Limited role of macrophages in generation of nerve injury-induced mechanical allodynia

The purpose of this study was to investigate the role of peripheral macrophages in the generation of mechanical allodynia utilizing a modification of the Chung rat model of neuropathy. Three distinct methods were used: (1) systemic and perineural macrophage inhibition utilizing CNI-1493; (2) depletion of the peripheral macrophage population by liposome-encapsulated clodronate; and (3) perineural administration of activated or inactivated bone marrow-derived macrophages (BMDM) in sham-surgery rats. Mechanical allodynia was tested on days 1, 3, 5, 7, and 10 post-intervention or surgery using von Frey monofilaments. In order to assess the role of spinal glia following these interventions, microglial (CNS macrophages) and astrocytic activation was assessed using immunohistochemistry. CNI-1493 did not attenuate mechanical allodynia, or spinal glial expression as compared to the saline control group. Similarly, the clodronate depletion of peripheral macrophages prior to nerve injury did not have any effect on the resultant mechanical allodynia or spinal glial activation. Perineural administration of activated or inactivated BMDM did not evoke mechanical allodynia in sham surgery rats. Of interest, we observed an ipsilateral, dorsal horn increase in microglial expression following perineural administration of activated macrophages. In summary, these data suggest a limited role of activated macrophages in the onset of mechanical allodynia in an animal model of neuropathy.     

Expression of the N-methyl-D-aspartate receptor NR1 splice variants and NR2 subunit subtypes in the rat colon

N-methyl-D-aspartate (NMDA) receptors and the expression of their different splice variants and subunits were previously characterized in the brain and spinal cord. However, knowledge on the NMDA receptor expression and function in the enteric nervous system is limited. Previous work suggested that NMDA receptors were involved in a rat model of visceral hypersensitivity. The aim of this study was to characterize the expression of the NMDA receptor NR1 splice variants and the NR2 subunit subtypes in the rat colon. We visualized the expression of NR1 protein in the rat submucosal and myenteric plexuses. The NR1 splice variants found in the colon of rats lacked the N1 and C1 cassettes and contained the C2 and C2' cassettes (NR1(000) and NR1(001)). The NR2B and NR2D subunits were also found in the rat colon. Moreover, NMDA receptors in the rat colon were heteromeric, since NR1 was co-localized with NR2B and NR2D subunits using fluorescent immunohistochemistry. The identification of the NMDA receptors in the enteric nervous system could lead to the development of drugs that selectively modulate bowel function.     

Intrathecal S-nitroso-N-acetylpenicillamine and L-cysteine attenuate nerve injury-induced allodynia through noradrenergic activation in rats

Spinal norepinephrine release and activation of spinal alpha(2)-adrenergic receptors represent important components of descending control of nociception. Recent studies have shown that nitric oxide is capable of stimulating neuronal norepinephrine release in the presence of thiol-containing compounds such as L-cysteine. In the present study, we tested a hypothesis in a rodent model of neuropathic pain that intrathecal injection of the nitric oxide donor S-nitroso-N-acetylpenicillamine and L-cysteine produces an antiallodynic action mediated by the spinal alpha(2)-adrenergic receptors. Allodynia was induced in rats by ligation of the left lumbar L5/L6 spinal nerves. Mechanical allodynia was quantified by application of von Frey filaments to the left hindpaw. Intrathecal injection of 20-100microg of S-nitroso-N-acetylpenicillamine in the presence of 200microg of L-cysteine, but not D-cysteine, dose-dependently attenuated the allodynia. Intrathecal injection of a combination of 100microg of S-nitroso-N-acetylpenicillamine and 50-200microg of L-cysteine also inhibited the allodynia in a dose-dependent manner. Pretreatment with a nitric oxide scavenger, carboxy-PTIO, or depletion of norepinephrine with a specific neurotoxin, N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine, prevented the antiallodynic action of intrathecal S-nitroso-N-acetylpenicillamine and L-cysteine. Furthermore, the antiallodynic effect produced by intrathecal injection of a combination of S-nitroso-N-acetylpenicillamine and L-cysteine was abolished by pretreatment with intrathecal injection of a non-specific alpha-adrenergic receptor antagonist, phentolamine, or an alpha(2) receptor antagonist, idazoxan.This study provides the first functional evidence that spinal nitric oxide interacts with the thiol-containing compounds to produce an antiallodynic effect in neuropathic pain. We propose that such an action is mediated by endogenous norepinephrine and spinal alpha(2)-adrenergic receptors.     

