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The role of spinal pathways in dopamine mediated alteration in the tail-flick reflex in rats
NeuropharmacologyVol. 23, No. 2A, pp. 149 153, 1984
0028-3908/84 $3.00 + 0.00 Copyright ~f5 1984 Pergamon Press Ltd
Printed in Great Britain. All rights reserved
THE ROLE OF SPINAL PATHWAYS IN DOPAMINE MEDIATED ALTERATION IN THE TAIL-FLICK REFLEX IN RATS T. S. JENSEN*,H. D. SCHRODERt and D. F. SMITH~ Departments of Neurology* and Anatomyt, University of Aarhus, DK-8000 Aarhus C, and Psychopharmacology Research Unit+, Psychiatric Hospital, DK-8240 Risskov, Denmark
(Accepted 28 June 1983) Summary--The latency of the tail-flick, following intrathecal infusion of the dopamine (DA) agonist, R-apomorphine was measured in rats with intact spinal cord or with spinal cord lesions. Apomorphine failed to influence the tail-flick response in intact rats, whereas it elevated the latency of the tail-flick in rats with either total spinal cord transection, or bilateral lesions of the dorsolaterat funiculi (DLF), but not in rats with lesions in either the dorsal columns or the medioventral parts of the lateral funiculi. The findings suggest that pathways in the dorsolateral funiculi modulate DA-mediated sensory processes and possible mechanisms are discussed. Key words: apomorphine, spinal dopamine mechanisms, nociceptive reflex, spinal cord lesions, dorsolateral funiculus, supraspinal control. The discovery of endogenous pain-inhibitory mechanisms has stimulated interest in the physiological and pharmacological properties of pain-modulatory systems. Previous studies have shown there to be several nociceptive inhibitory systems in the brain and spinal cord (for review, see Fields and Basbaum, 1978; Wall, 1978; Yaksh, Hammond and Tyce, 1981; Watkins and Mayer, 1982). Access of nociceptive information from the spinal cord to the brain may be reduced either directly, by an action exerted at the segmental level (Wall, 1978) or indirectly, by an effect mediated by descending fibre systems from the brain to the spinal cord (Wall, 1967; Hillman and Wall, 1969; Brown, 1971: Oliveras, Besson, Guilbaud and Liebeskind, 1974; Fields, Basbaum, Clanton and Anderson, 1977: Duggan and Griersmith, 1979). Several chemical substances such as enkephalin, noradrenaline, serotonin and substance P are known to play a role in mediating these segmental and suprasegmental modulating effects on spinal nociceptive processes (Headley, Duggan and Griersmith, 1978; Yaksh and Wilson, 1979; Yaksh, Farb, Leeman and Jessell, 1979; Proudfit and Hammond, 1980; Reddy and Yaksh, 1980). This report concerns the role of dopamine (DA) in the spinal cord (Barasi and Roberts, 1977; Commissiong and Neff, 1979; Demenge, Mouchet, Guerin and Feuerstein, 1981; Carp and Anderson, 1982; Skagerberg, Bj6rklund, Lindvall and Schmidt, 1982) on a spinal nociceptive reflex. Previously, it was reported that direct stimulation of spinal DA receptors (Demenge et al., 1981) by intrathecal (i.t.) infusion of DA agonists inhibited the thermonociceptive tail-flick response in spinal rats but not in rats with an intact neuraxis (Jensen and Smith, 1982, 1983b). Thus, these findings suggest that DA mech-
anisms may play a role in segmental nociceptive processes and that DA mechanisms in the spinal cord, involved in nociceptive processes at the segmental level, may be under the influence of descending inhibitory pathways. The present experiments were carried out to determine which spinal pathways are responsible for the differential effects of DA agonists on the tail-flick response in rats with an intact neuraxis and in rats with total spinal cord transection. METHODS
Animals Male Wistar rats, weighing 300-350 g were used. They were kept individually in clear plastic cages in a thermostatically controlled room (20°C) on a 12 hr light-dark cycle (lights on at 6 a.m.) and had free access to food and water.
Surgical procedures Rats under pentobarbital anaesthesia were implanted with an intrathecal catheter, using a modified version of the method described by Yaksh and Rudy (1976). Briefly, a PE-10 catheter was inserted through a slit in the cisterna magna and extended 7.5 cm down to the rostral edge of the lumbar enlargement. After catheterization and during the same anaesthesia, a laminectomy was done at T8-10. With the aid of a dissecting microscope, the dura was reflected carefully and either no cord lesion or various subtotal spinal cord lesions were made by crushing the cord tissue with a pair of microdissecting forceps.
Spinal nociceptive reflex measurement The spinal-mediated tail-flick test (D'Amour and Smith, 1941; Jensen and Smith, 1981) was assessed 149
150
T.S. JENSENet al.
