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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., Carleton University, 1973 B.Sc, Dalhousie University, 1976 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE- OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Department, of Physiology) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA August, 1979 © T. P h i l i p Hicks, 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 f o r 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 t h a t the L i b r a r y 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 g r a n t e d 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 . I t i s u n d e r s t o o d 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 not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f FMSlOUXtf The U n i v e r s i t y o f 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 D E - 6 B P 75-5 I I E i i ABSTRACT The e f f e c t s o f a range of neuronal e x c i t a n t s were exam-i n e d on the f i r i n g o f c e n t r a l neurones of the c e r e b r a l c o r t e x , v e n t r o b a s a l thalamus, dentate gyrus 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 of urethane a n a e s t h e t i z e d r a t s . These responses were 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 on the b a s i s o f t h e i r s u s c e p t i b i l i t i e s t o a number of a n t a g o n i s t s and from these r e s u l t s , i n f e r e n c e s were made concerning probable r e c e p t o r mechanisms employed by the a g o n i s t s . Throughout these experiments the technique of i o n t o p h o r e s i s was found to be an i d e a l one f o r e v a l u a t i n g the e f f e c t s o f a g o n i s t s and antagon-i s t s on s i n g l e neurones. Neurones i n the cortex , thalamus and Renshaw c e l l s o f the s p i n a l 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 . These r e s -ponses were e l i c i t e d a l s o by both n i c o t i n i c and m u s c a r i n i c c h o l i n o m i m e t i c s . E x c i t a t i o n s produced by a c e t y l c h o l i n e and a c e t y l - B - m e t h y l c h o l i n e were antagonized by a t r o p i n e and those of a c e t y l c h o l i n e and n i c o t i n i c a g o n i s t s were b l o c k e d by n i c o t -i n i c a n t a g o n i s t s . The r e s u l t s may be i n t e r p r e t e d as r e v e a l i n g a d i f f e r e n c e between e x c i t a t o r y c h o l i n e r g i c r e c e p t o r s i n the r a t and i n the c a t ; the nature of these r e c e p t o r s i s discussed. to The e x c i t a t o r y responses of v e n t r o b a s a l thalamic neurones i o n t o p h o r e t i c a l l y a p p l i e d amino a c i d s r e l a t e d to glutamate i i i and a s p a r t a t e c o u l d be b l o c k e d both by glutamate d i e t h y l e s t e r and a-aminoadipate. These two a n t a g o n i s t s were found to pos-sess d i f f e r e n t mechanisms of a c t i o n however, as the r a n k i n g orders of s u s c e p t i b i l i t y o f the a g o n i s t s d i f f e r e d f o r each an-t a g o n i s t . An a n a l y s i s o f these orders l e d to the p r o p o s a l t h a t more than one and p o s s i b l y as many as three d i f f e r e n t r e c e p t o r s f o r the e x c i t a t o r y amino a c i d s e x i s t on c e n t r a l neurones. A number of a d d i t i o n a l compounds were t e s t e d f o r an e v a l u a t i o n of t h e i r a n t a g o n i s t i c p r o p e r t i e s a g a i n s t the amino a c i d induced responses, and these r e s u l t s are d i s c u s s e d i n l i g h t o f p o s s i b l e s t e r i c requirements of the r e c e p t o r s . Granule c e l l s of the dentate gyrus were e x c i t e d by the amino a c i d s and by t h e i r s y n a p t i c responses to s t i m u l a t i o n of p e r f o r a n t path and commissural i n p u t s . A d i f f e r e n t i a l e f f e c t -i v eness o f glutamate d i e t h y l e s t e r and a-aminoadipate was sug-g e s t i v e that two d i s t i n c t e x c i t a t o r y amino a c i d r e c e p t o r s , both of which appear to be of s y n a p t i c s i g n i f i c a n c e , c o e x i s t on the same neurones. The e f f e c t s o f octopamine were compared w i t h those of catecholamines on neurones of the c o r t e x and d o r s a l horn of the s p i n a l cord. Both e x c i t a t i o n and d e p r e s s i o n of neuronal f i r i n g was observed w i t h octopamine and these responses appeared not\to.,be c o r r e l a t e d w i t h those e f f e c t e d by the catecholamines. A f u r t h e r s e p a r a t i o n of the a c t i o n s of octopamine and the 1 a I V catecholamines was evident when the amine induced responses were compared i n the presence of the antagonists, propranolol and a-flupenthixol. These blocking compounds were e f f e c t i v e i n attenuating the effects of the catecholamines, but had no eff e c t upon the octopamine induced changes i n f i r i n g rate. The r e s u l t s suggest that receptors sensitive to octopamine and which appear to be pharmacologically d i s t i n c t from those pre-viously categorized as catecholamine receptors, may exist on central neurones of the rat. On the basis of the present findings, i t was evident that when the technique of iontophoresis i s combined with standard neurophysiological methods of i d e n t i f y i n g central neurones by their responses to synaptic stimulation, valuable information can be obtained concerning the nature of the synaptic trans-mitters employed by these c e l l s . V TABLE OF CONTENTS C e r t i f i c a t e o f Examination i A b s t r a c t i i L i s t of Tables i x L i s t o f F i g u r e s x i L i s t of A b b r e v i a t i o n s x i v Sources of Drugs x v i i Acknowledgements x v i i i Chapter I: General I n t r o d u c t i o n 1 a) Chemical Tran s m i s s i o n 1 b) C r i t e r i a For T r a n s m i t t e r s 5 c) I o n t o p h o r e s i s 8 d) The Present Study 16 Chapter I I : M a t e r i a l s and Methods 17 a) S u p r a s p i n a l Experiments 19 b) S p i n a l Cord Experiments 20 ' c) S t i m u l a t i o n and Recording Procedures 21 d) Recording From S i n g l e Neurones and Drug D e l i v e r y Technique 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 A n a l y s i s 29 g) H i s t o l o g i c a l V e r i f i c a t i o n o f E l e c t r o d e S i t e s 29 Chapter I I I : A c e t y l c h o l i n e 31 a) I n t r o d u c t i o n 31 1) H i s t o c h e m i c a l 32 2) Recovery 33 v i 3) I o n t o p h o r e s i s 34 i / Cortex 34 i i / Thalamus - 41 i i i / S p i n a l Cord 46 b) R e s u l t s 51 i / Cortex 51 C h o l i n e r g i c A g o n i s t s 51 C h o l i n e r g i c A n t a g o n i s t s 56 i i / V e n t r o b a s a l Thalamus.... 61 C h o l i n e r g i c A g o n i s t s 61 C h o l i n e r g i c A n t a g o n i s t s 69 i i i / Renshaw C e l l s 74 E x c i t a t o r y Responses 74 C h o l i n e r g i c A n t a g o n i s t s 77 c) D i s c u s s i o n 82 P o s s i b l e Receptor Mechanisms 84 Chapter IV: Amino A c i d s 88 (Ve n t r o b a s a l Thalamus) a) I n t r o d u c t i o n 88 1) Uptake and Release 88 2) H i s t o l o g i c a l F i n d i n g s 91 3) I o n t o p h o r e s i s . 94 A g o n i s t s 94 An t a g o n i s t s 105 -4) Experimental R a t i o n a l e s 106 i / A n t a g o n i s t s : GDEE 107 i i / A n t a g o n i s t s : aAA 108 v i i i i i / A n t a g o n i s t s : KDEE, 2A3P, 2A4P and BAA 109 i v / A g o n i s t s : ADCP and BAG 110 v/ A g o n i s t s : K a i n i c a c i d 112 b) R e s u l t s 113 i / E x c i t a n t Mechanisms. 113 i i / E f f e c t i v e A n t a g o n i s t s . . 116 i i i / Other A n t a g o n i s t s 135 i v / BAG 139 c) D i s c u s s i o n 144 E f f e c t s o f Other Analogues 150 Amino A c i d s (Continued) Dentate Gyrus 155 a) I n t r o d u c t i o n . 155 b) R e s u l t s 157 c) D i s c u s s i o n 170 Chapter V: Octopamine 172 a) General Overview 172 1) Proposed F u n c t i o n s 173 2) Receptors For Octopamine 178 i / I n v e r t e b r a t e s . . 178 i i / V e r t e b r a t e s 179 b) R e s u l t s . . 182 i / E f f e c t s of Octopamine and Nor a d r e n a l i n e 185 i i / Potency E s t i m a t i o n s 185 i i i / E f f e c t s o f Dopamine 188 i v / I d e n t i f i e d C o r t i c a l Neurones 192 v i i i v/ D o r s a l Horn Neurones 197 v i / E f f e c t s o f 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 200 v i i / E f f e c t s o f C l o z a p i n e And Metoclopramide 205 c) D i s c u s s i o n 208 Chapter VI: Summary and Concluding Remarks 214 References 218 i x LIST OF TABLES Table Page 1 Drug S o l u t i o n s 24 2 Numbers of c h o l i n o c e p t i v e neurones of the 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 drugs . . 52 3 Numbers of c h o l i n o c e p t i v e neurones o f the v e n t r o b a s a l complex of the thalamus responding to n i c o t i n i c and m u s c a r i n i c drugs 68 4 R e l a t i v e i o n t o p h o r e t i c "doses" o f a-amino-a d i p i c a c i d and glutamate 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 of each of a range of neuronal e x c i t a n t s 117 5 Summary of e f f e c t s of L-glutamic a c i d d i e t h y l e s t e r a g a i n s t p a i r s of neuronal e x c i t a n t s . 122 6 Summary of e f f e c t s of a-aminoadipate a g a i n s t p a i r s of n euronal e x c i t a n t s • 128 7 Summary of the r e s u l t s of potency d e t e r -minations and antagonism o f 3-amino-g l u t a r a t e 143 8 R e l a t i v e p o t e n c i e s .of L-glutamate and L - a s p a r t a t e t e s t e d on dentate granule c e l l s 160 9 Numbers of u n i d e n t i f i e d c o r t i c a l neurones t e s t e d w i t h both octopamine and norad-r e n a l i n e 189 10 Numbers o f u n i d e n t i f i e d c o r t i c a l neurones t e s t e d w i t h both octopamine and dopamine 190 11 Numbers of i d e n t i f i e d c o r t i c a l neurones t e s t e d w i t h both octopamine and norad-r e n a l i n e 191 12 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 octop-amine and n o r a d r e n a l i n e 195 X Table (cont'd) Page 13 C h a r a c t e r i z a t i o n o f neuronal responses of 26 s p i n a l neurones t e s t e d w i t h noradren-a l i n e , dopamine and octopamine 196 x i LIST OF FIGURES F i g u r e Page 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 A3MC, and the e f f e c t s o f the n i c o t i n i c a n t a g o n i s t , DH3E 54 2 Responses of two c o r t i c a l neurones to ACh and cholinomimetics v.; '. . . , 58 3 Responses of a c o r t i c a l neurone to ACh and the e f f e c t o f the n i c o t i n i c a n t a g o n i s t , mecamylamine 60 4 E f f e c t of a t r o p i n e on c e l l f i r i n g induced by ACh and glutamate 63 5 E f f e c t o f a t r o p i n e on cholinomimetics and ACh 65 6 Responses of a VB thalamic neurone to the 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 -i z e neuronal responses 67 7 Responses o f a thalamic neurone to ACh and cholinomimetics i n the presence of 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 8 Responses of a s i n g l e thalamic neurone to the a d m i n i s t r a t i o n of ACh, glutamate, A(3MC and c a r b a c h o l : e f f e c t o f curare 73 9 Responses o f a thalamic neurone to ACh, cholinomimetics and the e f f e c t s o f a t r o p i n e 76 10 The a c t i v a t i o n o f a Renshaw c e l l whose resp-onse to a v o l l e y i n the L5 v e n t r a l r o o t i s shown, to ACh, c a r b a c h o l and A3MC 79 11 E f f e c t s o f curare and a t r o p i n e on the c h o l -i n e r g i c e x c i t a t i o n of two Renshaw c e l l s 81 12 Diagrammatic r e p r e s e n t a t i o n o f the mole 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 13 T y p i c a l r e c o r d i n g s of thalamic responses to the amino a c i d e x c i t a n t s used i n this study 115 x i i F i g u r e (cont'd) Page 14 E f f e c t o f GDEE a g a i n s t the stereoisomers of a s p a r t a t e 120 15 T y p i c a l responses used i n c o n s t r u c t i n g the r a n k i n g orders of amino a c i d e x c i -t a n t s when GDEE was the a n t a g o n i s t employed 125 16 T y p i c a l responses used i n c o n s t r u c t i n g the r a n k i n g orders of amino a c i d e x c i -t a n t s when aAA was the a n t a g o n i s t employed 131 17 E f f e c t s o f the 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 of a spontaneously d i s -c h a r g i n g VB thalamic neurone 134 18 E f f e c t s o f the phosphonic analogues as amino a c i d a n t a g o n i s t s 138 19 E f f e c t s o f GDEE and aAA a g a i n s t e x c i t a t i o n by 3AG and a comparison w i t h the responses to L-glutamate and L - a s p a r t a t e . . . 141 20 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 granule c e l l s , and a schematic o f a granule c e l l i n r e l a t i o n s h i p to the dentate gyrus 159 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 o f a granule c e l l 163 22 Averaged EPSP f i e l d responses evoked by stim-u l a t i n g PP and COMM i n p u t s to the dentate granule c e l l s 166 23 The effect: o f b i c u c u l l i n e on the de p r e s s i o n of a granule c e l l by GABA and i t s a c t i v -a t i o n by aAA 169 24 Examples of the e f f e c t s o f octopamine and n o r a d r e n a l i n e on the f i r i n g r a t e s o f neurones of the c e r e b r a l c o r t e x 184 25 Responses of a c o r t i c a l neurone to dopamine and n o r a d r e n a l i n e 187 26 A dose-response curve o f octopamine depres-s i o n s on a s p i n a l i n t e r n e u r o n e a c t i v a t e d by v e n t r a l r o o t v o l l e y s 194 x i i i F i g u r e (cont'd) Page 27 Responses of two neurones t e s t e d w i t h octop-amine and n o r a d r e n a l i n e , and octopamine and dopamine, and e f f e c t s o f a n t a g o n i s t s 199 28 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 neurone responding to octopamine and noradren-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 29 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 neurone depressed by octopamine and n o r a d r e n a l i n e 204 30 E f f e c t s o f c l o z a p i n e and metoclopramide on the f i r i n g o f c o r t i c a l and s p i n a l neurones 207 LIST OF ABBREVIATIONS > ACh A3MC AChE ADCP 2A4P 2A3P A A ASP BICUC ca r b a c h o l (CARB) °C cm. CNS COMM curare I c y c l i c ..AMP D-DH3E DMPP DC DLH DOP GLUT GDEE g or gm Hz hr HA966 i . p . a c e t y l c h o l i n e a c e t y l - 3 - m e t h y l c h o l i n e a c e t y l c h o l i n e s t e r a s e (±)-cis-1-amino-1,3-dicarboxycyclopentane 2-amino-4-phosphonobutyrate 2-amino-3-phosphonoproprionate angstrom ' a n t e r i o r a s p a r t a t e b i c u c u l l i n e c a r b a m i n o y l c h o l i n e c e n t i g r a d e degrees centimetre c e n t r a l nervous system commissural p r o j e c t i o n (from c o n t r a l a t e r a l pocampus) D-tubocurarine c u r r e n t c y c l i c adenosine-3,5 1-monophosphate dextro d i h y d r o - 3 - e r y t h r o i d i n e 1,1-dimethy1-4-phenylpiperazinium i o d i d e d i r e c t c u r r e n t DL-homocysteate dopamine glutamate glutamate d i e t h y l e s t e r gram h e r t z hour l-hydroxy-3-aminopyrrolid-2-'one i n t r a p e r i t o n e a l XV KDEE k a i n a t e d i e t h y l e s t e r Kg k i l o g r a m K H Z k i l o h e r t z kilo-ohms L- laevo L l a t e r a l LGN l a t e r a l g e n i c u l a t e nucleus m mega-ohms mg m i l l i g r a m s ml m i l l i l i t r e s mm m i l l i m e t r e s msec m i l l i s e c o n d s mV m i l l i v o l t s min minutes M molar nA nanoamperes NIC n i c o t i n e NMA N-methyl-DL-aspartate NOR n o r a d r e n a l i n e n. nucleus n number ng nanogram OCT octopamine E" para PP p e r f o r a n t path pmol picomole P p o s t e r i o r P p r o b a b i l i t y sec second SE standard e r r o r of the mean VB v e n t r o b a s a l V v e r t i c a l V v o l t s w/v weight per volume x v i aAA a-aminoadipate (both D and DL forms were used) 3AA 3-aminoadipate (racaemic mixture o n l y was used) 3AG 3-aminoglutarate GABA y-aminobutyrate ym micrometre yv m i c r o v o l t X 2 c h i squared SOURCES OF DRUGS x v i i ICN Pharmaceuticals Calbiochem L-glutamic a c i d L - a s p a r t i c a c i d D - a s p a r t i c a c i d N-methyl-DL-aspartic a c i d y-aminobutyric a c i d DL-a-aminoadipic a c i d carbamylcholine c h l o r i d e a c e t y l - 3 - m e t h y l c h o l i h e • Merck d i h y d r o - 3 - e r y t h r o i d i n e bromide 1, l - d i m e t h y l - 4 - p h e n y l p i p e r a z i n i u m i o d i d e mecamylamine h y d r o c h l o r i d e Lundbeck a - f l u p e n t h i x o l h y d r o c h l o r i d e Koch-Light and Mann Research L-glutamic a c i d d i e t h y l e s t e r Sandoz a t r o p i n e sulphate DL-homocysteic a c i d n o r a d r e n a l i n e • H C l dopamine-HCl 2-amino-4-phosphonobut-y r i c a c i d D-glutamic a c i d N u t r i t i o n a l Biochem. a c e t y l c h o l i n e bromide D-tubocurarine • HCl Sigma k a i n i c a c i d octopamine•HCl BDH Biochemicals n i c o t i n e a c i d t a r t r a t e ICN-K&K:rare & f i n e chem. d i v i s i o n 2-amino-3-phosphonoprop-r i o n i c a c i d B. R o u f o g a l i s (Vancouver) M. Walker (Vancouver). c l o z a p i n e h y d r o c h l o r i d e Drugs r e c e i v e d as g i f t s : metoclopramide•HCl Dr. p r o p r a n o l o l - H C l Dr, b i c u c u l l i n e methochloride• Dr. G.A.R. Johnston (Canberra). . 3-aminoglutaric a c i d " D-a-aminoadipic a c i d Dr. J.C. Watkins ( B r i s t o l ) . 3-aminoadipic a c i d Dr. J.C. Watkins v i a Dr. G.A.R. Johnston ADCP was s y n t h e s i z e d by Drs D.G. C l a r k and V. G u j r a l (Vancouver). x v i i i ACKNOWLEDGEMENTS The s u c c e s s f u l completion of 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 achieved without the he l p , support, advice and encouragement of many people. I th e r e f o r e wish to extend my g r a t i t u d e to those who took p a r t i n the var i o u s aspects of the progress and development of my graduate research p r o j e c t , as w e l l as i n my education i n the p r i n c i p l e s of physiology. Drs. N. Wilson, J . M i l l e r , R. Pederson and D. Randall formed my graduate committee and were very h e l p f u l w i t h t h e i r support and advice. For c o l l a b o r a t i o n and val u a b l e d i s c u s -sions I wish to thank Drs. S. Assaf, J . H a l l , T. Richardson, D. West, H. Wheal and Mr. G. Hutchinson. T e c h n i c a l support was always c h e e r f u l l y provided by Mrs. Y. Heap, who a s s i s t e d i n the 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 the h i s t o l o g y ; Mr. K. Henze, who d i d a l l the photography, and h i s s t a f f who rendered t e c h n o l o g i c a l a s s i s -tance. Mrs. M. Forsythe and her s t a f f are 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 Medical Research C o u n c i l f i n a n c i a l l y supported the present study. The encouragement and confidence of my w i f e , Debbie, has been of great help to me and ther e f o r e m e r i t s s p e c i a l mention. L a s t , but c e r t a i n l y not l e a s t , I wish to o f f e r my most s i n c e r e thanks to Professor H. McLennan who has been the i d e a l s u p ervisor; by gran t i n g me independence i n my research endeav-ours, a l l o w i n g me to l e a r n from my own e r r o r s , yet being always there w i t h guidance to help me out of d i f f i c u l t y when i t was r e q u i r e d . His example w i l l continue to be a standard of excellence 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 I GENERAL INTRODUCTION a) Chemical Tra n s m i s s i o n The b a s i c f u n c t i o n a l u n i t of the nervous system i s the nerve c e l l , or neurone. Much of the c u r r e n t i n v e s t i g a t i o n i n the neurosciences i s concerned w i t h e l u c i d a t i n g the mechanisms whereby neurones i n t e r a c t w i t h each'other to produce i n t e g r a t e d c o n t r o l of b o d i l y f u n c t i o n s and p a t t e r n s of behaviour. I t i s now w e l l e s t a b l i s h e d t h a t neurones are capable of gen e r a t i n g and conducting e l e c t r i c a l a c t i v i t y , and t h a t t h i s a c t i v i t y i n f l u e n c e s other neurones. In 1897 S h e r r i n g t o n c o i n e d the term " s y n a p s i s " , l a t e r changed to "synapse", to d e s c r i b e the narrow r e g i o n of anatomical j u x t a p o s i t i o n between neurones which he b e l i e v e d to be the s i t e of f u n c t i o n a l i n t e r -connection. S h o r t l y t h e r e a f t e r , E l l i o t t (1905) made the important o b s e r v a t i o n that s y s t e m i c a l l y administered a d r e n a l i n e and s t i m u l a t i o n of the p e r i p h e r a l sympathetic nervous system produced q u a l i t a t i v e l y s i m i l a r e f f e c t s . He a l s o noted t h a t the e f f e c t of a d r e n a l i n e was l o c a l i z e d to the j u n c t i o n s o f nerve and smooth muscle, and although E l l i o t t was not e x p l i c i t on the p o i n t , the i m p l i c a t i o n was that nerve impulses r e l e a s e ' minute amounts of a d r e n a l i n e or an a d r e n a l i n e - l i k e substance 2 onto those t i s s u e s which are s y m p a t h e t i c a l l y i n n e r v a t e d . The involvement of chemical substances a f f e c t i n g p e r i p h -e r a l l y i n n e r v a t e d t i s s u e s was not r e s t r i c t e d s o l e l y to a d r e n a l i n e , however. Dale (1914) was l e d to the c o n c l u s i o n t h a t a c e t y l c h o l i n e (ACh) possessed two d i f f e r e n t a c t i o n s on b i o l o g i c a l t i s s u e s ; those mimicked by muscarine and b l o c k e d by a t r o p i n e (muscarinic e f f e c t s ) and those e l i c i t e d by n i c o t i n e and which c o u l d be b l o c k e d by i n c r e a s e d concentra-t i o n s of t h i s a g o n i s t ( n i c o t i n i c e f f e c t s ) . Despite these e a r l y demonstrations of the p h a r m a c o l o g i c a l s e n s i t i v i t y of b i o l o g i c a l t i s s u e s , i t remained f o r Loewi (1921) to c o r r e l a t e d i r e c t l y these o b s e r v a t i o n s w i t h nerve s t i m u l a t i o n , and so p r o v i d e the f i r s t p h y s i o l o g i c a l evidence t h a t nerve t e r m i n a l s r e l e a s e chemical substances which can then e x e r t p h a r m a c o l o g i c a l e f f e c t s upon an i n n e r v a t e d t i s s u e . Loewi s t i m u l a t e d the vagus nerve of a p e r f u s e d f r o g h e a r t , c o l l e c t e d the e f f l u e n t f l u i d and t r a n s f e r r e d t h i s to the medium p e r f u s i n g a second h e a r t which had not been s t i m u l a t e d . He observed a slowing of the second h e a r t , and t h i s e f f e c t was a t t r i b u t e d to the presence i n the p e r f u s a t e of a substance r e l e a s e d from the vagus of the s t i m u l a t e d h e a r t . Loewi-r e f e r r e d to t h i s m a t e r i a l as " V a g u s s t o f f " , which was l a t e r i d e n t i f i e d as ACh (Bacq and Brown, 1937; Dale, 1938; f o r review see McLennan, 1963). 3 Langley (1905, 1907) i n t r o d u c e d the term " r e c e p t i v e substance", l a t e r shortened by C l a r k e (1926) to " r e c e p t o r " , to designate a s p e c i f i c although h y p o t h e t i c a l moiety on e f f e c t o r 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 produce a c h a r a c t e r i s t i c response. I t was not u n t i l the work of C l a r k e (1926) that the concept of a d i s c r e t e , pharmaco-l o g i c a l l y s p e c i f i c r e c e p t o r was examined q u a n t i t a t i v e l y . F o l l o w i n g the p i o n e e r i n g work of Loewi and Langley, C l a r k e attempted to estimate the number of r e c e p t o r s f o r ACh per u n i t area of c a r d i a c muscle c e l l from co n c e n t r a t i o n - r e s p o n s e curves. From h i s s t u d i e s the b a s i c p r i n c i p l e s of a g o n i s t -r e c e p t o r k i n e t i c s were d e r i v e d , and a t l a s t a b a s i c d e s c r i p t i o n of the r o l e of s y n a p t i c t r a n s m i t t e r s was made p o s s i b l e . B r i e f l y , i t i s b e l i e v e d t h a t t r a n s m i t t e r s are s t o r e d i n nerve t e r m i n a l s , from which they are r e l e a s e d i n t o the s y n a p t i c c l e f t upon i n v a s i o n of the t e r m i n a l by an a c t i o n p o t e n t i a l . Through d i f f u s i o n , the t r a n s m i t t e r s a r r i v e a t and r e a c t w i t h p o s t s y n a p t i c r e c e p t o r s on the membrane s u r f a c e of the e f f e c t o r t a r g e t (Dale, 1938). The p a r t i c u l a r response evoked i n the t a r g e t organ i s dependent on the changes induced i n the membrane of the p o s t s y n a p t i c c e l l by the t r a n s -m i t t e r . In a manner s t i l l not known p r e c i s e l y , the t r a n s m i t t e r - r e c e p t o r r e a c t i o n causes m o l e c u l a r changes i n the p o s t s y n a p t i c membrane. I t i s p r e s e n t l y b e l i e v e d however, (Katz, 1966; E c c l e s , 1964,1.977) t h a t such changes i n v o l v e 4 a l t e r a t i o n s of membrane p e r m e a b i l i t y which permit s e l e c t i v e passage of the i n t r a c e l l u l a r and e x t r a c e l l u l a r i o n s . T h i s i o n i c t r a n s f e r i s not permanent, but may continue f o r as long as the a g o n i s t binds to the r e c e p t o r molecule (Katz, 1962; 1966; Katz and M i l e d i , 1973). The c e l l u l a r response to the i o n i c f l u x across the membrane may induce secondary e f f e c t s , such as f o r example the i n i t i a t i o n of s e c r e t o r y p r o c e s s e s , or the formation of a new a c t i o n p o t e n t i a l , f o l l o w e d i n c e r t a i n i n s t a n c e s by c o n t r a c t i o n of the i n n e r v a t e d t i s s u e , or any other event c h a r a c t e r i s t i c of the t a r g e t c e l l . Although i n t e r n e u r o n a l t r a n s f e r of i n f o r m a t i o n at some synapses may i n v o l v e e l e c t r i c a l r a t h e r than chemical t r a n s m i s s i o n (Bennett, 1966) the l a t t e r remains the predominant mechanism f o r neuronal i n t e g r a t i o n i n the nervous system. Thus the r o l e of t r a n s m i t t e r s may be seen as c a r r i e r s of i n f o r m a t i o n , c o n t r o l l i n g b i o l o g i c a l events by means of the t r a n s d u c t i o n of e l e c t r i c a l s i g n a l s i n t o d i f f e r e n t chemical s i g n a l s , each w i t h an apparent s p e c i f i c i t y of a c t i o n . Subsequent to the e a r l y work of Langley and C l a r k e d e f i n i n g the nature of r e c e p t o r s , i n v e s t i g a t i o n s by others u s i n g both a g o n i s t s and a n t a g o n i s t s as p h a r m a c o l o g i c a l t o o l s gave r i s e to the i n t e r p r e t a t i o n of dose-response data i n terms of a g o n i s t - r e c e p t o r i n t e r a c t i o n s , although these analyses n e c e s s i t a t e d the f o r m u l a t i o n of a number of assump-t i o n s t hat c o u l d not be t e s t e d d i r e c t l y . In a d d i t i o n to these pharmacokinetic s t u d i e s , A h l q u i s t (1948) was a b l e to e s t a b l i s h f u r t h e r t h a t a l l of the adrenoceptors mediating responses i n a v a r i e t y of s y m p a t h e t i c a l l y i n n e r v a t e d organs and t i s s u e s i n a number of s p e c i e s c o u l d be d i v i d e d i n t o two c l a s s e s , namely a- and $-receptors. The s u b d i v i s i o n of c h o l i n e r g i c e f f e c t s i n t o m u s c a r i n i c and n i c o t i n i c by Dale had long been recognized by t h i s time. Although most of the s t u d i e s c i t e d above d e a l t e x c l u -s i v e l y w i t h the p h a r m a c o l o g i c a l a n a l y s i s of p e r i p h e r a l systems, at pr e s e n t i t i s w i d e l y b e l i e v e d t h a t s y n a p t i c t r a n s m i t t e r s e x e r t t h e i r e f f e c t s on s p e c i f i c p o s t s y n a p t i c r e c e p t o r s o f neurones of the mammalian c e n t r a l nervous system (CNS). Since there i s l i t t l e doubt that there are a v a r i e t y of chemical compounds which f u n c t i o n as t r a n s m i t t e r s , i t i s reasonable to expect t h a t the s y n a p t i c r e c e p t o r s f o r these substances possess a r e s t r i c t e d p h a r m a c o l o g i c a l s e n s i t i v i t y . T h i s allows f o r the p r e s e r v a t i o n of f u n c t i o n a l s p e c i f i c i t y , which may be p a r t i c u l a r l y important i n view of the marked degree o f com-p l e x i t y of neuronal i n t e r a c t i o n i n the CNS. b) C r i t e r i a For T r a n s m i t t e r s A s e r i e s of c r i t e r i a has been developed from the c l a s s i c a l s t u d i e s on the p e r i p h e r a l nervous system which e s t a b l i s h e d ACh as a t r a n s m i t t e r at neuromuscular and g a n g l i o n i c synapses. In order f o r a substance to be a s s i g n e d a r o l e of a s y n a p t i c 6 t r a n s m i t t e r i t must f u l f i l l the f o l l o w i n g requirements: i t must be p r e s e n t (1) i n the neurone w i t h the necessary enzymes (2) and p r e c u r s o r s (3) f o r i t s s y n t h e s i s ; i t must be shown to be r e l e a s e d (4) f o l l o w i n g neuronal d e p o l a r i z a t i o n ; there must be a process f o r the i n a c t i v a t i o n (5) of the substance and f i n a l l y i t must mimic the a c t i o n (6) of the n a t u r a l t r a n s m i t t e r and i n t e r a c t w i t h s u b c e l l u l a r elements i n an i d e n t i c a l f a s h i o n as the t r a n s m i t t e r i t s e l f (Werman, 1966; S a l m o i r a g h i and S t e f a n i s 1967; McLennan, 1970; Storm-Mathisen, 1977). A l l of these c r i t e r i a have been f u l f i l l e d f o r ACh and n o r a d r e n a l i n e at c e r t a i n p e r i p h e r a l synapses, and evidence i s r a p i d l y accumulating i n favour of c e n t r a l r o l e s f o r these and other substances, n o t a b l y dopamine, s e r o t o n i n , c a r n o s i n e , glutamine, y-aminobutyrate, g l y c i n e , glutamate, a s p a r t a t e , p r o l i n e , a d r e n a l i n e and h i s t a m i n e . More r e c e n t r e s e a r c h has p o i n t e d towards p o s s i b l e f u n c t i o n s i n s y n a p t i c p h y s i o l o g y f o r substance P, enkephaline and r e l a t e d "neuropeptides", phenyl-ethylamine and i t s d e r i v a t i v e s , t a u r i n e and adenosine t r i p h o s -phate, whether these w i l l a l l e v e n t u a l l y " f i t " the c r i t e r i a as c o n v e n t i o n a l t r a n s m i t t e r s or the c r i t e r i a themselves w i l l r e q u i r e m o d i f i c a t i o n i s at p r e s e n t a s u b j e c t of much specu-l a t i o n (Burnstock, 1976; B a l d e s s a r i n i and F i s c h e r , 1978). In an attempt to c l a s s i f y and d i f f e r e n t i a t e between the a c t i o n s of these substances and the more e s t a b l i s h e d t r a n s m i t t e r s :: of s y n a p t i c a c t i o n , a new terminology has been proposed which 7 includes f a l s e transmitter, co-transmitter, neurohormone, neuromodulator, etc. This nomenclature i s not yet widely accepted however, and indeed remains to be adequately defined. Relevant to the issue of this "new wave" of possible mediators of synaptic transmission i s the discussion by Werman (1966) and Freeman (1976) of the techniques for asses-sing the roles of such compounds i n synaptic action. A change i n discharge frequency of a neurone induced by the d i r e c t administration of a compound i s a frequently used but possibly i n s u f f i c i e n t paradigm for assessment of i t s mode of action. For example ACh may, i n addition to changing the rate of f i r i n g , possess trophic actions (Drachman, 1974; Purves, 1976). There i s also growing evidence for changes i n the s e n s i t i v i t y of neuronal receptors following denervation (Cannon and Rosenbluth, 1949; K u f f l e r , Dennis and Harris, 1971; Purves, 1976; Roper, 1976). Furthermore i n t r a c e l l u l a r "second messengers" activated by the transmitter may function i n c e l l u l a r processes .distinct from those subserving the generation of synaptic potentials (Greengard and Kebabian, 1974; Daly, 1976). Although synaptic transmission does mediate the transfer of action potentials from one c e l l to another (or prevent the formation of an action p o t e n t i a l , as i s the case with i n h i b i -tory substances), synapses do much more than follow r i g i d pat-terns of a c t i v i t y . They are p l a s t i c and dynamic structures whose components are capable of a range of q u a l i t a t i v e l y 8 d i f f e r e n t responses (Drachman, 1974; and other a r t i c l e s i n this volume). For example some neurones do not generate action poten-t i a l s , yet influence adjoining neurones s o l e l y by graded release of transmitter (Rail and Shepherd, 1968; Werblin and Dowling, 1969; Pearson and Fourtner, 1975). Furthermore, postsynaptic ' receptors of neurones or muscle may p r o l i f e r a t e or undergo alterations i n t h e i r s e n s i t i v i t y to the transmitter when they are denervated (Ungerstedt, 1971). Other examples of synaptic p l a s t i c i t y are provided by the phenomena of f a c i l i t a t i o n , adap-tatio n and habituation: processes which are believed to involve changes i n synaptic function (Kandel, 1976). In summary, i t i s becomming clear that as more i s learned about transmitters and the ways they are u t i l i z e d by synapses, the more refinement w i l l be required of p r e v a i l i n g concepts concerning the organi-zation of the nervous system. c) Iontophoresis One means of i d e n t i f i c a t i o n of synaptic transmitters i s the demonstration of the presence of neuronal receptors sensitive to the compounds, and t h i s forms one of the more important c r i t e r i a as outlined i n the previous section. One of the chief advantages to studying the actions of drugs using the iontophoretic method i s that i t i s possible to examine t h e i r e f f e cts upon single neurones i n vivo without a f f e c t i n g the whole of the nervous system or other p h y s i o l o g i c a l processes 9 ( r e s p i r a t i o n , b l o o d p r e s s u r e , etc.) such as may occur when drugs are a d m i n i s t e r e d s y s t e m i c a l l y . Avoidance of the blood-b r a i n b a r r i e r and other s y n a p t i c b a r r i e r s ( C u r t i s and E c c l e s , 1958b) i s another advantage p r o v i d e d by t h i s method of admini-s t r a t i o n , as i s the r e l a t i v e ease of r a p i d l y t e s t i n g and comparing the e f f e c t s of a s e r i e s of compounds upon neuronal f i r i n g . The b a s i c theory of i o n t o p h o r e s i s and d e t a i l s concerning the p r a c t i c a l use of the technique have been p u b l i s h e d ( C u r t i s , 1964; 1976; S a l m o i r a g h i and S t e f a n i s , 1967; K r n j e v i c , 1971; Bloom, 1974) but a b r i e f overview of some of the more s a l i e n t c o n s i d e r a t i o n s i s warranted here. The i o n t o p h o r e t i c method was f i r s t i n t r o d u c e d by Suh, '. Wang and Lim (1936) who a p p l i e d ACh to the f l o o r of the 4th v e n t r i c l e of dogs i n an e f f o r t to map out the p r e s s o r area. However the technique was used most e f f e c t i v e l y by Nastuk (1953') and l a t e r by C u r t i s and E c c l e s (1958) f o l l o w i n g refinements • i n t r o d u c e d by the l a t t e r authors f o r t e s t i n g c e n t r a l neurones. I t i s known t h a t i f an e l e c t r i c a l c u r r e n t i s a p p l i e d to an i o n i c s o l u t i o n , there w i l l occur a m i g r a t i o n of ions toward or away from the source of the imposed f i e l d . T h i s i s the fundamental p r i n c i p l e of i o n t o p h o r e s i s , f o r when the source of the e l e c t r i c a l f i e l d i s p o s i t i o n e d a t the mouth of a b a r r e l of a m u l t i p i p e t t e assembly, the r e s u l t a n t m i g r a t i o n of ions away from the source w i l l e f f e c t t h e i r e j e c t i o n a t the open t i p of the b a r r e l . I f the p i p e t t e assembly i s p o s i t i o n e d 10 i n c l o s e p r o x i m i t y to a neurone, such t h a t r e c o r d i n g s of the c e l l u l a r e l e c t r i c a l a c t i v i t y can be made through a NaCl s o l u t i o n i n one of the p i p e t t e b a r r e l s , drugs may be e j e c t e d and 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 observed on the r a t e of c e l l u l a r f i r i n g . Although the amount a c t u a l l y e j e c t e d i s somewhat l e s s than the p r e d i c t e d v a l u e due to complex i n t e r -a c t i o n s o c c u r r i n g a t the p i p e t t e t i p , the d e l i v e r y i s l i n e a r over the range of c u r r e n t s normally employed, and i s propor-t i o n a l to the magnitude of c u r r e n t a p p l i e d (see r e f e r e n c e s above, and Zieglgansberger,. Herz and Teschemacher, 1969; H a l l , H i c k s, McLennan, Richardson and Wheal, 1979). I t i s n e c e s s a r y i n some experiments to ensure that s u c c e s s i v e i o n t o p h o r e t i c a p p l i c a t i o n s of a compound d e l i v e r approximately the same dose i n order f o r t e s t s to be made under e q u i v a l e n t c o n d i t i o n s . Since the a b s o l u t e amount e j e c t e d i s not known w i t h any accuracy, the u s u a l procedure i s to r e g u l a t e the time of e j e c t i o n and r e t e n t i o n , keeping constant the i n t e r v a l between e j e c t i o n s . U s u a l l y a r e t a i n i n g c u r r e n t of 10-15 nA (rianoamperes) i s s u f f i c i e n t to prevent "leakage". With such a constant schedule of e j e c t i o n and r e t e n t i o n , the c o n c e n t r a t i o n of the drug s o l u t i o n a t the p i p e t t e t i p becomes s t a b l e and no "warming up" p e r i o d i s r e q u i r e d when the e j e c t i n g c u r r e n t i s switched on (MacDonald and N i s t r i , 1978). Te s t s can t h e r e f o r e be made to assess the a c t i v i t y f o r example, of an a n t a g o n i s t upon the e f f e c t s 11 induced by two or more c y c l i c a l l y a d m i n i s t e r e d a g o n i s t s . Another p o i n t to c o n s i d e r when t e s t i n g the a c t i v i t y of an a n t a g o n i s t i s the l e v e l of e x c i t a t i o n produced by the agonists. The r e l a t i o n s h i p t h a t e x i s t s between dose of a g o n i s t and response e l i c i t e d i s