Development of a scalable methodology for imaging neuropeptide release in the brain

开发一种可扩展的大脑神经肽释放成像方法

• 批准号: 9056190
• 负责人: David J Anderson
• 金额: $ 25.01万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 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 2017-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9056190
• 关键词: Affect; Aggressive behavior; Antibodies; Anxiety; Beds; Behavior; Behavioral Assay; Biogenic Amines; Biological Models; Brain; Brain region; Cellular biology; Chemicals; Chemistry; Code; Confocal Microscopy; Crustacea; Dense Core Vesicle; Development; Disease; Drosophila genus; Drosophila melanogaster; Emotions; Fluorescence; Functional disorder; Ganglia; Genetic; Goals; Human; Hunger; Image; Imaging Device; In Vitro; Individual; Label; Lead; Maternal Behavior; Measures; Mental Depression; Mental disorders; Metaphor; Methodology; Methods; Microdialysis; Microscopy; Mission; Molecular Genetics; Monitor; Moods; Motivation; Motor output; Nerve; Neurobiology; Neuromodulator; Neurons; Neuropeptides; Neurosciences; Neurosecretory Granule; Outcome; PHluorin; Pattern; Pharmaceutical Preparations; Play; Post-Traumatic Stress Disorders; Proteins; Public Health; Reporter; Reporting; Reproduction; Research; Resolution; Scanning; Series; Serotonin; Signal Transduction; Social Behavior; Synapses; Synaptic Vesicles; System; Techniques; Technology; Testing; Thirst; Time; Transgenic Organisms; Work; brain electrical activity; expression vector; fly; imaging modality; improved; in vivo; innovation; instrumentation; millisecond; mind control; neural circuit; neuronal circuitry; neuroregulation; new technology; novel; novel strategies; optical imaging; optogenetics; public health relevance; relating to nervous system; sensor; spatiotemporal; supercomputer; technology development; temporal measurement; tool; transmission process; two-photon; vesicular release

 DESCRIPTION (provided by applicant): A common metaphor to describe the brain is that it is like a supercomputer. Consequently, current efforts at improving technologies for large-scale recording of brain function are primarily focused on measuring its electrical activity. However, unlike a supercomputer, the brain is an electrochemical machine. Superimposed upon its network of synaptic connections is a "chemical connectome," a largely invisible network of neuromodulators, such as serotonin and neuropeptides (NPs), which exert a profound influence on brain function. Neuromodulators influence brain states that alter the computations performed by neural circuits, and are central to emotion, mood and affect. An understanding of neuromodulatory influences is therefore relevant to psychiatric disorders in humans. Without the ability to measure and manipulate the release of specific neuromodulators, our understanding of neuronal circuit function will be fundamentally incomplete. Nevertheless, a gap remains between our ability to monitor brain electrical activity, and our ability to monitor chemical activty with commensurate spatio-temporal resolution. Specifically, there is no method to visualize the release of specific NPs in the brain, at individual synapses. To fill this gap, we propose to develop a method for imaging the release of specific NPs at the level of nerve terminals, in vivo. The long-term goal is to develop new methods for visualizing, detecting and inhibiting NP release in vivo, and to apply these methods to understanding the dynamics of neuromodulation of specific, behaviorally relevant neural circuits. The overall goal of this proposal is to developa novel approach for time-resolved imaging of NP release from nerve terminals. The central objective of this proposal is to tag components of large dense core vesicles (LDCVs), and specific NPs, with pH-sensitive fluorescent proteins and to determine whether these reporters can be used to image neurosecretory granule release. Drosophila melanogaster provides a useful test-bed for this technology because of its genetic manipulability and sophisticated imaging methodologies. To achieve our objective, in Aim 1 we will fuse different pH-sensitive fluorescent reporters to the protein coding sequences of several LDCV-specific proteins and NPs, and generate transgenic flies. In Aim 2, we will use optogenetic activation of specific neuropeptidergic neurons containing these reporters in vivo, to determine whether activation-dependent increases in fluorescence can be detected, and distinguished from synaptic vesicle (SV) release. The contribution will be to determine the feasibility of the proposed approach, and to achieve a proof- of-principle application of the method. This contribution is significant, because it has the potential to create a transformative new technology with broad general applications. The contribution is innovative, because it combines expertise from cell biology, molecular genetics and neural circuit analysis to develop a novel methodology. The work proposed in this application will therefore benefit the field of neuroscience as a whole, and also enable studies of neural chemistry and circuitry whose dysfunction may underlie psychiatric disorders.

 描述（由应用程序提供）：描述大脑的常见隐喻是它就像超级计算机。因此，当前改善大脑功能大规模记录技术的努力主要集中在测量其电活动上。但是，与超级计算机不同，大脑是一台电化学机器。叠加在其突触连接网络上的是一种“化学连接组”，它在很大程度上看不见的神经调节剂网络，例如5-羟色胺和神经肽（NPS），对大脑功能产生了深远的影响。神经调节剂会影响大脑状态，该状态改变了神经元电路执行的计算，并且是情绪，情绪和情感的核心。因此，对神经调节影响的理解与人类的精神疾病有关。没有测量和操纵特定神经调节剂的释放的能力，我们对神经元电路功能的理解将是根本上不完整的。然而，我们监测脑电活动的能力与我们通过相应的空间分辨率监测化学活动的能力之间仍然存在差距。具体而言，没有方法可以在单个突触下可视化大脑中特定的NP的释放。为了填补这一空白，我们建议开发一种在体内在神经末端级别释放特定NP的方法。长期目标是开发新方法，以在体内可视化，检测和抑制NP释放，并应用这些方法来了解特定，行为相关的神经电路的神经调节的动力学。该提案的总体目标是开发新颖的方法，用于从神经终端释放NP的时间分辨成像。该提案的核心目的是标记具有pH敏感荧光蛋白的大型核心蔬菜（LDCV）和特定的NP的标记成分，并确定这些记者是否可以使用这些记者来对神经分泌颗粒的释放进行成像。果蝇Melanogaster为该技术提供了有用的测试床，因为其基因操纵和复杂的成像方法。为了实现我们的目标，在AIM 1中，我们将融合不同的pH敏感荧光记者与几种LDCV特异性蛋白质和NP的蛋白质编码序列，并产生转基因蝇。在AIM 2中，我们将使用在体内包含这些记者的特定神经肽神经元的光遗传激活，以确定是否可以检测到荧光的激活依赖性增加，并与突触场地（SV）释放区分开。贡献将是确定所提出的方法的可行性，并实现该方法的原则应用。这项贡献很重要，因为它有可能通过广泛的一般应用创建一种变革性的新技术。该贡献具有创新性，因为它结合了细胞生物学，分子遗传学和神经元电路分析的专业知识来开发一种新颖的方法。因此，本应用程序中提出的工作将使整个神经科学领域受益，还可以研究神经元化学和电路的研究，其功能障碍可能是精神疾病的基础。