Optogenetic Tools for in vivo Analysis of Cortical Circuit Plasticity

神经可塑性是大脑基本过程的中心，包括学习，以及使用滥用物质导致的神经回路的长期变化。与细胞/分子水平的重大进展相比，我们对大脑回路的功能组织和重组的了解仍然有限。越来越多的体内证据表明，单细胞在成年期和成年期保持可塑性。然而，我们对单个细胞的功能特性的知识主要是描述性的。这限制了我们对在可塑性过程中组装和修改神经元调谐功能的潜在机制的理解。为了了解细胞在可塑性过程中的特性是如何变化的，必须从体内相互连接的神经元群体中进行记录。皮层中超过一半的突触接触来自距离靶细胞-200 j.lm半径内的神经元。重要的是，大脑皮层细胞之间的连接主要沿着皮质深度进行。因此，我们需要同时监测皮质体积内所有相邻神经元的活动，从而记录在三维空间中。到目前为止，还没有实验工具可以让我们做到这一点。这个项目寻求建立新的活体方法，使我们能够在三维中分析神经电路。为此，我们将提出两项技术来记录和操纵哺乳动物皮质中的电路：(1)超高速三维双光子成像；(2)使用光遗传学工具以单细胞和单棘波分辨率对神经活动进行光学操纵。由于超快多光子显微镜和光激活离子通道的最新技术进步，拟议的发展成为可能。简而言之：无惯性近红外激光扫描技术允许体内快速的结构和功能成像以及高分辨率的光学刺激。拟议的项目将结合这些关键技术来产生基础设施和实验技能，以研究皮质微电路的功能和可塑性，从而帮助研究人员了解完整大脑的神经可塑性机制。

Neuroplasticity is central to fundamental processes in the brain, including learning, and long-lasting changes in neural circuits that result from employing substances of abuse. In contrast to significant advances at the cellular/molecular level, our understanding of the functional organization and reorganization of brain circuits remains limited. Increasing in vivo evidence suggests that single cells remain plastic into and during adulthood. However, our knowledge of the functional properties of single cells is primarily descriptive. This limits our understanding of the underlying mechanisms involved in assembling and modifying neuronal tuning functions during plasticity. In order to understand how the properties of cells change during plasticity, it is imperative to record from a population of interconnected neurons in vivo. More than half of all synaptic contacts in the cortex arise from neurons within a -200 j.lm radius from the target cell. Importantly, connections between cells in the cerebral cortex are predominantly along cortical depth. Therefore, we need to monitor simultaneously the activity of all adjacent neurons in a cortical volume, and thus record in three dimensions. To date, no experimental tool exists that would allows us to do this. This project seeks to establish novel in vivo methods that will allow us to analyze neural circuits in three dimensions. For this purpose, we will advance two technologies to record from and manipulate circuits in the mammalian cortex: (1) Ultra-fast three-dimensional two-photon imaging, and (2) Optical manipulation of neural activity with single-cell and single-spike resolution using optogenetic tools. The proposed developments have become possible because of recent technical advances in ultra-fast multi-photon microscopy and light-activated ion channels. In short: Inertia-free nearinfrared laser scanning technology allows for in vivo fast structural and functional imaging as well as for high resolution optical stimulation. The proposed project will combine these key technologies to generate the infrastructure and the experimental skills to study the function and plasticity of cortical microcircuits and thus will help researchers to understand mechanisms of neuroplasticity in the intact brain.