During social interactions an individual’s behavior is largely governed by the subset of signals emitted by others. Discrimination of ‘self’ from ‘other’ regulates the territorial urine countermarking behavior of mice. To identify the cues for this social discrimination and understand how they are interpreted, we designed an olfactory-dependent countermarking assay. We find Major Urinary Proteins (MUPs) sufficient to elicit countermarking, and unlike other vomeronasal ligands that are detected by specifically tuned sensory neurons, MUPs are detected by a combinatorial strategy. A chemosensory signature of ‘self’ that modulates behavior is developed via experience through exposure to a repertoire of MUPs. In contrast, aggression can be elicited by MUPs in an experience-independent but context dependent manner. These findings reveal that individual-emitted chemical cues can be interpreted based on their combinatorial permutation and relative ratios, and they can transmit both fixed and learned information to promote multiple behaviors.

SUMMARY Females may display dramatically different behavior depending on their state of ovulation. This is thought to occur through sex-specific hormones acting on behavioral centers in the brain. Whether incoming sensory activity also differs across the ovulation cycle to alter behavior has not been investigated. Here, we show that female mouse vomeronasal sensory neurons (VSNs) are temporarily and specifically rendered “blind” to a subset of male-emitted pheromone ligands during diestrus yet fully detect and respond to the same ligands during estrus. VSN silencing occurs through the action of the female sex-steroid progesterone. Not all VSNs are targeted for silencing; those detecting cat ligands remain continuously active irrespective of the estrous state. We identify the signaling components that account for the capacity of progesterone to target specific subsets of male-pheromone responsive neurons for inactivation. These findings indicate that internal physiology can selectively and directly modulate sensory input to produce state-specific behavior.

A variety of social behaviors like intermale aggression, fear, and mating rituals are important for sustenance of a species. In mice, these behaviors have been implicated to be mediated by peptide pheromones that are sensed by a class of G protein-coupled receptors, vomeronasal receptor type 2 (V2Rs), expressed in the pheromone detecting vomeronasal organ. Matching V2Rs with their cognate ligands is required to learn what receptors the biologically relevant pheromones are acting on. However, this feat has been greatly limited by the unavailability of appropriate heterologous tools commonly used to study ligand receptor specificity, because this family of receptors fails to traffic to the surface of heterologous cells. Here we show that calreticulin, a housekeeping chaperone commonly expressed in most eukaryotic cells, is sparsely expressed in the vomeronasal sensory neurons (VSNs). Correspondingly, knockdown of calreticulin in commonly available cell lines enables V2Rs to efficiently target to the cell membrane. Using this knowledge, we have now been able to successfully surface express receptors and functionally identify cognate ligands. Additionally, calreticulin4, a homolog of calreticulin shows restricted and enriched expression in the VSNs. Interestingly, in heterologous cells, calreticulin4 does not inhibit surface expression of V2Rs and can in part carry out functions of calreticulin. On the basis of our data, we postulate that V2Rs may use a unique trafficking mechanism whereby an important and more commonly expressed chaperone is deleterious for membrane export and is replaced by a functionally equivalent homolog that does not inhibit export while carrying out its functions.

Circadian rhythms synchronize physiological processes with the light-dark cycle and are regulated by a hierarchical system initiated in the suprachiasmatic nucleus, a hypothalamic region that receives direct photic input. The suprachiasmatic nucleus then entrains additional oscillators in the periphery. Circadian rhythms are maintained by a molecular transcriptional feedback loop, of which brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein 1 (BMAL1) is a key member. Disruption of circadian rhythms by deletion of the BMAL1 gene (Bmal1 knockout [KO]) induces a variety of disease states, including infertility in males, due to unidentified mechanisms. We find that, despite normal sperm function, Bmal1 KO males fail to mate with receptive females, indicating a behavioral defect. Mating is dependent on pheromone detection, as are several other behaviors. We determined that Bmal1 KO males also fail to display aggression and avoidance of predator scent, despite intact main olfactory function. Moreover, the vomeronasal organ, a specialized pheromone-responsive organ, was also functionally intact, as determined by calcium imaging in response to urine pheromone stimulus. However, neural circuit tracing using c-FOS activation revealed that, although Bmal1 KO males displayed appropriate activation in the olfactory bulb and accessory olfactory bulb, the bed nucleus of the stria terminalis and the medial preoptic area (areas responsible for integration of copulatory behaviors) failed to activate highly in response to the female scent. This indicates that neural signaling in select behavioral centers is impaired in the absence of BMAL1, likely underlying Bmal1 KO male copulatory defects, demonstrating the importance of the BMAL1 protein in the maintenance of neural circuits that drive pheromone-mediated mating behaviors.

