Brain Research, 559 (1991) 17-21 © 1991 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/91/$03.50 ADONIS 000689939116923B

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BRES 16923

Substance P and N M D A receptors mediate a slow nociceptive ventral root potential in neonatal rat spinal cord Scott J. Woodley* and Joan J. Kendig Department of Anesthesia, Stanford University School of Medicine, Stanford, CA 94305-5123 (U.S.A.) (Accepted 16 April 1991)

Key words: N-Methyl-D-aspartate; Nociception; Substance P; Spinal cord; Ventral root potential; Windup

Substance P and glutamate actions have separately been implicated in the generation of nociceptive-related slow ventral root potentials (slow VRPs). We report that slow VRPs are dependent on both substance P and NMDA receptor-mediated neurotransmission. Slow VRPs of 10--40 s duration were evoked by electrically stimulating a lumbar dorsal root and recorded at the corresponding ipsilateral ventral root in spinal cords isolated from 1- to 5-day-old rats; the monosynaptic reflex was also recorded. The NMDA receptor antagonist APV (5-20 #M) and the substance P antagonist spantide (10-20 #M) both reversibly depressed the slow VRP without affecting the monosynaptie reflex; spantide and APV applied together nearly abolished the slow VRP. The quisqualate-kainate receptor antagonist CNQX (1-5 #M) reduced the monosynaptic reflex and an early component of the slow VRP. A slow VRP could be elicited by brief (0.1-1.0 s) focal applications of either substance P (2-20 #M) or NMDA (10/~M), and also by CGRP (2-20 gM). Substance P-evoked and NMDA-evoked responses were blocked by their respective antagonists spantide and APV. Each was also cross-sensitive to the other antagonist. Both excitatory amino adds, acting on an NMDA receptor, and substance P, acting on a tachykinin receptor, thus appear to be involved in generating this slow potential. Both NMDA and taehykinin receptors are necessary to generate a full response. INTRODUCTION Slow v e n t r a l r o o t p o t e n t i a l s (slow V R P s ) of 1 0 - 4 0 s d u r a t i o n c a n be r e c o r d e d f r o m the v e n t r a l roots of b o t h adult frog 1° a n d n e o n a t a l rat spinal cords is. Slow V R P s c a n b e elicited b y e i t h e r n o x i o u s stimuli to the p e r i p h ery 3° o r electrical s t i m u l a t i o n o f dorsal roots at a n i n t e n sity c o r r e s p o n d i n g to the t h r e s h o l d of s m a l l - d i a m e t e r noc i c e p t i v ~ ' a f f e r e n t s ; they have b e e n f u r t h e r l i n k e d to n o c i c e p t i o n b y sensitivity to a variety o f analgesic agents 14,3°. I n s e p a r a t e studies, slow V R P s have b e e n ascribed alt e r n a t i v e l y to excitatory a m i n o acids 5 a n d to p e p t i d e s such as s u b s t a n ~ , P Le°. T h e r e has b e e n c o n s i d e r a b l e deb a t e c o n c e r n i n g the respective roles of excitatory a m i n o acids a n d s u b s t a n c e P in nociceptive n e u r o t r a n s m i s s i o n 2' 22. W e r e p o r t that b o t h e x c i t a t o r y a m i n o acids, acting o n a g l u t a m a t e r e c e p t o r of the N M D A type, a n d p e p t i d e s such as s u b s t a n c e P a n d calcitonin g e n e - r e l a t e d p e p t i d e ( C G R P ) , a p p e a r to be i n v o l v e d in g e n e r a t i n g slow v e n tral root p o t e n t i a l s in n e o n a t a l rat spinal cord.

