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Facilitation of the tail-flick reflex by noxious cutaneous stimulation in the rat: antagonism by a substance P analogue
Brain Research, 462 (1988) 15-21
15
Elsevier BRE 13961
Facilitation of the tail-flick reflex by noxious cutaneous stimulation in the rat: antagonism by a substance P analogue Ruth A. Cridland and James L. Henry Departments of Psychiatry and Physiology, McGill University, Montreal, Que. (Canada) (Accepted 26 April 1988)
Key words: Substance P antagonist; Tail flick; Intrathecal; Nociception; Spinal cord; Rat
Effects of noxious cutaneous stimulation on the tail flick reflex were examined in the anaesthetized rat. Noxious stimulation was applied by immersing the distal 4 cm of the tail in water at 55 °C for 1.5 min. The tail flick reflex was tested at 3 min intervals by applying a noxious radiant heat stimulus to a region of the tail 10 cm proximal to the tip. Tail immersion reduced reaction time to tail flick by 30% and 20% at 0.5 and 3.5 min after immersion, respectively. Reaction time returned to control at 6.5 min and tended to increase above baseline values at 9.5 and 12.5 min. Naloxone (10 mg/kg, i.p.) potentiated the effects of tail immersion on reaction time and prevented the increase above baseline. When the surface temperature of the skin used to evoke the tail flick reflex was raised by I0 °C using innocuous radiant heat, reaction time was not significantlydifferent from the control, suggesting that an increase in skin temperature per se is insufficient to account for the response to immersion. Intrathecal administration of a substance P antagonist (1 nmol) attenuated the response to tail immersion. These results indicate that noxious cutaneous stimulation may release an agent in the spinal cord which facilitates the tail flick reflex, and that this agent is antagonized by a substance P antagonist.
INTRODUCTION Anatomical, biochemical and physiological evidence suggests that substance P may be involved in regulating the excitability of spinal neurones involved in the transmission of nociceptive information (for review see ref. 20). Iontophoretic application of substance P onto dorsal horn n e u r o n e s in the cat excites those neurones which are activated by application of a noxious radiant heat stimulus to the cutaneous receptive field t2'21.24.25, and in our earlier e x periments we have observed that intrathecal administration of substance P in the rat produces a transient decrease in reaction time to tail withdrawal from a noxious radiant heat stimulus4'3°-32. The subsequent report that noxious thermal stimulation of the skin releases substance P-like immunoreactivity in the dorsal horn 7 prompted an extension of our earlier work, to determine the effects of noxious cutaneous stimulation on reaction time in the tail flick test.
MATERIALS AND METHODS
Anaesthesia Experiments were done on lightly anaesthetized male S p r a g u e - D a w l e y rats (250-350 g). Anaesthesia was induced by an i.p. injection of a solution of sodium pentobarbital (20 mg/kg) and chloral hydrate (120 mg/kg) mixed just prior to injection. A supplemental dose of sodium pentobarbital (9 mg/kg) and chloral hydrate (20 mg/kg) was given in the rare cases in which the initial dose was insufficient to produce the desired level of anaesthesia.
Measurement of reaction time in the tail flick test To elicit the tail withdrawal reflex, the tail was placed above a projector bulb which was focussed 10-12 cm proximal to the tip of the tail; this region was blackened to promote the absorption of heat. Hick of the tail exposed the light beam to a photodetector which in turn stopped the beam and a timer,
Correspondence: J.L. Henry, Departments of Psychiatry and Physiology, McGill University, 3655 Drummond Street, Montreal, Que. H3G 1Y6, Canada. 0006-8993/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
16 displaying the reaction time m e a s u r e d to one hundredth of a second j3. Reaction time to tail withdrawal was measured at 3 min intervals. Three successive readings were taken to establish the baseline reaction time. The experiment was not continued unless t h e s e three reaction times had a S.D. less than 10% of the mean. The intensity of the bulb was set so that the baseline reaction time was 5 - 6 s and was not adjusted thereafter. Trials were terminated automatically if a tail flick did not occur within 10 s.
Prolonged noxious cutaneous stimulation by tail immersion A f t e r 3 readings were taken to establish the baseline reaction time, intense noxious stimulation was applied to the tail by immersing the distal 4 cm in water maintained at 55 + 1 °C for 1.5 min. T h e r e was usually an initial withdrawal reflex but this subsided rapidly so that the tail r e m a i n e d flaccid in the water for the r e m a i n d e r of the immersion period. I m m e r sion was timed to end 0.5 min prior to the next test for reaction time. Five more readings were then taken, and the animal was sacrificed with an overdose of halothane before recovery from the anaesthesia. In a group of animals, naloxone hydrochloride (10 mg/kg) was administered i.p. 5 rain prior to establishing the mean baseline reaction time and the e x p e r i m e n t was carried out as above except that, after 4 readings had been taken, the tail was i m m e r s e d a second time and an additional 4 readings were taken. In a separate series of experiments, the skin temperature of the base of the tail was m o n i t o r e d to determine to what extent the surface t e m p e r a t u r e had increased following immersion of the tail. A thermistor (Yellow Springs) was taped to the ventral surface of the tail in the same position where the light was focussed to elicit the tail flick reflex in earlier experiments (10 cm from the tip of the tail). Baseline skin t e m p e r a t u r e was established and the distal 4 cm of the tail was i m m e r s e d in water at 55 °C as described above. Readings of skin t e m p e r a t u r e were taken during tail immersion and thereafter at 30 s intervals for a 10 rain period. As the experiments indicated that tail immersion increased skin t e m p e r a t u r e at the site from which the tail flick reflex was elicited, experiments were done in which the skin t e m p e r a t u r e was raised by innocuous means using a 250 W infrared heat lamp. This
was done to determine if an increase in skin temperature per se could account for the decrease in reaction time. A thermistor, shielded with reflecting material except for the portion held against the skin, was positioned on the ventral surface of the tail immediately adjacent to the region where the light was focussed to elicit the tail flick reflex. Three baseline reaction times were taken, and the skin t e m p e r a t u r e was raised using the heat lamp. (It was necessary to use a heat lamp rather than a warm bath because of the cooling effect of the water when the tail was removed from the bath.) W h e n the skin t e m p e r a t u r e was raised to 10 °C above the baseline t e m p e r a t u r e , reaction time was again determined.
