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Monoaminergic influences on various inhibitions of the spinal monosynaptic reflex Sastry, Bhagavatula Sree Rama 1975

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M0N0AKINE11G1C INFLUENCES ON VARIOUS INHIBITIONS OF THE SPINAL MONOSYNAPTIC REFLEX by BHAGAVATULA SREE RAMA S AS TRY A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENT FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Division of Pharmacology and Toxicology of the Faculty of Pharmaceutical Sciences We accept t h i s t hesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA December, 1975 In presenting th is thesis in par t ia l fu l f i lment of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library shal l make i t f reely avai lable for reference and study. I further agree that permission for extensive copying of th is thesis for scholar ly purposes may be granted by the Head of my Department or by his representat ives. It is understood that copying or pub l icat ion of th is thesis for f inanc ia l gain shal l not be allowed without my wri t ten permission. Department of Pharmaceutical Sciences The Universi ty of B r i t i s h Columbia 2075 Wesbrook P l a c e Vancouver, Canada V6T 1W5 Date January 23, 1976 i i ABSTRACT The f u n c t i o n a l s i g n i f i c a n c e of bulbospinal 5-hydroxytryptamine (5-HT) and noradrenaline neurones i s not w e l l understood. Therefore i n t h i s study, the e f f e c t s of various drugs that a l t e r monoaminergic synaptic a c t i v i t y were tested on bulbospinal, presynaptic, recurrent and r e c i p r o c a l l a i n h i b i t i o n s of an extensor (quadriceps, QUAD) and a f l e x o r (posterior biceps-semitendi-nosus, PBST) monosynaptic r e f l e x (MSR) i n unanaesthetized decerebrate cats. The following agents were employed i n the i n v e s t i g a t i o n : a biogenic amine neuronal uptake blocker, imipramine HCI (0.125 - 5 mg/kg); a 5-HT neuronal uptake blocker, fluoxetine HCI ( L i l l y 110140, 0.25 - 6 mg/kg); a 5-HT precursor, 5-hydroxytryptophan (75 mg/kg); a tryptophan hydroxylase i n h i b i t o r that depletes 5-HT, DL-p_-chlorophenylalanine (300 mg/kg i . p . , i n -jected on two consecutive days before the experiment); a tyrosine hydroxy-lase i n h i b i t o r that depletes noradrenaline, DL-a-methyl-p_-tyrosine methyl ester HCI (125 mg/kg i . p . , administered 16 and 4 hours p r i o r to the experi-ment) ; a 5-HT antagonist, cyproheptadine HCI (2.5 - 5 mg/kg); an adrenergic blocker, phenoxybenzamine HCI (2.5 - 5 mg/kg); and clonidine (2.5 - 40 u g/kg), reported to be a s p e c i f i c a-adrenergic agonist. A thoracic cold block, which prevents supraspinal inputs to the caudal s p i n a l cord, was applied to test whether a drug acts through the descending systems and to determine i f the i n h i b i t i o n s of the MSR under study are influenced by the supraspinal tonic pathways. The p h y s i o l o g i c a l and the pharmacological studies have l e d to the following conclusions: presynaptic and recurrent i n h i b i t i o n s of the QUAD-i i i but not of the PBST-MSR are under a tonic i n h i b i t o r y influence of a de-scending system which involves 5-HT and noradrenaline; bulbospinal i n h i -b i t i o n of the QUAD-MSR involves both presynaptic and postsynaptic types of i n h i b i t i o n and both types of i n h i b i t i o n are antagonized by a t o n i c a l l y active 5-HT system; a t o n i c a l l y active descending system antagonizes recip-rocal l a i n h i b i t i o n of the extensor but not of the fle x o r r e f l e x ; the ex-c i t a b i l i t y of QUAD l a afferents i s decreased by a descending t o n i c a l l y ac-tiv e 5-HT system; and a t o n i c a l l y active supraspinal system has an ov e r a l l excitatory influence on the extensor motoneurones. Imipramine was more potent i n antagonizing bulbospinal and recurrent i n h i b i t i o n s of the MSR when administered i n t r a - a r t e r i a l l y to the spinal cord than when injected intravenously or i n t r a - a r t e r i a l l y to the brain stem. Therefore, the 5-HT nerve terminals proposed to be involved i n antagonizing bulbospinal and recurrent i n h i b i t i o n s are l i k e l y located i n the spinal cord. Clonidine antagonized a l l the i n h i b i t i o n s of the extensor and the flexor MSRs tested i n this study. However, iontophoretically applied c l o -nidine blocked the depressant effects of glycine and Y -aminobutyric acid on approximately 50% of the spinal neurones tested. This finding suggests that clonidine i s not a s p e c i f i c a-adrenergic agonist and may have blocked the i n h i b i t i o n s by antagonizing glycine and y-aniinobutyric acid. John G. S i n c l a i r S u p e r v i s o r . i v TABLE OF CONTENTS Page LIST OF FIGURES v LIST OF DRUGS . . . . . . . . . . . . i . v i i i ABSTRACT i x INTRODUCTION . . . . . . . 1 LITERATURE SURVEY 3 Bulbospinal monoaminergic neurones 3 The s p i n a l monosynaptic r e f l e x 4 The e f f e c t s on the MSR of agents that a l t e r monoaminergic synaptic transmission 6 The e f f e c t s of i o n t o p h o r e t i c a l l y applied 5-HT and noradrenaline on motoneurones 8 Presynaptic i n h i b i t i o n of the MSR 9 Postsynaptic i n h i b i t i o n of the MSR 14 a. Reciprocal l a i n h i b i t i o n 15 b. Recurrent i n h i b i t i o n 16 Bulbospinal i n h i b i t i o n of the MSR 19 EXPERIMENTAL 23 Sur g i c a l procedures 23 Stimulation and recording procedures 25 Cold block 27 Pharmacological studies 27 l a a f f e r e n t antidromic action p o t e n t i a l s and motoneurone f i e l d p o t e n t i a l s 29 Preparation of test drug solutions 31 Calculations 31 Iontophoretic study 32 Preparation of the micropipettes 33 RESULTS . . . . . . . . . 34 Presynaptic i n h i b i t i o n 34 Bulbospinal i n h i b i t i o n 45 Recurrent i n h i b i t i o n 55 Reciprocal l a i n h i b i t i o n 56 Control experiments 56 The unconditioned MSR 64 Blood pressure ' 66 Iontophoretic study 67 DISCUSSION 73 Presynaptic i n h i b i t i o n of the MSR 73 Bulbospinal i n h i b i t i o n of the MSR - 77 Recurrent i n h i b i t i o n of the MSR 82 Reciprocal l a i n h i b i t i o n of the MSR . ." 85 The unconditioned MSR 85 Clonidine e f f e c t s 89 CONCLUSIONS 93 REFERENCES . . . . . 95 v i LIST OF FIGURES Figure Page 1. The effects of imipramine HCI on presynaptic i n h i b i t i o n of the QUAD- and the PBST-MSRs . . . . . . . . 36 2. The actions of fluoxetine HCI on presynaptic i n h i b i t i o n of the QUAD- and the PBST-MSRs 37 3. The effects of a cold block and imipramine HG1. on presynaptic i n h i b i t i o n of the QUAD-MSR i n cats pretreated with £-CPA or a-MPT 38 4. The effects of a small dose of imipramine HCI, 5-HTP,vand cyproheptadine HCI on presynaptic i n h i b i t i o n of the QUAD-MSR . 39 5. The blockade of presynaptic i n h i b i t i o n of the QUAD- and the PBST-MSRs by clonidine HCI . 40 6. The time course of presynaptic i n h i b i t i o n of the QUAD-MSR and the f a c i l i t a t i o n of QUAD-AP during conditioning by PBST group I afferents 41 7. The blockade of presynaptic i n h i b i t i o n of the QUAD-MSR and the corresponding f a c i l i t a t i o n of QUAD-AP by imipramine HCI 42 8. The effects of imipramine HCI on the unconditioned QUAD-MSR, presynaptic i n h i b i t i o n of the QUAD-MSR, and the unconditioned and the conditioned QUAD-AP . . . . 43 9. The enhancement of presynaptic i n h i b i t i o n of the QUAD-MSR by cyproheptadine HCI and phenoxybenzamine HCI . 44 10. The blockade of bulbospinal i n h i b i t i o n of the QUAD-MSR by fluoxetine HCI and a reversal of the blockade by'cyproheptadine HCI . 47 11. The effects of fluoxetine HCI and cyproheptadine HCI on bulbospinal i n h i b i t i o n of the QUAD- and the PBST-MSRs .' 48 12. The effects of imipramine HCI,.5-HTP.and:cyproheptadine HCI on bulbospinal i n h i b i t i o n of the QUAD-MSR 49 v i i 13. The blockade of bulbospinal and recurrent-i n h i b i t i o n s of the QUAD-MSR by clonidine HC1 .' 50 14. The enhancement of bulbospinal i n h i b i t i o n of the QUAD- and the PBST-MSRs by cyproheptadine HC1 . . . . . . 51 15. The blockade of bulbospinal i n h i b i t i o n of the dorsal root - ventral root MSR by imipramine HC1 when administered through intravenous and i n t r a -a r t e r i a l routs . 52 16. The blockade of bulbospinal i n h i b i t i o n of the QUATHMSR, the associated reduction i n the a n t i -dromic f i e l d p otential and the f a c i l i t a t i o n of the QUAD-AP by imipramine HC1 . 53 17. The blockade of bulbospinal i n h i b i t i o n of the QUAD-MSR and the associated f a c i l i t a t i o n of the QUAD-AP and i n h i b i t i o n of the antidromic f i e l d p otential by imipramine HC1 54 18. The actions of fluoxetine HC1 on recurrent i n h i b i t i o n of the QUAD- and the PBST-MSRs 58 19. The effects of a cold block and imipramine HC1 on recurrent i n h i b i t i o n of the QUAD-MSR i n cats pretreated with p_-CPA or a-MPT 59 20. The effects of imipramine HC1, 5-HTP and cyproheptadine HC1 on recurrent i n h i b i t i o n of the QUAD-MSR . . . 60 21. The enhancement of recurrent i n h i b i t i o n of the QUAD-MSR by cyproheptadine HC1 and phenoxybenzamine HC1 . . . . . . . . . . . . . 61 22. The antagonism of recurrent i n h i b i t i o n of the dorsal root - ventral root MSR by imipramine HC1 when injected through i n t r a -venous and i n t r a - a r t e r i a l routs . . 62 23. The blockade of reciprocal l a i n h i b i t i o n of the QUAD- and the PBST-MSRs by clonidine HC1 . 63 24. The effects of a cold block, imipramine HC1 and cyproheptadine HC1 on the QUAD-MSR, QUAD-AP and the antidromic f i e l d p otential 65 25. The blockade by clonidine #,g":;ttie depression of a spinal neurone discharge rate induced by Y~amino-butyric acid 70 26. The blockade of the effects .of y-aminobutyric acid and glycine by clonidine 72 v i i i 27. A diagrammatic neuronal arrangement i l l u s t r a t i n g the s u p r a s p i n a l system, i n v o l v i n g 5-HT and n o r -a d r e n a l i n e , i m p l i c a t e d i n t o n i c a l l y antagonizing p r e s y n a p t i c i n h i b i t i o n of the QUAD-MSR 76 28. A diagrammatic neuronal c i r c u i t r y r e p r e s e n t i n g the t o n i c a l l y a c t i v e 5-HT system that i s proposed to antagonize p r e s y n a p t i c and p o s t s y n a p t i c types of b u l b o s p i n a l i n h i b i t i o n of the QUAD-MSR 81 29. A diagrammatic neuronal scheme p o r t r a y i n g the t o n i c a l l y a c t i v e s u p r a s p i n a l system, i n v o l v i n g n o r a d r e n a l i n e and 5-HT, i m p l i c a t e d i n antagonizing r e c u r r e n t i n h i b i t i o n of the QUAD-MSR 84 30. ' A schematic neuronal arrangement i l l u s t r a t i n g : 1. the proposed descending t o n i c a l l y a c t i v e 5-HT system that decreases the e x c i t a b i l i t y o f QUAD l a a f f e r e n t s and 2. the descending t o n i c f a c i l i t a t o r y system that impinges on the extensor motoneurones 88 ix LIST OF DRUGS Drugs Mechanism of action References y-Aminobutyric acid (GAB A) Suggested to be a synaptic transmitter involved i n presynaptic i n h i b i t i o n . Barker and N i c o l l , 1972.; B e l l and Ander-son, 1972; Davidoff, 1972; Eccles et a l . , 1963. Clonidine HCI DL-p_- ch lo r opheny 1-alanine (p_-CPA) Suggested to be a s p e c i f i c a-adrenergic agonist i n the central nervous system. Tryptophan hydroxylase i n h i -b i t o r . Depletes 5-hydroxy-tryptamine i n the spinal cord of the cat. Anden et a l . , 19 70; Finch, 1974; Kobinger and P i c h l e r , 1975. Taber and Anderson, 1973. Cyproheptadine HCI Fluoxetine HCI ( L i l l y 110140) Glycine 5-Hydroxytryptophan (5-HTP) Imipramine HCI 5-Hydroxytryptamine an-tagonist. Blocks 5-hydroxy-tryptophan effects on the monosynaptic reflex. Reported to be a s p e c i f i c 5-hydroxytryptamine neuronal uptake blocking agent. Probably the in h i b i t o r y transmitter of the recurrent and reciprocal l a i n h i b i t o r y pathways i n the spinal cord. A 5-hydroxytryptamine pre-cursor. Reported to elevate 5-hydroxytryptamine levels i n the spinal cord of the cat. 5-Hydroxytryptamine and nor-adrenaline neuronal uptake blocker. Banna and Anderson, 1968. F u l l e r et a l . , 1975; Wong et a l . , 1975. Curtis et a l . , 1968j 1971; Werman e_t a l . , 1968. Anderson and Shi-buya, 1966. Carlsson et a l . 1969a, b. DL- a-me thy l-_p_- ty r o-sine methyl ester HCI (a-MPT) Tyrosine hydroxylase i n h i -b i t o r . Reported to deplete noradrenaline i n the cat's spinal cord. King and Jewett, 1971. Phenoxybenzamine HCI Adrenergic blocker. X ACKNOWLEDGEMENTS The author i s deeply indebted to Dr. John G. S i n c l a i r for his guid-ance and encouragement throughout the course of this work. He i s grateful to Dr. D.M.J. Quastel for his valuable and construc-tive comments and c r i t i c i s m . He i s thankful to Dr. H. McLennan, Dr. J.H. McNeill and Dean B.E. Riedel for reviewing the thesis. The encouragement, i n s p i r a t i o n and help of Judith Anne are invaluable. This study was supported by a grant from the Medical Research Council of Canada to Dr. S i n c l a i r as well as a Medical Research Council Studentship, a Research on Drug Abuse summer Scholarship and a Warner-Lambert Research Fellowship i n Pharmacy to the author. The author wishes to thank the following for t h e i r generous contribu-tion of drugs used i n this investigation: Drs. F u l l e r and Hosley of E l i L i l l y and Co. for fluoxetine HC1, Geigy Ltd. for imipramine HC1, Merck Sharp and Dohme Ltd. for cyproheptadine HC1 and Smith Kline and French Ltd. for phenoxybenzamine HC1. To my parents and brother 1 INTRODUCTION The functional significance of the bulbospinal 5-hydroxytryptamine (5-HT) and noradrenaline neurones i s not w e l l established. We have therefore been investigating the effects of various drugs, which a l t e r monoaminergic synaptic a c t i v i t y , on i n h i b i t i o n s of the lumbosacral mono-synaptic r e f l e x (MSR) i n unanaesthetized decerebrate cats. Previous findings from our laboratory suggest that a 5-HT system antagonizes bulbospinal i n h i b i t i o n of the MSR and a supraspinal system involving 5-HT and noradrenaline decreases recurrent i n h i b i t i o n of the extensor quadriceps MSR (Sastry, 1973; S i n c l a i r and Sastry, 1974, 1974a). In the present investigation experiments were designed to further test the above hypotheses and to determine the location of 5-HT terminals implicated i n antagonizing bulbospinal and recurrent i n h i b i t i o n s . Bulbospinal i n h i b i t i o n of the MSR i s evoked by stimulation i n the ventromedial bulbar r e t i c u l a r formation (Magoun and Rhines, 1946). This i n h i b i t i o n involves a postsynaptic i n h i b i t i o n of the MSR (Llinas and Terzuolo, 1964, 1965; Jankowska et a l . , 1968). Carpenter et a l . (1966) suggested that activation of the dorsal but not the above ventromedial r e t i c u l a r formation evoked dorsal root potentials on spinal l a afferents. However, Chan and Barnes (1972) reported that stimulation i n the ventral bulbar r e t i c u l a r formation could evoke presynaptic i n h i b i t i o n of the MSR. There appears to be a discordance between the l a t t e r two studies. There-fore, experiments were conducted to test whether bulbospinal i n h i b i t i o n of the MSR involves presynaptic and postsynaptic types of i n h i b i t i o n and, i f 2 so, to determine whether 5-HT systems antagonize both types. Imipramine blocked spinal presynaptic inhibition of the MSR in de-cerebrate cats but had l i t t l e effect in animals with a spinal transection (Tan and Henatsch, 1969). Moreover, this agent more effectively antago-nized the inhibition of an extensor than of a flexor MSR. Imipramine is known to block the neuronal uptake of 5-HT and noradrenaline (Carlsson et a l . , 1969a, b). Therefore, the involvement of these biogenic amines in influencing presynaptic inhibition of the extensor and the flexor MSRs was tested. Studies were also undertaken to determine whether monoaminer-gic systems affect reciprocal l a inhibition of the MSR. 3 LITERATURE SURVEY Bulbospinal monoaminergic neurones: There i s strong evidence for the presence of 5-hydroxytryptamine (5-HT) and noradrenaline neurones i n the spinal cord. Stimulation of the isolated r o s t r a l spinal cord of frogs or mice releases 5-HT and noradrenaline (Anden et a l . , 1964, 1965) while chronic transection of the rabbit spinal cord depletes these amines i n the caudal spinal cord (Magnusson and Rosen-gren, 1963; Carlsson et a l . , 1963). Therefore, these monoamines i n the spinal cord are associated with the descending neurones. Histochemical fluorescence studies of Dahlstrom and Fuxe (1964, 1965) indicate that almost a l l the bulbospinal 5-HT neuronal somata are located i n the caudal raphe nuclei of the ventromedial caudal brain stem (Raphe obscurus, pallidus and magnus) and the noradrenaline containing neurones originate mainly i n the medulla oblongata between the r o s t r a l t i p of the i n f e r i o r olivary nucleus and the pyramidal decussation. Some of the 5-HT and noradrenaline containing neurones i n the brain stem make mutual contact while some synapse with other c e l l s (Dahlstrom and Fuxe, 1965; Fuxe, 1965). The bulbospinal 5-HT and noradrenaline axons i n i t i a l l y descend v e n t r a l l y , l a t e r a l to the pyramidal decussation, i n the brain stem. In the spinal cord, the 5-HT fibres descend i n the dorsolateral and the ventromedial f u n i c u l i while the noradrenaline fibres run through the dorsolateral and the ventrolateral f u n i c u l i . Some of the monoaminergic fibres cross to the opposite side (Dahlstrom and Fuxe, 1965). Both the 5-HT and the noradrenaline nerve terminals i n the lumbosacral spinal cord 4 of the cat were found to be dense i n the substantia gelatinosa of the dorsal horn and the dorsolateral and the ventrolateral motor nuclei of the ventral horn (Dahlstrom and Fuxe, 1965; Fuxe, 1965). While some 5-HT and noradrenaline nerve terminals synapse with interneurones, some synapse with motoneurones. The monoaminergic fibres are unmyelinated and about 1um i n diameter (Dahlstrom and Fuxe, 1965). The spinal monosynaptic re f l e x : The group l a afferents which convey impulses from the annulospiral