EXCITOTOXIN EFFECTS ON SPINAL CORD IN VITRO

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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.