What Reaches the Antenna? How to Calibrate Odor Flux and Ligand-Receptor Affinities

Physiological studies on olfaction frequently ignore the airborne quantities of stimuli reaching the sensory organ. We used a gas chromatography-calibrated photoionization detector to estimate quantities released from standard Pasteur pipette stimulus cartridges during repeated puffing of 27 compounds and verified how lack of quantification could obscure olfactory sensory neuron (OSN) affinities. Chemical structure of the stimulus, solvent, dose, storage condition, puff interval, and puff number all influenced airborne quantities. A model including boiling point and lipophilicity, but excluding vapor pressure, predicted airborne quantities from stimuli in paraffin oil on filter paper. We recorded OSN responses of Drosophila melanogaster, Ips typographus, and Culex quinquefasciatus, to known quantities of airborne stimuli. These demonstrate that inferred OSN tuning width, ligand affinity, and classification can be confounded and require stimulus quantification. Additionally, proper dose-response analysis shows that Drosophila AB3A OSNs are not promiscuous, but highly specific for ethyl hexanoate, with other earlier proposed ligands 10- to 10 000-fold less potent. Finally, we reanalyzed published Drosophila OSN data (DoOR) and demonstrate substantial shifts in affinities after compensation for quantity and puff number. We conclude that consistent experimental protocols are necessary for correct OSN classification and present some simple rules that make calibration, even retroactively, readily possible.