Vomeronasal Input Research Articles

In vivo stimulus presentation to the mouse vomeronasal system: Surgery, experiment, setup, and software

BackgroundAchieving controlled stimulus delivery is a major challenge in the physiological analysis of the vomeronasal system (VNS). New methodWe provide a comprehensive description of a setup allowing controlled stimulus delivery into the vomeronasal organ (VNO) of anesthetized mice. VNO suction is achieved via electrical stimulation of the sympathetic nerve trunk (SNT) using cuff electrodes, followed by flushing of the nasal cavity. Successful application of this methodology depends on several aspects including the surgical preparation, fabrication of cuff electrodes, experimental setup modifications, and the stimulus delivery and flushing. Here, we describe all these aspects in sufficient detail to allow other researchers to readily adopt it. We also present a custom written MATLAB based software with a graphical user interface that controls all aspects of the actual experiment, including trial sequencing, hardware control, and data logging. ResultsThe method allows measurement of stimulus evoked sensory responses in brain regions that receive vomeronasal inputs. An experienced investigator can complete the entire surgical procedure within thirty minutes. Comparison with existing methodsThis is the only approach that allows repeated and controlled stimulus delivery to the intact VNO, employing the natural mode of stimulus uptake. The approach is economical with respect to stimuli, requiring stimulus volumes as low as 1–2μl. ConclusionsThis comprehensive description will allow other investigators to adapt this setup to their own experimental needs and can thus promote our physiological understanding of this fascinating chemosensory system. With minor changes it can also be adapted for other rodent species.

Characteristic Response to Chemosensory Signals in GABAergic Cells of Medial Amygdala Is Not Driven by Main Olfactory Input.

Chemosensory stimuli from same species (conspecific) and different species (heterospecific) elicit categorically different immediate-early gene (IEG) response patterns in medial amygdala in male hamsters and mice. All heterospecific stimuli activate anterior medial amygdala (MeA) but only especially salient heterospecific stimuli, such as those from predators activate posterior medial amygdala (MeP). We previously reported that characteristic patterns of response in separate populations of cells in MeA and MeP distinguish between different conspecific stimuli. Both gamma aminobutyric acid (GABA)-immunoreactive (ir) cells and GABA-receptor-ir cells make this distinction. Here, using zinc sulfate lesions of the main olfactory epithelium, we show evidence that main olfactory input does not contribute to the characteristic patterns of response in GABA-ir cells of male hamster amygdala, either for conspecific or heterospecific stimuli. Some GABAergic cells are output neurons carrying information from medial amygdala to behavioral executive regions of basal forebrain. Thus, the differential response to different conspecific signals can lead to differential activation of downstream circuits based on nonolfactory input. Finally, we show that an intact vomeronasal organ is necessary and sufficient to produce the characteristic patterns of response to conspecific and heterospecific chemosensory stimuli in hamster medial amygdala. Although main olfactory input may be critical in species with less prominent vomeronasal input for equivalent medial amygdala responses, work presented here suggests that hamster medial amygdala uses primarily vomeronasal input to discriminate between important unlearned conspecific social signals, to distinguish them from the social signals of other species, and may convey that information to brain circuits eliciting appropriate social behavior.

Functional organization of glomerular maps in the mouse accessory olfactory bulb.

SummaryThe mammalian accessory olfactory system (AOS) extracts information about species, sex, and individual identity from social odors, but its functional organization remains unclear. We imaged presynaptic Ca2+ signals in vomeronasal inputs to the accessory olfactory bulb (AOB) during peripheral stimulation using light sheet microscopy. Urine- and steroid-responsive glomeruli densely innervated the anterior AOB. Glomerular activity maps for sexually mature female mouse urine overlapped maps for juvenile and/or gonadectomized urine of both sexes, whereas maps for sexually mature male urine were highly distinct. Further spatial analysis revealed a complicated organization involving selective juxtaposition and dispersal of functionally-grouped glomerular classes. Glomeruli that were similarly tuned to urines were often closely associated, whereas more disparately tuned glomeruli were selectively dispersed. Maps to a panel of sulfated steroid odorants identified tightly-juxtaposed groups that were disparately tuned and dispersed groups that were similarly tuned. These results reveal a modular, non-chemotopic spatial organization in the AOB.