Mutation analysis of the N-methyl-D-aspartate receptor NR1 subunit gene (GRIN1) in schizophrenia

Dysfunction of N-methyl-D-aspartate (NMDA) type ionotropic glutamate receptors has been implicated in the etiology of schizophrenia based on psychotomimetic properties of the antagonist phencyclidine (PCP) and observation that mice expressing low levels of the N-methyl-D-aspartate receptor NR1 subunit exhibit behavioral alterations that may be ameliorated by neuroleptic drugs. Based on the hypothesis that some schizophrenic patients have functionally deficient mutation(s) of the gene encoding N-methyl-D-aspartate receptor NR1 subunit (GRIN1), we screened 48 Japanese patients with schizophrenia for mutations in the coding region of the GRIN1 gene. Four variants, IVS2-22T>C, IVS2-12G>A, IVS4-34C>T, and 1719G/A (Pro516Pro), were identified. No non-synonymous mutation was detected. No significant association was suggested by case-control comparisons. Results indicate that genomic variations of the GRIN1 gene are not likely to be involved substantially in the etiology of schizophrenia.     

Turnover analysis of N-methyl-D-aspartate receptor subunit NR1 protein in PC12 cells.

The post-translational fate of N-methyl-D-aspartate receptor (NMDAR) subunit NR1 was characterized in PC12 cells using pulse-chase labeling, block of protein synthesis by cyclohexamide and deglycosylation by endoglycosidase H. Metabolic labeling of NR1 protein indicated a biphasic degradation of NR1 protein with half-lives of 1.6 and 16.1 h for a rapidly (78%) and a slowly (22%) degrading population. Immunoprecipitation of NR1 following the block of protein synthesis by cyclohexamide revealed that the rapidly and slowly degrading pools mainly consisted of the NR1 splice variants NR1-4a and NR1-2a. Sensitivity of NR1 protein to deglycosylation by endoglycosidase H indicated the presence of an immature form of NR1 that was retained in the endoplasmic reticulum. PC12 cells serve as a useful model for the elucidation of translational and post-translational mechanisms of NMDAR expression.     

The ultrastructural localisation of the N-methyl-D-aspartate NR2B receptor subunit in rat lumbar spinal cord

Glutamate together with its N-methyl-d-aspartate (NMDA) receptors has an important role in the transmission of stimuli in the spinal cord. Whilst the expression of the various NMDA receptor subunits within the spinal cord has been investigated the subcellular location of the NMDA NR2B subunit has yet to be definitively established. Both mRNA and light microscopical studies have failed to unequivocally demonstrate the proposed pre-synaptic location of this subunit. This has been proposed from pharmacological data and is thought to underlie the apparent analgesic properties of selective NR2B antagonists. Using pre-embedding immunohistochemistry combined with electron microscopy our findings provide the first definitive morphological evidence for both a pre- and post-synaptic localisation of NR2B/containing NMDA receptors, and suggest expression by astrocytes, in the rat lumbar spinal cord.     

Expression of the N-methyl-D-aspartate receptor subunit NR3B regulates dendrite morphogenesis in spinal motor neurons

