Fig. 1: Volumetric imaging of neuronal activity during visual stimulation and identification of activity patterns.

a Mice are head-fixed and GCaMP6s signals from pyramidal neurons are recorded through a cranial window via volumetric two-photon microscopy while visual stimuli are displayed on a monitor. Mouse running speed on a wheel and mouse whisking captured through an infrared camera are also recorded. Drawing of the experimental preparation adapted from Jesús Pérez-Ortega, Tzitzitlini Alejandre-García & Rafael Yuste (2021) Long-term stability of cortical ensembles eLife 10:e64449. https://doi.org/10.7554/eLife.64449. b Three sequentially recorded planes of layer 2/3 visual cortex at ~37 fps (i.e., a period of ~81 ms for 3 planes). Scale bar: 100 µm. c Neuronal regions of interest (ROIs) above 10 dB of peak signal-to-noise ratio (PSNR). Colored ROIs indicate neurons tuned to oriented drifting gratings (see Supplementary Fig. 1). Scale bar: 100 µm. d Raster plot of all neurons recorded simultaneously during a five-minute session of visual stimulation (left) accompanied by timestamps for random repetitions of drifting gratings along eight directions. Neurons are sorted by ensemble activity patterns, identified through clustering analysis applied to all frames (right). Stimulus presentation times are organized based on the clustering of activity patterns (bottom-right). Significance of each activity pattern’s similarity within a cluster is tested using a one-sided z-test (p < 0.5). Significant activity patterns are identified as ensembles. Ensemble p values from left to right p = 3 × 10^–5, p = 1 × 10^–4, p = 5 × 10^–10, p = 3 × 10^–5, p = 0.04. *p < 0.05, ***p < 0.001. e Mouse running speed and whisking motion energy (left), and they are sorted according to the activity pattern clustering detected in d on the right. f Ensembles timestamps (left) are extracted from the activity pattern clustering (right).