The characteristics of the electrovomeronasogram: its loss following vomeronasal axotomy in the garter snake

Electrovomeronasogram (EVG) recordings were made from adult garter snakes, Thamnophis sirtalis. Stimulation of vomeronasal epithelium with a stimulus prepared from prey, earthworm electric shock secretion (ESS), evoked EVG response in a dose-dependent manner. The magnitude of the EVG response to ESS was remarkably larger than n-amyl acetate and glutamate, which elicited insignificant responses, supporting the idea that the vomeronasal system is differentially sensitive to liquid delivery of biologically significant chemical stimuli. Fourteen days following vomeronasal axotomy, the magnitudes of the EVG responses of animals which received bilateral axotomy without cauterization or with cauterization was -0.19+/-0.07 mV or -0.05+/-0.02 mV respectively, compared with the normal EVG response of -0.41+/-0.10 mV. The epithelia of animals which received bilateral axotomy without cauterization exhibited remarkable degeneration of the bipolar neurons. Maximal depletion of bipolar neurons occurred in the epithelia denervated with cauterization, though the difference between cell densities in vomeronasal neuron layers in these epithelia was not statistically significant. The present results clearly indicate that the fewer neurons the epithelium contains, the smaller EVG response it generates, suggesting that the receptor neurons are the primary origin of EVG responses.      

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.     

Purification and characterization of a chemoattractant from electric shock-induced earthworm secretion, its receptor binding, and signal transduction through the vomeronasal system of garter snakes

Following shocks with low voltage electric current, earthworms, Lumbricus terrestris, secrete a yellow mucus that has alarm properties for conspecifics and chemoattractive properties for garter snakes, Thamnophis sirtalis. A proteinaceous chemoattractant for garter snakes has been isolated and purified to homogeneity from such secretions by means of permeation chromatography and semipreparative nondenaturing polyacrylamide gel electrophoresis. The purified protein is highly attractive to garter snakes; it loses its activity after proteolytic digestion. It is a glycoprotein consisting of a single polypeptide chain with an NH2-terminal alanine. This chemoattractant has a minimum molecular mass of 15.4 kDa calculated from its amino acid and carbohydrate contents and an apparent molecular mass of about 20 kDa as estimated from sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It has a pI of about 4.0, and it binds wheat germ agglutinin but not concanavalin A. This chemoattractant shows a protein to carbohydrate ratio of 2.0 +/- 0.08 (n = 5) and a ratio of total sugar to amino sugar of 1.9 +/- 0.08 (n = 3). The sequence of its NH2-terminal 15 amino acid residues has been determined. Studies were also conducted on the chemosignal transduction through the vomeronasal sensory system of the garter snake. Dot blot analysis showed that the purified chemoattractant bound to snake vomeronasal sensory epithelial membrane fractions. It did not bind to membrane extracts of the nonsensory epithelium of the vomeronasal mushroom body. The chemoattractant also bound specifically to vomeronasal sensory epithelial membrane in a reversible and saturable fashion with Kd and Bmax values of about 0.3 microM and 0.4 nmol/mg of protein, respectively. In electrophysiological studies, the chemoattractant applied to the vomeronasal epithelium caused an increase in firing rate of individual neurons in the accessory olfactory bulb of garter snakes, the projection site for vomeronasal neurons. The present results are the first clear biochemical and electrophysiological evidence for a vomeronasal epithelium response to a purified nonvolatile odorant, and this makes the garter snake vomeronasal system ideal for studying the mechanisms of chemosignal transduction.     

Developmental changes in cytochrome oxidase histochemistry in the main and accessory olfactory bulbs of embryonic and neonatal garter snakes (Thamnophis sirtalis spp.)

