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

项目摘要 神经系统疾病，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-作为单元运行以产生单个输出的神经元组。通常， 尽管血清素信号没有受到干扰， 被明确识别。有趣的是，产生降钙素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，C. elegans有一个 广泛研究的产卵电路，具有产卵的定量行为输出，使其成为理想的 模型电路进行研究。这个回路被血清素和神经肽NLP-3激活， 雌雄同体特异性神经元（HSN）。以前的工作表明，这两种信号都需要 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 特征在于C.神经肽NLP-3最近才被发现参与了线虫的产卵回路， 下蛋在我的初步工作中，我已经确定了一个假定的NLP-3 G蛋白偶联受体（GPCR），F10D7.1。 在这个建议中，我将测试HSN向产卵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，以确保电路的鲁棒激活。 我的第一个目标是验证F10D7.1作为C中NLP-3激活的G蛋白偶联受体。线虫卵， 铺设电路我将从基因上验证F10D7.1与NLP-3活性相关。我将分析两个功能丧失 F10D7.1突变体与各种遗传背景杂交，用于产蛋缺陷。此外，我将表达 F10D7.1在异源细胞中的结合，以确定NLP-3肽的结合潜力及其活性水平。 我的第二个目标是识别C.表达F10D7.1的线虫产卵回路， 接收NLP-3信号以激活产卵电路。我将创建一个GFP结构， F10D7.1的启动子片段。将其注入C.我可以用它来识别那些表达 F10D7.1，假设由NLP-3激活。然后我可以选择性地敲除这些细胞中的F10D7.1， 并测定卵滞留。 我的第三个目标是确定NLP-3信号传导对产卵回路活动的影响， 动物钙成像肝钙显像。elegans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。我将在细胞中表达钙指示剂， 表达在Aim 2a中发现的F10D7.1，并量化受体的丢失如何改变Aim 2a的活性模式。 F10D7.1与血清素的双重敲除提高了这一水平。 总的来说，这个项目将解决为什么血清素和神经肽被用于相同的问题。 电路.

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.