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: 9146349
• 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 2018-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9146349
• 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; Health; 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; 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; connectome; expression vector; fly; imaging modality; improved; in vivo; innovation; instrumentation; millisecond; mind control; neural circuit; neuronal circuitry; neuroregulation; new technology; novel; novel strategies; novel therapeutics; optical imaging; optogenetics; 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（由申请人提供）：描述大脑的一个常见比喻是它就像一台超级计算机。因此，目前改进大规模脑功能记录技术的努力主要集中在测量其电活动。然而，与超级计算机不同的是，大脑是一台电化学机器。叠加在其突触连接网络上的是“化学连接体”，这是一种基本上不可见的神经调节剂网络，例如血清素和神经肽（NP），对大脑功能产生深远的影响。神经调节剂影响大脑状态，从而改变神经回路执行的计算，并且是情绪、心境和情感的核心。因此，了解神经调节影响与人类精神疾病相关。如果没有测量和操纵特定神经调节剂释放的能力，我们对神经元回路功能的理解从根本上来说是不完整的。然而，我们监测脑电活动的能力和我们以相应的时空分辨率监测化学活动的能力之间仍然存在差距。具体来说，没有方法可以可视化大脑中单个突触处特定纳米粒子的释放。为了填补这一空白，我们建议开发一种体内成像特定纳米粒子在神经末梢水平释放的方法。长期目标是开发用于可视化、检测和抑制体内 NP 释放的新方法，并应用这些方法来了解特定的、行为相关的神经回路的神经调节动态。该提案的总体目标是开发一种对神经末梢释放的 NP 进行时间分辨成像的新方法。该提案的中心目标是用 pH 敏感荧光蛋白标记大型致密核心囊泡 (LDCV) 和特定 NP 的成分，并确定这些报告基因是否可用于对神经分泌颗粒释放进行成像。由于其遗传可操作性和复杂的成像方法，果蝇为这项技术提供了一个有用的测试平台。为了实现我们的目标，在目标 1 中，我们将把不同的 pH 敏感荧光报告基因融合到几种 LDCV 特异性蛋白和 NP 的蛋白编码序列中，并生成转基因果蝇。在目标 2 中，我们将利用光遗传学激活体内含有这些报告基因的特定神经肽能神经元，以确定是否可以检测到激活依赖性荧光增加，并将其与突触小泡 (SV) 释放区分开来。贡献将是确定所提出方法的可行性，并实现该方法的原理验证应用。这一贡献意义重大，因为它有潜力创造一种具有广泛通用应用的变革性新技术。这项贡献具有创新性，因为它结合了细胞生物学、分子遗传学和神经回路分析的专业知识，开发了一种新颖的方法。因此，本申请中提出的工作将有利于整个神经科学领域，并且还能够研究神经化学和电路，其功能障碍可能是精神疾病的基础。