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Studies on noradrenergic supersensitivity of the cyclic AMP response in rat cerebral cortex Kallstrom, Elizabeth 1979

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STUDIES ON NORADRENERGIC SUPERSENSITIVITY OF THE CYCLIC AMP RESPONSE IN RAT CEREBRAL CORTEX by E l i z a b e t h K a l l s t r o m M.Sc, The U n i v e r s i t y of B r i t i s h Columbia, 1979 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n 'THE FACULTY ,OF GRADUATE STUDIES (Department of Pathology) We accept t h i s t h e s i s as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA October 1979 E l i z a b e t h K a l l s t r o m , 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 t h e r e q u i r e m e n t s an a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h t h e L i b r a r y s h a l l m a k e 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 a n d s t u d y . I f u r t h e r a g r e e 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 p u r p o s e s may be g r a n t e d by t h e H e a d o f my D e p a r t m e n t o 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 o f 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 n o t 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 . D e p a r t m e n t o f Pathology  The U n i v e r s i t y o f B r i t i s h C o l u m b i a 2075 Wesbrook P l a c e Vancouver, Canada V6T 1W5 D a t e October 15. 1979 6 ABSTRACT I n t r a c e r e b r a l i n j e c t i o n s o f the n e u r o t o x i n 6-OHDA i n t o t h e d o r s a l b u n d l e (DB) c a u s e s s e l e c t i v e d e p l e t i o n o f c o r t i c a l n o r a d r e n a l i n e (NA) s t o r e s . The c o r t i c a l n e u r o n s may t h e n d e v e l o p s u p e r s e n s i t i v i t y t o NA and t h i s may be m e a s u r a b l e by t h e l e v e l o f cAMP a c c u m u l a t i o n . S e v e n days was c h o s e n as a p e r i o d o f t i m e f r o m i n j e c t i o n to the d e v e l o p m e n t o f t h e s u p e r s e n s i t i v e r e s p o n s e , and t e n weeks was t a k e n as t h e l o n g - t e r m p e r i o d t o m e a s u r e p e r m a n e n t e f f e c t s o f t h i s t r e a t m e n t . A t s e v e n days t h e r e was a s i g n i f i c a n t i n c r e a s e i n m a x i m a l s t i m u l a t i o n and a s l i g h t , b u t n o t s i g n i f i c a n t , s h i f t o f t h e d o s e -r e s p o n s e c u r v e . The b a s e l i n e v a l u e s o f cAMP r e m a i n e d u n c h a n g e d . The e f f e c t o f t h e cAMP s y s t e m a f t e r t e n weeks p o s t - i n j e c t i o n c o n s i s t e d o f a s i g n i f i c a n t s h i f t o f t h e d o s e - r e s p o n s e c u r v e to the l e f t , c o r r e s p o n d i n g t o a l o w e r i n g o f K p , and a s i g n i f i c a n t i n c r e a s e i n b o t h b a s e l i n e and m a x i m a l s t i m u l a t i o n l e v e l s , o r V , o f cAMP. max The v e r y h i g h r e s p o n s i v e n e s s o f t h e a d e n y l a t e c y c l a s e s y s t e m d u r i n g t h e end o f t h e s e c o n d p o s t - n a t a l week was c h a r a c t e r i z e d by h i g h e r b a s e l i n e l e v e l s o f cAMP and g r e a t e r cAMP a c c u m u l a t i o n i n r e s p o n s e t o a l l NA c o n c e n t r a t i o n s t e s t e d . H o w e v e r , t h e r e was no s i g n i f i c a n t s h i f t o f t h e d o s e - r e s p o n s e c u r v e . K i n d l i n g had no e f f e c t on the N A - s t i m u l a t e d cAMP r e s p o n s e , s h o w i n g u n c h a n g e d b a s a l and m a x i m a l s t i m u l a t i o n l e v e l s i n b o t h a n t e r i o r and p o s t e r i o r c o r t i c a l s l i c e s . T h e s e r e s u l t s a r e d i s c u s s e d i n terms o f o u r p r e s e n t know-l e d g e o f t h e r o l e o f cAMP as a component o f t h e p o s t - s y n a p t i c r e c e p t o r c o m p l e x . - i i i -TABLE OF CONTENTS ABSTRACT i i TABLE OF CONTENTS i i i LIST OF FIGURES v LIST OF ABBREVIATIONS v i i ACKNOWLEDGEMENT i x I . INTRODUCTION 1 1. Catecholamine Systems 1 a) Descending NA path 1 b) Ascending NA path 3 c) NA paths o r i g i n a t i n g i n LC 3 2. Some Well Known Drugs Which A f f e c t Noradrenergic and Dopaminic 5 Systems 3. cAMP and Neural Function 8 4. S u p e r s e n s i t i v i t y 16 5. cAMP i n Disease 24 a) Ethanol, Morphine and cAMP 24 b) Mental Disease 27 c) E p i l e p s y 30 d) Miscellaneous C l i n i c a l Conditions 33 e) cAMP and C l i n i c a l Chemistry 33 6. Assay Methods for cAMP 33 a) Phosphorylase A c t i v a t i o n Assay 34 b) Enzymatic C y c l i n g Procedures 34 c) P r o t e i n Kinase Assay 35 d) P r o t e i n Binding Assays 35 e) P r e l a b e l l i n g Techniques 36 7. Statement of the Problem 37 - i v -I I . EXPERIMENTAL PROCEDURE 39 1. M a t e r i a l s 39 2. Methods 39 a) Treatment of Animals 39 i ) K i n d l i n g Procedure 39 i i ) 6-OHDA I n j e c t i o n s 40 i i i ) FLA-63 I n j e c t i o n s 41 b) Treatment of B r a i n Tissue 41 i ) Tissue preparation 41 i i ) Incubation Procedure and I s o l a t i o n of cAMP 42 i i i ) Determination of cAMP 43 i v ) Measurement of NA l e v e l s 45 v) P r o t e i n Determination 48 3. A n a l y s i s of Data 48 I I I . RESULTS 50 1. FLA-63 50 2. Development of Method and E a r l y Findings 51 3. Kindl e d Rats 63 4. Developmental E f f e c t on the cAMP System 68 5. S u p e r s e n s i t i v i t y 70 IV. DISCUSSION 76 V. REFERENCES 85 VI. APPENDIX 94 1. Table I 94 2. Table I I 95 - v -LIST OF FIGURES 1. S a g i t t a l p r o j e c t i o n s of NA pathways 2 2. S a g i t t a l p r o j e c t i o n s of DA pathways 4 3. Schematic diagram of synaptic connections 6 4. Proposed r o l e f o r cAMP and p r o t e i n phosphorylation 11 5. Schematic diagram of noradrenergic nerve ending 13 6. Dose-response curve of 6 hr FLA-63-treated animals 52 7. Dose-response curve of 24 hr FLA-63-treated animals 53 8. Dose-response curve of animals s a c r i f i c e d by c e r v i c a l f r a c t u r e 54 and ether 9. Standard curve f or the determination of cAMP content at 1:200 57 d i l u t i o n 10. Standard curve for the determination of cAMP content at 1:60 58 d i l u t i o n 11. Time course of the s t i m u l a t i o n of cAMP formation by NA 60 12. The response of d i f f e r e n t c o r t i c a l regions to NA 61 13. The response of d i f f e r e n t c o r t i c a l regions to NA with l e f t and 62 r i g h t hemispheres combined 14. R e l a t i v e recovery of NA from d i f f e r e n t alumina pH 64 15. R e l a t i v e i n t e n s i t y of spectrophotofluorimeter readings 65 16. S t i m u l a t i o n of cAMP formation by NA i n a n t e r i o r cortex of 66 k i n d l e d animals 17. S t i m u l a t i o n of cAMP formation by NA i n p o s t e r i o r cortex of 67 k i n d l e d animals - vi -Dose-response curve of 15 day o l d animals Dose-response curve of v e h i c l e - i n j e c t e d animals Dose-response curve of 7 day 6-OHDA-injected animals Dose-response curve of 10 week 6-OHDA-injected animal - v i i -LIST OF ABBREVIATIONS Ach — a c e t y l c h o l i n e Ct-MT - alpha-methyl-p-tyrosine A-P - a n t e r i o - p o s t e r i o r ATP - adenosine triphosphate BSA - bovine serum albumin CA - catecholamine cAMP - c y c l i c 3 1,5 1-adenosine monophosphate cGMP - c y c l i c 3 5 1 - g u a n o s i n e monophosphate CNS - c e n t r a l nervous system CPZ - chlorpromazine DA - dopamine DB - d o r s a l bundle DPH - diphenylhydantoin DPG - 3,4-dihydroxyphenyl glucolaldehyde E C 5 0 - concentration of drug producing half-maximal response EPSP - f a s t e x c i t a t o r y post-synaptic p o t e n t i a l 5-HT - s e r o t o n i n GABA - Y -aminobutyric a c i d [ 3H] - t r i t i a t e d compound LC - locus coeruleus M - molar MFB - medial f o r e b r a i n bundle M-L _ m e d i o - l a t e r a l - v i i i -NA - noradrenaline PDE - phosphodiesterase PK - p r o t e i n kinase PKI - p r o t e i n kinase i n h i b i t o r PPi - inorganic pyrophosphate SCG - superior c e r v i c a l ganglion s-IPSP - slow i n h i b i t o r y post-synaptic p o t e n t i a l 6-OHDA - 6-hydroxydopamine SN - s u b s t a n t i a n i g r a TB - toothbar TCA - t r i c h l o r o a c e t i c a c i d TFP - t r i f l u o p e r a z i n e - i x -ACKNOWLEDGEMENTS I would l i k e to express my sincere g r a t i t u d e to Dr. E.G. McGeer, whose advice, encouragement and patience made the completion of t h i s t h e s i s p o s s i b l e . I would l i k e to thank Dr. S.W. French f or i n t r o d u c i n g me to the t o p i c of t h i s t h e s i s . I am a l s o indebted to Dr. J . Nagy who provided many va l u a b l e suggestions and ideas during the experiments. F i n a n c i a l a s s i s t a n c e from the N a t i o n a l I n s t i t u t e of Health (grant to Dr. S.W. French), U.B.C. Summer Studentship and the Department of Pathology are g r a t e f u l l y acknowledged. F i n a l l y I would l i k e to thank Dr. R.H. Pearce and the Department of Pathology f o r t h e i r understanding, a d m i n i s t r a t i v e f l e x i b i l i t y and a s s i s t a n c e i n the completion of t h i s t h e s i s . - x -"Problems worthy of attack prove t h e i r worth by h i t t i n g back." P i e t Hein - 1 -I. INTRODUCTION This thesis is concerned with the possible role of noradrenaline-stimulated adenylate cyclase a c t i v i t y i n various pathological conditions such as epilepsy and the development of tolerance or addiction to c e r t a i n drugs such as ethanol. Adenylate cyclase is the name for the enzyme or enzymes which catalyze the reactions: Mg2+ ATP > cAMP + PPi Noradrenaline-stimulated adenylate cyclase refers to that form of the enyzme which shows increased a c t i v i t y i n the presence of noradrenaline. In this introduction the anatomy of cen t r a l noradrenergic and dopaminergic systems w i l l f i r s t be described b r i e f l y as well as the i n t e r a c t i o n of various well known drugs with these systems i n so far as such information is necessary for the sections that follow on the possible roles of cAMP i n neural function, s u p e r s e n s i t i v i t y and pathology. 1. Catecholamine Systems The anatomy of cen t r a l noradrenergic and dopaminergic systems was i n i t i a l l y demonstrated by using histochemical fluorescent techniques and confirmed and extended by le s i o n and immunohistochemical studies (1,2). The Ungerstedt (3) map describes the following noradrenergic (NA) neuronal systems i n the rat bra i n ( F i g . 1): a) Descending NA path: The most caudal c e l l group (Al) gives r i s e to one system of fibers des-cending i n the anterior funiculus and ve n t r a l part of the l a t e r a l funiculus to terminate i n the ventral horn, and a second system descending i n the dorsal part of the l a t e r a l funiculus to terminate i n the dorsal horn. F i g . 1. S a g i t t a l p r o j e c t i o n s of ascending NA pathways i n the r a t . The descending pathways are not included. The s t r i p e s i n d i c a t e the major nerve terminal areas. (From Ungerstedt 1971). - 3 -b) Ascending NA path ("ventral bundle"): C e l l bodies i n the pons and medulla oblongata ( A l , A2, A5, A7) give r i s e to one of the major ascending NA pathways. The axons of t h i s v e n t r a l bundle t r a v e l through the r e t i c u l a r formation, continue through the MFB and terminate i n the lower b r a i n stem, mesencephalon and diencephalon. This system innervates the whole hypothalamus, arcuate and supraoptic n u c l e i , p r e o p t i c area and v e n t r a l part of s t r i a t e r m i n a l i s . c) NA paths o r i g i n a t i n g i n LC: The locus coeruleus, A6, gives r i s e to three d i f f e r e n t NA systems inne r -v a t i n g almost a l l areas of the b r a i n . One NA t r a c t descends from A6 to innervate lower b r a i n stem n u c l e i . A second t r a c t t r a v e l s l a t e r a l l y to enter the cerebellum and terminates i n the c e r e b e l l a r cortex. A t h i r d t r a c t , the " d o r s a l bundle", ascends i n the MFB and septum together w i t h the v e n t r a l bundle. The d o r s a l bundle gives o f f branches to the g e n i c u l a t e bodies, hypothalamus and the thalamic n u c l e i before terminating i n the c e r e b r a l cortex and hippocampus. F i b e r s i n n e r v a t i n g the hypothalamus are p a r t l y crossed, w h i l e those t r a v e l l i n g to the cortex are a l l uncrossed. L e s i o n studies i n d i c a t e that c e r t a i n b r a i n areas, such as the medial p r e o p t i c nucleus, v e n t r a l s t r i a t e r m i n a l i s , i n f e r i o r o l i v e , habenular and some parts of the thalamus, receive few i f any f i b e r s from the locus coeruleus (4-6) but t h i s nucleus i s c e r t a i n l y r e s p o n s i b l e for a l l , or almost a l l , of the c o r t i c a l noradrenergic i n n e r v a t i o n (7,8). I t i s worth re-emphasis that the NA systems to the cortex are i p s i l a t e r a l . Dopaminergic systems are not the c h i e f concern of t h i s t h e s i s but the chemistry and drug e f f e c t s are often c l o s e l y r e l a t e d and frequent mention w i l l be made of the best known and most thoroughly studied dopaminergic t r a c t . This F i g . 2. S a g i t t a l p r o j e c t i o n s of dopamine pathways i n the r a t . S t r i p e d areas i n d i c a t e dense terminal f i e l d s . (From Ungerstedt 1971). - 5 -a r i s e s i n the pigmented c e l l s of the s u b s t a n t i a n i g r a and ascends r o s t r a l l y through the medial f o r e b r a i n bundle to innervate the e n t i r e s t r i a t u m (caudate and putamen) ( F i g . 2). A s i m i l a r t r a c t a r i s e s i n the v e n t r a l tegmentum j u s t medial to the su b s t a n t i a n i g r a and innervates various l i m b i c n u c l e i , p a r t i c u l a r l y the nucleus accumbens. The other dopaminergic systems i n b r a i n are not p e r t i n e n t to t h i s t h e s i s . 2. Some Well Known Drugs Which A f f e c t Dopaminergic and Noradrenergic Systems A schematic diagram of a noradrenergic nerve ending i s shown i n F i g . 3 with an i n d i c a t i o n of the s i t e s of a c t i o n of various drugs described below. 6-Hydroxydopamine (6-OHDA) i s a s e l e c t i v e neurotoxin which, depending upon the method of a d m i n i s t r a t i o n , can be used to destroy noradrenergic and/or dopaminergic systems i n b r a i n . Because of i t s s e l e c t i v i t y , i t has been a very u s e f u l t o o l i n a wide v a r i e t y of i n v e s t i g a t i o n s on the anatomy, biochemistry, pharmacology and f u n c t i o n a l i m p l i c a t i o n s of the catecholaminergic systems ( 9 ) . 6-OHDA i s a s t r u c t u r a l analog of the catecholamines and i s accumulated i n t o catecholamine neurons by the a c t i v e uptake processes which recover much of the dopamine and noradrenaline released from such neurons (10,11). The s p e c i f i c i t y of 6-OHDA probably depends upon the s p e c i f i c i t y of neuronal uptake mechanisms - i . e . i t does not k i l l c h o l i n e r g i c or serotonergic neurons because i t i s not accumulated by them. The molecular mechanism of the neurotoxic a c t i o n of 6-OHDA remains specul-a t i v e but probably depends upon i t s easy o x i d a t i o n which may lead to o- and p-quinones (12), to peroxides or, p o s s i b l y , to the formation of a superoxide r a d i c a l . Any one of se v e r a l h i g h l y r e a c t i v e r a d i c a l s might be the neurotoxic agent (13). P a r g y l i n e B B - 6 -Amphetamine N o r a d r e n a l i n e 6-OHDA oL-methyl-p tyrosine FLA-63 Disulfiram Reserpine Cocaine Amphetamine Chlorpromazine F i g . 3. Schematic diagram of noradrenergic nerve ending. White rectangles and white arrows show endogenous metabolites and t h e i r l o c a t i o n s . Black rectangles and black arrows show the s i t e of a c t i o n of v a r i o u s noradrenergic drugs. (Modified from McGeer, Eccles and McGeer, 1978). - 1 -The neurotoxic e f f e c t s of 6-OHDA f o l l o w i n g i n t r a v e n t r i c u l a r i n j e c t i o n are i n i t i a l l y seen i n nerve terminal areas, w i t h s t r u c t u r a l damage and loss of uptake mechanisms appearing as e a r l y as two hours a f t e r the a d m i n i s t r a t i o n of the 6-OHDA (14-18). The neuronal p e r i k a r y a seem less s u s c e p t i b l e to the neurotoxic actions than the nerve endings, but both e l e c t r o n microscopic and biochemical evidence i n d i c a t e that the pe r i k a r y a i n the s u b s t a n t i a n i g r a and locus coeruleus begin to degenerate w i t h i n 1 to 2 days (19-22). Ot-Methyl-p- tyros ine i s a s e l e c t i v e i n h i b i t o r of ty r o s i n e hydroxylase, the key enzyme i n the synthesis of both dopamine and noradrenaline. Treatment of ra t s w i t h t h i s m a t e r i a l r e s u l t s i n large and s e l e c t i v e depletions of these amines from both p e r i p h e r a l and c e n t r a l stores (23). Reserpine i s an agent which destroys the cap a c i t y of v e s i c l e s to bind dopamine, noradrenaline and c e r t a i n other neurotransmitters. Lacking v e s i c u l a r p r o t e c t i o n , the released amines are metabolized r a p i d l y . Hence, reserpine causes profound depletions i n the l e v e l s of these t r a n s m i t t e r s . The e f f e c t l a s t s for s e v e r a l days a f t e r a s i n g l e dose of reserpine because the v e s i c l e b i n d i n g capacity i s i r r e v e r s i b l y destroyed and new v e s i c u l a r p r o t e i n must be synthesized and transported from the p e r i k a r y a to the nerve endings before e f f e c t i v e storage can be res t o r e d . Chlorpromazine and other phenothiazines block noradrenaline, dopamine and sero t o n i n r e c e p t o r s , w i t h d i f f e r e n t members of the s e r i e s showing some v a r i -a b i l i t y i n t h e i r a c t i v i t y at the various receptors. H a l o p e r i d o l and other butyrophenones are receptor blockers which tend to be p a r t i c u l a t l y a c t i v e at dopaminergic as opposed to noradrenergic or seroto-nergic receptors. - 8 -Amphetamine i n h i b i t s the reuptake of the catecholamines, stimulates some catecholamine release and i n h i b i t s MAO s l i g h t l y . A l l three actions tend to p o t e n t i a t e the a c t i o n of catecholamines i n the synaptic c l e f t . The t r i c y c l i c antidepressants are i n h i b i t o r s of the reuptake of noradren-a l i n e and s e r o t o n i n , w i t h some v a r i a t i o n i n the a c t i v i t y of the various drugs on the two systems. Since reuptake i s a major mechanism for removing the amines from the synaptic c l e f t , the reuptake i n h i b i t o r s p o t e n t i a t e g r e a t l y the synaptic a c t i o n of these amines. Cocaine i n h i b i t s the reuptake of noradrenaline and dopamine in t o synaptic nerve endings and thus p o t e n t i a t e s t h e i r a c t i o n s . P r o p r a n o l o l i s a 8-adrenergic receptor b l o c k e r . I s o p r o t e r e n o l i s a d i r e c t noradrenergic agonist a c t i v e at ^-adrenergic receptors. Phenoxybenzamine i s an a-adrenergic receptor b l o c k e r . Apomorphine i s a d i r e c t dopaminergic agonist. 