Simultaneous in vivo imaging of Ca ²⁺ signals in periarteriolar cholinergic axonal varicosities and arteriole diameter changes in the mouse cerebral cortex

Basal forebrain cholinergic neurons project widely to the cerebral cortex and participate in cerebrovascular regulation. Although cholinergic axons are distributed around the cerebrovasculature, their functional relationship with arteriolar dynamics remains unclear. In this study, we established an in vivo two-photon imaging approach to simultaneously measure Ca ²⁺ signals in cholinergic axonal varicosities and arteriolar diameters in urethane-anesthetized mice. An adeno-associated virus (AAV) vector (rAAV-ChAT-jGCaMP8s) was injected into the nucleus basalis of Meynert. In vivo imaging of the frontal cortex revealed bead-shaped GCaMP signals around the arterioles. Pinch stimulation transiently increased Ca ²⁺ signals in periarteriolar varicosities, followed by arteriolar dilation, with an approximately 2-s delay between their peaks. Linear regression analysis disclosed a significant relationship between the magnitudes of these changes. This approach enabled simultaneous evaluation of cholinergic axonal activity and arteriolar dynamics in vivo , providing a tool to investigate the cholinergic regulation of cerebrovasculature.