e d ,  + acetyl low Na2S  CoA, pH  FIGURE 13. a) E f f e c t o f p r e - i n c u b a t i o n . b) C o n t r o l r e s u l t . N o t e homogeneous n o n - s p e c i f i c p r e c i p i t a t i o n f o r m i n g t h e background a g a i n s t which the o u t l i n e o f t h e motoneurone c a n b e s e e n (b) c y t o p l a s m i s f r e e o f r e a c t i o n e n d p r o d u c t . Mo -  motoneurone  -3S-  Non-specific degree that a  light  cell  i t appeared  could  be  faintly  occasion  t h e methods  ground  (Fig.  fixation  reduced  adsorption  the a c t i v i t y  of lead  Inclusion  as n o t e d  This o f ChAc  and  and c e l l s  was  of  lead  pattern  III.  Sodium  fixation.  of sodium  of  sodium sulphide  (pH a b o v e 8.0)  sections  for a  i n i t ( F i g . 12b, 1 4 a ) . adsorption  sulphide  found  that  solutions  of tissue  sections  became v e r y  at the beginning  fragile  yellow  and t h e  T h e pH w a s  o f each  therefore  incubation;  taken at the conclusion  of the experiments  the  approximately  Only  t o be  developed  i n each  6.6-6.7.  100 m l b e a k e r  a t Na S; 2  two s e c t i o n s this  to  sections  treated with  t o come o f f t h e c o v e r s l i p s . 6.5  i s known  measurements pH  ChAc  to distinguish.  to s t a i n except  and f r a g i l i t y  I t was  pH  formalin  The  to variable non-specific  (LEWIS, 1 9 6 1 ) .  adjusted.to  that  the back-  sulphide  cause marked d i s t o r t i o n  tended  both  1970).  Alkalinity  tissue  that  described  (1970).  of p r e c i p i t a t i o n scattered  t h o u g h t t o be due (BURT,  negative  on  non-specific  a l s o b y BURT  were d i f f i c u l t  The w h i t e m a t t e r was  It  as  o f CuSOi* i n t h e m e d i u m a s a n i n h i b i t o r  pale  peculiar  attempted  confirmed  gave a s i m i l a r r e s u l t t o t h e f o r m a l i n were very  granules  (Fig. 13b).  I t c a n be n o t e d  are paler.  to- t h e  the outline of the control  f i x a t i o n was  14b).  only  fine black  which  distinguished  formalin  and t h e c e l l  present  as w i d e s p r e a d  brown background; a g a i n s t  On in  p r e c i p i t a t i o n was  procedure  showed were allowed  b)  O l d e r e n z y m e 1*5 h o u r 1 % f o r m a l i n i n 25mM HEPES 5 m i n . w a s h e d i n 1 0 % s u s c r o s e 25mM H E P E S p H 6 . 0 , f i l t e r e d l o w N a 2 S pH ±h h o u r i n c u b a t i o n .  FIGURE 14. E f f e c t o f f o r m a l i n f i x a t i o n . N o t e t h a t b o t h n o n - s p e c i f i c p r e c i p i t a t i o n and c e l l u l a r p r e c i p i t a t i o n i s reduced. Mo - m o t o n e u r o n e s  WM  - white  matter  much n o n - s p e c i f i c volume and t h u s Developing at  45 s e c .  destruction was of  time  i n the Na S 2  s o l u t i o n was  t i m e p o s s i b l e was  of the integrity  were d i f f i c u l t  containing  important therefore accurate  that  method  neurones  (Fig.  staining  f o r ChAc  immediately  magnification.  results since  The  16)  orientation  simple.  t o mount w h o l e and  fragmentation to their  However, often  be  that  l i m i t e d to permit  respective  determinations  cells  I t  nuclei.  o f AChE  o f KARNOVSKY a n d ROOTS  on t h e s p i n a l c o r d .  limit  Fragmentation  relatively  localization  Acetylcholinesterase thiocholine  employed t o  t h e d e e p n u c l e i was m o u n t e d .  a l l o c a t i o n of cells  Histochemical  large  established  of the t i s s u e .  i n the s p i n a l cord  sections  away- i n t o a  the q u a l i t y of s t a i n i n g .  shortest  the portion  IV.  not  to f a l l  a n t e r i o r a n d p o s t e r i o r h o r n s was  only  an  improved  not a problem  cerebellar  was  The  end p r o d u c t  by the d i r e c t  (1964) were a l s o  stained  were  large  done  moto-  s i m i l a r i n s i z e and shape t o t h e ones  ( F i g . 15) a l t h o u g h  the correspondence i s  o b v i o u s b e c a u s e F i g . 15 w a s  taken  at a  higher  b) FIGURE 15.  3 hour  incubation  Typical result. Note n u c l e a r and c y t o p l a s m i c staining and s m a l l e r c e l l s a d j a c e n t t o motoneurones t h a t appear r e f r a c t o r y t o end-product p r e c i p i t a t i o n (both a & b ) . Nc-  nucleus  C - cytoplasm  NS  - negative  to  stain  - I l l -  FIGURE 16. A c e t y l c h o l i n e s t e r a s e NS  - negative  to  Mo -  motoneurone  Nc-  nucleus  result.  stain  Note s i m i l a r small c e l l s adjacent t o large n e g a t i v e t o s t a i n as i n F i g . 15b.  motoneurones  - 4 3 -  B.  Cerebellum The  conditions  of the incubation  s t u d i e s were i d e n t i c a l spinal  cord,  staining mainly  except  t h e enzyme  appeared p a l e r .  cerebellar  nuclei  were a l s o  found  ' The m i d d l e  1/3  third  f o r the  had aged and t h e r e f o r e o f ChAc was  of the rostrocaudal  ( F i g . 17), a l t h o u g h  was  as o p t i m a l  The d i s t r i b u t i o n  i n the medial  where t h e n u c l e u s with  t o those described  that  i n the middle  employed i n the c e r e b e l l a r  extent  some c e l l s  of the  t o l e v e l s c & d of F i g . 5  i n t e r p o s i t u s and m e d i a l i s  A  i n the brain  merge v e n t r a l l y  serial  were s t a i n e d w i t h  similar  nerve  stem.  set of transverse  nuclei  ChAc  (Fig. 18).  t h e v e s t i b u l a r complex and t h e genu o f t h e f a c i a l  is.visible  studied  containing  n u c l e u s more c a u d a l l y  taken t o correspond  the  sections  of the r a t cerebellar  H a e m a t o x y l i n - D a r r o w Red and showed  cytoarchitectnic pattern  to that  reported  i n the  a  l i t -  erature.  I.  Nucleus The  nucleus were medially (Fig.  Medialis  neurones- t h a t  s m a l l , and l o c a t e d  and v e n t r a l l y n e x t  cell  lying  Violet  to the roof  adjacent  ( F i g . 20 & 2 1 ) .  2/3  of the nucleus ventricle  f o r comparison  stained  a  known  o f the vagus  of corresponding  i n sections  and AChE t a k e n  activity  Cells  i n the medial  of the IV  i n the motor nucleus  (FRIZELL e t a l . , 1970). t o be p r e s e n t  activity  i n the caudal  1 8 ) . , I n F i g . 19 i s i l l u s t r a t e d  cholinergic  found  showed ChAc  dimensions  separately  to those which  by  showed  were  Cresyl ChAc  FIGURE 17.  Middle nuclei  1/3 o f r o s t r o - c a u d a l e x t e n t o f c e r e b e l l a r o f t h e r a t . C r e s y l V i o l e t . Tr«.Ksy«r*e section.  C o r r e s p o n d s t o l e v e l c a n d d o f F i g . 