Developmental studies examining the changes in oxidative metabolic activity are useful for understanding how and if the vomeronasal and olfactory systems respond to stimulation during embryogenesis. Garter snakes are good candidates for examining the potential functionality of the vomeronasal system in utero. In adult garter snakes, the vomeronasal system mediates many behaviors. Neonatal garter snakes exhibit these same behaviors, and the vomeronasal system has been shown to mediate feeding behavior in neonates. Using cytochrome oxidase histochemistry, we examined changes in the oxidative metabolic activity of main and accessory olfactory bulbs of embryonic and neonatal garter snakes (Thamnophis sirtalis sirtalis and T. s. parietalis). Cytochrome oxidase staining is greater in the accessory olfactory bulb than in the main olfactory bulb of embryonic garter snakes. However, neonates show no differences in the staining of the accessory and main olfactory bulbs, suggesting a change in the stimulation of the main olfactory bulb after birth. This is the first report of cytochrome oxidase histochemistry in reptiles and in the vomeronasal system of embryonic vertebrates. © 1993 Wiley-Liss, Inc.     

Signal transduction in the vomeronasal organ of garter snakes: ligand-receptor binding-mediated protein phosphorylation.

The vomeronasal (VN) system of garter snakes plays an important role in several species-typical behaviors, such as prey recognition and responding to courtship pheromones. We (X.C. Jiang et al., J. Biol. Chem. 265 (1990) 8736-8744 and Y. Luo et al., J. Biol. Chem. 269 (1994) 16867-16877) have demonstrated previously that in the snake VN sensory epithelium, the chemoattractant ES20, a 20-kDa glycoprotein derived from electric shock-induced earthworm secretion, binds to its receptor which is coupled to PTX-sensitive G-proteins. Such binding results in elevated levels of IP3. We now report that ES20-receptor binding regulates the phosphorylation of two membrane-bound proteins with molecular masses of 42- and 44-kDa (p42/44) in both intact and cell-free preparations of the VN sensory epithelium. ES20 and DAG regulate the phosphorylation of p42/44 in a similar manner. ES20-receptor binding-mediated phosphorylation of p42/44 is rapid and transient, reaching a peak value within 40 seconds and decaying thereafter. Phosphorylation of p42/44 appears to be regulated by the countervailing actions of a specific membrane-bound protein kinase and a protein phosphatase. The phosphorylation of these membrane-bound proteins significantly reduces the activity of G-proteins as evidenced by a decrease in GTPase activity, but has little effect on ligand-receptor binding. These findings suggest that p42/44 play a role in modulating the signal transduction induced by ES20 in the vomeronasal system.     

Embryonic and neonatal development of the vomeronasal and olfactory systems in garter snakes (Thamnophis spp.)

Newborn, unfed garter snakes (Thamnophis spp.) respond preferentially to aqueous extracts of natural prey items, and these responses are mediated by the vomeronasal system (VNS). Since the VNS, and possibly the olfactory system (OS), are functional at birth, we examined the ontogeny of VNS and OS structures in four embryonic stages and two postnatal ages in garter snakes. The results of this study show 1) significant changes in thickness of the receptor epithelia for both systems; 2) temporal differences in the innervation of the telencephalon for each system; and 3) concurrent development of primary and secondary projection sites in both systems. Possible interactions between different cell populations and their significance for morphogenesis are discussed.     

Differential histochemical localization patterns of reduced nicotinamide adenine dinucleotide phosphate-diaphorase and neuronal nitric oxide synthase during postnatal development of the rat vomeronasal organ

The present study was undertaken to examine the localization patterns of nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) by enzyme histochemistry and neuronal nitric oxide synthase (NOS) by immunohistochemistry in the vomeronasal organ of rat from postnatal day 0 for 8 weeks (adult). Nicotinamide adenine dinucleotide phosphate-diaphorase activity was not observed in the sensory epithelium of the vomeronasal organ at postnatal day 0 (the day of birth) and at day 1. At postnatal day 2, NADPH-d activity was observed in several vomeronasal neurons and on the surface of the sensory epithelium. From 25 days through adulthood, the number of vomeronasal neurons having NADPH-d activity increased gradually. On the other hand, neuronal NOS immunoreactivity was not observed in the sensory epithelium of the vomeronasal organ in newborns or in the adult rat. In this study, it is suggested that the nitric oxide pathway in the sensory epithelium of the vomeronasal organ comes into play beyond postnatal day 3. Moreover, it was found that NADPH-d and neuronal NOS are not colocalized in the sensory epithelium of the developing rat vomeronasal organ.     

