The biochemical correlates of sleep homeostasis

睡眠稳态的生化相关性

• 批准号: 8576571
• 负责人: Anna Kalinchuk
• 金额: $ 31.74万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-08-15 至 2017-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8576571
• 关键词: AMPA Receptors; Acute; Adenosine; Affect; Alleles; Animal Model; Apoptotic; Behavioral; Biochemical; Biochemical Pathway; Brain; Cell Death; Cells; Chronic; Clinical; Cognition; Cognitive deficits; Data; Development; Feedback; Gene Expression; Generations; Glutamates; Health; Homeostasis; Immunoglobulin G; Immunohistochemistry; Impaired cognition; Indium; Investigation; Knowledge; Lesion; Life; Measures; Mediating; Mental Health; Microdialysis; Moods; N-Methylaspartate; Neurons; Nitrates; Nitrites; Performance; Pharmacological Treatment; Phosphorylation; Production; Recovery; Research; Role; Sampling; Site; Sleep; Sleep Deprivation; Source; Staging; Synaptic plasticity; System; Testing; Time; Transgenic Mice; Work; adeno-associated viral vector; alertness; basal forebrain; cholinergic; combat; density; extracellular; frontal lobe; human NOS2A protein; inhibitor/antagonist; interdisciplinary approach; novel; postsynaptic; pressure; prevent; receptor; recombinase; trafficking; treatment strategy; vesicular glutamate transporter 2

DESCRIPTION (provided by applicant): Sleep homeostasis is a powerful mechanism, which forces us to sleep over and above daily requirements after a sleep loss or sleep deprivation (SD). SD increases homeostatic sleep pressure (HSP), which is manifested as increased sleepiness and impaired cognition, while recovery sleep (RS) reverses these consequences. The investigation of the biochemical changes, triggered in the brain during SD, is one of the key steps towards counteracting the deleterious effects of sleep loss. Extensive research to date has identified the cholinergic basal forebrain (BF) as an important site in sleep homeostasis. Recently, we discovered a biochemical cascade, which is triggered in the BF during SD and promotes RS: it begins with a rapid induction of inducible nitric oxide synthase (iNOS) followed by the increases in NO and extracellular adenosine [AD]ex, first in the BF (1-3h SD) and later in the prefrontal/frontal cortex (PFC/FC) (4-6h SD). The current proposal aims to extend our knowledge on biochemical correlates of sleep homeostasis. We propose that the SD is a qualitatively and quantitatively distinctive state, which is generated by the cross-talk between the BF and PFC/FC nitrinergic, adenosinergic, glutamatergic and cholinergic systems. We see this interaction as central in building up HSP, increase in sleepiness and in impaired performance. We propose following studies to test our hypothesis. Specific Aim 1 (SA1). To investigate whether the increase in extracellular glutamate [Glu]ex is the trigger for iNOS NO AD cascade in the BF and PFC/FC. Our preliminary data (PD) show that the BF [Glu]ex increases within 30min after the beginning of SD, preceding the increases in nitrate/nitrite [NOx-]ex and [AD]ex. Also, we will investigate the consequences of genetically manipulated decrease in BF [Glu]ex. We also show that SD- induced [AD]ex prevents progressive increase in [Glu]ex during SD and thus protects brain from excitotoxic damage. SA2. To investigate whether cholinergic (ChBF) cells are key elements in triggering iNOS->NO->AD cascade in the BF and PFC/FC. Our PD show that iNOS is induced mostly in ChBF cells. Also our PD show that the lesion of ChBF cells using 192 IgG-saporin blocks increase in the BF [NOx-]ex and prevents triggering Glut iNOS NO AD cascade in PFC/FC. We will also identify the cellular source of PFC/FC iNOS/NO using Diaminofluorescein-2 Diacetate and immunohistochemistry. SA3. To investigate whether iNOS NO AD cascade in the PFC/FC contribute towards synaptic plasticity as determined by AMPA receptor (AMPAR) subunit phosphorylation-dependent trafficking. We will infuse iNOS inhibitor or A1R antagonist to the PFC/FC during SD and measure the changes in GluR1 and GluR2 phosphorylation. Our PD show that iNOS blocking reverses SD-induced effects. SA4. To investigate whether chronic SD (CSD) triggers iNOS NO AD cascade in the BF and PFC/FC. Our PD indicate that apart from daily SD-induced increases in [Glut]ex, [NOx-]ex and [AD]ex, the basal levels of [NOx-]ex and [AD]ex increase during 5 days CSD and correlate with the increase in sleepiness. We will investigate whether iNOS or A1R block will affect sleepiness and AMPAR trafficking.

