A single vomeronasal receptor promotes intermale aggression through dedicated hypothalamic neurons.

The pheromonal information received by the vomeronasal system plays a crucial role in regulating social behaviors such as aggression in mice. Despite accumulating knowledge of the brain regions involved in aggression, the specific vomeronasal receptors and the exact neural circuits responsible for pheromone-mediated aggression remain unknown. Here, we identified one murine vomeronasal receptor, Vmn2r53, that is activated by urine from males of various strains and is responsible for evoking intermale aggression. We prepared a purified pheromonal fraction and Vmn2r53 knockout mice and applied genetic tools for neuronal activity recording, manipulation, and circuit tracing to decipher the neural mechanisms underlying Vmn2r53-mediated aggression. We found that Vmn2r53-mediated aggression is regulated by specific neuronal populations in the ventral premammillary nucleus and the ventromedial hypothalamic nucleus. Together, our results shed light on the hypothalamic regulation of male aggression mediated by a single vomeronasal receptor.

Sex differences in main olfactory system pathways involved in psychosexual function.

We summarize literature from animal and human studies assessing sex differences in the ability of the main olfactory system to detect and process sex-specific olfactory signals ("pheromones") that control the expression of psychosexual functions in males and females. A case is made in non primate mammals for an obligatory role of pheromonal signaling via the main olfactory system (in addition to the vomeronasal-accessory olfactory system) in mate recognition and sexual arousal, with male-specific as well as female-specific pheromones subserving these functions in the opposite sex. Although the case for an obligatory role of pheromones in mate recognition and mating among old world primates, including humans, is weaker, we review the current literature assessing the role of putative human pheromones (eg, AND, EST, "copulin"), detected by the main olfactory system, in promoting mate choice and mating in men and women. Based on animal studies, we hypothesize that sexually dimorphic effects of putative human pheromones are mediated via main olfactory inputs to the medial amygdala which, in turn, transmits olfactory information to sites in the hypothalamus that regulate reproduction.

Identification of an Intra- and Inter-specific Tear Protein Signal in Rodents.

Rodents use the vomeronasal olfactory system to acquire both inter- and intra-specific information from the external environment and take appropriate actions. For example, urinary proteins from predator species elicit avoidance in mice, while those from male mice attract female mice. In addition to urinary proteins, recent studies have highlighted the importance of lacrimal proteins for intra-specific communications in mice. However, whether the tear fluid of other species also mediates social signals remains unknown. Here, we show that a lacrimal protein in rats (predators of mice), called cystatin-related protein 1 (ratCRP1), activates the vomeronasal system of mice. This protein is specifically produced by adult male rats in a steroid hormone-dependent manner, activates the vomeronasal system of female rats, and enhances stopping behavior. When detected by mice, ratCRP1 activates the medial hypothalamic defensive circuit, resulting in decreased locomotion coupled with lowered body temperature and heart rate. Notably, ratCRP1 is recognized by multiple murine type 2 vomeronasal receptors, including Vmn2r28. CRISPR/Cas9-mediated deletion of vmn2r28 impaired both ratCRP1-induced neural activation of the hypothalamic center and decrease of locomotor activity in mice. Taken together, these data reveal the neural and molecular basis by which a tear fluid compound in rats affects the behavior of mice. Furthermore, our study reveals a case in which a single compound that mediates an intra-specific signal in a predator species also functions as an inter-specific signal in the prey species.

Afferent projections to the different medial amygdala subdivisions: a retrograde tracing study in the mouse.

