Adolescent sleep shapes social novelty preference in mice

Abstract

Sleep disturbances frequently occur in neurodevelopmental disorders such as autism, but the developmental role of sleep is largely unexplored, and a causal relationship between developmental sleep defects and behavioral consequences in adulthood remains elusive. Here, we show that in mice, sleep disruption (SD) in adolescence, but not in adulthood, causes long-lasting impairment in social novelty preference. Furthermore, adolescent SD alters the activation and release patterns of dopaminergic neurons in the ventral tegmental area (VTA) in response to social novelty. This developmental sleep function is mediated by balanced VTA activity during adolescence; chemogenetic excitation mimics, whereas silencing rescues, the social deficits of adolescent SD. Finally, we show that in Shank3-mutant mice, improving sleep or rectifying VTA activity during adolescence ameliorates adult social deficits. Together, our results identify a critical role of sleep and dopaminergic activity in the development of social interaction behavior.

Extended Data Fig. 1. Developmental shaping of social preferences and adolescent sleep disruption (SD).

a, Schematics of the three-chamber social interaction assay. b, c, Naïve WT C57BL/6J mice showed age-dependent increases of sociability (b) and social novelty preference (c) in three-chamber social interactions over the developmental course (n = 10 each). Preference indices were calculated as indicated in each graph. One-way ANOVA, b, F (3, 36) = 5.33, P = 0.004; c, F (3, 36) = 4.23, P = 0.01, Dunnett’s post-hoc comparisons to P28, b, P56, ** P = 0.007, P84, ** P = 0.005; c, P56, ** P = 0.004. d – f, Hourly percentage of Wake (d), NREM (e) and REM (f) sleep in adolescent mice (P35 – 42) over a 24-hr light-dark cycle before (Baseline, black), during (red), and after (blue) the 5 days with daily SD sessions (ZT 2 – 6, indicated by the grey stripe). The 3 groups of data were from the same mice (n = 4). RM 2-way ANOVA followed by Bonferroni’s post-tests, Wake, Time × Treatment F (46, 207) = 3.18, P = 9.5 × 10⁻⁹, post-test During SD vs. Baseline, ZT2-3 ** P = 0.008, ZT3-4 ** P = 0.001, ZT4-5 ** P = 0.005, ZT5-6 * P = 0.02; NREM, Time × Treatment F (46, 207) = 2.90, P = 1.4 × 10⁻⁷, post-test During SD vs. Baseline, ZT2-3 * P = 0.01, ZT3-4 *** P = 0.0007, ZT4-5 ** P = 0.007, ZT5-6 * P = 0.03; After SD vs. Baseline, ZT15-16 * P = 0.02; REM, Time × Treatment F (46, 207) = 3.23, P = 5.9 × 10⁻⁹, post-test During SD vs. Baseline, ZT2-3 *** P = 0.0008, ZT3-4 ** P = 0.002, ZT4-5 *** P = 0.0004, ZT5-6 ** P = 0.02. g – k, Total amount of time (g), bout number (h) and average bout length (i) of Wake, NREM and REM states between ZT 2 – 6, total amount of 3 states during the following 6 hrs (ZT 6 – 12, j) and over the 24-hour light-dark cycle (k) (n = 9). One-way ANOVA within each state, g, Wake, F (1.48, 11.83) = 230.2, P = 0.000000001; NREM, F (1.28, 10.24) = 167.7, P = 0.00000005; REM, F (1.78, 14.21) = 79.99, P = 0.00000003; h, Wake, F (1.72, 13.75) = 54.78, P = 0.0000005; NREM, F (1.87, 14.94) = 70.18, P = P = 0.00000003; REM, F (1.42, 11.38) = 58.42, P = 0.000003; i, Wake, F (1.01, 8.07) = 24.47, P = 0.001; NREM, F (1.40, 11.18) = 67.54, P = P=0.000002; j, Wake, F (1.60, 12.81) = 11.30, P = 0.002; NREM, F (1.49, 11.94) = 11.41, P = 0.003; REM, F (1.48, 11.87) = 5.01, P = 0.03; k, Wake, F (1.93, 15.40) = 10.27, P = 0.002; NREM, F (1.80, 14.40) = 7.72, P = 0.006; REM, F (1.93, 15.43) = 7.59, P = 0.005. Dunnett’s multiple comparisons to Baseline, * P < 0.05; ** P < 0.01; *** P < 0.001. Unpaired t-test for REM in i, P = 0.31. l, m, EEG power spectrum of NREM (l) and REM (m) sleep was not altered after 5 days of SD compared to Baseline (n = 9). Both P > 0.05 by Kolmogorov-Smirnov test. n, Plasma corticosterone level of adolescent mice (P35 – 42) immediately after the SD session on 1st day and 5th day of SD compared to naïve mice of same age receiving no shake (n = 10 each). One-way ANOVA, F (2, 27) = 2.30, P = 0.12, followed by Tukey’s multiple comparisons test, all n.s. Data are shown as mean ± s.e.m. All tests were two-sided. n.s., not significant.

