細胞内カルシウムと睡眠 Intracellular calcium and sleep

細胞内カルシウム濃度を制御するチャネルやポンプが睡眠の長さを決めるということ（睡眠制御遺伝子であること）を提唱した論文が出ました。睡眠の本質があぶり出されつつあると感じます。

Tatsuki F, Sunagawa GA, Shi S, Susaki EA, Yukinaga H, Perrin D, Sumiyama K, Ukai-Tadenuma M, Fujishima H, Ohno R, Tone D, Ode KL, Matsumoto K, Ueda HR. 

Involvement of Ca2+-dependent hyperpolarization in sleep duration in mammals. 

Neuron 2016 AOP  doi: 10.1016/j.neuron.2016.02.032.

プレスリリースはこちら

This paper proposed the idea that intracellular calcium is involved in animal's sleep time, which may be the essential of this biological function.

Press release in English

「平均的ニューロンモデル」を作成し、睡眠時・覚醒時の神経活動の状態をシミュレーションしました。シミュレーターに組み込まれた分子群を仮想ノックアウトしていくと、上記の分子群が睡眠制御遺伝子候補として引っかかってきました。

だいたいこういう遺伝子は「似た者」がいくつかあるのが通常です。そこで、最近話題のCRISPR技術を使って片っ端からファミリー分子のノックアウトマウスを作製し、表現型解析を行いました。

さらに、胎生致死になってしまう遺伝子については、阻害剤の投与で薬理学的に検証。細胞内カルシウムの流入を抑えると、脳全体で神経活動がupregulateされる（つまり寝にくくなってしまう）ことを示しています。

このような大規模な解析を実現するため、近年開発された「個体レベルのシステム生物学」のための技術が活用されました。一世代でノックアウトマウスの作製が可能なTriple-CRISPRシステム [Sunagawa et al. Cell Reports 2016]、マウスを測定チャンバーに入れるだけで睡眠測定が可能なSSS [同上]、全脳スケールで神経細胞の活動をイメージングし解析するCUBIC [Susaki et al. Cell 2014] などです。これらの技術は既存法のスループットを1オーダー以上改善できるものばかりです。

The idea was examined by simulations of an averaged neuron model and following animal sleep analysis of KO mice of 21 genes (!) and a high throughput and non-invasive sleep analysis system. Upregulated neural activities by inhibiton of Ca channel were observed by whole-brain imaging. These technologies has been developed for 'organism-level systems biology' in mammal.

このような規模の研究は、これらの技術がより普及するにつれ、長期的には一般的なものになっていく可能性があります。

ちなみに、「カルシウムを摂れば不眠に効く！？」みたいな感想がちらほらありますが、これはちょっと違う話です ^^;

Reference :

Susaki EA et al. 

Whole-Brain Imaging with Single-Cell Resolution Using Chemical Cocktails and Computational Analysis. 

Cell 157, 726–739, 2014. 

Sunagawa GA et al.

Mammalian Reverse Genetics without Crossing Reveals Nr3a as a Short-Sleeper Gene.

Cell reports 14, 662-677, 2016.

関連記事/related article

Proc Natl Acad Sci U S A. 2010;107(8):3829-33

Thalamic deactivation at sleep onset precedes that of the cerebral cortex in humans.

Magnin M, Rey M, Bastuji H, Guillemant P, Mauguiere F, Garcia-Larrea L.

Thalamic and cortical activities are assumed to be time-locked throughout all vigilance states. Using simultaneous intracortical and intrathalamic recordings, 

we demonstrate here that the thalamic deactivation occurring at sleep onset most often precedes that of the cortex by several minutes, whereas reactivation of both structures during awakening is synchronized. Delays between thalamus and cortex deactivations can vary from one subject to another when a similar cortical region is considered. In addition, heterogeneity in activity levels throughout the cortical mantle is larger than previously thought during the descent into sleep. Thus, asynchronous thalamo-cortical deactivation while falling asleep probably explains the production of hypnagogic hallucinations by a still-activated cortex and the common self-overestimation of the time needed to fall asleep.

＝＝＝

●入眠時にThalamusのほうがcortexより活動が早く（数分～数十分）ことを、ヒト検体を用いて検証。覚醒時はほぼ同時。

●the dimention of activation (DA)という、波形解析のアルゴリズムを用いて脳波を解析→大きな同期性徐波が出る睡眠時は数値が低下、ランダムで細かな波がでる覚醒時は数値が増加

Molecular Psychiatry 15:154-165, 2010

Conditional corticotropin-releasing hormone overexpression in the mouse forebrain enhances rapid eye movement sleep

