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Ca(2+)-activated Cl(−) currents have been observed in many physiological processes, including sensory transduction in mammalian olfaction. The olfactory vomeronasal (or Jacobson's) organ (VNO) detects molecular cues originating from animals of the same species or from predators. It then triggers innate behaviors such as aggression, mating, or flight. In the VNO, Ca(2+)-activated Cl(−) channels (CaCCs) are thought to amplify the initial pheromone-evoked receptor potential by mediating a depolarizing Cl(−) efflux. Here, we confirmed the co-localization of the Ca(2+)-activated Cl(−) channels anoctamin 1 (Ano1, also called TMEM16A) and Ano2 (TMEM16B) in microvilli of apically and basally located vomeronasal sensory neurons (VSNs) and their absence in supporting cells of the VNO. Both channels were expressed as functional isoforms capable of giving rise to Ca(2+)-activated Cl(−) currents. Although these currents persisted in the VNOs of mice lacking Ano2, they were undetectable in olfactory neuron-specific Ano1 knockout mice irrespective of the presence of Ano2. The loss of Ca(2+)-activated Cl(−) currents resulted in diminished spontaneous and drastically reduced pheromone-evoked spiking of VSNs. Although this indicated an important role of anoctamin channels in VNO signal amplification, the lack of this amplification did not alter VNO-dependent male–male territorial aggression in olfactory Ano1/Ano2 double knockout mice. We conclude that Ano1 mediates the bulk of Ca(2+)-activated Cl(−) currents in the VNO and that Ano2 plays only a minor role. Furthermore, vomeronasal signal amplification by CaCCs appears to be dispensable for the detection of male-specific pheromones and for near-normal aggressive behavior in mice.

In contrast to the main olfactory system that detects volatile chemicals in the nasal air, the vomeronasal system can detect nonvolatile chemicals as well as volatiles. In the vomeronasal system, chemicals are perceived by the vomeronasal organ (VNO) projecting axons to the accessory olfactory bulb (AOB). Beavers (Castor spp.) are semiaquatic mammals that have developed chemical communication. It is possible that the beaver's anal gland secretions, nonvolatile and insoluble substances, may work as a messenger in the water and that beavers may detect the nonvolatile chemicals floating on the water surface via the VNO. The present study aimed to clarify the specificities of the beaver vomeronasal system by histologically and immunohistochemically analyzing the VNO and AOB of 12 Eurasian beavers (C. fiber). The VNO directly opened to the nasal cavity and was independent of a narrow nasopalatine duct connecting the oral and nasal cavities. The VNO comprised soft tissues including sensory and nonsensory epithelium, glands, a venous sinus, an artery, as well as cartilage inner, and bone outer enclosures. The AOB had distinct six layers, and anti-G protein α-i2 and α-o subunits were, respectively, immunoreactive in rostral and caudal glomeruli layers indicating expressions of V1Rs and V2Rs. According to gene repertories analysis, the beavers had 23 and six intact V1R and V2R genes respectively. These findings suggested that beavers recognize volatile odorants and nonvolatile substances using the vomeronasal system. The beaver VNO was developed as well as in other rodents, and it had two specific morphological features, namely, disadvantaged contact with the oral cavity because of a tiny nasopalatine duct, and a double bone and cartilage envelope. Our results highlight the importance of the vomeronasal system in beaver chemical communication and support the possibility that beavers can detect chemicals floating on the water surface via the VNO.

Vomeronasal information is critical in mice for territorial behavior. Consequently, learning the territorial spatial structure should incorporate the vomeronasal signals indicating individual identity into the hippocampal cognitive map. In this work we show in mice that navigating a virtual environment induces synchronic activity, with causality in both directionalities, between the vomeronasal amygdala and the dorsal CA1 of the hippocampus in the theta frequency range. The detection of urine stimuli induces synaptic plasticity in the vomeronasal pathway and the dorsal hippocampus, even in animals with experimentally induced anosmia. In the dorsal hippocampus, this plasticity is associated with the overexpression of pAKT and pGSK3β. An amygdalo-entorhino-hippocampal circuit likely underlies this effect of pheromonal information on hippocampal learning. This circuit likely constitutes the neural substrate of territorial behavior in mice, and it allows the integration of social and spatial information., Male and female mice need to generate spatial maps that integrate vomeronasal signals of territory owners in the hippocampus-dependent memory. The authors show that vomeronasal information influences learning-related activity in the hippocampus via the amygdaloid PMCo, lateral entorhinal cortex, and dorsal CA1.

Lungfish are the fish related most closely to tetrapods. The olfactory organ of lungfish contains two distinct sensory epithelia: the lamellar olfactory epithelium (OE) and the recess epithelium (RecE). Based on their ultrastructural and histological characteristics, the lamellar OE and the RecE are considered to correspond respectively to the teleost OE and a primitive vomeronasal organ (VNO). In tetrapods, the OE and VNO have been shown to express different families of olfactory receptors; for example, in mammals, the OE expresses odorant receptors and trace amine-associated receptors, while the VNO expresses type 1 (V1Rs) and type 2 (V2Rs) vomeronasal receptors. In the present study, we examined the expression of V1Rs in the olfactory organs of two African lungfish, Protopterus annectens and Protopterus amphibius. RNA sequencing and phylogenetic analyses identified 29 V1R genes in P. annectens and 50 V1R genes in P. amphibius. Most V1Rs identified in these lungfish were classified as the tetrapod-type V1Rs initially found in tetrapods and distinct from fish-type V1Rs. In teleost, which all lack a VNO, all olfactory receptors are expressed in the OE, while in Xenopus V1Rs are expressed exclusively in the OE, and not in the VNO. In situ hybridization analysis indicated that lungfish V1Rs were expressed mainly in the lamellar OE and rarely in the RecE. These results imply that V1R expression in lungfish represents an intermediate step toward the complete segregation of V1R expression between the OE and VNO, reflecting the phylogenetic position of lungfish between teleosts and amphibians.

The ability of terrestrial vertebrates to find food and mating partners, and to avoid predators, relies on the detection of chemosensory information. Semiochemicals responsible for social and sexual behaviors are detected by chemosensory neurons of the vomeronasal organ (VNO), which transmits information to the accessory olfactory bulb. The vomeronasal sensory epithelium of most mammalian species contains a uniform vomeronasal system; however, rodents and marsupials have developed a more complex binary vomeronasal system, containing vomeronasal sensory neurons (VSNs) expressing receptors of either the V1R or V2R family. In rodents, V1R/apical and V2R/basal VSNs originate from a common pool of progenitors. Using single cell RNA-sequencing, we identified differential expression of Notch1 receptor and Dll4 ligand between the neuronal precursors at the VSN differentiation dichotomy. Our experiments show that Notch signaling is required for effective differentiation of V2R/basal VSNs. In fact, Notch1 loss of function in neuronal progenitors diverts them to the V1R/apical fate, whereas Notch1 gain of function redirects precursors to V2R/basal. Our results indicate that Notch signaling plays a pivotal role in triggering the binary differentiation dichotomy in the VNO of rodents.

