DEVELOPMENTAL EXPRESSION OF N-METHYL-D-ASPARTATE (NMDA)-INDUCED NEUROTOXICITY, NMDA RECEPTOR FUNCTION, AND THE NMDAR1 AND GLUTAMATE-BINDINGPROTEIN SUBUNITS IN CEREBELLAR GRANULE CELLS IN PRIMARY CULTURES

Cerebellar granule cells maintained in vitro as primary cultures are a relatively homogeneous neuronal population that can be used to evalua te the developmental expression of neurotransmitter receptors and to a ssess their role in cell survival and degeneration. The toxicity induc ed by N-methyl-D-aspartate (NMDA) in granule cells maintained under pa rtially depolarizing conditions and in the presence of physiologic ext racellular concentrations of Mg2+ was greatest for the neurons maintai ned for 14 days in vitro (DIV). However, following NMDA receptor activ ation neurons as young as 5 DIV exhibited increases in the concentrati on of intracellular free Ca2+ which were as large as those achieved wi th cells at 8-9 or 13-14 DN. The less mature neurons exhibited a ''dow n-regulation'' of responses to increasing concentrations of NMDA and t he more mature cells maintained elevated intracellular Ca2+ levels dur ing the inter-stimulus periods. Immunochemical analyses of the express ion of the NMDA receptor-associated proteins NMDAR1 and glutamate-bind ing protein (GBP) in granule cells indicated a developmental increase in both proteins, albeit the pattern of expression of NMDAR1 was the m ore complex. No definite correlation has yet been established between toxicity induced by NMDA and the expression of these two proteins. Fin ally, although the developmental expression of nitric oxide synthase, an enzyme that catalyzes the formation of the potentially neurotoxic r adicals nitric oxide and superoxide anion, increased progressively wit h the maturation of neurons in culture, an inhibitor of this enzyme di d not protect neurons from NMDA-induced toxicity. Therefore, the devel opmental changes in granule cells that lead to increased vulnerability following excessive activation of NMDA receptors are not yet complete ly defined.