C I SEROTONIN INVOLVEMENT IN THE BLOCKADE OF BULBOSPINAL AND RECURRENT INHIBITION OF THE MONOSYNAPTIC REFLEX by BHAGAVATULA SREE RAMA SASTRY A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in the D i v i s i o n of Pharmacology and Toxicology of the Faculty of Pharmaceutical Sciences We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA September, 1973. In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and study. I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying o f t h i s t h e s i s f o r s c h o l a r l y purposes may by h i s r e p r e s e n t a t i v e s . be granted by PAVAA4VMJLCC^C-^V J ; The U n i v e r s i t y o f B r i t i s h Vancouver 8, Canada Date s h a l l not be permission. Department o f U to - Department or I t i s understood t h a t copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l g a i n written the Head of my <x^Ji Columbia (^i2> I allowed without my ii ABSTRACT Sastry, B.S.R., D i v i s i o n of Pharmacology and Toxicology, Faculty of Pharmaceutical Sciences, U n i v e r s i t y of B r i t i s h Columbia; September, 1973. Serotonin Involvement i n the Blockade of Bulbospinal and Recurrent I n h i b i t i o n of the Monosynaptic Reflex. The monoamine uptake blocking agents, imipramine HC1 (5 mg/kg i.v.) and desipramine HC1 (4.8 mg/kg i . v . ) , and the monoamine oxidase i n h i b i t o r , pargyline HC1 (30 mg/kg i.v.) antagonized bulbospinal i n h i b i t i o n (BSI) of the monosynaptic r e f l e x (MSR) i n unanaesthetized cats decerebrated at the m i d - c o l l i c u l a r l e v e l . The e f f e c t of imipramine was q u a n t i t a t i v e l y more on BSI of the quadriceps (QUAD)-MSR compared to that on BSI of the posterior biceps-semitendinosus (PBST)-MSR . Imipramine's a c t i o n on t h i s i n h i b i t i o n was also q u a n t i t a t i v e l y greater compared to that of the equimolar dose of desipramine. Pretreatment of the animals with the tryptophan hydroxylase i n h i b i t o r , DL-p_-chlorophenylalanine (p_-CPA) (300 mg/kg i . p . for 3 consecutive days) completely eliminated the blocking a c t i o n of imipramine. However, pretreatment of the animals with the tyrosine hydroxylase i n h i b i t o r , DL-0(-methyl-p_-tyrosine methyl ester HC1 (0(-MPT) (126 mg/kg i . p . given 16 and 4 hours before the recording ) had no e f f e c t on imipramine's a c t i o n . These findings strongly suggest that a 5-hydroxytryptamine (5-HT, serotonin) system antagonizes BSI of the MSR. They do not support the proposal of Clineschmidt and Anderson (1970) that the bulbospinal i n h i b i t o r y pathway involves a 5-HT interneurone i n the s p i n a l cord. Imipramine HC1 (5 mg/kg i.v.) and pargyline HC1 (30 mg/kg i.v.) blocked recurrent i n h i b i t i o n (RI) of the MSR evoked by stimulation of a dorsal root. Imipramine blocked RI of the QUAD-MSR but had no e f f e c t on RI of Ill the PBST-MSR. Pretreatment of the animals with either JJ-CPA or o(-MPT prevented the blocking action of imipramine on RI. Application of a 'cold block' which potentiated RI of the QUAD-MSR also eliminated the blocking action of imipramine on t h i s i n h i b i t i o n . These observations suggest that a supraspinal monoaminergic system which involves 5-HT and noradrenaline l i n k s has a tonic i n h i b i t o r y e f f e c t on RI of the QUAD-MSR. John G. S i n c l a i r , Ph.D., Supervisor. iv TABLE OF CONTENTS V LIST OF FIGURES ABSTRACT ii INTRODUCTION 1 SURVEY OF LITERATURE 5 Organization of the Lumbosacral Spinal Cord of the Cat . . . 5 The Segmental Monosynaptic Reflex 7 Postsynaptic I n h i b i t i o n i n the Spinal Cord . . . . . . . . 8 a. Reciprocal I n h i b i t i o n 10 b. Recurrent I n h i b i t i o n of the Spinal Motoneurones . . 11 Presynaptic I n h i b i t i o n i n the Spinal Cord 12 The Raphe Nuclei 13 The Descending Monoaminergic Fibres i n the Spinal Cord . . 16 Bulbospinal I n h i b i t i o n of the Monosynaptic Reflex 19 Supraspinal E f f e c t s on Renshaw C e l l s 22 EXPERIMENTAL 25 RESULTS 31 Bulbospinal I n h i b i t i o n of the Monosynaptic Reflex 31 Recurrent I n h i b i t i o n of the Monosynaptic Reflex 35 The Unconditioned Monosynaptic Reflex 41 Blood Pressure E f f e c t s 42 DISCUSSION REFERENCES 43 . 52. V LIST OF FIGURES Figure 1. Page A diagrammatic representation o f the experimental set up 2. The e f f e c t of imipramine 26 on bulbospinal i n h i b i t i o n . of the DR-MSR i n A. non-pretreated cats; B. i n cats pretreated with £~CPA; C. i n cats pretreated with 3. oC-MPT 32 The e f f e c t of imipramine on bulbospinal i n i n b i t l o n of the QUAD-MSR and the PBST-MSR 4. The blockade of bulbospinal i n h i b i t i o n of the . .. PBST-MSR by imipramine 5. 34 The e f f e c t of desipramine on bulbospinal i n h i b i t i o n of the MSR 6. 36 The e f f e c t of pargyline on bulbospinal i n h i b i t i o n of the MSR 7. 33 The e f f e c t of imipramine 37 on recurrent i n h i b i t i o n of the DR-MSR i n A. non-pretreated cats; B. i n cats pretreated with _p_-CPA; C. i n cats pretreated with 8. 0(-MPT The e f f e c t of imipramine on recurrent i n h i b i t i o n of the QUAD-MSR and the PBST-MSR 9. 38 39 The e f f e c t of pargyline on recurrent i n h i b i t i o n of the MSR 40 VI ACKNOWLEDGEMENTS The author i s deeply indebted to Dr. John G. S i n c l a i r f o r h i s invaluable guidance and i n s p i r a t i o n throughout the course of t h i s investigation. He i s g r a t e f u l to Mrs. Marjorie Chaplin f o r her help i n preparing the f i g u r e s . This work was supported by a grant from the Medical Research Council to Dr. S i n c l a i r and a U n i v e r s i t y of B r i t i s h Columbia summer research scholarship to the author. 1 INTRODUCTION Recently, several investigators have suggested that the putative neurotransmitters i n the c e n t r a l nervous system mine (CNS), 5-hydroxytrypta- (5-HT, serotonin) and noradrenaline (NA) are involved i n motor c o n t r o l (Cranmer et a l . , 1959; Fuxe, 1965; Anden et a l . , 1966; Anderson, 1972). Chronic transection of the s p i n a l cord depletes 5-HT and NA caudal to the transection (Carlsson et^ a l . , 1963; Magnusson and Rosengren, 1963). Stimulation of the s p i n a l cord gives r i s e to a release of 5-HT (Anden et a l . , 1964, and NA 1965). These observations suggest that the s p i n a l cord contains descending 5-HT and NA f i b r e s of supraspinal o r i g i n . The raphe n u c l e i are located along the m i d - s a g i t t a l plane i n the midbrain, pons and medulla oblongata (Taber et^ al^., 1960). Histochemical (Dahlstrom and Fuxe, 1964, 1965) and pharmacological (Fuxe, 1965) studies indicate that almost the e n t i r e 5-HT neuronal population i n the CNS i s located i n the raphe n u c l e i . Neurones containing NA are most densely located i n the medulla oblongata but also exist i n the pons and midbrain. Noradrenergic c e l l s have a more scattered d i s t r i b u t i o n than 5-HT Most of the descending 5-HT cells. and NA f i b r e s originate i n the medulla oblongata (Dahlstrom and Fuxe, 1965). These monoaminergic f i b r e s descend i n the d o r s o l a t e r a l and ventromedial f u n i c u l i of the spinal cord and terminate i n the substantia gelatinosa i n the dorsal horn and on the v e n t r o l a t e r a l and d o r s o l a t e r a l motor n u c l e i i n the v e n t r a l horn 1965). (Fuxe, 2 Existence of the descending monoaminergic systems r a i s e s the question of their f u n c t i o n a l s i g n i f i c a n c e i n the s p i n a l cord. In unanaesthetized cats with an acute s p i n a l transection, 3,-tryptophan, 5-hydroxytryptophan (5-HTP) and J--~3 ,4-dihydroxyphenylalanine (1-dopa) increased the MSR (Anderson and Shibuya, 1966; Baker and Anderson, 1970a). Pretreatment of the cats with pargyline potentiated the e f f e c t s of 5-HTP, _l-tryptophan and L-dopa on the MSR (Anderson e_t al_. , 1967) . In cats with a chronic s p i n a l transection, pargyline and JL-tryptophan did not a l t e r the MSR (Shibuya and Anderson, 1968). Moreover, imipramine, a 5-HT neuronal uptake blocking agent, potentiated the e f f e c t of 5-HTP and pargyline on the MSR i n cats with an acute s p i n a l transection but not i n cats with a~ chronic s p i n a l transection (Clineschmidt et a l . , 1971; Clineschmidt, 1972). Pretreatment of the animals with the tryptophan hydroxylase i n h i b i t o r , DL-pj-chlorophenylalanine (p_-CPA) , blocked MSR (Taber, 1971). These observations the e f f e c t s of 5-HTP on the indicate that a descending 5-HT system i n the s p i n a l cord has a f a c i l i t a t o r y action on the MSR. Bulbospinal i n h i b i t o r y systems were also suggested to e x i s t . In decerebrate-decerebellate cats, stimulation i n the nucleus raphe magnus produced long latency negative d o r s a l root p o t e n t i a l s (DRPs) i n the lumbosacral s p i n a l cord. These DRPs were blocked by the 5-HT antagonists, methysergide and cinanserin (Proudfit and Anderson, 1973). S i m i l a r l y , bulbospinal i n h i b i t i o n of the MSR, as f i r s t described by Magoun and Rhines (1946), was also blocked by the 5-HT antagonists, methysergide, d_-lysergic acid diethylamide (LSD) , 2««bromo-LSD (BOL) and cinanserin (Clineschmidt and Anderson, 1970). These authors found, using c l o s e a r t e r i a l i n j e c t i o n s to the brain stem and the s p i n a l cord, that LSD and methysergide act at the s p i n a l cord l e v e l . Hence, they suggested that 3 the bulbospinal i n h i b i t o r y system contains a 5-HT interneurone i n the s p i n a l cord. To investigate further the p o s s i b i l i t y that the l a t t e r bulbospinal i n h i b i t o r y system contains a 5-HT l i n k i t i s reasoned that, i f the hypo- thesis of Clineschmidt and Anderson (1970) i s correct, drugs which i n c r ease the a v a i l a b i l i t y of 5-HT i n the synaptic c l e f t s should bulbospinal i n h i b i t i o n (BSI) of the MSR. the uptake of 5-HT of NA increase Imipramine p r e f e r e n t i a l l y blocks whereas desipramine p r e f e r e n t i a l l y blocks the uptake (Ross and Renyi, 1969; Carlsson et^ al_. , 1969; Shaskan and Snyder, 1970). In the s p i n a l cord, pargyline elevates the l e v e l s of 5-HT but does not s i g n i f i c a n t l y increase the l e v e l s of NA and the e f f e c t s of t h i s drug on the MSR are very l i k e l y mediated through 5-HT (Anderson et a^L., 1967) . Therefore, i n the present study, imipramine, desipramine and pargyline are tested on BSI of the MSR. Stimulation of the ventromedial mesencephalic r e t i c u l a r formation, v e n t r o l a t e r a l bulbar r e t i c u l a r formation, v e n t r a l thalamus (Fields of F o r e l and Zona Incerta) and p e r i c r u c i a t e cortex decreased the rate of .. Renshaw c e l l discharges evoked by antidromic a c t i v a t i o n of the motor axons (Koizumi et a l . , 1959; man, Haase and Meulen, 1961; Mac Lean and Le£f- 1967). In a similar study by Haase and Meulen (1961), stimulation of the anterior lobe of the cerebellum was found to f a c i l i t a t e the Ren- shaw c e l l discharges. These findings i n d i c a t e that the Renshaw c e l l d i s charges are influenced by supraspinal inputs. In decerebrate cats, imipramine blocked recurrent i n h i b i t i o n (RI) of the MSR (Von Tan and Henatsch, 1968). However, t h i s drug had no effect on the i n h i b i t i o n i n cats with the s p i n a l cord transected at the thoracic l e v e l (Von Tan and Henatsch, 1969). These authors suggested that a mono- 4 aminergic pathway has a strong curbing e f f e c t on RI of the MSR. The pre- sent i n v e s t i g a t i o n i s an extention of the above work and i s designed to determine whether RI of the flexor and extensor MSRs are equally affected by imipramine and whether the drug's e f f e c t s are mediated through a supraspinal 5-HT or NA system. 