Multiple sleep alterations in mice lacking cannabinoid type 1 receptors

Abstract

Cannabinoid type 1 (CB1) receptors are highly expressed in the brain and play a role in behavior control. Endogenous cannabinoid signaling is modulated by high-fat diet (HFD). We investigated the consequences of congenital lack of CB1 receptors on sleep in mice fed standard diet (SD) and HFD. CB1 cannabinoid receptor knock-out (KO) and wild-type (WT) mice were fed SD or HFD for 4 months (n = 9-10 per group). Mice were instrumented with electroencephalographic (EEG) and electromyographic electrodes. Recordings were performed during baseline (48 hours), sleep deprivation (gentle handling, 6 hours), sleep recovery (18 hours), and after cage switch (insomnia model paradigm, 6 hours). We found multiple significant effects of genotype on sleep. In particular, KO spent more time awake and less time in non-rapid-eye-movement sleep (NREMS) and rapid-eye-movement sleep (REMS) than WT during the dark (active) period but not during the light (rest) period, enhancing the day-night variation of wake-sleep amounts. KO had slower EEG theta rhythm during REMS. REMS homeostasis after sleep deprivation was less effective in KO than in WT. Finally, KO habituated more rapidly to the arousing effect of the cage-switch test than WT. We did not find any significant effects of diet or of diet x genotype interaction on sleep. The occurrence of multiple sleep alterations in KO indicates important roles of CB1 cannabinoid receptors in limiting arousal during the active period of the day, in sleep regulation, and in sleep EEG in mice.

Figure 1. Body weight as a function of age from onset of dietary treatment to surgery in cannabinoid type 1 (CB₁) receptor knock-out mice (KO) and wild-type (WT) mice.

Fed standard diet (SD, A) or high-fat diet (HFD, B). Age of the mice at surgery is reported as the abscissa of the rightmost data points and did not differ significantly between groups (P = 0.219, ANOVA). Data are means ± SEM with n = 9–10 per group. * and §, P<0.05, ANOVA, main effect of genotype on mice fed SD and HFD, respectively.

Figure 2. Percentage of baseline recording time spent in wakefulness (W), non-rapid-eye-movement sleep (NREMS), and rapid-eye-movement sleep (REMS) as a function of the time of day (A, D, and G) and as 24-hour average (B, E, and H).

Panels C, F, and I show the difference in the amount of time spent in each wake-sleep state between the light and dark period expressed as a percentage of the 24-h amount of time spent in each state. Zeitgeber time is time from lights on. Statistical analysis in panels A, D, and G was performed on average values over 6-hour periods (horizontal lines). Data are means ± SEM in KO and WT fed SD or HFD, with n = 9–10 per group. * and §: P<0.05, WT-SD vs. KO-SD and WT-HFD vs. KO-HFD, respectively. Abbreviations have the same meaning as in Figure 1.

Figure 3. Percentage of baseline recording time spent in W, NREMS, and REMS as a function of episode duration.

Data are means ± SEM in KO and WT fed SD or HFD, with n = 9–10 per group. Abbreviations and symbols have the same meaning as in Figure 2.

Figure 4. Electroencephalographic (EEG) rhythms during sleep in KO and WT mice.

Panels A and B show EEG power spectral density in all REMS and NREMS epochs during the 48-hour baseline recordings expressed as a percentage of the respective total EEG spectral power. The inset in A shows the frequency of the EEG spectral peak. The inset in B shows magnification of NREMS spectral power at frequencies <4 Hz. Panel C shows power in the delta frequency range (1–4 Hz, EEG slow-wave activity, SWA) during NREMS in baseline conditions. EEG SWA was normalized to values in the last 4 hours of the light period. Data are means ± SEM in KO and WT fed SD or HFD, with n = 9–10 per group. Abbreviations and symbols have the same meaning as in Figure 2.

Figure 5. Wake-sleep profile during and after sleep deprivation in KO and WT mice.

Panels A, B, and C show the percentage of recording time spent in W, NREMS, and REMS as a function of the time of day during sleep deprivation and subsequent sleep recovery. Statistical analysis was performed on average values over 6-hour periods (horizontal lines). Panels D and E show the percentage of NREMS and REMS time lost during sleep deprivation, respectively, which was recovered at the end of the sleep recovery period. Panel F shows EEG SWA in NREMS during recovery after sleep deprivation with the same scale as in Figure 3. Data are means ± SEM in KO and WT fed SD or HFD, with n = 9–10 per group. Abbreviations and symbols have the same meaning as in preceding figures.

Figure 6. Wake-sleep profile during 6 h cage-switch test in KO and WT mice.

Percentage of recording time spent in W (A), NREMS (B), and REMS (C) as a function of the time during a cage-switch test. Statistical analysis was performed on the first hour, the average of the second and third hours (horizontal lines), and the average of the last three hours of the cage-switch test. Insets show differences (Δ) in the percentage of recording time spent in a given wake-sleep state between the last 3 hours of the cage-switch test and the corresponding time period during baseline recordings. Data are means ± SEM in KO and WT fed SD or HFD, with n = 9–10 per group. a, P<0.05 vs. baseline recordings. †, P<0.05, KO-HFD vs. KO-SD. Other abbreviations and symbols have the same meaning as in preceding figures.