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Studies on serotonin involvement in nucleus raphe inhibition and morphine depression of spinal cord neurones Khanna, Sanjay 1986

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STUDIES ON SEROTONIN INVOLVEMENT IN NUCLEUS RAPHE INHIBITION AND MORPHINE DEPRESSION OF SPINAL CORD NEURONES By SANJAY KHANNA M.Pharm., Panjab Univers i ty , 1983 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Div is ion of Pharmacology and Toxicology in the Faculty of Pharmaceutical Sciences) We accept th i s thesis as conforming to the required-standard THE UNIVERSITY OF BRITISH COLUMBIA Apr i l 1986 © Sanjay Khanna, 1986 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an advanced degree a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I agr e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e head o f my department o r by h i s o r h e r r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f pHPiR MA CE UTI CAL GCit-AICES-The U n i v e r s i t y o f B r i t i s h C o l u m b i a 2075 Wesbrook P l a c e V ancouver, Canada V6T 1W5 Date - i i -ABSTRACT It is not clear whether 5-hydroxytryptamine (5-HT) mediates, at least partly, the nucleus raphe magnus (NRM) stimulation-produced inhibition of deep dorsal horn wide dynamic range (WDR) neuronal nociceptive activity. The role of 5-HT in the NRM phasic inhibition is suggested by the presence of 5-HT in some of the descending fibres from the NRM, the release of 5-HT into spinal perfusates upon NRM stimulation and the fact that the iontophoretic application of 5-HT in the spinal cord depresses the nociceptive activity of the deep dorsal horn WDR neurones. However, systemic administration or iontophoretic application of methysergide, a putative 5-HT antagonist in the spinal cord did not reduce the NRM phasic inhibition of the deep dorsal horn WDR neurones. Experiments were therefore performed to determine whether 5-HT mediates the NRM phasic inhibition of WDR neurones by comparing the inhibition of the neuronal nociceptive activity to the NRM stimulation before and after administering the selective 5-HT uptake blocker, fluoxetine (6.0 mg/kg, i .v . ) , or the monoamine oxidase inhibitor, pargyline (30.0 mg/kg, i .v . ) . The NRM phasic inhibition following the drug treatment with fluoxetine or pargyline was decreased. Thus, 5-HT does not mediate but appears to reduce the NRM phasic inhibition of the deep dorsal horn WDR neurones. Fluoxetine administration did not affect the noxious heat-evoked activity of the dorsal horn WDR neurones. This lack of effect of fluoxetine on neuronal nociceptive activity probably reflects a lack of 5-HT involvement in the tonic control of dorsal horn WDR neuronal nociceptive a c t i v i t y , as suggested by some authors. Pargyline treatment produced an increase in the heat-evoked a c t i v i t y of the dorsal horn neurones studied. The mechanism responsible for th i s effect i s not known. Experiments were performed to re-examine the effect of morphine on the NRM phasic inh ib i t i on of spinal cord nociceptive transmiss ion. A controversy ex is ts as to whether morphine e l i c i t s a supraspinal attenuation of spinal nociceptive transmission. Some invest igators claim that morphine act ivates a NRM descending inh ib i t i on since micro in ject ion of the drug into th is nucleus produces a decrease in the nociceptive a c t i v i t y of the spinal cord dorsal horn WDR neurones. However, systemic morphine fa i l ed to enhance the NRM phasic i nh ib i t i on of the dorsal horn WDR neurones. To test the hypothesis that morphine act ivates a descending serotonergic inh ib i to ry system from the NRM impinging on the deep dorsal horn WDR neurones, morphine and f luoxetine were given concurrent ly. However, with th i s treatment a decrease in the NRM phasic i nh ib i t i on was observed which was greater than seen with f luoxet ine alone. Thus, these experimental results do not favour the above hypothesis. Morphine also suppressed the noxious heat-evoked a c t i v i t y of the deep dorsal horn WDR neurones. When th i s drug was administered concurrently with f luoxet ine, the observed decrease in the nociceptive a c t i v i t y of the WDR neurones was not s t a t i s t i c a l l y d i f ferent from that observed with morphine alone. This f inding suggests that 5-HT does not - i v m e d i a t e morphine's s u p p r e s s i v e e f f e c t on t h e deep d o r s a l horn WDR neurones and i s a g a i n s t t h e h y p o t h e s i s t h a t 5-HT i s i n t i m a t e l y i n v o l v e d i n m e d i a t i n g morphine i n h i b i t i o n o f s p i n a l c o r d n o c i c e p t i v e t r a n s m i s s i o n . Dr. J.G. S i n c l a i r , Ph.D. S u p e r v i s o r LIST OF ABBREVIATIONS CPA 4-chloroamphetamine DA dopamine DLF dorsolateral funiculus DLH DL-homocysteic acid 5,7-DHT 5,7-dihydroxytryptamine DC PS dorsal column postsynaptic spinomedullary ELI enkephal in- l ike EPSP exc i tatory postsynaptic potential GABA Y-aminobutyric acid HTM high threshold mechanoreceptive 5-HIAA 5-hydroxyindole acet ic acid 5-HT 5 - hyd roxy t ry pt am i n e HRP horseradish peroxidase i .p . intraper i toneal i .v . i ntravenous kg k i l ogram K potassi um LTM low threshold mechanoreceptive LSD lyserg ic acid diethylamide mg mil 1igram min minute MAO I monoamine oxidase inh ib i to r NS nociceptive spec i f i c - vi NA noradrenaline NRM nucleus raphe magnus PCPA p-chlorophenylalanine PAG periaqueductal gray PMN polymodal nociceptor PAD primary afferent depolar izat ion sec second S.E.M. standard error of mean SG substantia gelat inosa SG.. inner substantia gelat inosa SG outer substantia gelatinosa o WDR wide dynamic range - v i i -TABLE OF CONTENTS Page ABSTRACT i i LIST OF ABBREVIATIONS v LIST OF TABLES i x LIST OF FIGURES x ACKNOWLEDGEMENT x i i INTRODUCTION 1 REVIEW OF LITERATURE 5 Noc i cep t i ve Mechanism of Spinal Cord Dorsal Horn 5 Cutaneous Noc iceptors 5 Dorsal Horn Neurones Responding to Noxious Input 9 The Nucleus Raphe Magnus-Spinal Cord Neuraxis i n the C a t . . . . 20 Phys io l ogy of Descending I n h i b i t i o n by S t imu la t i on of NRM in the Cat 25 Pharmacology of the Nucleus Raphe Magnus S t i m u l a t i o n -Produced I n h i b i t i o n of Dorsal Horn Neuronal A c t i v i t y i n the Cat 29 Pharmacology of Morphine on the Deep Dorsal Horn WDR Neurones 33 Tonic Descending I n h i b i t i o n : Involvement of Endogenous Opio id Pept ides and the E f f e c t of Morphine 33 Intravenous Morphine on Noxious St imulus-Evoked Responses of the Deep Dorsal Horn WDR Neurones 36 Ion topho re t i c Adm in i s t r a t i on of Morphine 39 Suprasp ina l Mediated E f f e c t s o f Morphine on the Deep Dorsal Horn WDR Neuronal A c t i v i t y 43 Role of 5-HT in Morphine Ac t ion 45 Pharmacology of F l uoxe t i ne and Pa rgy l i ne 47 - v i i i Page METHOD AND MATERIALS 50 Surgical Procedures 50 St imulat ion, Recording and Testing of Dorsal Horn Neurones.. 51 Supraspinal Stimulation 53 Preparation of Recording Microelectrodes 53 Computer Programmes 54 Pharmacological Studies 55 Calculat ions 57 RESULTS 59 Serotonin Involvement in NRM Stimulation-Produced Inh ib i t ion 59 Effect of Fluoxetine 59 Effect of Pargyline 62 Effect of Pargyline plus Fluoxetine 67 Effect of Morphine 70 Serotonin Involvement in Morphine Action 77 Control Experiments 84 DISCUSSION 89 Involvement of 5-HT in the NRM Phasic Inh ib i t ion of Dorsal Horn Neurones 89 Role of Serotonin in Morphine Depression of Nociceptive Response in the Dorsal Horn Neurones 102 CONCLUSION 108 BIBLIOGRAPHY 110 - ix LIST OF TABLES Page Table 1 Portion of analysis of variance for effect of morphine on noxious heat-evoked a c t i v i t y of individual dorsal horn neurones at different time periods 74 Table 2 Portion of analysis of variance and Newman-keuls test of significance for morphine effect on heat-evoked response averaged for five dorsal horn neurones 75 Table 3 Effect of morphine on the nucleus raphe magnus phasic i n h i b i t i o n of dorsal horn neuronal nociceptive a c t i v i t y in fluoxetine pretreated animals 80 X -LIST OF FIGURES Figure Page 1 Osci l loscope traces of neuronal response to noxious radiant heat and heat plus nucleus raphe magnus st imulat ion 60 2 Computer generated histograms i l l u s t r a t i n g the effect of f luoxet ine and f luoxet ine plus morphine, respect ive ly , on the noxious heat-evoked and heat plus NRM stimulation-produced effect on dorsal horn neuronal nociceptive a c t i v i t y 61 3 The effect of f luoxet ine on the nucleus raphe magnus stimulation-produced inh ib i t i on of the nociceptive a c t i v i t y of the dorsal horn neurones 63 4 Lack of ef fect of intravenous infusion of f luoxet ine on the noxious heat-evoked a c t i v i t y of the dorsal horn neurones 64 5 Lack of effect of f luoxet ine on the spontaneous a c t i v i t y of the dorsal horn neurones 65 6 The effect of pargyline and pargyline plus f luoxet ine on the nucleus raphe magnus stimulation-produced i nh ib i t i on of the dorsal horn neuronal nociceptive a c t i v i t y 66 7 The effect of pargyline and pargyline plus f luoxet ine on the noxious heat-evoked a c t i v i t y of dorsal horn neurones 68 8 The effect of pargyline and pargyline plus f luoxet ine on the spontaneous a c t i v i t y of dorsal horn neurones 69 9 Lack of effect of morphine on the nucleus raphe magnus stimulation-produced inh ib i t i on of nociceptive a c t i v i t y of dorsal horn neurones 71 10 The suppressive effect of cumulative doses of morphine on the noxious heat-evoked a c t i v i t y of dorsal horn neurones 73 11 Inh ib i t ion of spontaneous a c t i v i t y of dorsal horn neurones by cumulative doses of morphine 76 12 The block of nucleus raphe magnus stimulation-produced inh ib i t i on by morphine administered to f luoxet ine pretreated animals. The data i s expressed as percent of control 78 - xi -Figure Page 13 The ef fect of morphine on the nucleus raphe magnus stimulation-produced inh ib i t i on in f luoxetine pretreated animal. The data i s expressed as actual percent i nh ib i t i on 79 14 The inh ib i to ry ef fect of cumulative doses of morphine on noxious heat-evoked a c t i v i t y of dorsal horn WDR neurones in f luoxet ine pretreated animals 82 15 The inh ib i t i on of spontaneous a c t i v i t y of dorsal horn neurones by morphine in f luoxetine pretreated an ima ls . . . . 83 16 Lack of ef fect of the infusion of the vehic le on the nucleus raphe magnus phasic i nh ib i t i on of dorsal horn neuronal nociceptive a c t i v i t y 85 17 Lack of ef fect of intravenous infusion of the vehic le on the noxious heat-evoked a c t i v i t y of dorsal horn neurones 86 18 Lack of ef fect of intravenous infusion of the vehic le on spontaneous a c t i v i t y of dorsal horn neurones 87 19 A comparison of the percentage inh ib i t i on and noxious heat-evoked a c t i v i t y of dorsal horn neurones with noxious temperature applied at level of 45°C and 5 2 ° C . . . 88 20 A schematic neuronal arrangement i l l u s t r a t i n g some possible synaptic connections which would produce nucleus raphe magnus phasic inh ib i t i on of deep dorsal horn wide dynamic range neurone 92 21 A schematic neuronal arrangement i l l u s t r a t i n g the nucleus raphe magnus phasic inh ib i to ry impingement of deep dorsal horn wide dynamic range neurone mediated v ia an inh ib i to ry interneurone in the substantia gelatinosa 93 22 A schematic arrangement i l l u s t r a t i n g some possible synaptic connections by which the serotonergic bulbospinal neurones attenuate the nucleus raphe magnus phasic inh ib i t i on of deep dorsal horn neurone.. . . 95 23 A schematic neuronal arrangement i l l u s t r a t i n g the serotonergic impingement on the inh ib i to ry interneurone in the substantia gelatinosa which blocks the nucleus raphe magnus phasic i nh ib i t i on of deep dorsal horn neurone 97 - x i i -ACKNOWLEDGEMENT The author is indebted to Dr. John G. S i nc l a i r for his patient guidance and encouragement throughout the course of th i s study. He is also grateful to Drs. R.J. Ensom, G. Bell ward, J . Diamond and J .A . Pearson for reviewing the thes i s . This study was supported by a grant from the B r i t i s h Columbia Health Care Research foundation and the Medical Research Council of Canada to Dr. S i n c l a i r . Special thanks are extended to the author's parents in India for words of encouragement they wrote to the author. F ina l l y the author would l i k e to thank Ms. Grace Lo for her invaluable assistance during the course of th is study. Dedicated to my parents - 1 -INTRODUCTION Many studies have demonstrated that e l e c t r i c a l st imulat ion of the nucleus raphe magnus (NRM) in the cats can i nh ib i t the noxious s t imu l i -evoked a c t i v i t y (Fields et a l . 1977; Guilbaud et a l . 1977; McCreery et a l . 1979; Kajander et a l . 1984; Gray and Dostrovsky 1983), and C-f ibre stimulation-produced responses (Morton et a l . 1983) of the deep dorsal horn wide dynamic range (WDR) neurones. This i nh ib i t i on of evoked a c t i v i t y i s by a f ib re system from the NRM which descends in the dorsolateral funiculus (DLF). DLF les ions , but not ventrolateral t ract les ions made rostra l to the s i t e of recording, reduced the effect in the cat (F ie lds et a l . 1977). This i s further substantiated by the anatomical study of Basbaum et a l . (1978) who found that f ibres from the NRM large ly descend in the DLF to the spinal cord. Some of the descending f ibres from the NRM to the cat spinal cord are serotonergic (Fung et a l . 1985). These serotonergic f ibres can make synaptic contacts on the dendrites and soma of postsynaptic targets in the spinal cord (Ruda and Gobel 1980; Gobel et a l . 1982). Stimulation of the NRM produces an increased release of 5-hydroxytryptamine (5-HT) in the spinal cord perfusates of rats (Hammond et a l . 1985). When 5-HT i s iontophoresed e i ther in the v i c i n i t y of the ce l l body of deep dorsal horn neurones (Belcher et a l . 1978) or in the region of substantia gelat inosa (SG) of the spinal cord (Headley et a l . 1978), i nh i b i t i on of the nociceptive a c t i v i t y of the deep dorsal horn neurones occurs. These l ines of evidence suggest that 5-HT might - 2 -mediate, at least par t l y , the NRM phasic i nh ib i t i on of the nociceptive a c t i v i t y of the dorsal horn neurones. Contrary to th i s speculat ion, Griersmith et a l . (1981) reported that methysergide, a 5-HT antagonist, intravenously administered or iontophoret ica l ly applied in the SG, f a i l ed to reduce the NRM phasic i nh ib i t i on of dorsal horn neuronal nociceptive a c t i v i t y . However, the study by Griersmith et a l . (1981) i s subject to c r i t i c i sm on the choice of antagonist used to ident i fy the role of 5-HT. Methysergide, when microiontophoret ica l ly applied on neurones in areas of the CNS known to be innervated by 5-HT containing terminals , f a i l ed to block but instead mimicked the inh ib i to ry effect of iontophoretic 5-HT (Haigler and Aghajanian 1977). Therefore, experiments were designed to determine whether 5-HT plays a role in the NRM phasic i nh ib i t i on of dorsal horn neuronal nociceptive a c t i v i t y . These experiments on NRM phasic i nh ib i t i on involved the use of f luoxet ine , a se lect ive neuronal 5-HT uptake blocker, and pargyl ine, a monoamine oxidase i nh i b i t o r . The rat ionale followed was that , i f 5-HT mediates the NRM stimulation-produced inh ib i t i on of dorsal horn WDR neurones, then f luoxet ine and pargyline treatment should enhance 5-HT synaptic transmission and increase the extent of NRM inh ib i t i on of neuronal nociceptive a c t i v i t y . There i s evidence that the NRM is involved in morphine ant inoc icept ion. For example, lesions of the NRM can block the increase in the rat t a i l - f l i c k latency seen with systemic morphine (Proudfit and Anderson 1975; Yaksh et a l . 1977). Micro inject ion of morphine into the NRM produces an increase in the threshold for nociception-induced - 3 -voca l i za t ion in the rats (Dickenson et a l . 1979). Behavioral studies also suggest that 5-HT might be involved in morphine ant inoc icept ion. For example, Taiwo et a l . (1985) observed that , in the presence of intrathecal monoamine uptake blockers, subthreshold doses of parenteral morphine produced ant inoc icept ion. This potent iat ion was prevented by deplet ion of 5-HT by p-chlorophenylalanine (PCPA) pretreatment or by intrathecal administration of the putative 5-HT antagonist, methysergide. Vasko et a l . (1984) have reported that micro inject ion of morphine into the NRM of the rats produced ant inocicept ion which could be attenuated by depletion of spinal cord 5-HT using the 5-HT neurotoxin, 5,7-dihydroxytryptamine. This evidence would suggest that morphine ant inocicept ion might be mediated via the NRM through release of spinal 5-HT. E lectrophysio logica l studies have also attempted to ident i fy whether morphine attenuates spinal cord nociceptive transmission through a NRM s i te of ac t ion. The evidence i s c on f l i c t i n g . Le Bars et a l . (1976) studied the effect of intravenous morphine (2.0 mg/kg) on the NRM phasic i nh ib i t i on of deep dorsal horn neuronal nociceptive a c t i v i t y in decerebrate ca t s . Morphine was found to have no effect on th i s i n h i b i t i o n . However, when morphine (10-20 ug) was microinjected into the NRM of anaesthetized cats there was a suppression of noxious heat-evoked a c t i v i t y of the deeper dorsal horn WDR neurones (Du et a l . 1984). To re-examine the question of whether the NRM mediates morphine i nh ib i t i on of spinal cord dorsal horn neuronal nociceptive a c t i v i t y , the ef fect of morphine on the NRM stimulation-produced inh ib i t i on of dorsal - 4 -horn neuronal a c t i v i t y was evaluated. The rat ionale was that i f morphine act ivates a descending i nh i b i t i o n , from the NRM onto the deep dorsal horn neurones, then i t should potentiate the NRM phasic i n h i b i t i o n . Experiments were also designed to test whether morphine inh ib i t i on of neuronal nociceptive a c t i v i t y was affected by enhanced 5-HT synaptic transmiss ion. If morphine inh ib i t i on of deep dorsal horn neurones i s in part mediated by 5-HT, then i t should be enhanced by f luoxet ine treatment. Furthermore, i f 5-HT i s involved in NRM phasic i nh ib i t i on and morphine enhances th i s i nh i b i t i on , then morphine and f luoxet ine administered concurrently should increase the inh ib i t i on more than e i ther drug alone. - 5 -REVIEW OF LITERATURE Nociceptive Mechanism of Spinal Cord Dorsal Horn CUTANEOUS NOCICEPTORS: Sensory information from the skin enters the CNS via cutaneous nerves consist ing of several f ibre types. These include large diameter myelinated Ao.3— f i b res , the small diameter A6-f ibres and the unmyelinated C- f ib res . Of these various types of cutaneous nerve f i b re s , i t i s the A6- and C-f ibres which convey noxious information from the skin nociceptors to the spinal cord s i tes in the CNS (Perl 1984). The cutaneous nociceptor units have been c l a s s i f i ed into two types, namely, high threshold mechanoreceptor units (HTM) with A6- and C-axons (Burgess and Perl 1967; Bessou and Perl 1969) and polymodal nociceptor units (PMN) with C-axons (Bessou and Perl 1969). The properties of these nociceptor units have been characterized in the cat using ex t race l lu la r electrodes to record unit a c t i v i t y from s ing le primary afferent axons (Burgess and Perl 1967; Bessou and Perl 1969). It was observed that HTM unit axons, which general ly had no background a c t i v i t y , responded to strong noxious pressure applied to the i r receptive f i e l d . Pressure thresholds required to excite these HTM axons varied from unit to un i t . A l inear re lat ionsh ip between discharge rate of units and stimulus intens i ty was observed. These units were res is tant to act ivat ion from noxious radiant heat of the i r receptive f i e l d s . However, in the both cat and monkey i t has been observed that - 6 -app l icat ion of noxious radiant heat to the receptive f i e l d of HTM units with A<5- axons frequently sensit ized these units so that they became responsive to subsequent heat st imulat ion (F i tzgera ld and Lynn 1977; Campbell et a l . 