Glial Chloride Homeostasis Under Transient Ischemic Stress

High water permeabilities permit rapid adjustments of glial volume upon changes in external and internal osmolarity, and pathologically altered intracellular chloride concentrations ([Cl(–)](int)) and glial cell swelling are often assumed to represent early events in ischemia, infections, or traumatic brain injury. Experimental data for glial [Cl(–)](int) are lacking for most brain regions, under normal as well as under pathological conditions. We measured [Cl(–)](int) in hippocampal and neocortical astrocytes and in hippocampal radial glia-like (RGL) cells in acute murine brain slices using fluorescence lifetime imaging microscopy with the chloride-sensitive dye MQAE at room temperature. We observed substantial heterogeneity in baseline [Cl(–)](int), ranging from 14.0 ± 2.0 mM in neocortical astrocytes to 28.4 ± 3.0 mM in dentate gyrus astrocytes. Chloride accumulation by the Na(+)-K(+)-2Cl(–) cotransporter (NKCC1) and chloride outward transport (efflux) through K(+)-Cl(–) cotransporters (KCC1 and KCC3) or excitatory amino acid transporter (EAAT) anion channels control [Cl(–)](int) to variable extent in distinct brain regions. In hippocampal astrocytes, blocking NKCC1 decreased [Cl(–)](int), whereas KCC or EAAT anion channel inhibition had little effect. In contrast, neocortical astrocytic or RGL [Cl(–)](int) was very sensitive to block of chloride outward transport, but not to NKCC1 inhibition. Mathematical modeling demonstrated that higher numbers of NKCC1 and KCC transporters can account for lower [Cl(–)](int) in neocortical than in hippocampal astrocytes. Energy depletion mimicking ischemia for up to 10 min did not result in pronounced changes in [Cl(–)](int) in any of the tested glial cell types. However, [Cl(–)](int) changes occurred under ischemic conditions after blocking selected anion transporters. We conclude that stimulated chloride accumulation and chloride efflux compensate for each other and prevent glial swelling under transient energy deprivation.