Control of glycinergic input to spinal dorsal horn neurons by distinct muscarinic receptor subtypes revealed using knockout mice

Muscarinic acetylcholine receptors (mAChRs) play an important role in the tonic regulation of nociceptive transmission in the spinal cord. However, how mAChR subtypes contribute to the regulation of synaptic glycine release is unknown. To determine their role, glycinergic spontaneous inhibitory postsynaptic currents (sIPSCs) were recorded in lamina II neurons by using whole-cell recordings in spinal cord slices of wild-type (WT) and mAChR subtype knockout (KO) mice. In WT mice, the mAChR agonist oxotremorine-M dose-dependently decreased the frequency of sIPSCs in most neurons, but it had variable effects in other neurons. In contrast, in M3-KO mice, oxotremorine-M consistently decreased the glycinergic sIPSC frequency in all neurons tested, and in M2/M4 double-KO mice, it always increased the sIPSC frequency. In M2/M4 double-KO mice, the potentiating effect of oxotremorine-M was attenuated by higher concentrations in some neurons through activation of GABA(B) receptors. In pertussis toxin-treated WT mice, oxotremorine-M also consistently increased the sIPSC frequency. In M2-KO and M4-KO mice, the effect of oxotremorine-M on sIPSCs was divergent because of the opposing functions of the M3 subtype and the M2 and M4 subtypes. This study demonstrates that stimulation of the M2 and M4 subtypes inhibits glycinergic inputs to spinal dorsal horn neurons of mice, whereas stimulation of the M3 subtype potentiates synaptic glycine release. Furthermore, GABA(B) receptors are involved in the feedback regulation of glycinergic synaptic transmission in the spinal cord. This study revealed distinct functions of mAChR subtypes in controlling glycinergic input to spinal dorsal horn neurons.     

Functional activity of the M2 and M4 receptor subtypes in the spinal cord studied with muscarinic acetylcholine receptor knockout mice

Stimulation of spinal muscarinic acetylcholine receptors (mAChRs) produces potent analgesia. Both M(2) and M(4) mAChRs are coupled to similar G proteins (G(i/o) family) and play a critical role in the analgesic action of mAChR agonists. To determine the relative contribution of M(2) and M(4) subtypes to activation of G(i/o) proteins in the spinal cord, we examined the receptor-mediated guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPgammaS) binding in M(2) and M(4) subtype knockout (KO) mice. Basal [(35)S]GTPgammaS binding in the spinal cord was similar in the wild-type controls, M(2) and M(4) single-KO, and M(2)/M(4) double-KO mice. The spinal [(35)S]GTPgammaS binding stimulated by either muscarine or oxotremorine-M was not significantly different among three groups of wild-type mouse strains. In M(2) single-KO and M(2)/M(4) double-KO mice, the agonist-stimulated [(35)S]GTPgammaS binding was completely abolished in the spinal cord. Furthermore, the agonist-stimulated [(35)S]GTPgammaS binding in the spinal cord of M(4) single-KO mice was significantly reduced ( approximately 15%), compared with that in wild-type controls. On the other hand, the spinal [(35)S]GTPgammaS binding stimulated by a mu-opioid agonist was not significantly different between wild-type and M(2) and M(4) KO mice. This study provides complementary new evidence that M(2) is the most predominant mAChR subtype coupled to the G(i/o) proteins in the spinal cord. Furthermore, these data suggest that a small but functionally significant population of M(4) receptors exists in the mouse spinal cord. The functional activity of these M(4) receptors seems to require the presence of M(2) receptors.     

人食管下括约肌M2和M3受体的基因表达

目的 探讨人食管下括约肌的胆碱能受体基因表达和分布的情况，从分子水平揭示食管下括约肌的抗返流机制。方法 应用RT-PCR方法，研究钩状纤维和套索纤维中M2、M3的分布与表达。结果 钩状纤维、套索纤维M2和M3均高表达，M2的表达水平分别为0．411±0．023和0．409±0．044，M3的表达水平分别为0．141±0．016和0．145±0．023，而且M2比M3有较高的表达，两者差异有统计学意义（P〈0．01）．结论 在钧状纤维、套索纤维中．存在丰富的M2、M3,同时与M3相比，M2处于优势。     

