Fig. 3: Identification of cortical ensembles by spatially coded visual stimuli.

a Task and training schematic diagram. Chronic two-photon microscopy was used to obtain the cortical ensembles linked to each spatially-coded square on the screen. The eyeblink task of mice was trained by repeating the synchronized CS (the flash of a specific square (3, C)) and US (a periorbital air puff) to the head-fixed mouse on a freely moving treadmill. A high-speed camera was used to record the eyeblink and other movements of the mouse. b The 5\(\times\)6 square array on the full screen was spatially encoded. It sequentially illuminated, one by one, for 5 s each with a 5 s interval (indicated by blue arrows). c The flicker of square (3, C) at different durations activated ‘On’ neurons 1-2 and ‘Off’ neurons 3-5 that were consistently related to the opening and closing of square (3, C), respectively. n = 12 mice. Scale bar: 50 μm. d The ratio of ‘On’ and ‘Off’ neurons belonged to ‘On’ and ‘Off’ ensembles and other neurons outside of ensembles in the observed neurons (n = 1737 neurons from 4 mice). e The mapping of neuronal ensembles linked to each square (color-coded) by stitching 6 FOVs together to cover the entire V1. n = 6 mice. A, anterior; P, posterior; M, medial; L, lateral. Scale bar: 200 μm. f Representative receptive field of two neurons corresponds to the ‘On’ and ‘Off’ of several squares, respectively. Lower panel: the average Ca²⁺ response of the neuron to the ‘On’(left) or ‘Off’(right) of each square. g The air puff activated neurons in the ‘On’ ensemble (neurons 8-9) to square (3, D) and other neurons outside the ensemble (neurons 10-11). Scale bar: 50 μm. Lower-left: correlation of the neurons inside (On neurons 3-9; Off 15-17) and outside the ensemble. Right: Ca²⁺ activities of the neurons responding to the ‘On’ (O) and ‘Off’ (F) of the square (3, D) and air puff (A). n = 21 neurons, 6 mice. Source data are provided as a Source Data file.