Abstract

Lesions of the globus pallidus externa (GPe) produce a profound sleep loss (∼45%) in rats, suggesting that GPe neurons promote sleep. As GPe neuronal activity is enhanced by dopamine (DA) from the substantia nigra pars compacta (SNc), we hypothesized that SNc DA via the GPe promotes sleep. To test this hypothesis, we selectively destroyed the DA afferents to the caudoputamen (CPu) using 6-hydroxydopamine and examined changes in sleep-wake profiles in rats. Rats with 80–90% loss of SNc neurons displayed a significant 33.7% increase in wakefulness (or sleep reduction). This increase significantly correlated with the extent of SNc DA neuron loss. Furthermore, these animals exhibited sleep-wake fragmentation and reduced diurnal variability of sleep. We then optogenetic-stimulated SNc DA terminals in the CPu and found that 20-Hz stimulation from 9 to 10 PM increased total sleep by 69% with high electroencephalograph (EEG) delta power. We finally directly optogenetic-stimulated GPe neurons and found that 20-Hz stimulation of the GPe from 9 to 10 PM increased total sleep by 66% and significantly increased EEG delta power. These findings elucidate a novel circuit for DA control of sleep and the mechanisms of abnormal sleep in BG disorders such as Parkinson's disease and Huntington's disease.

Introduction

Although it has been known that the basal ganglia (BG) are involved in an array of functions including emotional, cognitive, and motor control, a role for BG in sleep-wake behavior has only been recently recognized. In rats, cell-body lesions of the dorsal striatum (caudate-putamen, CPu) produce a 15% increase per day in sleep and fragmentation of sleep-wake states as well as flattened circadian sleep rhythms (Qiu et al. 2010). Notably, lesions of the globus pallidus externa (GPe), which receives inhibitory inputs from the CPu, produce a marked reduction (∼45%) in sleep, while lesions of other BG structures such as the subthalamic nucleus, globus pallidus interna (GPi), and substantia nigra pars reticulata (SNr) do not significantly alter the sleep-wake cycle. These observations suggest that the GPe promotes sleep.

Dopamine (DA) plays a critical role in modulating GPe activity. The CPu receives DA innervation from the substantia nigra pars compacta (SNc), and this DA acts on D2 receptors in GABAergic striatopallidal neurons to increase GPe neuronal activity (Cooper and Stanford 2001; Querejeta et al. 2001). Nonselective lesions in the substantia nigra region, including both SNc and SNr, result in profound insomnia in rats and cats (Lai et al. 1999; Gerashchenko et al. 2006). Conversely, systemic DA D2 agonists produce sleepiness and sleep attacks in humans (Hirayama et al. 2008; Lipford and Silber 2012). Collectively, these results suggest that SNc DA neurons projecting to the CPu may play a role in sleep-wake control by disinhibiting the GPe.

To test this hypothesis, we examined sleep-wake profiles of rats with selective SNc DA neurons lesions using the specific neurotoxin, 6-hydroxydopamine (6-OHDA). Next, we used an optogenetics approach to excite SNc DA terminals in the CPu and examined sleep-wake state changes. Finally, we tested the sleep-wake effects of direct optogenetic stimulation of the GPe.

Methods and Materials

Animals

Pathogen-free, adult, male Sprague-Dawley rats (275–300 g, Harlan) were individually housed and had free access to food and water. All animals were housed under controlled conditions (12-h light starting at 07:00 AM, 100 lux) in an isolated ventilated chamber maintained at 20–22 °C. All protocols were approved by the Institutional Animal Care and Use Committee of Beth Israel Deaconess Medical Center.

6-OHDA Experiment

Surgery

In order to ablate DA afferents in the CPu, rats were injected under anesthesia (i.p., 800 mg/kg ketamine, 80 mg/kg xylazine, Med-Vet) with 6-OHDA (50–90 nL of 6% solution; Sigma, USA) into the ventral GPe (coordinate: anterioposterior (AP) = 0.8 mm, mediolateral (ML) = ±3.0 mm, dorsoventral (DV) = −7.0 mm) (Paxinos and Watson 2009) bilaterally using a pressure injection system (Lu et al. 2000). These coordinates were chosen because the DA neurons innervating the striatum pass through this region, and our pilot studies confirm selective destruction of DA terminals in the CPu (Fig. 1D). Following these injections, rats were implanted with 4 screw electrodes for recording electroencephalograph (EEG) (2 on the frontal bone and 2 on the parietal bone) and 2 electromyography (EMG) wire electrodes on to the nuchal muscles for recording EMG. The other ends of the electrodes were connected to a 6-pin pedestal (Plastics One, USA) that was then secured on to the skull using dental cement.

Figure 1.

Selective loss of SNc DA neurons and projections to the CPu. TH-immunostained sections at the level of either SNc (A–C) or CPu (D–F) from a control rat (A and E), from a rat with partial lesion (B), from a rat with near complete loss of DA neurons in SNc and DA terminals in the CPu (C and F) and from a rat with unilateral 6-OHDA injections (D). The loss of DA cells and their projections were seen in SNc (B and C) and CPu (F) but did not extend to DA neurons in the VTA (B and C) and their terminals in the nucleus of accumbens (NAc) (D, left hemisphere and F).