5 SURVEY OF THE LITERATURE Organization of the Lumbosacral Spinal Cord of the Cat The c e l l bodies of the lumbosacral afferent f i b r e s are located i n the dorsal root ganglia. A l l central processes of these c e l l s enter the s p i n a l cord. The peripheral processes innervate various structures such as s k e l e t a l muscles, skin, e t c . , and convey impulses c e n t r a l l y . These f i b r e s are c l a s s i f i e d into various groups according to their thickness, myelination and function. Group I afferents are 12 - 20 |im i n diameter and are heavily myelinated. These f i b r e s are s u b c l a s s i f i e d into l a and l b a f f e r e n t s . The l a f i b r e s convey impulses from the annulospiral endings of the muscle spindles, whereas, the l b f i b r e s convey impulses from the Golgi tendon organs. The conduction v e l o c i t y of the group I f i b r e s i s high (about 120 m/sec) compared to that of the other f i b r e s . Group I I (diameter 6 - 1 2 pm) and group I I I ( 1 - 6 pm) f i b r e s are c a l l e d high threshold muscle a f f e r e n t s . They are t h i n l y myelinated and their conduct i o n v e l o c i t y i s f a i r l y low (about 5 - 3 0 m/sec). The t h i n l y myelinated cutaneous alpha ( 6 - 1 7 Jim) and d e l t a ( 1 - 6 pm) f i b r e s convey impulses from cutaneous o r i g i n . Both muscle nerves and cutaneous nerves contain unmyelinated group IV f i b r e s (Ruch and Patton, 1960). Rexed (1952, 1954, 1964) subdivided the spinal gray matter into nine laminae and a tenth region surrounding the c e n t r a l canal. This type of subdivision of the c e l l groups i s based on the appearance of the c e l l s and the boundaries are only zones of t r a n s i t i o n . Lamina I i s a thin sheath that caps the surface and bends around the margins of the dorsal horn. 6 Lamina I I i s situated immediately v e n t r a l to the lamina I. I t contains t i g h t l y packed small c e l l s and i s traversed by many strands of spinal afferent f i b r e s . This lamina corresponds to the substantia gelatinosa. Lamina I I I i s a band of large neurones scattered across the dorsal horn and i s situated v e n t r a l to lamina I I . Lamina IV i s the broadest of the f i r s t four layers. I t i s a heterogenous zone with small to large c e l l s of v a r i a b l e shapes. Lamina V i s a broad zone extending across the neck of the dorsal horn and i s subdivided into medial and l a t e r a l zones. Many f i b r e s pass through the l a t e r a l zone to give i t a r e t i c u l a t e d appearance. Lamina VI i s located at the base of the dorsal horn. I t i s subdivided into the small medial and the larger l a t e r a l zones. The medial zone i s less compact and contains large triangular or star shaped neurones. Descending supraspinal pathways project to c e l l s i n the l a t e r a l zone. Lamina VII occupies most of the intermediate zone of the gray matter. The v e n t r a l part of t h i s lamina extends into the v e n t r a l horn. Medium sized c e l l s predominate i n t h i s zone. The dorsal nucleus of Clarke, the intermediolateral and intermediomedial n u c l e i are seen i n t h i s layer. The v e n t r a l part of t h i s layer includes Renshaw c e l l s , interneurones and Y~motoneurones, Lamina VIII contains a heterogeneous mixture of small and medium sized c e l l s with scattered large neurones; and i s not sharply separated from lamina VII. Lamina VIII i s confined to the medial part of the v e n t r a l horn. Vestibulospinal and r e t i c u l o s p i n a l f i b r e s , and the medial l o n g i t u d i n a l f a s c i c u l u s terminate i n t h i s zone. Lamina IX i s composed of the largest c e l l s of the spinal cord, the 0{-motoneurones, situated i n the v e n t r o l a t e r a l region of the v e n t r a l horn. The medial nuclear masses have a d i f f u s e border with lamina VIII. A considerable number of the t h i c k l y myelinated afferent f i b r e s terminate i n the dorsal nucleus of Clarke (Grant and Rexed, 1958). The 7 c o l l a t e r a l branches of the afferent f i b r e s that are d i s t r i b u t e d to parts of the anterior horn become concentrated mostly i n the c e n t r a l part of the lamina VI. These c o l l a t e r a l s i n numerous small bundles pass into lamina IX where they arborize about the soma and dendrites of the motoneurones (Sprague and Ha, 1964). The dorsal root f i b r e s also give off c o l l a t e r a l s which pass into lamina VII. Since, c o l l a t e r a l s of dorsal root f i b r e s passing to lamina VII and IX traverse broad regions of lamina VII, some f i b r e s end upon interneurones i n t h i s zone, as well as on dendrites of motor n u c l e i which extend beyond the l i m i t s of lamina IX (Sprague and Ha, 1964). Group l b , II and I I I muscle nerve afferents and cutaneous afferents generate synaptic p o t e n t i a l s i n the c e n t r a l parts of laminae V, VI and VII (Sprague and Ha, 1964). The motoneurones are organized according to their f u n c t i o n a l inner- vation. The c e l l s that innervate the extensor muscles are located i n the v e n t r o l a t e r a l horn l a t e r a l to those that innervate the f l e x o r muscles. The f l e x o r motoneurones are arranged i n a number of subgroups such that each group of c e l l s innervate muscles which move a p a r t i c u l a r j o i n t l i e i n the same h o r i z o n t a l plane i n the v e n t r a l horn; the more d i s t a l the muscle, the more dorsal the p o s i t i o n of the c e l l s (Romains, 1964). The Segmental Monosynaptic Reflex When a s p i n a l dorsal root i s stimulated with minimal i n t e n s i t i e s of current, a small v e n t r a l root discharge can be observed. As the strength i s increased, the amplitude of the early v e n t r a l root stimulus discharge increases and reaches a maximal value and upon further increase i n the stimulus strength l a t e discharges are observed. The early sharp spike r e f l e c t s the synchronous a c t i v a t i o n of OC-motoneurones through l a a f f e r ents and i s referred to as the monosynaptic r e f l e x (MSR). The l a t e 8 asynchronous discharges r e f l e c t the f i r i n g of motoneurones through polysynaptic r e f l e x (PSR) pathways mediated by a c t i v a t i o n of high threshold afferent f i b r e s . The latency between stimulation of the d o r s a l root and recording the MSR from the v e n t r a l root includes the conduction i n the l a f i b r e s , one synaptic delay and the conduction from the motoneu- rone c e l l bodies to the v e n t r a l root recording electrode. The time to the peak of MSR time total i s about 2 msec. The c e n t r a l delay i n transmission across a single synapse i s approximately 0.5 msec (Eccles, 1961). The predominant feature of the afferent f i b r e s i s divergence. That i s , a s i n g l e afferent f i b r e branches and p a r t i c i p a t e s i n f i r i n g of many motoneurones. However, f o r a motoneurone to f i r e , many presynaptic knobs impinging on the motoneurone must be a c t i v a t e d . The amplitude of the MSR e l i c i t e d by stimulation of a d o r s a l root i s an index of the number of motoneurones that are r e c r u i t e d into the discharge zone. But, many motoneurones are depolarized only to a subthreshold l e v e l and are said to be excited s u b l i m i n a l l y . These motoneurones do not contribute to the amplitude of the MSR. During the process of f a c i l i t a t i o n , however, the motoneurones that are excited s u b l i m i n a l l y and those that are not excited by the test stimulus w i l l be a v a i l a b l e for recruitment into the discharge zone. Hence, as more motoneurones are r e c r u i t e d , the amplitude of the MSR increases. During i n h i b i t i o n of motoneurones, the e x c i t a b i l i t y of these c e l l s i s reduced and they are eliminated from the discharge zone, hence, the amplitude of the MSR decreases. Postsynaptic I n h i b i t i o n i n the Spinal Cord Postsynaptic i n h i b i t i o n i s an index of depression of the neuronal e x c i t a b i l i t y which occurs independently v i t y (Brock £t al_. , 1952; of the excitatory synaptic a c t i - Coombs ^jt ad. , 1955a, b) . This process involves 9 i n h i b i t i o n of a neurone by d i r e c t synaptic impingement. Examples of postsynaptic i n h i b i t i o n include r e c i p r o c a l ( d i r e c t , l a ) 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 afferents a c t i v a t i n g i n h i b i t o r y interneurones which impinge on ^-motoneurones of antagonistic muscles (Eccles e£ a^l. , 1956). Recurrent i n h i b i t i o n i s brought about by v o l l e y s i n the motor axon c o l l a t e r a l s which a c t i v a t e i n h i b i t o r y interneurones known as Renshaw c e l l s , which inturn, i n h i b i t the motoneurones (Eccles et a l . , 1954). The membrane p o t e n t i a l of a mammalian motoneurone i s about -70 mV and i s c a l l e d the r e s t i n g membrane p o t e n t i a l (RMP) (Frank and Fourtes, 1955) . During i n h i b i t i o n of motoneurones a hyperpolarization of the motoneurone membrane occurs which i s known as the i n h i b i t o r y postsynaptic p o t e n t i a l (IPSP) (Brock et a l ., 1952). The equilibrium p o t e n t i a l f o r the IPSP i s about -80 mV (Brock et a l . , 1952). During r e c i p r o c a l i n h i b i t i o n , the IPSP i n the motoneurone i s observed about 1.5 to 2.0 msec a f t e r i t s onset. The decay of the IPSP i s about 3.0 msec (Brock et a l . , 1952a). During hyperpolarization the i n h i b i t o r y transmitter increases the membrane permeability to ions having a hydrated diameter l e s s than 1.4 times that of potassium ion. Thus, there i s said to be a decrease i n membrane resistance or an increase i n membrane conductance. I t was suggested (Coombs e t ^ a l . , 1955a, b) that during the IPSP there i s an inward d i f f u s i