Extended Data Fig. 2. Comparisons of same-sex social interaction behavior between sexes.

a, Male (left) and female (right) test mice received Ctrl or SD between P35 – 42 and the three-chamber assay were performed using gender-matched stimulus mice, respectively, at P56. b – e, Interaction time with the empty cup (E, b), stranger 1 (S1) in Trial 1(c), S1 in Trial 2 (d), and stranger 2 (S2) in Trial 2 (e) during the three-chamber test was binned every 2 min and presented for male and female test mice separately. n = 10 mice each. RM 3-way ANOVA. P value of each variable (Sex, Time and Treatment) was indicated in the graph. f – j, Total interaction time (f), number of interaction bouts (g), average length of interaction bouts (h), total time spent in each chamber (i), and number of entries of each chamber (j) were presented separately for male-male interactions and female-female interactions and compared directly considering Sex, Stimulus and Treatment as 3 independent variables using RM 3-way ANOVA. n = 10 mice each. ANOVA P values are indicated in the graphs. In all parameters, Sex does not show a significant contribution to total variation (P > 0.05). Additional 2-way ANOVA was performed for total interaction time (f) and number of interaction bouts (g) in Trial 2, S1 and S2 categories (box insets) followed by Bonferroni’s post-tests comparing sexes within treatment. ANOVA P values were indicated in the box insets. All post-tests, male vs. female, P > 0.05. Data are shown as mean ± s.e.m. All tests used were two-tailed. Data of male and female subjects in this Extended Data Figure were combined and presented in Fig. 1c–i. Data are shown as mean ± s.e.m. All tests were two-sided.

Extended Data Fig. 3. Behavioral probing of adolescent SD mice.

a, Male mice received Ctrl or SD between P42 – 49 and were tested at P52 – 56 using the three-chamber assay with male stimulus mice (n = 10 each). Interaction time, RM 2-way ANOVA, Stimulus × Treatment F (3, 54) = 1.20, P = 0.32, Tukey’s post-test, Trial 1 Ctrl * P = 0.03, SD ** P = 0.004, Trial 2 Ctrl ** P = 0.009, SD P = 0.11. Preference indices, Welch’s t-test, ** P = 0.007. b, Male test mice received Ctrl or SD between P35 – 42 and the three-chamber assay using female stimulus mice at P56 (n = 10 each). Interaction time, RM 2-way ANOVA, Stimulus × Treatment F (3, 54) = 0.28, P = 0.84, Tukey’s post-test, Trial 1 Ctrl ** P = 0.002, SD *** P = 0.0003, Trial 2 Ctrl * P = 0.046, SD *** P = 0.0002; Preference indices, Welch’s t-test, both P > 0.05. c, d, Sociability preference (c) and social novelty preference (d) were not significantly different between mice receiving Ctrl protocol during adolescence and naïve mice with normal, undisturbed sleep. Only male mice were included in comparisons. Ctrl data were the same as that in Extended Data Fig. 2 (n = 10 each). Welch’s t-test. e, Ctrl and SD mice spent equal time in the two side chambers during the habituation phase of three-chamber assay (n = 7 each). Paired t-test. f, Comparison of social interaction performance between male mice sleep-deprived using the gentle-touch method with those deprived by automated SD in 3 cohorts that were wean at approximately same time. Samples were combined and included in the SD group in Fig. 1g. g, h, Mice receiving previous Ctrl or SD (n = 13 in Ctrl; 12 in SD) at P35 – 42 showed similar locomotion dynamics in the three-chamber apparatus (g) and comparable total distance traveled during the whole 20-min session of three-chamber assay (h). g, RM 2-way ANOVA followed by Bonferroni’s post-test, all n.s. h, Welch’s t-test. i, Time spent in self-grooming during the three-chamber assay was similar between groups (n = 15 in Ctrl; 14 in SD). Welch’s t-test P = 0.42. j, Mice receiving SD at P35 – 42 showed similar latency to their first entry to the open arm when placed in an elevated plus maze at P70 – 84 (n = 9 in Ctrl; 8 in SD). Welch’s t-test P = 0.83. The two mice showing latency of 300 sec did not enter the open arm at all during the whole 5-min session. k, The novel object recognition test with reduced memory requirement. The Test trial was performed immediately after 10 min of Training session. m, Interaction time with each object during the Test trial was plotted to the left Y-axis, and the ratio of time spent with the novel object over total interaction time was plotted to the right Y-axis. n = 10 in Ctrl; 9 in SD. Left, paired t-test, Ctrl, ** P = 0.008, SD, * P = 0.03. Right, Welch’s t-test, * P = 0.04. l, The social memory tests. Upper, the 1 stimulus paradigm, where the same stimulus mouse S1 was re-presented to the test mouse in Trial 2 after a 30-min interval; Lower, the 2 stimuli paradigm in which Trial 2 contains a novel stimulus mouse S2 in addition to S1. n, Interaction time with the single stimulus mouse in each trial of the 1 stimulus test. n = 10 each. Paired t-test, both * P = 0.02. o, Interaction time with the 2 stimulus mice during the 2 stimulus test. n = 10 each. RM one-way ANOVA, Ctrl, F (1.96, 17.66) = 18.94, P < 0.0001; SD, F (1.68, 15.16) = 4.44, P = 0.04, Tukey’s post-test, Ctrl, S1, Trial 1 vs trial 2, *** P = 0.0007; Trial 2, S1 vs. S2, * P = 0.03, SD, S1, Trial 1 vs. Trial 2, * P = 0.03. p, Comparisons of ratio of S1 interaction (Trial 2/Trial 1) between paradigms. n = 10 each. Welch’s t-test, Ctrl, ** P = 0.009; SD, P = 0.68. Data are shown as mean ± s.e.m. All tests were two-sided.