Pitviper sensory perception incorporates diverse stimuli through the integration of trichromatic color vision, bifocal heat-sensing, and dual-system chemoperception. Chemoperception, or olfaction, is mediated by chemoreceptors in the olfactory bulb and the vomeronasal organ, but the true genomic complexity of the gene families and their relative contributions is unknown. A full genomic accounting of pitviper chemoperception directly complements our current understanding of their venoms by generating a more complete polyphenic representation of their predatory arsenal. To characterize the genetic repertoire of pitviper chemoperception, we analyzed a full-genome assembly for Crotalus adamanteus, the eastern diamondback rattlesnake. We identified hundreds of genes encoding both olfactory receptors (ORs; 362 full-length genes) and type-2 vomeronasal receptors (V2Rs; 430 full-length genes). Many chemoreceptor genes are organized into large tandem repeat arrays. Comparative analysis of V2R orthologs across squamates demonstrates how gene array expansion and contraction underlies the evolution of the chemoreceptor repertoire, which likely reflects shifts in life history traits. Chromosomal assignments of chemosensory genes identified sex chromosome specific chemoreceptor genes, providing gene candidates underlying observed sex-specific chemosensory-based behaviors. We detected widespread episodic evolution in the extracellular, ligand-binding domains of both ORs and V2Rs, suggesting the diversification of chemoreceptors is driven by transient periods of positive selection. We provide a robust genetic framework for studying pitviper chemosensory ecology and evolution.

Activating transcription factor 5 (ATF5) is a stress-responsive transcription factor that belongs to the cAMP response element-binding protein (CREB)/ATF family, and is essential for the differentiation and survival of sensory neurons in murine olfactory organs. However, the study of associated proteins and target genes for ATF5 has been hampered due to the limited availability of immunoprecipitation-grade ATF5 antibodies. To overcome this issue, we generated hemagglutinin (HA)-tag knock-in mice for ATF5 using CRISPR/Cas9-mediated genome editing with one-step electroporation in oviducts (i-GONAD). ATF5-HA fusion proteins were detected in the nuclei of immature and some mature olfactory and vomeronasal sensory neurons in the main olfactory epithelium and vomeronasal organ, respectively, as endogenous ATF5 proteins were expressed, and some ATF5-HA proteins were found to be phosphorylated. Chromatin immunoprecipitation (ChIP) experiments revealed that ATF5-HA bound to the CCAAT/enhancer-binding protein (C/EBP)-ATF response element site in the promotor region of receptor transporting protein 1 (Rtp1), a chaperone gene responsible for proper olfactory receptor expression. These knock-in mice may be used to examine the expression, localization, and protein-protein/-DNA interactions of endogenous ATF5 and, ultimately, the function of ATF5 in vivo.

The vomeronasal organ (VNO) is sensory organ located in the ventral region of the nasal cavity in rodents. The VNO develops from the olfactory placode during the secondary invagination of olfactory pit. The embryonic vomeronasal structure appears as a neurogenic area where migratory neuronal populations like endocrine gonadotropin-releasing hormone-1 (GnRH-1) neurons form. Even though embryonic vomeronasal structures are conserved across most vertebrate species, many species including humans do not have a functional VNO after birth. The vomeronasal epithelium (VNE) of rodents is composed of two major types of vomeronasal sensory neurons (VSNs): (1) VSNs distributed in the apical VNE regions that express vomeronasal type-1 receptors (V1Rs) and the G protein subunit Gαi2, and (2) VSNs in the basal territories of the VNE that express vomeronasal type-2 receptors (V2Rs) and the G subunit Gαo. Recent studies identified a third subclass of Gαi2 and Gαo VSNs that express the formyl peptide receptor family. VSNs expressing V1Rs or V2Rs send their axons to distinct regions of the accessory olfactory bulb (AOB). Together, VNO and AOB form the accessory olfactory system (AOS), an olfactory subsystem that coordinates the social and sexual behaviors of many vertebrate species. In this review, we summarize our current understanding of cellular and molecular mechanisms that underlie VNO development. We also discuss open questions for study, which we suggest will further enhance our understanding of VNO morphogenesis at embryonic and postnatal stages.

In the rodent brain, the central processing of ecologically relevant chemical stimuli involves many different areas located at various levels within the neuraxis: the main and accessory olfactory bulbs, some nuclei in the amygdala, the hypothalamus, and brainstem. These areas allow the integration of the chemosensory stimuli with other sensory information and the selection of the appropriate neurohormonal and behavioral response. This review is a brief introduction to the processing of intraspecific chemosensory stimuli beyond the secondary projection, focusing on the activity of the relevant amygdala and hypothalamic nuclei, namely the medial amygdala and ventromedial hypothalamus. These areas are involved in the appropriate interpretation of chemosensory information and drive the selection of the proper response, which may be behavioral or hormonal and may affect the neural activity of other areas in the telencephalon and brainstem.Recent data support the notion that the processing of intraspecific chemical signals is not unique to one chemosensory system and some molecules may activate both the main and the accessory olfactory system. Moreover, both these systems have mixed projections and cooperate for the correct identification of the stimuli and selection of relevant responses.

Pheromonal communication is an ancient and pervasive sensory modality in urodelan amphibians. One family of salamander pheromones (the sodefrin precursor-like factor (SPF) family) originated 300 million years ago, at the origin of amphibians. Although salamanders are often thought of as relatively simple animals especially when compared to mammals, the pheromonal systems are varied and complex with nuanced effects on behavior. Here, we review the function and evolution of pheromonal signals involved in male-female reproductive interactions. After describing common themes of salamander pheromonal communication, we describe what is known about the rich diversity of pheromonal communication in each salamander family. Several pheromones have been described, ranging from simple, invariant molecules to complex, variable blends of pheromones. While some pheromones elicit overt behavioral responses, others have more nuanced effects. Pheromonal signals have diversified within salamander lineages and have experienced rapid evolution. Once receptors have been matched to pheromonal ligands, rapid advance can be made to better understand the olfactory detection and processing of salamander pheromones. In particular, a large number of salamander species deliver pheromones across the skin of females, perhaps reflecting a novel mode of pheromonal communication. At the end of our review, we list some of the many intriguing unanswered questions. We hope that this review will inspire a new generation of scientists to pursue work in this rewarding field.

Variations in gene expression patterns represent a powerful source of evolutionary innovation. In a rodent living about 70 million years ago, a genomic accident led an immune formyl peptide receptor (FPR) gene to hijack a vomeronasal receptor regulatory sequence. This gene shuffling event forced an immune pathogen sensor to transition into an olfactory chemoreceptor, which thus moved from sensing the internal world to probing the outside world. We here discuss the evolution of the FPR gene family, the events that led to their neofunctionalization in the vomeronasal organ and the functions of immune and vomeronasal FPRs.

