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Spinal NK2 receptors contribute to the increased excitability of the nociceptive flexor reflex during persistent peripheral inflammation
Brain Research 751 Ž1997. 169–174
Short communication
Spinal NK 2 receptors contribute to the increased excitability of the nociceptive flexor reflex during persistent peripheral inflammation Yu-ping Jia, Virginia S. Seybold
)
Department of Cell Biology and Neuroanatomy, UniÕersity of Minnesota, 4-144 Jackson Hall, 321 Church St. S.E., Minneapolis, MN 55455, USA Accepted 23 December 1996
Abstract The role of endogenous neurokinin A in changes in the excitability of spinal neurons during adjuvant-induced, peripheral inflammation was examined by determining the effect of a selective NK 2 receptor antagonist, GR103537, on the nociceptive flexor reflex in rats. Intrathecal administration of GR103537 Ž1.4–14 nmol. dose-dependently attenuated the increased activity of the flexor reflex ipsilateral to the inflamed paw. The activity of GR103537 at NK 2 receptors was confirmed by blockade of the facilitation of the reflex by neurokinin A but not substance P in normal rats. These results indicate that endogenous neurokinin A increases the excitability of spinal neurons during persistent peripheral inflammation. Keywords: Tachykinin; Neurokinin receptor; Nociception; Hyperalgesia; Inflammation; Neurokinin A; Flexor reflex; Spinal cord
Compelling evidence suggests that tachykinins are involved in the processing of nociceptive information in the spinal cord. The activity of neurokinin A ŽNKA. in the spinal cord parallels that of substance P. NKA is released into the dorsal horn following noxious thermal, mechanical, electrical and chemical stimuli applied to the skin w5,10,11x, and intrathecal administration of NKA facilitates nociceptive reflexes w2,6,30x. Conversely, NK 2 receptors bind NKA with the highest affinity, and antagonists of this receptor attenuate the prolonged responses of dorsal horn neurons that occur acutely following cutaneous application of noxious chemical stimuli w4,15x. Furthermore, NKA may contribute to the increased excitability of spinal neurons that accompanies peripheral inflammation. There is both direct w10x and indirect w18x evidence that release of NKA is increased during the development of peripheral inflammation, and an antagonist of NK 2 receptors attenuates the hyperexcitability of spinal neurons that accompanies acute inflammation w17x. Since peripheral inflammation results in long-term changes in the expression of tachykinins in primary afferent neurons w3,14x as well as neurokinin receptors in the
) Corresponding author. Fax: q1 Ž612. 624-8118; E-mail: [email protected]
spinal cord w13,25x, it is important to determine whether endogenous tachykinins contribute to the increase in excitability of spinal neurons that occurs several days after the induction of peripheral inflammation. We have shown that the activity of the electrically-evoked nociceptive flexor reflex is increased in rats at 2–3 days after induction of peripheral inflammation with Complete Freund’s Adjuvant w20x. The aim of the present study was to examine the effect of a selective NK 2 receptor antagonist on the nociceptive flexor reflex to determine whether NK 2 receptors contribute to the maintenance of the increased activity of this reflex during persistent inflammation. Experiments were performed on male Sprague–Dawley rats Ž250–330 g.. Experimental procedures were approved by the Institutional Animal Use and Care Committee. An emulsion of Complete Freund’s adjuvant Ž CFA, adjuvant:saline, 1:1, 130 ml. was injected s.c., under anesthesia, into the plantar surface of the left hindpaw. Overt signs of peripheral inflammation occurred only in the injected paw and persisted for several days. Two to 3 days after injection of CFA into the left hind paw, edema was evident in animals used in this study: CFA-treated paws were approximately twice as thick in the dorsal-ventral dimension as untreated hindpaws ŽFig. 1A.. In addition, inflamed paws exhibited a decrease in paw withdrawal latency to a radiant heat stimulus w9x indicating thermal hyperalgesia ŽFig. 1B..
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Y.-p. Jia, V.S. Seyboldr Brain Research 751 (1997) 169–174
The nociceptive flexor reflex was used as an index of the excitability of spinal neurons. The reflex was measured in decerebrate, spinalized rats as previously described w20x. Rats were rendered decerebrate by aspiration of the forebrain at mid collicular level, and the spinal cord was transected at T8–9. Experiments began at least 1 h after spinal transection. The flexor reflex was elicited in the hamstring muscles by stimulating the sural nerve with a single electrical pulse Ž0.5 ms. of constant current at an interval of 4 min. The stimulus intensity was defined by the recording conditions within each experiment such that the stimulus was sufficient to activate C fibers. Compound action potentials of the sural nerve were recorded using a pair of silver hook electrodes placed distal to the stimulating electrode. There were no differences between normal and CFA-treated rats in the thresholds for activation of A b-, A d- or C-fibers ŽTable 1.. The values confirm that peripheral inflammation did not result in a change in peripheral axon excitability. In the present experiments, there was no difference in the stimulus intensity used to evoke the reflex in normal animals or rats with inflamed hind paws Ž1.0 " 0.1 mA for each group, mean " S.E.M...
Fig. 1. Two to 3 days after injection of CFA into the left hindpaw, the ipsilateral hind paw exhibited edema, as indicated by an increase in dorsalrventral paw thickness ŽA., and hyperalgesia, as indicated by a decrease in latency of withdrawal of the hind paw to a thermal stimulus w9x ŽB.. ) P - 0.05, compared to the contralateral paw and † P - 0.05, compared to the same side of untreated animals Žtwo-way ANOVA, with Duncan’s multiple range test.. The number in parentheses within the bar are numbers of animal tested.
Table 1 Thresholds for activation of A b-, A d- and C-fibers in the sural nerve in normal and CFA-treated rats Fiber-type a
Normal
CFA-treated
Ab Ad C
5.6 "0.3 mA Ž21. 100.5 "9.4 mA Ž21. 0.46"0.3 mA Ž16.
5.3 " 0.3 mA Ž14. 101.3 "11.9 mA Ž14. 0.48" 0.3 mA Ž11.
a
Pulse width was 0.1 ms for detection of A b-fibers an‘ 0.5 ms for detection of A d- and C-fibers. Values represent the mean"S.E.M. The n of each sample is given in parentheses.