1979). They did not respond to i r r i t a n t chemicals (Burgess and Perl 1967). The unmyelinated PMN units respond to noxious pressure, noxious heat and the appl icat ion of i r r i t a n t chemicals to the i r receptive f i e l d s (Bessou and Perl 1969). The authors observed a graded increase in response of these units to increasing skin temperature. Heat sens i t i za t ion of these units also occurred after a single strong heating of the receptive f i e l d . Beck et a l . (1974) have described thermal nociceptors in the cat which respond well to noxious heat but poorly to pressure. However, doubts have been expressed as to whether these f ibres are representative of a c lass of nociceptors d i s t i n c t from PMN un i t s . For example, Lynn (1984) has suggested that the thermal nociceptors might be PMN at the most insens i t i ve end of the mechanical s ens i t i v i t y range for the l a t t e r un i t s . Further, these putative thermal nociceptors were not tested for t he i r responsiveness to i r r i t a n t chemicals. Course and termination of primary af ferents: Detailed analysis of the spinal cord termination of primary afferents has been done by Light and Perl (1977 and 1979). These authors combined anterograde transport of horseradish peroxidase (HRP) by cut dorsal roots with l i gh t microscopy to trace the spinal cord region of termination of these - 7 -roots . The experiments were done in three d i f ferent species, namely, ca t , rat and monkey. Simi lar pattern of termination of the dorsal root f ib res in the spinal cord was observed for a l l three species. Fine diameter f ibres were observed to enter the dorsal horn through the t rac t of Lissauer whereas the large diameter f ibres were observed to run medial ly and form a bundle in the dorsal columns. Co l la tera ls from f ine f ib res were seen to terminate mainly in the super f i c ia l laminae of the dorsal horn, namely, laminae I and II (also ca l led the marginal zone and the substantia ge lat inosa, respec t i ve ly ) . Based on the diameter of the co l l a t e ra l s terminating in the super f i c ia l laminae the authors suggested that f ibres terminating in the marginal zone were mainly of the A5- axon type and those terminating in the SG (substantia gelatinosa) were mainly of the C-axon type. These authors extended the i r experiments to monkeys in which they se lec t i ve ly lesioned e i ther the medial or the la te ra l sect ion of the dorsal roots and studied the pattern of termination of the intact section of the dorsal roots. It has been shown that in monkeys the la te ra l section of the dorsal root consists mainly of unmyelinated and th in l y myelinated f ibres whereas the medial portion of the dorsal root consists of myelinated f ibres with medium to large diameter. When the medial d i v i s i on of the dorsal roots i s lesioned the HRP stained f ibres of the la te ra l d i v i s i on showed presumed synaptic enlargements concentrated in the marginal zone and in the SG, with some endings also appearing at the base of dorsal horn. In contrast , with the l a te ra l d i v i s i on cut and the medial l e f t in tac t , HRP stained terminals were found to be densely d is t r ibuted in the nucleus proprius - 8 -and in the deeper l ayers . The termination of the two d iv i s ions of the dorsal root were found to overlap in lamina III of the primate dorsal horn. The termination pattern of s ing le , funct iona l ly ident i f i ed primary afferents have also been character ized. For example, Light and Perl (1979a) iontophoresed HRP in t race l1u la r l y into phys io log ica l l y i den t i f i ed s ingle A6- afferents and observed that the HTM units gave off co l l a te ra l s which ended in terminal arbors in laminae I and V. Sometimes the terminal arbors of the HTM units also penetrated the outer lamina II (also refered to as lamina 11 0 ) . Delta hair afferent co l l a t e ra l s arborised and terminated mainly in ventral or inner lamina II (also referred to as lamina 11-j) and laminae I I I - V. Attempts have also been made to ident i fy the course and termination of C-primary afferents in the spinal cord using i n t r a ce l l u l a r HRP. For example, Perl (1984a) have reported that a small sample of such units studied gave off co l l a t e ra l s which terminated mainly in the SG. Some co l l a te ra l s were observed to pass deep and have terminals ventral to SG. Coupling HRP histochemistry to l i gh t and electron microscopy, Rethelyi et a l . (1982) observed that the co l l a t e ra l s of the A6- HTM units had en passant enlargements along the i r course. These co l l a te ra l s terminated in a bouton enlargement and synaptic connections (pre or post) were found at these boutons. Such boutons e i ther made simple axo-dendr i t i c contact or were central terminals in glomeruli being both pre-or postsynaptic to dendrites and axons in the g lomerul i . Such termination pattern for the A6- HTM units were observed both in laminae - 9 -I and V. On the basis of above observations i t might be concluded that the zone of termination of the small diameter myelinated and unmyelinated primary af ferents , some of which are nociceptors, terminate p r i n c i pa l l y in the super f i c ia l dorsal horn (laminae I and I I ) . In contrast the large diameter primary afferents terminate mainly in lamina III and in the deeper laminae of the spinal cord dorsal horn in adult cats (see Brown 1981 for review). DORSAL HORN NEURONES RESPONDING TO NOXIOUS INPUT: Noxious information carr ied by the somatic nociceptors i s conveyed f i r s t to the spinal cord s i t e s , where i t i s processed and relayed to supraspinal region in the CNS and also i n i t i a t e s segmental re f lexes. There are at least two types of spinal cord dorsal horn neurones which are excited by the noxious information entering the spinal cord. These are the wide dynamic range (WDR) or mult i recept ive neurones and noc iceptor-spec i f i c (NS) neurones. The former c lass of neurones i s excited by both noxious and innocuous st imul i whereas NS neurones are excited only by noxious s t imu l i . Light and Durkovic (1984) have mapped the laminar organization of dorsal horn neurones which respond to the noxious st imul i of the sk in . The experiments were done in decerebrate and spinal ized cats . Cel ls which responded to the noxious pinch of the i r peripheral receptive f i e l d were found to be d is t r ibuted throughout laminae I- VII of the spinal cord dorsal horn. The most heavy concentration of such neurones was - 10 -determined to be in the region of laminae I- II and V-VII . WDR neurones: Light and Durkovic (1984) have reported that , in the cat , neurones of th i s class are concentrated in the spinal cord deep dorsal horn laminae V - VI I . The e lectrophys io logica l charac ter i s t i cs of these deep dorsal horn WDR neurones have been described by a number of authors (Handwerker et a l . 1975; Pr ice and Browe 1973). These neurones, as mentiond ea r l i e r , were excited by both innocuous mechanical st imulat ion and noxious pinch of the i r peripheral receptive f i e l d s . A high proportion of such neurones were also found to be stimulated by noxious heating of the i r receptive f i e l d s and there was a graded increase in the response of these neurones to increasing skin temperature. A prominent after-discharge was seen in these neurones after the noxious thermal stimulus was removed. Some of these neurones also discharged to innocuous cooling or warming of the i r receptive f i e l d s . The deep dorsal horn WDR neurones have been reported to receive inputs from both large diameter (AaB) and small diameter (A6, C-f ibre) afferents (Handwerker et a l . 1975; Light and Durkovic 1984). Based on the ca lcu lat ions of the latency of responses in these neurones to large diameter afferent s t imulat ion, the above authors have suggested that a monosynaptic Aa3-afferent input to the deep dorsal horn WDR neurones i s poss ib le . This i s consistent with h is to log ica l f indings that dendrites of deep dorsal horn neurones and axonal arbor izat ion of large diameter afferents are co-terminus in these deeper laminae of dorsal horn - 11 -(Brown 1982). Polysynaptic input from Aa3-afferents to these neurones has also been described (Handwerker et a l . 1975). However, the d i rect ac t i va t ion of these deep dorsal horn neurones by nociceptive afferents i s uncerta in. This i s espec ia l ly true for the C-nociceptor a f ferents , most of which apparently terminate in the super f i c ia l laminae. It i s poss ib le , therefore, that many of the WDR neurones are in polysynaptic re la t ion to nociceptors. Various inh ib i to ry influences on the deep dorsal horn WDR neurones in the cat have also been reported. For example, Hi 11 man and Wall (1969) observed an inh ib i t i on of a c t i v i t y of these neurones by l i g h t t a c t i l e s t imul i applied to areas which were adjacent to the exc i tatory receptive f i e l d s of these neurones. Handwerker et a l . (1975) described a segmental i nh ib i t i on of heat-evoked responses in these neurones by st imult ion of large diameter nerve trunk afferents or the dorsal columns. Consistent ly , when A-f ibres of nerve are po lar izat ion blocked, the C-f ibre discharge of WDR neurones to st imulat ion of th i s nerve grows markedly larger (Gregor & Zimmermann 1972). Du et a l . (1984a) reported an i nh ib i t i on of high in tens i ty hindlimb nerve stimulation-produced and noxious heat-evoked response in dorsal horn neurones by A-afferent st imulat ion of forelimb nerves. This inh ib i t i on was mediated by supraspinal structures as i t could be reduced by micro inject ion of the local anaesthetic, l i doca ine , in supraspinal s i tes including the NRM. Du et a l . (1984a) did not c l a s s i f y the spinal cord dorsal horn neurones studied. However, i t i s possible that they were of the WDR type for Le Bars et al . (1979) had reported that in rats only the WDR - 12 -type of neurones could be inh ib i ted by st imulat ion in the fore-region of the animals body. A supraspinal tonic inh ib i to ry control on the noxious stimulus-evoked and C-f ibre evoked response in WDR neurones has also been described (Handwerker et a l . 1975; Soja and S inc l a i r 1983a). Some of the deep dorsal horn WDR neurones have axons which ascend the spinal cord to supraspinal structures. For example, some of the spinocervical t ract (Cervero et a l . 1977 ) , spinothalamic t ract (Rucker et a l . 1984), dorsal column postsynaptic spinomedul1ary (DCPS) t ract (Brown & Fyffe 1981) and spinomesencephalic t ract neurones (Yezierski & Schwartz 1984) have WDR cha rac te r i s t i c s . F ina l ly , a morpho-functional study on the deep dorsal horn WDR neurones have also been attempted. In such experiments phys io log ica l l y indent i f ied neurones were marked in t race l1u la r l y with the HRP. This procedure allows a study of the morphology of neurones with known functional propert ies. For example, R i tz & Greenspan (1985) have described lamina V, WDR neurones with large soma size and dendr i t i c spread which extended considerably in rostro-caudal and mediolateral d i r e c t i on . In the dorso-ventral d i rect ion the dendrites of these neurones were large ly confined to Lamina III to lamina VII . In very few cases did the dendrites penetrate the super f i c ia l laminae. These authors also described a small sample of lamina VII WDR neurones. These neurones had a small soma size compared to neurones in the lamina V and the i r dendrites were mostly confined to the ventral horn. Bennett et a l . (1984) have described dorsal DCPS neurones located in the laminae III - IV and having WDR cha rac te r i s t i c s . These neurones had - 13 -dendr i t i c spread extending from laminae III - V, whereas those DCPS neurones which were not responsive to noxious st imul i had a dendr i t i c spread confined to laminae III and IV. WDR neurones have also been reported in laminae I & II of the cat dorsal horn (Bennett et a l . 1981; Bennett et a l . 1979; Cervero et a l . 1976; Craig & Kn i f fk i 1985; F i tzgera ld 1981 and Cervero et a l . 1979a). The number of WDR neurones recorded from the super f i c ia l laminae, as a percentage of the total ce l l population in th i s region, var ies . For example, WDR neurones formed the majority of the neurones (64%) recorded by F i tzgera ld (1981) from th i s region of the spinal cord in decerebrate ca ts , whereas, such neurones constituted a very small proportion of the sample recorded by Cervero et a l . (1979a) in anaesthetized ca t s . F i tzgera ld (1981) has studied extensively the properties of WDR neurones of super f i c ia l laminae in ca ts . Most of the neurones studied had large receptive f i e l d s and responded to both brushing and noxious pinch applied to the i r receptive f i e l d s . Many of the neurones showed habituation to repeated brushing of the same area of the i r receptive f i e l d . Habituation to noxious pinch was less common. A prominent after-discharge general ly developed in these neurones to pinching in receptive f i e l d s . The response to a noxious heat stimulus was not tes ted . Most of the WDR neurones recorded by F i tzgera ld (1981) had C-f ibre input . Convergent input on these neurones of e i ther A6 or As or both A5 and A3 f ibres was also reported. Unlike the deeper dorsal horn neurones, the C-f ibre response in the WDR neurones of the super f i c ia l - 14 -laminae was reported to occur at extremely regular latencies giving time locked spikes on st imulat ion of the sural nerve. The authors suggested that such time locked C-f ibre response could be due to a monosynaptic C-afferent input to these neurones. No 'wind up' on C- f ibre s t imu la t ion , which i s typ ica l of deeper dorsal horn WDR neurones, was seen in these neurones. The authors also calculated the latency of response in these neurones to A3 and AS s t imu la t ion . Based on these ca lcu lat ions the author suggested that A6 response occurred at la tenc ies which could be monosynaptic whereas A3 response was at latencies which might be polysynaptic. The inh ib i to ry responses of the super f i c ia l laminae WDR neurones also d i f fered from that of deeper dorsal horn WDR neurones. Po la r i sa t ion block of A- f ibres did not enhance the C-f ibre response in the former neurones (F i tzgera ld 1981) which i s unlike that reported for the l a t t e r group of neurones (see above). F i tzgera ld (1981) also studied the ef fect of sural nerve st imulation at A3 strength on the response of WDR neurones to bradykinin injected subcutaneously in the i r receptive f i e l d s . Whereas in lamina V WDR neurones the response to bradykinin was inh ib i ted by a condit ioning stimulus in the sural nerve at A3 f ibre in tens i t y , no inh ib i t i on was observed in the super f i c ia l laminae neurones. Whether these neurones project to supraspinal structures i s not known. Morpho-functional studies, s imi lar to that described before, have also been attempted for super f i c ia l laminae WDR neurones (Bennett et a l . 1981; Bennett et a l . 1979; Light et a l . 1979; Rethelyi et a l . - 15 -1983). Some of the WDR neurones thus described have charac te r i s t i c s of Waldeyer c e l l s and stalked c e l l s of the super f i c ia l laminae. Generally the dendrites of WDR neurones were confined to the super f ic ia l laminae and in some cases penetrated lamina I I I , with the axon also terminating in super f i c ia l laminae. The axons of some WDR Waldeyer c e l l s could be traced to the ventral horn of the spinal cord. There might also ex ist a d i f ference in the laminar arrangement of neurones in the SG such that c e l l s in SGn are excited predominantly by HTM and polymodal receptors innervated by A6- and C- f ib res , while c e l l s in the SG-j are driven pr imar i ly by LTM (Light et a l . 1979b). NS neurones: Ce l l s which are driven so le ly by noxious st imulat ion of the i r receptive f i e lds have been described in ca ts , by various authors (Christensen & Perl 1970; Bennett et a l . 1981; Bennett et a l . 1979; Cervero et a l . 1976; Craig and Kn i f fk i 1985; F i tzgerald 1981; and Light & Durkovic 1984). Although NS neurones have also been reported in the deep dorsal horn of the spinal cord (Light and Durkovic 1984), much of the work on these type of neurones has concentrated in the region of laminae I and II of the super f i c ia l dorsal horn. The response charac te r i s t i c of these neurones to noxious st imulat ion of the i r receptive f i e lds has been studied. Christensen & Perl (1970) described NS neurones of lamina I which e i ther responded to noxious pinch or both noxious pinch and heat applied to the i r receptive f i e l d s . The former type has been cal led class 3a and the l a t t e r c lass 3b by Cervero et a l . (1976). The class 3a NS neurones were desribed as - 16 -receiving input from only A<S-cutaneous afferents whereas class 3b neurones received both A6-and C-f ibre afferent input (Cervero et al . 1976). F i tzgera ld (1981), found a number of NS neurones of the super f i c i a l laminae that developed long after-discharges las t ing seconds af ter pinching the receptive f i e l d . On the contrary, Steedman et a l . (1985) reported on super f i c ia l laminae NS neurones in which increased f i r i n g to noxious pinch of the i r RF was followed by tota l s i lence or decreased f i r i n g after removal of exc i tatory inf luence. Inhib i tory influences on the NS neurones of the super f i c ia l laminae of spinal cord dorsal horn have also been reported. For example, Christensen and Perl (1970), noted an inh ib i to ry receptive f i e l d for NS ce l l s which was general ly found adjacent to the i r exci tatory f i e l d s in a surround fashion. Only strong noxious mechanical or noxious thermal s t imul i applied to the inh ib i to ry RF of these ce l l s was able to i nh ib i t the a c t i v i t y in the NS neurones. However, recently Steedman et a l . (1985) also reported an inh ib i to ry receptive f i e l d of these neurones, which in some cases overlapped the exc i tatory f i e l d , and where appl icat ion of innocuous mechanical st imulation produced the i n h i b i t i o n . Stimulation of large diameter afferents of the nerve supplying the RF of NS neurones inh ib i ted the discharge in these neurones to noxious st imulat ion (Cervero et a l . 1976; Steedman et a l . 1985). These neurones could be inh ib i ted by st imulat ion of large afferent f ibres in dorsal column at in tens i ty su f f i c i en t to excite large afferent f ibres (Cervero et a l . 1979d). Cervero et a l . (1976) reported a supraspinal tonic inh ib i to ry control on the a c t i v i t y in the - 17 -super f i c i a l laminae NS neurones. However, the tonic i nh ib i t i on exercised by supraspinal structures on these neurones was reported to be weaker than the apparently s imi la r descending tonic i nh ib i t i on of deeper dorsal horn WDR c e l l s . Cervero et a l . (1979c) have also examined the extent of descending tonic i nh ib i t i on on NS, SG neurones. In a small sample of six such neurones, there was no change in the background a c t i v i t y of four neurones and, in the remaining two, only small or t rans ient changes were observed on appl icat ion of spinal cold b lock. Using i n t r a ce l l u l a r recording from the super f i c ia l laminae NS neurones, Steedman et a l . (1985) have calculated that the central delay before a response in these neurones could be due to the act ivat ion of A3 or A6 afferent f i b r e s . Based on these ca l cu la t ions , the authors suggested that these neurones received a predominantly exc i tatory monosynaptic A6 input and a inh ib i to ry polysynaptic A 3 input. Like super f i c ia l laminae WDR neurones, the response to C-f ibre st imulat ion in the super f i c ia l laminae NS neurones occurred at constant latency with no 'wind up' phenomenon. Some of the NS lamina I neurones of the cat might be project ing to supraspinal s t ructures. For example, Craig and Kn i f f k i (1985) reported that a number of NS neurones from lamina I could be ant idromical ly act ivated from the contra latera l thalamus. However, the number of NS lamina I neurones which might project supraspinal ly might be smal l . Thus, Kumazawa and Perl (1975) could ant idromical ly act ivate only one-third of these neurones from the contralateral spinal cord. S im i l a r l y Cervero et a l . (1979d and 1979b) fa i led to backf i re two-third - 18 -of the lamina I NS and SG neurones, respect ive ly , by st imulat ion at a spinal cord s i te three spinal segments rostra l to the recording s i t e . Morpho-functional analysis of the super f i c ia l laminae NS neurones have revealed at least three d i f fe rent morphological types (Bennett et a l . 1981; Bennett et a l . 1979; Steedman et a l . 1985; Molony et a l . 1981; Rethelyi et a l . 1983 and Light et a l . 