Real-time passive volume responses of astrocytes to acute osmotic and ischemic stress in cortical slices and in vivo revealed by two-photon microscopy

The brain swells over the several minutes that follow stroke onset or acute hypo-osmotic stress because cells take up water. Measuring the volume responses of single neurons and glia has necessarily been confined to isolated or cultured cells. Two-photon laser scanning microscopy enables real-time visualization of cells functioning deep within living neocortex in vivo or in brain slices under physiologically relevant osmotic and ischemic stress. Astrocytes and their processes expressing green fluorescent protein in murine cortical slices swelled in response to 20 min of overhydration (-40 mOsm) and shrank during dehydration (+40 or +80 mOsm) at 32-34°C. Minute-by-minute monitoring revealed no detectable volume regulation during these osmotic challenges, particularly during the first 5 min. Astrocytes also rapidly swelled in response to elevated [K⁺]_o for 3 min or oxygen/glucose deprivation (OGD) for 10 min. Post-OGD, astroglial volume recovered quickly when slices were re-supplied with oxygen and glucose, while neurons remained swollen with beaded dendrites. In vivo, rapid astroglial swelling was confirmed within 6 min following intraperitoneal water injection or during the 6-12 min following cardiac arrest. While the astrocytic processes were clearly swollen, the extent of the astroglial arbor remained unchanged. Thus, in contrast to osmo-resistant pyramidal neurons (Andrew et al., 2007) that lack known aquaporins, astrocytes passively respond to acute osmotic stress, reflecting functional aquaporins in their plasma membrane. Unlike neurons, astrocytes better recover from brief ischemic insult in cortical slices, probably because their aquaporins facilitate water efflux.