o n of c h l o r i d e ions and an outward d i f f u sion of potassium ions through the neuronal membrane. However, Lux et a l . (1970), Lux (1971) and L l i n a s and Baker (1972) proposed that the IPSP i s generated by a s e l e c t i v e permeability increase to chloride ions i n the outward d i r e c t i o n . They also reported that a potassium ion permeability change i s probably not s i g n i f i c a n t l y involved i n this process. When the membrane p o t e n t i a l i s lowered (depolarized), the IPSP 10 increases, The IPSP decreases or reverses when the membrane i s hyperpol a r i z e d by passage of cathodal current. The IPSP may following an iontophoretic also be reversed i n j e c t i o n of chloride ions into the neurone (Coombs et^ a l . , 1955a) . a. Reciprocal I n h i b i t i o n Discharges i n group l a afferents not only excite the motoneurones of the s y n e r g i s t i c muscles but also i n h i b i t the motoneurones of the antagonistic muscles through an interneurone. The i n h i b i t o r y transmitter released from the l a i n h i b i t o r y interneurone axon terminal i s suggested to be glycine (Werman et a l . , 1968; was C u r t i s et a l . , 1968). When glycine administered i o n t o p h o r e t i c a l l y i n the v i c i n i t y of the motoneurone, t h i s amino acid hyperpolarized and reduced the resistance of the neuro- n a l membrane. P r i o r hyperpolarization of the membrane reduced or reversed the e f f e c t of glycine. Comparison of the equilibrium p o t e n t i a l s of ionic events associated with the IPSP and the hyperpolarization produced by glycine indicate that they were s i m i l a r (Werman et_ a_l. , 1968; ejt ail. , 1968). Strychnine, the Curtis which blocks the l a i n h i b i t o r y pathway, spe- c i f i c a l l y blocks the e f f e c t s of glycine on the motoneurone (Curtis et^ a l . , 1971). Hultborn et_ al_. (1971) found that impulses i n the motor axon c o l l a t e r a l s i n h i b i t the interneurones of the l a i n h i b i t o r y pathway. Thus, they suggested that increased motoneuronal f i r i n g i n h i b i t s the discharges from the l a i n h i b i t o r y interneurones to the antagonistic motoneurones. Hultborn and Udo (1972) have shown that the disynaptic i n h i b i t o r y e f f e c t s on the motoneurones from the descending c o r t i c o - , rubro- and v e s t i b u l o spinal t r a c t s involve the l a i n h i b i t o r y interneurones. Thus, there seems to be a convergence of supraspinal and l a afferent inputs e x c i t i n g the 11 la inhibitory interneurones. b. Recurrent I n h i b i t i o n of the Spinal Motoneurones Volleys i n the motor axon c o l l a t e r a l s s y n a p t i c a l l y a c t i v a t e the • i n h i b i t o r y interneurones, Renshaw c e l l s , which inturn impinge on motoneu- rones. Renshaw c e l l s , activated by v e n t r a l root stimulation, discharge i n a c h 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 second (Eccles et^ al_., 1954) . The reason f o r such a high f r e quency of f i r i n g of these neurones was attributed to a convergence of excitatory input from the c o l l a t e r a l s of many motoneurone axons ( Eccles et^ a l . , 1956a ; R y a l l et a l . , 1970). When s y n a p t i c a l l y activated, the duration of Renshaw c e l l discharge i s about 50 msec (Eccles elt a l . , 1956a) . R y a l l (1970) found that i n cats anaesthetized with chloralose, a n t i - dromic v o l l e y s i n the motoneurone axons may evoke a postsynaptic inhibi- t i o n of Renshaw c e l l s instead of e x c i t a t i o n . The latency observed f o r t h i s i n h i b i t i o n suggested that the e f f e c t i s brought about by a disynarp t i c pathway and the other interneurone involved i n t h i s pathway was found to be a Renshaw c e l l (Ryall, 1970). Thus, some Renshaw c e l l s inhi- b i t the discharges of other Renshaw c e l l s and t h i s i s probably responsible for recurrent f a c i l i t a t i o n . Renshaw c e l l s are also involved i n i n h i b i t i n g the l a i n h i b i t o r y interneurones as discussed i n the previous section, but there seems to be no input from l a i n h i b i t o r y interneurones to Renshaw c e l l s (Ryall and Piercey, 1971). Stimulation of i p s i l a t e r a l afferent f i b r e s i n the group I I and I I I muscle nerves and cutaneous afferents have excitatory input to Renshaw c e l l s through polysynaptic chains. However, stimulation of the same afferents on the c o n t r a l a t e r a l side brought about i n h i b i t i o n of these c e l l s without e x c i t a t i o n (Ryall and Piercey, 1971). Renshaw c e l l s are more strongly excited by the c o l l a t e - 12 Q(-motoneurone axons r a l s of large phasic rone axons than the small tonic motoneu- However, 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 a l . , 1972). Several pharmacological and p h y s i o l o g i c a l studies indicate that the chemical transmitter released at the motor axon collateral-Renshaw c e l l synapse i s acetylcholine (Curtis and R y a l l , 1966a, b, c ) . D i h y d r o - p erythroidine, which blocks c h o l i n e r g i c transmission at the n i c o t i n i c receptors, depressed the response of Renshaw c e l l s to synaptic stimulation (Eccles et a l . , 1956a; C u r t i s and R y a l l , 1966b). Eserine, an a n t i c h o l i nesterase drug, greatly prolonged the discharges of Renshaw c e l l s induced by synaptic e x c i t a t i o n (Eccles et a l . , 1954, 1956a). I n t r a - a r t e r i a l i n j e - c t i o n of acetylcholine or n i c o t i n e excites Renshaw c e l l s (Eccles et^ a l . , 1956a; C u r t i s and R y a l l , 1966a). The but not n i c o t i n e , i s increased excitatory action of a c e t y l c h o l i n e , by eserine while dihydro-p-erythroidine decreased the excitatatory action of both the substances (Eccles e£ a l . , 1956a). The i n h i b i t o r y transmitter released at the Renshaw cell-motoneu- rone synapse was 1968; suggested to be glycine (Eccles, 1966; Curtis et^ a l . , 1968; Presynaptic C u r t i s , 1969; Werman et a l . , C u r t i s et^ al_., 1971). I n h i b i t i o n i n the Spinal Cord Presynaptic i n h i b i t i o n i n the s p i n a l cord was on and Matthews (1938). Stimulation d i f f e r e n c e along the stimulated f i r s t shown by Barr- of a dorsal root produces a p o t e n t i a l or the adjacent dorsal root. The poten- t i a l developed i s c a l l e d negative dorsal root p o t e n t i a l (DRP). The represents a primary afferent depolarization t h i s DRP i n the s p i n a l cord neuronal output due (PAD), and DRP generation of induces a net i n h i b i t o r y action on the moto- to depression of the presynaptic excitatory impulses 13 (Renshaw, 1946; Brooks et^ a l . , 1948). Powerful presynaptic i n h i b i t i o n can be obtained i n cats anaesthetized with pentobarbital and usually t h i s i n h i b i t i o n i s l e s s intense i n decerebrate cats (Eccles et_ al_., 1963b) . In decerebrate cats v o l l e y s i n l a and l b afferents of f l e x o r muscle nerves depolarize group l a f i b r e s of both flexor and extensor muscle nerves. But group l a and l b v o l l e y s i n exten- sor muscle nerves, except those of the quadriceps muscle, do not have any depolarizing e f f e c t s on the f l e x o r or extensor l a afferents 1964). There was no e f f e c t from l a afferents group l b (Eccles et 21I., (Eccles, of any muscle nerves on 1963a) and cutaneous afferents (Eccles et a l . , 1963c). Group l b and II v o l l e y s from a l l muscle nerves and v o l l e y s exert a presynaptic cutaneous i n h i b i t i o n on group l b afferents of f l e x o r or extensor o r i g i n . Of those, the most powerful e f f e c t was found to be from group l b afferents (Eccles^et a l . , 1963a). Cutaneous v o l l e y s to a greater extent and group l b and extent produce a DRP on the cutaneous afferents (Eccles et a l . , 1963c). In producing presynaptic l a and II v o l l e y s from muscle nerves to a lesser i n h i b i t i o n on l a afferents by a c t i v a t i n g l b a f f e r e n t s , there i s a latent period of about 4 msec between the conditioning stimulus and maximum at about 20 msec and onset of the PAD. The PAD reaches i t s p e r s i s t s for about 200 msec or more (Eccles et a l . , 1962). Since the synaptic delay i n the mammalian c e n t r a l nervous system (CNS) i s about 0.5 msec (Eccles, 1961), i t was the c e n t r a l delay during presynaptic through at least two Eccles synaptic postulated i n h i b i t i o n involves transmission s e r i a l l y arranged interneurones (Eccles et. al.. , 1962) . (1963, 1964) suggested that the PAD i s due to a chemical transmitter released near the primary afferent terminal l a s t interneurone i n the chain. He also postulated of the presynaptic that terminals reduce the that from the depolarization magnitude of the p o t e n t i a l i n 14 these terminals, thereby l i m i t i n g the output of the synaptic transmitter (Eccles, 1963). Picrotoxin (Eccles et_ a l . , 1963a) and b i c u c u l l i n e (Curtis e_t al. , 1970, 1971a) were found to reduce the DRP. Since these agents s e l e c t i v e l y block the effects of $-aminobutyric acid (GABA), this amino acid was proposed as the neurotransmitter mediating the PAD (Eccles, 1964a). Consis- tent with this proposal, Barker and N i c o l l (1972) showed a sodium i o n dependent depolarizing c.ction of GABA on the primary afferent terminals and a s p e c i f i c blockade of i t s e f f e c t by b i c u c u l l i n e i n the i s o l a t e d s p i n a l cord of the frog. Recently, Krnjevic and Morris (1972) showed that the negative DRP produced i n the lumbar s p i n a l cord of the cat during stimulation of the s p i n a l afferent f i b r e s i s associated with an increase i n the e x t r a c e l l u l a r potassium ion concentration. These authors suggested that this r i s e i n the e x t r a c e l l u l a r potassium ion concentration i s either due to the a c t i v i t y of the unmyelinated nerve terminals or to a release of this i o n from the postsynaptic structures. The Raphe Nuclei The raphe n u c l e i are situated along the mid-sagittal plane i n the midbrain, pons and medulla oblongata (Taber et a l . , 1960). These c e l l s are separated from other c e l l u l a r aggregations by f i b r e masses. The r o s t r a l end of the raphe complex i s found i n the r o s t r a l mesencephalon and the caudal end i n the caudal h a l f of the medulla oblongata. Depending upon the location and type of c e l l s , the raphe n u c l e i are c l a s s i f i e d as f o l l o ws: nucleus raphe obscurus, nucleus raphe p a l l i d u s , nucleus raphe magnus, nucleus raphe pontis, nucleus c e n t r a l i s superior, nucleus l i n e a r i s medius and nucleus l i n e a r i s r o s t r a l i s (Taber et a l . , 1960). inter- 15 The nucleus raphe obscurus i s located