Selectivity of agonists for the active state of M1 to M4 muscarinic receptor subtypes

We measured the intrinsic relative activity (RA(i)) of muscarinic agonists to detect possible selectivity for receptor subtypes and signaling pathways. RA(i) is a relative measure of the microscopic affinity constant of an agonist for the active state of a GPCR expressed relative to that of a standard agonist. First, we estimated RA(i) values for a panel of agonists acting at the M(4) muscarinic receptor coupled to three distinct G-protein pathways: G(i) inhibition of cAMP accumulation, G(s) stimulation of cAMP accumulation, and G alpha(15) stimulation of phosphoinositide hydrolysis. Our results show similar RA(i) values for each agonist, suggesting that the same active state of the M(4) receptor triggers the activation of the three G proteins. We also estimated RA(i) values for agonists across M(1) to M(4) muscarinic subtypes stably transfected in Chinese hamster ovary cells. Our results show selectivity of McN-A-343 [4-I-[3-chlorophenyl]carbamoyloxy)-2-butynyltrimethylammnonium chloride] for the M(1) and M(4) subtypes and selectivity of pilocarpine for the M(1) and M(3) subtypes. The other agonists tested lacked marked selectivity among M(1) to M(4) receptors. Finally, we estimated RA(i) values from published literature on M(1), M(2), and M(3) muscarinic responses and obtained results consistent with our own studies. Our results show that the RA(i) estimate is a useful receptor-dependent measure of agonist activity.     

Interferon-gamma induced disruption of GABAergic inhibition in the spinal dorsal horn in vivo

The proinflammatory cytokine interferon-gamma (IFN-gamma), which can be present in elevated levels in the central nervous system during pathological conditions, may be involved in the generation of persistent pain states by inducing neuronal hyperexcitability. The aim of the present study was to examine whether loss of dorsal horn GABAergic inhibition may underlie this IFN-gamma-mediated neuronal hyperexcitability. Repetitive intrathecal injections of recombinant rat IFN-gamma (1000 U) or control buffer were administered to rats every second day for eight days. Electrophysiological recordings from lumbar dorsal horn neurons (n=46) were performed under halothane anaesthesia. Cellular responses were recorded before, during and after microiontophoretic application of the GABA antagonist bicuculline. In control animals, all cellular responses studied were significantly enhanced in the presence of bicuculline, including increased spontaneous activity, enhanced responses to innocuous and noxious mechanical stimulation and reduced paired-pulse depression. In contrast, in IFN-gamma-treated animals, bicuculline ejection had little or no facilitating effect on neuronal responses and instead a significant proportion of neurons displayed reduced responses. Seventy-four percent of cells from IFN-gamma treated animals showed a reduction in the response to noxious stimulation and 47% of the cells showed increased rather than reduced paired-pulse depression in the presence of bicuculline, thus suggesting IFN-gamma-induced excitatory actions by GABA. These findings show that the prolonged presence of increased levels of IFN-gamma in the central nervous system may contribute to the generation of central sensitization and persistent pain by reducing inhibitory tone in the dorsal horn. This implies a potential link between disinhibition and cytokine action in the spinal cord.     

Effects of spinal kappa-opioid receptor agonists on the responsiveness of nociceptive superficial dorsal horn neurons.

Spinal cord application of the kappa-opioid receptor agonists dynorphin (50 nmol) or (1S,2S)U-50,488H (0.19-1.9 mumol) produced changes in the excitability of some superficial dorsal horn nociceptive neurons. One-third of the cells exhibited expansion of their receptive fields as defined using mechanical stimuli following a spinal kappa agonist (dynorphin or U-50,488H); receptive field expansions were of the same order as those observed immediately after a conditioning electrical stimulus applied to a peripheral nerve. In addition, spinal U-50,488H produced changes in mechanical and thermal thresholds of the majority of superficial dorsal horn neurons. These changes were dose-dependent. Facilitation of responses occurred at lower doses and inhibition occurred primarily at higher doses, but these effects were not reversed by subsequent administration of naloxone. The data are consistent with the hypothesis that one action of increases in spinal dynorphin levels due to peripheral inflammation, tissue injury or nerve damage, is to contribute to enhanced neuronal excitability in superficial dorsal horn neurons.     

