Calcium entry causes a prolonged refractory period in peptidergic neurons of Aplysia

Abstract

A brief train of electrical stimuli to the pleuroabdominal connective of Aplysia produces a cumulative depolarization in the peptidergic bag cell neurons within the abdominal ganglion. This response is followed by an afterdischarge which lasts for about 30 min, and then by a prolonged refractory period lasting for several hours. During the refractory period the cumulative depolarization in response to stimulation is attenuated, and stimulation either fails to initiate afterdischarges or produces discharges of much shorter duration. We have used the cationophore X537A to test the hypothesis that the prolonged refractory period is caused by calcium entry into the bag cell neurons during the afterdischarge. Exposure of intact bag cell clusters to X537A at concentrations from 1 to 10 microM for a period of 20 min in calcium-containing media produced no change in their resting potentials or in their ability to generate action potentials, but induced a state resembling natural refractoriness in response to subsequent stimulation. Both natural refractoriness and that induced by X537A could be overcome by extracellular tetraethylammonium ions (90 mM). Dose response data showed that concentrations of ionophore of 2.5 to 5.0 microM produce an attenuation of afterdischarge that is similar to that following a stimulated afterdischarge. These concentrations of X537A also produced an enhancement of 3H-labeled peptide release from these cells that is comparable to that observed on stimulation of an afterdischarge. Moreover, the time course of recovery from exposure to 5.0 microM X537A parallels that of natural refractoriness, recovery being essentially complete about 20 hr after X537A exposure or stimulation. The ionophore did not affect the mean duration of afterdischarge when applied in calcium-deficient media. The electrical effects of X537A were investigated using isolated bag cell neurons in cell culture. After treatment with the adenylate cyclase activator, forskolin, and theophylline, such isolated cells show many of the electrical changes occurring in intact bag cell clusters at the onset of afterdischarge, including the enhancement of action potentials, as well as increased input resistance and the emergence of oscillations in membrane potential. None of these parameters was significantly affected by concentrations of ionophore that induce refractoriness. In response to repetitive intracellular stimulation, however, some forskolin-treated cells undergo a cumulative depolarization which is similar to that seen at the onset of afterdischarge in intact clusters. This cumulative depolarization was found to be attenuated or abolished by 5.0 microM X537A.(ABSTRACT TRUNCATED AT 400 WORDS)