Chemogenetic activation of NAc A 2A R neurons increases sleep

We first used a chemogenetic approach to clarify the neurobehavioural and electroencephalographic outcomes of activating A 2A R-expressing indirect pathway neurons in the NAc. To target excitatory “designer receptors exclusively activated by designer drugs” (DREADD)19 in A 2A R neurons located in the NAc, we used transgenic mice in which Cre-recombinase was expressed under the A 2A R promoter (A 2A R-Cre)20, 21. Adeno-associated viral vectors (AAV) carrying Cre-recombinase-dependent hM3Dq DREADD (AAV-hSyn-DIO-hM3Dq-mCherry) were stereotaxically injected bilaterally into the NAc of A 2A R-Cre mice (NAc-hM3Dq mice; Fig. 1a, b). To investigate the effect of NAc A 2A R neuron stimulation on behavioural activity, we measured locomotor activity after intraperitoneal injections of vehicle or clozapine-N-oxide (CNO), a hM3Dq ligand that evokes neuronal excitation. Locomotor activity was decreased in NAc-hM3Dq mice for 5 h after the administration of CNO at 20:00, i.e., at the beginning of the dark period when mice usually show high levels of arousal (Fig. 1c). We then analysed electroencephalogram (EEG) and electromyogram (EMG) recordings made after vehicle or CNO injections at 20:00 to measure sleep. Compared with vehicle injection, injection of CNO led to a dose-dependent and 3 h-long increase in slow-wave sleep (SWS), which is also known as non-rapid eye movement (non-REM) sleep, the major part of sleep characterised by slow and high-voltage brain waves (Fig. 1d, e and Supplementary Fig. 1a). The number of prolonged SWS episodes (duration between 120–470 s) was also significantly increased (Fig. 1f). Changes in the mean episode number of wakefulness and SWS, and the number of stage transitions from wakefulness to SWS or from SWS to wakefulness did not differ significantly between mice treated with vehicle or CNO for 3 h after the intraperitoneal injection, as assessed by paired Student’s t-test (Supplementary Fig. 1b, c). To assess whether EEG activity was altered by chemogenetic activation, we compared the normalised EEG power spectrum of SWS in NAc-hM3Dq mice at baseline (1 day prior to vehicle injection) and after treatment with vehicle or CNO, and in A 2A R-Cre mice without AAV injections at baseline (Fig. 1g). The EEG activity in the frequency range of 0.5–25 Hz during SWS was indistinguishable between chemogenetically induced and natural (baseline or vehicle treatment) sleep. We also compared the absolute EEG power spectrum of SWS in each condition, but detected no statistical differences (Supplementary Fig. 1d). Moreover, mice treated with vehicle and CNO had similar SWS spindle frequency (Supplementary Fig. 1e). These analyses suggest that the induced sleep was likely physiological sleep rather than abnormal sleep.

Fig. 1 Chemogenetic stimulation of A 2A R neurons in the NAc induced SWS. a A 2A R-Cre mice were injected with AAV-hSyn-DIO-hM3Dq-mCherry into the NAc and implanted with somnographic electrodes. b A brain section was stained against mCherry to confirm that hM3Dq-mCherry protein was expressed in the NAc. Scale bar: 500 μm. c Time course of locomotor activity. Black and white bars indicate dark and light periods, respectively. Two-way repeated measures ANOVA was performed followed by Bonferroni’s post hoc comparisons. *P < 0.05, **P < 0.01, compared with vehicle. d Typical examples of EEG (power spectrogram and wave traces), EMG, and hypnograms after the administration of vehicle or CNO. e Dose-dependent changes in SWS time normalised to the SWS time of the vehicle control. One-way ANOVA was performed followed by Bonferroni’s post hoc comparisons. f SWS episode spectrum. *P < 0.05, compared with vehicle, assessed by paired two-tailed Student’s t-test. g EEG power density of SWS between 20:00 and 23:00. Data are presented as the mean ± SEM (n = 6). Each pair of grey dots indicates data from one mouse Full size image

