代谢型谷氨酸受体5mRNA与脑梗死的相关性研究

目的：观察急性脑缺血大鼠脑组织中mGluR5 mRNA在不同时段的表达变化的规律,探讨mGluR5 mRNA与脑梗死的相关性。方法：75只雄性Wistar大鼠按照随机数字表法分为正常对照组、手术组1、3、6、12、24 h及3 d、14 d组及其相对应的假手术对照组,采用大鼠MCAO模型,利用原位杂交技术,检测各组mGluR5 mRNA的表达。结果：与正常对照组相比,假手术对照组mGluR5 mRNA表达无明显改变（P〉0.05）。手术组mGluR5 mRNA各时间段表达均显著高于假手术对照组及正常对照组,缺血1 h其表达即有升高,比较差异均有统计学意义（P〈0.01）。mGluR5 mRNA表达6 h达到高峰（F=5.328,P〈0.00）,3 d后表达开始下降（P〈0.01）,14 d基本降至正常对照水平（P〉0.05）。结论：mGluR5在脑缺血后升高,提示mGluR5在脑梗死中有着重要作用,从而对我们的临床研究指出一条途径。     

脑型和味型代谢性谷氨酸受体的不同功能

目的　利用脑型及味型mGluR4在体内外的表达 ,检测两种受体结构功能上的差异 ,从而证实它们在信号转导功能上的不同。　方法　将克隆得到的脑型及味型mGluR4分别通过脂质体介导转染CHO细胞。在L MSG和L AP4的作用下 ,检测两种受体的功能。利用WesternBlot及原位杂交技术 ,分别检测它们在体内外的表达。　结果　转染脑型及味型mGluR4的CHO细胞 ,表达两种不同分子量的免疫活性蛋白 ,其中脑型分子量约为 12 0kD而味型约为 6 8kD。在L MSG和L AP4作用下 ,味型mGluR4对刺激物反应的浓度远高于脑型mGluR4。对大鼠脑组织及舌味蕾的原位杂交证明 ,mGluR4在小脑颗粒细胞有很高的表达 ,而在味蕾细胞表达率较低 ,阳性细胞仅占味蕾细胞的 5 %。　结论　味组织中的mGluR4与脑组织中mGluR4在结构和功能上有明显不同。味型mGluR4特异性表达于味蕾细胞 ,在高于脑内的浓度下 ,结合细胞外的谷氨酸 ,通过降低cAMP的浓度引发味觉信号转导。是谷氨酸味觉的特异性受体     

Glutamate transporters alterations in the reorganizing dentate gyrus are associated with progressive seizure activity in chronic epileptic rats.

The expression of glial and neuronal glutamate transporter proteins was investigated in the hippocampal region at different time points after electrically induced status epilepticus (SE) in the rat. This experimental rat model for mesial temporal lobe epilepsy is characterized by cell loss, gliosis, synaptic reorganization, and chronic seizures after a latent period. Despite extensive gliosis, immunocytochemistry revealed only an up-regulation of both glial transporters localized at the outer aspect of the inner molecular layer (iml) in chronic epileptic rats. The neuronal EAAC1 transporter was increased in many somata of individual CA1-3 neurons and granule cells that had survived after SE; this up-regulation was still present in the chronic epileptic phase. In contrast, a permanent decrease of EAAC1 immunoreactivity was observed in the iml of the dentate gyrus. This permanent decrease in EAAC1 expression, which was only observed in rats that experienced progressive spontaneous seizure activity, could lead to abnormal glutamate levels in the iml once new abnormal glutamatergic synaptic contacts are formed by means of sprouted mossy fibers. Considering the steady growth of reorganizing mossy fibers in the iml, the absence of a glutamate reuptake mechanism in this region could contribute to progression of spontaneous seizure activity, which occurs with a similar time course.     

Acute effects of acamprosate and MPEP on ethanol Drinking-in-the-Dark in male C57BL/6J mice

Background: Recently, a simple procedure in mice, Drinking‐in‐the‐Dark (DID), was hypothesized to have value for medication development for human alcoholism. In DID, mice are offered intermittent, limited access to ethanol over a series of days during the dark phase that results in rapid drinking to intoxication in predisposed genotypes. Methods: We measured the effects of acamprosate or MPEP, metabotropic glutamate 5 receptor (mGluR5) antagonist, on intake of 20% ethanol, plain tap water or 10% sugar water using the DID procedure in male C57BL/6J mice. Results: Acamprosate (100, 200, 300, or 400 mg/kg) dose dependently decreased ethanol drinking with 300 mg/kg reducing ethanol intake by approximately 20% without affecting intake of plain water or 10% sugar water. MPEP (1, 3, 5, 10, 20, or 40 mg/kg) was more potent than acamprosate with 20 mg/kg reducing ethanol intake by approximately 20% and for longer duration without affecting intake of plain water or 10% sugar water. Conclusions: These results support the hypothesis that mGluR5 signaling plays a role in excessive ethanol intake in DID and suggest DID may have value for screening novel compounds that reduce overactive glutamate signaling for potential pharmaceutical treatment of excessive ethanol drinking behavior.     

