Src family kinases mediate the inhibition of substance P release in the rat spinal cord by μ‐opioid receptors and GABAB receptors,but not α2 adrenergic receptors

GABA_B, μ‐opioid and adrenergic α₂ receptors inhibit substance P release from primary afferent terminals in the dorsal horn. Studies in cell expression systems suggest that μ‐opioid and GABA_B receptors inhibit transmitter release from primary afferents by activating Src family kinases (SFKs), which then phosphorylate and inhibit voltage‐gated calcium channels. This study investigated whether SFKs mediate the inhibition of substance P release by these three receptors. Substance P release was measured as neurokinin 1 receptor (NK1R) internalization in spinal cord slices and in vivo. In slices, NK1R internalization induced by high‐frequency dorsal root stimulation was inhibited by the μ‐opioid agonist DAMGO and the GABA_B agonist baclofen. This inhibition was reversed by the SFK inhibitor PP1. NK1R internalization induced by low‐frequency stimulation was also inhibited by DAMGO, but PP1 did not reverse this effect. In vivo, NK1R internalization induced by noxious mechanical stimulation of the hind paw was inhibited by intrathecal DAMGO and baclofen. This inhibition was reversed by intrathecal PP1, but not by the inactive PP1 analog PP3. PP1 produced no effect by itself. The α₂ adrenergic agonists medetomidine and guanfacine produced a small but statistically significant inhibition of NK1R internalization induced by low‐frequency dorsal root stimulation. PP1 did not reverse the inhibition by guanfacine. These results show that SFKs mediate the inhibition of substance P release by μ‐opioid and GABA_B receptors, but not by α₂ receptors, which is probably mediated by the binding of G protein βγ subunits to calcium channels.     

Substance P release in the dorsal horn assessed by receptor internalization: NMDA receptors counteract a tonic inhibition by GABA(B) receptors

Inhibitory amino acids have antinociceptive actions in the spinal cord that may involve inhibition of neurotransmitter release from primary afferents. Rat spinal cord slices with dorsal roots were used to study the effect of GABA and glycine on substance P release, assessed by the internalization of neurokinin 1 receptors. After electrical stimulation of the dorsal root at 100 Hz, about half of neurokinin 1 receptor-immunoreactive neurons in laminae I-IIo showed internalization. This internalization was inhibited by GABA (100 microM) and the GABA(B) agonist R-baclofen (10 microM), but not by the GABA(A) agonist muscimol (20 microM) or glycine (100 microM). The GABA(B) antagonist 2-hydroxysaclofen (100 microM) reversed the inhibitory effect of GABA, but not the GABA(A) antagonist bicuculline (100 microM). These findings demonstrate that GABA(B) receptors, but not GABA(A) or glycine receptors, inhibit substance P release induced by dorsal root stimulation. In contrast, R-baclofen did not inhibit the internalization produced by NMDA (100 microM), indicating that the stimulatory effect of NMDA receptors on substance P release is able to surmount the inhibitory effect of GABA(B) receptors. In the presence of the GABA(B) antagonist 2-hydroxysaclofen (100 microM), but not in its absence, stimulation of the dorsal root at 1 or 10 Hz was able to elicit internalization, which was not inhibited by the NMDA receptor antagonist AP-5 (50 microM) or the channel blocker MK-801 (10 microM). Therefore, inhibition of substance P release by GABA(B) receptors is tonic, and in its absence SP release no longer requires NMDA receptor activation.     

