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Illness-induced hyperalgesia is mediated by spinal neuropeptides and excitatory amino acids
BRAIN RESEARCH ELSEVIER
Brain Research 664 (1994) 17-24
Research report
Illness-induced hyperalgesia is mediated by spinal neuropeptides and excitatory amino acids Linda R. Watkins a,, Eric P. Wiertelak b Linda E. F u r n e s s
a
S t e v e n F. Maier a
a Department of Psychology, Unit~ersity of Colorado at Boulder, Campus Box 345, Boulder, CO 80309, USA b Department of Psychology, Macalester College, St. Paul, MN, USA Accepted 9 August 1994
Abstract
The spinal cord dorsal horn contains neural mechanisms which can greatly facilitate pain. We have recently shown that 'illness'-inducing agents, such as intraperitoneally administered lipopolysaccharide (LPS; bacterial endotoxin), can produce prolonged hyperalgesia. This hyperalgesic state is mediated at the level of the spinal cord via activation of the NMDA-nitric oxide cascade. However, prolonged neuronal depolarization is required before such a cascade can occur. The present series of experiments were aimed at identifying spinal neurotransmitters which might be responsible for creating such a depolarized state. These studies show that LPS hyperalgesia is mediated at the level of the spinal cord by substance P, cholecystokinin and excitatory amino acids acting at non-NMDA sites. No apparent role for serotonin or kappa opiate receptors was found.
Keywords: Spinal cord; Substance P; Cholecystokinin; Serotonin; Opiate; Excitatory amino acid; Hyperalgesia; CP-96345; L-365260; Methysergide; Norbinaltorphimine; Naltrexone
I. Introduction
The spinal dorsal horn contains mechanisms which can increase nociceptive transmission when activated (for reviews, see [9,34,38,65]). These mechanisms enhance dorsal horn excitability and are thought to at least partially mediate the facilitation of nociception (hyperalgesia) that is produced by conditions as diverse as sciatic nerve ligature [36,68], heat injury [10] and subcutaneous injection of irritants such as mustard oil [66], formalin [34] and carageenen [45]. Facilitation of dorsal horn excitability and resultant hyperalgesia appears to be produced by the same NMDA-NO cascade that is involved in producing sustained enhancement of neural excitability in other regions of the nervous system [7,29]. The spinal NMDANO cascade is initiated by substance P (SP) acting at NK-1 receptors and excitatory amino acids (EAAs) acting at non-NMDA ionotropic and metabotropic
* Corresponding author. 0006-8993/94/$07.00 © 1994 Elsevier Science B.V, All rights reserved SSDI 0 0 0 6 - 8 9 9 3 ( 9 4 ) 0 0 9 8 0 - 5
EAA receptors on N M D A receptor-containing neurons. Thus spinal administration of EAAs and substance P can produce behavioral hyperalgesia and increased dorsal horn neural excitability and these effects of EAAs and substance P are blocked by N M D A antagonists [2,33,37]. In addition, the dorsal horn windup produced by C-fiber stimulation and the hyperalgesia induced by prolonged nociceptive stimulation are eliminated by spinal NK-1 antagonists and non-NMDA EAA antagonists [10,33,68]. Many of the conditions which lead to hyperalgesia are known to produce sustained primary afferent Cfiber a n d / o r A~ nociceptive spinal input. Terminals of these primary afferents contain both substance P and glutamate [19,50] and at least some of the hyperalgesia-inducing manipulations noted above have been shown to produce the release of substance P and glutamate in the dorsal horn [19,50]. This set of facts has led to the view that the the spinal NMDA-NO cascade is set into motion by the prolonged release of substance P and glutamate from primary afferents. However, we have recently demonstrated a form of hyperalgesia that is difficult to explain in this fashion. The intraperitoneal (i.p.) injection of 'illness'-inducing
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I..R. Watkins et al. / Brain Researctl 604 (1994) ] 7-24
agents lithium chloride (LiCI) and endotoxin (lipopolysaccharide, LPS) p r o d u c e a p r o l o n g e d hyperalgesia as m e a s u r e d by both tail-flick to r a d i a n t heat and subcut a n e o u s formalin tests [31,57,58,62,64]. T h e hyperalgesia is m e d i a t e d by spinal N M D A - N O m e c h a n i s m s in that it is completely blocked by spinal application of the competitive N M D A receptor a n t a g o n i s t APV, the n o n - c o m p e t i t i v e N M D A antagonist MK-801 and the N O synthesis inhibitor L - N A M E [62 (previous article in this issue)]. Importantly, the hyperalgesia p r o d u c e d by L i C I and LPS does not a p p e a r to be m e d i a t e d by primary afferent p a i n i n p u t to the spinal cord from the viscera. Nociceptive afferents from the viscera to the spinal cord are carried in the splanchnic nerve [8] and t r a n s e c t i o n of the splanchnic nerve has no effect on the hyperalgesia p r o d u c e d by illness-inducing agents [57]. Instead, the e n h a n c e m e n t of nociceptive behavior produced by LiCI a n d LPS is likely to be m e d i a t e d by d e s c e n d i n g circuitry, since it is blocked by both hepatic vagotomy a n d dorsolateral funiculus lesions [57] a n d is not blocked by t r a n s e c t i o n of the splanchnic nerve [57]. T h e r e are a n u m b e r of possibilities. Activation of the spinal N M D A - N O cascade could be p r o d u c e d by m e a n s o t h e r t h a n substance P and g l u t a m a t e release. Alternatively, LiCI and LPS might lead to spinal substance P and g l u t a m a t e release from a source other than primary afferent terminals. Circuitry involved in pain m o d u l a t i o n descends to the dorsal horn from the rostro-ventral m e d u l l a [6], with the n u c l e u s r a p h e magnus ( N R M ) being a primary site [6]. I n d e e d , descending systems play a role in pain facilitation [17,18,31], as well as their b e t t e r k n o w n involvement in pain inhibition. Interestingly, d e s c e n d i n g axons from the N R M c o n t a i n both substance P [39] a n d g l u t a m a t e [40] and N R M lesions block the hyperalgesia p r o d u c e d by illn e s s - i n d u c i n g agents [57]. T h u s LiC1 and LPS might activate spinal N M D A - N O m e c h a n i s m s by stimulating a d e s c e n d i n g release of substance P and glutamate. T h u s one p u r p o s e of the p r e s e n t e x p e r i m e n t s was to d e t e r m i n e w h e t h e r spinal substance P a n d E A A s are involved in the m e d i a t i o n of illness-induced hyperalgesia. In addition, there are o t h e r t r a n s m i t t e r s k n o w n to be involved in d e s c e n d i n g pain facilitation. Serotonin, cholecystokinin (CCK) a n d d y n o r p h i n are most promin e n t . F o r example, electrical s t i m u l a t i o n of sites in the R V M can facilitate nociceptive r e s p o n d i n g [71] a n d this effect can be blocked by spinal a d m i n i s t r a t i o n of the the non-selective 5 - H T receptor a n t a g o n i s t methysergide [71]. Similarly, pain facilitation can be blocked by spinal application of CCKB antagonists [63] a n d k a p p a opiate antagonists [46]. T h u s a n o t h e r p u r p o s e of the p r e s e n t e x p e r i m e n t s was to d e t e r m i n e w h e t h e r L P S - i n d u c e d hyperalgesia could be blocked by spinal a d m i n i s t r a t i o n of either serotonin, C C K or kappa opiate antagonists.