The nociceptive flexor reflex was recorded using a pair of stainless steel needle electrodes inserted into the hamstring muscles. The muscle potentials elicited during the reflex were processed by a window discriminator with the level set just above background noise. Output from the window discriminator was collected by computer for the generation of peristimulus time histograms. The number of compound potentials elicited in the hamstring muscles during the reflex was integrated over 1-s intervals. All of the data for the flexor reflex reported in this study were based on the number of muscle potentials that occurred above ongoing activity and are reported as the mean " S.E.M.. The baseline reflex was stable for at least 30 min before initiation of an experiment. Labview software Žver. 3.0 for Macintosh, National Instruments, Austin, TX. was used to control the timing of electrical stimulation and to perform analog to digital conversions of muscle potentials. The nociceptive flexor reflex in CFA-treated rats exhibited characteristics that were consistent with those described in an earlier study w20x. Ž1. There was a significant increase in CFA-treated rats in the number of potentials summed over 120 s following the electrical stimulus Žnormal: 2404 " 466, n s 14; CFA: 4702 " 387, n s 16; P 0.01, unpaired Student’s t-test., but, there was no change in the number of muscle potentials summed over the first 5 s of the reflex Žnormal: 412 " 37, n s 14; CFA: 485 " 30, n s 16; Fig. 2.. Ž2. CFA treatment resulted in a 2-fold increase in the duration of the evoked reflex Ž143 " 21 s in CFA-treated animals and 64 " 7 s in normal animals, P - 0.05, unpaired Student’s t-test.. Ž3. A higher proportion of CFA-treated rats Ž13r16. exhibited ongoing activity compared to untreated rats Ž5r21, P - 0.01, Fisher’s exact test.. Ongoing activity was defined as activity that lasted longer than 10 min at the beginning of the recording session and also continued between evoked reflexes. Based upon the results from animals included in two different studies, the increase in activity of the nociceptive flexor reflex in persistent peripheral inflammation is reproducible. In both normal and CFA-treated rats, the number of potentials evoked in the hamstring muscles by a stimulus that activated A d-fibers Ž0.1 mA. was small relative to the response to a stimulus that activated C-fibers w5.7 " 1.4% Ž n s 7. and 6.5 " 1.5% Ž n s 5., respectively, of the
Y.-p. Jia, V.S. Seyboldr Brain Research 751 (1997) 169–174
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reflex evoked by inclusion of C-fibersx, and the duration was less than 5 s. Because the response to stimulation of A d-fibers was small under our recording conditions, the effects of drugs on this component of the reflex were not determined. No evoked potentials were measured in the hamstring muscles in response to stimulation of A b-fibers in the sural nerve of normal or CFA-treated rats. The NK 2 receptor antagonist GR103537 ŽGlaxo, UK., was used to determine whether endogenous NKA contributed to the enhanced nociceptive flexor reflex that was observed in CFA-treated animals. GR103537, a peptide analog of NKA, is a selective antagonist of NK 2 receptors w8x. An intrathecal cannula ŽPE-10., inserted caudally from Fig. 3. Time course of the effect of GR103537 on the number of potentials evoked in the hamstring muscles of CFA-treated animals by electrical stimulation of the ipsilateral sural nerve. Data were integrated from 0 to 120 s and are presented as a percentage of the baseline reflex Ži.e. the average of five reflexes evoked immediately prior to drug delivery.. The number of times each dose was tested is indicated in parentheses. ) P - 0.05, compared to the baseline reflex prior to drug administration Žtimes 0. and † P - 0.05, compared to the response to vehicle alone Žveh.. at the same time point Žtwo-way ANOVA with Duncan’s multiple range test..
Fig. 2. Effect of GR103537 on the number of potentials in the hamstring muscles evoked by electrical stimulation of the ipsilateral sural nerve. Data were 20 min after i.t. administration of the drug. Drug or vehicle alone were injected in a volume of 10 ml followed by 10 ml saline to flush the cannula. Data were integrated from 0 to 5 s ŽA. and 0–120 s ŽB.. Baseline reflexes in individual animals were determined immediately prior to delivery of drug and were an average of five evoked reflexes. The number of times each dose was tested is indicated within the bar. veh., vehicle. ) P - 0.05, compared to untreated animals and † P - 0.05, compared to the baseline reflex and the response to vehicle for CFAtreated animals Žtwo-way ANOVA with Duncan’s multiple range test..
the level of the transection of the spinal cord to the lumber enlargement was used for drug delivery. The location of the cannula tip was verified after each experiment. Data were included only if the tip of the cannula laid adjacent to the dorsal half of spinal segments L 4-L 5 . GR103537 had no effect on the short latency Ž5 s. activities evoked in the hamstring muscles in either untreated or CFA-treated rats ŽFig. 2A.. In contrast, the antagonist attenuated long latency activity in CFA-treated rats in a dose-dependent manner but had no inhibitory effect on the flexor reflex in untreated rats ŽFig. 2B.. A dose of 4.7 nmol GR103537 inhibited increased activity of the flexor reflex in CFA-treated rats by approximately 50%. The inhibitory effect of GR103537 on the reflex was observed from 10 to 35 min, with a peak effect at approximately 20 min ŽFig. 3.. Ongoing activity was inhibited as well in 7 of 8 CFA-treated animals treated with 14 nmol of GR103537. Fig. 4 illustrates the time course of the inhibition in response to 14 nmol GR103537, the selective effect of the antagonist on the long latency activity, and recovery of the reflex after 30 min. Several factors indicate that the inhibitory effect of GR103537 was mediated by activity of the drug at NK 2 receptors. Firstly, the effect of the drug could not be attributed to the vehicle as the vehicle for GR103537 Ž2.5 mM acetic acid. had no effect on the reflex evoked in normal or CFA-treated rats ŽFigs. 2 and 3.. Secondly, the inhibitory effect of the drug was not due to nonspecific damage of the spinal cord because there was recovery of the spinal reflex after each application of the antagonist ŽFigs. 3 and 4.. Thirdly, the pharmacology of GR103537
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Y.-p. Jia, V.S. Seyboldr Brain Research 751 (1997) 169–174
Fig. 4. Representative record of evoked flexor reflexes before and after 14 nmol GR103537 in a CFA-treated animal. The vertical scale represents the number of potentials ŽP. summed at 1-s intervals. The arrow indicates the time of delivery of GR103537. v indicates delivery of an electrical stimulus Ž1.0 mA, 0.5 ms..
