1 trillion odors, 1,000 genes, 1 nose: The scent-sational mysteries of smell

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1 1 trillion odors, 1,000 genes, 1 nose: The scent-sational mysteries of smell Kayla Huber Department of Biology Lake Forest College Lake Forest, Illinois Abstract The mammalian olfactory system can recognize an enormous number of odors (with current estimates at one trillion). My lab has sought to ask how the diversity and specificity of olfactory perception is accomplished. We discovered a large family of genes that produce odorant receptors (ORs) selectively in the olfactory epithelium (OE). Each cell selects a type of receptor to express based on where it is located in the OE, and all cells expressing the same receptor send their information to the same part of the olfactory bulb. We found that odorant identities are encoded by the unique combination of ORs that are activated. My lab has also pioneered work on pheromone detection, which occurs primarily in the vomeronasal organ (VNO) and involves three families of chemosensory receptors. Utilizing a genetic transneuronal tracer that we developed, we found that VNO neurons activate hormone-secreting neurons in the hypothalamus, which can affect sexual behaviors. Our work has furthered the understanding of how odorants and pheromones are detected, coded, and affect physiology and behavior. We now seek to elucidate how the OR repertoire can be adapted to the environment and how odorants are deconstructed and then reconstructed in the brain in order to create perceptions. Introduction Chemodetection the ability to detect chemicals related to smell or taste is the most ancient sense, and it is essential for survival, reproductive behaviors, memory, and interspecies communication (1). Every organism possesses a way to detect the chemical composition of its surroundings. In mammals, the sense of smell is mediated by the olfactory system. In this, odorous ligands from the environment enter the nose and come into contact with a specialized olfactory neuroepithelium (OE). This epithelium is primarily composed of three different types of cells: olfactory sensory neurons, supporting cells, and basal cells (which are stem cells that constantly produce new olfactory neurons throughout an organism s life) (2). Olfactory sensory neurons are unique in that they are bipolar; their dendrites travel through the mucus membrane and produce cilia, which are large, hair-like structures that are capable of interacting with incoming odorant molecules (3). At the other end of the bipolar neuron is an axon that projects into the glomerular layer of the olfactory bulb (OB). The ends of axons cluster together in spherical structures called glomeruli, which project into various regions of the brain for higher-level processing of sensory information (Figure 1). The mammalian olfactory system is also capable of detecting pheromones, which are chemicals that transmit messages between members of the same species and are implicated in aggression, copulation, and reproduction. Microvilli of sensory neurons in the vomeronasal organ (VNO) detect these chemicals and send their axons to the accessory olfactory bulb (AOB), which go on to project into distinct regions of the brain (4). The olfactory system is remarkable in its discriminatory power and can perceive a multitude of chemicals (even ones with very similar structures) as distinct odors. My lab has sought to elucidate how the diversity and specificity of olfactory perception is accomplished. We also wish to understand how chemicals in the environment are translated into particular behaviors that are advantageous for members of a species. The Search for Odorant Receptor Genes Our initial experiments sought to isolate the genes responsible for producing odorant receptors (ORs) in the OE. Previous research had determined that the binding of odorants to olfactory sensory neurons (OSNs) produced GTP-dependent increases in adenylyl cyclase in the cilia (5), which indicated that G protein-coupled receptors (GPCRs) might be involved in the first stages of olfactory signal transduction. Therefore, we conducted our search for OR genes in the rat OE while operating under three main assumptions: 1) ORs must be expressed exclusively in the OE, 2) due to the fact that odorants vary in structure, OR genes must be varied, but still belong to the same multigene family, and 3) ORs must be related to other types of GPCRs. In our first experiment, we attempted to identify receptors in the rat OE that resembled GPCRs that had been sequenced previously (6). We crafted degenerate oligonucleotide primers that would attach to the amino acid sequences of transmembrane domains 2 and 7 of previously identified GPCRs. We then exposed these primers to rat OE cdna, ran a series of polymerase chain reactions (PCR) to amplify the sequences of interest, and obtained 64 products. In order to determine if any of our products contained members of a multigene family, we mixed a restriction enzyme in with our PCR products (which scans DNA and cuts the mol- Figure 1. Information relay in the olfactory system. When an odorous ligand from the environment binds to particular odorant receptors, intracellular cascades lead to the activation of olfactory sensory neurons. These neurons eventually converge onto certain glomeruli in the olfactory bulb and then project into a wide array of brain areas.