18 hr after surgery when no residual anaesthesia was present. In the version of the test used here, the latency of withdrawal of the tail of a rat from a radiant heat source focussed 4 cm proximal to the tip of the tail was recorded. The intensity of the thermal stimulus was adjusted to provide a latency of 5.5-6sec in intact, untreated rats. Three tail-flick latencies were measured at 60 sec intervals both before and 5,10,20 and 90 min after intrathecal infusion of drug. The means of the tail-flick latencies were used for statistical analysis, carried out by multiple comparisons with unlesioned rats (Dunnett, 1964), unless otherwise stated.
Drugs The DA agonist R-apomorphine hydrochloride (Sandoz) (0.98#mol/kg) (And~n, Rubenson, Fuxe and H6kfelt, 1967), or its vehicle (0.001 M ascorbic acid), was administered by intrathecal injection in a volume of 50/al/kg, followed by 15#1 saline (0.9~ NaC1), to ensure delivery of the drug into the intrathecal space.
His t ology After testing was completed the rats were killed by an overdose of pentobarbital. Spinal cord segments, containing the lesions, were removed and the location of the catheter in the intrathecal space, without additional damage to the spinal cord, was checked upon removal. The thoracic cord segments, fixed in 2.5~ formalin solution were soaked in a 30~o (w/v) sucrose solution for 24 hr, frozen with CO2-snow and sectioned transversely at 30 #m with a cryostat. Alternate sections were stained with thionin for identification of cell bodies, while remaining sections were stained with a modified Fink-Heimer technique (Hjort-Simonsen, 1970) for study of fibre architecture. The extent of spinal cord lesions were reconstructed from superimposed serial sections. On the basis of location and extent of subtotal lesions, rats were assigned to one of 6 groups. Assignment was done by an investigator (H.D.S.) uninformed of the experimental findings in the tail-flick test. The following lesion groups were made: group 1: dorsal column lesion; group 2: dorsal column lesion with bilateral quadrant lesion, including the dorsolateral funiculi (DLF); group 3: bilateral quadrant lesion including the dorsolateral funiculi; group 4: bilateral quadrant lesion without involvement of dorsolateral funiculi; group 5: dorsal column lesion, and bilateral quadrant lesion without involvement of dorsolateral funiculi: and group 6: unilateral lesion of dorsolateral funiculi with or without dorsal column lesion. Thirty-eight rats were operated and tested. In 2 rats spinal cord lesions could not be assessed due to technical reasons and in another 6 rats the histological sections did not permit an accurate assessment of the extent of spinal cord lesions. These rats were eliminated from the study, so complete data were
obtained from 30 rats with subtotal spinal cord lesions.
RESULTS
Spinal lesions On the basis of histological examination, 4. rats were assigned to group 1, 6 to group 2, 5 to group 3, 4 to group 4, 6 to group 5, and 5 to group 6. Figure 1 presents a schematic drawing of the maximum extent of the spinal lesions. Table 1 shows the effects of both complete and subtotal spinal cord lesions on the tail-flick response latencies. The analysis of variance for baseline tail-flick latencies was significant ( F = 6.11, df7/38; P < 0.001), due to a signiticant reduction in latencies in rats with total spinal cord transection and in rats with bilateral quadrant lesions including dorsolateral funiculi. No other spinal cord lesions influenced the baseline tail-flick latencies significantly.
Effect of intrathecal administration of apomorpnine in the intact and spinally-lesioned rat Figure 2 shows the time course and magnitude of the effects of intrathecal infusion of either Rapomorphine or vehicle on the tail-flick latency in both spinal and intact rats. As shown previc)usly (Jensen and Smith, 1982, 1983a, b) R-apomorphine increased the tail-flick latency significantly in spinal rats at 5, 10 and 20min after intrathecal infusion, while it failed to influence the tail-flick latency significantly in intact rats. Table 1 presents the results observed in rats assigned to the different lesion groups after intrathecal infusion of apomorphine. Analysis of variance showed a main effect of spinal lesions at 5, 10 and 90 min after intrathecal infusion of drug (F = 113.49, 6.56 and 8.27, respectively; df7/38; P < 0.01). Further analysis indicated that in groups 2 and 3, apomorphine produced a significant increase of the tail-flick latency which did not differ from the effects produced by intrathecal injection of apomorphine after total spinal cord transection. Examination of the 2 groups indicated that the common factor was bilateral lesions of the dorsolateral funiculi. In contrast, apomorphine, given intrathecally, failed to influence the tail-flick latency significantly in rats in which the dorsolateral funiculus was left intact on either one or both sides of the spinal cord.
