Face validation and pharmacologic analysis of Sik3Sleepy mutant mouse as a possible model of idiopathic hypersomnia.

Idiopathic hypersomnia (IH) is a chronic neurologic disorder with unknown mechanisms that result in long night-time sleep, daytime sleepiness, long non-refreshing naps, and difficult awakening presenting as sleep drunkenness. IH patients are typically diagnosed by shorter sleep latency on multiple sleep latency test (MSLT) along with long sleep time. Only symptomatic drug treatments are currently available for IH and no animal model to study it. Sleepy mice carry a splicing mutation in the Sik3 gene, leading to increased sleep time and sleep need. Here we used a mouse version of MSLT and a decay analysis of wake EEG delta power to validate the Sleepy mutant mouse as an animal model for IH. Sleepy mice had shorter sleep latency in the dark (active) phase than wild-type mice. They also showed lower decay of EEG delta density during wakefulness, possibly reflecting increased sleep inertia. These data indicate that the Sleepy mouse may have partial face validity as a mouse model for idiopathic hypersomnia. We then investigated the effect of orexin-A and the orexin receptor 2-selective agonist YNT-185 on the sleepiness symptoms of the Sleepy mouse. Intracerebroventricular orexin-A promoted wakefulness for 3 h and decreased wake EEG delta density after injection in Sleepy mice and wild-type mice. Moreover, Sleepy mice but not wild-type mice showed a sleep rebound after the orexin-A-induced wakefulness. Intraperitoneal YNT-185 promoted wakefulness for 3 h after injection in Sleepy mice, indicating the potential of using orexin agonists to treat not only orexin deficiency but hypersomnolence of various etiologies.

Positive allosteric adenosine A2A receptor modulation suppresses insomnia associated with mania- and schizophrenia-like behaviors in mice.

Background: Insomnia is associated with psychiatric illnesses such as bipolar disorder or schizophrenia. Treating insomnia improves psychotic symptoms severity, quality of life, and functional outcomes. Patients with psychiatric disorders are often dissatisfied with the available therapeutic options for their insomnia. In contrast, positive allosteric modulation of adenosine A2A receptors (A2ARs) leads to slow-wave sleep without cardiovascular side effects in contrast to A2AR agonists. Methods: We investigated the hypnotic effects of A2AR positive allosteric modulators (PAMs) in mice with mania-like behavior produced by ablating GABAergic neurons in the ventral medial midbrain/pons area and in a mouse model of schizophrenia by knocking out of microtubule-associated protein 6. We also compared the properties of sleep induced by A2AR PAMs in mice with mania-like behavior with those induced by DORA-22, a dual orexin receptor antagonist that improves sleep in pre-clinical models, and the benzodiazepine diazepam. Results: A2AR PAMs suppress insomnia associated with mania- or schizophrenia-like behaviors in mice. A2AR PAM-mediated suppression of insomnia in mice with mania-like behavior was similar to that mediated by DORA-22, and, unlike diazepam, did not result in abnormal sleep. Conclusion: A2AR allosteric modulation may represent a new therapeutic avenue for sleep disruption associated with bipolar disorder or psychosis.

Substrate-induced product-release mechanism of lipocalin-type prostaglandin D synthase.

Prostaglandin D2 (PGD2), an endogenous somnogen, is a unique PG that is secreted into the cerebrospinal fluid. PGD2 is a relatively fragile molecule and should be transported to receptors localized in the basal forebrain without degradation. However, it remains unclear how PGD2 is stably carried to such remote receptors. Here, we demonstrate that the PGD2-synthesizing enzyme, Lipocalin-type prostaglandin D synthase (L-PGDS), binds not only its substrate PGH2 but also its product PGD2 at two distinct binding sites for both ligands. This behaviour implys its PGD2 carrier function. Nevertheless, since the high affinity (Kd = âˆ¼0.6 µM) of PGD2 in the catalytic binding site is comparable to that of PGH2, it may act as a competitive inhibitor, while our binding assay exhibits only weak inhibition (Ki = 189 µM) of the catalytic reaction. To clarify this enigmatic behavior, we determined the solution structure of L-PGDS bound to one substrate analog by NMR and compared it with the two structures: one in the apo form and the other in substrate analogue complex with 1:2 stoichiometry. The structural comparisons showed clearly that open or closed forms of loops at the entrance of ligand binding cavity are regulated by substrate binding to two sites, and that the binding to a second non-catalytic binding site, which apparently substrate concentration dependent, induces opening of the cavity that releases the product. From these results, we propose that L-PGDS is a unique enzyme having a carrier function and a substrate-induced product-release mechanism.

