Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
5737838 | Neuroscience | 2017 | 12 Pages |
Abstract
Newly formed ectopic hair-cell-like cells (EHCLCs) induced by overexpression of atonal homolog 1 (Atoh1) in vitro were found to possess features of endogenous hair cells (HCs) in previous reports and in the present study. However, limited information is available regarding whether EHCLCs and native spiral ganglion neurons (SGNs) form afferent synapses, which are important for the restoration of hearing. In the current study, we focused on the afferent synaptogenesis between EHCLCs and SGN-derived dendrites. Cochlear explants of auditory epithelia with native SGNs retained were cultured in vitro, and human adenovirus serotype 5 (Ad5) vectors encoding Atoh1 were used to overexpress Atoh1 and induce EHCLCs. We observed that the neurites of the original SGNs extended toward the lesser epithelial ridge (LER) and innervated the EHCLCs. Immunohistochemical analyses revealed the expression of presynaptic ribbon C-terminal-binding protein 2 (CtBP2) and postsynaptic density protein (PSD)-95 in the nerve endings of SGN-derived neurons adjacent to EHCLCs. PSD-95 was located directly opposite CtBP2-positive puncta in the terminals of branches of SGNs, demonstrating that the neurites of SGNs formed afferent-like synaptic connections with EHCLCs. However, the expression of glutamate receptor type 2 (GluR2) could not be detected in the terminals of branches of SGNs surrounding EHCLCs. In addition, we found that the presynaptic ribbon (CtBP2) formation in EHCLCs preceded neural innervation. Furthermore, CtBP2-positive puncta increased and then decreased in EHCLCs, similar to the changes observed in endogenous HCs in terms of their number and distribution. Our finding of the generation of cochlear afferent synapses between EHCLCs and original SGNs will lay the foundation for regenerative approaches to restoring hearing after hair cell loss.
Keywords
CTBP2lesser epithelial ridgeTuj1SGNsHuman adenovirus serotype 5ATOH1Ad5MYO7AGluR2NF-200OHCPBSHCsRT-PCRDVIPSDeGFPmembrane-associated guanylate kinaseMAGUKEDTAEthylenediaminetetraacetic acidIHCpostsynaptic densitystandard error of the meanLERouter hair cellInner hair cellHair cellsPhosphate-buffered salineSEMSpiral ganglion neuronsatonal homolog 1real-time polymerase chain reactionenhanced green fluorescent proteinAMPA receptor
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Authors
Wen-wei Luo, Rui Ma, Xiang Cheng, Xiao-yu Yang, Zhao Han, Dong-dong Ren, Ping Chen, Fang-lu Chi, Juan-mei Yang,