Enhancement of NMDA receptor phosphorylation of the spinal dorsal horn and nucleus gracilis neurons in neuropathic rats

NR1 is an essential component of functional NMDA receptors and can be activated by phosphorylation. It is suggested that phosphorylation of NR1 (pNR1) contributes to central sensitization after intradermal capsaicin injection. The present study investigates whether increases of spinal pNR1 are correlated to central sensitization and thus pain behaviors in neuropathic pain. Neuropathic rats were produced by L5 spinal nerve ligation, mechanical thresholds of the paw were measured, and then the L4/5 spinal cords and the nucleus gracilis (NG) were removed and immunostained for pNR1. The results showed that the number of pNR1-immunoreactive neurons was significantly increased in the ipsilateral cord, at 3, 7, and 28 days after nerve ligation and these increases coincide with mechanical allodynia. The increase of pNR1-immunoreactive neurons in the NG was observed only at 28 days after the nerve ligation. Western blot analyses confirmed the significant increase of pNR1 protein in spinal dorsal horn after nerve ligation. A protein kinase A inhibitor, H89, moderately reversed mechanical allodynia in 7 day neuropathic rats. Many pNR1-immunoreactive neurons were identified as projection neurons by retrograde tracer. The data suggest that PKA mediated NMDA receptor phosphorylation plays an important role in spinal nerve ligation induced neuropathic pain.     

Characteristics of spinal dorsal horn neurons after partial chronic deafferentation by dorsal root transection

Unilateral transections of 1-3 lumbar dorsal roots were performed in 13 adult cats to investigate the effect of partial deafferentation on dorsal horn neurons. Eleven to 45 days after deafferentation various parameters of spontaneous and evoked activity of 169 neurons were measured and compared to the data of 168 neurons from previous experiments recorded under identical experimental conditions except that these animals had not been deafferented. Eighty-six of the units encountered were located in the segment of transected dorsal root(s) and 82 in the caudally adjacent segment. No significant differences could be observed in the functional properties of these two samples of units. Most parameters measured indicate that either no change at all in responsiveness or signs of decreased excitability occurred in the partially deafferented neurons compared to units recorded in control animals. Discharges evoked by noxious skin heating indicate a linear relationship between discharge frequency and skin temperature. This kind of encoding curve could also be measured during a reversible cold block of the spinal cord at segment L1. The mean encoding curves before and during spinal blockade were not different in deafferented compared to corresponding curves measured in control animals. The only finding that could be interpreted as an indication for increased excitability of partially deafferented neurons was that the mean frequency of spontaneous discharges of a subsample of heat-sensitive neurons was higher in deafferented compared to control animals. Possible mechanisms are discussed.     

Upregulation and redistribution of ephrinB and EphB receptor in dorsal root ganglion and spinal dorsal horn neurons after peripheral nerve injury and dorsal rhizotomy

EphrinB–EphB receptor signaling plays diverse roles during development, but recently has been implicated in synaptic plasticity in the matured nervous system and in pain processes. The present study investigated the correlation between expression of ephrinB and EphB receptor proteins and chronic constriction injury (CCI) of the sciatic nerve and dorsal rhizotomy (DR) in dorsal root ganglion (DRG) and spinal cord (SC); and interaction of CCI and DR on expression of these signals. Adult, male Sprague–Dawley rats were employed and thermal sensitivity was determined in the sham operated CCI and DR rats. Western blot and immunobiochemistry analysis and immunofluorescence staining techniques were used to detect the expression and location of the ephrinB–EphB receptor proteins in DRG and SC. The results showed that expression of ephrinB1 and EphB1 receptor proteins was significantly upregulated in DRG and SC in a time‐dependent manner corresponding to the development of thermal hyperalgesia after CCI. The increased expression is predominately located in the medium‐ and small‐sized DRG neurons, the superficial layers of spinal dorsal horn (DH) neurons, and the IB4 positive nociceptive terminals. DR increases ephrinB1 in DRG, not SC and EphB receptor in SC, not DRG. DR suppressed CCI‐induced upregulation of ephrinB1 in SC and EphB1 receptor in DRG and SC. These findings indicate that ephrinB–EphB receptor activation and redistribution in DRG and DH neurons after nerve injury could contribute to neuropathic pain. This study may also provide a new mechanism underlying DR‐induced analgesia in clinic.     

