Endocannabinoid Signaling Regulates Sleep Stability

<div><p>The hypnogenic properties of cannabis have been recognized for centuries, but endogenous cannabinoid (endocannabinoid) regulation of vigilance states is poorly characterized. We report findings from a series of experiments in mice measuring sleep with polysomnography after various systemic pharmacological manipulations of the endocannabinoid system. Rapid, unbiased scoring of vigilance states was achieved using an automated algorithm that we devised and validated. Increasing endocannabinoid tone with a selective inhibitor of monoacyglycerol lipase (JZL184) or fatty acid amide hydrolase (AM3506) produced a transient increase in non-rapid eye movement (NREM) sleep due to an augmentation of the length of NREM bouts (NREM stability). Similarly, direct activation of type 1 cannabinoid (CB1) receptors with CP47,497 increased NREM stability, but both CP47,497 and JZL184 had a secondary effect that reduced NREM sleep time and stability. This secondary response to these drugs was similar to the early effect of CB1 blockade with the antagonist/inverse agonist AM281, which fragmented NREM sleep. The magnitude of the effects produced by JZL184 and AM281 were dependent on the time of day this drug was administered. While activation of CB1 resulted in only a slight reduction in gamma power, CB1 blockade had dramatic effects on broadband power in the EEG, particularly at low frequencies. However, CB1 blockade did not significantly reduce the rebound in NREM sleep following total sleep deprivation. These results support the hypothesis that endocannabinoid signaling through CB1 is necessary for NREM stability but it is not necessary for sleep homeostasis.</p></div

FAAH Inhibition with AM3506 Increases NREM Sleep Time and Stability while Decreasing REM Sleep.

<p><b>A,</b> Diagram of experimental protocol for recording sleep after administration of the long-lasting FAAH inhibitor AM3506. <b>B,</b> Quantification of NREM sleep time and architecture for the AM3506 experiment (<i>N</i> = 9). <b>C,</b> Quantification of REM sleep time and architecture. In all graphs, the grey shaded region denotes the dark photoperiod. Symbols represent mean±SEM for 3 Hr time bins. Asterisks denote significant difference from vehicle baseline. All injections administered at onset of dark photoperiod (ZT 12:00).</p

MAGL Inhibition with JZL184 Attenuates Gamma Frequency Oscillations During Sleep.

<p><b>A-C</b>, Average power spectra for epochs of different vigilance states across the entire DP (left hand) and LP (right hand). Solid lines denote means and shaded region around lines denotes SEM. <b>A</b>, Wake. <b>B</b>, NREM. <b>C</b>, REM. <b>D-F</b>, Change over the day in summated power in different frequency bandwidths from the power spectra: delta (left hand column), theta (middle column), and gamma (right hand column). In these graphs, results from epochs of wake are denoted in red (<b>D</b>), NREM are in blue (<b>E</b>), and REM are in green (<b>F</b>). Symbols/Bars represent mean±SEM for 3 hr time bins (<i>N</i> = 10). Grey background in graphs shows dark photoperiod. Asterisks denote significant difference from vehicle baseline. All injections administered at onset of DP (ZT 12:00).</p

Direct Activation of CB1 with the Full Agonist, CP47,497, Has Biphasic Effects on NREM Sleep that are Mediated by the CB1 Receptor.

<p><b>A,</b> 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>B,</b> Quantification of NREM sleep time and architecture (<i>N</i> = 9). <b>C,</b> Quantification of REM sleep time and architecture. <b>D,</b> Diagram of experimental protocol for recording sleep after co-administering CP47 with AM281. <b>E,</b> 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±SEM for 3 Hr time bins. Asterisks denote significant difference from vehicle baseline.</p

Differential Effects of Increasing 2-AG Tone on NREM and REM Sleep Based on Circadian Timing of Drug Administration.

<p><b>A,</b> Diagram of experimental protocol for recording sleep after administration of the MAGL inhibitor, JZL184. <b>B,</b> Quantification of NREM (top row) and REM (bottom row) sleep time and architecture for experiment where JZL was administered prior to the DP (<i>N</i> = 10). <b>C</b>, Quantification of NREM (top row) and REM (bottom row) sleep time and architecture for experiment where JZL was administered prior to the LP (<i>N</i> = 8). In all graphs, the grey shaded region denotes the dark photoperiod. Symbols represent mean±SEM for 3 Hr time bins. Asterisks denote significant difference from vehicle baseline.</p

Endocannabinoid Signaling Is Necessary for the Rebound in NREM Duration Following Sleep Deprivation, but Blockade of CB1 Has only a Weak Effect on the Rebound in Total Sleep Time.

<p><b>A</b>, Schematic overview of sleep deprivation experimental paradigm. <b>B</b>, Time course of NREM sleep time across all days of sleep deprivation experiment. Horizontal red bar on the <i>Sleep Dep</i> day indicates when sleep deprivation took place. Vertical dotted lines denote boundaries between experimental phases. Blue symbols/lines show AM281 group (<i>N</i> = 9) while the vehicle group (<i>N</i> = 11) is depicted in black. Downward facing arrows () indicate time of drug administration. <b>C</b>, Comparisons of total NREM sleep time (top), NREM bout duration (middle), and the number of NREM bouts (bottom) on the first baseline day (vehicle administration all groups) and first day of recovery (vehicle or AM281 administration). Asterisks (*) denote significant pair-wise comparisons within-groups between drug conditions and measures obtained during vehicle baseline. Daggers (<b>†</b>) denote significant pair-wise comparisons between groups on the recovery day. In <b>B</b> & <b>C</b>, Grey shaded regions indicate the DP, and symbols/bars represent means±SEM across all subjects for each 3 Hr time bin.</p

Example EEG/EMG Traces on Different Time Scales Following Vehicle or CP47,497 Administration.