MATERIALS AND METHODS Newborn (1- to 5-day-old) Sprague-Dawley rat pups were anes

thetized with isoflurane or diethyl ether and decapitated. The spinal cord from the upper thoracic to the sacral level was rapidly removed, placed in a chamber and superfused with artificial cerebrospinal fluid (ACSF) at a rate of approximately 2.5 ml/min. The ACSF consisted of (in mM) NaCI 123, KCI 5, NaH2PO4.H20 1.2, CaCI2 2, MgSO4.7H20 1.3, NaHCO 3 26, glucose 30, equilibrated with 95% 02/5% CO 2 to bring the pH to 7.4, and warmed to a temperature of 27-28 °C as measured by a thermistor in the chamber near the cord. A suction-stimulating electrode was placed on a large dorsal root, most commonly the 4th lumbar root, and a suction-recording electrode on the corresponding ipsil ateral ventral root at its exit from the cord. Stimuli were single square wave pulses 0.2 ms in duration. Stimulus intensity was adjusted to be well supramaximal for eliciting the slow VRP (7-40 V), and frequency was maintained at 0.02 Hz throughout each experiment. In some experiments substance P (2-20 /~M), N-methyl-v-aspartate (NMDA) (10 #M) or calcitonln gene-related peptide (CGRP) (5-20 gM) was applied directly to the cord by pressure ejection from a micropipette positioned near the insertion of the dorsal root. Responses were amplified by a high-gain amplifier with a band width set at DC to 30 kI-Iz and monitored on an oscilloscope screen. For measurement and later analysis, responses were digitized, averaged (n = 5), and stored on diskette. Responses to application of neurotransmitters were recorded similarly but not averaged. The monosynaptic reflex was measured and plotted for illustrations without further manipulation; electrically evoked slow VRPs, recorded at higher gain, were digitally filtered by a single pass through an RC filter with a 20 ms rise time in order to minimize the faster noise components of the record; transmitter-evoked slow VRPs were similarly filtered with a falter rise time of 100 ms. Slow VRP shape and amplitude was not affected by the filter. Ventral root potenrials were measured from baseline to peak of the monosynaptic reflex and the transmitter-evoked slow VRPs; slow VRPs elicited by

* Present address: Department of Psychology, University of California, Davis, CA 95616, U.S.A. Correspondence: J.J. Kendig, Department of Anesthesia, Stanford University School of Medicine, Stanford, CA 94305-5123, U.S.A.

18 dorsal root stimulation were measured at a point 3 s after the stimulus. Data acquisition and analysis were handled by commercially available software (pCLAMP, Axon Instruments). Preparations were allowed to equilibrate for 30 min and were accepted if two control measurements 15 min apart showed less than 10% change in monosynaptic and slow ventral root reflexes. Under control conditions, responses from such preparations remained stable for 3 h or more. Neurotransminer antagonists were made up as stock solutions in distilled water, diluted to the desired concentration in ACSF, and applied to the cord in the perfusate. All compounds were obtained from commercial sources.

RESULTS Typical slow ventral root potentials elicited by electrical stimulation of a lumbar dorsal root are shown in Fig. 1. The slow VRPs were reversibly depressed by both the

A

I 0.16mV

CONTROL

APV 20 IJ,M

WASH

B

CONTROL

SPANTIDE 16 gM

CONTROL

SPANTIDE 15 p.M

WASH

C

substance P antagonist spantide (5-20/~M) (n = 5) and the N M D A receptor antagonist oe-2-amino-5-phosphonovalerate (APV) (5-20 #M) (n = 9) (Fig. 1). Application of the two antagonists together nearly abolished the slow VRP, leaving only a 5-10% remnant apparently not sensitive to either antagonist (n = 2) (Fig. 1). The faster monosynaptic reflex was unaffected by either A P V or spantide (n = 14) (Fig. 1). The time courses of onset and recovery from block were different for the two antagonists. A P V block of the slow V R P was detectable within 5 min and rapidly and completely reversible on washing with drug-free solution (30 min). Spantide's effects were slower (15-20 min) to appear and prolonged washing with drug-free solution was required to effect partial reversibility. The two antagonists also appeared different in their effects on the slow VRP. Spantide was somewhat selective for the late components, whereas an early component appeared more sensitive to A P V (Fig. 1). However, there was extensive overlap, and the entire slow V R P was depressed by both agents in the concentrations tested. The effects of the selective kainate and quisqualate receptor blocking agent 6-cyano-7-nitroquinoxaline-2,3dione ( C N Q X ) 7 were also examined. C N Q X reversibly depressed the monosynaptic reflex (n = 3). There was also some depression of the early component of the slow V R P (Fig. 2). Brief applications of either N M D A (10/aM) (n = 10) or substance P (2-20 #M) (n = 9) elicited dose-dependent slow ventral root potentials (Fig. 3) which were sensitive to their respective antagonists A P V (n = 2) and spantide (n = 3) over the same concentration range as the slow V R P evoked by dorsal root stimulation (Fig. 4). Calcitonin gene-related peptide ( C G R P ) (5-20 #M) also evoked a slow V R P (n = 3). Stable substance P-evoked