Effects of glutamate receptor antagonists on spinal dorsal horn neurons during zymosan-induced inflammation in rats.

These experiments examined the effects of spinal administration of the N-methyl-D-aspartate (NMDA) receptor antagonist DL-2-amino-5-phosphonovaleric acid (APV), the non-NMDA receptor antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX), or the metabotropic glutamate receptor antagonist DL-2-amino-3-phosphonoproprionic acid (AP3) on responses of spinal dorsal horn neurons evoked by thermal and mechanical stimuli applied to the rat hindpaw in either an inflamed or noninflamed state. Administration of APV, DNQX, or AP3 decreased heat-evoked neuronal discharges of wide dynamic range (WDR) neurons that were previously augmented by zymosan-induced inflammation. APV and DNQX also decreased heat-evoked discharges of WDR neurons that were previously unaffected by saline injection. Administration of either APV or DNQX, but not AP3, decreased heat-evoked neuronal discharges of nociceptive-specific (NS) neurons in both zymosan- and saline-injected rats. These data suggest that NMDA and non-NMDA receptors contribute to spinal processing of thermal stimuli in both the inflamed and noninflamed state, whereas metabotropic glutamate receptors might serve a role that is unique to WDR neurons in the inflamed state. Only DNQX consistently increased mechanical response thresholds and decreased slopes of the mechanical stimulus response functions (SRFs) of NS and WDR neurons, but this effect was observed in both inflamed and noninflamed states. These data suggest that spinal processing of mechanical stimuli is preferentially mediated by glutamate acting at non-NMDA receptors in either the inflamed or noninflamed state.     

Multiple binding affinities of N-methylscopolamine to brain muscarinic acetylcholine receptors: differentiation from M1 and M2 receptor subtypes

The properties of the specific binding of the muscarinic receptor ligands [3H]quinuclidinyl benzilate and N-[3H]methylscopolamine in rat brain were compared. The specific binding of both ligands was affected equally by heat, phospholipase A2 and trypsin. N-[3H]methylscopolamine labeled only a fraction of the total muscarinic receptors recognized by [3H]quinuclidinyl benzilate in different brain areas and in the heart. Evidence is presented that N-[3H]methylscopolamine, in fact, binds to a subpopulation of [3H]quinuclidinyl benzilate binding sites. The distribution of the high-affinity binding sites of N-[3H]methylscopolamine did not show a different tissue dependence as compared to the total receptor population, and did not parallel the distribution of the pirenzepine-sensitive M1 receptor subtype. Similarly, the affinity of both [3H]quinuclidinyl benzilate and N-[3H]methylscopolamine varied from one tissue to another by a maximum of 2-fold. Although (-)-quinuclidinyl benzilate competed for the specific binding of [3H]quinuclidinyl benzilate in different tissues according to the law of mass-action, N-methylscopolamine showed an anomalous interaction with two binding sites. The low-affinity binding sites of N-methylscopolamine showed saturability of [3H]quinuclidinyl benzilate binding and stereoselectivity. When the binding characteristics of these N-methylscopolamine-inaccessible binding sites of [3H]quinuclidinyl benzilate in the brain were investigated further, it was found that N-methylscopolamine bound exclusively with a single low affinity, whereas pirenzepine still interacted with two receptor populations incorporated in these sites. It is concluded from several lines of evidence that the heterogeneity of binding of N-methylscopolamine to muscarinic receptors does not represent an interaction with the muscarinic M1 and M2 receptor subtypes defined by pirenzepine. Thus, the unique binding profile of pirenzepine to muscarinic receptors cannot be explained merely on the basis of its hydrophilic nature.     

