Neural basis for pheromone signal transduction in mice.

Pheromones are specialized chemical messengers used for inter-individual communication within the same species, playing crucial roles in modulating behaviors and physiological states. The detection mechanisms of these signals at the peripheral organ and their transduction to the brain have been unclear. However, recent identification of pheromone molecules, their corresponding receptors, and advancements in neuroscientific technology have started to elucidate these processes. In mammals, the detection and interpretation of pheromone signals are primarily attributed to the vomeronasal system, which is a specialized olfactory apparatus predominantly dedicated to decoding socio-chemical cues. In this mini-review, we aim to delineate the vomeronasal signal transduction pathway initiated by specific vomeronasal receptor-ligand interactions in mice. First, we catalog the previously identified pheromone ligands and their corresponding receptor pairs, providing a foundational understanding of the specificity inherent in pheromonal communication. Subsequently, we examine the neural circuits involved in processing each pheromone signal. We focus on the anatomical pathways, the sexually dimorphic and physiological state-dependent aspects of signal transduction, and the neural coding strategies underlying behavioral responses to pheromonal cues. These insights provide further critical questions regarding the development of innate circuit formation and plasticity within these circuits.

Deciphering the chemical language of inbred and wild mouse conspecific scents.

In most mammals, conspecific chemosensory communication relies on semiochemical release within complex bodily secretions and subsequent stimulus detection by the vomeronasal organ (VNO). Urine, a rich source of ethologically relevant chemosignals, conveys detailed information about sex, social hierarchy, health, and reproductive state, which becomes accessible to a conspecific via vomeronasal sampling. So far, however, numerous aspects of social chemosignaling along the vomeronasal pathway remain unclear. Moreover, since virtually all research on vomeronasal physiology is based on secretions derived from inbred laboratory mice, it remains uncertain whether such stimuli provide a true representation of potentially more relevant cues found in the wild. Here, we combine a robust low-noise VNO activity assay with comparative molecular profiling of sex- and strain-specific mouse urine samples from two inbred laboratory strains as well as from wild mice. With comprehensive molecular portraits of these secretions, VNO activity analysis now enables us to (i) assess whether and, if so, how much sex/strain-selective 'raw' chemical information in urine is accessible via vomeronasal sampling; (ii) identify which chemicals exhibit sufficient discriminatory power to signal an animal's sex, strain, or both; (iii) determine the extent to which wild mouse secretions are unique; and (iv) analyze whether vomeronasal response profiles differ between strains. We report both sex- and, in particular, strain-selective VNO representations of chemical information. Within the urinary 'secretome', both volatile compounds and proteins exhibit sufficient discriminative power to provide sex- and strain-specific molecular fingerprints. While total protein amount is substantially enriched in male urine, females secrete a larger variety at overall comparatively low concentrations. Surprisingly, the molecular spectrum of wild mouse urine does not dramatically exceed that of inbred strains. Finally, vomeronasal response profiles differ between C57BL/6 and BALB/c animals, with particularly disparate representations of female semiochemicals.

Phosphodiesterase 5A regulates the vomeronasal pump in mice.

The vomeronasal organ (VNO) is a specialized chemoreceptive structure in many vertebrates that detects chemical stimuli, mostly pheromones, which often elicit innate behaviors such as mating and aggression. Previous studies in rodents have demonstrated that chemical stimuli are actively transported to the VNO via a blood vessel-based pumping mechanism, and this pumping mechanism is necessary for vomeronasal stimulation in behaving animals. However, the molecular mechanisms that regulate the vomeronasal pump remain mostly unknown. In this study, we observed a high level of expression of phosphodiesterase 5A (PDE5A) in the vomeronasal blood vessel of mice. We provided evidence to support the potential role of PDE5A in vomeronasal pump regulation. Local application of PDE5A inhibitors-sildenafil or tadalafil-to the vomeronasal organ (VNO) reduced stimulus delivery into the VNO, decreased the pheromone-induced activity of vomeronasal sensory neurons, and attenuated male-male aggressive behaviors. PDE5A is well known to play a role in regulating blood vessel tone in several organs. Our study advances our understanding of the molecular regulation of the vomeronasal pump.

