Intrathecal high-dose histamine induces spinally-mediated nociceptive behavioral responses through a polyamine site of NMDA receptors

Previous research has demonstrated that a high dose of histamine (1600 pmol) injected i.t. in mice can evoke nociceptive behaviors consisting of biting/licking along with occasional scratching. The present study was undertaken to examine the involvement of spinal N-methyl-d-aspartate (NMDA) and histamine H(1) and H(2) receptors in the nociceptive behaviors evoked by high-dose histamine. Co-administration of the histamine H(1) receptor antagonists, d-chlorpheniramine and pyrilamine, or the histamine H(2) receptor antagonists, ranitidine and zolantidine, failed to suppress the histamine-evoked nociceptive behaviors. Moreover, following histamine administration, nociceptive behaviors in histamine H(1) receptor-knockout and histamine H(2) receptor-knockout mice were indistinguishable from those in wild-type mice, suggesting that histamine-induced nociceptive behaviors are not mediated through histamine H(1) and H(2) receptors in the spinal cord. The histamine-induced nociceptive behaviors were inhibited by co-administration of the competitive NMDA receptor antagonists, d-(-)-2-amino-5-phosphonovaleric acid (D-APV) and 3-((+)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPPA), and the ion channel blocker, (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cycloheptene-5,10-imine maleate (MK-801). Co-administration of ifenprodil, an antagonist for both the polyamine site and the NR2B subunit of NMDA receptors, also inhibited the histamine-induced nociceptive behaviors. (R-[R, S])-alpha-(4-hydroxyphenyl)-beta-methyl-4-(phenylmethyl)-1-piperidinepropanol hydrochloride (Ro25-6981), an antagonist of the NMDA receptor subtype containing the NR2B subunit, did not inhibit histamine-induced nociceptive behaviors, whereas these behaviors were attenuated by pretreatment with an antisense oligodeoxynucleotide against the mRNA for the NR1 subunit of the NMDA receptor. Moreover, agmatine and arcaine, antagonists for a polyamine site on the NMDA receptor, inhibited nociceptive behaviors induced by histamine. These results suggest that a polyamine site on spinal NMDA receptors is involved in eliciting the nociceptive behavioral episode following intrathecal injection of histamine.     

Intrathecally administered D-cycloserine produces nociceptive behavior through the activation of N-methyl-D-aspartate receptor ion-channel complex acting on the glycine recognition site

Intrathecal (i.t.) administration of D-cycloserine (100 and 300 fmol), a partial agonist of the glycine recognition site on the N-methyl-D-aspartate (NMDA) receptor ion-channel complex, produced a behavioral response mainly consisting of biting and/or licking of the hindpaw and the tail along with slight hindlimb scratching directed toward the flank in mice, which peaked at 5 - 10 min and almost disappeared at 15 min after the injection. The behavior induced by D-cycloserine (300 fmol) was dose-dependently inhibited by an intraperitoneal injection of morphine (0.5-2 mg/kg), suggesting that the behavioral response is related to nociception. The nociceptive behavior was also dose-dependently inhibited by i.t. co-administration of 7-chlorokynurenic acid (0.25-4 nmol), a competitive antagonist of the glycine recognition site on the NMDA receptor ion-channel complex; D-(-)-2-amino-5-phosphonovaleric acid (62.5-500 pmol), a competitive NMDA receptor antagonist; MK-801 (62.5-500 pmol), an NMDA ion-channel blocker; ifenprodil (0.5-8 nmol); arcaine (31-125 pmol); and agmatine (0.1-10 pmol), all being antagonists of the polyamine recognition site on the NMDA receptor ion-channel complex. However, [D-Phe7,D-His9]-substance P(6-11), a specific antagonist for substance P (NK1) receptors, and MEN-10,376, a tachykinin NK2-receptor antagonist, had no effect on D-cycloserine-induced nociceptive behavior. These results in the mouse spinal cord suggest that D-cycloserine-induced nociceptive behavior is mediated through the activation of the NMDA receptor ion-channel complex by acting on the glycine recognition site and that it does not involve the tachykinin receptor mechanism.     

