Participation of central descending nociceptive facilitatory systems in secondary hyperalgesia produced by mustard oil

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Kc}word!: Hyperalgesia: Mustard oil: Neulmtensin:Chol.xystokinin: Rostml wntromedid medulla: Descending pain tlcilitation

1. Introduction P

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sponses to subsequent noxious stimuli (hyperdgesia) [36]. The development of hyperalgesia is believed to be the result of both a sensitization of primary afferent nociceptors and a change in excitability of central nociceptive dorsal horn neurons, termed central sensitization. Thai hyperalgesia is often observed distant from the site of prolonged noxious stimulation (secondary Ihyperalgesia) supports a role for the central nervous system (CNS) in the production of this phenomenon [3,4]. While both peripheral and local spinal mech:inisrns have been the primary focus of most studies investigating central sensitization, an involvement of central descending pain facilitator systcms has received little attention. T existence of descending systems that enhance spinal nrsci-

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ceptive responses has been well documented. Electrical stimulation, glutamate injection, or neurotensin injection into the rostral ventromedial medulla (RVM) have all been shown to facilitate spinal behavioral and dorsal horn neuron responses to noxious stimulation via descending neuronal projections [28–30,39–41 ]. Additionally, physiologically distinct classes of neurons have been identified within the RVM which are believed to mediate facilitation of spinal nociception [7]. Finally, facilitation of spinal nociception observed in studies involving vagal nerve stimulation [25–27], illness-induced hyperalgesia [33], and formalin pain [34] all appear to involve pain facilitator systems which descend from supraspinal sites. The current series of experiments were designed to further elucidate the role of descending pain facilitator systems in central sensitization following sustained noxious peripheral input. Mustard oil (allyl isothiocyanate), a chemical irritant which produces a neurogenic inflammation and selectively excites C-fibers [11,37], was utilized in the current study to produce a secondary hyperalgesia. The

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tail-flick reflex was used as the nociceptive measure since the reflex remains intact in spinal transected anilma]s and therefore the contribution of supr:~spinal involvement to hyperalgesia may be assessed. Previous work has identified one specific descending system that facilitates spinal nociception involving neurotensin in the IRVMand spinal CCK [3I]. Thus, a potential in~olvemcnt (ofthis system in central sensitization and secondary hyperalgesia produced by mustard oil was investigated.

neural tissue, and a heat cautery to seal major blood vessels. Complete transections were confirmed visually and surgical wounds were closed using silk sutures. Shan-operated animals received the laminectomy but no neural damage. Animals were tested approximately 24 h following the procedure. Bladders of spinally transected animals were expressed as needed. Spinal transected animals that received intrathecal drug injections were implanted with intrathecal catheters and received subsequent spinal transection 5–7 days later.

2. Materials and methods

2.2.4. Electt-ol>ticlesions in the RVIVI Electrolytic lesions were made by passing cathodal current (5 mA, 60 s) through a stereotaxically placed krngsten microelectrode which was cut flat on the tip. Three electrolytic lesions were made medially and 1 mm bilaterally in the RVM. The coordinates of the lesions relative to the intermural line were – 2.0 mm (rostralcaudal); – 1.0, 0, + 1.0 mm (medial-lateral), and – 9.5 mm (dorsal-ventral) [23]. Surgical wounds were closed using silk sutures. In sham-operated animals, the electrode was lowered into the RVM but no current was passed. Animals were given 5–7 days to recover from the procedure.

