. CP47 produced a large decrease in bout duration across all time points of the LP (CP47 vs vehicle, ZT00-12: t(182.30) -2.69, p 0.025) that was blocked by co-administration of AM281 (CP47+AM281 vs CP47, ZT00-09:: t(184.77) ! 3.30, p 0.003). CP47+AM281 did not change NREM bout duration relative to vehicle. The number of NREM bouts was affected by a significant overall interaction (treatment x time of day within photoperiod, F(18, 192.19) = 5.20, p < 0.001) and main effects of both photoperiod (F(1, 155.99) = 11.18, p = 0.001) and drug treatment (F(2, 54.76) = 4.79, p = 0.012). Across the entire recording day, CP47 produced an increase in the number of NREM bouts (t(48.65) = 2.98, p = 0.013), but the only specific time point with significant differences between treatments was the first quarter of the DP where both CP47 (t(176.71) = 4.58, p < 0.001) and CP47+AM281 (t(178.81) = -4.57, p < 0.001) produced a significant increase in NREM bouts. As discussed later, AM281 alone increases the number NREM bouts during the first quarter of the DP, so the increased number of NREM bouts is confounded by AM281's effect. Thus, CP47's effects on sleep architecture are largely mediated through the CB1 receptor.Inhibition of Monoacylglycerol Lipase Stabilizes NREM and Suppresses REMSleep Measurements. Considering that activation of CB1 receptors with exogenous ligands can facilitate sleep, we next sought to test the hypothesis that eCBs could similarly promote NREM sleep. Increasing endogenous 2-AG tone with JZL, a selective MAGL inhibitor, reduced phasic EMG activity and increased the amount of low-frequency high-voltage EEG activity characteristic of NREM sleep (Fig 5). To quantify vigilance states after pharmacologically increasing 2-AG levels, subjects were sequentially given a 1.6 (low), 8.0 (moderate), and 16.0 (high) mg/kg doses of JZL, after which an additional 24 Hr recording with no injection (recovery) was obtained (Fig 6A). Within-subject comparisons were made using sleep measures obtained during a 24 Hr baseline recording that followed a vehicle injection. Several reports have suggested that endocannabinoid levels fluctuate across the circadian cycle [33, 34, 42], but it is unclear how or if this may be related to sleep. Therefore, two experiments were performed with JZL in separate groups of mice. In one, JZL was administered before the DP, when mice are most active (Fig 6B), and in the other, JZL was administered prior jir.2012.0140 to the LP (Fig 6C). When given just before the onset of the DP (between fpsyg.2017.00209 ZT 11:30 and 12:00), JZL administration had obvious effects on NREM sleep that mirrored those seen with CP47 (Fig 6B; top row).PLOS ONE | DOI:10.1371/journal.pone.0152473 March 31,14 /Endocannabinoid Signaling Regulates Sleep StabilityFig 5. Example EEG/EMG Traces on Different Time Scales Following Vehicle or 6-Methoxybaicalein msds AZD0156 web JZL184 Administration. EEG and EMG traces are from the same subject at the same stage of the circadian cycle after administration of either vehicle (A, A’, and left get 6-Methoxybaicalein column of C) or 16 mg/kg JZL184 (B, B’, and right column of C). Data are from experiment with JZL administration before the LP. Panels A and B show a 2 Hr 15 min window from ZT 02:00?4:15, AG-221 supplement roughly 2 Hr after drug administration, coinciding with peak effects observed on sleep. Panels A’ and B’ show a 15 min long segment expanded from the region in A and B highlighted by the dashed orange box. Panel C shows representative 18 sec long data segments corresponding to NREM and wake obtained fo.. CP47 produced a large decrease in bout duration across all time points of the LP (CP47 vs vehicle, ZT00-12: t(182.30) -2.69, p 0.025) that was blocked by co-administration of AM281 (CP47+AM281 vs CP47, ZT00-09:: t(184.77) ! 3.30, p 0.003). CP47+AM281 did not change NREM bout duration relative to vehicle. The number of NREM bouts was affected by a significant overall interaction (treatment x time of day within photoperiod, F(18, 192.19) = 5.20, p < 0.001) and main effects of both photoperiod (F(1, 155.99) = 11.18, p = 0.001) and drug treatment (F(2, 54.76) = 4.79, p = 0.012). Across the entire recording day, CP47 produced an increase in the number of NREM bouts (t(48.65) = 2.98, p = 0.013), but the only specific time point with significant differences between treatments was the first quarter of the DP where both CP47 (t(176.71) = 4.58, p < 0.001) and CP47+AM281 (t(178.81) = -4.57, p < 0.001) produced a significant increase in NREM bouts. As discussed later, AM281 alone increases the number NREM bouts during the first quarter of the DP, so the increased number of NREM bouts is confounded by AM281's