Temperature dependency of basal and evoked release of amino acids and calcitonin gene-related peptide from rat dorsal spinal cord

Moderate hypothermia significantly diminishes consequences of spinal and cerebral anoxia. One component of this neuroprotection has been hypothesized to be suppression of excitotoxic transmitter release. Whether this suppression is attributable to reduced hypoxic injury that induces release or an alteration of the release process itself is unclear. We sought to characterize the temperature sensitivity (Q10) of basal and evoked calcitonin gene-related peptide (CGRP) and amino acid release from dorsal horn slices of rat spinal cord over a range of temperatures from 40 to 8 degrees C. At 40 degrees C, potassium (60 mM) and capsaicin (10 microM) evoked a 21- and 32-fold increase in basal CGRP concentrations, respectively. Capsaicin had no effect on glutamate release, but potassium evoked a 2.7-fold increase. Release evoked by either potassium or capsaicin was reduced in a biphasic fashion with declining temperature. Over the range of 40 to 34 degrees C, the Q10 values for evoked release for CGRP were 11.3 (potassium) and 39.7 (capsaicin) and for glutamate, 5. 5 (potassium). Over the range of 34 to 8 degrees C, Q10 values were near unity for all evoked release (0.8 and 1.3 for CGRP and 1.2 for glutamate). Although serine, glycine, glutamine, taurine, and citrulline showed no evoked release, basal levels were reduced at temperatures below 34 degrees C. The pronounced temperature dependency of evoked transmitter release between 40 and 34 degrees C is consistent with the profound cerebral protection observed with mild hypothermia in which metabolic activity is only slightly depressed.     

Region-specific increase in glutamate release from dorsal horn of rats with adjuvant inflammation

Glutamate is considered an important pain transmitter and responsible for inflammatory hyperalgesia, but quantitative and topographical changes in glutamate release in the dorsal horn during peripheral inflammation have not been characterized. To address this issue, image analysis with a confocal laser scanning microscope was performed for quantitatively mapping capsaicin-evoked glutamate release from the lumbar cord slice of rats following unilateral adjuvant inoculation to the hind-paw. Capsaicin induced glutamate release from laminae I, II and X in the spinal cord of the adjuvant-treated and untreated sides, without apparent release from laminae III-V. The concentration of released glutamate in laminae I, II and X was higher on the adjuvant-treated side than on the untreated side. The results suggest that adjuvant inflammation increases glutamate release from capsaicin-sensitive primary afferents in laminae I, II and X.     

Activation of spinal N-methyl-D-aspartate or neurokinin receptors induces long-term potentiation of spinal C-fibre-evoked potentials

The use-dependent increase in synaptic strength between primary afferent C-fibres and second-order neurons in superficial spinal dorsal horn may be an important cellular mechanism underlying central hyperalgesia. This long-term potentiation can be blocked by antagonists of the N-methyl-D-aspartate subtype of glutamate receptor, the neurokinin 1 or the neurokinin 2 receptor. We have tested here whether activation of these receptors by superfusion of the spinal cord with corresponding agonists in the absence of presynaptic activity is sufficient to induce long-term potentiation. In urethane anaesthetized rats C-fibre-evoked field potentials were elicited in superficial laminae of lumbar spinal cord by electrical stimulation of the sciatic nerve. In rats with intact spinal cord, controlled superfusion of the spinal cord at recording segments for 60 min with N-methyl-D-aspartate, substance P or neurokinin A never induced long-term potentiation. Spinal superfusion with a mixture of N-methyl-D-aspartate, substance P and neurokinin A also failed to induce long-term potentiation in four rats tested. In spinalized rats, however, long-term potentiation was induced by either N-methyl-D-aspartate (at 10 microM, to 173 +/- 16% of control) substance P (at 10 microM, to 176 +/- 13% of control) or by neurokinin A (at 1 microM, to 198 +/- .20% of control). The induction of long-term potentiation by N-methyl-D-aspartate, substance P or neurokinin A was blocked by intravenous application of the receptor antagonists dizocilpine maleate (0.5 mg/kg), RP67580 (2 mg/kg) or SR48968 (0.2 mg/kg), respectively. Thus, activation of N-methyl-D-aspartate or neurokinin receptors may induce long-lasting plastic changes in synaptic transmission in afferent C-fibres and this effect may be prevented by tonic descending inhibition.     