Type 2 vomeronasal receptor-A4 subfamily: Potential predator sensors in mice.

Sensory signals detected by olfactory sensory organs are critical regulators of animal behavior. An accessory olfactory organ, the vomeronasal organ, detects cues from other animals and plays a pivotal role in intra- and inter-species interactions in mice. However, how ethologically relevant cues control mouse behavior through approximately 350 vomeronasal sensory receptor proteins largely remains elusive. The type 2 vomeronasal receptor-A4 (V2R-A4) subfamily members have been repeatedly detected from vomeronasal sensory neurons responsive to predator cues, suggesting a potential role of this receptor subfamily as a sensor for predators. This review focuses on this intriguing subfamily, delving into its receptor functions and genetic characteristics.

Following the p63/Keratin5 basal cells in the sensory and non-sensory epithelia of the vomeronasal organ.

The vomeronasal organ (VNO) is a part of the accessory olfactory system, which detects pheromones and chemical factors that trigger a spectrum of sexual and social behaviors. The vomeronasal epithelium (VNE) shares several features with the epithelium of the main olfactory epithelium (MOE). However, it is a distinct neuroepithelium populated by chemosensory neurons that differ from the olfactory sensory neurons in cellular structure, receptor expression, and connectivity. The vomeronasal organ of rodents comprises a sensory epithelium (SE) and a thin non-sensory epithelium (NSE) that morphologically resembles the respiratory epithelium. Sox2-positive cells have been previously identified as the stem cell population that gives rise to neuronal progenitors in MOE and VNE. In addition, the MOE also comprises p63 positive horizontal basal cells, a second pool of quiescent stem cells that become active in response to injury. Immunolabeling against the transcription factor p63, Keratin-5 (Krt5), Krt14, NrCAM, and Krt5Cre tracing experiments highlighted the existence of horizontal basal cells distributed along the basal lamina of SE of the VNO. Single cell sequencing and genetic lineage tracing suggest that the vomeronasal horizontal basal cells arise from basal progenitors at the boundary between the SE and NSE proximal to the marginal zones. Moreover, our experiments revealed that the NSE of rodents is, like the respiratory epithelium, a stratified epithelium where the p63/Krt5+ basal progenitor cells self-replicate and give rise to the apical columnar cells facing the lumen of the VNO.

Synchronized Expansion and Contraction of Olfactory, Vomeronasal, and Taste Receptor Gene Families in Hystricomorph Rodents.

Chemical senses, including olfaction, pheromones, and taste, are crucial for the survival of most animals. There has long been a debate about whether different types of senses might influence each other. For instance, primates with a strong sense of vision are thought to have weakened olfactory abilities, although the oversimplified trade-off theory is now being questioned. It is uncertain whether such interactions between different chemical senses occur during evolution. To address this question, we examined four receptor gene families related to olfaction, pheromones, and taste: olfactory receptor (OR), vomeronasal receptor type 1 and type 2 (V1R and V2R), and bitter taste receptor (T2R) genes in Hystricomorpha, which is morphologically and ecologically the most diverse group of rodents. We also sequenced and assembled the genome of the grasscutter, Thryonomys swinderianus. By examining 16 available genome assemblies alongside the grasscutter genome, we identified orthologous gene groups among hystricomorph rodents for these gene families to separate the gene gain and loss events in each phylogenetic branch of the Hystricomorpha evolutionary tree. Our analysis revealed that the expansion or contraction of the four gene families occurred synchronously, indicating that when one chemical sense develops or deteriorates, the others follow suit. The results also showed that V1R/V2R genes underwent the fastest evolution, followed by OR genes, and T2R genes were the most evolutionarily stable. This variation likely reflects the difference in ligands of V1R/V2Rs, ORs, and T2Rs: species-specific pheromones, environment-based scents, and toxic substances common to many animals, respectively.

