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Characterization of neuropharmacological systems in the mammalian central nervous system Hicks, T. Philip 1979

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CHARACTERIZATION OF NEUROPHARMACOLOGICAL SYSTEMS IN THE MAMMALIAN CENTRAL NERVOUS SYSTEM  T. PHILIP HICKS B.A., B.Sc,  Carleton  University,  1973  Dalhousie U n i v e r s i t y ,  1976  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE- OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Department, o f P h y s i o l o g y )  We accept t h i s t h e s i s as conforming to the r e q u i r e d  standard  THE UNIVERSITY OF BRITISH COLUMBIA August, 1979 ©  T. P h i l i p H i c k s , 1979  In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s an advanced degree a t the U n i v e r s i t y of B r i t i s h C o l u m b i a , I agree the L i b r a r y  for  that  s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y .  I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department o r by h i s r e p r e s e n t a t i v e s .  It  i s understood t h a t c o p y i n g o r p u b l i c a t i o n  of t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l written permission.  Department o f  FMSlOUXtf  The U n i v e r s i t y of B r i t i s h Columbia 2075 Wesbrook P l a c e V a n c o u v e r , Canada V6T 1W5  DE-6  B P 75-5 I I E  not be a l l o w e d w i t h o u t my  ii  ABSTRACT  The ined  e f f e c t s o f a range o f n e u r o n a l e x c i t a n t s  on t h e f i r i n g  ventrobasal  were exam-  o f c e n t r a l neurones o f the c e r e b r a l  thalamus,  d e n t a t e g y r u s and d o r s a l  and v e n t r a l  horns o f the s p i n a l cords o f urethane anaesthetized T h e s e r e s p o n s e s were p h a r m a c o l o g i c a l l y basis  of their susceptibilities  from these r e s u l t s , i n f e r e n c e s receptor  ideal ists  cholinomimetics.  and antagon-  t h a l a m u s a n d Renshaw  cells  of a c e t y l c h o l i n e antagonists.  a l s o by b o t h n i c o t i n i c  agonists  The r e s u l t s may  d i f f e r e n c e between e x c i t a t o r y  be i n t e r p r e t e d  applied  and those  as  cholinergic receptors  e x c i t a t o r y responses of ventrobasal  iontophoretically  amino a c i d s  and  were b l o c k e d by n i c o t -  and i n t h e c a t ; t h e n a t u r e o f t h e s e r e c e p t o r s  The  res-  and m u s c a r i n i c  were a n t a g o n i z e d b y a t r o p i n e  and n i c o t i n i c  o f the  These  E x c i t a t i o n s p r o d u c e d by a c e t y l c h o l i n e  acetyl-B-methylcholine  to  f o u n d t o be an  c o r d were r e a d i l y e x c i t e d by a c e t y l c h o l i n e .  p o n s e s were e l i c i t e d  rat  these  on s i n g l e n e u r o n e s .  spinal  a  was  and  probable  Throughout  the e f f e c t s o f agonists  Neurones i n t h e c o r t e x ,  inic  were made c o n c e r n i n g  the technique of iontophoresis  one f o r e v a l u a t i n g  on t h e  of antagonists  mechanisms e m p l o y e d b y t h e a g o n i s t s .  experiments  rats.  characterized  t o a number  cortex,  is  thalamic  revealing i n the discussed.  neurones  r e l a t e d to glutamate  iii  and  a s p a r t a t e c o u l d be b l o c k e d b o t h by g l u t a m a t e  and  a-aminoadipate.  T h e s e two a n t a g o n i s t s were  s e s s d i f f e r e n t mechanisms orders of s u s c e p t i b i l i t y tagonist.  An a n a l y s i s  o f the a g o n i s t s d i f f e r e d  acid light  amino a c i d s  of possible  Granule amino a c i d s  requirements  iveness o f glutamate  on  on  were  different central tested  f o r an  a g a i n s t t h e amino  o f the r e c e p t o r s .  inputs.  diethylester  two d i s t i n c t  o f which appear  t h e same  an-  are discussed i n  synaptic responses  p e r f o r a n t p a t h and c o m m i s s u r a l  both  f o r each  o f t h e d e n t a t e g y r u s were e x c i t e d by t h e  and b y t h e i r  that  compounds  and t h e s e r e s u l t s  steric  cells  exist  antagonistic properties  induced responses,  gestive  as many as t h r e e  A number o f a d d i t i o n a l  evaluation of their  t o pos-  of these orders l e d to the proposal  f o r the e x c i t a t o r y  neurones.  found  o f a c t i o n however, as t h e r a n k i n g  t h a t more t h a n one and p o s s i b l y receptors  diethylester  to s t i m u l a t i o n of  A differential  and a-aminoadipate  excitatory  effect-  was  sug-  amino a c i d r e c e p t o r s ,  t o be o f s y n a p t i c s i g n i f i c a n c e ,  coexist  neurones.  The e f f e c t s catecholamines  o f o c t o p a m i n e were compared w i t h t h o s e o f  on n e u r o n e s Both  o f t h e c o r t e x and d o r s a l h o r n o f  the s p i n a l  cord.  excitation  and d e p r e s s i o n o f n e u r o n a l  f i r i n g was  o b s e r v e d w i t h o c t o p a m i n e and t h e s e r e s p o n s e s  appeared  not\to.,be  c o r r e l a t e d w i t h t h o s e e f f e c t e d by t h e c a t e c h o l a m i n e s .  A further  s e p a r a t i o n of the a c t i o n s  o f o c t o p a m i n e and t h e  1  a  IV  catecholamines was evident when the amine induced  responses  were compared i n the presence o f the a n t a g o n i s t s ,  propranolol  and a - f l u p e n t h i x o l . i n attenuating  These b l o c k i n g compounds were e f f e c t i v e  the e f f e c t s o f the catecholamines, but had no  e f f e c t upon the octopamine induced  changes i n f i r i n g r a t e .  The  s e n s i t i v e to octopamine and  r e s u l t s suggest that r e c e p t o r s  which appear to be p h a r m a c o l o g i c a l l y  d i s t i n c t from those pre-  v i o u s l y c a t e g o r i z e d as catecholamine r e c e p t o r s , may e x i s t on c e n t r a l neurones o f the r a t .  On the b a s i s o f the present  f i n d i n g s , i t was evident  when the technique o f i o n t o p h o r e s i s  i s combined w i t h  that  standard  n e u r o p h y s i o l o g i c a l methods o f i d e n t i f y i n g c e n t r a l neurones by t h e i r responses to s y n a p t i c s t i m u l a t i o n , v a l u a b l e can be obtained  concerning  m i t t e r s employed by these  information  the nature o f the s y n a p t i c cells.  trans-  V  TABLE OF  Certificate  CONTENTS  of Examination  i  Abstract  i i  List  of Tables  ix  List  of Figures  xi  List  of Abbreviations  xiv  S o u r c e s o f Drugs  xvii  Acknowledgements  xviii  Chapter I: General  Introduction  1  a)  Chemical Transmission  1  b)  C r i t e r i a For  5  c)  Iontophoresis  d)  The  Chapter  Present  Transmitters  8  Study  II: Materials  and  16 Methods  17  a)  Supraspinal  Experiments  19  b)  S p i n a l Cord Experiments  20  Stimulation  21  ' c) d)  and  Recording Procedures  R e c o r d i n g From S i n g l e N e u r o n e s  and  Drug D e l i v e r y T e c h n i q u e  22  e)  E l e c t r i c a l Equipment  28  f)  S t a t i s t i c a l Analysis  29  g)  Histological Verification Electrode  Chapter a)  Sites  of 29  III: Acetylcholine  31  Introduction 1) H i s t o c h e m i c a l 2) R e c o v e r y  31 32 33  vi  3) I o n t o p h o r e s i s i/ ii/ iii/  34  Cortex  34  Thalamus Spinal  -  Cord  46  b) R e s u l t s i/  ii/  iii/  51  Cortex  51  Cholinergic  Agonists  51  Cholinergic  Antagonists  56  Ventrobasal  Thalamus....  61  Cholinergic  Agonists  61  Cholinergic  Antagonists  69  Renshaw C e l l s  74  E x c i t a t o r y Responses  74  Cholinergic  77  Antagonists  c) D i s c u s s i o n  82  P o s s i b l e R e c e p t o r Mechanisms Chapter  41  IV: Amino A c i d s  84 88  (Ventrobasal  Thalamus)  a) I n t r o d u c t i o n  88  1) U p t a k e and R e l e a s e  88  2) H i s t o l o g i c a l  91  Findings  3) I o n t o p h o r e s i s .  94  Agonists  94  Antagonists -4) E x p e r i m e n t a l  105  Rationales  106  i/  Antagonists:  GDEE  107  ii/  Antagonists:  aAA  108  vii  iii/  Antagonists:  iv/  KDEE,  2A3P,  2A4P a n d BAA  109  Agonists:  ADCP a n d BAG  110  Kainic  112  v/ A g o n i s t s :  acid  b) R e s u l t s i/ ii/ iii/ iv/  113  E x c i t a n t Mechanisms.  113  E f f e c t i v e Antagonists..  116  Other Antagonists  135  BAG  139  c) D i s c u s s i o n Effects Amino A c i d s  144 o f Other Analogues  (Continued) Dentate Gyrus  a) I n t r o d u c t i o n  .  150 155 155  b) R e s u l t s  157  c) D i s c u s s i o n  170  C h a p t e r V: O c t o p a m i n e a) G e n e r a l  172  Overview  172  1) P r o p o s e d F u n c t i o n s  173  2) R e c e p t o r s F o r O c t o p a m i n e  178  i/ ii/  Invertebrates..  178  Vertebrates  179  b) R e s u l t s . . i/  ii/ iii/ iv/  Effects  182 o f Octopamine and  Noradrenaline  185  Potency Estimations  185  Effects  188  o f Dopamine  Identified  C o r t i c a l Neurones  192  viii  v/ D o r s a l H o r n N e u r o n e s vi/ vii/  197  Effects of Propranolol a-Flupenthixol Effects  of Clozapine  And 200 And  Metoclopramide  205  c) D i s c u s s i o n C h a p t e r V I : Summary and C o n c l u d i n g References  208 Remarks  214 218  ix  L I S T OF  TABLES  Table  Page  1  Drug S o l u t i o n s  24  2  Numbers o f c h o l i n o c e p t i v e n e u r o n e s o f t h e c e r e b r a l c o r t e x responding to n i c o t i n i c and m u s c a r i n i c d r u g s ..  52  Numbers o f c h o l i n o c e p t i v e n e u r o n e s o f t h e v e n t r o b a s a l complex o f t h e t h a l a m u s r e s p o n d i n g t o n i c o t i n i c and m u s c a r i n i c drugs  68  3  4  5  6 7  8  9  10 11  12  R e l a t i v e i o n t o p h o r e t i c " d o s e s " o f a-aminoa d i p i c a c i d and g l u t a m a t e d i e t h y l e s t e r r e q u i r e d to antagonize the e f f e c t s o f each o f a range o f n e u r o n a l e x c i t a n t s  117  Summary o f e f f e c t s o f L - g l u t a m i c diethylester against pairs of excitants.  122  acid neuronal  Summary o f e f f e c t s o f a-aminoadipate against p a i r s of neuronal excitants  •  128  Summary o f t h e r e s u l t s o f p o t e n c y d e t e r m i n a t i o n s and a n t a g o n i s m o f 3-aminoglutarate  143  R e l a t i v e p o t e n c i e s .of L - g l u t a m a t e and L - a s p a r t a t e t e s t e d on d e n t a t e g r a n u l e cells  160  Numbers o f u n i d e n t i f i e d c o r t i c a l n e u r o n e s t e s t e d w i t h b o t h o c t o p a m i n e and n o r a d renaline  189  Numbers o f u n i d e n t i f i e d c o r t i c a l n e u r o n e s t e s t e d w i t h b o t h o c t o p a m i n e and dopamine  190  Numbers o f i d e n t i f i e d c o r t i c a l n e u r o n e s t e s t e d w i t h b o t h o c t o p a m i n e and n o r a d renaline  191  C l a s s i f i c a t i o n of d o r s a l horn neurones a c c o r d i n g to t h e i r responses to octopamine and n o r a d r e n a l i n e  195  X  Table 13  (cont'd) C h a r a c t e r i z a t i o n of neuronal responses of 26 s p i n a l n e u r o n e s t e s t e d w i t h n o r a d r e n a l i n e , dopamine and o c t o p a m i n e  Page  196  xi  L I S T OF FIGURES  Figure 1  2 3  4 5 6  7  8  9  10  11 12  13  Page Responses o f a c e r e b r a l c o r t i c a l neurone to ACh, n i c o t i n e a n d A3MC, a n d t h e e f f e c t s o f t h e n i c o t i n i c a n t a g o n i s t , DH3E  54  R e s p o n s e s o f two c o r t i c a l n e u r o n e s t o ACh and c h o l i n o m i m e t i c s v.; '. ..,  58  R e s p o n s e s o f a c o r t i c a l n e u r o n e t o ACh a n d the e f f e c t o f t h e n i c o t i n i c a n t a g o n i s t , mecamylamine  60  E f f e c t o f a t r o p i n e on c e l l by ACh a n d g l u t a m a t e  63  Effect ACh  firing  induced  o f a t r o p i n e on c h o l i n o m i m e t i c s a n d 65  R e s p o n s e s o f a VB t h a l a m i c n e u r o n e t o t h e s e r i e s o f e x c i t a n t s used to c h a r a c t e r ize neuronal responses  67  R e s p o n s e s o f a t h a l a m i c n e u r o n e t o ACh a n d cholinomimetics i n the presence o f n i c o t i n i c and m u s c a r i n i c b l o c k e r s  71  Responses o f a s i n g l e t h a l a m i c neurone t o t h e a d m i n i s t r a t i o n o f ACh, g l u t a m a t e , A(3MC a n d c a r b a c h o l : e f f e c t o f c u r a r e  73  R e s p o n s e s o f a t h a l a m i c n e u r o n e t o ACh, c h o l i n o m i m e t i c s and t h e e f f e c t s o f atropine  76  The a c t i v a t i o n o f a Renshaw c e l l whose r e s p onse t o a v o l l e y i n t h e L5 v e n t r a l r o o t i s shown, t o ACh, c a r b a c h o l a n d A3MC  79  E f f e c t s o f c u r a r e a n d a t r o p i n e on t h e c h o l i n e r g i c e x c i t a t i o n o f two Renshaw c e l l s  81  Diagrammatic r e p r e s e n t a t i o n o f the m o l e c u l a r s t r u c t u r e s o f glutamate and a s p a r t a t e  98  T y p i c a l recordings o f thalamic responses to t h e amino a c i d e x c i t a n t s used i n t h i s s t u d y  115  xii Figure 14 15  16  17  18 19  20  21 22  23  24  25 26  (cont'd)  Page  E f f e c t o f GDEE a g a i n s t t h e s t e r e o i s o m e r s of a s p a r t a t e  120  T y p i c a l responses used i n c o n s t r u c t i n g t h e r a n k i n g o r d e r s o f amino a c i d e x c i t a n t s when GDEE was t h e a n t a g o n i s t employed  125  T y p i c a l responses used i n c o n s t r u c t i n g t h e r a n k i n g o r d e r s o f amino a c i d e x c i t a n t s when aAA was t h e a n t a g o n i s t employed  131  E f f e c t s o f t h e amino a c i d a n t a g o n i s t s on the f i r i n g r a t e o f a spontaneously disc h a r g i n g VB t h a l a m i c n e u r o n e  134  E f f e c t s o f t h e p h o s p h o n i c a n a l o g u e s as amino a c i d a n t a g o n i s t s  138  E f f e c t s o f GDEE a n d aAA a g a i n s t e x c i t a t i o n by 3AG a n d a c o m p a r i s o n w i t h t h e r e s p o n s e s t o L - g l u t a m a t e and L - a s p a r t a t e . . .  141  E f f e c t s o f amino a c i d a n t a g o n i s t s on g r a n u l e c e l l s , and a schematic o f a g r a n u l e c e l l i n r e l a t i o n s h i p t o t h e dentate gyrus  159  The a c t i o n s o f aAA a n d GDEE on s y n a p t i c a n d amino a c i d e x c i t a t i o n o f a g r a n u l e c e l l  163  A v e r a g e d EPSP f i e l d r e s p o n s e s e v o k e d b y s t i m u l a t i n g PP a n d COMM i n p u t s t o t h e d e n t a t e granule c e l l s  166  The e f f e c t : o f b i c u c u l l i n e on t h e d e p r e s s i o n o f a g r a n u l e c e l l b y GABA a n d i t s a c t i v a t i o n b y aAA  169  Examples o f t h e e f f e c t s o f o c t o p a m i n e and n o r a d r e n a l i n e on t h e f i r i n g r a t e s o f neurones o f the c e r e b r a l c o r t e x  184  R e s p o n s e s o f a c o r t i c a l n e u r o n e t o dopamine and n o r a d r e n a l i n e  187  A dose-response curve o f octopamine depress i o n s on a s p i n a l i n t e r n e u r o n e activated by v e n t r a l r o o t v o l l e y s  194  xiii Figure 27  28  29 30  (cont'd)  Page  R e s p o n s e s o f two n e u r o n e s t e s t e d w i t h o c t o p amine and n o r a d r e n a l i n e , and o c t o p a m i n e and dopamine, and e f f e c t s o f a n t a g o n i s t s  199  E f f e c t o f p r o p r a n o l o l on a s p i n a l n e u r o n e r e s p o n d i n g t o o c t o p a m i n e and n o r a d r e n a l i n e w i t h o p p o s i t e changes i n r a t e  202  E f f e c t o f p r o p r a n o l o l on a s p i n a l n e u r o n e d e p r e s s e d by o c t o p a m i n e and n o r a d r e n a l i n e  204  E f f e c t s o f c l o z a p i n e and m e t o c l o p r a m i d e on t h e f i r i n g o f c o r t i c a l and s p i n a l n e u r o n e s  207  L I S T OF ABBREVIATIONS  ACh  acetylcholine  A3MC  acetyl-3-methylcholine  AChE  acetylcholinesterase  ADCP  (±)-cis-1-amino-1,3-dicarboxycyclopentane  2A4P  2-amino-4-phosphonobutyrate  2A3P  2-amino-3-phosphonoproprionate  A  angstrom  A  anterior  ASP  aspartate  BICUC  bicuculline  carbachol > (CARB)  carbaminoylcholine  °C  centigrade  cm.  centimetre  CNS  c e n t r a l nervous  COMM  commissural p r o j e c t i o n pocampus)  curare  D-tubocurarine  I  current  c y c l i c ..AMP  cyclic  D-  dextro  DH3E  dihydro-3-erythroidine  DMPP  1,1-dimethy1-4-phenylpiperazinium  DC  direct  DLH  DL-homocysteate  DOP  dopamine  GLUT  glutamate  GDEE  glutamate  g  gram  o r gm  '  degrees system (from c o n t r a l a t e r a l  adenosine-3,5 -monophosphate 1  current  diethylester  Hz  hertz  hr  hour  HA966  l-hydroxy-3-aminopyrrolid-2-'one  i.p.  intraperitoneal  iodide  XV  KDEE  kainate diethylester  Kg  kilogram  KHZ  kilohertz kilo-ohms  L-  laevo  L  lateral  LGN  lateral  m  mega-ohms  mg  milligrams  ml  millilitres  mm  millimetres  msec  milliseconds  mV  millivolts  min  minutes  M  molar  nA  nanoamperes  NIC  nicotine  NMA  N-methyl-DL-aspartate  NOR  noradrenaline  n.  nucleus  n  number  ng  nanogram  OCT  octopamine  E" PP  para  pmol  picomole  P  posterior  P  probability  sec  second  SE  s t a n d a r d e r r o r o f t h e mean  VB  ventrobasal  V  vertical  V  volts  w/v  weight  geniculate nucleus  perforant  path  p e r volume  xvi aAA  a-aminoadipate  ( b o t h D and DL  3AA  3-aminoadipate  ( r a c a e m i c m i x t u r e o n l y was  3AG  3-aminoglutarate  GABA  y-aminobutyrate  ym  micrometre  yv  microvolt  X  c h i squared  2  forms  were u s e d ) used)  xvii SOURCES OF DRUGS ICN  Pharmaceuticals  Calbiochem  L-glutamic  acid  atropine  L-aspartic  acid  DL-homocysteic  D-aspartic  acid  noradrenaline•HCl  N-methyl-DL-aspartic y-aminobutyric  acid  carbamylcholine  acid  dopamine-HCl  acid  DL-a-aminoadipic  sulphate  2-amino-4-phosphonobutyric acid  acid  D-glutamic  chloride  acid  acetyl-3-methylcholihe • Nutritional Merck  acetylcholine  dihydro-3-erythroidine  bromide  D-tubocurarine  bromide  1,l-dimethyl-4-phenylpiperazinium  •  HCl  iodide  mecamylamine h y d r o c h l o r i d e  Sigma kainic  Lundbeck  acid  octopamine•HCl  a-flupenthixol  hydrochloride  Koch-Light  a n d Mann R e s e a r c h  L-glutamic  acid  BDH  Biochemicals  nicotine  acid  tartrate  ICN-K&K:rare & f i n e chem. division  diethylester  Sandoz clozapine  Biochem.  2-amino-3-phosphonoprop-  hydrochloride  rionic  acid  Drugs r e c e i v e d as g i f t s : metoclopramide•HCl  Dr. B. R o u f o g a l i s  propranolol-HCl  Dr, M. W a l k e r  b i c u c u l l i n e methochloride• . 3-aminoglutaric  (Vancouver)  (Vancouver).  D r . G.A.R. J o h n s t o n  acid  (Canberra).  "  D-a-aminoadipic a c i d  Dr. J.C. Watkins  3-aminoadipic  D r . J . C . W a t k i n s v i a D r . G.A.R.  acid  (Bristol).  ADCP was s y n t h e s i z e d b y D r s D.G. C l a r k a n d V. G u j r a l  Johnston (Vancouver).  xviii ACKNOWLEDGEMENTS The s u c c e s s f u l c o m p l e t i o n o f a s c i e n t i f i c p r o j e c t , such as t h i s d o c t o r a l t h e s i s , cannot be a c h i e v e d w i t h o u t t h e h e l p , s u p p o r t , a d v i c e and encouragement o f many p e o p l e . I therefore w i s h t o extend my g r a t i t u d e t o those who took p a r t i n t h e v a r i o u s a s p e c t s o f t h e p r o g r e s s and development o f my graduate r e s e a r c h p r o j e c t , as w e l l as i n my e d u c a t i o n i n t h e p r i n c i p l e s of physiology. Drs. N. W i l s o n , J . M i l l e r , R. Pederson and D. R a n d a l l formed my graduate committee and were v e r y h e l p f u l w i t h t h e i r support and a d v i c e . F o r c o l l a b o r a t i o n and v a l u a b l e d i s c u s s i o n s I w i s h t o thank Drs. S. A s s a f , J . H a l l , T. R i c h a r d s o n , D. West, H. Wheal and Mr. G. H u t c h i n s o n . T e c h n i c a l support was always c h e e r f u l l y p r o v i d e d by Mrs. Y. Heap, who a s s i s t e d i n t h e s u r g i c a l p r e p a r a t i o n s ; Mr. R. Walker, who d i d t h e h i s t o l o g y ; Mr. K. Henze, who d i d a l l t h e photography, and h i s s t a f f who r e n d e r e d t e c h n o l o g i c a l a s s i s t a n c e . Mrs. M. F o r s y t h e and h e r s t a f f a r e thanked f o r t h e i r e x c e l l e n t and e f f i c i e n t s e c r e t a r i a l s e r v i c e s . The M e d i c a l Research C o u n c i l f i n a n c i a l l y supported t h e p r e s e n t s t u d y . The encouragement and c o n f i d e n c e o f my w i f e , Debbie, has been o f g r e a t h e l p t o me and t h e r e f o r e m e r i t s s p e c i a l mention. L a s t , b u t c e r t a i n l y n o t l e a s t , I w i s h t o o f f e r my most s i n c e r e thanks t o P r o f e s s o r H. McLennan who has been t h e i d e a l s u p e r v i s o r ; by g r a n t i n g me independence i n my r e s e a r c h endeavo u r s , a l l o w i n g me t o l e a r n from my own e r r o r s , y e t b e i n g always t h e r e w i t h guidance t o h e l p me o u t o f d i f f i c u l t y when i t was r e q u i r e d . H i s example w i l l c o n t i n u e t o be a s t a n d a r d of e x c e l l e n c e f o r me i n my f u t u r e s c i e n t i f i c endeavours.  1  CHAPTER  GENERAL  a) The  Chemical basic  cell,  I  INTRODUCTION  Transmission  f u n c t i o n a l u n i t o f the nervous  or neurone.  neurosciences  Much o f t h e c u r r e n t  i s concerned with  system i s the nerve  i n v e s t i g a t i o n i n the  e l u c i d a t i n g t h e mechanisms  whereby n e u r o n e s i n t e r a c t w i t h e a c h ' o t h e r control  of bodily  It  activity  and c o n d u c t i n g  influences  other  the narrow region  n e u r o n e s w h i c h he b e l i e v e d connection. important  Shortly  that  neurones.  I n 1897  later  e f f e c t of adrenaline  the p o i n t ,  and t h a t  this  Sherrington  changed t o "synapse", t o  o f a n a t o m i c a l j u x t a p o s i t i o n between t o be t h e s i t e  systemically  of functional  was l o c a l i z e d  administered  adrenaline  He a l s o n o t e d  system that  to the j u n c t i o n s o f  and a l t h o u g h E l l i o t t  t h e i m p l i c a t i o n was t h a t n e r v e  m i n u t e amounts o f a d r e n a l i n e  inter-  (1905) made t h e  sympathetic nervous  similar effects.  n e r v e and smooth m u s c l e , on  activity,  s t i m u l a t i o n of the p e r i p h e r a l  produced q u a l i t a t i v e l y the  that neurones a r e capable o f  thereafter, E l l i o t t  observation  integrated  of behaviour.  electrical  t h e term " s y n a p s i s " ,  describe  and  and p a t t e r n s  i s now w e l l e s t a b l i s h e d  generating  coined  functions  to produce  was n o t e x p l i c i t impulses r e l e a s e  o r an a d r e n a l i n e - l i k e  substance  '  2  onto those The erally  t i s s u e s which are  involvement of chemical  innervated  adrenaline, that  t i s s u e s was  however.  Dale  a c e t y l c h o l i n e (ACh)  biological by  (muscarinic  nicotine  and  tions  this  Despite sensitivity  these  blocked  (nicotinic  the  so p r o v i d e  those by  these the  was  observed a slowing  effects  fluid  of  and  the  released  from the vagus of  referred  to  the  t h i s m a t e r i a l as as ACh  see McLennan,  (Bacq and  1963).  actions  on  blocked by  concentra-  the  pharmacological f o r Loewi with  nerve  physiological  evidence  substances which  upon an  innervated  of a perfused  frog  transferred this not  second h e a r t ,  a t t r i b u t e d to the presence  identified  conclusion  increased  medium p e r f u s i n g a s e c o n d h e a r t w h i c h h a d He  to  elicited  observations  first  the vagus n e r v e  periph-  effects).  release chemical  effluent  the  t i s s u e s , i t remained  then exert pharmacological  collected  solely  different  e a r l y demonstrations of  that nerve terminals  Loewi s t i m u l a t e d  l e d to  two  e f f e c t s ) and  (1921) t o c o r r e l a t e d i r e c t l y and  restricted  m i m i c k e d by m u s c a r i n e and  of b i o l o g i c a l  stimulation,  not  (1914) was  w h i c h c o u l d be agonist  innervated.  substances a f f e c t i n g  possessed  t i s s u e s ; those  atropine  of  sympathetically  and  of a  stimulated heart.  "Vagusstoff", Brown, 1937;  effect substance  Loewi-  w h i c h was  Dale,  the  stimulated.  this  i n the p e r f u s a t e  tissue.  heart,  to  been  can  1938;  later f o r review  3  Langley substance",  (1905, later  to designate effector  1907)  introduced  s h o r t e n e d by  a specific  the  Clarke  (1926) t o  "receptor", on  c e l l s w i t h which p h a r m a c o l o g i c a l agents i n t e r a c t to  of Clarke  (1926) t h a t  logically  specific  Following  the  attempted  to e s t i m a t e  of  "receptive  although h y p o t h e t i c a l moiety  produce a c h a r a c t e r i s t i c response.  area  term  the  concept of  receptor  pioneering  was  from  curves.  From h i s  receptor  k i n e t i c s were d e r i v e d ,  description  of  the  r o l e of  the  until  the  work  examined q u a n t i t a t i v e l y . Langley,  number o f r e c e p t o r s  cell  studies  not  a d i s c r e t e , pharmaco-  work o f L o e w i and  the  c a r d i a c muscle  I t was  Clarke  f o r ACh  per  unit  concentration-response  b a s i c p r i n c i p l e s of and  at  synaptic  last  a  agonist-  basic  transmitters  was  made  possible.  Briefly,  i t is believed  nerve terminals, synaptic  cleft  potential.  upon i n v a s i o n o f  the  T h r o u g h d i f f u s i o n , the  effector target  evoked i n the  transmitters  from which they are  react with postsynaptic the  that  target  receptors  (Dale,  1938).  released terminal  are into  by  o r g a n i s d e p e n d e n t on  the  i n t h e membrane o f  the  postsynaptic  mitter.  I n a manner s t i l l  not  known p r e c i s e l y ,  reaction  postsynaptic  membrane.  (Katz,  Eccles,  1966;  a r r i v e at  1964,1.977) t h a t  of  changes  cell  by  the  trans-  the  causes m o l e c u l a r changes i n  It i s presently  and  p a r t i c u l a r response  induced  transmitter-receptor  action  t h e membrane s u r f a c e  The  in  the  an  transmitters on  stored  believed  such changes  however, involve  the  4  alterations passage of ionic as  o f membrane p e r m e a b i l i t y w h i c h p e r m i t the  t r a n s f e r i s not  the  agonist binds  1966;  K a t z and  ionic  flux  s u c h as the  event  1966)  o f a new  controlling electrical an  of  specificity  to the  the n a t u r e  using both agonists to the  response  followed  e v e n t s by  of  role  or  any  may (Bennett, neuronal  of  information,  chemical  the  transduction  s i g n a l s , each  of  with  of a c t i o n .  e a r l y work o f L a n g l e y  and  antagonists  others  as p h a r m a c o l o g i c a l  tools  dose-response data  formulation  n e c e s s i t a t e d the  Clarke  i n v e s t i g a t i o n s by  analyses  be  or  transmission  means o f  i n t e r p r e t a t i o n of  could not  effects,  a t some s y n a p s e s  i n t e r a c t i o n s , although  that  the  Although  terms o f a g o n i s t - r e c e p t o r  tions  to  in certain  tissue,  Thus t h e  carriers  long  1962;  p r e d o m i n a n t mechanism f o r  of receptors, and  (Katz,  target c e l l .  system.  s e e n as  f o r as  secondary  innervated  the  This  secretory processes,  than chemical  remains the  biological  induce of  ions.  continue  cellular  information  i n the nervous  Subsequent  gave r i s e  the  signals into different  apparent  defining  rather  be  may  action potential,  t r a n s f e r of  t r a n s m i t t e r s may  The  initiation  characteristic  latter  integration  1973).  t h e membrane may  electrical  the  extracellular  receptor molecule  c o n t r a c t i o n of  interneuronal involve  to the  f o r example t h e  i n s t a n c e s by  and  permanent, but  Miledi,  across  formation  other  intracellular  selective  these  o f a number o f  tested directly.  in  assump-  In a d d i t i o n to  these  pharmacokinetic further  that  a l l of  a v a r i e t y of a number o f a-  and  studies, Ahlquist  species  this  The  and  at present  the  by  their  the  e f f e c t s on  cited  that  doubt  the  that  synaptic  preservation  particularly  as  of neuronal  s e r i e s of on  For  there  are  namely  recognized  the  exclu-  transmitters,  receptors  receptors  system  of  (CNS).  Since  chemical  i t i s reasonable  f o r these  to  substances  sensitivity.  functional specificity, i n view of  systems,  transmitters  a v a r i e t y of  This  allows  w h i c h may  t h e marked d e g r e e o f  i n t e r a c t i o n i n the  be  com-  CNS.  Transmitters  criteria  has  been d e v e l o p e d from the  p e r i p h e r a l nervous  a transmitter  order  of  important  Criteria  studies  been  synaptic  specific postsynaptic  possess a r e s t r i c t e d pharmacological  A  long  above d e a l t  t h e mammalian c e n t r a l n e r v o u s  expect that  b)  classes,  in  cholinergic effects  D a l e had  studies  compounds w h i c h f u n c t i o n as  plexity  in  tissues  pharmacological analysis of p e r i p h e r a l  is little  the  o r g a n s and  i n t o two  s u b d i v i s i o n of  i t i s widely believed  neurones of  for  to e s t a b l i s h  time.  sively with  there  divided  nicotinic  A l t h o u g h most o f  exert  innervated  c o u l d be  $-receptors.  able  adrenoceptors mediating responses  sympathetically  into muscarinic by  the  (1948) was  at neuromuscular  f o r a s u b s t a n c e t o be  classical  system which e s t a b l i s h e d  ACh  and  In  assigned  ganglionic a r o l e of  a  synapses. synaptic  6  transmitter be and  present  (1)  i n the  precursors  released  (4)  a process it  i t must f u l f i l l  f o r the  for i t s synthesis;  action  itself  McLennan, 1970; All  of  other  at  i n favour  phate,  whether  (Burnstock,  an  attempt  of  these  of  synaptic  functions  to  synapses,  classify  s u b s t a n c e s and  finally and  the  as  Stefanis  and  evidence i s  and  aspartate, research  a subject  adenosine  t e r m i n o l o g y has  for  phenyltriphos-  criteria  themselves o f much  Fischer,  more e s t a b l i s h e d  has  physiology  " f i t " the  criteria  and  carnosine,  will  specu-  1978).  d i f f e r e n t i a t e between the  the  a c t i o n , a new  and  i n synaptic  B a l d e s s a r i n i and and  and  f o r ACh  More r e c e n t  a l l eventually or  be  fashion  r e l a t e d "neuropeptides",  i s at present  1976;  must  1977).  g l y c i n e , glutamate,  transmitters  require modification  identical  be  c e n t r a l r o l e s f o r these  histamine.  these w i l l  there  Salmoiraghi  i t s derivatives, taurine  conventional  lation  of  shown t o  transmitter  dopamine, s e r o t o n i n ,  e n k e p h a l i n e and  e t h y l a m i n e and  as  and  towards p o s s i b l e  s u b s t a n c e P,  i n an  (2)  s u b s t a n c e and  have been f u l f i l l e d  glutamine, y-aminobutyrate, adrenaline  the  i t must  enzymes  i t must be  natural  Storm-Mathisen,  substances, notably  pointed  the  certain peripheral  accumulating  proline,  of  of  (Werman, 1966;  these c r i t e r i a  noradrenaline rapidly  (6)  necessary  depolarization;  s u b c e l l u l a r elements  transmitter  1967;  the  i n a c t i v a t i o n (5)  the  interact with  following requirements:  neurone w i t h  following neuronal  must m i m i c  the  (3)  the  In  actions  transmitters  been proposed  ::  which  7  includes f a l s e transmitter,  co-transmitter,  neurohormone,  neuromodulator, e t c .  T h i s nomenclature i s not yet w i d e l y  accepted however, and  indeed remains to be adequately  Relevant to the i s s u e of t h i s "new mediators of s y n a p t i c  transmission  defined.  wave" of p o s s i b l e  i s the d i s c u s s i o n  by  Werman (1966) and Freeman (1976) of the techniques f o r s i n g the r o l e s of such compounds i n s y n a p t i c a c t i o n . i n discharge  frequency of a neurone induced by  administration  the  asses-  A change  direct  of a compound i s a f r e q u e n t l y used but  possibly  i n s u f f i c i e n t paradigm f o r assessment of i t s mode of a c t i o n . For example ACh  may,  i n a d d i t i o n to changing the r a t e of  f i r i n g , possess t r o p h i c a c t i o n s  (Drachman, 1974;  Purves, 1976).  There i s a l s o growing evidence f o r changes i n the of neuronal r e c e p t o r s Rosenbluth, 1949; 1976;  f o l l o w i n g denervation  sensitivity  (Cannon  and  K u f f l e r , Dennis and H a r r i s , 1971;  Purves,  Roper, 1976). Furthermore i n t r a c e l l u l a r "second messengers"  a c t i v a t e d by the t r a n s m i t t e r may  function i n c e l l u l a r  processes . d i s t i n c t from those subserving  the g e n e r a t i o n  s y n a p t i c p o t e n t i a l s (Greengard and Kebabian, 1974; Although s y n a p t i c  transmission  of a c t i o n p o t e n t i a l s from one formation  Daly, 1976).  does mediate the  terns of a c t i v i t y .  transfer  c e l l to another (or prevent  of an a c t i o n p o t e n t i a l , as i s the case w i t h  t o r y substances),  of  inhibi-  synapses do much more than f o l l o w r i g i d They are p l a s t i c and  the  pat-  dynamic s t r u c t u r e s  whose components are capable of a range of  qualitatively  8  d i f f e r e n t responses (Drachman, 1974; volume).  and  other  For example some neurones do not  articles in this  generate a c t i o n poten-  t i a l s , yet i n f l u e n c e a d j o i n i n g neurones s o l e l y by graded release of t r a n s m i t t e r 1969;  ( R a i l and  Shepherd, 1968;  Pearson and Fourtner,  receptors  1975).  of neurones or muscle may  W e r b l i n and  Dowling,  Furthermore, p o s t s y n a p t i c ' p r o l i f e r a t e or undergo  a l t e r a t i o n s i n t h e i r s e n s i t i v i t y to the t r a n s m i t t e r when they are denervated (Ungerstedt, 1971). Other examples o f p l a s t i c i t y are p r o v i d e d  by  synaptic  the phenomena of f a c i l i t a t i o n ,  t a t i o n and h a b i t u a t i o n : processes which are b e l i e v e d to changes i n s y n a p t i c  f u n c t i o n (Kandel, 1976).  adap-  involve  In summary, i t i s  becomming c l e a r that as more i s l e a r n e d about t r a n s m i t t e r s the ways they are u t i l i z e d by  synapses, the more refinement  w i l l be r e q u i r e d of p r e v a i l i n g concepts concerning zation  c) One  of  and  the  organi-  the nervous system.  Iontophoresis means of i d e n t i f i c a t i o n of s y n a p t i c  transmitters  demonstration o f the presence o f neuronal r e c e p t o r s to the compounds, and  t h i s forms one  c r i t e r i a as o u t l i n e d i n the p r e v i o u s c h i e f advantages to studying  i s the sensitive  of the more important section.  One  of  the  the a c t i o n s of drugs u s i n g  the  i o n t o p h o r e t i c method i s t h a t i t i s p o s s i b l e to examine t h e i r e f f e c t s upon s i n g l e neurones i n v i v o without a f f e c t i n g the whole o f the nervous system or other p h y s i o l o g i c a l processes  9  (respiration,  blood pressure,  drugs are a d m i n i s t e r e d brain barrier  and  as  comparing  the e f f e c t s  concerning  the p r a c t i c a l (Curtis,  Krnjevic,  1971;  Lim  ventricle  later  technique  by  i n t r o d u c e d by  an  Curtis the  was  and  solution,  source  of the  a barrel of  electrical  occur  source  open t i p o f t h e b a r r e l .  the p r e s s o r  '.  4th  area.  Nastuk  central  (1953')  neurones.  c u r r e n t i s a p p l i e d to ions  imposed f i e l d .  o f i o n t o p h o r e s i s , f o r when field  Suh,  o f the  a migration of  of the  This i s the  i s p o s i t i o n e d a t t h e mouth o f  of a m u l t i p i p e t t e assembly,  i o n s away f r o m t h e  here.  floor  for testing  electrical  source  fundamental p r i n c i p l e  o f some o f  i n t r o d u c e d by  out  1967;  (1958) f o l l o w i n g r e f i n e m e n t s •  authors  there w i l l  neuronal  Stefanis,  overview  to the  t o map  and  details  u s e d most e f f e c t i v e l y by  Eccles  latter  t o w a r d o r away f r o m t h e the  effort  i s known t h a t i f an  ionic  first  a p p l i e d ACh  o f dogs i n an  However t h e  a brief  admini-  have been  and  c o n s i d e r a t i o n s i s warranted  (1936) who  blood-  and E c c l e s ,  testing  technique  Salmoiraghi  but  of the  o f compounds upon  the  i o n t o p h o r e t i c method was  Wang and  It  1976;  Bloom, 1974)  t h e more s a l i e n t  and  1964;  of  when  t h i s method o f  o f i o n t o p h o r e s i s and use  occur  (Curtis  ease o f r a p i d l y  of a s e r i e s  b a s i c theory  published  The  synaptic barriers  i s the r e l a t i v e  The  as may  Avoidance  a d v a n t a g e p r o v i d e d by  stration,  firing.  systemically.  other  1958b) i s a n o t h e r  e t c . ) such  will  the r e s u l t a n t  effect  their  migration  ejection  I f the p i p e t t e assembly  at  the  i s positioned  10  in  close proximity  to a neurone,  cellular  electrical  solution  i n one  and  of  activity the  firing.  be  actions over  occurring  the  tional  range of  to  the  amount a c t u a l l y e j e c t e d  is  normally  Zieglgansberger,.  It  i s necessary  successive  approximately  the  under e q u i v a l e n t ejected  i s not  to r e g u l a t e  constant current  the of  "leakage". retention, pipette  conditions.  the  the  references 1969;  Hall,  that  of  can  f o r example, o f an  t o be  the  the  usual  retention,  no  drug  Usually to  procedure keeping a retaining prevent  s o l u t i o n at  "warming up"  period  i s s w i t c h e d on  therefore  antagonist  be  made  amount  schedule of e j e c t i o n  ejecting current Tests  i s propor-  absolute  (rianoamperes) i s s u f f i c i e n t  concentration  1978).  (see  for tests  accuracy,  t i p becomes s t a b l e and  Nistri,  activity  Since  such a constant  r e q u i r e d when t h e  and  to ensure  i n t e r v a l between e j e c t i o n s .  With  linear  1979).  t i m e o f e j e c t i o n and  nA  delivery is  a p p l i c a t i o n s o f a compound d e l i v e r  known w i t h any  10-15  inter-  Teschemacher,  Wheal,  same dose i n o r d e r  the  the  complex  applied  i n some e x p e r i m e n t s  iontophoretic  to  employed,  H e r z and  McLennan, R i c h a r d s o n and  due  t i p , the  current  the  ejected of  pipette  currents  be  rate  predicted value  the magnitude of  above, and Hicks,  at  the  of  made t h r o u g h a N a C l  p i p e t t e b a r r e l s , d r u g s may  Although  somewhat l e s s t h a n t h e  and  can  recordings  t h e i r p h a r m a c o l o g i c a l e f f e c t s o b s e r v e d on  cellular  is  such t h a t  and the is  (MacDonald  made t o a s s e s s  upon t h e  effects  the  11  induced  by  two  o r more c y c l i c a l l y  Another p o i n t antagonist The  i s the  use  the  i s sigmoidal,  activity the  drug, w i l l  be  antagonist  and  will  starting points  the  determination  ejecting  due  to  approximately  a n t a g o n i s m may responses of  not  the  the  two  i n the presence  f o r the  firing  claimed agonists  the  two  theory  a c t i o n of due  and  the  the  to  differ-  rate i s often  inactivation.  