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Culturing hippocampal neurons

Abstract

We provide protocols for preparing low-density dissociated-cell cultures of hippocampal neurons from embryonic rats or mice. The neurons are cultured on polylysine-treated coverslips, which are suspended above an astrocyte feeder layer and maintained in serum-free medium. When cultured according to this protocol, hippocampal neurons become appropriately polarized, develop extensive axonal and dendritic arbors and form numerous, functional synaptic connections with one another. Hippocampal cultures have been used widely for visualizing the subcellular localization of endogenous or expressed proteins, for imaging protein trafficking and for defining the molecular mechanisms underlying the development of neuronal polarity, dendritic growth and synapse formation. Preparation of glial feeder cultures must begin 2 weeks in advance, and it takes 5 d to prepare coverslips as a substrate for neuronal growth. Dissecting the hippocampus and plating hippocampal neurons takes 2–3 h.

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Figure 1
Figure 2: Phase-contrast images of hippocampal neurons during the first 4 d of culture.
Figure 3: Low-magnification phase-contrast images of hippocampal cultures plated after 1 d (a) and 13 d in culture (b).
Figure 4: Using selective markers to visualize axons and dendrites.
Figure 5: Initial segment markers can be used to visualize the origin of the axon.
Figure 6: Expressing GFP enables visualization of the complete arborization of an individual cell.
Figure 7: A Stage 5 neuron in a 3-week-old culture.

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Acknowledgements

Research in our laboratory is supported by US National Institutes of Health grants NS17112 and MH66179. Our thanks to all past and present members of the lab, who have contributed so much to these protocols and whose work has underscored the truth of the ancient maxim, in vitro veritas.

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Correspondence to Gary Banker.

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Kaech, S., Banker, G. Culturing hippocampal neurons. Nat Protoc 1, 2406–2415 (2006). https://doi.org/10.1038/nprot.2006.356

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