During postnatal development, the dendrites of spinal motor neurons are refined in an activity-dependent manner that can be influenced by blocking activation of N-methyl-D-aspartate (NMDA) receptors. In late postnatal life, dendritic refinement ceases, and dendrite architecture is unaffected by NMDA antagonists; however the molecular substrate for limiting dendritic plasticity is not understood. During late postnatal development, expression of the NR3B NMDA receptor subunit, a putative dominant-negative subunit that reduces glutamate-induced ionic currents, is upregulated within motor neurons. To investigate whether increasing NR3B expression may contribute to the loss in late development of activity-dependent dendritic reorganization in the spinal cord, we over-expressed NR3B in cultured rat spinal motor neurons, and compared its effects on dendrite morphology with the effects of pharmacological blockade of NMDA receptors. We found that over-expression of the NR3B receptor subunit increased the length and complexity of dendritic arbor, and increased numbers of dendritic filopodia, suggesting that NR3B promotes the addition of branch segments in developing motor neurons. In contrast, blockade of NMDA receptor activity by the NMDA antagonist DL-2-amino-5-phosphonovalerate (AP5) had little effect on the overall length or complexity of dendritic arbor. Instead, treatment with AP5 resulted in significant reorganization of dendritic arbor in a manner that favored addition of dendritic segments of high branch orders, at the expense of those closer to the cell body. These results suggest that expression of the NR3B subunit may participate in activity-dependent reorganization of dendritic architecture, but via a mechanism that may be inconsistent with loss of NMDA receptor activity.     

An association study of the N-methyl-D-aspartate receptor NR1 subunit gene (GRIN1) and NR2B subunit gene (GRIN2B) in schizophrenia with universal DNA microarray

Dysfunction of the N-methyl-D-aspartate (NMDA) receptors has been implicated in the etiology of schizophrenia based on psychotomimetic properties of several antagonists and on observation of genetic animal models. To conduct association analysis of the NMDA receptors in the Chinese population, we examined 16 reported SNPs across the NMDA receptor NR1 subunit gene (GRIN1) and NR2B subunit gene (GRIN2B), five of which were identified in the Chinese population. In this study, we combined universal DNA microarray and ligase detection reaction (LDR) for the purposes of association analysis, an approach we considered to be highly specific as well as offering a potentially high throughput of SNP genotyping. The association study was performed using 253 Chinese patients with schizophrenia and 140 Chinese control subjects. No significant frequency differences were found in the analysis of the alleles but some were found in the haplotypes of the GRIN2B gene. The interactions between the GRIN1 and GRIN2B genes were evaluated using the multifactor-dimensionality reduction (MDR) method, which showed a significant genetic interaction between the G1001C in the GRIN1 gene and the T4197C and T5988C polymorphisms in the GRIN2B gene. These findings suggest that the combined effects of the polymorphisms in the GRIN1 and GRIN2B genes might be involved in the etiology of schizophrenia.European Journal of Human Genetics (2005) 13, 807-814. doi:10.1038/sj.ejhg.5201418 Published online 20 April 2005.     

AMPA receptor subunit expression in the cuneate nucleus of adult squirrel monkeys after peripheral nerve injury

The primate somatosensory system provides an excellent model system with which to investigate adult neural plasticity. Here, we report immunohistochemical staining data for the GluR1 and GluR2/3 AMPA receptor subunits in the cuneate nucleus of adult squirrel monkeys one week after median nerve compression. These data are compared to subunit changes in the area 3b cortex of the same animals. We report differences between control and deprived brainstem implying that deprivation induced changes in subunit expression mirror those reported in the cortex. There are significant increases in GluR1 receptor subunit staining intensity and significant decreases in GluR2/3 receptor subunit staining intensity. This pattern of expression resembles receptor configurations reported in developing sensory systems. Taken together, these results suggest that the brainstem and the cortex initially progress through a phase of developmental recapitulation prior to the onset of NMDA mediated adult somatosensory reorganization.     

The role of peripheral N-methyl-D-aspartate receptors in Freund's complete adjuvant induced mechanical hyperalgesia in rats

