The expression of long lasting afterdischarge by isolated Aplysia bag cell neurons

Abstract

The cluster of neuropeptidergic bag cells in the abdominal ganglion of Aplysia is able to generate a long lasting afterdischarge of compound action potentials following brief electrical stimulation of the pleurovisceral connective nerve or extracellular addition of cyclic adenosine 3':5'-monophosphate (cAMP) analogues. We have now examined the response of individual cultured bag cells to the cAMP analogue, 8-benzylthio-cAMP, applied extracellularly or intracellularly. In common with bag cells in an intact cluster, individual cultured bag cells respond to extracellular application of 0.5 mM-8-benzylthio-cAMP by generating a long lasting discharge of action potentials (mean duration = 123 min). The discharge, in cultured cells, started after a mean delay of 26 min following addition of the cAMP analogue. This long delay suggests an intracellular site of action for the cAMP analogue. The discharge could be blocked by cobalt ions but not by tetrodotoxin. The frequency of the 8-benzylthio-cAMP-induced discharge often could be increased by brief repetitive intracellular stimulation. Moreover, if, after addition of benzylthio-cAMP, discharge was prevented by a hyperpolarizing bias current, a short burst of spikes (5 to 15 sec), produced by releasing the hyperpolarization, could induce a prolonged afterdischarge in the presence of the bias current. The onset of the discharge induced in cultured cells by 8-benzylthio-cAMP was preceded by an increase in membrane resistance (mean increase = 68%) and a decrease in the threshold for spikes evoked by depolarizing current pulses. The width of single action potentials evoked by a threshold depolarizing pulse of constant current was augmented following 8-benzylthio-cAMP. Moreover, frequency-dependent spike broadening also was enhanced markedly. Fully broadened action potentials generated by repetitive current pulses (~1 pulse/sec) were approximately double the width of controls and were characterized by a prominent shoulder on their falling phase. This enhancement of spike broadening preceded the onset of the discharge. In 3 out of 17 experiments, a regenerative hyperpolarizing response was also observed following 8-benzylthio-cAMP. This transient response was associated with an increase in membrane conductance. Direct intracellular microinjection of 8-benzylthio-cAMP into bag cell neurons also enhanced spike genesis, increased membrane resistance, and produced subthreshold oscillations in membrane potential and spontaneous discharge. The response of cultured bag cells to 8-benzylthio-cAMP qualitatively resembles the response of intact bag cell systems to either cAMP analogues or electrical stimulation. Quantitative differences exist, however, particularly in the frequency of firing and mode of onset of discharge in these two systems. The response of the cultured bag cells is consistent with the hypothesis that cAMP induces a decrease in the membrane conductance to potassium ions.