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.