John Y. Lin, PhD - US grants

? DESCRIPTION (provided by applicant): This proposal aims to develop new molecular techniques to map activities of neurons, manipulate the strength of communication between neurons and disrupt intracellular signaling. These 'optogenetic' approaches will be used to further our understandings of brain function on behavior and have important implications in our understandings of neurological conditions and neurodegenerative diseases. The first goal is to develop a technique where the researchers can use optical approach to identify synaptic connections that were active during the performance of a behavior task. This reporter system can be turned on with light, which defines the window of activity reporting, and fluorescence signal can be detected if there is significant activity between two defined cell groups. Many existing approaches can only be used to map excitatory connections, whereas the proposed approach can be used to identify activities between synapses utilizing any neurotransmitters. The approach will utilize a split fluorescent protein approach where its complementation and the generation of fluorescent signal is activity dependent. This approach will test whether a defined synaptic connection is involved in the performance of a behavior. The second goal is to develop a technique where the researchers can use light to modulate the strength of synaptic communication between neurons. Increasing synaptic strength is believed to underlie memory and learning, and its disruption has been implicated in drug addiction and many neurological conditions. Having the ability to modulate the synaptic strength experimentally can be used to interrogate how changes in synaptic strength alter learning and memory, leading to the observed adaptive behavior in the animals in both normal and pathological conditions. Many small protein fragments can alter synaptic strengths between neurons. A light-responsive protein can be used to functionally mask these protein fragments in the dark and light can be used to functionally release these protein fragments. This will permit rapid experimental control of synaptic strength and their functional effects can be studied in the behaving animals. This tool can be used to understand how alteration in synaptic strength changes during learning and adaption. The third goal of the project is to develop a technique where G-protein coupled receptor mediated second messenger pathway is inhibited by light. G-protein coupled receptors mediate the effects of neuromodulator and neuropeptides in the nervous system and they have great importance in modulating and/or mediating behaviors. Using a similar approach as described above, competitive binding peptides that disrupt G-protein coupled receptor-G protein interactions or peptides that directly inhibit the effectors of G- protein pathways can be masked and unmasked with light-responsive protein and light illumination. With this approach, light will turn off G protein activation or effectors of G-protein pathway rapidly to interrogate the behavioral effects of neuromodulators or neuropeptides in specific cells with defined temporal resolution.

Project Summary: This proposal outlines the development of a fundamentally new optogenetic technology capable of flexibly manipulating the activity of thousands of neurons contributing to the dynamic activity of distributed neural circuits with single neuron resolution. No method that currently exists even remotely meets the need of flexible, selective control of thousands of neurons distributed across large volumes of the brain. Filling this methodological gap is a central research objective of the BRAIN Initiative, because doing so will transform our ability to investigate how the nervous system encodes, processes, utilizes, stores, and retrieves information. The overall objective for this application is to acquire critical structural knowledge of photoactive states of a red-shifted channelrhodopsin and use these to engineer a photoselectable channel prototype that demonstrates the potential of our approach for future development in behaving animals. This would allow opsin-expressing neurons to be flexibly selected, activated, and deselected with light. By leveraging new structural knowledge, we anticipate that we can develop a fundamentally new approach to optogenetics that takes us beyond genetically targeted control and into an era of functionally targeted, flexible control of any neural ensemble. The aims of our research are to obtain the first atomic structures of red-shifted channelrhodopsin mutants in three channel states, engineer a three-state ReaChR mutant with high open conductance and optimized action spectra, and demonstrate reversible photoselective control of neurons in vivo with PReaChR prototypes. We anticipate that completion of these aims will yield the following expected outcomes. First, it will produce new knowledge of the underlying structural transformations between channelrhodopsin photostates that will enable efficient computational design of photoselectable optogenetic tools. Second, it will produce the first examples of photoselective channelrhodopsins useful for neural excitation. Third, it will assess the utility of these new opsins for flexible control of distributed sets of neurons. Collectively, these will provide a roadmap to extending the transformative new trait of photoselectabilty to a wide range of existing optogenetic tools for excitation, inhibition and modulation of neural activity. Further research in this direction should ultimately enable flexible control of spatially complex distributions of neurons in head-fixed and freely moving animals during behavior, a key to furthering our understanding of the intricate neural dynamics that underlie our thoughts, feeling, and actions and how circuit dynamics are disrupted by neurological disorders.

Project Summary: Neuropeptides are key components of modulation across the central nervous system. These short peptides are released from neurons and non-neuronal cells and have powerful modulatory effect on neuronal activity leading to changes in sensory perception, motor output and complex behaviors. Currently there are no experimental tools that can manipulate the release and functions of these important neuromodulators with high spatial and temporal resolutions. As such, the main objective of the proposed project is to develop optogenetic approaches to inhibit the release neuropeptides from neurons without disrupting the release of non-peptide neurotransmitters. In addition, we will also develop an improved approach to suppress the release of all synaptic vesicles non-selectively. To achieve these goals, we will use photosensitizing fluorescent proteins in combination with chromophore assisted light inactivation (CALI) and light induced protein dimerization (CRY2/CIB1) to disrupt the release of secretory vesicles from neurons. By targeting these proteins to vesicles containing neuropeptides, we will be able to achieve the selective disruption of the release of neuropeptides without affecting synaptic transmission. These tools can be used to selectively turn off the release of neuropeptides at a specific region, at a specific synaptic connection or onto specific cells with unprecedented temporal resolution. We expect these tools to drastically change the way we, as a community of neurophysiologists, approach the study of neuromodulation, eventually gathering new knowledge to understand the underlying circuits for human thoughts, feeling, and actions and its disruption in neurological disorders.