Intrathecal administration Trp9]substance P
of
[D-Pro 2, o-Phe 7,D-
In an additional series of experiments, rats (225-250 g) were i m p l a n t e d with an indwelling intrathecal catheter under chloral hydrate anaesthesia (300 mg/kg). A polyethylene catheter (Intramedic, PE-10) was inserted through an incision in the dura at the atlanto-occipital junction. Spinous processes were used as landmarks to position the catheter so that the inner tip lay at the 5th lumbar vertebral level. This level of administration was chosen because it allowed access of the injectate into the grey matter of the sacrococcygeal regions of the spinal cord 6 where the afferent fibres of the tail flick reflex have been shown to terminate 11. The catheter was fixed with dental cement to a screw e m b e d d e d in the skull. Postmortem examination confirmed that the inner catheter tip lay at the lower l u m b a r vertebral level. The rats were allowed to recover over 4 days following implantation of the catheter, and only those animals which were free of any neurological deficit were used in subsequent experiments. [D-Pro2,D-Phe7,D-Trpg]substance P was purchased from Institut A r m a n d - F r a p p i e r (lot A15563); the purity was 97.7% (as d e t e r m i n e d by H P L C ) and the peptide content (as d e t e r m i n e d by amino acid analysis) was 95.0%. The solution of the substance P analogue was divided into 100 ktl aliquots on the day of preparation and kept frozen a t - 4 °C until used. During the experiment, the aliquot of solution was kept on ice until use. Baseline reaction time was established after pretreatment with naloxone (10 mg/kg, i.p.) as de-
17 scribed above.
125
[D-Pro2,D-Phe7,D-Trp9]substance P
(1.0 nmol as d e t e r m i n e d from the p e p t i d e content), was delivered via the intrathecal c a t h e t e r in 10 ktl of artificial cerebrospinal fluid (CSF; aqueous solution of 128.6 m M NaC1, 2.6 m M KC1, 2.0 m M MgC12 and 1.4 m M CaC12; p h o s p h a t e - b u f f e r e d , p H 7.33). This was followed by an additional injection of 10/~1 of artificial C S F to flush the catheter. The catheter volume was a p p r o x i m a t e l y 8/A. The intrathecal injection was delivered over a period of 1.5 min and was timed to end 1 min prior to the next test for reaction time. Three m e a s u r e m e n t s of reaction time were taken, and the tail was i m m e r s e d in water at 55 °C as described above. In control experiments, 20/~1 of artificial C S F were given in place of the substance P analogue/CSF injections.
Analysis of data D a t a were analyzed by an analysis of variance ( A N O V A ) , with intrathecal administration (substance P analogue vs CSF) as the between-subject factor and time as the within-subject factor. D a t a from experiments without intrathecal injections were analyzed with a one-way repeated measure A N O V A . Post-hoc comparisons between means were m a d e using T u k e y ' s Wholly Significant Difference test ~s. A paired Student's t-test was used to c o m p a r e reaction times before and after increasing skin t e m p e r a t u r e . RESULTS The level of anaesthesia was sufficient to prevent any overt sign of discomfort to the rat during experimentation, yet a stable response could be o b t a i n e d in the tail flick test for about 45 min. Pinching of the tail or hindleg p r o d u c e d only a withdrawal response; there was no vocalization, spontaneous m o v e m e n t or visible sign of distress.
Effects of tail immersion on reaction time in the tail flick test I m m e r s i o n of the tip of the tail decreased reaction time to 69.6 _+ 2.8% (mean + S . E . M . ; n = 5) and 81.5 _+ 5.1% of the mean control value at 0.5 and 3.5 min after tail immersion, respectively, as illustrated in Fig. 1. The baseline reaction time prior to tail immersion was 5.92 _+ 0.20 s. The r e p e a t e d - m e a s u r e s A N O V A yielded a significant trial factor (F5,20 =
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Time (3 min intervals) Fig. 1. Effects on reaction time in the tail flick test of immersion of the tip of the tail in water at 55 °C. Reaction time was measured at 3 rain intervals. The ordinate shows reaction time expressed as a mean percent of pretreatment values + S.E.M. (n = 5). The dashed vertical line represents the time of removal of the tail following immersion for 1.5 min in heated water (0.5 min prior to the next test for reaction time). **, P < 0.01, significantly different from mean baseline reaction time.
14.51, P < 0.001). T u k e y ' s test revealed that the tail flick latencies were significantly different from pretreatment values at 0.5 and 3.5 min after tail immersion (P < 0.01). Reaction time had r e t u r n e d to control values by 6.5 min after tail immersion ( P > 0.05) and thereafter increased to above control values, although the increase was not significant ( P > 0.05). Immersion of tip of the tail in water at 55 °C increased the surface skin t e m p e r a t u r e at the region where the tail flick reflex was elicited to 6 - 8 °C above the baseline skin t e m p e r a t u r e (n = 4). However, raising the skin t e m p e r a t u r e of the same region by application of innocuous radiant heat failed to alter the reaction time significantly (t = 1.49, P > 0.05), even when the t e m p e r a t u r e was raised by 10 °C. Reaction time in this case was 91.1 + 7.3% of the mean baseline value (n = 6). Baseline t e m p e r a t u r e s of the surface of the skin were 2 0 - 2 3 °C. Immersion of the tip of the tail in water at 55 °C was without effect on core body t e m p e r a t u r e as measured by a rectal t h e r m o m e t e r (n = 4). Baseline core body t e m p e r a t u r e s were 3 6 - 3 7 °C. Following the decrease in reaction time induced by
18 tail immersion, there was a tendency for reaction time to increase above control values. In our previous experiments with substance P, it was observed that the full response to intrathecal administration of substance P included an indirect activation of an opioid mechanism which counteracted the facilitatory effects of substance P on reaction time 3°. Therefore, in the present study, animals were pretreated with naloxone. Naloxone pretreatment potentiated the decrease in reaction time following tail immersion and prevented the subsequent overshoot, allowing two tests of tail immersion within the same experiment. The reproducibility of the effects of repeated tail immersion in animals pretreated with 10 mg/kg of naloxone i.p. are illustrated in Fig. 2. Naloxone treatment was without effect on baseline reaction time (see absolute values for reaction times in different treatment groups). The repeated-measures A N O V A revealed a sig-
Effects of administration of [t)-Pro2,/~-Phe',/~Trpg]substance P on the response to tail immersion In pilot experiments, a n u m b e r of different doses (up to 6.5 nmol) of the substance P antagonist were surveyed. After intrathecal administration of the higher closes of the substance P antagonist, many of the animals failed to withdraw the tail in the tail flick test. These animals were not tested with tail immersion, and upon waking they exhibited a flaccid paralysis similar to that reported by others r'2~'31. For the present study, a dose of 1 nmol was selected because preliminary results indicated that this dose has minimal effects on motor control. Nonetheless, some rats exhibited altered baseline reaction times as a result of the substance P antagonist alone; of the 15 rats injected, 4 failed to flick the tail at 1-7 rain after the injection. These rats were not included in the results. In the remaining 11 rats, the tail flick response was unaltered by the intrathecal administration of the
nificant trial factor (Fs.40 = 15.16, P < 0.001). Reaction time was 5.20 + 0.45 s prior to tail immersions.
