High-affinity olfactory receptor for the death-associated odor cadaverine Ashiq Hussaina,1,2, Luis R. Saraivaa,2,3, David M. Ferrerob,2, Gaurav Ahujaa,2, Venkatesh S. Krishnaa, Stephen D. Liberlesb,4, and Sigrun I. Korschinga,4 aInstitut für Genetik, Universität zu Köln, 50674 Cologne, Germany; and bDepartment of Cell Biology, Harvard University, Boston, MA 02115 Edited* by Cornelia I. Bargmann, The Rockefeller University, New York, NY, and approved October 18, 2013 (received for review October 2, 2013) Carrion smell is strongly repugnant to humans and triggers distinct Results innate behaviors in many other species. This smell is mainly carried Zebrafish Avoid a Cadaverine Odor Source. The zebrafish has in by two small aliphatic diamines, putrescine and cadaverine, which recent years emerged as an important model system for un- are generated by bacterial decarboxylation of the basic amino derstanding olfaction in vertebrates because of a remarkable acids ornithine and lysine. Depending on the species, these diamines similarity in the basic principles of olfactory representation (9) may also serve as feeding attractants, oviposition attractants, or and some technical advantages over the mammalian system (10). social cues. Behavioral responses to diamines have not been in- However, behavioral responses of zebrafish to diamines have not vestigated in zebrafish, a powerful model system for studying been described. We report here that zebrafish, like humans, show vertebrate olfaction. Furthermore, olfactory receptors that detect pronounced innate aversion behavior for cadaverine (Fig. 1). cadaverine and putrescine have not been identified in any species We developed a valence assay to measure behavioral re- so far. Here, we show robust olfactory-mediated avoidance behav- sponses of zebrafish to olfactory cues. Zebrafish were habituated to iorofzebrafish to cadaverine and related diamines, and concomi- a rectangular tank, and the position of each fish was recorded before tant activation of sparse olfactory sensory neurons by these diamines. and after odor delivery. Shifts in average position toward or away The large majority of neurons activated by low concentrations of from the odor source were recorded as attraction and avoidance, fi cadaverine expresses a particular olfactory receptor, trace amine- respectively. Food odor, an attractant for zebra sh, caused a mean displacement of 0.25 tank lengths (TLs, P < 0.01) toward the odor associated receptor 13c (TAAR13c). Structure-activity analysis indi- B C cates TAAR13c to be a general diamine sensor, with pronounced source (Fig. 1 ), but tank water alone had no effect (Fig. 1 and Table S1). In contrast, cadaverine caused a mean displacement of selectivity for odd chains of medium length. This receptor can also P < be activated by decaying fish extracts, a physiologically relevant 0.28 TLs ( 0.01) away from the odor source, and was thus aversive (Fig. 1 A and C,andTable S1). Furthermore, the fish spent several- source of diamines. The identification of a sensitive zebrafish fold less time in close approach (distances < 0.05 TL) to the stimulus olfactory receptor for these diamines provides a molecular basis application site (P < 0.0001), although this area was not completely for studying neural circuits connecting sensation, perception, and avoided (P < 0.03) (Fig. 1A and Table S1), suggesting that the innate behavior. zebrafish did for short periods of time investigate the area, where stimulus was given. Mean velocity or total distance traveled was not NEUROSCIENCE Danio rerio | aversion | heterologous expression | polyamines altered during avoidance behavior (Table S1). Thus, the displace- ment observed is not caused by changes in motility but may result adaverine, putrescine, and other biogenic diamines are strongly from an assessment of odor valence by the fish. Crepulsive odors to humans, for whom these odors presumably Next, we analyzed whether related diamines were similarly signal bacterial contamination. It may be expected that animal aversive. We tested diamines with different carbon chain lengths, species feeding on carcasses attribute a more positive valence to ranging from C3 (diaminopropane) to C10 (diaminodecane). C diamines, and indeed both putrescine and cadaverine have been Avoidance behavior was observed (Fig. 1 ) to putrescine (C4), reported to be feeding attractants for rats (1) as well as goldfish (2). cadaverine (C5), diaminohexane (C6), diaminoheptane (C7), Similarly, insects depositing their eggs in carcasses or other pro- fi teineacous materials are attracted by these diamines (3). Beyond Signi cance signaling danger or food, putrescine and cadaverine also serve as social cues in several vertebrate species, both for marking of