Optogenetic silencing tools for precise, all-optical analysis of synaptic circuits

神经活动的光遗传学操作已经成为研究突触可塑性、神经元回路功能和特定脑区域在动物行为中的作用的不可或缺的策略。与光遗传激活剂并行，神经元沉默工具正在成熟，近年来取得了重大进展。例如，有效的工程和天然阴离子传导通道视紫红质（ACRs）的开发和鉴定为有效的体内光遗传沉默开辟了新的途径。然而，在长时间沉默神经元和抑制突触传递方面仍然存在显著的局限性。与光遗传激活不同，神经元活动的可靠抑制通常需要在整个沉默期间持续照明，这限制了用光学指示器同时成像神经元活动，通过视觉干扰干扰动物行为，并且在高强度下可能具有细胞毒性。此外，最近的研究表明acr不适合用于突触末端的光遗传沉默。为了解决第一个限制，我们将在第一个资助期设计的acr的基础上开发增强型双稳态acr，这些acr 1)用短光脉冲激活，2)在没有光的情况下保持激活数分钟，3)可以在指定的时间点以高时间精度灭活。我们将进一步将它们定位到不同的亚细胞区室，以便允许对神经元活动进行电路特异性和亚细胞操作。关于第二个限制，迫切需要开发一种工具，可以在不抑制神经元活动本身的情况下特异性地抑制预期目标区域的突触传递。由于acr不适合这种方法，我们将利用Gi/o蛋白偶联受体（gpcr）的高效能来抑制突触释放。我们将探索合适的天然光敏视紫红质，并生成嵌合光学激活的gpcr（光- gpcr）来识别特异性偶联到Gi/o的变异。这种方法将允许识别适合持续和重复激活的光gpcr，并有望成为突触沉默的有效光遗传学工具。我们将在哺乳动物和无脊椎动物模型系统中表征我们新生成的工具，以验证其普遍适用性。最后，我们将通过应用我们的工具来测试果蝇伤害感受网络、海马切片培养和小鼠丘脑皮质系统中的特定电路功能，来解决这些系统中的新问题。

Optogenetic manipulation of neural activity has become an indispensable strategy to interrogate synaptic plasticity, neuronal circuit function and the role of defined brain regions in animal behavior. In parallel to optogenetic activators, neuronal silencing tools are coming of age and significant progress has been made in recent years. For example, the development and identification of potent engineered and natural anion conducting Channelrhodopsins (ACRs) have opened new avenues for efficient optogenetic silencing in vivo. However, significant limitations remain for silencing neurons over extended periods and for inhibiting synaptic transmission. Unlike optogenetic activation, reliable suppression of neuronal activity generally requires continuous illumination throughout the entire silencing period, which limits simultaneous imaging of neuronal activity with optical indicators, disturbs animal behavior by visual interference and at high intensities can be cytotoxic. Moreover, recent work has shown that ACRs are not suitable for optogenetic silencing of synaptic terminals. To address the first limitation, we will build on our ACRs engineered during the first funding period and develop enhanced bistable ACRs that are 1) activated with a short light pulse, 2) remain active in absence of light for minutes and 3) can be inactivated with high temporal precision at a defined time point. We will further target them to different subcellular compartments in order to allow circuit-specific as well as sub-cellular manipulation of neuronal activity. Regarding the second limitation, there is an urgent need to develop tools that can specifically suppress synaptic transmission in a desired target area without inhibiting neuronal activity per se. As ACRs are not suitable for this approach, we will take advantage of the high potency of Gi/o-protein coupled receptors (GPCRs) to inhibit synaptic release. We will explore suitable natural light-sensitive rhodopsins and generate chimeric optically activated GPCRs (opto-GPCRs) to identify variants coupling specifically to Gi/o. This approach will allow identifying an opto-GPCR suitable for sustained and repeated activation over time and holds promise as a potent optogenetic tool for synaptic silencing. We will characterize our newly generated tools in mammalian and invertebrate model systems in vivo to verify their universal applicability. Lastly, we will address novel questions in these systems by applying our tools to test specific circuit functions in the Drosophila nociceptive network, hippocampal slice cultures and the mouse thalamocortical system.