Figure 1.

Selective loss of SNc DA neurons and projections to the CPu. TH-immunostained sections at the level of either SNc (A–C) or CPu (D–F) from a control rat (A and E), from a rat with partial lesion (B), from a rat with near complete loss of DA neurons in SNc and DA terminals in the CPu (C and F) and from a rat with unilateral 6-OHDA injections (D). The loss of DA cells and their projections were seen in SNc (B and C) and CPu (F) but did not extend to DA neurons in the VTA (B and C) and their terminals in the nucleus of accumbens (NAc) (D, left hemisphere and F).

Sleep Recording and Analysis

After a period of postoperative recovery (10–14 days), the animals were transferred to the recording room and habituated to the recording cables and conditions for 2 days. Following this habituation period, 48-h EEG/EMG recordings (AM systems, USA) were performed on all the rats. The sleep-wake data collected from the rats were divided into 10-s epochs and scored into one of the 3 sleep-wake states—Wake, NREM sleep and REM sleep using SleepSign (Kissei Comtec) according the criteria previously described (Lu et al. 2000).

Histology

On completion of sleep-wake recordings, the rats were deeply anesthetized by chloral hydrate (500 mg/kg), perfused with 50 mL saline followed by 500 mL 10% formalin through the heart. The brains were removed, post-fixed for 4 h in 10% formalin, and then equilibrated in 20% sucrose in PBS overnight. The brains were sectioned on a freezing microtome at 40 µm into 4 series. One series of sections were processed for tyrosine hydroxylase (TH)-immunocytochemistry to verify the loss of the DA terminals in the striatum and DA neuronal cell bodies in SNc. The sections were washed in 0.1 M phosphate-buffered saline (PBS), pH 7.4 (2 changes) and then incubated in the primary antiserum (Anti-TH, 1:20 000, Diasorin). Sections were then washed in PBS and incubated in biotinylated secondary antiserum (against appropriate species IgG, 1:1000 in PBS) for 1 h and washed in PBS and incubated in avidin–biotin–horseradish peroxidase conjugate (Vector labs) for 1 h. Sections were then washed again and incubated in a 0.06% solution of 3,3-diaminobenzidin tetrahydrochloride (DAB, Sigma) plus 0.02% H2O2. We then counted the remaining number of TH+ neurons in the SNc in order to quantify the extent of lesions. Cell counting was performed on 3 adjacent sections (separated by 160 µm) on both sides and presented as number/section/side. According to the extent of lesions, we grouped animals into a partial lesion group with 50–80% DA cell loss in the SNc (PL) and a complete lesion group with >80% DA cell loss in the SNc (CL).

Optogenetics Experiments

Surgery

Under anesthesia (i.p., 800 mg/kg ketamine, 80 mg/kg xylazine), a burr hole was made and a fine glass pipette (1-mm glass stock, tapering slowly to a 10–20 µm tip) containing recombinant AAV10 carrying Ef1α::ChR2–mCherry (1–2 × 1012 infectious particles/mL) was lowered to the SNc (AP = −5.0 mm, ML = ±1.8 mm, DV = −7.6 mm) or GPe (AP = −0.85 mm, ML = ±2.9 mm, DV = −6.0 mm) bilaterally, as per the atlas of Paxinos and Watson (2009). A total of 50 nL virus was delivered over a 5-min period per hemisphere via nitrogen gas pulses of 20–40 psi using an air compression system previously described (Lu et al. 2000). After 2 additional minutes, the pipette was slowly withdrawn, for SNc DA terminal stimulation, 2 cannulas (18 mm long, 0.37 mm diameter) were placed at CPu (AP = +0.2 mm, ML = ±2.9 mm, DV = −4.5 mm). For GPe stimulation, the cannulas were placed 0.5 mm dorsal to the AAV injection site (AP = −0.85 mm, ML = ±2.9 mm, DV = −5.5 mm) on both sides. Electrodes for recording EEG and EMG were then implanted for polysomnographic recordings. Both the cannulas and the EEG/EMG electrodes were affixed to the skull with dental cement.

Photostimulation

After surgical procedures, rats were allowed to recover in individual housing for at least 2 weeks. Animals were then acclimated to flexible EEG/EMG connection cable for 3 days within individual recording chambers.