Optogenetic activation of NAc A 2A R neurons induces SWS

We then used optogenetic stimulation of channelrhodopsin-2 (ChR2), a blue-light-gated cation channel, expressed in NAc A 2A R neurons to further explore the temporal properties of sleep responses evoked by activating A 2A R neurons in the NAc. After AAV carrying Cre-dependent ChR2 (AAV-EF1α-DIO-ChR2-mCherry) was stereotaxically injected into the NAc of A 2A R-Cre mice (NAc-ChR2 mice; Fig. 2a, b), we performed whole-cell patch-clamp recordings of acutely prepared tissue slices containing the NAc (Fig. 2c–i) to test the response of ChR2-expressing neurons, presumably A 2A R neurons, to optogenetic stimulation. Brief pulses of light (5–15 ms) evoked single action potentials in ChR2-expressing A 2A R neurons, whereas pulses longer than 15 ms resulted in two spikes (Fig. 2f, g). Light pulses evoked action potentials with high frequency fidelity between 1 Hz and 30 Hz (Fig. 2h, i). We then stimulated the NAc in vivo in NAc-ChR2 mice with 5-ms pulses of blue light in the frequency range of 5–40 Hz at 22:00 when the mice were awake. The latency to sleep onset, defined as the time from the first light pulse to the appearance of the first SWS episode lasting longer than 20 s, was dependent on the pulse frequency as well as on whether the stimulation was unilateral or bilateral, with NAc-ChR2 mice showing the shortest sleep latency when stimulated bilaterally at 20 Hz (Fig. 2j and Supplementary Movies 1, 2). Photostimulation at 20 Hz induced profound SWS, resulting in more than 80% SWS during the illumination period between 22:00 and 23:00 (Figs. 2k–m). After the last light pulse, NAc-ChR2 mice exhibited a significant rebound in wakefulness over the next hour compared with A 2A R-Cre mice injected with AAV expressing only mCherry (NAc-mCherry mice; Fig. 2l, m). The mice essentially behaved normally during the hour after photostimulation (Supplementary Fig. 2). We used whole-cell patch-clamp recordings in acute brain slices to confirm that optical stimulation of ChR2-expressing A 2A R neurons at 20 Hz evoked action potentials during a 1-h period (Supplementary Fig. 3). The optogenetically induced firing was sustained at 20 Hz during the initial 5−15 min, depending on the individual neuron, and then progressively decreased to 3.0 ± 1.8 Hz after 1-h stimulation.

Fig. 2 Optogenetic stimulation of A 2A R neurons in the NAc evoked a rapid and robust SWS response. a A 2A R-Cre mice injected with AAV-EF1α-DIO-ChR2-mCherry into the NAc. b A brain section stained against ChR2-mCherry in the NAc. Scale bar: 500 μm. c–e A typical section of a NAc-ChR2 mouse for patch-clamp electrophysiology showing a ChR2-mCherry-expressing neuron (white arrowheads) c, the patch pipette (white arrow) and the recorded neuron in phase contrast d, and the recorded neuron with Lucifer yellow e. f Recorded neuron showed typical membrane properties and a spiking pattern of medium spiny neurons in response to 500-ms current injections from –40 to 80 pA with 40-pA steps (shown in red). g Brief 5-ms to 15-ms light pulses at 5-ms intervals evoked single action potentials in ChR2-expressing neurons (shown in black). A pulse longer than 15 ms resulted in 2 spikes (shown in grey). h Responses of an A 2A R neuron to 20 pulses of blue light at various frequencies. Each vertical blue bar represents a 5-ms light pulse. i Fidelity responses of ChR2-expressing neurons to light pulses at frequencies up to 100 Hz (n = 5 cells from 2 mice). j Sleep latency after photostimulation in the frequency range of 5–40 Hz. Light was illuminated at least until the mice slept. **P < 0.01 compared between unilateral and bilateral photostimulation, assessed by paired two-tailed Student’s t-test. Statistical differences between pulse frequency conditions were assessed by one-way repeated measures ANOVA followed by Bonferroni’s test. k Typical examples of EEG (power spectrogram and wave traces), EMG, and hypnograms of two mice injected with AAV-EF1α-DIO-ChR2-mCherry (lower panel) or AAV-EF1α-DIO-mCherry (upper panel). l, m The time course l and hourly SWS amount m during optogenetic experiments. n, o SWS episode spectrum n and EEG power density o during photostimulation. Data are presented as the mean ± SEM (n = 6). Each grey dot indicates data from one mouse. *P < 0.05, **P < 0.01 compared between mouse groups, assessed by unpaired two-tailed Student’s t-test n or mixed model ANOVA followed by Bonferroni’s post hoc comparisons l, m. Blue bars indicate the period of light illumination Full size image

