Journal of Neurocytology最新文献

The present study examined interspecies, intersexual, and age-related changes in size of the vomeronasal neuroepithelium (VNNE) of two species of greater bushbabies (genus Otolemur, Infraorder Lorisiformes, Suborder Strepsirrhini). Tissue blocks containing the vomeronasal organs of nine O. crassicaudatus (8 adults, 1 neonate) and ten O. garnettii (9 adults, 1 neonate) were studied by means of serial paraffin sectioning and computer-based reconstruction of VNNE volume. In addition, the immunoreactivity of the VNNE to two neuronal markers, neuron-specific beta tubulin (BT) and olfactory marker protein (OMP) was compared between species, sexes, and ages. Results indicated that a clear VNNE is present at birth in both species, and OMP immunoreactivity was verified in O. garnettii at birth. Male and female adults of both species showed OMP-immunoreactive and BT-immunoreactive neurons in the VNNE. Immunohistochemical findings indicated that all males and the youngest females had the thickest VNNE, especially at the marginal junctions with the receptor-free epithelium. Results of a 2-way Analysis of Variance (ANOVA, species x sex) revealed no significant differences in VNNE length or volume between species, but O. crassicaudatus had significantly (p < 0.05) greater palatal length. Significant (p < 0.05) differences also were found between sexes in VNNE volume, but no significant differences in palatal length or VNNE length. The distribution of VNNE volume against age indicated that the sex differences were more pronounced in O. crassicaudatus than O. garnettii. For both species and sexes, distribution of VNNE volume against age suggested an age-related reduction in volume. These findings demonstrate postnatal plasticity in VNNE size in Otolemur that is reminiscent of that found for olfactory structures in some rodents. Bushbabies or other strepsirrhine primates may offer an opportunity for further understanding of behavioral correlates of VNNE postnatal plasticity, which may represent primitive functional characteristics of the order Primates.

Many diabetic individuals develop anosmia but the mechanism(s) causing the dysfunction in the olfactory system is (are) unknown. Glial fibrillary acidic protein expression is reduced in diabetic retinopathy and is also reduced, with unknown consequences, in other brain regions of diabetic rats. We used immunohistochemistry and immunoblotting from untreated control and streptozotocin-induced type 1 (insulin dependent) diabetic rats to investigate main olfactory epithelial mitotic rate and glial fibrillary acidic protein expression in the lamina propria of the sensory epithelium and in the olfactory bulb. Numbers of bromodeoxyuridine-positive cells were significantly lower in the diabetic sensory epithelium compared to non-diabetic controls. Immunohistochemical observations suggested a qualitative difference in glial fibrillary acidic protein expression in both regions examined especially in the olfactory bulb external plexiform layer and the lamina propria. Immunoblot analysis confirmed that the diabetic olfactory bulb and lamina propria expressed less glial fibrillary acidic protein compared to the non-diabetic control group. The lower expression levels in the olfactory bulb external plexiform layer suggested by immunohistochemistry do not reflect a change in the number of astrocytes since the numbers of S100B(+) cells were not different between the two groups.

Binding sites of Griffonia simplicifolia I-B4 isolectin (GS-I-B4), which recognizes terminal alpha-galactose residues of glycoconjugates, were examined in the juxtaluminal region of the rat vomeronasal sensory epithelium and its associated glands of the vomeronasal organ, using a lectin cytochemical technique. Lowicryl K4M-embedded ultra-thin sections, which were treated successively with biotinylated GS-I-B4 and streptavidin-conjugated 10 nm colloidal gold particles, were observed under a transmission electron microscope. Colloidal gold particles, which reflect the presence of terminal alpha-galactose-containing glycoconjugates, were present in vomeronasal receptor neurons in the sensory epithelium and secretory granules of acinar cells of associated glands of the epithelium. Quantitative analysis demonstrated that the density of colloidal gold particles associated with sensory cell microvilli that projected from dendritic endings of vomeronasal neurons was considerably higher than that of microvilli that projected from neighboring sustentacular cells. The same was true for the apical cytoplasms of these cells just below the microvilli. These results suggest that of the sensory microvilli and dendritic endings contained a much larger amount of the alpha-galactose-containing glycoconjugates, compared with those in sustentacular microvilli. Further, biochemical analyses demonstrated several vomeronasal organ-specific glycoproteins with terminal alpha-galactose.

The main and accessory olfactory epithelia of the mouse are composed of many cell populations. Each sensory neuron is thought to express one allele of one of the approximately 1000 odorant or approximately 300 vomeronasal receptor genes. Sensory neurons die and are replaced by new neurons that differentiate from precursor cells throughout the lifetime of the individual. Neuronal replacement is asynchronous, resulting in the co-existence of cells at various stages of differentiation. Receptor gene diversity and ongoing neuronal differentiation produce complex mosaics of gene expression within these epithelia. Accurate description of gene expression patterns will facilitate the understanding of mechanisms of gene choice and differentiation. Here we report a detailed protocol for two- and three-color fluorescent RNA in situ hybridization (ISH) and its combination with immunohistochemistry, or detection of bromodeoxyuridine (BrdU)-incorporated DNA after labeling. The protocol is applied to cryosections of the main and accessory olfactory epithelia in mouse.

