Neurogenesis in the vomeronasal epithelium of adult rats: evidence for different mechanisms for growth and neuronal turnover

The pattern of cell migration during neuronal turnover in the vomeronasal sensory epithelium (VN-SE) is controversial. In mice, proliferating cells were detected at the edges and were described as migrating to the center of the VN-SE. In rats, in addition to proliferating cells at the margins of the epithelium, dividing cells are also present along the entire basal lamina of the VN-SE. In marsupials, dividing cells have also been observed in the margins and in the center of the VN-SE, the latter of which migrate vertically and become neurons. To investigate whether the process of neuronal turnover in placental mammals consists of horizontal and/or vertical migration, and whether or not this process is common to mammals, adult rats were injected with bromodeoxyuridine (BrdU) and allowed to survive for different periods of time. The distribution of BrdU-labeled cells in the horizontal and vertical dimension of the VN-SE was analyzed as a function of time. Both horizontal and vertical migrations of BrdU-labeled cells were detected. Since cells in the center of the VN-SE migrate vertically, and, as demonstrated by coexpression of markers of neuronal maturity and BrdU, become mature one day after undergoing mitosis, it is very likely that these cells participate in neuronal turnover. Conversely, because cells in the margins of the VN-SE stop migrating horizontally on day 14 before they have reached the center of the VN-SE, and since the VN-SE continues to grow during adulthood, it is likely that most of these latter cells constitute pools for growth.     

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     

Maturation of vomeronasal receptor neurons in vitro by coculture with accessory olfactory bulb neurons

To analyze the mechanisms of perception and processing of pheromonal signals in vitro, we previously developed a new culture system for vomeronasal receptor neurons (VRNs), referred to as the vomeronasal pocket (VN pocket). However, very few VRNs were found to express the olfactory marker protein (OMP) and to have protruding microvilli in VN pockets, indicating that these VRNs are immature and that VN pockets are not appropriate for pheromonal recognition. To induce VRN maturation in VN pockets, we here attempted to coculture VN pockets with a VRN target-accessory olfactory bulb (AOB) neurons. At 3 weeks of coculture with AOB neurons, the number of OMP-immunopositive VRNs increased. By electron microscopy, the development of microvilli in VRNs was found to occur coincidentally with OMP expression in vitro. These results indicate that VRN maturation is induced by coculture with AOB neurons. The OMP expression of VRNs was induced not only by AOB neurons but also by neurons of other parts of the central nervous system (CNS). Thus, VRN maturation requires only CNS neurons. Since the maturation of VRNs was not induced in one-well separate cultures, the nonspecific induction of OMP expression by CNS neurons suggests the involvement of a direct contact effect with CNS in VRN maturation.     

Neurogenesis, migration, and apoptosis in the vomeronasal epithelium of adult mice

The location of neurogenesis and the direction of migration of neurons in the adult mouse vomeronasal organ is controversial. Cell division occurs at the center, and particularly, at the edges of the epithelium. Newly generated cells at the center of the epithelium participate in neurogenesis, however, it is unknown to what extent dividing cells at the edges participate in growth, become apoptotic or mature into neurons. Premitotic cells were labeled with bromodeoxyuridine (BrdU) in adult mice and animals allowed to survive for different postinjection periods. The terminal deoxynucleotidyl transferase-mediated biotinylated dUTP nick end-labeling (TUNEL) method was used to show the distribution of apoptotic cells. The vertical and horizontal position of BrdU-labeled cells was analyzed as a function of postinjection survival time. Vertical and horizontal migration of BrdU-labeled cells were detected. Cells in the central portions of the epithelium migrated vertically to become neurons as demonstrated by co-expression of olfactory marker protein. Cells at the edges migrated horizontally very slowly (less than 10% of the distance from the edge to the center of the epithelium per month), thus indicating that these cells participate in cell renewal exclusively in marginal regions. Neural turnover in the mouse vomeronasal epithelium, therefore appears to occur through a process of vertical migration. Data on the distribution of apoptotic cells indicate that a number of dividing cells throughout the epithelium, but particularly at the edges, die before becoming functional neurons. Accordingly, most dividing cells at the edges probably constitute a reservoir of stem cells dying before differentiation.     