A strong shift to prolonged SWS episodes with a duration of 60 s or more was observed in NAc-ChR2 mice (Fig. 2n), resulting in a large increase in the mean duration of SWS episodes (Supplementary Fig. 4a). Moreover, optogenetic stimulation of A 2A R neurons in the NAc significantly increased the mean number of SWS episodes and the number of wake-to-SWS or SWS-to-wake transitions (Supplementary Fig. 4b, c). The EEG activity in the frequency range of 0.5–25 Hz during SWS episodes was indistinguishable between NAc-ChR2 and NAc-mCherry mice with photostimulation at 20 Hz (Fig. 2o and Supplementary Fig. 4d) as well as between NAc-ChR2 mice with no, 5-Hz, or 10-Hz photostimulation (Supplementary Fig. 4e). Moreover, the number of SWS spindles during photostimulation was similar between NAc-ChR2 and NAc-mCherry mice (Supplementary Fig. 4f). These data suggest that optogenetic activation of A 2A R neurons in the NAc induced physiological sleep rather than abnormal sleep.

Activation of NAc core A 2A R neurons induces SWS

The two components of the NAc, the core and the shell, have anatomically and functionally distinct projections to the forebrain, diencephalon and brainstem22,23,24. We therefore examined whether sleep is differentially affected by specific stimulation of ChR2-expressing neurons in the core or shell. We unilaterally injected AAV-EF1α-DIO-ChR2-mCherry into the NAc core or shell of A 2A R-Cre mice and examined the effect of photostimulation on SWS between 22:00 and 23:00. Typical examples of EEG, EMG and hypnograms from mice in the ‘no light’ or ‘light’ conditions are shown in Fig. 3a, b. Photostimulation of ChR2 in A 2A R neurons in the NAc core strongly increased SWS by 2.9-fold, as compared to the baseline amount of sleep in mice without light stimulation between 22:00 and 23:00 (Fig. 3a, c, d), while SWS was not changed by photostimulation of neurons in the NAc core that expressed only mCherry (Fig. 3f). In contrast, SWS tended to be reduced when only A 2A R neurons of the medial portion of the NAc shell were stimulated (Fig. 3c, e), although there was no statistically significant difference (P = 0.065, paired Student’s t-test, n = 6).

Fig. 3 Optogenetic stimulation of A 2A R neurons in the NAc core, but not the shell, produced SWS. a, b Typical examples of EEG, EMG and hypnograms of two mice injected with AAV-EF1α-DIO-ChR2-mCherry in the NAc core a or shell b. Photostimulation of the NAc core, but not the shell, induced SWS (bottom panels), as compared to baseline sleep of the two mice without photostimulation (top panels). c Typical sections from mice with AAV injections in the core (left panel) or shell (right panel) were stained for mCherry and Nissl. Scale bars: 500 μm. Drawings of superimposed AAV injection sites in the core (in red, n = 6) and shell (in green, n = 6) are shown below the photomicrographs. d–f Hourly amount of SWS during optogenetic stimulation. Data are presented as the mean ± SEM. Each pair of grey dots indicates data from one mouse. **P < 0.01 compared to baseline sleep without photostimulation, assessed by paired two-tailed Student’s t-test. g–j Brain sections of a mouse expressing ChR2 in the NAc core cell bodies stained with an antibody against mCherry show ChR2-mCherry positive nerve terminals in the VP g, LH h, TMN i, and VTA j. High-magnification photomicrographs of the black rectangular area are shown as insets in h–j. Scale bars: g–j, 200 μm; insets in h–j, 10 μm. Other abbreviations: ac, anterior commissure; IC, islands of Calleja; 3 V, third ventricle; IP, interpeduncular nucleus. N.S., not significant Full size image