2 ecule at particular places). A majority of our products were cut into only a few pieces. However, one product was cut into many pieces, hinting that it might be composed of several members of a multigene family. After cloning and sequencing this particular product, we determined that each piece encoded its own unique protein (all of which resembled GPCRs). Using this product as probes allowed us to find even more proteins, expressed exclusively in the OE, that resembled typical GPCRs. Interestingly, all of the proteins were related, but varied extensively in particular portions of their amino acid sequences. This satisfied our second assumption and suggested that each OR in this family would be able to interact with different odorant molecules. Replication of this experiment with the genome of the channel catfish revealed a smaller family of OR genes than in the rat (7). In addition, we were able to determine through RNA in situ hybridizations that a given OR is expressed in only 0.5%-2.0% of olfactory neurons. This indicated each cell in the OE possesses a unique identity based on which receptor it expresses. When more complete genome sequences of particular organisms were released by the National Center for Biotechnology Information, we once again replicated our experiments in order to determine the true size of the OR gene family and its distribution upon the chromosomes. In humans, we were able to identify 339 OR genes that were capable of encoding functional proteins, which could be divided into 172 subfamilies (meaning they possess more related sequences, and presumably discriminate amongst odorants with similar structures) (8). Interestingly, these OR genes are distributed among 51 different loci on 21 chromosomes, and most subfamilies are encoded by a single chromosomal locus. We found far more OR genes and subfamilies in the mouse (913 and 241, respectively) (9, 10), which suggests that mice may be able to discriminate a larger number of odors than humans. and the dendrites of OB neurons. Due to the fact that there are approximately 2,000 glomeruli in the mouse OB and upwards of 4 million OSNs in the nasal cavity, we predicted that glomeruli could serve as processing units of olfactory information, and odors might be mapped onto particular glomeruli. Utilizing various OR probes, we performed in situ hybridization on sections of mice OBs. We determined that OSNs that express the same receptor project their axons to the same glomeruli. Conversely, OSNs that express different receptors project their axons to different glomeruli, even if two receptor genes are expressed within the same spatial zone of the OE (12). This indicates that olfactory information that is quite distributed in the nose becomes highly organized once it reaches its next destination the olfactory bulb. Due to the intimate relationship that exists between the OE and the OB, we wondered if epithelial and bulbar maps evolve independently or if they are linked, for example, by the retrograde influences of the bulb on the epithelium. We proceeded to analyze the onset of OR gene expression in developing mouse embryos using in situ hybridization. OR gene expression began at embryonic day 11.5 and increased dramatically between embryonic day 12.5 and In addition, spatial zones of OR gene expression were observed as early as day 13 (13). Previous research has indicated that the first synapses between OSN axons and the dendrites of OB neurons are formed at embryonic day 14 (14, 15). This provided evidence against the hypothesis that signals from the OB induce OR gene expression in the OE. To further disprove this hypothesis, we examined OR gene expression in extra-toesj mutant mice that lack OBs. We found that such mutant mice had identical levels of OR gene expression, and all ORs were expressed in a zonally appropriate manner. Therefore, the expression and organization of receptors appears to be an intrinsic property of OSNs. Figure 2. Structurally related odorants activate different combinations of OSNs. Seven ligands belonging to the n-aliphatic group interact with various types of olfactory sensory neurons, producing radically different odorant perceptions in humans (displayed on left). Organization of Olfactory Sensory Information Within the OE and OB Given the large number of genes involved in the production of ORs and the sheer number of odorants that exist in the environment, our lab asked the question: do any organizational strategies underlie the discriminatory capacity of the olfactory system? In order to answer this question, we isolated and cloned select members of mouse OR gene subfamilies. We then performed in situ hybridization on sections of mice nasal cavities using labeled RNA probes created from our clones. We found that the expression of each subfamily is limited