was consistent with its activity at NK 2 receptors. The potency of GR103537 in the present assay was comparable to its activity in blocking the activity of an NK 2 receptor agonist in rat brain in vivo w8x. Even facilitation of the flexor reflex that was observed in CFA-treated rats at 5 min following injection of GR103537 ŽFig. 3. is consistent with reports of transient excitatory effects of other NK 2 receptor antagonists that are peptide analogs ŽMEN 10207 w30x; GR94800 w4x.. The excitatory effect has been attributed to partial agonist activity of the peptide w4,30x. Finally, we confirmed the differential antagonism of NK 1 and NK 2 receptors by GR103537 by testing its effectiveness in inhibiting tachykinin-induced facilitation of the reflex in normal animals. A dose of GR103537 Ž4.7 nmol. that inhibited the increased activity of the nociceptive flexor reflex in CFA-treated animals also inhibited NKAinduced facilitation of the reflex Ž7 nmol NKA. but had no effect on the SP-induced facilitation Ž7 nmol SP. ŽFig. 5.. The IC 50 of GR103537 at rat NK 2 receptors is equivalent to the EC 50 of NKA at the same receptor in vitro w8x, and 500 nM GR103537 does not attenuate formation of inositol phosphate by 10 nM substance P at rat NK 1 receptor w19x. Furthermore, the IC 50 of GR103537 at NK 1 and NK 3 receptors was greater than 3 mM in radioreceptor assays performed on rat brain ŽSeybold, Abrahams and Linden, unpubl. obs... Therefore, the activity of GR103537 in attenuating the increased excitability of the nociceptive flexor reflex in CFA-treated rats was most likely mediated by blockade of NK 2 receptors. Thus, the present study confirms an increase in the activity of the electrically-evoked nociceptive flexor reflex during persistent peripheral inflammation and extends our understanding of the role of neurokinin receptors in the maintenance of the increased reflex activity. The ability of GR103537 to decrease the activity of the nociceptive reflex 2–3 days after induction of peripheral inflammation indicates that NK 2 receptors contribute to the maintenance of the hyperactivity of the reflex in addition to NK 1 receptors w20x. The observation that the NK 2 receptor antagonist decreased the long latency activity of the reflex is consistent with data that exogenous NKA causes an
increase in the duration of the electrically evoked flexor reflex in normal rats w29x. The report that pretreatment with a different antagonist of NK 2 receptors, SR48968, blocks the development of hyperexcitable spinal neurons that occurs in response to peripheral injection of carrageenan and kaolin w17x indicates that NK 2 receptors contribute to the development of the increased excitability of spinal neurons during acute inflammation as well. The lack of an effect of the NK 2 receptor antagonist on the electrically evoked nociceptive reflex in untreated rats is not consistent with reports that NK 2 receptor antagonists attenuate responses to noxious thermal w7x as well as noxious and innocuous mechanical stimuli w17x in electrophysiological studies of dorsal horn neurons conducted in normal animals. In addition, two groups w16,27x have reported that NK 2 receptor antagonists decrease the prolonged phase of the ventral root potential evoked by C-fiber stimulation in normal neonatal rat spinal cord, however, data obtained in this model may reflect greater expression of neurokinin receptors in neonatal spinal cord compared to adult w12x as well as altered receptor phar-
Fig. 5. GR103537 Ž4.7 nmol. antagonized facilitation of the flexor reflex induced by NKA Ž7 nmolr10 ml, i.t.. but not the facilitation induced by SP Ž7 nmolr10 ml, i.t... Data were integrated from 0 to 60 s and are presented as a percentage of the baseline reflex. ) P - 0.05, compared to the response prior to GR103537 Žpaired Student’s t test, ns 7..
Y.-p. Jia, V.S. Seyboldr Brain Research 751 (1997) 169–174
macology w19,24x. Our results are also at variance with another report based upon the electrically-evoked nociceptive flexor reflex w30x. The inhibitory effect of MEN10207 observed in this report w30x could reflect greater release of endogenous NKA by virtue of the 10-fold greater current that was used to evoke the reflex. It is noteworthy that opposing results have been reported for NK 2 receptor antagonists in the tail flick assay in rats w6,21x. Thus, the ability to detect participation of NK 2 receptors in spinal cord physiology in normal animals is dependent upon the assay, and we cannot exclude the possibility that the robust electrical stimulus used in our assay obscured subtle effects of NK 2 receptor activation in normal rats. The ability of an NK 2 receptor antagonist to attenuate the activity of the nociceptive flexor reflex in CFA-treated rats 2–3 days after induction of inflammation implies that increased activation of NK 2 receptors is maintained during peripheral inflammation. Blockade of NK 2 receptors also attenuates increased activity of spinal neurons that occurs acutely following stimulation of unmyelinated primary afferent neurons either chemically w4,15,16x, electrically w30x, or several hours after induction of inflammation w17x. During persistent peripheral inflammation, increased activation of NK 2 receptors may occur as a consequence of increased release of the peptide w10x. However, post-synaptic changes of increased receptor expression or increased affinity of the receptor for tachykinins, as has been described for NK 1 receptors during adjuvant-induced inflammation w13,25x, must also be considered. The cellular location of the NK 2 receptors mediating the effects described in this study remains to be resolved. Although NK 2 receptor mRNA is not detectable in adult rat spinal cord w26,28x, NK 2 binding sites have been localized to the dorsal horn of the spinal cord w31x, and NKA modulates the firing of dorsal horn neurons evoked by peripheral stimuli w1x. Together, these data suggest that NK 2 receptors may occur on terminals of neurons that are extrinsic to the spinal cord. Presynaptic NK 2 receptors have been localized immunohistochemically on neurons in the enteric nervous system w22x. However, NKA modulation of postsynaptic responses to excitatory amino acids w1,23x suggests that NK 2 receptors may also occur on spinal neurons. Since the increased activity of nociceptive flexor reflex in CFA-treated rats is most likely due to increased activity of interneurons w20x, the effect of GR103537 in our study may be mediated by blockade of NK 2 receptors at synapses of primary afferent neurons onto interneurons. In conclusion, the present results confirm that the nociceptive flexor reflex exhibits increased activity 2–3 days after injection of CFA into the hind paw of the rat. The pharmacological data indicate that spinal NK 2 receptors contribute to the increased activity of the reflex. NK 2 receptors at synapses onto interneurons in the dorsal horn may contribute to hyperalgesia observed in animals with inflamed hind paws as well as to increased motor reflexes.