Histochemical and immunocytochemical study of the migration of neurons from the rat olfactory placode

Immunocytochemical and histochemical methods have been used to describe the neuronal population migrating from the rat olfactory placode and to analyze the spatio-temporal evolution of this neuronal migration during development. Several neuronal markers, such as binding to the lectin Ulex europaeus (UEA I) and the presence of neuron-specific enolase (NSE), olfactory marker protein (OMP), and luteinizing hormone-releasing hormone (LHRH), have been tested in order to determine whether migrating neurons originate from both the medial and the lateral parts of the placode and whether they all express LHRH. Our data show that a large population of differentiated migrating neurons can be identified with an antibody against NSE from the 14th day of gestation and with UEA I one day later. Migrating neurons are closely associated with both the vomeronasal axon fascicles emerging from the medial pit and the olfactory axons originating from the lateral pit. However, the neuron migration from the lateral pit appears to be more discrete than that from the medial pit. No LHRH immunoreactivity has been detected among neurons migrating from the lateral pit. Some neurons accompanying the olfactory axon fascicles exhibit a high level of maturation as shown by their OMP-positivity. Numerous neurons positive for both NSE and UEA I have also been observed within the presumptive olfactory nerve layer in early embryonic stages.     

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.     

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.     

Selective G protein beta gamma-subunit compositions mediate phospholipase C activation in the vomeronasal organ

Chemosensory neurons of the vomeronasal organ (VNO) are supposed to detect pheromones controlling social and reproductive behavior in most terrestrial vertebrates. Recent studies indicate that pheromone signaling in VNO neurons is mediated via phospholipase C (PLC) activation generating the two second messengers inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). Since G alpha(i) and G alpha(o) predominantly expressed in VNO neurons are usually not involved in activating PLC, it was explored if PLC activation may be mediated by G beta gamma subunits. It was found that a scavenger for beta gamma dimers reduced the urine-induced IP3 formation in VNO preparations in a dose-dependent manner indicating a role for G beta gamma complexes. Towards an identification of the relevant G beta and G gamma subunit(s), PCR approaches as well as immunohistochemical experiments were performed. It was found that out of the five known G beta subtypes, only G beta2 was expressed in both G alpha(i) as well as G alpha(o) neurons. Experimental approaches focusing on the spatial expression profile of identified G gamma subtypes revealed that G gamma8-positive neurons are preferentially localized to the basal region of the vomeronasal epithelium, whereas G gamma2-reactive cells are restricted to the apical G alpha(i)-positive layer of the sensory epithelium. As IP3 formation induced upon stimulation with volatile urinary compounds was selectively blocked by G gamma2-specific antibodies whereas second messenger formation elicited upon stimulation with alpha2u globulin was inhibited by antibodies recognizing G gamma8, it is conceivable that PLC activation in the two populations of chemosensory VNO neurons is mediated by different G beta gamma complexes.     

Retinoic acid selectively inhibits death of basal vomeronasal neurons during late stage of neural circuit formation

In mouse, sexual, aggressive, and social behaviors are influenced by G protein-coupled vomeronasal receptor signaling in two distinct subsets of vomeronasal sensory neurons (VSNs): apical and basal VSNs. In addition, G protein-signaling by these receptors inhibits developmental death of VSNs. We show that cells of the vomeronasal nerve express the retinoic acid (RA) synthesizing enzyme retinal dehydrogenase 2. Analyses of transgenic mice with VSNs expressing a dominant-negative RA receptor indicate that basal VSNs differ from apical VSNs with regard to a transient wave of RA-regulated and caspase 3-mediated cell death during the first postnatal week. Analyses of G-protein subunit deficient mice indicate that RA and vomeronasal receptor signaling combine to regulate postnatal expression of Kirrel-2 (Kin of IRRE-like), a cell adhesion molecule regulating neural activity-dependent formation of precise axonal projections in the main olfactory system. Collectively, the results indicate a novel connection between pre-synaptic RA receptor signaling and neural activity-dependent events that together regulate neuronal survival and maintenance of synaptic contacts.     

