Interactions between GABAergic and cholinergic processes in the nucleus pontis oralis: neuronal mechanisms controlling active (rapid eye movement) sleep and wakefulness

Abstract

The cholinergic system within the nucleus pontis oralis (NPO) of the pontine tegmentum is critically involved in the generation of active (rapid eye movement) sleep. Previously, we demonstrated that a GABAergic system in the NPO also plays an important role in the control of the behavioral states of wakefulness as well as active sleep. The present study examined interactions between these two neuronal systems vis-a-vis the occurrence of these behavioral states. Accordingly, cholinergic and GABAergic agonists and antagonists were injected into the NPO, and their combined effects on sleep and waking states of chronic, unanesthetized cats were examined. Microinjections of carbachol into the NPO elicited active sleep with a short latency. However, a preinjection of muscimol (a GABA(A) agonist) completely blocked the active sleep-inducing effects of carbachol. The induction of active sleep by carbachol was also suppressed by a subsequent injection of muscimol. On the other hand, the microinjection of scopolamine (a muscarinic receptor antagonist) did not block the induction of active sleep by bicuculline (a GABA(A) antagonist). We conclude that the excitatory cholinergic control of NPO neurons that are involved in the generation of active sleep is gated by a pontine GABAergic system that exerts its effects postsynaptically by inhibiting NPO neurons, resulting in the suppression of active sleep and the generation of wakefulness. In the absence of the activation of this GABAergic gating mechanism, active sleep occurs. These results reveal that specific interactions between cholinergic and GABAergic processes in the NPO play a critical role in the generation of active sleep and wakefulness.

Figure 1.

Anatomical location of injection sites (n = 8) in the nucleus pontis oralis. The effective injections of both cholinergic and GABAergic agonists and antagonists were all within the NPO. Two schematic frontal planes of the cat brainstem are illustrated at levels posterior (P) 2.5 and P 3.0, respectively. Circles indicate sites where injections were delivered to the left and right side, respectively. The boxed area (A) is enlarged and presented in a photomicrograph (A′). The arrow points to the injection site, which is marked with the Chicago sky blue dye. bc, Brachium conjunctivum; bp, brachium pontis; cp, cerebral peduncle; DR, dorsal raphe nucleus; FTP, paralemniscal tegmental field; IC, inferior colliculus.

Figure 2.

An injection of muscimol into the NPO blocked carbachol-induced active sleep. A, Representative polygraphic recordings after the combined injection of muscimol and carbachol. Note that the injection of muscimol induced wakefulness with a latency of 2.3 min. The injection of muscimol was performed during a spontaneous episode of quiet sleep. Twenty-one minutes after the injection of muscimol, carbachol was injected into the same area of the NPO; however, the injection of carbachol failed to induce active sleep. B, C, Hypnograms presented from recording sessions during the combined injection of muscimol and carbachol or saline and carbachol, respectively. Note that the injection of carbachol into the NPO induced a short-latency, long-lasting episode of active sleep (C). However, when muscimol was injected into the NPO, 21 min before the injection of carbachol, it blocked the induction of active sleep by carbachol (B). Control injections of saline into the same area of the NPO did not block active sleep that occurred after the injection of carbachol, indicating that the effect of muscimol was produced by the pharmacological action of this drug and was not an artifact of the injection procedure (C). EEG, Electroencephalogram; EOG, electro-oculogram; EMG, electromyogram. Vertical calibration bars, 100 μV.

Figure 3.

Pie charts presenting the mean percentage of time spent in wakefulness, quiet sleep, and active sleep during the first hour (top) and the 3 hr recording period (bottom) after the combined injection of muscimol and carbachol or saline and carbachol, respectively. Data within parentheses are means ± SEM. Compared with the percentages observed after the injection of saline and carbachol (n = 6), the injection of muscimol and carbachol significantly increased the percentage of time spent in wakefulness (p < 0.01) and significantly decreased the percentage of time spent in active sleep (p < 0.01) during the first hour of recording. During the entire 3 hr recording period, the injection of muscimol and carbachol also induced significant changes in the percentage of time spent in wakefulness (p < 0.01) and active sleep (p < 0.01).

Figure 4.

Effects of the combined injection of muscimol and carbachol on the latency, frequency, and duration of the longest episodes of active sleep, quiet sleep, and wakefulness. Each bar represents the mean value; error bars indicate the SEM of each population. A, The effect of the injection of muscimol and carbachol on the mean latency of active sleep. The latency was measured from the second injection to the onset of the first episode of the indicated behavioral state. Injections of muscimol and carbachol significantly increased the mean latency of active sleep. B, The effect of the injection of muscimol and carbachol on the mean frequency (the number of episodes of a specific behavioral state per hour) of active sleep (B1), quiet sleep (B2), and wakefulness (B3). The injection of muscimol and carbachol significantly reduced the frequency of active sleep. C, The effect of the injection of muscimol and carbachol on the duration of the longest episode of active sleep (C1), quiet sleep (C2), and wakefulness (C3). The injection of muscimol and carbachol significantly decreased the mean duration of active sleep and increased the mean duration of wakefulness. Asterisks indicate the levels of statistical significance of the difference between means; ^**p < 0.01.