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Acknowledgements The authors thank Dr. Ann Parsons for helpful discussions and comments on the manuscript. The work was supported by a grant from the National Institutes of Health ŽNS 17702, V.S. Seybold.. References w1x Cumberbatch, M.J., Chizh, B.A. and Headley, P.M., Modulation of excitatory amino acid responses by tachykinins and selective tachykinin receptor agonists in the rat spinal cord, Br. J. Pharmacol., 115 Ž1995. 1005–1012. w2x Cridland, R.A. and Henry, J.L., Comparision of the effects of substance P, neurokinin A, physalaemin, and eledoisin in facilitating a nociceptive reflex in the rat, Brain Res., 381 Ž1986. 93–99. w3x Donaldson, L.F., Harmar, A.J., McQueen, D.S. and Seckl, J.R., Increased expression of preprotachykinin, calcitonin gene-related peptide, but not vasoactive intestinal peptide messenger RNA in dorsal root ganglia during the development of adjuvant monoarthritis in the rat, Molec. Brain Res., 16 Ž1992. 143–149. w4x Dougherty, P.M., Palecek, J., Paleckova, ´ V. and Willis, W.D., Neurokinin 1 and 2 antagonists attenuate the responses and NK1 antagonists prevent the sensitization of primate spinothalamic tract neurons after intradermal capsaicin, J. Neurophysiol., 72 Ž1994. 1464–1475. w5x Duggan, A.W., Hope, P.J., Jarrott, B., Schaible, H.-G. and Fleetwood-Walker, S.M., Release, spread and persistence of immunoreactive neurokinin A in the dorsal horn of the cat following noxious cutaneous stimulation. Studies with antibody microprobes, Neuroscience, 35 Ž1990. 195–202. w6x Fleetwood-Walker, S.M., Mitchell, R., Hope, P.J., El-Yassir, N., Molony, V. and Bladon, C.M., The involvement of neurokinin receptor subtypes in somatosensory processing in the superficial dorsal horn of the cat, Brain Res., 519 Ž1990. 169–182. w7x Fleetwood-Walker, S.M., Parker, R.M.C., Munro, R.E., Young, M.R., Hope, P.J. and Mitchell, R., Evidence for a role of tachykinin NK2 receptors mediating brief nociceptive inputs to rat dorsal horn Žlaminae III–V. neurons, Eur. J. Pharmacol., 242 Ž1993. 173–181. w8x Hagan, R.M., Beresford, I.J.M., Stables, J., Dupere, J., Stubbs, C.M., Elliott, P.J., Sheldrick, R.L.G., Chollet, A., Kawashima, A., McElroy, A.B. and Ward, P., Characterization, CNS distribution and function of NK 2 receptors studied using potent NK 2 receptor antagonists, Regul. Pepttides, 46 Ž1993. 9–19. w9x Hargreaves, K., Dubner, R., Brown, F., Flores, C. and Joris, J., A new and sensitive method for measuring thermal nociception in cutaneous hyperalgesia, Pain, 32 Ž1988. 77–88. w10x Hope, P.J., Jarrott, B., Schaible, H.-G., Clarke, R.W. and Duggan, A.W., Release and spread of immunoreactive neurokinin A in the cat spinal cord in a model of acute arthritis, Brain Res., 533 Ž1990. 292–299. w11x Hua, X.-Y., Saria, A., Gamse, R., Theodorsson-Norheim, E., Brodin, E. and Lundburg, J.M., Capsaicin induced release of multiple tachykinins Žsubstance P, neurokinin A, and eledoisin-like material. from guinea-pig spinal cord and ureter, Neuroscience, 19 Ž1986. 313–319. w12x Kar, S. and Quirion, R., Neuropeptide receptors in developing and adult rat spinal cord: An in vitro quantitative autoradiography study of calcitonin gene-related peptide, neurokinins, m-opioid, galanin, somatostatin, neurotensin and vasoactive intestinal polypeptide receptors, J. Comp. Neurol., 354 Ž1995. 253–281. w13x McCarson, K.E. and Krause, J.E., NK-1 and NK-3 type tachykinin receptor mRNA expression in the rat spinal cord dorsal horn is increased during adjuvant or formalin-induced nociception, J. Neurosci., 14 Ž1994. 712–720.
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w14x Minami, M., Kuraishi, Y., Kawamura, M., Yamaguchi, T., Masu, Y., Nakanishi, S. and Satoh, M., Enhancement of preprotachykinin A gene expression by adjuvant-induced inflammation in the rat spinal cord: possible involvement of substance P-containing spinal neurons in nociception, Neurosci. Lett., 98 Ž1989. 105–110. w15x Munro, F.E., Fleetwood-Walker, S.M., Parker, R.M.C. and Mitchell, R., The effects of neurokinin receptor antagonists on mustard oilevoked activation of rat dorsal horn neurons, Neuropeptides, 25 Ž1993. 299–305. w16x Nagy, I., Miller, B.A. and Woolf, C.J., NK 1 and NK 2 receptors contribute to C-fibre evoked slow potentials in the spinal cord, Neuroreport, 5 Ž1994. 2105–2108. w17x Neugebauer, V., Rumenapp, P. and Schaible, H.-G., The role of spinal neurokinin-2 receptors in the processing of nociceptive information from the joint and the generation and maintenance of inflammation-evoked hyperexcitability of dorsal horn neurons in the rat, Eur. J. Neurosci., 8 Ž1996. 249–260. w18x Parker, R.M.C., Fleetwood-Walker, S.M., Rosie, R., Munro, R.E. and Mitchell, R., Inhibition by NK 2 but not NK 1 antagonists of carrageenan-induced preprodynorphin mRNA expression in rat dorsal horn lamina I neurons, Neuropeptides, 25 Ž1993. 213–222. w19x Parsons, A.M., El-Fakahany, E.E. and Seybold, V.S., Tachykinins alter inositol phosphate formation, but not cAMP levels, in primary cultures of neonatal rat spinal neurons through activation of neurokinin receptors, Neuroscience, 68 Ž1995. 855–865. w20x Parsons, A.M., Honda, C.N., Jia, Y.-P., Budai, D., Xu, X.-J., Wiesenfeld-Hallin, Z. and Seybold, V.S., Spinal NK 1 receptors contribute to the increased excitability of the nociceptive flexor reflex during persistent peripheral inflammation, Brain Res., 739 Ž1996. 263–275. w21x Picard, P., Boucher, S., Regoli, D., Gitter, B.D., Howbert, J.J. and Couture, R., Use of non-peptide tachykinin receptor antagonists to substantiate the involvement of NK 1 and NK 2 receptors in a spinal nociceptive reflex in the rat, Eur. J. Pharmacol., 232 Ž1993. 255– 261.