endings of the muscle spindles make synaptic contact with the a-motoneurones i n the ventrolateral spinal motor nuclei which, i n turn, innervate the extrafusal muscle fibres belonging to a synergistic muscle. This r e f l e x i s called the spinal monosynaptic re f l e x (MSR). Experimentally the MSR i s usually evoked by stimulation of a spinal dorsal root or a peripheral a f f e r -ent nerve and recorded from an appropriate ventral root. The synaptic delay across l a afferent - a-motoneurone synapses i s about 0.5 msec (Eccles, 1961). The latency between the stimulation of a dorsal root and the i n i -t i a t i o n of the compound action potential (MSR) on the ventral root i s about 1 msec and includes the conduction time along the afferent f i b r e , the syn-aptic delay and the conduction along the ventral root. The MSR peaks about 0.5 msec after i t s i n i t i a t i o n (Renshaw, 1940). The magnitude of the MSR i s an index of the number of motoneurones that discharge simultaneously. How-ever, the l a afferents depolarize many motoneurones only to a subthreshold l e v e l . These motoneurones are said to be excited subliminally and do not contribute to the size of the MSR. During f a c i l i t a t i o n of the MSR, the motoneurones that were excited subliminally or those that were not a c t i -vated by the test stimulus are recruited into the discharge zone. As more 5 motoneurones discharge, the size of the MSR enhances. The i n h i b i t i o n of the MSR involves elimination of some motoneurones from the discharge zone resulting i n a reduction of the magnitude of the reflex. The synaptic transmitter at the l a afferent - a-motoneurone synapses i s unknown. Substance P, which includes a group of polypeptides, i s more concentrated i n the dorsal than i n the ventral spinal gray matter (Taka-hashi and Otsuka, 1975). This agent was reported to be a potent excitant of a-motoneurones. Li o r e s a l , which blocked the synaptically induced ex-c i t a t i o n of the motoneurones, antagonized the effect of substance P (Saito et a l . , 1975). However, Krnjevic and Morris (1973) reported that the de-pol a r i z a t i o n of c e l l s i n the cuneate nucleus produced by substance P was slow and concluded that this agent may not be the transmitter released by the primary afferents. The levels of L-glutamate i n the dorsal spinal gray matter were re-ported to be higher than i n the ventral gray matter (Davidoff et a l . , 1967). The spinal neurones were excited by iontophoretically applied glutamate (Curtis et a l . , 1960; Haldeman and McLennan, 1972). This action of gluta-mate was reversed by iontophoretically administered glutamic acid diethyl ester which was suggested to be a s p e c i f i c glutamate antagonist (Haldeman et a l . , 1972; Haldeman and McLennan, 1972). However, Zieglgahsberger and P u i l (1973) reported that glutamic acid d i e t h y l ester increased the mem-brane conductance of spinal motoneurones and suggested that this agent i s not a s p e c i f i c glutamate antagonist. Therefore, the p o s s i b i l i t y that glu-tamate i s the transmitter released at the primary afferent terminals de-serves further investigation. 6 The effects on the MSR of agents that a l t e r monoamiriergic synaptic  transmission: In unanaesthetized cats with an acute spinal transection, the 5-HT precursors, 5-hydroxytryptophan (5-HTP, 75 mg/kg i.v.) and l-tryptophan (100 mg/kg i.v.) and the monoamine oxidase i n h i b i t o r , pargyline (30 mg/kg i . v . ) , enhanced the MSR to about 310, 170 and 190% of control, respective-l y . The 5-HT levels i n the spinal cord of the cat were enhanced by about 300% by 5-HTP and 70% by pargyline, at the above doses (Anderson and Shi-buya, 1966; Anderson et a l . , 1967). Pargyline did not s i g n i f i c a n t l y a l t e r the levels of noradrenaline i n the spinal cord (Anderson e t a l . , 1967). The effects of 5-HTP and l-tryptophan on the MSR were potentiated by pre-treatment of the animals with pargyline (Anderson et^ a l . , 1967). Methy-sergide, cinanserin, d-lysergic acid diethylamide (LSD), 2-bromo-LSD and cyproheptadine reversed the f a c i l i t a t o r y effects of 5-HTP and l-tryptophan (Banna and Anderson, 1968). The effect of pargyline was blocked by methy-sergide but not by the ct-adrenergic blocking agent, phenoxybenzamine (An-derson e_t _al. , 1967) . In cats with a chronic spinal transection, l-tryptophan and pargyline did not enhance the MSR, however, 5-HTP s t i l l f a c i l i t a t e d the ref l e x (Shi-buya and Anderson, 1968). In the spinal cord caudal to a chronic tran-section, 25% of the control 5-HT levels (Shibuya and Anderson, 1968), 17% of the tryptophan hydroxylase a c t i v i t y (Clineschmidt et_ _al. , 1971a) and 20% of the dopa decarboxylase a c t i v i t y (Anden, 1965) remained. Therefore, Shibuya and Anderson (1968) suggested that the enhancement of the MSR by 5-HTP i n the animals with a chronic spinal transection might be due to i t s conversion into 5-HT within the 5-HT interneurones of the spinal cord. However, there i s no good evidence for the presence of 5-HT interneurones 7 i n the spinal cord (Dahlstrom and Fuxe, 1965; Fuxe, 1965). Evidence exists to indicate that 5-HT synthesis from 5-HTP can occur extraneuronally: (Kuhar et a l . , 1971). Moreover, 5-HTP can enter adrenergic terminals and displace catecholamines (Ng e_t a l . , 1972) or d i r e c t l y activate adrenergic receptors (Innes, 1962). These observations o f f e r alternative explanations for the enhancement of the MSR by 5-HTP i n animals with a chronic spinal transection as observed by Shibuya and Anderson (1968). Taber and Anderson (1973) reported that the f a c i l i t a t o r y effect of 5-HTP on the MSR was prevented i n cats pretreated with the tryptophan hydroxylase i n h i b i t o r , DL-p_-chlorophenylalanine (p_-CPA, 300 mg/kg i.p. for two consecutive days). The lumbosacral 5-HT levels were depleted by about 90% i n these animals. These authors suggested that i n the above animals 5-HT, synthesized from 5-HTP, entered the empty synaptic vesicles but did not overflow into the synaptic c l e f t s to stimulate the receptors. The t r i c y c l i c antidepressants, a m i t r i p t y l i n e , imipramine and desipra-mine, potentiated the f a c i l i t a t o r y effect of 5-HTP on the MSR i n cats with a s p i n a l transection (Clineschmidt et a l . , 1971). Furthermore, imipramine augmented the actions of 5-HTP and pargyline on the re f l e x i n cats with an acute but not a chronic spinal transection (Clineschmidt et a l . , 1971; Clineschmidt, 1972). The above findings indicate that a descending 5-HT system i n the spinal cord of the cat has a f a c i l i t a t o r y effect on the MSR. We observed that a thoracic cold block, which eliminates supraspinal inputs to the caudal s p i -n a l cord, reduced the quadriceps but not the posterior biceps-semitendinosus MSR by about 45 per cent (Sastry, 1973). Baker and Anderson (1970) reported that 1-3,4-dihydroxyphenylalanine (1-dopa, 30 mg/kg i.v.) enhanced the MSR to about 210% of control and 8 pargyline pretreatment potentiated t h i s e f f e c t of ^ -dopa. Phenoxybenzamine, chlorpromazine and ethobutamoxane antagonized the f a c i l i t a t i o n of the re-f l e x by l^dopa (Baker and Anderson, 1970a). After an i n j e c t i o n of 1^ -dopa (20 mg/kg i . v . ) , dopamine levels i n the spinal cord were elevated by about 30 f o l d , however, noradrenaline levels were not s i g n i f i c a n t l y increased. Therefore, Baker and Anderson (1970) concluded that the effect of 1-dopa on the MSR might be mediated through dopamine. However, there i s no e v i -dence for the presence of dopaminergic pathways i n the spinal cord (Dahl-strom and Fuxe, 1965; Fuxe, 1965). The effects of iontophoretically applied 5-HT and noradrenaline on  motoneurones: Iontophoretically applied 5-HT and noradrenaline hyperpolarized many motoneurones and blocked the soma-dendritic component of the motoneurone antidromic action potentials. However, a few c e l l s were excited by these amines ( P h i l l i s et a l . , 1968). Engberg and Ryall (1966) reported that nor-adrenaline reduced the DL-homocysteate induced f i r i n g rate of the moto-neurones. Engberg and Marshall (1971) found that noradrenaline increased the motoneurone membrane resistance and the hyperpolarization produced by thi s agent was augmented by a conditioning hyperpolarization and reduced by a depolarization. Hence, these authors suggested that noradrenaline might reduce the permeability of the motoneurone membrane to Na +. However, i n a subsequent report Marshall and Engberg (1973) reported that H +, when iontophoretically applied near the motoneurones, produced effects s i m i l a r to those of iontophoretically applied noradrenaline. Engberg and Marshall (1971) were passing iontophoretic currents of about 100 nA to eject nor-adrenaline from a low pH solution. P h i l l i s and coworkers ( P h i l l i s , 1974; 9 P h i l l i s et a l . , 1973; Yarbrough et a l . t 1974) postulated that the depres-sant effects of 5-HT and noradrenaline on the cerebral c o r t i c a l neurones I | might be mediated by Ca . I t i s unknown whether a s i m i l a r s i t u a t i o n ex-i s t s for the effects of these amines on the spinal motoneurones. Krnjevic and Kisiewicz (1972) reported that i n j e c t i o n of Ca into the motoneurones resulted i n a hyperpolarization of these neurones accompanied by an increase i n membrane conductance i n some c e l l s and no a l t e r a t i o n i n the membrane conductance of other c e l l s . These authors suggested that the i n t r a c e l l u l a r Ca might increase the e f f l u x of K . From the above studies i t i s apparent that further investigation i s necessary to understand the mechanisms of ac-t i o n of 5-HT and noradrenaline on the motoneurones. Presynaptic i n h i b i t i o n of the MSR: When a spinal dorsal root was stimulated an exponentially decaying positive p o t e n t i a l , the P wave or the cord dorsum p o t e n t i a l , was recorded from an electrode positioned on the spinal cord near the dorsal root entry zone (Gasser and Graham, 1933). Barron and Matthews (1938) reported that stimulation of a dorsal root e l i c i t e d an exponentially decreasing negative p o t e n t i a l , the negative dorsal root potential (DRP), on a rootlet of the above root and on anaadjacent dorsal root. These authors postulated that the source of the P wave and the DRP was the same. The DRP represents an increase i n the e x c i t a b i l i t y of the primary afferents (Wall, 1958) r e s u l t -ing from a depolarization of these fibres (Eccles and Krnjevic, 1959; Eccles et a l . , 1962, 1962a, b). Although a measurement of the potential change i n -side the afferent terminals was not possible, i t was inferred that the ob-served increase of the e x c i t a b i l i t y originated at the terminals since the afferent fibres were depolarized (Eccles e_t ' a l . , 1962, 1962a, b) . 10 When the spinal MSR was conditioned with a p r i o r stimulation of the group I afferents belonging to a flexor or the extensor quadriceps muscle, the r e f l e x was gradually i n h i b i t e d . This i n h i b i t i o n of the MSR began about 5 msec after the conditioning stimulus, peaked after 20 msec and had a du-ration of approximately 200 msec. A s i m i l a r time course was observed for the DRP (Brooks et a l . , 1948; Frank and Fuortes, 1957). During the above i n h i b i t i o n of the MSR the excitatory postsynaptic potentials (EPSPs) of a p a r t i c i p a t i n g motoneurone were reduced. However, stimulation of the con-ditioning afferents did not a l t e r the motoneurone membrane potential at i t s normal resting l e v e l or at a depolarized or hyperpolarized state (Frank and Fuortes, 1957; Frank, as cited by Eccles, 1964). Therefore, i t i s generally believed that the depression of the motoneurone EPSPs during the above i n h i -b i t i o n of the MSR i s due to a reduction i n the efficacy of the presynaptic function. The i n h i b i t i o n i s thus referred to as presynaptic i n h i b i t i o n (Frank and Fuortes, 1957). However, Frank (1959) suggested that i f the i n -h i b i t i o n of the motoneurones i s on the dendrites, a microelectrode situated within the motoneurone soma might not be able to record the hyperpolariza-t i o n and hence he called the i n h i b i t i o n "remote" i n h i b i t i o n . Depolarization of the motor nerve terminals i n the rat diaphragm re-sulted i n a decrease i n the size of the end plate potentials, however, the magnitude of the miniature end plate potentials was unaltered. Therefore, i t was suggested that the reduction of the end plate potential was due to a decrease i n the number of quanta of the transmitter liberated by the nerve impulse (Hubbard and W i l l i s , 1962). Moreover, Takeuchi and Takeuchi (1962) observed that, at the giant synapses of Loligo, the magnitude of the post-synaptic potential was dependent on the size but not the l e v e l of the peak of the presynaptic action p o t e n t i a l . Therefore, Eccles (1964) postulated 11 that an action potential reaching a depolarized primary afferent terminal would release less synaptic transmitter than control, thereby, producing a smaller motoneurone EPSP. Since the latency between the conditioning stimulus and the onset of i n h i b i t i o n of the MSR i s about 5 msec and the synaptic delay at a single central synapse i s approximately 0.5 msec (Eccles, 1961), i t was hypothe-sized that the presynaptic i n h i b i t o r y pathway involves two or more s e r i a l l y arranged interneurones (Eccles et a l . , 1962). Gray (1962, 1963) discovered axo-axonal synapses i n the spinal cord. Hence, Eccles (1964) suggested that the l a s t interneurone i n the above i n h i b i t o r y pathway makes axo-axonal synapse with the primary afferent terminal. A neutral amino acid, Y _a minobutyric acid (GABA), and the enzyme that decarboxylates glutamic acid to form GABA, glutamic acid decarboxylase, were found i n the f e l i n e spinal cord with higher levels i n the dorsal than i n the ventral gray matter (Graham et a l . , 1967; Graham and Aprison, 1969; Albers and Brady, 1959). In the cat, loss of spinal interneurones resulted i n a decrease i n the levels of GABA (Miyata and Otsuka, 1972; Davidoff ++ 3 ejt _al. , 1967). A Ca dependent release of previously loaded H-GABA was observed i n the isolat e d amphibian spinal cord when the r o s t r a l s pinal cord was stimulated ( C o l l i n s , 1974). When GABA was t o p i c a l l y applied on the fe l i n e spinal cord, the DRP was blocked but the afferent fibres were depo-l a r i z e d (Eccles e_t a l . , 1963) . Similar effects were observed on the spinal ecofd of the frog (Schmidt, 1963; Barker and N i c o l l , 1972; Davidoff, 1972). Depletion of the spinal cord GABA by pretreatment of cats with semicarbazide resulted i n a blockade of the DRP ( B e l l and Anderson, 1972). Based on the observation that Mg blocked the DRP but did not a l t e r the depolarization of the primary afferent filaments by GABA, Davidoff (1972) and Barker and 12 N i c o l l (1972) suggested that the above e f f e c t of GABA was on the a f f e r e n t t e r m i n a l s . Levy (1974) reported that i n t r a v e n o u s l y i n j e c t e d GABA (100 mg/kg) d e p o l a r i z e d the f e l i n e d o r s a l root f i laments that were e i t h e r cut p e r i p h e r a l l y at the d o r s a l root e x i t through the dura or s e c t i o n e d c e n t r a l l y at the d o r s a l root entry zone on the s p i n a l c o r d . Furthermore the GABA a n t a g o n i s t s , b i c u c u l l i n e and p i c r o t o x i n , blocked the DRPs and a l s o the a c -t i o n o f GABA on the primary a f f e r e n t s (Eccles eit a l . , 1963; D a v i d o f f , 1972; Levy, 1974). Degroat et a l . (1972) reported that GABA d e p o l a r i z e d the d o r -s a l r o o t g a n g l i a of the c a t . The d e p o l a r i z a t i o n of amphibian sensory gan-g l i a produced by t h i s amino a c i d was r e p o r t e d to be dependent on the e x t r a -c e l l u l a r C l c o n c e n t r a t i o n ( N i s h i ^ i t a l . , 1974). Although GABA d e p o l a r i z e d the primary a f f e r e n t s when t o p i c a l l y a p p l i e d on the s p i n a l cord or i n t r a -venously i n j e c t e d , t h i s agent reduced the e x c i t a b i l i t y of these f i b r e s when i o n t o p h o r e t i c a l l y a p p l i e d at the a f f e r e n t terminals ( C u r t i s and R y a l l , 1966). T h e r e f o r e , although GABA appears to be i n v o l v e d i n the p r o d u c t i o n of p r e -s y n a p t i c i n h i b i t i o n , i t s s i t e o f a c t i o n i s s t i l l u n c e r t a i n . During the s t i m u l a t i o n of the primary a f f e r e n t s , the e x t r a c e l l u l a r K + c o n c e n t r a t i o n i n the s p i n a l cord i n c r e a s e d ( K r i z et a l . , 1974; Singer and Lux, 1973; K r n j e v i c and M o r r i s , 1972; ten Bruggencate e_t a l . , 1974). K r n j e -v i c and M o r r i s (1972) and K r i z et_ a l . (1974) suggested that t h i s i n c r e a s e i n the K + l e v e l s might be r e s p o n s i b l e f o r the p r o d u c t i o n of p r e s y n a p t i c i n h i b i t i o n . However, ten Bruggencate e_t a l . (1974) found that p i c r o t o x i n + R b l o c k e d the DRPs and i n c r e a s e d the e x t r a c e l l u l a r K l e v e l s whereas Nembutal enhanced the DRPs but reduced the e x t r a c e l l u l a r K + c o n c e n t r a t i o n . C u r t i s e t a l . (1971a) suggested that GABA, w h i l e i n h i b i t i n g the s p i n a l i n t e r n e u r o n e s , may r e l e a s e K + at the i n h i b i t o r y synapses. These i o n s may d e p o l a r i z e the primary afferent t e r m i n a l s . T h i s suggestion i s not only 13 consistent with the depressant effects of GABA i n the cat spinal cord but also with i t s presumably indirect excitatory action on the afferent termi-nals. However, the above proposal i s not supported by any experimental evidence. Carpenter e_t a l . (1963) reported that i n unanaesthetized decerebrate cats, the DRP, evoked by stimulation of the flexor or the extensor group I afferents, was either unchanged or s l i g h t l y reduced after a spinal tran-section. Presynaptic i n h i b i t i o n of the MSR was