Dopaminergic ventral tegmental area (VTA) neurons are critically involved in a variety of behaviors that rely on heightened arousal, but whether they directly and causally control the generation and maintenance of wakefulness is unknown. We recorded calcium activity using fiber photometry in freely behaving mice and found arousal-state-dependent alterations in VTA dopaminergic neurons. We used chemogenetic and optogenetic manipulations together with polysomnographic recordings to demonstrate that VTA dopaminergic neurons are necessary for arousal and that their inhibition suppresses wakefulness, even in the face of ethologically relevant salient stimuli. Nevertheless, before inducing sleep, inhibition of VTA dopaminergic neurons promoted goal-directed and sleep-related nesting behavior. Optogenetic stimulation, in contrast, initiated and maintained wakefulness and suppressed sleep and sleep-related nesting behavior. We further found that different projections of VTA dopaminergic neurons differentially modulate arousal. Collectively, our findings uncover a fundamental role for VTA dopaminergic circuitry in the maintenance of the awake state and ethologically relevant sleep-related behaviors.
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We thank T. Davidson for assistance in the set-up of the fiber photometry rig, A. Whittle for assistance in core body temperature measurements and for feedback on the manuscript, A. Rolls and P. Bonnavion for early training in sleep–wake recording and viral manipulations and discussion and J. Garner for discussion. This work was supported by National Institutes of Health (NIH) grants RO1-MH087592, RO1-MH102638 and RO1-AG04767 (L.d.L.), a Brain and Behavior Research Foundation (NARSAD) grant (L.d.L.), the US Israel Binational Science Foundation and the Klarman Family Foundation (L.d.L.), Edmond and Lily Safra Center of Brain Science (ELSC) postdoctoral fellowships (A.E.-R.) and NIH F32 AA022832 (W.J.G.). We thank the Stanford Neuroscience Microscopy Service, supported by NIH NS069375.
The authors declare no competing financial interests.
Integrated supplementary information
(a-g) Transient detection. (a) Representative fluorescence trace. (b) Filtered ΔF/F signals. Pink line, signal low-pass filtered at 0 – 4 Hz. Green line, signal low-pass filtered at 0 – 40 Hz. (c) The derivative of the squared difference between the filtered signals. Pink line depicts the thresholding at the mean + 2 s.d. (d) Prospective transients (pink circles). (e) Prospective transients occurring during high ΔF/F periods. Blue line depicts the mean + 2 s.d. threshold. (f,g) Transient detected (red circles). The exact peak time of each transient was identified by taking the maximal value of the thresholded signal within each transient. (h-k) Paired comparison of (h) fluorescence, (i) transient rate, (j) transient amplitude and (k) transient rate * transient amplitude, across the different arousal states. n = 4 animals, 8 trials per animal. Different colors depict different mice. The P values were obtained from paired t-tests. (l) Durations of arousal states following a state transition. Note most of wake episodes following NREM sleep were shorter than 15 s; the animals transitioned back to NREM sleep following these brief arousals. In contrast, NREM sleep and REM sleep episodes tended to be longer. (m-o) We stereotaxically injected the control virus AAV-DJ-EF1α-DIO-GFP into the VTA of a Th-Cre mouse and implanted a fiber optic (400 µm) and EEG/EMG electrodes. (n) Representative fluorescence trace, EEG and EMG trace across spontaneous sleep-wake states. (o,p) Mean (± s.e.m.) (o) fluorescence and (p) calcium transient rate during wake, NREM sleep and REM sleep. n = 5 trials, one-way ANOVA.
Supplementary Figure 2 Spontaneous sleep–wake states of Th-Cre mice transduced with the different viral constructs.
