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Neuronal swelling during cytotoxic edema is triggered by Na + and Cl - entry and is Ca 2+ independent. However, the causes of neuronal death during swelling are unknown. Here, we investigate the role of large-conductance Pannexin-1 (Panx1) channels in neuronal death during cytotoxic edema. Panx1 channel inhibitors reduce and delay neuronal death in swelling triggered by voltage-gated Na + entry with veratridine. Neuronal swelling causes downstream production of reactive oxygen species (ROS) that opens Panx1 channels. We confirm that ROS activates Panx1 currents with whole-cell electrophysiology and find scavenging ROS is neuroprotective. Panx1 opening and subsequent ATP release attract microglial processes to contact swelling neurons. Depleting microglia using the CSF1 receptor antagonist PLX3397 or blocking P2Y 12 receptors exacerbates neuronal death, suggesting that the Panx1-ATP-dependent microglia contacts are neuroprotective. We conclude that cytotoxic edema triggers oxidative stress in neurons that opens Panx1 to trigger death but also initiates neuroprotective feedback mediated by microglia contacts., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Intracellular chloride ion concentration ([Cl - ] i ) homeostasis is critical for excitatory/inhibitory balance and volume regulation in neurons. We quantitatively map spatiotemporal dendritic [Cl - ] i dynamics during N-methyl-d-aspartate (NMDA) excitotoxicity to determine how Cl - changes contribute to localized dendritic swelling (blebbing) in stroke-like conditions. Whole-cell patch clamp electrophysiology combined with simultaneous fluorescence lifetime imaging (FLIM) of the Cl - dye N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE; MQAE-FLIM) reliably report resting and dynamic [Cl - ] i shifts in dendrites. NMDA application generates spatially restricted and persistent high [Cl - ] i subdomains at dendritic blebs in a process that requires Ca 2+ influx and the subsequent opening of small-conductance Ca 2+ -activated K + (SK) channels. We propose sustained and localized K + efflux increased extracellular K + concentrations ([K + ] o ) sufficiently at discrete regions to reverse K + -Cl - cotransporter (KCC2) transport and trigger synaptic swelling. Together, our data establish a mechanism for KCC2 to generate pathological [Cl - ] i microdomains in blebbing with relevance for multiple neurological disorders., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Neuronal activation is fundamental to information processing by the brain and requires mitochondrial energy metabolism. Mitochondrial Ca 2+ uptake by the mitochondrial Ca 2+ uniporter (MCU) has long been implicated in the control of energy metabolism and intracellular Ca 2+ signalling, but its importance to neuronal function in the brain remains unclear. Here, we used in situ electrophysiology and two-photon imaging of mitochondrial Ca 2+ , cytosolic Ca 2+ , and NAD(P)H to test the relevance of MCU activation to pyramidal neuron Ca 2+ signalling and energy metabolism during action potential firing. We demonstrate that mitochondrial Ca 2+ uptake by the MCU is tuned to enhanced firing rate and the strength of this relationship varied between neurons of discrete brain regions. MCU activation promoted electron transport chain activity and chemical reduction of NAD + to NADH. Moreover, Ca 2+ buffering by mitochondria attenuated cytosolic Ca 2+ signals and thereby reduced the coupling between activity and the slow afterhyperpolarization, a ubiquitous regulator of excitability. Collectively, we demonstrate that the MCU is engaged by accelerated spike frequency to facilitate neuronal activity through simultaneous control of energy metabolism and excitability. As such, the MCU is situated to promote brain functions associated with high frequency signalling and may represent a target for controlling excessive neuronal activity., (© 2022. The Author(s).)

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., (Copyright © 2016 the American Physiological Society.)