s i g m o i d a l , and the i d e a l dose of a g o n i s t to use i s t h a t which giv e s a half-maximal response (ED50) and which i s on the l i n e a r p o r t i o n of the curve. In theory i t i s n e c e s s a r y f i r s t to determine the ED^Q'S of two a g o n i s t s , and any r e l a t i v e d i f f e r e n c e s to the responses observed to the a g o n i s t s t e s t e d at t h i s dose, i n the presence of a b l o c k i n g drug, w i l l be a s c r i b a b l e to a d i f f e r e n t i a l a c t i o n of the a n t a g o n i s t and w i l l not r e f l e c t unequal s h i f t s due to d i f f e r -ent s t a r t i n g p o i n t s along the curves. In p r a c t i c e however, the d e t e r m i n a t i o n of a neurone's maximal f i r i n g r a t e i s o f t e n i m p o s s i b l e due to such f a c t o r s as s p i k e i n a c t i v a t i o n . A more p r a c t i c a l approach which i s used w i d e l y i s to e s t a b l i s h e j e c t i n g c u r r e n t s f o r the two a g o n i s t s which e l i c i t s t a b l e and approximately equal f i r i n g r a t e s . A s e l e c t i v i t y of antagonism may be claimed i f the percentage decreases i n the responses of the a g o n i s t s d i f f e r c o n s i d e r a b l y . When u s i n g the i o n t o p h o r e t i c approach to determine the p o t e n c i e s of compounds, a number of a d d i t i o n a l v a r i a b l e s r e q u i r e c o n s i d e r a t i o n , of which the t r a n s p o r t number assumes c o n s i d e r a b l e importance. T h i s number r e p r e s e n t s the q u a n t i t y of i o n a c t u a l l y r e l e a s e d from the p i p e t t e as a f r a c t i o n of the 12 amount which should be expelled by a given current. This r a t i o , always less than 1, i s determined by a number of factors including the i o n i c strength of the solution and the degree of i o n i c i n t e r a c t i o n with the pipette surface which gives r i s e to various potentials at the t i p . Gent, Morgan and Wolstencroft (1974) found that the apparent differences i n potency of two excitant amino acids, L-glutamate and L-aspartate, were sometimes considerably biased due to differences i n the transport numbers of the two compounds from d i f f e r e n t pipettes. These potency estimations had to be corrected for the transport numbers i n order to obtain an accurate assessment of the r e l a t i v e potencies. Zieglgansberger et a l . (1969) also measured e f f l u x of 3[H]-glutamate under c a r e f u l l y controlled conditions and found that release was proportional to the amount of current applied, even with i n t e n s i t i e s well beyond those normally employed. No d i r e c t r elationship existed between the diameter of the t i p of the electrode and transport number. Solutions of low i o n i c strength possessed a l i n e a r r e l a t i o n s h i p between current i n t e n s i t y and amount ejected, although they displayed lower transport numbers. Since i t i s necessary for compounds to be ionized i n solution for t h e i r iontophoretic delivery from micropipettes, i t i s often necessary to adjust the pH of the solutions with NaOH or HCl. The simultaneous release of H + or Na+ from such 13 s o l u t i o n s together w i t h p h a r m a c o l o g i c a l agents i n c a t i o n i c form may complicate i n t e r p r e t a t i o n of r e s u l t s , s i n c e i t i s known that H + by i t s e l f can cause neuronal e x c i t a t i o n (Freder-i c k s d n , Jordan and P h i l l i s , 1971; Stone, 1972). T h i s problem can be avoided by employing a number of t e s t s o l u t i o n s at d i f f e r e n t pH v a l u e s w i t h i n the c a t i o n i c range, or by t e s t i n g the e f f e c t of the suspected i o n i t s e l f . T h i s i s done by e x p e l l i n g from an HCl c o n t a i n i n g s o l u t i o n (or from a NaCl c o n t a i n i n g s o l u t i o n , i f the e f f e c t s of N a + are of i n t e r e s t ) the corresponding i o n and o b s e r v i n g the e f f e c t f o r comparison w i t h t h a t e l i c i t e d by the drug compound. Of course the r e v e r s e procedure w i l l a l l o w the t e s t i n g of p o s s i b l e a n i o n i c e f f e c t s . L i k e a l l m i c r o e l e c t r o d e experiments on c e n t r a l neurones, an unavoidable problem i s t h a t of sampling b i a s , and t h i s can be ma n i f e s t e d i n two ways. '. F i r s t , s i n c e the p o t e n t i a l r e c o r d e d a t the e l e c t r o d e t i p i s a d i r e c t r e s u l t of c u r r e n t flow i n the p e r i n e u r o n a l space, l a r g e r c e l l bodies w i t h c o r r e s p o n d i n g l y g r e a t e r s u r f a c e areas w i l l have a l a r g e r c u r r e n t from the c e l l d u r i n g an a c t i o n p o t e n t i a l . As these c e l l s w i l l generate h i g h amplitude e l e c t r i c a l p o t e n t i a l s they w i l l be r e a d i l y d e t e c t e d by m i c r o e l e c t r o d e s and can be rec o r d e d e a s i l y over g r e a t e r d i s t a n c e s from the c e l l body. T h i s b i a s w i l l be r e i n f o r c e d by another f a c t o r , and t h a t i s th a t l a r g e r c e l l s are more f r e q u e n t l y encountered simply on a 14 s t a t i s t i c a l b a s i s , u n l e s s the area under i n v e s t i g a t i o n i s comprised mostly of small c e l l s . Secondly, i n the case of i o n t o p h o r e t i c experiments on l y those c e l l s w i l l be d e t e c t e d which f i r e spontaneously or which are a c t i v a t e d by the e j e c t i o n of an e x c i t a t o r y substance; nor m a l l y an amino a c i d i s used. In the l a t t e r case, d i f f i -c u l t i e s may a r i s e i f the amino a c i d p r e f e r e n t i a l l y e x c i t e s l o c a l i n h i b i t o r y mechanisms. The r e s u l t may be t h a t many neurones w i l l appear to be unresponsive, or i n h i b i t e d , or not d e t e c t e d a t a l l , when i n f a c t an e x c i t a t o r y response may be masked ( F e l i x and McLennan, 1971; McLennan, 1971). These b i a s i n g f a c t o r s may be avoided i n p a r t by a d e l i b e r a t e s e l e c -t i v e sampling, t h a t i s by s t u d y i n g only responses from a known p o p u l a t i o n of neurones i d e n t i f i e d 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 (Stone, 1972; Hutchinson, McLennan and Wheal, 1978). A r e c e n t development i n neuropharmacological s t u d i e s which employ i o n t o p h o r e s i s i s the use of m u l t i p i p e t t e assem-b l i e s which are c o n s t r u c t e d w i t h the drug c o n t a i n i n g p i p e t t e s a t t a c h e d a l o n g s i d e the r e c o r d i n g b a r r e l , but which are broken back to v a r i o u s d i s t a n c e s behind the r e c o r d i n g b a r r e l 'is'.'.tip In t h i s manner the a d m i n i s t r a t i o n of compounds can be made at known d i s t a n c e s away from the s i t e of r e c o r d i n g , a l l o w i n g comparisons to be made of the t o p o g r a p h i c a l l o c a l i z a t i o n of d e n d r i t i c s e n s i t i v i t y ( Z i e g l g a n s b e r g e r and Champagnat, 1977; 15 1978). This approach promises to be rewarding i n i n v e s t i g -a t i o n s of CNS regions where neurone somata and t h e i r d e n d r i t i c processes are organized 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 cortex or s p i n a l cord. Another i n n o v a t i v e approach used i n recent stud i e s of pharmacological aspects of synaptic physiology concerns the simultaneous 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 two m u l t i p i p e t t e assemblies (Duggan, H a l l and Headley, 1977; Engberg, Flatman and Lambert, 1979). One of these i s placed near or i n t o the soma of the neurone i n the usual f a s h i o n , w h i l e the second assembly i s advanced through the t i s s u e u n t i l i t i s p o s i t i o n e d somewhere w i t h i n the d e n d r i t i c a r b o u r i z a t i o n of the same neurone. The e f f e c t s of compounds e j e c t e d from the l a t t e r p i p e t t e assembly can then be t e s t e d against a number of experimental v a r i a b l e s , such as: the d i f f e r e n t e f f e c t s upon f i r i n g of perisomal 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 neuronal f i r i n g of d e n d r i t i c de- or h y p e r p o l a r i z a t i o n i n response to the f i r i n g to synaptic a c t i v a t i o n . With t h i s type of twin 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 to use i o n t o p h o r e s i s i n a very s o p h i s t i c a t e d f a s h i o n , enabling the concurrent 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 processes and the pharmacological character-i z a t i o n of r e c e p t o r s , a l l at the l e v e l of the s i n g l e neurone i n the mammalian CNS. 16 d) The Present Study The experiments to be d e s c r i b e d were undertaken w i t h the aim of 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 o f s e v e r a l d i f f e r e n t p o p u l a t i o n s of neuronal r e c e p t o r s i n the mammalian CNS. Neuronal responses to i o n t o p h o r e t i c a l l y a d m i n i s t e r e d a g o n i s t s and a n t a g o n i s t s were used to analyze the r e c e p t o r s f o r the f o l l o w i n g systems: 1) ACh i n the c e r e b r a l c o r t e x , VB thalamus and v e n t r a l s p i n a l cord; 2) amino a c i d s i n the VB thalamus and dentate gyrus; and 3) amines i n the c e r e b r a l c o r t e x and d o r s a l s p i n a l cord. Each o f the three s e r i e s o f i n v e s t i g a t i o n s employed a common experimental approach and where p o s s i b l e , the c h a r a c t e r i z e d r e c e p t o r s were a s s o c i a t e d w i t h neurones i d e n t i f i e d s y n a p t i c a l l y through c o n v e n t i o n a l n e u r o p h y s i o l o g i c a l methods. Receptors f o r ACh appear to possess unique p r o p e r t i e s , not d e s c r i b e d b e f o r e f o r c e n t r a l neurones; the amino a c i d s were found to r e a c t w i t h at l e a s t two and p o s s i b l y three d i s -t i n c t r e c e p t o r s ; and i t i s proposed that octopamine e x e r t s i t s e f f e c t s through r e c e p t o r s d i f f e r e n t from those used by the catecholamines. A,more thorough d i s c u s s i o n o f the r a t i o n a l e s f o r these experiments and the r e l e v a n t background l i t e r a t u r e appears i n the r e s p e c t i v e i n t r o d u c t i o n s f o r each of the systems examined. 17 CHAPTER I I MATERIALS AND METHODS A l l of the experiments to be d e s c r i b e d i n t h i s t h e s i s were performed on r a t s of e i t h e r sex, weighing between 137 and 420 gm. The animals were a n a e s t h e t i z e d w i t h urethane (1.5 g/Kg, i.p.) i n a manner which appeared to generate minimal, i f any dis c o m f o r t on the p a r t of the animal. A n a e s t h e s i a was main-t a i n e d by a d d i t i o n a l doses of the same a n a e s t h e t i c . A d d i t i o n a l doses were gi v e n whenever the animal e x h i b i t e d any of the f o l l o w i n g s i g n s : 1) b u l g i n g of the eyes; 2) pronounced t w i t c h i n e s s of v i b r i s s a e ; 3) h y p e r s e n s i t i v i t y of the f l e x i o n r e f l e x i n response to hindpaw pinches; or 4) spontaneous body movements, u s u a l l y of the limbs. In many of the experiments, cannulae were p l a c e d i n the blood v e s s e l s f o r maintenance of a n a e s t h e s i a or f o r the t e s t i n g of p h a r m a c o l o g i c a l compounds, as p a r t of the experimental p r o t o c o l . The femoral v e i n or o c c a s i o n a l l y the j u g u l a r v e i n were used. The cannulae comprised f i n e p o l y e t h y l e n e t u b i n g of 0.9mm o u t s i d e diameter, and duri n g t h e i r placement i n the bl o o d v e s s e l s , were f i l l e d w i t h a small q u a n t i t y of h e p a r i n -i z e d s a l i n e . The employment of 3-way stopcocks p e r m i t t e d the use of the same cannula f o r the a d m i n i s t r a t i o n of more than 18 one material. To accomplish t h i s , the usual procedure was to i n j e c t the compound of inter e s t between two administrations of saline. This permitted the comparison of the drug e f f e c t with a control (the f i r s t saline injection) and further ensured the delivery of the entire dose of the compound (the "chaser" i n j e c t i o n ) . With cannula lengths of approximately 5 cm, 0.5 ml for each saline i n j e c t i o n was s u f f i c i e n t for the above procedures. Following anaesthesia, the i n i t i a l s u r g i c a l procedures involved the placement of cannulae and i n many experiments, the i s o l a t i o n and cutting of the peroneal or s c i a t i c nerve. The skin flaps around the cannula were closed by coarse s t i t c h i n g , while those around the exposed hindlimb nerve were raised to form a pool by th e i r attachment to a plexiglass r i n g fastened to the frame which supported the animal. This pool was f i l l e d with warmed l i q u i d p a r a f f i n o i l within which were immersed the ends of bipolar s i l v e r electrodes. These were bent i n a manner so as to allow the nerve to be placed i n contact with each pole, and to be supported within the pool. Body temperature was maintained at 3 7 . 5°C by a heating pad regulated by a temperature control unit (EKEG Electronics) equipped with a r e c t a l thermistor probe. 19 a) S u p r a s p i n a l Experiments F o l l o w i n g the i n i t i a l a n a e s t h e t i c and s u r g i c a l procedures, the animals were p o s i t i o n e d i n a head-holder frame (Kopf Stereo-t a x i c ; model 1204) and f i x e d i n p l a c e by ear-bars mounted on the frame's s i d e s . With the i n c i s o r bar i n p o s i t i o n 4.8 nim beneath the h o r i z o n t a l plane at the i n t e r - a u r a l l i n e ( s t e r e o -t a x i c zero: v e r t i c a l ) and the head midway between the frame's s i d e s judged by the c a l i b r a t i o n s on the ear-bars ( s t e r e o t a x i c zero: l a t e r a l ) , the animal's b r a i n was f i x e d i n p l a c e i n an o r i e n t a t i o n corresponding to t h a t of the a t l a s of Konig and K l i p p e l (1974). A f t e r a m i d l i n e s a g g i t a l i n c i s i o n through the s c a l p , the s k u l l was exposed 'by the g e n t l e s c r a p i n g back of s c l e r a and' o v e r l y i n g t i s s u e s w i t h a p e r i o s t e a l e l e v a t o r . T h i s procedure r e v e a l e d bregma, lambda and the s a g g i t a l suture. One or two bone f l a p s o v e r l y i n g the b r a i n area to be i n v e s t i g a t e d were removed through the use of a hand d r i l l f i t t e d w i t h a s m a l l round d e n t a l b u r r , and claw-tooth f o r c e p s . T h i s procedure exposed the dura mater. B l e e d i n g from s k i n or dura was c o n t r o l l e d by a p p l y i n g small p l e d g e t s of Gelfoam (Upjohn) w h i l e b l e e d i n g from bone was stopped w i t h bone wax. The dura was c a r e f u l l y r e f l e c t e d , w i t h e x t r a . c a u t i o n whenever the s u p e r i o r s a g g i t a l sinus was exposed. I f a p o o l was c o n s t r u c t e d the exposed nervous t i s s u e was covered w i t h warmed l i q u i d p a r a f f i n o i l ; otherwise a few drops of warmed Locke s o l u t i o n 20 were used to keep the t i s s u e moist. For experiments on the c o r t e x and VB thalamus, the exposed b r a i n r e g i o n s roughly corresponded to AP boundaries 1.5 mm and 6.0 mm p o s t e r i o r to bregma, and L boundaries 1.0 mm to 4.5 mm l a t e r a l to the m i d l i n e . For experiments on the hippocampus, the exposed r e g i o n s on the i p s i l a t e r a l ( e x p e r i -mental) s i d e had boundaries 1.5 mm and 8.5 mm p o s t e r i o r to bregma and 4.5 mm l a t e r a l to the m i d l i n e . The c o n t r a l a t e r a l s i d e was exposed 0.5 mm to 2.5 mm p o s t e r i o r to bregma and 3.0 mm l a t e r a l to the m i d l i n e . The bone o v e r l y i n g the s a g g i t a l sinus was removed e n t i r e l y . b) S p i n a l Cord Experiments F o l l o w i n g the i n i t i a l a n a e s t h e t i c procedures and placement of cannulaej the animals were p l a c e d on a r a i s e d p l a t f o r m f o r a d d i t i o n a l s u r g i c a l p r e p a r a t i o n . A long (approximately 10 cm) s k i n i n c i s i o n was made along the d o r s a l m i d l i n e from the second lumbar to the f i r s t s a c r a l spinous p r o c e s s . The e r r e c t o r spinae muscles were d i s s e c t e d from both s i d e s of the spine, and the spinous processes and laminae of the second lumbar to the f i r s t s a c r a l v e r t e b r a e were removed w i t h bone f o r c e p s . The surrounding t i s s u e was h e l d away from the v e r t e b r a e by t i s s u e r e t r a c t o r s . The dura mater was then c a r e f u l l y cut away making the s p i n a l c o r d and s p i n a l r o o t s e a s i l y a c c e s s a b l e . Extreme care was taken to a v o i d touching the s p i n a l c o r d and 21 to keep a l l t i s s u e s moist w i t h warmed Locke s o l u t i o n . F o l l o w i n g completion of the laminectomy, d o r s a l r o o t s were cut. V e n t r a l r o o t s to the hindlimb were i d e n t i f i e d by the t w i t c h induced f o l l o w i n g t h e i r severance. The animals were then suspended upon a frame by means of clamps secured to two of the remaining spinous pro c e s s e s . The animal's head was supported and a p o o l f o r l i q u i d p a r a f f i n c o n s t r u c t e d by r a i s i n g the s k i n f l a p s as a l r e a d y d e s c r i b e d . D o r s a l and v e n t r a l r o o t s were suspended w i t h i n t h i s p o o l by b i p o l a r s i l v e r s t i m u l a t i n g e l e c t r o d e s . c) S t i m u l a t i n g And Recording Procedures S t i m u l a t i n g e l e c t r o d e s were lowered s t e r e o t a x i c a l l y i n t o v a r i o u s r e g i o n s o f the CNS u s i n g Kopf micromanipulators. P l a c e -ments were as f o l l o w s : A 3.2, L 2.6, V 2.6 f o r crus c e r e b r i ; A 4.2 L 2.8, V 3.9 f o r VB thalamus; P 0.5, L 4.4, V 1.3 f o r angular bun-d l e ; A 3.7, L 1.5, V 1.7 f o r the dentate gyrus, a c c o r d i n g to the a t l a s of Konig and K l i p p e l (1974). C o n c e n t r i c b i p o l a r e l e c t r o d e s (SNE 100, Rhodes Instruments) having a t i p s e p a r a t i o n o f 0.5 mm and DC r e s i s t a n c e i n normal s a l i n e o f 75-100 Kfi were used f o r e l e c t r i c a l b r a i n s t i m u l a t i o n . S i n g l e monophasic r e c t a n g u l a r p u l s e s o f 0.1 msec d u r a t i o n were used i n the s t i m u l a t i o n of b r a i n s i t e s ; s p i n a l r o o t s and p e r i -p h e r a l nerves were a l s o s t i m u l a t e d at these parameters, but through s i l v e r w i r e s . 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-eded 20 V, and more t y p i c a l l y was i n the range of 2-8 V. 22 M i c r o p i p e t t e s used f o r drug 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 a c t i v i t y were p l a c e d w i t h an AB T r a n s v e r t e x microdrive and a l a r g e micromanipulator mounted on a bar a t t a c h e d to the s t e r e o t a x i c frame. E l e c t r o d e s were a t t a c h e d to the micromanip-u l a t o r i n f i x e d o r i e n t a t i o n by a custom made h o l d i n g device which allowed the easy removal and replacement o f the assembly to the h o l d e r . The m i c r o d r i v e was used to lower the micr o p i p e t t e assemblies i n 4ym steps through the employment of an e l e c t r o n i -c a l l y 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 taken from the r a t b r a i n a t l a s o f Konig and K l i p p e l (1974) and were v e r i f i e d by comparison w i t h the 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 the m i d l i n e pos-i 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 su r f a c e of the c o r t e x f o r v e r t i c a l c o - o r d i n a t e s . T h e . r e c o r d i n g s i t e s f o r c o r t i c a l experiments were i n the somatosensory area: (A 4:4, L 2.6, V 4.3-2.8) and f o r the VB thalamus were: A 4.4, L 2.6, V -0.6; and f o r dentate gyrus: A 3.7, L 1.5, V 1.8. d) Recording From S i n g l e Neurones And Drug D e l i v e r y Technique E x t r a c e l l u l a r r e c o r d i n g s from neurones of the c e r e b r a l c o r t e x , VB thalamus, dentate gyrus 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 c o r d were made through the c e n t r a l b a r r e l o f m u l t i -b a r r e l p i p e t t e assemblies. The g l a s s m u l t i b a r r e l blanks were obt a i n e d from Vancouver S c i e n t i f i c Glassblowing as a r r a y s o f 7 fused g l a s s c a p i l l a r i e s , heat-drawn to an o v e r a l l diameter of 2.5-3.5 mm. These blanks were p l a c e d i n a v e r t i c a l Canberra type m i c r o e l e c t r o d e p u l l e r , heated, and p u l l e d to f i n e t i p s . 23 The r e s u l t i n g assembly was then t r a n s f e r r e d to a L e i t z Wetzlar l i g h t microscope where the t i p s were viewed under 16x magni-f i c a t i o n . With the a i d of an eyepiece g r a t i c u l e and a t h i n , needle-shaped s t e e l bar c o n t r o l l e d by a j o y - s t i c k , the t i p s of the m i c r o p i p e t t e s were broken back to a t o t a l diameter of 5-10 um. The p i p e t t e s were then f i l l e d w i t h g l a s s - d i s t i l l e d water by b o i l i n g f o r 20 minutes or u n t i l a l l b a r r e l s were seen to be f i l l e d . The assemblies were then allowed to c o o l and the d i s t i l l e d water w i t h i n the b a r r e l s was removed from the s h a f t s w i t h a s y r i n g e and 30 gauge f l e x i b l e needle. The water was r e p l a c e d by one of the s o l u t i o n s l i s t e d i n Table 1. 4 M NaCl was used to f i l l the r e c o r d i n g b a r r e l and o c c a s i o n a l l y e i t h e r 2 M or 4 M NaCl was used i n a separate c u r r e n t n e u t r a -l i z a t i o n b a r r e l . The s o l u t i o n s l i s t e d i n Table 1 were made wi t h d i s t i l l e d water or 0.15 M NaCl. The pH was ad j u s t e d w i t h e i t h e r HCl or NaOH to b r i n g i t w e l l away from the i s o e l e c t r i c p o i n t . The s o l u t i o n s were f i l t e r e d through a 0.8 um-passing M i l l i p o r e f i l t e r and c e n t r i f u g e d f o r 1 minute i n a Beckman microfuge "B". . A f t e r t h e i r placement i n the m i c r o p i p e t t e bar-r e l s , the s o l u t i o n s were allowed to d i f f u s e to the t i p s f o r 24 hours b e f o r e being used f o r the experiment. Care was always taken to keep away dust and d i r t p a r t i c l e s from -the m i c r o p i p e t t e s , and when not i n use they were r e f r i d g e r a t e d (+6°C) w i t h t h e i r t i p s immersed i n d i s t i l l e d water. When i n use, a pl a t i n u m wire extended i n t o the NaCl of the c e n t r a l b a r r e l and was connected to the a m p l i f i e r probe. S i l v e r wires TABLE 1 Drug S o l u t i o n s Compound Con c e n t r a t i o n Solvent pH A c t i v e i o n Na-L-Glutamate 0.05-0.50 M 0.15 M NaCl and H2O 7.0 c a t i o n metoclopramide monochloride 0.02-0.20 M 0.15 M NaCl and H2O 5.8 c a t i o n c l o z a p i n e a c e t i c a c i d and H2O 6.8-6.9 a t r o p i n e s u l f a t e 0 . 0 2 M 0 . , 1 5 J M NaCl 5 . 0 c a t i o n mecamylamine h y d r o c h l o r i d e 0 . 0 5 M 0 , , 1 5 M NaCl 5 . 5 c a t i o n d i h y d r o - 3 - e r y t h r o i d i n e bromide 0 , . 0 1 - 0 , . 0 2 M 0 . . 1 5 M NaCl 7 . 5 c a t i o n d-tubocurarine h y d r o c h l o r i d e 0 . 0 2 M 0 . 1 5 M NaCl 5 . 0 c a t i o n 1 , 1 - d i m e t h y l - 4 - p h e n y l p i p e r a z i n i u m i o d i d e 0 . 0 1 M 0 . , 1 5 M NaCl 6 . 0 c a t i o n n i c o t i n e hydrogen t a r t r a t e 0 , . 0 5 - 0 . 1 0 M 0 . . 1 5 M NaCl 4 . 5 c a t i o n b i c u c u l l i n e methochloride 0 . 0 1 M 0 . , 1 5 M NaCl 7 . 0 c a t i o n k a i n i c a c i d 0 . 0 5 M 0 . . 1 5 M NaCl 8 . 0 - 8 . , 2 anion L-glutamate d i e t h y l e s t e r 0 . 5 0 M H 2 0 3 . 4 - 3 , , 5 c a t i o n DL-octopamine h y d r o c h l o r i d e 0 . 5 0 M H 20 3 . 0 - 5 , , 2 c a t i o n DL-noradrenaline b i t a r t r a t e 0 . 5 0 M H 2 0 2 . 7 - 5 , , 2 c a t i o n DL-dopamine h y d r o c h l o r i d e 0 . 5 0 "M H 20 3 . 5 - 5 . , 0 c a t i o n ( ±)-cis- 1 -amino- 1 , 3-dicarboxycyclopentane 0 . 2 0 M H 2 0 8 . 2 anion TABLE 1 (cont'd) Drug Solutions Compound Concentration Solvent PH Active acetylcholine bromide 1.00 M H 20 4.5 cation carbamylcholine chloride 0. 20 M H 20 4.5 cation acetyl-3-methylcholine bromide 0.20 M H 20 4.5 cation Na-D-glutamate 0.50 M H20 8.0 anion Na-L-aspartate 0.50 M H 20 8.0 anion Na-D-aspartate 0.50 M H 20 8.0 anion DL-homocysteate 0.20 M H20 7.6 anion N-me thy1-DL-aspartate 0.10 M H20 8.0 anion DL-a-aminoadipate 0.50 M H 20 8.0 anion D-a-aminoadipate 0.20 M H20 8.0 anion 3-aminoadipate 0.20 M H 20 8.3 anion 3-aminoglutarate 0.20 M H 20 8.1 anion 2-amino-3-phosphonoproprionate 0.20 M H 20 8.0 anion 2-amino-4-phosphonobutyrate 0.20 M H 20 8.0 anion a-flupenthixol 0.20 M H20 3.4 cation DL-propranolol hydrochloride 0.50 M H20 3.4 cation y-aminobutyrate 0.50 M H 20 3.5 cation N3 L n 2 6 extended i n t o the remaining drug c o n t a i n i n g b a r r e l s , connec-t i n g 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 c u r r e n t source. A l l b a r r e l s were checked f o r adequate c u r r e n t flow b e f o r e each experiment, the u s u a l range of the DC r e s i s t a n c e being 70-100 Mfi, w h i l e the r e c o r d i n g b a r r e l t y p i c a l l y e x h i b i t e d v a l u e s w i t h i n the range of 1-5 Mfi. In order to prevent the d i f f u s i o n o f i o n s from the t i p s of the 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 to those used to e j e c t the ions f o r each of the outer drug c o n t a i n i n g b a r r e l s . The magnitude of these r e t a i n i n g c u r r e n t s v a r i e d w i t h i n the range of 7-15 nA. Currents used to r e t a i n or pass drugs 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 ten t u r n potentiometers - one f o r each b a r r e l - and were passed to the b a r r e l s of the p i p e t t e assemblies through 1000 Mfi s e r i e s r e s i s t o r s . R e l a t i v e l y s m a l l v a r i a t i o n s i n r e s i s t a n c e a t the e l e c t r o d e t i p s t h e r e f o r e d i d not a p p r e c i a b l y a f f e c t the flow of c u r r e n t . For most types 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 or more compounds i n order to permit comparisons of t h e i r e f f e c t s upon neuronal f i r i n g i n the absence and presence of an a d d i t i o n a l compound, u s u a l l y an a n t a g o n i s t . To accomplish t h i s an automatic t i m i n g device c o n t r o l l i n g the p e r i o d s of e j e c t i o n and r e t e n t i o n was employed. The r e l a t i v e apparent p o t e n c i e s of compounds were assessed by comparing the magnitudes of the e j e c t i n g c u r r e n t s 27 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 of the f i r i n g f r e q u e n c i e s (see d i s c u s s i o n o f t h i s matter i n Chapter!). The assessment of ph a r m a c o l o g i c a l d i f f e r e n c e s between the e f f e c t s of two a g o n i s t s was accomplished f o r each of the present i n v e s t i g a t i o n s through the u t i l i z a t i o n o f a n t a g o n i s t s . Although i n a l l the s t u d i e s the e f f e c t s of more than two ag o n i s t s were of i n t e r e s t , f o r the g r e a t e r p a r t comparisons were made i n each i n s t a n c e w i t h o n l y two. With d i f f e r e n t combinations of a g o n i s t p a i r s a r a n k i n g scheme c o u l d be estab-l i s h e d , and t h i s procedure forms the b a s i s f o r the experiments d e s c r i b e d i n Chapter 4. When a s u i t a b l e neurone was encountered, two of the ag o n i s t s were e j e c t e d a t f i x e d i n t e r v a l s f o r constant d u r a t i o n s by the automatic timer. F o l l o w i n g the r e c o r d i n g of at l e a s t three s u i t a b l e c y c l e s of responses which produced a p p r o x i -mately equal but submaximal changes i n f i r i n g r a t e s , antagon-i s t s were a p p l i e d and the r e g u l a r c y c l i n g o f a g o n i s t s c o n t i n -ued. In cases where the responses produced by two ag o n i s t s were both reduced i n the presence of an a n t a g o n i s t , the c r i t e r i o n f o r s e l e c t i v e a c t i o n was t h a t the peak response of one a g o n i s t was at l e a s t 25% l e s s than t h a t of the second, i n three s u c c e s s i v e c y c l e s . In a l l cases, r e c o v e r y to c o n t r o l v a l u e s a f t e r a d m i n i s t r a t i o n of an a n t a g o n i s t was ended, was r e q u i r e d f o r the r e s u l t to be accepted. 28 e) E l e c t r i c a l Equipment The s t i m u l a t i n g e l e c t r o d e s were connected through a s e l e c t i o n p anel and stimulus i s o l a t i o n u n i t s to e i t h e r a Grass S8 or an Ortec 4651 s t i m u l a t o r . The s i g n a l from the r e c o r d i n g b a r r e l of the m u l t i p i p e t t e assembly was f e d through a p r e a m p l i f i e r or an impedence matcher. The r e c o r d e d e l e c t r i c a l a c t i v i t y was passed through a type 3A9 or 2A63 T e k t r o n i x d i f f e r e n t i a l a m p l i f i e r and the output d i s p l a y e d on a type KM 565 Dual beam o s c i l l o s c o p e which c o u l d be t r i g g e r e d from the s t i m u l a t o r . A second p a r a l l e l dual beam o s c i l l o s c o p e which was not t r i g g e r e d from the s t i m u l a t o r monitored the continuous e l e c t r i c a l a c t i -v i t y . E x t r a c e l l u l a r neuronal a c t i v i t y was f i l t e r e d u s i n g a 100 Hz - 10 K H Z bandpass, w h i l e evoked p o t e n t i a l r e c o r d i n g s were observed w i t h f i l t e r s w i t h i n the 10 Hz - 3 K H Z range. The a m p l i f i e d s i g n a l from the o s c i l l o s c o p e was f e d to a l o u d -speaker and an EKEG ratemeter, the i n t e g r a t e d ratemeter out-put being 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 paper r e c o r d e r . The a m p l i f i e d s i g n a l s were a l s o l e d i n t o a v o l t a g e d i s c r i m i n a t o r which compared the r e c o r d e d s i g n a l s w i t h a manually a d j u s t a b l e t r i g g e r i n g p o t e n t i a l so t h a t a c t i o n poten-t i a l s above a c e r t a i n amplitude f i r e d a schmidt t r i g g e r . The r e s u l t i n g output p u l s e s c o u l d be subsequently l e d i n t o a PDP-8/L D i g i t a l computer or a PDP-11/10 computer programmed to d i s p l a y post stimulus time, l a t e n c y and ratemeter prog-rammes. The analyzed data were p l o t t e d on a v i s u a l d i s p l a y t e r m i n a l or on an i n t e r a c t i v e d i g i t a l p l o t t e r ( T e k t r o n i x 4462) . 29 f ) S t a t i s t i c a l A n a l y s i s The s t a t i s t i c a l t e s t s which were used f o r comparisons of data were both parametric i n nature and were designed to d i s p r o v e the N u l l Hypothesis. For comparisons between two independent measures, such as the magnitudes of c e l l u l a r responses to d i f f e r e n t e x c i t a n t s , a Student's t t e s t f o r the comparison of a s i n g l e s m a l l sample w i t h a known standard was used. For t e s t s of s t a t i s t i c a l independence, such as comparing the types of responses e l i c i t e d from a sample of neurones by d i f f e r e n t a g o n i s t s , a x 2 t e s t was employed. g) H i s t o l o g i c a l V e r i f i c a t i o n Of E l e c t r o d e S i t e s The l o c a t i o n s of s t i m u l a t i n g s i t e s were marked by p a s s i n g low DC anodal c u r r e n t (10-15 yA) f o r 10-20 seconds through the s t a i n l e s s s t e e l e l e c t r o d e s causing the d e p o s i t i o n of Fe"^" at the t i p . T h i s was f o l l o w e d by the p e r f u s i o n of the animal wi t h a s o l u t i o n of 1% potassium f e r r o c y a n i d e i n 10% f o r m a l i n . The i r o n d e p o s i t s were subsequently v i s u a l i z e d as blue-green spots i n h i s t o l o g i c a l s e c t i o n s . The cyanide r e a c t s w i t h Fe"*-1" to form the P r u s s i a n blue r e a c t i o n product which i s e a s i l y viewed under the l i g h t microscope. C o n f i r m a t i o n 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 was achieved by the i o n t o p h o r e t i c e j e c t i o n of Pontamine sky b l u e dye (20% w/v i n 4 M NaCl)(Thomas and Wilson, 1965) at the con-c l u s i o n of the experiment. Upon t e r m i n a t i o n of the r e c o r d i n g 30 p e r i o d , animals were p l a c e d i n deep a n a e s t h e s i a and subsequ-e n t l y p e r f u s e d t r a n s c a r d i a l l y w i t h 200 ml of 0.9% NaCl f o l l o w e d by 200 mi of 10% f o r m a l i n . The b r a i n s were e x c i s e d and kept i n f o r m a l i n u n t i l h i s t o l o g i c a l procedures c o u l d be conducted. Frozen s e c t i o n s of 50 um t h i c k n e s s were cut and s t a i n e d w i t h c r e s y l v i o l e t or s a f f r a n i n . 31 CHAPTER I I I ACETYLCHOLINE a) I n t r o d u c t i o n Of a l l the endogenous chemical substances which have been proposed as s y n a p t i c t r a n s m i t t e r s , ACh has r e c e i v e d the most complete experimental support and indeed, was the f i r s t comp-ound f o r which t h i s r o l e was confirmed. Dale (1914) found t h a t i n t e s t i n a l smooth muscle bore predominately m u s c a r i n i c r e c e p t o r s i n that muscarine was a potent e x c i t a n t and mimicked the a c t i o n of ACh i t s e l f . The e x c i t a t i o n caused by muscar-i n i c a g o n i s t s c o u l d be b l o c k e d by a t r o p i n e . The v e r t e b r a t e neuromuscular j u n c t i o n and g a n g l i o n c e l l s w i t h i n the gut were s e n s i t i v e only to n i c o t i n e and ACh and not to muscarine, and the a c t i o n of these e x c i t a n t s was s u s c e p t i b l e to antagonism by c u r i f o r m drugs, of which D-tubocurarine (curare) i s an example. T h i s dual nature of c h o l i n e r g i c r e c e p t o r s i n d i f f e r e n t p e r i p h e r a l t i s s u e s has been subsequently extended to areas i n the mammalian CNS ( f o r reviews see F e l d b e r g , 1945 and Pradhan and Dutta, 1971). Three techniques have proven most v a l u a b l e i n the i d e n t i -f i c a t i o n of c h o l i n e r g i c neurones i n the CNS: 1) s t a i n i n g procedures f o r the ACh h y d r o l y z i n g enzyme, a c e t y l c h o l i n e s -t e r a s e (AChE), which has been shown to be a marker f o r some 32 c e n t r a l c h o l i n e r g i c neurones ( C s i l l i k , 1975); 2) r e c o v e r y of ACh from d i s c r e t e b r a i n r e g i o n s f o l l o w i n g v a r i o u s experimen-t a l i n t e r v e n t i o n s designed to promote or d i m i n i s h the r e l e a s e of t r a n s m i t t e r ; and 3) d i r e c t p r e s e n t a t i o n of cholinomimetic drugs to the v i c i n i t y of s i n g l e neurones by way of iontophor-e t i c e j e c t i o n from m i c r o p i p e t t e s w h i l e s i m u l t a n e o u s l y r e c o r -ding the ongoing c e l l u l a r e l e c t r i c a l a c t i v i t y . 1) H i s t o c h e m i c a l H i s t o c h e m i c a l s t u d i e s d i r e c t e d towards AChE have demonstrated the presence of c h o l i n e r g i c neurones i n l a y e r V of the c e r e b r a l c o r t e x ( K r n j e v i d and S i l v e r , 1965), cerebellum ( F r i e d e and Flemming, 1964; A u s t i n and P h i l l i s , 1965; S i l v e r , 1967), b a s a l g a n g l i a , hippocampus and hypothalamus (Lewis and Shute, 1967), v a r i o u s thalamic n u c l e i ( K o e l l e , 1954; Lewis and Shute, 1963, F r i e d e , 1966) as w e l l as numerous other r e g i o n s . Although AChE has been found to e x i s t i n many d i f f e r e n t areas of the nervous system, i t i s b e l i e v e d that i n c e r t a i n cases t h i s may not 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 occurs' a t these s i t e s ( S i l v e r , 1967; Gwyn and F l u m e r f e l t , 1971). Caution has t h e r e f o r e been r e q u i r e d when a s s e s s i n g the s i g n i f -i c a n c e of r e s u l t s o b tained by t h i s h i s t o c h e m i c a l technique, and the c o n c l u s i o n s reached concerning the p o s s i b l e s y n a p t i c r o l e of ACh w i t h i n r e g i o n s o f the CNS based on AChE l o c a l i -z a t i o n alone have n e c e s s a r i l y been l i m i t e d (McLennan, 1970). 33 2) Recovery-Macintosh and Oborin (1953) e s t a b l i s h e d the technique of c o l -l e c t i n g and measuring ACh i n f l u i d o b t a i n e d from the s u r f a c e of the c e r e b r a l cortex. T h i s approach was continued by M i t c h e l l (1960, 1961, 1963), McLennan (1964), Jasper, Khan and E l l i o t t (1965), Dudar and Szerb (1969) and Dudar (1977), among ot h e r s , u s i n g c o l l e c t i n g cups p l a c e d on exposed c o r t i c a l or hippocampal t i s s u e , and w i t h the p u s h - p u l l cannula technique (Gaddum, 1961). The r e l e a s e of ACh was observed to d i m i n i s h w i t h i n c r e a s e d depth of a n a e s t h e s i a and c h r o n i c u n d e r c u t t i n g of c o r t i c a l s l a b s (Hebb, K r n j e v i c and S i l v e r , 1963; K r n j e v i c and S i l v e r , 1965; C o l l i e r and M i t c h e l l , 1967); was enhanced from sensory areas f o l l o w i n g a p p r o p r i a t e s t i m u l a t i o n ( C o l l i e r and Murray-Brown, 1968; Neal, Hemsworth and M i t c h e l l , 1968); and was shown to c o r r e l a t e w i t h EEG a c t i v a t i o n ( B a r t o l i n i and Pepeu, 1967). These data are of s i g n i f i c a n c e i n c o n s i d e r a t i o n of the b i o l o g i c a l f u n c t i o n f o r ACh s i n c e i t s r e l e a s e from neurones i s an important c r i t e r i o n f o r the establishment of i t s r o l e as a s y n a p t i c t r a n s m i t t e r . The i n v e r s e r e l a t i o n s h i p of AChi output and depth o f a n a e s t h e s i a , the decreased r e l e a s e f o l -lowing c o r t i c a l u n d e r c u t t i n g , and stimulus-evoked r e l e a s e a l l s t r o n g l y suggest that ACh i s c o n t a i n e d i n neurones, and t h a t upon a p p r o p r i a t e s t i m u l a t i o n i t i s r e l e a s e d from them. Since many other areas of the CNS have been examined f o r ACh r e l e a s e and comparable r e s u l t s obtained, these w i l l not be d e s c r i b e d 34 here, but f o r a summary of these and r e l a t e d i n v e s t i g a t i o n s , r e f e r e n c e i s made to Pepeu (1973). 