NR2B containing NMDA receptor dependent windup of single spinal neurons

Windup, the frequency dependent build-up of spinal neuronal responses, is implicated in the development of central sensitization of nociceptive pathways. N-methyl-D-aspartate (NMDA) receptors have been shown to be involved in these processes but the role of various receptor subtypes at the spinal level is not fully understood. In our experiments, we compared the inhibitory effect of MK-801 (a nonselective NMDA receptor antagonist, 0.01-3 mg/kg i.v.) and CI-1041 (an NR2B subunit specific NMDA receptor antagonist, 0.3-10 mg/kg i.v.) on the formation of dorsal horn neuronal windup in spinalized rats, in vivo. Both types of antagonist blocked windup considerably at doses not affecting the normal synaptic transmission. These results are in agreement with the well-documented effectivity of NR2B subtype selective NMDA receptor antagonists in chronic pain models and give the first direct evidence that spinal mechanisms are involved in this effect.     

Possible involvement of tachykinin NK(1) and NMDA receptors in histamine-induced hyperalgesia in mice.

Intrathecal (i.t.) injection of histamine elicited a significant hyperalgesic response as assayed by the tail-flick test. This hyperalgesic effect peaked at 15 min following i.t. administration of histamine (800 pmol) and returned to control level with 30 min. Hyperalgesia produced by histamine was inhibited dose-dependently by i.t. co-administration of the histamine H(1) receptor antagonist, d-chlorpheniramine, but not the histamine H(2) receptor antagonist, ranitidine. The tachykinin NK(1) receptor antagonists, (+)-[(2S,3S)-3-(2-methoxy-benzyl-amino)-2-phenylpiperidine] (CP-99,994), and [Tyr(6), D-Phe(7), D-His(9)]substance P-(6-11) (sendide), inhibited histamine-induced hyperalgesic response in a dose-dependent manner. A significant antagonistic effect of [D-Phe(7), D-His(9)]substance P-(6-11), a selective antagonist for substance P receptors, was observed against histamine-induced hyperalgesic response. The tachykinin NK(2) receptor antagonist, Asp-Tyr-D-Trp-Val-D-Trp-D-Trp-Lys-NH(2) (MEN-10,376), had no effect on hyperalgesia elicited by histamine. The competitive N-methyl-D-aspartate (NMDA) receptor antagonists, and D-(-)-2-amino-5-phosphonovaleric acid (D-APV), (+/-)-3-(2-carboxypiperazin-yl)propyl-1-phosphoric acid (CPP), the noncompetitive NMDA receptor antagonist dizocilpine (MK-801), and L-N(G)-nitro arginine methyl ester (L-NAME), a nitric oxide (NO) synthase inhibitor, markedly inhibited histamine-induced hyperalgesic response. The present results suggest that hyperalgesic response induced by i.t. injection of histamine may be mediated by tachykinin NK(1) receptors, but not NK(2) receptors in the spinal cord. In addition, spinal NMDA receptor-NO system may also contribute to elicitation of hyperalgesia following i.t. injection of histamine.     

Spinal ERK activation via NO-cGMP pathway contributes to nociceptive behavior induced by morphine-3-glucuronide

Intrathecal (i.t.) injection of morphine-3-glucuronide (M3G), a major metabolite of morphine without analgesic actions, produces a severe hindlimb scratching followed by biting and licking in mice. The pain-related behavior evoked by M3G was inhibited dose-dependently by i.t. co-administration of tachykinin NK₁ receptor antagonists, sendide, [D-Phe⁷, D-His⁹] substance P(6-11), CP-99994 or RP-67580 and i.t. pretreatment with antiserum against substance P. The competitive NMDA receptor antagonists, D-APV and CPP, the NMDA ion-channel blocker, MK-801 or the competitive antagonist of the polyamine recognition site of NMDA receptor ion-channel complex, ifenprodil, produced inhibitory effects on i.t. M3G-evoked nociceptive response. The NO-cGMP-PKG pathway, which involves the extracellular signal-regulated kinase (ERK), has been implicated as mediators of plasticity in several pain models. Here, we investigated whether M3G could influence the ERK activation in the NO-cGMP-PKG pathway. The i.t. injection of M3G evoked a definite activation of ERK in the lumbar dorsal spinal cord, which was prevented dose-dependently by U0126, a MAP kinase-ERK inhibitor. The selective nNOS inhibitor N^ω-propyl-l-arginine, the selective iNOS inhibitor W1400, the soluble guanylate cyclase inhibitor ODQ and the PKG inhibitor KT-5823 inhibited dose-dependently the nociceptive response to i.t. M3G. In western blotting analysis, inhibiting M3G-induced nociceptive response using these inhibitors resulted in a significant blockade of ERK activation induced by M3G in the spinal cord. Taken together, these results suggest that activation of the spinal ERK signaling in the NO-cGMP-PKG pathway contributes to i.t. M3G-evoked nociceptive response.     

