LRRC8A-dependent volume-regulated anion channels contribute to ischemia-induced brain injury and glutamatergic input to hippocampal neurons

Volume-regulated anion channels (VRACs) are critically involved in regulating cell volume, and leucine-rich repeat-containing protein 8A (LRRC8A, SWELL1) is an obligatory subunit of VRACs. Cell swelling occurs early after brain ischemia, but it is unclear whether neuronal LRRC8a contributes to ischemia-induced glutamate release and brain injury. We found that Lrrc8a conditional knockout (Lrrc8a-cKO) mice produced by crossing Nestin(Cre+/-) with Lrrc8a(flox+/+) mice died 7-8 weeks of age, indicating an essential role of neuronal LRRC8A for survival. Middle cerebral artery occlusion (MCAO) caused an early increase in LRRC8A protein levels in the hippocampus in wild-type (WT) mice. Whole-cell patch-clamp recording in brain slices revealed that oxygen-glucose deprivation significantly increased the amplitude of VRAC currents in hippocampal CA1 neurons in WT but not in Lrrc8a-cKO mice. Hypotonicity increased the frequency of spontaneous excitatory postsynaptic currents (sEPSCs) in hippocampal CA1 neurons in WT mice, and this was abolished by DCPIB, a VRAC blocker. But in Lrrc8a-cKO mice, hypotonic solution had no effect on the frequency of sEPSCs in these neurons. Furthermore, the brain infarct volume and neurological severity score induced by MCAO were significantly lower in Lrrc8a-cKO mice than in WT mice. In addition, MCAO-induced increases in cleaved caspase-3 and calpain activity, two biochemical markers of neuronal apoptosis and death, in brain tissues were significantly attenuated in Lrrc8a-cKO mice compared with WT mice. These new findings indicate that cerebral ischemia increases neuronal LRRC8A-dependent VRAC activity and that VRACs contribute to increased glutamatergic input to hippocampal neurons and brain injury caused by ischemic stroke.