2.1. Animu[,\ Adult male Sprague–Dawley rats (400-450 g.: Harlan, Indianapolis, IN) were used in al] experiments. The animals were housed in the AAALAC apprc,ved animal care facility in the Bowen Science Building. LJniversity of Iowa, where they were singly housed with free access to food and water. The procedures described below were approved by (he Institutional Animal Care and Use Committee at the University of Iowa. 2.2. Surgical preparation

2..~.Experitnenta[ nocicepti(e testing protocol All animals received at least one of tha following four surgical procedures under pentobarbital scldium anesthesia (45-50 mg/kg, i.p.). 2.2. J. Implantation of’ int)(~((’t-(l?tal,quide cunnulaj Animals were stereotaxically implanted (David Kopf. stereotaxic apparatus) with microinjection guide cannulas 3 mm dorsal to the RVM. The coordinates for placement of cannulas relative to the interaural line were —2.0 mrn (rostral-caudal). O mm (Imedial-laleral). and –6.5 mm (dorsal-ventral) [23]. The slainless steel in{.racerebralguide cannulas (26 gauge needle shaft) had a length of 17.5 mm and were kept in place with acrylic dental cement secured by two stainless steel skull screws. Each cannula was fitted with a 33 gauge stylet to prevent the cannula from becoming clogged. Animals were given 5–7 days to recover from the procedure prior to testing. 2.2.2. Implantation of intruthecol Lathetet-s Animals were implanted with intrathecal spinal catheters as previously described [38]. Spinal catheter-swere inserted into the spinal subarachnoid space by passing an 8.5 cm length of PE- 10 tubing through a slit in the atlanto-occipital membrane to the level of the rostral lumbar enlargcrnent. Animals were given 5–7 days to recover from the procedure prior to testing. 2.2.3. Spinal transection The spinal cord was exposed by Iaminectomy and transected at spinal segments T8-T9 using scissors to cut the

The left hind leg was shaved, and rats were placed in Plexiglas cylinders in which they were awake, loosely restrained, and allowed to acclimate for 45 min prior to testing. The noxious thermal tail-flick reflex was used to measure nociceptive responsiveness [5]. The ventral surface of the animal’s tail was exposed to focused radiant heat and the time required for the animal to remove its tail was expressed as the tail-flick latency (TFL). Four baseline values were obtained for each animal and the mean was designated the predrug latency. Baseline values were in the range of 3.()–4.0 s, and a 10 s cut-off time was used to prevent tail skin tissue damage. Following determination of predrug latency, 20 WI of mustard oil (100~o) was applied topically to the leg approximately 30 mm above the ankle. Tail-flick latencies were measured at 5-rein intervals both prior to and post-mustard oil application for 60 min following mustard oil. 2.4. Drug injection protocol Intracerebral microinjections were performed by lowering a 33 gauge injection needle through the guide cannula and delivering 1.0 ~1 of drug in 30 s into the RVM. The injection needle was connected to a Hamilton 10 pl syringe using polyethylene tubing (PE1O) and an air bubble was maintained in the tubing to monitor flow of the drug solution. It. injections were made using a 30 gauge needle connected to a Hamilton 50 LI syringe with PE20 tubing. Drug (5 PI) was delivered over 30 s followed by a 10 K1

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flush of saline. Intraccrebral or it. injection of antagonists occurred 10 min prior to mustard oil application. In the experiments involving it. injection of CCK receptor antagonists, each animal received only one dose of Antagonist, and complete dose-inhibition curves were generated in each experiment using multiple animals. Following e~ch experiment animals were injected with methylene blue in the RVM and spinal cord for subsequent histological analysis of sites of drug injection. 2.5. Histology The locations of intracerebral guide cannulas and electrolytic lesions were verified by removing the brain and placing it in 10’% formalin overnight followed by 30’% sucrose for an additional 24 h. The brains were frozen, and 40 ~m cryostat sections were cut, mounted on slides, and stained with cresyl violet for microscopic examination of injection and lesion sites. Photomicrogt-aphs of the electrolytic lesion sites within the RVM were prepared. Laminectomy revealed it. injections of methylene blue to be confined to the rostra] lumbar spinal cord. 2.6. Da~a analysis Sample sizes for each experimentiil group consisted of’ 5 7 animals and in most cases the data is rwprescnted as tail-flick iatencics [TFL (s)]. To cmnpare tk overall hyperalgesic response (o mustard oil in normal and spinal of the retransected animals, the area under the c sponse was used to represent the overall response. This was calculated as the change in post-drug tail--flicklatency from the pre-drttg tail-flick latency plotted against time using the trapezoidal rule (ATFL X 60 rein). The dose-inhibition curves involving it. CCK receptor antagonists were generated by recording the response [ATFL (70)] at the time of maximal facilitation of the tail-flick reflex by mustard oil (20 min following mustard oil application). Tail-flick Iatencies were converted to percent change in latency [ATFL (%)] using the formula:

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2.7. Dru&s Drugs used in the present experiments were mustard oil (allyl isothiocyanate; Fluka, Buchs, Switzerland); proglumide (Sigma Chemical Co., St. Louis, MO); SR48692 (provided as a gift from Sanofi Recherche, France); and devazepide, and L-365260 (provided as gifts from Merck Sharp & Dohme, UK). Proglumide and SR48692 were dissolved in 4 and 100% DMSO, respectively. L-365260 and devazepide were dissolved in polyethylene glycol and stock solutions were made from subsequent 1:1000 dilutions with distilled water. The lack of effect of the vehicle controls was determined in preliminary experiments.

3. Results 3.1. Effect.s of’mustat-d oil in normal and spinal transected animals Immediately following topical application of mustard oil (20 wl, 10070)to the lateral surface of the left hind leg the animals appeared agitated with frequent biting and vocalizations. This response lasted approximately 5–7 min after which the animals behaved normally for the duration of the cxpcrimcul. Mustal-d oil additionally produced a slgnif’icant facilitation of the tail-flick reflex that was appal”cnt 5 min following application and las~ed 60 min (big. I). This effect wtis maximal approximately 20 min following application (ATFL = – 30.7 + 3.5%) and the

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Apparent ID50+ S.E.M. values were defined aS the dose of antagonist required to inhibit 5070 of the hyperalgesic response and were calculated from the dose-inhibition curves using a non-linear regression curve fitting computer program (Graphpad Inplot). Statistical analysis for multiple comparisons was carried out using ANOVA with Fisher’s test for post hoc comparisons. Comparisons between two groups were performed using a t-test. A P value less than 0.05 was considered statistically significant in all tests.

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Time(rein) Fig. 1. Topical application of mustard oil (20 wI, 100%) to the left hind leg significantly facilitates the tail-flick reflex compared to pre-mustard oil corm-ok (ANOVA, P < 0.05). The facilitation of the tail-flick reflex prwducedby mustard oil was inhibited in spinal transected animals (areas under the cu]-vesfor MO and (spimdized) were significantly different: f-test, P < ().05). Data expressed as mean ~ S.E.M. of the TFL (s) for the hascline (B) and over time following mustard oil application.

magnitude of effect remained f:iirly constant over the 60 min time course. In the spinal transected animals, prcdrtrg tail-flick latencies were similar to normal animals (3. I ~z0.4 und 3.3 f 0.2, respectively, for spinal transected and normal animals). Following application of mustard oil. however, t’acilita[ion of the tail-flick reflex was found to be significantly less in both magnitude and duration in spinal transected animals (Fig. I). Using area under (he c a measure of overall hyperalgesic response, spinal transection reduced the hyperalgesic response by approximately SO%. The X rein) in normal and areas under the curves ( spinal transected animals were —52.3 ~ 8.6 and —I I.2 f 4.1, respectively. No difference in response to mustard oil was observed in sham spinal h-ansected animals as conpared to normal animals. of’RVM [esions ml !t[ustatrloil induced )L)pct”-

Three electro]ylic lesions were made in the RVM nledially and I mm bilaterally that affected a large volume o(’ lissue including nucleus raphe magnus, nucleus reticulw”is paragigantocellularis, and nuclctrs reticularis partigigantocellularis latertilis (Fig. 2). Animals with lesions appeareci to behave normally and predrug tail-flick lat.encies were