Activation of peripheral cannabinoid CB1 receptors inhibits mechanically evoked responses of spinal neurons in noninflamed rats and rats with hindpaw inflammation

The presence of cannabinoid1 (CB1) receptors on primary afferent fibres may provide a novel target for cannabinoid analgesics. The present study investigated the ability of peripheral CB1 receptors to modulate innocuous and noxious transmission in noninflamed rats and rats with peripheral carrageenan inflammation. Effects of peripheral injection of arachidonyl-2-choroethylamide (ACEA; 10 and 30 micro g in 50 micro L), a selective CB1 receptor agonist, on mechanically evoked responses of dorsal horn neurons were studied in noninflamed rats and rats with peripheral carrageenan inflammation. Peripheral injection of ACEA (30 micro g in 50 micro L) significantly inhibited innocuous (12 g) mechanically evoked responses of spinal neurons in noninflamed (27 +/- 4% of control; P < 0.01) and inflamed (12 +/- 8% of control; P < 0.05) rats. Similarly, noxious (80 g) mechanically evoked responses of spinal neurons were inhibited by peripheral injection of ACEA (30 micro g in 50 micro L) in noninflamed rats (51 +/- 9% of control; P < 0.01) and rats with peripheral carrageenan inflammation (21 +/- 8% of control; P < 0.01). Inhibitory effects of ACEA were significantly greater in rats with peripheral carrageenan inflammation than in noninflamed rats (P < 0.05). Inhibitory effects of ACEA were significantly blocked by coadministration of the CB1 receptor antagonist SR141716A in both groups of rats. Peripheral injection of SR141716A alone did not alter mechanically evoked responses of spinal neurons in either group of rats. These data demonstrate that activation of peripheral CB1 receptors can inhibit innocuous and noxious somatosensory processing. Furthermore, following peripheral inflammation there is an enhanced inhibitory effect of a peripherally administered CB1 receptor agonist on both innocuous and noxious mechanically evoked responses of spinal neurons.     

Low-speed subcellular fractionation method for determining noxious stimulus-evoked spinal neurokinin-1 receptor internalization

Substance P release from nociceptive primary afferents activates post-synaptic neurokinin-1 (NK-1) receptors causing subsequent NK-1 receptor internalization. Fluorescent immunohistochemistry is typically used to quantify NK-1 receptor internalization, an indirect measure of substance P (SP) release. However, this technique entails several limitations that restrict its application. Using simple subcellular fractionation and immunoblotting methods, we demonstrate that intrathecal SP invokes a rapid and dose-dependent increase in dorsal horn cytoplasmic NK-1 receptors. We also show that hind paw compression and noxious thermal stimulation increase cytoplasmic NK-1 receptor, when compared to sham stimulations. Fluorescent immunohistochemistry confirmed that increases in cytoplasmic NK-1 corresponded with increased NK-1 receptor internalization. Herein, we report that low-speed centrifugation and Western immunoblotting provide NK-1 internalization results consistent with those obtained by more traditional methods. These data support previous findings demonstrating a role for spinal NK-1 receptors in nociceptive processing.     

Inhibitory effects of CB1 and CB2 receptor agonists on responses of DRG neurons and dorsal horn neurons in neuropathic rats

Cannabinoid 2 (CB2) receptor mediated antinociception and increased levels of spinal CB2 receptor mRNA are reported in neuropathic Sprague-Dawley rats. The aim of this study was to provide functional evidence for a role of peripheral, vs. spinal, CB2 and cannabinoid 1 (CB1) receptors in neuropathic rats. Effects of the CB2 receptor agonist, JWH-133, and the CB1 receptor agonist, arachidonyl-2-chloroethylamide (ACEA), on primary afferent fibres were determined by calcium imaging studies of adult dorsal root ganglion (DRG) neurons taken from neuropathic and sham-operated rats. Capsaicin (100 nm) increased [Ca2+]i in DRG neurons from sham and neuropathic rats. JWH-133 (3 microm) or ACEA (1 microm) significantly (P<0.001) attenuated capsaicin-evoked calcium responses in DRG neurons in neuropathic and sham-operated rats. The CB2 receptor antagonist, SR144528, (1 microm) significantly inhibited the effects of JWH-133. Effects of ACEA were significantly inhibited by the CB1 receptor antagonist SR141716A (1 microm). In vivo experiments evaluated the effects of spinal administration of JWH-133 (8-486 ng/50 microL) and ACEA (0.005-500 ng/50 microL) on mechanically evoked responses of neuropathic and sham-operated rats. Spinal JWH-133 attenuated mechanically evoked responses of spinal neurons in neuropathic, but not sham-operated rats. These inhibitory effects were blocked by SR144528 (0.001 microg/50 microL). Spinal ACEA inhibited mechanically evoked responses of neuropathic and sham-operated rats, these effects were blocked by SR141716A (0.01 microg/50 microL). Our data provide evidence for a functional role of CB2, as well as CB1 receptors on DRG neurons in sham and neuropathic rats. At the level of the spinal cord, CB2 receptors have inhibitory effects in neuropathic, but not sham-operated rats suggesting that spinal CB2 may be an important analgesic target.     

