Extracellular ions during veratridine-induced neurotoxicity in hippocampal slices: neuroprotective effects of flunarizine and tetrodotoxin

Abstract

Veratridine, by blocking Na + channel inactivation and shifting activation to more negative membrane potentials 5, causes Na +-influx and a persistent tendency for depolarization. Veratridine is neurotoxic to cultured neurones 43, and this neurotoxicity can be blocked by the class IV calcium antagonist, flunarizine 37. We were interested to know whether similar effects could be found in a functional differentiated tissue containing adult neurones and glial cells. We examined this in hippocampal slices using extracellular potential recordings and ion-selective microelectrodes sensitive to [Na +] o, [Ca 2+] o and [K +] o. Veratridine blocked synaptic transmission in CA1, and induced several episodes of spreading depression (SD). This was followed by a long-lasting increase in [K +] o and a continuous decrease in [Ca +] o. Following veratridine exposure to hypoxia only revealed a small negative DC shift and small shifts in extracellular ions; indicating that the cells had lost the ability to maintain ion homeostasis before the hypoxia, and that veratridine had been neurotoxic. In hippocampal slices obtained from guinea pigs which had been pretreated with 40 mg/kg × 2 flunarizine orally the time before the first SD induced by veratridine was doubled. Although the ion shifts during the first SD were similar to controls, flunarizine reduced the time of recovery of [Ca 2+] o, [K +] o and DC potential. The increase in [K +] o baseline and the massive decrease in [Ca 2+] o baseline seen following the SDs in the solvent group were smaller in the flunarizine-treated slices. During the subsequent hypoxic period the negative DC shift was 8× larger in the flunarizine group, and the shifts in [K +] o, [Na +] o and [Ca 2+] o were bigger. Tetrodotoxin also delayed the first SD during veratridine and increased the size of the DC shift during the subsequent hypoxic period. Both flunarizine and tetrodotoxin therefore protected adult brain tissue containing glia from the neurotoxicity of veratridine. These findings suggest that persistent Na +-influx and the consequent Ca 2+-influx produce neurotoxicity, and that the ability to attenuate this neurotoxicity may be important in the mechanism of action of cerebroprotective drugs from different pharmacological classes.