Chronic stress promotes NREM sleep in mice

To evaluate the influence of chronic stress on sleep, we first exposed mice to 2 weeks of stressful stimuli (foot shock, 20 times/day; air puff, 30 times/day; physical restraint, 1 h/day) (Fig 1A). Chronic stress significantly increased depressive-like behaviors in the sucrose preference test (SPT), forced swimming test (FST), and tail suspension test (TST), but did not significantly alter the locomotor activity evaluated by the open field test (OFT) (Fig 1B). These results suggest that 2 weeks of chronic stress increased depressive-like behaviors without affecting motor function. In addition, we evaluated the effects of exposure to chronic stress on diurnal locomotor activity assessed by the wheel-running test (WRT) (Fig 1C). We found that 2 weeks of chronic stress significantly reduced the fragmented light-phase activity but increased the fragmented dark-phase activity (Fig 1C), suggesting that chronic stress may influence the sleep/wake states of mice.

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Fig 1. Chronic stress promotes NREM sleep in mice.

(A) Schematic of the experimental design. (B) Depressive–like behaviors in different experimental groups (n = 7 animals/group). Non–CS: mice that received no exposure to chronic stress stimuli (CS); CS: mice that received exposure to CS. (C) Left: wheel–running activities of mice in Non–CS and CS groups. Middle: activity onsets of mice in Non–CS (n = 12 animals) and CS (n = 8 animals) groups during a period of 8 days. Right: the number of activity bouts in light and dark phases of mice in Non–CS (n = 12 animals) and CS (n = 8 animals) groups. (D) Representative EEG spectrograms, EEG and EMG traces (recorded from ZT 1 to ZT 2) of mice in Non–CS and CS groups. (E) Left: time course changes of NREM and REM sleep and wakefulness of mice in Non–CS (n = 6 animals) and CS (n = 7 animals) groups. Middle: total sleep–wake amounts during the whole day (24 h) of mice in Non–CS (n = 6 animals) and CS (n = 7 animals) groups. Right: total sleep–wake amounts during light phase (ZT 0 –ZT 12) and dark phase (ZT 12 –ZT 24) of mice in Non–CS (n = 6 animals) and CS (n = 7 animals) groups. (F) Number of transitions between different pair of brain states during the light phase (ZT 0 –ZT 12), dark phase (ZT 12 –ZT 24), and the whole day (24 h) of mice in Non–CS (n = 6 animals) and CS (n = 7 animals) groups. W–N: Wake to NREM; N–W: NREM to Wake; N–R: NREM to REM. For all figures: one–way ANOVA with Sidak’s multiple comparisons test, *, P < 0.05; **, P < 0.001; ***, P < 0.0001; ns = no significant difference. Error bars indicate the SEM. Underlying data can be found in S1 Data. EEG, electroencephalography; EMG, electromyography; FST, forced swimming test; NREM, nonrapid eye movement; OFT, open field test; SPT, sucrose preference test; TST, tail suspension test; WRT, wheel-running test; ZT, Zeitgeber time.

https://doi.org/10.1371/journal.pbio.3002282.g001

Next, we evaluated the influences of chronic stress on the sleep/wake states of mice by electromyography (EMG) and electroencephalography (EEG) after exposure to 2 weeks of chronic stress (Fig 1D). We found that chronic stress caused a marked increase in NREM sleep duration and a complementary decrease in wakefulness time during both the daytime and nighttime (Fig 1D and 1E). In contrast, the rapid eye movement (REM) sleep duration was not significantly affected by chronic stress (Fig 1D and 1E). To determine whether the increase in NREM sleep duration was caused by an increase in the initiation or maintenance of NREM sleep, we analyzed the transitions between different pairs of brain states. We found that chronic stress significantly increased the number of wake-NREM transitions (Fig 1F) without significantly affecting the number of NREM-wake and NREM-REM transitions (Fig 1F), indicating enhancement of the initiation but not the maintenance of NREM sleep. The above results indicate that chronic stress promotes NREM sleep in mice.

Activation of the LHb mediates the chronic stress-induced increase in NREM sleep duration

Accumulating evidence suggests that the LHb encodes stressful signals and plays a pivotal role in the regulation of sleep [10,16–21]. This prompted us to explore the role of the LHb in mediating the influences of chronic stress on sleep/wake states. We induced the expression of the neuronal inhibitor DREADD hM4Di-EGFP in LHb neurons by injecting AAV2/9-hSyn-hM4Di-EGFP into the bilateral LHb (Fig 2A). LHb neurons expressing hM4Di-EGFP were inhibited daily via i.p. injection of CNO (1 mg/kg) for 14 days (Figs 2B and S1A). We found that although the long-term inhibition of RMTg-projecting LHb neurons did not significantly affect sleep/wake states in wild-type mice (S1B–S1D Fig), inhibiting LHb neurons during exposure to stressful stimuli reduced not only the chronic stress-induced increase in NREM sleep duration but also the effects of chronic stress on wakefulness and the number of wake-NREM transitions (Figs 2C–2E, S1E, and S1F). These results indicate the indispensable role played by the LHb in mediating the effects of chronic stress on sleep/wake states.

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Fig 2. Activation of the LHb mediates the chronic stress–induced increase in NREM sleep duration.

(A) Specific labeling of LHb neurons with hM4Di–EGFP or EGFP. (B) Current–evoked action potentials in a representative hM4Di infected LHb neuron recorded before, during and after CNO perfusion (10 μM). (C) Schematic of the experimental design. (D) Representative EEG spectrograms, EEG and EMG traces (recorded from ZT 1 to ZT 2) of mice in different experimental groups. All animals received exposure to chronic stress stimuli (CS) and i.p. injection of CNO (1 mg/kg). CS+EGFP–CNO: mice that received LHb injection of AAV2/9–hSyn–EGFP; CS+hM4Di–CNO: mice that received LHb injection of AAV2/9–hSyn–hM4Di–EGFP. (E) Left: time course changes of NREM sleep of mice in CS+EGFP–CNO and CS+hM4Di–CNO groups (n = 6 animals/group). Middle–left: total NREM sleep amounts during the whole day (24 h) of mice in CS+EGFP–CNO and CS+hM4Di–CNO groups (n = 6 animals/group). Middle–right: NREM sleep amounts during light phase (ZT 0 –ZT 12) and dark phase (ZT 12 –ZT 24) of mice in CS+EGFP–CNO and CS+hM4Di–CNO groups (n = 6 animals/group). Right: number of transitions between different pair of brain states during the whole day (24 h) of mice in CS+EGFP–CNO and CS+hM4Di–CNO groups (n = 6 animals/group). (F) Specific labeling of LHb neurons with hM3Dq–EGFP or EGFP. (G) LHb neurons expressing hM3Dq can be activated by bath application of CNO (10 μM, 100 s). (H) Schematic of the experimental design. (I) Representative EEG spectrograms, EEG and EMG traces (recorded from ZT 1 to ZT 2) of mice in different experimental groups. All animals received i.p. injection of CNO (1 mg/kg). EGFP–CNO: mice that received LHb injection of AAV2/9–hSyn–EGFP. hM3Dq–CNO: mice that received LHb injection of AAV2/9–hSyn–hM3Dq–EGFP. (J) Left: time course changes of NREM sleep of mice in EGFP–CNO (n = 6 animals) and hM3Dq–CNO (n = 7 animals) groups. Middle–left: total NREM sleep amounts during the whole day (24 h) of mice in EGFP–CNO (n = 6 animals) and hM3Dq–CNO (n = 7 animals) groups. Middle–right: NREM sleep amounts during light phase (ZT 0 –ZT 12) and dark phase (ZT 12 –ZT 24) of mice in EGFP–CNO (n = 6 animals) and hM3Dq–CNO (n = 7 animals) groups. Right: number of transitions between different pair of brain states during the whole day (24 h) of mice in EGFP–CNO (n = 6 animals/group) and hM3Dq–CNO groups (n = 7 animals/group). W–N: Wake to NREM; N–W: NREM to Wake; N–R: NREM to REM. For all figures: one–way ANOVA with Sidak’s multiple comparisons test, **, P < 0.001; ***, P < 0.0001. Error bars indicate the SEM. Underlying data can be found in S1 Data. ACSF, artificial cerebrospinal fluid; EEG, electroencephalography; EMG, electromyography; LHb, lateral habenula; NREM, nonrapid eye movement; ZT, Zeitgeber time.