substance P analogue alone. Intrathecal administration of [D-Pro2,D-PheT,D-
At 0.5 and 3.5 min after the first tail immersion, reac-
Trpg]substance P significantly attenuated the effects
tion time decreased to 58.2 + 3.6% and 76.1 _+ 7.9%, respectively, of the mean control value (P < 0.01, n = 6). Reaction time was not significantly different
of tail immersion on reaction time (Fig. 3). The A N O V A yielded a significant intrathecal administration x time interaction (F2,128 = 5.55, P < 0.005). Tukey's test revealed that the animals which received
from pretreatment values over the next two readings (P > 0.05). Immersion of the tail a second time produced a similar response in terms of both magnitude
125
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Fig. 2. Effects on reaction time of two successive noxious stimulations of the tail in rats pretreated with naloxone (10 mg/kg, i.p. ; n = 6). The dashed vertical lines represent the times of removal of the tail following immersion (0.5 min prior to the next for reaction time). **, P < 0.01, significantly different from mean baseline reaction time. Other details are as in Fig. 1.
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Fig. 3. Effects of intrathecal administration of 1 nmol of [DPro2,D_PheT,D.Trpg]substance P (©: n = 11) and of artificial cerebrospinal fluid (0, n = 7) on the decrease in reaction time induced by tail immersion. The arrow represents the time of intrathecal injection (1 min prior to the next test for reaction time) and the vertical dashed line represents the time of tail removal following tail immersion (0.5 min prior to the next test for reaction time). **, P < 0.01, *, P < 0.05, significantly different from respective values in CSF-treated animals. Other details are as in Fig. 1.
19 the substance P analogue had different reaction times from those which received CSF at 0.5 min (P < 0.01), 3.5 rain (P < 0.05) and 6.5 min (P < 0.01) after tail immersion but not at any other time tested (P >
0.05). In rats treated with the substance P analogue, reaction time decreased to 85.4 __ 4.3% and 89.2 + 5.1% of the mean baseline value at 0.5 min and 3.5 min after tail immersion, respectively (n = 11). Prior to administration of the substance P antagonist, reaction time was 5.21 + 0.16 s. Reaction time had returned to control values by 6.5 min after tail immersion. When 20/A of CSF was given in the place of the substance P analogue solution, reaction time decreased to 53.2 + 3.9% and 77.3 + 5.2% of the mean control values at 0.5 min and 3.5 min after tail immersion, respectively (n = 7). Prior to administration of CSF, reaction time was 5.36 + 0.35 s. By 9.5 min after tail immersion, reaction time had returned to control values. DISCUSSION The use of the lightly anaesthetized rat to study the tail flick reflex is a well established technique, particularly for those experiments that require invasive surgery 3'1°'26. In fact, other investigators 3'1° have reported that the tail flick reflex in the anaesthetized rat is more pronounced than that in the awake rat. Our experience confirms this observation. In the present study, immersion of the tip of the tail in water at 55 °C significantly decreased reaction time when the tail flick reflex was evoked from a more proximal region. This decrease in reaction time could be elicited a second time within 12 rain, suggesting that the immersion did not produce any longstanding changes in sensitivity in the tail which would affect the response to tail immersion, at least within the time frame of this experiment. Opioid involvement in the response Following the initial decrease in reaction time after tail immersion, reaction time increased to above baseline values. In previous studies, we have observed that intrathecal administration of substance P in the awake rat produces effects which are similar in time course to those observed in the present study, a decrease in reaction time followed by a naloxone-
reversible overshoot 3°. The results of the present study suggest that the increase in reaction time observed after tail immersion may be mediated by an opioid factor. They show further the'similarity between the substance P-induced decrease in reaction time and that observed upon noxious cutaneous stimulation. Substance P involvement in the response Substance P antagonists have been used effectively to block the responses mediated by exogenous substance P within the CNS 1'22'31. Furthermore, a substance P antagonist markedly depresses a reflex in the isolated spinal cord-tail preparation of the rat which is induced by high-intensity electrical stimulation of the dorsal root 29 or by the application of capsaicin 28. [o-Pro2,D-Phe7,D-Trp9]substance P was used in the present study because previously it has been found to block the effects of intrathecally administered substance P on reaction time in the tail flick test 31 and on a caudally directed scratching and biting behaviour 22. The ability of this analogue to attenuate the decrease in reaction time suggests that endogenously released substance P may have been involved in the immersion-induced facilitation of the tail flick reflex. Of course the possibility must be considered that the attenuation of the response to tail immersion by the substance P antagonist may be due to a neurotoxic action of this analogue17,23; however, this is unlikely because the analogue was without effect on baseline reaction time in the animals tested. Therefore, the results from the present study are interpreted as adding further support to the suggestion that substance P is involved in the transmission of thermal nociceptive information within the spinal cord 12. The small decrease in reaction time in rats pretreated with the substance P antagonist may suggest that there was an incomplete block of substance P receptors by the analogue or that another chemical besides substance P may be involved in the mediation of this response. The possibility that noxious cutaneous stimulation induces the release of substance P within the spinal cord is consistent with earlier studies. Electrical stimulation of the sciatic nerve in the cat at intensities sufficient to recruit A 6 and C fibres has been shown to release substance P-like immunoreactivity (-LI) into a spinal cord superfusate 27 and in the dorsal horn as
20
measured by microdialysis 2. Immersion of the hind paw of the cat in water at 50 and 52 °C but not at 37 °C releases substance P in the substantia gelatinosa of the spinal cord as m e a s u r e d by the inhibition of binding of 125I-substance P to antibody-laden microprobes 7. Noxious mechanical 15 and chemogenic 14 stimulation of primary afferents have also been reported to increase substance P-LI in the dorsal horn. Mediation by a peripheral or central action