Calcitonin gene-related peptide promotes mechanical nociception by potentiating release of substance P from the spinal dorsal horn in rats

In vitro superfusion with capsaicin (5 X 10(-7) M) of slices of the dorsal half of the rat spinal cord produced a significant increase in a release of immunoreactive substance P (iSP). Calcitonin gene-related peptide (CGRP: 10(-6) M) significantly potentiated the capsaicin-induced release of iSP. On the other hand, when CGRP (5 nmol/rat) was intrathecally injected, the peptide produced a significant hyperalgesia to mechanical noxious stimuli (pinching the hind paw), but aversive responses and potentiation of substance P-induced aversive responses were never observed. These findings suggest that in the rat spinal dorsal horn, CGRP potentiates the release of substance P from the primary afferent terminal and promotes the transmission of nociceptive information induced by mechanical noxious stimuli.     

Opposing roles for cannabinoid receptor type-1 (CB?) and transient receptor potential vanilloid type-1 channel (TRPV1) on the modulation of panic-like responses in rats

The midbrain dorsal periaqueductal gray (dPAG) has an important role in orchestrating anxiety- and panic-related responses. Given the cellular and behavioral evidence suggesting opposite functions for cannabinoid type 1 receptor (CB?) and transient receptor potential vanilloid type-1 channel (TRPV1), we hypothesized that they could differentially influence panic-like reactions induced by electrical stimulation of the dPAG. Drugs were injected locally and the expression of CB? and TRPV1 in this structure was assessed by immunofluorescence and confocal microscopy. The CB?-selective agonist, ACEA (0.01, 0.05 and 0.5?pmol) increased the threshold for the induction of panic-like responses solely at the intermediary dose, an effect prevented by the CB?-selective antagonist, AM251 (75?pmol). Panicolytic-like effects of ACEA at the higher dose were unmasked by pre-treatment with the TRPV1 antagonist capsazepine (0.1?nmol). Similarly to ACEA, capsazepine (1 and 10?nmol) raised the threshold for triggering panic-like reactions, an effect mimicked by another TRPV1 antagonist, SB366791 (1?nmol). Remarkably, the effects of both capsazepine and SB366791 were prevented by AM251 (75?pmol). These pharmacological data suggest that a common endogenous agonist may have opposite functions at a given synapse. Supporting this view, we observed that several neurons in the dPAG co-expressed CB? and TRPV1. Thus, the present work provides evidence that an endogenous substance, possibly anandamide, may exert both panicolytic and panicogenic effects via its actions at CB? receptors and TRPV1 channels, respectively. This tripartite set-point system might be exploited for the pharmacotherapy of panic attacks and anxiety-related disorders.     

Intrathecal administration of sigma-1 receptor agonists facilitates nociception: involvement of a protein kinase C-dependent pathway

