EXCITOTOXIN EFFECTS ON SPINAL CORD IN VITRO

兴奋性氨基酸（EAA）作用于特定的膜受体， 在许多神经系统疾病中促进神经元变性或死亡， 与运动神经元疾病的发病机制有特别的关系。 EAA介导的对解剖学和组织化学定义的神经元的损伤可以 在出生后大鼠脊髓的器官型滚管培养中进行检查 软线（OTC-SC）。 我们的研究表明， OTC-SC通过兴奋毒素，N-甲基-D-天冬氨酸（NMDA）。 这个项目 检查NMDA和其他EAA激动剂对胆碱能标记物的作用， 使用OTC-SC系统的体外脊髓。 首先，将用NMDA处理OTC-SC，用或不用特异性NMDA。 对手。 脊髓胆碱能系统的损伤将通过以下方法进行评估： 染色培养物后的组织学和形态测定分析 乙酰胆碱酯酶活性（AChE）和胆碱乙酰转移酶（CHAT）- 免疫反应性，并通过ACHE和CHAT活性的生化分析。 将检查培养物的乳酸脱氢酶流出，以获得 神经元损伤的总体评估。 接下来，剂量-反应曲线和 将确定影响的时间方面。 第三，将培养物用EAA激动剂红藻氨酸或 使君子酸 使用特定的NMDA受体拮抗剂或非NMDA受体拮抗剂， 受体拮抗剂，损害的程度是通过介导的 将确定不同的EAA受体亚型。 在OTC-SC中，与红藻氨酸或NMDA相比，L-谷氨酸的损伤较小 比在许多培养系统中，更密切地反映其在体内的作用。 因此，第四个具体目标是研究两种可能的 降低谷氨酸作用的机制：谷氨酸的作用 摄取和犬尿烯酸作为内源性EAA拮抗剂的作用。 第五，我们将确定一种有效的生长抑素类似物SMS 201- 995，可以改变NMDA，L-谷氨酸或其他 兴奋毒素对OTC-SC中神经元的影响。 这项研究的长期目标是更好地了解 脊髓神经元变性和死亡的兴奋性毒素。

Excitatory amino acids (EAAs) acting at specific membrane receptors may promote neuron degeneration or death in many nervous system diseases and have been specifically implicated in pathogenesis of motor neuron diseases. EAA-mediated damage to anatomically and histochemically defined neurons can be examined in organotypic roller tube cultures of postnatal rat spinal cord (OTC-SCs). Our studies show damage to ventral and dorsal horns in OTC-SCs by the excitotoxin, N-methyl-D-aspartic acid (NMDA). This projects examines actions of NMDA and other EAA agonists on cholinergic markers in spinal cord in vitro using the OTC-SC system. First, OTC-SCs will be treated with NMDA, with or without specific antagonists. Damage to spinal cord cholinergic systems will be assessed by histological and morphometric analysis after staining cultures for acetylcholinesterase activity (AChE) and choline acetyltransferase (CHAT) - immunoreactivity, and by biochemical analysis of ACHE and CHAT activities. Lactate dehydrogenase efflux from cultures will be examined to obtain an overall assessment of neuronal damage. Next, dose-response curves and temporal aspects of the effects will be determined. Third, cultures will be treated with the EAA agonists, kainic acid or quisqualic acid. Using specific NMDA-receptor antagonists or non-NMDA- receptor antagonists, the extent to which damage is mediated through different EAA receptor subtypes will be determined. In OTC-SCs, damage by L-glutamate compared with kainate or NMDA is less than in many culture systems, reflecting more closely its effect in vivo. Therefore, the fourth specific aim is to investigate two possible mechanisms for the decreased effect of glutamate: the role of glutamate uptake and the role of kynurenic acid as an endogenous EAA antagonist. Fifth, we will determine whether a potent somatostatin analogue, SMS 201- 995, can alter the toxic effects of NMDA, L-glutamate, or other excitotoxins on neurons in OTC-SCs. The long-term objective of this research is to understand better the role of excitotoxins in spinal neuron degeneration and death.