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

神经调节剂诱导的细胞内的空间特异性、时间动力学和上下文依赖性 信号是解释神经调节器功能的关键。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 人们对此知之甚少。长期的目标是揭示细胞和亚细胞的特异性，时间上的 动力学，以及神经调节剂诱导的细胞内信号的上下文依赖性。总体目标 本研究旨在确定乙酰胆碱(ACh)介导的蛋白激酶A的特征和突触功能 (PKA)在海马区的活动。中心假说是ACh通过空间，时间， 以及对突触可塑性至关重要的上下文相关的特异性。这个项目的基本原理来自于 从多条证据中。首先，G-αQ偶联的M受体(MAChRs)可提高PKA活性。 第二，PKA活动表现出丰富的空间、时间和上下文相关特征。第三，扰动 PKA活动的传播和持续时间改变了细胞和行为功能，说明了其重要性 时空动力学。最后，mAChRs和PKA都是突触可塑性的强大调节者。这个 中枢假说将在急性海马片和头部固定的小鼠身上进行测试，有三个特定的目标 为了确定亚细胞隔间(目标1)、时间动力学(目标2)和上下文- ACh激活PKA的依赖性(目标3)以及这些特征在突触可塑性中的作用。至 确定mAChR介导的PKA活性的特征，光遗传学将被用来诱导ACh的释放，并 将使用新型生物传感器和双光子荧光寿命来测量ACh水平和PKA活性 成像显微镜(2pFLIM)。为了确定这些特征对突触可塑性的贡献，亚细胞 针对隔室的、光激活的致动器将被用来干扰PKA的空间和时间活动 精确度和电生理学将被用来测量突触传递。拟议的研究是 创新是因为从概念上讲，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 表明他们的贡献的信号。在方法上，这项研究使用了尖端技术来 诱导神经调节剂的释放，并用空间和时间测量和扰动细胞内信号 精确度-这些方法将在神经调节剂以外的细胞信号传递中得到广泛应用 研究。这项拟议的研究具有重要意义，因为它将提供对功能的解释力，而不是 只是细胞内信号的识别，塑造细胞的生理和行为。这些结果将揭示新的 神经调节剂的作用原理，并提供对分子机制如何普遍行为的见解 相关功能。从长远来看，这些结果将有助于设计更好的针对相关功能的疗法 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.