2. Materials and methods 2.1. Subjects
Adult Sprague-Dawley rats (Holtzman Laboratories) were used in all experiments and were 450-600 g at the time of testing. They were singly housed and maintained on a 12 h/12 h light/dark cycle. Experiments occurred during the light part of the cycle. All procedures were in accord with protocols approved by the University of Colorado Institutional Animal Care and Use Committee, 2.2. Tai(flick testing and experimental procedure
Tailflick (TF) latencies to radiant heat were assessed using a modification [4] of the procedure developed by D'Amour and Smith [14]. All TF trials were conducted in a dimly lit experimental room maintained at 27°C. The room was maintained at this temperature because this minimizes tail temperature effects on TF latencies. We have monitored tail temperature and have not observed drug-induced changes at this room temperature. During testing, rats were loosely restrained in Plexiglascylinders designed such that their tails protruded from the rear allowing testing without disturbance of the subject. The rats were placed in the tubes 30 min before testing. This 30 min adaptation was followed by measurement of baseline (BL) TF latencies. The tail was placed over a radiant heat source adjusted to produce BL latencies in untreated animals in the 6-s range. This goal latency was chosen to allow observation of both analgesia and hyperalgesia [31] with the last three averaged to form the BL for that subject. Trials were automatically terminated at 18 s if no tail flick occurred, in order to prevent tissue damage. Following assessment of baseline TF latencies drug or vehicle was administered intrathecally (IT) and a second post-drug BL determined as above beginning 10 min after injection. LPS (i.p.) was then administered. TF latencies were then measured at 5-min intervals beginning 5 min after LPS injection for 55 min. TF measurements were made by blind experimenters unaware of group membership. Animals were carefully observed for possible impairments of motor function. None were observed except as noted below. 2.3. lntrathecal surgery and injection
Surgery was performed under sodium pentobarbital anesthesia (Nembutal, Abbott Laboratories, 55 mg/kg, i.p.), supplemented with methoxyflurane (Metofane, Pitman-Moore) as required. Intrathecal (IT) catheters were implanted into the subdural space surrounding the spinal cord at the lumbosacral enlargement. Construction of catheters and implantation procedures have been described in detail elsewhere [55,67]. Subjects were given a 1-2-week recovery period after surgery. Penicillin-streptomycin was given post-operatively to prevent infection. During testing, drug or vehicle was injected into the IT catheter, followed by a 10 /zl physiological saline flush delivered slowly over 30 s (for further details see [55]). 2.4. Drugs
In order to test the potential involvement of EAAs, substance P, CCK and opioids in LPS hyperalgesia, the effects of CNQX (AMPA receptor antagonist), CP-96345 (substance P antagonist), L-365,260 (antagonist of the CCK-B receptor), methysergide (serotonin antagonist), nor-binaltorphimine (kappa opiate receptor antagonist), and naltrexone (broad spectrum opiate antagonist) were assessed in separate experiments. It should be noted that CP-96345 may interact with calcium channels and therefore have non-selective effects [20]. CP-96345 (generously provided by Pfizer; Lot #19797-57-1) was dissolved in physiological saline at a concentration of 2 mg/ml,
L.R. Watkinset al. / Brain Research 664 (1994) 17-24 aliquoted and frozen at -70°C until used. On the day of testing, an aliquot was slowlythawed and kept cold until used. Each aliquot was only used once. Ten/zl was injected IT, thereby delivering20/xg, 15 rain prior to i.p. LPS. This dose was based on the work of Malmberg and Yaksh [33]. A dose of 200 /~g was also tested, but testing was discontinued upon observation of motor deficits. Vehicle controls received equivolume saline. L-365,260 (generously provided by Merck; Lot #0005007) was dissolved at a concentration of 0.12 and 0.012 mg/ml in dimethysulfoxide (DMSO; buffered to pH = 7.0 using 10 mM PBS; Sigma), aliquoted and frozen at - 70°C until used. Each aliquot was handled as described above for CP-96345. At the time of testing, 1 p,l was injected, thereby delivering either 0.12 or 0.012 p.g, 15 min before LPS. These doses were in accordance with our earlier work [63]. Vehicle controls received equivolume DMSO (pH = 7.0). CNQX (Research Biochemicals, Lot #LR-II-12) was made fresh on the day of testing by dissolving it in a concentration of 0.68 mg/ml in 45% CDEX (Research Biochemicals, Lot #WC1290A) in physiological saline. At the time of testing, 10 p,l was injected, thereby delivering 6.8 p,g, 15 min prior to LPS. This dose was in accordance with the work of Malmberg and Yaksh [33]. Vehicle controls received equivolume 45% CDEX-saline. Methysergide (Research Biochemicals, Lot #NA591A) was dissolved at a concentration of 3.01 mg/ml in 30% DMSO-70% PBS (pH 7.0). Aliquots were stored at -70°C until used. At the time of testing, 10 /~1 was injected, thereby delivering 30.1 /zg, 15 min prior to LPS This dose was in accordance with the work of Zhou and Gebhart [71]. Vehicle controls received equivolume 30% DMSO-70% PBS. Nor-binaltorphimine (Research Biochemicals, LOt #BFW-I-31) was dissolved at a concentration of 19.4 mg/ml in DMSO (pH 7.0, using PBS). Aliquots w e r e stored at - 7 0 ° C until used. At the time of testing, 1 p,l was injected, thereby delivering 19.4/.Lg, 15 min prior to LPS. This dose is comparable to the dose used in our previous studies [60]. Vehicle controls received equivolume buffered DMSO. Naltrexone (Research Biochemicals, Lot #M1089A) was made fresh at a concentration of 7 mg/ml in physiological saline. At the time of testing 1 p.1 was injected, thereby delivering 7/zg, 15 rain prior to LPS. This dose is in accordance with our prior work [60]. Vehicle controls received equivolume saline. To induce hyperalgesia, LPS was injected at a dose of 0.2 mg/kg in a volume of 20 ml/kg i.p. This regimen was based on prior work from this lab [31,57,58,62,64].
2.5. Data analysis BL and post-LPS latencies were separately analyzed by ANOVA to determine whether there were group differences. Subsequent comparisons were made by Scheffe test with alpha level set at 0.05. Paired t-tests were used to determine whether tailflick latencies were reliably elevated above baseline.
3. R e s u l t s
3.1. Effects of IT CNQX on LPS-induced hyperalgesia Our previous work demonstrated that blockade of the N M D A receptor abolished LPS-induced hyperalgesia [62 (previous article in this issue)]. The first experiment in the present study was designed to determine whether this hyperalgesia could also be blocked by spinal application of CNQX, a drug which blocks nonN M D A EAA receptors. One group received CNQX followed by LPS (CNQX + LPS; n = 8), one received
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Fig. 1. Blockade of LPS hyperalgesia by intrathecal CNQX, a nonNMDA EAA receptor antagonist. Prolonged LPS-induced hyperalgesia was observed in vehicle controls (open triangles). CNQX blocked this LPS-induced hyperalgesic state (filled squares). CNQX, in the absence of LPS, had no effect on pain responsivity (open squares).
the CNQX vehicle only followed by LPS (Vehicle + LPS; n = 8) and one received CNQX followed by the LPS vehicle only (CNQX + Saline; n = 8). A group administered only the two vehicles was not necessary, since we have previously found T F latencies to be stable across a 55 min testing period using the identical set of procedures [61] and TF to be unaffected by IT saline [61]. IT CNQX by itself had no effect on TF latency (Fig. 1). BL T F latencies did not differ between groups either before (B1) (F2,21 = 1.33, P > 0.05) or after (B2) (F2,21 = 3.052, P > 0.05) CNQX administration. As in previous experiments [31,57,58,64], the administration of LPS resulted in a reduction in T F latencies below BL. This effect was evident 10 min after LPS and lasted throughout the 55 min timecourse. Importantly, CNQX completely blocked the hyperalgesia produced by LPS. A N O V A revealed a reliable difference between groups (Fz,zl = 12.037, P < 0.0005). Post hoc Scheffe tests indicated that the Vehicle + LPS group differed from the CNQX + LPS and CNQX + Saline groups which did not differ from each other.
3.2. Effects of IT CP-96345 on LPS-induced hyperalgesia The design of this study was identical to that of the previous experiment. One group received the substance P antagonist CP-96345 followed by LPS (CP96345 + LPS; n -- 9), one received the CP-96345 vehicle only followed by LPS (Vehicle + LPS; n = 9) and one received CP-96345 followed by the LPS vehicle only (CP-96345 + Saline; n = 8). CP-96345 had no effect on T F latencies (Fig. 2). There were no BL differences between groups either before ( F < 1.0) or after ( F < 1.0) CP-96345. Moreover, the CP-96345 + Saline group remained stable for the 55 min of testing, with latencies at BL levels. LPS again led to a reduction in T F latencies, which here was apparent at the 10 min point and persisted for most of the 55 min of testing. CP-96345 blocked this hyperalgesia. A N O V A yielded a
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Fig. 2. Blockade of LPS hyperalgesia by intrathecal CP-96345, a substance P receptor antagonist. Prolonged LPS-induced hyperalgesia was observed in vehicle controls (open triangles). CP-96345 blocked this LPS-induced hyperalgesic state (filled squares). CP96345. in the absence of LPS, had no effect on pain responsivity (open squares). reliable difference between g r o u p s (F2,23 = 6.401, P < 0.01). Post hoc Scheffe tests indicated that Vehicle + LPS group differed reliably from the other two. The CP-96345 + LPS and CP-96345 + Saline groups did not differ reliably. 3.3. Effects of I T L-365,260 on LPS-induced hyperalgesia The design of this experiment also followed the pattern above. One group received 0.012 /zg of the CCK B receptor antagonist L-365,260 followed by LPS (0.012/xg L-365,260 + LPS; n = 8), one group received 0.12 /xg of the CCK B receptor antagonist L-365,260 followed by LPS (0.12 /zg L-365,260 + LPS; n = 10), one group received 0.12 /xg of the CCK B receptor antagonist L-365,260 followed by LPS vehicle (L365,260 + Saline; n = 7), and one received vehicle followed by LPS (Vehicle + LPS; n = 16: due to a time delay between testing the two L-365,260 drug groups, complete control groups were run with each and the data pooled for analysis) (Fig. 3). Baseline TF latencies did not differ between groups either before (B1, F < 1.0) or after (B2, F < 1.0) intrathecal drug injection. LPS produced hyperalgesia in vehicle + LPS controls. This hyperalgesia was blocked by the 0.12 mg L-365,260. A N O V A yielded a reliable difference between groups (F3,37 = 12.274, P < 0.0001). Scheffe tests indicated that the Vehicle + LPS group differed from the 0.12 /zg L - 3 6 5 , 2 6 0 + L P S group and from the 0.12 /ag L365,260 + Saline group. These latter two groups did not differ from each other. Scheffe tests also indicated that the Vehicle + LPS group did not differ from the 0.012 /.tg L-365,260 + LPS group, indicating that L365,260 exerted a dose-dependent blockade of LPS hyperalgesia. This low dose group reliably differed from both the 0.12 /xg L-365,260 + LPS group and from the 0.12 /zg L-365,260 + Saline group. Of course, conclusions concerning dose dependency should be tempered, since only two doses were employed.
