This video illustrates the procedure for making primary neuronal cultures from
midgastrula stage Drosophila embryos. The methods for collecting embryos and their
dechorionation using bleach are demonstrated. Using a glass pipet attached to a mouth
suction tube, we illustrate the removal of all cells from single embryos. The method for
dispersing cells from each embyro into a small (5 l) drop of medium on an uncoated glass
coverslip is demonstrated. A view through the microscope at 1 hour after plating
illustrates the preferred cell density. Most of the cells that survive when grown in
defined medium are neuroblasts that divide one or more times in culture before extending
neuritic processes by 12-24 hours. A view through the microscope illustrates the level of
neurite outgrowth and branching expected in a healthy culture at 2 days in vitro. The
cultures are grown in a simple bicarbonate based defined medium, in a 5% CO2
incubator at 22-24°C. Neuritic processes continue to elaborate over the first week in
culture and when they make contact with neurites from neighboring cells they often form
functional synaptic connections. Neurons in these cultures express voltage-gated sodium,
calcium, and potassium channels and are electrically excitable. This culture system is
useful for studying molecular genetic and environmental factors that regulate neuronal
differentiation, excitability, and synapse formation/function.
