Hydrogen peroxide and phosphoinositide metabolites synergistically regulate a cation current to influence...

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 (H₂ O₂ ) and activation of phospholipase C (PLC), which hydrolyses phosphatidylinositol-4,5-bisphosphate into diacylglycerol (DAG) and inositol trisphosphate (IP₃ ). We previously demonstrated that H₂ O₂ gates a voltage-dependent cation current and evokes spiking in bag cell neurons. The present study tests if DAG and IP₃ impact the H₂ O₂ -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 H₂ O₂ -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 H₂ O₂ -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 H₂ O₂ -induced current. Furthermore, PKC activation followed by H₂ O₂ 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 H₂ O₂ production. H₂ O₂ 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 H₂ O₂ -induced current, which is mimicked by the PLC activator, N-(3-trifluoromethylphenyl)-2,4,6-trimethylbenzenesulfonamide. The frequency of H₂ O₂ -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 H₂ O₂ -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.