Functional microanatomy of the vomeronasal complex of bats.

Recently, Yohe and Krell (The Anatomical Record, vol. 306:2765-2780) lamented the incongruence between genetics and morphology in the vomeronasal system of bats. Here, we studied 105 bat species from 19 families using histology, iodine-enhanced computed tomography (CT), and/or micro-CT. We focused on structural elements that support a functional peripheral vomeronasal receptor organ (vomeronasal organ [VNO]), together comprising the "vomeronasal complex." Our results support prior studies that describe a functional VNO in most phyllostomid bats, miniopterids, and some mormoopids (most known Pteronotus spp.). All of these species (or congeners, at least) have vomeronasal nerves connecting the VNO with the brain and some intact genes related to a functional VNO. However, some bats have VNOs that lack a neuroepithelium and yet still possess elements that aid VNO function, such as a "capsular" morphology of the vomeronasal cartilages (VNCs), and even large venous sinuses, which together facilitate a vasomotor pump mechanism that can draw fluid into the VNO. We also show that ostensibly functionless VNOs of some bats are developmentally associated with ganglionic masses, perhaps involved in endocrine pathways. Finally, we demonstrate that the capsular VNC articulates with the premaxilla or maxilla, and that these bones bear visible grooves denoting the location of the VNC. Since these paraseptal grooves are absent in bats that have simpler (bar-shaped or curved) VNCs, this trait could be useful in fossil studies. Variable retention of some but not all "functional" elements of the vomeronasal complex suggests diverse mechanisms of VNO loss among some bat lineages.

Incubation time, embryonic development and the vomeronasal organ of the Laysan albatross (Phoebastria immutabilis).

The effect of lengthened incubation periods on embryonic development, especially vestigial structures, is poorly understood. An example of which is the avesuchian vomeronasal organ (VNO), a nasal chemosensory organ found in many tetrapods but absent in adult avesuchians (crocodilians and birds) in whom it is presumed to be a transitory fetal structure. The Laysan Albatross (Phoebastria immutabilis) has an incubation period of their eggs of about 65 days. This incubation period is twice that of domestic fowl, wherein a putative VNO has been documented as an epithelial thickening. The purpose of this study is to document the development of a putative VNO in the albatross. Serial histological sections of nine albatross embryonic heads, across 6 stages (representing days 19 to 32: stages 31-39), were examined. A paired putative VNO was present as a short, tubular structure in the anterodorsal aspect on either side of the nasal septum from stage 32 onwards, getting steadily longer in later specimens. At the earliest stages, the epithelial walls of the tube resemble a neuroepithelium, but then becomes thinner and simpler in morphology. Based on our available age range, it is unclear whether it persists as a rudimentary structure (like that of the human) or if it is a transitory structure (like in chickens) in these mid embryonic stages. Though future studies must determine the fate of the Laysan albatross VNO (e.g., is it retained postnatally?), the role of incubation period length on embryonic development is a bigger question to be explored.

Enhanced Thermoelectric Properties of Coated Vanadium Oxynitride Nanoparticles/PEDOT:PSS Hybrid Films.

Thermoelectric (TE) materials transform thermal energy into electricity, which can play an important role for global sustainability. Conducting polymers are suitable for the preparation of flexible TE materials because of their low-cost, lightweight, flexible, and easily synthesized properties. Here, we fabricate organic-inorganic hybrids by combining vanadium oxynitride nanoparticles coated with nitrogen-doped carbon (NC@VNO) and poly(3,4-ethylenedioxy thiophene):poly(styrene sulfonate) (PEDOT:PSS). We find that the electrical conductivity, Seebeck coefficient, and power factor of the NC@VNO/PEDOT:PSS film can be enhanced up to 4158 S/cm, 45.8 µV/K, and 873 µW/mK2 at 380 K, respectively. The large enhancement of the power factor may be due to the facilitation of the interfacial charge transport tunnel between the NC@VNO nanoparticles and PEDOT:PSS. The improvement of the Seebeck coefficient may be due to the energy filter effect as induced by interfacial contact and internal electric field between the NC@VNO nanoparticles and PEDOT:PSS. Our measurement suggests that the high binding energy of pyrrolic-N enhances the Seebeck coefficient, and the high binding energy of oxide-N increases electrical conductivity.

