A genetic approach to elucidate the role of NMDA in the development and function of horizontal connections in ferret visual cortex

项目摘要 这项探索性提议的目标是在雪貂模型系统中尽早递送和表达遗传物质 参与发展.我们的策略是基于现有的方法在小鼠中开发的快速和空间 在有丝分裂后的细胞中定位表达DNA质粒，这是一种称为“离子穿孔”的技术。我们的目标是 将CRISPR/Cas9表达到雪貂初级视觉皮层（V1）中，以操纵特定基因的表达 对视觉电路的发展和功能至关重要。雪貂是一个理想的模式系统，因为它们是天生的 相对不成熟，视觉电路的发展是很好的特点。在本申请中，我们建议 优化并验证雪貂中的离子穿孔以敲减N-甲基-D-天冬氨酸的表达 受体（NMDAR），以阐明其在V1电路的形成和功能中的功能作用。我们将 在体外和体内验证该技术，并开始获得合作R 01的初步数据， NMDAR在局部和远程（列间）水平方向的发展和功能中的作用 连接在雪貂V1的第2-3层。这种方法的成功开发将打开遗传学的大门。 在具有类似于灵长类动物的新皮层的皮层柱状组织的高等哺乳动物中的操纵， 人类它还将允许开发诸如精神分裂症等大脑病理学的遗传模型。

Project Summary The goal of this exploratory proposal is to deliver and express genetic material in the ferret model system early in development. Our strategy is based on an existing method developed in mouse for the rapid and spatially localized expression of DNA plasmids in postmitotic cells, a technique termed ‘iontoporation’. We aim to express CRISPR/Cas9 into ferret primary visual cortex (V1) to manipulate the expression of specific genes critical for the development and function of visual circuitry. Ferret is an ideal model system, as they are born relatively immature and visual circuit development is well-characterized. In this application, we propose to optimize and validate iontoporation in ferret to knockdown the expression of N-Methyl-D-aspartic acid receptors (NMDARs) to elucidate their functional role in the formation and function of V1 circuitry. We will validate the technique in vitro and in vivo, and start obtaining preliminary data for a collaborative R01, on the role of NMDARs in the development and function of local and long-range (between columns) horizontal connections in layers 2-3 of ferret V1. Successful development of this approach will open the door to genetic manipulation in a higher mammal with cortical columnar organization of the neocortex similar to primates and humans. It will also allow the development of genetic models of brain pathologies such as Schizophrenia.