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.     

Nerve injury-induced mechanical but not thermal hyperalgesia is attenuated in neurokinin-1 receptor knockout mice

Mice lacking the gene encoding for substance P and neurokinin A, or the NK-1 receptor, exhibit alterations in behavior to various acute nociceptive stimuli. However, behavioral responses of NK-1 mutant animals have not been well characterized in models of chronic pain. We studied the behavioral responses of NK-1 knockout and wild-type control mice to thermal and mechanical stimuli before and after inducing chronic neuropathic pain by unilateral ligation of the L5 spinal nerve. Mechanical hyperalgesia was evaluated by determining the frequency of withdrawal to von Frey monofilaments applied to the hind paws. Nerve injury-induced hyperalgesia to thermal stimuli was examined by determining responses to radiant heat and cooling stimuli. The contribution of the sympathetic nervous system to mechanical hyperalgesia was evaluated by administering 3 mg/kg phentolamine, an alpha-adrenergic antagonist, subcutaneously. Following spinal nerve injury, withdrawal frequencies to mechanical stimulation increased in wild-type mice within 1 day and persisted during the 9-week observation period, whereas in the knockout mice, withdrawal frequencies did not increase significantly. In contrast, withdrawal latencies to radiant heat decreased up to 2 weeks after nerve injury in both the NK-1 and the wild-type mice. Similarly, the increase in withdrawal frequency to the cooling stimuli following the nerve injury was not different in the NK-1 knockout and wild-type mice. Mechanical hyperalgesia in the wild-type mice was not reversed by systemic administration of phentolamine, suggesting that the pain is not sympathetically maintained. The results indicate that NK-1 receptors contribute to the development of mechanical, but not thermal, hyperalgesia in neuropathic pain.     

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.     

Cellular localization and distribution of neurokinin-1 receptors in the rat stomach

In the stomach, the majority of substance P's effects are mediated by the activation of neurokinin-1 (NK1) receptors. The gastric cellular distribution of these receptors in Wistar and Sprague-Dawley rats was determined using immunocytochemistry. The localization of the NK1 receptors with respect to von Willebrand's factor, protein gene product 9.5, substance P, vasoactive intestinal peptide, and calcitonin gene-related peptide was also determined. Results show that NK1 receptor immunoreactivity was dependent on the duration of fixation. In corpus and antrum tissues that were fixed in 4% paraformaldehyde for 30 min, the presence of NK1 receptor immunoreactivity was demonstrated on nerve fibers throughout the stomach, on the surface and in the cytoplasm of myenteric cell bodies, on circular smooth muscle cells, and on vascular endothelial cells. This was observed in tissues from both rodent strains. Overnight fixation in the same fixative, however, demonstrated the presence of NK1 receptor immunoreactivity only on nerve fibers and cell bodies of the myenteric plexus, and on circular smooth muscle cells. In 30-min fixed tissues, the localization of NK1R immunoreactivity on vascular endothelial cells and nerve fibers was confirmed by co-localization with von Willebrand's factor and protein gene product 9.5 immunoreactivity, respectively. In both rodent strains, NK1 receptor immunoreactivity was co-localized with substance P immunoreactivity on nerve fibers of the longitudinal and circular muscle. In the Wistar rat, NK1 receptor immunoreactivity was co-localized with vasoactive intestinal peptide immunoreactivity or calcitonin gene-related peptide immunoreactivity throughout the stomach. However, in the Sprague-Dawley rat, NK1 receptor immunoreactivity was only co-localized with calcitonin gene-related peptide immunoreactivity in a minority of fibers of the circular muscle. The overall results of this study show that the antigenic epitopes of the NK1 receptor are sensitive to overfixation. When tissues were not overfixed, NK1 receptor immunoreactivity was distributed more extensively throughout the rat stomach than has been described previously. The results of this study provide the anatomical basis for many of the actions of substance P in the rat stomach.     

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.     

Illness-induced hyperalgesia is mediated by spinal neuropeptides and excitatory amino acids

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.     

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.     

Disruption of the substance P receptor (neurokinin-1) gene does not prevent upregulation of preprotachykinin-A mRNA in the spinal cord of mice following peripheral inflammation

The neuropeptide substance P is thought to play an important role in nociception, although the function of the peptide remains controversial. Following peripheral inflammation there is a pronounced upregulation of substance P expression both in sensory neurons and in postsynaptic neurons within the spinal cord. We have examined the levels of expression of mRNA encoding substance P and dynorphin following the development of inflammatory hyperalgesia in mice in which the substance P receptor gene, also known as the neurokinin-1 receptor gene, has been disrupted by homologous recombination. We show that inflammatory hyperalgesia following injection of complete Freund's adjuvant develops normally in animals that lack the neurokinin-1 receptor and that expression of mRNAs encoding substance P and the neuropeptide dynorphin are upregulated regardless of the genotype of the mouse. This suggests that substance P activity is not required for the development and maintenance of inflammatory hyperalgesia and that the upregulation of substance P expression is mediated by neurotransmitters other than substance P.     

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.     

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.     

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.     

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.