m i d - s a g i t t a l l y i n the caudal medulla. This nucleus extends r o s t r a l l y upto the caudal pole of the i n f e r i o r o l l v e r y complex. In the dorsoventral plane, the nucleus i s s i t u ated primarily on the dorsal side. The c e l l s are medium to small i n s i z e . The nucleus raphe p a l l i d u s i s situated v e n t r a l to nucleus raphe obscurus and extends from the l e v e l of f a c i a l nucleus to the caudal medulla, sli- ghtly r o s t r a l to the caudal end of raphe obscurus nucleus. The nucleus raphe p a l l i d u s i s boardered by the pyramidal tract v e n t r a l l y and divided i n t o dorsal and v e n t r a l masses at the caudal part. The nucleus raphe magnus extends from the r o s t r a l end of the nucleus p a l l i d u s to the l e v e l of trapazoid body. The raphe magnus consists of a comparatively large mass of c e l l s which extend l a t e r a l l y into the r e t i c u l a r formation. At many l e v e l s , these c e l l s are separated from the r e t i c u l a r formation by l o n g i t u d i n a l l y running f i b r e s . The nucleus raphe pontis occupies the mid-sagittal part of the pons; however, some c e l l s are found more l a t e r a l l y . Most of the c e l l s of the nucleus raphe pontis are separated by dorsoventrally running f i b r e s . The nucleus c e n t r a l i s superior i s boardered by the decussation of brachium conjunctivum i n the d o r s o l a t e r a l plane and the nucleus i n t e r peduncularis i s situated v e n t r a l to these c e l l s . The nucleus raphe dorsal i s i s situated i n the v e n t r a l part of the periaqueductal gray and extends from the l e v e l of the dorsal tegmental nucleus to the caudal pole of the occulomotor nucleus. Ventral to the nucleus raphe d o r s a l i s , the medial l o n g i t u d i n a l fasciculus i s situated on either side. The nucleus l i n e a r i s intermedius i s composed of scattered c e l l s of small s i z e located i n the caudal midbrain. The nucleus l i n e a r i s r o s t r a l i s includes c e l l s of large, medium and small s i z e and i s situated medial to the occulomotor outflow. 16 The Descending Monoamlnergic Fibres i n the Spinal Cord In rabbits, a f t e r chronic s p i n a l transection at the second thoracic l e v e l , both 5-hydroxytryptamine (5-HT) and noradrenaline decrease caudal to the transection (Carlsson et_ al_., (NA) l e v e l s 1963; Magnusson and Rosengren, 1963). In mice and frogs, stimulation of the s p i n a l cord i n v i t r o induces a release of 5-HT these authors concluded that 5-HT and NA (Anden et a l . , 1964, 1965). Thus and NA are associated with the descend- ing neuronal pathways. Using histochemical (1964, 1965) fluorescence techniques, Dahlstrom and Fuxe showed the existence of monoaminergic neurones i n the of r a t s , guinea pigs, rabbits and cats. They found two CNS d i s t i n c t types of nerve c e l l s showing e i t h e r yellow or green fluorescence. The cells e x h i b i t i n g yellow fluorescence are medium to large i n s i z e and round to oval i n shape. The d i s t r i b u t i o n of these c e l l s that give r i s e to descending axons i s almost e n t i r e l y l i m i t e d to the caudal raphe n u c l e i (Raphe obscurus, raphe p a l l i d u s and raphe magnus). The c e l l s showing green fluorescence are scattered as small masses composed of neurones small to medium i n s i z e , multipolar and round to oval i n shape. Of these, cells that give r i s e to descending f i b r e s are located mostly i n the medulla oblongata from the r o s t r a l end of the i n f e r i o r o l i v e r y complex to the l e v e l of pyramidal decussation. Pharmacological studies (Fuxe, 1965) i n d i c a t e that c e l l s showing yellow fluorescence are those containing 5-HT green fluorescence are those containing NA. following observations: whereas those e x h i b i t i n g The evidence i s based on the The monoamine depleting agent, reserpine, almost completely removes both the yellow and green fluorescence; the monoamine oxidase (MAO) i n h i b i t o r , nialamide, enhances the yellow fluorescence; the tyrosine hydroxylase i n h i b i t o r , O^-methy1-m-tyrosine, s e l e c t i v e l y and reduces the green fluorescence. Fluorescence microscopic studies revealed that 5-HT and NA nerve terminals i n the region of raphe n u c l e i make contacts with c e l l s of each other as w e l l as with some nonfluorescent c e l l s (Fuxe, 1965). The 5-HT - axons from the caudal raphe n u c l e i run i n a v e n t r o l a t e r a l d i r e c t i o n and almost reach the v e n t r a l surface of the b r a i n stem l a t e r a l to the pyramidal t r a c t (Dahlstrom and Fuxe, 1965). Spinal lesion studies revealed that the 5-HT f i b r e s descend i n the d o r s o l a t e r a l and ventromedial f u n i - c u l i of the s p i n a l cord (Dahlstrom and Fuxe, 1965). Some of these f i b r e s cross to the other side of the s p i n a l cord. Most of the f i b r e s descend- ing d o r s o l a t e r a l l y i n the lumbosacral s p i n a l cord terminate i n the subs t a n t i a gelatinosa, and the ventromedially descending f i b r e s terminate i n the v e n t r o l a t e r a l and d o r s o l a t e r a l motor n u c l e i of the lamina IX. In cats, the density of termination of 5-HT f i b r e s i n substantia gelatinosa and the motornuclei i s almost the same. The NA f i b r e s also run v e n t r a l l y i n the b r a i n stem to reach the v e n t r o l a t e r a l part. In the s p i n a l cord, these f i b r e s descend i n the v e n t r o l a t e r a l and d o r s o l a t e r a l f u n i c u l i with some f i b r e s crossing to the opposite side. The NA terminals were found to be most dense i n substan- t i a gelatinosa and dense i n the v e n t r o l a t e r a l and d o r s o l a t e r a l motor n u c l e i of lamina IX. However, regions other than the above also receive NA terminals (Fuxe, 1965). Both the descending 5-HT and NA f i b r e s are unmyelinated and of 0.3 to 1.0 urn i n diameter (Dahlstrom and Fuxe, 1965). The observation that monoamine neurones of the caudal b r a i n stem give r i s e to descending pathways raises the question of t h e i r functional s i g n i f i c a n c e . In unanaesthetized cats with an acute spinal transection, 5-hydroxytryptophan (5-HTP) (75 mg/kg i . v . ) , J_-tryptophan (100 mg/kg i.v.) and l_-3,4-dihydroxyphenylalanine (1-dopa) (30 mg/kg i.v.) increased the 18 MSR to 310 %, 172 % and 212 % of the control levels respectively (Anderson and Shibuya, 1966; Baker and Anderson, 1970a). The 5-HT l e v e l s i n the s p i n a l cord of the cat increase a f t e r treatment with 5-HTP (Anderson and Shibuya, 1966). Four hours a f t e r the i n j e c t i o n of pargyline (30 mg/kg i . v the levels of 5-HT were elevated by 70 % but the NA levels were not s i g n i f i c a n t l y increased i n the cat's s p i n a l cord (Anderson et a l . , 1967). Pargyline also increased the MSR and i t s effects are blocked by the 5-HT antagonists but not by 0(-adrenergic blocking agents; thus, the e f f e c t s of t h i s MAO i n h i b i t o r on the MSR are very l i k e l y mediated through 5-HT (Anderson et a l . , 1967). However, pretreatment of cats with pargyline potentiated the e f f e c t s of L-dppa as w e l l as 5-HTP and ^-tryptophan on the MSR (Anderson et a l , , 1967). But, the action of L-dopa on the MSR was blocked by the 5-HT antagonists (Anderson and Banna, 1968). In cats with a chronic s p i n a l transection, pargyline and 1-tryptophan did not a l t e r the MSR; however, 5-HTP s t i l l enhanced the MSR i n thes animals (Shibuya and Anderson, 1968). In the s p i n a l cord, caudal to a chronic transection, 25 % of the control 5-HT l e v e l s (Shibuya and Anderson, 1968), 20 % of the dopa decarboxylase activity (Anden et a l . , 1964) and 17 % of the tryptophan hydroxylase a c t i v i t y remained (Clineschmidt et a l . , 1971a). Thus, Shibuya and Anderson (1968) suggested that the 5-HTP enhancement of the MSR i n cats with a chronic s p i n a l transection might be due to the presence of 5-HT interneurones i n the s p i n a l cord. However, evidence exists to show that 5-HT synthesis from 5-HTP can occur extraneuronally (Kuhar et a l . , 1971). Furthermore, 5-HTP can e i t h e r enter the adrenergic terminals and displace catecholamines (Ng ejt a l . , 1972) or d i r e c t l y a c t i v a t e the adrenergic receptors (Innes, 1962) These findings o f f e r a l t e r n a t i v e explanations for the work of Shibuya and Ande rson (1968) wliich shows a 5—HTP enhancement of the MSR i n chronic 19 s p i n a l animals. Clineschmidt jet a l . (1971) and Clineschmidt, (1972) showed that the neuronal uptake blocking agent, imipramine, potentiated the e f f e c t of 5-HTP and pargyline i n cats with an acute s p i n a l transection but not i n the animals with a chronic s p i n a l transection. Thus, a descending 5-HT system exists i n the s p i n a l cord of the cat whose o v e r a l l e f f e c t on the MSR is facilitation. Pretreatment of the cats with the tryptophan hydroxylase i n h i b i t o r , DL-£-chlorophenylalanine (p_-CPA) (300 mg/kg i . p . f o r 2 consecutive days) blocks the e f f e c t s of 5-HTP on the MSR ested that, although 5-HTP was (Taber, 1971). Hence, Taber sugg- converted into 5-HT i n these animals, 5-HT i s taken up by the empty synaptic v e s i c l e s but did not overflow into the synaptic c l e f t and activate the receptors. The increase of the MSR uced by 5-HTP i n cats with an acute s p i n a l cord transection was blocked by the following 5-HT cyproheptadine Clineschmidt ejt a l . , 1971). Bulbospinal I n h i b i t i o n of the Monosynaptic Magoun and Rhines also antagonists: methysergide, d-lysergic acid diethylamide (LSD), 2-bromo-LSD (BOL), cinanserin and (Banna and Anderson, 1968; ind- Reflex (1946) reported that the ventromedial bulbar r e t i c u l a r formation contains a descending neuronal system which exerts a general i n h i b i t o r y influence on the segmental MSR. a concomitant They also observed melting of the decerebrate r i g i d i t y . The caudal raphe n u c l e i f a l l within the i n h i b i t o r y area described by Magoun and Rhines (1946). The mechanism of bulbospinal i n h i b i t i o n (BSI) of the MSR was died by Llinas (1964a, b), L l i n a s and Terzuolo (1964, 1965) stu- and Jankow- ska et a l - (1968). These studies involved stimulation i n the bulbar r e t i c u l a r formation while recording the MSR and the i n t r a c e l l u l a r mem- 20 brane p o t e n t i a l of a p a r t i c i p a t i n g 0(-extensor motoneurone. Under these conditions,the MSR was reduced, the membrane was hyperpolarized, the resistance of the motoneurone membrane was reduced and the soma-dendrit i c (SD) component of the action p o t e n t i a l was blocked when the motoneurone was activated antidromically. These findings i