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Nucleofection and Primary Culture of Embryonic Mouse Hippocampal and Cortical Neurons
Published on: January 24, 2011
Culturing pyramidal neurons from the early postnatal mouse hippocampus and cortex
Gerard M J Beaudoin1, Seung-Hye Lee, Dipika Singh
1Department of Physiology, University of California-San Francisco (UCSF), San Francisco, California, USA.
Nature Protocols
|September 1, 2012
Summary
We developed a protocol for culturing mouse hippocampal and cortical neurons. This method supports neuronal survival, development, and synaptic function for up to four weeks, aiding neuroscience research.
Area of Science:
- Neuroscience
- Cell Biology
- Developmental Biology
Background:
- Culturing postnatal mouse neurons is crucial for studying genetically engineered models.
- Existing methods may have limitations in supporting long-term neuronal health and functionality.
Purpose of the Study:
- To present an optimized protocol for isolating and culturing early postnatal mouse hippocampal and cortical pyramidal neurons.
- To enable robust neuronal cultures for various downstream applications in neuroscience research.
Main Methods:
- Isolation and low-density dissociation of pyramidal neurons from postnatal day 0-1 mouse hippocampus and cortex.
- Culture on poly-L-lysine-coated glass substrates without feeder layers.
- Preparation of glass substrates initiated 5 days prior to neuronal plating.
Main Results:
- Cultured neurons exhibit excellent survival, extensive axonal and dendritic arborization, and expression of neuronal/synaptic markers.
- Functional synaptic connections are formed in vitro.
- Neurons are amenable to low- and high-efficiency transfection and time-lapse imaging.
Conclusions:
- This optimized cell culture technique provides a reliable method for maintaining mouse hippocampal and cortical neurons for up to 4 weeks.
- The protocol supports diverse applications including immunocytochemistry, biochemical assays, shRNA knockdown, and live imaging.