Enhancement of NMDA-induced functional responses without concomitant NMDA receptor changes following chronic ethanol exposure in cerebellar granule cells

Primary cultures of rat cerebellar granule cells were used to investigate the effects of chronic ethanol exposure (50-100 mM for 3 days) on NMDA receptor functions (Ca2+ fluxes and neurotoxicity), binding parameters of the non-competitive NMDA receptor antagonist [3H]MK-801, relative abundance of mRNAs coding for NMDA receptor subunits, and expression of NMDA receptor subunit proteins. Ethanol exposure caused a marked increase in NMDA-produced neurotoxicity but produced a differential pattern of effects on NMDA-induced Ca2+ fluxes with a marked enhancement of NMDA-stimulated free cytoplasmic Ca2+ concentrations ([Ca2+]i), but no changes in NMDA-induced 45Ca2+ uptake. As shown by [3H]MK-801 binding experiments, chronic ethanol had no effect on affinity or number of the NMDA receptors. Furthermore, ethanol exposure had no effect on the relative abundance of the mRNAs for any of the NMDA receptor subunits (four splice variants of NR1, or NR2A-C), or on the expression of NMDA receptor subunit proteins. Our data confirm previous observations that chronic ethanol exposure enhances NMDA receptor-mediated neurotoxicity and elevation of [Ca2+]i, but also suggest that the increased responsiveness of NMDA receptors is not necessarily associated with alterations in the subunit composition or the ligand binding properties of NMDA receptors.     

Supraspinal vs spinal sites of the antinociceptive action of the subtype-selective NMDA antagonist ifenprodil

The N-methyl-D-aspartate (NMDA) antagonist ifenprodil and several structurally related compounds are highly selective for the NR2B-containing receptor subtype. This selectivity could provide an explanation for the reported difference of the analgesic and side-effect profile of ifenprodil-like compounds from other NMDA antagonists. In this work, we have queried if the ifenprodil-induced antinociception can be attributed to the block of NMDA receptors in the spinal cord. Ifenprodil and some other NMDA antagonists (MK-801, memantine) were tested in a model of inflammatory pain (Randall-Selitto) in rats. The in vivo NMDA antagonism was assessed in anaesthetised rats on responses of spinal dorsal horn (DH) neurones to iontophoretic NMDA and in the model of single motor unit (SMU) wind-up. Ifenprodil, MK-801 and memantine dose-dependently increased nociceptive thresholds in the Randall-Selitto model. Antinociceptive doses of the channel blockers selectively antagonised NMDA responses of DH neurones and inhibited wind-up. In contrast, antinociceptive doses of ifenprodil did not show any NMDA antagonism in electrophysiological tests. Although ifenprodil did not inhibit the SMU responses to noxious stimuli in spinalised rats, it markedly and dose-dependently inhibited nociceptive SMU responses in sham-spinalised rats. These results argue against the spinal cord being the principal site of antinociceptive action of ifenprodil; supraspinal structures seem to be involved in this effect.     

