(A) Illustration of the forced locomotion experiment with simultaneous calcium imaging of MLIs. Mice can walk freely but the treadmill is motorized during a 20 s period, corresponding to the area shaded in purple in C and D between 6 and 26 s (dark purple corresponds to the 15 s maximal speed motorization period between 10 and 25 s of the recordings). At the right, a simplified circuit diagram of the cerebellar molecular and Purkinje cell layer illustrating craniotomy, 2 photon scanning and drug delivery. (B) Horizontal view of viral-mediated GCaMP3 expression in MLIs of a Pvalb-Cre mouse. Example ROIs are colored (see panels C,D,E,F). (C) Ca2+ responses during three non-consecutive locomotion recordings (recording number upper left; recordings repeated every 2–4 min, see Materials and methods). Top black traces show the distance traveled by the mouse during the recordings. Bottom traces show fluorescence for 7 randomly chosen example ROIs corresponding to individual cell bodies (see color-coded regions in B). The time period used for analysis is shaded in dark purple. (D) Same depiction as in (C) but after the application of CPCCOEt. (E) Calcium responses before and after CPCCOEt application. The 75th percentile of the fluorescence trace during the maximal-speed locomotion period [10, 25] s, dark purple shaded region in (C,D) is shown as a function of the recording number. For this example mouse (animal #2), 6 recordings before- and 8 after drug delivery were performed. (F) Calcium activity normalized to pre-drug baseline. The pre-drug decay in fluorescence was subtracted from all points per ROI (see Materials and methods for details). Data for the same ROIs is shown in the same color in panels (B-F). Data points before CPCCOEt application are in pale color, data after drug delivery in dark color. (G) Change in fluorescence with CPCCOEt vs. pre-drug baseline data from all animals (Nanimals = 4) and ROIs (NROIs = 313). The change in fluorescence is shown as a function of the pre-drug baseline fluorescence during locomotion. Data from each animal is plotted with the same symbols. The color of each symbol indicates whether a significant increase (red, p<0.01, T-test), decrease (blue, p<0.01, T-test) or no change (gray) has been observed when comparing baseline locomotion fluorescence with fluorescence after CPCCOEt application (see Materials and methods for more details). The linear regression on all points yields a correlation of −0.547 with p-value<0.0001, explaining 30.0% of the variance in the data.