5. D o r s o l a t e r a l p r o t u b e r a n c e o f Goodman (1963) e v i d e n t . L  - lateral  I  - interpositus  M  - medial  IP  nucleus nucleus  nucleus  - inferior  peduncle  V - vestibular nuclear  complex  FIGURE 18.  C a u d a l 2/3 o f r o s t r o - c a u d a l e x t e n t o f t h e c e r e b e l l a r nuclei of the r a t . Section i s c u t o b l i q u e l y so that medial s t r u c t u r e s r e p r e s e n t more c a u d a l l e v e l s . Medial nucleus theref o r e c o r r e s p o n d s t o l e v e l f and g o f F i g . 5 w h i l e l a t e r a l n u c l e u s c o r r e s p o n d s t o l e v e l c and d. Note that dorsolateral protuberance i s missing. L  - lateral  I  - interpositus  M - medial IP  nucleus nucleus  - inferior  V - vestibular IV - f o u r t h  nucleus  peduncle nuclear  ventricle  complex  FIGURE  19.  a) V a g a l  Cells  b) C e l l s  from medial  Comparison of c e l l s medial nuclei.  nucleus staining  f o r ChAc i n v a g a l and  Note s i m i l a r i t y o f i n t e n s i t y o f s t a i n i n g i n a and b. C e l l s f r o m m e d i a l n u c l e u s shown i n b a r e s m a l l . N -  neurone  -4?  b) FIGURE  Same a s a b o v e ,  20. C r e s y l  acetylcholinesterase.  V i o l e t a n d AChE s t a i n i n g i n t h e c a u d a l m e d i a l  nucleus.  C e l l c o r r e s p o n d i n g i n s i z e t o t h o s e s t a i n e d b y ChAc i n F i g . 19b and AChE i n F i g . 20b a r e f o u n d i n l o w e r l e f t c o r n e r o f a b o v e , o t h e r l a r g e r c e l l s o f m e d i a l n u c l e u s c a n a l s o be seen i n a. N -  neurone  a) C h A c s t a i n i n g  medial  nucleus  b) C o n t r o l - o m i s s i o n o f b o t h c h o l i n e a n d a c e t y l - C o A f r o m i n c u b a t i o n medium. C o r n e r o f w h i t e m a t t e r included (left upper). FIGURE  21.  C o m p a r i s o n o f ChAc s t a i n i n g s e c t i o n control i n the medial nucleus. Note p a l e o u t l i n e s e c t i o n b. N - neurone  WM  of small - white  and i t s c o r r e s p o n d i n g  and l a r g e r matter  cells  i n the  control  -M-  for al.  I t was- n o t p o s s i b l e  t o determine  ChAc were  t o those  (1961),  small et  although  cells  a l .  Nucleus  GOODMAN  n u c l e i was t a k e n  (1963).  nucleus  (Fig. 23).  VILLE,  cells  between t h e l a t e r a l and  t o t h e "hump" a s  i srepresented i n  shows  I thas been suggested  small  depending  described  both  round and  ventrolaterally i nthe lateral  cells  that  areidentical  on t h e angle  these  spindle  and appear round o r  a t which  they  a r e c u t (COUR-  1970).  The v e n t r o l a t e r a l p o r t i o n correspond  cells  predominantly  o f t h epars that  t h e CV s e c t i o n s ChAc were  were o f t e n pointing  reacted  ( L c ) o f VOOGD  positively  i n s i z e t o those (Fig. 24).  found  Other  scattered  and on t h eborder  Those c e l l s  convexa  t h a t were  thought  found  of thelateral  described slightly  nucleus  above as seen i n larger cells  i nthedorsal  part  staining  of the lateral  o f t h e i n t e r p o s i t u s and l a t e r a l  s i t u a t e d on t h eborder  theorigin  was  (1964).  f o r ChAc were  f l a t t e n e d i n appearance with  toward  nucleus  (1961) a n d t h e s a m l l  i ntheventrolateral portion  corresponded  nucleus  of thelateral  t o S L P o f FLOOD a n d J A N S E N  portion  The  for  lateral  T h e CV s e c t i o n t h a t  small  and round  flattened  celled  b y MATSUSHITA  and L a t e r a l i s  17 w h e n e x a m i n e d m i c r o s c o p i c a l l y shaped  and  as described  i n F i g . 22 t h e b o r d e r  spindle  to  b e l o n g i n g t o SMP o f FLOOD e t  nucleus  Interpositus  shown  interpositus  shaped  stained  t h e y w e r e o f t h e same m a g n i t u d e a s t h e  o f t h emedial  As  Fig.  which  (1971).  II.  by  identical  i f thecells  their  o f t h ebrachium  nuclei.'  o f t h e two n u c l e i long  axes  conjunctivum  (Fig.  25).  - S o -  FIGURE 22. R e p r e s e n t a t i v e s e c t i o n showing d i s t r i b u t i o n o f c e l l s e x h i b i t i n g ChAc a c t i v i t y . N o t e a g g r e g a t i o n s o f c e l l s i n SLP and on of i n t e r p o s i t u s and l a t e r a l n u c l e i . L  - nucleus  lateralis  I  - nucleus  interpositus  border  M - nucleus medialis IP  - inferior  IV - f o u r t h  peduncle  ventricle  V - vestibular  nuclear  SLP  - subnucleus  £3?  -  parvi  complex cellularis  lateralis.  white matter pattern of staining cellular staining.  i  obscuring  57-  a)  CV - s m a l l s p i n d l e s h a p e d a n d r o u n d c e l l s the v e n t r a l p o r t i o n o f l a t e r a l n u c l e u s .  found i n  50^  b)  A C h E same a r e a  as above  FIGURE 2 3 . C r e s y l V i o l e t a n d AChE s t a i n i n g i n s m a l l portion of the l a t e r a l nucleus.  celled  N o t e t h a t many o f t h e c e l l s a r e c u t t o a p p e a r s h a p e d b u t some a p p e a r r o u n d . N -  neurones  ventral  spindle  b) AChE  - ventrolateral  portion  of l a t e r a l nucleus  FIGURE 24. ChAc and AChE s t a i n i n g i n s m a l l c e l l e d portion of the l a t e r a l nucleus.  ventrolateral  N o t e t h a t more o f t h e c e l l s a p p e a r r o u n d as opposed t o F i g . 23. N NS  neurone - neurones  negative  t o t h e ChAc  stain.  i n this  figure  a)  ChAc - f l a t t e n e d c e l l on b o r d e r o f i n t e r p o s i t u s l o n g a x i s o r i e n t e d t o w a r d o r i g i n o f BC.  with  2o^  b)  AChE s t a i n i n g more m e d i a l l y  FIGURE 25. I n t e r p o s i t u s  cells  staining  situated  interpositus  cell.  f o r ChAc and AChE.  I n b n o t e t h e d i f f e r e n c e i n s t a i n i n g i n t e n s i t y f o r AChE between c e l l s . I n t h i s study c e l l s were s a i d t o s t a i n f o r AChE when t h e i r i n t e n s i t y o f s t a i n i n g c o r r e s p o n d e d to t h a t o f t h e s p i n d l e shaped c e l l i n b. WM  - white matter  N - neurone  B - border of interpositus anrl "I a 1 - v a 1 n u c l e i  Acetylcholinesterase showed that  studies  a similar distribution  described  above f o r ChAc  done on t h e c e r e b e l l a r of intensely stained (Fig. 22).  nuclei cells  as  -55DISCUSSION A. E v a l u a t i o n  I.  of the Method  .Histochemical  Histochemistry and  biochemical  techniques one  i s based  are designed  Although  to deal  of  o f enzyme a c t i v i t y ,  i s complicated  cytoplasm  out  reactions.  