DISCUSSION As in previously reported work it has been demonstrated that apomorphine given intrathecall~, in spinally transected, but not intact rats increased the latency to generate a thermally-evoked, spinallymediated reflex (Jensen and Smith, 1982, 1983a, b). This effect appears to be mediated by spinal DA
Role of spinal pathways
151
Group 1
Group 2
Group 3
Group 4
Group 5
Group 6
Fig. 1. Maximal extent of spinal cord lesions, reconstructed from superimposed serial sections. Group 1: dorsal column lesion; Group 2: dorsal column lesion including dorsolateral funiculi (DLF); Group 3: bilateral quadrant lesion including DLF; Group 4: bilaterai quadrant lesion without DLF lesion: Group 5: dorsal column lesion and bilateral quadrant lesion without DLF involvement: Group 6: unilateral DLF lesion with or without dorsal column lesion.
receptors (Demenge et al., 1981) as the action of a p o m o r p h i n e was counteracted in a dose-dependent a n d stereospecific m a n n e r by D A antagonists but not by serotonergic, ~-adrenergic,/~-adrenergic or opioid antagonists. While the D A - m e d i a t e d inhibition of the tail-flick response in spinal transected rats could be due to a direct effect on the m o t o r c o m p o n e n t of the response, it is more likely that spinal D A receptor systems are primarily associated with afferent sensory function. Thus, in previous work (Jensen and Smith,
1983b) it was f o u n d t h a t stimulation of spinal D A receptors in the spinal animal facilitated tail withdrawal reflexes in response to light tactile stimuli, in addition to inhibiting the tail withdrawal response evoked by thermal, noxious stimuli. Moreover, in recent experiments it was observed t h a t a p o m o r p h i n e given intrathecally produced a dose-dependent elevation of the hot-plate response in the intact animal, which was counteracted by D A antagonists in a stereospecific way (Jensen and Yaksh, 1984). T a k e n
Table 1. Effects of spinal cord lesions on the tail-flick latency in rats before (baseline) and 5, 10, 20 and 90 min after the intrathecal injection of R-apomorphine (0.98 #mol/kg) Tail-flick latency (sec):~ N Baseline 5 10 Treatment groupt 8 5190_+0.11 5.38_+0.31 5.53_+0.30 Intact (sham operated) 8 4.31 _+0.06** 9.44_+0.70** 7.41 _+0.46** Total spinal cord transection Subtotal spinal cord transection 4 5.80-+0.11 5.68_+0.29 5.80+0.31 Group 1 6 5.25 _+0.40 8.53 + 0.72** 7.52 + 0.50** Group 2 5 4.44-+0.31"* 8.58_+0.50** 7.22_+0.48* Group 3 4 5.95_+0.19 5.95_+0.49 5.75_+0.10 Group 4 6 5.32_+0.45 5.03-+0.22 4.93-+0.31 Group 5 5 5.60_+0.24 6.74_+0.66 6.38_+0.22 Group 6 * and ** indicate P < 0.05 and P < 0.01, respectively, by one-way ANOVA with comparisons made to by Dunnett's test. t See text for surgical procedure and group assignment :~Mean+ SEM.
20 5.90_+0.13 6.49_+0.49
90 5.80_+0.08 4.20_+0.16"*
5.90_+0.11 5.65+0.13 5.92 -+0.60 5.03 _+0.30 5.94_+0.34 4.26_+0.22** 5.98-+0.18 5.83_+0.13 5.52_+0.34 5.72_+0.38 6.10_+0.25 5.52_+0.39 corresponding intact group values
152
T . S . JENSEN et al.
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t Intrathecal injection T
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Fig. 2. Time course of tail-flick response latency in rats with intact spinal cord (circles) and in rats with total spinal cord transection (squares) given either 0.98#mol/kg Rapomorphine (filled symbols) or vehicle (unfilled symbols) by intrathecal injection. Each value represents mean _+SEM of 8 rats. *P < 0.001 compared to corresponding vehicletreated values by t-tests. together, these observations suggest that spinal DA receptors may serve to modulate afferent sensory transmission in the spinal cord. As part of continuing experiments designed to investigate the substrate by which the effect of apomorphine is made manifest, selective lesions of various parts of the spinal cord were carried out. In the present study, the effects of total spinal cord transection were found to be mimicked by partial lesions which bilaterally incorporated the dorsolateral funiculi. The fact that damage to the dorsal columns, and/or the medioventral aspects of the lateral funiculi had no effect rules out the possibility that nonspecific surgical trauma of the cord played a role in the alteration of tail-flick latency following intrathecal injection of apomorphine, and suggest that pathways in these systems are not relevant to the response in the spinal rats. While the present findings suggest that blockade of activity in the dorsolateral funiculi resulted in a lowering of the nociceptive threshold and in the appearance of inhibition of a spinal reflex mediated by a DA receptor they are unable to indicate the identity of the fibre systems responsible for these effects. Some previous observations, however, suggest that monoaminergic fibre systems originating in the lower brainstem and descending to the spinal cord may be involved. Thus, spinopetal serotonergic and noradrenergic systems, coursing in the dorsolateral funiculi are known to exert a tonic inhibitory effect on spinal nociceptive reflexes (Proudfit and Hammond, 1980; Zemlan, Corrigan and Pfaff, 1980; Jensen and Smith, 1983a). The present observation that bilateral lesions of the dorsolateral funiculi lowered nociceptive threshold is consistent with the modulating effect of these systems on nociceptive processes. In addition, the recent observations that inhibition of the tail-flick response induced by apomorphine can be produced in the intact animal by either systemic (Jensen and Smith,