Sigma sites, originally proposed as opioid receptor subtypes, are currently thought to represent unique receptors with a specific pattern of drug selectivity, a well-established anatomical distribution and broad range of functional roles including potential involvement in nociceptive mechanisms. We have recently demonstrated that intrathecal (i.t.) treatment with a sigma-1 receptor antagonist reduced formalin-induced pain behavior. In the present study, we investigated the potential role of spinal sigma-1 receptor agonists in peripherally initiated nociception and attempted to elucidate intracellular signaling mechanisms associated with spinal cord sigma-1 receptor activation in mice. The i.t. injection of the sigma-1 receptor agonists PRE-084 (PRE) or carbetapentane (CAR) significantly decreased tail-flick latency (TFL) and increased the frequency of paw withdrawal responses to mechanical stimulation (von Frey filament, 0.6 g) as well as the amount of Fos expression in the spinal cord dorsal horn induced by noxious paw-pinch stimulation. These PRE- or CAR-induced facilitatory effects on nociception were significantly blocked by i.t. pretreatment with the sigma-1 receptor antagonist, BD-1047, the phospholipase C (PLC) inhibitor, U-73,122, the Ca(2+)-ATPase inhibitor, thapsigargin, and the protein kinase C (PKC) inhibitor, chelerythrine. Western blot analysis further revealed that i.t. PRE or CAR injection significantly increased pan-PKC as well as the PKCalpha, epsilon, and zeta isoforms in the dorsal horn. Collectively, these findings demonstrate that calcium-dependent second messenger cascades including PKC are involved in the facilitation of nociception associated with spinal sigma-1 receptor activation.     

De novo expression of neurokinin‐1 receptors by spinoparabrachial lamina I pyramidal neurons following a peripheral nerve lesion

Lamina I of the spinal dorsal horn is a major site of integration and transmission to higher centers of nociceptive information from the periphery. One important primary afferent population that transmits such information to the spinal cord expresses substance P (SP). These fibers terminate in contact with lamina I projection neurons that express the SP receptor, also known as the neurokinin‐1 receptor (NK‐1r). Three types of lamina I projection neurons have been described: multipolar, fusiform, and pyramidal. Most neurons of the first two types are thought to be nociceptive and express the NK‐1r, whereas most pyramidal neurons are nonnociceptive and do not express the NK‐1r. In this immunocytochemical and behavioral study, we induced a neuropathic pain‐like condition in the rat by means of a polyethylene cuff placed around in the sciatic nerve. We document that this lesion led to a de novo expression of NK‐1r on pyramidal neurons as well as a significant increase in SP‐immunoreactive innervation onto these neurons. These phenotypic changes were evident at the time of onset of neuropathic pain‐related behavior. Additionally, we show that, after a noxious stimulus (intradermal capsaicin injection), these NK‐1r on pyramidal neurons were internalized, providing evidence that these neurons become responsive to peripheral noxious stimulation. We suggest that the changes following nerve lesion in the phenotype and innervation pattern of pyramidal neurons are of significance for neuropathic pain and/or limb temperature regulation. J. Comp. Neurol. 521:1915–1928, 2013. © 2012 Wiley Periodicals, Inc.     

Mechanisms of antinociception of spinal galanin: how does galanin inhibit spinal sensitization?

Galanin by a spinal action has been shown to have an antihyperalgesic action. Thus, in rats with lumbar intrathecal (IT) catheters, the thermal hyperalgesia evoked by carrageenan paw injection was blocked by IT delivery of galanin(1-29) (Gal(1-29)) and galanin(2-11) (Gal(2-11)) with the rank order of activity being Gal(1-29)>Gal(2-11). We sought to determine whether this spinal action reflects an effect upon afferent transmitter release, e.g., substance P (SP), and/or on secondary neurons, e.g., signaling postsynaptic to neurokinin 1 (NK1) receptor activation. To address the question on afferent release, we investigated the effect of IT administration of galanin on tissue injury-induced spinal NK1 internalization (an indicator of SP release). Noxious stimulation (paw compression) produced an increase in NK1 internalization in dorsal horn lamina I. IT pretreatment of rats with Gal(1-29) and Gal(2-11) significantly attenuated the evoked NK1 internalization, with the rank order of activity being Gal(1-29)>Gal(2-11)>saline. To address the question of postsynaptic action, we examined the effects of IT galanin upon IT SP-induced thermal hyperalgesia and spinal PGE2 release. Application of SP (30 nmol) directly to spinal cord led to a decrease in thermal thresholds and a profound increase in PGE(2) concentration in spinal dialysates. Both phenomena were reversed by Gal(1-29) and Gal(2-11) (10nmol, IT). These findings suggest that the antihyperalgesic effect of spinal galanin is due to its action on sites both presynaptic (inhibition of SP release) and postsynaptic (blockade of SP-evoked hyperalgesia and PGE2 production) to the primary afferents.     