attributed to only group I afferent activation (Eccles et a l . , 1961). Therefore, Carpenter et a l . (1963) suggested that presynaptic i n h i b i t i o n of the MSR i s not under a tonic influence of a supraspinal system. However, group l a and lb afferents can produce DRPs on lb afferents as wel l (Eccles et a l . , 1962). Furthermore, i t i s not known whether, i n the study of Carpenter et al. (1963), the DRPs were evoked on the flexor or the extensor afferents or on both. Stimulation of the group I I and I I I , cutaneous and the high threshold j o i n t afferents, c o l l e c t i v e l y called the fl e x i o n r e f l e x afferents (Eccles and Lundberg, 1959), results i n i n h i b i t i o n of an extensor and f a c i l i t a t i o n of a flexor MSR. In decerebrated cats, the above effects of the fl e x i o n re-f l e x afferents are suppressed but reappear after a spinal transection (Holm-qvist and Lundberg, 1961). In order to evoke presynaptic i n h i b i t i o n of the MSR, without the interference of the effects of the fl e x i o n r e f l e x afferents, i t would be necessary to use a conditioning stimulating strength that a c t i -vates only group I afferents. Eccles et a l . (1962) suggested that at a conditioning i n t e r v a l that exceeds 25 msec the interference of postsyn-aptic i n h i b i t i o n with presynaptic i n h i b i t i o n of the MSR was minimal. In decerebrate cats, the monoamine neuronal uptake blocking agent, imipramine, antagonized presynaptic i n h i b i t i o n of the MSR. This effect of 14 imipramine was not present after a spinal transection (Tan and Henatsch, 1969). Therefore, imipramine i s very l i k e l y acting through a supraspinal system. Moreover, this agent had a greater blocking action on the i n h i b i -tion of an extensor than on that of a flexor MSR (Tan and Henatsch, 1969). Postsynaptic i n h i b i t i o n of the MSR: Postsynaptic i n h i b i t i o n of the MSR involves a direct synaptic i n h i b i -tion of the a-motoneurones. Examples of postsynaptic i n h i b i t i o n include reciprocal ( d i r e c t , la) i n h i b i t i o n (Lloyd, 1941) and recurrent (antidromic) i n h i b i t i o n (Renshaw, 1941). Reciprocal i n h i b i t i o n i s exerted by l a af f e r -ents activating l a i n h i b i t o r y interneurones which impinge on a-motoneurones of antagonistic muscles (Eccles et a l . , 1956). Recurrent i n h i b i t i o n i s produced by discharges i n the motor axon c o l l a t e r a l s that activate i n h i b i -tory interneurones, Renshaw c e l l s , which i n turn i n h i b i t the motoneurones (Eccles et a l . , 1954a). The resting membrane potential of the fe l i n e a-motoneurones i s about -70 mV (Frank and Fuortes, 1955). During postsynaptic i n h i b i t i o n of the MSR the motoneurones are hyperpolarized producing the i n h i b i t o r y postsynap-t i c potential (IPSP, Brock et d . , 1952). During the IPSP the membrane conductance i s increased. The equilibrium potential for the IPSP i s about -80 mV (Coombs et a l . , 1955). An inward d i f f u s i o n of CI and an e f f l u x of K + were suggested to occur during the IPSP (Coombs et a l . , 1955). However, Lux et a l . (1970), Lux (1971) and Llinas and Baker (1972) proposed that the IPSP i s generated by a selective increase i n permeability to CI . They also reported that K + permeability change i s probably not involved i n this process. However, i n a recent lecture Eccles (1975) stated that i n the hippocampal pyramidal c e l l s the IPSP i s generated by an i n f l u x of CI and 15 probably an e f f l u x of K1", supporting the suggestion of Coombs et a l . (1955) . a. Reciprocal l a i n h i b i t i o n : Reciprocal l a i n h i b i t i o n of a MSR i s produced by stimulation of group l a afferents belonging to an antagonistic muscle. This i n h i b i t o r y pathway involves an interneurone interposed between the conditioning l a afferent terminal and the a-motoneurone (Eccles e_t a l . , 1956) . The i n h i b i t i o n i s observed when the conditioning stimulus preceeds the test stimulus by less than 1 msec, i s maximal when the conditioning i n t e r v a l i s approximately 1 msec and i s present for about 2 msec (Lloyd, 1941). Glycine, a basic amino acid, i s present i n the sp i n a l cord and i s more concentrated i n the ventral than i n the dorsal gray matter (Graham et_ a l . , 1967). Destruction of spinal interneurones resulted, i n a reduction i n the levels of this amino acid (Davidoff e_t a l . , 1967). Iontophoretically ap-p l i e d glycine hyperpolarized the motoneurones and reduced the motoneurone membrane resistance. P r i o r hyperpolarization of the membrane reduced or reversed the glycine induced hyperpolarization. The equilibrium potentials of the i n h i b i t o r y postsynaptic potential and the hyperpolarization produced by glycine were s i m i l a r (Wermari et a l . , 1968; Curtis et a l . , 1968). Strych-nine blocks reciprocal l a i n h i b i t i o n of the MSR (Eccles et a l . , 1954a) and i s reported to be a s p e c i f i c glycine antagonist i n the f e l i n e s pinal cord (Curtis et^ al., 1971). Therefore, the transmitter released from the l a in h i b i t o r y interneurones i s probably glycine. The l a i n h i b i t o r y interneurones were i n h i b i t e d by the activation of the motor axon c o l l a t e r a l s (Hultborn et a l . , 1971). Therefore, Hultborn et a l . (1971) suggested that the enhancement of motoneuronal discharges results i n a reduction i n the f i r i n g of the l a i n h i b i t o r y interneurones 16 that innervate the antagonistic motoneurones. The disynaptic i n h i b i t i o n of the a-motoneurones by the descending vestibulo-, rubro-, and corti c o -spinal tracts was found to involve the l a in h i b i t o r y interneurones. More-over, these interneurones were also excited by high threshold muscle a f f e r -ents and cutaneous impulses (Eccles et a l . , 1956). Therefore, there seems to be a convergence of supraspinal and spinal afferent excitatory input to the l a i n h i b i t o r y interneurones. b. Recurrent i n h i b i t i o n : As described previously, discharges i n motor axon c o l l a t e r a l s synap-t i c a l l y excite Renshaw c e l l s which i n turn i n h i b i t the motoneurones. Ren-shaw c e l l s , activated i n the above fashion, f i r e i n a ch a r a c t e r i s t i c burst with an i n i t i a l frequency of greater than 1000 spikes per sec which gradu-a l l y decreases to control f i r i n g over a period of 50 msec (Eccles et_ a l . , 1954a, 1956a). A high convergence of excitatory input from the c o l l a t e r a l s of many motor axons was suggested to be responsible for the above mentioned high frequency discharge of Renshaw c e l l s (Eccles e_t a l . , 1956a; R y a l l et a l . , 1972). Recurrent i n h i b i t i o n begins about 2 - 3 msec af t e r the condi-tioning stimulation of the motor axons, peaks after approximately 5 msec and decays over a period of about 50 msec (Renshaw, 1941). The pharmacological and physiological studies indicate that the motor axon c o l l a t e r a l - Renshaw c e l l synapse i s cholinergic (Curtis and R y a l l , 1966a, b, c; Eccles et a l . , 1956a). Dihydro- (3-erythroidine, which blocks cholinergic transmission at the n i c o t i n i c receptors, reduced the Renshaw c e l l response to synaptic activation (Eccles et a l . , 1956a; Curtis et a l . , 1966b). Eserine, an anticholinesterase drug, greatly prolonged the d i s -charges of Renshaw c e l l s induced by synaptic excitation (Eccles et a l . , 17 1954a, 1956a). I n t r a - a r t e r i a l l y injected acetylcholine or nicotine excite Renshaw c e l l s (Eccles et a l . , 1956a; Curtis and R y a l l , 1966a). The above action of acetylcholine, but not nicotine, i s enhanced by eserine whereas dihydro-6-erythroidine reduced the excitatory action of both these sub-stances (Eccles et a l . , 1956a). The i n h i b i t o r y transmitter at the Renshaw c e l l - motoneurone synapses was suggested to be glycine (Curtis e_t a l . , 1968; C u r t i s , 1969; Curtis et a l . , 1971; Werman et a l . , 1968). In cats anaesthetized with chloralose, antidromic volleys i n the motor axons were reported to sometimes produce an i n h i b i t i o n of Renshaw c e l l s i n -stead of an excitation ( R y a l l , 1970). The latency observed for this i n h i -b i t i o n suggested that the effect was brought about by a disynaptic pathway involving a Renshaw c e l l . Therefore, some Renshaw c e l l s i n h i b i t the f i r i n g of other Renshaw c e l l s ( R y a l l , 1970). The l a i n h i b i t o r y interneurones were shown to be i n h i b i t e d by Renshaw c e l l s , as mentioned previously. However, there seems to be no input from the l a i n h i b i t o r y interneurones to Renshaw c e l l s (Ryall and Piercey, 1971). Volleys i n the i p s i l a t e r a l flexon r e f l e x afferents excite Renshaw c e l l s through a polysynaptic chain, whereas, d i s -charges i n the above afferents on the contralateral side i n h i b i t these c e l l s (Ryall and Piercey, 1971). The c o l l a t e r a l s of large phasic a-motoneurone axons more strongly excite Renshaw c e l l s than those of the small tonic motoneurone axons. The small tonic motoneurones rather than the large phasic motoneurones are more e f f e c t i v e l y i n h i b i t e d by Renshaw c e l l s (Ryall et al., 1972). Wilson et a l . (1960) reported that recurrent i n h i b i t i o n of extensor or flexor motoneurones was more e f f e c t i v e l y produced by antidromic activation of motor axons belonging to the same or synergistic muscles. In cats anaesthetized with chloralose, stimulation of the ventromedial mesencephalic r e t i c u l a r formation at the l e v e l of substantia nigra (A 6.5 -18 1, In the stereotaxic atlas of Snider and Niemer, 1964) or i n decerebrate cats, activation of the ventrolateral bulbar r e t i c u l a r formation at the l e v e l of the hypoglossal nucleus (P 9.5 - 11) was found to i n h i b i t the rate of Renshaw c e l l discharges evoked by antidromic volleys i n the motor axons (Koizumi et a l . , 1959; Haase and Van der Meulen, 1961; MacLean and Leffman, 1967). The above i n h i b i t i o n could be produced by stimulating either side of the r e t i c u l a r formation but was stronger when the contralateral r e t i c u -l a r formation was stimulated (Haase and Van der Meulen, 1961). The latency between stimulation of the mesencephalic r e t i c u l a r formation and the onset of the i n h i b i t i o n of Renshaw c e l l f i r i n g rate was found to be about 9 msec. This i n h i b i t i o n lasted at i t s maximum strength for approximately 25 msec. While the i n h i b i t i o n of Renshaw c e l l discharge rate by activation of the mesencephalic r e t i c u l a r formation was stronger, the i n h i b i t i o n produced by stimulation of the bulbar r e t i c u l a r formation had a longer duration (Mac-Lean and Leffman, 1967). Following the stimulation of the ventral thalamus ( f i e l d s of f o r e l , zona incerta or the pericruciate Cortex) the Renshaw c e l l discharges, evoked by activation of the motor axons, were reduced i n number. (MacLean and Leffman, 1967). On the other hand, stimulation of the cere-b e l l a r anterior lobe f a c i l i t a t e d the synaptically induced Renshaw c e l l d i s -charge rate (Haas and Van der Meulen, 1961). In addition, stimulation of the ventral thalamus or the pericruciate cortex could sometimes activate the Renshaw c e l l s (MacLean and Leffman, 1967). The above findings indicate that Renshaw c e l l discharges are influenced by supraspinal and spi n a l afferent inputs. Recurrent i n h i b i t i o n of the quadriceps MSR i n decerebrate cats was en-hanced during a thoracic cold block, a procedure that reversibly blocks supraspinal inputs to the lumbosacral spinal cord (Sastry, 1973; S i n c l a i r 19 and Sastry, 1974a). Therefore, we proposed that the above i n h i b i t i o n i s under a tonic i n h i b i t o r y influence of a descending system. Tan and Henatsch (1969) reported that imipramine antagonized recurrent i n h i b i t i o n of the MSR i n decerebrate cats. They also found that imipramine f a i l e d to a l t e r the i n h i b i t i o n when the spinal cord was transected at the thoracic l e v e l . This agent had a greater blocking effect on recurrent i n -h i b i t i o n of an extensor than of a flexor r e f l e x . In a follow up study we found that i n decerebrate cats, imipramine antagonized recurrent i n h i b i t i o n of the quadriceps but not of the posterior biceps-semitendinosus MSR. The monoamine oxidase i n h i b i t o r , pargyline, also antagonized the i n h i b i t i o n of the MSR. A thoracic cold block completely reversed the blockade by imipra-mine. Moreover, depletion of 5-HT or noradrenaline completely prevented the action of imipramine on the i n h i b i t i o n of the quadriceps MSR. There-fore, we proposed that a supraspinal system, which involves 5-HT and nor-adrenaline, antagonizes recurrent i n h i b i t i o n of the extensor but not of the flexor MSR (Sastry, 1973; S i n c l a i r and Sastry, 1974a). Iontophoretically applied 5-HT and noradrenaline either increased or decreased Renshaw c e l l f i r i n g but the predominant effect was i n h i b i t i o n (Biscoe and Curtis, 1966; Engberg and R y a l l , 1966; Weight and Salmoiraghi, 1966). Bulbospinal i n h i b i t i o n of the MSR: The lumbosacral MSR i s i n h i b i t e d following a stimulation i n the ven-tromedial bulbar r e t i c u l a r formation (Magoun and Rhines, 1946). During this bulbospinal i n h i b i t i o n of the MSR the motoneurones were hyperpolarized, the resistance of the motoneurone membrane was reduced and the soma-dendrit-i c component of the action potential was blocked when the motoneurones were 20 activated antidromically (Llinas and Terzuolo, 1964, 1965; Jankowska et a l . , 1968). These findings indicate that bulbospinal i n h i b i t i o n of the MSR i n -volves a postsynaptic type of i n h i b i t i o n . When chloride ions were ionto-phor e t i c a l l y injected into the extensor motoneurones, the hyperpolarization produced during stimulation of the bulbar r e t i c u l a r formation was reversed (Llinas and Terzuolo, 1964; Jankowska et a l . , 1968). Therefore, the i o n i c mechanisms responsible for bulbospinal i n h i b i t i o n of the extensor MSR ap-pear to be s i m i l a r to those of reciprocal l a i n h i b i t i o n . Llinas and Ter-zuolo (1965) found that i n j e c t i o n of Cl into the flexor a-motoneurones did not reverse the hyperpolarization during bulbospinal i n h i b i t i o n . Therefore, these authors suggested that the bulbospinal i n h i b i t o r y synapses on the flexor motoneurones are on the dendrites. However, Jankowska et a l . (1968) did not observe any difference between the flexor and extensor motoneurones i n this regard. The discrepancy between the two above studies may be due to the difference i n the experimental preparations. Jankowska et a l . (1968) performed t h e i r experiments on decerebrate cats with a contralateral hemi-sected and an i p s i l a t e r a l dorsal transected spinal cord. The spinal cord was intact i n the study of Llinas and Terzuolo (1965). The bulbospinal i n h i b i t o r y pathway, which descends i n the ventral quadrant of the s p i n a l cord, l i k e l y has a disynaptic linkage involving an interneurone i n the spinal cord (Jankowska et a l . , 1968; Clineschmidt and Anderson, 1970). The conduction vel o c i t y of this pathway was reported to be high. Stimulation of the medial r e t i c u l a r formation i n the caudal brain stem, 1 mm below the f l o o r of the fourth v e n t r i c l e (V about -5 to -6 i n the stere-otaxic atlas of Snider and Niemer, 1964), produced negative DRPs on flexor and extensor l a afferents (Carpenter et a l . , 1966). These r e t i c u l o s p i n a l 21 fibres descend i n the ventromedial spinal cord. Carpenter et a l . (1966) also reported that stimulation of the ventral caudal bulbar r e t i c u l a r formation (about 4 mm below the f l o o r of the fourth ventricle) did not produce DRPs on these afferents. However, Chan and Barnes (1972) found that stimulation i n the ventral caudal bulbar r e t i c u l a r formation, 2 mm l a t e r a l from the mid-sagittal l i n e , resulted i n short and long latency DRPs on l a afferents. Chan and Barnes also noted a time correlation be-tween the e x c i t a b i l i t y of the l a afferents, the DRP and the i n h i b i t i o n of the MSR while stimulating i n the bulbar area. Therefore, bulbospinal i n -h i b i t i o n of the MSR appears to involve a presynaptic type of i n h i b i t i o n as we l l . Llinas (1964) found that strychnine (0.15 and 0.5 mg/kg i.v.) de-creased the hyperpolarization of the extensor motoneurones during bulbo-spinal i n h i b i t i o n but did not antagonize the i n h i b i t i o n of the MSR. This f a i l u r e of strychnine to reverse the i n h i b i t i o n of the MSR may be due to the presence of the presynaptic type of bulbospinal i n h i b i t i o n which may not be affected by strychnine. Clineschmidt and Anderson (1970) reported that methysergide, cinanserin, ^ - l y s e r g i c acid diethylamide (LSD) and 2-bromo-LSD antagonized bulbospinal i n h i b i t i o n . The results of i n t r a - a r t e r i a l i n j e c t i o n of methysergide and LSD to the spinal cord and the brain stem suggested that these agents block bulbospinal i n h i b i t i o n at the spinal cord l e v e l . Therefore, Clineschmidt and Anderson (1970) proposed that a 5-HT interneurone i n the spinal cord i s involved i n the bulbospinal i n h i b i t o r y pathway. However, there i s no good evidence for the existence of 5-HT c e l l bodies i n the spinal cord (Dahlstrom and Fuxe, 1965; Fuxe, 1975). In a subsequent study, Proudfit and Anderson (1973) suggested that the blockade of bulbospinal i n h i b i t i o n by cinanserin and methysergide could result from an enhancement of bulbo-22 spinal f a c i l i t a t i o n i f t h i s f a c i l i t a t o r y pathway i s under i n h i b i t o r y i n -fluence of a t o n i c a l l y active 5-HT system. However, we found that methy-sergide converted bulbospinal i n h i b i t i o n into a 3 - 4 f o l d f a c i l i t a t i o n i n animals that were pretreated with p-chlorophenylalanine (Sastry, 1973; S i n c l a i r and Sastry, 1974). Hence, methysergide does not appear to act as a 5-HT antagonist i n the system. Imipramine (5 mg/kg i . v . ) , desipramine (4.8 mg/kg i.v.) and pargyline (30 mg/kg i . v . ) , drugs that would be expected to enhance 5-HT a c t i v i t y , antagonized bulbospinal i n h i b i t i o n of the MSR i n unanaesthetized decerebrate cats. Moreover, the above effect of imipramine was completely prevented by a depletion of 5-HT but not noradrenaline. Therefore, we proposed that bulbospinal i n h i b i t i o n of the MSR i s under an in h i b i t o r y influence of a 5-HT system (Sastry, 1973; S i n c l a i r and Sastry, 1974). Proudfit and Anderson (1974) reported that cinanserin and methysergide blocked DRPs on presumably group l a afferents evoked by bulbar stimulation. Therefore, they proposed that a 5-HT system depolarizes these afferent terminals. 