(A) The Ca2+ indicator GCaMP6f was locally injected into the POm (bottom) which sends axonal projections to layers 1 and 5 of the forepaw S1 (top). Inset, in vivo two-photon Ca2+ image of POm axonal projections in forepaw S1 (depth, 60 μm; scale bar, 10 μm). (B) Two-photon Ca2+ imaging of GCaMP6f-expressing POm axons in forepaw S1 was performed in head-restrained mice trained to report the detection of a tactile stimulus (200 Hz, 500 ms) by licking a reward port. Correct responses (HIT) were rewarded with sucrose water reward (10 μl, 10% sucrose). (C) Top, raster plot showing a typical behavioral response (licks) sorted into correct HIT performance and Catch (no-stimulus) trials. Gray, spontaneous; red, tactile stimulus; green, response epoch; blue, reward epoch. Blue line, reward delivery. Bottom, example of Ca2+ activity pattern during correct performance and Catch trials from the POm axon in (A). Each row represents a single trial, sorted according to trial number. (D) Mass average with standard error of the mean (SEM; shaded area) of all stimulus-evoked Ca2+ transients in all axons during correct goal-directed performance (HIT; black). Behavioral epochs indicated by color bars (red, stimulus; green, response; blue, reward). (E) Probability of evoking a Ca2+ response during correct HIT behavior (black) compared with tactile-evoked activity in the naive state (gray, n = 113 axons; Mann–Whitney test). (F) Top, Ca2+ activity pattern during HIT performance in the tactile goal-directed task. Each row is an independent axon normalized to maximum fluorescence and sorted by the timing of the peak amplitude (gray, baseline; red, stimulus; green, response epoch; blue, reward epoch). Red lines, stimulus delivery. Dashed line, reward delivery. Bottom, average Ca2+ response in POm axons active during the stimulus and response epoch (green), reward epoch (blue); baseline (no behavior; gray). (G) The probability of a Ca2+ transient in POm axons during baseline (gray), response epoch (green), reward epoch (blue). n = 418 axons, 11 mice. Friedman test + Dunn’s multiple comparisons test. (H) The amplitude of Ca2+ transients in POm axons evoked during baseline (gray), response epoch (green), and reward epoch (blue). n = 239 axons, 11 mice with evoked Ca2+ transients. Friedman test + Dunn’s multiple comparisons test. (I) Top, average lick frequency during spontaneous (gray), stim/response (green), and reward (blue) epochs during correct HIT behavior. Bottom, histogram of Ca2+ transient probability in POm axons. *p < 0.05, ***p < 0.001, ****p < 0.0001.