A more  i s to e s t a b l i s h  agonists which e l i c i t A  stable  selectivity  i f the p e r c e n t a g e differ  i t is  I n p r a c t i c e however,  spike  rates.  and  of a b l o c k i n g  o f a neurone's maximal f i r i n g s u c h f a c t o r s as  agonist  agonists,  unequal s h i f t s  curves.  an  agonists.  (ED50)  responses observed to  reflect  along  equal  be  When u s i n g  require  dose,  In  of  and  dose of  curve.  approach which i s used widely currents  potencies  the  a s c r i b a b l e to a d i f f e r e n t i a l  ent  practical  ideal  t h e ED^Q'S o f  d i f f e r e n c e s to the this  the  a half-maximal response  to determine  t e s t e d at  impossible  and  l i n e a r p o r t i o n of  first  relative  agonists  decreases  of in  the  considerably.  i o n t o p h o r e t i c approach to determine  the  o f compounds, a number o f a d d i t i o n a l v a r i a b l e s  c o n s i d e r a t i o n , of which the  considerable of  the  o f e x c i t a t i o n p r o d u c e d by  i s that which gives  necessary  and  agonists.  r e l a t i o n s h i p t h a t e x i s t s b e t w e e n dose o f a g o n i s t  w h i c h i s on  any  t o c o n s i d e r when t e s t i n g level  response e l i c i t e d to  administered  importance.  t r a n s p o r t number  T h i s number r e p r e s e n t s  i o n a c t u a l l y r e l e a s e d f r o m t h e p i p e t t e as  the  assumes quantity  a fraction  of  the  12  amount which should be e x p e l l e d by a g i v e n c u r r e n t .  This  r a t i o , always l e s s than 1, i s determined by a number of f a c t o r s i n c l u d i n g the i o n i c s t r e n g t h of the s o l u t i o n and degree of i o n i c i n t e r a c t i o n w i t h  the  the p i p e t t e s u r f a c e which  g i v e s r i s e to v a r i o u s p o t e n t i a l s at the t i p . Gent, Morgan and W o l s t e n c r o f t  (1974) found that  apparent d i f f e r e n c e s i n potency of two L-glutamate and L - a s p a r t a t e , b i a s e d due  e x c i t a n t amino a c i d s ,  were sometimes  considerably  to d i f f e r e n c e s i n the t r a n s p o r t numbers of the  compounds from d i f f e r e n t p i p e t t e s . had  the  These potency  estimations  to be c o r r e c t e d f o r the t r a n s p o r t numbers i n order  o b t a i n an accurate Zieglgansberger  assessment of the r e l a t i v e  glutamate under c a r e f u l l y c o n t r o l l e d c o n d i t i o n s and p r o p o r t i o n a l to the amount of  No  of [H]3  found  current  a p p l i e d , even w i t h i n t e n s i t i e s w e l l beyond those employed.  normally  d i r e c t r e l a t i o n s h i p e x i s t e d between the  of the t i p of the e l e c t r o d e and  to  potencies.  et a l . (1969) a l s o measured e f f l u x  that r e l e a s e was  two  t r a n s p o r t number.  diameter  Solutions  of low i o n i c s t r e n g t h possessed a l i n e a r r e l a t i o n s h i p between c u r r e n t i n t e n s i t y and  amount e j e c t e d , although they d i s p l a y e d  lower t r a n s p o r t numbers. Since  i t i s necessary f o r compounds to be i o n i z e d i n  s o l u t i o n f o r t h e i r i o n t o p h o r e t i c d e l i v e r y from m i c r o p i p e t t e s , it  i s o f t e n necessary to a d j u s t the pH of the s o l u t i o n s w i t h  NaOH or HCl.  The  simultaneous r e l e a s e of H  +  or Na+  from such  13  solutions f o r m may  together complicate  known t h a t H icksdn, can  interpretation  agents i n c a t i o n i c  of r e s u l t s ,  by i t s e l f c a n c a u s e n e u r o n a l 1971; S t o n e ,  be a v o i d e d  by e m p l o y i n g  a number o f t e s t  pH v a l u e s w i t h i n  effect  o f the suspected  1972).  ion i t s e l f .  solution,  corresponding  i f the e f f e c t s  i o n and o b s e r v i n g  that e l i c i t e d  by t h e d r u g  reverse procedure w i l l  (Freder-  This  problem  solutions at  range,  o r by  testing  T h i s i s done by  f r o m an H C l c o n t a i n i n g s o l u t i o n  containing  with  the c a t i o n i c  since i t i s  excitation  and P h i l l i s ,  expelling  the  +  pharmacological  Jordan  different the  with  of Na  (or from a NaCl are of i n t e r e s t )  +  the e f f e c t  compound.  f o r comparison  Of c o u r s e  the  allow  the t e s t i n g  of possible anionic  a l l microelectrode  experiments  on c e n t r a l  effects. Like  an u n a v o i d a b l e can  be m a n i f e s t e d  recorded flow  i n the p e r i n e u r o n a l  current cells  greater  from the c e l l  will  generate  be r e a d i l y  recorded  easily  This bias w i l l that  i n two ways.  space,  high  result  will  amplitude  greater  electrical  distances  be r e i n f o r c e d by a n o t h e r  larger cells  with  have a l a r g e r  an a c t i o n p o t e n t i a l .  d e t e c t e d by m i c r o e l e c t r o d e s over  of current  l a r g e r c e l l bodies  surface areas  during  b i a s , and t h i s  '. F i r s t , s i n c e t h e p o t e n t i a l  a t the e l e c t r o d e t i p i s a d i r e c t  correspondingly  will  problem i s t h a t o f sampling  neurones,  As  these  p o t e n t i a l s they  a n d c a n be  from t h e c e l l factor,  body.  and t h a t i s  a r e more f r e q u e n t l y e n c o u n t e r e d  simply  on a  14  statistical  basis, unless  comprised mostly of Secondly, those are  be  a c t i v a t e d by  normally  an  c u l t i e s may local  arise  case of  masked  which f i r e  e j e c t i o n o f an  i f the  and  sampling,  population synaptic Wheal,  be  an  avoided  t h a t i s by  of neurones  latter  r e s u l t may  case,  or  excites  t h a t many  inhibited,  McLennan,  i n p a r t by  studying  only  i d e n t i f i e d by  s t i m u l a t i o n (Stone,  be  diffi-  or  e x c i t a t o r y r e s p o n s e may  McLennan, 1971;  f a c t o r s may  the  unresponsive,  when i n f a c t  which  e x c i t a t o r y substance;  In  The  only  spontaneously or  amino a c i d p r e f e r e n t i a l l y  a p p e a r t o be  at a l l ,  (Felix  biasing  1972;  1971).  not be  These  a deliberate selec-  responses  their  f r o m a known  responses  to  H u t c h i n s o n , McLennan  and  1978).  A recent  development i n n e u r o p h a r m a c o l o g i c a l  w h i c h employ i o n t o p h o r e s i s b l i e s which are attached  comparisons dendritic  the  drug c o n t a i n i n g p i p e t t e s  the  but  which are  made o f t h e  sensitivity  site  broken  r e c o r d i n g b a r r e l 'is'.'.tip  a d m i n i s t r a t i o n o f compounds can  away f r o m t h e  t o be  the  studies  o f m u l t i p i p e t t e assem-  recording barrel,  distances behind  t h i s manner t h e  known d i s t a n c e s  i s the use  constructed with  alongside  back to v a r i o u s In  i o n t o p h o r e t i c experiments  i n h i b i t o r y mechanisms.  detected  under i n v e s t i g a t i o n i s  cells.  detected  the  area  amino a c i d i s u s e d .  neurones w i l l  tive  small  i n the  cells will  the  of recording,  topographical  (Zieglgansberger  and  be  made a t  allowing  localization Champagnat,  of 1977;  15  1978).  T h i s approach promises to be r e w a r d i n g i n i n v e s t i g -  a t i o n s of CNS  r e g i o n s where neurone somata and  their dendritic  p r o c e s s e s are o r g a n i z e d i n d i f f e r e n t but  d i s c r e t e l a y e r s , such  as i n the hippocampus, c e r e b e l l a r c o r t e x  or s p i n a l c o r d .  Another i n n o v a t i v e  approach used i n r e c e n t s t u d i e s  p h a r m a c o l o g i c a l a s p e c t s of s y n a p t i c  p h y s i o l o g y concerns  s i m u l t a n e o u s t e s t i n g of a s i n g l e c e n t r a l neurone w i t h multipipette  a s s e m b l i e s (Duggan, H a l l and  Engberg, Flatman and  of  Lambert, 1979).  One  Headley,  the  two  1977;  of these i s p l a c e d  near or i n t o the soma of the neurone i n the u s u a l  fashion,  w h i l e the second assembly i s advanced through the t i s s u e i t i s positioned  somewhere w i t h i n the d e n d r i t i c  of the same neurone.  The  arbourization  e f f e c t s of compounds e j e c t e d  the l a t t e r p i p e t t e assembly can  then be  different  e f f e c t s upon f i r i n g of p e r i s o m a l drug a d m i n i s t r a t i o n  compared  w i t h d e n d r i t i c a p p l i c a t i o n , or the e f f e c t s on n e u r o n a l  to s y n a p t i c  or h y p e r p o l a r i z a t i o n  activation.  fashion,  to use  i n the mammalian  CNS.  i n response to the  firing  iontophoresis  i n a very  e n a b l i n g the c o n c u r r e n t i n v e s t i g a t i o n  of p h y s i o l o g i c a l p r o c e s s e s and i z a t i o n of r e c e p t o r s ,  firing  W i t h t h i s type of t w i n m i c r o p i p e t t e  arrangement, i t i s p o s s i b l e sophisticated  from  tested against a  number of e x p e r i m e n t a l v a r i a b l e s , such as: the  of d e n d r i t i c de-  until  the p h a r m a c o l o g i c a l  character-  a l l a t the l e v e l of the s i n g l e neurone  16  d)  The P r e s e n t  The of  experiments  t o be d e s c r i b e d  were u n d e r t a k e n w i t h  c h a r a c t e r i z i n g the p h a r m a c o l o g i c a l p r o p e r t i e s  different CNS.  populations  of neuronal receptors  Neuronal responses  agonists for  Study  the f o l l o w i n g  systems:  investigations  Receptors  were  series of  before  receptors;  associated  conventional  methods.  f o r ACh a p p e a r t o p o s s e s s u n i q u e f o r c e n t r a l neurones;  and i t i s p r o p o s e d  A,more  properties,  t h e amino  a t l e a s t two and p o s s i b l y  through receptors  catecholamines. for  receptors  s y n a p t i c a l l y through  were f o u n d t o r e a c t w i t h  effects  Each o f the three  the c h a r a c t e r i z e d  neurophysiological  tinct  i n t h e VB  a n d 3) a m i n e s i n t h e c e r e b r a l  s p i n a l cord.  with neurones i d e n t i f i e d  described  2) amino a c i d s  VB  employed a common e x p e r i m e n t a l a p p r o a c h a n d  where p o s s i b l e ,  not  receptors  1) ACh i n t h e c e r e b r a l c o r t e x ,  thalamus and d e n t a t e gyrus; and d o r s a l  administered  were u s e d t o a n a l y z e t h e  thalamus and v e n t r a l s p i n a l c o r d ;  cortex  of several  i n t h e mammalian  to i o n t o p h o r e t i c a l l y  and a n t a g o n i s t s  the aim  that  acids  three  dis-  octopamine exerts i t s  d i f f e r e n t from those u s e d by t h e  thorough d i s c u s s i o n o f the r a t i o n a l e s  these experiments and the r e l e v a n t  appears  i n the r e s p e c t i v e  systems  examined.  introductions  background  literature  f o r each o f the  17  CHAPTER  MATERIALS AND  All  of the experiments  performed gm.  on r a t s  METHODS  t o be d e s c r i b e d  of either  i n this  sex, w e i g h i n g  thesis  i n a manner w h i c h  discomfort  on t h e p a r t  t a i n e d by a d d i t i o n a l Additional  twitchiness  signs:  A n a e s t h e s i a was  of the eyes;  3) h y p e r s e n s i t i v i t y  2)  pronounced  of the f l e x i o n  f o r maintenance  o f p h a r m a c o l o g i c a l compounds,  c a n n u l a e were p l a c e d i n t h e  of a n a e s t h e s i a or f o r the t e s t i n g as p a r t  of the e x p e r i m e n t a l  The f e m o r a l v e i n o r o c c a s i o n a l l y  were u s e d .  The c a n n u l a e  comprised  the j u g u l a r  and d u r i n g t h e i r placement  b l o o d v e s s e l s , were f i l l e d  with a small quantity  saline.  vein  fine polyethylene tubing  o f 0.9mm o u t s i d e d i a m e t e r ,  use  body  u s u a l l y o f the limbs.  blood vessels  ized  main-  t o h i n d p a w p i n c h e s ; o r 4) s p o n t a n e o u s  I n many o f t h e e x p e r i m e n t s ,  protocol.  i f any  o f t h e same a n a e s t h e t i c .  1) b u l g i n g  of vibrissae;  i n response  movements,  to generate minimal,  of the animal.  doses  (1.5 g/Kg,  d o s e s were g i v e n whenever t h e a n i m a l e x h i b i t e d any  the f o l l o w i n g  reflex  appeared  were  b e t w e e n 137 a n d 420  The a n i m a l s were a n a e s t h e t i z e d w i t h u r e t h a n e  i.p.)  of  II  i n the  of heparin-  The employment o f 3-way s t o p c o c k s p e r m i t t e d t h e  o f t h e same c a n n u l a f o r t h e a d m i n i s t r a t i o n o f more  than  18  one m a t e r i a l .  To accomplish t h i s ,  the u s u a l procedure was  i n j e c t the compound of i n t e r e s t between two of  saline.  to  administrations  T h i s p e r m i t t e d the comparison of the drug e f f e c t  with a control  (the f i r s t  s a l i n e i n j e c t i o n ) and  further  ensured the d e l i v e r y of the e n t i r e dose of the compound (the "chaser" i n j e c t i o n ) . With cannula l e n g t h s of approximately 5 cm,  0.5  ml  f o r each s a l i n e i n j e c t i o n was  s u f f i c i e n t f o r the  above procedures. F o l l o w i n g a n a e s t h e s i a , the i n i t i a l  s u r g i c a l procedures  i n v o l v e d the placement of cannulae and i n many experiments, the  i s o l a t i o n and c u t t i n g of the p e r o n e a l or s c i a t i c nerve.  The  s k i n f l a p s around the cannula were c l o s e d by coarse  s t i t c h i n g , w h i l e those around the exposed h i n d l i m b nerve were r a i s e d to form a p o o l by t h e i r attachment to a p l e x i g l a s s r i n g f a s t e n e d to the frame which supported the animal. p o o l was  This  f i l l e d w i t h warmed l i q u i d p a r a f f i n o i l w i t h i n which  were immersed the ends of b i p o l a r s i l v e r e l e c t r o d e s .  These  were bent i n a manner so as to a l l o w the nerve to be p l a c e d in  c o n t a c t w i t h each p o l e , and to be supported w i t h i n the  pool.  Body temperature was maintained at 3 7 . 5 ° C by a h e a t i n g  pad r e g u l a t e d by a temperature c o n t r o l u n i t equipped w i t h a r e c t a l t h e r m i s t o r probe.  (EKEG E l e c t r o n i c s )  19  a)  Supraspinal  Following  the  Experiments  initial  anaesthetic  and  s u r g i c a l procedures,  a n i m a l s were p o s i t i o n e d i n a h e a d - h o l d e r  frame  t a x i c ; m o d e l 1204)  i n p l a c e by  ear-bars  incisor  i n p o s i t i o n 4.8  the  frame's  and  sides.  fixed  With the  beneath the h o r i z o n t a l p l a n e taxic  zero:  vertical)  and  at  the  bar  inter-aural  the  calibrations  zero:  the  a n i m a l ' s b r a i n was  orientation Klippel  corresponding  a midline e x p o s e d 'by  overlying  tissues with  r e v e a l e d bregma, bone f l a p s  the  bleeding  carefully  gentle  the  use  fixed  the  nim  (stereoframe's  i n place  atlas  on  (stereotaxic in  of Konig  and  applying  s c r a p i n g back of  an and  Bleeding  reflected,  a few  One  This  or  two  a  small  procedure  dura  of Gelfoam  was  (Upjohn)  bone wax.  I f a p o o l was  covered  and'  procedure  The  e x t r a . c a u t i o n whenever  exposed.  the  i n v e s t i g a t e d were  from s k i n or  stopped with  with  This  f i t t e d with  forceps.  small pledgets  f r o m bone was  otherwise  t o be  scalp,  sclera  s a g g i t a l suture.  claw-tooth  s a g g i t a l s i n u s was  oil;  through the  o f a hand d r i l l  e x p o s e d n e r v o u s t i s s u e was  paraffin  line  ear-bars  a periosteal elevator.  dura mater.  c o n t r o l l e d by  superior  the  that of  o v e r l y i n g the b r a i n area  round d e n t a l burr, the  the  lambda and  removed t h r o u g h t h e  was  to  on  saggital incision  s k u l l was  while  mounted  (1974).  After  exposed  Stereo-  the h e a d midway b e t w e e n t h e  s i d e s j u d g e d by lateral),  (Kopf  the  w i t h warmed  dura  the  constructed liquid  d r o p s o f warmed L o c k e s o l u t i o n  20  were u s e d For  t o keep  the t i s s u e  e x p e r i m e n t s on t h e c o r t e x  exposed b r a i n r e g i o n s 1.5 mm to  a n d 6.0 mm  4.5 mm  lateral  hippocampus,  roughly  to the m i d l i n e .  lateral  s i d e was e x p o s e d 0.5 mm lateral  c o r r e s p o n d e d t o AP  the exposed r e g i o n s  bregma and 4.5 mm  and VB t h a l a m u s , t h e boundaries  p o s t e r i o r t o bregma, a n d L b o u n d a r i e s  mental) s i d e had b o u n d a r i e s  mm  moist.  mm  F o r e x p e r i m e n t s on t h e on t h e i p s i l a t e r a l  1.5 mm  and 8.5 mm  to the midline.  t o 2.5 mm  to the m i d l i n e .  1.0  (experi-  p o s t e r i o r to  The c o n t r a l a t e r a l  p o s t e r i o r t o bregma a n d 3.0  The bone o v e r l y i n g t h e s a g g i t a l  s i n u s was removed e n t i r e l y . b)  S p i n a l Cord  Experiments  Following  the i n i t i a l  cannulaej  t h e a n i m a l s were p l a c e d  additional skin  anaesthetic  surgical preparation.  i n c i s i o n was made a l o n g  on a r a i s e d p l a t f o r m f o r A long  (approximately  the d o r s a l midline  spinae  m u s c l e s were d i s s e c t e d f r o m b o t h s i d e s o f t h e s p i n e ,  the  first  The  surrounding  tissue  processes  sacral vertebrae  process.  from the second  to the f i r s t  the spinous  spinous  10 cm)  lumbar  and  sacral  p r o c e d u r e s and placement o f  The e r r e c t o r  a n d l a m i n a e o f t h e s e c o n d lumbar t o were removed w i t h  bone  forceps.  t i s s u e was h e l d away f r o m t h e v e r t e b r a e  retractors.  The d u r a m a t e r was  then c a r e f u l l y  making t h e s p i n a l c o r d and s p i n a l r o o t s E x t r e m e c a r e was t a k e n t o a v o i d  touching  easily  by  c u t away  accessable.  t h e s p i n a l c o r d and  21  to keep a l l t i s s u e s m o i s t w i t h  warmed L o c k e s o l u t i o n .  Following  c o m p l e t i o n o f t h e laminectomy,  d o r s a l r o o t s were c u t .  Ventral  roots  to the hindlimb  following  their  were i d e n t i f i e d by t h e t w i t c h  severance.  The a n i m a l s were t h e n  upon a f r a m e by means o f clamps spinous processes. for  within  c)  described.  The a n i m a l ' s h e a d was  this  pool  Stimulating  Stimulating various  Dorsal  by b i p o l a r s i l v e r  And R e c o r d i n g  2.8, V 3.9  stimulating  atlas  Concentric  Kopf m i c r o m a n i p u l a t o r s . f o r crus  f o r the dentate gyrus,  bipolar electrodes  saline  o f 75-100 K f i were u s e d f o r e l e c t r i c a l  o f 0.5 mm  S i n g l e monophasic r e c t a n g u l a r  e d e d 20 V, and more  f o r angular according  bun-  to the  Instruments)  a n d DC r e s i s t a n c e i n n o r m a l  pulses  used i n the s t i m u l a t i o n of b r a i n  through s i l v e r wires.  c e r e b r i ; A 4.2  (SNE 100, Rhodes  a t i p separation  n e r v e s were a l s o  Place-  (1974).  having  pheral  electrodes.  stereotaxically into  f o r VB t h a l a m u s ; P 0.5, L 4.4, V 1.3  o f K o n i g and K l i p p e l  suspended  Procedures  o f t h e CNS u s i n g  d l e ; A 3.7, L 1.5, V 1.7  pool  t h e s k i n f l a p s as  a n d v e n t r a l r o o t s were  ments were as f o l l o w s : A 3.2, L 2.6, V 2.6 L  s u p p o r t e d and a  by r a i s i n g  e l e c t r o d e s were l o w e r e d  regions  suspended  s e c u r e d t o two o f t h e r e m a i n i n g  l i q u i d p a r a f f i n constructed  already  induced  o f 0.1 msec  sites;  stimulated  brain  stimulation.  duration  spinal roots  were  and p e r i -  at these parameters, but  The i n t e n s i t y o f s t i m u l a t i o n r a r e l y exce-  t y p i c a l l y was  i n t h e r a n g e o f 2-8  V.  22 M i c r o p i p e t t e s u s e d f o r d r u g a d m i n i s t r a t i o n and r e c o r d i n g neuronal and  a c t i v i t y were p l a c e d w i t h  a large micromanipulator  stereotaxic ulator  frame.  i n fixed  which allowed  cally  microdrive  mounted on a b a r a t t a c h e d  E l e c t r o d e s were a t t a c h e d  to the  t o t h e micromanip-  o r i e n t a t i o n b y a c u s t o m made h o l d i n g  device  the easy removal and replacement o f t h e assembly  to the h o l d e r . assemblies  an AB T r a n s v e r t e x  The m i c r o d r i v e was u s e d t o l o w e r t h e m i c r o p i p e t t e  i n 4ym s t e p s  t h r o u g h t h e employment o f an e l e c t r o n i -  c o n t r o l l e d s t e p p i n g motor. A l l measurements f o r e l e c t r o d e  p o s i t i o n s were t a k e n  from the r a t b r a i n a t l a s  o f Konig a n d K l i p p e l  (1974) and were v e r i f i e d b y c o m p a r i s o n w i t h t h e known p o s i t i o n o f bregma f o r a n t e r i o r - p o s t e r i o r c o - o r d i n a t e s ; w i t h t h e m i d l i n e posi t i o n of the s a g g i t a l  sinus  f o r l a t e r a l c o - o r d i n a t e s and w i t h the  surface of the cortex f o r v e r t i c a l sites  f o rcortical  co-ordinates.  The.recording  e x p e r i m e n t s were i n t h e s o m a t o s e n s o r y  area:  (A 4:4, L 2.6, V 4.3-2.8) a n d f o r t h e VB t h a l a m u s were: A 4.4, L 2.6, V -0.6; a n d f o r d e n t a t e  d)  Recording  spinal  A 3.7, L 1.5, V 1.8.  From S i n g l e N e u r o n e s And D r u g D e l i v e r y T e c h n i q u e  Extracellular VB t h a l a m u s ,  gyrus:  recordings dentate  from neurones o f t h e c e r e b r a l c o r t e x ,  gyrus and d o r s a l and v e n t r a l h o r n s o f t h e  c o r d were made t h r o u g h t h e c e n t r a l b a r r e l  b a r r e l p i p e t t e assemblies. obtained  The g l a s s m u l t i b a r r e l b l a n k s  from Vancouver S c i e n t i f i c  7 fused glass c a p i l l a r i e s , o f 2.5-3.5 mm.  puller,  Glassblowing  were p l a c e d  were  as a r r a y s o f  h e a t - d r a w n t o an o v e r a l l  These b l a n k s  type m i c r o e l e c t r o d e  of multi-  diameter  i n a vertical  h e a t e d , and p u l l e d t o f i n e  Canberra tips.  23  The  resulting  assembly  was t h e n t r a n s f e r r e d  l i g h t m i c r o s c o p e where t h e t i p s were v i e w e d fication.  With  needle-shaped the  water to the  to a t o t a l  by b o i l i n g  f o r 20 m i n u t e s  with  or u n t i l  and a t h i n , the t i p s of  diameter o f glass-distilled  a l l b a r r e l s were  d i s t i l l e d water w i t h i n  t h e b a r r e l s was removed f r o m t h e  r e p l a c e d b y one o f t h e s o l u t i o n s  4 M N a C l was u s e d either  with  to f i l l  barrel.  distilled  The s o l u t i o n s  water  filter  (+6°C) w i t h t h e i r use,  away f r o m  f o r 1 minute  i n the micropipette  into  C a r e was  particles  immersed i n d i s t i l l e d the NaCl  a n d was c o n n e c t e d t o t h e a m p l i f i e r  bar-  to the t i p s f o r  and when n o t i n u s e t h e y were tips  um-passing  i n a Beckman  f o r the experiment.  a p l a t i n u m wire extended  barrel  the i s o e l e c t r i c  t h r o u g h a 0.8  t a k e n t o keep away d u s t a n d d i r t  micropipettes,  and o c c a s i o n a l l y  i n T a b l e 1 were made  t h e s o l u t i o n s were a l l o w e d t o d i f f u s e  always  i n T a b l e 1.  The pH was a d j u s t e d w i t h  t h e i r placement  24 h o u r s b e f o r e b e i n g u s e d  the  i t well  and c e n t r i f u g e d  m i c r o f u g e "B". . A f t e r rels,  listed  The s o l u t i o n s were f i l t e r e d  Millipore  The w a t e r  i n a separate current neutra-  o r 0.15 M N a C l .  e i t h e r H C l o r NaOH t o b r i n g point.  listed  the recording b a r r e l  2 M o r 4 M N a C l was u s e d  lization  seen  The a s s e m b l i e s were t h e n a l l o w e d t o c o o l a n d  s h a f t s w i t h a s y r i n g e a n d 30 gauge f l e x i b l e n e e d l e . was  Wetzlar  16x m a g n i -  s t e e l b a r c o n t r o l l e d by a j o y - s t i c k ,  The p i p e t t e s were t h e n f i l l e d  be f i l l e d .  under  t h e a i d o f an e y e p i e c e g r a t i c u l e  m i c r o p i p e t t e s were b r o k e n b a c k  5-10 um.  to a L e i t z  from refridgerated  water.  When i n  o f the c e n t r a l  probe.  Silver  wires  TABLE 1 Drug Compound  Solutions Concentration  Na-L-Glutamate metoclopramide  monochloride  Active ion  0.15 M N a C l and H 2 O 7.0  cation  0.02-0.20 M  0.15 M N a C l and H 2 O 5.8  cation  a c e t i c a c i d and H 2 O 6.8-6.9 sulfate  mecamylamine  hydrochloride  dihydro-3-erythroidine d-tubocurarine  bromide  hydrogen  0.02  M  0., 15 J M  NaCl  5.0  cation  0.05  M  0,, 1 5  M  NaCl  5.5  cation  0.. 1 5  M  NaCl  7.5  cation  0,. 0 1 - 0 , . 0 2  hydrochloride  1,1-dimethyl-4-phenylpiperazinium nicotine  pH  0.05-0.50 M  clozapine atropine  Solvent  iodide  tartrate  M  0.02  M  0. 15  M  NaCl  5.0  cation  0.01  M  0., 1 5  M  NaCl  6.0  cation  0.. 1 5  M  NaCl  4.5  cation  7.0  cation  0,. 0 5 - 0 . 1 0  M  b i c u c u l l i n e methochloride  0.01  M  0., 1 5  M  NaCl  kainic  0.05  M  0.. 1 5  M  NaCl  0.50  M  hydrochloride  0.50  DL-noradrenaline b i t a r t r a t e DL-dopamine  acid  L-glutamate DL-octopamine  diethylester  hydrochloride  (±)-cis-1-amino-1,3-dicarboxycyclopentane  8 . 0 - 8 . ,2  anion  H 0  3 . 4 - 3 , ,5  cation  M  H 0  3 . 0 - 5 , ,2  cation  0.50  M  H 0  2 . 7 - 5 , ,2  cation  0.50  "M  H 0  3 . 5 - 5 . ,0  cation  0.20  M  H 0  2  2  2  2  2  8.2  anion  TABLE 1 (cont'd) Drug Compound acetylcholine  Solutions  Concentration bromide  PH  Active  M  H0 2  4.5  cation  0. 20 M  H0 2  4.5  cation  a c e t y l - 3 - m e t h y l c h o l i n e bromide  0.20  M  H0 2  4.5  cation  Na-D-glutamate  0.50  M  H0 2  8.0  anion  Na-L-aspartate  0.50  M  H0 2  8.0  anion  Na-D-aspartate  0.50  M  H0 2  8.0  anion  DL-homocysteate  0.20  M  H0 2  7.6  anion  N-me  0.10  M  H0 2  8.0  anion  DL-a-aminoadipate  0.50  M  H0 2  8.0  anion  D-a-aminoadipate  0.20  M  H0 2  8.0  anion  3-aminoadipate  0.20  M  H0 2  8.3  anion  3-aminoglutarate  0.20  M  H0 2  8.1  anion  2-amino-3-phosphonoproprionate  0.20  M  H0 2  8.0  anion  2-amino-4-phosphonobutyrate  0.20  M  H0 2  8.0  anion  a-flupenthixol  0.20  M  H0 2  3.4  cation  DL-propranolol hydrochloride  0.50  M  H0 2  3.4  cation  y-aminobutyrate  0.50  M  H0  3.5  cation  carbamylcholine  chloride  thy1-DL-aspartate  1.00  Solvent  2  N3 Ln  26  extended i n t o ting All  the remaining  drug  containing barrels,  the s o l u t i o n s t h e r e i n c o n t a i n e d w i t h  a current  b a r r e l s were c h e c k e d f o r a d e q u a t e c u r r e n t f l o w  connecsource.  before  each experiment,  t h e u s u a l r a n g e o f t h e DC r e s i s t a n c e b e i n g  70-100 Mfi, w h i l e  the r e c o r d i n g b a r r e l  typically exhibited  v a l u e s w i t h i n t h e r a n g e o f 1-5 Mfi.  In o r d e r of  to prevent  the d i f f u s i o n  o f i o n s from the t i p s  t h e p i p e t t e s , c u r r e n t s were employed o p p o s i t e  used to e j e c t barrels. within  the i o n s f o r each o f the outer  The m a g n i t u d e o f t h e s e  t h e r a n g e o f 7-15 nA.  retaining  Currents  to  drug  those  containing  currents varied  used to r e t a i n  or pass  d r u g s i o n t o p h o r e t i c a l l y were i n d i v i d u a l l y  c o n t r o l l e d by t e n  turn potentiometers  - and were p a s s e d  - one f o r e a c h b a r r e l  the b a r r e l s o f the p i p e t t e a s s e m b l i e s resistors. electrode of  Relatively tips  through  small variations  to  1000 Mfi s e r i e s  i n r e s i s t a n c e a t the  therefore d i d not appreciably a f f e c t  the flow  current.  F o r most t y p e s  of experiments  i t was  d e s i r e d to e j e c t i n  a s e q u e n t i a l manner,  two o r more compounds  i n order  comparisons o f t h e i r  effects  firing  absence and p r e s e n c e  o f an a d d i t i o n a l  antagonist. controlling employed. assessed  To a c c o m p l i s h  upon n e u r o n a l  this  an a u t o m a t i c  the p e r i o d s o f e j e c t i o n The r e l a t i v e  by c o m p a r i n g  apparent  compound,  to permit  i n the  u s u a l l y an  timing  device  and r e t e n t i o n was  potencies  o f compounds  the magnitudes o f the e j e c t i n g  were  currents  27  required firing  to e l i c i t  frequencies  The the  (see d i s c u s s i o n o f t h i s matter  Although  combinations  described  o f more t h a n  only  two.  of agonist pairs a ranking  and t h i s  procedure  With  timer.  intervals  Following  f o r constant  b u t submaximal changes i n f i r i n g  In cases  reduced  criterion  f o r selective  successive cycles. after  required  rates,  of agonists  antagoncontin-  i n t h e p r e s e n c e o f an a n t a g o n i s t , t h e  a g o n i s t was a t l e a s t  values  approxi-  where t h e r e s p o n s e s p r o d u c e d by two a g o n i s t s  were b o t h  three  durations  the recording of at l e a s t  were a p p l i e d a n d t h e r e g u l a r c y c l i n g  ued.  estab-  two o f t h e  s u i t a b l e c y c l e s o f responses which produced  mately equal  one  different  i n C h a p t e r 4.  the automatic  ists  comparisons  forms t h e b a s i s f o r t h e e x p e r i m e n t s  a g o n i s t s were e j e c t e d a t f i x e d  three  two  scheme c o u l d be  When a s u i t a b l e n e u r o n e was e n c o u n t e r e d ,  by  of antagonists.  f o r the greater part  were made i n e a c h i n s t a n c e w i t h  i n Chapter!).  f o r each o f the  the u t i l i z a t i o n  i n a l l the studies the e f f e c t s  a g o n i s t s were o f i n t e r e s t ,  o f the  d i f f e r e n c e s between  o f two a g o n i s t s was a c c o m p l i s h e d  i n v e s t i g a t i o n s through  lished,  a n d submaximal l e v e l s  assessment o f p h a r m a c o l o g i c a l  effects  present  s t a b l e , equal  a c t i o n was t h a t t h e peak r e s p o n s e o f 25% l e s s  than  t h a t o f the second, i n  I n a l l cases, r e c o v e r y  to c o n t r o l  a d m i n i s t r a t i o n o f an a n t a g o n i s t was ended, was  f o r the r e s u l t  t o be  accepted.  28  e)  Electrical  The  stimulating  p a n e l and  e l e c t r o d e s were c o n n e c t e d  stimulus i s o l a t i o n  O r t e c 4651 of  Equipment  stimulator.  the m u l t i p i p e t t e  o r an was  The  amplifier  through and  signal  assembly  impedence m a t c h e r .  passed  a type  the output  from  parallel  was  to e i t h e r from  o r 2A63 T e k t r o n i x  from  vity.  E x t r a c e l l u l a r n e u r o n a l a c t i v i t y was  100  - 10  The  amplified  speaker  and  signal  from  not  filtered  The  amplified  t h e 10 Hz  manually tials  - 3 KHZ  resulting  f e d to a loud-  led into  amplitude  fired  o u t p u t p u l s e s c o u l d be  so t h a t  a schmidt  subsequently  computer o r a PDP-11/10 computer  to d i s p l a y post  s t i m u l u s time,  The  latency  a n a l y z e d d a t a were p l o t t e d  t e r m i n a l o r on an  interactive  digital  out-  paper a voltage a  action  poten-  trigger.  led into  PDP-8/L D i g i t a l  rammes.  range.  the r e c o r d e d s i g n a l s w i t h  adjustable triggering potential  above a c e r t a i n  using a  the i n t e g r a t e d r a t e m e t e r  s i g n a l s were a l s o  d i s c r i m i n a t o r w h i c h compared  acti-  potential recordings  p u t b e i n g r e c o r d e d on an E s t e r l i n e - A n g u s R e c t i l i n e a r recorder.  A  triggered  electrical  t h e o s c i l l o s c o p e was  an EKEG r a t e m e t e r ,  D u a l beam  the s t i m u l a t o r .  the continuous  within  an  activity  565  d u a l beam o s c i l l o s c o p e w h i c h was  were o b s e r v e d w i t h f i l t e r s  or  differential  on a t y p e KM  K H Z bandpass, w h i l e evoked  S8  preamplifier  3A9  the s t i m u l a t o r m o n i t o r e d  Hz  a  recorded e l e c t r i c a l  triggered  selection  the r e c o r d i n g b a r r e l  f e d through  displayed  a  a Grass  The  o s c i l l o s c o p e w h i c h c o u l d be second  units  through  The  a  programmed  and r a t e m e t e r  prog-  on a v i s u a l  display  plotter  ( T e k t r o n i x 4462) .  29  f)  Statistical  The  statistical  Analysis t e s t s w h i c h were u s e d f o r c o m p a r i s o n s  were b o t h p a r a m e t r i c i n n a t u r e and were d e s i g n e d t o the N u l l H y p o t h e s i s .  F o r c o m p a r i s o n s b e t w e e n two  measures,  s u c h as t h e m a g n i t u d e s  different  excitants,  a single tests  small  a Student's t t e s t  sample  of s t a t i s t i c a l  independence,  g)  t e s t was  locations  stainless  steel  the t i p .  T h i s was  with The  a solution iron  spots  f o r the comparison  (10-15  of neurones  by  Of E l e c t r o d e  f o r 10-20  causing  f o l l o w e d by  seconds  different  o f Fe"^" a t  the p e r f u s i o n of the animal i n 10%  sections.  The  formalin.  as b l u e - g r e e n  c y a n i d e r e a c t s w i t h Fe"* " -1  to form the P r u s s i a n b l u e r e a c t i o n p r o d u c t which  is easily  microscope.  Confirmation of r e c o r d i n g m i c r o e l e c t r o d e placements a c h i e v e d by dye  ( 2 0 % w/v  clusion  low  through the  the d e p o s i t i o n  o f 1% p o t a s s i u m f e r r o c y a n i d e  the l i g h t  the types  Sites  d e p o s i t s were s u b s e q u e n t l y v i s u a l i z e d  viewed under  For  s i t e s were m a r k e d by p a s s i n g  yA)  electrodes  in histological  used.  of  employed.  of stimulating  anodal current  independent  s u c h as c o m p a r i n g  f r o m a sample  Histological Verification  The DC  a x2  disprove  responses to  w i t h a known s t a n d a r d was  of responses e l i c i t e d agonists,  of c e l l u l a r  of data  the i o n t o p h o r e t i c  ejection  i n 4 M NaCl)(Thomas  of the experiment.  o f Pontamine  and W i l s o n , 1965)  Upon t e r m i n a t i o n o f t h e  was  sky b l u e a t the conrecording  30  period, ently  a n i m a l s were p l a c e d  perfused  transcardially  f o l l o w e d by 200 mi and  kept  i n deep a n a e s t h e s i a with  o f 10% f o r m a l i n .  and subsequ-  200 ml o f 0.9% N a C l The b r a i n s were  i n formalin u n t i l h i s t o l o g i c a l procedures  conducted. stained with  Frozen  excised  c o u l d be  s e c t i o n s o f 50 um t h i c k n e s s were c u t and  c r e s y l v i o l e t or  saffranin.  31  CHAPTER I I I  ACETYLCHOLINE  a)  Introduction  Of a l l t h e endogenous c h e m i c a l s u b s t a n c e s w h i c h h a v e b e e n proposed  as  s y n a p t i c t r a n s m i t t e r s , ACh  complete  e x p e r i m e n t a l s u p p o r t and  ound f o r w h i c h that  intestinal  receptors  confirmed.  itself.  was The  sensitive  and  o n l y to n i c o t i n e  the a c t i o n  and ACh  and n o t  susceptible  different  peripheral  i n t h e mammalian CNS  P r a d h a n and D u t t a ,  Three fication  terase  by  muscar-  to  (AChE),  and  antagonism  ( c u r a r e ) i s an receptors i n extended  see F e l d b e r g ,  1945  1971).  t e c h n i q u e s h a v e p r o v e n most v a l u a b l e i n t h e  f o r t h e ACh  mimicked  t h e g u t were  been s u b s e q u e n t l y  ( f o r reviews  of c h o l i n e r g i c neurones  procedures  and  to muscarine,  of which D-tubocurarine  t i s s u e s has  found  The v e r t e b r a t e  ganglion c e l l s within  This dual nature of c h o l i n e r g i c  and  caused  atropine.  example.  to a r e a s  (1914)  comp-  predominately muscarinic  excitation  o f t h e s e e x c i t a n t s was  c u r i f o r m drugs,  Dale  t h e most  the f i r s t  a potent excitant  a g o n i s t s c o u l d be b l o c k e d by junction  received  i n d e e d , was  smooth m u s c l e b o r e  o f ACh  neuromuscular  by  r o l e was  i n that muscarine  the a c t i o n inic  this  has  i n t h e CNS:  h y d r o l y z i n g enzyme,  w h i c h has  b e e n shown t o be  1)  identi-  staining  acetylcholinesa m a r k e r f o r some  32  central  c h o l i n e r g i c neurones  (Csillik,  ACh f r o m d i s c r e t e b r a i n r e g i o n s tal of  i n t e r v e n t i o n s designed  following various  to the v i c i n i t y  experimen-  t o promote o r d i m i n i s h  t r a n s m i t t e r ; a n d 3) d i r e c t p r e s e n t a t i o n  drugs  of  the release  cholinomimetic  o f s i n g l e n e u r o n e s by way o f  etic  e j e c t i o n from m i c r o p i p e t t e s  ding  the ongoing c e l l u l a r  1)  1975); 2) r e c o v e r y o f  while  electrical  iontophor-  simultaneously  recor-  activity.  Histochemical  Histochemical  s t u d i e s d i r e c t e d t o w a r d s AChE h a v e  demonstrated  the presence o f c h o l i n e r g i c neurones i n l a y e r V o f the c e r e b r a l cortex  ( K r n j e v i d and S i l v e r ,  Flemming,  1964; A u s t i n  1965), c e r e b e l l u m  and P h i l l i s ,  1965; S i l v e r ,  g a n g l i a , h i p p o c a m p u s and h y p o t h a l a m u s thalamic  Friede,  1966) as w e l l a s numerous o t h e r to e x i s t  regions.  sites  Caution icance and role  i n many d i f f e r e n t  i t i s b e l i e v e d that  areas  (Silver,  of results  1967; Gwyn and F l u m e r f e l t ,  obtained  the conclusions  by t h i s h i s t o c h e m i c a l  reached concerning  o f ACh w i t h i n r e g i o n s  of the  i n c e r t a i n cases  may  1971). the s i g n i f technique,  the p o s s i b l e  o f t h e CNS b a s e d on AChE  have n e c e s s a r i l y b e e n l i m i t e d  this  occurs' a t  h a s t h e r e f o r e b e e n r e q u i r e d when a s s e s s i n g  z a t i o n alone  1967),  Although  n o t mean t h a t f u n c t i o n a l c h o l i n e r g i c t r a n s m i s s i o n these  basal  n u c l e i ( K o e l l e , 1954; L e w i s and S h u t e , 1963,  AChE h a s b e e n f o u n d system,  1967),  ( L e w i s and S h u t e ,  various  nervous  ( F r i e d e and  synaptic locali-  (McLennan,  1970).  33 2)  Recovery-  M a c i n t o s h and lecting of  the  and  Oborin  (1953) e s t a b l i s h e d t h e  m e a s u r i n g ACh  cerebral cortex.  Mitchell  (1960,  1961,  This  ( 1 9 6 5 ) , Dudar and  others,  using  collecting  t i s s u e , and  (Gaddum, 1961). with  increased  The  a p p r o a c h was  Szerb  cups p l a c e d with  on  observed to  depth of anaesthesia  and  chronic  and  1965;  Collier  and  and  Silver,  Mitchell,  and  Murray-Brown,  and  was  EEG  among or  technique diminish  1963;  Krnjevic enhanced  stimulation  Hemsworth and  and  (Collier  Mitchell,  1968);  activation (Bartolini  and  1967).  biological an  important  as  a synaptic  o u t p u t and  significance in consideration  criterion  since  f o r the The  upon a p p r o p r i a t e  undercutting, t h a t ACh  i t s release  and  the  the  AChi fol-  stimulus-evoked release a l l i n n e u r o n e s , and f r o m them.  h a v e b e e n examined f o r ACh  comparable r e s u l t s o b t a i n e d ,  the  i t s role  decreased release  i s contained  CNS  of  r e l a t i o n s h i p of  stimulation i t i s released  areas of  of  from neurones  establishment  inverse  depth o f anaesthesia,  suggest  many o t h e r  of  transmitter.  