We investigated the role of excitatory amino acid receptors in mechanical hyperalgesia induced by subcutaneous injection of Freund's complete adjuvant (FCA) into the rat hind paw. In normal rats, an intraplantar (i.pl.) injection of L-glutamate, but not of D-glutamate (3 pmol/0.1 ml each) produced a mechanical hyperalgesia in the hind paw with a lowered paw-withdrawal threshold to pressure. In rats that developed mechanical hyperalgesia associated with inflammation in the hind paw following i.pl. injection of FCA (0.15 ml), the injection of N-methyl-D-aspartate (NMDA) receptor antagonist, MK-801 (1 pmol/0.1 ml) into the inflamed paw increased the paw pressure threshold. On the other hand, the injection of non-NMDA receptor antagonist, 6-cyano-7-nitroqiunoxaline-2,3-dione (CNQX, 10 pmol/0.1 ml) into the inflamed paw had no effect on FCA-induced lowering of the paw pressure threshold. The results suggest that NMDA, but not non-NMDA receptors play a substantial role in mediating the development of mechanical hyperalgesia induced in the inflamed paw following i.pl. FCA injection.     

Excitatory amino acid receptors of the electrosensory system: the NR1/NR2B N-methyl-D-aspartate receptor

The amino acid sequence of the N-methyl-D-aspartate (NMDA) receptor subunit NR2B from the brown ghost knife fish Apteronotus leptorhynchus has been determined and compared with the sequence of the murine NR2B. This comparison revealed high levels of sequence conservation throughout the ligand binding and membrane spanning segments. The functional properties of the NR1 and NR2B receptor complex were examined by coexpression in HEK cells. The recombinant AptNR1/NR2B receptors produced robust currents after stimulation with glutamate or NMDA in the presence of glycine. Measurements of the concentration dependencies for these agonists indicated that the agonist binding sites on the apteronotid receptor are highly conserved, with nearly identical agonist affinities to those of the murine NR1/NR2B receptor. The kinetic responses of the fish receptor were also highly conserved, with deactivation rates for the AptNR2B receptor matching those of the murine NR2B containing receptor. Evidently, most of the unique functional properties that reside in the NR2B receptor subunit have been well conserved in teleost NMDA receptors. On the other hand, the apteronitid receptor displayed a lowered sensitivity to voltage-dependent Mg(2+) block and a reduced affinity for the NR2B-specific noncompetitive antagonist ifenprodil. We conclude that the functional properties that result from the incorporation of the NR2B receptor in the NMDA receptor complex have been maintained since the evolutionary divergence of teleost and mammalian organisms.     

低氧对脑N-甲基-D-天冬氨酸受体亚基2B的表达影响

<正>N-甲基-D-天冬氨酸(N-methyl-D-aspartate,NMDA)受体广泛存在于神经细胞的突触后膜上,有较高的钙离子通透性,是与学习记忆及精神性疾病密切相关的一类谷氨酸门控离子通道受体。该受体是由7种类型的亚基NR1,NR2A-D,NR3A、B组成的四聚体或五聚体,其中NR1与NR2、NR3之间不具有明显的同源关系。NMDAR亚基在突触形成和活性反应中发挥不同的调节作用,NR1/NR2A在成年大鼠神经系统突触可塑性     

Identification of single-nucleotide polymorphisms in the human N-methyl-D-aspartate receptor subunit NR2D gene, GRIN2D, and association study with schizophrenia

OBJECTIVES: The glutamatergic dysfunction is one of the main hypotheses for the pathophysiology of schizophrenia. N-methyl-D-aspartate receptors are of major interest because phencyclidine, a non-competitive antagonist of N-methyl-D-aspartate receptors, produces a schizophrenia-like psychosis. Therefore, the genes encoding N-methyl-D-aspartate receptor subunits are strong candidates for schizophrenia susceptibility genes. We focused on the N-methyl-D-aspartate receptor subunit NR2D gene in the case-control study of schizophrenia. METHODS: We screened for polymorphisms in exons, exon-intron boundaries and the 5' upstream region of GRIN2D by direct sequencing in 32 Japanese patients. Out of the total 13 single-nucleotide polymorphisms identified, we genotyped 200-201 Japanese patients and 219-221 controls for nine common single-nucleotide polymorphisms (minor allele frequency over 0.05). RESULTS: None of the nine single-nucleotide polymorphisms showed significant differences in genotype and allele frequencies between cases and controls. We observed significant associations of pairwise haplotypes in three combinations of four single-nucleotide polymorphisms, INT10SNP-EX13SNP2, EX13SNP2-EX13SNP3 and EX6SNP-EX13SNP2, with the disease even after the Bonferroni correction (P=1.094 x 10(-6), Pcorrected=2.297 x 10(-5), P=2.825 x 10(-6), Pcorrected=5.933 x 10(-5) and P=2.02 x 10(-4), Pcorrected=4.242 x 10(-3), respectively). The same results were also obtained using the false discovery rate (BL) method at the threshold P value, 2.908 x 10(-3). CONCLUSIONS: We conclude that the GRIN2D locus is a possible genomic region contributing to schizophrenia susceptibility in the Japanese population.     