Evidence for the interaction of glutamate and NK1 receptors in the periphery

It is known that Substance P (SP) enhances glutamate- and N-methyl-D-aspartate (NMDA)-induced activity in spinal cord dorsal horn neurons and that this enhancement is important in the generation of wind-up and central sensitization. It is now known that SP and glutamate receptors are present on sensory axons in rat glabrous skin. This raises the issue as to whether SP and glutamate interact in the periphery. Using the tail skin in rats, the present study demonstrates 1) that unmyelinated axons at the dermal-epidermal junction immunostain for antibodies directed against NMDA, non-NMDA or SP (NK1) receptors; 2) that glutamate injected into the tail skin results in dose-dependent nociceptive behaviors interpreted as mechanical hyperalgesia, mechanical allodynia and thermal hyperalgesia, which are blocked following co-injection with glutamate antagonists; 3) that peripheral injection of SP potentiates glutamate-induced nociceptive behaviors in that the co-injection of SP+glutamate results in a significantly longer duration of behavioral responses compared to the responses seen following injection of either substance alone. These data provide support for the hypothesis that primary afferent neurons might well be subject to similar mechanisms that result in wind-up or central sensitization of spinal cord neurons.     

ATP P2x receptors and sensory synaptic transmission between primary afferent fibers and spinal dorsal horn neurons in rats

ATP P2x receptors and sensory synaptic transmission between primary afferent fibers and spinal dorsal horn neurons in rats. J. Neurophysiol. 80: 3356-3360, 1998. Glutamate is a major fast transmitter between primary afferent fibers and dorsal horn neurons in the spinal cord. Recent evidence indicates that ATP acts as another fast transmitter at the rat cervical spinal cord and is proposed to serve as a transmitter for nociception and pain. Sensory synaptic transmission between dorsal root afferent fibers and neurons in the superficial dorsal horn of the lumbar spinal cord were examined by whole cell patch-clamp recording techniques. Experiments were designed to test if ATP could serve as a transmitter at the lumbar spinal cord. Monosynaptic excitatory postsynaptic currents (EPSCs) were completely abolished after the blockade of both glutamatergic alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate and N-methyl--aspartate receptors. No residual current was detected, indicating that glutamate but not ATP is a fast transmitter at the dorsal horn of the lumbar spinal cord. Pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), a selective P2x receptor antagonist, produced an inhibitory modulatory effect on fast EPSCs and altered responses to paired-pulse stimulation, suggesting the involvement of a presynaptic mechanism. Intrathecal administration of PPADS did not produce any antinociceptive effect in two different types of behavioral nociceptive tests. The present results suggest that ATP P2x2 receptors modulate excitatory synaptic transmission in the superficial dorsal horn of the lumbar spinal cord by a presynaptic mechanism, and such a mechanism does not play an important role in behavioral responses to noxious heating. The involvement of other P2x subtype receptors, which is are less sensitive to PPADS, in acute nociceptive modulation and persistent pain remains to be investigated.     

Human and rat primary C-fibre afferents store and release secretoneurin, a novel neuropeptide

Secretoneurin is a recently discovered neuropeptide derived from secretogranin II (SgII). Since this peptide could be detected in the dorsal horn of the spinal cord we studied whether it is localized in and released from primary afferent neurons. Secretoneurin was investigated with immunocytochemistry and radioimmunoassay in spinal cord, dorsal root ganglia and peripheral organs. SgII mRNA was determined in dorsal root ganglia. Normal rats and rats pre-treated neonatally with capsaicin to destroy selectively polymodal nociceptive (C-) fibres were used. Slices of dorsal spinal cord were perfused in vitro for release experiments. Immunocytochemistry showed a distinct distribution of secretoneurin-immunoreactivity (IR) in the spinal cord and, lower brainstem. A particularly high density of fibres was found in lamina I and outer lamina II of the caudal trigeminal nucleus and of the spinal cord. This distribution was qualitatively identical in rat and human post-mortem tissue. Numerous small diameter and some large dorsal root ganglia neurons were found to contain SgII mRNA. Capsaicin treatment led to a marked depletion of secretoneurin-IR in the substantia gelatinosa, but not in other immunopositive areas of the spinal cord and to a substantial loss of small (< 25 microns) SgII-mRNA-containing dorsal root ganglia neurons. Radioimmunoassay revealed a significant decrease of secretoneurin-IR in the dorsal spinal cord, the trachea, heart and urinary bladder of capsaicin-treated rats. Perfusion of spinal cord slices with capsaicin as well as with 60 mM potassium led to a release of secretoneurin-IR. In conclusion, secretoneurin is a neuropeptide which is stored in and released from capsaicin-sensitive, primary afferent (C-fibre) neurons.(ABSTRACT TRUNCATED AT 250 WORDS)     

