Mechanism of activation and function of the odorant receptor expressed at the axon terminus-growth cone of olfactory sensory neurons

A unique feature in the topographic organization of the olfactory bulb is the “dual role” of the odorant receptor. It detects odorants and it has been suggested to play a critical role in the axonal convergence of olfactory sensory neurons to form glomeruli in specific loci of the olfactory bulb. This spatial segregation of sensory afferents results in the sensory map. A role of the odorant receptors in axon guidance was suggested by genetic experiments demonstrating that manipulations of odorant receptor sequences perturb the sensory map (Wang et al., 1998). This hypothesis was confirmed by subsequent works (Barnea et al., 2004, Strotmann et al., 2004) that revealed the presence of the olfactory receptor in the most distal portion of the axon and at the growth cone. The open question to be addressed was whether the odorant receptor expressed at the axon terminal was functional and if yes, what was the signalling pathway coupled to its activation. In a previous study, Maritan et al., 2009, demonstrated, for the first time, that the odorant receptor expressed at the axon terminus - growth cone is functional and coupled to local increases of Ca2+ and cAMP. Although cAMP and Ca2+ are the primary second messengers produced upon activation of the odorant receptor, cGMP is also synthesized and takes part in several key processes such as adaptation, neuronal development and long term cellular responses to odorant stimulation. Many aspects of the regulation of cGMP in olfactory sensory neurons (OSNs) were still unknown, as the mechanism of coupling to odorant receptors (ORs) and downstream targets. To address these points, we investigated the dynamics and the intracellular distribution of cGMP in living rat OSNs in culture transfected with a genetically encoded sensor for cGMP. We demonstrated that OSNs treated with pharmacological stimuli able to activate particulate or soluble guanylyl cyclases (pGC and sGC) presented an increase in cGMP in the whole neuron, from cilia - dendrite to the axon terminus - growth cone. Upon odorant stimulation, a rise in cGMP was again found in the entire neuron, including the axon terminal, where it is locally synthesized. The odorant - dependent rise in cGMP is due to sGC activation by NO and requires an increase of cAMP. The link between cAMP and NO synthase appears to be the rise in [Ca2+]c elicited by either plasma membrane Ca2+ channel activation and Ca2+ mobilization from stores via the guanine nucleotide exchange factor Epac. Finally we show that a cGMP rise can elicit the phosphorylation of nuclear CREB both in vitro and in vivo. The local synthesis of cGMP, coupled to the OR expressed at the axon terminal, suggested that not only cAMP, but also cGMP can contribute to OSN axonal convergence. The question then arose on the mechanism of activation, i.e. the possible natural ligands, of the olfactory receptor at the axon terminal. We hypothesized that a few molecules expressed in gradient in the olfactory bulb could bind and activate the odorant receptor expressed at the axon terminal, regulating in this way the axon pathfinding to its final target. To test our hypothesis we studied the spatio - temporal dynamics of Ca2+ and cAMP in response to molecules from the bulb. We found that a pool of these molecules is capable of eliciting a rise in Ca2+ and cAMP in the axon terminus - growth cone of OSNs loaded with fura-2 or transfected with the sensor for cAMP. To assess whether the Ca2+ and cAMP rises were due to the activation of the olfactory receptor at the axon terminal, we expressed specific odorant receptors in HEK cells. The Ca2+ rise was observed only in HEK cells transfected with specific receptors, but not in HEK cells transfected with the empty vector (controls). All together, our data demonstrate the presence of a pool of active molecules in the bulb able to activate the OR expressed at the axon terminus - growth cone. To assess the physiological meaning of the variation in Ca2+ and cAMP levels on the turning behaviour of the olfactory sensory neuron axons, real - time imaging experiments on isolated olfactory sensory neurons were performed. We analyzed the behaviour of olfactory sensory neuron growth cone in response to gradients of molecules capable of modulating Ca2+ and cAMP levels at the axon terminus - growth cone, such as forskolin, a generic activator of adenylyl cyclase, odors and the active pool of molecules from the olfactory bulb. We found that all these molecules, including the ones from the bulb, were able to regulate the turning behaviour of the olfactory sensory neuron axons. All together our data suggest that molecules from the olfactory bulb, via activation of the odorant receptor expressed at the axon terminus – growth cone, contribute in providing the olfactory sensory neuron axons with instruction to reach the proper target in the olfactory bul