n d i c a t e that BSI of the MSR i s of the postsynaptic type. When chloride ions were iontop h o r e t i c a l l y i n j e c t e d into the motoneurone the hyperpolarization produced during stimulation of the bulbar r e t i c u l a r formation was reversed (Llinas and Terzuolo, 1964). Thus the i o n i c mechanisms responsible f o r BSI are s i m i l a r to those of the r e c i p r o c a l i n h i b i t i o n . Bulbospinal i n h i b i t i o n of the 0 ( - f l e x o r motoneurones also involves a synaptic i n h i b i b i t o r y impingement on these motoneurones (Llinas and Terzuolo, 1965; Jankowska e^t a l . , 1968). Llinas and Terzuolo concluded (1965) that the i n h i b i t o r y synapses of the pathway are on the dendri-r t i c tree of the motoneurone, f a r from the soma. This conclusion was based on the observation that i n j e c t i o n of chloride ions i n t o the motoneurone did not reverse the hyperpolarization produced during BSI. However, Jankowska ejt a l . (1968) did not observe any difference between f l e x o r and extensor motoneurones i n this regard. The difference i n these two studies may be due to the difference i n the experimental Jankowska et_ a l . (1968) used decerebrated preparations. cats with a c o n t r a l a t e r a l hemisected and an i p s i l a t e r a l dorsal transected s p i n a l cord. The s p i n a l cord was i n t a c t i n the study of L l i n a s and Terzuolo (1965). The bulbospinal i n h i b i t o r y pathway descends i n the v e n t r a l quadrant of the s p i n a l cord. This pathway most l i k e l y has a disynaptic linkage and i t s conduction v e l o c i t y i s high (Jankowska et a l . , 1968). Llinas (1964b) found that strychnine (0.15 and 0.5 mg/kg i . v . ) decreased the hyperpolarization of the extensor motoneurone membrane caused by BSI but this drug did not block the i n h i b i t i o n of the MSR. He also found that picrotoxin (1 mg/kg i.v.) and mephenesin (120 mg/kg i.v.) did not block BSI and suggested that BSI i s probably not of presynaptic type. However, p i c r o t o x i n blocks the segmental DRP but does not block the heterosegmental and heterosensory DRPs (Besson and Abdelmoumene, 19 70; Besson et a l . , 1971; Benoish et a l . , 1972). Thus the p o s s i b i l i t y for a presynaptic type of BSI to exist cannot be e n t i r e l y ruled out. Although neither strychnine nor mephenesin blocked BSI when administered i n d i v i d u a l l y , a combination of low doses of these two drugs blocked the i n h i b i t i o n (Llinas, 1964b). In an attempt to explain these findings, L l i n a s (1964b) suggested the following two possible mechanisms: 1. Stry- chnine may increase a c t i v i t y i n the i n h i b i t o r y pathway while depressing the i n h i b i t o r y action. Mephenesin may antagonize this increased a c t i v i t y i n the pathway. Thus, the blocking e f f e c t of the combined i n j e c t i o n can be observed. 2. When strychnine blocks BSI i t may increase the background excitatory impingement on the motoneurone and mephenesin may block this excitatory influence. Stimulation of the medial r e t i c u l a r formation i n the caudal b r a i n 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 Stereotaxic Atlas of Snider and Niemer, 1964) produced negative DRPs on l a afferents of both flexor and extensor muscle nerves (Carpenter et a l . , 1966). These r e t i c u l o s p i n a l fibres descend i n the ventromedial s p i n a l cord. Carpenter at a l . (1966) also reported that stimulation of the v e n t r a l 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 v e n t r i c l e ) did not produce negative DRPs on l a afferents. However, Chan and Barnes (19 72) reported that stimulation of the v e n t r a l 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 ) produced both short and long latency negative DRPs 22 on l a afferents. Thus, a possible involvement of a presynaptic type of BSI can not be ruled out. I t i s not understood why there i s a controvercy between the above two reports. Several pharmacological studies have been carried out on BSI of the MSR. The following drugs were shown to block BSI: Strychnine (0.05 mg/kg i.v) dichloroisoproterenol ( 7 mg/kg i.v.) and reserpine (0.5mg/kg i.p ) (McLennan, 1961); mephenesin (2o - 30 mg/kg i.v.) (Kaada, 1950); morphine and meperidine (0.5 - 16 mg/kg i.v.) ( S i n c l a i r , 1973). B i c u c u l l i n e , a s p e c i f i c GABA antagonist, blocked BSI of the flexor MSR but had no e f f e c t on BSI of the extensor MSR (Huffman and McFadin, 1972). The 5-HT antagonists: methysergide (0.5 mg/kg i . v . ) , LSD (0.25 mg/kg i . v . ) , BOL (1.0 - 1.5 mg/kg i.v.) and cinanserin (4.0 mg/kg i.v.) but not cyproheptadine (5.0 mg/kg i.v.) also blocked BSI (Clineschmidt and Anderson, 1970). The results of c l o s e - a r t e r i a l i n j e c t i o n of methysergide and LSD to the s p i n a l cord and the b r a i n stem suggested that they act at the s p i n a l cord l e v e l . Thus, Clineschmidt and Anderson (19 70) proposed that the bulbospinal i n h i b i t o r y pathway contains a 5-HT interneurone i n the s p i n a l cord. Supraspinal E f f e c t s on Renshaw C e l l s Stimulation of the ventromedial mesencephalic r e t i c u l a r formation at the l e v e l of substantia n i g r a (A 6.5 - 1.0) or the v e n t r o l a t e r a l bulbar r e t i c u l a r formation at the l e v e l of hypoglossal nucleus (P 9.5 - 11.0) was found to have an i n h i b i t o r y e f f e c t on the number of Renshaw c e l l discharges evoked by antidromic volleys i n the motoneurone axons (Koizumi et a l . , 1959; Haase and Meulen, 1961; Mac Lean and Leffman, 1967). This i n h i b i t o r y e f f e c t can be obtained by stimulating either side of the r e t i cular formation but i s stronger when the side c o n t r a l a t e r a l to the Ren- 23 shaw c e l l was stimulated (Haase and Meulen, 1961). The latency between stimulation of the mesencephalic r e t i c u l a r formation and the onset of i t s i n h i b i t o r y e f f e c t on the Renshaw c e l l discharge i s about 9 msec and t h i s e f f e c t l a s t s at i t s maximum strength f o r about 20 to 25 msec. While stimulation of the mesencephalic r e t i c u l a r formation has a stronger i n h i b i t o r y e f f e c t on Renshaw c e l l discharges, the stimulation of bulbar r e t i c u l a r formation has a longer duration of action (MacLean and L e f f man, 1967). Stimulation of v e n t r a l thalamus (Fields of F o r e l , Zona Incerta) or the p e r i c r u c i a t e cortex i n h i b i t s the Renshaw c e l l discharges evoked by antidromic stimulation of the motor axons. Such an i n h i b i t i o n i s rapid i n onset and of short duration (MacLean and Leffman, 1967). The descen- ding f i b r e s from p e r i c r u c i a t e cortex pass through the pyramids i n the brain stem (MacLean and Leffman, 1967). Stimulation of the anterior lobe of the cerebellum has a f a c i l i t a tory e f f e c t on Renshaw c e l l discharges evoked by antidromic stimulation of the motoneurone axons. Stimulation i n the b r a i n stem r e t i c u l a r formation had no e f f e c t on these c e l l s activated i n the above manner (Haase and Meulen, 1961). However, when the Renshaw c e l l discharges were evoked by stimulation of a dorsal root, a c t i v a t i o n of the r e t i c u l a r formation with a conditioning i n t e r v a l of 12 msec, increased the disch*- arge rate of the Renshaw c e l l s (Haase and Meulen, 1961). In addition, stimulation of the v e n t r a l thalamus or the p e r i c r u c i a t e cortex could activate Renshaw c e l l s to discharge (MacLean and Leffman, 1967). The work of Haase and Meulen (1961) and MacLean and Leffman (1967) indicate that the Renshaw c e l l discharges are influenced by supraspinal inputs. Von Tan and Henatsch (1968) have studied the e f f e c t s of imipramine (0.5 to 2.0 mg/kg i.v.) on recurrent i n h i b i t i o n of the MSR i n decerebrate cats. They also showed that this drug blocks the i n h i b i t i o n at these doses. However, when the s p i n a l cord of the animal was sectioned at the thoracic l e v e l the drug did not block recurrent i n h i b i t i o n ( Von Tan and Henatsch, 1969). They suggested that a supraspinal monoaminergic pathway has a strong curbing effect on recurrent i n h i b i t i o n of the MSR. 25 EXPERIMENTAL Adult cats of e i t h e r sex (2.0 - 4.0 kg) were anaesthetized with ether. The trachea was cannulated and the animal was a r t i f i c i a l l y respired using a respiratory pump (Type AC; HP 1/4; C.F.Palmer (Lond.) Ltd.). The l e f t c a r o t i d artery was cannulated with No. 160 polyethylene tubing (Clay Adams, Div. of Becton, Dickinson and company) f i l l e d with d i l u t e d sodium heparin (Upjohn Company of Canada). This tubing was connected to a P-1000-A pressure transducer (Narco-Bio- Systems) which i n turn was connected to a type DMP-4A desk model physiograph (Narco-B±o-Systems) for recording blood pressure. The other carotid artery was l i g a t e d . A cephalic vein was cannulated with No. 90 polyethylene tubing f i l l e d with normal s a l i n e . This cannula was used f o r intravenously i n j e c t i n g the test drugs. The animal's head was mounted on a stereotaxic frame (Narishige S c i e n t i f i c Instrument Laboratory). The s k u l l overlying the f r o n t a l and p a r i e t a l lobes of the cerebral cortex was removed. The animal was decerebrated at the m i d - c o l l i c u l a r l e v e l ( F i g . 1), the b r a i n tissue above the transection was removed and the s k u l l cavity was packed with gauze. The cut edges of the bone were covered with bone wax to control bleeding and prevent a i r embolism. Blood loss during decerebration was replaced by i n j e c t i n g dextran ( 6 ^ w / v ) immediately a f t e r decerebration. The o c c i p i t a l bone overlying the cerebellum was removed and the dura was cut to expose the cerebellum. A laminectomy was performed i n the lumbosacral region of the s p i n a l 26 Dorsal root L 7 Ventral root F i g . 