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

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 set), 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/set) 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 8benzylthio-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. It is known that the principal function of the peptidergic bag cell neurons of the abdominal ganglion of Aplysia is to “command” the onset of egg laying and its associated behavior by releasing their neurosecretory product, egg-laying hormone (ELH), during a long lasting afterdischarge (Kupfermann, 1970; Kupfermann and Kandel, 1970; Strumwasser et al., 1980; Stuart et al., ’ This work was supported by National Institutes of Health Grant NS 13896 to F. S. We thank Mr. John Scotese and Mr. Daniel P. Viele for preparing the primary cultures of bag cells used in these studies. ’ To whom reprint requests should be addressed. 1980). In the absence of external stimulation, the bag cells within an intact abdominal ganglion have relatively high resting potentials and show no spontaneous activity. Following either brief electrical stimulation of a presumed afferent pathway in the pleuroabdominal connectives or the extracellular application of peptides from the atrial gland (Dudek and Blankenship, 1977a; Heller et al., 1979, 1980; Kaczmarek et al., 1978; Kupfermann and Kandel, 1970), the bag cells depolarize and discharge repetitively, and in synchrony, for a duration of about 30 min, after which, the cells become relatively refractory to further stimulation (Dudek and Blankenship, 1977b; The Journal of Neuroscience CAMP Effect on Bag Cell Neurons 627 Kaczmarek et al., 1978; Kupfermann and Kandel, 1970). A variety of lines of evidence suggests that these electrical properties are controlled by the intracellular concentration of adenosine 3’:5’-monophosphate (CAMP) (Kaczmarek et al., 1978). Initiation of afterdischarge is associated with an -200% increase in CAMP levels within the bag cells which peaks around 2 min into afterdischarge and, thereafter, declines to control levels. The afterdischarge may be initiated by a number of CAMP analogues (Kaczmarek et al., 1978) including 8-benzylthio-CAMP, 8-methylthio-CAMP, and o-NOz-benzylCAMP ester (whereas analogues of guanosine 3’:5’-monophosphate are without effect; L. K. Kaczmarek and J. Nerbonne, unpublished results). In addition, the duration of the afterdischarge may be greatly prolonged by phosphodiesterase inhibitors (Kaczmarek et al., 1978). Our present studies are an attempt to determine how much of the afterdischarge property is intrinsic to single bag cells isolated in cell culture (Kaczmarek et al., 1979; Strumwasser et al., 1978). In the intact abdominal ganglion, the processes of the bag cell neurons form a complex network that is coupled by gap junctions (Kaczmarek et al., 1979). Cell culture offers the advantage of making electrical measurements and intracellular injection possible without the complications of the electrical, and metabolic, coupling that results from such interconnections (Kaczmarek et al., 1979; Blankenship and Haskins, 1979). We have studied the effects of the membranepermeant, phosphodiesterase-resistant CAMP analogue, 8-benzylthio-CAMP (8BT-CAMP) (Meyer and Miller, 1974; Treistman and Levitan, 1976) on the properties of isolated bag cell neurons and show that such isolated neurons respond to this analogue with enhanced spike electrogenesis, increased membrane resistance, and the onset of oscillations in membrane potential that drive a long lasting discharge. Moreover, in the presence of this analogue, a brief bout of spike production may generate a prolonged afterdischarge. We suggest that these changes are the result of a long lasting decrease in potassium ion conductance. Materials and Methods Aplysia californica was collected locally (southern California coast) by Mr. John Scotese. The animals were kept at 14°C and all electrophysiological experiments were carried out at this temperature. Abdominal ganglia were dissected out and incubated at 22°C for 6 hr in filtered seawater containing 1.25% neutral protease with neomycin sulfate (100 pg/ml) (Kaczmarek et al., 1979; Strumwasser et al., 1978). After removing the connective tissue capsules, the bag cells were disaggregated using a Pasteur pipette and seeded into 35-mm Falcon tissue culture dishes containing modified L-15 medium (Leibovitz, 1963) obtained from Gibco and made up in artificial seawater containing 15 mM Hepes (4(2-hydroxyethyl) lpiperazine-ethanesulfonic acid, pH 7.8) and neomycin sulfate (50 pg/ml). The cells rapidly attached to the bottom of the culture dishes and within 2 or 3 days, had developed elaborate neuritic branches (Strumwasser et al., 1978, 1980). Electrophysiological experiments usually were carried out within 1 week of seeding. Glass microelectrodes were pulled on a Brown-Flaming electrode puller and filled with 2 M potassium citrate. These electrodes had resistances of 30 to 100 megohms. Cultured bag cell neurons were penetrated, under visual control, on a Leitz Diavert microscope. Recording of membrane potential was through an M701-WPI electrometer and the intensity of current applied to depolarize or hyperpolarize the cell was monitored using either the current monitor circuit of the electrometer or an independent virtual ground circuit. When cells were impaled with a single electrode, which was used for both voltage recording and passage of current, the input resistance of the cells was estimated by measuring the maximum voltage displacements to a range of small hyperpolarizing currents after correction for bridge imbalance. In some experiments, however, the cells were penetrated with a second microelectrode which was used for the passage of imposed currents. For pressure microinjection of 8BT-CAMP, the tips of microelectrodes were brushed against a ground glass plate, to produce final tip diameters of -1.0 pm. These electrodes were filled with 20 mM 8BT-CAMP, 0.3 M KC1 and mounted in an electrode holder connected to a cylinder of Nz gas so that pulses of pressure (15 to 45 psi, 2 set to 2 min) could be applied to the interior of the electrodes under control of a solenoid-operated threeway valve (McCaman et al., 1977). Pressure injection into bag cells was carried out as described elsewhere (Kaczmarek et al., 1980). To achieve the final concentrations of pharmacological agents used in this study, 30 to 300 ~1 of a more concentrated solution were added directly to the 3 ml of L-15 medium bathing the cultured cells in a 35-mm Falcon culture dish. 8BT-CAMP was obtained from ICN (standard and high pressure liquid chromatography (HPLC) pure grades).