Erved 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 jasp.12117 to NREM and Wake obtained following vehicle and CP47 administration. These data segments were taken from the segments shown in A and B. The color-coded hypnogram shown at the bottom of A, B, A’, and B’ represents consecutive 2 sec epochs shown as wake (red), NREM (blue), unclassified (grey). No REM purchase ��-Amatoxin occurred during this period. Black traces depict EEG, red traces depict EMG. A and B are AC220 molecular weight identically scaled. A’ and B’ are identically scaled. All traces in C are identically scaled. doi:10.1371/journal.pone.0152473.gCP47+AM281; Fig 4D). As an internal positive control, the experiment was continued for a third day when CP47 (1 mg/kg) was administered alone. There was an overall interaction for NREM sleep time (Fig 4E; treatment x time of day within photoperiod, F(18, 198.22) = 11.31, p < 0.001), a secondary interaction (treatment x photoperiod, F(2, 133.28) = 43.47, p < 0.001), and a main effect of photoperiod (F(1, 160.97) = 364.21, p < 0.001). While CP47 alone increased sleep time during the DP (ZT12-15 ZT15-18: t(263.34) ! 6.43, p < 0.001) and reduced it during the LP (ZT00-03, 03?6, and 06?9: t(263.34) -3.12, p 0.006), co-administering AM281 significantly attenuated the CP47 effects during the DP (AM281+CP47 vs CP47, ZT12-15, 15?8, and 18?1: t(263.34) -2.94, p 0.011) and reversed them completely during the LP (ZT00-03 and 03?6: t(263.34) ! 2.57, p 0.033). Compared to vehicle, CP47 +AM281 had mixed effects on NREM in the DP (ZT12-15 (increased): t(263.34) = 4.14, p < 0.001; ZT18-21(decreased): t(263.34) = -2.79, p = 0.017), but there were no differencesPLOS ONE | DOI:10.1371/journal.pone.0152473 March 31,12 /Endocannabinoid Signaling Regulates Sleep StabilityPLOS ONE | DOI:10.1371/journal.pone.0152473 March 31,13 /Endocannabinoid Signaling Regulates Sleep StabilityFig 4. Direct Activation of CB1 with the Full fpsyg.2017.00209 Agonist, CP47,497, Has Biphasic Effects on NREM Sleep that are Mediated by the CB1 Receptor. A, Diagram of experimental protocol for recording sleep after administration of the CB1 agonist, CP47,497. All injections given at the onset of the dark photoperiod (ZT 12:00). B, Quantification of NREM sleep time and architecture (N = 9). C, Quantification of REM sleep time and architecture. D, Diagram of experimental protocol for recording sleep after co-administering CP47 with AM281. E, Quantification of NREM sleep in experiments with co-administration of CP47 and AM281. In all graphs, the grey shaded region denotes the dark photoperiod. Symbols represent mean EM for 3 Hr time bins. Asterisks denote significant difference from vehicle baseline. doi:10.1371/journal.pone.0152473.gduring the LP. These results replicated our previous findings on CP47’s biphasic effect on sleep, and co-administration of AM281 blocked this effect suggesting that CP47’s effects on NREM sleep are mediated through CB1. The CP47-induced changes in sleep architecture were similarly blunted by co-administration of AM281. For NREM bout duration, there was a significant overall interaction (treatment x time of day within photoperiod, F(18, 202.64) = 5.487, p < 0.001), secondary interaction (treatment x photoperiod, F(2, 103.25) = 13.06, p < 0.001), and main effects of both drug treatment (F(2, 61.707) = 4.376, p = 0.017) and photoperiod (F(1, 169.28) = 45.05, p < 0.001).Erved 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 jasp.12117 to NREM and Wake obtained following vehicle and CP47 administration. These data segments were taken from the segments shown in A and B. The color-coded hypnogram shown at the bottom of A, B, A’, and B’ represents consecutive 2 sec epochs shown as wake (red), NREM (blue), unclassified (grey). No REM occurred during this period. Black traces depict EEG, red traces depict EMG. A and B are identically scaled. A’ and B’ are identically scaled. All traces in C are identically scaled. doi:10.1371/journal.pone.0152473.gCP47+AM281; Fig 4D). As an internal positive control, the experiment was continued for a third day when CP47 (1 mg/kg) was administered alone. There was an overall interaction for NREM sleep time (Fig 4E; treatment x time of day within photoperiod, F(18, 198.22) = 11.31, p < 0.001), a secondary interaction (treatment x photoperiod, F(2, 133.28) = 43.47, p < 0.001), and a main effect of photoperiod (F(1, 160.97) = 364.21, p < 0.001). While CP47 alone increased sleep time during the DP (ZT12-15 ZT15-18: t(263.34) ! 6.43, p < 0.001) and reduced it during the LP (ZT00-03, 03?6, and 06?9: t(263.34) -3.12, p 0.006), co-administering AM281 significantly attenuated the CP47 effects during the DP (AM281+CP47 vs CP47, ZT12-15, 15?8, and 18?1: t(263.34) -2.94, p 0.011) and reversed them completely during the LP (ZT00-03 and 03?6: t(263.34) ! 2.57, p 0.033). Compared to vehicle, CP47 +AM281 had mixed effects on NREM in the DP (ZT12-15 (increased): t(263.34) = 4.14, p < 0.001; ZT18-21(decreased): t(263.34) = -2.79, p = 0.017), but there were no differencesPLOS ONE | DOI:10.1371/journal.pone.0152473 March 31,12 /Endocannabinoid Signaling Regulates Sleep StabilityPLOS ONE | DOI:10.1371/journal.pone.0152473 March 31,13 /Endocannabinoid Signaling Regulates Sleep StabilityFig 4. Direct Activation of CB1 with the Full fpsyg.2017.00209 Agonist, CP47,497, Has Biphasic Effects on NREM Sleep that are Mediated by the CB1 Receptor. A, Diagram of experimental protocol for recording sleep after administration of the CB1 agonist, CP47,497. All injections given at the onset of the dark photoperiod (ZT 12:00). B, Quantification of NREM sleep time and architecture (N = 9). C, Quantification of REM sleep time and architecture. D, Diagram of experimental protocol for recording sleep after co-administering CP47 with AM281. E, Quantification of NREM sleep in experiments with co-administration of CP47 and AM281. In all graphs, the grey shaded region denotes the dark photoperiod. Symbols represent mean EM for 3 Hr time bins. Asterisks denote significant difference from vehicle baseline. doi:10.1371/journal.pone.0152473.gduring the LP. These results replicated our previous findings on CP47’s biphasic effect on sleep, and co-administration of AM281 blocked this effect suggesting that CP47’s effects on NREM sleep are mediated through CB1. The CP47-induced changes in sleep architecture were similarly blunted by co-administration of AM281. For NREM bout duration, there was a significant overall interaction (treatment x time of day within photoperiod, F(18, 202.64) = 5.487, p < 0.001), secondary interaction (treatment x photoperiod, F(2, 103.25) = 13.06, p < 0.001), and main effects of both drug treatment (F(2, 61.707) = 4.376, p = 0.017) and photoperiod (F(1, 169.28) = 45.05, p < 0.001).