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。神经调节剂通过通过G蛋白偶联受体（GPCR）作用来影响神经元功能，以改变神经元令人兴奋和合成的传播，从而影响电路功能。 GPCR是用于治疗各种疾病（包括神经系统疾病）的主要药物靶标。体内亚细胞信号传导机制和行为之间的因果关系很差，因为可用于监视自由行为的动物信号的工具有限。 GPCR的激活刺激G蛋白信号传导，以增加或减少环状单磷酸（CAMP）积累，双向控制蛋白激酶A（PKA）和由CAMP（EPAC）信号直接激活的交换蛋白。尽管GPCR是潜水员，但下游的第二使者系统受到限制。因此，该提案的总体假设是GPCR通过生成CAMP介导的信号传导的不同空间时间模式来控制基本的神经节电路函数来解码传入的调节输入。 To test this hypothesis, I propose two innovative specific aims: Specific Aim 1 – I will determine the spatial temporal dynamics in real-time of A-kinase phosphorylation using virtually 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.为了执行此目标，我将使用转基因小鼠靶向特定的神经元细胞类型和两光子荧光寿命成像显微镜（FLIM）来量化FRET活性。这些结果将基于我以前发表的发现，并将引起基底神经节领域的广泛关注。特定目标2 - 我将在赞助的机动运动中，在自由移动小鼠的特定纹状体细胞类型中监视CAMP和PKA临时信号传导曲线，并使用实际上表达AKAR和EPAC传感器和深脑在体内纤维光度测定仪上的AKAR ROTAROD上的运动技巧和运动技能学习。该提案将是第一个确定突触可塑性和学识渊博行为期间cAMP介导的信号动力学的一个提案。在我的职业生涯中，我一直有兴趣确定合成可塑性与行为之间的因果关系。在职业生涯的每个阶段，我都提高了技术能力，并完善了我的科学实验设计。当我和导师一起训练时，博士。 Lovinger和Vogel，我将进一步扩大我的技术能力，并提高我的科学成熟社会化，以提出有影响力的问题并设计适当的实验以解决这些问题。此外，我的导师将训练我有效地传达我的科学发现，成功实验室，并向学生进行导师。我已经招募了两名壁外调查员，博士。为担任咨询委员会成员的欢呼和格莱默尔（Gremel），并帮助我成功过渡到独立教师职位。我的导师将共同确保我接受了获得终身教师职位所需的技能，并成为独立的研究调查员。