The zebrafish, (Danio rerio) is an important model organism for the analysis of molecular mechanisms that govern neuronal circuit development. The neuronal circuitry that mediates olfaction is crucial for the development and survival of all teleost fishes. In concert with other sensory systems, olfaction is functional at early stages in zebrafish development and mediates important behavioral and survival strategies in the developing larva. Odorant cues are transduced by an array of signaling molecules from receptors in olfactory sensory neurons. The scaffolding protein family known as Homer is well placed to orchestrate this signaling cascade by interacting with and coupling membrane bound receptors to cytosolic signaling partners. To date, Homer has not been demonstrated in the zebrafish. Here we report that the Homer 1b/c isoform was prominent in the olfactory system from the earliest stages of differentiation. We describe the spatial and temporal distribution of Homer in the zebrafish olfactory system. At 24 hours post fertilization (hpf), Homer expression delineated the boundary of the presumptive olfactory placode. Subsequent expression steadily increased throughout the developing olfactory placode, with a prominent localization to the dendritic knobs of the olfactory sensory neurons. Homer expression in the developing olfactory bulb was punctate and prominent in the glomeruli, displaying an apparent synaptic localization. This work supports the hypothesis that Homer is an important molecule in neuronal circuit development, necessary for crucial behaviors required for development and survival.

Brain-derived neurotrophic factor (BDNF) and Neurotrophin 3 (NT-3) are members of the neurotrophin family and are expressed in the developing and adult tongue papillae. BDNF null-mutated mice exhibit specific impairments related to innervation and development of the gustatory system while NT-3 null mice have deficits in their lingual somatosensory innervation. To further evaluate the functional specificity of these neurotrophins in the peripheral gustatory system, we generated double BDNF/NT-3 knockout mice and compared the phenotype to BDNF(-/-) and wild-type mice. Taste papillae morphology was severely distorted in BDNF(-/-) xNT-3(-/-) mice compared to single BDNF(-/-) and wild-type mice. The deficits were found throughout the tongue and all gustatory papillae. There was a significant loss of fungiform papillae and the papillae were smaller in size compared to BDNF(-/-) and wild-type mice. Circumvallate papillae in the double knockouts were smaller and did not contain any intraepithelial nerve fibers. BDNF(-/-) xNT-3(-/-) mice exhibited additive losses in both somatosensory and gustatory innervation indicating that BDNF and NT-3 exert specific roles in the innervation of the tongue. However, the additional loss of fungiform papillae and taste buds in BDNF(-/-) xNT-3(-/-) mice compared to single BDNF knockout mice indicate a synergistic functional role for both BDNF-dependent gustatory and NT-3-dependent somatosensory innervations in taste bud and taste papillae innervation and development.

Neurotrophins, neurotrophin receptors and sensory neurons are required for the development of lingual sense organs. For example, neurotrophin 3 sustains lingual somatosensory neurons. In the traditional view, sensory axons will terminate where neurotrophin expression is most pronounced. Yet, lingual somatosensory axons characteristically terminate in each filiform papilla and in each somatosensory prominence within a cluster of cells expressing the p75 neurotrophin receptor (p75NTR), rather than terminating among the adjacent cells that secrete neurotrophin 3. The p75NTR on special specialized clusters of epithelial cells may promote axonal arborization in vivo since its over-expression by fibroblasts enhances neurite outgrowth from overlying somatosensory neurons in vitro. Two classical observations have implicated gustatory neurons in the development and maintenance of mammalian taste buds--the early arrival times of embryonic innervation and the loss of taste buds after their denervation in adults. In the modern era more than a dozen experimental studies have used early denervation or neurotrophin gene mutations to evaluate mammalian gustatory organ development. Necessary for taste organ development, brain-derived neurotrophic factor sustains developing gustatory neurons. The cardinal conclusion is readily summarized: taste buds in the palate and tongue are induced by innervation. Taste buds are unstable: the death and birth of taste receptor cells relentlessly remodels synaptic connections. As receptor cells turn over, the sensory code for taste quality is probably stabilized by selective synapse formation between each type of gustatory axon and its matching taste receptor cell. We anticipate important new discoveries of molecular interactions among the epithelium, the underlying mesenchyme and gustatory innervation that build the gustatory papillae, their specialized epithelial cells, and the resulting taste buds.

Geniculate ganglion axons arrive in the lingual mesenchyme on embryonic day 13 (E13), 3-4 days before penetrating fungiform papilla epithelium (E17). This latency may result from chemorepulsion by epithelial Sema3A (Dillon et al. (2004) Journal of Comparative Neurology 470, 13-24), or Sema3F, which we report is also expressed in this epithelium. Sema3A and Sema3F repelled or suppressed geniculate neurite outgrowth, respectively, and these effects were stage and neurotrophic factor dependent. BDNF-stimulated outgrowth is repelled by Sema3A until E17, but insensitive to Sema3F from E16. NT-4-stimulated neurite outgrowth is sensitive to Sema3A and Sema3F through E18, but NT-4 has not been detected in E15-18 tongue. E15-18 tongue explants did not exhibit net chemorepulsion of geniculate neurites, but the ability of tongue explants to support geniculate neurite outgrowth fluctuates: E12-13 (Rochlin et al. (2000), Journal of Comparative Neurology, 422, 579-593) and E17-18 explants promote and may attract geniculate neurites, but stages corresponding to intralingual arborization do not. The E18 trophic and tropic effects were evident even in the presence of BDNF or NT-4, suggesting that some other factor is responsible. Intrinsic neurite outgrowth capability (without exogenous neurotrophic factors) fluctuated similarly: ganglia deteriorated at E15, but exhibited moderate outgrowth at E18. The chemorepulsion studies are consistent with a role for Sema3A, not Sema3F, in restricting geniculate axons from the epithelium until E17, when axons penetrate the epithelium. The transient inability of tongue explants to promote geniculate neurite outgrowth may signify an alternative mechanism for restricting geniculate axons from the epithelium: limiting trophic factor access.