The present experiment tested whether the serotonin antagonist methysergide could block LPS hyperalgesia. Pilot work failed to find any effect of methysergide on LPS hyperalgesia at any of a range of doses. The present experiment therefore presents a complete group using the most effective dose found to block hyperalgesia in prior studies investigating a different hyperalgesia paradigm [71]. Because pilot data strongly suggested that methysergide would not reduce hyperalgesia a control to assess possible analgesic effects of methysergide by itself was not included. The purpose of such a control would be to counter the possibility that reversal of hyperalgesia was caused by an analgesic effect of the agent itself summating with the hyperalgesic effect of LPS, rather than a blockade of the hyperalgesia produced by LPS. Since a reversal of hyperalgesia was not expected it was felt that animal usage for this control was not justified. Thus, the present experiment contained only two groups. One received methysergide followed by LPS (methysergide; n = 8) and one received equivolume vehicle followed by LPS (vehicle; n = 8). As expected, the baseline latencies did not differ between groups either before ( B 1 ; FI,I4 = 1.094, P > 0.05) or after (B2; FIA 4 = 2.054, P > 0.05) intrathecai drug delivery (Fig. 4). LPS again resulted in a persistent reduction in TF latencies and this reduction was not altered by methysergide ( F <
1.0). 3.5. Effects of I T nor-binaltorphimine on LPS-induced hyperalgesia Pilot work here also did not find an effect of nor-binaltorphimine at any of a variety of doses. What is presented here is one log greater than the intrathecal
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Fig. 3. Blockade of LPS hyperalgesia by 0.12 ~Lg intrathecal L-365,260, a C C K 8 receptor antagonist. Prolonged LPS-induced hyperalgesia was observed in vehicle controls (open triangles). The 0.12 p.g L-365,260 dose blocked this LPS-induced hyperalgesic state (open squares) whereas 0.012 p,g L-365,260 (filled circles) was without effect. The 0.12 p.g L-365,260 dose had no effect on pain responsivity in the absence of LPS (filled triangles).
L.R. Watkins et aL /Brain Research 664 (1994) 17-24
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Fig. 4. Lack of effect of methysergide on LPS hyperalgesia. LPS produced prolonged hyperalgesiain vehicle controls (open triangles). Methysergide (filled squares) did not block this hyperalgesicstate.
dose previously found to block tail-shock induced analgesia in our laboratory [60]. A group that received only nor-binaltorphimine was not included for the same reasons as detailed above. Thus two groups were tested: one group that received intrathecal vehicle followed by LPS (n = 8) and one group that received intrathecal nor-binaltorphimine followed by LPS (n = 8). As expected, the baseline latencies did not differ between groups either before (B1; F1,14 = 0.019, P > 0.05) or after (B2; F1,14 = 1.303, P > 0.05) intrathecal drug delivery (Fig. 5). LPS again resulted in a persistent reduction in T F latencies and this reduction was not altered by methysergide ( F < 1.0).
3.6. Effects of IT naltrexone on LPS-induced hyperalgesia Pilot work here also did not find an effect of naltrexone at any of a variety of doses. Thus what is presented here is the intrathecal dose previously found to be effective in blocking/x, ~ and K opiate receptor mediated tail-shock induced analgesias in our laboratory [59,60]. A group that received only naltrexone was not included for the same reasons as detailed above. Thus two groups were tested: one group that received intrathecal vehicle followed by LPS (n = 8) and one group that received intrathecal naltrexone followed by LPS (n---8). As expected, the baseline latencies did
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Fig. 6. Lack of effect of naltrexone on LPS hyperalgesia. LPS produced prolonged hyperalgesia in vehicle controls (open triangles). Nor-binaltorphimine (filled squares) did not block this hyperalgesic state.
not differ between groups either before (B1; F < 1.0) or after (B2; F < 1.0) intrathecal drug delivery (Fig. 6). LPS again resulted in a persistent reduction in TF latencies and this reduction was not altered by naltrexone ( F < 1.0).
4. D i s c u s s i o n
The present series of experiments suggest that the hyperalgesia produced by LPS is: (a) mediated at the level of the spinal cord by substance P, CCK and EAAs acting at non-NMDA sites, since this hyperalgesia was blocked by CP-96345, L-365260 and CNOX, respectively; and (b) apparently not mediated at the level of the spinal cord by either opiate receptors or serotonin, since this hyperalgesia was not blocked by either naltrexone or nor-binaltorphimine or by methysergide, respectively. Although these latter experiments each tested a single dose of antagonist, the doses chosen were ones that effectively blocked spinal pain modulation in previous studies [59,60,71]. Involvement of EAAs in the NMDA-mediated hyperalgesia induced by i.p. LPS is supported by prior work. EAAs can act both at N M D A and non-NMDA sites. Our previous studies have demonstrated that N M D A receptors are involved in LPS hyperalgesia, since MK-801 (an N M D A channel blocker) and APV (an N M D A receptor antagonist) both abolish this hyperalgesic state [62 (previous article in this issue)]. The present experiments extend this finding by demonstrating that non-NMDA EAA receptors are also critical, since CNOX also abolished LPS hyperalgesia. EAAs acting through non-NMDA receptors is a major pathway by which neurons can become sufficiently depolarized to cause Mg 2÷ to be expelled from the N M D A channel [38]. Iontophoretically applied EAAs in the spinal cord dorsal horn increase the size of cutaneous receptive fields in nociceptive neurons [72] and activate nociceptive projection neurons [1,28]. Additionally, when subthreshold currents of EAA agonists are ion-
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L.R. Watkins et al. /Brain Research 064 (1994) 17 24
tophoresed near neurons, an enhanced neuronal response is observed to peripheral nociceptive stimuli [21]. At a behavioral level, non-NMDA agonists elicit pain behaviors when injected intrathecally [3] and produce hyperalgesia on the T F test [3]. Similarly, nonNMDA EAA receptor antagonists block hyperalgesias produced by nerve ligatures, burns and s.c. formalin [10,61,68]. Given that 40% of nociceptive projection neurons respond to EAAs acting at N M D A receptors but n o t to EAAs acting at non-NMDA receptors [28], such neurons clearly require depolarization by substances other than EAAs before N M D A receptors can be fully activated. Substance P could fulfill such a role. The present experiments indicate that substance P may be critically involved in NMDA-mediated hyperalgesia, since this hyperalgesic state was abolished by the substance P antagonist, CP-96,345. Such a role for substance P is supported by previous studies which showed that substance P selectively facilitates nociceptive responses of spinal cord dorsal horn neurons [22] and produces prolonged (up to 2 h) increases in responses of nociceptive spinothalamic neurons to iontophoresed N M D A [15,44]. Intrathecal substance P elicits behavioral indices of pain [16,25,26] and produces hyperalgesia on the TF [13,69,70] and foot withdrawal tests [10]. Furthermore, concurrent intrathecal injection of substance P and N M D A enhances nociceptive responses in the formalin test, over and above the actions of either intrathecal drug alone [41]. Blockade of spinal substance P blocks hyperalgesias induced by intrathecal galanin, subcutaneous chemical irritants, burns and cold stress [12,27,43,47,48,61,69]. Substance P does not appear to be the only neuropeptide mediating LPS hyperalgesia. CCK was also found to be important, since LPS hyperalgesia was blocked by the selective CCK B receptor antagonist L-365,260. However, conflicting evidence exists regarding the role of CCK in pain modulation at the level of the spinal cord. At an electrophysioiogical level, CCK has been observed to either modestly enhance [30] or have no effect on [52] responses of spinal cord dorsal horn neurons to nociceptive stimuli. Likewise, behavioral studies indicate that CCK and CCK antagonists can either enhance [23], inhibit [5,23] or have no effect on [5,32,42,53,54,63] pain threshold. In contrast to such conflicting results on basal pain responsivity, it seems clear that CCK plays a key role in pain facilitation due to its apparent anti-opiate effects on analgesia [5,32,54,63]. However, these studies concluded that CCK opposes opiate analgesia rather than inducing a true hyperalgesic state [5,32,54,63]. Perhaps the deciding factor in whether anti-analgesia or hyperalgesia is observed at a behavioral level rests in whether neuronal excitation is sufficiently prolonged to enable N M D A activation to occur. Prolonged activation could
presumably be attained either through continued release of CCK or by concomitant release of substance P a n d / o r EAAs acting via non-NMDA sites, as seems to occur during LPS-induced hyperalgesia. Given the weak excitatory effect of CCK on dorsal horn neurons [30] and the general lack of effect of exogenous CCK or CCK antagonists on basal pain sensitivity [5,32,54,63], it seems unlikely that CCK directly creates prolonged depolarization on its own. Such modulatory actions would have gone undetected in previous studies of CCK anti-analgesia. The involvement of spinal neuropeptides raises the issue of the neuronal source of these substances. Most studies have produced hyperalgesia by creating input to the spinal cord dorsal horn via activation of primary nocieeptive afferents in dorsal root ganglia. Thus, involvement of substance P and glutamate in such hyperalgesic states did not appear surprising, since both substance P and glutamate are present in nociceptive primary afferent pathways [24]. The involvement of substance P and glutamate in LPS hyperalgesia is therefore noteworthy, since i.p. LPS does n o t appear to produce hyperalgesia via direct spinal input. Therefore, substance P, CCK and glutamate involvement in this phenomenon probably do not arise from nociceptive neurons of dorsal root ganglia. Indeed, transection of the splanehnic nerve (the pathway for visceral information to reach the spinal cord [8]) has no effect on i.p. LPS hyperalgesia [57]. The data suggest that afferent information generated by i.p. LPS reaches the brain via the vagus nerve and produces hyperalgesia at the level of the spinal cord via activation of a centrifugal pathway arising in the nucleus raphe magnus and descending via the dorsolateral funiculi [57]. Given this anatomical information, substance P, glutamate and CCK could arise either from intrinsic dorsal horn neurons or via descending projections from supraspinal sites, but not likely from neurons in the dorsal root ganglia [11,24,35,40,49,51]. This suggests the question as to whether other hyperalgesias might involve EAAs and neuropeptides derived from sources o t h e r t h a n primary afferents. Perhaps the strongest evidence for this concerns the effects of s.c. formalin. This irritant produces hyperalgesia and does so even at distant sites such as the distal tail. This hyperalgesia is not only blocked by intrathecally administered antagonists of substance P and glutamate [61], but is also blocked by either nucleus raphe magnus lesions or spinal transection, indicating release, either directly or indirectly, of these substances following activation of centrifugal circuitry (Watkins, L.R., Wiertelak, E., Furness, L.E., Horan, R. and Maier, S.F., Formalin induced hyperalgesia is mediated via a centrifugal pathway, unpublished results). Thus, centrifugal circuitry might actually mediate diverse forms of hyperalgesia and these centrifugal pathways may enhance pain at the level of
L.R. Watkins et al. / Brain Research 664 (1994) 17-24
the spinal cord by inducing the release of EAAs and neuropeptides independent of primary afferent input.
