Ephrin-A3 is required for tonotopic map precision and auditory functions in the mouse auditory brainstem.

Tonotopy is a prominent feature of the vertebrate auditory system and forms the basis for sound discrimination, but the molecular mechanism that underlies its formation remains largely elusive. Ephrin/Eph signaling is known to play important roles in axon guidance during topographic mapping in other sensory systems, so we investigated its possible role in the establishment of tonotopy in the mouse cochlear nucleus. We found that ephrin-A3 molecules are differentially expressed along the tonotopic axis in the cochlear nucleus during innervation. Ephrin-A3 forward signaling is sufficient to repel auditory nerve fibers in a developmental stage-dependent manner. In mice lacking ephrin-A3, the tonotopic map is degraded and isofrequency bands of neuronal activation upon pure tone exposure become imprecise in the anteroventral cochlear nucleus. Ephrin-A3 mutant mice also exhibit a delayed second wave in auditory brainstem responses upon sound stimuli and impaired detection of sound frequency changes. Our findings establish an essential role for ephrin-A3 in forming precise tonotopy in the auditory brainstem to ensure accurate sound discrimination.

Ephrin-B/EphB Signaling Is Required for Normal Innervation of Lingual Gustatory Papillae.

The innervation of taste buds is an excellent model system for studying the guidance of axons during targeting because of their discrete nature and the high fidelity of innervation. The pregustatory epithelium of fungiform papillae is known to secrete diffusible axon guidance cues such as BDNF and Sema3A that attract and repel, respectively, geniculate ganglion axons during targeting, but diffusible factors alone are unlikely to explain how taste axon terminals are restricted to their territories within the taste bud. Nondiffusible cell surface proteins such as Ephs and ephrins can act as receptors and/or ligands for one another and are known to control axon terminal positioning in several parts of the nervous system, but they have not been studied in the gustatory system. We report that ephrin-B2 linked ß-galactosidase staining and immunostaining was present along the dorsal epithelium of the mouse tongue as early as embryonic day 15.5 (E15.5), but was not detected at E14.5, when axons first enter the epithelium. Ephrin-B1 immunolabeling was barely detected in the epithelium and found at a somewhat higher concentration in the mesenchyme subjacent to the epithelium. EphB1 and EphB2 were detected in lingual sensory afferents in vivo and geniculate neurites in vitro. Ephrin-B1 and ephrin-B2 were similarly effective in repelling or suppressing outgrowth by geniculate neurites in vitro. These in vitro effects were independent of the neurotrophin used to promote outgrowth, but were reduced by elevated levels of laminin. In vivo, mice null for EphB1 and EphB2 exhibited decreased gustatory innervation of fungiform papillae. These data provide evidence that ephrin-B forward signaling is necessary for normal gustatory innervation of the mammalian tongue.

Neurotrophin-4 is more potent than brain-derived neurotrophic factor in promoting, attracting and suppressing geniculate ganglion neurite outgrowth.

The geniculate ganglion, which provides innervation to taste buds in the anterior tongue and palate, is unique among sensory ganglia in that its neurons depend on both neurotrophin-4 (NT4) and brain-derived neurotrophic factor (BDNF) for survival. Whereas BDNF is additionally implicated in taste axon guidance at targeting stages, much less is known about the guidance role of NT4 during targeting, or about either neurotrophin during initial pathfinding. NT4 and BDNF have distinct expression patterns in vivo, raising the possibility of distinct roles. We characterized the influence of NT4 and BDNF on geniculate neurites in collagen I gels at early embryonic through postnatal stages. During early pathfinding to the tongue (embryonic days 12-13; E12-13), NT4 and BDNF promote significantly longer outgrowth than during intralingual targeting (E15-18). NT4 is more potent than BDNF at stimulating neurite outgrowth and both factors exhibit concentration optima, i.e. intermediate concentrations (0.25 ng/ml NT4 or 25 ng/ml BDNF) promote maximal neurite extension and high concentrations (10 ng/ml NT4 or 200 ng/ml BDNF) suppress it. Only partial suppression was seen at E12 (when axons first emerge from the ganglion in vivo) and postnatally, but nearly complete suppression occurred from E13 to E18. We show that cell death is not responsible for suppression. Although blocking the p75 receptor reduces outgrowth at the optimum concentrations of NT4 and BDNF, it did not reduce suppression of outgrowth. We also report that NT4, like BDNF, can act as a chemoattractant for geniculate neurites, and that the tropic influence is strongest during intralingual targeting (E15-18). NT4 does not appear to act as an attractant in vivo, but it may prevent premature invasion of the epithelium by suppressing axon growth.

Brain-derived neurotrophic factor attracts geniculate ganglion neurites during embryonic targeting.

Geniculate axons are initially guided to discrete epithelial placodes in the lingual and palatal epithelium that subsequently differentiate into taste buds. In vivo approaches show that brain-derived neurotrophic factor (BDNF) mRNA is concentrated in these placodes, that BDNF is necessary for targeting taste afferents to these placodes, and that BDNF misexpression disrupts guidance. We used an in vitro approach to determine whether BDNF may act directly on geniculate axons as a trophic factor and as an attractant, and whether there is a critical period for responsiveness to BDNF. We show that BDNF promotes neurite outgrowth from geniculate ganglion explants dissected from embryonic day (E) 15, E18, infant, and adult rats cultured in collagen gels, and that there is a concentration optimum for neurite extension. Gradients of BDNF derived from slow-release beads caused the greatest bias in neurite outgrowth at E15, when axons approach the immature gustatory papillae. Further, neurites advanced faster toward the BDNF bead than away from it, even if the average amount of neurotrophic factor encountered was the same. We also found that neurites that contact BDNF beads did not advance beyond them. At E18, when axons would be penetrating pregustatory epithelium in vivo, BDNF continued to exert a tropic effect on geniculate neurites. However, at postnatal and adult stages, the influence of BDNF was predominantly trophic. Our data support a role for BDNF acting as an attractant for geniculate axons during a critical period that encompasses initial targeting but not at later stages.