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.     

Ciliated cells in vitamin A-deprived cultured hamster tracheal epithelium do divide

Summary The pseudostratified tracheal epithelium, composed of a heterogeneous phenotypically varying cell population, was studied with respect to the in vitro cell proliferative activity of differentiated epithelial cells. Ciliated tracheal epithelial cells so far have been considered to be terminally differentiated, nonproliferating cells. Tracheal organ cultures obtained from vitamin A-deprived Syrian Golden hamsters were cultured in a vitamin A-deficient, serum-free, hormone-supplemented medium. In vitamin A-deprived tracheal epithelium treated with physiologically active all-trans retinol and low cigarette-smoke condensate concentrations it is possible to stimulate the cell proliferation of both basal and columnar cells. Therefore, the probability of finding proliferating columnar cells was increased compared with the in vivo and the vitamin A-deprived situation in which cell proliferative activity is relatively low. In the presence of cigarette-smoke condensate in a noncytotoxic concentration, basal, small mucous granule, ciliated, and indifferent tracheal epithelial cells incorporated [methyl-³H]-thymidine into the DNA during the S phase. The finding that ciliated cells were labeled was supported by serial sections showing the same labeled ciliated cell in two section planes separated by 2 to 3 μm, without labeled epithelial cells next to the ciliated cell. Furthermore, a ciliated tracheal epithelial cell incorporating [methyl-³H]thymidine into DNA was also seen in tracheal cultures of vitamin A-deprived hamsters treated with all-trans retinol in a physiologic concentration. The present study was financially supported by the Scientific Advisory Committee on Smoking and Health (Dutch Cigarette Industry Foundation) and the Ministry of Welfare, Health and Cutural Affairs.     

Ontogenetic change in blood oxygen capacity and maximum activity in garter snakes (Thamnophis sirtalis)

Summary There is an ontogenetic increase in the time that garter snakes (Thamnophis s. sirtalis) can maintain maximum activity at 25°C. Newborn snakes are exhausted by 3–5 min of activity while adults can be active for 20–25 min. The increased endurance of adult snakes results from ontogenetic increases in both aerobic and anaerobic energy generation. At rest juvenile and adult snakes have the same whole-body lactic acid concentrations, but at exhaustion adult lactic acid concentrations are 1.5 times those of juveniles. This increase in anaerobic energy production accounts for part of the endurance of adult snakes, but increased aerobic metabolism appears to be more important. Among the mechanisms increasing aerobic metabolism are more effective pulmonary ventilation and a 3-fold ontogenetic increase in blood oxygen capacity.The rapid exhaustion of small garter snakes probably limits the microhabitats they can occupy and the sorts of hunting methods they can employ. Small garter snakes feed only on small prey that are easily subdued. There is an ontogenetic increase in the relative size of prey eaten by garter snakes that parallels the ontogenetic increase in endurance. Adult feeding habits are adopted at the same body size at which adult blood oxygen capacity and endurance are attained.     

