Dopamine Receptor Type 2-Expressing Medium Spiny Neurons in the Ventral Lateral Striatum Have a Non-REM Sleep-Induce Function

Analysis of sleep structure before and after the VLS/VS D2-MSNs ablation. a, Experimental setup. We conducted 24 h EEG/EMG recording in a soundproof box and set a 12/12 h light/dark cycle using LED light. b, Total amount of wake, NREM sleep, and REM sleep time in control (DOX-off day −1) and DOX-off days 5, 7, and 10 (n = 5 mice). Repeated-measures ANOVA was followed by the Tukey–Kramer post hoc test (Results of statistical tests is shown in Table 1). c, Time spent in each sleep state during the light and dark phases in control (DOX-off day −1) and DOX-off mice on days 5, 7, and 10 (n = 5 mice). Repeated-measures ANOVA was followed by the Tukey–Kramer post hoc test (Results of statistical tests is shown in Table 2). d, Daily variations in wake time (left), NREM sleep (middle), REM sleep (right) every hour in control (DOX-off day −1), and DOX-off day 10 (n = 5 mice). Two-way repeated-measures ANOVA was followed by the Tukey–Kramer post hoc test (Results of statistical tests is shown in Table 3). e, Episode numbers of wake, NREM sleep, and REM sleep for 24 h, light, and dark phases in control (DOX-off day −1) and DOX-off days 5, 7, and 10 (n = 5 mice). Repeated-measures ANOVA. Wake (24 h): p = 0.62; wake (light), p = 0.36; wake (dark), p = 0.89; NREM (24 h): p = 0.60; NREM (light), p = 0.33; NREM (dark), p = 0.88; REM (24 h): p = 0.30; REM (light), p = 0.42; REM (dark), p = 0.88. f, Daily variations in number of episodes during each sleep/wake state in control (DOX-off day −1), DOX-off day 10 (n = 5 mice). Two-way repeated-measures ANOVA was followed by the Tukey–Kramer post hoc test). Results of statistical tests is shown in Table 4. g, Mean duration of each bout of wake, NREM sleep, and REM sleep for 24 h, light, and dark phases in control (DOX-off day −1) and DOX-off days 5, 7, and 10 (n = 5 mice). Repeated-measures ANOVA. Wake (24 h): p = 0.21, Wake (light): p = 0.22, Wake (dark): p = 0.04 (no significant difference between each group using the Tukey–Kramer post hoc test), NREM (24 h): p = 0.20, NREM (light): p = 0.29, NREM (dark): p = 0.16, REM (24 h): p = 0.14, REM (light): p = 0.37, REM (dark): p = 0.16. h, Daily variations in mean duration during each sleep-wake state in control (DOX-off day −1) and DOX-off day 10 (n = 5 mice). Two-way repeated-measures ANOVA was followed by the Tukey–Kramer post hoc test). Results of statistical tests is shown in Table 5. Error bars indicate SEM; *p < 0.05.

Comparison of EEG power spectrum before and after the VS D2-MSNs ablation. EEG spectra before (DOX-off day −1) and after (DOX-off day 10) the VS D2-MSNs ablation during each sleep-wake state (n = 5 mice). Two-way repeated-measures ANOVA was followed by the Tukey–Kramer post hoc test). Results of statistical tests is shown in Table 6. Colored shades indicate SEM; *p < 0.05.