w22x Portbury, A.L., Furness, J.B., Southwell, B.R., Wong, H.C., Walsh, J.H. and Bunnett, N.W., Distribution of neurokinin-2 receptors in the guinea-pig gastrointestinal tract, Cell Tissue Res., 286 Ž1996. 281– 292. w23x Rusin, K.I., Ryu, P.D. and Randic, M., Modulation of excitatory amino acid responses in rat dorsal horn neurons by tachykinins, J. Neurophysiol., 68 Ž1992. 265–286. w24x Suzuki, H., Yoshioka, K., Maehara, T., Hagan, R.M., Nakanishi, S. and Otsuka, M., Pharmacological characterization of tachykinin receptors mediating acetylcholine release from neonatal rat spinal cord, Eur. J. Pharmacol., 241 Ž1993. 105–110. w25x Stucky, C.L., Galeazza, M.T. and Seybold, V.S., Time-dependent changes in Bolton-Hunter-labeled 125 I-substance P binding in rat spinal cord following unilateral adjuvant-induced peripheral inflammation, Neuroscience, 57 Ž1993. 397–409. w26x Takeda, Y. and Krause, J.E., Pharmacological and molecular biological studies on the diversity of rat tachykinin NK 2 receptor subtypes in rat central nervous system, duodenum, vas deferens and urinary bladder, Ann. N.Y. Acad. Sci., 632 Ž1991. 479–482. w27x Thompson, S.W.N., Urban, L. and Dray, A., Contribution of NK 1 and NK 2 receptor activation to high threshold afferent fibre evoked ventral root responses in the rat spinal cord in vitro, Brain Res., 625 Ž1993. 100–108. w28x Tsuchida, K., Shigemoto, R., Yokota, Y. and Nakanishi, S., Tissue distribution and quantitation of the mRNA for three rat tachykinin receptors, Eur. J. Biochem., 193 Ž1990. 751–757. w29x Wiesenfeld-Hallin, Z. and Xu, X.-J., The differential roles of substance P and neurokinin A in spinal cord hyperexcitability and neurogenic inflammation, Regul. Peptides, 46 Ž1993. 165–173. w30x Xu, X.-J., Maggi, C.A., Wiesenfeld-Hallin, Z., On the role of NK-2 tachykinin receptors in the mediation of spinal reflex excitability in the rat, Neuroscience, 44 Ž1991. 483–490. w31x Yashpal, K., Dam, T.V. and Quirion, R., Quantitative autoradiographic distribution of multiple neurokinin binding sites in rat spinal cord, Brain Res., 506 Ž1990. 259–266.
Short communication
Spinal NK 2 receptors contribute to the increased excitability of the nociceptive flexor reflex during persistent peripheral inflammation Yu-ping Jia, Virginia S. Seybold
)
Department of Cell Biology and Neuroanatomy, UniÕersity of Minnesota, 4-144 Jackson Hall, 321 Church St. S.E., Minneapolis, MN 55455, USA Accepted 23 December 1996
Abstract The role of endogenous neurokinin A in changes in the excitability of spinal neurons during adjuvant-induced, peripheral inflammation was examined by determining the effect of a selective NK 2 receptor antagonist, GR103537, on the nociceptive flexor reflex in rats. Intrathecal administration of GR103537 Ž1.4–14 nmol. dose-dependently attenuated the increased activity of the flexor reflex ipsilateral to the inflamed paw. The activity of GR103537 at NK 2 receptors was confirmed by blockade of the facilitation of the reflex by neurokinin A but not substance P in normal rats. These results indicate that endogenous neurokinin A increases the excitability of spinal neurons during persistent peripheral inflammation. Keywords: Tachykinin; Neurokinin receptor; Nociception; Hyperalgesia; Inflammation; Neurokinin A; Flexor reflex; Spinal cord
Compelling evidence suggests that tachykinins are involved in the processing of nociceptive information in the spinal cord. The activity of neurokinin A ŽNKA. in the spinal cord parallels that of substance P. NKA is released into the dorsal horn following noxious thermal, mechanical, electrical and chemical stimuli applied to the skin w5,10,11x, and intrathecal administration of NKA facilitates nociceptive reflexes w2,6,30x. Conversely, NK 2 receptors bind NKA with the highest affinity, and antagonists of this receptor attenuate the prolonged responses of dorsal horn neurons that occur acutely following cutaneous application of noxious chemical stimuli w4,15x. Furthermore, NKA may contribute to the increased excitability of spinal neurons that accompanies peripheral inflammation. There is both direct w10x and indirect w18x evidence that release of NKA is increased during the development of peripheral inflammation, and an antagonist of NK 2 receptors attenuates the hyperexcitability of spinal neurons that accompanies acute inflammation w17x. Since peripheral inflammation results in long-term changes in the expression of tachykinins in primary afferent neurons w3,14x as well as neurokinin receptors in the
) Corresponding author. Fax: q1 Ž612. 624-8118; E-mail: [email protected]
spinal cord w13,25x, it is important to determine whether endogenous tachykinins contribute to the increase in excitability of spinal neurons that occurs several days after the induction of peripheral inflammation. We have shown that the activity of the electrically-evoked nociceptive flexor reflex is increased in rats at 2–3 days after induction of peripheral inflammation with Complete Freund’s Adjuvant w20x. The aim of the present study was to examine the effect of a selective NK 2 receptor antagonist on the nociceptive flexor reflex to determine whether NK 2 receptors contribute to the maintenance of the increased activity of this reflex during persistent inflammation. Experiments were performed on male Sprague–Dawley rats Ž250–330 g.. Experimental procedures were approved by the Institutional Animal Use and Care Committee. An emulsion of Complete Freund’s adjuvant Ž CFA, adjuvant:saline, 1:1, 130 ml. was injected s.c., under anesthesia, into the plantar surface of the left hindpaw. Overt signs of peripheral inflammation occurred only in the injected paw and persisted for several days. Two to 3 days after injection of CFA into the left hind paw, edema was evident in animals used in this study: CFA-treated paws were approximately twice as thick in the dorsal-ventral dimension as untreated hindpaws ŽFig. 1A.. In addition, inflamed paws exhibited a decrease in paw withdrawal latency to a radiant heat stimulus w9x indicating thermal hyperalgesia ŽFig. 1B..