1979). Two of these have a small per ikaraya. One of these has dendrites directed in rostro-caudal fashion in lamina I and SG 0 . The other type has dendrites arranged s im i l a r l y as to above type plus i t might have dendr i t ic arbor isat ion running transversely across lamina II and lamina I I I . The axons of these neurones are also general ly directed rostro-caudal ly in laminae I and I I . The axons branched outside the l im i t of the dendr i t i c tree of t h i s parent neurone. The th i rd morphological type of NS neurone has a r e l a t i ve l y larger ce l l body with dendrites also confined to lamina I but with some dendrite branches fol lowing the la te ra l curvature of lamina I into the intermediate zone of the dorsal horn. The axons of t h i s type of neurone could be traced into lamina I and also were seen directed vent ra l l y towards the ventral horn of the spinal cord. In an attempt to provide a spec i f i c function for neurones in the SG in sensory transmission, Price et a l . (1979) recorded from neurone pairs in the marginal zone of the monkey spinal cord. The f i r s t neurone encountered was ident i f i ed as a spinothalamic t ract neurone in the marginal zone while the more ventral neurone was located in the SG Q. Both neurones of each pair had s imi lar afferent inputs with the SG0 neurones having smaller receptive f i e l d s and shorter latenc ies to - 19 -appropriate nociceptive s t imu l i . These invest igators concluded that the SG0 c e l l s may relay afferent nociceptive input to lamina I neurones which, in turn project to supraspinal areas. Miscellaneous neurones: F i na l l y , neurones in the spinal cord have been ident i f i ed which respond d i f f e ren t l y from WDR or NS neurones to the appl icat ion of noxious st imul i to the i r cutaneous receptive f i e l d s . For example, Cervero et a l . (1977 ) have described deep dorsal horn spino-cerv ica l t ract neurones which were inhib i ted by noxious st imul i applied to the i r receptive f i e l d s (I-Class neurones). Another c lass of neurones described by the above authors were c l a s s i f i ed as E-I c e l l s . These were excited vigorously by the f i r s t noxious stimulus to the i r receptive f i e l d s , the a c t i v i t y of the neurones remained high on the termination of the stimulus and when a subsequent noxious stimulus was applied the neuronal a c t i v i t y was inh ib i t ed . The spontaneous a c t i v i t y of these neurones was high and was increased s l i gh t l y and t rans ient ly on cold blocking the spinal cord. Cervero et a l . (1979a) have also described SG neurones which have s imi lar charac ter i s t i cs to the above deep dorsal horn neurones in that a noxious stimulus applied to the receptive f i e l d inh ib i ted the a c t i v i t y of the neurone. The authors ca l led th i s neuronal type as c lass inverse-3 (3~). This c lass of SG neurone could be excited by innocuous s t imu l i . Another class of SG neurones described by the above authors, and ca l led class inverse-2 ("_"), could be inh ib i ted by both noxious and innocuous stimulus applied to the i r receptive f i e l d s . - 20 -The Nucleus Raphe Magnus-Spinal Cord Neuraxis in the Cat The nucleus raphe magnus (NRM) i s a midl ine medullary nucleus extending from the rostra l pole of the i n f e r i o r o l ive to the level of the rostra l pole of the superior o l i v e . Detailed cytoarchitectural features of th i s nucleus have been described by Taber et a l . (1960). These authors i den t i f i ed three d i f fe rent ce l l types in the NRM: big and medium sized polygonal c e l l s present in large numbers; smal l , round, p i r i form or spindle shaped c e l l s , and giant c e l l s . Fibres or ig inat ing within the nucleus and those crossing the nucleus were also i den t i f i ed as well as the presence of abundant boutons. Dhalstrom and Fuxe (1964), using histofluorescence techniques, demonstrated that many of these c e l l s in the NRM have a yel low fluorescence d i s t i n c t i v e for 5-hydroxytryptamine (5-HT, serotonin). Wiklund et a l . (1981) have estimated that the serotonergic neurones in the NRM const i tute about 15% of the tota l neuronal population of t h i s nucleus in the ca t . This group iden t i f i ed serotonergic neurones also by using the histofluorescence technique. At least some of these neurones project to the spinal cord since a par t ia l les ion in th i s nucleus resu l ts in a pronounced decrease in the level of 5-HT in the dorsal horn of the spinal cord (Oliveras et a l . 1977). Employing a double label ing technique in the rat , Bowker et a l . (1981) mapped the serotonin containing ce l l s of the brainstem that project to the spinal cord. These ce l l s were mainly in the nucleus raphe obscurus, raphe pa l l i dus , raphe magnus and the adjacent re t i cu la r formation. Of the serotonin containing ce l l s in the raphe nuclei at least 73.4% projected to the - 21 -spinal cord. Conversely, of sp ina l l y project ing c e l l s in these nuc le i , at least 88.6% contained serotonin. In cats , of the tota l number of sp ina l l y project ing neurones from the above brainstem regions 69% were serotonergic (Fung et a l . 1985). West and Wolstencroft (1977) have estimated that in the cat only 10% of the NRM f ibres projecting to the spinal cord are serotonergic. Their ca lcu lat ions were based on the assumption that the bulbospinal 5-HT units could be ident i f i ed by the i r ant ic ipated slow conduction ve loc i t y , due to the i r unmyelinated axons. However, not a l l bulbospinal 5-HT axons in the cat are unmyelinated (Ruda and Gobel 1980) and therefore the number of NRM bulbospinal serotonergic f ibres might have been underestimated by West and Wolstencroft (1977). Indeed, using a more str ingent means of i d en t i f i c a t i o n , that of suscep t i b i l i t y to 5,7-dihydroxytryptamine, Wessendorf et a l . (1981) found that in rats 40% of the bulbospinal units in the NRM were probably serotonergic. Descending f ibres from the NRM to the spinal cord have also been demonstrated in the cat using retrograde flow technique involving horseradish peroxidase (Basbaum et a l . 1978). These f ibres travel in the dorsolateral funiculus of the spinal cord and terminate in laminae I, I I , and in parts of laminae V, VI, and VII . Some of these descending f ib res are serotonergic since (H)-amino acid microinjected into the NRM i s anterogradely transported into morphologically i dent i f i ed 5-HT axonal endings in the spinal cord of cats (Ruda et a l . 1981). Furthermore, thorac ic transect ion resu l ts in a loss of spinal 5-HT below the level of section without a l ter ing that found in the cerv ica l enlargement - 22 -(OTiveras et a l . 1977). The above observations suggest that much or a l l of the spinal serotonin i s contributed by a descending f ib re system from the nucleus raphe. A quant i tat ive analysis of the concentration of serotonin in d i f fe rent regions of the spinal cord was performed by Oliveras et a l . (1977). Chemical assay was used to determine the serotonin content of the spinal cord t issue obtained by punching out microdiscs from h is to log ica l sections of the spinal cord. The concentration of 5-HT was at least twice as high in the gray matter as in the white matter. The regions of the spinal cord gray matter containing the most serotonin included the motor nucleus, lamina X and the dorsolateral most part of the dorsal horn ( in the region of SG). Axonal terminals which take up labe l led 5-HT have also been demonstrated in the super f ic ia l laminae I and II of the dorsal horn (Ruda et a l . 1981). The 5-HT axonal endings in the spinal cord synapse on dendrites and perikaya of postsynaptic targets (Ruda and Gobel 1980). Very few such axonal endings synapse on terminals of primary afferent f i b r e s . The postsynaptic targets of 5-HT terminals include projection neurones in lamina I and stalked c e l l s in the SG (Gobel et a l . 1982). Some of these targets have enkephalin-1ike immunoreactivity (ELI; Glazer and Basbaum 1984). This group also found that the majority of 5-HT axonal endings did not have any de f i n i t i v e synaptic c l e f t . These anatomical observations would favour a postsynaptic action of descending 5-HT f ibres and a non-synaptic action by release of 5-HT into the v i c i n i t y of target c e l l s . Other descending projections from the NRM are - 23 -to the spinal trigeminal nucleus (pars caudal i s ) , dorsal motor nucleus of the vagus and the so l i t a r y nucleus (Basbaum et a l . 1978). Ascending projections from th i s nucleus travel along the ventromedial tegmentum and the medio-longitudinal fasc iculus and project to the periaqueductal gray (PAG), cen t ra l i s medial i s , paracentral i s , cen t ra l i s l a t e r a l i s and dorsomedial thalamus, la te ra l and dorsal hypothalamic nucleus and zona incerta (Basbaum et a l . 1976). The NRM receives l i t t l e or no d i rect input from the spinal cord. The input to the nucleus includes that from the PAG. Anatomical connections between these nuclei have been shown (Abols and Basbaum 1981) and e lectrophysio logica l evidence also ex ists for such a project ion (Shah and Dostrovsky 1980). The input from the PAG to the NRM i s predominantly exc i tatory for both raphe-spinal and non-spinal project ing raphe ce l l s (Lovic et a l . 1978; Maciewicz et a l . 1984). There is also a large input from the nearby gigantocel1ular re t i cu la r formation (Rgc; Mehler 1964). Stimulation in the medullary re t i cu l a r formation produces a graded depolar izat ion of raphe c e l l s . The NRM, in tu rn , projects to the medullary and pontine re t i cu la r formation ( Bob i l l i e r et a l . 1976) and st imulat ion in the nucleus raphe magnus i s known to i nh ib i t tooth pulp responses in the medullary re t i cu la r formation (Lovic and Wolstencroft 1979). Reciprocal connection between NRM and dorsal column nuclei have also been suggested (Saade et a l . 1982; Jundi et a l . 1982). An exci tatory input to the NRM from the sensorimotor cortex has also been described (West and Wolstencroft 1978). - 24 -The raphe c e l l s are spontaneously act ive in decerebrate cats and are excited by noxious and innocuous st imul i applied to the i r receptive f i e l d (Anderson et a l . 1977). Auerbach et a l . (1985) have reported that in the conscious cat , presumed serotonergic neurones in the NRM showed slow, regular rates of discharge. The authors also reported that some of these neurones could be excited by cutaneous noxious pinch and noxious heat. However, these neurones did not spec i f i c a l l y respond to noxious st imul i as they could also be activated by non-noxious auditory and visual s t imu l i . Further, no di f ference was observed in the e f f i cacy of low in tens i ty and high in tens i ty st imulat ion of the i n fe r i o r a lveolar nerve to excite these neurones. The lower in tens i ty range used to stimulate the above nerve was adjusted to produce only Ae - f ib re ac t ivat ion whereas high in tens i ty st imulat ion of the i n fe r i o r alveolar nerve produced aversive behaviour in the animal. However, i t i s not c lear whether the presumed 5-HT neurones studied by the authors contributed to the raphe-spinal serotonergic f ibre system, though c e l l s with s imi la r charac ter i s t i cs have been shown to project to the spinal cord (Light 1981). Serotonin i s also released into the spinal cord on intense e l e c t r i c a l st imulat ion of peripheral nerves (Yaksh and Tyce 1981). Some of these f ind ings, namely (1) act ivat ion of presumed serotonergic neurones by noxious s t imu l i , and, (2) act ivat ion of raphe-spinal f ib res by noxious st imul i (some of which might be serotonergic) and release of 5-HT into the spinal cord on intense peripheral nerve st imulat ion, may suggest a neuronal substrate for a negative feedback loop involv ing - 25 -5-HT. Noxious information may, through structures l i k e the medullary re t i cu l a r formation (a major receiving area for pain transmission pathways in cat) stimulate the NRM to produce a i nh ib i t i on at the spinal 1eve l . Co-existence of 5-HT with enkephalins in neurons of the ventral NRM has been demonstrated (Glazer et a l . 1981), however the i r functional s ign i f i cance i s not known. Physiology of Descending Inh ib i t i on by St imulat ion of NRM in the Cat The NRM-spinal cord neuraxis described above i s one of the descending systems by which supraspinal centres exert control over the sensory information through the spinal cord. The control by the NRM on the a c t i v i t y of spinal cord dorsal horn neurones to peripheral noxious st imul i has been described by Fields et a l . (1977). Other supraspinal regions involved in descending control of dorsal horn neurones to noxious st imul i includes the periaqueductal gray (PAG; Liebeskind et a l . 1973), the nucleus g igantoce l lu la r i s (McCreery and Bloedel 1975) the red nucleus (Gray and Dostrovsky 1984), dorsolateral pons in the region of locus coerleus (Hodge et a l . 1983), the la te ra l r e t i cu la r nucleus (Morton et a l . 1983), the preoptic area and basal forebrain (Carstens et a l . 1983), the ventromedial septal area (Carstens et a l . 1982) and the l a te ra l tegmental f i e l d (Edeson and Ryall 1983). The supraspinal influence on the response of dorsal horn neurones to peripheral noxious st imul i can e i ther be tonic or phasic. The tonic control manifests as a powerful i nh ib i t i on of a c t i v i t y in WDR dorsal horn neurones. This tonic i nh ib i t i on of neuronal a c t i v i t y appears to be - 26 -se lec t ive for input from nociceptors vs . low threshold mechanoreceptors (LTM; Handwerker et a l . 1975; Soja and S inc l a i r 1983a). The anatomical l o c i for the or ig in of th i s tonic i nh ib i t i on appears to be at the level of brainstem, in the region of the la te ra l r e t i cu l a r nucleus. Lesions in th i s region reduces or abolishes the tonic descending i nh ib i t i on on WDR dorsal horn neurones (Hall et a l . 1982). Lesions in other brainstem areas including the NRM do not block th i s tonic i nh ib i t i on (Hall et a l . 1982). However, the NRM is involved in stimulation-produced (phasic) i nh i b i t i on of noxious stimuli-evoked a c t i v i t y in the dorsal horn neurones. For example, e l e c t r i ca l st imulation in NRM inhib i ted the response of dorsal horn neurones in laminae I, V and VI to high threshold mechanical st imulat ion (presumably in the noxious range) (F ie lds et al . 1977). Guilbaud et a l . (1977) reported a s imi lar i nh ib i t i on of evoked a c t i v i t y to pinch or intense e l e c t r i ca l st imulat ion in WDR interneurones in ca ts . These authors and Fields et a l . (1977) also reported that NRM st imulat ion produced a preferent ia l i nh ib i t i on of evoked a c t i v i t y of the dorsal horn neurones to noxious st imul i without af fect ing the response of these neurones to innocuous s t imu l i . But a wide body of evidence does not favour the spe c i f i c i t y of i nh ib i t i on on NRM st imulat ion. LTM and HTM cat spinothalamic neurones are both inh ib i ted by NRM st imulat ion (McCreery et a l . 1979). The non-noxious st imul i were more e f fec t i ve l y inh ib i ted when i t was applied for a sustained period of time rather than repe t i t i ve l y or randomly. Evoked a c t i v i t y in NS, WDR, and LTM spinocervical t ract neurones were inh ib i ted on st imulat ion of NRM - 27 -(Kajander et a l . 1984). S im i l a r l y , there was a lack of spec i f i c i t y in i nh ib i t i on of evoked a c t i v i t y in WDR laminae IV-V spinal cord interneurones in the cat to e i ther C-f ibre st imulat ion of the t i b i a l nerve or short a i r puffs to move hairs (Morton et a l . 1983). Gray and Dostrovsky (1983) showed a nonselective i nh ib i t i on of evoked a c t i v i t y in NS, WDR and LTM neurones having the same degree of afferent input. Thus a preponderance of evidence would suggest a nonspeci f ic , nonselective i nh ib i t i on of evoked a c t i v i t y in dorsal horn neurones by NRM s t imu la t ion . Evidence also ex ists that e l ec t r i ca l st imulat ion of the NRM decreases the spontaneous f i r i n g of cat dorsal horn neurones (Belcher et a l . 1978). This i nh ib i t i on of evoked and spontaneous a c t i v i t y i s by a f i b re system from the NRM which descends in the dorsolateral funiculus (DLF). DLF l es ions , but not ventro latera l les ions made rostra l to the s i t e of recording, reduced the ef fect in the cat (F ie lds et a l . 1977). This would be further substantiated by the anatomical study of Basbaum et a l . (1978) who found that f ibres from the NRM large ly descend in DLF to the spinal cord. There i s evidence that th i s descending inh ib i t i on can occur via a presynaptic s i t e of ac t ion. Martin et a l . (1979) reported that st imulat ion of the NRM in cats resul t in primary afferent depolar izat ion (PAD) of myelinated Aag- cutaneous f ibres innervating rapidly and slowly adapting receptors as well as A6- f ibres innervating hair f o l l i c l e mechanoreceptors and nociceptors. They tested terminal e x c i t a b i l i t y of s ingle sural nerve f i b re s , innervating care fu l l y characterized cutaneous - 28 -receptors. They concluded that st imulat ion of the NRM resul ts in nonselective PAD in a l l types of myelinated cutaneous af ferents . These ef fects would account for a nonselective depression of sensory inputs noted by some authors. Paradoxica l ly , NRM st imulat ion produces a hyperpolar izat ion of C-f ibres innervating polymodal nociceptors and C-mechanoreceptors and consequently the threshold for intraspinal (antidromic) exc i ta t ion for these f ibres i s increased (Hentall and Fie lds 1979). This would suggest an enhancement of transmitter release to peripheral st imulat ion of C-primary af ferents . Carstens et a l . (1981), however, suggests that presynaptic i nh ib i t i on of nociceptive C-afferents may occur by mechanisms other than those thought to occur for large af ferents . They point to the s im i l a r i t y of action of opiates and NRM st imulat ion both increasing the threshold for intraspinal act ivat ion of primary afferents and inh ib i t i ng dorsal horn neurones (Carstens et a l . 1979; Sastry 1979). Curt is et a l . (1983) questioned these f ind ings. They found that iontophoret ic 5-HT, the putative neurotransmitter involved in NRM act ion , increased the threshold for antidromic act ivat ion of Ia afferent terminals whereas GABA, K or tetanic st imulat ion of f lexor muscle afferents decreased the threshold for antidromic ac t i va t i on . They speculated that the 5-HT ef fect may not be related to presynaptic control of transmitter re lease. NRM st imulat ion may also produce postsynaptic i nh ib i t i on of dorsal horn neurones. Belcher et a l . (1978) reported that neuronal responses evoked by the excitant amino ac id , DL- homocysteic acid (DLH), was - 29 -reduced by NRM stimulation. Using intracellular recording techniques Giesler et al . (1981) reported that in f i v e of seven primate spinothalamic neurones tested, stimulation of the NRM produced a membrane hyperpolarization. They also found that NRM induced a block of antidromic action potentials in spinothalamic tract neurones. This evidence would support a postsynaptic inhibition of dorsal horn neurones by NRM stimulation. Finally, not all dorsal horn neurones are inhibited by electrical stimulation of NRM. Dubisson and Wall (1980) reported excitation of laminae I and II cells upon NRM stimulation. These cells responded to pressure, touch and brushing. Based on these findings Dubisson and Wall (1980) speculated that descending inhibition produced by electrical stimulation of the NRM may occur via an excitatory input to SG cells which, in turn, inhibit deep dorsal horn neurones. Pharmacology o f the Nucleus Raphe Magnus S t imu la t i on -P roduced I n h i b i t i o n  o f Dorsa l Horn Neuronal A c t i v i t y i n the Cat Essentially, three approaches have been used in elucidating whether 5-HT is involved in brainstem inhibition of spinal cord neuronal activity: (1) blocking synaptically released 5-HT with putative 5-HT antagonists, (2) selectively depleting 5-HT stores with p-chlorophenylalanine (PCPA), a tryptophan hydroxylase inhibitor or (3) increasing the concentration of synaptically released 5-HT using selective serotonergic uptake blockers. It has already been mentioned that the NRM is thought not to be - 30 -involved in tonic i nh ib i t i on of deep dorsal horn neuronal a c t i v i t y . Pharmacological evidence against a 5-HT involvement in tonic i nh ib i t i on comes from the work of Soja and S i n c l a i r (1980). Neither depletion of 5-HT stores by pretreatment with PCPA nor increasing the concentration of synapt ica l ly released 5-HT by f luoxet ine, a serotonin uptake i nh i b i t o r , affected the response of dorsal horn c e l l s to noxious st imul i in cats with spinal cord conduction intact or cold blocked. Since most of the spinal 5-HT i s located in the descending f ib re system or ig inat ing in the NRM, these observations and those mentioned ea r l i e r do not support a role for the NRM in tonic descending inh ib i t i on of deep dorsal horn WDR neurones. However, pharmacological manipulations suggest that 5-HT has a role in phasic i nh ib i t i on of deep dorsal horn ce l l s e l i c i t e d by NRM or PAG st imulat ion. PAG inh ib i t i on of fe l ine WDR dorsal horn neurones, which i s part ly relayed through the NRM, i s blocked by 5-HT antagonists, methysergide (Carstens et a l . 1981a; Barnes et a l . 1979) and LSD (Guilbaud et a l . 1973) and reduced by PCPA pretreatment (Carstens et a l . 1981a). The antagonism by methysergide i s reversed by intravenous 5-hydroxytryptophan (Barnes et a l . 