In contrast to the single sensory surface present in teleost fishes, several spatially segregated subsystems with distinct molecular and functional characteristics define the mammalian olfactory system. However, the evolutionary steps of that transition remain unknown. Here we analyzed the olfactory system of an early diverging tetrapod, the amphibian Xenopus laevis, and report for the first time the existence of two odor-processing streams, sharply segregated in the main olfactory bulb and partially segregated in the olfactory epithelium of pre-metamorphic larvae. A lateral odor-processing stream is formed by microvillous receptor neurons and is characterized by amino acid responses and Gαo/Gαi as probable signal transducers, whereas a medial stream formed by ciliated receptor neurons is characterized by responses to alcohols, aldehydes, and ketones, and Gαolf/cAMP as probable signal transducers. To reveal candidates for the olfactory receptors underlying these two streams, the spatial distribution of 12 genes from four olfactory receptor gene families was determined. Several class II and some class I odorant receptors (ORs) mimic the spatial distribution observed for the medial stream, whereas a trace amine-associated receptor closely parallels the spatial pattern of the lateral odor-processing stream. Other olfactory receptors (some class I odorant receptors and vomeronasal type 1 receptors) and odor responses (to bile acids, amines) were not lateralized, the latter not even in the olfactory bulb, suggesting an incomplete segregation. Thus, the olfactory system of X. laevis exhibits an intermediate stage of segregation and as such appears well suited to investigate the molecular driving forces behind olfactory regionalization.

Mammalian olfactory receptor families are segregated into different olfactory organs, with type 2 vomeronasal receptor (v2r) genes expressed in a basal layer of the vomeronasal epithelium. In contrast, teleost fish v2r genes are intermingled with all other olfactory receptor genes in a single sensory surface. We report here that, strikingly different from both lineages, the v2r gene family of the amphibian Xenopus laevis is expressed in the main olfactory as well as the vomeronasal epithelium. Interestingly, late diverging v2r genes are expressed exclusively in the vomeronasal epithelium, whereas "ancestral" v2r genes, including the single member of v2r family C, are restricted to the main olfactory epithelium. Moreover, within the main olfactory epithelium, v2r genes are expressed in a basal zone, partially overlapping, but clearly distinct from an apical zone of olfactory marker protein and odorant receptor-expressing cells. These zones are also apparent in the spatial distribution of odor responses, enabling a tentative assignment of odor responses to olfactory receptor gene families. Responses to alcohols, aldehydes, and ketones show an apical localization, consistent with being mediated by odorant receptors, whereas amino acid responses overlap extensively with the basal v2r-expressing zone. The unique bimodal v2r expression pattern in main and accessory olfactory system of amphibians presents an excellent opportunity to study the transition of v2r gene expression during evolution of higher vertebrates. To what extent such differences influence the coding and discrimination abilities of the respective olfactory systems is unclear, and the evolutionary path toward such segregation is unknown.

In olfactory receptor neurons (ORNs) of aquatic animals amino acids have been shown to be potent stimuli. Here we report on calcium imaging experiments in slices of the olfactory mucosa of Xenopus laevis tadpoles. We were able to determine the response profiles of 283 ORNs to 19 amino acids, where one profile comprises the responses of one ORN to 19 amino acids. 204 out of the 283 response profiles differed from each other. 36 response spectra occurred more than once, i.e., there were 36 classes of ORNs identically responding to the 19 amino acids. The number of ORNs that formed a class ranged from 2 to 13. Shape and duration of amino acid-elicited [Ca2+]i transients showed a high degree of similarity upon repeated stimulation with the same amino acid. Different amino acids, however, in some cases led to clearly distinguishable calcium responses in individual ORNs. Furthermore, ORNs clearly appeared to gain selectivity over time, i.e., ORNs of later developmental stages responded to less amino acids than ORNs of earlier stages. We discuss the narrowing of ORN selectivity over stages in the context of expression of olfactory receptors.