Sleep and the Functional Genomics of Synaptic Modulation

7.项目摘要/摘要许多研究一致表明， 在睡眠/清醒状态下的转录组。我们基于RNAseq技术的初步证据表明， 超过2800个基因在恢复睡眠中响应于4小时的睡眠而差异表达。 与对照组相比，这些DEG富含与突触功能相关的基因 以及控制突触强度的转录因子，肌细胞增强因子2（MEF 2C）的靶点。 一个关于睡眠功能的主要建议假定在清醒时皮层突触强度的整体建立 在随后的睡眠过程中，神经元的经验和突触强度的下降。Mef 2活性形式的表达 降低突触强度我们的初步证据表明，四个小时的睡眠不足会增加 MEF 2（活性形式）与pMEF 2（抑制形式）的比率。这让我们想到了睡眠的假设- 需要调节转录因子MEF 2C的活性，以改变下游mRNA的表达， 降低前脑皮层谷氨酸神经元上的谷氨酸突触强度。 我们计划通过首先建立行为基因组中不同转录组的表达来验证这一假设。 在野生型小鼠中的状态条件下，然后将其在这些相同条件下的表达与 条件性Mef 2c敲除突变体（ko将限于谷氨酸前脑神经元）。 我们计划使用三个睡眠指标来描述Mef 2c突变体的睡眠需求/解决表型 需要的将在基线和睡眠剥夺（SD）条件下检查突变体和野生型对照。 我们的初步证据表明，稳态睡眠反应在突变体中丢失。 MEF 2的磷酸化状态决定其活性，因此我们将评估MEF 2的磷酸化状态 与行为状态条件相关。此外，由于高去甲肾上腺素能活性可导致卵裂， HDAC 4的N-末端抑制MEF 2转录活性，我们将评估该N-末端产物 在睡眠清醒状态下也是如此。 我们将描述睡眠相关的，Mef 2依赖的突触结构和功能变化，包括 形态学定义的棘数、树突结构和mEPSC频率和振幅记录自 在体外培养的大鼠大脑前扣带回皮质2-3层和5-6层锥体神经元。 该提案将提供第一个全面的基于RNAseq的睡眠/清醒状态分析之一 我们是一个有用的资源，为调查人员，以帮助调查和了解大 长时间清醒后发生的转录组学变化。它可以提供一个重要的开始， 睡眠相关细胞自主信号级联和分子靶点的鉴定， 对大脑健康很重要。

7. Project Summary/Abstract Numerous studies have consistently shown a remarkably large change in the transcriptome across sleep/wake states. Our preliminary evidence based on RNAseq technology suggests more than 2800 genes are differentially expressed in recovery sleep in response to four hours of sleep deprivation, compared to control conditions. These DEG's are enriched for genes related to synaptic function and for targets of a transcription factor, myocyte enhancing factor 2 (MEF2C), that controls synaptic strength. A leading proposal for the function of sleep posits an overall buildup of cortical synaptic strength during waking experience and a decrease of synaptic strength during ensuing sleep. Expression of the active form of Mef2 decreases synaptic strength. Our preliminary evidence suggests that four hours of sleep deprivation increases the MEF2 (the active form) to pMEF2 (the repressive form) ratio. This has lead us to the hypothesis that sleep- need modulates the activity of the transcription factor, MEF2C, to alter the expression of downstream mRNA to reduce glutamate synapse strength on forebrain cortical glutamate neurons. We plan to test this hypothesis by first establishing the differential transcriptome expression across behavioral state conditions in wildtype mice and then to compare it across these same conditions to the expression in conditional Mef2c knockout mutants (the ko will be restricted to glutamate forebrain neurons). We plan to characterize a sleep need/resolution phenotype in the Mef2c mutant using three indices of sleep need. Mutants and wildtype controls will be examined under baseline and sleep deprivation (SD) conditions. Our preliminary evidence shows that the homeostatic sleep response is lost in the mutants. The phosphorylation state of MEF2 determines its activity so we will assess the phosphorylation state of MEF2 in correlation with behavioral state condition. Additionally, since high noradrenergic activity can cause cleavage of the N-terminal of HDAC4 to inhibit MEF2 transcriptional activity, we will assess this N-terminal product across sleep wake states as well. We will characterize sleep-related, Mef2 dependent structural and functional changes of synapses, including morphologically defined spine number, dendritic structure and mEPSC frequency and amplitude recorded from layer 2-3 & 5-6 pyramidal neurons, in frontal cortical lobe (the anterior cingulate) in vitro. This proposal will provide one of the first comprehensive RNAseq based analyses across sleep/wake states that we be a useful resource for investigators to aid in the investigation and understanding of the large transcriptomic change that takes place in response to prolonged waking. It can provide an essential starting point for the identification of sleep related cell autonomous signaling cascades and molecular targets, important to brain health.