BRAIN EAGER: Development of Robotic Microscopy to monitor the longitudinal molecular dynamics of single neurons and circuits in situ in mammalian brain

该项目旨在开发一种新技术，机器人显微镜（RM），以“桥接多个空间，时间和组织尺度，为最终导致行为和认知的神经回路的新兴特性提供基本见解。“拟议的研究有两个主要好处。首先，这些实验将导致RM仪器的开发，该仪器可以在数周内一次监测活脑组织切片中单个神经元的分子动力学。其次，实验将首次使用RM研究学习和记忆如何改变特定神经元的基本特性，以增强突触可塑性，为学习和记忆机制提供新的见解。在过去，RM被用于研究培养中的神经元。在拟议的研究中的主要活动将是验证RM的效用，以监测保持其自然的生理连接和架构的脑切片中的神经元动力学。其他好处将是科学界获得一种技术，可以纵向检查神经元和其他细胞类型的生物化学，并开发新的仪器，可用于教授学生的学术课程，博士后研究员和研究科学家研究脑回路的新成像方法。需要解决的问题是RM是否可以用于研究在原位表达Arc基因的神经元，海马脑切片。ARC对于长期记忆巩固和突触可塑性是重要的，并且其活性在影响学习的刺激下在特定神经元群体中大大增强。所采用的方法将涉及使用新的Arc基因探针和转基因小鼠，以确定RM是否可以识别通过长时程增强（LTP）和长时程抑制（LTD）在脑切片中激活的选择性Arc表达神经元或在学习和记忆巩固期间在体内脑中激活的选择性Arc表达神经元。这些研究的目的是确定不同的Arc神经元回路是否介导LTP和LTD，以及在学习过程中在大脑中体内激活的相同Arc表达神经元是否可以在脑切片中原位通过RM监测。研究的范围将确定RM是否可以识别和研究通过不同形式的学习和记忆巩固在体外和体内激活的不同Arc神经元回路。

This project is directed at developing a novel technology, robotic microscopy (RM), to "bridge multiple spatial, temporal, and organizational scales to provide fundamental insights into the emergent properties of neural circuitry that ultimately lead to behavior and cognition." There are two primary benefits of the proposed studies. First, the experiments will result in the development of an RM instrument that can monitor the molecular dynamics of individual neurons in slices of living brain tissue over weeks at a time. Second, the experiments will use RM to study for the first time how learning and memory change the fundamental properties of specific neurons in situ to enhance synaptic plasticity, providing new insights into the mechanisms involved in learning and memory. In the past, RM was used to study neurons in culture. The primary activity in the proposed studies will be to validate the utility of RM to monitor neuronal dynamics in brain slices that maintain their natural physiological connectivity and architecture. Other benefits will be access of the scientific community to a technology that can examine the biochemistry of neurons and other cell types longitudinally and the development of new instrumentation that can be used in academic courses that teach students, postdoctoral fellows and research scientists novel imaging approaches to study brain circuits.The problem to be addressed is whether RM can be used to study neurons expressing the Arc gene in situ in hippocampal brain slices. Arc is important for long-term memory consolidation and synaptic plasticity, and its activity is greatly enhanced in specific neuron populations by stimuli that affect learning. The methods to be employed will involve the use of novel Arc genetic probes and transgenic mice to determine if RM can identify selective Arc-expressing neurons activated in brain slices by long-term potentiation (LTP) and long-term depression (LTD) or in brain in vivo during learning and memory consolidation. The goals of the studies will be to determine if different Arc neuronal circuits mediate LTP and LTD, and if the same Arc expressing neurons activated in vivo in the brain during learning can be monitored by RM in situ in brain slices. The scope of the studies will determine if RM can identify and study different Arc neuronal circuits activated in vitro and in vivo by different forms of learning and memory consolidation.