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u hyperalgesi~ was significantly inhibited in animals Fig. 3. N trecciiing lesions in t RVM compared to sham controls (ANOVA, P < ().()5). Dii[a expressed as mean+ S.E.M. of the TFL (s) for the hLselinc (~) and uver time fu]lowing mustard oil application.

similar to the sham lesion group (4.() f 0.4 and 3.6 ~ 0.2, respectively, for lesion and sham animals). Mustard oil induced facilitation of the tail-flick reflex, however, was

the medid and hilateml electrolytic Icsitms (5 mA. 60 s) in the RVM. The arrows indicate the specific lesion sites. Fig.2 Photomicrugraph illustrating

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Time (nlin) Urg.5. MIIS(A oil hypctmlg,csiiiwas kigaificandy reduceci hy pr{)glumidc I s trarIsuIcd atlil)ud~ compared to vehicle ct~nlr~)ls(ANOVA, 1’.-:0.05). Dal:j cxpwswxl as mean+ S.U.M. of the TFI. (s) fr~r the h:~sclinc(B) at]d {)ic! Iinlu (ijllowing mustard oil applicalinn.

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significantly inhibited in animals that had mccived lesions in the RVM as compared to sham lesion controls (Fig. 3). 3.3. Effects of”it. administtwtion of CCK t-eceplor antagonists on mustard oil h.yperalgesia in normal und spinal transected animals Administration of the non-selective CCK receptor antagonist proglumide (10 p.g) it. 10 m prior to mustard oil application resulted in u complete inhibition of the hyperalgesic effect produced by mustard oil in both normal (Fig. 4) and spinal transected animals (Fig. 5). Additionally, proglumide was found to produce z significant inhibition of’the tail-flick reflex (Fig. 4) in normal animals. In contrast, this inhibition of the reflex by proglumide was not observed in spinal transected animals (Fig. 5). Proglumide was without effect in the absence of mustard oil. The effects of it. administration of multiple doses of the selective CCK,\ (devazepidc) and CCK~ (L-365260) receptor antagonists on mustard oil-induced hyperalgesia were determined in normal animals. It. injection of L365260 (1–600 ng) 10 min prior to mustard oil application dose-dependently inhibited mustard oil hyperalgesia with an apparent ID~Ovalue of 364 i 33 ng (Fig. 6). Similar to proglumide, the greatest dose of L-365260 (600 ng) was found to produce an inhibition of the tail-flick. retlex (Fig. 6). Administration of devazepide (60(J-3000 ng) it. dosedependently inhibited mustard oil hypm-algesia at doses approximately 5-fold greater than that observed with L365260 (Fig. 6). The apparent ID~(lvalue was 1760 f 188

3.4. Efltict.s of t~zicroinje~ti(]ttoj’ neurotensin receptor antagonists in the RVM on mustard oil hyperalgesia Microinjection of the non-peptide neurotensin receptor antagonist SR48692 (3.5 p,g) into the RVM (Fig. 7) 10 40

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byselective Fig. 6. Dose-dependerr[inhibition of mustard oilhyperalgesia CCK.~(devazcpide) met CCK ~ (L-365260) receptrrr’ arrtagonists injected il. I() min prior 10 mustard oil application. Data expressed as mean+ S.E.M. of’ the A TFL (%) at the time of maximal facilitation of the tail-flick reflex (20 min following mustard oil application).

Fig. 7. Illustration U1 microi]qjcction sites of SR4M92 (. ) within the RVM ( – 2.0 mm intcmural).

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Time (rein) Fig. 8. SR48692 (3.5 +g) microinjccled into the RVM 10 Iminprim tu mustard oil application significantly reducccl[he facilitation ofthc t~il-flick reflex compared tn \chicle controls (ANOVA. [) < 0.05). SR48f192 produced a significant inhihitiun 01’ [he tail-flick refkx compared to pre-mustard oil controls (ANOVA. 1) ~:0.05). Dtila expmsscd as mean ~ S.E.M. of the TFL (s) for the haX]ilW and over [ime fullmving mustard oil application.

min prior to mustard oil application completely inhibited the facilitation of the tail-flick reflex produced by mustard oil (Fig, 8). Similar to it. CCK receptor antagonists, SR48692 in the RVM significantly inhibited the tail-tlick reflex in mustard oil treated anireals (Fig. 8), while having no effect in the absence of mustard oil.

