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Neuroscience. 2017 Aug 15;357:185-196. doi: 10.1016/j.neuroscience.2017.05.040. Epub 2017 May 31.

Afferent synaptogenesis between ectopic hair-cell-like cells and neurites of spiral ganglion induced by Atoh1 in mammals in vitro.

Author information

1
Department of Otology and Skull Base Surgery, EYE & ENT Hospital of Fudan University, Shanghai 200031, PR China; Shanghai Clinical Medical Center of Hearing Medicine, Shanghai 200031, PR China; Key Laboratory of Hearing Medicine, Ministry of Health, Shanghai, 200031 PR China; Research Institute of Otolaryngology, Fudan University, Shanghai, 200031 PR China.
2
Shanghai Clinical Medical Center of Hearing Medicine, Shanghai 200031, PR China; Key Laboratory of Hearing Medicine, Ministry of Health, Shanghai, 200031 PR China; Research Institute of Otolaryngology, Fudan University, Shanghai, 200031 PR China.
3
Department of Cell Biology, Emory University, Atlanta, GA 30322, USA.
4
Department of Otology and Skull Base Surgery, EYE & ENT Hospital of Fudan University, Shanghai 200031, PR China; Shanghai Clinical Medical Center of Hearing Medicine, Shanghai 200031, PR China; Key Laboratory of Hearing Medicine, Ministry of Health, Shanghai, 200031 PR China; Research Institute of Otolaryngology, Fudan University, Shanghai, 200031 PR China. Electronic address: chifanglu@126.com.
5
Department of Otology and Skull Base Surgery, EYE & ENT Hospital of Fudan University, Shanghai 200031, PR China; Shanghai Clinical Medical Center of Hearing Medicine, Shanghai 200031, PR China; Key Laboratory of Hearing Medicine, Ministry of Health, Shanghai, 200031 PR China; Research Institute of Otolaryngology, Fudan University, Shanghai, 200031 PR China. Electronic address: yangjuanmei1982@126.com.

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:

Atoh1; C-terminal-binding protein (CtBP2); afferent synaptogenesis; ectopic hair-cell-like cells (EHCLCs); lesser epithelial ridge (LER); postsynaptic density protein 95 (PSD-95)

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