Augmentation of bursting pacemaker activity by egg-laying hormone in Aplysia neuron R 15 is mediated by a cyclic AMP-dependent increase in Ca 2 " and K + currents ( neuropeptide / intraceliular messenger / voltage clamp )

Release of the neuropeptide egg-laying hormone (ELH) from Aplysia bag cell neurons augments the endogenous bursting pacemaker activity of neuron R15. We have studied the ionic mechanisms underlying the effect ofELH in voltage-clamped R15 neurons. Both electrical discharge of the bag cells, which releases endogenous ELH, and application of synthetic ELH on cell R15 result in an increase in two discrete ionic currents. One of these currents activates with hyperpolarization, reverses near the K+ equilibrium potential, is sensitive to the external K+ concentration, and is blocked by addition of 5 mM Rb+ or 1 mM Ba21 to the bathing medium. This current appears to be identical to the inwardly rectifying K+ current IR The other current activates with depolarization and is blocked by replacement of external Ca2+ with Co2+ or Mn2+. This current appears to be a voltage-gated Ca2+ current ICa. Both ICa and IR in R15 have previously been shown to be enhanced by the neurotransmitter serotonin, acting via intracellular cyclic AMP. We now report that increasing cyclic AMP in R15, by applying either serotonin or the adenylate cyclase activator forskolin together with a phosphodiesterase inhibitor, mimics and occludes the action of ELH on neuron R15. Furthermore, application of ELH increases the cyclic AMP content of single R15 neurons, as measured by radioimmunoassay. Finally, the effects ofELH are potentiated by a phosphodiesterase inhibitor. These results suggest that ELH augments bursting activity in R15 by causing cyclic AMPmediated increases in IR and Ic.. The electrical activity of bursting pacemaker neurons can be modulated by a variety of neurotransmitters and hormones (1-3). Often the effect of neuromodulatory substances on bursting neurons is neither simply excitatory nor simply inhibitory. Instead, neuromodulators often cause changes in the pattern of rhythmic activity. The ionic and intracellular mechanisms of these complex changes in electrical activity of bursting cells are not well understood. The endogenously generated bursting activity of neuron R15 from the abdominal ganglion of Aplysia can be modulated by biogenic amines (4-6), neuropeptides (7, 8), and synaptic stimuli (9, 10). The bursting activity of R15 is also modulated by repetitive firing ("afterdischarge") of an electrically coupled cluster of neurosecretory "bag cell" neurons, also found in the abdominal ganglion (11). The bag cell neurons synthesize several neuropeptides, which are released during the afterdischarge. These peptides initiate egg-laying and associated behaviors by affecting both the reproductive organs and central neurons of Aplysia (12-15). Release of the neuropeptide egg-laying hormone (ELH) from bag cell neurons, or bath application of purified native ELH, results in a long-lasting (up to 3 hr) increase in the number and frequency of spikes during bursts in R15 (7, 14). In addition, ELH induces an increase in the amplitude of the interburst hyperpolarization (7, 14). Hence, ELH augments the bursting activity of R15, apparently by affecting both the depolarizing and hyperpolarizing phases of the burst cycle. In this study, the effects of ELH on ionic currents were examined in voltage-clamped R15 neurons. We show here that two ionic currents are enhanced by ELH: an inward (or anomalously) rectifying K+ current (IR) and a voltage-gated Ca2l current (ICa). It has been demonstrated previously that both IR and ICa are enhanced by serotonin (5-HT; 5hydroxytryptamine) and that the increase in these currents is mediated by a 5-HT-stimulated increase in the intracellular level of cyclic AMP in cell R5 (5, 6, 16). Here we present evidence that the enhancement of ICa and IR by ELH is likewise mediated by an increase in cyclic AMP. MATERIALS AND METHODS Aplysia californica (200-300 g) were obtained from Alacrity Marine Biologicals (Redondo Beach, CA). Individuals that had laid eggs during the previous 1 or 2 days were not used. The animals were injected with 100 ml of 400 mM MgCl2, the abdominal ganglion was removed and pinned to the Sylgard base of a 1-ml chamber, and the connective tissue sheath overlying the neuronal cell bodies was removed. In bag cell discharge experiments, only the portion of the sheath covering the soma of R15 was removed. The preparation was continuously superfused at 1 ml/min (unless indicated otherwise) with saline containing 460 mM NaCl, 55 mM MgCl2, 11 mM CaCl2, 10 mM KCl, and 10 mM Na Hepes (pH 7.4). In some experiments, the saline contained 100 mM MgCl2 to reduce spontaneous synaptic activity, and NaCl was lowered to 392 mM to maintain osmolarity. Neuron R15 (17) was impaled with one or two electrodes (2-10 Mfl) containing either 0.5 M K2SO4 or 1.5 M KCl. Steady-state current vs. voltage (I-V) curves were constructed from ionic currents measured with the use of a conventional oneor twoelectrode voltage clamp system. All experiments were performed at least three times unless otherwise indicated. In most experiments, ELH was applied by "puffing" 1 dul of saline containing 40 A.M synthetic ELH (kindly provided by F. Strumwasser, Boston University) over the soma of R15. Synthetic ELH produces egg-laying behavior indistinguishable from that seen after injection of native purified ELH into Aplysia (18). The ELH saline was applied from the drawn-out tip of polyethylene tubing positioned 400 ,um away from the soma of R15. Tracer dye indicated that the ELH Abbreviations: ELH, egg-laying hormone; 5-HT, serotonin; ICa' voltage-gated Ca2" current; IR, inward rectifying K+ current. *Present address: Medical Research Council Molecular Neurobiology Unit, University of Cambridge Medical School, Hills Road, Cambridge CB2 2QH, England. tTo whom reprint requests should be addressed. 6307 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 6308 Neurobiology: Levitan et al. saline was washed away from R15 in 1-2 min. Puffing normal saline at R15 produced no effects. Bag cell afterdischarges were elicited by electrically stimulating the left bag cell cluster with a suction electrode. Occurrence ofthe discharge was confirmed by extracellularly monitoring with a second suction electrode the activity of the right bag cell cluster, which is electrically coupled to the left cluster. Application of ELH in experiments in which cyclic AMP content was measured was done by one of two protocols. In two experiments, R15 was voltage-clamped to -75 mV in a 200-,ul chamber. Then 100 /.l of a 24 ,iM ELH solution was injected into the chamber. In a third experiment, 3 tkl of 40 tkM ELH was puffed over spontaneously bursting R15 neurons. In all three experiments, the preparation was allowed to incubate without superfusion for 10 min and then quickly frozen by addition of 70% propylene glycol/30% normal saline (vol/vol) cooled with dry ice to below -20'C (19). R15 neurons were removed from the ganglion and placed in a 98% ethanol/2% 0.2 M HCl solution (vol/vol) at 40C. The cells were stored in this solution at -700C and pools of five cells were assayed for cyclic AMP content by radioimmunoassay (DuPont RIANEN cyclic AMP RIA kit). Forskolin was obtained from Calbiochem. 3-Isobutyl-1methylxanthine was obtained from Sigma. Ro-20-1724 was kindly provided by W. Burkhard (Hoffman-LaRoche).