Figure 3.

Two-Step ACC Calcium Imaging

(A) Example GRIN lens placement in ACC.

(B) Fluorescence signal from a neuronal region of interest (ROI) identified by CNMF-E (top panel, blue) and fitted trace (orange) due to the inferred deconvolved neuronal activity (bottom panel).

(C) Histogram showing the distribution of average event rates across the population of recorded neurons. Events were defined as any video frame on which the inferred activity was non-zero.

(D) Average trial aligned activity for all recorded neurons, sorted by the time of peak activity. No normalization was applied to the activity. The gray bars under (D), (E), and (G) between choice and outcome indicate the time period that was warped to align trials of different duration.

(E) Regression analysis predicting activity on each trial from a set of predictors coding the choice (top or bottom), second step (left or right), outcome (rewarded or not) that occurred in each trial, and their interactions. Lines show the population coefficient of partial determination (CPD) as a function of time relative to trial events. Circles indicate where CPD is significantly higher than expected by chance, assessed by permutation test with Benjamini-Hochberg correction for comparison at multiple time points.

(F) Representation of the second-step state before and after the trial outcome. Points show second-step predictor loadings for individual neurons at a time point halfway between choice and outcome (x axis) and a time point 250 ms after trial outcome (y axis).

(G) Time course of pre- and post-outcome representations of second-step state, obtained by projecting the second step predictor loadings at each time point onto the pre- and post-outcome second-step representations. The red and blue triangles indicate the time points used to define the projection vectors.

(H) Representation of trial outcomes (reward or not) obtained at the left and right poke. Points show predictor loadings for individual neurons 250 ms after trial outcome in a regression analysis in which outcomes at the left and right poke were coded by separate predictors. The regression analysis was identical to that shown in (E) except that the outcome and second-step x outcome predictors were replaced by left outcome and right outcome predictors, which coded reward/non-reward in trials that reached the left or right second-step state, respectively.