While we have been assuming that the response to tail immersion is due to a facilitation of nociceptive transmission within the spinal cord, alternative possibilities must be considered which might explain the observed decrease in reaction time. For example, if the skin t e m p e r a t u r e at the site activated to p r o v o k e the tail flick reflex were increased, the respective thermal nociceptors would require less heat to reach threshold and consequently the latency of the response should decrease. Immersion of the tip of the tail did increase skin t e m p e r a t u r e at this site by 6 - 8 °C from 2 0 - 2 3 °C. H o w e v e r , raising the skin t e m p e r a t u r e at this site by innocuous means by even 10 °C failed to alter reaction time. Thus, an increase in skin t e m p e r a t u r e per se is insufficient to account for the m a r k e d facilitation of the tail flick reflex. As r e p e a t e d application of noxious thermal stimuli has been shown to sensitize the responses of unmyelinated 7A6"E9 and m y e l i n a t e d nociceptive afferents 9'19, prolonged heating of the tail in the present study could have sensitized the thermal nociceptors in the tail, thereby lowering the threshold and de-
REFERENCES 1 Akerman, B., Rosell, S. and Folkers, K., Intrathecal [DProZ,D-Trp7'9]Sp elicits hypoalgesia and motor blockade in the rat and antagonizes noxious responses induced by substance P, Acta Physiol. Scand., 114 (1982) 631-633. 2 Brodin, E., Linderoth, B., Gazelius, B. and Ungerstedt, U., In vivo release of substance P in cat dorsal horn studied with microdialysis, Neurosci. Lett., 76 (1987) 357-362. 3 Cargill, C.L., Steinman, J.L. and Willis, W.D., A fictive tail flick reflex in the rat, Brain Research, 345 (1985) 45-53. 4 Cridland, R.A. and Henry, J.L., Comparison of the effects of substance P, neurokinin A, physalaemin and eledoisin in facilitating a nociceptive reflex in the rat, Brain Research, 381 (1986) 93-99. 5 Cridland, R.A. and Henry, J.L., Intrathecal administration
creasing reaction time. F u r t h e r m o r e , immersion of the tail may have p r o d u c e d tissue injury and oedema. With massive tissue damage or stimulation of a large receptive field, sensitization may spread to an area which lies outside the stimulated area s~
of substance P in the rat: spinal transection or morphine blocks the behavioural responses but not the facilitation of the tail flick reflex, Neurosci. Lett., 84 (1988) 203-208. 6 Cridland, R.A., Yashpal, K., Romita, V.V., Gauthicr, S. and Henry, J.L., Distribution of label after intrathecal administration of 125I-substanceP in the rat, Peptides, 8 (1987) 213-221. 7 Duggan, A.W., Morton, C.R., Zhao, Z.Q. and Hendry, I.A., Noxious heating of the skin releases immunoreactive substance P in the substantia gelatinosa of the cat: a study with antibody microprobes, Brain Research, 403 (1987) 345-349. 8 Fitzgerald, M., The sensitization of cutaneous nociceptors by spread from a nearby injury and its blockade by local anaesthesia, Proc. Physiol. Soc., 278 (1978) 44P-45P. 9 Fitzgerald, M. and Lynn, B., The sensitization of high
21 threshold mechanoreceptors with myelinated axons by repeated heating, J. Physiol., 265 (1977') 549-563. 10 Gebhart, G.F. and Ossipov, M.H., Characterization of inhibition of the spinal nociceptive tail-flick reflex in the rat from the medullary lateral reticular nucleus, J. Neurosci., 6 (1986) 701-713. 11 Grossman, M.L., Basbaum, A.I. and Fields, H.L., Afferent and efferent connections of the rat tail flick reflex (a model used to analyze pain control mechanisms), J. Comp. Neurol., 206 (1982) 9-16. 12 Henry, J.L., Effects of substance P on functionally identified units in cat spinal cord, Brain Research, 114 (1976) 439-451. 13 Isabel, G., Wright, D.M. and Henry, J.L., Design for an inexpensive unit for measuring tail flick latencies, J. Pharmacol. Methods, 5 (1981) 241-247. 14 Kantner, R.M., Kirby, M.L. and Goldstein, B.D., Increase in substance P in the dorsal horn during a chemogenic nociceptive stimulus, Brain Research, 338 (1985) 196-199. 15 Kuraishi, Y., Hirota, N., Sugimoto, M., Satoh, M. and Takagi, H., Effects of morphine on noxious stimuli-induced release of substance P from rabbit dorsal horn in vivo, Life Sci., 33 (1983) 693-696. 16 Lynn, B., Cutaneous hyperalgesia, Brit. Med. Bull., 33 (1977) 103-108. 17 Matsumura, H., Sakurada, T., Hara, A., Kuwahara, H., Ando, R., Sakurada, S. and Kisara, K., Intrathecal substance P analogue causes motor dysfunction in the rat, Neuropharmacology, 24 (1985) 811-813. 18 Myers, J.L., Fundamentals of Experimental Design, 3rd edn., Allyn and Bacon, Boston, 1979. 19 Perl, E.R., Kumazawa, T., Lynn, B. and Kenins, P., Sensitization of high threshold receptors with unmyelinated (C) afferent fibers, Progr. Brain Res., 43 (1976) 263-276. 20 Pernow, B., Substance P, Pharmacol. Rev., 35 (1983) 85-141. 21 Piercey, M.F., Einspahr, F.J., Dobry, P.J.K., Schroeder, L.A. and Hollister, R.P., Morphine does not antagonize the substance P mediated excitation of dorsal horn neurons,
Brain Research, 186 (1980) 421-434. 22 Piercey, M.F., Schroeder, L.A., Folkers, K., Xu, J.-C. and Horig, J., Sensory and motor functions of spinal cord substance P, Science, 214 (1981) 1361-1363. 23 Post, C. and Paulsson, I., Antinociceptive and neurotoxic actions of substance P analogues in the rat's spinal cord after intrathecal administration, Neurosci. Lett., 57 (1985) 159-164. 24 Randi6, M. and Mileti6, V., Effect of substance P in cat dorsal horn neurones activated by noxious stimuli, Brain Research, 128 (1977) 164-169. 25 Sastry, B.R., Substance P effects on spinal nociceptive neurones, Life Sci., 24 (1979) 2169-2178. 26 Vanegas, H., Barbaro, N.M. and Fields, H.L., Midbrain stimulation inhibits tail-flick only at currents sufficient to excite rostral medullary neurons, Brain Research, 321 (1984) 127-133. 27 Yaksh, T.L., Jessel, T.M., Gamse, R., Mudge, A~W. and Leeman, S.E., Intrathecal morphine inhibits substance P release from mammalian spinal cord in vivo, Nature (Lond.), 286 (1980) 155-157. 28 Yanagisawa, M., Otsuka, M., Konishi, S., Akagi, H., Folkers, K. and Rosell, S., A substance P antagonist inhibits a slow reflex response in the spinal cord of the newborn rat, A cta Physiol. Scand. , 116 (1982) 109-112. 29 Yanagisawa, M. and Otsuka, M., The effect of a substance P antagonist on chemically induced nociceptive reflex in the isolated spinal cord-tail preparation of the newborn rat, Proc. Jap. Acad., 60 (1984) 427-430. 30 Yashpal, K. and Henry, J.L., Endorphins mediate overshoot of substance P-induced facilitation of a spinal nociceptive reflex, Can. J. Physiol. Pharmacol., 61 (1983) 303-307. 31 Yashpal, K. and Henry, J.L., Substance P analogue blocks SP-induced facilitation of a spinal nociceptive reflex, Brain Res. Bull., 13 (1984) 597-600. 32 Yashpal, K., Wright, D.M. and Henry, J.L., Substance P reduces tail-flick latency: implication for chronic pain syn-, dromes, Pain, 14 (1982) 155-167.
