Function of the neuropeptide NLP-3 in relationship to serotonin signaling within C. elegans egg-laying circuit

神经肽 NLP-3 与线虫产卵回路中血清素信号传导的关系

• 批准号: 10460939
• 负责人: Allison Marie Butt
• 金额: $ 3.16万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-01 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10460939
• 关键词: Address; Affect; Anabolism; Anatomy; Animal Model; Animals; Behavioral; Binding; Biochemical; Biological Assay; Bipolar Disorder; Caenorhabditis elegans; Calcium; Cells; Cellular Assay; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Control Animal; Coupled; Defect; Double-Stranded RNA; Ensure; G-Protein-Coupled Receptors; GPR3 gene; GTP-Binding Proteins; Genes; Genetic; Hermaphroditism; Hyperactivity; Image; Imaging Techniques; Individual; Knock-out; Knowledge; Label; Libraries; Link; Mammals; Measures; Mental Depression; Modeling; Mood Disorders; Muscle; Nematoda; Nervous system structure; Neurons; Neuropeptide Receptor; Neuropeptides; Neurotransmitters; Output; Pattern; Peptides; Pharmaceutical Preparations; Phenocopy; Phenotype; Plasmids; Positioning Attribute; RNA Interference; Reporter; Reporting; Serotonin; Signal Transduction; Signaling Molecule; Substance P; System; Testing; Transgenes; Work; calcium indicator; egg; experimental study; fluorescence imaging; improved; knock-down; loss of function; mutant; nervous system disorder; neural circuit; overexpression; promoter; receptor; tool

Project Summary Neurological disorders, such as depression and bipolar disorder, are thought to be caused by compromised neural circuits – groups of neurons that operate as units to generate a single output. Frequently, drugs that aIter serotonin signaling seem to positively impact mood disorders even though no disruption in serotonin has been clearly identified. Interestingly, serotonin-producing neurons frequently also produce a neuropeptide known as Substance P, but why this occurs is not known. The nematode Caenorhabditis elegans is ideal for studying circuitry with its simple nervous system and well-known anatomy; additionally, C. elegans has an extensively studied egg-laying circuit that has a quantitative behavioral output of egg laying making it an ideal model circuit to study. This circuit is activated by serotonin and a neuropeptide NLP-3 being released from the Hermaphrodite Specific Neurons (HSNs). Previous work has shown that both of these signals are required for proper activation levels; however, it is unclear why two signals are required. While serotonin has been well characterized in the C. elegans egg-laying circuit, the neuropeptide NLP-3 was only recently implicated in egg laying. In my preliminary work I have identified a putative NLP-3 G protein coupled receptor (GPCR), F10D7.1. In this proposal I will test the hypothesis that the HSN signals to the muscles of the egg-laying circuit using NLP-3 and serotonin as partially redundant signals to ensure robust activation of the circuit. My first aim is to vet F10D7.1 as an NLP-3-activated G protein coupled receptor in the C. elegans egg- laying circuit. I will genetically verify that F10D7.1 is coupled to NLP-3 activity. I will assay two loss-of-function F10D7.1 mutants crossed with various genetic backgrounds for egg laying defects. Additionally, I will express F10D7.1 in heterologous cells to determine the binding potential of NLP-3 peptides and their activity levels. My second aim is to identify the cells within the C. elegans egg-laying circuit that express F10D7.1 and receive an NLP-3 signal to activate the egg-laying circuit. I will create a GFP construct driven by a promoter fragment of F10D7.1. After injecting this into C. elegans, I can use this to identify cells that express F10D7.1 and hypothetically are activated by NLP-3. I can then selectively knock down F10D7.1 in these cells and assay for egg retention. My third aim is to determine the effects of NLP-3 signaling on the egg-laying circuit activity using live- animal calcium imagining. Calcium imaging in live C. elegans is an excellent tool developed by my lab to directly observe how a signal affects a circuit's activation. I will express a calcium indicator in the cells that express F10D7.1 found in Aim 2a and quantify how the loss of the receptor changes the activity pattern of the circuit, heightened by the dual knockout of F10D7.1 with serotonin. Overall, this project will address the question of why serotonin and neuropeptides are utilized in the same circuit.

项目概要 神经系统疾病，如抑郁症和双相情感障碍，被认为是由受损引起的 神经回路——作为单元运行以生成单个输出的神经元组。通常，药物 尽管血清素没有受到干扰，但改变血清素信号似乎会对情绪障碍产生积极影响。 被明确识别。有趣的是，产生血清素的神经元也经常产生神经肽 被称为物质 P，但发生这种情况的原因尚不清楚。线虫秀丽隐杆线虫是理想的 研究电路及其简单的神经系统和众所周知的解剖学；此外，线虫有一个 广泛研究的产蛋电路，具有定量的产蛋行为输出，使其成为理想的产蛋电路 模型电路进行研究。该回路由血清素和从神经元释放的神经肽 NLP-3 激活。 雌雄同体特定神经元 (HSN)。先前的工作表明，这两个信号都是 适当的激活水平；然而，尚不清楚为什么需要两个信号。虽然血清素一直很好 神经肽 NLP-3 是线虫产卵回路中的特征，最近才与卵有关 铺设。在我的初步工作中，我鉴定了一个假定的 NLP-3 G 蛋白偶联受体 (GPCR)，F10D7.1。 在这个提案中，我将测试 HSN 向产蛋回路的肌肉发出信号的假设： NLP-3 和血清素作为部分冗余信号，以确保电路的稳健激活。 我的第一个目标是验证 F10D7.1 作为线虫卵中 NLP-3 激活的 G 蛋白偶联受体 - 敷设电路。我将从基因角度验证 F10D7.1 与 NLP-3 活性偶联。我将检测两种功能丧失 F10D7.1 突变体与产蛋缺陷的各种遗传背景杂交。另外，我要表达 F10D7.1 在异源细胞中测定 NLP-3 肽的结合潜力及其活性水平。 我的第二个目标是鉴定线虫产卵回路中表达 F10D7.1 和 接收 NLP-3 信号以激活产卵电路。我将创建一个由 F10D7.1 的启动子片段。将其注射到秀丽隐杆线虫中后，我可以用它来识别表达 F10D7.1 和假设被 NLP-3 激活。然后我可以选择性地敲除这些细胞中的 F10D7.1 并测定卵子保留情况。 我的第三个目标是使用活体确定 NLP-3 信号传导对产卵回路活动的影响 动物钙想象。活体线虫中的钙成像是我的实验室开发的一个出色的工具 直接观察信号如何影响电路的激活。我将在细胞中表达钙指示剂 表达目标 2a 中发现的 F10D7.1 并量化受体的丢失如何改变 F10D7.1 的活动模式 电路，通过 F10D7.1 与血清素的双重敲除而增强。 总体而言，该项目将解决为什么血清素和神经肽在同一环境中使用的问题 电路。