Induction of narcolepsy-like symptoms by orexin receptor antagonists in mice.

Orexins/hypocretins are hypothalamic neuropeptides that promote and stabilize wakefulness by binding to the orexin receptor type-1 (OX1R) and type-2 (OX2R). Disruption of orexinergic signaling results in the sleep disorder narcolepsy in mice, rats, dogs, and humans. The orexin receptor antagonist suvorexant promotes sleep by blocking both OX1R and OX2R. Whereas suvorexant has been clinically approved for the treatment of insomnia because it is well tolerated in experimental animals as well as in human patients, a logical question remains as to why orexin receptor antagonists do not induce overt narcolepsy-like symptoms. Here we show that acute and chronic suvorexant promotes both rapid eye movement (REM) and non-rapid eye movement (NREM) sleep without inducing cataplexy in mice. Interestingly, chronic suvorexant increases OX2R mRNA and decreases orexin mRNA and peptide levels, which remain low long after termination of suvorexant administration. When mice are chronically treated with suvorexant and then re-challenged with the antagonist after a 1-week washout, however, cataplexy and sleep-onset REM (SOREM) are observed, which are exacerbated by chocolate administration. Heterozygous orexin knockout mice, with lower brain orexin levels, show cataplexy and SOREM after acute suvorexant administration. Furthermore, we find that acute suvorexant can induce cataplexy and SOREM in wild-type mice when co-administered with chocolate under stress-free (temporally anesthetized) conditions. Taken together, these results suggest that suvorexant can inhibit orexin synthesis resulting in susceptibility to narcolepsy-like symptoms in mice under certain conditions.

Acute Social Defeat Stress Increases Sleep in Mice.

Social conflict is a major source of stress in humans. Animals also experience social conflicts and cope with them by stress responses that facilitate arousal and activate sympathetic and neuroendocrine systems. The effect of acute social defeat (SoD) stress on the sleep/wake behavior of mice has been reported in several models based on a resident-intruder paradigm. However, the post-SoD stress sleep/wake effects vary between the studies and the contribution of specific effects in response to SoD or non-specific effects of the SoD procedure (e.g., sleep deprivation) is not well established. In this study, we established a mouse model of acute SoD stress based on strong aggressive mouse behavior toward unfamiliar intruders. In our model, we prevented severe attacks of resident mice on submissive intruder mice to minimize behavioral variations during SoD. In response to SoD, slow-wave sleep (SWS) strongly increased during 9 h. Although some sleep changes after SoD stress can be attributed to non-specific effects of the SoD procedure, most of the SWS increase is likely a specific response to SoD. Slow-wave activity was only enhanced for a short period after SoD and dissipated long before the SWS returned to baseline. Moreover, SoD evoked a strong corticosterone response that may indicate a high stress level in the intruder mice after SoD. Our SoD model may be useful for studying the mechanisms and functions of sleep in response to social stress.

Octacosanol and policosanol prevent high-fat diet-induced obesity and metabolic disorders by activating brown adipose tissue and improving liver metabolism.

Brown adipose tissue (BAT) is an attractive therapeutic target for treating obesity and metabolic diseases. Octacosanol is the main component of policosanol, a mixture of very long chain aliphatic alcohols obtained from plants. The current study aimed to investigate the effect of octacosanol and policosanol on high-fat diet (HFD)-induced obesity. Mice were fed on chow, or HFD, with or without octacosanol or policosanol treatment for four weeks. HFD-fed mice showed significantly higher body weight and body fat compared with chow-fed mice. However, mice fed on HFD treated with octacosanol or policosanol (HFDo/p) showed lower body weight gain, body fat gain, insulin resistance and hepatic lipid content. Lower body fat gain after octacosanol or policosanol was associated with increased BAT activity, reduced expression of genes involved in lipogenesis and cholesterol uptake in the liver, and amelioration of white adipose tissue (WAT) inflammation. Moreover, octacosanol and policosanol significantly increased the expression of Ffar4, a gene encoding polyunsaturated fatty acid receptor, which activates BAT thermogenesis. Together, these results suggest that octacosanol and policosanol ameliorate diet-induced obesity and metabolic disorders by increasing BAT activity and improving hepatic lipid metabolism. Thus, these lipids represent promising therapeutic targets for the prevention and treatment of obesity and obesity-related metabolic disorders.

The Leptomeninges Produce Prostaglandin D2 Involved in Sleep Regulation in Mice.

