This work was supported by the National Natural Science Foundation of China (81770997, 81771016, 81830030); the joint funding project of Beijing Natural Science Foundation and Beijing Education Committee (KZ201810025040); the Beijing Natural Science Foundation (7174291); and the China Postdoctoral Science Foundation (2016M601067).

All procedures were carried out in accordance with the NRC/ILAR Guide for the Care and Use of Laboratory Animals (8th Edition). The study protocol was approved by the Institutional Animal Care and Use Committee of Capital Medical University, Beijing, China.
1. Animal Selection
<ol>
	<li>For all experiments, use adult C57BL/6J male mice (8 weeks old) as the animal model. 
	NOTE: C57BL/6J mice carrying a splice variant of the Cdh23 exhibit accelerated senescence ...

<ol>
	<li>Lie, A., Skogstad, M., Johnsen, T. S., Engdahl, B., Tambs, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+prevalence+of+notched+audiograms+in+a+cross-sectional+study+of+12,055+railway+workers.">The prevalence of notched audiograms in a cross-sectional study of 12,055 railway workers.</a> Ear and Hearing. 36 (3), 86-92 (2015).</li><li>Fettiplace, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hair+cell+transduction,+tuning,+and+synaptic+transmission+in+the+mammalian+cochlea.">Hair cell transduction, tuning, and synaptic transmission in the mammalian cochlea.</a> Comprehensive Physiology. 7 (4), 1197-1227 (2017).</li><li>Liberman, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cochlear+frequency+map+for+the+cat:+labeling+auditory-nerve+fibers+of+known+characteristic+frequency.">The cochlear frequency map for the cat: labeling auditory-nerve fibers of known characteristic frequency.</a> Journal of the Acoustical Society of America. 72 (5), 1441-1449 (1982).</li><li>Fettiplace, R., Kim, K. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+physiology+of+mechanoelectrical+transduction+channels+in+hearing.">The physiology of mechanoelectrical transduction channels in hearing.</a> Physiological Reviews. 94 (3), 951-986 (2014).</li><li>Muller, M., von Hunerbein, K., Hoidis, S., Smolders, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+physiological+place-frequency+map+of+the+cochlea+in+the+CBA/J+mouse.">A physiological place-frequency map of the cochlea in the CBA/J mouse.</a> Hearing Research. 202 (1-2), 63-73 (2005).</li><li>Viberg, A., Canlon, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+guide+to+plotting+a+cochleogram.">The guide to plotting a cochleogram.</a> Hearing Research. 197 (1-2), 1-10 (2004).</li><li>Paquette, S. T., Gilels, F., White, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noise+exposure+modulates+cochlear+inner+hair+cell+ribbon+volumes,+correlating+with+changes+in+auditory+measures+in+the+FVB/nJ+mouse.">Noise exposure modulates cochlear inner hair cell ribbon volumes, correlating with changes in auditory measures in the FVB/nJ mouse.</a> Scientific Reports. 6, 25056 (2016).</li><li>Fernandez, K. A., Jeffers, P. W., Lall, K., Liberman, M. C., Kujawa, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aging+after+noise+exposure:+acceleration+of+cochlear+synaptopathy+in+"recovered"+ears.">Aging after noise exposure: acceleration of cochlear synaptopathy in "recovered" ears.</a> Journal of Neuroscience. 35 (19), 7509-7520 (2015).</li><li>Wichmann, C., Moser, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relating+structure+and+function+of+inner+hair+cell+ribbon+synapses.">Relating structure and function of inner hair cell ribbon synapses.</a> Cell and Tissue Research. 361 (1), 95-114 (2015).</li><li>Matthews, G., Fuchs, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+diverse+roles+of+ribbon+synapses+in+sensory+neurotransmission.">The diverse roles of ribbon synapses in sensory neurotransmission.</a> Nature Reviews Neuroscience. 11 (12), 812-822 (2010).