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

基本信息

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
项目类别：
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

项目摘要

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（适用提供）：神经元之间发生了两种主要的化学通信模式：快速突触传播，例如由谷氨酸和GABA介导的直接控制神经元的电活动以及缓慢的合成传递，例如由无甲甲肾上腺素和多巴胺介导的，这些传播是由Norepinephrine and Dopamine介导的，这些肾上腺素和多巴胺是无法直接测量Neurony equist firical equctial firctical neuron firctical timal firctical timal timal timal timal timal timal timal timal realital timal realital timal hearon的介导的。缓慢的突触传播也称为神经调节，在确定兴奋性，突触可塑性和神经元功能的其他方面起着重要的调节作用，有时无法对快速突触传播的功能进行强有力的控制。但是，与快速合成传播不同，可以通过越来越多的现代方法（例如多电极记录，电压成像和钙成像方法）进行监控，而对生存动物中的精确神经调节事件的了解少得多，因为没有一种可靠地记录相关活动的神经神经触发的viv fiv in nys nyuronsection ny nyuronsection in nys nyurons in n nysty nyurons in ny nyurons in ny nyurons of单个Neuromons in viv in viv in viv in viv in ny neuromons中。为了克服这个问题，我们提出了一种新的方法，用于通过成像循环AMP（CAMP）和蛋白激酶A（PKA）的活性来检查体内单神经元分辨率的神经调节活性。 CAMP/PKA途径是多巴胺和去甲肾上腺素的常见下游信号转移途径。尽管基于Förster共振能量转移（FRET）的遗传编码的CAMP/PKA传感器已用于体外实验，但由于在体内挑战性较小的情况下，由于较低的信噪比，它们在体内的应用很困难。我们提出了一种多收益的方法，以消除使用当前FRET成像方法遇到的几种瓶颈，以最大程度地提高信噪比。我们的方法包括：1）开发和改进CAMP/PKA传感器，2）实施FRET成像模式，该模式比在光散发脑组织中更有效的FRET测量更有效，3）纠正与体内成像条件相关的光差，以及4）为高对比度，可重复可重复的FET成像而开发新颖的小鼠试剂。我们将通过确定使用光遗传学方法和使用不同的压力刺激的小鼠在麻醉小鼠中的去甲肾上腺素作用的时空模式来验证这种方法来监测神经调节活性。如果成功，我们的努力将提供以前无法实现的能力，以对细胞和电路水平的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完成快速突触传播的测量，以增强我们对动物行为基础的脑功能的理解。