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 thought to produce Ca²⁺ influx into neurons only indirectly by Na⁺-dependent depolarization. However, Ca²⁺ entry via AMPA/KA receptors has now been demonstrated in several neuronal types, including cerebellar Purkinje cells. We have investigated whether such Ca²⁺ influx is sufficient to 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, neurons were exposed to agonists for 20 min and cell survival was evaluated by a fluorescence assay 24 hr later. KA (100 μM) reduced neuronal survival relative to controls to 43 ± 3% when applied in Na⁺-containing solution and to 45 ± 3% in Na⁺-free solution. This toxicity was blocked completely by CNQX but only slightly by 100 μM Cd²⁺ and 50 μM D-(-)-2-amino-5-phosphonovaleric acid. Both Purkinje neurons and non-Purkinje cell types present in the cultures were similarly vulnerable to toxic KA exposure, but the population marked by KA-induced Co²⁺ uptake was selectively diminished by the excitotoxicity. Na⁺-independent excitotoxicity could also be induced by domoate, AMPA, or glutamate. Compared to KA, NMDA was relatively ineffective in inducing cell death. Most of the KA-induced excitotoxicity could be blocked by removal of extracellular Ca²⁺ during the KA exposure and for a 5 min period thereafter. Furthermore, antagonists of the Ca²⁺-activated enzymes nitric 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 involving Na⁺ influx, but rather depending on direct Ca²⁺ permeation and activation of Ca²⁺-dependent enzymatic processes.