NMDA receptor activation modulates evoked release of substance P from rat spinal cord

The possible modulation exerted by glutamate on substance P (SP) release from the rat spinal cord has been investigated. The N-methyl-D-aspartate (NMDA) receptor agonist, NMDA (1 microM), increased SP basal outflow by 46.5+/-10.9% (n = 3, P<0.01) without changing the evoked release of the peptide. Conversely, NMDA antagonists but not 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) inhibited both electrically-evoked and capsaicin-induced release of SP. In particular, D-2-amino-5-phosphonopentanoate (D-AP5; 50 microM) inhibited electrically-evoked and capsaicin-induced release of SP by 93+/-2.4% and 93.2+/-3.8% (n = 12, P<0.01), respectively. Functional pharmacological evidence is provided for glutamate exerting a positive feedback on SP release evoked by C fibre stimulation via NMDA receptor activation.     

Altered c-fos expression in the parabrachial nucleus in a rodent model of CFA-induced peripheral inflammation

Increases in the expression of immediate early genes have been shown to occur in the lumbar spinal cord dorsal horn after peripheral inflammation. Given that the pontine parabrachial nucleus has been implicated in nociceptive as well as antinociceptive processes and is reciprocally connected with the spinal cord dorsal horn, it seems likely that peripheral inflammation will cause alterations in immediate early gene expression in this nucleus. To test this hypothesis we examined cFos-like immunoreactivity in a rodent complete Freund's adjuvant-induced peripheral inflammatory model of persistent nociception. Unilateral hind paw injections of complete Freund's adjuvant produced inflammation, hyperalgesia of the affected limb, and alterations in open field behaviors. Immunocytochemical analysis demonstrated a bilateral increase in cFos-like immunoreactivity in the lateral and Kolliker-Fuse subdivisions of the parabrachial nucleus at 6 and 24 hours postinjection and an ipsilateral decrease below basal levels in the Kolliker-Fuse subdivision at 96 hours postinjection when compared to saline controls. Taken together, these results suggest that select parabrachial neurons are activated by noxious somatic inflammation. These active parabrachial neurons are likely to participate in ascending nociceptive and/or descending antinociceptive pathways.     

Interactions between excitatory amino acids and tachykinins in the rat spinal dorsal horn

Whole-cell patch-clamp technique of freshly isolated rat spinal dorsal horn (DH) neurons, intracellular recording from DH neurons in a slice preparation, and high performance liquid chromatography with fluorimetric detection of release of endogenous glutamate and aspartate from spinal cord slice following activation of primary afferent fibers were employed to investigate interactions between excitatory amino acids (EAA) and tachykinins [substance P (SP) and neurokinin A (NKA)]. Potentiation of N-methyl-D-aspartate (NMDA)-, quisqualate (QA)- and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-, but not kainate-induced currents by SP and NKA was found. Spantide II, a claimed novel nonselective tachykinin antagonist, effectively blocked the SP (2 nM)-induced potentiation of the responses of DH neurons to NMDA. In the presence of glycine (0.1 microM), the SP-evoked increase of the NMDA-induced current was prevented. However, 7-chlorokynurenic acid (2 microM), a competitive antagonist at the glycine allosteric site of the NMDA receptor, led to the reestablishment of the SP effect. Brief high frequency electrical stimulation of primary afferent fibers produced a long-lasting potentiation of presumed monosynaptic and polysynaptic excitatory postsynaptic potentials and sustained enhanced release of endogenous glutamate (218.3 +/- 66.1%) and aspartate (286.3 +/- 58.0%). Possible functional implications of the observed phenomena are discussed in relation to transmission and integration of sensory information, including pain.     