NMDA受体亚单位NR1、NR2A和NR2B在大鼠海马的免疫组织化学表达

目的 :观察N 甲基 D 门冬氨酸受体亚单位 1 (N methyl D aspartatereceptorsubunit 1 ,NR1 )、亚单位 2A(NR2A)和亚单位 2B(NR2B)在成年大鼠海马结构各区的表达特点 ,为研究三者在海马生理和病理过程中的作用提供形态学资料。方法 :大鼠脑 2 0 μm厚冰冻切片 ,免疫组织化学ABC法显色 ,图像分析。结果 :NR1、NR2A和NR2B在海马CA1～CA3区锥体细胞以及齿状回颗粒细胞普遍表达 ,三者中以NR1免疫组织化学反应最强 ,NR2A最弱 ,NR2B居中。NR1与NR2A在海马各区间的表达水平都无显著差异 ;NR2B在海马CA1区的表达明显强于其在CA3区及齿状回的表达 ,尤其是CA1区锥体细胞的顶树突在贯穿辐射层及腔隙分子层的全长中都呈高表达。结论 :NR1、NR2A和NR2B在正常海马结构各区的表达强度和形式存在差异 ,提示各区间天然N 甲基 D 门冬氨酸(N methyl D aspartate ,NMDA)受体的亚单位构成比例可能有所不同     

Molecular characterization and embryonic expression of the family of N-methyl-D-aspartate receptor subunit genes in the zebrafish.

We present the cloning of 10 N-methyl-D-aspartate (NMDA) receptor subunits from the zebrafish. These subunits fall into five subtypes, each containing two paralogous genes. Thus, we report two NMDAR1 genes (NR1.1 and NR1.2), and eight NMDAR2 genes, designated NR2A.1 and NR2A.2, NR2B.1 and NR2B.2, NR2C.1 and NR2C.2, and NR2D.1 and NR2D.2. The predicted sequences of the NR1 paralogs display 90% identity to the human protein. The NR2 subunits show less identity, differing most at the N- and C-termini. The NR1 genes are both expressed embryonically, although in a nonidentical manner. NR1.1 is found in brain, retina, and spinal cord at 24 hours postfertilization (hpf). NR1.2 is expressed in the brain at 48 hpf but not in the spinal cord. NR2 developmental gene expression varies: both paralogs of the NR2A are expressed at 48 hpf in the retina, only one paralog of the NR2B is expressed at low levels in the heart at 48 hpf. Neither of the NR2C is expressed embryonically. Both paralogs of the NR2D are expressed: 2D.1 is in the forebrain, retina, and spinal cord at 24 hpf, whereas the 2D.2 is only found in the retina. Our findings demonstrate that the zebrafish can serve as a useful model system for investigating the role of NMDA receptors in the development of the nervous system.     

Preferential relocation of the N-methyl-D-aspartate receptor NR1 subunit in nucleus accumbens neurons that contain dopamine D1 receptors in rats showing an apomorphine-induced sensorimotor gating deficit

