Persistent AMPA receptor stimulation alters [Ca²⁺]_i homeostasis in cultures of embryonic dopaminergic neurons

The effect of the ionotropic glutamate receptor agonist, AMPA, on intracellular Ca²⁺ concentrations ([Ca²⁺]_i) was studied in dopaminergic neurons present in primary cultures of ventral tegmental mesencephalon of 14 day rat embryos. Exposure of cells to 10 μM AMPA for 1 min increased [Ca²⁺]_i by 2-3 fold in dopaminergic and other neurons and this response was obliterated within 5 min by superfusion with AMPA-free incubation buffer. In dopaminergic neurons, 1 min or 5 min exposure to 50 μM AMPA increased [Ca²⁺]_i 3 to 5 times over control values. This rise in [Ca²⁺]₁ persisted even after a 20 min superfusion with AMPA-free media, whereas, [Ca²⁺]_i in non-dopaminergic neurons was reversed to control values during this time. Preincubation (2 min) of cultured cells with NBQX or the L-type channel blocker, nifedipine, but not with MK-801 blunted the rise of [Ca²⁺]_i in dopaminergic and other neurons. Pretreatment with 2 μM NBQX shifted the dose response curve for AMPA to the right without changing the basal [Ca²⁺]_i. The presence of 10 μM dantrolene, a blocker of Ca²⁺ release from intracellular stores, did not alter the initial rise of [Ca²⁺]_i elicited by 50 μM AMPA, but prevented the destabilization of Ca²⁺ homeostasis by facilitating the recovery to normal of basal [Ca²⁺]_i. Exposure to 50 μM AMPA (5 min) caused an irreversible increase of [Ca²⁺]_i in dopaminergic neurons and cell death was manifested by propidium iodide uptake 6-7 h after AMPA exposure. The present results show that in dopaminergic neurons the increase of [Ca²⁺]_i elicited by activation of AMPA receptors is mediated by sustained Ca²⁺ flux through voltage-gated channels and through a Ca²⁺-dependent Ca²⁺ release from intracellular stores. Protracted stimulation of AMPA and kainate receptors caused death of dopaminergic neurons before that of other neurons present in the same culture. The increased vulnerability of dopaminergic neurons to AMPA may represent a mechanism of pathological relevance during ontogenesis.