Combination administration of alprazolam and N-Ethylmaleimide synergistically enhances sleep behaviors in mice with no potential CNS side effects

Experimental animals

C57BL/6J male mice (4-week, 19 ± 1 g) were obtained from the Beijing HFK Bioscience Co., Ltd. (Beijing, China). Prior to any experiments, the mice were kept in group housing (3–5 animals per cage) at 25 °C under a 12-hour light/dark cycle (lights turning on at 8 a.m.), with food and water provided ad libitum. The animal welfare and experimentation project were approved by the Animal Ethics Committee of State Key Laboratory of NBC Protection for Civilian (LAE-2022-03-001). At the end of experiments, the mice placed in the CO₂ chamber were immediately euthanized by gradually increasing concentrations of CO₂ at 10% displacement rate and continued to be ventilated for 2 min after cessation of breathing (Sengar et al., 2018). During the sleep behaviors tests, mice meeting the criteria of surviving surgery and exhibiting typical EEG/EMG signals were selected for the study, otherwise they were excluded. In other experiments, mice with regular spontaneous activity were included.

During the experiment, each mouse was assigned a temporary random number within the weight range and divided into each group (Li et al., 2019). The study was conducted in a double-blind fashion, where neither the experimental operator nor the data analyst could see each other’s results.

Compound preparation and administration

The Alp tablets was acquired from Yimin Pharmaceutical Co., Ltd, Beijing, China. NEM (97.0%) and sodium carboxymethylcellulose (CMC Na) were purchased from Sigma, USA.

Following our preliminary experiment and a random number table method, a total of 32 mice (eight mice per group) were randomly divided into the following treatment groups: (1) the control group treated with 1% CMC Na; (2) the Alp alone group treated with 1.84 mg/kg Alp in suspended with 1% CMC Na; (3) the NEM alone group treated with 1.0 mg/kg NEM in dissolved with 1% CMC Na; (4) the combination Alp and NEM (Alp@NEM) group treated with 1.84 mg/kg Alp and 1.0 mg/kg NEM in suspended with 1% CMC Na. All mice were intragastrically administered with a constant volume of 10 mL/kg body weight.

Sleep behaviors tests

The sleep behaviors tests were performed with reference to previously established protocols with slight improvements (Zhang et al., 2014; Li et al., 2021). Following different treatments, the mice were placed freely in a separate cage, where the ambient temperature was maintained at 37 °C. The mouse was gently rotated until it exhibited the signs of loss of righting reflex (LORR) and was unable to prostrate itself on limbs, a period defined as sleep latency. In addition, the mouse was unable to maintain LORR with at least three paws touching the bottom, a period defined as sleep duration. The sleep latency and sleep duration of each mouse were measured and recorded precisely with a chronometer (Fig. S1). Additionally, the sleep ratio was calculated as the percentage of mice that exhibited signs of LORR out of the total number of mice in each group.

EEG/EMG devices implantation surgery

Under 1.5% isoflurane inhalation anesthesia, mice were prepared for implantation of electrodes for electroencephalogram and electromyography (EEG/EMG) (Pinnacle Technologies, Lawrence, KS, USA) The electrodes were implanted in the skull of mice with reference to the previous surgical protocol (Sharma, Sahota & Thakkar, 2018; Jones et al., 2022). Shortly, three screw electrodes were implanted into the mouse’s skull and a head cap was placed to record EEG. The two flexible stainless Teflon-coated wires of the head cap were secured bilaterally into the trapezius muscle to record muscle activity (EMG). The entire setup was then fixed onto the skull using dental cement, with the wound closure achieved through suturing. Subsequently, the mouse was placed on a heated pad to aid recovery until it regained normal mobility. During the following week, postoperative anti-infective penicillin (40,000 units intraperitoneally per day) and analgesic meloxicam (4 mg/kg subcutaneously per day) were utilized.

Sleep recording and analysis

After one week habituation following surgery, the mouse’s head cap with a multichannel electrode pedestal was connected to an amplifier by a flexible cable and commutator. The acquired electrical signal was digitally converted using Sleep Sign (Kissei Comtec, Nagano, Japan). Wake-sleep status was scored automatically and manually in 4-second epochs. Wakefulness was defined by the presence of an asynchronous EEG and high-phasic EMG activity. Additionally, non-rapid eye movement (NREM) sleep was defined by a high-amplitude slow-δ wave EEG (0.5–4.0 Hz) together with a low EMG compared to wakefulness. Moreover, rapid eye movement (REM) sleep was defined by the presence of a high-power EEG θ wave (6.0–8.0 Hz) coupled with low EMG activity (suggestive of muscle atonia) compared to NREM. A chemical treatment was administered approximately 15 min before the EEG/EMG recording took place (Masuda et al., 2023).