Extended Data Fig. 4. Fiber photometry recording of VTADA activity during social interactions.

a, Social performance of DAT-Cre mice in the fiber photometry setup (NS, normal sleep. n = 14 in Ctrl/NS; 9 in SD, including mice with poor GCaMP signals). RM 2-way ANOVA, Stimulus × Treatment F (3, 63) = 4.49, P = 0.006, followed by Tukey’s post-test, Trial 1, *** P < 0.0001, ** P = 0.004; Trial 2, ** P = 0.002. b, The mean amplitude of Ca2+ transients detected during the 20 min session (Trials 1 + 2) were similar between Ctrl and SD groups (n = 6 mice, transient peak Z-score were first averaged within each animal). Welch’s t-test, P = 0.41. c, d, Representative traces of GCaMP signals in Ctrl and SD mice during Trial 1 (c) and Trial 2 (d) of the social interaction assay. Colored stripes indicate interaction bouts. Red circles on trace indicate Ca2+ transients detected. e, Example GCaMP traces of the first, second, third and last interaction bout of each category. Time 0 s indicates bout onset, and yellow stripes indicate bout duration. f, g, Ctrl and SD mice in their home-cages were given a food pellet, and the VTA GCaMP6f signals were recorded and aligned to the time point when they first contacted the food pellets. GCaMP6f traces of 10 – 12 trials from n = 4 mice (2 – 3 trials each animal). across all trials were averaged and shown for Ctrl (f) and SD (g) animals. Shaded area indicates s.e.m. Insets show area under curve (AUC) of GCaMP6f signals within 0 – 10 s upon contact with the food pellet or a neutral object of similar size (e.g., a Q-tip head), n = 4 mice, paired t-test, Ctrl, * P = 0.04; SD, ** P = 0.002. h, Relative changes in AUC, n = 4 mice, Welch’s t-test, P = 0.56. Data are shown as mean ± s.e.m. All tests were two-sided.

Extended Data Fig. 5. Adolescent SD increased the membrane resistance but did not affect the intrinsic excitability of or synaptic inputs to VTADA neurons.

a, Representative image of a horizontal acute brain slice from a DAT-Cre::Ai14 mouse. VTADA neurons were filled with neurobiotin during whole-cell recording. The slices were subsequently fixed in 4% PFA and stained with Streptavidin (Alexa FluorTM 488 conjugate, Invitrogen) and antibodies against TH. Scale bar, 100 μm. b, Representative traces of spontaneous firing measured in a cell from a Ctrl (top, black) and SD animal (bottom, red; scale 50 mV and 5 s). c, Evoked firing in response to depolarizing current (90 pA, upper square pulses in gray) in a Ctrl (top black trace) and SD animal (red top trace; scale bars 50 mV and 0.1 s). The bottom two traces and current steps in Ctrl and SD conditions illustrate the hyperpolarizing response to a −20 pA step used for measurements of membrane resistance (indicated by the connected gray dashed [baseline] and vertical solid lines [deflection amplitude]). d, There were no observed differences in spontaneous firing frequency (n = 23 Ctrl and 27 SD neurons from 8 mice in each group, t(14) = 0.6394, P = 0.5329; data transformed with a square root, nested t-test) between the control (gray) and sleep-deprived animals (red). e, f, There were no effects of sleep deprivation (red) on the number of action potentials (APs) fired (n = 23 Ctrl and 26 SD neurons, F(16, 752) = 0.1899, P = 0.9998; two-way repeated measure ANOVA) or the firing frequency (n = 23 Ctrl and 26 SD neurons, F(16, 752) = 0.4933, P = 0.9511; two-way repeated measure ANOVA) in response to current pulse injections after adolescent sleep deprivation. g, h, There were no observed differences in hyperpolarization-activated (Ih) currents (n = 22 neurons from 7 Ctrl mice and 27 neurons from 8 SD mice, t(13) = 0.6605, P = 0.5204; nested t-test) or capacitance (n = 23 Ctrl and 27 SD neurons from 8 mice in each group, t(14) = 1.364, P = 0.1941; nested t-test) between Ctrl (left, gray) and SD animals (right, red). i, Membrane resistance was significantly increased in SD animals (M = 769.7 ± 54.32 MΩ; red) relative to Ctrl (M = 574.8 ± 36.01 MΩ; gray; n = 23 Ctrl and 27 SD neurons from 8 mice in each group, t(14) = 2.396, *P = 0.0311; nested t-test). j, k, SD did not have a significant effect on the coefficient of variation (CV) of rostrally evoked excitatory (n = 13 neurons from 9 mice in each group, t(24) = 0.9020, P = 0.3760; data transformed with square root, nested t-test) or inhibitory synaptic inputs (n = 10 neurons from 8 SD mice and 13 neurons from 10 Ctrl mice, t(21) = 1.795, P = 0.0870; nested t-test) to VTADA neurons. l, m, No observed differences between Ctrl (gray) and SD animals (red) in the paired pulse ratio (PPR) of rostrally evoked excitatory (n = 13 neurons from 9 mice in each group, t(24) = 0.0684, P = 0.9460; nested t-test) or inhibitory inputs (n = 10 neurons from 8 SD mice and 13 neurons from 10 Ctrl mice, t(16) = 1.927, P = 0.0720; data transformed with square root, nested t-test) to VTADA neurons. n, o, SD had no effect on the frequency (n = 10 neurons from 9 Ctrl mice and 9 neurons from 8 SD mice, t(15) = 0.0400, P = 0.9686; nested t-test) or amplitude (n = 10 neurons from 9 Ctrl mice and 9 neurons from 8 SD mice, t(17) = 1.544, P = 0.1410; nested t-test) of miniature excitatory postsynaptic currents (mEPSCs) in VTADA neurons. p, q, No significant differences in the amplitude (n = 10 neurons from 8 mice in each group, t(14) = 0.4431, P = 0.6645; nested t-test) or frequency (n = 10 neurons from 8 mice in each group, t(14) = 0.9301, P = 0.3681; nested t-test) of miniature inhibitory postsynaptic currents (mIPSCs) recorded in VTADA neurons from Ctrl (gray) or SD (red) animals. For all bar graphs, each bar represents 1 animal while the individual data points are from neurons recorded from that animal. Data are shown as mean ± s.e.m. All tests were two-sided.

