Cytomegalovirus infection inhibits the expression of N-methyl-d-aspartate receptors in the developing mouse hippocampus and primary neuronal cultures

Cytomegalovirus (CMV) is the most significant infectious cause of developmental brain disorders in humans. The infection occasionally persists and causes neurological disorders. The N-methyl-d-aspartate (NMDA) subtype of glutamate receptors is essential for the development and plasticity of synapses, but also is involved in neuronal excitotoxicity during viral infection. Here we investigated the effects of murine CMV (MCMV) infection on the expression of NMDA receptors in the hippocampal neurons of neonatal mice and primary neuronal cultures. Viral antigen was mostly found in hippocampal pyramidal neurons from the CA1 to CA3. Image analysis of immunohistochemistry demonstrated that the expression of NMDA receptor subunit 1 (NMDA-R1) protein in CA1 neurons of MCMV-infected brain was reduced to 40% of that in uninfected brain. The signal of in situ hybridization for NMDA-R1 mRNA was also decreased in CA1 neurons of MCMV-infected brain. In primary neuronal cultures, reduction of NMDA-R1 expression in MCMV-infected neurons was also detected by immunocytochemistry and Western blotting. These results suggest that reduction of NMDA receptor expression by MCMV infection may cause a decrease in the susceptibility of the neurons to excitotoxic cell death, and may be related to the establishment of viral persistence and functional disturbances in MCMV-infected neurons.     

神经细胞膜NMDA受体蛋白激光共聚焦显微镜亚细胞定位研究

目的明确神经元膜NMDA受体蛋白分子在神经元表面的分布规律，对单个NMDA受体簇进行定位。方法应用免疫荧光标记激光共聚焦显微镜方法，对原代培养大鼠皮层神经元进行观测。结果与细胞核标记的碘化丙啶红色荧光对比，NMDA受体蛋白分子标记的FITC绿色荧光主要分布于神经元树突干膜和胞体近树突起始部分的胞膜上，呈明显的颗粒点状分布。结论NMDA受体簇在单细胞上主要与突触后膜分布一致，位于树突起始部及树突干膜上，激光共聚焦技术可以精确地定位标记后单个膜蛋门分子。     

AR-R15896AR blocks the early NMDA-induced loss of MAP2 in primary cortical cultures

Abstract

The low-affinity use-dependent N-methyl-D-aspartate (NMDA) receptor antagonist AR-R15896AR is neuroprotective in primary rat cortical cultures exposed to toxic concentrations of NMDA and reduces the magnitude of NMDA-triggered increases in [Ca²⁺],. Here we show using fluorescence staining and measurements of microtubule-associated protein-2 (MAP2) levels, that AR-R15896AR inhibits the NMDA- induced loss of MAP2 that occurs within 2 min following NMDA exposure. Understanding the multiple, Ca²⁺-triggered intracellular events that occur following NMDA receptor stimulation is important to the development of safe and effective neuroprotective agents. [Neurol Res 1999; 21: 524–528]     

Contribution of the glycine site of NMDA receptors in rostral and intermediate-caudal parts of the striatum to the regulation of muscle tone in rats

The aim of the present study was to assess the contribution of the glycine site of NMDA receptors in the striatum to the regulation of muscle tone. Muscle tone was examined using a combined mechano and electromyographic method, which measured simultaneously the muscle resistance (MMG) of the rat's hind foot to passive extension and flexion in the ankle joint and the electromyographic activity (EMG) of the antagonistic muscles of that joint: gastrocnemius and tibialis anterior. Muscle rigidity was induced by haloperidol (2.5 mg/kg i.p.). 5,7-dichlorokynurenic acid (5,7-DCKA), a selective glycine site antagonist, injected in doses of 2.5 and 4.5 μg/0.5 μl bilaterally, into the rostral region of the striatum, decreased both the haloperidol-induced muscle rigidity (MMG) and the enhanced electromyographic activity (EMG). 5,7-DCKA injected bilaterally in a dose of 4.5 μg/0.5 μl into the intermediate-caudal region of the striatum of rats not pretreated with haloperidol had no effect on the muscle tone. The present results suggest that blockade of the glycine site of NMDA receptors in the rostral part of the striatum may be mainly responsible for the antiparkinsonian action of this drug.     

In vitro and in vivo antagonistic activities of SM-31900 for the NMDA receptor glycine-binding site

The purpose of this study was to clarify the in vitro pharmacological profile and the in vivo activity of (3S)-7-chloro-3-[2-((1R)-1-carboxyethoxy)-4-aminomethylphenyl]aminocarbonylmethyl-1,3,4,5-tetrahydrobenz[c,d]indole-2-carboxylic acid hydrochloride (SM-31900). SM-31900 inhibited the binding of [3H]glycine and [3H]5,7-dichlorokynurenic acid, radioligands for the N-methyl-D-aspartate (NMDA) receptor glycine-binding site, to rat brain membranes in a competitive manner, with K(i) values of 11+/-2 and 1.0+/-0.1 nM, respectively, and completely prevented the binding of [3H]dizocilpine (MK-801), a radioligand for the NMDA receptor channel site. In cultures of rat cortical neurons, SM-31900 markedly prevented the neuronal cell death induced by transient exposure to glutamate, in a concentration-dependent manner. Its neuroprotective potency was much stronger than those of other glycine-binding site antagonists (4-trans-2-carboxy-5,7-dichloro-4-phenylaminocarbonylamino-1,2,3,4-tetrahydroquinoline (L-689,560), 5,7-dichlorokynurenic acid, and 7-chlorokynurenic acid). Furthermore, SM-31900 showed anticonvulsant activity when administered systemically, unlike other antagonists. These data indicate that SM-31900 is a systemically active antagonist with high affinity for the NMDA receptor glycine-binding site.     

神经元膜NMDA受体蛋白免疫复合物单分子纳米结构及定位AFM比较研究

目的探讨NMDAr(N-甲基-D-天冬氨酸受体)在神经细胞膜表面定位，对比不同成像方法的特点，建立纳米尺度神经细胞膜表面蛋白单分子三维标记的免疫细胞化学新方法。方法应用原子力显微镜，对分布在云母表面的抗NMDAR1亚单位IgG-葡萄球菌蛋白A-胶体金颗粒免疫标记复合物分子进行扫描，明确其特定的三维结构作为对照标准，然后扫描结合了免疫标记复合物分子的神经细胞膜表面，对表面形貌作出对比后确定目的抗原的定位和复合物三维结构。并与光镜、激光共聚焦、扫描电镜等方法进行对比。结果在空白云母表面，免疫复合物分子在原子力显微镜下呈现出均匀分散粒径为49nm的特征性球形结构。在神经元表面结合免疫标记物后可以发现有大量的粒径为53nm的球形或双球形(短棒状)结构，颗粒均匀，截面为双峰或单峰。光镜下染色成片状结果，在共聚焦显微镜下荧光呈颗粒点状分布，扫描电镜结果为单个或结合颗粒，缺乏三维成像能力。结论原子力显微镜下免疫胶体金标记技术可以在纳米尺度对目的膜受体蛋白进行定位和表面三维结构测定。NM-DA受体蛋白可以结合一个或两个胶体金复合物，与传统成像手段相比，原子力显微镜下胶体金标记方法可以对单个膜NMDA受体蛋白抗原进行定位、定性、定量标记测定。