Focal lesions within the ventral striato-pallidum abolish attraction for male chemosignals in female mice

In rodents, socio-sexual behaviour is largely mediated by chemosensory cues, some of which are rewarding stimuli. Female mice display an innate attraction towards male chemosignals, dependent on the vomeronasal system. This behaviour likely reflects the hedonic value of sexual chemosignals. The anteromedial aspect of the olfactory tubercle, along with its associated islands of Calleja, receives vomeronasal inputs and sexually-dimorphic vasopressinergic innervation. Thus, we hypothesised that this portion of the ventral striato-pallidum, known to be involved in reward processing, might be important for sexual odorant-guided behaviours. In this study, we demonstrate that lesions of this region, but not of regions in the posterolateral striato-pallidum, abolish the attraction of female mice for male chemosignals, without affecting significantly their preference for a different natural reward (a sucrose solution). These results show that, at least in female mice, the integrity of the anterior aspect of the medioventral striato-pallidum, comprising a portion of the olfactory tubercle and associated islands of Calleja, is necessary for the attraction for male chemosignals. We suggest that this region contributes to the processing of the hedonic properties of biologically significant odorants.

Both olfactory epithelial and vomeronasal inputs are essential for activation of the medial amygdala and preoptic neurons of male rats

Chemosensory inputs signaling volatile and nonvolatile molecules play a pivotal role in sexual and social behavior in rodents. We have demonstrated that olfactory preference in male rats, that is, attraction to receptive female odors, is regulated by the medial amygdala (MeA), the cortical amygdala (CoA), and the preoptic area (POA). In this paper, we investigated the involvement of two chemosensory organs, the olfactory epithelium (OE) and the vomeronasal organ (VNO), in olfactory preference and copulatory behavior in male rats. We found that olfactory preferences were impaired by zinc sulfate lesion of the OE but not surgical removal of the VNO. Copulatory behaviors, especially intromission frequency and ejaculation, were also suppressed by zinc sulfate treatment. Neuronal activation in the accessory olfactory bulb (AOB), the MeA, the CoA, and the POA was analyzed after stimulation by airborne odors or soiled bedding of estrous females using cFos immunohistochemistry. Although the OE and VNO belong to different neural systems, the main and accessory olfactory systems, respectively, both OE lesion and VNO removal almost equally suppressed the number of cFos-immunoreactive cells in those areas that regulate olfactory preference. These results suggest that signals received by the OE and VNO interact and converge in the early stage of olfactory processing, in the AOB and its targets, although they have distinct roles in the regulation of social behaviors.

High convergence of olfactory and vomeronasal influence in the telencephalon of the terrestrial salamander Plethodon shermani

Previous work suggested that the telencephalic pathways of the main olfactory and vomeronasal systems of vertebrates are mostly isolated from each other, with the possible exception of convergence of the two systems into a small part of the olfactory amygdala. We tested the hypothesis of convergence between the main olfactory and vomeronasal systems by investigating the physiology of telencephalic olfactory responses in an in vitro brain preparation of the salamander Plethodon shermani. This animal was chosen because its olfactory and vomeronasal nerves can be separated and stimulated independently. The nerves were stimulated by short current pulses delivered through suction electrodes. Evoked field potentials and intracellular responses were systematically recorded in the telencephalon. The results showed an abundant overlap of olfactory and vomeronasal nerve-evoked field potentials in the ipsilateral lateral telencephalon and the amygdala. Single neurons receiving bimodal main olfactory and vomeronasal input were found in the dorsolateral telencephalon and amygdala. A classification of response latencies suggested that a subset of these neurons received direct input from both the main and accessory olfactory bulbs. Unimodal excitatory main olfactory responses were mostly found in neurons of the caudal telencephalic pole, but were also present in the striato-pallial transition area/lateral pallium region and striatum. Unimodal excitatory vomeronasal responses were found in neurons of the striato-pallial transition area, vomeronasal amygdala, and caudal amygdala. We conclude that the main olfactory and vomeronasal systems are extensively integrated within the salamander telencephalon and probably act in concert to modulate behavior.

