Deep brain live imaging of cAMP and protein kinase A activities underlying synaptic- and circuit-level mechanisms during learned behaviors

学习行为过程中突触和回路水平机制下的 cAMP 和蛋白激酶 A 活动的深部脑部实时成像

• 批准号: 10546580
• 负责人: Shana M Augustin
• 金额: $ 24.9万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-03-01 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10546580
• 关键词: Address; Adenylate Cyclase; Advisory Committees; Affect; Anatomy; Animals; Basal Ganglia; Behavior; Behavioral; Brain; Cell Culture Techniques; Cells; Cognition; Committee Members; Corpus striatum structure; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Data; Decision Making; Development; Disease; Dorsal; Drug Targeting; Drug usage; Energy Transfer; Ensure; Equilibrium; Etiology; Experimental Designs; Extramural Activities; Faculty; Fiber; Fluorescence Resonance Energy Transfer; Fostering; G-Protein-Coupled Receptors; GTP-Binding Proteins; Hormones; Image; Imaging Techniques; Impairment; Lateral; Learning; Learning Skill; Ligand Binding; Ligands; Link; Lipids; Location; Locomotion; Long-Term Depression; Measures; Mediating; Memory; Mentors; Molecular Profiling; Molecular Target; Monitor; Moods; Motor; Mus; Neuromodulator; Neurons; Outcome; Parkinson Disease; Pathway interactions; Pattern; Performance; Periodicity; Pharmaceutical Preparations; Phosphorylation; Phosphotransferases; Photometry; Photons; Physiological; Physiological Processes; Positioning Attribute; Preparation; Process; Proteins; Publishing; Reporter; Research; Research Personnel; Running; Second Messenger Systems; Signal Pathway; Signal Transduction; Signaling Protein; Smell Perception; Specificity; Students; Synapses; Synaptic Transmission; Synaptic plasticity; System; Testing; Time; Tissue imaging; Training; Transgenic Mice; Viral; adenylyl cyclase type V; base; behavioral response; brain tissue; career; cell type; design; experimental study; fluorescence lifetime imaging; improved; in vivo; information processing; innovation; interest; learned behavior; motor skill learning; nervous system disorder; neural circuit; neuronal excitability; neuroregulation; neurotransmission; optical imaging; postsynaptic; receptor; recruit; relating to nervous system; response; sensor; skills; small hairpin RNA; spatiotemporal; tenure track; tool; two-photon

Neuromodulation is crucial for information processing throughout the brain. Neuromodulators influence neuronal function by acting through G protein-coupled receptors (GPCRs) to alter neuronal excitability and synaptic transmission, which can then affect circuit functions. GPCRs are major drug targets used to treat a variety of diseases, including neurological disorders. The causal link between in vivo subcellular signaling mechanisms and behaviors is poorly understood due to the limited tools available to monitor signaling in freely behaving animals. Activation of GPCRs stimulates G-protein signaling to increase or decrease cyclic monophosphate (cAMP) accumulation and bidirectionally control Protein Kinase A (PKA) and Exchange Protein directly Activated by cAMP (EPAC) signaling. Although GPCRs are diverse, the downstream second messenger systems are limited. Therefore, the overarching hypothesis of this proposal is that GPCRs decode incoming modulatory inputs by generating distinct spatiotemporal patterns of cAMP-mediated signaling to control basal ganglia circuit functions. To test this hypothesis, I propose two innovative specific aims: Specific Aim 1 – I will determine the spatiotemporal dynamics in real- time of A-kinase phosphorylation using virally expressed A-kinase activity reporter (AKAR) and cAMP using the EPAC Föster resonance energy transfer (FRET) - based sensors before and after the induction of striatal long- term depression (LTD) in specific cell types. To execute this Aim, I will use transgenic mice to target specific neuronal cell types and two-photon fluorescence lifetime imaging microscopy (FLIM) to quantify FRET activity. These results will build on my previous published findings and will be of broad interest to the basal ganglia field. Specific Aim 2 – I will monitor cAMP and PKA temporal signaling profiles in specific striatal cell types in freely-moving mice during spontaneous locomotion and motor-skill learning on the accelerated rotarod using virally expressed AKAR and EPAC sensors and deep brain in vivo fiber photometry. This proposal will be the first to determine the cAMP mediated signaling dynamics in striatum during synaptic plasticity and learned behaviors. Throughout my career, I have been interested in determining the causal link between synaptic plasticity and behaviors. At every stage of my career, I have advanced in my technical abilities and refined my scientific experimental design. As I train with my mentors, Drs. Lovinger and Vogel, I will further expand my technical abilities and increase my scientific sophistication to ask impactful questions and design appropriate experiments to address these questions. Additionally, my mentors will train me to communicate my scientific findings effectively, run a successful lab, and mentor to students. I have recruited two extramural investigators, Drs. Cheer and Gremel to serve as advisory committee members and aid in my successful transition to an independent faculty position. Together, my mentors will ensure that I am trained in the skills required to attain a tenure-track faculty position and succeed as an independent research investigator.

神经调节对于整个大脑的信息处理至关重要。神经调节剂通过 G 蛋白偶联受体 (GPCR) 改变神经元兴奋性和突触传递来影响神经元功能，从而影响 GPCR 的回路功能，从而成为治疗多种疾病的主要药物靶点。由于可用于监测自由行为动物的信号传导的工具有限，因此对体内亚细胞信号传导机制和行为之间的因果关系知之甚少。虽然 GPCR 多种多样，但下游第二信使系统是有限的。该提议的主要内容是，GPCR 通过生成 cAMP 介导的信号传导的不同时空模式来解码传入的调制输入，以控制基底神经节回路功能。为了检验这一假设，我提出了两个创新的具体目标：目标 1 – 我将使用病毒表达的 A 激酶活性报告基因 (AKAR) 和基于 EPAC Föster 共振能量转移 (FRET) 的传感器的 cAMP 来实时确定 A 激酶磷酸化前后的时空动态。为了实现这一目标，我将使用转基因小鼠来靶向特定的神经元细胞类型和双光子荧光寿命成像显微镜。 (FLIM) 来量化 FRET 活性。这些结果将建立在我之前发表的研究结果的基础上，并将引起基底神经节领域的广泛兴趣。具体目标 2 – 我将自由监测特定纹状体细胞类型中的 cAMP 和 PKA 时间信号传导谱。使用病毒表达的 AKAR 和 EPAC 传感器以及深部脑体内光纤光度测定法在加速旋转棒上进行自发运动和运动技能学习期间移动小鼠。该提案将是第一个确定 cAMP 介导的信号动力学的提案。在我的职业生涯中，我一直对确定突触可塑性和行为之间的因果关系感兴趣。在我职业生涯的每个阶段，我都提高了我的技术能力并完善了我的科学实验设计。与我的导师 Lovinger 和 Vogel 博士一起培训，我将扩展我的技术能力并提高我的科学水平，以提出有影响力的问题并设计适当的实验来进一步解决这些问题，我将训练我传达我的科学导师的发现。我聘请了两位校外研究人员 Cheer 博士和 Gremel 博士作为顾问委员会成员，并帮助我成功过渡到独立教职职位。我接受过获得终身教授职位和成功成为一名独立研究调查员所需的技能培训。