Inhibition of spinal protein kinase C blocks substance P-mediated hyperalgesia

Substance P (SP) is an important neuromediator in the spinal processing of nociceptive afferent information. Our previous study has shown that spinal (intrathecal, IT) application of SP produces thermal hyperalgesia that is mediated by activation of the G-protein coupled NK1 receptor. The activation of some classes of the G-protein coupled receptors is known to produce diacylglycerol with consequent activation of protein kinase C (PKC). In the present study, we have demonstrated that intrathecal administration of a selective PKC inhibitor GF109203X (GF, 0.73 nmol) in rats chronically implanted with intrathecal catheters 15 min prior to IT-SP (48 nmol) completely blocked the SP-induced thermal hyperalgesia. The effect of GF was dose-dependent (0.073-0.73 nmol). Bisindolymaleimide V, the inactive homolog of GF, had no effect. Pretreatment with GF 3 h, but not 24 h, prior to SP still produced antinociception. Moreover, intrathecal treatment with GF (0.73 nmol) attenuated the formalin paw injection-induced flinching, preferentially at the 2nd phase, that is known to be associated with the release of endogenous SP at the spinal cord. These data suggest that activation of spinal PKC is involved in the SP-mediated hyperalgesia. Thus, SP, which is released in the spinal cord subsequent to persistent stimulation of small sensory afferents after tissue injury, may contribute to spinal hyperexcitability and persistent pain by enhancement of PKC-mediated phosphorylation of target molecules such as NMDA receptors.     

Involvement of tachykinin NK1 receptors in nociceptin-induced hyperalgesia in mice

Intrathecal (i.t.) injection of nociceptin at small doses (3.0 and 30.0 fmol) produced a significant hyperalgesic response as assayed by the tail-flick test. This hyperalgesic effect peaked at 15 min following i.t. administration of nociceptin (3.0 fmol) and returned to control level within 30 min. Hyperalgesia elicited by nociceptin was inhibited dose-dependently by i.t. co-administration of tachykinin NK₁ receptor antagonists, CP-99,994 and sendide. A significant antagonistic effect of [ -Phe⁷, -His⁹] substance P (6–11), a selective antagonist for substance P, was observed against the nociceptin-induced hyperalgesia. Pretreatment with i.t. substance P antiserum and i.t. capsaicin resulted in a complete block of the reduced threshold produced by nociceptin. The NK₂ receptor antagonist, MEN-10,376 and pretreatment with neurokinin A antiserum did not alter the behavioural effect of nociceptin. The N-methyl- -aspartate (NMDA) receptor antagonists, dizocilpine (MK-801) and (−)-2-amino-5-phosphonovaleric acid ( -APV), and -N^G-nitro arginine methyl ester ( -NAME), a nitric oxide synthase inhibitor, failed to inhibit nociceptin-induced hyperalgesia. The results obtained suggest that the hyperalgesic effect of nociceptin may be mediated through tachykinin NK₁ receptors in the spinal cord.     

Effects of aging on hyperalgesia and spinal dynorphin expression in rats with peripheral inflammation

The aging process is associated with various morphological and biochemical changes in the nervous system that may affect the processing of noxious inputs. This study showed greater hyperalgesia and up-regulation of spinal dynorphin (DYN) expression in aging than in young adult rats during CFA-induced peripheral inflammation. These data indicate that nociception is regulated differently in aging individuals, a fact that should be considered when selecting treatment strategies for aging populations with persistent pain.     

Recognition of neurokinin 1 receptor (NK1-R): an antibody to a peptide sequence from the third extracellular region binds to brain NK1-R

Substance P (SP) can produce cytokine-like responses by astrocytes and mononuclear cells. In an effort to identify neurokinin-1-receptors (NK1-R), an antibody to NK1-R was generated by using a linear peptide sequence from the deduced third extracellular region (ECR) corresponding to the seven transmembrane rat brain NK1-R. The ECR-3 peptide was coupled to keyhole-limpet hemocyanin and the antisera produced in rabbits was purified by binding to a peptide-affinity matrix. The specificity for the anti-peptide antibody was shown by its reactivity to the ECR-3 peptide by ELISA. The anti-ECR-3 peptide antibody could detect, by Western blot analysis of SDS-PAGE-separated rat brain membranes, a single band with an apparent molecular weight (MW) of 53–54 kDa. An affinity matrix made from the anti-ECR-3 antibody was used to isolate NK1-R from rat brain membranes which exhibited two products on SDS-PAGE with apparent MW of 54 and 44 kDa. The C6 astrocytes were shown to express NK1-R as determined by [¹²⁵I]Bolten-Hunter SP binding to intact cells with a K_d = 0.32 nM. These C6 cells did not co-express either NK2-R or NK3-R when analyzed at the mRNA level. The anti-ECR-3 peptide antibody could inhibit [¹²⁵I]Bolten-Hunter SP binding to intact C6 astrocytes and CHO cells expressing NK1-R by greater than 95% when compared to normal rabbit IgG which failed to inhibit radiolabeled SP binding. Thus, an antibody which recognizes surface determinants to the NK1-R could be generated upon immunization with an NK1-R peptide.     