Cannabinoid CB(1) receptor expression in rat spinal cord

While evidence implicates the endogenous cannabinoid system as a novel analgesic target at a spinal level, detailed analysis of the distribution of the cannabinoid receptor CB(1) in spinal cord has not been reported. Here, immunocytochemical studies were used to characterize the CB(1) receptor expression in rat spinal cord. Staining was found in the dorsolateral funiculus, the superficial dorsal horn (a double band of CB(1) immunoreactivity (ir) in laminae I and II inner/III transition), and lamina X. Although CB(1)-ir was present in the same laminae as primary afferent nociceptor markers, there was limited colocalization at an axonal level. Interruption of both primary afferent input by dorsal root rhizotomy and descending input by rostral spinal cord hemisection produced minor changes in CB(1)-ir. This and colocalization of CB(1)-ir with interneurons expressing protein kinase C subunit gamma-ir suggest that the majority of CB(1) expression is on spinal interneurons. These data provide a framework and implicate novel analgesic mechanisms for spinal actions of cannabinoids at the CB(1) receptor.     

BDNF released during neuropathic pain potentiates NMDA receptors in primary afferent terminals

NMDA receptors in primary afferent terminals can contribute to hyperalgesia by increasing neurotransmitter release. In rats and mice, we found that the ability of intrathecal NMDA to induce neurokinin 1 receptor (NK1R) internalization (a measure of substance P release) required a previous injection of BDNF. Selective knock‐down of NMDA receptors in primary afferents decreased NMDA‐induced NK1R internalization, confirming the presynaptic location of these receptors. The effect of BDNF was mediated by tropomyosin‐related kinase B (trkB) receptors and not p75 neurotrophin receptors (p75^NTR), because it was not produced by proBDNF and was inhibited by the trkB antagonist ANA‐12 but not by the p75^NTR inhibitor TAT‐Pep5. These effects are probably mediated through the truncated form of the trkB receptor as there is little expression of full‐length trkB in dorsal root ganglion (DRG) neurons. Src family kinase inhibitors blocked the effect of BDNF, suggesting that trkB receptors promote the activation of these NMDA receptors by Src family kinase phosphorylation. Western blots of cultured DRG neurons revealed that BDNF increased Tyr¹⁴⁷² phosphorylation of the NR2B subunit of the NMDA receptor, known to have a potentiating effect. Patch‐clamp recordings showed that BDNF, but not proBDNF, increased NMDA receptor currents in cultured DRG neurons. NMDA‐induced NK1R internalization was also enabled in a neuropathic pain model or by activating dorsal horn microglia with lipopolysaccharide. These effects were decreased by a BDNF scavenger, a trkB receptor antagonist and a Src family kinase inhibitor, indicating that BDNF released by microglia potentiates NMDA receptors in primary afferents during neuropathic pain.     

Increased neuronal expression of neurokinin‐1 receptor and stimulus‐evoked internalization of the receptor in the rostral ventromedial medulla of the rat after peripheral inflammatory injury

