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Honokiol (HNK) is a small-molecule lignin extracted from Magnolia Officinalis, demonstrating high potency in promoting nonrapid eye movement (NREM) sleep by modulating the benzodiazepine site of the GABAA receptor. However, the clinical use of HNK in the treatment of insomnia is restricted by its extremely low oral bioavailability. In the present work, enhanced oral bioavailability of HNK was achieved by loading it into poly lactide–glycolide acid microparticles (HNK-MP). After oral administration, HNK-MP demonstrated 15-fold increase of AUC0–12 h in comparison to free HNK. The maximum blood concentration (Cmax) of HNK in HNK-MP-treated rats was 3.6 μg/mL at 2 h after oral administration, which was 6.5-fold of that in free HNK-treated rats. Oral administration of HNK-MP (20 mg/kg) efficiently increased NREM sleep by 60% by enhancing the transition from wakefulness to NREM sleep in rats. The biosafety of HNK-MP was assessed in vivo, and no damage occurred in the gastrointestinal tract. The present study pr...

The aim of this work was to develop a simple computer-based sleep-scoring algorithm to detect the three vigilance states—rapid eye movement (REM) sleep, non-REM (NREM) sleep, and wakefulness—using the entire frequency domain with relative thresholds. A variety of frequencies and time-domain features were extracted from each 4-s epoch in retrospective 24-h sleep data sets from mice using an algorithm developed in Matlab version 7.0. This algorithm is composed of five steps: (1) determining the EMG–power ratio, (2) determining the three energy areas (high, middle, and low) using EMG–power ratio thresholds (e.g., 5.5 and 6), (3) determining the θ/δ ratio, (4) distinguishing wakefulness from NREM sleep using the θ/δ ratio in the middle-energy area, and (5) distinguishing REM from NREM sleep using the θ/δ ratio in the low-energy area. We were able to achieve a high degree (92%) of agreement between the results of this algorithm and the results of a waveform-recognition procedure. This algorithm should overcome the inconsistencies inherent in manual scoring and reduce the time required for expert analysis. This algorithm is a reliable and efficient tool for automated detection of the three vigilance states.

Ethanol has extensive effects on sleep and daytime alertness, causing premature disability and death. Adenosine, as a potent sleep-promoting substance, is involved in many cellular and behavioral responses to ethanol. However, the mechanisms of hypnotic effects of ethanol remain unclear. In this study, we investigated the role of adenosine in ethanol-induced sleep using C57BL/6Slac mice, adenosine A2A receptor (A2AR) knockout mice, and their wild-type littermates. The results showed that intraperitoneal injection of ethanol (3.0 g/kg) at 21:00 decreased the latency to non-rapid eye movement (NREM) sleep and increased the duration of NREM sleep for 5 h. Ethanol dose-dependently increased NREM sleep, which was consistent with decreases in wakefulness in C57BL/6Slac mice compared with their own control. Caffeine (5, 10, or 15 mg/kg), a nonspecific adenosine receptor antagonist, dose-dependently and at high doses completely blocked ethanol-induced NREM sleep when administered 30 min prior to (but not after) ethanol injection. Moreover, ethanol-induced NREM sleep was completely abolished in A2AR knockout mice compared with wild-type mice. These findings strongly indicate that A2AR is a key receptor for the hypnotic effects of ethanol, and pretreatment of caffeine might be a strategy to counter the hypnotic effects of ethanol.

