Longitudinal two-photon calcium imaging of S1BF. (A) Water release probability during cage ACC. (B) Trial structure. A nose poke into the IR beam initiates the onset of a trial. A random delay period (0.2–0.8 sec) is followed by a fixed 1 sec delay before water delivery. (C) Schematic of two-photon calcium imaging setup, with an animal head-fixed on a wheel. (D, Top) Experimental time line for longitudinal calcium imaging. (Bottom) Longitudinal imaging of the same field of view (FOV) across ACC days. Scale bar, 30 µm. (E) Post hoc labeling of imaging site with methyl blue. Scale bar, 500 µm. (F) Cell masks of an example imaging FOV. Scale bar, 20 µm. (G) Example traces of airpuff-evoked responses and the mean calcium traces from the same cell across all trials on ACC day 6. F (fluorescence) is reported in arbitrary units. Light gray lines indicate individual trials, and black lines indicate the mean response to airpuff. The gray shaded area indicates the duration of airpuff. (H) Example trace of a cell within an imaging session. Blue lines indicate airpuff onset and offset. Black lines indicate blank trial onset and offset. Airpuff lasted for 0.5 sec. Airpuff trials and blank trials were randomly interleaved. The intertrial interval was 20 sec.