This study examined possible mechanisms by which Substance P (Sub P) assumes a pronociceptive role in the rostral ventromedial medulla (RVM) under conditions of peripheral inflammatory injury, in this case produced by intraplantar (ipl) injection of complete Freund's adjuvant (CFA). In saline‐ and CFA‐treated rats, neurokinin‐1 receptor (NK1R) immunoreactivity was localized to neurons in the RVM. Four days after ipl injection of CFA, the number of NK1R‐immunoreactive neurons in the RVM was increased by 30%, and there was a concomitant increase in NK1R‐immunoreactive processes in CFA‐treated rats. Although NK1R immunoreactivity was increased, tachykinin‐1 receptor (Tacr1) mRNA was not increased in the RVM of CFA‐treated rats. To assess changes in Sub P release, the number of RVM neurons that exhibited NK1R internalization was examined in saline‐ and CFA‐treated rats following noxious heat stimulation of the hind paws. Only CFA‐treated rats that experienced noxious heat stimulation exhibited a significant increase in the number of neurons showing NK1R internalization. These data suggest that tonic Sub P release is not increased as a simple consequence of peripheral inflammation, but that phasic or evoked release of Sub P in the RVM is increased in response to noxious peripheral stimulation in a persistent inflammatory state. These data support the proposal that an upregulation of the NK1R in the RVM, as well as enhanced release of Sub P following noxious stimulation, underlie the pronociceptive role of Sub P under conditions of persistent inflammatory injury. J. Comp. Neurol. 522:3037–3051, 2014. © 2014 Wiley Periodicals, Inc.     

Insulin-like growth factor-II/Mannose-6-phosphate receptor in the spinal cord and dorsal root ganglia of the adult rat

The insulin-like growth factor-II/mannose-6-phosphate (IGF-II/M6P) receptor is a multifunctional transmembrane glycoprotein, which interacts with a number of molecules, including IGF-II and M6P-containing lysosomal enzymes. The receptor is widely distributed throughout the brain and is known to be involved in lysosomal enzyme trafficking, cell growth, internalization and degradation of IGF-II. In the present study, using autoradiographic, Western blotting and immunocytochemical methods, we provide the first report that IGF-II/M6P receptors are discretely distributed at all major segmental levels of the spinal cord and dorsal root ganglia of the adult rat. In the spinal cord, a high density of [(125)I]IGF-II binding sites was evident in the ventral horn (lamina IX) and in areas around the central canal (lamina X), whereas intermediate grey matter and dorsal horn were associated with moderate receptor levels. The dorsal root ganglia exhibited rather high density of [(125)I]IGF-II binding sites. Interestingly, meninges present around the spinal cord displayed highest density of [(125)I]IGF-II binding compared to any given region of the spinal grey matter or the dorsal root ganglia. Western blot results indicated the presence of the IGF-II/M6P receptor at all major levels of spinal cord and dorsal root ganglia, with little segmental variation. At the cellular level, spinal motorneurons demonstrated the most intense IGF-II/M6P receptor immunoreactivity, followed by interneurons in the intermediate region and deeper dorsal horn. Some scattered IGF-II/M6P immunoreactive fibers were found in the superficial laminae of the dorsal horn and dorsolateral funiculus. The meninges of the spinal cord also seemed to express IGF-II receptor immunoreactivity. In the dorsal root ganglia, receptor immunoreactivity was evident primarily in a subset of neurons of all diameters. These results, taken together, provide anatomical evidence of a role for the IGF-II/M6P receptor in general cellular functions such as transport of lysosomal enzymes and/or internalization followed by clearance of IGF-II in the spinal cord and dorsal root ganglia.     

The effects of intracerebroventricular AM-251, a CB1-receptor antagonist, and ACEA, a CB1-receptor agonist, on penicillin-induced epileptiform activity in rats

