Developmental expression of N-methyl-d-aspartate (NMDA)-induced neurotoxicity, NMDA receptor function, and the NMDAR1 and glutamate-binding protein subunits in cerebellar granule cells in primary cultures

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Abstract
Cerebellar granule cells maintained in vitro as primary cultures are a relatively homogeneous neuronal population that can be used to evaluate the developmental expression of neurotransmitter receptors and to assess their role in cell survival and degeneration. The toxicity induced by N-methyl-d-aspartate (NMDA) in granule cells maintained under partially depolarizing conditions and in the presence of physiologic extracellular concentrations of Mg2+ was greatest for the neurons maintained for 14 days in vitro (DIV). However, following NMDA receptor activation neurons as young as 5 DIV exhibited increases in the concentration of intracellular free Ca2+ which were as large as those achieved with cells at 8–9 or 13–14 DIV. The less mature neurons exhibited a “down-regulation” of responses to increasing concentrations of NMDA and the more mature cells maintained elevated intracellular Ca2+ levels during the inter-stimulus periods. Immunochemical analyses of the expression of the NMDA receptor-associated proteins NMDAR1 and glutamatebinding protein (GBP) in granule cells indicated a developmental increase in both proteins, albeit the pattern of expression of NMDAR1 was the more complex. No definite correlation has yet been established between toxicity induced by NMDA and the expression of these two proteins. Finally, although the developmental expression of nitric oxide synthase, an enzyme that catalyzes the formation of the potentially neurotoxic radicals nitric oxide and superoxide anion, increased progressively with the maturation of neurons in culture, an inhibitor of this enzyme did not protect neurons from NMDA-induced toxicity. Therefore, the developmental changes in granule cells that lead to increased vulnerability following excessive activation of NMDA receptors are not yet completely defined.