Characterization of neuronal population of the raphe nucleus, and establishing their role in autism

中缝核神经元群的表征及其在自闭症中的作用

• 批准号: 9360829
• 负责人: Abhignya Subedi
• 金额: --
• 依托单位: U.S. NATIONAL INST/CHILD HLTH/HUMAN DEV
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-10-01 至 2020-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9360829
• 关键词: Address; Affect; Aggressive behavior; Amino Acids; Animals; Antibodies; Anxiety; Area; Arousal; Autistic Disorder; Behavior; Behavior Disorders; Behavioral; Biological Models; Brain; Brain Stem; Butyric Acids; CRISPR/Cas technology; Calcium; Candidate Disease Gene; Cell Nucleus; Cells; Clutch Size; Collection; Data; Defect; Development; Disease; Dorsal; Enhancers; Family; Fright; GABA transporter; GTP-Binding Protein alpha Subunits, Gs; Genes; Genetic; Glutamates; Human; Image; In Situ Hybridization; Individual; Knowledge; Label; Laboratories; Larva; Link; Mammals; Maps; Mental Depression; Mental disorders; Methods; Midbrain structure; Modification; Molecular; Molecular Profiling; Monitor; Mus; Mutate; Mutation; Neurons; Neuropeptides; Neurotransmitters; Obsessive-Compulsive Disorder; Outcomes Research; Pain; Partner in relationship; Pathologic; Pattern; Population; Predisposition; Prosencephalon; RNA; Research; Research Design; Rewards; Role; Schizophrenia; Sensory; Serotonin; Siblings; Sleep; Source; Spinal Cord; Staining method; Stains; Stimulus; Structure; Symptoms; System; Testing; Therapeutic Intervention; Transgenes; Transgenic Organisms; Variant; Vertebrates; Zebrafish; calcium indicator; experimental study; gamma-Aminobutyric Acid; interest; intersectionality; member; mutant; nervous system development; nervous system disorder; neurotransmission; paralogous gene; population based; raphe nuclei; screening; sensory stimulus; serotonergic regulation; solute; transcriptome sequencing; water flow

Project Summary/Abstract The mid-brain raphe nucleus is a highly conserved structure present throughout vertebrates and holds a widespread interest because it is the source of the serotonergic network of the entire brain. Although variation exists among serotonergic neuronal population, still little is known about their differences. Even less is known about the non-serotonergic neuronal population. The precise projection of each population of neurons is also not identified. Studies in various animals have implicated raphe nucleus in functions including; sleep, arousal, fear, reward, aggression, and pain. Also, serotonin imbalance has been found to increase susceptibility to neurological disorders such as autism. However, which population of neurons is responsible for modulating any of these behaviors, and what are their roles is yet to identify. Therefore in this application, I will address some of these issues using six day old larval zebrafish. A much simpler but conserved brain structure to mammals, transparency in the earlier stages, huge clutch size and amenability to genetic modification make them an ideal system for the proposed research. Using the Burgess laboratory collection of transgenic and enhancer trap lines, I will identify various serotonergic and non-serotonergic population of neurons in the raphe nucleus in aim 1 by imaging. I will further characterize them with RNA-seq experiments followed by the in situ hybridization and generate their molecular profile. My preliminary data already suggest that there is multiple sub-populations of non- serotonergic neurons exist. In aim 2, I will generate the projectome profile by stochastically labeling individual neurons in each of these sub populations using Gal4/Cre intersectional approach. To identify functional subregions, genetically encoded calcium indicator (GCaMP) will be expressed in the lines used in aim 1 and will be imaged while subjecting the larvae to flow stimulus. Neurons in the same functional subregions will show similar Ca++ activity. I will test the hypothesis that mutation in an autism candidate gene, solute carrier family 6, member 4 (slc6a1), a γ-amino acid butyric acid (GABA) transporter, causes persistent GABA neurotransmission resulting in serotonin imbalance and manifests autism like behavior in aim 3. First, I will generate mutants in the lines characterized in aim 1 and 2. I will check developmental defects by imaging population of the raphe neurons in mutants and comparing them to their wild type siblings. The functional defects will be tested similar to aim 2 because heightened sensory responsiveness is a common autism symptom. By the end of this study, I aim to generate clear functional subdivision of raphe neurons and identify their role in the autism that may be applicable for the therapeutic intervention.

项目概要/摘要 中脑中缝核是整个脊椎动物中高度保守的结构，具有 广泛的兴趣，因为它是整个大脑血清素网络的来源。虽然 血清素能神经元群之间存在差异，但人们对它们的差异仍知之甚少。甚至 关于非血清素能神经元群体知之甚少。每个人口的精确预测 神经元的数量也没有被识别。对各种动物的研究表明中缝核的功能 包括;睡眠、觉醒、恐惧、奖赏、攻击性和疼痛。此外，还发现血清素失衡 增加对自闭症等神经系统疾病的易感性。然而，哪些神经元群体 负责调节任何这些行为，但它们的作用尚未确定。所以 在此应用程序中，我将使用六天大的斑马鱼幼虫来解决其中一些问题。一个更简单的 但哺乳动物的大脑结构是保守的，早期阶段是透明的，窝的大小和 基因改造的适应性使它们成为拟议研究的理想系统。使用 伯吉斯实验室收集转基因和增强子陷阱线，我将识别各种血清素能 通过成像观察目标 1 中中缝核中的非血清素神经元群。我会进一步 通过 RNA-seq 实验表征它们，然后进行原位杂交并生成它们 分子概况。我的初步数据已经表明存在多个非非人群亚群 存在血清素能神经元。在目标 2 中，我将通过随机标记生成项目组概况 使用 Gal4/Cre 交叉方法对每个亚群中的单个神经元进行分析。识别 功能分区，基因编码钙指示剂（GCaMP）将在细胞系中表达 用于目标 1，并将在幼虫受到流动刺激时成像。神经元在同一 功能分区将显示出类似的 Ca++ 活性。我将检验自闭症突变的假设 候选基因，溶质载体家族 6，成员 4 (slc6a1)，γ-氨基酸丁酸 (GABA) 转运蛋白，引起持续的 GABA 神经传递，导致血清素失衡并表现出来 目标 3 中的类似自闭症的行为。首先，我将生成目标 1 和 2 中所表征的品系中的突变体。我将 通过对突变体中缝神经元进行成像并比较来检查发育缺陷 他们与他们的野生型兄弟姐妹。功能缺陷的测试将与目标 2 类似，因为提高了 感觉反应是一种常见的自闭症症状。在本研究结束时，我的目标是产生清晰的 中缝神经元的功能细分并确定它们在自闭症中的作用，这可能适用于 治疗干预。