Population Ca²⁺ signals of the VLS D2-MSNs across the sleep-wake states. a, Schematic illustration of the fiber photometry system for monitoring intracellular Ca²⁺ signals of VLS D2-MSNs in D2-YC mice. The EEG and EMG were recorded simultaneously. PMT, photomultiplier tube. b, YCnano50 fluorescent expression in D2-YC mice (left) and a schematic of the fiber photometric recording site (right) in the VLS. The asterisks indicate the tip of the optical fiber. Scale bar, 1 mm. c, Representative examples of EEG signal trace and power spectrogram, EMG signal, and population Ca²⁺ signal trace and spectrogram of VLS D2-MSNs across the sleep-wake states during the light (upper panel) and dark (lower panel) phases. The green line shows wakefulness, the blue line shows NREM sleep and the red line shows REM sleep. d, Peak frequency of Ca²⁺ signals in VLS D2-MSNs during each sleep-wake state during the light (left)/dark (right) phases (28 recordings from 7 mice and 11 recordings from 5 mice, respectively; one-way ANOVA followed by Tukey–Kramer post hoc test, wake vs NREM, p = 0.13 and p = 0.37, respectively; NREM vs REM, p = 0.47 and p = 0.72, respectively; wake vs REM, p = 0.67 and p = 0.82, respectively). e, Correlation coefficient between the Ca²⁺ signals and each frequency band (δ, θ, σ, β, and γ) of EEG power or EMG power during wake and NREM sleep, each frequency band (δ, θ, σ, β, and γ) of EEG power during REM sleep during light (left)/dark (right) phases. f, Cross-correlation between EMG power and Ca²⁺ signals during wake, between EEG γ power and Ca²⁺ signals during wake, between EEG β and γ power and Ca²⁺ signals during NREM sleep, and between EEG γ power and Ca²⁺ signals during REM sleep during light (left)/dark (right) phases. g, Mean Ca²⁺ signal levels of the VLS D2-MSNs across the sleep-wake states during light (left)/(right) phases (28 recordings from 7 mice and 11 recordings from 5 mice, respectively; one-way ANOVA followed by Tukey–Kramer post hoc test, wake vs NREM, p = 1.7 × 10⁻³ and p = 0.08, respectively; NREM vs REM, p = 0.80 and p = 1.2 × 10⁻⁵, respectively; wake vs REM, p = 7.0 × 10⁻³ and p = 4.1 × 10⁻⁴, respectively). h, The Ca²⁺ signal of the VLS D2-MSNs aligned with each sleep/wake transition during light (left)/dark (right) phases. Upper panel, Ca²⁺ signal fluctuations during individual transitions with color-coded fluorescence intensity (NREM to wake, n = 386 and 183; wake to NREM, n = 450 and 188; NREM to REM, n = 93 and 7; REM to wake, n = 53 events and 7 from 7 and 5 mice, respectively). Middle panel, Average Ca²⁺ signals from all transitions. Lower panel, Mean of Ca²⁺ signal levels before and after 20 s from each stage transition (VLS: p_NREM-wake = 3.7 × 10⁻³ and p = 0.10, respectively, p_wake-NREM = 0.03 and p = 0.01, respectively, p_NREM-REM = 0.33 and p = 0.96, respectively, p_REM-wake = 0.02 and p = 3.3 × 10⁻³, respectively; paired t test). Gray shading and error bars indicate SEM; *p < 0.05. W, wake; N, NREM sleep; R, REM sleep.

Sleep-wake state change by optogenetic activation of the VLS D2-MSNs. a, Schematic illustration of optogenetic activation of VLS D2-MSNs in Drd2-ChR2(C128S) mice (hereafter referred to as D2-ChR2 mice). Scale bar, 1 mm. The blue and yellow shades indicate the illumination times. b, Representative examples of EEG signal traces, power spectrograms, and EMG signals under the photostimulation of VLS D2-MSNs during the wake state in D2-ChR2 mice. Vertical blue and yellow shades indicate illumination times. The upper panel shows the optogenetic manipulation and the lower panel shows the control experiment. c, NREM sleep induction rate which indicates whether NREM sleep occurred within 100 s after light illumination. Upper column shows a light phase and lower column shows a dark phase. In each column, left side shows the activation group (blue light illumination) and right side shows control group [yellow light illumination; n = 4 mice, 5–10 manipulations/recordings, a total of 49 trials for activation (light phase), 51 trials for control (light phase), a total of 38 trials for activation (dark phase), 39 trials for control (dark phase)]. d, Representative examples of EEG signal trace, power spectrogram, and EMG signal under the photostimulation of VLS D2-MSNs during the NREM sleep in D2-ChR2 mouse. Vertical blue shade indicates illumination times. e, NREM sleep latency after the initiation of optogenetic activation of VLS D2-MSNs during the wake state. The columns on the left and right show the light and dark phases, respectively. In each column, left bar shows the activation group, right bar shows the control group [n = 4 mice, 5–10 manipulations/recordings, a total of 49 trials for activation (light phase), 51 trials for control (light phase), a total of 38 trials for activation (dark phase), 39 trials for control (dark phase); p_light = 6.9 × 10⁻⁵, p_dark = 1.5 × 10⁻³, independent t test]. f, Percentage of NREM sleep time for 100 s after the initiation of VLS D2-MSNs photoactivation during the wake state. The columns on the left and right show the light and dark phases, respectively. In each column, left bar shows the activation group, light bar shows the control group [n = 4 mice, 5–10 manipulations/recordings, a total of 49 trials for activation (light phase), 51 trials for control (light phase), a total of 38 trials for activation (dark phase), 39 trials for control (dark phase); p_light = 1.2 × 10⁻⁴, p_dark = 2.4 × 10⁻⁴, independent t test]. g, EEG spectrum of D2-ChR2 mice during natural NREM sleep (blue line; we extracted and averaged 200 s of NREM sleep before optogenetic activation) and optogenetically induced sleep (black line) during the light (left panel) and dark phases (right panel; n = 4 mice, the same mice in c–e; δ: p_light = 0.63, p_dark = 0.94; θ: p_light = 0.59, p_dark = 0.75, independent t test). Colored shades and error bars indicate SEM; *p < 0.05.