Open Collections

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 Division of Pharmacology and Toxicology of the Faculty of Pharmaceutical Sciences We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA September, 1973. In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis fo r scholarly purposes may be granted by the Head of my Department or by h i s representatives. It i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of PAVAA4VMJLCC^C-^V;J <x^Ji I The University of B r i t i s h Columbia Vancouver 8, Canada Date U to - (^i2> i i ABSTRACT Sastry, B.S.R., Division of Pharmacology and Toxicology, Faculty of Pharmaceutical Sciences, University of Br i t i s h Columbia; September, 1973. Serotonin Involvement in the Blockade of Bulbospinal and Recurrent  Inhibition 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 inhibitor, pargyline HC1 (30 mg/kg i.v.) antagonized bulbospinal inhibition (BSI) of the monosynaptic reflex (MSR) in unanaesthetized cats decerebrated at the mid-collicular level. The effect of imipramine was quantitatively more on BSI of the quadriceps (QUAD)-MSR compared to that on BSI of the posterior biceps-semitendinosus (PBST)-MSR . Imipramine's action on this inhibition was also quantitatively greater compared to that of the equimolar dose of desipramine. Pretreatment of the animals with the tryptophan hydroxylase inhibitor, DL-p_-chlorophenylalanine (p_-CPA) (300 mg/kg i.p. for 3 consecutive days) completely eliminated the blocking action of imipramine. However, pretreatment of the animals with the tyrosine hydroxylase inhibitor, 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 effect on imipramine's action. 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 inhibitory pathway involves a 5-HT interneurone in the spinal cord. Imipramine HC1 (5 mg/kg i.v.) and pargyline HC1 (30 mg/kg i.v.) blocked recurrent inhibition (RI) of the MSR evoked by stimulation of a dorsal root. Imipramine blocked RI of the QUAD-MSR but had no effect on RI of I l l 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 this inhibition. These observations suggest that a supraspinal monoaminergic system which involves 5-HT and noradrenaline links has a tonic inhibitory effect on RI of the QUAD-MSR. John G. Sinclair, Ph.D., Supervisor. i v TABLE OF CONTENTS LIST OF FIGURES V ABSTRACT ii INTRODUCTION 1 SURVEY OF LITERATURE 5 Organization of the Lumbosacral Spinal Cord of the Cat . . . 5 The Segmental Monosynaptic Reflex 7 Postsynaptic Inhibition in the Spinal Cord . . . . . . . . 8 a. Reciprocal Inhibition 10 b. Recurrent Inhibition of the Spinal Motoneurones . . 11 Presynaptic Inhibition in the Spinal Cord 12 The Raphe Nuclei 13 The Descending Monoaminergic Fibres in the Spinal Cord . . 16 Bulbospinal Inhibition of the Monosynaptic Reflex 19 Supraspinal Effects on Renshaw Cells 22 EXPERIMENTAL 25 RESULTS 31 Bulbospinal Inhibition of the Monosynaptic Reflex 31 Recurrent Inhibition of the Monosynaptic Reflex 35 The Unconditioned Monosynaptic Reflex 41 Blood Pressure Effects 42 DISCUSSION 43 REFERENCES . 52. V LIST OF FIGURES Figure Page 1. A diagrammatic representation of the experimental set up 26 2. The effect of imipramine on bulbospinal inhibition . of the DR-MSR in A. non-pretreated cats; B. i n cats pretreated with £~CPA; C. in cats pretreated with oC-MPT 32 3. The effect of imipramine on bulbospinal ininbitlon of the QUAD-MSR and the PBST-MSR 33 4. The blockade of bulbospinal inhibition of the . .. PBST-MSR by imipramine 34 5. The effect of desipramine on bulbospinal inhibition of the MSR 36 6. The effect of pargyline on bulbospinal inhibition of the MSR 37 7. The effect of imipramine on recurrent inhibition of the DR-MSR in A. non-pretreated cats; B. in cats pretreated with _p_-CPA; C. in cats pretreated with 0(-MPT 38 8. The effect of imipramine on recurrent inhibition of the QUAD-MSR and the PBST-MSR 39 9. The effect of pargyline on recurrent inhibition of the MSR 40 V I ACKNOWLEDGEMENTS The author is deeply indebted to Dr. John G. Sinclair for his invaluable guidance and inspiration throughout the course of this investigation. He i s grateful to Mrs. Marjorie Chaplin for her help in preparing the figures. This work was supported by a grant from the Medical Research Council to Dr. Sinclair and a University of Br i t i s h Columbia summer research scholarship to the author. 1 INTRODUCTION Recently, several investigators have suggested that the putative neurotransmitters in the central nervous system (CNS), 5-hydroxytrypta-mine (5-HT, serotonin) and noradrenaline (NA) are involved in motor control (Cranmer et a l . , 1959; Fuxe, 1965; Anden et a l . , 1966; Anderson, 1972). Chronic transection of the spinal cord depletes 5-HT and NA caudal to the transection (Carlsson et^ a l . , 1963; Magnusson and Rosengren, 1963). Stimulation of the spinal cord gives r i s e to a release of 5-HT and NA (Anden et a l . , 1964, 1965). These observations suggest that the spinal cord contains descending 5-HT and NA fibres of supraspinal origin. The raphe nuclei are located along the mid-sagittal plane in 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 entire 5-HT neuronal population in the CNS i s located in the raphe nuclei. Neurones containing NA are most densely located in the medulla oblongata but also exist in the pons and midbrain. Noradrenergic c e l l s have a more scattered distribution than 5-HT c e l l s . Most of the descending 5-HT and NA fibres originate in the medulla oblongata (Dahlstrom and Fuxe, 1965). These monoaminergic fibres descend in the dorsolateral and ventromedial funiculi of the spinal cord and terminate i n the substantia gelatinosa in the dorsal horn and on the ventrolateral and dorsolateral motor nuclei in the ventral horn (Fuxe, 1965). 2 Existence of the descending monoaminergic systems raises the question of their functional significance in the spinal cord. In unanaesthetized cats with an acute spinal 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 effects of 5-HTP, _l-tryptophan and L-dopa on the MSR (Anderson e_t al_. , 1967) . In cats with a chronic spinal transection, pargyline and JL-tryptophan did not alter the MSR (Shibuya and Anderson, 1968). Moreover, imipramine, a 5-HT neuronal uptake blocking agent, potentiated the effect of 5-HTP and pargyline on the MSR in cats with an acute spinal transection but not in cats with a~ chronic spinal transection (Clineschmidt et a l . , 1971; Clineschmidt, 1972). Pretreatment of the animals with the tryptophan hydroxylase inhibitor, DL-pj-chlorophenylalanine (p_-CPA) , blocked the effects of 5-HTP on the MSR (Taber, 1971). These observations indicate that a descending 5-HT system i n the spinal cord has a f a c i l i t a t o r y action on the MSR. Bulbospinal inhibitory systems were also suggested to exist. In decerebrate-decerebellate cats, stimulation in the nucleus raphe magnus produced long latency negative dorsal root potentials (DRPs) in the lumbosacral spinal cord. These DRPs were blocked by the 5-HT antagonists, methysergide and cinanserin (Proudfit and Anderson, 1973). Similarly, bulbospinal inhibition 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 close-a r t e r i a l injections to the brain stem and the spinal cord, that LSD and methysergide act at the spinal cord level. Hence, they suggested that 3 the bulbospinal inhibitory system contains a 5-HT interneurone in the spinal cord. To investigate further the poss i b i l i t y that the latter bulbospinal inhibitory system contains a 5-HT link i t i s reasoned that, i f the hypo-thesis of Clineschmidt and Anderson (1970) is correct, drugs which incr-ease the a v a i l a b i l i t y of 5-HT in the synaptic clefts should increase bulbospinal inhibition (BSI) of the MSR. Imipramine preferentially blocks the uptake of 5-HT whereas desipramine preferentially blocks the uptake of NA (Ross and Renyi, 1969; Carlsson et^ al_. , 1969; Shaskan and Snyder, 1970). In the spinal cord, pargyline elevates the levels of 5-HT but does not significantly increase the levels of NA and the effects of this drug on the MSR are very l i k e l y mediated through 5-HT (Anderson et a^ L., 1967) . Therefore, in the present study, imipramine, desipramine and pargyline are tested on BSI of the MSR. Stimulation of the ventromedial mesencephalic reticular formation, ventrolateral bulbar reticular formation, ventral thalamus (Fields of Forel and Zona Incerta) and pericruciate cortex decreased the rate of .. Renshaw c e l l discharges evoked by antidromic activation of the motor axons (Koizumi et a l . , 1959; Haase and Meulen, 1961; Mac Lean and Le£f-man, 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 indicate that the Renshaw c e l l dis-charges are influenced by supraspinal inputs. In decerebrate cats, imipramine blocked recurrent inhibition (RI) of the MSR (Von Tan and Henatsch, 1968). However, this drug had no effect on the inhibition in cats with the spinal cord transected at the thoracic level (Von Tan and Henatsch, 1969). These authors suggested that a mono-4 aminergic pathway has a strong curbing effect on RI of the MSR. The pre-sent investigation is an extention of the above work and is designed to determine whether RI of the flexor and extensor MSRs are equally affected by imipramine and whether the drug's effects 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 fibres are located in the dorsal root ganglia. A l l central processes of these c e l l s enter the spinal cord. The peripheral processes innervate various structures such as skeletal muscles, skin, etc., and convey impulses centrally. These fibres are classified into various groups according to their thickness, myelination and function. Group I afferents are 12 - 20 |im in diameter and are heavily myelinated. These fibres are subclassified into l a and lb afferents. The l a fibres convey impulses from the annulospiral endings of the muscle spindles, whereas, the lb fibres convey impulses from the Golgi tendon organs. The conduction velocity of the group I fibres i s high (about 120 m/sec) compared to that of the other fibres. Group II (diameter 6-12 pm) and group III ( 1 - 6 pm) fibres are called high threshold muscle afferents. They are thinly myelinated and their conduc-tion velocity i s f a i r l y low (about 5 - 3 0 m/sec). The thinly myelinated cutaneous alpha ( 6 - 1 7 Jim) and delta ( 1 - 6 pm) fibres convey impulses from cutaneous origin. Both muscle nerves and cutaneous nerves contain unmyelinated group IV fibres (Ruch and Patton, 1960). Rexed (1952, 1954, 1964) subdivided the spinal gray matter into nine laminae and a tenth region surrounding the central canal. This type of subdivision of the c e l l groups is based on the appearance of the c e l l s and the boundaries are only zones of transition. Lamina I is a thin sheath that caps the surface and bends around the margins of the dorsal horn. 6 Lamina II is situated immediately ventral to the lamina I. It contains tightly packed