Lighting up the brain: Optogenetic tools to record, trace, and manipulate brain circuits at cellular resolution

点亮大脑：以细胞分辨率记录、追踪和操纵大脑回路的光遗传学工具

• 批准号: 10244755
• 负责人: Ahmed Abdelfattah
• 金额: $ 142.83万
• 依托单位: BROWN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-16 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10244755
• 关键词: Action Potentials; Amino Acids; Animals; Behavior; Brain; Cells; Chemicals; Communication; Coupled; Coupling; Directed Molecular Evolution; Disease; Exhibits; Fluorescence; Health; Heart; Human; Image; Lighting; Longitudinal Studies; Maps; Measures; Memory; Microdialysis; Microscope; Molecular Conformation; Network-based; Neurons; Neuropeptides; Phage Display; Process; Protein Engineering; Proteins; Rabies virus; Reporter; Resolution; Role; Shapes; Signal Transduction; Spectrum Analysis; Structure; Synaptic Potentials; Tracer; Translating; Visualization; brain cell; detection method; empowered; genetic payload; improved; nanobodies; neural circuit; neural network; neurotoxicity; novel; optogenetics; relating to nervous system; sensor; spatiotemporal; tool; two-photon; voltage

PROJECT SUMMARY Brain circuits are dynamic networks of neurons that process information in the form of electrical and chemical signals to form memories and shape behaviors. To investigate how brain circuits instantiate fundamental computations underlying behaviors, we need to map their wiring diagrams coupled with functional analysis at cellular resolution. However, the electrical (voltage) and chemical (e.g. neuropeptides) signals are not directly visible, and current circuit tracing tools are insufficient for meaningful functional analysis. Using protein engineering this proposal aims to develop a toolbox of genetically-encoded fluorescent reporters and tracers specifically tailored to study neural circuits. At the electrical level, voltage sensors can image the precise timing of action potentials and subthreshold voltage not detectable by other means. However, even the latest voltage sensors do not perform well with high-resolution microscopes that use 2-photon illumination for imaging deep in the brain. To overcome these limitations, we are taking a two-pronged approach by evolving amino acids at the mechanistic heart of voltage sensor proteins and by using spectroscopy to aid our protein engineering efforts. We believe directed evolution will improve voltage sensitivity and 2-photon functionality >10 fold, enabling us to image currently invisible signals, like synaptic potentials, deep inside the brain. At the chemical level, neuropeptides are highly expressed in almost all cortical neurons, but their role and impact in animals can only be inferred because current detection methods, like microdialysis, are invasive and lack spatiotemporal resolution. We are using phage display to evolve nanobodies capable of recognizing neuropeptides and coupling their conformational changes to fluorescence changes from reporter molecules. These sensors will provide visualization of neuropeptide release at cellular resolution throughout an animal’s brain during behavior paradigms that mimic human health and disease states. At the cellular connectivity level, current tools for circuit- mapping, like rabies virus, exhibit substantial neurotoxicity, prohibiting meaningful functional analyses. We are engineering proteins with a natural propensity to assemble into structures capable of delivering a genetic payload to specific cells to produce more effective and less toxic tools to map and manipulate brain circuits. Effective and robust tools to map the brain will bridge functional and structural analysis and finally allow long- term studies of neural networks based on their connectivity. Overall, the optogenetic tools developed in this proposal will translate the chemical and electrical signals between neural circuits into fluorescence that can be easily measured. Consequently, they can be used to unravel the functional basis and causes of neuronal disorders at a level of detail that has not been accessible to date and empower us to develop novel treatments.

项目总结