The forked tongue and edge detection in snakes (Crotalus oreganus): An experimental test.

Many stimulus-detection systems are lateralized to allow for simultaneous comparison of paired stimuli. It has been hypothesized that the deeply forked tongue of snakes and some derived lizards functions as a chemical edge detector where cues gathered by each tine are kept separate to provide two points of lateral odor assessment by the central nervous system via vomeronasal input. While following a chemical trail, one time can be on the trail, the other off, and such differential information prompts the snake to turn back to the trail. The authors tested this hypothesis in rattlesnakes within a predatory context by unilaterally severing the vomeronasal nerves. If edge detection is used by snakes during prey trailing, then unilateral denervation should disrupt trailing ability. The authors found no change in the seven separate trailing parameters measured. Therefore, they found no support for the edge detection hypothesis as it applies to prey trailing behavior. Instead, the deeply forked tongue may represent a chemosensory specialization to increase odor-sampling area, with snakes and derived lizards detecting only the concentration of chemical trails.

Convergence of olfactory and vomeronasal projections in the rat basal telencephalon

Olfactory and vomeronasal projections have been traditionally viewed as terminating in contiguous non-overlapping areas of the basal telencephalon. Original reports, however, described areas such as the anterior medial amygdala where both chemosensory afferents appeared to overlap. We addressed this issue by injecting dextran amines in the main or accessory olfactory bulbs of rats and the results were analyzed with light and electron microscopes. Simultaneous injections of different fluorescent dextran amines in the main and accessory olfactory bulbs were performed and the results were analyzed using confocal microscopy. Similar experiments with dextran amines in the olfactory bulbs plus FluoroGold in the bed nucleus of the stria terminalis indicate that neurons projecting through the stria terminalis could be integrating olfactory and vomeronasal inputs. Retrograde tracing experiments using FluoroGold or dextran amines confirm that areas of the rostral basal telencephalon receive inputs from both the main and accessory olfactory bulbs. While both inputs clearly converge in areas classically considered olfactory-recipient (nucleus of the lateral olfactory tract, anterior cortical amygdaloid nucleus, and cortex-amygdala transition zone) or vomeronasal-recipient (ventral anterior amygdala, bed nucleus of the accessory olfactory tract, and anteroventral medial amygdaloid nucleus), segregation is virtually complete at posterior levels such as the posteromedial and posterolateral cortical amygdalae. This provides evidence that areas so far considered receiving a single chemosensory modality are likely sites for convergent direct olfactory and vomeronasal inputs. Therefore, areas of the basal telencephalon should be reclassified as olfactory, vomeronasal, or mixed chemosensory structures, which could facilitate understanding of olfactory-vomeronasal interactions in functional studies.

The vomeronasal system and pheromones

The vomeronasal system and pheromones

Feedback Loops Link Odor and Pheromone Signaling with Reproduction

Pheromones can have profound effects on reproductive physiology and behavior in mammals. To investigate the neural circuits underlying these effects, we used a genetic transneuronal tracer to identify neurons that synapse with GnRH (LHRH) neurons, the key regulators of reproduction. We then asked whether the connected neurons are presynaptic or postsynaptic to GnRH neurons and analyzed their responses to chemosensory cues. Surprisingly, these experiments indicate that GnRH neurons receive pheromone signals from both odor and pheromone relays in the brain and may also receive common odor signals. Moreover, feedback loops are evident whereby GnRH neurons could influence both odor and pheromone processing. Remarkably, approximately 800 GnRH neurons communicate with approximately 50,000 neurons in 53 functionally diverse brain areas, with some connections exhibiting sexual dimorphism. These studies reveal a complex interplay between reproduction and other functions in which GnRH neurons appear to integrate information from multiple sources and modulate a variety of brain functions.