The medial amygdaloid nucleus (Me) is a key node in the socio-sexual brain, composed of anterior (MeA), posteroventral (MePV) and posterodorsal (MePD) subdivisions. These subdivisions have been suggested to play a different role in reproductive and defensive behaviours. In the present work we analyse the afferents of the three Me subdivisions using restricted injections of fluorogold in female outbred CD1 mice. The results reveal that the MeA, MePV and MePD share a common pattern of afferents, with some differences in the density of retrograde labelling in several nuclei. Common afferents to Me subdivisions include: the accessory olfactory bulbs, piriform cortex and endopiriform nucleus, chemosensory amygdala (receiving direct inputs from the olfactory bulbs), posterior part of the medial bed nucleus of the stria terminalis (BSTM), CA1 in the ventral hippocampus and posterior intralaminar thalamus. Minor projections originate from the basolateral amygdala and amygdalo-hippocampal area, septum, ventral striatum, several allocortical and periallocortical areas, claustrum, several hypothalamic structures, raphe and parabrachial complex. MeA and MePV share minor inputs from the frontal cortex (medial orbital, prelimbic, infralimbic and dorsal peduncular cortices), but differ in the lack of main olfactory projections to the MePV. By contrast, the MePD receives preferential projections from the rostral accessory olfactory bulb, the posteromedial BSTM and the ventral premammillary nucleus. In summary, the common pattern of afferents to the Me subdivisions and their interconnections suggest that they play cooperative instead of differential roles in the various behaviours (e.g., sociosexual, defensive) in which the Me has been shown to be involved.

Innate Predator Odor Aversion Driven by Parallel Olfactory Subsystems that Converge in the Ventromedial Hypothalamus.

The existence of innate predator aversion evoked by predator-derived chemostimuli called kairomones offers a strong selective advantage for potential prey animals. However, it is unclear how chemically diverse kairomones can elicit similar avoidance behaviors. Using a combination of behavioral analyses and single-cell Ca(2+) imaging in wild-type and gene-targeted mice, we show that innate predator-evoked avoidance is driven by parallel, non-redundant processing of volatile and nonvolatile kairomones through the activation of multiple olfactory subsystems including the Grueneberg ganglion, the vomeronasal organ, and chemosensory neurons within the main olfactory epithelium. Perturbation of chemosensory responses in specific subsystems through disruption of genes encoding key sensory transduction proteins (Cnga3, Gnao1) or by surgical axotomy abolished avoidance behaviors and/or cellular Ca(2+) responses to different predator odors. Stimulation of these different subsystems resulted in the activation of widely distributed target regions in the olfactory bulb, as assessed by c-Fos expression. However, in each case, this c-Fos increase was observed within the same subnuclei of the medial amygdala and ventromedial hypothalamus, regions implicated in fear, anxiety, and defensive behaviors. Thus, the mammalian olfactory system has evolved multiple, parallel mechanisms for kairomone detection that converge in the brain to facilitate a common behavioral response. Our findings provide significant insights into the genetic substrates and circuit logic of predator-driven innate aversion and may serve as a valuable model for studying instinctive fear and human emotional and panic disorders.

Observations on the effects of odours on the homeopathic response.

Samuel Hahnemann described incidences where the homeopathic response was disrupted by noxious smells in the environment. An earlier paper proposed that homeopathic medicines may be sensed by vomeronasal cells (VNCs) i.e. microvillus or brush cells in the vomeronasal organ (VNO), the taste buds and associated with the trigeminal nerve and nervus terminalis. This paper proposes an extension to the theory and suggests that a subset of solitary chemosensory cells (SCCs) in the diffuse chemosensory system (DCS) that is morphologically similar to VNCs might also be receptive to homeopathic medicines. The types of odours that may interfere with this process are described. Two clinical cases of disruption of the homeopathic response are given as examples, showing that successful re-establishment of remedy action can be produced by timely repetition of the medicine. The ramifications on clinical homeopathic practice are discussed.

Human body scents: do they influence our behavior?

Pheromonal communication in the animal world has been of great research interest for a long time. While extraordinary discoveries in this field have been made, the importance of the human sense of smell was of far lower interest. Humans are seen as poor smellers and therefore research about human olfaction remains quite sparse compared with other animals. Nevertheless amazing achievements have been made during the past 15 years. This is a collection of available data on this topic and a controversial discussion on the role of putative human pheromones in our modem way of living. While the focus was definitely put on behavioral changes evoked by putative human pheromones this article also includes other important aspects such as the possible existence of a human vomeronasal organ. If pheromones do have an influence on human behavior there has to be a receptor organ. How are human body scents secreted and turned into odorous substances? And how can con-specifics detect those very odors and transmit them to the brain? Apart from that the most likely candidates for human pheromones are taken on account and their impact on human behavior is shown in various detail.

Sexual behaviour and neuronal activation in the vomeronasal pathway and hypothalamus of food-deprived male rats.

