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.
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 new techniques can be used to understand how neurons encode and store information, with potential implications for ameliorating Alzheimer's disease, addiction, traumatic brain injury, and neurodegeneration.
|Rodriguez, Erik A; Campbell, Robert E; Lin, John Y et al. (2017) The Growing and Glowing Toolbox of Fluorescent and Photoactive Proteins. Trends Biochem Sci 42:111-129|
|Rodriguez, Erik A; Tran, Geraldine N; Gross, Larry A et al. (2016) A far-red fluorescent protein evolved from a cyanobacterial phycobiliprotein. Nat Methods 13:763-9|