SPANTIDE 15 pM APV 20 p.M

A D

CONTROL

CONTROL

SPANTIDE 15 p.M

CNQX 1 p.M

S P A N T i D E15 p.M APV 20 llM

Fig. 1. The slow ventral root potential (slow VRP) is sensitive to both the NMDA receptor antagonist APV and the substance P antagonist spantide. A: the depression produced by 20 min exposure to 20/~M APV is rapidly and completely reversed on 30 rain washing. B: spantide (16/~M) produces a depression of slower onset that is slowly and only partially reversed on washing for one hour in drug-free ACSF. C: application of spantide (15/~M) followed by APV (20/~M) abolishes most of the slow VRP, leaving a remnant of -10% of the original amplitude. D: the monosynaptic reflex from the same preparation shown in C is unaffected by spantide and APV.

B

CONTROL

CNOX 1 p.M

WASH

Fig. 2. A: the monosynaptic reflex is reversibly depressed by the kainate-quisqualate receptor antagonist CNQX (1 #M). B: the slow VRP from the same preparation is only slightly affected by CNQX.

19

A

A

~SP 200 ms

~ SP 400 ms

~ SP 800 ms

=¢-

SP CONTROL

APV 10 I4M

WASH

CONTROL

SPANTiOES.U

WASH

a

B

~NMDA200 ms

NMDA 300 ms

~ NMDA 500 ms

Fig. 3. Dose-dependent slow VRPs evoked by brief focal application of either substance P (SP) (A) or NMDA (B) to the spinal cord. Neurotransmitters were applied at the times indicated by the arrows by application of a fixed pressure to a pipette containing the transmitter for the period shown beneath each response. Concentration of SP in the pipette was 2 #M; of NMDA, 10 #M.

potentials could be readily elicited by r e p e a t e d applications at 5-rain intervals. Systematic exploration o f alternative locations for the pipette tip r e v e a l e d m a x i m u m sensitivity to substance P in an a r e a on t h e dorsal surface of the cord n e a r the insertion of the dorsal root; the ventral half of the cord surface was insensitive to substance P. It was m o r e difficult to achieve stable responses to N M D A . Ten-minute intervals were r e q u i r e d b e t w e e n applications, and even at this interval, responses often decreased in a m p l i t u d e o v e r time. Location of m a x i m u m

A

f CONTROL

WASH

DISCUSSION

~v

CONTROL

sensitivity to N M D A was m o r e difficult to ascertain because o f the p r o l o n g e d desensitization to r e p e a t e d applications; however, the shortest latency responses could usually be elicited b y application just ventral to the insertion of the dorsal root. We e x p l o r e d the interactions b e t w e e n the two neurotransmitters by examining cross-sensitivity to the antagonists for each receptor. A P V (10 # M ) rapidly and reversibly inhibited the response to substance P (Fig. 5) (n = 4). Spantide a p p e a r e d to block the response to N M D A (Fig. 5) (n = 4), but reversibility was difficult to establish; the effect o f spantide on the N M D A - e v o k e d response was partially reversed on washing in one out of 4 attempts.

¢ SPANTIDE 5/~M

B

N.o2k'. "

Fig. 5. Substance P and NMDA-evoked VRPs are cross-sensitive to each other's antagonist. A: substance P at a concentration of 2 /~M in the pipette applied for 1 s at the arrow produced a response which was reversibly depressed by 10 pM APV. B: NMDA at a concentration of 10/~M in the pipette was applied for 300 ms in the control and evoked a slow VRP. In the presence of spentide (5 pM) application of NMDA for 1 s failed to evoke a response. On washing, there was only a partial recovery; application for 2 s evoked a slow VRP.

