Lingual deficits in neurotrophin double knockout mice

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 −/− x NT-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 −/− x NT-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 −/− x NT-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.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/47465/1/11068_2005_Article_3330.pd

Neurothrophins in the development of the gustatory system and teeth

Flavors of taste are detected by a set of microscopical cellular aggregates calledthe taste buds, present in different arrangements on the upper surface of the tongueand other areas in the mouth. Taste buds are chemosensors that can detect differenttaste qualities such as sweet, sour, bitter, salt and umami. Information from thetaste buds is carried via nerve fibers to the brain where these signals are furtherprocessed, finally leading to the experience of taste. Taste buds not only help usenjoy food, they are also part of an important control and warning system, enablingus to avoid consuming hazardous substances or spoiled food. During the past severalyears, more light has been shed on many aspects of the gustatory system, includingthe local epithelial origin of taste buds and taste transduction mechanisms. However,the manner in which taste buds develop and become connected to the brain by nervefibers has long been a matter of debate. The present work now demonstrates the crucialimportance of two proteins closely related to nerve growth factor (NGF) in tastebud development and proper taste and sensory functions of the mouth. Messenger RNAcoding for the NGF-related proteins BDNF (brain derived neuro trophic factor) andNT-3 (neurotrophin-3) were found to be present in precisely the right locations inand around the taste buds to suggest that they are of vital importance for controllingthe arrival of nerve fibers to these structures. These hypotheses were borneout bystudies of mice in which either the gene for BDNF or NT-3 had been selectively turnedoff. Mice that developed without BDNF had very few and abnormal taste buds and wereunable to discriminate between primary tastes such as sweet, salt or bitter. Animalsin which the NT-3 gene had instead been turned off appeared to have a normal developmentof their taste bud system, but instead demon strated severe impairment of sensoryfunction of mucous membranes of the oral cavity. These results demonstrate key roles for the two neurotrophic proteins BDNF andNT-3 in con trolling the development of the gustatory and somatosensory apparatusesof the mouth. These neurotrophins also seem to be important in oral gustatory andsomatosensory innervation in humans. It remains to be seen whether or not disturbancesof the genes for these neurotrophic proteins might underlie rare human conditionssuch as familial dysautonomia in which the ability to discriminate tastes is lost.These novel findings may also have implications in more common cases in problemsof loss of taste and sensation in the mouth such as those seen after injury to thenerves, either by accident or following oral or facial surgery. Knowledge about whichproteins are needed to stimulate nerve fibers to grow into mucous membranes of theoral cavity during development suggests that these same proteins might become helpfulin stimulating regeneration of injured nerves in adult patients, perhaps helpingthem to regain lost taste and sensory function. Neurotrophins are also expressed in the developing teeth. They all show temporospatiallyspecific expression patterns. We suggest that neurotrophins are involved in earlymorphogenetic events during tooth development and that NGF and BDNF are involvedin the innervation of the dental pulp. Further experiments are required to determinethe precise roles of neurotrophins in tooth development. The GDNF family of ligands and receptors are also expressed in developing tongueand teeth. They might be involved in the autonomic innervation ot the tongue. Inteeth, they might partici pate both in morphogenetic events during tooth developmentand participate in tooth innervation. Keywords: Gustation, gustatory, taste buds, neurotrophins, NGF, BDNF, NT-3, NT-4,GDNF, GFRalpha-l, GFRalpha-2, knockout, innervation, development, tooth, tooth development,odontogene sis. Christopher A. Nosrat, 1997 ISBN 91-628-2795-

Expression patterns of neurotrophic factor mRNAs in developing human teeth

Neurotrophic factors regulate survival, differentiation, growth and plasticity in the nervous system. In addition, based on their specific and shifting temporospatial expression patterns, neurotrophic factors have been implicated in morphogenetic events during tooth development in rodents. To determine whether these findings in rodents could be related to humans, we have now studied nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), glial cell-line derived neurotrophic factor (GDNF), and neurturin (NTN) mRNA expression patterns in developing human teeth during gestational weeks 6.5–11. Using in situ hybridization histochemistry, we found distinct and specific patterns of neurotrophin and GDNF mRNA expression in the developing human teeth. NGF mRNA labeling was weak and confined predominantly to the dental papilla. BDNF mRNA labeling was stronger than NGF mRNA and was seen in the mesenchyme located lateral to the dental organ, as well as in epithelial structures (inner dental epithelium and enamel knot). NT-3 mRNA was observed in the dental papilla and in the area of the cervical loop. NT-4 mRNA was expressed in both oral and dental epithelia in all stages studied. GDNF mRNA was found in the dental follicle and at different sites in the inner dental epithelium. Weak NTN mRNA labeling was also found in the developing teeth. Based on these findings, we suggest that neurotrophins, GDNF and NTN might be involved in morphogenetic events during early stages of tooth development in humans. Protein gene product (PGP) 9.5-immunoreactive nerve fibers were observed in the dental follicle by 11 weeks coinciding with the labeling for neurotrophic factor mRNAs in this structure. This suggests that these neurotrophic factors might be involved in the innervation of dental structures. The rich expression of neurotrophic factors in developing dental tissues suggests that developing, or possibly adult, dental tissue might be used as an allograft source of trophic support for diseases of the nervous system.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/42275/1/s00441-002-0618-8.pd