small c e l l s and is traversed by many strands of spinal afferent fibres. This lamina corresponds to the substantia gelatinosa. Lamina III is a band of large neurones scattered across the dorsal horn and i s situated ventral to lamina II. Lamina IV is the broadest of the f i r s t four layers. It i s a heterogenous zone with small to large c e l l s of variable shapes. Lamina V i s a broad zone extending across the neck of the dorsal horn and i s subdivided into medial and late r a l zones. Many fibres pass through the lateral zone to give i t a reticulated appearance. Lamina VI is located at the base of the dorsal horn. It is subdivided into the small medial and the larger lateral zones. The medial zone is less compact and contains large triangular or star shaped neurones. Descending supraspinal pathways project to c e l l s in the lateral zone. Lamina VII occupies most of the intermediate zone of the gray matter. The ventral part of this lamina extends into the ventral horn. Medium sized c e l l s predominate in this zone. The dorsal nucleus of Clarke, the intermediolateral and intermediomedial nuclei are seen in this layer. The ventral part of this 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 is not sharply separated from lamina VII. Lamina VIII is confined to the medial part of the ventral horn. Vestibulospinal and reticulospinal fibres, and the medial longitudinal fasciculus terminate in this zone. Lamina IX i s composed of the largest c e l l s of the spinal cord, the 0{-motoneurones, situated in the ventrolateral region of the ventral horn. The medial nuclear masses have a diffuse border with lamina VIII. A considerable number of the thickly myelinated afferent fibres terminate in the dorsal nucleus of Clarke (Grant and Rexed, 1958). The 7 collateral branches of the afferent fibres that are distributed to parts of the anterior horn become concentrated mostly in the central part of the lamina VI. These collaterals in numerous small bundles pass into lamina IX where they arborize about the soma and dendrites of the moto-neurones (Sprague and Ha, 1964). The dorsal root fibres also give off collaterals which pass into lamina VII. Since, collaterals of dorsal root fibres passing to lamina VII and IX traverse broad regions of lamina VII, some fibres end upon interneurones in this zone, as well as on dendrites of motor nuclei which extend beyond the limits of lamina IX (Sprague and Ha, 1964). Group lb, II and III muscle nerve afferents and cutaneous afferents generate synaptic potentials in the central parts of laminae V, VI and VII (Sprague and Ha, 1964). The motoneurones are organized according to their functional inner-vation. The c e l l s that innervate the extensor muscles are located in the ventrolateral horn late r a l to those that innervate the flexor muscles. The flexor motoneurones are arranged in a number of subgroups such that each group of c e l l s innervate muscles which move a particular joint l i e in the same horizontal plane in the ventral horn; the more d i s t a l the muscle, the more dorsal the position of the c e l l s (Romains, 1964). The Segmental Monosynaptic Reflex When a spinal dorsal root is stimulated with minimal intensities of current, a small ventral root discharge can be observed. As the stimulus strength is increased, the amplitude of the early ventral root discharge increases and reaches a maximal value and upon further increase in the stimulus strength late discharges are observed. The early sharp spike reflects the synchronous activation of OC-motoneurones through la affer-ents and i s referred to as the monosynaptic reflex (MSR). The late 8 asynchronous discharges reflect the f i r i n g of motoneurones through poly-synaptic reflex (PSR) pathways mediated by activation of high threshold afferent fibres. The latency between stimulation of the dorsal root and recording the MSR from the ventral root includes the conduction time in the l a fibres, one synaptic delay and the conduction from the motoneu-rone c e l l bodies to the ventral root recording electrode. The total time to the peak of MSR i s about 2 msec. The central delay in transmission across a single synapse i s approximately 0.5 msec (Eccles, 1961). The predominant feature of the afferent fibres i s divergence. That i s , a single afferent fibre branches and participates in f i r i n g of many motoneurones. However, for a motoneurone to f i r e , many presynaptic knobs impinging on the motoneurone must be activated. The amplitude of the MSR e l i c i t e d by stimulation of a dorsal root i s an index of the number of motoneurones that are recruited into the discharge zone. But, many motoneurones are depolarized only to a subthreshold level and are said to be excited subliminally. 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 subliminally and those that are not excited by the test stimulus w i l l be available for recruitment into the discharge zone. Hence, as more motoneurones are recruited, the amplitude of the MSR increases. During inhibition 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 Inhibition in the Spinal Cord Postsynaptic inhibition i s an index of depression of the neuronal excitability which occurs independently of the excitatory synaptic a c t i -v i t y (Brock £t al_. , 1952; Coombs ^jt ad. , 1955a, b) . This process involves 9 inhibition of a neurone by direct synaptic impingement. Examples of post-synaptic inhibition include reciprocal (direct, la) inhibition (Lloyd, 1941) and recurrent (antidromic) inhibition (Renshaw, 1941). Reciprocal inhibition i s exerted by l a afferents activating inhibitory interneurones which impinge on ^-motoneurones of antagonistic muscles (Eccles e£ a^l. , 1956). Recurrent inhibition i s brought about by volleys in the motor axon collaterals which activate inhibitory interneurones known as Renshaw c e l l s , which inturn, inhibit the motoneurones (Eccles et a l . , 1954). The membrane potential of a mammalian motoneurone is about -70 mV and i s called the resting membrane potential (RMP) (Frank and Fourtes, 1955) . During inhibition of motoneurones a hyperpolarization of the moto-neurone membrane occurs which i s known as the inhibitory postsynaptic potential (IPSP) (Brock et a l ., 1952). The equilibrium potential for the IPSP i s about -80 mV (Brock et a l . , 1952). During reciprocal inhibition, the IPSP in the motoneurone i s obser-ved about 1.5 to 2.0 msec after i t s onset. The decay of the IPSP i s about 3.0 msec (Brock et a l . , 1952a). During hyperpolarization the inhibitory transmitter increases the membrane permeability to ions having a hydrated diameter less than 1.4 times that of potassium ion. Thus, there i s said to be a decrease in membrane resistance or an increase in membrane con-ductance. It was suggested (Coombs et ^ a l . , 1955a, b) that during the IPSP there i s an inward diffusion of chloride 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 Llinas and Baker (1972) proposed that the IPSP i s generated by a selective permeability increase to chloride ions in the outward direction. They also reported that a potassium ion permeability change i s probably not significantly involved in this process. When the membrane potential i s lowered (depolarized), the IPSP 10 increases, The IPSP decreases or reverses when the membrane is hyperpo-larized by passage of cathodal current. The IPSP may also be reversed following an iontophoretic injection of chloride ions into the neurone (Coombs et^ a l . , 1955a) . a. Reciprocal Inhibition Discharges in group l a afferents not only excite the motoneurones of the synergistic muscles but also inhibit the motoneurones of the antagonistic muscles through an interneurone. The inhibitory transmitter released from the l a inhibitory interneurone axon terminal is suggested to be glycine (Werman et a l . , 1968; Curtis et a l . , 1968). When glycine was administered iontophoretically in the v i c i n i t y of the motoneurone, this amino acid hyperpolarized and reduced the resistance of the neuro-nal membrane. Prior hyperpolarization of the membrane reduced or reversed the effect of glycine. Comparison of the equilibrium potentials of the ionic events associated with the IPSP and the hyperpolarization produced by glycine indicate that they were similar (Werman et_ a_l. , 1968; Curtis ejt ail. , 1968). Strychnine, which blocks the l a inhibitory pathway, spe-c i f i c a l l y blocks the effects of glycine on the motoneurone (Curtis et^ a l . , 1971). Hultborn et_ al_. (1971) found that impulses in the motor axon c o l l a -terals inhibit the interneurones of the la inhibitory pathway. Thus, they suggested that increased motoneuronal f i r i n g inhibits the discharges from the l a inhibitory interneurones to the antagonistic motoneurones. Hultborn and Udo (1972) have shown that the disynaptic inhibitory effects on the motoneurones from the descending cortico-, rubro- and vestibulo-spinal tracts involve the la inhibitory interneurones. Thus, there seems to be a convergence of supraspinal and l a afferent inputs exciting the 11 la inhibitory interneurones. b. Recurrent Inhibition of the Spinal Motoneurones Volleys in the motor axon collaterals synaptically activate the • inhibitory interneurones, Renshaw c e l l s , which inturn impinge on motoneu-rones. Renshaw c e l l s , activated by ventral root stimulation, discharge in a characteristic burst with an i n i t i a l frequency of greater than 1000 spikes per second (Eccles et^ al_., 1954) . The reason for such a high fre-quency of f i r i n g of these neurones was attributed to a convergence of excitatory input from the collaterals of many motoneurone axons ( Eccles et^ a l . , 1956a ; Ryall et a l . , 1970). When synaptically activated, the duration of Renshaw c e l l discharge i s about 50 msec (Eccles elt a l . , 1956a) . Ryall (1970) found that in cats anaesthetized with chloralose, anti-dromic volleys in the motoneurone axons may evoke a postsynaptic i n h i b i -tion of Renshaw c e l l s instead of excitation. The latency observed for this inhibition suggested that the effect i s brought about by a disynar-ptic pathway and the other interneurone involved in this pathway was found to be a Renshaw c e l l (Ryall, 1970). Thus, some Renshaw c e l l s i n h i -bit the discharges of other Renshaw c e l l s and this i s probably responsible for recurrent f a c i l i t a t i o n . Renshaw ce l l s are also involved in inhibiting the l a inhibitory interneurones as discussed in the previous section, but there seems to be no input from l a inhibitory interneurones to Renshaw cel l s (Ryall and Piercey, 1971). Stimulation of i p s i l a t e r a l afferent fibres in the group II and III muscle nerves and cutaneous afferents have excitatory input to Renshaw ce l l s through polysynaptic chains. However, stimulation of the same afferents on the contralateral side brought about inhibition of these c e l l s without excitation (Ryall