Immunolocalization of water channel aquaporins in the vomeronasal organ of the rat: expression of AQP4 in neuronal sensory cells

The vomeronasal organ comprises a pair of narrow tubes in the mammalian nasal septum, serving as a chemosensory system for pheromones. We examined the expression and localization of water channel aquaporins (AQPs) in the rat vomeronasal organ. AQP1 was localized in blood vessels, being particularly abundant in cavernous tissues of the nonsensory mucosa. AQP5 was found in the apical membrane of the gland acinar cells in the vomeronasal organ. AQP3 was detected in the basal cells of the nonsensory epithelium, whereas it was absent in the sensory epithelium. AQP4 was found in both the sensory and the nonsensory epithelia. Interestingly, AQP4 was highly concentrated in the sensory cells of the sensory epithelium. Immunoelectron microscopic examination clearly showed that AQP4 was localized at the plasma membrane in the cell body and lateral membrane of the dendrite, except for the microvillous apical membrane. Nerve fiber bundles emanating from neuronal sensory cells were positive for AQP4, whereby the plasma membrane of each axon was positive for AQP4. These observations clearly show that neuronal sensory cells in the vomeronasal organ are unique in that they express abundant AQP4 at their plasma membrane. This is in marked contrast to the olfactory and central nervous systems, where AQPs are not detectable in neurons, and instead, AQP4 is abundant in the supporting cells and astrocytes surrounding them. The present findings suggest a unique water-handling feature in neuronal sensory cells in the vomeronasal organ.     

Use of a double-label method to detect rapid changes in the rate of cell proliferation.

We developed a double-label method to directly measure the rate at which cells enter S-phase of the cell cycle. All cells in S-phase were first labeled with a short pulse of [3H]-thymidine. This was followed by a longer incubation in bromodeoxyuridine (BrdU), a thymidine analogue. Nuclei labeled with [3H]-thymidine were detected by autoradiography and those labeled with BrdU by immunocytochemistry. Cells labeled only with BrdU must have entered S-phase at some time after the end of the [3H]-thymidine pulse. Thus, the rate of entry of cells into S-phase could be determined. This method was shown to be more accurate and more sensitive than determining changes in the rate at which cells entered S-phase with a continuous labeling protocol. It was possible to detect changes in proliferative activity that occurred in less than 1 hr. We used this double-label technique to study changes in the cell cycle during the terminal differentiation of chicken embryo lens fiber cells. These studies revealed differences in the effects of several treatments known to stimulate fiber cell differentiation. They also demonstrated the presence in the embryonic eye of factors that stimulate and prevent lens cell proliferation and differentiation.     

Identification of an olfactory receptor neuron subclass: cellular and molecular analysis during development

Development of olfactory receptor neuron populations was studied using the previously described monoclonal antibody (Mab) 2B8 which binds to cell surface glycoproteins of presumptive olfactory receptor neurons. In order to definitively demonstrate that the cells recognized were olfactory receptor neurons and to better characterize these cells during development, a well-established receptor cell marker, olfactory marker protein (OMP), was studied at the same time as the 2B8 antigens in double-label immunofluorescence analyses of olfactory structures in rats from Day 13 of gestation (E13) to the early postnatal period. Olfactory epithelium cryostat sections of E13 rats showed binding of the 2B8 Mab to bipolar cells in caudal regions of the nasal cavity. The 2B8 Mab also recognized a large number of cells in the vomeronasal organ (VNO) at this stage. No specific binding of anti-OMP was seen until E15. At this time approximately half of the 2B8 reactive cells also expressed OMP. By birth, greater than 90% of the 2B8 reactive cells expressed OMP. The percentage of total fluorescent labeled cells which are double labeled remained relatively constant at 23-33% as the total number of cells increased between E15 and 2 days postnatal. 2B8 immunoreactivity can be found in the olfactory nerve layer of the olfactory bulb and the presumptive accessory olfactory bulb at E15. In double-label experiments the 2B8 Mab did not bind to all anti-OMP-labeled glomeruli of postnatal to adult rats. In summary, the 2B8 Mab recognizes cells early during development and appears to recognize a subclass of olfactory receptor cells and their axon terminals. Developmental changes in the electrophoretic profile of the olfactory 2B8 antigens were also studied. In the olfactory epithelium a single band at Mr of 200,000 was seen at E19. After birth three bands at 220,000, 180,000 and 110,000 were observed but in adults only two bands of Mr 215,000 and 163,000 were detected. During olfactory bulb development the Mr of the two major 2B8 reactive bands did not change but remained the same as the two major bands seen in the adult olfactory epithelium. The olfactory bulb band at Mr of 215,000 showed a 3 to 4-fold increase and the band at 163,000 showed a 10-fold increase in specific activity from birth to adulthood.     