GABA transporter subtype 1 (GAT1) constructs high affinity reuptake sites for GABA in the CNS and regulates GABAergic transmission. Compounds that inhibit GAT1 are targets often used for the treatment of epilepsy; however sedation has been reported as a side effect of these agents, indicating potential sedative and/or hypnotic uses for these compounds. In the current study, we observed the sleep behaviors of mice treated with NO-711, a selective GAT1 inhibitor, in order to elucidate the role of GAT1 in sleep-wake regulation during the active phase. The data revealed that NO-711 at a high dose of 10 mg/kg caused a marked enhancement of EEG activity in the frequency ranges of 3-25 Hz during wakefulness as well as rapid eye movement (REM) sleep. During the non-REM (NREM) sleep, NO-711 (10 mg/kg) elevated EEG activity in the frequency ranges of 1.5-6.75 Hz. Similar changes were found in mice treated with a low dose of 3 mg/kg. NO-711 administered i.p. at a dose of 1, 3 or 10 mg/kg significantly shortened the sleep latency of NREM sleep, increased the amount of NREM sleep and the number of NREM sleep episodes. NO-711 did not affect the sleep latency and the amount of REM sleep. NO-711 dose-dependently increased c-Fos expression in sleep-promoting nucleus of the ventrolateral preoptic area and median preoptic area. However, c-Fos expression was decreased in the wake-promoting nuclei, tuberomammillary nucleus and lateral hypothalamus. These results indicate that NO-711 can increase NREM sleep in mice.

The Dempster–Shafer (D–S) theory of evidence provides a powerful tool for combination of uncertainty information, and has been extensively applied to deal with uncertainty and vagueness. This paper shows a new approach to combine weighted interval-valued belief structures based on Atanassov’s intuitionistic fuzzy sets (A-IF) theory. Two numerical examples are provided to illustrate the rule of combination of weighted interval-valued belief structures. It is then found that this combination can lead to a good result in dealing with the corresponding belief structures in accordance with people’s cognitive thinking.

The suprachiasmatic nucleus (SCN) is a master circadian pacemaker. It regulates the 24 h timing of many physiological processes, including sleeping and waking. After bilateral SCN lesions, the circadian rhythm of the sleep wake cycle was completely eliminated, but the characteristics of the sleep wake profile were not well clarified. In the present study, bilateral suprachiasmatic nucleus lesions (SCNX) were created in rats to determine the effects of SCNX on the sleep-wake profile in terms of number of episodes, mean duration of each stage and number of transitions between stages. Although bilateral SCN lesions completely eliminated the circadian rhythm in the sleepwake cycle, there was no significant change in the total amount of daily sleep or wakefulness before and after lesions. However, the SCN-lesioned rats exhibited anincreased episode number of wake and non-rapid eye movement (NREM) sleep during dark period and a decreased one during light period. The mean duration of wakefulness decreased significantly during the dark period and increased during the light period. The number of transitions between NREM sleep and wakefulness increased during the dark period and decreased during the light period. The NREM sleep power density and total number of episodes, number of transitions, and mean duration were not significantly different from those of sham controls when viewed over 24 h. These results indicate that lesions of suprachiasmatic nucleus did not alter the total amount of daily sleep but did modify the structure of sleepwake profiles in rats.

SUMMARY Aims: Safranal (2,6,6-trimethyl-1,3-cyclohexadiene-1-carboxaldehyde, C10H14O) is an active ingredient in the saffron, which is used in traditional medicine. It has been reported to have sedative and anti-epileptic effects, but its hypnotic effects remain uncertain. The aim of this study was to evaluate effects of safranal on sleep-wake cycle. Methods: We established hypnotic-model mice treated with a low dose of pentobarbital 20 mg/kg, and administered different doses of safranal, vehicle, or diazepam. The change of sleep-wake cycle was assessed by sleep recording and c-Fos expression in the brain was analyzed by immunohistochemistry. Results: Safranal increased the duration of non-rapid eye movement (NREM) sleep, shortened NREM sleep latency, and enhanced the delta power activity of NREM sleep. Immunohistochemical evaluation revealed that safranal increased c-Fos expression in the ventrolateral preoptic nucleus (VLPO), one of the putative sleep centers, and decreased it in the arousal histaminergic tuberomammillary nuclei (TMN). Conclusion: These findings indicate that safranal enhances NREM sleep in pentobarbital-treated mice. The hypnotic effects of safranal may be related to the activation of the sleep-promoting neurons in the VLPO and the simultaneous inhibition of the wakefulness-promoting neurons in the TMN, suggesting that safranal may be a hypnotic substance.

