Mechanisms and Effects of Intracellular Calcium Buffering on Neuronal Survival in Organotypic Hippocampal Cultures Exposed to Anoxia/Aglycemia or to Excitotoxins

摘要

Neuronal calcium loading attributable to hypoxic/ischemic injury is believed to trigger neurotoxicity. We examined in organotypic hippocampal slice cultures whether artificially and reversibly enhancing the Ca²⁺ buffering capacity of neurons reduces the neurotoxic sequelae of oxygen–glucose deprivation (OGD), whether such manipulation has neurotoxic potential, and whether the mechanism underlying these effects is pre- or postsynaptic. Neurodegeneration caused over 24 hr by 60 min of OGD was triggered largely by NMDA receptor activation and was attenuated temporarily by pretreating the slices with cell-permeant Ca²⁺ buffers such as 1,2 bis(2-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid acetoxymethyl ester (BAPTA-AM). This pretreatment produced a transient, reversible increase in intracellular buffer content as demonstrated autoradiographically using slices loaded with ¹⁴C-BAPTA-AM and by confocal imaging of slices loaded with the BAPTA-AM analog calcium green-acetoxymethyl ester (AM). The time courses of¹⁴C-BAPTA retention and of neuronal survival after OGD were identical, indicating that increased buffer content is necessary for the observed protective effect. Protection by Ca²⁺buffering originated presynaptically because BAPTA-AM was ineffective when endogenous transmitter release was bypassed by directly applying NMDA to the cultures, and because pretreatment with the low Ca²⁺ affinity buffer 2-aminophenol-N,N,O-triacetic acid acetoxymethyl ester, which attenuates excitatory transmitter release, attenuated neurodegeneration. Thus, in cultured hippocampal slices, enhancing neuronal Ca²⁺ buffering unequivocally attenuates or delays the onset of anoxic neurodegeneration, likely by attenuating the synaptic release of endogenous excitatory neurotransmitters (excitotoxicity).