The “olfactostriatum” of snakes: A basal ganglia vomeronasal structure in tetrapods

The olfactostriatum is a portion of the basal ganglia of snakes situated ventromedially to the nucleus accumbens proper. It receives a major vomeronasal input from the nucleus sphericus, the primary target of accessory olfactory bulb efferents. Recently, the ophidian olfactostriatum has been characterized on the basis of chemoarchitecture (distribution of serotonin, neuropeptide Y and tyrosine hydroxylase) and hodology (afferent and efferent connections). In contrast to the nucleus accumbens proper, the olfactostriatum is densely immunoreactive for serotonin and neuropeptide Y and sparsely immunoreactive for tyrosine hydroxylase. The nucleus accumbens proper and the olfactostriatum share most afferent connections except those originating in the nucleus sphericus, which are exclusively directed to the olfactostriatum. Similarly, the nucleus accumbens proper and the olfactostriatum show a similar pattern of efferent connections including those going to the ventral pallidum, although the olfactostriatum alone projects to the main and accessory olfactory bulbs as well as some amygdaloid nuclei. On the basis of its chemoarchitecture, the olfactostriatum resembles the mammalian ventral pallidum (but also the shell of the nucleus accumbens). Its connections, however, suggests that the olfactostriatum could be a specialized portion of the shell of nucleus accumbens extended more ventromedially than previously believed and devoted to processing vomeronasal information. Comparative data suggest that a similar structure is present in the basal ganglia of amphibians and mammals.

Pre-exposure to female chemosignals or intracerebral GnRH restores mating behavior in naive male hamsters with vomeronasal organ lesions.

Chemosensory input is essential for mating in male hamsters and the vomeronasal organ is critical to mating in naive males. In studies to investigate the convergence of vomeronasal chemosensory input and the neurohormone gonadotrophin releasing hormone (GnRH), we have unexpectedly found that pre-exposure to pheromone-containing chemosignals from female hamsters will also eliminate mating deficits normally seen in naive male hamsters with vomeronasal organs removed (VNX). In the present studies, naive-intact and naive-VNX male hamsters were given intracerebroventricular injections of GnRH or saline and exposed to female pheromones found in hamster vaginal fluid (HVF) or to water 40 min prior to a 5 min mating test. VNX males given saline injections and exposed to water had severe mating deficits, but VNX males given saline injections and exposed to HVF mated normally. As shown previously, males given GnRH injections and exposed to water also mated normally. HVF exposure prior to a mating test apparently acted to compensate for the lack of vomeronasal input in these males.

Activation of accessory olfactory bulb neurons during copulatory behavior after deprivation of vomeronasal inputs in male rats

We examined whether the vomeronasal organ (VNO) is the sole receptor for pheromonal cues for male sexual behavior. Males carrying surgical removal of the VNO (VNOx) mated with stimulus females as sham-operated males, with a comparable number of mounts but a prolonged latency for ejaculation. In sham-operated males, mating increased cFos immunoreactivity in the granule and mitral cell layers of the accessory olfactory bulb and in the medial amygdala. VNOx diminished baseline as well as mating-induced cFos in the granule cell layer and in the medial amygdala; VNOx had no effect on either basal or induced cFos immunoreactivity in the mitral cell layer. Thus, during mating encounter, cFos expression in the mitral cell layer did not depend on VNO inputs. The medial amygdala may be modulated by impulses other than of mitral cell origin.