As feeding and mating are mutually-exclusive goal-orientated behaviours, we investigated whether brief food deprivation would impair the display of sexual behaviour of male rats. Analysis of performance in a sexual incentive motivation test revealed that, similar to fed males, food-deprived males preferred spending time in the vicinity of receptive females rather than nonreceptive females. Despite this, food-deprived males were more likely to be slow to mate than normally-fed males, and a low dose of the satiety peptide α-melanocyte-stimulating-hormone attenuated the effect of hunger. Using Fos immunocytochemistry, we compared neuronal activity in the vomeronasal projection pathway in response to oestrous cues from receptive females between food-deprived and fed males. As in fed males, more Fos expression was seen in the rostral part of the bed nucleus of the stria terminalis and in the medial preoptic area in food-deprived males, confirming that food-deprived males can recognise and respond to female oestrous cues. However, although there was also an increase in Fos expression in the bed nucleus of the accessory tract and in the posteromedial amygdala in fed males, no increases were seen in these areas in food-deprived rats. We also found selective attenuation in the activation of lateral posterior paraventricular nucleus (lpPVN) oxytocin neurones in food-deprived males. Taken together, the data show that, although food-deprived males can still become sexually motivated, copulation is delayed, and this is accompanied by variations in neuronal activity in the vomeronasal projection pathway. We propose that, in hungry rats, the lpPVN oxytocin neurones (which project to the spinal cord and are involved in maintaining penile erection) facilitate the transition from motivation to intromission, and their lack of activation impairs intromission, and thus delays mating.

Modulation of gonadotrophin-releasing hormone pulse generator activity by the pheromone in small ruminants.

In small ruminants, such as goats and sheep, a primer pheromone produced by males induces an out-of-seasonal ovulation in anoestrous females, a phenomenon known as the male effect. The male effect is unique in that an external chemical stimulus can immediately modulate the activity of the hypothalamic gonadotrophin-releasing hormone (GnRH) pulse generator. We have established a monitoring method of the GnRH pulse generator activity in Shiba goat. Using this method as a sensitive bioassay to assess the male effect pheromone activity, we have shown that the male effect pheromone is synthesised in an androgen-dependent manner in the sebaceous glands or their vicinity in specific body regions in goats. Although chemical identity of the pheromone is yet to be determined, analyses of male goat hair extracts by gas chromatography fractionation suggest that the male effect pheromone is a volatile substance with relatively small molecular weight. From morphological and molecular biological studies in goats, it is suggested that the pheromone molecule is detected by a member of the V1R family located on both the olfactory neurones and the vomeronasal sensory neurones, and the pheromone signal is conveyed to the medial nucleus of amygdala via the main olfactory and vomeronasal pathways and, subsequently, to the hypothalamic GnRH pulse generator to enhance its activity.

Pheromone signal transduction in humans: what can be learned from olfactory loss.

Because humans seem to lack neuronal elements in the vomeronasal organ (VNO), many scientists believe that humans are unable to detect pheromones. This view is challenged by the observations that pheromone-like compounds, 4,16-androstadien-3-one (AND) and oestra-1,3,5(10),16-tetraen-3-ol (EST), activate the human hypothalamus. Whether these activations are mediated via VNO, venous blood or olfactory mucosa is presently unknown. To disentangle between the three alternatives, we conducted activation studies in 12 heterosexual males with chronic anosmia because of nasal polyps. Polyposis hampers signal transduction via the olfactory mucosa without interfering with the VNO or the pheromone transport via venous blood. Twelve healthy men served as controls. Subjects were investigated with (15)O-H(2)O PET during smelling of odorless air (base line), AND, EST, vanillin, and acetone. Smelling of EST activated the anterior hypothalamus in controls, but not anosmics. Neither did the anosmics display cerebral activations with AND or vanillin. Clusters were detected only with the trigeminal odorant acetone, and only in the thalamus, brainstem, the anterior cingulate, and parts of the sensorimotor cortex. Direct comparisons with controls (controls-anosmics) showed clusters in the olfactory cortex (amygdala and piriform cortex) with AND, vanillin, and acetone, and in the anterior hypothalamus with EST. The observed absence of olfactory and presence of trigeminal activations in anosmics indicates that polyposis primarily affected signal processing via the olfactory mucosa. The anosmics inability to activate the hypothalamus with EST, therefore, suggests that in healthy men EST signals were primarily transmitted via the olfactory system.

