In various neurons, including neuroendocrine cells, non‐selective cation channels elicit plateau potentials and persistent firing. Reproduction in the marine snail Aplysia californica is initiated when the neuroendocrine bag cell neurons undergo an afterdischarge, that is, a prolonged period of enhanced excitability and spiking during which egg‐laying hormone is released into the blood. The afterdischarge is associated with both the production of hydrogen peroxide (H2O2) and activation of phospholipase C (PLC), which hydrolyses phosphatidylinositol‐4,5‐bisphosphate into diacylglycerol (DAG) and inositol trisphosphate (IP3). We previously demonstrated that H2O2 gates a voltage‐dependent cation current and evokes spiking in bag cell neurons. The present study tests if DAG and IP3 impact the H2O2‐induced current and excitability. In whole‐cell voltage‐clamped cultured bag cell neurons, bath‐application of 1‐oleoyl‐2‐acetyl‐sn‐glycerol (OAG), a DAG analogue, enhanced the H2O2‐induced current, which was amplified by the inclusion of IP3 in the pipette. A similar outcome was produced by the PLC activator, N‐(3‐trifluoromethylphenyl)‐2,4,6‐trimethylbenzenesulfonamide. In current‐clamp, OAG or OAG plus IP3, elevated the frequency of H2O2‐induced bursting. PKC is also triggered during the afterdischarge; when PKC was stimulated with phorbol 12‐myristate 13‐acetate, it caused a voltage‐dependent inward current with a reversal potential similar to the H2O2‐induced current. Furthermore, PKC activation followed by H2O2 reduced the onset latency and increased the duration of action potential firing. Finally, inhibiting nicotinamide adenine dinucleotide phosphate oxidase with 3‐benzyl‐7‐(2‐benzoxazolyl)thio‐1,2,3‐triazolo[4,5‐d]pyrimidine diminished evoked bursting in isolated bag cell neuron clusters. These results suggest that reactive oxygen species and phosphoinostide metabolites may synergize and contribute to reproductive behaviour by promoting neuroendocrine cell firing. Key points Aplysia bag cell neurons secrete reproductive hormone during a lengthy burst of action potentials, known as the afterdischarge. During the afterdischarge, phospholipase C (PLC) hydrolyses phosphatidylinositol‐4,5‐bisphosphate into diacylglycerol (DAG) and inositol trisphosphate (IP3). Subsequent activation of protein kinase C (PKC) leads to H2O2 production. H2O2 evokes a voltage‐dependent inward current and action potential firing. Both a DAG analogue, 1‐oleoyl‐2‐acetyl‐sn‐glycerol (OAG), and IP3 enhance the H2O2‐induced current, which is mimicked by the PLC activator, N‐(3‐trifluoromethylphenyl)‐2,4,6‐trimethylbenzenesulfonamide. The frequency of H2O2‐evoked afterdischarge‐like bursting is augmented by OAG or OAG plus IP3. Stimulating PKC with phorbol 12‐myristate 13‐acetate shortens the latency and increases the duration of H2O2‐induced bursts. The nicotinamide adenine dinucleotide phosphate oxidase inhibitor, 3‐benzyl‐7‐(2‐benzoxazolyl)thio‐1,2,3‐triazolo[4,5‐d]pyrimidine, attenuates burst firing in bag cell neuron clusters.

Many behaviors and types of information storage are mediated by lengthy changes in neuronal activity. In bag cell neurons of the hermaphroditic sea snail, Aplysia californica, a transient cholinergic synaptic input triggers an ~30 min afterdischarge. This causes these neuroendocrine cells to release egg laying hormone and elicit reproductive behavior. When acetylcholine is pressure-ejected onto a current-clamped bag cell neuron, the evoked depolarization is far longer than the current evoked by acetylcholine under voltage-clamp, suggesting recruitment of another conductance. Our earlier studies found bag cell neurons to display a voltage-dependent persistent Ca2+ current. Hence, we hypothesized that this current is activated by the acetylcholine-induced depolarization, and sought a selective Ca2+ current blocker. Rapid Ca2+ current evoked by 200-ms depolarizing steps in voltage-clamped, cultured bag cell neurons demonstrated a concentration-dependent sensitivity to Ni2+, Co2+, Zn2+, and verapamil but not Cd2+ or -conotoxin GIVa. Leak subtraction of Ca2+ current evoked by 10-s depolarizing steps using the IC100 of Ni2+, Co2+, Zn2+, or verapamil revealed persistent Ca2+ current, demonstrating persistent current block. Only Co2+ and Zn2+ did not suppress the acetylcholine-induced current, although Zn2+ appeared to impact additional channels. When Co2+ was applied during an acetylcholine-induced depolarization, the amplitude was reduced; furthermore, protein kinase C activation, previously established to enhance the persistent Ca2+ current, extended the depolarization. Therefore, the persistent Ca2+ current sustains the acetylcholine-induced depolarization and may translate brief cholinergic input into afterdischarge initiation. This could be a general mechanism of triggering long-term change in activity using a short-lived input.