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.