Sleep is a systemic physiological phenomenon in the biological world (Tononi et al., 2024), which can reduce the decomposition of body energy, promote the removal of metabolic wastes from the brain, and play a crucial role in maintaining wakefulness, cognition, learning, and memory. It also has an important impact on regulating the secretion of physiological hormones and the immune function of the body (Baranwal et al., 2023). Normal sleep phases can be divided into non-rapid eye movement (NREM) sleep and rapid eye movement (REM) sleep, with distinct electroencephalogram (EEG) and behavioral characteristics in different states. NREM sleep plays a key role in physical recovery, energy reserve, and memory consolidation, while REM sleep is associated with dreaming and cognitive processing (Sun et al., 2020). The sleep-wake state is regulated by the brain’s neurotransmitter systems. The wake-promoting pathways involve monoaminergic neurons in the anterior brainstem and caudal hypothalamus that directly project to the cerebral cortex, hypothalamus, and thalamus to regulate wakefulness. These monoaminergic neurons include norepinephrineergic neurons in the locus coeruleus (LC) region (Bajwa and Kulshrestha, 2013), serotonergic neurons in the dorsal raphe nucleus (DRN) (Oikonomou et al., 2019), dopaminergic neurons in the ventral tegmental area (VTA) (Montero et al., 2021), and histaminergic neurons in the tuberomammillary nucleus (TMN) (Scammell et al., 2019). These central neurons generate wakefulness through simultaneous stimulation of cortical activation and an increase in muscle tone, with their firing frequency highest during the awake state and decreasing during NREM sleep. Additionally, cholinergic neurons in the laterodorsal tegmental nucleus (LDT) and pedunculopontine nucleus (PPT) are the most critical components of the ascending arousal system (Jones, 2020), while glutamatergic neurons mediating amino acid neurotransmission drive wakefulness in the thalamocortical circuit. During NREM sleep, gamma-aminobutyric acidergic (GABAergic) neurons in the ventrolateral preoptic nucleus (VLPO) of the anterior hypothalamus inhibit wakefulness and arousal via direct projections to the histaminergic TMN. However, with the continuous development of society, factors such as the accelerated pace of life and heightened stress levels have contributed to the widespread occurrence of sleep disorders. These disorders are typically manifested as difficulty falling asleep, maintaining sleep, frequent awakenings, or early morning awakenings, which result in decreased daytime functioning and significantly disrupt individuals’ daily lives and occupational performance (Hughes et al., 2021). Currently, cognitive behavioral therapy (CBT) is the first-line treatment for insomnia disorders, but due to limitations including protracted treatment duration and delayed therapeutic onset, sedative-hypnotic drugs remain the preferred intervention for most insomnia patients. Nevertheless, the medication patterns for sleep disorders are relatively homogeneous, characterized by low efficacy and high side effects. Long-term application can disrupt the brain’s reward system, leading to addictive behaviors and subsequently causing cognitive dysfunction (Crowe and Stranks, 2018; He et al., 2019). It can also interfere with the normal function of neurotransmitters, thereby affecting mood regulation (Ciucă Anghel et al., 2023). Additionally, it significantly increases the risk of developing neurological diseases (He et al., 2019; Zheng et al., 2023). Therefore, it is essential to conduct studies on drug combinations for sleep regulation to minimize the potential adverse effects associated with the use of single drugs at high doses.