23 EXPERIMENTAL S u r g i c a l procedures: These experiments were c a r r i e d out i n a s h i e l d e d room on a t o t a l of 119 cats o f e i t h e r sex weighing between 2.3 and 3.7 kg. The animals were anaesthet ized w i t h ether s o l v e n t U . S . P . and the t r a c h e a was then cannulated to a r t i f i c i a l l y r e s p i r e and maintain the animal under anaesthesia with the help of a Palmer r e s p i r a t o r y pump (type AC; HP 1/4). The l e f t c a r o t i d a r -t e r y was cannulated w i t h a No. 160 polyethylene tubing (Clay Adams) f i l l e d w i t h d i l u t e d sodium h e p a r i n (Upjohn Company of Canada). In some e x p e r i -ments t h i s tubing was connected to a P-1000-A pressure transducer which was i n t u r n connected to a DAM-4A physiograph (Narco-Bio-Systerns) f o r r e -c o r d i n g the b l o o d p r e s s u r e . In other experiments the tubing was attached to a Statham P23AC pressure transducer which was connected to a 79D p o l y -graph (Grass Instruments Company) to monitor the b l o o d p r e s s u r e . The r i g h t c a r o t i d a r t e r y was l i g a t e d . The a n i m a l ' s head was f i x e d to a N a r i s h i g e s t e r e o t a x i c head h o l d e r . The s k u l l bone o v e r l y i n g the f r o n t a l and the p a r i e t a l c e r e b r a l cortex was then removed and the animal decerebrated at the m i d - c o l l i c u l a r l e v e l . The b r a i n t i s s u e r o s t r a l to the t r a n s e c t i o n was removed and the s k u l l c a v i t y packed w i t h gauze. The cut edges o f the bone were f i l l e d with bone wax to prevent a i r embolism and b l e e d i n g . About 5 ml o f dextran (average mol. wt. 170,000, 6% W/V; Sigma Chemical Co.) were i n f u s e d immediately a f t e r d e c e r e -b r a t i o n to compensate f o r b l o o d l o s s . The o c c i p i t a l bone covering the c e r -ebellum was removed and the dura was s e c t i o n e d to expose the cerebellum. 24 A lumbosacral s p i n a l laminectomy was performed and the L 6 , L7 and SI v e n t r a l roots were s e c t i o n e d b i l a t e r a l l y . In twelve animals the c o r r e s p o n d -i n g d o r s a l roots were cut b i l a t e r a l l y but i n the r e s t of the animals the l e f t d o r s a l roots were l e f t i n t a c t and the p e r i p h e r a l nerves l e a d i n g to the extensor quadriceps (QUAD) and the f l e x o r p o s t e r i o r biceps-semitendinosus (PBST) muscles were i s o l a t e d and cut . B i p o l a r p l a t i n u m e l e c t r o d e s were attached to the c e n t r a l ends of the above nerves. The s k i n f l a p s on the a n i m a l ' s back were used to make a p o o l f o r h o l d i n g m i n e r a l o i l which p r e -vents d r y i n g o f the exposed s p i n a l cord and stops the spread of c u r r e n t d u r i n g s t i m u l a t i o n of the s p i n a l r o o t s . The temperature o f the m i n e r a l o i l p o o l and the body of the animal was maintained at 36 ± 1° C w i t h the a i d of automatic D.C. temperature r e g u l a t o r s (Richardson et a l . , 1965) or a h e a t -i n g lamp. In most of the animals an a d d i t i o n a l s p i n a l laminectomy was performed at the lower t h o r a c i c l e v e l (T10 - T12), the s p i n a l cord was exposed and kept warm w i t h m i n e r a l o i l so that a f u n c t i o n a l c o l d b l o c k (a procedure d e s c r i b e d l a t e r i n t h i s s e c t i o n ) could be a p p l i e d . In some animals that were used f o r m i c r o e l e c t r o d e s t u d i e s a b i l a t e r a l pneumothorax was performed to minimize the r e s p i r a t i o n induced movement of the s p i n a l c o r d . To i n t r a - a r t e r i a l l y administer a t e s t drug to the s p i n a l c o r d , a No. 90 p o l y e t h y l e n e t u b i n g f i l l e d w i t h 0.9% NaCl (W/V) was i n s e r t e d i n four animals through the l e f t femoral a r t e r y i n t o the abdominal a o r t a such that the t i p of the cannula l a y about 1 cm caudal to the e x i t of the r e n a l a r t e r i e s . A l l the major a r t e r i e s below the t i p o f the cannula except those l e a d i n g to the s p i n a l cord were l i g a t e d as d e s c r i b e d by Holmstedt and Skog-lund (1953). 25 In another four animals, to i n j e c t a drug i n t r a - a r t e r i a l l y to the brain stem, a No. 60 polyethylene tubing f i l l e d with 0.9% NaCl was inserted through the a x i l l a r y artery so that the t i p of the cannula lay at the branching of the vertebral artery from the l e f t subclavian artery. The omocervical and the i n t e r n a l thoracic arteries were ligate d (Clineschmidt and Anderson, 1970). To i n j e c t the test drugs intravenously, a cephalic vein was cannulated with a No. 90 tubing f i l l e d with 0.9% NaCl. Ether was discontinued following surgery and about three hours were allowed for the elimination of the anaesthetic. The animal was maintained on a r t i f i c i a l respiration throughout the experiment. Stimulation and recording procedures: The central end of a cut dorsal root, usually L7, and the correspond-ing ventral root were placed on bipolar platinum hook electrodes. The dorsal root - ventral root monosynaptic r e f l e x (MSR) was evoked every 5 sec by stimulation of the dorsal root (0.1 msec square wave pulse delivered from the S2 unit of a S8 Grass stimulator and passed through a S1U5 stimu-l a t i o n i s o l a t i o n u n i t ) . The stimulation strength was supramaximal for the MSR. To evoke the QUAD- or the PBST-MSR the respective peripheral nerve was stimulated using the stimulation parameters described for the dorsal root - ventral root MSR. The resulting compound action potential was recorded from the appropriate ventral root, amplified with the help of a Tektronix 2A61 d i f f e r e n t i a l amplifier and displayed on a Tektronix R564B storage oscilloscope. A concentric bipolar stainless s t e e l electrode with a t i p diameter of about 0.5 mm and a separation of 0.5 mm between the two poles was positioned 26 i n the ventromedial bulbar r e t i c u l a r formation (P 7.5 to 13.5; L 0 to 0.5; V -6 to -10 i n the stereotaxic atlas of Snider and Niemer, 1964). Bulbo-spinal i n h i b i t i o n of the MSR was evoked by a t r a i n of square wave pulses (300 msec t r a i n , 150 Hz, 0.5 msec pulses) delivered through the above elec-trode. The i n t e r v a l between the end of the t r a i n and the stimulus to evoke the MSR was 7.5 msec. Bulbar s i t e s and stimulus i n t e n s i t i e s were chosen which did not e l i c i t movement of the f a c i a l , neck and the forelimb muscles or disturb the blood pressure. Recurrent i n h i b i t i o n of the MSR was produced by stimulation of a ven-t r a l root (0.5 msec pulse) adjacent to the root from which the MSR was recorded. The conditioning i n t e r v a l was 7.5 msec. Reciprocal l a i n h i b i t i o n of the QUAD- or the PBST-MSR was induced by stimulation of the nerve belonging to the respective antagonistic muscle 1 msec p r i o r to evoking the MSR. The stimulation strength to evoke bulbospinal, recurrent or reciprocal l a i n h i b i t i o n of the MSR was adjusted to reduce the MSR to about 40 per cent of i t s control value. Presynaptic i n h i b i t i o n of the QUAD- or the PBST-MSR was produced by stimulation of the nerve from the respective antagonistic muscle 30 msec before evoking the MSR. The conditioning stimulus strength was adjusted so that only group I afferents were activated. This was determined by the presence of a single compound action potential recorded from the dorsal root that carried the impulses from the above conditioning nerve. The bulbospinal, recurrent, reciprocal l a or presynaptic conditioning s t i m u l i were delivered from the SI unit of the S8 stimulator and fed through a S1U5 stimulation i s o l a t i o n unit and a switch box. This switch box was u t i l i z e d to connect or disconnect the conditioning s t i m u l i to any 27 of the above s t i m u l a t i o n s i t e s so that a l l the above i n h i b i t i o n s o f the MSR c o u l d be evoked i n s u c c e s s i o n i n the same animal. Cold b l o c k : To prevent s u p r a s p i n a l inputs to the lumbosacral s p i n a l c o r d , about 1 cm cubes of frozen mammalian Ringer s o l u t i o n or a r t i f i c i a l c e r e b r o s p i n a l f l u i d were p l a c e d on the exposed t h o r a c i c s p i n a l c o r d . T h i s procedure w i l l be r e f e r r e d to as a c o l d b l o c k . The blockade of b u l b o s p i n a l i n h i b i t i o n d u r i n g the c o l d b l o c k was taken as the c r i t e r i o n f o r a f u n c t i o n a l blockade of the s u p r a s p i n a l i n p u t s to the s p i n a l c o r d . To reverse the c o l d b l o c k the cubes were removed, the c o l d s o l u t i o n was a s p i r a t e d and warm m i n e r a l o i l was then added and changed repeatedly u n t i l b u l b o s p i n a l i n h i b i t i o n of the MSR returned to the p r e - c o l d b l o c k l e v e l . Pharmacological s t u d i e s : Imipramine HCI (5 mg/kg i . v . ; Geigy L t d . ) was t e s t e d on p r e s y n a p t i c i n h i b i t i o n o f the QUAD- and the PBST-MSR. The e f f e c t of a c o l d b l o c k was t e s t e d before and a f t e r the i n j e c t i o n of imipramine. S i x animals were p r e t r e a t e d on two consecutive days w i t h DL-p_-chloro-p h e n y l a l a n i n e (p_-CPA, 300 mg/kg i . p . ; Sigma Chemical Co.) and the e x p e r i -ment was conducted on the t h i r d day when the 5-hydroxytryptamine (5-HT) l e v e l s i n the s p i n a l cord of the cat were r e p o r t e d to be depleted by about 90 per cent of c o n t r o l (Taber and Anderson, 1973). S i x other animals were p r e t r e a t e d w i t h DL-a-methyl-p_-tyrosine methyl e s t e r HCI (a-MPT, 125 mg/kg i . p . ; Sigma Chemical Co.) 16 and 4 hours p r i o r to the experiment. A s i m i l a r pretreatment was reported to deplete n o r -a d r e n a l i n e i n the s p i n a l cord of the cat to immeasurable l e v e l s (King and 28 Jewett, 1971). The e f f e c t s of a c o l d b l o c k , imipramine (5 mg/kg i . v . ) and a second c o l d b l o c k were t e s t e d i n sequence on p r e s y n a p t i c and r e c u r r e n t i n h i b i t i o n s of the QUAD-MSR i n the above £-CPA o r a-MPT p r e t r e a t e d animals. F l u o x e t i n e HC1 ( L i l l y 110140; E l i L i l l y and C o . ) , reported to be a s p e c i f i c 5-HT n e u -r o n a l uptake b l o c k i n g agent ( F u l l e r e_t a l . , 1975; Wong et a l . , 1974, 1975), was administered at 10 min i n t e r v a l s i n i n c r e a s i n g doses. The i n i t i a l dose was 0.25 mg/kg i . v . and the cumulative dose administered over 50 min was 6 mg/kg. The a c t i o n s of t h i s agent were determined on b u l b o s p i n a l , r e c u r r e n t , r e c i p r o c a l l a and p r e s y n a p t i c i n h i b i t i o n s of the extensor and the f l e x o r MSRs. A c o l d b l o c k was a p p l i e d before and a f t e r the i n j e c t i o n of f l u o x e t i n e . In p r e l i m i n a r y s t u d i e s 5-hydroxytryptophan (5-HTP; Sigma Chemical Co.) produced somewhat v a r i a b l e e f f e c t s on the uncondit ioned MSR as reported by Clineschmidt et a l . (1971). However, when the animals were p r e t r e a t e d w i t h a s m a l l dose of imipramine (0.25 mg/kg i . v . ) the a c t i o n of 5-HTP was l e s s v a r i a b l e . T h e r e f o r e , 5-HTP (75 mg/kg i . v . ) was administered 10 min a f t e r completing the i n j e c t i o n of imipramine and i t s e f f e c t s on b u l b o s p i n a l , r e -c u r r e n t , r e c i p r o c a l l a and p r e s y n a p t i c i n h i b i t i o n s of the QUAD-MSR were determined. The 5-HT a n t a g o n i s t , cyproheptadine HC1 (5 mg/kg i . v . ; Merck Sharp and Dbhme L t d . ) , was i n j e c t e d 40 and 60 min a f t e r completing the a d m i n i s t r a t i o n of f l u o x e t i n e or 5-HTP, r e s p e c t i v e l y . C l o n i d i n e HC1 (Boehringer Ingleheim L t d . ) , reported to be a s p e c i f i c a - a d r e n e r g i c agonist i n the c e n t r a l nervous system (Anden et a l . , 1970; K o -b i n g e r and P i c h l e r , 1975), was administered at 10 min i n t e r v a l s i n geometri-c a l l y i n c r e a s i n g cumulative doses. The i n i t i a l dose was 2 . 5 u g / k g i . v . and the f i n a l cumulative dose over 40 min was 40 ug/kg. A l l the p r e v i o u s l y 29 mentioned i n h i b i t i o n s of the flexor and the extensor MSRs were tested dur-ing clonidine i n j e c t i o n s . A cold block was applied before and after the in j e c t i o n of clonidine. The a-adrenergic blocking agent, phenoxybenzamine HCI (5 mg/kg; Smith Kline French) was injected 30 min after the completion of the administration of clonidine and the i n h i b i t i o n s of the flexor MSR were tested. Cyproheptadine (2.5 mg/kg) or phenoxybenzamine (2.5 mg/kg) was i n j e c t -ed twice at 10 min intervals and a l l the in h i b i t i o n s of the QUAD- and the PBST-MSR described previously were tested. In four of the twelve animals used for these experiments the in h i b i t i o n s of the extensor and the flexor MSRs were evoked i n the same animal. In order to determine the s i t e of action of imipramine i n blocking bulbospinal and recurrent i n h i b i t i o n s of the MSR (Sastry, 1973; S i n c l a i r and Sastry, 1974, 1974a), cumulative dose-response curves were established for the blockade of the i n h i b i t i o n s when the drug was injected i n t r a - a r t e r -i a l l y to the spinal cord, i n t r a - a r t e r i a l l y to the brain stem or intraven-ously. In these experiments the i n i t i a l dose of imipramine was 0.125 mg/kg when administered i n t r a - a r t e r i a l l y and 0.5 mg/kg when administered i n t r a -venously. Subsequent doses, increasing by geometric progression, were administered slowly at 10 min in t e r v a l s . The f i n a l cumulative dose was 4 mg/kg. Ia afferent antidromic action potentials and motoneurone f i e l d potentials: In eight cats, a tungsten (5 um exposed t i p , about 5 MQ) or a glass microelectrode f i l l e d with 3M NaCl (about 2 vim and 3 MR) was directed to-wards the QUAD motor nucleus while recording the orthodromic and the a n t i -dromic f i e l d potentials from the electrode t i p as described by Eccles et a l . 30 (1954). These f i e l d potentials were evoked by stimulation of the peripheral QUAD nerve (0.5 msec pulse) and the L6 ventral root (0.02 msec pulse). The t i p of the electrode was assumed to be i n the QUAD motor nucleus where the magnitudes of the f i e l d potentials were maximal. This position was usually 2 mm l a t e r a l to the midline and about 4.1 mm ventral from the top of the spinal cord as reported by Eccles e_t a l . (1954). The QUAD l a afferents were antidromically activated by stimulating i n the QUAD motor nucleus (Wall, 1958). The resulting compound action poten-t i a l (QUAD-AP) was recorded from the peripheral nerve. The magnitude of the QUAD-AP was adjusted to less than 50 per cent of i t s maximal siz e . In a l l of the experiments the same microelectrode was used to record the maxi-mum antidromic f i e l d potential and to evoke the QUAD-AP. The antidromic f i e l d p otential and the QUAD-AP were tested during bulbospinal i n h i b i t i o n of the extensor MSR. In three experiments, the time course of spinal presynaptic i n h i b i t i o n of the MSR and the corresponding f a c i l i t a t i o n of the QUAD-AP was followed by a l t e r i n g the conditioning i n t e r v a l . The i n t e r v a l where the QUAD-AP was maximally f a c i l i t a t e d (11.5 - 13.5 msec) was chosen to study the drug and the cold block effects i n a l l eight experiments. The conditioning i n t e r v a l selected for these studies on presynaptic i n h i b i t i o n of the QUAD-MSR was 30 msec where interference of a postsynaptic i n h i b i t i o n was reported to be minimal (Eccles et^ a l . , 1962). The stimulation pulses to evoke the antidromic f i e l d p otential or the QUAD-AP were delivered from the S2 unit of the S8 stimulator and passed through a S1U5 stimulation i s o l a t i o n unit. The QUAD-MSR, the antidromic f i e l d potential and the QUAD-AP were fed to a switch box which was used to connect one of the above inputs to a recording device. The f i e l d p o t e ntial 31 was fed through a DAM-5 d i f f e r e n t i a l p r e a m p l i f i e r o r a VF-1 vol tage f o l l o w e r (W.P. Instruments) and a T e k t r o n i x 2A61 d i f f e r e n t i a l a m p l i f i e r and d i s p l a y e d on the storage o s c i l l o s c o p e . The QUAD-AP was recorded as d e s c r i b e d f o r the MSR. The e f f e c t s of imipramine (2 mg/kg i . v . ; i n j e c t e d twice) and c y p r o -heptadine (5 mg/kg i . v . ) were t e s t e d on the unconditioned QUAD-MSR, QUAD-AP and the f i e l d p o t e n t i a l as w e l l as the above responses during b u l b o s p i -n a l and s p i n a l p r e s y n a p t i c i n h i b i t i o n s of the MSR. In a d d i t i o n , the a c t i o n o f a c o l d b l o c k was t e s t e d before and a f t e r imipramine on the uncondit ioned and p r e s y n a p t i c c o n d i t i o n e d MSR, QUAD-AP and the f i e l d p o t e n t i a l . P r e p a r a t i o n of t e s t drug s o l u t i o n s : A l l the t e s t drugs except cyproheptadine and phenoxybenzamine were d i s s o l v e d i n 0.9% NaCl j u s t before i n j e c t i o n . Cyproheptadine (5 mg/kg) was d i s s o l v e d i n 0.5 ml o f propylene g l y c o l and the r e s u l t i n g s o l u t i o n was d i l u t e d to 1 ml with 0.9% N a C l . PPhenoxybenzamine (5 mg/kg) was d i s s o l v e d i n 1 ml of propylene g l y c o l . The v e h i c l e , propylene g l y c o l (1 ml i n j e c t e d over 2 m i n ) , was t e s t e d on the i n h i b i t i o n s of the MSR i n three animals. Dextran (6% W/V) was sometimes used to maintain the b l o o d volume and F l a x e d i l (Poulenc L t d . ) was r o u t i n e l y used to prevent movements d u r i n g nerve s t i m u l a t i o n . A l l agents were administered w i t h the a i d of an i n f u -s i o n pump. C a l c u l a t i o n s : In most of the experiments the s i g n a l output from the storage o s c i l l o -scope was fed through an Ortec 4623 s i g n a l averager and d i s p l a y e d on a T e k -t r o n i x R5030 dual beam o s c i l l o s c o p e . P i c t u r e s of the s i g n a l s were taken 32 on P o l a r o i d l a n d f i l m , type 107, u s i n g a C-27 T e k t r o n i x o s c i l l o s c o p e camera. The magnitudes of the uncondit ioned and the c o n d i t i o n e d responses (MSR, a n t i d r o m i c f i e l d p o t e n t i a l o r the QUAD-AP) were determined at 10 min i n t e r -v a l s by averaging 8 consecutive uncondit ioned and the f o l l o w i n g 4 c o n d i -t i o n e d responses. The average uncondit ioned response before and a f t e r a drug a d m i n i s t r a t i o n was expressed as a per cent o f the f i n a l c o n t r o l r e -sponse. The a c t u a l per cent i n h i b i t i o n o r f a c i l i t a t i o n of the response on the f i n a l c o n t r o l t e s t was c a l c u l a t e d and equated to 100 per cent i n h i b i t i o n or f a c i l i t a t i o n , r e s p e c t i v e l y . The a c t u a l per cent i n h i b i t i o n or f a c i l i -t a t i o n o f the previous c o n t r o l t e s t s and t e s t s subsequent to a drug i n j e c -t i o n were adjusted on the b a s i s o f t h i s f i n a l c o n t r o l f i g u r e . T h e r e f o r e , a procedure which enhanced the i n h i b i t i o n or f a c i l i t a t i o n of a response would give a value of greater than 100 p e r cent whereas a blockade o f the above would r e s u l t i n a r e d u c t i o n i n the per cent f i g u r e . I o n t o p h o r e t i c study: In s i x animals a f i v e b a r r e l m i c r o p i p e t t e (3 - 7 um t i p d i a m e t e r ) , mounted on a micromanipulator, was i n s e r t e d i n t o the s p i n a l gray matter. The c e n t r a l b a r r e l of the m i c r o p i p e t t e was f i l l e d with 3M NaCl ( 3 - 1 0 MQ) and each of the outer b a r r e l s was f i l l e d w i t h one of the f o l l o w i n g s o l u -t i o n s : g l y c i n e (0.5M, pH 3.5 HC1; Sigma Chemical C o . ) , Y - a m i n o b u t y r i c a c i d (GABA, 0.5M, pH 3.5 HC1; Sigma Chemical C o . ) , c l o n i d i n e HC1 (0.01M i n 0.1M N a C l , pH 4 HC1) and Na glutamate (0.2M, pH 7.5 NaOH; Sigma Chemical C o . ) . The NaCl b a r r e l was connected i n s e r i e s to a VF-1 Voltage f o l l o w e r , a T e k t r o n i x 2A61 d i f f e r e n t i a l a m p l i f i e r and an o s c i l l o s c o p e . The e x t r a -c e l l u l a r a c t i o n p o t e n t i a l s of 34 u n i d e n t i f i e d s i n g l e s p i n a l neurones were recorded through the NaCl b a r r e l . The s i g n a l s were fed through a Ferch 33 r a t e meter and recorded on a polygraph as spikes per second. The drug ions were h e l d i n the p i p e t t e by a backing current of about 10 nA/ The e f f e c t of c l o n i d i n e on the f i r i n g rate of the c e l l s was t e s t e d by p a s s i n g a p o s i t i v e c u r r e n t ( u s u a l l y from 0 backing current to about 30 n A ) . The maximum c l o n i d i n e current that d i d not s i g n i f i c a n t l y a l t e r the neurone f i r i n g frequency was then determined. G l y c i n e or GABA was ejected by p a s s i n g 2 - 20 nA p o s i t i v e current and the e f f e c t on the c e l l f i r i n g r a t e was determined b e f o r e , during and a f t e r an e j e c t i o n o f c l o n i d i n e that d i d not s i g n i f i c a n t l y a l t e r the c o n t r o l f i r i n g r a t e . A negative current (5 - 40 nA) was passed through the glutamate b a r r e l to i n c r e a s e and m a i n t a i n the f i r i n g r a t e of most o f the c e l l s at a reasonably constant l e v e l . C u r -rent e f f e c t s were checked by p a s s i n g a negative current through the c l o n i d i n e b a r r e l and a p o s i t i v e current through the glutamate b a r r e l . P r e p a r a t i o n of the m i c r o p i p e t t e s : The m i c r o p i p e t t e s (Vancouver S c i e n t i f i c Glass Blowers) were p u l l e d using a N a r i s h i g e v e r t i c a l m i c r o p i p e t t e p u l l e r . The m i c r o p i p e t t e t i p was then broken during o b s e r v a t i o n through a microscope so that the t i p diameter was 3 - 7 um. A l l s o l u t i o n s were passed through a m i l l i p o r e f i l t e r before use. A few o f the m i c r o p i p e t t e s were f i l l e d w i t h the drug s o l u t i o n s by c e n -t r i f u g i n g at 9000 r e v o l u t i o n s per min f o r about 10 min. Other p i p e t t e s were immersed i n g e n t l y b o i l i n g d i s t i l l e d water f o r about an hour. The water was then cooled and the m i c r o p i p e t t e s were found to be f i l l e d . The d i s t i l l e d water from the stems of the p i p e t t e s was removed using a 31 gauge hypodermic needle and s y r i n g e . Then the p i p e t t e s were f i l l e d with the drug s o l u t i o n s and l e f t overnight i n a c o l d room w i t h the t i p s immersed i n d i s t i l l e d water. The next day these p i p e t t e s were c e n t r i f u g e d at 6000 r e v o l u t i o n s per min f o r 5 min before use. 34 RESULTS Presynaptic i n h i b i t i o n : The thoracic cold block enhanced presynaptic i n h i b i t i o n of the QUAD-MSR but did not a l t e r the i n h i b i t i o n of the PBST-MSR (Fig. 1,2,5,7 and 8). Imipramine HC1 (5 mg/kg i . v . ; Fig. 1 and 8) and fluoxetine HC1 ( L i l l y 110140, 0.25 - 6 mg/kg i . v . ; Fig. 2) antagonized the i n h i b i t i o n of the extensor but not of the flexor MSR. The blockade of the i n h i b i t i o n by imipramine or fluoxetine was completely eliminated by a cold block. The 5-HT antagonist, cyproheptadine HC1 (5 mg/kg i . v . ) , also reversed the blocking action of fluoxetine. In animals that were pretreated with £-CPA or a-MPT, neither a cold block nor imipramine had an effect on the i n h i b i t i o n of the QUAD-MSR (Fig. 3). The small dose of imipramine (0.25 mg/kg i.v.) had no s i g n i f i c a n t effect on presynaptic i n h i b i t i o n of the extensor MSR. However, when 5-HTP was administered 10 min after the completion of the i n j e c t i o n of imipramine, the i n h i b i t i o n was gradually antagonized. This blocking effect reached maximum i n about 60 min. Cyproheptadine (5 mg/kg i.v.) p a r t i a l l y reversed the above action of 5-HTP (Fig. 4). Clonidine HC1 (2.5 - 40 ug/kg i.v.) antagonized presynaptic i n h i b i t i o n of the extensor and the flexor MSRs. A cold block did not a l t e r the above effects of clonidine. Phenoxybenzamine HC1 (5 mg/kg i.v.) also had no effect on the above blockade of the PBST-MSR (Fig. 5). Fig. 6 depicts a parall e l i s m between the time course of the f a c i l i -35 t a t i o n of the QUAD-AP and that of the i n h i b i t i o n of the QUAD-MSR, suggesting a cause - e f f e c t r e l a t i o n s h i p between the two events. The c o l d b l o c k enhanced p r e s y n a p t i c i n h i b i t i o n of the QUAD-MSR as w e l l as the uncondit ioned and the c o n d i t i o n e d QUAD-AP ( F i g . 8 ) . T h i s e f f e c t on the c o n d i t i o n e d QUAD-AP was not apparent from F i g . 7 but was observed i n s i x of e i g h t experiments. Imipramine (2 mg/kg i . v . , administered twice) reduced the uncondit ioned QUAD-AP, antagonized p r e s y n a p t i c i n h i b i t i o n of the QUAD-MSR and blocked the f a c i l i t a t i o n of the QUAD-AP d u r i n g the c o n d i t i o n i n g ( F i g . 7 and 8 ) . A c o l d b l o c k and cyproheptadine (5 mg/kg i . v . ) p a r t l y reversed the above e f f e c t s of imipramine. Cyproheptadine (2.5 mg/kg i . v . ) or phenoxybenzamine (2.5 mg/kg i . v . ) , i n j e c t e d t w i c e , enhanced the i n h i b i t i o n of the extensor but p a r t i a l l y antag-onized the i n h i b i t i o n of the f l e x o r MSR i n the experiments i n which the i n -h i b i t i o n s of the above MSRs were evoked i n the same animal and i n those i n which the i n h i b i t i o n of e i t h e r MSR was produced ( F i g . 9 ) . 36 PBST Time (min) F i g . 1. The e f f e c t s of imipramine HC1 (IMI, 5 mg/kg) on p r e s y n a p t i c i n h i -b i t i o n o f the QUAD- (n=6) and the PBST- (n=6) MSRs. A c o l d b l o c k (CB) was a p p l i e d before and a f t e r the a d m i n i s t r a t i o n of imipramine as i n d i c a t e d on the a b s c i s s a . In t h i s and the subsequent graphs: 1. each p o i n t equals the mean ± S . E . M . and 2. the d u r a t i o n o f a drug i n j e c t i o n o r a c o l d b l o c k a p -p l i c a t i o n i s i n d i c a t e d on the a b s c i s s a . 37 50 m x z 100 1 5 0 L Q U A D Li l ly 110140 0-25 05 10 20 4<3 60 P B S T - 3 0 £ S ,t t t ,t t t • £ B 3 0 60 90 J 120 - 3 0 TIME(min) Li l ly 110140 025 05 10 20 4 0 60 fift .t t t .t t * C B YP-5 0 3 0 60 90 120 Fig. 2. The actions of fluoxetine HCI ( L i l l y 110140, increasing cumulative doses i n mg/kg as indicated on the abscissa) on presynaptic i n h i b i t i o n of the QUAD- (n=5) and the PBST- (n=5) MSRs. A cold block (CB) was applied p r i o r and subsequent to the in j e c t i o n of fluoxetine. Cyproheptadine HCI (CYP, 5 mg/kg) was administered 40 min aft e r the completion of the i n j e c -tion of fluoxetine. 38 T I M E (MIN) Fig. 3. The effects of a cold block (CB), imipramine HC1 (IMI, 5 mg/kg) and a second cold block, tested i n sequence, on presynaptic i n h i b i t i o n of the QUAD-MSR i n cats pretreated with p-CPA (n=6) or a-MPT (n=6) . 39 T IME (min) Fig. 4. The effects of a small dose of imipramine HCI (IMI, 0.25 mg/kg), 5-HTP (75 mg/kg) and cyproheptadine HCI (CYP, 5 mg/kg) on presynaptic i n h i -b i t i o n of the QUAD-MSR (n=6). 40 P I - Q U A D 100 2.5 5 10 20 40 PXB-5 -30 0 30 60 90 120 (MIN) F i g . 5. The blockade of presynaptic i n h i b i t i o n of the QUAD- (PI-QUAD, n=6) and the PBST- (PI-PBST, n=4) MSRs by clonidine HCI administered i n geo-m e t r i c a l l y increasing cumulative doses i n u g / k g as in d i c a t e d on the abscis-sa. A cold block (CB) was applied before and a f t e r the i n j e c t i o n of c l o n i -dine. The action of phenoxybenzamine HCI (PXB, 5 mg/kg) was tested on the i n h i b i t i o n of the f l e x o r r e f l e x a f t e r the second cold block. 41 ^ J 2 m v •— 2 m s e c QUAD u n c o n d J V_ 5 10 12 20 30 50 70 0-2 mv ^^^p^^^ ^^^^ ~ S e s T S e X t 2 i e f t f r a " S i l l u s t r a t e t h e unconditioned ( S c o n d ) re! o T e a c h " f r a ^ e C^X^UI81lnterValfl ±n M e c are i n d i c a t e d a t t h e b o t t L f r a m e . Each s x g n a l r e p r e s e n t s t h e a v e r a g e d r e s p o n s e of f o u r s w e e p s 42 M S R Q U A D A P C O N T R O L 2 m v 2 msec 0*2mv C B I M I 4 C B C Y P - 5 F i g . 7. The blockade bf p r e s y n a p t i c i n h i b i t i o n of the QUAD-MSR and the corresponding f a c i l i t a t i o n of the QUAD-AP by imipramine HCI (IMI, 2 mg/kg i n j e c t e d twice over 20 m i n ) . In each panel the top frames represent the QUAD-MSR (MSR) and the bottom frames represent the QUAD-AP. In each p a n e l the l e f t s i g n a l s i l l u s t r a t e the uncondit ioned responses and the r i g h t ones p o r t r a y the responses c o n d i t i o n e d by PBST group I a f f e r e n t s . A c o l d b l o c k (CB) was a p p l i e d 10 min before and 10 min a f t e r the i n j e c t i o n o f 4 mg/kg of imipramine. Cyproheptadine HCI (CYP, 5 mg/kg) was administered 20 min a f t e r the i n j e c t i o n of imipramine. Each s i g n a l represents the averaged response of four sweeps. 43 r. TIME (MIN) Fig. 8. The effects of imipramine HC1 (IMI, 2 mg/kg administered twice) on the unconditioned QUAD-MSR (bottom l e f t graph), presynaptic inhibition of the QUAD-MSR (top left) , the unconditioned QUAD-AP (bottom right) and the conditioned QUAD-AP (top right). A cold block (CB) was applied before and after imipramine. Cyproheptadine HC1 (CYP, 5 mg/kg) was administered after the second cold block as indicated on the abscissa.(n=8). 44 50r PBST PBST 100h ® fr-CD QUAD X s-s 100 150 L QUA D -20 0 20 4 0 I -20 20 40 T I M E (min) F i g . 9. The enhancement of p r e s y n a p t i c i n h i b i t i o n of the QUAD-MSR by cypro-heptadine HCI (CYP, 2.5 mg/kg administered t w i c e ; bottom l e f t graph) and phenoxybenzamine HCI (PXB, 2.5 mg/kg i n j e c t e d twice; bottom r i g h t ) . The i n h i b i t i o n of the PBST-MSR was p a r t i a l l y b l o c k e d by cyproheptadine (top l e f t ) and phenoxybenzamine (top r i g h t ) . For each graph n=4. 45 Bulbospinal i n h i b i t i o n : Fluoxetine (0.25 - 6 mg/kg i.v.) antagonized bulbospinal i n h i b i t i o n of both the extensor and the flexor MSRs, although the effect was greater on the i n h i b i t i o n of the extensor reflex. This blockade by fluoxetine was p a r t i a l l y reversed by cyproheptadine (5 mg/kg i . v . ; Fig. 10 and 11). Imipramine (0.25 mg/kg i.v.) did not s i g n i f i c a n t l y a l t e r the i n h i b i -t i o n of the QUAD-MSR but the subsequent 5-HTP (75 mg/kg i.v.) administra-tion converted the i n h i b i t i o n into a f a c i l i t a t i o n i n four of s i x animals tested (Fig. 12). The antagonism gradually reached maximum about 60 min after the s t a r t of in j e c t i o n . Cyproheptadine (5 mg/kg i.v.) p a r t i a l l y reversed the blockade by 5-HTP (Fig. 12). Clonidine (2.5 -.40 jig/kg i.v.) converted the i n h i b i t i o n of the QUAD-MSR into a f a c i l i t a t i o n i n four of s i x experiments (Fig. 13, l e f t panel). Bulbospinal i n h i b i t i o n of the PBST-MSR was s i m i l a r l y blocked by this agent. Phenoxybenzamine (5 mg/kg i.v.) did not a l t e r the blockade of the flexor MSR. Fig. 14 i l l u s t r a t e s that cyproheptadine (2.5 - 5 mg/kg i.v.) enhanced and phenoxybenzamine (2.5 - 5 mg/kg i.v.) antagonized the i n h i b i t i o n of both the QUAD- and the PBST-MSRs. The magnitude of cyproheptadine's action was greater on the i n h i b i t i o n of the extensor MSR than on that of the f l e x -or MSR. The dose-response curve for i n t r a - a r t e r i a l i n j e c t i o n of imipramine to the brain stem was not s i g n i f i c a n t l y different from the curve for i n t r a -venous administration of th i s agent (Fig. 15). However, the dose-response curve for imipramine was shifte d to the l e f t of the other two curves when the drug was administered i n t r a - a r t e r i a l l y to the spinal cord. The points after the doses 0.25 - 2 mg/kg on the dose-response curve for i n t r a - a r t e r i a l 46 i n j e c t i o n to the s p i n a l cord were s i g n i f i c a n t l y d i f f e r e n t (P < 0.05) than the corresponding p o i n t s on the other two curves ( F i g . 15). T h e r e f o r e , imipramine was more potent when i n j e c t e d i n t r a - a r t e r i a l l y to the s p i n a l cord than when administered by the other two r o u t e s . During b u l b o s p i n a l i n h i b i t i o n of the QUAD-MSR, the antidromic f i e l d p o t e n t i a l was reduced and the QUAD-AP f a c i l i t a t e d ( F i g . 16). Imipramine (2 mg/kg i . v . , administered twice) antagonized the above i n h i b i t i o n of the QUAD-AP w h i l e b l o c k i n g b u l b o s p i n a l i n h i b i t i o n of the QUAD-MSR ( F i g . 16 and 17). Cyproheptadine (5 mg/kg i . v . ) p a r t i a l l y reversed t h i s blockade. 47 2msec CONTROL L i l l y 110140-6 CYP-5 Fig. 10. The blockade of bulbospinal i n h i b i t i o n of the QUAD-MSR by fluoxe-tine HC1 ( L i l l y 110140, t o t a l dose of 6 mg/kg injected over 50 min) and a reversal of the blockade by cyproheptadine HC1 (CYP, 5 mg/kg) administered 50 min after the completion of fluoxetine i n j e c t i o n . The l e f t frame i n each panel represents the unconditioned and the right the conditioned MSR. Each signal represents the averaged response of four sweeps. 48 Fig. 11. The effects of fluoxetine HC1 ( L i l l y 110140, administered i n i n -creasing cumulative doses i n mg/kg as indicated on the abscissa) and cypro-heptadine HC1 (CYP 5 mg/kg) on bulbospinal i n h i b i t i o n of the QUAD- (n=5) and the PBST- (n=5) MSRs. • 49 Fig. 12. The effects of imipramine HCI (IMI, 0.25 mg/kg), 5-HTP (75 mg/kg) and cyproheptadine HCI (CYP, 5 mg/kg), tested i n sequence, on bulbospinal i n h i b i t i o n of the QUAD-MSR (n=6). 50 -100 50 2 /° o CQ X ? 50 100 B S I - Q U A D R I - Q U A D •30 2.5 5 10 20 40 i t • .• • 10 20 40 CB 30 60 90 T I M E •30-CB (MIN) Fig. 13. The blockade of bulbospinal (BSI-QUAD, n=6) and recurrent (RI-QUAD, n=6) in h i b i t i o n s of the QUAD-MSR by clonidine HC1 (administered i n geometri-c a l l y increasing cumulative doses i n u g/kg as indicated,on the abscissa). The effects of a cold block (CB) application was tested on recurrent i n h i -b i t i o n before and after the i n j e c t i o n of clonidine. 51 TIME(min) F i g . 14. The enhancement of bulbospinal i n h i b i t i o n of the QUAD- (top l e f t graph) and the PBST- (bottom l e f t ) MSRs by cyproheptadine HCI (CYP, 2.5 mg/kg administered twice) . The antagonism of the i n h i b i t i o n of the QUAD-(top right) and the PBST- (bottom right) MSRs by phenoxybenzamine HCI (PXB, 2.5 mg/kg i n j e c t e d twice) i s also i l l u s t r a t e d . For each graph n=4. 52 | IMI (mg/kg) 0 . 1 2 5 0 - 2 5 0 - 5 1.0 2 . 0 4 0 I * • !• • A • I | -20 0 20 40 60 80 TIME (min) Fig. 15. The blockade of bulbospinal i n h i b i t i o n of the dorsal root - ven-t r a l root MSR by imipramine HC1 (IMI). The drug was administered i n t r a -venously ( i . v . , 0.5 - 4 mg/kg), i n t r a - a r t e r i a l l y to the spinal cord ( i . a . -s . c , 0.125 - 4 mg/kg) and i n t r a - a r t e r i a l l y to the brain stem ( i . a . - b.s., 0.125 - 4 mg/kg) i n geometrically increasing cumulative doses. For each curve n=4. 