Bradykinin increases the proportion of neonatal rat dorsal root ganglion neurons that respond to capsaicin and protons

A number of studies have examined bradykinin-induced sensitization of primary afferent neurons to mechanical or thermal stimuli. However, bradykinin-induced sensitization to other chemical stimuli has not been systematically addressed. We used primary cultures of dorsal root ganglion neurons from neonatal rats to determine whether bradykinin alters the responsiveness of individual neurons to capsaicin and protons. An increase in the concentration of free intracellular Ca2+ was used as a measure of a response to capsaicin or low pH. Pretreatment with bradykinin (30 nM) increased the proportion of "intermediate-size" (240-320 microm2) dorsal root ganglion neurons that responded to capsaicin (100 nM) or low pH (6.1). However, among "small-size" (160-239 microm2) neurons, bradykinin increased the proportion of neurons that responded to low pH (6.1) but not to capsaicin (10 or 100 nM). Because treatment with arachidonic acid (10 microM) did not mimic the effect of bradykinin and inhibition of cyclo-oxygenase and lipoxygenase with 5,8,11,14-eicosatetraynoic acid (10 microM) did not inhibit the effect of bradykinin on the response to capsaicin, it is not likely that the bradykinin-induced enhancement of neuronal responsiveness is mediated by arachidonic acid or its metabolites in this model. These results support the hypothesis that bradykinin sensitizes primary afferent neurons to other chemicals such as protons that are present in inflamed tissue, particularly by recruiting additional sensory neurons to respond to a given chemical stimulus. An increase in the number of responsive nociceptors that innervate inflamed tissue would contribute to hyperalgesia via spatial summation on spinal neurons in the pathway for pain. Furthermore, since bradykinin enhanced the responsiveness of small-size neurons that responded to protons but not to capsaicin, these data suggest that bradykinin-induced sensitization to protons and capsaicin occur by different mechanisms.     

ELISA detection of IL-1 beta in human sera needs independent confirmation. False positives in hospitalized patients

Electrical field stimulation evoked a reproducible outflow of calcitonin gene-related peptide-like immunoreactivity (CGRP-LI) from the dorsal half of the rat spinal cord, an effect which was abolished by prior application of capsaicin, tetrodotoxin or removal of extracellular Ca2+. Adenosine (EC50 3.2 microM) and the selective adenosine A1 receptor agonist N6-cyclohexyladenosine (EC50 8.2 nM) inhibited evoked CGRP-LI outflow, while the selective adenosine A2 receptor agonist CGS-21680 was ineffective up to 10 microM. The action of adenosine was prevented by the adenosine A1 receptor selective antagonist 8-cyclopentyl-1,3-dipropylxanthine (30 microM), which did not affect CGRP-LI release on its own.     

Metabotropic glutamate receptor antagonists, like GABA(B) antagonists, potentiate dorsal root-evoked excitatory synaptic transmission at neonatal rat spinal motoneurons in vitro

Recordings of whole-cell synaptic current responses elicited by electrical stimulation of dorsal roots were made from motoneurons, identified by antidromic invasion, in isolated spinal cord preparations from five- to eight-day-old Wistar rats. Supramaximal electrical stimulation of the dorsal root evoked complex excitatory postsynaptic currents with mean latencies (+/- S.E.M.) of 6.1 +/- 0.26 ms, peak amplitude of -650 +/- 47 pA and duration of 4.30 +/- 0.46 s (n=34). All phases of excitatory postsynaptic currents were potentiated to approximately 20% above control levels in the presence of the metabotropic glutamate receptor antagonists S-2-amino-2-methyl-4-phosphonobutanoate (MAP4; 200 microM; n=15) and 2S, 1'S,2'S-2-methyl-2-(carboxycyclopropyl)glycine (MCCG; 200 microM; n=9). A similar level of potentiation was produced by the GABA(B) receptor antagonist 3-N[1-(S)-(3,4-dichlorophenyl)ethyl]amino-2-(S)-hydroxypropyl-P-benzyl-p hosphinic acid (CGP55845; 200 nM; n=5). MAP4 (200 microM) produced a six-fold rightward shift in the concentration-effect plot for the depressant action of the metabotropic glutamate receptor agonist S-2-amino-4-phosphonobutanoate (L-AP4), whereas CGP55845 produced no significant change in the potency of L-AP4. MAP4 did not antagonize the depressant actions of baclofen (n=8), 1S,3S-1-aminocyclopentane-1,3-dicarboxylate (n=4) or 2-S,1'S,2'S-2-(carboxycyclopropyl)glycine (n=4). The metabotropic glutamate receptor antagonists produced no change in the holding current of any of the neurons, indicating that they had no significant postsynaptic excitatory actions. These results are the first to indicate a possible physiological role for metabotropic glutamate receptors in the spinal cord. Like GABA(B) receptors, they control glutamatergic synaptic transmission in the segmental spinal pathway to motoneurons. This is likely to be a presynaptic control mechanism.     

