Trans-synaptic optical control of user-defined synaptic connections

摘要 据估计，人类大脑包含超过1000亿个神经元，这些神经元由超过100万亿个突触连接在一起。这些突触连接增加了神经回路的计算能力，对于感觉、感知、学习和记忆以及不同行为状态的选择和表达至关重要。虽然现在存在许多工具来激活，抑制或调节大脑中的特定细胞类型，但目前没有一种能够操纵用户定义的突触前和突触后细胞类型之间的活动。因此，我们对特定突触连接所扮演的角色以及它们如何促进信息处理和行为的知识仍然非常有限。在这里，我们提出了一种从根本上独特的方法来光学控制特定细胞类型之间的用户定义的突触连接的活动。我们将开发一个双组分系统，使光学可逆的，突触选择性抑制使用突触前抑制GPCR（trans-OptoGi）的跨突触激活。在目标1中，我们将使用高通量GPCR筛选来工程化不在哺乳动物大脑中表达的光敏配体：GPCR对。我们将使用跨细胞成像分析功能验证这些候选人，优化其神经元运输，测试其在急性脑切片中兴奋性和抑制性突触连接的活性，并验证其在体内调节小鼠行为的功效。在目标2中，我们将建立在这种方法的基础上，使用类似的验证步骤来光学控制大脑中内源性突触前GPCR的活性。重要的是，这些遗传编码的跨突触工具将使用常见的腺相关病毒（AAV）基因递送方法和目前用于许多神经科学实验室标准光遗传学实验的光源。所有生成的结构将与研究社区免费共享。这些工具的成功开发将使可逆的，突触特异性的操作成为可能，这在当前的神经科学工具箱中是明显缺乏的。

Abstract The human brain is estimated to contain over 100 billion neurons that are wired together by more than 100 trillion synapses. These synaptic connections increase the computational capabilities of neural circuits and are essential for sensation, perception, learning and memory, and the selection and expression of distinct behavioral states. While many tools now exist to activate, inhibit, or modulate specific cell types in the brain, none are currently capable of manipulating activity between user-defined pre- and postsynaptic cell types. As a result, our knowledge of the roles played by specific synaptic connections and how they contribute to information processing and behavior is still quite limited. Here we propose a fundamentally unique approach to optically control the activity of user-defined synaptic connections between specific cell types. We will develop a two-component system to enable optically reversible, synapse-selective inhibition using trans-synaptic activation of presynaptic inhibitory GPCRs (trans-OptoGi). In Aim 1, we will use high-throughput GPCR screens to engineer light-sensitive ligand: GPCR pairs not expressed in the mammalian brain. We will functionally validate these candidates using trans-cellular imaging assays, optimize their neuronal trafficking, test their activity at both excitatory and inhibitory synaptic connections in acute brain slices, and validate their efficacy in vivo to modulate mouse behavior. In Aim 2, we will build upon this approach to optically control the activity of endogenous presynaptic GPCRs in the brain using similar validation steps. Importantly, these genetically-encoded trans-synaptic tools will use common adeno-associated viral (AAV) gene delivery methods and light sources currently used for standard optogenetic experiments in many neuroscience labs. All constructs generated will be freely shared with the research community. Successful development of these tools will enable reversible, synapse-specific manipulations, which are noticeably lacking in the current neuroscience toolbox.