Effect of MK-801 on the antinociceptive effect of [D-Ala(2),N-MePhe(4), Gly-ol(5)]enkephalin in diabetic mice

The role of N-methyl-D-aspartate (NMDA) receptors in supraspinal and spinal sites on the reduction of supraspinal micro-opioid receptor-induced antinociception in diabetic mice was examined. The antinociceptive effect of i.c.v. [D-Ala(2), N-MePhe(4), Gly-ol(5)]enkephalin (DAMGO, 20 pmol) in diabetic mice was significantly less than that in non-diabetic mice. The antinociceptive effect of i.c.v. DAMGO (20 pmol) was significantly and dose dependently reduced by i.c.v. (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801) in both non-diabetic (0.03-0.3 nmol) and diabetic mice (0.1-3.0 nmol). While the antinociceptive effect of i.c.v. DAMGO (10 pmol) was significantly enhanced by i.c.v NMDA (0.01-0.1 nmol) in non-diabetic mice, the same doses of i.c.v. NMDA had no significant effect on the antinociceptive effect of i.c.v. DAMGO (20 pmol) in diabetic mice. In non-diabetic mice, the antinociceptive effect of DAMGO (20 pmol, i.c.v.) was dose dependently reduced by intrathecal administration of MK-801 (0.1-1.0 nmol). The antinociceptive effect of DAMGO (20 pmol, i.c.v.) was dose-dependently enhanced by MK-801 (0.1-1.0 nmol, i.t.) in diabetic mice. Furthermore, NMDA (0.1 nmol, i.t.) significantly enhanced the antinociceptive effect of DAMGO (10 pmol, i.c.v.) in non-diabetic mice. However, in non-diabetic mice, the antinociceptive effect of DAMGO (40 pmol, i.c.v.) was dose dependently reduced by NMDA (0.03-0.3 nmol, i.t.). These results suggest that NMDA receptor function in supraspinal and spinal sites appear to be modulated differently by the diabetic state, and this functional modulation may play an important role in the reduction of supraspinal micro-opioid receptor-induced antinociception in diabetic animals.     

Up-regulation of glutamate receptors in nucleus tractus solitarii underlies potentiation of baroreceptor reflex by heat shock protein 70

Whereas induction of the 70-kDa heat shock protein (HSP70) in the nucleus tractus solitarii (NTS), the terminal site in the brain stem for primary baroreceptor afferents, augments baroreceptor reflex (BRR) response, the underlying cellular and molecular mechanism is essentially unexplored. In Sprague-Dawley rats, we evaluated the hypothesis that HSP70 may potentiate BRR response by up-regulating the molecular synthesis and functional expression of glutamate receptors in the NTS. Animals subjected to brief hyperthermic heat shock (HS; 42 degrees C for 15 min) exhibited augmented expression of NR1 or NR2A subunit of N-methyl-D-aspartate (NMDA) receptors, GluR1 or GluR4 subunits of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate receptors and KA1 subunit of kainate receptors in the NTS. Intriguingly, this up-regulation of glutamate receptors was preceded by an increase in HSP70 expression at the NTS. The HS-induced augmentation in responsiveness of barosensitive NTS neurons to transient hypertension or potentiation of BRR response was discernibly blunted by MK-801 or 6-cyano-7-nitroquinoxaline-2,3-dione. Bilateral microinjection into the NTS of an antisense hsp70 oligonucleotide (50 pmol) before HS significantly suppressed the induced expression of HSP70 or the increase in glutamate receptor subunits in the dorsal medulla and discernibly attenuated the potentiation of BRR response. Control microinjection into the NTS of sense or scrambled hsp70 oligonucleotide (50 pmol) was ineffective. These findings suggest that HSP70 induced by HS may enhance BRR response by up-regulating the molecular synthesis and functional expression of NR1 or NR2A subunit of NMDA receptors and GluR1, GluR4, or KA1 subunit of non-NMDA receptors in the NTS.     

胍丁胺对吗啡长期处理引起的NMDA受体蛋白表达改变的影响

目的:观察胍丁胺对吗啡长期处理引起的NMDA受体蛋白改变的影响。方法:采用吗啡递增给药制备大鼠慢性依赖模型,并观察依赖状态下大鼠海马和伏隔核NMDA受体NR1和NR2B亚基蛋白表达量的变化,以及胍丁胺对吗啡作用的影响。结果:与对照组相比,吗啡慢性处理大鼠在纳洛酮催促下能出现典型的戒断综合征,提示依赖模型建立成功。用免疫印记(Western blotting)技术发现,海马部位的NR2B亚基明显下调,而NR1亚基未见显著性变化;吗啡慢性处理不引起伏隔核NR2B亚基的明显变化,但NR1亚基却显著上调。胍丁胺与吗啡伴随给药能逆转吗啡对两脑区NMDA受体蛋白表达的调节作用。结论:胍丁胺调节阿片依赖可能与其逆转吗啡对NMDA受体亚基数量和构成的调节有关。     