Cloning and tissue distribution of two new potassium channel alpha-subunits from rat brain

The release of excitatory amino acids from Schwann cell cultures in the rat was monitored using high-performance liquid chromatography. The basal concentration of glutamate and aspartate was 33 +/- 4 nM (mean +/- S.E.M., n = 12) and 8 +/- 1 nM (mean +/- S.E.M., n = 12), respectively. ATP (100 microM) caused a receptor-mediated increase in release of glutamate and aspartate from Schwann cell cultures. Bath application of adenosine (100 microM) was without effect on release of excitatory amino acids suggesting involvement of P2 receptors. Suramin, a competitive antagonist at P2 receptors, prevented the response to ATP. The release of excitatory amino acids evoked by ATP was not abolished in calcium-depleted saline. Pretreatment of the Schwann cultures with 50 microM 1,2-bis(2-aminophenoxy)ethane-N,N,N'N'-tetracetic acid-acetoxymethyl ester (BAPTA-AM) abolished the effect of ATP. ATP-evoked release of glutamate from cultured Schwann cells was significantly reduced by thapsigargin (1 microM), an inhibitor of Ca(2+)-ATPase of the Ca2+ pump of internal stores. U73122, a selective inhibitor of receptor-coupled phospholipase C-dependent processes, abolished stimulatory effect of ATP suggesting that ATP's action is mediated through an inositol 1,4,5-triphosphate-sensitive calcium store. The action of ATP was not blocked by L-trans-pyrrolidine-2,4-dicarboxylate, an inhibitor of the electrogenic glutamate transporter, nor was it blocked in Na(+)-free medium, and glutamate release was not stimulated by a depolarizing stimulus, suggesting that ATP-evoked release of glutamate from Schwann cells is not due to the reversal of the glutamate uptake. An anion transport blocker, furosemide, reduced ATP-induced glutamate release. These results suggest that ATP-stimulated glutamate and aspartate release from Schwann cells may be through a calcium-dependent furosemide-sensitive mechanism.     

Increasing isoflurane from 0.9 to 1.1 minimum alveolar concentration minimally affects dorsal horn cell responses to noxious stimulation

BACKGROUND: The spinal cord appears to be the site at which isoflurane suppresses movement that occurs in response to a noxious stimulus. In an attempt to localize its site of suppressant action, the authors determined the effect of isoflurane on dorsal horn neuronal responses to supramaximal noxious stimulation at end-tidal concentrations that just permitted and just prevented movement. METHODS: Rats (n = 14) were anesthetized with isoflurane, and after lumbar laminectomy, the minimum alveolar concentration (MAC) for each rat was determined using a supramaximal mechanical stimulus. In these same rats, after extracellular microelectrode placement in the lumbar spinal cord, dorsal horn neuronal responses to the supramaximal stimulus were determined at the concentrations of isoflurane that bracketed each rat's MAC (0.1% higher and lower than MAC). The MAC of isoflurane was then re-determined. RESULTS: Dorsal horn neuronal response was 1,757+/-892 impulses/min at 0.9 MAC and 1,508+/-988 impulses/min at 1.1 MAC, a 14% decrease (P < 0.05). Cell responses varied, with some cells increasing their response at the higher concentration of isoflurane. The MAC of isoflurane was 1.38+/-0.2% before and 1.34+/-0.2% after determination of dorsal horn neuronal responses. CONCLUSIONS: Isoflurane, at concentrations that bracket MAC, has a variable and minimal depressant effect on dorsal horn cell responses to noxious mechanical stimulation. These data suggest that the major action of isoflurane to suppress movement evoked by a noxious stimulus might occur primarily at a site other than the dorsal horn.     