Extant anuran amphibians originate from an evolutionary intersection eventually leading to fully terrestrial tetrapods. In many ways, they have to deal with exposure to both terrestrial and aquatic environments: (i) phylogenetically, as derivatives of the first tetrapod group that conquered the terrestrial environment in evolution; (ii) ontogenetically, with a development that includes aquatic and terrestrial stages connected via metamorphic remodeling; and (iii) individually, with common changes in habitat during the life cycle. Our knowledge about the structural organization and function of the amphibian olfactory system and its relevance still lags behind findings on mammals. It is a formidable challenge to reveal underlying general principles of circuity-related, cellular, and molecular properties that are beneficial for an optimized sense of smell in water and air. Recent findings in structural organization coupled with behavioral observations could help to understand the importance of the sense of smell in this evolutionarily important animal group. We describe the structure of the peripheral olfactory organ, the olfactory bulb, and higher olfactory centers on a tissue, cellular, and molecular levels. Differences and similarities between the olfactory systems of anurans and other vertebrates are reviewed. Special emphasis lies on adaptations that are connected to the distinct demands of olfaction in water and air environment. These particular adaptations are discussed in light of evolutionary trends, ontogenetic development, and ecological demands.

Olfactory marker protein (OMP) was first described as a protein expressed in olfactory receptor neurons (ORNs) in the nasal cavity. In particular, OMP, a small cytoplasmic protein, marks mature ORNs and is also expressed in the neurons of other nasal chemosensory systems: the vomeronasal organ, the septal organ of Masera, and the Grueneberg ganglion. While its expression pattern was more easily established, OMP’s function remained relatively vague. To date, most of the work to understand OMP’s role has been done using mice lacking OMP. This mostly phenomenological work has shown that OMP is involved in sharpening the odorant response profile and in quickening odorant response kinetics of ORNs and that it contributes to targeting of ORN axons to the olfactory bulb to refine the glomerular response map. Increasing evidence shows that OMP acts at the early stages of olfactory transduction by modulating the kinetics of cAMP, the second messenger of olfactory transduction. However, how this occurs at a mechanistic level is not understood, and it might also not be the only mechanism underlying all the changes observed in mice lacking OMP. Recently, OMP has been detected outside the nose, including the brain and other organs. Although no obvious logic has become apparent regarding the underlying commonality between nasal and extranasal expression of OMP, a broader approach to diverse cellular systems might help unravel OMP’s functions and mechanisms of action inside and outside the nose.

The nasolacrimal apparatus (NLA) is a feature common to many sauropsid amniotes. It consists of an orbital Harderian gland (HG)whose secretions drain into the nasal cavity, in the vicinity of the vomeronasal organ (VNO), an accessory olfactory organ derived from the olfactory epithelium, and a connecting nasolacrimal duct (NLD). Though not all features are present in all posthatchling sauropsids (i.e., no VNO in crocodilomorphs), it is not clear if this system either never existed or failed to develop during the embryonic stages. The purpose of this study is to histologically describe the ontogeny of the NLA and the main olfactory organ in Alligator mississippiensis. Alligator specimens, from embryonic stage 9 to hatchling, were serially histologically sectioned, stained, photographed, and segmented into different tissues using Abobe Photoshop and then reconstructed using Amira for 3D analysis and quantitative nasal epithelial distribution. Though there was no evidence of a VNO, the rest of the NLA was present. The development of the NLA could be subdivided into four phases: (1) inception of NLD, (2) establishment of orbitonasal connections of NLD, (3) bone development, and (4) nasal cavity growth. Glands mature during this last phase and the nasal region rapidly grows, rotates, and is displaced anteriorly. The gradual proportional increase in nonolfactory epithelial distribution during ontogeny is consistent with the literature. Alligator embryonic nasal and NLD growth differs from that of mammals and squamates. The NLD is connected to the anterior third of the nasal region during its initial attachment, but as anterior nasal growth exceeds posterior growth, it is gradually displaced into the posterior third of the nasal region by hatching. It is unknown whether this is a derived archosaur condition or just another example of the morphological variation seen within sauropsid amniotes.

Background. For neurodegenerative diseases such as Huntington’s disease (HD), early diagnosis is essential to treat patients and delay symptoms. Impaired olfaction, as observed as an early symptom in Parkinson´s disease, may also constitute a key symptom in HD. However, there are few reports on olfactory deficits in HD. Therefore, we aimed to investigate, in a transgenic rat model of HD: (1) whether general olfactory impairment exists and (2) whether there are disease-specific dynamics of olfactory dysfunction when the vomeronasal (VNE) and main olfactory epithelium (MOE) are compared. Methods. We used male rats of transgenic line 22 (TG22) of the bacterial artificial chromosome Huntington disease model (BACHD), aged 3 days or 6 months. Cell proliferation, apoptosis and macrophage activity were examined with immunohistochemistry in the VNE and MOE. Results. No differences were observed in cellular parameters in the VNE between the groups. However, the MOE of the 6-month-old HD animals showed a significantly increased number of mature olfactory receptor neurons. Other cellular parameters were not affected. Conclusions. The results obtained in the TG22 line suggest a relative stability in the VNE, whereas the MOE seems at least temporarily affected.

In the olfactory organ of lungfish, recesses at the bases of lamellae comprise sensory and nonsensory epithelia. The sensory epithelium of the recesses, the recess epithelium, is distinguished from the olfactory epithelium covering the lamella by the absence of ciliated olfactory receptor cells. Therefore, it has been suggested that the recess epithelium is a primordium of the vomeronasal organ of tetrapods. However, developmental changes in the number and distribution of recesses in the olfactory organ of lungfish were unknown. We examined four Protopterus aethiopicus specimens of body lengths 215-800 mm to determine the localization of recesses in their olfactory organs. Histological examination showed recesses at the bases of lamellae in all individuals examined. The recesses were localized mainly in the medial and caudal parts of the olfactory organs, especially in juveniles. Compared to smaller fish, larger fish had a larger number of recesses, distributed more broadly in their olfactory organs. Significance of the recess localization and its relationship to the function of lungfish olfactory organ warrants further investigation.