On the chemosensory nature of the vomeronasal epithelium in adult humans

In contrast to many lower vertebrates, the vomeronasal epithelium (VNE) in humans has long been regarded as absent or functionally irrelevant. For example, the neural connection between the VNE and the accessory olfactory bulb has been reported to degenerate during the second half of pregnancy and its presence has not been demonstrated in adults. Further, reports on the organ's occurrence in adult humans have been contradictory. The aims of this study were to collect immunohistochemical data on the neurogenic or epithelial character of the VNE [for example, with antibodies against protein gene product 9.5 (PGP 9.5), olfactory marker protein (OMP), beta-tubulin, and cytokeratin], determine its proliferative capacity (for example, proliferating cell nuclear antigen), as well as to examine the differentiation activity of VNE cells and their interactions with extracellular matrix components (for example, hyaluronan receptor CD44, galectins, and caveolin). To this end, we studied the vomeronasal organs (VNOs) of 22 human cadavers, three adult biopsies, one embryo (week 8) and one fetus (week 13) by means of immunohistochemistry. The histology of the VNE appeared extremely heterogeneous. There were sections of stratified, respiratory, and typical "pseudostratified" vomeronasal epithelia consisting of slender bipolar cells. Mostly negative immunohistochemical results for OMP indicated that the human VNE does not function like the mature olfactory epithelium. In addition, the investigations did not support the hypothesis that neural connections between the VNE and central brain structures might be present. On the other hand, the presence of some bipolar cells positive for both PGP 9.5 and soybean lectin (SBA) pointed to a neuron-like activity of a small subset of VNE cells. Proliferation antigens located in the nuclei of basally located cells of the VNE were not regularly expressed. However, positive reactions for CD44 demonstrated a high activity of VNE cells in terms of differentiation and migration. Some bipolar cells showed immunoreactivity for caveolin indicating its possible role in signal transduction and differentiation. In summary, the reaction patterns of most antibodies in the adult human VNE are different from those obtained in the olfactory epithelium and the VNO of the rat. However, the VNE shows a specific pattern of activity unique to the mucosa of the nasal cavity. Considering the histologically well differentiated epithelium and its steady maintenance, the VNE of the adult human appears to be a highly differentiated structure the function of which remains unclear.     

The Efferent Connections of the Olfactory Bulb and Accessory Olfactory Bulb in the Snakes,Thamnophis sirtalis and Thamnophis radix

The efferent connections of the olfactory bulb and accessory olfactory bulb of two species of garter snakes, Thamnophis sirtalis and T. radix were studied with experimental anterograde degeneration techniques. Axons of cells located in the olfactory bulb terminate ipsilaterally in all parts of the anterior olfactory nucleus, olfactory tubercle and lateral pallium. In addition, some axons enter the ipsilateral stria medullaris thalami, cross the midline in the habenular commissure, enter the contralateral stria medullaris thalami and terminate in the contralateral lateral pallium. The axons of cells in the accessory olfactory bulb course through the telencephalon completely separated from the fibers of olfactory bulb origin and terminate predominantly in the nucleus sphericus. These results confirm previous reports of the separation between the central projections of the olfactory and vomeronasal systems in a variety of vertebrates. The totality of the separation between these two systems coupled with the extensive development of the vomeronasal-accessory bulb system in these snakes suggests that they may be ideal subjects for further research on the functional significance of the vomeronasal system.     