1979). However, in studies by Guilbaud et a l . (1973) and Barnes et a l . (1979) a substant ia l increase in background and noxious stimulus evoked a c t i v i t y was seen after administrat ion of the 5-HT antagonists. It i s , therefore, not c lear whether the reduction in the degree of i nh ib i t i on was due to a blockade of synapt ica l ly released 5-HT at the terminals of the bulbospinal neurones or to these changes in background or evoked a c t i v i t y . - 31 -However, in experiments using methysergide, no reduction of i nh ib i t i on by NRM st imulat ion was seen. Methysergide was applied iontophoret ica l ly e i ther in the substantia gelatinosa (SG; Griersmith et a l . 1981) or on ce l l bodies of dorsal horn neurones (Belcher et a l . 1978) or administered intravenously (Griersmith et a l . 1981). Belcher et a l . (1978) also observed that methysergide inhib i ted exc i tatory responses to NRM st imulat ion. However, methysergide per se decreased the e x c i t a b i l i t y of the c e l l s and th i s may have influenced the observed r e su l t s . Another technique which has been adopted to unravel the role of bulbospinal 5-HT neurones i s the use of iontophoresis. Randic and Yu (1976) examined neurones in laminae I and II of the decerebrate cat which were excited by noxious cutaneous s t imu l i . Iontophoretic 5-HT depressed 70% of these neurones examined with the depression las t ing up to 10 min. When applied in the SG, 5-HT se lec t i ve l y reduced responses of dorsal horn laminae IV-V neurones to peripheral noxious st imul i (Headley et a l . 1978). It also pre fe rent ia l l y inh ib i ted evoked a c t i v i t y in NS neurones without af fect ing a c t i v i t y in LTM neurones when applied in the v i c i n i t y of the ce l l bodies (Belcher et a l . 1978). These authors found, in add i t ion, that 5-HT iontophoret ica l ly released onto ce l l bodies of WDR neurones nonselect ively inh ib i ted the i r evoked a c t i v i t y . Thus, evidence would favour a se l e c t i v i t y of action of 5-HT depending on the region of the spinal cord to which i t i s applied as well as the ce l l type. Interest ing ly , the effect of 5-HT in the SG i s reversed by methysergide iontophoresed into the SG pr ior to or - 32 -concurrently with 5-HT administrat ion but not when applied after 5-HT iontophoresis (Griersmith and Duggan 1980). These authors suggested that 5-HT may have a modulatory role such that , once the ef fect of 5-HT i s i n i t i a t e d , other changes may take place which go beyond a simple drug-receptor interact ion and, therefore, methysergide can prevent but not reverse 5-HT e f fec t s . Davies and Roberts (1981) suggested that neuromodulation may occur at substance P receptors since neuronal exc i ta t ion by substance P was antagonized by iontophoretic 5-HT, an ef fect only weakly antagonized by c inanser in , a putative 5-HT antagoni s t . However, not a l l neuronal responses are reduced or inhib i ted by iontophoretic 5-HT. Iontophoretic 5-HT was found to increase the spontaneous or excitatory amino acid induced neuronal f i r i ng in the spinal cord dorsal horn (Belcher et a l . 1978; Todd and M i l l a r 1983). Todd and M i l l a r (1983) examined the effect of iontophoretic 5-HT on interneurones located in laminae I, II and III of the cat spinal dorsal horn. The interneurones were characterized by the i r responsiveness to peripheral natural st imulation as LTM, HTM or WDR interneurones. Iontophoretic 5-HT excited 68% of the units examined and exc i tat ion was observed in a l l three classes of interneurones. The authors suggested that these interneurones were inh ib i to ry to deep dorsal horn neurones. Thus exc i ta t ion of these SG interneurones by 5-HT would i nh i b i t a c t i v i t y in the deep;, dorsal horn c e l l s . Indeed, Headley et a l . (1978) have shown that 5-HT, iontophoresed into the SG, inh ib i t s deep dorsal horn neurones. If 5-HT i s important in mediating the - 33 -i nh i b i t i on of deep dorsal horn neuronal a c t i v i t y upon NRM st imulat ion, i t i s possible that i t does so by exc i t ing inh ib i to ry interneurones in the super f i c ia l laminae of the dorsal horn. This idea would be supported by the work of Dubisson and Wall (1980) who observed that st imulat ion of the NRM excited laminae I and II interneurones of the cat spinal cord dorsal horn. The c e l l s studied by th i s group were character ized as being responsive to peripheral noxious s t imu l i . On the other hand, M i l e t i c et a l . (1984) reported that st imulat ion of the NRM markedly suppressed the response of the laminae I and II interneurones of the dorsal horn to peripheral noxious st imulat ion. The neurones which were inh ib i ted also had considerable 5-HT l i k e immunoreactive contacts onto the i r dendr i tes. However, the authors did not attempt to study whether appl icat ion of 5-HT on these neurones mimicked the ef fect of the NRM st imulat ion. Pharmacology of Morphine on the Deep Dorsal Horn WDR Neurones TONIC DESCENDING INHIBITION: INVOLVEMENT OF ENDOGENOUS OPIOID PEPTIDES  AND THE EFFECT OF MORPHINE: Whether the endogenous opioid peptides mediate the tonic supraspinal i nh ib i t i on of nociceptive a c t i v i t y of deep: doral horn WDR neurones has been investigated using opiate antagonists. The rat ionale used in these studies i s that , i f the endogenous opioid peptides mediate the tonic descending inh ib i t i on of these neurones, then administration of an opiate antagonist would be expected to reduce th i s i n h i b i t i o n . Duggan et a l . (1977a) reported that - 34 -intravenous administrat ion of the opiate antagonist, naloxone (1.3 - 3.2 mg/kg) fa i l ed to a l te r the tonic supraspinal i nh ib i t i on impinging on these neurones. The tonic i nh ib i t i on was measured as the increase in noxious stimuli-evoked response of a neurone during the cold block state of the spinal cord. A decrease in the tonic supraspinal i nh ib i t i on would be expected to result in a smaller increase in the neuronal f i r i n g rate to noxious st imul i in the cold block state of the spinal cord. Furthermore, one would also expect to see an increase in the spontaneous f i r i n g of these neurones in the warm, intact state of the spinal cord. Naloxone, however, fa i led to produce e i ther of these changes. Thus, these observations would suggest that endogenous opioid peptides do not mediate the tonic descending supraspinal inh ib i t i on impinging on the deep dorsal horn WDR neurones. The same conclusion was reached by S inc l a i r et a l . (1980) based on a s imi lar study. The ef fect of morphine on the tonic descending inh ib i t i on of the dorsal horn WDR neurones has also been invest igated. For example, Duggan et a l . (1980) have reported that intravenous morphine (0.5 - 1.0 mg/kg), in anaesthetized cats , reduced the tonic descending i nh ib i t i on of dorsal horn neurones excited by impulses in C-f ibre af ferents . The extent of tonic i nh ib i t i on on these neurones was measured as explained in the preceding paragraph. A decrease in the di f ference in the evoked neuronal discharge with the spinal cord in the normal vs cold-blocked state was taken as ind ica t i ve of a decrease in the tonic i n h i b i t i o n . However, Soja and S inc l a i r (1983) have questioned these f ind ings. These authors have suggested that the decrease in the di f ference of the evoked - 35 -discharge, as measured by Duggan et a l . 1980, was due to a reduction in the e x c i t a b i l i t y of the neurones in the presence of morphine. Jurna and Grossman (1976) have also suggested that intravenous morphine decreased the tonic i nh ib i t i on on ventrolateral t ract axons excited by A6- and C-f ibre inputs. They observed that intravenous morphine reduced the A6- and C-f ibre evoked a c t i v i t y in the cold block state of the spinal cord but increased the a c t i v i t y in these axons in the normal, warm state of the spinal cord. The authors suggested that the inh ib i to ry ef fect of morphine on the axonal a c t i v i t y in the cold blocked state of the spinal cord was due to a spinal action of morphine, whereas, in the warm, intact state of the spinal cord morphine depressed the tonic descending i nh ib i t i on on these axons which masked the spinal inh ib i to ry act ions . The above experiments do not provide conclusive evidence that morphine has an effect on the tonic descending inh ib i t i on impinging on the deep dorsal horn neurones and the ascending axons in the anterolatera l quadrant of the spinal cord. The resu l ts obtained by Duggan et a l . (1980) are complicated by a change in the e x c i t a b i l i t y of the neurones. The experiment of Jurna and Grossman (1976) also does not provide d i rec t evidence of an ef fect of morphine on tonic descending i n h i b i t i o n . The exc i tatory ef fect of morphine on the ascending axons of the spinal cord might have resulted by an exc i ta t ion of supraspinal f a c i l i t a t o r y inputs to these axons or a combination of morphine's ef fect on the tonic descending inh ib i t i on and f a c i l i t a t o r y inputs to these axons. - 36 -INTRAVENOUS MORPHINE ON NOXIOUS STIMULUS-EVOKED RESPONSES OF THE DEEP DORSAL HORN WDR NEURONES Normal, in tact state of the Spinal Cord: Conf l i c t ing reports have appeared on morphine's ef fect on dorsal horn WDR neuronal a c t i v i t y when using anaesthetized or decerebrated cat preparations in which the spinal cord was i n tac t . For example, Soja and S inc l a i r (1983b) reported that , in chloralose anaesthetized cats , morphine (1.0 mg/kg, tota l dose) decreased the noxious heat evoked a c t i v i t y in dorsal horn WDR neurones. S imi lar ef fects have been reported by Belcher and Ryall (1978) and Davies and Dray (1978). This l a t t e r group also reported that the inh ib i to ry effect of morphine was reversed by intravenous naloxone. Using decerebrate ca ts , Hanaoka et a l . (1978) observed that the nociceptive response of dorsal horn lamina V NS neurones was decreased by morphine in a dose dependent manner (morphine 0.5 - 2.0 mg/kg, i . v . ) . However, other authors have reported a lack of ef fect or var iable ef fects of intravenous morphine on natural noxious stimuli-evoked responses of the deep dorsal horn neurones. Thus, Le Bars et a l . (1976b) observed a lack of ef fect of morphine (2.0 mg/kg, i . v . ) on noxious pinch evoked responses in WDR, lamina V neurones in the decerebrate cat . The authors suggested that th i s lack of ef fect of morphine might be due to an exaggerated descending tonic i nh ib i t i on observed on these neurones which might mask the effect of morphine. However, Jurna and Grossman (1976) suggested an alternate explanat ion. Also using the decerebrate cat preparation, they observed that morphine - 37 -(0.5 - 2.0 mg/kg, i . v . ) produced no c lear effect on f i r i n g of ventro latera l t ract axons to sural A6- and C-f ibre s t imulat ion. They postulated that morphine might reduce the tonic descending inh ib i t i on on the spinal dorsal horn neurones and thereby release the a c t i v i t y in the i r ascending axons, but at the same time i t might i nh i b i t the a c t i v i t y of these axons by i t s spinal inh ib i to ry ef fect on the f i r i n g of the ce l l body of or ig in of these axons. These counter action of morphine might balance each other so that no clear response to morphine i s seen. That morphine might se lec t i ve ly reduce the nociceptive responses on the deep dorsal horn neurones has been suggested by Belcher and Ryall (1976). They reported that morphine, reduced responses to noxious st imul i of these c e l l s but did not ef fect a c t i v i t y in non-nociceptive c e l l s to peripheral innocuous s t imu l i . S imi lar observations have been made by Davies and Dray (1978). F i na l l y , morphine (0.3 - 1.6 mg/kg, i . v . ) also reduces the C-f ibre. stimulation-evoked responses of dorsal horn laminae IV-VII WDR neurones (Duggan et al . 1980). This ef fect of morphine was reversed by naloxone administered intravenously. Transected state of the spinal cord: In anaesthetized or decerebrate cats with the spinal cord transected, the effect of intravenous morphine on the noxious stimulus-evoked a c t i v i t y of WDR, NS and HTM neurones have been cons istent ly reduced by intravenous morphine (Kitahata et a l . 1974; Le Bars et a l . 1975; Zieglgansberger and Bayerl 1976; Hanaoka et al . 1978; Davies and Dray 1978; C a l v i l l o et al . 1979; - 38 -Einspahr and Piercey 1980; Piercey et a l . 1980; Sastry and Goh 1983). In the study by Ca l v i l l o et a l . (1979), the inh ib i to ry ef fect of morphine on noxious heat-evoked responses in the laminae IV-V c e l l s was also mimicked by other opiate analgesics, namely, meperidine and fentany l . In a few studies mentioned above, the inh ib i to ry effect of morphine on nociceptive responses of dorsal horn WDR neurones was shown to be reversed by the intravenous administrat ion of the narcotic antagonist, naloxone (Le Bars et a l . 1975; Zieglgansberger and Bayerl 1976; C a l v i l l o et a l . 1979; Piercey et a l . 1980; Einspahr and Piercey 1980). Zieglgansberger and Bayerl (1976) and Ca l v i l l o et a l . (1979) also iontophoresed naloxone near the ce l l being recorded in the few instances. Again naloxone reversed the inh ib i to ry ef fects of morphine. The experiments quoted in the preceding paragraph would suggest the fo l lowing: (1) morphine has a spinal s i te of action where i t can act to i nh i b i t the noxious stimulus-evoked responses in the dorsal horn WDR, NS and HTM neurones, and (2) the ef fect of morphine on dorsal horn neuronal f i r i n g might be mediated v ia an opioid receptor since i t can be reversed by the opiate receptor antagonist, naloxone. However, intravenous morphine may not se lec t i ve ly i nh i b i t nociceptive responses of deep dorsal horn WDR neurones in the spinal cat preparat ion. For example, Einsphar and Piercey (1980) observed a reduction of both the noxious and non-noxious stimulus evoked responses. These effects of morphine were reversed by intravenous naloxone. Morphine, however, did not affect the evoked responses of the neurones which could be excited only by non-noxious s t imu l i . - 39 -In the spinal cat preparation intravenous morphine also reduced the la te component of the A6- and C-f ibre mediated discharge evoked in WDR neurones by e l e c t r i c a l st imulat ion of peripheral nerves (Le Bars et a l . 1976a; Le Bars et a l . 1975; Johnson and Duggan 1981). This ef fect of morphine was naloxone revers ib le . The early component of the discharge mediated by Ag-f ibre exc i ta t ion was unaffected by morphine (Le Bars et a l . 1975; Le Bars et a l . 1976a). Intravenous morphine in the spinal cat preparation also reduced the A6- and C-f ibre st imulat ion evoked responses in the ascending ventrolateral t ract axons (Jurna and Grossman 1976). Again, th i s ef fect of morphine was reversed by intravenous naloxone. IONTOPHORETIC ADMINISTRATION OF MORPHINE Morphine has been iontophoret ica l ly applied in the spinal cord dorsal horn in the fol lowing manner: (1) in the v i c i n i t y of the ce l l body of the deep dorsal horn neurone being recorded, or (2) in the SG region of the dorsal horn while recording from a deep dorsal horn neurone. When iontophoresed in the v i c i n i t y of the ce l l body, morphine has produced inconsistent ef fects on the noxious stimuli-evoked a c t i v i t y . For example, Ca l v i l l o et a l . (1974 and 1979) reported that the narcot ics morphine, meperidine and fentanyl reduced the noxious heat and pinch-evoked discharge in the majority of the neurones. S im i l a r l y , Belcher and Ryall (1978) and Zieglgansberger and Bayerl (1976) reported that morphine and levorphanol depressed the nociceptive resonses in most neurones studied. The ana lges ica l ly inact ive isomer of levorphanol, - 40 -dextrorphan, was found to have no or a very weak effect on the nociceptive responses of these deep dorsal horn neurones. This i nh ib i to ry effect of morphine was reversed by iontophoretic naloxone administered near the recording s i t e (Ca l v i l l o et a l . 1974; Belcher and Ryall 1978; Zieglgansberger and Bayerl 1976). Contrary to the above reports, morphine, when iontophoresed near the region of the ce l l bodies, was reported to excite rather than i nh i b i t the noxious st imul i evoked responses in the deep dorsal horn WDR neurones (Duggan et a l . 1977; Davies and Dray 1978; Piercey et a l . 1980). This affect of iontophoretic morphine was naloxone i n sens i t i ve . It has been suggested that th i s lack of uniformity of effect of morphine might result from a l im i t a t i on of the iontophoretic technique (Duggan and North 1984). These authors have suggested that the receptors of importance for morphine action might be only sparsely present near the ce l l body. Whether iontophoretic morphine has a s i t e of action other than in the region of the ce l l body of the deep dorsal horn neurones has also been invest igated. For example, Duggan et a l . (1977), Davies and Dray (1978), and Sastry and Goh (1983) observed that iontophoretic appl icat ion of morphine in the region of the SG of the spinal cord reduced the peripheral noxious st imul i evoked-responses in the majority of the dorsal horn WDR neurones. This effect of morphine was reversed or reduced by simultaneous or pr ior iontophoresis of naloxone in the SG (Duggan et a l . 1977; Davies and Dray 1978). Intravenous naloxone (0.1 mg/kg) also antagonized the ef fects of the iontophoretic morphine (Duggan et a l . 1977). - 41 -When iontophoresed in the region of the SG morphine has a se lect ive action in that i t depresses the nociceptive responses of the deep dorsal horn WDR neurones without af fect ing the innocuous stimuli-evoked responses of these neurones (Duggan et a l . 1977; Davies and Dray 1978). S imi lar s e l e c t i v i t y of morphine action has also been suggested when i t i s iontophoresed near the region of the ce l l body of deep dorsal horn neurones. For example, Ca l v i l l o et a l . (1979) reported that morphine fa i l ed to affect the response of these units to innocuous peripheral s t imu l i , whereas, the nociceptive responses were reduced. But Zieglgansberger and Bayerl (1976) observed that morphine depressed both the innocuous and noxious stimuli-evoked responses in these neurones. The non-noxious stimulus evoked responses in the LTM units was e i ther unaffected (Ca l v i l l o et a l . 1979; Zieglgansberger and Bayerl 1976), or enhanced (Belcher and Ryall 1978). The possible mechanism by which morphine a l ters the nociceptive responses of the deep dorsal horn WDR neurones has also been invest igated. Both postsynaptic and presynaptic mediated effects have been suggested. Zieglgansberger and Bayerl (1976) noted that iontophoretic morphine, applied in the v i c i n i t y of the ce l l body, led to a reduction in the rate of r i se of the EPSP recorded i n t r a c e l l u l a r l y . This occurred in the absence of detectable changes in the membrane potential or conductance of the ce l l membrane. The invest igators proposed that a reduction in the rate of r i se of the EPSP would reduce f i r i n g to exc i tatory inputs impinging on these neurones. They also noted that morphine applied in th i s manner reduced depolar izat ions by - 42 -L-glutamate and that intravenous morphine (2.0 mg/kg) produced a hyperpolar izat ion in 50% of the c e l l s tested. These observations would suggest a postsynaptic mechanism through which morphine exerts i t s suppressive ef fects on the deep dorsal horn neurones. This suggested postsynaptic mechanism of action of morphine i s strengthened by the observation made by other workers that morphine decreased the exc i ta t ion of the deep dorsal horn neurones by L-glutamate (Ca l v i l l o et a l . 1974 and 1979; Dostrovsky and Pomeranz 1976; Belcher and Ryall 1978; Piercey et a l . 1980). The neurones tested by these authors were recorded ex t race l l u l a r l y and morphine and L-glutamate were applied iontophoret ica l ly in the v i c i n i t y of the ce l l body of the neurone tested. However, the depressive effect of morphine in these experiments was e i ther potentiated or unaffected by naloxone iontophoresed at the same s i t e (Belcher and Ryall 1978; Piercey et al . 1980). Furthermore, intravenous morphine fa i led to reduce the exc i tatory amino acid induced f i r i ng of these neurones (Dostrovsky and Pomeranz 1976; Piercey et a l . 1980). These l a t t e r observations do not support the suggestion that morphine exerts a suppressive effect through a postsynaptic mechanism. A presynaptic depressive effect of morphine on the noxious st imul i evoked a c t i v i t y in the deep dorsal horn neurones has also been suggested. For example, in decerebrate, spinal cord sectioned ca ts , morphine was applied iontophoret ica l ly near the terminals of cutaneous sural nerve A6- and C-f ibres in the super f i c ia l laminae of the dorsal horn or administered systemica l ly . The terminal e x c i t a b i l i t y of these - 43 -f ib res was decreased but the PAD of these f ibres produced by st imulat ion of small diameter afferents of the super f i c ia l peroneal nerve was enhanced (Sastry 1978 and 1979). Sastry (1979) proposed that morphine decreased the transmission of impulses from small diameter primary afferents to dorsal horn neurones by an enhancement of the presynaptic i nh ib i t i on that these f ibres exert on each other. The decrease in e x c i t a b i l i t y during morphine appl icat ion was interpreted as being due to hyperpolar izat ion of terminals and th i s was considered responsible for the observed increase in PAD. SUPRASPINAL MEDIATED EFFECTS OF MORPHINE ON THE DEEP DORSAL HORN WDR  NEURONAL ACTIVITY: The ef fects of morphine on the tonic descending i nh ib i t i on of dorsal horn WDR neurones has already been considered. It i s su f f i ce to mention here that a decrease of the tonic inh ib i t i on by intravenous morphine had been suggested (Jurna and Grossman 1976; Duggan et a l . 