Cutaneous Squamous Cell Carcinoma Arising in Immunosuppressed Patients: A Systematic Review of Tumor Profiling Studies.

As solid organ transplantation becomes more prevalent, more individuals are living as members of the immunosuppressed population with an elevated risk for cutaneous squamous cell carcinoma (cSCC). Although great progress has been made in understanding the pathogenesis of cSCC in general, little is known about the drivers of tumorigenesis in immunosuppressed patients and organ-transplant recipients, specifically. This systematic review sought to synthesize information regarding the genetic and epigenetic alterations as well as changes in protein and mRNA expression that place this growing population at risk for cSCC, influence treatment response, and promote tumor aggressiveness. This review will provide investigators with a framework to identify future areas of investigation and clinicians with additional insight into how to best manage these patients.

Analysis of the vomeronasal organ transcriptome reveals variable gene expression depending on age and function in rabbits.

The vomeronasal organ (VNO) is a chemosensory organ specialized in pheromone detection that shows a broad morphofunctional and genomic diversity among mammals. However, its expression patterns have only been well-characterized in mice. Here, we provide the first comprehensive RNA sequencing study of the rabbit VNO across gender and sexual maturation stages. We characterized the VNO transcriptome, updating the number and expression of the two main vomeronasal receptor families, including 128 V1Rs and 67 V2Rs. Further, we defined the expression of formyl-peptide receptor and transient receptor potential channel families, both known to have specific roles in the VNO. Several sex hormone-related pathways were consistently enriched in the VNO, highlighting the relevance of this organ in reproduction. Moreover, whereas juvenile and adult VNOs showed significant transcriptome differences, male and female did not. Overall, these results contribute to understand the genomic basis of behavioural responses mediated by the VNO in a non-rodent model.

Embryology of the naso-palatal complex in Gekkota based on detailed 3D analysis in Lepidodactylus lugubris and Eublepharis macularius.

The vomeronasal organ (VNO), nasal cavity, lacrimal duct, choanal groove, and associated parts of the superficial (soft tissue) palate are called the naso-palatal complex. Despite the morphological diversity of the squamate noses, little is known about the embryological basis of this variation. Moreover, developmental data might be especially interesting in light of the morpho-molecular discordance of squamate phylogeny, since a 'molecular scenario' implies an occurrence of unexpected scale of homoplasy also in olfactory systems. In this study, we used X-ray microtomography and light microscopy to describe morphogenesis of the naso-palatal complex in two gekkotans: Lepidodactylus lugubris (Gekkonidae) and Eublepharis macularius (Eublepharidae). Our embryological data confirmed recent findings about the nature of some developmental processes in squamates, for example, involvement of the lateral nasal prominence in the formation of the choanal groove. Moreover, our study revealed previously unknown differences between the studied gekkotans and allows us to propose redefinition of the anterior concha of Sphenodon. Interpretation of some described conditions might be problematic in the phylogenetic context, since they represent unknown: squamate, nonophidian squamate, or gekkotan features.

The Nasopalatine Ducts Are Required for Proper Pheromone Signaling in Mice.

The vomeronasal organ (VNO) specializes in detection of chemosignals, mainly pheromones, which control social communication and reproduction in many mammals. These pheromones must solubilize with nasal fluids before entering the VNO, and it was suggested that they are delivered to and cleared from the VNO by active pumping. Yet, the details of this pheromone delivery process are unclear. In this study, we first constructed a high-resolution 3D morphological image of the whole adult mouse snout, by using ultra-high-resolution micro-CT. We identified a net of micro tunnels starting from the nostrils and extending around and through the VNO. These micro tunnels connect the nasal cavity with the VNO and the oral cavity via the nasopalatine ducts (NPD). Other micro tunnels connect the nasal cavity to the main olfactory epithelium. We next demonstrated that physical obstruction of the NPD severely impairs the clearance of dissolved compounds from the VNO lumen. Moreover, we found that mice with blocked NPD display alterations in chemosignaling-evoked neuronal activation in brain regions associated with the vomeronasal system. Finally, NPD-blocked male mice exhibit reduced preference for female chemosignals, and impaired social interaction behavior. Taken together, our findings indicate that the NPD in mice are connected to both the nasal and oral cavity, serving an essential role in regulating the flow of soluble chemosignals through the VNO, and are required for proper pheromone-mediated social communication.