One of the major inconveniences encountered in sleep studies is the time-consuming labor involved in electroencephalography (EEG) analysis. The choice of epoch duration is of prime importance and is highly related to the outcome of EEG analysis. This study was designed to find the optimal EEG epoch duration that accurately reveals the sleep-wake profiles of animals and relieve researchers of the laborious analysis of rodent sleep-wake parameters. We analyzed mouse and rat EEG signals with commonly used epoch durations (4, 8, 10, 20, and 30 s) and compared the resulting sleep-wake profiles in terms of amounts of sleep and wakefulness, number and duration of episodes, number of sleep-wake stage transitions, and EEG power spectra. There were no statistical differences in the amount and EEG power density of wakefulness, rapid eye movement (REM) and non-REM sleep among the 5 epoch durations we used. However, the shorter the epoch, the more numerous the stage transitions and the larger the episode number. The purpose of an experiment should be a main criterion for determining the optimal epoch length for the experiment. When the amount of each stage and power density of EEG signals are desired, a 30-s epoch duration is appropriate, to save time; and the results obtained are no different from those found by analysis of other epoch durations. If stage transition and duration and number of episodes are to be considered, a 4-s epoch duration is suggested as the best choice for analysis.

To avoid the stress encountered during oral drug administration, we implanted chronically a catheter into the stomach, and recorded electroencephalogram (EEG) and electromyogram, in freely moving rats to evaluate their sleep–wake pattern. Rats with catheters in their stomach did not exhibit any changes in sleep–wake profiles in terms of sleep amount, number of episodes and EEG power spectra. When administered through the catheter, caffeine (6 mg/kg) statistically increased wakefulness, as compared with the vehicle control. However, when given orally by hand restraint and gavage, it caused no increase in wakefulness, owing to the masking effect of this method, which caused increased wakefulness when saline was used by handling animals. These results indicate that oral administration through a chronic stomach catheter is a useful way for evaluating wake-promoting components.

Modafinil is a wake-promoting compound with low abuse potential used in the treatment of narcolepsy. Although the compound is reported to affect multiple neurotransmitter systems such as catecholamines, serotonin, glutamate, GABA, orexin, and histamine, however, the molecular mechanism by which modafinil increases wakefulness is debated. Herein we used dopamine (DA) D2receptor (D2R)-deficient mice combined with D1R- and D2R-specific antagonists to clarify the role of DA receptors in the arousal effects of modafinil. In wild-type mice, intraperitoneal modafinil induced wakefulness in a dose-dependent manner. Pretreatment with either D1R antagonist SCH23390 [R-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine] at 30 μg/kg or D2R antagonist raclopride at 2 mg/kg blocked the arousal effects of low-dose modafinil at 22.5 and 45 mg/kg. When modafinil was given at 90 and 180 mg/kg, pretreatment of D1R antagonist did not affect the wakefulness at all, whereas D2R antagonist significantly attenuated the wakefulness to the half level compared with vehicle control. Similarly, D2R knock-out (KO) mice exhibited attenuated modafinil-induced wakefulness. However, pretreatment of D2R KO mice with D1R antagonist completely abolished arousal effects of modafinil. These findings strongly indicate that dopaminergic D1R and D2R are essential for the wakefulness induced by modafinil.