The sense of smell employs some of the largest gene families in the genome to detect and distinguish a multitude of different odors. Within vertebrates, 4 major olfactory receptor families have been described; of which, only 3 (OR, TAAR-like, and V1R) were found already in lamprey, a jawless vertebrate. The forth family (V2R) was believed to have originated later, in jawed vertebrates. Here we have delineated the entire vomeronasal receptor repertoire in 3 lamprey species. We report the presence of 6 v1r and 2 v2r genes in Lethenteron camtschaticum, arctic lamprey, and Lampetra fluviatilis, river lamprey (6 and 1, respectively, in sea lamprey, Petromyzon marinus). Three v1r genes but no v2r genes were found to be expressed in olfactory sensory neurons in the characteristic sparse expression pattern. Our results show the olfactory function of some V1Rs already in lamprey and, unexpectedly, an early origin of the V2R family in the shared ancestor of jawed and jawless vertebrates. However, lamprey v2r genes appear not to have acquired an olfactory function yet, thus dissociating the evolutionary origin of the family from the onset of a function as olfactory receptor.

application/pdf, Artiodactyl livestock animals have a vomeronasal system that detects pheromones. Vomeronasal receptors comprise type 1 (V1R) coupled with G protein α-i2 (Gαi2) and type 2 (V2R) coupled with G protein α-o (Gαo). Laboratory rodents have two segregated V1R and V2R pathways that reach separately to the accessory olfactory bulb (AOB). In contrast, the AOBs of goats and sheep are entirely positive for Gαi2, indicating that they have only the V1R pathway. However, we detected a few V2R genes in the genome of cattle, goats, sheep and pigs by genome assembly. Thus, we immunohistochemically analyzed the AOBs of cattle and pigs to confirm which type of the vomeronasal system is present in artiodactyl livestock species. The glomerular layer of the AOB in cattle and pigs was entirely positive for anti-Gαi2 and weakly positive for anti-Gαo, as in the V1R uniform type of vomeronasal system in other mammal species. These findings indicated that artiodactyl livestock species have a uniform type of vomeronasal system composing the V1R pathway. Therefore, caution is advised when extrapolating knowledge of laboratory rodents with two vomeronasal pathways to livestock animals that have one. © 2022 Elsevier Ltd

To be social, the ability to recognize and discriminate conspecific individuals is indispensable in social animals, including primates, rodents, birds, fish, and social insects which live in societies or groups. Recent studies using molecular biology, genetics, in vivo and in vitro physiology, and behavioral neuroscientific approaches have provided detailed insights into how animals process and recognize the information of individuals. Here, we review the most distinct sensory modalities for individual recognition in animals, namely, olfaction and vision. In the case of rodents, two polymorphic gene complexes have been identified in their urine as the key and essential pheromonal components for individual recognition: the major histocompatibility complex (MHC) and the major urinary protein (MUP). Animals flexibly utilize MHC and/or MUP, which are detected by the main olfactory epithelium (MOE) and/or the vomeronasal organ (VNO) for various types of social recognition, such as strain recognition, kin recognition, and individual recognition. In contrast, primates, including humans, primarily use facial appearance to identify others. Face recognition in humans and other animals is naturally unique from genetic, cognitive, developmental, and functional points of view. Importantly note that nurture effects during growth phase such as social experience and environment can also shape and tune this special cognitive ability, in order to distinguish subtle differences between individuals. In this review, we address such unique nature and nurture mechanisms for individual recognition.

Most mammals rely on chemosensory cues for individual recognition, which is essential to many aspects of social behavior, such as maternal bonding, mate recognition, and inbreeding avoidance. Both volatile molecules and nonvolatile peptides secreted by individual conspecifics are detected by olfactory sensory neurons in the olfactory epithelium and the vomeronasal organ. The pertinent cues used for individual recognition remain largely unidentified. Here we show that nonformylated, but not N-formylated, mitochondrially encoded peptides—that is, the nine N-terminal amino acids of NADH dehydrogenases 1 and 2—can be used to convey strain-specific information among individual mice. We demonstrate that these nonformylated peptides are sufficient to induce a strain-selective pregnancy block. We also observed that the pregnancy block by an unfamiliar peptide derived from a male of a different strain was prevented by a memory formed at the time of mating with that male. Our findings also demonstrate that pregnancy-blocking chemosignals in the urine are maternally inherited, as evidenced by the production of reciprocal sons from two inbred strains and our test of their urine’s ability to block pregnancy. We propose that this link between polymorphic mitochondrial peptides and individual recognition provides the molecular means to communicate an individual’s maternal lineage and strain.

The vomeronasal organ (VNO) is an accessory olfactory device related to reproductive behavior. The soft tissue of the tubular organ is composed of sensory/non-sensory epithelia and a highly developed vasculature, which in the latter the dilation and contraction of blood vessels are thought to contribute to pumping in and out luminal fluid or air, like penile erectile tissue. The present histological observation of the murine VNO revealed a more complicated vasculature than previously evaluated ones with large differences along the rostro-caudal axis. An immunohistochemical study for vasoactive substances displayed extremely dense innervation by cholinergic nerves containing nitric oxide synthase and VIP/PHI in the thick smooth muscle layer surrounding venous sinuses at light and electron microscopic levels. Furthermore, the differential distribution of cholinergic nerves and adrenergic nerves may provide a novel insight into the pumping mechanism of VNO.

This article studies the morphological remodeling of olfactory organs in the fire salamander (Salamandridae, Caudata), from the larval stages of ontogeny to the adult and throughout the course of the annual cycle. The fire salamander exhibits adaptations to the aquatic environment during premetamorphic life and terrestrial adaptations after metamorphosis. During adulthood, the annual activity of this species is divided into three seasonal periods: a breeding period, a nonbreeding period, and hibernation. We observed significant differences in morphology of olfactory organs between developmental stages as well as between each period within the annual cycle. For the first time in caudates, we examined the morphology of olfactory organs during the winter period (wintering larvae, hibernating adults). The results show that the remodeling of olfactory organs during the life of the fire salamander occurs both on macro- and micromorphological levels. Macromorphological ontogenetic variability includes the shape of the main olfactory chamber (MOC) and the distribution of olfactory epithelium (OE) in the MOC and in the vomeronasal organ (VNO). In larvae, the vomeronasal epithelium (VNE) is in a separate cavity, while in the post-metamorphic stages of ontogeny, the VNE occurs in the diverticulum of the MOC. In adult fire salamanders, both olfactory organs are most developed during the breeding season and reduced during hibernation. The VNE and OE in the MOC are also reduced during hibernation. Micro-morphological changes included different types/subtypes of olfactory receptor neurons (ORNs) in the OE in particular stages of ontogeny and periods within the annual cycle, for example, ciliate ORNs are present in the VNE only in the larval stages and giant ORNs occur only in nonbreeding adults. Also, there was a variable set of types of olfactory supporting cells in the VNO of the fire salamander during pre- and postmetamorphic life stages.