Purpose: Several results support the conclusion that the cannabinoid system has a role in generation and cessation of epileptic seizures. The aim of this study was to evaluate the effects of intracerebroventricular AM‐251 [N‐(piperidin‐1‐yl)‐5‐(4‐iodophenyl)‐1‐(2,4‐dichlorophenyl)‐4‐methyl‐1H‐pyrazole‐3‐carboxamide], a CB1‐receptor antagonist, and ACEA (arachidonyl‐2‐chloroethylamide), a CB1‐receptor agonist, on penicillin‐induced epileptiform activity in rats. Methods: In the first set of experiments, 30 min after penicillin injection, AM‐251, at doses of 0.125, 0.25, 0.5, and 1 μg, was administered intracerebroventricularly (i.c.v.). In the second set of experiments, 30 min after penicillin injection, ACEA, at doses of 2.5, 5, 7.5, and 15 μg (i.c.v.), was administered. In the third set of experiments, AM‐251, at doses of 0.125 and 0.25 μg (i.c.v.), was administered 10 min before ACEA (7.5 μg, i.c.v.) injection. Results: ACEA, at a dose of 7.5 μg, significantly decreased the frequency of penicillin‐induced epileptiform activity without changing the amplitude. ACEA, at doses of 2.5, 5, and 15 μg, had no impact on either frequency or amplitude of epileptiform activity. AM‐251, at doses of 0.25 and 0.50 μg, significantly increased the frequency of epileptiform activity. AM‐251, at a dose of 0.25 μg (i.c.v.), was the most effective in changing the frequency of penicillin‐induced epileptiform activity, and it also caused status epilepticus–like activity. AM‐251, at doses of 0.125 and 0.25 μg, 10 min before ACEA (7.5 μg), reversed the anticonvulsant action of ACEA. Discussion: The results of the present study provide electrophysiologic evidence for the role of CB1 receptors in regulating the frequency of epileptiform activity in the model of penicillin‐induced epilepsy. To elucidate the precise mechanism of cannabinoid action in the brain during seizure, more advanced electrophysiologic and neurochemical studies are required.     

Upregulation of neurokinin-1 receptor expression in rat spinal cord by an N-terminal metabolite of substance P

Chronic inflammatory conditions are associated with an upregulation of both substance P (SP) and neurokinin-1 (NK-1) receptors in the dorsal spinal cord. These receptors have been implicated in hyperalgesia as well as stress-induced analgesia. On the basis of the release of SP during chronic pain, and its rapid metabolism, we tested the hypothesis that SP metabolites regulate the synthesis of either SP or NK-1 receptors in the spinal cord. We measured expression of preprotachykinin mRNA and NK-1 receptor mRNA following intrathecally administered substance P(1-7) (SP1-7), the major metabolite of SP in rat, and following capsaicin, a compound known to induce release of endogenous SP. SP(1-7) and capsaicin each increased NK-1 receptor mRNA in the spinal cord (6 h) followed by an increase in NK-1 receptor-immunoreactivity (24 h and 1 week). D-SP(1-7), a D-isomer and antagonist of SP(1-7), did not mimic the effect of SP(1-7), indicating stereoselectivity. Instead, D-SP(1-7) prevented the upregulation of NK-1 receptor immunoreactivity that was induced by capsaicin injected intrathecally, suggesting that the effect of capsaicin is also mediated by SP N-terminal metabolites. In contrast, the decrease in SP synthesis produced by capsaicin was not dependent on SP metabolites as SP(1-7) failed to decrease either preprotachykinin mRNA content in dorsal root ganglia (6 h) or SP immunoreactivity in the lumbar spinal cord (24 h and 1 week). In addition, the effects of capsaicin on SP synthesis were not prevented by D-SP(1-7). Thus, SP metabolites, at times and doses that are antinociceptive, appear to enhance SP-mediated signal transduction by upregulating NK-1 receptor expression without affecting SP synthesis.     

Immunocytochemical localization of the vanilloid receptor 1 (VR1): relationship to neuropeptides, the P2X3 purinoceptor and IB4 binding sites