Allosteric interactions at the m1, m2 and m3 muscarinic receptor subtypes

The purpose of our study was to investigate the interactions of allosteric antagonists at the individual m1, m2 and m3 muscarinic receptor subtypes. This was achieved through the use of transformed Chinese hamster ovary cells stably expressing the rat m1 or m3 receptor genes. A homogeneous population of the m2 subtype was obtained from rat heart tissue. Our data indicate that the cardioselective antagonists (gallamine, methoctramine, AF-DX 116 and himbacine) display the following rank order of potency for both displacing ligand binding to the primary site on the receptor and allosterically decelerating ligand dissociation: m2 greater than m1 greater than m3. Schild analysis showed the following rank order of the magnitude of gallamine's cooperative interactions with the three receptor subtypes: m3 greater than m1 greater than m2. By comparison, the ion-channel blockers (verapamil, phencyclidine and quinidine) exhibited a rank order of potency for cooperative effects similar to that of cardioselective antagonists; however, these blockers did not show appreciable specificity in their interaction with the receptor primary binding site. There was a lack of correlation between the displacement of ligand binding and the allosteric potencies of the allosteric antagonists at each of the three muscarinic receptor subtypes, thus revealing the complex nature of interaction (both competitive and allosteric) between many of these compounds with the muscarinic receptor. Despite the fact that the majority of allosteric muscarinic antagonists are also K+ channel blockers, the use of pertussis toxin did not support the notion that this channel represents the allosteric site coupled to the receptor.     

Expression of 5-HT1A receptor mRNA in rat lumbar spinal dorsal horn neurons after peripheral inflammation

The present study observed the expression of the 5-hydroxytryptamine (5-HT) (1A) receptor mRNA in the lumbar spinal dorsal horn neurons following carrageenan inflammation using in situ hybridization (ISH). We also studied the co-localization of 5-HT(1A) receptor mRNA and gamma-amino butyric acid (GABA) or enkephalin (ENK) immunoreactivities using a combined fluorescent ISH and immunofluorescent histochemical double-staining technique. The finding of this study demonstrated that 5-HT(1A) receptor mRNA was widely distributed in the spinal dorsal horn with the highest density in laminae III-VI. Following carrageenan-induced inflammation, the 5-HT(1A) receptor mRNA expression in all layers of ipsilateral dorsal horn was significantly enhanced, and the peak occurred after 8h. Furthermore, the number of 5-HT(1A) receptor mRNA and GABA or ENK immunoreactive double-labeled cells was also markedly increased 8h after carrageenan injection. These findings suggested that following peripheral inflammation, the synthesis of 5-HT(1A) receptor was increased in the lumbar spinal dorsal horn neurons, especially in spinal GABA and ENK neurons.     

Interferon-gamma induces characteristics of central sensitization in spinal dorsal horn neurons in vitro

Hyperexcitability of spinal dorsal horn neurons, also known as 'central sensitization', is a component of pain associated with pathological conditions in the nervous system. The aim of the present study was to analyze if the pro-inflammatory cytokine, interferon-gamma (IFN-gamma), which can be released for extended periods of time in the nervous system during inflammatory and infectious events, can alter synaptic activity in dorsal horn neurons and thereby contribute to such hyperexcitability. Treatment of cultured dorsal horn neurons with IFN-gamma for 2 weeks resulted in a significantly reduced clustering of alpha-amino-3-hydroxy-5-methylisoxazole (AMPA) receptor subunit 1 (GluR1) that was dependent on nitric oxide. The neurons displayed an increased frequency and amplitude of excitatory postsynaptic currents (EPSCs) upon IFN-gamma treatment. Treated dorsal horn neurons also exhibited increased responsiveness to stimulation of dorsal root ganglia (DRG) axons in a two-compartment model. Furthermore, disinhibition by the GABA(A) receptor antagonist picrotoxin (PTX) significantly increased EPSC frequency and induced bursting in untreated cultures but did not significantly increase the frequency in treated neurons, which displayed bursting even without PTX. GABA(A) agonists reduced activity more strongly in treated cultures and immunochemical staining for GABA(A) receptors showed no difference from controls. Since GluR1-containing AMPA receptors (AMPARs) occur predominantly on inhibitory neurons in the dorsal horn, we suggest that the IFN-gamma-mediated increase in spontaneous activity and responsiveness to DRG axon stimulation, decrease in sensitivity to PTX and tendency for EPSC bursting result from a reduced expression of GluR1 on these neurons and not from a reduction in active GABA(A) receptors in the network. IFN-gamma thereby likely causes disinhibition of synaptic activity and primary afferent input in the dorsal horn, which consequently results in central sensitization.     

