CA2+ ENTRY VIA AMPA KA RECEPTORS AND EXCITOTOXICITY IN CULTURED CEREBELLAR PURKINJE-CELLS/

Initial studies of glutamate receptors activated by kainate (KA) found them to be Ca2+ impermeable. Activation of these receptors was though t to produce Ca2+ influx into neurons only indirectly by Na+-dependent depolarization. However, Ca2+ entry via AMPA/KA receptors has now bee n demonstrated in several neuronal types, including cerebellar Purkinj e cells. We have investigated whether such Ca2+ influx is sufficient t o induce excitotoxicity in cultures of cerebellar neurons enriched for Purkinje cells. Agonists at non-NMDA receptors induced Ca2+ influx in the majority of these cells, as measured by whole-cell voltage clamp and by fura-2 [Ca2+], microfluorimetry. To assess excitotoxicity, neur ons were exposed to agonists for 20 min and cell survival was evaluate d by a fluorescence assay 24 hr later. KA (100 mu M) reduced neuronal survival relative to controls to 43 +/- 3% when applied in Na+-contain ing solution and to 45 +/- 3% in Na+-free solution. This toxicity was blocked completely by CNQX but only slightly by 100 mu M Cd2+ and 50 m u M D-(-)-2-amino-5-phosphonovaleric acid. Both Purkinje neurons and n on-Purkinje cell types present in the cultures were similarly vulnerab le to toxic KA exposure, but the population marked by KA-induced Co2uptake was selectively diminished by the excitotoxicity. Na+-independe nt excitotoxicity could also be induced by domoate, AMPA, or glutamate . Compared to KA, NMDA was relatively ineffective in inducing cell dea th. Most of the KA-induced excitotoxicity could be blocked by removal of extracellular Ca2+ during the KA exposure and for a 5 min period th ereafter. Furthermore, antagonists of the Ca2+-activated enzymes nitri c oxide synthase and calpain significantly reduced the KA-induced cell death. These results show that non-NMDA receptor activation can cause excitotoxicity in cerebellar Purkinje neurons by mechanisms not invol ving Na+ influx, but rather depending on direct Ca2+ permeation and ac tivation of Ca2+-dependent enzymatic processes.