Background Many animals rely heavily on olfaction to navigate their environment. Among rodents, olfaction is crucial for a wide range of social behaviors. The vomeronasal olfactory system in particular plays an important role in mediating social communication, including the detection of pheromones and recognition signals. In this study we examine patterns of vomeronasal type-1 receptor (V1R) evolution in the house mouse and related species within the genus Mus. We report the extent of gene repertoire turnover and conservation among species and clades, as well as the prevalence of positive selection on gene sequences across the V1R tree. By exploring the evolution of these receptors, we provide insight into the functional roles of receptor subtypes as well as the dynamics of gene family evolution. Results We generated transcriptomes from the vomeronasal organs of 5 Mus species, and produced high quality V1R repertoires for each species. We find that V1R clades in the house mouse and relatives exhibit distinct evolutionary trajectories. We identify putative species-specific gene expansions, including a large clade D expansion in the house mouse. While gene gains are abundant, we detect very few gene losses. We describe a novel V1R clade and highlight candidate receptors for future study. We find evidence for distinct evolutionary processes across different clades, from largescale turnover to highly conserved repertoires. Patterns of positive selection are similarly variable, as some clades exhibit abundant positive selection while others display high gene sequence conservation. Based on clade-level evolutionary patterns, we identify receptor families that are strong candidates for detecting social signals and predator cues. Our results reveal clades with receptors detecting female reproductive status are among the most conserved across species, suggesting an important role in V1R chemosensation. Conclusion Analysis of clade-level evolution is critical for understanding species’ chemosensory adaptations. This study provides clear evidence that V1R clades are characterized by distinct evolutionary trajectories. As receptor evolution is shaped by ligand identity, these results provide a framework for examining the functional roles of receptors.

Generally, the olfactory organ of vertebrates consists of the olfactory epithelium (OE) and the vomeronasal organ (VNO). The OE contains ciliated olfactory receptor neurons (ORNs), while the VNO contains microvillous ORNs. The ORNs in the OE express odorant receptors (ORs), while those in the VNO express type 1 and type 2 vomeronasal receptors (V1Rs and V2Rs). In turtles, the olfactory organ consists of the upper (UCE) and lower chamber epithelia (LCE). The UCE contains ciliated ORNs, while the LCE contains microvillous ORNs. Here we investigated the distribution of cells expressing vomeronasal receptors in the olfactory organ of turtles. The turtle vomeronasal receptors were encoded by two V1R genes and two V2R genes. Among them, V2R1 and V2R26 were mainly expressed in the LCE, while V1R3 was expressed both in the UCE and LCE. Notably, vomeronasal receptors were expressed by a limited number of ORNs, which was confirmed by the expression of the gene encoding TRPC2, an ion channel involved in the signal transduction of vomeronasal receptors. Furthermore, expression of ORs by the majority of ORNs was suggested by the expression of the gene encoding CNGA2, an ion channel involved in the signal transduction of ORs. Thus, olfaction of turtle seems to be mediated mainly by the ORs rather than the vomeronasal receptors. More importantly, the relationship between the fine structure of ORNs and the expression of olfactory receptors are not conserved among turtles and other vertebrates.

The vomeronasal system (VNS) has been extensively studied within specific animal families, such as Rodentia. However, the study of the VNS in other families, such as Canidae, has long been neglected. Among canids, the vomeronasal organ (VNO) has only been studied in detail in the dog, and no studies have examined the morphofunctional or immunohistochemical characteristics of the VNS in wild canids, which is surprising, given the well‐known importance of chemical senses for the dog and fox and the likelihood that the VNS plays roles in the socio‐reproductive physiology and behaviours of these species. In addition, characterising the fox VNS could contribute to a better understanding of the domestication process that occurred in the dog, as the fox would represent the first wild canid to be studied in depth. Therefore, the aim of this study was to analyze the morphological and immunohistochemical characteristics of the fox VNO. Tissue dissection and microdissection techniques were employed, followed by general and specific histological staining techniques, including with immunohistochemical and lectin‐histochemical labelling strategies, using antibodies against olfactory marker protein (OMP), growth‐associated protein 43 (GAP‐43), calbindin (CB), calretinin (CR), α‐tubulin, Gαo, and Gαi2 proteins, to highlight the specific features of the VNO in the fox. This study found significant differences in the VNS between the fox and the dog, particularly concerning the expression of Gαi2 and Gαo proteins, which were associated with the expression of the type 1 vomeronasal receptors (V1R) and type 2 vomeronasal receptors (V2R), respectively, in the vomeronasal epithelium. Both are immunopositive in foxes, as opposed to the dog, which only expresses Gαi2. This finding suggests that the fox possesses a well‐developed VNO and supports the hypothesis that a profound transformation in the VNS is associated with domestication in the canid family. Furthermore, the unique features identified in the fox VNO confirm the necessity of studying the VNS system in different species to better comprehend specific phylogenetic aspects of the VNS.

Cancer is often treated with broad-spectrum cytotoxic drugs that not only eradicate cancerous cells but also have detrimental side effects. One of these side effects, disruption of the olfactory system, impedes a patient’s ability to smell, perceive flavor, and ultimately may interfere with their nutritional intake and recovery from cancer. Recent studies reported that the chemotherapy drug, cyclophosphamide (CYP), can damage gustatory epithelia and disrupt cell proliferation in olfactory epithelia. In this study, we asked if CYP altered globose and horizontal basal cell proliferation in the murine main olfactory epithelium (MOE) and vomeronasal organ (VNO). We used antibodies for Ki67, a marker strictly associated with cell proliferation, and Keratin 5, a marker for the cytoskeleton of horizontal basal cells. Our results revealed a significant CYP-induced decrease in the number of proliferative cells in both epithelia, especially globose basal cells in the MOE, within the first 1–2 days postinjection. Recovery of cell renewal was apparent 6 days after injection. The immunohistochemical markers showed significantly higher levels of globose and horizontal basal cell proliferation in CYP-injected mice at 14 and 30 days postinjection compared with control mice. The prolonged proliferative activation of globose and horizontal basal cells suggests that, besides altering proliferation of olfactory epithelia, the epithelial substrate needed for successful cell renewal may be adversely affected by CYP.

The accessory olfactory bulb (AOB), the first neural circuit in the mouse accessory olfactory system, is critical for interpreting social chemosignals. Despite its importance, AOB information processing is poorly understood compared with the main olfactory bulb (MOB). Here, we sought to fill gaps in the understanding of AOB interneuron function. We used 2-photon GCaMP6f Ca2+ imaging in an ex vivo preparation to study chemosensory tuning in AOB external granule cells (EGCs), interneurons hypothesized to broadly inhibit activity in excitatory mitral cells (MCs). In ex vivo preparations from mice of both sexes, we measured MC and EGC tuning to natural chemosignal blends and monomolecular ligands, finding that EGC tuning was sparser, not broader, than upstream MCs. Simultaneous electrophysiological recording and Ca2+ imaging showed no differences in GCaMP6f-to-spiking relationships in these cell types during simulated sensory stimulation, suggesting that measured EGC sparseness was not due to cell type-dependent variability in GCaMP6f performance. Ex vivo patch-clamp recordings revealed that EGC subthreshold responsivity was far broader than indicated by GCaMP6f Ca2+ imaging, and that monomolecular ligands rarely elicited EGC spiking. These results indicate that EGCs are selectively engaged by chemosensory blends, suggesting different roles for EGCs than analogous interneurons in the MOB. SIGNIFICANCE STATEMENT The mouse accessory olfactory system (AOS) interprets social chemosignals, but we poorly understand AOS information processing. Here, we investigate the functional properties of external granule cells (EGCs), a major class of interneurons in the accessory olfactory bulb (AOB). We hypothesized that EGCs, which are densely innervated by excitatory mitral cells (MCs), would show broad chemosensory tuning, suggesting a role in divisive normalization. Using ex vivo GCaMP6f imaging, we found that EGCs were instead more sparsely tuned than MCs. This was not due to weaker GCaMP6f signaling in EGCs than in MCs. Instead, we found that many MC-activating chemosignals caused only subthreshold EGC responses. This indicates a different role for AOB EGCs compared with analogous cells in the main olfactory bulb.

