IN VITRO MODEL DEVELOPMENT OF SEROTONIN NEURONS

血清素神经元的体外模型开发

• 批准号: 8357741
• 负责人: CYNTHIA Louise BETHEA
• 金额: $ 3.63万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-05-01 至 2012-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8357741
• 关键词: Action Potentials; Affinity; Binding Sites; Biological Models; Brain; Characteristics; ES Cell Line; Erinaceidae; Estrogen Receptor beta; Exhibits; Funding; GABA Receptor; Grant; In Vitro; Macaca; Macaca mulatta; Methods; National Center for Research Resources; Neurons; Physiological; Primates; Principal Investigator; Progesterone Receptors; Protocols documentation; Psychotropic Drugs; Research; Research Infrastructure; Resources; Sequential Treatment; Serotonin; Site; Source; Staging; Study models; Testing; Time; Tryptophan 5-monooxygenase; United States National Institutes of Health; cost; embryonic stem cell; immunocytochemistry; in vitro Model; model development; proto-oncogene protein kfgf; reuptake

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. We sought an in vitro primate model for studying serotonin neurons. Using the rhesus embryonic stem cell (ESC) line 366.4, a protocol as developed which generates a high percentage of serotonin neurons as determined by immunocytochemistry for tryptophan hydroxylase and the serotonin reuptake transporter. In addition, the ESC-derived neurons express estrogen receptor beta (ERbeta) and progesterone receptors (PR) in a manner similar to serotonin neurons in the macaque brain. We further optimized yield and obtained functional characteristics of the derived serotonin neurons. Sequential treatments of ESC 366.4 during expansion stage with fibroblast growth factor 4 and sonic hedgehog markedly increased the yield of serotonin neurons. These serotonin neurons propagated action potentials and expressed GABA receptors. Also for the first time we demonstrate that these ESC-derived serotonin neurons exhibit functional high affinity transporter sites, as well as high-affinity serotonin (5HT1A) binding sites, which are essential targets of common psychoactive drugs. Finally, to test the generality of this method, we utilized another rhesus ESC line, ORMES-22, which efficiently differentiated into serotonin neurons. Together these findings demonstrate the feasibility of our protocol to direct different primate neuronal ESC lines to serotonin neurons with physiological characteristics, which makes them a useful in vitro model system.

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 子项目的主要研究者可能是由其他来源提供的， 包括其他NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 我们寻求一种体外灵长类动物模型来研究5-羟色胺神经元。使用恒河猴胚胎干细胞（ESC）系366.4，开发了一种产生高百分比5-羟色胺神经元的方案，如通过色氨酸羟化酶和5-羟色胺再摄取转运蛋白的免疫细胞化学所确定的。此外，ESC衍生的神经元以与猕猴脑中的5-羟色胺神经元类似的方式表达雌激素受体β（ER β）和孕酮受体（PR）。我们进一步 优化产量并获得衍生的5-羟色胺神经元的功能特性。在扩增阶段用成纤维细胞生长因子4和音刺猬蛋白顺序处理ESC 366.4显著增加了5-羟色胺神经元的产量。这些5-羟色胺神经元传播动作电位并表达GABA受体。我们还首次证明，这些ESC衍生的5-羟色胺神经元表现出功能性高亲和力转运位点，以及高亲和力5-羟色胺（5-HT 1A）结合位点，这是常见的精神活性药物的基本目标。最后，为了测试这种方法的通用性，我们利用另一种恒河猴ESC系ORMES-22，其有效地分化为5-羟色胺神经元。总之，这些研究结果表明，我们的方案的可行性，指导不同的灵长类神经元ESC线的5-羟色胺神经元的生理特性，这使得他们在体外模型系统是一个有用的。