3) I o n t o p h o r e s i s The d i r e c t a p p l i c a t i o n o f cholinomimetic agents to s i n g l e neurones has proved to be the method o f choice f o r determining 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 of c e n t r a l c h o l i n o c e p t i v e c e l l s , and has p r o v i d e d a powerful technique a i d i n g i n the i d e n t i f i c a t i o n of neurones which possess r e c e p t o r s f o r t h i s compound. The p r i n c i p l e of i o n t o p h o r e s i s and the techniques f o r i t s use were d e s c r i b e d i n Chapters I (c) and I I (d). i / Cortex The e a r l y p h a r m a c o l o g i c a l s t u d i e s on the a c t i o n of ACh i n the CNS have mainly used the cat as an experimental animal, and the e f f e c t s of a c e t y l c h o l i n e as w e l l as of cholinomimetics and a n t a g o n i s t s were f i r s t s t u d i e d on c e r e b r a l c o r t i c a l neurones by K r n j e v i c and P h i l l i s (1961, 1962, 1963a,b,c), Spehlmann (1963), Randic, S i m i n o f f and Straughan, (1964) and Crawford and C u r t i s (1966). Although many neurones appeared i n s e n s i t i v e to ACh, the g r e a t e s t p r o p o r t i o n of c h o l i n o c e p t i v e c e l l s o c c u r r e d between the depths 0.8 and 1.3 mm from the c o r t i c a l s u r f a c e , corresponding to l a y e r s IV and V. . C h o l i n o c e p t i v e c e l l s t y p i c a l l y were e x c i t e d by iontopho-r e t i c a l l y - a p p l i e d ACh w i t h a slow onset and a prolonged a f t e r -e f f e c t : t h i s was i n c o n t r a s t to the f a s t onset and o f f s e t 35 e x c i t a t o r y a c t i o n observed w i t h L-glutamate ( K r n j e v i c and P h i l l i s , 1963a; Randic et al.-, 1964). Betz c e l l s were id e n -t i f i e d by t h e i r a n t i d r o m i c discharge f o l l o w i n g s t i m u l a t i o n of the medullary pyramids and were shown to be e x c i t e d by m u s c a r i n i c c h o l i n o m i m e t i c s , w h i l e the a c t i o n of ACh was shown to be b l o c k e d by such a n t a g o n i s t s as a t r o p i n e and b e n a c t y z i n e (Crawford and C u r t i s , 1966). A c e t y l - g - m e t h y l c h o l i n e (A3MC) was as or more e f f e c t i v e than ACh i t s e l f on many c o r t i c a l neurones although i t s a c t i o n s c l o s e l y resembled those of ACh, s p e c i f i c a l l y concerning l a t e n c y of e f f e c t , d u r a t i o n of a f t e r -discharge and l a c k of d e s e n s i t i z a t i o n d e s p i t e prolonged a d m i n i s t r a t i o n . Other e f f e c t i v e c h o l i n e e s t e r s i n c l u d e d carb-a m i n o y l c h o l i n e ( c a r b a c h o l ) , p r o p i o n y l c h o l i n e , s u c c i n y l c h o l i n e and n i c o t i n y l c h o l i n e , although some of these had weaker a c t i o n s than ABMC or ACh. Muscarine and muscarone were ve r y e f f e c t i v e e x c i t a t o r y agents on c h o l i n o c e p t i v e neurones, even on c e l l s which were only weakly e x c i t e d by ACh. A r e c o l i n e , p i l o c a r p i n e and oxotremorine e x e r t e d t h e i r e x c i t a t o r y e f f e c t s w i t h a slower onset than ACh (>1 min) and e l i c i t e d p rolonged a f t e r - d i s c h a r g e s l a s t i n g f o r s e v e r a l minutes. V a r i o u s a n t i c h o l i n e s t e r a s e s a p p l i e d i o n t o p h o r e t i c a l l y appeared to i n c r e a s e n e u r onal f i r i n g r a t e s , and both K r n j e v i c and P h i l l i s (1963c) and Spehlmann (1963) found t h a t p r o s t i g m i n e and t e n s i l o n e x c i t e d some neurones. T h i s was b e l i e v e d to be due to the parasympatho-mimetic a c t i o n s of these compounds, i n a d d i t i o n to t h e i r pos-s i n g a c t i v i t y as a n t i c h o l i n e s t e r a s e s . When ACh i t s e l f caused 36 e x c i t a t i o n , 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 both 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 . K r n j e v i c and P h i l l i s (1963b) a t t r i b u t e d the e x c i t a t o r y a c t i o n o c c a s i o n a l l y observed w i t h n i c o t i n e as " u n s p e c i f i c " due to the p r o p e r t y of t h i s compound to a f f e c t neurones o n l y a f t e r a comparatively long a p p l i c a t i o n and w i t h an abrupt onset, producing "a paroxysmal discharge which i s q u i t e d i f -f e r e n t from the e f f e c t s of ACh on c h o l i n o c e p t i v e neurones". N i c o t i n e appeared to be e f f e c t i v e i r r e s p e c t i v e of the s e n s i -t i v i t y to ACh, and d i d not i t s e l f antagonize the a c t i o n s of ACh. T h i s f i n d i n g was not confirmed by Crawford and C u r t i s (1966) who observed that n i c o t i n e was a c t i v e as a n e u r o n a l e x c i t a n t only.on those neurones which were r e s p o n s i v e to ACh although not a l l of the c h o l i n o c e p t i v e c e l l s c o u l d be e x c i t e d by i t . 1 ,l-Dimethyl-4-phenylpiperazinium i o d i d e (DMPP), a n i c o t i n i c a g o n i s t on p e r i p h e r a l neurones (Chen, Portman and Wickel, 1951), was found r e l a t i v e l y i n a c t i v e . Some of the substances which had been used w i t h success i n 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 a c t i o n s of ACh were found by K r n j e v i c and P h i l l i s (1963c) to be l a r g e l y i n a c t i v e as c e n t r a l b l o c k e r s of the e f f e c t s of ACh and a l l cholinomimetics. Curare and d i h y d r o - B - e r y t h r o i d i n e (DHBE) were among these compounds; weak e x c i t a t o r y a c t i o n s of curare were sometimes observed - a p r o p e r t y incompatible w i t h i t s known a n t a g o n i s t i c a c t i o n although others have r e p o r t e d t h i s a l s o (Feldberg and 37 F l e i s c h h a u e r , 1962). D H B E was onl y modestly e f f e c t i v e as an ACh b l o c k e r on a few c o r t i c a l neurones; Spehlmann (1963) and Crawford and C u r t i s (1966) found t h a t some s y n a p t i c a l l y - e x c i t e d c e l l s c o u l d have t h e i r a c t i v a t i o n s b l o c k e d by t h i s a n t a g o n i s t however, and no e x p l a n a t i o n was o f f e r e d f o r these anomalous .^results . Gallamine, mecamylamine and hexamethonium, a l l n i c o t i n i c b l o c k e r s , d i d e x h i b i t some a n t a g o n i s t i c e f f e c t s , although the s p e c i f i c i t y f o r ACh-induced responses was not marked. The ob s e r v a t i o n s of a c e r t a i n degree of a c t i o n of the n i c o t i n i c a n t a g o n i s t s seems a t v a r i a n c e w i t h the l a c k of a s p e c i f i c e f f e c t i v e n e s s of n i c o t i n i c a g o n i s t s on the f e l i n e c o r t i c a l neurones ( K r n j e v i c and P h i l l i s , 1963c) although the r e s u l t s o f Crawford and C u r t i s (1966) showing n i c o t i n i c e x c i t -a t i o n s may be s i g n i f i c a n t i n t h i s r e s p e c t . The most e f f e c t i v e agents i n causing a s e l e c t i v e , r e v e r -s i b l e b l o c k of c h o l i n e r g i c e x c i t a t i o n were those, p o s s e s s i n g a n t i - m u s c a r i n i c p r o p e r t i e s ; thus a t r o p i n e and e s p e c i a l l y hyos-c i n e were s p e c i f i c at low doses ( i n th a t the ne u r o n a l responses to glutamate were i n t e r f e r e d w i t h to a l e s s e r extent than those to ACh - see below). Benactyzine and caramiphen a l s o demonstrated a t r o p i n e - l i k e p r o p e r t i e s , although caramiphen was somewhat l e s s e f f e c t i v e i n t h i s r e gard. A l l of these antag-o n i s t s possessed a l o c a l a n a e s t h e t i c a c t i o n i n th a t at moderate to h i g h doses, n o n - s e l e c t i v e , g e n e r a l depressant e f f e c t s were observed. Recovery of the e f f e c t s o f glutamate always prece-eded those of ACh however, and when a p p l i e d i o n t o p h o r e t i c a l l y 38 at low e j e c t i n g c u r r e n t s (<60 nA), a g r e a t e r s p e c i f i c i t y of a c t i o n a g a i n s t ACh was o b t a i n e d ( K r n j e v i c and P h i l l i s , 1963c). In c o n s i d e r a t i o n of these r e s u l t s , o b t a i n e d through the use of an e x t e n s i v e range o f c h o l i n e r g i c drugs, K r n j e v i c and P h i l l i s and most subsequent i n v e s t i g a t o r s (Crawford and C u r t i s , 1966; Legge, Randic and Straughan, 1966; Spehlmann and Downes, 1974; Spehlmann and Smathers, 1974) concluded that the r e c e p t o r s on f e l i n e c o r t i c a l neurones are almost e x c l u -s i v e l y m u s c a r i n i c i n n a t u r e . T h i s c o n t r a s t s w i t h the c h o l i n e r -g i c pharmacology of some p e r i p h e r a l synapses as w e l l as the s t a t e of a f f a i r s of c h o l i n e r g i c r e c e p t o r s of Renshaw c e l l s , to be d i s c u s s e d p r e s e n t l y . In a d d i t i o n to the e x c i t a t o r y a c t i o n of ACh on c o r t i c a l neurones, t h i s compound has been observed to cause d e p r e s s i o n o f the f i r i n g r a t e s of some c e l l s . The depressant e f f e c t c o u l d be observed on neurones w i t h h i g h spontaneous r a t e s o f d i s c h a r g e , or on those whose f i r i n g r a t e s were e l e v a t e d by i o n t o p h o r e t i c a p p l i c a t i o n of glutamate. Randic et a l . (1964) found t h a t a c o n s i d e r a b l e number (about 25% of the c o r t i c a l neurones t e s t e d were d i r e c t l y e x c i t e d by ACh, and t h a t few (5%) were i n h i b i t e d . S i x neurones which were depressed by ACh had the i n h i b i t i o n s p a r t i a l l y antagonized by a t r o p i n e . These f i n d i n g s were c o r r o b o r a t e d by Crawford and C u r t i s (1966), who found t h a t among those neurones depressed by ACh, many co u l d be i d e n t i f i e d as b eing Betz c e l l s . I t was p o s s i b l e on 2 39 of 15 c e l l s however, to antagonize the e f f e c t s of ACh w i t h the n i c o t i n i c a n t a g o n i s t DH3E whereas most c e l l s t e s t e d w i t h a t r o p i n e e x h i b i t e d a reduced ACh s e n s i t i v i t y . P h i l l i s and York (1967; 1968a,b,c,), Jordan and P h i l l i s (1972) and Spehl-mann and Downes (1974) a l s o have found ACh-induced i n h i b i t i o n s i n c e r e b r a l c o r t e x , and observed i n h i b i t o r y s y n a p t i c a c t i o n s w i t h s t i m u l a t i o n of a f f e r e n t f i b r e s from the co r t e x i t s e l f , l a t e r a l hypothalamus and r e t i c u l a r f o r m a t i o n . Both m u s c a r i n i c and n i c o t i n i c a n t a g o n i s t s have been r e p o r t e d to reduce these c o r t i c a l i n h i b i t i o n s ( P h i l l i s and York, 1968c) and the p r e c i s e p h a r m a c o l o g i c a l nature of these i n h i b i t o r y c h o l i n e r g i c p r ocesses t h e r e f o r e remains u n c e r t a i n . A l l the above r e p o r t s have been based on r e s u l t s o b t a i n e d from f e l i n e neurones. Stone (1972) examined the responses of c o r t i c a l neurones i n the r a t somatosensory area to ion t o p h o r -e t i c a l l y - a p p l i e d cholinomimetics and a n t i c h o l i n e r g i c substan-ces. ACh e x c i t e d 80% of i d e n t i f i e d pyramidal t r a c t neurones and showed no i n h i b i t o r y a c t i o n s on t h i s p o p u l a t i o n . Physo-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 and AfBMC, ca r b a c h o l and p i l o c a r p i n e mimicked the e f f e c t s of ACh, but n i c o t i n e proved i n e f f e c t i v e . Of the c h o l i n e r g i c a n t a g o n i s t s t e s t e d , a t r o p i n e and hyoscine but not cur a r e , b l o c k e d the e f f e c t s o f ACh and A3MC. ACh and m u s c a r i n i c a g o n i s t s i n h i b -i t e d 25% of the non-pyramidal t r a c t neurones, most of which l a y between 0.5 to 1.6 mm below the c o r t i c a l s u r f a c e ; these 40 depressions were b l o c k e d by a t r o p i n e or hyoscine. Non-pyramidal t r a c t c e l l s l o c a t e d above 0.6 mm depth were e x c i t e d by ACh and n i c o t i n e , and these e f f e c t s were a b o l i s h e d by c u r a r a . Thus Stone (1972) concluded t h a t the c h o l i n o c e p t i v e pyramidal t r a c t c e l l s of r a t c e r e b r a l c o r t e x possess e x c l u s i v e l y m u s c a r i n i c e x c i t a t o r y r e c e p t o r s , those of the non-pyramidal t r a c t neurones which occur below 0.6 mm are m u s c a r i n i c , and the more s u p e r f i c i a l l y l o c a t e d neurones possess n i c o t i n i c r e c e p t o r s . When c o n t r a s t e d w i t h those of the c a t , these r e s u l t s d i f -f e r i n c e r t a i n r e s p e c t s which are worthy of mention. The s e g r e g a t i o n of n i c o t i n i c responses w i t h i n the upper 0.6 mm of the c o r t e x as observed by Stone (1972) has not been noted by others who observed e f f e c t s w i t h t h i s cholinomimetic i n c a t s ( K r n j e v i c and P h i l l i s , 1963} Spehlmann, 1963; Crawford and C u r t i s , 1966), although among these authors there was d i s a g r e -ement concerning the s i g n i f i c a n c e of n i c o t i n i c e x c i t a t i o n s . The o b s e r v a t i o n s of P h i l l i s and York (1968) t h a t ACh e l i c i t e d i n h i b i t o r y responses of f e l i n e neurones and t h a t these c o u l d be b l o c k e d both by n i c o t i n i c and by m u s c a r i n i c a n t a g o n i s t s was a f u r t h e r p o i n t of disagreement w i t h the r e s u l t s on r a t neurones, as Stone found evidence o n l y f o r a m u s c a r i n i c i n h i b -i t o r y e f f e c t . Because of these c o n f l i c t i n g r e p o r t s of the pharmacology of c h o l i n o c e p t i v e neurones between cat and r a t , and due to other i n c o n s i s t e n c i e s i n the l i t e r a t u r e (see below) i t was 41 f e l t worthwhile to i n v e s t i g a t e the nature of the r e c e p t o r s f o r ACh on r a t c e n t r a l neurones u s i n g a range of c h o l i n o -mimetic a g o n i s t s and a n t a g o n i s t s i n order to c h a r a c t e r i z e t h e i r p r o p e r t i e s p h a r m a c o l o g i c a l l y , and to thereby permit comparisons w i t h those of the w e l l c h a r a c t e r i z e d f e l i n e neurones. i i / Thalamus V e n t r o b a s a l thalamic r e l a y neurones which were a c t i v a t e d s y n a p t i c a l l y a t s h o r t l a t e n c y (6-8 msec) by s t i m u l -a t i o n of hindlimb nerves and a n t i d r o m i c a l l y by s t i m u l a t i o n of the i p s i l a t e r a l primary sensory c o r t e x (Andersen and Sears, 1974) were e x c i t e d by amino a c i d s and ACh (Andersen and C u r t i s , 1964a). The mean l a t e n c y to e x c i t a t i o n of thalamic neurones by 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 was longer than t h a t observed f o r Renshaw c e l l s (to be d i s c u s s e d below) , and the e x c i t a t i o n s were o f t e n preceeded by a r e d u c t i o n i n the spont-aneous r a t e suggesting a p o s s i b l e d i f f e r e n c e i n the pharma-c o l o g i c a l p r o p e r t i e s of these two n e u ronal p o p u l a t i o n s . There was f r e q u e n t l y observed a cumulative decrease i n neuronal s e n s i t i v i t y toward constant a d m i n i s t r a t i o n s of ACh, i n d i c a t i n g p o s s i b l e r e c e p t o r d e s e n s i t i z a t i o n (Katz and T h e s l e f f 1958); and a delay (5-60 sees) was o f t e n observed i n the o f f -set of f i r i n g when the e j e c t i n g c u r r e n t was terminated, s i m i l a r to t h a t observed i n the c o r t e x w i t h m u s c a r i n i c e x c i t -ants ( K r n j e v i c and P h i l l i s , 1963b). Those neurones which were e x c i t e d both by m u s c a r i n i c and n i c o t i n i c a g o n i s t s 42 responded w i t h a s t r o n g e r e x c i t a t i o n to ACh i n the presence of i n h i b i t o r s of AChE (Andersen and C u r t i s , 1964b). DH3E, and to a l e s s e r degree a t r o p i n e , were e f f e c t i v e i n antagon-i z i n g the c h o l i n e r g i c e x c i t a t i o n s , although the s y n a p t i c a l l y induced a c t i v a t i o n by cutaneous nerve s t i m u l a t i o n was u n a f f e c -ted by e i t h e r b l o c k i n g agent. The s u g g e s t i o n was made th a t the r e c e p t o r s mediating the a c t i o n s of ACh are of a pharmaco-l o g i c a l type i n t e r m e d i a t e between n i c o t i n i c and m u s c a r i n i c : they are "not as n i c o t i n i c as those of Renshaw c e l l s and not as m u s c a r i n i c as those of c o r t i c a l neurones" (Andersen and C u r t i s , 1964b). These r e s u l t s were confirmed (McCance, P h i l l i s and Westerman, 1966; 1968 a, b) and extended to the l a t e r a l and medial g e n i c u l a t e n u c l e i (Curtis and Davis, 1963, P h i l l i s , Tebecis and York, 1967; T e b e c i s , 1970), and n. r e t i c u l a r i s where mixed 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-a t i n g i n h i b i t i o n s were d e s c r i b e d (Ben-Ari, D i n g l e d i n e , Kanazawa and K e l l y , 1976). There have been s e v e r a l d i f f e r e n c e s of note among these v a r i o u s i n v e s t i g a t o r s however, s e r v i n g to emphasize that d i f f e r e n t thalamic n u c l e i are p h a r m a c o l o g i c a l l y h e t e r o -genous to an extent. C u r t i s and Davis (1963) found DH3E to be i n e f f e c t i v e i n a n t a g o n i z i n g s y n a p t i c e x c i t a t i o n s of LGN neurones but capable o f r e d u c i n g the ACh induced f i r i n g , w h i l e curare and g a l l a m i n e had no observable a n t a g o n i s t i c e f f e c t s . I t was a l s o noted f r e q u e n t l y t h a t A3MC e l i c i t e d depressant e f f e c t s . In a 43 subsequent r e p o r t on v e n t r o l a t e r a l neurones (Davis, 1966) i t was mentioned that a t r o p i n e c o u l d b l o c k completely the e x c i t a t o r y e f f e c t s of ACh when the a n t a g o n i s t was admi n i s t e r e d a t low doses (10 nA). Higher doses of a t r o p i n e (30 nA) antagonized not o n l y the s y n a p t i c responses of thalamic neurones to s t i m u l a t i o n of the c e r e b e l l o - t h a l a m i c t r a c t , but the DL-homocysteate (DLH) e l i c i t e d e x c i t a t i o n s as w e l l . P h i l l i s et a l f (1967) confirmed the dual nature of c h o l i n -e r g i c r e c e p t o r s i n the LGN, found a t r o p i n e to be a n t a g o n i s t i c towards some ACh responses (8 of 21 neurones) and found my lot-on, a n i c o t i n i c b l o c k i n g drug, to be e f f e c t i v e i n preven-t i n g ACh induced changes i n f i r i n g (12 of 18 neurones). ACh depressed the f i r i n g of only 4% of the g e n i c u l a t e neurones t e s t e d . The ph a r m a c o l o g i c a l nature of t h i s i n h i b i t i o n was not i n v e s t i g a t e d f u r t h e r , presumably due to the few neurones which were l o c a t e d i n t h i s r e g i o n and were i n h i b i t e d by ACh. A l a t e r study by Tebecis (1970) on medial g e n i c u l a t e neurones i d e n t i f i e d by s t i m u l a t i o n of the a u d i t o r y c o r t e x r e v e a l e d t h a t o f those c e l l s responding to ACh, 37% were c l e a r l y i n h i b i t e d . These i n h i b i t o r y responses d i d not e x h i b i t apparent d e s e n s i t i z a t i o n as d i d the e x c i t a t i o n s , an observa-t i o n r e p o r t e d i n i t i a l l y by Andersen and C u r t i s (1964b) f o r VB neurones. Tebecis noted f u r t h e r t h a t n i c o t i n i c and musca-r i n i c cholinomimetics e l i c i t e d both e x c i t a t i o n and i n h i b i t i o n . The e f f e c t s of ACh, whether of an e x c i t a t o r y or i n h i b i t o r y 44 nature were antagonized by both types o f c h o l i n e r g i c b l o c k e r s , although the antagonism was not always c l e a r - c u t on a l l neurones. The b l o c k e r s were hot t e s t e d on the responses produced by other cholinomimetics, and t h e r e f o r e a d e f i n i t i v e c h a r a c t e r i z a t i o n of the r e c e p t o r p r o p e r t i e s of these c e l l s was not made p o s s i b l e . In a l a t e r s e r i e s of experiments, M a r s h a l l and McLennan (1972) s t u d i e d the c h o l i n e r g i c pharmacology of f e l i n e v e n t r o -l a t e r a l t h alamic neurones and compared t h e i r r e s u l t s w i t h the phar m a c o l o g i c a l p r o p e r t i e s o f v a r i o u s s y n a p t i c i n p u t s from other b r a i n r e g i o n s . They found t h a t both DHgE and a t r o p i n e s e l e c t i v e l y b l o c k e d ACh induced e x c i t a t i o n s ; the concomitant s y n a p t i c a c t i v a t i o n of these c e l l s by 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 pathway was a l s o s e n s i t i v e to antagonism by both these b l o c k i n g agents, although a t r o p i n e was the more e f f e c t i v e . The tendency towards a s l i g h t l y g r e a t e r m u s c a r i n i c than n i c o t i n i c n a ture was a l s o noted f o r neurones of the VB complex (McCance et a l . , 1966). G o d f r a i n d (1975) found t h a t w i t h i n the p u l v i n a r complex there e x i s t s a tendency f o r ACh to e x c i t e the more p o s t e r i o r l y l o c a t e d neurones, w h i l e 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 of ACh were l e s s c l e a r - c u t . These l a t t e r e f f e c t s were manifes-ted by more f r e q u e n t l y - o c c u r r i n g i n h i b i t i o n s and l e s s d i s t i n c t changes i n the f i r i n g r a t e s . Changes i n f i r i n g p a t t e r n , r a t h e r than e x c i t a t i o n or i n h i b i t i o n were noted, coupled w i t h 45 an a p p a r e n t l y lower potency of ACh. A t r o p i n e , but not DH3E or mecamylamine, r e a d i l y b l o c k e d ACh induced f i r i n g . The e a r l i e r o b s e r v a t i o n s of a g r e a t e r s e n s i t i v i t y to ACh and g l u t -amate wit h i n c r e a s i n g depth f o r v a r i o u s thalamic n u c l e i was confirmed (McCance et a l . , 1968a; McLennan, Huffman and M a r s h a l l , 1968), and G o d f r a i n d concluded that p u l v i n a r neurones are predominately m u s c a r i n i c . Ben-Ari et a l . (1976) examined the p h a r m a c o l o g i c a l charac-t e r i s t i c s of c h o l i n e r g i c i n h i b i t i o n s u s i n g neurones of s t i l l another thalamic r e g i o n , the nucleus r e t i c u l a r i s . Although d i s a g r e e i n g about the exact mechanisms i n v o l v e d , i t i s note-worthy t h a t t h i s group and Duggan and H a l l (1975) concurred i n f i n d i n g t h a t i n h i b i t o r y e f f e c t s of ACh were prevented both by n i c o t i n i c and m u s c a r i n i c a n t a g o n i s t s . These s t u d i e s on the v a r i o u s n u c l e i of the f e l i n e thalamus r e v e a l e d the e x i s t e n c e of ACh r e c e p t o r s which appeared to pos-sess, 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 between n i c o -t i n i c and, m u s c a r i n i c . However there has been no s y s t e m a t i c i n v e s t i g a t i o n of the e f f e c t s of the a n t a g o n i s t s a g a i n s t e x c i t -a t i o n s by the c h o l i n o m i m e t i c s , and so the q u e s t i o n of whether two .separate p o p u l a t i o n s of ACh r e c e p t o r s , one m u s c a r i n i c and the other n i c o t i n i c , 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 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 i s r e s p o n s i b l e f o r m e d i a t i n g the e f f e c t s of ACh r e q u i r e d f u r t h e r r e s e a r c h . T h i s problem, coupled w i t h the l a c k of any p h a r m a c o l o g i c a l s t u d i e s on r a t thalamic neurones, prompted the present i n v e s t i g a t i o n . 46 i i i / S p i n a l Cord The e a r l i e s t s t u d i e s on the pharmacology of c e n t r a l c h o l i n o c e p t i v e neurones which used the i o n t o p h o r e t i c technique were conducted on s p i n a l neurones by C u r t i s and E c c l e s (1958a,b), C u r t i s and P h i l l i s (1960) and C u r t i s , P h i l l i s and Watkins (1961). C u r t i s and E c c l e s (1958a,b) examined the responses of Renshaw c e l l s to s y n a p t i c a c t i v a t i o n by the ionto-p h o r e t i c a d m i n i s t r a t i o n of ACh and a range of 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 , and to a n t i c h o l i n e s t e r a s e compounds, and v a r i o u s c h o l i n e r g i c a n t a g o n i s t s . Renshaw c e l l s were r e a d i l y e x c i t e d by ACh and n i c o t i n e . S u e c i n y l c h o l i n e , A3MC, ca r b a c h o l and a r e c o l i n e were a l l some-what l e s s e f f e c t i v e than ACh, although c a r b a c h o l was sometimes observed to be equipotent. Prostigmine a d m i n i s t e r e d alone or concomitantly w i t h ACh g r e a t l y i n c r e a s e d both the spontaneous and the s y n a p t i c a l l y evoked dis c h a r g e s of Renshaw c e l l s , an a c t i o n c o n s i s t e n t w i t h i t s known anti-AChE p r o p e r t y . Curare had somewhat v a r i a b l e e f f e c t s on the ACh induced and s y n a p t i c f i r i n g of these neurones; i t was u s u a l l y r e l a t i v e l y i n e f f e c t i v e , although on some c e l l s a r e d u c t i o n of the response to ACh g r e a t e r than 50% was observed. A much more pronounced e f f e c t a g a i n s t Renshaw c e l l e x c i t a t i o n s was obt a i n e d when DH3E was employed as an a n t a g o n i s t . In a d d i t i o n to b l o c k i n g the e f f e c t o f 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 and n i c o t i n e , t h i s compound n e a r l y a b o l i s h e d the s y n a p t i c a l l y evoked e x c i t a t i o n . 47 In a l a t e r r e p o r t , C u r t i s et a l . , (1961) s t u d i e d a wide range of c h o l i n e r g i c drugs i n an e f f o r t to d e s c r i b e f u l l y the pha 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 of ACh r e c e p t o r s on Renshaw c e l l s . A s e r i e s of c h o l i n e 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 . In comparison w i t h the b r i e f a f t e r - d i s c h a r g e observed w i t h ACh, these e s t e r s e l i c i t e d q u i t e prolonged e f f e c t s f o l l o w i n g the t e r m i n a t i o n of the i o n t o p h o r e t i c c u r r e n t . T h i s phenomenon was c o n s i d e r e d due, i n p a r t , to the i n e f f e c t i v e n e s s of AChE toward the e s t e r s and the r e l a t i v e s t a b i l i t i e s of the a g o n i s t -r e c e p t o r complexes formed: a parameter which presumably d i f -f e r s f o r 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 . The experiments i n d i c a t e d that there e x i s t n o n - c h o l i n o c e p t i v e r e c e p t o r s on Renshaw c e l l s , s i n c e DH3E f a i l e d to antagonize, and even some-times p o t e n t i a t e d the responses of the neurones when these were a c t i v a t e d by f i r i n g the d o r s a l r o o t s . C u r t i s and R y a l l (1966a,b,c) confirmed the r e s u l t s of the above r e p o r t s , r e a c h i n g the c o n c l u s i o n t h a t Renshaw c e l l s i n the c at possess both n i c o t i n i c and m u s c a r i n i c ACh r e c e p t o r s , the l a t t e r m a n i f e s t i n g an e x c i t a t o r y a c t i o n some 500-700 msec, l a t e r than the i n i t i a l , n i c o t i n i c a c t i o n . The " f a s t " , or " e a r l y " component of the s y n a p t i c d i s c h a r g e s r e f l e c t e d the n i c o t i n i c a c t i o n . In f a c t , the l a t e r d i s c h a r g e s observed w i t h ACh may r e f l e c t the presence of m u s c a r i n i c r e c e p t o r s u n i n v o l v e d i n the " t r u e " s y n a p t i c a c t i o n , s i n c e , on o c c a s i o n , a t r o p i n e was found to p a r t i a l l y antagonize the a c t i o n of ACh. 48 When DHgE antagonized the s y n a p t i c e x c i t a t i o n and the e x c i t a t i o n by i o n t o p h o r e t i c n i c o t i n e , the responses to A8MC were u n a l t e r e d . The ACh induced e x c i t a t i o n was c o n s i d e r a b l y a t t e n u a t e d as w e l l . By c o n t r a s t , when a t r o p i n e p a r t i a l l y reduced the e f f e c t i v e n e s s of v e n t r a l r o o t v o l l e y s i n synap-t i c a l l y e x c i t i n g the c e l l s , A3MC induced e x c i t a t i o n s were antagonized, ACh induced e x c i t a t i o n s were reduced to 607c1 of c o n t r o l s , w h i l e those produced by n i c o t i n e were u n a f f e c t e d . Since no c r o s s o v e r between n i c o t i n i c a n t a g o n i s t s and muscar-i n i c a g o n i s t s (or v i c e v e r s a) was observed, and s i n c e the e x c i t a t i o n s caused by ACh were a composite of both the n i c o t -i n i c and m u s c a r i n i c e f f e c t s (as n e i t h e r a n t a g o n i s t completely a b o l i s h e d i t s a c t i o n ) , C u r t i s and R y a l l (1966a,b,c,) reached the c o n c l u s i o n t h a t two separate p o p u l a t i o n s of ACh s e n s i t i v e r e c e p t o r s e x i s t on Renshaw c e l l s of the c a t . The i n f r e q u e n t l y observed responses of s p i n a l c o r d i n t e r -neurones other than Renshaw c e l l s to ACh and c h o l i n o m i m e t i c s , are weakly e x c i t a t o r y or i n h i b i t o r y and are not b e l i e v e d to p l a y any s i g n i f i c a n t r o l e i n p rocesses of s y n a p t i c t r ansmission ( C u r t i s , R y a l l and Watkins, 1966; Engberg and R y a l l , 1966). Headley, Lodge and B i s c o e (1975) examined the responses of Renshaw c e l l s i n r a t s 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 c h o l i n o -mimetics and c h o l i n e r g i c a n t a g o n i s t s and found s e v e r a l d i f f e r -ences between the responses observed and those r e p o r t e d f o r the c a t . The antagonism by a t r o p i n e of ACh responses on most 49 c e l l s was h i g h l y e f f e c t i v e , although the s e l e c t i v i t y f o r ACh ag a i n s t other e x c i t a n t s was not as pronounced as t h a t observed w i t h DHBE. A t r o p i n e m o d i f i e d s y n a p t i c a l l y evoked responses' i n a s i m i l a r manner as d i d DH3E; thus the t o t a l number of sp i k e s evoked i n a given 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 stimulus was reduced, and the l a t e n c y to the f i r s t and s u c c e s s i v e s p i k e s was i n c r e a s e d . DH3E and a t r o p i n e only o c c a s i o n a l l y antagonized the e f f e c t s of A8MC, ca r b a c h o l and n i c o t i n e w i t h -out m a n i f e s t i n g n o n - s p e c i f i c e f f e c t s . D i f f e r e n c e s between these r e s u l t s and those r e p o r t e d f o r f e l i n e Renshaw c e l l s were: 1) l a c k of o b s e r v a t i o n of a l a t e m u s c a r i n i c e x c i t a t i o n f o l l o w i n g a c t i v a t i o n of the 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 , 1966b,c); 2) e f f e c t i v e n e s s o f both a t r o p i n e and DH3E i n b l o c k i n g the v e n t r a l r o o t a c t i v a t i o n ; 3) s e l e c t i v e blockade by both DH3E and a t r o -pine of ACh responses without a f f e c t i n g responses to amino a c i d e x c i t a n t s ; and 4) a marked q u a l i t a t i v e d i f f e r e n c e i n the degree of s p e c i f i c i t y of the a n t a g o n i s t s i n b l o c k i n g the e x c i t a t o r y a c t i o n s of n i c o t i n i c and m u s c a r i n i c cholinomimetics; the 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 m u s c a r i n i c a n t a g o n i s t s a g a i n s t A3MC on the one hand, and wi t h n i c o t i n i c a n t a g o n i s t s a g a i n s t n i c o t i n e on the other, w i t h no o v e r l a p p i n g antagonism. That r a t Renshaw c e l l s possess a mixture of n i c o t i n i c and m u s c a r i n i c p o p u l a t i o n s o f r e c e p t o r s as i s true f o r the c a t , 50 seemed u n l i k e l y due to the o b s e r v a t i o n s t h a t both DHgE and a t r o p i n e b l o c k e d completely the ACh e x c i t a t i o n s and t h a t DH6E c o u l d b l o c k the a c t i o n o f A3MC. Headley et a l . (1975) conclu-ded that there i s a q u a l i t a t i v e d i f f e r e n c e between c h o l i n e r g i c r e c e p t o r s of cat and r a t Renshaw c e l l s , and that f o r the r a t , they cannot be c a t e g o r i z e d i n the t r a d i t i o n a l manner. 51 b) R e s u l t s i / Cortex Neurones of the c e r e b r a l c o r t e x were e x c i t e d by glutamate, ACh or any of the s e v e r a l cholinomimetics used i n the p r e s e n t study (see Table 1), and those e x c i t e d by ACh were l o c a t e d at a l l depths from the s u r f a c e , from ca. 0.2 - 2.0 mm. (see depth summary, Table 2). I n h i b i t o r y e f f e c t s of ACh and cholinomimetics were not s t u d i e d i n the presen t i n v e s t i g a t i o n . As the m i c r o p i p e t t e was lowered through the cortex , ACh or occas-i o n a l l y an e x c i t a t o r y amino a c i d was e j e c t e d c o n t i n u o u s l y to l o c a t e neurones which were not spontaneously a c t i v e . C h o l i n e r g i c A g o n i s t s . I t was observed c o n s i s t e n t l y t h a t neurones which were respon-s i v e to ACh were e x c i t e d a l s o by A3MC, n i c o t i n e , c a r b a c h o l , or DMPP a t a l l l e v e l s i n the c e r e b r a l c o r t e x . A t o t a l of 61 neurones a f f e c t e d by ACh were t e s t e d f o r t h e i r r e s p onsiveness to both 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 , and Table 2 i n d i c -ates t h a t the m a j o r i t y were e x c i t e d by both. The upper t r a c e i n F i g u r e 1 shows the responses o f a neurone to ACh, n i c o t i n e and ABMC. ACh t y p i c a l l y d i s p l a y e d a medium f a s t onset o f a c t i o n , a c h i e v i n g a peak f i r i n g r a t e between 5 - 2 0 sees. I t was d i f f i c u l t to c h a r a c t e r i z e the responses o f c o r t i c a l neurones to the a g o n i s t s and e s p e c i a l l y f o r n i c o t i n e , on the b a s i s o f the f i r i n g p a t t e r n evoked by these compounds, as both shor t l a t e n c y and long l a t e n c y on-and o f f s e t responses were obtained. Such responses o c c a s i o n a l l y 52 TABLE 2. Numbers o f c h o l i n o c e p t i v e neurones of 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 drugs. ACh N i c o t i n i c M u s c a r i n i c + 0 + 0 + 58 51 7 0 53 3 2 — 3 . 0 0 3 0 2 1 Depths where c h o l i n o c e p t i v e neurones were found. • 10-H C o 0 01 02 0.3 0.4 05 0.6 0.7 OS 0.9 10 11 1.2 13 14 15 Depth i n mm. 5 3 Figure 1. Responses of a cerebral c o r t i c a l neurone to ACh, nicotine and AgMC, and the effects of the n i c o t i n i c antagonist DHgE. Ordinate: f i r i n g rate of the c e l l i n s p i k e s / s e c ; abcis-sa: time, i n this and every other Figure of rate recordings. Horizontal bars beneath the traces represent periods of drug application and the numbers are the iontophoretic currents used to expel the compounds, measured i n nA. This c e l l was located i n the somatosensory area, at a depth of 1.10 mm from the c o r t i c a l surface. Applied for 9.5 min. at an ejecting current of 30 nA, DHgE antagonized the excitations produced by ACh and nicotine (middle record) and the i n i t i a l responses recovered 3.5 min. after cessation of DH$E administration. 54 Cortex Depth: 1.10 mm 100 r 0 d 100 o> c o \ CO CD 'CL CO 0 ACH 50 NIC 35 ApMC 25 i i I Min. 100 0 55 resembled those e l i c i t e d by the m u s c a r i n i c a g o n i s t AgMC (see Fi g u r e 2), although the response p a t t e r n i l l u s t r a t e d i n Fig u r e 1 was more t y p i c a l . Carbachol, a compound which has been r e p o r t e d to possess 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 as a n i c o t i n i c a g o n i s t on those c e l l s t e s t e d i n the r a t CNS. T h i s p o i n t w i l l be d i s c u s s e d f u r t h e r w i t h r e s p e c t to the a c t i o n s of the c h o l i n -e r g i c a n t a g o n i s t s . Carbachol e l i c i t e d v e r y f a s t onset e x c i t a -t o r y e f f e c t s upon neurones which responded to ACh. DMPP was t e s t e d on neurones which responded to n i c o t i n e or c a r b a c h o l , and i n every i n s t a n c e i t s e f f e c t was e q u i v a l e n t to th a t of n i c o t i n e . A3MC was the s o l e m u s c a r i n i c e x c i t a n t employed s i n c e t h i s compound i s known to have a very s p e c i f i c a c t i o n on m u s c a r i n i c r e c e p t o r s ( K r n j e v i c and P h i l l i s , 1963c) and was e a s i l y e j e c t e d 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 h i g h c u r r e n t s . AgMC e x c i t e d neurones w i t h a slow onset and o f t e n had a peak e f f e c t immediately upon s w i t c h i n g the c u r r e n t from e j e c t i n g to r e t a i -n i n g , even when p l a t e a u responses had been a t t a i n e d ( F i g u r e 1). The d u r a t i o n of a f t e r e f f e c t was u s u a l l y l e s s prolonged than was the r i s e time to peak a c t i v i t y ( F i g u r e s 1, 2, 4 and 5). These 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 to those d e s c r i b e d by K r n j e v i c and P h i l l i s (1963c) and Crawford and C u r t i s (1966) f o r f e l i n e c o r t i c a l neurones. 