cortical  strongly  are  f u n c t i o n f o r ACh  is  and  Neal,  shown t o c o r r e l a t e w i t h  These data  lowing  1968;  Khan  undercutting  1967); was  from sensory areas f o l l o w i n g a p p r o p r i a t e  Pepeu,  cannula  was  (Hebb, K r n j e v i c  by  (1977),  o f ACh  slabs  surface  continued  Dudar  col-  exposed c o r t i c a l  the p u s h - p u l l  release  from the  (1964), J a s p e r ,  (1969) and  of c o r t i c a l Silver,  obtained  1963), McLennan  Elliott  hippocampal  in fluid  technique of  these w i l l not  be  that Since release  described  34  here,  b u t f o r a summary o f t h e s e a n d r e l a t e d  reference 3) The  i s made t o P e p e u  (1973).  Iontophoresis direct  application  neurones has proved  o f c h o l i n o m i m e t i c agents  cells,  and has p r o v i d e d a p o w e r f u l  identification compound.  i/ ACh  of central cholinoceptive technique  o f neurones which possess  The p r i n c i p l e  The e a r l y p h a r m a c o l o g i c a l  i n t h e CNS h a v e m a i n l y  animal,  and t h e e f f e c t s  cholinomimetics  used  I (c) and I I  t h e c a t as an e x p e r i m e n t a l  of acetylcholine  as w e l l  and P h i l l i s  1963a,b,c),  Randic,  neurones appeared of  (1963),  and C u r t i s  insensitive  cholinoceptive cells  1.3 mm  from  as o f  s t u d i e d on c e r e b r a l (1961, 1962,  S i m i n o f f and  (1966).  Although  Straughan,  many  t o ACh, t h e g r e a t e s t p r o p o r t i o n  o c c u r r e d between t h e depths  the c o r t i c a l  (d).  s t u d i e s on t h e a c t i o n o f  c o r t i c a l neurones by K r n j e v i c  (1964) a n d C r a w f o r d  i n the  receptors f o r this  and a n t a g o n i s t s were f i r s t  Spehlmann  aiding  o f i o n t o p h o r e s i s and t h e t e c h n i q u e s  i t s u s e were d e s c r i b e d i n C h a p t e r s Cortex  to single  t o b e t h e method o f c h o i c e f o r d e t e r m i n i n g  the p h a r m a c o l o g i c a l c h a r a c t e r i s t i c s  for  investigations,  surface, corresponding  0.8 a n d  t o l a y e r s IV  and V.  . Cholinoceptive cells  t y p i c a l l y were e x c i t e d b y i o n t o p h o -  r e t i c a l l y - a p p l i e d ACh w i t h a s l o w effect:  onset  t h i s was i n c o n t r a s t t o t h e f a s t  and a p r o l o n g e d onset  after-  and o f f s e t  35  excitatory Phillis, tified  a c t i o n observed with L-glutamate  1963a; R a n d i c e t  by  their  a l . - , 1964).  antidromic  t h e m e d u l l a r y p y r a m i d s and muscarinic t o be  blocked  by  as  while  such a n t a g o n i s t s  C u r t i s , 1966).  neurones although i t s a c t i o n s  discharge  concerning  and  lack of  administration. aminoylcholine and  latency  (carbachol),  of e f f e c t ,  o n l y w e a k l y e x c i t e d by  (>1  some o f  ACh.  their  min)  and  elicited  i o n t o p h o r e t i c a l l y appeared both Krnjevic  and  (1963) f o u n d t h a t p r o s t i g m i n e T h i s was  mimetic actions sing  activity  as  of  ACh,  of a f t e r -  included  carb-  succinylcholine weaker  effective excitatory c e l l s w h i c h were  pilocarpine  and  excitatory effects with a  applied  neurones.  cortical  prolonged  t h e s e had  Arecoline,  f o r s e v e r a l minutes.  and  (A3MC)  those of  esters  n e u r o n e s , e v e n on  lasting  rates,  shown  benactyzine  duration  propionylcholine,  m u s c a r o n e were v e r y  oxotremorine exerted  was  ACh.  cholinoceptive  o n s e t t h a n ACh  on many  desensitization despite  t h a n ABMC o r  a g e n t s on  itself  Other e f f e c t i v e c h o l i n e  M u s c a r i n e and  and  of  by  a c t i o n o f ACh  c l o s e l y resembled  n i c o t i n y l c h o l i n e , although  actions  excited  atropine  iden-  stimulation  Acetyl-g-methylcholine  o r more e f f e c t i v e t h a n ACh  specifically  following  the as  and  B e t z c e l l s were  were shown t o be  cholinomimetics,  ( C r a w f o r d and was  discharge  (Krnjevic  believed  prolonged  Various  and t o be  after-discharges  anticholinesterases  to  Phillis  increase  neuronal  (1963c) and  due  to  the  firing  Spehlmann  tensilon excited  some  parasympatho-  t h e s e compounds, i n a d d i t i o n t o  anticholinesterases.  slower  When ACh  their  itself  pos-  caused  36  excitation,  p r o s t i g m i n e was  found  to p o t e n t i a t e the  heightened  a c t i v i t y when b o t h were a d m i n i s t e r e d c o n c u r r e n t l y . Krnjevic action due  and  occasionally  observed  a comparatively  onset,  producing  ferent  from  Nicotine tivity ACh.  and  observed  only.on  although not it.  o f ACh  t o be  on  effective  d i d not  T h i s f i n d i n g was  excitant  Wickel,  not  itself  irrespective antagonize  confirmed  by  t h a t n i c o t i n e was  active  a g o n i s t on p e r i p h e r a l n e u r o n e s  1951), was  found  relatively  substances  by K r n j e v i c and  and  observed  of the  - a property  action although  sensi-  the a c t i o n s of  as  and a  Curtis  neuronal  c o u l d be  to  ACh  excited  i o d i d e (DMPP), a (Chen, P o r t m a n  been used w i t h  and  (1963c) t o be o f ACh  and  largely  were  inactive  as  a l l cholinomimetics.  a c t i o n s o f c u r a r e were  incompatible with  success  a c t i o n s o f ACh  d i h y d r o - B - e r y t h r o i d i n e (DHBE) were among  compounds; weak e x c i t a t o r y  dif-  inactive.  which had  Phillis  only  abrupt  t h o s e n e u r o n e s w h i c h were r e s p o n s i v e  c e n t r a l b l o c k e r s of the e f f e c t s Curare  an  Crawford  a n t a g o n i z i n g the p e r i p h e r a l n i c o t i n i c  found  neurones  c h o l i n o c e p t i v e neurones".  a l l o f the c h o l i n o c e p t i v e c e l l s  Some o f t h e  excitatory  "unspecific"  and w i t h  1,l-Dimethyl-4-phenylpiperazinium  nicotinic  as  compound t o a f f e c t  long a p p l i c a t i o n  the e f f e c t s  t o ACh,  with nicotine  the  "a p a r o x y s m a l d i s c h a r g e w h i c h i s q u i t e  appeared  (1966) who  in  (1963b) a t t r i b u t e d  to the p r o p e r t y o f t h i s  after  by  Phillis  these  sometimes  i t s known a n t a g o n i s t i c  o t h e r s have r e p o r t e d t h i s  also  (Feldberg  and  37  Fleischhauer, ACh  blocker  could  (1966) f o u n d  blockers,  some  the s p e c i f i c i t y  marked.  The o b s e r v a t i o n s  nicotinic  antagonists  specific  effectiveness  c o r t i c a l neurones  synaptically-excited  f o r these  antagonist anomalous  some a n t a g o n i s t i c  o f a c e r t a i n degree o f a c t i o n  seems a t v a r i a n c e  with  of n i c o t i n i c agonists  (Krnjevic  and P h i l l i s ,  properties;  thus a t r o p i n e ( i n that  t o g l u t a m a t e were i n t e r f e r e d w i t h  demonstrated a t r o p i n e - l i k e  a selective,  rever-  and e s p e c i a l l y h y o s the neuronal  responses than  B e n a c t y z i n e and caramiphen properties,  anaesthetic  doses, n o n - s e l e c t i v e ,  observed.  1963c) a l t h o u g h t h e  to a l e s s e r extent  general  also  a l t h o u g h c a r a m i p h e n was  somewhat l e s s e f f e c t i v e i n t h i s r e g a r d . onists possessed a l o c a l  on t h e f e l i n e  e x c i t a t i o n were those, p o s s e s s i n g  c i n e were s p e c i f i c a t low d o s e s  t h o s e t o ACh - s e e b e l o w ) .  of a  respect.  most e f f e c t i v e a g e n t s i n c a u s i n g  sible block of cholinergic  the lack  o f the  (1966) s h o w i n g n i c o t i n i c e x c i t -  may be s i g n i f i c a n t i n t h i s  anti-muscarinic  effects,  f o r A C h - i n d u c e d r e s p o n s e s was n o t  o f Crawford and C u r t i s  The  to h i g h  (1963) and  b l o c k e d by t h i s  was o f f e r e d  did exhibit  although  ations  Spehlmann  G a l l a m i n e , mecamylamine and hexamethonium, a l l  nicotinic  results  that  have t h e i r a c t i v a t i o n s  however, a n d no e x p l a n a t i o n .^results .  m o d e s t l y e f f e c t i v e as an  on a few c o r t i c a l n e u r o n e s ;  C r a w f o r d and C u r t i s cells  D H B E was o n l y  1962).  A l l o f these  action  i n that  antag-  a t moderate  d e p r e s s a n t e f f e c t s were  Recovery o f the e f f e c t s o f glutamate always  e d e d t h o s e o f ACh however, a n d when a p p l i e d  prece-  iontophoretically  38  at  low  ejecting currents  action against In use  ACh  was  consideration  o f an  extensive  Phillis  and  Curtis,  1966;  (<60  nA),  obtained of  the  receptors  on  range o f  s i v e l y muscarinic  c h o l i n e r g i c drugs, K r n j e v i c  and  investigators  Smathers,  state of a f f a i r s  cholinergic receptors  of  the  this  firing  c o u l d be  the  s y n a p s e s as w e l l  as  o f Renshaw  e x c i t a t o r y a c t i o n o f ACh  compound has  the  cells,  on  of  some c e l l s .  on  to cause  cortical depression  spontaneous r a t e s  or  rates  been observed  t h o s e whose f i r i n g  a considerable  were i n h i b i t e d .  number  (about  e x c i t e d by  Randic 25%  of  ACh,  f i n d i n g s were c o r r o b o r a t e d  by  a n t a g o n i z e d by C r a w f o r d and  by  e t a l . (1964) the  and  cortical that  as  being  Betz c e l l s .  atropine.  Curtis  among t h o s e n e u r o n e s d e p r e s s e d by  identified  of  few  S i x n e u r o n e s w h i c h were d e p r e s s e d by  inhibitions partially  found that  r a t e s were e l e v a t e d  a p p l i c a t i o n of glutamate.  n e u r o n e s t e s t e d were d i r e c t l y  c o u l d be  the c h o l i n e r -  o b s e r v e d on n e u r o n e s w i t h h i g h  found that  the  with  exclu-  depressant e f f e c t  iontophoretic  had  almost  The  discharge,  (5%)  concluded that  presently.  In a d d i t i o n to neurones,  and  Spehlmann  1974)  contrasts  some p e r i p h e r a l  discussed  1966;  c o r t i c a l neurones are  g i c pharmacology of  t o be  (Crawford  Straughan,  i n nature. This  of  P h i l l i s , 1963c). the  Spehlmann and feline  ( K r n j e v i c and  of  through  L e g g e , R a n d i c and  Downes, 1974;  specificity  these r e s u l t s , obtained  most s u b s e q u e n t  and  a greater  ACh,  I t was  ACh  These  (1966),  who  many p o s s i b l e on  2  39  of  15 c e l l s  however, t o a n t a g o n i z e  the n i c o t i n i c  (1967;  o f ACh w i t h  a n t a g o n i s t DH3E w h e r e a s most c e l l s  atropine e x h i b i t e d a reduced York  the e f f e c t s  ACh s e n s i t i v i t y .  1968a,b,c,), Jordan  and P h i l l i s  tested with  P h i l l i s and  (1972) a n d S p e h l -  mann and Downes (1974) a l s o h a v e f o u n d A C h - i n d u c e d in  cerebral  with  c o r t e x , and o b s e r v e d  stimulation of afferent  inhibitory  fibres  l a t e r a l h y p o t h a l a m u s and r e t i c u l a r and  nicotinic  cortical  pharmacological  All from  synaptic actions  the cortex  formation.  Both  itself, muscarinic  a n t a g o n i s t s have been r e p o r t e d t o reduce  inhibitions  processes  from  inhibitions  (Phillis  nature  and York,  o f these  these  1968c) and t h e p r e c i s e  inhibitory  cholinergic  t h e r e f o r e remains u n c e r t a i n .  t h e above r e p o r t s h a v e b e e n b a s e d  f e l i n e neurones.  Stone  on r e s u l t s  obtained  (1972) e x a m i n e d t h e r e s p o n s e s  of  c o r t i c a l neurones i n the r a t somatosensory area t o i o n t o p h o r etically-applied ces. and  cholinomimetics  ACh e x c i t e d  and a n t i c h o l i n e r g i c  80% o f i d e n t i f i e d p y r a m i d a l  showed no i n h i b i t o r y  tract  and p i l o c a r p i n e mimicked  n i c o t i n e proved  neurones  a c t i o n s on t h i s p o p u l a t i o n .  stigmine p o t e n t i a t e d the ACh-induced e x c i t a t i o n s carbachol  substan-  ineffective.  the e f f e c t s  Physo-  a n d AfBMC,  o f ACh, b u t  Of t h e c h o l i n e r g i c  antagonists  tested,  a t r o p i n e and h y o s c i n e b u t n o t c u r a r e , b l o c k e d t h e  effects  o f ACh a n d A3MC.  ited  ACh a n d m u s c a r i n i c  25% o f t h e n o n - p y r a m i d a l  l a y b e t w e e n 0.5 t o 1.6 mm  agonists  inhib-  t r a c t n e u r o n e s , most o f w h i c h  below the c o r t i c a l  surface;  these  40  d e p r e s s i o n s were b l o c k e d by pyramidal by ACh  tract  and n i c o t i n e ,  Thus S t o n e tract  cells  muscarinic  l o c a t e d above 0.6 and  these  (1972) c o n c l u d e d  cells  a t r o p i n e or hyoscine.  of r a t cerebral  that  cortex possess  in certain  t h e c o r t e x as o b s e r v e d o t h e r s who (Krnjevic Curtis,  observed and  was  by  responses Stone  (1972) has  Phillis,  1963}  this  observations of P h i l l i s responses  and  York  as  Stone  found  evidence  with  and  t h e r e was  disagre-  excitations.  that  elicited  these  the r e s u l t s  could  on r a t  only for a muscarinic  r e p o r t s of the  c h o l i n o c e p t i v e n e u r o n e s b e t w e e n c a t and  other  by  inhib-  effect. Because of these c o n f l i c t i n g  of  of  and by m u s c a r i n i c a n t a g o n i s t s  a f u r t h e r p o i n t of disagreement  neurones,  mm  i n cats  Crawford  (1968) t h a t ACh  o f f e l i n e n e u r o n e s and  b l o c k e d b o t h by n i c o t i n i c  itory  cholinomimetic  of n i c o t i n i c  dif-  The  not been noted  Spehlmann, 1963;  significance  the  nicotinic receptors.  w i t h i n t h e u p p e r 0.6  1966), a l t h o u g h among t h e s e a u t h o r s  inhibitory be  a r e m u s c a r i n i c , and  those o f the c a t , these r e s u l t s  effects with  ement c o n c e r n i n g t h e The  mm  non-pyramidal  r e s p e c t s which are worthy of mention.  segregation of n i c o t i n i c  curara .  exclusively  o f the  l o c a t e d neurones possess  When c o n t r a s t e d w i t h  excited  the c h o l i n o c e p t i v e p y r a m i d a l  t r a c t n e u r o n e s w h i c h o c c u r b e l o w 0.6  fer  d e p t h were  e f f e c t s were a b o l i s h e d by  e x c i t a t o r y r e c e p t o r s , those  more s u p e r f i c i a l l y  mm  Non-  inconsistencies  i n the l i t e r a t u r e  pharmacology  r a t , and  due  to  ( s e e b e l o w ) i t was  41  f e l t worthwhile f o r ACh  on  to  i n v e s t i g a t e the  nature  r a t c e n t r a l neurones using  mimetic agonists  and  antagonists  their properties  pharmacologically,  comparisons w i t h  those of  of  a range of  i n order and  the w e l l  the  to  to  receptors cholino-  characterize  thereby  characterized  permit  feline  neurones. ii/  Thalamus  activated ation the  Ventrobasal  thalamic  s y n a p t i c a l l y at  short  o f h i n d l i m b n e r v e s and  ipsilateral  1974)  primary  were e x c i t e d by  Curtis,  1964a).  n e u r o n e s by  The  r e l a y n e u r o n e s w h i c h were  latency  cortex  amino a c i d s mean l a t e n c y  and  ( t o be  cological properties  T h e r e was neuronal  a possible  of  these  frequently  sensitivity  set  of  similar ants  (Andersen  a delay  (5-60  that  s e e s ) was  were e x c i t e d b o t h by  often  and  that  the spont-  pharma-  populations.  decrease of  and  in ACh,  Thesleff  o b s e r v e d i n the was  off-  terminated,  cortex with muscarinic  1963b).  muscarinic  than  i n the  administrations  ejecting current  Phillis,  thalamic  d i f f e r e n c e i n the neuronal  Sears,  b e l o w ) , and  a reduction  of  and  longer  d e s e n s i t i z a t i o n (Katz  observed i n the  ( K r n j e v i c and  was  discussed  toward c o n s t a n t  f i r i n g when t h e to  ACh  observed a cumulative  indicating possible receptor 1958); and  two  stimulation  to e x c i t a t i o n of  e x c i t a t i o n s were o f t e n p r e c e e d e d by  stimul-  ( A n d e r s e n and  i o n t o p h o r e t i c a l l y a p p l i e d ACh  suggesting  msec) by  a n t i d r o m i c a l l y by  sensory  o b s e r v e d f o r Renshaw c e l l s  aneous r a t e  (6-8  excit-  Those neurones which nicotinic  agonists  42  responded w i t h of i n h i b i t o r s and  a stronger o f AChE  to a l e s s e r  izing  the  induced t e d by  ( A n d e r s e n and  type "not  Phillis the  those  1964b). and  lateral  1963,  1964b).  as  Phillis,  medial Tebecis  unaffec-  made t h a t  a r e o f a pharmacoand  muscarinic:  o f Renshaw c e l l s  a, b)  and  and  (Andersen  T h e s e r e s u l t s were c o n f i r m e d 1968  synaptically  s u g g e s t i o n was  of c o r t i c a l neurones"  Westerman, 1966; and  those  DH3E,  i n antagon-  the  between n i c o t i n i c  as n i c o t i n i c as  The  presence  s t i m u l a t i o n was  t h e a c t i o n s o f ACh  intermediate  as m u s c a r i n i c Curtis,  Curtis,  cutaneous nerve  e i t h e r b l o c k i n g agent.  are  i n the  c h o l i n e r g i c e x c i t a t i o n s , although  a c t i v a t i o n by  logical  t o ACh  d e g r e e a t r o p i n e , were e f f e c t i v e  the r e c e p t o r s m e d i a t i n g  they  excitation  not  and  (McCance, extended  to  geniculate n u c l e i (Curtis  and  and  1970), and  York,  1967;  Tebecis,  Davis, n.  r e t i c u l a r i s where m i x e d n i c o t i n i c / m u s c a r i n i c r e c e p t o r s  medi-  ating  Kanazawa  and  i n h i b i t i o n s were d e s c r i b e d  Kelly,  1976).  among t h e s e that  genous t o an  Curtis  had  no  thalamic  Davis  nuclei  are pharmacologically  (1963) f o u n d  DH3E t o be  s y n a p t i c e x c i t a t i o n s o f LGN t h e ACh  observable  frequently  i n v e s t i g a t o r s however, s e r v i n g t o  note  emphasize hetero-  extent.  and  antagonizing of reducing  T h e r e have b e e n s e v e r a l d i f f e r e n c e s o f  various  different  (Ben-Ari, Dingledine,  induced  firing,  while  antagonistic effects.  t h a t A3MC e l i c i t e d  depressant  ineffective  neurones but curare I t was  and  capable gallamine  also  effects.  in  In  noted a  43  s u b s e q u e n t r e p o r t on v e n t r o l a t e r a l n e u r o n e s was m e n t i o n e d t h a t a t r o p i n e excitatory at  effects  low d o s e s  antagonized  could block  (Davis,  completely  o f ACh when t h e a n t a g o n i s t  (10 n A ) . not only  Higher  the s y n a p t i c responses o f  DL-homocysteate  Phillis  (DLH) e l i c i t e d  thalamic  i n t h e LGN, f o u n d a t r o p i n e  my lot-on, a n i c o t i n i c t i n g ACh i n d u c e d  not  drug,  changes i n f i r i n g of only  t o be a n t a g o n i s t i c  The p h a r m a c o l o g i c a l  t o be e f f e c t i v e  i n preven-  (12 o f 18 n e u r o n e s ) .  4% o f t h e g e n i c u l a t e nature  of this  ACh  neurones  inhibition  was  i n v e s t i g a t e d f u r t h e r , p r e s u m a b l y due t o t h e few n e u r o n e s  w h i c h were l o c a t e d i n t h i s r e g i o n  A  later  study  by T e b e c i s  a n d were i n h i b i t e d b y ACh.  (1970) on m e d i a l  geniculate  n e u r o n e s i d e n t i f i e d by s t i m u l a t i o n o f t h e a u d i t o r y revealed clearly apparent  t h a t o f those inhibited.  cells  VB n e u r o n e s .  Tebecis  cholinomimetics  responses  effects  b y A n d e r s e n and C u r t i s  d i dnot exhibit observa-  (1964b) f o r  noted further that n i c o t i n i c elicited  cortex  t o ACh, 37% were  d e s e n s i t i z a t i o n as d i d t h e e x c i t a t i o n s , an initially  rinic  responding  These i n h i b i t o r y  tion reported  The  of cholin-  (8 o f 21 n e u r o n e s ) and f o u n d  blocking  depressed the f i r i n g tested.  the dual nature  f  t o w a r d s some ACh r e s p o n s e s  t r a c t , but  e x c i t a t i o n s as w e l l .  e t a l (1967) c o n f i r m e d  ergic receptors  administered  (30 nA)  neurones t o s t i m u l a t i o n o f the c e r e b e l l o - t h a l a m i c the  i t  the  was  doses o f a t r o p i n e  1966)  and musca-  b o t h e x c i t a t i o n and i n h i b i t i o n .  o f ACh, w h e t h e r o f an e x c i t a t o r y o r i n h i b i t o r y  44  n a t u r e were a n t a g o n i z e d b y b o t h although the antagonism neurones. produced  clear-cut  on t h e r e s p o n s e s  of the r e c e p t o r p r o p e r t i e s  (1972) s t u d i e d  series  of experiments,  o f these  t h a l a m i c neurones  other brain regions.  activation  of various synaptic inputs  They f o u n d  The t e n d e n c y  to  antagonism  towards a s l i g h t l y  greater muscarinic  f o r neurones  o f t h e VB  (McCance e t a l . , 1966).  Godfraind there e x i s t s  (1975) f o u n d  a tendency  l o c a t e d neurones, o f ACh were l e s s  that within  the pulvinar  f o r ACh t o e x c i t e  t h e more  complex  posteriorly  while i n the a n t e r i o r r e g i o n s , the e f f e c t s clear-cut.  These l a t t e r  t e d by more f r e q u e n t l y - o c c u r r i n g changes i n t h e f i r i n g rather  sensitive  o f the  a l t h o u g h a t r o p i n e was t h e more  t h a n n i c o t i n i c n a t u r e was a l s o n o t e d complex  the concomitant  by s t i m u l a t i o n  c e r e b e l l o - t h a l a m i c pathway was a l s o agents,  from  t h a t b o t h DHgE a n d a t r o p i n e  o f these c e l l s  these b l o c k i n g  ventro-  a n d compared t h e i r r e s u l t s w i t h t h e  s e l e c t i v e l y b l o c k e d ACh i n d u c e d e x c i t a t i o n s ;  effective.  cells  M a r s h a l l a n d McLennan  the c h o l i n e r g i c pharmacology o f f e l i n e  pharmacological properties  by b o t h  definitive  possible.  In a l a t e r  synaptic  on a l l  by o t h e r c h o l i n o m i m e t i c s , and t h e r e f o r e a  n o t made  lateral  was n o t a l w a y s  The b l o c k e r s were h o t t e s t e d  characterization was  types o f c h o l i n e r g i c b l o c k e r s ,  rates.  e f f e c t s were m a n i f e s -  inhibitions  Changes  and l e s s  in firing  distinct  pattern,  t h a n e x c i t a t i o n o r i n h i b i t i o n were n o t e d , c o u p l e d w i t h  45  an  apparently  l o w e r p o t e n c y o f ACh.  o r mecamylamine, r e a d i l y b l o c k e d earlier  observations  amate w i t h  Atropine,  ACh  of a g r e a t e r  induced  not  thalamic  Marshall,  1 9 6 8 ) , and  are predominately  Ben-Ari teristics another  worthy  region,  about  that this  the  t h a t p u l v i n a r neurones  and  u s i n g neurones of  the n u c l e u s  g r o u p and  reticularis.  Duggan and  effects  muscarinic  T h e s e s t u d i e s on revealed  Hall  o f ACh  of the  tinic  and,  the  two . s e p a r a t e the  However t h e r e has  effects  of  cholinomimetics, populations  other n i c o t i n i c ,  feline  effects  coupled thalamic  with  o f ACh the  by  o f ACh  the and  b e e n no  antagonists so  against  the q u e s t i o n  r e c e p t o r s , one  required further research.  neurones, prompted  pharmacological the p r e s e n t  pos-  systematic excit-  of whether  muscarinic  or a s i n g l e r e c e p t o r p o s s e s s i n g  l a c k o f any  thalamus  between n i c o -  nicotinic/muscarinic properties is responsible for the  in  both  r e c e p t o r s which appeared to  i n g e n e r a l , mixed p r o p e r t i e s i n t e r m e d i a t e  the  note-  (1975) c o n c u r r e d  sess,  i n v e s t i g a t i o n of  still  antagonists.  o f ACh  muscarinic.  charac-  Although  were p r e v e n t e d  the v a r i o u s n u c l e i  the e x i s t e n c e  a t i o n s by  and  e x a c t mechanisms i n v o l v e d , i t i s  that i n h i b i t o r y  nicotinic  was  e t a l . (1976) e x a m i n e d t h e p h a r m a c o l o g i c a l  thalamic  finding  glut-  muscarinic.  of c h o l i n e r g i c i n h i b i t i o n s  disagreeing  and  nuclei  (McCance e t a l . , 1968a; McLennan, Huffman concluded  The  t o ACh  confirmed  Godfraind  DH3E  firing.  sensitivity  i n c r e a s i n g depth f o r v a r i o u s  but  This  and  mixed  mediating problem,  s t u d i e s on  investigation.  rat  46  iii/  S p i n a l Cord  central  The e a r l i e s t  cholinoceptive  studies  on t h e p h a r m a c o l o g y o f  neurones which used  the i o n t o p h o r e t i c  t e c h n i q u e were c o n d u c t e d on s p i n a l n e u r o n e s by C u r t i s and Eccles and  ( 1 9 5 8 a , b ) , C u r t i s and P h i l l i s  Watkins  (1961).  C u r t i s and E c c l e s  r e s p o n s e s o f Renshaw c e l l s phoretic  administration  muscarinic various  agonists,  cholinergic  (1960) a n d C u r t i s ,  to synaptic  Phillis  (1958a,b) e x a m i n e d t h e a c t i v a t i o n by t h e i o n t o -  o f ACh and a r a n g e o f n i c o t i n i c  and t o a n t i c h o l i n e s t e r a s e  and  compounds, a n d  antagonists.  Renshaw c e l l s were r e a d i l y e x c i t e d b y ACh and n i c o t i n e . Suecinylcholine, what  A3MC, c a r b a c h o l  l e s s e f f e c t i v e t h a n ACh, a l t h o u g h  o b s e r v e d t o be e q u i p o t e n t . concomitantly and  w i t h ACh g r e a t l y  consistent with  was  administered  increased  b o t h the spontaneous  o f Renshaw c e l l s ,  i t s known a n t i - A C h E p r o p e r t y .  o f these neurones;  a l t h o u g h on some c e l l s  i t was u s u a l l y r e l a t i v e l y  a reduction  greater  t h a n 5 0 % was o b s e r v e d .  against  Renshaw c e l l  of the response  abolished  Curare synaptic ineffective,  t o ACh  In a d d i t i o n  when DH3E was  to b l o c k i n g  i o n t o p h o r e t i c a l l y a p p l i e d ACh a n d n i c o t i n e , t h i s  nearly  an  A much more p r o n o u n c e d e f f e c t  e x c i t a t i o n s was o b t a i n e d  e m p l o y e d as an a n t a g o n i s t .  sometimes alone or  somewhat v a r i a b l e e f f e c t s on t h e ACh i n d u c e d and  firing  of  carbachol  Prostigmine  the s y n a p t i c a l l y evoked d i s c h a r g e s  action had  and a r e c o l i n e were a l l some-  the s y n a p t i c a l l y evoked e x c i t a t i o n .  the e f f e c t compound  47  In a l a t e r  report,  C u r t i s e t a l . , (1961) s t u d i e d  r a n g e o f c h o l i n e r g i c d r u g s i n an e f f o r t pharmacological cells. In  c h a r a c t e r i s t i c s o f ACh r e c e p t o r s  A series of choline  comparison w i t h  these e s t e r s termination was  fers  for nicotinic  indicated  that  after-discharge  and m u s c a r i n i c  and R y a l l  in  than the i n i t i a l ,  some-  these  the r e s u l t s o f the  that  nicotinic  action.  Renshaw c e l l s i n ACh  receptors,  the l a t e r  action,  The " f a s t " , o r  discharges  since,  r e f l e c t e d the  discharges  the presence o f muscarinic  found to p a r t i a l l y  and e v e n  on  an e x c i t a t o r y a c t i o n some 500-700 msec,  In fact,  synaptic  dif-  roots.  and m u s c a r i n i c  component o f t h e s y n a p t i c  the " t r u e "  receptors  to antagonize,  the conclusion  l a t t e r manifesting  ACh may r e f l e c t  of the agonist-  The e x p e r i m e n t s  (1966a,b,c) c o n f i r m e d  reaching  action.  agonists.  the dorsal  the  nicotinic  phenomenon  t h e r e s p o n s e s o f t h e n e u r o n e s when  cat possess both n i c o t i n i c  "early"  stabilities  exist non-cholinoceptive  the  later  This  formed: a parameter which presumably  were a c t i v a t e d by f i r i n g  above r e p o r t s ,  o b s e r v e d w i t h ACh,  t o t h e i n e f f e c t i v e n e s s o f AChE  Renshaw c e l l s , s i n c e DH3E f a i l e d  Curtis  current.  and t h e r e l a t i v e  there  times p o t e n t i a t e d  on Renshaw  quite prolonged e f f e c t s f o l l o w i n g the  due, i n p a r t ,  complexes  f u l l y the  e s t e r s were e f f e c t i v e as e x c i t a n t s .  of the i o n t o p h o r e t i c  toward the e s t e r s receptor  the b r i e f  elicited  considered  to describe  a wide  observed  receptors  on o c c a s i o n ,  a n t a g o n i z e t h e a c t i o n o f ACh.  with  uninvolved  atropine  was  48  When DHgE a n t a g o n i z e d e x c i t a t i o n by  iontophoretic  were u n a l t e r e d .  The  ACh  a t t e n u a t e d as w e l l . reduced the tically  the  a n t a g o n i z e d , ACh  S i n c e no inic  abolished the  The  that  e x i s t on  any  (Curtis,  Renshaw c e l l s  m i m e t i c s and  the  cat.  The  in rats  cholinergic  ences between the  observed,  1966;  Biscoe to  of  607c o f  and  1  muscar-  since  the  of b o t h the  nicot-  completely  o f ACh  sensitive  cat.  t o ACh  and  are of  spinal cord  believed  synaptic  and  r e s p o n s e s o b s e r v e d and  found  to  transmission  Ryall,  (1975) e x a m i n e d t h e  iontophoretically  inter-  cholinomimetics,  not  E n g b e r g and  atropine  were  (1966a,b,c,) r e a c h e d  the  antagonists  a n t a g o n i s m by  synap-  and  antagonist  Ryall  i n processes  Watkins,  in  unaffected.  antagonists  i n h i b i t o r y and  significant role  partially  were  separate populations  or  t o A8MC  were r e d u c e d t o  nicotine  the  considerably  volleys  observed responses of  H e a d l e y , L o d g e and of  and  t h a n Renshaw c e l l s  and  root  neither  Renshaw c e l l s  infrequently  Ryall  (as  Curtis  two  responses  were a c o m p o s i t e  effects  weakly e x c i t a t o r y  play  ACh  i t s action),  the  and  A3MC i n d u c e d e x c i t a t i o n s  t h o s e p r o d u c e d by  muscarinic  neurones other are  cells,  c a u s e d by  excitation  when a t r o p i n e  of v e n t r a l  ( o r v i c e v e r s a ) was  conclusion  receptors  contrast,  c r o s s o v e r between n i c o t i n i c  agonists  and  nicotine,  induced e x c i t a t i o n s  while  excitations inic  By  synaptic  i n d u c e d e x c i t a t i o n was  effectiveness  exciting  controls,  the  1966).  responses  applied  cholino-  several  differ-  those r e p o r t e d  o f ACh  r e s p o n s e s on  for most  49  c e l l s was h i g h l y e f f e c t i v e , against with  other  DHBE.  although  the s e l e c t i v i t y  e x c i t a n t s was n o t a s p r o n o u n c e d a s t h a t  Atropine modified  synaptically  a s i m i l a r manner a s d i d DH3E; t h u s  t h e t o t a l number o f s p i k e s  was r e d u c e d , a n d t h e l a t e n c y t o t h e f i r s t s p i k e s was i n c r e a s e d . the e f f e c t s  and s u c c e s s i v e  o f A8MC, c a r b a c h o l  and n i c o t i n e w i t h -  effects.  D i f f e r e n c e s between t h e s e  results  and those  excitation  following activation  effectiveness  o f both  activation;  pine  o f ACh r e s p o n s e s w i t h o u t  acid  e x c i t a n t s ; a n d 4) a marked q u a l i t a t i v e  the  late  1 9 6 6 b , c ) ; 2)  a t r o p i n e a n d DH3E i n b l o c k i n g t h e v e n t r a l  root  3) s e l e c t i v e b l o c k a d e  degree o f s p e c i f i c i t y  excitatory  of a  o f t h e c e l l s by  v e n t r a l r o o t v o l l e y s ( c f . C u r t i s and R y a l l ,  the  reported f o r  Renshaw c e l l s w e r e : 1) l a c k o f o b s e r v a t i o n  muscarinic  stimulus  DH3E a n d a t r o p i n e o n l y o c c a s i o n a l l y  out m a n i f e s t i n g n o n - s p e c i f i c  feline  observed  e v o k e d responses' i n  evoked i n a g i v e n p e r i o d f o l l o w i n g the v e n t r a l r o o t  antagonized  f o r ACh  affecting  by b o t h  responses  t o amino  difference i n  of the antagonists  actions of n i c o t i n i c  DH3E a n d a t r o -  i n b l o c k i n g the  and m u s c a r i n i c  cholinomimetics;  f e l i n e neurones r e v e a l i n g a g r e a t e r b l o c k i n g a c t i o n w i t h  muscarinic  antagonists  a g a i n s t A3MC on t h e one hand, a n d  with n i c o t i n i c  antagonists  no  antagonism.  overlapping  T h a t r a t Renshaw c e l l s muscarinic  populations  a g a i n s t n i c o t i n e on t h e o t h e r ,  possess  of receptors  a mixture  with  o f n i c o t i n i c and  as i s t r u e f o r t h e c a t ,  50  seemed u n l i k e l y due t o t h e o b s e r v a t i o n s atropine blocked could block ded  that  receptors they  completely  t h e ACh e x c i t a t i o n s a n d t h a t DH6E  t h e a c t i o n o f A3MC.  there  t h a t b o t h DHgE a n d  i s a qualitative  H e a d l e y e t a l . (1975)  conclu-  d i f f e r e n c e between c h o l i n e r g i c  o f c a t a n d r a t Renshaw c e l l s ,  and t h a t  cannot be c a t e g o r i z e d i n t h e t r a d i t i o n a l  f o r the r a t ,  manner.  51  b) i/  Results Cortex  Neurones o f t h e c e r e b r a l  g l u t a m a t e , ACh o r any o f t h e s e v e r a l the  present  located (see  study  (see Table  a t a l l depths  2).  1 ) , a n d t h o s e e x c i t e d b y ACh were f r o m c a . 0.2 - 2.0 mm.  I n h i b i t o r y e f f e c t s o f ACh a n d  c h o l i n o m i m e t i c s were n o t s t u d i e d  ionally  were e x c i t e d b y  cholinomimetics used i n  from t h e s u r f a c e ,  d e p t h summary, T a b l e  As t h e m i c r o p i p e t t e  cortex  i n the present  was l o w e r e d t h r o u g h t h e c o r t e x , ACh o r o c c a s -  an e x c i t a t o r y  amino a c i d was e j e c t e d  l o c a t e n e u r o n e s w h i c h were n o t s p o n t a n e o u s l y Cholinergic  investigation.  continuously active.  Agonists.  I t was o b s e r v e d c o n s i s t e n t l y t h a t n e u r o n e s w h i c h were s i v e t o ACh were e x c i t e d  a l s o b y A3MC, n i c o t i n e ,  DMPP a t a l l l e v e l s i n t h e c e r e b r a l  cortex.  n e u r o n e s a f f e c t e d b y ACh were t e s t e d to b o t h n i c o t i n i c ates  that The  and m u s c a r i n i c  the majority upper t r a c e  a medium f a s t between 5 - 2 0  i n Figure  and  o f 61  responsiveness  and T a b l e  2 indic-  1 shows t h e r e s p o n s e s o f a ACh t y p i c a l l y  achieving  I t was d i f f i c u l t  responses o f c o r t i c a l neurones n i c o t i n e , on t h e b a s i s  t h e s e compounds,  or  were e x c i t e d b y b o t h .  onset o f a c t i o n , sees.  respon-  carbachol,  A total  for their  agonists,  n e u r o n e t o ACh, n i c o t i n e a n d ABMC.  for  to  as b o t h  a peak f i r i n g  to the agonists  latency  rate  to c h a r a c t e r i z e the  o f the f i r i n g  short  displayed  and e s p e c i a l l y  pattern  and l o n g  e v o k e d by l a t e n c y on-  o f f s e t r e s p o n s e s were o b t a i n e d . S u c h r e s p o n s e s o c c a s i o n a l l y  52  TABLE 2. cortex  Numbers  responding  ACh  o f c h o l i n o c e p t i v e neurones o f t h e . c e r e b r a l to n i c o t i n i c  and m u s c a r i n i c  Nicotinic  +  0  drugs.  Muscarinic  +  0  +  58  51  7  0  53  3  2  —  3  . 0  0  3  0  2  1  D e p t h s where c h o l i n o c e p t i v e n e u r o n e s were  found.  • 10-H C  o  0  01  02 0.3 0.4 05 0.6 0.7 OS  0.9 10  D e p t h i n mm.  11 1.2 13  14 15  53  Figure  1.  Responses o f a c e r e b r a l c o r t i c a l neurone to ACh,  n i c o t i n e and AgMC, and the e f f e c t s o f the n i c o t i n i c DHgE. Ordinate:  firing  r a t e o f the c e l l i n s p i k e s / s e c ;  sa: time, i n t h i s and every other F i g u r e of r a t e H o r i z o n t a l bars beneath the t r a c e s r e p r e s e n t  the compounds, measured i n nA.  l o c a t e d i n the somatosensory area, the c o r t i c a l  Applied  abcis-  recordings.  periods  a p p l i c a t i o n and the numbers are the i o n t o p h o r e t i c used to expel  antagonist  o f drug  currents  T h i s c e l l was  a t a depth o f 1.10 mm  from  surface.  f o r 9.5 min. a t an e j e c t i n g c u r r e n t o f 30 nA,  DHgE antagonized the e x c i t a t i o n s produced by ACh and n i c o t i n e (middle r e c o r d ) and the i n i t i a l responses recovered a f t e r c e s s a t i o n o f DH$E a d m i n i s t r a t i o n .  3.5 min.  54  Cortex Depth: 1.10 mm 100 r  0 ACH 50  NIC 35  ApMC 25  d 100 o> co \ CO CD  'CL  CO  0 i  i  I Min.  100  0  55  resembled Figure  those  elicited  2), although  1 was more  by t h e m u s c a r i n i c  the response p a t t e r n  a g o n i s t AgMC ( s e e illustrated  typical.  Carbachol,  a compound w h i c h h a s b e e n r e p o r t e d  mixed n i c o t i n i c / m u s c a r i n i c p r o p e r t i e s , a c t e d agonist be  on t h o s e  discussed  ergic  cells  respect  Carbachol  This point  elicited  very  fast  onset  upon n e u r o n e s w h i c h r e s p o n d e d t o ACh.  instance  will  to the actions o f the cholin-  t e s t e d on n e u r o n e s w h i c h r e s p o n d e d i n every  to possess  as a n i c o t i n i c  t e s t e d i n t h e r a t CNS.  further with  antagonists.  tory effects  and  i n Figure  excita-  DMPP was  to n i c o t i n e or carbachol,  i t s e f f e c t was e q u i v a l e n t  to that of  nicotine. A3MC was t h e s o l e m u s c a r i n i c  e x c i t a n t employed  since  compound i s known t o have a v e r y  specific  receptors  1963c) a n d was e a s i l y  ( K r n j e v i c and P h i l l i s ,  from m i c r o p i p e t t e  b a r r e l s even w i t h  e x c i t e d neurones with  a slow onset  i m m e d i a t e l y upon s w i t c h i n g ning,  high  currents.  duration  of after  was  the r i s e  t i m e t o peak a c t i v i t y  and P h i l l i s  feline  cortical  ( F i g u r e 1).  e f f e c t was u s u a l l y l e s s p r o l o n g e d (Figures  than  1, 2, 4 a n d 5 ) .  to those  d e s c r i b e d by  (1963c) a n d C r a w f o r d a n d C u r t i s neurones.  effect  from e j e c t i n g t o r e t a i -  T h e s e e x c i t a t o r y p r o p e r t i e s were s i m i l a r Krnjevic  ejected  AgMC  e v e n when p l a t e a u r e s p o n s e s h a d b e e n a t t a i n e d  The  for  a c t i o n on m u s c a r i n i c  and o f t e n had a peak  the current  this  (1966)  56  Some n e u r o n e s were f o u n d w h i c h were e x c i t e d by ACh a n d A3MC a n d n o t b y t h e n i c o t i n i c others by  (8 c e l l s ) were u n r e s p o n s i v e  ACh, DMPP, c a r b a c h o l  were l e s s  responsive  to e l i c i t  Cholinergic  (11 c e l l s )  while  t o AgMC b u t were e x c i t e d  and n i c o t i n e .  Sixcortical  t o ACh t h a n t o a l l  judged by t h e i n t e n s i t y red  excitants  neurones  cholinomimetics,  o f e j e c t i n g c u r r e n t s w h i c h were r e q u i -  excitations of equivalent  magnitude.  Antagonists.  As t h e e x c i t a t i o n o f t h e same n e u r o n e s b y b o t h n i c o t i n i c a n d muscarinic  cholinomimetics  was f r e q u e n t l y o b s e r v e d ,  several  antagonists  were employed i n o r d e r  specificity  o f t h e e x c i t a t o r y a c t i o n s o f A3MC, n i c o t i n e a n d  carbachol.  Figures  tinic  antagonists,  elicited  the  1 and 3 i l l u s t r a t e  1 that  the n i c o t i n i c  excitatory effects  o r no e f f e c t those  the e f f e c t  o f two n i c o -  DH3E a n d mecamylamine, on e x c i t a t i o n s  by c h o l i n e r g i c a g o n i s t s .  in Figure  t o determine the degree o f  I t i s c l e a r f r o m t h e example  antagonist  o f ACh a n d n i c o t i n e w h i l e  upon t h a t p r o d u c e d b y A3MC.  i l l u s t r a t e d were o b t a i n e d  When a d m i n i s t e r e d  blocked  with  completely exerting l i t t l e  Similar results to  the n i c o t i n i c  blocker curare.  