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（gpcr）改变神经元的兴奋性和突触传递，进而影响神经回路功能，从而影响神经元功能。gpcr是治疗包括神经系统疾病在内的多种疾病的主要药物靶点。体内亚细胞信号传导机制和行为之间的因果关系尚不清楚，因为在自由行为的动物中监测信号传导的工具有限。gpcr的激活刺激g蛋白信号通路增加或减少环单磷酸腺苷（cAMP）的积累，并双向控制蛋白激酶A （PKA）和cAMP直接激活的交换蛋白（EPAC）信号通路。虽然gpcr是多种多样的，但下游的第二信使系统是有限的。因此，该建议的总体假设是，gpcr通过产生不同的camp介导信号的时空模式来解码传入的调节输入，以控制基底神经节回路功能。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 -我将使用病毒表达的a激酶活性报告因子（AKAR）和使用EPAC Föster共振能量转移（FRET）传感器的cAMP，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激酶磷酸化的时空动态。为了实现这一目标，我将使用转基因小鼠靶向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活性。这些结果将建立在我以前发表的发现的基础上，并将对基底神经节领域产生广泛的兴趣。具体目标2 -我将使用病毒表达的AKAR和EPAC传感器和深部脑体内纤维测光技术，监测自由运动小鼠自发运动和运动技能学习过程中特定纹状体细胞类型的cAMP和PKA时间信号谱。本研究将首次确定cAMP介导的纹状体突触可塑性和学习行为过程中的信号动力学。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很感兴趣。在我职业生涯的每一个阶段，我的技术能力都在不断提高，我的科学实验设计也在不断完善。当我和导师们一起训练时。Lovinger和Vogel，我将进一步扩展我的技术能力，提高我的科学成熟度，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传达我的科学发现，管理一个成功的实验室，并指导学生。我已经招募了两名校外调查员。齐尔和格莱梅尔担任咨询委员会成员，帮助我成功过渡到独立教师的职位。我的导师们将共同确保我获得获得终身教职职位所需的技能培训，并成功地成为一名独立的研究人员。

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.