Fig. 3: DRN^GAD2 neurons display wakefulness-dependent populational activities.

a Schematic of fiber photometry and simultaneous EEG/EMG recordings. AAV-EF1α-DIO-jGCaMP7b was injected into the DRN of GAD2-Cre mice. b, c Representative images showing the expression of jGCaMP7b in the DRN (b) and the colocalization of jGCaMP7b with GAD2 mRNA (c). Aq, aqueduct. d Quantitative analysis of the efficiency and specificity of jGCaMP7b to label DRN^GAD2 neurons. n = 3 mice. e Representative EEG spectrogram, EEG/EMG traces, color-coded brain states and Ca²⁺ fluorescence trace across different wakefulness/sleep states. Freq., frequency; W, wake; NR: NREM; R: REM. f Quantification of ΔF/F during wake, NREM sleep and REM sleep. n = 9 mice, one way ANOVA followed by Tukey post hoc test, F_2,24 = 8.38, wake vs NREM: P = 0.032, wake vs REM: P = 0.002. g Comparison of ΔF/F during long- and short-bout wakefulness. n = 9 mice, two tailed paired t test, t₈ = 4.687, P = 0.00157. h–k Ca²⁺ activities of DRN^GAD2 neurons during transitions from wake to NREM sleep (h), NREM sleep to wake (i), NREM to REM sleep (j), and REM sleep to wake (k). n = 9 mice, two tailed paired t test (h–j). For (h), t₈ = 12.075, P = 2.04 × 10^–6; for (i), t₈ = 5.865, P = 3.76 × 10⁻⁴; for (j), t₈ = 1.273, P = 0.251. For k, Wilcoxon signed rank test, W = 45, P = 0.004. Shading represents ±SEM. l Schematic experimental setup for simultaneous photometry and pupil recording in awake head-fixed mice. m Representative pupil (top) and Ca²⁺ (bottom) traces during spontaneous pupil size recording. Pupil_Max, max pupil size. n Heatmaps showing all pupil dilation bouts (top) and corresponding Ca²⁺ signals (bottom) extracted from 6 recorded mice. The vertical red lines indicate the onset of pupil dilation. Pupil_Norm., normalized pupil size. o Average traces of pupil size (red) and Ca²⁺ signals (dark) aligned to onset of pupil dilation. Shading represents ±SEM. p Cumulative probability distribution of all the Pearson correlation coefficients of Ca²⁺ signals of DRN^GAD2 neurons with pupil size. The gray area indicates the non-significant Pearson correlation coefficients. *P < 0.05, **P < 0.01, ***P < 0.001, n.s., not significant. Data (d, f–k) are presented as mean ± SEM.