15
Elsevier BRE 13961
Facilitation of the tail-flick reflex by noxious cutaneous stimulation in the rat: antagonism by a substance P analogue Ruth A. Cridland and James L. Henry Departments of Psychiatry and Physiology, McGill University, Montreal, Que. (Canada) (Accepted 26 April 1988)
Key words: Substance P antagonist; Tail flick; Intrathecal; Nociception; Spinal cord; Rat
Effects of noxious cutaneous stimulation on the tail flick reflex were examined in the anaesthetized rat. Noxious stimulation was applied by immersing the distal 4 cm of the tail in water at 55 °C for 1.5 min. The tail flick reflex was tested at 3 min intervals by applying a noxious radiant heat stimulus to a region of the tail 10 cm proximal to the tip. Tail immersion reduced reaction time to tail flick by 30% and 20% at 0.5 and 3.5 min after immersion, respectively. Reaction time returned to control at 6.5 min and tended to increase above baseline values at 9.5 and 12.5 min. Naloxone (10 mg/kg, i.p.) potentiated the effects of tail immersion on reaction time and prevented the increase above baseline. When the surface temperature of the skin used to evoke the tail flick reflex was raised by I0 °C using innocuous radiant heat, reaction time was not significantlydifferent from the control, suggesting that an increase in skin temperature per se is insufficient to account for the response to immersion. Intrathecal administration of a substance P antagonist (1 nmol) attenuated the response to tail immersion. These results indicate that noxious cutaneous stimulation may release an agent in the spinal cord which facilitates the tail flick reflex, and that this agent is antagonized by a substance P antagonist.
INTRODUCTION Anatomical, biochemical and physiological evidence suggests that substance P may be involved in regulating the excitability of spinal neurones involved in the transmission of nociceptive information (for review see ref. 20). Iontophoretic application of substance P onto dorsal horn n e u r o n e s in the cat excites those neurones which are activated by application of a noxious radiant heat stimulus to the cutaneous receptive field t2'21.24.25, and in our earlier e x periments we have observed that intrathecal administration of substance P in the rat produces a transient decrease in reaction time to tail withdrawal from a noxious radiant heat stimulus4'3°-32. The subsequent report that noxious thermal stimulation of the skin releases substance P-like immunoreactivity in the dorsal horn 7 prompted an extension of our earlier work, to determine the effects of noxious cutaneous stimulation on reaction time in the tail flick test.
MATERIALS AND METHODS
Anaesthesia Experiments were done on lightly anaesthetized male S p r a g u e - D a w l e y rats (250-350 g). Anaesthesia was induced by an i.p. injection of a solution of sodium pentobarbital (20 mg/kg) and chloral hydrate (120 mg/kg) mixed just prior to injection. A supplemental dose of sodium pentobarbital (9 mg/kg) and chloral hydrate (20 mg/kg) was given in the rare cases in which the initial dose was insufficient to produce the desired level of anaesthesia.
Measurement of reaction time in the tail flick test To elicit the tail withdrawal reflex, the tail was placed above a projector bulb which was focussed 10-12 cm proximal to the tip of the tail; this region was blackened to promote the absorption of heat. Hick of the tail exposed the light beam to a photodetector which in turn stopped the beam and a timer,
Correspondence: J.L. Henry, Departments of Psychiatry and Physiology, McGill University, 3655 Drummond Street, Montreal, Que. H3G 1Y6, Canada. 0006-8993/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
16 displaying the reaction time m e a s u r e d to one hundredth of a second j3. Reaction time to tail withdrawal was measured at 3 min intervals. Three successive readings were taken to establish the baseline reaction time. The experiment was not continued unless t h e s e three reaction times had a S.D. less than 10% of the mean. The intensity of the bulb was set so that the baseline reaction time was 5 - 6 s and was not adjusted thereafter. Trials were terminated automatically if a tail flick did not occur within 10 s.
Prolonged noxious cutaneous stimulation by tail immersion A f t e r 3 readings were taken to establish the baseline reaction time, intense noxious stimulation was applied to the tail by immersing the distal 4 cm in water maintained at 55 + 1 °C for 1.5 min. T h e r e was usually an initial withdrawal reflex but this subsided rapidly so that the tail r e m a i n e d flaccid in the water for the r e m a i n d e r of the immersion period. I m m e r sion was timed to end 0.5 min prior to the next test for reaction time. Five more readings were then taken, and the animal was sacrificed with an overdose of halothane before recovery from the anaesthesia. In a group of animals, naloxone hydrochloride (10 mg/kg) was administered i.p. 5 rain prior to establishing the mean baseline reaction time and the e x p e r i m e n t was carried out as above except that, after 4 readings had been taken, the tail was i m m e r s e d a second time and an additional 4 readings were taken. In a separate series of experiments, the skin temperature of the base of the tail was m o n i t o r e d to determine to what extent the surface t e m p e r a t u r e had increased following immersion of the tail. A thermistor (Yellow Springs) was taped to the ventral surface of the tail in the same position where the light was focussed to elicit the tail flick reflex in earlier experiments (10 cm from the tip of the tail). Baseline skin t e m p e r a t u r e was established and the distal 4 cm of the tail was i m m e r s e d in water at 55 °C as described above. Readings of skin t e m p e r a t u r e were taken during tail immersion and thereafter at 30 s intervals for a 10 rain period. As the experiments indicated that tail immersion increased skin t e m p e r a t u r e at the site from which the tail flick reflex was elicited, experiments were done in which the skin t e m p e r a t u r e was raised by innocuous means using a 250 W infrared heat lamp. This
was done to determine if an increase in skin temperature per se could account for the decrease in reaction time. A thermistor, shielded with reflecting material except for the portion held against the skin, was positioned on the ventral surface of the tail immediately adjacent to the region where the light was focussed to elicit the tail flick reflex. Three baseline reaction times were taken, and the skin t e m p e r a t u r e was raised using the heat lamp. (It was necessary to use a heat lamp rather than a warm bath because of the cooling effect of the water when the tail was removed from the bath.) W h e n the skin t e m p e r a t u r e was raised to 10 °C above the baseline t e m p e r a t u r e , reaction time was again determined.