Dexmedetomidine (DMED), a highly selective α₂-adrenergic receptor (α₂-AR) agonist, exerts pharmacological effects by acting on α₂-AR in the central nervous system and the periphery (Chima et al., 2022), Its effects include sedation, analgesia, hypnosis, the antisympathetic effects, anxiolysis, delirium reduction, anti-inflammatory properties, and protection of vital organs. In recent years, DMED has been increasingly used in the treatment of chronic insomnia (Ramaswamy et al., 2021). It is generally believed that DMED induces sleep by activating presynaptic α₂ receptors on norepinephrine (NE) neurons in the LC, thereby reducing NE release through a G_i-coupled mechanism (Fan et al., 2023). Studies have also demonstrated that DMED exerts sleep effects by activating GABAergic neurons in the lateral preoptic area (LPO) (Zhang et al., 2015). Compared with other sedative drugs, the sleep induced by DMED is similar to physiologic sleep, with no respiratory depression and increased sleep depth (Wu et al., 2016; Ren et al., 2018; Bao et al., 2021; Dong et al., 2022). At the same time, patients can be easily awakened when DMED exerts its sedative effect. Although the neural mechanism underlying this rapid arousability during DMED sedation remains unclear and may involve VTA dopaminergic neurons (Qiu et al., 2020), DMED is widely used in clinical anesthesia and intensive care unit (ICU) sedation due to this unique property (Wang et al., 2023; Duan et al., 2024). In addition, several studies have shown that DMED can prolong the total duration of NREM, increase the depth of sleep, and improve the quality of sleep (Feng et al., 2018; Fan et al., 2023). However, DMED has some limitations. It may trigger cardiovascular side effects such as hypertension, bradycardia, and hypotension caused by postsynaptic α₂-AR activation (Weerink et al., 2017), as well as dose-dependence risk, possible tolerance and withdrawal reactions with long-term use, and higher economic costs, all of which limit its use in clinical practice to some extent.

Eszopiclone (ESZ), as a third-generation novel non-benzodiazepine drug (NBZD), belongs to the pyrrolidine derivatives of the cyclopyrrolone family (Pinto et al., 2016), and is a GABA receptor agonist, capable of selective binding to GABA-A receptors coupled to benzodiazepine receptors, enhancing the inhibitory effect of GABA on neurons and exerting anxiolytic, sedative, and hypnotic effects (Hanson et al., 2008). Additionally, studies have demonstrated that inhibition of hypocretin (HCRT) neurons in the lateral hypothalamus (LH), alongside suppression of basal forebrain and DRN, represents a potential mechanism underlying the sleep-promoting effects of ESZ. ESZ typically induces sleep, which is characterized by a shorter NREM latency, prolonged NREM sleep duration, and unchanged REM sleep duration (Gerashchenko et al., 2017). It facilitates NREM sleep by enhancing GABAergic transmission in the thalamic reticular nucleus, thereby inducing sleep spindle generation (Wamsley et al., 2013; Dimitrios et al., 2020). ESZ has the advantages of rapid onset of action, short half-life, and few side effects, especially in improving sleep maintenance disorders. Therefore, ESZ is commonly used in the treatment of insomnia and can effectively improve sleep onset and maintenance in patients. Despite the significant advantages of ESZ in the treatment of insomnia, there are some limitations and application drawbacks. It may cause common adverse effects, such as dry mouth, dizziness, fatigue, abnormal taste, and daytime sleepiness (Liang et al., 2019), and long-term use may produce drug dependence and withdrawal reactions. In addition, the tolerability and higher economic cost issues of ESZ have limited its use in clinical settings.

Although DMED and ESZ each have well-defined roles and advantages in regulating sleep, studies of their combined use have not been reported. Since DMED mainly acts in regions such as the brainstem and hypothalamus, it regulates the noradrenergic system by agonizing α₂-AR, which in turn inhibits sympathetic activity to induce sleep (Nelson et al., 2003), whereas ESZ mainly acts in regions such as the cerebral cortex, the limbic system, and the thalamus, and promotes sleep by augmenting GABAergic inhibitory neurotransmission, and decreasing the excitability of the cortex and the limbic system (Hanson et al., 2008). The two are complementary in their sites of action and neurotransmitter regulatory pathways, and their combined application may further enhance sleep depth and improve sleep quality. Therefore, the combination is expected to enhance the therapeutic effect while reducing the dose of a single drug, decrease the occurrence of side effects, and optimize the economic cost of treatment.

For this reason, the present study focused on the combination of DMED and ESZ as an entry point, integrating isobolographic analysis, electroencephalogram/electromyogram (EEG/EMG) sleep monitoring, c-Fos immunofluorescence staining, and liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) to investigate the interaction between these two drugs on sleep in mice and their potential mechanisms of action. Our results demonstrate that the combination of DMED and ESZ exhibits a synergistic effect on sleep in mice. The underlying mechanism may involve prolonging NREM sleep, modulating neuronal activity in the VLPO and TMN brain regions, and altering brain neurotransmitter levels. These findings provide a robust theoretical basis for clinical combination therapy to improve sleep quality.