The Role of Urine in Semiochemical Communication between Females and Males of Domestic Dog (Canis familiaris) during Estrus.

This study aimed to assess the mechanisms of semiochemical signal detection in dogs. In the first experiment, five males were exposed to volatile semiochemicals emitted by a live female in estrus and the female's urine sample collected during estrus. The odor of canine food and clean air were used as controls. In the second experiment, 25 males could directly sniff and lick the urine samples from females in estrus, from females in anestrus, from males and from humans, placed in a lineup. Sniffing, licking and salivation, as well as keeping dogs at different distances from the source of odor, were recorded in both experiments. Experiment 1 showed that food odor was sniffed by males longer than estrous urine. Volatile semiochemicals from females in estrus evoked interest in males but without visual cues did not cause overt symptoms of sexual arousal. In Experiment 2, the estrous urine evoked interest in males and provoked significantly longer sniffing. Licking accompanied by salivation was observed in all instances only during direct contact with estrous urine. The results suggest a complex character of detection of female reproductive status, in which both volatile and nonvolatile compounds emitted by females and present in female urine are involved.

Development of the squamate naso-palatal complex: detailed 3D analysis of the vomeronasal organ and nasal cavity in the brown anole Anolis sagrei (Squamata: Iguania).

BACKGROUND: Despite the diverse morphology of the adult squamate naso-palatal complex - consisting of the nasal cavity, vomeronasal organ (VNO), choanal groove, lacrimal duct and superficial palate - little is known about the embryology of these structures. Moreover, there are no comprehensive studies concerning development of the nasal cavity and VNO in relation to the superficial palate. In this investigation, we used X-ray microtomography and histological sections to describe embryonic development of the naso-palatal complex of iguanian lizard, the brown anole (Anolis sagrei). The purpose of the study was to describe the mechanism of formation of adult morphology in this species, which combines the peculiar anole features with typical iguanian conditions. Considering the uncertain phylogenetic position of the Iguania within Squamata, embryological data and future comparative studies may shed new light on the evolution of this large squamate clade. RESULTS: Development of the naso-palatal complex was divided into three phases: early, middle and late. In the early developmental phase, the vomeronasal pit originates from medial outpocketing of the nasal pit, when the facial prominences are weakly developed. In the middle developmental phase, the following events can be noted: the formation of the frontonasal mass, separation of the vestibulum, appearance of the lacrimal duct, and formation of the choanal groove, which leads to separation of the VNO from the nasal cavity. In late development, the nasal cavity and the VNO attain their adult morphology. The lacrimal duct establishes an extensive connection with the choanal groove, which eventually becomes largely separated from the oral cavity. CONCLUSIONS: Unlike in other tetrapods, the primordium of the lacrimal duct in the brown anole develops largely beyond the nasolacrimal groove. In contrast to previous studies on squamates, the maxillary prominence is found to participate in the initial fusion with the frontonasal mass. Moreover, formation of the choanal groove occurs due to the fusion of the vomerine cushion to the subconchal fold, rather than to the choanal fold. The loss or significant reduction of the lateral nasal concha is secondary. Some features of anole adult morphology, such as the closure of the choanal groove, may constitute adaptations to vomeronasal chemoreception.

MRI Features of the Vomeronasal Organ in Dogs (Canis Familiaris).