56 Some neurones were found which were e x c i t e d by ACh and A3MC and not by the n i c o t i n i c e x c i t a n t s (11 c e l l s ) w h i l e others (8 c e l l s ) were unresponsive to AgMC but were e x c i t e d by ACh, DMPP, c a r b a c h o l and n i c o t i n e . S i x c o r t i c a l neurones were l e s s r e s p o n s i v e to ACh than to a l l c h o l i n o m i m e t i c s , judged by the i n t e n s i t y of e j e c t i n g c u r r e n t s which were r e q u i -r e d to e l i c i t e x c i t a t i o n s o f e q u i v a l e n t magnitude. C h o l i n e r g i c A n t a g o n i s t s . As the e x c i t a t i o n o f the same neurones by both n i c o t i n i c and mu s c a r i n i c cholinomimetics was f r e q u e n t l y observed, s e v e r a l a n t a g o n i s t s were employed i n order to determine the degree of s p e c i f i c i t y o f the e x c i t a t o r y a c t i o n s of A3MC, n i c o t i n e and car b a c h o l . F i g u r e s 1 and 3 i l l u s t r a t e the e f f e c t o f two n i c o -t i n i c a n t a g o n i s t s , DH3E and mecamylamine, on e x c i t a t i o n s e l i c i t e d by c h o l i n e r g i c a g o n i s t s . I t i s c l e a r from the example i n F i g u r e 1 t h a t the n i c o t i n i c a n t a g o n i s t b l o c k e d completely the e x c i t a t o r y e f f e c t s of ACh and n i c o t i n e w h i l e e x e r t i n g l i t t l e or no e f f e c t upon that produced by A3MC. S i m i l a r r e s u l t s to those i l l u s t r a t e d were ob t a i n e d w i t h the n i c o t i n i c b l o c k e r curare. When ad m i n i s t e r e d at low doses, a t r o p i n e was a l s o e f f e c t i v e i n a b o l i s h i n g the e x c i t a t i o n o f c o r t i c a l neurones by ACh, as i l -l u s t r a t e d i n F i g u r e .4, and by^A3MC. The a c t i v a t i o n o f neurones by car b a c h o l ( F i g u r e 5) or n i c o t i n e was maintained d e s p i t e the pres-ence 'of a t r o p i n e . 57 F i g u r e 2. Responses of two c o r t i c a l neurones to ACh and cholino-mimetics. Neurone at 0.24 mm responds w i t h f a s t onset and o f f s e t to a l l e x c i t a n t s w h i l e a slower onset and o f f s e t response pat-t e r n c h a r a c t e r i z e s the responses of another c e l l at 1.64 mm. 58 Cortex 50 r L M A IWl 0 . 2 4 m m u ^ 4 0 40 45 1 m i F l Ixl £ 5 0 r >v 1.64mm 35 "zo" "Ed' 59 F i g u r e 3. Responses of a c o r t i c a l neurone to ACh a d m i n i s t e r e d w i t h 50 nA, and the e f f e c t of the n i c o t i n i c a n t a g o n i s t , meca-mylamine. A n t a g o n i s t was a d m i n i s t e r e d l e s s than 2 min b e f o r e an e f f e c t was observed. T h i s c e l l was l o c a t e d at a depth of 0.98 mm from the c o r t i c a l s u r f a c e . Cortex n .. n Q Q Depth:0.98 mm M E C A M Y L A M I N E 37 50 50 50 I I Min . 61 I t was noted p r e v i o u s l y that c a r b a c h o l possesses 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 at c e r t a i n s i t e s (Goth, 1976); however i t s a c t i o n i n the p r e s e n t study was n i c o t i n i c , i n agreement w i t h others who have t e s t e d t h i s compound on c e n t r a l neurones ( K o e l l e , 1965; Davis, 1966; Horn, 1975; T a n g r i , Sinha, M i s r a and Bhargava, 1976). As F i g u r e 5 i l l u s t r a t e s , the a c t i o n of c a r b a c h o l was u n a f f e c t e d by a dose o f a t r o p i n e s u f -f i c i e n t to b l o c k e x c i t a t i o n s e l i c i t e d by A8MC and ACh. Since the a c t i o n of an a g o n i s t i s d e f i n e d p r i n c i p a l l y by i t s antag-o n i s t , and as c a r b a c h o l e l i c i t e d e x c i t a t i o n s were r e g u l a r i l y antagonized by DH8E or curare but not by a t r o p i n e , i t was reasonable 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 a c t i o n of c a r b a c h o l as being n i c o t i n i c . i i / V e n t r o b a s a l (VB) Thalamus C h o l i n e r g i c A g o n i s t s The r e s u l t s summarized i n Table 3 were ob t a i n e d from the admin-i s t r a t i o n of ACh, c a r b a c h o l , n i c o t i n e and A8MC to neurones of the VB thalamus 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 hindlimb nerves. The range of l a t e n c i e s f o r t h i s s y n a p t i c a c t i v a t i o n was 5-9 msec. On every c e l l t e s t e d , c a r b a c h o l appeared to exert a more potent e x c i t a t o r y e f f e c t than d i d either ACh or AgMC, judged by the same c r i t e r i a as t h a t a l r e a d y d e s c r i -bed f o r c o r t i c a l neurones. T y p i c a l thalamic responses to the e x c i t a n t s are i l l u s t r a t e d i n F i g u r e 6. 6 2 F i g u r e 4. E f f e c t of a t r o p i n e on c e l l f i r i n g induced by ACh and glutamate. A t r o p i n e (55nA) s e l e c t i v e l y antagonized the e x c i t a t o r y a c t i o n o f ACh w h i l e having no e f f e c t upon t h a t e l i c i t e d by glutamate. Each arrow r e p r e s e n t s a time i n t e r v a l of 3.0 min. 63 Cortex DEPTH : 1.34 mm 50 r . ATROPINE 55 GLUT30 ACH 30 i 1 i Min. 64 •Figure 5. E f f e c t of atropine on cholinomimetics and ACh. The effects of A3MC and of ACh are markedly attenuated i n the presence of 23 nA of atropine applied for 3.5 min., whereas those of carbachol, a n i c o t i n i c agonist, remain unaffected. 65 Cortex 0.64mm A§MC CARB ACh 75 40 75 </> 1 min. 66 F i g u r e 6. Responses of a VB thalamic neurone to the s e r i e s of e x c i t a n t s used to c h a r a c t e r i z e neuronal responses. Note the d i f f e r e n c e i n l a t e n c y to peak e x c i t a t i o n achieved w i t h ABMC as compared wi t h ACh, glutamate, c a r b a c h o l and n i c o t i n e . C e l l was evoked by s t i m u l a t i o n of the p e r o n e a l nerve. 67 ACh GLUT A B M C 25 12 43 CARB NIC 20 31 68 TABLE 3. Numbers of c h o l i n o c e p t i v e neurones o f the v e n t r o -b a s a l complex of the thalamus responding to n i c o t i n i c and m u s c a r i n i c drugs. ACh N i c o t i n i c M u s c a r i n i c + 0 — + 0 — + 42 41 1 0 36 6 0 — 3 0 2 1 0 2 1 + : E x c i t a t i o n — : I n h i b i t i o n 0 : No E f f e c t Pontamine sky b l u e spot i n d i -c a t i n g r e g i o n o f VB thalamus where c e l l s were recorded. 69 C h o l i n e r g i c A n t a g o n i s t s . As was found f o r c e r e b r a l c o r t i c a l neurones, the e x c i t a t i o n s produced by ACh, ca r b a c h o l and n i c o t i n e were antagonized by both DHBE and curare, as i s shown i n F i g u r e s 7 and 8. At r o p i n e 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 by glutamate as w e l l as those produced by ACh. To a v o i d t h i s n o n - s p e c i f i c type o f e f f e c t ( d e s c r i b e d by C u r t i s and P h i l l i s , 1960; K r n j e v i c and P h i l l i s , 1963c), only low e j e c t i n g c u r r e n t s ( 1 - 1 0 nA) or moderate c u r r e n t s (10 - 30 nA) used f o r s h o r t p e r i o d s of time, were employed. Under these circumstances the e x c i t a t i o n of 18 neurones by ABMC was a b o l i s h e d or s t r o n g l y reduced by a t r o p i n e , and on 14 of these, ACh induced e x c i t a -t i o n s were a l s o markedly attenuated. An example of the blocking a c t i o n of a t r o p i n e i s i l l u s t r a t e d i n F i g u r e 7, w h i l e the a c t i o n of n i c o t i n e was u n a f f e c t e d . The r a t h e r p a r a d o x i c a l f i n d i n g on both c o r t i c a l and VB thalamic neurones t h a t DHBE, curare and a t r o p i n e c o u l d i n some cases completely b l o c k the e x c i t a t i o n s produced by ACh wh i l e at the same time s p a r i n g the e x c i t a t o r y e f f e c t s o f the n o n - s u s c e p t i b l e cholinomimetic, prompted the experiments i l l u s t r a t e d i n F i g u r e s 8 and 9. C o n t r o l responses of a neurone were e l i c i t e d by the e j e c t i o n of ACh, L-glutamate, ABMC and ca r b a c h o l , as shown i n the upper t r a c e o f F i g u r e 8. In the presence of a dose of curare which j u s t antagonized the e f f e c t s of ACh and ca r b a c h o l but not L-glutamate or ABMC, i t was observed t h a t an i n c r e a s e i n the c u r r e n t e j e c t i n g ACh or 70 F i g u r e 7. Responses of a thalamic neurone to ACh and c h o l i n o -mimetics 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 . During the a p p l i c a t i o n o f DHBE w i t h an i o n t o p h o r e t i c c u r r e n t of 17 nA, neuronal e x c i t a t i o n by ABMC was u n a f f e c t e d , w h i l e during a t r o p i n e (25 nA), e x c i t a t i o n by ABMC was a b o l i s h e d w h i l e the response to n i c o t i n e remained unchanged. In the presence of both a n t a g o n i s t s , ACh induced e x c i t a t i o n was prevented. DHBE was o f t e n observed to have an e x c i t a t o r y e f f e c t i t s e l f , and when t h i s e f f e c t was prominent, c u r a r e c o u l d be used to demonstrate a s i m i l a r s e l e c t i v e antagonism as DHBE, without a concomitant i n c r e a s e i n the b a s e l i n e f i r i n g r a t e . DHBE was a p p l i e d f o r 5.5 min., and rec o v e r y was obt a i n e d a f t e r 4.0 min. A t r o p i n e was a p p l i e d f o r 1.5 min., and rec o v e r y o c c u r r e d 2.5 min. 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 c u r r e n t . VB Thalamus Depth: 4.63 mm Atropine 72 F i g u r e 8. Responses of a s i n g l e thalamic neurone to the admin-i s t r a t i o n of ACh, glutamate, A3MC and c a r b a c h o l w i t h the c u r r e n t s i n d i c a t e d . The middle r e c o r d was o b t a i n e d 4.5 min. a f t e r the s t a r t of e j e c t i o n of curare from another b a r r e l of the e l e c t r o d e ; the lower r e c o r d was o b t a i n e d 1.5 min. a f t e r the end of the curare c u r r e n t . Note i n the c e n t r e t r a c e the i n c r e a s e d c u r r e n t s of ACh and c a r b a c h o l , and the concurrent a d m i n i s t r a t i o n of these two e x c i t a n t s at " c o n t r o l " doses. 73 V B Thalamus Depth : 5 . 6 mm 5 0 r A C H 2 5 G L U T 7 A p M C 2 0 C A R B 5 C U R A R E 4 5 74 ca r b a c h o l caused a r e v e r s a l of the e f f e c t of the a n t a g o n i s t (centre t r a c e ) . A s i m i l a r r e s u l t was obt a i n e d w i t h the e x c i t -a t i o n s produced by ACh or AgMC when the a n t a g o n i s t was atropine ( F i g u r e 9). T h i s f i n d i n g suggested as p o s s i b i l i t i e s t h a t : 1) the i n c r e a s e d c o n c e n t r a t i o n of a g o n i s t allowed a c t i v a t i o n of more d i s t a n t r e c e p t o r s to those which were b l o c k e d by the an t a g o n i s t , 2) the a d d i t i o n a l a g o n i s t i n t e r a c t e d w i t h muscar-i n i c r e c e p t o r s not b l o c k e d by cu r a r e ( f o r F i g u r e 8), or 3) the a d d i t i o n a l a g o n i s t c o m p e t i t i v e l y d i s p l a c e d the a n t a g o n i s t from the r e c e p t o r s . To t e s t these p o s s i b i l i t i e s , ACh and c a r b a c h o l were e j e c -ted s i m u l t a n e o u s l y w i t h 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 F i g u r e 8, and the antagonism by curare was overcome. The bot-tom t r a c e of the F i g u r e demonstrates the r e v e r s i b i l i t y of the b l o c k i n g a c t i o n of the a n t a g o n i s t . Analogous e f f e c t s were obt a i n e d when ACh and AgMC were used i n the presence of a t r o p i n e . i i i / Renshaw C e l l s E x c i t a t o r y Responses. Renshaw c e l l s were i d e n t i f i e d by t h e i r c h a r a c t e r i s t i c h i g h frequency discharge which f o l l o w s a n t i d r o m i c s t i m u l a t i o n of the c e n t r a l end of severed v e n t r a l r o o t s ( F i g u r e 10) (Renshaw, 1946; E c c l e s , F a t t and Koketsu, 1954; E c c l e s , E c c l e s and F a t t , 1956; Frank and Fuo.rtes, 1956; C u r t i s and E c c l e s , 1958a,b; C u r t i s and R y a l l , 1966c). 75 F i g u r e 9. Responses o f a thalamic neurone to ACh, c h o l i n o -mimetics, and the e f f e c t s o f a t r o p i n e . At a dose o f a t r o p i n e s u f f i c i e n t to a b o l i s h the e x c i t a t i o n by ACh and ABMC, the antagonism was overcome w i t h concurrent a d m i n i s t r a t i o n of these two e x c i t a n t s . Responses to n i c o t i n e remained unaf-f e c t e d throughout the a d m i n i s t r a t i o n o f the a n t a g o n i s t . The cen t r e t r a c e was ob t a i n e d 1.0 - 2.0 min. a f t e r the s t a r t of the s t a r t o f the a t r o p i n e e j e c t i o n . The double bars i n the cen t r e t r a c e forilACh r e p r e s e n t s e j e c t i o n at 56 nA, w h i l e t h a t f o r A3MC re p r e s e n t s e j e c t i o n at 70 nA; the l a s t response o f the c e n t r e t r a c e r e p r e s e n t s the concomitant a d m i n i s t r a t i o n o f these two a g o n i s t s , at c o n t r o l c u r r e n t s . V B Tha lamus 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 to moderate f r e q u -ency r a t e o f spontaneous discharge (ca. 20 spi k e s / s e c ) but co u l d be s t i m u l a t e d to f i r e a t ve r y constant f r e q u e n c i e s up to 90 or 100 s p i k e s / s e c by the e j e c t i o n of glutamate, without any e f f e c t upon sp i k e amplitude. When ad m i n i s t e r e d to Renshaw c e l l s i n small amounts, ACh, n i c o t i n i c and m u s c a r i n i c e x c i -t a n t s and L-glutamate were observed to cause e x c i t a t o r y responses. The r e d u c t i o n i n the magnitude of the c u r r e n t r e t a i n i n g c a r b a c h o l w i t h i n a m i c r o p i p e t t e b a r r e l from 10 nA to 5 nA was o f t e n s u f f i c i e n t to e l i c i t a powerful e x c i t a t i o n ( F i g u r e 10). The onset o f the AgMC induced e x c i t a t i o n was long by comparison w i t h ACh or c a r b a c h o l . C h o l i n e r g i c A n t a g o n i s t s . Both 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 a b o l i s h e d the f i r i n g o f Renshaw c e l l s e l i c i t e d by ACh (F i g u r e 11). Curare or DH3E a l s o antagonized the 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 , but had no a n t a g o n i s t i c e f f e c t towards;that o f A3MC. A t r o p i n e substan-t i a l l y reduced the e f f e c t s o f A3MC i n p a r a l l e l w i t h the antag-onism of the ACh responses, but had no apparent e f f e c t upon the car b a c h o l e x c i t a t i o n s . In agreement w i t h the f i n d i n g s o f Headley et a l . (1975), the e a r l y responses o f s y n a p t i c a l l y evoked Renshaw c e l l s were reduced by a t r o p i n e as w e l l as by DH3E or cur a r e . 78 F i g u r e 10. The a c t i v a t i o n of a Renshaw c e l l , whose response to a v o l l e y i n the L5 v e n t r a l r o o t i s shown above, to ACh, ca r b a c h o l and A$MC u s i n g e j e c t i n g c u r r e n t s as i n d i c a t e d . In the case of the c a r b a c h o l e x c i t a t i o n s , the r e t a i n i n g c u r r e n t i n the drug c o n t a i n i n g b a r r e l was reduced from 10 to 5 nA, the subsequent "leakage" from the p i p e t t e being s u f f i c i e n t to induce a potent e f f e c t . ™ J " 1 0 0 / J V 1 0 msec Renshaw 2 5 r o cu co \ CO cu C L CO o 1 A C H 5 C A R B - 5 A p M C 2.5 1.0 Min. 80 F i g u r e 11. Responses of two Renshaw c e l l s to ACh and c h o l i n o -mimetics. Curare (45 nA) antagonized the e f f e c t s of ACh and carb a c h o l w h i l e having no e f f e c t a g a i n s t those o f ABMC or glutamate. 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 min.; r e c o v e r y of c o n t r o l responses o c c u r r e d a f t e r 6.0 min. The second c e l l was t e s t e d w i t h the same e x c i t a n t s i n the presence o f a t r o p i n e (16 nA); the centre t r a c e was reco r d e d 3.5 min. a f t e r the c o n t r o l (upper) t r a c e . Recovery was ob t a i n e d a f t e r 8.0 min. Note the s p e c i f i c i t y o f a c t i o n o f a t r o p i n e i n a n t a g o n i z i n g o n l y the responses to ACh and i;to~ ABMC". .. Note a l s o the d i f f e r -ent s c a l e o f the two f i g u r e s . S P I K E S / S E C . T 8 82 c) D i s c u s s i o n From the r e s u l t s of the pr e s e n t i n v e s t i g a t i o n , i t appears t h a t the c h o l i n e r g i c r e c e p t o r s which mediate e x c i t a t i o n of r a t c e n t r a l neurones d i f f e r from those p r e v i o u s l y c h a r a c t e r i z e d i n the c a t , i n t h a t the e x c i t a t i o n s by ACh possess both muscarinic and n i c o t i n i c p r o p e r t i e s at a l l s i t e s examined. 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 o f c h o l i n o c e p t i v e c e n t r a l neurones i n the c a t as e s t a b l i s h e d by the s t u d i e s of K r n j e v i c and P h i l l i s (1961; 1962; 1963a,b,c), Spehlmann (1963); Randic et a l . (1964) and Crawford and C u r t i s (1966), are u s u a l l y regarded as e x c l u s i v e l y m u s c a r i n i c i n nature. In view of the present f i n d i n g s which support the concept t h a t r a t c o r t i c a l neurones e x h i b i t both n i c o t i n i c and m u s c a r i n i c e f f e c t s , i t i s perhaps r e l e v a n t to r e c a l l t h a t i n the c a t a l s o some evidence has been p r e s e n t e d which showed e x c i t a t o r y e f f e c t s of n i c o t i n e and c a r b a c h o l , and an a c t i o n of DH$E as a s y n a p t i c a n t a g o n i s t (Spehlmann, 1963; Crawford and C u r t i s , 1966). The present o b s e r v a t i o n s d i f f e r a l s o i n some r e s p e c t s from those of Stone (1972) who found m u s c a r i n i c a c t i o n s on c o r t i c a l pyramidal t r a c t neurones of the r a t l o c a t e d e x c l u s -i v e l y below 0.6 mm. whereas other s u p e r f i c i a l (above 0.6 mm) c h o l i n o c e p t i v e c e l l s appeared to possess on l y n i c o t i n i c r e c e p t o r s . Although no attempt was made i n the pr e s e n t experi-ments to c l a s s i f y neurones as pyramidal or non-pyramidal t r a c t , i t i s u n c l e a r why n i c o t i n e - s e n s i t i v e c e l l s were encountered 83 at a l l l e v e l s of the c e r e b r a l c o r t e x whereas Stone was unable to show an e f f e c t w i t h n i c o t i n e or n i c o t i n i c a n t a g o n i s t s at depths below 0.6 mm. The r e s u l t s from VB thalamic neurones are i n good agree-ment w i t h those o b t a i n e d from p r e v i o u s experiments i n the cat (Andersen and C u r t i s , 1964a,b; Davis, 1966; M a r s h a l l and McLennan, 1972), where i t was r e p o r t e d that both 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 and a n t a g o n i s t s are p h a r m a c o l o g i c a l l y a c t i v e . One minor d i f f e r e n c e however was the o b s e r v a t i o n i n an e a r l i e r r e p o r t (McCance et a l . , 1966) that f e l i n e VB thalamic neurones possess somewhat more m u s c a r i n i c than n i c o t i n i c prop-e r t i e s , i n t h a t the e f f e c t s of ABMC and a t r o p i n e were more c l e a r cut upon c e l l a c t i v i t y than were the n i c o t i n i c compounds. No such tendency towards a g r e a t e r m u s c a r i n i c than n i c o t i n i c p r o p e r t y of VB thalamic neurones was noted i n the p r e s e n t experiments w i t h r a t s . More r e c e n t l y , King and R y a l l (1979) have shown i n the cat t h a t a t r o p i n e , although a s p e c i f i c a n t a g o n i s t f o r c h o l i n -e r g i c r e c e p t o r s , does not adequately d i s t i n g u i s h between the a c t i o n s of n i c o t i n i c and m u s c a r i n i c cholinomimetics on Renshaw c e l l s . T h i s f i n d i n g supports the s u g g e s t i o n (Headley et a l . , 1975; McLennan and H i c k s , 1978) t h a t the d i v i s i o n between the two types of r e c e p t o r i s l e s s r i g i d than was p r e v i o u s l y sup-posed. B i r d and Aghajanian (1976) reached a s i m i l a r conclusion from t h e i r experiments on hippocampal c e l l s , where these 84 neurones were found to respond to n i c o t i n i c and m u s c a r i n i c e x c i t a n t s , and the responses to ACh c o u l d be b l o c k e d by both type of a n t a g o n i s t . • P o s s i b l e Receptor Mechanisms As both A3MC and n i c o t i n i c a g o n i s t s e x c i t e d the same c h o l i n o -c e p t i v e neurones i n the c e r e b r a l c o r t e x , VB thalamus and vent-r a l horn o f the s p i n a l cord, and as ACh induced e x c i t a t i o n s of these c e l l s were antagonized both by a t r o p i n e and the n i c o -t i n i c b l o c k e r s , none of these neuronal p o p u l a t i o n s possess e x c l u s i v e l y n i c o t i n i c or m u s c a r i n i c r e c e p t o r s . Indeed, s i n c e the e x c i t a t o r y responses e l i c i t e d by ACh, n i c o t i n e and ABMC were observed to resemble each other c l o s e l y i n terms o f resp-onse l a t e n c y and p a t t e r n on some neurones ( F i g u r e 2), i t c o u l d be suggested t h a t a common r e c e p t o r i s i n v o l v e d i n the c h o l i n -e r g i c a c t i o n . T h i s p r o p o s a l r e c e i v e s some support a l s o from the o b s e r v a t i o n that on VB thalamic neurones, the combined a c t i o n o f ACh and c a r b a c h o l , which by themselves are antagonized by a c h o l i n e r g i c b l o c k e r , can a c t a d d i t i v e l y to overcome the antagonism. The p r o p o s a l that a s i n g l e r e c e p t o r which has both n i c o t -i n i c 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 i s u n l i k e l y by i t s e l f to account f u l l y f o r the observed a c t i o n s o f ACh and cholinomime-t i c s . T h i s i s apparent from the r e s u l t s showing a complete antagonism o f ACh responses at a time when one or the other type of cholinomimetic i s s t i l l e f f e c t i v e i n e l i c i t i n g n euronal 85 responses. I f o n l y a s i n g l e , p h a r m a c o l o g i c a l l y u n s p e c i f i c c h o l -i n e r g i c r e c e p t o r p o p u l a t i o n i s r e s p o n s i b l e f o r mediating the e f f e c t s of ACh and chol i n o m i m e t i c s , then the a c t i o n o f muscarinic a n t a g o n i s t s should reduce the e x c i t a t i o n s o f n i c o t i n i c a g o n i s t s , and v i c e v e r s a . The evidence from t h i s study i s not wh o l l y i n acc o r d w i t h e i t h e r o f the two p o s s i b i l i t i e s which have been v a r -i o u s l y proposed f o r f e l i n e c h o l i n o c e p t i v e neurones: 1) t h a t there e x i s t two p o p u l a t i o n s o f separate r e c e p t o r s both s e n s i t i v e to ACh, one which i s a c t i v a t e d by m u s c a r i n i c and the other by n i c o t i n i c a g o n i s t s ( C u r t i s and R y a l l , 1966c); or 2) t h a t there e x i s t s one p o p u l a t i o n o f "mixed" r e c e p t o r s responding to both n i c o t i n i c and mu s c a r i n i c cholinomimetics (Andersen and C u t t i s , 1964b). The experiments where a r e v e r s a l o f the antagonism by curare was caused by both doubling the e j e c t i n g c u r r e n t a p p l i e d to the ACh or c a r b a c h o l c o n t a i n i n g b a r r e l and a d m i n i s t e r i n g the two ag o n i s t s c o n c u r r e n t l y ( F i g u r e 8), p r o v i d e a r e s u l t d i f f i c u l t to r e c o n c i l e w i t h e i t h e r a one or two r e c e p t o r h y p o t h e s i s . The a d d i t -i v e e f f e c t o f ACh and c a r b a c h o l i n overcoming the a c t i o n of curare i m p l i e s c o - o p e r a t i v i t y a g a i n s t the c o m p e t i t i v e a n t a g o n i s t , i m p l i c a -t i n g the e x i s t e n c e o f a s i n g l e r e c e p t o r or r e c e p t i v e s i t e shared by 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 constants o f ;the two substances are the same whether a d m i n i s t e r e d together or alone; y e t when e j e c t e d alone, the e f f e c t s o f each are blocked. However the maintained e f f e c t i v e n e s s o f A3MC at a time when the e f f e c t s of ACh and c a r b a c h o l are antagonized by cu r a r e i s su g g e s t i v e o f the presence o f two p o p u l a t i o n s o f r e c e p t o r s , although an 86 explanation i s not obvious why ACh i s completely unable to produce an excitatory e f f e c t through the muscarinic population a of receptors which are unaffected by curare. Certain alternative proposals may be made which might have a bearing upon the observed r e s u l t s . For example, i t may be that a single cholinergic receptor with more than one recep-t i v e s i t e e xists. Perhaps curare exerts an action upon one of the s i t e s which i s activated by n i c o t i n i c agonists but which cannot be activated by muscarinic ones, and i t may not be pos-s i b l e for ACh to exert an eff e c t unless both the n i c o t i n i c and the muscarinic s i t e s are available. Another p o s s i b i l i t y i s that "spare receptors" or receptors distant from those affected by the antagonist become activated by the administration of additional agonist. This l a t t e r proposal seems u n l i k e l y how-ever, because: 1) ACh and carbachol at doses which are normally antagonized, should not diffuse over a greater distance when applied concurrently than when applied separately; and 2) such an explanation w i l l not account for the maintenance of a mus-ca r i n i c action i n the presence of a n i c o t i n i c antagonist, at the same time as ACh i s antagonized. Two probable conclusions a r i s i n g from these r e s u l t s are that: 1) the receptors i n the rat are not ea s i l y described as being either of the n i c o t i n i c , muscarinic or mixed types: instead they rather appear to lack s e l e c t i v i t y toward the pharm-acologic a l agents with which they interact; or 2) the cholinergic 87 r e c e p t o r s on r a t c e n t r a l neurones c o n s i s t of a s i n g l e popula-t i o n p o s s e s s i n g two 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 , and indepen-dent a c t i v e s i t e s , both which i n t e r a c t w i t h t h e i r r e s p e c t i v e cholinomimetic a g o n i s t s and a n t a g o n i s t s , and both o f which are n e c e s s a r y i n a c o - o p e r a t i v e sense f o r the i n t e g r a t e d r e s -ponse to ACh i n e x c i t i n g neurones. Although the one c o n c l u s i o n - that the e x c i t a t o r y c h o l i n e r -g i c 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 appear to demonstrate l e s s s e l e c t i v i t y f o r r e a c t i n g w i t h a g o n i s t s than do the f e l i n e r e c e p t o r s - may seem warranted on the b a s i s of the evidence o b t a i n e d w i t h a n t a g o n i s t s , i t i s not intended to propose a t o t a l l y e q u i v a l e n t mechanism f o r ACh r e c e p t o r s throughout a l l r e g i o n s o f the r a t CNS. Carbachol was c l e a r l y a more e f f e c t i v e e x c i t a t o r y agent upon Renshaw c e l l s than upon thalamic neurones, and was of even weaker apparent potency when t e s t e d on c o r t i c a l c e l l s . Furthermore, c o r t i c a l c e l l s were found which responded to ACh and one c l a s s of cholinomimetic only. The suggestion of Headley et a l . (1975) t h a t the d i v i s i o n o f n i c o t i n i c and m u s c a r i n i c a c t i o n s of ACh should not p r o p e r l y be a p p l i e d to the CNS i s s u b s t a n t i a t e d by t h i s i n v e s t i g a t i o n . Although the c o n c l u s i o n s support the concept o f Headley et a l . and B i r d and Aghajanian (1976) t h a t ACh r e c e p t o r s i n the r a t CNS d i f f e r from those of the c a t , the p r e s e n t p h a r m a c o l o g i c a l r e s u l t s are not o f a nature which a l l o w a c l e a r - c u t d e s c r i p t i o n of t h e i r p r o p e r t i e s i n t o t o . 87a One further p o s s i b i l i t y which must be considered i s that the apparent difference between rat and cat ACh receptors may be influenced by the type of anaesthetic employed by various investigators. However, although Krnjevid and P h i l l i s (1963a,b,c) i n cats used a mixture of d i a l l y l - b a r b i t u r i c acid (Dial) and urethane, Crawford and Curtis (1966) used pentobarbitone sodium, while Spehlmann and Downes (1974) used a mixture of methoxyflurane, nitrous oxide and oxygen i n encephale i s o l e preparations, there was nevertheless a general agreement on the r e a c t i v i t y of the f e l i n e receptors. In the experiments on rats, Stone (1972) used urethane, D i a l or halothane i n oxygen, while Headley et a l . (1975) employed pentobarbitone sodium, and again the e f f e c t of the various anaesthetics did not appear to a l t e r the r e s u l t s . However urethane, the drug used i n a l l of the present experi-ments, does possess anticholinesterase properties and therefore conceivably could a l t e r certain of the cholinomimetic responses; and i f t h i s were to be the case the apparently d i f f e r e n t patterns of reaction of the receptors of rat and cat could be affected. 88 CHAPTER IV AMINO ACIDS VENTROBASAL THALAMUS a) I n t r o d u c t i o n The amino a c i d s are r e l a t i v e newcomers to the l i s t of subs-tances b e l i e v e d to a c t as s y n a p t i c t r a n s m i t t e r s i n mammals. T h e i r i m p l i c a t i o n i n s y n a p t i c f u n c t i o n has d e r i v e d from sev-e r a l d i f f e r e n t but r e l a t e d f i e l d s of i n v e s t i g a t i o n , i n c l u d i n g neurochemistry, h i s t o l o g y , p h y s i o l o g y and pharmacology, as i n d i c a t e d by the r e s u l t s of such s t u d i e s , summarized below. 1) Uptake and Release Since there i s an uneven d i s t r i b u t i o n i n the mammalian CNS of L-glutamate, L - a s p a r t a t e , y-aminobutyrate, g l y c i n e and t a u r i n e (Graham, Shank, A p r i s o n and Werman, 1967), i t has been-suggested t h a t these compounds may have f u n c t i o n s other than t h a t of s e r v i n g i n m e t a b o l i c p r o c e s s e s . S t u d i e s concerning the c e n t r a l f u n c t i o n of glutamate and a s p a r t a t e i n p a r t i c u l a r have r e v e a l e d that changes i n the d e n s i t y of h i g h a f f i n i t y uptake s i t e s f o r these a c i d s , a f t e r l e s i o n s , may be used to map p o s s i b l e glutamate pathways. High a f f i n i t y uptake has been suggested as a p o s s i b l e 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 the CNS where the r e s p e c t i v e compound a c t s as a s y n a p t i c t r a n s m i t t e r (Storm-Mathisen, 1977; f o r a d i s c u s -s i o n of uptake mechanisms, see reviews by Johnston, 1978; 1979). Two independent groups of i n v e s t i g a t o r s (Nadler, Vaca, White, Lynch and Cotman, • 1976; White, Nadler, Hamberger, Cotman and Cummins, 1977; Storm-Mathisen, 1977) have shown th a t a l o s s of h i g h a f f i n i t y uptake of glutamate occurs i n the molecular ( s y n a p t i c ) l a y e r of the dentate gyrus a f t e r l e s i o n s are made i n the p e r f o r a n t path - a source of a f f e r e n t terminals to t h i s r e g i o n (Hjorth-Simonsen and Jeune, 1972). No other change i n amino a c i d content i n the dentate gyrus was observed a f t e r the l e s i o n s other than a r e d u c t i o n of glutamate. Auto-r a d i o g r a p h i c a n a l y s i s of hippocampal s l i c e s i n c u b a t e d i n H 3H]-glutamate r e v e a l e d a s i g n i f i c a n t l o s s of s i l v e r g r a i n s from the area known to r e c e i v e t e r m i n a l s of the p e r f o r a n t path, a f t e r l e s i o n s were made of t h i s i n p u t (Storm-Mathisen, 1977a). Since h i s t o l o g i c a l s t u d i e s have shown no evidence of a l o s s of hippocampal neurones f o l l o w i n g such l e s i o n s , i t seems reasonable to a t t r i b u t e the l o s s of glutamate uptake and the lowering of i t s l e v e l s i n the dentate gyrus to the disappearance of p e r f o r a n t path t e r m i n a l s , which by i m p l i c -a t i o n c o n t a i n glutamate as a t r a n s m i t t e r substance. These s t u d i e s improved upon p r e v i o u s attempts to l i n k glutamate l e v e l s w i t h anatomical t r a c t s (see review by Johnson, 1972) i n t h a t i t was p o s s i b l e to exclude the l o s s of neurones and t e r m i n a l s i n t r i n s i c to the hippocampus as a cause of the 90 glutamate depletions, due to the well organized anatomical arrangement of this structure and i t s afferent pathways. A similar approach has been possible for studies on the c o r t i c o - s t r i a t a l pathway (Divac, Fonnum and Storm-Mathisen, 1977; McGeer, McGeer, Scherer and Singh, 1977; Kim, Hassler, Huag and Paik, 1977; Kim, Hassler, Piak and Schroder, 1977). E a r l i e r studies had shown that the cortex sends a large, excit-atory projection to the caudate nucleus (Webster, 1961; Buch-wald, Price, Vernon and H u l l , 1973) which i s probably gluta-mate containing (Spencer, 1976). A p a r t i a l deafferentation of the caudate by c o r t i c a l undercutting or aspiration resulted i n a loss of glutamate content and uptake si t e s from th i s basal ganglionic structure (McGeer et al., 1977). Neurochemical studies on altered uptake of glutamate following lesions have suggested the presence of t r a n s c a l -l o s a l connections and a cortico-thalamic pathway which may be mediated by glutamate (Divac et a l . , 1977; Lund-Karlsen and Fonnum, 1978). It should be noted however that since glutamate and aspartate share the same high a f f i n i t y uptake system i n mammalian CNS (Balcar and Johnston, 1972; 1973) this evidence does not exclude the p o s s i b i l i t y that aspartate i s the trans-mitter of some or a l l of these systems, and evidence of other types (eg: pharmacological - see below) which allows d i f f e r e n -t i a t i o n between glutamate and aspartate mediated synapses i s required. For example, microdissection methods have shown that 91 i n the c e r e b e l l a of r a t s , L-glutamate i s a s s o c i a t e d w i t h gran-u l e c e l l s (Nadi, McBride and A p r i s o n , 1977) and L - a s p a r t a t e w i t h c l i m b i n g f i b r e s (Nadi, Kanter, McBride and A p r i s o n , 1977). In the cat there i s evidence t h a t L-glutamate i s a s s o c i a t e d w i t h some primary a f f e r e n t f i b r e s (Shank, Graham, A p r i s o n and Werman, 1967) w h i l e L - a s p a r t a t e appears to be c o n t a i n e d i n interneurones ( D a v i d o f f , Graham, Shank, Werman and A p r i s o n , 1967). Studi e s on t h e i r r e l e a s e have a l s o p r o v i d e d experimental support f o r a r o l e as t r a n s m i t t e r s of the e x c i t a t o r y amino a c i d s . Koyama (1972), Dodd and B r a d f o r d (1976) and B r a d f o r d and Dodd (1978) have observed glutamate r e l e a s e from the c o r t e x f o l l o w i n g l i g h t - e v o k e d s t i m u l a t i o n , d u r i n g c o r t i c a l e p i l e p t i c f o c i and i n response to s t i m u l a t i o n of hindlimb nerves. In v i t r o r e l e a s e of glutamate from the p y r i f o r m 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 l a t e r a l o l f a c t o r y t r a c t ( Bradford and R i c h a r d s , 1976) and from the dentate gyrus f o l -lowing s t i m u l a t i o n of the p e r f o r a n t path (Wieraszko and Lynch, 1979) coupled w i t h the evidence that a s p a r t a t e i s r e l e a s e d from the commissural a f f e r e n t s to the dentate gyrus (Nadler et a l . , 1976) has strengthened the view t h a t these two amino a c i d s are t r a n s m i t t e r s of s y n a p t i c e x c i t a t i o n i n some r e g i o n s of the CNS. 2) H i s t o l o g i c a l F i n d i n g s Although glutamate and a s p a r t a t e are now known to cause e x c i t -a t i o n of n e a r l y a l l c e n t r a l neurones, the o b s e r v a t i o n of a 92 heterogenous p a t t e r n of d i s t r i b u t i o n w i t h i n the CNS (Shank and A p r i s o n , 1970) and a d i f f e r e n c e i n l e v e l s between these a c i d s i n c e r t a i n b r a i n r e g i o n s (Graham et a l . , 1967; D a v i d o f f et a l . , 1967; Johnson, 1972; 1977; Wenthold, 1978) has r e i n f o r c e d the i d e a t h a t these e x c i t a t o r y amino a c i d s are t r a n s m i t t e r s of d i f f e r e n t synapses. In the s p i n a l c o r d of the c a t glutamate i s r e l a t i v e l y more co n c e n t r a t e d i n d o r s a l r o o t s and i n the d o r s a l horn, w h i l e a s p a r t a t e l e v e l s are g r e a t e r i n the v e n t r a l horn (Duggan and Johnston, 1970). Furthermore temporary i s c h a e -mia. of the c o r d r e s u l t s i n a p r e f e r e n t i a l l o s s of a s p a r t a t e concomitant w i t h a l o s s of i n t e r n e u r o n e s , s u p p o r t i n g the sug-g e s t i o n t h a t glutamate might be the t r a n s m i t t e r of primary a f f e r e n t s , w h i l e a s p a r t a t e may e x e r t an e x c i t a t o r y s y n a p t i c a c t i o n when r e l e a s e d from s p i n a l i n t e r n e u r o n e s . Evidence has been pre s e n t e d to suggest t h a t a s p a r t a t e and p o s s i b l y glut-amate may be t r a n s m i t t e r s of c o c h l e a r a f f e r e n t s of the c a t and guinea p i g , s i n c e these compounds and no other candidate trans-m i t t e r s a l t e r e d the compound a c t i o n p o t e n t i a l s of the c o c h l e a r nerve i n a manner K l i n k e and O e r t e l (1977) suggested the n a t u r a l t r a n s m i t t e r should. L e v e l s of a s p a r t a t e , and to a less marked extent those of glutamate, decrease i n the c o c h l e a r nucleus a f t e r l e s i o n s of the a u d i t o r y nerve (Wenthold, 1978). These decreases are g r e a t e s t i n those s u b d i v i s i o n s of the nucleus which r e c e i v e the g r e a t e s t primary i n n e r v a t i o n (Went-h o l d and G u l l e y , 1977; 1978). 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 of glutamate mediated synapses through a u t o r a d i o g r a p h i c l o c a l i -z a t i o n of t r i t i a t e d glutamate have been u n s u c c e s s f u l (McLennan, 1976) although the v i g o r o u s p a r t i c i p a t i o n of g l i a l elements of the thalamus and c o r t e x i n the uptake of t h i s a c i d was noted. Uptake i n t o g l i a has been demonstrated a l s o i n s l i c e s of the s p i n a l c o r d (Fagg, Jones and Jordan, 1978), n e o n a t a l mouse b r a i n (Herz, Schoiisboe, Boechler, M u k e r j i and F e d e r o f f , 1978), r e t i n a (White and Neal, 1976) and other r e g i o n s , s u g g e s t i n g that t h i s type of uptake may be as important a mechanism of c e s s a t i o n of a c t i o n as degradation by s p e c i f i c enzymes (eg: ACh) or re-uptake i n t o a f f e r e n t t e r m i n a l s (eg: catecholamines). The q u e s t i o n of whether the more important mechanism f o r t e r m i n a t i o n of a c t i o n of a s y n a p t i c a l l y r e l e a s e d amino a c i d t r a n s m i t t e r i s n euronal uptake (Logan and Snyder, 1971; 1972; Bennett, Logan and Snyder, 1972; 1973; L e v i and R a i t e r i , 1973; Lakshmanan and Padmanaban, 1974; M i c h a e l i s , M i c h a e l i s and Boy-a r s k i , 1974; Beart, 1976; Iversen and Storm-Mathisen, 1976; Takagaki, 1976; White and Neal,1976) or g l i a l uptake (Faeder and S a l t p e t e r , 1970; Ehninger and F a l k , 1971; Hamberger, 1971; H o k f e l t and Ljundgdahl, 1972; Roberts and Keen, 1974; Salem, Hammerschlag, Brancho and Orkland, 1975; Snodgrass and I v e r -sen, 1974; Henn, 1976; H o s l i and H o s l i , 1976; Logan, 1976; B a l c a r , Borg and Mandel, 1977) i s at p r e s e n t c o n t r a v e r s i a l , and i s d i s c u s s e d a t l e n g t h elsewhere (Johnson, 1978; Schousboe, 1978; Shank and Graham, 1978; Watkins, 1978). For a more 94 d e t a i l e d overview of the anatomical d i s t r i b u t i o n of mammalian c e n t r a l amino a c i d t r a n s m i t t e r s , the reviews by C u r t i s and Johnston (1974) and K r n j e v i c (1974) are v a l u a b l e . 