NAc A 2A R neurons are thought to release the neurotransmitter γ-aminobutyric-acid (GABA) upon activation and may thus induce sleep by inhibiting postsynaptic targets. Brain sections of A 2A R-Cre mice injected with AAV-EF1α-DIO-ChR2-mCherry into the NAc core that were stained with an antibody against mCherry revealed that ChR2-mCherry-expressing core neurons heavily innervated the VP (Fig. 3g). These neurons also made sparse to moderate projections to well-known arousal-promoting areas, such as the lateral hypothalamus (LH), which produces orexin; the tuberomammillary nucleus (TMN), which produces histamine; and the ventral tegmental area (VTA), which produces dopamine (Fig. 3h–j). We therefore determined the extent to which the VP, LH, TMN and VTA contribute to the sleep control mediated by the NAc core. We first performed whole-cell patch-clamp experiments in acute slices to test the responses to optogenetic stimulation of ChR2-expressing NAc core terminals in the VP (Fig. 4a–e). Stimulation (5-ms pulses, 10 Hz) of the NAc core terminals decreased the firing rate and evoked inhibitory postsynaptic currents (IPSC) in most of the recorded VP neurons (Fig. 4b–e and Supplementary Fig. 5a–d), while no such responses were observed in the LH, TMN or VTA (Fig. 4e), possibly due to the low density of synaptic connections on hypothalamic (i.e., LH and TMN) and midbrain VTA neurons. These findings suggest prominent functional synaptic connectivity between the ChR2-expressing NAc core and VP neurons.

Fig. 4 Optogenetic stimulation of ChR2-expressing NAc core axonal terminals in the VP evoked a SWS response. a A 2A R-Cre mice were injected with AAV-EF1α-DIO-ChR2-mCherry into the NAc core and photostimulation occurred at the target side, e.g., the VP. b Typical cell-attached recording of a VP neuron in a NAc-ChR2 mouse. Brief light pulses decreased the firing rate. c Optical stimulations elicited IPSC in a VP neuron (measured in voltage-clamp at –70 mV). The individual trace is shown in grey, whereas the averaged trace is shown in black. d Optogenetically evoked IPSC were completely blocked in the presence of picrotoxin (PTX: 100 μM; shown in pink). e Number and proportion of neurons in the VP, LH, VTA and TMN that responded to optical stimulation of ChR2-expressing NAc terminals. f Typical examples of EEG, EMG and hypnograms of a NAc-ChR2 mouse, in which photostimulation of ChR2-expressing NAc terminals in the VP induced SWS (bottom panel), as compared to baseline sleep in a mouse without photostimulation (top panel). g Hourly amount of SWS during optogenetic stimulation. Only photostimulation of terminals in the VP induced SWS in NAc-ChR2 mice. h Sequential brain sections of a VP-lesioned NAc-ChR2 mouse stained with neuronal marker NeuN (left panel) and mCherry (right panel) show neuronal cell loss in the VP portion, while this area is innervated by ChR2/mCherry-expressing NAc core terminals. Scale bars: 500 μm. i Hourly amount of SWS during VP photostimulation in VP-lesioned NAc-ChR2 mice. Data are presented as the mean ± SEM. Each pair of grey dots indicates data from one mouse. **P < 0.01 compared to baseline sleep of NAc-ChR2 mice without photostimulation, assessed by paired two-tailed Student’s t-test (g, i). Blue bars indicate the period of light illumination. Other abbreviation: OT, olfactory tubercle; ac, anterior commissure; 3 V, third ventricle. N.S., not significant Full size image