Representations and Transformations of Odor Information in the Mouse Olfactory System

For a wide variety of organisms on the planet, the sense of smell is of critical importance for survival. The mouse olfactory system mediates both learned and innate odor-driven behaviors, including activities as diverse as the localization of food sources, the avoidance of predators, and the selection of mates. How a chemical stimulus in the environment ultimately leads to the generation of an appropriate behavioral response, however, remains poorly understood. All of these behaviors begin with the binding of an odorant in the external environment to receptors on sensory neurons in the olfactory epithelium. These sensory neurons transmit this odor information to neurons in the olfactory bulb via spatially stereotyped axonal projections, and a subset of these bulbar neurons, mitral and tufted cells, in turn transmit this information to a number of higher brain regions implicated in both learned and innate odor-driven behaviors, including the piriform cortex and amygdala. Previous work has revealed that odorants drive activity in unique, sparse ensembles of neurons distributed across the piriform cortex without apparent spatial preference. The patterns of neural activity observed, however, do not reveal whether mitral and tufted cell projections from a given glomerulus to piriform are segregated or distributed, or whether they are random or determined. Distinguishing between these possibilities is important for understanding the function of piriform cortex: a random representation of odor identity in the piriform could accommodate learned olfactory behaviors, but cannot specify innate odor-driven responses. In addition, behavioral studies in which the function of the amygdala has been compromised have found that innate odor-driven behaviors are disrupted by these manipulations while learned odor-driven behaviors are left intact, strongly suggesting a role for the amygdala in innate olfactory responses. How odor information is represented in the amygdala, as well as the amygdala's exact role in the generation of olfactory responses, however, remain poorly understood. We therefore developed a strategy to trace the projections from identified glomeruli in the olfactory bulb to these higher olfactory centers. Electroporation of TMR dextran into single glomeruli has permitted us to define the neural circuits that convey olfactory information from specific glomeruli in the olfactory bulb to the piriform cortex and amygdala. We find that mitral and tufted cells from every glomerulus elaborate similar axonal arbors in the piriform. These projections densely fan out across the cortical surface in a homogeneous manner, and quantitative analyses fail to identify features that distinguish the projection patterns from different glomeruli. In contrast, the cortical amygdala receives spatially stereotyped projections from individual glomeruli. The stereotyped projections from each glomerulus target a subregion of the posterolateral cortical nucleus, but may overlap extensively with projections from other glomeruli. The apparently random pattern of projections to the piriform and the determined pattern of projections to the amygdala are likely to provide the anatomic substrates for distinct odor-driven behaviors mediated by these two brain regions. The dispersed mitral and tufted cell projections to the piriform provide the basis for the generation of previously observed patterns of neural activity and suggest a role for the piriform cortex in learned olfactory behaviors, while the pattern of mitral and tufted cell projections to the posterolateral amygdala implicate this structure in the generation of innate odor-driven behaviors. We have also developed high-throughput methods for imaging odor-evoked activity in targeted populations of neurons in multiple areas of the olfactory system to investigate how odor information is represented and transformed by the mouse brain. We have used a modified rabies virus that drives expression of GCaMP3, a calcium-sensitive indicator of neural activity, to image odor-evoked responses from mitral and tufted cells, as well as a modified adenoassociated virus that drives expression of GCaMP3 to image odor-evoked responses from neurons in piriform cortex. These imaging methods have permitted us to examine odor-evoked responses in a transgenic mouse where 95% of sensory neurons express a single kind of olfactory receptor (M71). In these mice, there is a 1,000-fold increase in sensory neurons expressing the M71 receptor ligand acetophenone, and a 20-fold reduction in neurons expressing olfactory receptors from the endogenous repertoire. These M71 transgenic mice provide a useful tool for examining the role that the normally stereotyped pattern of sensory neuron input to the bulb plays in olfactory processing, as well as how odor information is transformed as is moves from the sensory periphery to the cortex. In control mice, odors evoke activity in unique ensembles of spatially distributed, narrowly tuned mitral and tufted cells, and the number of cells responding to odor increases linearly with stimulus concentration. Surprisingly, despite the fact that there is a significant decrease in sensory neuron activity in response to odors other than acetophenone in M71 transgenics, a wide variety of odorants are able to evoke mitral and tufted cell activity in these mice. Furthermore, the number of cells responding to these odors as well as the magnitude of these odor-evoked responses are higher in M71 transgenics compared to controls. However, despite a massive increase in acetophenone-evoked sensory neuron input to the bulb in M71 transgenics, mitral and tufted cell responses to acetophenone are similar in M71 transgenics and controls. Our results provide evidence for excitatory mechanisms that amplify weak sensory neuron input as well as inhibitory mechanisms that suppress strong, pervasive odor-evoked input, suggesting that a major role of the olfactory bulb is to aid in the comprehensive detection and refinement of olfactory signals from the environment. Despite the fact that the representation of odor in the olfactory bulb of M71 transgenic mice differs from that observed in controls, we find the representations of odor in the piriform cortex of M71 transgenic mice and controls is quantitatively indistinguishable. Our results suggest that circuits intrinsic to the piriform significantly transform the representation of odor information as it moves from the olfactory bulb to the piriform cortex. Moreover, in comparison to the olfactory bulb, the piriform encodes odor in a more sparse, distributed manner within a much narrower dynamic range. The nature of the representation of odor we observe in piriform cortex further supports a role for this area in mediating odor discrimination and associative odor-driven behaviors. The work described in this thesis has provided insight into the way odor is represented in several areas of the mouse olfactory system, clues about how odor information is transformed as it passes through the brain, and the role that different areas of the olfactory system play in odor-driven perception and behavior. In the future, the novel techniques and methods described in this thesis can be applied to the study of many different areas of the mammalian brain, giving our work the potential to have a significant impact on our understanding of how patterns of neural activity may ultimately underlie the generation of perceptions, emotions, and behaviors