S100Z is expressed in a lateral subpopulation of olfactory receptor neurons in the main olfactory system of Xenopus laevis.

In contrast to other S100 protein members, the function of S100 calcium-binding protein Z (S100Z) remains largely uncharacterized. It is expressed in the olfactory epithelium of fish, and it is closely associated with the vomeronasal organ (VNO) in mammals. In this study, we analyzed the expression pattern of S100Z in the olfactory system of the anuran amphibian Xenopus laevis. Using immunohistochemistry in whole mount and slice preparations of the larval olfactory system, we found exclusive S100Z expression in a subpopulation of olfactory receptor neurons (ORNs) of the main olfactory epithelium (MOE). S100Z expression was not co-localized with TP63 and cytokeratin type II, ruling out basal cell and supporting cell identity. The distribution of S100Z-expressing ORNs was laterally biased, and their average number was significantly increased in the lateral half of the olfactory epithelium. The axons of S100Z-positive neurons projected exclusively into the lateral and intermediate glomerular clusters of the main olfactory bulb (OB). Even after metamorphic restructuring of the olfactory system, S100Z expression was restricted to a neuronal subpopulation of the MOE, which was then located in the newly formed middle cavity. An axonal projection into the ventro-lateral OB persisted also in postmetamorphic frogs. In summary, S100Z is exclusively associated with the main olfactory system in the amphibian Xenopus and not with the VNO as in mammals, despite the presence of a separate accessory olfactory system in both classes.

Sensory neurobiology: Muscles power pheromone sensation.

While we understand how the five main sensory organs enable and facilitate stimulus detection, little is known about how the vomeronasal organ enables pheromone sensation. A new study finds specialized muscles poised to coordinate stimulus delivery, dynamics, and arousal.

Histological evaluation of the alpaca (Vicugna pacos) vomeronasal organ.

Little is known about the neuronal structure of the vomeronasal organ (VNO), a receptor organ responsible for pheromone perception, in the alpaca (Vicugna pacos). This study was performed to determine the localization of neuronal elements, including protein gene product 9.5 (PGP 9.5), a pan-neuronal marker, olfactory marker protein (OMP), a marker of mature olfactory receptor cells, and phospholipase C beta 2 (PLC-ß2), a marker of solitary chemoreceptor cells (SCCs), in the VNO. OMP was identified in receptor cells of the vomeronasal sensory epithelium (VSE), while PGP 9.5 and PLC-ß2 were localized in both the VSE and vomeronasal non-sensory epithelium. Collectively, these results suggested that the alpaca VNO possesses SCCs and olfactory receptor cells, which recognize both harmful substances and pheromones.

Nescient helix-loop-helix 1 (Nhlh1) is a novel activating transcription factor 5 (ATF5) target gene in olfactory and vomeronasal sensory neurons in mice.

Activating transcription factor 5 (ATF5) is a transcription factor that belongs to the cAMP-response element-binding protein/ATF family and is essential for the differentiation and survival of sensory neurons in mouse olfactory organs. However, transcriptional target genes for ATF5 have yet to be identified. In the present study, chromatin immunoprecipitation-quantitative polymerase chain reaction (ChIP-qPCR) experiments were performed to verify ATF5 target genes in the main olfactory epithelium and vomeronasal organ in the postnatal pups. ChIP-qPCR was conducted using hemagglutinin (HA)-tagged ATF5 knock-in olfactory organs. The results obtained demonstrated that ATF5-HA fusion proteins bound to the CCAAT/enhancer-binding protein-ATF response element (CARE) site in the enhancer region of nescient helix-loop-helix 1 (Nhlh1), a transcription factor expressed in differentiating olfactory and vomeronasal sensory neurons. Nhlh1 mRNA expression was downregulated in ATF5-deficient (ATF5-/-) olfactory organs. The LIM/homeobox protein transcription factor Lhx2 co-localized with ATF5 in the nuclei of olfactory and vomeronasal sensory neurons and bound to the homeodomain site proximal to the CARE site in the Nhlh1 gene. The CARE region of the Nhlh1 gene was enriched by the active enhancer marker, acetyl-histone H3 (Lys27). The present study identified Nhlh1 as a novel target gene for ATF5 in murine olfactory organs. ATF5 may upregulate Nhlh1 expression in concert with Lhx2, thereby promoting the differentiation of olfactory and vomeronasal sensory neurons.