V1R and V2R segregated vomeronasal pathways to the hypothalamus.

The vomeronasal system is segregated from the epithelium to the bulb. Two classes of receptor neurons are apically and basally placed in the vomeronasal epithelium, express Gi2alpha and Goalpha proteins and V1R and V2R receptors and project to the anterior and posterior portions of the accessory olfactory bulb, respectively. Apart from common vomeronasal recipient structures in the amygdala, only the anterior accessory olfactory bulb projects to the bed nucleus of the stria terminalis and only the posterior accessory olfactory bulb projects to the dorsal anterior amygdala. The efferent projections from these two amygdaloid structures to the hypothalamus were investigated. These two vomeronasal subsystems mediated by V1R and V2R receptors were partially segregated, not only in amygdala, but also in the hypothalamus.

Fibroblast growth factor 8 signaling through fibroblast growth factor receptor 1 is required for the emergence of gonadotropin-releasing hormone neurons.

GnRH neurons are essential for the onset and maintenance of reproduction. Mutations in both fibroblast growth factor receptor (Fgfr1) and Fgf8 have been shown to cause Kallmann syndrome, a disease characterized by hypogonadotropic hypogonadism and anosmia, indicating that FGF signaling is indispensable for the formation of a functional GnRH system. Presently it is unclear which stage of GnRH neuronal development is most impacted by FGF signaling deficiency. GnRH neurons express both FGFR1 and -3; thus, it is also unclear whether FGFR1 or FGFR3 contributes directly to GnRH system development. In this study, we examined the developing GnRH system in mice deficient in FGF8, FGFR1, or FGFR3 to elucidate the individual contribution of these FGF signaling components. Our results show that the early emergence of GnRH neurons from the embryonic olfactory placode requires FGF8 signaling, which is mediated through FGFR1, not FGFR3. These data provide compelling evidence that the developing GnRH system is exquisitely sensitive to reduced levels of FGF signaling. Furthermore, Kallmann syndrome stemming from FGF signaling deficiency may be due primarily to defects in early GnRH neuronal development prior to their migration into the forebrain.

Courtship pheromone-induced c-Fos-like immunolabeling in the female salamander brain.

Plethodontid salamanders display intricate courtship behaviors. Proteinaceous courtship pheromones were recently discovered in the submandibular (mental) gland of the male Plethodon shermani, the red-legged salamander. Behavioral studies showed that these male pheromones are delivered by direct contact to the female snout and modulate her receptivity during courtship. Previous reports demonstrated that experimental application of courtship pheromones activates vomeronasal sensory neurons in P. shermani. The present study investigated the CNS response to courtship pheromones in that species using immunocytochemical detection of the immediate-early gene product c-Fos. The results show that application of a male gland extract to females activated Fos-like immunolabeling in the extended vomeronasal amygdala of the accessory olfactory system, as well as in the preoptic area and ventromedial hypothalamus; regions of the brain known to mediate reproductive responses in vertebrates. The gland extract additionally activated Fos-like labeling in the raphe median, possibly indicating a serotonergic activation. Application of individual purified courtship pheromone proteins resulted in increases in Fos-like labeling in some of the regions activated by the complete submandibular gland extract, but the pattern of labeling was not as clear as that of the complete extract. Unlike other known vertebrate reproductive pheromones, courtship pheromones in P. shermani were effective only at a high concentration. This could result from the particular mode of pheromone transfer in that species, which involves sustained direct contact between male and female. It is concluded that salamander courtship pheromones exert their influence on behavior through the vomeronasal pathway and its direct projections to the preoptic and hypothalamic regions.

The vomeronasal system in mice: from the nose to the hypothalamus- and back!

The vomeronasal pathway in rodents runs parallel to the main olfactory pathway and mediates responses to different classes of chemosensory stimuli. Both olfactory systems can converge and synergize to control reproductive behaviors and hormonal changes triggered by chemosensory cues. Novel experimental approaches expressing genetic transneuronal tracers in hypothalamic neurons regulating reproduction have set the stage to analyze how chemosensory inputs are integrated in the brain to elicit reproductive behaviors and hormonal changes, and how neuroendocrine status might modulate susceptibility to chemosensory cues.

