Fast Ca²⁺ responses in astrocyte end-feet and neurovascular coupling in mice

Cerebral blood flow (CBF) is regulated by the activity of neurons and astrocytes. Understanding how these cells control activity-dependent increases in CBF is crucial to interpreting functional neuroimaging signals. The relative importance of neurons and astrocytes is debated, as are the functional implications of fast Ca²⁺ changes in astrocytes versus neurons. Here, we used two-photon microscopy to assess Ca²⁺ changes in neuropil, astrocyte processes, and astrocyte end-feet in response to whisker pad stimulation in mice. We also developed a pixel-based analysis to improve the detection of rapid Ca²⁺ signals in the subcellular compartments of astrocytes. Fast Ca²⁺ responses were observed using both chemical and genetically encoded Ca²⁺ indicators in astrocyte end-feet prior to dilation of arterioles and capillaries. A low dose of the NMDA receptor antagonist (5R,10s)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine-hydrogen-maleate (MK801) attenuated fast Ca²⁺ responses in the neuropil and astrocyte processes, but not in astrocyte end-feet, and the evoked CBF response was preserved. In addition, a low dose of 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP), an agonist for the extrasynaptic GABA_A receptor (GABA_A R), increased CBF responses and the fast Ca²⁺ response in astrocyte end-feet but did not affect Ca²⁺ responses in astrocyte processes and neuropil. These results suggest that fast Ca²⁺ increases in the neuropil and astrocyte processes are not necessary for an evoked CBF response. In contrast, as local fast Ca²⁺ responses in astrocyte end-feet are unaffected by MK801 but increase via GABA_A R-dependent mechanisms that also increased CBF responses, we hypothesize that the fast Ca²⁺ increases in end-feet adjust CBF during synaptic activity.