Neuromodulators are essential signaling molecules that regulate many neural processes, including cognition, mood, memory, and sleep, through their influence on brain circuits. Monitoring the release and distribution of neuromodulators in behaving animals is critical for understanding the diverse functions of these molecules. A major impediment to developing this understanding is the lack of tools that can monitor these compounds at the temporal, spatial and concentration scales relevant to these brain processes. Filling this technological gap is one of the most pressing needs in neuroscience research. Our proposal directly bridges this gap by developing a platform of new tools for chronic, non- invasive monitoring of neuromodulators at millisecond, subcellular, and nanomolar resolution. Genetically-encoded fluorescent indicators for calcium and glutamate have transformed investigation of dynamic brain processes in the major model systems, including worms, flies, rodents, and increasingly primates. Building on our prior experience in developing these tools, we now propose to build a new suite of GPCR-activation-based (GRAB) genetically-encoded fluorescent indicators for neuromodulators. Our preliminary data shows we can generate GRABs with >500% fluorescence change and nanomolar affinity in mammalian cells. We propose to further develop and validate these prototypes in cultured neurons, flies, rodent brain slices, anesthetized and behaving mice to maximize their utility.
In Aim 1, we will develop GRAB indicators for acetylcholine, serotonin, and norepinephrine by iteratively screening libraries that systematically vary in insertion site, linkers, cpGFP sequence, and FP-GPCR protein surface interface. The dimensions of optimization will be dF/F, membrane surface expression, affinity, and non-disruption of endogenous signaling. Our targeted performance levels are >10x dF/F, nanomolar range affinity and <10 millisecond on-rates in vitro.
In Aim 2, performance of top candidate GRAB indicators from the in vitro screen will be validated following long-term expression in drosophila olfactory system, in brain slice, in anesthetized and behaving mouse cortex. Feedback from these experiments will guide iterative optimization in Aim 1. Successful completion of our Aims will yield a suite of powerful molecular constructs, cell-type specific viral tools and technical approaches that will be broadly disseminated to the neuroscience community. The GRAB indicators can be easily integrated with existing mouse models of human mental disorders. Since these probes for neuromodulators are well-suited for a wide range of preparations, and a large number of investigators, they will have a multiplicative impact on our understanding of neural circuit function and dysfunction when combined with other advances supported by the BRAIN Initiative.

Public Health Relevance

The proposed research is relevant to public health because it will produce a powerful platform of new tools for chronic, non-invasive monitoring of neuromodulation in the brain during behavior. This is relevant to NIH?s mission because these tools will permit new classes of experiments that reveal the role these important molecules play in regulating cognition, mood, memory, and sleep, and how aberrant neuromodulation causes many neurological disorders.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project--Cooperative Agreements (U01)
Project #
5U01NS103558-02
Application #
9525449
Study Section
Special Emphasis Panel (ZNS1)
Program Officer
Talley, Edmund M
Project Start
2017-09-30
Project End
2020-06-30
Budget Start
2018-07-01
Budget End
2019-06-30
Support Year
2
Fiscal Year
2018
Total Cost
Indirect Cost
Name
University of Southern California
Department
Type
Schools of Arts and Sciences
DUNS #
072933393
City
Los Angeles
State
CA
Country
United States
Zip Code
90089
Sun, Fangmiao; Zeng, Jianzhi; Jing, Miao et al. (2018) A Genetically Encoded Fluorescent Sensor Enables Rapid and Specific Detection of Dopamine in Flies, Fish, and Mice. Cell 174:481-496.e19
Wang, Huan; Jing, Miao; Li, Yulong (2018) Lighting up the brain: genetically encoded fluorescent sensors for imaging neurotransmitters and neuromodulators. Curr Opin Neurobiol 50:171-178
Zhang, Guang-Wei; Sun, Wen-Jian; Zingg, Brian et al. (2018) A Non-canonical Reticular-Limbic Central Auditory Pathway via Medial Septum Contributes to Fear Conditioning. Neuron 97:406-417.e4
Zhang, Guang-Wei; Shen, Li; Zhong, Wen et al. (2018) Transforming Sensory Cues into Aversive Emotion via Septal-Habenular Pathway. Neuron 99:1016-1028.e5
Jing, Miao; Zhang, Peng; Wang, Guangfu et al. (2018) A genetically encoded fluorescent acetylcholine indicator for in vitro and in vivo studies. Nat Biotechnol 36:726-737
Tanaka, Masashi; Sun, Fangmiao; Li, Yulong et al. (2018) A mesocortical dopamine circuit enables the cultural transmission of vocal behaviour. Nature 563:117-120
Chou, Xiao-Lin; Wang, Xiyue; Zhang, Zheng-Gang et al. (2018) Inhibitory gain modulation of defense behaviors by zona incerta. Nat Commun 9:1151