effect. Thus, CP47's effects on sleep architecture are largely mediated through the CB1 receptor.Inhibition of Monoacylglycerol Lipase Stabilizes NREM and Suppresses REMSleep Measurements. Considering that activation of CB1 receptors with exogenous ligands can facilitate sleep, we next sought to test the hypothesis that eCBs could similarly promote NREM sleep. Increasing endogenous 2-AG tone with JZL, a selective MAGL inhibitor, reduced phasic EMG activity and increased the amount of low-frequency high-voltage EEG activity characteristic of NREM sleep (Fig 5). To quantify vigilance states after pharmacologically increasing 2-AG levels, subjects were sequentially given a 1.6 (low), 8.0 (moderate), and 16.0 (high) mg/kg doses of JZL, after which an additional 24 Hr recording with no injection (recovery) was obtained (Fig 6A). Within-subject comparisons were made using sleep measures obtained during a 24 Hr baseline recording that followed a vehicle injection. Several reports have suggested that endocannabinoid levels fluctuate across the circadian cycle [33, 34, 42], but it is unclear how or if this may be related to sleep. Therefore, two experiments were performed with JZL in separate groups of mice. In one, JZL was administered before the DP, when mice are most active (Fig 6B), and in the other, JZL was administered prior jir.2012.0140 to the LP (Fig 6C). When given just before the onset of the DP (between fpsyg.2017.00209 ZT 11:30 and 12:00), JZL administration had obvious effects on NREM sleep that mirrored those seen with CP47 (Fig 6B; top row).PLOS ONE | DOI:10.1371/journal.pone.0152473 March 31,14 /Endocannabinoid Signaling Regulates Sleep StabilityFig 5. Example EEG/EMG Traces on Different Time Scales Following Vehicle or JZL184 Administration. EEG and EMG traces are from the same subject at the same stage of the circadian cycle after administration of either vehicle (A, A’, and left column of C) or 16 mg/kg JZL184 (B, B’, and right column of C). Data are from experiment with JZL administration before the LP. Panels A and B show a 2 Hr 15 min window from ZT 02:00?4:15, roughly 2 Hr after drug administration, coinciding with peak effects observed on sleep. Panels A’ and B’ show a 15 min long segment expanded from the region in A and B highlighted by the dashed orange box. Panel C shows representative 18 sec long data segments corresponding to NREM and wake obtained fo.. CP47 produced a large decrease in bout duration across all time points of the LP (CP47 vs vehicle, ZT00-12: t(182.30) -2.69, p 0.025) that was blocked by co-administration of AM281 (CP47+AM281 vs CP47, ZT00-09:: t(184.77) ! 3.30, p 0.003). CP47+AM281 did not change NREM bout duration relative to vehicle. The number of NREM bouts was affected by a significant overall interaction (treatment x time of day within photoperiod, F(18, 192.19) = 5.20, p < 0.001) and main effects of both photoperiod (F(1, 155.99) = 11.18, p = 0.001) and drug treatment (F(2, 54.76) = 4.79, p = 0.012). Across the entire recording day, CP47 produced an increase in the number of NREM bouts (t(48.65) = 2.98, p = 0.013), but the only specific time point with significant differences between treatments was the first quarter of the DP where both CP47 (t(176.71) = 4.58, p < 0.001) and CP47+AM281 (t(178.81) = -4.57, p < 0.001) produced a significant increase in NREM bouts. As discussed later, AM281 alone increases the number NREM bouts during the first quarter of the DP, so the increased number of NREM bouts is confounded by AM281's effect. Thus, CP47's effects on sleep architecture are largely mediated through the CB1 receptor.Inhibition of Monoacylglycerol Lipase Stabilizes NREM and Suppresses REMSleep Measurements. Considering that activation of CB1 receptors with exogenous ligands can facilitate sleep, we next sought to test the hypothesis that eCBs could similarly promote NREM sleep. Increasing endogenous 2-AG tone with JZL, a selective MAGL inhibitor, reduced phasic EMG activity and increased the amount of low-frequency high-voltage EEG activity characteristic of NREM sleep (Fig 5). To quantify vigilance states after pharmacologically increasing 2-AG levels, subjects were sequentially given a 1.6 (low), 8.0 (moderate), and 16.0 (high) mg/kg doses of JZL, after which an additional 24 Hr recording with no injection (recovery) was obtained (Fig 6A). Within-subject comparisons were made using sleep measures obtained during a 24 Hr baseline recording that followed a vehicle injection. Several reports have suggested that endocannabinoid levels fluctuate across the circadian cycle [33, 34, 42], but it is unclear how or if this may be related to sleep. Therefore, two experiments were performed with JZL in separate groups of