Extended Data Fig. 6. Axonal projections of VTADA neurons in NAc and mPFC.

a, Numbers of infected VTADA neurons in DAT-Cre mice (n = 5 in Ctrl; 2 in SD). b, Representative image showing layer 1 – 3 of mPFC (anterior cingulate area). Only very few SYP-mRuby puncta were present in layer 1 compared to deeper layers. Scale bar, 50 μm. c, Quantification of areal density of SYP-mRuby puncta in NAc as well as layer 2/3 and layer 5/6 of mPFC. 17 – 24 fields were imaged in each region from 3 animals, and SYP-mRuby puncta were counted. F (2, 55) = 19.79, P < 0.0001 by one-way ANOVA, followed by Tukey’s post-test, *** P < 0.0001. d-f, A single plane of Z-stack confocal image in separate and merged channel views showing the soma of a mPFC pyramidal neuron (same neuron in Fig. 4f, Ctrl). Arrows indicate SYP-mRuby puncta and corresponding “pits” on the cytoplasmic membrane of the target neuron. Scale bar, 5 μm. g, k, n, Representative examples of MSN dendrites in NAc (g) and apical oblique dendrites (k) or basal dendrites (n) from mPFC pyramidal neurons in Ctrl and SD animals. Scale bar, 5 μm. h-j, Total spine density of NAc MSNs (h, n = 48 neurons from 5 mice in Ctrl; 39 neurons from 4 mice in SD), percentage of dendritic spines in contact with SYP-mRuby puncta (i, n = 48 neurons from 5 mice in Ctrl; 39 neurons from 4 mice in SD) and normalized integrated intensity of SYP-mRuby puncta that colocalized with labeled spines (j, n = 25 neurons from 4 mice in Ctrl; 21 neurons from 3 mice in SD) were not changed by adolescent SD. l, m, Total spine density (l) and percentage of SYP-mRuby-contacting spines (m) on apical oblique dendrites of mPFC pyramidal neurons (n = 37 neurons from 5 mice in Ctrl; 33 neurons from 4 mice in SD). o, p, Total spine density (o) and percentage of SYP-mRuby-contacting spines (p) on basal dendrites of mPFC pyramidal neurons (n = 40 neurons from 6 mice in Ctrl; 31 neurons from 4 mice in SD). h – p, all P > 0.05 by Welch’s t-test. Data are shown as mean ± s.e.m. All tests were two-sided.

Extended Data Fig. 7. VTADA neurons were overexcited by SD during adolescence.

DAT-Cre mice received AAV-DJ-EF1α-DIO-GCaMP6f injections in VTA at P21 and implantations for fiber photometry and EEG/EMG recording at P30. Simultaneous fiber photometry and EEG/EMG recordings were performed at P37 – 38. a – d, Representative GCaMP (top, black), EEG (middle, red) and EMG (bottom, blue) signals during spontaneous NREM sleep (a), wake (b), REM sleep (c) and SD sessions (d). Red circles on GCaMP traces indicate Ca2+ transients detected. e, f, Quantification of frequency (e) and amplitude (peak Z-score, f) of Ca2+ transients detected in each state. n = 4 mice. e, RM one-way ANOVA, F (3, 9) = 5.68, P = 0.02, followed by Dunnett’s multiple comparisons to NREM, ** P = 0.007; NREM vs. SD, # P = 0.01 by paired t-test. f, RM one-way ANOVA, F (3, 9) = 2.57, P = 0.12; NREM vs. Wake, # P = 0.02, NREM vs SD, # P = 0.02, by paired t-test. Data are shown as mean ± s.e.m. All tests were two-sided.

Extended Data Fig. 8. CNO administration in adolescent hM3DqDAT and hM4DiDAT mice.

