Suprasternal gland secretion of male short-tailed opossum induces IP3 generation in the vomeronasal organ.

Chemical communication is an important component of mammalian social behaviors. Gray short-tailed opossums (Monodelphis domestica) communicate by scent marking. The male opossum possesses a prominent suprasternal scent gland, extracts of which strongly attract female opossums. This attractivity remains unaltered following repeated lyophilization. The suprasternal gland secretion functions in a sexually dimorphic manner, i.e., it elicits elevated levels of IP(3) in the vomeronasal (VN) sensory epithelium of female opossums, but suppressed the levels of IP(3) in the VN sensory epithelium of male opossums. The elevated levels of IP(3) induced by suprasternal gland secretion in female vomeronasal sensory epithelium is inhibited by the G(i/o) specific inhibitor, NF023, but not its inactive analogue, NF007. It is also suppressed by specific antibodies to the alpha subunits of G(i) and G(o) proteins, by the phospholipase C inhibitor, U73122, as well as by GDPbetaS. Surprisingly, GDPbetaS itself enhances basal levels of IP(3) in female VN sensory epithelium. This GDPbetaS-induced increase in levels of IP(3) is reduced by the PLC inhibitor, U73122, but not by the G(i/o) inhibitor, NF023. In addition, GDP also enhances basal levels of IP(3). GDPbetaS, a known inhibitor of G-protein activation, thus appears to have dual functions: as both stimulator and inhibitor of IP(3) production in the VN sensory epithelium of opossums. In contrast, this nucleotide analogue functions as an inhibitor in the VN sensory epithelium of mice. The mechanism of signal transduction underlying the suprasternal gland secretion-elicited signals in the VN sensory epithelium of opossums appears to involve signals that are generated through activation of G-protein-coupled receptors and transduced via activation of G(i/o)-proteins and the effector, phospholipase C, resulting in an increased production of the second messenger, IP(3). The extracellular signals are thus amplified.

Firing properties of accessory olfactory bulb mitral/tufted cells in response to urine delivered to the vomeronasal organ of gray short-tailed opossums.

In comparison with many mammals, there is limited knowledge of the role of pheromones in conspecific communication in the gray short-tailed opossum. Here we report that mitral/tufted (M/T) cells of the accessory olfactory bulb (AOB) of male opossums responded to female urine but not to male urine with two distinct patterns: excitation followed by inhibition or inhibition. Either pattern could be mimicked by application of guanosine 5'-O-3-thiotriphosphate and blocked by guanosine 5'-O-2-thiodiphosphate, indicating that the response of neurons in this pathway is through a G-protein-coupled receptor mechanism. In addition, the inhibitor of phospholipase C (PLC), U73122, significantly blocked urine-induced responses. Male and female urine were ineffective as stimuli for M/T cells in the AOB of female opossums. These results indicate that urine of diestrous females contains a pheromone that directly stimulates vomeronasal neurons through activation of PLC by G-protein-coupled receptor mechanisms and that the response to urine is sexually dimorphic.

Female snake sex pheromone induces membrane responses in vomeronasal sensory neurons of male snakes.

The vomeronasal organ (VNO) is important for activating accessory olfactory pathways that are involved in sexually dimorphic mating behavior. The VNO of male garter snakes is critically important for detection of, and response to, female sex pheromones. In the present study, under voltage-clamp conditions, male snake VNO neurons were stimulated with female sexual attractiveness pheromone. Thirty-nine of 139 neurons exhibited inward current responses (reversal potential: -10.6 +/- 2.8 mV). The amplitude of the inward current was dose dependent, and the relationship could be fitted by the Hill equation. Under current-clamp conditions, application of pheromone produced membrane depolarizing responses and increases in firing frequency. These results suggest that the female pheromone directly affects male snake VNO neurons and results in opening of ion channels, thereby converting the pheromone signal to an electrical signal. The response to female pheromone is sexually dimorphic, that is, the pheromone does not evoke responses in VNO neurons of female snakes. An associated finding of the present study is that the female sex pheromone, which is insoluble in aqueous solutions, became soluble in the presence of Harderian gland homogenate.

Efferent connections of the main olfactory bulb in the opossum (Monodelphis domestica): a characterization of the olfactory entorhinal cortex in a marsupial.