1980; but see Soja and S i n c l a i r 1983b). This effect of morphine was suggested to be mediated by a supraspinal s i te of action of morphine (Jurna and Grossman 1976). However, other authors have suggested that morphine might act ivate descending pathways that i nh ib i t noxious stimuli-evoked discharges in the deep, dorsal horn WDR neurones. For example, Hanaoka et a l . (1978) observed that intravenous morphine produced a greater percentage decrease of the nociceptive responses of the dorsal horn neurones during normal spinal cord conduction than when the spinal cord was transected. But, Soja and S inc l a i r (1983b) questioned these resul ts and suggested that the dif ference in the - 44 -percentage i nh ib i t i on of the dorsal horn neuronal a c t i v i t y by intravenous morphine in the intact vs transected state of the spinal cord might have resulted from differences in the control values for neuronal response in these d i f ferent state of the spinal cord. They further suggested that the inh ib i to ry ef fect of intravenous morphine (1.0 mg/kg) on the dorsal horn nociceptive a c t i v i t y could be explained by a spinal s i t e of action of the drug. S i nc l a i r (1985) has investigated the effect of perfusing morphine between the th i rd vent r i c l e and the c isterna magna on the nociceptive a c t i v i t y of the dorsal horn neurones. Concentration of 10- 5 M and 10" 4 M morphine were without ef fect on the nociceptive a c t i v i t y of the dorsal horn neurones. Higher concentration of perfused morphine ( IO - 3 M) resulted in an increase in the spinal cord dorsal horn neuronal responses to peripheral noxious s t imu l i . But th i s ef fect of morphine may have been nonspecif ic since i t was not usual ly reversed by naloxone. The resul ts of th i s study do not support the hypothesis that morphine attenuates nociceptive a c t i v i t y of the dorsal horn neurones through a supraspinal mechanism. Other approaches have been used to test whether morphine attenuates spinal cord nociceptive transmission through a supraspinal s i t e of ac t ion . Le Bars et a l . (1976b) studied the effects of intravenous morphine (2.0 mg/kg) on the NRM stimulation-evoked inh ib i t i on of deep dorsal horn neuronal nociceptive a c t i v i t y in decerebrated cats . Morphine was found to be i ne f f ec t i ve . Du et a l . (1984) microinjected morphine (10-20 ug) into the NRM of anaesthetized cats while recording - 45 -the a c t i v i t y of deep dorsal horn neurones to noxious peripheral s t imu l i . The result was a suppression of noxious heat-evoked a c t i v i t y of the dorsal horn WDR neurones. The authors suggested that morphine act ivates a descending inh ib i to ry system from the NRM to suppress nociceptive transmission at the spinal cord l e v e l . But the dose of morphine microinjected in the above experiment by Du et a l . (1984) might not be relevant to ant inocicept ive mechanism. Clark et a l . (1983) have calculated that micro inject ion of 10 pg of morphine at a brainstem s i t e would y i e ld morphine concentrations at a distance within 2 mm of the in ject ion s i te which would be far in excess of concentrations necessary to produce antinociception by systemic administration of the drug. Role of 5-HT in Morphine Action There i s evidence that brainstem s i t e s , espec ia l ly NRM and PAR may mediate morphine analgesia. For example, les ion of the NRM can block the increase in the t a i l - f l i c k latency seen with systemic morphine (Proudfit and Anderson 1975; Yaksh et a l . 1977). Micro inject ion of morphine into the NRM produced an increase in the threshold for nociception-induced voca l i zat ion in rats (Dickenson et a l . 1979). These experiments suggest that the NRM might be involved in mediating the analgesic ef fects of morphine. Also i t i s known that f ibres from the NRM descend to the spinal cord and some of these f ibres are serotonergic. These f ibres may mediate NRM st imulat ion induced behavioral analgesia by release of 5-HT into the spinal cord (Hammond and Yaksh 1984). This, and also the fact - 46 -that NRM has a postulated role in morphine ant inoc icept ion, has resulted in considerable research in invest igat ing the role of 5-HT in morphine ant inoc icept ion. Systemical ly injected morphine produced an increase in spinal dorsal horn leve ls of 5-hydroxyindole acet ic acid (5-HIAA), a metabolite of 5-HT (Shiomi et a l . 1978). Spinal transect ion blocked the elevat ion of 5-HIAA e l i c i t ed by morphine below the level of les ion without a l ter ing the elevation above the l e s i on . This suggested that the increase in leve ls of 5-HIAA acid was due to the supraspinal act ion of morphine, possibly at the NRM locus which i s the major source of spinal 5-HT. The increase in the spinal dorsal horn leve ls of 5-HIAA might be due to increased turnover of 5-HT by morphine. It i s possible that increase release of 5-HT in the spinal cord dorsal horn by morphine might mediate i t s ant inocicept ive act ion. Micro inject ion of morphine into the NRM of the rats produced antinociception which could be attenuated by depletion of spinal cord 5-HT with 5,7-dihydroxytryptamine (Vasko et a l . 1984). In the presence of intrathecal monoamine uptake blockers, am i t r i p t y l i ne , desipramine and se r t r a l i ne , subthreshold doses of parenteral morphine produced antinociception (Taiwo et a l . 1985). Depletion of 5-HT by PCPA pretreatment or intrathecal administration of the putative 5-HT antagonist, methysergide, prevented the ant inoc icept ive effect of subthreshold dose of morphine in presence of uptake blockers. When given parentera l ly , f luoxet ine, a se lect ive 5-HT uptake blocker, also potentiated the analgesic effect of parenteral morphine in rat t a i l - f l i c k test (Larson & Takemori 1977). Depletion of 5-HT v ia systemic pretreatment with PCPA (Tenen 1968) or - 47 -in t ravent r i cu la r 5,6-DHT (Vogt 1974) antagonized the increase in t a i l - f l i c k latencies produced by systemic morphine. However, intrathecal methysergide, in the absence of any 5-HT uptake blockers, f a i l ed to block the increase in t a i l - f l i c k latency produced by systemic morphine (Proudfit and Hammond 1981). Pharmacology of Fluoxetine and Pargyl ine Serotonin released in synaptic transmission i s mainly inact ivated by high a f f i n i t y uptake into presynaptic terminals (Shasken and Snyder 1970). Brain synaptosomes have been e f fec t i ve l y used for studying the uptake mechanism of 5-HT and i t s pharmacological manipulation. For example, Wong et a l . (1974), using rat brain synaptosomes, have studied the effect of f luoxet ine on 5-HT uptake in th i s preparation. Fluoxetine was found to produce an i nh ib i t i on of 5-HT uptake in the synaptosomal preparat ion. The uptake inh ib i t i on was found to be se lect ive for 5-HT. The i nh ib i to r constant (K-j) of f luoxet ine was 5 x 1 0 - 8 M for 5-HT in contrast to 1.5 x 10~5 M and 1.0 x 1 0 - 5 M for dopamine and noradrenaline, respect ive ly . Fu l le r et a l . (1975) compared equal doses of several 5-HT uptake blockers with f luoxet ine, for the i r re l a t i ve a b i l i t i e s to antagonize the depletion of 5-HT in the rat brain synaptosomes by 4-chloroamphetamine (CPA). Fluoxetine completely antagonized the action of CPA whereas chlorimipramine and imipramine had no e f fec t . Further, f luoxet ine antagonized the H75/12 induced depletion of 5-HT but not noradrenaline. In, in vivo experiments in ra t s , - 48 -f luoxet ine decreased 5-HIAA leve ls in the brain which i s suggestive of 5-HT uptake blockade (Fu l le r and Perry 1974). This ef fect of f luoxet ine occurred in the absence of any i nh ib i t i on of monoamine oxidase system. Further, f luoxet ine has no a f f i n i t y for serotonergic, alpha-adrenergic, muscarinic and histaminergic receptors (Wong et a l . 1983) and therefore i t s uptake block of 5-HT might not be mediated through any receptors. In vivo uptake block of 5-HT in the rats by f luoxet ine has been suggested because of f luoxet ine 's a b i l i t y to decrease 5-HIAA concentration in bra in , antagonize serotonin depletion of the brain by CPA treatment and potentiate ef fects of the serotonin precursor (Fu l le r 1982). Fluoxetine has been used in analgesic experiments. For example, Messing et a l . (1975) studied the effect of f luoxet ine on foot shock thresholds in ra t s . It was observed that , in rats pretreated with f luoxet ine , the threshold currents required to produce a jump response was elevated compared to control animals. The analgesic effect of f luoxet ine in t a i l - j e r k test has also been reported (Hynes et a l . 1985). In contrast , f luoxet ine had no s ign i f i can t ef fect on hot plate react ion times in rats (Malec and Langwinski 1980). A l l three groups, however, demonstrated a potent iat ing effect of f luoxet ine on morphine-induced ant inoc icept ion. Fluoxetine produced th i s ef fect without a l te r ing the a f f i n i t y of morphine for i t s receptor (Hynes et a l . 1985). This i s consistent with the hypothesis that an endogenous serotonergic system might play an important role in morphine - 49 -ant inoc icept ion. Pargyl ine i s an i r reve rs ib l e i nh ib i to r of monoamine oxidase (Taylor et a l . 1960). It produces a rapid and long- last ing accumulation of monoamines in the brain (Spector et a l . 1960; Pletscher et a l . 1961). Further, i t has been reported to produce a rapid accumulation of monoamine, espec ia l ly 5-HT, in the cat spinal cord (Anderson et a l . 1967). This ef fect of pargyline on spinal 5-HT leve ls i s suggested to be responsible for the enhanced spinal cord monosynaptic ref lex in cats (S i nc l a i r and Sastry 1974). Whether pargyline has any analgesic action i s not known. It i s also not clear whether pargyline treatment can potentiate the ant inocicept ive effect of f luoxet ine . However, the potent iat ion of f luoxet ine effect on body temperature by treatment with a monoamine oxidase inh ib i to r (MA0I) has been demonstrated in rabbits (S inc la i r and Lo 1977). The authors suggested that t h i s was due to great ly enhanced 5-HT a c t i v i t y due to neuronal uptake block by f luoxet ine in MA0I pre-treated animals compared to a lesser ef fect of f luoxet ine on 5-HT a c t i v i t y in animals not pre-treated with MAOI. - 50 -METHODS AND MATERIALS Surgical Procedures These experiments were carr ied out in a Faraday radio frequency shielded room on adult cats of e i ther sex. The animals were i n i t i a l l y anaesthetized with a 3% halothane (Fluothane®)/oxygen mixture which was del ivered at a rate of four L/min via a gas anaesthetic machine (Ohio). To monitor blood pressure, a carot id artery was cannulated with a polyethylene tubing (No. 160) which was p re f i l l ed with d i l u te sodium heparin (Upjohn Co., Canada). A cephal ic vein was cannulated with No. 90 polyethylene tubing p r e f i l l e d with sa l ine for intravenous administrat ion of drugs. In every experiment, a-chloralose (60 mg/kg, i . v . , Sigma Chemical Co.) was injected over a 10 min period while the halothane was slowly reduced to a concentration of 1.0% and maintained during surgery. Adequacy of anaesthesia was determined by a lack of sudden blood pressure or pup i l l a ry diameter change during noxious s t imulat ion. Supplementary doses of chloralose were administered as required. The trachea was exposed and cannulated with a ribbed polyethylene Y-shaped hose connector for a r t i f i c i a l resp i ra t ion . The animals were f i rmly secured to a stereotaxic animal frame (Narashigi S c i en t i f i c Instruments Labs.) , the hindlimbs were shaved and fastened with gypsum to an adjustable wooden platform with the toe pads up. The lumbosacral region of the spinal cord was care fu l l y exposed by performing a laminectomy on the l\-S1 segments. The dura mater was cut - 51 -and pinned back to surrounding t issue in a hammock-like fashion, gently suspending the spinal cord. In some experiments the l e f t L 7 dorsal root was iso lated and mounted intact on a s i l ve r bipolar hook electrode which was connected to one channel of an Ortec (Model 4600 Series) or a Grass (SD-9 Model) s t imulator . A small hole was made in the skul l above the cerebellum through which the supraspinal st imulat ion electrode was inser ted . The animals were paralyzed with gallamine t r i e th iod ide (Flaxedil®, Poulenc) and a r t i f i c i a l l y respired using a respirator (Harvard Apparatus). End-tidal C0 2 leve ls were continuously monitored and maintained between 3.5 and 4.5% using a cal ibrated medical C0 2 gas analyzer (Beckman, Model LB-2). A b i l a te ra l pneumothorax was rout inely performed so as to minimize respirat ion-induced spinal cord movement during recording. The ent ire preparation was iso lated from the f loor by i n f l a t i ng the supporting table (N 2 40 p s i , Zero-G Iso lat ion Table). This " f l oa t ing" table minimized inherent f loor v ibrat ions from being transmitted through the animal frame and microcarr ier to the recording e lectrode. S t i m u l a t i o n , Record ing and Tes t i ng o f Dorsa l Horn Neurones Using a stereoscope (Olympus) and a pair of f ine watchmaker's forceps, a small hole was care fu l l y made in the pia mater at the l e f t L 7 dorsal root entry zone. The recording microelectrode (glass or carbon f ibre) was secured to a hydraul ic microdrive holder (David Kopf Instruments), which in turn was mounted onto a f ine adjusting electrode - 52 -ca r r i e r (Narashig i) . The electrode was then placed perpendicular to the spinal cord surface over the pial opening and lowered by remote control in 10 um steps. The signal picked up by the recording microelectrode was fed into a high impedance preamplif ier (M707, W.P. Instruments), amplif ied and subsequently displayed on a s p l i t beam storage osc i l loscope (Tektronix, Model 5111). The output of the osc i l loscope was fed to a window d iscr iminator , audio monitor and spike integrator , whose output, in tu rn , was connected to a polygraph (Grass 79D) and microcomputers (Rockwell Aim-65, Apple II p lus ) . A search stimulus (1.5 V, 0.1 ms, 1.0 Hz) was applied to the L 7 dorsal root. Neurones encountered were c l a s s i f i ed according to t he i r response to natural forms of st imul i and general ly, they f e l l into two categor ies. One type responded to l i gh t mechanical s t imulat ion, such as touch or hair movement of the receptive f i e l d . A second type responded to these st imul i and, in add i t ion, they responded with a sustained discharge to a maintained pinch or a 10-15 sec pulse of noxious radiant heat. These neurones were term low threshold mechanoreceptive (LTM) and WDR neurones, respect ive ly . The l a t t e r group of neurones were used in th i s study. Noxious heat was applied using a quartz halogen projector lamp focused on the receptive f i e l d of WDR neurones studied. The noxious temperature was set at 45-55°C and applied for 10-15 sec. A miniature thermocouple, placed on the receptive f i e l d was used to feed back control the temperature appl ied. In a l l experiments noxious radiant heat was applied automatical ly using a microcomputer at two - 53 -minute i n t e r va l s . The temperature and duration of noxious radiant heat was ca re fu l l y adjusted for each neurone so as to evoke reproducible responses. Suprasp ina l S t imu l a t i o n A bipolar electrode was used to stimulate the NRM. The stereotaxic co-ordinates chosen were, AP = -7, DV = -10 and L = 0. The electrode was inserted in the sag i t ta l plane at an angle of 14° from v e r t i c a l . The nucleus was stimulated by pulse t ra ins of 333 msec length at 100 Hz for 25 sec. The st imulat ion in tens i ty was adjusted to produce about 50% inh ib i t i on of the neuronal nociceptive a c t i v i t y . The st imulat ion voltage used in the study ranged from 2.5 v to 7.0 v. In a l l experiments NRM st imulat ion was applied automatical ly, using a micro-computer, at four minute in te rva l s ; the onset being at the start of the heat pulse. P repa ra t i on o f Record ing M i c roe l e c t r odes Microelectrodes were constructed from glass c ap i l l a r i e s with 1.5 mm outside diameter (Glass Co. of America, Omega Dot Brand). They were pul led on a ver t i ca l electrode pul ler (Narashig i ) . Under a microscope the t i p diameters were broken back to 1.0 - 1.5 ym and the electrode was then f i l l e d by cap i l l a r y action with a 4 M NaCl so lu t ion . Most electrodes used had a DC resistances of 15 megohms or l e s s . Carbon f ib re electrodes were prepared according to the method of Armstrong-James and M i l l a r (1979). A carbon f ib re of su i tab le length - 54 -was inserted into a glass cap i l l a r y (as used above) and pulled on the ver t i ca l electrode pu l l e r . The micropipette formed had several centimeters of carbon f ib re protruding from the t i p . Under a l i gh t microscope, the f ibre was trimmed and etched in chromic acid at 0.12 mA current so that only 15 pm of carbon f ibre protruded from the micropipette. The micropipette was then f i l l e d with 4 M NaCl or sa l ine so lut ion (0.9%) and used for ex t race l lu la r recording. Computer Programmes A microcomputer (Rockwell Aim-65) was programmed to receive the output from the ratemeter. In addi t ion, outputs from the microcomputer were used to t r igger the noxious radiant heat st imulator and the supraspinal st imulat ion apparatus. These t r igger ing pulses were applied every two minutes to the noxious radiant heat stimulator and every four minutes to the supraspinal st imulat ion apparatus. The computer counted and printed the number of neuronal action potent ia ls 25 sec before and 25 sec after the onset of each stimulus ( f i l e name = SINCL). In add i t ion, the Aim-65 microcomputer tr iggered an Apple II plus computer 25 sec before the heat pulse. This l a t t e r computer also received an input d i r e c t l y from the ratemeter and counted the number of action potent ia ls for 55 sec beginning 25 sec before the onset of the heat pulse ( f i l e name = New.TS). These data were stored on d i s c s . A program was avai lable which allowed for the averaging of several responses ( f i l e name = New.GTS). - 55 -Pharmacological Studies Experiments were designed to test whether 5-HT was involved in mediating the NRM stimulation produced inh ib i t i on of the deep dorsal horn WDR neurones. One WDR neurone was studied per animal. The supraspinal st imulat ion was adjusted so as to produce approximately 50% i nh i b i t i on of evoked neuronal a c t i v i t y to heat. Drugs were administered after stable control responses to heat and heat plus supraspinal st imulat ion had been achieved. The involvement of 5-HT in the NRM phasic inh ib i t i on was investigated e i ther by the administrat ion of f luoxet ine or pargyl ine. The dose of f luoxet ine used was 6.0 mg/kg. This dose of f luoxet ine enhances the spinal cord monosynaptic ref lex in ca t s , an ef fect believed to be due to an enhanced level of synaptic 5-HT (Sastry and S inc l a i r 1976). The drug was administered by intravenous infus ion over a period of 13 minutes to avoid any marked change in the blood pressure. The dose of pargyline used in th i s study was 30.0 mg/kg. This dose of pargyline is known to increase the spinal cord leve ls of 5-HT (Anderson et a l . 