Profile of neuronal excitation following selective activation of the neurokinin-1 receptor in rat deep dorsal horn in vitro

The excitatory actions of the selective neurokinin-1 receptor (NK1R) agonist [Sar⁹,Met(O₂)¹¹]substance P (SP) were tested on a sample (n=50) of deep dorsal horn neurones in the isolated and hemisected young rat spinal cord. Superfusion of the NK1R agonist (2 μM) elicited a prolonged membrane depolarisation (6.6±0.5 mV) and an increase in action potential firing in 41/50 (82%) neurones. These [Sar⁹,Met(O₂)¹¹]SP-induced depolarisations were attenuated by the selective NK1R antagonist GR82334 (1 μM). An increased neuronal excitability after [Sar⁹,Met(O₂)¹¹]SP application was indicated by an augmented spike frequency generated in response to long duration, step depolarisations. In order to assess whether a direct excitatory action existed, [Sar⁹,Met(O₂)¹¹]SP was re-tested on a sample of TTX-treated neurones (n=14). The majority (9/14) retained agonist sensitivity although the amplitude of the depolarisation was reduced to 48% of the control value. A sample of neurones (n=7) that responded to the NK1R agonist were morphologically characterised after filling with the intracellular dye, biocytin. Dorsal dendrites that clearly penetrated lamina II and that could receive a direct C-afferent input, were identified in only 2/7 neurones. These electrophysiological and neuroanatomical data demonstrate that deep dorsal horn neurones possess functional NK1Rs. The implications of the existence of these NK1Rs in the context of spinal somatosensory systems and SP is considered.     

Spinoparabrachial tract neurons showing substance P receptor-like immunoreactivity in the lumbar spinal cord of the rat

By using substance P receptor (SPR) immunofluorescence histochemistry combined with fluorescent retrograde labeling, SPR-like immunoreactive (SPR-LI) neurons sending their axons to the lateral parabrachial region were observed in the lumbar spinal cord of the rat. After injection of Fluoro-Gold into lateral parabrachial region, retrogradely labeled neurons with SPR-LI were seen frequently in lamina I and the lateral spinal nucleus, and occasionally in laminae IV and V, with a predominantly contralateral distribution. Some of these neurons, especially those in lamina I, may convey nociceptive information to the lateral parabrachial region.     

A selective and extremely potent antagonist of the neurokinin-1 receptor

Sendide [Tyr6,D-Phe7,D-His9]-substance P(6-11) has been examined by measurements of ligand binding to crude membrane fractions and by functional tests on the spinally mediated behavioral response. Sendide potently displaced [3H]-labeled substance P (SP) binding to mouse spinal cord membranes in a competitive manner. In vivo, sendide, intrathecally co-injected with SP, competitively antagonized SP-induced scratching, biting and licking. The behaviors elicited by physalaemin, septide and [Sar9, Met(O2)11]-SP were also reduced by co-administration of sendide. Large doses of sendide were needed to reduce the action of neurokinin A, D-septide, neurokinin B and eledoisin. The in vitro and in vivo pharmacological profile of sendide demonstrated that it is a selective and extremely potent antagonist of the neurokinin-1 receptor.     