Changes of colon epithelium proliferation due to individual aging with cyclin proliferating cell nuclear antigen (PCNA/cyclin) immunostaining compared to [3H]-thymidine radioautography

We report a change in the proliferative activity of mouse colonic epithelium due to development and aging. In order to measure the proliferative activity, colonic epithelium was immunostained for cyclin proliferating cell nuclear antigen (PCNA/cyclin), which appears from the Gl to the S phase of the cell cycle, and compared with labeling obtained by [³H]-thymidine radioautography. Litter mice of six age groups from the fetal period (embryonic day 19), newborn period (postnatal day 1), suckling period (postnatal day 5), weaning period (postnatal dy 21), adult period (2 month old) to the senescent period (11 month old) were examined by immunohistochemistry. The descending colons were fixed in methacarn (method-Carnoy) and embedded in paraffin. Sections were stained for PCNA/cyclin activity using 19A2 monoclonal antibody and the avidin-biotin peroxidase complex (ABC) technique. For radioautography, litter mice of nine age groups using in vivo intraperitoneal administration of [³H]-thymidine. The labeling indices of colonic epithelial cells in the proliferative zone were then analyzed and compared between the two investigative methods. Our results show that the prliferative activity of mice colon was high in the fetal and newborn periods and almost constant from the suckling period to senescence, as demonstrated by both PCNA/cyclin immunohistochemistry and [³H]-thymidine radioautography. The labeling index seen by PCNA/cyclin immunohistochemistry was, however, higher than that seen by [³H]-thymidine radioautography.     

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 IP₃ in the vomeronasal (VN) sensory epithelium of female opossums, but suppressed the levels of IP₃ in the VN sensory epithelium of male opossums. The elevated levels of IP₃ 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 GDPβS. Surprisingly, GDPβS itself enhances basal levels of IP₃ in female VN sensory epithelium. This GDPβS-induced increase in levels of IP₃ is reduced by the PLC inhibitor, U73122, but not by the G_i/o inhibitor, NF023. In addition, GDP also enhances basal levels of IP₃. GDPβS, a known inhibitor of G-protein activation, thus appears to have dual functions: as both stimulator and inhibitor of IP₃ 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, IP₃. The extracellular signals are thus amplified.     

Neurogenesis in subclasses of vomeronasal sensory neurons in adult mice

The vomeronasal sensory epithelium contains two distinct populations of vomeronasal sensory neurons. Apical neurons express G_i2α‐linked V1R vomeronasal receptors and project to the anterior portion of the accessory olfactory bulb, while basal neurons express G_oα‐linked V2R receptors and project to the posterior portion. Sensory neurons expressing V1R and V2R vomeronasal receptors are sensitive to different stimuli. Neurons in the vomeronasal system undergo continuous cell turnover during adulthood. To analyze over time neurogenesis of the different sensory cell populations, adult mice were injected with bromodeoxyuridine (BrdU) and sacrificed at postinjection days 1, 3, 5, 7, and 11. Newborn vomeronasal neurons were revealed by antibodies against BrdU while subclasses of vomeronasal neurons were identified using antibodies against G_oα or G_i2α proteins. To ascertain whether G proteins are early expressed during neurogenesis, multiple labeling experiments using PSA‐NCAM and doublecortin were performed. Distribution of BrdU‐labeled cells was analyzed in angular segments from the margin of the sensory epithelium. No sexual differences were found. Within survival groups, BrdU‐G_oα labeled cells were found more marginally when compared with BrdU‐G_i2α labeled cells. The number of BrdU‐positive cells decreased from day 1 to day 3 to remain constant afterwards. The relative proportions of BrdU‐G_i2α and BrdU‐G_oα labeled cells remained similar and constant from postinjection day 1 onwards. This rate was also comparable with BrdU‐positive cells starting day 3. These results indicate an early, constant, and similar rate of neurogenesis in the two major subclasses of vomeronasal neurons, which suggests that both cell populations maturate independently. © 2010 Wiley Periodicals, Inc. Develop Neurobiol 70: 961–970, 2010