The vomeronasal organ (VNO) plays a key role in sensing pheromonal cues, which elicits social and reproductive behaviors. Although the VNO is highly conserved across mammals, it has been lost in some species that have evolved alternate sensing systems during diversification. In this study, we investigate a newly identified VNO-specific gene, ancV1R, in the extant 261 species of mammals to examine the correlation between genotype (ancV1R) and phenotype (VNO). As a result, we found signatures for the relaxation of purifying selection (inactivating mutations and the elevation of dN/dS) on ancV1Rs in VNO-lacking mammals, such as catarrhine primates, cetaceans, the manatees, and several bat lineages, showing the distinct correlation between genotype and phenotype. Interestingly, we further revealed signatures for the relaxation of purifying selection on ancV1R in true seals, otters, the fossa, the owl monkey, and alcelaphine antelopes in which the existence of a functional VNO is still under debate. Our additional analyses on TRPC2, another predictive marker gene for the functional VNO, showed a relaxation of purifying selection, supporting the possibility of VNO loss in these species. The results of our present study invite more in-depth neuroanatomical investigation in mammals for which VNO function remains equivocal.

Neuronal identity dictates the position in an epithelium, and the ability to detect, process, and transmit specific signals to specified targets. Transcription factors (TFs) determine cellular identity via direct modulation of genetic transcription and recruiting chromatin modifiers. However, our understanding of the mechanisms that define neuronal identity and their magnitude remain a critical barrier to elucidate the etiology of congenital and neurodegenerative disorders. The rodent vomeronasal organ provides a unique system to examine in detail the molecular mechanisms underlying the differentiation and maturation of chemosensory neurons. Here, we demonstrated that the identity of postmitotic/maturing vomeronasal sensory neurons (VSNs), and vomeronasal-dependent behaviors can be reprogrammed through the rescue of

The study of the α-subunit of Gi2 and Go proteins in the accessory olfactory bulb (AOB) was crucial for the identification of the two main families of vomeronasal receptors, V1R and V2R. Both families are expressed in the rodent and lagomorph AOBs, according to a segregated model characterized by topographical anteroposterior zonation. Many mammal species have suffered from the deterioration of the Gαo pathway and are categorized as belonging to the uniform model. This scenario has been complicated by characterization of the AOB in the tammar wallaby, Notamacropus eugenii, which appears to follow a third model of vomeronasal organization featuring exclusive Gαo protein expression, referred to as the intermediate model, which has not yet been replicated in any other species. Our morphofunctional study of the vomeronasal system (VNS) in Bennett’s wallaby, Notamacropus rufogriseus, provides further information regarding this third model of vomeronasal transduction. A comprehensive histological, lectin, and immunohistochemical study of the Bennett’s wallaby VNS was performed. Anti-Gαo and anti-Gαi2 antibodies were particularly useful because they labeled the transduction cascade of V2R and V1R receptors, respectively. Both G proteins showed canonical immunohistochemical labeling in the vomeronasal organ and the AOB, consistent with the anterior–posterior zonation of the segregated model. The lectin Ulex europaeus agglutinin selectively labeled the anterior AOB, providing additional evidence for the segregation of vomeronasal information in the wallaby. Overall, the VNS of the Bennett’s wallaby shows a degree of differentiation and histochemical and neurochemical diversity comparable to species with greater VNS development. The existence of the third intermediate type in vomeronasal information processing reported in Notamacropus eugenii is not supported by our lectin-histochemical and immunohistochemical findings in Notamacropus rufogriseus Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. This work was partially supported by a University of Santiago de Compostela Grant [1551-8179] to P.S.Q. SI

Chronic wasting disease (CWD) is a contagious and fatal transmissible spongiform encephalopathy affecting species of the cervidae family. CWD has an expanding geographic range and complex, poorly understood transmission mechanics. CWD is disproportionately prevalent in wild male mule deer and male white-tailed deer. Sex and species influences on CWD prevalence have been hypothesized to be related to animal behaviours that involve deer facial and body exocrine glands. Understanding CWD transmission potential requires a foundational knowledge of the cellular prion protein (PrPC) in glands associated with cervid behaviours. In this study, we characterized the presence and distribution of PrPC in six integumentary and two non-integumentary tissues of hunter-harvested mule deer (Odocoileus hemionus) and white-tailed deer (O. virginianus). We report that white-tailed deer expressed significantly more PrPC than their mule deer in the parotid, metatarsal, and interdigital glands. Females expressed more PrPC than males in the forehead and preorbital glands. The distribution of PrPC within the integumentary exocrine glands of the face and legs were localized to glandular cells, hair follicles, epidermis, and immune cell infiltrates. All tissues examined expressed sufficient quantities of PrPC to serve as possible sites of prion initial infection, propagation, and shedding.

Differential expression of cell adhesion molecules in neuronal populations is one of the many mechanisms promoting the formation of functional neural circuits in the developing nervous system. The IgLON family consists of five cell surface immunoglobulin proteins that have been associated with various developmental disorders, such as autism spectrum disorder, schizophrenia, and major depressive disorder. However, there is still limited and fragmented information about their patterns of expression in certain regions of the developing nervous system and how their expression contributes to their function. Utilizing an in situ hybridization approach, we have analyzed the spatiotemporal expression of all IgLON family members in the developing mouse brain, spinal cord, eye, olfactory epithelium, and vomeronasal organ. At one prenatal (E16) and two postnatal (P0 and P15) ages, we show that each IgLON displays distinct expression patterns in the olfactory system, cerebral cortex, midbrain, cerebellum, spinal cord, and eye, indicating that they likely contribute to the wiring of specific neuronal circuitry. These analyses will inform future functional studies aimed at identifying additional roles for these proteins in nervous system development.