1. A diagramatic representation of the experimental set up. I 27 cord. In some preparations L6, L7 and SI dorsal roots were cut b i l a t e r a l l y . In other preparations the dorsal roots on the l e f t were l e f t intact and the nerves on this side leading to the posterior biceps-semitendinosus (PBST) and quadriceps (QUAD) muscles were i s o l a t e d and cut. The c e n t r a l ends of these nerves were attached to b i p o l a r stimulating electrodes. The v e n t r a l roots L6, L7 and SI on the l e f t side were sectioned i n a l l preparations. The skin flaps on the back of the animal were used to make a pool f o r holding mineral o i l which prevented drying of the s p i n a l cord and spread of the current. The temperature of the mineral o i l pool and the body of the animal was maintained at 36 + 1^ C using automatic D.C. temperature regulators (Richardson et a l . , 1965) or a heating lamp. In animals that were used i n the 'cold block' experiments, the s p i n a l cord was exposed at T10 - T12, the dura-matter was cut and warm o i l was poured on the cord to maintain the temperature of the exposed s p i n a l cord at about 37^ C. Ether was discontinued following surgery and three hours were allowed for elimination of ether before recordings were taken. The animal was maintained on a r t i f i c i a l r e s p i r a t i o n throughout the experiment. The c e n t r a l end of the dorsal roots on the l e f t side ( i n most of the cases L7) and the corresponding v e n t r a l root were placed on b i p o l a r platinum hook electrodes, the monosynaptic r e f l e x (MSR) was evoked every 5 sec by stimulation of the dorsal root (DR-MSR) with a square wave pulse (0.1 msec) from the S2 unit of a Grass stimulator and which passes through a SIU5 stimulation i s o l a t i o n unit. The stimulus strength used was supramaximal f o r the MSR. In the animals with i n t a c t dorsal roots on the l e f t side and cut nerves to QUAD and PBST muscles the central end of one of the muscle nerves was stimulated to evoke the QUAD-MSR or PBST-MSR using the above mentioned parameters. The compound action p o t e n t i a l i n the v e n t r a l 28 root was amplified using a Tektronix 2A61 d i f f e r e n t i a l a m p l i f i e r and displayed on a Tektronix 560 model oscilloscope. A b i p o l a r coaxial s t a i n l e s s s t e e l electrode (0.5 mm 0.5 mm exposed t i p ) was directed s t e r e o t a x i c a l l y to the v i c i n i t y of the caudal raphe n u c l e i (P 7.5 parations; V -6 to -10 1964) separation, to 13.5; L o.o i n most and 0.5 i n a few pre- i n the Stereotaxic Atlas of Snider and Niemer, (Fig. 1). A locus i n this area was stimulated by a t r a i n (300 msec duration) of square wave pulses of 0.5 msec duration and at 150 Hz using the SI unit of a Grass S8 stimulator and a SIU5 stimulation i s o l a t i o n unit. This t r a i n was delivered so that the end of the t r a i n occurred msec before a stimulus was delivered to evoke the MSR. 7.5 The l o c a t i o n of the electrode i n the b r a i n stem and the stimulus strength (usually less than 5.0 V) were adjusted so that the MSR was i n h i b i t e d to about 40 % of i t s o r i g i n a l s i z e . Furthermore, the electrode was considered to be p l a - ced only i f there were no tonic contractures of the neck, back and the forelimbs or marked changes i n the blood pressure during the stimulation. Recurrent i n h i b i t i o n (RI) of the MSR was obtained by stimulation of the c e n t r a l ends of two v e n t r a l roots (usually L6 and SI) 7.5 msec before evoking the MSR. was In some preparations, however, a s i n g l e ventral root stimulated. Square wave pulses of 0.5 msec duration using the SI unit of a S8 Grass stimulator and a SIU5 stimulation i s o l a t i o n unit were delivered, the stimulus strength was adjusted so that the MSR ted to approximately 40 % of the unconditioned was inhibi- value. In some animals, a f t e r obtaining stable recordings of bulbospinal i n h i b i t i o n (BSI) and RI of the MSR, was F l a x e d i l (gallamine t r i e t h i o d i d e ) i n j e c t e d to minimize the e f f e c t of movement of the animal on the recordings. Bulbospinal and recurrent i n h i b i t i o n of the MSR were tested at 10 29 min i n t e r v a l s . The MSR was q u a n t i f i e d by averaging 10 consecutive spikes. The average MSR before and a f t e r the drug administration was expressed as a percentage of the f i n a l control average MSR. The degree of e i t h e r BSI or RI was q u a n t i f i e d by averaging the amplitude of 10 consecutive MSR spikes as w e l l as the following 5 spikes conditioned with e i t h e r BSI or RI. The percentage difference between these values on the f i n a l control test was equal to 100 % i n h i b i t i o n . Percent i n h i b i t i o n of previous and subsequent tests were c a l c u l a t e d based on t h i s f i g u r e . ft Following control recordings, imipramine HCl (5 mg/kg i.v.) or an ft equimolar amount of desipramine HCl (4.8 mg/kg i.v.) was administered ftft over a 10 min period. In other animals pargyline HCl (30 mg/kg i . v . ) was i n j e c t e d over a 30 min period. In two preparations methysergide ftftft bimaleate (0.5 mg/kg i.v.) was i n j e c t e d over a 5 min period. A l l i n j e c t i o n s were given using an i n f u s i o n pump (Harvard Model 975}. To block supraspinal inputs to the s p i n a l cord, 1 cm cubes of frozen mammalian Ringer s o l u t i o n were placed on the s p i n a l cord which was exposed at the T10 - T12 l e v e l (Wall, 1967). The absence of BSI of the MSR 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 block of supraspinal inputs. In the subsequent discussion these animals w i l l be referred to as the 'cold block' preparations. To reverse the 'cold block', Ringer cubes were removed and the cold Ringer s o l u t i o n was sucked out using an a s p i r a tor. Warm o i l (about 37^ C) was poured on the cord repeatedly u n t i l BSI of the MSR returned to i t s pre-'cold block' l e v e l . This r e v e r s i b l e 'cold block' technique was used to test whether the effects of imipramine on the MSR and RI of the MSR were mediated through a supraspinal neuronal system. <e f </cf ifc «fc Some animals were pretreated with DL-JJ-*chlorophenylalanine (p_-CPA} (300 mg/kg i . p . f o r 3 consecutive days) and prepared f o r record- 30 ings 24 hours a f t e r the l a s t dose. Two doses of p_-CPA 300 mg/kg i . p . given two consecutive days reduced 5-hydroxytryptamine levels to 10 to 20 % of control values i n the s p i n a l cord (Taber, 19 71). Other animals were pretreated with DL~o(-methyl-p_-tyrosine methyl **** ester HC1 (126 mg/kg i.p.) 16 and 4 hours before preparing the animal for recordings. A f t e r a s i m i l a r treatment noradrenaline was reported to be depleted to an immeasurable quantity i n the s p i n a l cord (King and Jewett, 1971). Foot Note * Geigy Limited. ** Abbott Pharmaceuticals ***Sandoz Pharmaceuticals. ****Sigma Chemical Company. 31 RESULTS Bulbospinal I n h i b i t i o n of the Monosynaptic Reflex Imipramine HC1 (5.0 mg/kg i.v.) completely blocked bulbospinal i n h i b i t i o n (BSI) of the DR-MSR. The e f f e c t was rapid i n onset and was maximal a f t e r . 2 0 - 3 0 min. The above and subsequent time refer to the s t a r t of the i n j e c t i o n . The blocking action of imipramine started to decrease around 50 min and.was completely absent a f t e r about 2 hours (Fig. 2A). Bulbospinal i n h i b i t i o n of the QUAD-MSR and the PBST-MSR were blocked by. the above dose of imipramine. However, the e f f e c t on the i n h i b i t i o n of the QUAD-MSR was more rapid i n onset and greater i n magnitude. At 10 min imipramine had converted the i n h i b i t i o n into f a c i l i t a t i o n i n 5 of 6 QUAD experiments (% i n h i b i t i o n = -10.3+ 6.3 S.E.M.). Convertion of the i n h i b i t i o n to f a c i l i t a t i o n at 10 min occurred i n only 1 of 8 PBST experiments (% i n h i b i t i o n = 56.8 + 15.2). The maximal e f f e c t of imipramine was -87.9 + 19.4 % (50 min) i n the QUAD experiments and -15.5 + 26.5 % (30 min) i n the PBST experiments ( F i g . 3 and 4). Pretreatment of the cats with DL-p_-chlorophenylalanine (p_-CPA) completely prevented the e f f e c t of imipramine i n blocking BSI of the DR-MSR ( F i g . 2B). However, pretreatment of the animals with DL-P(-methylp_-tyrosine methyl ester HC1 (0^-MPT) had no e f f e c t on the blocking action of imipramine ( F i g . 2C). The effects of desipramine HC1 (4.8 mg/kg i.v.) on BSI of the DRMSR were q u a l i t a t i v e l y s i m i l a r to those of imipramine but, desipramine's 32 -20 0 20 40 60 80 100 -20 0 20 40 60 -20 0 20 40 60 Time (min) F i g , 2. The e f f e c t of imipramine CLMI) on bulbospinal i n h i b i t i o n of the DR-MSR i n A. non-pretreated c a t s , n = 6; B. cats pretreated with JJ.-CPA (300 rag/kg i . p . f o r 3 consecutive days), n = 7; and C. cats-pretreated with C^-MPT (126 mg/kg i . p . 16 and 4 hours p r i o r to recording), n = 6. Imipramine HCl (5 mg/kg i.v.) was injected over 10 min as indicated on the abscissa. Each point i n t h i s and subsequent graphs reprents the mean + - S.E.M. of the conditioned (lower graph). (upper graph) and the unconditioned MSR 33 " -20 i 1 0 Time 20 40 a 60 (min) F i g . 3. The e f f e c t of imipramine HC1 CIMI) C5 mg/kg i.v.) on bulbospinal i n h i b i t i o n .of the QUAD-MSR, n = 6; and the PBST-MSR, n = 8. 34 F i g . 4. The blockade of bulbospinal i n h i b i t i o n by imipramine. A. The large spike represents the unconditioned PBST-MSR and the small spike represents i n h i b i t i o n of t h i s r e f l e x produced by a conditioning stimulus i n the v i c i n i t y of the raphe magnus nucleus. B. A p a r t i a l blockade of the i n h i b i t i o n at the end of a 10 min i n j e c t i o n of imipramine HC1 (5 mg/kg i . v . ) . C. Complete blockade of the i n h i b i t i o n 10 min a f t e r B. Each frame represents 3 unconditioned and 3 conditioned sweeps of the MSR. 35 action was quantitatively less and of longer duration compared to that of imipramine (Fig. 5). Pargyline HCl (30 mg/kg i.v.) blocked BSI of the DR-MSR. The onset of this e f f e c t was gradual, and the effectwas prolonged. The blocking action was maximal at about 90 min and there was no i n d i c a t i o n of recovery w i t h i n 3 1/2 hours (Fig. 6). In two £-CPA pretreated animals i n which imipramine f a i l e d to block BSI of the MSR, methysergide (0.5 mg/kg i.v.) converted the i n h i b i t i o n to a 3 - 4 fold facilitation. To test whether BSI and recurrent i n h i b i t i o n (Rl)were stable, these i n h i b i t i o n s were recorded f o r 3 1/2 hours i n two experiments. The maximum deviation from the c o n t r o l values was 13.7 % (BSI) and 17.4 % (RI). Recurrent I n h i b i t i o n of the Monosynaptic Reflex Imipramine HCl (5.0 mg/kg i.v.) blocked RI of the DR-MSR. The e f f e c t was gradual and reached maximum in 50 min and p e r s i s t e d for longer than 90 min ( F i g . 7). Recurrent i n h i b i t i o n of the QUAD-MSR was blocked by imipramine, the e f f e c t was rapid i n onset and reached maximum i n about 10 min ( F i g . 8A). However, the drug did not block RI of the PBST-MSR ( F i g . 8B). Application of a 'cold block 1 s i g n i f i c a n t l y enhanced RI of the QUAD- MSR (RI % of control = 115.42 + 0.7, n = 6) but not RI of the PBST-MSR (RI % of control = 106.38 + 10.52, n = 5). The e f f e c t of imipramine on RI of the QUAD-MSR was completely eliminated and RI was a c t u a l l y enhanced when a 'cold block' was applied 30 min a f t e r the i n j e c t i o n of the drug (RI % of control = 116.02 + 5.4, n = 6) ( F i g . 