Sleep is an essential biological process that maintains and improves physical and mental health, such as consolidating memories, strengthening the immune system, regulating hormones, maintaining cardiovascular health, and removing central nervous system (CNS) waste via the glymphatic system [1,2]. A good rhythmic sleep that includes an alternation between three non-rapid eye movement (NREM) phases and a rapid eye movement (REM) phase of sleep contains four-to-five cycles in one night [3]. Any factors influencing this sleep architecture are important for the quality of sleep. These factors include endogenous factors, such as neurotransmitters and hormones, and exogenous factors, such as social cues and light/dark exposures [4]. More and more studies have focused on the REM mechanism in the brain during sleep [5,6]. However, the endogenous hormone influencing NREM mechanism remains unclear.
Melatonin (MLT), an endogenous indoleamine hormone that is secreted during NREM sleep, is referred to as the “hormone of darkness” [7]. MLT has a regulatory role on sleep with a rhythmic elevation in the evening and maximum level in the early morning. Exogenous MLT is associated with a circadian-dependent improvement of sleep. Selective prevention of MLT synthesis entrains the circadian rhythm and disrupts sleep during the dark phase [8]. Since >90% of all hormones are secreted during NREM, MLT may control wakefulness/sleep via the regulation of the NREM stage.
MLT acts on two types of MLT receptors (MLTRs) to exert its role in sleep (MLT type 1 receptor [MT1R] and MLT type 2 receptor [MT2R]. Therefore, the neurons expressing MLTR in the brain may be targets for MLT. Previous studies have demonstrated that the paraventricular thalamus (PVT) neurons express both MT1R and MT2R [9,10]. Because genetic knockout of MT1R or MT2R exerts changes in NREM and REM sleep, the PVT may control sleep via the expression of MLTR. There is substantial evidence showing that inhibition of PVT increases NREM sleep, while activation of the PVT produces a transition from wakefulness to sleep [9,11,12]. In addition, PVT activity is associated with daily changes in sleep cycles. Taken together, these studies suggest that MLT possibly targets PVT neurons by acting on MLTRs [13]. However, the link between MLT and PVT in the regulation of sleep remains to be established.
Recently, Wang et al. [14] reported that MLT selectively acts on the PVT neurons to control the NREM stages of sleep. The findings clarified the link between MLT and PVT neurons, as well as the role in NREM sleep.
Wang et al. first used C3H/HeJ mice as the major subjects in their study. This kind of transgenic mouse has an intact MLT synthesis machinery that facilitates studying the role of MLT on sleep. After the model of light and dark cycles, the authors examined the expression of MLTRs in the PVT in C3H/HeJ mice. They confirmed that both MT1R and MT2R were expressed in the PVT and changed associated with zeitgeber times (ZTs) in the cycle. Furthermore, Wang et al. clarified that two kinds of positive PVT neurons (MTNR1A and MTNR1B) expressed different patterns and exhibited circadian-dependent changes. These results suggested that MLTRs expressed on PVT neurons are potential targets for MLT. The authors further investigated how MLT changes the activities of PVT neurons in vitro. The excitability of PVT neurons was examined by performing whole-cell recordings. Two indicators (firing rate and hyperpolarized membrane potential) were decreased in a dose-dependent fashion by bath-application of MLT. The best effective concentration was 10 nM in the Wang et al. study. To further identify which types of MLTR exerts an important role in PVT neurons, A selective MT2R antagonist (cis-4-phenyl-2-propionamidotetralin [4P-PDOT]) and an MT1R selective MT1R antagonist (S26131. 4P-PDOT alone) but not S26131 prevented the inhibitory effects of MLT on PVT neurons. Taken together, these data suggested that MLT targets postsynaptic MLTRs and directly inhibits the excitability of PVT neurons.
To elucidate the effects of MLT on the activities of PVT neurons, Wang et al. performed c-Fos immunostaining experiments and showed that the number of c-Fos-positive neurons were decreased in the MLT group compared to the vehicle group. Measurement of calcium activity in vivo is a method to reflect the activity of neurons accompanied by behavior change. Wang et al. conducted fiber photometry with a calcium indicator in the PVT brain area and reported that an MLT infusion through an implanted cannula decreased spontaneous Ca2+ fluctuations in free-behaving mice. Furthermore, a multi-channel electrophysiologic recording also confirmed that exogenous application of MLT decreased the activity of single PVT neurons.
