Results
Chemogenetic stimulation of the PVH increases wakefulness
Rats (N=8) with AAV10-hSyn-DIO-hM3Dq-mCherry (AAV-M3-mCherry) injections
in bilateral PVH showed a normal pattern of sleep-wake behavior
following saline injection at 8:00 (Fig. 1B). However, following CNO
(0.2 mg/kg) administration at 8:00 that stimulated the PVH neurons, the
same rats showed long bouts of wake, with NREM sleep latency
(F[2,23]=19.74, P<0.001) significantly
lengthened (CNO= 95.2±5.0min; saline= 23.4±2.8min) (Fig.1B, D, E).
Sleep-wake transition analysis (Fig. 1D) showed that CNO decreased the
number of state transitions from NREM to REM sleep, REM sleep to wake
and from wake to NREM. Wake, NREM, REM bouts number were significantly
decreased and wake duration increased over 6 hours (Fig.1E). The mean
number of REM bouts was significantly decreased from 20.3±1.8 to 6.4±2.0
by CNO (p< 0.001), and wake duration increased from 60.3±4.9
to 163.1±28.8 s (p< 0.001) (Fig.1E). REM and NREM sleep
duration were not significantly influenced (Fig.1E).
Sleep and wake amount analysis revealed a significant increase in hourly
waking amounts by CNO
(Fig. 2),
lasting about 6 hours by CNO at 8:00
(Fig. 2A)
and 4 hours by CNO at 20:00
(Fig. 2C).
The amount of wakefulness during 6, 12 hours by CNO at 8:00 or 20:00
also significantly increased
(Fig. 2B,
D).
PVH-PB pathway activation increase wakefulness
To examine if the PVH promotes wakefulness via its projections to the
PB, we injected cre dependent AAV-M3-mCherry into the PVH and AAV6-Cre
bilaterally in the PB in 4 rats (Fig. 1C). In this model system,
AAV6-cre injections resulted in cre expression in neurons that project
to the PB, and cre was in PVH neurons that were exposed to the
cre-dependent AAV-M3-mCherry, M3 was inserted in the PVH neurons that
project to the PB. Only neurons with both AAV-M3-mCherry and cre
expressed the M3 DREADD; CNO then activated the subset of PVH neurons
that project to the PB, i.e., selectively activating the PVH-PB pathway.
Activation of the PVH-PB pathway was confirmed by Fos expression in the
PVH and cre in the PB (Fig. 1C, F1-3). PVH-PB pathway activation by CNO
induced prolonged wakefulness (Fig. 1G) compared to saline injection at
8:00. CNO injection (0.2 mg/kg)
at 8:00 significantly increased wake duration, but decreased NREM and
REM duration and bouts (Fig. 1H). Consistent with this, wake and NREM
amounts were also significantly different between the CNO and saline
groups (Fig. 3) when CNO was injected at 8:00 or 20:00. These results
indicate that the PVH-PB pathway promotes arousal during active and
inactive period.
RCA activation increases wakefulness
Like the PVH, the RCA also receives input from the SCN and projects to
the PB; the PVH together with the RCA may relay SCN signals to the PB
for control of arousal. We next examined the role of the RCA in arousal
by injecting AAV-M3 into the RCA bilaterally. We had 8 rats with
AAV-M3-mCherry in bilateral RCA without the SCN expression of mCherry
(Fig. 4A). Compared to saline injection, CNO injections in these animals
at 8:00 significantly increased wakefulness and reduced NREM sleep and
REM sleep in first three hours post CNO injections (Fig. 5A). Wake
increase by CNO at 20:00 was only seen in the first hour post CNO
injection (Fig. 5C). No sleep rebound was seen after the CNO arousal
effects at either time. The amount of wakefulness during the first 3 and
12 hours by CNO at 8:00 significantly increased
(Fig. 5B),
but the amount of wakefulness only increase 3 hours at 20:00 (Fig. 5D).
Consistent with these results, CNO injection increased wake duration or
NREM sleep latency (F[2,23]=14.46,
P<0.001, saline=15.5±2.0min , CNO= 66.2±6.0min.), and
decreased number of bouts and stage transition of wake, NREM and REM
sleep (Fig.4C-D).
SCN activation increases wakefulness
We injected 18 nl AAV-M3-mCherry bilaterally in more than 20 rats, but
only 5 rats were successfully expressed in the SCN (Fig. 4E). The
location and size of the SCN made it difficult to achieve relatively
confined and complete bilateral injections of AAV-M3. As a result, we
only obtained 5 rats with unilateral transfection in the SCN. Despite
the unilateral nature of the expression in these animals, CNO injection
at 8:00 but not at 20:00 significantly increased wake amounts during the
first three hours compared to saline injection (Fig. 6A,C) . CNO
significantly increased the NREM sleep latency (p< 0.01,
saline=16.2±2.7 min, CNO=60.1±17.7 min, Fig. 4F,G). As compared with
saline-injected controls, CNO markedly increased the amount of wake,
decreased the amount of NREM sleep and REM sleep during light condition
(Fig. 6B). There was no disruption of the sleep architecture during the
subsequent period (Fig. 6A). Of the rats where injections missed the SCN
completely as the sham control, AAV-M3-mCherry was often expressed in
the third ventricle; CNO also did not affect sleep in this group (n=5),
similar to saline-injected controls.
Dual immunostaining for Fos, a marker of neuronal activation, and
mCherry showed transfected neurons and Fos in the SCN and Fos in the PVH
and RCA ipsilateral to SCN activation (compared to the missed injection
side) (Fig. 7). SCN activation significantly increased expression of Fos
in the SCN, RCA and PVH (Fig. 7C) compared to the sham group (Fig.
7A,B). We obtained a few unilateral injection cases, which showed that
the one side SCN activates other side SCN (Fig.7.D-F); the PVH
stimulation did not affect the SCN or RCA Fos expression (Fig.7. G-I);
ipsilateral RCA activation potently stimulates both side SCN (Fig. 7
J-L).