the proper biochemical  histochemical  technique  s a m e t i m e may  alter  Analysis  i n the  be  lost  controls  t h a t may  localization  at the  intracellular  influences  from  exposes  and a c e r t a i n  the c e l l  to diffuse  Care must be t a k e n  to carry  f o r any m o d i f i c a t i o n s  improve  localization  the characteristics or a c t i v i t y  of the  but at the  of the  consideration. o f the mechanisms of h i s t o c h e m i c a l  shown t h a t v a r i o u s terization  bio-  tissue sectioning  to external environmental may  enzyme  The  In addition  particularly  i n t o t h e i n c u b a t i o n medium.  enzyme u n d e r  a l l the other  and a c t i v e .  by t h e f a c t t h a t  amount o f enzyme a c t i v i t y freely  system,  must always/ be aware o f t h e  of interfering  the  histochemical  and c h a r a c t e r i z e an enzyme s y s t e m  possibility the site  histological  a s i n g l e enzyme  i n the tissue are also present  tube but the histochemist  level,  with  of  particular  sight of the fact that  chemist can i s o l a t e test  on a c o m b i n a t i o n  principles*.  must n o t l o s e  systems  localization  factors can a f f e c t l o c a l i z a t i o n  o f t h e enzyme o f i n t e r e s t .  based  on s u b s t r a t e  types  of systems  reactions and  Histochemical  h y d r o l y s i s can be d i v i d e d  has  charac-  methods  into four  main  as f o l l o w s :  *•Histochemical p r i n c i p l e s obtained a n d G.G-. G L E N N E R (1965).  f r o m C.W.M. ADAMS  (1965)  -SL-  i)  Multiple  sequence  ii)  Non-cpupling  iii)  simultaneous  iv) The  coupling  post-incubation  choline  taneous  coupling  acetylase  coupling  system most c l o s e l y resembles  system,  schematically  represented  , . . G> e n z y m i c . ... , .. , Substrate ~.—> i n i t i a l r e a c t i o n p r o d u c t reaction *  final  r e a c t i o n productT^2i£H£—* v i s i b l e ^ developer  r  simul-  as follows:  ®capture ^ " •—-4 reaction  v  JL  a  precipitate *  . ,' ® ChAc _ _ © P b ( N 0 ) ., . „ @ N a S Acetyl-CoA » CoA-SH 1——tL$ l e a d - C p A i - * PbS A  c  This  system w i l l  II.  HEBB  i n terms  choline  as t h eacceptor.  for  ;  the  biochemical  The-  (1957) o u t l i n e s a n d r e v i e w s  acetylations  I t was e s t a b l i s h e d  pool  substances  and that  i ncellular c a n donate  i t can i nturn  synthesizing  as t h edonor  (1970),  thesubject  group  article of  of choline  i n 1954 b y Lipman t h a t t h e for various  i t was t h e c e n t r e metabolism.  reaction  of theacetyl  comprehensive  coenzyme a c e t y l - C o A was r e s p o n s i b l e  that  of  Substrates  acetylation.  tion  2  a & b, 1972).  acetylcholine, Acetyl-CoA  and  2  ( 1 9 6 9 , 1 9 7 0 ) a n d HEBB e t a l .  There a r e two s u b s t r a t e s of  3  be considered  e x p e r i m e n t s d o n e b y BURT KASA e t a l . (1970  u  o f an a c e t y l  I tfollows  an a c e t y l group  biological  then  distribu-  that  several  t o form acetyl-CoA and  b e c o m e t h e common s o u r c e  of acetyl  groups.'  The the  histochemical  formation  method under c o n s i d e r a t i o n  o f f r e e CoA-SH f r o m h y d r o l y s i s  ChAc and i t s s u b s e q u e n t p r e c i p i t a t i o n localization limited  t o that-produced  of  the rate  is  used BURT  i t i s essential that  as a measure o f (1969,1970)  acetate  found  doubled.  formation  This is  formed p r i o r  which to  suggest  evidence of  ChAc, a l s o  of  choline.  hydrolysis  given  operates  occurs  than that  mechanism.  an acetyl-enzyme complex  + +  ,  choline  which  this  intermediate  at a faster rate the latter  explanation  of acetate  view  the activity  non-specific  i n t h e absence o f c h o l i n e . and mentions  the following,  of acetyl-CoA,  (3) a c t i o n o f a c e t o - a c e t y l - C o A  enzymes t h a t  Addition  with the  i n t h e absence  i s that  (1) n o n - e n z y m a t i c h y d r o l y s i s  CoA-SH b u t t h e y w o u l d  i s hydrolyzed  i s known t o i n h i b i t  the formation  action of esterases,  incubation  medium  phenomena was  as a two-step  An a l t e r n a t i v e e x p l a n a t i o n  (1970) f a v o u r s  substrate.  i n the complete  f o rt h i s  BURT s u p p o r t s  inhibits  There a r e other  the  observed  of choline the  t o t r a n s f e r o f t h e a c e t y l group t o c h o l i n e and  0.IM C u  possibilities: (2)  that  acetate,  that  The dependence  o r absence of c h o l i n e  i n t h e absence  i n t h e absence o f t h e acceptor  liberate  HEBB  that  that  The e x p l a n a t i o n  would  o f f r e e CoA be  m e a s u r e d r a d i o m e t r i c a l l y was more  cholineacetyltransferase  accurate  specificity.  ChAc r e a c t i o n was 45-50% and  F o r most  by t h e a c t i o n o f ChAc.  o f r e a c t i o n on t h e presence  on  of acetyl-CoA by  by l e a d .  formation  i s based  require  could  lead  t o release  of free  the addition of another  o f an AChE i n h i b i t o r  (10  - l t  medium i n t h e a b s e n c e o f c h o l i n e  thiolase.  second  M eserine)  to  d i dn o t reduce  the  non-specific  the  concentration  cholinesterases in  h y d r o l y s i s b y more t h a n of eserine  •'(ioViPFPV^than t h a t the  non-specific  This  part  Cu  could  + +  of the choline part  CoA  hydrolase.  by C u  bellar  10  - l t  that  considered  that  esterases  there d i d  sections  1 0 " M DFP o r t h o s e 3  employing  o f DFP.  Various  a f f e c t accurate i n turn.  also  However, i n t h e c e r e -  Localization i n d i c a t e the presence  t h e enzyme o f i n t e r e s t b u t i t must a l s o  reaction  that  were more v a r i a b l e when  method must n o t o n l y  location.  that  d e p e n d e n c e was  d i f f e r e n c e between  -Mechanism o f H i s t o c h e m i c a l  anatomical  h y d r o l y s i s are  s u t d y a n d i t was f o u n d  r e s u l t s obtained  concentration  activity.  by p o s t u l a t i n g  Choline  M DFP i n t h e s p i n a l c o r d .  A histochemical  ChAc  DFP t h e h i s t o c h e m i c a l  i n t h e complete media c o n t a i n i n g  studies  lower  III.  be  dependent.  appear t o be a s i g n i f i c a n t  containing  of control.  T h e r e f o r e we c a n c o n c l u d e  Thus, in.media c o n t a i n i n g  i n the present  incubated  of  non-specific .  