Hearing Loss Controlled by Optogenetic Stimulation of Nonexcitable Nonglial Cells in the Cochlea of the Inner Ear.

Mitsuo Satõ ,Hidenori Inohara,Hiroshi Hibino,Kenji F Tanaka,Taiga Higuchi,Kaoru Ogawa,Sho Kanzaki,Genki Ogata,Hirohide Takebayashi,Shuichi Sakamoto,Satoru Uetsuka,Samuel Choi,Shizuo Komune,Fumiaki Nin,Seishiro Sawamura,T Ōta ,Kunio Doi ,Masataka Masuda ,Katsuo Hori ,Tsukihisa Yoshida ,Toshihisa Watabe

doi:10.3389/fnmol.2017.00300

Abstract

Light-gated ion channels and transporters have been applied to a broad array of excitable cells including neurons, cardiac myocytes, skeletal muscle cells and pancreatic β-cells in an organism to clarify their physiological and pathological roles. Nonetheless, among nonexcitable cells, only glial cells have been studied in vivo by this approach. Here, by optogenetic stimulation of a different nonexcitable cell type in the cochlea of the inner ear, we induce and control hearing loss. To our knowledge, deafness animal models using optogenetics have not yet been established. Analysis of transgenic mice expressing channelrhodopsin-2 (ChR2) induced by an oligodendrocyte-specific promoter identified this channel in nonglial cells—melanocytes—of an epithelial-like tissue in the cochlea. The membrane potential of these cells underlies a highly positive potential in a K+-rich extracellular solution, endolymph; this electrical property is essential for hearing. Illumination of the cochlea to activate ChR2 and depolarize the melanocytes significantly impaired hearing within a few minutes, accompanied by a reduction in the endolymphatic potential. After cessation of the illumination, the hearing thresholds and potential returned to baseline during several minutes. These responses were replicable multiple times. ChR2 was also expressed in cochlear glial cells surrounding the neuronal components, but slight neural activation caused by the optical stimulation was unlikely to be involved in the hearing impairment. The acute-onset, reversible and repeatable phenotype, which is inaccessible to conventional gene-targeting and pharmacological approaches, seems to at least partially resemble the symptom in a population of patients with sensorineural hearing loss. Taken together, this mouse line may not only broaden applications of optogenetics but also contribute to the progress of translational research on deafness.

Highlights

In biomedical research, opsins, which are light-gated ion channels or transporters such as cation-selective channelrhodopsin and archaerhodopsin and anion-permeable halorhodopsin, have been expressed primarily in neuronal cell types in vivo (Deisseroth, 2015; Glock et al, 2015)
To confirm the expression of ChR2(C128S) in the cochlea, we carried out western blot analysis with an anti-green fluorescent protein (GFP) antibody, which can detect enhanced yellow fluorescence protein (EYFP) fused to the channel (Figure 1 and Supplementary Figure S1)
We demonstrated that genetic introduction of ChR2(C128S) driven by the PLP promoter results in expression of the functional channels in cochlear intermediate cells (IC) that are classified into nonexcitable cells (Figure 2)

Summary

Introduction

Opsins, which are light-gated ion channels or transporters such as cation-selective channelrhodopsin and archaerhodopsin and anion-permeable halorhodopsin, have been expressed primarily in neuronal cell types in vivo (Deisseroth, 2015; Glock et al, 2015). Technical advances have allowed researchers to induce light-gated channels in cardiac myocytes, skeletal muscle cells and pancreatic β-cells in live animals and to electrically manipulate the cells in a particular region and/or timing with illumination (Bruegmann et al, 2015; Vogt et al, 2015; Johnston et al, 2016) These experiments have provided insights into novel therapies for heart diseases, muscle paralysis and diabetes. Tanaka et al (2012) developed a transgenic methodology that can stably and strongly induce the channelrhodopsin-2 (ChR2) protein in the mouse; this approach is called ‘‘the knockin-mediated enhanced gene expression by improved tetracycline-controlled gene induction (KENGE-tet) system’’ In this system, a promoter that is active in a different cell population is selectable; the ChR2 gene is reported to be driven in glial cells of astrocytes, oligodendrocytes, or microglia. In a mouse line harboring ChR2 in oligodendrocytes, photodepolarization of these cells causes early- and late-onset acceleration of axonal conduction and affects short- and long-term functional plasticity in the hippocampus (Yamazaki et al, 2014)

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Results

Conclusion