EXPRESS: It's not all rapid dilations: on contributions of constrictions, biphasic and minute-long vessel responses to brain functional hyperemia.

Though neurovascular coupling has long been studied, a comprehensive characterization of the extent, kinetics, and variability of individual microvessels' responses is lacking. To address this, we employed our NOVAS3D deep learning pipeline to analyze microvascular volumes acquired in mice under alpha-chloralose sampled every ~4 s for 5 minutes before and after either somatosensory stimulation or optogenetic actuation, tuned to evoke commensurate response magnitudes. Our findings revealed a variety of responses, including both constrictions and dilations, with arteries exhibiting notable, termed biphasic responses. During our 5-min post-stimulation period, we observed protracted responses across all vessel types with an onset time of 102 (IQR 29-185) s following optogenetic stimulation and a response duration of 29 (IQR 12-89) s. Interestingly, we found low recruitment rates (28%) in the capillary bed; however, capillaries that responded had dilations of 0.8 (0.6-1.1) μm and constrictions of 1(IQR 0.6-1.9). Forepaw stimulation showed similar ranges with an onset time of 252 (IQR 118-229) s, and a duration of 16 (IQR 8-32) s. This study demonstrates that the timeframe in which microvessels are showing caliber changes is much longer than previously appreciated and also significantly longer than the period over which mesoscopic CBF signal shows deviation.