Involvement of brain-derived neurotrophic factor (BDNF) in the development of periodontal Ruffini endings.

The periodontal Ruffini ending has been reported to show immunoreactivity for tyrosine kinase B (trkB), the high-affinity receptor for brain-derived neurotrophic factor (BDNF), in the periodontal ligament of the rat incisor. Furthermore, adult heterozygous BDNF-mutant mice showed malformation and reduction of the periodontal Ruffini endings. To investigate further roles of BDNF in these structures, the development, distribution, and terminal morphology of Ruffini endings were examined in the incisor periodontal ligament of heterozygous and homozygous BDNF mutant mice, as well as in the wild-type littermate by immunohistochemistry for protein gene product (PGP) 9.5, a general neuronal marker. A similar distribution and terminal formation of PGP 9.5-immunoreactive nerve fibers was recognized in the periodontal ligament of all phenotypes at postnatal week (PW) 1. At this stage, the nerve fibers had a beaded appearance, but did not form the periodontal Ruffini endings. At PW2, the heterozygous and wild-type mice started to show ramified nerve fibers resembling the mature shape of periodontal Ruffini endings. At PW3, the Ruffini endings occurred in the periodontal ligament of the wild-type and heterozygous mice. While the Ruffini endings of the wild-type mice appeared either ruffled or smooth, as reported previously, most of these structures showed a smooth outline in the heterozygous mice. The homozygous mice lacked the typical Ruffini endings at PW3. In the quantitative analysis, homozygous mice had the smallest percentages of PGP 9.5-immunoreactive areas at the same postnatal periods, but there were no significant differences between wild-type and heterozygous mice during PW1-3. These findings suggest a possible involvement of BDNF during the postnatal development and, in particular, the maturation of periodontal Ruffini endings. Furthermore, other neurotrophins may play a role in the development and/or early maturation of the periodontal nerve fibers, as indicated by the presence of nerve fibers in the BDNF-homozygous mice.

The involvement of brain-derived neurotrophic factor (BDNF) in the regeneration of periodontal Ruffini endings following transection of the inferior alveolar nerve.

The present study employed immunohistochemistry for protein gene product 9.5 (PGP 9.5) to examine the regeneration process of Ruffini endings, the primary mechanoreceptor in the periodontal ligament, in heterozygous mice with targeted disruption of the brain-derived neurotrophic factor (BDNF) gene and their littermates, following transection of the inferior alveolar nerve. When immunostained for PGP 9.5, periodontal Ruffini endings appeared densely distributed in the periodontal ligament of the heterozygous mice, but the density of the positively stained nerve fibers in the ligament was 20% lower than that in the control littermates. At 3 days after surgery, the PGP 9.5-positive neural elements had disappeared; they began to appear in the periodontal ligament of both animals at 7 days. However, the recovery pattern of the PGP 9.5-positive nerves differed between heterozygous and wild type mice, typical periodontal Ruffini endings morphologically identical to those in the control group appeared in the wild-type mice at 7 days, whereas such Ruffini endings were detectable in the heterozygous mice at 28 days, though much smaller in number. On day 28, when PGP 9.5-positive nerves were largely regenerated in wild type mice, their distribution was much less dense in the ligament of the heterozygous mice than in the non-treated heterozygous mice. The density of PGP 9.5-positive nerve fibers was significantly lower in the heterozygous mice than in wild type mice at any stage examined. These data showing that a reduced expression of BDNF causes delayed regeneration of the periodontal Ruffini endings suggest the involvement of BDNF in the regeneration process of these mechanoreceptors.

The periodontal Ruffini endings in brain derived neurotrophic factor (BDNF) deficient mice.

Innervation and terminal morphology in the lingual periodontal ligament of the incisor were investigated in brain derived neurotrophic factor (BDNF) heterozygous mice and littermate wild-type mice (aged two months) using immunohistochemistry for protein gene product 9.5 (PGP 9.5), a general neuronal marker. In addition, computer-assisted quantitative analysis was performed for a comparison of neuronal density in the periodontal ligament between heterozygous and wild-type mice. In wild-type mice, the periodontal ligament was found to be richly innervated by the mechanoreceptive Ruffini endings and nociceptive free nerve endings in the alveolus-related part of the periodontal ligament. The periodontal Ruffini endings in the wild-type mice incisor ligament were classified into two types: type I with ruffled outlines, and type II with a smooth outline. BDNF heterozygous mice showed malformations of the type I Ruffini endings which included fewer nerve fibers and fewer ramifications than those in wild-type mice as well as smooth outlines of the axon terminals. Quantitative analysis under a confocal microscope showed a roughly 18% reduction in neuronal density in the periodontal ligament of the heterozygous mice. These findings suggest that the development and maturation of the periodontal Ruffini endings require BDNF.

Comprometimento do primeiro molar permanente após 1 ano de sua erupçäo / The possible demage of the firts permanent molar during one year after your eruption: elaboration of a prevention plan

Os autores examinaram escolares de primeiro grau, de ambos os sexos, que tinham tido seus primeiros molares permanentes erupcionados pelo período decorrido de até um ano, observando o grau de comprometimento daqueles dentes. Ao final da pesquisa e de acordo com os resultados, elaboraram proposta preventiva para os referidos dentes