Distribution of PSA-NCAM expression in the amygdala of the adult rat

Synaptic plasticity in the amygdala appears to be necessary for the generation of emotional memories. However, the molecular bases of this plasticity are not fully understood. Because the polysialylated form of the neural cell adhesion molecule (PSA-NCAM) has been implicated in memory consolidation in the hippocampus and temporal cortex, we have studied in detail the expression of this molecule in the adult rat amygdala with an antibody against PSA-NCAM. Our results demonstrate for the first time the presence of PSA-NCAM in the adult rat amygdala. Immunoreactive somata and processes are abundant in the amygdalo-hippocampal transition area, central nucleus, intra-amygdaloid bed nucleus of the stria terminalis, anterior and posterior cortical nuclei, periamygdaloid cortex and medial nucleus of the amygdala. In addition PSA-NCAM immunoreactive neuronal somata and processes exist in the lateral, basal and accessory basal nuclei, anterior amygdaloid area and amygdalo-striatal area. The presence of this molecule in areas that receive olfactory or vomeronasal input could reflect the intrinsic plasticity of these chemosensory systems. PSA-NCAM expression in the lateral amygdala could indicate its participation in the plastic events that lead to the generation of emotional memories such as those related to fear conditioning.

Experience facilitates vomeronasal and olfactory influence on Fos expression in medial preoptic area during pheromone exposure or mating in male hamsters

Chemosensory stimuli are essential for mating in male hamsters but either main olfactory or vomeronasal input is sufficient in sexually experienced males. Activation in central chemosensory pathways and medial preoptic area, after stimulation with female chemosignals or after mating, was estimated by counting neurons expressing Fos protein in experienced and naive males, with or without vomeronasal organ lesions. Regions counted included main and accessory olfactory bulbs, corticomedial amygdala, bed nucleus stria terminalis and medial preoptic area. Chemosensory stimulation was more effective in activating medial preoptic area in experienced than in naive males. In experienced males with vomeronasal organs removed, main olfactory input was as effective in activating medial preoptic area as was the combination of main and accessory input available to intact animals. As previously reported, the main olfactory input remaining after vomeronasal lesions in naive males was poorly effective in activating medial preoptic area, and these animals had impaired mating behavior. The change in access of chemosensory input to medial preoptic area after experience suggests that an experience-dependent synaptic modulation in this pathway, perhaps in the amygdala, may underlie some changes in mating behavior with experience.

Sensory requirements for noncontact penile erection in the rat.

Physical contact is not necessary for male rats to display penile erection when placed in the vicinity of females in estrus (noncontact erection, NCE). The modality and reception of sensory stimuli for the induction of NCE were analyzed. Olfactory bulbectomy eliminated NCE, and destruction of olfactory mucosa by zinc sulfate solution caused a significant suppression of NCE, whereas removal of the vomeronasal organ had no effect. Fresh urine collected from estrous females, but not fresh feces, induced NCE. Neither devocalization of estrous females by cutting the inferior laryngeal nerve nor deprivation of visual cues by placing an opaque barrier between the subjects and stimulus females disrupted NCE. The results indicate that olfactory cues emitted by estrous females induce NCE via the olfactory epithelium. Contrary to what had been suggested by previous experiments, vomeronasal inputs are not indispensable for the induction of NCE.

Vomeronasal, olfactory, hormonal convergence in the brain. Cooperation or coincidence?

This paper briefly reviews three topics: (a) Convergent vomeronasal and olfactory chemosensory input into a common circuit considered important for mating in male golden hamsters; (b) Apparent activation of this circuit by intracerebral luteinizing hormone-releasing hormone (LHRH) and (c) Interaction of the LHRH and the two chemosensory inputs. Mapping of c-fos protein in male hamsters mating with females or exposed to female pheromones indicates intense activation in the central vomeronasal pathways, induced by both olfactory and vomeronasal input. This input appears to activate the medial preoptic area, where chemosensory activation is greater in experienced than in inexperienced animals. Intracerebral injection of LHRH facilitates mating behavior and increases Fos expression in the medial preoptic area of mating males. Despite evidence that the release of LHRH in response to female chemosignals is dependent on vomeronasal input, no activation of LHRH neurons has been demonstrated in males exposed to such chemosignals. Electrical stimulation of the vomeronasal organ can induce Fos expression in chemosensory pathways and in medial preoptic LHRH neurons.

On the nature of mammalian and human pheromones.