2+ FLA-63 i s a very potent Cu c h e l a t i n g d i s u l f i d e . This drug i n h i b i t s dopamine-8-hydroxylase a c t i v i t y and causes a very r a p i d and complete d e p l e t i o n of c e n t r a l NA stores (24). 3. cAMP and Neural Function cAMP i s now be l i e v e d to be an i n t r a c e l l u l a r r e g u l a t o r y agent for a large number of c e l l u l a r processes. I t s occurrence has been demonstrated i n a l l animal species and t i s s u e s i n v e s t i g a t e d (25). The c r i t i c a l enzyme for i t s synthesis i s adenylate c y c l a s e . The highest l e v e l s of both adenylate c y c l a s e a c t i v i t y and cAMP i n most adult animals are found i n the b r a i n (26). Therefore - 9 -a great deal of a t t e n t i o n has been focussed on the problem of how cAMP functi o n s i n nervous t i s s u e . E a r l y support f or a r o l e for cAMP i n neurotransmission i n the CNS came from the discovery that e l e c t r i c a l s t i m u l a t i o n of b r a i n s l i c e s r e s u l t e d i n increased cAMP l e v e l s and from the demonstration of the s u b c e l l u l a r l o c a l i z a t i o n i n the synaptosomal f r a c t i o n of the enzymes in v o l v e d i n the cAMP generating system (27). A r o l e for cAMP has been postulated i n three aspects of neuronal func-t i o n i n g : mediation of neurotransmitter a c t i o n on postsynaptic membranes, r e g u l a t i o n of neurotransmitter b i o s y n t h e s i s (28), and f u n c t i o n i n g of micro-tubules (29). The f i r s t of these r o l e s i s the one f o r which there i s the most experimental evidence and i t i s t h i s w i t h which t h i s t h e s i s w i l l be p r i m a r i l y concerned. When neurotransmitters are released from presynaptic t e r m i n a l s , they are thought to induce a change i n postsynaptic membrane p o t e n t i a l by r e a c t i n g w i t h s p e c i f i c receptors on the postsynaptic membrane. In the case of some t r a n s -m i t t e r s and some type of rec e p t o r s , such as a c e t y l c h o l i n e at the neuromuscular j u n c t i o n or with GABA i n the c e n t r a l nervous system, the a c t i o n i s b e l i e v e d to in v o l v e a r a p i d change i n i o n i c fluxes through the membrane. In other i n s t a n c e s , i t i s b e l i e v e d that the postsynaptic a c t i o n of the neurotransmitter may be the i n d u c t i o n of a s e r i e s of chemical r e a c t i o n s which render the c e l l membrane more or less s e n s i t i v e to other inputs. Substances with t h i s type of a c t i o n have been termed by some neuromodulators (30) and by others "metabo-t r o p i c n e u rotransmitters" as opposed to the " i o n o t r o p i c n e u r o t r a n s m i t t e r s " e x e m p l i f i e d by the c l a s s i c a l case of a c e t y l c h o l i n e at the neuromuscular j u n c t i o n (31). There i s considerable evidence that the c y c l i c n u c l e o t i d e s - 10 -(cAMP and cGMP), may play key r o l e s i n the postsynaptic a c t i o n of many of these s o - c a l l e d neuromodulators or metabotropic neurotransmitters. The best evidence i s a v a i l a b l e w i t h regard to a key r o l e f o r cAMP i n the f u n c t i o n i n g of the catecholaminergic neurotransmitters, dopamine and noradrenaline, but there i s a l s o evidence that cGMP i s inv o l v e d i n the a c t i o n of a c e t y l c h o l i n e at muscarinic, as opposed to n i c o t i n i c , r e c e ptors. The i n i t i a l , and perhaps s t i l l the best, evidence f o r a receptor r o l e of cAMP comes from studies of the superior c e r v i c a l ganglion (SCG), which contains c h o l i n e r g i c p r e g a n g l i o n i c f i b e r s and a dopaminergic interneuron synapsing on the pos t g a n g l i o n i c neuron ( F i g . 4). When pr e g a n g l i o n i c f i b e r s are stimu l a t e d , a c e t y l c h o l i n e i s released at the po s t - g a n g l i o n i c n i c o t i n i c receptor causing a f-EPSP; t h i s a c t i o n probably does not i n v o l v e any c y c l i c n u c l e o t i d e . These f i b e r s , however, a l s o synapse at muscarinic receptors on the pos t g a n g l i o n i c neuron and the a c t i o n of a c e t y l -choline at these receptors causes a s-EPSP which i s b e l i e v e d to be mediated by cGMP. The preganglionic c h o l i n e r g i c f i b e r s also synapse on dopaminergic i n t e r n u n c i a l neurons; the release of dopamine from these neurons onto the a-adrenergic p o s t g a n g l i o n i c receptors generates a s-IPSP which i s thought to be mediated by cAMP (32-34). Evidence f o r such a mechanism comes from the f a c t that both e l e c t r i c a l s t i m u l a t i o n of the preganglionic f i b e r s and the a p p l i -c a t i o n of exogenous dopamine to g a n g l i o n i c s l i c e s r e s u l t i n an increased cAMP l e v e l i n the pos t g a n g l i o n i c neuron (35-37). A dopamine-sensitive adenylate cyclase has been found i n the ganglion as w e l l as i n the caudate nucleus and other areas of the CNS which c o n t a i n extensive dopaminergic i n n e r v a t i o n (38). Exogenous a p p l i c a t i o n of cAMP to the ganglion causes p o s t g a n g l i o n i c hyper-p o l a r i z a t i o n (39). The postulated sequence of steps i s i n d i c a t e d i n F i g . 5. Muiconmc Cholinergic P R E -G A N G L I O N I C C H O L I N E R G I C F I B R E S A C h b l o c k e d by h e i o m t t h o n i u m N i c o t i n i c C ^ e P O S T G A N G L I O N I C N E U R O N E b l o c k e d by o t r o p m e - I - b l o c k e d by a - o d r e n t r g i c ' a n t a g o n i s t s D O P A M I N E R° 'C\ CAMP P O S T G A N G L I O N I C S Y N A P T I C P O T E N T I A L S I N T E R N E U R O N E - | - b l o c k e d by a t r o p i n e A C h T F i g . 4. Schematic diagram of synaptic connections i n the superior c e r v i c a l , ganglion and postulated r o l e of c y c l i c n u c l e o t i d e s i n the genesis of postganglionic synaptic p o t e n t i a l s . (From Greengard 1976). - 12 -Dopamine stimulates a s p e c i f i c adenylate cyclase to produce increased l e v e l s of cAMP. The cAMP i n turn i s b e l i e v e d to stimulate the phosphorylation of s p e c i f i c substrate p r o t e i n s i n the membrane by cAMP-dependent p r o t e i n kinases (PK), such phosphorylation a f f e c t i n g membrane p e r m e a b i l i t y c h a r a c t e r i s t i c s . Such cAMP-dependent p r o t e i n kinases have been found i n microsomal f r a c t i o n s of bovine b r a i n and shown to be capable of c a t a l y z i n g the phosphorylation of endogenous membrane-bound substrate p r o t e i n s from synaptic membrane f r a c t i o n s (40,41). The s p e c i f i c i t y of cAMP a c t i o n i n d i f f e r e n t c e l l types could be accounted f o r by the s p e c i f i c i t y of various phosphokinases and the substrate p r o t e i n s . Two proteins have already been found which seem to be s p e c i f i c to nervous t i s s u e and are phosphorylated w i t h i n 5 sec by endogenous phospho-kinases a f t e r cAMP a c t i v a t i o n . These proteins are concentrated i n the synaptic membrane f r a c t i o n on s u b c e l l u l a r f r a c t i o n a t i o n and t h e i r c o n c e n t r a t i o n i n t i s -sue increases i n the immediate p o s t n a t a l period during the time when synaptic s t r u c t u r e s develop. Moreover, there i s evidence that there i s a good cor-r e l a t i o n between the degree of phosphorylation of one of these p r o t e i n s and the degree of i o n i c f l u x obtained i n response to the a c t i o n of 8-agonists (42,43). In the c e n t r a l nervous system there i s evidence that cAMP i s involved i n the postsynaptic actions of both noradrenaline and dopamine and i t i s w i t h the former catecholamine that t h i s t h e s i s i s p r i m a r i l y concerned. As i n d i c a t e d i n s e c t i o n 1-1 ( F i g . 1), the noradrenergic systems of b r a i n have very l i m i t e d s i t e s of o r i g i n i n the b r a i n stem but ramify very broadly to innervate most of the b r a i n i n c l u d i n g the cerebellum and c e r e b r a l cortex. Most of the a v a i l a b l e evidence upon the probable importance of cAMP i n the noradrenergic receptor comes from work on the cerebellum and c e r e b r a l cortex i n v i v o or i n v i t r o . Altered Microtubular Function Cyclic A Protein Kinase PRESYNAPTIC NEURON PR0TEIN-P04-ion c o n d u c t a n c e ? e l t c t r o g e n i c pump ? to I POSTSYNAPTIC NEURON / ATP 5 ' A M P F i g . 5. Proposed r o l e s f o r cAMP and p r o t e i n phosphorylation i n neuronal f u n c t i o n . A key element of t h i s model i s that the second messenger, dopamine, a f f e c t s the membrane p o t e n t i a l of the c e l l through the p r o t e i n kinase system and the substrate p r o t e i n c o n t r o l s the permeability of the post-synaptic membrane. (From Greengard 1976). - 14 -In v i v o i t has been shown that the spontaneous discharge of c e r e b e l l a r P u r k i n j e c e l l s i s i n h i b i 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 s of noradrenaline or cAMP which cause a h y p e r p o l a r i z a t i o n of the P u r k i n j e c e l l membrane. The i n h i b i t o r y e f f e c t of noradrenaline on P u r k i n j e c e l l s i s p o t e n t i a t e d by phospho-d i e s t e r a s e i n h i b i t o r s ; such i n h i b i t i o n i s co n s i s t e n t w i t h a mediation by cAMP since degradation of cAMP i s accomplished by a phosphodiesterase ( F i g . 5) (44). S i m i l a r l y , 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 noradrenaline or cAMP to pyramidal t r a c t neurons of the c e r e b r a l cortex cause an i n h i b i t i o n of these neurons, while a p p l i c a t i o n of e i t h e r a c e t y l c h o l i n e or cGMP r e s u l t s i n pyramidal e x c i t a t i o n (45,46). In i n v i t r o work, i t has been shown that incubation of s l i c e s of r a t c e r e b r a l c o r t e x w i t h 10 noradrenaline causes a two-fold increase i n the cAMP l e v e l and that t h i s e f f e c t can be enhanced by pretreatment with r e s e r p i n e . -4 Maximal s t i m u l a t i o n occurred at 10 M noradrenaline a f t e r a s i x minute i n c u b a t i o n (47). S i m i l a r increases i n cAMP l e v e l s on in c u b a t i o n w i t h nor-adrenaline have also been found i n c e r e b r a l c o r t i c a l s l i c e s from guinea pigs (48), r a b b i t s (49,50), and humans (51). The p r e f e r r e d time of in c u b a t i o n v a r i e d from 2 to 6 min and the concentrations g i v i n g maximal response from - 5 - 4 . . . approximately 10 to 10 M. In some species, such as the guinea p i g , cAMP l e v e l s i n the s l i c e s a l s o increased on incu b a t i o n w i t h other biogenic amines such as histamine and se r o t o n i n (48); i n s l i c e s from other species such as the human (51) and r a t (48), these other neurotransmitters had no e f f e c t . I t seems probable, i n any case, that the e f f e c t s of d i f f e r e n t t r a n s m i t t e r s are not mediated through e x a c t l y the same adenylate cyclase but e i t h e r through d i f f e r e n t forms of the enzyme or through the same enzyme l i n k e d i n v a r y i n g fashion to other components of the membrane which confer receptor s p e c i f i c i t y . cAMP formation i n b r a i n s l i c e s can a l s o be stimulated by other m a t e r i a l s which are not now ge n e r a l l y accepted as neurotransmitters. Adenosine, f o r example, which has been proposed as a neurotransmitter but i s not yet g e n e r a l l y accepted, can apparently also stimulate cAMP formation by a c t i v a t i o n of mem-brane receptors; i t can al s o be incorporated i n t o the cAMP precursor pool (52). D e p o l a r i z i n g agents, such as potassium, v e r a t r i d i n e and ouabain, a l s o stimulate cAMP production and i t has been suggested that these may act through r e l e a s e of adenosine as an intermediary process. D e p o l a r i z i n g agents cause an increase i n i n t r a c e l l u l a r sodium concentration and t h i s e f f e c t can be blocked by mem-brane s t a b i l i z e r s such as cocaine which prevent both the increase i n sodium p e r m e a b i l i t y and the r i s e i n cAMP l e v e l s (53). In human b r a i n s l i c e s , the e f f e c t s of noradrenaline and v e r a t r i d i n e on cAMP l e v e l s are reported to be a d d i t i v e (54). Sodium f l u o r i d e i s another agent which w i l l s t i m ulate increases i n cAMP formation i n b r a i n homogenates; i n t h i s case, however, adenosine has been reported to i n h i b i t the e f f e c t (55). Increases i n cAMP l e v e l s induced by e i t h e r e l e c t r i c a l s t i m u l a t i o n or 2+ d e p o l a r i z i n g agents are dependent upon the presence of Ca . Low concen-2+ . . . t r a t i o n s of Ca can, by themselves, stimulate cAMP accumulation, while high concentrations have an i n h i b i t o r y e f f e c t (56). E x c i t a t i o n of c e l l s r e s u l t s i n 2+ increased i n t r a c e l l u l a r concentrations of Ca and these increased concen-t r a t i o n s r e s u l t i n a c t i v a t i o n of the phosphodiesterase a c t i v i t y which provides a r a p i d c o n t r o l mechanism for cAMP l e v e l s (57,58). As might be expected f o r an enzyme b e l i e v e d to be invo l v e d i n t i m a t e l y i n neuronal transmission, adenylate cyclase a c t i v i t y i n b r a i n changes with matur-a t i o n . In r a t s , f o r example, the c e r e b r a l cortex undergoes considerable post-- 16 -n a t a l development reaching adult morphology at about 15 days of age (59). Basal adenylate cyclase a c t i v i t y i n t h i s s t r u c t u r e increases approximately f o u r - f o l d from b i r t h to about 21 days of age w h i l e the maximum response to the 2+ catecholamines, to Ca and to sodium f l u o r i d e i s reached at approximately two weeks (60). Receptor s p e c i f i c i t y a l s o seems to develop p o s t n a t a l l y ; c o r t i c a l adenylate cyclase of newborn r a t s responds to both dopamine and sero t o n i n but t h i s responsiveness decreases w i t h age. Responsiveness to noradrenaline i s markedly decreased i n senescent animals (61); t h i s probably r e f l e c t s a l o s s of d e n d r i t i c spines and the synaptic contacts thereon. I t i s only part of the general p a t t e r n which i s beginning to emerge i n d i c a t i n g a lo s s of neuronal f u n c t i o n i n g i n normal aging that may account f o r many of the decrements i n behavior and changes i n pharmacological s e n s i t i v i t y which have been noted i n senescent animals and humans. 4. S u p e r s e n s i t i v i t y S u p e r s e n s i t i v i t y has been defined as the phenomenon i n which the amount of an agonist required to produce a given response i s le s s than normal. I t i s measured by a s h i f t of the dose-response curve for that agonist to the l e f t . This s h i f t may sometimes be accompanied by an increase i n the maximum response and/or a change i n the slope of the dose-response curve. S u b s e n s i t i v i t y i s the opposite phenomenon with the opposite m a n i f e s t a t i o n s . Cannon's 'Law of Denervation' (62) has been modified by Fleming (63) to become the 'Law of Inne r v a t i o n ' : "When f u n c t i o n a l nerve a c t i v i t y i s c h r o n i c a l l y increased or decreased ( s u r g i c a l l y , p h y s i o l o g i c a l l y , p a t h o l o g i c a l l y or pha r m a c o l o g i c a l l y ) , the s e n s i t i v i t y of most d i s t a l e f f e c t o r s to any process which i n i t i a t e s a response i n the e f f e c t o r i s slowly a l t e r e d i n a d i r e c t i o n which w i l l compensate for the a l t e r e d neural input". The procedures by which s u p e r s e n s i t i v i t y can be achieved i n c l u d e : 1) s u r g i c a l denervation and d e c e n t r a l i z a t i o n , 2) pharmacological denervation, 3) blockade of receptor or e f f e c t o r organ, 4) prevention of release of t r a n s m i t t e r , 5) a l t e r a t i o n of sensory s t i m u l i , 6) s u r g i c a l a b l a t i o n of a f f e r e n t pathways. The f i r s t t i s s u e f o r which a mechanism of s u p e r s e n s i t i v i t y was proposed was s k e l e t a l muscle (64). Only the end p l a t e i s s e n s i t i v e to acetycholine i n an innervated muscle, but s e v e r a l days a f t e r denervation, the area of s e n s i -t i v i t y slowly spreads outward u n t i l the e n t i r e surface of the f i b e r responds to a c e t y l c h o l i n e . S u p e r s e n s i t i v i t y was p o s t u l a t e d to: (a) be due to a spread of receptors, (b) represent a return to a pre-innervated state of the muscle, and (c) to be due to a loss of contact between t r a n s m i t t e r and end organ. S u p e r s e n s i t i v i t y i n s k e l e t a l muscle i s f u r t h e r c h a r a c t e r i z e d by an increased membrane r e s i s t a n c e and capacitance, an increase i n the duration of the a c t i o n p o t e n t i a l and a s p e c i f i c i t y f o r a c e t y l c h o l i n e (65). Denervated s k e l e t a l muscle w i l l accept i n n e r v a t i o n by transplanted nerve, w h i l e innervated muscle w i l l not (66). S u p e r s e n s i t i v i t y i n smooth muscle, u n l i k e that i n s k e l e t a l muscle, i s not s p e c i f i c f o r a s i n g l e t r a n s m i t t e r . I t depends on a change i n p e r m e a b i l i t y or 2+ c o n t r a c t i l e mechanisms and a change i n Ca b i n d i n g (67). Exocrine glands a l s o e x h i b i t s u p e r s e n s i t i v i t y , which tends to resemble that i n smooth muscle rather than that i n s k e l e t a l muscle. S a l i v a r y glands, f o r example, e x h i b i t - 18 -no n - s p e c i f i c s u p e r s e n s i t i v i t y . Furthermore, when the gland i s exposed to increased amounts of a c e t y l c h o l i n e by the a d m i n i s t r a t i o n of a c h o l i n e s t e r a s e i n h i b i t o r , such as physostigmine, the gland becomes s u b s e n s i t i v e . S u p e r s e n s i t i v i t y to noradrenaline i n v o l v i n g changes i n the cAMP system has been e x t e n s i v e l y studied i n the p i n e a l gland. The p i n e a l gland i s under c i r c a d i a n i n f l u e n c e , so that during the dark c y c l e noradrenergic input from the SCG increases. NA a c t i v a t e s a cAMP system which, i n t u r n , a c t i v a t e s an N- a c e t y l t r a n s f e r a s e that i s a r a t e - c o n t r o l l i n g enzyme i n the b i o s y n t h e s i s of melatonin. L i g h t , denervation, ganglionectomy and 6-OHDA i n j e c t i o n a l l produce a s u p e r s e n s i t i v e cAMP response to NA by d e p l e t i n g the noradrenergic input (68). S u p e r s e n s i t i v i t y of the cAMP system i s accompanied by a super-i n d u c t i o n of the N- a c e t y l t r a n s f e r a s e (69,70). The enzyme i n d u c t i o n , but not the cAMP response, i s dependent on p r o t e i n s y n t h e s i s . The s u p e r s e n s i t i v i t y response can be abolished by i n j e c t i o n s of the NA agonist, i s o p r o t e r e n o l , or of NA i t s e l f . Repeated i n j e c t i o n s of i s o p r o t e r e n o l or extended periods of dark render the p i n e a l cAMP system s u b s e n s i t i v e . The development of s e n s i t i v i t y changes i n the p i n e a l gland i s very r a p i d . S u b s e n s i t i v i t y , a f t e r i s o -p r o t e r e n o l a d m i n i s t r a t i o n , develops a f t e r 6 hours and s u p e r s e n s i t i v i t y , a f t e r denervation, w i t h i n 24 hours. The changes i n s e n s i t i v i t y of the cAMP generating system are p a r a l l e l e d by changes i n the number of $-adrenergic b i n d i n g s i t e s (71,72). Measurement of the number and a f f i n i t y of sodium-independent, saturable b i n d i n g s i t e s i s now widely accepted as a biochemical index of the density and s e n s i t i v i t y of receptors (73). Although i t i s w e l l e s t a b l i s h e d that denervation produces s u p e r s e n s i t i v i t y i n p e r i p h e r a l e f f e c t o r organs, i t i s not p o s s i b l e to extr a p o l a t e p e r i p h e r a l mechanisms to the CNS. Sharpless (74) asks what kind of f u n c t i o n a l changes - 19 -would be expected i n CNS pathways i f neurons responded to disuse. P e r i p h e r a l s t r u c t u r e s are r e l a t i v e l y simple to study, while CNS s t r u c t u r e s are very complicated, w i t h elaborate feed-back inputs on each c e l l . However, there are f u n c t i o n a l as w e l l as neurochemical observations suggesting that CNS systems also respond to disuse. The temperature-regulating center of the hypothalamus slowly develops a r e v e r s i b l e s u p e r s e n s i t i v i t y as a r e s u l t of scopolamine admin-i s t r a t i o n (75). F r o n t a l lobectomy r e s u l t s i n an increase i n c o n v u l s i b i l i t y and thalamic l e s i o n s r e s u l t i n h y p e r e x c i t a b i l i t y of the i p s i l a t e r a l c o r t e x (76). Many be h a v i o r a l reports do not define the t r a n s m i t t e r systems which are i n v o l v e d i n the s u p e r s e n s i t i v i t y phenomenon. There are, however, few data i n d i c a t i n g the development of s u p e r s e n s i t i v i t y to other t r a n s m i t t e r substances but the most extensive and p e r t i n a n t l i t e r a t u r e , however, i s concerned w i t h changes i n receptor s e n s i t i v i t y to dopamine i n the s t r i a t u m and to nor-adrenaline i n the c e r e b r a l cortex. There are considerable b e h a v i o r a l (77), p h y s i o l o g i c a l (78) and biochemical (79,80) data i n d i c a t i n g s u p e r s e n s i t i v i t y of s t r i a t a l dopaminergic receptors a f t e r denervation or chronic treatment w i t h amine-depleting or b l o c k i n g agents. Most groups who have studied the problem f i n d that s u p e r s e n s i t i v i t y i s accompanied by an enhanced response of adenylate cyclase i n the s t r i a t a l homogenates to exogenous dopamine (81-83) but there i s at l e a s t one group who has f a i l e d to f i n d any change i n dopamine-stimulated adenylate cyclase a c t i v i t y i n r a t s showing be h a v i o r a l s u p e r s e n s i t i v i t y (84). Studies of p o s s i b l e changes i n s e n s i t i v i t y i n noradrenergic receptors have been more dependent upon chemical as opposed to b e h a v i o r a l measures because there has been no c l e a r d e f i n i t i o n of the b e h a v i o r a l r o l e of the c e r e b r a l c o r t i c a l noradrenergic systems. S u p e r s e n s i t i v i t y , as measured by an enhanced response of c o r t i c a l adenylate cyclase a c t i v i t y to noradrenaline i n ^n v i t r o - 20 -incub a t i o n s , has been found i n r a t s f o l l o w i n g denervation by a b l a t i o n of the locus coeruleus (85), chemical l e s i o n s w i t h 6-OHDA (86,87), or treatment w i t h reserpine (93). The s u p e r s e n s i t i v e response i s s p e c i f i c f o r a and 8 a g o n i s t s , r e v e r s i b l e and cha r a c t e r i z e d by a s h i f t to the l e f t of the dose-response curve. The i n t e r p r e t a t i o n of the data i n le s i o n e d animals can be complex at c e r t a i n times a f t e r the l e s i o n s because of lesion-induced changes i n the presynaptic nerve t e r m i n a l s . This i s p a r t i c u l a r l y true w i t h 6-OHDA l e s i o n s , because of the pronounced s e n s i t i v i t y of the terminals to 6-OHDA (see s e c t i o n 1-2). Noradrenaline i s g e n e r a l l y removed from the synaptic c l e f t l a r g e l y by the high a f f i n i t y (uM range) neuronal reuptake system. An e a r l y e f f e c t of 6-OHDA l e s i o n i n g i s the degeneration of the nerve terminals w i t h impairment of the amine uptake mechanisms. One consequence i s that exogenous noradrenaline added to incubated s l i c e s may remain i n contact w i t h the postsynaptic receptors longer than would be the case i n s l i c e s with i n t a c t nerve terminals. This may r e s u l t i n an apparent " s u p e r s e n s i t i v i t y " . This presynaptic e f f e c t i s s i m i l a r to that o c c u r r i n g i f cocaine (an uptake i n h i b i t o r ) i s added to the incubates and develops 24 to 72 hours a f t e r production of the l e s i o n i n 6-OHDA-treated animals. The true s u p e r s e n s i t i v i t y which i s due to postsynaptic changes i s not p o t e n t i a t e d by cocaine and was found to develop 72 to 96 hours a f t e r 6-OHDA-treatment (89). K a l i s k e r e_t a l . (89) d i d f u r t h e r studies on the e a r l y and l a t e components of the changes i n the cAMP system i n the cor t e x f o l l o w i n g 6-OHDA-induced l e s i o n s . Consistent with the postulate that the e a r l y component r e f l e c t s f a i l u r e of the presynaptic reuptake mechanisms, they found that the e a r l y p o t e n t i a t i o n of the s t i m u l a t i o n of cAMP formation was not apparent w i t h i s o p r o t e r e n o l , was l i m i t e d to low NA concentrations and followed the same time course as the reduction of high a f f i n i t y [ JH]-NA accumulation. I s o p r o t e r e n o l i s a NA agonist which i s not accumulated by the high a f f i n i t y uptake system. High concentrations of NA (30 uM) accumulate by a low a f f i n i t y uptake system which i s not i n h i b i t e d by cocaine and does not in v o l v e s p e c i f i c , presynaptic transport systems. The e f f e c t s of both i s o p r o t e r e n o l and NA on cAMP formation were p o t e n t i a t e d by 72 to 96 hrs a f t e r the l e s i o n , i n d i c a t i n g the postsynaptic mechanism at that time. I t was not c l e a r , however, from K a l i s k e r ' s data, whether the postsynaptic e f f e c t i n v o l v e d a or $ rece p t o r s , as both phentol-amine and pro p r a n o l o l i n h i b i t e d the e f f e c t to the same degree. Recent reports suggest that 8 receptors are more l i k e l y than a to be involved i n the changes i n responsiveness of the cAMP-generating system a f t e r 6-OHDA treatment. Membrane preparations of r a t c e r e b r a l cortex were incubated 3 3 wit h the a and 8 r a d i o l i g a n d s , [ H]-WB-4101 and [ H]-dihydroalprenolone r e s p e c t i v e l y , a f t e r i n t r a v e n t r i c u l a r i n j e c t i o n s of 6-OHDA. Treated animals e x h i b i t e d a greater degree of 8 l i g a n d binding (90). The slopes of the binding curves were i d e n t i c a l f o r c o n t r o l and treated animals, p o i n t i n g to an increase i n the number of b i n d i n g s i t e s of a s i n g l e h i g h - a f f i n i t y receptor c l a s s . The time course of the increase i n receptor density c o r r e l a t e d w e l l w i t h the time course of the increase i n NA-stimulated cAMP l e v e l s . Receptor density s t a r t e d to increase a f t e r 4 days and reached a maximum of 150% a f t e r 16 days. NA-stimulated cAMP was 150% of c o n t r o l l e v e l s a f t e r 4 days and reached a maximum of 200% a f t e r 11 days, at which time the EC,.^  had also reached a minimum (91). Both studies (90,91) show a discrepancy between the percent increase i n receptor d e n s i t y (25 to 50%) and the percent increase i n the cAMP response (approx. 100%). The reason f o r t h i s remains unclear but i t may w e l l depend upon the f a c t that the r a d i o a c t i v e ligands used bind to - 22 -both the postsynaptic receptor and presynaptic autoreceptors while cAMP i s asso c i a t e d w i t h only the postsynaptic receptors. There i s l i t t l e evidence f o r t h i s d i s t i n c t i o n i n c o r t i c a l noradrenergic systems but e x c e l l e n t evidence i n s t r i a t a l dopaminergic systems. The a d m i n i s t r a t i o n of 6-OHDA to r a t s on the f i r s t day a f t e r b i r t h prevents the development of presynaptic nerve t e r m i n a l s , but does not prevent the development of 8 noradrenergic receptors. The time course of the increase i n 8 receptors c o r r e l a t e d w e l l w i t h the time course of the development of the i s o p r o t e r e n o l - s t i m u l a t e d cAMP response, both reaching adult l e v e l s on day 16 i n both c o n t r o l and 6-OHDA-treated animals. The 6-OHDA-treated animals showed a 45 to 75% higher receptor density and a 40 to 65% higher cAMP response (92) which i s another instance of s u p e r s e n s i t i v i t y developing as a consequence of disuse. S u b s e n s i t i v i t y of NA-stimulated cAMP accumulation i n c o r t i c a l s l i c e s has also been demonstrated i n mice or r a t s treated w i t h an agent (d-amphetamine or a t r i c y c l i c antidepressant) which would cause excessive noradrenergic s t i m u l a t i o n (93). In studies on p e r i p h e r a l organs, such as the cat n i c t i t a t i n g membrane, Trendelenburg described what he c a l l e d two q u a l i t a t i v e l y d i f f e r e n t types of s u p e r s e n s i t i v i t y (94). One type was produced by d e c e n t r a l i z a t i o n (pre-g a n g l i o n i c blockade of the neuronal input) and r e s u l t e d i n a moderate degree of n o n - s p e c i f i c s u p e r s e n s i t i v i t y which developed rather slowly over 7-14 days. The other type was produced by denervation and had two components. The e a r l y component resembled the e f f e c t of cocaine and developed r a p i d l y w i t h i n 24-48 hours. The l a t e component was s i m i l a r to the e f f e c t of d e c e n t r a l -i z a t i o n . Again, as i n the case of 6-OHDA-induced c e n t r a l l e s i o n s , there i s - 23 -considerable pharmacological evidence supporting the view that the e a r l y component i s a presynaptic e f f e c t rather than true postsynaptic super-s e n s i t i v i t y . Cocaine, an uptake i n h i b i t o r , mimicked the e a r l y , but not the l a t e , component and only the l a t t e r was revealed by i s o p r o t e r e n o l as t h i s 8 agonist i s not taken up i n t o the presynaptic membrane. Since a c e t y l c h o l i n e i s not removed from i t s receptor by reuptake mechanisms, parasympathetic denervation a l s o i n v o l v e s only a slow-developing postsynaptic component. Because of the saturable nature of amine uptake mechanisms, the amine concentration at the receptor i s not a l i n e a r f u n c t i o n of the e x t e r n a l amine concentration. This non-linear r e l a t i o n s h i p i s r e s p o n s i b l e for changes i n slope and n o n - p a r a l l e l s h i f t s of the slopes of dose-response curves. Langer found that the slope increased w i t h a decrease i n the potency (an increase i n EC^Q) of d i f f e r e n t amines (95). This e f f e c t was only noticed i n innervated or d e c e n t r a l i z e d organs where the amine-uptake, presynaptic systems were i n t a c t . Cocaine treatment or denervation abolished t h i s e f f e c t , i n d i c a t i n g i t s dependence upon such uptake systems. The p o s s i b l e occurrence of such pre-synaptic changes i s only one of the complicating f a c t o r s that must be con-sidered i n e v a l u a t i o n of data purporting to i n d i c a t e sub- or s u p e r - s e n s i t i v i t y . Some of the problems encountered when attempting to f i n d the exact mechanism of s e n s i t i v i t y changes are the v a r i a t i o n s i n r e s u l t s obtained by d i f f e r e n t i n v e s t i g a t o r s . A l l s e n s i t i v i t y changes are manifested by s h i f t s i n the dose-response curves. However, some studies show an increased maximum response, others not. Some authors also report a change i n b a s e l i n e values, but u s u a l l y t h i s does not occur. And some reports state a change i n V max but constant K for adenylate c y c l a s e , w h i l e others state a change i n the K w i t h constant V . Furthermore, the d i s c o n c e r t i n g reports that super-m max & r - 24 -s e n s i t i v i t y of the NA-stimulated cAMP system never develops i n the guinea p i g c e r e b r a l cortex (96) or f o l l o w i n g 6-OHDA-induced l e s i o n s add to the d i f f i c u l t y of e s t a b l i s h i n g a general model of s e n s i t i v i t y changes i n the CNS. .5 . cAMP i n Disease Mental disease, e p i l e p s y and drug a d d i c t i o n are major unsolved problems and can be regarded as challenges f o r neuropathologists. I t seems very probable that perturbations i n neurotransmission are fundamental and i t i s therefore not s u r p r i s i n g that extensive work has been done attempting to i d e n t i f y such p e r t u r b a t i o n s . The l i t e r a t u r e on the p o s s i b l e r o l e of cAMP i n these c o n d i t i o n s i s reviewed here. I t must be remembered, however, that there i s as much or more l i t e r a t u r e on the p o s s i b l e r o l e of almost every known neurotransmitter and r e l a t e d compound i n each of these diseases. I t i s becoming c l e a r that the CNS i s a rather p l a s t i c s t r u c t u r e and that any p e r t u r b a t i o n i n one neuronal system may cause compensating and secondary changes i n others. Hence i t has proven d i f f i c u l t to pinpoint the e t i o l o g y , e s p e c i a l l y i n those c o n d i t i o n s which show no c o n s i s t e n t histopathology. Only more and be t t e r research data may give the necessary c l u e s . A few references are a l s o c i t e d on the p o s s i b l e involvement of cAMP i n var i o u s other neuropathological c o n d i t i o n s and the p o s s i b l e uses of cAMP assays i n CSF and urine as d i a g n o s t i c a i d s . a) Ethanol, Morphine and cAMP: V o l i c i e r et_ al. (97) noticed a decrease i n cAMP l e v e l s a f t e r acute a l c o h o l i n g e s t i o n . This decrease was mainly a t t r i b u t e d to a marked decrease i n c e r e b e l l a r cAMP. Redos e_t a l . (98), however, observed no change i n cAMP l e v e l s i n any of the seven regions of b r a i n assayed a f t e r acute ethanol, chronic ethanol or ethanol withdrawal. - 25 -There are also some, not e n t i r e l y c o n s i s t e n t , observations on the e f f e c t s of ethanol on the cAMP generating system i n rat b r a i n (99). I s r a e l et a l . (100) found that chronic a l c o h o l a d m i n i s t r a t i o n increased basal adenylate cyclase a c t i v i t y as measured i n both c o r t i c a l s l i c e s and homogenates. Acute a l c o h o l treatment had no e f f e c t . Chronic a l c o h o l a d m i n i s t r a t i o n also increased the adenylate cyclase response to NaF i n homogenates but abolished the response to NA i n c o r t i c a l s l i c e s . The phosphodiesterase (PDE) a c t i v i t y of the c e r e b r a l cortex was unchanged by chronic a l c o h o l treatment or by a d d i t i o n of ethanol to s l i c e s i n v i t r o (101). French et a l . (102,103) observed a s h i f t i n the s e n s i t i v i t y of c o r t i c a l adenylate cyclase to NA during chronic a l c o h o l treatment and withdrawal. A l c o h o l i n g e s t i o n f o r four months, w i t h s a c r i f i c e two hours a f t e r the l a s t d r i n k , r e s u l t e d i n a s h i f t to the r i g h t i n the dose-response curve compared to dextrose-fed c o n t r o l s , i . e . an apparent s u b s e n s i t i v e response of the adrenergic receptor. This could i n d i c a t e an increase i n the r e l e a s e and turnover of NA during chronic ethanol treatment w i t h a compensatory change i n the receptor (true s u b s e n s i t i v i t y ) , or an apparent s u b s e n s i t i v i t y could be due to decreased r e t e n t i o n of NA at the postsynaptic s i t e because of increased metabolism or reuptake. On the t h i r d day of withdrawal a f t e r four months of ethanol i n g e s t i o n , the dose-response curve s h i f t e d to the l e f t , i . e . the adrenergic receptor e x h i b i t e d an apparent s u p e r s e n s i t i v e response to NA s t i m u l a t i o n . The onset of d e l i r i u m tremens i n humans and foot-shock h y p e r s e n s i t i v i t y i n r a t s both occur on the t h i r d day of withdrawal from chronic a l c o h o l . The adrenergic s u p e r s e n s i t i v i t y was shown to i n v o l v e 8 but not a-adrenergic receptors i n the c e r e b r a l c o r t e x , - 26 -as p r o p r a n o l o l but not phenoxybenzamine blocked the response to NA (10 4M f o r maximal s t i m u l a t i o n ) (102,104). P r o p r a n o l o l a l s o diminished the c l i n i c a l symptoms of d e l i r i u m tremens. French et a l . (105) f u r t h e r e s t a b l i s h e d that the s u p e r s e n s i t i v i t y response a l s o occurred during histamine and s e r o t o n i n s t i m u l a t i o n , but not during GABA or a c e t y l c h o l i n e s t i m u l a t i o n . These f i n d i n g s led French to postulate a non-s p e c i f i c p o s t j u c t i o n a l s u p e r s e n s i t i v i t y phenomenon as the underlying mechanism of a l c o h o l withdrawal. Other i n v e s t i g a t o r s have examined the s e n s i t i v i t y of the dopamine receptors i n the s t r i a t u m and nucleus accumbens of r a t s a f t e r chronic ethanol treatment. Some beha v i o r a l evidence has been reported i n d i c a t i n g a s u p e r s e n s i t i v i t y (106, 107) during treatment and a s u b s e n s i t i v i t y (77) during withdrawal, but measurements of dopamine-sensitive adenylate cyclase i n s t r i a t a l homogenates from eth a n o l - t r e a t e d rats have not supported the hypothesis of s e n s i t i v i t y changes i n the postsynaptic receptor (109). Some evidence of s u b s e n s i t i v i t y i n mice during withdrawal has, however, been found i n s i m i l a r in v i t r o s tudies of dopamine-sensitive adenylate cyclase (110). There are a l s o a number of reports suggesting that some of the e f f e c t s of morphine may be r e l a t e d to changes i n a c t i v i t y of a catecholamine-sensitive adenylate cyclase although, i n t h i s i n