Spinal substance P release in vivo during the induction of long-term potentiation in dorsal horn neurons

Long-term potentiation (LTP) in wide dynamic range (WDR) neurons in the dorsal horn has been suggested to contribute to central sensitization and the development of chronic pain. Indirect experimental evidence indicates an involvement of substance P (SP), in this respect. The aim of the present study was to monitor the extracellular level of substance P-like immunoreactivity (SP-LI) in the dorsal horn of the rat during and after induction of LTP in WDR neurons in vivo. Electrophysiological recordings of single (WDR) neurons were performed in parallel with microdialysis in the dorsal horn under urethane-anaesthesia. The amount of SP-LI in the microdialysate was determined by radioimmunoassay. As previously shown, high frequency conditioning stimulation of the sciatic nerve induced an increased firing response of WDR neurons. An increased response to C-fibre stimulation, but not A-fibre stimulation, could be determined. A significant increase of the extracellular level of SP-LI in the dorsal horn was detected during, but not after, induction of LTP. These data suggest that SP may be involved in the induction of LTP by high frequency stimulation. However, the maintenance of spinal LTP following high frequency peripheral nerve stimulation does not seem to depend on an increased release of SP.     

Ethanol potentiation of GABAergic transmission in cultured spinal cord neurons involves gamma-aminobutyric acidA-gated chloride channels

The interaction of ethanol with gamma-aminobutyric acid (GABA)-mediated 36-Cl-influx and its modulation by various drugs was investigated in C57 mice spinal cord cultured neurons. Ethanol (5-100 mM) potentiated the effect of GABA on 36Cl-influx; whereas at concentrations greater than or equal to 50 mM ethanol activated Cl- channels directly. The effect of ethanol was specific for GABAA receptor-gated Cl- channels, as ethanol did not potentiate glycine-induced 36Cl-influx in the same neurons. Both the enhancing and direct effects of ethanol on 36Cl-influx were blocked by GABA antagonists like bicuculline, picrotoxinin and inverse agonists of the benzodiazepine site like the imidazodiazepine R015-4513 (ethyl-8-azido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo [1,5 alpha], [1,4]benzodiazepine-3-carboxylate) and N-methyl-beta-carboline-3-carboxamide (FG-7142). Ethanol potentiating effect of GABA-induced 36Cl-influx was also reversed by methyl-6,7-dimethyl-4-ethyl-beta-carboline-3-carboxylate. The effects of the inverse agonists were blocked by the benzodiazepine receptor antagonist R015-1788. Both R015-4513 and FG-7142 reversed direct and GABA potentiating effects of ethanol effect at concentrations lower than those that exhibit inverse agonistic activity in the 36Cl-influx assay in cultured neurons. These results suggest that ethanol facilitation of GABAAergic transmission involves GABA receptor-gated Cl- channels and that this interaction may be responsible for some of the pharmacological effects of ethanol.     

WDR response profiles of spinal dorsal horn neurons may be unmasked by barbiturate anesthesia

The number of WDR (convergent, multireceptive) neurons encountered in the spinal dorsal horn of physiologically intact, awake, drug-free cats has been much smaller than expected (9% in intact, drug-free animals). Control studies in barbiturate-anesthetized or spinal cord transected animals indicate that the dearth of WDR neurons was not just an artifact of the chronic recording technique. In those preparations WDR neurons represented 34% and 61% of the sample, respectively. Initial studies in which the effects of light barbiturate anesthesia on spinal dorsal horn neurons (n = 12) have been examined revealed that a 20 mg/kg dose of pentobarbital can, in some neurons (n = 4), unmask thermally evoked activity that was not present in the intact, drug-free animal. Responses to noxious mechanical stimuli were also enhanced following barbiturate administration. These changes resulted in a reclassification of neural type from low threshold in the intact, awake, drug-free animal to WDR in the anesthetized animal.     