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The nociceptive flexor reflex was used as an index of the excitability of spinal neurons. The reflex was measured in decerebrate, spinalized rats as previously described w20x. Rats were rendered decerebrate by aspiration of the forebrain at mid collicular level, and the spinal cord was transected at T8–9. Experiments began at least 1 h after spinal transection. The flexor reflex was elicited in the hamstring muscles by stimulating the sural nerve with a single electrical pulse Ž0.5 ms. of constant current at an interval of 4 min. The stimulus intensity was defined by the recording conditions within each experiment such that the stimulus was sufficient to activate C fibers. Compound action potentials of the sural nerve were recorded using a pair of silver hook electrodes placed distal to the stimulating electrode. There were no differences between normal and CFA-treated rats in the thresholds for activation of A b-, A d- or C-fibers ŽTable 1.. The values confirm that peripheral inflammation did not result in a change in peripheral axon excitability. In the present experiments, there was no difference in the stimulus intensity used to evoke the reflex in normal animals or rats with inflamed hind paws Ž1.0 " 0.1 mA for each group, mean " S.E.M...
Fig. 1. Two to 3 days after injection of CFA into the left hindpaw, the ipsilateral hind paw exhibited edema, as indicated by an increase in dorsalrventral paw thickness ŽA., and hyperalgesia, as indicated by a decrease in latency of withdrawal of the hind paw to a thermal stimulus w9x ŽB.. ) P - 0.05, compared to the contralateral paw and † P - 0.05, compared to the same side of untreated animals Žtwo-way ANOVA, with Duncan’s multiple range test.. The number in parentheses within the bar are numbers of animal tested.
Table 1 Thresholds for activation of A b-, A d- and C-fibers in the sural nerve in normal and CFA-treated rats Fiber-type a
Normal
CFA-treated
Ab Ad C
5.6 "0.3 mA Ž21. 100.5 "9.4 mA Ž21. 0.46"0.3 mA Ž16.
5.3 " 0.3 mA Ž14. 101.3 "11.9 mA Ž14. 0.48" 0.3 mA Ž11.
a
Pulse width was 0.1 ms for detection of A b-fibers an‘ 0.5 ms for detection of A d- and C-fibers. Values represent the mean"S.E.M. The n of each sample is given in parentheses.
The nociceptive flexor reflex was recorded using a pair of stainless steel needle electrodes inserted into the hamstring muscles. The muscle potentials elicited during the reflex were processed by a window discriminator with the level set just above background noise. Output from the window discriminator was collected by computer for the generation of peristimulus time histograms. The number of compound potentials elicited in the hamstring muscles during the reflex was integrated over 1-s intervals. All of the data for the flexor reflex reported in this study were based on the number of muscle potentials that occurred above ongoing activity and are reported as the mean " S.E.M.. The baseline reflex was stable for at least 30 min before initiation of an experiment. Labview software Žver. 3.0 for Macintosh, National Instruments, Austin, TX. was used to control the timing of electrical stimulation and to perform analog to digital conversions of muscle potentials. The nociceptive flexor reflex in CFA-treated rats exhibited characteristics that were consistent with those described in an earlier study w20x. Ž1. There was a significant increase in CFA-treated rats in the number of potentials summed over 120 s following the electrical stimulus Žnormal: 2404 " 466, n s 14; CFA: 4702 " 387, n s 16; P 0.01, unpaired Student’s t-test., but, there was no change in the number of muscle potentials summed over the first 5 s of the reflex Žnormal: 412 " 37, n s 14; CFA: 485 " 30, n s 16; Fig. 2.. Ž2. CFA treatment resulted in a 2-fold increase in the duration of the evoked reflex Ž143 " 21 s in CFA-treated animals and 64 " 7 s in normal animals, P - 0.05, unpaired Student’s t-test.. Ž3. A higher proportion of CFA-treated rats Ž13r16. exhibited ongoing activity compared to untreated rats Ž5r21, P - 0.01, Fisher’s exact test.. Ongoing activity was defined as activity that lasted longer than 10 min at the beginning of the recording session and also continued between evoked reflexes. Based upon the results from animals included in two different studies, the increase in activity of the nociceptive flexor reflex in persistent peripheral inflammation is reproducible. In both normal and CFA-treated rats, the number of potentials evoked in the hamstring muscles by a stimulus that activated A d-fibers Ž0.1 mA. was small relative to the response to a stimulus that activated C-fibers w5.7 " 1.4% Ž n s 7. and 6.5 " 1.5% Ž n s 5., respectively, of the
Y.-p. Jia, V.S. Seyboldr Brain Research 751 (1997) 169–174
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reflex evoked by inclusion of C-fibersx, and the duration was less than 5 s. Because the response to stimulation of A d-fibers was small under our recording conditions, the effects of drugs on this component of the reflex were not determined. No evoked potentials were measured in the hamstring muscles in response to stimulation of A b-fibers in the sural nerve of normal or CFA-treated rats. The NK 2 receptor antagonist GR103537 ŽGlaxo, UK., was used to determine whether endogenous NKA contributed to the enhanced nociceptive flexor reflex that was observed in CFA-treated animals. GR103537, a peptide analog of NKA, is a selective antagonist of NK 2 receptors w8x. An intrathecal cannula ŽPE-10., inserted caudally from Fig. 3. Time course of the effect of GR103537 on the number of potentials evoked in the hamstring muscles of CFA-treated animals by electrical stimulation of the ipsilateral sural nerve. Data were integrated from 0 to 120 s and are presented as a percentage of the baseline reflex Ži.e. the average of five reflexes evoked immediately prior to drug delivery.. The number of times each dose was tested is indicated in parentheses. ) P - 0.05, compared to the baseline reflex prior to drug administration Žtimes 0. and † P - 0.05, compared to the response to vehicle alone Žveh.. at the same time point Žtwo-way ANOVA with Duncan’s multiple range test..