Nucleus accumbens NMDA receptor subunit expression and function is enhanced in morphine-dependent rats

We have previously shown, using radioligand binding studies, that N-methyl-d-aspartate (NMDA) NR1 and NR2A receptor subunits density was decreased in the forebrain of morphine-dependent rats. We have now determined if morphine-dependent rats display regional differences in NMDA receptor expression and whether such changes are functionally relevant. In morphine-dependent rats, the expression of NR1 and NR2A subunits protein, as determined by Western blotting with NMDA receptor subunit antibodies, were decreased in frontal cortex and hippocampus but significantly increased in the nucleus accumbens. The expression of the NR2B subunit was unchanged in all regions examined. In separate groups of morphine-dependent rats, MK-801-induced hyperactivity (thought to be mediated via modulation of nucleus accumbens dopamine release) was significantly enhanced in morphine-dependent animals. Similarly, the MK-801-induced increase of dopamine metabolism was significantly increased in the nucleus accumbens of morphine-dependent animals as compared to sham controls. Results provide both biochemical and behavioural evidence to suggest that NMDA receptor function in the nucleus accumbens, at least with respect to an interaction with the limbic dopamine system, is markedly enhanced in morphine-dependent rats. This increase in function may be associated with an enhanced expression of NMDA receptors, particularly those in the nucleus accumbens containing the NR2A subunit. Taken together, these data support several studies in the literature indicating that NMDA receptors in the nucleus accumbens are involved in the process of opiate dependence.     

NR2B subunit-specific NMDA antagonist Ro25-6981 inhibits the expression of conditioned fear: a comparison with the NMDA antagonist MK-801 and fluoxetine

N-methyl-D-asparate (NMDA)-mediated glutamatergic neurotransmission is strongly involved in the development of trauma-induced behavioral dysfunctions, and indirect evidence suggests that NR2B subunit-expressing NMDA receptors are primarily involved in this process. Earlier studies showed that NR2B blockers inhibit the acquisition of conditioned fear, a frequently used model of post-traumatic stress disorder, but their effects on the expression of conditioned fear was poorly studied. We investigated here the effects of the selective serotonin reuptake blocker, fluoxetine, the NMDA blocker, MK-801, and the NR2B subunit blocker, Ro25-6981 on the expression of conditioned fear. Rats received 10 foot shocks administered over 5 min and were tested 24 h later in the shocking context. Treatments were administered 1 h before testing. Shocks dramatically increased freezing and reduced exploration. MK-801 and Ro25-6981 significantly ameliorated both changes. The effects of fluoxetine were less pronounced. In the open field, MK-801 increased locomotion, ataxia, and stereotypy (effects typical of NMDA blockade). Neither fluoxetine nor Ro25-6981 affected locomotion in the open field. Thus, the NR2B-specific NMDA blockade preserved the beneficial effects of general NMDA antagonists on the expression of conditioned fear but did not produce the locomotor side-effects typical of the latter. These findings warrant further studies on the effects of NR2B antagonists in models of post-traumatic stress disorder.     

Spinal cord ionotropic glutamate receptors function in formalin-induced nociception in preweaning rats

