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.

项目摘要/摘要 中脑中缝核是一种高度保守的结构，存在于脊椎动物中，具有 广泛的兴趣，因为它是整个大脑5-羟色胺能网络的来源。虽然 5-羟色胺能神经元种群之间存在差异，但对它们之间的差异知之甚少。连 对非5-羟色胺能神经元群体知之甚少。对每个人口的准确预测 神经元的数量也没有确定。对各种动物的研究表明，中缝核团具有一定的功能。 包括睡眠、唤醒、恐惧、奖励、攻击性和痛苦。此外，还发现5-羟色胺失衡 以增加对自闭症等神经疾病的易感性。然而，哪一组神经元 负责调节这些行为中的任何一种，他们的角色还没有确定。因此 在这个应用程序中，我将使用六天大的斑马鱼幼体来解决其中的一些问题。一个简单得多的 但哺乳动物保守的大脑结构，早期阶段的透明度，巨大的卵子大小和 对基因修改的适应性使它们成为拟议研究的理想系统。使用 伯吉斯实验室收集的转基因和增强子诱捕剂品系，我将鉴定各种5-羟色胺 目的1的中缝核内非5-羟色胺能神经元的数量。我会进一步 用rna-seq实验和原位杂交对它们进行表征，并产生它们的 分子图谱。我的初步数据已经表明，有多个亚种群的非 5-羟色胺能神经元存在。在目标2中，我将通过随机标记生成项目组配置文件 使用Gal4/Cre交叉切片法对每个亚群中的单个神经元进行检测。要确定 功能亚区，基因编码的钙指示剂(GCaMP)将在这些品系中表达 在目标1中使用，并将在对幼虫进行流动刺激时进行成像。神经元在相同的 功能亚区将表现出相似的钙离子活性。我要测试一下自闭症患者突变的假设 候选基因，溶质载体家族6，成员4(Slc6a1)，γ-氨基酸丁酸(Gba) 转运蛋白，导致持续性的GABA神经传递，导致5-羟色胺失衡和表现 目标3中类似自闭症的行为。首先，我将在目标1和2中描述的线路中产生突变。我将 通过对突变型中缝神经元群进行成像和比较来检查发育缺陷 给他们的野生型兄弟姐妹。功能缺陷将进行类似于目标2的测试，因为 感觉反应是自闭症的一种常见症状。在这项研究结束时，我的目标是产生明确的 对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 治疗性干预。