Department of Neurobiology and Behavior, University of California Irvine, Irvine, California 92697, USA.
Department of Neurobiology and Behavior, University of California Irvine, Irvine, California 92697, USA
Cold Spring Harb Protoc. 2020 Nov 2;2020(11):pdb.prot106823. doi: 10.1101/pdb.prot106823.
In the developing tadpole visual system, the targeting and branching of optic axons in the brain is a dynamic process that is closely intertwined with the morphological differentiation and maturation of their postsynaptic neurons and with the formation, stabilization, and elimination of functional synapses. The coordinated addition and retraction of axonal and dendritic branches guides the gradual recognition between pre- and postsynaptic neuronal partners, which subsequently allows synaptic connections to be formed. Axon and dendrite branching and selective synapse formation and stabilization are developmental mechanisms largely orchestrated by an array of signaling molecules that interact in vivo for the proper formation of functional visual circuits. In vivo real-time imaging of individual fluorophore-labeled neurons in living tadpoles has allowed investigation of molecular and cellular mechanisms mediating circuit assembly at a cellular level in the intact organism. In this protocol, we describe the use of bulk and single-cell electroporation to rapidly and efficiently transfect individual retinal ganglion cells (RGCs) with different reagents and to simultaneously visualize optic axon arbor morphology and presynaptic sites in real time. Similar techniques for labeling and visualizing RGC axons can be combined with the use of morpholino antisense oligonucleotides, as we describe here, to alter gene expression cell autonomously.
在发育中的蝌蚪视觉系统中，视神经轴突在大脑中的靶向和分支是一个动态过程，与它们的突触后神经元的形态分化和成熟以及功能突触的形成、稳定和消除密切交织在一起。轴突和树突分支的协调添加和缩回指导着前突触和后突触神经元伙伴之间的逐渐识别，随后允许形成突触连接。轴突和树突的分支以及选择性突触的形成和稳定是由一系列信号分子协调的发育机制，这些信号分子在体内相互作用，以形成功能性视觉回路。在活体蝌蚪中对单个荧光标记神经元进行体内实时成像，使得可以在完整生物体中以细胞水平研究介导回路组装的分子和细胞机制。在本方案中，我们描述了使用批量和单细胞电穿孔技术快速有效地转染单个视网膜神经节细胞（RGC）不同试剂，并实时可视化视神经轴突树突形态和突触前位点。我们在这里描述的用于标记和可视化 RGC 轴突的类似技术可以与使用抗基因表达的 morpholino 反义寡核苷酸结合使用，以自主改变细胞中的基因表达。
