Date on Master's Thesis/Doctoral Dissertation

8-2014

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Anatomical Sciences and Neurobiology

Committee Chair

Krimm, Robin Frances

Committee Co-Chair (if applicable)

Petruska, Jeffrey

Committee Member

Petruska, Jeffrey

Committee Member

Cai, Jun

Committee Member

Bickford, Martha

Committee Member

Lundy, Robert

Subject

Nervous system--Wounds and injuries; Regeneration (Biology); Taste buds

Abstract

Brain derived neurotropic factor (BDNF) is required for the gustatory neuron survival,target innervation, and taste bud maintenance during development. However, whether BDNF has any function in the adult gustatory system in normal conditions or after nerve injury is unclear. To address these issues, I inducibly removed BDNF in all cells expressing BDNF in adult mice. In the experimental animals, Bdnf expression decreased to 5% of control mice in the lingual epithelium and geniculate ganglion (p< 0.01) at both two weeks and ten weeks after tamoxifen administration. I found no effect on taste bud morphology at four weeks following Bdnf gene deletion. However, ten weeks following Bdnf gene deletion, P2X3-positive and TUJ1-positive gustatory innervation to individual taste buds was reduced by nearly half (each with p < 0.01) and both taste bud volume and taste cell number decreased 30% (each with p< 0.01). These experiments demonstrate that BDNF is required for maintenance of normal levels of taste innervation and normal taste bud morphology in adulthood. In addition, taste cells expressing PLCß2 (phospholipase C ß2), a marker for taste cells that respond to sweet, bitter and umami, did not decrease after Bdnf gene deletion in the adult. Thus, the missing taste cells are of another type. This indicates that taste cell loss is not uniform across the various taste cell types, even if nearly all taste cell types receive the P2X3 and TUJ1 innervation. Since BDNF is required for initial innervation of the taste system and supports taste bud innervation and size in adulthood, it could also be required for nerve reinnervation after injury. To determine if Bdnf is still expressed following nerve section, the chorda tympani nerve (taste nerve) was sectioned and Bdnf level was detected with Real Time RT-PCR. Bdnf continued to be expressed at normal levels from two days to two months post-surgery in both geniculate ganglion and tongue epithelium. Therefore, BDNF could be involved with chorda tympani regeneration. To determine if this was the case, the Bdnf gene was deleted in adult inducible transgenic mice (under the control of a Ubiquitin promoter) two weeks before chorda tympani nerve section. Taste bud number was reduced by half in all genotypes at two weeks post-surgery (p < 0.01). For the remaining taste buds, gustatory innervation was nearly gone with only a little innervation from the trigeminal nerve remaining in the taste bud (p < 0.01). Taste bud volume (p < 0.01) and taste cell number (p< 0.01) were reduced by half for both control and experimental genotypes. Eight weeks post-surgery, taste bud number recovered in mice without Bdnf gene deletion, but did not recover in mice following Bdnf gene deletion (p < 0.01). Gustatory nerve innervation returned in 70% of the taste buds in control mice (p< 0.01). For those reinnervated taste buds, both taste bud volume and taste cell number increased to normal levels. However, in mice lacking the Bdnf gene, gustatory fibers only reinnervated 7.8% of the taste buds (p < 0.01); for most uninnervated taste buds, both taste bud volume and taste cell number remained small. These experiments demonstrate that BDNF is crucial for promoting regeneration of gustatory nerve fibers in adulthood. Following gustatory nerve section, considerable adult plasticity has been observed on the contralateral side including enlarged taste buds with more cells (Guagliardo and Hill, 2007). To determine if this anatomical change was associated with alter Bdnf expression. I examined Bdnf level in the geniculate ganglion and tongue epithelium on the contralateral side following chorda tympani nerve section. Results showed Bdnf expression increased two fold at two weeks post-surgery in geniculate ganglion on the contralateral side (p < 0.05), indicating BDNF may involve with the observed plastic changes. To determine if the increase in taste bud size was associated with increased innervation and/or regulated by BDNF, the Bdnf gene was then deleted in inducible knockout mice before nerve surgery, and taste bud size and amount of innervation were measured on the contralateral side. The results showed taste bud volume, taste cell number and a marker for nerve fibers all increased on the contralateral side in mice without Bdnf gene deletion at eight weeks post-surgery. This indicates that larger taste buds could be supported by increased TUJ1 positive fibers from trigeminal nerve. In addition, in mice lacking Bdnf, taste bud volume, taste cell number and innervation did not increase on the contralateral side after surgery, which indicates that Bdnf may contribute to larger taste buds on the contralateral side following nerve section by supporting increased innervation to the larger taste buds.

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