The dose-response relationships for sleep induction by DMED and ESZ administered individually were determined using the LORR experiment. The results demonstrated that both DMED and ESZ significantly shortened sleep latency (Figures 1A,D), prolonged the total sleep time in mice (Figures 1B,E), and the sleep rate increased markedly with higher doses (Figures 1C,F). The ED₅₀ for DMED was 124 μg/kg, and for ESZ, it was 48 mg/kg. In the combination drug experiment, the ED₅₀ for sleep induction was achieved at a DMED dose of 11.0 μg/kg and an ESZ dose of 4.4 mg/kg. Compared to individual administration, the doses required to achieve the same effect in the combined treatment were reduced by more than 11-fold, indicating a significantly greater effect than simple additive interactions. To further clarify the interaction between DMED and ESZ in combination, we employed the isobolographic analysis, a classical method for evaluating drug interactions. This approach offers the advantage of providing a visual and quantitative assessment of the interaction between two or more drugs, making it particularly valuable in pharmacological research (Tallarida, 2011; Foucquier and Guedj, 2015). The isobolographic analysis revealed that the ED₅₀ of the combined treatment fell to the left of the additive line, indicating a synergistic effect between the two drugs in inducing sleep (Figure 2C). To the best of our knowledge, this is the first preclinical study to demonstrate synergistic sleep regulation by combined DMED and ESZ administration in mouse models. Although this specific drug combination has not yet been clinically implemented for human sleep disorders, our findings provide a mechanistic foundation for translational research. From a clinical perspective, such combination therapy may substantially reduce required doses of individual agents, thereby minimizing side effects associated with high-dose monotherapy. Furthermore, for patients with sleep disorders refractory to monotherapy, the DMED-ESZ combination constitutes a novel therapeutic strategy that may enhance clinical efficacy and improve sleep quality.

The sleep-wake state is mainly divided into three states: NREM, REM, and Wake, each characterized by distinct electroencephalogram (EEG) features. During NREM sleep, EMG signals nearly disappear, the overall EEG frequency slows down, the amplitude increases, and Delta waves become significantly enhanced. During REM sleep, EMG signals also nearly disappear, but the overall EEG frequency increases, the amplitude decreases, and Theta waves become notably enhanced. In contrast, during the Wake phase, EMG signals are significantly enhanced, the overall EEG frequency increases, and the amplitude decreases (Scammell et al., 2017; Claar et al., 2023). Based on the EEG/EMG monitoring results, the combined administration of DMED and ESZ significantly reduced the duration of wakefulness (Figure 3A), prolonged the duration of NREM sleep (Figure 3B), and this effect was markedly superior to that of individual drug administration. This aligns with previous studies indicating that hypnotic drugs typically alter total sleep time and NREM sleep rather than REM sleep (Bajwa and Kulshrestha, 2013; Hajiaghaee et al., 2016; Yoon and Cho, 2018; Kim et al., 2019; Shi et al., 2019). From a functional perspective, NREM sleep plays a critical role in bodily recovery, energy conservation, and memory consolidation (Sun et al., 2020). The extension of NREM sleep by the combined treatment suggests its significant potential in promoting physiological maintenance and enhancement. Adequate NREM sleep facilitates protein synthesis and the repair of damaged tissues, which are essential for growth, development, and daily recovery (Tononi and Cirelli, 2014). This indicates that the combined treatment may improve overall sleep quality by enhancing the physiological functions associated with NREM sleep. Furthermore, the prolongation of NREM sleep may positively impact cognitive functions. DMED has been shown to protect neurocognition by reducing interleukin (IL)-6 and IL-1β in the hippocampus (Wang et al., 2019; Mei et al., 2021). It also improves cognitive function by reducing inflammation via α₂-AR-mediated decreases in IL-1β and NF-κB levels (Li et al., 2020). ESZ can effectively improve the sleep quality in patients with sleep disorders, reduce the number of nocturnal awakenings, and thereby enhance cognitive function (Huo et al., 2022). The significant increase in NREM sleep in this study suggests that the combined treatment may improve cognitive ability in mice. This finding provides a new theoretical basis for the clinical application of combined drug therapy. In the sleep cycle, NREM and REM sleep alternate, and the balance between the two is crucial for maintaining normal sleep and physiological functions. Although the combined treatment in this study did not significantly affect REM sleep (Figure 3C), the extension of NREM sleep may indirectly influence the functions related to REM sleep. Future studies could further explore the effects of combined treatment on the alternating patterns of NREM and REM sleep throughout the sleep cycle.