This study aimed to provide researchers with an atlas of the survey anatomy, histology, and imaging of the nasal cavity of Tupaia belangeri chinensis. Seven T. b. chinensis adult males were euthanized and scanned using micro-computed tomography (CT). The nose was separated, and tissue sections were made on the coronal and axial planes to observe the survey anatomy and histological and imaging characteristics of the nose. T. b. chinensis contains one maxilloturbinal and three ethmoturbinals, one nasoturbinal, one interturbinal, two frontoturbinals, and one lamina semicircularis in the unilateral nasal cavity. Other identified structures were the ostiomeatal complex, vomeronasal organ, superior nasal vault, maxillary sinus, and frontal recess. The drainage pathways of the sinuses and nasal airflow in T. b. chinensis were confirmed. The vault epithelium consisted of the squamous epithelium, respiratory epithelium, transitional epithelium, and olfactory epithelium. Micro-CT confirmed our findings of the coronal tissue sections. The nasal cavity anatomy of T. b. chinensis is similar to that of some strepsirrhine primates. However, the airflow and olfactory function are quite different from that of humans. Our gross and histological atlas of the nasal septum, turbinals, maxillary sinus, and frontal recess provides a reference for researchers to use T. b. chinensis for nasal cavity functional research.

The accessory olfactory bulb (AOB) is the first integrative center of the vomeronasal system (VNS), and the general macroscopic, microscopic, and neurochemical organizational patterns of the AOB differ fundamentally among species. Therefore, the low degree of differentiation observed for the dog AOB is surprising. As the artificial selection pressure exerted on domestic dogs has been suggested to play a key role in the involution of the dog VNS, a wild canid, such as the fox, represents a useful model for studying the hypothetical effects of domestication on the AOB morphology.A comprehensive histological, lectin-histochemical, and immunohistochemical study of the fox AOB was performed. Anti-Gαo and anti-Gαi2 antibodies were particularly useful, as they label the transduction cascade of the vomeronasal receptor types 1 (V1R) and 2 (V2R), respectively. Other employed antibodies included those against proteins such as microtubule-associated protein 2 (MAP-2), tubulin, glial fibrillary acidic protein, growth-associated protein 43 (GAP-43), olfactory marker protein (OMP), calbindin, and calretinin.The cytoarchitecture of the fox AOB showed a clear lamination, with neatly differentiated layers; a highly developed glomerular layer, rich in periglomerular cells; and large inner cell and granular layers. The immunolabeling of Gαi2, OMP, and GAP-43 delineated the outer layers, whereas Gαo and MAP-2 immunolabeling defined the inner layers. MAP-2 characterized the somas of AOB principal cells and their dendritic trees. Anti-calbindin and anti-calretinin antibodies discriminated neural subpopulations in both the mitral-plexiform layer and the granular cell layer, and the lectin Ulex europeus agglutinin I (UEA-I) showed selectivity for the AOB and the vomeronasal nerves.The fox AOB presents unique characteristics and a higher degree of morphological development compared with the dog AOB. The comparatively complex neural basis for semiochemical information processing in the fox compared with that observed in dogs suggests loss of AOB anatomical complexity during the evolutionary history of dogs and opens a new avenue of research for studying the effects of domestication on brain structures.

Patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019, suffer from respiratory and non-respiratory symptoms. Among these symptoms, the loss of smell has attracted considerable attention. The objectives of this study were to determine which cells are infected, what happens in the olfactory system after viral infection, and how these pathologic changes contribute to olfactory loss. For this purpose, Syrian golden hamsters were used. First, we verified the olfactory structures in the nasal cavity of Syrian golden hamsters, namely the main olfactory epithelium, the vomeronasal organ, and their cellular components. Second, we found angiotensin-converting enzyme 2 expression, a receptor protein of SARS-CoV-2, in both structures and infections of supporting, microvillar, and solitary chemosensory cells. Third, we observed pathological changes in the infected epithelium, including reduced thickness of the mucus layer, detached epithelia, indistinct layers of epithelia, infiltration of inflammatory cells, and apoptotic cells in the overall layers. We concluded that a structurally and functionally altered microenvironment influences olfactory function. We observed the regeneration of the damaged epithelium, and found multilayers of basal cells, indicating that they were activated and proliferating to reconstitute the injured epithelium.

During lactation, adult female mice display aggressive responses toward male intrud-ers, triggered by male- derived chemosensory signals. This aggressive behavior is not shown by pup- sensitized virgin females sharing pup care with dams. The genetic mechanisms underlying the switch from attraction to aggression are unknown. In this work, we investigate the differential gene expression in lactating females ex-pressing maternal aggression compared to pup- sensitized virgin females in the me-dial amygdala (Me), a key neural structure integrating chemosensory and hormonal information. The results showed 197 genes upregulated in dams, including genes encoding hormones such as prolactin, growth hormone, or follicle- stimulating hor-mone, neuropeptides such as galanin, oxytocin, and pro- opiomelanocortin, and genes related to catecholaminergic and cholinergic neurotransmission. In contrast, 99 genes were downregulated in dams, among which we find those encoding for inhibins and transcription factors of the Fos and early growth response families. The gene set analysis revealed numerous Gene Ontology functional groups with higher expres-sion in dams than in pup- sensitized virgin females, including those related with the regulation of the Jak/Stat cascade. Of note, a number of olfactory and vomeronasal receptor genes was expressed in the Me, although without differences between dams and virgins. For prolactin and growth hormone, a qPCR experiment comparing dams, pup- sensitized, and pup- naïve virgin females showed that dams expressed higher lev-els of both hormones than pup- naïve virgins, with pup- sensitized virgins showing intermediate levels. Altogether, the results show important gene expression changes in the Me, which may underlie some of the behavioral responses characterizing ma-ternal behavior.

The accessory olfactory bulb (AOB) plays a critical role in classifying pheromonal signals. Here we identify two previously undescribed sources of aromatase signaling in the AOB: (1) a population of aromatase-expressing neurons in the AOB itself; (2) a tract of aromatase-expressing axons which originate in the ventral medial amygdala (MEA) and terminate in the AOB. Using a retrograde tracer in conjunction with a transgenic strategy to label aromatase-expressing neurons throughout the brain, we found that a single contiguous population of neurons in the ventral MEA provides the only significant feedback by aromatase-expressing neurons to the AOB. This population expresses the estrogen receptor alpha (ERα) and displayed anatomical sex differences in the number of neurons (higher in male mice) and the size of cell bodies (larger in females). Given the previously established relationship between aromatase expression, estrogen signaling, and the function of sexually dimorphic circuits, we suggest that this feedback population is well-positioned to provide neuroendocrine feedback to modulate sensory processing of social stimuli in the AOB.

(1) Background: Hormone-dependent events that occur throughout spermatogenesis during postnatal testis maturation are significant for adult male fertility. Any disturbances in the T/DHT ratio in male progeny born from females fertilized by finasteride-treated male rats (F0:Fin) can result in the impairment of testicular physiology. The goal of this work was to profile the testicular transcriptome in the male filial generation (F1:Fin) from paternal F0:Fin rats. (2) Methods: The subject material for the study were testis from immature and mature male rats born from females fertilized by finasteride-treated rats. Testicular tissues from the offspring were used in microarray analyses. (3) Results: The top 10 genes having the highest and lowest fold change values were mainly those that encoded odoriferous (Olfr: 31, 331, 365, 633, 774, 814, 890, 935, 1109, 1112, 1173, 1251, 1259, 1253, 1383) and vomeronasal (Vmn1r: 50, 103, 210, 211, Vmn2r: 3, 23, 99) receptors and RIKEN cDNA 5430402E10, also known as odorant-binding protein. (4) Conclusions: Finasteride treatment of male adult rats may cause changes in the testicular transcriptome of their male offspring, leading to a defective function of spermatozoa in response to odorant-like signals, which are recently more and more often noticed as significant players in male fertility.

