(a) Task timeline during widefield calcium imaging. To distinguish activity associated with initial wheel movement from activity driven by visual motion, we introduced an open-loop period 0.5–1.2 s after grating onset, when wheel movements did not move the grating. Trials were excluded post-hoc if choices were made after this period. (b) Stimulus-locked wheel velocity for one example session. Thin lines are individual trials for all correct Left or Right choices (velocity sign flipped for Left choices so that correct is always positive), smoothed with a 10 ms Gaussian window. Thick line: mean. (c) Stimulus-triggered mean calcium fluorescence at five regions of interest (ROIs) for one example session. Thick lines: mean response to correct trials with only contralateral stimuli. Thin lines: same for correct trials with only ipsilateral stimuli. Shaded regions: standard deviation of fluorescence across trials. Black circle: response latency, defined as time for mean fluorescence to reach 30% of the peak. Right: distribution of calcium fluorescence for each ROI across all sessions, taken over a 100 ms time window aligned to response latency (shaded regions on left), separated for contralateral (black) and ipsilateral (gray) stimulus trials. Error bar is 95% confidence interval for the mean fluorescence. (d) Summary of fluorescence response latencies to contralateral stimuli for six mice (see Materials and methods for genotype information; three mice were excluded due to having too few trials to reliably measure onset latency). Rows show data from individual mice, with dots showing each ROI’s response latency for trials pooled across sessions. Bottom row: response latencies and reaction time (RT) averaged across mice (dots and lines: median ± m.a.d.). (e) Response latencies to contralateral and ipsilateral stimuli for each region, averaged across mice (closed and open circles). Significance was determined by a two-tailed paired t-test, t(5) = –10.38, –12.00, −5.46, –1.97, −4.72. Colors as in (c). (f) Stimulus-locked calcium fluorescence at VISp (blue) and MOs (green) ROIs for three different GCaMP6 mouse genotypes (see Materials and methods). Lines indicate the session-averaged fluorescence for trials with only contralateral stimuli, pooling sessions from Snap25-G6s (solid), tetO-G6s (light dashed), and Vglut1-Cre;Ai95 (heavy dashed) mice. Shaded regions indicate the 95% confidence interval. Right inset: fluorescence averaged over time windows depicted by the gray bars on the left, separated for different contralateral contrast values and normalized to the same maximum and minimum across all genotypes. (g) Widefield data were preprocessed by singular value decomposition (SVD), which compresses, smooths, and denoises the calcium signals (Peters et al., 2021). The spatial smoothing that SVD causes depends on the signal statistics and is not spatially homogeneous or isotropic. This pseudocolor image shows the equivalent smoothing kernel resulting from SVD preprocessing, that is, the SVD-smoothed imaging that would result from a raw image with a single pixel over Right VISp (black square), and zero everywhere else. The kernel has ~0.1–0.2 mm diameter. White: scale bar.