According to current knowledge, the vomeronasal organ (VNO, Jacobson's organ) is the structure responsible for semiochemical signal detection. In dogs and other mammals, it is located close to the vomer and palatine processes of the incisive and maxillary bones. Although there are reports describing the anatomy and histology of this structure, there are limited available reports assessing this organ in live individuals and no direct visualization reports in dogs. The aim of this study was 2-fold: (1) preparation and optimization of a protocol for magnetic resonance imaging (MRI) examination of the VNO in a cadaver study with precise visualization and localization, and (2) characterization of the physiological VNO image features in MRI of live dogs. The first part of the study was performed on 10 beagle cadavers, the second on 8 live beagle dogs. For the VNO visualization, a 1.5T MRI (Philips® Ingenia) scanner and 20-channel digital head-neck spine coil were used (Philips®, Holland). The cadaver study allowed confirmation of the organ's location by the topical application of an MRI contrast agent (gadolinium) via the external entrance of the VNO canal. Accurate delineation of the VNO was obtained using a high resolution submillimeter three-dimensional T1-fast field echo (FFE) 3D sequence. Imaging of the VNO in 8 living dogs allowed the description of the morphological MRI features and direct evaluation of its shape and size. The results obtained demonstrate the ability to visualize the VNO in vivo and to evaluate its structure in dogs.

Puberty is a Critical Period for Vomeronasal Organ Mediation of Socio-sexual Behavior in Mice.

Genetic disruption of the vomeronasal organ (VNO), an organ responsible for pheromone processing, drastically alters socio-sexual behavior in mice. However, it is not known whether the VNO has a role during the pubertal organizational period when sex-typical socio-sexual behaviors emerge, or if disruption of the organ in adulthood is sufficient to alter socio-sexual behavior. To bypass the lifelong VNO disruption of genetic knockout models, we surgically ablated the VNO of male and female mice either during the peripubertal period [postnatal day (PND) 28-30] or adulthood (PND 58-60), with sham controls at both ages. We ruled out anosmia via the buried food test and assessed sexual odor preferences by simultaneously exposing mice to same- and opposite-sex soiled-bedding. We then measured territorial aggression with the resident-intruder paradigm and assessed sexual behavior in response to an encounter with an estrus-induced female. Neural activity approximated by FOS-immunoreactivity along the VNO-accessory olfactory pathway was measured in response to opposite-sex odors. We found that peripubertal VNO ablation decreased sexual odor preferences and neural activity in response to opposite-sex odors, and drastically reduced territorial aggression in male mice. Conversely, adult VNO ablation resulted in subtle differences in sexual odor preferences compared with sham controls. Regardless of the VNO condition, mice displayed sex-typical copulatory behaviors. Together, these results suggest that puberty is a critical period in development whereby the VNO contributes to the sexual differentiation of behavior and neural response to conspecific odors.

Virus-Mediated Overexpression of Vomeronasal Receptors and Functional Assessment by Live-Cell Calcium Imaging.

The mammalian vomeronasal organ (VNO) detects and transduces molecular cues emitted by other individuals that influence social behaviors such as mating and aggression. The detection of these chemosignals involves recognition of specific ligands by dedicated G protein-coupled receptors. Here, we describe recent methodological advances using a herpes virus-based amplicon delivery system to overexpress vomeronasal receptor genes in native, dissociated VNO neurons and to characterize corresponding cell responses to potential ligands through Ca2+ imaging. This methodology enables us to analyze the response patterns of single vomeronasal receptors to a large number of chemosensory stimuli.

Biologically meaningful scents: a framework for understanding predator-prey research across disciplines.

Fear of predation is a universal motivator. Because predators hunt using stealth and surprise, there is a widespread ability among prey to assess risk from chemical information - scents - in their environment. Consequently, scents often act as particularly strong modulators of memory and emotions. Recent advances in ecological research and analytical technology are leading to novel ways to use this chemical information to create effective attractants, repellents and anti-anxiolytic compounds for wildlife managers, conservation biologists and health practitioners. However, there is extensive variation in the design, results, and interpretation of studies of olfactory-based risk discrimination. To understand the highly variable literature in this area, we adopt a multi-disciplinary approach and synthesize the latest findings from neurobiology, chemical ecology, and ethology to propose a contemporary framework that accounts for such disparate factors as the time-limited stability of chemicals, highly canalized mechanisms that influence prey responses, and the context within which these scents are detected (e.g. availability of alternative resources, perceived shelter, and ambient physical parameters). This framework helps to account for the wide range of reported responses by prey to predator scents, and explains, paradoxically, how the same individual predator scent can be interpreted as either safe or dangerous to a prey animal depending on how, when and where the cue was deposited. We provide a hypothetical example to illustrate the most common factors that influence how a predator scent (from dingoes, Canis dingo) may both attract and repel the same target organism (kangaroos, Macropus spp.). This framework identifies the catalysts that enable dynamic scents, odours or odorants to be used as attractants as well as deterrents. Because effective scent tools often relate to traumatic memories (fear and/or anxiety) that cause future avoidance, this information may also guide the development of appeasement, enrichment and anti-anxiolytic compounds, and help explain the observed variation in post-traumatic-related behaviours (including post-traumatic stress disorder, PTSD) among diverse terrestrial taxa, including humans.

