Spatial, temporal, and context-dependent features of GPCR-mediated protein kinase A activity

神经调质诱导的细胞内信号转导的空间特异性、时间动态和背景依赖性 信号是解释神经调节剂功能的关键。然而，尽管许多信号的身份 神经调节剂受体下游的分子是已知的，这些特征的性质和功能是已知的。 不太了解。长期目标是揭示细胞和亚细胞特异性，时间特异性， 动力学和神经调节剂诱导的细胞内信号的上下文依赖性。总体目标 本文旨在探讨乙酰胆碱（ACh）介导的蛋白激酶A（PKA）的特性和突触功能 (PKA)海马体的活动。中心假设是ACh通过空间、时间、 和对突触可塑性至关重要的上下文依赖特异性。这个项目的基本原理来自于 从多条证据线上首先，G α q偶联的毒蕈碱ACh受体（mAChRs）提高PKA活性。 第二，PKA活动表现出丰富的空间，时间和上下文相关的功能。第三，扰动 PKA活性的扩散和持续时间改变细胞和行为功能，说明其重要性。 时空动力学最后，mAChR和PKA都是突触可塑性的强大调节剂。的 中心假设将在急性海马切片和头部固定的小鼠中进行测试，有三个具体目标 以确定亚细胞区室（目标1），时间动态（目标2）和上下文- 依赖性（目的3）PKA激活ACh和突触可塑性的这些功能的作用。到 确定mAChR介导的PKA活性的特征，光遗传学将用于诱导ACh释放， ACh水平和PKA活性将用新型生物传感器和双光子荧光寿命测量 成像显微镜（2pFLIM）。为了确定这些特征对突触可塑性的贡献， 隔室靶向，光激活致动器将用于干扰PKA的活动与空间和时间 精确度和电生理学将被用来测量突触传递。拟议的研究是 创新，因为在概念上，它超越了分子的身份，揭示了它们的行为， 超越静态快照，揭示信号动态，并超越了解一个 揭示他们的贡献。在方法上，研究采用了尖端技术， 诱导神经调节剂释放，以及测量和干扰具有空间和时间 精确-这些方法将在神经调节剂以外的细胞信号传导中找到广泛的应用 research.拟议的研究是重要的，因为它将提供解释能力，如何功能，而不是 仅仅是细胞内信号的同一性，塑造细胞的生理和行为。这些结果将揭示新的 原则的神经调节剂的行动，并提供深入了解如何分子机制一般行为 相关特征。从长远来看，这些结果将有助于设计针对相关特征的更好疗法 在神经和精神疾病方面。

The spatial specificity, temporal dynamics, and context dependence of neuromodulator-induced intracellular signals are essential to explain neuromodulator function. However, although the identity of many signaling molecules downstream of neuromodulator receptors are known, the nature and functions of these features are poorly understood. The long-term goal is to uncover the cellular and subcellular specificity, the temporal dynamics, and the context-dependence of neuromodulator-induced intracellular signals. The overall objective here is to determine the features and synaptic functions of acetylcholine (ACh)-mediated protein kinase A (PKA) activity in the hippocampus. The central hypothesis is that ACh regulates PKA with spatial, temporal, and context-dependent specificity that is essential to synaptic plasticity. The rationale for this project came from multiples lines of evidence. First, Gαq-coupled muscarinic ACh receptors (mAChRs) elevate PKA activity. Second, PKA activity demonstrates rich spatial, temporal, and context-dependent features. Third, perturbations of the spread and duration of PKA activity alter cellular and behavior functions, illustrating the importance of its spatiotemporal dynamics. Finally, mAChRs and PKA are both powerful regulators of synaptic plasticity. The central hypothesis will be tested in both acute hippocampal slices and head-fixed mice, with three specific aims to determine the subcellular compartments (Aim 1), the temporal dynamics (Aim 2), and the context- dependence (Aim 3) of PKA activation by ACh and the roles of these features for synaptic plasticity. To determine the features of mAChR-mediated PKA activity, optogenetics will be used to induce ACh release, and ACh level and PKA activity will be measured with novel biosensors and two-photon fluorescence lifetime imaging microscopy (2pFLIM). To determine the contribution of these features to synaptic plasticity, subcellular compartment-targeted, light-activated actuators will be used to perturb PKA activity with spatial and temporal precision, and electrophysiology will be used to measure synaptic transmission. The proposed research is innovative because conceptually, it goes beyond the identity of molecules to revealing their actions, goes beyond static snapshots to revealing signaling dynamics, and goes beyond knowing the involvement of a signal to revealing their contributions. Methodologically, the research employs cutting-edge technology to induce neuromodulator release, and to measure and perturb intracellular signals with spatial and temporal precision – these approaches will find widespread application in cellular signaling beyond neuromodulator research. The proposed research is significant because it will offer explanatory power on how features, and not just identity of intracellular signals, shape cellular physiology and behavior. These results will reveal new principles of neuromodulator action, and provide insights into how molecular mechanisms general behaviorally relevant features. In the long run, these results will help design better therapies that target the relevant features in neurological and psychiatric disorders.