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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).)