旋磁场对锂-匹鲁卡品癫痫大鼠模型的影响

目的 观察旋磁场对锂-匹鲁卡品癫痫大鼠模型癫痫发作和海马mGluR1、mGluR5表达的影响。方法 将癫痫组大鼠按60mg／kg体重腹腔注射氯化锂24h后，再给予匹鲁卡品（35mg／kg体重）腹腔注射致痫。观察旋磁场（场强为20mT）作用不同时间后对癫痫大鼠模型癫痫发作程度、潜伏期及终止时间的影响．并同时记录癫痫发作时的伴随症状及脑电数据；分析旋磁场对海马mGluR1、mGluR5表达的影响。用电泳图像分析系统计算mGluR1、mGluR5mRNA水平的相对含量，计算mGluR1mRNA与mGluR5mRNA的比值。结果 经为期8d的旋磁场作用后，癫痫大鼠发作终止时间明显提前，发作次数减少，其伴随症状减轻；模型组大鼠mGluR1mRNA水平明显增高，mGluR5mRNA水平明显降低，其mGluR1与mGluR5比值明显大于其它各组；各磁场作用组癫痫大鼠模型的基因表达与模型组正好相反，其mGluR5水平明显增高而mGluR1水平显著降低，尤其是经磁场作用8d后，这种变化更加显著。结论 20mT旋磁场对大鼠癫痫发作有一定的抑制作用，长时间的磁场作用对癫痫大鼠海马mGluR1mRNA和mGluR5mRNA表达有显著影响，但mGluR5表达增强的意义还有待进一步探讨。     

Co-activation of nAChR and mGluR induces V oscillation in rat medial septum diagonal band of Broca slices

Aim： To examine whether co-activation of nAChR and mGluR1 induced y oscillation （20-60 Hz） in rat medial septum diagonal band of Broca （MSDB） slices. Meth6ds： Rat brain sagittal slices containing the MSDB were prepared. Extracellular field potentials were recorded with glass microelectrodes. The nAChR and mGluR1 agonists were applied to the slices to induce network activity. Data analysis was performed off-line using software Spike 2. Results： Co-application of the nAChR agonist nicotine （1 pmol/L） and the mGluR1 agonist dihydroxyphenylglycine （DHPG, 25 pmol/L） was able to induce y oscillation in MSDB slices. The intensity of nAChR and mGluR1 activation was critical for induction of network oscillation at a low （8 oscillation） or high frequency （y oscillation）： co-application of low concentrations of the two agonists only increased the power and frequency of oscillation within the range of 8, whereas y oscillation mostly appeared when high concentrations of the two agonists were applied. Conclusion： Activation of mGluR1 and nAChR is able to program slow or fast network oscillation by altering the intensity of receptor     

Expression of the metabotropic glutamate receptor 5 (mGluR5) induces melanoma in transgenic mice

Glutamate is the major excitatory neurotransmitter in the mammalian CNS and mediates fast synaptic transmission upon activation of glutamate-gated ion channels. In addition, glutamate modulates a variety of other synaptic responses and intracellular signaling by activating metabotropic glutamate receptors (mGluRs), which are G protein-coupled receptors. The mGluRs are also expressed in nonneuronal tissues and are implicated in a variety of normal biological functions as well as diseases. To study mGluR-activated calcium signaling in neurons, we generated mGluR5 transgenic animals using a Thy1 promoter to drive expression in the forebrain, and one founder unexpectedly developed melanoma. To directly investigate the role of mGluR5 in melanoma formation, we generated mGluR5 transgenic lines under a melanocyte-specific promoter, tyrosinase-related protein 1. A majority of the founders showed a severe phenotype with early onset. Hyperpigmentation of the pinnae and tail could be detected as early as 3-5 d after birth for most of the mGluR5 transgene-positive mice. There was 100% penetrance in the progeny from the tyrosinase-related protein 1-mGluR5 lines generated from founders that developed melanoma. Expression of mGluR5 was detected in melanoma samples by RT-PCR, immunoblotting, and immunohistochemistry. We evaluated the expression of several cancer-related proteins in tumor samples and observed a dramatic increase in the phosphorylation of ERK, implicating ERK as a downstream effector of mGluR5 signaling in tumors. Our findings show that mGluR5-mediated glutamatergic signaling can trigger melanoma in vivo. The aggressive growth and severe phenotype make these mouse lines unique and a potentially powerful tool for therapeutic studies.