1983a) or intrathecal (Jensen and Yaksh, 1984) pretreatment with serotonergic and noradrenergic receptor blockers, suggest that the modulation of spinal DA mechanisms by the dorsolateral funiculi is mediated in part by spinopetal serotonergic and noradrenergic pathways. It is tempting to speculate on the mechanisms by which spinopetal serotenergic and noradrenergic fibre systems coursing in the dorsolateral funiculi may modulate spinal DA-ergic mechanisms. There is evidence which shows that serotonin and noradrenaline may facilitate activity in the motor horn (McCall and Aghajanian, 1979; White and Neuman, 1980; Astrachan and Davis, 1981; Bell and Matsumiya, 1981). Thus, local application of serotonin and noradrenaline into the ventral horn by microinjection (Bell and Matsumiya, 1981) or by intrathecal injection (Astrachan and Davis, 1981) will facilitate the afferent-evoked ventral root reflex and the auditoryevoked startle response, respectively. It is suggested that the facilitatory influences exerted either directly or indirectly on the motor system will appear to physiologically antagonize the attenuation produced by the spinal DA receptor systems on the processing of the afferent stimulus in the dorsal horn which evokes the ventral root reflex. In the intact animal this makes it likely that a facilitation of activity in the motor horn is exerted by spinopetal monoamine systems. The present experiments suggest that, insofar as a spinal nociceptive reflex is concerned, the inhibitory effects mediated by the DA receptor would presumably be insufficient to depress the afterent drive and consequently block the reflex. Upon, spinal transection, bilateral lesions of the dorsolateral funiculi or spinal antagonism of the serotonergic and noradrenergic receptor systems (Yaksh and Wilson, 1979; Reddy and Yaksh, 1980), the loss of facilitation of motor horn cells would render the reflex pathway sensitive to the modulatory influences of the DA receptor. Acknowledgements--We thank Dr T. L. Yaksh for his
helpful suggestions in preparing this manuscript as well as Mrs Elin Kristensen, Mrs Lone Munko and Mr Bjarne Krunderup for technical assistance. This study was. supported by grants from Aarhus University Research Fund and Institute for Experimental Clinical Research, Aarhus University, to T.S.J.
REFERENCES D'Amour F. E. and Smith D. L. (1941) A method for determining loss of pain sensation. J. Pharmac. exp. Ther.
72: 74-79. And6n N. E., Rubenson A., Fuxe K. and H6kfelt T. (1967) Evidence for dopamine receptor stimulation by apomorphine. J. Pharm. Pharmac. 19: 627-629. Astrachan D. I. and Davis M. (1981) Spinal modulation of the acoustic startle response: The role of norepinephrine, serotonin and dopamine. Brain Res. 206: 223-228. Barasi S. and Roberts M. H. T. (1977) Responses of motoneurones to electrophoretically applied dopamine. Br. J. Pharmac. 60: 29-34.
Role of spinal pathways Bell J. A. and Matsumiya T. (1981) Inhibitory effects of dorsal horn and excitant effects of ventral horn intraspinal microinjections of norepinephrine and serotonin in the cat Life Sci. 29: 1507-1514. Brown A. G. (1971) Effects of descending impulses on transmission through the spinocervical tract. J. Physiol., Lond. 219: 103-125. Carp J. S. and Anderson R. J. (1982). Dopamine receptormediated depression of spinal monosynaptic transmission. Brain Res. 242: 247-254. Commissiong J. W. and Neff N. H. (1979) Current status of dopamine in the mammalian spinal cord. Biochem. Pharmac. 28: 1569-1573. Demenge P., Mouchet P., Guerin B. and Feuerstein C. (1981) Identification and distribution of neuroleptic binding sites in the rat spinal cord. J. Neurochem. 37: 53-59. Duggan A. W. and Griersmith B. T. (1979) Inhibition of the spinal transmission of nociceptive information by supraspinal stimulation in the cat. Pain 6: 147-161. Dunnett C. W. (1964) New table for multiple comparisons with a control. Biometrics 20: 482-491. Fields H. L., Basbaum A. I., Clanton C. H. and Anderson S. D. (1977) Nucleus raphe magnus inhibition of spinal cord dorsal horn neurons. Brain Res. 126: 441-453. Fields H. L. and Basbaum A. I. (1978) Brainstem control of spinal pain-transmission neurons. A. Rev. Physiol. 40: 217-248. Headley P. M., Duggan A. W. and Griersmith B. T. (1978) Selective reduction by noradrenaline and 5-hydroxytryptamine of nociceptive responses of cat dorsal horn neurones. Brain Res. 145: 185-189. Hillman P. and Wall P. B. (1969) Inhibitory and excitatory factors influencing the receptive fields of lamina 5 spinal cord cells. Expl Brain Res. 9: 284-306. Hjort-Simonsen A. (1970) Silver impregnation of degenerating axons and terminals on mounted cryostat sections of fresh and fixed brains. Stain Tech. 45: 199-204. Jensen T. S. and Smith D. F. (1981) The role of consciousness in stress-induced analgesia. J. Neural Trans. 52:55 60. Jensen T. S. and Smith D. F. and Smith D. F. (1982) Dopaminergic effects on tail-flick response in spinal rats. Eur. J. Pharmac. 79: 129-133. Jensen T. S. and Smith D. F. (1983a) The role of 5 HT and NA in spinal dopaminergic analgesia. Eur. J. Pharmac. 86: 65-70, Jensen T. S. and Smith D. F. (1983b) Stimulation of spinal dopaminergic receptors: Differential effects on tail reflexes in rats. Neuropharmacology 22: 477-483. Jensen T. S. and Yaksh T. L. (1984) Effects of an intrathecal
153
dopamine agonist, apomorphine, on thermal and chemical evoked noxious responses in rats. Brain Res. In press. McCall R. B. and Aghajanaian G. K. (1979) Serotonergic facilitation of facial motoneuron excitation. Brain Res. 169:11-27.