A revised conceptual framework for mouse vomeronasal pumping and stimulus sampling.

The physiological performance of any sensory organ is determined by its anatomy and physical properties. Consequently, complex sensory structures with elaborate features have evolved to optimize stimulus detection. Understanding these structures and their physical nature forms the basis for mechanistic insights into sensory function. Despite its crucial role as a sensor for pheromones and other behaviorally instructive chemical cues, the vomeronasal organ (VNO) remains a poorly characterized mammalian sensory structure. Fundamental principles of its physico-mechanical function, including basic aspects of stimulus sampling, remain poorly explored. Here, we revisit the classical vasomotor pump hypothesis of vomeronasal stimulus uptake. Using advanced anatomical, histological, and physiological methods, we demonstrate that large parts of the lateral mouse VNO are composed of smooth muscle. Vomeronasal smooth muscle tissue comprises two subsets of fibers with distinct topography, structure, excitation-contraction coupling, and, ultimately, contractile properties. Specifically, contractions of a large population of noradrenaline-sensitive cells mediate both transverse and longitudinal lumen expansion, whereas cholinergic stimulation targets an adluminal group of smooth muscle fibers. The latter run parallel to the VNO's rostro-caudal axis and are ideally situated to mediate antagonistic longitudinal constriction of the lumen. This newly discovered arrangement implies a novel mode of function. Single-cell transcriptomics and pharmacological profiling reveal the receptor subtypes involved. Finally, 2D/3D tomography provides non-invasive insight into the intact VNO's anatomy and mechanics, enables measurement of luminal fluid volume, and allows an assessment of relative volume change upon noradrenergic stimulation. Together, we propose a revised conceptual framework for mouse vomeronasal pumping and, thus, stimulus sampling.

Somatic ion channels and action potentials in olfactory receptor cells and vomeronasal receptor cells.

Olfactory receptor cells are primary sensory neurons that catch odor molecules in the olfactory system, and vomeronasal receptor cells catch pheromones in the vomeronasal system. When odor or pheromone molecules bind to receptor proteins expressed on the membrane of the olfactory cilia or vomeronasal microvilli, receptor potentials are generated in their receptor cells. This initial excitation is transmitted to the soma via dendrites, and action potentials are generated in the soma and/or axon and transmitted to the central nervous system. Thus, olfactory and vomeronasal receptor cells play an important role in converting chemical signals into electrical signals. In this review, the electrophysiological characteristics of ion channels in the somatic membrane of olfactory receptor cells and vomeronasal receptor cells in various species are described and the differences between the action potential dynamics of olfactory receptor cells and vomeronasal receptor cells are compared.

Vomeronasal organ volume increases with body size and is dissociated with the loss of a visual signal in Sceloporus lizards.

