Primary Exploration Of Auditory Potential Induced By Optogenetic Stimulation

Background:Deafness has a significant influence on people's life. Artificial cochlear implants, which stimulate auditory nerve directly, are the main therapy for severe or extremely severe deafness at present. However, artificial cochlear implant is expensive and brings huge financial burden to our society. It is desiderate to find an economic and long-term alternative method. ChR2 is widely studied in the neural activity of brain cells. There is no direct experiment data about the expression and regulation of ChR2 in cochlear ganglion cells. In this study, we introduce ChR2 gene into remnant cochlear ganglion cells in inner ear. Use the photo electricity switch to realize the translation between audio energy and electroneurographic signal. The cochlea in the ear converts sound waves to electrical impulses that the brain processes as sound. We planned to provide new theoretical foundation by starting from study of the excitability regulation in spiral ganglion cell, which will be of great importance in improving deaf people's health and living quality.Purpose:Design safe and efficient ChR2 viral vector. Explore the conditions of ChR2 gene expression in basement membrane in vitro. Make sure the conditions of the ChR2 gene expression in cochlea in vivo.Acquire cochlea potentials by using 470nm blue laser to irradiate ChR2Methods:Use molecular biological technique to construct ChR2 lentivirus and adeno-associated virus vectors.Observe the expression of ChR2 on the SGNs of Guinea pigs basement membrane. Use 470nm blue laser to irradiate the transfected SGNs and record the generated electrical activity by patch clamp, finding the optimum transfection titer.Introduce AAV-hSyn-hChR2(H134R)-EYFP by a self-made tubing through round window. Analyze the transfection by immunohistochemical, colocalization, PCR and 470nm blue laser irradiation and explore the best conditions to produce potentials.Results:Completed the construction of plenti-hSyn-hChR2(H134R)-EYFP vector andAAV-hSyn-hChR2(H 134R)-EYFP vector. The optimum transfection titer is 6.25x105 PV.Action potentials were found in 470nm blue laser recording by patch clamp. Introduce virus by using a self-made tubing through round window, which can promote the efficiency of transfection and alleviate the hearing loss. Construct the adult animal models whose cochlea was transfected by ChR2 gene. The cochlea potentials below 470nm irritation should be non-liner characteristic. Meanwhile the best laser intensity of 470nm blue laser was 3.70 mW,with less tissue injury.Conclusions:Completed the ChR2 gene transfection in adult animal models. Found out the conditions of lentivirus and adeno-associated virus transfection. Established the adult animal models with transfection of ChR2 gene. Furthermore, we record the cochlea potentials below 470nm blue laser as a beginner in China.