Oliveras J. L., Besson J. M., Guilbaud G, and Liebeskind J. C. (1974) Behavioral and electrophysiological evidence of pain inhibition from midbrain stimulation in the cat. Expl Brain Res. 20: 32-44. Proudflt H. K. and Hammond D. L. (1980) Alterations in nociceptive threshold and morphine-induced analgesia produced by intrathecally administered amine antagonists. Brain Res. 218:393 399. Reddy S. V. R. and Yaksh T. L. (1980) Spinal noradrenergic terminal system mediates antinociception. Brain Res. 189: 391-401. Skagerberg G., Bj6rklund A., Lindvall O. and Schmidt R. H. (1982) Origin and termination of the diencephalospinal dopamine system in the rat. Brain Res. Bull. 9" 237-244. Wall P. D. (1967) The laminar organization of dorsal horn and effects of descending impulses. J. Physiol., Lond. 188: 403-423. Wall P. D. (1978) The gate control theory of pain mechanisms: A reexamination and re-statement. Brain 101: 1-18. Watkins L. R. and Mayer D. J, (1982) Organization of endogenous opiate and nonopiate pain control systems. Science 216:1185-1192. White S. R. and Neuman R. S. (1980) Facilitation of spinal motoneurone excitability by 5-hydroxytryptamine and noradrenaline. Brain Res. 188: 119-127. Yaksh T. L. and Rudy T. A. (1976) Chronic catheterization of the spinal subarachnoid space. Physiol. Behav. 17" 1031-1036. Yaksh T. L. and Wilson P. R. (1979) Spinal serotonin terminal system mediates antinociception. J. Pharmac. exp. Ther. 208: 446-453. Yaksh T. L., Farb D., Leeman S. and Jessell T. (1979) Intrathecal capsaicin depletes substance P in the rat spinal cord and produces prolonged thermal analgesia. Science 206: 481-483. Yaksh T. L., Hammond D. L. and Tyce G. M. (1981) Functional aspects of bulbospinal monoaminergic projections in modulating processing of somatosensory information. Fedn Proc. Fedn Am. Socs. exp. Biol. 40: 2786-2794. Zemlan F. P., Corrigan S. A. and Pfaff D. W. (1980) Norad-renergic and serotonergic mediation of spinal analgesia mechanisms. Eur. J. Pharmac. 61:111 124.
0028-3908/84 $3.00 + 0.00 Copyright ~f5 1984 Pergamon Press Ltd
Printed in Great Britain. All rights reserved
THE ROLE OF SPINAL PATHWAYS IN DOPAMINE MEDIATED ALTERATION IN THE TAIL-FLICK REFLEX IN RATS T. S. JENSEN*,H. D. SCHRODERt and D. F. SMITH~ Departments of Neurology* and Anatomyt, University of Aarhus, DK-8000 Aarhus C, and Psychopharmacology Research Unit+, Psychiatric Hospital, DK-8240 Risskov, Denmark
(Accepted 28 June 1983) Summary--The latency of the tail-flick, following intrathecal infusion of the dopamine (DA) agonist, R-apomorphine was measured in rats with intact spinal cord or with spinal cord lesions. Apomorphine failed to influence the tail-flick response in intact rats, whereas it elevated the latency of the tail-flick in rats with either total spinal cord transection, or bilateral lesions of the dorsolaterat funiculi (DLF), but not in rats with lesions in either the dorsal columns or the medioventral parts of the lateral funiculi. The findings suggest that pathways in the dorsolateral funiculi modulate DA-mediated sensory processes and possible mechanisms are discussed. Key words: apomorphine, spinal dopamine mechanisms, nociceptive reflex, spinal cord lesions, dorsolateral funiculus, supraspinal control. The discovery of endogenous pain-inhibitory mechanisms has stimulated interest in the physiological and pharmacological properties of pain-modulatory systems. Previous studies have shown there to be several nociceptive inhibitory systems in the brain and spinal cord (for review, see Fields and Basbaum, 1978; Wall, 1978; Yaksh, Hammond and Tyce, 1981; Watkins and Mayer, 1982). Access of nociceptive information from the spinal cord to the brain may be reduced either directly, by an action exerted at the segmental level (Wall, 1978) or indirectly, by an effect mediated by descending fibre systems from the brain to the spinal cord (Wall, 1967; Hillman and Wall, 1969; Brown, 1971: Oliveras, Besson, Guilbaud and Liebeskind, 1974; Fields, Basbaum, Clanton and Anderson, 1977: Duggan and Griersmith, 1979). Several chemical substances such as enkephalin, noradrenaline, serotonin and substance P are known to play a role in mediating these segmental and suprasegmental modulating effects on spinal nociceptive processes (Headley, Duggan and Griersmith, 1978; Yaksh and Wilson, 1979; Yaksh, Farb, Leeman and Jessell, 1979; Proudfit and Hammond, 1980; Reddy and Yaksh, 1980). This report concerns the role of dopamine (DA) in the spinal cord (Barasi and Roberts, 1977; Commissiong and Neff, 1979; Demenge, Mouchet, Guerin and Feuerstein, 1981; Carp and Anderson, 1982; Skagerberg, Bj6rklund, Lindvall and Schmidt, 1982) on a spinal nociceptive reflex. Previously, it was reported that direct stimulation of spinal DA receptors (Demenge et al., 1981) by intrathecal (i.t.) infusion of DA agonists inhibited the thermonociceptive tail-flick response in spinal rats but not in rats with an intact neuraxis (Jensen and Smith, 1982, 1983b). Thus, these findings suggest that DA mech-
