Extended Data Fig. 3: Two-photon imaging of cortical neuronal activities across brain states.
From: Cortical regulation of two-stage rapid eye movement sleep
a, Top: fluorescence image of RSC in a RBP4Cre mouse injected with AAV-DIO-GCaMP6s. Scale bar: 200 μm. Bottom: example field of view for two-photon imaging. Scale bar: 50 μm. b, Example Ca2+ traces of RSC L5 neurons together with color-coded brain states. c, Average Ca2+ activity of RSC L5 neurons across sleep states. Each line indicates averaged Ca2+ activity for all identified neurons from one recording session (n = 9 sessions from 4 mice). P(Wake vs NREM) = 0.72; P(Wake vs REM) = 0.0023; P(NREM vs REM) = 0.0012; **P < 0.005, two-sided paired t test. d-f, Similar to a-c, but for L2/3 neurons in the M2 of Thy1-GCaMP6s mice. Data were from n = 6 sessions in 4 mice. P > 0.1 for all comparisons. P(Wake vs NREM) = 0.22; P(Wake vs REM) = 0.85; P(NREM vs REM) = 0.10. Scale bar in d: 50 μm. g-h, Scatter (g) or line (h) plots showing the distribution of the activity difference between REM sleep and other states, for RSC L2/3 neurons (n = 1238 neurons from 6 mice), RSC L5 neurons (n = 289 from 4 mice) and M2 L2/3 neurons (n = 193 from 4 mice). i, Percentages of REM-active, REM-inactive and non-modulated neurons in RSC L2/3 (n = 12 sessions from 6 mice), RSC L5 (n = 9 sessions from 4 mice) or M2 L2/3 (n = 6 sessions from 4 mice). * P < 0.05, ** P < 0.01, *** P < 0.001, two-sided unpaired t test. j, Averaged Ca2+ activities for REM-active (n = 706 neurons), REM-inactive (n = 188) and non-modulated (n = 344) neurons in the L2/3 of RSC, at NREM → REM (top) or REM → wake (bottom) transitions (from 6 mice). The same dataset were used as the top panel in g. Vertical lines, transition time points. Dotted lines above the averaged traces indicate time points that are significantly different from baseline (−30s to −6s before NREM → REM, or 6 s to 30 s after REM → wake). Closed dots: P < 0.05 for REM-active neurons; open dots: P < 0.05 for REM-inactive neurons. P > 0.1 for non-modulated neurons. Kruskal-Wallis tests with Dunn’s correction for multiple comparison. Note that the latency for significant activity increase of REM-active neurons was 2.3 s. Error bars or shadings, SEM.
