Genomic Bases of Behavioral Learning: Single Cell Approaches

描述（由申请人提供）：经验依赖的突触可塑性和潜在的基因调控对正常的大脑发育和学习至关重要，并且在许多发育和学习障碍中被破坏，如精神分裂症，阿尔茨海默病，药物成瘾和年龄相关的记忆丧失。迄今为止，大多数关于突触可塑性和基因调控的细胞和分子机制的研究都采取了高度还原的方法，在体外进行了非常简单的制备。然而，对于完整的大脑在实际行为学习过程中发生的记忆的基因组机制，我们几乎一无所知。特别是，还原论的方法使得研究不同输入和途径的整合变得困难或不可能，而这些输入和途径对学习的许多方面至关重要，并且是神经发育和联想学习的标志。基于这些原因，我们的长期目标是在行为学习过程中单个识别神经元水平上表征长期可塑性的全基因组机制。对于这项具有挑战性的任务，我们将使用定义良好的应用撤回反射模型系统，并在简化的行为准备中对简单的记忆形成电路进行几乎完整的映射。我们将记录在非联想形式的学习（敏化）和联想形式的学习（经典条件反射）过程中，该回路中单个识别的关键神经元的活动以及它们之间的突触连接。而且，我们将第一次在特定的单个神经元学习和记忆时监测整个基因组的运作。因此，我们将把神经活动与基因表达、可塑性和行为联系起来。我们还将确定在致敏和条件作用过程中神经元类型特异性的细胞信号机制和基因调控途径，并测试它们在长期可塑性中的作用。基于我们之前的研究结果，我们假设三种信号通路（5-HT、NO和活性）协同作用，在条件反射过程中比敏化过程中产生更特异性和更持久的记忆痕迹。此外，5-HT和NO各自可以改变至少1000个基因的表达（其中一些重叠）并诱导大规模的染色质重塑。这些发现提出了一个基本问题：这些不同的输入是如何在调节回路中单个神经元的全基因组基因调控水平上整合的？我们将确定导致这种整合的关键分子靶点（包括启动子、增强子和相关转录因子），并研究它们在形成持久记忆中的决策点作用。这些研究将极大地促进我们对突触和基因组机制的理解，这些机制有助于电路的形成、学习和记忆，以及它们在影响神经发育和记忆的疾病中可能出现的功能障碍。

DESCRIPTION (provided by applicant): Experience-dependent synaptic plasticity and underlying gene regulation are crucial for normal brain development and learning, and are disrupted in a broad array of disorders of development and learning such as schizophrenia, Alzheimer's disease, drug addiction and age related memory loss. To date, most studies of the cellular and molecular mechanisms of synaptic plasticity and gene regulation have taken a highly reductionist approach in very simplified preparations in vitro. However, practically nothing is known about genomic mechanisms of memory that occur during actual behavioral learning in the intact brain. In particular, the reductionist approach makes it difficult or impossible to study the integration of different inputs and pathways that is critical for many aspects of learning, and is a hallmark of neurodevelopment and associative learning. For these reasons, our long-term objectives are to characterize genome-wide mechanisms of long-term plasticity at the level of single identified neurons during behavioral learning. For this challenging task we will use the well-defined model system of the Aplysia withdrawal reflex, with a nearly complete mapping of a simple memory-forming circuit in a simplified behavioral preparation. We will record the activity of key individually identified neurons in that circuit and the synaptic connections between them during both a nonassociative form of learning (sensitization) and an associative form of learning (classical conditioning). And, for the first time, we will monitor the operation of the entire genome within specific individual neurons as they learn and remember. As a result, we will link neural activity to gene expression, plasticity, and behavior. We will also identify neuron type specific cellular signaling mechanisms and gene regulatory pathways during sensitization and conditioning, and test their roles in long-term plasticity. Based on our previous results, we hypothesize that three signaling pathways (5-HT, NO, and activity) act synergistically to produce more specific and longer-lasting memory traces during conditioning than during sensitization. Furthermore, 5-HT and NO can each change expression at least a thousand genes (some of which overlap) and induce large-scale chromatin remodeling. These findings have raised a fundamental question: how are these different inputs integrated at the level of genome-wide gene regulation in individual neurons in the circuit for conditioning? We will identify critical molecular targets (including promoters, enhancers and relevant transcription factors) leading to such integration, and examine their roles as decision points in the formation of long-lasting memories. These studies will significantly advance our understanding of synaptic and genomic mechanisms that contribute to circuit formation, learning, and memory, and their possible dysfunction in diseases that affect neurodevelopment and memory.