氯胺酮对甲醛致痛诱导大鼠脊髓背角神经元兴奋性的抑制

背景:氯胺酮是否可通过影响脊髓水平的伤害性信息的传递而发挥抗伤害作用尚不清楚;一氧化氮在脊髓水平主要参与痛觉过敏的形成和发展,可诱导Fos表达,但其是否参与了氯胺酮对痛信号的转导或调控的机制不明。目的:观察大鼠脊髓对甲醛痛刺激的反应及氯胺酮的影响。设计:均衡随机的动物实验。单位:徐州医学院附属医院麻醉科和江苏省麻醉学重点实验室。材料:实验于2000-01/03在徐州医学院江苏省麻醉学重点实验室进行。取SD大鼠30只,用均衡随机方法分为6组熏甲醛组6只,甲醛 氯胺酮组6只,氯胺酮 甲醛组6只,氯胺酮组6只,甲醛 生理盐水组3只,生理盐水组3只,各组雌雄比例相同。方法:①甲醛组:体积分数为0.05的甲醛200μL一侧前爪掌心皮下注射,刺激1h。②甲醛 氯胺酮组:甲醛痛刺激10min后腹腔注射100mg/kg氯胺酮1h。③氯胺酮 甲醛组:腹腔注射氯胺酮10min后再行甲醛痛刺激1h。④氯胺酮组:腹腔注射同等剂量氯胺酮1h。⑤甲醛 生理盐水组:甲醛痛刺激10min后腹腔注射等容(10mL/kg)的生理盐水1h。⑥生理盐水组:腹腔注射等容生理盐水1h。主要观察指标:①各组大鼠行为学表现。②取脊髓切片,用c-fos基因免疫组化法和NADPH-d组化技术染色,观察大鼠脊髓背角4层(Ⅰ～Ⅱ层,Ⅲ～Ⅳ层,Ⅴ～Ⅵ层,Ⅶ～Ⅹ层)切片Fos样免疫阳性神经元(FLI)和FLI/NOS双标记神经元的数目变化。结果:30只大鼠全部进入结果分析。①行为学变化:甲醛组及甲醛 生理盐水组大鼠注射甲醛后,出现痛反应;注射氯胺酮的大鼠,注射后数分钟内翻正反射消失,无明显的痛行为表现,而呈持续睡眠状态,至灌注时翻正反射仍未恢复。②FLI神经元表达:甲醛组及甲醛 生理盐水组大鼠注射侧脊髓背角出现大量FLI阳性神经元,主要分布在脊髓背角Ⅰ～Ⅱ层;氯胺酮 甲醛组、甲醛 氯胺酮组大鼠脊髓FLI细胞的分布与甲醛组及甲醛 生理盐水组基本相似,但FLI阳性细胞数量显著减少(P<0.01);氯胺酮组和生理盐水组大鼠脊髓未见或偶见FLI阳性细胞。③FLI/NOS双标记神经元表达:氯胺酮 甲醛组、甲醛 氯胺酮组脊髓背角Ⅰ～Ⅱ层双标记神经元数目显著少于甲醛组及甲醛 生理盐水组眼(1±1),(1±1),(7±3),(8±3)个/切片,P<0.01演,氯胺酮组和生理盐水组无表达。结论:同侧相应脊髓节段的某些神经元参与了化学性致痛信息的传导和调控,氯胺酮通过抑制这些神经元的活动而产生抗伤害作用;此作用与抑制脊髓内一氧化氮合酶阳性神经元的活性有关。     

Effect of morphine on deep dorsal horn projection neurons depends on spinal GABAergic and glycinergic tone: implications for reduced opioid effect in neuropathic pain

The mu opioid agonist morphine has distinct effects on spinal dorsal horn neurons in the superficial and deep laminae. However, it is not clear if the inhibitory effect of morphine on dorsal horn projection neurons is secondary to its potentiating effect on inhibitory interneurons. In this study, we tested the hypothesis that removal of GABAergic and glycinergic inhibitory inputs attenuates the effect of morphine on dorsal horn projection neurons and the reduced spinal GABAergic tone contributes to attenuated morphine effect in neuropathic pain. Single-unit activity of deep dorsal horn projection neurons was recorded in anesthetized normal/sham controls and L(5) and L(6) spinal nerve-ligated rats. Spinal application of 10 microM morphine significantly inhibited the evoked responses of dorsal horn neurons in both normal/sham controls, and this effect was abolished by the specific mu opioid antagonist. However, the effect of morphine on dorsal horn projection neurons was significantly reduced in nerve-injured rats. Furthermore, topical application of the GABA(A) receptor antagonist bicuculline (20 microM) almost abolished the effect of morphine in normal/sham control rats but did not significantly attenuate the morphine effect in nerve-injured rats. On the other hand, the glycine receptor antagonist strychnine (4 microM) significantly decreased the effect of morphine in both nerve-injured and control animals. These data suggest that the inhibitory effect of opioids on dorsal horn projection neurons depends on GABAergic and glycinergic inputs. Furthermore, reduced GABAergic tone probably contributes to diminished analgesic effect of opioids in neuropathic pain.     

