P < 0.001). The AM281 group had reduced journal.pone.0158910 NREM stability compared to the

P < 0.001). The AM281 group had APTO-253 web reduced NREM stability compared to the vehicle group (t(76.81) = -2.67, p = 0.009), with specific differences evident at two time AZD0156 chemical information points of recovery (ZT06-09: t(256.30) = -3.63, p < 0.001; ZT00-03: t(256.30) = -2.47, p = 0.014). There were no between-group differences during the baseline recordings. Relative to their own baseline, the vehicle group had increased NREM stability during the first 3 Hr of recovery (ZT06-09: t(265.32) = 3.95, p < 0.001), but the AM281 treated mice did not show increased NREM stability following TSD. Rather, the AM281 group had decreased NREM stability during the second half of the recording (ZT18-21 00?3: t(265.32) -1.99, p 0.048). For the number of NREM bouts (Fig 12C, bottom), there was an overall interaction (group x time of day within photoperiod within experimental phase, F(24, 220.93) = 2.95, p < 0.001) and a main effect of photoperiod (F(1, 174.17) = 17.77, p < 0.001). There were no differences between groups during baseline, but the AM281 group had significantly more NREM bouts than the vehicle group during the first three hours of recovery (ZT06-09: t(169.85) = 2.16, p = 0.032). Relative to their own baseline, the vehicle group increased the number of NREM bouts during the first quarter of the DP (ZT12-15: t(177.13) = 2.20, p = 0.029). These findings show that AM281 blocks the increase in NREM stability during recovery from TSD and increases the number of NREM bouts to compensate. REM sleep was also affected by TSD with reduced REM early in recovery in both the AM281 and vehicle groups (S10 Fig). Late in the recovery, there was increase in REM sleep in the AM281 treated group due to an increase in the number of REM bouts. Consistent with our previous experiments, blockade of CB1 receptors produced a large increase in low frequency oscillations that occluded any changes due to sleep deprivation (S11 Fig). However, wake theta power did increase during the sleep deprivation period (S11 Fig, panel B). Thus, power spectral results cannot be used as an index of homeostatic drive in this experiment as they are confounded by the baseline effects of CB1 antagonism.DiscussionThe major findings of this work are that eCB signaling through the CB1 receptor is necessary and sufficient for the stability of NREM sleep. Direct activation of CB1 with CP47 or increasing eCB tone with JZL or AM3506 augmented the time spent in NREM primarily due to increased NREM bout length. This suggests that increasing eCB signaling stabilizes the fpsyg.2017.00209 NREM state. Notably these effects were biphasic, where the initial increase in sleep and NREM stability gave way to a secondary reduction and destabilization of NREM bouts suggesting that homeostatic regulation of sleep remained intact. Further support for the role of eCBs in regulating NREM stability comes from experiments with the CB1 antagonist AM281, where blockade of CB1 reduced the duration of NREM bouts. Importantly, this effect was largely offset by a concomitant increase in the number of NREM bouts resulting in minimal changes in overall sleep time. Again, the opposing effects of reduced NREM bout duration and number suggest homeostatic regulation of sleep remains intact in the absence of CB1 signaling. However, CB1 blockade consistently resulted in substantial broadband changes to EEG power spectra, particularly in low frequency bandwidths that are often used as correlates of sleep drive. To test the involvement of eCB signaling in sleep homeostat.P < 0.001). The AM281 group had reduced NREM stability compared to the vehicle group (t(76.81) = -2.67, p = 0.009), with specific differences evident at two time points of recovery (ZT06-09: t(256.30) = -3.63, p < 0.001; ZT00-03: t(256.30) = -2.47, p = 0.014). There were no between-group differences during the baseline recordings. Relative to their own baseline, the vehicle group had increased NREM stability during the first 3 Hr of recovery (ZT06-09: t(265.32) = 3.95, p < 0.001), but the AM281 treated mice did not show increased NREM stability following TSD. Rather, the AM281 group had decreased NREM stability during the second half of the recording (ZT18-21 00?3: t(265.32) -1.99, p 0.048). For the number of NREM bouts (Fig 12C, bottom), there was an overall interaction (group x time of day within photoperiod within experimental phase, F(24, 220.93) = 2.95, p < 0.001) and a main effect of photoperiod (F(1, 174.17) = 17.77, p < 0.001). There were no differences between groups during baseline, but the AM281 group had significantly more NREM bouts than the vehicle group during the first three hours of recovery (ZT06-09: t(169.85) = 2.16, p = 0.032). Relative to their own baseline, the vehicle group increased the number of NREM bouts during the first quarter of the DP (ZT12-15: t(177.13) = 2.20, p = 0.029). These findings show that AM281 blocks the increase in NREM stability during recovery from TSD and increases the number of NREM bouts to compensate. REM sleep was also affected by TSD with reduced REM early in recovery in both the AM281 and vehicle groups (S10 Fig). Late in the recovery, there was increase in REM sleep in the AM281 treated group due to an increase in the number of REM bouts. Consistent with our previous experiments, blockade of CB1 receptors produced a large increase in low frequency oscillations that occluded any changes due to sleep deprivation (S11 Fig). However, wake theta power did increase during the sleep deprivation period (S11 Fig, panel B). Thus, power spectral results cannot be used as an index of homeostatic drive in this experiment as they are confounded by the baseline effects of CB1 antagonism.DiscussionThe major findings of this work are that eCB signaling through the CB1 receptor is necessary and sufficient for the stability of NREM sleep. Direct activation of CB1 with CP47 or increasing eCB tone with JZL or AM3506 augmented the time spent in NREM primarily due to increased NREM bout length. This suggests that increasing eCB signaling stabilizes the fpsyg.2017.00209 NREM state. Notably these effects were biphasic, where the initial increase in sleep and NREM stability gave way to a secondary reduction and destabilization of NREM bouts suggesting that homeostatic regulation of sleep remained intact. Further support for the role of eCBs in regulating NREM stability comes from experiments with the CB1 antagonist AM281, where blockade of CB1 reduced the duration of NREM bouts. Importantly, this effect was largely offset by a concomitant increase in the number of NREM bouts resulting in minimal changes in overall sleep time. Again, the opposing effects of reduced NREM bout duration and number suggest homeostatic regulation of sleep remains intact in the absence of CB1 signaling. However, CB1 blockade consistently resulted in substantial broadband changes to EEG power spectra, particularly in low frequency bandwidths that are often used as correlates of sleep drive. To test the involvement of eCB signaling in sleep homeostat.