APV 20 I~M

WASH

Fig. 4. The responses evoked by SP and NMDA are reversibly depressed in the presence of their respective antagonists spantide and APV. Neurotransmitters were applied at the points shown by the arrows. A: substance P at a concentration of 20/~M in the pipette applied for 2 s evoked a response which was reversibly depressed by 5/xM spantide. B: NMDA at a concentration of 10 #M in the pipette applied for 800 ms evoked a response which was depressed by 20/~M APV; recovery on washing was only partial.

Different laboratories have previously described slow ventral root potentials o f very long time course (10-40 s duration). O n e group has implicated tachykinins such as substance P in slow V R P generation, b a s e d on sensitivity to the antagonist substance P analogue spantide 2° and on the ability of substance P to e v o k e such a response 1. A second group ascribes the slow ventral r o o t potential to the N M D A r e c e p t o r for excitatory a m i n o a d d s , based on sensitivity to A P V s. We confirm b o t h results. The slow V R P is sensitive to both spantide a n d APV, and slow V R P s can be e v o k e d b y b o t h substance P and N M D A . T h e fact that 5-10% o f the response r e m a i n e d after application of both spantide and A P V t o g e t h e r sug-

20 gests that other receptors may be involved in generating a small part of the slow VRP. In the neonatal rat spinal cord 15, as in the adult frog 7 and rat H, the fast EPSP which generates the monosynaptic reflex is mediated by non-NMDA excitatory amino acid receptors as evidenced by its sensitivity to CNQX and insensitivity to APV. The present experiments were done on cords from rats 5 days old or less. We have been able to record slow VRPs in thick spinal cord slices from rats up to 17 days old (L. Gibbs and J.J. Kendig, unpublished data). Since the slow VRP increases in amplitude dramatically on repetitive stimulation, it may be related to the 'windup' phenomenon observed in adult spinal cord in response to repeated noxious or C-fiber stimulation 2s'29. Recent evidence shows that an APV-sensitive prolonged depolarization is elicited in young rat ventral horn neurons by the same stimulus pattern which evokes windup 2s. The pathway involved in generating the slow VRP is largely unknown. The neurotransmitters glutamate, substance P and C G R P are contained in primary afferent neurons of various types and may be co-localized in some nerve terminals 4. Excitatory amino acids may be released as transmitters both from primary afferent nerve terminals and from first or higher order interneurons; N M D A receptors, however, may be localized to higher order interneurons 22. In the neonatal cord capsaicin, reported to specifically depolarize C-fibers 6'27, induces a release of substance P and C G R P (J.J. Kendig, S.J. Woodley and T. Yaksh, unpublished data). Primary afferent stimulation generates intrinsically slow potentials in interneurons 3x and motor neurons 2s. Excitatory amino acids generate intrinsically slow depolarizing potentials in interneurons 8"9A7. Both N M D A and non-NMDA receptors participate in the generation of the early components of slow potentials. Substance P also directly depolarizes interneurons ~6'17 and possibly also motor neurons 19, generating a slow depolarization in interneurons 26. Substance P may act in part by potentiating the release of endogenous glutamate from spinal cord 12'13. Substance P has also been shown to potentiate NMDAreceptor-mediated glutamate responses in spinal dorsal horn neurons 23.

REFERENCES 1 Akagi, H., Konishi, S., Otsuka, M. and Yanagisawa, M., The role of substance P as a neurotransmitter in the reflexes of slow time courses in the neonatal rat spinal cord, Br. J. Pharmacol., 84 (1985) 663-673. 2 Bossut, D., Frenk, H. and Mayer, D.J., Is substance P a primary afferent neurotransmitter for nociceptive input? IV. 2-Amino-5-phosphonovalerate (APV) and (DPro-2,D-Trp-7,9)substance P exert different effects on behaviors induced by intratheeal substance P, strychnine and kainic acid, Brain Research, 455 (1988) 247-253.