Structure, function and context : the impact of morphometry and ecology on olfactory sensitivity

Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at the Massachusetts Institute of Technology and the Woods Hole Oceanographic Institution February, 2005In this thesis, the relationships of olfactory sensitivity to three biological variables were tested. The sensitivity of a marine mammal, the sea otter (Enhydra lutris) was measured in order to determine whether a marine lifestyle results in impaired olfaction. The effect of dietary relevance on sensitivity to specific odorants was evaluated. Finally, a new morphometric model of olfactory uptake efficiency was developed and tested against behavioral measurements of olfactory sensitivity in twelve mammalian species from five orders. Olfactory thresholds were obtained for the first time from two sea otters for seven odorant compounds from various natural sources. Otters were trained using operant conditioning to participate in direct behavioral testing. Sea otter olfactory sensitivity was comparable to that of previously studied terrestrial mammals. The incidence of an odorant in the diet of the olfactor was found to influence specific sensitivity to that compound but to varying degrees among different mammalian orders. Nasal cavity specimens were measured using radiologic (CT scan) and histologic (light microscopy) techniques. Surface areas and volumes of the nasal cavity were used to calculate the Olfactory Uptake Efficiency (OUE). OUE is significantly related to olfactory bulb volume. A possible relationship was found between OUE and general olfactory sensitivity.I am grateful to the Oregon Zoo and the Oregon Coast Aquarium, whose exhibit animals provided the olfactory threshold data, as well as the Monterey Bay Aquarium, the Point Defiance Zoo and the New England Aquarium, which also participated. Nasal cavity specimens were generously donated by the American Museum of Natural History, the Whitehead Institute at the Massachusetts Institute of Technology, the Biology Department of MIT, the California Oiled Wildlife Network, the Harvard Museum of Comparative Zoology, the Institute for Hydrology and Ecology at Monk's Hood, Tufts Veterinary School, the New England Regional Primate Research Center, Lion Country Safari Zoo, and the Cameron Park Zoo. Funding was provided by the Woods Hole Oceanographic Institution's Education Department, Biology Department and Ocean Ventures Fund, the National Science and Engineering Research Council of Canada, the Gen Foundation, the Massachusetts Institute of Technology's Student Assistance Fund, the European Chemoreception Research Organization, the Society for Experimental Biology, the Company of Biologists, and the Office of Naval Research

