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 GABAA receptor (GABAAR), 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 GABAAR‐dependent mechanisms that also increased CBF responses, we hypothesize that the fast Ca2+ increases in end‐feet adjust CBF during synaptic activity. We separate fast Ca2+responses in astrocyte processes and end‐feet. An astrocyte‐specific genetically encoded indicator and a chemical Ca2+ indicator were used. Fast Ca2+ increases in astrocyte end‐feet are linked to the functional hyperemic response.