53 MS R 2 m i 2 m s e c FP V /\ 11 m v 1 m s e c QUAD AP 0 * 2 m v 2 m s e c CONTROL IMI-4 CYP-5 Fig. 16. The blockade of bulbospinal i n h i b i t i o n of the QUAD-MSR (MSR), and the associated reduction i n the antidromic motoneurone f i e l d p o t ential (FP) and the f a c i l i t a t i o n of QUAD-AP by imipramine HCI (IMI, 2 mg/kg administered twice over 20 min). A reversal of the blockade by cyproheptadine HCI (CYP, 5 mg/kg injected 20 min after the completion of imipramine administration). The l e f t signals i n each p a i r represent the unconditioned responses and the right signals i l l u s t r a t e the conditioned responses. Each signal represents the averaged response of 4 sweeps. / ^ 1 / \ / \l I-20 60 0 20 TIME(mln) -20 60 F i g . 17. The blockade o f b u l b o s p i n a l i n h i b i t i o n o f the QUAD-MSR (BSI-MSR) , and the a s s o c i a t e d f a c i l i t a t i o n of the QUAD-AP and i n h i b i t i o n o f the antidromic motoneurone f i e l d p o t e n t i a l (BSI-FP) by imipramine HC1 (IMI, 2 mg/kg a d m i n i s t e r e d t w i c e ) . The. e f f e c t s of cyproheptadine HC1 (CYP, 5 mg/kg) are a l s o shown (n=8). 55 Recurrent i n h i b i t i o n : Clonidine (2.5 - 40 ug/kg i.v.) blocked the i n h i b i t i o n of the QUAD-(Fig. 13, right panel) and the PBST- (not shown) MSRs. A cold block did not a l t e r the above action of clonidine. Also, phenoxybenzamine (5 mg/kg i.v.) f a i l e d to reverse clonidine's effect on the i n h i b i t i o n of the flexor MSR. When supraspinal inputs to the spinal cord were eliminated by the ap-p l i c a t i o n of a thoracic cold block, recurrent i n h i b i t i o n of the QUAD-MSR was f a c i l i t a t e d (Fig. 13, right panel and Fig. 18, l e f t panel). The i n h i -b i t i o n of the PBST-MSR was, however, unaffected (Fig. 18). Fluoxetine (0.25 - 6 mg/kg i . v . ) , given following the recovery of the cold block, an-tagonized the i n h i b i t i o n of the QUAD- but not of the PBST-MSR. A second cold block, applied a f t e r the i n j e c t i o n of fluoxetine, completely reversed the above blockade. Cyproheptadine (5 mg/kg i.v.) also p a r t i a l l y reversed the action of fluoxetine on the i n h i b i t i o n of the extensor MSR (Fig. 18). In the cats pretreated with n_-CPA or a-MPT neither a cold block nor imipramine (5 mg/kg i.v.) altered recurrent i n h i b i t i o n of the QUAD-MSR (Fig. 19). The i n h i b i t i o n of the extensor MSR was unaltered by the small dose of imipramine (0.25.'mg/kg i . v . ) , however, 5-HTP (75 mg/kg i.v.) gradually blocked the i n h i b i t i o n . This blockade reached maximum i n about 60 min. Cyproheptadine (5 mg/kg i.v.) reversed the blockade by 5-HTP (Fig. 20). Cyproheptadine (2.5 - 5 mg/kg i.v.) and phenoxybenzamine (2.5 - 5 mg/kg i.v.) enhanced the i n h i b i t i o n of the extensor but p a r t i a l l y reversed the i n h i b i t i o n of the flexor MSR i n the experiments i n which recurrent i n -h i b i t i o n of the QUAD- and the PBST-MSRs was evoked i n the same animal and i n those i n which the i n h i b i t i o n of either MSR was produced (Fig. 21). 56 The dose^response curve for i n t r a - a r t e r i a l administration of imipra-mine to the spinal cord was shift e d to the l e f t of the curves for i n t r a -a r t e r i a l i n j e c t i o n to the brain stem or intravenous administration (Fig. 22). The points after the doses 0.25 - 2 mg/kg on the curve for i n t r a -a r t e r i a l i n j e c t i o n to the spinal cord were s i g n i f i c a n t l y different (P < 0. 05) from the corresponding points on the other two curves. These re-sults indicate that imipramine antagonized recurrent i n h i b i t i o n by a spinal s i t e of action. Reciprocal l a i n h i b i t i o n : Imipramine (5 mg/kg i . v . , n=6) , fluoxetine (0.25 - 6 mg/kg i . v . , n=5), cyproheptadine (2.5 - 5 mg/kg i . v . , n=4) and phenoxybenzamine (2.5 - 5 mg/kg 1. v., n=4) had no s i g n i f i c a n t effect on reciprocal l a i n h i b i t i o n of the ex-tensor and the flexor MSRs. Moreover, 5-HTP (75 mg/kg i . v . , n=6) also f a i l e d to a l t e r the i n h i b i t i o n of the extensor MSR. A cold block enhanced the i n h i b i t i o n of the extensor but not of the flexor MSR. Clonidine (2.5 - 40u g/kg i.v.) antagonized reciprocal l a i n -h i b i t i o n of both the QUAD- and the PBST-MSRs (Fig. 23). A subsequent cold block did not a l t e r clonidine's effect. Furthermore, phenoxybenzamine (5 mg/kg i.v.) also f a i l e d to a l t e r the blockade of the PBST r e f l e x by c l o n i -dine. Control experiments: To test whether the i n h i b i t i o n s were stable throughout the time course of experiments, bulbospinal and recurrent i n h i b i t i o n s of the dorsal root -ventral root MSR were recorded for 2^ hours i n two experiments. The maxi-mum mean deviation from the control values was 7.8% for bulbospinal and 9.3% f o r r e c u r r e n t i n h i b i t i o n . In f i v e experiments the s i z e of the QUAD-MSR was reduced to about 50 per cent of i t s c o n t r o l value by d e c r e a s i n g the s t i m u l a t i o n s t r e n g t h . In these animals r e c u r r e n t and p r e s y n a p t i c i n h i b i t i o n s were tested, on the c o n t r o l and the reduced MSR without a l t e r i n g the c o n d i t i o n i n g s t i m u l a t i o n s t r e n g t h . The i n h i b i t i o n s of the reduced MSR were not s i g n i f i c a n t l y a l -t e r e d from those of the c o n t r o l ( r e c u r r e n t : 103 ± 4.2% S . E . M . ; p r e s y n a p - r i t i c : 105 ± 6.7% S . E . M . ) . However, i n these animals a c o l d b l o c k reduced the MSR by 46 ± 5.9% S . E . M . and i n c r e a s e d r e c u r r e n t i n h i b i t i o n to 122 ± 5.2% S . E . M . and p r e s y n a p t i c i n h i b i t i o n to 128 ± 7.1% S . E . M . of c o n t r o l . These r e s u l t s suggest that the enhancement of the i n h i b i t i o n s d u r i n g a c o l d b l o c k i s not due to a r e d u c t i o n i n the s i z e of the uncondit ioned MSR. Propylene g l y c o l (1 ml) enhanced b u l b o s p i n a l , r e c u r r e n t and p r e s y n -a p t i c i n h i b i t i o n s of the QUAD-MSR. However, the i n h i b i t i o n s returned to c o n t r o l values about 1 min a f t e r the i n j e c t i o n , i n d i c a t i n g that the a c -t i o n s of cyproheptadine and phenoxybenzamine on the above i n h i b i t i o n s were independent of the e f f e c t s of the v e h i c l e . 58 Z 50 O CO X z ~. 100 150 Q U A D L i l l y 110140 0 - 2 5 0 5 10 2 0 4 0 6 0 PBST -30 SB. * M ,t t t • £YE-5 30 60 90 120 TIME (min) -30 L i l l y 110140 0 250-5 10 2 0 4 0 6 0 fia.ttt.ttt 30 60 Sir" 5 90 120 Fig. 18. The actions of fluoxetine HCI ( L i l l y 110140, administered i n i n -creasing cumulative doses i n mg/kg as indicated on the abscissa) on recur-rent i n h i b i t i o n of the QUAD- (n=5) and the PBST- (n=5) MSRs. A cold block (CB) was applied before and after the i n j e c t i o n of fluoxetine. The effects of cyproheptadine HCI (CYP, 5 mg/kg) were also tested. 59 )r PC PA r a M P T C 100F C B IMI •20 C B 2 0 40 C B IMI 2 0 xCB 20 T i m e (min) Fig. 19. The effects of a cold block (CB) , imipramine HC1 (IMI, 5 mg/kg) and a second cold block, tested i n sequence, on recurrent i n h i b i t i o n of the QUAD-MSR i n cats pretreated with JD-CPA (n=6) or ct-MPT (n=6) . 60 o CO 100 0 3 0 6 0 9 0 T I M E ( m i n ) 120 150 Fig. 20. The effects of a small dose of imipramine HCI (IMI, 0.25 mg/kg), 5-HTP (75 mg/kg) and cyproheptadine HCI (CYP, 5 mg/kg) on recurrent i n h i -b i t i o n of the QUAD-MSR (n=6). 61 PBST PBST 50r T TIME (min) F i g . 21. The enhancement of recurrent i n h i b i t i o n of the QUAD-MSR by cypro-heptadine HCI (CYP, 2.5 mg/kg administered twice; bottom l e f t graph) and phenoxybenzamine HCI (PXB, 2.5 mg/kg i n j e c t e d twice; bottom r i g h t ) . The i n h i b i t i o n of the PBST-MSR was p a r t i a l l y blocked by cyproheptadine (top l e f t ) and phenoxybenzamine (top r i g h t ) . For each graph n=4. 62 Fig. 22. The antagonism of recurrent i n h i b i t i o n of the dorsal root - ven-t r a l root MSR by imipramine HC1 (IMI). The drug was injected intravenously ( I . V . , 0.5 - 4 mg/kg), i n t r a - a r t e r i a l l y to the spinal cord ( i . a . - s.c. 0.125 - 4 mg/kg) and i n t r a - a r t e r i a l l y to the brain stem ( i . a . - b.s., 0.125 -4 mg/kg) i n geometrically increasing cumulative doses. For each curve n=4. 63 T I M E (MIN) F i g . 23. The blockade of r e c i p r o c a l l a i n h i b i t i o n of the QUAD- (top l e f t , n=6) and the PBST- (top r i g h t , n=4) MSRs by clonidine HCI administered i n geometrically increasing cumulative doses i n u g/kg as ind i c a t e d on the ab-s c i s s a . The depression of the QUAD- (bottom l e f t , n=6) and the PBST- (bot-tom r i g h t , n=4) MSRs by clonidine i s also i l l u s t r a t e d . A cold block (CB) was tested before and a f t e r the clo n i d i n e i n j e c t i o n . The action of phenoxy-benzamine HCI (PXB, 5 mg/kg) was tested on the i n h i b i t i o n of the f l e x o r r e f l e x a f t e r the second cold block. 64 The uncondit ioned MSR: Imipramine (5 mg/kg i . v . ) decreased the unconditioned QUAD- and the PBST-MSRs by about 45 per cent. This o b s e r v a t i o n i s c o n s i s t e n t w i t h p r e -vious reports ( S a s t r y , 1973; S i n c l a i r and S a s t r y , 1974). Imipramine a l s o reduced the extensor MSR i n p_-CPA p r e t r e a t e d animals. A c o l d b l o c k decreased the extensor MSR i n unpretreated animals ( F i g . 23 and 24) but f a i l e d to a l t e r t h i s MSR i n animals p r e t r e a t e d w i t h £-CPA. The f l e x o r r e f l e x was u n a l t e r e d by the c o l d b l o c k a p p l i c a t i o n ( F i g . 23) . A c o l d b l o c k enhanced the QUAD-AP but reduced the f i e l d p o t e n t i a l ( F i g . 24). Imipramine ( 2 - 4 mg/kg i . v . ) reduced the QUAD-MSR, the QUAD-AP and the f i e l d p o t e n t i a l . A c o l d b l o c k p a r t l y reversed the above a c t i o n of imipramine on the QUAD-AP but not on the MSR and the f i e l d p o t e n t i a l ( F i g . 24). In 4 of 5 experiments the submaximal f i e l d p o t e n t i a l was aug-mented by imipramine (2 mg/kg i . v . ) but was depressed by a second dose. F l u o x e t i n e ( t o t a l dose of 6 mg/kg i . v . ) enhanced the extensor and the f l e x o r MSR to 139 ± 10.5% S . E . M . and 130 ± 4% S . E . M . of c o n t r o l v a l u e , r e -s p e c t i v e l y (n=5). In s i x animals, imipramine (0.25 mg/kg i . v . ) had no s i g n i f i c a n t e f f e c t on the QUAD-MSR but i n three of the above animals 5-HTP (75 mg/kg i . v . ) , administered subsequent to imipramine, enhanced t h i s extensor r e f l e x (190.6, 306.7 and 238.1% o f c o n t r o l ; 90 min a f t e r i n j e c t i o n ) . I n two animals, 5-HTP i n i t i a l l y reduced (71.4 and 50.1%; 20 min a f t e r the s t a r t of i n j e c t i o n ) but l a t e r enhanced (207.1 and 248%; 90 min a f t e r i n j e c t i o n ) the MSR. In the remaining animal the MSR was depressed throughout the time t e s t e d (61.9%; 90 min a f t e r 5-HTP). Cyproheptadine blocked the above f a c i l i t a t i o n of the QUAD-MSR by 5-HTP (91.9 ± 11.2 S . E . M . , MSR % of c o n t r o l ) and converted the enhancement of the 65 TIME (min) Fig. 24. The effects of a cold block (CB), imipramine HCI (IMI, 2 mg/kg administered twice), a second cold block and cyproheptadine HCI (CYP, 5 mg/kg), tested i n sequence, on the QUAD-MSR, the QUAD-AP and the a n t i -dromic f i e l d p o t e ntial (FP). For each graph n=8. 66 QUAD and the PBST r e f l e x e s produced by f l u o x e t i n e i n t o a depression (QUAD: 48.4 ± 8.7% S . E . M . ; PBST: 73.5 ± 9.5% S . E . M . ) . When administered a l o n e , cyproheptadine (2.5 - 5 mg/kg i . v . ) reduced the extensor and the f l e x o r MSR to 62.8 ± 13.7 S . E . M . and 70 ± 12.7 S . E . M . ,*?% of c o n t r o l ) , r e s p e c t i v e -l y (n=4). Phenoxybenzamine a l s o , at the same dose, depressed these r e -f lexes (QUAD: 61 ± 8.9% S . E . M . ; PBST: 45 ± 3.7% S . E . M . ; n=4). Blood p r e s s u r e : F l u o x e t i n e (0.25 - 6 mg/kg i . v . ) d i d not a l t e r the blood pressure of the animals. 5-HTP u s u a l l y had a b i p h a s i c a c t i o n , an i n i t i a l enhancement of the s y s t o l i c pressure by about 23 per cent that l a s t e d over 30 min f o l -lowed by a f a l l i n the d i a s t o l i c pressure by about 12.5 per cent that u s u -a l l y l a s t e d throughout the experiment. A s i m i l a r o b s e r v a t i o n was made by Anderson and Shibuya (1966). However, there was no c o r r e l a t i o n between these b l o o d p r e s s u r e changes and the b l o c k i n g a c t i o n of t h i s agent on the i n h i b i t i o n s o f the extensor MSR. Imipramine d i d not s i g n i f i c a n t l y a l t e r the b l o o d p r e s s u r e when given i n g e o m e t r i c a l l y i n c r e a s i n g doses at 10 min i n t e r v a l s (the t o t a l dose over 50 min was 4 mg/kg). This agent, however, reduced the mean b l o o d pressure to about 75 and 85 per cent o f c o n t r o l when administered at a dose of 5 mg/kg over 10 min and at a t o t a l dose of 4 mg/kg over 20 m i n , r e s p e c t i v e l y . These e f f e c t s of imipramine on the b l o o d pressure very l i k e l y d i d not con-t r i b u t e to t h i s agent 's a c t i o n on p r e s y n a p t i c i n h i b i t i o n of the MSR s i n c e imipramine blocked the i n h i b i t i o n of the QUAD- but not the PBST-MSR and a c o l d b l o c k which had no e f f e c t on the blood pressure completely e l i m i n a t e d the above e f f e c t of imipramine. C l o n i d i n e (2.5 - 40 y g / k g i . v . ) had an i n i t i a l t r a n s i e n t hypertensive 67 e f f e c t followed by a r e d u c t i o n of about 15% of the mean b l o o d p r e s s u r e which p e r s i s t e d throughout the experiment. Cyproheptadine (5 mg/kg i . v . ) and phenoxybenzamine (5 mg/kg i . v . ) reduced the mean b l o o d pressure by about 18.5 and 25%, r e s p e c t i v e l y . T h i s e f f e c t i n both cases l a s t e d throughout the course of the experiment. The depressant e f f e c t s of these agents on b l o o d p r e s s u r e would not appear to be r e l a t e d to t h e i r a c t i o n on b u l b o s p i n a l , r e c u r r e n t and p r e s y n a p t i c i n h i -b i t i o n s o f the MSR s i n c e cyproheptadine enhanced whereas phenoxybenzamine antagonized b u l b o s p i n a l i n h i b i t i o n . Moreover, both the agents enhanced p r e s y n a p t i c and r e c u r r e n t i n h i b i t i o n s of the QUAD- but not of the PBST-MSR. Noradrenal ine and 5-HT had been i m p l i c a t e d i n antagonizing these i n h i b i -t i o n s of the extensor r e f l e x (see d i s c u s s i o n and S i n c l a i r and S a s t r y , 1974a). I o n t o p h o r e t i c study: I o n t o p h o r e t i c a l l y a p p l i e d c l o n i d i n e had v a r i e d e f f e c t s on the discharge r a t e of s p i n a l neurones that were e i t h e r spontaneously f i r i n g o r whose d i s -charge rate was i n c r e a s e d by glutamate. C l o n i d i n e c u r r e n t s of l e s s than 10 nA u s u a l l y had no s i g n i f i c a n t e f f e c t ; h i g h e r currents i n c r e a s e d , decreased or d i d not a l t e r the f i r i n g r a t e . The discharge r a t e of 33 c e l l s t e s t e d was decreased by y - a m i n o b u t y r i c a c i d (GABA; F i g . 25 and 26). C l o n i d i n e , at currents that d i d not s i g n i f i -c a n t l y change the c o n t r o l f i r i n g r a t e , antagonized the e f f e c t of GABA on 49 p e r cent of the neurones ( F i g . 25 and 26). However, i n 12 p e r cent of the cases c l o n i d i n e p o t e n t i a t e d the a c t i o n of GABA. C l o n i d i n e f a i l e d to a l t e r the amino a c i d ' s e f f e c t on 39 per cent of the neurones. Glycine a l s o reduced the discharge rate o f 31 neurones t e s t e d ( F i g . 26). The e f f e c t of t h i s amino a c i d on 42 per cent of the above c e l l s was 68 antagonized by clonidine (Fig. 26). Clonidine augmented the effect of glycine on 18 per cent of the cells but did not alter the amino acid's action on 40 per cent of the neurones. On 4 cells clonidine antagonized the effect of GABA but had no effect on the action of glycine when these amino acids were tested on the same neurone. Similarly, in three cases the action of glycine but not of GABA was blocked by clonidine. 69 F i g . 25. The blockade by c l o n i d i n e (10 nA) o f the depression of a spon-taneously f i r i n g s p i n a l neurone discharge r a t e by y - a m i n o b u t y r i c a c i d (GABA, 5 n A ) . Both agents were a p p l i e d i o n t o p h o r e t i c a l l y . A : c o n t r o l . G: i l l u s t r a t e s the e f f e c t of p o s i t i v e c u r r e n t (15 n A ) . + ve current-15 2 sec F i g . 25 F i g . 26. The blockade of the e f f e c t s o f A: y - a m i n o b u t y r i c a c i d (GABA, i o n t o p h o r e t i c a l l y a p p l i e d , 5 - 2 5 nA) and B: g l y c i n e (5 - 15 nA) on u n i d e n t i f i e d s p i n a l neurones by c l o n i d i n e (5 or 10 nA as i n d i c a t e d ) . The c e l l i n A was spontaneously f i r i n g ; the discharge rate o f the n e u -rone i n B was increased and maintained at an approximately constant l e v e l by glutamate (15 nA). 