a, Cre- and hM3Dq^DAT mice were placed in an open-field arena 30 min following the i.p. injection of CNO (2 mg/kg). Total distance traveled in the arena during an 8-min session was measured (n = 5). * P = 0.01 by Welch’s t-test. b, c, The 2-bottle free-choice drinking test. Cre- and hM3Dq^DAT mice were kept in a cage where they have free access to the bottles containing H₂O and CNO solution (50 mg/L), respectively, on opposite sides of the cage. The volume of H₂O and CNO solution consumed over 24 hours were recorded. Compared to Ctrl mice, SD mice drank more CNO solution than H₂O (b), and the preference for CNO (c), measured by the portion of CNO volume consumed over the total liquid consumed (H₂O + CNO), was increased in the SD group (n = 5). b, * P = 0.01 by paired t-test; c, ** P = 0.003 by Welch’s t-test. d, Social performance of Cre- and mCherry^DAT animals in the three-chamber assay at P56. e – g, DAT-Cre mice were injected with AAV-DJ-EF1α--DIO-hM3Dq in VTA at P21 and EEG recording was performed at P35 – 42. CNO (2mg/kg, i.p.) or saline was administered to the animal at ZT2. Hourly percentage of Wake (e), NREM (f) and REM (g) states during the 12-hr light phase are shown. Control group includes one mCherry^DAT mouse with CNO injection and three hM3Dq^DAT mice with saline injections (total n = 4); hM3Dq^DAT + CNO, n = 5. RM 2-way ANOVA followed by Bonferroni’s post-test, e, f, both ** P = 0.006. h – j, DAT-Cre mice were injected with AAV-DIO-hM4Di in VTA at P21 and EEG recording was performed at P35 – 42. Mice were subjected to SD protocol during ZT 2 – 6, and CNO (1mg/kg, i.p.) was administered to the animal at ZT2 and ZT4. Compared to the baseline recording (1 day before SD + CNO treatment) where the animals did not receive SD or CNO, the SD protocol can still effectively deprive both NREM (i) and REM (j) sleep even with the presence of CNO (n = 4). RM 2-way ANOVA followed by Bonferroni’s post-test, h, ZT 2 – 3 *** P = 0.0005, ZT 3 – 4 *** P < 0.0001, ZT 4 – 5 ** P = 0.009, ZT 5-6 * P =0.04; i, ZT 2 – 3 *** P = 0.0002, ZT 3 – 4 *** P < 0.0001, ZT 4 – 5 * P = 0.01; j, ZT 3 – 4 ** P = 0.001. k, l, Quantification of each state during the 4 hours after the CNO injection in hM3Dq^DAT mice (k) and in hM4Di^DAT mice with concurrent SD (l) as well as respective controls. k, n = 4 in control; 5 in hM3Dq^DAT + CNO, Wake, * P = 0.03; NREM, * P = 0.02 by Welch’s t-test. l, n = 4 mice each, Wake, ** P = 0.003; NREM, ** P = 0.005 by Welch’s t-test. REM, * P = 0.03 by Mann-Whitney test. m, No significant difference was found in the social memory test between Cre– and hM3Dq^DAT mice after adolescent CNO injections (n = 5). Left, * P = 0.02, ** P = 0.005 by paired t-test. Right, P = 0.67 by Welch’s t-test. n, The DAT-Cre mice that received AAV-DJ-EF1α-DIO-hM4Di-mCherry (hM4Di^DAT) or mCherry control virus (mCherry^DAT) on water regulation were given a single injection of CNO (2 mg/kg) and subsequently allowed access to both H₂O and sucrose solution (1%, w/v) for 4 hours. The mCherry^DAT mice showed strong preference to the sucrose, whereas the hM4Di^DAT mice, although still consuming more sucrose solution than water, showed a significantly lowered sucrose preference, which was calculated as the volume of sucrose solution consumed divided by the total liquid volume consumed (H₂O + sucrose, n = 5). Left, mCherry^DAT, ** P = 0.001, hM4Di^DAT, ** P = 0.005 by paired t-test. Right, ** P = 0.006 by Welch’s t-test. o, Novel object recognition test with reduced memory requirement was performed on adult mCherry^DAT mice and hM4Di^DAT mice (P56 – 60) with prior adolescent SD and CNO injections (P37 – 41). n = 7 in mCherry^DAT; 9 in hM4Di^DAT. Left, Wilcoxon matched-pairs signed rank test, mCherry^DAT, * P = 0.02; hM4Di^DAT, P = 0.10. Right, Welch’s t-test, P = 0.81. Data are shown as mean ± s.e.m. All tests were two-sided. n.s., not significant.

Extended Data Fig. 9. Adolescent sleep defects and adult in Shank3 InsG3680 mice.

a – c, Three-chamber social test was performed in InsG3680+/+ mutants (Homo, n = 8) and WT littermates (n = 9) at P56. Absolute interaction time and percentage of each interaction category are shown in a and b, respectively. Preference indices are shown in c. a, RM 2-way ANOVA, Stimulus × Genotype F (3, 45) = 2.18, P = 0.10 followed by Tukey’s post-test, P values from the post-test are indicated on the graph; b, RM 2-way ANOVA, Stimulus × Genotype F (3, 45) = 1.97, P = 0.13, Tukey’s post-test, *** P < 0.0001, ** P = 0.004, * P = 0.01; c, Sociability, Mann-Whitney test, P = 0.13, Social Novelty, Welch’s t-test, * P = 0.04. d – f, Hourly percentage of Wake (d), NREM (e) and REM (f) states over the 24-hr light-dark cycle at P35 – 42 in adolescent InsG3680 mice (n = 5 in WT; 8 in Homo). g, h, Amount of Wake, NREM and REM states in InsG3680 mice at P35 – 42 (n = 5 in WT; 8 in Homo) in the light phase (g) and the dark phase (h). Welch’s t-test, g, Wake, * P = 0.02 (t = 3.06, df = 8.24), NREM, * P = 0.04 (t = 2.32, df = 9.26), REM, * P = 0.03 (t = 2.46, df = 10.86). h, all P > 0.05. i – l, EEG Power spectrum of NREM (i) and REM(k) sleep at P35 – 42 (n = 5 in WT; 8 in Homo). Kolmogorov-Smirnov test, i, P = 0.12; k, P = 0.39. Relative EEG powers of each frequency band are shown in j for NREM and l for REM. Welch’s t-test, j, Delta, P = 0.10, Theta, P = 0.63, Alpha, * P = 0.02, Beta, P = 0.43; l, Delta, P = 0.08, Theta, * P = 0.03, Alpha, P = 0.15, Beta, P = 0.90. m, The pAAV-mTH-Cre construct. Cre expression is under control of mouse tyrosine hydroxylase (TH) promoter. n – q, Representative images of a InsG3680 mouse which received injection of AAV-DJ-mTH-Cre + AAV-DJ-EF1α-DIO-mCherry in VTA and after two weeks of viral expression. Red, mCherry fluorescence; Green, TH immunostaining; Blue, DAPI. Scale bars, 200 μm. r, Dual labeling strategy in InsG3680 mice. s, Quantification showing the efficiency (% of TH/mCherry double positive neurons in total TH+ neurons) and specificity (% of double positive neurons in total mCherry+ neurons) of dopaminergic neuron labeling within the VTA area using co-injection of AAV-DJ-mTH-Cre and AAV-DJ-EF1α-DIO-mCherry (or -hM4Di-mCherry). n = 3 animals. t, The VTA axonal terminals in NAc were examined in WT (n = 40 neurons from 4 animals) and Homo (n = 38 neurons from 4 animals) InsG3680 mice at P70, using the dual labeling strategy with AAV-mTH-Cre. Welch’s t-test, P = 0.0000003. Data are shown as mean ± s.e.m. All tests were two-sided. n.s., not significant.

