Using Primary Neuron Cultures of Drosophila to Analyze Neuronal Circuit Formation and Function

For many decades, primary neuron cultures of Drosophila have been used complementary to work in vivo. Primary cultures were instrumental for the analysis of physiological properties of Drosophila neurons and synapses, and they were used for the analysis of developmental processes. Recent developments have established Drosophila primary neurons based on Schneider’s culture media as a means to investigate the neuronal cytoskeleton, opening up novel opportunities for research into cellular mechanisms of axonal growth, synapse formation, and perhaps even neuronal degeneration. These cell cultures provide readouts for cytoskeletal dynamics that are difficult or impossible to access in vivo, and which turned out to be highly conserved with mammalian or other vertebrate neurons. Therefore, the same genetic manipulations in Drosophila can now be studied synergistically in culture and in vivo, to address cell biological principles of neuronal circuit formation and function. Here, we describe in detail how these cell cultures are generated and discuss principal considerations for the experimental design and the solution of common problems. Furthermore, we describe in detail how to generate Schneider’s media with adjustable inorganic ion concentrations. These media have been shown to promote the physiological maturation of neurons, thus expanding the use of the primary neuron cultures into the synaptic stage. The culture strategies described here recapitulate in vivo development with impressive accuracy and provide a promising means for Drosophila research on neuronal development and function.