4. Discussion The data from the present series of experiments demonstrate that topical application t)f mustard oil to the lateral

s t h l a 30 mm above the unkle, produces a facilitation of’ the tail-flick reflex (secondary hyperalgesia). Mustard oil, a chemical irritant which .sclcctively activates C-fibers, was used in the present experiments to provide a prolonged, peripheral nociceptive input [11,37]. Cutaneous application to the lateral surface of the hind leg was chosen based on previous studies from this laboratory that have demonstrated mustard oil application to this area produces secondary hyperalgesia measuring thermal foot withdrawal latencies and electromyogram responses from the knee flexor musculature [2,13]. In the current study, the facilitation of the tail-flick reflex produced by mustard oil was significantly, but not completely inhibited in magnitude and duration in spinal transected animals. This suggests that mustard oil induced facilitation of the tail-tlick reflex involves both mechanisms intrinsic to the spinal cord as well as a central descending pain t’acilitatory component involving supraspinal sites. In the current study, spinal transection was found to have no effect on baseline tail-flick latencies which apparently contradicts certain prior studies suggesting that spired transection does affect this spinal reflex [9]. There appears to be somewhat conflicting data on this subject, however, since other previous studies have similarly reported tailtlick latencies to be unaffected by spinal transection [33,34]. While it is unclear why conflicting results have been reported on this subject, that spinal transection did not affect baseline tail-flick latency in our studies precludes the possibility that differences in these groups may be attributed to changes in baseline tail-flick latency. That the majority of the hyperalgesic response (80%) was inhibited in spinal transected animals suggests a prominent role for central descending pain facilitator systems in mustard oil-induced secondary hyperalgesia. These results apparently reveal another mechanism of central sensitization following sustained noxious peripheral input in addition to the previously established notions of sensitization of primary afferent nociceptors and dorsal horn nociceptive neurons via peripheral and intrinsic spinal mechanisms. The area of the rostral ventromedial medulla (RVM) is one supraspinal site that has received much attention in terms of its ability to facilitate spinal nociceptive responses via descending neuronal projections [6,8]. Low intensity electrical stimulation or low doses of glutamate microinjected into the RVM have been shown to facilitate the spinal tail-flick reflex and enhance spinal dorsal horn neuron responses to noxious thermal stimulation via descending neuronal projections [39,41]. This facilitation of spinal nociception by RVM stimulation is mediated, at least in part, by spinal 5HT1 receptors [40]. Neurotensin microinjected into the RVM has also been shown to facilitate the tail-flick reflex and spinal dorsal horn neuron responses to noxious thermal stimulation [28–30]. Additionally, electrical stimulation of vagal afferent fibers has b shown to facilitate the spinal tail-flick reflex and dorsal horn neuron responses to noxious stimulation via