and Piercey, 1971). Renshaw cells are more strongly excited by the collate-12 rals of large phasic Q(-motoneurone axons than the small tonic motoneu-rone axons However, the small tonic motoneurones rather than the large phasic motoneurones are more effectively inhibited by Renshaw c e l l s (Ryall et a l . , 1972). Several pharmacological and physiological studies indicate that the chemical transmitter released at the motor axon collateral-Renshaw c e l l synapse is acetylcholine (Curtis and Ryall, 1966a, b, c). Dihydro - p -erythroidine, which blocks cholinergic transmission at the nicotinic receptors, depressed the response of Renshaw ce l l s to synaptic stimulation (Eccles et a l . , 1956a; Curtis and Ryall, 1966b). Eserine, an anticholi-nesterase drug, greatly prolonged the discharges of Renshaw c e l l s induced by synaptic excitation (Eccles et a l . , 1954, 1956a). Intra-arterial inje-ction of acetylcholine or nicotine excites Renshaw c e l l s (Eccles et^ a l . , 1956a; Curtis and Ryall, 1966a). The excitatory action of acetylcholine, but not nicotine, is increased by eserine while dihydro - p-erythroidine decreased the excitatatory action of both the substances (Eccles e£ a l . , 1956a). The inhibitory transmitter released at the Renshaw cell-motoneu-rone synapse was suggested to be glycine (Eccles, 1966; Werman et a l . , 1968; Curtis et^ a l . , 1968; Curtis, 1969; Curtis et^ al_., 1971). Presynaptic Inhibition in the Spinal Cord Presynaptic inhibition in the spinal cord was f i r s t shown by Barr-on and Matthews (1938). Stimulation of a dorsal root produces a potential difference along the stimulated or the adjacent dorsal root. The poten-t i a l developed is called negative dorsal root potential (DRP). The DRP represents a primary afferent depolarization (PAD), and generation of this DRP in the spinal cord induces a net inhibitory action on the moto-neuronal output due to depression of the presynaptic excitatory impulses 13 (Renshaw, 1946; Brooks et^ a l . , 1948). Powerful presynaptic inhibition can be obtained in cats anaesthetized with pentobarbital and usually this inhibition i s less intense in decere-brate cats (Eccles et_ al_., 1963b) . In decerebrate cats volleys in la and lb afferents of flexor muscle nerves depolarize group la fibres of both flexor and extensor muscle nerves. But group l a and lb volleys in exten-sor muscle nerves, except those of the quadriceps muscle, do not have any depolarizing effects on the flexor or extensor l a afferents (Eccles, 1964). There was no effect from l a afferents of any muscle nerves on group lb (Eccles et 21I., 1963a) and cutaneous afferents (Eccles et a l . , 1963c). Group lb and II volleys from a l l muscle nerves and cutaneous volleys exert a presynaptic inhibition on group lb afferents of flexor or extensor origin. Of those, the most powerful effect was found to be from group lb afferents (Eccles^et a l . , 1963a). Cutaneous volleys to a greater extent and group lb and II volleys from muscle nerves to a lesser extent produce a DRP on the cutaneous afferents (Eccles et a l . , 1963c). In producing presynaptic inhibition on l a afferents by activating l a and lb afferents, there is a latent period of about 4 msec between the conditioning stimulus and onset of the PAD. The PAD reaches i t s maximum at about 20 msec and persists for about 200 msec or more (Eccles et a l . , 1962). Since the synaptic delay in the mammalian central nervous system (CNS) i s about 0.5 msec (Eccles, 1961), i t was postulated that the central delay during presynaptic inhibition involves transmission through at least two s e r i a l l y arranged interneurones (Eccles et. al.. , 1962) . Eccles (1963, 1964) suggested that the PAD i s due to a chemical synaptic transmitter released near the primary afferent terminal from the last interneurone in the chain. He also postulated that depolarization of the presynaptic terminals reduce the magnitude of the potential in 14 these terminals, thereby limiting the output of the synaptic transmitter (Eccles, 1963). Picrotoxin (Eccles et_ a l . , 1963a) and bicuculline (Curtis e_t al. , 1970, 1971a) were found to reduce the DRP. Since these agents selectively block the effects of $-aminobutyric acid (GABA), this amino acid was pro-posed as the neurotransmitter mediating the PAD (Eccles, 1964a). Consis-tent with this proposal, Barker and Nico l l (1972) showed a sodium ion-dependent depolarizing c.ction of GABA on the primary afferent terminals and a specific blockade of i t s effect by bicuculline in the isolated spinal cord of the frog. Recently, Krnjevic and Morris (1972) showed that the negative DRP produced in the lumbar spinal cord of the cat during stimulation of the spinal afferent fibres i s associated with an increase in the extracellular potassium ion concentration. These authors suggested that this rise in the extracellular potassium ion concentration is either due to the activity of the unmyelinated nerve terminals or to a release of this ion from the postsynaptic structures. The Raphe Nuclei The raphe nuclei are situated along the mid-sagittal plane in the midbrain, pons and medulla oblongata (Taber et a l . , 1960). These cells are separated from other cellular aggregations by fibre masses. The rost-r a l end of the raphe complex is found in the rostral mesencephalon and the caudal end in the caudal half of the medulla oblongata. Depending upon the location and type of c e l l s , the raphe nuclei are classified as f o l l o -ws: nucleus raphe obscurus, nucleus raphe pallidus, nucleus raphe magnus, nucleus raphe pontis, nucleus centralis superior, nucleus linearis inter-medius and nucleus linearis rostralis (Taber et a l . , 1960). 15 The nucleus raphe obscurus is located mid-sagittally in the caudal medulla. This nucleus extends rostrally upto the caudal pole of the inferior ollvery complex. In the dorsoventral plane, the nucleus i s s i t u -ated primarily on the dorsal side. The cells are medium to small in size. The nucleus raphe pallidus i s situated ventral to nucleus raphe obscurus and extends from the level of f a c i a l nucleus to the caudal medulla, s l i -ghtly rostral to the caudal end of raphe obscurus nucleus. The nucleus raphe pallidus is boardered by the pyramidal tract ventrally and divided into dorsal and ventral masses at the caudal part. The nucleus raphe magnus extends from the rostral end of the nucleus pallidus to the level of trapazoid body. The raphe magnus consists of a comparatively large mass of cells which extend laterally into the reticu-lar formation. At many levels, these cells are separated from the reticu-lar formation by longitudinally running fibres. The nucleus raphe pontis occupies the mid-sagittal part of the pons; however, some cells are found more laterally. Most of the cells of the nucleus raphe pontis are sepa-rated by dorsoventrally running fibres. The nucleus centralis superior i s boardered by the decussation of brachium conjunctivum in the dorsolateral plane and the nucleus inter-peduncularis i s situated ventral to these c e l l s . The nucleus raphe dorsa-l i s is situated in the ventral part of the periaqueductal gray and exten-ds from the level of the dorsal tegmental nucleus to the caudal pole of the occulomotor nucleus. Ventral to the nucleus raphe dorsalis, the medial longitudinal fasciculus i s situated on either side. The nucleus linearis intermedius i s composed of scattered cells of small size loca-ted in the caudal midbrain. The nucleus linearis rostralis includes cells of large, medium and small size and is situated medial to the occulo-motor outflow. 16 The Descending Monoamlnergic Fibres in the Spinal Cord In rabbits, after chronic spinal transection at the second thoracic level, both 5-hydroxytryptamine (5-HT) and noradrenaline (NA) levels decrease caudal to the transection (Carlsson et_ al_., 1963; Magnusson and Rosengren, 1963). In mice and frogs, stimulation of the spinal cord in vitro induces a release of 5-HT and NA (Anden et a l . , 1964, 1965). Thus these authors concluded that 5-HT and NA are associated with the descend-ing neuronal pathways. Using histochemical fluorescence techniques, Dahlstrom and Fuxe (1964, 1965) showed the existence of monoaminergic neurones in the CNS of rats, guinea pigs, rabbits and cats. They found two distinct types of nerve cells showing either yellow or green fluorescence. The cells exhibiting yellow fluorescence are medium to large in size and round to oval in shape. The distribution of these cells that give rise to descend-ing axons is almost entirely limited to the caudal raphe nuclei (Raphe obscurus, raphe pallidus and raphe magnus). The cells showing green fluorescence are scattered as small masses composed of neurones small to medium in size, multipolar and round to oval in shape. Of these, cells that give rise to descending fibres are located mostly in the medulla oblongata from the rostral end of the inferior olivery complex to the level of pyramidal decussation. Pharmacological studies (Fuxe, 1965) indicate that cells showing yellow fluorescence are those containing 5-HT whereas those exhibiting green fluorescence are those containing NA. The evidence is based on the following observations: The monoamine depleting agent, reserpine, almost completely removes both the yellow and green fluorescence; the monoamine oxidase (MAO) inhibitor, nialamide, enhances the yellow fluorescence; and the tyrosine hydroxylase inhibitor, O^-methy1-m-tyrosine, selectively reduces the green fluorescence. Fluorescence microscopic studies revealed that 5-HT and NA nerve terminals in the region of raphe nuclei make contacts with cells of each other as well as with some nonfluorescent cells (Fuxe, 1965). The 5-HT -axons from the caudal raphe nuclei run in a ventrolateral direction and almost reach the ventral surface of the brain stem la t e r a l to the pyra-midal tract (Dahlstrom and Fuxe, 1965). Spinal lesion studies revealed that the 5-HT fibres descend in the dorsolateral and ventromedial funi-c u l i of the spinal cord (Dahlstrom and Fuxe, 1965). Some of these fibres cross to the other side of the spinal cord. Most of the fibres descend-ing dorsolaterally in the lumbosacral spinal cord terminate in the sub-stantia gelatinosa, and the ventromedially descending fibres terminate in the ventrolateral and dorsolateral motor nuclei of the lamina IX. In cats, the density of termination of 5-HT fibres in substantia gelatinosa and the motornuclei i s almost the same. The NA fibres also run ventrally in the brain stem to reach the ventrolateral part. In the spinal cord, these fibres descend in the ventrolateral and dorsolateral funiculi with some fibres crossing to the opposite side. The NA terminals were found to be most dense in substan-t i a gelatinosa and dense in the ventrolateral and dorsolateral motor nuclei of lamina IX. However, regions other than the above also receive NA terminals (Fuxe, 1965). Both the descending 5-HT and NA fibres are unmyelinated and of 0.3 to 1.0 urn in diameter (Dahlstrom and Fuxe, 1965). The observation that monoamine neurones of the caudal brain stem give rise to descending pathways raises the question of their functional significance. 