did inhibit  h y d r o l y s i s o f an u n i d e n t i f i e d a c e t y l -  r e a c t i o n becaomes c h o l i n e  the  be e x p l a i n e d  + +  concentration  i n d e p e n d e n t h y d r o l y s i s may b e d u e t o  to non-specific  confirmed  acetate  t o 11% that  o f DFP was shown n o t t o i n h i b i t  to inhibition  and  not  of acetyl-CoA  enzyme o r enzymes c a u s i n g  sensitive  of free  a l l esterases  increasing non-specific  H o w e v e r , DFP a t a h i g h e r  hydrolysis  concentration  N o r .did  to inhibit  the formation  required, t o i n h i b i t  BURT'S r e s u l t s w i t h the  sufficiently  f u r t h e r reduce  t h e absence of c h o l i n e .  2%.  steps  indicate i t s correct  i n the simultaneous  localization  o f t h e enzyme  coupling will  -51-  In  order  assure of  that  zero  substrate  enzyme. is  to reflect  breakdown  These  high  be a c h i e v e d  that  the concentration  is  to that  initial a first  only  apply that  accurately  are operant,  order  concentration  i e ., that  the rate  on t h e c o n c e n t r a t i o n  i t i s not rate i s present  reaction product.  limiting  Equally  of  concentration  i n large  and t h i s  excess  over  important i s  o f t h e c a p t u r e reagent be i n l a r g e  o f t h e p r o d u c t o f t h e enzyme  speed  one must  when t h e s u b s t r a t e  i fthe substrate  of the i n i t i a l  the  depends  such  that  excess  kinetics  conditions  sufficiently  can  order  enzymic a c t i v i t y  of formation  of the final  reaction, i e .i twill  of the i n i t i a l  reaction  so that  reaction  product  depend  both  reaction product  on t h e  and on i t s c o u p l i n g  characteristics. The c a p t u r e and  i t must form  investigation gent.  BURT  maximal at  reagent must n o t i n t e r f e r e a stable  lead  has been  that  f r o m p H 4-5 a n d t h a t  a concentration  o f 1.8  ions(KASA  a radiometric  assay  a concentration partially  et a l  value.  KASA  control  activity  optimal  trapping  i s known  reduced  preparation  e t al,(1972)  of lead  rea-  CoA was  efficiency  t o be s e n s i t i v e  ' 1 9 7 2 ) , b u t BURT  of acetylcholine  o f 2mM  purified  v  In this  chosen as t h e c a p t u r e  the precipitation  activity  occurred  mM.  Cholineacetyltransferase metal  t h e enzyme  insoluble precipitate.  nitrate  (1970) n o t e d  with  the specific  stated  was o b t a i n e d  (1970) r e p o r t e d  synthesis, lead  o f ChAc merely  t o heavy  activity  t o only that  i n the presence  that i n  nitrate i n of a  92% o f t h e c o n t r o l  over  60% o f t h e  o f 0.1 mM  lead  nitrate  solution.  In the.present  concentration  was  approximating  the conditions  therefore  used'because  increased  Another  source  investigation the'stronger  i t o f f e r e d the adVantage'of  for first  the probability  order  kinetics  of- a c c u r a t e  localization.  o f h i s t o c h e m i c a l m i s i n t e r p r e t a t i o n c a n be  caused  by n o n - s p e c i f i c a d s o r p t i o n  of the capture  tissue  (ADAMS, 1 9 6 5 ) .  to a minimal extent  partly  responsible  but  generally  especially of  This  occurs  f o r background  i s not the cause  i f the i n t r a c e l l u l a r  high the  summary,  and i s  s t a i n i n g i n the present  for false  localization  localization  i t c a n be seen t h a t  concentraiton enzyme  will  of substrate  system w i l l  case,  o f ChAc  of a c t i v i t y  i t i s important  i s not  and o f capture  be s a t u r a t e d  IV. It  reagent  and a t any g i v e n  The  of f i n a l  reaction  i s o f paramount importance  when c h a r a c t e r i z i n g an enzyme. however optimum a c t i v i t y facilitate Optimal  localization. conditions  a phosphate buffered  ions.  so  a  that  time  there  and a  product.  Enzyme-ChAc i n a biochemical  approximate p h y s i o l o g i c a l conditions  this  t o have  be a m i n i m a l amount o f i n i t i a l r e a c t i o n p r o d u c t  maximal c o n c e n t r a t i o n  in  agent t o the  concern. In  in  and  as c l o s e l y  as p o s s i b l e  In a histochemical  i s often  sacrificed  The ChAc  system  preparation  i n order  i s no  solution,in was  to  exception.  f o r C h A c a r e t h e f o l l o w i n g : pH  H o w e v e r , a p H o f 6.0  7.5,  the absence of heavy  u s e d ((KASA e t a l . , 1 9 7 2 )  r e d u c e s n o n - s p e c i f i c s t a i n i n g and i s a l s o more  terms of the capture  system:to  r e a c t i o n as a l r e a d y  metal because  advantageous  mentioned  (BURT, 19 7 0 )  Phosphate B u f f e r  i s not  phosphate  organic  and  so  et  a l . (1970) and  of  displacement of  increasing and  salt  therefore  greater  the  has  the  again  optima.  that  the  same as  The  on  i t s characterization.  d e p e n d e n c e on  studied In  support the choline  i n the this  of  with  FONNUM  the  capture  the of  heavy  reagents.  the  (already mentioned),  system  two  for metal  histochemical  biochemical  specificity  the  sacrificed  ChAc by  case  (1967)  The  less precise  of  i n the  HEBB  proportion  medium i s u s e d .  inhibition  i n terms  by  sites  b e e n shown by  optima are  enzyme s t u d i e d  that  cacodylate  Increasing  enzyme t h e  biochemical  the  that  the  interacts with  i t s binding  incubation  of  is  evidence  has  lead  used,  (1970).  concentrations  so  are  enzyme f r o m  been d i s c u s s e d  Proof  BURT  a hypotonic  histochemical salts  buffers  HEPES by  solubility  localization  s u i t a b l e because  main  system depends  lines  enzyme a r e  and  of  i t s  i t s response  to  inhibitors. KASA e t  a l . (1972) has  studied  on  ChAc a c t i v i t y  i n homogenates of  on  histochemical  sections.  inhibit  ACh  synthesis  and  i n the  the  appearance of  to  histochemical end  the  e f f e c t s of  nervous  t i s s u e as  CM-oroacetylcholine  80%  that  system  of  three  was  c o n t r o l i n the  chloroacetylcholine  product obtained  i n DFP  inhibitors  well  as  found  to  homogenates, eliminated  treated  rat  spinal  and  false  cord. Immobilization localization h o w e v e r was product  of  the  enzyme t o p r e v e n t d i f f u s i o n  i s desirable. found  in this  to  reduce  The  usual  the  amount o f  i n v e s t i g a t i o n , and  method of  others  formalin  histochemical have  fixation end  reported.that  the  enzyme, a c t i v i t y  Therefore,  f o r m a l i n f i x a t i o n was  d i f f u s i o n was nuclear  i s considerably  accepted  as  a  staining  s i n c e AChE i s n o t  (KOELLE,  1951).  