the RVM [25–27]. Neurons within the RVhl have been physiologically characterized based on their activity during various conditions of nociceptive responsiveness [7], and those neurons within the RVM believed to play a role in facilitation of nociception (on-cells) have been implicated in enhancement of nociception produced by spatially remote noxious stimuli [20,2 1]. Finally, the RlrM has been implicated in the facilitation of the tail-flick reflex observed in models hyperalgesia involving illness-inducing agents and formalin [33,34]. Given (hat the RVM appears to be an important supraspinal site in descending pain facilitation, the effects of electrolytic lesions ir this area on mustard oil hyperalgesia were examined. The RVM is composed of a number of nuclei, including the n. raphe magnus, n. reticularis gigantocellularis, and In. reticularis paragigantocellularis Iateralis. These nuclei have all been implicated in descending pain facilitation [30,39,41], and thus three large electrolytic lesions were made in the RVM so that a large volume of tissue would be affl~cted.While mustard oil-induced facilitation of the tail-flick reflex was completely blocked by these RVM lesions, animals that inadvertently received lesions outside of the RVM (which were not included in the data) continued to exhibit hyperalgesia, illustrating the specificity of the RVM in mediating this effect. These results suggest that the RVM plays a prominent role in secondary hyperalgesia and central sensitization of dorsal horn neurons following prolonged noxious peripheral input. A number of neurotransmitters have been localized in medullo-spinal projections that have also been implicated in pain modulation, including serotonin, substance excitatory amino acids, and cholecystokinin [18,22]. One specific descending pain facilitator system that we have focused on involves neurotensin in the RVhl and spinal CCK. Neurotensin is localized within the RVM, and neurotensinergic afferent projections from a number of supraspinal sites terminate in the RVM [1]. Administration of neurotensin into the RVM has recently been shown to facilitate spinal nociceptive responses via spinal CCK~ receptors [31]. Spinal CCK has been implicated in facilitation of nociception [12,24] and has been found to be localized in neurons intrinsic to the spinal cord, in primary afferent fibers, and descending medullo-spinal projections [18,19,32]. In the current study, spinal administration of the non-selective CCK receptor antagonist proglumide completely inhibited mustard oil hyperalgesia in both normal and spinal transected animals. The fact that spinal proglumide completely blocked mustard oil hyperalgesia in spinally-intact animals, an effect largely due to central descending systems, suggests an involvement of spinal CCK in descending pain facilitation, ~lossibly via medullo-spinal CCK projections. The lesser hyperalgesic effect observed in spinal transected animals was also blocked by proglumide, implicating spinal CCK in primary afferent fibers and/or intrinsic spinal neurons in this effect. That the selective CCK~ receptor antagonist L-365260

[15] was found to be 5 times more potent than the CCKA receptor antagonist devazepide [16] in inhibiting mustard oil hyperalgesia implicates spinal CCK~ receptors in mediating this effect. Similar to spinal proglumide, microinjection of the non-peptide neurotensin receptor antagonist SR48692 [10] into the RVM completely inhibited mustard oil hyperalgesia. These results are consistent with previous data demonstrating that neurotensin in the RVM facilitates spinal nociception, and support a physiological role for a previously described descending neurotensinCCK pain facilitator system in mediating central sensitization produced by sustained nociceptive input. In addition to blocking mustard oil induced hyperalgesia, administration of spinal proglumide and supraspinal SR48692 produced a significant inhibition of the tail-flick reflex. Since these receptor antagonists were without effect in the absence of mustard oil, it appears that mustard oil concurrently activates both descending pain facilitator and inhibitory systems. While the pain facilitator component is most prominent, a selective block of this descending system reveals an apparently active descending pain inhibitory system. These results are not surprising given the large amount of evidence demonstrating noxious stimuli to activate descending pain inhibitory systems - a phenomenon referred to a diffuse noxious inhibitory controls [14]. That stimuli such as environmental cues or endogenous/exogenous opioids may concurrently activate both descending inhibitory and facilitator systems from the RVM has received recent attention [6,17]. Additionally, in models of both illness-induced and formalin hyperalgesia involving descending pain facilitator systems, a selective block of pain facilitation resulted in antinociception measuring the tail-flick reflex [34,35]. These results are consistent with data in the current study and support concurrent activation of descending facilitator and inhibitory systems by noxious stimuli. It is also noteworthy that the non-selective blocks of mustard oil hyperalgesia observed in spinal transected and RVM lesion animals did not result in an inhibition of the tail-flick reflex. One may conclude that such non-selective manipulations inhibit both descending facilitation and inhibition, and further demonstrate a specific role for supraspinal neurotensin and spinal CCK in descending facilitation of nociception. In summary, we have shown that cutaneous application of mustard oil to the hind leg produces a facilitation of the tail-tlick reflex that involves both intrinsic spinal cord mechanisms and supraspinal sites, including the RVM. Both neurotensin in the RVM and spinal CCK via CCK~ receptors appear to be important mediators of mustard oil hyperalgesia, offering evidence of a physiological role for a specific descending pain facilitator system which has been previously described. These results suggest that secondary hyperalgesia and central sensitization produced by prolonged nociceptive input are the result of not only peripheral and intrinsic spinal cord mechanisms, but also of descending pain facilitator systems within the CNS.