Olfactory projections have been investigated for decades, but few reports using modern, sensitive neural tracers are available. In marsupials, only lesion-degeneration studies exist and they are restricted to the genera Didelphis and Trichosurus. Some of the territories described as olfactory-recipient such as the upper portion of the rhinal fissure and the vomeronasal amygdala are, however, controversial. Also, the characterization of the olfactory portion of the entorhinal cortex is far from clear in acallosal mammals. The present report investigates, using biotinylated dextran-amine, the olfactory connections in the short-tailed opossum (Monodelphis domestica) and characterizes the olfactory portion of the entorhinal cortex in non-placental mammals. The data indicate that olfactory projections do not reach the upper portion of the rhinal fissure, but partially end in the vomeronasal amygdala, i.e., the medial and posteromedial cortical amygdaloid nuclei; thus, although olfactory and vomeronasal system have largely segregated outputs, areas of overlap should be restudied. The olfactory portion of the entorhinal cortex is much larger than previously described, extending up to the occipital pole of the cerebral hemisphere. Collectively, these data contribute to our understanding of the organization of the hippocampal formation in marsupials.

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.

Efferent connections of the "olfactostriatum": a specialized vomeronasal structure within the basal ganglia of snakes.

The olfactostriatum is a portion of the basal ganglia of snakes that receives substantial vomeronasal afferents through projections from the nucleus sphericus. In a preceding article, the olfactostriatum of garter snakes (Thamnophis sirtalis) was characterized on the basis of chemoarchitecture (distribution of serotonin, neuropeptide Y and tyrosine hydroxylase) and pattern of afferent connections [Martinez-Marcos, A., Ubeda-Banon, I., Lanuza, E., Halpern, M., 2005. Chemoarchitecture and afferent connections of the "olfactostriatum": a specialized vomeronasal structure within the basal ganglia of snakes. J. Chem. Neuroanat. 29, 49-69]. In the present study, its efferent connections have been investigated. The olfactostriatum projects to the main and accessory olfactory bulbs, lateral cortex, septal complex, ventral pallidum, external, ventral anterior and dorsolateral amygdalae, bed nucleus of the stria terminalis, preoptic area, lateral posterior hypothalamic nucleus, ventral tegmental area, substantia nigra and raphe nuclei. Tracer injections in the nucleus accumbens proper, a structure closely associated with the olfactostriatum, result in a similar pattern of efferent connections with the exception of those reaching the main and accessory olfactory bulbs, lateral cortex, external, ventral anterior and dorsolateral amygdalae and bed nucleus of the stria terminalis. These data, therefore, help to characterize the olfactostriatum, an apparently specialized area of the nucleus accumbens. Double labeling experiments after tracer injections in the nucleus sphericus and the lateral posterior hypothalamic nucleus demonstrate a pathway between these two structures through the olfactostriatum. Injections in the olfactostriatum and in the medial amygdala show parallel projections to the lateral posterior hypothalamic nucleus. Since this hypothalamic nucleus has been previously described as projecting to the hypoglossal nucleus, both, the medial amygdala and the olfactostriatum may mediate vomeronasal influence on tongue-flick behavior.

Modification of odor investigation and discrimination in female opossums (Monodelphis domestica) following the ablation of the accessory olfactory bulbs.

To determine whether the vomeronasal system of the Brazilian short-tailed opossum (Monodelphis domestica) is important to the response to conspecific chemical signals, the authors tested female opossums with conspecific odors, before and after ablation of their accessory olfactory bulbs (AOBs). Anesthesia and sham treatments did not modify females' discrimination of conspecific odors when tested against water, between male and female odors, or between different odors from the same male donors. Odor investigation was partially diminished following partial ablation of the AOB, and complete ablation of the AOBs further impaired the ability of females to discriminate between certain odors. These findings provide the first evidence for the importance of the vomeronasal system in the detection of chemosignals of known origin in opossums.

Food preferences of captive gray short-tailed opossums (Monodelphis domestica).

The gray short-tailed opossum has been a subject in behavioral and biomedical studies for the last quarter century, but researchers know little about its preferred diet. The authors describe a study designed to determine food preferences of this species for the purpose of identifying suitable rewards to be used in behavioral studies.