Olfactory inputs to hypothalamic neurons controlling reproduction and fertility.

In order to gain insight into sensory processing modulating reproductive behavioral and endocrine changes, we have aimed at identifying afferent pathways to neurons synthesizing luteinizing hormone-releasing hormone (LHRH, also known as gonadotropin-releasing hormone [GnRH]), a key neurohormone of reproduction. Injection of conditional pseudorabies virus into the brain of an LHRH::CRE mouse line led to the identification of neuronal networks connected to LHRH neurons. Remarkably, and in contrast to established notions on the nature of LHRH neuronal inputs, our data identify major olfactory projection pathways originating from a discrete population of olfactory sensory neurons but fail to document any synaptic connectivity with the vomeronasal system. Accordingly, chemosensory modulation of LHRH neuronal activity and mating behavior are dramatically impaired in absence of olfactory function, while they appear unaffected in mouse mutants lacking vomeronasal signaling. Further visualization of afferents to LHRH neurons across the brain offers a unique opportunity to uncover complex polysynaptic circuits modulating reproduction and fertility.

Imaging of olfaction and gustation.

Positron emission tomographic (PET) scanning can be used to assess the cortical processing of responses to different odorants in man. The olfactory bulb receives the first projections on an ipsilateral basis from the nose, but then the cortex and the limbic system respond bilaterally. More irritating, burning odors (e.g., acetone) project via the trigeminal rather than the olfactory nerve and induce changes in the brain stem and the cortex. Sweet and salty tastes are readily distinguishable, but a bitter or painful stimulus leads to a deactivation of the primary gustatory cortex, which is in keeping with the need to focus on an avoidance response. The complex discrimination of odors, however, seems to involve the insula cortex, cerebellum, and hypothalamus, with pheromones seemingly sensed via the olfactory system in humans.

Molecular cloning and characterization of protein phosphatase 2C of vomeronasal sensory epithelium of garter snakes.

The earthworm-derived chemoattractant ES20 interacts with its G-protein-coupled receptors on the plasma membrane of vomeronasal (VN) sensory neurons of garter snakes, resulting in an increase in inositol trisphosphate [J. Biol. Chem. 269 (1994) 16867] and a rapid phosphorylation of the membrane-bound proteins, p42/44 [Biochim. Biophys. Acta 1450 (1999) 320]. The phosphorylation of p42/44 proteins are countervailingly regulated by a protein kinase and an okadaic acid-insensitive but fluoride-sensitive protein phosphatase (PPase) [J. Liu et al. (loc. cit.)]. The phosphorylation of p42/44 induced by ES20 appears to play a role in the regulation of signal transduction pathways by modulating the GTPase activity [J. Liu et al. (loc. cit.)]. A 564-bp fragment of cDNA was obtained from VN RNA of garter snakes by reverse transcription polymerase chain reaction with degenerate primers. The 564-bp fragment was amplified, cloned, and sequenced. Northern blot analysis revealed that both the VN organ (VNO) and brain contained the gene of PPase 2C. A full-length complementary 4119-bp DNA containing an open reading frame of 1146bp that encodes a protein of 382 amino acids with a molecular mass of 49,123Da was obtained from the VN cDNA library of garter snakes. The deduced amino acid sequence showed 88% amino acid identity to bovine protein phosphatase 2C alpha and 87% identity to human and rat PP2C alpha and to Mg(2+)-dependent protein phosphatase 1A of rat and rabbit. In situ hybridization revealed that the mRNA of VN protein phosphatase 2C is expressed in the vomeronasal sensory epithelium. This is the first report of the identification of a type 2C serine/threonine protein phosphatase in the VN system.

Neural substrates for processing chemosensory information in snakes.