https://doi.org/10.1371/journal.pbio.3002282.g002

To further determine the contribution of the LHb to chronic stress-induced sleep alterations, we next evaluated the impacts of long-term activation of LHb neurons on depressive-like behaviors and sleep/wake states. We induced the expression of the neuronal activator DREADD hM3Dq-EGFP in LHb neurons by injecting AAV2/9-hSyn-hM3Dq-EGFP into the bilateral LHb (Fig 2F). LHb neurons were activated daily via i.p. injection of CNO (1 mg/kg) for 14 days (Fig 2G and 2H). We found that long-term activation of LHb neurons not only significantly increased depressive-like behaviors in the SPT, FST, and TST (S2A Fig) but also resulted in an increase in the duration of NREM sleep, a higher number of transitions to NREM sleep, and reduced wakefulness (Figs 2I, 2J, S2B, and S2C). Taken together, the above results suggest that the LHb is both necessary and sufficient for the effects of chronic stress on sleep/wake states.

Activation of RMTg-projecting LHb neurons mimics the effects of chronic stress on sleep

It is well established that LHb neurons can directly innervate several brain regions implicated in sleep regulation, including the DRN, VTA, and RMTg [10,15]. Given that previous observations suggest that DRN-projecting, VTA-projecting, and RMTg-projecting LHb neurons form largely separate circuits [8,22,23] and in light of our finding that activation of LHb neurons is indispensable for the NREM sleep-promoting effects of chronic stress, we explored which subgroup of LHb neurons might be important for the regulation of NREM sleep. We first delivered the monosynaptic retrograde transport virus rAAV2/2-Retro-Cre [24] into the DRN, VTA, or RMTg (Fig 3B, 3D and 3F), respectively. Next, we induced the expression of hM3Dq-EGFP specifically in DRN-projecting LHb neurons, VTA-projecting LHb neurons, or RMTg-projecting LHb neurons by injecting a Cre-dependent virus encoding hM3Dq (AAV2/9-DIO-hM3Dq-EGFP) into the LHb (Fig 3B, 3D and 3F). DRN-projecting LHb neurons, VTA-projecting LHb neurons, or RMTg-projecting LHb neurons were activated daily via i.p. injection of CNO (1 mg/kg) for 14 days (Fig 3A). We found that activation of DRN-projecting LHb neurons or VTA-projecting LHb neurons only increased the duration of NREM sleep in the daytime but not the nighttime (Figs 3C, 3E, and S3A–S3D). In contrast, activation of RMTg-projecting LHb neurons significantly increased the duration and initiation of NREM sleep during both the daytime and nighttime (Figs 3G, S3E and S3F). These results suggest that activation of RMTg-projecting LHb neurons better mimics the sleep-regulating effects of global activation of LHb neurons or chronic stress than activation of DRN-projecting LHb neurons or VTA-projecting LHb neurons.

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Fig 3. Activation of RMTg–projecting LHb neurons mimics the effects of chronic stress on sleep.

(A) Schematic of the experimental design. (B, D, F) Specific labeling of DRN–projecting LHb neurons (B) or VTA–projecting LHb neurons (D) or RMTg–projecting LHb neurons (F) with hM3Dq–EGFP or EGFP. (C, E, G). Left: time course changes of NREM sleep of mice in different experimental groups. All mice received i.p. injection of CNO (1 mg/kg). EGFP–CNO: mice that received DRN (C) or VTA (E) or RMTg (G) injection of rAAV2/2–Retro–Cre and LHb injection of AAV2/9–DIO–EGFP (n = 6 animals/group); hM3Dq–CNO: mice that received DRN (C, n = 6 animals) or VTA (E, n = 6 animals) or RMTg (G, n = 7 animals) injection of rAAV2/2–Retro–Cre and LHb injection of AAV2/9–DIO–hM3Dq–EGFP. Middle: NREM sleep amounts during light phase (ZT 0 –ZT 12) and dark phase (ZT 12 –ZT 24) of mice in EGFP–CNO and hM3Dq–CNO groups. Right: number of transitions between different pair of brain states during the whole day (24 h) of mice in EGFP–CNO and hM3Dq–CNO groups. W–N: Wake to NREM; N–W: NREM to Wake; N–R: NREM to REM. Scale bars: 200 μm (B, D, F). For all figures: one–way ANOVA test, *, P < 0.05; **, P < 0.01; ***, P < 0.0001. Error bars indicate the SEM. Underlying data can be found in S1 Data. DRN, dorsal raphe nucleus; LHb, lateral habenula; NREM, nonrapid eye movement; RMTg, rostromedial tegmental nucleus; VTA, ventral tegmental area; ZT, Zeitgeber time.

https://doi.org/10.1371/journal.pbio.3002282.g003

LHb CaMKIIα neurons activate RMTg GABA neurons through direct projections

To determine the structure of the LHb-RMTg pathway in mice, we first delivered rAAV2/2-Retro-Cre into the RMTg (S4A Fig). Next, a Cre-dependent virus encoding the yellow fluorescent protein eYFP (AAV2/9-DIO-eYFP) was injected into the LHb (S4A Fig). We found that RMTg-projecting LHb neurons labeled with eYFP were mainly located in the medial and lateral part of the LHb (S4A Fig), with approximately 94.64% of eYFP-labeled RMTg-projecting neurons being immunopositive for CaMKIIα (S4A Fig). Next, to determine the identity of RMTg neurons receiving direct LHb inputs, we delivered the monosynaptic anterograde transport virus AAV2/1-Cre [25] into the LHb and AAV2/9-DIO-eYFP into the RMTg of C57BL/6 mice (S4B Fig). We found that approximately 91.86% of RMTg neurons receiving direct LHb inputs were immunopositive for GABA (S4B Fig). These results suggest that a subset of CaMKIIα-expressing LHb neurons directly innervate GABA-expressing RMTg neurons in mice.

To determine how LHb neurons regulate neuronal activity in the RMTg, we delivered rAAV2/2-Retro-Cre into the RMTg of C57BL/6 mice and a Cre-dependent virus encoding channelrhodopsin-2 and mCherry (AAV2/9-DIO-ChR2-mCherry) into the LHb (Fig 4A). Next, we optogenetically activated LHb-RMTg projections and recorded postsynaptic currents from RMTg neurons (Fig 4B). Optogenetically activating LHb-RMTg projections evoked exclusively excitatory postsynaptic currents (EPSCs) in 59.26% of recorded neurons (Fig 4C). In addition, the recorded postsynaptic currents were completely blocked by the application of TTX and restored by the application of TTX/4-AP (Fig 4D), indicating that the postsynaptic currents recorded in RMTg neurons were elicited by direct synaptic connections between RMTg-projecting LHb neurons and the recorded RMTg neurons. Furthermore, the excitatory effects of LHb-RMTg projections could be blocked by the AMPA/kainate receptor antagonist NBQX (Fig 4E). The above results indicate that LHb neurons activate RMTg neurons through direct projections.

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Fig 4. LHb CaMKIIα neurons activate RMTg GABA neurons through direct projections.