s t a n c e , the i n v e s t i g a t i o n s have been concentrated on the dopamine-sensitive adenylate cyclase i n the s t r i a t u m and l i m b i c system rather than on the NA-sensitive enzyme i n the cortex. Rat s t r i a t a l DA and cAMP l e v e l s are markedly increased during morphine dependence, and cAMP l e v e l s are markedly decreased during withdrawal (101,111, 112). Both acute and chronic a d m i n i s t r a t i o n of morphine are s a i d to increase - 27 -basa l adenylate cyclase a c t i v i t y but not to change s i g n i f i c a n t l y the degree of dopamine s t i m u l a t i o n (113). During withdrawal, however, the s t i m u l a t o r y e f f e c t s of both dopamine and apomorphine on the enzyme seem to be markedly decreased (101,111,113,114). The a d d i t i o n of morphine or r e l a t e d enkephalins to i n v i t r o assay systems has a l s o been reported to i n h i b i t the s t i m u l a t i o n of adenylate cyclase by dopamine (115,116) or noradrenaline (117). This f i n d i n g may complicate i n t e r p r e t a t i o n of r e s u l t s obtained on drug-treated animals, however, i t i s s t i l l p o s s i b l e that n a r c o t i c analgesics exert t h e i r a ctions through the r e g u l a t i o n of adenylate cyclase a c t i v i t y i n c e n t r a l nervous system s t r u c t u r e s . b) Mental Disease: The search for biochemical f a c t o r s i n the e t i o l o g y of the psychoses has been going on f o r many decades and has inv o l v e d extensive i n v e s t i g a t i o n s of the blood, urine and other t i s s u e s of mentally i l l persons i n an attempt to define some pathogenetic biochemical abnormality. Each d i s -covery of a new class of compounds important to b r a i n f u n c t i o n has i n i t i a t e d a new s e r i e s of such i n v e s t i g a t i o n s . Hormones, v i t a m i n s , trace elements, neuro-t r a n s m i t t e r s and c y c l i c n u c l e o t i d e s have a l l been e x t e n s i v e l y studied. The work has produced many reports of abnormalities p e c u l i a r to schizophrenia or a f f e c t i v e i l l n e s s but none have been c o n s i s t e n t l y confirmed. There are serious problems of i n t e r p r e t a t i o n because of the p o s s i b l e e f f e c t s of d i e t , s t r e s s and s i m i l a r f a c t o r s other than the psych o t i c i l l n e s s . The advent of the a n t i p s y c h o t i c drugs provided both a new stimulus and a new approach to the search for biochemical e t i o l o g i c a l f a c t o r s . I t was argued that i d e n t i f i c a t i o n of the b r a i n systems modified by the c l i n i c a l l y a c t i v e drugs would allow the presumption that i t was perturbations i n those systems - 28 -which l e d to the psychotic symptomatology. This assumption i s not n e c e s s a r i l y v a l i d - a n t i c h o l i n e r g i c s were the treatment of choice f o r many years i n Parkinsonism, f or example, but there i s now overwhelming evidence that the problem l i e s i n the dopaminergic and not i n the c h o l i n e r g i c systems. I t a l s o now seems probable that various disturbances i n any one of a number of i n t e r -connected neuronal systems i n b r a i n could lead to s i m i l a r symptomatology, so that schizophrenia and a f f e c t i v e i l l n e s s may each comprise a group of diseases rather than an e t i o l o g i c a l e n t i t y . Despite these d i f f i c u l t i e s , some hypotheses have been formulated w i t h regard to the psychoses which are based on the ge n e r a l l y accepted modes of a c t i o n of a n t i p s y c h o t i c drugs and are of i n t e r e s t here because they suggest a key r o l e for the catecholamine-stimulated adenylate c y c l a s e s . A p r e s e n t l y favored hypothesis on schizophrenia i s that i t involv e s hyper-a c t i v i t y of a dopaminergic system of b r a i n . A key piece of evidence l e a d i n g to t h i s hypothesis was the e x c e l l e n t c o r r e l a t i o n found between the c l i n i c a l potency of various phenothiazines and t h e i r a b i l i t y to i n h i b i t the s t i m u l a t i o n by dopamine of adenylate cyclase a c t i v i t y i n s t r i a t a l homogenates (118). No such c o r r e l a t i o n e x i s t e d between c l i n i c a l potency and the e f f e c t s on other neurotransmitter systems studied. U n f o r t u n a t e l y , although there was a s i m i l a r c o r r e l a t i o n w i t h i n the fa m i l y of a n t i p s y c h o t i c butyrophenone d e r i v a t i v e s , the c o r r e l a t i o n d i d not hold when members of the two groups of drugs were compared. Thus, f o r example, h a l o p e r i d o l i s much more potent c l i n i c a l l y i n comparison w i t h chiorpromazine than t h e i r r e l a t i v e e f f e c t s on dopamine-stimulated adenylate cyclase would suggest. I t has been found, however, that the two groups of drugs f a l l i n t o place i f b i n d i n g assays rather than adenylate cyclase measurements are used as the biochemical index of dopamine receptors - 29 -(119) . Whether t h i s i n d i c a t e s that the important a n t i p s y c h o t i c a c t i o n i s at l e a s t p a r t i a l l y at a presynaptic dopamine receptor which does not i n v o l v e cAMP or i n some n o n - s t r i a t a l locus having a dopamine-stimulated adenylate c y c l a s e w i t h p r o p e r t i e s which are somewhat d i f f e r e n t than those of the s t r i a t a l enzyme i s not c l e a r . Whatever the outcome of t h i s debate, a c t i v i t y w i t h i n any one s t r u c t u r a l c l a s s of receptor blockers does seem to c o r r e l a t e w i t h c l i n i c a l potency and has been used as a screening t e s t i n the development of new drugs (120) . An important side e f f e c t of chronic drug treatment i n schizophrenia i s the development of t a r d i v e d y s k i n e s i a , a movement dis o r d e r b e l i e v e d to be due to s u p e r s e n s i t i v i t y of the s t r i a t a l dopaminergic receptors. The enhanced r e s -ponsiveness of s t r i a t a l dopamine-sensitive adenylate cyclase i n animals chron-i c a l l y treated w i t h n e u r o l e p t i c s , presumably i n d i c a t i n g a s u p e r s e n s i t i v i t y induced by the prolonged dopamine receptor blockade, has been suggested as a model of c l i n i c a l l y observed t a r d i v e d y s k i n e s i a (121). The t r i c y c l i c antidepressants which are agents of choice f o r the treatment of depression are a l l amine uptake b l o c k e r s . Compounds which p r e f e r e n t i a l l y i n h i b i t dopamine uptake are r e l a t i v e l y i n e f f e c t i v e c l i n i c a l l y , suggesting that the dopamine systems are probably not p r i m a r i l y involved i n t h i s d i s o r d e r . Some p a t i e n t s w i t h depression respond b e t t e r to noradrenaline uptake i n h i b i t o r s than to s e r o t o n i n uptake i n h i b i t o r s while the converse i s true i n other cases. There i s now evidence from s e v e r a l centers that some cases of depression show low l e v e l s of noradrenaline metabolites i n the pre-treatment stage, w h i l e others show low l e v e l s of the s e r o t o n i n m e t a b o l i t e , 5-hydroxyindoleacetic a c i d . Reportedly, the pre-treatment l e v e l s have p r e d i c t i v e value as to the type of drug which w i l l be e f f e c t i v e i n a p a r t i c u l a r p a t i e n t (122). This has - 30 -led to the hypothesis that there are at l e a s t two e t i o l o g i c a l l y d i s t i n c t forms of depression, one i n which there i s hypofunctioning of noradrenergic systems and another i n which serotonergic systems are d e f e c t i v e . Since cAMP seems to play a key r o l e i n the postsynaptic a c t i o n of these t r a n s m i t t e r s , i t would be u s e f u l to have more data than are p r e s e n t l y a v a i l a b l e on p o s s i b l e a b n o r m a l i t i e s of t h i s substance i n these d i s o r d e r s . Urine e x c r e t i o n of both NA and cAMP has been reported to be elevated during mania (123,124). Manic-depressive p a t i e n t s are often treated with L i + s a l t s w i t h considerable success. P a t i e n t s who improve on L i + therapy r e p o r t e d l y show a decreased u r i n a r y e x c r e t i o n of cAMP (125). L i + reduces the rel e a s e of NA and 5-HT from b r a i n s l i c e s and antagonized both NA- and histamine-induced adenylate cyclase a c t i v i t y i n b r a i n s l i c e s and NaF s t i m u l a t i o n i n homogenates (126). These, however, are only a few of the many biochemical e f f e c t s reported for L i + and i t i s d i f f i c u l t to e x p l a i n the chemical e f f e c t i v e n e s s of L i + i n both mania and depression on the basis of any of the biochemical data so f a r reported. Nor can u r i n a r y l e v e l s of e i t h e r NA or cAMP be taken as good i n d i c e s of c o n t r o l noradrenergic a c t i v i t y . Drugs which i n h i b i t or reduce PDE a c t i v i t y i n the CNS have al s o been reported to reduce anxiety quite e f f e c t i v e l y (127). C a f f e i n e , t h e o p h y l l i n e and theobromine belong to t h i s group and t h e i r e f f e c t i s mimicked by a d m i n i s t r a t i o n of di-butyryl-cAMP. c) E p i l e p s y E p i l e p s y i s a group of n e u r o l o g i c a l disorders c h a r a c t e r i z e d by an abnormal s u s c e p t i b i l i t y of neuronal aggregates to generate and propagate a c t i o n poten-t i a l s . Factors i n f l u e n c i n g the e l e c t r i c a l state and metabolism of neuronal systems are c e r t a i n to c o n t r i b u t e to t h i s abnormality. In order to i n v e s t i g a t e - 31 -the morphological, biochemical, p a t h o l o g i c a l and n e u r o p h y s i o l o g i c a l changes before, during and a f t e r the development of convulsive s e i z u r e s , experimental techniques to provoke seizures have been developed. These techniques i n c l u d e a u d i t o r y , photogenic, pharmacologic and e l e c t r o g e n i c s t i m u l a t i o n . K i n d l i n g i s a form of chronic e l e c t r i c a l s t i m u l a t i o n of the amygdala or other s e n s i t i v e areas designed to produce i n animals a chronic seizure s u s c e p t i b i l i t y which c l o s e l y resembles the human c o n d i t i o n (128). In attempting to l o c a l i z e those areas of the b r a i n which a c t i v e l y p a r t i c i p a t e i n the expression of s e i z u r e s , i t has been observed that rhythmic, sharp waves develop concurrent with c l i n i c a l m a n i festations of amygdaloid convulsions (129). By l e s i o n i n g various areas of the c e r e b r a l c o r t e x , i t has also been e s t a b l i s h e d that only those animals with p r e f r o n t a l and o r b i t a l l e s i o n s r e q u i r e d more s t i m u l a t i o n s to develop k i n d l e d convulsions. Motor, c i n g u l a t e and p o s t e r i o r c o r t i c a l l e s i o n s were without e f f e c t . S t i m u l a t i o n of the f r o n t a l lobe of e p i l e p t i c p a t i e n t s a l s o evokes s e i z u r e s i d e n t i c a l to those spontaneously suffered by the p a t i e n t s . These observations suggest that i t i s worthwhile examining c o r t i c a l areas f o r biochemical changes relevant to seizure phenomena. There i s an enormous l i t e r a t u r e on the p o s s i b l e r o l e of v a r i o u s neuro-t r a n s m i t t e r s i n convulsive behavior and i t seem c l e a r that pharmacological or lesion-produced changes i n a number of t r a n s m i t t e r s can modify the development or p o t e n t i a t i o n of s e i z u r e s . In p a r t i c u l a r , a great body of evidence suggests that catecholamines exert a suppressant e f f e c t on seizure a c t i v i t y . Mice which were g e n e t i c a l l y prone to audiogenic s e i z u r e s had lower l e v e l s of b r a i n NA than d i d normal mice (128). C i r c a d i a n f l u c t u a t i o n s i n NA l e v e l s i n the r a t - 32 -have also been shown to be i n v e r s e l y r e l a t e d to seizure s e n s i t i v i t y (129). Hippocampal and amygdalar NA l e v e l s were depleted i n k i n d l e d cats (130). The suppressant r o l e of NA i n seizure development i s also supported by pharmacological s t u d i e s . Drugs which deplete NA by i n h i b i t i n g synthesis or by destroyin g NA neurons, g e n e r a l l y tend to increase seizure s u s c e p t i b i l i t y and s e v e r i t y (131-136). Conversely, i n t r a c e r e b r a l i n j e c t i o n s of NA reduced s u s c e p t i b i l i t y to audiogenic as w e l l as chemically and e l e c t r i c a l l y induced s e i z u r e s (137). Precursors of NA and MAO i n h i b i t o r s had the same e f f e c t (130,131). Some i n v e s t i g a t o r s have postulated s u p e r s e n s i t i v i t y as an underlying mechanism of e p i l e p t i c s e i z u r e a c t i v i t y . E p i l e p t o g e n i c f o c i have been ob-served to c o n s i s t of aggregates of p a r t i a l l y denervated neurons i n the c e r e b r a l cortex (138). These neurons a l s o have very unstable r e s t i n g membrane poten-t i a l s , a feature often observed i n s u p e r s e n s i t i v e nerve c e l l s . Undercut slabs of cortex very q u i c k l y become e p i l e p t o g e n i c , i . e . , showed increased c a p a c i t y to s u s t a i n paroxysmal a c t i v i t y ( e p i l e p t i f o r m a f t e r discharge). Several i n v e s t i g a t i o n s have al s o i m p l i c a t e d cAMP i n the expression of seizure a c t i v i t y . A v a r i e t y of agents capable of i n c r e a s i n g cAMP l e v e l s i n the rat cortex were found to be e p i l e p t o g e n i c (139,140). Symptom-free e p i l e p t i c s have been reported to have lower concentrations of cAMP i n the CSF than found i n p a t i e n t s who had undergone convulsive a t t a c k s w i t h i n 0 to 3 days (141). In more d e t a i l e d measurements of cAMP l e v e l s i n the CSF of e p i l e p t i c p a t i e n t s , i t was found that the cAMP l e v e l was highest during s e i z u r e , then declined g r a d u a l l y over 7 days a f t e r seizure and then remained at a normal l e v e l (142). This author suggested that changes i n the cAMP l e v e l s i n the CSF was a f a i r l y accurate measure of changes i n n e u r o n a l / g l i a l - 33 -cAMP concentrations and would not be a f f e c t e d by t o n i c and c l o n i c muscular c o n t r a c t i o n s . d) Miscellaneous c l i n i c a l c o n d i t i o n s A d m i n i s t r a t i o n of i s o p r e n a l i n e or adrenaline r e s u l t s i n elevated blood l e v e l s of cAMP. This response i s much lower i n pati e n t s w i t h b r o n c h i a l asthma. A l l e r g i c c o n d i t i o n s may be ass o c i a t e d w i t h a d e f e c t i v e B receptor response of the cAMP system to adrenergic s t i m u l a t i o n . Low concentrations of Pb i n h i b i t s both basal and hormone-stimulated l e v e l s of adenylate cyclase a c t i v i t y . Concentrations needed to e l i c i t t h i s e f f e c t approach those which are t o x i c and cause n e u r o l o g i c a l abnormalities i n v i v o (143). e) cAMP and c l i n i c a l chemistry cAMP assays may also have some uses i n c l i n i c a l chemistry. P a r a t h y r o i d s t i m u l a t i o n of kidney c o r t i c a l adenylate cyclase r e s u l t s i n increased u r i n a r y e x c r e t i o n of cAMP. Lack of t h i s response i s d i a g n o s t i c f or pseudo-hypoparathyroidism (144,145). Vasopressin stimulates kidney medullary adenylate cyclase r e s u l t i n g i n an increase i n the l e v e l of cAMP excreted i n the u r i n e . Lack of t h i s response i s d i a g n o s t i c f o r re n a l diabetes i n s i p i d u s . P a t i e n t s w i t h cerebrovascular i n f a r c t i o n showed an elevated l e v e l of cAMP i n both CSF and systemic f l u i d compared to c o n t r o l s (146). Comatose p a t i e n t s showed a lower CSF l e v e l of cAMP; the more severe the coma the lower the cAMP l e v e l . A change i n coma status was accompanied by a change i n CSF cAMP l e v e l . A l l plasma cAMP l e v e l s were normal (147,148). 