Acknowledgements This work was supported by NIH Grant MH45045, NIH Grant NS31569, and the Undergraduate Research Opportunities Program. We wish to thank Pfizer for the gift of CP-96345 and Merck for the gift of L-365,260. References [1] Aanonsen, L.M., Lei, S. and Wilcox, G.L., Excitatory amino acid receptors and nociceptive neurotransmission in rat spinal cord, Pain, 41 (1990) 309-321. [2] Aanonsen, L.M. and Wilcox, G.L., Phencyclidine selectively blocks a spinal action of N-methyl-D-asparate in mice, Neurosci. Lett., 67 (1986) 191-197. [3] Aanonsen, L.M. and Wilcox, G.L., Nociceptive action of excitatory amino acids in the mouse: effects of spinally administered opioids, J. Pharmacol. Exp. Ther., 243 (1987) 9-10. [4] Akil, H. and Mayer, D.J., Antagonism of stimulation produced analgesia by p-CPA, a serotonin synthesis inhibitor, Brain Res., 44 (1982) 692-697. [5] Baber, N.S., Dourish, C.T. and Hill, D.R., The role of CCK, caerulein and CCK antagonists in nociception, Pain, 39 (1989) 307-328. [6] Basbaum, A.I. and Fields, H.L., Endogenous pain control systems: brainstem spinal pathways and endorphin circuitry, Ann. Rev. Neurosci., 7 (1984) 309-338. [7] Bliss, T.V., Douglas, R.M., Errington, M.L. and Lynch, M.A., Correlation between long-term potentiation and release of endogenous amino acids from dentate gyrus of anesthetized rats, J. PhysioL, 377 (1986) 391-408. [8] Cervero, F. and Tattersall, J.E.H., Somatic and visceral sensory integration in the thoracic spinal cord. Cervero and Morrison, Progress in Brain Research, Elsevier Science, Amsterdam, 1986, 189-202. [9] Coderre, T.J., Katz, J., Vaccarino, A.L. and Melzack, R., Contribution of central neuroplasticity to pathological pain: review of clinical and experimental evidence, Pain, 52 (1993) 259-285. [10] Coderre, T.J. and Melzack, R., Central neural mediators of secondary hyperalgesia following heat injury in rats: neuropeptides and excitatory amino acids, Neurosci. Lett., 131 (1991) 71-74. [11] Conrath-Verrier, M., Dietl, M. and Tramu, G., Cholecystokinin-like immunoreactivity in the dorsal horn of the spinal cord of the rat: a light and electron microscopic study, Neuroscience, 13 (1984) 871-885. [12] Cridland, R.A. and Henry, J.L., Facilitation of the tail-flick reflex by noxious cutaneous stimulation in the rat: antagonism by a substance P analogue, Brain Res., 462 (1988) 15-21. [13] Cridland, R.A. and Henry, J.L., lntrathecal administration of substance P in the rat: spinal transection or morphine blocks the behavioural respones but not the facilitation of the tail flick reflex, Neurosci. Lett., 84 (1988) 203-208. [14] D'Amour, F.E. and Smith, D.L., A method for determining loss of pain sensation, J. Pharrnacol. Exp. Ther., 72 (1941) 74-79. [15] Dougherty, P. and Willis, W.D., Enhancement of spinothalamic neuron reponses to chemical and mechanical stimuli following combined microiontophoreitc application of NMDA and substance P, Pain, 47 85-93.
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[16] Eide, P.K. and Hole, K., Interactions between serotonin and substance P in the spinal regulation of nociception, Brain Res., 550 (1991) 225-230. [17] Fields, H.L., Is there a facilitating component to central pain modulation, Am. Pain Soc. Z, 1 (1992) 71-79. [18] Gebhart, G.F. and Randich, A., Vagal modulation of nociception, Am. Pain Soc. J., 1 (1992) 26-33. [19] Go, V.L.W. and Yaksh, T.L., Release of substance P from the cat spinal cord, J. Physiol., 391 (1987) 141-167. [20] Guard, S., Boyle, S.J., Tang, K.W., Wafting, K.J., McKnight, A.T. and Woodruff, G.M., The interaction of the NK-1 receptor antagonist CP-96,345 with L-type calcium channels and its functional consequence, Br. J. Pharmacol., 110 (1993) 385-391. [21] Haley, J.E. and Wilcox, G.L., Involvement of excitatory amino acids and peptides in the spinal mechanisms underlying hyperalgesia. In Willis (Ed.), Hyperalgesia and Allodynia, Raven Press, New York, 1992, pp. 281-293. [22] Henry, J.L., Effects of substance P on functionally identified units in cat spnal cord, Brain Res., 114 (1976) 439-451. [23] Hill, R,G., CCK analgesia and hyperalgesia after intrathecal administration in the rat: comparison with CCK related peptides, Neuropeptides, 10 (1987) 87-108. [24] Hokfelt, T., Ljungdahl, A., Terenius, L., Elde, R. and Nilsson, G., Immunohistochemical analysis of peptide pathways possibly related to pain and analgesia: enkephalin and substance P, Proc. Natl. Acad. Sci. USA, 74 (1977) 3081-3085. [25] Hunskaar, S., Fasmer, O.B. and Hole, K., Acetylsalicylic acid, paracetamol and morphine inhibit behavioral responses to intrathecally admiistered substance P or capsaicin, Life Sci., 37 (1985) 1835-1841. [26] Hylden, J.L.K. and Wilcox, G.L., Intrathecal substance P elicits a caudally directed biting and scratching behavior in mice, Brain Res., 217 (1981) 212-215. [27] Kuraishi, Y., Kawabata, S., Matsumoto, T., Nakamura, A., Fujita, H. and Satoh, M., Involvement of substance P in hyperalgesia induced by intrathecal galanin, Neurosci. Res., 11 (1991) 276-285. [28] Lei, S. and Wilcox, G.L., Excitatory amino acid receptor regulation of spinal nociceptive neurotransmission, Eur. J. Pharmacol., 183 (1990) 1438. [29] Madison, D.V., Malenka, R.C. and Nicoll, R.A., Mechanisms underlying long-term potentiation of synaptic transmission, Annu. Rev. Neurosci., 14 (1991) 379-397. [30] Magnuson, D.S.K., Sullivan, A.F., Simonne, G., Roues, B.P. and Dickenson, A.H., Differential interactions of cholecystokinin and FLFQPQRF-NH2 with mu and delta opioid antinocicetion in the rat spinal cord, Neuropeptides, 16 (1990) 213-218. [31] Maier, S.F., Wiertelak, E.P., Martin, D. and Watkins, L.R., Interleukin-1 mediates the behavioral hyperalgesia produced by lithium chloride and endotoxin, Brain Res., 623 (1993) 321-325. [32] Maier, S.F., Wiertelak, E.P. and Watkins, L.R., Endogenous pain facilitory systems: antianalgesia and hyperalgesia, Am. Pain Soc. J., 1 (1992) 191-198. [33] Malmberg, A.B. and Yaksh, T.L., Hyperalgesia mediated by spinal glutamate or substance P receptor blocked by spinal cyclooxygenase inhibition, Science, 257 (1992) 1276-1279. [34] Malmberg, A.B. and Yaksh, T.L., Spinal nitric oxide synthesis inhibition blocks NMDA-induced thermal hyperalgesia and produces antinociception in the formalin test in rats, Pain, 54 (1993) 291-300. [35] Mantyh, P.W. and Hunt, S.P., Evidence for choleystokinin-like immunoreactive neurons in the rat medulla oblongata which project to the spinal cord, Brain Res., 291 (1984) 49-54. [36] Mao, J., Price, D.D., Mayer, D.J., Lu, J. and Hayes, R.L., Intrathecal MK-801 and local nerve anesthesia synergistically reduce nociceptive behaviours in rats with experimental peripheral mononeuropathy, Brain Res., 576 (1992) 254-262.