Involvement of oxytocin in spinal antinociception in rats with inflammation

The present study was conducted on rats with inflammation induced by subcutaneous injection of carrageenan into the left hindpaw. Intrathecal administration of oxytocin produced dose-dependent increases in the hindpaw withdrawal latency (HWL) to thermal and mechanical stimulation in rats with inflammation. The antinociceptive effect of oxytocin was blocked by intrathecal administration of atosiban, a selective oxytocin antagonist, indicating that oxytocin receptor mediates oxytocin-induced antinociception in the spinal cord. The oxytocin-induced antinociceptive effect was attenuated by intrathecal administration of the opioid antagonist naloxone, suggesting an involvement of the endogenous opioid system in oxytocin-induced antinociception in the spinal cord of rats with inflammation. Furthermore, the antinociceptive effect of oxytocin was attenuated by intrathecal injections of the mu-receptor antagonist beta-funaltrexamine and the kappa-receptor antagonist nor-binaltorphimine, but not by the delta-receptor antagonist naltrindole, illustrating that mu- and kappa-receptors, but not delta-receptor, are involved in oxytocin-induced antinociception in the spinal cord of rats with inflammation. Moreover, intrathecal administration of atosiban alone induced a hyperalgesia in rats with inflammation, indicating that endogenous oxytocin is involved in the transmission and regulation of nociceptive information in the spinal cord of rats with inflammation. The present study showed that both exogenous and endogenous oxytocin displayed antinociception in the spinal cord in rats with inflammation, and mu- and kappa-receptors were involved in oxytocin-induced antinociception.     

Neurokinin-1 receptor immunoreactivity in hypotension sensitive sympathetic preganglionic neurons

Substance P activation of neurokinin-1 (NK1) receptors on spinal sympathetic preganglionic neurons (SPN) influences blood pressure. We identified SPN likely to subserve the baroreceptor reflex and established if these neurons showed NK1 receptor-immunoreactivity. Nitroprusside (NP) infusion or inferior vena cava (IVC) constriction activated similar numbers of SPN. Of these, about 40% were NK1 receptor-immunoreactive after NP infusion, but only about 20% were NK1 receptor-immunoreactive after IVC constriction. The distribution of Fos/NK1 receptor SPN suggested that substance P may preferentially target sympathoadrenal SPN.     

Time-course of the internalization and recycling of neurokinin 1 receptors in rat dorsal horn neurons

Neurokinin 1 receptor (NK1R) internalization in dorsal horn neurons is important for intracellular signaling in nociception. Since the rates of NK1R internalization and recycling vary substantially, particularly between cultured and native cells, it is imperative to characterize them in dorsal horn neurons. When rat spinal cord slices were incubated at 35 degrees C with 1 microM substance P (SP), NK1Rs in lamina I neurons internalized rapidly following apparent exponential association kinetics (half-life=71 s). Confocal images of neuronal somas at different incubation times revealed that NK1Rs were uniformly distributed at the cell surface up to 30 s and formed aggregates at the membrane by 60 s. NK1R-containing endosomes migrated to the cell interior at 90-120 s, and were found throughout the cytoplasm at 300 s and thereafter. Upon elimination of SP, NK1Rs recycled back to the cell surface following an apparent linear time-course. Recycling was slower than internalization, being completed in 60-90 min. Confocal microscopy revealed that NK1R-containing endosomes docked at the cell surface 45 min after the elimination of SP. NK1Rs still formed aggregates at the cell surface at 60 min, but were once again uniformly distributed along the membrane by 90 min. NK1R internalization and recycling also occurred in lamina I dendrites. NK1R-containing endosomes in dendrites did not migrate to the cytoplasm. These results show that NK1R internalization and recycling are considerably faster in dorsal horn neurons than in cultured cells, and that most NK1Rs in dorsal horn neurons are internalized when NK1R-mediated hyperalgesia is more severe.     

Increased delayed type hypersensitivity in rats subjected to unilateral mononeuropathy is mediated by neurokinin-1 receptors

An animal model of peripheral mononeuropathy was utilized in the present study to investigate the potential role of substance P (SP) in modifying immune responses associated with chronic pain conditions. Animals subjected to unilateral sciatic ligation and sham-operated animals were sensitized with keyhole limpet hemocyanin (KLH) and subsequently challenged in the ipsilateral or contralateral hind paw to produce a delayed-type hypersensitivity (DTK) response. Subcutaneous microdialysis and radioimmunoassay were used to measure interstitial fluid SP levels in the challenged tissue prior to and following immune challenge in control and neuropathic animals. Following immune challenge, there was a significant increase in the concentration of SP in tissue dialysate samples from the challenged paw of both sham-operated and neuropathic animals. However, tissue SP levels in neuropathic animals were more than two-fold higher than those obtained from sham-operated controls following challenge. SP concentration remained elevated for 2.5 h following immune challenge in neuropathic animals compared to 90 min in sham-operated animals. Compared with controls, neuropathic animals also exhibited an increased DTH response that was reversed, in a dose-related fashion, by the non-peptide NK-1 receptor blocker L-703,606. The same antagonist had no effect in sham-operated animals. These data suggest that the increased DTH response in animals subjected to unilateral mononeuropathy involves SP and NK-1 receptors present in the challenged tissue.     