a t l o w d o s e s , a t r o p i n e was a l s o  effective  i n a b o l i s h i n g t h e e x c i t a t i o n o f c o r t i c a l n e u r o n e s b y ACh, a s lustrated  i n F i g u r e .4,  carbachol  ( F i g u r e 5) o r n i c o t i n e was m a i n t a i n e d  ence 'of  atropine.  il-  a n d by^A3MC. The a c t i v a t i o n o f n e u r o n e s by despite  t h e pres-  57  Figure  2.  mimetics.  R e s p o n s e s o f two N e u r o n e a t 0.24  c o r t i c a l n e u r o n e s t o ACh mm  responds  to a l l e x c i t a n t s while a slower onset tern characterizes  the responses  with fast and  offset  of another  cell  and  cholino-  o n s e t and  offset  response  pat-  a t 1.64  mm.  58  Cortex 50 r LM  IWl  A  0.24mm  u ^  40  40  45  1  m  i  F  l  Ixl  £  5 0  r  >v  35  1.64mm  "zo"  "Ed'  59  Figure with  3.  50 nA,  mylamine. an  R e s p o n s e s o f a c o r t i c a l n e u r o n e t o ACh and  A n t a g o n i s t was  e f f e c t was  0.98  mm  the e f f e c t  from  observed. the  o f the n i c o t i n i c administered  T h i s c e l l was  c o r t i c a l surface.  less  administered  a n t a g o n i s t , mecathan  2 min  before  l o c a t e d at a depth  of  Cortex  n  MECAMYLAMINE  50  ..  n Q Q  Depth:0.98 mm  50  37  50 I  I  Min.  61  I t was  noted  p r e v i o u s l y that carbachol possesses  n i c o t i n i c / m u s c a r i n i c p r o p e r t i e s at c e r t a i n however i t s a c t i o n i n t h e p r e s e n t agreement w i t h neurones Misra  (Koelle,  and  action ficient  o t h e r s who  Bhargava,  to block  and  as  antagonized reasonable carbachol ii/  1976).  excitations  Horn,  1975;  elicited  elicited  (VB)  summarized  o f ACh,  i n Table  5-9  The  by  e x c i t a t i o n s were n o t by  Sinha,  the  ACh.  sufSince  i t s antagregularily  atropine, i t  was  a c t i o n of  3 were o b t a i n e d  msec.  On  every  ACh  o r AgMC, j u d g e d  by  the  bed  f o r c o r t i c a l neurones. illustrated  cell  from the  Typical i n Figure  for this  tested,  as  thalamic 6.  of  a c t i v a t e d by s t i m u l a t i o n synaptic  carbachol  excitatory effect  same c r i t e r i a  admin-  A8MC t o n e u r o n e s  range of l a t e n c i e s  a p p e a r e d t o e x e r t a more p o t e n t  are  Tangri,  by A8MC and  c a r b a c h o l , n i c o t i n e and  nerves.  a c t i v a t i o n was  central  a dose o f a t r o p i n e  thalamus w h i c h were s y n a p t i c a l l y  of hindlimb  in  Thalamus  The  results  1976);  nicotinic.  Agonists  excitants  compound on  to c a t e g o r i z e the p h a r m a c o l o g i c a l  Cholinergic  t h e VB  nicotinic,  agonist i s defined p r i n c i p a l l y  as b e i n g  (Goth,  Figure 5 i l l u s t r a t e s ,  DH8E o r c u r a r e b u t  Ventrobasal  istration  As  1966;  u n a f f e c t e d by  carbachol  by  s t u d y was  have t e s t e d t h i s  Davis,  o f c a r b a c h o l was  t h e a c t i o n o f an onist,  1965;  sites  mixed  than  did  that already responses  to  either  descrithe  62  F i g u r e 4. and  Effect  glutamate.  o f a t r o p i n e on  Atropine  e x c i t a t o r y a c t i o n o f ACh elicited of  3.0  by  min.  glutamate.  cell  firing  (55nA) s e l e c t i v e l y w h i l e h a v i n g no  i n d u c e d by  ACh  antagonized  the  effect  upon  Each arrow r e p r e s e n t s a time  that interval  63  Cortex DEPTH : 1.34 mm  50 r  .  GLUT30  ATROPINE  55  ACH 30  i  1  i Min.  64  •Figure 5.  E f f e c t of a t r o p i n e on cholinomimetics  and ACh.  The e f f e c t s o f A3MC and o f ACh are markedly attenuated  i n the  presence o f 23 nA o f a t r o p i n e a p p l i e d f o r 3.5 min., whereas those of c a r b a c h o l , a n i c o t i n i c agonist, remain u n a f f e c t e d .  65  Cortex  A§MC 75  0.64mm  CARB ACh 40 75  </>  1 min.  66  Figure  6.  R e s p o n s e s o f a VB  e x c i t a n t s used difference  to c h a r a c t e r i z e neuronal responses.  i n l a t e n c y to peak e x c i t a t i o n  compared w i t h ACh, was  t h a l a m i c neurone to the s e r i e s  e v o k e d by  glutamate,  stimulation  Note  the  a c h i e v e d w i t h ABMC as  c a r b a c h o l and n i c o t i n e .  of the p e r o n e a l  of  nerve.  Cell  67  ACh 25  GLUT 12  ABMC 43  CARB 20  NIC 31  68  TABLE 3. basal  Numbers o f c h o l i n o c e p t i v e n e u r o n e s  complex o f t h e t h a l a m u s r e s p o n d i n g  muscarinic  o f the ventro-  to n i c o t i n i c  and  drugs.  ACh  Nicotinic  Muscarinic  +  0  —  +  0  —  +  42  41  1  0  36  6  0  —  3  0  2  1  0  2  1  +  : Excitation  —  : Inhibition  0  : No  Effect  Pontamine sky b l u e c a t i n g r e g i o n o f VB where c e l l s  were  spot  indi-  thalamus  recorded.  69  Cholinergic  Antagonists.  As was f o u n d f o r c e r e b r a l c o r t i c a l p r o d u c e d b y ACh, c a r b a c h o l b o t h DHBE a n d c u r a r e , Atropine by  the e x c i t a t i o n s  a n d n i c o t i n e were a n t a g o n i z e d  a s i s shown  i n Figures  by  7 a n d 8.  f r e q u e n t l y tended to reduce e x c i t a t i o n s produced  g l u t a m a t e a s w e l l as t h o s e  non-specific  neurones,  type  of effect  1960; K r n j e v i c a n d P h i l l i s ,  p r o d u c e d b y ACh.  To a v o i d  this  ( d e s c r i b e d by C u r t i s and P h i l l i s , 1963c), o n l y  low e j e c t i n g c u r r e n t s  (1-10  nA) o r m o d e r a t e c u r r e n t s  (10 - 30 nA) u s e d f o r s h o r t  periods  o f t i m e , were employed.  Under t h e s e  excitation  the  o f 18 n e u r o n e s b y ABMC was a b o l i s h e d o r s t r o n g l y  r e d u c e d by a t r o p i n e ,  and on 14 o f t h e s e ,  t i o n s were a l s o m a r k e d l y a t t e n u a t e d . action  circumstances  of atropine  i s illustrated  ACh i n d u c e d  excita-  An example o f t h e b l o c k i n g  i n Figure  7, w h i l e  the action  o f n i c o t i n e was u n a f f e c t e d .  The VB  rather paradoxical  thalamic  some c a s e s while  finding  n e u r o n e s t h a t DHBE, c u r a r e completely  block  non-susceptible  cholinomimetic,  i n Figures  and a t r o p i n e  could i n  t h e e x c i t a t i o n s p r o d u c e d b y ACh  a t t h e same t i m e s p a r i n g  illustrated  on b o t h c o r t i c a l a n d  8 a n d 9.  the e x c i t a t o r y e f f e c t s prompted  o f the  the experiments  C o n t r o l r e s p o n s e s o f a neurone  were e l i c i t e d b y t h e e j e c t i o n o f ACh, L - g l u t a m a t e , ABMC a n d carbachol,  as shown  i n t h e upper t r a c e o f F i g u r e  p r e s e n c e o f a dose o f c u r a r e w h i c h j u s t o f ACh a n d c a r b a c h o l  8.  antagonized  In the the effects  b u t n o t L - g l u t a m a t e o r ABMC, i t was  o b s e r v e d t h a t an i n c r e a s e  i n the current  e j e c t i n g ACh o r  70  Figure  7.  mimetics During of  t h e a p p l i c a t i o n o f DHBE w i t h  atropine  response  t o n i c o t i n e remained unchanged. ACh i n d u c e d  occurred  while  selective  increase  curare  Atropine  itself,  c o u l d be u s e d t o  a n t a g o n i s m a s DHBE,  i n the baseline  while  prevented.  t o have an e x c i t a t o r y e f f e c t  firing  DHBE was a p p l i e d f o r 5.5 m i n . , a n d r e c o v e r y 4.0 m i n .  current  In the presence  e x c i t a t i o n was  e f f e c t was p r o m i n e n t ,  demonstrate a s i m i l a r a concomitant  an i o n t o p h o r e t i c  blockers.  (25 nA), e x c i t a t i o n b y ABMC was a b o l i s h e d  DHBE was o f t e n o b s e r v e d when t h i s  and m u s c a r i n i c  e x c i t a t i o n b y ABMC was u n a f f e c t e d ,  of both antagonists,  and  n e u r o n e t o ACh a n d c h o l i n o -  i n the presence o f n i c o t i n i c  17 nA, n e u r o n a l  during the  Responses o f a t h a l a m i c  without  rate.  was o b t a i n e d  after  was a p p l i e d f o r 1.5 m i n . , a n d r e c o v e r y  2.5 m i n . a f t e r  the c e s s a t i o n o f the e j e c t i n g current.  VB  Thalamus Depth: 4.63 mm  Atropine  72  Figure  8.  Responses o f a s i n g l e t h a l a m i c  istration currents after  o f ACh,  indicated.  the  start  the  electrode;  end  o f the  carbachol,  the  centre and  The  the  carbachol  m i d d l e r e c o r d was  of e j e c t i o n of  curare  Note i n the  excitants  g l u t a m a t e , A3MC and  curare  l o w e r r e c o r d was  neurone to the with  obtained  admin-  the 4.5  min.  from another b a r r e l of  obtained  1.5  min.  after  current. t r a c e the concurrent  a t " c o n t r o l " doses.  increased  currents  a d m i n i s t r a t i o n of  o f ACh these  and two  the  73  VB  Thalamus Depth: 5 . 6  50 r  ACH25  GLUT 7  ApMC20  CARB5  CURARE  45  mm  74  carbachol (centre  caused a r e v e r s a l o f the e f f e c t  trace).  A similar  result  o f the antagonist  was o b t a i n e d w i t h  the e x c i t -  a t i o n s p r o d u c e d b y ACh o r AgMC when t h e a n t a g o n i s t was a t r o p i n e (Figure  9).  This  finding  suggested  as p o s s i b i l i t i e s  1) t h e i n c r e a s e d c o n c e n t r a t i o n o f a g o n i s t o f more  distant receptors  antagonist, inic  the  i n t e r a c t e d w i t h muscar(for Figure  8 ) , o r 3) t h e  competitively displaced the antagonist  test  these  possibilities,  simultaneously  Figure tom  by c u r a r e  by the  from  receptors.  To ted  2) t h e a d d i t i o n a l a g o n i s t  agonist  activation  t o t h o s e w h i c h were b l o c k e d  receptors not blocked  additional  allowed  that:  with  ACh a n d c a r b a c h o l were e j e c -  c o n t r o l c u r r e n t s as i l l u s t r a t e d i n  8, a n d t h e a n t a g o n i s m b y c u r a r e was o v e r c o m e .  trace of the Figure  blocking  The b o t -  demonstrates the r e v e r s i b i l i t y  a c t i o n o f the antagonist.  Analogous e f f e c t s  o f the were  o b t a i n e d when ACh a n d AgMC were u s e d i n t h e p r e s e n c e o f atropine. iii/  Renshaw  Excitatory Renshaw  Responses.  cells  frequency the  Cells  were i d e n t i f i e d b y t h e i r  discharge  central  which f o l l o w s antidromic  end o f severed  ventral roots  1946;  E c c l e s , F a t t and K o k e t s u ,  1956;  F r a n k a n d Fuo.rtes,  Curtis  and R y a l l ,  characteristic  1966c).  high  stimulation of  ( F i g u r e 10) (Renshaw,  1954; E c c l e s , E c c l e s a n d F a t t ,  1956; C u r t i s and E c c l e s , 1958a,b;  75  Figure  9.  mimetics, sufficient  Responses o f a t h a l a m i c  and the e f f e c t s o f a t r o p i n e .  two e x c i t a n t s . throughout  centre  t r a c e was o b t a i n e d  start  centre  the administration  o f the atropine  trace  centre  ejection.  unaf-  o f the antagonist.  The d o u b l e b a r s  The  i n the  e j e c t i o n a t 56 nA, w h i l e  e j e c t i o n a t 70 nA; t h e l a s t  trace represents  t h e s e two a g o n i s t s ,  administration of  1.0 - 2.0 m i n . a f t e r t h e s t a r t o f  forilACh represents  f o r A3MC r e p r e s e n t s the  concurrent  Responses t o n i c o t i n e remained  fected  the  A t a dose o f a t r o p i n e  t o a b o l i s h t h e e x c i t a t i o n by ACh a n d ABMC, t h e  a n t a g o n i s m was overcome w i t h these  n e u r o n e t o ACh, c h o l i n o -  that  response o f  the concomitant a d m i n i s t r a t i o n o f  at control  currents.  VB  Thalamus  77  Renshaw c e l l s n o r m a l l y  d i s p l a y e d a low t o moderate f r e q u -  ency r a t e o f spontaneous d i s c h a r g e c o u l d be s t i m u l a t e d to  to f i r e  90 o r 100 s p i k e s / s e c  any  effect  ( c a . 20 s p i k e s / s e c ) b u t  a t very  constant  f r e q u e n c i e s up  by t h e e j e c t i o n o f glutamate,  upon s p i k e a m p l i t u d e .  When a d m i n i s t e r e d  t o Renshaw  cells  i n small  tants  a n d L - g l u t a m a t e were o b s e r v e d t o c a u s e e x c i t a t o r y  responses. retaining to  amounts, ACh, n i c o t i n i c  without  The r e d u c t i o n carbachol  5 nA was o f t e n  (Figure  10).  and m u s c a r i n i c  i n the magnitude o f the c u r r e n t  within a micropipette  sufficient  The o n s e t  exci-  to e l i c i t  b a r r e l from  a powerful  o f t h e AgMC i n d u c e d  10 nA  excitation  e x c i t a t i o n was  l o n g b y c o m p a r i s o n w i t h ACh o r c a r b a c h o l .  Cholinergic  Antagonists.  Both n i c o t i n i c  and m u s c a r i n i c  blockers  abolished  o f Renshaw c e l l s  elicited  also  t h e e x c i t a t i o n e l i c i t e d by c a r b a c h o l ,  antagonized  no a n t a g o n i s t i c e f f e c t tially  b y ACh ( F i g u r e 1 1 ) .  the f i r i n g  t o w a r d s ; t h a t o f A3MC.  reduced the e f f e c t s  o n i s m o f t h e ACh r e s p o n s e s , carbachol  excitations.  Headley e t a l . (1975),  Atropine  o f A3MC i n p a r a l l e l w i t h  I n agreement w i t h  but had substan-  the antag-  b u t h a d no a p p a r e n t e f f e c t  upon the  the findings o f  the e a r l y responses o f s y n a p t i c a l l y  e v o k e d Renshaw c e l l s were r e d u c e d b y a t r o p i n e DH3E o r c u r a r e .  C u r a r e o r DH3E  as w e l l as b y  78  Figure to  10.  a volley  The a c t i v a t i o n  o f a Renshaw c e l l ,  i n t h e L5 v e n t r a l  c a r b a c h o l a n d A$MC u s i n g  root  ejecting  whose  i s shown above, currents  as  response t o ACh,  indicated.  In  the case of the carbachol e x c i t a t i o n s ,  the r e t a i n i n g  in  the drug c o n t a i n i n g  f r o m 10 t o 5 nA, t h e  subsequent  b a r r e l was r e d u c e d  " l e a k a g e " from the p i p e t t e  induce a potent  effect.  being  current  s u f f i c i e n t to  ™ J "  1 0 msec  Renshaw 25  r  ocu \co cu CO  CL  CO  o  1  ACH 5 CARB-5  ApMC 1.0  2.5 Min.  100/JV  80  Figure  11.  mimetics.  R e s p o n s e s o f two Renshaw c e l l s Curare  (45 nA) a n t a g o n i z e d  carbachol while having glutamate. of  control  no e f f e c t  a g a i n s t those  responses  occurred  after  6.0 m i n .  (upper) t r a c e .  Note the s p e c i f i c i t y  ent  o f ACh a n d  o f ABMC o r  The s e c o n d  t h e same e x c i t a n t s i n t h e p r e s e n c e  (16 n A ) ; t h e c e n t r e t r a c e was r e c o r d e d  only  the e f f e c t s  T h i s a n t a g o n i s t was a p p l i e d f o r 4.5 m i n . ; r e c o v e r y  was t e s t e d w i t h  control  t o ACh and c h o l i n o -  the responses  cell  of atropine  3.5 m i n . a f t e r t h e  R e c o v e r y was o b t a i n e d  after  8.0 m i n .  of action of atropine i n antagonizing  t o ACh a n d i;to~ ABMC". .. N o t e a l s o t h e d i f f e r -  s c a l e o f t h e two  figures.  SPIKES /  T8  SEC.  82  c)  Discussion  From t h e r e s u l t s o f t h e p r e s e n t the  cholinergic receptors  i n v e s t i g a t i o n , i t appears  which mediate e x c i t a t i o n o f r a t  c e n t r a l neurones  differ  the  cat,  t h e e x c i t a t i o n s b y ACh p o s s e s s b o t h  and  nicotinic  The  i n that  from those p r e v i o u s l y  properties  at a l l sites  pharmacological properties  characterized i n  et  Phillis  (1961;  of cholinoceptive  1962; 1 9 6 3 a , b , c ) ,  a l . (1964) a n d C r a w f o r d and C u r t i s  r e g a r d e d as e x c l u s i v e l y m u s c a r i n i c present  f i n d i n g s which  support  Spehlmann (1966),  Randic  In view of the  that r a t c o r t i c a l  and m u s c a r i n i c  perhaps r e l e v a n t  i n the c a t a l s o  that  of Krnjevic  are usually  i n nature.  the concept  central  (1963);  neurones e x h i b i t both n i c o t i n i c to r e c a l l  muscarinic  examined.  neurones i n t h e c a t as e s t a b l i s h e d by t h e s t u d i e s and  that  effects, i t i s some  evidence  has  b e e n p r e s e n t e d w h i c h showed e x c i t a t o r y e f f e c t s o f n i c o t i n e  and  carbachol,  a n d an a c t i o n o f DH$E as a s y n a p t i c  (Spehlmann, 1963; C r a w f o r d a n d C u r t i s ,  The  present  observations  from those o f Stone cortical  1966).  also  i n some  (1972) who f o u n d m u s c a r i n i c  respects  actions  pyramidal t r a c t neurones o f the r a t l o c a t e d  i v e l y b e l o w 0.6 mm. cholinoceptive receptors.  cells  whereas o t h e r  superficial  appeared to possess only  i s unclear  on  exclus-  ( a b o v e 0.6  mm)  nicotinic  A l t h o u g h no a t t e m p t was made i n t h e p r e s e n t  ments t o c l a s s i f y it  differ  antagonist  neurones as p y r a m i d a l o r n o n - p y r a m i d a l  why n i c o t i n e - s e n s i t i v e c e l l s were  experitract,  encountered  83  at  a l l levels  of the  to  show an e f f e c t w i t h n i c o t i n e  d e p t h s b e l o w 0.6 The  results  cerebral  f r o m VB  (Andersen  and  McLennan,  1972), where i t was  an  o b t a i n e d from  Curtis,  earlier  clear No  such  tendency o f VB  experiments  King  and  Ryall  adequately  This finding  supports  from  McLennan and  their  noted  a specific  cells.  Hicks,  and A g h a j a n i a n  experiments  the  1978)  of receptor i s l e s s  Bird  prop-  than  i n the  compounds.  nicotinic present  (1979) h a v e shown i n t h e  and m u s c a r i n i c  posed.  thalamic  rats.  of n i c o t i n i c  types  and  a t r o p i n e were more  t h a n were t h e n i c o t i n i c  actions  two  cat  and  than n i c o t i n i c  towards a g r e a t e r m u s c a r i n i c  r e c e p t o r s , does n o t  1975;  Marshall  t h a t f e l i n e VB  o f ABMC and  activity  that atropine, although  ergic  i n the  the o b s e r v a t i o n i n  somewhat more m u s c a r i n i c  More r e c e n t l y , cat  agree-  reported that both n i c o t i n i c  t h a l a m i c n e u r o n e s was  with  at  antagonists are p h a r m a c o l o g i c a l l y  i n t h a t the e f f e c t s  property  1966;  r e p o r t (McCance e t a l . , 1966)  c u t upon c e l l  i n good  experiments  m i n o r d i f f e r e n c e however was  neurones possess erties,  previous  1964a,b; D a v i s ,  a g o n i s t s and  One  antagonists  thalamic neurones are  those  active.  or n i c o t i n i c  unable  mm.  ment w i t h  muscarinic  c o r t e x w h e r e a s S t o n e was  antagonist for  d i s t i n g u i s h between cholinomimetics  suggestion  t h a t the  rigid  cholin-  (1976) r e a c h e d  on h i p p o c a m p a l  on Renshaw  (Headley  et a l . ,  d i v i s i o n between  t h a n was  the  p r e v i o u s l y sup-  a similar  cells,  the  where  conclusion  these  84  neurones  were f o u n d  excitants,  to respond  and the responses  to n i c o t i n i c  and m u s c a r i n i c  t o ACh c o u l d be b l o c k e d b y b o t h  type o f a n t a g o n i s t . • P o s s i b l e R e c e p t o r Mechanisms As b o t h A3MC a n d n i c o t i n i c c e p t i v e neurones ral of  i n the c e r e b r a l  horn o f the s p i n a l these c e l l s  tinic  c o r t e x , VB t h a l a m u s  c o r d , a n d as ACh i n d u c e d  cholinoand v e n t -  excitations  b l o c k e r s , none o f t h e s e n e u r o n a l p o p u l a t i o n s p o s s e s s or muscarinic receptors.  e x c i t a t o r y responses  were o b s e r v e d onse l a t e n c y  ergic  and p a t t e r n  action.  Indeed,  b y ACh, n i c o t i n e  each o t h e r c l o s e l y on some n e u r o n e s  (Figure  This proposal receives  o f ACh a n d c a r b a c h o l , w h i c h  by a c h o l i n e r g i c b l o c k e r ,  of resp-  2), i t could  i n the c h o l i n -  some s u p p o r t a l s o  on VB t h a l a m i c n e u r o n e s ,  from  t h e combined  by themselves  can a c t a d d i t i v e l y  since  a n d ABMC  i n terms  a common r e c e p t o r i s i n v o l v e d  observation that  action  elicited  to resemble  be s u g g e s t e d t h a t  the  t h e same  were a n t a g o n i z e d b o t h b y a t r o p i n e a n d t h e n i c o -  exclusively nicotinic the  agonists excited  are antagonized  t o overcome t h e  antagonism.  The inic  fully This  antagonism of  a s i n g l e r e c e p t o r which  and m u s c a r i n i c c h a r a c t e r i s t i c s  account tics.  proposal that  from  o f ACh r e s p o n s e s  cholinomimetic i s s t i l l  nicot-  i s u n l i k e l y by i t s e l f t o  f o r the observed actions  i s apparent  has both  o f ACh a n d c h o l i n o m i m e -  the r e s u l t s  showing  a  complete  a t a t i m e when one o r t h e o t h e r type effective  i neliciting  neuronal  85  responses.  I f only a single,  pharmacologically unspecific  chol-  inergic  receptor population i s r e s p o n s i b l e f o rmediating the  effects  o f ACh a n d c h o l i n o m i m e t i c s ,  antagonists and  should reduce the e x c i t a t i o n s  vice versa.  accord with  The e v i d e n c e  one  from t h i s  two p o p u l a t i o n s  of nicotinic  study  of separate  receptors both  ( C u r t i s and R y a l l ,  muscarinic  cholinomimetics  ACh  concurrently  reconcile with effect  implies ting  doubling  either  and C u t t i s ,  s e n s i t i v e t o ACh,  n i c o t i n i c and 1964b).  o f t h e a n t a g o n i s m by c u r a r e  the e j e c t i n g  or carbachol containing b a r r e l  agonists  ive  (Andersen  e x p e r i m e n t s where a r e v e r s a l by b o t h  current a p p l i e d to the  a n d a d m i n i s t e r i n g t h e two  ( F i g u r e 8), p r o v i d e  a result  d i f f i c u l t to  a one o r two r e c e p t o r h y p o t h e s i s .  o f ACh a n d c a r b a c h o l  i n overcoming  The a d d i t -  the a c t i o n of curare  c o - o p e r a t i v i t y against the competitive  antagonist, implica-  the existence o f a s i n g l e receptor or r e c e p t i v e s i t e  the a g o n i s t s . T h i s i s because the d i f f u s i o n substances  a r e t h e same w h e t h e r a d m i n i s t e r e d  y e t when e j e c t e d a l o n e , t h e e f f e c t s the m a i n t a i n e d  s h a r e d by  c o n s t a n t s o f ;the two together  or alone;  o f each a r e blocked.  However  e f f e c t i v e n e s s o f A3MC a t a t i m e when t h e e f f e c t s  o f ACh a n d c a r b a c h o l a r e a n t a g o n i z e d the p r e s e n c e  there  1 9 6 6 c ) ; o r 2) t h a t t h e r e e x i s t s one  o f "mixed" r e c e p t o r s r e s p o n d i n g t o b o t h  was c a u s e d  1) t h a t  var-  and t h e o t h e r b y n i c o t i n i c  population  The  agonists,  i s not wholly i n  c h o l i n o c e p t i v e neurones:  w h i c h i s a c t i v a t e d by m u s c a r i n i c  agonists  the a c t i o n o f muscarinic  e i t h e r o f t h e two p o s s i b i l i t i e s w h i c h h a v e b e e n  i o u s l y proposed f o r f e l i n e exist  then  o f two p o p u l a t i o n s  by c u r a r e i s s u g g e s t i v e o f  o f receptors, although  an  86  e x p l a n a t i o n i s n o t obvious why ACh i s completely  unable to  produce an e x c i t a t o r y e f f e c t through the m u s c a r i n i c  population  a  o f r e c e p t o r s which are u n a f f e c t e d by curare. C e r t a i n a l t e r n a t i v e p r o p o s a l s may be made which might have a b e a r i n g upon the observed r e s u l t s . be  For example, i t may  that a s i n g l e c h o l i n e r g i c r e c e p t o r with more than one recep-  tive site exists.  Perhaps curare exerts an a c t i o n upon one o f  the s i t e s which i s a c t i v a t e d by n i c o t i n i c a g o n i s t s but which cannot be a c t i v a t e d by m u s c a r i n i c  ones, and i t may not be pos-  s i b l e f o r ACh to e x e r t an e f f e c t unless both the n i c o t i n i c and the m u s c a r i n i c that "spare  s i t e s are a v a i l a b l e .  Another  possibility is  r e c e p t o r s " or r e c e p t o r s d i s t a n t from those a f f e c t e d  by the a n t a g o n i s t become a c t i v a t e d by the a d m i n i s t r a t i o n of a d d i t i o n a l agonist.  This l a t t e r proposal  seems u n l i k e l y how-  ever, because: 1) ACh and c a r b a c h o l a t doses which a r e normally antagonized,  should n o t d i f f u s e over a g r e a t e r d i s t a n c e when  a p p l i e d c o n c u r r e n t l y than when a p p l i e d s e p a r a t e l y ; and  2) such  an e x p l a n a t i o n w i l l n o t account f o r the maintenance o f a musc a r i n i c a c t i o n i n the presence o f a n i c o t i n i c a n t a g o n i s t , a t the same time as ACh i s antagonized. Two probable that:  c o n c l u s i o n s a r i s i n g from these r e s u l t s are  1) the r e c e p t o r s i n the r a t are not e a s i l y d e s c r i b e d as  b e i n g e i t h e r o f the n i c o t i n i c , m u s c a r i n i c  or mixed  types:  i n s t e a d they r a t h e r appear to l a c k s e l e c t i v i t y toward the pharma c o l o g i c a l agents w i t h which they  i n t e r a c t ; or 2) the c h o l i n e r g i c  87 receptors  on  r a t c e n t r a l neurones  t i o n p o s s e s s i n g two dent  active  sites,  cholinomimetic are necessary p o n s e t o ACh Although gic  of a s i n g l e  pharmacologically distinct, both which i n t e r a c t w i t h  a g o n i s t s and  in exciting t h e one  and  both  the evidence  f o r the  integrated res-  neurones.  c o n c l u s i o n - t h a t the e x c i t a t o r y  selectivity  receptors -  throughout  f o r reacting with  may  obtained with  propose a t o t a l l y  seem w a r r a n t e d  agonists  on  e q u i v a l e n t m e c h a n i s m f o r ACh  excitatory  agent  C a r b a c h o l was  The  and m u s c a r i n i c  a p p l i e d t o t h e CNS  i s s u b s t a n t i a t e d by  the c o n c l u s i o n s support  and  Bird  and A g h a j a n i a n  CNS  differ  of  their  class  a c t i o n s o f ACh  Although  results  one  cortical  than  from  are not  those  the  cells  o f the  toto.  division  should not properly this  investigation.  of Headley et a l .  r e c e p t o r s i n the r a t  c a t , the present  pharmacological  o f a nature which a l l o w a c l e a r - c u t  properties in  were  of cholinomimetic only.  concept  (1976) t h a t ACh  upon  p o t e n c y when  s u g g e s t i o n o f H e a d l e y e t a l . (1975) t h a t t h e  of n i c o t i n i c be  clearly  upon Renshaw c e l l s  Furthermore,  t o ACh and  to  receptors  t e s t e d on  found which responded  than  intended  o f even weaker apparent  cells.  to  the b a s i s of  t h a l a m i c n e u r o n e s , and was cortical  choliner-  appear  a n t a g o n i s t s , i t i s not  a l l r e g i o n s o f t h e r a t CNS.  a more e f f e c t i v e  indepen-  o f which  r e c e p t o r s i n s e v e r a l r e g i o n s o f the r a t b r a i n  the f e l i n e  popula-  their respective  a n t a g o n i s t s , and  i n a c o - o p e r a t i v e sense  demonstrate l e s s do  consist  description  87a One  f u r t h e r p o s s i b i l i t y which must be c o n s i d e r e d  the apparent d i f f e r e n c e between r a t and  c a t ACh  receptors  be i n f l u e n c e d by the type of a n a e s t h e t i c employed by investigators.  However, although  K r n j e v i d and  (1963a,b,c) i n c a t s used a mixture of acid  i s that may  various  Phillis  diallyl-barbituric  (Dial) and urethane, Crawford and C u r t i s (1966) used  pentobarbitone  sodium, w h i l e Spehlmann and Downes (1974) used  a mixture of methoxyflurane, n i t r o u s oxide and oxygen i n encephale i s o l e p r e p a r a t i o n s , there was  nevertheless  agreement on the r e a c t i v i t y of the f e l i n e  a general  receptors.  In the experiments on r a t s , Stone (1972) used urethane, D i a l or halothane i n oxygen, while Headley e t a l . (1975) employed pentobarbitone  sodium, and again the e f f e c t of  v a r i o u s a n a e s t h e t i c s d i d not appear to a l t e r the  the  results.  However urethane, the drug used i n a l l of the present  experi-  ments, does possess a n t i c h o l i n e s t e r a s e p r o p e r t i e s and  therefore  c o n c e i v a b l y could a l t e r c e r t a i n of the cholinomimetic and  i f t h i s were to be the case the apparently  of r e a c t i o n of the r e c e p t o r s of r a t and  responses;  different  patterns  cat c o u l d be a f f e c t e d .  88  CHAPTER IV  AMINO ACIDS  VENTROBASAL THALAMUS  a) The  Introduction amino a c i d s  tances b e l i e v e d Their eral  a r e r e l a t i v e newcomers t o t h e l i s t t o a c t as s y n a p t i c  implication i n synaptic  transmitters  summarized  below.  Uptake and R e l e a s e  Since  there  i s an u n e v e n d i s t r i b u t i o n i n t h e mammalian CNS  of L-glutamate, L-aspartate, taurine  y-aminobutyrate,  (Graham, Shank, A p r i s o n  suggested that  the  sev-  and pharmacology, as  i n d i c a t e d by the r e s u l t s o f such s t u d i e s ,  that  from  of investigation, including  neurochemistry, h i s t o l o g y , physiology  1)  i n mammals.  f u n c t i o n has d e r i v e d  d i f f e r e n t but r e l a t e d f i e l d s  o f subs-  g l y c i n e and  and Werman, 1967),  t h e s e compounds may h a v e f u n c t i o n s  of serving  i n metabolic processes.  Studies  c e n t r a l f u n c t i o n o f glutamate and a s p a r t a t e  have r e v e a l e d uptake  sites  map p o s s i b l e  that  changes i n t h e d e n s i t y  f o r these a c i d s ,  other  than  concerning i nparticular  of high  affinity  a f t e r l e s i o n s , may be u s e d t o  glutamate pathways.  been s u g g e s t e d as a p o s s i b l e  i t h a s been-  High a f f i n i t y  uptake has  index f o r the l o c a l i z a t i o n of  89  those r e g i o n s  i n t h e CNS  where t h e r e s p e c t i v e  as a s y n a p t i c  transmitter  (Storm-Mathisen,  compound  1977;  acts  for a  discus-  s i o n o f u p t a k e mechanisms, see r e v i e w s by J o h n s t o n , 1978; Two White,  independent groups  L y n c h and Cotman, • 1976;  Cotman and Cummins, 1977; that  of investigators  a loss  molecular  White,  (synaptic) layer  Vaca,  N a d l e r , Hamberger,  Storm-Mathisen,  of high a f f i n i t y  (Nadler,  1979).  1977)  have  shown  uptake o f glutamate o c c u r s i n the  o f the dentate gyrus a f t e r  lesions  a r e made i n t h e p e r f o r a n t p a t h - a s o u r c e o f a f f e r e n t  terminals  to t h i s  other  region  ( H j o r t h - S i m o n s e n and J e u n e ,  change i n amino a c i d after  the l e s i o n s  radiographic  analysis  of hippocampal  f r o m t h e a r e a known t o r e c e i v e  1977a).  Since h i s t o l o g i c a l  slices loss  input  of s i l v e r  seems r e a s o n a b l e t o a t t r i b u t e the l o w e r i n g o f i t s l e v e l s  (Storm-Mathisen, evidence of  such l e s i o n s , i t  the l o s s o f glutamate  c o n t a i n g l u t a m a t e as a t r a n s m i t t e r  studies  i m p r o v e d upon p r e v i o u s a t t e m p t s  levels with anatomical tracts in  that  i t was  terminals  possible  intrinsic  uptake  i n the dentate gyrus to the  d i s a p p e a r a n c e o f p e r f o r a n t p a t h t e r m i n a l s , w h i c h by ation  grains  perforant  s t u d i e s h a v e shown no following  Auto-  incubated i n  t e r m i n a l s of the  l e s i o n s were made o f t h i s  a l o s s of hippocampal neurones  and  observed  o t h e r than a r e d u c t i o n o f glutamate.  3  after  No  c o n t e n t i n t h e d e n t a t e g y r u s was  H H]-glutamate r e v e a l e d a s i g n i f i c a n t  path,  1972).  implic-  substance. to l i n k  These  glutamate  ( s e e r e v i e w by J o h n s o n ,  to e x c l u d e the l o s s  1972)  of neurones  t o t h e hippocampus as a c a u s e o f t h e  and  90  glutamate d e p l e t i o n s , due  to the w e l l organized  arrangement of t h i s s t r u c t u r e and  anatomical  i t s a f f e r e n t pathways.  A s i m i l a r approach has been p o s s i b l e f o r s t u d i e s on c o r t i c o - s t r i a t a l pathway (Divac, Fonnum and 1977;  McGeer, McGeer, Scherer and  Huag and Paik,  1977;  E a r l i e r s t u d i e s had  Kim,  Piak and  shown that the c o r t e x  a t o r y p r o j e c t i o n to the caudate nucleus wald, P r i c e , Vernon and mate c o n t a i n i n g the caudate by  H u l l , 1973)  (Spencer, 1976).  ganglionic structure  Kim,  Hassler,  Schroder, 1977).  sends a l a r g e , e x c i t -  (Webster, 1961;  Buch-  which i s probably g l u t a -  A p a r t i a l deafferentation  c o r t i c a l undercutting  a l o s s of glutamate content and  Storm-Mathisen,  Singh, 1977;  Hassler,  the  of  or a s p i r a t i o n r e s u l t e d i n  uptake s i t e s from t h i s  basal  (McGeer et a l . , 1977).  Neurochemical s t u d i e s on a l t e r e d uptake of glutamate f o l l o w i n g l e s i o n s have suggested the presence of t r a n s c a l l o s a l connections and  a c o r t i c o - t h a l a m i c pathway which may  mediated by glutamate (Divac et a l . , 1977; Fonnum, 1978). and  aspartate  mammalian CNS does not  Lund-Karlsen  be  and  I t should be noted however that s i n c e glutamate share the same h i g h a f f i n i t y uptake system i n (Balcar and  Johnston, 1972;  1973)  exclude the p o s s i b i l i t y that a s p a r t a t e  m i t t e r of some or a l l of these systems, and  this  evidence  i s the  evidence of  transother  types (eg: p h a r m a c o l o g i c a l - see below) which allows d i f f e r e n t i a t i o n between glutamate and required.  a s p a r t a t e mediated synapses i s  For example, m i c r o d i s s e c t i o n methods have shown that  91 in  the  ule  c e r e b e l l a of r a t s ,  cells  with  ( N a d i , M c B r i d e and  climbing fibres  In the with  L-glutamate i s a s s o c i a t e d with  cat there  (Nadi,  Aprison, Kanter,  i s evidence  some p r i m a r y  1977)  and  gran-  L-aspartate  M c B r i d e and  Aprison,  1977).  that L-glutamate i s a s s o c i a t e d  afferent fibres L-aspartate  (Shank, Graham, A p r i s o n  Werman, 1967)  while  a p p e a r s t o be  contained  interneurones  ( D a v i d o f f , Graham, Shank, Werman and  and  in  Aprison,  1967). Studies  on  their  support  for a role  acids.  Koyama  and  Dodd  cortex  r e l e a s e have a l s o p r o v i d e d  as  t r a n s m i t t e r s of the  ( 1 9 7 2 ) , Dodd and  (1978) h a v e o b s e r v e d  Bradford  nerves.  foci  and  In v i t r o  ( B r a d f o r d and  i n response to s t i m u l a t i o n of r e l e a s e of glutamate  coupled  of  are  the  2)  1976)  the  has  and  evidence  lateral  from the  t r a n s m i t t e r s of  the  cortical hindlimb  pyriform  olfactory dentate  tract  gyrus  ( W i e r a s z k o and  that aspartate  a f f e r e n t s to the  strengthened  Bradford  dentate  Lynch,  i s released gyrus  the view t h a t these  synaptic excitation  fol-  two  (Nadler  et  amino  i n some r e g i o n s  CNS.  Histological  Although ation  with  commissural  1976)  acids  Richards,  from the  s t i m u l a t i o n of the p e r f o r a n t path  from the al.,  (1976) and  glutamate r e l e a s e from  c o r t e x f o l l o w i n g s t i m u l a t i o n of the  1979)  e x c i t a t o r y amino  following light-evoked stimulation, during  epileptic  lowing  experimental  Findings  g l u t a m a t e and  aspartate  a r e now  of n e a r l y a l l c e n t r a l neurones,  the  known t o c a u s e observation of  excita  92  heterogenous p a t t e r n Aprison, in  1970)  distribution within  Johnson,  idea  that  1972;  (Graham e t a l . , 1967;  1977;  Wenthold,  synapses.  In the  d o r s a l horn, while (Duggan and  mia.  of the  aspartate  Johnston,  while  amate may  be  guinea p i g ,  nerve  since  a l t e r e d the  glutamate  and  greater  in  i n the  of  transmitter  and  should.  the  a f t e r l e s i o n s of  Gulley,  transmitter  exert  that  an  of  excitatory  aspartate  cochlear  the  greatest  1977;  the  ventral  and  other  synaptic  p o s s i b l y glutthe  cat  candidate the  Levels  of aspartate,  d e c r e a s e i n the  and  trans-  cochlear the  and  to a less  cochlear  (Wenthold,  i n those s u b d i v i s i o n s of  greatest primary innervation  1978).  sug-  primary  (1977) s u g g e s t e d  auditory nerve  ischae-  Evidence  a f f e r e n t s of no  the  Oertel  those of glutamate,  the  aspartate  supporting  compound a c t i o n p o t e n t i a l s o f  nucleus which r e c e i v e and  cat  the  of  Furthermore temporary  t h e s e compounds and  These decreases are  hold  may  to suggest  transmitters  marked e x t e n t nucleus  the  from s p i n a l i n t e r n e u r o n e s .  i n a manner K l i n k e  natural  are  interneurones,  aspartate  been p r e s e n t e d  mitters  1970).  a l o s s of  a c t i o n when r e l e a s e d has  levels  et a l . ,  transmitters  of  and  acids  reinforced  i n dorsal roots  t h a t g l u t a m a t e m i g h t be  afferents,  (Shank  cord r e s u l t s i n a p r e f e r e n t i a l loss of  concomitant with gestion  CNS  Davidoff  has  are  s p i n a l cord  r e l a t i v e l y more c o n c e n t r a t e d  horn  1978)  t h e s e e x c i t a t o r y amino a c i d s  different  the  a d i f f e r e n c e i n l e v e l s between t h e s e  c e r t a i n b r a i n regions  1967;  is  and  of  1978). the (Went-  93  Attempts  to i d e n t i f y n e u r o a n a t o m i c a l l y the s i t e s  glutamate mediated  synapses  zation of t r i t i a t e d 1976)  g l u t a m a t e have been  and  c o r t e x i n the uptake  Uptake  into  glia  spinal  cord  (Fagg, Jones  brain  has b e e n  unsuccessful of g l i a l  of t h i s  demonstrated a l s o  elements  a c i d was  t y p e o f u p t a k e may  or re-uptake i n t o  The  be  transmitter  in slices  terminals  enzymes  of a s y n a p t i c a l l y  ( L o g a n and  Snyder,  1974;  Michaelis, Michaelis  Takagaki,  1976;  Saltpeter,  Hokfelt  White 1970;  and N e a l , 1 9 7 6 )  and L j u n d g d a h l , 1972;  1974;  Henn, 1976;  Hosli  B o r g and M a n d e l ,  1971;  and H o s l i ,  and  1976;  (Faeder  1974;  1971;  Salem,  and  Logan,  Boy-  1976;  Hamberger,  Snodgrass  1973;  Iver-  1976;  i s at present contraversial,  elsewhere  Shank and Graham, 1978;  uptake  R o b e r t s and Keen,  1977)  i s discussed at length  1978;  or g l i a l  and O r k l a n d , 1975;  1972;  and R a i t e r i ,  Storm-Mathisen,  E h n i n g e r and F a l k ,  Hammerschlag, B r a n c h o  Balcar,  I v e r s e n and  Levi  acid  1971;  Lakshmanan and Padmanaban, B e a r t , 1976;  (eg:  r e l e a s e d amino  1973;  1974;  1978),  (eg: catecholamines).  1972;  arski,  and  specific  Snyder,  and  of the  t h e more i m p o r t a n t m e c h a n i s m f o r  i s n e u r o n a l uptake  Bennett, Logan  sen,  noted.  as i m p o r t a n t a m e c h a n i s m o f  afferent  q u e s t i o n o f whether  termination of action  of  and o t h e r r e g i o n s , s u g g e s t i n g  c e s s a t i o n o f a c t i o n as d e g r a d a t i o n by  and  (McLennan,  and J o r d a n , 1978), n e o n a t a l mouse  ( W h i t e and N e a l , 1976)  this  ACh)  locali-  ( H e r z , S c h o i i s b o e , B o e c h l e r , M u k e r j i and F e d e r o f f ,  retina that  through autoradiographic  although the v i g o r o u s p a r t i c i p a t i o n  the thalamus  of  ( J o h n s o n , 1978;  Watkins,  1978).  