Cadaverine and putrescine, two diamines emanating from de- fl territories—for example, in feline species (4)—and for burial of caying esh, are strongly repulsive odors to humans but serve as conspecifics (5). innate attractive or social cues in other species. Here we show fi Very little is known about the molecular and cellular basis of that zebra sh, a vertebrate model system, exhibit powerful and cadaverine-driven behaviors. Cadaverine and putrescine evoke innate avoidance behavior to both diamines, and identify a high- electrophysiological responses in the olfactory epithelium of two affinity olfactory receptor for cadaverine. fish species (2, 6) and cadaverine-responsive olfactory sensory neurons and glomeruli have been identified in the mouse (7, 8). Author contributions: S.D.L. and S.I.K. designed research; A.H., L.R.S., D.M.F., G.A., and V.S.K. performed research; A.H., S.D.L., and S.I.K. analyzed data; and A.H., L.R.S., G.A., However, chemosensory receptors that detect cadaverine or re- S.D.L., and S.I.K. wrote the paper. lated diamines are unknown in any species, and could provide The authors declare no conflict of interest. valuable tools to study how the olfactory system mediates innate *This Direct Submission article had a prearranged editor. aversion or attraction. 1 Here, we show that cadaverine is a major product of zebrafish Present address: Department of Molecules, Signaling, and Development, Max-Planck- fi Institut für Neurobiologie, 82152 Martinsried, Germany. tissue decay, activates a zebra sh olfactory receptor (trace amine- 2 associated receptor 13c, TAAR13c) with high affinity, and elicits A.H., L.R.S., D.M.F., and G.A. contributed equally to this work. 3Present address: European Molecular Biology Laboratory–European Bioinformatics Insti- a strong, low-threshold, and olfactory-mediated avoidance response – fi fi tute (EMBL EBI) and Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, in zebra sh. In vivo measurements indicate that high af nity Hinxton-Cambridge CB10 1SD, United Kingdom. cadaverine responses occur primarily in TAAR13c-expressing 4To whom correspondence may be addressed. E-mail: [email protected] olfactory sensory neurons. These findings provide an important or [email protected]. foundation for understanding the molecular basis of a powerful This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. odor-driven behavior. 1073/pnas.1318596110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1318596110 PNAS | November 26, 2013 | vol. 110 | no. 48 | 19579–19584 Downloaded by guest on September 26, 2021 Cadaverine A mean displacement 1.0 pre post (stimulus tracks) Fig. 1. Aversive behavioral response of zebrafish to x 0.5 diamines. (A and B) Cadaverine-evoked aversion and food odor-evoked attraction of individual zebrafish fish position (y) as visualized by movement patterns before (orange 0 tracks) and after (brown tracks) stimulus addition 0 0.25 0.50 0.75 1.0 (1 mM, 180 μL). The position of stimulus deposition fish position (x coordinate) B Food Odor (blue X), as well as the mean location before (blue 1.0 pre post (stimulus tracks) circle) and after (red, green circles) stimulus addition are indicated. The axes represent tank dimensions. (C) Mean displacement was expressed as a percent- age of tank length (n = 6 ± SEM). Addition of tank x 0.5 water alone had no significant effect, whereas food odor elicited strong attraction. Similar avoidance fish position (y) behavior was observed in male and female fish. 0 Significance was evaluated by Student t test, *P < 0 0.25 0.50 0.75 1.0 0.05, **P < 0.01. (D) Aversion behavior requires ol- C ** D ** E factory input. Nostril closure of zebrafish eliminates ** aversion to 1 mM cadaverine or putrescine. Black 30 ** 60 ** * ** bars, no treatment; white bars, nostril closure; **P < ** ** 20 40 45 ** 0.01, n = 3 ± SEM. (E) Avoidance behavior to dia- ** mines was dose-dependent. Responses to cadaverine 10 20 Aversion 30 and diaminoheptane were measured over a broad 0 0 ** concentration range (1 μMto1M,n = 3 ± SEM). The * behavior is clearly saturable at 1-mM stimulus con- 10 Water 20 15 centration. Significance was evaluated by Student Putrescine anosmic anosmic < < Putrescine Cadaverine Attraction t test, asterisks refer to cadaverine, *P 0.05, **P 20 Cadaverine 40 mean displacement (% of TL) mean displacement (% of Diaminoheptane mean displacement (% of TL) mean displacement (% of TL) mean displacement (% of 0.01. The slight decrease at higher concentrations is Diaminooctane 0 Diaminohexane Diaminoheptane 0.001 0.1 10 1000 30 Diaminopropane not significant except for putrescine (P < 0.05). (F) Diaminodecane concentration (mM) Cadaverine-evoked aversion of an individual zebra- Food Putrescine ** Cadaverine fish in a flow-through