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

项目摘要 大脑皮层的突触接线图是在发育过程中建立的， 成年人大脑中的网络对于皮层可靠地编码有关大脑的信息的能力至关重要。 感官世界然而，可塑性也是哺乳动物大脑的一个基本特征， 有证据表明，即使是神经元反应特性的核心特征，如方向调谐， 在成人大脑中，视觉皮层神经元的可塑性比以前认为的更强。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？ 维持在成年人的大脑中神经元活性依赖的转录和翻译在神经细胞的增殖和分化中发挥重要作用。 在发展过程中的皮质网络的组织，这些过程有助于持久的 成人大脑中神经元结构和功能的可塑性。弧是最重要的活动之一， 由于Arc蛋白直接在突触处起内吞作用， AMPA型谷氨酸受体，诱导长期抑制（LTD）和输入特异性突触消除。 成年人初级视皮层Arc基因敲除损害感受野可塑性，而过表达Arc则影响感受野可塑性。 Arc增强了这种可塑性。这些数据提出了一种有趣的可能性，即 活动依赖的Arc表达可能起着在皮质的柔韧性和稳定性之间建立平衡的作用。 表示。然而，没有先前的研究已经有实验手段来选择性地操纵 在成人大脑中，像Arc这样的基因的活性依赖性调节，以确定 皮质可塑性Arc的活性依赖性转录是由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7 kB。我们已经证明，基于CRISPR的 将dCas 9-染色质调节融合蛋白募集至活性依赖性基因调节元件可 用于选择性调节基因表达的活性依赖性组分。在目标1中，我们将 使用我们已经表征的两种新的dCas 9/CRISPR小鼠品系， Arc的转录，并确定Arc蛋白的光依赖性调节的后果 在体内初级视皮层（V1）中的表达。在目标2中，我们将使用慢性体内钙成像 评估削弱电弧感应对取向稳定性和可塑性的影响的方法 调整到V1。揭示了活性依赖性转录的表观遗传调控与 成人视觉皮层的突触可塑性有可能改变神经科学家研究 对健康和疾病中大脑皮层功能的研究。

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.