anisms may play a role in segmental nociceptive processes and that DA mechanisms in the spinal cord, involved in nociceptive processes at the segmental level, may be under the influence of descending inhibitory pathways. The present experiments were carried out to determine which spinal pathways are responsible for the differential effects of DA agonists on the tail-flick response in rats with an intact neuraxis and in rats with total spinal cord transection. METHODS
Animals Male Wistar rats, weighing 300-350 g were used. They were kept individually in clear plastic cages in a thermostatically controlled room (20°C) on a 12 hr light-dark cycle (lights on at 6 a.m.) and had free access to food and water.
Surgical procedures Rats under pentobarbital anaesthesia were implanted with an intrathecal catheter, using a modified version of the method described by Yaksh and Rudy (1976). Briefly, a PE-10 catheter was inserted through a slit in the cisterna magna and extended 7.5 cm down to the rostral edge of the lumbar enlargement. After catheterization and during the same anaesthesia, a laminectomy was done at T8-10. With the aid of a dissecting microscope, the dura was reflected carefully and either no cord lesion or various subtotal spinal cord lesions were made by crushing the cord tissue with a pair of microdissecting forceps.
Spinal nociceptive reflex measurement The spinal-mediated tail-flick test (D'Amour and Smith, 1941; Jensen and Smith, 1981) was assessed 149
150
T.S. JENSENet al.
18 hr after surgery when no residual anaesthesia was present. In the version of the test used here, the latency of withdrawal of the tail of a rat from a radiant heat source focussed 4 cm proximal to the tip of the tail was recorded. The intensity of the thermal stimulus was adjusted to provide a latency of 5.5-6sec in intact, untreated rats. Three tail-flick latencies were measured at 60 sec intervals both before and 5,10,20 and 90 min after intrathecal infusion of drug. The means of the tail-flick latencies were used for statistical analysis, carried out by multiple comparisons with unlesioned rats (Dunnett, 1964), unless otherwise stated.
Drugs The DA agonist R-apomorphine hydrochloride (Sandoz) (0.98#mol/kg) (And~n, Rubenson, Fuxe and H6kfelt, 1967), or its vehicle (0.001 M ascorbic acid), was administered by intrathecal injection in a volume of 50/al/kg, followed by 15#1 saline (0.9~ NaC1), to ensure delivery of the drug into the intrathecal space.
His t ology After testing was completed the rats were killed by an overdose of pentobarbital. Spinal cord segments, containing the lesions, were removed and the location of the catheter in the intrathecal space, without additional damage to the spinal cord, was checked upon removal. The thoracic cord segments, fixed in 2.5~ formalin solution were soaked in a 30~o (w/v) sucrose solution for 24 hr, frozen with CO2-snow and sectioned transversely at 30 #m with a cryostat. Alternate sections were stained with thionin for identification of cell bodies, while remaining sections were stained with a modified Fink-Heimer technique (Hjort-Simonsen, 1970) for study of fibre architecture. The extent of spinal cord lesions were reconstructed from superimposed serial sections. On the basis of location and extent of subtotal lesions, rats were assigned to one of 6 groups. Assignment was done by an investigator (H.D.S.) uninformed of the experimental findings in the tail-flick test. The following lesion groups were made: group 1: dorsal column lesion; group 2: dorsal column lesion with bilateral quadrant lesion, including the dorsolateral funiculi (DLF); group 3: bilateral quadrant lesion including the dorsolateral funiculi; group 4: bilateral quadrant lesion without involvement of dorsolateral funiculi; group 5: dorsal column lesion, and bilateral quadrant lesion without involvement of dorsolateral funiculi: and group 6: unilateral lesion of dorsolateral funiculi with or without dorsal column lesion. Thirty-eight rats were operated and tested. In 2 rats spinal cord lesions could not be assessed due to technical reasons and in another 6 rats the histological sections did not permit an accurate assessment of the extent of spinal cord lesions. These rats were eliminated from the study, so complete data were
obtained from 30 rats with subtotal spinal cord lesions.