Population genetics of mouse lemur vomeronasal receptors: current versus past selection and demographic inference.

BACKGROUND: A major effort is underway to use population genetic approaches to identify loci involved in adaptation. One issue that has so far received limited attention is whether loci that show a phylogenetic signal of positive selection in the past also show evidence of ongoing positive selection at the population level. We address this issue using vomeronasal receptors (VRs), a diverse gene family in mammals involved in intraspecific communication and predator detection. In mouse lemurs, we previously demonstrated that both subfamilies of VRs (V1Rs and V2Rs) show a strong signal of directional selection in interspecific analyses. We predicted that ongoing sexual selection and/or co-evolution with predators may lead to current directional or balancing selection on VRs. Here, we re-sequence 17 VRs and perform a suite of selection and demographic analyses in sympatric populations of two species of mouse lemurs (Microcebus murinus and M. ravelobensis) in northwestern Madagascar. RESULTS: M. ravelobensis had consistently higher genetic diversity at VRs than M. murinus. In general, we find little evidence for positive selection, with most loci evolving under purifying selection and one locus even showing evidence of functional loss in M. ravelobensis. However, a few loci in M. ravelobensis show potential evidence of positive selection. Using mismatch distributions and expansion models, we infer a more recent colonisation of the habitat by M. murinus than by M. ravelobensis, which most likely speciated in this region earlier on. CONCLUSIONS: These findings suggest that the analysis of VR variation is useful in inferring demographic and phylogeographic history of mouse lemurs. In conclusion, this study reveals a substantial heterogeneity over time in selection on VR loci, suggesting that VR evolution is episodic.

Vomeronasal signal deficiency enhances parental behavior in socially isolated male mice.

We previously reported that social isolation promotes parental care in sexually naïve male mice. This effect was blocked by exposure to chemosensory and auditory social signals derived from males in an adjacent compartment. In the present study, we examined whether the chemosensory signals detected in the vomeronasal organ (VNO) are involved in parental behavior by using mice deficient for a VNO-specific ion channel (Trpc2-/-) and thus impaired in VNO-input signaling. We housed virgin homozygous Trpc2-/- and heterozygous Trpc2± males for 3weeks during puberty (5-8weeks old) alone or in groups of 3-5 males. At 8weeks of age, the mice were placed with three pups in an observation cage and tested for parental behavior. The Trpc2-/- males housed under isolated conditions spent significantly longer in the vicinity of pups than did the Trpc2-/- males than had been group housed, whereas no isolation effect was observed in heterozygous Trpc2± males. Both Trpc2 knockout and isolation housing significantly increased the time males spent licking pups and crouching (arched back posture over pups to enable nursing), whereas only isolation housing increased the incidence of retrieval behavior. These results demonstrated that social signals transmitted not only through the VNO but also from other modalities, independent of each other, suppress the expression of parental behavior during puberty in sexually naïve males.

Vomeronasal Receptors in Vertebrates and the Evolution of Pheromone Detection.

Pheromones were identified as chemical signals used for intraspecific communication in insects (e.g., sexual attraction) in the 1950s. However, only almost 40 years later the vomeronasal receptors type-1 (V1R) and type-2 (V2R) were identified, usually associated with the presence of a vomeronasal organ (VNO). VRs are widespread in amphibians, reptiles, and mammals, but birds lost the VNO. Similarly, fishes lack VRs and a VNO but can still detect pheromones, instead using the olfactory receptors related to class A and class C G protein-coupled receptors. Here, we review recent evidence on VR repertoire contraction/expansion in vertebrates. We assess the association between VNO development and VR repertoire size. Phylogenetic relationships and selective pressures illuminate the dynamic evolutionary history of the VRs in vertebrates.