Figure 5.

Dose-dependent effects of a preinjection of muscimol on the mean latency to the first episode of active sleep (A) and the mean percentage of time spent in active sleep (B) during the 3 hr recording period after an injection of carbachol (22 mm). All four doses of muscimol were injected in a saline solution of 0.25 μl. Data were obtained from 18 injections (100 μm, n = 3; 1 mm, n = 4; 5 mm, n = 4; 10 mm, n = 7). Each point presents the mean for each dose of muscimol; error bars indicate the SEM.

Figure 6.

Carbachol-induced active sleep was suppressed by a subsequent injection of muscimol. A, Representative polygraphic recordings from an experimental session that consisted of the combined injection of muscimol and carbachol. An injection of carbachol into the NPO induced, with a short latency, a long-duration episode of active sleep. The injection of muscimol, which was performed 25 min after the injection of carbachol, immediately induced wakefulness. B, C, Hypnograms of recording sessions of the combined injection of carbachol and muscimol or carbachol and saline. The induction of active sleep by carbachol was suppressed by a subsequent injection of muscimol and a prolonged episode of wakefulness occurred after an injection of muscimol (B). Control injections of saline into the same area of the NPO did not have an effect on carbachol-induced active sleep (C). Vertical calibration bars, 100 μV.

Figure 7.

Pie charts presenting the mean percentage of time spent in wakefulness, quiet sleep, and active sleep during the first hour (top) and the 3 hr recording period (bottom) after the combined injection of carbachol and muscimol or carbachol and saline, respectively. The injection of carbachol and muscimol significantly increased the percentage of time spent in wakefulness (p < 0.01) and significantly decreased the percentage of time spent in active sleep (p < 0.01) compared with these parameters after the injection of carbachol and saline during the first hour of recording. The injection of carbachol and muscimol also increased the percentage of time spent in wakefulness and decreased the percentage of time spent in active sleep (p < 0.01) during the 3 hr recording period.

Figure 8.

Effects of the combined injection of carbachol and muscimol on the latency, frequency, and duration of active sleep, quiet sleep, and wakefulness. Each bar represents the mean value; error bars indicate the SEM of each population. A, The effect of the injection of carbachol and muscimol on the latency of active sleep and wakefulness. Latency was measured from the second injection to the onset of the first episode of active sleep or wakefulness, respectively. The injection of carbachol and muscimol significantly increased the mean latency of active sleep and reduced the mean latency of wakefulness. B, The injection of carbachol and muscimol significantly reduced the frequency of active sleep. C, The injection of carbachol and muscimol significantly decreased the mean duration of active sleep and increased the mean duration of wakefulness. Asterisks indicate the levels of statistical significance of the difference between means; ^*p < 0.05; ^**p < 0.01.

Figure 9.

The injection of scopolamine into the NPO failed to block active sleep induced by a subsequent injection of bicuculline. A, Representative polygraphic recordings from an experimental session that consisted of the combined injection of scopolamine and bicuculline. Note that, 30 min after the injection of scopolamine, an injection of bicuculline into the same region of the NPO induced, immediately, an episode of active sleep. B, C, Hypnograms from recording sessions of the combined injection of scopolamine and bicuculline or scopolamine and carbachol, respectively. Note that an injection of scopolamine into the NPO, which was delivered 30 min before the injection of bicuculline, did not block the occurrence of active sleep induced by a subsequent injection of bicuculline (B). However, an injection of scopolamine did block the induction of active sleep by a subsequent injection of carbachol, indicating an antagonistic effect of scopolamine on cholinergic receptors (C). EEG, Electroencephalogram; EOG, electro-oculogram; EMG, electromyogram. Vertical calibration bars, 100 μV.

Figure 10.

Pie charts presenting the mean percentage of time spent in wakefulness, quiet sleep, and active sleep during the first hour (top) and the 3 hr recording period (bottom) after the combined injection of scopolamine and bicuculline or scopolamine and carbachol, respectively. Compared with the percentages observed after the injection of scopolamine and carbachol (n = 5), the injection of scopolamine did not block the increase in the percentage of time spent in active sleep (p < 0.01) or the reduction in the percentage of time spent in quiet sleep (p < 0.05) and wakefulness (p < 0.05) induced by bicuculline (n = 6) during the first hour of recording. During the 3 hr recording period, the injection of scopolamine and bicuculline produced significant changes only in the percentage of the time spent in active sleep (p < 0.01).

Figure 11.

Effects of the injection of scopolamine and bicuculline on the latency, frequency, and duration of active sleep, quiet sleep, and wakefulness. Each bar represents the mean value; error bars indicate the SEM of each population. A, The injection of scopolamine and bicuculline significantly reduced the mean latency of active sleep (p < 0.01) and increased the mean latency of wakefulness (p < 0.05) compared with these parameters after the injection of scopolamine and carbachol. B, The injection of scopolamine and bicuculline significantly increased the frequency of active sleep (p < 0.05). C, The injection of scopolamine and bicuculline significantly increased the mean duration of active sleep. Asterisks indicate the levels of statistical significance of the difference between means; ^*p < 0.05; ^**p < 0.01.