Injection of nanomolar amounts of prostaglandin D2 (PGD2) into the rat brain has dose and time-dependent somnogenic effects, and the PGD2-induced sleep is indistinguishable from physiologic sleep. Sleep-inducing PGD2 is produced in the brain by lipocalin-type PGD2 synthase (LPGDS). Three potential intracranial sources of LPGDS have been identified: oligodendrocytes, choroid plexus, and leptomeninges. We aimed at the identification of the site of synthesis of somnogenic PGD2 and therefore, generated a transgenic mouse line with the LPGDS gene amenable to conditional deletion using Cre recombinase (flox-LPGDS mouse). To identify the cell type responsible for producing somnogenic PGD2, we engineered animals lacking LPGDS expression specifically in oligodendrocytes (OD-LPGDS KO), choroid plexus (CP-LPGDS KO), or leptomeninges (LM-LPGDS KO). We measured prostaglandins and LPGDS concentrations together with PGD synthase activity in the brain of these mice. While the LPGDS amount and PGD synthase activity were drastically reduced in the OD- and LM-LPGDS KO mice, they were unchanged in the CP-LPGDS KO mice compared with control animals. We then recorded electroencephalograms, electromyograms, and locomotor activity to measure sleep in 10-week-old mice with specific knockdown of LPGDS in each of the three targets. Using selenium tetrachloride, a specific PGDS inhibitor, we demonstrated that sleep is inhibited in OD-LPGDS and CP-LPGDS KO mice, but not in the LM-LPGDS KO mice. We concluded that somnogenic PGD2 is produced primarily by the leptomeninges, and not by oligodendrocytes or choroid plexus.

A gain-of-function study of amelioration of pentylenetetrazole-induced seizures by endogenous prostaglandin D2.

We previously showed that knockout mice of hematopoietic prostaglandin (PG) D synthase (H-PGDS) produce less PGD2 to exacerbate pentylenetetrazole (PTZ)-induced seizures. Here, we adopted a gain-of-function strategy and used transgenic mice that over-express human H-PGDS enzyme, to elucidate the role of overproduction of endogenous PGD2 in PTZ-induced seizures. H-PGDS-transgenic mice showed the elevated level of a urinary metabolite of PGD2, tetranor-PGDM, 3.3- and 2.8-fold higher than the wild-type littermates under the basal condition and after the PTZ administration, respectively, without significantly changing the urinary concentration of a PGE2-metabolite, tetranor-PGE2. The intensity of PTZ-induced seizures was decreased in H-PGDS-transgenic mice as evident by the increased seizure onset latency, and a decrease in total duration of generalized tonic-clonic seizures and a total number of EEG seizure spikes during the postictal period (84 s, 17 s, and 5.3/min, respectively), as compared to wild-type mice (53 s, 24 s, and 12.6/min, respectively). These results indicate that overproduction of endogenous PGD2 decreased PTZ-induces seizures.

Continuous intrathecal orexin delivery inhibits cataplexy in a murine model of narcolepsy.

Narcolepsy-cataplexy is a chronic neurological disorder caused by loss of orexin (hypocretin)-producing neurons, associated with excessive daytime sleepiness, sleep attacks, cataplexy, sleep paralysis, hypnagogic hallucinations, and fragmentation of nighttime sleep. Currently, human narcolepsy is treated by providing symptomatic therapies, which can be associated with an array of side effects. Although peripherally administered orexin does not efficiently penetrate the blood-brain barrier, centrally delivered orexin can effectively alleviate narcoleptic symptoms in animal models. Chronic intrathecal drug infusion through an implantable pump is a clinically available strategy to treat a number of neurological diseases. Here we demonstrate that the narcoleptic symptoms of orexin knockout mice can be reversed by lumbar-level intrathecal orexin delivery. Orexin was delivered via a chronically implanted intrathecal catheter at the upper lumbar level. The computed tomographic scan confirmed that intrathecally administered contrast agent rapidly moved from the spinal cord to the brain. Intrathecally delivered orexin was detected in the brain by radioimmunoassay at levels comparable to endogenous orexin levels. Cataplexy and sleep-onset REM sleep were significantly decreased in orexin knockout mice during and long after slow infusion of orexin (1 nmol/1 µL/h). Sleep/wake states remained unchanged both quantitatively as well as qualitatively. Intrathecal orexin failed to induce any changes in double orexin receptor-1 and -2 knockout mice. This study supports the concept of intrathecal orexin delivery as a potential therapy for narcolepsy-cataplexy to improve the well-being of patients.

Natural (∆9-THC) and synthetic (JWH-018) cannabinoids induce seizures by acting through the cannabinoid CB1 receptor.