Brief applications of either N M D A or substance P evoked slow potentials at the ventral root. Sensitivity of the substance P-evoked response to the glutamate N M D A receptor blocker APV was clear-cut; recent findings show that an N M D A receptor antagonist also blocks contralateral slow VRPs elicited by substance p3. Sensitivity of substance P responses to glutamate receptor antagonists can be accounted for by the evidence outlined above that substance P acts in part by releasing glutamate. The substance P antagonist spantide appeared to block the slow potential evoked by NMDA, although this result is less certain because of difficulty in reversing the depression. Substance P analogues such as spantide may have effects of their own rather than functioning as pure substance P antagonists. Local anesthetic actions have been reported for tachykinin antagonists, although at higher concentrations than those used in the present study 21. If the apparent antagonism of NMDAevoked responses by spantide is real, the simplest explanation is that depolarization by active tachykinin receptors relieves N M D A receptor block; other synergistic actions are also possible 23. Cross-sensitivity to antagonists implies that active receptors of both types are required for the full expression of a response to either neurotransmitter. Both glutamate and substance P have been implicated in the generation of nociceptive responses; in vivo, the behaviors evoked by intrathecal injection of either glutamate or substance P exhibit crossdesensitization 24. In conclusion, we have shown that a slow ventral root potential in intact neonatal rat spinal cord relies on both neuropeptide and excitatory amino acid neurotransmission. Both components are necessary to the generation of a full response. The results may be relevant to nociceptive neurotransmission in vivo, and specifically to the prolonged increase in excitability which may underlie postinjury hyperalgesia.

Acknowledgements. Supported by NIH Grant NS13108 to J.J.K. The results were presented in part at the 1990 annual meeting of the Society for Neuroscience.

3 Brugger, E, Evans, R.H. and Hawkins, N.S., Effects of N-methyl-D-aspartate antagonists and spantide on spinal reflexes and responses to substance P and capsaicin in isolated spinal cord preparations from mouse and rat, Neuroscience, 36 (1990) 611622. 4 De Biasi, S. and Rustioni, A., Glutamate and substance P coexist in primary afferent terminals in the superficial laminae of spinal cord, Proc. Natl. Acad. Sci. U.S.A., 85 (1988) 78207824. 5 Evans, R.H., A slow spinal synaptic respop~se mediated at NMA receptors, Br. J. Pharmacol., 88 (1986) 269P.

6 Fitzgerald, M. and Woolf, C.J., The time course and specificity

21 of the changes in the behavioural and dorsal horn cell responses to noxious stimuli following peripheral nerve eapsaicin treatment in the rat, Neuroscience, 7 (1982) 2051-2056. 7 Fletcher, E,J., Martin, D., Aram, J.A., Lodge, D. and Honore, T., Quinoxalinediones selectively block quisqualate and kainate receptors and synaptic events in rat neocortex and hippocampus and frog spinal cord in vitro, Br. J. Pharmacol., 95 (1988) 585-597. 8 Gerber, G. and Randic, M., Excitatory amino acid-mediated components of synaptieally evoked input from dorsal roots to deep dorsal horn neurons in the rat spinal cord slice, Neurosci. Lea., 106 (1989) 211-219. 9 Gerber, G. and Randic, M., Participation of excitatory amino acid receptors in the slow excitatory synaptic transmission in the rat spinal dorsal horn in vitro, Neurosci. Lett., 106 (1989) 220228. 10 Holohean, A.M., Vega, J.L., Hackman, J.C. and Davidoff, R.A., Excitatory transmitters, ventral root potentials and [K +]o in the isolated frog leg-spinal cord preparation, Neurosci. Lett., 95 (1988) 173-178. 11 Jessell, T.M., Amino acid receptor-mediated transmission at primary afferent synapses in rat spinal cord, J. Exp. Biol., 124 (1986) 239-258. 12 Kangrga, I., Larew, J.S. and Randie, M., The effects of substance P and ealcitonin gene-related peptide on the efflux of endogenous glutamate and aspartate from the rat spinal dorsal horn in vitro, Neurosci. Lett., 108 (1990) 155-160. 13 Kangrga, I. and Randie, M., Tachykinins and ealcitonin generelated peptide enhance release of endogenous glutamate and aspartate from the spinal dorsal horn slice, J. Neurosci., 10 (1990) 2026-2038. 14 Kendig, J.J., Savola, M.K.T., Woodley, S.J. and Maze, M., Alpha-2 adrenoceptors inhibit a nociceptive response in neonatal rat spinal cord, Eur. J. Pharmacol., 192 (1991) 293-300. 15 Long, S.K., Smith, D.A., Siarey, R.J. and Evans, R.H., Effect of 6-cyano-2,3-dihydroxy-7-nitro-quinoxaline(CNQX) on dorsal root-, NMDA-, kainate- and quisqualate-mediated depolarization of rat motoneurones in vitro, Br. J. Pharmacol., 100 (1990) 850-854. 16 Murase, K., Nedeljkov, V. and Randic, M., The actions of neuropeptides on dorsal horn neurons in the rat spinal cord slice preparation: an intracellular study, Brain Research, 234 (1982) 170-176. 17 Murase, K., Ryu, P.D. and Randic, M., Excitatory and inhibitory amino acids and peptide-induced responses in acutely isolated rat spinal dorsal horn neurons, Neurosci. Lett., 103 (1989)