Rationale Neonates respond to noxious stimuli at or before birth, but the organization of nociceptive systems changes well into postnatal life. It is unknown how nociceptive information is processed in the immature animal and, specifically, whether noxious stimulation is transmitted by glutamatergic circuits, known to play an important role in nociception in the adult. Both N-methyl-d-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are found within the neonatal spinal cord, but in immature form, and when they become involved in pain processing in vivo is not known. Objectives The objective was to determine the age-related changes in the involvement of spinal NMDA and AMPA receptors in formalin-induced nociception during early life. Because the formalin test provides a measure of immediate nociceptive responding (first phase) and of peripheral and central sensitization (second phase), a second aim was to determine if there is specificity of the effects to either phase. Materials and methods NMDA antagonists (MK801, AP5) or an AMPA antagonist (YM872) was administered intrathecally, and pups were assessed in the formalin test behaviorally and by Fos expression within the spinal cords of 3-, 10-, and 21-day-old rats. Results The NMDA antagonists attenuated formalin-induced behavioral responses at the youngest age tested with some selectivity for the second phase of responding. MK-801 did not induce motor impairment at any age. YM872 also attenuated formalin-induced nociceptive responses at all ages throughout the test session, although there was some motor impairment in the 3-day-old subjects. Spinal administration of either YM872 or MK-801 reduced Fos expression in the spinal cord at all ages. Conclusions These data suggest that spinal NMDA and AMPA receptor are functional and involved in formalin-induced nociception throughout development.     

Protection by imidazol(ine) drugs and agmatine of glutamate-induced neurotoxicity in cultured cerebellar granule cells through blockade of NMDA receptor.

This study was designed to assess the potential neuroprotective effect of several imidazol(ine) drugs and agmatine on glutamate-induced necrosis and on apoptosis induced by low extracellular K+ in cultured cerebellar granule cells. Exposure (30 min) of energy deprived cells to L-glutamate (1-100 microM) caused a concentration-dependent neurotoxicity, as determined 24 h later by a decrease in the ability of the cells to metabolize 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) into a reduced formazan product. L-glutamate-induced neurotoxicity (EC50=5 microM) was blocked by the specific NMDA receptor antagonist MK-801 (dizocilpine). Imidazol(ine) drugs and agmatine fully prevented neurotoxicity induced by 20 microM (EC100) L-glutamate with the rank order (EC50 in microM): antazoline (13)>cirazoline (44)>LSL 61122 [2-styryl-2-imidazoline] (54)>LSL 60101 [2-(2-benzofuranyl) imidazole] (75)>idazoxan (90)>LSL 60129 [2-(1,4-benzodioxan-6-yl)-4,5-dihydroimidazole](101)>RX82 1002 (2-methoxy idazoxan) (106)>agmatine (196). No neuroprotective effect of these drugs was observed in a model of apoptotic neuronal cell death (reduction of extracellular K+) which does not involve stimulation of NMDA receptors. Imidazol(ine) drugs and agmatine fully inhibited [3H]-(+)-MK-801 binding to the phencyclidine site of NMDA receptors in rat brain. The profile of drug potency protecting against L-glutamate neurotoxicity correlated well (r=0.90) with the potency of the same compounds competing against [3H]-(+)-MK-801 binding. In HEK-293 cells transfected to express the NR1-1a and NR2C subunits of the NMDA receptor, antazoline and agmatine produced a voltage- and concentration-dependent block of glutamate-induced currents. Analysis of the voltage dependence of the block was consistent with the presence of a binding site for antazoline located within the NMDA channel pore with an IC50 of 10-12 microM at 0 mV. It is concluded that imidazol(ine) drugs and agmatine are neuroprotective against glutamate-induced necrotic neuronal cell death in vitro and that this effect is mediated through NMDA receptor blockade by interacting with a site located within the NMDA channel pore.     

An increase in spinal dehydroepiandrosterone sulfate (DHEAS) enhances NMDA-induced pain via phosphorylation of the NR1 subunit in mice: Involvement of the sigma-1 receptor

