A novel approach to examine slow synaptic transmission in vivo

一种检查体内缓慢突触传递的新方法

• 批准号: 9327081
• 负责人: Tianyi Mao
• 金额: $ 31.71万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-30 至 2018-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9327081
• 关键词: Accelerometer; Adrenergic Antagonists; Animal Behavior; Animals; Axon; Behavior; Benchmarking; Brain; Calcium; Cells; Chemicals; Communication; Complement; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Defect; Development; Dopamine; Electrodes; Energy Transfer; Event; Exposure to; Foxes; Functional disorder; Future; Glutamates; Habitats; Head; Image; In Situ; In Vitro; Individual; Investigation; Light; Measurement; Measures; Mediating; Methods; Microdialysis; Modernization; Monitor; Mus; Neuromodulator; Neurons; Noise; Norepinephrine; Pathway interactions; Pattern; Performance; Periodicity; Play; Reagent; Regulation; Reporting; Reproducibility; Resolution; Role; Scanning; Signal Transduction; Signal Transduction Pathway; Smell Perception; Somatosensory Cortex; Source; Stress; Synaptic Transmission; Synaptic plasticity; Technology; Urine; Walking; adaptive optics; base; brain tissue; contrast imaging; experimental study; fluorescence lifetime imaging; gamma-Aminobutyric Acid; hippocampal pyramidal neuron; imaging approach; imaging modality; improved; in vivo; in vivo imaging; light scattering; locus ceruleus structure; nervous system disorder; neuroregulation; noradrenergic; novel; novel strategies; optogenetics; public health relevance; response; sensor; sound; spatiotemporal; treadmill; two-photon; voltage

DESCRIPTION (provided by applicant): Two primary modes of chemical communication occur between neurons in the brain: fast synaptic transmission, such as that mediated by glutamate and GABA, which directly control the electrical activities of neurons, and slow synaptic transmission, such as that mediated by norepinephrine and dopamine, which regulate subcellular signaling events that cannot be measured directly from neuronal electrical activities. Slow synaptic transmission, which is also called neuromodulation, plays important modulatory roles in regulating excitability, synaptic plasticity and other aspects of neuronal function, and eventually imposes powerful control over the function of fast synaptic transmission. However, unlike fast synaptic transmission, which can be monitored directly via an increasing number of modern approaches such as multi-electrode recording, voltage imaging and calcium imaging methods, much less is known about the precise neuromodulatory events that occur in living animals because there has not been an established method to reliably record the relevant activities triggered by neuromodulation in individual neurons in vivo. To overcome this problem, we propose a novel approach for examining neuromodulatory activities with single-neuron resolution in vivo by imaging the activity of cyclic AMP (cAMP) and protein kinase A (PKA). The cAMP/PKA pathway is a common downstream signal transduction pathway for both dopamine and norepinephrine. Although genetically encoded cAMP/PKA sensors based on Förster resonance energy transfer (FRET) have been used for experiments in vitro, their application in vivo has been difficult due to lower signal-to-noise ratios under the more challenging in vivo imaging conditions. We propose a multipronged approach to eliminate several bottlenecks encountered with current FRET imaging approaches to maximize the signal-to-noise ratio. Our approach includes: 1) developing and improving cAMP/PKA sensors, 2) implementing a FRET imaging modality that is more effective than conventional FRET measures in light-scattering brain tissue, 3) correcting light aberrations associated with in vivo imaging conditions, and 4) developing novel mouse reagents for high-contrast, reproducible FRET imaging. We will validate the utility of this method for monitoring neuromodulatory activities by determining the spatiotemporal patterns of norepinephrine action in anesthetized mice using optogenetic approaches and in behaving mice using different stress stimulations. If successful, our efforts will provide a previously unattainable ability to conduct large- scale monitoring of neuromodulatory activities in the brain at the cellular and circuitry levels. This ability to quantitate neuromodulation will complement the measurements of fast synaptic transmission to enhance our understanding of brain function underlying animal behavior.

描述（由申请人提供）：在脑中的神经元之间发生两种主要的化学通信模式：快速突触传递，例如由谷氨酸和GABA介导的突触传递，其直接控制神经元的电活动;以及缓慢突触传递，例如由去甲肾上腺素和多巴胺介导的突触传递，其调节不能从神经元电活动直接测量的亚细胞信号传导事件。慢突触传递（Slow synaptic transmission）又称神经调制（neuromodulation），在调节神经元兴奋性、突触可塑性等方面起着重要的调节作用，并最终对快突触传递的功能产生强有力的控制。然而，与快速突触传递不同，快速突触传递可以通过越来越多的现代方法（如多电极记录，电压成像和钙成像方法）直接监测，对活体动物中发生的精确神经调节事件知之甚少，因为还没有建立方法来可靠地记录体内单个神经元中神经调节触发的相关活动。为了克服这个问题，我们提出了一种新的方法，通过成像环磷酸腺苷（cAMP）和蛋白激酶A（PKA）的活性，在体内检查单神经元分辨率的神经调节活动。cAMP/PKA通路是多巴胺和去甲肾上腺素的共同下游信号转导通路。尽管基于Förster共振能量转移（FRET）的基因编码的cAMP/PKA传感器已用于体外实验，但由于在更具挑战性的体内成像条件下的较低信噪比，它们在体内的应用一直很困难。我们提出了一个多管齐下的方法，以消除目前的FRET成像方法遇到的几个瓶颈，以最大限度地提高信噪比。我们的方法包括：1）开发和改进cAMP/PKA传感器，2）在光散射脑组织中实施比常规FRET测量更有效的FRET成像模式，3）校正与体内成像条件相关的光像差，以及4）开发用于高对比度、可再现FRET成像的新型小鼠试剂。我们将验证这种方法的实用性，通过确定的时空模式的去甲肾上腺素的作用在麻醉小鼠使用光遗传学方法和行为小鼠使用不同的应激刺激监测神经调节活动。如果成功，我们的努力将提供一种以前无法实现的能力，在细胞和电路水平上对大脑中的神经调节活动进行大规模监测。这种定量神经调节的能力将补充快速突触传递的测量，以增强我们对动物行为背后的脑功能的理解。