Figure 7. Forced and spontaneous walk elicit highly similar global brain activity.

(A) Z-stacks of map of brain regions activated by walk in flies expressing cytosolic GCaMP (pan-Gal4;UAS-GCaMP6m) or synaptically tagged GCaMP (pan-Gal4;UAS-sytGCaMP6m) (R² of $\frac{∆F}{F}=f\left(walk\right)$). (B) Time series of components during spontaneous walk (green) or rest (magenta). Box plots show: center line, median; box limits, upper and lower quartiles; whiskers, ×1.5 interquartile range; points, outliers. (C) Time series of active components during forced walk (green) or forced rest (magenta). (D) R² for behavior regression at different conditions (all regions were pooled, spontaneous, forced, forced with severed connection between ventral nerve cord (VNC) and brain). (E) R² for behavior regression for different active components for forced (red) vs. spontaneous (green) walk. Empty markers correspond to adjusted (Benjamini–Hochberg correction) p-value >0.05 for comparison to 0. Bars are 95% CI. (F) R² difference between brain region activity during forced turn on the ipsilateral side and forced turn on the contralateral side (orange). Forced turning speeds ranged from 0.3 to 2 rad/s. Only data with N ≥ 4 components were analyzed. Lilac shows the difference for spontaneous turns. N = 58 flies for spontaneous walk/turn of different genotypes and N = 26 flies for forced walk/turn, see table in methods for details. Empty markers correspond to adjusted (Benjamini–Hochberg correction) p-value >0.05 for comparison to 0. Star corresponds to a significant difference between spontaneous and forced turns: Mann–Whitney U-test adjusted p-value <0.05 (Benjamini–Hochberg correction). Box plots show: center line, median; box limits, upper and lower quartiles; whiskers, ×1.5 interquartile range; points, outliers. Bars are 95% CI. (G) Comparison between activity at walk onset for spontaneous (green) and forced (red) walk for additional components. Individual trials were normalized and averaged for each component. Shaded regions represent the trial SEM (standard error of the mean), nf: number of flies, nt: total number of trials. Gray shaded area indicate walk onset. Stars show the significance of difference in the integral between −0.5 s and the start of walk, and is corrected for multiple comparison (Benjamini–Hochberg): *p < 0.05, ***p < 0.001.

Figure 7—figure supplement 1. Additional evidence for most activity being induced by walk.

(A) Comparison of activity during walk for cytosolic and synaptically tagged GCaMP. Left: expression patterns (baseline fluorescence), Right: Coefficient in linear models of regional activity as a function of walk. (B) Regression coefficients with forced or spontaneous walk for components. Bar is 60 µm. (C) R² and coefficient for ∆F/F regression with forced and spontaneous walk for different brain regions for dopaminergic neurons. (D) R² and coefficient for ∆F/F regression with forced and spontaneous walk for different brain regions for octopaminergic neurons. (E) R² and coefficient for ∆F/F regression with forced and spontaneous walk for different brain regions for serotonergic neurons. TH/DDC-Gal4: spontaneous, N = 9, forced, N = 5; Tdc2-Gal4: spontaneous, N = 7, forced, N = 6; Trh-Gal4: spontaneous, N = 6, forced, N = 6 flies. (B-E): Empty markers correspond to adjusted (Benjamini–Hochberg correction) p-value >0.05 for comparison to 0. Bars are 95% CI. (F) Panel showing GCaMP-fluorescence relative to onset of walk for pan-neuronally expressed GCaMPs in different regions relative to the GNG. Piece-wise linear curves were fitted to fluorescence traces 0.5 s before to 0.5 s after the onset of walk. The onset time was determined as the point of change from one linear piece to the next. Note that all medians are positive. Inset: Fit of activity onset. TH/DDC-Gal4: N = 9, Tdc2-Gal4: N = 7, Trh-Gal4: N = 6 flies. (G) Comparison between activity at walk onset for spontaneous (green) and forced (red) walk for additional components. Individual trials were normalized and averaged for each component. Shaded regions represent the trial SEM, nf: number of flies, nt: total number of trials. Stars show the significance of difference in the integral between −500 ms to start of walk, and is corrected for multiple comparison (Benjamini–Hochberg): **p < 0.01, ***p < 0.01.