DESCRIPTION (provided by applicant): Optogenetic technologies, designed for control of defined elements of neural circuitry with high temporal precision and within intact systems, have recently come into broad use. Key components of this approach include light-activated regulators of transmembrane ion conductance such as channelrhodopsins (ChRs) which can be temporally precise in action, and readily targeted to specific brain regions (e.g. by viral vectors. Now an unexpected alignment in results from our laboratory has opened up a path to optogenetic tools which will make defined circuit elements susceptible to potent control even in deep brain circuits and without genetics. We propose to develop, distribute, and apply (to anxiety research) these technologies, bringing to bear tools from biophysics and structural biology, through which brain circuits may be targeted for versatile inhibition or excitation. In Ai 1, we propose to leverage new structural information to create a potent inhibitory ChR. This is a long-sought goal of optogenetics, not yet achieved. Current fast optogenetic inhibitory tools are ion pumps, not potent enough to reliably interfere with nerve signaling in large tissue volumes or in illuminated axons. These pumps are limited in efficacy since only a single ion is moved across the membrane for each photon absorbed. But channels instead move many ions per photon and can also shunt and influence input resistance. To enable robust interception of spikes in deep-brain or axonal settings for loss-of-function experiments essential to the field, here we will integrate our novel structural information to create inhibitory optogenetic channels. In Aim 2, we extend this rational design of ChRs to include altered color-sensitivity in the settin of novel ion selectivity. Despite the fact that only a handful of ChRs have been identified, major advances have come from comparisons, comparative mutagenesis, and chimeras. We therefore will integrate structural information with a new set of ChRs to modulate photocurrent properties, creating and testing ChRs with diverse color-sensitivity properties in each class for versatile optogenetic control. Finally in Aim 3, we will validate and refine these novel tools for mammalian behavior in anxiety models. This technology cannot be developed optimally and distributed to the community without a phase of validation and refinement in a real-world setting. Now launching from our recently published work on anxiety, we will employ the tools developed here to probe behavioral consequences of deep-brain optogenetic control in experiments of high significance for understanding the generation and control of anxiety-related states. Together, these efforts will generate versatile and powerful new optogenetic tools, and provide fundamental insight into casual dynamics of anxiety- related behaviors based on precise control of distinct neural circuit pathways.

Optogenetic technologies for controlling brain circuits are now widely applied to the study of neurological and psychiatric conditions such as Parkinson's Disease and anxiety in animal models. This is important work for human health since due to the complexity of the brain we do not know precisely which circuits must be modulated, and in what manner, to understand, diagnose, and treat neuropsychiatric disease; moreover, current tools such as nonselective electrodes and drugs do not allow us to understand how complex functions arise from interactions of diverse cells. The efforts proposed here will generate versatile and powerful new optogenetic tools for high precision control, and also provide direct insights into the dynamics of anxiety-related behaviors controlled by specific neural circuits.