Fig 1.
SR9009 remains effective at three-hour interval administration and causes sleep rebound.
(A) Schematic of experimental paradigm to assess efficacy of the REV-ERB agonist SR9009. Mice were administered with SR9009 at ZT6, ZT9 and ZT12 consecutively in order to assess efficacy of the drug when administered at three-hour intervals. (B) SR9009 increases wakefulness when dosed at ZT6 (F(1,14) = 17.3, p<0.005, ZT7 p = 0.002, ZT8 p = 0.03) and ZT9 (F(1,14) = 4.7, p<0.05, ZT10 p = 0.005, ZT11 N.S., but not ZT12 (F(1,14) = 0.54, N.S.). SR9009 inhibits SWS at ZT6 (F(1,14) = 11.0, p<0.01, ZT7 p = 0.001, ZT8 p = 0.004); but not ZT9 (F(1,14) = 1.5, N.S.) and ZT12 (F(1,14) = 0.95, N.S.); and SR9009 inhibits REM sleep at ZT6 (F(1,14) = 16.0, p<0.005, ZT8 p = 0.0002, ZT9 p = 0.001), ZT9 (F(1,14) = 13.9, p<0.005, ZT10 p = 0.03, ZT11 p = 0.006, ZT12 p = 0.007), and ZT12 (F(1,14) = 7.7, p < 0.05, ZT13 p = 0.04, ZT15 P = 0.007). There was no rebound in wakefulness (F(1,14) = 2.7, N.S.) or REM (F(1,14) = 8.9, N.S.) during the following dark phase, but there was a significant SWS rebound (F(1,14) = 13.7, p<0.005, ZT16 p = 0.004, ZT17 p = 0.001, ZT18 p = 0.01, and ZT24 p = 0.007). (C) SR9009 significantly increased REM sleep latency (F(1,8) = 6.8, p<0.05) at ZT6 (p = 0.003) and ZT12 (p = 0.002). *P<0.05, ** P<0.01, *** P<0.005.
Fig 2.
Acute daily administration of SR9009 does not induce short-term tolerance (A) Schematic of experimental paradigm to assess short-term tolerance in response to the REV-ERB agonist SR9009. Mice were injected with SR9009 (i.p. 100mg kg-1) at ZT6 on three consecutive days in order to assess tolerance. (B) SR9009 increased wakefulness, but only reached significance on day 3 (p<0.05), decreased SWS on days 2 and 3 (p<0.05), and decreased REM on day 3 (C) Overall, SR9009 increased SWS latency (F(1,5) = 7.7, p<0.05, only significant on Day 2 p = 0.02). SR9009 increased REM sleep latency, reaching significance on all days tested (F(1,5) = 183.4, p<0.0001, Day 1 p = 0.0004, Day 2 p<0.0001, and Day 3 p<0.0001). *P<0.05, *** P<0.005.
Fig 3.
Time-Response EEG activity of SR9009 during the light phase.
(A) Schematic of experimental paradigm to assess SR9009 time-dependent behavioral activity of mice EEG. Mice were dosed with SR9009 at different time points (ZT3, ZT6, and ZT9) on separate days in order to generate a Time-Response Curve. (B) Injections with SR9009 (i.p. 100mg kg-1) induced wakefulness at ZT6 (p<0.05), but not ZT3 and ZT9, reduced SWS at ZT6 (p<0.05), but not ZT3 and ZT9, and decreased significantly REM sleep at ZT3 (p<0.05), and ZT6 (p<0.05), but not at ZT9. The maximal effect on wakefulness was achieved when the animals were dosed at ZT6 compared to ZT3 and ZT9. *P<0.05
Fig 4.
Time-Response EEG activity of SR9009 during the dark phase Mice were dosed with SR9009 at different time points (ZT15, ZT18, ZT21) on separate days in order to generate a Time-Response Curve.
Injections with SR9009 (i.p. 100mg kg-1) failed to induce wakefulness at ZT15 and ZT18. but rather decreased wakefulness at ZT21 (p<0.05). SR9009 increased SWS at ZT21 (p<0.05) and had no effect on REM at any of these time points. *P<0.05.
Fig 5.
SR9009 effect during transitions from light-to-dark and dark-to-light.
There was no effect of SR9009 on wakefulness or SWS at ZT0/24; however REM was significantly decreased at this time (p<0.05). There were trends toward increased wakefulness and decreased SWS and REM by SR9009 at ZT12 (all p’s = 0.06), but did not reach statistical significance. *P<0.05.
Fig 6.
SR9009 SWS and REM sleep latency during a 24h period.
SR9009 injections increased the latency to enter REM sleep (F(1,4) = 16.9, p<0.05, ZT6 p = 0.003, ZT15, p = 0.005) in a circadian manner. The effects on SWS were not significant in the overall ANOVA. Latencies to enter SWS and REM following the acute injections at the three-hour intervals presented in Figs 3–6 are graphed across a 24h period. ** P<0.01.