Natural cannabinoids and their synthetic substitutes are the most widely used recreational drugs. Numerous clinical cases describe acute toxic symptoms and neurological consequences following inhalation of the mixture of synthetic cannabinoids known as "Spice." Here we report that an intraperitoneal administration of the natural cannabinoid Δ9-tetrahydrocannabinol (10 mg/kg), one of the main constituent of marijuana, or the synthetic cannabinoid JWH-018 (2.5 mg/kg) triggered electrographic seizures in mice, recorded by electroencephalography and videography. Administration of JWH-018 (1.5, 2.5 and 5 mg/kg) increased seizure spikes dose-dependently. Pretreatment of mice with AM-251 (5 mg/kg), a cannabinoid receptor 1-selective antagonist, completely prevented cannabinoid-induced seizures. These data imply that abuse of cannabinoids can be dangerous and represents an emerging public health threat. Additionally, our data strongly suggest that AM-251 could be used as a crucial prophylactic therapy for cannabinoid-induced seizures or similar life-threatening conditions.

Octacosanol restores stress-affected sleep in mice by alleviating stress.

Octacosanol, a component of various food materials, possesses prominent biological activities and functions. It fights against cellular stress by increasing glutathione level and thus scavenging oxygen reactive species. However, its anti-stress activity and role in sleep induction remained elusive. We hypothesize that octacosanol can restore stress-affected sleep by mitigating stress. Cage change strategy was used to induce mild stress and sleep disturbance in mice, and effects of octacosanol administration on amount of sleep and stress were investigated. Results showed that octacosanol did not change rapid eye movement (REM) or non-REM (NREM) sleep compared to vehicle in normal mice. However, in cage change experiment, octacosanol induces significant increase in NREM sleep at doses of 100 and 200 mg/kg (75.7 ± 14.9 and 82.7 ± 9.3 min/5 h) compared to vehicle (21.2 ± 5.1 min/5 h), and decreased sleep latency. Octacosanol induced sleep by increasing number of sleep episodes and decreasing wake episode duration. Plasma corticosterone levels were significantly reduced after octacosanol (200 mg/kg) administration, suggesting a decrease in stress level. Octacosanol-induced changes in sleep-wake parameters in stressed-mice were comparable to the values in normal mice. Together, these data clearly showed that, though octacosanol does not alter normal sleep, it clearly alleviates stress and restore stress-affected sleep.

Triethylene glycol, an active component of Ashwagandha (Withania somnifera) leaves, is responsible for sleep induction.

Insomnia is the most common sleep complaint which occurs due to difficulty in falling asleep or maintaining it. Most of currently available drugs for insomnia develop dependency and/or adverse effects. Hence natural therapies could be an alternative choice of treatment for insomnia. The root or whole plant extract of Ashwagandha (Withania somnifera) has been used to induce sleep in Indian system of traditional home medicine, Ayurveda. However, its active somnogenic components remain unidentified. We investigated the effect of various components of Ashwagandha leaf on sleep regulation by oral administration in mice. We found that the alcoholic extract that contained high amount of active withanolides was ineffective to induce sleep in mice. However, the water extract which contain triethylene glycol as a major component induced significant amount of non-rapid eye movement sleep with slight change in rapid eye movement sleep. Commercially available triethylene glycol also increased non-rapid eye movement sleep in mice in a dose-dependent (10-30 mg/mouse) manner. These results clearly demonstrated that triethylene glycol is an active sleep-inducing component of Ashwagandha leaves and could potentially be useful for insomnia therapy.

Specific Targeting of the Basolateral Amygdala to Projectionally Defined Pyramidal Neurons in Prelimbic and Infralimbic Cortex.

Adjacent prelimbic (PL) and infralimbic (IL) regions in the medial prefrontal cortex have distinct roles in emotional learning. A complete mechanistic understanding underlying this dichotomy remains unclear. Here we explored targeting of specific PL and IL neurons by the basolateral amygdala (BLA), a limbic structure pivotal in pain and fear processing. In mice, we used retrograde labeling, brain-slice recordings, and adenoviral optogenetics to dissect connectivity of ascending BLA input onto PL and IL neurons projecting to the periaqueductal gray (PAG) or the amygdala. We found differential targeting of BLA projections to PL and IL cortex. Activating BLA projections evoked excitatory and inhibitory responses in cortico-PAG (CP) neurons in layer 5 (L5) of both PL and IL cortex. However, all inhibitory responses were polysynaptic and monosynaptic BLA input was stronger to CP neurons in IL cortex. Conversely, the BLA preferentially targeted corticoamygdalar (CA) neurons in layer 2 (L2) of PL over IL cortex. We also reveal that BLA input is projection specific by showing preferential targeting of L5 CP neurons over neighboring L3/5 CA neurons in IL cortex. We conclude by showing that BLA input is laminar-specific by producing stronger excitatory responses CA neurons in L3/5 compared with L2 in IL cortex. Collectively, this study reveals differential targeting of the BLA to PL and IL cortex, which depends both on laminar location and projection target of cortical neurons. Overall, our findings should have important implications for understanding the processing of pain and fear input by the PL and IL cortex.