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[37] Meller, S.T., Dykstra, C. and Gebhart, G.F., Production of endogenous nitric oxide and activation of soluble guanylate cyclase arc required for N-methyI-D-aspartate produced facilitation of the nociceptive tail-flick reflex, Eur. J. Pharmacol., 214 (19921 93-96. [38] Meller, S.T. and Gebhart, G.F., Nitric oxide (NO) and nociceprive processing in the spinal cord, Pain, 52 (19931 127 136. [39] Menetry, D. and Basbaum, A.I., The distribution of substance P-, enkephalin- and dynorphine-immunoreactive neurons inthe medulla of the rat and their contribution to bulbospinal pathways, Neurosci., 23 (1987) 173-187. [40] Minson, J., Pilowsky, P., Llewellyn-Smith, I., Kaneko, T., Kapoor, V. and Chalmers, J., Glutamate in spinally projecting neurons of the rostral ventral medulla, Brain Res., 555 (1991) 326-331. [41] Mjellem-Joly, N., Lund, A., Berge, O.G. and Hole, K., lntrathecal co-administration of substance P and NMDA augments nociceptive responses in the formalin test, Pain. 51 (19921 195-198. [42] O'Neill, F., Dourish, C.T. and lversen, S.D., Morphine-induced analgesia in the rat paw pressure test is blocked by CCK and enhanced by the CCK antagonist MK-329, J. Neuropharmacol., 28 (1989) 243-247. [43] Ohkubo, T., Shibata, M., Takahashi, H. and lnoki, R., Roles of substance P and somatostatin on transmission of nociceptive information induced by formalin in spinal cord, J. Pharmacol. Exp. Ther., 252 (1990) 1261-1268. [44] Randic, M., Hecimovic, H. and Ryu, P.D., Substance P modulates glutamate-induced currents in acutely isolated rat spinal dorsal horn neurons, Neurosci. Lett., 117 (1990)74-80. [45] Ren, K., ttylden, J.L.K., Williams, G.M., Ruda, M.A. and Dubnet, R., The effects of a non-competitive NMDA receptor antagonist, MK-80I, on behavioral hyperalgesia and dorsal horn neuronal activity in rats with unilateral inflammation, Pain, 50 (1992) 331-344. [46] Ren, K., Randich, A. and Gebhart, G.F., Spinal serotonergic and kappa opioid receptors mediate facilitation of the nociceptive tail flick reflex by vagal afferent stimulation, Pain, 45 (1991) 321-329. [47] Sakurada, T., katsumata, K., Yogo, H., Tan-No, K., Skaurada, S. and Kisara, K., Antinociception induced by CP 96,345, a nonpeptide NK-I receptor antagonist, in the mouse formalin and capsaicin tests, Neurosci. Lett., 151 (19931 142-145. [48] Satoh, M., Kuraishi, Y. and Kawamura, M., Effects of intrathecal antibodies of substance P, calcitonin gene-related peptide and galanin on repeated cold stress-induced hyperalgesia: comparison with carrageenan-induced hyperalgesia, Pain, 49 (1992) 273-278. [49] Schiffmann, S.N., Teugels, E., Halleux, P., Menu, R. and Vanderhaeghen, J.-J., Cholecystokinin mRNA detection in rat spinal cord motoneurons but not in dorsal root ganglia neurons, Neurosci. Lett., 123 (1991) 123-126. [50] Skilling, S.R., Smulling, D.H., Beitz, A. and Larsson, A.A., Extracellular amino acide concentrations in the dorsal sinai cord of freely moving rats following veratridine and nociceptive stimulation, J. Neurochem., 51 (1988) 127-132. [51] Skirboll, L., Hokfelt, T., Dockray, G., Rehfield, J., Brownstein, M. and Cuello, A.C., Evidence for periaqueductal cbolecystokinin-substance P neurons projecting to the spinal cord, J. Neurosci., 3 (19831 1151- 1157. [52] Suberg, S.N. and Watkins, L.R., Interaction of cholecystokinin and opioids in pain modulation. In Akil and Lewis (Eds.), Pain and Headache, Karger, Basel, 1987, 247-265. [53] Wang, X.-J., Wang, X.-H. and Han, J.-S., Cholecystokinin octapeptide antagonized opioid analgesia mediated by mu and kappa but not delta receptors in the spinal cord of rat, Brain Res., 523 (1990) 5-1(I.
[54] Watkins, L.R., Kinscheck, I.B. and Mayer, l).,h, Potentialion ol opiate analgesia and apparent reversal of morphine tolernacc by proglumide, Science. 224 (1984) 395-396. [55] Watkins, L.R. and Mayer. 1)J., lnvolvemenl oi spinal opioid systems in footshock induced analgesia: antagonism by naloxone is possible only before induction of analgesia. Brain Res., 242 ( 19821 3119-316. [56] Reference deleted. [57] Watkins, L.R., Wiertelak, E.P., Goehler, L.E., Mooney-Heiberger, K., Martinez, J., Furness, L., Smith, K.P. and Maier, S.F., Neurocircuitry of illness-induced hyperalgesia, Brab~ Res., 634 (1994) 214 226. [58] Watkins. L.R.. Wiertelak, E.P., Goehler, LK., Smith, K.P., Martin, D. and Maier, S.F., Characterization of eytokine induced hyperalgesia, Brain Res., 654 (1994) 15-26. [59] Watkins. L.R., Wiertelak, E.P., Grisel. J.E., Silbert, L.tt. and Maier, S.F., Parallel activation of multiple spinal opiate systems appears to mediate 'non-opiate stress-induced analgesias', Brain Res., 594 (19921 99-11118. [60] Watkins, LR., Wiertelak, E.P. and Maier, S.F., Kappa opiate receptors mediate analgesia at spinal levels, Brain Res., 582 (19921 1-9. [61] Wiertelak, E.P., Furness, L.E,, Horan, R., Martinez, J., Maier, S.F. and Watkins, L.R., Subcutaneous formalin produces centrifugal hyperalgesia at a non-injected site via the NMDA-nitric oxide cascade, Brain Res., 649 (1994) 19-26. [62] Wiertelak, E.P., Furness, L.E., Watkins, L.R. and Maier, S.F., Illness-induced hyperalgesia is mediated by a spinal NMDAnitric oxide cascade, Brain Res., 664 (1994) 9-16. [63] Wiertelak, E.P., Maier, S.F. and Watkins, L.R., Cholecystokinin anti-analgesia: light cues abolish morphine analgesia, Science, 256 (1992) 830-833. [64] Wiertelak, E.P., Smith, K.P., Furness, L, Mooney-Heiberger, K., Mayr, T., Maier, S.F. and Watkins, E.R., Acute and conditioned hyperalgesic responses to illness, Pain, 56 (19941 227-234. [65] Wilcox, G.L., Spinal mediators of nociceptive neurotransmission and hyperalgesia: relationships among synaptic plasticity, analgesic tolerance and blood flow, Am. Pain Soc. J., 2 (19931 265-275. [66] Woolf, C.J. and Thompson, S.W.N., The induction and maintenance of central sensitization is dependent on N-methyl-oaspartic acid receptor activation: implications for the treatment of post-injury pain hypersensitivity states, Pain, 44 (1991) 293299. [67] Yaksh, T.L. and Rudy, T.A,, Chronic catheterization of the spinal subarachnoid space, Physiol. Behat~., 17 ( t 976) 1031 - 1036. [68] Yamamoto, T. and Yaksh, T.E., Spinal pharmacology of thermal hyperesthesia induced by incomplete ligation of sciatic nerve: excitatory amino acids, Pain, 49 (1992) 121-128. [69] Yashpal, K., Radhakrishnan, V., Coderre, T.J. and Henry, J.L., CP-96,345, but not its stereoisomer, CP-96,344, blocks the nociceptive responses to intrathecally administered substance P and to noxious thermal and chemical stimuli in the rat, Neuroscience, 52 (1993) 1039-1047. [70] Yashpal, K., Wright, D.M. and Henry, J.L., Substance P reduces tail flick latency: implication for chronic pain syndromes, Pain, 15 (1982) 155-167. [71] Zhou, M. and Gebhart, G.B., Spinal serotonin receptors mediate descending facilitation of a nociceptive reflex from the nuclei reticularis gigantocellularis and gigantocellularis pars alph in the rat, Brain Res., 550 (1991) 35-48. [72] Zieglg~insberger, W. and Herz, A., Changes of cutaneous receptive fields of spinocervical tract neurones and other dorsal horn neurons by microelectrophoretically administered amino acids, Exp. Brain Res., 13 (19711 111-126.
Brain Research 664 (1994) 17-24
Research report
Illness-induced hyperalgesia is mediated by spinal neuropeptides and excitatory amino acids Linda R. Watkins a,, Eric P. Wiertelak b Linda E. F u r n e s s
a
S t e v e n F. Maier a
a Department of Psychology, Unit~ersity of Colorado at Boulder, Campus Box 345, Boulder, CO 80309, USA b Department of Psychology, Macalester College, St. Paul, MN, USA Accepted 9 August 1994
Abstract
The spinal cord dorsal horn contains neural mechanisms which can greatly facilitate pain. We have recently shown that 'illness'-inducing agents, such as intraperitoneally administered lipopolysaccharide (LPS; bacterial endotoxin), can produce prolonged hyperalgesia. This hyperalgesic state is mediated at the level of the spinal cord via activation of the NMDA-nitric oxide cascade. However, prolonged neuronal depolarization is required before such a cascade can occur. The present series of experiments were aimed at identifying spinal neurotransmitters which might be responsible for creating such a depolarized state. These studies show that LPS hyperalgesia is mediated at the level of the spinal cord by substance P, cholecystokinin and excitatory amino acids acting at non-NMDA sites. No apparent role for serotonin or kappa opiate receptors was found.
