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. 2023 Apr 17;12:e85202. doi: 10.7554/eLife.85202

Figure 1. Global brain activation during walk.

(A) Schematic overview of the preparation and analysis method. Please see methods for details. (B) Raster plot of the activity of regions. Top panels depict walking bouts in green and rest or grooming in magenta. Lower panel shows calcium activity elicited throughout the experiment. The brighter the higher the calcium transients. Mean forward speed: 5.6 mm/s, mean angular velocity: 0.4 rad/s. Bar is 60 μm. (C) Sample traces (ΔF/F) for different brain regions relative to walk (green). (D) First two principal components from whole brain activity color coded with behavior (see additional examples in Figure 1—figure supplement 1). (E) R2 for regression of single regions with single behaviors (all regions were pooled, but p-values are obtained after averaging regions for each fly. Walk: N = 16, Flail: N = 7, Groom: N = 6). Mann–Whitney U-test Bonferroni adjusted p-values: Walk vs. Flail: 0.085, Walk vs. Groom: 0.011, Flail vs. Groom: 0.26. Center line, median; box limits, upper and lower quartiles; whiskers, ×1.5 interquartile range; points, outliers. (F) R2 of single region activity regression with walk, walk onset and walk offset (all regions were pooled, nsyb-Gal4: N = 16, GMR57C10-Gal4: N = 4). Regressors for walk onset or offset are Dirac functions convolved with the GCaMP response (see Methods). Box plots show: center line, median; box limits, upper and lower quartiles; whiskers, ×1.5 interquartile range; points, outliers. Mann–Whitney U-test for the two genotypes grouped (comparison of fly-wise averages): walk vs. walk onset: p = 3 × 10−5, walk vs. walk offset: p = 6 × 10−5. (G) Z-stack map of R2 median (Walk: N (flies) = 16, Flail: N = 7, Groom: N = 6) for regression between single region activity and walk, flail or groom (see values in Figure 1—figure supplement 1D-F). (H) Coefficient of single region’ activity regression with walk. All regions’ 95% CI ( bars) are above zero and all adjusted p-values <0.001 (Benjamini–Hochberg correction). N = 16.

Figure 1.

Figure 1—figure supplement 1. Correlation of regional activity with walk.

Figure 1—figure supplement 1.

(A) R2 during walk at different recording frequencies for pan-neuronally expressed GCaMP6f or GCaMP6m (all regions were pooled). No significant difference was found for walk between the different frequencies. Box plots show: center line, median; box limits, upper and lower quartiles; whiskers, ×1.5 interquartile range; points, outliers. (B) R2 for walking with eyes free (not painted, N = 5) and eyes covered (painted, N = 5, p-value: 0.34). Box plots show: center line, median; box limits, upper and lower quartiles; whiskers, ×1.5 interquartile range; points, outliers. (C) Additional examples (two different flies) of whole brain activity reduced to the two first principal component space. (D) R2 obtained with regressing activity of individual regions with walk (pan-Gal4;UAS-GCaMP). Adjusted p-values (Benjamini–Hochberg correction) were all <0.01. Bars are 95% CI. (E) R2 obtained with regressing activity of individual regions with flail (pan-Gal4;UAS-GCaMP). Empty markers correspond to adjusted (Benjamini–Hochberg correction) p-value >0.05 for comparison to 0. Bars are 95% CI. (F) R2 obtained with regressing activity of individual regions with groom (pan-Gal4;UAS-GCaMP). Bars are 95% CI. (G) Correlation coefficient obtained with regressing activity of individual regions with flail (pan-Gal4;UAS-GCaMP). Bars are 95% CI. (H) Correlation coefficient obtained with regressing activity of individual regions with grooming behavior (pan-Gal4;UAS-GCaMP). Bars are 95% CI. (I) R2 obtained with regressing activity of individual regions with walking on an air-supported (N = 19) vs. a styrofoam ball (N = 13), p-value = 0.02. Box plots show: center line, median; box limits, upper and lower quartiles; whiskers, ×1.5 interquartile range; points, outliers. (J) R2 obtained with regressing activity of individual regions with walking on an air-supported (N = 19) vs. a Styrofoam ball (N = 13). Box plots show: center line, median; box limits, upper and lower quartiles; whiskers, ×1.5 interquartile range; points, outliers. (K) Top: Examples of brain activity maps (R2 as z-stacks) and names of individual brain regions. Bottom: Schematic showing brain regions in adult fly brain adapted from Ito et al., 2014.