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.

该子项目是利用资源的众多研究子项目之一 由 NIH/NCRR 资助的中心拨款提供。子项目的主要支持 并且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。 子项目可能列出的总成本 代表子项目使用的中心基础设施的估计数量， NCRR 赠款不直接向子项目或子项目工作人员提供资金。 我们寻找一种体外灵长类动物模型来研究血清素神经元。使用恒河猴胚胎干细胞 (ESC) 系 366.4，这是一种已开发的方案，通过色氨酸羟化酶和血清素再摄取转运蛋白的免疫细胞化学测定，可产生高比例的血清素神经元。此外，ESC衍生的神经元以与猕猴大脑中的血清素神经元类似的方式表达雌激素受体β（ERβ）和孕激素受体（PR）。我们进一步优化了产量并获得了衍生的血清素神经元的功能特征。在扩增阶段用成纤维细胞生长因子 4 和音刺猬连续处理 ESC 366.4 显着增加了血清素神经元的产量。这些血清素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 衍生的血清素神经元表现出功能性高亲和力转运蛋白位点，以及高亲和力血清素 (5HT1A) 结合位点，这些是常见精神活性药物的重要靶点。最后，为了测试该方法的通用性，我们利用了另一种恒河猴 ESC 系 ORMES-22，它能有效分化为血清素神经元。这些发现共同证明了我们的方案将不同灵长类神经元 ESC 系引导至具有生理特征的血清素神经元的可行性，这使它们成为有用的体外模型系统。