56--63. 18 Otsuka, M. and Konishi, S., Electrophysiology of mammalian spinal cord in vitro, Nature, 252 (1974) 733-734. 19 Otsuka, M. and Yanagisawa, M., The effects of substance P and badofen on motoneurones of isolated spinal cord of the newborn rat, J. Exp. Biol., 89 (1980) 201-214. 20 Otsuka, M. and Yanagisawa, M., Effect of a tachykinin antagonist on a nociceptive reflex in the isolated spinal cord-tail preparation of the newborn rat, J. Physiol., 395 (1988) 255-270. 21 Post, C., Butterworth, J.F., Strichartz, G.R., Karlsson, J.-A. and Persson, C.G.A., Tachykinin antagonists have potent local anaesthetic actions, Eur. I. Pharmacol., 117 (1985) 347-354. 22 Raigorodsky, G. and Urea, G., Intrathecal N-methyl-o-aspartate (NMDA) activates both nociceptive and antinociceptive systems, Brain Research, 422 (1987) 158-162. 23 Randic, M., Hecimovic, H. and Ryu, ED., Substance P modulates glutamate-induced currents in acutely isolated rat spinal dorsal horn neurones, Neurosci. Lett., 117 (1990) 74-80. 24 Sun, X. and Larson, A.A., Desensitization and sensitization to substance P- and excitatory amino acid-induced behaviors in the mouse, Soc. Neurosci. Abstr., 16 (1990) 377. 25 Thompson, S.W.N., King, A.E. and Woolf, C.J., Activity-dependent changes in rat ventral horn neurons in vitro; summation of prolonged afferent evoked post-synaptic depolarizations produce a D-2-amino-5-phosphonovaleric acid sensitive windup, Eur. J. Neurosci., 2 (1990) 638-649. 26 Urban, L. and Randic, M., Slow excitatory transmission in rat dorsal horn: possible mediation by peptides, Brain Research, 290 (1984) 336-341. 27 Williams, J.T. and Zieglgansberger, W., The acute effects of capsaicin on rat primary afferents and spinal neurons, Brain Reseach, 253 (1982) 125-131. 28 Woolf, C. and Wiesenfeld-Hallin, Z., Substance P and ealcitonin gene-related peptide synergistically modulate the gain of the nociceptive flexor withdrawal reflex in the rat, Neurosci. Lett., 66 (1986) 226-230. 29 Woolf, C.J. and Wall, P.D., Relative effectiveness of C primary afferent fibers of different origins in evoking a prolonged facilitation of the flexor reflex in the rat, J. Neurosci., 6 (1986) 1433-1443. 30 Yanagisawa, M., Murakoshi, T., Tamai, S. and Otsuka, M., Tail-pinch method in vitro and the effects of some anti-nociceptive compounds, Eur. J. Pharmacol., 106 (1985) 231-239. 31 Yoshimura, M. and Jessell, T.M., Primary afferent-evoked synaptic responses and slow potential generation in rat substantia gelatinosa neurons in vitro, J. Neurophysiol., 62 (1989) 96-108.