(a) Distribution of trial-wise responses on 50+ neuron stimulation trials calculated from raw (Raw; black) and neuropil subtracted (Sub; red) traces (N = 30,894 neurons, 1312 trials). A large fraction of negative responses are observed even in raw traces without neuropil subtraction. Inset: Difference between each neuron’s trial-wise response when responses were calculated from neuropil subtracted or raw traces (Sub – Raw). Responses are slightly reduced when traces are neuropil subtracted (−0.16 ± 0.58 ΔF/σF, p=0 Wilcoxon signed-rank test). (b) Neuropil-subtracted trial-wise responses are highly correlated with raw trial-wise responses (R2 = 0.87, p=0), although slightly reduced (β0 = -0.10 ± 5.32 x 10−4, p=0) with this reduction mainly limited to large positive going responses (β1 = 0.82 x 10−1±3.31 × 10−4, p=0). Red: linear fit to data, Black: neuropil-subtracted data binned into deciles by raw trial-wise response and averaged within decile. Data are mean ± s.d. (c) Trial-wise responses classified as suppressed, activated and no response using neuropil-subtracted traces also show negative (−1.75 ± 1.05 ΔF/σF, p=0 Wilcoxon signed-rank test), roughly zero (0.16 ± 0.94 ΔF/σF, p=0, Wilcoxon signed-rank test) and positive (3.72 ± 3.18 ΔF/σF, p=0 Wilcoxon signed-rank test) trial-wise responses calculated from raw traces in the same trials. These groups also differed significantly from each other (p=0 Kruskal-Wallis test; Suppressed vs No response, p=0, Suppressed vs Activated, p=0, No response vs Activated, p=0, Bonferroni corrected for multiple comparisons) indicating that our thresholds on neuropil subtracted traces reliably separate different classes of trial-wise responses even in the absence of neuropil-subtraction. (d) Example raw (light red, light blue) and neuropil-subtracted (dark red, dark blue) traces for neurons reliably activated (red) and suppressed (blue) across trials (using neuropil-subtracted trial-wise magnitudes). Neuropil subtraction slightly decreases response magnitudes in both groups, though does not induce large changes in response time-course. Negative responses can be seen with and without neuropil-subtraction. Shading is mean ± s.e.m across neurons. Traces have been linearly interpolated across the photostimulation epoch (grey box) to avoid the photostimulation artefact for display only. All box-plots are median (mid-line) with 25th and 75th percentiles (box) and 5th and 95th percentiles (whiskers). N = 3.68 × 106 trial-wise responses, 30894 neurons, 11 sessions, 6 mice, 1–2 sessions each.