Adenosine is a purinergic neuromodulator that influences neuronal activity, metabolism, and vascular tone. While its vasoactive effects are well established, its contribution to neurovascular coupling (NVC) in unanesthetized animals remains poorly understood. Here, we studied how adenosine regulates brain hemodynamics in unanesthetized, head-fixed mice. Using widefield fluorescence imaging through a polished thin-skull cranial window, we simultaneously measured cortical blood volume, tissue oxygenation, and cortical metabolic signals across the dorsal cortex. In the somatosensory cortex, both voluntary locomotion and whisker stimulation induced robust increases of cerebral blood volume and brain tissue oxygenation. Blocking adenosine signaling with systemic caffeine (10 mg/kg), a non-selective adenosine receptor antagonist, had no measurable effect on resting-state hemodynamics. However, during sensory stimulation, the cortical blood volume response exhibited a faster onset time, while sustained vasodilation during prolonged activation was reduced. These results indicate that adenosine primarily shapes the temporal profile and persistence of NVC rather than basal vascular tone. To elucidate the mechanisms underlying these effects, we measured the extracellular adenosine levels using a newly developed genetically encoded adenosine sensor (GRABAdo) and fiber photometry. We observed a delayed rise in extracellular adenosine following locomotion onset, suggesting that adenosine contributes to stabilizing and sustaining the vascular response during longer neural activity events. Funding: American Physiological Society SURF program This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.
Claypool et al. (Fri,) studied this question.