to one of four zones in the OE and such zones display bilateral symmetry in the two nasal cavities (11). However, particular receptors are expressed fairly randomly throughout their designated zone. Such a result suggested an initial organization of olfactory sensory information, whereby OSNs that express the same or related receptors are grouped together. Such groups may recognize identical or highly similar odorants. We next asked whether olfactory information remains fairly distributed in the OB, as it is within particular zones of the OE, or if it becomes more organized, whereby identical receptors project to the same portion of the bulb. We turned our attention to glomeruli, which are spheres located in the OB and the site of synapse formation between the axons of OSNs Cracking the Odor Receptor Code The next piece of the olfaction puzzle was to determine how a limitless number of odorous ligands can interact with a limited number of odorant receptors and eventually be perceived as unique scents. Previous research had demonstrated that single ORs can be activated by multiple odorants (16), though the ligand specificity of only two ORs had been identified at the time (17, 18). In order to identify ORs that recognize particular odorants, we exposed individual mouse OSNs to various aliphatic odorants and monitored their activity using calcium imaging. We then determined the OR genes expressed by each OSN using single-cell real-time PCR. In line with previous studies, we found that a single OR can recognize multiple odorants, and a single odorant can be recognized by multiple ORs. However, individual odorants appear to be recognized by the unique combination of ORs that they activate (19). We also found that the slightest of changes in an odorant s structure or concentration was capable of altering the combination of receptors that recognize the odorant (Figure 2). Oddly enough, particular ligands can be perceived as different odors at different concentrations. For example, thioterpineol is often perceived as tropical fruit at low concentrations and stench at very high concentrations. Therefore, the receptor code for odorants appears to be quite mal-

3 leable. To gain a more comprehensive understanding of odor coding, we exposed 3,000 mouse OSNs to 125 odorants with diverse structures and perceived odors (20). We discovered that the OSN repertoire is biased, with particular odorant mixtures (particularly aldehydes) activating far more OSNs than others. However, a vast majority of OSNs are narrowly tuned, with 44.7% of the neurons responding to only one mixture of structurally related odorants. Of those neurons that responded to only one mixture, 92.1% of them were selectively activated by 1-3 odorants. Coupled with this, a small proportion of OSNs appeared to be broadly tuned and responded to a large number of mixtures. The considerable scale of this second experiment allowed us to definitively answer the question of how sizeable the code for various odorants is. It appears as though the number of ORs activated by a single odorant can vary extensively, from 2-33 receptors in aldehydes to 1-6 in cyclic alkanes. Therefore, there is extreme diversity in the receptors used to encode particular ligands, even among those that are highly related in structure. The Search for Pheromone Receptor Genes Our lab has also sought to illuminate the process by which pheromones are detected in the accessory olfactory organ, termed the vomeronasal organ (VNO). VNO neurons (VNs) appear to be distinct from OSNs, as they lack particular olfactory signal transduction molecules, including the G protein α subunit Gαolf (21). However, certain subsets of VNs have been shown to express high levels of two other G protein α subunits, Gαo and Gαi2 (22). Taking up the baton from Dulac and Axel, who identified a family of genes that encode for pheromone receptors expressed only on the Gαi2 subset (23), we sought to identify the unique family of genes that encode for pheromone receptors expressed on the Gαo subset (VRs). Using the PCR-based differential screening approach, we attempted to identify genes that were expressed in one VR but not another. We eventually identified a novel family of proteins that belong to the GPCR superfamily (24). The family possessed very unusual features, such as an incredibly long and variable N-terminal extracellular domain, which is presumably the site of ligand binding. Twelve years after our identification of such a family, we returned to the subject of chemodetection in the VNO. While it had been firmly established that pheromones interact with two families of chemoreceptors, V1Rs (which are coexpressed with Gαi2 in the apical zone) and V2Rs (which are coexpressed with Gαo in the basal zone), we wished to search for additional chemoreceptor