Next, we examined the effects of photostimulation of ChR2-expressing NAc core terminals in the VP, LH, TMN or VTA on sleep/wake behaviour in A 2A R-Cre mice. Light stimulation for 1 h in the VP portion containing ChR2-expressing NAc core terminals remarkably increased SWS by 4.0-fold (Fig. 4f, g), an effect that was similar to the SWS increase evoked by photostimulation of cell bodies in the NAc core (Fig. 3d), although stimulation of ChR2-expressing NAc core terminals in the LH, TMN or VTA did not induce significant changes in SWS (Fig. 4g). IPSC recordings of VP neurons in acute brain slices during 1-h photostimulation revealed that the IPSC frequency was 4.8-fold higher than the baseline at the onset of photostimulation, but returned to the baseline level after 40–50 min (Supplementary Fig. 5e–g). Because the distance between the AAV injection site in the NAc and photostimulation site in the VP is only 0.8 mm, it is conceivable that the changes in SWS resulted from photostimulation of ChR2 in the NAc core cell bodies rather than their axonal terminals in the VP. Hence, we also stimulated an area in the olfactory tubercle 0.8 mm anterior to the NAc core, which served as an anatomical control site. The amount of SWS sleep, however, remained unchanged (P > 0.05, Wilcoxon signed-rank test, n = 4; Fig. 4g). To exclude the possibility that VP terminal stimulation increased SWS by antidromic activation of the NAc core cell bodies, we unilaterally lesioned VP cells using the neurotoxin ibotenic acid in mice expressing ChR2 in the ipsilateral NAc core (Fig. 4h, left panel). Photostimulation of ChR2-expressing NAc terminals in the VP of those mice (Fig. 4h, right panel) did not affect SWS (Fig. 4i), indicating that the VP is necessary for inducing SWS. Optical stimulation of ChR2 in NAc A 2A R neurons also evoked IPSC in NAc neurons that did not express ChR2 (Supplementary Fig. 6a–d). This observation may indicate collateral innervation between NAc neurons25, which likely include VP-projecting GABAergic neurons of the direct pathway26. GABAergic neurons are the main neuronal population in the VP27 and therefore, we tested whether disinhibition of these neurons induces SWS. We chemogenetically activated VP GABAergic neurons using AAV-mediated expression of DREADD hM3Dq in mice expressing Cre-recombinase under the promoter of the glutamate decarboxylase 2 (Gad2) gene which is expressed in neurons that use GABA as a neurotransmitter, to catalyse the decarboxylation of glutamate to GABA (Supplementary Fig. 6e, f). The total amount of SWS, however, was suppressed for 3 h after administering 1 mg kg−1 CNO to VP-hM3Dq mice, suggesting that SWS is not likely to be induced after disinhibition of VP neurons, for example, by inhibiting VP-projecting GABAergic NAc neurons.

NAc core A 2A R neurons are necessary for SWS

To examine the necessity of NAc core A 2A R neurons for physiological sleep/wake behaviour, we chemogenetically inhibited these neurons using AAV-mediated expression of inhibitory DREADD hM4Di, which suppresses neuronal activity when CNO is applied19, in the NAc core of A 2A R-Cre mice (NAc-hM4Di mice; Fig. 5a, b). We intraperitoneally administered CNO in NAc-hM4Di mice at 20:00, and CNO dose-dependently reduced SWS for up to 4 h (Fig. 5c, d). The total amount of SWS was suppressed by 80% for 4 h with the highest dose of CNO (i.e., 0.3 mg kg−1) compared to vehicle treatment (Fig. 5d). The number of SWS episodes was decreased for 4 h after CNO administration (Fig. 5e), whereas the mean duration of SWS was not changed (Fig. 5f). These results suggest that NAc core A 2A R neuron activity is necessary to induce SWS under baseline conditions. Moreover, we investigated animal behaviours for over 1 h after CNO administration (Supplementary Fig. 7) and found that CNO significantly increased foraging behaviour (including digging and sniffing) compared to vehicle treatment. CNO administration tended to decrease other behaviours, such as grooming and eating, but the difference did not reach statistical significance (P = 0.084 for grooming, P = 0.35 for eating, mixed model analysis of variance (ANOVA) followed by Bonferroni’s test, n = 6).