The role of the vomeronasal system in food preferences of the gray short-tailed opossum, Monodelphis domestica.

Although feeding deficits have been reported in snakes and lizards following vomeronasal system disruption, no deficit has been previously reported in a mammal. We tested gray short-tailed opossums with items from four different food categories prior to occluding access to the vomeronasal organ. Preoperatively, opossums preferred meat to fruit or vegetables. Following occlusion of the nasopalatine canal, but not after control treatment, opossums failed to demonstrate food preferences.

Chemoarchitecture and afferent connections of the "olfactostriatum": a specialized vomeronasal structure within the basal ganglia of snakes.

The olfactostriatum, a portion of the striatal complex of snakes, is the major tertiary vomeronasal structure in the ophidian brain, receiving substantial afferents from the nucleus sphericus, the primary target of accessory olfactory bulb efferents. In the present study, we have characterized the olfactostriatum of garter snakes (Thamnophis sirtalis) on the basis of chemoarchitecture (distribution of serotonin, neuropeptide Y and tyrosine hydroxylase) and hodology (afferent connections). The olfactostriatum is densely immunoreactive for serotonin and neuropeptide Y and shows moderate-to-weak immunoreactivity for tyrosine hydroxylase. In addition to afferents from the nucleus sphericus, the olfactostriatum receives inputs from the dorsal and lateral cortices, nucleus of the accessory olfactory tract, external and dorsolateral amygdalae, dorsomedial thalamic nucleus, ventral tegmental area and raphe nuclei. Double labeling experiments demonstrated that the distribution of serotonin and neuropeptide Y in this area almost completely overlaps the terminal field of projections from the nucleus sphericus. Also, serotonergic and dopaminergic innervation of the olfactostriatum likely arise, respectively, from the raphe nuclei and the ventral tegmental area, whereas local circuit neurons originate the neuropeptide Y immunoreactivity. These results indicate that the olfactostriatum of snakes could be a portion of the nucleus accumbens, with features characteristic of the accumbens shell, devoted to processing vomeronasal information. Comparative data suggest that a similar structure is present in the ventral striatum of amphibians and mammals.

Calbindin D28k, parvalbumin, and calretinin immunoreactivity in the main and accessory olfactory bulbs of the gray short-tailed opossum, Monodelphis domestica.

The vertebrate main and accessory olfactory bulbs (MOB and AOB) are the first synaptic sites in the olfactory pathways. The MOB is a cortical structure phylogenetically well conserved in its laminar structure and overall synaptic organization, while the AOB has significant species variation in size. In order to better understand signal processing in the two olfactory systems and the species differences, immunocytochemical staining and analysis were done of the neuronal expression patterns of the calcium-binding proteins calbindin D28k (CB), parvalbumin (PV), and calretinin (CR) in the MOB and AOB in a marsupial species, the gray short-tailed opossum, Monodelphis domestica. In the MOB, antibody to CB labeled periglomerular cells, superficial short axon cells / Van Gehuchten cells; antibody to PV labeled Van Gehuchten cells; and antibody to CR immunostained periglomerular cells, superficial short axon cells / Van Gehuchten cells, and granule cells. In the AOB, CB immunoreactivity was detected in periglomerular cells and a subpopulation of granule cells; antibody to PV labeled the superficial short axon cells / Van Gehuchten cells and granule cells; and antibody to CR labeled a small number of periglomerular cells, superficial short axon cells / Van Gehuchten cells, and granule cells. These results showed that the patterns of CB, PV, and CR expression differ in the opossum main and accessory olfactory bulbs and differ from that in other animal species. These varying patterns of neuronal immunostaining may be related to the different functions of the main and accessory olfactory bulbs and to the differing signal processing features.

Conspecific odor investigation by gray short-tailed opossums (Monodelphis domestica).