In  the  not  source  staining i s considered  found  on  that  t o tke e n d o p l a s m i c  than.to  the  intracellular  nuclear  s o m e t i m e s o b s e r v e d may  but  has  The affect  not  been  post-mortem  in  the  that  nervous  there  freezing The of  most r i g o r o u s  a  and  s t a i n i n g has of  systems  the  indication  not  ChAc i n d i c a t e s  (FONNUM,  1967)  nuclear  of  immediate post-mortem  (HEBB, 1957)  of  also  diffusion  the  conceivably Fresh  to reduce handling  and  frozen  of  tissue  l o s s of  Green et  to  activity  a l . (1970)  i n ChAc a c t i v i t y  with  showed repeated  tissue.  criteria  f o r the  following, according  substrate  AChE  membrane  localization.  apparent decrease  thawing  the  nuclear  reticulum  an  the  is specific  for a  histochemical to Glenner specific  localization  (1965):  enzyme^group  enzymes. (ii)  as  and  the  slight  of d i f f u s e  localization  be  i n order  i s no  tissue no  enzyme a r e (i)  of  was  evidence  t i s s u e p r e p a r a t i o n may  s e c t i o n s were employed There  In  a l . (1970b).  proven.  enzyme c h a r a c t e r i s t i c s  a minimum.  error.  membrane, t h e r e f o r e  staining this  and  ChAc s y s t e m n u c l e a r  The  rather  KASA e t  employed  of  t o be  been evaluated. i t i s bound  reduced  a histochemical  substrate  i t s naturally occurring (iii)  the  the  same  activities  analogue.  a n a t u r a l l y - i n s o l u b l e (undenatured)  component and (iv)  identifies  enzyme has  a  soluble  vice-versa. reagents  m e d i u m h a v e no  effect  added on  the  to  the  histochemical  characteristics  of  incubating enzymic  -Ll-  activity  demonstrated.  (v) effect (vi) tissue  the steps  of histochemical  on t h e l o c a l i z a t i o n absence  of enzymic  activity  i n d i c a t e s the absence  c a n be c o n c l u d e d from  the substrate the preceding  ( i ) , ( i i ) and  limitations  of the system w i t h described.  toward  either i n vivo  assumptions  have been  have  and c h a r a c t e r i z a t i o n o f enzyme  enzyme c a p a b l e o f h y d r o l y z i n g It  tissue preparation  a substrate or i n vitro at that  respect  activity.  i n a o f an  site.  discussion  ( i i i ) have been proven  no  that  and t h a t t h e  t o ( i v ) , (v) a n d ( v i )  B.  The C e r e b e l l o - t h a l a m i c Pharmacological  Pathway  i n v e s t i g a t i o n s suggest that  thalamic  p a t h w a y may b e i n p a r t  cholinergic.  observed  that  BC e v o k e d  response  i n the nucleus v e n t r a l i s l a t e r a l i s  be  abolished  the short  latency  o r reduced by a t r o p i n e  but not i o n t o p h o r e t i c a l l y . et  a l . , 1966) a l s o  markedly  spike  application  o f t h e BC e v o k e d  and f i e l d  o f t h e thalamus can  administered  investigations  t h e BC e v o k e d  field  o f a system  s o l e l y on t h e b a s i s  'pharmacological (1966)  establishment  field  response by  identity'  as being  response  was  o f ACh o r an  iontophoretic  b y McLENNAN  of 'identity  t h e most important function.  (1963,1970)  include  i snot  of action' or  although these are stressed  of transmitter  criteria  Other  by  f o r the  criteria  as  the following: from which i t  released. (2) t h e n e u r o n e  for  (FRIGYESI  as c h o l i n e r g i c however  (1) t h e s u b s t a n c e m u s t b e p r e s e n t i n n e u r o n e s is  intravenously  o f ACh.  Identification conclusive  (1971)  I n a d d i t i o n McCANCE e t a l . , ( 1 9 6 8 a & b) n o t e d  augmentation  listed  showed t h a t  MARSHALL  a f f e c t e d by the i n t r a c a r o t i d a d m i n i s t r a t i o n  atropine.  WERMAN  Previous  the cerebello-  manufacture (3) T h e r e  transmitter  t h e necessary enzymic  mechanisms  of transmitter.  must be a system  f o r inactivation of the liberated  w h i c h may o r may n o t b e  (4) d u r i n g  enzymic  s t i m u l a t i o n i t may b e p o s s i b l e ^ t o  proposed; t r a n s m i t t e r synapses.  must possess  substance  i n the region  collect the  of termination  of  The  main t h r u s t of  the  second The  the  criterion  two  i n v e s t i g a t i o n was  i . e . presence  enzymes t h a t  are'key  of  there  these  was  no  of ChAc as  there  animals  ( i n the  1964)  was  f o r AChE  distribution dog,  r a t , KOELLE, suggests  most areas  (cerebellar In  the  Fig.  &  26  of  the  inactivating  these  the  are  exception  there  or  no  appear  t o be  In addition there  the  the in  activity  nervous  the  as  guinea  nucleus  stained  appeared  of AChE, p l u s  s t r u c t u r e s such  conclusive  of  ChAc however  of  a  Studies  a l . ,  1951;  etaal.,  the  correlated  cerebellum  the  the  as  c h o l i n e r g i c system  uniform.  fact  presence  i s considered  and  i n the  appearance  discrepancies  be  found  absence cannot  more r e l i a b l e  In  s t a i n i n g of  a c h o l i n e r g i c system.  1961;  of  cat  i n the  These  o f AChE a l o n e  (HEBB, 1957,  1967  between s p e c i e s .  t h a t AChE can  t o be  AChE  variation  f o r AChE a l t h o u g h  t o be  of  i t (GOLDBERG,  variation  erythrocytes  evidence  q u a n t i t i e s of  p i g , r a t , p i g e o n and  a l . , 1964), considerable  ChAc, i n d i c a t e s t h a t taken  of  cerebellar cortex  lateral  demonstration  various  i s considerable  study  of  1970  closely  of  high  ChAc accompanying  a  et  until  particular).  between c e r e b e l l a r f o l i a  (FRIEDE  AChE.  BURGEN e t  fairly  are  enzyme  a l . ; 1963,' F R I E D E .  AChE a c t i v i t y comparative  enzymes i n  a l . , 1948;  activities  brain with  in  little  27).  of both  their  enzyme.  (KOELLE, e t a a l . 1 9 4 9 ) .  FELDBERG e t  cerebellum  with  synthesizing  a v a i l a b l e f o r the  1954*, SHUTE e t  that  cortex  present  the  a c h o l i n e r g i c system  the  h i s t o c h e m i c a l method  the  the  s u b s t a n t i a t i o n of  have been measured b i o c h e m i c a l l y but  done on  in  of  to  s y n t h e s i z i n g enzyme ChAc and  Both  in  this  as  PHILLIS,  in  o f ACh  nonor  be The'  presence  indicative 1965b).because  TABLE I A c t i v i t y * of Choline A c e t y l t r a n s f e r a s e in the C e r e b e l l u m  Rat  Pigeon  Guinea-pig  Cat  <0. 1 (8)  0. 71±0.19 (6)  1. 60±0.22 (13)  1.52±0. 14 (8)  1. 23±0.18 (6)  1 38±0.22 (8)  2. 60±0.08 (5)  1.23±0. 