Intrathecal administration Trp9]substance P
of
[D-Pro 2, o-Phe 7,D-
In an additional series of experiments, rats (225-250 g) were i m p l a n t e d with an indwelling intrathecal catheter under chloral hydrate anaesthesia (300 mg/kg). A polyethylene catheter (Intramedic, PE-10) was inserted through an incision in the dura at the atlanto-occipital junction. Spinous processes were used as landmarks to position the catheter so that the inner tip lay at the 5th lumbar vertebral level. This level of administration was chosen because it allowed access of the injectate into the grey matter of the sacrococcygeal regions of the spinal cord 6 where the afferent fibres of the tail flick reflex have been shown to terminate 11. The catheter was fixed with dental cement to a screw e m b e d d e d in the skull. Postmortem examination confirmed that the inner catheter tip lay at the lower l u m b a r vertebral level. The rats were allowed to recover over 4 days following implantation of the catheter, and only those animals which were free of any neurological deficit were used in subsequent experiments. [D-Pro2,D-Phe7,D-Trpg]substance P was purchased from Institut A r m a n d - F r a p p i e r (lot A15563); the purity was 97.7% (as d e t e r m i n e d by H P L C ) and the peptide content (as d e t e r m i n e d by amino acid analysis) was 95.0%. The solution of the substance P analogue was divided into 100 ktl aliquots on the day of preparation and kept frozen a t - 4 °C until used. During the experiment, the aliquot of solution was kept on ice until use. Baseline reaction time was established after pretreatment with naloxone (10 mg/kg, i.p.) as de-
17 scribed above.
125
[D-Pro2,D-Phe7,D-Trp9]substance P
(1.0 nmol as d e t e r m i n e d from the p e p t i d e content), was delivered via the intrathecal c a t h e t e r in 10 ktl of artificial cerebrospinal fluid (CSF; aqueous solution of 128.6 m M NaC1, 2.6 m M KC1, 2.0 m M MgC12 and 1.4 m M CaC12; p h o s p h a t e - b u f f e r e d , p H 7.33). This was followed by an additional injection of 10/~1 of artificial C S F to flush the catheter. The catheter volume was a p p r o x i m a t e l y 8/A. The intrathecal injection was delivered over a period of 1.5 min and was timed to end 1 min prior to the next test for reaction time. Three m e a s u r e m e n t s of reaction time were taken, and the tail was i m m e r s e d in water at 55 °C as described above. In control experiments, 20/~1 of artificial C S F were given in place of the substance P analogue/CSF injections.
Analysis of data D a t a were analyzed by an analysis of variance ( A N O V A ) , with intrathecal administration (substance P analogue vs CSF) as the between-subject factor and time as the within-subject factor. D a t a from experiments without intrathecal injections were analyzed with a one-way repeated measure A N O V A . Post-hoc comparisons between means were m a d e using T u k e y ' s Wholly Significant Difference test ~s. A paired Student's t-test was used to c o m p a r e reaction times before and after increasing skin t e m p e r a t u r e . RESULTS The level of anaesthesia was sufficient to prevent any overt sign of discomfort to the rat during experimentation, yet a stable response could be o b t a i n e d in the tail flick test for about 45 min. Pinching of the tail or hindleg p r o d u c e d only a withdrawal response; there was no vocalization, spontaneous m o v e m e n t or visible sign of distress.
Effects of tail immersion on reaction time in the tail flick test I m m e r s i o n of the tip of the tail decreased reaction time to 69.6 _+ 2.8% (mean + S . E . M . ; n = 5) and 81.5 _+ 5.1% of the mean control value at 0.5 and 3.5 min after tail immersion, respectively, as illustrated in Fig. 1. The baseline reaction time prior to tail immersion was 5.92 _+ 0.20 s. The r e p e a t e d - m e a s u r e s A N O V A yielded a significant trial factor (F5,20 =
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Time (3 min intervals) Fig. 1. Effects on reaction time in the tail flick test of immersion of the tip of the tail in water at 55 °C. Reaction time was measured at 3 rain intervals. The ordinate shows reaction time expressed as a mean percent of pretreatment values + S.E.M. (n = 5). The dashed vertical line represents the time of removal of the tail following immersion for 1.5 min in heated water (0.5 min prior to the next test for reaction time). **, P < 0.01, significantly different from mean baseline reaction time.
14.51, P < 0.001). T u k e y ' s test revealed that the tail flick latencies were significantly different from pretreatment values at 0.5 and 3.5 min after tail immersion (P < 0.01). Reaction time had r e t u r n e d to control values by 6.5 min after tail immersion ( P > 0.05) and thereafter increased to above control values, although the increase was not significant ( P > 0.05). Immersion of tip of the tail in water at 55 °C increased the surface skin t e m p e r a t u r e at the region where the tail flick reflex was elicited to 6 - 8 °C above the baseline skin t e m p e r a t u r e (n = 4). However, raising the skin t e m p e r a t u r e of the same region by application of innocuous radiant heat failed to alter the reaction time significantly (t = 1.49, P > 0.05), even when the t e m p e r a t u r e was raised by 10 °C. Reaction time in this case was 91.1 + 7.3% of the mean baseline value (n = 6). Baseline t e m p e r a t u r e s of the surface of the skin were 2 0 - 2 3 °C. Immersion of the tip of the tail in water at 55 °C was without effect on core body t e m p e r a t u r e as measured by a rectal t h e r m o m e t e r (n = 4). Baseline core body t e m p e r a t u r e s were 3 6 - 3 7 °C. Following the decrease in reaction time induced by
18 tail immersion, there was a tendency for reaction time to increase above control values. In our previous experiments with substance P, it was observed that the full response to intrathecal administration of substance P included an indirect activation of an opioid mechanism which counteracted the facilitatory effects of substance P on reaction time 3°. Therefore, in the present study, animals were pretreated with naloxone. Naloxone pretreatment potentiated the decrease in reaction time following tail immersion and prevented the subsequent overshoot, allowing two tests of tail immersion within the same experiment. The reproducibility of the effects of repeated tail immersion in animals pretreated with 10 mg/kg of naloxone i.p. are illustrated in Fig. 2. Naloxone treatment was without effect on baseline reaction time (see absolute values for reaction times in different treatment groups). The repeated-measures A N O V A revealed a sig-
Effects of administration of [t)-Pro2,/~-Phe',/~Trpg]substance P on the response to tail immersion In pilot experiments, a n u m b e r of different doses (up to 6.5 nmol) of the substance P antagonist were surveyed. After intrathecal administration of the higher closes of the substance P antagonist, many of the animals failed to withdraw the tail in the tail flick test. These animals were not tested with tail immersion, and upon waking they exhibited a flaccid paralysis similar to that reported by others r'2~'31. For the present study, a dose of 1 nmol was selected because preliminary results indicated that this dose has minimal effects on motor control. Nonetheless, some rats exhibited altered baseline reaction times as a result of the substance P antagonist alone; of the 15 rats injected, 4 failed to flick the tail at 1-7 rain after the injection. These rats were not included in the results. In the remaining 11 rats, the tail flick response was unaltered by the intrathecal administration of the
nificant trial factor (Fs.40 = 15.16, P < 0.001). Reaction time was 5.20 + 0.45 s prior to tail immersions.
