Using epigenome editing of transcriptional enhancers to regulate adult visual cortical plasticity

项目摘要 大脑皮质的突触接线图是在发育过程中建立的，这一稳定性 成人大脑中的网络对于大脑皮质可靠地编码关于大脑的信息的能力是重要的 感官世界。然而，可塑性也是哺乳动物大脑和不断增长的身体的一个基本特征 证据表明，即使是神经元反应特性的核心特征，如定向调节 在成年人的大脑中，视觉皮质中的神经元比之前认为的更具动态可塑性。这些 数据提出了一个问题，突触连接的稳定性和可塑性之间是如何平衡的 维持在成人大脑中吗？神经元活性依赖的转录和翻译在 发育期间大脑皮层网络的组织，这些过程有助于持久 成人大脑中神经元结构和功能的可塑性。ARC是该活动中最重要的活动之一 皮质发育过程中受调控的基因，因为Arc蛋白直接在突触与内吞作用 AMPA型谷氨酸受体，诱导长期抑制(LTD)和输入特异性突触消除。 成人初级视皮层Arc基因敲除会损害感受野的可塑性，而过度表达 Arc的引入增强了这种可塑性。这些数据提出了一种有趣的可能性，即 依赖活动的Arc表达可能在大脑皮质的灵活性和稳定性之间设定平衡 申述。然而，以前的研究还没有实验手段来选择性地操纵 在成人大脑中对Arc等基因的活性依赖调节，以确定 皮质可塑性。Arc的活性依赖转录是由Arc启动子的相互作用介导的 远端增强子元件位于Arc上游约7kB。我们已经证明了基于CRISPR的 将dCas9-染色质调节融合蛋白招募到活性依赖的基因调控元件可以 被用来选择性地调节基因表达的活性依赖部分。在Aim 1中，我们将 使用我们表征的两个新品系的dCas9/CRISPR小鼠来滴定活性依赖的 Arc的转录并确定Arc蛋白的光依赖调控的后果 在体内初级视皮层(V1)的表达。在目标2中，我们将使用慢性活体钙成像 评估引弧障碍对定向稳定性和可塑性的影响的方法 在V1中进行调整。揭示了活性依赖转录的表观遗传调控之间的这种关系 成人视皮层中的突触可塑性有可能改变神经科学家的研究方法 对健康和疾病中的皮质功能的研究。

Project Summary The synaptic wiring diagram of the cerebral cortex is established during development, and the stability of this network in the adult brain is important for the ability of the cortex to reliably encode information about the sensory world. However, plasticity is also a fundamental feature of the mammalian brain, and a growing body of evidence is revealing that even core features of neuronal response properties, such as the orientation tuning of neurons in the visual cortex, are more dynamically plastic in the adult brain than previously thought. These data raise the question, how is the balance between the stability and the plasticity of synaptic connections maintained in the adult brain? Neuronal activity-dependent transcription and translation play essential roles in the organization of cortical networks during development, and these processes contribute to long-lasting plasticity of neuronal structure and function in the adult brain. Arc is among the most important of the activity regulated genes during cortical development, because Arc protein functions directly at synapses to endocytose AMPA-type glutamate receptors, inducing long-term depression (LTD) and input-specific synaptic elimination. Genetic knockout of Arc in adult primary visual cortex impairs receptive field plasticity, whereas overexpression of Arc enhances this plasticity. These data raise the interesting possibility that the molecular mechanisms of activity-dependent Arc expression may act to set the balance between flexibility and stability of cortical representations. However, no prior study has had the experimental means to selectively manipulate the activity-dependent regulation of genes like Arc in the adult brain in order to determine the consequences for cortical plasticity. The activity-dependent transcription of Arc is mediated by the interaction of the Arc promoter with a distal enhancer element located ~7kB upstream of Arc. We have shown that the CRISPR-based recruitment of dCas9-chromatin regulator fusion proteins to activity-dependent gene regulatory elements can be used to selectively modulate the activity-dependent component of gene expression. Here in Aim 1 we will use two novel strains of dCas9/CRISPR mice we have characterized to titrate the activity-dependent transcription of Arc and determine the consequences for the light-dependent regulation of Arc protein expression in the primary visual cortex (V1) in vivo. In Aim 2 we will use chronic in vivo calcium imaging methods to assess the consequences of impairing Arc induction on the stability and plasticity of orientation tuning in V1. Revealing such a relationship between epigenetic regulation of activity-dependent transcription and synaptic plasticity in the adult visual cortex has the potential to transform how neuroscientists approach the study of cortical function in health and disease.