10 (7)  0. 44±0.10 (4)  10 5±1. 5 (7)  10. 6±1. 5 (5)  0. 98±0 27 (8)  1. 55±0.30  Granular layer  1. 92±0 37 (5)  1. 36±0.19  White  2 75±0 48 (.4)  Nuclei  6. 90±0 51 (6)  26. ChAc  +  0. 62±0.15 (11)  Molecular layer  FIGURE  Rabbit  (9)  (9)  i n the cerebellum  3 78±1.08 (4)  (from G o l d b e r g ,  1967)  * t h e a c t i v i t y i s expressed as umoles o f a c e t y l choline synthesized/g dry weight/hr. Each v a l u e r e p r e s e n t s t h e ± s t a n d a r d e r r o r (number o f sections) + from data  o f McCaman & H u n t  (1965).  -LI-  TABLE n A c t i v i t y * of C h o l i n e s t e r a s e in the C e r e b e l l u m  Rat  Pigeon  Molecular layer  1000±123 (4)  4293±316 (6)  3757±285 (7)  2087±216 (6)  2330±288 (7)  Granular layer  1472±89 (5)  2399±190 (8)  2390±131 (6)  3702±643 (6)  3694±282 (5)  White  1620±223 (4)  775±132 (7)  1192±69 (7)  1270±126 (5)  1193±211 (6)  Nuclei  152 5±232  610±58 (4)  1354±189 (5)  (5)  Guinea-pig  FIGURE 27. AChE i n t h e c e r e b e l l u m  (from  Rabbit  Cat  1790±276 (9)  Goldberg]1967).  * The a c t i v i t y e x p r e s s e d a s y m o l e s o f a c e t y l c h o l i n e hydrolyzed/g d r y weight 1 h r . Each"value represents t h e ± s t a n d a r d e r r o r (number o f s e c t i o n s ) .  its'  activity  AChE.  i s always  I t i s desirable  paralleled  then t o demonstrate  ChAc i n c o n j u n c t i o n w i t h pathway The  in  that  i s c o n s i d e r e d t o be  afferent the  -fibres)  superior  b e e n made the  nuclei  cerebellar  peduncle input  superior  (containing  attributed  peduncle  the BC). that  the audio-visual  cerebellar  peduncle  to a cholinergic  1968) and  appears  (containing  to a  centres.  The a c t i v i t y i n  on t h e o t h e r hand has been  c o m p o n e n t o f t h e B.C.  that  some n e u r o n e s  intracellularly retrograde  peduncle.  in  the lateral  activity specify  portion  (PHILLIS,1968)  of the interpositus  nucleus of the cerebellum that  f o r A C h E , a r e t h e same c e l l s  I t was c o n f i r m e d i n t h i s  that  i n t h e deep c e r e b e l l a r which  the cells  study that  n u c l e u s and on t h e b o r d e r  f o r b o t h AChE and ChAc.  On c l o s e r that  i n the lateral  (PHILLIS,1965a).  stain  evidence  d e g e n e r a t i o n and a c c u m u l a t i o n o f AChE on s e c t i o n o f  the  stained  found i n  cholinergic  i s supported by t h e o b s e r v a t i o n i n t h e c a t  and i n t h e l a t e r a l  as  A suggestion has  This  nucleus  t o be h e a v i e s t  the activity  of the r a t i s related  from  and  t h e same a m o u n t o f a c t i v i t y  (ARVY,1966; ODUTOLA.1970)  middle  the  and t h e middle  shows a l m o s t  peduncle  nervous  cholinergic.  r a t ( S P E R T I , 1 9 5 9 a ,&,~b; K O E L L E , 1 9 5 4 ) nuclei,  of  o f AChE b e f o r e a p a r t i c u l a r  i n t h e c a t (SNELL,1961; PHILLIS,1965a,  the lateral  o f ACh and  the presence  c o n c e n t r a t i o n o f AChE i n t h e c e r e b e l l a r  peduncles the  by t h e presence  nuclei  BURT nuclei  cells  found  of the interpositus  (1970)  also  noted  ChAc  of the r a t but d i dnot  contained i t .  e x a m i n a t i o n i t was a l s o staining  observed  f o r AChE and ChAc were  i n thU study predominantly  small and  (10-15y)  monkey  cells  were  and middle  (EAGER;1968; found  (20-25y)  sized cells.  COURVILLE.et a l . , 1970) s c a t t e r e d  was made t h a t  contribute  t o e f f e r e n t p r o j e c t i o n systems.  Matsushita  small  cells  taposition  probably that  to large  Having ating the  fibres  (87%)  remains  found  there  by the onset  would  t h e thalamus  The  possible  other  Study  origin-  of  of cholinergic that  a high  t o Ach and t h a t  o f AChE and ChAc FRIEDE,1966)  proportion  the limits of (1959) a r e  o f marked ACh Biochemical  ( K O E L L E , 1954"; F E L D B E R G , show  n u c l e i i s o f medium corroborated  the cholinergic portion  afferent-to  tothe  and t e r m i n a t i o n  and disappearance  o f enzymes- a s a l s o  indicate that  which  system.  (McLENNAN e t a l . , 1 9 6 8 ) .  i n the thalamic  The d i s t r i b u t i o n  jux-  I t i s suggested  p o s t e r i o r o f HASSLER  BURGEN a n d C H I P M A N , 1 9 5 1 ,  distribution  nucleus  study belong  o f t h e c a t show  neurones respond  studies  these  may b e a c h o l i n e r g i c s y s t e m  t o be r e s o l v e d .  of the cells  confirmed  i n close  (VOOGD 1 9 6 4 ) .  i n the present  ventralis oralis  defined  was  However,  of the lateral  v i a t h e BC  i n the thalamus  histochemical  1948;  portion  indicated that  sensitivity and  celled  cells  of thalamic  sharply  This  a n d n o m e n t i o n was made o f t h e  joins the efferent projection  nucleus  a x o n s a n d may n o t  as being  i n the cerebellar n u c l e i the course  mechanisms  the  small  cells,  does p r o j e c t s  that  short  e t a l . (1971) i n t h e c a t a n d r a t .  the small  group  they possess  were c o n s i s t e n t l y d e s c r i b e d  exclusively  small  throughout a l l c e r e b e l l a r n u c l e i and t h e  suggestion  by  In the cat  that  their  intensity. by t h i s  of the fibre  study system  i s not strong.  termination  site  of the cholinergic  portion  of  BC i s t h e r e d n u c l e u s .  large  and small  cells  Evidence  has been found  i n the r e d nucleus  (WALDRON e t a l . , 1969 ) M c L E N N A N , 1 9 6 9 ) .  AChE  confirm  a strong  cerebello-rubral  interpositus  nucleus  nucleus  cells  and from  to magnocellular red  been found  RN.  t o be  Some  excited  (McLENNAN,1970).  Possible presence  future  investigations  of a cholinergic  to substantiate  component  i n BC c o u l d  further the  consist of  following: (1)  bution such  studies  (COURVILLE, 1966;  to parvocellular  i n t h e r e d nucleus have a l s o  ACh  the  lateral  both  intensely f o r  Anatomical  projection  VOOGD,1964) f r o m n u c l e u s  by  stain  that  Extend o f ChAc  the investigation of the histochemical i n t h e deep c e r e b e l l a r  nuclei  t o other  distrianimals  as t h e c a t and monkey. (2)  Note t h e e f f e c t  distribution caudal  o f ChAc  and r o s t r a l  the proportion thalamus.  of transection  i n the cerebellar  t o r e d nucleus  of cholinergic  could  neurones  o f the peduncle nuclei. give that  on t h e  Transection  an i n d i c a t i o n o f project  tothe  -71-  C.  