Communication by chemical (pheromone) signals is important in many species, including mammals. Chemosensory and hormonal systems can interact in at least two ways: (i) chemosensory input, especially but not exclusively that through the vomeronasal organ, may elicit hormonal release, which, in turn, may facilitate behavioral or physiological responses; and (ii) hormones, especially steroids, may be essential for some responses to chemosensory, including vomeronasal, input. Recent, still controversial reports, suggest that chemosensory communication may occur in humans via a residual vomeronasal organ and that chemosensory/hormonal interactions also operate in humans. In this symposium these matters are examined critically. Johnston explores the concept of pheromone communication and suggests that the notion of a single-chemical 'magic bullet' irresistibly leading to a preprogrammed result is too simplistic despite documented examples of special stimuli acting via the vomeronasal organ. Meredith briefly reviews evidence for hormonal mediation of the effects of vomeronasal input; including a situation where vomeronasal and hormonal facilitation of a behavior appear to be interchangeable--but where vomeronasal input appears important only in inexperienced animals. Wood discusses evidence that the effectiveness of chemosensory input to particular brain nuclei depends critically on the simultaneous presence of a steroid hormone within the same nucleus. Monti-Bloch presents his evidence that steroids may act as gender-specific chemical signals in humans, exciting an electrical response from the residual human vomeronasal organ and affecting human hormone levels.

Role of the vomeronasal input in maternal behavior.

This article reviews the role of the vomeronasal system in the induction of parental behavior in female and male rats, using, primarily, the sensitization model. The following questions are addressed: (1) Is the vomeronasal system sexually dimorphic? (2) Do the sex differences found in the VNS underlie those seen in behavior? (3) Do mechanisms, other than the classical 'organizational' effects of perinatal gonadal steroids, play a role in the organization of behavioral phenotypes in parental behavior? and (4) Does vomeronasal input play a role in the formation of the mother infant bond in humans? The first question has been answered throughout the 1980's in various studies of the organizational actions of postnatal exposure to gonadal steroids. The second aim has been addressed in a functional approach by lesion and neural activation studies. The experiments which lead us to consider the hypothesis that nonsteroidal factors in development, and specifically GABA, could account for the expression of parental care are reviewed. Finally, research relevant to the existence of a vomeronasal organ in humans and a possible pheromonal input in the formation of mother-infant bonds in humans is reviewed.

Efferents and Centrifugal Afferents of the Main and Accessory Olfactory Bulbs in the Snake Thamnophis sirtalis

This study reinvestigates the efferent projections of the main and accessory olfactory bulbs and describes for the first time the centrifugal projections to the main and accessory olfactory bulbs in the snake Thamnophis sirtalis, using the intraaxonal transport of the anterograde and retrograde tracer biotinylated dextran amine and the retrograde tracer horseradish peroxidase. The olfactory projection consists of three tracts: the lateral olfactory tract, which projects bilaterally to the lateral cortex and the rostral amygdala, crossing the midline through the stria medullaris-habenular commissure system; the intermediate olfactory tract, which projects ipsilaterally to the olfactory tubercle and contributes to the contralateral projection; and the medial olfactory tract, which projects ipsilaterally to the dorsomedial retrobulbar formation. The vomeronasal projection is formed by a single tract, the accessory olfactory tract, that projects ipsilaterally to the nucleus of the accessory olfactory tract, the medial amygdala and the nucleus sphericus. The centrifugal projections to the main and accessory olfactory bulb are composed of two components: one that arises in areas that receive the olfactory or vomeronasal input (neurons in the olfactory tubercle, retrobulbar formation and lateral cortex project to the main olfactory bulb; and neurons in the nucleus of the accessory olfactory tract, the medial amygdala and the nucleus sphericus project to the accessory olfactory bulb), and another that arises in areas not directly implicated in processing the chemosensory information (the nucleus of the diagonal band of Broca and the dorsal cortex). These data allow the recognition of the general pattern of organization of the reptilian olfactory and vomeronasal systems.