The role of spinal cholecystokinin B receptors in thermal allodynia and hyperalgesia in diabetic mice

We examined the tail-flick response to various heat intensities in diabetic and non-diabetic mice. Heat intensities were set to one of six values by adjusting the source of voltage for a 50-W projection bulb to 20, 25, 35, 50, 65 and 80 V. Tail-flick latencies at source voltages of 35 and 50 V in diabetic mice were significantly shorter than those in non-diabetic mice. However, tail-flick latencies at 25, 65 and 80 V in diabetic mice were not significantly altered. Although tail-flick latencies in non-diabetic mice were not affected by i.t. pre-treatment with CI-988, a selective cholecystokinin B (CCK(B)) receptor antagonist, those at 35 and 50 V in diabetic mice were significantly increased. In non-diabetic mice, i.t. pre-treatment with cholecystokinin octapeptide (CCK-8), at a dose of 0.3 ng, decreased tail-flick latencies at 35 and 50 V. Furthermore, the attenuation of tail-flick latencies induced by i.t. pre-treatment with CCK-8 in non-diabetic mice was reversed by i.t. pre-treatment with CI-988. Protein kinase C (PKC) activator phorbol-12, 13-dibutyrate (PDBu)-induced reduction in the tail-flick latencies at heat intensities of 35 and 50 V in non-diabetic mice was dose-dependently and significantly reversed by i.t. pre-treatment with CI-988. On the other hand, the CCK-8-induced thermal hyperalgesia and allodynia at heat intensities of 35 and 50 V in non-diabetic mice were inhibited when PKC activity was inhibited by i.t. pre-treatment with calphostin C. These results indicate that the thermal allodynia and hyperalgesia in diabetic mice may be due, at least in part, to the activation of CCK(B) receptors followed by the activation of PKC in the spinal cord.     

Occurrence and distribution of substance P receptors in the cerebral blood vessels of the rat

The distribution of immunoreactivity to the receptor for substance P was examined in the cerebral blood vessels of the rat. Substance P immunoreactivity has been demonstrated in the nerve fibers of the cerebral blood vessels. Recently, the production of substance P receptor specific antibody has enabled the detection of localization of the substance P receptor in the central nervous system. In this study, we examined the existence of nerve fibers with substance P receptor immunoreactivity in the cerebral blood vessels and the cranial ganglia innervating the cerebral blood vessels. Sprague–Dawley rats were perfused with fixative and the pial arteries and the cranial ganglia known to innervate the cerebral blood vessels, i.e., trigeminal, sphenopalatine, internal carotid, otic and superior cervical ganglia, were dissected. All specimens were incubated with anti-substance P receptor IgG, then stained by the avidin–biotin–peroxidase complex method. Numerous nerve fibers with varicosities forming plexuses, with substance P receptor immunoreactivity were observed on the walls of the major extracerebral arteries forming the circle of Willis and its branches. Substance P receptor immunoreactivity was also detected in the endothelium of the cerebral arteries. Substance P receptor immunoreactivity was positive in many neurons of the sphenopalatine ganglion, otic ganglion, trigeminal ganglion, superior cervical ganglion and internal carotid ganglion. The present study demonstrated the existence of nerve fibers with substance P receptor immunoreactivity in the cerebral blood vessels and the cranial ganglia that innervate the cerebral blood vessels. These findings are important in understanding the responsiveness of the cerebral blood vessels to substance P.     

Role of intracellular calcium in thermal allodynia and hyperalgesia in diabetic mice

We examined the involvement of cytosolic calcium on thermal hyperalgesia and allodynia seen in diabetic mice. Tail-flick latencies at source voltages of a 50-W projection bulb to 35 and 50 V in diabetic mice were significantly shorter than those in non-diabetic mice. Tail-flick latencies at 35 and 50 V in diabetic mice were increased by pretreatment with ryanodine, which blocks Ca2+ release from Ca2+/caffeine-sensitive microsomal pools. On the other hand, intrathecal (i.t.) pretreatment with thapsigargin, which inhibits Ca2+ uptake into the inositol-1,4,5-trisphosphate-sensitive microsomal Ca2+ pool, decreased tail-flick latencies at 35 and 50 V in non-diabetic mice. Thus, it seems likely that thermal allodynia and hyperalgesia in diabetic mice may be due, in part, to the enhancement of intracellular calcium level in the spinal cord.     