8A). Pretreatment of the cats with e i t h e r JJ-CPA or Q^MPT completely eliminated the blocking action of imipramine on RI of the DR-MSR (Fig. 7). 36 0 O 5 c 50 100 - 2 100 c o o OC 50 L S DM I -20 0 20 40 T i m e F i g . 5. The e f f e c t of desipramine HC1 s p i n a l i n h i b i t i o n of the MSR abscissa. 80 100 120 (min) (DMI) C4.8 mg/kg i.v.) on bulbo- Cupper graph) and the unconditioned MSR (lower graph), n = 6. Desipramine was on the 60 injected over 10 min as indicated 37 -20 0 30 60 90 7120 150 180 210 Time (min) F i g . 6. The e f f e c t of pargyline EC1 (PARG) (30 mg/kg i.v.) on bulbospinal i n h i b i t i o n of the MSR Cupper graph) and the unconditioned MSR (lower graph), n = 6. Pargyline was injected-over 30 min as indicated on the abscissa. l 38 -20 0 20 Time 40 60 (min) F i g . 7. The e f f e c t of imipramine (IMI) on recurrent i n h i b i t i o n of the DR-MSR i n non-pretreated cats, n = 5, (A); cats pretreated with _p_-CPA (300 mg/kg i . p . f o r 3 consecutive days), n = 6, (B); and cats pretreated with C^-MPT (126 mg/kg i . p . 16 and 4 hours p r i o r to recording), n = 6, (C). 39 F i g . 8. The e f f e c t of imipramine HCl (LMI) (5 mg/kg i.v.) on recurrent i n h i b i t i o n of the QUAD-MSR, n = 6, CA); and the PBST-MSR, n = 6, (B). A 'cold block' (CB) was applied over 10 min as indicated on the abscissa. AO F i g . 9. The e f f e c t of pargyline HCl CPAUG) (30 mg/kg i.v.) on recurrent i n h i b i t i o n of the DR-MSR, n = 6. i 41 Pargyline also antagonized RI of the DR-MSR i n 4 of 6 animals. The e f f e c t was gradual and reached maximum i n about 90 min. Although quant i t a t i v e l y the e f f e c t of pargyline on t h i s i n h i b i t i o n was less than that produced by imipramine, the former's e f f e c t was longer l a s t i n g ( F i g . 9). The Unconditioned Monosynaptic Reflex Imipramine depressed the DR-MSR to about 60 % of i t s control value. The e f f e c t was maximal at about 30 min and the MSR started to return to c o n t r o l l e v e l s about 30 min l a t e r . Approximately 2 hours a f t e r the i n j e c t ion of the drug the MSR returned to i t s control value ( F i g . 2A). The QUADand PBST-MSR were also depressed by imipramine ( F i g . 8A, B). A p p l i c a t i o n of a 'cold block' reduced the QUAD-MSR to about 60 % of i t s c o n t r o l value (MSR % of c o n t r o l = 57.9 + 6.9, n = 5) but did not have a s i g n i f i c a n t e f f e c t on the PBST-MSR (MSR % of control = 96.5 + 1.74, n = 5). A f t e r the i n j e c t i o n of imipramine, when a 'cold block' was applied at 30 min, the QUAD-MSR was not depressed further ( F i g . 8A). Imipramine depressed the DR-MSR i n the animals pretreated with e i t h e r jpj-CPA or 0(-MPT and the depressant e f f e c t at 30 min was not s i g n i f i c a n t l y d i f f e r e n t from that of the DR-MSR i n the non-pretreated animals ( F i g . 2). Desipramine also reduced the DR-MSR to about 65 % of i t s c o n t r o l value. The drug e f f e c t reaches maximum i n about 40 min and the spike did not return to control value within 2 hours ( F i g . 5). Pargyline exerted a biphasic e f f e c t on the DR-MSR. The drug i n i t i a l l y depressed the MSR to about 88 % of i t s control value. However, at about 90 min the spike was f a c i l i t a t e d to about 106 % of the c o n t r o l . This late f a c i l i t a t o r y e f f e c t was variable from animal to animal and reached i t s max i n about 150 min and p e r s i s t e d upto 210 min at which time the experiment was discontinued ( F i g . 6). 42 Blood Pressure E f f e c t s Imipramine depressed the mean pulse pressure to about 69 % of i t s control value. In animals that were pretreated with js-CPA or 0(-MPT, imipramine depressed the mean pulse pressure to about 95 % and 77 % of the control values respectively. The time course of depression of blood pressure i s s i m i l a r to that of the blocking action of imipramine on BSI. However, the l a t t e r e f f e c t of the drug i s not related to i t s e f f e c t oh the blood pressure, because of the reasons narrated i n the next section (Discussion). Desipramine at the given dose d i d not have a s i g n i f i c a n t e f f e c t on blood pressure of most of the animals, however, t h i s drug s i g n i f i c a n t l y reduced blood pressure i n two. animals. Pargyline had no appreciable e f f e c t on blood pressure. : A3 DISCUSSION The finding that imipramine, desipramine and pargyline blocked bulbospinal i n h i b i t i o n (BSI) of the monosynaptic r e f l e x (MSR) i s not consistent with the proposal by Clineschmidt and Anderson (1970) that the bulbospinal i n h i b i t o r y system contains a 5-hydroxytryptamine (5-HT) link. Since imipramine f a i l e d to antagonize BSI of the MSR treated with the tryptophan hydroxylase i n h i b i t o r , i n cats pre- DL-£-chlorophenylala- nine (JJ-CPA) but s t i l l maintained i t s blocking action i n animals pretreated with the tyrosine hydroxylase i n h i b i t o r , DL-0(-methyl-p_-tyrosine (O^-MPT), i t i s assumed that the action of imipramine i s mediated through 5-HT. This assumption i s strengthened by the observation that pargyline, which, at the given dose, s i g n i f i c a n t l y elevates the l e v e l s of 5-HT but not noradrenaline (NA) i n the s p i n a l cord of the cat (Anderson et^ a l . , 1967), blocked BSI. Furthermore, desipramine at an equimolar dose to that of imipramine was q u a n t i t a t i v e l y less e f f e c t i v e than imipramine i n blocking BSI. I t i s known that imipramine p r e f e r e n t i a l l y blocks the uptake of 5-HT whereas desipramine p r e f e r e n t i a l l y blocks the uptake of NA (Carlsson et a l . , 1969; Ross and Renyi, 1969; Shaskan and Snyder, 1970). Thus, the findings i n the present i n v e s t i g a t i o n strongly suggest that 5-HT i s involved i n antagonizing rather than producing BSI. It i s i n t e r e s t i n g to note that the experiments c a r r i e d out by Anderson and coworkers (Anderson and Shibuya, 1966; Anderson et a l . , 1967; Shibuya and Anderson, 1968; Banna and Anderson, 1968) very strongly 44 indicate that the 5-HT axons descending i n the s p i n a l cord have 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. I t i s tempting to speculate that at least a part of the f a c i l i t a t o r y e f f e c t of 5-HT on the MSR i s due to a tonic i n h i b i t o r y action of a 5-HT system on the i n h i b i t o r y pathways ( d i s i n h i b i t i o n ) , Thus, the net e f f e c t on the MSR i s f a c i l i t a t i o n . The following observations support the above suggestion: 1. Imipramine blocked BSI of the MSR. 2. This drug blocked recurrent i n h i b i t i o n (RI) (see subsequent discussion) and presynaptic i n h i b i t i o n (unpublished observations) of the QUAD-MSR. 3. Engberg-et a l . (1968) suggested that a descending 5-HT system has a tonic i n h i b i t o r y action on lb and flexor r e f l e x afferents. Although Clineschmidt and Anderson (1970) proposed that a 5-HT i n t e r neurone i n the s p i n a l cord i s contained i n the bulbospinal i n h i b i t o r y pathway, there i s no evidence to suggest that there are 5-HT interneurones i n the s p i n a l cord. Furthermore, d - l y s e r g i c a c i d diethylamide was used (LSD), which as a 5-HT antagonist by the above workers, i s known to stimu- l a t e 5-HT receptors (Costa, 1956; Horita and Gogerty, 1958; Anden et a l . , 1968) and the e f f e c t s may be dose dependent; stimulating at low doses and blocking at high doses (Costa, 1956). The e f f e c t i v e dose of LSD i n blocking BSI (0.25 mg/kg) produced an enhancement of the unconditioned MSR (Clineschmidt and Anderson, 1970). This i s consistent with a 5-HT stimulant e f f e c t as seen following the i n j e c t i o n of 5-HT precursors (Anderson and Shibuya, 1966). Moreover, the dose of LSD required to block the 5-HTP induced enhancement of the MSR was higher than that which was e f f e c t i v e i n blocking BSI of the MSR (Banna and Anderson, 1968). The e f f e c t of 2-bromo-LSD (BOL) on BSI was transient; cyproheptadine, which blocked the 5-HTP enhancement of the MSR at a lower dose, did not block BSI (Clineschmidt and Anderson, 1970). Also, the 5-HT antagonists 45 blocked the action of Jb-3,4-dihydroxyphenylalanine (i-dopa) on the MSR (Banna and Anderson, 1968). In the present study, when methysergide was tested on BSI i n two animals that were pretreated with p_-CPA and i n which imipramine f a i l e d to block BSI, methysergide converted the i n h i b i t i o n to a 3 - 4 f o l d f a c i l i t a t i o n ; this drug may be acting on a non-serotonergic system. I t i s i n t e r e s t i n g that imipramine, pargyline and LSD a l l depressed the f i r i n g of the mesencephalic raphe neurones i n rats (Sheard e_t a l . , 1972; Aghajanian et a l . , 1970; Aghajanian et a l . , 1968). Furthermore, this depressant e f f e c t of imipramine i s absent i n rats pretreated with p_-CPA (Sheard et a l . , 1972). H o s l i et a l . (1971) reported that 5-HT had a general excitatory e f f e c t when i o n t o p h o r e t i c a l l y applied on the bulbos p i n a l neurones. Thus, i f the 5-HT neurones i n the raphe n u c l e i excite the bulbospinal i n h i b i t o r y neurones, i t may be possible that BSI i s blorcked by imipramine and pargyline since the 5-HT neurones i n the raphe n u c l e i stop f i r i n g a f t e r these drugs. However, Clineschmidt and Anderson (1970) found that LSD was more e f f e c t i v e i n blocking BSI of the MSR when administered by c l o s e - a r t e r i a l i n j e c t i o n to the s p i n a l cord than by closea r t e r i a l i n j e c t i o n to the b r a i n stem. Thus the s i t e of action of LSD i s most l i k e l y i n the s p i n a l cord. Also, the bulbospinal i n h i b i t o r y pathway can not have a 5-HT neurone descending from the raphe n u c l e i to the s p i n a l cord since, the conduction v e l o c i t y of the i n h i b i t o r y pathway was found to be high (Jankowska et a l . , 1968; Clineschmidt and Anderson, 1970) the unmyelinated 5-HT and fibres of 0.3 to 1.0 fm diameter (Dahlstrom and Fuxe, 1965) can not conduct impulses that f a s t . As mentioned there i s no evidence f o r 5-HT earlier, interneurones to exist i n the s p i n a l cord. Bulbospinal i n h i b i t i o n of the flexor and the extensor MSRs was found to be of the postsynaptic type ( L l i n a s , 1964a; Llinas and Terzuolo, 1964, 46 1965; Jankowska et a l . , 1968). the i n h i b i t o r y pathway most l i k e l y invol- ves a disynaptic l i n k and the conduction v e l o c i t y of the pathway i s high (Jankowska et a l . , 1968). While Jankowska et a l . (1968) did not f i n d any difference between the i o n i c mechanisms involved i n BSI of the f l e x o r and extensor MSRs, Llinas and Terzuolo (1965) noted differences between the two and suggested that the i n h i b i t o r y synapses of the bulbospinal i n h i b i t o r y pathway