| Objective: In the present study,the hearing sensitivity of zebrafish was examined by analysing the auditory evoked potentials(AEPs)over a range of total lengths(TLs)from 12 to 46 mm.Morphological changes in the hair cells(HCs)of the saccule(the main auditory end organ)and their synapses with primary auditory neurons were investigated to lay the foundation for NIHL zebrafish model.This study also intends to verify the change of auditory function and morphological changes of ribbon synapse which include ribbon,postsynaptic membrane,afferent nerve fibers after 105 dB SPL broadband noise exposure 2h,explore the relationship between ribbon synaptic damage and supra-threshold hearing function,and define the mechanism of NIHL occurrence and development induced by ribbon synaptic damageMethods: Zebrafish was divided into 7 groups according to age and TLs.Subsequently,adult zebrafish hearing frequency range and the change of auditory brainstem response(ABR)threshold at the development process were investigated.Meanwhile,the distribution,density and morphological changes of saccule HCs and ribbon synapses were studied by immunofluorescence and ultrastructural methods.Animal noise experiments were consisted of control group and noise group which is divided into 1DPN,1WPN and 1MPN subgroups and experienced 105 dB SPL broadband noise exposure for 2 hours.The study investigate the auditory function(ABR,DPOAE,CAP)of experimental guinea pig,immunofluorescence and ultrastructural change of the ribbon synapses,and mRNA expression changes of ribbon synapse which included Ribeye A-domian,Cav1.3,piccolo,Bassoon,RIMs1.Results: In the present study,we examined the hearing sensitivity in zebrafish with a TL ranging from 12 to 46 mm(corresponding to an age from 40 dpf to 20 months post-fertilization)and assessed the age-related morphological variations in saccular HCs.The major findings are summarized as follows: 1,the hearing sensitivity,but not the hearing frequency range,of zebrafish changes with TL(or age);2,HC number continues to increase until late in adulthood,and more than twothirds of new HCs are located in the posterior saccule;3,there are evident regionspecific differences in HC height and diameter and TL(age)-related differences in HC density,ribbon protein expression pattern and ribbon size,which are consistent with the AEP threshold change;As to noise experiment,ABR and DPOAE threshold reversible recovery,but the amplitude of CAP can't be returned to normal after noise exposure.The number of synaptic ribbon was largely recovered,but the number of Ribeye A-domian at 8kHz site in the basement membrane was not restored.With repair of synaptic ribbon after injury is partially reversible,damage mainly occurred at the modiolar side in IHC,and mRNA expression of Ribeye A-domian and Piccolo can't be recovered.Conclusions: The AEPs were detected up to a much higher frequency limit(12 kHz)than previously reported.No significant difference in the frequency response range was observed across the TL range examined.However,the AEP thresholds demonstrated both developmental improvement and age-related loss of hearing sensitivity.The changes in hearing sensitivity were roughly consistent with the morphological changes in the saccule,including(1)the number and density of HCs,(2)the organization of stereocilia,and(3)the quantity of a main ribbon protein,Ribeye b.The results of this study established a clear baseline for the hearing ability of zebrafish and revealed that the changes in the saccule contribute to the observed changes in TL(age)-related hearing sensitivity.Temporary threshold shift was brought by the massive damage to ribbon synapse caused by transient low noise exposure.The decrease of CAP amplitude suggests the deficits in both intensity and temporal processing,revealing the nature of synaptopathy in hidden hearing loss.Ultrastructural results suggested that the injury mainly occurring at IHC modiolar side,and part of mRNA expression of ribbon synaptic protein was abnormal after noise. |
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