Neural Circuit Dynamics and Ensemble Coding in the Locust and Fruit Fly Olfactory System

Raw sensory information is usually processed and reformatted by an organism’s brain to carry out tasks like identification, discrimination, tracking and storage. The work presented in this dissertation focuses on the processing strategies of neural circuits in the early olfactory system in two insects, the locust and the fruit fly. Projection neurons (PNs) in the antennal lobe (AL) respond to an odor presented to the locust’s antennae by firing in slow information-carrying temporal patterns, consistent across trials. Their downstream targets, the Kenyon cells (KCs) of the mushroom body (MB), receive input from large ensembles of transiently synchronous PNs at a time. The information arrives in slices of time corresponding to cycles of oscillatory activity originating in the AL. In the first part of the thesis, ensemble-level analysis techniques are used to understand how the AL-MB system deals with the problem of identifying odors across different concentrations. Individual PN odor responses can vary dramatically with concentration, but invariant patterns in PN ensemble responses are shown to allow odor identity to be extracted across a wide range of intensities by the KCs. Second, the sensitivity of the early olfactory system to stimulus history is examined. The PN ensemble and the KCs are found capable of tracking an odor in most conditions where it is pulsed or overlapping with another, but they occasionally fail (are masked) or reach intermediate states distinct from those seen for the odors presented alone or in a static mixture. The last part of the thesis focuses on the development of new recording techniques in the fruit fly, an organism with well-studied genetics and behavior. Genetically expressed fluorescent sensors of calcium offer the best available option to study ensemble activity in the fly. Here, simultaneous electrophysiology and two-photon imaging are used to estimate the correlation between G-CaMP, a popular genetically expressible calcium sensor, and electrical activity in PNs. The sensor is found to have poor temporal resolution and to miss significant spiking activity. More generally, this combination of electrophysiology and imaging enables explorations of functional connectivity and calibrated imaging of ensemble activity in the fruit fly.</p

The unfolded protein response couples neuronal identity to circuit formation in the developing mouse olfactory system

Complex genetic mechanisms both endow developing neuronal subtypes with distinct molecular identities and translate those identities into the signatures of cell surface axon guidance molecules that direct neural circuit assembly. The final steps of this process, where axon guidance molecules instruct circuit outcomes, are well-understood. However, the upstream identity molecules that define guidance molecule signatures, and the molecular mechanisms by which cell type identity is transformed into these signatures, remain enigmatic. The murine olfactory system contains nearly 1,5000 olfactory sensory neuron (OSN) subtypes which are intermixed in the olfactory epithelium (OE). Each OSN subtype expresses a unique olfactory receptor (OR) protein which both tunes its response properties to odorants in the environment and acts as an identity molecule that ensures all axons of a given OSN type converge to a single set of target glomeruli in the olfactory bulb (OB). Using a combination of bioinformatic and mouse genetic approaches, we have discovered an unanticipated role for endoplasmic reticulum stress (ER stress) and the unfolded protein response (UPR) in the translation of OR identity to OSN axon guidance molecule expression and glomerular targeting. We find that slight differences in OR amino acid sequences lead to differential activation of the ER stress sensor PERK in different OSN subtypes. Graded patterns of the UPR are then interpreted through a master regulator transcription factor, Ddit3, which controls a set of stress-responsive axon guidance molecules that orchestrate the process of glomerular segregation in the OB. Our results define a novel paradigm for axon guidance in which graded activation of a canonical stress response pathway is leveraged towards the conversion of discrete neuronal identities into discrete circuit formation outcomes. These findings may be widely relevant for the formation of neural circuits across a variety of systems