Fig. 2. Effect of GR103537 on the number of potentials in the hamstring muscles evoked by electrical stimulation of the ipsilateral sural nerve. Data were 20 min after i.t. administration of the drug. Drug or vehicle alone were injected in a volume of 10 ml followed by 10 ml saline to flush the cannula. Data were integrated from 0 to 5 s ŽA. and 0–120 s ŽB.. Baseline reflexes in individual animals were determined immediately prior to delivery of drug and were an average of five evoked reflexes. The number of times each dose was tested is indicated within the bar. veh., vehicle. ) P - 0.05, compared to untreated animals and † P - 0.05, compared to the baseline reflex and the response to vehicle for CFAtreated animals Žtwo-way ANOVA with Duncan’s multiple range test..
the level of the transection of the spinal cord to the lumber enlargement was used for drug delivery. The location of the cannula tip was verified after each experiment. Data were included only if the tip of the cannula laid adjacent to the dorsal half of spinal segments L 4-L 5 . GR103537 had no effect on the short latency Ž5 s. activities evoked in the hamstring muscles in either untreated or CFA-treated rats ŽFig. 2A.. In contrast, the antagonist attenuated long latency activity in CFA-treated rats in a dose-dependent manner but had no inhibitory effect on the flexor reflex in untreated rats ŽFig. 2B.. A dose of 4.7 nmol GR103537 inhibited increased activity of the flexor reflex in CFA-treated rats by approximately 50%. The inhibitory effect of GR103537 on the reflex was observed from 10 to 35 min, with a peak effect at approximately 20 min ŽFig. 3.. Ongoing activity was inhibited as well in 7 of 8 CFA-treated animals treated with 14 nmol of GR103537. Fig. 4 illustrates the time course of the inhibition in response to 14 nmol GR103537, the selective effect of the antagonist on the long latency activity, and recovery of the reflex after 30 min. Several factors indicate that the inhibitory effect of GR103537 was mediated by activity of the drug at NK 2 receptors. Firstly, the effect of the drug could not be attributed to the vehicle as the vehicle for GR103537 Ž2.5 mM acetic acid. had no effect on the reflex evoked in normal or CFA-treated rats ŽFigs. 2 and 3.. Secondly, the inhibitory effect of the drug was not due to nonspecific damage of the spinal cord because there was recovery of the spinal reflex after each application of the antagonist ŽFigs. 3 and 4.. Thirdly, the pharmacology of GR103537
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Fig. 4. Representative record of evoked flexor reflexes before and after 14 nmol GR103537 in a CFA-treated animal. The vertical scale represents the number of potentials ŽP. summed at 1-s intervals. The arrow indicates the time of delivery of GR103537. v indicates delivery of an electrical stimulus Ž1.0 mA, 0.5 ms..
was consistent with its activity at NK 2 receptors. The potency of GR103537 in the present assay was comparable to its activity in blocking the activity of an NK 2 receptor agonist in rat brain in vivo w8x. Even facilitation of the flexor reflex that was observed in CFA-treated rats at 5 min following injection of GR103537 ŽFig. 3. is consistent with reports of transient excitatory effects of other NK 2 receptor antagonists that are peptide analogs ŽMEN 10207 w30x; GR94800 w4x.. The excitatory effect has been attributed to partial agonist activity of the peptide w4,30x. Finally, we confirmed the differential antagonism of NK 1 and NK 2 receptors by GR103537 by testing its effectiveness in inhibiting tachykinin-induced facilitation of the reflex in normal animals. A dose of GR103537 Ž4.7 nmol. that inhibited the increased activity of the nociceptive flexor reflex in CFA-treated animals also inhibited NKAinduced facilitation of the reflex Ž7 nmol NKA. but had no effect on the SP-induced facilitation Ž7 nmol SP. ŽFig. 5.. The IC 50 of GR103537 at rat NK 2 receptors is equivalent to the EC 50 of NKA at the same receptor in vitro w8x, and 500 nM GR103537 does not attenuate formation of inositol phosphate by 10 nM substance P at rat NK 1 receptor w19x. Furthermore, the IC 50 of GR103537 at NK 1 and NK 3 receptors was greater than 3 mM in radioreceptor assays performed on rat brain ŽSeybold, Abrahams and Linden, unpubl. obs... Therefore, the activity of GR103537 in attenuating the increased excitability of the nociceptive flexor reflex in CFA-treated rats was most likely mediated by blockade of NK 2 receptors. Thus, the present study confirms an increase in the activity of the electrically-evoked nociceptive flexor reflex during persistent peripheral inflammation and extends our understanding of the role of neurokinin receptors in the maintenance of the increased reflex activity. The ability of GR103537 to decrease the activity of the nociceptive reflex 2–3 days after induction of peripheral inflammation indicates that NK 2 receptors contribute to the maintenance of the hyperactivity of the reflex in addition to NK 1 receptors w20x. The observation that the NK 2 receptor antagonist decreased the long latency activity of the reflex is consistent with data that exogenous NKA causes an
increase in the duration of the electrically evoked flexor reflex in normal rats w29x. The report that pretreatment with a different antagonist of NK 2 receptors, SR48968, blocks the development of hyperexcitable spinal neurons that occurs in response to peripheral injection of carrageenan and kaolin w17x indicates that NK 2 receptors contribute to the development of the increased excitability of spinal neurons during acute inflammation as well. The lack of an effect of the NK 2 receptor antagonist on the electrically evoked nociceptive reflex in untreated rats is not consistent with reports that NK 2 receptor antagonists attenuate responses to noxious thermal w7x as well as noxious and innocuous mechanical stimuli w17x in electrophysiological studies of dorsal horn neurons conducted in normal animals. In addition, two groups w16,27x have reported that NK 2 receptor antagonists decrease the prolonged phase of the ventral root potential evoked by C-fiber stimulation in normal neonatal rat spinal cord, however, data obtained in this model may reflect greater expression of neurokinin receptors in neonatal spinal cord compared to adult w12x as well as altered receptor phar-
Fig. 5. GR103537 Ž4.7 nmol. antagonized facilitation of the flexor reflex induced by NKA Ž7 nmolr10 ml, i.t.. but not the facilitation induced by SP Ž7 nmolr10 ml, i.t... Data were integrated from 0 to 60 s and are presented as a percentage of the baseline reflex. ) P - 0.05, compared to the response prior to GR103537 Žpaired Student’s t test, ns 7..
