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名为机器人显微镜(RM)的新技术，以“连接多个空间、时间和组织尺度，以提供对最终导致行为和认知的神经回路新特性的基本见解。”拟议的研究有两个主要好处。首先，这些实验将导致一种RM仪器的开发，该仪器可以一次监测几周内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来研究学习和记忆如何改变特定神经元的原位基本属性，以增强突触可塑性，为学习和记忆涉及的机制提供新的见解。在过去，RM被用来研究培养中的神经元。在拟议的研究中，主要活动将是验证RM在监测脑片中的神经元动力学方面的效用，以保持其自然的生理连通性和结构。其他好处将是科学界可以获得一种可以纵向检查神经元和其他类型细胞的生物化学的技术，以及开发可以在学术课程中使用的新仪器，这些课程向学生、博士后研究员和研究科学家传授研究大脑回路的新的成像方法。需要解决的问题是，RM是否可以用于研究在海马脑片中原位表达Arc基因的神经元。ARC对长期记忆巩固和突触可塑性非常重要，在特定的神经元群体中，它的活性因影响学习的刺激而大大增强。将采用的方法包括使用新的Arc基因探针和转基因小鼠来确定RM是否能够在学习和记忆巩固过程中识别大脑切片中通过长时增强(LTP)和长时抑制(LTD)激活的选择性Arc表达神经元或在活体大脑中。这项研究的目标将是确定不同的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激活的同一Arc表达神经元是否可以通过脑片上的RM原位监测。研究的范围将决定RM是否能够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.