Nociceptin inhibits non-adrenergic non-cholinergic contraction in guinea-pig airway

1. Electrical field stimulation (EFS) of guinea-pig isolated main bronchi induced a non-adrenergic non-cholinergic (NANC) contractile response. Nociceptin (0.01-1 microm) significantly inhibited the contractile response to EFS (P<0.01), but not to capsaicin (P>0.05). 2. The mu-, delta- and kappa-opioid receptor antagonists, naloxone (0.3 microM), naltrindole (3 microM) and norbinaltorphimine (1 microm), respectively, did not significantly affect the inhibitory effect of nociceptin (0.03 microM; P>0.05). 3. The novel nociceptin antagonist, [Phe1psi(CH2-NH)Gly2]nociceptin(1-13)NH2 (0.03-1 microM); the sigma ligands, carbetapentane (30 microM), 3-phenylpiperidine (30-100 microM) and (+)-cyclazocine (10-100 microM) significantly reversed the inhibitory effect of nociceptin (0.03 microM, P<0.05). In contrast, rimcazole, did not significantly reverse the inhibitory effect of nociceptin (0.03 microM) at any concentration tested (P>0.05). 4. EFS of guinea-pig bronchial preparations significantly increased SP-LI release above basal SP-LI (P<0.05). In the presence of nociceptin (1 microM), EFS induced a significant increase in SP-LI release above basal SP-LI release (P<0.05). Nociceptin caused a 59+11% (n=5) inhibition of EFS-induced release of SP-LI. 5. Nociceptin reduces the release of sensory neuropeptides induced by EFS, but not capsaicin, from guinea-pig airways. These experiments provide further evidence for a role for nociceptin in regulating the release of sensory neuropeptides in response to EFS.     

alpha2,3-sialyltransferase mRNA and alpha2,3-linked glycoprotein sialylation are increased in malignant gliomas

Microinjection of a substance P analogue (1 mM; 7 or 10 nl) into laminae I and II of the L7 dorsal horn of decerebrate cats significantly potentiated (P < 0.05) the increase in arterial pressure evoked by microinjection of L-glutamate (109 mM; 7 or 10 nl) into these spinal sites. Microinjection of the substance P analogues (i.e., GR73638 and [Sar9,Met(O2)11]-substance P) which were selective NK-1 receptor agonists, had no impact on the cardioacceleration evoked by microinjection of L-glutamate (P > 0.05). In addition, microinjection of these analogues had no effect on the modest and non-significant increase in phrenic nerve discharge evoked by L-glutamate. We conclude that stimulation of NK-1 receptors in the superficial laminae of the dorsal horn potentiates the pressor responses to microinjection of L-glutamate.     

Slow intrinsic optical signals in the rat spinal dorsal horn in slice

Tetanic stimulation of high-threshold primary afferent fibers in the dorsal root was found to elicit intrinsic optical signals (IOSs) in transverse slices of 11- to 20-day-old rat spinal cords. The IOS, lasting for 30 s or longer, was most prominent in the lamina II of the dorsal horn. Treatment with a Na+-K+-2Cl- co-transport blocker, furosemide, abolished the IOS, suggesting that the origin of the IOS is the cellular swelling due to an activity-dependent rise in extracellular K+. Substance P antagonist spantide, glutamate antagonists 2-amino-5-phosphonovaleric acid and 6-cyano-7-nitroquinoxaline-2,3-dione, and the mu-opioid agonist [d-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin suppressed IOSs. Thus, IOSs represent at least in part the slow excitatory response that is known to be generated in dorsal horn neurons after tetanic activation of unmyelinated afferent fibers.     

Endomorphin-2 is an endogenous opioid in primary sensory afferent fibers

Evidence is presented that the recently discovered endogenous mu-selective agonist, endomorphin-2, is localized in primary sensory afferents. Endomorphin-2-like immunoreactivity was found to be colocalized in a subset of substance P- and mu opiate receptor-containing fibers in the superficial laminae of the spinal cord and spinal trigeminal nucleus. Disruption of primary sensory afferents by mechanical (deafferentation by dorsal rhizotomy) or chemical (exposure to the primary afferent neurotoxin, capsaicin) methods virtually abolished endomorphin-2-like immunoreactivity in the dorsal horn. These results indicate that endomorphin-2 is present in primary afferent fibers where it can serve as the endogenous ligand for pre- and postsynaptic mu receptors and as a major modulator of pain perception.