The c-Fos protein, a product of the immediate-early gene c-fos, serves as a marker of neuronal activity and is widely used to study functional anatomy and neuronal activation states in the nervous system (Zant et al., 2016). As studies have indicated, the VLPO and TMN play central and well-defined roles in regulating the sleep-wake cycle (Chung et al., 2017; Scammell et al., 2019). By focusing on the VLPO and TMN, we can target the core components of the sleep-wake regulatory network. In this study, using c-Fos immunofluorescence staining, we investigated the effects of combined DMED and ESZ administration on neuronal activity in the VLPO and TMN brain regions. VLPO neurons primarily release inhibitory neurotransmitters, GABA and glycine. Existing studies have comprehensively demonstrated the central role of the VLPO in sleep initiation and maintenance through various perspectives, including neurotransmitter regulation (Gaus et al., 2002), environmental influences (Gong et al., 2000), and circuit mechanisms (Cirelli and Tononi, 2000), as evidenced by c-Fos immunohistochemistry. In contrast to the VLPO, the TMN is a key nucleus for maintaining wakefulness and is the sole source of histaminergic (His) neurons (Yu et al., 2014). In 2005, Saper and colleagues proposed the “flip-flop switch model” for sleep-wake transitions, which hypothesizes that sleep-promoting neurons in the VLPO and wake-promoting neurons in the TMN mutually inhibit each other to facilitate sleep-wake transitions (Saper et al., 2005). Our results showed that the combined treatment increased the number of c-Fos positive neurons in the VLPO (Figures 4C,E) while decreasing the number of c-Fos positive neurons in the TMN (Figures 4D,F). These findings align with the “flip-flop switch model,” further supporting the hypothesis that the combined administration of DMED and ESZ promotes sleep by modulating neuronal activity in these two brain regions. Additionally, the effect of sleep-inducing drugs on the VLPO is closely associated with the prolongation of NREM sleep. During NREM sleep, VLPO neuronal activity increases, leading to elevated expression of c-Fos protein, the product of the c-fos gene. Studies using optogenetics have shown that inhibiting the excitability of histaminergic neurons in the TMN during wakefulness can induce NREM sleep but not REM sleep (Venner et al., 2016; Yamashita and Yamanaka, 2017), which is consistent with our experimental results showing that the combined treatment suppresses REM sleep while enhancing NREM sleep.