Keywords: Spinal cord; Substance P; Cholecystokinin; Serotonin; Opiate; Excitatory amino acid; Hyperalgesia; CP-96345; L-365260; Methysergide; Norbinaltorphimine; Naltrexone
I. Introduction
The spinal dorsal horn contains mechanisms which can increase nociceptive transmission when activated (for reviews, see [9,34,38,65]). These mechanisms enhance dorsal horn excitability and are thought to at least partially mediate the facilitation of nociception (hyperalgesia) that is produced by conditions as diverse as sciatic nerve ligature [36,68], heat injury [10] and subcutaneous injection of irritants such as mustard oil [66], formalin [34] and carageenen [45]. Facilitation of dorsal horn excitability and resultant hyperalgesia appears to be produced by the same NMDA-NO cascade that is involved in producing sustained enhancement of neural excitability in other regions of the nervous system [7,29]. The spinal NMDANO cascade is initiated by substance P (SP) acting at NK-1 receptors and excitatory amino acids (EAAs) acting at non-NMDA ionotropic and metabotropic
* Corresponding author. 0006-8993/94/$07.00 © 1994 Elsevier Science B.V, All rights reserved SSDI 0 0 0 6 - 8 9 9 3 ( 9 4 ) 0 0 9 8 0 - 5
EAA receptors on N M D A receptor-containing neurons. Thus spinal administration of EAAs and substance P can produce behavioral hyperalgesia and increased dorsal horn neural excitability and these effects of EAAs and substance P are blocked by N M D A antagonists [2,33,37]. In addition, the dorsal horn windup produced by C-fiber stimulation and the hyperalgesia induced by prolonged nociceptive stimulation are eliminated by spinal NK-1 antagonists and non-NMDA EAA antagonists [10,33,68]. Many of the conditions which lead to hyperalgesia are known to produce sustained primary afferent Cfiber a n d / o r A~ nociceptive spinal input. Terminals of these primary afferents contain both substance P and glutamate [19,50] and at least some of the hyperalgesia-inducing manipulations noted above have been shown to produce the release of substance P and glutamate in the dorsal horn [19,50]. This set of facts has led to the view that the the spinal NMDA-NO cascade is set into motion by the prolonged release of substance P and glutamate from primary afferents. However, we have recently demonstrated a form of hyperalgesia that is difficult to explain in this fashion. The intraperitoneal (i.p.) injection of 'illness'-inducing
Is
I..R. Watkins et al. / Brain Researctl 604 (1994) ] 7-24
agents lithium chloride (LiCI) and endotoxin (lipopolysaccharide, LPS) p r o d u c e a p r o l o n g e d hyperalgesia as m e a s u r e d by both tail-flick to r a d i a n t heat and subcut a n e o u s formalin tests [31,57,58,62,64]. T h e hyperalgesia is m e d i a t e d by spinal N M D A - N O m e c h a n i s m s in that it is completely blocked by spinal application of the competitive N M D A receptor a n t a g o n i s t APV, the n o n - c o m p e t i t i v e N M D A antagonist MK-801 and the N O synthesis inhibitor L - N A M E [62 (previous article in this issue)]. Importantly, the hyperalgesia p r o d u c e d by L i C I and LPS does not a p p e a r to be m e d i a t e d by primary afferent p a i n i n p u t to the spinal cord from the viscera. Nociceptive afferents from the viscera to the spinal cord are carried in the splanchnic nerve [8] and t r a n s e c t i o n of the splanchnic nerve has no effect on the hyperalgesia p r o d u c e d by illness-inducing agents [57]. Instead, the e n h a n c e m e n t of nociceptive behavior produced by LiCI a n d LPS is likely to be m e d i a t e d by d e s c e n d i n g circuitry, since it is blocked by both hepatic vagotomy a n d dorsolateral funiculus lesions [57] a n d is not blocked by t r a n s e c t i o n of the splanchnic nerve [57]. T h e r e are a n u m b e r of possibilities. Activation of the spinal N M D A - N O cascade could be p r o d u c e d by m e a n s o t h e r t h a n substance P and g l u t a m a t e release. Alternatively, LiCI and LPS might lead to spinal substance P and g l u t a m a t e release from a source other than primary afferent terminals. Circuitry involved in pain m o d u l a t i o n descends to the dorsal horn from the rostro-ventral m e d u l l a [6], with the n u c l e u s r a p h e magnus ( N R M ) being a primary site [6]. I n d e e d , descending systems play a role in pain facilitation [17,18,31], as well as their b e t t e r k n o w n involvement in pain inhibition. Interestingly, d e s c e n d i n g axons from the N R M c o n t a i n both substance P [39] a n d g l u t a m a t e [40] and N R M lesions block the hyperalgesia p r o d u c e d by illn e s s - i n d u c i n g agents [57]. T h u s LiC1 and LPS might activate spinal N M D A - N O m e c h a n i s m s by stimulating a d e s c e n d i n g release of substance P and glutamate. T h u s one p u r p o s e of the p r e s e n t e x p e r i m e n t s was to d e t e r m i n e w h e t h e r spinal substance P a n d E A A s are involved in the m e d i a t i o n of illness-induced hyperalgesia. In addition, there are o t h e r t r a n s m i t t e r s k n o w n to be involved in d e s c e n d i n g pain facilitation. Serotonin, cholecystokinin (CCK) a n d d y n o r p h i n are most promin e n t . F o r example, electrical s t i m u l a t i o n of sites in the R V M can facilitate nociceptive r e s p o n d i n g [71] a n d this effect can be blocked by spinal a d m i n i s t r a t i o n of the the non-selective 5 - H T receptor a n t a g o n i s t methysergide [71]. Similarly, pain facilitation can be blocked by spinal application of CCKB antagonists [63] a n d k a p p a opiate antagonists [46]. T h u s a n o t h e r p u r p o s e of the p r e s e n t e x p e r i m e n t s was to d e t e r m i n e w h e t h e r L P S - i n d u c e d hyperalgesia could be blocked by spinal a d m i n i s t r a t i o n of either serotonin, C C K or kappa opiate antagonists.
2. Materials and methods 2.1. Subjects
Adult Sprague-Dawley rats (Holtzman Laboratories) were used in all experiments and were 450-600 g at the time of testing. They were singly housed and maintained on a 12 h/12 h light/dark cycle. Experiments occurred during the light part of the cycle. All procedures were in accord with protocols approved by the University of Colorado Institutional Animal Care and Use Committee, 2.2. Tai(flick testing and experimental procedure
Tailflick (TF) latencies to radiant heat were assessed using a modification [4] of the procedure developed by D'Amour and Smith [14]. All TF trials were conducted in a dimly lit experimental room maintained at 27°C. The room was maintained at this temperature because this minimizes tail temperature effects on TF latencies. We have monitored tail temperature and have not observed drug-induced changes at this room temperature. During testing, rats were loosely restrained in Plexiglascylinders designed such that their tails protruded from the rear allowing testing without disturbance of the subject. The rats were placed in the tubes 30 min before testing. This 30 min adaptation was followed by measurement of baseline (BL) TF latencies. The tail was placed over a radiant heat source adjusted to produce BL latencies in untreated animals in the 6-s range. This goal latency was chosen to allow observation of both analgesia and hyperalgesia [31] with the last three averaged to form the BL for that subject. Trials were automatically terminated at 18 s if no tail flick occurred, in order to prevent tissue damage. Following assessment of baseline TF latencies drug or vehicle was administered intrathecally (IT) and a second post-drug BL determined as above beginning 10 min after injection. LPS (i.p.) was then administered. TF latencies were then measured at 5-min intervals beginning 5 min after LPS injection for 55 min. TF measurements were made by blind experimenters unaware of group membership. Animals were carefully observed for possible impairments of motor function. None were observed except as noted below. 2.3. lntrathecal surgery and injection
Surgery was performed under sodium pentobarbital anesthesia (Nembutal, Abbott Laboratories, 55 mg/kg, i.p.), supplemented with methoxyflurane (Metofane, Pitman-Moore) as required. Intrathecal (IT) catheters were implanted into the subdural space surrounding the spinal cord at the lumbosacral enlargement. Construction of catheters and implantation procedures have been described in detail elsewhere [55,67]. Subjects were given a 1-2-week recovery period after surgery. Penicillin-streptomycin was given post-operatively to prevent infection. During testing, drug or vehicle was injected into the IT catheter, followed by a 10 /zl physiological saline flush delivered slowly over 30 s (for further details see [55]). 2.4. Drugs