It has been shown that inhibition of PVT neurons increases NREM sleep [9]. Therefore, the potent downregulation of MLT on the activity of PVT neurons indicated that MLT and MLTRs in the PVT exert a regulatory role in sleep. A local infusion of MLT into the PVT and simultaneous EEG/EMG monitoring in wakefulness/sleep was performed. It was found that MLT induced an increase in NREM sleep but not REM sleep. MLT also induced a reduction in wakefulness of 22.24%. To further verify the key role of PVT neurons, MLT was applied to another brain area (ventrobasal [VB] complex of the thalamus). The data showed that the time spent in wakefulness/sleep was not changed by MLT. Finally, the authors applied a mixture of S26131 and 4P-PDOT into the PVT and found that MLTR antagonists also decreased NREM sleep but not REM sleep. These results demonstrated a selective link between MLT and PVT neurons in NREM.
Furthermore, Wang et al. used an RNA interference (RNAi) method to selectively knock down MLTRs in PVT neurons. This RNAi strategy targets both MT1R and MT2R. C3H/HeJ mice injected with the shRNA virus was designated as the shMTR group and the control virus was designated as the shCTRL group. After virus injection, the wakefulness and sleep states were monitored using EEG/EMG. Wang et al. found that compared to the shCTRL group, the shMTR group had a decrease in NREM but an increase in wakefulness, suggesting that alterations in wakefulness and sleep states changed. Interestingly, both NREM and REM sleep were noted to decrease with the onset of dark and light phases, respectively. Importantly, the episode number but not the duration in NREM sleep was significantly decreased, suggesting that the transition between wakefulness and NREM sleep was impaired. These results suggest that the endogenous MLT signaling in the PVT is important to the transition period of NREM sleep.
Previous studies have shown that both endogenous and exogenous MLT are used to benefit sleep and circadian rhythm-related sleep disorders [15]. For example, endogenous MLT secretion is associated with immune, and antioxidant defenses, and glucose regulation. Endogenous MLT secretion also strengthens the coupling of circadian rhythms, especially core temperature and sleep-wake rhythms [16]. Exogenous MLT supplementation is used for the clinical treatment of sleep disorders, such as insomnia and circadian rhythm disorders. However, the optimal therapeutic dose and dosage form of exogenous MLT are inconsistent across studies [17]. Moreover, exogenous MLT has been shown to inhibit the secretion and release of endogenous MLT, potentially leading to various impairments [18]. Despite these findings for MLT in diverse clinical applications, there is limited research on the specific doses of exogenous MLT that may pose a risk for disease development. However, the potential targets of endogenous MLT signaling in the brain are unknown. Wang et al. found that PVT neurons expressing MLTRs are a target for MLT signaling controlling NREM sleep. Wakefulness and sleep are regulated by homeostatic factors, circadian cues, and allostatic factors. The authors showed that selective knockdown of MLTRs in PVT neurons increases wakefulness in the transition between light and dark periods. Given that MLT-MLTR signaling stabilizes this sleep architecture, some pathologic factors, such as stress, influence MLTR neurons in the PVT. Indeed, inhibition of PVT neurons attenuates stress-induced increase of wakefulness [11], suggesting the potential significance of the neural mechanism underlying MLTR neurons in the PVT of pathologic conditions. Moreover, MTNR-positive neurons in the PVT are more active in the dark phase, indicating the PVT neurons are sensitive and associated with rhythmic MLT signaling during NREM sleep [13]. Future studies are needed to clarify how MLT signaling controls PVT neurons to regulate sleep.
PVT neurons are divided into PVT1, PVT2, PVT3, PVT4, and PVT5 neuronal types. There are many neuromodulator receptors across the five PTV subtypes. For example, dopamine Drd2 and low-density lipoprotein receptors predominate in the PVT1 subtype. The Drd1 receptor and neuropeptide FF receptor 1 are represented in the PVT3 subtype, whereas the Drd3 receptor is expressed in the PVT5 subtype. These results suggest that the differences in gene expression in PVT subtypes may have different functions. Wang et al. showed that MLTR1 and MLTR2 have roles on neuronal excitability. However, the expression of MLTR in these five PVT subtypes is unclear. Therefore, the potential differences in MLTR expression across distinct neuronal types, as well as the varied biological functions, warrant further investigation. Furthermore, the authors have determined the MTNR1A and MTNR1B expression profiles in the PVT. Further research is needed to elucidate specific MTNR1A and MTNR1B function in the PVT during sleep. This study revealed that specific MLT signaling expression in the PVT controls NREM sleep and provides evidence for a novel neuronal mechanism underlying MLT regulation on NREM.