Snakes interact with their chemical environment through their olfactory and vomeronasal systems. The present report summarizes advances on neural substrates for processing chemosensory information. First, the efferent and centrifugal afferent connections of the main and accessory olfactory bulbs were reinvestigated. Second, the afferent and efferent connections of the nucleus sphericus, the main target of the accessory olfactory bulb, were characterized. The nucleus sphericus gives rise to a very small projection to the hypothalamus, but it does project to other telencephalic structures where olfactory and vomeronasal information could converge. Third, the intra-amygdaloid circuitry and the amygdalo-hypothalamic projections were described. The medial amygdala, for instance, receives both vomeronasal and olfactory inputs and projects to the hypothalamus, namely, to the lateral posterior hypothalamic nucleus. Fourth, because the lateral posterior hypothalamic nucleus projects to the hypoglossal nucleus, the motor center controlling the tongue musculature, this projection could constitute a pathway for chemosensory information to influence tongue-flicking behavior. In summary, vomeronasal information is mostly relayed to the hypothalamus not via the nucleus sphericus but through other telencephalic structures. Convergence of olfactory and vomeronasal information appears to occur at different levels in the telencephalon. A neural substrate for the chemosensory control of tongue-flicking behavior is provided.

Neural substrates for tongue-flicking behavior in snakes.

Snakes deliver odorants to the vomeronasal organ by means of tongue-flicks. The rate and pattern of tongue-flick behavior are altered depending on the chemical context. Accordingly, olfactory and vomeronasal information should reach motor centers that control the tongue musculature, namely, the hypoglossal nucleus (XIIN); however, virtually nothing is known about the circuits involved. In the present work, dextran amines were injected into the tongue of garter snakes (Thamnophis sirtalis) to identify the motoneurons of the XIIN. Tracers were then delivered into the XIIN to identify possible afferents of chemical information. Large injections into the XIIN yielded retrograde labeling in two chemosensory areas: the medial amygdala (MA) and the lateral posterior hypothalamic nucleus (LHN). Smaller injections only yielded labeled neurons in the LHN. In fact, the MA, which receives afferents from the accessory olfactory bulb, the rostroventral lateral cortex, and the nucleus sphericus, projects to the LHN. Injections into the MA did not show terminal labeling in the XIIN but in an area lateral to it. However, injections into the LHN gave rise not only to labeled fibers in the XIIN but also to retrograde labeling in the MA, thus confirming the chemosensory input to LHN. Injecting different fluorescent tracers into the tongue and into the LHN corroborated the projection from the LHN to the XIIN. The present report investigates further connections of the olfactory and vomeronasal systems and describes the afferent connections to XIIN in a nonmammalian vertebrate. The circuit for tongue-flicking behavior described herein should be evaluated using functional studies.

Combined c-fos and 14C-2-deoxyglucose method to differentiate site-specific excitation from disinhibition: analysis of maternal behavior in the rat.

On the basis of evidence that 14C-2-deoxyglucose (2-DG) autoradiography indicates activity at axonal terminals, whereas c-fos immunocytochemistry indicates activity of neuronal cell bodies, we combined these techniques in adjacent histological brain sections to assess excitatory and disinhibitory synaptic relations in selected sites in female rats in which maternal behavior was elicited by natural parturition, sensitization (7- to 10-day cohabitation with foster pups), or hysterectomy. All individuals in these three groups expressed maternal behavior immediately before 2-DG injection. Controls were non-maternal virgins. Parturient and Hysterectomized groups: elevation (compared with controls) in both 2-DG and c-fos activity in medial preoptic area (MPOA) indicated an increase in its input and output activity, i.e., an excitatory interaction; the MPOA was previously shown to be critical for maternal behavior. Sensitized group: a decrease in 2-DG activity of vomeronasal nuclei (bed nucleus of the accessory olfactory tract, BAOT, and medial amygdala, ME, replicating our previous study) and an elevation in c-fos activity, jointly indicate disinhibition of these nuclei, that were previously shown to modulate pup-chemostimulation-induced sensitization. All other sites showed evidence of excitatory input-output relationships (i.e., joint increase in both 2-DG and c-fos activity), e.g., bed nucleus of the stria terminalis (BNST), lateral habenula (LHAB), central gray (CG), thalamus (THAL), septum (SEPT), and ventral tegmental area (VTA). The present study demonstrates the feasibility of measuring 2-DG and c-fos activity jointly in adjacent sections of the same brain, thereby providing evidence to distinguish between localized excitation and disinhibition.