In mammalian reproduction, sexually active males seek female conspecifics, while estrous females try to approach males. This sex-specific response tendency is called sexual preference. In small rodents, sexual preference cues are mainly chemosensory signals, including pheromones. In this article, we review the physiological mechanisms involved in sexual preference for opposite-sex chemosensory signals in well-studied laboratory rodents, mice, rats, and hamsters of both sexes, especially an overview of peripheral sensory receptors, and hormonal and central regulation. In the hormonal regulation section, we discuss potential rodent brain bisexuality, as it includes neural substrates controlling both masculine and feminine sexual preferences, i.e., masculine preference for female odors and the opposite. In the central regulation section, we show the substantial circuit regulating sexual preference and also the influence of sexual experience that innate attractants activate in the brain reward system to establish the learned attractant. Finally, we review the regulation of sexual preference by neuropeptides, oxytocin, vasopressin, and kisspeptin. Through this review, we clarified the contradictions and deficiencies in our current knowledge on the neuroendocrine regulation of sexual preference and sought to present problems requiring further study.

Chemotherapy patients often experience chemosensory changes during and after drug therapy. The chemotherapy drug, cyclophosphamide (CYP), has known cytotoxic effects on sensory and proliferating cells of the taste system. Like the taste system, cells in the olfactory epithelia undergo continuous renewal. Therefore, we asked if a single injection of 75 mg/kg CYP would affect cell proliferation in the anterior dorsomedial region of the main olfactory epithelium (MOE) and the vomeronasal organ (VNO) from 0 to 125 days after injection. Both epithelia showed a decrease in Ki67-labeled cells compared to controls at day 1 and no Ki67+ cells at day 2 postinjection. In the sensory layer of the MOE, cell proliferation began to recover 4 days after CYP injection and by 6 days, the rate of proliferation was significantly greater than controls. Ki67+ cells peaked 30 days postinjection, then declined to control levels at day 45. Similar temporal sequences of initial CYP-induced suppression of cell proliferation followed by elevated rates peaking 30–45 days postinjection were seen in the sustentacular layer of the MOE and all 3 areas (sensory, sustentacular, marginal) of the VNO. CYP affected proliferation in the sensory layer of the MOE more than the sustentacular layer and all 3 areas of the VNO. These findings suggest that chemotherapy involving CYP is capable of affecting cell renewal of the olfactory system and likely contributes to clinical loss of function during and after chemotherapy.

The accessory olfactory bulb (AOB) is the first neural integrative centre of the vomeronasal system (VNS), which is associated primarily with the detection of semiochemicals. Although the rabbit is used as a model for the study of chemocommunication, these studies are hampered by the lack of knowledge regarding the topography, lamination, and neurochemical properties of the rabbit AOB. To fill this gap, we have employed histological stainings: lectin labelling with Ulex europaeus (UEA-I), Bandeiraea simplicifolia (BSI-B4), and Lycopersicon esculentum (LEA) agglutinins, and a range of immunohistochemical markers. Anti-G proteins Gαi2/Gαo, not previously studied in the rabbit AOB, are expressed following an antero-posterior zonal pattern. This places Lagomorpha among the small groups of mammals that conserve a double-path vomeronasal reception. Antibodies against olfactory marker protein (OMP), growth-associated protein-43 (GAP-43), glutaminase (GLS), microtubule-associated protein-2 (MAP-2), glial fibrillary-acidic protein (GFAP), calbindin (CB), and calretinin (CR) characterise the strata and the principal components of the BOA, demonstrating several singular features of the rabbit AOB. This diversity is accentuated by the presence of a unique organisation: four neuronal clusters in the accessory bulbar white matter, two of them not previously characterised in any species (the γ and δ groups). Our morphometric study of the AOB has found significant differences between sexes in the numerical density of principal cells, with larger values in females, a pattern completely opposite to that found in rats. In summary, the rabbit possesses a highly developed AOB, with many specific features that highlight the significant role played by chemocommunication among this species.

Sensory gene families are of special interest for both what they can tell us about molecular evolution and what they imply as mediators of social communication. The vomeronasal type-1 receptors (V1Rs) have often been hypothesized as playing a fundamental role in driving or maintaining species boundaries given their likely function as mediators of intraspecific mate choice, particularly in nocturnal mammals. Here, we employ a comparative genomic approach for revealing patterns of V1R evolution within primates, with a special focus on the small-bodied nocturnal mouse and dwarf lemurs of Madagascar (genera Microcebus and Cheirogaleus, respectively). By doubling the existing genomic resources for strepsirrhine primates (i.e. the lemurs and lorises), we find that the highly speciose and morphologically cryptic mouse lemurs have experienced an elaborate proliferation of V1Rs that we argue is functionally related to their capacity for rapid lineage diversification. Contrary to a previous study that found equivalent degrees of V1R diversity in diurnal and nocturnal lemurs, our study finds a strong correlation between nocturnality and V1R elaboration, with nocturnal lemurs showing elaborate V1R repertoires and diurnal lemurs showing less diverse repertoires. Recognized subfamilies among V1Rs show unique signatures of diversifying positive selection, as might be expected if they have each evolved to respond to specific stimuli. Furthermore, a detailed syntenic comparison of mouse lemurs with mouse (genus Mus) and other mammalian outgroups shows that orthologous mammalian subfamilies, predicted to be of ancient origin, tend to cluster in a densely populated region across syntenic chromosomes that we refer to as a V1R “hotspot.”

Innate immune chemoreceptors of the formyl peptide receptor (Fpr) family are expressed by vomeronasal sensory neurons (VSNs) in the accessory olfactory system. Their biological function and coding mechanisms remain unknown. We show that mouse Fpr3 (Fpr-rs1) recognizes the core peptide motif f-MKKFRW that is predominantly present in the signal sequence of the bacterial protein MgrB, a highly conserved regulator of virulence and antibiotic resistance in Enterobacteriaceae. MgrB peptide can be produced and secreted by bacteria, and is selectively recognized by a subset of VSNs. Exposure to the peptide also stimulates VSNs in freely behaving mice and drives innate avoidance. Our data shows that Fpr3 is required for neuronal detection and avoidance of peptides derived from a conserved master virulence regulator of enteric bacteria., The role of chemoreceptors on vomeronasal neurons are not fully understood. Here the authors show that in mice, the vomeronasal chemoreceptor Fpr3 responds to peptides from the bacterial MgrB protein, and that exposure to these peptides drives an avoidance response.