Olfactory marker protein during ontogeny: Immunohistochemical localization

Specific immunohistochemical staining for the olfactory marker protein (OMP) is first demonstrated in rat olfactory receptor neurons at embryonic day 18, at which age no OMP can be seen in the olfactory bulb or vomeronasal epithelium. At 21 days OMP staining in the olfactory epithelium is more extensive and is evident in the fibrous and glomerular layers of the bulb as well. Staining intensity increases progressively until the full adult pattern is seen by 1 month postnatally. In the vomeronasal organ, staining is not observed until the fourth postnatal day and, even then, only with higher antiserum concentrations. In mice, very similar results are obtained, except for a much earlier appearance of OMP, on embryonic day 14. Olfactory epithelium from 12- and 13-day rat embryos maintained in organ culture for up to 2 weeks did not exhibit OMP staining, nor did several neural or nonneural tissues from adult animals. The temporal and causal interrelationships between OMP and other indicators of olfactory receptor cell maturation are considered.     

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.     

Calcium-activated chloride channels in the apical region of mouse vomeronasal sensory neurons

The rodent vomeronasal organ plays a crucial role in several social behaviors. Detection of pheromones or other emitted signaling molecules occurs in the dendritic microvilli of vomeronasal sensory neurons, where the binding of molecules to vomeronasal receptors leads to the influx of sodium and calcium ions mainly through the transient receptor potential canonical 2 (TRPC2) channel. To investigate the physiological role played by the increase in intracellular calcium concentration in the apical region of these neurons, we produced localized, rapid, and reproducible increases in calcium concentration with flash photolysis of caged calcium and measured calcium-activated currents with the whole cell voltage-clamp technique. On average, a large inward calcium-activated current of -261 pA was measured at -50 mV, rising with a time constant of 13 ms. Ion substitution experiments showed that this current is anion selective. Moreover, the chloride channel blockers niflumic acid and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid partially inhibited the calcium-activated current. These results directly demonstrate that a large chloride current can be activated by calcium in the apical region of mouse vomeronasal sensory neurons. Furthermore, we showed by immunohistochemistry that the calcium-activated chloride channels TMEM16A/anoctamin1 and TMEM16B/anoctamin2 are present in the apical layer of the vomeronasal epithelium, where they largely colocalize with the TRPC2 transduction channel. Immunocytochemistry on isolated vomeronasal sensory neurons showed that TMEM16A and TMEM16B coexpress in the neuronal microvilli. Therefore, we conclude that microvilli of mouse vomeronasal sensory neurons have a high density of calcium-activated chloride channels that may play an important role in vomeronasal transduction.     

Perinatal size and maturation of the olfactory and vomeronasal neuroepithelia in lorisoids and lemuroids

Explanations for the chemosensory abilities of newborn mammals focus primarily on food (milk) acquisition and communication (e.g., maternal-infant bonding). However, the relative importance of the main and accessory (vomeronasal) olfactory systems is hypothesized to differ at birth between altricial and precocial mammals. Strepsirrhines (lemurs and lorises) possess main and accessory olfactory systems, and vary in life-history traits related to infant dependency and maturation. Accordingly, this study examines the size and maturational characteristics of vomeronasal (VNNE) and olfactory (OE) neuroepithelia in strepsirrhines. Serially sectioned heads of 18 infant cadavers were examined microscopically for neuroepithelial distribution. Measurements were taken on the length of the nasal fossa on one side that was occupied by VNNE and OE. The data were corrected for body size using the cranial length or body mass, and were then examined for correlation with several life-history variables, as well as activity pattern. In addition, immunohistochemistry was used to identify cells in the VNNE and OE that express olfactory marker protein (OMP), a marker of mature olfactory neurons. Relative OE extent was not significantly correlated with any of the life-history variables. Relative VNNE length was negatively correlated with relative gestation length and relative neonatal mass (P<0.05). However, when we corrected for phylogenetic relationships, we found no significant correlations between either of the neuroepithelial measurements and life-history variables. Immunohistochemical findings suggest that OE has more OMP-reactive cells than VNNE in all species. OMP-reactive cells appear to be less numerous in diurnal species compared to most nocturnal species. These results indicate that the VNNE may be relatively longer at birth in altricial species. However, it remains uncertain how phylogeny and/or ontogeny may explain these findings.     

