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Culturing pyramidal neurons from the early postnatal mouse hippocampus and cortex

Abstract

The ability to culture and maintain postnatal mouse hippocampal and cortical neurons is highly advantageous, particularly for studies on genetically engineered mouse models. Here we present a protocol to isolate and culture pyramidal neurons from the early postnatal (P0-P1) mouse hippocampus and cortex. These low-density dissociated cultures are grown on poly-L-lysine–coated glass substrates without feeder layers. Cultured neurons survive well, develop extensive axonal and dendritic arbors, express neuronal and synaptic markers, and form functional synaptic connections. Further, they are highly amenable to low- and high-efficiency transfection and time-lapse imaging. This optimized cell culture technique can be used to culture and maintain neurons for a variety of applications including immunocytochemistry, biochemical studies, shRNA-mediated knockdown and live imaging studies. The preparation of the glass substrate must begin 5 d before the culture. The dissection and plating out of neurons takes 3–4 h and neurons can be maintained in culture for up to 4 weeks.

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Figure 1: Overview of the technique.
Figure 2: Illustration of the technique to remove brains from P0 mice.
Figure 3: Steps for dissection of the hippocampus from the intact brain.
Figure 4: Trituration of isolated hippocampi/cortex.
Figure 5: Images of cultured hippocampal neurons.
Figure 6: Morphology and immunostaining of neurons for synaptic markers.
Figure 7: Synaptic functional assay on cultured neurons.
Figure 8: Neurons in culture are amenable to time-lapse imaging.

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Acknowledgements

We thank the past and present members of the Reichardt laboratory for their support and insight. This work was supported by US National Institutes of Health Grant F32-MH079661 (G.M.J.B.), the Simons Foundation (L.F.R.) and start-up funds from the Munroe-Meyer Institute, University of Nebraska Medical Center (J.A.).

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Authors and Affiliations

Authors

Contributions

G.M.J.B., S.-H.L. and J.A. designed experiments. G.M.J.B., S.-H.L., D.S., Y.Y., Y.-G.N. and J.A. performed the experiments. G.M.J.B., S.-H.L., D.S., Y.Y. and J.A. collected, analyzed and interpreted data. J.A. and L.F.R. supervised the experiments. G.M.J.B., S.-H.L., L.F.R. and J.A. wrote the manuscript.

Corresponding author

Correspondence to Jyothi Arikkath.

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Competing interests

Seung-Hye Lee is currently an employee of Genentech; however, all her contributions to this manuscript were from studies conducted while she was at UCSF. The other authors have no competing financial interests.

Supplementary information

Supplementary Figure 1

Neuronal morphology and immunostaining. (PDF 1645 kb)

Supplementary Figure 2

Immunostaining for glia and neurons. (PDF 785 kb)

Supplementary Figure 3

Immunostaining for inhibitory neurons. (PDF 234 kb)

Supplementary Figure 4

Western blot analysis on high-efficiency–transfected neurons. (PDF 549 kb)

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Beaudoin, G., Lee, SH., Singh, D. et al. Culturing pyramidal neurons from the early postnatal mouse hippocampus and cortex. Nat Protoc 7, 1741–1754 (2012). https://doi.org/10.1038/nprot.2012.099

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