Effects of pre-emptively administered nociceptin on the development of thermal hyperalgesia induced by two models of experimental mononeuropathy in the rat

Pre-emptive analgesia is thought to be produced by the prevention of spinal facilitation evoked by nociceptive input to the spinal cord. Opioid receptor-like 1 (ORL1) receptor agonist has been reported to inhibit the development of spinal facilitation. We investigated the effect of nociceptin, an ORL1 receptor agonist, on the development of thermal hyperalgesia and the expression of Fos-like immunoreactivity (Fos-LI) in the spinal dorsal horn induced by two neuropathic pain models, the chronic constriction injury model and the partial sciatic nerve injury model. Chronic constriction injury is created by placing four loosely tied ligatures around the right sciatic nerve. Partial sciatic nerve injury was created by tight ligation of one third to one half of the right sciatic nerve. All drugs were injected intrathecally 10 min before the nerve injury. The anti-hyperalgesic effect of drugs was evaluated by the measurement of the paw withdrawal latency (PWL) against thermal nociceptive stimulation. The PWLs of the injured paws were measured 7, 14 and 21 days after the nerve injury. Expression of Fos-LI was examined 2 h after the nerve injury. Intrathecal injection of nociceptin significantly delayed the development of thermal hyperalgesia and decreased the expression of Fos-LI induced by chronic constriction injury, but not that induced by partial sciatic nerve injury. These data indicate that pre-emptive administration of nociceptin might be one strategy for the prevention of the development of neuropathic pain.     

Neurokinin-1 receptor in the basolateral nuclear group of the canine amygdala--comparative study in normal and aggressive dogs

Substance P and its NK-1 receptor are involved in the modulation of aggressive behavior. Because of the role of the basolateral nuclear group (BNG) of the amygdala in canine aggression, neurokinin-1 receptor (NK-1) immunoreactivity in this brain region was assessed stereologically in 7 normally behaving and 6 pathologically aggressive dogs. The first aim of this study was to obtain information about the absolute number of neurons expressing the NK-1 receptor in the canine BNG because absolute numbers of neurons expressing the NK-1 receptor are not documented in literature. Additionally, an exploratory comparison was made between NK-1 expressing neurons in the BNGs of normally behaving and aggressive dogs. Results showed a very low amount (1-2%) of BNG neurons containing the NK-1 receptor in both groups. Aggressive dogs had significantly more NK-1-receptor-positive BNG neurons than normal dogs, but the numerical densities and fractions of receptor-positive neurons did not differ significantly between both groups. Combined with the fact that aggressive dogs have 27% more neurons in their BNGs than normal dogs, as reported in a previous study, these findings suggest a limited role for the NK-1-receptor-positive neurons within the BNG in the modulation of canine aggression. The present report of absolute numbers of neurons expressing the NK-1 receptor in the canine BNG could however be useful for further quantitative studies.     

Stress-induced dura vascular permeability does not develop in mast cell-deficient and neurokinin-1 receptor knockout mice

Migraine headaches are often precipitated by stress and seem to involve neurogenic inflammation (NI) of the dura mater associated with the sensation of throbbing pain. Trigeminal nerve stimulation had been reported to activate rat dura mast cells and increase vascular permeability, effects inhibited by neonatal pretreatment with capsaicin implicating sensory neuropeptides, such as substance P (SP). The aim of the present study was to investigate NI, assessed by extravasation of 99-Technetium-gluceptate (⁹⁹Tc-G), as well as the role of mast cells, SP and its receptor (NK-1R) in dura mater of mice in response to acute stress. Restraint stress for thirty min significantly increased ⁹⁹Tc-G extravasation in the dura mater of C57BL mice. This effect was absent in W/W^v mast cell-deficient mice and NK-1 receptor knockout mice (NK-1R−/−), but was unaltered in SP knockout mice (SP−/−). Acute restraint stress also resulted in increased dura mast cell activation in C57BL mice, but not in NK-1R−/− mice. These data demonstrate for the first time that acute stress triggers NI and mast cell activation in mouse dura mater through the activation of NK-1 receptors. The fact that SP−/− mice had intact vascular permeability response to stress indicates that some other NK-1 receptor agonist may substitute for SP. These results may help explain initial events in pathogenesis of stress-induced migraines.     