1976) which i s l i k e l y responsible for the enhanced monosynaptic ref lex in cats (S inc la i r & Sastry 1974). Again, th i s drug was infused slowly over a period of 30 minutes to avoid changes in the blood pressure of the ca ts . The effect of drug treatment on the NRM phasic inh ib i t i on was followed for a period of one hour. In the experiment with pargyl ine, f luoxet ine was administered one hour after the start of the pargyline in fus ion . This protocol was followed to study whether, in presence of both f luoxet ine and pargyl ine, the change in the NRM phasic inh ib i t i on was more than observed with administrat ion - 56 -of f luoxet ine or pargyline alone. The effect of drug treatment on the spontaneous a c t i v i t y and heat evoked a c t i v i t y of the neurones was also studied in these experiments. Experiments were also designed to study the effect of morphine treatment on the NRM phasic i nh ib i t i on and the heat-evoked a c t i v i t y of the deep dorsal horn WDR neurones. The experimental design was as mentioned above. Morphine was administered in doses of 0.5 mg/kg, 0.5 mg/kg and 1.0 mg/kg to a cumulative dose of 2.0 mg/kg. The f i r s t dose of 0.5 mg/kg of morphine was administered after stable controls to heat and heat plus supraspinal st imulation had been achieved. The next doses of morphine were administered after stable neuronal responses to heat and heat plus supraspinal st imulation in presence of the preceding doses were seen. Morphine, 2.0 mg/kg cumulative dose, was also administered to f luoxet ine treated cats to test whether 5-HT was involved in mediating the ef fect of morphine on heat-evoked a c t i v i t y and on NRM phasic i nh i b i t i on of WDR dorsal horn neurones. Morphine was administered in these experiments 15 min after a second dose (6.0 mg/kg) of f luoxet ine . The experimental protocol was as above. F i na l l y , control experiments were also done in which the effect of in fus ion of drug vehic le on the NRM phasic i nh ib i t i on and heat-evoked a c t i v i t y of the deep dorsal horn WDR neurones was studied. In some experiments the NRM phasic i nh ib i t i on was tested on d i f ferent leve ls of e x c i t a b i l i t y of the dorsal horn neurones. In these experiments the neurones were f i r s t excited by noxious heat at a temperature of 45°C - 57 -applied to the i r receptive f i e l d s . The NRM stimulus in tens i ty was adjusted to produce approximately 50% inh ib i t i on of heat-evoked a c t i v i t y . The neurones were then excited by an appl icat ion of noxious heat at a temperature of 52°C and the extent of the NRM phasic i nh i b i t i on was observed without a l te r ing the NRM stimulus i n tens i t y . On completion of the experiments the animals were k i l l ed using an intravenous in jec t ion of saturated potassium chlor ide so lu t ion . A les ion was then made at the supraspinal st imulation s i te by passing 1 mA of d i rec t anodal current for 15 sec. The medullary region of the brain was then excised and stored in formalin for at least 48 hrs. The t i ssue was la te r sectioned using a cryostat (Damon/IEC Divis ion) and slide-mounted for h i s to log ica l ve r i f i c a t i on of the st imulation s i t e . Ca lcu lat ions The data in these experiments were quantif ied using a microcomputer (Rockwell Aim-65) which was programmed to count ce l l discharges 25 sec before and after the onset of noxious radiant heat. The number of ex t race l lu la r action potent ia ls recorded 25 sec before the onset of noxious stimulus was designated as the "spontaneous a c t i v i t y " of the recorded ce l l and the ce l l discharge count recorded 25 sec from the onset of the noxious stimulus was designated as the "noxious heat-evoked" a c t i v i t y of the c e l l . Inh ib i t ion of noxious heat-evoked a c t i v i t y by the NRM st imulat ion was calculated as percentage of control response to noxious heat in absence of any NRM st imulat ion. The spontaneous a c t i v i t y , noxious heat-evoked a c t i v i t y and the inh ib i t i on of - 58 -nociceptive ce l l a c t i v i t y to NRM st imulat ion for each animal were calculated as percentage of control (where control was equated to 100%) for d i f fe rent time points and used either to plot a time course curve or averaged to plot histograms as i l l u s t r a t ed in the Results sec t ion . S t a t i s t i c a l analysis of the observations during d i f ferent time periods was made using two way analysis of variance followed by Newman-keuls test for comparisons of means (using "U.B.C. GENLIN" which i s a general least square analysis of variance program). Absolute values of observations were used except where noted otherwise. The analysis i s discussed in deta i l for F i g . 10 (page72 to 75). - 59 -RESULTS The experiments in th i s study were done on dorsal horn neurones character ized as WDR neurones. They could be excited by innocuous touch or brushing of the receptive f i e l d and also by noxious pinch or noxious radiant heat. The ex t race l lu la r action potent ia ls recorded were usually very large indicat ing that the recording was from the ce l l body region or a proximal dendrite of the neurone. The depth at which such recordings were made ranged from 1400 pm to 2300 urn from the surface of the spinal cord. Although actual h is to log ica l ve r i f i c a t i on of the spinal cord laminar corresponding to th i s electrode depth range was not made in th i s study, previous experience in our laboratory indicates that th i s depth range corresponded to the spinal laminae IV-VI. The resu l ts of various experiments on these neurones are detai led below. F ig . 1 consists of osc i l loscope traces i l l u s t r a t i n g the response of WDR neurone to noxious radian heat (A) and NRM phasic i nh ib i t i on (B) . A heat pulse of 50°C i s indicated by the so l id bars. Nucleus raphe magnus st imulat ion i s indicated by the broken l ines in B. It is c lear that , the response of the neurone to noxious radiant heat was inh ib i ted by NRM, s t imu la t ion . Serotonin Involvement in NRM Stimulation-Produced Inh ib i t i on EFFECT OF FLUOXETINE Experiments with f luoxet ine were done on seven neurones, one neurone per animal. F i g . 2 i s comprised of computer generated - 60 -F I G : 1 O s c i l l o s c o p e t r a c e of n e u r o n a l response t o n o x i o u s r a d i a n t heat and n o x i o u s heat p l u s n u c l e u s raphe magnus s t i m u l a t i o n . The n o x i o u s heat was a p p l i e d d u r i n g the time p e r i o d i n d i c a t e d by t h e bar and t h e n u c l e u s raphe was s t i m u l a t e d d u r i n g t h e p e r i o d i n d i c a t e d by b r o k e n l i n e . The i n c r e a s e i n t h e n o x i o u s heat evoked f i r i n g o f t h e neurone ( i n d i c a t e d i n t r a c e A ) was c o n s i d e r a b l y r educed by c o n c o m i t a n t s t i m u l a t i o n of the n u c l e u s raphe ( t r a c e B )• - 61 -FIG: 2 Computer g e n e r a t e d h i s t o g r a m s , each r e p r e s e n t i n g t h e n e u r o n a l d i s c h a r g e o v e r 55 sec around a n o x i o u s heat p u l s e . In each h i s t o g r a m , n o x i o u s heat was a p p l i e d o v er t h e p e r i o d i n d i c a t e d by the c o n t i n u o u s b a r and t h e n u c l e u s raphe magnus was s t i m u l a t e d d u r i n g t h e b r o k e n l i n e . Each h i s t o g r a m was c o m p i l e d from 4 c o n s e c u t i v e n o x i o u s heat p u l s e s . Heat was a p p l i e d a t a t e m p e r a t u r e o f 55*C f o r 15 s e c . The numbers a t t h e upper r i g h t p o s i t i o n a r e t h e number o f d i s c h a r g e s o f t h e c e l l o v e r 25 sec from t h e o n s e t o f t h e heat p u l s e . The n u c l e u s raphe magnus was s t i m u l a t e d a t 100 Hz , 2.8 v i n t r a i n s o f 333 msec f o r 25 s e c . P a n e l A r e p r e s e n t s t h e c o n t r o l r e s ponse o f neurone i n absence o f d r u g t r e a t m e n t . P a n e l B r e p r e s e n t s the r e s p o n s e o f t h e neurone i n t h e p r e s e n c e o f f l u o x e t i n e ( 6.0 mg/kg, i . v . ). F l u o x e t i n e reduced t h e i n h i b i t i o n o f n e u r o n a l d i s c h a r g e by n u c l e u s raphe s t i m l a t i o n . P a n e l C r e p r e s e n t s t h e e f f e c t o f two c u m u l a t i v e dose o f morphine ( 1.0 mg/kg t o t a l dose, i . v . ). Morphine d e c r e a s e d t h e d i s c h a r g e of t h e neurone t o heat and t o heat i n presence o f n u c l e u s raphe s t i m u l a t i o n . - 62 -histograms t yp i c a l l y obtained from neurones used in th i s study. Each histogram i l l u s t r a t e s the neuronal discharge over 55 sec around a noxious heat pulse ( indicated by the bar ) . The NRM was stimulated during the broken l i n e s . Each histogram is compiled of four consecutive noxious heat pulses. Panel A represents the control response of a neurone in the absence of drug treatment whereas panel B represents the response of the neurone in the presence of f luoxet ine (6.0 mg/kg, i . v . ) . The number at the upper r ight posit ion of each histogram i s the average number of neuronal discharges over 25 sec from the onset of the heat pulse. It i s apparent from these numbers that f luoxet ine treatment had l i t t l e or no effect on the response of the neurone to noxious heat whereas i t attenuated the inh ib i t i on of the nociceptive a c t i v i t y produced by the NRM s t imu l t i on . The mean response of seven neurones to f luoxet ine i s i l l u s t r a ted in F igs. 3, 4 and 5. F ig . 3 i s a graph i l l u s t r a t i n g the time course of f luoxet ine on the NRM phasic i n h i b i t i o n . A s t a t i s t i c a l l y s ign i f i can t decrease in the NRM phasic i nh ib i t i on was observed at times 4, 20 and 28 minutes after the start of f luoxet ine in fus ion . However, these points were not d i f ferent from each other. The noxious heat-evoked a c t i v i t y ( F i g . 4) and the spontaneous a c t i v i t y of these neurones (F ig . 5) were not changed by f luoxet ine treatment. EFFECT OF PARYGLINE The ef fect of pargyline (30.0 mg/kg, i . v . ) infused over 30 minutes on the NRM st imulat ion i s i l l u s t r a t ed in F i g . 6. Pargyline produced a - 63 -FIG: 3 The e f f e c t of the fluoxetine ( 6.0 mg/kg, i . v . ) on the nucleus raphe magnus stimulation-produced i n h i b i t i o n of the nociceptive a c t i v i t y of wide dynamic neurones. The drug was infused over the time period indicated by the bar. The value at time four minutes before the s t a r t of the infusion ( or -4 min ) was equated to 100%. Values before and a f t e r time -4 min were calculated based on t h i s f i g u r e . Each point represents the mean 4 S.E.M., n=7. The i n h i b i t i o n at time +4, +20 and + 28 min was found to be s i g n i f i c a n t l y decreased from the 100% control (* P < 0.05 ) - 64 -150n 0 100' t c 0 0 c*. 0 N. 50-( O ) v v o h a d o c t l v l t y C N - 7 > -20 1 0 t i m e 1 — 20 C i n m i n ) 40 FIG: 4 Lack of e f f e c t of intravenous i n f u s i o n of fl u o x e t i n e ( 6.0 mg/kg, i . v . ) on the noxious heat evoked-activity of wide dynamic range neurones. The drug was infused over the time period indicated by the bar. Data are expressed as i n Fig.3. Each point represents the mean + S . E . M . , n=7. - 65 -FLU6m0fcg r o t i n 1 — 2 0 mlrO — l 4 0 - 2 0 ( i n FIG: 5 Lack o f e f f e c t o f i n t r a v e n o u s i n f u s i o n o f f l u o x e t i n e ( 6.0 mg/kg, i . v . ) on t h e spontaneous a c t i v i t y o f wide dynamic range neurones. The drug was i n f u s e d o v e r the time p e r i o d i n d i c a t e d by the b a r . Data a r e e x p r e s s e d as i n F i g . 3 . Each p o i n t r e p r e s e n t s t h e mean + S . E . M . , n=6. - 66 -125-0 L A B C "D FIG: 6 The e f f e c t of p a r g y l i n e ( 30.0 mg/kg, i . v . ) and p a r g y l i n e p l u s f l u o x e t i n e ( 6.0 mg/kg, i . v . ) on the nucleus raphe magnus st i m u l a t i o n - p r o d u c e d i n h i b i t i o n of the d o r s a l horn neuronal a c t i v i t y . P a r g y l i n e was i n f u s e d over a time p e r i o d of 30 min. T h i r t y minutes l a t e r f l u o x e t i n e was i n f u s e d over a time p e r i o d of 13 min. Bar A r e p r e s e n t s the mean i n h i b i t i o n between the time p e r i o d 12 and 4 min b e f o r e the s t a r t of p a r g y l i n e i n f u s i o n . T h i s value was equated t o 100% and the value s of subsequent bars were c a l c u l a t e d based on t h i s f i g u r e . Bar B r e p r e s e n t s the mean i n h i b i t i o n d u r i n g the p a r g y l i n e i n f u s i o n whereas bar C r e p r e s e n t s the mean i n h i b i t i o n up t o 26 min f o l l o w i n g p a r g y l i n e i n f u s i o n . Bar D r e p r e s e n t s the mean i n h i b i t i o n d u r i n g the f l u o x e t i n e i n f u s i o n and the bar E r e p r e s e n t s the mean i n h i b i t i o n i n the next 24 min. The f o l l o w i n g comparisons were made and * i n d i c a t e s a s i g n i f i c a n t d i f f e r e n c e ( P < 0.05 ): A-B *, A-C *, A-D *, A-E *, B-C *, C-D, and C-E. N=4. - 67 -time-dependent decrease in the NRM phasic i n h i b i t i o n . The decrease in the NRM phasic i nh ib i t i on with pargyline in the time period of up to 20 minutes post- infus ion was more than the decrease observed for the NRM phasic i nh ib i t i on during pargyline in fus ion . Pargyline also produced a time-dependent increase in the noxious heat-evoked a c t i v i t y of these neurones such that the increase in the post- infusion period was more than observed during pargyline infusion (F ig . 7) . L i t t l e or no effect of pargyline was seen on the spontaneous a c t i v i t y of these neurones ( F i g . 8 ) . EFFECT OF PARGYLINE PLUS FLUOXETINE In these experiments, animals treated with pargyline were then administered f luoxet ine. Fluoxetine was administered about one hour after pargyline administrat ion. Administration of f luoxet ine did not produce any further change in the NRM phasic i nh ib i t i on compared to the i nh ib i t i on with pargyline alone in the post infusion period although i t was s i gn i f i c an t l y less compared to the 100% control value (F ig . 6 ) . However, f luoxet ine increased the noxious radiant heat-evoked a c t i v i t y of the neurones (F ig . 7) . The increase observed with f luoxet ine was s t a t i s t i c a l l y s i gn i f i c an t l y d i f ferent from the mean of the evoked a c t i v i t y observed with pargyline during the infusion and the post-infusion time period as well as from pre-drug control value. The change in evoked a c t i v i t y with f luoxet ine during the infusion and post- infusion time period was no d i f ferent from each other. Fluoxetine administered to pargyline treated animals produced a - 68 -200-1 A B C D FIG: 7 The e f f e c t o f p a r g y l i n e ( 30.0 mg/kg, i . v . ) and p a r g y l i n e p l u s f l u o x e t i n e ( 6.0 mg/kg, i . v . ) on t h e n o x i o u s heat-evoked a c t i v i t y o f d o r s a l horn wide dynamic range neurones. Data a r e e x p r e s s e d as i n d i c a t e d i n F i g . 6. The f o l l o w i n g comparisons were made and * i n d i c a t e s a s i g n i f i c a n t d i f f e r e n c e ( P < 0.05 ): A-B *, A-C *, A-D *, A-E *, B-C *, C-D * and D-E. N=4. - 69 -FIG: 8 The e f f e c t o f p a r g y l i n e ( 30.0 mg/kg, i . v . ) and p a r g y l i n e p l u s f l u o x e t i n e ( 6.0 mg/kg, i . v . ) on t h e spontanous a c t i v i t y o f d o r s a l h o rn neurones. Data a r e e x p r e s s e d as i n d i c a t e d as i n F i g . 6. The f o l l o w i n g comparisons were made and * i n d i c a t e s a s i g n i f i c a n t d i f f e r e n c e ( P < 0.05 ): A-B, A-C, A-D *, A-E *, D-E *, C-D *, and C-E *. N=4. - 70 -decrease in the spontaneous a c t i v i t y of the neurones studied ( F i g . 8 ) . The decrease was s t a t i s t i c a l l y s ign i f i cant from the pre-drug control values and from pargyline treated values. This ef fect of f luoxet ine in pargyline treated animals was also time-dependent in that the mean spontaneous a c t i v i t y in the f luoxet ine post- infusion time period was s i gn i f i c an t l y less than the mean spontaneous a c t i v i t y during the infus ion of f luoxet ine . Ef fect of Morphine Morphine was given in three divided doses of 0.5 mg/kg, 0.5 mg/kg and 1.0 mg/kg, respect ively to a cumulative dose of 2.0 mg/kg. The resu l ts of the experiments are i l l u s t r a t ed in F igs . 9, 10 and 11. The ef fect of morphine on the inh ib i t i on of nociceptive a c t i v i t y by the NRM st imulat ion was inconsistent in that i t e i ther increased or decreased the NRM phasic i nh ib i t i on of the ce l l and no s t a t i s t i c a l l y s i gn i f i can t change in e i ther d i rec t ion was found. The increase in i nh ib i t i on was observed in 3 neurones and the inh ib i t i on was decreased in 2 neurones. The maximum increase in i nh ib i t i on was observed with a morphine cumulative dose of 2.0 mg/kg. This increase ranged from 113 to 145 percent of the pre-drug control value. The maximum decrease in i nh ib i t i on was also seen with a morphine cumulative dose of 2.0 mg/kg. The decreases observed in these 2 neurones were -35 ( i . e . , rather than observing an i nh i b i t i o n , a f a c i l i t a t i o n of noxious heat-evoked a c t i v i t y was observed upon NRM stimulat ion) and 50 percent of the pre-drug control value. The grouped data on these f ive neurones i s i l l u s t r a ted in F i g . 9. - 71 -125-0 L A B C D FIG: 9 Lack o f e f f e c t o f morphine ( 2.0 mg/kg t o t a l dose, i . v . ) on t h e n u c l e u s raphe magnus s t i m u l a t i o n - p r o d u c e d i n h i b i t i o n o f n o c i c e p -t i v e a c t i v i t y o f d o r s a l h o rn neurones. Morphine was i n f u s e d i n t h r e e c u m u l a t i v e doses bf 0.5, 0.5, and 1.0 mg/kg r e s p e c t i v e l y . Bar A r e p r e s e n t s t h e mean i n h i b i t i o n between t h e time p e r i o d 12 and 4 min. b e f o r e s t a r t o f morphine i n f u s i o n . T h i s v a l u e was equ a t e d t o 100./.. V a l u e s r e p r e s e n t e d by b a r s f o l l o w i n g t h i s were c a l c u l a t e d based on t h i s f i g u r e . B a r s B, C and D r e p r e s e n t s the mean i n h i b i t i o n , between t h e ti m e p e r i o d 14 t o 22 min, 10 t o 22 min and 6 t o 14 min p o s t - i n f u s i o n , t o t h e t h r e e c u m u l a t i v e doses of morphine r e s p e c t i v e l y . N=5. - 72 -Morphine produced a decrease in the heat-evoked a c t i v i t y of the neurones (F ig . 10). The decrease was observed to be dose-dependent with a decrease observed with a dose of 0.5 mg/kg of morphine and a maximal decrease of evoked ac t i v i t y with morphine cumulative dose of 2.0 mg/kg. On visual inspection of the F ig . 10 the dose-dependent ef fect of cumulative doses of morphine is not apparent. This i s because F i g . 10 i s based on the average of noxious heat-evoked a c t i v i t y and i t s S.E.M. of 5 neurones, from d i f ferent animals, which exhib i t considerable animal to animal va r i a t i on . However, use of analysis of variance e f fec t i ve l y allows removal of animal to animal var ia t ion and permits examination of treatment ef fects on a given animal for which measured a c t i v i t y is quite stable for a given treatment. For example, Table 1, animal 1, treatment 1 (control) the mean a c t i v i t y is 1124.5 and standard deviat ion of 87.64. In the same animal for treatment 2 (morphine 0.5 mg/kg), the value was 699.33 with a standard deviat ion of 57.49. Thus, these standard deviat ions are reasonable and the same is.seen for a l l treatments within th i s animal and with d i f ferent animals. However, when a given treatment is compared for animals 1 to 5 considerable animal to animal var iat ion in the mean of heat-evoked ac t i v i t y i s observed. Table 2 shows that when observations are averaged for a l l 5 animals, there i s a dose-dependent effect of morphine treatment. This i s shown by the four subsets of the Newman-keuls t e s t . Morphine also decreased the spontaneous a c t i v i t y of the neurones ( F i g . 11). The decrease in spontaneous a c t i v i t y by morphine was - 73 -125-n 0 L *J C 0 u u-0 N •P u 0 "D 0 X 0 > 0 1 0 0 -75-50-25-FIG: 10 The e f f e c t o f morphine ( 2.0 mg/kg t o t a l dose, i . v . ) on t h e n o x i o u s heat-evoked a c t i v i t y o f d o r s a l horn neurones. Morphine was a d m i n i s t e r e d i n t h r e e c u m u l a t i v e doses as i n F i g . 9. Data a r e e x p r e s s e d as i n d i c a t e d i n F i g . 9. Morphine a d m i n i s t r a t i o n d e c r e a s e d t h e h e a t - e v o k e d a c t i v i t y o f t h e n e u r o n e s . The f o l l o w i n g c o mparisons were made and * i n d i c a t e s a s i g n i f i c a n t d i f f e r e n c e ( P < 0.05 ): A-B *, A-C *, A-D *, B-C *, B-D *, and C-D *. N=5. - 74 Table 1 P o r t i o n of a n a l y s i s of variance for e f f e c t of morphine on noxious heat-evoked a c t i v i t y of i n d i v i d u a l d o r s a l horn neurones at d i f f e r e n t time periods 1 " 6* 6 n 6° 6 n n 6 0 MEAN 1124 .5 480.50 1269.5 249.50 1384.0 0 87.G44 40.004 59.288 62.788 9.2952 2 D 6° 6 n 6° 6 n n 6 0 MEAN 699.33 501.OO 503.67 102.67 1067.7 0 * r 57.497 76.407 40.849 34.448 75.197 3 en 8 n 8° 6 n 8 n 0 MEAN 388.83 715.12 508.50 92.167 676.00 0 f l > D V 18.519 70.516 32.196 36.706 73.644 4 6 n 6 n 6 n 6 n 12 n 0 MEAN 272.33 760.17 459.50 89.000 551.50 0 STDV 65.534 66.189 47.627 31.279 78.151 M u l t i p l e ' range t e s t s Newman-Keuls t e s t at 5% p r o b a b i l i t y l e v e l T h ere a r e 10 homogeneous s u b s e t s which a r e l i s t e d as f o l l o w s : 4 4 , 4 3. 