(A) Experimental setup: a mouse with a cranial window is placed on a treadmill in front of a blue screen monitor, and it is head-fixed under the two-photon microscope for calcium imagin. (B) Static blue screen was used to record spontaneous activity during 5 min, three sessions per day, 5 min apart between them. (C) Visual stimulation protocol constituted of 50 repetitions of a 2 s single-orientation drifting gratings with a mean static screen between each of them during 1–5 s randomly to record evoked activity for 5 min, three sessions per day, 5 min apart between them. (D) Strategy to image the same neurons in the field of view on different days: (left) a single plane was carefully located in a reference recorded position (day 1), then two extra planes also were imaged separated 5 µm up and down. Three planes were imaged in a period of 81 ms. (Right) Maximum intensity projection was obtained from the three planes generating a single frame. Scale bar: 50 µm. (E left) Detection of regions of interest (ROIs; gray shapes) based on Suite2P algorithm, green ROI is used as a representative example, scale bar: 50 µm; (up right) extraction of calcium signal (ΔF/F0) with peak signal-to-noise ratio (PSNR) >18 dB; (middle right) spike inference using foopsi algorithm; (bottom right) a binary signal obtained by thresholding spike inference, which is used to represent the active frames of the neuron. (F) Raster plot built with binary signals from active neurons recorded simultaneously. Each row represents the activity of a single neuron, black dots represent the activity of the neuron. (G) Center of an example image at 4× zoom (white square on D right) recorded in the same location up to 46 days after the first day of recording. Note that image at day 46 is noisier than on the first days. Scale bar: 12.5 µm. (H) Count of active neurons in different days. The number of active neurons identified on day 1 decreased significantly on days 43–46 during spontaneous and evoked activity (p=0.023 and p=0.013, respectively). (I) Percentage of single-neuron activity, that is, percentage of frames a neuron was active. The average activity of all neurons was ~15% on all days during spontaneous and evoked activity. (J) Merge of active neuron ROIs from two sessions: first session from day 1 (green in four panels) versus a second single session from same day 1 (5 min later), one session from days 2, 10, and 46 (from left to right, respectively, magenta). The intersection of active neurons in both sessions is in gray color. Scale bar: 50 µm. (K) Percentage of active neurons between two sessions: first session from day 1 (green) versus second sessions from days 1, 2, 10, and 46 (magenta). Common active neurons (gray) in both sessions were 42% ± 2% during spontaneous and 37% ± 4% during evoked activity. There were no significant differences on common active neurons between days 1 and 2, but a significant decrease on days 10 and 43–46 during spontaneous (p=2 × 10–6 and p=4 × 10–9, respectively) and evoked activity (p=2 × 10–4 and p=4 × 10–9, respectively). Data are presented as mean ± SEM. Kruskal–Wallis test with post hoc Tukey–Kramer: *p<0.05, **p<0.01, and ***p<0.001. See Figure 1—source data 2.