氯胺酮对甲醛致痛诱导大鼠脊髓背角神经元兴奋性的抑制

背景：氯胺酮是否可通过影响脊髓水平的伤害性信息的传递而发挥抗伤害作用尚不清楚；一氧化氮在脊髓水平主要参与痛觉过敏的形成和发展，可诱导Fos表达，但其是否参与了氯胺酮对痛信号的转导或调控的机制不明。 目的：观察大鼠脊髓对甲醛痛刺激的反应及氯胺酮的影响。 设计：均衡随机的动物实验。 单位：徐州医学院附属医院麻醉科和江苏省麻醉学重点实验室。 材料：实验于2000-01／03在徐州医学院江苏省麻醉学重点实验室进行。取SD大鼠30只，用均衡随机方法分为6组。甲醛组6只，甲醛＋氯胺酮组6只，氯胺酮＋甲醛组6只，氯胺酮组6只。甲醛＋生理盐水组3只，生理盐水组3只，各组雌雄比例相同。 方法：④甲醛组：体积分数为0．05的甲醛200μL一侧前爪掌心皮下注射，刺激1h。②甲醛＋氯胺酮组：甲醛痛刺激10min后腹腔注射100mg／kg氯胺酮1h。③氯胺酮＋甲醛组：腹腔注射氯胺酮10min，后再行甲醛痛刺激1h。④氯胺酮组：腹腔注射同等剂量氯胺酮1h。⑤甲醛＋生理盐水组：甲醛痛刺激10min后腹腔注射等容（10mL／kg）的生理盐水1h。⑥生理盐水组：腹腔注射等容生理盐水1h。 主要观察指标：①各组大鼠行为学表现。②取脊髓切片，用c-fos基因免疫组化法和NADPH-d组化技术染色，观察大鼠脊髓背角4层（Ⅰ～Ⅱ层，Ⅲ～Ⅳ层，Ⅴ～Ⅵ层，Ⅶ～Ⅹ层）切片Fos样免疫阳性神经元（FLI）和FLI／NOS双标记神经元的数目变化。 结果：30只大鼠全部进入结果分析。①行为学变化：甲醛组及甲醛＋生理盐水组大鼠注射甲醛后，出现痛反应；注射氯胺酮的大鼠，注射后数分钟内翻正反射消失，无明显的痛行为表现，而呈持续睡眠状态。至灌注时翻正反射仍未恢复。②FLI神经元表达：甲醛组及甲醛＋生理盐水组大鼠注射侧脊髓背角出现大量FLI阳性神经元，主要分布在脊髓背角Ⅰ～Ⅱ层；氯胺酮＋甲醛组、甲醛＋氯胺酮组大鼠脊髓FLI细胞的分布与甲醛组及甲醛＋生理盐水组基本相似，但FLI阳性细胞数量显著减少（P〈0．01）；氯胺酮组和生理盐水组大鼠脊髓未见或偶见FLI阳性细胞。③FLI／NOS双标记神经元表达：氯胺酮＋甲醛组、甲醛＋氯胺酮组脊髓背角Ⅰ～Ⅱ层双标记神经元数目显著少于甲醛组及甲醛＋生理盐水组[（1&;#177;1），（1&;#177;1），（7&;#177;3），（8&;#177;3）个／切片，P〈0．01]，氯胺酮组和生理盐水组无表达。 结论：同侧相应脊髓节段的某些神经元参与了化学性致痛信息的传导和调控，氯胺酮通过抑制这些神经元的活动而产生抗伤害作用；此作用与抑制脊髓内一氧化氮合酶阳性神经元的活性有关。