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的 cAMP 和蛋白激酶 A 活动的深部脑部实时成像

• 批准号: 10580090
• 负责人: Shana M Augustin
• 金额: $ 24.78万
• 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/10580090
• 关键词: Acceleration; Address; Adenylate Cyclase; Advisory Committees; Affect; Anatomy; Animals; Basal Ganglia; Behavior; Behavioral; Brain; Cell Culture Techniques; Cells; Cognition; Committee Members; Communication; Corpus striatum structure; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Data; Decision Making; Development; Disease; Dissociation; Dorsal; Drug Targeting; Drug usage; Energy Transfer; Ensure; Equilibrium; Etiology; Experimental Designs; Extramural Activities; Faculty; Fiber; Fiber Optics; 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; Medicine; 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; 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; neuronal excitability; neuroregulation; neurotransmission; optical fiber; optical imaging; postsynaptic; receptor; recruit; 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。神经调质通过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，从而影响电路功能。GPCR是用于治疗包括神经系统疾病在内的多种疾病的主要药物靶标。由于可用于监测自由行为动物信号的工具有限，因此对体内亚细胞信号传导机制与行为之间的因果关系了解甚少。GPCR的激活刺激G蛋白信号传导以增加或减少环磷酸（cAMP）积累并双向控制蛋白激酶A（PKA）和由cAMP直接激活的交换蛋白（EPAC）信号传导。虽然GPCR是多样的，下游的第二信使系统是有限的。因此，该提议的首要假设是GPCR通过产生cAMP介导的信号传导的不同时空模式来解码传入的调节输入以控制基底神经节电路功能。为了验证这一假设，我提出了两个创新的具体目标：具体目标1 -我将确定时空动态的真实的时间的A-激酶磷酸化使用病毒表达的A-激酶活性报告（AKAR）和cAMP使用EPAC福斯特共振能量转移（FRET）为基础的传感器之前和之后的纹状体长期抑郁症（LTD）在特定的细胞类型。为了实现这一目标，我将使用转基因小鼠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）定量FRET活性。这些结果将建立在我以前发表的研究结果，并将广泛的兴趣，基底神经节领域。特异性目标2 - I将使用病毒表达的AKAR和EPAC传感器和深部脑体内纤维光度测定法，在自发运动和加速旋转杆上的运动技能学习期间，监测自由移动小鼠中特定纹状体细胞类型中的cAMP和PKA时间信号传导谱。这一提议将是第一个确定突触可塑性和学习行为过程中纹状体cAMP介导的信号动力学。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。在我职业生涯的每一个阶段，我都在提高我的技术能力，完善我的科学实验设计。当我和我的导师Lovinger博士和Vogel博士一起训练时，我将进一步扩展我的技术能力，提高我的科学素养，提出有影响力的问题，并设计适当的实验来解决这些问题。此外，我的导师将训练我有效地交流我的科学发现，运行一个成功的实验室，并指导学生。我已经招募了两名校外调查员，Cheer博士和Gremel博士担任咨询委员会成员，并帮助我成功地过渡到一个独立的教师职位。我的导师们将共同确保我接受获得终身教职所需技能的培训，并成功地成为一名独立的研究调查员。