(A) Left: scheme for specific labeling of RMTg–projecting LHb neurons with ChR2–mCherry. Right: representative images of the LHb and RMTg 2 weeks after virus injections. (B) Scheme for recording the optogenetically evoked postsynaptic currents in RMTg neurons. (C) Left: pie chart indicates whether the absolute amplitude was greater for evoked EPSCs or no response (NonR). Right: absolute amplitude of optogenetically evoked IPSCs and EPSCs in RMTg neurons (n = 54 cells). (D) Optogenetically evoked postsynaptic currents were completely blocked by the application of TTX and recovered by the application of TTX/4–AP. (E) Optogenetically evoked EPSCs were blocked by the application of NBQX. (F) Upper–left: scheme for specific infection of RMTg–projecting LHb neurons with GCaMP7s. Upper–right: representative images of the LHb 2 weeks after virus injections. Bottom–left: heat maps showing the Ca²⁺ signals evoked by foot shock (6 trials) and air puff (6 trials) in RMTg–projecting LHb neurons. Bottom–middle: averaged responses of mice in different groups. Bottom–right: stress–related stimuli significantly increased the amplitude of Ca²⁺ signals in RMTg–projecting LHb neurons (n = 6 animals). (G) Upper–left: scheme for specific infection of RMTg neurons receiving direct LHb inputs with GCaMP7s. Upper–right: representative images of the RMTg 2 weeks after virus injections. Bottom–left: heat maps showing the Ca²⁺ signals evoked by foot shock (6 trials) and air puff (6 trials) in RMTg postsynaptic neurons. Bottom–middle: averaged responses of mice in different groups. Bottom–right: stress–related stimuli significantly increased the amplitude of Ca²⁺ signals in RMTg postsynaptic neurons (n = 8 animals). Scale bars: 200 μm (A, F, G). For all figures: one–way ANOVA test, ***, P < 0.0001. Error bars indicate the SEM. Underlying data can be found in S1 Data. 4-AP, 4-aminopyridine; ACSF, artificial cerebrospinal fluid; EPSC, excitatory postsynaptic current; LHb, lateral habenula; RMTg, rostromedial tegmental nucleus; TTX, tetrodotoxin.

https://doi.org/10.1371/journal.pbio.3002282.g004

The LHb encodes stressful signals [8,10–14]. To determine whether neurons in the LHb-RMTg pathway also encode stressful signals, we first injected rAAV2/2-Retro-Cre into the RMTg and infected RMTg-projecting LHb neurons with a Cre-dependent virus encoding the Ca²⁺ indicator GCaMP7s (AAV2/9-DIO-GCaMP7s) (Fig 4F). Next, we used fiber photometry [26,27] to quantitatively measure stressful stimulus (foot shock and air puff)-induced changes in Ca²⁺ signals in RMTg-projecting LHb neurons (Fig 4F). We observed that stressful stimuli reliably evoked robust activation of RMTg-projecting LHb neurons (Fig 4F). Next, we used fiber photometry to quantitatively measure stressful stimulus (foot shock and air puff)-induced changes in Ca²⁺ signals in RMTg neurons receiving direct LHb inputs (Fig 4G). Stressful stimuli also evoked robust activation of RMTg postsynaptic neurons (Fig 4G). The above results indicate that the LHb-RMTg pathway encodes stressful signals.

Activation of the LHb-RMTg pathway promotes NREM sleep

Given that activation of RMTg-projecting LHb neurons promotes NREM sleep (Figs 3F, 3G, S3E and S3F) and that the LHb can activate RMTg neurons through direct projections (Fig 4B–4E), we next tested whether direct activation of LHb-RMTg projections can also promote NREM sleep. We first delivered rAAV2/2-Retro-Cre into the bilateral RMTg and infected RMTg-projecting LHb neurons with AAV2/9-DIO-ChR2-mCherry (Fig 5A). An optic fiber was implanted above the RMTg (Fig 5A). Laser stimulation (20 Hz, 2 min per trial) was applied during the wake state in freely moving mice, and REM and NREM sleep, and wakefulness were classified based on EEG and EMG recordings (Fig 5B). We found that optogenetic activation of LHb-RMTg projections caused a marked increase in NREM sleep duration and a decrease in wakefulness time (Fig 5B and 5C), suggesting that short-term activation of LHb-RMTg projections promotes NREM sleep.

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Fig 5. Activation of the LHb–RMTg pathway promotes NREM sleep.

(A) Scheme for specific infection of RMTg–projecting LHb neurons with ChR2–mCherry. (B) An example optogenetic activation experiment in a mouse received RMTg injection of rAAV2/2–Retro–Cre and LHb injection of AAV2/9–DIO–ChR2–mCherry. Shown are the EEG spectrogram, EEG and EMG traces. Blue shading represents laser stimulation (20 Hz, 2 min). (C) Left: percentage of time in NREM, REM, or the wake state before, during, and after laser stimulation, averaged from 7 animals. Shading represents the 95% confidence interval, and the blue stripe indicates the laser stimulation period. Right: laser stimulation significantly increased NREM sleep and decreased wakefulness and did not significantly affect REM sleep. (D) Schematic of the experimental design. (E) Specific labeling of RMTg neurons receiving direct LHb inputs with hM3Dq–EGFP or EGFP. (F) RMTg postsynaptic neurons expressing hM3Dq can be activated by bath application of CNO (10 μM, 100 s). (G) Representative EEG spectrograms, EEG and EMG traces (recorded from ZT 1 to ZT 2) of mice in different experimental groups. All mice received LHb injection of AAV2/1–Cre and i.p. injection of CNO (1 mg/kg). EGFP–CNO: mice that received RMTg injection of AAV2/9–DIO–EGFP. hM3Dq–CNO: mice that received RMTg injection of AAV2/9–DIO–hM3Dq–EGFP. (H) Left: time course changes of NREM sleep of mice in EGFP–CNO (n = 6 animals) and hM3Dq–CNO (n = 7 animals) groups. Middle–left: total sleep–wake amounts during the whole day (24 h) of mice in EGFP–CNO (n = 6 animals) and hM3Dq–CNO groups (n = 7 animals). Middle–right: total sleep–wake amounts during light phase (ZT 0 –ZT 12) and dark phase (ZT 12 –ZT 24) of mice in EGFP–CNO (n = 6 animals) and hM3Dq–CNO (n = 7 animals) groups. Right: number of transitions between different pair of brain states during the whole day (24 h) of mice in EGFP–CNO (n = 6 animals) and hM3Dq–CNO (n = 7 animals) groups. W–N: Wake to NREM; N–W: NREM to Wake; N–R: NREM to REM. Scale bar: 200 μm (A). For all figures: one–way ANOVA with Sidak’s multiple comparisons test, *, P < 0.05; **, P < 0.001; ***, P < 0.0001; ns = no significant difference. Error bars indicate the SEM. Underlying data can be found in S1 Data. ACSF, artificial cerebrospinal fluid; EEG, electroencephalography; EMG, electromyography; LHb, lateral habenula; NREM, nonrapid eye movement; REM, rapid eye movement; RMTg, rostromedial tegmental nucleus; ZT, Zeitgeber time.

https://doi.org/10.1371/journal.pbio.3002282.g005

To further evaluate the contribution of the LHb-RMTg pathway to the regulation of sleep, we induced the expression of hM3Dq-EGFP specifically in RMTg postsynaptic neurons through injection of AAV2/1-Cre into the bilateral LHb and injection of AAV2/9-DIO-hM3Dq-EGFP into the bilateral RMTg (Fig 5D and 5E). RMTg neurons expressing hM3Dq-EGFP were activated daily via i.p. injection of CNO (1 mg/kg) for 14 days (Fig 5D and 5F). Long-term activation of RMTg neurons also significantly increased the duration of NREM sleep and the number of transitions to NREM sleep (Figs 5G, 5H and S5). The above results confirm that specific activation of the LHb-RMTg pathway promotes NREM sleep.