4 2 ) 4 1 , 1 4 ) 1 3 ) 3 4 , 2 1 , 2 2, 3 2, 3 3 ) 2 1 , 2 2, 3 2, 3 3, 5 4 ) 5 3, 1 2, 2 3 ) 1 2 , 2 3, 2 4 ) 5 2, 1 1 ) 3 1 ) 5 1 ) B.: a s i g n i f i e s a n i m a l s such t h a t ia i n d i c a t e s 1st animal, 2a means 2nd animal and so on. b s i g n i f i e s t r e a t m e n t such t h a t l b i s c o n t r o l , 2 n i s res p o n s e t o morphine 0.5 mg/kg, 3 D i s response t o morphine c u m u l a t i v e dose o f 1.0 mg/kg and 4 D i s res p o n s e t o 2.0 mg/kg c u m u l a t i v e dose o f mor p h i n e . n s i g n i f i e s number o f o b s e r v a t i o n s made. - 75 -Table 2 Portion of analysis of variance and Newman-keuls test of significance for morphine effect on heat-evoked response averaged for five dorsal horn neurones 3 0 n 30 n 3 6 n 36° 0 MEAN 901.60 574.86 502.30 447.33 0 STDV 462.60 323.73 225.45 226.73 Mul t i p l e range tests Newman-Keuls test at 5% p r o b a b i l i t y level There are 4 homogeneous subsets which are l i s t e d as follows: ( .4 ) ( .3 ) ( .2 ) ( .1 ) Time for multiple range test was 0.64974E-02 seconds. Cumulative time 1s 0.17889 seconds. N.B.: n s i gn i f i e s number of observations made. - 76 -FIG: 11 The e f f e c t of morphine ( 2.0 mg/kg t o t a l dose, i . v . ) on t h e spontaneous a c t i v i t y o f d o r s a l horn neurones. Morphine was a d m i n i s t e r e d as i n F i g . 9. Data a r e e x p r e s s e d as i n d i c a t e d i n F i g . 9. Morphine d e c r e a s e d t h e spontaneous a c t i v i t y of the neurones. The f o l l o w i n g comparisons were made and * i n d i c a t e s s i g n i f i c a n t d i f f e r e n c e ( P < 0.05 ): A-B *, A-C *, A-D *, B-C B-D *, and C-D. N=5. - 77 -observed with a dose of 0.5 mg/kg and was maximal with the cumulative dose of 1.0 mg/kg. Sero ton in Involvement i n Morphine A c t i o n In these experiments, af ter obtaining stable neuronal responses to f luoxet ine , the animals were administered morphine in three cumulative doses as described in the Material and Methods sect ion. The rat ionale was to study whether enhanced serotonergic synaptic transmission in the presence of f luoxet ine affected the neuronal response to morphine. A result typ ica l of th i s study i s i l l u s t r a t ed in F ig . 2 panel C. Morphine, when administered in a cumulative dose of 1.0 mg/kg to f luoxet ine treated animals, reduced the NRM phasic i nh ib i t i on of the neurones nociceptive a c t i v i t y . The grouped data for f i ve neurones tested in th i s manner i s i l l u s t r a t ed in F ig . 12. Morphine, in f luoxet ine treated animals, produced a s t a t i s t i c a l l y s ign i f i can t reduction of the NRM phasic inh ib i t i on at a l l dose levels when compared to the NRM phasic i nh ib i t i on in the presence of f luoxet ine alone or with the pre-drug cont ro l . A decrease in inh ib i t i on was observed with the morphine dose of 0.5 mg/kg which was the same as observed with morphine cumulative dose of 1.0 mg/kg, and the maximal decrease was with the cumulative dose of 2.0 mg/kg. The va r i a t i on , par t i cu la r l y with the morphine cumulative dose of 2.0 mg/kg, i s due in part to the method of expressing the data as percentage of cont ro l . The same data plotted as the actual percent i nh i b i t i on i s shown in F i g . 13. The values for the above f igures of the indiv idual neurones are reported in Table 3. Normally, converting the - 78 -FIG: 12 The b l o c k o f n u c l e u s raphe magnus s t i m u l a t i o n - p r o d u c e d i n h i b i t i o n by morphine ( 2.0 mg/kg t o t a l dose, i . v . ). Morphine was a d m i n i s t e r e d as i n F i g . 9. At t h e time of i n f u s i o n o f the f i r s t dose o f morphine t h e a n i m a l s had a l r e a d y been t r e a t e d w i t h f l u o x e t i n e ( 6.0 mg/kg. i . v . ). Bar A r e p r e s e n t s t h e mean i n h i b i t i o n t o f l u o x e t i n e between time 10 and 4 min b e f o r e t h e s t a r t o f morphine i n f u s i o n . T h i s v a l u e was eq u a t e d t o 100 %. B a r s B, C and D r e p r e s e n t t h e mean i n h i b i t i o n , between t h e time p e r i o d s i n d i c a t e d i n F i g . 9, t o t h e t h r e e c u m u l a t i v e doses o f morphine r e s p e c t i v e l y . Each o f t h e t h r e e b a r s i s r e p r e s e n t e d as % of t h e f l u o x e t i n e c o n t r o l . The f o l l o w i n g c omparisons were made and * i n d i c a t e s a s i g n i f i c a n t d i f f e r e n c e ( P < 0.05 ): A-B *, A-C *, A-D *, B-C, B-D *, and C-D *. N=5. - 79 -6 0 - t F I G : 1 3 T h e e f f e c t o f m o r p h i n e ( 2 . 0 m g / k g t o t a l d o s e , i . v . ) o n t h e n u c l e u s r a p h e m a g n u s s t i m u l a t i o n - p r o d u c e d i n h i b i t i o n o f d o r s a l h o r n n e u r o n a l n o c i c e p t i v e a c t i v i t y . M o r p h i n e w a s a d m i n i s t e r e d a s i n F i g . 9 t o f l u o x e t i n e p r e t r e a t e d a n i m a l s . T h e d a t a p l o t t e d i s s a m e a s i n F i g . 1 2 , e x c e p t i n t h i s F i g . a c t u a l p e r c e n t a g e i n h i b i t i o n a r e p l o t t e d . T h e f o l l o w i n g c o m p a r i s o n s w e r e m a d e a n d * i n d i c a t e s a s i g n i f i c a n t d i f f e r e n c e ( P < 0 . 0 5 ) : A - B * , A - C * , A - D * , B - C , B - D * , a n d C - D * . N = 5 . - 80 -Table 3 Ef fect of morphine on the NRM phasic i nh ib i t i on of dorsal horn neuronal nociceptive a c t i v i t y in f luoxet ine pretreated animals % Inh ib i t ion to NRM Stimulation of a Noxious Neurone Heat Evoked Ac t i v i t y Control Morphine Cumulative Doses (0.5 mg/kg (1.0 mg/kg) (2.0 mg/kg) -10 to -2 min +14 to +22 min +10 to +22 min +6 to +14 min 1 35.4 (100) 14.6 (41.4) 11.5 (32.5) 16.2 (45.7) 2 61.4 (100) 48.7 (79.3) 52.9 (86.2) 50.2 (81.7) 3 27.8 (100) 7.6 (27.2) -17.1 (-61.3) -107.0 (-332.4) 4 51.7 (100) 57.5 (111.3) 51.6 (99.9) 39.5 (76.4) 5 53.8 (100) 38.4 (71.3) 45.4 (84.4) 59.4 (110.3) Mean ± SEM 46.0 (100 33.9* (66.1 28.9* (48.3 14.5* • (-3.6 + + + + + + + + 6.2 0.0) 9.6 14.2) 13.7 29.7) 27.7 82.8) N.B.: Control values represent the response of neurones to f luoxet ine pr io r to infusion of morphine and i s averaged for time period on the tab le . The averaged response of neurones to d i f fe rent cumulative doses of morphine for the time period mentioned fol lowing drug infusion are also given above. The values in brackets represent the inh ib i t i on expressed as percent of control (with pre-morphine response equated to 100%) whereas non-bracketed values are actual percent i nh i b i t i on s . Aster isk indicates a s ign i f i can t di f ference of the morphine response from the con t ro l . When the response to morphine was compared with each other, the change with morphine cumulative dose of 0.5 mg/kg and 1.0 mg/kg was ident ica l but d i f ferent from change seen with dose of 2.0 mg/kg. - 81 -data to percent of control decreased the apparent va r i a t i on . However, in th i s case i t was increased. Converting the actual percent i nh ib i t i on to percent of control caused an ampl i f icat ion of changes observed due to drug treatment, the ampl i f icat ion being greatest for the neurone having the smallest control percent i nh ib i t i on ( i . e . , neurone 3 ) . This neurone also showed the greatest change in the inh ib i t i on fol lowing morphine treatment. Thus, the var ia t ion shown in F i g . 12 i s exceedingly l a rge . Morphine, in f luoxet ine treated animals produced a dose-dependent decrease in the noxious heat-evoked a c t i v i t y compared to pre-drug control values or with the f luoxet ine control (F ig . 14). This group was also compared with the group where the effect of morphine alone was determined on the noxious heat-evoked a c t i v i t y . This was done to determine whether enhanced 5-HT synaptic transmission, in the presence of f luoxet ine , affected the inh ib i to ry action of morphine on the neuronal nociceptive a c t i v i t y . Since th i s was an unpaired comparison i t was necessary to normalize morphine ef fects in both the groups. This was done by equating the control pre-drug response in both the groups to 100%. The ef fect of the three cumulative doses of morphine in both the test groups was normalized to the i r respective 100% control and then compared s t a t i s t i c a l l y . When thus compared, the effect of morphine at a l l three dose leve ls on neuronal nociceptive a c t i v i t y in f luoxetine treated groups was not s t a t i s t i c a l l y d i f fe rent from the effect of the three cumulative doses of morphine alone. Morphine in f luoxetine treated animals also produced a dose-dependent decrease in spontaneous a c t i v i t y of the neurones when compared to the i r paired controls (F ig . 15). - 82 -125-1 0 L A B C FIG: 14 The i n h i b i t o r y e f f e c t o f morphine ( 2.0 mg/kg, i . v . ) on n o x i o u s heat-evoked a c t i v i t y o f d o r s a l horn neurones. Morphine was a d m i n i s t e r e d as i n F i g . 9. At t h e t i m e of i n f u s i o n o f the f i r s t dose of morphine t h e a n i m a l s had a l r e a d y been t r e a t e d w i t h f l u o x e t i n e ( 6.0 mg/kg, i . v . ). Data a r e e x p r e s s e d as i n d i c a t e d i n F i g . 12. The i n h i b i t i o n o f evoked a c t i v i t y by morphine was dose-dependant. The f o l l o w i n g c omparisons were made and * i n d i c a t e s a s i g n i f i c a n t d i f f e r e n c e ( P < 0.05 ): A-B *, A-C *, A-D *, B-C *, B-D *, and C-D *. N=5. - 83 -FIG: 15 The i n h i b i t i o n o f spontaneous a c t i v i t y o f d o r s a l horn neurones by morphine ( 2.0 mg/kg t o t a l dose, i . v . ). Morphine was a d m i n i s t e r e d as i n F i g . 9. At t h e time o f i n f u s i o n o f t h e f i r s t dose o f morphine t h e a n i m a l s had a l r e a d y been t r e a t e d w i t h f l u o x e t i n e ( 6.0 mg/kg, i . v . ). Data a r e e x p r e s s e d as i n d i c a t e d i n F i g . 12. The d e c r e a s e o f spontaneous a c t i v i t y by morphine from t h e f l u o x e t i n e c o n t r o l was dose-dependent. The f o l l o w i n g c o mparisons were made and * i n d i c a t e s a s i g n i f i c a n t d i f f e r e n c e ( P < 0.05 ): A-B *, A-C *, A-D *, B-C *, B-D *, and C-D *. N=5. - 84 -Control Experiments In these experiments the response of WDR neurones to heat and heat plus NRM st imulat ion was studied with the infusion of the drug vehic le ( d i s t i l l e d water). The resu l ts of these experiments are i l l u s t r a t ed in F igs. 16, 17 and 18. The NRM phasic i nh i b i t i on , evoked a c t i v i t y and the spontaneous a c t i v i t y of the neurones was not affected by infus ion of the veh i c l e . Control experiments were also designed to test whether changing the e x c i t a b i l i t y of neurones affected the NRM phasic i nh ib i t i on of the ce l l a c t i v i t y . The resul ts are i l l u s t r a t ed in F ig . 19. The heat-evoked response of the neurones at 52°C (986.12 ± 179.37) was more than the response at 45°C (634.01 ± 141.96). This increase in ce l l a c t i v i t y at 52°C vs 45°C was s t a t i s t i c a l l y s i gn i f i c an t . The NRM phasic i nh ib i t i on showed a decl ine with the increasing ce l l a c t i v i t y . The mean % inh ib i t i on at 52° was 46.79 ± 16.04 compared to 56.79 ± 16.04 at 45°C. This decrease in the NRM phasic i nh ib i t i on was also s t a t i s t i c a l l y s igni f i c an t . - 85 -l S O - i < 0 > i n h i b i t i o n CN-5> 0 100-L c 0 u 0 \ 5 0 -V & K L E O H - 2 0 1 o t i m e C l n 1 — 2 0 m i n ) — i 4 0 FIG: 16 The e f f e c t o f t h e v e h i c l e ( d i s t i l l e d w a t e r , 1.5 ml/kg, i . v ) on th e n u c l e u s raphe magnus s t i m u l a t i o n - p r o d u c e d i n h i b i t i o n o f t h e n o c i c e p t i v e a c t i v i t y o f t h e d o r s a l h o r n neurones. The v e h i c l e was i n f u s e d o v e r the time p e r i o d i n d i c a t e d by t h e b a r . The v a l u e a t time f o u r m i n u t e s b e f o r e t h e s t a r t o f t h e i n f u s i o n ( o r -4 min ) was equated t o 100%. V a l u e s b e f o r e and a f t e r t i m e -4 min were c a l c u l a t e d based on t h i s f i g u r e . Each p o i n t r e p r e s e n t s t h e mean + S.E.M.,n=5. The i n h i b i t i o n a t each p o i n t was found not t o be d i f f e r e n t from t h e 100% c o n t r o l . - 86 -150-1 C 0> a v o W a d o c t l v l t y CN -5> o ioo L c 0 u \ 5 0 -1 0 t. i ma 1 , — 2 0 Cin min) — i 40 - 2 0 FIG: 17 Lack of e f f e c t of i n t r a v e n o u s i n f u s i o n of the v e h i c l e on the noxious heat-evoked a c t i v i t y of the d o r s a l horn neurones. The v e h i c l e was i n f u s e d over the time p e r i o d i n d i c a t e d by the bar. Data are expressed as i n F i g . 15. Each p o i n t r e p r e s e n t s the mean + S.E.M., n=5. V E H C L E — I D t i m e C i n 1 — 20 m i n ) —I 40 -20 FIG: 18. Lack of e f f e c t of i n t r a v e n o u s i n f u s i o n of v e h i c l e on the spontaneous a c t i v i t y of d o r s a l horn neurones. The v e h i c l e was i n f u s e d over the time p e r i o d i n d i c a t e d by the bar. Data are expressed as i n F i g . 15. Each p o i n t r e p r e s e n t s the mean + S . E . M . , n=5. - 88 -2 0 0 — i F I G : 19 A comparison of the percentage i n h i b i t i o n of noxious heat-evoked a c t i v i t y of d o r s a l horn neurones with noxious heat a p p l i e d at 45* C and 52*C. The p a i r of histograms on the l e f t compare the percentage i n h i b i t i o n at the same stim u l u s i n t e n s i t y of the nucleus raphe magnus whereas the p a i r on the r i g h t compares the heat-evoked a c t i v i t y . The mean i n h i b i t i o n at 52*C was s i g n i f i c a n t l y decreased compared to the i n h i b i t i o n a t 45 C and the heat-evoked response at 52* C was s i g n i f i c a n t l y i n c r e a s e d compared to the reponse a t 45*C ( P < 0.05 ). N=6. - 89 -DISCUSSION Involvement o f 5-HT i n the NRM Phas i c I n h i b i t i o n o f Dorsa l Horn Neurones It has been speculated that 5-HT might mediate, at least par t l y , the NRM phasic i nh ib i t i on of deep dorsal horn WDR neurones. To test th i s hypothesis, experiments were designed to enhance synaptic transmission of 5-HT in the cat spinal cord using f luoxet ine, a 5-HT uptake blocker, and pargyl ine, a monoamine oxidase i nh i b i t o r . The rat iona le of such a design was that , under condit ions of enhanced spinal 5-HT synaptic transmission, the NRM phasic i nh ib i t i on of dorsal horn neurones, i f mediated by 5-HT, should be increased. However, treatment with f luoxet ine and pargyl ine, contrary to the expressed speculation produced a decrease in the NRM phasic inh ib i t i on of deep: dorsal horn WDR neurones. The involvement of serotonin in th i s drug effect and the possib le impl icat ion for the ro le of 5-HT in the NRM st imulat ion-produced inh ib i t i on of the deep dorsal horn WDR neurones i s discussed bel ow. There i s both biochemical and functional evidence to suggest that f luoxet ine and pargyline can enhance 5-HT synaptic transmission. Relevant biochemical evidence has been mentioned ea r l i e r in the t ex t . B r i e f l y , f luoxet ine i s reported to be a very se lect ive uptake blocker of 5-HT in rat synaptosomes and only in very high doses does i t block the uptake of noradrenaline (NA) and dopamine (DA; Wong et a l . 1974; Fu l le r et a l . 1975). S im i l a r l y , pargyline i s reported to markedly increase 5-HT, but not NA l eve l s , in the cat spinal cord (Anderson et a l . 1967). - 90 -This evidence would suggest that the drugs, f luoxet ine and pargyl ine, would have a greater ef fect on serotonergic systems as compared to the other monoaminergic systems. S i n c l a i r and Sastry (1974) have provided evidence that the bulbospinal i nh ib i t i on of the cat spinal monosynaptic re f lex (MSR) i s under an inh ib i to ry inf luence of a 5-HT system. In th i s study, imipramine, which pre fe rent ia l l y blocks the uptake of 5-HT, antagonized the bulbospinal i nh ib i t i on of the MSR and the effect of imipramine was blocked in 5-HT depleted animals. Pargyline and f luoxet ine , administered systemical ly and in same doses used in the present study, also reduced the bulbospinal i nh ib i t i on of the MSR in cats (S inc l a i r and Sastry 1974; Sastry and S inc l a i r 1976). By extrapolat ing from the above evidence i t can be postulated that administrat ion of e i ther f luoxet ine or pargyline to the experimental animals in th i s study would have resulted in enhanced 5-HT synaptic transmiss ion. Since both drug treatments resulted in an antagonism of the NRM phasic i nh ib i t i on of the deep, dorsal horn WDR neurones, th i s drug effect might be due to the increased 5-HT synaptic a c t i v i t y . There i s other supporting evidence which does not suggest the idea that 5-HT mediates the NRM phasic inh ib i t i on of the deep dorsal horn WDR neurones in the cat . For example, Griersmith et a l . (1981) have reported that the putative 5-HT antagonist, methysergide, administered e i ther intravenously or by iontophoresis in the region of SG, fa i l ed to reduce the NRM stimulation-produced inh ib i t i on of nociceptive a c t i v i t y of dorsal horn neurones. S im i l a r l y , Belcher et a l . (1978) fa i led to observe a reversal of the NRM phasic i nh ib i t i on of dorsal horn neuronal - 91 -nociceptive a c t i v i t y by methysergide iontophoresed in the v i c i n i t y of the neuronal ce l l body. The neuronal c i r cu i t r y upon which f luoxet ine and pargyline act to exert the ef fects reported in th i s study i s not known. Hypothetical neuronal c i r c u i t s are proposed in F igs . 20 and 21 to explain the mechanism of the NRM phasic i n h i b i t i o n . The NRM stimulation-produced inh ib i t i on of nociceptive a c t i v i t y of the deep doral horn WDR neurones may occur by several mechanisms: (1) It may operate via a d i rect inh ib i to ry input onto the deep; dorsal horn neurones. Descending f ibres from the NRM have been observed to make soma-dendritic contacts in the deep * dorsal horn laminae V-VII (Light and Kavookjian 1985). (2) The nociceptive afferent drive to deepi dorsal horn WDR neurones may also be reduced by an inh ib i t i on of an interneurone in the super f i c ia l laminae which may be part of the exc i tatory polysynaptic afferent input onto these deep dorsal horn neurones. Consistent with th i s idea, soma-dendritic synaptic contacts made by descending NRM f ibres have also been observed in the super f i c ia l laminae of spinal cord dorsal horn in cats (Light and Kavookjian 1985; Ruda et a l . 1981). (3) Presynaptic i nh ib i t i on of nociceptor primary afferent terminals or interneurones in the Afferent pathway produced by the NRM st imulat ion would also reduce the nociceptive dr ive to the deep dorsal horn WDR neurones. Martin et a l . (1979) reported that st imulat ion of the NRM in cats resulted in primary afferent depolar izat ion (PAD) of afferent terminals of A5-nociceptors. F i na l l y , (4) NRM st imulat ion may exci te interneurones in the super f i c ia l laminae NRM FIBRE A A A -A SG 0 WDR NEURONE NEURONE PRIMARY AFFERENT FIG: 20 A s c h e m a t i c n e u r o n a l arrangement i l l u s t r a t i n g some p o s s i b l e s y n a p t i c c o n n e c t i o n s which would produce n u c l e u s raphe magnus p h a s i c i n h i b i t i o n o f deep d o r s a l horn wide dynamic range neurones. In t h i s and t h e f o l l o w i n g f i g u r e s , f i l l e d c e l l b o d i e s i n d i c a t e t h a t t h e neurones a r e i n h i b i t o r y whereas open c e l l b o d i e s i n d i c a t e t h a t the neurones have e x c i t a t o r y e f f e c t on the p o s t s y n a p t i c t a r g e t s ( see t e x t f o r d e t a i l s ). 93 W D R N E U R O N E S G P R I M A R Y N E U R O N E S A F F E R E N T FIG: 21 A schematic neuronal arrangement i l l u s t r a t i n g the nucleus raphe magnus phasic i n h i b i t o r y impingement of deep dorsal horn neurones mediated v i a an i n h i b i t o r y interneurone i n the substantia gelatinosa. - 94 -which are inh ib i to ry to the WDR neurones of the deep dorsal horn (Dubisson and Wall 1980). The chemical nature of the transmitter mediating the NRM phasic i nh ib i t i on of deep dorsal horn WDR neurones i s not known. E l ec t r i ca l st imulat ion of the NRM i s l i k e l y to excite both serotonergic and non-serotonergic bulbospinal f i b r e s . The balance of such act ivat ion resu l ts in i nh ib i t i on of WDR neurones. However, since f luoxet ine and pargyline antagonized rather than enhance the i nh i b i t i on , i t appears that 5-HT i s not the pr inc ip le mediator of the i n h i b i t i o n . Furthermore, these resul ts suggest that 5-HT may be involved in antagonzing th is bulbospinal i n h i b i t i o n . A possible mechanism of action of 5-HT in the spinal cord to block the NRM phasic inh ib i t i on i s discussed below. One of the most obvious s i tes of 5-HT action i s in the substantia gelat inosa of the spinal cord dorsal horn since the concentration of 5-HT in the spinal cord of the cat i s highest in th i s region (Oliveras et a l . 1977). Iontophoretic appl icat ion of 5-HT in the v i c i n i t y of the ce l l bodies of laminae I and II neurones i s known to excite these neurones (Todd and M i l l a r 1983), some of which were characterized to be of the WDR type. Therefore, i t i s possible that these super f i c ia l laminae interneurones are a l i nk in the polysynaptic nociceptive afferent input to deep dorsal horn WDR neurones. NRM st imulat ion, through 5-HT release, may block the concurrent NRM phasic i nh ib i t i on by ac t iva t ing these super f i c ia l laminae exci tatory interneurones (F ig . 