Analysis of the vomeronasal receptor repertoire, expression and allelic diversity in swine.

Here we report a comprehensive analysis of the vomeronasal receptor repertoire in pigs. We identified a total of 25 V1R sequences consisting of 10 functional genes, 3 pseudogenes, and 12 partial genes, while functional V2R and FPR genes were not present in the pig genome. Pig V1Rs were classified into three subfamilies, D, F, and J. Using direct high resolution sequencing-based typing of all functional V1Rs from 10 individuals of 5 different breeds, a total of 24 SNPs were identified, indicating that the allelic diversity of V1Rs is much lower than that of the olfactory receptors. A high expression level of V1Rs was detected in the vomeronasal organ (VNO) and testes, while a low expression level of V1Rs was observed in all other tissues examined. Our results showed that pigs could serve as an interesting large animal model system to study pheromone-related neurobiology because of their genetic simplicity.

Recognition of bacterial signal peptides by mammalian formyl peptide receptors: a new mechanism for sensing pathogens.

Formyl peptide receptors (FPRs) are G-protein-coupled receptors that function as chemoattractant receptors in innate immune responses. Here we perform systematic structure-function analyses of FPRs from six mammalian species using structurally diverse FPR peptide agonists and identify a common set of conserved agonist properties with typical features of pathogen-associated molecular patterns. Guided by these results, we discover that bacterial signal peptides, normally used to translocate proteins across cytoplasmic membranes, are a vast family of natural FPR agonists. N-terminally formylated signal peptide fragments with variable sequence and length activate human and mouse FPR1 and FPR2 at low nanomolar concentrations, thus establishing FPR1 and FPR2 as sensitive and broad signal peptide receptors. The vomeronasal receptor mFpr-rs1 and its sequence orthologue hFPR3 also react to signal peptides but are much more narrowly tuned in signal peptide recognition. Furthermore, all signal peptides examined here function as potent activators of the innate immune system. They elicit robust, FPR-dependent calcium mobilization in human and mouse leukocytes and trigger a range of classical innate defense mechanisms, such as the production of reactive oxygen species, metalloprotease release, and chemotaxis. Thus, bacterial signal peptides constitute a novel class of immune activators that are likely to contribute to mammalian immune defense against bacteria. This evolutionarily conserved detection mechanism combines structural promiscuity with high specificity and enables discrimination between bacterial and eukaryotic signal sequences. With at least 175,542 predicted sequences, bacterial signal peptides represent the largest and structurally most heterogeneous class of G-protein-coupled receptor agonists currently known for the innate immune system.

Physiological characterization of formyl peptide receptor expressing cells in the mouse vomeronasal organ.

The mouse vomeronasal organ (VNO) is a chemosensory structure that detects both hetero- and conspecific social cues. Based on largely monogenic expression of either type 1 or 2 vomeronasal receptors (V1Rs/V2Rs) or members of the formyl peptide receptor (FPR) family, the vomeronasal sensory epithelium harbors at least three neuronal subpopulations. While various neurophysiological properties of both V1R- and V2R-expressing neurons have been described using genetically engineered mouse models, the basic biophysical characteristics of the more recently identified FPR-expressing vomeronasal neurons have not been studied. Here, we employ a transgenic mouse strain that coexpresses an enhanced variant of yellow fluorescent protein together with FPR-rs3 allowing to identify and analyze FPR-rs3-expressing neurons in acute VNO tissue slices. Single neuron electrophysiological recordings allow comparative characterization of the biophysical properties inherent to a prototypical member of the FPR-expressing subpopulation of VNO neurons. In this study, we provide an in-depth analysis of both passive and active membrane properties, including detailed characterization of several types of voltage-activated conductances and action potential discharge patterns, in fluorescently labeled vs. unmarked vomeronasal neurons. Our results reveal striking similarities in the basic (electro) physiological architecture of both transgene-expressing and non-expressing neurons, confirming the suitability of this genetically engineered mouse model for future studies addressing more specialized issues in vomeronasal FPR neurobiology.