It is unknown whether neurons can dynamically control the capacity for secretion by promptly changing the number of plasma membrane voltage-gated Ca(2+) channels. To address this, we studied peptide release from the bag cell neurons of Aplysia californica, which initiate reproduction by secreting hormone during an afterdischarge. This burst engages protein kinase C (PKC) to trigger the insertion of a covert Ca(2+) channel, Apl Cav2, alongside a basal channel, Apl Cav1. The significance of Apl Cav2 recruitment to secretion remains undetermined; therefore, we used capacitance tracking to assay secretion, along with Ca(2+) imaging and Ca(2+) current measurements, from cultured bag cell neurons under whole-cell voltage-clamp. Activating PKC with the phorbol ester, PMA, enhanced Ca(2+) entry, and potentiated stimulus-evoked secretion. This relied on channel insertion, as it was occluded by preventing Apl Cav2 engagement with prior whole-cell dialysis or the cytoskeletal toxin, latrunculin B. Channel insertion reduced the stimulus duration and/or frequency required to initiate secretion and strengthened excitation-secretion coupling, indicating that Apl Cav2 accesses peptide release more readily than Apl Cav1. The coupling of Apl Cav2 to secretion also changed with behavioral state, as Apl Cav2 failed to evoke secretion in silent neurons from reproductively inactive animals. Finally, PKC also acted secondarily to enhance prolonged exocytosis triggered by mitochondrial Ca(2+) release. Collectively, our results suggest that bag cell neurons dynamically elevate Ca(2+) channel abundance in the membrane to ensure adequate secretion during the afterdischarge., (Copyright © 2015 the authors 0270-6474/15/352747-19$15.00/0.)

Electrical transmission is a dynamically regulated form of communication and key to synchronizing neuronal activity. The bag cell neurons of Aplysia are a group of electrically coupled neuroendocrine cells that initiate ovulation by secreting egg-laying hormone during a prolonged period of synchronous firing called the afterdischarge. Accompanying the afterdischarge is an increase in intracellular Ca2+ and the activation of protein kinase C (PKC). We used whole cell recording from paired cultured bag cell neurons to demonstrate that electrical coupling is regulated by both Ca2+ and PKC. Elevating Ca2+ with a train of voltage steps, mimicking the onset of the afterdischarge, decreased junctional current for up to 30 min. Inhibition was most effective when Ca2+ entry occurred in both neurons. Depletion of Ca2+ from the mitochondria, but not the endoplasmic reticulum, also attenuated the electrical synapse. Buffering Ca2+ with high intracellular EGTA or inhibiting calmodulin kinase prevented uncoupling. Furthermore, activating PKC produced a small but clear decrease in junctional current, while triggering both Ca2+ influx and PKC inhibited the electrical synapse to a greater extent than Ca2+ alone. Finally, the amplitude and time course of the postsynaptic electrotonic response were attenuated after Ca2+ influx. A mathematical model of electrically connected neurons showed that excessive coupling reduced recruitment of the cells to fire, whereas less coupling led to spiking of essentially all neurons. Thus a decrease in electrical synapses could promote the afterdischarge by ensuring prompt recovery of electrotonic potentials or making the neurons more responsive to current spreading through the network., (Copyright © 2015 the American Physiological Society.)

Although the contribution of Ca(2+) buffering systems can vary between neuronal types and cellular compartments, it is unknown whether distinct Ca(2+) sources within a neuron have different buffers. As individual Ca(2+) sources can have separate functions, we propose that each is handled by unique systems. Using Aplysia californica bag cell neurons, which initiate reproduction through an afterdischarge involving multiple Ca(2+)-dependent processes, we investigated the role of endoplasmic reticulum (ER) and mitochondrial sequestration, as well as extrusion via the plasma membrane Ca(2+)-ATPase (PMCA) and Na(+)/Ca(2+) exchanger, to the clearance of voltage-gated Ca(2+) influx, Ca(2+)-induced Ca(2+)-release (CICR), and store-operated Ca(2+) influx. Cultured bag cell neurons were filled with the Ca(2+) indicator, fura-PE3, to image Ca(2+) under whole-cell voltage clamp. A 5 Hz, 1 min train of depolarizing voltage steps elicited voltage-gated Ca(2+) influx followed by EGTA-sensitive CICR from the mitochondria. A compartment model of Ca(2+) indicated the effect of EGTA on CICR was due to buffering of released mitochondrial Ca(2+) rather than uptake competition. Removal of voltage-gated Ca(2+) influx was dominated by the mitochondria and PMCA, with no contribution from the Na(+)/Ca(2+) exchanger or sarcoplasmic/endoplasmic Ca(2+)-ATPase (SERCA). In contrast, CICR recovery was slowed by eliminating the Na(+)/Ca(2+) exchanger and PMCA. Last, store-operated influx, evoked by ER depletion, was removed by the SERCA and depended on the mitochondrial membrane potential. Our results demonstrate that distinct buffering systems are dedicated to particular Ca(2+) sources. In general, this may represent a means to differentially regulate Ca(2+)-dependent processes, and for Aplysia, influence how reproductive behavior is triggered.