The vanilloid receptor (VR1) protein functions both as a receptor for capsaicin and a transducer of noxious thermal stimuli. To determine the expression and targetting of this protein, we have generated antisera against both the amino and carboxy termini of VR1. Within the dorsal root and trigeminal ganglia of rats, VR1-immunoreactivity (VR1-ir) was restricted to small and medium sized neurons. VR1-ir was transported into both the central and peripheral processes of these primary afferent neurons, as evidenced by: (i) the presence of VR1-ir in nerve fibres and terminals in lamina I and lamina II of the superficial dorsal horn, and the association of VR1-ir with small diameter nerve fibres in the skin and cornea; (ii) the reduction of VR1-ir in the spinal cord after dorsal rhizotomy; and (iii) the accumulation of VR1-ir proximal to sciatic nerve ligation. At the ultrastructural level, VR1-ir was associated with plasma membranes of neuronal perikarya in dorsal root ganglia and nerve terminals in the dorsal horn. VR1-ir was also seen in nerve fibres and terminals in the spinal trigeminal nucleus and nucleus of the solitary tract. Within a large proportion of dorsal root ganglion neurons and the terminals of their axons, VR1-ir was colocalized with staining for the P2X3 purinoceptor, and with binding sites for the lectin IB4. Surprisingly, VR1-ir did not coexist substantially in nerve fibres and terminals that contain substance P and calcitonin gene-related peptide, suggesting complex mechanisms for the release of these neuropeptides in response to capsaicin application.     

Effects of dorsal rhizotomy on neurokinin receptor sub-types in the rat spinal cord: a quantitative autoradiographic study.

Although abundant evidence suggests a major role for substance P (SP) and other neurokinins (NK) in the transmission of nociceptive information, it is not known whether the various NK receptor classes are differentially located in the substantia gelatinosa of the spinal cord where primary afferent fibres mostly terminate. In order to investigate this issue, we studied the effects of unilateral dorsal rhizotomy on binding of 125I-Bolton-Hunter-SP, (2-[125I]iodohistidyl1)-neurokinin A, and 125I-Bolton-Hunter-eledoisin as respective radioligands for the NK-1, NK-2 and NK-3 receptor sub-types. Seven, 14, 21 and 28 days following unilateral lumbosacral dorsal horn deafferentiation, NK receptor binding parameters were evaluated using quantitative receptor autoradiography. Rhizotomy produced an increase in the densities of NK-1, NK-2 and NK-3 binding sites in the superficial laminae of the dorsal horn. Increases were maximal at 14 days, post-operatively, for both NK-1 and NK-2 sites; slight recovery being observed thereafter. For NK-3 sites, unilateral rhizotomy induced a progressive increase in binding without evidence of recovery over time, at least up to 28 days post-lesion. NK-1 receptor binding parameters around the central canal and in the ventral horn were not affected by the dorsal rhizotomy. These data suggest that all 3 NK receptor classes are located post-synaptically to afferent fiber terminals in laminae I, II and X of the dorsal horn of the spinal cord.     

AM251, a selective antagonist of the CB1 receptor, inhibits the induction of long-term potentiation and induces retrograde amnesia in rats

Long-term potentiation (LTP) has a long history as putative mechanism of memory formation, specially in the hippocampus, a structure essential for memory formation. Endocannabinoids are one of the endogenous systems that modulate this plasticity event: the activation of hippocampal CB1 receptors may inhibit local GABA release. Here, we have studied both (1) the role of the selective CB1 antagonist AM251 upon LTP induction in a hippocampal slice preparation, and (2) the effect of its intrahippocampal administration in the step-down inhibitory avoidance (IA) and the open field habituation tasks (OF). Standard extracellular electrophysiology techniques were used to record field excitatory postsynaptic potentials from the dendritic region of CA1 neurons in response to a high frequency stimulation of Schaffer's collaterals; a micropipette ejected 0.2 microM of AM251 (in DMSO/PBS) 2 min before the stimulus: LTP was induced and lasted more than 30 min in the control, but not in the AM251-treated group. Immediately after training, either in IA (footshock, 0.5 mA) or OF, animals received a bilateral infusion of 0.55 or 5.5 ng/side of AM251 or its vehicle in the CA1 region, and test was performed 24 h later. AM251 has caused a significative decrease in the test step-down latency when compared to the control group, but no differences were detected in the OF task, including the number of crossings, i.e., there were no motor effects. The LTP supression could be caused by AM251 acting over GABAergic interneurons that modulate the LTP-bearing glutamatergic neurons. Endocanabinoids would then be the natural dis-inhibitors of local plasticity in the dorsal hippocampus, and the amnestic action of AM251 would be due to a disruption of this endogenous modulatory system.