The regulation of sleep and wakefulness is widely recognized to be related to the levels of corresponding neurotransmitters (Holst and Landolt, 2018). And NE, DA, Ach, Glu, and His as excitatory neurotransmitters play a role in initiating and maintaining wakefulness in the sleep-wake cycle, and 5-HT, 5-HIAA, and GABA as inhibitory neurotransmitters can induce and maintain sleep state. The results of the present study showed that the combination of DMED and ESZ significantly decreased the levels of DA, NE, Ach, Glu, and His, while increasing the levels of 5-HT, 5-HIAA, and GABA, suggesting that the combination synergistically promotes the molecular mechanism of sleep through the bi-directional regulation of excitatory-inhibitory neurotransmitter balance. DMED, as an α2-AR agonist, negatively feeds back by inhibiting the activity of NEergic neurons in the LC and reduces NE release (Bajwa and Kulshrestha, 2013), while ESZ may indirectly reduce NE levels by enhancing GABAergic inhibition. NE levels were significantly reduced after the combination in this study (Figure 5A), suggesting that the two drugs may reduce arousal drive by synergistically inhibiting the LC-NE pathway. The dopaminergic neurons are mainly located in the VTA and substantia nigra compacta (SNc) of the midbrain, with extensive fiber connections to the sleep-wake brain regions, and their dopaminergic signals projected to the forebrain play a key role in maintaining arousal and motivational behaviors (Montero et al., 2021). DA levels were significantly reduced in the present study (Figure 5B), suggesting that the combination may reduce arousal drive by inhibiting dopaminergic neuronal activity in the VTA and SNc. Noradrenergic neurons project to the VTA via the medial forebrain bundle (MFB). During wakefulness, VTA dopaminergic neurons exhibit activation states, whereas inhibition of these neurons promotes sleep (Eban-Rothschild et al., 2016). DMED, through its suppression of noradrenergic signaling, attenuates excitatory drive to VTA-DA neurons, thereby reducing dopamine (DA) release. ESZ, a GABA-A receptor agonist, exerts its effects by potentiating GABAergic inhibitory signaling. DA neurons in the VTA and SNc receive GABAergic projections from the ventral pallidum and nucleus accumbens, which directly suppress DA neuronal activity. Concurrently, enhanced GABAergic tone inhibits cortical and limbic dopaminergic systems, further reducing DA release (Tan et al., 2012). His is secreted only by histaminergic neurons in the TMN brain region, whose nerve fibers project widely throughout the brain and are essential for maintaining arousal (Scammell et al., 2019), and the reduction in His levels after the combination is consistent with the reduction in c-Fos positive neurons in the TMN (Figure 5C). This further supports the finding that inhibition of TMN activity is an important mechanism for sleep promotion. Acetylcholinergic neurons are mainly located in the LDT and PPT of the brainstem. Acetylcholine (ACh), which is involved in sleep regulation, is released from the terminals of these neurons and widely projects to the cerebral cortex, amygdala, hippocampus, thalamus, brainstem, and midbrain to promote arousal (Jones, 2020). Glu drives arousal in the thalamo-cortical loop (Dash et al., 2009). The significant decrease in Ach and Glu after the combination suggests that the two drugs may synergistically attenuate arousal signaling through inhibition of thalamo-cortical glutamatergic transmission and basal forebrain acetylcholinergic activity (Figures 5D,E). GABA, as a major inhibitory neurotransmitter in the central nervous system (CNS), mediates sleep via GABA-A receptors in areas such as the VLPO and basal forebrain (Scammell et al., 2017; Cai et al., 2023), ESZ acts as a GABA-A receptor agonist and directly enhances GABAergic inhibition, whereas DMED may have a synergistic effect by inhibiting noradrenergic release and deregulating inhibition of GABAergic neurons. The GABA content in this study was significantly elevated after the combination (Figure 5F), suggesting that the two drugs amplify GABAergic signaling through a dual mechanism (direct activation of receptors and deregulation of upstream inhibition). 5-HT neurons in the DRN exhibit complex modulatory roles in sleep-wake transitions, with their primary metabolite being 5-HIAA (Oikonomou et al., 2019). Accumulation of 5-HT may promote sleep through activation of 5-HT2A receptors. The elevation of 5-HT and 5-HIAA in the present study may reflect the modulation of the DRN-5-HT system by the combination of drugs (Figures 5G,H), but the exact mechanism needs to be further investigated. The initiation of REM sleep is associated with suppression of 5-HT and NE neurons, potentially mediated by activation of the lateral habenula (LHb) (Zhao et al., 2015; Takahashi et al., 2022). In this study, the sustained inhibition of NE may establish permissive neurochemical conditions for REM sleep. Notably, 5-HT has been demonstrated to activate the claustrum while inhibiting theta rhythm-promoting brain regions (Hajos et al., 2003; Nuñez and Buño, 2021). This dual action aligns with the insignificant REM sleep changes observed in the current study, potentially explaining why REM sleep remained unaffected.

In summary, this study reveals for the first time that the combination of DMED and ESZ has a synergistic effect in regulating sleep in mice, and elucidates the potential mechanism of the synergistic effect in terms of changes in sleep phases, c-Fos protein expression, and neurotransmitter content. The finding provides a significant theoretical foundation for the clinical application of combination therapy in treating sleep disorders and lays the groundwork for the development of novel sleep-regulating drugs.