![]() ![]() Two-photon imaging, light transmittance (LT) changes, and electrophysiological field recordings followed by electron microscopy in hippocampal CA1 st. Four non-excitatory AA (l-alanine, glycine, l-glutamine, l-serine: AGQS) at plasmatic concentrations were applied to slices from mice expressing EGFP in pyramidal neurons or astrocytes during normoxia or hypoxia. Hypoxia-induced synaptic depression in hippocampal slices becomes irreversible with non-excitatory AA, alongside their intracellular accumulation and increased tissue electrical resistance. Although excitotoxic AA were extensively studied, little is known about non-excitatory AA during hypoxic injury. Bleeding in hemorrhagic stroke and TBI also release plasma AA. In ischemic stroke and post-traumatic brain injury (TBI), blood-brain barrier disruption leads to leaking plasma amino acids (AA) into cerebral parenchyma. These new findings refute the notion that VRAC-mediated glutamate release plays significant role in ischemic brain injury. Het mice had full swelling-activated glutamate release, while KO animals showed its virtual absence. However, despite identical brain damage, Het and KO mice dramatically differed in their VRAC activities. In contrast, the brain-wide LRRC8A knockout reduced ischemic infarction by ~50% in both heterozygotes (Het) and the full Lrrc8a knockout (KO) as compared to the control Lrrc8a(flox/+) genotype. The inducible deletion of astrocytic LRRC8A yielded no histological or behavioral protection. To produce stroke, genetically modified mice were subjected to a 40-minute occlusion of the middle cerebral artery. We used two molecular genetic strategies to ablate LRRC8A expression in either brain astrocytes only (inducible deletion of Lrrc8a(flox/flox) with Aldh1l1-CreERT2) or the majority of brain cells (neurons, astrocytes, and oligodendrocytes with Nestin-Cre). In this work, we tested the widely accepted idea that harmful effects of VRACs in the brain are mediated by pathological release of the excitatory transmitter glutamate. Prior studies demonstrated that treatment with non-specific VRAC blockers, or brain-specific deletion of the essential VRAC subunit LRRC8A, are highly protective in rodent stroke. The ubiquitous volume-regulated anion channels (VRACs), which are composed of LRRC8 proteins, facilitate cell volume homeostasis, and contribute to many other physiological processes. ![]() However, in at least one brain cell type, astrocytes, this transporter is paradoxically stimulated by cell swelling and contributes to sustained astrocytic edema in CNS pathologies (asterisk). Another electroneutral transporter, NKCC1, drives the net uptake of Na + /K + /Cl − under basal conditions, and is additionally strongly activated by cellular shrinkage. Many cells types additionally activate KCl loss via electroneutral K +, Cl − transporters (KCC, also outlined in box). This facilitates cooperative loss of K + and Cl −, which is coupled with release of osmotically obligated water. RIGHT: In the response to osmotic swelling cells dramatically increase membrane permeability to Cl − and other anions due to activation of volume regulated anion channels (VRAC, outlined in box). Membrane mechanisms responsible for regulatory volume decrease (RVD) in osmotically swollen cells LEFT: Under steady-state conditions, membrane permeability is dominated by K + channels (outlined in box) and transmembrane water fluxes are equilibrated. ![]()
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