Summary a n d  (1) of can  IN  The method d e v e l o p e d  C h A c b y BURT be u s e d  central THE  localization  ( 1 9 7 0 ) a n d H E B B e t a l . (1970) i s s p e c i f i c o f ChAc  and  i n the  system.  RAT: ChAc d i s t r i b u t i o n  deep c e r e b e l l a r (3)  Medium  most abundantly lateral  scattered (4)  (5)  The  The  and s m a l l  s i z e d ChAc  caudal  There were  that  of the medial stained  of a l l three  were  found  nucleus  a l s o a few  nucleus  darkly  contained  cells  a  f o r ChAc.  n u c l e i h a v e b e e n shown  of the thalamus v i a the brachium  The r e s u l t s o b t a i n e d  cholinergic.  i n the  the nucleus i n t e r p o s i t u s . part  cells  cells  that  staining cells  i n the ventral" portion of the l a t e r a l  i n t h e VL  indicating  AChE d i s t r i b u t i o n  nuclei.  throughout  terminate  parallels  portion of interpositus.  group of small  (6)  f o r histochemical  as an i n d i c a t i o n o f t h e presence  nervous  (2)  and  Conclusions  are consistent with  conjunctivum.  other  a portion of the cerebello-thalamic  to  studies  pathway i s  ->2BIBLIOGRAPHY  A d a m s , C.W.M. 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H u n t (1965) Microdetermination of choline acetylase i n nervous t i s s u e . J . Neurochem. 12, 253-259. M c L e n n a n , H. ( 1 9 6 3 ) 'Synaptic Transmission'. Philadelphia: W.B. S a u n d e r s C o . . p p . 1 3 4 . F i r s t e d i t i o n . McLennan, J.  H. ( 1 9 6 9 ) Cholinesterase i n the red nucleus. N e u r o p h a r m a c o l . £, 489-490.  Int.  M c L e n n a n , H. ( 1 9 7 0 ) 'Synaptic Transmission'. Philadelphia: W.B. S a u n d e r s C o . . p p . 1 7 8 . S e c o n d e d i t i o n . M c L e n n a n , H. ; R.D. H u f f m a n , K.C. M a r s h a l l ( 1 9 6 8 ) Patterns of e x c i t a t i o n o f •'thalamic n e u r o n e s by amino a c i d s and by acetylcholine. N a t u r e 219, 387-388. ;  N i e u w e n h u y s , R. ( 1 9 6 7) C o m p a r a t i v e anatomy o f t h e c e r e b e l l u m , in: P r o g r e s s i n B r a i n Res. 25, 1-93, e d . by C A . Fox and R . S . S n i d e r . E l s e v i e r P u b l i s h i n g C o . , New Y o r k . O ' B r i e n , J . H . ; H. C o n d e ( 1 9 7 0 ) Functional organization of the anterior red nucleus. B r a i n R e s . 21_, 3 4 5 - 3 6 5 . O d u t o l a , A.B. (1970) cholinesterase re-appraisal.  The t o p o g r a p h i c a l l o c a l i z a t i o n o f a c e t y l i n the adult r a t cerebellum: A H i s t b c h e m i e .23., 9 8 - 1 0 6 .  P h i l l i s , J.W. (1965a) Cholinesterase i n the cat cerebellar c o r t e x , deep n u c l e i and p e d u n c l e s . E x p e r i e n t i a 21, 266-268. P h i l l i s , J.W. ( 1 9 6 5 b ) C h o l i n e r g i c mechanisms B r i t . Med. B u l l . 2 1 , 26-28.  i n the cerebellum.  P h i l l i s , J.W. (1968) Acetylcholinesterase i n the feline cerebellum. J . Neurochem. 15, 691-698.  P o m p e i a n o , 0. ( 1 9 6 7 ) Functional organization of the cerebellar projections t o the s p i n a l cord. Progress i n Brain Res. 25, 2 8 2 - 3 2 1 e d . b y C A . F o x a n d R.S. S n i d e r . Elsevier P u b l i s h i n g C o . , New Y o r k . P o m p e i a n o , 0.; A . B r o d a l ( 1 9 5 7 ) Experimental demonstration of a somatotopical origin of rubrospinal fibres i n the c a t . J . Comp. N e u r o l . 1 0 8 , 2 2 5 - 2 5 2 . R a n d , R.W. (1954) Anatomical and experimental study o f the c e r e b e l l a r n u c l e i and t h e i r e f f e r e n t pathways i nt h e monkey. J . Comp.. N e u r o l . 1 0 1 , 1 6 7 - 2 2 4 . S h u t e , C C D . ; P.R. L e w i s ( 1 9 6 3 ) Cholinesterase containing systems o f t h e b r a i n o f t h e r a t . N a t u r e 199, 1160-1164 Snell,  R.S. (1961) The h i s t o c h e m i c a l l o c a l i z a t i o n o f c h o l i n e s t e r ase i n t h e c e n t r a l nervous system. B i b l . A n a t . 2_, 5 0 - 5 8 .  Sperti,  L . ; S. S p e r t i ( 1 9 5 9 a ) E f f e c t s o£chronic l e s i o n s o f t h e c e r e b e l l a r peduncles on c e r e b e l l u m cholinesterase activity i n the albino rat. E x p e r i e n t i a 15_, 4 4 1 - 4 4 2 .  Sperti,  L . ; S. S p e r t i ( 1 9 5 9 b ) Effects of midline cerebellar s p l i t t i n g and o f l e s i o n s i n c e r e b r a l c o r t e x on cerebellum cholinesterase a c t i v i t y , i n the albino r a t . E x p e r i e n t i a 15_, 4 4 2 .  T o y a m a , K.; N . T s u k a h a r a , K. K o s a k a , K. M a t s u n a m i ( 1 9 7 0 ) S y n a p t i c e x c i t a t i o n o f r e d n u c l e u s neurones by f i b r e s from i n t e r p o s i t u s nucleus. E x p t l . B r a i n R e s . 11^, 1 8 7 - 1 9 8 . V e r h a a r t , W.J.C..(1970) 'Comparative Anatomical Aspects o f Mammalian B r a i n Stem and S p i n a l C o r d ' . VolII. I l l u s t r a t i o n s and Tables. P r o e f s c h r . Van Gorcum a n d Co. N.V., A s s e n . V o o g d , J . (1964) 'The C e r e b e l l u m o f t h e C a t . Fiber Connections' Thesis. Proefschr. N.V., A s s e n .  S t r u c t u r e and Van Gorcum a n d Co.  V o o g d , J . (196 7) Comparative aspects o f t h e s t r u c t u r e and f i b e r c o n n e c t i o n s o f t h e mammalian c e r e b e l l u m in: Progress i n B r a i n Res..25_, 9 4 - 1 3 4 e d . b y C A . F o x a n d R.S. S n i d e r . E l s e v i e r P u b l i s h i n g C o . , New Y o r k . f  W a l d r o n , H.A.; D.G. Gwyn (1969) Acetylcholinesterase activity i n t h e r e d nucleus o f t h e r a tand i t s response t o axotomy. B r a i n R e s . 1_3, 1 4 6 - 1 5 4 . W e i d e n r e i c h , F. (1899) Zur Anatomie der z e n t r a l e n K l e i n h i r n kerne d e r Sauger. Z t s c h r . M o r p h o l . u . A n t h r o p o l . 1_, 259-312. (quoted by Voogd, 1964).  W e r m a n , R. ( 1 9 6 6 ) A review- c r i t e r i a f o r identification of c e n t r a l nervous system t r a n s m i t t e r . Comp. B i o c h e m . and P h y s i o l . 18, 745-766.  a  Y o s h i d a , . M . ; K. Y a j i m a , M. Uno ( 1 9 6 6 ) Different activation of t h e two t y p e s o f t h e p y r a m i d a l t r a c t n e u r o n e s t h r o u g h t h e c e r e b e l l o - t h a l a m o c o r t i c a l pathway. E x p e r i e n t i a 22 , 3 3 1 - 3 3  APPENDIX  I  Cholineacetyltransferase Stock  Solutions m.w.  solute  solvent"  1.  25 mM H E P E S p H 10% sucrose  7.0  238.3  1.4893 g 10 g  250  mis  H 0  2.  25 mM  6.0  238.3  1.4893 g or 2.9786 g  250 500  mis mis  H 0 H 0  3.  10 mM  HEPES pH  Choline  4 . 4.5 mM L e a d  Chloride  Nitrate  5. c a . 0 . 0 5 4 M  DFP  2  2 2  139.63  1 3 9 . 6 3 mg  100 m i s o f 25 mM H E P E S p H 6.0  331.20  1 4 9 . 