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 (Ander-son and Shibuya, 1966; Baker and Anderson, 1970a). The 5-HT levels in the spinal cord of the cat increase after treatment with 5-HTP (Anderson and Shibuya, 1966). Four hours after the injection of pargyline (30 mg/kg i.v the levels of 5-HT were elevated by 70 % but the NA levels were not sig-nificantly increased in the cat's spinal 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 effects of this MAO inhibitor 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 effects of L-dppa as well 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 spinal transection, pargyline and 1-trypto-phan did not alter the MSR; however, 5-HTP s t i l l enhanced the MSR in thes animals (Shibuya and Anderson, 1968). In the spinal cord, caudal to a chronic transection, 25 % of the control 5-HT levels (Shibuya and Ander-son, 1968), 20 % of the dopa decarboxylase activity (Anden et a l . , 1964) and 17 % of the tryptophan hydroxylase activity remained (Clineschmidt et a l . , 1971a). Thus, Shibuya and Anderson (1968) suggested that the 5-HTP enhancement of the MSR in cats with a chronic spinal transection might be due to the presence of 5-HT interneurones in the spinal 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 either enter the adrenergic terminals and displace catecholamines (Ng ejt a l . , 1972) or directly activate the adrenergic receptors (Innes, 1962) These findings offer alternative explanations for the work of Shibuya and Ande rson (1968) wliich shows a 5—HTP enhancement of the MSR in chronic 19 spinal animals. Clineschmidt jet a l . (1971) and Clineschmidt, (1972) show-ed that the neuronal uptake blocking agent, imipramine, potentiated the effect of 5-HTP and pargyline in cats with an acute spinal transection but not in the animals with a chronic spinal transection. Thus, a descen-ding 5-HT system exists in the spinal cord of the cat whose overall effect on the MSR is f a c i l i t a t i o n . Pretreatment of the cats with the tryptophan hydroxylase inhibitor, DL-£-chlorophenylalanine (p_-CPA) (300 mg/kg i.p. for 2 consecutive days) blocks the effects of 5-HTP on the MSR (Taber, 1971). Hence, Taber sugg-ested that, although 5-HTP was converted into 5-HT in these animals, 5-HT i s taken up by the empty synaptic vesicles but did not overflow into the synaptic c l e f t and activate the receptors. The increase of the MSR ind-uced by 5-HTP in cats with an acute spinal cord transection was also blocked by the following 5-HT antagonists: methysergide, d-lysergic acid diethylamide (LSD), 2-bromo-LSD (BOL), cinanserin and cyproheptadine (Banna and Anderson, 1968; Clineschmidt ejt a l . , 1971). Bulbospinal Inhibition of the Monosynaptic Reflex Magoun and Rhines (1946) reported that the ventromedial bulbar reticular formation contains a descending neuronal system which exerts a general inhibitory influence on the segmental MSR. They also observed a concomitant melting of the decerebrate r i g i d i t y . The caudal raphe nuclei f a l l within the inhibitory area described by Magoun and Rhines (1946). The mechanism of bulbospinal inhibition (BSI) of the MSR was stu-died by Llinas (1964a, b), Llinas and Terzuolo (1964, 1965) and Jankow-ska et al- (1968). These studies involved stimulation in the bulbar reticular formation while recording the MSR and the intracellular mem-20 brane potential of a participating 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-dendri-t i c (SD) component of the action potential was blocked when the moto-neurone was activated antidromically. These findings indicate that BSI of the MSR is of the postsynaptic type. When chloride ions were ionto-phoretically injected into the motoneurone the hyperpolarization produ-ced during stimulation of the bulbar reticular formation was reversed (Llinas and Terzuolo, 1964). Thus the ionic mechanisms responsible for BSI are similar to those of the reciprocal inhibition. Bulbospinal inhibition of the 0(-flexor motoneurones also involves a synaptic inhibibitory impingement on these motoneurones (Llinas and Terzuolo, 1965; Jankowska e^ t a l . , 1968). Llinas and Terzuolo (1965) concluded that the inhibitory synapses of the pathway are on the dendri-r t i c tree of the motoneurone, far from the soma. This conclusion was based on the observation that injection of chloride ions into the moto-neurone did not reverse the hyperpolarization produced during BSI. However, Jankowska ejt a l . (1968) did not observe any difference between flexor and extensor motoneurones in this regard. The difference in these two studies may be due to the difference in the experimental preparations. Jankowska et_ a l . (1968) used decerebrated cats with a contralateral hemisected 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 inhibitory pathway descends in the ventral quadrant of the spinal cord. This pathway most li k e l y has a disynaptic linkage and i t s conduction velocity is 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 inhibition 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 is probably not of presynaptic type. However, picrotoxin 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 possi b i l i t y for a presynaptic type of BSI to exist cannot be entirely ruled out. Although neither strychnine nor mephenesin blocked BSI when administered individually, a combination of low doses of these two drugs blocked the inhibition (Llinas, 1964b). In an attempt to explain these findings, Llinas (1964b) suggested the following two possible mechanisms: 1. Stry-chnine may increase activity i n the inhibitory pathway while depressing the inhibitory action. Mephenesin may antagonize this increased activity in the pathway. Thus, the blocking effect of the combined injection 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 reticular formation i n the caudal brain stem 1 mm below the floor of the fourth ventricle ( V about -5 to -6 in 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 reticulospinal fibres descend in the ventromedial spinal cord. Carpenter at a l . (1966) also reported that stimulation of the ventral caudal bulbar reticular formation ( about 4 mm below the floor of the fourth ventricle) did not produce negative DRPs on l a afferents. However, Chan and Barnes (19 72) reported that stimulation of the ventral caudal bulbar reticular formation ( 2 mm later a l from the mid-sagittal line) 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. It i s not understood why there is a controver-cy 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); morph-ine and meperidine (0.5 - 16 mg/kg i.v.) (Sinclair, 1973). Bicuculline, a specific GABA antagonist, blocked BSI of the flexor MSR but had no effect 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 cypro-heptadine (5.0 mg/kg i.v.) also blocked BSI (Clineschmidt and Anderson, 1970). The results of close-arterial injection of methysergide and LSD to the spinal cord and the brain stem suggested that they act at the spinal cord level. Thus, Clineschmidt and Anderson (19 70) proposed that the bulbospinal inhibitory pathway contains a 5-HT interneurone in the spinal cord. Supraspinal Effects on Renshaw Cells Stimulation of the ventromedial mesencephalic reticular formation at the level of substantia nigra (A 6.5 - 1.0) or the ventrolateral bulbar reticular formation at the level of hypoglossal nucleus (P 9.5 -11.0) was found to have an inhibitory effect on the number of Renshaw c e l l discharges evoked by antidromic volleys in the motoneurone axons (Koizumi et a l . , 1959; Haase and Meulen, 1961; Mac Lean and Leffman, 1967). This inhibitory effect can be obtained by stimulating either side of the r e t i -cular formation but is stronger when the side contralateral to the Ren-23 shaw c e l l was stimulated (Haase and Meulen, 1961). The latency between stimulation of the mesencephalic reticular formation and the onset of i t s inhibitory effect on the Renshaw c e l l discharge i s about 9 msec and this effect lasts at i t s maximum strength for about 20 to 25 msec. While stimulation of the mesencephalic reticular formation has a stronger inhibitory effect on Renshaw c e l l discharges, the stimulation of bulbar reticular formation has a longer duration of action (MacLean and Leff-man, 1967). Stimulation of ventral thalamus (Fields of Forel, Zona Incerta) or the pericruciate cortex inhibits the Renshaw c e l l discharges evoked by antidromic stimulation of the motor axons. Such an inhibition i s rapid in onset and of short duration (MacLean and Leffman, 1967). The descen-ding fibres from pericruciate cortex pass through the pyramids in 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 effect on Renshaw c e l l discharges evoked by antidromic stimulation of the motoneurone axons. Stimulation in the brain stem reticular formation had no effect on these cells activated in the above manner (Haase and Meulen, 1961). However, when the Renshaw c e l l discharges were evoked by stimulation of a dorsal root, activation of the reticular formation with a conditioning interval of 12 msec, increased the disch*-arge rate of the Renshaw cells (Haase and Meulen, 1961). In addition, stimulation of the ventral thalamus or the pericruciate cortex could activate Renshaw cells 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 effects of imipramine (0.5 to 2.0 mg/kg i.v.) on recurrent inhibition of the MSR in decerebrate cats. They also showed that this drug blocks the inhibition at these doses. However, when the spinal cord of the animal was sectioned at the thoracic level the drug did not block recurrent inhibition ( Von Tan and Henatsch, 1969). They suggested that a supraspinal monoaminergic pathway has a strong curbing effect on recurrent inhibition of the MSR. 25 EXPERIMENTAL Adult cats of either 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 carotid artery was cannulated with No. 160 polyethylene tubing (Clay Adams, Div. of Becton, Dickinson and company) f i l l e d with diluted sodium heparin (Upjohn Company of Canada). This tubing was conne-cted to a P-1000-A pressure transducer (Narco-Bio- Systems) which in turn was connected to a type DMP-4A desk model physiograph (Narco-B±o-Systems) for recording blood pressure. The other carotid artery was ligated. A cephalic vein was cannulated with No. 90 polyethylene tubing f i l l e d with normal saline. This cannula was used for intravenously injecting the test drugs. The animal's head was mounted on a stereotaxic frame (Narishige Scientif i c Instrument Laboratory). The skull overlying the frontal and parietal lobes of the cerebral cortex was removed. The animal was decerebrated at the mid-collicular level (Fig. 1), the brain tissue