families that might be present in the VNO. We conducted a high throughput screen for GPCRs expressed in mouse VNO neurons, utilizing primers for 365 GPCRs never before implicated in olfaction or taste. We identified several members of the formyl peptide receptor (FPR) family selectively expressed in subsets of VNO neurons (25). Such receptors are typically involved in the detection of formylated peptides that are released from bacteria or mitochondria at the site of infection or tissue damage (26). We hypothesize that such receptors confer a selective advantage upon organisms, as it may enable the detection of decay in a food source or bacterial infection in a member of the same species. It is also plausible that formylated peptides found in feces may enable individuals to detect the presence of members of their own species or potential predators. Bringing the OE into Pheromone Detection & the VNO into Odorant Detection While it is widely assumed that the OE strictly interacts with odorants and the VNO strictly interacts with pheromones, our lab has stumbled across some data throughout the years that paints a starkly different picture. When it came to our attention that a small number of functional OSNs lack Gαolf (27), and peptides that ordinarily interact with major histocompatibility complex proteins are capable of stimulating OSNs (28), we began to postulate about the existence of a unique family of peptide receptors in the OE. We proceeded to conduct a high throughput screen for GPCRs expressed in mouse OSNs, utilizing primers for GPCRs never before implicated in olfaction or taste. Subsequent in situ hybridizations confirmed the existence of two GPCR genes Taar7d and Taar9 that are expressed in a small portion of OSNs. Such genes encode for members of the trace amine-associated receptor (TAAR) family, and were found to respond to compounds derived from urine (a major social cue in rodents) (29). We have also identified TAARs in the OE of macaque monkeys. Such receptors appear to activate when exposed to an amine that smells like rotten fish, implicating the receptors in producing aversive signals that discourage the consumption of spoiled food (30). This preliminary data has led us to believe that pheromone detection is not a function served solely by the VNO. Our next question was whether odorants could be detected in the VNO. We exposed single mouse VNO neurons to various odorant mixtures and monitored their response with calcium imaging. We found that VNO neurons were activated by various odorants, and were also capable of distinguishing between structurally related ligands. Even more surprising, VNO neurons responded to odorants at much lower concentrations than OSNs (31). While the exact function of odorant detection in the VNO still remains elusive, we hypothesize that particular odors in the environment may signal to mice the presence of predators or the acceptability of a site for feeding or nesting. The OB, VNO, and Beyond In order to determine the fate of axons beyond the OB and VNO, it was necessary to develop a tool that would allow us to chart neural circuits. Realizing that current neuronal tracers were incredibly limited, we set out to create a tracer that would be produced by the neurons of interest themselves. In our transgenic mice, the expression of a barley lectin (BL) cdna was controlled by the promoter of the rat OMP gene (a protein that is expressed almost exclusively by neurons in the OE and VNO). Staining of tissue samples with an antibody that recognizes BL revealed that BL was present not only in the OB and VNO, but also in the main olfactory bulb, accessory olfactory bulb, and brain regions connected to the bulbs, indicating that it was successful in tracing connections in the olfactory system and beyond (32). Employing this tracing method, we investigated the neurons that synapse with gonadotropin-releasing hormone (GnRH) neurons, a small collection of cells in the hypothalamus that mediate endocrine responses to pheromones (33). We were particularly interested in how GnRH neurons produce reproductive physiology and behavior. We were shocked to find that GnRH neurons received pheromone signals from not only pheromone relays, but also odor relays. In addition, approximately 800 GnRH neurons went on to connect with about 50,000 neurons in 53 brain areas, many of which have been implicated in sexual behaviors, including the MEA and BSTMPM (pheromone processing centers) and portions of the hypothalamus involved in female sexual posturing, mounting, and ejaculation latency (34). These results indicate that GnRH neurons act as a processing center, receiving inputs from several sources and sending axons to a multitude of brain regions, many of which are integral in reproductive behavior. Filling in the Gaps When we entered the