Functional assay of ARNO and ARF6 in neurite elongation and branching

During development of the nervous system, neurite outgrowth is necessary for the formation of connections between nerve cells. Neurons are highly polarized cells that send out distinct processes, axons, and dendrites; however, the molecular regulation of the differential growth of these processes remains incompletely understood. Primary cultures of rat hippocampal neurons have been used to study many aspects of neuronal cell biology, including neurite extension, establishment of polarity, biogenesis of synapses, and membrane trafficking. After attachment to the substrate, hippocampal neurons begin sending out multiple processes by approximately 12 h after plating. The axonal process is derived from one of these processes, and is evident after 48 h in culture. Complete polarity of axons and dendrites is established after 7 days in culture. The establishment of these cultures and the ability to transfect them with potential regulatory genes allows the researcher to dissect out the pathways relevant to neurite extension. To study the role of small GTPases in neurite extension and branching, we describe methods for culture of hippocampal neurons, for transfection of these cells, and assessment of neurite extension and branching.