The effects of artificial calcium buffers on calcium responses and glutamate-mediated excitotoxicity in cultured hippocampal neurons

After loading cultured rat hippocampal neurons with the acetoxymethyl ester of the Ca²⁺ buffer BAPTA, or its dimethyl analogue DMB, the magnitudes of transient (20-25 s) depolarization- or excitatory amino acid- induced Ca²⁺ responses were reduced, as were the rates of increase and recovery of [Ca²⁺](i). In contrast, during prolonged (3-30 min) stimulation, the magnitudes of the Ca²⁺ responses were not reduced in buffer-loaded neurons, even though the rates of increase and recovery were still much slower compared to neurons loaded with the control molecule half- BAPTA-AM. The potential consequences of this action of BAPTA and DMB were then examined in an in vitro model of excitotoxicity in which we found that, in both fetal and postnatal cultures, glutamate-induced excitotoxicity was enhanced, rather than reduced. An additional and unexpected observation was that during exposure of neurons to solutions containing BAPTA-AM, dimethyl- BAPTA-AM, or half-BAPTA-AM, we observed a rapid but reversible increase in intracellular [Ca²⁺] that appeared to be mediated via an activation of voltage-operated Ca²⁺ channels; most probably due to a direct depolarizing effect. We suggest that the presence of artificial Ca²⁺ buffers interferes with the normal Ca²⁺-dependent mechanisms for limiting Ca²⁺ entry during stimulation and thereby leads to an enhanced net Ca²⁺ influx. One consequence of this action is to enhance the potency of glutamate as an excitotoxic agent. These results agree with previous observations that excitotoxicity is better correlated with the total net flux of Ca²⁺, rather than measurements of intracellular ionic Ca²⁺. Our results do not support a potential use of artificial Ca²⁺ buffers as neuroprotective agents.