In order to perform their designated functions, proteins require precise subcellular localizations. For cell-surface proteins, such as receptors and channels, they are able to transduce signals only when properly targeted to the cell membrane. Calreticulin is a multi-functional chaperone protein involved in protein folding, maturation, and trafficking. However, evidence has been accumulating that calreticulin can also negatively regulate the surface expression of certain receptors and channels. In these instances, depletion of calreticulin enhances cell-surface expression and function. In this review, we discuss the role of calreticulin with a focus on its negative effects on the expression of cell-surface proteins.

Although mammalian odorant receptors (ORs) were identified over 15 years ago, we still do not understand how odorant molecules interact with ORs at a molecular level. Previous studies of mammalian ORs have tested small numbers of ORs against large numbers of odorants. Some fundamental properties of the olfactory system, however, require investigation of a wide panel of diverse ORs with a large number of chemically diverse odorants. Previously, we identified OR accessory proteins, RTP1 and RTP2. They are expressed specifically in olfactory neurons, are associated with OR proteins and facilitate the OR trafficking to the plasma membrane when coexpressed in mammalian cell lines. Using this approach, high-throughput screening using a large repertoire of mammalian ORs is now possible. The activation profiles can be used to develop a predictive model relating physicochemical odorant properties, receptor sequences, and their interactions, enabling us to predict a tested receptor's response to a novel odorant and a novel receptor's response to a tested odorant. This will provide a basis for understanding how structurally diverse odorant molecules activate the mammalian OR repertoire. Similarly, two families of vomeronasal receptors, V1Rs and V2Rs, are also notoriously difficult to functionally express in heterologous cells. However, coexpression of the RTP family members with V1Rs or V2Rs does not seem to facilitate trafficking of the receptor proteins. This suggests that the vomeronasal organ has a unique biosynthetic pathway for membrane proteins.Since mammalian odorant receptors (ORs) were identified over 15 years ago, many chemosensory (olfactory and taste) receptors have been identified. These include ORs and TAARs expressed by the olfactory sensory neurons in the olfactory epithelium, V1Rs and V2R expressed in the vomeronasal organ, T1Rs and T2Rs expressed in the taste buds [1][2][3][4][5][6][7][8][9]. These are seven transmembrane, G-protein coupled receptors. Insects use a completely different set of chemoreceptors, the Ors and the Grs, to detect chemicals [10][11][12][13][14]. These proteins likely function primarily as ligand-gated channels [15,16].The vast majority of chemosensory receptors must form a complex with a partner protein to functionally respond to stimuli. Taste receptors T1R1, T1R2 and T1R3 show little response to taste stimuli when expressed alone in a heterologous system, however coexpression of T1R2 and T1R3 results in the formation of a sweet taste receptor and coexpression of T1R1 and T1R3 results in the formation of an umami (l-amino acid) taste receptor [17][18][19]. Similarly, candidate sour taste receptors PKD2L1 and PKD1L3 need to be coexpressed in the same cells to be efficiently trafficked to the cell surface in HEK293T cells [20]. In the case of V2Rs, a nonclassical MHC class 1b molecules and ß2-microglobulin may form a complex with some V2Rs [21]. Finally, in insect olfaction, all Ors require Or83b as a partner for proper trafficking and function [22].Following this pattern,...