Our laboratory has recently demonstrated that an increase in the spinal neurosteroid, dehydroepiandrosterone sulfate (DHEAS) facilitates nociception via the activation of sigma-1 receptors and/or the allosteric inhibition GABA_A receptors. Several lines of evidence have suggested that DHEAS positively modulates N-methyl-d-aspartate (NMDA) receptor activity within the central nervous system. Moreover, we have demonstrated that the activation of sigma-1 receptors increases NMDA receptor activity. Since NMDA receptors play a key role in the enhancement of pain perception, the present study was designed to determine whether spinally administered DHEAS modulates NMDA receptor-mediated nociceptive activity and whether this effect is mediated by sigma-1 or GABA_A receptors. Intrathecal (i.t.) DHEAS was found to significantly potentiate i.t. NMDA-induced spontaneous pain behaviors. Subsequent immunohistochemical analysis demonstrated that i.t. DHEAS also increased protein kinase C (PKC)- and protein kinase A (PKA)-dependent phosphorylation of the NMDA receptor subunit NR1 (pNR1), which was used as a marker of NMDA receptor sensitization. The sigma-1 receptor antagonist, BD-1047, but not the GABA_A receptor agonist, muscimol, dose-dependently suppressed DHEAS’s facilitatory effect on NMDA-induced nociception and pNR1 expression. In addition, pretreatment with either a PKC or PKA blocker significantly reduced the facilitatory effect of DHEAS on NMDA-induced nociception. Conversely the GABA_A receptor antagonist, bicuculline did not affect NMDA-induced pain behavior or pNR1 expression. The results of this study suggest that the DHEAS-induced enhancement of NMDA-mediated nociception is dependent on an increase in PKC- and PKA-dependent pNR1. Moreover, this effect of DHEAS on NMDA receptor activity is mediated by the activation of spinal sigma-1 receptors and not through the inhibition of GABA_A receptors.     

Channel blockers acting at N-methyl-D-aspartate receptors: differential effects of mutations in the vestibule and ion channel pore

A large number of structurally diverse compounds act as open-channel blockers of NMDA receptors. They may share discrete or overlapping binding sites within the channel. In this study, the effects of mutations in and around the membrane-spanning and pore-forming regions of NMDA receptor subunits were studied with three blockers, MK-801, memantine, and TB-3-4, using recombinant NMDA receptors expressed in Xenopus laevis oocytes. Mutations at the critical asparagine residues in the M2 loop of NR1 and NR2B and at a tryptophan residue in M2 of NR2B reduced block by MK-801, memantine, and TB-3-4. Mutations at residues in the pre-M1, M1, M3, post-M3, and post-M4 regions had differential effects on the three blockers. Many mutations in these regions reduced block by MK-801 and TB-3-4 but had no effect on block by memantine. The differential effects on block by memantine and MK-801 are unlikely to be caused by differences in the size of these blockers. Benzyl rings in MK-801 and TB-3-4 may make hydrophobic interactions with aromatic and hydrophobic amino acid residues in the pore. Some mutations in the pre-M1 and M3 regions generated constitutively open channels, characterized by large holding currents. The effects of the various mutants are discussed in the context of models based on the known structure of the pore of the KcsA potassium channel and on previous studies dealing with solvent accessible residues in NMDA receptor subunits as determined by modification after cysteine mutagenesis.     

Delayed postoperative latent pain sensitization revealed by the systemic administration of opioid antagonists in mice

The long-lasting post-surgical changes in nociceptive thresholds in mice, indicative of latent pain sensitization, were studied. The contribution of kappa opioid and N-methyl-d-aspartate (NMDA) receptors was assessed by the administration of nor-binaltorphimine or MK-801; dynorphin levels in the spinal cord were also determined. Animals underwent a plantar incision and/or a subcutaneous infusion of remifentanil (80μg/kg), and mechanical thresholds (von Frey) were evaluated at different times. On day 21, after complete recovery of mechanical thresholds and healing of the wound, one of the following drugs was administered subcutaneously: (-)-naloxone (1mg/kg), (+)-naloxone (1mg/kg), naloxone-methiodide (3mg/kg), or nor-binaltorphimine (5mg/kg). Another group received subcutaneous MK-801 (0.15mg/kg) before nor-binaltorphimine administration. Dynorphin on day 21 was determined in the spinal cord by immunoassay. In mice receiving remifentanil during surgery, the administration of (-)-naloxone or nor-binaltorphimine induced significant hyperalgesia even 5months after manipulation. Nociceptive thresholds remained unaltered after (+)-naloxone or naloxone-methiodide. On day 21 after manipulation, the administration of MK-801 prevented nor-binaltorphimine-induced hyperalgesia. No changes in dynorphin levels were observed before or after opioid antagonist administration. In conclusion, surgery produced latent pain sensitization evidenced by opioid antagonist-precipitated hyperalgesia. The effect was stereospecific, centrally originated, and mediated by kappa opioid receptors. The blockade of nor-binaltorphimine-induced hyperalgesia by MK-801, suggests that NMDA receptors are also involved. Our results show for the first time that surgery induces latent, long-lasting changes in the processing of nociceptive information that can be induced by non-nociceptive stimuli such as the administration of opioid antagonists.     