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

Encoding of Odorants by Olfactory Sensory Neurons

The olfactory system relies on a combinatorial code where a given odorant receptor (OR) detects multiple odorants, and a given odorant is detected by multiple ORs (Malnic, Hirono et al. 1999). Prior attempts to decipher the code have emphasized linking genetic sequence to functional profile, but this approach has led to deorphanization of only ~85 out of ~1200 ORs in mouse (Zhang and Firestein 2007). With such a narrow window onto the combinatorial code, even the deorphaned ORs effectively remain stranded. High throughput calcium imaging of olfactory sensory neurons (OSNs) can provide the missing context. With this method, it is possible to survey the population response patterns while still preserving information on the individual receptive fields that contribute to the ensemble. I have used this technique to gain a more comprehensive view of the combinatorial code. Octanal is an odorant capable of recruiting many OSNs, but how functionally diverse are they? Screening with a panel of odorants made the subdivisions among this large suite of OSNs clear, revealing that nearly half uniquely parse the test panel. Expanding upon this, I show that such rare response patterns can be used like a fingerprint to assess, via physiology, that an OSN expresses a given OR. Population level analysis of the combinatorial code led me to two driving concepts. One is that the OR repertoire, despite its diversity, is nevertheless markedly constrained in its ability to discriminate certain series of odorants. For example, an OSN cannot respond to an alcohol and acid without also responding to an aldehyde. Exploring potential mechanisms, I used designer aldehydes that were trapped in an intermediate polar anchor state. I found that a previously discounted binding mode correlated with the ability of OSNs to selectively respond to aldehydes while excluding alcohols. The other key finding is that odorants can often adopt high energy conformations when activating OSNs. Initially, this was noted for aromatic odorants during a general screen. To probe the phenomenon in greater detail, I used a series of cyclized compounds that mimic rarely assumed states of the flexible tail of octanal. Comparing the activation strength of each analog to that elicited by unconstrained octanal demonstrated extensive co-recognition. This suggests that the flexibility of octanal contributes to its promiscuity in terms of recruiting a high number of OSNs. This study led to the realization that rings could often be treated as merely preserving a particular trajectory of a hydrocarbon backbone. Guided by this concept, I developed new panels with odorants that previously would have been considered discrepant. Hedione is an odorant where a ring imparts specialized geometry that greatly impacts perception. Yet at the OR combinatorial code level, I found that the ring was not critical and flexible but related odorants were still effective. I also demonstrated that OSNs readily accept odorants where an aromatic ring has been substituted with specific alkyl fragments. Thus, aromatic rings too, despite their unique electronics, are sometimes better viewed from a strictly architectural perspective. Using population analysis to identify what the ORs deem the important features of odorants can clarify the trends that sculpt the combinatorial code. This knowledge can help us consolidate seemingly broad receptive fields to better understand what information the OR repertoire extracts from the external chemical environment