Activation of the LHb-RMTg pathway is required for the NREM sleep-promoting effects of chronic stress

Given that neurons in the LHb-RMTg pathway encode stressful signals (Fig 4F and 4G) and that activation of the LHb-RMTg pathway promotes NREM sleep (Figs 3F, 3G and 5), we postulated that activation of the LHb-RMTg pathway is important for the NREM sleep-promoting effects of chronic stress. To test this possibility, we used chemogenetics to specifically inhibit neurons in the LHb-RMTg pathway during daily exposure to stressful stimuli (Fig 6A). We first delivered rAAV2/2-Retro-Cre into the bilateral RMTg and infected RMTg-projecting LHb neurons with AAV2/9-DIO-hM4Di-EGFP (Fig 6B). RMTg-projecting LHb neurons were chemogenetically inhibited during daily exposure to stressful stimuli (Fig 6A and 6C). Inhibition of RMTg-projecting LHb neurons during exposure to chronic stress significantly impaired the NREM sleep-promoting effects of chronic stress (Figs 6D, 6E, S6A and S6B). Next, to determine whether the activation of RMTg neurons receiving direct LHb inputs is required for the NREM sleep-promoting effects of chronic stress, we injected AAV2/1-Cre into the bilateral LHb and infected postsynaptic RMTg neurons with AAV2/9-DIO-hM4Di-EGFP (Fig 6F). LHb postsynaptic neurons were chemogenetically inhibited during exposure to chronic stress (Fig 6A and 6G). We found that chemogenetic inhibition of RMTg postsynaptic neurons also impaired the NREM sleep-promoting effects of chronic stress (Figs 6H, 6I, S6C and S6D). Thus, activation of the LHb-RMTg pathway is required for the NREM sleep-promoting effects of chronic stress.

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Fig 6. Activation of the LHb–RMTg pathway is required for the NREM sleep–promoting effects of chronic stress.

(A) Schematic of the experimental design. (B) Specific labeling of RMTg–projecting LHb neurons with hM4Di–EGFP or EGFP. (C) Current–evoked action potentials in a representative hM4Di infected LHb neuron recorded before, during, and after CNO perfusion (10 μM). (D) Representative EEG spectrograms, EEG and EMG traces (recorded from ZT 1 to ZT 2) of mice in different experimental groups. All animals received exposure to chronic stress stimuli (CS), RMTg injection of rAAV2/2–Retro–Cre and i.p. injection of CNO (1 mg/kg). CS+EGFP–CNO: mice that received LHb injection of AAV2/9–DIO–EGFP. CS+hM4Di–CNO: mice that received LHb injection of AAV2/9–DIO–hM4Di–EGFP. (E) Left: time course changes of NREM sleep of mice in CS+EGFP–CNO (n = 6 animals) and CS+hM4Di–CNO (n = 7 animals) groups. Middle–left: total NREM sleep amounts during the whole day (24 h) of mice in CS+EGFP–CNO (n = 6 animals) and CS+hM4Di–CNO (n = 7 animals) groups. Middle–right: NREM sleep amounts during light phase (ZT 0 –ZT 12) and dark phase (ZT 12 –ZT 24) of mice in CS+EGFP–CNO (n = 6 animals) and CS+hM4Di–CNO (n = 7 animals) groups. Right: number of transitions between different pair of brain states during the whole day (24 h) of mice in CS+EGFP–CNO (n = 6 animals) and CS+hM4Di–CNO (n = 7 animals) groups. W–N: Wake to NREM; N–W: NREM to Wake; N–R: NREM to REM. (F) Specific labeling of RMTg neurons receiving direct LHb inputs with hM4Di–EGFP or EGFP. (G) Current–evoked action potentials in a representative RMTg neuron infected with hM4Di recorded before, during, and after CNO perfusion (10 μM). (H) Representative EEG spectrograms, EEG and EMG traces (recorded from ZT 1 to ZT 2) of mice in different experimental groups. All animals received exposure to chronic stress stimuli (CS), LHb injection of AAV2/1–Cre and i.p. injection of CNO (1 mg/kg). CS+EGFP–CNO: mice that received RMTg injection of AAV2/9–DIO–EGFP. CS+hM4Di–CNO: mice that received RMTg injection of AAV2/9–DIO–hM4Di–EGFP. (I) Left: time course changes of NREM sleep of mice in CS+EGFP–CNO and CS+hM4Di–CNO groups (n = 7 animals/group). Middle–left: total NREM sleep amounts during the whole day (24 h) of mice in CS+EGFP–CNO and CS+hM4Di–CNO groups (n = 7 animals/group). Middle–right: NREM sleep amounts during light phase (ZT 0 –ZT 12) and dark phase (ZT 12 –ZT 24) of mice in CS+EGFP–CNO and CS+hM4Di–CNO groups (n = 7 animals/group). Right: number of transitions between different pair of brain states during the whole day (24 h) of mice in CS+EGFP–CNO and CS+hM4Di–CNO groups (n = 7 animals). For all figures: one–way ANOVA with Sidak’s multiple comparisons test, *, P < 0.05; **, P < 0.001; ***, P < 0.0001; ns = no significant difference. Error bars indicate the SEM. Underlying data can be found in S1 Data. ACSF, artificial cerebrospinal fluid; EEG, electroencephalography; EMG, electromyography; LHb, lateral habenula; NREM, nonrapid eye movement; RMTg, rostromedial tegmental nucleus; ZT, Zeitgeber time.

https://doi.org/10.1371/journal.pbio.3002282.g006

Bright light treatment reduces the NREM sleep-promoting effects of chronic stress

Bright light signals encoded by GABA neurons in the vLGN/IGL can inhibit neuronal activity in the LHb and ameliorate chronic stress-induced depressive-like symptoms [8]. We assessed whether vLGN/IGL neurons directly innervate the LHb-RMTg pathway through a modified rabies virus-based di-synaptic retrograde tracing strategy. LHb neurons were infected with AAV expressing the rabies glycoprotein and histone-tagged green fluorescent protein (Helper) (Fig 7A and 7B), which is required for the replication of rabies virus [28]. Next, we injected SAD-ΔG-DsRed (EnvA) into the RMTg to infect Helper⁺ RMTg-projecting LHb neurons via their presynaptic terminals (Fig 7A and 7B). The rabies-DsRed⁺/Glyco-EGFP⁺ double-infected LHb relay neurons (starter cells) produced infectious ΔG-rabies-DsRed that propagated transneuronally to infect vLGN/IGL neurons that formed synapses with them (Fig 7C). We observed that starter cells were mainly distributed in the LHb (Fig 7C). In the vLGN/IGL, approximately 314 neurons/animal were labeled with rabies virus (n = 4 animals). We found that most of the rabies virus-labeled vLGN/IGL neurons were immunopositive for GABA (approximately 92.64%), suggesting that GABA neurons in the vLGN/IGL can form di-synaptic connections with the RMTg through the LHb.

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Fig 7. Bright light treatment reduces the NREM sleep–promoting effects of chronic stress.