Gray short-tailed opossums (Monodelphis domestica) are small marsupials, which have recently become the subjects of numerous laboratory investigations. While these opossums have well-developed olfactory systems and complex scent-marking behaviors, the significance of their use of odors in conspecific communication is still poorly understood. Investigation of body odors by male and female opossums was examined in the present study. Males investigated flank and urine odors of nonestrous adult females significantly more than controls, but not urine from sexually inexperienced juvenile females or urine of females at cytological estrus. Since in this species females have an induced estrus, it would be advantageous for males to investigate and follow the odors of urine of diestrous females, which become receptive in proximity to males. Female opossums investigated odors of male mandibles and suprasternal glands significantly more than controls but not odors of male urine. We suggest that the use of glandular secretions is more common and more effective than urine for intraspecific communication between gray short-tailed opossums: In the semiarid conditions inhabited by the opossums, glandular secretions are less volatile and are effective for longer periods than urine and would be of greater value in intraspecific communication if, as suggested in the literature, these opossums are nomadic and meet one another infrequently.

Structure and function of the vomeronasal system: an update.

Several developments during the past 15 years have profoundly affected our understanding of the vomeronasal system (VNS) of vertebrates. In the mid 1990s, the vomeronasal epithelium of mammals was found to contain two populations of receptor cells, based on their expression of G-proteins. These two populations of neurons were subsequently found to project their axons to different parts of the accessory olfactory bulb (AOB), forming the basis of segregated pathways with possibly heterogeneous functions. A related discovery was the cloning of members of at least two gene families of putative vomeronasal G-protein-coupled receptors (GPRs) in the vomeronasal epithelium. Ligand binding to these receptors was found to activate a phospholipase C (PLC)-dependent signal transduction pathway that primarily involves an increase in intracellular inositol-tris-phosphate and intracellular calcium. In contrast to what was previously believed, neuron replacement in the vomeronasal epithelium appears to occur through a process of vertical migration in most mammals. New anatomical studies of the central pathways of the olfactory and vomeronasal systems indicated that these two systems converge on neurons in the telencephalon, providing an anatomical substrate for functional interactions. Combined anatomical, physiological and behavioral studies in mice provided new information that furthered our understanding of one of the most striking pheromonal phenomena, the Bruce effect. Finally, contrary to prior observations, new anatomical studies indicated that a vomeronasal organ (VNO) was present in human adults and reports were published indicating that this system might be functional. These latter observations are still controversial and require confirmation from independent laboratories.

Calbindin D28K immunoreactive neurons in vomeronasal organ and their projections to the accessory olfactory bulb in the rat.

The vomeronasal system is a nasal chemosensory system involved in pheromone detection. The chemosensory receptor neurons are located in the sensory epithelium of the vomeronasal organ (VNO). Their axons terminate in the glomeruli of the accessory olfactory bulb (AOB). In this study, we examined the expression of calbindin D28k (CB) in the rat VNO and AOB. In the VNO, a subpopulation of receptor neurons in the middle layer of the sensory epithelium was immunostained with antibodies to CB. Their axons could be traced to terminate in a group of glomeruli in the anterior half of the AOB glomerular layer. This group of CB-immunostained glomeruli in the anterior half of the AOB included a few large glomeruli close to the boundary between the anterior and posterior halves of the AOB, and several small glomeruli scattered in the anterior region of the AOB glomerular layer. The positions of the CB-immunostained glomeruli in the AOB, especially those close to the anterior-posterior boundary, were similar in the two bulbs and in different rats. No sex difference was found. A developmental study showed that the CB-immunoreactive receptor neurons in the middle layer of the VNO sensory epithelium and CB-immunoreactive glomeruli in the anterior AOB were present on the 14th postnatal day and older. The distribution pattern of the CB-immunostained receptor neurons and their localized projection suggest the possibility that these neurons may express the same or functionally related pheromone receptor genes.

Differential effects of lesions of the vomeronasal and olfactory nerves on garter snake (Thamnophis sirtalis) responses to airborne chemical stimuli.

The roles of the main (MOS) and accessory (AOS) olfactory systems of garter snakes in response to airborne chemicals were investigated. Preoperatively, all snakes responded to airborne odors with increased tongue-flick rate and duration. Postoperatively, sham-operated snakes responded to airborne odors with increased tongue-flick rates, but snakes with main olfactory nerve cuts failed to respond to the odors, and snakes with vomeronasal nerve cuts responded to nonprey odors only. Preoperatively, exposure to earthworm odor produced more frequent and shorter duration tongue-flicks during locomotion compared with exposure to water. Postoperatively, only sham-lesioned snakes exhibited differential responding to earthworm odors. This study demonstrates that the MOS is critical for the initiation of tongue-flick behavior in response to airborne odors and that discrimination of odors with biological significance requires a functional AOS.