The Fiber-optic cables (0.25 mm diameter) were placed inside the implanted cannula 2 days prior to stimulation. The other end of the optic fiber (FC connection) was attached to a rotating optical joint (FRJ_FC-FC, Doric Lenses, USA) to relieve torque. The joint was connected via a fiber to a blue-light laser (473 nm, CL473–050, Crystalaser, USA), light pulse trains 120, 60, or 30-ms pulses for 10 s at 5, 10, 20 Hz, and at 30-s intervals (10 s on/20 s off) were programmed using an electronic stimulator (SEN-7103 Nihon Kohden, Japan) and output via an isolator (ss-102J, Nihon Kohden, Japan). Light stimulation was conducted from 9 PM to 10 PM or 9 AM to 10 AM. The EEG/EMG recorded from 9 PM to 10 PM or 9 AM to 10 AM without light stimulation and with light stimulation was served as baseline control and experiment group, respectively. The sleep-wake cycle parameters (the amount, the bouts and mean duration of wake, NREM and REM sleep, and sleep-wake transitions) were quantified by off-line scoring of the entire hour at 9 PM or 9 AM for each animal.

The locations of AAV injections and cannula tips were confirmed by immunolabeling with rabbit anti-mCherry (1:10 000; Clontech). The histology and immunostaining were performed as described above.

Statistical Analysis

The quantitative data were presented as the mean ± standard error of mean (SEM). Statistical significance was assessed with the paired t-test, with P < 0.05 taken as the threshold of significance.

Results

The Sleep-Wake Effects of 6-OHDA Lesions

Lesions of SNc DA Neurons and DA Terminals in the CPu

The CPu (or dorsal striatum) and ventral striatum (or the nucleus accumbens; NAc) are neuroanatomically distinct structures that play independent, likely opposite roles in sleep-wake control (Qiu et al. 2010; Lazarus et al. 2012). To elucidate the role of DA in CPu, it was critical to restrict the loss of DA terminals to the CPu. Injections of 6-OHDA into the ventral GPe led to selective destruction of DA neurons in the SNc and loss of DA terminals in the CPu, as evidenced from the loss of TH+ neurons and terminals in the respective regions (Fig. 1). On the other hand, DA neurons in the ventral tegmental area (VTA; A10 cell group) medial to the SNc and VTA DA projections in the NAc were mostly unaffected by these injections (Fig. 1B–D,F). Similarly, other regions with dense DA terminals, such as the preoptic area, hypothalamus, basal forebrain, and central nucleus of amygdala, were intact (data not shown). We quantified the extent of lesions by counting the number of remaining TH+ neurons in the SNc and grouping lesioned animals into 2 categories: Rats with partial lesions which with 50–80% DA cell loss in the SNc (PL; n = 7, Fig. 1B) and rats with near complete lesions which with >80% DA cell loss in the SNc (CL; n = 6, Fig. 1C). To confirm the integrity of VTA, we counted TH-ir neurons in the VTA in a section containing the highest number of DA neurons in complete SNc lesion group and controls and found no difference (control group = 147.2 ± 9.5/side vs. SNc lesion control = 144.8 ± 5.6/side).

Sleep-Wake Changes

Sleep-wake parameters, including time spent in each sleep-wake state, bout numbers, and average bout durations from the CL and PL animals were compared with saline-injected control animals (sham-L; n = 5). On average, CL rats showed a 33.7% increase in wakefulness (909.1 min ± 80.9 CL vs. 679.8 min ± 11.5 control, P = 0.004) and 25.8% and 54.1% reductions in NREM (477.4 min ± 65.7 CL vs. 643.7 min ± 11.6 control, P = 0.037) and REM sleep (53.5 min ± 17.1 CL vs. 116.5 min ± 3.9 control, P = 0.019) respectively (Fig. 2A). This increase in wake was more prominent during the day than during the night (Fig. 2B). The Circadian index (CI = (meannight − meanday)⁄mean24 h) is used to estimate the circadian amplitude (Lu et al. 2001). In the CL group, wake, NREM, and REM sleep CI (34.0 ± 16.5%, 68.3 ± 29.2%, and 18.1 ± 33.01% of control, respectively, P < 0.05) were all significantly reduced compared with controls, indicating that SNc lesions reduce circadian amplitude of sleep-wake behavior.

Figure 2.

Loss of DA terminals in CPu leads to sleep alterations (A and B) Changes in daily amounts of wake, NREM, REM sleep, and hourly percentages of wake across 24 h in animals with complete SNc lesions (CL; >80% DA cell loss). (C and D) Changes in daily amounts of wake, NREM, REM sleep, and hourly percentages of wake across 24 h in animals with partial SNc lesions (PL; 50–80% DA cell loss). Both sets of animals showed a significant increase in wake and decrease in NREM sleep. While REM sleep was decreased in CL animals, it was unaltered in PL animals. When compared with the control animals, the CL rats displayed attenuation diurnal variation of wake due to higher increase in wakefulness during daytime (B) but there was no change in the diurnal pattern of wake in the PL animals as the wake increase was evenly distributed throughout the day (D). *P < 0.05, **P < 0.01.

Figure 2.