above the transection was removed and the skull cavity was packed with gauze. The cut edges of the bone were covered with bone wax to control bleeding and prevent air embolism. Blood loss during decerebration was replaced by injecting dextran (6^w/v) immediately after decerebration. The occipital bone overlying the cerebellum was removed and the dura was cut to expose the cerebellum. A laminectomy was performed in the lumbosacral region of the spinal 26 Dorsal root L 7 Ventral root Fig. 1. A diagramatic representation of the experimental set up. I 27 cord. In some preparations L6, L7 and SI dorsal roots were cut bilatera-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 isolated and cut. The central ends of these nerves were attached to bipolar stimulating electrodes. The ventral roots L6, L7 and SI on the l e f t side were sectioned in a l l preparations. The skin flaps on the back of the animal were used to make a pool for holding mineral o i l which prevented drying of the spinal 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 in the 'cold block' experiments, the spinal 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 spinal 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 respiration throughout the experiment. The central end of the dorsal roots on the l e f t side (in most of the cases L7) and the corresponding ventral root were placed on bipolar platinum hook electrodes, the monosynaptic reflex (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 isolation unit. The stimulus strength used was supra-maximal for the MSR. In the animals with intact dorsal roots on the le 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 potential in the ventral 28 root was amplified using a Tektronix 2A61 d i f f e r e n t i a l amplifier and displayed on a Tektronix 560 model oscilloscope. A bipolar coaxial stainless steel electrode (0.5 mm separation, 0.5 mm exposed tip) was directed stereotaxically to the v i c i n i t y of the caudal raphe nuclei (P 7.5 to 13.5; L o.o in most and 0.5 in a few pre-parations; V -6 to -10 in the Stereotaxic Atlas of Snider and Niemer, 1964) (Fig. 1). A locus in this area was stimulated by a train (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 isolation unit. This train was delivered so that the end of the train occurred 7.5 msec before a stimulus was delivered to evoke the MSR. The location of the electrode in the brain stem and the stimulus strength (usually less than 5.0 V) were adjusted so that the MSR was inhibited to about 40 % of i t s original size. Furthermore, the electrode was considered to be pla-ced only i f there were no tonic contractures of the neck, back and the forelimbs or marked changes in the blood pressure during the stimulation. Recurrent inhibition (RI) of the MSR was obtained by stimulation of the central ends of two ventral roots (usually L6 and SI) 7.5 msec before evoking the MSR. In some preparations, however, a single ventral root was stimulated. Square wave pulses of 0.5 msec duration using the SI unit of a S8 Grass stimulator and a SIU5 stimulation isolation unit were delivered, the stimulus strength was adjusted so that the MSR was in h i b i -ted to approximately 40 % of the unconditioned value. In some animals, after obtaining stable recordings of bulbospinal inhibition (BSI) and RI of the MSR, Flaxedil (gallamine triethiodide) was injected to minimize the effect of movement of the animal on the recordings. Bulbospinal and recurrent inhibition of the MSR were tested at 10 29 min intervals. The MSR was quantified by averaging 10 consecutive spikes. The average MSR before and after the drug administration was expressed as a percentage of the f i n a l control average MSR. The degree of either BSI or RI was quantified by averaging the amplitude of 10 consecutive MSR spikes as well as the following 5 spikes conditioned with either BSI or RI. The percentage difference between these values on the f i n a l control test was equal to 100 % inhibition. Percent inhibition of previous and subsequent tests were calculated based on this figure. 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 injected over a 30 min period. In two preparations methysergide ftftft bimaleate (0.5 mg/kg i.v.) was injected over a 5 min period. A l l injections were given using an infusion pump (Harvard Model 975}. To block supraspinal inputs to the spinal cord, 1 cm cubes of frozen mammalian Ringer solution were placed on the spinal cord which was expo-sed at the T10 - T12 level (Wall, 1967). The absence of BSI of the MSR was taken as the criterion for a functional 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 solution was sucked out using an aspira-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' level. This reversible '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. <fe </fc ifc «fc Some animals were pretreated with DL-JJ-*chlorophenylalanine (p_-CPA} (300 mg/kg i.p. for 3 consecutive days) and prepared for record-30 ings 24 hours after the last 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 in the spinal 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. After a similar treatment noradrenaline was reported to be depleted to an immeasurable quantity in the spinal cord (King and Jewett, 1971). Foot Note * Geigy Limited. ** Abbott Pharmaceuticals ***Sandoz Pharmaceuticals. ****Sigma Chemical Company. 31 RESULTS Bulbospinal Inhibition of the Monosynaptic Reflex Imipramine HC1 (5.0 mg/kg i.v.) completely blocked bulbospinal inhibition (BSI) of the DR-MSR. The effect was rapid in onset and was maximal after . 2 0-30 min. The above and subsequent time refer to the start of the injection. The blocking action of imipramine started to decrease around 50 min and.was completely absent after about 2 hours (Fig. 2A). Bulbospinal inhibition of the QUAD-MSR and the PBST-MSR were blocked by. the above dose of imipramine. However, the effect on the inhibition of the QUAD-MSR was more rapid i n onset and greater i n magnitude. At 10 min imipramine had converted the inhibition into f a c i l i t a t i o n in 5 of 6 QUAD experiments (% inhibition = -10.3+ 6.3 S.E.M.). Convertion of the inhibition to f a c i l i t a t i o n at 10 min occurred in only 1 of 8 PBST expe-riments (% inhibition = 56.8 + 15.2). The maximal effect of imipramine was -87.9 + 19.4 % (50 min) in the QUAD experiments and -15.5 + 26.5 % (30 min) in the PBST experiments (Fig. 3 and 4). Pretreatment of the cats with DL-p_-chlorophenylalanine (p_-CPA) completely prevented the effect of imipramine in blocking BSI of the DR-MSR (Fig. 2B). However, pretreatment of the animals with DL-P(-methyl-p_-tyrosine methyl ester HC1 (0^-MPT) had no effect on the blocking action of imipramine (Fig. 2C). The effects of desipramine HC1 (4.8 mg/kg i.v.) on BSI of the DR-MSR were qualitatively similar 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) Fig, 2. The effect of imipramine CLMI) on bulbospinal inhibition of the DR-MSR in A. non-pretreated cats, n = 6; B. cats pretreated with JJ.-CPA (300 rag/kg i.p. for 3 consecutive days), n = 7; and C. cats-pretreated with C^ -MPT (126 mg/kg i.p. 16 and 4 hours prior to recording), n = 6. Imipramine HCl (5 mg/kg i.v.) was injected over 10 min as indicated on the abscissa. Each point in this and subsequent graphs reprents the mean + - S.E.M. of the conditioned (upper graph) and the unconditioned MSR (lower graph). 33 " 1 i a -20 0 20 40 60 Time (min) Fig. 3. The effect of imipramine HC1 CIMI) C5 mg/kg i.v.) on bulbospinal inhibition .of the QUAD-MSR, n = 6; and the PBST-MSR, n = 8. 34 Fig. 4. The blockade of bulbospinal inhibition by imipramine. A. The large spike represents the unconditioned PBST-MSR and the small spike represents inhibition of this reflex produced by a conditioning stimulus in the v i c i n i t y of the raphe magnus nucleus. B. A parti a l blockade of the inhibition at the end of a 10 min injection of imipramine HC1 (5 mg/kg i . v . ) . C. Complete blockade of the inhibition 10 min after 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 effect was gradual, and the effectwas prolonged. The blocking action was maximal at about 90 min and there was no indication of reco-very within 3 1/2 hours (Fig. 6). In two £-CPA pretreated animals in which imipramine failed to block BSI of the MSR, methysergide (0.5 mg/kg i.v.) converted the inhibition to a 3 - 4 fold f a c i l i t a t i o n . To test whether BSI and recurrent inhibition (Rl)were stable, these inhibitions were recorded for 3 1/2 hours in two experiments. The maximum deviation from the control values was 13.7 % (BSI) and 17.4 % (RI). Recurrent Inhibition of the Monosynaptic Reflex Imipramine HCl (5.0 mg/kg i.v.) blocked RI of the DR-MSR. The effect was gradual and reached maximum in 50 min and persisted for longer than 90 min (Fig. 7). Recurrent inhibition of the QUAD-MSR was blocked by imipramine, the effect was rapid in onset and reached maximum in about 10 min (Fig. 8A). However, the drug did not block RI of the PBST-MSR (Fig. 8B). Application of a 'cold block 1 significantly 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 effect of imipramine on RI of the QUAD-MSR was completely eliminated and RI was actually enhan-ced when a 'cold block' was applied 30 min after the injection of the drug (RI % of control = 116.02 + 5.4, n = 6) (Fig. 8A). Pretreatment of the cats with either 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 5 0 100 -2 100 c o o OC S 5 0 L DM I - 2 0 0 2 0 4 0 6 0 8 0 1 0 0 1 2 0 T i m e ( m i n ) Fig. 5. The effect of desipramine HC1 (DMI) C4.8 mg/kg i.v.) on bulbo-spinal inhibition of the MSR Cupper graph) and the unconditioned MSR (lower graph), n = 6. Desipramine was injected over 10 min as indicated on the abscissa. 37 - 2 0 0 3 0 6 0 9 0 7120 1 5 0 1 8 0 2 1 0 Time (min) Fig. 6. The effect of pargyline EC1 (PARG) (30 mg/kg i.v.) on bulbospinal inhibition 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 - 2 0 0 2 0 4 0 6 0 T i m e ( m i n ) Fig. 7. The effect of imipramine (IMI) on recurrent inhibition of the DR-MSR in non-pretreated cats, n = 5, (A); cats pre-treated with _p_-CPA (300 mg/kg i.p. for 3 consecutive days), n = 6, (B); and cats pretreated with C^ -MPT (126 mg/kg i.p. 16 and 4 hours prior to recording), n = 6, (C). 