RESULTS
Spinal lesions On the basis of histological examination, 4. rats were assigned to group 1, 6 to group 2, 5 to group 3, 4 to group 4, 6 to group 5, and 5 to group 6. Figure 1 presents a schematic drawing of the maximum extent of the spinal lesions. Table 1 shows the effects of both complete and subtotal spinal cord lesions on the tail-flick response latencies. The analysis of variance for baseline tail-flick latencies was significant ( F = 6.11, df7/38; P < 0.001), due to a signiticant reduction in latencies in rats with total spinal cord transection and in rats with bilateral quadrant lesions including dorsolateral funiculi. No other spinal cord lesions influenced the baseline tail-flick latencies significantly.
Effect of intrathecal administration of apomorpnine in the intact and spinally-lesioned rat Figure 2 shows the time course and magnitude of the effects of intrathecal infusion of either Rapomorphine or vehicle on the tail-flick latency in both spinal and intact rats. As shown previc)usly (Jensen and Smith, 1982, 1983a, b) R-apomorphine increased the tail-flick latency significantly in spinal rats at 5, 10 and 20min after intrathecal infusion, while it failed to influence the tail-flick latency significantly in intact rats. Table 1 presents the results observed in rats assigned to the different lesion groups after intrathecal infusion of apomorphine. Analysis of variance showed a main effect of spinal lesions at 5, 10 and 90 min after intrathecal infusion of drug (F = 113.49, 6.56 and 8.27, respectively; df7/38; P < 0.01). Further analysis indicated that in groups 2 and 3, apomorphine produced a significant increase of the tail-flick latency which did not differ from the effects produced by intrathecal injection of apomorphine after total spinal cord transection. Examination of the 2 groups indicated that the common factor was bilateral lesions of the dorsolateral funiculi. In contrast, apomorphine, given intrathecally, failed to influence the tail-flick latency significantly in rats in which the dorsolateral funiculus was left intact on either one or both sides of the spinal cord.
DISCUSSION As in previously reported work it has been demonstrated that apomorphine given intrathecall~, in spinally transected, but not intact rats increased the latency to generate a thermally-evoked, spinallymediated reflex (Jensen and Smith, 1982, 1983a, b). This effect appears to be mediated by spinal DA
Role of spinal pathways
151
Group 1
Group 2
Group 3
Group 4
Group 5
Group 6
Fig. 1. Maximal extent of spinal cord lesions, reconstructed from superimposed serial sections. Group 1: dorsal column lesion; Group 2: dorsal column lesion including dorsolateral funiculi (DLF); Group 3: bilateral quadrant lesion including DLF; Group 4: bilaterai quadrant lesion without DLF lesion: Group 5: dorsal column lesion and bilateral quadrant lesion without DLF involvement: Group 6: unilateral DLF lesion with or without dorsal column lesion.
receptors (Demenge et al., 1981) as the action of a p o m o r p h i n e was counteracted in a dose-dependent a n d stereospecific m a n n e r by D A antagonists but not by serotonergic, ~-adrenergic,/~-adrenergic or opioid antagonists. While the D A - m e d i a t e d inhibition of the tail-flick response in spinal transected rats could be due to a direct effect on the m o t o r c o m p o n e n t of the response, it is more likely that spinal D A receptor systems are primarily associated with afferent sensory function. Thus, in previous work (Jensen and Smith,
1983b) it was f o u n d t h a t stimulation of spinal D A receptors in the spinal animal facilitated tail withdrawal reflexes in response to light tactile stimuli, in addition to inhibiting the tail withdrawal response evoked by thermal, noxious stimuli. Moreover, in recent experiments it was observed t h a t a p o m o r p h i n e given intrathecally produced a dose-dependent elevation of the hot-plate response in the intact animal, which was counteracted by D A antagonists in a stereospecific way (Jensen and Yaksh, 1984). T a k e n
Table 1. Effects of spinal cord lesions on the tail-flick latency in rats before (baseline) and 5, 10, 20 and 90 min after the intrathecal injection of R-apomorphine (0.98 #mol/kg) Tail-flick latency (sec):~ N Baseline 5 10 Treatment groupt 8 5190_+0.11 5.38_+0.31 5.53_+0.30 Intact (sham operated) 8 4.31 _+0.06** 9.44_+0.70** 7.41 _+0.46** Total spinal cord transection Subtotal spinal cord transection 4 5.80-+0.11 5.68_+0.29 5.80+0.31 Group 1 6 5.25 _+0.40 8.53 + 0.72** 7.52 + 0.50** Group 2 5 4.44-+0.31"* 8.58_+0.50** 7.22_+0.48* Group 3 4 5.95_+0.19 5.95_+0.49 5.75_+0.10 Group 4 6 5.32_+0.45 5.03-+0.22 4.93-+0.31 Group 5 5 5.60_+0.24 6.74_+0.66 6.38_+0.22 Group 6 * and ** indicate P < 0.05 and P < 0.01, respectively, by one-way ANOVA with comparisons made to by Dunnett's test. t See text for surgical procedure and group assignment :~Mean+ SEM.
