Fig. S3

2P Imaging of Brainwide Responses during BC, Related to Figures 3and 5

(A) Modified BC causes immobility in freely swimming fish. Fish that are exposed to 50 ms shocks delivered with a uniformly distributed random inter-shock interval between 15 and 25 s enter a reduced mobility state (n = 10; pink shading indicates shock period; p = 10⁻⁵, paired Student’s t test comparing average speed in 5 min pre-shock to 5 min post-shock). (B) Max-projection of two representative fish with the ROI of neurons with significant shock-triggered activation colored according to their Δf/f response (red and blue indicate positive and negative responses, respectively; see STAR Methods). Significant responses were distributed throughout the brain with clusters of large responses in the posterior portion of the vHb (white arrow on left) and the area lateral of the Neuropil Region 6 (white arrows on right). (C) Shock-triggered Δf/f response of significantly activated neurons in several anatomical regions throughout the brain. (D) Images displaying the fit between the regressor and the Δf/f Ca²⁺ trace for all neurons across all z-slices for five fish from the control, shocked, and re-exposed conditions. The ROI of each neuron is colored by the value of the Pearson correlation coefficient (r) between the regressor and its Δf/f calcium trace. The ROIs are rendered partially transparent (alpha = 0.2) such that all planes can be perceived. The control and shocked fish in the top row represent the same fish that are shown in Figure 3F for comparison. (E) Shocks activate vHb neurons. The shock-triggered average Δf/f trace of a vHb neuron. The time of shock is indicated by a dashed red line. To determine the significance of the shock-triggered response, 400 bootstrapped average responses were generated using randomized shock times (see STAR Methods). The shaded areas indicate s.e.m. of shock-triggered responses (green) and bootstraps (beige). (F) The percentage of neurons in the vHb that showed a significant shock-triggered activation in response to individual shocks was not significantly reduced by ketamine treatment in either the left or right hemisphere (p = 0.52 and 0.60, respectively, Student’s t test). The percentage of activated neurons was also not significantly different between the hemispheres in either the shocked, ketamine, or re-exposed conditions (p = 0.51, 0.66, and 0.28, respectively, paired Student’s t test). (G) The average baseline-subtracted Δf/f response for all vHb neurons separately for the left and right hemispheres in the control (left), shocked (center), and ketamine-treated (right) conditions. Activity was not significantly different between the two hemispheres (p = 0.81, p = 0.08, and p = 0.20, respectively, paired Student’s t test on activity in final two min). Shaded area and error bars are s.e.m.