The present work evaluated the reproducibility and variance of the cerebral blood flow (CBF) response to natural whisker stimulation in the barrel cortex of awake behaving mice. The animal was placed on an air float ball that allowed the animal to walk, while the head of the animal was fixed in a custom-made stereotactic apparatus. Dynamic CBF changes in the barrel cortex and animal locomotion were simultaneously monitored with laser-Doppler flowmetry (LDF) and an optical motion sensor that detected the rotation distance of the ball, respectively. Whisker stimulation-induced CBF measured under daytime and nighttime conditions showed consistent responses (24% and 23% of the pre-stimulus baseline, respectively), whereas the amount of locomotion was 1.4 times higher during nighttime relative to daytime. Repeated longitudinal experiments over 7 days showed a reproducible, evoked CBF (13-26% relative to the baseline among 7 animals). The mean of the variance coefficient (i.e., standard deviation divided by mean) across multiple days was 0.11 and 0.75 for evoked CBF and locomotion, respectively. These results showed reproducible and reliable measurements of longitudinal CBF response in behaving mice regardless of day-to-day variations in locomotion. Furthermore, we confirmed that the CBF response to whisker stimulation was well localized and reproducible, measured with laser speckle imaging under awake condition. The results further show the capability of long-term hemodynamic imaging in normal and disease-model mice, which is of particular importance for understanding the longitudinal changes and plasticity of neurovascular coupling and behavioral performances such as during growth, development and aging.
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