Prostaglandin (PG) D 2 has been proposed to be essential for the initiation and maintenance of the physiological sleep of rats because intracerebroventricular administration of selenium tetrachloride (SeCl 4 ), a selective inhibitor of PGD synthase (PGDS), was shown to reduce promptly and effectively the amounts of sleep during the period of infusion. However, gene knockout (KO) mice of PGDS and prostaglandin D receptor (DP 1 R) showed essentially the same circadian profiles and daily amounts of sleep as wild-type (WT) mice, raising questions about the involvement of PGD 2 in regulating physiological sleep. Here we examined the effect of SeCl 4 on the sleep of WT and KO mice for PGDS and DP 1 R and that of a DP 1 R antagonist, ONO-4127Na, on the sleep of rats. The i.p. injection of SeCl 4 into WT mice decreased the PGD 2 content in the brain without affecting the amounts of PGE 2 and PGF 2α . It inhibited sleep dose-dependently and immediately after the administration during the light period when mice normally sleep, increasing the wake time; and the treatment with this compound resulted in a distinct sleep rebound during the following dark period. The SeCl 4 -induced insomnia was observed in hematopoietic PGDS KO mice but not at all in lipocalin-type PGDS KO, hematopoietic and lipocalin-type PGDS double KO or DP 1 R KO mice. Furthermore, the DP 1 R antagonist ONO-4127Na reduced sleep of rats by 30% during infusion into the subarachnoid space under the rostral basal forebrain at 200 pmol/min. These results clearly show that the lipocalin-type PGDS/PGD 2 /DP 1 R system plays pivotal roles in the regulation of physiological sleep.

To investigate tobacco-related knowledge, attitudes and analyze related factors among the college students in Guangzhou.In May 2012, 11 593 college students from six universities in Guangzhou were selected and investigated by stratified cluster random sampling.Investigation content includes social demographic information, smoking behaviors, tobacco-related knowledge and attitudes. Chi-square test was used to analyze the difference about knowledge of harm of tobacco and awareness of tobacco control related legislation as well as tobacco-related attitudes between smokers and non-smokers. Factors on tobacco-related knowledge were analyzed by using rank sum test.Current smoking rate among undergraduates was 6.1% (706/11 593) , 11.5% (622/5388) for males and 1.4% (84/6205) for females. The awareness rate of that smoking addiction was a chronic disease in non-smokers(82.6%, 8954/10 836) was higher than that in smokers (73.1%, 509/696) (χ(2) = 40.09, P0.01). The awareness rate about smoking could cause emphysema in college students was 78.6% (8986/11 427) , and the rate was higher in non-smokers(79.3%, 8522/10 741) than that in smokers(67.6%, 464/686)(χ(2) = 52.57, P0.01). The awareness rate about passive smoking could cause lung cancer in college students was 84.6% (9636/11 391) , and the rate was higher in non-smokers (85.2%, 9125/10 706) than that in smokers (74.6%, 511/685) (χ(2) = 55.86, P0.01). The awareness about Framework Convention on Tobacco Control was 25.7% (2966/11 554) , and the rate was lower in non-smokers (25.3%, 2751/10 856) than that in smokers (30.7%, 215/700) (χ(2) = 9.80, P0.01). Among smokers, 54.8% (377/688) considered that smoking was enjoyful. This was higher than that in non-smokers (16.8%, 1802/10 752) (χ(2) = 606.92, P0.05). Among non-smokers, 92.2% (9935/10 781) considered that government should strengthen smoking control. The percentage was higher than that in smokers (74.2%, 515/694) (χ(2) = 258.13, P0.05). College students who were females, at high-grade and with high parental educational level, high monthly household income, high living expenses per month in school and household registration in towns and places out of Guangdong province showed higher tobacco-related knowledge score (all P values0.01).The awareness of tobacco-related knowledge was not high generally among college students in Guangzhou, but the awareness was significantly higher in non-smokers than that of smokers. The related factors which influenced the awareness rate of tobacco-related knowledge include sex, grade, place of household registration, parent's educational level and household income.