Heterogeneity in the accessory olfactory system

The mammalian accessory olfactory bulb (AOB) is chemoarchitecturally heterogeneous in that it stains differentially with a number of markers; the receptor cells that project to the AOB are similarly heterogeneous. What is the significance of this heterogeneity? We have found that the AOB of the gray, short-tailed opossum, Monodelphis domestica, stains differentially with a number of 'markers': antibodies to olfactory marker protein (OMP) and the alpha subunit of the G protein Gi2, the lectin of Vicia villosa and NADPH-diaphorase. These markers stain the rostral AOB more strongly than the caudal AOB whereas, the G protein subunit G(o) alpha is located predominantly in the posterior subdivision of the AOB. This heterogeneity in the chemoarchitecture of the AOB may reflect a fundamental organizational dichotomy within the vomeronasal system that corresponds to a functional dichotomy. The vomeronasal sensory epithelium also exhibits a chemoarchitectural heterogeneity: receptor cells in the basal third are G(o) alpha-immunoreactive whereas the cells in the middle third are Gi2 alpha-immunoreactive. Tracing studies using WGA-HRP demonstrate that the neurons in the middle third of the vomeronasal sensory epithelium project their axons to the anterior AOB whereas those in the basal third appear to project to the posterior AOB.      

Inhibition of apoptotic signaling cascades causes loss of trophic factor dependence during neuronal maturation

During development, neurons are acutely dependent on target-derived trophic factors for survival. This dependence on trophic support decreases dramatically with maturation in several neuronal populations, including sympathetic neurons. Analyses of nerve growth factor deprivation in immature and mature sympathetic neurons indicate that maturation aborts the cell death pathway at a point that is mechanistically indistinguishable from Bax deletion. However, neither the mRNA nor protein level of BAX changes with neuronal maturation. Therefore, BAX must be regulated posttranslationally in mature neurons.Nerve growth factor deprivation in immature sympathetic neurons induces two parallel processes: (a) a protein synthesis-dependent, caspase-independent translocation of BAX from the cytosol to mitochondria, followed by mitochondrial membrane integration and loss of cytochrome c; and (b) the development of competence-to-die, which requires neither macromolecular synthesis nor BAX expression. Activation of both signaling pathways is required for caspase activation and apoptosis in immature sympathetic neurons. In contrast, nerve growth factor withdrawal in mature sympathetic neurons did not induce the translocation of either BAX or cytochrome c. Moreover, mature neurons did not develop competence-to-die with cytoplasmic accumulation of cytochrome c. Therefore, inhibition of both BAX-dependent cytochrome c release and the development of competence-to-die contributed to the loss of trophic factor dependence associated with neuronal maturation.     

The vomeronasal cavity in adult humans

We observed the surface of the anterior part of the nasal septum of living subjects using an endoscope. In approximately 13% of 1842 patients without pathology of the septum, the vomeronasal pit was clearly observed on each side of the septum, and in 26% it was observed only on one side. The remaining observations indicated either the presence of putative pits or no visible evidence of a pit. However, repetitive observations on 764 subjects depicted changes over time, from nothing visible to well-defined pits and vice versa. Based on 130 subjects observed at least four times, we estimate that approximately 73% of the population exhibits at least one clearly defined pit on some days. By computer tomography, the vomeronasal cavities were located at the base of the most anterior part of the nasal septum. Histological studies indicated that the vomeronasal cavities consisted of a pit generally connected to a duct extending in a posterior direction under the nasal mucosa. Many glands were present around the duct, which contained mucus. There was no sign of the pumping elements found in other mammalian species. Most cells in the vomeronasal epithelium expressed keratin, a protein not expressed by olfactory neurons. Vomeronasal epithelial cells were not stained by an antibody against the olfactory marker protein, a protein expressed in vomeronasal receptor neurons of other mammals. Moreover, an antibody against protein S100, expressed in Schwann cells, failed to reveal the existence of vomeronasal nerve bundles that would indicate a neural connection with the brain. Positive staining was obtained with the same antibodies on specimens of human olfactory epithelium. The lack of neurons and vomeronasal nerve bundles, together with the results of other studies, suggests that the vomeronasal epithelium, unlike in other mammals, is not a sensory organ in adult humans.     