<p>EEG and EMG traces are from the same subject at the same stage of the circadian cycle after administration of either vehicle (<b>A</b>, <b>A’</b>, and left column of <b>C</b>) or 1 mg/kg CP47,497 (<b>B</b>, <b>B’</b>, and right column of <b>C</b>). Panels <b>A</b> and <b>B</b> show a 2 Hr 15 min window from ZT 14:00–16:15, roughly 2 Hr after drug administration, coinciding with peak effects observed on sleep. Panels <b>A’</b> and <b>B’</b> show a 15 min long segment expanded from the region in <b>A</b> and <b>B</b> highlighted by the dashed orange box. Panel <b>C</b> shows representative 18 sec long data segments corresponding to NREM and Wake obtained following vehicle and CP47 administration. These data segments were taken from the segments shown in <b>A</b> and <b>B</b>. The color-coded hypnogram shown at the bottom of <b>A</b>, <b>B</b>, <b>A’</b>, and <b>B’</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. <b>A</b> and <b>B</b> are identically scaled. <b>A’</b> and <b>B’</b> are identically scaled. All traces in <b>C</b> are identically scaled.</p

Example EEG/EMG Traces on Different Time Scales Following Vehicle or JZL184 Administration.

<p>EEG and EMG traces are from the same subject at the same stage of the circadian cycle after administration of either vehicle (<b>A</b>, <b>A’</b>, and left column of <b>C</b>) or 16 mg/kg JZL184 (<b>B</b>, <b>B’</b>, and right column of <b>C</b>). Data are from experiment with JZL administration before the LP. Panels <b>A</b> and <b>B</b> show a 2 Hr 15 min window from ZT 02:00–04:15, roughly 2 Hr after drug administration, coinciding with peak effects observed on sleep. Panels <b>A’</b> and <b>B’</b> show a 15 min long segment expanded from the region in <b>A</b> and <b>B</b> highlighted by the dashed orange box. Panel <b>C</b> shows representative 18 sec long data segments corresponding to NREM and wake obtained following vehicle and JZL administration. These data segments were taken from the segments shown in <b>A</b> and <b>B</b>. The color-coded hypnogram shown at the bottom of <b>A</b>, <b>B</b>, <b>A’</b>, and <b>B’</b> represents consecutive 2 sec epochs shown as wake (red), NREM (blue), REM (green), and unclassified (grey). Note the loss of REM sleep following JZL administration. Black traces depict EEG, red traces depict EMG. <b>A</b> and <b>B</b> are identically scaled. <b>A’</b> and <b>B’</b> are identically scaled. All traces in <b>C</b> are identically scaled.</p

Example Results of State-Scoring.

<p><b>A</b>, Example of scored state-space with color-coded state clusters. <b>B</b>, Example of power spectra derived from the scored epochs. Power spectra are color-coded according to the state they were derived from. Solid lines indicate borders of 95% confidence interval of power spectra for all epochs of associated state across the day. <b>C</b>, Distribution of time spent in each classification criteria over the day. <b>D</b>, Pattern of sleep-wake states over the day shown as percent time of 3 Hr bins. Grey background denotes the dark photoperiod. <b>E,</b> Aligned time-frequency power spectrum, raw EEG, raw EMG, and color-coded hypnogram for a single recording day. Time of day denoted as zeitgeber time underneath the hypnogram. <b>F</b>, Expanded view of hashed yellow box in panel E. Small yellow hashed boxes highlight times with state transitions and correspond to subpanels F1 –F3. <b>F1</b>, Wake to NREM transition. <b>F2,</b> NREM to wake transition. <b>F3,</b> Transition from NREM to REM and transition from REM to wake. For A-D and all hypnograms shown in E–F, wake is indicated in red, NREM is indicated in blue, and REM is indicated in green. For all periodograms shown in E and F, absolute power specified in the heat map is given by the colorbar between panel F and subpanel F3.</p

Blockade of CB1 Has Minimal Effects on NREM Sleep Time but Fragments NREM, Resulting in Reduced REM Sleep.

<p><b>A,</b> Schematic overview of experimental paradigm. <b>B,</b> Effect of administering different doses of AM281 at the onset of the DP (ZT 12:00, <i>N</i> = 12). <b>C,</b> Effect of administering different doses of AM281 at the onset of the LP (ZT 00:00, <i>N</i> = 9). <b>B & C,</b> Measures of NREM (top row) and REM (bottom row) sleep time and architecture are shown. In all graphs, grey shaded regions denote the the DP. Asterisks (*) denote significant pair-wise comparisons between drug conditions and measures obtained during vehicle baseline. Symbols represent means±SEM across all subjects for each 3 Hr time bin.</p