Fentanyl and Morphine Cause Dose‐Dependent Sleep Disruption in C57BL/6J (B6) Mice

Abstract

Opioids are important for pain management but have the unwanted side effect of sleep disruption in humans (Sleep Med Clin 13:271, 2018), rats (Sci Rep 9:14122, 2019), and mice (Neurosci Abstr Online 502.02, 2019). Dose-response data are needed for efforts to develop countermeasures to the undesired effects of opioids. Therefore, this study is testing the hypothesis that the sleep-disrupting effects of opioids are dose dependent. Experiments were conducted in accordance with the FASEB Statement of Principles for the use of Animals in Research and Education. Adult male B6 mice (n=8) were housed in a temperature- and humidity-controlled environment and 12:12h light:dark cycle. Mice were implanted with telemeters (DSI HD-X02) to record cortical electroencephalogram (EEG) and neck muscle electromyogram. These measures made it possible to objectively quantify the effects of opioids on states of wakefulness, rapid eye movement (REM) sleep, and non-REM (NREM) sleep. One week after recovery from surgery, each mouse began receiving subcutaneous injections (0.3 mL) within 60 min of light onset. States of sleep and wakefulness were recorded for 4 h after each injection. Repeated injections into the same mouse were separated by 3 or 4 days. Saline (vehicle control) was followed by increasing, half log doses (mg/kg) of morphine (0.01 to 30) or fentanyl (0.001 to 3). Every 10 s of each 4-h recording was scored by two individuals, one of whom was blinded to the treatment condition. Sleep scoring concordance between the scorers was >90%. Data were analyzed using repeated measures ANOVA and Tukey's multiple comparisons test (GraphPad Prism V9). Morphine (F(8, 56) = 104.4, p <0.0001) and fentanyl (F(8, 56) = 94.65, p < 0.0001) dose-dependently increased wakefulness, decreased NREM sleep (F(8, 56) = 110.2, p <0.0001; F(8, 56) = 96.30, p < 0.0001) and decreased REM sleep (F(8, 56) = 34.00, p <0.0001; F(8, 56)= 51.80, p <0.0001). The lowest dose of morphine that significantly increased wakefulness and decreased NREM sleep was 1 mg/kg (p = 0.04, p = 0.03); and 3 mg/kg for decreasing REM sleep (p = 0.0004). For fentanyl, the lowest dose that significantly increased wakefulness and decreased NREM and REM sleep was 0.3 mg/kg (p = 0.001; p = 0.0016; p = 0.0007). Higher doses of morphine and fentanyl eliminated NREM and REM sleep. Both opioids also significantly and differentially altered EEG power during wakefulness (see companion abstract by O’Brien et al.). Nonlinear regression was used to calculate the half-maximal dose (mg/kg) for increasing wakefulness (ED50) and inhibiting sleep (ID50). ED50 and ID50 data were used to compare drug potency. Morphine ED50 was 2.2 to increase wakefulness, ID50 2.3 to decrease NREM sleep, and 1.3 to decrease REM sleep. Fentanyl ED50 was 0.2 to increase wakefulness, ID50 0.2 to decrease NREM sleep, and 0.1 to decrease REM sleep. These data demonstrate that opioid-induced sleep disruption is dose-dependent, and that fentanyl is more potent than morphine to disrupt sleep.