Involvement of a spinal brain-derived neurotrophic factor/full-length TrkB pathway in the development of nerve injury-induced thermal hyperalgesia in mice

Partial sciatic nerve ligation in mice caused a marked and persistent decrease in the latency of paw withdrawal from a thermal stimulus only on the ipsilateral side. This thermal hyperalgesia was abolished by repeated intrathecal pretreatment with a specific antibody to brain-derived neurotrophic factor (BDNF), but not neurotrophin-4, just before and after the nerve ligation. These results provide direct evidence that BDNF within the spinal cord may contribute to the development of thermal hyperalgesia caused by nerve injury in mice. We previously reported that protein level of full-length TrkB, which contains the cytoplasmic protein tyrosine kinase domain, were clearly increased on the ipsilateral side of spinal cord membranes obtained from sciatic nerve-ligated mice. In the present study, we further demonstrated that the increased in the protein level of full-length TrkB is completely reversed by concomitant intrathecal injection of BDNF antibody. Furthermore, thermal hyperalgesia induced by nerve ligation was completely suppressed by repeated intrathecal injection of a specific antibody to full-length TrkB and an inhibitor of the protein tyrosine kinase activity for the neurotrophin receptor, K-252a. However, repeated intrathecal injection of a specific antibody to truncated TrkB, which lacks the cytoplasmic protein tyrosine kinase domain, failed to reverse thermal hyperalgesia observed in nerve-ligated mice. These findings suggest the possibility that the binding of BDNF to full-length TrkB and subsequent its activation may play a critical role in the development of neuropathic pain-like thermal hyperalgesia induced by nerve injury in mice.     

Expression of the type 1 and type 2 receptors for tumor necrosis factor after traumatic spinal cord injury in adult rats

Posttraumatic inflammation has been implicated in secondary tissue damage after spinal cord injury (SCI). Tumor necrosis factor-alpha (TNF-alpha) is a key inflammatory mediator that is increasingly expressed after SCI. The effect of TNF-alpha is mediated through its receptors TNFR1 (p55) and TNFR2 (p75). However, whether these two receptors are expressed after SCI has not been demonstrated. In the present study, the temporo-spatial expression of TNFR1 and TNFR2 was examined in rats that had received a 10 g impact injury dropped at a height of 12.5 mm using the New York University impact device. In sham operates, no detectable TNFR1 or TNFR2 immunoreactivity (IR) was observed. In contused spinal cord, TNFR1 protein expression and immunoreactivity (IR) were detected as early as 15 min postinjury, reached its peak at 8 h, and declined markedly after 1 and 3 days postinjury. The temporal pattern of TNFR2 expression was similar to that of TNFR1 but its expression peaked at 4 h postinjury. During peak expression, TNFR1- and TNFR2-IR were most intense at the site of injury and decreased gradually from the injury epicenter. TNFR1- and TNFR2-positive cells included neurons, astrocytes, and oligodendrocytes. Methylprednisolone (MP), a synthetic glucocorticoid, partially inhibited the injury-induced expression of TNFR1 and TNFR2, an effect which could be reversed by RU486, an antagonist of glucocorticoid receptors. We suggest that the expression of TNFR1 and TNFR2 after SCI may contribute to posttraumatic inflammatory responses of TNF-alpha.     

Expression and localization of p80 interleukin-1 receptor protein in the rat spinal cord

The biological effects of interleukin (IL)-1 are mediated by two distinct receptors, the p80 or type I (IL-1RI) and p68 or type II (IL-1RII) receptors. Because IL-1RII has a short, 29-amino acid cytoplasmic domain which may not be sufficient for signaling, there is considerable evidence indicating that IL-1 may signal exclusively through the IL-1RI receptor. Here, we report the expression, distribution, and cellular localization of the IL-1RI protein in the adult rat spinal cord in vivo and embryonic spinal cord in vitro. We found that IL-1RI was expressed in both the gray and white matter throughout the entire length of the spinal cord and was localized in neurons of the anterior horn, astrocytes, oligodendrocytes, and central canal ependymal cells. Interestingly, resting microglia were negative for IL-1RI. In primary cultures obtained from the embryonic day (E) 15 rats, IL-1RI was expressed in eeurons, astrocytes, and oligodendrocytes as well as microglia. These data provide both in vivo and in vitro evidence that neurons and glial cells express the IL-1RI proteins. The differential expression of IL-1RI in the developing, but not mature, microglia may indicate the difference of these cells in response to IL-1 stimuli during maturation. The distribution and cellular localization of IL-1RI proteins in the spinal cord provide a molecular basis for understanding the reciprocal interaction between the immune and the central nervous systems.