Primary Cultures of Moth Olfactory Receptor Neurons

Insects critically depend on their olfaction for many behaviors related to mating, feeding, and social interactions. They have evolved highly sensitive olfactory receptor neurons (ORNs). Although in situ extracellular recordings are commonly used to analyze the response profile, coding properties, and physiology of these cells, a detailed analysis of their signal transduction pathways requires patch-clamp recordings. To facilitate the use of this electrophysiological technique and a good control of the composition of intra- and extracellular ORN solutions, this paper describes a protocol for culturing insect ORNs. As insect ORNs are closely enveloped by accessory cells, preventing any acute dissociation of adult ORNs and direct access to their membranes, this protocol describes the dissection of antennal cells from moth pupae, before the differentiation of ORNs. Developing antennae are enzymatically and mechanically dissociated and then cultured upside down with the 'hanging column technique' in a conditioned medium that supports the differentiation and survival of ORNs for several weeks. ORNs cultured by this method are suitable for functional assays such as patch-clamp recordings.