field in 1991, only rudimentary facts about the anatomy of the olfactory system were characterized. Twenty-four years later, we have made leaps and bounds in our understanding of olfaction. We now know that odorous ligands, or even peptides, from the environment enter the nasal cavity and activate a unique combination of odorant receptors located on cilia. Such receptors are members of the G protein-coupled receptor superfamily, though the sequence of each odorant receptor possesses considerable variability, which enables them to interact with a wide array of odorants. The expression of these unique odorant receptors is restricted to particular zones of the olfactory epithelium. Olfactory sensory information undergoes an additional level of organization once it enters the main olfactory bulb, as all neurons expressing the same odorant receptor project to the same glomeruli (which are spherical structures located within the bulb). In a parallel process, pheromones (and occasionally odorants or peptides) enter the vomeronasal organ and attach to specialized pheromone receptors located on microvilli. Such receptors are also members of the G protein-coupled receptor superfamily, though they are unique in that they possess an extensive N-terminal extracellular domain (which provides ample surface for ligand binding). Neurons of the vomeronasal organ project to the accessory olfactory bulb and then to a large number of brain areas, many of which are implicated in aggression, flight from danger, sexual behaviors, avoidance, reproduction, and territoriality. This knowledge has fueled efforts by my colleagues to fill in the gaps that remain in the field of olfaction. Richard Axel s lab has sought to elucidate the mechanism by which one OR gene from a massive gene family is selected and subsequently expressed in a particular region of the OE. Using chromosome conformation capture, they discovered an H

4 enhancer that is capable of associating with several gene promoters on different chromosomes, but only associating with one OR gene in any given olfactory neuron (35). However, it is still unknown how such an enhancer is capable of consistently producing the zonal organization that is characteristic of the OE throughout an organism s lifetime. Joseph Lewcock and Randall Reed have also added an interesting piece to the gene choice puzzle, indicating that the expression of a functional OR produces a feedback mechanism that inhibits the expression of any other OR alleles within a particular olfactory sensory neuron (36). Following suit with the findings of Randall Reed, Richard Axel s lab developed a gene targeting strategy that allows one to examine the stability of receptor gene choice over the life of an OSN. They found that early on in development, a small proportion of OSNs switched the receptor that they expressed. However, neurons that expressed a mutated, nonfunctional OR switched the receptor that they expressed with much greater frequency (37). The exact molecular feedback signal that functional ORs release in order to cease gene switching remains to be characterized. Haiqing Zhao and Randall Reed have also determined that the survival of particular OSNs and their receptors depends not only upon their functionality, but their level of activity. After creating mutant mice that lacked the OCNC1 subunit in some populations of neurons, which is an integral portion of the olfactory cyclic nucleotide-gated channel that initiates the olfactory signal transduction pathway, they found that OCNC1-deficient neurons were specifically depleted from the OE over time (38). It still remains to be seen whether odorant-evoked activity provides differentiating OSNs with an enhanced capacity (in comparison to their less active peers) to acquire vital neurotropic factors. As we move forward in the field of olfaction research, I wish to focus my efforts on identifying the process by which diverse odorant molecules are deconstructed and subsequently reconstructed in the brain in order to create distinct perceptions. Every odor that we cherish, whether it be wet grass after a rainstorm, a lover s perfume, or a campfire, can reduced to a chemical. This chemical is transformed into the language of the brain electricity, neurotransmitters, ions and eventually a conscious experience. Learning this language from the outside in is one of the most challenging pursuits one can undertake. Acknowledgements I would like to thank Dr. DebBurman for all of his guidance and support throughout this project. I will be forever indebted to my classmates for their wit and all of the joy they have brought into my life. Finally, I am so grateful for every member of my lab, past and present, as well as my colleagues for pushing me towards greatness. 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