In order to test the potential involvement of EAAs, substance P, CCK and opioids in LPS hyperalgesia, the effects of CNQX (AMPA receptor antagonist), CP-96345 (substance P antagonist), L-365,260 (antagonist of the CCK-B receptor), methysergide (serotonin antagonist), nor-binaltorphimine (kappa opiate receptor antagonist), and naltrexone (broad spectrum opiate antagonist) were assessed in separate experiments. It should be noted that CP-96345 may interact with calcium channels and therefore have non-selective effects [20]. CP-96345 (generously provided by Pfizer; Lot #19797-57-1) was dissolved in physiological saline at a concentration of 2 mg/ml,
L.R. Watkinset al. / Brain Research 664 (1994) 17-24 aliquoted and frozen at -70°C until used. On the day of testing, an aliquot was slowlythawed and kept cold until used. Each aliquot was only used once. Ten/zl was injected IT, thereby delivering20/xg, 15 rain prior to i.p. LPS. This dose was based on the work of Malmberg and Yaksh [33]. A dose of 200 /~g was also tested, but testing was discontinued upon observation of motor deficits. Vehicle controls received equivolume saline. L-365,260 (generously provided by Merck; Lot #0005007) was dissolved at a concentration of 0.12 and 0.012 mg/ml in dimethysulfoxide (DMSO; buffered to pH = 7.0 using 10 mM PBS; Sigma), aliquoted and frozen at - 70°C until used. Each aliquot was handled as described above for CP-96345. At the time of testing, 1 p,l was injected, thereby delivering either 0.12 or 0.012 p.g, 15 min before LPS. These doses were in accordance with our earlier work [63]. Vehicle controls received equivolume DMSO (pH = 7.0). CNQX (Research Biochemicals, Lot #LR-II-12) was made fresh on the day of testing by dissolving it in a concentration of 0.68 mg/ml in 45% CDEX (Research Biochemicals, Lot #WC1290A) in physiological saline. At the time of testing, 10 p,l was injected, thereby delivering 6.8 p,g, 15 min prior to LPS. This dose was in accordance with the work of Malmberg and Yaksh [33]. Vehicle controls received equivolume 45% CDEX-saline. Methysergide (Research Biochemicals, Lot #NA591A) was dissolved at a concentration of 3.01 mg/ml in 30% DMSO-70% PBS (pH 7.0). Aliquots were stored at -70°C until used. At the time of testing, 10 /~1 was injected, thereby delivering 30.1 /zg, 15 min prior to LPS This dose was in accordance with the work of Zhou and Gebhart [71]. Vehicle controls received equivolume 30% DMSO-70% PBS. Nor-binaltorphimine (Research Biochemicals, LOt #BFW-I-31) was dissolved at a concentration of 19.4 mg/ml in DMSO (pH 7.0, using PBS). Aliquots w e r e stored at - 7 0 ° C until used. At the time of testing, 1 p,l was injected, thereby delivering 19.4/.Lg, 15 min prior to LPS. This dose is comparable to the dose used in our previous studies [60]. Vehicle controls received equivolume buffered DMSO. Naltrexone (Research Biochemicals, Lot #M1089A) was made fresh at a concentration of 7 mg/ml in physiological saline. At the time of testing 1 p.1 was injected, thereby delivering 7/zg, 15 rain prior to LPS. This dose is in accordance with our prior work [60]. Vehicle controls received equivolume saline. To induce hyperalgesia, LPS was injected at a dose of 0.2 mg/kg in a volume of 20 ml/kg i.p. This regimen was based on prior work from this lab [31,57,58,62,64].
2.5. Data analysis BL and post-LPS latencies were separately analyzed by ANOVA to determine whether there were group differences. Subsequent comparisons were made by Scheffe test with alpha level set at 0.05. Paired t-tests were used to determine whether tailflick latencies were reliably elevated above baseline.
3. R e s u l t s
3.1. Effects of IT CNQX on LPS-induced hyperalgesia Our previous work demonstrated that blockade of the N M D A receptor abolished LPS-induced hyperalgesia [62 (previous article in this issue)]. The first experiment in the present study was designed to determine whether this hyperalgesia could also be blocked by spinal application of CNQX, a drug which blocks nonN M D A EAA receptors. One group received CNQX followed by LPS (CNQX + LPS; n = 8), one received
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Fig. 1. Blockade of LPS hyperalgesia by intrathecal CNQX, a nonNMDA EAA receptor antagonist. Prolonged LPS-induced hyperalgesia was observed in vehicle controls (open triangles). CNQX blocked this LPS-induced hyperalgesic state (filled squares). CNQX, in the absence of LPS, had no effect on pain responsivity (open squares).
the CNQX vehicle only followed by LPS (Vehicle + LPS; n = 8) and one received CNQX followed by the LPS vehicle only (CNQX + Saline; n = 8). A group administered only the two vehicles was not necessary, since we have previously found T F latencies to be stable across a 55 min testing period using the identical set of procedures [61] and TF to be unaffected by IT saline [61]. IT CNQX by itself had no effect on TF latency (Fig. 1). BL T F latencies did not differ between groups either before (B1) (F2,21 = 1.33, P > 0.05) or after (B2) (F2,21 = 3.052, P > 0.05) CNQX administration. As in previous experiments [31,57,58,64], the administration of LPS resulted in a reduction in T F latencies below BL. This effect was evident 10 min after LPS and lasted throughout the 55 min timecourse. Importantly, CNQX completely blocked the hyperalgesia produced by LPS. A N O V A revealed a reliable difference between groups (Fz,zl = 12.037, P < 0.0005). Post hoc Scheffe tests indicated that the Vehicle + LPS group differed from the CNQX + LPS and CNQX + Saline groups which did not differ from each other.
3.2. Effects of IT CP-96345 on LPS-induced hyperalgesia The design of this study was identical to that of the previous experiment. One group received the substance P antagonist CP-96345 followed by LPS (CP96345 + LPS; n -- 9), one received the CP-96345 vehicle only followed by LPS (Vehicle + LPS; n = 9) and one received CP-96345 followed by the LPS vehicle only (CP-96345 + Saline; n = 8). CP-96345 had no effect on T F latencies (Fig. 2). There were no BL differences between groups either before ( F < 1.0) or after ( F < 1.0) CP-96345. Moreover, the CP-96345 + Saline group remained stable for the 55 min of testing, with latencies at BL levels. LPS again led to a reduction in T F latencies, which here was apparent at the 10 min point and persisted for most of the 55 min of testing. CP-96345 blocked this hyperalgesia. A N O V A yielded a
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Fig. 2. Blockade of LPS hyperalgesia by intrathecal CP-96345, a substance P receptor antagonist. Prolonged LPS-induced hyperalgesia was observed in vehicle controls (open triangles). CP-96345 blocked this LPS-induced hyperalgesic state (filled squares). CP96345. in the absence of LPS, had no effect on pain responsivity (open squares). reliable difference between g r o u p s (F2,23 = 6.401, P < 0.01). Post hoc Scheffe tests indicated that Vehicle + LPS group differed reliably from the other two. The CP-96345 + LPS and CP-96345 + Saline groups did not differ reliably. 3.3. Effects of I T L-365,260 on LPS-induced hyperalgesia The design of this experiment also followed the pattern above. One group received 0.012 /zg of the CCK B receptor antagonist L-365,260 followed by LPS (0.012/xg L-365,260 + LPS; n = 8), one group received 0.12 /xg of the CCK B receptor antagonist L-365,260 followed by LPS (0.12 /zg L-365,260 + LPS; n = 10), one group received 0.12 /xg of the CCK B receptor antagonist L-365,260 followed by LPS vehicle (L365,260 + Saline; n = 7), and one received vehicle followed by LPS (Vehicle + LPS; n = 16: due to a time delay between testing the two L-365,260 drug groups, complete control groups were run with each and the data pooled for analysis) (Fig. 3). Baseline TF latencies did not differ between groups either before (B1, F < 1.0) or after (B2, F < 1.0) intrathecal drug injection. LPS produced hyperalgesia in vehicle + LPS controls. This hyperalgesia was blocked by the 0.12 mg L-365,260. A N O V A yielded a reliable difference between groups (F3,37 = 12.274, P < 0.0001). Scheffe tests indicated that the Vehicle + LPS group differed from the 0.12 /zg L - 3 6 5 , 2 6 0 + L P S group and from the 0.12 /ag L365,260 + Saline group. These latter two groups did not differ from each other. Scheffe tests also indicated that the Vehicle + LPS group did not differ from the 0.012 /.tg L-365,260 + LPS group, indicating that L365,260 exerted a dose-dependent blockade of LPS hyperalgesia. This low dose group reliably differed from both the 0.12 /xg L-365,260 + LPS group and from the 0.12 /zg L-365,260 + Saline group. Of course, conclusions concerning dose dependency should be tempered, since only two doses were employed.
