The majority of the brain’s vasculature is comprised of intricate capillary networks lined by capillary pericytes. However, it remains unclear whether capillary pericytes influence blood flow. Using two-photon microscopy to observe and manipulate brain capillary pericytes in vivo , we find that their optogenetic stimulation decreases lumen diameter and blood flow, but with slower kinetics than similar stimulation of mural cells on upstream pial and pre-capillary arterioles. This slow vasoconstriction was inhibited by the clinically-used vasodilator fasudil, a Rho-kinase inhibitor that blocks contractile machinery. Capillary pericytes were also slower to constrict back to baseline following hypercapnia-induced dilation, and slower to dilate towards baseline following optogenetically-induced vasoconstriction. Optical ablation of single capillary pericytes led to sustained local dilation and a doubling of blood cell flux selectively in capillaries lacking pericyte contact. These data indicate that capillary pericytes contribute to basal blood flow resistance and slow modulation of blood flow throughout the brain.

The majority of the brain’s vasculature is comprised of intricate capillary networks lined by capillary pericytes. However, it remains unclear whether capillary pericytes contribute to blood flow control. Using two-photon microscopy to observe and manipulate single capillary pericytes in vivo, we find their optogenetic stimulation decreases lumen diameter and blood flow, but with slower kinetics than mural cells of upstream pial and pre-capillary arterioles. This slow, optogenetically-induced vasoconstriction was inhibited by the clinically-used vasodilator fasudil, a Rho kinase inhibitor that blocks contractile machinery. Capillary pericytes were also slower to constrict back to baseline following hypercapnia-induced dilation, and relax towards baseline following optogenetically-induced vasoconstriction. In a complementary approach, optical ablation of single capillary pericytes led to sustained local dilation and a doubling of blood cell flux in capillaries lacking pericyte contact. Altogether these data indicate that capillary pericytes contribute to basal blood flow resistance and slow modulation of blood flow throughout the capillary bed.

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. Significance: Two-photon microscopy is a powerful tool for in vivo imaging of the mammalian brain at cellular to subcellular resolution. However, resources that describe methods for imaging live newborn mice have remained sparse. Aim: We describe a non-invasive cranial window procedure for longitudinal imaging of neonatal mice. Approach: We demonstrate construction of the cranial window by iterative shaving of the calvarium of P0 to P12 mouse pups. We use the edge of a syringe needle and scalpel blades to thin the bone to thickness. The window is then reinforced with cyanoacrylate glue and a coverslip to promote stability and optical access for at least a week. The head cap also includes a light-weight aluminum flange for head-fixation during imaging. Results: The resulting chronic thinned-skull window enables in vivo imaging to a typical cortical depth of without disruption of the intracranial environment. We highlight techniques to measure vascular structure and blood flow during development, including use of intravenous tracers and transgenic mice to label the blood plasma and vascular cell types, respectively. Conclusions: This protocol enables direct visualization of the developing neurogliovascular unit in the live neonatal brain during both normal and pathological states.

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