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.

神经肽调节突触和回路以控制情绪和各种行为 包括食欲、痛觉和昼夜节律。尽管神经肽的重要性， 用目前的方法不可能在完整的活脑中检测突触神经肽的释放。 然而，我们最近开发了一种新的光学方法，成像胞吐的神经肽含有致密核心囊泡（DCV）在完整的生活果蝇突触。这种办法基于 在插入荧光团激活蛋白（FAP），它赋予荧光的正常 非荧光染料孔雀石绿色（MG）转化为前神经肽，从而将FAP靶向DCV 流明在细胞外应用小分子膜不渗透性MG衍生物后， 足以通过融合孔，单个DCV的活性诱发融合可以容易地被 在果蝇的神经肌肉接头处得到了解决。此外，我们检测到一种新的DCV模式， 一种自发的胞吐作用，其特征是对分泌细胞的扰动敏感 设备（例如，破伤风毒素抗性）。最后，初步研究表明，神经肽-FAP方法适用于研究完整成年果蝇的昼夜节律肽能神经元 个脑袋因此，为了证明FAP成像的实用性，该方法将用于回答 关于果蝇昼夜节律回路功能的基本问题。比如说， 我们将确定多个神经元释放神经肽的时间，并解决是否存在一个神经肽的问题。 一种新发现的自发释放模式解释了破伤风毒素抵抗行为效应 一种果蝇神经肽此外，我们将使用新的染料，第二种光谱不同的FAP变体， 基因组工程使两种神经肽的突触释放的同时成像 天然转录控制。除了测试关于肽能传递的特定假设外， 在成年果蝇大脑的昼夜节律回路中，这些研究将作为原理的证明， 将真实的时间神经肽-FAP成像应用于其他系统，包括哺乳动物中枢神经系统， 神经系统