Schousboe,  F o r a more  94  d e t a i l e d overview of central  anatomical  amino a c i d t r a n s m i t t e r s ,  Johnston 3)  the  (1974) and  Krnjevic  d i s t r i b u t i o n of  the  r e v i e w s by  (1974) a r e  mammalian  Curtis  and  valuable.  Iontophoresis  Agonists. One  of  the  first  i n d i c a t i o n s that  g l u t a m a t e and  might possess p h a r m a c o l o g i c a l a c t i v i t y was  the  Whipple  observation  thereafter, number o f recorded  ylic  by Madden, Woods, S h i l l ,  i n dogs, a v o m i t i n g  B r o o k s , R a n s m e i e r and  from the  work o f H a y a s h i  (1954;  amino a c i d s  f i r m l y b a s e d on to the  i n the  activity  brains  the  function of  the not  cerebral  cortex.  p o w e r f u l e x c i t a t o r y and  effects  on  until  and  other  and  general  G l u t a m a t e and  became admin-  range  ACh,  electrical aspartate  topically  or i n t r a v e n t r i c u l a r l y .  Curtis  and  colleagues  were unaware o f  these  of  and  y-aminobutyrate i n h i b i t o r y  b r a i n , w h e t h e r t h e y were a p p l i e d  pos-  dicarbox-  Hayashi  extensive  aspartate  a  the  the  t h e mammalian CNS  e f f e c t s upon t h e  Soon  potentials  however, t h a t  dogs an  glutamate,  exerted  the  induced.  I t was  aspartate  o f apes and  their various of  administered  d i r e c t experimental evidence.  compounds w h i c h i n c l u d e d described  1956)  and  (1949) f o u n d t h a t  influenced  isolated frog brain.  involvement of glutamate,  istered  Gerard  CNS  Remington  r e s p o n s e was  d i c a r b o x y l i c amino a c i d s  pioneering sible  i n t h e mammalian  (1945) t h a t when t h e s e compounds were  intravenously  aspartate  findings  95  (cf. Watkins,  1978)  same c o n c l u s i o n s  and  therefore  of Hayashi,  independently  that  glutamate,  cysteate  possess e x c i t a t o r y a c t i v i t y  Watkins,  1960;  confirmation  1961a,b; C u r t i s and of  the  earlier  observations  experiments wherein the newly phoresis  (Suh  e t a l . , 1936;  a d a p t e d f o r use pharmacology. three  ent  Among t h e  from that of  P e r r i n and  calcium  currents  and  1960).  t o an  extent  used to pass  - 0.50  lasted until  the  the  the  ance b e t w e e n t h e p o i n t d e p o l a r i z a t i o n was  the  current  the the  spinal rate  interof of  pipettes. short  laten-  e x c i t a n t s , and iontophoretic the  magnitude of  u s e d and the  the  the  current.  onset.  amino a c i d s w i t h  o f e j e c t i o n and  shown t o be  the  Curtis,  the magnitudes  r a p i d as  t i m e c o u r s e and  d e p e n d e n t upon t h e  to  r e p e t i t i v e l y with  as  differ-  C u r t i s , 1959;  compounds f r o m t h e  cessation of  o f a c t i o n was  actions,  cholinomimetics  increased  i n response to  Motoneurones responded to depolarization,  was  p r o d u c e d marked  d e p e n d e n t on  sec)  being  1958;  e x c i t a t o r y amino a c i d s  termination  C h a p t e r I)  When a d m i n i s t e r e d  cies  The  see  ionto-  a manner c o n s i d e r a b l y  Eccles,  tested f i r e d  effect  1953;  cysteate)  by  A l l * interneurones (0.25  This  arose from a s e r i e s of  c h e a l a t i n g agents,  ( C u r t i s and  neuronal f i r i n g the  aspartate  Watkins,  neurones the  1963).  compounds t e s t e d f o r t h e i r  i n neuronal f i r i n g  or convulsants  1960;  and  i n i n v e s t i g a t i n g mammalian c e n t r a l n e u r o -  (glutamate,  increases  and  Phillis  developed technique of  Nastuk,  the  aspartate  (Curtis,  Watkins,  reached  a membrane  this  action  estimated  dist-  c e l l membrane.  associated with  changes  in  The  96  postsynaptic potentials, increase  t h e amino a c i d s  caused  an  i n t h e c e l l ' s e x c i t a b i l i t y as m e a s u r e d by  direct  current  t h e amino  acid  injection.  Renshaw c e l l s  agonists. acids,  a l s o were e x c i t e d by  DH3E d i d n o t a n t a g o n i z e t h e r e s p o n s e s  indicating  nicotinic  i n that  that  cholinergic  their  actions  receptors.  t o t h e amino  are not mediated  T h i s b e l i e f was  the o b s e r v a t i o n that  the a c i d s  of  i n t e r n e u r o n e s , which  are n o n - c h o l i n o c e p t i v e (see Chapter III).  structural  and W a t k i n s  ently et  aspartate.  c o n f i r m e d and  al.,  1964)  1961;  and  extended  Krnjevic  depolarization  basic  o f t h e amino a c i d r e c e p t o r t h r o u g h  examination o f the a c t i v i t y and  a  (1960) were a b l e t o deduce t h e  requirements  to glutamate  caused  reinforced  by  Curtis  also  through  o f compounds  structurally  related  T h e i r m a j o r r e s u l t s were (Curtis  and P h i l l i s ,  and W a t k i n s ,  subsequ-  1963;  1963a; C r a w f o r d  and  t h e f o l l o w i n g p r o v i d e s a summary o f t h o s e  Curtis Curtis,  findings.  Most o f the s t r u c t u r a l m o d i f i c a t i o n s o f L - g l u t a m a t e L-aspartate yielded Those v a r i a t i o n s ional in  (refer  amino group in  were  except-  t o F i g u r e 12, n e x t p a g e ) i n t h a t  they  resulted  a r e as f o l l o w s :  1) a l k y l a t i o n  of aspartate with a s i n g l e methyl  artate  3 times  the potency  i s about  replacement  potency.  i n molecular s t r u c t u r e which  group,  of  10  o f the  times  o f D L - a s p a r t a t e and the potency  3 - c a r b o x y l group  the  especially  t h e c a s e o f t h e D i s o m e r where N - m e t h y l - D L - a s p a r t a t e  about  or  compounds w i t h a t t e n u a t e d e x c i t a t o r y  an e n h a n c e d a c t i v i t y  an  is  N-methyl-D-asp-  o f D - a s p a r t a t e ; 2) o f D-glutamate  the  with a  97  Figure  12.  Diagrammatic  s t r u c t u r e s o f glutamate sesses but  two t e r m i n a l  differ  length  r e p r e s e n t a t i o n o f the molecular and a s p a r t a t e .  carboxyl  i n that glutamate  than can a s p a r t a t e ,  carbon chain.  groups  due t o t h e e x t r a  et  a l . , 1979).  c a n be l e s s  greater  length of i t s  F o r t h e same r e a s o n , g l u t a m a t e  distance  pos-  and an amino s u b s t i t u e n t ,  can extend over a  more " f o l d e d " c o n f i g u r a t i o n i n t h a t carboxyl  Each molecule  c a n assume a  i t s minimal a to w  than that o f aspartate  (Hall  GLUTAMATE N(+) w  la  99  s u l p h o n i c a c i d group,  forming D-homocysteate, having a potency  on the order of 5 - 10 times as great as D-glutamate; 3) a l k y l a t i o n of the amino group of D-aspartate w i t h an s u b s t i t u e n t to form N-iminomethyl-D-aspartate,  iminomethyl  or w i t h an  s u b s t i t u e n t to form N - e t h y l - D - a s p a r t a t e , both which are  ethyl about  twice as potent as D-aspartate. Many compounds c l o s e l y r e l a t e d i n s t r u c t u r e to  L-glutamate  and L - a s p a r t a t e share p o t e n c i e s of a s i m i l a r o r d e r of magnitude and among these are: L - c y s t e a t e , L-homocysteate, D-aspartate, 3-aminoglutarate, L - c y s t e i n e s u l p h i n a t e , N-methyl-L-glutamate and  3-hydroxyglutamate. I t was  p o s s i b l e to summarize i n an o v e r a l l f a s h i o n the  s t r u c t u r e - a c t i v i t y r e l a t i o n s o f the above i n f o r m a t i o n , as done by C u r t i s and o t h e r s .  was  The f o l l o w i n g p r e s e n t s a b r i e f  summary of these o b s e r v a t i o n s .  Peak a c t i v i t y occurs w i t h a  2 or 3 carbon s e p a r a t i o n between amino and c a r b o x y l t e r m i n a l groups,  and there i s v a r i a t i o n i n a c t i v i t y w i t h the i s o m e r i c  forms.  Some a c i d groups s u b s t i t u t i n g f o r the t e r m i n a l c a r b o x y l  of a s p a r t a t e r e t a i n e x c i t a t o r y potency, w h i l e o t h e r s ( p h e n o l i c or phosphoryl s u b s t i t u t i o n s ) r e p l a c i n g the t e r m i n a l c a r b o x y l of glutamate  reduce potency.  amino group reduces potency glutarate  Changing the p o s i t i o n of the although some compounds, 3-amino-  (3AG) and 2-aminomethy1-succinate f o r example, are  reduced only somewhat.  I n t r o d u c t i o n of s m a l l groups i n t o  the  carbon c h a i n , involvement o f the a and/or to c a r b o x y l t e r m i n a l s  100  in  ester  o r amide l i n k a g e s ,  amino group leads  of  (the n o t a b l e e x c e p t i o n b e i n g  to i n a c t i v i t y  t i v e which  or a l k y l a t i o n  lack  t h e amino g r o u p  amino  More r e c e n t l y  has  been c o n s i d e r a b l y  have been t e s t e d expanded.  Some o f t h e s e compounds i n c l u d e  a l . , 1974;  Although  varying  degrees  extent  b e d by  activity  i t i s believed their  that  their  domoate, q u i s q u a l a t e , i b o t e n a t e ,  several  Curtis  Johnston 1975;  et a l . ,  1976;  c a r b o x y l groups  groups  1974;  of i n v e s t i g a t o r s  the s t r u c t u r e - a c t i v i t y  and W a t k i n s  c o n c l u s i o n s from  McCulloch Wheal,  compounds h a v e  some a r e  c a n be  differences  (ADCP)  McLennan and  Other molecules  These  this  enhanced p o t e n c i e s .  ( w h i c h i s known t o e x i s t  "boat" forms).  to extend  The  and  of molecular constraint,  as N - m e t h y l a s p a r t a t e .  attempt  series  glutamate  for excitatory  these v e r y potent e x c i t a t o r y  s u c h as ADCP  to which  prompted  1970;  Biscoe et a l . ,  1978).  and  Loss  (± )-cis_-1-amino -1, 3 - d i c a r b o x y c y c l o p e n t a n e  ( S h i n o z a k i and K o n i s h i ,  "chair"  carboxyl groups.  Many o f t h e s e newer m a t e r i a l s  c o n f e r s upon t h e m o l e c u l e s  inflexible  inac-  o f compounds r e s e m b l i n g  are c o n f o r m a t i o n a l l y r e s t r i c t e d ,  et  Compounds a r e  l e a d s to the f o r m a t i o n o f the  the l i s t  a s p a r t a t e which  k a i n a t e and  the  acids.  and  attribute  or both  of  N-methylaspartate)  or reduced a c t i v i t y .  t h e a - c a r b o x y l group  of i n h i b i t o r y  or a c y l a t i o n  remarkably i n both  are l i m i t e d separated,  i n structure (cited  i n the such  have  above) to  relationships  descri-  (1960)  these experiments, which  are  based  101 upon t h e  r e l a t i v e magnitudes of  required  to e l i c i t  to  s i m i l a r e f f e c t s must be  certain technical considerations  of which have a l r e a d y  observations  often necessary  t o use  since  be  there  istics  of  may  two  (proportion active known.  A  several  the  current  must be  constant  1964;  consideration relative the  for  1972;  Cox,  the  due  i n the  upon t h e  some  equal,  assumed, y e t  i t is  character-  transport  i f not  at  a l . , 1973;  numbers the  actually  l e a s t on  to e s t i m a t i o n s  1977).  This  dose A  final  of potency concerns  v a r i a b l e c a n n o t be  studies to  the  on  H e a d l e y and  known  absence of  Watkins,  1977).  (Balcar This  postsynaptic  only  transient,  surface  will  ces  of e x c i t a t o r y potency which are  the  perineuronal  space w i l l  locally  be  there  is  a f f e c t e d memband  Johnston,  a high  affinity  restricted  o b s e r v e d , whereas  not  p e r s i s t and  uptake  i s known b e c a u s e  of uptake processes p o s s e s s i n g  transmitter,  each  specific  d i f f e r e n c e i n areas of amino a c i d s  the  accurately  c e n t r a l n e u r o n e s and  p r e s e n c e or  and  theoreti-  a f f e c t the  Purves,  a  assemblies  drug d e l i v e r y the  due  Since  f o r accuracy,  Furthermore  f o r the v a r i o u s  presence  the  et  a considerable  rane obtains  I).  a r e a s o f n e u r o n a l membrane a f f e c t e d by  iontophoretic that  care  r e l a t i o n s h i p between time of e j e c t i o n  relevant  compounds.  processes,  for  required  assumed t o be  Clarke  surface  evidence  in  (Chapter  grounds, p i p e t t e n o n - l i n e a r i t i e s w i l l  (Curtis,  currents  a c t u a l l y engaged i n e x p e l l i n g  amount e j e c t e d must a l s o be cal  drawn w i t h  different pipette  differences  micropipettes.  of  ions)  are  ejecting  (Gent e t a l . , 1 9 7 4 ) ,  been d e a l t w i t h  l a r g e number o f  of  iontophoretic  r a p i d l y removed produce a  effects substanfrom  relatively  102  long and  lasting thereby  ston,  Cox  Despite  with  diffusing  (Curtis  these  difficulties,  1976).  measurements o f  r e s p e c t to time  approximately  (see a l s o Z i e g l g a n s b e r g e r issue f i r s t  a c i d s by  Curtis  raised  and  concluded  i t s receptor  and  current  i n the  early  e x p e r i m e n t s on  and  i t s receptor.  seemed most r e a s o n a b l e findings.  Curtis,  E v a n s , H e a d l e y , M a r t i n and Tateishi  react with  least  and  Umeda, 1975)  three molecules  Buu,  and M c C u l l o c h ,  with  1975;  the  appear  agonist  their  been accepted 1971;  Watkins,  the  These  i n view of  T h i s v i e w has  Davies  amino  t h e mechanism o f  most s u b s e q u e n t i n v e s t i g a t o r s (van G e l d e r ,  that at  employed  e t a l . , 1969).  colleagues concerned  Johnston,  1979)  of r e l e a s e  t h a t a t h r e e - p o i n t attachment o f the  structure-activity  Ichishita,  and  e x c i t a n t s , and  l i n e a r nature  of e j e c t i o n  r e a c t i o n between t h e amino a c i d  Gelder;  vitro  kainate (Hall et a l . ,  s m a l l d i f f e r e n c e s among t h e s e  both with  An  as when  a t known c o n c e n t r a t i o n s i n  a s p a r t a t e and  as w e l l t h e  vision  potency  B i s c o e , Evans, Headley, M a r t i n  indicated  Biscoe,  John-  F u r t h e r m o r e , measurements o f t r a n s p o r t num-  f o r glutamate,  h a v e shown o n l y  van  and  o b t a i n e d by more c o n v e n t i o n a l means, s u c h  e t a l . , 1961;  Watkins,  with  (Lodge, C u r t i s  i o n t o p h o r e t i c t e s t i n g s h a v e b e e n shown t o a g r e e w e l l  those  authors  a g r e a t e r a r e a o f membrane  e t a l . , 1977).  compounds a r e a d m i n i s t e r e d  bers  over  a c t i v a t i n g more r e c e p t o r s  1978;  through  effect,  Puil  the r e c e p t o r to produce i t s a c t i v a t i o n  and  1974; Shimizu,  additional  t o be  by  pro-  r e q u i r e d to (McLennan  103  and  Wheal,  1976a).  This  contrasts  with  the  i n v e r t e b r a t e s , where i t a p p e a r s t h a t up required  (Dudel,  shown t h a t One  two  final  discussed  by  1975), w h i l e Diamond and  m o l e c u l e s must r e a c t aspect  of  C u r t i s and  the  be  Their  conclusion  are  specific  synaptic  identical implying the  of  the  or  i t was  of  specific  the  these  question  the  processes;  suggesting  action  effects);  the  high  CNS  s u g g e s t e d a r o l e s o l e l y as  tances  finally  throughout  intermediates  As  the  rather  discussed  by  as  t o more r e c e n t c h e m i c a l and  McLennan  synaptic  of  the  (1975) and  i n our  of  and  transmitter  o f f s e t actions  need not  i n a c t i v a t i o n processes,  metabolic  C u r t i s and  Johnston  the  no  dicarboxylic  longer  understanding of  o f L and  subs-  excitation.  neuropharmacological properties equivalence  these  synaptic  acceptance of are  a  extra-synaptic  concentrations  transmitters  developments  In p a r t i c u l a r , the r a p i d on-  in  t h a n as m e d i a t o r s o f  (1974) t h e s e b a r r i e r s t o amino a c i d s  as  of:  enantiomorphs,  degradative  excitants, (such  L  c e r t a i n n o n s p e c i f i c i t y of and  of  substances  observations  D and  enzymatic  u n i v e r s a l i t y of a c t i o n of  the  s p i n a l neurones.  b a s e d upon t h e  time c o u r s e s o f a c t i o n of lack of  acids  cysteate. or r e l a t e d substances  u n l i k e l y that  was  are  (1973) h a v e  amino  co-workers r e l a t e d to  transmitters  the  Roper  excitatory transmitters that  s i x molecules  in teleosts.  actions  whether glutamate, a s p a r t a t e could  to  situation in  of  tenable the  these  due  neuro-  excitants.  D isomers i n producing  be  an  i n d i c a t i o n of  f o r as  already  lack  noted,  104  (see  above) d i f f u s i o n  away f r o m t h e  d e p e n d e n t u p t a k e a r e now  widely  isms f o r the  removal of  Universality  o f a c t i o n i s an  f u n c t i o n as  1972; cord 1978) is  thalamus  and  1974;  variability  Gent and  (Von  of neurones.  T h i s f i n d i n g has  both synaptic  and  point w i l l  The not  be  been r e s t r i c t e d  lated  restricted  these  acids  conformations.  i n an  separation  of  1976), s p i n a l  that  1972)  exist  and  o f amino a c i d e x c i t a n t s  and  the  their  The  there  f o r some g r o u p s  Johnson,  this  of  the  of  carboxyl  must  stimu-  structurally-  p o s i t i o n s of  compounds a r e known w i t h those  precise  aspartate  r e c e p t o r ( s ) has  relative  the a  t h e more f l e x i b l e an  pres-  greater trans-  amino a c i d  (that i s , with groups),  has  amino a c i d s ,  question  I f glutamate r e a c t s with  terminal  Morgan e t a l . ,  section.  extended conformat i o n the  a  shown  r e c e p t o r s may  of e x c i t a t o r y substances w i t h  candidates.  been  the h y p o t h e s i s  to the n a t u r a l l y o c c u r i n g  degree of c e r t a i n t y than are  receptor  of  in a later  umed a c t i v e g r o u p s o f t h e s e  mitter  Wolstencroft,  McLennan, 1970;  when i n t e r a c t i n g w i t h use  1979). •  argument a g a i n s t  c o n f i g u r a t i o n w h i c h g l u t a m a t e and  the  1978;  s i n c e i t has  prompted  as b r i e f l y m e n t i o n e d e a r l i e r ,  assume  mechan-  Baumgarten e t a l . , 1963)  examination of e f f e c t s  molecular  (Johnston,  extra-synaptic neuronal  discussed  acceptable  -  e t a l . , 1976b; H u t c h i n s o n e t a l . ,  i n the p o t e n c i e s  McLennan e t a l . , 1968;  a c t i v e Na  (McLennan e t a l . , 1968;  Biscoe  o l f a c t o r y bulb  t o be  insufficient  transmitter  Gent e t a l . , 1974; (Duggan,  held  transmitter  a synaptic  t h a t w i t h i n the  s y n a p s e and  then  maximal  aspartate  105  and  i t s potent  this  analogue, N-methylaspartate cannot r e a c t  receptor.  quisqualate  Equally,  and k a i n a t e  would r e a c t w i t h o f compounds traint  et  an a s p a r t a t e - p r e f e r r i n g r e c e p t o r .  which possess v a r y i n g  characteristics  (Johnston  by  o f t h e e x c i t a t o r y amino a c i d  e t a l . , 1976a; 1978; McLennan  1978) e s p e c i a l l y when s u c h s t u d i e s a r e a c c o m p a n i e d  antagonists  and  recep-  e t a l . , 1974; M c C u l l o c h e t a l . , 1974; B i s c o e  an e x a m i n a t i o n o f t h e s p e c i f i c i t y  Haefely,  res-  i n s t u d i e s o f t h e pharma-  a l . , 1975; S h i m i z u e t a l . , 1975; B i s c o e  Wheal,  The u s e  degrees o f molecular  H u t c h i n s o n e t a l . , 1978; M a c D o n a l d a n d N i s t r i , and  ibotenate,  c a n n o t assume a c o n f i g u r a t i o n w h i c h  has t h e r e f o r e proved o f v a l u e  cological tors  e a c h o f t h e amino a c i d s  with  (Shimizu,  1977; H a l l ,  McLennan,  Ichishita Hicks  o f a c t i o n o f amino a c i d  and Umeda, 1975; P o l e and  a n d McLennan,  1978; H i c k s ,  Hall  1978).  Antagonists. The p o s s i b l e r o l e transmission  o f t h e amino a c i d s i n mammalian  c o u l d n o t be p r o p e r l y  i n v e s t i g a t e d u n t i l the  development o f s u i t a b l e a n t a g o n i s t s . in  1972, w i t h  and  McLennan  antagonistic compound of  synaptic  This  development began  t h e d e s c r i p t i o n b y Haldeman, H u f f m a n , M a r s h a l l (1972) and Haldeman a n d McLennan a c t i o n s of glutamate  reduced  the synaptic  d i e t h y l e s t e r (GDEE).  and g l u t a m a t e i n d u c e d  c e n t r a l n e u r o n e s . The e f f e c t s  were u n a f f e c t e d  o f DLH, c y s t e a t e  or a f f e c t e d to a l e s s e r extent  glutamate responses,  but this  (1972) o f t h e This  excitations  or aspartate  t h a n were t h e  s e l e c t i v i t y was n o t o b s e r v e d  106  f o r a l l neurones.  When the e f f e c t s of GDEE were c o n t r a s t e d  w i t h those of methionine i t was  sulphoximine  (Curtis et a l . ,  1972),  found t h a t although the l a t t e r a n t a g o n i s t d i d b l o c k  amino a c i d responses,  t h i s e f f e c t was  less selective for glut-  amate than t h a t observed w i t h the d i e t h y l e s t e r .  I t soon  became c l e a r t h a t not a l l the substances proposed o n i s t s are e f f e c t i v e with equal s p e c i f i c i t y Wheal, 1976b). proposed  oxyaporphine  antag-  (McLennan and  Some of the other substances which have been  as a n t a g o n i s t s of glutamate  pyrrolid-2-one  as  i n c l u d e l-hydroxy-3-amino-  (HA966) (Davies and Watkins, 1972); 5,6-dimeth-  ( n u c i f e r i n e ) ('Duggan, Lodge, B i s c o e and  Headley,  1973) ; a w a t e r - s o l u b l e d e r i v a t i v e of A - 1 - t e t r a h y d r o c a n n a b i n o l (SP111) (Segal, 1978); 2-amino-4-phosphonobutyrate (2A4P) (Cull-Candy, Donnellan, James and Lunt, ester  1976); k a i n a t e d i e t h y l -  (KDEE) (Padjen, 1976); a,e-diaminopimelate  (Biscoe,  Davies, Dray, Evans, F r a n c i s , M a r t i n and Watkins, 1977) D-a-aminoadipate all  (aAA)  and  ( H a l l , McLennan and Wheal, 1977).  these a n t a g o n i s t s , the most u s e f u l appear to be aAA,  and HA966 (McLennan and Wheal, 1976b; B i s c o e e t a l . ,  Of GDEE  1977;  Hicks et a l . , 1978) , an'd t h i s matter w i l l be d e a l t w i t h more e x t e n s i v e l y i n the d i s c u s s i o n s e c t i o n of t h i s  4) The  chapter.  Experimental R a t i o n a l e s experiments  to be d e s c r i b e d were performed  o b j e c t i v e s i n mind, and although each addresses  with various a somewhat  d i f f e r e n t problem, the o v e r a l l theme which serves to u n i f y each i n v e s t i g a t i o n concerns  the e l u c i d a t i o n of the a c t i o n s of  107  the e x c i t a t o r y the r a t i o n a l e i/  amino a c i d s .  f o r conducting the present  A n t a g o n i s t s : GDEE  first  described  GDEE a g a i n s t  The f o l l o w i n g  a relatively  the e x c i t a t i o n  of effect.  some o f w h i c h tested  specific  o f c e n t r a l neurones  berg,  1973; A l t m a n n ,  1976),  of a s s e s s i n g tudes  C l a r k e and Straughan,  1973; C u r t i s  Pickelmann  1973; C u r t i s  1973; D o s t r o v s k y  c u r r e n t magni-  1977).  t h e r e h a v e b e e n many c o n f i r m a findings  that  GDEE i s an  i n d u c e d and s y n a p t i c a l l y  e t a l . , 1972, 1973; D a v i e s and a n d Pomeranz, 1973; S t o n e , 1973; 1974; Wheal  1974; 1975; McLennan a n d Wheal, 1976; S e g a l , 1976;  Spencer  e t a l . , 1976a,b; H i c k s a n d McLennan,  Miller,  1979).although  glutamate  1972;  and Padjen,  1976; L o w a g i e and G e r s c h e n f e l d , 1974; McLennan, and K e r k u t ,  and S t e i n -  et a l . ,  19 77; M a c D o n a l d , N i s t r i  e f f e c t i v e b l o c k e r both o f glutamate  Watkins,  different  1972; D a v i e s and  and c h o i c e o f e j e c t i n g  (Curtis  1976;  i n t e c h n i q u e s u c h a s i n methods  these d i s c l a i m e r s ,  excitations  and P h i l l i s ,  et al.,  Ten Bruggencate,  t i o n s ^ o f Haldeman and McLennan's  evoked  by glutamate,  of factors,  t e s t s b e i n g made f r o m  or d i f f e r e n c e s antagonism  1975; B a i l e y  ( Z i e g l g a n s b e r g e r and P u i l ,  Despite  action of  agreement r e g a r d i n g i t s s e l e c t -  and C o n s t a n t ! ,  regions (Tebecis,  Watkins,  (1972)  s p e c i e s d i f f e r e n c e s between t h e a n i m a l s  Shank and Freeman, 1976); brain  antagonistic  T h i s may be due t o a v a r i e t y  include  (Nistri  experiments.  A l t h o u g h Haldeman a n d McLennan  t h e r e has n o t been u n i v e r s a l ivity  summaries p r e s e n t  as opposed  i t s specificity  1979; Wheal a n d  f o r the e f f e c t s o f  t o t h o s e o f a s p a r t a t e , DLH o r ACh  still  108  r e m a i n e d a m a t t e r o f some c o n t r o v e r s y . idered apropriate  t o examine i n a c a r e f u l l y  ( s e e C h a p t e r s I and neuronal excitants tibilities  group  Antagonists:  o f GDEE a g a i n s t  and  selectively to perhaps  against  that  aAA  r a c a e m i c m i x t u r e was  evidence,  i t was  a-aminoadipate  inactive,  Hall  and  the v a l i d i t y  further  a marked s e l e c t i v i t y  of this  Evans  and H a l l ,  ferent  Biscoe,  1978).  groups  seemed t o be  Evans  and W a t k i n s ,  GDEE and aAA  B a s e d upon  DLH  et a l . ,  1978;  Hall  and NMA  1977;  o f amino a c i d s , a logical  appeared  et a l . ,  action.  con-  the  (Biscoe,  Davies  Watkins,  McLennan  indicated  as a n t a g o n i s t s  a comparison  course of  against  1978;  acids.  described  D a v i e s and  In view o f the r e s u l t s which  both possessed a c t i v i t y  this  o f t h e amino  o f aAA  spon-  the D isomer o f  d e d u c t i o n , and w h i c h of e f f e c t  the  suppressed the  a number o f c o m m u n i c a t i o n s  e x c i t a t i o n s produced b y aspartate,  1978;  in fact  amino  (1977) n o t i c e d  excitatory,  s h o u l d a c t as an a n t a g o n i s t  firming  1977;  weakly  a number  occurring  p r o p o s e d t h a t when i s o l a t e d ,  thereafter,  al.,  et a l . ,  o f the t h a l a m i c neurones.  Shortly  et  or a  . During the p r o c e s s of s c r e e n i n g  a l t h o u g h L - a - a m i n o a d i p a t e was  activity  to  other,investigators.  f o r potency evaluations,  taneous  suscep-  hoped one  of  a c c o u n t f o r some o f t h e  o f a n a l o g u e s and homologues o f t h e n a t u r a l l y acids  cons-  a range  relative  I n t h i s manner, i t was  GDEE a c t e d  of  therefore  c o n t r o l l e d manner  i n order to e v a l u a t e t h e i r  of the e x c i t a n t s ,  anomalous r e s u l t s  ii/  I I ) the e f f e c t s  to antagonism.  e s t a b l i s h whether  I t was  of t h e i r  that  for difactions  109  iii/  A n t a g o n i s t s : KDEE, 2A3P, 2A4P, BAA  earlier as  that  antagonists  amate. the  a variety  to that  earlier  depolarizing  of f e l i n e  of a l o c a l  n o t . t o be u s e f u l report  effects  and o f t h e a c t i o n  e t a l . (1977) r e p o r t e d  spontaneous f i r i n g  similar  anaesthetic.  as an a n t a g o n i s t ,  an a n t a g o n i s t - l i k e  responses of frog  ation  excitatory  although Padjen effect  However, 2A4P and a homologue, (2A3P), were r e p o r t e d effects neurones  (Watkins, C u r t i s  amplitude  1976).  lumbar  1977), and W h i t e  the  spinal et a l . ,  (1978) f o u n d t h a t  2A4P  reduced the amplitude o f the field  response of granule  2A4P d i d n o t a p p e a r t o a l t e r  or form o f the f i e l d  stimulation  competitive  i n antagonizing  on f e l i n e  and B r a n d ,  i n the dentate gyrus.  depolariz-  2-amino-3-phosphonoproprionate  excitants  and r e v e r s i b l y  a  (Cull-Candy et a l . ,  p e r f o r a n t p a t h (PP) e v o k e d d e n d r i t i c cells  o f KDEE upon t h e  o b s e r v e d upon t h e m i n i a t u r e  (1977) and D u n w i d d i e , M a d i s o n and L y n c h substantially  (1976) d i d  2-amino-4-phosphonobutyrate  t o be i n e f f e c t i v e  o f any amino a c i d  said  motoneurones.  e f f e c t was  junction potentials  glut-  I t t h e r e f o r e was  i n a manner r e s e m b l i n g  a n t a g o n i s m , a l t h o u g h no  of  t h a t KDEE d e p r e s s e s  found to reduce the glutamate induced  o f l o c u s t muscle  to act  s p i n a l n e u r o n e s i n a manner  Another analogue of glutamate, (2A4P), was  mentioned  o f compounds h a v e b e e n r e p o r t e d  of synaptic  MacDonald  I t was  o f the granule c e l l  t h e r e an o b s e r v a b l e a l t e r a t i o n  r e s p o n s e s e v o k e d by axons  (mossy  antidromic  fibres),  i n the p r e s y n a p t i c  the  nor  was  f i b r e poten-  t i a l s , t h u s r u l i n g o u t t h e p o s s i b i l i t y o f an a c t i o n o f r e d u c i n g  110  the  excitability  of the granule  cells  through n o n - s p e c i f i c  effects.  3AA equal  to act  l e n g t h b e t w e e n t h e amino and c o - c a r b o x y l  to that o f glutamate, while  carboxyl oxyl  has a c h a i n  separation  distance  equivalent  of aspartate.  that of the antagonist, a s an a n t a g o n i s t ,  on n e u r o n a l  I t s close structural aAA a l s o s u g g e s t e d  similarity  that i t might  and so an e x a m i n a t i o n o f i t s e f f e c t s  In view o f r e p o r t s  the  a n amino t o a — -  t o t h a t o f t h e amino - w-carb-  f i r i n g was c o n s i d e r e d  inconsistent,  possessing  of interest.  i n the l i t e r a t u r e ,  some o f w h i c h a r e  a s e r i e s o f e x p e r i m e n t s was c o n d u c t e d t o t e s t  a c t i o n s o f KDEE, 3AA and t h e p h o s p h o n i c a c i d d e r i v a t i v e s  upon t h e e x c i t a t i o n o f t h a l a m i c  neurones  induced  by a v a r i e t y  o f amino a c i d s .  iv/ the  Agonists:  ADCP a n d 3AG  excitatory activities  Q u a n t i t a t i v e measurements o f  of conformational^  restricted  amino a c i d s upon c e n t r a l n e u r o n e s h a v e p r o v i d e d information  t o w a r d s an u n d e r s t a n d i n g  amino a c i d - r e c e p t o r  Wheal  excitant rigid  (1978) f o u n d t h a t ADCP a c t s i n the r a t thalamus.  s t r u c t u r e which allows  groups. aspartate  o f the nature  o f the  r e a c t i o n a d d r e s s e d b y C u r t i s and W a t k i n s  (1960; J o h n s t o n e t a l . , 1974; B i s c o e and  some u s e f u l  I f the intercharge a r e compared,  e t a l . , 1976a). as a p o w e r f u l  McLennan neuronal  T h i s m o l e c u l e has a n e a r l y r o t a t i o n of only  the carboxyl  distances which occur  i t i s clear that  they  i n ADCP a n d  are u n l i k e l y to  Ill  s h a r e < t h e same a c t i v e s i t e s a three p o i n t attachment. possible possess Hall  o f a n amino a c i d r e c e p t o r , a s s u m i n g  By c o n t r a s t ,  a very  good f i t i s  f o r ADCP a n d g l u t a m a t e upon a r e c e p t o r w h i c h w o u l d a p p r o p r i a t e l y spaced a c t i v e s i t e s  e t a l . , 1979).  macologically  (Buu e t a l . , 1976;  I f ADCP i s i n f a c t a v e r y  specific  agonist  potent  and p h a r -  o f g l u t a m a t e due t o i t s i n t e r -  c a r b o x y l s e p a r a t i o n , b u t does n o t m i m i c t h e a c t i o n o f a s p a r t a t e , t h e n i t w o u l d be e x p e c t e d glutamate i n response and  t o b e h a v e i n a. s i m i l a r  to antagonists.  I f the  fashion to  effects  one o f t h e d i c a r b o x y l i c t r a n s m i t t e r c a n d i d a t e s  separable  i n terms o f t h e i r  antagonists, support  different  then t h i s would p r o v i d e  o f the hypothesis  o f ADCP  were t o be  susceptibilities  to the  a d d i t i o n a l evidence i n  o f two r e c e p t o r s  f o r t h e amino a c i d s .  A n o t h e r compound w h i c h m i g h t be u s e f u l i n d e t e r m i n i n g nature  o f amino a c i d r e c e p t o r s  tested  originally  and  as g l u t a m a t e  favourable"  This  s u b s t a n c e was  f o r c e n t r a l a c t i v i t y b y C u r t i s e t a l . , (1961)  C u r t i s and Watkins  active  i s 3AG.  (1960) a n d was f o u n d  i n e x c i t a t o r y potency,  situation  the 3-carbon, r a t h e r  is  interesting  i n this  t o be n e a r l y a s despite  the "less  o f t h e amino g r o u p w h i c h i s p o s i t i o n e d  on  t h a n on t h e a - c a r b o n regard  amino t o w - c a r b o x y l s p a c i n g  that  (page 9 9 ) . I t  3AG p o s s e s s e s  as does a s p a r t a t e ,  amate; t h e r e f o r e t h e p h a r m a c o l o g i c a l  i f t h e amino t o w - c a r b o x y l d i s t a n c e  determinant o f s p e c i f i c i t y .  t h e same  but not glut-  properties of this  m o l e c u l e might be e x p e c t e d t o mimic a s p a r t a t e glutamate,  the  but not i s an  important  112  v/ A g o n i s t s : ring was  Kainic acid  Kainic acid is a naturally  compound f o u n d i n t h e m a r i n e a l g a D i g e n e a s i m p l e x , first  Takemoto, potent  isolated 1978).  and  by  Takemoto  anthelmintic  e x c i t a n t of neurones  Nistri,  (Shinozaki  Biscoe  ( f o r review,  p r o p e r t i e s and and  structure should  receptors  cological  Konishi,  (Hall  react only with  e t a l . , 1979),  analysis of  Furthermore,  observations  of Shinozaki  the  Shinozaki  and  Shibuya,  amino a c i d  useful  and  1974), t h a t k a i n a t e  two  suggested a form of  e x c i t a n t s a t the  does p r o d u c e n e u r o n a l  glutamate r e c e p t o r s , are  Konishi  of k a i n i c a c i d  (see  then i t i s reasonable antagonist  discussion)  a c t i o n upon p o s t s y n a p t i c 1974;  McGeer and  H e r n d o n and kainic  1977)  a c i d i n these  that  If kainic  interaction  of  this  a  effects  direct  (Olney  F r a n k h u y z e n and  et a l . ,  Mulder,  a f u r t h e r i n c e n t i v e to  s t u d i e s i n v o l v i n g the  with  receptor.  the n e u r o t o x i c  a r e m e d i a t e d by  provided  crayfish  to suppose t h a t i t s  glutamate receptors  McGeer, 1976;  Coyle,  of  the  c o - o p e r a t i v i t y between  e x c i t a t i o n v i a an  f r e q u e n t l y s t a t e d assumption  (1970)  potentiates  amino a c i d r e c e p t o r .  s u s c e p t i b l e t o an  infor-  receptor.  g l u t a m a t e r e s p o n s e s o f r a t c o r t i c a l n e u r o n e s and muscle f i b r e s ,  rigid  glutamate-prefer-  i t s a c t i o n might p r o v i d e the  The  and  s u g g e s t e d t h a t a pharma-  s t r u c t u r e of  effects  1970;  e t a l . , 1976a; C o n s t a n t i  m a t i o n about the  acid  see  is a  h i g h p o t e n c y o f k a i n i c a c i d , whose r e l a t i v e l y  molecular  these  and  1976).  The  ring  identified  I t has  J o h n s t o n e t a l . , 1974;  and  occur-  antagonists.  1977; include  113  b) i/  Results E x c i t a n t mechanisms  e x c i t e d by  the  N e u r o n e s o f t h e VB  iontophoretic a p p l i c a t i o n of  D-glutamate, L - a s p a r t a t e , ADCP and  ACh,  among t h e s e action  although  typical  of that f i r s t  As  for  s p i n a l Renshaw c e l l s ,  equally Figure  to the  e x c i t a t i o n s induced  an  intermediate  glutamate or a s p a r t a t e , appears not  t o ,be  equivalent  due  and  inactivation,  but  t h i s may  actions of  t h a t no be  these  potencies.  onset  NMA  had  elicited this  applied  illustrated  smaller  NMA  As  o r ADCP.  currents  in  lengthy  materials  (Cox  of  to  materials,  that used f o r  subsequent  spike  latency of  onset  uptake process  a f a c t o r i n the  excited  effect  required  to e q u a l  i s true f o r kainate  cellular  ACh  The  t h e more p o t e n t  e x c i t a t i o n and  a continued  and  time, between t h a t  ejecting currents  with  e t a l . , 1979).  is likely  high  and  study  f i g u r e t h a t DLH  excitations with  increasing their  and  glutamate  to those  by ADCP, as  kainate,  to the  L-glutamate y i e l d e d intense  it  kainate  i n the p r e s e n t  i s c l e a r a l s o from t h i s  w i t h what was  (Hall  of  13.  It  elicit  C u r t i s e t a l . , (1960)  i n comparison  and  of  H u t c h i n s o n e t a l . , (1978)  e x c i t a t i o n s by  aspartate  DLH,  a rapid termination  stereoisomers  n o t e d e a r l i e r by  p r o t r a c t e d time courses  g l u t a m a t e and  and  NMA,  of e x c i t a t i o n d i f f e r e d  d e s c r i b e d by  a c t i o n s o f the  aspartate.  for  onset  L-glutamate,  kainate,  time courses  Fast  and  by  D-aspartate,  the  compounds.  char.acterizeld the  very  t h a l a m u s were  and  NMA  f o r ADCP  slow time courses  in  however, exists, the  e t a l . , 1 9 7 7 ) . a s w e l l as  the  114  Figure  13.  Typical  r e c o r d i n g s o f the r a t e  t h a l a m i c neurones to the e x c i t a n t s s e r i e s o f experiments. a rapid  onset  increase.  i n onset  of e x c i t a t i o n  Glutamate and  o f e x c i t a t i o n w h i l e ACh  more m o d e r a t e r a t e much s l o w e r  and  following  The  and  of kainate.  text).  of  present induced  elicited  a  compounds were  a prolonged  the c e s s a t i o n of e j e c t i o n ;  currents enables  (see  DLH  remaining  decrease  this  A comparison  an e s t i m a t e o f p o t e n c y  although other c o n s i d e r a t i o n s are c e r t a i n estimates  in' the  aspartate both  e x e r t e d as w e l l ,  e s p e c i a l l y marked i n the case ejecting  tested  of response  was of  the  t o be made,  to i n f l u e n c e  such  116  ii/  E f f e c t i v e Antagonists  excitatory  actions of  investigation of  their  the  decreasing The  aAA  the o v e r a l l r e s u l t s  order  of  of t h e i r  f i g u r e s i n the  two  the  the  orders  to each a n t a g o n i s t .  Table  c e l l s w h i c h were  e x c i t a t o r y compounds, i n to each  antagonist.  t h e mean i o n t o p h o r e t i c  antagonists  sufficiently  of each of the  to  excitants, rela-  o f t h e most s u s c e p t i b l e .  These f i g u r e s a l s o i l l u s t r a t e  t h a t when c o m p a r i s o n s  the  c u r r e n t s r e q u i r e d to d i f f e r e n t i a t e  p r o d u c e d by  to the  antagonists,  dose o f a n t a g o n i s t  absolute but  d u c e d by  aAA  and  was  used to reduce  the  same t i m e as  a relatively selective  administered;  a relative GDEE. the  the  higher  the r a n k i n g  The  specificity one  effect  before  of  antagonism o f the  the a s p a r t a t e  D-aspartate  was  that i s , there o f the  exception  on L - a s p a r t a t e  dose o f GDEE was one  i t i s appar-  is chiefly  was  antagonism  at  m o n i t o r e d ; here  respect  e x c i t a t i o n s was t o be  pro-  D-aspartate  Thus a c o n s i d e r a b l e  recognized  i s not  r e q u i r e d to produce  excitant with  induced  related  o c c u r r e d when GDEE  e x c i t a t i o n s p r o d u c e d by  susceptible stereoisomer.  of both  v  are  the e x c i t a t i o n s  a d j o i n i n g compounds w i t h i n e a c h r a n k ,  that f o r both  less  parallel  to t h a t needed f o r d e p r e s s i o n  made o f  an  the  to e s t a b l i s h ranking  from those  columns a r e  demonstrate a s e l e c t i v e b l o c k  ent  through the  susceptibility  c u r r e n t s r e q u i r e d to e x p e l  tive  and  susceptibilities  used to o b t a i n a ranking  p o s s i b l e to separate  amino a c i d s  o f GDEE and  relative  4 summarizes  I t was  to  a the  reduction  required  t h e more s u s c e p t i b l e .  