Many organisms communicate using signals in different sensory modalities (multicomponent or multimodal). When one signal or component is lost over evolutionary time, it may be indicative of changes in other characteristics of the signalling system, including the sensory organs used to perceive and process signals. Sceloporus lizards predominantly use chemical and visual signals to communicate, yet some species have lost the ancestral ventral colour patch used in male-male agonistic interactions and exhibit increased chemosensory behaviour. Here, we asked whether evolutionary loss of this sexual signal is associated with larger vomeronasal organ (VNO) volumes (an organ that detects chemical scents) compared with species that have retained the colour patch. We measured VNO coronal section areas of 7-8 adult males from each of 11 Sceloporus species (4 that lost and 7 that retained the colour patch), estimated sensory and total epithelium volume, and compared volumes using phylogenetic analysis of covariance, controlling for body size. Contrary to expectations, we found that species retaining the ventral patch had similar relative VNO volumes as did species that had lost the ancestral patch, and that body size explains VNO epithelium volume. Visual signal loss may be sufficiently compensated for by increased chemosensory behaviour, and the allometric pattern may indicate sensory system trade-offs for large-bodied species.

Glycoconjugate-Specific Developmental Changes in the Horse Vomeronasal Organ.

The vomeronasal organ (VNO) is a tubular pheromone-sensing organ in which the lumen is covered with sensory and non-sensory epithelia. This study used immunohistochemistry and lectin histochemistry techniques to evaluate developmental changes, specifically of the glycoconjugate profile, in the horse VNO epithelium. Immunostaining analysis revealed PGP9.5 expression in some vomeronasal non-sensory epithelium (VNSE) cells and in the vomeronasal receptor cells of the vomeronasal sensory epithelium (VSE) in fetuses, young foals, and adult horses. Olfactory marker protein expression was exclusively localized in receptor cells of the VSE in fetuses, young foals, and adult horses and absent in VNSE. To identify the glycoconjugate type, lectin histochemistry was performed using 21 lectins. Semi-quantitative analysis revealed that the intensities of glycoconjugates labeled with WGA, DSL, LEL, and RCA120 were significantly higher in adult horse VSE than those in foal VSE, whereas the intensities of glycoconjugates labeled with LCA and PSA were significantly lower in adult horse VSE. The intensities of glycoconjugates labeled with s-WGA, WGA, BSL-II, DSL, LEL, STL, ConA, LCA, PSA, DBA, SBA, SJA, RCA120, jacalin, and ECL were significantly higher in adult horse VNSE than those in foal VNSE, whereas the intensity of glycoconjugates labeled with UEA-I was lower in adult horse VNSE. Histochemical analysis of each lectin revealed that various glycoconjugates in the VSE were present in the receptor, supporting, and basal cells of foals and adult horses. A similar pattern of lectin histochemistry was also observed in the VNSE of foals and adult horses. In conclusion, these results suggest that there is an increase in the level of N-acetylglucosamine (labeled by WGA, DSL, LEL) and galactose (labeled by RCA120) in horse VSE during postnatal development, implying that they may influence the function of VNO in adult horses.

Connectivity and molecular profiles of Foxp2- and Dbx1-lineage neurons in the accessory olfactory bulb and medial amygdala.

In terrestrial vertebrates, the olfactory system is divided into main (MOS) and accessory (AOS) components that process both volatile and nonvolatile cues to generate appropriate behavioral responses. While much is known regarding the molecular diversity of neurons that comprise the MOS, less is known about the AOS. Here, focusing on the vomeronasal organ (VNO), the accessory olfactory bulb (AOB), and the medial amygdala (MeA), we reveal that populations of neurons in the AOS can be molecularly subdivided based on their ongoing or prior expression of the transcription factors Foxp2 or Dbx1, which delineate separate populations of GABAergic output neurons in the MeA. We show that a majority of AOB neurons that project directly to the MeA are of the Foxp2 lineage. Using single-neuron patch-clamp electrophysiology, we further reveal that in addition to sex-specific differences across lineage, the frequency of excitatory input to MeA Dbx1- and Foxp2-lineage neurons differs between sexes. Together, this work uncovers a novel molecular diversity of AOS neurons, and lineage and sex differences in patterns of connectivity.