Impaired sleep and enhanced stress hormone secretion are the hallmarks of stress-related disorders, including major depression. The central neuropeptide, corticotropin-releasing hormone (CRH), is a key hormone that regulates humoral and behavioral adaptation to stress. Its prolonged hypersecretion is believed to play a key role in the development and course of depressive symptoms, and is associated with sleep impairment. To investigate the specific effects of central CRH overexpression on sleep, we used conditional mouse mutants that overexpress CRH in the entire central nervous system (CRH-COE-Nes) or only in the forebrain, including limbic structures (CRH-COE-Cam). Compared with wild-type or control mice during baseline, both homozygous CRH-COE-Nes and -Cam mice showed constantly increased rapid eye movement (REM) sleep, whereas slightly suppressed non-REM sleep was detected only in CRH-COE-Nes mice during the light period. In response to 6-h sleep deprivation, elevated levels of REM sleep also became evident in heterozygous CRH-COE-Nes and -Cam mice during recovery, which was reversed by treatment with a CRH receptor type 1 (CRHR1) antagonist in heterozygous and homozygous CRH-COE-Nes mice. The peripheral stress hormone levels were not elevated at baseline, and even after sleep deprivation they were indistinguishable across genotypes. As the stress axis was not altered, sleep changes, in particular enhanced REM sleep, occurring in these models are most likely induced by the forebrain CRH through the activation of CRHR1. CRH hypersecretion in the forebrain seems to drive REM sleep, supporting the notion that enhanced REM sleep may serve as biomarker for clinical conditions associated with enhanced CRH secretion.

＝＝＝

●Camk2a-CRE x R26-CRHの系で、BFにCRHを発現
●ストレス→corticosterone系について。しかしデータは微妙。上がるのがREMってのも・・・

PNAS 103:17949-17954, 2006

 Lipocalin-type prostaglandin D synthase produces prostaglandin D2 involved in regulation of physiological sleep

Prostaglandin (PG) D2 has been proposed to be essential for the initiation and maintenance of the physiological sleep of rats because intracerebroventricular administration of selenium tetrachloride (SeCl4), 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 (DP1R) showed essentially the same circadian profiles and daily amounts of sleep as wild-type (WT) mice, raising questions about the involvement of PGD2 in regulating physiological sleep. Here we examined the effect of SeCl4 on the sleep of WT and KO mice for PGDS and DP1R and that of a DP1R antagonist, ONO-4127Na, on the sleep of rats. The i.p. injection of SeCl4 into WT mice decreased the PGD2 content in the brain without affecting the amounts of PGE2 and PGF2α. 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 SeCl4-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 DP1R KO mice. Furthermore, the DP1R 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/PGD2/DP1R system plays pivotal roles in the regulation of physiological sleep.

 　＝＝＝

●SeCl4（PGD synthaseの阻害剤）を入れると、sleep↓

●DP1R antagonistのONO-4127Naを入れると、sleep↓

●しかし、DP1R-KOマウスでは、sleepの量もcircadianも差がない

●L-PGDS-KOマウスでも、sleepの量もcircadianも差がない

  (Ref.)

↓

●PGD2以外のalternative pathwayによるcompensationの問題/阻害剤のspecificityの問題

●DP1R-KOマウスでSeCl4を入れるとどうなる？

↓

●H-PGDS、L-PGDS、DP1R-KOマウスでSeCl4を入れたら、、L-PGDS、DP1R-KOマウスではsleep↓の効果が見えなくなった。ということは、acuteの効果はやはり見える。

●sleep↓の後のリバウンドは、L&H-PGDS-DKOにすると消える。

●いずれにしても、circadianはがっつり残る→睡眠ホメオスタシスと日内変動のメカニズムがどう絡み合うのか？？

 

Current Opinion in Pharmacology 7:33-38, 2007 (Review)

Prostaglandins and adenosine in the regulation of sleep and wakefulness

Prostaglandin (PG) D2 and adenosine are potent humoral sleep-inducing factors that accumulate in the brain during prolonged wakefulness. PGD2 is produced in the brain by lipocalin-type PGD synthase, which is localized mainly in the leptomeninges, choroid plexus and oligodendrocytes, and circulates in the cerebrospinal fluid as a sleep hormone. It stimulates DP1 receptors on leptomeningeal cells of the basal forebrain to release adenosine as a paracrine signaling molecule to promote sleep. Adenosine activates adenosine A2A receptor-expressing sleep-active neurons in the basal forebrain and the ventrolateral preoptic area. Sleep-promoting neurons in the ventrolateral preoptic area send inhibitory signals to suppress the histaminergic neurons in the tuberomammillary nucleus, which contribute to arousal through histamine H1 receptors. Increased knowledge of the molecular mechanisms by which PGD2 induces sleep through activation of adenosine A2A receptors and inhibition of the histaminergic arousal system will be useful both for a better understanding of sleep/wake regulation and for the development of novel types of sleeping pills or anti-doze drugs.