3) I o n t o p h o r e s i s A g o n i s t s . One of the f i r s t i n d i c a t i o n s that glutamate and a s p a r t a t e 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 i n the mammalian CNS was the o b s e r v a t i o n by Madden, Woods, S h i l l , Remington and Whipple (1945) t h a t when these compounds were ad m i n i s t e r e d i n t r a v e n o u s l y i n dogs, a v o m i t i n g response was induced. Soon t h e r e a f t e r , Brooks, Ransmeier and Gerard (1949) found t h a t a number of d i c a r b o x y l i c amino a c i d s i n f l u e n c e d the p o t e n t i a l s r e c o r d e d from the i s o l a t e d f r o g b r a i n . I t was not u n t i l the p i o n e e r i n g work o f Hayashi (1954; 1956) however, that the pos-s i b l e involvement of glutamate, a s p a r t a t e and other dicarbox-y l i c amino a c i d s i n the f u n c t i o n of the mammalian CNS became f i r m l y based on d i r e c t experimental evidence. Hayashi admin-i s t e r e d to the b r a i n s of apes and dogs an e x t e n s i v e range of compounds which i n c l u d e d glutamate, a s p a r t a t e and ACh, and d e s c r i b e d t h e i r v a r i o u s e f f e c t s upon the g e n e r a l e l e c t r i c a l a c t i v i t y of the c e r e b r a l cortex. Glutamate and a s p a r t a t e e x e r t e d powerful e x c i t a t o r y and y-aminobutyrate i n h i b i t o r y e f f e c t s on the b r a i n , whether they were a p p l i e d t o p i c a l l y or i n t r a v e n t r i c u l a r l y . C u r t i s and c o l l e a g u e s were unaware of these f i n d i n g s 95 ( c f . Watkins, 1978) and t h e r e f o r e independently reached the same c o n c l u s i o n s of Hayashi, t h a t glutamate, a s p a r t a t e and c y s t e a t e possess e x c i t a t o r y a c t i v i t y ( C u r t i s , P h i l l i s and Watkins, 1960; 1961a,b; C u r t i s and Watkins, 1960; 1963). T h i s c o n f i r m a t i o n of the e a r l i e r o b s e r v a t i o n s arose from a s e r i e s of experiments wherein the newly developed technique of i o n t o -p h o r e s i s (Suh et a l . , 1936; Nastuk, 1953; see Chapter I) was adapted f o r use i n i n v e s t i g a t i n g mammalian c e n t r a l neuro-pharmacology. Among the compounds t e s t e d f o r t h e i r a c t i o n s , three (glutamate, a s p a r t a t e and c y s t e a t e ) produced marked i n c r e a s e s i n n e u ronal f i r i n g by a manner c o n s i d e r a b l y d i f f e r -ent from that of c a l c i u m c h e a l a t i n g agents, cholinomimetics or convulsants ( C u r t i s and E c c l e s , 1958; C u r t i s , 1959; C u r t i s , P e r r i n and Watkins, 1960). When ad m i n i s t e r e d to s p i n a l i n t e r -neurones the e x c i t a t o r y amino a c i d s i n c r e a s e d the r a t e of neuronal f i r i n g to an extent dependent on the magnitudes of the c u r r e n t s used to pass the compounds from the p i p e t t e s . A l l * i n t erneurones t e s t e d f i r e d r e p e t i t i v e l y w i t h s h o r t l a t e n -c i e s (0.25 - 0.50 sec) i n response to the e x c i t a n t s , and the e f f e c t l a s t e d u n t i l the c e s s a t i o n of the i o n t o p h o r e t i c current. The t e r m i n a t i o n o f a c t i o n was as r a p i d as the onset. Motoneurones responded to the amino a c i d s w i t h a membrane d e p o l a r i z a t i o n , the time course and magnitude of t h i s a c t i o n being dependent upon the c u r r e n t used and the estimated d i s t -ance between the p o i n t of e j e c t i o n and the c e l l membrane. The d e p o l a r i z a t i o n was shown to be a s s o c i a t e d w i t h changes i n 96 p o s t s y n a p t i c p o t e n t i a l s , i n t h a t the amino a c i d s caused an i n c r e a s e i n the c e l l ' s e x c i t a b i l i t y as measured by d i r e c t current i n j e c t i o n . Renshaw c e l l s a l s o were e x c i t e d by the amino a c i d a g o n i s t s . DH3E d i d not antagonize the responses to the amino a c i d s , i n d i c a t i n g t h at t h e i r a c t i o n s are not mediated through n i c o t i n i c c h o l i n e r g i c r e c e p t o r s . T h i s b e l i e f was r e i n f o r c e d by the o b s e r v a t i o n that the a c i d s a l s o caused a d e p o l a r i z a t i o n 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). C u r t i s and Watkins (1960) were able to deduce the b a s i c s t r u c t u r a l requirements of the amino a c i d r e c e p t o r through an examination of the a c t i v i t y of compounds s t r u c t u r a l l y r e l a t e d to glutamate and a s p a r t a t e . T h e i r major r e s u l t s were subsequ-e n t l y confirmed and extended ( C u r t i s and Watkins, 1963; C u r t i s e t a l . , 1961; K r n j e v i c and P h i l l i s , 1963a; Crawford and C u r t i s , 1964) and the f o l l o w i n g p r o v i d e s a summary of those f i n d i n g s . Most of 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-glutamate or L - a s p a r t a t e y i e l d e d 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 potency. Those v a r i a t i o n s i n molecular s t r u c t u r e which were except-i o n a l ( r e f e r to F i g u r e 12, next page) i n that they r e s u l t e d i n an enhanced a c t i v i t y are as f o l l o w s : 1) a l k y l a t i o n of the amino group of a s p a r t a t e w i t h a s i n g l e methyl group, e s p e c i a l l y i n the case of the D isomer where N-methyl-DL-aspartate i s about 3 times the potency of DL-aspartate and N-methyl-D-asp-a r t a t e i s about 10 times the potency of D-aspartate; 2) the replacement of the 3-carboxyl group of D-glutamate w i t h a 97 F i g u r e 12. Diagrammatic r e p r e s e n t a t i o n o f the mo 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 . Each molecule pos-sesses two t e r m i n a l c a r b o x y l groups and an amino su b s t i t u e n t , but d i f f e r i n t h a t glutamate can extend over a g r e a t e r l e n g t h than can a s p a r t a t e , due to the e x t r a l e n g t h o f i t s carbon c h a i n . For the same reason, glutamate can assume a more " f o l d e d " c o n f i g u r a t i o n i n that i t s minimal a to w ca r b o x y l d i s t a n c e can be l e s s than t h a t o f a s p a r t a t e ( H a l l et a l . , 1979). G L U T A M A T E N(+) w l a 99 sulphonic acid group, forming D-homocysteate, having a potency on the order of 5 - 10 times as great as D-glutamate; 3) al k y l -ation of the amino group of D-aspartate with an iminomethyl substituent to form N-iminomethyl-D-aspartate, or with an ethyl substituent to form N-ethyl-D-aspartate, both which are about twice as potent as D-aspartate. Many compounds closely r e l a t e d i n structure to L-glutamate and L-aspartate share potencies of a similar order of magnitude and among these are: L-cysteate, L-homocysteate, D-aspartate, 3-aminoglutarate, L-cysteine sulphinate, N-methyl-L-glutamate and 3-hydroxyglutamate. It was possible to summarize i n an o v e r a l l fashion 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 of the above information, as was done by Curtis and others. The following presents a b r i e f summary of these observations. Peak a c t i v i t y occurs with a 2 or 3 carbon separation between amino and carboxyl terminal groups, and there i s v a r i a t i o n i n a c t i v i t y with the isomeric forms. Some acid groups substituting for the terminal carboxyl of aspartate r e t a i n excitatory potency, while others (phenolic or phosphoryl substitutions) replacing the terminal carboxyl of glutamate reduce potency. Changing the p o s i t i o n of the amino group reduces potency although some compounds, 3-amino-glutarate (3AG) and 2-aminomethy1-succinate for example, are reduced only somewhat. Introduction of small groups into the carbon chain, involvement of the a and/or to carboxyl terminals 100 i n e s t e r or amide l i n k a g e s , or a l k y l a t i o n or a c y l a t i o n of the amino group (the n o t a b l e e x c e p t i o n being N-methylaspartate) leads to i n a c t i v i t y or reduced a c t i v i t y . Compounds are i n a c -t i v e which l a c k the amino group or both carboxyl groups. Loss of the a - c a r b o x y l group leads to the formation of the s e r i e s of i n h i b i t o r y amino a c i d s . More r e c e n t l y the l i s t of compounds resembling glutamate and a s p a r t a t e which have been t e s t e d f o r e x c i t a t o r y a c t i v i t y has been c o n s i d e r a b l y expanded. Many of these newer m a t e r i a l s 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 , and i t i s b e l i e v e d t h a t t h i s a t t r i b u t e c o n f e r s upon the molecules t h e i r enhanced potencies. Some of these compounds i n c l u d e domoate, q u i s q u a l a t e , ibotenate, k a i n a t e and (± )-cis_-1-amino -1, 3-dicarboxycyclopentane (ADCP) (S h i n o z a k i and K o n i s h i , 1970; Johnston e t a l . , 1974; McCulloch et a l . , 1974; B i s c o e et a l . , 1975; 1976; McLennan and Wheal, 1978). Although these v e r y potent e x c i t a t o r y compounds have v a r y i n g degrees of molecular c o n s t r a i n t , some are remarkably i n f l e x i b l e such as ADCP (which i s known to e x i s t i n both " c h a i r " and "boat" forms). Other molecules are l i m i t e d i n the extent to which t h e i r c a r b o x y l groups can be separated, such as N-methylaspartate. These d i f f e r e n c e s i n s t r u c t u r e have prompted s e v e r a l groups of i n v e s t i g a t o r s ( c i t e d above) to attempt to extend 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 h i p s d e s c r i -bed by C u r t i s and Watkins (1960) The c o n c l u s i o n s from these experiments, which are based 101 upon the r e l a t i v e magnitudes of i o n t o p h o r e t i c 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 i m i l a r e f f e c t s must be drawn w i t h care due to c e r t a i n t e c h n i c a l c o n s i d e r a t i o n s (Gent et a l . , 1974), some of which have a l r e a d y been d e a l t w i t h (Chapter I ) . Since a l a r g e number of o b s e r v a t i o n s are r e q u i r e d f o r accuracy, i t i s o f t e n necessary to use s e v e r a l d i f f e r e n t p i p e t t e assemblies s i n c e there may be d i f f e r e n c e s i n the drug d e l i v e r y c h a r a c t e r -i s t i c s of two m i c r o p i p e t t e s . Furthermore the t r a n s p o r t numbers ( p r o p o r t i o n of the c u r r e n t a c t u a l l y engaged i n e x p e l l i n g the a c t i v e i o n s ) must be assumed to be equal, i f not a c t u a l l y known. A constant r e l a t i o n s h i p between time of e j e c t i o n and amount e j e c t e d must a l s o be assumed, yet at l e a s t on t h e o r e t i -c a l 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 a f f e c t the dose ( C u r t i s , 1964; C l a r k e et a l . , 1973; Purves, 1977). A f i n a l c o n s i d e r a t i o n r e l e v a n t to e s t i m a t i o n s of potency concerns the r e l a t i v e s u r f a c e areas of n e u r o n a l membrane a f f e c t e d by each of the compounds. T h i s v a r i a b l e cannot be known a c c u r a t e l y f o r i o n t o p h o r e t i c s t u d i e s on c e n t r a l neurones and there i s evidence that due to the presence or absence of s p e c i f i c uptake processes, a c o n s i d e r a b l e d i f f e r e n c e i n areas of a f f e c t e d memb-rane ob t a i n s f o r the v a r i o u s amino a c i d s ( B a l c a r and Johnston, 1972; Cox, Headley and Watkins, 1977). T h i s i s known because i n the presence of uptake processes p o s s e s s i n g a h i g h a f f i n i t y f o r the t r a n s m i t t e r , only t r a n s i e n t , l o c a l l y r e s t r i c t e d e f f e c t s upon the p o s t s y n a p t i c s u r f a c e w i l l be observed, whereas substan-ces of e x c i t a t o r y potency which are not r a p i d l y removed from the p e r i n e u r o n a l space w i l l p e r s i s t and produce a r e l a t i v e l y 102 long l a s t i n g e f f e c t , d i f f u s i n g over a g r e a t e r area of membrane and thereby a c t i v a t i n g more r e c e p t o r s (Lodge, C u r t i s and John-ston, 1978; Cox et a l . , 1977). Des p i t e these d i f f i c u l t i e s , measurements of potency through i o n t o p h o r e t i c t e s t i n g s have been shown to agree w e l l w i t h those o b t a i n e d by more c o n v e n t i o n a l means, such as when compounds are a d m i n i s t e r e d at known c o n c e n t r a t i o n s i n v i t r o ( C u r t i s et a l . , 1961; B i s c o e , Evans, Headley, M a r t i n and Watkins, 1976). Furthermore, measurements of t r a n s p o r t num-bers f o r glutamate, a s p a r t a t e and k a i n a t e ( H a l l et a l . , 1979) have shown o n l y s m a l l d i f f e r e n c e s among these e x c i t a n t s , and i n d i c a t e d as w e l l the approximately l i n e a r nature of r e l e a s e both w i t h r e s p e c t to time of e j e c t i o n and c u r r e n t employed (see a l s o Z i e g l g a n s b e r g e r et a l . , 1969). An i s s u e f i r s t r a i s e d i n the e a r l y experiments on amino a c i d s by C u r t i s and c o l l e a g u e s concerned the mechanism of the r e a c t i o n between the amino a c i d and i t s r e c e p t o r . These authors concluded that a t h r e e - p o i n t attachment of the a g o n i s t w i t h i t s r e c e p t o r seemed most reasonable i n view of t h e i r s t r u c t u r e - a c t i v i t y f i n d i n g s . T h i s view has been accepted by most subsequent i n v e s t i g a t o r s (van Gelder, 1971; Buu, P u i l and van Gelder; Johnston, C u r t i s , Davies and McCulloch, 1974; B i s c o e , Evans, Headley, M a r t i n and Watkins, 1975; Shimizu, I c h i s h i t a , T a t e i s h i and Umeda, 1975) w i t h the a d d i t i o n a l pro-v i s i o n t h a t at l e a s t three molecules appear to be r e q u i r e d to r e a c t w i t h the r e c e p t o r to produce i t s a c t i v a t i o n (McLennan 103 and Wheal, 1976a). T h i s c o n t r a s t s w i t h the s i t u a t i o n i n i n v e r t e b r a t e s , where i t appears t h a t up to s i x molecules are r e q u i r e d (Dudel, 1975), w h i l e Diamond and Roper (1973) have shown that two molecules must r e a c t i n t e l e o s t s . One f i n a l aspect of the a c t i o n s of the amino a c i d s d i s c u s s e d by C u r t i s and co-workers r e l a t e d to the q u e s t i o n of whether glutamate, a s p a r t a t e or cysteate. or r e l a t e d substances c o u l d be s p e c i f i c e x c i t a t o r y t r a n s m i t t e r s of s p i n a l neurones. T h e i r c o n c l u s i o n t h a t i t was u n l i k e l y t h a t these substances are s y n a p t i c t r a n s m i t t e r s was based upon the o b s e r v a t i o n s o f : i d e n t i c a l time courses o f a c t i o n of the D and L enantiomorphs, im p l y i n g the l a c k of s p e c i f i c enzymatic degradative processes; the u n i v e r s a l i t y of a c t i o n of the e x c i t a n t s , s u g g e s t i n g a c e r t a i n n o n s p e c i f i c i t y of a c t i o n (such as i n e x t r a - s y n a p t i c e f f e c t s ) ; and f i n a l l y the h i g h c o n c e n t r a t i o n s of these subs-tances throughout the CNS suggested a r o l e s o l e l y as metabolic i n t e r m e d i a t e s r a t h e r than as mediators of s y n a p t i c e x c i t a t i o n . As d i s c u s s e d by McLennan (1975) and C u r t i s and Johnston (1974) these b a r r i e r s to the acceptance of the d i c a r b o x y l i c amino a c i d s as s y n a p t i c t r a n s m i t t e r s are no longer tenable due to more r e c e n t developments i n our understanding o f the neuro-chemical and neuropharmacological p r o p e r t i e s of these excitants. In p a r t i c u l a r , the e q u i v a l e n c e of L and D isomers i n p r o d u c i n g r a p i d on- and o f f s e t a c t i o n s need not be an i n d i c a t i o n of l a c k of t r a n s m i t t e r i n a c t i v a t i o n processes, f o r as a l r e a d y noted, 104 (see above) d i f f u s i o n away from the synapse and a c t i v e Na -dependent uptake are now w i d e l y h e l d to be a c c e p t a b l e mechan-isms f o r the removal of t r a n s m i t t e r (Johnston, 1978; 1979). • U n i v e r s a l i t y of a c t i o n i s an i n s u f f i c i e n t argument a g a i n s t a f u n c t i o n as a s y n a p t i c t r a n s m i t t e r s i n c e i t has been shown th a t w i t h i n the thalamus (McLennan e t a l . , 1968; Morgan et a l . , 1972; Gent et a l . , 1974; Gent and W o l s t e n c r o f t , 1976), s p i n a l c o r d (Duggan, 1974; B i s c o e et a l . , 1976b; Hutchinson e t a l . , 1978) and o l f a c t o r y bulb (Von Baumgarten et a l . , 1963) there i s v a r i a b i l i t y i n the p o t e n c i e s of these a c i d s f o r some groups of neurones. T h i s f i n d i n g has prompted the h y p o t h e s i s t h a t both s y n a p t i c and e x t r a - s y n a p t i c neuronal r e c e p t o r s may e x i s t McLennan et a l . , 1968; McLennan, 1970; Johnson, 1972) and t h i s p o i n t w i l l be d i s c u s s e d i n a l a t e r s e c t i o n . The examination of e f f e c t s of amino a c i d e x c i t a n t s has not been r e s t r i c t e d to the n a t u r a l l y o c c u r i n g amino a c i d s , as b r i e f l y mentioned e a r l i e r , and the q u e s t i o n of the p r e c i s e molecular c o n f i g u r a t i o n which glutamate and a s p a r t a t e must assume when i n t e r a c t i n g w i t h t h e i r r e c e p t o r ( s ) has stimu-l a t e d the use of e x c i t a t o r y substances w i t h s t r u c t u r a l l y -r e s t r i c t e d conformations. The r e l a t i v e p o s i t i o n s of the p r e s -umed a c t i v e groups of these compounds are known w i t h a g r e a t e r degree of c e r t a i n t y than are those of the more f l e x i b l e t r a n s -m i t t e r candidates. I f glutamate r e a c t s w i t h an amino a c i d r e c e p t o r i n an extended conformat i o n ( t h a t i s , w i t h maximal s e p a r a t i o n of the t e r m i n a l c a r b o x y l groups), then a s p a r t a t e 105 and i t s potent analogue, N-methylaspartate cannot r e a c t w i t h t h i s r e c e p t o r . E q u a l l y , each of the amino a c i d s i b o t e n a t e , q u i s q u a l a t e and k a i n a t e cannot assume a c o n f i g u r a t i o n which would r e a c t w i t h 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 . The use of compounds which possess v a r y i n g degrees of molecular r e s -t r a i n t has t h e r e f o r e proved o f value i n s t u d i e s of the pharma-c o l o g i c a l c h a r a c t e r i s t i c s of the e x c i t a t o r y amino a c i d recep-t o r s (Johnston e t a l . , 1974; McCulloch et a l . , 1974; Biscoe et a l . , 1975; Shimizu et a l . , 1975; Biscoe et a l . , 1976a; Hutchinson e t a l . , 1978; MacDonald and N i s t r i , 1978; McLennan and Wheal, 1978) e s p e c i a l l y when such s t u d i e s are accompanied by an examination o f the s p e c i f i c i t y o f a c t i o n of amino a c i d a n t a g o n i s t s (Shimizu, I c h i s h i t a and Umeda, 1975; Pole and Haefel y , 1977; H a l l , Hicks and McLennan, 1978; Hi c k s , H a l l and McLennan, 1978). A n t a g o n i s t s . The p o s s i b l e r o l e of the amino a c i d s i n mammalian s y n a p t i c t r a n s m i s s i o n c o u l d not 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 of s u i t a b l e a n t a g o n i s t s . T h i s development began i n 1972, w i t h the d e s c r i p t i o n by Haldeman, Huffman, M a r s h a l l and McLennan (1972) and Haldeman and McLennan (1972) of the a n t a g o n i s t i c a c t i o n s of glutamate d i e t h y l e s t e r (GDEE). T h i s compound reduced the s y n a p t i c and glutamate induced e x c i t a t i o n s of c e n t r a l neurones. The e f f e c t s of DLH, c y s t e a t e or a s p a r t a t e were u n a f f e c t e d or a f f e c t e d to a l e s s e r extent than were the glutamate responses, but t h i s s e l e c t i v i t y was not observed 106 for a l l neurones. When the effects of GDEE were contrasted with those of methionine sulphoximine (Curtis et a l . , 1972), i t was found that although the l a t t e r antagonist did block amino acid responses, this e f f e c t was less s e l e c t i v e for glut-amate than that observed with the diethylester. It soon became clear that not a l l the substances proposed as antag-onists are e f f e c t i v e with equal s p e c i f i c i t y (McLennan and Wheal, 1976b). Some of the other substances which have been proposed as antagonists of glutamate include l-hydroxy-3-amino-pyrrolid-2-one (HA966) (Davies and Watkins, 1972); 5,6-dimeth-oxyaporphine (nuciferine) ('Duggan, Lodge, Biscoe and Headley, 1973) ; a water-soluble derivative of A -1-tetrahydrocannabinol (SP111) (Segal, 1978); 2-amino-4-phosphonobutyrate (2A4P) (Cull-Candy, Donnellan, James and Lunt, 1976); kainate diethyl-ester (KDEE) (Padjen, 1976); a,e-diaminopimelate (Biscoe, Davies, Dray, Evans, Francis, Martin and Watkins, 1977) and D-a-aminoadipate (aAA) (Hall, McLennan and Wheal, 1977). Of a l l these antagonists, the most useful appear to be aAA, GDEE and HA966 (McLennan and Wheal, 1976b; Biscoe et a l . , 1977; Hicks et a l . , 1978) , an'd this matter w i l l be dealt with more extensively i n the discussion section of this chapter. 4) Experimental Rationales The experiments to be described were performed with various objectives i n mind, and although each addresses a somewhat di f f e r e n t problem, the o v e r a l l theme which serves to unify each investigation concerns the elucidation of the actions of 107 the e x c i t a t o r y amino a c i d s . The f o l l o w i n g summaries prese n t the r a t i o n a l e f o r conducting the prese n t experiments. i / A n t a g o n i s t s : GDEE Although Haldeman and McLennan (1972) f i r s t d e s c r i b e d a r e l a t i v e l y s p e c i f i c a n t a g o n i s t i c a c t i o n of GDEE a g a i n s t the e x c i t a t i o n of c e n t r a l neurones by glutamate, there has not been u n i v e r s a l agreement r e g a r d i n g i t s s e l e c t -i v i t y of e f f e c t . T h i s may be due to a v a r i e t y o f f a c t o r s , some of which i n c l u d e s p e c i e s d i f f e r e n c e s between the animals t e s t e d ( N i s t r i and Constant!, 1975; B a i l e y and P h i l l i s , 1976; Shank and Freeman, 1976); t e s t s b e i n g made from d i f f e r e n t b r a i n r e g i o n s (Te b e c i s , 1973; C u r t i s et a l . , 1972; Davies and Watkins, 1973; Altmann, Ten Bruggencate, Pickelmann and S t e i n -berg, 1976), or d i f f e r e n c e s i n technique such as i n methods of a s s e s s i n g antagonism and ch o i c e of e j e c t i n g c u r r e n t magni-tudes ( Z i e g l g a n s b e r g e r and P u i l , 1973; C u r t i s et a l . , 1972; Cl a r k e and Straughan, 19 77; MacDonald, N i s t r i and Padjen, 1977). D e s p i t e these d i s c l a i m e r s , there have been many confirma-t i o n s ^ o f Haldeman and McLennan's f i n d i n g s t h a t GDEE i s an e f f e c t i v e b l o c k e r both of glutamate induced and s y n a p t i c a l l y evoked e x c i t a t i o n s ( C u r t i s et a l . , 1972, 1973; Davies and Watkins, 1973; Dostrovsky and Pomeranz, 1973; Stone, 1973; 1976; Lowagie and Ge r s c h e n f e l d , 1974; McLennan, 1974; Wheal and Kerkut, 1974; 1975; McLennan and Wheal, 1976; Segal, 1976; Spencer et a l . , 1976a,b; Hicks and McLennan, 1979; Wheal and M i l l e r , 1979).although i t s s p e c i f i c i t y f o r the e f f e c t s of glutamate as opposed to those of a s p a r t a t e , DLH or ACh s t i l l 108 remained a matter of some c o n t r o v e r s y . I t was t h e r e f o r e cons-i d e r e d a p r o p r i a t e to examine i n a c a r e f u l l y c o n t r o l l e d manner (see Chapters I and II) the e f f e c t s of GDEE a g a i n s t a range o f neuronal e x c i t a n t s i n order to e v a l u a t e t h e i r r e l a t i v e suscep-t i b i l i t i e s to antagonism. In t h i s manner, i t was hoped to e s t a b l i s h whether GDEE ac t e d s e l e c t i v e l y a g a i n s t one or a group of the e x c i t a n t s , and to perhaps account f o r some of the anomalous r e s u l t s of o t h e r , i n v e s t i g a t o r s . i i / A n t a g o n i s t s : aAA . During the process of s c r e e n i n g a number of analogues and homologues of the n a t u r a l l y o c c u r r i n g amino a c i d s f o r potency e v a l u a t i o n s , H a l l et a l . , (1977) n o t i c e d t h a t although L-a-aminoadipate was weakly e x c i t a t o r y , the racaemic mixture was i n a c t i v e , and i n f a c t suppressed the spon-taneous a c t i v i t y o f the thalamic neurones. Based upon t h i s evidence, i t was proposed that when i s o l a t e d , the D isomer of a-aminoadipate should a c t as an a n t a g o n i s t of the amino acids. S h o r t l y t h e r e a f t e r , a number of communications appeared con-f i r m i n g the v a l i d i t y of t h i s deduction, and which d e s c r i b e d f u r t h e r a marked s e l e c t i v i t y of e f f e c t o f aAA a g a i n s t the e x c i t a t i o n s produced b y a s p a r t a t e , DLH and NMA (Biscoe, Davies et a l . , 1977; B i s c o e , Evans et a l . , 1977; Davies and Watkins, 1978; Evans and Watkins, 1978; H a l l et a l . , 1978; McLennan and H a l l , 1978). In view of the r e s u l t s which i n d i c a t e d t hat GDEE and aAA both possessed a c t i v i t y as a n t a g o n i s t s f o r d i f -f e r e n t groups of amino a c i d s , a comparison of t h e i r a c t i o n s seemed to be a l o g i c a l course of a c t i o n . 109 i i i / A n t a g o n i s t s : KDEE, 2A3P, 2A4P, BAA I t was mentioned e a r l i e r t h a t a v a r i e t y of compounds have been r e p o r t e d to act as a n t a g o n i s t s of s y n a p t i c e f f e c t s and of the a c t i o n of g l u t -amate. MacDonald et a l . (1977) r e p o r t e d t h a t KDEE depresses the spontaneous f i r i n g o f f e l i n e s p i n a l neurones i n a manner s i m i l a r to that of a l o c a l a n a e s t h e t i c . I t t h e r e f o r e was s a i d n o t . t o be u s e f u l as an a n t a g o n i s t , although Padjen (1976) d i d r e p o r t e a r l i e r an a n t a g o n i s t - l i k e e f f e c t o f KDEE upon the d e p o l a r i z i n g responses of f r o g motoneurones. Another analogue of glutamate, 2-amino-4-phosphonobutyrate (2A4P), was found to reduce the glutamate induced d e p o l a r i z -a t i o n of l o c u s t muscle i n a manner resembling a c o m p e t i t i v e antagonism, although no e f f e c t was observed upon the m i n i a t u r e e x c i t a t o r y j u n c t i o n p o t e n t i a l s (Cull-Candy et a l . , 1976). However, 2A4P and a homologue, 2-amino-3-phosphonoproprionate (2A3P), were r e p o r t e d to be i n e f f e c t i v e i n a n t a g o n i z i n g the e f f e c t s of any amino a c i d e x c i t a n t s on f e l i n e lumbar s p i n a l neurones (Watkins, C u r t i s and Brand, 1977), and White et a l . , (1977) and Dunwiddie, Madison and Lynch (1978) found t h a t 2A4P s u b s t a n t i a l l y and r e v e r s i b l y reduced the amplitude of the p e r f o r a n t path (PP) evoked d e n d r i t i c f i e l d response of granule c e l l s i n the dentate gyrus. 2A4P d i d not appear to a l t e r the amplitude or form o f the f i e l d responses evoked by a n t i d r o m i c s t i m u l a t i o n of the granule c e l l axons (mossy f i b r e s ) , nor was there an observable a l t e r a t i o n i n the p r e s y n a p t i c f i b r e poten-t i a l s , thus r u l i n g out the p o s s i b i l i t y of an a c t i o n of r e d u c i n g 110 the e x c i t a b i l i t y of the granule c e l l s through n o n - s p e c i f i c e f f e c t s . 3AA has a c h a i n l e n g t h between the amino and co-carboxyl equal to that of glutamate, w h i l e p o s s e s s i n g an amino to a — -c a r b o x y l s e p a r a t i o n e q u i v a l e n t to t h a t o f the amino - w-carb-o x y l d i s t a n c e of a s p a r t a t e . I t s c l o s e s t r u c t u r a l s i m i l a r i t y to t h a t of the a n t a g o n i s t , aAA a l s o suggested that i t might act as an a n t a g o n i s t , and so an examination of i t s e f f e c t s on neuronal f i r i n g was c o n s i d e r e d of i n t e r e s t . In view of r e p o r t s i n the l i t e r a t u r e , some of which are i n c o n s i s t e n t , a s e r i e s o f experiments was conducted to t e s t the a c t i o n s o f KDEE, 3AA and the phosphonic a c i d d e r i v a t i v e s upon the e x c i t a t i o n of thalamic neurones induced by a v a r i e t y of amino a c i d s . i v / A g o n i s t s : ADCP and 3AG Q u a n t i t a t i v e measurements of the e x c i t a t o r y a c t i v i t i e s o f c o n f o r m a t i o n a l ^ r e s t r i c t e d amino a c i d s upon c e n t r a l neurones have p r o v i d e d some u s e f u l i n f o r m a t i o n towards an understanding o f the nature of the amino a c i d - r e c e p t o r r e a c t i o n addressed by C u r t i s and Watkins (1960; Johnston et a l . , 1974; Biscoe et a l . , 1976a). McLennan and Wheal (1978) found that ADCP a c t s as a powerful neuronal e x c i t a n t i n the r a t thalamus. T h i s molecule has a n e a r l y r i g i d s t r u c t u r e which allows r o t a t i o n o f onl y the c a r b o x y l groups. I f the i n t e r c h a r g e d i s t a n c e s which occur i n ADCP and as p a r t a t e are compared, i t i s c l e a r t h a t they are u n l i k e l y to I l l share < the same a c t i v e s i t e s of an amino a c i d receptor, assuming a three p o i n t attachment. By c o n t r a s t , a very good f i t i s p o s s i b l e f o r ADCP and glutamate upon a r e c e p t o r which would possess 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 (Buu et a l . , 1976; H a l l et a l . , 1979). I f ADCP i s i n f a c t a v e r y potent and phar-m a c o l o g i c a l l y s p e c i f i c a g o n i s t o f glutamate due to 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 , but does not mimic the a c t i o n of a s p a r t a t e , then i t would be expected to behave i n a. s i m i l a r f a s h i o n to glutamate i n response to a n t a g o n i s t s . I f the e f f e c t s o f ADCP and one of the d i c a r b o x y l i c t r a n s m i t t e r candidates were to be separable i n terms of t h e i r d i f f e r e n t s u s c e p t i b i l i t i e s to the a n t a g o n i s t s , then t h i s would p r o v i d e a d d i t i o n a l evidence i n support o f the hyp o t h e s i s o f two r e c e p t o r s f o r the amino acids. Another compound which might be u s e f u l i n determining the nature of amino a c i d r e c e p t o r s i s 3AG. T h i s substance was t e s t e d o r i g i n a l l y f o r c e n t r a l a c t i v i t y by C u r t i s et a l . , (1961) and C u r t i s and Watkins (1960) and was found to be n e a r l y as a c t i v e as glutamate i n e x c i t a t o r y potency, d e s p i t e the " l e s s f a v o u r a b l e " s i t u a t i o n of the amino group which i s p o s i t i o n e d on the 3-carbon, r a t h e r than on the a-carbon (page 99). I t i s i n t e r e s t i n g i n t h i s r e g a r d t h a t 3AG possesses the same amino to w-carboxyl spacing as does a s p a r t a t e , but not g l u t -amate; t h e r e f o r e the ph a r m a c o l o g i c a l p r o p e r t i e s o f t h i s molecule might be expected to mimic a s p a r t a t e but not glutamate, i f the amino to w-carboxyl d i s t a n c e i s an important determinant o f s p e c i f i c i t y . 112 v/ A g o n i s t s : K a i n i c a c i d K a i n i c a c i d i s a n a t u r a l l y occur-r i n g compound found i n the marine a l g a Digenea simplex, and was f i r s t i s o l a t e d and i d e n t i f i e d by Takemoto ( f o r review, see Takemoto, 1978). I t has a n t h e l m i n t i c p r o p e r t i e s and i s a potent e x c i t a n t of neurones ( S h i n o z a k i and K o n i s h i , 1970; Johnston et a l . , 1974; B i s c o e e t a l . , 1976a; C o n s t a n t i and N i s t r i , 1976). The h i g h potency of k a i n i c a c i d , whose r e l a t i v e l y r i g i d m o l e c u l a r s t r u c t u r e should r e a c t o n l y w i t h g l u t a m a t e - p r e f e r -r i n g r e c e p t o r s ( H a l l et a l . , 1979), suggested t h a t a pharma-c o l o g i c a l a n a l y s i s of i t s a c t i o n might p r o v i d e u s e f u l i n f o r -mation about the s t r u c t u r e of the amino a c i d r e c e p t o r . Furthermore, the o b s e r v a t i o n s of S h i n o z a k i and K o n i s h i (1970) and S h i n o z a k i and Shibuya, 1974), t h a t k a i n a t e p o t e n t i a t e s the glutamate responses of r a t c o r t i c a l neurones and of c r a y f i s h muscle f i b r e s , suggested a form of c o - o p e r a t i v i t y between these two e x c i t a n t s at the amino a c i d r e c e p t o r . I f k a i n i c a c i d does produce neuronal e x c i t a t i o n v i a an i n t e r a c t i o n w i t h glutamate r e c e p t o r s , then i t i s reasonable to suppose t h a t i t s e f f e c t s are s u s c e p t i b l e to an a n t a g o n i s t of t h i s r e c e p t o r . The f r e q u e n t l y s t a t e d assumption t h a t the n e u r o t o x i c e f f e c t s of k a i n i c a c i d (see d i s c u s s i o n ) are mediated by a d i r e c t a c t i o n upon p o s t s y n a p t i c glutamate r e c e p t o r s (Olney et a l . , 1974; McGeer and McGeer, 1976; Frankhuyzen and Mulder, 1977; Herndon and Coyle, 1977) p r o v i d e d a f u r t h e r i n c e n t i v e to include k a i n i c a c i d i n these s t u d i e s i n v o l v i n g the a n t a g o n i s t s . 113 b) R e s u l t s i / E x c i t a n t mechanisms Neurones of the VB thalamus were e x c i t e d by the i o n t o p h o r e t i c a p p l i c a t i o n of L-glutamate, D-glutamate, L - a s p a r t a t e , D-aspartate, k a i n a t e , NMA, DLH, ADCP and ACh, although the time courses of e x c i t a t i o n d i f f e r e d among these compounds. F a s t onset and a r a p i d t e r m i n a t i o n of a c t i o n t y p i c a l of t h a t f i r s t d e s c r i b e d by C u r t i s et a l . , (1960) char.acterizeld the a c t i o n s of the stereoisomers of glutamate and a s p a r t a t e . As noted e a r l i e r by Hutchinson et a l . , (1978) f o r s p i n a l Renshaw c e l l s , e x c i t a t i o n s by k a i n a t e and NMA had very p r o t r a c t e d time courses i n comparison to those e l i c i t e d by glutamate and a s p a r t a t e and i n the p r e s e n t study t h i s applied e q u a l l y to the e x c i t a t i o n s induced by ADCP, as i l l u s t r a t e d i n F i g u r e 13. I t i s c l e a r a l s o from t h i s f i g u r e t h a t DLH and ACh excited w i t h what was an i n t e r m e d i a t e onset time, between that of glutamate or a s p a r t a t e , and k a i n a t e , NMA or ADCP. The e f f e c t appears not to ,be due to the s m a l l e r c u r r e n t s r e q u i r e d to e l i c i t e q u i v a l e n t e x c i t a t i o n s w i t h the more potent m a t e r i a l s , f o r i n c r e a s i n g t h e i r e j e c t i n g c u r r e n t s to equal that used f o r L-glutamate y i e l d e d i n t e n s e e x c i t a t i o n and subsequent spike i n a c t i v a t i o n , but w i t h a continued lengthy l a t e n c y of onset ( H a l l et a l . , 1979). As i s true f o r k a i n a t e and NMA however, i t i s l i k e l y t h a t no c e l l u l a r uptake process f o r ADCP e x i s t s , and t h i s may be a f a c t o r i n the slow time courses i n the a c t i o n s of these m a t e r i a l s (Cox et a l . , 1977).as w e l l as the h i g h p o t e n c i e s . 114 F i g u r e 13. T y p i c a l r e c o r d i n g s of the r a t e of response of thalamic neurones to the e x c i t a n t s t e s t e d i n ' the p r e s e n t s e r i e s o f experiments. Glutamate and a s p a r t a t e both induced a r a p i d onset of e x c i t a t i o n w h i l e ACh and DLH e l i c i t e d a more moderate r a t e i n c r e a s e . The remaining compounds were much slower i n onset and e x e r t e d as w e l l , a prolonged decrease of e x c i t a t i o n f o l l o w i n g the c e s s a t i o n of e j e c t i o n ; t h i s was e s p e c i a l l y marked i n the case of k a i n a t e . A comparison of the e j e c t i n g c u r r e n t s enables an estimate of potency to be made, although other c o n s i d e r a t i o n s are c e r t a i n to i n f l u e n c e such estimates (see t e x t ) . 