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Molecular tools for labeling and manipulating functional brain circuits

Award Number: R01MH119353
ORGANIZATION: NATIONAL INSTITUTE OF MENTAL HEALTH
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)
PERIOD OF PERFORMANCE START DATE: 04/01/2019
PERIOD OF PERFORMANCE END DATE: 01/31/2024

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Molecular tools for labeling and manipulating functional brain circuits - Project Summary Molecular tools for labeling and manipulating functional brain circuits A fundamental goal of neuroscience is to discover the subpopulations of neurons that are associated with specific behaviors. For example, what neurons in the brain do we utilize when we experience fear, or thirst? What neurons are utilized when we learn to associate a specific context or stimulus with the feeling of fear? The field has experienced a revolution with the emergence of tools such as channelrhodopsins, DREADDS, real-time calcium indicators, and multiphoton microscopes that enable monitoring and manipulation of neuronal activity in awake, behaving animals. However, these tools are most useful when the experimentalist already has a specific hypothesis for which neuronal subpopulations may be relevant to the behavior of interest. What is lacking is a technology to guide the researcher to specific brain regions and neuronal subpopulations, when prior information about the behavior under study is absent or incomplete. Here we propose to develop a family of molecular tools, collectively called “FLARE”, for Fast Light- and Activity-Regulated Expression, that may be highly useful for the study of the neural circuit basis of behaviors. FLARE is a coincidence detector of light and elevated cytosolic calcium (a proxy for neuronal activity) that gives expression of any reporter gene of choice as its output. If FLARE components are expressed throughout a brain region, and light is delivered to that region via an implanted fiber, coincident with a stimulus of interest, then transgene expression should be selectively turned on only in the subpopulation of neurons that fired during the moment of light delivery, which could be as short as a few minutes to seconds. By using a transgene such as GFP-channelrhodopsin, it would be possible to both image the neuronal subpopulation of interest, and drive its activity, thereby examining the causal relationship to behavior. Our preliminary results show that second-generation FLARE2 marks activated neurons in culture with a tagging time window of only 60 seconds. In this project, we propose to fully characterize and validate FLARE2 and its variants (including single chain FLARE, scFLARE) in vivo (mouse and fly) while simultaneously applying protein engineering techniques and directed evolution (with which our laboratory has extensive experience), to iteratively improve and optimize the family of FLARE tools. To permit brain-wide mapping of neural circuits, we also propose a FLARE variant, called “nanoFLARE”, that can be uncaged by either light or a small-molecule that can be delivered throughout the entire brain via IP injection into the animal. NanoFLARE features a luciferase moiety fused to the protease component of the tool, that we discovered can uncage the light-sensitive LOV domain via proximity-dependent BRET. Hence, nanoFLARE can be uncaged either by direct blue light illumination, or by delivery of the small-molecule luciferase substrate furimazine to the brain. A successful outcome of this proposal would create a toolkit that will empower neuroscientists to discover neural subpopulations that underlie a wide range of behaviors and cognitive processes, with unprecedented speed and accuracy, and provide a means to interrogate the mechanisms by which these circuits encode function.


Issue Date FY	Funding FY	Legal Entity Name	Legal Entity Address	Legal Entity City	Legal Entity State	Legal Entity Zip Code	Legal Entity COUNTY	Legal Entity COUNTRY	Assistance Listing	Award Code	Budget Year	Action Date	Action Type	Action Amount

Issue Date FY: 2024 ( Subtotal = $0 )
2024	2023	THE LELAND STANFORD JUNIOR UNIVERSITY	450 JANE STANFORD WAY	STANFORD	CA	94305	SANTA CLARA	USA	Mental Health Research Grants	000	5	6/3/2024	NON-COMPETING CONTINUATION	$0
														Subtotal = $0

Issue Date FY: 2023 ( Subtotal = $636,953 )
2023	2023	LELAND STANFORD JUNIOR UNIVERSITY, THE	450 JANE STANFORD WAY	STANFORD	CA	94305	SANTA CLARA	USA	Mental Health Research Grants	000	5	1/13/2023	NON-COMPETING CONTINUATION	$636,953
														Subtotal = $636,953

Issue Date FY: 2022 ( Subtotal = $651,179 )
2022	2022	LELAND STANFORD JUNIOR UNIVERSITY, THE	450 JANE STANFORD WAY	STANFORD	CA	94305	SANTA CLARA	USA	Mental Health Research Grants	001	4	5/23/2022	NON-COMPETING CONTINUATION	$65,120
2022	2022	LELAND STANFORD JUNIOR UNIVERSITY, THE	450 JANE STANFORD WAY	STANFORD	CA	94305	SANTA CLARA	USA	Mental Health Research Grants	000	4	1/21/2022	NON-COMPETING CONTINUATION	$586,059
														Subtotal = $651,179

Issue Date FY: 2021 ( Subtotal = $722,715 )
2021	2021	LELAND STANFORD JUNIOR UNIVERSITY, THE	450 JANE STANFORD WAY	STANFORD	CA	94305	SANTA CLARA	USA	Mental Health Research Grants	000	3	1/22/2021	NON-COMPETING CONTINUATION	$722,715
														Subtotal = $722,715

Issue Date FY: 2020 ( Subtotal = $774,652 )
2020	2020	LELAND STANFORD JUNIOR UNIVERSITY, THE	450 JANE STANFORD WAY	STANFORD	CA	94305	SANTA CLARA	USA	Mental Health Research Grants	000	2	1/10/2020	NON-COMPETING CONTINUATION	$774,652
														Subtotal = $774,652

Issue Date FY: 2019 ( Subtotal = $788,339 )
2019	2019	LELAND STANFORD JUNIOR UNIVERSITY, THE	450 SERRA MALL	STANFORD	CA	94305	SANTA CLARA	USA	Mental Health Research Grants	001	1	9/24/2019	NEW	$0
2019	2019	LELAND STANFORD JUNIOR UNIVERSITY, THE	450 SERRA MALL	STANFORD	CA	94305	SANTA CLARA	USA	Mental Health Research Grants	000	1	2/26/2019	NEW	$788,339
														Subtotal = $788,339

Grand Total All Awards = $3,573,838

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Molecular tools for labeling and manipulating functional brain circuits

Award Number: R01MH119353

ORGANIZATION: NATIONAL INSTITUTE OF MENTAL HEALTH

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)

PERIOD OF PERFORMANCE START DATE: 04/01/2019

PERIOD OF PERFORMANCE END DATE: 01/31/2024

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