39 Fig. 8. The effect of imipramine HCl (LMI) (5 mg/kg i.v.) on recurrent inhibition 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 Fig. 9. The effect of pargyline HCl CPAUG) (30 mg/kg i.v.) on recurrent inhibition of the DR-MSR, n = 6. i 41 Pargyline also antagonized RI of the DR-MSR in 4 of 6 animals. The effect was gradual and reached maximum in about 90 min. Although quan-ti t a t i v e l y the effect of pargyline on this inhibition was less than that produced by imipramine, the former's effect was longer lasting (Fig. 9). The Unconditioned Monosynaptic Reflex Imipramine depressed the DR-MSR to about 60 % of i t s control value. The effect was maximal at about 30 min and the MSR started to return to control levels about 30 min later. Approximately 2 hours after the inject-ion of the drug the MSR returned to i t s control value (Fig. 2A). The QUAD-and PBST-MSR were also depressed by imipramine (Fig. 8A, B). Application of a 'cold block' reduced the QUAD-MSR to about 60 % of i t s control value (MSR % of control = 57.9 + 6.9, n = 5) but did not have a significant effect on the PBST-MSR (MSR % of control = 96.5 + 1.74, n = 5). After the injection of imipramine, when a 'cold block' was applied at 30 min, the QUAD-MSR was not depressed further (Fig. 8A). Imipramine depressed the DR-MSR in the animals pretreated with either jpj-CPA or 0(-MPT and the depressant effect at 30 min was not significantly different from that of the DR-MSR in the non-pretreated animals (Fig. 2). Desipramine also reduced the DR-MSR to about 65 % of i t s control value. The drug effect reaches maximum in about 40 min and the spike did not return to control value within 2 hours (Fig. 5). Pargyline exerted a biphasic effect 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 fa c i l i t a t e d to about 106 % of the control. This late f a c i l i t a t o r y effect was variable from animal to animal and reached i t s max in about 150 min and persisted upto 210 min at which time the experi-ment was discontinued (Fig. 6). 42 Blood Pressure Effects 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 similar to that of the blocking action of imipramine on BSI. However, the latter effect of the drug i s not related to i t s effect oh the blood pressure, because of the reasons narrated in the next section (Discussion). Desipramine at the given dose did not have a significant effect on blood pressure of most of the animals, however, this drug significantly reduced blood pressure in two. animals. Pargyline had no appreciable : effect on blood pressure. A3 DISCUSSION The finding that imipramine, desipramine and pargyline blocked bulbospinal inhibition (BSI) of the monosynaptic reflex (MSR) is not consistent with the proposal by Clineschmidt and Anderson (1970) that the bulbospinal inhibitory system contains a 5-hydroxytryptamine (5-HT) link. Since imipramine failed to antagonize BSI of the MSR in cats pre-treated with the tryptophan hydroxylase inhibitor, DL-£-chlorophenylala-nine (JJ-CPA) but s t i l l maintained i t s blocking action in animals pretrea-ted with the tyrosine hydroxylase inhibitor, DL-0(-methyl-p_-tyrosine (O^-MPT), i t i s assumed that the action of imipramine is mediated through 5-HT. This assumption i s strengthened by the observation that pargyline, which, at the given dose, significantly elevates the levels of 5-HT but not noradrenaline (NA) in the spinal cord of the cat (Anderson et^ a l . , 1967), blocked BSI. Furthermore, desipramine at an equimolar dose to that of imipramine was quantitatively less effective than imipramine in block-ing BSI. It is known that imipramine preferentially blocks the uptake of 5-HT whereas desipramine preferentially blocks the uptake of NA (Carlsson et a l . , 1969; Ross and Renyi, 1969; Shaskan and Snyder, 1970). Thus, the findings in the present investigation strongly suggest that 5-HT is involved in antagonizing rather than producing BSI. It i s interesting to note that the experiments carried 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 in the spinal cord have an overall f a c i l i t a t o r y effect on the MSR. It is tempting to speculate that at least a part of the fa c i l i t a t o r y effect of 5-HT on the MSR is due to a tonic inhibitory action of a 5-HT system on the inhibitory pathways (disinhibition), Thus, the net effect 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 inhibition (RI) (see subsequent discussion) and presynaptic inhibition (unpublished observations) of the QUAD-MSR. 3. Engberg-et a l . (1968) suggested that a descending 5-HT system has a tonic inhibitory action on lb and flexor reflex afferents. Although Clineschmidt and Anderson (1970) proposed that a 5-HT inter-neurone in the spinal cord i s contained in the bulbospinal inhibitory pathway, there i s no evidence to suggest that there are 5-HT interneurones in the spinal cord. Furthermore, d-lysergic acid diethylamide (LSD), which was used as a 5-HT antagonist by the above workers, i s known to stimu-late 5-HT receptors (Costa, 1956; Horita and Gogerty, 1958; Anden et a l . , 1968) and the effects may be dose dependent; stimulating at low doses and blocking at high doses (Costa, 1956). The effective dose of LSD i n blocking BSI (0.25 mg/kg) produced an enhancement of the unconditioned MSR (Clineschmidt and Anderson, 1970). This is consistent with a 5-HT stimulant effect as seen following the injection 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 effective in blocking BSI of the MSR (Banna and Anderson, 1968). The effect 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 in two animals that were pretreated with p_-CPA and in which imipramine failed to block BSI, methysergide converted the inhibition to a 3 - 4 fold f a c i l i t a t i o n ; this drug may be acting on a non-seroto-nergic system. It i s interesting that imipramine, pargyline and LSD a l l depressed the f i r i n g of the mesencephalic raphe neurones in rats (Sheard e_t a l . , 1972; Aghajanian et a l . , 1970; Aghajanian et a l . , 1968). Furthermore, this depressant effect of imipramine is absent i n rats pretreated with p_-CPA (Sheard et a l . , 1972). Hosli et a l . (1971) reported that 5-HT had a general excitatory effect when iontophoretically applied on the bulbo-spinal neurones. Thus, i f the 5-HT neurones in the raphe nuclei excite the bulbospinal inhibitory neurones, i t may be possible that BSI is blor-cked by imipramine and pargyline since the 5-HT neurones in the raphe nuclei stop f i r i n g after these drugs. However, Clineschmidt and Anderson (1970) found that LSD was more effective in blocking BSI of the MSR when administered by close-arterial injection to the spinal cord than by close-a r t e r i a l injection to the brain stem. Thus the site of action of LSD is most lik e l y i n the spinal cord. Also, the bulbospinal inhibitory pathway can not have a 5-HT neurone descending from the raphe nuclei to the spinal cord since, the conduction velocity of the inhibitory pathway was found to be high (Jankowska et a l . , 1968; Clineschmidt and Anderson, 1970) and the unmyelinated 5-HT fibres of 0.3 to 1.0 fm diameter (Dahlstrom and Fuxe, 1965) can not conduct impulses that fast. As mentioned earlier, there is no evidence for 5-HT interneurones to exist in the spinal cord. Bulbospinal inhibition of the flexor and the extensor MSRs was found to be of the postsynaptic type (Llinas, 1964a; Llinas and Terzuolo, 1964, 46 1965; Jankowska et a l . , 1968). the inhibitory pathway most lik e l y invol-ves a disynaptic link and the conduction velocity of the pathway i s high (Jankowska et a l . , 1968). While Jankowska et a l . (1968) did not find any difference between the ionic mechanisms involved in BSI of the flexor and extensor MSRs, Llinas and Terzuolo (1965) noted differences between the two and suggested that the inhibitory synapses of the bulbospinal inhibitory pathway with the flexor motoneurones are on the dendrites, far from the soma. Huffman and McFadin (1972) found that bicuculline, a spe-c i f i c t^-aminobutyric acid antagonist, blocked BSI of the flexor MSR but had no effect on BSI of the extensor MSR. It is interesting that i n the present study imipramine's blocking action on BSI of the QUAD-MSR was quantitatively greater than that on BSI of the PBST-MSR. This difference in the blocking action of the drug may be due to the differences in the mechanism by which the bulbospinal inhibitory pathway exerts i t s action on the QUAD-MSR and the PBST-MSR or due to the difference in the 5-HT input to these two types of the MSRs. Carpenter et a l . (1966) found that stimulation in the medial caudal bulbar reticular formation (1 mm below the floor of the fourth ventricle) produced negative dorsal root potentials (DRPs) on the l a afferents. These authors also reported that stimulation in the above reticular forma-tion about 4 mm below the floor of the fourth ventricle did not produce negative DRPs on the l a afferents. These observations suggest that the Magoun and Rhine's (1946) inhibitory area, the ventromedial caudal bulbar reticular formation, may not have a presynaptic type of bulbospi-nal inhibitory pathway. However, Chan and Barnes (19 72) observed that s t i -mulation in the ventral caudal bulbar reticular formation (2 mm lateral from the mid-sagittal line) produced negative DRPs on l a afferents; these authors also noted a time correlation between the primary afferent depolarization (PAD), the negative DRP and the inhibition of the MSR while stimulating in the above bulbar area. Since a negative DRP reflects PAD and presynaptic inhibition, these findings may suggest, contrary to those of Carpenter et a l . (1966), that a presynaptic type of inhibitory component is present in BSI of the MSR. In the present study only the ventromedial caudal bulbar reticular formation was stimulated ( V -6 to -10, L 0.0 in the Stereotaxic Atlas of Snider and Niemer, 1964). This area of stimulation is not exactly the same as that used in the study of Chan and Barnes'(1972) and i t i s not clear whether a presynaptic in h i b i -tory component was involved in the present study of the MSR.. Assuming that in the present study only the postsynaptic type of the bulbospinal inhibitory pathway was stimulated and that the inhibitory pathway contains a disynaptic link (Jankowska e_t a l . , 1968) and that the interneurone is located i n the spinal cord, It 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 in the spinal cord. It seems unlikely that the inhibitory 5-HT neurone ends on the soma of the bulbospinal neurone since 5-HT was found to have a general excitatory effect on the latter neurones (Hosli et a l . , 1971). It is known that the 5-HT neurones in the raphe nuclei send axons that descend in the spinal cord and terminate in the dorso-later a l and the ventrolateral motor nuclei of the ventral horn of the spinal cord (Fuxe, 1965). Thus i t seems more likely that an inhibitory 5-HT neurone may end on the axon terminal of the bulbospinal neurone or on the interneurone in the spinal cord. It is not known what other neuronal systems send inputs to the interneurones connected with the bulbospinal inhibitory pathway. Recently i t has been reported that some interneurones in the spinal cord which 48 receive inputs from the primary afferents also receive supraspinal inputs (Koizumi et a l . , 1959; Engberg et a l . , 1968). For example, the l a in h i -bitory interneurones receive supraspinal excitatory input (Hultborn and Udo, 19 72). Llinas reported that strychnine, a specific glycine antagonist reduced the hyperpolarization of the motoneurone membrane produced by BSI. Strychnine blocks