Fig. 3

Fig. 3

2P Imaging of Cellular-Resolution Brainwide Responses during BC Reveal the Dynamics of Experience-Encoding Activity in the vHb

(A) Head-fixed volumetric 2P calcium imaging during BC while monitoring tail movement.

(B) Diagram of imaged brain area (yellow rectangle).

(C) Head-fixed fish exhibit a reduction in tail movement rate as a result of BC (black: control, n = 6; blue: shocked, n = 8; p = 0.030, one-sided Student’s t test). Prior exposure to acute ketamine eliminates this response (pink, n = 4, p = 0.283, n.s.).

(D) Regressor consisting of linearly rising activity following the onset of shocks was used to search for cells with activity correlated to the extent of BC.

(E) Example of the fit between the regressor and the Δf/f Ca²⁺ trace for four neurons (bottom; locations at top; black dashed line indicates start of shocks).

(F) Region of interest (ROI) of each neuron colored by the correlation (r) between its Δf/f Ca²⁺ trace and the regressor (scale bar, 25 μm). See also Figure S3.

(G) Average baseline-subtracted Δf/f response for all neurons in each of several brain regions (see also Figure S4). In the vHb, the average activity over the final 2 min was increased in shocked fish (n = 8) compared to control (n = 6) and ketamine-treated (n = 4) fish (p = 0.0189 and p = 0.0432, respectively; two-way repeated-measures ANOVA with Tukey HSD post hoc comparison). Shocked fish also showed a decrease in raphe activity (p = 0.012 and p = 0.007) and dorsal thalamus activity (p = 0.007 and p = 0.006) compared to controls and ketamine-treated fish.

(H) Example of MultiMAP registration to identify serotonergic neurons in the superior raphe (see STAR Methods).

(I and J) Raster plot of baseline-subtracted Δf/f activity of all 5-HT⁺ neurons (I) (n = 158 neurons from 3 fish) and 5-HT^– neurons (J) (n = 332 neurons) sorted based on hierarchical clustering with top 8 flat clusters demarcated by coloring scheme of left. 5-HT⁺ neurons, on average, exhibited a significantly larger response to BC onset compared to 5-HT^– neurons (60-s window following onset; p = 0.026, paired Student’s t test) and compared to 5-HT⁺ neurons in non-shocked controls (n = 4 non-shocked control fish; p = 0.027, Student’s t test). 5-HT^– neurons did not have a significant response to BC onset (p = 0.37, Student’s t test).