睡眠を誘発する神経システムの解明

科学研究費助成事業 研究成果報告書：若手研究(B)2015-2017課題番号 : 15K1835

Involvement of S-nitrosylation of actin in inhibition of neurotransmitter release by nitric oxide

<p>Abstract</p> <p>Background</p> <p>The role of the diffusible messenger nitric oxide (NO) in the regulation of pain transmission is still a debate of matter, pro-nociceptive and/or anti-nociceptive. <it>S</it>-Nitrosylation, the reversible post-translational modification of selective cysteine residues in proteins, has emerged as an important mechanism by which NO acts as a signaling molecule. The occurrence of <it>S</it>-nitrosylation in the spinal cord and its targets that may modulate pain transmission remain unclarified. The "biotin-switch" method and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry were employed for identifying <it>S</it>-nitrosylated proteins.</p> <p>Results</p> <p>Here we show that actin was a major protein <it>S</it>-nitrosylated in the spinal cord by the NO donor, <it>S</it>-nitroso-<it>N</it>-acetyl-DL-penicillamine (SNAP). Interestingly, actin was <it>S</it>-nitrosylated, more in the S2 fraction than in the P2 fraction of the spinal homogenate. Treatment of PC12 cells with SNAP caused rapid <it>S</it>-nitrosylation of actin and inhibited dopamine release from the cells. Just like cytochalasin B, which depolymerizes actin, SNAP decreased the amount of filamentous actin cytoskeleton just beneath the membrane. The inhibition of dopamine release was not attenuated by inhibitors of soluble guanylyl cyclase and cGMP-dependent protein kinase.</p> <p>Conclusion</p> <p>The present study demonstrates that actin is a major <it>S</it>-nitrosylated protein in the spinal cord and suggests that NO directly regulates neurotransmitter release by <it>S</it>-nitrosylation in addition to the well-known phosphorylation by cGMP-dependent protein kinase.</p

Gating and the Need for Sleep: Dissociable Effects of Adenosine A1 and A2A Receptors

Roughly one-third of the human lifetime is spent in sleep, yet the reason for sleep remains unclear. Understanding the physiologic function of sleep is crucial toward establishing optimal health. Several proposed concepts address different aspects of sleep physiology, including humoral and circuit-based theories of sleep-wake regulation, the homeostatic two-process model of sleep regulation, the theory of sleep as a state of adaptive inactivity, and observations that arousal state and sleep homeostasis can be dissociated in pathologic disorders. Currently, there is no model that places the regulation of arousal and sleep homeostasis in a unified conceptual framework. Adenosine is well known as a somnogenic substance that affects normal sleep-wake patterns through several mechanisms in various brain locations via A1 or A2A receptors (A1Rs or A2ARs). Many cells and processes appear to play a role in modulating the extracellular concentration of adenosine at neuronal A1R or A2AR sites. Emerging evidence suggests that A1Rs and A2ARs have different roles in the regulation of sleep. In this review, we propose a model in which A2ARs allow the brain to sleep, i.e., these receptors provide sleep gating, whereas A1Rs modulate the function of sleep, i.e., these receptors are essential for the expression and resolution of sleep need. In this model, sleep is considered a brain state established in the absence of arousing inputs

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

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

L-PGDS-produced PGD2 in premature, but not in mature, adipocytes increases obesity and insulin resistance

Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) is responsible for the production of PGD2 in adipocytes and is selectively induced by a high-fat diet (HFD) in adipose tissue. In this study, we investigated the effects of HFD on obesity and insulin resistance in two distinct types of adipose-specific L-PGDS gene knockout (KO) mice: fatty acid binding protein 4 (fabp4, aP2)-Cre/L-PGDS flox/flox and adiponectin (AdipoQ)-Cre/L-PGDS flox/flox mice. The L-PGDS gene was deleted in adipocytes in the premature stage of the former strain and after maturation of the latter strain. The L-PGDS expression and PGD2 production levels decreased in white adipose tissue (WAT) under HFD conditions only in the aP2-Cre/L-PGDS flox/flox mice, but were unchanged in the AdipoQ-Cre/L-PGDS flox/flox mice. When fed an HFD, aP2-Cre/L-PGDS flox/flox mice significantly reduced body weight gain, adipocyte size, and serum cholesterol and triglyceride levels. In WAT of the HFD-fed aP2-Cre/L-PGDS flox/flox mice, the expression levels of the adipogenic, lipogenic, and M1 macrophage marker genes were decreased, whereas those of the lipolytic and M2 macrophage marker genes were enhanced or unchanged. Insulin sensitivity was improved in the HFD-fed aP2-Cre/L-PGDS flox/flox mice. These results indicate that PGD2 produced by L-PGDS in premature adipocytes is involved in the regulation of body weight gain and insulin resistance under nutrient-dense conditions

Slow-wave sleep is controlled by a subset of nucleus accumbens core neurons in mice

Sleep control is ascribed to a two-process model, a widely accepted concept that posits homoeostatic drive and a circadian process as the major sleep-regulating factors. Cognitive and emotional factors also influence sleep–wake behaviour; however, the precise circuit mechanisms underlying their effects on sleep control are unknown. Previous studies suggest that adenosine has a role affecting behavioural arousal in the nucleus accumbens (NAc), a brain area critical for reinforcement and reward. Here, we show that chemogenetic or optogenetic activation of excitatory adenosine A2A receptor-expressing indirect pathway neurons in the core region of the NAc strongly induces slow-wave sleep. Chemogenetic inhibition of the NAc indirect pathway neurons prevents the sleep induction, but does not affect the homoeostatic sleep rebound. In addition, motivational stimuli inhibit the activity of ventral pallidum-projecting NAc indirect pathway neurons and suppress sleep. Our findings reveal a prominent contribution of this indirect pathway to sleep control associated with motivation

Sleep and Wakefulness Are Controlled by Ventral Medial Midbrain/Pons GABAergic Neurons in Mice

Sleep–wake behavior is controlled by a wide range of neuronal populations in the mammalian brain. Although the ventral midbrain/pons (VMP) area is suggested to participate in sleep–wake regulation, the neuronal mechanisms have remained unclear. Here, we found that nonspecific cell ablation or selective ablation of GABAergic neurons by expressing diphtheria toxin fragment A in the VMP in male mice induced a large increase in wakefulness that lasted at least 4 weeks. In contrast, selective ablation of dopaminergic neurons in the VMP had little effect on wakefulness. Chemogenetic inhibition of VMP GABAergic neurons also markedly increased wakefulness. The wake-promoting effect of the VMP GABAergic neuron ablation or inhibition was attenuated to varying degrees by the administration of dopamine D1 or D2/3 receptor antagonists and abolished by the administration of both antagonists together. In contrast, chemogenetic activation of VMP GABAergic neurons very strongly increased slow-wave sleep and reduced wakefulness. These findings suggest that VMP GABAergic neurons regulate dopaminergic actions in the sleep–wake behavior of mice