Innate social behaviors like intermale aggression, fear, and mating rituals are important for survival and propagation of a species. In mice, these behaviors have been implicated to be mediated by peptide pheromones that are sensed by a class of G protein-coupled receptors, vomeronasal receptor type 2 (V2Rs), expressed in the pheromone-detecting vomeronasal organ (VNO) (Chamero et al., Nature 450:899–902, 2007; Haga et al., Nature 466:118–122, 2010; Kimoto et al., Curr Biol 17:1879–1884, 2007; Leinders-Zufall et al., Nat Neurosci 12:1551– 1558, 2009; Papes et al., Cell 141:692–703, 2010) Matching V2Rs with their cognate ligands is required to understand what receptors the biologically relevant pheromones are acting on. However, this goal has been greatly limited by the unavailability of appropriate heterologous tools commonly used to carry out receptor deorphanization, due to the fact that this family of receptors fails to traffic to the surface of heterologous cells. We have demonstrated that calreticulin, a housekeeping chaperone commonly expressed in most eukaryotic cells, is sparsely expressed in the vomeronasal sensory neurons (VSNs). Stable knock down of calreticulin in a HEK293T derived cell line (R24 cells) allows us to functionally express V2Rs on the surface of heterologous cells. In this chapter we describe protocols for maintenance and expansion of the R24 cell line and functional assays for V2Rs using these cells.

The vivid world of odors is recognized by the sense of olfaction. Olfaction in mice is mediated by a repertoire of about 1200 G Protein Coupled Receptors (GPCRs) 1 that are postulated to bind volatile odorant molecules and converting the extracellular signal into an intracellular signal by coupling with G protein Gαolf. Binding of the odorants to the receptors is thought to follow a combinatorial rule, that is, one odorant may bind several receptors and one receptor may bind several odorants to varying degrees 2 . Biochemical, signaling and ligand binding studies have been conveniently carried out for most GPCRs using heterologous cells. However use of heterologous cells for study of odorant receptors, was precluded for a long time since on transfection they failed to export to the surface. Saito et al have demonstrated single membrane pass Receptor Transporting Protein (RTP) family chaperones show enhanced expression in the olfactory sensory neurons and act as chaperones to traffic odorant receptors to the surface in heterologous cells, when co transfected together 3 . To carry out biochemical assays for receptors using heterologous cells, one must first determine if the receptor shows robust surface expression in the cell line. This can be assayed by overexpressing the receptors with the chaperone RTP1S followed by live cell staining to fluorescently label the extracellular domain or a tag in the extracellular domain exclusively. Here we demonstrate a protocol to carry out live cell staining that can be used to detect odorant receptors on the surface of HEK293T cells conveniently. In addition, it may also be used to assay for surface expression of other chemosensory receptors or GPCRs. Protocol Procedure:The procedure comprises of three parts completed over a total time of 3 days: transferring cells, transfection and immunocytochemistry, one step carried out per day. Transfer and transfection of cells over the first two days must be carried out in sterile conditions in a laminar flow chamber. 2. Grow cells to a desired confluency in a 100mm culture plate, depending on the number of plates required to set up. It is important to determine the fraction of cells to be transferred to avoid overgrown or sparse cells: for instance, from a 100% confluent 100 mm dish, one may transfer about 3% to a 35 mm dish to obtain approximately 30% confluency in the latter. 3. Prior to transferring cells, in the sterile laminar flow chamber, coat 22 X 22 mm glass cover slips with sterile poly D lysine (1mg/ mL) and place one in each 35 mm cell culture dish, coated side up for plating cells (Figure 1). Allow the solution to dry for 5-10 minutes. During this interval, the UV source in the laminar chamber may be turned on to sterilize the cover slips, if desired. Poly D lysine helps heterologous cells stick to the cover slips. 4. To transfer cells, aspirate out the existing media in the 100mm dish, wash by carefully adding 8 mL sterile PBS, aspirate PBS and trypsinize the cells using 3 mL 0.05% Trypsin-EDTA; when cells detach...