INDUCTION OF THE PERMEABILITY TRANSITION PORE IN CULTURED STRIATAL NEURONS UPON CHRONIC NMDA RECEPTOR ACTIVATION

Conrad C. Alano (1), Gisela Beutner (1), Robert A. Gross (1,2), Shey-Shing Sheu (1)
(1) Department of Pharmacology and Physiology
(2) Department of Neurology, University of Rochester School of Medicine and Dentistry, Rochester, NY

Impairment of bioenergetics and calcium homeostasis may play a role in the pathogenesis of neurodegenerative states, such as Parkinson's and Huntington's diseases. In the present study, we evaluated the effects of N-methyl-D-aspartic acid- (NMDA) on cytosolic and mitochondrial calcium homeostasis in cultured striatal neurons. Striatal neurons were treated with NMDA (1mM) and [Ca2+]m and [Ca2+]c fluorescence were assayed using the calcium fluorescent indicators rhod-2 and either fura-2 (high affinity) or BTC (low affinity), respectively. NMDA receptor activation resulted in a sudden increase in [Ca2+]c in both fura-2 and BTC loaded cells, as expected. This rapid increase in [Ca2+]c was followed by a gradual increase. Approximately 20 min after continuous exposure of NMDA, a second sudden increase in [Ca2+]c was observed. This second sudden increase in [Ca2+]c was not observed in cells pretreated with CsA (100nM). Interestingly, the cells loaded with BTC showed a larger increase in [Ca2+]c compared to those in cells loaded with fura-2 upon activation of the NMDA receptor. Under similar experimental conditions, treatment with NMDA resulted in an increase in [Ca2+]m, which is followed by a precipitous drop in signal enfin. This decrease is also prevented by CsA. These results suggest that NMDA receptor activation in living neurons can induce a permeability transition across the mitochondrial membrane. These results also suggest that using the high-affinity dye fura-2 may lead to an underestimation of [Ca2+]c levels upon prolonged activation of NMDA receptor, and that this can be corrected for by using the low affinity dye BTC.

This work was supported by grants National Institute of Health Grant HL-33333 and DA/0D10J14, the Council for Tobacco Research Grant 4299, and the Pharmacological Sciences Training Grant GM 08427.