Extended Data Fig. 10. Effects of Flupirtine and DORA12 treatments on adolescent sleep and adult social interactions in Shank3 InsG3680 mice.

a – d, Hourly percentage of Wake (a) and REM (b) state over a 24-hr light-dark cycle and the quantification of each state during the light (c) or dark (d) phase in adolescent homozygous InsG3680 mice with Flupirtine treatment (n = 7 in Veh; 8 in Flup). RM 2-way ANOVA, a, Wake, Time x Treatment, F (23, 299) = 1.74, P = 0.02; b, REM, Time x Treatment, F (23, 299) = 0.90, P = 0.61, Bonferroni’s post-test, Wake ** P = 0.002, * P = 0.04. c, d, Welch’s t-test, NREM in Light Phase, * P = 0.048. e – h, Hourly percentage of Wake (f), NREM (g) and REM (h) state over a 24-hr light-dark cycle in adolescent homozygous InsG3680 mice with DORA treatment. Arrows indicate injections of DORA12 or vehicle (50% PEG400 in saline) at ZT2. n = 5 each. RM 2-way ANOVA with Bonferroni’s post-test. i – k, Quantification of each state during the light (i) or dark (j) phase and 4 hours following the injection of DORA12 or vehicle (k). n = 5 each. Welch’s t-test, k, NREM, * P = 0.04. l – n, Homozygous InsG3680 mice received daily DORA12 (20 mg/kg, i.p.) or vehicle (50% PEG400 in saline) for 5 consecutive days during P37 – 41, and three-chamber social interaction assay was performed at P56. n = 12 in Veh; 11 in DORA. Interaction time (l), RM 2-way ANOVA, Stimulus × Treatment F (3, 63) = 1.43, P = 0.24, Tukey’s post-test, Trial 1, Veh, ** P = 0.004, DORA, ** P = 0.003; Trial 2, Veh, P = 0.45, DORA, * P = 0.03. Preference indices of sociability (m) and social novelty (n), Welch’s t-test, m, t = 0.93, df = 19.22, P = 0.36, n, t = 3.01, df = 16.29, ** P = 0.008. Data are shown as mean ± s.e.m. All tests were two-sided. n.s., not significant.

Figure 1.. Adolescent sleep disruption (SD) induced loss of social novelty preference in adult social interactions.

a, Adolescent SD protocol and timeline of experimental procedures. b, Representative occupancy heatmaps of Ctrl and SD mice in the three-chamber apparatus. c-i, Interaction time with each stimulus during the three-chamber test at P56 was quantified. Binned interaction time is shown in c-f, total interaction time is shown in g, and preference indices of sociability and social novelty are shown in h and i, respectively. n = 20 mice (10 males + 10 females). Repeated-measure (RM)-2-way ANOVA, d, Time F(3.66, 139.0) = 16.83, P = 1.2 × 10⁻¹⁰; e, Time F(3.09, 117.3) = 3.98, P = 0.009, Treatment F(1, 38) = 6.27, P = 0.02; f, Time F(3.54, 134.7) = 13.67, P = 1.1 × 10⁻⁸, Treatment F(1, 38) = 7.81, P = 0.008; g, Stimulus × Treatment F(3, 114) = 6.23, P = 0.0006, followed by Tukey’s post-tests (P as indicated). Welch’s t-test, h, t(35.99) = 0.46, P = 0.64. Mann-Whitney test, i, P = 4.1 × 10^-7. j-l, Mice subjected to adolescent restraint (P35 – 42) were tested using the three-chamber assay at P56. n = 12 mice. j, RM-2-way ANOVA (Stimulus × Treatment F(3, 66) = 0.14, P = 0.94) with Tukey’s post-tests (P as indicated). Welch’s t-tests, k, t(21.47) = 0.13, P = 0.90; l, t(21.44) = 0.11, P = 0.92. m–p, Mice that received adolescent Ctrl or SD (P35 – 42) were tested using the three-chamber assay at P84. n = 13 mice. n, RM-2-way ANOVA (Stimulus × Treatment F(3, 72) = 10.76, P = 6.3 × 10⁻⁶) with Tukey’s post-tests (P as indicated). Welch’s t-tests, o, t(21.44)= 3.77, P = 0.001; p, t(16.44) = 4.64, P = 0.0003. q–t, Mice received Ctrl or SD protocol during P84 – 91 and the three-chamber assay was performed at P105. n = 13 mice. r, RM-2-way ANOVA (Stimulus × Treatment F(3, 72) = 0.74, P = 0.53) with Tukey’s post-tests (P as indicated). Welch’s t-tests, s, t(16.97) = 0.92, P = 0.37; t, t(23.93) = 0.22, P = 0.82. Data are shown as mean ± s.e.m. All tests were two-sided. For detailed statistics information, see Supplementary Table 1.

