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

点击翻译按钮获取中文摘要

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.