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M.(l. IJthanel al. \Br(ti71Rt,.\,,l/tc/t737 (1996)83–91

Acknowledgements

The authors wish to thank Michael Burcharn for preparation of the graphics. This work was supported by T32HL07121 and DA 02879.

References

[[] Bcitz, A.J.,The sites of origin of’ bmins(em rreumtensin anLIwrotrrnin projections to the rodent nucleus raphe magnus. J. IVctwrM[i., 2 (1982) 829-842. prevents [2] Cleland, C.L.. Lim, F-Y. md Gebhart, G.F., Pentobarbital in the rat, Pain, the development of C-fiber–in~uced hypcmlgesia 57 (1994) 3 I -43. [3] Coderre, T.J.. Katz, J., Vaccarino. A.L. and Mclzack.,R.. (:[,nlribw tion of central neurophrsticity 10 pathnlugicd pain:review of clinical and experimental evidcrrcx, P(:;w 52 (1993) 259–28.5. [4] Coderrc, T.J. and Melzack. R.. Increased pain sensiti~ity following heat injury involves a central mechanism, Beha[I. Bruin Re.\., 15 (1985) 259-262. [5] D’Amour, F.E. and Smith, D.L., A method fi)r determining loss of pain sensation, J. Pharma
[

Mantyh, P.W. and Hunt, S.P., Evidence for cholecystokinin-like immurroreactive neuronsin the rat medulla oblongata which project

