In vitro culture of neurons and glia cells from embryonic mouse hippocampus and neocortex



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In vitro culture of neurons and glia cells from embryonic mouse hippocampus and neocortex

E-protocol written by Alexandra Nothnagel

Responsible for cell culture, photographs and videos: Sabrina Colasse

Biology of the Synapse, Laboratory of Antoine Triller

Institute of Biology Ecole Normale Supérieure – Paris – France
I Introduction

Herein I present a protocol to culture mainly pyramidal neurons in a mixed culture with glia from embryonic (E17 or E18) mouse hippocampus and cortex for up to 4 weeks on Poly-DL-ornithine hydrobromide coated glass coverslips without additional astroglial feeder cell layers. The serum-free neuron maintenance medium does not conduce to the growth and survival of astrocytes [1]. Dissociated neuron cultures have been studied for more than 40 years now and in vitro neuron development is well-characterized [2-4]. The cultured neurons express neuronal markers, develop extensive axonal and dendritic arbors, establish spines and form functional synaptic connections [5]. Thus, dissociated neuron cultures became a model to investigate the cellular and molecular mechanisms underlying postnatal neuron development such as neuron polarity formation [6, 7], dendritic outgrowth [8], axon guidance [9] and synaptogenesis [5] in the CNS. This in vitro model allows more controlled manipulations than in vivo models and is suited for various applications including immunocytochemistry, biochemistry, electrophysiology, Calcium and Sodium imaging and protein and RNA isolation. I use this culture model to study the maturation of the extracellular matrix and its relation to synaptogenesis and receptor recruitment to synapses. The neuron culture is a sequence of the following processes: coverslip coating, dissection, dissociation, cell plating and culture maintenance with feeding.




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