Age-Related Hearing Loss:Gamma-amino Butyric Acid, Nicotinic Acetylcholine And N-Methyl-D-Aspartate Receptor Expression Changes In Spiral Ganglion Neurons Of The Mouse Cochlea

Age-related hearing loss (ARHL)-Presbycusis is one of the top three most common chronic health conditions affecting individuals aged65years and older. Applying a conservative estimate of the prevalence rate of ARHL (50%) among the population65years and older in China. There are approximately50million senior citizens in China with significant hearing loss at present, and this number will be double in next20years. There are currently no biomedical treatments to prevent or reverse this condition. Age-related hearing loss is a disorder caused by mixed pathology include both genetic and environmental factors. Schuknecht classified presbycusis into four subtypes:(1) sensory (loss of hair cell), neural (loss of spiral ganglion neurons, SGNs), metabolic (atrophy of the stria vascularis) and mechanical (thickening and stiffening of the basilar membrane)."Sensorineural" presbycusis refers to the high-frequency-hearing impairment resulting from loss of hair cells and degeneration of SGNs without major changes in the function of the stra vascularis and other accessory structures of the inner ear which is a major contributor to age-related hearing loss. Neuron degeneration during age significantly contributes to functional decline of the central nervous system. In cochlea, SGNs connect the hair cells with central auditory system, it is the first neurons in auditory pathway. Thus, to study the etiologies of SGNs degeneration during aging paves a way to prevent or slow down the progression of age-related hearing loss as well as other types of hearing impairment. The N-methyl-D-aspartate receptor (NMDAR) is a glutamate-gated ion channel ubiquitously distributed throughout the brain; it is fundamental for excitatory neurotransmission and normal central nervous system functions. NMDA receptors express at the same hair cell/afferent nerve synapse of a single spiral ganglion cell play a crucial complex role in normal physiological conditions for neurotransmission as well as for synaptic plasticity. NMDAR are important for neural communication, memory functions, learning processes, and mental performance and NMDARs are also involved in excitotoxicity in auditory system.Gamma-aminobutyric acid (GABA) is an important inhibitory neurotransmitter in the central nervous system. The GAB AA receptor (GABAAR) is one of the most important inhibitory receptors, It plays a critical role in regulating neuronal excitability and information processing in the neuron system. The majority of GABAARs are widely expressed in the auditory system and it is a very important protective receptor in auditory efferent system.Acetylcholine (ACh) is the main transmitter released by the medial olivocochlear efferent fibers nAChR mainly located at the synapse between efferent fibers and outer hair cells (OHC).It is now believed that the hair cell cholinergic receptor that mediates synaptic transmission between efferent olivocohlear fibers and hair cells of the cochlea, is formed by both a9and a10subunits. The activation of the hair cell nAChR leads to an increase in intracellular Ca+-activated K+(SK2) channels thus leading to hyperpolarization of hair cells and reduction of electromotility. The auditory system of CBA/CaJ strain is very similar to human physiologically and morphologically during aging, therefore it is a good model for studying sensorineural presbycusis with high-frequency hearing loss accompanied by hair cells loss, neuronal degeneration. A better understanding of the cochlear mechanisms underlying peripheral presbycusis will help lead to future treatments. Objectives of the present study were to investigate gamma-amino butyric acid A (GABAA) receptor subunit al, nicotinic acetylcholine (nACh) receptor subunit (32, and N-methyl-D-aspartate (NMDA) receptor subunit NR1mRNA and protein expression changes in spiral ganglion neurons (SGN) of the young adult and old mouse cochlea, utilizing quantitative immunohistochemistry and RT-PCR techniques and observe the hair cells and SGNs in cochlear mophyological change with age.Methods:ABR RECORDING PROCEDURES Young adult (Male3, Female3), old age (Male3, Female3) were anesthetized with a mixture of ketamine/xylazine (120and10mg/kg body weight, intraperitoneal injection) prior to all experimental sessions. All recording sessions were completed in a sound proof acoustic chamber (LAC lined with Sonex) with body temperature maintained with a heating pad. ABRs were measured using Biosig (TDT Gainesville, FL) data-acquisition system in response5ms tone pips (0.5-m rise fall times) with cos2onset envelop presented at rate of29/sec. Threshold was defined as the lowest intensity which elicited a clearly replicable response. IMMUNOCYCHEMISTRY Sacrificed the mice and dissected the cochlea. Cochlea were fixed in4%paraformaldehyde in PBS overnight at4℃and decalcified in10%EDTA in PBS for three days at4℃. After using cryoprotectant for cryoprotection overnight at4℃,the cochlea were embedded into degassed OCT overnight at4℃, oriented into the cryomold with OCT, degassed for1hour, then fast frozen. Cryosectioning was performed at5μm/section. The cochlea slides were stained with DAB using antibody GABAARa1,NMDARNR1or nAChRβ2. qRT-PCR10ng of total RNA was reverse transcribed and complementary DNA was amplified,and quantitative PCR was performed using Enhanced Avian HS RT-PCR100Kit.Results:(1) GABAARa1, nAChRβ2and NMDARNR1were detected in spiral ganglion neurons of CBA/CaJ mouse cochlea.(2) mRNA and protein expression of GABAARal and nAChRβ2decreased with age in spiral ganglion neurons of mouse cochlea. mRNA and protein expression of NMDARNR1increased with age in spiral ganglion neurons of mouse cochlea.(3) ABR thresholds shift over50dB from3-48kHz in old mice compared to young adult mice.(4)DPOAE thresholds shift over40of6,12,16,30,44kHz in old mice compared to young adult mice.12and16kHz of DPOAE amplitude decreased and the other frequency response were absent in the old mice.(5) SGN density decreased with age in basal, middle and apical turns, and SGN density of the basal turn decreased the most.(6) Correlations were observed between SGN density and ABR wave I Amplitude/ABR wave I Latency. There is a possive correlation between SGN density and ABR wave I Amplitude and a negative correlation between SGN density and ABR wave I Latency.Summary and Conclusions:Age-related changes of GABAARal, nAChR(32and NMDARNR1expression in CBA mouse SGNs were observed, which may be related to functional changes in cochlear synaptic transmission as a function of age. ABR thresholds and DPOAE shift over50dB from all the prequancy in old mice indicated that auditory function delince in all the level of auditory pathway, SGNs density and OHC significantly declined with age, all these are associated with age-related peripheral auditory system impairment and fianlly lead to age-related hearing loss. This finding leads to more insights into the function of these three cochlea receptors concerning their regulation in the aging mammalian auditory system, paving the way for novel biomedical interventions to prevent or slow down the progression of age-related hearing loss as well as other types of hearing impairment.