Microinjection of morphine in rat rostromedial tegmental nucleus (RMTg) decreased non-rapid eye movement (NREM) sleep in a dose-dependent manner.

(A) Nissl staining showed the implantation sites of the guide cannulas. (B, C) Morphine inhibited RMTg neurons in vitro. A typical trace showed that morphine induced hyperpolarization and inhibited spontaneous firing of a neuron in the RMTg (B) and changes of membrane potentials of RMTg neurons under whole-cell current-clamp with application of morphine. n = 4 cells, *p < 0.05 versus baseline using 1-way ANOVA followed by least significant difference (LSD) (C). (D) Time course of NREM and REM sleep and wakefulness during the light period (07:00–19:00 hours) in rats treated with artificial cerebrospinal fluid (ACSF) or morphine at 09:00 hour. (E) Sleep–wake quantities following ACSF or morphine microinjection in rats during post-injection period (10:00–13:00 hours). *p < 0.05, **p < 0.01 versus ACSF and #p < 0.05 versus 0.5 nmol/side morphine using 1-way ANOVA followed by LSD. (F–K) During the post-injection period (10:00–13:00 hours) after ACSF or morphine microinjection in rats, episode numbers of each stage (F); number of NREM (G) and REM (H) sleep bouts with different duration; mean duration of each stage (I); stage transitions between S (NREM sleep), W (wakefulness), and R (REM sleep) stages (J); and slow-wave activity (SWA) of NREM sleep (K). *p < 0.05, **p < 0.01 versus ACSF using unpaired t tests. MI, microinjection; Mor, morphine. ACSF (n = 6); morphine 0.5 nmol/side (n = 5); morphine 2 nmol/side (n = 5). The horizontal open bars on the x-axes indicate the 12-hour light period. Underlying data can be found in S1 Data.