Fig. 5 Chemogenetic inhibition of A 2A R neurons in the NAc core reduced sleep. a A 2A R-Cre mice were injected with AAV-hSyn-DIO-hM4Di-mCherry into the NAc core. b Brain sections were stained against mCherry to confirm that hM4Di-mCherry protein was expressed in the NAc core. Scale bar: 500 μm. c Time course of SWS. d CNO decreased dose-dependently the total amount of SWS. e, f CNO decreased number of SWS episodes e without affecting the mean duration f. g SWS amount for 7 h after sleep deprivation in the absence and presence of CNO. h, i SWS rebound after sleep deprivation h and CNO-induced SWS reduction i were not affected by CNO administration and sleep deprivation, respectively. Data are presented as the mean ± SEM (n = 6). Each pair of grey dots indicates data from one mouse. *P < 0.05, **P < 0.01 compared with vehicle, assessed by two-way c, g, one-way d–f repeated measures ANOVA followed by Bonferroni’s post hoc comparisons, or paired two-tailed Student’s t-test h, i. N.S., not significant Full size image

We also examined the role of NAc core A 2A R neurons for the homoeostatic sleep rebound after sleep deprivation by administering vehicle or CNO to NAc-hM4Di mice kept awake for 4 h prior to the drug treatment at 20:00. We observed that vehicle- and CNO-treated mice had a significant SWS rebound during the 7 h after sleep deprivation and that the CNO-induced decrease of SWS was similar in sleep-deprived and non-sleep-deprived animals during the same period (Fig. 5g). Two-way repeated measures ANOVA revealed no statistically significant interaction between sleep deprivation and CNO treatment (F (1,5) = 1.53, P = 0.272, n = 6). Moreover, the amount of SWS rebound after sleep deprivation or CNO-induced SWS reduction during 7 h was not affected by CNO treatment or sleep deprivation, respectively (Fig. 5h, i). These results suggest that the ability of NAc core A 2A R neurons to induce sleep is independent of homoeostatic sleep pressure.

Motivational stimuli suppress NAc neuron activity

Finally, we investigated the patterns of spontaneous activity of the indirect NAc core-VP pathway under various conditions of sleep and motivation using immunohistochemical examination of the expression of c-Fos, a marker of neuronal activation, in mice injected with the retrograde tracer cholera toxin subunit b (CTb) into the VP (Fig. 6a, b). Mice that were killed during the light phase at 12:00 spent more time (95 ± 4.1 min) asleep during the 3 h prior to tissue collection than mice that were killed during the dark phase at 24:00 when they slept 54 ± 6.2 min during the 3 h prior to tissue collection (Fig. 6d). Triple labelling for c-Fos, A 2A R, and CTb (Fig. 6c) revealed that mice killed during the day had a markedly increased number of VP-projecting NAc core A 2A R neurons expressing c-Fos (37 ± 5.0%) compared to mice that were killed during the night with c-Fos expression in 20 ± 2.0% of VP-projecting NAc core A 2A R neurons (Fig. 6e). To determine the role of homoeostatic sleep factors on the activity of the indirect NAc core-VP pathway, we deprived mice of sleep for 4 h before the dark period (Fig. 6f). The c-Fos expression in VP-projecting NAc core A 2A R neurons of sleep-deprived mice was not different from that in non-sleep-deprived mice (Fig. 6g), however, suggesting that homoeostatic sleep pressure does not induce the activity of VP-projecting NAc core A 2A R neurons. This observation is consistent with our finding that chemogenetic inhibition NAc core A 2A R neurons did not affect sleep rebound after sleep deprivation (Fig. 5g–i). Because the NAc is a critical brain area for motivational behaviour17, 18, we also examined SWS amount and activity of VP-projecting NAc core A 2A R neurons in the presence of motivational stimuli (e.g., toy, female mouse or chocolate) or stimuli that are unlikely associated with motivational behaviour (e.g., regular chow or bedding). The amount of SWS in mice was significantly lower in the presence of motivational stimuli compared to mice with non-motivational stimuli (Fig. 6h). Moreover, the number of NAc core neurons triple-labelled for c-Fos, A 2A R and CTb was lower in male mice that spent time with a toy, a female mouse, or chocolate compared to mice in the presence of non-motivational stimuli (Fig. 6i). These results suggest that motivational stimuli suppress the activity of VP-projecting NAc core A 2A R neurons and SWS.