22). A l te rnat ive ly , serotonin might also block the NRM phasic inh ib i t i on of deep dorsal horn neurones by inh ib i t i ng the super f i c ia l laminae 0 0 5-HT FIBRES A A WDR NEURONE SG NEURONE PRIMARY AFFERENT F I G : 22 A s c h e m a t i c n e u r o n a l arrangement i l l u s t r a t i n g some p o s s i b l e s y n a p t i c c o n n e c t i o n s by which t h e s e r o t o n e r g i c b u l b o s p i n a l neurones a t t e n u a t e the n u c l e u s raphe magnus p h a s i c i n h i b i t i o n of deep d o r s a l horn neurones. - 96 -interneurones, which are inh ib i to ry to deep dorsal horn neurones (F ig . 23). For example, Randic and Yu (1976) have reported that iontophoretic app l i cat ion of 5-HT inh ib i ted the majority of super f i c ia l laminae neurones in the i r study. Serotonin released in the SG might also act presynapt ical ly to decrease the NRM phasic i nh ib i t i on of the deep dorsal horn neurones. There i s some evidence in favour of t h i s postulate. For example, iontophoretic 5-HT increases the threshold for antidromic act ivat ion of AS- and C-f ibres in the cat spinal cord (Carstens et a l . 1981b). This ef fect of 5-HT on the primary afferent terminal e x c i t a b i l i t y i s opposite to that which occurs during PAD which i s thought to result in a decreased transmitter release during afferent ac t i va t i on . Therefore, the effect of 5-HT on the and AS- and C-afferent f ib re terminal e x c i t a b i l i t y might lead to an enhancement of transmitter release by peripheral act ivat ion of these af ferents . Since some of the AS- and C-f ibres are nociceptors (Burgess and Perl 1967; Bessou and Perl 1969) one can speculate that enhancement of transmitter release by nociceptor afferents in the presence of 5-HT would result in greater act ivat ion of dorsal horn neurones to peripheral noxious st imul i applied to the i r receptive f i e l d s . This increased nociceptive afferent dr ive on dorsal horn neurones might then decrease the inh ib i t i on of neurones by the NRM st imu la t ion . In the text above i t has been suggested that the decrease in the NRM phasic i nh ib i t i on of nociceptive a c t i v i t y of the deep dorsal horn neurones by f luoxet ine and pargyl ine, respect ive ly , might be due to the enhancement of 5-HT synaptic transmission in the spinal cord. - 97 -• 5-HT NRM FIBRE FIBRE I I WDR PRIMARY NEURONE NEURONES AFFERENT F I G : 23 A s c h e m a t i c n e u r o n a l arrangement i l l u s t r a t i n g t h e s e r o t o n e r g i c s y n a p t i c impingement on the i n h i b i t o r y i n t e r n e u r o n e i n t h e s u b s t a n t i a g e l a t i n o s a which a t t e n u a t e s t h e n u c l e u s raphe magnus i n h i b i t i o n o f deep d o r s a l horn neurones. - 98 -It i s also possible that the effect seen with these drug treatment might not only be mediated through a spinal s i t e of action but also through a supraspinal component. The supraspinal s i te(s) of drug action i s not known but i t i s possible that f luoxet ine and pargyline a l te r the e x c i t a b i l i t y of the NRM serotonergic neurones. Aghajanian (1978) postulated that monoaminergic neurones of the central nervous system are under feedback regulation whereby an increase in the a v a i l a b i l i t y of monoamine transmitter would be compensated by a decrease in the monoamine neuronal f i r i n g rate. In keeping with th i s hypothesis, i t has been observed that parenteral administration of drugs which enhance 5-HT a v a i l a b i l i t y , such as monoamine oxidase inh ib i to rs l i k e pargyline (Aghajanian et a l . 1970), and 5-HT uptake blockers l i k e chlorimipramine (Gallagher and Aghajanian 1975) and zimel idine (de Montigny et a l . 1981), depressed the f i r i n g rate of dorsal raphe serotonergic neurones. It i s possible that treatment with parenteral pargyline and f luoxet ine s im i l a r l y decreased the e x c i t a b i l i t y of serotonergic neurones of the NRM in the present study. Consistent with th i s view i s the f inding of Hammond et a l . (1985) that , in ra t s , parenteral administrat ion of f luoxet ine did not produce a quant i tat ive increase of basal or NRM stimulation-produced 5-HT release into the spinal perfusate. This can be explained by a scenario in which an increased release of spinal 5-HT by f luoxet ine , as a result of i t s serotonin uptake blocking a c t i v i t y , i s obscured by a reduction in the release of 5-HT due to i nh ib i t i on of the serotonergic raphe neurones f i r i ng rate. In th is hypothetical model, i f f luoxet ine and pargyline decreased the e x c i t a b i l i t y of the NRM - 99 -serotonergic neurones, then the number or the extent of exc i ta t ion of such neurones by the NRM st imulat ion might also decrease. This, in t u rn , would decrease the synaptic 5-HT input to the spinal cord. If 5-HT i s indeed the transmitter responsible for the NRM phasic i nh ib i t i on of the nociceptive a c t i v i t y of the deep dorsal horn WDR neurones then, in accordance with the above scenario, the NRM phasic i nh ib i t i on of neuronal a c t i v i t y would decrease. In the above model the speculated decreased e x c i t a b i l i t y of the NRM serotonergic neurones by f luoxet ine and/or pargyline might be due to an enhanced supraspinal 5-HT synaptic transmission. Consistent with th i s notion are the observations that iontophoretic appl icat ion of 5-HT in the NRM inh ib i ted the a c t i v i t y of NRM-spinal neurones in primates (Willcockson et a l . 1983), ident i f i ed NRM bulbospinal serotonergic neurones in rats (Wessendorf and Anderson 1983) and systemic administrat ion of 5-HT agonist, 5-methoxy-N-N-dimethyltryptamine, reduced the f i r i ng of presumed serotonergic neurones of the cat NRM (Fornal et a l . 1985). From the above discussion i t follows that the decrease in the NRM phasic i nh ib i t i on of dorsal horn neuronal nociceptive a c t i v i t y by administrat ion of f luoxet ine and pargyline might be due to the enhancement of 5-HT transmission at spinal and supraspinal s i t e s . The contr ibut ion of spinal vs supraspinal s i tes of drug action cannot be resolved from th i s study. Fluoxetine treatment did not ef fect the spontaneous and the heat-evoked a c t i v i t y of the neurones in th i s study. A s imi lar effect of - 100 -f luoxet ine have been ea r l i e r reported by Soja and S i n c l a i r (1980). However, treatment with pargyline produced an increase in the responsiveness of the deep dorsal horn WDR neurones to noxious heat applied to the i r receptive f i e l d s . This ef fect of pargyline cannot be s a t i s f a c t o r i l y explained. The increase i s possibly due to an action of the drug on the descending tonic i nh ib i t i on impinging on these neurones, however, we did not examine th i s po s s i b i l i t y in th i s study. Fluoxet ine, in the presence of pargyl ine, produced a small but s i gn i f i c an t increase in heat-evoked a c t i v i t y of neurones and a s ign i f i can t decrease in the spontaneous a c t i v i t y of the neurones studied. The mechanism of action of f luoxet ine in the presence of pargyline i s not known. Duggan and Griersmith (1979) have suggested that changes in the blood pressure with a drug treatment might affect the neuronal responsiveness to noxious heat. For example, these authors have reported that infusion of the vasodepressor agent, isobutyl methyl xanthine, produced a marked increase in ce l l f i r i n g to noxious heat. The authors suggested that the f a l l in B.P. might have decreased the d i ss ipa t ion of heat from the receptive f i e l d resul t ing in greater act ivat ion of nociceptors and an increase in the noxious drive to the spinal cord neurones. However, i t i s believed that the increase in the neuronal responsiveness to noxious heat with pargyline treatment in th i s study i s not a re f lec t ion of blood pressure changes because ( i ) the increase in the noxious heat-evoked a c t i v i t y by pargyline was accompanied by a small and gradual increase in blood pressure, whereas, - 101 -according to Duggan & Griersmith (1979), an increase in neuronal ce l l f i r i n g to heat i s accompanied with a marked f a l l in blood pressure, ( i i ) Clark and Ryall (1983), in cats , did not observe any change in ce l l f i r i n g to noxious heat applied to the receptive f i e l d when the blood pressure was made to suddenly f a l l or r ise with infus ion of hexamethonium or adrenal ine, respect ive ly , and ( i i i ) Lynn (1979), in rabb i t s , reported that noxious heat-evoked responses in polymodal nociceptors was independant of the loca l blood f low. It i s possible that the increase in the e x c i t a b i l i t y of neurones to noxious heat with pargyline treatment influenced the NRM phasic i nh i b i t i on of these neurones. To test th i s po s s i b i l i t y control experiments were done in which i t was observed that increasing the a c t i v i t y of neurones by increasing the temperature of noxious heat applied to the receptive f i e l d decreased the NRM phasic i nh i b i t i o n . In these control experiments, the average increase in the response of neurones to noxious heat was about 55% which was accompanied by an average decrease of 10% in the NRM phasic i n h i b i t i o n . In the pargyline treated group of neurones, the average increase in the neuronal response to noxious heat in the pargyline post- infusion period was about 44% with an average decrease of 14% in the NRM phasic i nh i b i t i o n . The decrease in the NRM phasic i nh ib i t i on with pargyline treatment, i f i t had been so le ly due to change in the e x c i t a b i l i t y of the neurones, should have been less than 10%. Because i t was otherwise i t suggests that the drug, pargyl ine, decreased the NRM phasic i nh i b i t i o n , par t l y , as the resul t of i t s pharmacological e f fec t , with the increase in ce l l e x c i t a b i l i t y also - 102 -contributing to its action. Finally, control experiments were run in which the NRM stimulation-produced inhibition of dorsal horn neuronal nociceptive activity was studied with the infusion of the vehicle. The inhibition was followed for period of up to one hour which is the same time period for which most of the drug effects were studied. In these control experiments the NRM phasic inhibition did not change significantly with the infusion of the vehicle nor with time. The noxious heat-evoked and the spontaneous activity of the neurones in this control experiment were also unaffected. Role o f Se ro ton in i n Morphine Depress ion o f Noc i cep t i ve Response i n the  Dorsa l Horn Neurones There is some evidence to suggest that the NRM serotonin might play an important role in morphine antinociception in the spinal cord (see text). It has been suggested that the systemic administration of morphine might activate descending serotonergic fibres originating in the NRM to produce an increase in the release of spinal cord serotonin. This spinally released serotonin might partly mediate, morphine antinociception. To investigate this idea, experiments were designed wherein morphine was administered to animals already treated with fluoxetine. It was reasoned that if 5-HT did mediate, at least partly, morphines inhibition of dorsal horn neuronal nociceptive activity, then the effect of morphine would be enhanced by fluoxetine. Clearly, this was not the case. Morphine, administered systemically in three - 103 -cumulative doses, produced a dose-dependent i nh ib i t i on of nociceptive a c t i v i t y of the deep dorsal horn WDR neurones which was no d i f fe rent from that observed when morphine and f luoxet ine were administered concurrent ly. Recent e lectrophys io logica l f indings also support the observation made in th i s study that 5-HT does not mediate morphine i nh ib i t i on of dorsal horn neuronal nociceptive a c t i v i t y . For example, Auerbach et a l . (1985) have observed that analgesic doses of morphine (2.0 mg/kg, i .p) did not increase the ac t i v i t y of presumed serotonergic neurones in the conscious cat . S im i l a r l y , Chiang and Pan (1985) have reported that , in the anaesthetized rats , administrat ion of morphine (5.0 mg/kg, i .p . ) did not affect the a c t i v i t y in the majority of presumed NRM-spinal project ing serotonergic neurones. C lear ly , i f morphine had increased the a c t i v i t y of th i s type of neurone, i t would have been strong evidence that the attenuation of dorsal horn neuronal nociceptive a c t i v i t y by morphine was mediated by 5-HT released synapt ica l ly in the spinal cord dorsal horn. A lack of exc i ta t ion of serotonergic neurones by morphine strengthens the observation made in th i s study. Furthermore, there i s no conclusive evidence that analgesic relevant doses of morphine attenuates the nociceptive a c t i v i t y of dorsal horn WDR neurones by act ivat ing a descending inh ib i tory system. Systemic morphine can inh ib i t dorsal horn neuronal nociceptive a c t i v i t y in both spinal cord intact (Soja and S inc l a i r 1983b) and transected cats (Hanaoka et a l . 1978). The l a t t e r authors also observed that systemic morphine produced a greater percentage decrease of the dorsal horn - 104 -neuronal nociceptive responses during normal spinal cord conduction than when the spinal cord was transected. Based on th i s observation the authors suggested that morphine act ivates descending pathways that i nh ib i t noxious stimuli-evoked discharges in deep dorsal horn WDR neurones. But, Soja and S inc l a i r (1983b) have suggested that the dif ference in the percentage i nh ib i t i on of the dorsal horn neuronal a c t i v i t y by systemic morphine in the intact vs transected state of the spinal cord might have resulted from differences in the control values for neuronal responses in these d i f ferent state of the spinal cord. Du et a l . (1984) observed that micro inject ion of 10-20ug of morphine into the NRM produced an inh ib i t i on of noxious heat-evoked a c t i v i t y of the dorsal horn WDR neurones. The authors also suggested that morphine act ivates a descending inh ib i tory system from the NRM to suppress nociceptive transmission at the spinal cord l e v e l . However, th i s ef fect of microinjected morphine might not re f l ec t the action of systemic morphine because, when given systemica l ly , the drug effect might be a re f l ec t i on of i t s action on a number of neuronal pools. Thus, when a larger supraspinal area was exposed to morphine by infusing the drug between the th i rd vent r i c le and the c is terna magna, no inh ib i t i on of dorsal horn neuronal nociceptive a c t i v i t y was observed (S inc l a i r 1985). Further, the doses of morphine microinjected by Du et a l . (1984) in the NRM would produce concentrations of the drug in the region around the nucleus which were calculated to be far in excess of concentrations necessary to produce antinociception by systemic administrat ion of the drug (Clark et a l . 1983). - 105 -The hypothesis that morphine act ivates a descending i nh ib i t i on to the spinal cord from the NRM was investigated in th i s study by considering the ef fects of the drug on the NRM phasic i nh ib i t i on of the deep dorsal horn WDR neurones. The rat ionale of th i s experiment was that i f morphine does act ivate a descending i nh ib i t i on from the NRM, then i t should potentiate the NRM phasic i nh ib i t i on of the dorsal horn neurones. However, the resul ts of th i s study were ambiguous. Morphine ei ther increased or decreased the NRM stimulation-produced i n h i b i t i o n . Further experiments were carr ied out to invest igate whether 5-HT might mediate or influence the morphine effect on the NRM phasic i n h i b i t i o n , by administering morphine to f luoxet ine treated animals. Here morphine cons istent ly produced a decrease in the NRM phasic inh ib i t i on which was greater than that observed with f luoxet ine. Thus, under condit ions of enhanced 5-HT synaptic transmission, morphine produced a decrease in the NRM phasic i nh ib i t i on which i s not consistent with the hypothesis that morphine act ivates a descending serotonergic system from the NRM to i nh i b i t spinal cord nociceptive transmission. There are a number of p o s s i b i l i t i e s as to how morphine might in teract with 5-HT systems, espec ia l ly of the NRM, to decrease the NRM stimulation-produced inh ib i t i on of the dorsal horn neurones. For example, morphine might enhance the 5-HT synaptic transmission of the NRM serotonergic neurones and therefore potentiate the ef fect of f luoxet ine in decreasing the NRM phasic i n h i b i t i o n . However, recent e lectrophys io logica l evidence does not support th is p o s s i b i l i t y . That i s , morphine given systemical ly does not affect the a c t i v i t y of the - 106 -presumed 5-HT interneurones in the NRM of conscious cats (Auerbach et a l . 1985) or of the NRM-spinal serotonergic neurones in anaesthetized rats (Chiang and Pan 1985). It i s also possible that morphine can interact with 5-HT in the spinal cord to block the NRM phasic i n h i b i t i o n . The possible mechanism of interact ion of 5-HT and morphine in the spinal cord i s not known but i t might occur at one or more of the s i tes described previously for spinal serotonin action on the descending i nh i b i t i o n . The decrease in the NRM phasic i nh ib i t i on by treatment with morphine in f luoxetine treated animals is un l ike ly due to the change in the e x c i t a b i l i t y of dorsal horn WDR neurones to noxious heat. This conclusion was reached based on control experiments which showed that a decrease in e x c i t a b i l i t y of neurones produced an enhancement of the NRM phasic i nh ib i t i on of nociceptive a c t i v i t y . The decrease in the e x c i t a b i l i t y of these neurones was accompanied by a decrease in the phasic i n h i b i t i o n . The e lectrophys io logica l results obtained in th i s study are not consistent with those obtained in certa in behavioural studies which mainly use the rat t a i l - f l i c k t e s t . Some of these studies provide evidence that morphine antinociception i s mediated in part v ia 5-HT systems. The fol lowing factors could be important in these con f l i c t i ng resu l t s , ( i ) The present experiments were performed on cats whereas most of the behavioural experiments were done in ra t s . Therefore, a species di f ference might explain the resul ts obtained, ( i i ) This study was done in anaesthetized animals. Anaesthesia may have altered the - 107 -resu l ts obtained. For example 3pentobarbital anaesthesia was found to diminish the ant inocicept ive potency of in t racran ia l morphine (Ossipov and Gebhart 1984). Thus, i t i s possible that anaesthesia blocked the supraspinal component of morphine's action in th i s study. ( i i i ) Morphine ant inocicept ion in behaving animals might occur by i t s action on d i f fe rent spinal or supraspinal neuronal pools than the one studied here. Some of these po s s i b i l i t i e s might also explain the resul ts obtained by LeBars et a l . (1980). These authors reported that micro inject ion of 5 pg of morphine into the NRM of rats was ant inocicept ive in behavioural experiments but excited or had no ef fect on the C-f ibre response of dorsal horn WDR neurones of the same anaesthetized animals. - 108 -CONCLUSION 1. The magnitude of the nucleus raphe magnus (NRM) phasic i nh ib i t i on of the deep dorsal horn wide dynamic range (WDR) neurones was not increased but rather decreased by the se lect ive 5-HT reuptake i nh i b i t o r , f luoxet ine, and by treatment with the monoamine oxidase i nh i b i t o r , pargyl ine. These resul ts indicate that enhancement of 5-HT transmission does not resul t in increased NRM phasic i nh ib i t i on of dorsal horn neuronal nociceptive a c t i v i t y . 2. Morphine did not produce a consistent ef fect on the NRM phasic i n h i b i t i o n . It e i ther produced an increase or a decrease of th i s i n h i b i t i o n . However, in the presence of f luoxet ine, morphine reduced the NRM phasic i n h i b i t i o n . These observations are not consistent with the hypothesis that morphine act ivates a descending serotonergic system from the NRM to i nh ib i t spinal cord nociceptive transmiss ion. 3. Fluoxetine treatment did not increase the noxious heat-evoked a c t i v i t y of dorsal horn WDR neurones. This observation i s s imi lar to the ea r l i e r reported ef fect of f luoxet ine by Soja and S inc l a i r (1980) who suggested, based on th i s and other evidence, that 5-HT i s not involved in the tonic control of nociceptive a c t i v i t y of these neurones. - 109 -4 . Morphine administrat ion produced an i nh ib i t i on of dorsal horn neuronal nociceptive a c t i v i t y . This inh ib i to ry effect of morphine was no d i f fe rent than that seen when morphine was administered to f luoxet ine pretreated animals. 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