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 Ca 2ϩ 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 preor 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 Ca 2ϩ buffers such as 1,2 bis(2-aminophenoxy)ethane-N,N,NЈ,NЈ-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 14 C-BAPTA-AM and by confocal imaging of slices loaded with the BAPTA-AM analog calcium green-acetoxymethyl ester (AM). The time courses of 14 C-BAPTA retention and of neuronal survival after OGD were identical, indicating that increased buffer content is necessary for the observed protective effect. Protection by Ca 2ϩ 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 Ca 2ϩ 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 Ca 2ϩ buffering unequivocally attenuates or delays the onset of anoxic neurodegeneration, likely by attenuating the synaptic release of endogenous excitatory neurotransmitters (excitotoxicity).

1 The effects of a range of structurally-dissimilar compounds which possess affinity for ai binding sites were examined on the responses of cultured hippocampal pyramidal neurones to the excitatory amino acid analogues N-methyl-D-aspartate (NMDA), kainate and (RS)-a-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA). 2 In mouse hippocampal neurones under whole-cell voltage-clamp, the compounds tested reversibly attenuated NMDA-, but not kainate-or AMPA-, evoked currents with a rank order potency (ICm3 The attenuation of NMDA-evoked responses was not mediated through interactions with the agonist, glycine (except haloperidol) or polyamine (except ifenprodil) binding sites on the NMDA receptor-channel complex but, in the light of the voltage-and, in some cases, use-dependent nature of their antagonism, an interaction with the ion channel appears to be a likely mechanism of action for many of the compounds. 4 Micromolar concentrations of selected ai site ligands also reduced NMDA-evoked rises in intracellular free calcium concentration in Fura-2-loaded cultured hippocampal neurones of the rat with the same rank order potency as observed in the electrophysiological studies. 5The data indicate that, at micromolar concentrations, the ai site ligands tested act as NMDA receptor antagonists, an action which does not appear to be mediated by high-affinity binding site(s). The functional effects of micromolar concentrations of ai site ligands cannot, therefore, be attributed exclusively to interactions with high-affinity ai binding sites.

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