Chemical Senses Advance Access originally published online on April 5, 2006
Chemical Senses 2006 31(6):497-504; doi:10.1093/chemse/bjj052
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Primary Culture of Gustatory Receptor Neurons from the Blowfly, Phormia regina
1 Department of Applied Physics and Chemistry, The University of Electro-Communications, Tokyo 182-8585, Japan 2 Department of Applied Biology, Faculty of Textile Science, Kyoto Institute of Technology, Kyoto 606-8585, Japan
Correspondence to be sent to: Tadashi Nakamura, Department of Applied Physics and Chemistry, The University of Electro-Communications, Tokyo 182-8585, Japan. e-mail: tad{at}pc.uec.ac.jp
Flies provide a powerful model system for exploring signaling systems in gustatory receptor neurons (GRNs). To elucidate the cellular and molecular bases of these signaling systems, we sought to develop techniques to dissociate GRNs. We developed a primary culture of GRNs isolated from the labella of the blowfly, Phormia regina, 4–5 days after pupation. Dissected labella were treated with papain in a low Ca2+ saline solution and shaken in Leibovitz's L-15 medium supplemented with 20-hydroxyecdysone, L-ascorbic acid, and trehalose with a test tube mixer. Released cells were plated and kept at 29°C in a medium containing fetal bovine serum. After a minimum of 2 days in culture, we observed survival or growth of bipolar cells with the characteristic morphology of GRNs. We also examined taste responsiveness by monitoring intracellular Ca2+ with a Ca2+-sensitive fluorescent dye, fluo-3. For some bipolar cells, application of sucrose, NaCl, or LiCl for 5–20 s transiently increased the intracellular Ca2+ levels in cell bodies for 20–30 s. The primary cell culture described here is useful for functional analysis of GRNs.
Key words: calcium imaging, fly, intracellular calcium ion concentration, taste cells