Distinctive spatiotemporal expression patterns for neurotrophins develop in gustatory papillae and lingual tissues in embryonic tongue organ cultures

Brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) mRNAs are expressed in the developing rat tongue and taste organs in specific spatiotemporal patterns. BDNF mRNA is present in the early lingual gustatory papilla epithelium, from which taste buds eventually arise, prior to the arrival of gustatory nerve fibers at the epithelium, whereas NT-3 initially distributes in the mesenchyme. However, a direct test for neural dependence of neurotrophin expression on the presence of innervation in tongue has not been made, nor is it known whether the patterns of neurotrophin expression can be replicated in an in vitro system. Therefore, we used a tongue organ culture model that supports taste papilla formation while eliminating the influence from sensory nerve fibers, to study neurotrophin mRNAs in lingual tissues. Rat tongue cultures were begun at embryonic day 13 or 14 (E13, E14), and BDNF, NT-3, nerve growth factor (NGF) and neurotrophin-4 (NT-4) mRNAs were studied at 0, 2, 3 and 6 days in culture. BDNF transcripts were localized in the gustatory epithelium of both developing fungiform and circumvallate papillae after 2 or 3 days in culture, and NT-3 transcripts were in the subepithelial mesenchyme. The neurotrophin distributions were comparable to those in vivo at E13–E16. In 6-day tongue cultures, however, BDNF transcripts in anterior tongue were not restricted to fungiform papillae but were more widespread in the lingual epithelium, while the circumvallate trench epithelium exhibited restricted BDNF labeling. The NT-3 expression pattern shifted in 6-day organ cultures in a manner comparable to that in the embryo in vivo, and was expressed in the lingual epithelium as well as mesenchyme. NGF mRNA expression was subepithelial throughout 6 days in cultures. NT-4 mRNA was not detected. The neurotrophin mRNA distributions demonstrate that temporospatial localization of neurotrophins observed during development in vivo is retained in the embryonic tongue organ culture system. Furthermore, initial neurotrophin expression in the developing lingual epithelium, mesenchyme, and/or taste papillae is not dependent on intact sensory innervation. We suggest that patterns of lingual neurotrophin mRNA expression are controlled by the influence of local tissue interactions within the tongue at early developmental stages. However, the eventual loss of restricted BDNF mRNA localization from fungiform papillae in anterior tongue suggests that sensory innervation may be important for restricting the localized expression of neurotrophins at later developmental stages, and for maintaining the unique phenotypes of gustatory papillae.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/42273/1/441-303-1-35_s004410000271.pd

Poly(ɛ-Caprolactone) and Poly (L-Lactic-Co-Glycolic Acid) Degradable Polymer Sponges Attenuate Astrocyte Response and Lesion Growth in Acute Traumatic Brain Injury

This study evaluated the response of rat brain to 2 degradable polymers (poly (L-lactic-co-glycolic acid) (PLGA), and poly(ɛ-caprolactone) (PCL)), two common materials in tissue engineering. PLGA has been extensively studied in the brain for controlled drug release as injectable microspheres and is generally accepted as biocompatible in that capacity. Biocompatibility in other forms and for different functions in the brain has not been widely studied. PCL was chosen as an alternative to PLGA for its slower degradation and less-acidic pH upon degradation. Porous scaffolds were made from both polymers and implanted into rat cerebral cortex for 1 and 4 weeks. Morphology, defect size, activation of microglia (OX-42) and astrocytes (glial fibrillary acidic protein (GFAP)), infiltration of activated macrophages (major histocompatibility complex (MHC)-II), and ingrowth of neurons (β-tubulin type III (Tuj-1)) and progenitor cells (nestin) were analyzed using hematoxylin and eosin staining and immunofluorescence. PCL induced a lower inflammatory response than PLGA, as demonstrated by lower MHC-II and GFAP expression and greater ingrowth. Both polymers alleviated astrocytic activation and prevented enlargement of the defect. Tuj-1-, nestin-, and GFAP-positive cells were observed growing on both polymers at the peripheries of the sponge implants, demonstrating their permissiveness to neural ingrowth. These findings suggest that both polymers attenuate secondary death and scarring and that PCL might have advantages over PLGA.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/63228/1/ten.2006.0440.pd

Confirmatory sequencing of full-genome amplicons generated by PCR.

<p>A. For each amplicons, the sequencing was carried out with primers other than those used for full-genome amplification. The linear sequencing template (black line), the PCR primers are shown at both ends of the amplicons. Bleu lines (amplicons 671F-892R), green lines (amplicons 6838F-6972R), red lines (amplicons 6838F-7076R) represent the sequenced regions attached to the corresponding sequencing primers. Numbers included in primer IDs represent the position relative to HPV_SD2 genome. B. Alignment of sequences generated by Sanger method along the HPV_SD2 generated by 454 sequencing. Bars in blue, green and red correspond to sequences shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058404#pone-0058404-g006" target="_blank">Figure 6A</a>. The newly generated sequences covered 4,622 nucleotides representing 63.3% of the total HPV_SD2 genome. C. Comparison of the sequences generated by Sanger method and HPV_SD2 contig generated by 454 sequencing. Size of each Sanger sequence and percentage identity are shown.</p

Composition of sample pools and methods for DNA and RNA extraction.

<p>Composition of sample pools and methods for DNA and RNA extraction.</p