MECHANISMS AND EFFECTS OF INTRACELLULAR CALCIUM BUFFERING ON NEURONALSURVIVAL IN ORGANOTYPIC HIPPOCAMPAL CULTURES EXPOSED TO ANOXIA AGLYCEMIA OR TO EXCITOTOXINS/

Neuronal calcium loading attributable to hypoxic/ischemic injury is be lieved to trigger neurotoxicity. We examined in organotypic hippocampa l slice cultures whether artificially and reversibly enhancing the Ca2 + buffering capacity of neurons reduces the neuro toxic sequelae of ox ygen-glucose deprivation (OGD), whether such manipulation has neurotox ic potential, and whether the mechanism underlying these effects is pr e- or postsynaptic, Neurodegeneration caused over 24 hr by 60 min of O GD was triggered largely by NMDA receptor activation and was attenuate d temporarily by pretreating the slices with cell-permeant Ca2+ buffer s such as 1,2 bis(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid ac etoxymethyl ester (BAPTA-AM), This pretreatment produced a transient, reversible increase in intracellular buffer content as demonstrated au toradiographically using slices loaded with C-14-BAPTA-AM and by confo cal imaging of slices loaded with the BAPTA-AM analog calcium green-ac etoxymethyl ester (AM). The time courses of C-14-BAPTA retention and o f neuronal survival after OGD were identical, indicating that increase d buffer content is necessary for the observed protective effect, Prot ection by Ca2+ buffering originated presynaptically because BAPTA-AM w as ineffective when endogenous transmitter release was bypassed by dir ectly applying NMDA to the cultures, and because pretreatment with the low Ca2+ affinity buffer 2-aminophenol-N,N,O-triacetic acid acetoxyme thyl ester, which attenuates excitatory transmitter release, attenuate d neurodegeneration. Thus, in cultured hippocampal slices, enhancing n euronal Ca2+ buffering unequivocally attenuates or delays the onset of anoxic neurodegeneration, likely by attenuating the synaptic release of endogenous excitatory neurotransmitters (excitotoxicity).