Sensorimotor gating as measured by prepulse inhibition (PPI) to startle-evoking auditory stimulation (AS) is disrupted in schizophrenia and in rodents receiving systemic administration of apomorphine, a dopamine D1/D2 receptor agonist, or MK-801, an N-methyl-d-aspartate (NMDA) receptor antagonist. The functional analogies and our prior results showing apomorphine- and AS-induced relocation of the dopamine D1 receptor (D1R) in the nucleus accumbens (Acb) shell suggest that apomorphine and AS may affect the subcellular distribution of the NMDA receptor NR1 subunit, a protein that forms protein-protein interactions with the D1R. We quantitatively compared the electron microscopic immunogold labeling for NR1 in dendritic profiles distinguished with respect to presence of D1R immunoreactivity and location in the Acb shell or core of rats receiving a single s.c. injection of vehicle (VEH) or apomorphine (APO) alone, or combined with AS (VEH+AS, APO+AS). The rats in the APO+AS group were previously shown to have PPI deficits, whereas the rats in the VEH+AS group had normal PPI. A significantly higher percentage of plasmalemmal and a lower percentage of cytoplasmic NR1 immunogold particles were seen in D1R-labeled dendritic spines in the Acb shell of the APO+AS group compared with all other groups. D1R-containing small dendrites in the Acb shell of the APO+AS group also showed a significantly higher density of plasmalemmal and a lower density of cytoplasmic NR1 immunogold particles compared with VEH or APO groups. In the Acb core, the APO+AS group had significantly fewer dendritic spines co-expressing NR1 and D1R compared with VEH or VEH+AS groups. These results, together with our earlier findings, suggest that NMDA receptors are preferentially mobilized in D1R-containing Acb neurons of rats showing apomorphine-induced disruption of PPI in a paradigm using acoustic stimulation.     

糖尿病机械性痛模型大鼠P2X3受体的时空表达

目的探讨糖尿病机械性痛(DMA)模型大鼠中P2X3受体(P2X3R)的时空表达变化。方法腹腔注射链脲菌素(STZ)建立大鼠DMA模型。采用von Frey细丝法测定DMA大鼠机械性痛阈,在不同时间点取腰髓4~5(L4~5)节段的脊髓背角(SDH)和背根神经节(DRG)以及足底皮肤,通过免疫荧光观察P2X3R阳性产物的表达变化。进一步Western blotting检测SDH和DRG中P2X3R蛋白的变化。结果与对照组(CON组)相比,注射STZ 7d后大鼠机械性痛阈显著降低,在14d时达到最低并持续至28d(P0.05)。免疫荧光和Western blotting结果均显示L4~5节段DRG中P2X3R表达在14d和21d时显著增多,与CON组相比差异有统计学意义(P0.05),28d时恢复至CON组水平;而在SDH及皮肤中,P2X3R表达上调出现在21d和28d(P0.05)。结论在STZ诱导的DMA大鼠机械性痛的不同时程中,DRG、SDH和皮肤中P2X3R的表达均呈现与痛敏变化几乎平行的变化趋势,但后两者比前者的变化在时间上略晚。这些结果说明P2X3R可能在DMA机械性痛敏的维持阶段起重要作用。     

NMDA受体NR1亚单位与GABA_A受体在精神分裂症小鼠海马齿状回颗粒细胞层的表达

目的:明确NMDA受体NR1亚单位、GABAA受体与精神分裂症(SP)小鼠海马齿状回颗粒细胞层新生颗粒细胞的共存模式;阐明NR1、GABAA受体在SP小鼠海马中的表达。方法:实验组小鼠腹腔注射MK-801(每日0.6 mg/kg),对照组注射等量的生理盐水,连续注射14 d后对两组动物分别进行BrdU标记,断头并进行如下处理:(1)免疫荧光染色,观察海马DG区中NR1和GABAA的表达以及与BrdU标记的新生颗粒细胞的共存模式;(2)利用RT-PCR技术,检测海马GABAA及NR1 mRNA表达水平的改变。结果:(1)停药后实验组与对照组比较,海马神经细胞的增殖率下降了23.1%(P0.05),GABAA、NR1两种神经细胞增殖数无差异性改变(P0.05);(2)实验组小鼠海马GABAAmRNA的表达量较对照组显著下降(P0.05),而NR1 mRNA的表达量较对照组明显增高(P0.05)。结论:(1)小鼠精神分裂症后可引起海马齿状回神经细胞增殖的降低;(2)小鼠精神分裂症后,在mRNA水平上,海马GABAA的表达降低,NR1的表达升高。