In humans, a first night effect (FNE) is characterized by increased sleep latency and decreased total sleep time in an unfamiliar environment, but the mechanism and treatment options for this universally experienced acute insomnia are unclear. We continuously recorded electroencephalography (EEG) and electromyogram (EMG) and measured plasma corticosterone levels to develop a mouse FNE model by inducing acute insomnia in mice that have been placed in unfamiliar cage environments. The sleep latency of mice 'moved to clean cages' (MCC) was longer than that for mice 'moved to dirty ones' (MDC). As compared to MDC mice, MCC mice showed stronger decreases in the amount of non-rapid eye movement (non-REM, NREM) and REM sleep, with a lower power density of NREM sleep, increased fragmentation and decreased stage transitions from NREM sleep to wake, and higher variation in plasma corticosterone levels. Treatment of MCC mice with zolpidem, diazepam, raclopride, pyrilamine, except SCH23390 shortened NREM sleep latency. In addition, zolpidem significantly increased NREM and REM sleep with the increase in slow wave activity (1.00-2.75 Hz), while raclopride significantly increased NREM and REM sleep without changing the EEG power density in MCC mice, whereas diazepam increased sleep with a drastic decrease in power density of the frequency band between 1.00 and 4.00 Hz, diazepam also increased the frequency band between 9.75 and 24.75 Hz during NREM sleep. These results indicate that a MCC mouse can mimic a FNE phenotype of humans and that zolpidem and raclopride may be useful drugs to prevent acute insomnia, including FNE.

GABA is the major inhibitory neurotransmitter in the mammalian central nervous system that has been strongly implicated in the regulation of sleep. GABA transporter subtype 1 (GAT1) constructs high affinity reuptake sites for GABA and regulates GABAergic transmission in the brain. However, the role of GAT1 in sleep-wake regulation remains elusive. In the current study, we characterized the spontaneous sleep-wake cycle and responses to sleep deprivation in GAT1 knock-out (KO) mice. GAT1 KO mice exhibited dominant theta-activity and a remarkable reduction of EEG power in low frequencies across all vigilance stages. Under baseline conditions, spontaneous rapid eye movement (REM) sleep of KO mice was elevated both during the light and dark periods, and non-REM (NREM) sleep was reduced during the light period only. KO mice also showed more state transitions from NREM to REM sleep and from REM sleep to wakefulness, as well as more number of REM and NREM sleep bouts than WT mice. During the dark period, KO mice exhibited more REM sleep bouts only. Six hours of sleep deprivation induced rebound increases in NREM and REM sleep in both genotypes. However, slow wave activity, the intensity component of NREM sleep was briefly elevated in WT mice but remained completely unchanged in KO mice, compared with their respective baselines. These results indicate that GAT1 plays a critical role in the regulation of REM sleep and homeostasis of NREM sleep.

Aims: Safranal (2,6,6‐trimethyl‐1,3‐cyclohexadiene‐1‐carboxaldehyde, C(10)H(14)O) is an active ingredient in the saffron, which is used in traditional medicine. It has been reported to have sedative and anti‐epileptic effects, but its hypnotic effects remain uncertain. The aim of this study was to evaluate effects of safranal on sleep‐wake cycle. Methods: We established hypnotic‐model mice treated with a low dose of pentobarbital 20 mg/kg, and administered different doses of safranal, vehicle, or diazepam. The change of sleep‐wake cycle was assessed by sleep recording and c‐Fos expression in the brain was analyzed by immunohistochemistry. Results: Safranal increased the duration of non‐rapid eye movement (NREM) sleep, shortened NREM sleep latency, and enhanced the delta power activity of NREM sleep. Immunohistochemical evaluation revealed that safranal increased c‐Fos expression in the ventrolateral preoptic nucleus (VLPO), one of the putative sleep centers, and decreased it in the arousal histaminergic tuberomammillary nuclei (TMN). Conclusion: These findings indicate that safranal enhances NREM sleep in pentobarbital‐treated mice. The hypnotic effects of safranal may be related to the activation of the sleep‐promoting neurons in the VLPO and the simultaneous inhibition of the wakefulness‐promoting neurons in the TMN, suggesting that safranal may be a hypnotic substance.

