Amine detection in aquatic organisms: receptor evolution, neuronal circuits and behavior in the model organism zebrafish

Olfactory cues are responsible for the generation of diverse behaviors in the animal kingdom. Olfactory receptors are expressed by specialized sensory neurons (OSNs) in the olfactory epithelium. Upon odorant binding to the olfactory receptor, these neurons are activated. The information is transferred to the olfactory bulb glomeruli, which represent the first relay station for olfactory processing in the brain. Most olfactory receptors are G-protein coupled receptors and form large gene families. One type of olfactory receptors is the trace amine-associated receptor family (TAAR). TAARs generally recognize amines. One particular member of the zebrafish TAAR family, TAAR13c, is a high-affinity receptor for the death-associated odor cadaverine, which induces aversive behavior. Here, we identified the cell type of amine-sensitive OSNs in the zebrafish nose, which show typical properties of ciliated neurons. We used OSN type-specific markers to unambiguously characterize zebrafish TAAR13c OSNs. Using the neuronal activity marker pERK we could show that low concentrations of cadaverine activate a specific, invariant glomerulus in the dorso-lateral cluster of glomeruli (dlG) in the olfactory bulb of zebrafish. This cluster was also shown to process amine stimuli in general, a feature that is conserved in the neoteleost stickleback. Apart from developing a technique to measure neuronal activity in the adult olfactory epithelium, we also established the use of GCaMP6-expressing zebrafish to measure neuronal activity in the larval brain. This will be helpful in deciphering neuronal circuits involved in odor processing in future experiments. Although adult zebrafish display aversive behavior in response to cadaverine, we found zebrafish larvae to be attracted to cadaverine in a similar behavioral assay. This shift of behavior in the ontogeny of zebrafish has to be further investigated. A TAAR13c gene knock-out could provide important insights into the neuronal processing of diamine stimuli and the role of TAAR13c for the generation of behavioral outputs. Here we used an alternative CRISPR/Cas9 approach to partially knock out the TAAR13c gene. The DNA sequence between two gRNA target sites was deleted from the genome. Further studies will have to characterize this knock-out. II The evolutionary origin of TAARs has not been conclusively described yet. Using a large scale analysis of 81 fish genomes we provide new insights into TAAR evolution. We found that TAARs together with its close sister group, TARLs, which drastically expanded in lamprey, originate in a duplication of the HTR4 gene after the emergence of chordates, but before the divergence of jawed from jawless fish. Class II TAARs are present only in the jawed vertebrate lineage. Contrary to our expectations we found TAAR13 to be retained in neoteleosts. Class II TAARs are characterized by early as well as late gene loss events at several points in fish evolution and single members often show family- or species-specific expansions

Olfactory function in the trace amine-associated receptor family (TAARs) evolved twice independently

Olfactory receptor families have arisen independently several times during evolution. The origin of taar genes, one of the four major vertebrate olfactory receptor families, is disputed. We performed a phylogenetic analysis making use of 96 recently available genomes, and report that olfactory functionality has arisen twice independently within the TAAR family, once in jawed and once in jawless fish. In lamprey, an ancestral gene expanded to generate a large family of olfactory receptors, while the sister gene in jawed vertebrates did not expand and is not expressed in olfactory sensory neurons. Both clades do not exhibit the defining TAAR motif, and we suggest naming them taar-like receptors (tarl). We have identified the evolutionary origin of both taar and tarl genes in a duplication of the serotonergic receptor 4 that occurred in the most recent common ancestor of vertebrates. We infer two ancestral genes in bony fish (TAAR12, TAAR13) which gave rise to the complete repertoire of mammalian olfactory taar genes and to class II of the taar repertoire of teleost fish. We follow their evolution in seventy-one bony fish genomes and report a high evolutionary dynamic, with many late gene birth events and both early and late gene death events

A single identified glomerulus in the zebrafish olfactory bulb carries the high-affinity response to death-associated odor cadaverine

The death-associated odor cadaverine, generated by bacteria-mediated decarboxylation of lysine, has been described as the principal activator of a particular olfactory receptor in zebrafish, TAAR13c. Low concentrations of cadaverine activated mainly TAAR13c-expressing olfactory sensory neurons, suggesting TAAR13c as an important element of the neuronal processing pathway linking cadaverine stimulation to a strongly aversive innate behavioral response. Here, we characterized the initial steps of this neuronal pathway. First we identified TAAR13c-expressing cells as ciliated neurons, equivalent to the situation for mammalian taar genes, which shows a high degree of conservation despite the large evolutionary distance between teleost fishes and mammals. Next we identified the target area of cadaverine-responsive OSNs in the olfactory bulb. We report that cadaverine dose-dependently activates a group of dorsolateral glomeruli, at the lowest concentration down to a single invariant glomerulus, situated at the medial border of the dorsolateral cluster. This is the first demonstration of a single stereotyped target glomerulus in the fish olfactory system for a non-pheromone odor. A mix of different amines activates many glomeruli within the same dorsolateral cluster, suggesting this area to function as a general amine response region