Involvement of spinal NK2 and NMDA receptors in aversive behavior induced by intra-arterial injection of capsaicin

The spinal processing by which intra-arterial injection of capsaicin (CAP) induces vocalization response (VOR) was investigated in guinea pigs. Intrathecal pre-treatment with CP-96,345 (a selective NK(1) receptor antagonist, 50 nmol) did not affect the CAP-induced VOR. However, significant attenuation of the VOR was observed by intrathecal pre-treatment with a selective NK(2) receptor antagonist MEN-10,376 (40 nmol) accompanied with a significant change in the response modality. MK-801 [an N-methyl-D-aspartate (NMDA) receptor antagonist, 20 and 40 nmol] inhibited the CAP-induced VOR dose-dependently without affecting the response modalities. Furthermore, intrathecal co-treatment with 40-nmol MEN-10,376 and 40-nmol MK-801 resulted in a marked inhibitory effect on the VOR followed by a significant alteration of response modalities. Intrathecal pre-treatment with neurokinin A (NKA; a tachykinin NK(2) receptor agonist, 1 nmol) enhanced the CAP-induced VOR. These behavioral results suggested that spinal NK(2) and NMDA receptors might have priority over NK(1) receptors in the spinal processing of nociceptive information from the CAP-sensitive nociceptor.     

Increased expression of neuronal Src and tyrosine phosphorylation of NMDA receptors in rat brain after systemic treatment with MK-801

We have observed that systemic treatment with the uncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 increases Src expression and NMDA receptor phosphorylation in rat brain. A partial cDNA encoding rat neuronal Src was isolated and its sequence was used to design specific oligonucleotide probes. Systemically administered MK-801 (5 mg/kg for 4 h) increased by 28+/-4% mRNA expression of neuronal Src in the superficial layers of the parietal cortex. This effect was observed at doses as low as 0.2 mg/kg. A similar, although more modest, induction was observed 6 h after phencyclidine (15 mg/kg) administration, but not after high doses of memantine and ketamine. The MK-801-induced effect was not blocked by pretreatment with clozapine. Consistent with the increase in mRNA levels, cortical Src protein was increased to 186 +/- 24% of control 24 h after MK-801 treatment. Total cellular Src activity was also increased in parietal cortex homogenates 4 h after MK-801 (5 mg/kg). Moreover, MK-801 treatment (0.5 mg/kg and 5 mg/kg for 4 h) increased tyrosine phosphorylation, but not protein levels, of the NMDA receptor subunit NR2A. These results provide evidence for a contribution of Src and tyrosine phosphorylation of NMDA receptors in the pharmacological actions of uncompetitive NMDA receptor antagonists.     

Histamine ameliorates spatial memory deficits induced by MK-801 infusion into ventral hippocampus as evaluated by radial maze task in rats

AIM: To investigate the role of histamine in memory deficits induced by MK-801 infusion into the ventral hippocampus in rats. METHODS: An 8-arm radial maze (4 arms baited) was used to assess spatial memory. RESULTS: Bilateral ventral intrahippocampal (ih) infusion of MK-801 (0.3 microg/site), an N-methyl-D-aspartate (NMDA) antagonist, impaired the retrieval process in both working memory and reference memory. Intrahippocampal injection of histamine (25 or 50 ng/site) or intraperitoneal (ip) injection of histidine (25, 50 or 100 mg/kg) markedly ameliorated the spatial memory deficits induced by MK-801. Both the histamine H1 antagonist pyrilamine (0.5 or 1.0 microg/site, ih) and the H2 antagonist cimetidine (2.5 microg/site, ih) abolished the ameliorating effect of histidine (100 mg/kg, ip) on reference memory deficits, but not that on working memory deficits induced by MK-801. CONCLUSION: The results indicate that histamine in the ventral hippocampus can ameliorate MK-801-induced spatial memory deficits, and that histamine's effect on reference memory is mediated by postsynaptic histamine H1 and H2 receptors.