Materials and Methods
Animals
Animal protocols were approved by Beth Israel Deaconess Medical Center Animal Care and Use committees. Male Sprague Dawley rats (300-325 g; Harlan Sprague Dawley, Indianapolis, IN) were housed under controlled conditions (LD=12:12, light on = 07:00 AM, 100 lux) in an isolated ventilated chamber maintained at 20–22oC, with ad libitum food and water.
Surgery
AAV injection and implantation for polysomnographic recording in rats were performed as described previously (Anaclet et al. , 2012; Anaclet et al. , 2014). Under deep ketamine/xylazine anesthesia, 18–60 nl of AAV10-hSyn-DIO-hM3Dq-mCherry (AAV-M3-mCherry) virus were injected into the PVH, RCA or SCN using glass micropipettes and a compressed air delivery system described previously (Anaclet et al. , 2014; Anaclet et al. , 2015; Gompf et al. , 2015; Chenet al. , 2016). Glass pipettes had small tip diameters (<20 mm) and did not cause visible tissue damage. Sham control rats were injected with 15 nl of saline. The coordinates for the injections were as follows: PVH [AP= −1.8 mm, ML= ± 0.4 mm, DV=−6.6 mm], RCA [AP=−0.8 mm, ML= ± 0.2 mm, DV= −8.3 mm] and SCN [AP= −1.0 mm, ML= ± 0.2 mm, DV= −8.3 mm]. The pathway experiment animals received AAV6-cre (Children Hospital, Boston) injection into the PB [AP= −9.0 mm, ML= ±2.0 mm, DV= −5.1 mm]. AAV6-cre has a unique property of retrograde transporting. In our case, cre is expected to be in the sites projecting to the PB. In the same surgery, cre dependent AAV-hM3Dq-mCherry virus was also injected into the PVH using the coordinates above. This approach selectively inserts M3 receptors in the PVH neurons projecting to the PB. All rats were implanted with EEG/EMG electrodes as described previously (Anaclet et al. , 2012; Anacletet al. , 2014).
Sleep–wake recording and analysis
In three weeks after surgery, the rats were connected via flexible recording cables and a commutator (Plastics One) to an analog amplifier (A-M Systems) and computer, with an analog-to-digital converter card and running Vital Recorder (Kissei Pharmaceutical). Rats were habituated to the recording cable for 2–3 days before starting EEG/EMG and video recording.
We used clozapine-N-oxide (CNO) to activate G protein-coupled receptors (cholinergic M3 receptors). Rat EEG/EMG were recorded for 48 hours. On day 1, the rats were treated with saline (i.p.), and on the other day rats were treated with CNO (Sigma-Aldrich; 0.2 mg/kg in saline, i.p.) injections at 8:00 (ZT 01, light-on at 7:00) or 20:00 (ZT13).  A dim red flashlight (8 lux at 25 cm) was used during injections performed in the dark. The vigilance states were automatically identified and analyzed off-line in 10 s epochs into REM sleep, NREM sleep, and wakefulness using SLEEPSIGN for Animal (Kissei Pharmaceutical, Nagano, Japan) (Chen et al., 2015). EEG/EMG signals were amplified and filtered (0.5–30 Hz for EEG, 40–200 Hz for EMG), and were then digitized at 128 Hz and recorded. Wakefulness was considered to have desynchronized EEG and high levels of EMG activity, NREM sleep was considered to have synchronized, high-amplitude, low-frequency (0.5–4 Hz) EEG signals in the absence of motor activity; and REM sleep was considered to have pronounced theta-like (4–9 Hz) EEG activity and muscle atonia. As a final step, defined sleep–wake stages were examined visually and corrected if necessary.
The percentage of time spent in W, NREM and REM sleep, as well as the number and the average durations of the episode was summarized for each group and each condition. The sleep latency is defined as the time between CNO or saline injection and the onset of NREM episode lasting >20 s.
Histology
At least one week after recordings, rats were perfused for histology and characterization of injection areas. Three hours after CNO injection rats were deeply anesthetized by chloral hydrate (500 mg/kg), perfused with 0.9% saline followed by 10% neutral buffered formalin (Sigma) through the heart. The brains were removed, and then equilibrated in PBS with 20% sucrose overnight. The brains were sectioned on a freezing microtome at 40μm into 4 series. For all c-Fos and mCherry immunohistochemical staining, visualization was based on diaminobenzidine reaction. On the first day, the sections were incubated overnight with rabbit anti-c-Fos, 1:50,000 (Oncogene) and then incubated in the secondary antibodies for 1h, followed by incubation in ABC reagents (1:1,000; Vector Laboratories) for 1h, then washed again and incubated in a 0.06% solution of 3,3-diaminobenzidinetetrahydrochloride (DAB, Sigma-Aldrich) with 0.05% CoCl2 and 0.01% NiSO4 (NH4) in PBS plus 0.02% H2O2 for 5-10 min. On the second day, the same sections were incubated overnight with anti-mCherry (1:10 000; Clontech), and follow the same protocol above mentioned. Then, the sections incubated in a 0.06% solution of DAB in PBS plus 0.02% H2O2 for 5-10 min. To delineate nuclear boundaries, sections mounted on glass slides were Nissl counterstained in 0.25% thionin in 0.2 M acetate buffer, pH 4.5, for 1 min, placed in 1% acetic acid for 1–5 min to differentiate stained structures, dehydrated in graded alcohols, cleared in xylene, and coverslipped (Chen et al., 2021). The pattern of Fos immunoreactivity was examined for SCN, PVH and RCA, bilateral counts were taken on three consecutive sections, 120 μm apart, that contained the largest nuclear areas, and these six counts were averaged.
Statistical analysis
All data were expressed as the means ± SEM. Statistical analysis was performed with SPSS 17.0 (SPSS Inc., Chicago, IL). Statistical analyses were performed with GraphPad Prism 5 (GraphPad Software, Inc., CA, USA). Time-course of the hourly amounts of each stage, histograms of sleep/wake amounts, sleep/wake stage transition number, number and duration of sleep/wake bouts were analyzed by the paired t -test, with each animal serving as its own control. For the sleep latency, total numbers of each vigilance stage were analyzed by two-way repeated measures analysis of variance (ANOVA) followed by the Fisher probable least-squares difference (PLSD) test to determine whether the difference among groups was statistically significant. The significance level was set at P<0.05 for all statistical tests.