Analysis of the signals modulated by serotonin in a C. elegans neural circuit

线虫神经回路中血清素调节的信号分析

基本信息

批准号：
10602839
负责人：
Shavanie Prashad
金额：
$ 3.26万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-05-01 至 2024-10-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10602839
关键词：
3-Dimensional Acetylcholine Animals Behavior Behavioral Biological Models Brain Brain Diseases Caenorhabditis elegans Calcium Cells Chemical Synapse Connexins Data Defect Dense Core Vesicle Electrical Synapse Electrons Event Fluorescence Microscopy Functional disorder Gap Junctions Genes Genetic Goals Green Fluorescent Proteins Human Image Knowledge Label Light Link Measures Mediating Mental Depression Mental disorders Methods Muscle Muscle Cells Muscle Contraction Nematoda Neurons Neuropeptides Neurotransmitters Organism Play Proteins RNA Interference Reporting Research Personnel Role Schizophrenia Series Serotonin Signal Transduction Source Structure Study models Synapses Synaptic Vesicles System Testing Varicosity Vesicle Work calcium indicator effective therapy egg experimental study gene function knock-down neural neural circuit neural model neuropsychiatric disorder neurotransmission optogenetics postsynaptic presynaptic prevent protein biomarkers ratiometric red fluorescent protein response sensor tool vesicle-associated membrane protein

项目摘要

Project Summary Altered serotonin signaling is linked to brain disorders like depression and schizophrenia. A current hypothesis is that serotonin 'modulates' the action of other neural signals with critical roles in depression. However, the vast complexity of the human brain makes it challenging to test this idea, and there is no neural circuit in any organism in which we understand in mechanistic detail how serotonin modulates other signals. This project seeks to fill this gap in knowledge by determining how serotonin modulates other signals in a powerful model system: the well-studied neural circuit that controls egg-laying in the C. elegans nematode. A pair of HSN neurons release serotonin to make the vulval type-1 and type-2 muscle cells (vm1s and vm2s) and the ventral cord type C (VCs) neurons more excitable. Simultaneous contraction of the vm1s and vm2s is required for egg laying. The VCs release acetylcholine to depolarize the vm2s; however, so far, no signal that could depolarize the vm1s and thus be potentiated by serotonin has been identified. I discovered that a pair of poorly-studied neurons called PVW produce branches that end in presynaptic termini over the vm1s. My preliminary data also shows that the PVW branches have strong calcium transients during egg laying, and that silencing PVW's activity mildly inhibits egg laying. Furthermore, weaker PVW calcium transients are observed when the vm1s produce small twitches that do not result in egg laying. My central hypothesis is that the PVW branches provide the unknown excitatory signal that triggers vm1 contractions and that serotonin potentiates this effect to result in egg laying. My first aim is to determine if the PVW branches form chemical and/or electrical synapses onto the vm1s. I will use fluorescent presynaptic protein markers to examine if the PVW varicosities contain small-clear vesicles, dense- core vesicles, and/or gap junctions. I will also analyze existing electron micrographs to reconstruct the ultrastructure and synaptic content of the PVW branch onto the vm1s. Next, I will use trans-synaptic labeling methods to determine if the PVW varicosities contact the vm1s. My second aim is to use a genetically-encoded fluorescent calcium indicator to record PVW neural activity within freely-behaving animals to determine if PVW is active when the vm1s are activated, and to study if the HSN or VC neurons modulate PVW's activity during egg laying. My third aim is to manipulate PVW activity with optogenetic and chemogenetic methods to test if the PVW neurons provide a serotonin-potentiated signal that excites the vm1s to induce egg laying. I will silence or induce PVW and VC activities to determine if both neurons are necessary and/or sufficient to induce egg laying in the presence and absence of serotonin. Lastly, I will use RNAi to disrupt presynaptic gene function in PVW to investigate if PVW uses neurotransmitter, neuropeptide, or gap junction signaling to induce egg laying and excite the vm1s, using ratiometric calcium imaging to read out vm1 activity. This study will provide the most detailed analysis to date of how serotonin modulates the response of a cell to an excitatory signal within a neural circuit, advancing our understanding of how serotonin dysfunction in brain circuits might contribute to mental disorders.

项目概要血清素信号改变与抑郁症和精神分裂症等大脑疾病有关。目前的假设血清素“调节”其他神经信号的作用，在抑郁症中发挥关键作用。然而，广阔的人脑的复杂性使得测试这个想法具有挑战性，并且任何生物体中都不存在神经回路其中我们详细了解了血清素如何调节其他信号的机制。该项目旨在填补通过确定血清素如何在强大的模型系统中调节其他信号来弥补这一知识差距：经过充分研究的控制线虫产卵的神经回路。一对 HSN 神经元释放血清素生成外阴 1 型和 2 型肌细胞（vm1s 和 vm2s）以及 C 型腹索（VC）神经元更兴奋。产蛋需要 vm1 和 vm2 同时收缩。风险投资公司释放乙酰胆碱使 vm2 去极化；然而，到目前为止，没有信号可以使 vm1 去极化，因此已被证实可被血清素增强。我发现了一对未被充分研究的神经元，称为 PVW 产生以 vm1 上的突触前末端结束的分支。我的初步数据还表明，PVW 枝条在产卵期间有强烈的钙瞬变，沉默 PVW 的活性会轻微抑制卵铺设。此外，当 vm1 产生小抽搐时，会观察到较弱的 PVW 钙瞬变不会导致产蛋。我的中心假设是 PVW 分支提供未知的兴奋性触发 vm1 收缩的信号，而血清素会增强这种效应，从而导致产蛋。我的第一个目标目的是确定 PVW 分支是否在 vm1 上形成化学和/或电突触。我会用荧光突触前蛋白标记物检查 PVW 静脉曲张是否含有小透明囊泡、致密囊泡核心囊泡和/或间隙连接。我还将分析现有的电子显微照片以重建 PVW 分支到 vm1 的超微结构和突触内容。接下来，我将使用跨突触标记确定 PVW 静脉曲张是否接触 vm1 的方法。我的第二个目标是使用基因编码的荧光钙指示剂记录自由行为动物内的 PVW 神经活动，以确定 PVW 是否当 vm1 被激活时处于活动状态，并研究 HSN 或 VC 神经元是否在 vm1 激活期间调节 PVW 的活动产蛋。我的第三个目标是通过光遗传学和化学遗传学方法操纵 PVW 活性，以测试是否 PVW 神经元提供血清素增强信号，激发 vm1 诱导产卵。我会沉默或者诱导 PVW 和 VC 活动以确定这两个神经元是否对于诱导产卵是必要和/或充分的在存在和不存在血清素的情况下。最后，我将使用 RNAi 来破坏 PVW 中的突触前基因功能，以研究 PVW 是否使用神经递质、神经肽或间隙连接信号传导来诱导产卵和兴奋 vm1s，使用比率钙成像来读出 vm1 活性。这项研究将提供最详细的迄今为止对血清素如何调节细胞对神经回路内兴奋信号的反应的分析，增进我们对大脑回路中血清素功能障碍如何导致精神障碍的理解。