0 4 mg  100 m i s  184.1  1 g  H 0 2  10 m i s isopentane + 90 m i s H 0 2  II.  Optimum I n c u b a t i o n 1.  Medium  Control Amount 10 m i s  25 mM  50 y l  DFP  59.57mg  dry  'make u p t o 10 m i s ' 2.  Final  Solution HEPES pH  6.0  25 mM  Concentrations HEPES pH  610  10 - if M  DFP  1.8 mM  Pb(NO )2  HEPES  1 5 m i s w i t h 2 5 mM H E P E S pH6.0 4.5mM, L e a d N i t r a t e  3  Test 10 m i s  10 mM  50 u l  DFP  . 5 9 . 5 7 mg make u p t o 10 m i s 4 . 4 0 5 mg  dry  Choline  Chloride  4mM  Choline  Chloride  10 - h D F P HEPES  15 m i s w i t h 25 mM H E P E S 2 5 mM H E P E S p H 6.0 p H 6.0 4.5 mM L e a d N i t r a t e 1.8 mM P b ( N 0 ) 2 3  Acetyl-CoA  0.2 mM  Acetyl  CoA  Appendix 3.  I  continued  Inhibitor 0.62  add  III.  Na S  CuSOi*  mg  cc  20%  saturated  0.56  cc  glacial  97.00  cc  distilled  aqueous  acetic  M  CuSCK  acetic  acid  Kaiser  glycerol  sodium  sulphide  acid  water  pH b e t w e e n 6 . 8 - 7 . 5 d e t e r m i n e d  by  adjusting  t h e amount o f  added.  jelly.  (Refr.  Distilled  1.47)  index  Gelatin Water  Glycerin  10  g  60  ml  70  ml  0.25  Phenol  V.  ^  10  solution  2  2.5  IV.  I X  to Test batch  g  DFP 1  g o f DFP  diluted solution  t o 100 placed  inactivate  must be done  dissolved  ml w i t h  a concentration to  was  i n 10  distilled  ml of isopentane  water.  i n 25 m i s o f i n c u b a t i o n o f c a . 10~  a n y DFP  k  M.  spilled  i n t h e fume  hood.  1.0  Then  50  and  u l of  then  this  medium r e s u l t s i n  N NaOH i s - k e p t  i n the process.  This  on  hand  procedure  -S2-  APPENDIX  II  Acetylcholinesterase 1.  Fixed text  frozen  sections,  under methods, were  medium f o r  2\  5 mg  medium as  following  are  between each  buffer  then  ml  of  0.2  of  1 ml  water  1 ml  of  30  solutions  final  incubation  M  mM  5 mM  Stock  iodide  incubation  (from N u t r i t i o n a l  i n 6.5  at  pH  ml  of  0.1  M  5.5  added i n o r d e r w i t h  stirring  potassium i n the  ferricyanide  cold  for  several  medium i s c l e a r , g r e e n i s h  for hours. the  I f an  i n place  2.  Float  & wash  in distilled  3.  Float  & wash  i n 0.5%  4.  Mount on  slide.  citrate'  CuSO^  keep  of  sodium  added  alcohol.  the  addition:  1 ml  stable  in  follows:  dissolved  sodium acetate  is  described  f l o a t e d i n the  acetylthiocholine  Biochemicals)  0.5  free  as  hours.  Incubation  The  prepared  1 ml  inhibitor of  The  i n colour  i s used  and  i t is  water.  water.  g e l a t i n 40%  Blot with  weeks.  filter  alcohol paper  solution. soaked  in  7.0%,  -«3Appendix  5.  II  continued  Dehydrate  a)  70%  b)  absolute  6.  Clear i n xylene  7.  M o u n t i n H.S.R.  5  a l c o h o l 10  sec.  a l c o h o l 1-5  minutes.  sec.  APPENDIX I I I Paraffin  Sections  Preparation  After is  of block  perfusion  excised  f o r cutting paraffin sections  of tissue i n situ  and p l a c e d  washed i n t a p water  with  10% f o r m a l i n i t  i n t h e same f i x a t i v e  f o r 3-4  days,  and t a k e n through t h e f o l l o w i n g  procedure: 1.  Dehydrate a ) .7 0;% b)  alcohol  95% a l c o h o l i) ii)  first second  c) M e t h y l i) ii) 2.  ii)  ii) iii)  4  hours  Benzoate  first second  overnight  change, change  3-4  hours  3-4  hours  3-4  hours  or u n t i l  first second  change change  first second third  overnight  change change change  2-3  hours  2-3  hours  Embed ' i n p a r a f f i n  Preparing 1.  hours  Impregnate i)  B.  change  4  Benzene i)  4.  change  Clear a)  3.  overnight  sections  t o be  stained.  Cut. s e c t i o n s , a t d e s i r e d microtome.  thickness  on t h e c l i n i c a l  saturated  - i s -  Appendix 2.  I I I continued Bring  sections  a) X y l e n e - t w o  3.  b)  Absolute  c)  70%  c)  water  Take  to water, hydration.  5 minutes  changes  alcohol  alcohol  through s t a i n i n g procedure  desired.  each  APPENDIX  Cresyl 1.  Violet  From f r e s h following 5 minutes  2.  IV  tissue, i t i s desirable  t o go  through  the  procedure p r i o r to standard procedure. each.  a)  water  b)  70%  c)  absolute  alcohol  d)  xylene -  two.changes  e)  absolute  alcohol  f)  70%  g)  water,  alcohol  alcohol  Standard procedure a)  Bring  section  b)  Place i n water  c)  Cresyl  for paraffin to water - 5  sections.  as d e s c r i b e d  III B  minutes.  V i o l e t s t a i n - approx.  B i o l e t A c e t a t e 0.25%  i n Appendix  aqueous  5 minutes. solution.  (Cresyl Allied  Chemicals). d)  Wash i n t a p  e)  70%  f)  CV  water.  alcohol. differentiator until  adequately d i f f e r e n t i a t e d .  2.  Appendix  IV  1-2  min.  10ml  3.  with agitation.  of Chloroform,  i)  95% a l c o h o l  j)  absolute  k)  xylene  1)  m o u n t i n H.S.R.  - two  changes  substance-purple.  (90ml o f 95%  3 drops g l a c i a l  a l c o h o l - two  Results Nissl  i  continued  changes  alcohol,  acetic  acid)  A P P E N D I X V-  H a e m a t o x y l i n and Darrow Red 1.  Standard procedure Bring  b)  Mordant  c)  Wash t h o r o u g h l y i n d i s t i l l e d  water  changing  or five  Stain 10%  the water  i n Lithium  Lithium  Wash w e l l by  f)  i n 1% a q u e o u s  as d e s c r i b e d I r o n Alum  bath  four  a final  Stain  i n A p p e n d i x I I I B 2.  f o r 30-60  minutes.  f o r 10 m i n u t e s , times.  Haematoxylin f o r 3 hours,  haematoxylin i n 95% a l c o h o l  0.15% e)  t o water  sections.  a)  d)  section  from p a r a f f i n  Carbonate  - 10 m i n u t e s ,  added  (5mls  t o 95 m i s  + 25g N a I 0 ) . 3  i n running t a p water;  bath of d i s t i l l e d  followed  water.  i n D a r r o w R e d f o r 10 m i n u t e s . Red  Stain Acetate  Buffer  p H 3.8  100 m i s  Darrow Red  25 mg.  Mixture brought for  just  to a boil,  1-2 m i n u t e s , c o o l e d t o r o o m  t u r e , •„ s h a k e n Mixture^ s t a b l e  occasionally  and  simmered tempera-  filtered.  f o r 1 month and c a n be  used  repeatedly. g)  Wash i n d i s t i l l e d  h)  Dehydrate alcohol  i)  Clear  water  f o r 5 minutes  and two changes  f o r 1 minute. each  i n two changes  of absolute alcohol.  i n x y l e n e , a n d m o u n t i n H.S.R.  o f 95%  Appendix V  2.  continued  Results Haematoxylin  stains myelin and  Darrow  Red  erythrocytes - blue  stains dark  sheaths,  neuronal  nuclei  red granules  cytoplasm granules.  - red  of  nucleoli black, light  red  with  chromatin,  - with dark  red  Nissl  

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