＝＝＝

●sleep deprivationでは、PGD2もadenosineも量が上がる。

●DP1R-KOでもcircadianに差がないそう・・・
adenosineの上流であることは分かった。

●A1R→BFのCholinergic neuron？
あまり重要な役回りではなさそう

●A2AR→caffeineのターゲット

●＜重要＞A1R-KOも、A2AR-KOも、normalなcircadian profilesを示し、NREM/REMの量もWTと変わらない。reboundについてはA2AR-KOでは差が出る（A1R-KOは差なし)。

●PGD2/A2ARアゴニスト投与後のc-Fos染色で、sleep-active neurons（VLPO）が同定された。
ref.: 32, 33

●NA, Ach, A1R signal---| VLPO

（serotonineとadenosineへの反応性の違いで、Type1/2に分けられている）
●5-HT ---| VLPO Type1 neuron
   ↓
VLPO Type2 neuron
●CGS21680　→　VLPO Type2 neuron

●Figure 1がよくまとまっている。

●全体的なインプレ：
・KOマウスで差が出ない～通常のS-Wサイクルで働いているというより、S-Wサイクルのコントロールをしているニューロンの活性を著しく変動させている、という感じ？Adenosineは日内変動がある不思議。

・Thresholdの形成要因は？細胞の外部/内部因子の関わり？

●VLPOのGABA neuron　→　TMNのヒスタミン、となっているが、それだけだろうか？

Sleep. 33(1):19-28, 2010.

Genetic evidence for a role for protein kinase A in the maintenance of sleep and thalamocortical oscillations.

STUDY OBJECTIVES: Genetic manipulation of cAMP-dependent protein kinase A (PKA) in Drosophila has implicated an important role for PKA in sleeplwake state regulation. Here, we characterize the role of this signaling pathway in the regulation of sleep using electroencephalographic (EEG) and electromyographic (EMG) recordings in R(AB) transgenic mice that express a dominant negative form of the regulatory subunit of PKA in neurons within cortex and hippocampus. Previous studies have revealed that these mutant mice have reduced PKA activity that results in the impairment of hippocampus-dependent long-term memory and long-lasting forms of hippocampal synaptic plasticity. DESIGN: PKA assays, in situ hybridization, immunoblots, and sleep studies were performed in R(AB) transgenic mice and wild-type control mice. MEASUREMENTS AND RESULTS: We have found that R(AB) transgenic mice have reduced PKA activity within cortex and reduced Ser845 phosphorylation of the glutamate receptor subunit GluR1. R(AB) transgenic mice exhibit non-rapid eye movement (NREM) sleep fragmentation and increased amounts of rapid eye movement (REM) sleep relative to wild-type mice. Further, R(AB) transgenic mice have more delta power but less sigma power during NREM sleep relative to wild-type mice. After sleep deprivation, the amounts of NREM and REM sleep were comparable between wild-type and R(AB) transgenic mice. However, the homeostatic rebound of sigma power in R(AB) transgenic mice was reduced. CONCLUSIONS: Alterations in cortical synaptic receptors, impairments in sleep continuity, and alterations in sleep oscillations in R(AB) mice imply that PKA is involved not only in synaptic plasticity and memory storage but also in the regulation of sleep/wake states.

＝＝＝

PKA-GluRの経路について、Tgマウスによる考察。

＝＝＝

Neurosci Lett.469(1):1-5, 2010

Sleep fragmentation reduces hippocampal CA1 pyramidal cell excitability and response to adenosine.

Sleep fragmentation (SF) impairs the restorative/cognitive benefits of sleep via as yet unidentified alterations in neural physiology. Previously, we found that hippocampal synaptic plasticity and spatial learning are impaired in a rat model of SF which utilizes a treadmill to awaken the animals every 2 min, mimicking the frequency of awakenings observed in human sleep apnea patients. Here, we investigated the cellular mechanisms responsible for these effects, using whole-cell patch-clamp recordings. 24h of SF decreased the excitability of hippocampal CA1 pyramidal neurons via decreased input resistance, without alterations in other intrinsic membrane or action potential properties (when compared to cage controls, or to exercise controls that experienced the same total amount of treadmill movement as SF rats). Contrary to our initial prediction, the hyperpolarizing response to bath applied adenosine (30 microM) was reduced in the CA1 neurons of SF treated rats. Our initial prediction was based on the evidence that sleep loss upregulates cortical adenosine A1 receptors; however, the present findings are consistent with a very recent report that hippocampal A1 receptors are not elevated by sleep loss. Thus, increased adenosinergic inhibition is unlikely to be responsible for reduced hippocampal long-term potentiation in SF rats. Instead, the reduced excitability of CA1 pyramidal neurons observed here may contribute to the loss of hippocampal long-term potentiation and hippocampus-dependent cognitive impairments associated with sleep disruption. (c) 2009 Elsevier Ireland Ltd. All rights reserved.

＝＝＝

電気生理のFigが2個だけ。