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

神经调节对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(GPCRs)改变神经元兴奋性和突触传递，进而影响神经回路功能，从而影响神经功能。GPCRs是用于治疗包括神经疾病在内的各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递的工具有限，体内亚细胞信号机制和行为之间的因果联系尚不清楚。GPCRs的激活刺激G蛋白信号通路增加或减少环单磷酸(CAMP)的蓄积，并双向调控蛋白激酶A(PKA)和由cAMP直接激活的交换蛋白(EPAC)信号。尽管GPCRs多种多样，但下游的第二信使系统是有限的。因此，这一建议的首要假设是，GPCRs通过产生不同的cAMP介导信号的时空模式来解码传入的调制输入，以控制基底节的电路功能。为了验证这一假说，我提出了两个创新的特定目标：特定目标1-I将使用病毒表达的A-激酶活性报告(AKAR)和使用基于EPAC Föster共振能量转移(FRET)的传感器在特定细胞类型诱导纹状体长期抑制(LTD)之前和之后实时确定A-Kinase磷酸化的时空动态。为了实现这一目标，我将使用转基因小鼠来定位特定的神经细胞类型，并使用双光子荧光寿命成像显微镜(FLIM)来量化FRET的活性。这些结果将建立在我之前发表的发现的基础上，并将引起基底节领域的广泛兴趣。特定目标2-I将使用病毒表达的AKAR和EPAC传感器以及活体脑深部纤维光度法，监测自由活动小鼠在自发运动和加速旋转棒上的运动技能学习过程中特定纹状体细胞类型中cAMP和PKA的时间信号谱。这将是首次确定纹状体在突触可塑性和习得行为过程中cAMP介导的信号动力学。在我的整个职业生涯中，我一直对确定突触可塑性和行为之间的因果联系感兴趣。在我职业生涯的每个阶段，我都提升了自己的技术能力，完善了我的科学实验设计。随着我与我的导师洛文杰博士和沃格尔博士一起训练，我将进一步扩大我的技术能力，提高我的科学成熟度，以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，我的导师将培训我有效地交流我的科学发现，运营一个成功的实验室，并指导学生。我已经招募了两名校外调查人员，乔尔博士和格雷梅尔博士担任顾问委员会成员，帮助我成功过渡到独立的教员职位。我的导师将一起确保我接受培训，掌握获得终身教职所需的技能，并作为一名独立的研究调查员取得成功。

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.