</li><li>Liberman, L. D., Liberman, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postnatal+maturation+of+auditory-nerve+heterogeneity,+as+seen+in+spatial+gradients+of+synapse+morphology+in+the+inner+hair+cell+area.">Postnatal maturation of auditory-nerve heterogeneity, as seen in spatial gradients of synapse morphology in the inner hair cell area.</a> Hearing Research. 339, 12-22 (2016).</li><li>Yang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maximal+number+of+pre-synaptic+ribbons+are+formed+in+cochlear+region+corresponding+to+middle+frequency+in+mice.">Maximal number of pre-synaptic ribbons are formed in cochlear region corresponding to middle frequency in mice.</a> Acta Oto-Laryngologica. 138 (1), 25-30 (2018).</li><li>Liberman, M. C., Kujawa, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cochlear+synaptopathy+in+acquired+sensorineural+hearing+loss:+Manifestations+and+mechanisms.">Cochlear synaptopathy in acquired sensorineural hearing loss: Manifestations and mechanisms.</a> Hearing Research. 349, 138-147 (2017).</li><li>Moser, T., Starr, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Auditory+neuropathy--neural+and+synaptic+mechanisms.">Auditory neuropathy--neural and synaptic mechanisms.</a> Nature Reviews Neurology. 12 (3), 135-149 (2016).</li><li>Yu, W. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Gata3-Mafb+transcriptional+network+directs+post-synaptic+differentiation+in+synapses+specialized+for+hearing.">A Gata3-Mafb transcriptional network directs post-synaptic differentiation in synapses specialized for hearing.</a> Elife. 2, 01341 (2013).</li><li>Buniello, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wbp2+is+required+for+normal+glutamatergic+synapses+in+the+cochlea+and+is+crucial+for+hearing.">Wbp2 is required for normal glutamatergic synapses in the cochlea and is crucial for hearing.</a> EMBO Molecular Medicine. 8 (3), 191-207 (2016).</li><li>Gilels, F., Paquette, S. T., Beaulac, H. J., Bullen, A., White, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Severe+hearing+loss+and+outer+hair+cell+death+in+homozygous+Foxo3+knockout+mice+after+moderate+noise+exposure.">Severe hearing loss and outer hair cell death in homozygous Foxo3 knockout mice after moderate noise exposure.</a> Scientific Reports. 7 (1), 1054 (2017).</li><li>Kane, K. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+background+effects+on+age-related+hearing+loss+associated+with+Cdh23+variants+in+mice.">Genetic background effects on age-related hearing loss associated with Cdh23 variants in mice.</a> Hearing Research. 283 (1-2), 80-88 (2012).</li><li>Jiang, X. W., Li, X. R., Zhang, Y. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+of+ribbon+synapses+number+of+cochlear+hair+cells+in+C57BL/6J+mice+with+age+(Delta).">Changes of ribbon synapses number of cochlear hair cells in C57BL/6J mice with age (Delta).</a> International Journal of Clinical and Experimental Medicine. 8 (10), 19058-19064 (2015).</li><li>Akil, O., Oursler, A., Fan, K., Lustig, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+auditory+brainstem+response+testing.">Mouse auditory brainstem response testing.</a> BIO-Protocol. 6 (6), (2016).</li><li>Zhou, X., Jen, P. H. -. S., Seburn, K. L., Frankel, W. N., Zheng, Q. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Auditory+brainstem+responses+in+10+inbred+strains+of+mice.">Auditory brainstem responses in 10 inbred strains of mice.</a> Brain Research. 1091 (1), 16-26 (2006).</li><li>Montgomery, S. C., Cox, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+mount+dissection+and+immunofluorescence+of+the+adult+mouse+cochlea.">Whole mount dissection and immunofluorescence of the adult mouse cochlea.</a> Journal of Visualized Experiments. (107), (2016).</li><li>Schmitz, F., Konigstorfer, A., Sudhof, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RIBEYE,+a+component+of+synaptic+ribbons:+a+protein's+journey+through+evolution+provides+insight+into+synaptic+ribbon+function.">RIBEYE, a component of synaptic ribbons: a protein's journey through evolution provides insight into synaptic ribbon function.