Pacary Emilie, Haas Matilda A, Wildner Hendrik, Azzarelli Roberta, Bell Donald M, Abrous Djoher Nora, Guillemot François
Division of Molecular Neurobiology, MRC National Institute for Medical Research.
J Vis Exp. 2012 Jul 26(65):4163. doi: 10.3791/4163.
In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system (1-5). To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex (6-8). Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions. Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method( 9). However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice (10-11) (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA (12). These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C). Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.
子宫内电穿孔（IUE）已成为研究胚胎神经系统不同区域发育的一种强大技术（1-5）。迄今为止，该工具已被广泛用于研究细胞增殖、分化和神经元迁移的调控，尤其是在发育中的大脑皮层（6-8）。在这里，我们详细介绍了子宫内电穿孔大脑皮层和海马体的方案，并提供证据表明这种方法可用于研究这两个脑区的树突和棘。原代培养神经元的可视化和操作有助于更好地理解树突、棘和突触发育所涉及的过程。然而，在体外生长的神经元并未暴露于正常发育过程中可能影响树突和/或棘形成与维持的所有生理信号。我们对野生型或突变型小鼠体内树突和棘结构的了解大多来自使用高尔基-考克斯方法的观察（9）。然而，高尔基染色被认为是不可预测的。确实，神经细胞群和纤维束是随机标记的，特定区域常常看起来完全被染色，而相邻区域却没有染色。最近的研究表明，荧光构建体的子宫内电穿孔是研究突变型/野生型小鼠树突、棘以及突触的一种有吸引力的替代方法（10-11）（图1A）。此外，与产生基因敲除小鼠相比，子宫内电穿孔是在特定时间窗口内对特定细胞群体进行功能获得和功能丧失研究的一种快速方法。另外，子宫内电穿孔已成功用于诱导基因表达或诱导RNA干扰方法，以优化对基因或短发夹RNA表达的时间控制（12）。子宫内电穿孔的这些优势因此为研究基因表达/抑制对树突和棘的影响开辟了新的维度——不仅在特定脑结构中（图B），而且在发育的特定时间点（图1C）。最后，子宫内电穿孔提供了一个有用的工具，用于识别参与树突、棘和/或突触发育的基因之间的功能相互作用。确实，与其他基因转移方法（如病毒）相比，在同一细胞群体中组合多个RNA干扰或转基因很简单。总之，子宫内电穿孔是一种强大的方法，已经有助于表征脑功能和疾病背后的分子机制，它在树突和棘的研究中也应该是有用的。