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.

Calcium transients in the garter snake vomeronasal organ.

The signaling cascade involved in chemosensory transduction in the VN organ is incompletely understood. In snakes, the response to nonvolatile prey chemicals is mediated by the vomeronasal (VN) system. Using optical techniques and fluorescent Ca(2+) indicators, we found that prey-derived chemoattractants produce initially a transient cytosolic accumulation of [Ca(2+)](i) in the dendritic regions of VN neurons via two pathways: Ca(2+) release from IP(3)-sensitive intracellular stores and, to a lesser extent, Ca(2+) influx through the plasma membrane. Both components seem to be dependent on IP(3) production. Chemoattractants evoke a short-latency Ca(2+) elevation even in the absence of extracellular Ca(2+), suggesting that in snake VN neurons, Ca(2+) release from intracellular stores is independent of a preceding Ca(2+) influx, and both components are activated in parallel during early stages of chemosensory transduction. Once the response develops in apical dendritic segments, other mechanisms can also contribute to the amplification and modulation of these chemoattractant-mediated cytosolic Ca(2+) transients. In regions close to the cell bodies of the VN neurons, the activation of voltage-sensitive Ca(2+) channels and a Ca(2+)-induced Ca(2+) release from intracellular ryanodine-sensitive stores secondarily boost initial cytosolic Ca(2+) elevations increasing their magnitude and durations. Return of intracellular Ca(2+) to prestimulation levels appears to involve a Ca(2+) extrusion mediated by a Na(+)/Ca(2+) exchanger mechanism that probably plays an important role in limiting the magnitude and duration of the stimulation-induced Ca(2+) transients.

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.

Conditioned discrimination of airborne odorants by garter snakes (Thamnophis radix and T. sirtalis sirtalis).

For snakes, the nasal chemical senses are critical in intraspecific communication and prey recognition. Although it is known that garter snakes can respond differentially to airborne odorants, no previous study has demonstrated that snakes can learn a task with airborne odors as discriminative stimuli. In Experiment 1, 7 plains garter snakes (Thamnophis radix) were trained in a two-choice apparatus to move into a compartment containing lemon-scented chips for a food reward. All 7 snakes improved performance when the first 10 and last 10 trials of the 100 trials of conditioning were compared. Four of the snakes were subsequently trained to move away from the scented compartment into the unscented compartment. The 4 snakes rapidly learned this reversal. In Experiment 2, 7 common garter snakes (T. sirtalis sirtalis) were trained to traverse a two-choice maze with the presence or absence of amyl acetate odor as the conditioned stimulus. The snakes were pretested for odor versus nonodor preference and were trained to go to the initially nonpreferred stimulus. Of the 7 snakes, 5 achieved a predetermined criterion (two training sessions with cumulative correct responding above the .05 confidence level) within 85 trials.

Light and electron microscopic observations on the normal structure of the vomeronasal organ of garter snakes.

The vomeronasal epithelium of adult garter snakes (Thamnophis sirtalis and T. radix) was studied by light and electron microscopy. The sensory epithelium is extraordinarily thick, consisting of a supporting cell layer, a bipolar cell layer, and an undifferentiated cell layer. The supporting cell layer is situated along the luminal surface and includes supporting cells and the peripheral processes (dendrites) of bipolar neurons. The luminal surfaces of both supporting cells and bipolar neurons are covered with microvilli. Specializations of membrane junctions are always observed between adjacent cells in the subluminal region. Below the supporting cell layer, the epithelium is characterized by a columnar organization. Each column contains a population of bipolar neurons and undifferentiated cells. These cells are isolated from the underlying vascular and pigmented connective tissue by the presence of a thin sheath of satellite cells and a basal lamina. Heterogeneity of cell morphology occurs within each cell column. Generative and undifferentiated cells occupy the basal regions and mature neurons occupy the apical regions. Transitional changes in cell morphology occur within the depth of each cell column. These observations suggest that the vomeronasal cell column is the structural unit of the organ and may represent the dynamic unit for cell replacement as well. A sequential process of cell proliferation, neuronal differentiation, and maturation appears to occur in the epithelium despite the adult state of the animal.