6. Assay Methods f o r cAMP Most i n v e s t i g a t i o n s of adenylate cyclase a c t i v i t y depend upon assay of the amount of cAMP formed during a standard i n c u b a t i o n p e r i o d . The various - 34 -methods which have been reported f o r cAMP determination are reviewed b r i e f l y here; choice of method for t h i s study was based on s e n s i t i v i t y and ease of performance. a) Phosphorylase a c t i v a t i o n assay H i s t o r i c a l l y , the f i r s t assay procedure f or cAMP was based on the a b i l i t y of cAMP to enhance the rate of a c t i v a t i o n of i n a c t i v e dog l i v e r phosphorylase. This assay evolved from the experiments that led to the discovery of cAMP (149,150). In the f i r s t stage, i n a c t i v e l i v e r phosphorylase i s converted to the a c t i v e enzyme; the rate of conversion i s dependent on the i n i t i a l cAMP concen-t r a t i o n . Dog l i v e r homogenate (11,000 x g supernatant f r a c t i o n ) i s incubated w i t h ATP, MgSO^, and c a f f e i n e together w i t h a known amount of cAMP or an unknown sample. In the second stage, the amount of a c t i v e phosphorylase i s determined by measuring the conversion of glucose-l-PO^ to glycogen during incubation at 37°C for 30 minutes. In the t h i r d stage, glycogen i s deter-mined by i t s c o l o r i m e t r i c r e a c t i o n w i t h i o d i n e . The assay i s s e n s i t i v e to 0.1 pmole of cAMP. The most serious disadvantage of t h i s assay i s i t s v a r i a b i l i t y from day to day; that i s , the absolute values for cAMP vary although the r e l a t i v e values are constant. Other disadvantages are the i n t e r f e r e n c e of various other agents w i t h the assay and the n e c e s s i t y to prepare the required enzymes (151,152). b) Enzymatic c y c l i n g procedures The cAMP can be i s o l a t e d by t h i n - l a y e r chromatography and converted to 5'-AMP with phosphodiesterase and then to ATP with myokinase and pyruvate kinase. The enzymatic c y c l i n g system w i t h ATP as the c a t a l y t i c component, generates glucose-6-P0^, which can be measured f l u o r i m e t r i c a l l y w i t h glucose-6-PO, - 35 -dehydrogenase (153). A l t e r n a t i v e l y , ATP can be determined by i t s luminescent r e a c t i o n w i t h f i r e f l y l u c i f e r i n and l u c i f e r a s e (154-156). The assay i s s e n s i -t i v e to 1 pmole cAMP and l i n e a r over three orders of magnitude. The main disadvantage i s the ri g o r o u s p u r i f i c a t i o n r e q u i r e d . c) P r o t e i n kinase assay Low concentrations of cAMP w i l l a c t i v a t e cAMP-dependent p r o t e i n kinase, which catal y z e s the phosphorylation of p r o t e i n substrates by ATP. The 32 incubation mixture includes [y- P]-ATP, Mg acetate, cAMP standard or unknown, histone (or casein) p r o t e i n , a c t i v a t e d p r o t e i n kinase and Na acetate b u f f e r . The r e a c t i o n i s terminated by the a d d i t i o n of T C A - t u n g s t a t e - s u l f u r i c a c i d , the p r e c i p i t a t e i s d i s s o l v e d i n NaOH and the r a d i o a c t i v i t y counted i n a s c i n t i l l a t i o n counter. The extent of histone (or casein) phosphorylation i s d i r e c t l y p r o p o r t i o n a l to the amount of cAMP present i n the in c u b a t i o n mixture. The s e n s i t i v i t y of the assay i s 0.3 pmole cAMP (157,158). d) P r o t e i n binding assays These depend upon the competition between cold cAMP i n unknown or standard w i t h a known amount of l a b e l l e d cAMP f o r s p e c i f i c b i n d i n g s i t e s on various p r o t e i n s . The amount of l a b e l l e d cAMP which i s bound i s i n v e r s e l y r e l a t e d to the amount of co l d cAMP. Radioimmunoassay: Of the competitive p r o t e i n binding s a t u r a t i o n assays, the radioimmunoassay i s the one g i v i n g the highest s e n s i t i v i t y . The method i s based on the competition of cAMP w i t h an i s o t o p i c a l l y l a b e l l e d s u c c i n y l a t e d c y c l i c n u c l e o t i d e d e r i v a t i v e f o r b i n d i n g on a s p e c i f i c antiserum. The 2'-O-succinylated cAMP i s conjugated to a p r o t e i n as the free carboxyl end and t h i s immunogenic c y c l i c n u c l e o t i d e d e r i v a t i v e i s i n j e c t e d i n t o r a b b i t s . A n t i s e r a produced c r o s s - r e a c t minimally with other n u c l e o t i d e s (159,160). The antiserum i s saturated w i t h an iod i n a t e d ( J"^ JI or " L J J'I) t y r o s i n e methyl ester d e r i v a t i v e of s u c c i n y l a t e d cAMP, which w i l l then compete w i t h the sample (or standard) cAMP f o r bindin g s i t e s on the an t i b o d i e s . Bound and free 125 . . . I d e r i v a t i v e can be separated by ammonium s u l f a t e p r e c i p i t a t i o n and the p r e c i p i t a t e counted i n a gamma spectrometer. The r a d i o a c t i v i t y i s i n v e r s e l y p r o p o r t i o n a l to the amount of sample cAMP and the s e n s i t i v i t y i s 0.01 to 2 pmole cAMP/tube. The prep a r a t i o n of the a n t i s e r a and the use of s h o r t - l i v e d i o d i n e isotopes poses t e c h n i c a l problems f o r r o u t i n e use. P r o t e i n kinase binding assay: Because of i t s s i m p l i c i t y and r e l a t i v e l y high s e n s i t i v i t y , the p r o t e i n kinase binding assay f o r cAMP i s a very widely used method (161,162). The r a t i o n a l e i s the same as f o r the radioimmunoassay. The binding p r o t e i n i s most fr e q u e n t l y s k e l e t a l muscle p r o t e i n kinase because of i t s high binding constant f o r cAMP. The t r i t i u m l a b e l l e d cAMP competes wi t h u n l a b e l l e d sample cAMP f o r bindin g s i t e s on the p r o t e i n kinase molecule. In a d d i t i o n , a bindin g enhancement f a c t o r , p r o t e i n kinase i n h i b i t o r p r o t e i n , i s added to increase the binding of the c y c l i c n u c l e o t i d e s . Unbound cAMP i s separated from bound cAMP-protein complex by adsorption on c e l l u l o s e e s t e r f i l t e r s or on charcoal (163,164). The p r o t e i n kinase i s so large a molecule that i t i s r e t a i n e d on the s p e c i a l m i l l i p o r e f i l t e r s used. The f i l t e r s or the charcoal supernatants are counted i n a s c i n t i l l a t i o n counter. The assay i s s e n s i t i v e to 0.05 to 0.1 pmole of cAMP. e) Pre l a b e l l i n g techniques When mammalian t i s s u e s and c e l l s are incubated i n an adenine co n t a i n i n g s o l u t i o n , t h i s purine i s converted i n t o 5'-AMP and f i n a l l y to ATP. This ATP i s then a c t i v e l y converted to cAMP. By p u l s e - l a b e l l i n g c e l l s with r a d i o a c t i v e adenine, the r e l a t i v e amounts of cAMP newly formed from ATP can be determined by i s o l a t i n g the cAMP and measuring i t s r a d i o a c t i v i t y (165). I t i s thus p o s s i b l e to measure changes i n the l e v e l s of cAMP but not absolute amounts of the c y c l i c n u c l e o t i d e (166). The s e n s i t i v i t y of t h i s method i s p r o p o r t i o n a l to the s p e c i f i c a c t i v i t y of the r a d i o a c t i v e adenine. 32 3 Homogenates can also be incubated with [y- P]ATP and [ H]-cAMP and co l d c a r r i e r cAMP. The samples are a p p l i e d to n e u t r a l hydrous alumina oxide columns, which s e l e c t i v e l y r e t a i n p o l y a n i o n i c n u c l e o t i d e s and P i . The e f f l u e n t , monovalent cAMP, can then be counted i n a three-channel Packard counter. This method i s extremely r a p i d and simple (167). 7. STATEMENT OF THE PROBLEM The o r i g i n a l aim of t h i s t h e s i s was to i n v e s t i g a t e changes i n noradrenergic responsiveness of the rat c e r e b r a l cortex t a k i n g place during the three days f o l l o w i n g chronic a l c o h o l withdrawal. The receptor was reported to d i s p l a y s u b s e n s i t i v i t y on day 0 but s u p e r s e n s i t i v i t y on day 3. I f these s e n s i t i v i t y changes were due to f u n c t i o n a l disuse, as a r e s u l t perhaps of a decrease i n NA contact w i t h the receptor or a decrease i n NA r e l e a s e , i t should be p o s s i b l e to mimic t h i s e f f e c t by b l o c k i n g NA s y n t h e s i s . The FLA-63-induced i n h i b i t i o n of dopamine-8-hydroxylase would h o p e f u l l y have produced the same f u n c t i o n a l disuse and consequent s u p e r s e n s i t i v i t y of the noradrenergic receptor. A l s o , the sequence of such s e n s i t i v i t y changes could be compared w i t h the a l c o h o l withdrawal-induced changes. In these experiments, the same assay was to be used f o r cAMP as had been used i n the a l c o h o l withdrawal studies (104). These sets of experiments were i n i t i a t e d but were not completed when the many problems w i t h the cAMP assay were recognized. The development of a more r e l i a b l e method i s o u t l i n e d i n the "RESULTS" s e c t i o n . Some of the major - 38 -dif f i c u l t i e s with the original method are listed below: 1) the 1 mm tissue slices were probably too thick for complete penetration of solutions 2) the pH of the Krebs-Ringer buffer was 6.0 at the onset of incubation and gradually changed to pH 7.2 at the c r i t i c a l time of neurohormone addition 3) the dilution of tissue with buffer was high so that the assay for cAMP was performed at a concentration where unreliable results might be expected 4) the binding assay was done at a very high concentration of salt which interfered with the binding to protein kinase. FLA-63 injected animals had a very high mortality rate and the literature contained very few reports on this drug at the time of experimentation. A NA depleting agent with better documented actions seemed preferable, so that any unusual results would not be attributed to di f f i c u l t i e s with the drug. There-fore, 6-OHDA, a drug which consistently depletes central NA stores, was chosen to demonstrate supersensitivity. By injecting 6-OHDA intracerebrally into the dorsal bundle, the NA stores of the cortex are selectively depleted while DA remains unchanged (168). The possibility that chronic supersensitivity of the cortical cAMP generating system might be induced in this fashion had not been investigated when this work was undertaken. The very high sensitivity of the adenylate cyclase system during the end of the second post-natal week (60) could be the result of supersensitive noradrenergic receptors, due to neonatal "disuse" as a result of a lag in the development of synapses compared to the development of 8 receptors. Finally, the possibility that supersensitivity might play a role in convulsive behavior was examined by studying possible receptor sensitivity changes in the kindled rat. - 39 -I I . EXPERIMENTAL PROCEDURE 1. M a t e r i a l s 3 . 3 Uniformly l a b e l l e d [ H]-cAMP (ammonium s a l t , 37.7 Ci/mmole) and [ H]-dopamine (12 Ci/mmole were obtained from New England Nuclear. P r o t e i n kinase i n h i b i t o r , cAMP-dependent p r o t e i n kinase, non-radioactive cAMP, 1-arterenol-D-b i t a r t r a t e and bovine serum albumin ( c r y s t a l l i n e ) were a l l purchased from the Sigma Chemical Company. Sodium p e n t o b a r b i t a l was bought from Abbott L a b o r a t o r i e s , 6-hydroxydopamine hydro-bromide from Regis Chemical Company, L-ascorbic a c i d from B r i t i s h Drug House and FLA-63 (bis-4-methyl-l-homopiperazinylthio-carbonyl d i s u l f i d e ) was a g i f t from AB A s t r a Lakemedel. Alumina ( a c i d washed, pH 3.0-5.0), Reagent grade, was purchased from the McArthur Chemical Company and DL-arterenol hydrochloride (B grade) from Calbiochem. A l l other chemicals, ACS grade, were obtained from e i t h e r M a l l i n c k r o d t or the F i s h e r Chemical Company. M i l l i p o r e f i l t e r s ( c e l l u l o s e e s t e r , HA 0.45, 24 mm) were purchased from the M i l l i p o r e Company. Adult male Wistar a l b i n o r a t s were purchased from Woodlyn L a b o r a t o r i e s , male Long-Evans hooded r a t s from the Canadian Breeding Farms and 15-day o l d Wistar a l b i n o r a t s , male and female, were supplied by the Vivarium of the U n i v e r s i t y of B r i t i s h Columbia. 2. Methods a) Treatment of Animals i ) K i n d l i n g Procedure Kindled rats were obtained from Dr. Corcoran and the procedure used was the f o l l o w i n g . Male Long-Evans hooded r a t s weighing 300 g were anaesthetized - 40 -with sodium pentobarbital (50 mg/kg, i.p.) 30 min p r i o r to the b i l a t e r a l implantation of electrodes into the medial amygdaloid area, for which the sterotaxic coordinates were: TB + 5mm + 5mm, D-V 8.5mm (from cortex), A-P -.8mm (from Bragma). E l e c t r i c a l stimulation through insulated bipolar electrodes was provided by a constant-current 60~Hz sine wave source of 160 uA in t e n s i t y . The reference electrode consisted of a male plug connected to a sur g i c a l screw i n the f r o n t a l bone v i a uninsulated wire (169). The rats were stimulated through the l e f t amygdaloid electrode for 1 sec/ day for 10-12 days, u n t i l stage 4 of ki n d l i n g was reached. This stage i s characterized by the development of generalized b i l a t e r a l clonus of the fore-limbs, rearing of the head and f a l l i n g . The animals were further stimulated to have at least 10 f u l l seizures, a f t e r which the entire k i n d l i n g procedure was repeated with stimulation through the ri g h t amygdaloid electrode. Any subsequent e l e c t r i c a l stimulation through the k i n d l i n g electrodes would always e l i c i t f u l l seizure responses, even i f the animals had been allowed a re s t i n g period of several months (170). i i ) 6-OHDA Injections Male Wistar rats weighing 300-350g were anaesthetized with sodium pento-b a r b i t a l (50 mg/kg, i.p.) 30 min p r i o r to the stereotaxic i n j e c t i o n s . The Konig and K l i p p e l A t l a s coordinates for the dorsal bundle (DB) noradrenergic tr a c t , which are based on 150g rats (171), had to be adjusted to allow for the greater weight of animals used i n this study, so that the f i n a l coordinates were: TB - 4.2mm, A-P + 2.6mm, M-L + 1.1mm, D-V 3.7mm. After the animals were placed i n position on the stereotaxic apparatus (David Kopf Stereotaxic Instruments), two burr holes were d r i l l e d for the b i l a t e r a l i n j e c t i o n of 2 ul/10 min of 2 ug/ul 6-OHDA, which was made up freshl y before use i n a solution - 41 -of 0.3 mg ascorbic acid/ml of isotonic s a l i n e . This very slow i n j e c t i o n rate, using a 10 u l Hamilton syringe, 34 gauge needle, ensured against d i f f u s i o n of 6-OHDA to proximal t r a c t s , i n p a r t i c u l a r the v e n t r a l bundle (VB) noradrenergic t r a c t (172). One group of 8 rats was l e f t for 10 weeks and a second group of 8 for 1 week before s a c r i f i c e and assay. A t h i r d group of 4 animals was i n j e c t e d with a control solution containing only the v e h i c l e , and was l e f t for 1 week before s a c r i f i c e . This was done to determine whether possible physical damage to the nerve tr a c t would a f f e c t the assay r e s u l t s . The i n t r a c e r e b r a l l y 6-OHDA-injected animals did not require tube-feeding, i n contrast to i n t r a v e n t r i c u l a r l y injected ones (172). i i i ) FLA-63 Injections FLA-63 was dissolved i n a few drops of g l a c i a l acetic acid (173) and d i l u t e d to volume i n 0.9% s a l i n e s o l u t i o n (the volume was as close to 1.5 ml/rat as p o s s i b l e ) . Control animals were injected with an equal volume of vehicle solution. b) Treatment of Brain Tissue i ) Tissue Preparation Animals were s a c r i f i c e d by c e r v i c a l d i s l o c a t i o n , the brains removed and put into a beaker containing i c e - c o l d Krebs-Ringer bicarbonate buffer (KRB-buffer), pH 7.4. The entire tissue preparation procedure was carried out on a bed of ice i n a cold-room at 4°C. The cerebral c o r t i c e s from both r i g h t and l e f t sides was dissected out, taking care not to include any white matter. The y i e l d was approximately 250 mg per adult r a t . The slabs of cortex were cut into 0.3 mm s l i c e s , using a Mcllwain tissue chopper (Brinkman - 42 -Instrument Company), y i e l d i n g sections of dimension 5 x 1.5 x .3 mm. The c o r t i c a l s l i c e s from one animal were then suspended i n KRB-buffer, the suspension vortexed and the s l i c e s d i s t r i b u t e d to six 25 ml Ehrlenmeyer f l a s k s , each containing 3 ml KRB-buffer, to achieve a random d i s t r i b u t i o n of the various areas of the cortex into each of the incubation f l a s k s . The KRB-buffer was kept on ice and aerated with 95% C>2-5% CC^ for one hour before and during the subsequent incubation (50). The composition of the KRB buffer was as follows: 25 mM sodium bicarbonate, 118 mM NaCl, 5mM KC1, 2.5 mM CaCl 0 , 2 mM KHLPO., 2 mM 2 2 4 MgSO^, 7 H 20 and 0.02 mM EDTA and 12 mM glucose. I t was prepared fres h l y each day from 10 x concentrated stock solutions. i i ) Incubation Procedure and I s o l a t i o n of cAMP The incubation flasks were placed i n a 37°C shaking waterbath (70 cycles/min), and gassed for 30 min with 95% 0^-5% CO2 through a i r - t i g h t rubber stoppers. This marked the start of the f i r s t preincubation period, which was terminated after 30 min by aspiration of the buffer. This pre-incubation period was necessary because there i s an immediate post-mortem r i s e in cAMP le v e l s and time must be allowed for the excess cAMP to be metabolized by phosphodiesterase to achieve constant baseline le v e l s (174,175). Fresh KRB-buffer, 2.9 ml, was added to each of the fla s k s , which again were gassed for 15 min with 95% 0 2"5% CO^ At this time, NA was added i n 100 u l of 30 x concentrated solutions to y i e l d f i n a l concentrations of 0 uM, 1 uM, 3 uM, 10 UM, 30 uM, and 100 uM. Stock solutions of 30 mM NA i n 0.004% (w/v) BSA were frozen i n aliquots and were s e r i a l l y d i l u t e d to 30 x concentrations before the additions to the fl a s k s . The presence of BSA has been found empirically to decrease the - 43 -oxidative decomposition of NA i n solution. 100 b l of BSA solution was added to one flask for each animal to allow the estimation of the i n d i v i d u a l baseline. Five minutes after these additions, the reaction was terminated by centrifugation of the samples i n the cold at 1000 x g for 30 sec, decantation of the buffer and addition of 1 ml of 10% TCA. The suspensions were homogenized with a Teflon pestle and the pestle was rinsed a f t e r each homogenization with 0.4 ml 10% TCA. The homogenates were centrifuged at 12,000 x g for 15 min i n a Sor v a l l centrifuge and the supernatants decanted o f f into 15 ml test tubes. The pe l l e t s were resuspended i n an additional 0.4 ml 10% TCA, recentrifuged and the two supernatants, containing the cAMP, were combined to give a t o t a l volume of 1.8 ml. The p e l l e t was dissolved i n 5 ml 2% (w/v) Na carbonate containing 0.1 N NaOH i n a warm bath for 4 hrs and used for determination of protein. The supernatant was extracted 6 times with 2 volumes of ether to remove the TCA. The remaining traces of ether were removed by placing the test tubes i n a warm water bath under a l i g h t stream of a i r . To each tube, 0.6 ml of 200 mM Na acetate buffer, pH 4.5, was added and the samples were stored at -20°C. Recovery of cAMP was determined by adding a known amount of radioactive cAMP p r i o r to homogenization. The samples were taken through the i d e n t i c a l procedures and aliquots taken for determination of r a d i o a c t i v i t y . Care was taken at a l l transfer steps to minimize losses of the sample. Despite t h i s care, however, transfer losses probably accounted for most of the experimental error i n the determination of cAMP. i i i ) Determination of cAMP A modification of the Gilman binding assay (161,162) was used for the measurement of cAMP levels i n c o r t i c a l t i s s u e . - 44 -a) cAMP b i n d i n g m i x t u r e 3 [ H]-cAMP ( s p . a c t . 