substance P analogue alone. Intrathecal administration of [D-Pro2,D-PheT,D-
At 0.5 and 3.5 min after the first tail immersion, reac-
Trpg]substance P significantly attenuated the effects
tion time decreased to 58.2 + 3.6% and 76.1 _+ 7.9%, respectively, of the mean control value (P < 0.01, n = 6). Reaction time was not significantly different
of tail immersion on reaction time (Fig. 3). The A N O V A yielded a significant intrathecal administration x time interaction (F2,128 = 5.55, P < 0.005). Tukey's test revealed that the animals which received
from pretreatment values over the next two readings (P > 0.05). Immersion of the tail a second time produced a similar response in terms of both magnitude
125
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Fig. 2. Effects on reaction time of two successive noxious stimulations of the tail in rats pretreated with naloxone (10 mg/kg, i.p. ; n = 6). The dashed vertical lines represent the times of removal of the tail following immersion (0.5 min prior to the next for reaction time). **, P < 0.01, significantly different from mean baseline reaction time. Other details are as in Fig. 1.
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Fig. 3. Effects of intrathecal administration of 1 nmol of [DPro2,D_PheT,D.Trpg]substance P (©: n = 11) and of artificial cerebrospinal fluid (0, n = 7) on the decrease in reaction time induced by tail immersion. The arrow represents the time of intrathecal injection (1 min prior to the next test for reaction time) and the vertical dashed line represents the time of tail removal following tail immersion (0.5 min prior to the next test for reaction time). **, P < 0.01, *, P < 0.05, significantly different from respective values in CSF-treated animals. Other details are as in Fig. 1.
19 the substance P analogue had different reaction times from those which received CSF at 0.5 min (P < 0.01), 3.5 rain (P < 0.05) and 6.5 min (P < 0.01) after tail immersion but not at any other time tested (P >
0.05). In rats treated with the substance P analogue, reaction time decreased to 85.4 __ 4.3% and 89.2 + 5.1% of the mean baseline value at 0.5 min and 3.5 min after tail immersion, respectively (n = 11). Prior to administration of the substance P antagonist, reaction time was 5.21 + 0.16 s. Reaction time had returned to control values by 6.5 min after tail immersion. When 20/A of CSF was given in the place of the substance P analogue solution, reaction time decreased to 53.2 + 3.9% and 77.3 + 5.2% of the mean control values at 0.5 min and 3.5 min after tail immersion, respectively (n = 7). Prior to administration of CSF, reaction time was 5.36 + 0.35 s. By 9.5 min after tail immersion, reaction time had returned to control values. DISCUSSION The use of the lightly anaesthetized rat to study the tail flick reflex is a well established technique, particularly for those experiments that require invasive surgery 3'1°'26. In fact, other investigators 3'1° have reported that the tail flick reflex in the anaesthetized rat is more pronounced than that in the awake rat. Our experience confirms this observation. In the present study, immersion of the tip of the tail in water at 55 °C significantly decreased reaction time when the tail flick reflex was evoked from a more proximal region. This decrease in reaction time could be elicited a second time within 12 rain, suggesting that the immersion did not produce any longstanding changes in sensitivity in the tail which would affect the response to tail immersion, at least within the time frame of this experiment. Opioid involvement in the response Following the initial decrease in reaction time after tail immersion, reaction time increased to above baseline values. In previous studies, we have observed that intrathecal administration of substance P in the awake rat produces effects which are similar in time course to those observed in the present study, a decrease in reaction time followed by a naloxone-
reversible overshoot 3°. The results of the present study suggest that the increase in reaction time observed after tail immersion may be mediated by an opioid factor. They show further the'similarity between the substance P-induced decrease in reaction time and that observed upon noxious cutaneous stimulation. Substance P involvement in the response Substance P antagonists have been used effectively to block the responses mediated by exogenous substance P within the CNS 1'22'31. Furthermore, a substance P antagonist markedly depresses a reflex in the isolated spinal cord-tail preparation of the rat which is induced by high-intensity electrical stimulation of the dorsal root 29 or by the application of capsaicin 28. [o-Pro2,D-Phe7,D-Trp9]substance P was used in the present study because previously it has been found to block the effects of intrathecally administered substance P on reaction time in the tail flick test 31 and on a caudally directed scratching and biting behaviour 22. The ability of this analogue to attenuate the decrease in reaction time suggests that endogenously released substance P may have been involved in the immersion-induced facilitation of the tail flick reflex. Of course the possibility must be considered that the attenuation of the response to tail immersion by the substance P antagonist may be due to a neurotoxic action of this analogue17,23; however, this is unlikely because the analogue was without effect on baseline reaction time in the animals tested. Therefore, the results from the present study are interpreted as adding further support to the suggestion that substance P is involved in the transmission of thermal nociceptive information within the spinal cord 12. The small decrease in reaction time in rats pretreated with the substance P antagonist may suggest that there was an incomplete block of substance P receptors by the analogue or that another chemical besides substance P may be involved in the mediation of this response. The possibility that noxious cutaneous stimulation induces the release of substance P within the spinal cord is consistent with earlier studies. Electrical stimulation of the sciatic nerve in the cat at intensities sufficient to recruit A 6 and C fibres has been shown to release substance P-like immunoreactivity (-LI) into a spinal cord superfusate 27 and in the dorsal horn as
20