reciprocal inhibition by antagonizing the action of the putative neurotransmitter, glycine that is presumably released from the l a inhibitory interneurone terminal at the interneurone-moto-neurone synapses. It may be possible that these interneurones are connec-ted with the bulbospinal inhibitory pathway. However, imipramine HCl (5 mg/kg i.v.) had no effect on reciprocal inhibition (unpublished obser-vations) suggesting that there is no 5-HT input to the l a inhibitory interneurones. Thus, i f these interneurones are involved i n BSI, a 5-HT inhibitory neurone must end on the axon terminal of the bulbospinal neurone. However, the idea that l a inhibitory interneurones are connected with the bulbospinal inhibitory pathway is purely speculative, Imipramine blocked RI of the QUAD-MSR and the effect of the drug is very l i k e l y mediated through a supraspinal monoaminergic system (see subsequent discussion). However, Renshaw cells do not seem to be connec-ted with the bulbospinal inhibitory pathway because imipramine's effect on BSI was eliminated by pretreatment of the cats with J3-CPA but not with Ot"MPT whereas imipramine's effect 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 of the cats with either p_-CPA or ClJ-MPT supports 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 is to the Renshaw cells involved in RI of the QUAD-MSR but not in RI of the PBST-MSR. Potentiation of RI of the MSR by application of a 'cold block' and complete removal of imipramine's effect on RI of the QUAD-MSR by a 'cold block 1 indicate that a supraspinal monoaminergic system has a tonic inhibitory effect on RI of the QUAD-MSR. Since pretreatment of the cats with either p_-CPA or P^ -MPT comple-tely 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 des-cending system must have links involving both 5-HT and NA. It i s intere-sting that the 5-HT and NA cells in the caudal brain stem have mutual synaptic contact and that there are both 5-HT and NA nerve terminals in the dorsolateral and ventrolateral motor nuclei of the ventral horn of the spinal cord (Fuxe. 1965). Application of a 'cold block' significantly reduced the QUAD-MSR but had no effect 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 injection of imipramine did not reduce the QUAD-MSR any further, This may suggest that the effect of imipramine on the QUAD-MSR is 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 effect on the PBST-MSR and imipramine reduced this MSR, the effect of the drug may be due to i t s action on neuronal systems at the segmental level. Eccles and Lundberg (1959) found that the exten-sor 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 significant effect on the depressant action of imipramine on the DR-MSR suggesting that this effect of imipramine on the DR-MSR is probably not mediated through a NA system. However, although pretreatment of the animals with JJ-CPA did not alter the action of imipramine on the DR-MSR upto 30 min of imipramine, the recovery of the MSR was faster than in nonpretreated animals. The reason for the faster recovery of the MSR in JJ-CPA pretrea-ted animals is not understood. However, since imipramine did depress the MSR in these animals, this effect of the drug can not be entirely due to a 5-HT input. The biphasic effect of pargyline on the MSR was previously reported by Anderson et a l . (1967). Based on the monoamine levels in the spinal cord after 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 in the present study is however smaller than that reported by Anderson ejt a l . (1967). This may be due to the difference i n the experimental prepara-tions. Anderson et a l . (1967) used cats with the spinal cord sectioned at the cervical level whereas in the present study cats decerebrated at the mid-collicular level were used. Thus the control MSR is consi-derably higher in most of the present experiments than the range (1.5 to 3.0 mV) used by Anderson et a l . (1967). Therefore in the present study, possibly fewer motoneurones are available for recruitment into the dis-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 surprising and not understood why this drug while blocking BSI, RI and presynaptic inhibition, reduces the MSR. The time course of the depressant action of imipramine on blood pressure is found to be similar to that of the drug's action on BSI, RI and the MSR. However, the possibility that imipramine's effects 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 significant effect 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. 'cold block' which had no effect on the blood pressure completely eliminated imipramine's effect on RI. As outlined above, the findings in the present investigation strongly suggest that a 5-HT system antagonizes BSI of the MSR and that a supraspinal monoaminergic system having both 5-HT and NA links has a tonic Inhibitory effect on RI of the QUAD-MSR. The 5-HT system that antagonizes BSI seems to be different from the monoaminergic system that blocks RI. Further investigation i s necessary to establish the s i t e of action of 5-HT involved in blocking these inhibitions. 52 REFERENCES AGHAJANIAN, G.K., FOOTE, W.F. and SHEARD, M.H. (1968). Lysergic acid diethylamide: sensitive neuronal units in the midbrain raphe. Science. 161: 706-708. AGHAJANIAN, G.K., GRAHAM, A.W. and SHEARD, M.H. (1970). Serotonin containing neurones in brain: Depression of f i r i n g by monoamine oxidase inhibitors. Science; 169: 1100-1102. ANDEN, N.E., CARLSSON, A., HILLARP, N.A. and MAGNUSSON, T. (1964). 5-hydroxytryptamine release by nerve stimulation of the spinal cord. Li f e Sci. 3: 473-478. ANDEN, N.E., CARLSSON, A., HILLARP, N.A. and MAGNUSSON, T. (1965). Noradrenaline release by nerve stimulation of the spinal cord. L i f e Sci. 4: 129-132. ANDEN, N.E., JUKES, M.G.M., LUNDBERG, A. and VYKLICKY, L. (1966). The effect of DOPA on the spinal cord. 1. Influence on transmission from primary afferents. Acta Physiol. Scand. 67: 373-386. ANDEN, N.E., CORRODI, H., FUXE, K. and HOKFELT, T. (1968). Evidence for a central 5-hydroxytryptamine receptor stimulation by lysergic acid diethylamide. Br. J. Pharmac. 34: 1-7. ANDERSON, E.G. and SHIBUYA, T. (1966). The effect of 5-hydroxytryptophan and L-tryptophan on spinal synaptic a c t i v i t y . J. Pharmac. exp. Ther. 153: 352-360. ANDERSON, E.G., BAKER, R.G. and BANNA, N.R. (1967). The effects of monoamine oxidase inhibitors on spinal synaptic ac t i v i t y . J. Pharmac. exp. Ther. 158: 405-415. BAKER, R.G. and ANDERSON, E.G. (1970). The effects of L-3,4-dihydroxy-phenylalanine on spinal reflex a c t i v i t y . J. Pharmac. exp. Ther. 173: 212-223. BANNA, N.R. and ANDERSON, E.G. (1968). The effects of 5-hydroxytryptamine antagonists on spinal neuronal ac t i v i t y . J. Pharmac. exp. Ther. 162: 319-325. BARKER, J.L. and NICOLL, R.A. (1972). Gamma-aminobutyric acid: role in primary afferent depolarization. Science. 176: 1043-1045. BARRON, D.H. and MATTHEWS, B.H.C. (1938). The interpretation of potential changes in the spinal cord. J. Physiol. 92: 276-321. BEN0ISH,J.M., BESSON, J.M., CONSEILLER, C. and LE BARS, D. (1972). Action of bicuculline on presynaptic inhibition of various origins in the cat's spinal cord. Brain Res. 43: 672-676. 53 BESSON, N.R.,and ABDELMOUMENE, M. £1970). Modifications of dorsal root potentials during c o r t i c a l seizures. Electroenceph. Clin. Neurophysiol. 2j9: 166-172. BESSON, J.M., RIVOT, J.P. and ALEONARD, P. (1971). Action of picrotoxin on presynaptic inhibition of various origins in the cat's spinal cord. Brain Res. .26: 212-216. BROCK, L.G., COOMBS, J.S. and ECCLES, J.C. (1952). The recording of potentials from motoneurones with an intracellular electrode. J. Physiol. 117: 431-460. BROCK, L.G.. COOMBS, J.S. and ECCLES, J.C. (1952a). The nature of the monosynaptic excitatory and inhibitory processes in the spinal cord. Proc. Roy. Soc. B. 140: 170-176. BROOKS, C.McC, Eccles, J.C. and MALCOLM, J.L. (1948). Synaptic potentials of inhibited motoneurones. .J Neurophysiol. 11: 417-430. CARLSSON, A., MAGNUSSON, T. and ROSENGREN, E. (1963). 5-hydroxytryptamine in spinal cord, normally and after transection. Experientia. 19: 359. CARLSSON, N., CORRODI, H., FUXE, K. and HOKFELT, T. (1969).Effect of . antidepressant drugs in the depletion of intraneuronal brain 5-hydroxy-tryptamine stores caused by 4-methyl-p-ethyl meta-tyramine. Eur. J.  Pharmac. _5: 357-363. CARPENTER, D., ENGBERG, I. and LUNDBERG, A. (1966). Primary afferent depolarization evoked from the brain stem and the cerebellum. Arch, i t a l . B i o l . 104: 73-85. CHAN, S.H.H. and BARNES, CD. (1972). A presynaptic mechanism evoked from brain stem reticular formation in the lumbar cord and i t s temporal significance. Brain Res. 45: 101-114. CLINESCHMIDT, B.V. (1972). Spinal monoamines and the toxic interaction between monoamine oxidase inhibitors and t r i c y c l i c antidepressants. Eur. J. Pharmac. 19: 126-129. CLINESCHMIDT, B.V. and ANDERSON, E.G. (1970). The blockade of bulbospinal inhibition by 5-hydroxytryptamine antagonists. Expl. Brain Res. 11: 175-186. CLINESCHMIDT, B.V., PIERCE, J.E. and SJOERDSMA, A. (1971). Interactions of t r i c y c l i c antidepressants and 5-hydroxyindolealkylamine precursors on spinal monosynaptic reflex transmission. J. Pharmac. exp. Ther. 179: 312-. 323. CLINESCHMIDT, B.V., PIERCE, J.E. and L0VENBERG, (1971a). Tryptophan hydroxylase and serotonin in spinal cord and brain stem before and after chronic transection. J. Neurochem. 18: 1593. COOMBS, J.S., ECCLES, J.C. and FATT, P. (1955a). The specific ionic 54 conductance and the ionic movements across the motoneuronal membrane that produce the inhibitory postsynaptic potential. J. Physiol. 130: 326-373. COOMBS, J.S., ECCLES, J.C. and FATT, P. (1955b). The inhibitory suppression of reflex discharges from motoneurones. J. Physiol.130: 396-413. COSTA, E. (1956). Effects of hallucinogenic and tranquilizing drugs on serotonin evoked uterine contractions. Proc. Soc. Exp. Bio l . Med. N.Y. 91: 39-41. CRANMER, J.J., BRANN, A.W. and BACH, L.M.N. (1959). An adrenergic basis for bulbar inhibition. Amer.J. Physiol. 197: 835. CURTIS, D.R. (1969). The pharmacology of spinal postsynaptic inhibition. Prog. Brain Res. 31: 171-189. CURTIS, D.R. and RYALL, R.W. (1966a). The excitation of Renshaw c e l l s by cholinomimetics. Expl. Brain Res. 2_: 49-65. CURTIS, D.R. and RYALL, R.W. (1966b). Acetylcholine receptors of Renshaw c e l l s . Expl. Brain Res. 1} 66-80. CURTIS, D.R. and RYALL, R.W. (1966c). The synaptic excitation of Renshaw c e l l s . Expl. Brain Res. 1} 81-96. CURTIS, D.R., HOSLI, L., JOHNSTON, G.A.R. and JOHNSTON, J.H. (1968). The hyperpolarization of spinal motoneurones by glycine and related amino acids. Expl. Brain Res. 5_: 238-262. CURTIS, D.R., DUGGAN, A.W., FELIX, D. and JOHNSTON, G.A.R. (1970). GABA, bicuculline and central inhibition. Nature.226: 1222-1224. CURTIS, D.R., DUGGAN, A.W. and JOHNSTON, G.A.R. (1971). The spe c i f i c i t y of strychnine as a glycine antagonist in the mammalian spinal cord. Expl. Brain Res. 12: 547-565. CURTIS, D.R., DUGGAN, A.W., FELIX, D. and JOHNSTON, G.A.R. (1971a). Bicuculline, an antagonist of GABA and synaptic inhibition in the spinal cord of the cat. Brain Res. 32: 69-96. DAHLSTROM, A. and FUXE, K. (1964). Evidence for the existence of mono-amine containing neurones in the central nervous system. I. Demonstration of monoamines in the c e l l bodies of brain stem neurones. Acta Physiol. Scand. 