We expressed GCaMP6f in ACC pyramidal neurons under the CaMKII promotor and imaged calcium activity through a gradient refractive index (GRIN) lens using a miniature fluorescence microscope (n = 4 mice, 21 sessions, 2,385 neurons, 3,732 trials) (Ghosh et al., 2011). Constrained non-negative matrix factorization for endoscope data (CNMF-E) (Zhou et al., 2018) was used to extract activity traces for individual neuron from the microscope video (Figure 3B). All subsequent analyses used the deconvolved activity inferred by CNMF-E. Activity was sparse, with an average event rate of 0.12 Hz across the recorded population (Figure 3C). We aligned activity across trials by time-warping the interval between the first-step choice and second-step port entry (labeled “outcome” in figures, as this is when outcome information becomes available) to match the median interval (Figure S4). Activity prior to choice and following outcome was not time warped.
