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

GPCR 介导的蛋白激酶 A 活性的空间、时间和上下文相关特征

• 批准号: 10441526
• 负责人: Yao Chen
• 金额: $ 39.38万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-01 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10441526
• 关键词: Acetylcholine; Acute; Animal Behavior; Behavior; Behavioral; Biochemical; Biological Assay; Biosensor; Brain; Calcium; Cell physiology; Cells; Cholinergic Receptors; Complex; Coupled; Cyclic AMP-Dependent Protein Kinases; Data; Dendritic Spines; Dependence; Electrophysiology (science); Event; Frequencies; G-Protein-Coupled Receptors; GTP-Binding Protein alpha Subunits, Gs; Glutamates; Goals; Head; Hippocampus (Brain); Image; Learning; Light; Link; Location; Measures; Mediating; Memory; Mental disorders; Methodology; Mission; Modeling; Molecular; Mus; Muscarinic Acetylcholine Receptor; Muscarinics; Nature; Neurodegenerative Disorders; Neuromodulator; Neuromodulator Receptors; Optical reporter; Pattern; Peptides; Pharmacologic Substance; Phosphorylation; Phosphotransferases; Population; Positioning Attribute; Protein Dynamics; Protein Kinase A Inhibitor; Psychiatric therapeutic procedure; Public Health; Recording of previous events; Regulation; Reporter; Research; Role; Shapes; Signal Pathway; Signal Transduction; Signaling Molecule; Sleep; Slice; Specificity; Synapses; Synaptic Transmission; Synaptic plasticity; System; Technology; Testing; Time; United States National Institutes of Health; Vertebral column; awake; base; cell behavior; cholinergic neuron; design; disability; drug of abuse; fluorescence lifetime imaging; imaging system; in vivo; innovation; insight; nervous system disorder; neural circuit; novel; optogenetics; personalized approach; protein activation; real-time images; receptor; response; spatiotemporal; synaptic function; targeted treatment; tool; two-photon

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.

神经调节剂诱导的细胞内的空间特异性，临时动力学和上下文依赖性 信号对于解释神经调节剂函数至关重要。但是，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 理解不佳。长期目标是揭示细胞和亚细胞特异性，临时性 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 这是确定乙酰胆碱（ACH）介导的蛋白激酶A的特征和突触功能 （PKA）海马中的活性。中心假设是ACH通过空间，临时， 和上下文依赖性特异性，这对于合成可塑性至关重要。这个项目的理由是 来自多种证据。首先，GαQ偶联的毒蕈碱ACH受体（MACHR）提高了PKA活性。 其次，PKA活动表明了丰富的空间，临时和上下文依赖性特征。第三，扰动 PKA活性的扩散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，MACHR和PKA都是突触可塑性的强大调节剂。这 中央假设将在急性海马切片和头部固定小鼠中进行测试，三个特定的目标 确定亚细胞隔室（AIM 1），临时动力学（AIM 2）和上下文 - PKA激活的依赖性（目标3）以及这些特征在突触可塑性方面的作用。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，并且 ACH水平和PKA活性将通过新型生物传感器和两光子荧光寿命进行测量 成像显微镜（2PFLIM）。确定这些特征对合成可塑性的贡献 针对隔室的，光激活的执行器将用于使用空间和临时的PKA活动 精度和电生理学将用于测量突触传播。拟议的研究是 创新性是因为从概念上讲，它超越了分子的身份来揭示其行为， 除了静态快照外，还可以揭示信号动力学，而不仅仅知道 信号以揭示他们的贡献。从方法上讲，研究员工的最先进技术 诱导神经调节剂释放，并测量和扰动具有空间和临时性的细胞内信号 精度 - 这些方法将在神经调节剂以外的细胞信号中找到宽度应用 研究。拟议的研究之所以重要，是因为它将提供有关功能的剥夺能力，而不是 仅具有细胞内信号的身份，形状的细胞生理和行为。这些结果将揭示新的 神经调节剂作用的原理，并提供有关分子机制在行为上如何普遍的见解 相关功能。从长远来看，这些结果将有助于设计针对相关功能的更好的疗法 在神经和精神疾病中。