Figure 2.. Adolescent SD attenuated the novelty-dependent response pattern of VTA^DA neurons in social interactions.

a, Social interaction test with simultaneous fiber photometry was performed on P56 DAT-Cre mice with prior adolescent Ctrl or SD (P35 – 42). Bottom, a representative trace of GCaMP signal recorded. Colored stripes indicate interaction bouts with E (grey), S1 in Trial 1(purple), S1 in Trial 2 (teal) and S2 (pink). Dotted line indicates Trial 2 onset. Red circles indicate transient peaks. b, Representative image showing AAV-delivered GCaMP6f expression and optical fiber placement in the VTA. Scale bar, 100 μm. c, Number of Ca²⁺ transients detected (Trials 1 + 2). n = 6 mice. Welch’s t-test, t(9.32)= 0.74, P = 0.48. d–g, GCaMP traces during the first interaction bouts with E (d), S1 in Trial 1 (e), S1 in Trial 2 (f) and S2 (g) aligned to the bout onset (time 0 s). n = 6 mice. Shaded area indicates s.e.m. h–k, Peak Z-score of GCaMP signals from first 10 bouts of interactions with E (h), S1 in Trial 1 (i), S1 in Trial 2 (j) and S2 (k) were fitted with one-phase exponential decay model and compared using extra-sum-of-squares F test. n = 6 mice. P is indicated on each graph. In j, P can’t be calculated due to both ambiguous fits. l, ΔZ-score of the first bout of each interaction category. n = 6 mice. RM-2-way ANOVA, Treatment F(1, 10) = 19.27, P = 0.001, followed by Tukey’s post-tests (within each group) and Bonferroni’s post-tests (between groups), post-test P as indicated. m–q Averaged area under curve (AUC) of first 3 interaction bouts of each category. n = 6 mice. Mann-Whitney tests, n, P = 0.009; o, P = 0.04. Welch’s t-tests, p, t(7.77) = 2.47, P = 0.04; q, t(7.82) = 1.43, P = 0.19. Data are shown as mean ± s.e.m. All tests were two-sided. For detailed statistics information, see Supplementary Table 1.

Figure 3.. Adolescent SD altered the dopamine release in NAc in response to social stimuli.

a, Mice with adolescent Ctrl or SD (P35 – 42) received AAVs carrying GRAB_DA sensor DA2m and optical fiber implantation at P42, and the social interaction test with simultaneous fiber photometry was performed at P56. b, Representative image showing DA2m immunostaining (green) and optical fiber placement to NAc. Scale bar, 500 μm. c–f, DA2m signals during the first interaction bouts with E (c), S1 in Trial 1 (d), S1 in Trial 2 (e) and S2 (f) aligned to the bout onset (time 0 s). n = 7 in Ctrl; 6 in SD. Shaded area indicates s.e.m. Upper insets show representative individual traces. g–j, Peak Z-score of DA2m signals from first 10 bouts of interactions with E (g), S1 in Trial 1 (h), S1 in Trial 2 (i) and S2 (j) were fitted with one-phase exponential decay model and compared using extra-sum-of-squares F test. n = 7 in Ctrl; 6 in SD. P is indicated on each graph. k, Calculation of ΔZ-score and AUC. l, ΔZ-score of the first bout of each interaction category. n = 7 in Ctrl; 6 in SD. RM-2-way ANOVA, Treatment x Stimulus, F(3, 33) = 3.24, P = 0.03, followed by Tukey’s post-tests within each group (P as indicated). m–p, Averaged AUC of first 3 interaction bouts of each category. n = 7 in Ctrl; 6 in SD. Mann-Whitney test, m, P = 0.02. Welch’s t-tests, n, t(16.97) = 0.92, P = 0.004; o, t(6.59) = 2.43, P = 0.048; p, t(8.20) = 0.73, P = 0.48. Data are shown as mean ± s.e.m. All tests were two-sided. For detailed statistics information, see Supplementary Table 1.

Figure 4.. Adolescent SD altered the projection profile of VTA^DA axons in NAc and mPFc.

a, Dual-color labeling strategy in DAT-Cre mice. b, Representative images showing the expression of SYP-mRuby (red) in VTA counter-stained with TH (green). Inset, magnified image of the yellow-boxed area. c, d, Representative images showing the labeling of a medium spiny neuron in NAc (c) and a pyramidal neuron in mPFC (d). e–m, Quantification of VTA^DA axonal termini on the somata of target neurons, i.e., NAc MSNs (e, h, i), mPFC PNs (f, j, k) and INs (g, l, m). Example images (e–g) are shown in single Z plane images (e1–g2’) and maximum intensity projection of Z-stack images (e3–g4’). Dotted contour indicates the soma area. SYP-mRuby puncta within the soma area were counted (h, 43 Ctrl/35 SD neurons from 4 mice each; j, 42 Ctrl/37 SD neurons from 5 mice each; l, 38 Ctrl/44 SD neurons from 5 mice each) and measured for their integrated intensity (i, 39 Ctrl/35 SD neurons from 4 mice each; k, 42 Ctrl/36 SD neurons from 5 mice each; m, 38 Ctrl/43 SD neurons from 5 mice each). Welch’s t-tests, h, t(63.67) = 3.42, P = 0.001; j, t(76.98) = 4.02, P = 0.0001; k, t(73.78) = 4.35, P = 0.00004. Mann-Whitney test, m, P = 0.002. Individual color channels of example images in b–g were adjusted for brightness/contrast before merged. Scale bars, b, 100 μm; c, d, 50 μm. e–m, 5 μm. Data are shown as mean ± s.e.m. All tests were two-sided. For detailed statistics information, see Supplementary Table 1.