Impact of morphometry and ecology on olfactory sensitivity

Thesis (Ph. D.)--Joint Program in Oceanography/Applied Ocean Science and Engineering (Massachusetts Institute of Technology, Dept. of Biology; and, the Woods Hole Oceanographic Institution), 2005.Handwritten on CDROMS: v. [1]. Appendix, histological images -- v. [2]. CT images. -- Table of contents refers to CDROMS as: Appendix and CT and histological images for all species, attached CD)Includes bibliographical references (v. 2, leaves 216-247).In this thesis, the relationships of olfactory sensitivity to three biological variables were tested. The sensitivity of a marine mammal, the sea otter (Enhydra lutris) was measured in order to determine whether a marine lifestyle results in impaired olfaction. The effect of dietary relevance on sensitivity to specific odorants was evaluated. Finally, a new morphometric model of olfactory uptake efficiency was developed and tested against behavioral measurements of olfactory sensitivity in twelve mammalian species from five orders. Olfactory thresholds were obtained for the first time from two sea otters for seven odorant compounds from various natural sources. Otters were trained using operant conditioning to participate in direct behavioral testing. Sea otter olfactory sensitivity was comparable to that of previously studied terrestrial mammals. The incidence of an odorant in the diet of the olfactor was found to influence specific sensitivity to that compound but to varying degrees among different mammalian orders. Nasal cavity specimens were measured using radiologic (CT scan) and histologic (light microscopy) techniques. Surface areas and volumes of the nasal cavity were used to calculate the Olfactory Uptake Efficiency (OUE). OUE is significantly related to olfactory bulb volume. A possible relationship was found between OUE and general olfactory sensitivity.by Jennifer Hammock.Ph.D

The teleost taar family of olfactory receptors: From rapidly evolving receptor genes to ligand-induced behavior

Trace amine-associated receptors (TAARs) have recently been shown to function as olfactory receptors in mammals. In this current study, the taar gene family has been delineated in jawless, cartilaginous, and bony fish (zero, 2, and >100 genes, respectively). I conclude that the taar genes are evolutionary much younger than the related OR and ORA/V1R olfactory receptor families, which are present already in lamprey, a jawless vertebrate. The 2 cartilaginous fish genes appear to be ancestral for 2 taar classes, each with mammalian and bony fish (teleost) representatives. Unexpectedly, a whole new clade, class III, of taar genes originated even later, within the teleost lineage. Taar genes from all 3 classes are expressed in subsets of zebrafish olfactory receptor neurons, supporting their function as olfactory receptors. The highly conserved TAAR1 (shark,mammalian, and teleost orthologs) is not expressed in the olfactory epithelium and may constitute the sole remnant of a primordial, non olfactory function of this family. Class III comprises three-fourths of all teleost taar genes and is characterized by the complete loss of the aminergic ligand-binding motif, stringently conserved in all 25 genes of the other 2 classes. Two independent intron gains in class III taar genes represent extraordinary evolutionary dynamics, considering the virtual absence of intron gains during vertebrate evolution. The dN/dS analysis suggests both minimal global negative selection and an unparalleled degree of local positive selection as another hallmark of class III genes. The accelerated evolution of class III teleost taar genes conceivably might mark the birth of another olfactory receptor gene family. Ligands have only been identified for a handful of olfactory receptors of mammals and insects, while only a single teleost olfactory receptor have been deorphanized, a member of the OlfC family, OlfCa. Zebrafish TAAR olfactory receptors of classI are good candidates for having amines as possible ligands, due to the presence of the aminergic ligand binding motifs. This study identifies diamines as specific ligands for a taar receptor, DrTAAR13c. These diamines activate a sparse subset of olfactory sensory neurons, as indicated by c-Fos expression in olfactory epithelium. Diamines, putrescine and cadaverine, are foul-smelling aliphatic polycations that occur naturally as a result of bacterial decarboxylation of amino acids lysine and arginine, respectively. The 15 concentration of diamines in their environment is correlated to the degree of putrefication. In the behavioral assay, zebrafish exposed to even low concentration of diamines show dramatic, quantifiable aversion, while it shows attraction towards food stimulus and no response for water. The ligand spectrum of TAAR13c closely parallels the behavioral effectiveness of these diamines. This data is consistent with the existence of a defined neuronal microcircuit that elicits a characteristic behavior upon activation of a single olfactory receptor, a novum in the vertebrate sense of smell