117  TABLE 4adipic  '.Relative'  iontophoretic  a c i d and glutamate  antagonize  "doses"  diethylester  t h e e f f e c t s o f each  of  .a-amino-  required  o f a range  to  of neuronal  excitants.-  a/AA  GDEE  (44)^  L-GLUT  1. 0  (75)  + 0 .1  (73)  D-ASP  1. 6 + 0 .1  (10)  +  1 .0  NMA  DLH/ADCP 1 .6 D-GLUT  2 .6  0 .1  (26)  L-ASP  1. 2 + 0 .2  (39)  L-ASP  3 .1 + 0 .2  (26)  D-GLUT  1. 3 + 0 .1  (16)  D-ASP  3 .9  0 .2  : (8)  ACh  1. 4 + 0 .2  (22)  L-GLUT  4 .6  + 0 .3  (16)  DLH  1. 5 + 0 .1  (15)  KAIN  5 .7 + 0 .2  (4)  KAIN  1. 8 + 0 .2  (10)  ADCP/NMA  ACh  a  Current expelling that  required  pound i n e a c h  antagonist i s expressed  t o antagonize rank.  Ranking  relative to  t h e most s u s c e p t i b l e  i s i n order of decreasing  susceptibility. b  Values  a r e mean ± S.E., number o f c e l l s i  parentheses.  com-  tested i n  118  This r e s u l t  is illustrated  i n F i g u r e 14, a n d F i g u r e s 15 a n d 16  present  some examples o f t y p i c a l  compile  the data.  A summary o f t h e r e s u l t s  r e s p o n s e s w h i c h were u s e d t o  used  to c o n s t r u c t the o r d e r i n g  s e q u e n c e o f t h e e x c i t a n t s when GDEE was appears  i n T a b l e 5.  sent r e l a t i v e another,  The r e s u l t s  antagonism  aAA was  for a full  effect  effectiveness  on a t o t a l  o f 187 VB  a t e were c l e a r l y and  i t was  to occur.  Thus, e x c i t a t i o n s preferentially  elicited  Glutamate  L-glutamate  by L - g l u t a m a t e  (7 o f 10 c e l l s ) ,  were  antagonized  As w o u l d be  t o t h o s e o f L - a s p a r t a t e (7 o f 12 c e l l s ) ,  extent  cells, than  '*'-',  those of  expected,  i n d u c e d e x c i t a t i o n s were a n t a g o n i z e d  others L-glutamate  acids.  a n d t h o s e o f L - a s p a r t a t e more  (8 o f 12 c e l l s ) .  exhibiting parallel  and a s p a r t -  the i s o m e r i c forms o f t h e s e  t o t h o s e o f D - a s p a r t a t e on 7 o f 3  than D-glutamate  examined  a n t a g o n i z e d o f t h e compounds,  D - a s p a r t a t e effects':.were d e p r e s s e d more r e a d i l y L-aspartate  from  pattern.  t h a l a m i c neurones,  to rank  several  Times f o r r e c o v e r y  o f GDEE as an a n t a g o n i s t was  t h e most r e a d i l y  possible  1 m i n . o f the  w h e r e a s GDEE o f t e n r e q u i r e d  the a n t a g o n i s t s f o l l o w e d a s i m i l a r The  T a b l e repre-  t h e o b s e r v a t i o n s p r e s e n t e d by  i n most c a s e s e f f e c t i v e w i t h i n  commencement o f e j e c t i o n , minutes  contained i n this  o f one e x c i t a n t w i t h r e s p e c t t o  and t h e s e d a t a e x t e n d  T a b l e 4.  t h e a n t a g o n i s t employed  preferentially  the remaining 5  r e d u c t i o n s o f e x c i t a t i o n , w h i l e on 8 o f 9 e f f e c t s were a n t a g o n i z e d  t h a n were t h o s e o f D - g l u t a m a t e ,  to a greater  w i t h the remaining  119  Figure  14.  artate.  Effect  o f GDEE a g a i n s t  Although D-aspartate  two i s o m e r s ,  this  example, following  t h e more  differential effect  observed u n t i l a considerable achieved.  was  the stereoisomers  GDEE was  s u s c e p t i b l e o f the  c o u l d u s u a l l y n o t be  reduction of both responses  applied with  a current  and t h e i l l u s t r a t e d r e s u l t t h e comencement  o f asp-  was  was  o f 35 nA i n t h i s  obtained  6.0 m i n .  o f e j e c t i o n o f the antagonist.  GDEE  121  Table  5.  Summary o f e f f e c t s  against pairs  of neuronal  column i n d i c a t e the  excitants.  t h e number o f c e l l s  compound l i s t e d  the  Therefore,  cells  the  angle  are  ranking  the  cells  the  excitants;  column was  lower  those  which d i d not  left  by  selectively  listed  at  the  triangle  are  the  i n the upper r i g h t  respond  according  tri-  to  the  scheme.  (5t-k- row)  were compared,  antagonism of L - a s p a r t a t e r e d u c t i o n of both 5^  i n each  antagonism f o l l o w e d  Thus f o r example, when L - a s p a r t a t e ACh  diethylester  figures  excitants  i n the  selective  acid  at which e x c i t a t i o n  other  figures  a t which the  rank o r d e r i n g o f the  The  a t the head o f the  b l o c k e d when compared w i t h left*  of L-glutamic  column and  effects  excitants.  3 * row) rc  5 cells  had  showed  and  Only t h e ACh  (third  one  three cell  effects  column)  and  preferential showed e q u i v a l e n t (intersection  of  preferentially  antagonized. On  no  Figures the  o c c a s i o n c o u l d NMA  i n parentheses  antagonist  d i d not  o r ADCP be  represent  antagonized  by  t h e number o f c e l l s  d i f f e r e n t i a t e between t h e  two  GDEE.  at which excitants.  L-GLUT  L-GLUT  D-ASP  L-ASP  0(2)  D-GLUT  ACh  DLH  KAIN  0(5)  0(1)  0  0  0(3)  0(3)  NT  NT  NT  2(2)  1(3)  0(1)  D-ASP  7(2)  L-ASP  7(5)  7(3)  D-GLUT  8(1)  NT  8(2)  5(3)  ACh  9  NT  DLH  6  NT  6  NT  8(2)  NT  NT  8  6(1)  KAIN  12(3)  NMA  0  0  NT  NT  NT  0  • NT  0  0  NT  1  NT  NT  NT  NT  KD  NT  0  0  4(2)  6(1)  ADCP  1(1)  " 3  4  ADCP  7  NT  6  NT  NT  6(1)  NMA  8  NT  6  NT  NT  NT  NT  5  5.  NT  123  cell  showing e q u a l  The  next  reductions.  compound  i n o r d e r w h i c h was most e a s i l y  o n i z e d b y GDEE was ACh.  When s e l e c t i v e  L - g l u t a m a t e and L - a s p a r t a t e in preference pectively,  to those  a n t a g o n i s m was  e x c i t a t i o n s were  observed  reduced  o f ACh on 9 o f 9 a n d 5 o f 9 c e l l s ,  and a t 3 o t h e r  a t i o n s were r e d u c e d  induced  antag-  cells  L-aspartate  a n d ACh  t o an e q u i v a l e n t degree.  res-  excit-  ACh was  ranked  below D-glutamate  s i n c e on 6 o f 7 n e u r o n e s , D - g l u t a m a t e  effects  were more r e a d i l y  antagonized.  with  k a i n a t e were made, ACh was 8 cells  (with 1 c e l l  cells  (with 2 c e l l s  tible  than  The  When c o m p a r i s o n s o f ACh  preferentially  showing e q u a l showing e q u a l  antagonized  r e d u c t i o n ) ; a t 8 o f 14 e f f e c t s ) ACh was more  remaining  active with very  amino a c i d  a g o n i s t s w h i c h were t e s t e d  antagonized.  low e j e c t i n g  T h e s e compounds were  GDEE a p p l i c a t i o n .  O n l y when h i g h  nA) o f GDEE were u s e d was  amplitude  effect during  whenever t h i s  Despite the  compounds  were more  the i n i t i a l  ejecting  currents  antagonism observed;  o f such h i g h  with often  c u r r e n t s , and t h e i r e x c i t a t o r y  e f f e c t s were f r e q u e n t l y p o t e n t i a t e d d u r i n g  more u s u a l  suscep-  DLH.  GDEE were o n l y r a r e l y  of  at 6 of  d o s e s was  period (40-60  however, t h e  a reduction i n spike  the p e r i o d o f e j e c t i o n o f the e x c i t a n t , and  occurred  these  the t r i a l  was abandoned.  p r o b l e m s some i n d i c a t i o n o f r a n k i n g  c o u l d be o b t a i n e d :  thus  s u s c e p t i b l e t h a n were t h o s e  DLH e l i c i t e d of kainate  among  effects  on 4 o f 7 c e l l s  124  Figure  15.  orders  o f amino a c i d e x c i t a n t s when GDEE was t h e a n t a g o n i s t  employed. required effects  Figures  i n t h e c e n t r a l column r e p r e s e n t  t o e j e c t GDEE i n o r d e r against  records, lows:  T y p i c a l responses used i n c o n s t r u c t i n g the ranking  the i l l u s t r a t e d  the e j e c t i n g currents  to e s t a b l i s h  differential  pairs of agonists.  For these  o f t h e e x c i t a n t s were as  L - g l u t a m a t e 41 nA, L - a s p a r t a t e  50 nA;  L-aspartate  75 nA;  35 nA, D - g l u t a m a t e  D - g l u t a m a t e 100 nA, D L - h o m o c y s t e a t e , kainate  the currents  13 nA, N - m e t h y l a s p a r t a t e ,  18 nA;  15 nA.  fol-  125  199 » n  126  while excitations by  GDEE when k a i n a t e  cells,  On no o c c a s i o n -  (5 o f 5  was t h e r e o b s e r v e d  a n t a g o n i s m b y GDEE o f t h e e x c i t a t i o n s  elicited  even  by ADCP  NMA. The  a  i n d u c e d e x c i t a t i o n s were r e d u c e d  i n each c a s e ) .  partial or  p r o d u c e d b y ADCP a n d NMA were n o t a f f e c t e d  total  effectiveness  o f 197 VB t h a l a m i c n e u r o n e s ,  those also Table  o f aAA a s an a n t a g o n i s t was e x a m i n e d on  6.  blocking  t e s t e d w i t h GDEE. Ejecting  currents  the e x c i t a t o r y  These r e s u l t s a r e p r e s e n t e d i n o f aAA w h i c h were e f f e c t i v e i n  actions  were l o w e r t h a n t h o s e r e q u i r e d that  those e x c i t a n t s  some o f w h i c h were among  o f most o f t h e a g e n t s  tested  f o r GDEE, and i t was a l s o  w h i c h were l e a s t s u s c e p t i b l e  o n i s m b y GDEE were most s u s c e p t i b l e  to the a c t i o n  with  elicited  the exception o f kainate.  NMA  to antago f aAA,  e f f e c t s were  a n t a g o n i z e d b y aAA more r e a d i l y t h a n t h o s e o f any o t h e r tant: that  thus  i t s a c t i o n was r e d u c e d  o f k a i n a t e on 7 o f 7 c e l l s ,  cells,  were compared d i r e c t l y , be d e t e c t e d  than  a n d o f ADCP on 7 o f 8 whose e f f e c t s were  however a t 12 c e l l s where t h e s e two  no c o n s i s t e n t l y  (at 5 c e l l s  selective  antagonism  t h e a n t a g o n i s m was e q u a l ,  4 and3 showed ADCP a n d DLH, r e s p e c t i v e l y tible).  extent  exci-  o f L - g l u t a m a t e on 7 o f 7  ADCP a n d DLH were t h e two e x c i t a n t s  n e x t most r e a d i l y b l o c k e d ;  could  to a greater  o f L - a s p a r t a t e on 6 o f 6 c e l l s  cells.  found  while  as t h e more s u s c e p -  127  Table  6.  Summary of e f f e c t s of a-aminoadipate (the separ-  ated D isomer) a g a i n s t p a i r s o f neuronal e x c i t a n t s . The Table f o l l o w s the same p a t t e r n as that o f Table L-aspartate  5; f o r example when  and ADCP were compared, 9 c e l l s were t e s t e d , 6 o f  which showed p r e f e r e n t i a l  antagonism o f ADCP e f f e c t s , 2 showed  e q u i v a l e n t r e d u c t i o n o f both e x c i t a n t s and one c e l l s e l e c t i v e r e d u c t i o n of the L - a s p a r t a t e On no o c c a s i o n  effects.  could ACh be antagonized by aAA.  showed a  NMA  NMA  DLH  ADCP  D-GLUT  L-ASP  0(1)  0(1)  NT  0  3(5)  NT  0(1)  DLH  6(1)  ADCP  8(1)  4(5)  NT  NT  4(1)  8(1)  6(2)  8(4)  D-GLUT  .  L-ASP  6  D-ASP  ' NT  NT  NT  NT  L-GLUT  7  10  8  6  KAIN  7  8  6  ACh  8  6 -  NT  D-ASP  L-GLUT  KAIN  ACh  0  NT  0  0  0(1)  NT  0  0  1(2)  NT  0  0  NT  1(4)  NT  0  NT  0  NT  0  NT  NT  0  0  0  0(2)  1  6  11(2)  7(1)  NT  NT  NT  10  7  7  NT  6  •  0  0  4  129  Glutamate of  a n d a s p a r t a t e f o l l o w DLH a n d ADCP i n t h e o r d e r  r a n k i n g e s t a b l i s h e d by aAA  cells,  ADCP was more  D-glutamate.  ( T a b l e s 4 and 6 ) .  susceptible  Selectivity  to antagonism  of antagonism  mers o f t h e s e a c i d s was more r e a d i l y onist  t h a n w i t h GDEE.  On 4 o f 5  than  was  among t h e s t e r e o i s o -  apparent w i t h t h i s  D - g l u t a m a t e e f f e c t s were f i r s t  antagon-  i z e d when compared w i t h t h o s e o f L - a s p a r t a t e ' ( 8 o f 13 4 reduced blocked  D-aspartate  i n t u r n was  L-glutamate  (7 o f 8  Other which  a more  (6 o f 6 c e l l s )  susceptible  compound  o f amino a c i d  results  of  10 c e l l s )  of  than  was  a n d ADCP  (8 o f 8 c e l l s )  by L - g l u t a m a t e ,  from  d e r i v e and w h i c h s e r v e , i n p a r t ,  c o n f i r m t h e above o r d e r : e x c i t a t i o n s p r o d u c e d  a n d ADCP  while  a g o n i s t s were made,  to  cells)  readily  cells).  comparisons  the f o l l o w i n g  produced  cells,  t o g e t h e r ) ; L - a s p a r t a t e e x c i t a t i o n s were more  than were t h o s e o f D - a s p a r t a t e  antag-  b y DLH  (10  than  those  r e d u c e d more  and t h o s e e l i c i t e d  by DLH  (8 o f 9  (6 o f 9 c e l l s ) were r e d u c e d more t h a n  those  L-aspartate.  Of aAA,  t h e two compounds  excitations  preference  caused  r e m a i n i n g w h i c h were t e s t e d  by k a i n a t e were r e d u c e d  to those e l i c i t e d  by ACh.  on 4 c e l l s i n  A l l o f the other  a n t s o f t h e s e r i e s w h i c h were t e s t e d were f o u n d more tible  to antagonism  (8 o f 8 c e l l s ) ,  t h a n k a i n a t e : ADCP  L-glutamate  (10 o f 10 c e l l s ) ;  o f aAA: DLH  (6 o f 6 c e l l s ) ,  excit-  suscep-  (6 o f 6 c e l l s ) ,  DLH  and t h e f o l l o w i n g  compounds when compared w i t h ACh were more r e a d i l y the presence  with  reduced i n  L - a s p a r t a t e (7 o f 7  130  Figure  16.  orders  o f amino  employed. required effects  a c i d e x c i t a n t s when aAA was  Figures  i n order  the i l l u s t r a t e d  the e j e c t i n g currents  DL-homocysteate L-aspartate  20,  to e s t a b l i s h  25.  currents  differential For  f o r t h e e x c i t a n t s w e r e as  42, L - g l u t a m a t e  10, ACh  ranking  antagonist  p a i r s of agonists.  L-aspartate  L - g l u t a m a t e 31, k a i n a t e Kainate  the  i n t h e c e n t r a l column r e p r e s e n t  t o e j e c t aAA against  records, lows:  T y p i c a l responses used i n c o n s t r u c t i n g the  13;  34;  36;  these fol-  131  100 tec.  132  cells),  and L - g l u t a m a t e  ming t h e l o c a t i o n of  the ranking Effects  (6  of 6 cells),  o f ACh a n d k a i n a t e  thus  further confir-  i n the lowest p o s i t i o n s  scheme.  o f GDEE and aAA on t h e s p o n t a n e o u s a c t i v i t y o f  VB t h a l a m i c n e u r o n e s were sometimes o b s e r v e d ,  although  were most p r o n o u n c e d o n l y when t h e a n t a g o n i s t s were at high  "doses"  susceptible appeared vity  i n efforts  agonists. Furthermore,  i n no way r e l a t e d  observed  observed  with  and  these  direct  employed  o f the l e a s t effects  to the pharmacological  the blockers.  selecti-  T h e r e was an i n c o n s i s t e n t l y  i n c r e a s e i n t h e s p i k e h e i g h t when h i g h e j e c t i n g  c u r r e n t s were u s e d is  to block e x c i t a t i o n s  these  t o e x p e l GDEE  (usually  r e l a t e d presumably to the o b s e r v a t i o n s (1973) who n o t e d  Puil  diethylester  >40  n A ) , and t h i s  of Zieglgansberger  a h y p e r p o l a r i z i n g a c t i o n of the  on t h e c e l l membrane o f s p i n a l  neurones.  B o t h aAA and GDEE c o u l d r e d u c e t h e s p o n t a n e o u s r a t e o f firing  on some n e u r o n e s ,  Figure  17  (depth:  illustrates  5.39  was d e p r e s s e d An  mm.  although  n o t a l w a y s on t h e same  an example o f a t h a l a m i c  from the c o r t i c a l  range o f that used f o r t h i s  s u r f a c e ) whose f i r i n g  spike amplitude.  w e l l beyond those  rate  o f aAA.  nA o f aAA w h i c h was i n t h e u p p e r antagonist, reduced  507o t h e s p o n t a n e o u s d i s c h a r g e , w i t h o u t on  neurone  i n a d o s e - r e l a t e d manner b y t h e e j e c t i o n  i o n t o p h o r e t i c dose o f 24  cells.  by a b o u t  an o b s e r v a b l e  effect  By c o n t r a s t GDEE a t d o s e s up t o 60  r e q u i r e d to antagonize  40  nA,  a l l b u t t h e most  -  133  Figure of  17.  firing  This  Effects  o f t h e amino a c i d  o f a spontaneously  d i s c h a r g i n g VB t h a l a m i c  c e l l was l o c a t e d a t a d e p t h o f 5.39 mm.  on  t h i s neurone  be  effective  to  the present  of  L-aspartate.  in  a d o s e - r e l a t e d manner a n d a t v e r y  depressant (Hall tested  (not i l l u s t r a t e d )  ranking  effect  scheme.  aAA a f f e c t s  showed b o t h  trials  GDEE a n d aAA t o according  Note the h i g h apparent  the r a t e o f f i r i n g  potency  of this  short latencies.  cell This  h a s b e e n shown t o be b i c u c u l l i n e - i n s e n s i t i v e The l a c k o f e f f e c t  ( w i t h i n the range o f those i n the lower  o f GDEE a t a l l d o s e s  used i n t h i s  t r a c e , which overlaps  ease o f comparison.  elevated  neurone.  Previous  i n a n t a g o n i z i n g amino a c i d r e s p o n s e s  e t a l . , 1977).  observed for  a n t a g o n i s t s on t h e r a t e  study)  the upper t r a c e  A t 60 nA, GDEE was o b s e r v e d  t h e s p i k e h e i g h t b y 157..  may be  t o have  134  135  resistant  amino a c i d e x c i t a n t s , l e f t  u n a l t e r e d b u t p o t e n t i a t e d by about  t h e spontaneous  rate  15% t h e a m p l i t u d e o f t h e  action potentials. iii/  Other Antagonists  The e f f e c t s  on  19 t h a l a m i c  to  t h e c r i t e r i a o u t l i n e d on p . 27) were o b t a i n e d  Considerable  neurones,  o f KDEE were  and a c c e p t a b l e  d i f f i c u l t y was e n c o u n t e r e d  compound due t o i t s e f f e c t s and  spike  amplitude.  doses,  height  (according from only  i n tests with  on s p o n t a n e o u s n e u r o n a l  KDEE i n v a r i a b l y d e p r e s s e d  of c e l l s which f i r e d low  results  evaluated'  spontaneously,  8.  this  firing  the a c t i v i t y  e v e n when a d m i n i s t e r e d a t  and i t caused b o t h i n c r e a s e s  of action potentials i n different  and d e c r e a s e s i n t h e phases o f i t s a c t i o n .  When e j e c t e d a t l o w (2 - 10 nA) c u r r e n t s , KDEE e x e r t e d an e f f e c t w h i c h a p p e a r e d t o be due t o a h y p e r p o l a r i z a t i o n o f t h e membrane: t h e s p o n t a n e i t y markedly p o t e n t i a t e d ling  was a b o l i s h e d a n d t h e s p i k e  (up t o 70%, c o n t r o l ) .  c u r r e n t s were m a i n t a i n e d  f o r long periods  min.)  o r i f l a r g e r c u r r e n t s were u s e d  usual  effect  amplitude. the  circumstances  o f time  reduction  full  although  still  ation o f a hindlimb Despite results  these  following  indeed the  o f t h e n e u r o n e t o amino a c i d s was o f t e n  i tcould  ( c a . 10  i n spike  recovery  c e s s a t i o n o f e j e c t i o n o f KDEE n e v e r o c c u r r e d ;  sensitivity  expel-  (15 - 30 n A ) , t h e more  o b s e r v e d was a c o n s i d e r a b l e Under t h e s e  When t h e s e  height  lost,  b e s y n a p t i c a l l y a c t i v a t e d by s t i m u l i ; :  nerve. p r o b l e m s i t was p o s s i b l e t o o b t a i n  of differential  antagonism with  some  KDEE, u n d e r c o n d i t i o n s  136  where low d o s e s were employed. produced on  by DLH more t h a n  the other c e l l  tible. ate, while  KDEE r e d u c e d  the e x c i t a t i o n s  t h o s e o f L - a s p a r t a t e on 2 o f 3 c e l l s ;  L - a s p a r t a t e e x c i t a t i o n s were t h e more  When c o m p a r i s o n s  were made w i t h L - a s p a r t a t e a n d k a i n -  t h e f o r m e r was a n t a g o n i z e d p r e f e r e n t i a l l y the e x c i t a t o r y  potentiated.  Three  suscep-  effects  on 2 o f 2  cells  o f k a i n a t e were c o n c o m i t a n t l y  other neurones  yielded  where DLH e f f e c t s were r e d u c e d more t h a n  acceptable  results,  those o f L-glutamate,  t h o s e o f k a i n a t e were b l o c k e d more t h a n DLH, w h i l e  L-glutamate  and k a i n a t e e x c i t a t i o n s were a n t a g o n i z e d i n p a r a l l e l . The onistic  final  series  activity  o f compounds t e s t e d  included  b o t h as an e x c i t a n t  for possible  3AA, 2A3P a n d 2A4P.  BAA was  and as an a n t a g o n i s t , on a l l 8  antaginactive  cells  examined. The  phosphonic  antagonists similar  analogues  (Figure  18) and t h e i r  example,  2A3P r e d u c e d  those o f L-glutamate of both  unaffected. amplitude height,  effects  some a c t i v i t y appeared  qualitative  the e x c i t a t i o n s on 3 c e l l s ,  caused  In c o n t r a s t  s e e n w i t h aAA,  by NMA  similar properties  i n that  compound was  rate  more  than  remained  the spike  frequently  of discharge.  i t selectively  or  on s p i k e  2A3P c o n s i s t e n t l y r e d u c e d  the spontaneous  For  w h e r e a s on 3 o t h e r s t h e  to the l a c k of e f f e c t  this  there  aspects.  e x c i t a n t s were e i t h e r p o t e n t i a t e d  and f u r t h e r m o r e  to i n h i b i t  i n their  as  t o be  t o t h o s e a l r e a d y d e s c r i b e d f o r aAA, a l t h o u g h  was a n o t a b l e d i f f e r e n c e  effects  d i d possess  observed  2A4P h a d  a n t a g o n i z e d t h e NMA  137  Figure  18.  antagonists.  Effects  2A4P p r e f e r e n t i a l l y  n e u r o n e s t o NMA pattern  o f the phosphonic  analogues  as amino a c  antagonized the responses  when compared w i t h g l u t a m a t e ,  o f e f f e c t was e v i d e n t f o r 2A3P.  and a  similar  1 min  r-  1  CO  139  responses but n o t those o f L-glutamate,  and reduced t h e a m p l i -  tude o f t h e s p i k e .  the a c t i v i t y  taneously firing  firing  rates  irregular iv/  was  thalamic neurones,  When t h e e x c i t a t i o n s  o f d i s c h a r g e t o a more  observed which  i n d u c e d b y BAG were  compared  or L-aspartate i n the presence o f  o f t h e two a n t a g o n i s t s ,  artate.  o f spon-  b o t h compounds r e d u c e d t h e  and changed t h e p a t t e r n  those o f L-glutamate  either  against  one ( s p i n d l e s ) .  BAG  with  When t e s t e d  correlated  On 6 o f 6 n e u r o n e s  GDEE o r aAA, a c o n s i s t e n t the e f f e c t s  o f BAG w i t h  where BAG a n d L - g l u t a m a t e  trend  L-aspwere  compared i n t h e p r e s e n c e o f GDEE, L - g l u t a m a t e was a n t a g o n i z e d but  n o t BAG.  When aAA was t h e a n t a g o n i s t t e s t e d ,  BAG e x c i t -  a t i o n s were r e d u c e d i n p r e f e r e n c e t o t h o s e o f L - g l u t a m a t e on 8 of 9 cells. regardless  Comparisons  o f BAG a n d L - a s p a r t a t e showed  o f t h e a n t a g o n i s t employed,  that  thepharmacological  b e h a v i o u r s o f t h e two e x c i t a n t s were t h e s a m e . ( F i g u r e 1 9 ) . Table  7 p r e s e n t s a summary  o f these f i n d i n g s ,  a p p a r e n t p o t e n c y o f BAG i n c o m p a r i s o n and L - a s p a r t a t e .  as w e l l  with that  as t h e  o f L-glutamate  140  Figure and  19.  E f f e c t s o f GDEE a n d aAA a g a i n s t  a comparison  tate.  w i t h the responses to L-glutamate  3AG more c l o s e l y a p p r o x i m a t e d  GDEE t h a n was  aspartate.  two r e s p o n s e s  response  decrement  bility  The c e n t r e  to L-glutamate  trace  on most o c c a s i o n s  of the upper  to i l l u s t r a t e  shown i s n o t a n o n - s p e c i f i c  but that  t o aAA.  appeared  aspartate  t o t h e a c t i o n o f aAA a n d l e s s s u s c e p t i b l e t o  includes  sitization,  and L - a s p a r -  the e x c i t a t i o n s of  than those o f glutamate, and i n f a c t , more s u s c e p t i b l e  e x c i t a t i o n by 3AG  i t reflects  a true  record  that the  o n s e t o f desen-  differential  suscepti-  142  Table  7.  Summary o f t h e r e s u l t s  o f potency  antagonism o f 8-aminoglutarate. based by  on 7 n e u r o n e s . w h e r e e q u a l  BAG, L - g l u t a m a t e  utive  trials  indicates against tate  t h e number  in  The lower  r a t e s were three  produced consec-  section o f the Table (B),  t h a t GDEE a n d aAA were t e s t e d  on t h e one hand, a n d BAG a n d a s p a r -  and as i n d i c a t e d by t h e p r e c e e d i n g  t o e x e r t an e f f e c t  preference  excitation  of occasions  BAG a n d L - g l u t a m a t e  BAG a p p e a r s  e v a l u a t i o n s were  and L - a s p a r t a t e , on a t l e a s t  f o r each c e l l .  on t h e o t h e r ,  Potency  d e t e r m i n a t i o n s and  u p o n t h e aAA s e n s i t i v e  to that antagonized  b y GDEE.  The t h r e e  Figure, receptor occasions  where GDEE a f f e c t e d L - a s p a r t a t e a n d BAG e q u a l l y , r e p r e s e n t where b o t h  e x c i t a n t s were r e l a t i v e l y  resistant  trials  to antagonism.  143  Effects  Of  3-Aminoglutarate  A. P o t e n c y . 3AG  Potency  Compared W i t h :  No.  Of  Cells:  L-glutamate  0.43  ± 0.13  7  L-aspartate  0.54  ±0.10  7  B. A n t a g o n i s m .  aAA  GDEE  3AG  8  0  L-glutamate  0  6  Both  1  0  3AG  0  0  L-aspartate  0  0  Both e q u a l l y  4  3  Antagonized:  equally  144  c)  Discussion  There a r e s e v e r a l d i f f e r e n t  b u t r e l a t e d m a t t e r s w h i c h c a n be  discussed  i n light  the  o f w h i c h i s t h a t a number o f amino a c i d e x c i t a n t s  first  of thalamic  susceptibility  specific  when compared w i t h been a s y s t e m a t i c  Chapter,  i n terms o f t h e i r  t o a n t a g o n i s m b y GDEE and aAA.  antagonist other  The p r e s e n t  GDEE c o u l d a n t a g o n i z e exceptions tible.  being  NMA  A ranking  effects  against  results  show c l e a r l y  i n Chapter I I .  result  DLH, ADCP and NMA  thus appears t h a t w h i l e  aspartate  suscep-  The e x c i t a t i o n s p r o -  specificity  of the  were remaining  c o u l d o n l y be r e d u c e d  l o w e r d o s e s o f GDEE c a n show  o f antagonism against  a n d ACh, l a r g e r c o n c e n t r a t i o n s  blockade.  although  also.  i n n o n - s p e c i f i c e f f e c t s probably  receptor  that,  neuronal  t h u s be e s t a b l i s h e d  the e f f e c t s  o f ACh were b l o c k e d  a pharmacological  a number o f  o f glutamate and a s p a r t a t e  b y GDEE w h i l e  amino a c i d s , k a i n a t e ,  It  anaesthetized  a n d ADCP), n o t a l l a r e e q u a l l y  duced by t h e s t e r e o i s o m e r s  when t h e e f f e c t s  acids  a l l t h e compounds t e s t e d ( t h e  o f the e x c i t a n t s c o u l d  antagonized  as a  t r a n s m i t t e r s , b u t t h e r e has never  nearly  t h e manner d e s c r i b e d  readily  f o r e x c i t a t o r y amino  i n v e s t i g a t i o n i n the urethane  of i t s d i f f e r e n t i a l  excitants.  by  i n this  e a r l i e r m e n t i o n e d , GDEE h a s b e e n g e n e r a l l y a c c e p t e d  relatively  rat  presented  n e u r o n e s and ACh a r e s e p a r a b l e  differential As  of the r e s u l t s  I t i s necessary  glutamate,  o f t h i s compound unrelated  therefore  s o l e l y to  to use c a r e f u l  145  c o n s i d e r a t i o n o f the range o f c u r r e n t s a l e s s e r extent as  from these resolve  o r a c t upon d i f f e r e n t alone,  question,  receptors  and a d d i t i o n a l e v i d e n c e as d i s c u s s e d  t h e same n e u r o n e s as GDEE p r o v i d e s i n the a n a l y s i s o f the types  w h i c h e a c h o f t h e amino a c i d s r e a c t . aspartate  t h e s e compounds  e x c i t a t i o n s were r e a d i l y  i s not evident i s required to  o f a n t a g o n i s m b y aAA  additional information of receptors  with  whereas g l u t a m a t e a n d  antagonized  o f GDEE, i t was t h e e x c i t a t o r y e f f e c t s w h i c h were e x t r e m e l y  share the  below.  examination o f the s p e c i f i c i t y  important  using  whether g l u t a m a t e and a s p a r t a t e  data  this  The on  aAA, a r e e f f e c t i v e b e f o r e  antagonists.  same r e c e p t o r  o v e r w h i c h GDEE, a n d to  o f NMA,  b y low d o s e s ADCP  and DLH  s e n s i t i v e t o a n t a g o n i s m b y aAA.  n o t e w o r t h y t h a t t h e a c t i o n s o f k a i n a t e were r e s i s t a n t blocker its  as t o GDEE.  effects,  difference  tions aAA  since  marked  The f i n a l  consid-  to a discussion of p o s s i b l e receptor  popula-  from a comparison o f the e f f e c t s  i s that w i t h i n  artate the orders ring  GDEE, r e v e a l a v e r y  i n the spectrum o f i t s a c t i o n s .  arising  to this  C l e a r l y aAA i s a u s e f u l a n t a g o n i s t  when c o n t r a s t e d w i t h  eration relevant  I ti s  o f GDEE and  the group o f isomers o f glutamate and aspa r e i n v e r t e d i n t h e two r a n k s ,  again  infer-  a d i f f e r e n c e i n t h e mechanisms o f a c t i o n o f t h e two  antagonists. The the  first  two r a n k i n g  a n d p e r h a p s most o b v i o u s p o i n t orders  i s that  arising  from  the r e c e p t o r which i s acted  146 upon b y GDEE p r e f e r e n t i a l l y  r e a c t s with L-glutamate,  t h a t w h i c h i s most s e n s i t i v e  t o the e x c i t a t o r y  i s most r e a d i l y b l o c k e d b y aAA. are  insensitive  conclusion  i s that L-glutamate  populations  that  actions a n d NMA  o f amino a c i d r e c e p t o r s .  c a n be e x t e n d e d exert  to the b l o c k i n g  S i n c e NMA  further  i t s excitatory of either  t o suggest  effect  action  induced  while o f NMA  excitations  o f GDEE, t h e l o g i c a l interact with  different  Moreover t h i s c o n c l u s i o n  that k a i n a t e appears to  through  a mechanism d i f f e r e n t  o f t h e above two a g o n i s t s , s i n c e n e i t h e r  n o r aAA a n t a g o n i z e d t h e e f f e c t s  of this  compound e x c e p t  from  GDEE when  t h e r e was e v i d e n c e o f n o n - s p e c i f i c  depression.  noted  o f c u r r e n t s o v e r w h i c h aAA  that  t h e r e was a w i d e r  range  c o u l d be u s e d b e f o r e n o n - s p e c i f i c  I t may be  e f f e c t s were o b s e r v e d ,  than  was t h e c a s e w i t h GDEE.  A further aspartate of  share  suggestion pertaining  t h e same r e c e p t o r a r i s e s  each rank.  A strict  may be i n t e r p r e t e d neuronal  firing  receptors.  inversion  as e v i d e n c e  that  an e x a m i n a t i o n  D-glutamate.  of the s u s c e p t i b i l i t i e s  two d i f f e r e n t  One r e c e p t o r may p o s s e s s  L-glutamate  the greatest  ( a n d i s t h e most r e a d i l y  groups o f sensitivity  a n t a g o n i z e d b y GDEE)  or weakly s e n s i t i v e  By c o n t r a s t ,  to the e f f e c t s  the ranking produced  b y aAA  shows D - g l u t a m a t e as b e i n g t h e most, a n d L - g l u t a m a t e susceptible  o f the i s o m e r i c forms,  acids  t h e s e compounds e l e v a t e  by i n t e r a c t i n g w i t h  while being only moderately of  from  t h e o r d e r i n g o f t h e i s o m e r i c f o r m s o f t h e s e amino  within  for  t o whether glutamate and  clearly  indicating  the least a  147  difference being  VB  of the receptor  a n t a g o n i z e d b y aAA.  It least  i n the pharmacological p r o f i l e  c a n be c o n c l u d e d  three  from a l l these o b s e r v a t i o n s  g r o u p s o f amino a c i d r e c e p t o r s  thalamic  neurones.  The r e c e p t o r  that at  p r o b a b l y e x i s t on  population  most s e n s i t i v e  t o a n t a g o n i s m b y aAA i s p r e f e r e n t i a l l y a c t i v a t e d b y NMA, this  amino a c i d i s t h e most  examined. and  In t h i s  context  susceptible  the ranking  receptors  s y s t e m o f aAA.  into GDEE-sensitive  a l . , 1974; D a v i e s  a c t i v a t e d by g l u t a m a t e ,  sibility since  matter w i l l  grounds  discussed  t o NMA b u t a l s o  acid  types  which  1977; E v a n s , F r a n c i s and On t h e one hand,  o n e does a p p e a r > i t o b e v m o s t u r e a d i l y  t h e aAA r e c e p t o r  which i s s e n s i t i v e  t o ADCP and DLH, c a n n o t be  an a s p a r t a t e - p r e f e r r i n g  does a p p e a r p l a u s i b l e  type,  (see next  t h e e x c i t a t i o n s p r o d u c e d by k a i n a t e  blocked  be  (Duggan, 1974; M c C u l l o c h  1977) r e m a i n s t o be e s t a b l i s h e d .  equated with  a h e a d o f glutamate  and^aAA s e n s i t i v e c o n f o r m s t o  and W a t k i n s ,  whereas t h e G D E E - s e n s i t i v e  only  i n fact  and a s p a r t a t e - p r e f e r r i n g  h a v e b e e n p r o p o s e d on o t h e r  not  This  only  a r e t h e n e x t most  Whether t h e c a t e g o r i z a t i o n o f amino  glutamate-preferring  Watkins,  receptor,  a n t a g o n i z e d compounds, b e i n g  f u r t h e r below.  et  t h a t DLH  ADCP, two compounds w h i c h m i g h t be e x p e c t e d t o r e a c t  readily  the  of a l l the agonists  i t i s i n t e r e s t i n g to note  with a "glutamate-preferring"  in  since  although  section).  exists.  the posFinally  are not r e a d i l y  b y e i t h e r GDEE o r aAA, i t may be t h a t  "kainate-preferring"receptor  easily  a third  type o f  148  Evidence populations sky  and  w h i c h may  (1977),  of s p i n a l  applications neurones  construed  o f r e c e p t o r s e x i s t has  Pomeranz  tization  be  who  t o a s p a r t a t e was  experiments,  found  t h a t t h e r e was  There are  evidence in fact  y e t the response  which lend support  to the p r e s e n t  number o f amino a c i d r e c e p t o r s e x i s t Duggan  (1974) o b s e r v e d  to a s p a r t a t e than  glutamate  preferentially  t h a t a s p a r t a t e and  transmitters tively.  excited  of interneurones  w h i l e NMA  These  Flatman  1979)  Steinberg  caused  last  a very  kainate a very  is significant  1975;  neurones.  while I t was  a c t as  sug-  synaptic  afferents  among t h e e f f e c t s  or a s p a r t a t e .  c o n d u c t a n c e and (Engberg,  that a  i n t h e c a t were more  two  t h e membrane t o p l a t e a u l e v e l s w i t h o u t  conductance,  it  primary  means  respec-  Measurement o f membrane c o n d u c t a n c e o f c a t motoneurones  glutamate  ized  and  observed.  suggestion  interneurones. might  present  different  interneurones,  glutamate  r e v e a l e d marked d i f f e r e n c e s and  spinal  same  been  central  t h a t Renshaw c e l l s  sensitive  gested  on  pulsatile  desensitization  o f d e s e n s i t i z a t i o n has  at least  Dostrov-  or  t h a t i n the  d a t a o b t a i n e d by  two  a desensi-  o f the  u n a f f e c t e d by p r i o r  other  that  to prolonged  However i t must be n o t e d no  indicating  b e e n p r e s e n t e d by  cord interneurones  of glutamate,  to glutamate.  as  and  Lambert,  that these  and A l t m a n n , Ten  o f NMA,  rapidly  kainate depolar-  marked changes  l a r g e decrease  of  in  l a r g e (immeasurable) i n c r e a s e 1978).  In t h i s  same i n v e s t i g a t o r s Bruggencate,  (1976) h a v e r e p o r t e d t h a t t h e  connection  too  (Engberg  etal.  Pickelmann conductance  and changes  149  e l i c i t e d i n motoneurones and r u b r o s p i n a l neurones by DLH and by glutamate are n o t i d e n t i c a l . Watkins and c o l l e a g u e s have shown t h a t by changing the c o n c e n t r a t i o n s of K  +  or N a  +  i n media b a t h i n g i s o l a t e d  spinal  cords o f f r o g and immature r a t , d i f f e r e n t d e p o l a r i z i n g e f f e c t s of amino a c i d s were observed,  and these f e l l  i n t o three groups:  1) L-glutamate and L - a s p a r t a t e ; 2) k a i n a t e and NMA; 3) L-homoc y s t e a t e and D-glutamate.(Evans e t a l . , 1977). type o f i o n i c m a n i p u l a t i o n ,  Davies  Using a s i m i l a r  and Watkins (1977) found  that f e l i n e s p i n a l neurones responded to k a i n a t e but not NMA i n the presence o f low c o n c e n t r a t i o n s o f Mg" ", w h i l e 1-1  L-aspar-  t a t e responses were more s e n s i t i v e than those o f L-glutamate to the b l o c k i n g a c t i o n o f t h i s i o n .  Presynaptic effects  could  be r u l e d out by the o b s e r v a t i o n of a s y n e r g i s t i c a c t i o n o f Ca" " with Mg"*", and i t i s known t h a t Ca" " antagonizes the 1-1  1  depressant  1-1  a c t i o n o f Mg" " i n the r e l e a s e o f t r a n s m i t t e r s from 1-1  presynaptic terminals. F u r t h e r evidence  i n support  o f the present r a n k i n g schemes  has been obtained from a study u s i n g s i m i l a r methods but employing d o u b l e - b l i n d t r i a l s , and Renshaw c e l l s  on f e l i n e s p i n a l  (McLennan and Lodge, 1979).  c o r r o b o r a t e d the p r e s e n t  interneurones Their results  schemata i n the main, and extended  these r e s u l t s by i n c l u d i n g a number of d i f f e r e n t e x c i t a n t s : i b o t e n a t e , L - c y s t e a t e , q u i s q u a l a t e and the separated D and L isomers o f homocysteate and N-methylaspartate.  Briefly,  L - c y s t e a t e and q u i s q u a l a t e appeared to f o l l o w c l o s e l y the  150  responses of L-glutamate w i t h regard to antagonism by aAA GDEE, while i b o t e n a t e and to a l e s s e r extent the  stereoisomers  of homocysteate more c l o s e l y mimicked the responses of r e p o r t e d here.  Both the D and L enantiomorphs of NMA  very s e n s i t i v e to antagonism by aAA  and  NMA, were  as w e l l as being  resistant  to the a n t a g o n i s t i c a c t i o n of GDEE, i n c o n f i r m a t i o n of the present r e s u l t s w i t h the racaemic Although obtained reached  mixture.  i t i s o f t e n i n a d v i s a b l e to compare r e s u l t s  from animals  of d i f f e r e n t p h y l a , the c o n c l u s i o n s here  have a n a l o g i e s among i n v e r t e b r a t e s , where  evidence  has been obtained f o r separate p o p u l a t i o n s of r e c e p t o r s f o r glutamate and  for aspartate  (Yarowski  f o r both s y n a p t i c and non-synaptic (Takeuchi and Onodera, 1975).  and Carpenter,  1976)  and  glutamate r e c e p t o r s  Furthermore, k a i n a t e may  only with the n o n - s y n a p t i c . r e c e p t o r s  (Cull-Candy,  react  1976).  E f f e c t s of Other Analogues Attempts t o demonstrate a s e l e c t i v e antagonism of the  effects  of k a i n a t e produced i n c o n s i s t e n t r e s u l t s , and were i n g e n e r a l unsuccessful. The r e s u l t s of the experiments which t e s t e d the other p o s s i b l e a n t a g o n i s t s , 3AA,  2A3P and  l i m i t e d amount of u s e f u l i n f o r m a t i o n .  three  2A4P y i e l d e d only a  For example,  3AA  appeared to be l a r g e l y i n a c t i v e , e i t h e r as an e x c i t a n t or as an a n t a g o n i s t , and the s i g n i f i c a n c e of t h i s o b s e r v a t i o n  will  151  be  d i s c u s s e d below.  did possess their  The  a limited  actions  the b l o c k i n g  degree  appeared  e x c i t a t i o n s produced  ists in  a c t i o n o f aAA,  t o be  low  since  T h i s p r o p e r t y may  i n fact  anomalous r e s u l t s  of others  1978)  synaptic  who  found  effects  they had  that  be  the phosphonic  response  analogues  antagonism  c o u l d be  t e s t e d by  by  i f they  alternative in  their  neurones. in  do,  e t a l . , 1977;  the r e p o r t  This  inactivity  amplitude induced as  possibility  o f the e f f e c t s  o f 2A3P  and  v i a intracellular recording  have a d e p o l a r i z i n g i s that  action.  