| Background and Objectives:Sensorineural deafness is the most common kind of hearing disability, The formation and the maintenance of auditory sense depend on the glutamate neurotransmitter released from the ribbon synaptic of the cochlear inner hail cells(IHC), which can complete mechanical – electrical transformation. The pathology change of cochlear inner hair cells which is the first tier auditory conduction synaptic is one of the important reasons causing sensorineural deafness.A lot of studies have shown the attenuation of audiologic function is closely related to the synaptic morphology、structure and quantity. Every IHC has to establish a synaptic connection with spiral neurons, and there were 10-30 ribbon synapses within a single hair. The neurotransmitter glutamate released from readily releasable pool(RRP) in the active region of the ribbon synapses in cochlear inner hair cells can activate glutamate receptor(AMPA) in postsynaptic membrane, and transmit the auditory information to the corresponding neurons further. So the most of the acoustic signal perception is done by IHC and synapses in the cochlear. What’s more, there are dozens of vesicles within every RRP so that the progress of release can be finished within a few microseconds. The synchronous release of mass of vesicles can be reflected by the excitatory postsynaptic potential(EPSPs).The quickly release of neurotransmitter in the IHCs needs effective Synaptic vesicle recycling mechanism. Synaptic vesicle recycling is the basis of the neurotransmitter sustained release, and further the rapid endocytosis is the precondition of recycling. In a word, both of them play an important role in promoting the message transfer. Clathrin mediated endocytosis is the important ways of the synaptic vesicle membrane reformation in central nervous system and AP- 2 is the key protein in different periods of endocytosis, which can bind clathrin and other proteins to formation the coated pits. AP-2 is consist of two large subunits(α and β2), one medium subunit(μ2) and a small subunit(σ2).The attachment of α subunit contacts with plasma membrane, combine with other proteins through DPF or DPW molecular signal. β2 subunit combine with β spiral structure of clathrin, also associated with the cargo); μ2 subunits recognition cyt oplasmic tail area by tyrosine receptor.IHC and its afferent neurons is a special kind of synapse, we guess that AP- 2 regulate the CME should play an important role in this area. However, researches on AP- 2 protein were limited to the central nervous system and its proteomics, the expression and function of AP- 2 in mice cochlea is unclear. This study using Laser scanning confocal microscopy and immune-fluroscencehistochemistry double labeling to investigate the expression of AP-2 in mice cochlea and explore the probable role in auditory physiology mechanism, then we will observe the expression of AP-2 in different development stages of mice cochlear through ABR and q RT – PCR, Finally, choosing the patch clamp method which use Tyrphostin A23 to inhibit AP- 2 protein function and make effect on endocytosis to discuss the probable mechanism in the hair cells and lay the foundation for the further research involved in the physiological role of CME and AP-2.Material and Methods:1)Male C57 mice(8 weeks) were used. Laser scanning confocal microscopy and immune-fluroscencehistochemistry double labeling to investigate the expression of AP-2 in mice cochlea2)Mice were divided into 4 experimental groups(7 、15 、35 days old and16 months old), which respectively represented the newborn, developmental, mature and old mice. The ABR, LSCM, immune-fluroscencehistochemistry and q RT- PCR were used in the experiment3)Through Tyrphostin A23 inhibition YxxФ molecular signals interact with AP- 2, analysis of the IHC electrophysiological changes.Result:1)AP-2 was found in the IHC cytoplasm, mainly expressed in the basal, nearby the ribbon synapse activity zone. The expression of morphological site is associated with function of CME pathway.2)For 15, 35 days and 16 months old groups, the ABR threshold were respective 18.67±1.21 d B n HL,13.83±1.47 d B n HL,37.83±7.68 d B n HL, however the 7 days old group wasn’t elicitated. What’s more, the AP-2 was found in the all stages of IHC cytoplasm, and the IMV of. 7, 15, 35 days and 16 months old groups were 190.91±17.27,494.06±27.63,838.41±38.23,682.65±72.22, and m RNA RQ is 0.53±0.09,1.03±0.02,1.00±0.09,1.03±0.06.3)Through Tyrphostin A23 inhibition, IHC voltage dependence Ca2+ channel was delayed, Ica2+ and △ Cm decreased, eventually influenced release of glutamate neurotransmitter.Conclusions:1)AP- 2 expressed mainly in the IHC synaptic activity area in adult mouse cochlear, which may play an important role in synaptic vesicle endocytosis.2)AP- 2 has different strength expression at different stages in mouse, and the expression level may be related with the auditory occur, formation and maintenance.3)inhibiting the function of AP – 2 may affect the CME, which means the AP- 2 plays a key role in synaptic vesicle recycling. |
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