(a,b) The percentage (mean ± s.e.m.) of time spent awake, in NREM sleep and REM sleep during 24 h of baseline sleep recording of Th-Cre mice transduced with mCherry (gray), hM4Di (red), eYFP (green) and ChR2 (blue) virus (n = 4 per group). (a) Summary of the entire 24 h period. No significant differences were found between the different groups (P > 0.24), one-way ANOVA tests. (b) Percentage per hour spent awake (left), in NREM sleep (middle) and REM sleep (right). No significant differences were found between the different groups (P > 0.86), two-way ANOVA between compound injected and time. These findings demonstrate that the viral manipulations themselves did not disrupt the normal sleep-wake cycle.
Supplementary Figure 3 Sleep–wake architecture following chemogenetic inhibition of Th+ VTA neurons.
(a,b) The percentage of time (mean ± s.e.m.) spent awake (left), in NREM sleep (middle) and REM sleep (right) during the 24 h starting with the saline or CNO (1 mg kg-1) administration to (a) mCherry and (b) hM4Di mice. n = 7 per group. * P < 0.05, *** P < 0.001, **** P < 0.0001, two-way RM ANOVA between compound injected and time, followed by Sidak’s post hoc tests. hM4Di: P(interaction) = 0.0008(wake), 0.0007(NREM), 0.058(REM). mCherry: P(interaction) > 0.2 for wake, NREM sleep and REM sleep. (c) Delta power as function of time during NREM sleep episodes following saline (left) and CNO (right) injections in hM4Di mice. n = 6 mice per group; summary of all episodes from all mice. P and R were obtained from a Pearson correlation.
(a) EEG power spectrum of NREM sleep episodes, in CNO-treated hM4Di mice, during high-fat chow (left), a female mouse (middle), and a component of fox odor (right) presentations. (b) EEG power spectrum of wake episodes during high-fat chow (left), a female mouse (middle), and a component of fox odor (right) presentations. (c) The percentage of time (mean ± s.e.m.) spent awake, in NREM sleep and REM sleep in the presence of high fat chow (left), female mouse (middle) and a component of fox odor (right). We implanted wild-type mice with EEG/EMG electrodes and presented them with salient stimuli (one stimulus at a time, at least 4 days apart) either the light or dark phases of the day. EEG/EMG activity was monitored during 2 h, in the presence of the salient stimuli. *** P < 0.001, **** P < 0.0001, paired t-tests. These findings demonstrate that a naturalistic increase drive for sleep reduces the capacity to maintain arousal in face of some saline stimuli.
(a) Representative hypnogram (left) and FFT-derived delta power (right) of a CNO-treated hM4Di mouse during the 1 h test period in a novel environment containing new nesting material. (b) Core body temperature (mean ± s.e.m.) in the course of 4 h following CNO administration in mCherry and hM4Di mice.
(a) Co-localization of Fos within TH cells. We delivered optical stimuli (25 Hz, during 5 s min-1, for 30 min) to the VTA of eYFP and ChR2 mice at the beginning of the light phase (ZT 2) and sacrificed the mice 90 min later. TH-positive neurons in midline cell groups from hypothalamus to hindbrain were scored for the presence of Fos. **** P < 0.0001, two-way RM ANOVA between distance and viral injection, followed by Bonferroni post hoc tests. The data demonstrate that optical stimulation produced a significant increase in Fos expression in TH+ cells throughout the anterior-posterior length of the VTA, but not in other TH+ neuronal populations. (b) Mean latency (± s.e.m.) to wake from NREM sleep following 5 s stimulation at 25 Hz, 3 days before and following sleep deprivation (SD; 4 h of gentle handling), at the same circadian time, of eYFP (left) and ChR2 (right) mice (n = 6 per group, 1 stim per mouse). P > 0.05, two-tailed paired sample t-tests. (c) EEG power spectrum during NREM sleep from eYFP (left; n = 7) and ChR2 (right; n = 8) mice, 4 s before the onset of stimulation (black) and 4 s into stimulation (gray). Quantification based on the average of 6 stimulations per mouse. (d) EEG power spectrum during wake episodes in eYFP (left) and ChR2 (right) mice during the 6-h stimulation period (ZT 0- ZT 6; light blue), the following 6 h of the light phase (ZT 6 - ZT 12; black), and the subsequent dark phase (ZT 12 – ZT 0; red). Insert represents EEG power spectrum during wake episodes across the 6 h of photostimulation in ChR2 animals. (e) EEG power spectrum of NREM sleep episodes following semi-chronic photostimulation. NREM sleep episodes EEG power spectrum during the 6 h following the termination of photostimulation in eYFP (left) and ChR2 (right) mice. Two-way RM ANOVA between frequency and time, followed by Tukey post-hoc tests. ChR2, P(time) < 0.0001, P(interaction) < 0.0001. eYFP, P(time) > 0.3. The EEG power spectrum during the 1st h following photostimulation differed significantly from the remaining 5 h of the light phase (P < 0.05 in all comparisons; Tukey post-hoc tests). (f) The percentage of time spent awake, in NREM sleep and REM sleep in mice pre-treated (45 min before) with saline / D1,D2 antagonists and undergoing a semi-chronic photostimulation (2 s per min, at 25 Hz, during 2 h; n = 4 per group). ** P < 0.01, **** P < 0.0001, two-way RM ANOVA between compound injected and viral transduction, followed by Sidak’s post hoc tests.
