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: 10066000
• 负责人: Allison Marie Butt
• 金额: $ 4.55万
• 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/10066000
• 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; Hyperactive behavior; 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.

项目摘要 神经性疾病，如抑郁症和双相情感障碍，被认为是由妥协引起的 神经回路--作为单位运作以产生单一输出的一组神经元。通常情况下，那些 5-羟色胺信号似乎对情绪障碍有积极影响，尽管5-羟色胺没有受到干扰。 已经被明确确认了。有趣的是，产生5-羟色胺的神经元也经常产生一种神经肽。 被称为P物质，但为什么会发生这种情况尚不清楚。线虫秀丽线虫是理想的 用其简单的神经系统和众所周知的解剖学研究电路；此外，线虫有一个 广泛研究了具有产蛋量化行为输出的产蛋回路，使其成为理想的产蛋回路 要研究的模型电路。这个回路是由5-羟色胺和神经肽NLP-3激活的。 两性特异性神经元(HSN)。先前的研究表明，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5-羟色胺一直很好 神经肽NLP-3是线虫产卵环路的特征，直到最近才被发现与卵子有关 躺着。在我的初步工作中，我已经确定了一个可能的NLP-3G蛋白偶联受体(GPCR)，F10D7.1。 在这个提案中，我将测试HSN向产卵回路的肌肉发出信号的假设 NLP-3和5-羟色胺作为部分冗余信号，确保电路的强劲激活。 我的第一个目标是研究F10D7.1作为线虫卵中NLP-3激活的G蛋白偶联受体- 铺设线路。我将从基因上验证F10D7.1与NLP-3活性的偶联。我要化验两个功能丧失 F10D7.1突变体与不同的产蛋缺陷遗传背景杂交。另外，我会表示 F10D7.1在异种细胞中测定NLP-3多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F10D7.1的细胞 接收NLP-3信号以激活产卵电路。我将创建一个GFP构造，由 F10D7.1启动子片段。在将其注射到线虫体内后，我可以用它来识别表达 F10D7.1和假设是被NLP-3激活的。然后我可以有选择地击倒这些细胞中的F10D7.1 以及卵子滞留的化验。 我的第三个目标是确定NLP-3信号对产卵回路活动的影响。 动物的钙质想象。活体线虫的钙成像是我的实验室开发的一个很好的工具 直接观察信号如何影响电路的激活。我将在细胞中表达一种钙指示剂 表达在Aim 2a中发现的F10D7.1，并量化该受体的丢失如何改变 回路，通过5-羟色胺双重敲除F10D7.1而增强。 总体而言，这个项目将解决为什么5-羟色胺和神经肽在相同的情况下被利用的问题 巡回赛。