37.7 Ci/mmole) was d i l u t e d w i t h 50 mM Na a c e t a t e b u f f e r , pH 4.5, t o g i v e 70 pmoles per ml or 5 x 10 DPM per ml. U n l a b e l l e d cAMP was d i s s o l v e d i n the same b u f f e r t o g i v e a s t o c k s o l u t i o n o f 40 nmoles per m l , wh i c h was l a t e r d i l u t e d 100 x t o g i v e a cAMP s t a n d a r d o f 400 pmoles p e r m l . P r o t e i n k i n a s e i n h i b i t o r ( P K I ) , was d i s s o l v e d i n water t o g i v e a c o n c e n t r a t i o n o f 0.1 mg per ml. P r o t e i n k i n a s e (PK) was d i s s o l v e d i n water t o g i v e a s t o c k s o l u t i o n o f 0.9 mg p e r m l , w h i c h was s t o r e d i n 600 u l a l i q u o t s . T h i s s t o c k s o l u t i o n was d i l u t e d to 87.1 mg per ml j u s t b e f o r e use. A l l r e a g e n t s were s t o r e d a t -20°C, e x c e p t f o r the 50 mM Na a c e t a t e b u f f e r , w h i c h was r e f r i g e r a t e d . b) cAMP b i n d i n g a s s ay The b i n d i n g a s s a y was conducted on i c e i n a f i n a l volume o f 250 u l con-t a i n i n g 50 u l PKI (5 u g ) , 50 u l [ 3H]-cAMP (3.5 pmoles) 5 t o 90 u l cAMP s t a n d a r d o r 100 u l sample. The r e a c t i o n was i n i t i a t e d by a d d i n g 50 u l PK (4.4 ug p r o t e i n ) . The c o n c e n t r a t i o n o f b i n d i n g p r o t e i n (PK) s h o u l d be s u f f i c i e n t 3 t o b i n d l e s s than 30% o f the [ H]-cAMP, t o ensure t h a t PK i s s a t u r a t e d when e q u i l i b r i u m i s reached a f t e r 1 h r of i n c u b a t i o n a t 0°C. A f t e r an a d d i t i o n a l 15 m in, 1 ml o f c o l d 20 mM p o t a s s i u m phosphate b u f f e r , pH 6.0 was added t o each assay tube. F o u r min l a t e r the a s s a y m i x t u r e s were passed t h r o u g h M i l l i p o r e f i l t e r s ( c e l l u l o s e e s t e r , HA 0.45, 24 mm) mounted on a s a m p l i n g m a n i f o l d ( M i l l i p o r e Company). The f i l t e r s were r i n s e d w i t h 10 ml o f c o l d Na a c e t a t e b u f f e r b e f o r e b e i n g d r i e d i n g l a s s c o u n t i n g v i a l s a t 65°C f o r 1 h r . The r a d i o a c t i v i t y was d e t e r m i n e d i n a r e f r i g e r a t e d N u c l e a r C h i c a g o L i q u i d s c i n t i l l a t i o n c o u n t e r , 300 s a m p l e r , u s i n g 10 ml o f s c i n t i l l a t i o n f l u i d c o n t a i n i n g 5 g PPO/1 t o l u e n e . The CPM r e c o r d e d was c o r r e c t e d t o DPM f o r each •ll? - 45 -3 sample from a quench curve, with the measured e f f i c i e n c y for [ H] varying between 34-39%. The blank count, i . e . assay mixture without PK, was always below 100 DPM and was ignored since test assays were always over 10,000 DPM. A standard curve was plotted on log-log paper, with t o t a l pmoles cAMP ( t r i t i a t e d + no n - t r i t i a t e d ) on the abscissae and DPM on the ordinate. The slope of the straight l i n e obtained between 5 to 40 pmoles varied inversely with the DPM recorded, so that for increasing amounts of unlabelled cAMP 3 competing with [ H]-cAMP for binding s i t e s , there i s a reduction i n DPM. The amount of cAMP i n a sample can therefore e a s i l y be read from the graph, i v ) Measurements of NA Levels The procedure followed for the estimation of NA was that of McGeer et^ a l . (176,177). a) Extraction of NA In order to evaluate the effectiveness of the 6-OHDA lesions, NA le v e l s were measured i n the hippocampi of control and lesioned animals. NA determin-ations could not be performed on the cerebral c o r t i c e s as these were used for cAMP estimations. An al t e r n a t i v e is to use one group of animals for NA and one for adenylate cyclase studies, but the v a r i a b i l i t y found by a l l investigators using 6-OHDA dictated our decision to use the same animals for both assays. Since innervation of both hippocampus and cortex i s from the same group of NA fib r e s (see F i g . I) the hippocampal NA levels should give a good index of c o r t i c a l depletion. The hippocampi were dissected out b i l a t e r a l l y and weighed. The y i e l d was usually 100 mg. The hippocampi were homogenized i n 1 ml 0.4 N per c h l o r i c containing 0.2 ace t i c acid (10 ml HCIO^ + 2.6 ml HAc + 437 ml ^ 0 ) , i n a Tri-R homogenizer with 20 passes of a Teflon pestle. The homogenate was l e f t standing i n ice for 20 min and then - 46 -c e n t r i f u g e d at 1000 x g f o r 10 min. The supernatant, c o n t a i n i n g NA, was decanted o f f i n t o a 5 ml test tube, the p e l l e t resuspended i n 0.5 ml p e r c h l o r i c a c e t i c a c i d reagent, r e c e n t r i f u g e d , and the two supernatants were pooled and stored at -20°C u n t i l assay. NA determinations were also performed on s u b c o r t i c a l t i s s u e from the k i n d l e d r a t s , i n the hope of d e t e c t i n g a change i n NA l e v e l s as a r e s u l t of the k i n d l i n g procedure. In t h i s case, the cerebellum was removed from the d e c o r t i c a t e d brains and discarded. The remaining s u b c o r t i c a l t i s s u e was d i v i d e d i n t o a n t e r i o r and p o s t e r i o r halves. The t i s s u e was weighed (500-600 mg/half b r a i n ) , and homogenized i n 2 ml of 0.4 N p e r c h l o r i c / a c e t i c a c i d using 20 passes of a T e f l o n p e s t l e i n a 10 ml T r i - R homogenizer. These preparations were subsequently treated i n the same way as those of the hippocampi. 3 Two ml of 0.2 M EDTA and a known amount of [ H]-DA, f o r i n t e r n a l recovery, was added to the c l e a r supernatant, which was adjusted to pH 8.5-9.0 wi t h 5 N NaOH before adding 300 mg of acid-washed alumina. As each batch of alumina v a r i e s s l i g h t l y , the pH at which optimal recovery was obtained was p r e v i o u s l y determined to be between 8.5-9.0 ( F i g . 12) f o r the p a r t i c u l a r alumina batch used i n these experiments. The alumina s l u r r y was s t i r r e d f o r 4 min before being introduced i n t o a glass column 10 cm long, which had pre-v i o u s l y been plugged w i t h glass wool. The column was washed twice w i t h 3 to 5 ml of water under gentle s u c t i o n . The NA was eluted w i t h 0.6 ml of 0.5 N a c e t i c a c i d without s u c t i o n . To t h i s eluant was added 0.6 ml of 1 M Na acetate b u f f e r , pH 6.0, and 25 u l of 0.5 N NaOH, to give a f i n a l pH of 6.0, which had p r e v i o u s l y been shown to give optimal fluorescence i n t e n s i t i e s ( F i g . 15). One blank and three NA standards (0.1 Ug, 0.2 ug, 0.4 Ug) were taken through the same procedure to allow comparison of the recovery of NA w i t h that of the - 47 -r a d i o a c t i v e dopamine. A 0.1 ml a l i q u o t of each pH-adjusted eluant was taken for r a d i o a c t i v i t y determination i n 10 ml of Bray's s c i n t i l l a t i o n mixture (180 g naphthalene, 12 g PPO, 0.45 g POPOP, 60 ml ethylene g l y c o l , 3 1 dioxane). Extreme care was taken throughout the e n t i r e procedure not to contaminate the eluants w i t h s c i n t i l l a t i o n f l u i d , as the phosphor contained i n these mixtures i n t e r f e r e s g r e a t l y with the s p e c t r o p h o t o f l u o r i m e t r i c i n t e n s i t y readings, b) The NA Assay A volume of 0.4 ml 1 M Na acetate b u f f e r , pH 6.0, was added to 0.6 ml of pH-adjusted eluant. Four min a f t e r the a d d i t i o n of 0.5 ml of an N i o d i n e s o l u t i o n (0.254 g I 2 + 5 g K l i n 5 ml water, d i l u t e d to 222 ml), the samples were treated w i t h 0.25 ml of 0.05 N sodium t h i o s u l f a t e followed by 0.5 ml of a mixture of 7 ml 5N NaOH and 3 ml 0.5% as c o r b i c a c i d . "Faded blanks" were done fo r each sample i n which 0.35 ml of 5 N NaOH was s u b s t i t u t e d f o r the NaOH/ asc o r b i c a c i d mixture. The test tubes were l e f t under l i g h t i n an open rack f o r 90 to 120 min. A f t e r t h i s p e r i o d , 0.15 ml of 0.5% asc o r b i c a c i d was added to the faded blanks. The fluorescence was read immediately afterwards i n an Aminco-Bowman spectrophotofluorimeter (American Instrument Company), at e x c i t a t i o n / e m i s s i o n wavelengths of 402 nm/502 nm. A l l assay steps were c a r r i e d out at room temperature. The NA content i n each sample was read o f f a l i n e a r standard curve (0.125 ug/ml-0.2 U g/ml, DL-arterenol-HCl). The NA standard was d i l u t e d to 0.2 Ug/ml from a stock s o l u t i o n of 10 ug/ml, which was stored at -20°C i n 1 ml a l i q u o t s . The measured values of NA per 0.6 ml eluant a l i q u o t (S^) were corrected f o r recovery and f o r eluant volume by using the i n d i v i d u a l r a d i o a c t i v e recovery (CPM/0.1 ml of eluant = C 1 ) , the mean co l d recovery per 0.6 ml - 48 -a l i q u o t i n the standards c a r r i e d through the column procedure (R £) and the mean CPM/0.1 ml i n these same standard samples ( C D ) . The t o t a l i n i t i a l NA per sample was then c a l c u l a t e d as S, .CD/C,Rr,. The t o t a l recovery of 1 K 1 L u n l a b e l l e d standards was 70 to 80%; r a d i o a c t i v e r ecoveries were l i n e a r l y r e l a t e d to c o l d r e c o v e r i e s but appreciably l e s s because of t r i t i u m exchange 14 during the procedure. The use of C - l a b e l l e d catecholamine permits c l o s e r r e a l c o r r e l a t i o n between hot and c o l d r ecoveries but was not used because a v a i l a b l e s p e c i f i c a c t i v i t i e s are so much lower than f or the t r i t i a t e d 14 d e r i v a t i v e s that the a d d i t i o n of s i g n i f i c a n t C CPM would add s u f f i c i e n t m a t e r i a l to i n f l u e n c e the fl u o r e s c e n t assay r e s u l t s . The l a b e l l e d r e coveries were u s u a l l y i n the range of 25 to 30% and the u n l a b e l l e d ones 70 to 80%. The data were expressed as ug NA/g wet b r a i n t i s s u e , v) P r o t e i n Determination A l l p r o t e i n determinations were done by the method of Lowry et a l . (178). The cAMP content had to be expressed as pmoles cAMP/mg p r o t e i n , as i t was never f e a s i b l e to weigh the t i s s u e s l i c e s before incubations. However, i f one assumes that adult r a t c e r e b r a l cortex contains 10% p r o t e i n , a l l data can e a s i l y be converted to pmoles cAMP/g wet weight b r a i n t i s s u e (179). 3. A n a l y s i s of Data The v a r i a t i o n s of NA l e v e l s were expressed as the means + S.E.M. (standard er r o r of the mean) and were analyzed using the student t - t e s t , w i t h d i f f e r e n c e s between groups considered to be s i g n i f i c a n t i f p<.05. In the s t a t i s t i c a l a n a l y s i s of the dose-response curves, i . e . the adenylate cyclase responses to d i f f e r e n t amounts of noradrenaline, q u a n t i t a t i v e assess-ment can be accomplished by comparing the doses producing equal responses i n - 49 -control and experimental preparations. Since the steepest part, i . e . the center, of a dose-response curve is the most accurate part, s t a t i s t i c a l analysis was done on the EC..Q values, i . e . the dose of agonist that produces a half-maximal response. The EC^Q values of agonists for most tissues appear to show a log normal d i s t r i b u t i o n . Therefore, a l l s t a t i s t i c a l compar-isons were done using log EC^Q data and the geometric means with t h e i r 95% confidence i n t e r v a l s have been presented. Geometric means were calculated from the EC^Q values estimated from the i n d i v i d u a l curves. Accurate estimates of changes i n s e n s i t i v i t y may be obtained by c a l c u l a t i n g the r a t i o s of geometric means of EC^Q's or by the determination of the d i f -ference between the mean logs, i . e . the log s h i f t , between control and experi-mental groups. This is probably the preferred method i n experimental designs i n which the same animal serves as i t s own con t r o l . In the present study such a design was not possible, as each animal could be used only once, either as a control or an experimental animal. In addition, EC^Q values were always graphical estimations. Maximal stimulations, baseline values and percents of maximal stimulation ( i . e . maximal stimulation-baseline value/ baseline value x 100) were also analysed on the o r i g i n a l dose-response curve data by the student's t - t e s t . - 50 -I I I . RESULTS 1. FLA-63 In an attempt to achieve noradrenergic s u p e r s e n s i t i v i t y of the c e r e b r a l cortex by pharmacological denervation, the drug FLA-63 was administered i n t r a -p e r i t o n e a l l y at a dose of 25 mg per kg (180). FLA-63 b i s ( 4 methyl-1-homopiperazinyl-thiocarbonyl) d i s u l f i d e i s a c h e l a t i n g agent and ther e f o r e . . 2+ i n h i b i t s the Cu -dependent dopamine-8-hydroxylase enzyme very e f f e c t i v e l y . The drug causes very r a p i d and very complete d e p l e t i o n of c e n t r a l NA s t o r e s , w i t h only minimal e f f e c t s on c e n t r a l DA stores (168,181). At a dose of 25 mg/kg, FLA-63 caused a 65% reduction of c o r t i c a l NA i n 4 hours while r a i s i n g DA l e v e l s by only 10% (173,182). FLA-63 u n f o r t u n a t e l y also a f f e c t s c e n t r a l thermoregulatory mechanisms (183) causing severe hypothermia w i t h i n 2 h r s , so that animals had to be kept at 30°C (184). Most of the animals died w i t h i n 12 hr i f the environmental temperature was not regulated. Their body temperature f e l l 3°C 2 hr a f t e r i n j e c t i o n . When the room temperature was adjusted to 30°C, the ra t s survived and had normal body temperatures. The v e h i c l e used to d i s s o l v e the drug may have a l s o c o n t r i b u t e d to the m o r t a l i t y r a t e . Although g l a c i a l a c e t i c a c i d used to d i s s o l v e the drug was d i l u t e d before i n j e c t i o n , the pH was very a c i d and could be expected to cause severe p e r i t o n e a l i n j u r y (185). The c o n t r o l s , however, received the v e h i c l e and d i d not show such a high m o r t a l i t y r a t e . The assay procedure employed i n t h i s e x p e r i - ment was the o r i g i n a l method described i n the f o l l o w i n g s e c t i o n ( s e c t i o n I I I - 2 ) . The animals were s a c r i f i c e d 6 hrs and 24 hrs p o s t - i n j e c t i o n . There appears to be a s u p e r s e n s i t i v e response a f t e r 6 h r s , as the dose response curve for the i n j e c t e d animals i s s h i f t e d to the l e f t of that f or the - 51 -c o n t r o l s . Both the baseline and maximal response cAMP l e v e l s are lower i n the experimental animals ( F i g . 6 ) . The dose response curve for the 24 hr animals was s h i f t e d s l i g h t l y to the r i g h t but the s h i f t was not s i g n i f i c a n t at p ( 0 . 0 5 ( F i g . 7 ) . The b a s e l i n e cAMP response was d r a s t i c a l l y lowered from the 6 hr l e v e l s , but the maximal response was s l i g h t l y higher than both c o n t r o l and 6 hr maximal l e v e l s . However, the c o n t r o l values appeared to be d i f f e r e n t f or 24 hr animals compared to 6 hr r a t s . The b a s e l i n e and cAMP l e v e l s were s i g n i f i c a n t l y lower at 24 hrs than at 6 hrs for c o n t r o l animals. The dose response curve a l s o appeared to s h i f t l e f t w a r d from 6 hrs to 24 h r s . I t should perhaps be pointed out that c o n t r o l animals were al s o kept at 30°C room temperature and t h e i r body temperature at 24 hrs was elevated 1 . 5 ° C . 2. Development of method and e a r l y f i n d i n g s Due to the v a r i a b i l i t y of the c o n t r o l animals i n the F L A - 6 3 experiments, and the lack of r e p r o d u c i b i l i t y of cAMP measurements i n mock experiments, i t was decided that the e n t i r e experimental procedure should be i n v e s t i g a t e d . The o r i g i n a l method ( 1 0 4 ) employed ether to s a c r i f i c e animals. Since ether i s a v o l a t i l e anaesthetic and therefore may a f f e c t the membrane of CNS c e l l s , the method of s a c r i f i c e seemed to be a good s t a r t i n g p o i n t . When the dose-response curves f or ra t s s a c r i f i c e d by ether and for r a t s s a c r i f i c e d by c e r v i c a l f r a c t u r e were compared, they were rather s i m i l a r , as shown i n F i g . 8. The slopes of the curves were almost i d e n t i c a l , but the ba s e l i n e cAMP response of the c e r v i c a l f r a c t u r e animals was s i g n i f i c a n t l y higher than that of the ether k i l l e d animals. The same was true f or the maximal response (here 3 0 0 uM NA) and the EC^Q a l s o seemed to be higher, but t h i s was not s i g n i f i c a n t . Although the two curves are very s i m i l a r , the data - 5 2 -F i g . 6. Dose-response curve of c e r e b r a l cortex to NA. C o r t i c a l s l i c e s from c o n t r o l ( O ) and 6 hr FLA-63 treated (•) r a t s were exposed to various concentrations of NA for 5 min. Tissue cAMP content i n the absence of NA (B) represents b a s e l i n e values. Each po i n t and v e r t i c a l bar represents the mean and S.E.M., r e s p e c t i v e l y , of 3 animals. Semilog p l o t . - 53 -F i g . 7. Dose-response curve of c e r e b r a l cortex to NA. C o r t i c a l s l i c e s from c o n t r o l ( O ) and 24 hr FLA-63 treated ( • ) r a t s were exposed to va r i o u s concentrations of NA f o r 5 min. Tissue cAMP content i n the absence of NA (B) represents b a s e l i n e v a l u e s . Each point and v e r t i c a l bar represents the mean and S.E.M., r e s p e c t i v e l y , of 4 animals. Semilog p l o t . - 54 -NA concentrat ion (/uM) F i g . 8. Dose-response curve of c e r e b r a l cortex to NA. C o r t i c a l s l i c e s from animals s a c r i f i c e d by c e r v i c a l f r a c t u r e (•) and by ether (O) were exposed to var i o u s concentrations of NA for 5 min. Tisue cAMP content i n the absence of NA (B) represents b a s e l i n e values. Each point and v e r t i c a l bar represents the mean and S.E.M., r e s p e c t i v e l y , of 4 animals. Semilog p l o t . - 55 -f o r the e t h e r - k i l l e d r a ts were always more i n c o n s i s t e n t than those using c e r v i c a l f r a c t u r e so that the bett e r procedure was used t h e r e a f t e r . O r i g i n a l l y , rather small pieces of cortex were removed with a razor blade and these pieces were s l i c e d i n t o twelve 1 mm t h i c k s e c t i o n s . Each incubation tube then contained two such s e c t i o n s . I t i s very u n l i k e l y that such t h i c k t i s s u e s l i c e s w i l l r e c e i v e uniform exposure to the bathing s o l u t i o n and the ion s , O2 and neurohormones i n i t (186). I t proved p o s s i b l e to d i s s e c t out much l a r g e r p o r t i o n s of t i s s u e by p e e l i n g c o r t i c a l slabs w i t h a very f i n e s c a l p e l . The y i e l d then became 250 to 300 mg per r a t . The new la b o r a t o r y f o r t u n a t e l y had a Mcllwain t i s s u e chopper, so the t i s s u e could be sectioned one-way i n t o 0.3 mm t h i c k s l i c e s . The t o t a l dimension of the s l i c e s were 5 mm long, 1.5 mm deep and 300 um t h i c k . This use of thinner s l i c e s should f a c i l i t a t e r e s p i r a t i o n and ensure more uniform exposure to inc u b a t i o n s o l u t i o n s and more r a p i d uptake of neurohormone from the medium. Krebs-Ringer biocarbonate b u f f e r must be aerated w i t h CO2 to achieve p h y s i o l o g i c a l pH but at atmospheric pressure (760 mm Hg), the proper mixture i s 95% 0 2 and 5% C0 2 to achieve pH 7.2 ( i d e a l l y i t should be 7.4). In the o r i g i n a l method, dry i c e (100% CC^) had been bubbled through the b u f f e r and t h i s r e s u l t e d i n a pH of 6.0. When t h i s pH 6.0 b u f f e r was t r a n s f e r r e d to incubation f l a s k s and aerated w i t h 95% 0^-5% CO^ mixture, the pH s t e a d i l y increased to 7.2 over about 20 minutes. During the second in c u b a t i o n p e r i o d , the neurohormone was added at 14 minutes and incubation continued for 6 minutes. The c r i t i c a l incubations t h e r e f o r e occurred at a time when the pH was changing. The Krebs-Ringer b u f f e r was therefore aerated with 95% 02 _5% C0„ before as w e l l as during the incubation. - 56 -NA was o r i g i n a l l y d i s s o l v e d i n BSA c o n t a i n i n g Krebs-Ringer bicarbonate b u f f e r , pH 7.2. This pH i s a b i t too b a s i c f o r NA, which w i l l decompose at a l k a l i n e pH and NA was subsequently d i s s o l v e d i n BSA-water instead. The d i s t i l l e d water i n the l a b o r a t o r y was pH 6. Termination of the neurohormone incubation was o r i g i n a l l y accomplished by the a d d i t i o n of TCA to the i n c u b a t i o n medium co n t a i n i n g the t i s s u e s l i c e s . The t i s s u e was separated by c e n t r i f u g a t i o n and the supernate extracted w i t h ether to remove the TCA and then l y o p h i l i z e d . The residue was taken up i n acetate b u f f e r . But high concentrations of s a l t s from the o r i g i n a l incubation medium were s t i l l present i n t h i s medium and these s a l t s would subsequently i n t e r f e r e w i t h cAMP bi n d i n g to PK. The termination procedure was therefore changed to incl u d e a 30 sec low-speed c e n t r i f u g a t i o n step and decantation of the b u f f e r before the a d d i t i o n of TCA. For the same reason, PK was d i s s o l v e d i n water rather than Krebs-Ringer bicarbonate b u f f e r , which had o r i g i n a l l y been the case, and the pH of the NaAc b u f f e r was r a i s e d from 4.0 to 4.5. Although binding i s more e f f i c i e n t at the lower pH, i n t e r f e r e n c e by ions i s minimized at the higher pH value. The minute amounts of cAMP which might have leaked out i n t o the medium were considered to be n e g l i g i b l e , and shown to be so by r a d i o a c t i v e recovery experiments. In the o r i g i n a l method the t i s s u e d i l u t i o n was 1:450. Figures 9 and 10 each show a Gilman binding assay standard curve with 100 u l acetate b u f f e r added to each tube and another standard curve with a t i s s u e sample c o n t a i n i n g cAMP i n 100 u l b u f f e r added to each tube. The amount of cAMP/100 u l of t i s s u e sample was 3.3 pmoles i n F i g . 