Figure 5.. Activity level of VTA^DA neurons during adolescence is critical for social novelty preference.

a, hM3Dq-mCherry expression in VTA. Sale bar, 100 μm. Virus combinations used are listed on the Right. b, Timeline of experimental procedures. CNO was administered at ZT2. c–e, Tree-chamber assay was performed on hM3Dq^DAT mice and control littermates at P56 – 60 (n = 8 Cre-/4 mCherry^DAT/9 hM3Dq^DAT mice). c, RM-2-way ANOVA (Stimulus × Treatment F(3, 45) = 3.93, P = 0.01) with Tukey’s post-tests (P as indicated). One-way ANOVA, d, F(2, 18) = 1.74, P = 0.20; e, F(2, 18) = 10.58, P = 0.0009, with Sidak’s post-tests, *** P = 0.0009, * P = 0.01. f, Timeline of experimental procedures. CNO was administered prior to SD onset. g–i, Three-chamber assay was performed on hM4Di^DAT and mCherry^DAT mice at P56 – 60. n = 8 mCherry^DAT/10 hM4Di^DAT mice. g, RM-2-way ANOVA (Stimulus × Treatment F(3, 48) = 1.95, P = 0.13) with Tukey’s post-tests (P as indicated). Welch’s t-test, h, t(14.60) = 0.61, P = 0.55, i, t(15.51) = 3.59, P = 0.003. j, m, Representative images showing NAc MSNs and mPFC PNs in hM3Dq^DAT (j) and SD + hM4Di^DAT mice (m) and respective controls. Scale bar, 5 μm. Dotted contour indicates the soma area. k, l, n, o, Quantification of SYP-mRuby puncta in hM3Dq^DAT (k, l) and SD + hM4Di^DAT (n, o) mice and control animals. k, n = 33 Control/35 hM3Dq^DAT MSNs from 4 mice each, Welch’s t-test, t(61.07) = 2.83, P = 0.006. l, n = 22 Control/33 hM3Dq^DAT PNs from 4 mice each, Mann-Whitney test, P = 0.14. n, n = 38 Control/42 hM4Di^DAT MSNs from 4 mice each, Mann-Whitney test, P = 0.008. o, n = 37 Control/39 hM4Di^DAT PNs from 4 mice each, Welch’s t-test, t(73.45) = 1.31, P = 0.19. Individual color channels of example images in a, j, and m were adjusted for brightness/contrast before merged. Data are shown as mean ± s.e.m. All tests were two-sided. For detailed statistics information, see Supplementary Table 1.

Figure 6.. Adolescent restoration of NREM sleep rescued the social interaction deficit in Shank3 InsG3680^+/+ mice.

a-c, Adolescent Flupirtine injections in InsG3680^+/+ mice. Hourly binned NREM sleep is shown in b, and quantification of time in each state within 4 h following the injection is shown in c. n = 8 in Flup/7 in Veh (0.3% DMSO in saline). b, RM-2-way ANOVA (Time × Treatment F(23, 299) = 2.61, P = 0.0001) with Bonferroni’s post-tests (P as indicated). c, Welch’s t-test, Wake t(7.88) = 3.09, P = 0.02; NREM t(8.85) = 3.33, P = 0.009. d–f, Flup- or Veh-treated InsG3680^+/+ mice were tested for social interactions at P56. n = 12 mice. d, RM-2-way ANOVA (Stimulus × Treatment F(3, 66) = 3.95, P = 0.01) with Tukey’s post-tests (P as indicated). Mann-Whitney test, e, P = 0.13, f, P = 0.0001. g, h, AAV-hSyn-ChR2-EYFP was injected to the PFC of InsG3680^+/+ mice (g, Scale bar, 500 μm), and 1-Hz light stimulation (ZT 2 – 6) was delivered daily between P37 – 41. i, j, Example heatmap (i) and quantification (j) of EEG power spectrum with or without 1-Hz stimulation. n = 11 mice in j, shaded area indicates s.e.m., Kolmogorov-Smirnov test, P = 0.0001. Paired t-test for frequency band, δ, t(10) = 2.35, P = 0.04; θ, t(10) = 3.03, P = 0.01; α, t(10) = 0.42, P = 0.68; β, t(10) = 1.88, P = 0.09. k, Quantification of time in each state with or without 1-Hz stimulation. n = 11 mice. Paired t-test, Wake, t(10) = 0.45, P = 0.66; NREM, t(10) = 0.19, P = 0.86; REM, t(10) = 0.68, P = 0.51. l–n, InsG3680^+/+ mice with or without adolescent 1-Hz stimulation were tested using the three-chamber assay at P56. n = 9 in No stim/10 in 1-Hz. l, RM 2-way ANOVA (Stimulus × Treatment F(3, 51) = 1.72, P = 0.18) with Tukey’s post-tests (P as indicated). Welch’s t-test, m, t(11.51) = 0.31, P = 0.77; n, t(14.52) = 2.21, P = 0.04. o-r, AAV-DIO-hM4Di-mCherry was injected to InsG3680^+/+; DAT-Cre^+/− mice for adolescent VTA inhibition, and three-chamber assay was performed at P56. n = 9 mice. p, RM-2-way ANOVA (Stimulus × Treatment F(3, 48) = 1.85, P = 0.15) with Tukey’s post-tests (P as indicated). q, Mann-Whitney test, P = 0.86; r, Welch’s t-test, t(15.20) = 3.60, P = 0.003. Data are shown as mean ± s.e.m. All tests were two-sided. For detailed statistics information, see Supplementary Table 1.