Brainstem control of the events of rem sleep

Serotonin (5-HT) is a key modulator/transmitter of the brain, and the dorsal (DR) and median (MR) raphe are the primary 5-HT-containing nuclei of the brainstem, providing widespread serotonergic innervation of the forebrain. These raphe nuclei are involved in several higher-order functions, most notably sleep and consciousness. The DR and MR share reciprocal connections with the brainstem, hypothalamus, preoptic area, and cortical structures fundamental to waking and sleep. While the electrophysiological properties of serotonergic DR/MR cells are indicative of a role in arousal/wakefulness, the diverse nature of serotonergic transmission, in part associated with the heterogeneity of 5-HT receptors, suggests that 5-HT also serves to regulate sleep. Emerging evidence now indicates that 5-HT DR neurons are a critical component of a complex neural circuitry in which (1) the DR exerts excitatory actions on the forebrain, driven in part by orexinergic input from the lateral hypothalamus; (2) the DR inhibits sleep-active neurons of the ventrolateral preoptic area (VLPO) during wakefulness; (3) the DR interacts with neurons that respond to sleep pressure and, as such, along with adenosine, may shift an organism from wakefulness to NREM through 5-HT excitation of a subpopulation of VLPO neurons; and (4) the DR/MR plays a permissive role in rapid eye movement (REM) sleep, as their virtual silencing during REM disinhibits REM-active neurons to thus trigger the state of REM sleep.

The pedunculopontine tegmental nucleus (PPT) represents a major aggregation of cholinergic neurons in the mammalian brainstem, which is important in the generation and maintenance of REM sleep.

We investigated the effects of unilateral and bilateral PPT lesions on sleep and all the conventional sleep-state related EEG frequency bands amplitudes, in an attempt to find the EEG markers for the onset and progression of PPT cholinergic neuronal degeneration.

The experiments were performed on 35 adult male Wistar rats, chronically implanted for sleep recording. During the surgical procedure for EEG and EMG electrodes implantation, the unilateral or bilateral PPT lesion was produced under ketamine/diazepam anesthesia, by the stereotaxically guided microinfusion of 100 nl 0.1 M ibotenic acid (IBO) into PPT. We applied Fourier analysis to signals acquired throughout 6 h of recordings, and each 10 s epoch was differentiated as a Wake, NREM or REM state. We also calculated the group probability density estimates (PDE) of all Wake, NREM and REM conventional EEG frequency amplitudes, and the number of all the transition states using MATLAB 6.5.

Our results show that the unilateral or bilateral PPT lesions did not change the sleep/wake architecture, but did change the sleep/wake state transitions structure and the sleep/state related “EEG microstructure”.

Unilateral or bilateral PPT lesions sustainably increased Wake/REM and REM/Wake transitions from 14 to 35 days after lesions. This was followed by decreased NREM/REM and REM/NREM transitions from 28 days only in the case of the bilateral PPT lesion.

The unilateral PPT lesion augmented both Wake theta and REM beta while it also attenuated the relative amplitude of the Wake delta frequency, with a delay of one week. Following a bilateral PPT lesion there was augmentation of the relative amplitude of the Wake, NREM, and REM beta and REM gamma frequency which occurred simultaneously to NREM and Wake delta attenuation.

We have shown that the PPT cholinergic neuronal loss sustainably increased the number of the Wake/REM and REM/Wake transitions and augmented sleep-states related cortical activation that was simultaneously expressed by the high frequency amplitude augmentation, as well as Wake and NREM delta frequency attenuation.

Sleep is composed of an alternating sequence of REM and non-REM episodes, but their respective roles are not known. We found that the overall firing rates of hippocampal CA1 neurons decreased across sleep concurrent with an increase in the recruitment of neuronal spiking to brief “ripple” episodes, resulting in a net increase in neural synchrony. Unexpectedly, within non-REM episodes, overall firing rates gradually increased together with a decrease in the recruitment of spiking to ripples. The rate increase within non-REM episodes was counteracted by a larger and more rapid decrease of discharge frequency within the interleaved REM episodes. Both the decrease in firing rates and the increase in synchrony during the course of sleep were correlated with the power of theta activity during REM episodes. These findings assign a prominent role of REM sleep in sleep-related neuronal plasticity.

Previously we indicated that the ventral tegmental area (VTA) may belong to the system regulating hippocampal theta rhythm. In the present study, we aimed at assessing the role of the GABAergic system of the VTA in regulation of hippocampal electric activity. Male Wistar rats received unilateral intra-VTA microinjection of either bicuculline (50 ng/0.5 μl, n = 9), muscimol (100 ng/0.5 μl, n = 10) or phaclofen (500 ng/0.5 μl, n = 9). 1-min tail pinch stimulations were applied at 10-min intervals to evoke theta rhythm episodes in hippocampus. We analysed peak power (P_max) and corresponding frequency (F_max) of EEG signal at delta and theta bands. Bicuculline induced theta rhythm in both hippocampi with 0 latency, continuous for ca. 33 min. Phaclofen also induced theta but in this group it appeared with latency (17.45 ± 3.16 min on average), lasted for ca. 33.6 min and during this time was interrupted by periods of irregular activity of variable length. Tail pinch was not applied in these groups. Muscimol induced an opposite effect: depression of theta P_max with simultaneous increase in delta P_max and a decrease in F_max delta during episodes of tail pinch-evoked theta. This effect had variable latency and no return to the control EEG could be observed. We propose that GABA activity in the VTA is of tonic character, so that abolition of this mechanism produces immediate effect, i.e. theta induction (strong by GABA_A and weak by GABA_B receptors blockade), whereas enhancing the already present GABAergic inhibition causes delayed, prolonged changes expressed as gradual loss of theta synchronisation.

Extraordinary strides have been made toward understanding the complexities and regulatory mechanisms of sleep over the past two decades thanks to the help of rapidly evolving technologies. At its most basic level, mammalian sleep is a restorative process of the brain and body. Beyond its primary restorative purpose, sleep is essential for a number of vital functions. Our primary research interest is to understand the cellular and molecular mechanisms underlying the regulation of sleep and its cognitive functions. Here I will reflect on our own research contributions to 50 years of extraordinary advances in the neurobiology of slow-wave sleep (SWS) and rapid eye movement (REM) sleep regulation. I conclude this review by suggesting some potential future directions to further our understanding of the neurobiology of sleep.