Loss of DA terminals in CPu leads to sleep alterations (A and B) Changes in daily amounts of wake, NREM, REM sleep, and hourly percentages of wake across 24 h in animals with complete SNc lesions (CL; >80% DA cell loss). (C and D) Changes in daily amounts of wake, NREM, REM sleep, and hourly percentages of wake across 24 h in animals with partial SNc lesions (PL; 50–80% DA cell loss). Both sets of animals showed a significant increase in wake and decrease in NREM sleep. While REM sleep was decreased in CL animals, it was unaltered in PL animals. When compared with the control animals, the CL rats displayed attenuation diurnal variation of wake due to higher increase in wakefulness during daytime (B) but there was no change in the diurnal pattern of wake in the PL animals as the wake increase was evenly distributed throughout the day (D). *P < 0.05, **P < 0.01.

Analysis of sleep-wake architecture showed that the CL rats also displayed fragmentation of sleep-wake states. The numbers of wake and NREM bouts were increased while NREM mean bouts durations were decreased both during the night and day, and wake bout durations were decreased during night (Table 1). On the other hand, the number of REM bouts decreased during the daytime but not during the night (Table 1). However, the mean durations of REM bouts did not differ significantly either during the day or night between CL rats and control rats (Table 1). Thus, the REM reduction in these animals was primarily due to a decrease in the number of REM bouts during the day.

Table 1

Changes in sleep-wake architecture following loss of dopaminergic afferents to the CPu

Group Wake
 
NREM
 
REM
 
Bout number Duration Bout number Duration Bout number Duration 
Lights-On 
 Sham-L 156.8 ± 7.7 86 ± 12.0 157.2 ± 8.1 156 ± 6.3 53.4 ± 4.4 95.6 ± 4.5 
 CL 294 ± 40.6* 87.3 ± 10.6 294.7 ± 40.4* 64.3 ± 11.1** 26 ± 8.5* 82.0 ± 9.3 
 PL 164.7 ± 12.4 87.4 ± 8.7 166.6 ± 11.9 137.1 ± 14.8 53.7 ± 4.7 98.6 ± 18.7 
Lights-Off 
 Sham-L 132.4 ± 9.6 211.8 ± 24 132.6 ± 9.3 106.2 ± 8.6 18.8 ± 4.2 72 ± 6.7 
 CL 230.3 ± 3.7** 114.3 ± 7.4* 231.3 ± 3.6** 59 ± 7.8* 25.7 ± 9.1 65.7 ± 5.1 
 PL 88.4 ± 3.1** 316.4 ± 26.6* 88 ± 3.4** 119 ± 15.7 22.6 ± 3.5 84.3 ± 3.2 
Group Wake
 
NREM
 
REM
 
Bout number Duration Bout number Duration Bout number Duration 
Lights-On 
 Sham-L 156.8 ± 7.7 86 ± 12.0 157.2 ± 8.1 156 ± 6.3 53.4 ± 4.4 95.6 ± 4.5 
 CL 294 ± 40.6* 87.3 ± 10.6 294.7 ± 40.4* 64.3 ± 11.1** 26 ± 8.5* 82.0 ± 9.3 
 PL 164.7 ± 12.4 87.4 ± 8.7 166.6 ± 11.9 137.1 ± 14.8 53.7 ± 4.7 98.6 ± 18.7 
Lights-Off 
 Sham-L 132.4 ± 9.6 211.8 ± 24 132.6 ± 9.3 106.2 ± 8.6 18.8 ± 4.2 72 ± 6.7 
 CL 230.3 ± 3.7** 114.3 ± 7.4* 231.3 ± 3.6** 59 ± 7.8* 25.7 ± 9.1 65.7 ± 5.1 
 PL 88.4 ± 3.1** 316.4 ± 26.6* 88 ± 3.4** 119 ± 15.7 22.6 ± 3.5 84.3 ± 3.2 

Values are mean ± SEM.

Sham-L, sham lesioned rats; CL, rats with >80% dopamine cell loss in the substania nigra pars compacta (SNc); PL, rats with 50–80% dopamine cell loss in the SNc.

*P < 0.05, **P < 0.01.

The animals with partial lesions (PL rats) also displayed changes in sleep-wake amounts, but the magnitude of those effects was much less compared with CL animals. PL rats displayed a 19.9% increase in wake (815.4 min ± 25.3 PL vs. 679.8 min ± 11.5 control, P = 0.007), a 21.7% decrease in NREM sleep (504.2 min ± 20.1 CL vs. 643.7 min ± 11.6 control, P = 0.002) and 3.3% increase in REM sleep (120.3 min ± 6.7 PL vs. 116.5 min ± 3.9 control, P = 0.644) (Fig. 2C). This increase in wake and NREM sleep mostly occurred during the night (Fig. 2D). Changes in wake were primarily due to increase of mean duration of the bouts while NREM sleep change was caused by reduction in bout number primarily (Table 1).

The large number of animals used in this study and variability in lesion extent allowed us to perform a correlation analysis of sleep-wake changes and lesion extent using both CL and PL groups. We found a significant negative correlation between the number of remaining SNc DA neurons and wakefulness, indicating that greater DA neuron loss in the SNc predicts greater sleep loss (r2 = 0.44, P = 0.006, Fig. 3). In general, the CL group appeared to be less active and showed loss in body weight, similar to lesions of the GPe (Qiu et al. 2010).

