Roppongi Reiko T, Champagne-Jorgensen Kevin P, Siddiqui Tabrez J
Department of Physiology and Pathophysiology, University of Manitoba; Kleysen Institute for Advanced Medicine, Health Sciences Centre.
Department of Physiology and Pathophysiology, University of Manitoba; Kleysen Institute for Advanced Medicine, Health Sciences Centre;
J Vis Exp. 2017 Apr 18(122):55000. doi: 10.3791/55000.
The ability to probe the structure and physiology of individual nerve cells in culture is crucial to the study of neurobiology, and allows for flexibility in genetic and chemical manipulation of individual cells or defined networks. Such ease of manipulation is simpler in the reduced culture system when compared to the intact brain tissue. While many methods for the isolation and growth of these primary neurons exist, each has its own limitations. This protocol describes a method for culturing low-density and high-purity rodent embryonic hippocampal neurons on glass coverslips, which are then suspended over a monolayer of glial cells. This 'sandwich culture' allows for exclusive long-term growth of a population of neurons while allowing for trophic support from the underlying glial monolayer. When neurons are of sufficient age or maturity level, the neuron coverslips can be flipped-out of the glial dish and used in imaging or functional assays. Neurons grown by this method typically survive for several weeks and develop extensive arbors, synaptic connections, and network properties.
在培养环境中探究单个神经细胞的结构和生理学特性的能力对于神经生物学研究至关重要，并且能够灵活地对单个细胞或特定网络进行基因和化学操作。与完整的脑组织相比，在简化的培养系统中进行这种操作更为简便。虽然存在许多分离和培养这些原代神经元的方法，但每种方法都有其自身的局限性。本方案描述了一种在玻璃盖玻片上培养低密度、高纯度啮齿动物胚胎海马神经元的方法，然后将其悬浮在单层胶质细胞上。这种“三明治培养法”能够使一群神经元实现独特的长期生长，同时获得来自下层胶质细胞单层的营养支持。当神经元达到足够的年龄或成熟水平时，含有神经元的盖玻片可以从胶质细胞培养皿中取出，用于成像或功能测定。通过这种方法培养的神经元通常能存活数周，并形成广泛的树突、突触连接和网络特性。