these antagonists and  l a t t e r p o s s i b i l i t y may  o f W a t k i n s e t a l . , (1977) where a  pharmacological t h e amino a c i d  for this  granule  have been i n t e r p r e t e d  a c t i o n s between d e n t a t e g r a n u l e c e l l s Support  on  to a d e p o l a r i z a t i o n  an e x a m i n a t i o n  interpretation  the  excitatory  due  these authors.  i n fact,  effects  Dunwiddie et  l o w e r i n g of the  2A4P upon membrane c o n d u c t a n c e see  of  The  might  antagon-  neurones.  the u n d e r l y i n g cause  stimulation  by  L-glut-  very noticeable  2A4P c o u l d b l o c k t h e  i n the dentate gyrus.  synaptic  to  reduced  of the thalamic  (White  the  a g o n i s t to  when compared w i t h  of p e r f o r a n t path  of the granule c e l l by  susceptible  N e v e r t h e l e s s the g e n e r a l u s e f u l n e s s of these  appears  cells  Thus  were p r e f e r e n t i a l l y  analogues  2A4P  as a n t a g o n i s t s , and  t h o s e o f aAA.  t h e most  r e d u c i n g the spike amplitudes  al.,  compounds, 2A3P and  of a c t i v i t y  to resemble  by NMA,  both of the phosphonic amate.  o t h e r two  f o r both phosphonic  induced responses  An  differ  thalamic be  found  complete analogues  o f c o r t i c a l n e u r o n e s was  upon seen.  152  A and  final  3AG.  comment i s r e q u i r e d r e g a r d i n g  Although  pharmacologically  the  racaemic mixture of  inactive,  mere p r e s e n c e o f e i t h e r present  i n aspartate,  glutamate,  does n o t  amino a c i d s .  this  the  or  finding  co-carboxyl  the  confer  remembered t h a t  o f amino a c i d s  can  possess  ties,  different  effects  separated  isomers of  the  before  lack of a c t i v i t y  the  A  similar  activity and  of  GDEE.  type  3AG Since  7),  is  the  ( H a l l e t a l . , 1979)  not  an  absolute  Furthermore, the  that  aAA  since  receptor  receptor w i l l  3AG  accept  excitants,  will  19)  especially  by  two  so  required  excitatory  antagonists,  (Table  - amino s e p a r a t i o n g l u t a m a t e and  to e x e r t be  of  aspartate  activity.  i t s effects  surmised  that  through this  a range of a-carboxyl  has  aAA  neurones, a l b e i t with  for excitatory  -  i m p l i c a t i o n s f o r some o f  observations  regarding  for  proper-  glutamate or a s p a r t a t e  i t may  This r e s u l t  be  to  forms  D-aAA; and  be made o f t h e  to the  agonists with  the p r e v i o u s l y r e p o r t e d  of  substantiated.  possessed  appeared  (Figure  amino s e p a r a t i o n s .  3AA  a-carboxyl  requirement  the  distance  pharmacological  did e x c i t e thalamic  compared w i t h  i t would appear  2.43A  is  be  that  stereoisomeric  o f L-ctAA and  i t s responses  3AG  a reduced potency  the  o f a n a l y s i s can  and  appeared to  - amino  3AA  excitatory properties  different  tests with  3AA  does s u g g e s t  of  c o n c l u s i o n w o u l d seem t o h o l d  DL-3AA, i t must be  v i z . the  effects  t o - c a r b o x y l - amino d i s t a n c e  in itself  Although t h i s  the  their  with  t h e more  a n t a g o n i s m by  potent aAA.  153  As was  noted  aAA-sensitive yet  these  fashion  receptor  agonists  to  paradoxical  result  sites The  the  the  is  molecule  can assume,  folded the then  it  is  by  configuration other  through both ability carboxyl  to -  If  to  GDEE,  this  since  molecule  situation is  assume  both  carboxyl  a wide  a  carboxyl  preferenactive  -  receptor.  co-carboxyl  which act  aspar-  in this  responsible  a A A a n d GDEE  w i t h the  i n an  can e x e r t  4),  (Table  is  the  and  effects  an e x c i t a t o r y  on of  effect  due  to  its  and a narrow s p a c i n g  of  the  substituents.  not  extended  GDEE r e c e p t o r ,  receptors  more  for  o n e h a n d w h y ADCP  fixed  a A A a n d GDEE p r e f e r r i n g  -  (contracted)  aAA c a n p a r t i a l l y a n t a g o n i z e  this  for  terminal  GDEE c a n a l s o  of  extended  seemingly  activated  minimum d i s t a n c e  ADCP i s  react  this  an a - c a r b o x y l  e x p l a i n on the  and cannot  since  the  a-carboxyl  i n an  carboxyl  their  confined  attain  effectiveness  h a n d why  glutamate,  "fit"  for  similar  same  when  the  receptor,  in a  the  upon  b y NMA a n d A D C P .  c o u l d be  and presumably  possible  antagonized  the  than  configuration.  differential  can  act  requirements  involves  structurally  s e p a r a t i o n much l e s s tate  steric  possessed  which can  act  possess  explanation the  range  to  glutamate  GDEE r e c e p t o r  a more  glutamate  they  to  GDEE-sensitive  expected  does  that  the  by m o l e c u l e s into  the  aAA r e c e p t o r  within  Furthermore,  as  One p o s s i b l e  reaction with separation  and not  since  distance  conformation.  ADCP a n d D L H a p p e a r  m i g h t be  glutamate,  to; co-carboxyl  tially  above,  154  Although the  does a p p e a r  observations herein presented,  experimental an  this hypothesis  support  t o e x p l a i n many o f  i n the absence o f d i r e c t  i t must r e m a i n s p e c u l a t i v e .  F o r example  e x p l a n a t i o n o f t h e l a c k o f a n t a g o n i s m b y aAA o f t h e e f f e c t s  o f k a i n a t e must be p r o v i d e d , pation of additional  although  the p o s s i b l e p a r t i c i -  t e r m i n a l groups p o s s e s s e d  by t h i s  mole-  cule  i n t h e a g o n i s t - r e c e p t o r r e a c t i o n may b e r e l e v a n t i n  this  regard  1979).  (Schwarcz,  Scholz and Coyle,  Indeed o t h e r evidence  in vitro,  although  L-glutamate,  may i n f a c t  a t i o n o f glutamate Johnston,  different  indicates  1978"; H a l l  that kainate binding  i n many r e s p e c t s f r o m  represent reaction with  receptors.(Simon,  et a l . ,  a  that of subpopul-  C o n t r e r a a n d K u h a r , 1976;  Kennedy and T w i t c h i n , 1978;' L o n d o n a n d C o y l e , 1979;  McLennan, 1 9 7 9 ) , a n d t h e p r e s e n t a GDEE-insensitive  subpopulation.  results  suggest  that this i s  155  DENTATE GYRUS  a) The  Introduction well-defined neuronal organization  formation the  provides  actions  Mathisen, is  the granule  in  a thin,  synaptic little and  an e x c e l l e n t t e s t i n g g r o u n d f o r  o f t h e amino a c i d s  1977b).  The p r i m a r y  cell,  and t h e i r cell  layer  (Lorente  Cotman,  Of  and Voorhoeve,  1966; L<J>mo, 1971; S t e w a r d , W h i t e a n d  p a r t i c u l a r i n t e r e s t to the present that  glutamate and a s p a r t a t e  study  mediate  groups o f a f f e r e n t f i b r e s  drites  o f the granule  cells.  a l . , (1977) h a v e r e p o r t e d  of glutamate and a s p a r t a t e  by  i s the the synaptic onto  t h e den-  N a d l e r e t a l . , (1976) a n d W h i t e that  a calcium-dependant  occurred  depolarization of slices  superior.  following  efflux  potassium-  o f r a t d e n t a t e gyrus and  S u r g i c a l i n t e r r u p t i o n o f the e n t o r h i n a l  way o f t h e p e r f o r a n t  path  (PP) r e d u c e d t h e r e l e a s e  amate f r o m t h e d e n t a t e g y r u s w h i l e pathway  anatomically  1956; 1958; A n d e r s e n ,  from d i f f e r e n t  regio  The  i n d i s c r e t e zones with  inputs  induced  tissue  1977).  likelihood  et  this  gyrus  de No, 1 9 3 4 ) .  and which have been s t u d i e d  e l e c t r o p h y s i o l o g i c a l ^ (Blackstad,  Holmqvist  (Storm-  type i n the dentate  t o these neurones occur  overlapping,  comparing  antagonists  which i s d i s t r i b u t e d w i t h i n  horseshoe-shaped  inputs  o f the hippocampal  input  of glut-  l e s i o n s o f the commissural  (COMM) f r o m t h e c o n t r a l a t e r a l h i p p o c a m p u s  diminished  156  the r e l e a s e o f a s p a r t a t e ( N a d l e r , White, Vaca, Cotman,  1978).  can p r e v e n t which a l s o  Wheal a n d M i l l e r  (1979) h a v e shown t h a t  t h e PP e v o k e d a c t i v a t i o n suggests  are mediated  by  that  of dentate granule  t h e synapses  f o r m e d b y t h e PP  cells  1966) a n d GDEE f o u n d  amino a c i d  induced  Cotman a n d L y n c h ,  firing  i n preventing the  ( S e g a l , 1976; S p e n c e r ,  1976).  i n the d e n d r i t i c  (Dudar,  These e f f e c t s  comparison  r e g i o n o f the neurones, site  acids  f o r glut-  1974) a n d i s t h e l o c u s f o r t h e s y n a p t i c i n p u t s .  of their  of the possible gyrus  using a  r e l a t i v e p o t e n c i e s on g r a n u l e c e l l s a n d  antagonism  I t was t h e i n t e n t i o n analysis,  f o r t h i s has  of action  o f t h e amino a c i d s i n t h e d e n t a t e  differential  Gribkoff,  o f amino a c i d s p r o b a b l y  However t h e r e h a s n o t y e t b e e n an a n a l y s i s involvement  fibres  the e x c i t a t i o n of  effective  b e e n shown t o be t h e most s e n s i t i v e amate  cells,  b y amino a c i d s h a s b e e n r e p o r t e d ( B i s c o e a n d  Straughan,  occur  GDEE  glutamate.  In o t h e r r e g i o n s o f t h e hippocampus, pyramidal  P e r r y and  o f t h e s y n a p t i c a n d amino a c i d  of the present  to investigate  study  the p o s s i b l e  effects.  t o p r o v i d e s u c h an  involvement  o f t h e amino  i n the s y n a p t i c o r g a n i z a t i o n o f t h e d e n t a t e g y r u s , and  to p r o v i d e a p h y s i o l o g i c a l logical Chapter.  testing  ground f o r t h e pharmaco-  r e s u l t s w i t h GDEE a n d aAA r e p o r t e d e a r l i e r  i n this  157  b)  Results  Neurones l y i n g i n the upper blade o f the dentate gyrus and which were s y n a p t i c a l l y a c t i v a t e d by s t i m u l a t i o n o f PP and COMM (Figure 20) were r e a d i l y e x c i t e d by the i o n t o p h o r e t i c administration DLH  and NMA.  o f the amino a c i d s , L-glutamate, Since  L-aspartate,  the p i p e t t e assembly was presumably  s i t u a t e d near the c e l l bodies any d i f f e r e n c e i n the apparent potency between L-glutamate and L - a s p a r t a t e d i s t r i b u t i o n of receptors  c o u l d r e f l e c t ;the  a f f e c t e d by the e j e c t e d  materials  r e l a t i v e to t h a t l o c a t i o n . The  r e l a t i v e apparent p o t e n c i e s  L-aspartate  o f L-glutamate and  upon granule c e l l s were obtained  f a s h i o n as that d e s c r i b e d  earlier;  i n a similar  that i s , by comparing the  magnitudes o f the e j e c t i n g c u r r e n t s r e q u i r e d to e l i c i t s t a b l e , equal and submaximal l e v e l s o f the f i r i n g  frequencies  of granule c e l l s , a v a l i d comparison s i n c e the t r a n s p o r t numbers f o r these two substances are e q u i v a l e n t The  ( H a l l e t a l . , 1979) .  r e s u l t s o f such determinations where r e p r o d u c i b l e  were made on a minimum o f three occasions p r e s e n t e d i n Table 8. average L - a s p a r t a t e  tests  p e r c e l l , are  Although n o t true f o r every c e l l , on  was twice as e f f e c t i v e as L-glutamate,  (p < 0.05) (Student's t t e s t ) .  Since  the PP input to the  granule c e l l s , which has been suggested to be glutamate mediated (White e t a l . , 1977; Wheal and M i l l e r , distally  l o c a t e d on the d e n d r i t e s  1979), i s  t h i s r e s u l t might i n d i c a t e  158  Figure on  20.  granule  granule  E f f e c t s of antagonists cell  cell  responses,  antagonist  responses, onist  GDEE was c l e a r l y more e f f e c t i v e  aAA was e f f e c t i v e  and a b l o c k e r  o f the e f f e c t s  Diagram r e p r e s e n t s and  dentate  tissue  the c e l l  only  t h a n aAA as  as an a s p a r t a t e  i s made.  Inset:  evoked  antag-  o f commissural s t i m u l a t i o n .  body l a y e r s o f t h e h i p p o c a m p u s  g y r u s a s v i e w e d when a c o r o n a l  Abbreviations:  gyrus and  o f glutamate e x c i t a t i o n s and p e r f o r a n t path  while  acids  and a s c h e m a t i c r e p r e s e n t a t i o n o f a  showing i t s r e l a t i o n s h i p t o t h e d e n t a t e  the hippocampus. an  o f t h e e x c i t a t o r y amino  section of this  P h o t o g r a p h o f P o n t a m i n e s k y b l u e spot.(arrow).  P  pyramidal  cell  HF  hippocampal  F  fimbria;  H  hilus;  G  granule  PP  area  o f perforant path  COMM  area  o f commissural a f f e r e n t t e r m i n a l s .  cell  layer;  fissure;  layer; afferent terminals;  1 5 9  ANTAGONIZED GDEE  aAA  6  1  field  3  0  excitation  6  0  0  9  0  3  0  5  PP s y n a p t i c  activation  PP e v o k e d EPSP Glutamate  COMM s y n a p t i c  activation  COMM e v o k e d EPSP Aspartate  BY  field  excitation  160  TABLE 8. tested  Relative potencies  on d e n t a t e  granule  o f L - g l u t a m a t e and L - a s p a r t a t e  cells.  Ratio No. of cells  I — asp  Range  -glut  Mean ± SE  aspartate >glutamate  9  1.2-8.0  2.8 ± 2.2  aspartate=glutamate  2  1.0  —  a s p a r t a t e <»glutamate  4  0.7-0.9  0.8 ± 0 . 1  161  the  existence  o f an a s p a r t a t e  more p r o x i m a l l y  The on  situated.  e f f e c t s o f GDEE and aAA were e x a m i n e d c o i n c i d e n t a l l y  amino a c i d i n d u c e d a n d s y n a p t i c  cells.  GDEE a d m i n i s t e r e d  attenuated  considerably  t h o s e o f NMA  (Figure  o f aAA on t h e o t h e r by  acids  or reducing (Figure  light  earlier to  a c t i v a t i o n s o f 30 g r a n u l e  with ejecting currents  o f 20 - 50 nA  the e f f e c t s o f L-glutamate but not  21, C - E ) , DLH o r L - a s p a r t a t e . hand, a l w a y s  The a c t i o n  spared e x c i t a t i o n s produced  L - g l u t a m a t e a t d o s e s up t o 30 nA b u t was e f f e c t i v e i n a b o l -  ishing  in  mediated i n n e r v a t i o n which i s  markedly  21, A - C ) .  i n this  Chapter.  three  s e l e c t i v i t y had been neurones,  This  ined pharmacologically, (1979) d i d r e p o r t  that  anticipated  However i t was n o t a l w a y s a c t i o n o f aAA s i n c e  property  amino  presented possible  on some  o f 30 c e l l s ) -amarked e x c i t a t o r y e f f e c t o f t h i s  i t s e l f was f o u n d .  rate  This  o f t h e f i n d i n g s on t h a l a m i c  t e s t the a n t a g o n i s t i c  (10  those o f the other  occasions  compound  has n o t been p r e v i o u s l y  exam-  a l t h o u g h Lodge, Headley and C u r t i s , aAA c o u l d  r a i s e the background  firing  a n d enhance t h e e f f e c t s o f L - g l u t a m a t e on s p i n a l n e u r o n e s .  This matter w i l l  be f u r t h e r  discussed  below.  When s e l e c t i v e a n t a g o n i s m o f amino a c i d i n d u c e d t i o n s had been e s t a b l i s h e d were examined.  the synaptic  excita-  responses o f the c e l l s  In the presence o f s u f f i c i e n t  to antagonize the e f f e c t s o f L-glutamate,  amounts o f GDEE  the response to  s t i m u l a t i o n o f PP b u t n o t o f COMM was a l s o b l o c k e d .  Conversely  162  F i g u r e 21.  The a c t i o n s o f aAA and GDEE on s y n a p t i c and amino  a c i d e x c i t a t i o n s of a s i n g l e granule were o b t a i n e d  cell.  The rows o f r e c o r d s  s e q u e n t i a l l y from above downwards and the photo-  graphs a r e o f f o u r superimposed sweeps. time s c a l e s o f the photographs.  Note the d i f f e r e n t  163  Spikes/sec.  50 ~\  COMM  A  PP  Control  0-<  B.  50 n ocAA 12  (1.5 min) o-J  C.  50 n Recovery  (8  min)  0->  D.  50 -i G D E E 40 (3 min)  o-< 50  n  Recovery (3 min)  0-1 NMA 10 GLUT 12  t-  (  30 sec.  Jo.2  10 msec  mV  )  Jo.2  4 msec  mV  164'  a  dose o f aAA w h i c h s e l e c t i v e l y  excitations  concomitantly  neurones, w h i l e Full  recovery  blocked  reduced  theresponse  L-aspartate  t h e COMM a c t i v a t i o n o f t h e  t o PP was l e f t  t o control l e v e l s o f both  unantagonized.  the synaptic  amino a c i d e f f e c t s was o b s e r v e d a f t e r c e s s a t i o n currents Figure eries  using  (lower block  e j e c t i n g the antagonists  21). S i m i l a r complete both blocking  section, Figure the synaptic  o r NMA  (centre  and t h e  of the  and bottom  traces,  s e r i e s o f antagonisms and recov-  a g e n t s were o b t a i n e d  20), while  on f i v e  i n 1 o f 6 cases,  e x c i t a t i o n o f a granule  cell  cells  aAA d i d  by PP.stimu-  lation . In a d d i t i o n t o s e l e c t i v e l y responses o f s i n g l e granule also the  for their  s t i m u l a t i o n o f PP a n d COMM.  13%, the  population  ized  i n tabular  tested  EPSPs e v o k e d b y  22 i l l u s t r a t e s ,  EPSP e v o k e d b y PP s t i m u l a t i o n b y By c o n t r a s t ,  aAA r e d u c e d  COMM b y 26%, w h i l e  The r e s u l t s p r e s e n t e d above a r e summar-  form i n F i g u r e 20).  r e m a r k e d t h a t when aAA was t e s t e d  t o a n t a g o n i z e amino a c i d i n d u c e d  the background r a t e  This  As F i g u r e  EPSP e v o k e d b y s t i m u l a t i n g  I t was e a r l i e r ability  the synaptic  GDEE a n d aAA were  the population  w h i l e aAA was w i t h o u t e f f e c t .  GDEE was i n e f f e c t i v e .  in  cells,  effects against  GDEE r e d u c e d t h e p o p u l a t i o n  antagonizing  o f discharge  firing,  for its  an i n c r e a s e  of.some c e l l s was o b s e r v e d .  h e i g h t e n e d a c t i v i t y was n o t e d e v e n when t h e s y n a p t i c  response  t o COMM s t i m u l a t i o n was r e d u c e d b y aAA. A l t h o u g h  165  Figure PP  22.  EPSP f i e l d  a n d COMM i n p u t s  trace  represents  r e s p o n s e s evoked by s t i m u l a t i n g t h e  to the dentate granule  t h e a v e r a g e o f 20 c o n s e c u t i v e  a r r o w on t h e u p p e r r i g h t h a n d the  height  o f the f i e l d  p r e s e n c e o f 70 nA GDEE the  r i g h t hand  cells.  column).  trace  Note  field.  ( c o n t r o l PP) i n d i c a t e s PP i n t h e  from t h e second r e c o r d o f  t h e complete l a c k o f e f f e c t  o f DaAA on t h e PP e v o k e d f i e l d , GDEE o n t h e COMM  sweeps. The  achieved by s t i m u l a t i n g (measured  Each  and t h e l a c k  of effect of  166  COMMISSURAL  PERFORANT  10 msec  167  L-a-aminoadipate Hall  i s a weak e x c i t a n t  e t a l . , 1979),  be w i t h o u t  effect  the D isomer  (Curtis  has u s u a l l y been r e p o r t e d t o  ( B i s c o e e t a l . , 1977; H a l l  see L o d g e e t a l . , 1 9 7 9 ) , y e t t h e p r e s e n t both with  the racaemic  It  seemed l i k e l y  of  direct  process  a n d W a t k i n s , 1960;  mixture  and w i t h  e t a l . , 1979; b u t  e f f e c t was o b t a i n e d the separated D  t h e r e f o r e t h a t some mechanism o t h e r  To examine t h i s  i m e n t i l l u s t r a t e d b y F i g u r e 23 was  A granule  effect,  a t a r a t e o f 35 - 50  ejection  o f L-glutamate  S u p e r i m p o s e d upon t h i s b a c k g r o u n d t h e i n h i b i t o r y tory effects  e l i c i t e d by e j e c t i o n s  t i v e l y were p r o d u c e d .  T h e r e a f t e r by r e d u c i n g  constant  GABA a n t a g o n i s t , b i c u c u l l i n e . t o GABA was l a r g e l y culline, by  level  return  and e x c i t a -  the L-glutamate  f r e q u e n c y was m a i n -  during the ejection  When t h e i n h i b i t o r y  a b o l i s h e d ( 1 - 2 min. a f t e r  of the  response  onset  of bicu-  25 nA, F i g u r e 2 3 B ) , t h e e x c i t a t i o n p r e v i o u s l y  aAA was c o n s i d e r a b l y a t t e n u a t e d ,  cessation  (25 nA).  o f GABA a n d o f aAA r e s p e c -  c u r r e n t the background f i r i n g  at a roughly  t h e exper-  performed.  c e l l was made t o f i r e  s p i k e s / s e c . by t h e c o n t i n u o u s  tained  t h a n one  e x c i t a t i o n m i g h t be i n v o l v e d , a n d a " d i s i n h i b i t o r y "  i s one p o s s i b i l i t y .  expelling  isomer.  of the b i c u c u l l i n e  of the i n h i b i t o r y  and reappeared  produced  following  current c o i n c i d e n t a l l y with the  response  t o GABA.  168  Figure of was  23.  The e f f e c t  a granule c e l l induced  of b i c u c u l l ine  (BICUC) on t h e d e p r e s s i o n  by GABA and i t s a c t i v a t i o n  to f i r e  by t h e c o n t i n u o u s  p r e s e r v e an a p p r o x i m a t e l y  charge.  (A) c o n t r o l ;  administration;  constant  (B) 2 m i n a f t e r  (C) 3% m i n a f t e r  The  administration of  amate, t h e a d m i n i s t e r i n g c u r r e n t b e i n g r e d u c e d to  by aAA.  by  cell  glut-  bicuculline  l e v e l o f background start of  dis-  bicuculline  cessation of bicuculline  current.  169  A.  GLUT  25  GLUT  15  75  0 »-  B  75  BICUC.25  o  CD CO \  to CD  J£ Q-  co o »GLUT 25 75 r  mm  0 «-  GABA 20  o;AA75  30  sec.  170  c) It  Discussion i s commonly a c c e p t e d  gyrus  that  thegranule  receive several afferent  s e p a r a t e upon t h e d e n d r i t i c  cells  inputs which a r e s p a t i a l l y  t r e e s ( B l a c k s t a d , 1956; 1 9 5 8 ) .  Among t h e s e a r e two w h i c h g i v e e x c i t a t o r y ally  situated projection  from  the  inputs  et  o f the dentate  are different,  acids.  The r e s u l t s  cells  although both  amate m e d i a t e d  1979)  findings  that  o f GDEE.  aspartate  and  Of the n a t u r a l l y  i s t h e most l i k e l y  excitation  and p h a r m a c o l o g i c a l t h e PP i n p u t i s g l u t -  ( c f . Nadler  would a c c o r d w i t h  pear  i s different  transmitter of this  e t a l . , 1978;  t h e more p r o x i m a l  COMM evoked  of a majority of the t o glutamate  location  o f t h e COMM  evidence  amino a c i d r e c e p t o r s , b o t h  t o be o f s y n a p t i c s i g n i f i c a n c e ,  acids,  White e t a l . , 1979),  These o b s e r v a t i o n s p r o v i d e d i r e c t excitatory  i s antagonized  effect  neurones examined t o a s p a r t a t e r a t h e r than  distinct  (Nadler  o c c u r r i n g amino  the apparently greater s e n s i t i v i t y  input.  that  t o be amino  biochemical  of granule c e l l s  aAA whose p a t t e r n o f p h a r m a c o l o g i c a l that  indicates  s i n c e i t c a n b e b l o c k e d b y GDEE; w h i l e on t h e  o t h e r h a n d COMM a c t i v a t i o n  from  the contralat-  o f t h e s e two  arelikely  confirm the e a r l i e r  (the  the excitatory  to stimulation  a l . , 1976;1978; W h i t e e t a l . , 1977)  (Wheal a n d M i l l e r ,  by  i n p u t from  which mediates  - a dist-  cortex  The e v i d e n c e h e r e p r e s e n t e d  t r a n s m i t t e r substance  responses  responses  the entorhinal  p e r f o r a n t p a t h ) a n d a more p r o x i m a l e r a l hippocampus.  o f the dentate  coexist  t h a t two  o f which ap-  on t h e same  cells.  171  The itself input  f i n d i n g that  o n some c e l l s  the antagonist  e x c i t e w h i l e a t t h e same t i m e b l o c k i n g requires  some comment.  The f a c t  c o u l d be p r e v e n t e d by an a n t a g o n i s t is  an i n d i r e c t  in  t h e hippocampus,  that  that  explanation  o f GABA i n d i c a t e s t h a t i t  c e r t a i n i n h i b i t o r y processes i n h i b i t i o n and  and Fukushima,  one o f t h e s e p r o c e s s e s  i s involved.  t o n i c d r i v e on i n h i b i t o r y i n t e r n e u r o n e s  Desmond a n d L e v y ,  cells  which give  (Assaf  1978), a n d s i n c e  t h e COMM f i b r e s a p p e a r s  plausible.  The s i m p l e s t  to therecurrent  1978; S t r u b l e ,  the e x c i t a t i o n o f the latter this proposal i s  whether t h e i n h i b i t o r y i n t e r n e u r o n e s rise  medi-  w h i c h a r e known  and M i l l e r ,  t o be a s p a r t e r g i c  or whether they a r e i n v o l v e d w i t h hilar  1978), i t i s  w o u l d be t h e r e m o v a l b y aAA o f an a s p a r t a t e  t o a c t upon t h e g r a n u l e  the  e x c i t a t i o n b y aAA  i n c l u d i n g both recurrent  e t a l . , 1970; T s u c h i y a  possible  by  and s i n c e  t h e COMM s y n a p t i c  e v o k e d b y c o n t r a l a t e r a l s t i m u l a t i o n a r e m e d i a t e d b y GABA  (Curtis  ated  effect,  that  aAA c o u l d  inhibition  o f granule  the processes  zone r e m a i n s t o b e e s t a b l i s h e d .  a r e those cells  occurring i n  172  CHAPTER  V  OCTOPAMINE  a)  General  Overview  Octopamine i s a n a t u r a l l y in  salivary  glands  o f octopus  Erspamer and B o r e t t i , widely  distributed  Juorioj  o c c u r r i n g amine f i r s t (Erspamer,  1948a,b,c;  1951a,b; E r s p a m e r a n d A s e r o ,  i n the animal  1976; H i c k s ,  discovered  1977).  kingdom  1952a,b; 1953) a n d  ( R o b e r t s o n and  Erspamer i d e n t i f i e d  the s t r u c t u r e  o f o c t o p a m i n e as L - p _ - h y d r o x y - 8 - p h e n y l e t h a n o l a m i n e a n d d e s cribed  a sympathomimetic  o r g a n s o f mammals. amine p o s s e s s e s dogs.  a c t i o n on b l o o d p r e s s u r e  Lands and Grant  a pressor  effect  (1952) f o u n d  acid,  traetment  with  (Armstrong,  a major m e t a b o l i t e inhibitors  that  levels  o f p_-hydroxy-  o f octopamine, f o l l o w i n g  o f t h e enzyme monoamine  Shaw a n d W a l l ,  octop-  2% t h a t o f n o r a d r e n a l i n e i n  Mammals i n c l u d i n g man e x c r e t e r a i s e d  mandelic  and i s o l a t e d  oxidase  1956; K a k i m o t o a n d A r m s t r o n g , 1960;  1962).  Correspondingly,  greater  q u a n t i t i e s o f o c t o p a m i n e u n d e r c o n d i t i o n s o f monoamine  oxidase  inhibition.  amine n o r m a l l y has rats  a very high  This  present  r a t and r a b b i t  contain 6-fold  demonstrated that n o t only  i n the tissues  turnover  tissues  r a t e as w e l l .  of these  i s octop-  animals,  Sympathetic  but i t  nerves o f  c o n t a i n d e t e c t a b l e amounts o f o c t o p a m i n e i n t h e a b s e n c e  o f monoamine o x i d a s e  inhibition,  indicating  t o M o l i n o f f and  173  Axelrod  (1969) the p o s s i b i l i t y that t h i s amine may  f u n c t i o n of i t s own 1) Proposed  in physiological  have a  processes.  Functions  A considerable  body of evidence has  accumulated which i s sug-  g e s t i v e of a f u n c t i o n f o r octopamine as a s y n a p t i c i n a v a r i e t y of i n v e r t e b r a t e 1974;  species  (Carpenter  transmitter  and  Gaubatz,  K r a v i t z , Talamo, Evans,. Wallace and B a t t e l l e ,  Robertson and  Carlson,  1976;  Robertson and J u o r i o , 1977;  Axelrod  and  Saavedra,  Nathanson, 1979).  1976; 1977;  Its role in  mammalian nervous t i s s u e however has been the source of erable speculation.  I t may  p e r i p h e r a l adrenoceptors and  Burks, 1974), and  consid-  a c t as a l e s s potent agonist  at  ( F i s c h e r , Horst and Kopin, 1965; K e l l y  s e v e r a l groups of i n v e s t i g a t o r s have pro-  posed t h a t octopamine f u n c t i o n s as a " f a l s e t r a n s m i t t e r " at these s i t e s  (Day  and Rand, 1963;  F i s c h e r , Mussachio and Horst,  Trendelenburg, 1963;  1964;  Kopin,  Kopin, F i s c h e r , Mussachio,  Horst and Weise, 1965). A f a l s e t r a n s m i t t e r has been d e f i n e d as a "substance which i s n o r m a l l y not present adrenergic  or present  i n only t r a c e amounts i n  nerves but which accumulates i n t e r m i n a l s  monoamine oxidase i n h i b i t i o n .  Thereafter  the f a l s e  t e r can be r e l e a s e d by nerve s t i m u l a t i o n but  elicits  responses at the s i t e of a c t i o n , compared w i t h (Kopin,  1966;  1968;1969; Kopin et a l . , 1966).  a l s o been termed a c o - t r a n s m i t t e r  after transmitdiminished  noradrenaline. Octopamine  ( M o l i n o f f and A x e l r o d ,  has 1972;  174  Jaim-Etcheverry (Boulton, t o be  and  Zieher,  1976), b u t  elucidated.  and  a definitive  The  proposal  modulates n o r a d r e n e r g i c merits  1975)  role  by  i n mammals has  Boulton  transmission  c o n s i d e r a t i o n i n view of  a neuromodulator  that  octopamine  a t c e n t r a l synapses  the p h y s i o l o g i c a l i m p l i c a -  tions  s u c h a mechanism w o u l d h a v e on  c u r r e n t views of  aptic  function.  a synaptic  of neuronal tigators and  In f a c t  a c t i v i t y has  i n various  K r a v i t z , 1976;  O'Shea and  Evans,  Briefly, including  receptors,  in  (Hoyle,  i n the p r o c e s s  direction.  1977;  these  on  transmission  of  mediated  noradrenaline.  t r a c e amines w h i c h a r e  of the neurone, might  amines  postsynaptic  modulation  t r a n s m i t t e r s s u c h as  as  trace  phenylethanolamine,  v i a presynaptic of synaptic  membranes i n s u c h a way activation  inves-  Battelle  the  act either d i r e c t l y  indirectly  cytoplasm  other  O'Shea,  (1976) s u g g e s t e d t h a t  tryptamine  or  modifier  1974;  E v a n s and  syn-  1979). '  Boulton  o r more o f the  b e e n d e m o n s t r a t e d by  invertebrates  by more " c o n v e n t i o n a l " One  as  octopamine, phenylethylamine,  t y r a m i n e and  release,  i t s role  L i n g l e , 1976;  yet  synthesized  interact with  to cause a continuous,  synaptic  partial  e i t h e r i n a d e p o l a r i z i n g or h y p e r p o l a r i z i n g This might  t h e n e u r o n e and  effectively  thereby  m i t t e r upon i t s r e l e a s e . d i s c u s s e d below i n l i g h t  " s e t the  modify  the  The  merits  of the  tone" of  action of of  results  with-  .  "prime"  the major  trans-  this hypothesis  w i l l be  of  study.  the present  175  Octopamine i s u n e v e n l y d i s t r i b u t e d brain with highest midbrain also  and  i n the  pocampus ng/g)  concentrations  spinal  cord  ( M o l i n o f f and  S a a v e d r a and Danielson,  Boulton  striatum,  as w e l l as  Axelrod,  Usdin,  1976; and  i n the  mammalian  hypothalamus,  ( c a . 10 n g / g ) .  cerebral cortex,  (3 - 10 ng/g)  i n the  I t has  been and  hip-  i n the p i n e a l g l a n d  (500  1972;  cerebellum  Saavedra,  1974;  Robertson,  1977).  The  secondary importance  i n t h e mammalian CNS.  ses  of t h i s  apparent  similarities  compound and  t h e s i z e d by  produces noradrenaline  (Pisano,  1960;  W i t k o p and  C r e v e l i n g , Daly,  appears the  same r e g i o n s aline in  of  C r e v e l i n g and  density gradients  reported  Horn,  t o be  1976)  and  i t a l s o sediments i n as  3  H—  suggesting  (Glowinski  and  noradrenaline  a  i n rough  has  and  M o l i n o f f and  1976;  Saldate  and  Orrego,  1976).  Finally,  1966;  been  from nervous  1971;  1972a,b; K o p i n e t a l . , 1965;  the  localization  t i s s u e upon s t i m u l a t i o n ( B a l d e s s a r i n i , Vogt,  of  noradren-  Iversen,  i t i s s t o r e d i n and  co-released with  It  terminals  Its distribution parallels  that of noradrenaline  Harmar and  1972),  1962).  i n nerve  sucrose  Axelrod,  syn-  Udenfriend,  Udenfriend,  and  arose  same enzyme w h i c h  system,  ( M o l i n o f f and  This view  of  Octopamine i s  nervous  synaptic vesicles..  fashion  the  to c o - e x i s t w i t h n o r a d r e n a l i n e  sympathetic  a role  i n p h y s i o l o g i c a l proces-  noradrenaline.  dopamine-3-hydroxylase,  1977;  o p i n i o n among  been t h a t octopamine p l a y s  the  Axelrod,  Buck, Murphy and M o l i n o f f ,  many r e s e a r c h e r s h a s  because of  detected  Baldessarini Buck,  octopamine i s  176  depleted  from t e r m i n a l s  (Garlsson Noach,  by t h e a d m i n i s t r a t i o n  a n d W a l d e c k , 1963; C h i n  of reserpine  A Paw, K n e g t - V e r p a l e n and  1968) o r 6 - h y d r o x y d o p a m i n e  ( B a l d e s s a r i n i , 1971).  Inasmuch a s t h e c e n t r a l a n d p e r i p h e r a l p r o p e r t i e s o f octopamine and n o r a d r e n a l i n e it  must n o t be t o o h a s t i l y  ties reflect  only  a p p e a r t o be c a u s a l l y r e l a t e d ,  assumed t h a t  a "metabolic  t h e s e common p r o p e r -  accident"  s e c o n d a r y r o l e f o r octopamine".  (Evans,  Few w o u l d d i s a g r e e  v i e w o f an i n d e p e n d e n t r o l e a s a s y n a p t i c dopamine, y e t t h i s  1978) o r  catecholamine  with the  transmitter f o r  i s a l s o t a k e n up b y t h e  same mechanism as i s n o r a d r e n a l i n e  and octopamine.  be  p r e s u m a b l y b y t h e same  stored  binding very  i n sympathetic  terminals  mechanism a s t h e o t h e r  much l i k e n o r a d r e n a l i n e  amines a n d i n s h o r t ,  i n a l l neurochemical  L a r g e l y because o f the advances a c h i e v e d cence h i s t o c h e m i s t r y ,  separate  neuronal  c a t e c h o l a m i n e s have been d e l i n e a t e d 1962; and  Falck,  Hillarp,  Dahlstrdm,  I t can  respects.  i n floures-  systems f o r t h e  i n the b r a i n  Thieme and T o r p ,  (Falck,  1962; H i l l a r p ,  1966; D a h l s t r o m and F u x e ,  are  u s e d due t o t h e a b s e n c e o f a s e c o n d h y d r o x y l  the  ring  sibility  therefore  CNS w h i c h n o r m a l l y mitter  Bjorklund  and L i n d v a l l ,  remains t h a t  there  methods  g r o u p on  1972).  The p o s -  e x i s t neurones i n the  employ o c t o p a m i n e a s a s y n a p t i c  but which respond  Fuxe  1964; 1 9 6 5 ) .  O c t o p a m i n e does n o t f l o u r e s c e when t h e s e b i o c h e m i c a l  (Axelsson,  functions  trans-  i n a s i m i l a r f a s h i o n a s do t h e  177  catecholamine  containing cells  to pharmacological  agents  ( e g : r e s e r p i n e , 6 - h y d r o x y d o p a m i n e , monoamine o x i d a s e  inhibi-  tors,  inver-  etc.).  tebrates  Indeed,  (Evans,  the  1978)  considerable evidence  f o r an  independant  physiological  f u n c t i o n o f o c t o p a m i n e p r o v i d e s an e x c e l l e n t conducting  experiments  physiological  and  i n mammalian  CNS.  pharmacological  o c t o p a m i n e i n mammalian CNS  a pharmacological vation  effects  for  Axelrod,  groups 1972;  Robertson, has  Harmar and  1977).  Horn,  1976;  These i n v e s t i g a t o r s  ence between the a l i n e having  two  i t s effect  1960;  found  possibly  many  reflect  concena  differ-  noradren-  p o o l than  u t i l i z e d whereas o n l y a p o r t i o n o f the  amine  noradren-  tissue  rapidly  1965).  obser-  M o l i n o f f and  that of  I n t h i s way  and A x e l r o d ,  as  available  amines i n s t o r a g e mechanism:  a much g r e a t e r i n t r a c e l l u l a r  is readily  The  that this  amine.  renaline  the  studies  b e e n made by  low p r e v a i l i n g  T h e s e o b s e r v a t i o n s may  of  D a n i e l s o n , B o u l t o n and  6 - f o l d g r e a t e r than  i n spite of r e l a t i v e l y  trations.  e f f e c t s has  for  substance  unknown.  ( K a k i m o t o and A r m s t r o n g ,  a rate of turnover  aline,  however, and  amounts o f o c t o p a m i n e a r e  i t to e x e r t p h y s i o l o g i c a l  different  of t h i s  physiological  agonist i s r e l a t i v e l y  that s u f f i c i e n t  rationale  aimed a t t h e c h a r a c t e r i z a t i o n  T h e r e h a v e b e e n e x c e e d i n g l y few with  from  octop-  a l l the newly s y n t h e s i z e d octopamine i s  available  f o r use  (Snyder,  tissue  norad-  Glowinski  178  2) R e c e p t o r s F o r i/  Octopamine  Invertebrates  as a s y n a p t i c port  The  likelihood  t r a n s m i t t e r has r e c e i v e d  specific  Octopamine  protein  phosphate  (cyclic  AMP)  Masden and B a r o n d e s , tained  stimulates  of molecular weight  a p p a r e n t l y by e l e v a t i n g  levels  1974).  These  action  and G r e e n g a r d  cyclic  AMP  of  120,000 i n A p l y s i a , adenosine-3,5'-mono1974;  Levitan,  i s main-  removal of octopamine  f r o m the  are i n accordance with a  consequence  of  transmitter  1978;  specificity cells  of e f f e c t  Gaubatz,  reveals  1977).  a marked  on some g a n g l i o n c e l l s  sensitivity i n Aplysia.  r e s p o n d by h y p e r p o l a r i z a t i o n v i a an i n c r e a s e i n  membrane c o n d u c t a n c e . renaline,  stimulation  (Robinson, Mobley,  Harmar and Horn,  I o n t o p h o r e s i s of octopamine  (1973),  (1976) , a l t h o u g h  o r c a u d a t e , t h e r e i s no  s y n t h e s i s by o c t o p a m i n e  S m i t h and S u l s n e r ,  which  the p h o s p h o r y l a t i o n  (1973), and N a t h a n s o n  i n mammalian h y p o t h a l a m u s  (1974)  on  of c y c l i c  results  sup-  out  d e s c r i b e d by G r e e n g a r d , K e b a b i a n and N a t h a n s o n  Nathanson  These  carried  This phosphorylation  c u r r e n t model o f p o s t s y n a p t i c  functions  t h e most t h o r o u g h  ( L e v i t a n and B a r o n d e s ,  f o r s e v e r a l hours a f t e r  i n c u b a t i o n medium.  and  octopamine  t h r o u g h t h e e x t e n s i v e work t h a t has b e e n  invertebrates.  of  that  This  effect  i s n o t e l i c i t e d by n o r a d -  p h e n y l e t h a n o l a m i n e o r dopamine 1974).  (Carpenter  S a a v e d r a , B r o w n s t e i n , C a r p e n t e r and  found asymmetrically  distributed  cells  and Axelrod,  i n t h i s mollusc  c o n t a i n no n o r a d r e n a l i n e o r dopamine, b u t p o s s e s s  179  quantities  o f o c t o p a m i n e as h i g h  finding provided  support  a s 3.7 p m o l / c e l l .  f o r the existence o f separate  tems f o r o c t o p a m i n e a n d n o r a d r e n a l i n e The  with  the s t r u c t u r e - a c t i v i t y  i t s r e c e p t o r , n o r d i d they  selective ists.  effects  Dougan a n d Wade  mollusc,  effects  1975).  adrenaline,  receptors. asing  apine  effects  o f another  t i s s u e by r e a c t i n g w i t h  dissimilar  receptors  noradrenaline  agonists  Octopamine i s e q u i p o t e n t  with  of the v e n t r i c u l a r  contractions.  a molar b a s i s with  ii/  Vertebrates  the  effects  cloz-  The o c t o p a m i n e  more s e n s i t i v e  t o t h e (+) f o r m ;  m e t h y l o c t o p a m i n e a n d ( - ) - a - m e t h y l o c t o p a m i n e were on  This  by metoelopramide,  20 t i m e s  o f (-) o c t o p a m i n e t h a n  a t these  dopamine i n i n c r e -  (Dougan a n d Wade, 1 9 7 8 b ) .  a p p e a r s t o be a b o u t  either  (Dougan, Wade a n d  catecholamines,  o f o c t o p a m i n e may be b l o c k e d  receptor  for possible stereo-  a r e much l e s s p o t e n t  or s u l p i r i d e  o f octopamine  beating ventricle  The o t h e r  the amplitude  effect  information  Dopamine may h a v e e x c i t a t o r y o r  on t h i s  o f two s t r u c t u r a l l y Mearrick,  test  species.  (1978a,b) c o n d u c t e d s u c h an i n v e s t i -  Tapes w a t l i n g i .  inhibitory  relations  sys-  o r examine t h e a c t i o n s o f amine a n t a g o n -  g a t i o n on the spontaneously  and  i n this  s t u d i e s on A p l y s i a d i d n o t p r o v i d e  regarding  This  tothe  (±)-Nequipotent  (±)-octopamine.  There have been o n l y  of phenylethylamines  a few s t u d i e s w h e r e i n  and c a t e c h o l a m i n e s  compared on n e u r o n e s o f t h e CNS, a n d t h e i r  results  were provide  180  little  a g r e e m e n t on t h e p o s s i b l e e x i s t e n c e  of separate  tor populations.  