with the f l e x o r motoneurones are on the dendrites, f a r from the soma. Huffman and McFadin (1972) found that b i c u c u l l i n e , a specific t^-aminobutyric acid antagonist, blocked BSI of the f l e x o r MSR but had no e f f e c t on BSI of the extensor MSR. I t i s i n t e r e s t i n g that i n the present study imipramine's blocking action on BSI of the QUAD-MSR was q u a n t i t a t i v e l y greater than that on BSI of the PBST-MSR. This difference i n the blocking action of the drug may be due to the differences i n the mechanism by which the bulbospinal i n h i b i t o r y pathway exerts i t s action on the QUAD-MSR and the PBST-MSR or due to the difference i n the 5-HT input to these two types of the MSRs. Carpenter et a l . (1966) found that stimulation i n the medial caudal bulbar r e t i c u l a r formation produced negative (1 mm below the f l o o r of the fourth v e n t r i c l e ) dorsal root potentials (DRPs) on the l a afferents. These authors also reported that stimulation i n the above r e t i c u l a r formation about 4 mm below the f l o o r of the fourth v e n t r i c l e did not produce negative DRPs on the l a afferents. These observations suggest that the Magoun and Rhine's (1946) i n h i b i t o r y area, the ventromedial bulbar r e t i c u l a r formation, may not have a presynaptic caudal type of bulbospi- n a l i n h i b i t o r y pathway. However, Chan and Barnes (19 72) observed that s t i mulation i n the v e n t r a l caudal bulbar r e t i c u l a r formation from the mid-sagittal l i n e ) produced negative (2 mm l a t e r a l DRPs on l a afferents; these authors also noted a time c o r r e l a t i o n between the primary afferent depolarization (PAD), the negative DRP and the i n h i b i t i o n of the while stimulating i n the above bulbar area. Since a negative DRP PAD and presynaptic i n h i b i t i o n , these findings may MSR reflects suggest, contrary to those of Carpenter et a l . (1966), that a presynaptic type of i n h i b i t o r y component i s present i n BSI of the MSR. ventromedial -10, L 0.0 In the present study only the caudal bulbar r e t i c u l a r formation was stimulated ( V -6 to i n the Stereotaxic Atlas of Snider and Niemer, 1964). This area of stimulation i s not exactly the same as that used i n the study of Chan and Barnes'(1972) and i t i s not tory component was clear whether a presynaptic i n h i b i - involved i n the present study of the MSR.. Assuming that i n the present study only the postsynaptic type of the bulbospinal i n h i b i t o r y pathway was stimulated and that the i n h i b i t o r y pathway contains a disynaptic l i n k (Jankowska e_t a l . , 1968) and that the interneurone i s located i n the s p i n a l cord, I t can be speculated that the blockade of BSI by 5-HT can be due to a 5-HT neurone that terminates either on the axon terminal or the soma of the bulbospinal neurone or the interneurone i n the s p i n a l cord. I t seems u n l i k e l y that the i n h i b i t o r y 5-HT neurone ends on the soma of the bulbospinal neurone since 5-HT was found to have a general excitatory e f f e c t on the l a t t e r neurones (Hosli et a l . , 1971). I t i s known that the 5-HT neurones i n the raphe n u c l e i send axons that descend i n the s p i n a l cord and terminate i n the dorso- l a t e r a l and the v e n t r o l a t e r a l motor n u c l e i of the v e n t r a l horn of the s p i n a l cord (Fuxe, 1965). Thus i t seems more l i k e l y that an i n h i b i t o r y 5-HT neurone may end on the axon terminal of the bulbospinal neurone or on the interneurone i n the s p i n a l cord. I t i s not known what other neuronal systems send inputs to the interneurones connected with the bulbospinal i n h i b i t o r y pathway. Recently i t has been reported that some interneurones i n the s p i n a l cord which 48 receive inputs from the primary afferents also receive supraspinal inputs (Koizumi et a l . , 1959; b i t o r y interneurones Udo, Engberg et a l . , 1968). For example, the l a i n h i - receive supraspinal excitatory input (Hultborn and 19 72). Llinas reported that strychnine, a s p e c i f i c glycine antagonist reduced the hyperpolarization of the motoneurone membrane produced by BSI. Strychnine blocks r e c i p r o c a l i n h i b i t i o n by antagonizing the action of the putative neurotransmitter, glycine that i s presumably released from the l a i n h i b i t o r y interneurone terminal at the interneurone-moto- neurone synapses. I t may be possible that these interneurones are connec- ted with the bulbospinal i n h i b i t o r y pathway. However, imipramine HCl (5 mg/kg i.v.) had no e f f e c t on r e c i p r o c a l i n h i b i t i o n (unpublished obser- vations) suggesting that there i s no 5-HT input to the l a i n h i b i t o r y interneurones. Thus, i f these interneurones are involved i n BSI, a 5-HT i n h i b i t o r y neurone must end on the axon terminal of the bulbospinal neurone. However, the idea that l a i n h i b i t o r y interneurones are connected with the bulbospinal i n h i b i t o r y pathway i s purely speculative, Imipramine blocked RI of the QUAD-MSR and the e f f e c t of the drug i s very l i k e l y mediated through a supraspinal monoaminergic system (see subsequent discussion). However, Renshaw c e l l s do not seem to be connected with the bulbospinal i n h i b i t o r y pathway because imipramine's e f f e c t on BSI was eliminated by pretreatment with of the cats with J3-CPA but not Ot"MPT whereas imipramine's e f f e c t on RI was prevented by pretreat-« ment of the cats with either JJ-CPA or 6L.-MPT. Thus, the 5-HT input to block BSI and the monoaminergic input to block RI are probably two d i f f - erent systems. Blockade of RI of the DR-MSR by imipramine and pargyline, and e l i mination of imipramine's blocking action by pretreatment with either p_-CPA or ClJ-MPT supports of the cats the finding of Von Tan and Henacsch 49 (1968) that a monoaminergic pathway antagonizes RI of the MSR. Imipramine's antagonism of RI of the QUAD-MSR but not RI of the PBST-MSR indicates that the monoamine input i s to the Renshaw c e l l s involved i n RI of the QUAD-MSR but not i n RI of the PBST-MSR. Potentiation of RI of the MSR by a p p l i c a t i o n of a 'cold block' and complete removal of imipramine's e f f e c t on RI of the QUAD-MSR by a 'cold block 1 indicate that a supraspinal monoaminergic system has a tonic i n h i b i t o r y e f f e c t on RI of the QUAD-MSR. Since pretreatment of the cats with e i t h e r p_-CPA or P^-MPT completely eliminated the blocking action of imipramine on RI, there can not be two sep -rate descending 5-HT and NA systems on RI. Instead, the descending system must have l i n k s i n v o l v i n g both 5-HT and NA. I t i s i n t e r e s t i n g that the 5-HT and NA c e l l s i n the caudal brain stem have mutual synaptic contact and that there are both 5-HT and NA nerve terminals i n the d o r s o l a t e r a l and v e n t r o l a t e r a l motor n u c l e i of the ventral horn of the s p i n a l cord (Fuxe. 1965). Application of a 'cold block' s i g n i f i c a n t l y reduced the QUAD-MSR but had no e f f e c t on the PBST-MSR suggesting that the QUAD-MSR receives a supraspinal tonic f a c i l i t a t o r y input. Imipramine decreased the DR-, QUAD- and PBST-MSRs. Application of a 'cold block' 30 min after the i n j e c t i o n of imipramine did not reduce the QUAD-MSR any further, This may suggest that the e f f e c t of imipramine on the QUAD-MSR i s due to the blockade of a supraspinal tonic f a c i l i t a t o r y system. Since, a 'cold block' had no e f f e c t on the PBST-MSR and imipramine reduced t h i s MSR, the e f f e c t of the drug may be due to i t s action on neuronal systems at the segmental l e v e l . Eccles and Lundberg (1959) found that the extensor motoneurones are under strong supraspinal influences and the flexor motoneurones are mostly independent of such influences. This agrees with 50 the present work. Pretreatment of the cats with cK-MPT did not have a s i g n i f i c a n t e f f e c t on the depressant action of imipramine on the DR-MSR suggesting that this e f f e c t of imipramine on the DR-MSR i s probably not mediated a NA system. However, although pretreatment of the animals with through JJ-CPA did not a l t e r the action of imipramine on the DR-MSR upto 30 min of imipramine, the recovery of the MSR was faster than i n nonpretreated animals. The reason f o r the faster recovery of the MSR i n JJ-CPA pretrea- ted animals i s not understood. However, since imipramine did depress the MSR i n these animals, t h i s e f f e c t of the drug can not be e n t i r e l y due to a 5-HT input. The biphasic e f f e c t of pargyline on the MSR was previously reported by Anderson et a l . (1967). Based on the monoamine levels i n the s p i n a l cord a f t e r pargyline and other pharmacological evidence they concluded that the enhancement phase of pargyline's action was mediated by increased endogenous levels of 5-HT. The enhancement of the MSR by pargyline i n the present study i s however smaller than that reported by Anderson ejt a l . (1967). This may be due to the difference i n the experimental preparations. Anderson et a l . (1967) used cats with the s p i n a l cord sectioned at the c e r v i c a l l e v e l whereas i n the present study cats decerebrated at the m i d - c o l l i c u l a r l e v e l were used. Thus the control MSR i s considerably higher i n most of the present experiments than the range (1.5 to 3.0 mV) used by Anderson et a l . (1967). Therefore i n the present study, possibly fewer motoneurones are a v a i l a b l e for recruitment into the d i s charge zone. The blocking action of imipramine on BSI and RI and the drug's depressant action on the MSR do not seem to be related since pargyline, which blocked BSI and RI f a c i l i t a t d the MSR. However, a part of the 51 action of imipramine on the MSR may be mediated through 5-HT and i t i s s u r p r i s i n g and not understood why this drug while blocking BSI, RI and presynaptic i n h i b i t i o n , reduces the MSR. The time course of the depressant action of imipramine on blood pressure i s found to be s i m i l a r to that of the drug's action on BSI, RI and the MSR. However, the p o s s i b i l i t y that imipramine's e f f e c t s on these are due to i t s action on the blood pressure i s ruled out because of the following reasons: 1. Pargyline which does not have a s i g n i f i c a n t e f f e c t on blood pressure blocked BSI and RI and had a biphasic action on the MSR. 2. Imipramine blocked RI of the QUAD-MSR but not RI of the PBST-MSR. 3. 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Serotonin involvement in the blockade of bulbospinal and recurrent inhibition of the monosynaptic reflex Sastry, Bhagavatual Sree Rama 1973
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