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.

项目总结/摘要 中脑中缝核是存在于脊椎动物中的高度保守的结构， 因为它是整个大脑神经元网络的来源。虽然 在多巴胺能神经元群体中存在差异，但对它们之间的差异知之甚少。甚至 对非多巴胺能神经元群体的了解较少。每个种群的精确预测 神经元也没有被识别出来。在各种动物中的研究表明中缝核在功能上 包括：睡眠、觉醒、恐惧、奖励、攻击和疼痛。此外，血清素失衡已被发现， 增加对神经系统疾病如自闭症的易感性。然而，哪一群神经元 负责调节任何这些行为，他们的角色是什么还有待确定。因此 在本申请中，我将使用六天大斑马鱼幼体来解决这些问题中的一些。简单得多的 但保留了哺乳动物的大脑结构，早期阶段的透明度，巨大的窝卵数， 对遗传修饰的适应性使它们成为所提出的研究的理想系统。使用 伯吉斯实验室收集的转基因和增强子的陷阱线，我将确定各种不同的转基因 在AIM 1中缝核中，通过成像观察到非多巴胺能神经元群体。我将进一步 用RNA-seq实验进行表征，然后进行原位杂交， 分子图谱我的初步数据已经表明，有多个非- 神经元存在。在aim 2中，我将通过随机标记生成projectome配置文件 使用Gal 4/Cre交叉方法在这些亚群中的每一个中的单个神经元。以识别 功能亚区，遗传编码的钙指示剂（GCaMP）将在品系中表达 在AIM 1中使用，并且将在使幼虫经受流动刺激的同时成像。同样的神经元 功能亚区将显示类似的Ca++活性。我将检验自闭症患者的基因突变 候选基因，溶质载体家族6，成员4（slc 6a 1），γ-氨基酸丁酸（GABA） 转运蛋白，导致持续的GABA神经传递，导致血清素失衡，并表现为 目标3中的自闭症样行为。首先，我将在目标1和2中描述的品系中产生突变体。我会 通过对突变体中缝核神经元群体进行成像并比较 它们的野生同胞。功能缺陷的测试与目标2相似，因为 感觉反应是一种常见的自闭症症状。到本研究结束时，我的目标是产生明确的 中缝神经元的功能细分，并确定其在自闭症中的作用，可能适用于 治疗干预