G i a r d i n a , Pedemonte and S a b e l l i  reported opposite  effects  ethylamine  on c o r t i c a l n e u r o n e s o f p h e n y l -  noradrenaline.  M o t o n e u r o n e s were  depres-  e q u i p o t e n t l y by s e r o t o n i n , i s o p r e n a l i n e , t y r a m i n e ,  mine, n o r a d r e n a l i n e  and o c t o p a m i n e  Kadzielawa,  O c t o p a m i n e was f o u n d  1976).  compared w i t h n o r a d r e n a l i n e Purkinje cells (Kostopoulos  provided  1975).  t o be a weak a g o n i s t the f i r i n g o f  f o r these  cells  octopamine a r e developed,  possible' explanation.  having  although  previous  (1971, 1973) noradrenergic  o f o c t o p a m i n e on  therefore not surprising,  receptors  However, s i n c e  S i g g i n s and Bloom  a smaller e f f e c t  noradrenaline  (Engberg, Flatman and  i n inhibiting  and Y a r b r o u g h ,  evidence  synapses,  dopa-  i n the c o r t e x o f the r a t c e r e b e l l a r vermis  e x p e r i m e n t s by H o f f e r ,  is  (1973)  and p h e n y l e t h a n o l a m i n e a p p l i e d i o n t o p h o r e t i c a l l y  when compared w i t h sed  recep-  until  the coexistence  adrenoceptors antagonists f o r  o f octopamine and  on t h e same n e u r o n e s r e m a i n s  another  S i m i l a r f i n d i n g s o f a weaker a c t i o n  o f o c t o p a m i n e compared w i t h n o r a d r e n a l i n e  o r dopamine on  n e u r o n e s o f t h e c e r e b r a l c o r t e x were r e p o r t e d by Henwood, Boulton  and P h i l l i s  e x c i t a t o r y responses  (1979).  nature  never  observed  t o any o f s e v e r a l amines t e s t e d ,  ding octopamine; n o r d i d they amine a n t a g o n i s t s  These authors  against  o f the receptors  examine t h e e f f e c t s  the depressions  involved.  inclu-  of specific  to determine the  181  The  e x t e n s i v e evidence  f o r a t r a n s m i t t e r r o l e o f octop-  amine i n i n v e r t e b r a t e s as w e l l as the r e l a t i v e l a c k o f pharmacological tigation.  s t u d i e s i n mammals prompted the p r e s e n t inves-  Neurones of the c e r e b r a l c o r t e x and d o r s a l horn o f  the s p i n a l cord were chosen f o r t e s t i n g w i t h octopamine and catecholamines  because the r e c e p t o r s f o r the l a t t e r compounds  i n these areas have been p h a r m a c o l o g i c a l l y c h a r a c t e r i z e d by many others Ryall,  ( K r n j e v i c and P h i l l i s ,  1963d; B i s c o e , C u r t i s , and  1966; Crawford and C u r t i s , 1966; Engberg and R y a l l ,  1966;  Roberts and Straughan, 1967; P h i l l i s ,  1968;  Johnson, Roberts,  Sobieszek  Tebecis and York,  and Straughan, 1969;  Engberg and M a r s h a l l , 1971; F r e d e r i c k s o n , Jordan  and P h i l l i s ,  1971;  Stone 1973; 1978; Jordan, McCrea, Steeves a n d M e n z i e s ,  1977;  Bradshaw, Bevan and Szabadi,  Phillis,  1978; f o r reviews see  1970; K r n j e v i c , 1974; T e b e c i s , 1974; Schwartz, 1975).  The r e s u l t s o b t a i n e d through the use o f pharmacologically s p e c i f i c amine a n t a g o n i s t s have g i v e n r i s e to the p r o p o s a l that octopamine e x e r t s an e f f e c t on c e n t r a l neurones which i s not dependent upon the presence of r e c e p t o r s f o r c a t e c h o l amines . (Hicks and McLennan, 1978a,b).  182  b)  Results  C e n t r a l n e u r o n e s were t e s t e d f o r t h e i r octopamine, n o r a d r e n a l i n e tion  of neurones i n the  cord  ( c a . 30%)  least  one  to only  150  The induced  cortical  and  firing  amine d i d n o t  and  8%  the  rates.  Those c e l l s  responded  to o n l y  of  the  one  40%  example o f  in this  i n the  this  cord.and  elicited  by  noradrenaline  c a t i o n o f the with  i n Figure  O c t o p a m i n e and  substance.  noradrenaline  was  the  considerably The  e j e c t i n g c u r r e n t ; by  by  octopamine l a s t e d o n l y maintained.  where t h e  d u r a t i o n of  the  as  contrast, long  as  the the  other  dopaonset An and  depression  o u t l a s t e d the  o b s e r v e d t o depend on  of  was  of n o r a d r e n a l i n e .  24b,  rate  cortex,  d i f f e r e n c e i n l a t e n c y of octopamine  i n given  at  responded  changes i n f i r i n g  account).  t i m e t h a n were t h o s e  noradrenaline  which  amines d i f f e r e d m a r k e d l y f r o m t h a t o f  Chapters  recovery  spinal  amine.  mine e f f e c t e d r a t e c h a n g e s w h i c h were more r a p i d i n and  propor-  n e c e s s a r i l y respond to a l l  compounds e m p l o y e d i n e x p e r i m e n t s (see o t h e r  ( c a . 20%)  responded to a l l three,  q u a l i t a t i v e nature by  Only a small  n e u r o n e s w h i c h were a f f e c t e d by  compound, 52%  two  to  r e s p o n d e d t o i o n t o p h o r e t i c a l l y a p p l i e d amines  r e s p o n d e d t o one of  dopamine.  cerebral cortex  t h r o u g h a change i n t h e i r  others:  and  responsiveness  appli-  after-effect the  magnitude  depression  caused  expelling current  183  Figure aline  24.  Examples  on t h e f i r i n g  of the e f f e c t s  c a t i o n o f octopamine respectively.  to both  with  T h i s c e l l was  and n o r a d r e n a l i n e  l o c a t e d 1.15  mm  (depth  o c t o p a m i n e and n o r a d r e n a l i n e .  d u r a t i o n o f e f f e c t between  in  (A).  (C) Neurone than  and i n h i b i t i o n  (B) A n o t h e r n e u r o n e  in  excitation  excitation  ( t h e open b a r s )  s u r f a c e o f the c o r t e x . responding  noradren-  r a t e s o f neurones of the c e r e b r a l c o r t e x .  (A) A n e u r o n e r e s p o n d i n g  bars)  o f o c t o p a m i n e and  t h e two  responding  to noradrenaline  (cell  d e p t h : 1.50  (closed  below the 1.34  mm)  Note d i f f e r e n c e  amines; o r d i n a t e  to octopamine w i t h  to appli-  a  s c a l e as  stronger  mm).  184  B  50  o  O) to  50  a m  50  75 75  to  I  'd CO  1  I min. 50  a  Octopamine  wm Noradrenaline  01 50  50  185  i/  Effects  and  Of  O c t o p a m i n e And  decreases  Noradrenaline  i n the r a t e s o f f i r i n g  neurone's were o b s e r v e d  i n response  Both  of cerebral  to the  u n i d e n t i f i e d neurones,  depressed  74  (Table was  9).  firing  The  different,  cells,  and  had  no  effect  significant  responses  opposite  t h e pH  o f the  effects.  octopamine.  their  firing  On  of less  26  cortical  depressed responses  of the  t h a n was  noradrenaline,  ejecting  c u r r e n t s r e q u i r e d to e l i c i t  magnitude.  The  in  lowering  the r a t e s o f f i r i n g  By  the  potent  two  particular  are by  shown  the  were u n a f f e c t e d  c e l l s which  effects t o be  o f the  two  noradrenaline  as a  intensities  same c r i t e r i o n n o r a d r e n a l i n e was than  no  neurones the  potency  the  amines a p p e a r e d  37  An  effects  a m i n e s , i t was  apparent by  9.  t h e r a n g e u s e d . (3.0  by b o t h  judged  46  shows  Of  I n F i g u r e 24a  The  rates decreased  t h a t o c t o p a m i n e was  test  (p > 0.36).  amine s o l u t i o n s o v e r  Potency E s t i m a t i o n s  of  the r e m a i n i n g  the x2  34  13  same n e u r o n e s  a s s o c i a t i o n between the  o f a n e u r o n e w h i c h was  e x c i t e d by  on  a  noradrenaline  the r a t e s o f f i r i n g  i s t h e o b s e r v a t i o n t h a t on  amines h a d  ii/  i t decreased  o c t o p a m i n e and n o r a d r e n a l i n e  note  and  as  From  i n c r e a s e d the r a t e s o f  o f c o n t i n g e n c y (Table 9) by  statistically of  and  a c t i o n o f o c t o p a m i n e upon t h e  e x c i t e d 42  analysis  of  cortical  amines.  sample p o p u l a t i o n o f 95 the  increases  of  other  -  by 5.2).  had  observed depressant  of  the  of equivalent similar 11  potency  neurones.  apparently  octopamine i n i n c r e a s i n g the n e u r o n a l  less f i r i n g rate  186  Figure  25.  dopamine  Responses o f a c o r t i c a l  neurone  ( d i v i d e d b a r ) and n o r a d r e n a l i n e  (depth  0.24  (solid bar).  mm)  to  187  OQ  Dopamine  188  of  6 cells.  When t h i s  An example o f t h i s  i s shown  type o f a c t i o n occurred,  other neurones t e s t e d with  artifact  illustrated  i s therefore  result.  Accurate  c a n n o t be o b t a i n e d w i t h o u t for  the e j e c t e d  iii/  to octopamine  ine  that  for this  duration,  usually  c a t i o n o f t h e amine.  responses.  u n l i k e l y to e x p l a i n the  q u a n t i t a t i v e measures o f potency numbers  The n e u r o n a l r e s p o n s i v e n e s s t o  responded  9 ) , i t was p o s s i b l e  the a l t e r a t i o n s i n f i r i n g  also  catecholamine.  e l i c i t e d by dopamine were o f  lasting  only  as l o n g  as t h e a p p l i -  Of 74 n e u r o n e s d e p r e s s e d by n o r a d r e n -  48 were d e p r e s s e d a l s o b y dopamine w h i l e (Figure  25).  were u n a f f e c t e d .  Of t h e s e  19 c e l l s ,  Of t h e 13 c e l l s  excited  not respond.  rate  amine were due t o an i n t e r a c t i o n  a l s o b y o c t o p a m i n e a n d 6 were d e p r e s s e d ;  did  could  (Chapter I ) .  Changes i n f i r i n g r a t e  9 were a l s o  that  t h e e f f e c t s o f o c t o p a m i n e a n d dopamine t o exam-  with a receptor  excited  evoked d e p r e s s a n t  (78 o f 95, s e e T a b l e  i n d u c e d by t h e p h e n o l i c  aline,  assembly  S i n c e many o f t h e s e n e u r o n e s a l s o  the p o s s i b i l i t y  short  observed  e x a m i n e d f o r a l l c e l l s w h i c h were a f f e c t e d by  noradrenaline.  contrast  24c.  a knowledge o f t r a n s p o r t  E f f e c t s Of Dopamine  dopamine was  to  compounds  also  t h e same p i p e t t e  y i e l d more p o w e r f u l n o r a d r e n a l i n e Electrode  i t was  i n Figure  10 were  Octopamine i n c r e a s e d  excited  the remaining  e x c i t e d by  by dopamine w h i l e  19 were  3  noradrenaline,  the other 4  cells  the f i r i n g o f 5  cells  189  TABLE 9.  Numbers o f u n i d e n t i f i e d  c o r t i c a l neurones  w i t h b o t h o c t o p a m i n e and n o r a d r e n a l i n e  tested  (n = 9 5 ) .  Octopamine  excitation Noradrenaline  depression no  effect  excitation  depression  no  effect  7:'  6  0  28  37  9  5  3  0  190  TABLE  10.  with both  Numbers o f u n i d e n t i f i e d o c t o p a m i n e a n d dopamine  c o r t i c a l neurones  tested  (n = 6 8 ) .  Octopamine  Dopamine  excitation  depression  excitation  6  10  4  depression  19  13  8  6  2  0  no  effect  no  effect  191  TABLE 11.  Numbers o f i d e n t i f i e d c o r t i c a l n e u r o n e s  w i t h b o t h o c t o p a m i n e and n o r a d r e n a l i n e  (n =  tested  31)  Octopamine excitation  Noradrenaline  excitation  4  depression  8  no  0  effect  depression  no 1  6  effect 3 8  1  0  192  which were e x c i t e d a l s o by both catecholamines, and one  other which d i d not  e x c i t e d by,dopamine.  respond to n o r a d r e n a l i n e  excited  but  was  Table 10 p r o v i d e s a more d e t a i l e d sum-  mary of the e f f e c t s of dopamine compared w i t h those of octopamine.  In s h o r t ,  the r e s u l t s i n d i c a t e that the e f f e c t s  of octopamine a l s o showed no p > 0.20)  significant association  w i t h those of dopamine, as was  of  noradrenaline.  iv/  I d e n t i f i e d C o r t i c a l Neurones  2  test  the case w i t h those  In an attempt to i d e n t i f y  those c o r t i c a l neurones which were a f f e c t e d by  low  doses of  the amines, s t i m u l a t i n g e l e c t r o d e s were p o s i t i o n e d t a x i c a l l y i n crus c e r e b r i and v e n t r o b a s a l thalamus (see Chapter I I ) .  (x  Thirty-one  stereo-  complex of  the  neurones a c t i v a t e d  by  a n t i d r o m i c s t i m u l a t i o n of crus c e r e b r i or which f i r e d at l a t e n c i e s of 5 to 12 msec, f o l l o w i n g thalamic  stimulation  were t e s t e d f o r t h e i r responses to i o n t o p h o r e t i c a l l y a p p l i e d amines, and  as the responses of these two  f i e d neurones to the a g o n i s t s r e s u l t s were pooled and the general  groups of  identi-  were i n d i s t i n g u i s h a b l e ,  are p r e s e n t e d i n Table 11.  the  Although  p a t t e r n of responses appears not markedly d i f f e r -  ent from that of the u n i d e n t i f i e d c e l l s o f Table 9, of the pyramidal t r a c t and e j e c t i o n currents  (up  thalamo-cortical  to 100  nA)  cells  were r e q u i r e d  even minimal e f f e c t s w i t h octopamine.  to  on a l l  high elicit  193  Figure  26.  A dose-response curve f o r depressant e f f e c t s  e l i c i t e d by octopamine a t a d o r s a l horn interneurone  acti-  vated a t short l a t e n c y by v o l l e y s t o the c e n t r a l end o f a severed d o r s a l r o o t . bars represent  Numbers i n parentheses i n d i c a t e t r i a l s ,  standard e r r o r o f the means.  r e c o r d o f 4 superimposed sweeps.  The t r a c e i s the  194  15 msec I  Current (nA)  ,  195  TABLE 12. to  their  C l a s s i f i c a t i o n o f d o r s a l horn neurones responses t o octopamine  according  and n o r a d r e n a l i n e .  (n=51)  Octopamine  excitation  depression  no  effect  excitation  8  2  0  depression  11  27  3  Noradrenaline  196  TABLE 13.  Characterization  of neuronal  responses  o f 26  s p i n a l n e u r o n e s t e s t e d w i t h n o r a d r e n a l i n e , dopamine a n d octopamine.  Dopamine  excitation  depression  Noradrenaline excitation depression  Numerator o f each f r a c t i o n e x c i t e d by o c t o p a m i n e w h i l e number  depressed.  were d e p r e s s e d these  2  1  T  T  4 5  7 5  indicates  t h e number o f c e l l s  the denominator r e p r e s e n t s the  F o r example, 9 o f t h e 26 c e l l s  tested  by n o r a d r e n a l i n e and e x c i t e d by dopamine; o f  9, o c t o p a m i n e e x c i t e d  4 and d e p r e s s e d  5.  197  v/ D o r s a l Horn Neurones  S i m i l a r f i n d i n g s to those w i t h  u n i d e n t i f i e d c o r t i c a l neurones were o b t a i n e d from d o r s a l horn neurones o f the s p i n a l cord.  These c e l l s were i d e n t i -  f i e d by t h e i r short l a t e n c y responses d o r s a l r o o t s (see Chapter  to s t i m u l i a p p l i e d to  I I and F i g u r e 26).  The p r i n c i p a l  f i n d i n g s are summarized i n Tables 12 and 13. Noradrenaline produced  p r i m a r i l y depressant  w h i l e the a c t i o n o f octopamine was more v a r i a b l e .  effects Opposite  e f f e c t s were o b t a i n e d on 37% o f the s p i n a l neurones responding both to n o r a d r e n a l i n e and octopamine.  Three c e l l s  depressed by n o r a d r e n a l i n e were not a f f e c t e d by octopamine a d m i n i s t e r e d w i t h i o n t o p h o r e t i c c u r r e n t s up to 100 nA. Dopamine was a l s o t e s t e d on 26 o f the 51 s p i n a l neurones, and the r e s u l t s are presented i n Table 13.  Again f o r a  p r o p o r t i o n o f the c e l l s the a c t i o n s e l i c i t e d by octopamine d i f f e r e d from those o b t a i n e d w i t h one or both o f the c a t e cholamines.  Due to the low number o f c e l l s i n some of the  c a r e g o r i e s i n contingency Tables 12 and 13, i t was not p o s s i b l e to analyze the r e s u l t s w i t h the x tical  independence.  2  test of s t a t i s -  However i t may be noted t h a t f o r the  data d i s p l a y e d i n Table 12 more than one t h i r d o f the c e l l s had o p p o s i t e responses  to octopamine and n o r a d r e n a l i n e ,  w h i l e Table 13 i l l u s t r a t e s c l e a r l y t h a t e f f e c t s w i t h  octop-  amine bore no g r e a t e r r e l a t i o n s h i p to those o f n o r a d r e n a l i n e than to those o f dopamine.  198  F i g u r e 27.  R e s p o n s e s o f two  (open b a r s ,  OCT)  upper t r a c e ; in  the  and  noradrenaline  o c t o p a m i n e and  dopamine  t h e u p p e r t r a c e , a n t a g o n i s m by  depressant  effect  of n o r a d r e n a l i n e  iontophoretic  c u r r e n t i s observed,  ses  c a u s e d by  octopamine  was  obtained  istration. the  NOR)  2.5  min  (40 nA)  after  N e u r o n e was  of  applied with the  the  40 nA  of  excitatory  below the  amines. of  respon-  Recovery  c e s s a t i o n o f the p r o p r a n o l o l mm  DOP)  (8 nA)  remain u n a f f e c t e d .  l o c a t e d 0.96  the  (divided bars,  propranolol  while  in  admin-  surface  of  cortex. The  thixol (75 nA) ses  ( s o l i d bars,  octopamine  lower t r a c e ; i n the p r e s e n c e of a n t a g o n i s t s  In the  and  neurones t e s t e d w i t h  lower  trace i l l u s t r a t e s  (55 nA) and  i n reducing  not  recovered  the  the  depressant  t h a t o f octopamine  to c o n t r o l l e v e l s  a-flupenthixol.(cell  d e p t h 0.45  selectivity  (also  effect 75 n A ) .  approximately mm).  5.5  of  a-flupenf  of  dopamine  The min  responafter  the  199  •  m  200  vi/  Effects  Of P r o p r a n o l o l And c t - F l u p e n t h i x o l  g a t i o n o f the e f f e c t s a n t a g o n i s t s was fically firing was  of noradrenaline  conducted  r a t e from those  effective  has  firing local  r a t e e l i c i t e d by octopamine. anaesthetic properties  neurones  ( o f 12 t e s t e d ) .  recovery  o f responses  those  27b),  28 a n d 2 9 ) .  depressions  one o t h e r was  dopamine w h i l e  and 7  spinal  achieved,  an  to both  noradrenaline.  from 5 c o r t i c a l  neurones  antagonist, a-flupenthixol.  the remaining  Of t h e s e  5 cells,  o c t o p a m i n e a n d dopamine  depressed  obser-  P r o p r a n o l o l was not  o f dopamine were a t t e n u a t e d  o f o c t o p a m i n e were s p a r e d .  ded w i t h  1966;  b u t n o t o f o c t o p a m i n e was  r e c o r d s were o b t a i n e d  the e f f e c t s  propranolol  i n a l l a t t e m p t s where a  t o c o n t r o l l e v e l s was  t h e dopamine r e c e p t o r  On a l l 5 c e l l s  decreases  t e s t s were achie-  ( o f 15 t e s t e d )  on n e u r o n e s w h i c h were e x c i t e d by  tested with  responses of  ( E n g b e r g and R y a l l ,  However,  against noradrenaline  Acceptable  Propranolol  Since  1973) c o m p l e t e  v e d . ( F i g u r e 27a, a n d F i g u r e s tested  changes i n  unaltered increases or  on o n l y 4 c o r t i c a l n e u r o n e s  effect  induced  o f the depressant  J o h n s o n e t a l . , 1969; S t o n e , ved  a n d dopamine r e c e p t o r  of the catecholamines.  as a b l o c k e r  investi-  t o d e t e r m i n e more s p e c i -  the independence o f octopamine  noradrenaline, which l e f t in  i n order  An  3  while respon-  (Figure  b y o c t o p a m i n e a n d e x c i t e d by c e l l was  and  depressed  b y dopamine.  the  responses  of s p i n a l neurones.  e x c i t e d by o c t o p a m i n e  a - F l u p e n t h i x o l was n o t t e s t e d on  201  Figure  28.  Rate r e c o r d  administered  with  15 nA  showing the  effect  of e j e c t i n g c u r r e n t  neurone, which responded to octopamine aline  (75 nA)  illustrates  min  and  opposite  selective  noradrenaline. t r a c e ) and  with  Time e l a p s e d  maximal e f f e c t  the  propranolol  to a s p i n a l  (75 nA)  changes i n r a t e .  antagonism of  time to r e c o v e r y  of  and The  depressant  noradrencentre effect  between c o n t r o l r e s p o n s e s  of antagonist  (middle  ( b o t t o m t r a c e ) was  5  inter-  of  (upper  t r a c e ) was min.  trace  8  202  203  Figure  29.  Rate r e c o r d showing  on d e p r e s s i o n s spinal  nolol  Antagonist  was  administered with  c u r r e n t o f 20 nA a p p l i e d f o r 2 min, w i t h  selective record.  of propranolol  e l i c i t e d by o c t o p a m i n e and n o r a d r e n a l i n e  interneurone.  ejecting  the e f f e c t  a n t a g o n i s m o b s e r v e d as i l l u s t r a t e d  Recovery occurred  ejection  phase.  approximately  3 min  the  i n the after  of a an resultant  centre the propra-  204  I  I min.  1  Noradrenaline  50 nA  ^3  Octopamine  65 nA  205  vii/  E f f e c t s Of  indicates  And  w h i c h can  clozapine  differentiate  phenylethylamine agonists  T a p e s w a t l i n g i (Dougan and two  Metoclopramide  t h a t m e t o c l o p r a m i d e and  antagonists and  Clozapine  on  effective  Accordingly,  effect  as  catecholamine  isolated ventricle  Wade, 1 9 7 8 b ) .  compounds were e x a m i n e d f o r an  are  between  the  A recent report  against  the  these amine  induced  responses o f ^ c o r t i c a l  and  ejected  i o n t o p h o r e t i c a l l y (14  cortical  and  3 s p i n a l c e l l s ) or  (4 c o r t i c a l  and  2 spinal  administered  intravenously  m e t o c l o p r a m i d e showed no the  amines.  recording  Clozapine  s p i n a l neurones.  of  Whether  cells),  a n t a g o n i s t i c a c t i o n t o w a r d any  was  from 8 c o r t i c a l  administered and  intravenously  5 s p i n a l neurones.  while  Regardless  o f the r o u t e  of a d m i n i s t r a t i o n f o r metoclopramide, both  compound and  clozapine  a l w a y s c a u s e d an  spontaneous r a t e of f i r i n g w i t h i n to  the  administration of  the  t i o n was higher as  abrupt  continued  f o r 5 or  Injections  6 min.  later.  These e f f e c t s  o f 0.9%  NaCl l e f t  of discharge,  and  the  while  unaffected.  i n neuronal  d o s e s were employed) w i t h  30 min.  rate  decrease  sees,  in the  amines s u p e r i m p o s e d on  e n h a n c e d b a c k g r o u n d a c t i v i t y was c a u s e d an  30  increase  firing  are  responses this  i f administra-  illustrated  amine i n d u c e d  the  time i f  o c c u r r i n g as  unaltered both  this  Metoclopramide  (or a s h o r t e r  recovery  of  the  long  i n Figure spontaneous  changes i n  firing.  30.  206  Figure  30.  Effects  firing  of c o r t i c a l  o f c l o z a p i n e a n d m e t o c l o p r a m i d e on t h e and s p i n a l neurones i n response  Upper t r a c e : A c o r t i c a l n e u r o n e r e s p o n d i n g sions  t o octopamine and n o r a d r e n a l i n e .  administered  i n t r a v e n o u s l y caused  the b a s e l i n e f i r i n g interpreted observed  without  as s e l e c t i v e  on s p o n t a n e o u s  w i t h weak  Clozapine  a l l 3 amines t e s t e d .  etically of  with  having  (due t o t h e d i r e c t  the c e l l  obtained  ceased,  with  depression  Metoclopramide administered  without  and r e c o v e r y  a l s o caused selective  terminated.  iontophor-  an enhancement  antagonism, and  f o r 5.5 m i n . , t h e f i r i n g o f the responses  f o r a s l o n g as 1.5 h r . a f t e r  m e t o c l o p r a m i d e was  effect  activity).  when a d m i n i s t r a t i o n was c o n t i n u e d of  (20 mg/kg)  a n e f f e c t w h i c h c o u l d be  antagonism  a c u r r e n t o f 12 nA  the background f i r i n g ,  depres-  a pronounced i n c r e a s e i n  Lower t r a c e : A s p i n a l n e u r o n e r e s p o n d i n g to  t o amines.  c o u l d n o t be  the a d m i n i s t r a t i o n of  Clozapine  20mq/kg  100  50  50  Q.  co50  Metoclopramide  0  25 25  12 nA  25  N3  1  i I min.  a  Octopamine  nn Dopamine  ™ Noradrenaline  >i  208  c) The  Discussion first  major c o n c l u s i o n  ments i s t h a t o c t o p a m i n e mammalian CNS, ound. of  b o t h as  Furthermore  this  arising  i s pharmacologically  an  e x c i t a t o r y and  i t i s clear  from the  s t u d y w h i c h compared t h e  catecholamines,  active in  a depressant results  actions  experi-  of  of  the  comp-  the  part  octopamine  and  t h e s e compounds d i f f e r  in their  pharma-  cological properties  as  in their  respon-  ses  O c t o p a m i n e t h e r e f o r e has  to a n t a g o n i s t s .  w h i c h c a n n o t be (Fischer  The  the  thereby  effector  by  Kopin,  The  which i s blocked  by  Noradrenaline be  a c t i v e as  both are the  synaptic  believed  cerebral cortex  Since  the  effects  r e l a t e d with  of  similar  to  I t must, m o r e o v e r , of  the  transmitters contained  (Tebecis,  transmitter.  the  synaptic  transmission  that  exerted  effect  transmitter  Emson and  at  therefore  i n f i b r e s which  the  as  a r e c e p t o r which  a  by  response  agent.  i n t h e mammalian CNS  1974;  of  hypothesis  generally considered  of octopamine are not  those of e i t h e r  actions  transmitter  t r a n s m i t t e r must  dopamine a r e  t o be  false  efficacy  antagonists  and  A  transmitter with  false  certain  transmitter  f o r the normal  produce a response q u a l i t a t i v e l y the normal t r a n s m i t t e r .  as  i t s a c t i o n on  decreases the  site.  false  1968).  specific false  as w e l l  the  (1968) e x e r t s  pharmacologically  receptor the  Kopin  r e a c t i o n of  agonists  explained  e t a l . , 1965;  d e f i n e d by is  that  from the p r e s e n t  to and  innervate  L i n d v a l l , 1979).  significantly  cor-  catecholamines, nor  are  209  they a f f e c t e d by adrenoceptor agents,  i t seems r e a s o n a b l e  pharmacological Indeed,  role  other  o r dopamine r e c e p t o r b l o c k i n g  t o a s c r i b e t o octopamine a than  as a f a l s e t r a n s m i t t e r .  s i n c e on many o c c a s i o n s  t h e r e were o b s e r v e d  of octopamine o p p o s i t e t o those this  indicates  a direct  effect  o f o c t o p a m i n e were i n d i r e c t , release  of catecholamines,  w i t h octopamine aline  cells  I f the a c t i o n  i . e . t o cause a p r e s y n a p t i c  then a g a i n  obtained with  the e f f e c t s those  observed  o f noradren-  the catecholamines  a n t i d r o m i c a l l y by p y r a m i d a l  t h a t most o f t h e s e  those  o f Stone  (Stone,  (1973).  c e l l s were d e p r e s s e d  t h e e f f e c t s were s l o w i n o n s e t  stimu-  This author  results  and those of c e l l s  cholamines,  and r e l a t i v e l y  induced  The  prolonged blocker  One d i f f e r e n c e b e t w e e n t h e p r e s e n t  o f Stone i s t h e o b s e r v a t i o n t h a t a s m a l l  o f the pyramidal  while  found  by n o r a d r e n a l i n e ,  1978) a n d t h a t p r o p r a n o l o l was a s e l e c t i v e  the depressions.  number  tract  on c o r t i c a l  o r s y n a p t i c a l l y b y s t i m u l a t i o n o f VB t h a l a m u s a r e i n  good agreement w i t h  on  on r e c e p t o r s .  s h o u l d be c o r r e l a t e d w i t h  results  activated  lation  of  o f n o r a d r e n a l i n e a n d dopamine  o r dopamine.  The  that  actions  t r a c t were e x c i t e d b y c a t e -  Stone d i d n o t d e s c r i b e such  noradrenaline  excitations.  o b s e r v a t i o n t h a t o c t o p a m i n e i s a weak a g o n i s t  the i d e n t i f i e d  not possess  cortical  neurones  suggests  only  t h a t t h e y do  r e c e p t o r s f o r o c t o p a m i n e and t h a t t h e e f f e c t s o f  210  this  amine a r e p e r h a p s  tors.  I t . was  that  t h e CNS  exist  f o r octopamine  the o b s e r v a t i o n t h a t  cells,  and Y a r b r o u g h ,  1975;  and w i t h more c l e a r l y  D o r s a l horn neurones  low  short doses  latencies  McLennan,  1978b).  at  (0.4  depths  t o a c t as  Ryall,  cortical  pyramidal  synaptic  Carlsson, 1966;  T h i s may  The  be  octopamine,  and  responded  (Biscoe,  Fuxe and H i l l a r p , 1977;  Curtis 1964;  receptors  cells,  sensitive  sarily predict  since  the  Jordan et a l . ,  presence  of responsiveness to  and  1977).  studies of  oct-  o f . neuronal  t o t h e c a t e c h o l a m i n e s does n o t  the degree  cord  Ryall,  Engberg  aimed a t d e t e r m i n i n g t h e p h a r m a c o l o g i c a l p r o p e r t i e s opamine s e n s i t i v e  and  believed  and  important c o n s i d e r a t i o n f o r future  by  might  r e c e p t o r s (Hicks  where c a t e c h o l a m i n e s a r e  N y g r e n and O l s o n , an  c o r d which  a c t i v a t i o n were a f f e c t e d  specific  transmitters  Falck,  sensitive  c e l l s were f o u n d i n t h e s p i n a l  mm)  tract  changes i n r a t e .  i n the s p i n a l  octopamine  by  t o c a t e c h o l a m i n e s more  defined  to synaptic  - 1.3  systems i n  ( c e r e b e l l a r P u r k i n j e cells,  and  o f t h e amines i n c l u d i n g  therefore possess  1966;  independent  H i c k s and McLennan, 1978a) a r e o n l y w e a k l y  readily  The:present  and n o r a d r e n a l i n e i s s u p p o r t e d  some n e u r o n e s  recep-  electrophysiological^  s e p a r a t e and  t o t h e p h e n o l i c amine b u t r e s p o n d  at  catecholamine  c e l l s most r e s p o n s i v e t o o c t o p a m i n e .  t h e r e may  Kostopoulos  through  not p o s s i b l e , to i d e n t i f y  those c o r t i c a l view  mediated  neces-  octopamine.  211  The for  results with  two a n t a g o n i s t s  offer  the e x i s t e n c e o f octopamine r e c e p t o r s  used by t h e c a t e c h o l a m i n e s . r e s p e c t i v e l y prevented decreases  attenuated  suggests  the e f f e c t s  amines.  The r e s u l t s  distinct  i n part  Neither  elicited  that receptors mediating  are p h a r m a c o l o g i c a l l y  noradrenaline  from those  a chlorbenzamide  of the catecholthe observations  few e x t r a p y r a m i d a l  Burki,  Ruch, A s p e r , B a g g i o l i n i and M i l l e r ,  antagonists lingi  derivative with  (Ahtee,  effects  of the receptors  as a n t a g o n i s t s  anti-  1975; J e n n e r ,  (Anden a n d S t o c k ,  and S t i l l e ,  1973;  1974; F o g , 1975;  f o r octopamine i n Tapes wat-  1978).  T h e s e two compounds h a v e no  f o r a n y o f t h e amines t e s t e d i n t h e  cortex or spinal  cord o f the r a t ; indeed  actions  of their  own on t h o s e  actions  c a n n o t be e x p l a i n e d on t h e b a s i s o f a  reaction with  with  1975) have b e e n d e s c r i b e d as e f f e c t i v e  (Dougan and Wade,  effect  effects of  1975) and c l o z a p i n e , a n e u r o l e p t i c  having  Kelly  This  neurones.  and c a t a l e p t i c p r o p e r t i e s  Marsden and P e r i n g e r ,  antag-  the a c t i o n s o f octopamine  a n d p h e n y l e t h a n o l a m i n e when compared  Metoclopramide, emetic  similar  by octopamine.  a r e i n agreement w i t h  on c o r t i c a l  those  induced  o f these  o f G i a r d i n a e t a l . , (1973) who r e p o r t e d o p p o s i t e phenylethylamine  from  a n d dopamine  rate, confirming  f i n d i n g s b y Bradshaw e t a l . , ( 1 9 7 8 ) . onists  distinct  evidence  P r o p r a n o l o l and a - f l u p e n t h i x o l  noradrenaline  i n the f i r i n g  further  octopamine  they had powerful  n e u r o n e s examined.  receptors.  These  competitive  212  In c o n s i d e r a t i o n o f the present to e v a l u a t e activator  the proposal  (Boulton,  1978).  This  exerts  direct  study  sitizing  t h a t o c t o p a m i n e a c t s as a s y n a p t i c  h a s shown t h a t o c t o p a m i n e most  from those  of the catecholamines.  e f f e c t by octopamine o f n o r a d r e n a l i n e  does n o t o f i t s e l f until  lator"  concerning  an a c c e p t a b l e  the r o l e  Caution that  provide  i s presented,  evidence  such experiments add l i t t l e  the  so f a r as c o r t i c a l  It is difficult  states e x p l i c i t l y  activator tic  information  to conceive  accepting  the view  since the e f f e c t of than  that o f norad-  e x c i t a t i o n s are o f a compound than  having  the sus-  n a t u r a l t r a n s m i t t e r , e s p e c i a l l y when t h e t u r n o v e r o f  compound i s so h i g h  1978)  role,  o f octopamine i n s y n a p t i c f u n c t i o n .  a m o d u l a t o r y r o l e when i t a p p e a r s more p o t e n t pected  (1975)  d e s c r i p t i o n o f t h e t e r m "neuromodu-  octopamine appears o c c a s i o n a l l y s t r o n g e r  involved.  sen-  evoked r e s -  f o r a modulatory  i s warranted f u r t h e r before  at least  A  and Y a r b r o u g h  octopamine i s a s y n a p t i c a c t i v a t o r ,  renaline,  probably  on r e c e p t o r s w h i c h c a n be d i s t i n g u i s h e d  p o n s e s as d e m o n s t r a t e d by K o s t o p o u l o s  and  i t is difficult  1976; 1978; B a l d e s s a r i n i a n d F i s c h e r ,  effects  pharmacologically  results,  should  i n the normal s t a t e . i n h i s hypothesis  readiness  f o r the c o n v e n t i o n a l  Further  experiments w i l l  (1976;'  that the synaptic  cause o n l y minor p o s t s y n a p t i c  effects while maintaining  Boulton  or presynap-  the synapse i n a s t a t e o f transmitter.  be r e q u i r e d b e f o r e  i t c a n be  213  concluded t h a t octopaminergic t r a n s m i s s i o n occurs i n mammali a n nervous t i s s u e .  However the r e s u l t s d e s c r i b e d here  do  o f f e r experimental support f o r the e x i s t e n c e of r e c e p t o r s s e n s i t i v e to octopamine which are independent  of those recep-  t i v e to the catecholamines, and i t i s c o n c e i v a b l e that may  be i n v o l v e d i n separate s y n a p t i c p r o c e s s e s .  they  214  CHAPTER V I  SUMMARY AND The  CONCLUDING REMARKS  results, presented i n this  thesis provide  p e r m i t s an a n a l y s i s o f s e v e r a l receptors  unclear, The  the exact  other  amino a c i d s  transmitters  the c h o l i n e r g i c  d i f f e r e n t regions  pharmacological properties although  The e x p e r i m e n t s w i t h ACh a n d  d r u g s h a v e shown t h a t  of neurones from s e v e r a l  and  d i f f e r e n t systems o f n e u r o n a l  i n t h e CNS o f t h e r a t .  cholinomimetic  evidence which  possess d i f f e r e n t  from those d e s c r i b e d  are presently  e x c e l l e n t c a n d i d a t e s as  i n mammalian CNS.  receptors  synaptic  regions  describing acids.  have c o n f i r m e d  and e v i d -  r o l e s as t r a n s m i t t e r s o f  o f mammalian CNS.  i n part,  the present  Several  results  the s e l e c t i v e antagonism o f the e x c i t a t o r y  amino  O c t o p a m i n e h a s y e t t o be p r o v e n as a compound o f i m p o r -  t a n c e i n t h e mammalian n e r v o u s  system,  a p p e a r s t o be a m a j o r t r a n s m i t t e r species.  synaptic  pharmacologically  i n t h e t h a l a m u s and s p i n a l c o r d ,  e x c i t a t i o n i n other  results. '  T h e s e compounds have b e e n shown  for their  reports  remains  i n v e s t i g a t o r s have c o n f i r m e d t h e p r e s e n t  ence i s r a p i d l y a c c u m u l a t i n g  recent  i n the c a t ,  nature o f these receptors  t o r e a c t w i t h a t l e a s t two, a n d p e r h a p s o t h e r distinct  receptors  of receptors  the f a c t  i n a number o f  The r e s u l t s o f t h e p r e s e n t  probable existence  despite  experiments  that i t  invertebrate showing t h e  f o r o c t o p a m i n e on c e n t r a l  215  neurones i n d i c a t e s that the  a more s y s t e m a t i c  investigation into  possible physiological roles for this  amine s h o u l d  be  conducted.  Despite  the  fact  that  there  of pharmacologically  specific  pounds t e s t e d i n t h e  present  ded  that  they are  mission. is  only  role  As one  as  the  a synaptic  compound. logical  can  experiments,  transmitter  the  r o l e of  Another experiments  may  be  used to b l o c k  synaptic  presence of fulfilled  i n Chapter  cortical  and  therefore  those  sampling of  the  were e x c i t e d by to  thalamic  do n o t  the  trans-  receptors before  a  o f known pharmacothe  f o r the  effects  of  dentate  f o r a p a r t i c u l a r com-  described  borne i n mind  h e r e i s the  i n Chapter  I.  with  influence  The  f r o m a sample o f  results  t o t a l population,  of repor-  somatosensory  n e u r o n e s w h i c h r e s p o n d e d t o ACh,  necessarily constitute a  ACh  conclu-  greatly.  I I I were o b t a i n e d  VB  com-  t o any p a r t i c u l a r  selectively  c o n s i d e r a t i o n w h i c h must be s u c h as  the  synaptic  assigned  transmitter  sampling b i a s , mentioned a l s o  equally  the  a c t i v a t i o n , s u c h as were d e s c r i b e d  pound i s s t r e n g t h e n e d  ted  i n processes of  i n Chapter I,  multiplicity  i t c a n n o t be  c r i t e r i a w h i c h must be  be  a  f o r many o f  However i n c a s e s where a n t a g o n i s t s  actions  synaptic gyrus,  of  receptors  a l l involved  discussed  a p p e a r s t o be  and  representative  for only  those  cells  which  were c h o s e n f o r s t u d y .  This  point  applies  experiments w i t h  the  a m i n e s , o u t l i n e d i n C h a p t e r V.  216  The  experiments with  different these  e m p h a s i s f r o m the  f o r the  from these ferences upon an  using  last  effect  antagonists.  As  dentate  gyrus  of agonists i t was  due  diffuse  the  t h a n an  The  for  absolute  conclusions  antagonists,  rather  i s encountered,  dif-  than  f o r example,  i s the p o s s i b l e i n f l u e n c e of  (Chapter may  there  to the  i n order  to the  administered  e x p e r i m e n t s on  IV,  reflect  part  2),  s u g g e s t e d t h a t the relative greater  agonists  granule  and  cells  in  sensiti-  differences i n innergreater  t o g l u t a m a t e on  apparent  these  cells  d i s t a n c e over which glutamate  f o r i t to r e a c t w i t h I t may  neuronal  differential  topographic  be  Perhaps n i c o t i n i c  cholinergic receptors  had  distally-located  t h a t a s i m i l a r phenomenon a c c o u n t s  d i f f e r e n c e s between n i c o t i n i c  c e n t r a l neurones. o n l y on  s u c h as  seen i n the  synaptic receptors. for  to the  responses  potency of aspartate c o u l d be  receptors  c o n s i d e r a t i o n r e l e v a n t t o t h e p r o b l e m s f a c e d when  iontophoresis,  vation;  rather  somewhat  c h o l i n e r g i c systems.  the  to  i n t h a t the  range o f e x c i t a n t s t e s t e d .  in susceptibility  m o r p h o l o g y on  vity  others,  a  e x p e r i m e n t s were b a s e d t h e r e f o r e upon r e l a t i v e  absolute  the A  the  amino a c i d s p o s s e s s e d  compounds d e m o n s t r a t e d a r e l a t i v e  affinity  with  the  and  muscarinic  excitants exert  l o c a t e d on  or near  actions their  to the  Compounds s u c h as A6MC must d i f f u s e  f u r t h e r i n order  excite  the neurone,  receptors  "muscarinic"  may  effects  cell  body.  s i n c e the  on  be  to  217  l o c a t e d d i s t a l l y to the c e l l body. f o r the d i f f e r e n c e s  I f this situation  of onset and o f f s e t  latencies  accounts  observed w i t h  the n i c o t i n i c and m u s c a r i n i c a g o n i s t s , t h e r e remains l e s s a r e a l p h a r m a c o l o g i c a l d i s t i n c t i o n between these  neverthetwo  c l a s s e s of compounds. 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