(A) Design of Helper virus and SAD–ΔG–DsRed (EnvA). (B) Experimental design of virus tracing in C57BL/6 mice. (C) Left: injection site of the LHb illustrating the location of starter cells (white). Right: a representative image of the vLGN/IGL showing RV–DsRed–labeled vLGN/IGL neurons that were immunopositive for GABA. Pie chart indicates the percentage of RV–DsRed–labeled vLGN/IGL neurons colabeled with GABA. (D) Schematic of the experimental design. (E) Representative EEG spectrograms, EEG and EMG traces (recorded from ZT 1 to ZT 2) of mice in different experimental groups. CS: mice that received exposure to chronic stress stimuli (CS). CS+LT: mice that received exposure to chronic stress stimuli and bright light treatment (3,000 lux, 2 h/day). (F) Left: time course changes of NREM and REM sleep and wakefulness of mice in CS (n = 6 animals) and CS+LT groups (n = 7 animals). Middle: total sleep–wake amounts during the whole day (24 h) of mice in in CS (n = 6 animals) and CS+LT groups (n = 7 animals). Right: total sleep–wake amounts during light phase (ZT 0 –ZT 12) and dark phase (ZT 12 –ZT 24) of mice in in CS (n = 6 animals) and CS+LT groups (n = 7 animals). (G) Number of transitions between different pair of brain states during the light phase (ZT 0 –ZT 12), dark phase (ZT 12 –ZT 24), and the whole day (24 h) of mice in CS (n = 6 animals) and CS+LT groups (n = 7 animals). W–N: Wake to NREM; N–W: NREM to Wake; N–R: NREM to REM. (H, I) The current–evoked action potentials recorded in RMTg–projecting LHb neurons (H) and RMTg neurons receiving direct LHb inputs (I) in different experimental groups. Non–CS: mice that did not receive exposure to chronic stress stimuli (n = 9 cells/group for H; n = 8 cells/group for I); CS: mice that receive exposure to chronic stress stimuli (n = 11 cells/group for H; n = 7 cells/group for I); CS+LT: mice that received exposure to chronic stress stimuli and bright light treatment (3,000 lux, 2 h/day) (n = 11 cells/group for H; n = 7 cells/group for I). Scale bars: 200 μm (C–LHb–left, C–vLGN/IGL–left), 20 μm (C–LHb–right, C–vLGN/IGL–right). For all figures: one–way ANOVA with Sidak’s multiple comparisons test, *, P < 0.05; **, P < 0.001; ns = no significant difference. Error bars indicate the SEM. Underlying data can be found in S1 Data. EEG, electroencephalography; EMG, electromyography; IGL, intergeniculate leaflet; LHb, lateral habenula; NREM, nonrapid eye movement; REM, rapid eye movement; RMTg, rostromedial tegmental nucleus; vLGN, ventral lateral geniculate nucleus; ZT, Zeitgeber time.

https://doi.org/10.1371/journal.pbio.3002282.g007

Given that activation of GABA neurons in the vLGN/IGL can inhibit the LHb [8], and inhibition of RMTg-projecting LHb neurons impairs the NREM sleep-promoting effects of chronic stress (Figs 6D, 6E, S6A and S6B), we tested the effects of long-term activation of LHb-projecting vLGN/IGL neurons on sleep/wake states. We delivered rAAV2/2-Retro-Cre into the bilateral LHb and infected LHb-projecting vLGN/IGL neurons with AAV2/9-DIO-hM3Dq-EGFP or AAV2/9-DIO-EGFP (S7A Fig). LHb-projecting vLGN/IGL neurons were activated daily via i.p. injection of CNO (1 mg/kg) for 14 days (S7B and S7C Fig). We found that long-term activation of LHb-projecting vLGN/IGL neurons did not significantly affect sleep/wake states in wild-type mice (S7D–S7F Fig). In contrast, chemogenetic activation of LHb-projecting vLGN/IGL neurons during daily exposure to stressful stimuli significantly reduced the NREM sleep-promoting effects of chronic stress (S7G–S7J Fig).

In light of our previous finding that bright light signals transmitted by GABA neurons in the vLGN/IGL can also inhibit the LHb [8], we hypothesized that an increase in the amount of light detected by vLGN/IGL neurons innervating the LHb-RMTg pathway can inhibit neural activity in the LHb-RMTg pathway and impair the NREM sleep-promoting effects of chronic stress. To test this possibility, during 14 days of exposure to stressful stimuli, animals received 2 h of daily bright light treatment (3,000 lux, 2 h/day) (Fig 7D). We found that bright light treatment significantly decreased the NREM sleep-promoting effects of chronic stress (Fig 7E–7G) and reduced the excitability of RMTg-projecting LHb neurons and RMTg neurons receiving direct LHb inputs (Fig 7H and 7I). The above results suggest that bright light treatment impairs the NREM sleep-promoting effects of chronic stress and reduces the aberrant activity of neurons in the LHb-RMTg pathway induced by chronic stress.

Activation of the vLGN/IGL-LHb-RMTg pathway is required for the ability of bright light treatment to reduce the NREM sleep-promoting effects of chronic stress

To evaluate the role of the vLGN/IGL-LHb-RMTg pathway in the effects of bright light treatment on NREM sleep, we first induced the expression of Cre recombinase in LHb-projecting vLGN/IGL neurons by injecting rAAV2/2-Retro-Cre into the bilateral LHb and infecting LHb-projecting vLGN/IGL neurons with AAV2/9-DIO-hM4Di-EGFP (Fig 8A and 8B). LHb-projecting vLGN/IGL neurons were chemogenetically inhibited during bright light treatment (3,000 lux, 2 h/day) (Fig 8A). Inhibition of LHb-projecting vLGN/IGL neurons significantly reduced the NREM sleep-reducing effects of bright light treatment in mice that received chronic stress (Figs 8C, 8D, S8A and S9B). Next, we injected rAAV2/2-Retro-Cre into the bilateral RMTg and infected RMTg-projecting LHb neurons with AAV2/9-DIO-hM3Dq-EGFP (Fig 8A and 8E). RMTg-projecting LHb neurons were chemogenetically activated during bright light treatment (3,000 lux, 2 h/day) (Fig 8A). Activation of RMTg-projecting LHb neurons also significantly reduced the NREM sleep-reducing effects of bright light treatment (Figs 8F, 8G, S8C and S8D). Finally, we injected AAV2/1-Cre into the bilateral LHb and infected RMTg neurons receiving direct LHb inputs with AAV2/9-DIO-hM3Dq-EGFP (Fig 8A and 8H). RMTg postsynaptic neurons were chemogenetically activated during bright light treatment (3,000 lux, 2 h/day) (Fig 8A). Activation of RMTg neurons receiving direct LHb inputs significantly impaired the NREM sleep-reducing effects of bright light treatment (Figs 8I, 8J, S8E and S8F). Taken together, these findings indicate that activation of the vLGN/IGL-LHb-RMTg pathway is required for the ability of bright light treatment to suppress the abnormal increase in NREM sleep duration induced by chronic stress.

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Fig 8. Activation of the vLGN/IGL-LHb-RMTg pathway is required for the ability of bright light treatment to reduce the NREM sleep-promoting effects of chronic stress.