Brain tissue preparation and immunofluorescence (IF)

After 30 min of administration, the mice in each group were quickly decapitated. Brain tissues were removed, fixed in 4% formaldehyde for 6 h, and then equilibrated in 20% sucrose in 1 × PBS for 48 h. Tissues were sectioned into 20 µm coronal sections using a Leica cryostat (CM1950; Leica Biosystems, Wetzlar, Germany) at −20 °C. The 20 µm frozen coronal sections were treated with 3.0% hydrogen peroxide for 25 min and rinsed by phosphate-buffered saline containing 0.1% Tween-20 (PBST). Sections were then blocked with bovine serum albumin containing 0.3% Triton x-100 for 2 h at 4 °C. After rinsing, sections were incubated with the primary antibody of rabbit anti-c-fos (1:1000 in PBS, ab222699, Abcam) overnight at 4 °C. Sections were rinsed and incubated with the anti-rabbit AlexaFluor-conjugated secondary antibody (1:500 in PBS, A32740; Thermo Fisher Scientific, Waltham, MA, USA) for 1 h at 37 °C and were treated with mounting medium containing DAPI (H-1500; Vectorlabs, Newark, CA, USA) before being sealed with a coverslip. The stained sections were then imaged using a confocal microscopy (Stellaris5 Leica, Wetzlar, Germany) with optical magnification of 20× and 40×. For each group, nine slices from three different mice were selected. In this experiments, c-fos expression in the ventrolateral preoptic nucleus (VLPO) and tuberomammillary nucleus (TMN) was quantified with ImageJ and the Cell Counter plugin. Cells with an immunofluorescence intensity exceedingly twice the background were classified as c-fos positive cells.

Statistics

Behavioral and imaging data statistics were analyzed using Student’s t-test or two-way ANOVA followed by Tukey’s multiple comparisons tests, as appropriate. All statistical analyses were conducted with GraphPad Prism version 9.0.2 for Windows (GraphPad Software, La Jolla, CA, USA). A significance threshold was set at p ≤ 0.01, with all values presented as Mean ± SEM.

NEM can enhance Alp-induced sleep behaviors

The sleep latency, sleep duration, and sleep ratio of mice were evaluated after orally administering different doses of Alp (1.0, 1.2, 1.6, 2.0, 2.2 mg/kg) through the sleep behaviors tests. The results indicated that Alp induced sleep in C57 mice in a dose-dependent manner, with an ED₅₀ of 1.84 mg/kg and an ED₉₅ of 2.87 mg/kg (Table 1).

Subsequently, gradient doses of Alp (0.8, 1.0, 1.4, 1.6 mg/kg) less than 1.84 mg/kg were combined with gradient doses of NEM (0.5, 1.0, 1.6 mg/kg) (Yang et al., 2023) to assess the sleep effect, as illustrated in Fig. 1. Firstly, compared with Alp alone, the combination of gradient doses of NEM significantly decreased the sleep latency (Fig. 1A; p < 0.001), extended sleep duration of mice (Fig. 1B; p < 0.001), and increased the sleep ratio in different groups (Fig. 1C). Secondly, as the dosage of NEM increased, the sleep effect of NEM enhancing Alp appeared to become more pronounced. Finally, a dose–response study (Fig. 1D) illustrated that ED₅₀ of sleep effect induced by different doses of NEM (0.5, 1.0, 1.6 mg/kg) were 1.41, 0.95, and 0.91 mg/kg, correspondingly. Surprisingly, there was no notable increase in efficacy when NEM doses exceeded 1.0 mg/kg. Therefore, 1.0 mg/kg NEM was selected for subsequent combination administration.

Notably, treatment with NEM alone did not exhibit any hypnotic effects (Fig. S3). Furthermore, the sleep behaviors tests indicated that a combination of Alp at 1.84 mg/kg (ED₅₀) and NEM at 1.0 mg/kg aligns with the effect of administering 2.87 mg/kg Alp (ED₉₅) alone, as confirmed in EEG/EMG recording analysis (Fig. S4).

NEM significantly affects the architecture of Alp-induced sleep

The sleep quality is a crucial factor in assessing the effectiveness of any treatment aimed at improving sleep (Liu et al., 2020). Upon initial visual inspection, no discernible disparities in sleep quality were observed in mice that were administered with Alp alone compared to those combinedly administered Alp and NEM. Subsequently, post-treatment analysis of EEG/EMG recordings was conducted.

