Analysis of photometric calcium signals

To visualize pathway-specific activity, the recorded fluorescence signals were expressed as percentage change in fluorescence ΔF/F = (F(t)-F₀)/F₀, where F(t) denotes the fluorescence value at time t across the entire trial time (from event 1 to event 11) and F₀ the baseline fluorescence level calculated as the mean value in the 1-s time window before the maintenance period (before event 5). For analysis across trials and mice, we z-scored the fluorescence signals for individual trials by calculating (F(t)-F₀)/σ, where σ is the standard deviation of the fluorescence values in the 1-s baseline window for F₀ calculation. Because mice behaved freely in the T-maze, trial phases varied in their duration. To temporally align trial-related signals we therefore resampled the z-scored signals for each trial phase, as defined above, to match the median duration of this phase across all trials. The median duration was used for plotting. This registration of the recorded signals by segment-wise temporal resampling permitted us to average and compare the activity patterns in the separate task phases across trials and mice. All mice contributed 4 expert sessions to the dataset, which in total comprised 1085 trials (544 rightward sample runs: 407 correct, 137 mistakes; 541 leftward sample runs: 405 correct, 136 mistake). The resampled traces were used for detailed analysis of the maintenance period (between events 5 and 7) in Figs. 2 and ​and44.

To control for potential motion artefacts and hemodynamic signal components in our fiber photometry data of mPFC→dmStr pathway activity, we used two experimental strategies: First, we recorded task-related photometric fluorescence signals in a separate cohort of GFP-expressing control mice (4 mice, 5 sessions each). Second, we performed simultaneous GCaMP6m measurements at two excitation wavelengths (488-nm and 425-nm excitation for calcium-dependent and calcium-independent fluorescence, respectively). To this end, both lasers were modulated at different carrier frequencies and the fluorescence signals were digitally demodulated (see above). The 425-nm excited control fluorescence traces were clearly smaller than the signals observed with 488-nm laser excitation and relatively flat (Supplementary Fig. ^{2a, b}). Control photometric signals from GFP mice were also small and relatively flat, with some negative-going fluorescence dips appearing for trial phases 3–4 and as mice approached the waterspout and licked to collect the reward. In summary, these control experiments exclude hemodynamic or motion-related artefacts as major confounds and indicate that the maintenance signals are of neuronal origin.