Fig. 1: Self-paced, spontaneous, left or right pedal-releasing task and effective LEC recording methods.

a Schematic diagram of a behavioral task that enabled us to monitor several events with high temporal resolution. A head-fixed rat pushed down both pedals for a short period (≥1 s) to start each trial, and subsequently released either pedal (e.g., right release) voluntarily and without an instruction cue to acquire a reward. The reward was dispensed with a random 300–700 ms delay. This task consisted of right-rewarded (R) and left-rewarded (L) blocks, which were alternated after the rat met the criteria (see Methods). b A typical example of task performance. The rat chose the correct pedal based on the reward. Large and small colored vertical bars (red represents right choice; blue represents left choice) indicate correct and incorrect trials, respectively. We averaged the number of right correct choices obtained from the previous 10 trials to calculate the proportion of correct choices. c Right-left pedal trajectories obtained from pre-trained (1^st day, top) and post-trained (14^th day, bottom) rats. d Learning curve over 14 training days. Inset, averaged proportion of correct choices on the 1^st and 14^th days. Black and red colors represent the pre- and post-training groups, respectively. ***p < 0.001, Mann–Whitney test. Error bars indicate SD. e LEC neurons projecting to the mPFC and hippocampus. Retrograde tracer (Fluoro-Gold) was injected into the mPFC, while retrograde viral tracer (mRFP-expressing G-deleted rabies viral vector) was injected into the hippocampus (Supplementary Fig. 2a). The distribution of retrogradely labeled neurons was subsequently examined in the LEC. Note that mPFC-projecting neurons (green) are found in layer Va while hippocampus-projecting neurons (red) are found in layers IIa and III of the LEC. f Example traces of simultaneous CA1 and LEC recording (left). Schema showing the position of optical fibers for identifying the two different projection neurons in the LEC (middle). The ipsilateral mPFC and CA1 were stimulated to identify mPFC- and CA1-projecting LEC neurons, respectively (Supplementary Fig. 2b). Example of recordings from a single CA1-projecting LEC neuron during optical stimulation (cyan area), with spike collisions. Black and red traces represent antidromic spikes in response to optical stimulation and spike collision tests, respectively. Black arrowheads indicate antidromic spikes. Red arrowheads indicate spontaneous spikes used as triggers for optical stimulation in collision tests (right). g The laminar position of the recording site for LEC cells was reconstructed using online and offline estimations. While recording, the recording depth was quickly determined by identifying CA1-projecting LEC neurons (f). After recording, probe shank tracks were visualized with DiI (Supplementary Fig. 2b).