The present experiment tested whether the serotonin antagonist methysergide could block LPS hyperalgesia. Pilot work failed to find any effect of methysergide on LPS hyperalgesia at any of a range of doses. The present experiment therefore presents a complete group using the most effective dose found to block hyperalgesia in prior studies investigating a different hyperalgesia paradigm [71]. Because pilot data strongly suggested that methysergide would not reduce hyperalgesia a control to assess possible analgesic effects of methysergide by itself was not included. The purpose of such a control would be to counter the possibility that reversal of hyperalgesia was caused by an analgesic effect of the agent itself summating with the hyperalgesic effect of LPS, rather than a blockade of the hyperalgesia produced by LPS. Since a reversal of hyperalgesia was not expected it was felt that animal usage for this control was not justified. Thus, the present experiment contained only two groups. One received methysergide followed by LPS (methysergide; n = 8) and one received equivolume vehicle followed by LPS (vehicle; n = 8). As expected, the baseline latencies did not differ between groups either before ( B 1 ; FI,I4 = 1.094, P > 0.05) or after (B2; FIA 4 = 2.054, P > 0.05) intrathecai drug delivery (Fig. 4). LPS again resulted in a persistent reduction in TF latencies and this reduction was not altered by methysergide ( F <
1.0). 3.5. Effects of I T nor-binaltorphimine on LPS-induced hyperalgesia Pilot work here also did not find an effect of nor-binaltorphimine at any of a variety of doses. What is presented here is one log greater than the intrathecal
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L.R. Watkins et aL /Brain Research 664 (1994) 17-24
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Fig. 4. Lack of effect of methysergide on LPS hyperalgesia. LPS produced prolonged hyperalgesiain vehicle controls (open triangles). Methysergide (filled squares) did not block this hyperalgesicstate.
dose previously found to block tail-shock induced analgesia in our laboratory [60]. A group that received only nor-binaltorphimine was not included for the same reasons as detailed above. Thus two groups were tested: one group that received intrathecal vehicle followed by LPS (n = 8) and one group that received intrathecal nor-binaltorphimine followed by LPS (n = 8). As expected, the baseline latencies did not differ between groups either before (B1; F1,14 = 0.019, P > 0.05) or after (B2; F1,14 = 1.303, P > 0.05) intrathecal drug delivery (Fig. 5). LPS again resulted in a persistent reduction in T F latencies and this reduction was not altered by methysergide ( F < 1.0).
3.6. Effects of IT naltrexone on LPS-induced hyperalgesia Pilot work here also did not find an effect of naltrexone at any of a variety of doses. Thus what is presented here is the intrathecal dose previously found to be effective in blocking/x, ~ and K opiate receptor mediated tail-shock induced analgesias in our laboratory [59,60]. A group that received only naltrexone was not included for the same reasons as detailed above. Thus two groups were tested: one group that received intrathecal vehicle followed by LPS (n = 8) and one group that received intrathecal naltrexone followed by LPS (n---8). As expected, the baseline latencies did
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not differ between groups either before (B1; F < 1.0) or after (B2; F < 1.0) intrathecal drug delivery (Fig. 6). LPS again resulted in a persistent reduction in TF latencies and this reduction was not altered by naltrexone ( F < 1.0).
4. D i s c u s s i o n
The present series of experiments suggest that the hyperalgesia produced by LPS is: (a) mediated at the level of the spinal cord by substance P, CCK and EAAs acting at non-NMDA sites, since this hyperalgesia was blocked by CP-96345, L-365260 and CNOX, respectively; and (b) apparently not mediated at the level of the spinal cord by either opiate receptors or serotonin, since this hyperalgesia was not blocked by either naltrexone or nor-binaltorphimine or by methysergide, respectively. Although these latter experiments each tested a single dose of antagonist, the doses chosen were ones that effectively blocked spinal pain modulation in previous studies [59,60,71]. Involvement of EAAs in the NMDA-mediated hyperalgesia induced by i.p. LPS is supported by prior work. EAAs can act both at N M D A and non-NMDA sites. Our previous studies have demonstrated that N M D A receptors are involved in LPS hyperalgesia, since MK-801 (an N M D A channel blocker) and APV (an N M D A receptor antagonist) both abolish this hyperalgesic state [62 (previous article in this issue)]. The present experiments extend this finding by demonstrating that non-NMDA EAA receptors are also critical, since CNOX also abolished LPS hyperalgesia. EAAs acting through non-NMDA receptors is a major pathway by which neurons can become sufficiently depolarized to cause Mg 2÷ to be expelled from the N M D A channel [38]. Iontophoretically applied EAAs in the spinal cord dorsal horn increase the size of cutaneous receptive fields in nociceptive neurons [72] and activate nociceptive projection neurons [1,28]. Additionally, when subthreshold currents of EAA agonists are ion-
22
L.R. Watkins et al. /Brain Research 064 (1994) 17 24
tophoresed near neurons, an enhanced neuronal response is observed to peripheral nociceptive stimuli [21]. At a behavioral level, non-NMDA agonists elicit pain behaviors when injected intrathecally [3] and produce hyperalgesia on the T F test [3]. Similarly, nonNMDA EAA receptor antagonists block hyperalgesias produced by nerve ligatures, burns and s.c. formalin [10,61,68]. Given that 40% of nociceptive projection neurons respond to EAAs acting at N M D A receptors but n o t to EAAs acting at non-NMDA receptors [28], such neurons clearly require depolarization by substances other than EAAs before N M D A receptors can be fully activated. Substance P could fulfill such a role. The present experiments indicate that substance P may be critically involved in NMDA-mediated hyperalgesia, since this hyperalgesic state was abolished by the substance P antagonist, CP-96,345. Such a role for substance P is supported by previous studies which showed that substance P selectively facilitates nociceptive responses of spinal cord dorsal horn neurons [22] and produces prolonged (up to 2 h) increases in responses of nociceptive spinothalamic neurons to iontophoresed N M D A [15,44]. Intrathecal substance P elicits behavioral indices of pain [16,25,26] and produces hyperalgesia on the TF [13,69,70] and foot withdrawal tests [10]. Furthermore, concurrent intrathecal injection of substance P and N M D A enhances nociceptive responses in the formalin test, over and above the actions of either intrathecal drug alone [41]. Blockade of spinal substance P blocks hyperalgesias induced by intrathecal galanin, subcutaneous chemical irritants, burns and cold stress [12,27,43,47,48,61,69]. Substance P does not appear to be the only neuropeptide mediating LPS hyperalgesia. CCK was also found to be important, since LPS hyperalgesia was blocked by the selective CCK B receptor antagonist L-365,260. However, conflicting evidence exists regarding the role of CCK in pain modulation at the level of the spinal cord. At an electrophysioiogical level, CCK has been observed to either modestly enhance [30] or have no effect on [52] responses of spinal cord dorsal horn neurons to nociceptive stimuli. Likewise, behavioral studies indicate that CCK and CCK antagonists can either enhance [23], inhibit [5,23] or have no effect on [5,32,42,53,54,63] pain threshold. In contrast to such conflicting results on basal pain responsivity, it seems clear that CCK plays a key role in pain facilitation due to its apparent anti-opiate effects on analgesia [5,32,54,63]. However, these studies concluded that CCK opposes opiate analgesia rather than inducing a true hyperalgesic state [5,32,54,63]. Perhaps the deciding factor in whether anti-analgesia or hyperalgesia is observed at a behavioral level rests in whether neuronal excitation is sufficiently prolonged to enable N M D A activation to occur. Prolonged activation could
presumably be attained either through continued release of CCK or by concomitant release of substance P a n d / o r EAAs acting via non-NMDA sites, as seems to occur during LPS-induced hyperalgesia. Given the weak excitatory effect of CCK on dorsal horn neurons [30] and the general lack of effect of exogenous CCK or CCK antagonists on basal pain sensitivity [5,32,54,63], it seems unlikely that CCK directly creates prolonged depolarization on its own. Such modulatory actions would have gone undetected in previous studies of CCK anti-analgesia. The involvement of spinal neuropeptides raises the issue of the neuronal source of these substances. Most studies have produced hyperalgesia by creating input to the spinal cord dorsal horn via activation of primary nocieeptive afferents in dorsal root ganglia. Thus, involvement of substance P and glutamate in such hyperalgesic states did not appear surprising, since both substance P and glutamate are present in nociceptive primary afferent pathways [24]. The involvement of substance P and glutamate in LPS hyperalgesia is therefore noteworthy, since i.p. LPS does n o t appear to produce hyperalgesia via direct spinal input. Therefore, substance P, CCK and glutamate involvement in this phenomenon probably do not arise from nociceptive neurons of dorsal root ganglia. Indeed, transection of the splanehnic nerve (the pathway for visceral information to reach the spinal cord [8]) has no effect on i.p. LPS hyperalgesia [57]. The data suggest that afferent information generated by i.p. LPS reaches the brain via the vagus nerve and produces hyperalgesia at the level of the spinal cord via activation of a centrifugal pathway arising in the nucleus raphe magnus and descending via the dorsolateral funiculi [57]. Given this anatomical information, substance P, glutamate and CCK could arise either from intrinsic dorsal horn neurons or via descending projections from supraspinal sites, but not likely from neurons in the dorsal root ganglia [11,24,35,40,49,51]. This suggests the question as to whether other hyperalgesias might involve EAAs and neuropeptides derived from sources o t h e r t h a n primary afferents. Perhaps the strongest evidence for this concerns the effects of s.c. formalin. This irritant produces hyperalgesia and does so even at distant sites such as the distal tail. This hyperalgesia is not only blocked by intrathecally administered antagonists of substance P and glutamate [61], but is also blocked by either nucleus raphe magnus lesions or spinal transection, indicating release, either directly or indirectly, of these substances following activation of centrifugal circuitry (Watkins, L.R., Wiertelak, E., Furness, L.E., Horan, R. and Maier, S.F., Formalin induced hyperalgesia is mediated via a centrifugal pathway, unpublished results). Thus, centrifugal circuitry might actually mediate diverse forms of hyperalgesia and these centrifugal pathways may enhance pain at the level of
L.R. Watkins et al. / Brain Research 664 (1994) 17-24
the spinal cord by inducing the release of EAAs and neuropeptides independent of primary afferent input.
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