描述（由申请人提供）：睡眠稳态是一种强大的机制，它迫使我们在睡眠不足或睡眠剥夺（SD）后睡眠超过日常需求。 SD 会增加稳态睡眠压力 (HSP)，表现为嗜睡增加和认知受损，而恢复睡眠 (RS) 则可逆转这些后果。对 SD 期间大脑中触发的生化变化的研究是抵消睡眠不足有害影响的关键步骤之一。迄今为止的广泛研究已确定胆碱能基底前脑（BF）是睡眠稳态的重要部位。最近，我们发现了一个生化级联，它在 SD 期间在 BF 中触发并促进 RS：它首先是诱导型一氧化氮合酶 (iNOS) 的快速诱导，然后是 NO 和细胞外腺苷 [AD]ex 的增加，首先是在BF (1-3h SD) 以及随后的前额叶/额叶皮层 (PFC/FC) (4-6h SD)。目前的提案旨在扩展我们对睡眠稳态生化相关性的了解。我们认为SD是一种定性和定量独特的状态，它是由BF和PFC/FC硝基能、腺苷能、谷氨酸能和胆碱能系统之间的串扰产生的。我们认为这种相互作用对于建立 HSP、增加困倦和降低表现至关重要。我们建议进行以下研究来检验我们的假设。具体目标 1 (SA1)。研究细胞外谷氨酸 [Glu]ex 的增加是否是 BF 和 PFC/FC 中 iNOS NO AD 级联的触发因素。我们的初步数据 (PD) 显示，BF [Glu]ex 在 SD 开始后 30 分钟内增加，先于硝酸盐/亚硝酸盐 [NOx-]ex 和 [AD]ex 增加。此外，我们还将研究基因控制 BF [Glu]ex 减少的后果。我们还表明，SD 诱导的 [AD]ex 可以防止 SD 期间 [Glu]ex 的逐渐增加，从而保护大脑免受兴奋性毒性损伤。 SA2。研究胆碱能 (ChBF) 细胞是否是触发 BF 和 PFC/FC 中 iNOS->NO->AD 级联的关键元件。我们的 PD 显示 iNOS 主要在 ChBF 细胞中诱导。我们的 PD 还表明，使用 192 IgG-皂草素对 ChBF 细胞的损伤可阻断 BF [NOx-]ex 的增加，并防止触发 PFC/FC 中的 Glut iNOS NO AD 级联。我们还将使用二氨基荧光素-2 二乙酸酯和免疫组织化学来鉴定 PFC/FC iNOS/NO 的细胞来源。 SA3。目的 研究 PFC/FC 中的 iNOS NO AD 级联是否有助于由 AMPA 受体 (AMPAR) 亚基磷酸化依赖性运输确定的突触可塑性。我们将在 SD 期间向 PFC/FC 注入 iNOS 抑制剂或 A1R 拮抗剂，并测量 GluR1 和 GluR2 磷酸化的变化。我们的 PD 表明，iNOS 阻断可逆转 SD 诱导的效应。 SA4。研究慢性 SD (CSD) 是否会触发 BF 和 PFC/FC 中的 iNOS NO AD 级联。我们的 PD 表明，除了每日 SD 诱导的 [Glut]ex、[NOx-]ex 和 [AD]ex 增加之外，[NOx-]ex 和 [AD]ex 的基础水平在 5 天 CSD 期间增加，并与嗜睡的增加。我们将调查 iNOS 或 A1R 阻断是否会影响嗜睡和 AMPAR 运输。