Figure 3.

Correlation of SNc dopamine neuron loss with total wakefulness. The increase in total wake amounts/24 h negatively correlated with the number of remaining DA neurons in the SNc by 6-OHDA lesions in both CL and PL groups.

Figure 3.

Correlation of SNc dopamine neuron loss with total wakefulness. The increase in total wake amounts/24 h negatively correlated with the number of remaining DA neurons in the SNc by 6-OHDA lesions in both CL and PL groups.

Optogenetic Stimulation of the SNc Dopamine Terminals in the CPu

To examine the effects of high DA level in the CPu on sleep, we injected AAV–ChR2–mCherry in the SNc and implanted fiber-optic stimulation cables bilaterally into the CPu in 5 rats. Laser stimulation (10 s on/20 s off) of SNc DA terminals in the CPu of the SNc–AAV–ChR2 rats were conducted from 9 PM to 10 PM, a time of high wakefulness in rodents; or from 9 AM to 10 AM, a time of sleep in rodents.

The locations of AAV injections, the nigrostriatal projections, and cannula tips were confirmed by immunolabeling with mCherry (Fig. 4A, a and b). We tried 5, 10, and 20-Hz stimulation, respectively; we found that 20 Hz appeared to be optimal for altering sleep during the dark phase. Twenty-Hz (10 s on/20 s off) stimulation in the CPu in a group of 5 rats during 9–10 PM significantly increased the amount of NREM sleep, compared with the same time period on a different day without stimulation (baseline: 14.6 ± 0.9 min; stimulation: 25.0 ± 1.1 min, P = 0.007) (Fig. 4B,C), while wakefulness was significantly reduced by DA fiber stimulation (baseline 40.6 ± 1.3 min; stimulation: 27.8 ± 1.5 min, P = 0.014) (Fig. 4B,E). We calculate the NREM sleep latency to see whether photostimulation the DA terminals in CPu will shorten the latency to NREM sleep and found that there was no significant difference between control and stimulation groups (data not shown). The NREM sleep increase was mainly due to an increase in duration of NREM bouts (Fig. 4H). Although mean REM duration was significantly increased (Fig. 4H), the total REM sleep time increase was not statistically significant (Fig. 4B). Sleep-wake transition (Fig. 4F) and bout numbers (Fig. 4G) were not significantly affected by stimulation. Twenty-Hz stimulation also resulted in a moderate increase in EEG delta power, but this increase did not reach statistical significance (Fig. 4I). To test whether there were any rebound effects after photostimulation, we analyzed the entire night of EEG/EMG recording. As showed in Figure 4C, compared with the baseline EEG, no rebound was found after photostimulation. Photostimulating the DA terminals in the CPu during 9–10 AM showed no sleep effects (Fig. 4D). None of the stimulations produced any detectable changes in motor behaviors.

Figure 4.

Optogenetic stimulating DA fibers in the CPu promotes sleep. (A) The expression of AAV–ChR2–mCherry and the location of the plastic optic fiber tip were confirmed after each experiment. mCherry immunostaining shows that the ChR2 proteins were expressed in cells mostly confined to the SNc but not SNr (a), and the mCherry-positive fibers were obviously seen in the CPu (b). The arrow in (b) shows the cannula tip in the CPu. (B) Total amount of wake, NREM and REM sleep in control and 20-Hz photostimulation during 9 PM. (C and D) The hourly amount of NREM sleep of baseline and 20-Hz stimulation group at dark or light phase, respectively. The blue columns indicate the photostimulation period. (E) Wake, NREM, and REM sleep stage changing of baseline, 5-, 10-, and 20-Hz photostimulation of DA fibers in the CPu during 9 PM. (F, G, and H) Sleep-wake state transitions, number and mean duration of wake, NREM, and REM bouts in baseline and 20-Hz photostimulation during 9 PM. Stimulation does not significantly change state transitions and sleep bouts, but mean duration of NREM sleep are significantly increased. (I) EEG power densities of NREM sleep of baseline and photostimulation at 5, 10, or 20 Hz. Power spectrum at 1–3 range of 20-Hz stimulation is increased over the baseline but the increase is not significant (p > 0.05). *P < 0.05, **P < 0.01.

Figure 4.

Optogenetic stimulating DA fibers in the CPu promotes sleep. (A) The expression of AAV–ChR2–mCherry and the location of the plastic optic fiber tip were confirmed after each experiment. mCherry immunostaining shows that the ChR2 proteins were expressed in cells mostly confined to the SNc but not SNr (a), and the mCherry-positive fibers were obviously seen in the CPu (b). The arrow in (b) shows the cannula tip in the CPu. (B) Total amount of wake, NREM and REM sleep in control and 20-Hz photostimulation during 9 PM. (C and D) The hourly amount of NREM sleep of baseline and 20-Hz stimulation group at dark or light phase, respectively. The blue columns indicate the photostimulation period. (E) Wake, NREM, and REM sleep stage changing of baseline, 5-, 10-, and 20-Hz photostimulation of DA fibers in the CPu during 9 PM. (F, G, and H) Sleep-wake state transitions, number and mean duration of wake, NREM, and REM bouts in baseline and 20-Hz photostimulation during 9 PM. Stimulation does not significantly change state transitions and sleep bouts, but mean duration of NREM sleep are significantly increased. (I) EEG power densities of NREM sleep of baseline and photostimulation at 5, 10, or 20 Hz. Power spectrum at 1–3 range of 20-Hz stimulation is increased over the baseline but the increase is not significant (p > 0.05). *P < 0.05, **P < 0.01.