116 i i / E f f e c t i v e A n t a g o n i s t s I t was p o s s i b l e to separate the e x c i t a t o r y a c t i o n s of the amino a c i d s through the p a r a l l e l i n v e s t i g a t i o n of GDEE and aAA and to e s t a b l i s h r a n k i n g orders of t h e i r r e l a t i v e s u s c e p t i b i l i t i e s to each a n t a g o n i s t . Table 4 summarizes the o v e r a l l r e s u l t s from those c e l l s which were used to o b t a i n a r a n k i n g of the e x c i t a t o r y compounds, i n d e c r e a s i n g order of t h e i r s u s c e p t i b i l i t y to each a n t a g o n i s t . The f i g u r e s i n the two columns are the mean i o n t o p h o r e t i c c u r r e n t s r e q u i r e d to e x p e l the a n t a g o n i s t s s u f f i c i e n t l y to demonstrate a s e l e c t i v e b l o c k of each of the e x c i t a n t s , r e l a -t i v e to that needed f o r d e p r e s s i o n of the 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 comparisons are made of 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 the e x c i t a t i o n s produced by a d j o i n i n g compounds w i t h i n each rank, i t i s appar-ent that f o r both a n t a g o n i s t s , the r a n k i n g i s c h i e f l y r e l a t e d to the dose of a n t a g o n i s t administered; that i s , there i s not an a b s o l u t e but a r e l a t i v e s p e c i f i c i t y v of the antagonism pro-duced by aAA and GDEE. The one e x c e p t i o n o c c u r r e d when GDEE was used to reduce the e x c i t a t i o n s produced by D-aspartate at the same time as the e f f e c t on L - a s p a r t a t e was monitored; here a r e l a t i v e l y h i g h e r dose of GDEE was r e q u i r e d to produce a s e l e c t i v e antagonism of the one e x c i t a n t w i t h r e s p e c t to the l e s s s u s c e p t i b l e stereoisomer. Thus a c o n s i d e r a b l e r e d u c t i o n of both of the a s p a r t a t e induced e x c i t a t i o n s was r e q u i r e d b e f o r e D-aspartate was r e c o g n i z e d to be the more s u s c e p t i b l e . 117 TABLE 4- '.Relative' i o n t o p h o r e t i c "doses" of .a-amino-a d i p i c a c i d and glutamate d i e t h y l e s t e r r e q u i r e d t o antagonize the e f f e c t s of each of a range of neuronal e x c i t a n t s . -a/AA GDEE NMA 1 .0 (44)^ DLH/ADCP 1 .6 + 0 .1 (73) D-GLUT 2 .6 + 0 .1 (26) L-ASP 3 .1 + 0 .2 (26) D-ASP 3 .9 0 .2 : (8) L-GLUT 4 .6 + 0 .3 (16) KAIN 5 .7 + 0 .2 (4) ACh L-GLUT 1. 0 (75) D-ASP 1. 6 + 0 .1 (10) L-ASP 1. 2 + 0 .2 (39) D-GLUT 1. 3 + 0 .1 (16) ACh 1. 4 + 0 .2 (22) DLH 1. 5 + 0 .1 (15) KAIN 1. 8 + 0 .2 (10) ADCP/NMA a Current e x p e l l i n g a n t a g o n i s t i s expressed r e l a t i v e t o t h a t r e q u i r e d t o antagonize the most s u s c e p t i b l e com-pound i n each rank. Ranking i s i n order of d e c r e a s i n g s u s c e p t i b i l i t y . b Values are mean ± S.E., number of c e l l s t e s t e d i n i parentheses. 118 Th i s r e s u l t i s i l l u s t r a t e d i n F i g u r e 14, and F i g u r e s 15 and 16 presen t some examples of t y p i c a l responses which were used to compile the data. A summary of the r e s u l t s used to c o n s t r u c t the o r d e r i n g sequence of the e x c i t a n t s when GDEE was the a n t a g o n i s t employed appears i n Table 5. The r e s u l t s c o n t a i n e d i n t h i s Table repre-sent r e l a t i v e antagonism of one e x c i t a n t w i t h r e s p e c t to another, and these data extend the o b s e r v a t i o n s p r e s e n t e d by Table 4. aAA was i n most cases e f f e c t i v e w i t h i n 1 min. of the commencement of e j e c t i o n , whereas GDEE o f t e n r e q u i r e d s e v e r a l minutes f o r a f u l l e f f e c t to occur. Times f o r r e c o v e r y from 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 p a t t e r n . The e f f e c t i v e n e s s of GDEE as an a n t a g o n i s t was examined on a t o t a l of 187 VB thalamic neurones, Glutamate and a s p a r t -ate were c l e a r l y the most r e a d i l y antagonized of the compounds, and i t was p o s s i b l e to rank the i s o m e r i c forms of these a c i d s . Thus, e x c i t a t i o n s e l i c i t e d by L-glutamate were antagonized p r e f e r e n t i a l l y to those of D-aspartate on 7 of 3 c e l l s , '*'-', D-aspartate effects':.were depressed more r e a d i l y than those of L - a s p a r t a t e (7 of 10 c e l l s ) , and those of L - a s p a r t a t e more than D-glutamate (8 of 12 c e l l s ) . As would be expected, L-glutamate induced e x c i t a t i o n s were antagonized p r e f e r e n t i a l l y to those of L - a s p a r t a t e (7 of 12 c e l l s ) , the remaining 5 e x h i b i t i n g p a r a l l e l r e d u c t i o n s of e x c i t a t i o n , w h i l e on 8 of 9 others L-glutamate e f f e c t s were antagonized to a g r e a t e r extent than were those of D-glutamate, w i t h the remaining 119 F i g u r e 14. E f f e c t of GDEE a g a i n s t the stereoisomers o f asp-a r t a t e . Although D-aspartate was the more s u s c e p t i b l e of the two isomers, t h i s d i f f e r e n t i a l e f f e c t c o u l d u s u a l l y not be observed u n t i l a c o n s i d e r a b l e r e d u c t i o n of both responses was achieved. GDEE was a p p l i e d w i t h a c u r r e n t o f 35 nA i n t h i s example, and the i l l u s t r a t e d r e s u l t was ob t a i n e d 6.0 min. f o l l o w i n g the comencement of e j e c t i o n of the a n t a g o n i s t . GDEE 121 Table 5. Summary of e f f e c t s o f L-glutamic a c i d d i e t h y l e s t e r a g a i n s t p a i r s of neuronal e x c i t a n t s . The f i g u r e s i n each column i n d i c a t e the number of c e l l s at which e x c i t a t i o n by the compound l i s t e d at the head of the column was s e l e c t i v e l y b l o c k e d when compared w i t h the other e x c i t a n t s l i s t e d at the l e f t * T h e r e f o r e , the f i g u r e s i n the lower l e f t t r i a n g l e are the c e l l s at which the s e l e c t i v e antagonism f o l l o w e d the rank o r d e r i n g of the e x c i t a n t s ; those i n the upper r i g h t t r i -angle are the c e l l s which d i d not respond a c c o r d i n g to the r a n k i n g scheme. Thus f o r example, when L - a s p a r t a t e ( t h i r d column) and ACh (5t-k- row) were compared, 5 c e l l s showed p r e f e r e n t i a l antagonism of L - a s p a r t a t e e f f e c t s and three showed e q u i v a l e n t r e d u c t i o n of both e x c i t a n t s . Only one c e l l ( i n t e r s e c t i o n of 5 ^ column and 3 r c* row) had the ACh e f f e c t s p r e f e r e n t i a l l y antagonized. On no o c c a s i o n c o u l d NMA or ADCP be antagonized by GDEE. F i g u r e s i n parentheses r e p r e s e n t the number of c e l l s a t which the a n t a g o n i s t d i d not d i f f e r e n t i a t e between the two e x c i t a n t s . L-GLUT D-ASP L-ASP D-GLUT ACh DLH KAIN ADCP NMA L-GLUT 0(2) 0(5) 0(1) 0 0 0(3) 0 0 D-ASP 7(2) 0(3) NT NT NT NT NT NT L-ASP 7(5) 7(3) 2(2) 1(3) 0 • NT 0 0 D-GLUT 8(1) NT 8(2) 0(1) NT 1 NT NT ACh 9 NT 5(3) 6(1) 4(2) 1(1) NT NT DLH 6 NT 6 NT 8(2) " 3 K D NT KAIN 12(3) NT NT 8 6(1) 4 0 0 ADCP 7 NT 6 NT NT 6(1) 5 NT NMA 8 NT 6 NT NT NT 5 . NT 123 c e l l showing equal r e d u c t i o n s . The next compound i n order which was most e a s i l y antag-o n i z e d by GDEE was ACh. When s e l e c t i v e antagonism was observed L-glutamate and L - a s p a r t a t e induced e x c i t a t i o n s were reduced i n p r e f e r e n c e to those of ACh on 9 of 9 and 5 of 9 c e l l s , r e s -p e c t i v e l y , and at 3 other c e l l s L - a s p a r t a t e and ACh e x c i t -a t i o n s were reduced to an e q u i v a l e n t degree. ACh was ranked below D-glutamate s i n c e on 6 of 7 neurones, D-glutamate e f f e c t s were more r e a d i l y antagonized. When comparisons of ACh w i t h k a i n a t e were made, ACh was p r e f e r e n t i a l l y antagonized a t 6 of 8 c e l l s (with 1 c e l l showing equal r e d u c t i o n ) ; at 8 of 14 c e l l s (with 2 c e l l s showing equal e f f e c t s ) ACh was more suscep-t i b l e than DLH. The remaining amino a c i d a g o n i s t s which were t e s t e d w i t h GDEE were onl y r a r e l y antagonized. These compounds were o f t e n a c t i v e w i t h v e r y low e j e c t i n g 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 the i n i t i a l p e r i o d of GDEE a p p l i c a t i o n . Only when h i g h e j e c t i n g c u r r e n t s (40-60 nA) of GDEE were used was antagonism observed; however, the more u s u a l e f f e c t of such h i g h doses was a r e d u c t i o n i n sp i k e amplitude d u r i n g the p e r i o d of e j e c t i o n of the e x c i t a n t , and whenever t h i s o c c u r r e d the t r i a l was abandoned. Desp i t e these problems some i n d i c a t i o n of r a n k i n g among the compounds c o u l d be obtained: thus DLH e l i c i t e d e f f e c t s were more s u s c e p t i b l e than were those of k a i n a t e on 4 of 7 c e l l s 124 F i g u r e 15. T y p i c a l responses used i n c o n s t r u c t i n g the r a n k i n g orders o f amino a c i d e x c i t a n t s when GDEE was the a n t a g o n i s t employed. F i g u r e s i n the c e n t r a l column r e p r e s e n t the c u r r e n t s r e q u i r e d to e j e c t GDEE i n order to e s t a b l i s h d i f f e r e n t i a l e f f e c t s a g a i n s t the i l l u s t r a t e d p a i r s o f a g o n i s t s . For these r e c o r d s , the e j e c t i n g c u r r e n t s o f the e x c i t a n t s were as f o l -lows: L-glutamate 41 nA, L - a s p a r t a t e 50 nA; L - a s p a r t a t e 35 nA, D-glutamate 75 nA; D-glutamate 100 nA, DL-homocysteate, 18 nA; k a i n a t e 13 nA, N-methylaspartate, 15 nA. 125 199 »n 126 wh i l e e x c i t a t i o n s produced by ADCP and NMA were not a f f e c t e d by GDEE when k a i n a t e induced e x c i t a t i o n s were reduced (5 of 5 c e l l s , i n each c a s e ) . On no occasion- was there observed even p a r t i a l antagonism by GDEE of the e x c i t a t i o n s e l i c i t e d by ADCP or NMA. The e f f e c t i v e n e s s of aAA as an a n t a g o n i s t was examined on a t o t a l of 197 VB thalamic neurones, some of which were among those a l s o t e s t e d w i t h GDEE. These r e s u l t s are pr e s e n t e d i n Table 6. E j e c t i n g c u r r e n t s o f aAA which were e f f e c t i v e i n b l o c k i n g the e x c i t a t o r y a c t i o n s of most of the agents t e s t e d were lower than those r e q u i r e d f o r GDEE, and i t was a l s o found t h a t those e x c i t a n t s which were l e a s t s u s c e p t i b l e to antag-onism by GDEE were most s u s c e p t i b l e to the a c t i o n of aAA, wi t h the ex c e p t i o n o f k a i n a t e . NMA e l i c i t e d e f f e c t s were antagonized by aAA more r e a d i l y than those o f any other e x c i -t a n t : thus i t s a c t i o n was reduced to a g r e a t e r extent than t h a t of k a i n a t e on 7 of 7 c e l l s , of L-glutamate on 7 of 7 c e l l s , o f L - a s p a r t a t e on 6 of 6 c e l l s and of ADCP on 7 of 8 c e l l s . ADCP and DLH were the two e x c i t a n t s whose e f f e c t s were next most r e a d i l y blocked; however at 12 c e l l s where these two were compared d i r e c t l y , no c o n s i s t e n t l y s e l e c t i v e antagonism c o u l d be det e c t e d (at 5 c e l l s the antagonism was equal, w h i l e 4 and3 showed ADCP and DLH, r e s p e c t i v e l y as the more suscep-t i b l e ) . 127 Table 6. Summary of effects of a-aminoadipate (the separ-ated D isomer) against pairs of neuronal excitants. The Table follows the same pattern as that of Table 5; for example when L-aspartate and ADCP were compared, 9 c e l l s were tested, 6 of which showed p r e f e r e n t i a l antagonism of ADCP ef f e c t s , 2 showed equivalent reduction of both excitants and one c e l l showed a select i v e reduction of the L-aspartate effects. On no occasion could ACh be antagonized by aAA. NMA DLH ADCP D-GLUT L-ASP D-ASP L-GLUT KAIN ACh NMA 0(1) 0(1) NT 0 NT 0 0 0 DLH 6(1) 3(5) NT 0(1) NT 0 0 • 0 ADCP 8(1) 4(5) . 0(1) 1(2) NT 0 0 NT D-GLUT NT NT 4(1) 1(4) NT 0 NT 0 L-ASP 6 8(1) 6(2) 8(4) 0 0(2) NT 0 D-ASP ' NT NT NT NT 6 1 NT NT L-GLUT 7 10 8 6 11(2) 7(1) 0 0 KAIN 7 8 6 NT NT NT 10 0 ACh 8 6 - NT 7 7 NT 6 4 129 Glutamate and a s p a r t a t e f o l l o w DLH and ADCP i n the order of r a n k i n g e s t a b l i s h e d by aAA (Tables 4 and 6). On 4 of 5 c e l l s , ADCP was more s u s c e p t i b l e to antagonism than was D-glutamate. S e l e c t i v i t y of antagonism among the s t e r e o i s o -mers of these a c i d s was more r e a d i l y apparent w i t h t h i s antag-o n i s t than w i t h GDEE. D-glutamate e f f e c t s were f i r s t antagon-i z e d when compared w i t h those of L - a s p a r t a t e ' ( 8 of 13 c e l l s , 4 reduced 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 r e a d i l y b l o c k e d than were those of D-aspartate (6 of 6 c e l l s ) w h i l e D-aspartate i n t u r n was a more s u s c e p t i b l e compound than was L-glutamate (7 of 8 c e l l s ) . Other comparisons of amino a c i d a g o n i s t s were made, from which the f o l l o w i n g r e s u l t s d e r i v e and which serve, i n p a r t , to c o n f i r m the above order: e x c i t a t i o n s produced by DLH (10 of 10 c e l l s ) and ADCP (8 of 8 c e l l s ) reduced more than those produced by L-glutamate, and those e l i c i t e d by DLH (8 of 9 c e l l s ) and ADCP (6 of 9 c e l l s ) were reduced more than those of L - a s p a r t a t e . Of the two compounds remaining which were t e s t e d w i t h aAA, e x c i t a t i o n s caused by k a i n a t e were reduced on 4 c e l l s i n p r e f e r e n c e to those e l i c i t e d by ACh. A l l of the other e x c i t -ants of the s e r i e s which were t e s t e d were found more suscep-t i b l e to antagonism than k a i n a t e : ADCP (6 of 6 c e l l s ) , DLH (8 of 8 c e l l s ) , L-glutamate (10 of 10 c e l l s ) ; and the 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 reduced i n the presence of aAA: DLH (6 o f 6 c e l l s ) , L - a s p a r t a t e (7 of 7 130 F i g u r e 16. T y p i c a l responses used i n c o n s t r u c t i n g the 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 the a n t a g o n i s t employed. F i g u r e s i n the c e n t r a l column r e p r e s e n t c u r r e n t s r e q u i r e d to e j e c t aAA i n order to e s t a b l i s h d i f f e r e n t i a l e f f e c t s a g a i n s t the i l l u s t r a t e d p a i r s of a g o n i s t s . For these r e c o r d s , the e j e c t i n g c u r r e n t s f o r the e x c i t a n t s were as f o l -lows: DL-homocysteate 20, L - a s p a r t a t e 36; L - a s p a r t a t e 42, L-glutamate 34; L-glutamate 31, k a i n a t e 13; Kainate 10, ACh 25. 131 100 tec. 132 c e l l s ) , and L-glutamate (6 of 6 c e l l s ) , thus f u r t h e r c o n f i r -ming the l o c a t i o n of ACh and k a i n a t e i n the lowest p o s i t i o n s of the r a n k i n g scheme. E f f e c t s o f GDEE and aAA on the spontaneous a c t i v i t y o f VB thalamic neurones were sometimes observed, although these were most pronounced only when the a n t a g o n i s t s were employed at h i g h "doses" i n e f f o r t s to b l o c k e x c i t a t i o n s o f the l e a s t s u s c e p t i b l e a g o n i s t s . Furthermore, these d i r e c t e f f e c t s appeared i n no way r e l a t e d to the pha r m a c o l o g i c a l s e l e c t i -v i t y observed w i t h the b l o c k e r s . There was an i n c o n s i s t e n t l y observed i n c r e a s e i n the spike 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 used to expel GDEE ( u s u a l l y >40 nA), and t h i s i s r e l a t e d presumably to the o b s e r v a t i o n s of Z i e g l g a n s b e r g e r and P u i l (1973) who noted a h y p e r p o l a r i z i n g a c t i o n of the d i e t h y l e s t e r on the c e l l membrane of s p i n a l neurones. Both aAA and GDEE c o u l d reduce the spontaneous r a t e o f f i r i n g on some neurones, although not always on the same c e l l s . F i g u r e 17 i l l u s t r a t e s an example of a thalamic neurone (depth: 5.39 mm. from the c o r t i c a l s u r f a c e ) whose f i r i n g r a t e was depressed i n a d o s e - r e l a t e d manner by the e j e c t i o n of aAA. An i o n t o p h o r e t i c dose of 24 nA of aAA which was i n the upper range of that used f o r t h i s antagonist, reduced by about 40 -507o the spontaneous d i s c h a r g e , without an observable e f f e c t on spike amplitude. By c o n t r a s t GDEE at doses up to 60 nA, w e l l beyond those r e q u i r e d to antagonize a l l but the most 133 F i g u r e 17. E f f e c t s o f the amino a c i d a n t a g o n i s t s on the r a t e of f i r i n g o f a spontaneously d i s c h a r g i n g VB thalamic neurone. T h i s c e l l was l o c a t e d at a depth of 5.39 mm. Previo u s t r i a l s on t h i s neurone (not i l l u s t r a t e d ) showed both GDEE and aAA to be e f f e c t i v e i n a n t a g o n i z i n g amino a c i d responses a c c o r d i n g to the pr e s e n t r a n k i n g scheme. Note the h i g h apparent potency of L - a s p a r t a t e . aAA a f f e c t s the r a t e o f f i r i n g o f t h i s c e l l i n a d o s e - r e l a t e d manner and at very s h o r t l a t e n c i e s . T h i s depressant e f f e c t has been shown to be b i c u c u l l i n e - i n s e n s i t i v e ( H a l l et a l . , 1977). The l a c k o f e f f e c t o f GDEE at a l l doses t e s t e d ( w i t h i n the range of those used i n t h i s study) may be observed i n the lower t r a c e , which o v e r l a p s the upper t r a c e f o r ease of comparison. At 60 nA, GDEE was observed to have e l e v a t e d the spike h e i g h t by 157.. 134 135 r e s i s t a n t amino a c i d e x c i t a n t s , l e f t the spontaneous r a t e u n a l t e r e d but p o t e n t i a t e d by about 15% the amplitude o f the a c t i o n p o t e n t i a l s . i i i / Other A n t a g o n i s t s The e f f e c t s o f KDEE were e v a l u a t e d ' on 19 thalamic neurones, and acc e p t a b l e r e s u l t s ( a c c o r d i n g to the 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 from o n l y 8. Cons i d e r a b l e d i f f i c u l t y was encountered i n t e s t s w i t h t h i s compound due to i t s e f f e c t s on spontaneous neuronal f i r i n g and s p i k e amplitude. KDEE i n v a r i a b l y depressed the a c t i v i t y o f c e l l s which f i r e d spontaneously, even when ad m i n i s t e r e d at low doses, and i t caused both i n c r e a s e s and decreases i n the hei g h t o f a c t i o n p o t e n t i a l s i n d i f f e r e n t phases o f i t s a c t i o n . When e j e c t e d a t low (2 - 10 nA) c u r r e n t s , KDEE ex e r t e d an e f f e c t which appeared to be due to a h y p e r p o l a r i z a t i o n o f the membrane: the sp o n t a n e i t y was a b o l i s h e d and the spi k e h e i g h t markedly p o t e n t i a t e d (up to 70%, c o n t r o l ) . When these e x p e l -l i n g c u r r e n t s were maintained f o r long p e r i o d s of time (ca. 10 min.) or i f l a r g e r c u r r e n t s were used (15 - 30 nA), the more u s u a l e f f e c t observed was a c o n s i d e r a b l e r e d u c t i o n i n s p i k e amplitude. Under these circumstances f u l l r e c o v e r y f o l l o w i n g the c e s s a t i o n o f e j e c t i o n o f KDEE never oc c u r r e d ; indeed the s e n s i t i v i t y o f the neurone to amino a c i d s was o f t e n l o s t , although i t c o u l d s t i l l be 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 ; : a t i o n o f a hindlimb nerve. De s p i t e these problems i t was p o s s i b l e to o b t a i n some r e s u l t s of d i f f e r e n t i a l antagonism w i t h KDEE, under c o n d i t i o n s 136 where low doses were employed. KDEE reduced the e x c i t a t i o n s produced by DLH more than those of L - a s p a r t a t e on 2 of 3 c e l l s ; on the other c e l l L - a s p a r t a t e e x c i t a t i o n s were the more suscep-t i b l e . When comparisons were made w i t h L - a s p a r t a t e and k a i n -ate, the former was antagonized p r e f e r e n t i a l l y on 2 of 2 c e l l s w h i l e the e x c i t a t o r y e f f e c t s of k a i n a t e were con c o m i t a n t l y p o t e n t i a t e d . Three other neurones y i e l d e d a c c e p t a b l e r e s u l t s , where DLH e f f e c t s were reduced more than those of L-glutamate, those of k a i n a t e were b l o c k e d more than 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 antagonized i n p a r a l l e l . The f i n a l s e r i e s of compounds t e s t e d f o r p o s s i b l e antag-o n i s t i c a c t i v i t y i n c l u d e d 3AA, 2A3P and 2A4P. BAA was i n a c t i v e both as an e x c i t a n t and as an a n t a g o n i s t , on a l l 8 c e l l s examined. The phosphonic analogues d i d possess some a c t i v i t y as a n t a g o n i s t s (Figure 18) and t h e i r e f f e c t s appeared to be s i m i l a r to those a l r e a d y d e s c r i b e d f o r aAA, although there was a no t a b l e d i f f e r e n c e i n t h e i r q u a l i t a t i v e a s p e c t s . For example, 2A3P reduced the e x c i t a t i o n s caused by NMA more than those of L-glutamate on 3 c e l l s , whereas on 3 others the e f f e c t s of both 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 or remained u n a f f e c t e d . In c o n t r a s t to the l a c k of e f f e c t on spike amplitude seen w i t h aAA, 2A3P c o n s i s t e n t l y reduced the spike h e i g h t , and furthermore t h i s compound was f r e q u e n t l y observed to i n h i b i t the spontaneous r a t e o f di s c h a r g e . 2A4P had s i m i l a r p r o p e r t i e s i n that i t s e l e c t i v e l y antagonized the NMA 137 F i g u r e 18. E f f e c t s o f the phosphonic analogues as amino ac a n t a g o n i s t s . 2A4P p r e f e r e n t i a l l y antagonized the responses neurones to NMA when compared w i t h glutamate, and a s i m i l a r p a t t e r n of e f f e c t was e v i d e n t f o r 2A3P. 1 min r-1 CO 139 responses but not those of L-glutamate, and reduced the ampli-tude of the s p i k e . When t e s t e d a g a i n s t the a c t i v i t y of spon-taneously f i r i n g thalamic neurones, both compounds reduced the f i r i n g r a t e s and changed the p a t t e r n of discharge to a more i r r e g u l a r one ( s p i n d l e s ) . i v / BAG When the e x c i t a t i o n s induced by BAG were compared w i t h those of L-glutamate or L - a s p a r t a t e i n the presence of e i t h e r of the two a n t a g o n i s t s , GDEE or aAA, a c o n s i s t e n t t r e n d was observed which c o r r e l a t e d the e f f e c t s o f BAG w i t h L-asp-a r t a t e . On 6 of 6 neurones where BAG and L-glutamate were compared i n the presence of GDEE, L-glutamate was antagonized but not BAG. When aAA was the 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 reduced i n p r e f e r e n c e to those of L-glutamate on 8 of 9 c e l l s . Comparisons of BAG and L - a s p a r t a t e showed t h a t r e g a r d l e s s of the a n t a g o n i s t employed, the ph a r m a c o l o g i c a l behaviours o f the two e x c i t a n t s were the same.(Figure 19). Table 7 prese n t s a summary of these f i n d i n g s , as w e l l as the apparent potency o f BAG i n comparison w i t h that o f L-glutamate and L - a s p a r t a t e . 140 F i g u r e 19. E f f e c t s o f GDEE and aAA a g a i n s t e x c i t a t i o n by 3AG and a comparison w i t h the responses to L-glutamate and L-aspar-t a t e . 3AG more c l o s e l y approximated the e x c i t a t i o n s of aspartate than those of glutamate, and i n f a c t , appeared on most occasions more s u s c e p t i b l e to the a c t i o n o f aAA and l e s s s u s c e p t i b l e to GDEE than was a s p a r t a t e . The c e n t r e t r a c e of the upper r e c o r d i n c l u d e s two responses to L-glutamate to i l l u s t r a t e t h a t the response decrement shown i s not a n o n - s p e c i f i c onset o f desen-s i t i z a t i o n , but that i t r e f l e c t s a t r u e d i f f e r e n t i a l s u s c e p t i -b i l i t y to aAA. 142 Table 7 . Summary of the r e s u l t s of potency determinations and antagonism of 8-aminoglutarate. Potency e v a l u a t i o n s were based on 7 neurones.where equal e x c i t a t i o n r a t e s were produced by BAG, L-glutamate and L - a s p a r t a t e , on at l e a s t three consec-u t i v e t r i a l s f o r each c e l l . The lower s e c t i o n o f the Table (B), i n d i c a t e s the number of occasions t h a t GDEE and aAA were t e s t e d a g a i n s t BAG and L-glutamate on the one hand, and BAG and aspar-t a t e on the other, and as i n d i c a t e d by the pr e c e e d i n g F i g u r e , BAG appears to e x e r t an e f f e c t upon the aAA s e n s i t i v e r e c e p t o r i n p r e f e r e n c e to t h a t antagonized by GDEE. The three o c c a s i o n s where GDEE a f f e c t e d L - a s p a r t a t e and BAG e q u a l l y , r e p r e s e n t t r i a l s where both e x c i t a n t s were r e l a t i v e l y r e s i s t a n t to antagonism. 143 E f f e c t s Of 3-Aminoglutarate A. Potency. 3AG Potency Compared With: No. Of C e l l s : L-glutamate 0.43 ± 0.13 7 L - a s p a r t a t e 0.54 ± 0 . 1 0 7 B. Antagonism. aAA GDEE Antagonized: 3AG 8 0 L-glutamate 0 6 Both e q u a l l y 1 0 3AG 0 0 L - a s p a r t a t e 0 0 Both e q u a l l y 4 3 1 4 4 c) D i s c u s s i o n There are s e v e r a l d i f f e r e n t but r e l a t e d matters which can be d i s c u s s e d i n l i g h t of the r e s u l t s p r e s e n t e d i n t h i s Chapter, the f i r s t of which i s that a number of amino a c i d e x c i t a n t s of thalamic neurones and ACh are separable i n terms of t h e i r d i f f e r e n t i a l s u s c e p t i b i l i t y to antagonism by GDEE and aAA. As e a r l i e r mentioned, GDEE has been g e n e r a l l y accepted as a r e l a t i v e l y s p e c i f i c a n t a g o n i s t f o r e x c i t a t o r y amino a c i d s when compared wi t h other t r a n s m i t t e r s , but there has never been a syst e m a t i c i n v e s t i g a t i o n i n the urethane a n a e s t h e t i z e d r a t of i t s d i f f e r e n t i a l e f f e c t s a g a i n s t a number of neuronal e x c i t a n t s . The presen t r e s u l t s show c l e a r l y t h a t , although GDEE c o u l d antagonize n e a r l y a l l the compounds t e s t e d (the exceptions being NMA and ADCP), not a l l are e q u a l l y suscep-t i b l e . A r a n k i n g o f the e x c i t a n t s c o u l d thus be e s t a b l i s h e d by the manner d e s c r i b e d i n Chapter I I . The e x c i t a t i o n s pro-duced by the stereoisomers of glutamate and a s p a r t a t e were r e a d i l y antagonized by GDEE w h i l e the e f f e c t s of the remaining amino a c i d s , k a i n a t e , DLH, ADCP and NMA c o u l d o n l y be reduced when the e f f e c t s of ACh were bl o c k e d a l s o . I t thus appears t h a t w h i l e lower doses of GDEE can show a p h a r m a c o l o g i c a l s p e c i f i c i t y o f antagonism a g a i n s t glutamate, a s p a r t a t e and ACh, l a r g e r c o n c e n t r a t i o n s of t h i s compound r e s u l t i n n o n - s p e c i f i c e f f e c t s probably u n r e l a t e d s o l e l y to r e c e p t o r blockade. I t i s necessary t h e r e f o r e 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 of c u r r e n t s over which GDEE, and to a l e s s e r extent aAA, are e f f e c t i v e b e f o r e u s i n g these compounds as a n t a g o n i s t s . whether glutamate and a s p a r t a t e share the same r e c e p t o r or a c t upon d i f f e r e n t r e c e p t o r s i s not e v i d e n t from these data alone, and a d d i t i o n a l evidence i s r e q u i r e d to r e s o l v e t h i s q u e s t i o n , as d i s c u s s e d below. The examination of the s p e c i f i c i t y o f antagonism by aAA on the same neurones as GDEE p r o v i d e s a d d i t i o n a l i n f o r m a t i o n important i n the a n a l y s i s of the types o f r e c e p t o r s w i t h which each of the amino a c i d s r e a c t . whereas glutamate and a s p a r t a t e e x c i t a t i o n s were r e a d i l y antagonized by low doses of GDEE, i t was the e x c i t a t o r y e f f e c t s o f NMA, ADCP and DLH which were extremely s e n s i t i v e to antagonism by aAA. I t i s noteworthy t h a t the a c t i o n s of k a i n a t e were r e s i s t a n t to this b l o c k e r as to GDEE. 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 since i t s e f f e c t s , when c o n t r a s t e d w i t h GDEE, r e v e a l a v e r y marked d i f f e r e n c e i n the spectrum of i t s a c t i o n s . The f i n a l c o n s i d -e r a t i o n r e l e v a n t to a d i s c u s s i o n o f p o s s i b l e r e c e p t o r popula-t i o n s a r i s i n g from a comparison of the e f f e c t s o f GDEE and aAA i s that w i t h i n the group of isomers of glutamate and asp-a r t a t e the orders are i n v e r t e d i n the two ranks, again i n f e r -r i n g a d i f f e r e n c e i n the mechanisms of a c t i o n o f the two a n t a g o n i s t s . The f i r s t and perhaps most obvious p o i n t a r i s i n g from the two ra n k i n g orders i s t h a t the r e c e p t o r which i s ac t e d 146 upon by GDEE p r e f e r e n t i a l l y r e a c t s w i t h L-glutamate, w h i l e t h a t which i s most s e n s i t i v e to the e x c i t a t o r y a c t i o n of NMA i s most r e a d i l y b l o c k e d by aAA. Since NMA induced e x c i t a t i o n s are i n s e n s i t i v e to the b l o c k i n g a c t i o n s of GDEE, the l o g i c a l c o n c l u s i o n i s t h a t L-glutamate and NMA i n t e r a c t w i t h d i f f e r e n t p o p u l a t i o n s of amino a c i d r e c e p t o r s . Moreover t h i s conclusion can be extended f u r t h e r to suggest that k a i n a t e appears to exe r t i t s e x c i t a t o r y e f f e c t through a mechanism d i f f e r e n t from that of e i t h e r o f the above two a g o n i s t s , s i n c e n e i t h e r GDEE nor aAA antagonized the e f f e c t s of t h i s compound except when there was evidence of n o n - s p e c i f i c d e p r e s s i o n . I t may be noted t h a t there was a wider range of c u r r e n t s over which aAA c o u l d be used b e f o r e n o n - s p e c i f i c e f f e c t s were observed, than was the case w i t h GDEE. A f u r t h e r suggestion p e r t a i n i n g to whether glutamate and a s p a r t a t e share the same r e c e p t o r a r i s e s from an examination of the o r d e r i n g of the i s o m e r i c forms of these amino a c i d s w i t h i n each rank. A s t r i c t i n v e r s i o n of the s u s c e p t i b i l i t i e s may be i n t e r p r e t e d as evidence t h a t these compounds e l e v a t e neuronal f i r i n g by i n t e r a c t i n g w i t h two d i f f e r e n t groups of r e c e p t o r s . One r e c e p t o r may possess the g r e a t e s t s e n s i t i v i t y f o r L-glutamate (and i s the most r e a d i l y antagonized by GDEE) whi l e being o n l y moderately or weakly s e n s i t i v e to the e f f e c t s of D-glutamate. By c o n t r a s t , the r a n k i n g produced by aAA shows D-glutamate as being the most, and L-glutamate the least s u s c e p t i b l e of the i s o m e r i c forms, c l e a r l y i n d i c a t i n g a 147 d i f f e r e n c e i n the ph a r m a c o l o g i c a l p r o f i l e of the r e c e p t o r being antagonized by aAA. I t can be concluded from a l l these o b s e r v a t i o n s t h a t at l e a s t three groups of amino a c i d r e c e p t o r s probably e x i s t on VB thalamic neurones. The r e c e p t o r p o p u l a t i o n most s e n s i t i v e to antagonism by 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 by NMA, s i n c e t h i s amino a c i d i s the most s u s c e p t i b l e of a l l the a g o n i s t s examined. In t h i s context i t i s i n t e r e s t i n g to note that DLH and ADCP, two compounds which might be expected to r e a c t only w i t h a " g l u t a m a t e - p r e f e r r i n g " r e c e p t o r , are the next most r e a d i l y antagonized compounds, be i n g i n f a c t ahead of glutamate i n the r a n k i n g system of aAA. Th i s matter w i l l be d i s c u s s e d f u r t h e r below. Whether the c a t e g o r i z a t i o n o f amino a c i d r e c e p t o r s i n t o GDEE-sensitive and^aAA s e n s i t i v e conforms to the g l u t a m a t e - p r e f e r r i n g and a s p a r t a t e - p r e f e r r i n g types which have been proposed on other grounds (Duggan, 1974; McCulloch et a l . , 1974; Davies and Watkins, 1977; Evans, F r a n c i s and Watkins, 1977) remains to be e s t a b l i s h e d . On the one hand, whereas the GDEE-sensitive o n e does appear >itobevmostureadily a c t i v a t e d by glutamate, the aAA r e c e p t o r which i s s e n s i t i v e not o n l y to NMA but a l s o to ADCP and DLH, cannot be e a s i l y equated w i t h an a s p a r t a t e - p r e f e r r i n g type, although the pos-s i b i l i t y does appear p l a u s i b l e (see next s e c t i o n ) . F i n a l l y s i n c e the e x c i t a t i o n s produced by k a i n a t e are not r e a d i l y b l o c k e d by e i t h e r GDEE or aAA, i t may be th a t a t h i r d type o f " k a i n a t e - p r e f e r r i n g " r e c e p t o r e x i s t s . 148 Evidence which may be construed as i n d i c a t i n g t h a t two p o p u l a t i o n s of r e c e p t o r s e x i s t has been pr e s e n t e d by Dostrov-sky and Pomeranz (1977), who found that there was a d e s e n s i -t i z a t i o n of s p i n a l c o r d interneurones to prolonged or p u l s a t i l e a p p l i c a t i o n s of glutamate, y e t the response of the same neurones to a s p a r t a t e was u n a f f e c t e d by p r i o r d e s e n s i t i z a t i o n to glutamate. However i t must be noted t h a t i n the p r e s e n t experiments, no evidence of d e s e n s i t i z a t i o n has been observed. There are i n f a c t other data o b t a i n e d by d i f f e r e n t means which l e n d support at l e a s t to the p r e s e n t suggestion t h a t a number of amino a c i d r e c e p t o r s e x i s t on c e n t r a l neurones. Duggan (1974) observed that Renshaw c e l l s i n the cat were more s e n s i t i v e to a s p a r t a t e than s p i n a l i n t e r n e u r o n e s , w h i l e glutamate p r e f e r e n t i a l l y e x c i t e d i n t e r n e u r o n e s . I t was sug-gested t h a t a s p a r t a t e and glutamate might a c t as s y n a p t i c t r a n s m i t t e r s of i n t e r n e u r o n e s and primary a f f e r e n t s r e s p e c -t i v e l y . Measurement of membrane conductance of c a t motoneurones r e v e a l e d marked d i f f e r e n c e s among the e f f e c t s of NMA, k a i n a t e and glutamate or a s p a r t a t e . These l a s t two r a p i d l y depolar-i z e d the membrane to p l a t e a u l e v e l s without marked changes of conductance, w h i l e NMA caused a v e r y l a r g e decrease i n conductance and k a i n a t e a v e r y l a r g e (immeasurable) i n c r e a s e (Engberg, Flatman and Lambert, 1978). In t h i s c o n n e c t i o n too i t i s s i g n i f i c a n t t h a t these same i n v e s t i g a t o r s (Engberg e t a l . 1975; 1979) and Altmann, Ten Bruggencate, Pickelmann and S t e i n b e r g (1976) have r e p o r t e d that the conductance changes 149 e l i c i t e d i n motoneurones and rubrospinal neurones by DLH and by glutamate are not i d e n t i c a l . Watkins and colleagues have shown that by changing the concentrations of K + or Na + i n media bathing i s o l a t e d spinal cords of frog and immature rat , d i f f e r e n t depolarizing effects of amino acids were observed, and these f e l l into three groups: 1) L-glutamate and L-aspartate; 2) kainate and NMA; 3) L-homo-cysteate and D-glutamate.(Evans et a l . , 1977). Using a similar type of io n i c manipulation, Davies and Watkins (1977) found that f e l i n e spinal neurones responded to kainate but not NMA in the presence of low concentrations of Mg"1-1", while L-aspar-tate responses were more sensitive than those of L-glutamate to the blocking action of this ion. Presynaptic effects could be ruled out by the observation of a synergistic action of Ca"1-1" with Mg"*-1", and i t i s known that Ca"1-1" antagonizes the depressant action of Mg"1-1" i n the release of transmitters from presynaptic terminals. Further evidence i n support of the present ranking schemes has been obtained from a study using similar methods but employing double-blind t r i a l s , on f e l i n e spinal interneurones and Renshaw c e l l s (McLennan and Lodge, 1979). Their r e s u l t s corroborated the present schemata i n the main, and extended these r e s u l t s by including a number of d i f f e r e n t excitants: ibotenate, L-cysteate, quisqualate and the separated D and L isomers of homocysteate and N-methylaspartate. B r i e f l y , L-cysteate and quisqualate appeared to follow c l o s e l y the 150 responses of L-glutamate with regard to antagonism by aAA and GDEE, while ibotenate and to a lesser extent the stereoisomers of homocysteate more cl o s e l y mimicked the responses of NMA, reported here. Both the D and L enantiomorphs of NMA were very sensitive to antagonism by aAA as well as being r e s i s t a n t to the antagonistic action of GDEE, in confirmation of the present results with the racaemic mixture. Although i t i s often inadvisable to compare res u l t s obtained from animals of d i f f e r e n t phyla, the conclusions here reached have analogies among invertebrates, where evidence has been obtained for separate populations of receptors for glutamate and for aspartate (Yarowski and Carpenter, 1976) and for both synaptic and non-synaptic glutamate receptors (Takeuchi and Onodera, 1975). Furthermore, kainate may react only with the non-synaptic.receptors (Cull-Candy, 1976). Effects of Other Analogues Attempts to demonstrate a sel e c t i v e antagonism of the e f f e c t s of kainate produced inconsistent r e s u l t s , and were i n general unsuccessful. The r e s u l t s of the experiments which tested the three other possible antagonists, 3AA, 2A3P and 2A4P yielded only a limited amount of useful information. For example, 3AA appeared to be largely i n a c t i v e , either as an excitant or as an antagonist, and the significance of t h i s observation w i l l 151 be d i s c u s s e d below. The other two compounds, 2A3P and 2A4P d i d possess a l i m i t e d degree of a c t i v i t y as a n t a g o n i s t s , and t h e i r a c t i o n s appeared to resemble those of aAA. Thus the e x c i t a t i o n s produced by NMA, the most s u s c e p t i b l e a g o n i s t to the b l o c k i n g a c t i o n of aAA, were p r e f e r e n t i a l l y reduced by both of the phosphonic analogues when compared wi t h L - g l u t -amate. 