9 and 4.2 pmoles i n F i g . 10. The t i s s u e d i l u t i o n was 1:200 i n F i g . 9 (a d i l u t i o n as low as 1:450 was not attempted) and 1:60 i n F i g . 10. The l i n e s are n o n - p a r a l l e l i n F i g . 9 and the amount of - 57 -c A M P c o n c e n t r a t i o n (pM) F i g . 9. Standard curve f o r the determination of cAMP content of b r a i n s l i c e s . A t i s s u e sample c o n t a i n i n g 3.3 pm cAMP i n 100 u l b u f f e r was added (• ) to the t y p i c a l standard curve ( O ) . Tissue d i l u t i o n 1:200. Log-log p l o t . - 58 -c A M P c o n c e n t r a t i o n ( p M ) F i g . 10. Standard curve f o r the determination of cAMP content of b r a i n s l i c e s . A t i s s u e sample c o n t a i n i n g 4.2 pm cAMP i n 100 u l b u f f e r was added (•) to the t y p i c a l standard curve ( o ) . Tissue d i l u t i o n 1:60. Log-log p l o t . cAMP i n the t i s s u e could therefore not be ac c u r a t e l y determined using these c o n d i t i o n s . The l i n e s are p a r a l l e l i n F i g . 10 although not c o i n c i d e n t a l , but the s l i g h t s h i f t i s w i t h i n experimental e r r o r . (A Scatchard p l o t gave a = 0.5 x 10~ 9M). Thus the experimental procedure f i n a l l y developed gave rep r o d u c i b l e values i n agreement w i t h the l i t e r a t u r e (163,187). The recovery of cAMP was approximately 73%. The time course of the accumulation of cAMP i n c o r t i c a l s l i c e s i s shown i n F i g . 11. The use of 100 uM NA f o r maximal s t i m u l a t i o n r e s u l t e d i n a very r a p i d increase i n cAMP apparent a f t e r a 1 min incubation and i n c r e a s i n g f u r t h e r a f t e r 5 min. The maximum was reached a f t e r 10 min but the d i f f e r e n c e between the values at 5 and 10 min was not s i g n i f i c a n t . The l e v e l s begin to de c l i n e on longer incubation periods due to the h y d r o l y t i c a c t i o n of PDE. The incubation period chosen was 6 min, so that a PDE i n h i b i t o r such as amino-p h y l l i n e or papaverine was not needed. F i v e minute incubations to which such an i n h i b i t o r was added d i d not give r e s u l t s s i g n i f i c a n t l y d i f f e r e n t from those i n d i c a t e d i n F i g . 11. The c e r e b r a l c o r t i c a l s l i c e s were mixed and randomly d i s t r i b u t e d to each of the s i x i n c u b a t i o n v i a l s so that any r e g i o n a l anatomical d i f f e r e n c e i n cAMP would not be detected. To v e r i f y the appropriateness of t h i s procedure, each region of the cortex was assayed separately as shown i n F i g . 12. There was no s i g n i f i c a n t d i f f e r e n c e between the c o r t i c a l lobes nor was there any d i f f e r e n c e between l e f t and r i g h t sides f o r e i t h e r b a s e l i n e or maximally stimulated values. When the l e f t and r i g h t sides were averaged, there was s t i l l no s i g n i f i c a n t d i f f e r e n c e between p a r i e t a l , f r o n t a l or o c c i p i t o - t e m p o r a l cortex. This i s shown i n F i g . 13 and was true for both b a s e l i n e and stimulated (100 uM NA) cAMP l e v e l s . - 60 -9 0 -.E 8 0 -0) J _ I i 1 1 —i— 0 1 5 10 2 0 3 0 T i m e ( m i n . ) F i g . 11. Time course of the s t i m u l a t i o n of cAMP formation by NA i n c o r t i c a l s l i c e s of adul t r a t s . B r a i n s l i c e s were incubated with 100 uM NA for the time periods i n d i c a t e d . Each poi n t and v e r t i c a l bar represents the mean and S.E.M., r e s p e c t i v e l y , of 4 animals. - 61 -140 h C o r t i c a l R e g i o n F i g . 12. The response of d i f f e r e n t c o r t i c a l regions to NA. The l e f t and r i g h t hemispheres were d i v i d e d i n t o F = f r o n t a l , P = p a r i e t a l and 0-T = o c c i p i t o -temporal c o r t i c a l r e g i o n s . B r a i n s l i c e s were exposed to 0 ^ uM ( b a s e l i n e ) and 100 uM NA f o r 5 min. Each po i n t and v e r t i c a l bar represents the mean and S.E.M., r e s p e c t i v e l y , of 6 animals. - 62 -120 r * 100 o CL c o E 8 0 < u 0) o E o o 60 4 0 r 20 1 0 0 yuM N A B a s e l i n e F P O - T C o r t i c a l R e g i o n F i g . 13. The response of d i f f e r e n t c o r t i c a l regions to NA. L e f t and r i g h t hemispheres were combined but d i v i d e d i n t o F = f r o n t a l , P = p a r i e t a l and 0-T = occ i p i t o - t e m p o r a l c o r t i c a l regions. B r a i n s l i c e s were exposed to 0 pK (base-l i n e ) and 100 ^ iM NA f o r 5 min. Each point and v e r t i c a l bar represents the mean and S.E.M., r e s p e c t i v e l y , of 6 animals. F i g . 13 i s a r e - p l o t of F i g . 12. - 63 -In order to e s t a b l i s h optimal c o n d i t i o n s for the NA determinations, the pH at which optimal recovery was obtained w i t h the batch of alumina used was e s t a b l i s h e d to be 8 . 5 - 9 . 0 ( F i g . 1 4 ) . S i m i l a r l y , maximal spectrophoto-f l u o r i m e t e r readings i n the NA assay were shown to be obtained i f the column eluant was adjusted to pH 6 .5 before a l i q u o t s were taken for a n a l y s i s ( F i g . 1 5 ) . 3. K i n d l e d Rats In order to te s t the hypothesis that permanent changes take place during the development of se i z u r e s u s c e p t i b i l i t y by k i n d l i n g , the NA s e n s i t i v e adenylate cyclase system was studied i n the e p i l e p t o g e n i c a l l y steady s t a t e of k i n d l e d r a t s . The c e r e b r a l cortex was d i v i d e d i n t o a n t e r i o r and p o s t e r i o r halves and incubated as described i n the "Methods" s e c t i o n . Because c o r t i c a l t i s s u e was scarce i n these experiments, only three concentrations of NA, 0 ( b a s e l i n e ) , 10 UM and 100 UM, were test e d . The average y i e l d was approximately 120 mg per c o r t i c a l h a l f . There was no s i g n i f i c a n t d i f f e r e n c e between k i n d l e d and c o n t r o l animals i n cAMP accumulation i n e i t h e r the a n t e r i o r or p o s t e r i o r c o r t i c a l s l i c e s at any NA concentration. Maximal s t i m u l a t i o n (100 uM NA) caused a 97% increase i n cAMP i n the a n t e r i o r cortex to y i e l d 66 .3 pmoles/mg p r o t e i n i n the k i n d l e d animals and a 92% increase to y i e l d 7 1 . 9 pmoles/mg p r o t e i n i n c o n t r o l s , as shown i n F i g . 16 and Table 1 (appendix). In the p o s t e r i o r s l i c e s ( F i g . 17) maximal NA s t i m u l a t i o n caused 121% increase i n cAMP ( 67 .3 pmoles/mg p r o t e i n ) i n k i n d l e d animals and 116% increase ( 6 6 . 6 pmoles/mg pr o t e i n ) i n c o n t r o l s (Table 1, appendix). F i g . 14. R e l a t i v e recovery of NA from alumina batches at various pH's. Each point and v e r t i c a l bar represents the mean and S.E.M., r e s p e c t i v e l y , of 4 determinations. - 65 -20 4 0 4-5 5-0 5.5 pH o f E l u a n t 6-0 6.5 7.0 7.5 F i g . 15. R e l a t i v e i n t e n s i t y of spectrophotofluorimeter readings as a f u n c t i o n of the pH of the Na Acetate b u f f e r eluant. Each point and v e r t i c a l bar represents the mean and S.E.M., r e s p e c t i v e l y , of 4 determinations. - 66 -B -fh 10 100 N A c o n c e n t r a t i o n (yuM) F i g . 16. S t i m u l a t i o n of cAMP formation by NA i n a n t e r i o r c o r t i c a l s l i c e s of k i n d l e d (• ) and c o n t r o l ( o ) r a t s . Tissue cAMP content i n the absence of NA (B) represents b a s e l i n e values. Each point and v e r t i c a l bar represents the mean and S.E.M., r e s p e c t i v e l y , of 7 k i n d l e d and 4 c o n t r o l animals. Semi-log p l o t . - 67 -B -fh 10 100 N A c o n c e n t r a t i o n (yuM) F i g . 17. S t i m u l a t i o n of cAMP formation by NA i n p o s t e r i o r c o r t i c a l s l i c e s of k i n d l e d (•) and c o n t r o l (o) r a t s . Tissue cAMP content i n the absence of NA (B) represents b a s e l i n e values. Each point and v e r t i c a l bar represents the mean and S.E.M., r e s p e c t i v e l y , of 7 k i n d l e d and 4 c o n t r o l animals. Semi-log p l o t . - 68 -There were also no s i g n i f i c a n t d i f f e r e n c e s between a n t e r i o r and p o s t e r i o r cortex i n e i t h e r k i n d l e d or c o n t r o l animals at any NA concentration t e s t e d . I d e a l l y , a f u l l dose-response curve should be constructed whenever receptor s e n s i t i v i t y changes are measured. I t i s p o s s i b l e that there i s a s h i f t of the midpoint of the curve i n s p i t e of there being no d i f f e r e n c e i n b a s e l i n e , 10 uM NA and 100 uM NA-stimulated cAMP values. Endogenous NA l e v e l s were measured i n the remaining s u b c o r t i c a l t i s s u e s . The average weight of s u b c o r t i c a l " h a l f - b r a i n s " was 500-600 mg. The NA l e v e l s of a n t e r i o r b r a i n halves were not s i g n i f i c a n t l y d i f f e r e n t , as shown i n Table 1 (appendix). However, NA l e v e l s of p o s t e r i o r b r a i n halves were s i g n i f i c a n t l y d i f f e r e n t at p<0.05 but not at p<0.025 (see Table 1, appendix). I t should be stressed that NA l e v e l s were measured on whole s u b c o r t i c a l t i s s u e s rather than on s p e c i f i c regions of the b r a i n thought to be i m p l i c a t e d i n seizure s u s c e p t i b i l i t y mechanisms. 4. Developmental E f f e c t on the cAMP System The responsiveness of the cAMP generating system to NA was studied i n s l i c e s from whole cortex i n 15 day o l d r a t s . This system i s i n s e n s i t i v e to NA at b i r t h but g r a d u a l l y develops a responsiveness which reaches a maximum at 2 weeks p o s t n a t a l and t h e r e a f t e r decreases to adult l e v e l s (about 50% of max) at 25 days. As shown i n F i g . 18, the baseline l e v e l of cAMP was s i g n i f i c a n t l y higher i n the young animals than i n the adult c o n t r o l s , 50.7 pmoles/mg p r o t e i n i n the young and 31.0 pmoles/mg p r o t e i n i n the a d u l t s . Every concentration of NA tested e l i c i t e d a s i g n i f i c a n t l y higher cAMP response i n the young r a t s as compared to the adult animals. This was e s p e c i a l l y true f or the maximal - 69 -N A c o n c e n t r a t i o n ( A J M ) F i g . 18. Dose-response curve of c e r e b r a l cortex to NA. C o r t i c a l s l i c e s from 15 day o l d (O ) and adult (•) r a t s were exposed to various concentrations of NA for 5 min. Tissue cAMP content i n the absence of NA (B) represents b a s e l i n e values. Each po i n t and v e r t i c a l bar represents the mean and S.E.M., r e s p e c t i v e l y , of 5 young and 6 adult animals. Semi-log p l o t . *Values s i g n i f i c a n t l y d i f f e r e n t from adults (p<0.05). - 70 -response, which was 125.7 pmoles/mg p r o t e i n i n the young and 59.4 pmoles/mg p r o t e i n i n the adult r a t s . These values correspond to an increase over b a s e l i n e values of 164% i n the 15 day o l d animals and 125% i n the adults i n response to 100 uM NA. However, the le f t w a r d h o r i z o n t a l s h i f t of the EC,-Q value i n the young as compared to the o l d group was not s i g n i f i c a n t . I t there f o r e appears that the very high responsiveness of the cAMP system at the end of the second post-n a t a l week i s not due to s u p e r s e n s i t i v i t y of the B receptor at t h i s stage of development of the b r a i n . 5. S u p e r s e n s i t i v i t y I n t r a v e n t r i c u l a r i n j e c t i o n of 6-OHDA i n animals r e s u l t s i n a severe deple-t i o n of both NA and DA throughout the b r a i n . Numerous studies have described the biochemical and h i s t o l o g i c a l e f f e c t s of such i n j e c t i o n s , but at the time of the experiments, no one had performed an experiment measuring NA-stimulated cAMP l e v e l s of the c e r e b r a l cortex a f t e r s p e c i f i c d e p l e t i o n of NA using s e l e c t i v e a p p l i c a t i o n s of 6-OHDA. I n t r a c e r e b r a l i n j e c t i o n s of 6-OHDA i n t o the do r s a l bundle s p e c i f i c a l l y deplete c o r t i c a l NA stor e s and leave almost a l l other catecholaminergic systems i n t a c t . I t had been reported i n the l i t e r a t u r e that c o r t i c a l NA stores were already decreased at 5 days f o l l o w i n g D.B. l e s i o n s w i t h 6-OHDA and anterograde degeneration was complete i n 15 days (188). I n t r a v e n t r i c u l a r 6-OHDA i n j e c t i o n s r e s u l t e d i n a 50% decrease i n NA stores a f t e r 7 days and an increase i n NA-stimulated cAMP accumulation a f t e r 5 days. The increased responsiveness of the cAMP system was s t i l l present a f t e r 4 weeks (189). The present i n v e s t i g a t i o n c o n s i s t e d of two s e r i e s of D.B. i n j e c t e d animals one group of 7 r a t s were assayed a f t e r 7 days and another group of 7 r a t s were s a c r i f i c e d a f t e r 10 weeks. Only r a t s w i t h a reduction i n hippocampal NA l e v e l s of at l e a s t 75% were included i n the experiment. A t h i r d group of 4 animals were i n j e c t e d w i t h the v e h i c l e only and a fo u r t h group of 6 animals served as untreated c o n t r o l s . F i g . 19 shows the dose-response curve f or v e h i c l e (mock) i n j e c t e d and untreated c o n t r o l animals. Although the v e h i c l e - i n j e c t e d animals seem to e x h i b i t a lower responsiveness to NA, t h i s i s not s t a t i s t i c a l l y s i g n i f i c a n t . When the NA l e v e l s were measured, 3 out of 4 v e h i c l e i n j e c t e d r a t s were severely depleted. The reason for t h i s remains obscure, although the p o s s i b i l i t y e x i s t s that t h i s group of animals were subjected to a n o n - s p e c i f i c s u r g i c a l i n j u r y or some i n t e r f e r e n c e w i t h axonal transport which led to temporary d e p l e t i o n of NA. There was no h i s t o l o g i c a l evidence of d e s t r u c t i o n of NA systems. The evidence of such n o n - s p e c i f i c i n j u r y from v e h i c l e i n j e c t i o n s complicate i n t e r p r e t a t i o n s of the r e s u l t s on 6-OHDA treated animals, although these were c l e a r l y d i f f e r e n t from the v e h i c l e - i n j e c t e d group As shown i n F i g . 20 animals s a c r i f i c e d 7 days a f t e r the 6-OHDA i n j e c t i o n s showed a s i g n i f i c a n t l y higher maximal s t i m u l a t i o n than c o n t r o l s . The bas e l i n e values of cAMP remained unchanged. There appeared to be a s l i g h t l e f t w a r d s h i f t i n the EC^Q of the dose response curve, i . e . a change i n K^, but t h i s s h i f t was not s t a t i s t i c a l l y s i g n i f i c a n t . There a l s o appeared to be a considerable change i n the slope of the curve. The e f f e c t on the cAMP system a f t e r 10 weeks p o s t - i n j e c t i o n i s shown i n F i g . 21 and Table 2 (appendix). There i s a s i g n i f i c a n t increase i n the baselin e l e v e l and maximum s t i m u l a t i o n , or V . There i s also a max - 72 -1 0 0 -c » 8 0 -o N A c o n c e n t r a t i o n (>uM) F i g . 19. Dose-response of c e r e b r a l cortex to NA. C o r t i c a l s l i c e s from v e h i c l e - i n j e c t e d ( O ) and c o n t r o l (•) animals were exposed to va r i o u s concentrations of NA for 5 min. Tissue cAMP content i n the absence of NA (B) represents b a s e l i n e values. Each po i n t and v e r t i c a l bar represents the mean and S.E.M., r e s p e c t i v e l y , of 3 mode-injected and 6 c o n t r o l animals. Semi-log p l o t . - 73 -T S h o r t - t e r m N A c o n c e n t r a t i o n (/uM) F i g . 2 0 . Dose-response curve of c e r e b r a l cortex to NA. C o r t i c a l s l i c e s from 6-OHDA i n j e c t e d ( 7 days) ( O ) and c o n t r o l (•) r a t s were exposed to va r i o u s concentrations of NA f o r 5 min. Tissue cAMP content i n the absence of NA (B) represents b a s e l i n e values. Each p o i n t and v e r t i c a l bar represents the mean and S.E.M., r e s p e c t i v e l y , of 7 short-term i n j e c t e d and 6 c o n t r o l animals. Semi-log p l o t . *Values s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l s . - 74 -120 c a> o k. Q. CO a < u 0) o E a 100 80 60 40 - t e r m 20* - 1 — / / ' 1 1 i _ B 1 3 10 30 N A c o n c e n t r a t i o n (>uM) 100 F i g . 