Role of NMDA receptors and voltage-activated calcium channels in an in vitro model of cerebral ischemia

Abstract

In an in vitro model of cerebral ischemia we investigated the functional consequences of repeated hypoxias and the potential protective effect of the N-methyl- d-aspartate (NMDA) receptor antagonist d-2-amino-5-phosphonovaleric acid ( d-APV) and the calcium channel blocker verapamil in preventing the expression of pathophysiological activity. Rat neocortical slices were exposed to nitrogen for 2–13 min and the hypoxia-induced functional modifications were monitored in layer II/III by recording the extracellular DC potential, the extracellular calcium concentration ([Ca 2+] o) and the stimulus-evoked synaptic responses. Hypoxia caused a reversible 2.4–24.6 mV negative shift in the extracellular DC potential associated with a [Ca 2+] o decrease from 1.2 to 0.2 mM and a complete loss of synaptic responsiveness. Repeating hypoxias induced an increase in the amplitude of this anoxic depolarization (AD) and a significant decrease in the AD onset latency. Synaptic responses partially recovered at 20 and 60 min intervals between subsequent hypoxic periods, indicating that the initial AD did not induce any short-term irreparable functional deficits. Verapamil (50 μM) caused an increase in the AD onset latency. However, in comparison to untreated controls, verapamil induced a reduction of excitatory and inhibitory responses during hypoxia probably by blocking voltage-activated calcium conductances. In addition, verapamil did not have any significant effect on the hypoxia-induced reduction of [Ca 2+] o. Bath application of d-APV (30 μM) prevented the significant reduction in the AD onset latency to the second hypoxia, but had no significant effect on the AD amplitude and duration. The hypoxia-induced decrease in [Ca 2+] o was not altered after addition of d-APV to the bathing medium. These data indicate that the influx of calcium through voltage-activated calcium channels and the NMDA receptor-gated ionophore does not significantly contribute to the massive depolarization observed under hypoxic conditions.