mice. In one, JZL was administered before the DP, when mice are most active (Fig 6B), and in the other, JZL was administered prior jir.2012.0140 to the LP (Fig 6C). When given just before the onset of the DP (between fpsyg.2017.00209 ZT 11:30 and 12:00), JZL administration had obvious effects on NREM sleep that mirrored those seen with CP47 (Fig 6B; top row).PLOS ONE | DOI:10.1371/journal.pone.0152473 March 31,14 /Endocannabinoid Signaling Regulates Sleep StabilityFig 5. Example EEG/EMG Traces on Different Time Scales Following Vehicle or JZL184 Administration. EEG and EMG traces are from the same subject at the same stage of the circadian cycle after administration of either vehicle (A, A’, and left column of C) or 16 mg/kg JZL184 (B, B’, and right column of C). Data are from experiment with JZL administration before the LP. Panels A and B show a 2 Hr 15 min window from ZT 02:00?4:15, roughly 2 Hr after drug administration, coinciding with peak effects observed on sleep. Panels A’ and B’ show a 15 min long segment expanded from the region in A and B highlighted by the dashed orange box. Panel C shows representative 18 sec long data segments corresponding to NREM and wake obtained fo.. CP47 produced a large decrease in bout duration across all time points of the LP (CP47 vs vehicle, ZT00-12: t(182.30) -2.69, p 0.025) that was blocked by co-administration of AM281 (CP47+AM281 vs CP47, ZT00-09:: t(184.77) ! 3.30, p 0.003). CP47+AM281 did not change NREM bout duration relative to vehicle. The number of NREM bouts was affected by a significant overall interaction (treatment x time of day within photoperiod, F(18, 192.19) = 5.20, p < 0.001) and main effects of both photoperiod (F(1, 155.99) = 11.18, p = 0.001) and drug treatment (F(2, 54.76) = 4.79, p = 0.012). Across the entire recording day, CP47 produced an increase in the number of NREM bouts (t(48.65) = 2.98, p = 0.013), but the only specific time point with significant differences between treatments was the first quarter of the DP where both CP47 (t(176.71) = 4.58, p < 0.001) and CP47+AM281 (t(178.81) = -4.57, p < 0.001) produced a significant increase in NREM bouts. As discussed later, AM281 alone increases the number NREM bouts during the first quarter of the DP, so the increased number of NREM bouts is confounded by AM281's effect. Thus, CP47's effects on sleep architecture are largely mediated through the CB1 receptor.Inhibition of Monoacylglycerol Lipase Stabilizes NREM and Suppresses REMSleep Measurements. Considering that activation of CB1 receptors with exogenous ligands can facilitate sleep, we next sought to test the hypothesis that eCBs could similarly promote NREM sleep. Increasing endogenous 2-AG tone with JZL, a selective MAGL inhibitor, reduced phasic EMG activity and increased the amount of low-frequency high-voltage EEG activity characteristic of NREM sleep (Fig 5). To quantify vigilance states after pharmacologically increasing 2-AG levels, subjects were sequentially given a 1.6 (low), 8.0 (moderate), and 16.0 (high) mg/kg doses of JZL, after which an additional 24 Hr recording with no injection (recovery) was obtained (Fig 6A). Within-subject comparisons were made using sleep measures obtained during a 24 Hr baseline recording that followed a vehicle injection. Several reports have suggested that endocannabinoid levels fluctuate across the circadian cycle [33, 34, 42], but it is unclear how or if this may be related to sleep. Therefore, two experiments were performed with JZL in separate groups of mice. In one, JZL was administered before the DP, when mice are most active (Fig 6B), and in the other, JZL was administered prior jir.2012.0140 to the LP (Fig 6C). When given just before the onset of the DP (between fpsyg.2017.00209 ZT 11:30 and 12:00), JZL administration had obvious effects on NREM sleep that mirrored those seen with CP47 (Fig 6B; top row).PLOS ONE | DOI:10.1371/journal.pone.0152473 March 31,14 /Endocannabinoid Signaling Regulates Sleep StabilityFig 5. Example EEG/EMG Traces on Different Time Scales Following Vehicle or JZL184 Administration. EEG and EMG traces are from the same subject at the same stage of the circadian cycle after administration of either vehicle (A, A’, and left column of C) or 16 mg/kg JZL184 (B, B’, and right column of C). Data are from experiment with JZL administration before the LP. Panels A and B show a 2 Hr 15 min window from ZT 02:00?4:15, roughly 2 Hr after drug administration, coinciding with peak effects observed on sleep. Panels A’ and B’ show a 15 min long segment expanded from the region in A and B highlighted by the dashed orange box. Panel C shows representative 18 sec long data segments corresponding to NREM and wake obtained fo.