(A) Schematic of the experimental design. (B) Specific labeling of LHb-projecting vLGN/IGL neurons with hM4Di-EGFP or EGFP. (C) Representative EEG spectrograms, EEG and EMG traces (recorded from ZT 1 to ZT 2) of mice in different experimental groups. All animals received exposure to CS, LT, LHb injection of rAAV2/2-Retro-Cre and i.p. injection of CNO (1 mg/kg). CS+LT+EGFP-CNO: mice that received vLGN/IGL injection of AAV2/9-DIO-EGFP. CS+LT+hM4Di-CNO: mice that received vLGN/IGL injection of AAV2/9-DIO-hM4Di-EGFP. (D) Left: time course changes of NREM sleep of mice in CS+LT+EGFP-CNO (n = 6 animals) and CS+LT+hM4Di-CNO (n = 7 animals) groups; Middle-left: total NREM sleep amounts during the whole day (24 h) of mice in CS+LT+EGFP-CNO (n = 6 animals) and CS+LT+hM4Di-CNO (n = 7 animals) groups; Middle-right: NREM sleep amounts during light phase (ZT 0 – ZT 12) and dark phase (ZT 12 – ZT 24) of mice in CS+LT+EGFP-CNO (n = 6 animals) and CS+LT+hM4Di-CNO (n = 7 animals) groups. Right: number of transitions between different pair of brain states during the whole day (24 h) of mice in CS+LT+EGFP-CNO (n = 6 animals) and CS+LT+hM4Di-CNO (n = 7 animals) groups. W-N: Wake to NREM; N-W: NREM to Wake; N-R: NREM to REM. (E) Specific labeling of RMTg-projecting LHb neurons with hM3Dq-EGFP or EGFP. (F) Representative EEG spectrograms, EEG and EMG traces (recorded from ZT 1 to ZT 2) of mice in different experimental groups. All animals received exposure to chronic stress stimuli (CS), bright light treatment (LT), RMTg injection of rAAV2/2-Retro-Cre and i.p. injection of CNO (1 mg/kg). CS+LT+EGFP-CNO: mice that received LHb injection of AAV2/9-DIO-EGFP. CS+LT+hM3Dq-CNO: mice that received LHb injection of AAV2/9-DIO-hM3Dq-EGFP. (G) Left: time course changes of NREM sleep of mice in CS+LT+EGFP-CNO and CS+LT+hM3Dq-CNO groups (n = 6 animals/group); Middle-left: total NREM sleep amounts during the whole day (24 h) of mice in CS+LT+EGFP-CNO and CS+LT+hM3Dq-CNO groups (n = 6 animals/group); Middle-right: NREM sleep amounts during light phase (ZT 0 – ZT 12) and dark phase (ZT 12 – ZT 24) of mice in CS+LT+EGFP-CNO and CS+LT+hM3Dq-CNO groups (n = 6 animals/group). Right: number of transitions between different pair of brain states during the whole day (24 h) of mice in CS+LT+EGFP-CNO and CS+LT+hM3Dq-CNO groups (n = 6 animals/group). (H) Specific labeling of RMTg neurons receiving direct LHb inputs with hM3Dq-EGFP or EGFP. (I) Representative EEG spectrograms, EEG and EMG traces (recorded from ZT 1 to ZT 2) of mice in different experimental groups. All animals received exposure to CS, LT, LHb injection of AAV2/1-Cre and i.p. injection of CNO (1 mg/kg). CS+LT+EGFP-CNO: mice that received RMTg injection of AAV2/9-DIO-EGFP. CS+LT+hM3Dq-CNO: mice that received RMTg injection of AAV2/9-DIO-hM3Dq-EGFP. (J) Left: time course changes of NREM sleep of mice in CS+LT+EGFP-CNO (n = 6 animals) and CS+LT+hM3Dq-CNO (n = 7 animals) groups; Middle-left: total NREM sleep amounts during the whole day (24 h) of mice in CS+LT+EGFP-CNO (n = 6 animals) and CS+LT+hM3Dq-CNO (n = 7 animals) groups; Middle-right: NREM sleep amounts during light phase (ZT 0 – ZT 12) and dark phase (ZT 12 – ZT 24) of mice in CS+LT+EGFP-CNO (n = 6 animals) and CS+LT+hM3Dq-CNO (n = 7 animals) groups. Right: number of transitions between different pair of brain states during the whole day (24 h) of mice in CS+LT+EGFP-CNO (n = 6 animals) and CS+LT+hM3Dq-CNO (n = 7 animals) groups. For all figures: One-way ANOVA with Sidak’s multiple comparisons test, P < 0.05; **, P < 0.001; ***, P < 0.0001 ns = no significant difference. Error bars indicate the SEM. EEG, electroencephalography; EMG, electromyography; LHb, lateral habenula; NREM, nonrapid eye movement; REM, rapid eye movement; RMTg, rostromedial tegmental nucleus; ZT, Zeitgeber time.

https://doi.org/10.1371/journal.pbio.3002282.g008

Ethics statement

All experiments were approved by the Jinan University Institutional Animal Care and Use Committee. The approval number of the protocol is 20220804–14. We also abide by the provisions of the Biosafety Law of the People’s Republic of China, the Regulations on the Administration of Experimental Animals, the National Standards for Experimental Animals (GB14925-2010), the Guidelines for Ethical Review of the Welfare of Experimental Animals (GBT 35892–2018), and the relevant rules and regulations formulated by Jinan University.

Animals

Adult (6 to 8 weeks old) male C57BL/6 mice were used in this study. The animals were housed in a 12 h:12 h light-dark cycle [light on at 7 AM, this time point is defined as Zeitgeber time (ZT) 0] with food and water provided ad libitum. The animals were randomly allocated to experimental and control groups. Experimenters were blind to the experimental group, and the order of testing was counterbalanced during behavioral experiments.

Surgery and intracranial injection

The mice were anesthetized (Avertin, 13 μl/g, i.p.) and placed in a stereotaxic instrument (RWD, Shenzhen, China). Erythromycin eye ointment was applied to prevent corneal drying and a heat pad (RWD, Shenzhen, China) was used to hold body temperature at 37°C. A small craniotomy hole was made using a dental drill (OmniDrill35, WPI, Sarasota, Florida, United States of America), and injections were performed via a micropipette connected to a Nanoliter Injector (NANOLITER 2010, WPI, Sarasota, Florida, USA) and its controller (Micro4, WPI, Sarasota, Florida, USA) at a slow flow rate of 0.1 μl/min to avoid potential damage to local brain tissue.

To specifically infect LHb neurons with hM3Dq-EGFP or hM4Di-EGFP or EGFP, AAV2/9-hSyn-hM3Dq-EGFP or AAV2/9-hSyn-hM4Di-EGFP or AAV2/9-hSyn-EGFP was injected into the LHb of C57BL/6 mice (virus titers: 3.5 × 10¹² GC/ml, 0.2 μl/injection; AP: −1.6 mm; ML: ± 0.25 mm; DV: −3.05 mm), respectively.

To specifically infect RMTg-projecting LHb neurons with ChR2-mCherry or mCherry or hM3Dq-EGFP or hM4Di-EGFP or EGFP or eYFP or GCaMP7s, rAAV2/2-Retro-Cre was injected into the RMTg of C57BL/6 mice (virus titers: 3 × 10¹² GC/ml; 0.1 μl/injection; AP: −4.3 mm; ML: ±0.35 mm; DV: −4.25 mm), AAV2/9-DIO-ChR2-mCherry or AAV2/9-DIO-mCherry or AAV2/9-DIO-hM3Dq-EGFP or AAV2/9-DIO-hM4Di-EGFP or AAV2/9-DIO-EGFP or AAV2/9-DIO-eYFP or AAV2/9-DIO-GCaMP7s was injected into the LHb (virus titers: 3.5 × 10¹² GC/ml, 0.2 μl/injection; AP: −1.6 mm; ML: ± 0.25 mm; DV: −3.05 mm), respectively.

To specifically infect DRN-projecting LHb neurons with hM3Dq-EGFP or EGFP, rAAV2/2-Retro-Cre was injected into the DRN of C57BL/6 mice (virus titers: 3 × 10¹² GC/ml; 0.2 ml/injection; AP: −5.5 mm; DV: −2.7 mm, with a 15°aangle), AAV2/9-DIO-hM3Dq-EGFP or AAV2/9-DIO-EGFP was injected into the LHb (virus titers: 3 × 10¹² GC/ml; 0.1 μl/injection), respectively.

To specifically infect VTA-projecting LHb neurons with hM3Dq-EGFP or EGFP, rAAV2/2-Retro-Cre was injected into the VTA of C57BL/6 mice (virus titers: 3 × 10¹² GC/ml; 0.2 ml/injection; AP: −3.2; ML: ± 0.5 mm; DV: −4.25 mm), AAV2/9-DIO-hM3Dq-EGFP or AAV2/9-DIO-EGFP was injected into the LHb (virus titers: 3 × 10¹² GC/ml; 0.1 μl/injection), respectively.