Dopamine (DA) and its D2receptor (R) are involved in cognition, reward processing, and drug addiction. However, their roles in sleep–wake regulation remain unclear. Herein we investigated the role of D2R in sleep–wake regulation by using D2R knock-out (KO) mice and pharmacological manipulation. Compared with WT mice, D2R KO mice exhibited a significant decrease in wakefulness, with a concomitant increase in non-rapid eye movement (non-REM, NREM) and REM sleep and a drastic decrease in the low-frequency (0.75–2 Hz) electroencephalogram delta power of NREM sleep, especially during the first 4 h after lights off. The KO mice had decreased mean episode duration and increased episode numbers of wake and NREM sleep, many stage transitions between wakefulness and NREM sleep during the dark period, suggesting the instability of the wake stage in these D2R KO mice. When the KO mice were subjected to a cage change or an intraperitoneal saline injection, the latency to sleep in the KO mice decreased to half of the level for WT mice. The D2R antagonist raclopride mimicked these effects in WT mice. When GBR12909, a dopamine transport inhibitor, was administered intraperitoneally, it induced wakefulness in WT mice in a dose-dependent manner, but its arousal effect was attenuated to one-third in the D2R KO mice. However, these 2 genotypes showed an identical response in terms of sleep rebound after 2, 4, and 6 h of sleep deprivation. These results indicate that D2R plays an essential role in the maintenance of wakefulness, but not in homeostatic regulation of NREM sleep.

L-stepholidine, an active ingredient of the Chinese herb Stephonia, is the first compound known to have mixed dopamine D1 receptor agonist/D2 antagonist properties and to be a potential treatment medication for schizophrenia. In schizophrenic patients insomnia is a common symptom and could be partly related to the presumed over-activity of the dopaminergic system. To elucidate whether stepholidine modulates sleep behaviors, we observed its effects on sleep–wake profiles in mice. The results showed that stepholidine administered i.p. at doses of 20, 40 or 80 mg/kg significantly shortened the sleep latency to non-rapid eye movement (non-REM, NREM) sleep, increased the amount of NREM sleep, and prolonged the duration of NREM sleep episodes, with a concomitant reduction in the amount of wakefulness. Stepholidine at doses of 40 and 80 mg/kg increased the number of state transitions from wakefulness to NREM sleep and subsequently from NREM sleep to wakefulness. However, stepholidine had no effect on either the amount of REM sleep or electroencephalogram power density of either NREM or REM sleep. Immunohistochemistry study showed that stepholidine dose-dependently increased c-Fos expression in neurons of the ventrolateral preoptic area, a sleep center in the anterior hypothalamus, as compared with the vehicle control. These results indicate that stepholidine initiates and maintains NREM sleep with activation of the sleep center in mice, suggesting its potential application for the treatment of insomnia.

To exam the effects of allopregnanolone and gamma-amino-butyric acid (GABA) on the excitotoxicity.The excitotoxicity was evoked by kainate (KA) in the primary culture of rat cerebral cortical cells. Effect of allopregnanolone or GABA on the excitotoxicity was examined by the measurement of lactate dehydrogenase (LDH) activity released in the culture medium.Either acute (3 h) or chronic (24 h) treatment with KA (0.01-1 mmol/L) produced a concentration-dependent increase in LDH activity released. The EC50 values were (0.16+/-0.03) mmol/L and (0.257+/-0.015) mmol/L, respectively. Acute treatment with allopregnanolone (10-1000 nmol/L) for 3 h did not significantly affect the 0.2 mmol/L KA-induced LDH activity. On the other hand, chronic treatment with allopregnanolone (10-1000 nmol/L) for 24 h, produced inhibition on the KA-induced LDH activity in a concentration-dependent manner. The EC50 value was (436+/-19) nmol/L. Acute treatment with GABA (0.1-100 micromol/L) exacerbate the 0.2 mmol/L KA-induced LDH activity in a concentration-dependent manner, with an EC50 value of (2.7+/-1.0) micromol/L; while chronic treatment with GABA had no significant effect.There were differential patterns between the effects of allopregnanolone and GABA on the KA-induced excitotoxicity.