72 73 DISCUSSION The neuronal c i r c u i t r y upon which drugs used i n t h i s study act i s not e n t i r e l y known. Diagrammatic neuronal arrangements have been proposed which i l l u s t r a t e the monoaminergic influences on presynaptic, bulbospinal and re-current i n h i b i t i o n s of the quadriceps (QUAD) monosynaptic r e f l e x (MSR), as shown i n Fig. 27, 28 and 29, respectively. A possible neuronal scheme for 5-HT influences on the unconditioned r e f l e x appears i n Fig. 30. The follow-ing discussion may be considered with reference to these figures. Presynaptic i n h i b i t i o n of the MSR: The blockade of presynaptic i n h i b i t i o n of the extensor QUAD but not of the flexor posterior biceps-semitendinosus (PBST) MSR by imipramine i s con-siste n t with a s i m i l a r finding by Tan and Henatsch (1969). The complete elimination of imipramine's blocking action on presynaptic i n h i b i t i o n by a thoracic cold block, which prevents supraspinal inputs to the caudal spinal cord, indicates that imipramine exerts this effect through a descending system. Since imipramine f a i l e d to antagonize presynaptic i n h i b i t i o n i n cats pretreated with either the tryptophan hydroxylase i n h i b i t o r , DL-p_-chloro-phenylalanine (p_-CPA) or the tyrosine hydroxylase i n h i b i t o r , DL-a-methyl-jD-tyrosine methyl ester HCI (a-MPT), the blockade of t h i s i n h i b i t i o n by imipramine i s l i k e l y mediated through 5-hydroxytryptamine (5-HT) and nor-adrenaline. Moreover, the complete elimination of imipramine's blocking action by either of the above pretreatments suggests that 5-HT and nor-74 a d r e n a l i n e neurones are s e r i a l l y arranged i n the same descending system through which imipramine exerts t h i s e f f e c t ( F i g . 27). At present we have no i n f o r m a t i o n concerning the o r d e r of the arrangement i n t h i s system. F l u o x e t i n e was r e p o r t e d to be a s p e c i f i c 5-HT neuronal uptake b l o c k i n g agent ( F u l l e r et a l . , 1975; Wong et a l . , 1974, 1975). 5-Hydroxytryptophan (5-HTP) e l e v a t e d 5-HT l e v e l s i n the s p i n a l cord of the cat (Anderson and Shibuya, 1966). In the present study, f l u o x e t i n e and 5-HTP antagonized p r e s y n a p t i c i n h i b i t i o n of the extensor MSR presumably by enhancing 5-HT s y n a p t i c a c t i v i t y . The f i n d i n g that the blockade of the i n h i b i t i o n by f l u -oxetine was reversed by a c o l d b l o c k i n d i c a t e s that t h i s agent produces i t s e f f e c t through a s u p r a s p i n a l system. F l u o x e t i n e d i d not a l t e r the i n h i b i -t i o n of the f l e x o r MSR s u p p o r t i n g the n o t i o n that 5-HT does not i n f l u e n c e t h i s i n h i b i t i o n . The enhancement of p r e s y n a p t i c i n h i b i t i o n of the QUAD- but not of the PBST-MSR by a t h o r a c i c c o l d b l o c k suggests that the i n h i b i t i o n of the ex-tensor MSR i s under a t o n i c i n h i b i t o r y i n f l u e n c e o f a descending system. Furthermore, the f i n d i n g that a c o l d b l o c k f a i l e d to enhance the i n h i b i t i o n o f the extensor MSR i n p_-CPA or a-MPT p r e t r e a t e d c a t s , suggests that the t o n i c i n h i b i t o r y system i n v o l v e s 5-HT and n o r a d r e n a l i n e . T h i s l a t t e r n o -t i o n i s strengthened by the enhancement of p r e s y n a p t i c i n h i b i t i o n of the QUAD-MSR by the 5-HT a n t a g o n i s t , cyproheptadine, and the adrenergic antag-o n i s t , phenoxybenzamine. Carpenter et a l . (1963) suggested t h a t , i n decerebrate c a t s , p r e s y n -a p t i c i n h i b i t i o n of the MSR i s not under a t o n i c i n h i b i t o r y i n f l u e n c e of a s u p r a s p i n a l system. This p r o p o s a l i s at v a r i a n c e w i t h the present hypothe-s i s that p r e s y n a p t i c i n h i b i t i o n of the QUAD-MSR i s t o n i c a l l y i n h i b i t e d by a descending monoaminergic system. The discrepancy may be due to d i f f e r e n t 75 experimental procedures adopted i n these two s t u d i e s . In the p r e s e n t i n -v e s t i g a t i o n the e f f e c t of a c o l d b l o c k was t e s t e d on the i n h i b i t i o n o f the MSR, whereas Carpenter e_t a l . (1963) observed the e f f e c t of a s p i n a l t r a n -s e c t i o n on the negative d o r s a l root p o t e n t i a l s (DRPs) evoked by s t i m u l a t i o n of group I a f f e r e n t s . E c c l e s et a l . (1962) reported that group I a f f e r e n t s can produce DRPs on both l a and l b a f f e r e n t s . Moreover, i n the study of Carpenter e_t a l . (1963) i t i s not known whether DRPs were generated on ex-tensor o r f l e x o r af ferents or on b o t h . The present study c l e a r l y i n v o l v e s i n h i b i t i o n of an extensor or a f l e x o r MSR. The r e f l e x was c o n d i t i o n e d by group I a f f e r e n t s and a c o n d i t i o n i n g i n t e r v a l of 30 msec was maintained to e l i m i n a t e the i n t e r f e r e n c e of p o s t s y n a p t i c i n h i b i t i o n (Eccles et_ a l . , 1962). Moreover i n the present experiments, u t i l i z i n g the primary a f f e r e n t e x c i t -a b i l i t y t e s t i n g procedure, a c o l d b l o c k augmented the f a c i l i t a t i o n of the a n t i d r o m i c compound a c t i o n p o t e n t i a l s on QUAD l a af ferents (QUAD-APs) evoked by a c o n d i t i o n i n g st imulus from the f l e x o r group I a f f e r e n t s . In the above experiments a c o l d b l o c k a l s o enhanced p r e s y n a p t i c i n h i b i t i o n of the QUAD-MSR. Imipramine antagonized the f a c i l i t a t i o n o f the QUAD-APs and blocked p r e s y n a p t i c i n h i b i t i o n . Both a c o l d b l o c k and cyproheptadine reversed the e f f e c t s of imipramine. These observations strengthen our p r o p o s a l i m p l i -c a t i n g a s u p r a s p i n a l monoaminergic system i n antagonizing p r e s y n a p t i c i n h i -b i t i o n of the QUAD-MSR. A c o l d b l o c k enhanced and imipramine decreased the uncondit ioned QUAD-AP/ T h i s a c t i o n of imipramine was reversed by a c o l d b l o c k and cyprohepta-d i n e . These r e s u l t s suggest that a t o n i c a l l y a c t i v e descending 5-HT system decreases the e x c i t a b i l i t y of QUAD l a a f f e r e n t s ( F i g . 30). It i s unknown how the above monoaminergic system exerts it's b l o c k i n g a c t i o n on p r e s y n a p t i c i n h i b i t i o n o f the extensor MSR. The s p i n a l i n t e r -T o n i c M O T O F i g . 27. A diagrammatic neuronal arrangement i l l u s t r a t i n g the s u p r a s p i n a l system, i n v o l v i n g 5-HT and n o r a d r e n a l i n e (NA), i m p l i c a t e d i n t o n i c a l l y a n -t a g o n i z i n g p r e s y n a p t i c i n h i b i t i o n of the QUAD-MSR. In t h i s and the subse-quent f i g u r e s the symbols + and - i n d i c a t e e x c i t a t o r y and i n h i b i t o r y s y n -apses, r e s p e c t i v e l y . 77 neurones activated by flex i o n r e f l e x afferents or group lb afferents were proposed to be t o n i c a l l y i n h i b i t e d by certain r e t i c u l o s p i n a l pathways (Eng-berg et_ al_. , 1968a, b: Holmqvist and Lundberg, 1959, 1961). In the present study, a cold block did not a l t e r the magnitude of the PBST-MSR suggesting that these flexor l a afferents are not under a tonic i n h i b i t o r y influence of a descending system. Therefore the monoaminergic terminals, proposed to t o n i c a l l y i n h i b i t the presynaptic i n h i b i t o r y pathway from the PBST group I afferents, l i k e l y synapse with interneurones i n the in h i b i t o r y pathway. We do not know why cyproheptadine and phenoxybenzamine antagonized the i n h i b i t i o n of the flexor MSR. I t i s unlikely that 5-HT and noradrenaline f a c i l i t a t e the above i n h i b i t i o n since imipramine and fluoxetine did not enhance th i s i n h i b i t i o n . The effects of clonidine on presynaptic i n h i b i t i o n of the MSR are d i s -cussed at the end of this section. In conclusion, the findings i n this study indicate that: 1. a de-scending 5-HT system t o n i c a l l y decreases the e x c i t a b i l i t y of the QUAD l a afferents (Fig. 30) and 2. a supraspinal system, involving both 5-HT and noradrenaline, t o n i c a l l y antagonizes presynaptic i n h i b i t i o n of the QUAD-MSR (Fig. 27). Bulbospinal i n h i b i t i o n of the MSR: The blockade of bulbospinal i n h i b i t i o n by the 5-HT precursor, 5-HTP, and the 5-HT neuronal uptake blocking agent, fluoxetine, as w e l l as a p a r t i a l reversal of the above blockade by the 5-HT antagonist, cyprohepta-dine, are consistent with our proposal implicating 5-HT i n antagonizing bulbospinal i n h i b i t i o n (Sastry, 1973: S i n c l a i r and Sastry, 1974). Bulbospinal i n h i b i t i o n of the QUAD-MSR was blocked by imipramine 78 ( S a s t r y , 1973; S i n c l a i r and S a s t r y , 1974) and f l u o x e t i n e and enhanced by cyproheptadine to a greater extent than the i n h i b i t i o n of the PBST-MSR. T h i s would suggest that the b u l b o s p i n a l i n h i b i t o r y pathway to the above extensor MSR r e c e i v e s a g r e a t e r i n h i b i t o r y i n f l u e n c e of the 5-HT system than that of the f l e x o r MSR. Assuming that the 5-HT system that i s proposed to antagonize b u l b o -s p i n a l i n h i b i t i o n i s t o n i c a l l y a c t i v e , where 5-HT i s s t e a d i l y r e l e a s e d i n t o the s y n a p t i c c l e f t s , imipramine and f l u o x e t i n e l i k e l y enhance the 5-HT s y n a p t i c a c t i v i t y by b l o c k i n g the uptake of t h i s amine i n t o the nerve t e r m i n a l s . This assumption i s strengthened by the f i n d i n g that cyprohepta-dine enhances b u l b o s p i n a l i n h i b i t i o n . Moreover, i n the _p_-CPA p r e t r e a t e d a n i m a l s , i n which the 5-HT nerve t e r m i n a l s are l i k e l y f u n c t i o n a l l y i n o p e r -a t i v e , imipramine f a i l e d to a l t e r the i n h i b i t i o n ( S a s t r y , 1973; S i n c l a i r and S a s t r y , 1974). Since imipramine was more potent i n antagonizing b u l b o s p i n a l i n h i b i -t i o n o f the MSR when administered i n t r a - a r t e r i a l l y to the s p i n a l cord than when i n j e c t e d i n t r a - a r t e r i a l l y to the b r a i n stem or i n t r a v e n o u s l y , the s p i -n a l cord i s very l i k e l y the s i t e o f i t s b l o c k i n g a c t i o n . This i m p l i e s that the 5-HT nerve t e r m i n a l s that are i n v o l v e d i n antagonizing b u l b o s p i n a l i n -h i b i t i o n are l o c a t e d i n the s p i n a l cord s i n c e imipramine very l i k e l y b l o c k s t h i s i n h i b i t i o n by b l o c k i n g the 5-HT neuronal uptake ( S i n c l a i r and S a s t r y , 1974). Phenoxybenzamine d i d not enhance b u l b o s p i n a l i n h i b i t i o n . Furthermore, pretreatment o f cats with a-MPT d i d not a l t e r the b l o c k i n g a c t i o n of i m i -pramine on t h i s i n h i b i t i o n ( S a s t r y , 1973; S i n c l a i r and S a s t r y , 1974). T h e r e f o r e , i t i s u n l i k e l y that a n o r a d r e n a l i n e system antagonizes b u l b o -s p i n a l i n h i b i t i o n . Phenoxybenzamine enhanced r e c u r r e n t (see d i s c u s s i o n on 79 r e c u r r e n t i n h i b i t i o n ) and p r e s y n a p t i c i n h i b i t i o n s o f the QUAD-MSR where n o r -a d r e n a l i n e was i m p l i c a t e d i n t o n i c a l l y antagonizing these i n h i b i t i o n s (see p r e s y n a p t i c i n h i b i t i o n - t h i s s e c t i o n ; S i n c l a i r and S a s t r y , 1974a). At present we have no e x p l a n a t i o n f o r the b l o c k i n g a c t i o n of phenoxybenza-mine on b u l b o s p i n a l i n h i b i t i o n . 1 I t i s improbable that a n o r a d r e n a l i n e s y s -tem mediates b u l b o s p i n a l i n h i b i t i o n s i n c e , i n p_-CPA p r e t r e a t e d cats i n which n o r a d r e n a l i n e would be expected to be p r e s e n t , imipramine d i d not enhance t h i s i n h i b i t i o n . Moreover.desipramine, an e f f e c t i v e n o r a d r e n a l i n e neuronal uptake b l o c k i n g agent, antagonized r a t h e r than enhanced the i n h i -b i t i o n ( S a s t r y , 1973; S i n c l a i r and S a s t r y , 1974). During b u l b o s p i n a l i n h i b i t i o n o f the QUAD-MSR, the QUAD-AP was f a c i l i -t a t e d and the antidromic motoneurone f i e l d p o t e n t i a l was reduced, i n d i c a t i n g that b u l b o s p i n a l i n h i b i t i o n of t h i s extensor MSR i n v o l v e s both p r e s y n a p t i c and p o s t s y n a p t i c types o f i n h i b i t i o n . This f i n d i n g would confirm s i m i l a r observations made by Chan and Barnes (1972). Imipramine antagonized both the above f a c i l i t a t i o n of the QUAD-AP and the r e d u c t i o n of the f i e l d poten-t i a l , w h i l e b l o c k i n g b u l b o s p i n a l i n h i b i t i o n of t h i s MSR. Moreover, cypro-heptadine p a r t i a l l y r e v e r s e d the above e f f e c t s o f imipramine. These r e s u l t s suggest that 5-HT i s i n v o l v e d i n antagonizing both the p r e s y n a p t i c and the p o s t s y n a p t i c types of b u l b o s p i n a l i n h i b i t i o n of the QUAD-MSR ( F i g . 28). In the majority of the experiments, minimal s t i m u l a t i o n s t r e n g t h u t i -l i z e d to evoke b u l b o s p i n a l i n h i b i t i o n reduced the antidromic f i e l d p o t e n -t i a l without a l t e r i n g the magnitude of the QUAD-AP. T h e r e f o r e , b u l b o s p i n a l i n h i b i t i o n of the MSR may be predominantly a p o s t s y n a p t i c t y p e ; o f i n h i b i t i o n . T h i s p o s t s y n a p t i c i n h i b i t o r y pathway l i k e l y contains a d i s y n a p t i c l i n k i n -v o l v i n g a s p i n a l interneurone (Jankowska:et a l . , 1968). H u l t b o r n and Udo (1972). showed that the l a i n h i b i t o r y i n t e r n e u r o n e s , a s s o c i a t e d w i t h r e c i p -80 rocal l a i n h i b i t i o n of the MSR, receive excitatory inputs from the descend-ing cortico-, rubro- and vestibulospinal t r a c t s . I t i s unknown whether the above interneurones are involved i n bulbospinal i n h i b i t i o n of the MSR. The hyperpolarization of motoneurones evoked by bulbospinal i n h i b i t i o n i s re-duced by strychnine (L l i n a s , 1964). This agent also blocks reciprocal l a i n h i b i t i o n by antagonizing the e f f e c t of the neurotransmitter, probably gly-cine, that i s released from the l a i n h i b i t o r y interneurones (Eccles et a l . , 1954a; Curtis and Duggan, 1969; Curtis et a l . , 1971). Therefore, i t i s pos-s i b l e that these interneurones are connected with the bulbospinal i n h i b i t o r y pathway. However, reciprocal l a i n h i b i t i o n does not appear to be inhi b i t e d by a 5-HT system (see discussion on this i n h i b i t i o n ) . I f the l a inhibitory, interneurones are involved i n the bulbospinal i n h i b i t o r y pathway, the 5-HT terminal i n the spinal cord must end on the axon terminals of the bulbospinal neurones. In the present investigation, since a t r a i n duration of 300 msec was used to evoke bulbospinal i n h i b i t i o n , we are unable to predict the conduction ve l o c i t y and the neuronal arrangement of the presynaptic bulbospinal i n h i b i -tory pathway. The tonic i n h i b i t i o n on various pathways activated by f l e x i o n reflex afferents or group lb afferents, i n decerebrate cats, were suggested to be mediated at the spinal interneurone l e v e l (Engberg et a l . , 1968a, b; Holmqvist and Lundberg, 1959, 1961). S i m i l a r l y , presynaptic type of bulbo-s p i n a l i n h i b i t i o n of the MSR might also be t o n i c a l l y i n h i b i t e d by the 5-HT system at an interneurone l e v e l . However, we have no evidence to support t h i s idea. Clineschmidt and Anderson (1970) found that methysergide, cinanserin, j l - l y s e r g i c acid diethylamide (LSD) and 2-bromo-LSD, but not cyproheptadine, antagonized bulbospinal i n h i b i t i o n of the MSR. These authors also found 81 M O T O F i g . 28. A diagrammatic neuronal c i r c u i t r y r e p r e s e n t i n g the t o n i c a l l y a c -t i v e 5-HT system that i s proposed to antagonize p r e - and p o s t s y n a p t i c types of b u l b o s p i n a l i n h i b i t i o n o f the QUAD-MSR. 82 that methysergide and LSD had a spinal s i t e of action. Therefore, they pro-posed that a 5-HT interneurone i n the spinal cord i s involved i n this i n h i b i -tory pathway. However, there i s no convincing evidence for the existence of 5-HT c e l l bodies i n the spinal cord (Dahlstrom and Fuxe, 1965; Fuxe, 1965; Proudfit and Anderson, 1973). Proudfit and Anderson (1973) suggested that the blockade of bulbospinal i n h i b i t i o n by cinanserin and methysergide could result from an enhancement of bulbospinal f a c i l i t a t i o n i f t h i s f a c i l i t a t o r y pathway i s under an i n h i b i t o r y influence of a t o n i c a l l y active 5-HT system. However, we observed that methysergide converted the i n h i b i t i o n into a f a c i l i -tation i n animals that were pretreated with p_-CPA (Sastry, 1973; S i n c l a i r and Sastry, 1974). Hence, methysergide i s probably not a 5-HT antagonist i n this system. In addition, our data do not f i t the proposals presented by Cline-schmidt and Ande rson (1970) or Proudfit and Anderson (1973). The action of clonidine on this i n h i b i t i o n i s discussed at the end of this section. In conclusion, the findings i n the present study along with our e a r l i e r observation (Sastry, 1973; S i n c l a i r and Sastry, 1974) indicate that a de-scending 5-HT system i n the spinal cord of the cat has a tonic i n h i b i t o r y influence on presynaptic and postsynaptic types of bulbospinal i n h i b i t i o n of the MSR (Fig. 28). Although i n Fig. 28 the 5-HT nerve terminals are shown to impinge on the interneurones i n the spinal cord, we do not know where i n the spinal cord these terminals exert t h e i r blocking action. Recurrent i n h i b i t i o n of the MSR: The antagonism of recurrent i n h i b i t i o n of the QUAD-MSR by 5-HTP and fluoxetine as w e l l as the p a r t i a l reversal of the above effects by cypro-heptadine are consistent with our proposal that a monoaminergic system which 83 i n v o l v e s s e r i a l l y arranged 5-HT and n o r a d r e n a l i n e neurones i n h i b i t s t h i s i n h i b i t i o n of the extensor MSR ( S a s t r y , 1973; S i n c l a i r and S a s t r y , 1974a). The f i n d i n g that the b l o c k i n g e f f e c t of f l u o x e t i n e was prevented by a c o l d b l o c k suggests that t h i s agent, l i k e imipramine ( S a s t r y , 1973; S i n c l a i r and S a s t r y , 1974a), i s e x e r t i n g i t s e f f e c t through a descending system. F l u o x e t i n e d i d not a l t e r r e c u r r e n t i n h i b i t i o n of the PBST-MSR. I t was p r e v i o u s l y shown that imipramine a l s o f a i l e d to a l t e r t h i s i n h i b i t i o n ( S i n -c l a i r and S a s t r y , 1974a). These f i n d i n g s suggest that r e c u r r e n t i n h i b i t i o n of t h i s f l e x o r MSR i s not i n f l u e n c e d by a 5-HT or a n o r a d r e n a l i n e system. The dose-response curves for imipramine's b l o c k i n g a c t i o n on r e c u r r e n t i n h i b i t i o n of the MSR, when the agent was administered i n t r a v e n o u s l y or i n t r a - a r t e r i a l l y to the s p i n a l cord or the b r a i n stem, i n d i c a t e that i m i p r a -mine very l i k e l y exerts i t s e f f e c t i n the s p i n a l c o r d . T h i s agent has been shown to p r e f e r e n t i a l l y b l o c k the uptake of 5-HT over that of n o r a d r e n a l i n e ( C a r l s s o n et a l . , 1969a, b ; Ross and R e n y i , 1969; Shaskan and Snyder, 1970). T h e r e f o r e , i n the proposed monoaminergic system that i n h i b i t s r e c u r r e n t i n h i b i t i o n , the 5-HT t e r m i n a l s are l i k e l y l o c a t e d i n the s p i n a l c o r d . Presuming that imipramine antagonized r e c u r r e n t i n h i b i t i o n by b l o c k i n g the neuronal uptake of the b i o g e n i c amines, the descending monoaminergic system must be t o n i c a l l y a c t i v e . This i d e a i s strengthened by the o b s e r v a -t i o n that imipramine acts through a s u p r a s p i n a l system but has a s p i n a l s i t e o f a c t i o n . Moreover, a c o l d b l o c k , which enhanced the i n h i b i t i o n of the QUAD-MSR i n c o n t r o l animals ( S a s t r y , 1973; S i n c l a i r and S a s t r y , 1974a), f a i l e d to a l t e r the i n h i b i t i o n i n animals p r e t r e a t e d w i t h p_-CPA o r a-MPT. The f i n d i n g that cyproheptadine and phenoxybenzamine enhanced the i n h i b i -t i o n of the extensor r e f l e x f u r t h e r i n d i c a t e s that the monoaminergic system i s t o n i c a l l y a c t i v e . 