To specifically infect postsynaptic RMTg neurons with EGFP or hM3Dq-EGFP or hM4Di-EGFP or GCaMP7s, AAV2/1-Cre (virus titers: 1.5 × 10¹³ GC/ml; 0.1 μl/injection) was injected into the LHb, AAV2/9-DIO-EGFP or AAV2/9-DIO-hM3Dq-EGFP or AAV2/9-DIO-hM4Di-EGFP or or AAV2/9-DIO-GCaMP7s was injected into the RMTg (0.15 μl/injection), respectively.

For di-synaptic tracing vLGN/IGL-LHb-RMTg pathway, 0.15 μl Helper virus (rAAV2/9-hSyn-EGFP-2a-TVA-2a-RVG-WPRE-pA) (virus titers: 2 × 10⁸ GC/ml) was injected into the LHb. Twenty-one days later, 0.1 μl of SAD-DG-DsRed (EnvA) (RV-DsRed, virus titers: 2 × 10⁸ GC/ml) was injected into RMTg.

Following injection, the micropipette was left in place for approximately 5 min and then extracted slowly (approximately 1 min to completely move the micropipette from the injection site to the surface of the brain) to minimize virus leakage in the track. Finally, the wound was sutured, antibiotics (bacitracin and neomycin) were applied to the surgical wound and ketoprofen (5 mg/kg) was injected subcutaneously; the animals were allowed to recover from anesthesia under a heat lamp.

Verification of the location of the RMTg

Previous studies have demonstrated that cocaine can selectively activate the RMTg after its rewarding effects have subsided [50–53]. In this study, we further assessed the location of the RMTg based on cocaine-induced c-Fos expression. Briefly, mice received an i.p. injection of cocaine (10 mg/kg). One hour later, the animals were anesthetized (13 μl/g avertin, i.p.) before receiving an intracardial infusion of 0.9% saline, followed by 4% paraformaldehyde in PBS. The brains were carefully removed thereafter. To label c-Fos⁺ cells, cryostat sections were blocked for 1 h and then incubated with a primary anti c-Fos antibody (rabbit, 1:500, 2250, Cell Signaling Technology) for 36 h at 4°C. The sections were subsequently incubated with a secondary antibody, DyLight 594-conjugated goat anti-rabbit IgG (DI-1 594, Vector Laboratories) at a dilution of 1:400 for 6 h at room temperature. Finally, all sections were washed in 0.1 M PBS and mounted with an antifade aqueous mounting medium containing DAPI (EMS, Hatfield, Pennsylvania, USA). Consistent with previous findings, we observed an increase in c-Fos expression in the RMTg, but not the VTA, 1 h after cocaine exposure (S10A Fig).

Injection site verification

After transcardial perfusion with 0.9% saline followed by 4% paraformaldehyde in 0.1 M PBS, the brain was removed and post-fixed with 4% paraformaldehyde overnight at 4°C, and then transferred into 30% sucrose until sectioning with a cryostat (CM1900, Leica Microsystems, Bannockburn, Illinois, USA). A series of 40 μm sections were collected for verification of injection sites. To confirm the injection sites of viruses that encoded a fluorescent protein (e.g., AAV2/9-DIO-hM3Dq-EGFP and AAV2/9-DIO-GCaMP7s) (S10B and S10C Fig), coronal brain sections were examined under a fluorescence microscope (Zeiss, Axioimager Z2 microscope). Only mice with verified fluorescent protein expression were used for analysis. Mice with virus injections that missed the target area, fluorescent protein expression that was too low in the targeted area or extensively extended beyond the targeted area were excluded from the study. To visualize the injection sites of viruses that did not encode fluorescent protein (i.e., rAAV2/2-Retro-Cre and AAV2/1-Cre), Alexa Fluor 647-conjugated cholera toxin subunit B (CTB-647, 0.05 μl/injection) was injected into the targeted regions along with the viruses. The location of the virus injection site was visualized with CTB-647 (S10D Fig). Only mice with verified injection sites were used for analysis.

EEG/EMG recordings and analysis of sleep-wake states

We implanted electrodes on day 7 of daily exposure to chronic stress or i.p. injection of CNO. The animals continued to receive daily exposure to chronic stress or i.p. injection of CNO for 7 days after electrode implantation. Two days before the end of the 14-day period of daily exposure to chronic stress or i.p. injection of CNO, the animals were transferred to the recording room and habituated to the recording cables and conditions for 2 days. Following this habituation period, EEG/EMG recordings (8400-K1, PINNACLE, USA) were performed on all animals for 24 h.

Original data were converted into European data format (edf). Sleep states were scored with sleep analysis software (SleepSign, Kissei Comtec). Cortical EEG and EMG signals were amplified, filtered (EEG, 0.5 to 30 Hz; EMG, 20 to 200 Hz), digitized at a sampling rate of 128 Hz, and recorded using Pinancle’s recoding equipment (8400-K1, PINNACLE, USA). When complete, polygraphic recordings were automatically scored offline by 4-s epochs as wake, NREM sleep, and REM sleep using SleepSign according to standard criteria. EEG power in the ranges from 0.5 to 4 Hz and from 5 to 8 Hz were defined as delta and theta power, respectively. The general criteria for classification of the different states were: wake = with locomotor activity, high EMG, and intermediate theta/delta wave ratio; NREM sleep = no locomotor activity, low EMG, and predominated with delta wave; REM sleep = no locomotor activity, low EMG, and predominated with theta wave. All classifications of states automatically assigned by SleepSign were examined visually and corrected manually. The total durations and episodes numbers of wake, NREM and REM were exported as a text file. Spectrograms were generated using Sirenia Sleep Pro software (PINNACLE, USA).

Fiber photometry

A fiber photometry system was used for recording Ca²⁺ signals from RMTg-projecting LHb neurons or RMTg neurons receiving direct LHb inputs. Briefly, 1 week after viruses injection, an optical fiber [O.D. = 230 mm, numerical aperture (NA) = 0.37] was placed in a ceramic ferrule and inserted towards the LHb (coordinates: AP: −1.6 mm; ML: ± 0.35 mm; DV: −2.6 mm) or RMTg (coordinates: AP: −4.3 mm; ML: ± 0.35 mm; DV: −4.2 mm) through the craniotomy. The ceramic ferrule was supported with a skull penetrating M1 screw and dental acrylic. Mice were individually housed and allowed to recover for at least 1 week. To record fluorescence signals, GCaMP7s fluorescence was detected through the optic fiber using a fiber photometry system (Inper, China). Calcium-dependent fluorescence signals were obtained by stimulating LHb or RMTg neurons expressing GCaMP7s with a 470 nm LED (40 μW at fiber tip) while calcium-independent signals were obtained by stimulating these cells with a 410 nm LED (20 μW at fiber tip), which was further used to correct for movement artifacts. The 410 nm signal was scaled using least-squares regression to minimize the difference between the 410 and 470 signal. The fitted 410 nm signal was then subtracted from the 470 nm signal to obtain the movement and bleaching-corrected signal. The values of fluorescence change (ΔF/F) by calculating (F−F0)/F0, where F0 was the averaged baseline fluorescence signal recorded before stressful stimuli.

For the foot shock stimulation, foot shocks (1 mA, 500 ms) were delivered with inter-trial intervals of 15 s. Shock delivery onset was used as the trigger event for data alignment. For the air puff stimulation, the body of the mouse was inserted into an acrylic “body tube,” with the mouse’s head extending out. A brief air puff was delivered to the face of the mouse by pressing a hand-pump air compressor (13 × 5 cm) attached to a PVC tube with its opening positioned approximately 5 cm away from the mouse’s nostril. One press of the hand pump generates a ~2-s-long gentle air puff. Air puffs were delivered with inter-trial intervals of approximately 15 s. Onset of air puff delivery was used as the trigger event for data alignment.

