Hydrogen Peroxide Gates a Voltage-Dependent Cation Current in Aplysia Neuroendocrine Cells.

Nonselective cation channels promote persistent spiking in many neurons from a diversity of animals. In the hermaphroditic marine-snail, Aplysia californica, synaptic input to the neuroendocrine bag cell neurons triggers various cation channels, causing an ∼30 min afterdischarge of action potentials and the secretion of egg-laying hormone. During the afterdischarge, protein kinase C is also activated, which in turn elevates hydrogen peroxide (H2O2), likely by stimulating nicotinamide adenine dinucleotide phosphate oxidase. The present study investigated whether H2O2 regulates cation channels to drive the afterdischarge. In single, cultured bag cell neurons, H2O2 elicited a prolonged, concentration- and voltage-dependent inward current, associated with an increase in membrane conductance and a reversal potential of ∼+30 mV. Compared with normal saline, the presence of Ca2+-free, Na+-free, or Na+/Ca2+-free extracellular saline, lowered the current amplitude and left-shifted the reversal potential, consistent with a nonselective cationic conductance. Preventing H2O2 reduction with the glutathione peroxidase inhibitor, mercaptosuccinate, enhanced the H2O2-induced current, while boosting glutathione production with its precursor, N-acetylcysteine, or adding the reducing agent, dithiothreitol, lessened the response. Moreover, the current generated by the alkylating agent, N-ethylmaleimide, occluded the effect of H2O2 The H2O2-induced current was inhibited by tetrodotoxin as well as the cation channel blockers, 9-phenanthrol and clotrimazole. In current-clamp, H2O2 stimulated burst firing, but this was attenuated or prevented altogether by the channel blockers. Finally, H2O2 evoked an afterdischarge from whole bag cell neuron clusters recorded ex vivo by sharp-electrode. H2O2 may regulate a cation channel to influence long-term changes in activity and ultimately reproduction.SIGNIFICANCE STATEMENT Hydrogen peroxide (H2O2) is often studied in a pathological context, such as ischemia or inflammation. However, H2O2 also physiologically modulates synaptic transmission and gates certain transient receptor potential channels. That stated, the effect of H2O2 on neuronal excitability remains less well defined. Here, we examine how H2O2 influences Aplysia bag cell neurons, which elicit ovulation by releasing hormones during an afterdischarge. These neuroendocrine cells are uniquely identifiable and amenable to recording as individual cultured neurons or a cluster from the nervous system. In both culture and the cluster, H2O2 evokes prolonged, afterdischarge-like bursting by gating a nonselective voltage-dependent cationic current. Thus, H2O2, which is generated in response to afterdischarge-associated second messengers, may prompt the firing necessary for hormone secretion and procreation.

A kainate receptor subunit promotes the recycling of the neuron-specific K+-Cl- co-transporter KCC2 in hippocampal neurons.

Synaptic inhibition depends on a transmembrane gradient of chloride, which is set by the neuron-specific K+-Cl- co-transporter KCC2. Reduced KCC2 levels in the neuronal membrane contribute to the generation of epilepsy, neuropathic pain, and autism spectrum disorders; thus, it is important to characterize the mechanisms regulating KCC2 expression. In the present study, we determined the role of KCC2-protein interactions in regulating total and surface membrane KCC2 expression. Using quantitative immunofluorescence in cultured mouse hippocampal neurons, we discovered that the kainate receptor subunit GluK2 and the auxiliary subunit Neto2 significantly increase the total KCC2 abundance in neurons but that GluK2 exclusively increases the abundance of KCC2 in the surface membrane. Using a live cell imaging assay, we further determined that KCC2 recycling primarily occurs within 1-2 h and that GluK2 produces an ∼40% increase in the amount of KCC2 recycled to the membrane during this time period. This GluK2-mediated increase in surface recycling translated to a significant increase in KCC2 expression in the surface membrane. Moreover, we found that KCC2 recycling is enhanced by protein kinase C-mediated phosphorylation of the GluK2 C-terminal residues Ser-846 and Ser-868. Lastly, using gramicidin-perforated patch clamp recordings, we found that the GluK2-mediated increase in KCC2 recycling to the surface membrane translates to a hyperpolarization of the reversal potential for GABA (EGABA). In conclusion, our results have revealed a mechanism by which kainate receptors regulate KCC2 expression in the hippocampus.

Ca2+ removal by the plasma membrane Ca2+-ATPase influences the contribution of mitochondria to activity-dependent Ca2+ dynamics in Aplysia neuroendocrine cells.

After Ca(2+) influx, mitochondria can sequester Ca(2+) and subsequently release it back into the cytosol. This form of Ca(2+)-induced Ca(2+) release (CICR) prolongs Ca(2+) signaling and can potentially mediate activity-dependent plasticity. As Ca(2+) is required for its subsequent release, Ca(2+) removal systems, like the plasma membrane Ca(2+)-ATPase (PMCA), could impact CICR. Here we examine such a role for the PMCA in the bag cell neurons of Aplysia californica CICR is triggered in these neurons during an afterdischarge and is implicated in sustaining membrane excitability and peptide secretion. Somatic Ca(2+) was measured from fura-PE3-loaded cultured bag cell neurons recorded under whole cell voltage clamp. Voltage-gated Ca(2+) influx was elicited with a 5-Hz, 1-min train, which mimics the fast phase of the afterdischarge. PMCA inhibition with carboxyeosin or extracellular alkalization augmented the effectiveness of Ca(2+) influx in eliciting mitochondrial CICR. A Ca(2+) compartment model recapitulated these findings and indicated that disrupting PMCA-dependent Ca(2+) removal increases CICR by enhancing mitochondrial Ca(2+) loading. Indeed, carboxyeosin augmented train-evoked mitochondrial Ca(2+) uptake. Consistent with their role on Ca(2+) dynamics, cell labeling revealed that the PMCA and mitochondria overlap with Ca(2+) entry sites. Finally, PMCA-dependent Ca(2+) extrusion did not impact endoplasmic reticulum-dependent Ca(2+) removal or release, despite the organelle residing near Ca(2+) entry sites. Our results demonstrate that Ca(2+) removal by the PMCA influences the propensity for stimulus-evoked CICR by adjusting the amount of Ca(2+) available for mitochondrial Ca(2+) uptake. This study highlights a mechanism by which the PMCA could impact activity-dependent plasticity in the bag cell neurons.