Optogenetics Stimulation of the GPe

As optogenetic stimulation of DA terminals in the CPu, which projects to the GPe, promote sleep, we next examined sleep-wake effects of direct GPe stimulation by bilaterally injecting AAV–ChR2–mCherry and implanting fiber-optic stimulation cables into the GPe (N = 6 rats). Figure 5A, a and b show the locations of AAV injections and cannula tips, c and d show the estimated stimulation field indicated by c-Fos expression. Direct stimulation of the GPe significantly increased NREM and REM sleep time compared with that during the same time period without stimulation (Fig. 5B–D). Both NREM sleep (baseline: 17.7 ± 1.8 min; stimulation: 27.0 ± 1.7 min, P = 0.004) and REM sleep times (baseline: 3.6 ± 0.8 min; stimulation 7.5 ± 0.5 min, P = 0.004) were significantly increased by 20-Hz stimulation; while wakefulness during stimulation was significantly reduced (baseline 38.5 ± 2.3 min; stimulation: 25.4 ± 1.6 min, P = 0.004) (Fig. 5B,C,D). No rebound was seen after the stimulation (Fig. 5C). No sleep effect was seen when the GPe was stimulated during 9–10 AM (Fig. 5C). The analysis of individual sleep and wake bouts showed that the NREM sleep increase was due to an increase in the duration of NREM bouts, while the REM sleep increase was due to an increase in the number of REM sleep bouts (Fig. 5F,G). Although the EEG power spectrum during 5- and 10-Hz stimulation was similar to nonstimulation sleep (Fig. 5H), 20-Hz stimulation significantly increased EEG delta power in 0.5–3 Hz range (Fig. 5H). Importantly, we did not observe aberrant motor behaviors during GPe stimulation.

Figure 5.

GPe stimulation increase sleep. (A) “a and b” sections were immunostained with mCherry to verify the expression and injection site of the AAV. “a” shows the location of AAV–ChR2–mCherry in the bilateral GPe. “b” higher magnification of the square region indicated in “a.” The arrows indicate the cannula tip above the GPe; “c and d” sections were immunostained with c-Fos to estimate the ranges of optogenetic stimulations. The arrow in “c” indicates the cannula tip above the GPe. Dotted line indicated the boundary of c-Fos expression. “d,” higher magnification of the square region indicated in “a.” (B) Total amount of wake, NREM, and REM sleep in control and 20-Hz photostimulation during 9 PM. (C) The hourly amount of NREM sleep of baseline and 20-Hz stimulation group at dark or light phase, respectively. The blue columns indicate the photostimulation period. (D) Wake, NREM, and REM sleep stage changes of baseline, 5-, 10-, and 20-Hz photostimulation in the GPe during 9 PM. (E, F, and G): Sleep-wake state transitions, number, and mean duration of wake, NREM, and REM bouts in control and 20-Hz photostimulation during 9 PM. GPe stimulation induces more transitions from NREM sleep into REM sleep and from REM sleep to wakefulness, that is, more number of REM sleep episodes occurred, while average duration of NREM sleep is significantly increase, on the contrary, the mean duration of wake bouts is decreased. (H) EEG power densities of NREM sleep of control and photostimulation at 5, 10, or 20 Hz. There was no essential difference in EEG power density during NREM sleep between 5-, 10-Hz photostimulation, and baseline. Twenty-Hertz photostimulation significantly increases slow delta power. The horizontal bar indicates where there is a statistical difference (P < 0.05). *P < 0.05, **P < 0.01.

Figure 5.