Y.-p. Jia, V.S. Seyboldr Brain Research 751 (1997) 169–174
macology w19,24x. Our results are also at variance with another report based upon the electrically-evoked nociceptive flexor reflex w30x. The inhibitory effect of MEN10207 observed in this report w30x could reflect greater release of endogenous NKA by virtue of the 10-fold greater current that was used to evoke the reflex. It is noteworthy that opposing results have been reported for NK 2 receptor antagonists in the tail flick assay in rats w6,21x. Thus, the ability to detect participation of NK 2 receptors in spinal cord physiology in normal animals is dependent upon the assay, and we cannot exclude the possibility that the robust electrical stimulus used in our assay obscured subtle effects of NK 2 receptor activation in normal rats. The ability of an NK 2 receptor antagonist to attenuate the activity of the nociceptive flexor reflex in CFA-treated rats 2–3 days after induction of inflammation implies that increased activation of NK 2 receptors is maintained during peripheral inflammation. Blockade of NK 2 receptors also attenuates increased activity of spinal neurons that occurs acutely following stimulation of unmyelinated primary afferent neurons either chemically w4,15,16x, electrically w30x, or several hours after induction of inflammation w17x. During persistent peripheral inflammation, increased activation of NK 2 receptors may occur as a consequence of increased release of the peptide w10x. However, post-synaptic changes of increased receptor expression or increased affinity of the receptor for tachykinins, as has been described for NK 1 receptors during adjuvant-induced inflammation w13,25x, must also be considered. The cellular location of the NK 2 receptors mediating the effects described in this study remains to be resolved. Although NK 2 receptor mRNA is not detectable in adult rat spinal cord w26,28x, NK 2 binding sites have been localized to the dorsal horn of the spinal cord w31x, and NKA modulates the firing of dorsal horn neurons evoked by peripheral stimuli w1x. Together, these data suggest that NK 2 receptors may occur on terminals of neurons that are extrinsic to the spinal cord. Presynaptic NK 2 receptors have been localized immunohistochemically on neurons in the enteric nervous system w22x. However, NKA modulation of postsynaptic responses to excitatory amino acids w1,23x suggests that NK 2 receptors may also occur on spinal neurons. Since the increased activity of nociceptive flexor reflex in CFA-treated rats is most likely due to increased activity of interneurons w20x, the effect of GR103537 in our study may be mediated by blockade of NK 2 receptors at synapses of primary afferent neurons onto interneurons. In conclusion, the present results confirm that the nociceptive flexor reflex exhibits increased activity 2–3 days after injection of CFA into the hind paw of the rat. The pharmacological data indicate that spinal NK 2 receptors contribute to the increased activity of the reflex. NK 2 receptors at synapses onto interneurons in the dorsal horn may contribute to hyperalgesia observed in animals with inflamed hind paws as well as to increased motor reflexes.
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Acknowledgements The authors thank Dr. Ann Parsons for helpful discussions and comments on the manuscript. The work was supported by a grant from the National Institutes of Health ŽNS 17702, V.S. Seybold.. References w1x Cumberbatch, M.J., Chizh, B.A. and Headley, P.M., Modulation of excitatory amino acid responses by tachykinins and selective tachykinin receptor agonists in the rat spinal cord, Br. J. Pharmacol., 115 Ž1995. 1005–1012. w2x Cridland, R.A. and Henry, J.L., Comparision of the effects of substance P, neurokinin A, physalaemin, and eledoisin in facilitating a nociceptive reflex in the rat, Brain Res., 381 Ž1986. 93–99. w3x Donaldson, L.F., Harmar, A.J., McQueen, D.S. and Seckl, J.R., Increased expression of preprotachykinin, calcitonin gene-related peptide, but not vasoactive intestinal peptide messenger RNA in dorsal root ganglia during the development of adjuvant monoarthritis in the rat, Molec. Brain Res., 16 Ž1992. 143–149. w4x Dougherty, P.M., Palecek, J., Paleckova, ´ V. and Willis, W.D., Neurokinin 1 and 2 antagonists attenuate the responses and NK1 antagonists prevent the sensitization of primate spinothalamic tract neurons after intradermal capsaicin, J. Neurophysiol., 72 Ž1994. 1464–1475. w5x Duggan, A.W., Hope, P.J., Jarrott, B., Schaible, H.-G. and Fleetwood-Walker, S.M., Release, spread and persistence of immunoreactive neurokinin A in the dorsal horn of the cat following noxious cutaneous stimulation. Studies with antibody microprobes, Neuroscience, 35 Ž1990. 195–202. w6x Fleetwood-Walker, S.M., Mitchell, R., Hope, P.J., El-Yassir, N., Molony, V. and Bladon, C.M., The involvement of neurokinin receptor subtypes in somatosensory processing in the superficial dorsal horn of the cat, Brain Res., 519 Ž1990. 169–182. w7x Fleetwood-Walker, S.M., Parker, R.M.C., Munro, R.E., Young, M.R., Hope, P.J. and Mitchell, R., Evidence for a role of tachykinin NK2 receptors mediating brief nociceptive inputs to rat dorsal horn Žlaminae III–V. neurons, Eur. J. Pharmacol., 242 Ž1993. 173–181. w8x Hagan, R.M., Beresford, I.J.M., Stables, J., Dupere, J., Stubbs, C.M., Elliott, P.J., Sheldrick, R.L.G., Chollet, A., Kawashima, A., McElroy, A.B. and Ward, P., Characterization, CNS distribution and function of NK 2 receptors studied using potent NK 2 receptor antagonists, Regul. Pepttides, 46 Ž1993. 9–19. w9x Hargreaves, K., Dubner, R., Brown, F., Flores, C. and Joris, J., A new and sensitive method for measuring thermal nociception in cutaneous hyperalgesia, Pain, 32 Ž1988. 77–88. w10x Hope, P.J., Jarrott, B., Schaible, H.-G., Clarke, R.W. and Duggan, A.W., Release and spread of immunoreactive neurokinin A in the cat spinal cord in a model of acute arthritis, Brain Res., 533 Ž1990. 292–299. w11x Hua, X.-Y., Saria, A., Gamse, R., Theodorsson-Norheim, E., Brodin, E. and Lundburg, J.M., Capsaicin induced release of multiple tachykinins Žsubstance P, neurokinin A, and eledoisin-like material. from guinea-pig spinal cord and ureter, Neuroscience, 19 Ž1986. 313–319. w12x Kar, S. and Quirion, R., Neuropeptide receptors in developing and adult rat spinal cord: An in vitro quantitative autoradiography study of calcitonin gene-related peptide, neurokinins, m-opioid, galanin, somatostatin, neurotensin and vasoactive intestinal polypeptide receptors, J. Comp. Neurol., 354 Ž1995. 253–281. w13x McCarson, K.E. and Krause, J.E., NK-1 and NK-3 type tachykinin receptor mRNA expression in the rat spinal cord dorsal horn is increased during adjuvant or formalin-induced nociception, J. Neurosci., 14 Ž1994. 712–720.
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