Expression patterns of the transcription factors Fezf1, Fezf2, and Bcl11b in the olfactory organs of turtle embryos.

Many tetrapod vertebrates have two distinct olfactory organs, the olfactory epithelium (OE) and vomeronasal organ (VNO). In turtles, the olfactory organ consists of two types of sensory epithelia, the upper chamber epithelium (UCE; corresponding to the OE) and the lower chamber epithelium (LCE; corresponding to the VNO). In many turtle species, the UCE contains ciliated olfactory receptor cells (ORCs) and the LCE contains microvillous ORCs. To date, several transcription factors involved in the development of the OE and VNO have been identified in mammals. Fez family zinc-finger protein 1 and 2 (Fezf1 and 2) are expressed in the OE and VNO, respectively, of mouse embryos, and are involved in the development and maintenance of ORCs. B-cell lymphoma/leukemia 11B (Bcl11b) is expressed in the mouse embryo OE except the dorsomedial parts of the nasal cavity, and regulates the expression of odorant receptors in the ORCs. In this study, we examined the expression of Fezf1, Fezf2, and Bcl11b in the olfactory organs of embryos in three turtle species, Pelodiscus sinensis, Trachemys scripta elegans, and Centrochelys sulcata, to evaluate their involvement in the development of reptile olfactory organs. In all three turtle species, Bcl11b was expressed in the UCE, Fezf2 in the LCE, and Fezf1 in both the UCE and LCE. These results imply that the roles of the transcription factors Fezf1, Fezf2, and Bcl11b in olfactory organ development are conserved among mammals and turtles.

Sensing and avoiding sick conspecifics requires Gαi2+ vomeronasal neurons.

BACKGROUND: Rodents utilize chemical cues to recognize and avoid other conspecifics infected with pathogens. Infection with pathogens and acute inflammation alter the repertoire and signature of olfactory stimuli emitted by a sick individual. These cues are recognized by healthy conspecifics via the vomeronasal or accessory olfactory system, triggering an innate form of avoidance behavior. However, the molecular identity of the sensory neurons and the higher neural circuits involved in the detection of sick conspecifics remain poorly understood. RESULTS: We employed mice that are in an acute state of inflammation induced by systemic administration of lipopolysaccharide (LPS). Through conditional knockout of the G-protein Gαi2 and deletion of other key sensory transduction molecules (Trpc2 and a cluster of 16 vomeronasal type 1 receptors), in combination with behavioral testing, subcellular Ca2+ imaging, and pS6 and c-Fos neuronal activity mapping in freely behaving mice, we show that the Gαi2+ vomeronasal subsystem is required for the detection and avoidance of LPS-treated mice. The active components underlying this avoidance are contained in urine whereas feces extract and two selected bile acids, although detected in a Gαi2-dependent manner, failed to evoke avoidance behavior. Our analyses of dendritic Ca2+ responses in vomeronasal sensory neurons provide insight into the discrimination capabilities of these neurons for urine fractions from LPS-treated mice, and how this discrimination depends on Gαi2. We observed Gαi2-dependent stimulation of multiple brain areas including medial amygdala, ventromedial hypothalamus, and periaqueductal grey. We also identified the lateral habenula, a brain region implicated in negative reward prediction in aversive learning, as a previously unknown target involved in these tasks. CONCLUSIONS: Our physiological and behavioral analyses indicate that the sensing and avoidance of LPS-treated sick conspecifics depend on the Gαi2 vomeronasal subsystem. Our observations point to a central role of brain circuits downstream of the olfactory periphery and in the lateral habenula in the detection and avoidance of sick conspecifics, providing new insights into the neural substrates and circuit logic of the sensing of inflammation in mice.

An updated synthesis of and outstanding questions in the olfactory and vomeronasal systems in bats: Genetics asks questions only anatomy can answer.

