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).