Expression patterns of homeobox genes in the mouse vomeronasal organ at postnatal stages

Homeodomain proteins are encoded by homeobox genes and regulate development and differentiation in many neuronal systems. The mouse vomeronasal organ (VNO) generates in situ mature chemosensory neurons from stem cells. The roles of homeodomain proteins in neuronal differentiation in the VNO are poorly understood. Here we have characterized the expression patterns of 28 homeobox genes in the VNO of C57BL/6 mice at postnatal stages using multicolor fluorescent in situ hybridization. We identified 11 homeobox genes (Dlx3, Dlx4, Emx2, Lhx2, Meis1, Pbx3, Pknox2, Pou6f1, Tshz2, Zhx1, Zhx3) that were expressed exclusively in neurons; 4 homeobox genes (Pax6, Six1, Tgif1, Zfhx3) that were expressed in all non-neuronal cell populations, with Pax6, Six1 and Tgif1 also expressed in some neuronal progenitors and precursors; 12 homeobox genes (Adnp, Cux1, Dlx5, Dlx6, Meis2, Pbx2, Pknox1, Pou2f1, Satb1, Tshz1, Tshz3, Zhx2) with expression in both neuronal and non-neuronal cell populations; and one homeobox gene (Hopx) that was exclusively expressed in the non-sensory epithelium. We studied further in detail the expression of Emx2, Lhx2, Meis1, and Meis2. We found that expression of Emx2 and Lhx2 initiated between neuronal progenitor and neuronal precursor stages. As far as the sensory neurons of the VNO are concerned, Meis1 and Meis2 were only expressed in the apical layer, together with Gnai2, but not in the basal layer.     

Herpes simplex virus US3 protein kinase regulates virus-induced apoptosis in olfactory and vomeronasal chemosensory neurons in vivo

A role for the US3 protein kinase of herpes simplex virus (HSV) in regulating virus-induced neuronal apoptosis was investigated in an experimental mouse system, in which wild-type HSV invades the central nervous system (CNS) via the olfactory and vomeronasal systems upon intranasal infection. Wild-type HSV-2 strain 186 infected a fraction of olfactory and vomeronasal chemosensory neurons without inducing apoptosis and was transmitted to the CNS, precipitating lethal encephalitis. In sharp contrast, an US3-disrupted mutant, L1BR1, induced neuronal apoptosis in these peripheral conduits upon infection, blocking viral transmission to the CNS and causing no signs of disease. An US3-repaired mutant, L1B(-)11, behaved similarly to the wild-type virus. Only 5 p.f.u. of L1BR1 was sufficient to compromise mice when the mutant virus was introduced directly into the olfactory bulb, a viral entry site of the CNS. These results suggest that the US3 protein kinase of HSV regulates virus-induced neuronal apoptosis in peripheral conduits and determines the neuroinvasive phenotype of HSV. Furthermore, virus-induced neuronal apoptosis of peripheral nervous system cells may be a protective host response that blocks viral transmission to the CNS.     

Timing of neuronal intermediate filament proteins expression in the mouse vomeronasal organ during pre- and postnatal development. An immunohistochemical study

Several types of intermediate filament proteins are expressed in developing and mature neurons; they cooperate with other cytoskeletal components to sustain neuronal function from early neurogenesis onward. In this work the timing of expression of nestin, peripherin, internexin, and the neuronal intermediate filament triplet [polypeptide subunits of low (NF-L), medium (NF-M), and high (NF-H) molecular weight] was investigated in the developing fetal and postnatal mouse vomeronasal organ (VNO) by means of immunohistochemistry. The results show that the sequence of expression of intermediate filament proteins is internexin, nestin, and NF-M in the developing vomeronasal sensory epithelium; internexin, peripherin, and NF-M in the developing vomeronasal nerve; and nestin, internexin and peripherin, NF-L, and NF-M in the nerve supply to accessory structures of the VNO. At sexual maturity (2 months) NF-M is only expressed in vomeronasal neurons and NF-M, NF-L and peripherin are expressed in extrinsic nerves supplying VNO structures. The differential distribution of intermediate filament proteins in the vomeronasal sensory epithelium and nerve is discussed in terms of the cell types present therein. It is concluded that several intermediate filament proteins are sequentially expressed during intrauterine development of the VNO neural structures in a different pattern according to the different components of the VNO.