Physiological recording from brain slices

For brain slice preparation, the mice were deeply anesthetized with isoflurane, and coronal sections (250 μm thick) containing the LHb or RMTg were cut using a vibratome (VT1200S; Leica Microsystems) in ice-cold artificial cerebrospinal fluid (ACSF, in mM: 119 NaCl; 2.5 KCl, 1 NaH₂PO₄, 11 glucose, 26.2 NaHCo₃, 2.5 CaCl₂, 1.3 MgCl₂, and 290 mOsm, at pH 7.4). The brain slices were recovered for approximately 1 h at room temperature in ACSF. After recovery, the slices were placed in the recording chamber and continuously perfused with ACSF.

Evoked postsynaptic currents were elicited by 2 ms blue light stimulation of axonal terminals of RMTg-projecting LHb neurons infected with ChR2-mCherry. Blue-light-evoked EPSCs and IPSCs were recorded when the membrane potential was held at −70 mV and 0 mV, respectively. To test whether the recorded EPSCs were mediated by the AMPA/kainate receptor, 10 μM NBQX was added to ACSF. To test whether the postsynaptic currents recorded in RMTg neurons were elicited by direct synaptic connections, 1 μM tetrodotoxin (TTX) and 100 μM 4-aminopyridine (4-AP) were added to ACSF. The recorded cells were intracellularly filled with biocytin for morphological evaluation.

To measure the excitability of RMTg-projecting LHb neurons or RMTg neurons receiving direct LHb inputs, electrodes were filled with K⁺-based peptide solution (in mM: 130 KMeSO₄, 10 KCl, 10 Na₂-phosphocreatine, 4 MgATP, 0.3 Na₃GTP, 10 HEPES, 290 mOsm, adjusted to 7.4 with KOH). Current steps were applied (for RMTg-projecting LHb neurons: 10 pA injection current per step, duration: 1 s; for RMTg neurons receiving direct LHb inputs: 20 pA injection current per step, duration: 1 s) from a membrane potential of −70 mV.

To measure the function of chemogenetic viruses, neurons expressing hM3Dq-EGFP or hM4Di-EGFP in the LHb or RMTg were recorded. For chemogenetic activation, hM3Dq-EGFP-labeled neurons were recorded in the current-clamp model. After 80 s of baseline recording, 10 μM CNO was washed into ACSF for 100 s, and the neurons were recorded for 4 min in total. For chemogenetic inhibition, hM4Di-EGFP-labeled neurons were injected with a 100 pA current, and the number of activated action potentials was calculated as the baseline. Then, 10 μM CNO was added to ACSF for 10 min, and the action potentials activated by 100 pA current injection were recorded. Finally, the CNO was washed out, and the activated action potentials were recorded.

All recordings were performed using a Multiclamp 700B amplifier (Molecular Devices). Traces were low-pass-filtered at 2 kHz and digitized at 10 kHz. For light stimulation, light pulses were delivered through digital commands from the Digidata 1550A and Digital stimulator (PG4000a, Cygnus Technology). The pipette resistance ranged from 4 to 6 MΩ. When stable whole-cell recordings were achieved with an access resistance below 25 MΩ, basic electrophysiological properties were recorded. Offline data analysis was performed using Clampfit 10.0 software (Molecular Devices).

Immunocytochemistry

All animals were anesthetized (Avertin, 13 ml/g, intraperitoneally) and perfused intracardially with 0.9% saline followed by 4% paraformaldehyde in phosphate-buffered saline (PBS). Brains were removed. For CaMKIIα labeling, 40 μm cryostat sections containing the LHb were placed in blocking solution for 1 h before incubation in primary antibody against CaMKIIα (rabbit, 1:500; ab5683, Abcam) (36 h at 4°C). Sections were then incubated with corresponding secondary antibody at a dilution of 1:400 for 6 h at room temperature: (Dylight 594) goat-anti-rabbit IgG (DI-1649, Vector Laboratories). For GABA labeling, 40 μm cryostat sections containing the RMTg were placed in blocking solution for 1 h before incubation in primary antibody against GABA (rabbit, 1:500; A2052, sigma) (36 h at 4°C). Sections were then incubated with corresponding secondary antibody at a dilution of 1:400 for 6 h at room temperature: (Dylight 594) goat-anti-rabbit IgG (DI-1649, Vector Laboratories).

Image analysis

Brain sections were imaged with a Zeiss 700 confocal microscope with 5× or 20× objectives, or a 40× oil immersion objective. For three-dimensional reconstruction of injected or virus labeled cells, optical sections were collected at 0.2 μm intervals. Using Image J and Photoshop CS5 (Adobe Corp., San Jose, California, USA), each stack of optical sections was montaged and projected to a 0° X-Y plane and a 90° Y-Z plane to obtain a three-dimensional reconstruction of the cell. Contrast and brightness were adjusted, and the red-green images had been converted to magenta-green.

Behavioral paradigms

Behavioral tests were performed during the light phase (ZT 6 to ZT 9) unless otherwise specified. Operators were blinded to the experimental group during scoring.

Chemogenetic manipulation

The designer drug CNO (1 mg/kg, i.p.; C0832, Sigma-Aldrich, St Louis, Missouri, USA) was administrated 30 min before exposure to stressful stimuli (ZT 5.5, Figs 2C, 6A and S7G) or bright light treatment (ZT 0.5, Fig 8A). For Figs 2H, 3A, 5D, S1A and S7C, CNO (1 mg/kg, i.p.) was administrated between ZT 3 to ZT 3.5.

Quantification of neurons infected with different viruses

To quantify percentage of RV-DsRed labeled vLGN/ IGL neurons co-labeled with GABA (Fig 7C), 4 C57BL/6 mice received LHb injection of helper virus and RV-DsRed were used. In each mouse, the number of RV-DsRed-labeled vLGN/IGL neurons and RV-DsRed/GABA double labeled vLGN/IGL neurons were counted in 4 serial brain sections (40 μm/section) across the vLGN/IGL. The percentage of RV-DsRed/GABA double labeled neurons was calculated as a percentage of the total number of RV-DsRed/GABA double labeled vLGN/IGL neurons counted in 4 mice from the total number of RV-DsRed-labeled neurons counted in 4 mice.

To quantify percentage of RMTg-projecting LHb neurons co-labeled with CaMKIIα (S4A Fig), 4 C57BL/6 mice received RMTg injection of rAAV2/2-Retro-Cre and LHb injection of AAV2/9-DIO-eYFP were used. In each mouse, the number of eYFP-labeled LHb neurons and eYFP/CaMKIIα double labeled LHb neurons were counted in 4 serial brain sections (40 μm/section) across the LHb. The percentage of eYFP/CaMKIIα double labeled neurons was calculated as a percentage of the total number of eYFP/CaMKIIα double labeled LHb neurons counted in 4 mice from the total number of eYFP neurons counted in 4 mice.

To quantify percentage of RMTg neurons receiving direct LHb inputs co-labeled with GABA (S4B Fig), 4 C57BL/6 mice received LHb injection of AAV2/1-Cre and RMTg injection of AAV2/9-DIO-eYFP were used. In each mouse, the number of eYFP-labeled RMTg neurons and eYFP/GABA double labeled LHb neurons were counted in 4 serial brain sections (40 μm/section) across the RMTg. The percentage of eYFP/GABA double labeled neurons was calculated as a percentage of the total number of eYFP/GABA double labeled RMTg neurons counted in 4 mice from the total number of eYFP neurons counted in 4 mice.

Statistics

All statistics were calculated using GraphPad Prism 7 software. Data analysis was done by experimenters blind to experimental conditions. Statistical details including the definitions and exact value of n (e.g., number of animals), p values, and the types of the statistical tests can be found in the Figures and Figure legends. One-way ANOVA and then Sidak’s multiple comparisons test was used to quantify the performance of the SPT, FST, TST, OFT tests, the amplitude of the IPSC and EPSC, and number of action potentials activated by current injection. For all figures, dot plots include horizontal line representing mean. Statistical significance was set at P < 0.05.