Supplementary Figure 7 Optogenetic interrogation of Th+ VTA efferent projections in modulation of sleep-to-wake transitions and nest-building behavior.
(a-h) Cumulative distribution of sleep-to-wake transitions following optogenetic stimulation at the NAc, mPFC, CeA and DLS at 1 and 25 Hz in eYFP and ChR2 mice during (a-d) NREM sleep and (e-h) REM sleep. Note that 25-Hz stimulation during NREM sleep in the NAc induced an almost immediate sleep-to-wake transition in about 70% of the stimulation trials. (i-l) Nesting behavior following semi-chronic optogenetic stimulation at the (i) NAc, (j) mPFC, (g) CeA and (l) DLS (2 s per min, for 3 h, at 25 Hz).The nesting score (mean ± s.e.m.) represents the amount of nesting material used and shape of the nest after a 3 h period (1, poor; 5, good). n = 4 mice per group. Wilcoxon matched-pairs signed rank test. No significant differences were found between the different groups (P > 0.5).
Supplementary Figures 1–7 (PDF 2099 kb)
hM4Di mouse injected with saline 45 min prior to the placement of inaccessible high fat palatable food in his home cage. (MP4 13207 kb)
hM4Di mouse injected with CNO 45 min prior to the placement of inaccessible high fat palatable food in his home cage. (MP4 10223 kb)
hM4Di mouse injected with saline 45 min prior to the placement of an adult female mouse in his home cage. (MP4 11650 kb)
hM4Di mouse injected with CNO 45 min prior to the placement of an adult female mouse in his home cage. (MP4 10973 kb)
hM4Di mouse injected with saline 45 min prior to the placement of an adult female mouse in his home cage. The female was restricted to a mesh cage. (MP4 18141 kb)
hM4Di mouse injected with CNO 45 min prior to the placement of an adult female mouse in his home cage. The female was restricted to a mesh cage. (MP4 14454 kb)
hM4Di mouse injected with CNO 45 min prior to his placement in a new cage containing ad-libitum food and water and a 3 g Nestlet. (MP4 18073 kb)
hM4Di mouse injected with saline 45 min prior to his placement in a new cage containing ad-libitum food and water and a 3 g Nestlet. (MP4 22172 kb)
hM4Di mouse injected with saline 45 min prior to his placement in a new cage together with his old nest, containing new nesting material. (MP4 9524 kb)
hM4Di mouse injected with CNO 45 min prior to his placement in a new cage together with his old nest, containing new nesting material. (MP4 7370 kb)
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Eban-Rothschild, A., Rothschild, G., Giardino, W. et al. VTA dopaminergic neurons regulate ethologically relevant sleep–wake behaviors. Nat Neurosci 19, 1356–1366 (2016). https://doi.org/10.1038/nn.4377
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