GPe stimulation increase sleep. (A) “a and b” sections were immunostained with mCherry to verify the expression and injection site of the AAV. “a” shows the location of AAV–ChR2–mCherry in the bilateral GPe. “b” higher magnification of the square region indicated in “a.” The arrows indicate the cannula tip above the GPe; “c and d” sections were immunostained with c-Fos to estimate the ranges of optogenetic stimulations. The arrow in “c” indicates the cannula tip above the GPe. Dotted line indicated the boundary of c-Fos expression. “d,” higher magnification of the square region indicated in “a.” (B) Total amount of wake, NREM, and REM sleep in control and 20-Hz photostimulation during 9 PM. (C) The hourly amount of NREM sleep of baseline and 20-Hz stimulation group at dark or light phase, respectively. The blue columns indicate the photostimulation period. (D) Wake, NREM, and REM sleep stage changes of baseline, 5-, 10-, and 20-Hz photostimulation in the GPe during 9 PM. (E, F, and G): Sleep-wake state transitions, number, and mean duration of wake, NREM, and REM bouts in control and 20-Hz photostimulation during 9 PM. GPe stimulation induces more transitions from NREM sleep into REM sleep and from REM sleep to wakefulness, that is, more number of REM sleep episodes occurred, while average duration of NREM sleep is significantly increase, on the contrary, the mean duration of wake bouts is decreased. (H) EEG power densities of NREM sleep of control and photostimulation at 5, 10, or 20 Hz. There was no essential difference in EEG power density during NREM sleep between 5-, 10-Hz photostimulation, and baseline. Twenty-Hertz photostimulation significantly increases slow delta power. The horizontal bar indicates where there is a statistical difference (P < 0.05). *P < 0.05, **P < 0.01.

Discussion

Selective destruction of SNc DA neurons and their afferents to the CPu led to a significant 33.7% increase in wakefulness (or sleep reduction) over a 24-h period. This wake increase negatively correlated with the remaining number of DA neurons in the SNc. Loss of SNc DA neurons also caused sleep-wake fragmentation and reduced diurnal sleep-wake variation. Conversely, 1-h (9 PM–10 PM) of optogenetic stimulation of SNc DA terminals in the CPu significantly increased sleep by 69%. One hour (9 PM–10 PM) of direct optogenetic stimulation of GPe neurons increased total sleep by 66% and increased delta EEG power during NREM sleep. These results delineate a novel sleep-regulating neural circuit in the BG mediated by DA.

Two previous studies have shown that nonselective lesions of both the SNc DA and SNr GABA neurons produced significant sleep reduction in cats and rats (Lai et al. 1999; Gerashchenko et al. 2006). As these studies used nonspecific orexin–saporin and glutamate agonists, it was not possible to distinguish the neuronal population within the SN contributing to sleep loss. However, our recent study (Qiu et al. 2010) showed that lesions restricted to the SNr by ibotenic acid did not result in alterations in sleep in rats. The current SNc lesion results indicate that SNc neurons, but not SNr neurons, are likely to be involved in circadian regulation of sleep and sleep promotion. This hypothesis is supported by a recent study in which MPTP induced SNc lesions in macaques fragmented sleep and reduced total sleep by 30% with a flattened circadian rhythm (Belaid et al. 2014). Although no studies have examined the total sleep time in de novo human Parkinson's subjects, the circadian alterations in sleep-wake behavior in rats with SNc lesions are consistent with a recent study of Parkinson's disease in nonhuman primate and humans (Fifel et al. 2014; Videnovic et al. 2014).The wake increase in the PL group mostly occurs during the night while the wake increase in the CL group occurs during both day and night. It is possible that low DA levels in the PL group can sustain the stability of sleep-wake behavior during the daytime, while complete DA depletion destabilizes sleep-wake control and reduces sleep during both day and night.

Consistent with the role of SNc DA neurons in sleep, administration of low-dose l-dopa and DA D2 agonists induces sleep in rats and mice (Dimpfel 2008; Laloux, Derambure, Houdayer, et al. 2008) and produces sleep attacks in human (Paus et al. 2003; Hirayama et al. 2008), although the effects of l-Dopa and D2 agonists in mice are relatively small (Laloux, Derambure, Kreisler, et al. 2008; Burgess et al. 2010).Although the SNc neurons do not change mean firing rates during sleep-wake cycle (Miller et al. 1983), they may switch to a burst mode in wake and REM sleep (Dahan et al. 2007). The burst firing is associated with DA release in the CPu (Grace and Bunney 1984). Our optogenetic stimulation of SNc DA terminals may not generate bursting firing, nevertheless optogenetics stimulation of DA terminals in the CPu increases DA release (Bass et al. 2013). Our results suggest that increased SNc firing rates promote a sleeping state.

In vitro DA increases GPe neuronal activity via D2 receptors at presynaptic sites of GABAergic striatopallidal terminals (Cooper and Stanford 2001; Querejeta et al. 2001). Both in vivo experimental depletion of striatal DA in animal models and DA loss in human cases of Parkinson's disease reduce GPe firing activity (Filion and Tremblay 1991; Hutchison et al. 1994). Interestingly, mice with selective lesions of cholinergic neurons in the striatum, reducing striatopallidal neuronal activity, show more wakefulness (Guzman et al. 2013). It has been long thought that the sleep effects of low-dose D2 agonists are due to D2 receptor mediated inhibition of the DA neurons in the VTA and SNc. The resulting reduction of DA, however, is hard to reconcile with the use of the same doses of D2 agonists that treat Parkinsonian symptoms and induce sleep attacks. On the other hand, clinical “low” doses of D2 agonists and l-Dopa are still likely much higher than the normal physiological range of DA levels.

