A New Method for Imaging Neuropeptide Release in the Brain

大脑中神经肽释放成像的新方法

• 批准号: 10016856
• 负责人: EDWIN S LEVITAN
• 金额: $ 19.67万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-15 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10016856
• 关键词: Address; Adult; Anatomy; Behavior; Behavior Control; Behavioral; Binding; Blood Circulation; Brain; Bypass; Circadian Rhythms; Clustered Regularly Interspaced Short Palindromic Repeats; Dense Core Vesicle; Desire for food; Detection; Drosophila genus; Dyes; Engineering; Exocytosis; Fluorescence; Genes; Genetic; Genomics; Image; Individual; Knock-in; Label; Mammals; Measurement; Measures; Membrane; Methodology; Methods; Monitor; Moods; Morphology; Neuraxis; Neuromuscular Junction; Neurons; Neuropeptides; Noise; Optics; PHluorin; Pain; Physiological; Pigments; Proteins; Resistance; Running; Signal Transduction; Site; Sleep; Synapses; System; Testing; Tetanus Toxin; Tetrodotoxin; Time; Tissues; Transcriptional Regulation; Transgenic Organisms; Variant; Vesicle; aqueous; base; circadian; experimental study; extracellular; fly; imaging approach; imaging modality; imaging study; insight; mind control; neuronal cell body; neuropeptide F; novel; pain perception; protein complex; targeted treatment; thiazole orange; tool; trafficking; transmission process; vesicle transport; vesicular release

Neuropeptides modulate synapses and circuits to control mood and a wide variety of behaviors including appetite, pain perception and circadian rhythms. Despite the importance of neuropeptides, it is not possible to detect synaptic neuropeptide release in the intact living brain with current methods. However, we recently developed a new optical approach for imaging exocytosis of neuropeptide-containing dense-core vesicles (DCVs) at intact living Drosophila synapses. This approach is based on inserting a fluorogen activating protein (FAP), which confers fluorescence on the normally nonfluorescent dye malachite green (MG), into a proneuropeptide, thus targeting the FAP to the DCV lumen. Following extracellular application of membrane impermeant MG derivatives that are small enough to pass through fusion pores, activity-evoked fusions of individual DCVs can readily be resolved at the Drosophila neuromuscular junction. Furthermore, we detected a novel mode of DCV spontaneous exocytosis that is distinguished by its sensitivity to perturbations of the secretory apparatus (e.g. resistance to tetanus toxin). Finally, preliminary studies show that the neuropeptide-FAP approach is applicable to studying circadian peptidergic neurons in the intact adult Drosophila brain. Therefore, to demonstrate the utility of FAP imaging, this approach will be used to answer fundamental questions regarding the function of the circadian circuit in the Drosophila. For example, we will determine the timing of neuropeptide release by multiple neurons and address whether a newly discovered mode of spontaneous release accounts for tetanus toxin resistant behavioral effects of a fly neuropeptide. Furthermore, we will use new dyes, a second spectrally distinct FAP variant and genomic engineering to enable simultaneous imaging of synaptic release of two neuropeptides under native transcriptional control. In addition to testing specific hypotheses about peptidergic transmission in the circadian circuit of the adult fly brain, these studies will serve as proof of principle examples for applying real time neuropeptide-FAP imaging to other systems including the mammalian central nervous system.

神经肽调节突触和电路以控制情绪和多种行为 包括食欲，疼痛感和昼夜节律。尽管神经肽很重要，但 无法通过当前方法检测完整活大脑中的突触神经肽释放。 但是，我们最近开发了一种新的光学方法，用于在完整活果蝇突触中对含神经肽的致密囊泡（DCV）进行成像胞吐作用。这种方法是基于的 插入荧光激活蛋白（FAP）时，该蛋白质赋予了正常的荧光 非荧光染料孔雀石绿（Mg），进入促尿肽，从而将FAP靶向DCV 流明。在细胞外应用膜不足mg衍生物的应用之后 足以通过融合孔，单个DCV的活动诱发融合很容易成为 在果蝇神经肌肉连接处解决。此外，我们检测到了DCV的新型模式 自发性胞吐作用以其对分泌的扰动的敏感性而区别 设备（例如对破伤风毒素的抗性）。最后，初步研究表明，神经肽-FAP方法适用于在完整的成年果蝇中研究昼夜节律肽基神经元 脑。因此，为了证明FAP成像的实用性，该方法将用于回答 有关果蝇中昼夜节律的功能的基本问题。例如， 我们将通过多个神经元确定神经肽释放的时机 新发现的自发释放模式是破伤风毒素的抗性行为效果 蝇神经肽。此外，我们将使用新染料，第二个频谱不同的FAP变体和 基因组工程能够同时成像两种神经肽的突触释放 天然转录控制。除了测试有关肽能传播的特定假设 在成人蝇脑的昼夜节律中，这些研究将作为原则示例的证明 将实时神经肽-FAP成像应用于包括哺乳动物中央的其他系统 神经系统。