measured by microdialysis 2. Immersion of the hind paw of the cat in water at 50 and 52 °C but not at 37 °C releases substance P in the substantia gelatinosa of the spinal cord as m e a s u r e d by the inhibition of binding of 125I-substance P to antibody-laden microprobes 7. Noxious mechanical 15 and chemogenic 14 stimulation of primary afferents have also been reported to increase substance P-LI in the dorsal horn. Mediation by a peripheral or central action
While we have been assuming that the response to tail immersion is due to a facilitation of nociceptive transmission within the spinal cord, alternative possibilities must be considered which might explain the observed decrease in reaction time. For example, if the skin t e m p e r a t u r e at the site activated to p r o v o k e the tail flick reflex were increased, the respective thermal nociceptors would require less heat to reach threshold and consequently the latency of the response should decrease. Immersion of the tip of the tail did increase skin t e m p e r a t u r e at this site by 6 - 8 °C from 2 0 - 2 3 °C. H o w e v e r , raising the skin t e m p e r a t u r e at this site by innocuous means by even 10 °C failed to alter reaction time. Thus, an increase in skin t e m p e r a t u r e per se is insufficient to account for the m a r k e d facilitation of the tail flick reflex. As r e p e a t e d application of noxious thermal stimuli has been shown to sensitize the responses of unmyelinated 7A6"E9 and m y e l i n a t e d nociceptive afferents 9'19, prolonged heating of the tail in the present study could have sensitized the thermal nociceptors in the tail, thereby lowering the threshold and de-
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creasing reaction time. F u r t h e r m o r e , immersion of the tail may have p r o d u c e d tissue injury and oedema. With massive tissue damage or stimulation of a large receptive field, sensitization may spread to an area which lies outside the stimulated area s~
of substance P in the rat: spinal transection or morphine blocks the behavioural responses but not the facilitation of the tail flick reflex, Neurosci. Lett., 84 (1988) 203-208. 6 Cridland, R.A., Yashpal, K., Romita, V.V., Gauthicr, S. and Henry, J.L., Distribution of label after intrathecal administration of 125I-substanceP in the rat, Peptides, 8 (1987) 213-221. 7 Duggan, A.W., Morton, C.R., Zhao, Z.Q. and Hendry, I.A., Noxious heating of the skin releases immunoreactive substance P in the substantia gelatinosa of the cat: a study with antibody microprobes, Brain Research, 403 (1987) 345-349. 8 Fitzgerald, M., The sensitization of cutaneous nociceptors by spread from a nearby injury and its blockade by local anaesthesia, Proc. Physiol. Soc., 278 (1978) 44P-45P. 9 Fitzgerald, M. and Lynn, B., The sensitization of high
21 threshold mechanoreceptors with myelinated axons by repeated heating, J. Physiol., 265 (1977') 549-563. 10 Gebhart, G.F. and Ossipov, M.H., Characterization of inhibition of the spinal nociceptive tail-flick reflex in the rat from the medullary lateral reticular nucleus, J. Neurosci., 6 (1986) 701-713. 11 Grossman, M.L., Basbaum, A.I. and Fields, H.L., Afferent and efferent connections of the rat tail flick reflex (a model used to analyze pain control mechanisms), J. Comp. Neurol., 206 (1982) 9-16. 12 Henry, J.L., Effects of substance P on functionally identified units in cat spinal cord, Brain Research, 114 (1976) 439-451. 13 Isabel, G., Wright, D.M. and Henry, J.L., Design for an inexpensive unit for measuring tail flick latencies, J. Pharmacol. Methods, 5 (1981) 241-247. 14 Kantner, R.M., Kirby, M.L. and Goldstein, B.D., Increase in substance P in the dorsal horn during a chemogenic nociceptive stimulus, Brain Research, 338 (1985) 196-199. 15 Kuraishi, Y., Hirota, N., Sugimoto, M., Satoh, M. and Takagi, H., Effects of morphine on noxious stimuli-induced release of substance P from rabbit dorsal horn in vivo, Life Sci., 33 (1983) 693-696. 16 Lynn, B., Cutaneous hyperalgesia, Brit. Med. Bull., 33 (1977) 103-108. 17 Matsumura, H., Sakurada, T., Hara, A., Kuwahara, H., Ando, R., Sakurada, S. and Kisara, K., Intrathecal substance P analogue causes motor dysfunction in the rat, Neuropharmacology, 24 (1985) 811-813. 18 Myers, J.L., Fundamentals of Experimental Design, 3rd edn., Allyn and Bacon, Boston, 1979. 19 Perl, E.R., Kumazawa, T., Lynn, B. and Kenins, P., Sensitization of high threshold receptors with unmyelinated (C) afferent fibers, Progr. Brain Res., 43 (1976) 263-276. 20 Pernow, B., Substance P, Pharmacol. Rev., 35 (1983) 85-141. 21 Piercey, M.F., Einspahr, F.J., Dobry, P.J.K., Schroeder, L.A. and Hollister, R.P., Morphine does not antagonize the substance P mediated excitation of dorsal horn neurons,
Brain Research, 186 (1980) 421-434. 22 Piercey, M.F., Schroeder, L.A., Folkers, K., Xu, J.-C. and Horig, J., Sensory and motor functions of spinal cord substance P, Science, 214 (1981) 1361-1363. 23 Post, C. and Paulsson, I., Antinociceptive and neurotoxic actions of substance P analogues in the rat's spinal cord after intrathecal administration, Neurosci. Lett., 57 (1985) 159-164. 24 Randi6, M. and Mileti6, V., Effect of substance P in cat dorsal horn neurones activated by noxious stimuli, Brain Research, 128 (1977) 164-169. 25 Sastry, B.R., Substance P effects on spinal nociceptive neurones, Life Sci., 24 (1979) 2169-2178. 26 Vanegas, H., Barbaro, N.M. and Fields, H.L., Midbrain stimulation inhibits tail-flick only at currents sufficient to excite rostral medullary neurons, Brain Research, 321 (1984) 127-133. 27 Yaksh, T.L., Jessel, T.M., Gamse, R., Mudge, A~W. and Leeman, S.E., Intrathecal morphine inhibits substance P release from mammalian spinal cord in vivo, Nature (Lond.), 286 (1980) 155-157. 28 Yanagisawa, M., Otsuka, M., Konishi, S., Akagi, H., Folkers, K. and Rosell, S., A substance P antagonist inhibits a slow reflex response in the spinal cord of the newborn rat, A cta Physiol. Scand. , 116 (1982) 109-112. 29 Yanagisawa, M. and Otsuka, M., The effect of a substance P antagonist on chemically induced nociceptive reflex in the isolated spinal cord-tail preparation of the newborn rat, Proc. Jap. Acad., 60 (1984) 427-430. 30 Yashpal, K. and Henry, J.L., Endorphins mediate overshoot of substance P-induced facilitation of a spinal nociceptive reflex, Can. J. Physiol. Pharmacol., 61 (1983) 303-307. 31 Yashpal, K. and Henry, J.L., Substance P analogue blocks SP-induced facilitation of a spinal nociceptive reflex, Brain Res. Bull., 13 (1984) 597-600. 32 Yashpal, K., Wright, D.M. and Henry, J.L., Substance P reduces tail-flick latency: implication for chronic pain syn-, dromes, Pain, 14 (1982) 155-167.