Our alternative hypothesis is that DA and D2 agonists directly promote sleep by indirectly increasing GPe activity, which promotes sleep. This hypothesis is supported by the fact that optogenetic stimulation of both SNc DA terminals in the CPu and GPe neurons enhances sleep, while CPu DA depletion using 6-OHDA reduces sleep. Despite the relatively focal region of optogenetic stimulation (0.5 mm radius) within the CPu and GPe, the sleep effects were robust, indicating that the DA and GPe strongly modulate sleep. In comparison, neither CPu nor GPe optogenetic stimulation results in abnormal gross motor behavior, although more specific testing is required to determine if fine motor control is affected. Of DA receptor agonists, D2 receptor agonists are most effect in treating Parkinson's disease, suggesting that D2 receptors in the striatum play more important role than D1 receptors in BG circuits.

How the GPe regulates sleep is an open question. We propose that the GABAergic GPe promotes sleep by inhibiting prefrontal cortical regions (Fig. 6), which in turn regulate the subcortical arousal centers like the medial parabrachial nucleus. The GABAergic GPe could inhibit the cortex via 3 potential routes—1) direct projections to the cortex (Saper 1984; Hur and Zaborszky 2005; Vetrivelan et al. 2010), 2) indirect projections through a relay in the thalamus (reticular, dorsomedial, and intralaminar thalamic nuclei), 3) indirect projections through additional relays in the GPi/SNr that project to the thalamus. Elevated cortical pyramidal neuronal activity has also been reported with loss of striatal DA both in vitro and in vivo (Wang et al. 2009; Zhang et al. 2010; Maillet et al. 2012). Notably, extensive thalamic lesions do not affect sleep-wake and motor behavior in rats and cats (Pritzel and Markowitsch 1980; Fuller et al. 2011), highlighting the possible role of extra-thalamocortical pathways (Fig. 6, dark lines). As the thalamus is the major relay for the direct pathway (CPu-SNr/GPi-thalamus-cortex), the role of CPu D1 receptors in sleep need to be evaluated by other approaches such as optogenetics. We propose a novel direct pallidocortical projection as the key circuit for BG control of sleep.

Figure 6.

Putative neural circuitry for SNc DA regulation of sleep. SNc DA acting on D2 receptors on striatopallidal neurons lead to disinhibition of GPe neurons, which inhibits the cerebral cortex via their direct and/or indirect projections. Arrowheads represent the excitatory inputs and circles represent the inhibitory inputs. The light lines indicate the primary proposed pathways by which SNc dopamine and basal ganglia regulate sleep, and dark lines represent the submissive pathways that regulate sleep by SNc dopamine and basal ganglia.

Figure 6.

Putative neural circuitry for SNc DA regulation of sleep. SNc DA acting on D2 receptors on striatopallidal neurons lead to disinhibition of GPe neurons, which inhibits the cerebral cortex via their direct and/or indirect projections. Arrowheads represent the excitatory inputs and circles represent the inhibitory inputs. The light lines indicate the primary proposed pathways by which SNc dopamine and basal ganglia regulate sleep, and dark lines represent the submissive pathways that regulate sleep by SNc dopamine and basal ganglia.

In contrast to dorsal striatum lesions, ventral striatal lesions (i.e., nucleus of accumbens, NAc) increase wakefulness and block the arousal effects of the stimulant modafinil (Qiu et al. 2010, 2012). Genetic deletions of adenosine A2A receptors from the NAc block the arousal effects of caffeine (Lazarus et al. 2011), suggesting that the NAc mediates sleep and wake effects of modafinil and caffeine. Given that D2 receptors and adenosine A2A receptors are co-localized, it is possible that D2 receptors in the NAc promote arousal. The NAc receives DA inputs mostly from the VTA and moderately from the ventral periaqueductal gray matter (vPAG) neurons (Hasue and Shammah-Lagnado 2002). The VTA receives orexin inputs (Hrabovszky et al. 2013), and the orexin–VTA pathway and direct orexin inputs to the NAc may underlie the role of orexin in reward and addiction as well as arousal (Mukai et al. 2009; Muschamp et al. 2014; Zarepour et al. 2014). The neural circuit underlying the NAc control of sleep-wake behavior is not known, but it is clear that the GPe is not involved as the NAc has no connection with the GPe.

Unlike the VTA and SNc, vPAG wake-active DA has strong projections to the basal forebrain, thalamus and hypothalamus, where DA and its receptors of D1 and D2 may directly promote arousal (Lu et al. 2006; Lazarus et al. 2012), which may underly the sleep increase in global D2 knockout mice (Qu et al. 2010). Elucidation of these DA circuits will be critical for understanding complexity of DA in sleep control and the mechanisms of abnormal sleep seen in BG disorders such as Parkinson's disease, Huntington's disease, and obsessive-compulsive disorder (Morton 2013; Paterson et al. 2013).

Funding

This work was supported by the National Institutes of Health (NS06184, NS062727), National Natural Science Foundation of China (31171049), Shanghai Committee of Science and Technology (11ZR1401800), and by the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry.

Notes

Conflict of Interest: None declared.

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