As shown in Fig. 2, compared to the control group, the duration of NREM (Fig. 2B; Alp vs. control, p = 0.0077; Alp@NEM vs. control, p < 0.0001) and REM (Fig. 2C; Alp vs. control, p < 0.0001; Alp@NEM vs. control, p < 0.0001) in the Alp and Alp@NEM groups was significantly prolonged, while the duration of awakening (Fig. 2A; Alp vs. control, p < 0.0001; Alp@NEM vs. control, p < 0.0001) was significantly reduced. Remarkably, the duration of REM in the Alp@NEM group was significantly longer than in the Alp group alone (Fig. 2C, p < 0.0001), suggesting that NEM enhanced the sleep-inducing effect of Alp by extending the REM portion of the sleep architecture.

Furthermore, the spatial distribution of mice in the home cage over a 24-hour period following each treatment was illustrated in Fig. 2D. Mice that treated with either the vehicle or NEM displayed random and frequent exploration of the entire cage. In contrast, mice that treated with Alp or a combination of Alp and NEM demonstrated markedly decreased activity (Fig. 2E; Alp vs. control, p < 0.0001; Alp@NEM vs. control, p < 0.0001) in comparison to the control group, indicating a pronounced sedative-hypnotic effect. This trajectory plot was almost consistent with our visual observations (Fig. S3).

NEM inhibits the neurons activation in the TMN instead of activating VLPO nucleus neurons in the hypothalamus

In order to assess the activation of neurons in various hypothalamic regions following exposure to either Alp or NEM, c-fos expression, which is a proto-oncogene that serves as a marker for neuronal activity (Luo et al., 2018), was examined through immunostaining of the hypothalamus. The examination centered on the VLPO and TMN crucial hypothalamic nuclei implicated in regulating the sleep-wake cycle.

Expressions of c-fos were assessed in the TMN and VLPO in all four groups. Prior research has shown that c-fos levels were increased in the VLPO and decreased in the TMN during sleep (Scammell et al., 2001). Our research findings revealed that c-fos expression in the VLPO was elevated in both the Alp and Alp@NEM groups in comparison to the control group (Figs. 3A and 3C; Alp vs. control, p = 0.0031; Alp@NEM vs. control, p = 0.0097). There were no significant differences observed in the NEM group. On the other hand, there was a significant decrease in c-fos levels in the TMN (Figd. 3B and 3D; Alp vs. control, p = 0.0092; NEM vs. control, p < 0.0001; Alp@NEM vs. control, p < 0.0001) in the Alp, NEM, and Alp@NEM groups compared to the control group. More importantly, the expression of c-fos in the Alp@NEM group was notably lower compared to the Alp group (p = 0.0042).

These findings suggested that NEM may impact sleep patterns by diminishing neuronal activity in regions associated with wakefulness like the TMN, as opposed to regions promoting sleep like the VLPO.

No side effects of the combination administration of Alp (ED₅₀) and NEM

The experiments demonstrated that combination administration 1.0 mg/kg NEM and 1.84 mg/kg (ED₅₀) Alp was nearly as effective as administrating 2.87 mg/kg Alp (ED₉₅) alone in inducing hypnotic effects (Fig. S3). However, the potential adverse effects were further investigated, including the impacts on general behaviors and well-being.

The results presented that the mice in the high-dose Alp (ED₉₅) groups had significant differences in climbing duration (Fig. 4A; p = 0.0095), locomotion duration (Fig. 4C; p = 0.0001), immobility duration (Fig. 4D; p = 0.0026), and distance (Fig. 4E, p = 0.01) in comparison to the control group. Conversely, no significant differences were observed in the remaining groups. The results from this study showed that the administration of Alp (ED₅₀), NEM, or a combination of both did not impact the spontaneous activity of locomotion. However, the highest dosage of Alp (ED₉₅) did hinder locomotor activity, correlating with the common side effects of drowsiness reported in clinical studies (Su et al., 2022).

Additionally, the results revealed significant differences in food intake (Fig. 5B; p < 0.0001) and water consumption (Fig. 5C; p < 0.0001) between the control group and the high-dose Alp (ED₉₅) groups. The results were consistent with the effects of dietary restriction (Haney et al., 1997) and increased water intake (Lobarinas & Falk, 2000) reported in Alp, and no significant differences were found in the other groups.

In summary, these findings suggested that the combined administration of Alp (ED₅₀) and NEM did not adversely affect overall behaviors and welfare in mice.