Single Cell Electroporation Method for Mammalian CNS Neurons in Organotypic Slice Cultures
Axon tracing is an essential technique to study the projection pattern of neurons in the CNS. Horse radish peroxidase and lectins have contributed to revealing many neural connection patterns in the CNS (Itaya and van Hoesen, 1982; Fabian and Coulter, 1985; Yoshihara, 2002). Moreover, a tracing method with fluorescent dye has enabled the observation of growing axons in living conditions, and demon strated a lot of developmental aspects in axon growth and guidance (Harris et al., 1987; O'Rourke and Fraser, 1990; Kaethner and Stuermer, 1992; Halloran and Kalil, 1994; Yamamoto et al., 1997). More recently, genetically encoded fluores cent proteins can be used as a powerful tool to observe various biological events. Several gene transfer techniques such as microinjection, biolistic gene gun, viral infection, lipofection and transgenic technology have been developed (Feng et al., 2000; Ehrengruber et al., 2001; O'Brien et al., 2001; Ma et al., 2002; Sahly et al., 2003). In particular, the electroporation technique was proved as a valuable tool, since it can be applied to a wide range of tissues and cell types with little toxicity and can be performed with relative technical easiness. Most methods, including a stand ard electroporation technique, are suitable for gene transfer to a large number of cells. However, this is not ideal for axonal tracing, because observation of individ ual axons is occasionally required. To overcome this problem, we have developed an electroporation method using glass micropipettes containing plasmid solutions and small current injection. Here we introduce the method in detail and exemplified results with some example applications and discuss its usefulness.
KeywordsHank Balance Salt Solution Silver Wire Cortical Slice Axon Arbor Electroporation Method
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- Halloran MC, Kalil K (1994) Dynamic behaviors of growth cones extending in the corpus callosum of living cortical brain slices observed with video microscopy. J Neurosci 14:2161–2177.Google Scholar
- Harris WA, Holt CE, Bonhoeffer F (1987) Retinal axons with and without their somata, growing to and arborizing in the tectum of Xenopus embryos: a time-lapse video study of single fibres in vivo. Development 101:123–133.Google Scholar
- Kaethner RJ, Stuermer CA (1992) Dynamics of terminal arbor formation and target approach of retinotectal axons in living zebrafish embryos: a time-lapse study of single axons. J Neurosci 12:3257–3271.Google Scholar
- Ma H, Zhu J, Maronski M, Kotzbauer PT, Lee VM, Dichter MA, Diamond SL (2002) Non-classical nuclear localization signal peptides for high efficiency lipofection of primary neurons and neuronal cell lines. Neuroscience 112:1–5.Google Scholar
- Yamamoto N, Higashi S, Toyama K (1997) Stop and branch behaviors of geniculocortical axons: a time-lapse study in organotypic cocultures. J Neurosci 17:3653–3663.Google Scholar