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

Methods are described for odor-stimulated labeling of olfactory receptor ne urons (ORNs) of the freshwater zebrafish Danio rerio and the marine spiny l obster Panulirus argus. Permeation of a cationic molecule, 1-amino-4-guanid obutane (= agmatine, AGE), through ion channels following odor stimulation, and its detection by an anti-AGE antibody, allow labeling of odor-stimulat ed ORNs. Parameters adjusted to optimize activity-dependent labeling includ ed labeling medium ionic composition, stimulation times, and AGE concentrat ion. For lobsters, 7% of ORNs were labeled by a complex odor, oyster mixtur e, under optimal conditions, which was stimulation for 5 s per min for 60 m in with 20 mM AGB in artificial seawater with reduced sodium and calcium co ncentrations. AGE was a weak odorant for lobsters; it elicited only a small electrophysiological response from ORNs and labeled < 1% of the ORNs durin g stimulation with AGE in the absence of odors. For the zebrafish, stimulat ion for 10 s per min for 10 min with 5 mM AGE plus odorant (L-glutamine) in fish Ringer's solution was the optimal labeling condition, resulting in la beling of 17% of the olfactory epithelial area. Approximately 6% of the olf actory epithelium was labeled during stimulation with a control stimulus, A GE alone. This labeling by AGE alone suggests it is an olfactory stimulus f or 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 AGE into ORNs. These results show that AGE can be used as an activity-dependent label for chemoreceptor neurons of diverse phyla living in a range of environmental conditions. (C) 1999 Elsevier Science B.V. All rights reserved.