84 0 N A A + 5 - H T T o n i c l a Q U A D M O T O Fig. 29. A diagrammatic neuronal scheme portraying the t o n i c a l l y active supraspinal system, involving noradrenaline (NA) and 5-HT, implicated i n antagonizing recurrent i n h i b i t i o n of the QUAD-MSR. 85 The present study does not i n d i c a t e where i n the s p i n a l cord the mono-aminergic system antagonizes the r e c u r r e n t i n h i b i t o r y pathway. Since the major e f f e c t of i o n t o p h o r e t i c a l l y a p p l i e d 5-HT and n o r a d r e n a l i n e on Renshaw c e l l s i s i n h i b i t i o n (Biscoe and C u r t i s , 1966; Engberg and R y a l l , 1966; Weight and S a l m o i r a g h i , 1966), perhaps the above system terminates on Ren-shaw c e l l b o d i e s . The e f f e c t s of c l o n i d i n e on r e c u r r e n t i n h i b i t i o n are discussed e l s e -where i n t h i s s e c t i o n . In c o n c l u s i o n these r e s u l t s , along with our e a r l i e r f i n d i n g s ( S a s t r y , 1973; S i n c l a i r and S a s t r y , 1974a), i n d i c a t e that r e c u r r e n t i n h i b i t i o n of the extensor QUAD- but not of the f l e x o r PBST-MSR i s under a t o n i c i n h i b i -t o r y i n f l u e n c e of a descending system which i n v o l v e s 5-HT and n o r a d r e n a l i n e . We a l s o propose that the 5-HT terminals i n t h i s monoaminergic system are l o c a t e d i n the s p i n a l cord ( F i g . 29). R e c i p r o c a l l a i n h i b i t i o n o f the MSR: Since imipramine, f l u o x e t i n e , 5-HTP, cyproheptadine and phenoxybenz-amine d i d not s i g n i f i c a n t l y a l t e r r e c i p r o c a l l a i n h i b i t i o n of the MSR, 5-HT and n o r a d r e n a l i n e do not l i k e l y a f f e c t t h i s i n h i b i t i o n . A c o l d b l o c k e n -hanced the i n h i b i t i o n of the QUAD-MSR but not of the PBST-MSR suggesting that the i n h i b i t i o n of the above extensor MSR i s under a t o n i c i n h i b i t o r y i n f l u e n c e of a descending system. The uncondit ioned MSR: The enhancement of the MSR by f l u o x e t i n e and 5-HTP as w e l l as a r e v e r -s a l of t h i s augmentation by cyproheptadine are c o n s i s t e n t with the r e p o r t s i n d i c a t i n g t h a t a 5-HT system has an o v e r a l l f a c i l i t a t o r y e f f e c t on the MSR 86 (Ande r s o n , 1972; Cl ineschmldt et a l . , 1971). In cats with an acute but not a c h r o n i c s p i n a l t r a n s e c t i o n , p a r g y l i n e and l - t r y p t o p h a n enhanced the MSR whereas imipramine p o t e n t i a t e d the f a c i l i t a t i o n of the MSR by 5-HTP ( C l i n e -schmidt et a l . , 1971; Shibuya and Anderson, 1968). These r e s u l t s i n d i c a t e that imipramine, p a r g y l i n e and JL-tryptophan act through a descending p a t h -way. In the present i n v e s t i g a t i o n , a c o l d b l o c k decreased the QUAD-MSR by about 45 per cent but d i d not a l t e r the PBST-MSR. T h e r e f o r e , a s u p r a s p i n a l system has an o v e r a l l t o n i c f a c i l i t a t o r y e f f e c t on t h i s extensor MSR. Since a c o l d b l o c k f a i l e d to a l t e r the QUAD-MSR i n JD-CPA p r e t r e a t e d animals, the above s u p r a s p i n a l system l i k e l y i n v o l v e s 5-HT. A c o l d b l o c k enhanced and imipramine decreased the QUAD-AP. This e f f e c t of imipramine was reversed by z. a c o l d b l o c k or cyproheptadine. These r e s u l t s suggest that a descending t o n i c a l l y a c t i v e 5-HT system decreases the e x c i t a b i l i t y of QUAD l a a f f e r -ents ( F i g . 30). The s i z e of the QUAD-AP i s i n v e r s e l y r e l a t e d to the magnitude of the QUAD-MSR ( F i g . 6 ) . T h e r e f o r e , the decrease i n the QUAD-AP by imipramine suggests that t h i s agent enhances the MSR. However, the r e f l e x was de-pressed r a t h e r than augmented by imipramine i n most of the experiments. The decrease i n the s i z e of the antidromic f i e l d p o t e n t i a l by t h i s agent may be r e s p o n s i b l e f o r the above r e d u c t i o n of the MSR. Barnes e t a l . (1962) r e p o r t e d t h a t , i n cats a n a e s t h e t i z e d w i t h p e n t o -b a r b i t a l , a f u n c t i o n a l blockade of s u p r a s p i n a l inputs to the s p i n a l cord r e s u l t e d i n a h y p e r p o l a r i z a t i o n o f the motoneurones. In the present s t u d y , the antidromic motoneurone f i e l d p o t e n t i a l was reduced by a p p l i c a t i o n o f a c o l d b l o c k . The above observations i n d i c a t e that the s u p r a s p i n a l systems have an o v e r a l l t o n i c e x c i t a t o r y e f f e c t on the motoneurones. I t i s unknown whether the motoneurones i n the study of Barnes e_t a l . (1962) i n n e r v a t e the 87 flexor or the extensor muscles. In the present study, although the f i e l d p o t e n tial might involve both flexor and extensor motoneurones, since a cold block decreased the extensor but not the flexor MSR, the descending tonic f a c i l i t a t o r y e f f e c t i s very l i k e l y on the extensor rather than the flexor motoneurones (Fig. 30). Imipramine augmented the submaximal f i e l d potential at a dose of 2 mg/kg but reduced this potential when a second dose was administered. I t i s unknown whether the above f a c i l i t a t o r y effect i s mediated through 5-HT. The maximal f i e l d p o t e ntial was decreased by imipramine. The MSR i n con-t r o l and i n p_-CPA pretreated animals was also depressed by this agent. Moreover, cyproheptadine did not reverse imipramine's action on the MSR and the f i e l d p o t e n t i a l . Therefore, the depressant effects of imipramine on the MSR and the f i e l d potential do not appear to be mediated through 5-HT. The decrease i n the MSR and the f i e l d potential produced by imipra-mine appears to be dependent on the descending inputs since l i t t l e depres-sion, by t h i s agent, occurred during a cold block (Fig. 24). The depressant effect of 5-HTP on the MSR, observed i n some experi-ments, i s probably not due to a 5-HT involvement as imipramine potentiated the f a c i l i t a t o r y action (Clineschmidt et a l . , 1971) but reduced the depres-sant effect of this agent. Furthermore, the reduction i n the MSR by 5-HTP was rapid i n onset and was not usually present 90 min after the s t a r t of i n j e c t i o n , when 5-HT levels i n the spinal cord were reported to be maximal (Anderson and Shibuya, 1966). The blockade of various i n h i b i t i o n s by fluoxetine and 5-HTP i s not due to t h e i r excitatory effect on the MSR since these compounds did not a l t e r reciprocal l a i n h i b i t i o n . Fluoxetine enhanced the flexor and the extensor MSR. However, this agent antagonized recurrent and presynaptic i n h i b i t i o n s 88 0 5 - H T \ / t o n i c + A A -l a Q U A D M O T O F i g . 30. A schematic neuronal arrangement i l l u s t r a t i n g : 1. the proposed descending t o n i c a l l y a c t i v e 5-HT system that decreases the e x c i t a b i l i t y of QUAD l a a f f e r e n t s and 2. the s u p r a s p i n a l t o n i c f a c i l i t a t o r y system that impinges on the extensor motoneurones. 89 of the extensor but not of the flexor r e f l e x . 5-HTP increased or decreased the MSR but always antagonized the i n h i b i t i o n s . In conclusion, the results i n the present investigation suggest that: 1. a t o n i c a l l y active 5-HT system decreases the e x c i t a b i l i t y of QUAD l a afferents and 2. a tonic supraspinal system has an excitatory effect on QUAD motoneurones (Fig. 30). Clonidine e f f e c t s : Clonidine was reported to be a s p e c i f i c a-adrenergic receptor stimu-lant i n the central nervous system (Anden et a l . , 1970; Finch, 1974; Ko-binger and Pi c h l e r , 1975). In support of these reports, Anderson and Stone (1974) found that this agent mimicked the depressant effects of nor-adrenaline on the majority of cerebral c o r t i c a l and medullary neurones tested. Moreover, bulbocapnine reversed the effects on some c e l l s of both noradrenaline and clonidine, but not of 5-HT. Therefore, clonidine was i n -cluded i n the present study to test the proposals implicating noradrenaline i n antagonizing presynaptic and recurrent i n h i b i t i o n s of the extensor MSR. A l l the in h i b i t i o n s of the QUAD- and the PBST-MSRs examined i n this investigation were antagonized by clonidine. Other observations i n the present study do not implicate noradrenaline i n antagonizing bulbospinal and reciprocal l a i n h i b i t i o n s of the extensor or the flexor MSRs or pre-synaptic and recurrent i n h i b i t i o n s of the flexor reflex. I t i s noteworthy that the adrenergic blocking agent, phenoxybenzamine, enhanced the i n h i b i -tions which were proposed to be under a tonic i n h i b i t o r y influence of nor-adrenaline but f a i l e d to increase other i n h i b i t i o n s . Moreover, this agent did not a l t e r the blocking action of clonidine on bulbospinal, reciprocal l a , presynaptic and recurrent i n h i b i t i o n s of the PBST-MSR. Therefore, the 90 blockade by clonidine of various i n h i b i t i o n s , where noradrenaline i s not suggested to be involved, i s puzzling. The finding that a cold block had no effect on clonidine's action on reciprocal l a , presynaptic and recurrent i n h i b i t i o n s of the QUAD and the PBST reflexes indicates that this agent i s acting i n the spinal cord. Although clonidine antagonizes the in h i b i t i o n s of the MSR, i t does not induce convulsions i n the animals. The flexor and the extensor MSRs were reduced by clonidine. The blockade of the i n h i b i t i o n s , the depression of the unconditioned MSR and the lack of convulsant effect by clonidine could be explained i f this agent decreases the release of synaptic transmitters. A l o c a l anaesthetic type of action by clonidine i s improbable because t h i s agent did not a l t e r the size of the neurone action potentials. The amino acid, y-aroinobutyric acid (GABA), has been implicated i n presynaptic i n h i b i t i o n of the MSR (Barker and N i c o l l , 1972; B e l l and Ander-son, 1974; Davidoff, 1972; Eccles £t a l . , 1963; Levy, 1974). Glycine i s probably the in h i b i t o r y transmitter involved i n reciprocal l a and recurrent i n h i b i t i o n s of the MSR (Curtis et a l . , 1968; Curtis, 1969; Curtis et a l . , 1971; Werman et a l . , 1968). The finding that strychnine, reported to be a s p e c i f i c glycine antagonist (Curtis et a l . , 1971), reduced the hyperpolar-i z a t i o n of motoneurones during bulbospinal i n h i b i t i o n ( L l i n a s , 1964) sug-gests that glycine may be involved i n th i s i n h i b i t i o n . Huffman and McFadin (1972) found that b i c u c u l l i n e , reported to be a s p e c i f i c GABA antagonist (Curtis et a l . , 1971a), blocked bulbospinal i n h i b i t i o n of the PBST-MSR but not of the QUAD-MSR. This observation suggests that GABA may be involved i n bulbospinal i n h i b i t i o n of the above flexor MSR. Hence, the p o s s i b i l i t y that clonidine antagonizes the effects of GABA and glycine was tested. Clonidine did antagonize the reduction of discharge irate produced by these 91 amino acids i n about 50 per cent of the s p i n a l neurones. Thus, c l o n i d i n e may have b l o c k e d the i n h i b i t i o n s by a n t a g o n i z i n g GABA and g l y c i n e . The reason f o r the f i n d i n g that i n some c e l l s c l o n i d i n e b l o c k e d the e f f e c t s o f e i t h e r g l y c i n e or GABA but not of both i s not c l e a r . I t should be mentioned that only c l o n i d i n e currents that had no s i g n i f i c a n t e f f e c t on the c o n t r o l c e l l f i r i n g r a t e were used to t e s t t h i s agent 's antagonism towards the a c t i o n s of the amino a c i d s . Higher c l o n i d i n e currents might have blocked the e f f e c t s o f both g l y c i n e and GABA on the above c e l l s but were not t e s t e d . C l o n i d i n e d i d not a l t e r the e f f e c t s of g l y c i n e and GABA on about 40 per cent of the c e l l s . Even when very h i g h currents were passed to e ject c l o n i d i n e , through one of the p i p e t t e s used i n t h i s study, the f i r i n g rate of 4 c e l l s tested was u n a l t e r e d . I t may be p o s s i b l e t h a t , i n the above case, the m i c r o p i p e t t e b a r r e l c o n t a i n i n g c l o n i d i n e was o b s t r u c t e d and t h i s agent could not be e j e c t e d . However i n the r e s t of the cases, although c l o n i d i n e f a i l e d to a l t e r the e f f e c t s of the amino a c i d s , t h i s agent i n -creased or decreased the discharge rate of the neurones when h i g h e r c u r -rents were passed, i n d i c a t i n g that c l o n i d i n e was r e l e a s e d . The present experiments were performed on u n i d e n t i f i e d s p i n a l neurones. A b e t t e r ap-proach would have been to t e s t the a c t i o n s of c l o n i d i n e , g l y c i n e and GABA on neurones that were i d e n t i f i e d to r e c e i v e endogenous g l y c i n e and GABA n e u r o n a l i n p u t s . At p r e s e n t , the reason f o r the p o t e n t i a t i o n of the amino a c i d e f f e c t s by c l o n i d i n e , observed i n a few cases, i s not c l e a r . The p o p u l a t i o n o f neurones t e s t e d i n t h i s study i s s m a l l . F u r t h e r i n v e s t i g a t i o n i s necessary to make more c o n c l u s i v e remarks. In view o f the present f i n d i n g s that c l o n i d i n e antagonized g l y c i n e 92 and GABA effects on some spinal neurones, this agent does not appear to be a s p e c i f i c a-adrenergic agonist. This idea i s supported by several reports i n the l i t e r a t u r e . Csongrady and Kobinger (1974) reported that clonidine activates h i s t a m u ^ receptors on the guinea pig a t r i a . Bloch elE a l . (1974) suggested that clonidine exerts i t s hypotensive effect through a dopaminer-gic, but not a noradrenergic, system i n the brain stem. Clonidine was pro-posed to antagonize the release of noradrenaline (Starke et a l . , 1972). Anderson and Stone (1974) reported that clonidine did not a l t e r the f i r i n g rate of 32 out of 185 c o r t i c a l and 5 out of 62 medullary c e l l s whose d i s -charge rate was reduced by noradrenaline. Furthermore, clonidine enhanced the f i r i n g rate of 15 c o r t i c a l and 4 medullary c e l l s which were depressed by noradrenaline. Therefore, the results that clonidine antagonized bulbospinal and re-ciprocal l a i n h i b i t i o n s of the flexor and the extensor MSRs, as w e l l as presynaptic and recurrent i n h i b i t i o n s of the flexor MSR, need not negate the hypotheses that the above i n h i b i t i o n s are not under a tonic influence of noradrenaline. 93 CONCLUSIONS In this investigation, the influences of bulbospinal 5-hydroxytrypt-amine (5-HT) and noradrenaline neurones on presynaptic, bulbospinal, recur-rent and reciprocal l a in h i b i t i o n s of the extensor quadriceps (QUAD) and the flexor posterior biceps-semitendinosus (PBST) monosynaptic reflexes (MSRs) were examined i n cats decerebrated at the mid - c o l l i c u l a r l e v e l . The observations led to the following proposals: 1. Presynaptic i n h i b i t i o n of the QUAD- but not of the PBST-MSR i s under a tonic i n h i b i t o r y influence of a descending system which involves 5-HT and noradrenaline (Fig. 27). 2. Stimulation i n the ventromedial bulbar r e t i c u l a r formation evokes both presynaptic and postsynaptic i n h i b i t i o n s of the extensor r e f l e x (Fig. 28). This finding supports a si m i l a r observation made by Chan and Barnes (1972). 3. A t o n i c a l l y active 5-HT system, i n the spinal cord, antagonizes presyn-aptic and postsynaptic types of bulbospinal i n h i b i t i o n of the QUAD re f l e x (Fig. 28). 4. A descending system involving 5-HT and noradrenaline has a tonic i n h i -b i t o r y influence on recurrent i n h i b i t i o n of the extensor MSR (Fig. 29). 5. The e x c i t a b i l i t y of QUAD l a afferents i s decreased by a descending t o n i -c a l l y active 5-HT system (Fig. 30). 6. A t o n i c a l l y active supraspinal system has an ov e r a l l excitatory influence on the extensor motoneurones (Fig. 30). 94 7. 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