62: Suppl.232: 1-55. DAHLSTROM, A. and FUXE, K. (1965). Evidence for the existence of mono-amine neurones in the central nervous system. II. Experimentally induced changes in the intraneuronal amine levels of bulbospinal neuron systems. Acta Physiol. Scand. 64: Suppl. 247: 1-36. ECCLES, J.C. (1961). The mechanisms of synaptic transmission. Ergebn. Physiol. 51: 229-430. ECCLES, J.C. (1963). Presynaptic and postsynaptic inhibitory actions in the spinal cord. Prog. Brain Res. jL: 1-18. 55 ECCLES, J.C. (1964). Presynaptic inhibition in the spinal cord. Prog. Brain Res. 12: 65. ECCLES, J.C. (1964a). The physiology of synapses. (Springer-Verlag), Berlin. ECCLES, J.C, FATT, P. and KOKETSU, K. (1954). Cholinergic and inhibitory synapses in a pathway from motor-axon collaterals to motoneurones. J_. Physiol. 126: 524-562. ECCLES, J.C, FATT, P. and LANDGREN, S. (1956). The central pathway for the direct inhibitory action of impulses in the largest afferent nerve fibres to muscles. J. Neurophysiol. 19: 75-98. ECCLES, J.C, ECCLES, R.M. and FATT, P. (1956a). Pharmacological investi-gations on a central synapse operated by acetyl choline. J. Physiol. 131: 154-169. ECCLES, J.C, SCHMIDT, R.F. and WILLIS, W.D. (1962). Presynaptic inhibition of the spinal monosynaptic reflex pathway. J. Physiol. 161: 282-297. ECCLES, J.C, KOSTYUK, P.G. and SCHMIDT, R.F. (1962a). Central pathways responsible for depolarization of primary afferent fibres. J. Physiol. 161: 237-257. ECCLES, J.C, SCHMIDT, R.F. and WILLIS, W.D. (1963a). Depolarization of central terminals of group lb afferent fibres of muscles^ J. Neuro- physiol. 26: 1 -27. ECCLES, J.C, SCHMIDT, R.F. and WILLIS, W.D. (1963b). Pharmacological studies on presynaptic inhibition. J. Physiol. 165: 403-420. ECCLES, J.C, SCHMIDT, R.F. and WILLIS, W.D. (1963c). Depolarization of central terminals of cutaneous afferent fibres. J. Neurophysiol.26: 646-661. ECCLES, R.M. and LUNDBERG, A. (1959). Supraspinal control of interneurones mediating spinal reflexes. J. Physiol. 147: 565-584. ENGBERG, I., LUNDBERG, A. and RYALL, R.W. (1968). Is the tonic decerebrate inhibition of reflex paths mediated by monoaminergic pathways? Acta Physiol. Scand. 72: 123-133. FRANK, K. and FOURTES, M.G.F. (1955). Potentials recorded from the spinal cord with microelectrodes. J. Physiol. 130: 625-654. FUXE, K. (1965). Evidence for the existence of monoamine neurones in the central nervous system. IV. Distribution of monoamine nerve terminals in the central nervous system. Acta Physiol. Scand. 64: Suppl. 247: 37-84. GRANT, G. and REXED, B. (1958). Dorsal spinal root afferents to Clark's column. Brain. 81: 567-57 6. HAASE, J. and VAN DER MEULEN, J.P. (1961). Effects of supraspinal stimul-56 ation on Renshaw ce l l s belonging to extensor motoneurones. J. Neurophysiol. 24: 510-520. HORITA, A. and GOGERTY, J.H. (1958). The pyretogenic effect of 5-hydroxy-tryptophan and i t s comparison with that of lysergic acid diethylamide. J. Pharmac. exp. Ther. 122: 195-200. HOSLI, L., TEBECIS, A.K. and SCHONWETTER, H.P. (1971). A comparison of the effects of monoamines on neurones of the bulbar reticular formation. Brain Res. 25: 357-370. HUFFMAN, R.D. and McFADIN, L.S. (1972). Effects of bicuculline on central inhibition. Neuropharmac. 11: 789-799. HULTBORN, H., JANKOWSKA, E. and LINDSTROM, S. (1971). Relative contribu-tion from different nerves to recurrent depression of l a IPSPs in motor neurones. J. Physiol. 215: 637-664. HULTBORN, H. and UDO, M. (1972). Recurrent depression from motor axon collaterals of supraspinal inhibition in motoneurones. Acta Physiol. Scand. 85: 44-57. INNES, I.R. (1962). An action of 5-hydroxytryptamine on adrenaline receptors. Br. J. Pharmac. Chemother. 19: 427-441. JANKOWSKA, E., LUND, S., LUNDBERG, A. and POMPEIANO, 0. (1968). Inhibitory effects evoked through ventral reticulospinal pathways. Arch, i t a l . B i o l .  106: 124-240. KAADA, B.R. (1950). Site of action of myahs±nv{.mephenesin, Tolserol) in the central nervous system. J. Neurophysiol. 13: 89-104. KING, D.and JEWETT, R < ( 1971). T h e e f f e c t s ofC<-methyltyrosine on sleep and brain norepinephrine in cats. J. Pharmac. exp. Ther. 177: 188-195. KOIZUMI, K., USHIYAMA, J. and BROOKS, CM. (1959). A study of reticular formation action on spinal interneurones and motoneurones. Jap. J. Physiol. 9: 282-303. KRNJEVIC, K. and MORRIS, M.E. (1972). Extracellular K + a c t i v i t y and slow potential changes in spinal cord and medulla. Can. J. Physiol. Pharmacol. 50: 1214-1217. KUHAR, M.J., ROTH, R.H. and AGHAJANIAN, G.K. (1971). Selective reduction of tryptophan hydroxylase act i v i t y in rat forebrain after midbrain raphe lesions. Brain Res. 35: 167-176. LLINAS, R. (1964a). Mechanisms of supraspinal actions upon spinal cord a c t i v i t i e s . Differences between reticular and cerebellar inhibitory actions upon alpha-extensor motoneurones. J. Neurophysiol. 27: 1117-1125. LLINAS, R. (1964b). Mechanisms of supraspinal action upon spinal cord 57 a c t i v i t i e s . Pharmacological studies on reticular inhibition of alpha-extensor motoneurones. J. Neurophysiol. 27: 1127-1137. LLINAS, R. and TERZUOLO, C.A. (1964). Mechanisms of supraspinal actions upon spinal cord a c t i v i t i e s . Reticular inhibitory mechanisms on alpha-extensor motoneurones. J. Neurophysiol-.- 27 : 579-591. LLINAS, R. and TERZUOLO, C.A. (1965). Mechanisms of supraspinal actions upon spinal cord a c t i v i t i e s . Reticular inhibitory mechanisms upon flexor motoneurones. J. Neurophysiol. 28: 413-421. LLINAS, R. and BAKER, R. (1972). A chloride-dependent inhibitory post-synaptic potential in cat trochlear motoneurones. J. Neurophysiol. 35: 484-492. LLOYD, D.P.C. (1941). A direct central inhibitory action of dromically conducted impulses. J. Neurophysiol. 4_: 184-190. LUX, H.D. (1971). Ammonium and chloride extrusion:.hyperpolarizing syna-ptic inhibition in spinal motoneurones. Science. 173: 555-557. LUX, H.D., LORACHER, C. and NEHER, E. (1970). The action of ammonium on postsynaptic inhibition of cat spinal motoneurones, Expl. Brain Res. 11: 431-447. MAC LEAN, J.B. and LEFFMAN, H. (1967). Supraspinal control of Renshaw c e l l s . Expl. Neurol. 18: 94-104. MAGNUSSON, T. and ROSENGREN, E. (1963). Catecholamines of the spinal cord normally and after transection. Experientia. 19: 229. MAGOUN, H.W. and RHINES, R. (1946). An inhibitory mechanism in the bulbar reticular formation. J. Neurophysiol. jh 165-171. McLENNAN, H. (1961). The effect of some catecholamines upon a monosynaptic reflex pathway in the spinal cord. J. Physiol. 158: 411. Ng, L.K.Y., CHASE, T.N., COLBURN, R.W. and KOPIN, I.J. (1972). Release of [ H] dopamine by L^5-hydroxytryptophan. Brain Res. 45: 499-505. PROUDFIT, H.K. and ANDERSON, E.G. (1973). Blockade by serotonin antago-nists of brain stem-evoked potentials recorded from spinal dorsal and ventral roots. Fed. Proc. 32'. 303 RENSHAW, B. (1941). Influence of discharge of motoneurones upon excitation of neighbouring motoneurones. J. Neurophysiol. h_: 167-183. REXED, B. (1952). The cytoarchitectonic organization of the spinal cord in the cat. J. Comp. Neurol. 96: 415-495. REXED, B. (1954). A cytoarchitectonic atlas of the spinal cord in the cat. J. Comp. Neurol. 100: 297^-379. REXED, B. (1964). Some aspects of the cytoarchitectonics and synaptology of the spinal cord. Prog. Brain Res.11: 58-92. 58 RICHARDSON, T.W., APRISON, M.H. and WERMAN, R. (1955). An automatic direct-current operating temperature-control device J. Appl. Physiol. 20: 1355-1356. i ROMANES, G.J. (1964). The motor pools of the spinal cord. Prog. Brain Res. 11: 93-119. ROSS, S.B. and RENYI, A.L. (1969). Inhibition of the uptake of t r i t i a t e d 5-hydroxytryptamine in brain tissue. Eur. J. Pharmac. _7_: 270-277. RUCH,T. and PATTON, H. (1960). Physoilogy and Biophysics. W.B. Saunders Company Pha. Nineteenth Edition. RYALL, R.W. (1970). Renshaw c e l l mediated inhibition of Renshaw c e l l s . Patterns of excitation and inhibition from inputs in motor axon collate-r a l s . J. Neurophysiol. 33: 257-270. RYALL, R.W. and PIERCEY, M.F. (1971). Excitation and inhibition of Ren-shaw c e l l s by impulses in peripheral afferent nerve fibres. J. Neurophy- s i o l . 34: 242-251. RYALL, R.W., PIERCEY, M.F., P0L0SA, C. and GOLDFARB (1972). Excitation of Renshaw c e l l s in relation to orthodromic and antidromic excitation of motoneurones. J. Neurophysiol.35: 137-148. SASTRY, B.S.R. and SINCLAIR, J.G. (1973). Unpublished observations. SHEARD, M.H., ZOLOVICK, A. and AGHAJANIAN, G.K. (1972). Raphe neurones: effect of t r i c y c l i c antidepressant drugs. Brain Res. 43: 690-694. SHASKAN, E.G. and SNYDER, S.H. (1970). Kinetics of serotonin uptake into slices of rat brain: Relationship to catecholamine uptake. J. Pharmac. exp. Ther. 175: 404-418. SHIBUYA, T. and ANDERSON, E.G. (1968). The influence of chronic cord transection on the effects of 5-hydroxytryptophan, 1^-tryptophan and par-gyline on spinal neuronal a c t i v i t y . J. Pharmac. exp. Ther. 164: 185-190. SINCLAIR, J.G. (1973). Morphine and meperidine on bulbospinal inhibition of the monosynaptic reflex. Eur. J. Pharmac. 21: 111-114. SNIDER, S. and NIEMER, T.W. (1964). A Stereotaxic Atlas of the Cat Brain. The University of Chicago Press, Second Edition. SPRAGUE, J.M.,and HA, H. (1964). The terminal fields of dorsal root fibres in the lumbosacral spinal cord of the cat and the dendritic organization of the spinal cord. Prog. Brain Res. 11: 120-152. TABER, C. (1971). p_-chlorophenylalanine blockade of the effects of 5-hy-droxytryptophan on spinal synaptic a c t i v i t y . Fed. Proc. 30: 317. TABER, E., BRODAL, A. and WALBERG, F. (1960). The raphe nuclei of the brain stem in the cat I. Normal topography and cytoarchitecture and gene-59 r a l discussion. J. Comp. Neurol. 114: 161-187. VON TAN,.U. and HENATSCH, H.D. (1968). Suppression of recurrent inhibition and of presynaptic inhibition of spinal motoneurones by the antidepressive agent imipramine ( Tofranil ). Pflugers Arch, ges. Physiol.300: R89. VON TAN, U. and HENATSCH, H.D. (1969). Differentiation of supraspinal and spinal sites of imipramine'action upon spinal inhibitory and excitato-ry systems. Pflugers Arch, ges. Physiol. 307: R120. WALL, P.D. (1967). The laminar organization of dorsal horn and effects of descending inputs. J. Physiol. 188: 403-423. WERMAN, R., DAVIDOFF, R.A. and APRISON, M.H. (1968). Inhibitory action of glycine on spinal neurones in the cat. J. Neurophysiol. 31: 81-95.