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 antagon-i s t s appears to be low s i n c e they had very n o t i c e a b l e e f f e c t s i n r e d u c i n g the spike amplitudes of the thalamic neurones. This p r o p e r t y may i n f a c t be the u n d e r l y i n g cause of the anomalous r e s u l t s of others (White et a l . , 1977; Dunwiddie et a l . , 1978) who found that 2A4P c o u l d b l o c k the e x c i t a t o r y s y n a p t i c e f f e c t s of p e r f o r a n t path s t i m u l a t i o n on granule c e l l s i n the dentate gyrus. The lowering of the amplitude of the granule c e l l response due to a d e p o l a r i z a t i o n induced by the phosphonic analogues might have been i n t e r p r e t e d as s y n a p t i c antagonism by these authors. T h i s p o s s i b i l i t y c o u l d be t e s t e d by an examination of the e f f e c t s of 2A3P and 2A4P upon membrane conductance v i a i n t r a c e l l u l a r r e c o r d i n g to see i f they do, i n f a c t , have a d e p o l a r i z i n g a c t i o n . An a l t e r n a t i v e i n t e r p r e t a t i o n i s t h a t these a n t a g o n i s t s d i f f e r i n t h e i r a c t i o n s between dentate granule c e l l s and thalamic neurones. Support f o r t h i s l a t t e r p o s s i b i l i t y may be found i n the r e p o r t of Watkins et a l . , (1977) where a complete p h a r m a c o l o g i c a l i n a c t i v i t y f o r both phosphonic analogues upon the amino a c i d induced responses of c o r t i c a l neurones was seen. 152 A f i n a l comment i s r e q u i r e d r e g a r d i n g the e f f e c t s of 3AA and 3AG. Although the racaemic mixture of 3AA appeared to be p h a r m a c o l o g i c a l l y i n a c t i v e , t h i s f i n d i n g does suggest t h a t the mere presence of e i t h e r the co-carboxyl - amino d i s t a n c e present i n a s p a r t a t e , or the to-carboxyl - amino d i s t a n c e of glutamate, does not i n i t s e l f c o n f e r e x c i t a t o r y p r o p e r t i e s to amino a c i d s . Although t h i s c o n c l u s i o n would seem to h o l d f o r DL-3AA, i t must be remembered that the s t e r e o i s o m e r i c forms of amino a c i d s can possess d i f f e r e n t p h a r m a c o l o g i c a l proper-t i e s , v i z . the d i f f e r e n t e f f e c t s of L-ctAA and D-aAA; and so t e s t s w i t h the separated isomers of 3AA w i l l be r e q u i r e d b e f o r e the l a c k of a c t i v i t y i s s u b s t a n t i a t e d . A s i m i l a r type of a n a l y s i s can be made of the e x c i t a t o r y a c t i v i t y of 3AG and i t s responses to the two a n t a g o n i s t s , aAA and GDEE. Since 3AG d i d e x c i t e thalamic neurones, a l b e i t w i t h a reduced potency compared w i t h glutamate or a s p a r t a t e (Table 7), i t would appear that the a - c a r b o x y l - amino s e p a r a t i o n of 2.43A ( H a l l et a l . , 1979) possessed by glutamate and a s p a r t a t e i s not an a b s o l u t e requirement f o r e x c i t a t o r y a c t i v i t y . Furthermore, s i n c e 3AG appeared to e x e r t i t s e f f e c t s through the aAA r e c e p t o r ( F i g u r e 19) i t may be surmised t h a t t h i s r e c e p t o r w i l l accept a g o n i s t s w i t h a range of a - c a r b o x y l -amino s e p a r a t i o n s . T h i s r e s u l t has i m p l i c a t i o n s f o r some of the p r e v i o u s l y r e p o r t e d o b s e r v a t i o n s w i t h the more potent e x c i t a n t s , e s p e c i a l l y r e g a r d i n g t h e i r antagonism by aAA. 153 A s w a s n o t e d a b o v e , ADCP a n d D L H a p p e a r t o a c t u p o n t h e a A A - s e n s i t i v e r e c e p t o r a n d n o t t h e G D E E - s e n s i t i v e r e c e p t o r , y e t t h e s e a g o n i s t s m i g h t b e e x p e c t e d t o a c t i n a s i m i l a r f a s h i o n t o g l u t a m a t e , s i n c e t h e y p o s s e s s t h e same a - c a r b o x y l t o ; c o - c a r b o x y l d i s t a n c e a s d o e s g l u t a m a t e w h e n i n a n e x t e n d e d c o n f o r m a t i o n . One p o s s i b l e e x p l a n a t i o n f o r t h i s s e e m i n g l y p a r a d o x i c a l r e s u l t i s t h a t t h e s t e r i c r e q u i r e m e n t s f o r a r e a c t i o n w i t h t h e aAA r e c e p t o r i n v o l v e s c a r b o x y l - c a r b o x y l s e p a r a t i o n w i t h i n t h e r a n g e p o s s e s s e d b y NMA a n d A D C P . F u r t h e r m o r e , t h e GDEE r e c e p t o r c o u l d b e a c t i v a t e d p r e f e r e n -t i a l l y b y m o l e c u l e s w h i c h c a n " f i t " t h e i r t e r m i n a l a c t i v e s i t e s i n t o a m o r e s t r u c t u r a l l y c o n f i n e d ( c o n t r a c t e d ) r e c e p t o r . T h e g l u t a m a t e m o l e c u l e c a n a t t a i n a n a - c a r b o x y l - c o - c a r b o x y l s e p a r a t i o n m u c h l e s s t h a n t h e m i n i m u m d i s t a n c e w h i c h a s p a r -t a t e c a n a s s u m e , a n d p r e s u m a b l y GDEE c a n a l s o a c t i n t h i s m o r e f o l d e d c o n f i g u r a t i o n . I f t h i s s i t u a t i o n i s r e s p o n s i b l e f o r t h e d i f f e r e n t i a l e f f e c t i v e n e s s o f aAA a n d GDEE ( T a b l e 4 ) , t h e n i t i s p o s s i b l e t o e x p l a i n o n t h e o n e h a n d why ADCP i s n o t a n t a g o n i z e d b y G D E E , s i n c e ADCP i s f i x e d i n a n e x t e n d e d c o n f i g u r a t i o n a n d c a n n o t r e a c t w i t h t h e GDEE r e c e p t o r , a n d o n t h e o t h e r h a n d why a A A c a n p a r t i a l l y a n t a g o n i z e t h e e f f e c t s o f g l u t a m a t e , s i n c e t h i s m o l e c u l e c a n e x e r t a n e x c i t a t o r y e f f e c t t h r o u g h b o t h a A A a n d GDEE p r e f e r r i n g r e c e p t o r s d u e t o i t s a b i l i t y t o a s s u m e b o t h a w i d e a n d a n a r r o w s p a c i n g o f t h e c a r b o x y l - c a r b o x y l s u b s t i t u e n t s . 154 Although t h i s h y p o t h e s i s does appear to e x p l a i n many of the o b s e r v a t i o n s h e r e i n presented, i n the absence of d i r e c t experimental support i t must remain s p e c u l a t i v e . For example an e x p l a n a t i o n of the l a c k o f antagonism by aAA of the e f f e c t s of k a i n a t e must be pr o v i d e d , although the p o s s i b l e p a r t i c i -p a t i o n of a d d i t i o n a l t e r m i n a l groups possessed by t h i s mole-cu l e i n the a g o n i s t - r e c e p t o r r e a c t i o n may be r e l e v a n t i n t h i s r e g a r d (Schwarcz, Scholz and Coyle, 1978"; H a l l e t a l . , 1979). Indeed other evidence i n d i c a t e s t h at k a i n a t e b i n d i n g i n v i t r o , although d i f f e r e n t i n many r e s p e c t s from t h a t o f L-glutamate, may i n f a c t r e p r e s e n t r e a c t i o n w i t h a subpopul-a t i o n o f glutamate receptors.(Simon, C o n t r e r a and Kuhar, 1976; Johnston, Kennedy and T w i t c h i n , 1978;' London and Coyle, 1979; McLennan, 1979), and the pr e s e n t r e s u l t s suggest t h a t t h i s i s a G D E E - i n s e n s i t i v e subpopulation. 155 DENTATE GYRUS a) I n t r o d u c t i o n The w e l l - d e f i n e d neuronal o r g a n i z a t i o n of the hippocampal formation p r o v i d e s an e x c e l l e n t t e s t i n g ground f o r comparing the a c t i o n s of the amino a c i d s and t h e i r a n t a g o n i s t s (Storm-Mathisen, 1977b). The primary c e l l type i n the dentate gyrus i s the granule c e l l , which i s d i s t r i b u t e d w i t h i n t h i s t i s s u e i n a t h i n , horseshoe-shaped l a y e r (Lorente de No, 1934). The s y n a p t i c i n p u t s to these neurones occur i n d i s c r e t e zones with l i t t l e o v e r l a p p i n g , and which have been s t u d i e d a n a t o m i c a l l y and e l e c t r o p h y s i o l o g i c a l ^ ( B l a c k s t a d , 1956; 1958; Andersen, Holmqvist and Voorhoeve, 1966; L<J>mo, 1971; Steward, White and Cotman, 1977). Of p a r t i c u l a r i n t e r e s t to the present study i s the l i k e l i h o o d t h a t glutamate and a s p a r t a t e mediate the s y n a p t i c i n p u t s from d i f f e r e n t groups of a f f e r e n t f i b r e s onto the den-d r i t e s of the granule c e l l s . Nadler e t a l . , (1976) and White et a l . , (1977) have r e p o r t e d that a calcium-dependant e f f l u x of glutamate and a s p a r t a t e o c c u r r e d f o l l o w i n g potassium-induced d e p o l a r i z a t i o n of s l i c e s o f r a t dentate gyrus and r e g i o s u p e r i o r . 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 input by way of the p e r f o r a n t path (PP) reduced the r e l e a s e of glut-amate from the dentate gyrus w h i l e l e s i o n s of the commissural pathway (COMM) from the c o n t r a l a t e r a l hippocampus d i m i n i s h e d 156 the r e l e a s e of a s p a r t a t e (Nadler, White, Vaca, P e r r y and Cotman, 1978). Wheal and M i l l e r (1979) have shown t h a t GDEE can prevent the PP evoked a c t i v a t i o n of dentate granule c e l l s , which a l s o suggests t h a t the synapses formed by the PP f i b r e s are mediated by glutamate. In other r e g i o n s of the hippocampus, the e x c i t a t i o n of pyramidal c e l l s by amino a c i d s has been r e p o r t e d (Biscoe and Straughan, 1966) and GDEE found e f f e c t i v e i n p r e v e n t i n g the amino a c i d induced f i r i n g (Segal, 1976; Spencer, G r i b k o f f , Cotman and Lynch, 1976). These e f f e c t s o f amino a c i d s probably occur i n the d e n d r i t i c r e g i o n of the neurones, f o r t h i s has been shown to be the most s e n s i t i v e s i t e o f a c t i o n f o r g l u t -amate (Dudar, 1974) and i s the locu s f o r the s y n a p t i c i n p u t s . However there has not y e t been an a n a l y s i s o f the p o s s i b l e involvement o f the amino a c i d s i n the dentate gyrus u s i n g a comparison of t h e i r r e l a t i v e p o t e n c i e s on granule c e l l s and d i f f e r e n t i a l antagonism of the s y n a p t i c and amino a c i d e f f e c t s . I t was the i n t e n t i o n o f the pr e s e n t study to pr o v i d e such an a n a l y s i s , to i n v e s t i g a t e the p o s s i b l e involvement of the amino a c i d s 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 the dentate gyrus, and to p r o v i d e a p h y s i o l o g i c a l t e s t i n g ground f o r the pharmaco-l o g i c a l r e s u l t s w i t h GDEE and aAA r e p o r t e d e a r l i e r i n t h i s Chapter. 157 b) Results Neurones ly i n g i n the upper blade of the dentate gyrus and which were synaptically activated by stimulation of PP and COMM (Figure 20) were r e a d i l y excited by the iontophoretic administration of the amino acids, L-glutamate, L-aspartate, DLH and NMA. Since the pipette assembly was presumably situated near the c e l l bodies any difference i n the apparent potency between L-glutamate and L-aspartate could r e f l e c t ;the d i s t r i b u t i o n of receptors affected by the ejected materials r e l a t i v e to that location. The r e l a t i v e apparent potencies of L-glutamate and L-aspartate upon granule c e l l s were obtained i n a similar fashion as that described e a r l i e r ; that i s , by comparing the magnitudes of the ejecting currents required to e l i c i t stable, equal and submaximal leve l s of the f i r i n g frequencies of granule c e l l s , a v a l i d comparison since the transport num-bers for these two substances are equivalent (Hall et al., 1979) . The r e s u l t s of such determinations where reproducible tests were made on a minimum of three occasions per c e l l , are presented i n Table 8. Although not true for every c e l l , on average L-aspartate 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 et a l . , 1977; Wheal and M i l l e r , 1979), i s d i s t a l l y located on the dendrites t h i s r e s u l t might indicate 158 F i g u r e 20. E f f e c t s of a n t a g o n i s t s o f the e x c i t a t o r y amino a c i d s on granule c e l l responses, and a schematic r e p r e s e n t a t i o n o f a granule c e l l showing i t s r e l a t i o n s h i p to the dentate gyrus and the hippocampus. GDEE was c l e a r l y more e f f e c t i v e than aAA as an a n t a g o n i s t 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 evoked responses, w h i l e aAA was e f f e c t i v e o n l y as an a s p a r t a t e antag-o n i s t and a b l o c k e r of the e f f e c t s o f commissural s t i m u l a t i o n . Diagram r e p r e s e n t s the c e l l body l a y e r s o f the hippocampus and dentate gyrus as viewed when a c o r o n a l s e c t i o n o f t h i s t i s s u e i s made. Ins e t : Photograph of Pontamine sky b l u e spot.-(arrow). A b b r e v i a t i o n s : P pyramidal c e l l l a y e r ; HF hippocampal f i s s u r e ; F f i m b r i a ; H h i l u s ; G granule c e l l l a y e r ; PP area o f p e r f o r a n t path a f f e r e n t t e r m i n a l s ; COMM area of commissural a f f e r e n t t e r m i n a l s . 1 5 9 ANTAGONIZED BY PP s y n a p t i c a c t i v a t i o n PP evoked EPSP f i e l d Glutamate e x c i t a t i o n GDEE 6 3 6 aAA 1 0 0 COMM s y n a p t i c a c t i v a t i o n COMM evoked EPSP f i e l d A s p a r t a t e e x c i t a t i o n 0 0 0 9 3 5 160 TABLE 8. R e l a t i v e p o t e n c i e s o f L-glutamate and L - a s p a r t a t e t e s t e d on dentate granule c e l l s . No. of c e l l s a s p a r t a t e >glutamate 9 1.2-8.0 2.8 ± 2.2 a s p a r t a t e = g l u t a m a t e 2 1.0 — as p a r t a t e <»glutamate 4 0.7-0.9 0.8 ± 0 . 1 R a t i o I — asp - g l u t Range Mean ± SE 161 the e x i s t e n c e of an a s p a r t a t e mediated i n n e r v a t i o n which i s more p r o x i m a l l y s i t u a t e d . The e f f e c t s of GDEE and aAA were examined c o i n c i d e n t a l l y on amino a c i d induced and s y n a p t i c a c t i v a t i o n s of 30 granule c e l l s . GDEE adm i n i s t e r e d w i t h e j e c t i n g c u r r e n t s of 20 - 50 nA att e n u a t e d c o n s i d e r a b l y the e f f e c t s of L-glutamate but not those of NMA ( F i g u r e 21, C-E), DLH or L - a s p a r t a t e . The a c t i o n of aAA on the other hand, always spared e x c i t a t i o n s produced by L-glutamate at doses up to 30 nA but was e f f e c t i v e i n a b o l -i s h i n g or r e d u c i n g markedly those of the other three amino a c i d s ( F i g u r e 21, A-C). Th i s s e l e c t i v i t y had been a n t i c i p a t e d i n l i g h t o f the f i n d i n g s on thalamic neurones, pr e s e n t e d e a r l i e r i n t h i s Chapter. However i t was not always p o s s i b l e to t e s t the a n t a g o n i s t i c a c t i o n of aAA s i n c e on some oc c a s i o n s (10 of 30 c e l l s ) -amarked e x c i t a t o r y e f f e c t of t h i s compound i t s e l f was found. T h i s p r o p e r t y has not been p r e v i o u s l y exam-i n e d p h a r m a c o l o g i c a l l y , although Lodge, Headley and C u r t i s , (1979) d i d r e p o r t t h a t aAA c o u l d r a i s e the background f i r i n g r a t e and enhance the e f f e c t s of L-glutamate on s p i n a l neurones. T h i s matter w i l l be f u r t h e r d i s c u s s e d below. When s e l e c t i v e antagonism of amino a c i d induced e x c i t a -t i o n s had been e s t a b l i s h e d the s y n a p t i c responses of the c e l l s were examined. In the presence o f s u f f i c i e n t amounts of GDEE to antagonize the e f f e c t s o f L-glutamate, the response to s t i m u l a t i o n o f PP but not of COMM was a l s o b l o c k e d . Conversely 162 Figure 21. The ac t i o n s of aAA and GDEE on synaptic 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 c e l l . The rows of records were obtained s e q u e n t i a l l y from above downwards and the photo-graphs are of four superimposed sweeps. Note the d i f f e r e n t time scales of the photographs. 163 Spikes/sec. A 50 ~\ Control 0-< 50 n ocAA 12 (1.5 min) B. o-J 50 n Recovery (8 min) C. 0-> 50 -i GDEE 40 (3 min) D. o-< 50 n Recovery (3 min) 0-1 NMA 10 GLUT 12 t- 30 sec. COMM ( Jo.2 mV 10 msec PP ) Jo.2 mV 4 msec 164' a dose of aAA which s e l e c t i v e l y b l o c k e d L - a s p a r t a t e or NMA e x c i t a t i o n s concomitantly reduced the COMM a c t i v a t i o n o f the neurones, w h i l e the response to PP was l e f t unantagonized. F u l l r e c o v e r y to c o n t r o l l e v e l s o f both the s y n a p t i c and the amino a c i d e f f e c t s was observed a f t e r c e s s a t i o n of the cu r r e n t s e j e c t i n g the an t a g o n i s t s (centre and bottom t r a c e s , F i g u r e 21). S i m i l a r complete s e r i e s of antagonisms and recov-e r i e s u s i n g both b l o c k i n g agents were o b t a i n e d on f i v e c e l l s (lower s e c t i o n , F i g u r e 20), w h i l e i n 1 of 6 cases, aAA d i d bl o c k the s y n a p t i c e x c i t a t i o n of a granule c e l l by PP.stimu-l a t i o n . In a d d i t i o n to s e l e c t i v e l y a n t a g o n i z i n g the s y n a p t i c responses o f s i n g l e granule c e l l s , GDEE and aAA were t e s t e d a l s o f o r t h e i r e f f e c t s a g a i n s t the p o p u l a t i o n EPSPs evoked by the s t i m u l a t i o n of PP and COMM. As F i g u r e 22 i l l u s t r a t e s , GDEE reduced the p o p u l a t i o n EPSP evoked by PP s t i m u l a t i o n by 13%, w h i l e aAA was without e f f e c t . By c o n t r a s t , aAA reduced the p o p u l a t i o n EPSP evoked by s t i m u l a t i n g COMM by 26%, w h i l e GDEE was i n e f f e c t i v e . The r e s u l t s presented above are summar-i z e d i n t a b u l a r form i n F i g u r e 20). I t was e a r l i e r remarked that when aAA was t e s t e d f o r i t s a b i l i t y to antagonize amino a c i d induced f i r i n g , an i n c r e a s e i n the background r a t e of discharge of.some c e l l s was observed. This heightened a c t i v i t y was noted even when the s y n a p t i c response to COMM s t i m u l a t i o n was reduced by aAA. Although 165 F i g u r e 22. EPSP f i e l d responses evoked by s t i m u l a t i n g the PP and COMM inp u t s to the dentate granule c e l l s . Each t r a c e r e p r e s e n t s the average o f 20 con s e c u t i v e sweeps. The arrow on the upper r i g h t hand t r a c e ( c o n t r o l PP) i n d i c a t e s the h e i g h t o f the f i e l d a c h i eved by s t i m u l a t i n g PP i n the presence o f 70 nA GDEE (measured from the second r e c o r d o f the r i g h t hand column). Note the complete l a c k o f e f f e c t of DaAA on the PP evoked f i e l d , and the l a c k o f e f f e c t o f GDEE on the COMM f i e l d . 166 COMMISSURAL PERFORANT 1 0 msec 167 L-a-aminoadipate i s a weak e x c i t a n t ( C u r t i s and Watkins, 1960; H a l l et a l . , 1979), the D isomer has u s u a l l y been r e p o r t e d to be without e f f e c t ( Biscoe et a l . , 1977; H a l l e t a l . , 1979; but see Lodge et a l . , 1979), y e t the pr e s e n t e f f e c t was o b t a i n e d both w i t h the racaemic mixture and w i t h the separated D isomer. I t seemed l i k e l y t h e r e f o r e t h a t some mechanism ot h e r than one of d i r e c t e x c i t a t i o n might be i n v o l v e d , and a " d i s i n h i b i t o r y " process i s one p o s s i b i l i t y . To examine t h i s e f f e c t , the exper-iment i l l u s t r a t e d by F i g u r e 23 was performed. A granule c e l l was made to f i r e a t a r a t e of 35 - 50 s p i k e s / s e c . by the continuous e j e c t i o n of L-glutamate (25 nA). Superimposed upon t h i s background the i n h i b i t o r y and e x c i t a -t o r y e f f e c t s e l i c i t e d by e j e c t i o n s o f GABA and of aAA respec-t i v e l y were produced. T h e r e a f t e r by r e d u c i n g the L-glutamate e x p e l l i n g c u r r e n t the background f i r i n g frequency was main-t a i n e d at a roughly constant l e v e l d u r i n g the e j e c t i o n of the GABA a n t a g o n i s t , b i c u c u l l i n e . When the i n h i b i t o r y response to GABA was l a r g e l y a b o l i s h e d ( 1 - 2 min. a f t e r onset of b i c u -c u l l i n e , 25 nA, F i g u r e 23B), the e x c i t a t i o n p r e v i o u s l y produced by aAA was c o n s i d e r a b l y attenuated, and reappeared f o l l o w i n g c e s s a t i o n of the b i c u c u l l i n e c u r r e n t c o i n c i d e n t a l l y w i t h the r e t u r n of the i n h i b i t o r y response to GABA. 168 F i g u r e 23. The e f f e c t of b i c u c u l l i n e (BICUC) on the depression of a granule c e l l by GABA and i t s a c t i v a t i o n by aAA. The c e l l was induced to f i r e by the continuous a d m i n i s t r a t i o n of g l u t -amate, the administering c u r r e n t being reduced by b i c u c u l l i n e to preserve an approximately constant l e v e l o f background d i s -charge. (A) c o n t r o l ; (B) 2 min a f t e r s t a r t of b i c u c u l l i n e a d m i n i s t r a t i o n ; (C) 3% min a f t e r c e s s a t i o n o f b i c u c u l l i n e current. 1 6 9 A . G L U T 2 5 75 0 »-G L U T 15 B 75 o CD CO \ to CD J£ Q-co B I C U C . 2 5 o »-75 r G L U T 2 5 mm 0 «-G A B A 2 0 o ; A A 7 5 30 sec. 170 c) D i s c u s s i o n I t i s commonly accepted that the granule c e l l s o f the dentate gyrus r e c e i v e s e v e r a l a f f e r e n t i n p u t s which are s p a t i a l l y separate upon the d e n d r i t i c t r e e s ( B l a c k s t a d , 1956; 1958). Among these are two which g i v e e x c i t a t o r y responses - a d i s t -a l l y s i t u a t e d p r o j e c t i o n from the e n t o r h i n a l c o r t e x (the p e r f o r a n t path) and a more proximal i n p u t from the c o n t r a l a t -e r a l hippocampus. The evidence here p r e s e n t e d i n d i c a t e s t h a t the t r a n s m i t t e r substance which mediates the e x c i t a t o r y responses o f the dentate c e l l s to s t i m u l a t i o n of these two inputs are d i f f e r e n t , although both are l i k e l y to be amino a c i d s . The r e s u l t s c o n f i r m the e a r l i e r b i o c h e m i c a l (Nadler et a l . , 1976;1978; White et a l . , 1977) and ph a r m a c o l o g i c a l (Wheal and M i l l e r , 1979) f i n d i n g s t h a t the PP in p u t i s g l u t -amate mediated s i n c e i t can be bl o c k e d by GDEE; w h i l e on the other hand COMM a c t i v a t i o n of granule c e l l s i s antagonized by aAA whose p a t t e r n of ph a r m a c o l o g i c a l e f f e c t i s d i f f e r e n t from t h a t o f GDEE. Of the n a t u r a l l y o c c u r r i n g amino a c i d s , a s p a r t a t e i s the most l i k e l y t r a n s m i t t e r of t h i s COMM evoked e x c i t a t i o n ( c f . Nadler et a l . , 1978; White et a l . , 1979), and the ap p a r e n t l y g r e a t e r s e n s i t i v i t y of a m a j o r i t y of the neurones examined to a s p a r t a t e r a t h e r than to glutamate would a c c o r d w i t h the more prox i m a l l o c a t i o n of the COMM inpu t . 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 evidence that two d i s t i n c t e x c i t a t o r y amino a c i d r e c e p t o r s , both of which ap-pear to be of s y n a p t i c s i g n i f i c a n c e , c o e x i s t on the same c e l l s . 171 The f i n d i n g that on some c e l l s the a n t a g o n i s t aAA c o u l d i t s e l f e x c i t e w h i l e at the same time b l o c k i n g the COMM s y n a p t i c input r e q u i r e s some comment. The f a c t t h a t e x c i t a t i o n by aAA co u l d be prevented by an a n t a g o n i s t of GABA i n d i c a t e s that i t i s an i n d i r e c t e f f e c t , and s i n c e c e r t a i n i n h i b i t o r y processes i n the hippocampus, i n c l u d i n g both r e c u r r e n t i n h i b i t i o n and th a t evoked by c o n t r a l a t e r a l s t i m u l a t i o n are mediated by GABA ( C u r t i s et a l . , 1970; Ts u c h i y a and Fukushima, 1978), i t i s p o s s i b l e t h a t one of these processes i s i n v o l v e d . The si m p l e s t e x p l a n a t i o n would be the removal by aAA of an a s p a r t a t e medi-ated t o n i c d r i v e on i n h i b i t o r y interneurones which are known to a c t upon the granule c e l l s (Assaf and M i l l e r , 1978; S t r u b l e , Desmond and Levy, 1978), and s i n c e the e x c i t a t i o n of the l a t t e r by the COMM f i b r e s appears to be a s p a r t e r g i c t h i s p r o p o s a l i s p l a u s i b l e . whether the i n h i b i t o r y i n t e r n e u r o n e s are those which g i v e r i s e to the r e c u r r e n t i n h i b i t i o n of granule c e l l s or whether they are i n v o l v e d w i t h the processes o c c u r r i n g i n the h i l a r zone remains to be e s t a b l i s h e d . 172 CHAPTER V OCTOPAMINE a) General Overview Octopamine i s a n a t u r a l l y o c c u r r i n g amine f i r s t d i s c o v e r e d i n s a l i v a r y glands o f octopus (Erspamer, 1948a,b,c; 1952a,b; Erspamer and B o r e t t i , 1951a,b; Erspamer and Asero, 1953) and wi d e l y d i s t r i b u t e d i n the animal kingdom (Robertson and J u o r i o j 1976; Hi c k s , 1977). Erspamer i d e n t i f i e d the s t r u c t u r e of octopamine as L-p_-hydroxy-8-phenylethanolamine and des-c r i b e d a sympathomimetic a c t i o n on b l o o d p r e s s u r e and i s o l a t e d organs of mammals. Lands and Grant (1952) found t h a t octop-amine possesses a p r e s s o r e f f e c t 2% t h a t o f n o r a d r e n a l i n e i n dogs. Mammals i n c l u d i n g man e x c r e t e r a i s e d l e v e l s of p_-hydroxy-mandelic a c i d , a major m e t a b o l i t e of octopamine, f o l l o w i n g traetment w i t h i n h i b i t o r s o f the enzyme monoamine oxidase (Armstrong, Shaw and W a l l , 1956; Kakimoto and Armstrong, 1960; 1962). Correspondingly, r a t and r a b b i t t i s s u e s c o n t a i n 6 - f o l d g r e a t e r q u a n t i t i e s of octopamine under c o n d i t i o n s of monoamine oxidase i n h i b i t i o n . T h i s demonstrated t h a t not only i s octop-amine nor m a l l y p r e s e n t i n the t i s s u e s of these animals, but i t has a ve r y h i g h turnover r a t e as w e l l . Sympathetic nerves of r a t s c o n t a i n d e t e c t a b l e amounts of octopamine i n the absence of monoamine oxidase i n h i b i t i o n , i n d i c a t i n g to 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 this amine may have a function of i t s own i n p h y s i o l o g i c a l processes. 1) Proposed Functions A considerable body of evidence has accumulated which i s sug-gestive of a function for octopamine as a synaptic transmitter i n a v a r i e t y of invertebrate species (Carpenter and Gaubatz, 1974; Kravitz, Talamo, Evans,. Wallace and B a t t e l l e , 1976; Robertson and Carlson, 1976; Axelrod and Saavedra, 1977; Robertson and Juorio, 1977; Nathanson, 1979). Its r o l e i n mammalian nervous tissue however has been the source of consid-erable speculation. It may act as a less potent agonist at peripheral adrenoceptors (Fischer, Horst and Kopin, 1965; Kelly and Burks, 1974), and several groups of investigators have pro-posed that octopamine functions as a " f a l s e transmitter" at these si t e s (Day and Rand, 1963; Trendelenburg, 1963; Kopin, Fischer, Mussachio and Horst, 1964; Kopin, Fischer, Mussachio, Horst and Weise, 1965). A f a l s e transmitter has been defined as a "substance which i s normally not present or present i n only trace amounts i n adrenergic nerves but which accumulates i n terminals a f t e r monoamine oxidase i n h i b i t i o n . Thereafter the f a l s e transmit-ter can be released by nerve stimulation but e l i c i t s diminished responses at the s i t e of action, compared with noradrenaline. (Kopin, 1966; 1968;1969; Kopin et a l . , 1966). Octopamine has also been termed a co-transmitter (Molinoff and Axelrod, 1972; 174 Jaim-Etcheverry and Z i e h e r , 1975) and a neuromodulator (Boulton, 1976), but a d e f i n i t i v e r o l e i n mammals has y e t to be e l u c i d a t e d . The p r o p o s a l by Boulton that octopamine modulates n o r a d r e n e r g i c t r a n s m i s s i o n a t c e n t r a l synapses m e r i t s c o n s i d e r a t i o n i n view of the p h y s i o l o g i c a l i m p l i c a -t i o n s such a mechanism would have on c u r r e n t views of syn-a p t i c f u n c t i o n . In f a c t i t s r o l e as a s y n a p t i c m o d i f i e r of neuronal a c t i v i t y has been demonstrated by other i n v e s -t i g a t o r s i n v a r i o u s i n v e r t e b r a t e s (Hoyle, 1974; B a t t e l l e and K r a v i t z , 1976; L i n g l e , 1976; Evans and O'Shea, 1977; O'Shea and Evans, 1979). ' B r i e f l y , Boulton (1976) suggested t h a t the t r a c e amines i n c l u d i n g octopamine, phenylethylamine, phenylethanolamine, tyramine and tryptamine a c t e i t h e r d i r e c t l y on p o s t s y n a p t i c r e c e p t o r s , or i n d i r e c t l y v i a p r e s y n a p t i c modulation of r e l e a s e , i n the process of s y n a p t i c t r a n s m i s s i o n mediated by more " c o n v e n t i o n a l " t r a n s m i t t e r s such as n o r a d r e n a l i n e . One or more of these t r a c e amines which are s y n t h e s i z e d w i t h -i n the cytoplasm of the neurone, might i n t e r a c t w i t h s y n a p t i c membranes i n such a way as to cause a continuous, p a r t i a l a c t i v a t i o n 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 . d i r e c t i o n . T h i s might e f f e c t i v e l y " s e t the tone" of "prime" the neurone and thereby modify the a c t i o n o f the major t r a n s -m i t t e r upon i t s r e l e a s e . The m e r i t s of t h i s h y p o t h e s i s w i l l be d i s c u s s e d below i n l i g h t of the r e s u l t s of the p r e s e n t study. 175 Octopamine i s unevenly d i s t r i b u t e d i n the mammalian b r a i n w i t h h i g h e s t c o n c e n t r a t i o n s i n the hypothalamus, midbrain and s p i n a l c o r d (ca. 10 ng/g). I t has been det e c t e d a l s o i n the c e r e b r a l c o r t e x , s t r i a t u m , cerebellum and h i p -pocampus (3 - 10 ng/g) as w e l l as i n the p i n e a l gland (500 ng/g) ( M o l i n o f f and A x e l r o d , 1972; Saavedra, 1974; A x e l r o d , Saavedra and Usdin, 1976; Buck, Murphy and M o l i n o f f , 1977; D a n i e l s o n , Boulton and Robertson, 1977). The o p i n i o n among many r e s e a r c h e r s has been t h a t octopamine p l a y s a r o l e of secondary importance i n the mammalian CNS. T h i s view arose because of the apparent s i m i l a r i t i e s i n p h y s i o l o g i c a l proces-ses of t h i s compound and n o r a d r e n a l i n e . Octopamine i s syn-t h e s i z e d by dopamine-3-hydroxylase, the same enzyme which produces n o r a d r e n a l i n e (Pisano, C r e v e l i n g and Udenfriend, 1960; C r e v e l i n g , Daly, Witkop and Udenfriend, 1962). I t appears 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 i n nerve t e r m i n a l s of the sympathetic nervous system, and i t a l s o sediments i n the same r e g i o n s of sucrose d e n s i t y g r a d i e n t s as 3H— noradren-a l i n e ( M o l i n o f f and A x e l r o d , 1972), suggesting a l o c a l i z a t i o n i n s y n a p t i c v e s i c l e s . . I t s d i s t r i b u t i o n p a r a l l e l s i n rough f a s h i o n t h a t of n o r a d r e n a l i n e (Glowinski and I v e r s e n , 1966; Harmar and Horn, 1976) and i t i s s t o r e d i n and has been r e p o r t e d to be c o - r e l e a s e d w i t h n o r a d r e n a l i n e from nervous 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 , 1971; B a l d e s s a r i n i and Vogt, 1972a,b; Kopin et a l . , 1965; M o l i n o f f and Buck, 1976; S a l d a t e and Orrego, 1976). F i n a l l y , octopamine i s 176 d e p l e t e d from t e r m i n a l s by the a d m i n i s t r a t i o n o f r e s e r p i n e ( G a r l s s o n and Waldeck, 1963; Chin A Paw, Knegt-Verpalen and Noach, 1968) or 6-hydroxydopamine ( B a l d e s s a r i n i , 1971). Inasmuch as the c e n t r a l and p e r i p h e r a l p r o p e r t i e s of octopamine and n o r a d r e n a l i n e appear to be c a u s a l l y r e l a t e d , i t must not be too h a s t i l y assumed t h a t these common proper-t i e s r e f l e c t only a "metabolic a c c i d e n t " (Evans, 1978) or secondary r o l e f o r octopamine". Few would di s a g r e e w i t h the view of an independent r o l e as a s y n a p t i c t r a n s m i t t e r f o r dopamine, y e t t h i s catecholamine i s a l s o taken up by the same mechanism as i s n o r a d r e n a l i n e and octopamine. I t can be s t o r e d i n sympathetic t e r m i n a l s presumably by the same b i n d i n g mechanism as the other amines and i n sho r t , functions very much l i k e n o r a d r e n a l i n e i n a l l neurochemical r e s p e c t s . L a r g e l y because of the advances achieved i n f l o u r e s -cence h i s t o c h e m i s t r y , separate n e u ronal systems f o r the catecholamines have been d e l i n e a t e d i n the b r a i n ( F a l c k , 1962; F a l c k , H i l l a r p , Thieme and Torp, 1962; H i l l a r p , Fuxe and Dahlstrdm, 1966; Dahlstrom and Fuxe, 1964; 1965). Octopamine does not f l o u r e s c e when these b i o c h e m i c a l methods are used due to the absence of a second h y d r o x y l group on the r i n g (Axelsson, B j o r k l u n d and L i n d v a l l , 1972). The pos-s i b i l i t y t h e r e f o r e remains t h a t there e x i s t neurones i n the CNS which normally employ octopamine as a s y n a p t i c t r a n s -m i t t e r but which respond i n a s i m i l a r f a s h i o n as do the 177 catecholamine c o n t a i n i n g c e l l s to p h a r m a c o l o g i c a l agents (eg: r e s e r p i n e , 6-hydroxydopamine, monoamine oxidase i n h i b i -t o r s , e t c . ) . Indeed, the c o n s i d e r a b l e evidence from i n v e r -t e b r a t e s (Evans, 1978) f o r an independant p h y s i o l o g i c a l f u n c t i o n of octopamine p r o v i d e s an e x c e l l e n t r a t i o n a l e f o r conducting experiments aimed at the c h a r a c t e r i z a t i o n of the p h y s i o l o g i c a l and p h a r m a c o l o g i c a l e f f e c t s of t h i s substance i n mammalian CNS. There have been e x c e e d i n g l y few p h y s i o l o g i c a l s t u d i e s w i t h octopamine i n mammalian CNS however, and i t s e f f e c t as a p h a r m a c o l o g i c a l a g o n i s t i s r e l a t i v e l y unknown. The obser-v a t i o n t h a t s u f f i c i e n t amounts of octopamine are a v a i l a b l e f o r i t to e x e r t p h y s i o l o g i c a l e f f e c t s has been made by many d i f f e r e n t groups (Kakimoto and Armstrong, 1960; M o l i n o f f and A x e l r o d , 1972; Harmar and Horn, 1976; D a n i e l s o n , Boulton and Robertson, 1977). These i n v e s t i g a t o r s found t h a t t h i s amine has a r a t e of turnover 6 - f o l d g r e a t e r than t h a t of noradren-a l i n e , i n s p i t e of r e l a t i v e l y low p r e v a i l i n g t i s s u e concen-t r a t i o n s . These o b s e r v a t i o n s may p o s s i b l y r e f l e c t a d i f f e r -ence between the two amines i n storage mechanism: noradren-a l i n e having a much g r e a t e r i n t r a c e l l u l a r p o o l than octop-amine. In t h i s way a l l the newly s y n t h e s i z e d octopamine i s r a p i d l y u t i l i z e d whereas only a p o r t i o n of the t i s s u e norad-r e n a l i n e i s r e a d i l y a v a i l a b l e f o r use (Snyder, G l o w i n s k i and A x e l r o d , 1965). 178 2) Receptors For Octopamine i / I n v e r t e b r a t e s The l i k e l i h o o d t h a t octopamine f u n c t i o n s as a s y n a p t i c t r a n s m i t t e r has r e c e i v e d the most thorough sup-p o r t through the e x t e n s i v e work that has been c a r r i e d out on i n v e r t e b r a t e s . Octopamine s t i m u l a t e s the p h o s p h o r y l a t i o n of s p e c i f i c p r o t e i n of molecular weight 120,000 i n A p l y s i a , a p p a r e n t l y by e l e v a t i n g l e v e l s o f c y c l i c adenosine-3,5'-mono-phosphate ( c y c l i c AMP) ( L e v i t a n and Barondes, 1974; L e v i t a n , Masden and Barondes, 1974). This p h o s p h o r y l a t i o n i s main-t a i n e d f o r s e v e r a l hours a f t e r removal of octopamine from the i n c u b a t i o n medium. These r e s u l t s are i n accordance w i t h a c u r r e n t model of p o s t s y n a p t i c consequence of t r a n s m i t t e r a c t i o n d e s c r i b e d by Greengard, Kebabian and Nathanson (1973), Nathanson and Greengard (1973), and Nathanson (1976) , although i n mammalian hypothalamus or caudate, there i s no s t i m u l a t i o n of c y c l i c AMP s y n t h e s i s by octopamine (Robinson, Mobley, Smith and Su l s n e r , 1978; Harmar and Horn, 1977). I o n t o p h o r e s i s of octopamine r e v e a l s a marked s e n s i t i v i t y and s p e c i f i c i t y of e f f e c t on some g a n g l i o n c e l l s i n A p l y s i a . These c e l l s respond 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 conductance. T h i s e f f e c t i s not e l i c i t e d by norad-r e n a l i n e , phenylethanolamine or dopamine (Carpenter and Gaubatz, 1974). Saavedra, Brownstein, Carpenter and Axelrod, (1974) found asymmetrically d i s t r i b u t e d c e l l s i n t h i s mollusc which c o n t a i n no n o r a d r e n a l i n e or dopamine, but possess 179 q u a n t i t i e s of octopamine as h i g h as 3.7 p m o l / c e l l . T h i s f i n d i n g p r o v i d e d support f o r the e x i s t e n c e o f separate sys-tems f o r octopamine and n o r a d r e n a l i n e i n t h i s s p e c i e s . The s t u d i e s on A p l y s i a d i d not p r o v i d e i n f o r m a t i o n r e g a r d i n g 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 of octopamine wit h i t s r e c e p t o r , nor d i d they t e s t f o r p o s s i b l e s t e r e o -s e l e c t i v e e f f e c t s or examine the a c t i o n s of amine antagon-i s t s . Dougan and Wade (1978a,b) conducted such an i n v e s t i -g a t i o n on the spontaneously b e a t i n g v e n t r i c l e of another m o l l u s c , Tapes w a t l i n g i . Dopamine may have e x c i t a t o r y or i n h i b i t o r y e f f e c t s on t h i s t i s s u e by r e a c t i n g w i t h e i t h e r of two s t r u c t u r a l l y d i s s i m i l a r r e c e p t o r s (Dougan, Wade and Mearri c k , 1975). The other catecholamines, n o r a d r e n a l i n e and a d r e n a l i n e , are much l e s s potent a g o n i s t s at these r e c e p t o r s . Octopamine i s equipotent w i t h dopamine i n i n c r e -a s i n g the amplitude of the v e n t r i c u l a r c o n t r a c t i o n s . T h i s e f f e c t of octopamine may be bl o c k e d by metoelopramide, c l o z -apine or s u l p i r i d e (Dougan and Wade, 1978b). The octopamine r e c e p t o r appears to be about 20 times more s e n s i t i v e to the e f f e c t s of (-) octopamine than to the (+) form; (±)-N-methyloctopamine and (-)-a-methyloctopamine were equipotent on a molar b a s i s w i t h (±)-octopamine. i i / V e r t e b r a t e s There have been on l y a few s t u d i e s wherein the e f f e c t s of phenylethylamines and catecholamines were compared on neurones of the CNS, and t h e i r r e s u l t s p r o v i d e 180 l i t t l e agreement on the p o s s i b l e e x i s t e n c e of separate recep-t o r p o p u l a t i o n s . G i a r d i n a , Pedemonte and S a b e l l i (1973) r e p o r t e d o p p o s i t e e f f e c t s on c o r t i c a l neurones of phenyl-ethylamine and phenylethanolamine 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 n o r a d r e n a l i n e . Motoneurones were depres-sed 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 , tyramine, dopa-mine, n o r a d r e n a l i n e and octopamine (Engberg, Flatman and Kadzielawa, 1976). Octopamine was found to be a weak agonist compared w i t h n o r a d r e n a l i n e i n i n h i b i t i n g the f i r i n g o f P u r k i n j e c e l l s 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 (Kostopoulos and Yarbrough, 1975). However, s i n c e p r e v i o u s experiments by H o f f e r , S i g g i n s and Bloom (1971, 1973) p r o v i d e d evidence f o r these c e l l s having n o r a d r e n e r g i c synapses, a s m a l l e r e f f e c t o f octopamine on adrenoceptors i s t h e r e f o r e not s u r p r i s i n g , although u n t i l a n t a g o n i s t s f o r octopamine are developed, the c o e x i s t e n c e of octopamine and n o r a d r e n a l i n e r e c e p t o r s on the same neurones remains another p o s s i b l e ' e x p l a n a t i o n . S i m i l a r f i n d i n g s of a weaker a c t i o n of octopamine compared w i t h n o r a d r e n a l i n e or dopamine on neurones of the 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 (1979). These authors never observed e x c i t a t o r y responses to any of s e v e r a l amines t e s t e d , i n c l u -ding octopamine; nor d i d they examine the e f f e c t s of s p e c i f i c amine a n t a g o n i s t s a g a i n s t the depressions to determine the nature o f the r e c e p t o r s i n v o l v e d . 