Ca_v1.3Calcium Channels Hearing Function And Active Cochlear Amplification Mechanism

PART ONE Expression Patterns of Cav1.3calcium channels in the rat cochleaObjective:To investigate the expression of Cav1.3calcium channels in the rat cochlea and study its role in auditory physiology and pathology.Methods:The distribution of Cav1.3calcium channels in the rat cochlea was detected by immunohistochemistry. The expression of Cav1.3calcium channels was measured with western bloting (WB) and reverse transcription-polymerase chain reaction (RT-PCR).Results:Immunohistochemistry revealed that Cav1.3calcium channels were localized in the outer hair cells (OHCs), inner hair cells (IHCs), limbus laminae spiralis, spiral ganglion cell, spiral ligament and stria vascularis. The results of RT-PCR and Western blotting demonstrated Cav1.3calcium channels had a tissue-specific expression pattern. Cav1.3mRNA and protein were intensively expressed in the basilar membrane and spiral ganglion while moderate level of Cav1.3was observed in spiral ligament and stria vascularis.Conclusion:The study preliminary reveals the expression patterns of Cav1.3calcium channels in the rat cochlea, providing a theoretical basis for further research on the role of Cav1.3calcium channels in the periphery auditory system. PART Two Downregulation of Cav1.3calcium channel expression in the cochlea is associated with Presbycusis in C57BL/6miceObjective:To investigate the expression of Cav1.3calcium channel in C57BL/6mice cochlea and study its correlation with presbycusis.Methods:Auditory function was measured with auditory brainstem response (ABR) in C57BL/6mice at4,14,24and48weeks. The expression of Cav1.3calcium channels was detected by immunofluorescence,Western Blotting and Real-time RT-PCR at various ages.Results:Immunofluorescence photographs reveal that Cav1.3channels were mainly localized in the hair cell, spiral ganglion cell, spiral ligment, stria vascularis, spiral limbus. Comparing with4-week group, the ABR threshold of4-,24-, and48-week increased (p<0.05). The ABR threshold at48-week group was significantly higher than others (p<0.01). Along with age, the missing hair cells and spiral ganglion cells increased, stria vascularis and spiral ligament atrophied, the expression of Cav1.3channels was gradually decreased(p<0.01).Conclusion:The expression of Cav1.3channels decreased along with age. Dysfunction or missing of Cavl.3calcium channels may be related to presbycusis. PART Three Contribution of connexin26to active cochlear amplification and underlying mechanisms for connexin26deficiency induced hearing lossObjective:To investigate Connexin26hearing function and contribution to active cochlear amplification and deafness mechanisms underlying Cx26deficiency induced hearing loss.Methods:We used loxP-Cre technique to delete Cx26expression in Deiter cells (DCs) and outer pillar cells (OPCs). The Cx26conditional knockout (cKO) mouse was generated by crossing Cx26loxP/loxp transgenic mice with a Proxl-CreERT2mouse line. Auditory function was measured with auditory brainstem response (ABR), DPOAE, CM and EP in Cx26cKO mice. The expression of Cx26, Cx30and Prestein were detected by immunofluorescence staining. Consecutive cochlear paraffin sections were stained with toluidine blue to account SG neuron. Prestin expression at the transcriptional level was measured by real-time RT-PCR.Results:Cx26labeling was absent in DCs and OPCs in Cx26cKO mice. The deletion of Cx26is visible in all three apical, middle, and basal turns. Cx26labeling in other cochlear structures, including the spiral limbus (SLM) and the lateral wall, remained normal. Cx30, which is another predominant connexin isoform in the cochlea, also retained normal expression. Targeted-deletion of Cx26in DCs and OPCs induced hearing loss The ABR threshold in homozygous mice was increased significantly and DPOAE was significantly reduced. CM and EP in Cx26cKO mice were normal. No reduction in prestin. No apparent cell degeneration in the Cx26cKO mouse cochlea. Conclusion:The study demonstrates that active cochlear amplification in vivo is dependent on supporting cell gap junctions. These new findings also show that connexin26deficiency can reduce active cochlear amplification to induce hearing loss.