20 5.90_+0.13 6.49_+0.49
90 5.80_+0.08 4.20_+0.16"*
5.90_+0.11 5.65+0.13 5.92 -+0.60 5.03 _+0.30 5.94_+0.34 4.26_+0.22** 5.98-+0.18 5.83_+0.13 5.52_+0.34 5.72_+0.38 6.10_+0.25 5.52_+0.39 corresponding intact group values
152
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Fig. 2. Time course of tail-flick response latency in rats with intact spinal cord (circles) and in rats with total spinal cord transection (squares) given either 0.98#mol/kg Rapomorphine (filled symbols) or vehicle (unfilled symbols) by intrathecal injection. Each value represents mean _+SEM of 8 rats. *P < 0.001 compared to corresponding vehicletreated values by t-tests. together, these observations suggest that spinal DA receptors may serve to modulate afferent sensory transmission in the spinal cord. As part of continuing experiments designed to investigate the substrate by which the effect of apomorphine is made manifest, selective lesions of various parts of the spinal cord were carried out. In the present study, the effects of total spinal cord transection were found to be mimicked by partial lesions which bilaterally incorporated the dorsolateral funiculi. The fact that damage to the dorsal columns, and/or the medioventral aspects of the lateral funiculi had no effect rules out the possibility that nonspecific surgical trauma of the cord played a role in the alteration of tail-flick latency following intrathecal injection of apomorphine, and suggest that pathways in these systems are not relevant to the response in the spinal rats. While the present findings suggest that blockade of activity in the dorsolateral funiculi resulted in a lowering of the nociceptive threshold and in the appearance of inhibition of a spinal reflex mediated by a DA receptor they are unable to indicate the identity of the fibre systems responsible for these effects. Some previous observations, however, suggest that monoaminergic fibre systems originating in the lower brainstem and descending to the spinal cord may be involved. Thus, spinopetal serotonergic and noradrenergic systems, coursing in the dorsolateral funiculi are known to exert a tonic inhibitory effect on spinal nociceptive reflexes (Proudfit and Hammond, 1980; Zemlan, Corrigan and Pfaff, 1980; Jensen and Smith, 1983a). The present observation that bilateral lesions of the dorsolateral funiculi lowered nociceptive threshold is consistent with the modulating effect of these systems on nociceptive processes. In addition, the recent observations that inhibition of the tail-flick response induced by apomorphine can be produced in the intact animal by either systemic (Jensen and Smith,
1983a) or intrathecal (Jensen and Yaksh, 1984) pretreatment with serotonergic and noradrenergic receptor blockers, suggest that the modulation of spinal DA mechanisms by the dorsolateral funiculi is mediated in part by spinopetal serotonergic and noradrenergic pathways. It is tempting to speculate on the mechanisms by which spinopetal serotenergic and noradrenergic fibre systems coursing in the dorsolateral funiculi may modulate spinal DA-ergic mechanisms. There is evidence which shows that serotonin and noradrenaline may facilitate activity in the motor horn (McCall and Aghajanian, 1979; White and Neuman, 1980; Astrachan and Davis, 1981; Bell and Matsumiya, 1981). Thus, local application of serotonin and noradrenaline into the ventral horn by microinjection (Bell and Matsumiya, 1981) or by intrathecal injection (Astrachan and Davis, 1981) will facilitate the afferent-evoked ventral root reflex and the auditoryevoked startle response, respectively. It is suggested that the facilitatory influences exerted either directly or indirectly on the motor system will appear to physiologically antagonize the attenuation produced by the spinal DA receptor systems on the processing of the afferent stimulus in the dorsal horn which evokes the ventral root reflex. In the intact animal this makes it likely that a facilitation of activity in the motor horn is exerted by spinopetal monoamine systems. The present experiments suggest that, insofar as a spinal nociceptive reflex is concerned, the inhibitory effects mediated by the DA receptor would presumably be insufficient to depress the afterent drive and consequently block the reflex. Upon, spinal transection, bilateral lesions of the dorsolateral funiculi or spinal antagonism of the serotonergic and noradrenergic receptor systems (Yaksh and Wilson, 1979; Reddy and Yaksh, 1980), the loss of facilitation of motor horn cells would render the reflex pathway sensitive to the modulatory influences of the DA receptor. Acknowledgements--We thank Dr T. L. Yaksh for his
helpful suggestions in preparing this manuscript as well as Mrs Elin Kristensen, Mrs Lone Munko and Mr Bjarne Krunderup for technical assistance. This study was. supported by grants from Aarhus University Research Fund and Institute for Experimental Clinical Research, Aarhus University, to T.S.J.
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