181 The extensive evidence for a transmitter role of octop-amine i n invertebrates as well as the r e l a t i v e lack of pharmacological studies i n mammals prompted the present inves-t i g a t i o n . Neurones of the cerebral cortex and dorsal horn of the spinal cord were chosen for testing with octopamine and catecholamines because the receptors for the l a t t e r compounds in these areas have been pharmacologically characterized by many others (Krnjevic and P h i l l i s , 1963d; Biscoe, Curtis, and Ryall, 1966; Crawford and Curtis, 1966; Engberg and R y a l l , 1966; Roberts and Straughan, 1967; P h i l l i s , Tebecis and York, 1968; Johnson, Roberts, Sobieszek and Straughan, 1969; Engberg and Marshall, 1971; Frederickson, Jordan and P h i l l i s , 1971; Stone 1973; 1978; Jordan, McCrea, Steeves andMenzies, 1977; Bradshaw, Bevan and Szabadi, 1978; for reviews see P h i l l i s , 1970; Krnjevic, 1974; Tebecis, 1974; Schwartz, 1975). The r e s u l t s obtained through the use of pharmacologically s p e c i f i c amine antagonists have given r i s e to the proposal that octopamine exerts an e f f e c t on central neurones which is not dependent upon the presence of receptors for catechol-amines . (Hicks and McLennan, 1978a,b). 182 b) R e s u l t s C e n t r a l neurones were t e s t e d f o r t h e i r responsiveness to octopamine, n o r a d r e n a l i n e and dopamine. Only a s m a l l propor-t i o n of neurones i n the c e r e b r a l c o r t e x (ca. 20%) and s p i n a l cord (ca. 30%) responded 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 through a change i n t h e i r f i r i n g r a t e s . Those c e l l s which responded to one amine d i d not n e c e s s a r i l y respond to a l l o t h e r s : of 150 c o r t i c a l neurones which were a f f e c t e d by at l e a s t one compound, 52% responded to a l l three, 40% responded to o n l y two and 8% responded to o n l y one amine. The q u a l i t a t i v e nature of the changes i n f i r i n g r a t e induced by the amines d i f f e r e d markedly from t h a t o f other compounds employed i n experiments i n the cord.and c o r t e x , (see other Chapters i n t h i s account). Octopamine and dopa-mine e f f e c t e d r a t e changes which were more r a p i d i n onset and r e c o v e r y time than were those of n o r a d r e n a l i n e . An example of t h i s d i f f e r e n c e i n l a t e n c y of octopamine and n o r a d r e n a l i n e i n g i v e n i n F i g u r e 24b, where the d e p r e s s i o n e l i c i t e d by n o r a d r e n a l i n e c o n s i d e r a b l y o u t l a s t e d the a p p l i -c a t i o n of the substance. The d u r a t i o n of the a f t e r - e f f e c t w i t h n o r a d r e n a l i n e was observed to depend on the magnitude of the e j e c t i n g c u r r e n t ; by c o n t r a s t , the d e p r e s s i o n caused by octopamine l a s t e d o n l y as long as the e x p e l l i n g c u r r e n t was maintained. 183 F i g u r e 24. Examples of the e f f e c t s of octopamine and noradren-a l i n e on the f i r i n g r a t e s of neurones of the c e r e b r a l c o r t e x . (A) A neurone responding w i t h e x c i t a t i o n and i n h i b i t i o n to a p p l i -c a t i o n of octopamine (the open bars) and n o r a d r e n a l i n e ( c l o s e d bars) r e s p e c t i v e l y . T h i s c e l l was l o c a t e d 1.15 mm below the s u r f a c e of the c o r t e x . (B) Another neurone (depth 1.34 mm) responding to both octopamine and n o r a d r e n a l i n e . Note d i f f e r e n c e i n d u r a t i o n of e f f e c t between the two amines; o r d i n a t e s c a l e as i n (A). (C) Neurone responding to octopamine w i t h a s t r o n g e r e x c i t a t i o n than to n o r a d r e n a l i n e ( c e l l depth: 1.50 mm). 184 50 B o O) to to ' d CO 50 50 50 01 a m 75 75 I 1 I min. a Octopamine wm Noradrenaline 50 50 185 i / E f f e c t s Of Octopamine And N o r a d r e n a l i n e Both i n c r e a s e s and decreases i n the r a t e s of f i r i n g o f c e r e b r a l c o r t i c a l neurone's were observed i n response to the amines. From a sample p o p u l a t i o n of 95 u n i d e n t i f i e d neurones, n o r a d r e n a l i n e depressed the f i r i n g of 74 and i n c r e a s e d the r a t e s of 13 (Table 9). The a c t i o n of octopamine upon the same neurones was d i f f e r e n t , as i t decreased the r a t e s of f i r i n g of 46 c e l l s , e x c i t e d 42 and had no e f f e c t on the remaining 9. An a n a l y s i s of contingency (Table 9) by the x 2 t e s t shows no s t a t i s t i c a l l y s i g n i f i c a n t a s s o c i a t i o n between the e f f e c t s of octopamine and n o r a d r e n a l i n e (p > 0.36). Of p a r t i c u l a r note i s the o b s e r v a t i o n t h a t on 34 c o r t i c a l neurones the two amines had o p p o s i t e e f f e c t s . In F i g u r e 24a are shown the responses of a neurone which was depressed by n o r a d r e n a l i n e and e x c i t e d by octopamine. The responses were u n a f f e c t e d by the pH of the amine s o l u t i o n s over the range used. (3.0 - 5.2). i i / Potency E s t i m a t i o n s On 26 of the 37 c e l l s which had t h e i r f i r i n g r a t e s decreased by both amines, i t was observed t h a t octopamine was of l e s s apparent potency as a depressant than was n o r a d r e n a l i n e , judged by the i n t e n s i t i e s of 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 e f f e c t s of e q u i v a l e n t magnitude. The two amines appeared to be of s i m i l a r potency i n lowering the r a t e s of f i r i n g o f the other 11 neurones. By the same c r i t e r i o n n o r a d r e n a l i n e was a p p a r e n t l y l e s s potent than octopamine i n i n c r e a s i n g the neuronal f i r i n g r a t e 186 F i g u r e 25. Responses of a c o r t i c a l neurone (depth 0.24 mm) to dopamine ( d i v i d e d bar) and n o r a d r e n a l i n e ( s o l i d b a r ) . 187 OQ Dopamine 188 of 6 c e l l s . An example of t h i s i s shown i n F i g u r e 24c. When t h i s type of a c t i o n o c c u r r e d , i t was a l s o observed that other neurones t e s t e d w i t h the same p i p e t t e assembly c o u l d y i e l d more powerful n o r a d r e n a l i n e evoked depressant responses. E l e c t r o d e a r t i f a c t i s t h e r e f o r e u n l i k e l y to e x p l a i n the i l l u s t r a t e d r e s u l t . Accurate q u a n t i t a t i v e measures of potency cannot be ob t a i n e d without a knowledge of t r a n s p o r t numbers f o r the e j e c t e d compounds (Chapter I ) . i i i / E f f e c t s Of Dopamine The neuronal responsiveness to dopamine was examined f o r a l l c e l l s which were a f f e c t e d by n o r a d r e n a l i n e . Since many of these neurones a l s o responded to octopamine (78 of 95, see Table 9), i t was p o s s i b l e a l s o to c o n t r a s t the e f f e c t s o f octopamine and dopamine to exam-in e the p o s s i b i l i t y t h a t the a l t e r a t i o n s i n f i r i n g r a t e induced by the p h e n o l i c amine were due to an i n t e r a c t i o n w i t h a r e c e p t o r f o r t h i s catecholamine. Changes i n f i r i n g r a t e e l i c i t e d by dopamine were of short d u r a t i o n , u s u a l l y l a s t i n g o n l y as long as the a p p l i -c a t i o n o f the amine. Of 74 neurones depressed by noradren-a l i n e , 48 were depressed a l s o by dopamine w h i l e 19 were e x c i t e d ( F i g u r e 25). Of these 19 c e l l s , 10 were e x c i t e d a l s o by octopamine and 6 were depressed; the remaining 3 were u n a f f e c t e d . Of the 13 c e l l s e x c i t e d by n o r a d r e n a l i n e , 9 were a l s o e x c i t e d by dopamine w h i l e the other 4 c e l l s d i d not respond. Octopamine i n c r e a s e d the f i r i n g of 5 c e l l s 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 t e s t e d w i t h both octopamine and n o r a d r e n a l i n e (n = 95). Octopamine e x c i t a t i o n d e p r e s s i o n no e f f e c t e x c i t a t i o n 7:' 6 0 Noradr e n a l i n e d e p r e s s i o n 28 37 9 no e f f e c t 5 3 0 190 TABLE 10. Numbers of u n i d e n t i f i e d c o r t i c a l neurones t e s t e d w i t h both octopamine and dopamine (n = 68). Octopamine e x c i t a t i o n Dopamine de p r e s s i o n no e f f e c t e x c i t a t i o n d e p r e s s i o n no e f f e c t 6 10 4 19 13 8 6 2 0 191 TABLE 11. Numbers of i d e n t i f i e d c o r t i c a l neurones t e s t e d w i t h both octopamine and n o r a d r e n a l i n e (n = 31) Octopamine e x c i t a t i o n d e p r e s s i o n no e f f e c t e x c i t a t i o n 4 1 3 No r a d r e n a l i n e d e p r e s s i o n 8 6 8 no e f f e c t 0 1 0 192 which were excited also by both catecholamines, and excited one other which did not respond to noradrenaline but was excited by,dopamine. Table 10 provides a more detailed sum-mary of the effects of dopamine compared with those of octopamine. In short, the r e s u l t s indicate that the effects of octopamine also showed no s i g n i f i c a n t association (x 2 test p > 0.20) with those of dopamine, as was the case with those of noradrenaline. i v / I d e n t i f i e d C o r t i c a l Neurones In an attempt to i d e n t i f y those c o r t i c a l neurones which were affected by low doses of the amines, stimulating electrodes were positioned stereo-t a x i c a l l y i n crus cerebri and ventrobasal complex of the thalamus (see Chapter I I ) . Thirty-one neurones activated by antidromic stimulation of crus cerebri or which f i r e d at latencies of 5 to 12 msec, following thalamic stimulation were tested for t h e i r responses to iontophoretically applied amines, and as the responses of these two groups of i d e n t i -f i e d neurones to the agonists were indistinguishable, the r e s u l t s were pooled and are presented i n Table 11. Although the general pattern 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 of Table 9, on a l l of the pyramidal tract and thalamo-cortical c e l l s high ejection currents (up to 100 nA) were required to e l i c i t even minimal effects with octopamine. 193 Figure 26. A dose-response curve for depressant e f f e c t s e l i c i t e d by octopamine at a dorsal horn interneurone a c t i -vated at short latency by volleys to the central end of a severed dorsal root. Numbers i n parentheses indicate t r i a l s , bars represent standard error of the means. The trace i s the record of 4 superimposed sweeps. 194 15 msec I , Current (nA) 195 TABLE 12. C l a s s i f i c a t i o n o f 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 . (n=51) Octopamine e x c i t a t i o n d e p r e s s i o n no e f f e c t e x c i t a t i o n 8 2 0 Noradr e n a l i n e d e p r e s s i o n 11 27 3 196 TABLE 13. C h a r a c t e r i z a t i o n o f neuronal responses of 26 s p i n a l neurones 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 octopamine. Dopamine e x c i t a t i o n depression Noradrenaline e x c i t a t i o n 2 1 T T d e p r e s s i o n 4 7 5 5 Numerator of each f r a c t i o n i n d i c a t e s the number of c e l l s e x c i t e d by octopamine w h i l e the denominator r e p r e s e n t s the number depressed. For example, 9 of the 26 c e l l s t e s t e d were depressed by n o r a d r e n a l i n e and e x c i t e d by dopamine; of these 9, octopamine e x c i t e d 4 and depressed 5. 197 v/ Dorsal Horn Neurones Similar findings to those with unid e n t i f i e d c o r t i c a l neurones were obtained from dorsal horn neurones of the spinal cord. These c e l l s were i d e n t i -f i e d by their short latency responses to st i m u l i applied to dorsal roots (see Chapter II and Figure 26). The p r i n c i p a l findings are summarized i n Tables 12 and 13. Noradrenaline produced primarily depressant effects while the action of octopamine was more variable. Opposite effects were obtained on 37% of the spinal neurones respon-ding both to noradrenaline and octopamine. Three c e l l s depressed by noradrenaline were not affected by octopamine administered with iontophoretic currents up to 100 nA. Dopamine was also tested on 26 of the 51 spinal neurones, and the res u l t s are presented i n Table 13. Again for a proportion of the c e l l s the actions e l i c i t e d by octopamine d i f f e r e d from those obtained with one or both of the cate-cholamines. Due to the low number of c e l l s i n some of the caregories i n contingency Tables 12 and 13, i t was not possible to analyze the re s u l t s with the x 2 test of s t a t i s -t i c a l independence. However i t may be noted that for the data displayed i n Table 12 more than one t h i r d of the c e l l s had opposite responses to octopamine and noradrenaline, while Table 13 i l l u s t r a t e s c l e a r l y that effects with octop-amine bore no greater relationship to those of noradrenaline than to those of dopamine. 198 F i g u r e 27. Responses of two neurones t e s t e d w i t h octopamine (open bars, OCT) and n o r a d r e n a l i n e ( s o l i d b ars, NOR) i n the upper t r a c e ; and octopamine and dopamine ( d i v i d e d b a r s , DOP) i n the lower t r a c e ; i n the presence of a n t a g o n i s t s of the amines. In the upper t r a c e , antagonism by p r o p r a n o l o l (8 nA) of the depressant e f f e c t of n o r a d r e n a l i n e a p p l i e d w i t h 40 nA of i o n t o p h o r e t i c c u r r e n t i s observed, w h i l e the e x c i t a t o r y respon-ses caused by octopamine (40 nA) remain u n a f f e c t e d . Recovery was obtained 2.5 min a f t e r c e s s a t i o n of the p r o p r a n o l o l admin-i s t r a t i o n . Neurone was l o c a t e d 0.96 mm below the s u r f a c e of the c o r t e x . The lower t r a c e i l l u s t r a t e s the s e l e c t i v i t y o f a - f l u p e n f t h i x o l (55 nA) i n r e d u c i n g the depressant e f f e c t of dopamine (75 nA) and not that o f octopamine ( a l s o 75 nA). The respon-ses r e c o v e r e d to c o n t r o l l e v e l s approximately 5.5 min a f t e r the a - f l u p e n t h i x o l . ( c e l l depth 0.45 mm). 1 9 9 • m 200 v i / E f f e c t s Of P r o p r a n o l o l And ct-Flupenthixol An i n v e s t i -g a t i o n of the e f f e c t s of n o r a d r e n a l i n e and dopamine receptor a n t a g o n i s t s was conducted i n order to determine more s p e c i -f i c a l l y the independence of octopamine induced changes i n f i r i n g r a t e from those of the catecholamines. P r o p r a n o l o l was e f f e c t i v e as a b l o c k e r of the depressant responses of noradrenaline, which l e f t u n a l t e r e d i n c r e a s e s or decreases i n f i r i n g r a t e e l i c i t e d by octopamine. Since p r o p r a n o l o l has l o c a l a n a e s t h e t i c p r o p e r t i e s (Engberg and R y a l l , 1966; Johnson et a l . , 1969; Stone, 1973) complete t e s t s were achie-ved on o n l y 4 c o r t i c a l neurones (of 15 t e s t e d ) and 7 s p i n a l neurones (of 12 t e s t e d ) . However, i n a l l attempts where a re c o v e r y o f responses to c o n t r o l l e v e l s was achieved, an e f f e c t a g a i n s t n o r a d r e n a l i n e but not of octopamine was obser-ved. ( F i g u r e 27a, and F i g u r e s 28 and 29). P r o p r a n o l o l was not t e s t e d on neurones which were e x c i t e d by n o r a d r e n a l i n e . A c c e p t a b l e r e c o r d s were ob t a i n e d from 5 c o r t i c a l neurones t e s t e d w i t h the dopamine r e c e p t o r a n t a g o n i s t , a - f l u p e n t h i x o l . On a l l 5 c e l l s the e f f e c t s of dopamine were a t t e n u a t e d w h i l e those of octopamine were spared. Of these 5 c e l l s , 3 respon-ded w i t h depressions to both octopamine and dopamine ( F i g u r e 27b), one other was depressed by octopamine and e x c i t e d by dopamine w h i l e the remaining c e l l was e x c i t e d by octopamine and depressed by dopamine. a - F l u p e n t h i x o l was not t e s t e d on the responses of s p i n a l neurones. 201 F i g u r e 28. Rate r e c o r d showing the e f f e c t of p r o p r a n o l o l a d m i n i s t e r e d w i t h 15 nA of e j e c t i n g c u r r e n t to a s p i n a l i n t e r -neurone, which responded to octopamine (75 nA) and noradren-a l i n e (75 nA) w i t h o p p o s i t e changes i n r a t e . The c e n t r e t r a c e i l l u s t r a t e s s e l e c t i v e antagonism of the depressant e f f e c t of n o r a d r e n a l i n e . Time ela p s e d between c o n t r o l responses (upper t r a c e ) and maximal e f f e c t of a n t a g o n i s t (middle t r a c e ) was 8 min and time to r e c o v e r y (bottom t r a c e ) was 5 min. 2 0 2 203 F i g u r e 29. Rate r e c o r d showing the e f f e c t o f p r o p r a n o l o l on depressions e l i c i t e d by octopamine and n o r a d r e n a l i n e of a s p i n a l interneurone. A n t a g o n i s t was a d m i n i s t e r e d w i t h an e j e c t i n g c u r r e n t of 20 nA a p p l i e d f o r 2 min, w i t h the r e s u l t a n t s e l e c t i v e antagonism observed as i l l u s t r a t e d i n the c e n t r e r e c o r d . Recovery o c c u r r e d approximately 3 min a f t e r the propra-n o l o l e j e c t i o n phase. 204 I 1 I min. Noradrenaline 50 nA ^ 3 Octopamine 65 nA 205 v i i / E f f e c t s Of Clozapine And Metoclopramide A r e c e n t r e p o r t i n d i c a t e s t h at metoclopramide and c l o z a p i n e are e f f e c t i v e as a n t a g o n i s t s which can d i f f e r e n t i a t e between catecholamine and phenylethylamine a g o n i s t s on the i s o l a t e d v e n t r i c l e of Tapes w a t l i n g i (Dougan and Wade, 1978b). A c c o r d i n g l y , these two compounds were examined f o r an e f f e c t a g a i n s t the amine induced responses o f ^ c o r t i c a l and s p i n a l neurones. Whether e j e c t e d i o n t o p h o r e t i c a l l y (14 c o r t i c a l and 3 s p i n a l c e l l s ) or a d m i n i s t e r e d i n t r a v e n o u s l y (4 c o r t i c a l and 2 s p i n a l c e l l s ) , metoclopramide showed no a n t a g o n i s t i c a c t i o n toward any of the amines. Cl o z a p i n e was a d m i n i s t e r e d i n t r a v e n o u s l y w h i l e r e c o r d i n g from 8 c o r t i c a l and 5 s p i n a l neurones. Regardless of 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 t h i s compound and c l o z a p i n e always caused an i n c r e a s e i n the spontaneous r a t e of f i r i n g w i t h i n 30 sees, w h i l e the responses to the a d m i n i s t r a t i o n of the amines superimposed on t h i s enhanced background a c t i v i t y was u n a f f e c t e d . Metoclopramide caused an abrupt decrease i n neuronal f i r i n g i f a d m i n i s t r a -t i o n was continued f o r 5 or 6 min. (or a s h o r t e r time i f h i g h e r doses were employed) w i t h r e c o v e r y o c c u r r i n g as long as 30 min. l a t e r . These e f f e c t s are i l l u s t r a t e d i n F i g u r e 30. I n j e c t i o n s of 0.9% NaCl l e f t u n a l t e r e d both the spontaneous r a t e of d i s c h a r g e , and the amine induced changes i n f i r i n g . 206 F i g u r e 30. E f f e c t s of c l o z a p i n e and metoclopramide on the f i r i n g of c o r t i c a l and s p i n a l neurones i n response to amines. Upper t r a c e : A c o r t i c a l neurone responding w i t h weak depres-sions to octopamine and n o r a d r e n a l i n e . C l o z a p i n e (20 mg/kg) adm i n i s t e r e d i n t r a v e n o u s l y caused a pronounced i n c r e a s e i n the b a s e l i n e f i r i n g without having an e f f e c t which c o u l d be i n t e r p r e t e d as s e l e c t i v e antagonism (due to the d i r e c t e f f e c t observed on spontaneous a c t i v i t y ) . Lower t r a c e : A s p i n a l neurone responding w i t h d e p r e s s i o n to a l l 3 amines t e s t e d . Metoclopramide a d m i n i s t e r e d iontophor-e t i c a l l y w i t h a c u r r e n t o f 12 nA a l s o caused an enhancement of the background f i r i n g , without s e l e c t i v e antagonism, and when a d m i n i s t r a t i o n was con t i n u e d f o r 5.5 min., the f i r i n g o f the c e l l ceased, and recover y o f the responses c o u l d not be obta i n e d f o r as long as 1.5 hr. a f t e r the a d m i n i s t r a t i o n of metoclopramide was terminated. 100 Q . co 50 0 Clozapine 20mq/kg 50 50 Metoclopramide 12 nA 25 25 25 N3 i 1 a Octopamine nn Dopamine ™ Noradrenaline >i I min. 208 c) D i s c u s s i o n The f i r s t major c o n c l u s i o n a r i s i n g from the p r e s e n t e x p e r i -ments i s t h a t octopamine i s p h a r m a c o l o g i c a l l y a c t i v e i n the mammalian CNS, both as an e x c i t a t o r y and a depressant comp-ound. Furthermore i t i s c l e a r from the r e s u l t s of the p a r t of t h i s study which compared the a c t i o n s of octopamine and catecholamines, that these compounds d i f f e r i n t h e i r pharma-c o l o g i c a l p r o p e r t i e s as a g o n i s t s as w e l l as i n t h e i r respon-ses to a n t a g o n i s t s . Octopamine t h e r e f o r e has c e r t a i n actions which cannot be e x p l a i n e d by the f a l s e t r a n s m i t t e r hypothesis ( F i s c h e r et a l . , 1965; Kopin, 1968). A f a l s e t r a n s m i t t e r as d e f i n e d by Kopin (1968) e x e r t s i t s a c t i o n on a r e c e p t o r which i s p h a r m a c o l o g i c a l l y s p e c i f i c f o r the normal t r a n s m i t t e r . The r e a c t i o n of the f a l s e t r a n s m i t t e r w i t h the s y n a p t i c r e c e p t o r thereby decreases the e f f i c a c y of t r a n s m i s s i o n a t the e f f e c t o r s i t e . The f a l s e t r a n s m i t t e r must t h e r e f o r e produce a response q u a l i t a t i v e l y s i m i l a r to that e x e r t e d by the normal t r a n s m i t t e r . I t must, moreover, e f f e c t a response which i s b l o c k e d by a n t a g o n i s t s of the t r a n s m i t t e r agent. Nor a d r e n a l i n e and dopamine are g e n e r a l l y c o n s i d e r e d to be a c t i v e as s y n a p t i c t r a n s m i t t e r s i n the mammalian CNS and both are b e l i e v e d to be c o n t a i n e d i n f i b r e s which i n n e r v a t e the c e r e b r a l c o r t e x ( T e b e c i s , 1974; Emson and L i n d v a l l , 1979). Since the e f f e c t s of octopamine are not s i g n i f i c a n t l y c o r -r e l a t e d w i t h those of e i t h e r of the catecholamines, nor are 209 they a f f e c t e d by adrenoceptor or dopamine r e c e p t o r b l o c k i n g agents, i t seems reasonable to a s c r i b e to octopamine a pha r m a c o l o g i c a l r o l e other than as a f a l s e t r a n s m i t t e r . Indeed, s i n c e on many occasions there were observed a c t i o n s of octopamine o p p o s i t e to those o f n o r a d r e n a l i n e and dopamine t h i s i n d i c a t e s a d i r e c t e f f e c t on r e c e p t o r s . I f the a c t i o n of octopamine were i n d i r e c t , i . e . to cause a p r e s y n a p t i c r e l e a s e of catecholamines, then a g a i n the e f f e c t s observed w i t h octopamine should be c o r r e l a t e d w i t h those o f noradren-a l i n e or dopamine. The r e s u l t s o b t a i n e d w i t h the catecholamines on c o r t i c a l c e l l s a c t i v a t e d a n t i d r o m i c a l l y by pyramidal t r a c t stimu-l a t i o n or s y n a p t i c a l l y by s t i m u l a t i o n of VB thalamus are i n good agreement w i t h those of Stone (1973). T h i s author found t h a t most of these c e l l s were depressed by n o r a d r e n a l i n e , that the e f f e c t s were slow i n onset and r e l a t i v e l y p rolonged (Stone, 1978) and that p r o p r a n o l o l was a s e l e c t i v e b l o c k e r of the dep r e s s i o n s . One d i f f e r e n c e between the pr e s e n t r e s u l t s and those o f Stone i s the o b s e r v a t i o n t h a t a s m a l l number of c e l l s o f the pyramidal t r a c t were e x c i t e d by c a t e -cholamines, w h i l e Stone d i d not d e s c r i b e such n o r a d r e n a l i n e induced e x c i t a t i o n s . The o b s e r v a t i o n t h a t octopamine i s a weak a g o n i s t o n l y on the i d e n t i f i e d c o r t i c a l neurones suggests t h a t they do not possess r e c e p t o r s f o r octopamine and t h a t the e f f e c t s of 210 t h i s amine are perhaps mediated through catecholamine recep-t o r s . It. was not p o s s i b l e , to i d e n t i f y e l e c t r o p h y s i o l o g i c a l ^ those c o r t i c a l c e l l s most r e s p o n s i v e to octopamine. The:present view that there may e x i s t separate and independent systems i n the CNS f o r octopamine and n o r a d r e n a l i n e i s supported by the o b s e r v a t i o n t h a t some neurones ( c e r e b e l l a r P u r k i n j e cells, Kostopoulos and Yarbrough, 1975; and c o r t i c a l pyramidal t r a c t c e l l s , Hicks and McLennan, 1978a) are o n l y weakly s e n s i t i v e to the p h e n o l i c amine but respond to catecholamines more r e a d i l y and w i t h more c l e a r l y d e f i n e d changes i n r a t e . D o r s a l horn neurones i n the s p i n a l c o r d which responded at s h o r t l a t e n c i e s to s y n a p t i c a c t i v a t i o n were a f f e c t e d by low doses of the amines i n c l u d i n g octopamine, and might t h e r e f o r e possess octopamine s p e c i f i c r e c e p t o r s (Hicks and McLennan, 1978b). The c e l l s were found i n the s p i n a l c o r d at depths (0.4 - 1.3 mm) where catecholamines are b e l i e v e d to a c t as s y n a p t i c t r a n s m i t t e r s (Biscoe, C u r t i s and R y a l l , 1966; C a r l s s o n , F a l c k , Fuxe and H i l l a r p , 1964; Engberg and R y a l l , 1966; Nygren and Olson, 1977; Jordan et a l . , 1977). T h i s may be an important c o n s i d e r a t i o n f o r f u t u r e s t u d i e s aimed at determining 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 of o c t -opamine s e n s i t i v e c e l l s , s i n c e the presence o f . neuronal r e c e p t o r s s e n s i t i v e to the catecholamines does not neces-s a r i l y p r e d i c t the degree of responsiveness to octopamine. 211 The r e s u l t s w i t h two a n t a g o n i s t s o f f e r f u r t h e r evidence f o r the e x i s t e n c e o f octopamine r e c e p t o r s d i s t i n c t from those used by the catecholamines. 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 r e s p e c t i v e l y prevented n o r a d r e n a l i n e and dopamine induced decreases i n the f i r i n g r a t e , c o n f i r m i n g i n p a r t s i m i l a r f i n d i n g s by Bradshaw et a l . , (1978). N e i t h e r of these antag-o n i s t s a t t e n u a t e d the e f f e c t s e l i c i t e d by octopamine. T h i s suggests t h a t r e c e p t o r s mediating the a c t i o n s of octopamine are 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 of the c a t e c h o l -amines. The r e s u l t s are i n agreement w i t h the o b s e r v a t i o n s of G i a r d i n a et a l . , (1973) who r e p o r t e d o p p o s i t e e f f e c t s of phenylethylamine and phenylethanolamine when compared w i t h n o r a d r e n a l i n e on c o r t i c a l neurones. Metoclopramide, a chlorbenzamide d e r i v a t i v e w i t h a n t i -emetic and c a t a l e p t i c p r o p e r t i e s (Ahtee, 1975; Jenner, Marsden and P e r i n g e r , 1975) and c l o z a p i n e , a n e u r o l e p t i c having few ext r a p y r a m i d a l e f f e c t s (Anden and Stock, 1973; B u r k i , Ruch, Asper, B a g g i o l i n i and S t i l l e , 1974; Fog, 1975; K e l l y and M i l l e r , 1975) have been d e s c r i b e d as e f f e c t i v e a n t a g o n i s t s o f the r e c e p t o r s f o r octopamine i n Tapes wat-l i n g i (Dougan and Wade, 1978). These two compounds have no e f f e c t as a n t a g o n i s t s f o r any of the amines t e s t e d i n the co r t e x or s p i n a l c o r d o f the r a t ; indeed they had powerful a c t i o n s of t h e i r own on those neurones examined. These a c t i o n s cannot be e x p l a i n e d on the b a s i s of a c o m p e t i t i v e r e a c t i o n w i t h octopamine r e c e p t o r s . 212 In c o n s i d e r a t i o n of the present r e s u l t s , i t i s d i f f i c u l t to evaluate the p r o p o s a l that octopamine a c t s as a s y n a p t i c a c t i v a t o r (Boulton, 1976; 1978; B a l d e s s a r i n i and F i s c h e r , 1978). This study has shown t h a t octopamine most probably e x e r t s d i r e c t e f f e c t s on r e c e p t o r s which can be d i s t i n g u i s h e d p h a r m a c o l o g i c a l l y from those of the catecholamines. A sen-s i t i z i n g e f f e c t by octopamine of n o r a d r e n a l i n e evoked r e s -ponses as demonstrated by Kostopoulos and Yarbrough (1975) does not of i t s e l f p r o v i d e evidence f o r a modulatory r o l e , and u n t i l an a c c e p t a b l e d e s c r i p t i o n of the term "neuromodu-l a t o r " i s presented, such experiments add l i t t l e i n f o r m a t i o n concerning the r o l e o f octopamine i n s y n a p t i c f u n c t i o n . Caution i s warranted f u r t h e r b e f o r e a c c e p t i n g the view t h a t octopamine i s a s y n a p t i c a c t i v a t o r , s i n c e the e f f e c t of octopamine appears o c c a s i o n a l l y s t r o n g e r than that o f norad-r e n a l i n e , at l e a s t so f a r as c o r t i c a l e x c i t a t i o n s are i n v o l v e d . I t i s d i f f i c u l t to conceive of a compound having a modulatory r o l e when i t appears more potent than the sus-p e c t e d 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 the turnover of the compound i s so h i g h i n the normal s t a t e . Boulton (1976;' 1978) s t a t e s e x p l i c i t l y i n h i s h y p o t h e s i s t h a t the s y n a p t i c a c t i v a t o r should cause o n l y minor p o s t s y n a p t i c or presynap-t i c e f f e c t s w h i l e m a i n t a i n i n g the synapse i n a s t a t e o f re a d i n e s s f o r the c o n v e n t i o n a l t r a n s m i t t e r . F u r t h e r experiments w i l l be r e q u i r e d b e f o r e i t can be 213 concluded that octopaminergic transmission occurs i n mammal-ian nervous tissue. However the re s u l t s described here do offer experimental support for the existence of receptors sensitive to octopamine which are independent of those recep-tive to the catecholamines, and i t i s conceivable that they may be involved i n separate synaptic processes. 214 CHAPTER VI SUMMARY AND CONCLUDING REMARKS The r e s u l t s , p r e s e n t e d i n t h i s t h e s i s p r o v i d e evidence which permits an a n a l y s i s of s e v e r a l d i f f e r e n t systems of neuronal r e c e p t o r s i n the CNS of the r a t . The experiments w i t h ACh and cholinomimetic drugs have shown that the c h o l i n e r g i c r e c e p t o r s of neurones from s e v e r a l d i f f e r e n t r e g i o n s possess d i f f e r e n t 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 from those d e s c r i b e d i n the c a t , and although the exact nature of these r e c e p t o r s remains u n c l e a r , other i n v e s t i g a t o r s have confirmed the present r e s u l t s . ' The amino a c i d s are p r e s e n t l y e x c e l l e n t candidates as s y n a p t i c t r a n s m i t t e r s i n mammalian CNS. These compounds have been shown to r e a c t w i t h at l e a s t two, and perhaps other 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 r e c e p t o r s i n the thalamus and s p i n a l cord, and e v i d -ence i s r a p i d l y accumulating f o r t h e i r r o l e s as t r a n s m i t t e r s of s y n a p t i c e x c i t a t i o n i n other r e g i o n s of mammalian CNS. Several r e c e n t r e p o r t s have confirmed i n p a r t , the presen t r e s u l t s d e s c r i b i n g the s e l e c t i v e antagonism of the e x c i t a t o r y amino a c i d s . Octopamine has y e t to be proven as a compound of impor-tance i n the mammalian nervous system, d e s p i t e the f a c t t h a t i t appears to be a major t r a n s m i t t e r i n a number of i n v e r t e b r a t e s p e c i e s . The r e s u l t s of the pr e s e n t experiments showing the probable e x i s t e n c e of r e c e p t o r s f o r octopamine on c e n t r a l 215 neurones i n d i c a t e s that a more syste m a t i c i n v e s t i g a t i o n i n t o the p o s s i b l e p h y s i o l o g i c a l r o l e s f o r t h i s amine should be conducted. Despite the f a c t t h at there appears to be a m u l t i p l i c i t y of p h a r m a c o l o g i c a l l y s p e c i f i c r e c e p t o r s f o r many of the com-pounds t e s t e d i n the present experiments, i t cannot be c o n c l u -ded t h a t they are a l l i n v o l v e d i n processes of s y n a p t i c t r a n s -m i s s i o n . As d i s c u s s e d i n Chapter I, the presence of r e c e p t o r s i s o n l y one of the c r i t e r i a which must be f u l f i l l e d b e f o r e a r o l e as a s y n a p t i c t r a n s m i t t e r may be a s s i g n e d to any p a r t i c u l a r compound. However i n cases where a n t a g o n i s t s of known pharmaco-l o g i c a l a c t i o n s can be used to b l o c k s e l e c t i v e l y the e f f e c t s of s y n a p t i c a c t i v a t i o n , such as were d e s c r i b e d f o r the dentate gyrus, the r o l e of s y n a p t i c t r a n s m i t t e r f o r a p a r t i c u l a r com-pound i s strengthened g r e a t l y . Another c o n s i d e r a t i o n which must be borne i n mind w i t h experiments such as those d e s c r i b e d here i s the i n f l u e n c e of sampling b i a s , mentioned a l s o i n Chapter I. The r e s u l t s r e p o r -ted i n Chapter I I I were ob t a i n e d from a sample of somatosensory c o r t i c a l and VB thalamic neurones which responded to ACh, and t h e r e f o r e do not n e c e s s a r i l y c o n s t i t u t e a r e p r e s e n t a t i v e sampling o f the t o t a l p o p u l a t i o n , f o r o n l y those c e l l s which were e x c i t e d by ACh were chosen f o r study. T h i s p o i n t a p p l i e s e q u a l l y to the experiments w i t h the amines, o u t l i n e d i n Chapter V. 216 The experiments w i t h the amino a c i d s possessed a somewhat d i f f e r e n t emphasis from the o t h e r s , i n t h a t the r e c e p t o r s f o r these compounds demonstrated a r e l a t i v e r a t h e r than an absolute a f f i n i t y f o r the range o f e x c i t a n t s t e s t e d . The c o n c l u s i o n s from these experiments were based t h e r e f o r e upon r e l a t i v e d i f -f e rences i n s u s c e p t i b i l i t y to the a n t a g o n i s t s , r a t h e r than upon an a b s o l u t e e f f e c t such as i s encountered, f o r example, wit h the c h o l i n e r g i c systems. A l a s t c o n s i d e r a t i o n r e l e v a n t to the problems f a c e d when u s i n g i o n t o p h o r e s i s , i s the p o s s i b l e i n f l u e n c e of neuronal morphology on the responses to the a d m i n i s t e r e d a g o n i s t s and a n t a g o n i s t s . As seen i n the experiments on granule c e l l s i n the dentate gyrus (Chapter IV, p a r t 2), d i f f e r e n t i a l s e n s i t i -v i t y o f a g o n i s t s may r e f l e c t topographic d i f f e r e n c e s i n i n n e r -v a t i o n ; i t was there suggested t h a t the g r e a t e r apparent potency of a s p a r t a t e r e l a t i v e to glutamate on these c e l l s c o u l d be due to the g r e a t e r d i s t a n c e over which glutamate had to d i f f u s e i n order f o r i t to r e a c t w i t h d i s t a l l y - l o c a t e d s y n a p t i c r e c e p t o r s . I t may be t h a t a s i m i l a r phenomenon accounts f o r the d i f f e r e n c e s between n i c o t i n i c and m u s c a r i n i c a c t i o n s on c e n t r a l neurones. Perhaps n i c o t i n i c e x c i t a n t s e x e r t t h e i r e f f e c t s o n l y on c h o l i n e r g i c r e c e p t o r s l o c a t e d on or near to the c e l l body. Compounds such as A6MC must d i f f u s e f u r t h e r i n order to e x c i t e the neurone, s i n c e the " m u s c a r i n i c " r e c e p t o r s may be 217 located d i s t a l l y to the c e l l body. I f this s i t u a t i o n accounts for the differences of onset and o f f s e t latencies observed with the n i c o t i n i c and muscarinic agonists, there remains neverthe-less a r e a l pharmacological d i s t i n c t i o n between these two classes of compounds. 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