21. Dose-response curve of c e r e b r a l cortex to NA. C o r t i c a l s l i c e s from 6-OHDA in jec ted (10 weeks) (O) and c o n t r o l ( • ) rats were exposed to various concentrat ions of NA for 5 min. Tissue cAMP content in the absence of NA (B) represents base l ine va lues . Each point and v e r t i c a l bar represents the mean and S . E . M . , r e s p e c t i v e l y , of 7 long-term injec ted and 6 c o n t r o l animals . Semi-log p l o t . *Values s i g n i f i c a n t l y d i f f e r e n t from contro l s (p<0.05). - 75 -s i g n i f i c a n t s h i f t to the l e f t of the dose-response curve, i . e . a lower Kp, and t h i s is probably the most r e l i a b l e evidence obtained for the manifestation of s u p e r s e n s i t i v i t y of the noradrenergic 6 receptor i n the cerebral cortex. The slope of the curve is also s l i g h t l y changed. - 76 -DISCUSSION  FLA-63 The causes of the high m o r t a l i t y among the i n j e c t e d animals were a combin-a t i o n of hypothermia and t o x i c i t y of the a c e t i c a c i d v e h i c l e (173). R a i s i n g the room temperature p a r t i a l l y solved the m o r t a l i t y problem and a d i f f e r e n t solvent f o r the drug might have f u r t h e r improved the experiment. However, the biochemical methods employed i n assaying cAMP accumulation were not r e l i a b l e as the dose response curve f o r c o n t r o l animals s h i f t e d and v a r i a b l e r e s u l t s were obtained with standards. Furthermore, both the basel i n e and the maximal response l e v e l s d i f f e r e n d markedly between the two sets of c o n t r o l animals. The s l i g h t increase i n body temperature seemed u n l i k e l y to account for such large changes i n the cAMP system. Rather, the experimental procedure seemed l i k e l y to be inadequate. The FLA-63 experiments were abandoned and the e n t i r e experimental procedure r e - i n v e s t i g a t e d i n the hope of developing a method which would y i e l d more cons i s t e n t r e s u l t s . Development of method The o r i g i n a l experimental procedure, as p r e v i o u s l y described (104), was i n c o n s i s t e n t i n the hands of t h i s i n v e s t i g a t o r . I t i s impossible to make any inferences about experimental treatments when the c o n t r o l treatments give v a r i a b l e r e s u l t s . The methodology of the e n t i r e cAMP f i e l d was undeveloped when the work for t h i s t h e s i s was done. Some p a r t i c u l a r l y serious flaws i n the method i n i t i a l l y used g r e a t l y exaggerated the inherent v a r i a b i l i t y of cAMP measurements. When working with b r a i n s l i c e preparations i t i s of paramount importance to keep co n d i t i o n s as p h y s i o l o g i c a l as p o s s i b l e . Therefore, the pH should be - 77 -very close to 7.4 and the t i s s u e t h i n enough to al l o w uniform p e n e t r a t i o n of substances added to the incubation medium and adequate r e s p i r a t i o n throughout the e n t i r e t i s s u e s l a b . I t i s also d e s i r a b l e to minimize a l l i n t e r f e r e n c e s when working with a s e n s i t i v e binding assay such as the Gilman method and i t i s mandatory to work at high enough concentrations of cAMP to allow accurate measurement. Even a f t e r a l l apparent precautions had been taken, the v a r i a b i l i t y i n the l e v e l s of cAMP measured was d i s c o n c e r t i n g . This i s g e n e r a l l y true i n the l i t e r a t u r e . The reason f o r t h i s v a r i a b i l i t y has been a s c r i b e d to i n d i v i d u a l d i f f e r e n c e s i n the cAMP system (47). K i n d l e d Rats The development of chronic seizure s u s c e p t i b i l i t y by the process known as k i n d l i n g i s marked by progressive stages of s e i z u r e a c t i v i t y . When the animal has reached the f i n a l stage, C - 5 , which i s ch a r a c t e r i z e d by loss of balance and g e n e r a l i z e d c l o n i c j e r k i n g , any subsequent e l e c t r i c a l s t i m u l a t i o n w i l l always e l i c i t f u l l seizure response even i f the animal has been r e s t i n g f o r se v e r a l months. This suggests that a permanent change, p o s s i b l y b i o c h e m i c a l , has taken place i n at l e a s t some s t r u c t u r e s of the b r a i n . Evidence i s c i t e d i n the i n t r o d u c t i o n ( s e c t i o n I - 5 c ) which i n d i c a t e s that noradrenaline exerts a suppressant e f f e c t on seizure a c t i v i t y . This evidence suggests that at l e a s t c e r t a i n areas of the k i n d l e d rat b r a i n might have c h r o n i c a l l y depleted NA s t o r e s . And i f NA exerts i t s e f f e c t upon neurons v i a the cAMP system i t was p l a u s i b l e that a change i n cAMP accumulation, i n response to i n v i t r o NA s t i m u l a t i o n , would be observed i n the present i n v e s t i -g a t i o n . I t was argued t h a t , i f the k i n d l e d b r a i n i s c h r o n i c a l l y depleted of NA, then areas where t h i s occurs should show a s u p e r s e n s i t i v e response to NA - 78 -s t i m u l a t i o n . This i s i n accord w i t h the suggestions of s e v e r a l i n v e s t i g a t o r s (reviewed i n s e c t i o n I-5c) that s u p e r s e n s i t i v i t y may be the mechanism under-l y i n g e p i l e p t i c a c t i v i t y . Since Corcoran (132) showed that the f r o n t a l cortex must be i n t a c t f o r the development of k i n d l e d s e i z u r e s to occur, the a n t e r i o r and p o s t e r i o r halves of the cortex were i n v e s t i g a t e d separately. I t was therefore d i s a p p o i n t i n g to f i n d no increase i n the cAMP response and no d i f f e r e n c e i n endogenous NA l e v e l s . I t i s l i k e l y that more d i s c r e t e regions of the cortex should have been employed i n the cAMP assay. S i m i l a r l y , NA l e v e l s should perhaps be measured e x c l u s i v e l y i n the hippocampal and amygdaloid regions. The present experiments were conducted during the steady-state e p i l e p t o -genic phase. A more f r u i t f u l approach might be to i n v e s t i g a t e s e q u e n t i a l changes i n both NA l e v e l s and cAMP response during C-1 to C-5 stages of k i n d l i n g . By studying these biochemical parameters during the development of s e i z u r e s u s c e p t i b i l i t y and i n more anatomical d e t a i l , more l i g h t might be shed on the p o s s i b l e r o l e of the NA-cAMP system i n t h i s p a r t i c u l a r neuropathological c o n d i t i o n . The development of responsiveness to NA of the cAMP generating system p a r a l l e l s the increase i n endogenous NA l e v e l s during the f i r s t two weeks of l i f e and reaches a maximum at t h i s time (190). Adenylate cyclase a c t i v i t y and receptor density a l s o reach maximum l e v e l s at the end of the second p o s t - n a t a l week (191). I t i s a very c r u c i a l time i n the development and maturation of the b r a i n . The c e r e b r a l cortex appears h i s t o l o g i c a l l y mature and the neuro-b l a s t has developed i n t o a mature neuron, d i s p l a y i n g adult EEG a c t i v i t y and a c t i o n p o t e n t i a l s at the end of the second week. Many enzyme systems develop at t h i s time and the formation of synapses i s almost complete (192). I t has - 79 -been suggested that NA might i n f l u e n c e morphogenesis by a mechanism i n v o l v i n g the increased s e n s i t i v i t y of the cAMP system (193). The observed increase i n endogenous cAMP l e v e l s during the f i r s t few weeks of l i f e could be the r e s u l t of an increase i n the amount of adenylate c y c l a s e , an increase i n the number of noradrenergic receptors, an increase i n the s e n s i t i v i t y of the receptors, an increase i n the concentration of NA at the receptor or a slower development of the PDE a c t i v i t y . The PDE a c t i v i t y i s not f u l l y developed u n t i l the t h i r d p o s t - n a t a l week and t h e r e f o r e the g r e a t l y augmented cAMP l e v e l s observed at two weeks are probably p a r t i a l l y due to a l e s s e f f i c i e n t breakdown of cAMP. Many of the other p o s s i b l e explanations mentioned above were not tested i n the present i n v e s t i g a t i o n but the r e s u l t s might i n d i c a t e that the increased cAMP l e v e l s were not due to a change i n s e n s i t i v i t y of the receptor, as the dose-response curve was not s h i f t e d to the l e f t . Responsiveness of the cAMP system develops at a s l i g h t l y d i f f e r e n t r a t e i n d i f f e r e n t regions of the cortex. P u r k i n j e c e l l s i n the cerebellum may e x i s t i n a h y p e r s e n s i t i v e state during the f i r s t neonatal weeks before the c e l l s have become f u l l y innervated (193). The present experiment assayed cAMP l e v e l s from whole cortex and p o s s i b l y masked some r e g i o n a l d i f f e r e n c e s i n receptor s e n s i t i v i t y . When esti m a t i n g s e n s i t i v i t y changes of b i o l o g i c a l receptors i t i s impor-tant to use complete dose-response curves rather than s i n g l e doses of drugs. Q u a n t i t a t i v e measurements of s u p e r s e n s i t i v i t y should always be based on the magnitude of h o r i z o n t a l s h i f t s of dose-response curves (194). The center of the curve i s the most accurate part and the h o r i z o n t a l s h i f t s should be measured on t h i s steep p o r t i o n of the curve. E q u i e f f e c t i v e doses of an - 80 -agonist are normally d i s t r i b u t e d on a log rather than on an a r i t h m e t i c s c a l e and therefore mean logs or geometric means of e q u i e f f e c t i v e agonist concentrations are the s t a t i s t i c a l l y v a l i d estimate of the mean s e n s i t i v i t i e s (195,196). A r i t h m e t i c means should not be used. The geometric mean should be presented w i t h i t s 95% confidence i n t e r v a l i n s t e a d of a standard e r r o r (197). Although the c r i t e r i o n for s u p e r s e n s i t i v i t y i s l e f t w a r d s h i f t of the dose-response curve, t h i s h o r i z o n t a l s h i f t i s sometimes accompanied by an increase i n baseline value, an increase i n the maximum response and a change i n the slope of the curve. A change i n the slope i s due to the saturable nature of the NA uptake mechanism, so that the concentration of NA at the receptor s i t e i s no longer a l i n e a r f u n c t i o n of the e x t e r n a l NA concentration (95). An increase i n the maximum response i s an i n d i c a t i o n of a more e f f i c i e n t coupling of the receptor to i n t r a c e l l u l a r mechanisms and a s h i f t to the l e f t i s a ma n i f e s t a t i o n of increased a f f i n i t y f o r the receptor by the agonist (92). The present experiments on 6-OHDA-treated r a t s showed an increase i n the maximum response i n the short-term animals and a considerable change i n the slope of the curve. This change i n slope was l e s s marked i n the long-term i n j e c t e d animals and occurs as the dose-response curve moves i n r e l a t i o n to the range of NA concentrations at which neuronal uptake i s saturated. The long-term i n j e c t e d r a t s also e x h i b i t e d a marked increase i n the maximum response as w e l l as an augmented b a s e l i n e l e v e l . I t i s i n t e r e s t i n g to f i n d that t h i s e f f e c t was evident a f t e r 10 weeks p o s t - i n j e c t i o n and t h i s i s the f i r s t p a r t i a l evidence that 6-OHDA-induced s u p e r s e n s i t i v i t y i s permanent. I t i s not p o s s i b l e to exclude a decrease i n PDE a c t i v i t y as an explanation f or the changes, although the incu b a t i o n c o n d i t i o n s were chosen to minimize any PDE i n t e r f e r e n c e . - 81 -However, the data on the long-term animals, p a r t i c u l a r l y the h o r i z o n t a l s h i f t to the l e f t of the dose-response curve, i s co n s i s t e n t w i t h the i n d u c t i o n of s u p e r s e n s i t i v i t y i n the adrenergic receptors. I t has been demonstrated numerous times that disuse of neuronal systems, i . e . f u n c t i o n a l denervation, i s u s u a l l y accompanied by an increase i n the number of 3 receptor b i n d i n g s i t e s (90,91). The time course of t h i s increase i n bindin g s i t e s p a r a l l e l s the time course of development of increase cAMP s e n s i t i v i t y to NA. The f a i l u r e to demonstrate s u p e r s e n s i t i v i t y of the c e n t r a l noradrenergic system i n the short-term i n j e c t e d animals i n p u z z l i n g since an increase i n the number of 8 bi n d i n g s i t e s has been observed to occur as r a p i d l y as 2 hours a f t e r disuse of neural input (198). I t i s q u i t e p o s s i b l e that the higher maximum s t i m u l a t i o n and the small s h i f t i n the curve were i n d i c a t i o n s of a trend towards s u p e r s e n s i t i v i t y of the noradrenergic receptor which might have been revealed had they been assayed at 2 weeks p o s t - i n j e c t i o n rather than 1 week. Anterograde degeneration i s complete at 15 days and i t i s p o s s i b l e t h a t i t would take that long or longer to develop a s u p e r s e n s i t i v e response. I t i s a l s o p o s s i b l e that the degenerative processes were slow and that 7 days was an unfortunate choice of time. K a l i s k e r e_t a l . (89) has shown a b i p h a s i c s e n s i t i v i t y change f o l l o w i n g i n t r a v e n t r i c u l a r 6-OHDA treatment. Perhaps 7 days i s intermediate between the development of the e a r l y presynaptic e f f e c t and the l a t t e r postsynaptic changes. The present i n v e s t i g a t i o n was l i m i t e d only to the e f f e c t on the NA-s e n s i t i v e cAMP generating system. P o s t j u n c t i o n a l s u p e r s e n s i t i v i t y i n smooth muscle p e r i p h e r a l system has been thought to be n o n - s p e c i f i c , w h i l e the s i t u a t i o n i n the CNS i s l e s s c l e a r . Reserpine-induced s u p e r s e n s i t i v i t y i n the - 82 -CNS does appear to be n o n - s p e c i f i c , w h i l e 6-OHDA i n j e c t i o n s r e s u l t i n receptor s e n s i t i v i t y changes i n response to catecholamines only (96). S u p e r s e n s i t i v i t y of the CNS could r e s u l t from any one, or a combination, of the f o l l o w i n g mechanisms: 1) an increase i n the number of receptors 2) a change i n the p r o p e r t i e s of receptors 3) a decrease i n PDE a c t i v i t y 4) an increase i n the amount of adenylate cyclase 5) an increase i n a c t i v i t y of adenylate cyclase 6) an increase i n the e f f i c i e n c y of coupling between receptor and i n t r a -c e l l u l a r mechanisms Recent binding studies point to the p o s s i b i l i t y that i n a s u p e r s e n s i t i v e p r e p a r a t i o n there are more 8 binding s i t e s . These could be synthesized de novo or they could be unmasked i f they e x i s t e d i n a sequestered s t a t e or they could simply move i n a f l u i d membrane. De novo synthesis can probably be excluded, as p r o t e i n synthesis i s not required for s u p e r s e n s i t i v i t y to develop (71). Decreased PDE a c t i v i t y has a l s o been suggested as a p o s s i b l e mechanism of s u p e r s e n s i t i v i t y . But i n t h i s preparation i t could probably not be the case, as i n c u b a t i o n i s terminated before the a c t i o n of PDE becomes evident. In a s u b s e n s i t i v e preparation t h i s s i t u a t i o n could obscure the i n t e r p r e t a t i o n of r e s u l t s , and i t would be p r e f e r a b l e i n such a study to include a PDE i n h i b i t o r i n the incubation medium. The coupling mechanisms involved i n the cAMP system are not completely understood. However, i f more receptors are a c t i v a t e d and there i s a one-to-one transmembranal coupling between the r e g u l a t o r y (R) subunit on the outer face of the membrane and the c a t a l y t i c (C) subunit on the inner aspect of the - 8 3 -membrane, an increase i n the amount of cAMP produced would r e s u l t . S i m i l a r l y , i f one receptor's c o n f i g u r a t i o n changed to become a one-to-two (or more) coupl i n g , a s u p e r s e n s i t i v e receptor would stimulate cAMP production by the increased number of a c t i v a t e d adenylate cyclase molecules. The choice of p r e p a r a t i o n employed always c a r r i e s both advantages and d i s -advantages. B r a i n s l i c e preparations are always used when studying NA-stimulated e f f e c t s on adenylate c y c l a s e , since the enzyme does not respond to NA i n homogenates. This i s , however, not the case for the DA-stimulated form of the enzyme. B r a i n s l i c e preparations are a l s o i n t u i t i v e l y c l o s e r to the b i o l o g i c a l s i t u a t i o n w i t h at l e a s t some of the anatomical features i n t a c t i n the c l o s e s t v i c i n i t y of the neurons, such as supporting a s t r o c y t e s and o l i g o -dendrocytes, although many axons and dendrites are severed i n b r a i n s l i c e preparations too. However, the s l i c e preparations are probably one of the most serious sources of the v a r i a b i l i t y seen i n these types of experiments. Homogenates, on the other hand, always y i e l d very uniform and reproducible r e s u l t s , probably because the homogenate p r e p a r a t i o n i s very uniform from assay to assay. This i s not p o s s i b l e i n s l i c e preparations. Although the utmost care was taken to e l i m i n a t e a l l white matter, there are always some f i b e r s i n t e r -spersed i n the grey matter. And although the d i s s e c t i o n of slabs of uniform thickness was attempted, i t i s not p o s s i b l e always to i n c l u d e a l l s i x lamina of the grey c o r t i c a l matter. I f d i f f e r e n t c e l l types are present i n d i f f e r e n t p r o p o r t i o n s , the cAMP response may d i f f e r too. When studying the e f f e c t of a neurotransmitter on cAMP accumulation, the " s t i m u l u s " and "response" are removed s e v e r a l steps. I d e a l l y , pharmacological - 84 -responses should be measured d i r e c t l y , since the more i n t e r v e n i n g steps that are present, the more l i k e l y the r e s u l t s are to show v a r i a b i l i t y . When the work on t h i s t h e s i s was s t a r t e d , cAMP studies represented one of very few approaches to receptor s t u d i e s . 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TABLE I <• The s t i m u l a t i o n of cAMP formation by NA i n a n t e r i o r and p o s t e r i o r cortex of k i n d l e d and c o n t r o l r a t s . £ a H x C o r t i c a l region cAMP pmoles/mg p r o t e i n % s t i m u l a t i o n NA ug/gm wet weight (100 uMNA)  Base l i n e 10 uMNA 100 UNA A n t e r i o r - k i n d l e d 33.7+5.0 62.0+9.98 66.3+6.3 196.7 0.471+0.054 - c o n t r o l 37.4+4.5 62.9+9.5 71.9+15.6 192.3 0.453+0.108 , <D P o s t e r i o r 1 - k i n d l e d 30.5+4.2 56.6+7.8 67.3+8.8 220.7 0.364+0.031 - c o n t r o l 30.9 + 4.1 48.8 + 4.9 66.6 + 10.1 215.5 0.268 + 0.052 TABLE I I The s t i m u l a t i o n of cAMP formation by NA i n 6-OHDA i n j e c t e d and c o n t r o l r a t s . Treatment cAMP pmoles/mg p r o t e i n % S t i m u l a t i o n EC 5 0N (uM MA) Baseline luMNA 3UMNA lOuMNA 30 uMNA 100 uMNA geometric 95% C. mean I. C o n t r o l 31.0+0.99 35.0+2.19 38.7+1.69 55.3+2.65 65.2+2.65 59.4+5.73 225.3 6.81 (5.58-8. 31) Mock-i n j ected 25 .9+0.96 26.4+1.8 34.2+2.2 47.1+2.1 50.4+0.93 50.2+2.7 202.3 4.44 (3.78-5. 22) 6-0HDA-1 week 32.0+0.95 39.6+2.94 51.7+3.9 75.3+4.3 89.5+7.1 88.8+5.2 291.3 4.97 (4/30-5. 76) 6-0HDA-10 weeks 39.3+2.6 48.7+4.3 69.1+4.8 78.3+5.6 87.9+6.0 94.2+5.0 253.7 3.31 (2.60-4. 24) The s t i m u l a t i o n of < cAMP formation by NA i n 15 day o l d and adult rats 15 day 50.7+2.16 58.5+5.1 74.2+6.47 111.2+12.73 124.7+5.86 125.7+9.5 264.4 5.85 (4.88-7. 02) 

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