tu the spinal cord, Brain Res., 291 (1984) 49–54. [[9] MarIcy. P.D., Nagy, J.I., Emsun, P.C. and Rehfeld, J.F., Cholecystokinin in the rat spinal cord: distribution and lack of effect of neonatal ca saicin treatment and rhizrrtomy, Brain Res., 238 (1982) 494-498. “ [X)] Morgan. M.M. and Fields, H.L., Pronounced changes in the activity of nuciceptive modulatory neurons in the rostral ventromedial medulla in response to prolonged thermal noxious stimuli, J. Neur-oplljsiol., 72 (1994) 1161–I 169. [21] Morgan. M.M., Heinricher, M.M. and Fields, H.L., Inhibition and Facilitation of’different nocifensor reflexes by spatially remote noxious stimuli, J. Nearophysio/., 72 ( 1994) I I52– 1160. [22] Nicholas, A.P.. Pieribone, V.A., Arvidsson, U. and Hokfelt, T., Semtonin, substance P and glcrtamate/aspartate-like immunoreactivities in mcdullrr-spinal pathways of rat and primate, Neuroscience, 48 (1992) 545-559. [23] Puxinos. G. md Watson, C. (Eds.), The Rat Brain in Stereotaxic C’omJimrre.\,2ncf eda., Academic Press, New York, 1986. [24] Pittaway, K.M., Rodriguez, R.E., Hughes, J. and Hill, R.G., CCK-8 analgesia and hyperalgesia after intrathecal administration in the rat: comparison with CCK-related peptides, Neur(~peptides, 10 (1987) 87– 108. [25] Ren, K., Randich, A. and Gebhart G.F., Vagal afferent modulation of spired nociceptive transmission in the rat, J. NeurophysioL, 62 (1989) 410-415. [26] Ken, K., Randich, A. and Gebhart, G.F., Electrical stimulation of cervial \,agal at’t’crents.I. Central relays for modulation of spinal mrciceptivc transmission, J. NewwphysioL, 64 (1990) 1098–1L14. [27] Ren, K., Randich, A. and Gebhart G.F., Spinal serotonergic and kappa opioid receptors mediate facilitation of the nociceptive tailtlick reflex by vagal afferent stimulation, Pain, 45 (1991) 321-329. [28] Urban, M.C). and Gebhart, G.F., Characterization of descending modulation uf spinal nociception by neurotensin in the rostral ventromedial medulla, J. Neurophysial. (1996) submitted. [29] LJrhan, M.0 and Smith, D.J., Role of neurotensin in the nucleus raphe magnus in opioid induced antinociception from the periaqueductal gray. J. Pharmcrcol.Ezp. Ther., 265 (1993) 580–586. [30] Urban, M.0. and Smith, D.J., Localization of the antinociceptive and antianalgesic eff’ectsof neurotensin within the rostral ventromedial medulla, Neurosci. Letf., 174 (1994) 21–25. [31] Urban, M.O., Smith, D,J. and Gebhart, G.F., Involvement of spinal choiecystokinin~ receptors in mediating neurotensin hypera~gesia from the mcdcdlary nucleus raphe magnus in the rat, J. Pharrrracol. E.xp Tker., 27X (1996). [32] Verge, V,M.K.. Wiesenfeld-Hallin, Z. and Hokfelt, T., Cholecystokinin in mammalian primary sensory neurons and spinal cord: in situ bybricii~ation studies in rat and monkey, Eur. J. Neurosci., 5 (1993) 240-250.” [33] Watkins. L.R,, Wicrtelak, E.P., Goehler, L.E., Mooney-Heiberger, K., Martinez, J., Furncss, L., Smith, K.P. and Maier, S.F., Neurocircuitry of illness-induced hyperalgesia, Brain Res., 639 (1994) 283– 299. [34] Wiertc]ak, E.P., Fumess, L.E., Horan, R., Martinez, J., Maier, S.F. and Watkins. L.R., Subcutaneous formalin produces centrifugal hypemlgesia at a non-injected site via the NMDA-nitric oxid cascade, Brain Res., 649 ( 1994) 19–26. [35] Wiertelak, E.P., Furness, L.E., Watkins, L.R. and Maier, S.F., Illness-induced hyperalgesia is mediated by a spinal NMDA-nitric oxide cascade, Brain Res., 664 ( 1994) 9– 16. [36] Willis, W.D. (Ed,), Hyperalgesia and Allodyrria, Raven Press, New York. 1992. [37] Woolf, C.J. and Wall, P.D., The relative effectiveness of C primary afferent fibers of’different origins in evoking a prolonged facilitation of tbc flexor reflex in the rat, J. Neuroxi., 6 (1986) 1433– 1443.

M.(2. 1/!-/1[1,! ?/ (d./’ Blrlill RCY(’C,).C}l 737 (1996) &-91

of the spinal [38] ‘r’aksh, T.L.and iludy,T.A,, Chroniccatheterization subwachnoid space. Ph?siol. i%hu(., 17 ( 1976) 1031–1036. [39] Zhuu, M. and Gcbharl, G.F.. Chatac[erization of descending inhibition and facilitation frurn the nuclei rcticularis gig:~lltc)cellul~tt.is mld gigantocellularis pars alpha in the rat. P[tin. 42 ( 1993) 337–350. [40] Zhuo, M. and Gebhart. G.F., Spinal serotouin receptnrs Imcdiale descending t’acilitatinnnt’v nuciceptivc rctlcx I’rnmthe nuclei reticu-

91

Iwis gigmtuccllulwis and gigantocellulzu-ispars alpha in the rat, flrai}~Rt,$., S50 ( 1991) 35–48. [41] Zhuo, M., and Gebhart, G.F., Characterization of descending facilitation and inhibition of spinal nociceptive transmission from the nuclei reticularis gigantnce]lularis and gigantocellnlaris pars alpha in [be rat, J. Ne~tt~~/,lt~.\i[~/., 67 (1992) 1599–1614.