Prostaglandin D(2) is crucial for seizure suppression and postictal sleep.

Epilepsy is a neurological disorder with the occurrence of seizures, which are often accompanied by sleep. Prostaglandin (PG) D2 is produced by hematopoietic or lipocalin-type PGD synthase (H- or L-PGDS) and involved in the regulation of physiological sleep. Here, we show that H-PGDS, L/H-PGDS or DP1 receptor (DP1R) KO mice exhibited more intense pentylenetetrazole (PTZ)-induced seizures in terms of latency of seizure onset, duration of generalized tonic-clonic seizures, and number of seizure spikes. Seizures significantly increased the PGD2 content of the brain in wild-type mice. This PTZ-induced increase in PGD2 was attenuated in the brains of L- or H-PGDS KO and abolished in L/H-PGDS KO mice. Postictal non-rapid eye movement sleep was observed in the wild-type and H-PGDS or DP2R KO, but not in the L-, L/H-PGDS or DP1R KO, mice. These findings demonstrate that PGD2 produced by H-PGDS and acting on DP1R is essential for seizure suppression and that the L-PGDS/PGD2/DP1R system regulates sleep that follows seizures.

Glutamate microinjection in the medial septum of rats decreases paradoxical sleep and increases slow wave sleep.

The role of the medial septum in suppressing paradoxical sleep and promoting slow wave sleep was suggested on the basis of neurotoxic lesion studies. However, these conclusions need to be substantiated with further experiments, including chemical stimulation studies. In this report, the medial septum was stimulated in adult male rats by microinjection of L-glutamate. Sleep-wakefulness was electrophysiologically recorded, through chronically implanted electrodes, for 2 h before the injection and 4 h after the injection. There was a decrease in paradoxical sleep during the first hour and an increase in slow wave sleep during the second hour after the injection. The present findings not only supported the lesion studies but also showed that the major role of the medial septum is to suppress paradoxical sleep.

Hypothalamic temperature: a key regulator in homeostatic restoration of sleep during chronic cold exposure in rats.

Exposure to cold ambient temperature (Ta) affects sleep-wake (S-W) state. The vigilance states on the other hand influence thermal status of the animals. Simultaneous recording of body temperature (Tb) with S-W is crucial to understand the homeostatic relationship between the two. In the present study we recorded both Tb and hypothalamic temperature (Thy) along with S-W, during acute and chronic exposure to mild cold (Ta). Electrooculogram (EOG), electroencephalogram (EEG) and electromyogram (EMG) electrodes were chronically implanted in rats to assess S-W. A thermocouple, near the preoptic area, and radio transmitter in the peritoneum, were implanted, to record Thy and Tb respectively. After three days of baseline recordings of S-W, Thy and Tb at Ta of 26 dergrees C, the rats were exposed to mild cold Ta (18 degrees C) for 28 days. All the parameters were recorded during cold exposure and also for five days after the termination of cold exposure. On the first day of cold exposure there was a decrease in slow wave sleep and paradoxical sleep, but they were restored by the 21st day of continued exposure. The Thy remained decreased throughout the cold exposure. Though the Tb showed a slight decrease on the first day of cold exposure, there was no appreciable change during the subsequent days. The Thy came back to near pre exposure level on termination of cod exposure. The decrease in Thy during mild cold exposure would have triggered cold defense mechanisms. Increase in wakefulness during acute cold exposure and non-shivering thermogenesis during chronic cold exposure are probably responsible for the maintenance of Tb. Decrease in Thy is probably the key trigger for initiating thermoregulatory measures to maintain Tb and homeostatic restoration of sleep.

Glutamate microinjection at the medial preoptic area enhances slow wave sleep in rats.

A large body of evidence has established the role of the medial preoptic area (mPOA) in regulation of slow wave sleep (SWS). Although the mPOA neurons contain excitatory neurotransmitter glutamate, its role in sleep-wakefulness is not known. In the present study microinjection of monosodium glutamate (40, 80 and 120 ng) into the mPOA augmented SWS. Earlier reports have shown enhancement of paradoxical sleep by glutamate in other brain areas.