The extensive diversity observed in bat nasal chemosensory systems has been well-documented at the histological level. Understanding how this diversity evolved and developing hypotheses as to why particular patterns exist require a phylogenetic perspective, which was first outlined in the work of anatomist Kunwar Bhatnagar. With the onset of genetics and genomics, it might be assumed that the puzzling patterns observed in the morphological data have been clarified. However, there is still a widespread mismatch of genetic and morphological correlations among bat chemosensory systems. Novel genomic evidence has set up new avenues to explore that demand more evidence from anatomical structures. Here, we outline the progress that has been made in both morphological and molecular studies on the olfactory and vomeronasal systems in bats since the work of Bhatnagar. Genomic data of olfactory and vomeronasal receptors demonstrate the strong need for further morphological sampling, with a particular focus on receiving brain regions, glands, and ducts.

Vomeronasal Receptors Associated with Circulating Estrogen Processing Chemosensory Cues in Semi-Aquatic Mammals.

In numerous animals, one essential chemosensory organ that detects chemical signals is the vomeronasal organ (VNO), which is involved in species-specific behaviors, including social and sexual behaviors. The purpose of this study is to investigate the mechanism underlying the processing of chemosensory cues in semi-aquatic mammals using muskrats as the animal model. Muskrat (Ondatra zibethicus) has a sensitive VNO system that activates seasonal breeding behaviors through receiving specific substances, including pheromones and hormones. Vomeronasal organ receptor type 1 (V1R) and type 2 (V2R) and estrogen receptor α and ß (ERα and ERß) were found in sensory epithelial cells, non-sensory epithelial cells and lamina propria cells of the female muskrats' VNO. V2R and ERα mRNA levels in the VNO during the breeding period declined sharply, in comparison to those during the non-breeding period, while V1R and ERß mRNA levels were detected reversely. Additionally, transcriptomic study in the VNO identified that differently expressed genes might be related to estrogen signal and metabolic pathways. These findings suggested that the seasonal structural and functional changes in the VNO of female muskrats with different reproductive status and estrogen was regulated through binding to ERα and ERß in the female muskrats' VNO.

Contactin-6-deficient male mice exhibit the abnormal function of the accessory olfactory system and impaired reproductive behavior.

INTRODUCTION: Contactin-6 (CNTN6), also known as NB-3, is a neural recognition molecule and a member of the contactin subgroup of the immunoglobulin superfamily. Gene encoding CNTN6 is expressed in many regions of the neural system, including the accessory olfactory bulb (AOB) in mice. We aim to determine the effect of CNTN6 deficiency on the function of the accessory olfactory system (AOS). METHODS: We examined the effect of CNTN6 deficiency on the reproductive behavior of male mice through behavioral experiments such as urine sniffing and mate preference tests. Staining and electron microscopy were used to observe the gross structure and the circuitry activity of the AOS. RESULTS: Cntn6 is highly expressed in the vomeronasal organ (VNO) and the AOB, and sparsely expressed in the medial amygdala (MeA) and the medial preoptic area (MPOA), which receive direct and/or indirect projections from the AOB. Behavioral tests to examine reproductive function in mice, which is mostly controlled by the AOS, revealed that Cntn6-/- adult male mice showed less interest and reduced mating attempts toward estrous female mice in comparison with their Cntn6+/+ littermates. Although Cntn6-/- adult male mice displayed no obvious changes in the gross structure of the VNO or AOB, we observed the increased activation of granule cells in the AOB and the lower activation of neurons in the MeA and the MPOA as compared with Cntn6+/+ adult male mice. Moreover, there were an increased number of synapses between mitral cells and granule cells in the AOB of Cntn6-/- adult male mice as compared with wild-type controls. CONCLUSION: These results indicate that CNTN6 deficiency affects the reproductive behavior of male mice, suggesting that CNTN6 participated in normal function of the AOS and its ablation was involved in synapse formation between mitral and granule cells in the AOB, rather than affecting the gross structure of the AOS.