High-resolution functional labeling of vertebrate and invertebrate olfactory receptor neurons using agmatine, a channel-permeant cation

Abstract

Methods are described for odor-stimulated labeling of olfactory receptor neurons (ORNs) of the freshwater zebrafish Danio rerio and the marine spiny lobster Panulirus argus. Permeation of a cationic molecule, 1-amino-4-guanidobutane (=agmatine, AGB), through ion channels following odor stimulation, and its detection by an anti-AGB antibody, allow labeling of odor-stimulated ORNs. Parameters adjusted to optimize activity-dependent labeling included labeling medium ionic composition, stimulation times, and AGB concentration. For lobsters, 7% of ORNs were labeled by a complex odor, oyster mixture, under optimal conditions, which was stimulation for 5 s per min for 60 min with 20 mM AGB in artificial seawater with reduced sodium and calcium concentrations. AGB was a weak odorant for lobsters; it elicited only a small electrophysiological response from ORNs and labeled<1% of the ORNs during stimulation with AGB in the absence of odors. For the zebrafish, stimulation for 10 s per min for 10 min with 5 mM AGB plus odorant ( l-glutamine) in fish Ringer’s solution was the optimal labeling condition, resulting in labeling of 17% of the olfactory epithelial area. Approximately 6% of the olfactory epithelium was labeled during stimulation with a control stimulus, AGB alone. This labeling by AGB alone suggests it is an olfactory stimulus for zebrafish; a conclusion supported by electrophysiological recordings. We used electrophysiological assays and channel blockers to examine, for each species, potential ion channels for entry of AGB into ORNs. These results show that AGB can be used as an activity-dependent label for chemoreceptor neurons of diverse phyla living in a range of environmental conditions.