Ion channels may be gated by Ca(2+) entering from the extracellular space or released from intracellular stores--typically the endoplasmic reticulum. The present study examines how Ca(2+) impacts ion channels in the bag cell neurons of Aplysia californica. These neuroendocrine cells trigger ovulation through an afterdischarge involving Ca(2+) influx from Ca(2+) channels and Ca(2+) release from both the mitochondria and endoplasmic reticulum. Liberating mitochondrial Ca(2+) with the protonophore, carbonyl cyanide-4-trifluoromethoxyphenyl-hydrazone (FCCP), depolarized bag cell neurons, whereas depleting endoplasmic reticulum Ca(2+) with the Ca(2+)-ATPase inhibitor, cyclopiazonic acid, did not. In a concentration-dependent manner, FCCP elicited an inward current associated with an increase in conductance and a linear current/voltage relationship that reversed near -40 mV. The reversal potential was unaffected by changing intracellular Cl(-), but left-shifted when extracellular Ca(2+) was removed and right-shifted when intracellular K(+) was decreased. Strong buffering of intracellular Ca(2+) decreased the current, although the response was not altered by blocking Ca(2+)-dependent proteases. Furthermore, fura imaging demonstrated that FCCP elevated intracellular Ca(2+) with a time course similar to the current itself. Inhibiting either the V-type H(+)-ATPase or the ATP synthetase failed to produce a current, ruling out acidic Ca(2+) stores or disruption of ATP production as mechanisms for the FCCP response. Similarly, any involvement of reactive oxygen species potentially produced by mitochondrial depolarization was mitigated by the fact that dialysis with xanthine/xanthine oxidase did not evoke an inward current. However, both the FCCP-induced current and Ca(2+) elevation were diminished by disabling the mitochondrial permeability transition pore with the alkylating agent, N-ethylmaleimide. The data suggest that mitochondrial Ca(2+) gates a voltage-independent, nonselective cation current with the potential to drive the afterdischarge and contribute to reproduction. Employing Ca(2+) from mitochondria, rather than the more common endoplasmic reticulum, represents a diversification of the mechanisms that influence neuronal activity.

Neurons may initiate behavior or store information by translating prior activity into a lengthy change in excitability. For example, brief input to the bag cell neurons of Aplysia results in an approximate 30-min afterdischarge that induces reproduction. Similarly, momentary stimulation of cultured bag cells neurons evokes a prolonged depolarization lasting many minutes. Contributing to this is a voltage-independent cation current activated by Ca(2+) entering during the stimulus. However, the cation current is relatively short-lived, and we hypothesized that a second, voltage-dependent persistent current sustains the prolonged depolarization. In bag cell neurons, the inward voltage-dependent current is carried by Ca(2+); thus we tested for persistent Ca(2+) current in primary culture under voltage clamp. The observed current activated between -40 and -50 mV exhibited a very slow decay, presented a similar magnitude regardless of stimulus duration (10-60 s), and, like the rapid Ca(2+) current, was enhanced when Ba(2+) was the permeant ion. The rapid and persistent Ca(2+) current, but not the cation current, were Ni(2+) sensitive. Consistent with the persistent current contributing to the response, Ni(2+) reduced the amplitude of a prolonged depolarization evoked under current clamp. Finally, protein kinase C activation enhanced the rapid and persistent Ca(2+) current as well as increased the prolonged depolarization when elicited by an action potential-independent stimulus. Thus the prolonged depolarization arises from Ca(2+) influx triggering a cation current, followed by voltage-dependent activation of a persistent Ca(2+) current and is subject to modulation. Such synergy between currents may represent a common means of achieving activity-dependent changes to excitability.