</a> Neuron. 28 (3), 857-872 (2000).</li><li>Suzuki, J., Corfas, G., Liberman, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Round-window+delivery+of+neurotrophin+3+regenerates+cochlear+synapses+after+acoustic+overexposure.">Round-window delivery of neurotrophin 3 regenerates cochlear synapses after acoustic overexposure.</a> Scientific Reports. 6, 24907 (2016).</li><li>Rutherford, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resolving+the+structure+of+inner+ear+ribbon+synapses+with+STED+microscopy.">Resolving the structure of inner ear ribbon synapses with STED microscopy.</a> Synapse. 69 (5), 242-255 (2015).</li><li>Liberman, L. D., Liberman, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+cochlear+synaptopathy+after+acoustic+overexposure.">Dynamics of cochlear synaptopathy after acoustic overexposure.</a> Journal of the Association for Research in Otolaryngology. 16 (2), 205-219 (2015).</li><li>Gilels, F., Paquette, S. T., Zhang, J., Rahman, I., White, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutation+of+Foxo3+causes+adult+onset+auditory+neuropathy+and+alters+cochlear+synapse+architecture+in+mice.">Mutation of Foxo3 causes adult onset auditory neuropathy and alters cochlear synapse architecture in mice.</a> Journal of Neuroscience. 33 (47), 18409-18424 (2013).</li><li>Wan, G., Gomez-Casati, M. E., Gigliello, A. R., Liberman, M. C., Corfas, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurotrophin-3+regulates+ribbon+synapse+density+in+the+cochlea+and+induces+synapse+regeneration+after+acoustic+trauma.">Neurotrophin-3 regulates ribbon synapse density in the cochlea and induces synapse regeneration after acoustic trauma.</a> Elife. 3, (2014).</li><li>Sergeyenko, Y., Lall, K., Liberman, M. C., Kujawa, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-related+cochlear+synaptopathy:+an+early-onset+contributor+to+auditory+functional+decline.">Age-related cochlear synaptopathy: an early-onset contributor to auditory functional decline.</a> Journal of Neuroscience. 33 (34), 13686-13694 (2013).</li><li>Furman, A. C., Kujawa, S. G., Liberman, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noise-induced+cochlear+neuropathy+is+selective+for+fibers+with+low+spontaneous+rates.">Noise-induced cochlear neuropathy is selective for fibers with low spontaneous rates.</a> Journal of Neurophysiology. 110 (3), 577-586 (2013).</li><li>Kujawa, S. G., Liberman, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adding+insult+to+injury:+cochlear+nerve+degeneration+after+"temporary"+noise-induced+hearing+loss.">Adding insult to injury: cochlear nerve degeneration after "temporary" noise-induced hearing loss.</a> Journal of Neuroscience. 29 (45), 14077-14085 (2009).</li><li>Valero, M. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noise-induced+cochlear+synaptopathy+in+rhesus+monkeys+(Macaca+mulatta).">Noise-induced cochlear synaptopathy in rhesus monkeys (Macaca mulatta).</a> Hearing Research. 353, 213-223 (2017).</li><li>Viana, L. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cochlear+neuropathy+in+human+presbycusis:+Confocal+analysis+of+hidden+hearing+loss+in+post-mortem+tissue.">Cochlear neuropathy in human presbycusis: Confocal analysis of hidden hearing loss in post-mortem tissue.</a> Hearing Research. 327, 78-88 (2015).</li><li>Tong, M., Brugeaud, A., Edge, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regenerated+synapses+between+postnatal+hair+cells+and+auditory+neurons.">Regenerated synapses between postnatal hair cells and auditory neurons.</a> Journal of the Association for Research in Otolaryngology. 14 (3), 321-329 (2013).</li><li>Landegger, L. D., Dilwali, S., Stankovic, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neonatal+murine+cochlear+explant+technique+as+an+in+vitro+screening+tool+in+hearing+research.">Neonatal murine cochlear explant technique as an in vitro screening tool in hearing research.</a> Journal of Visualized Experiments. (124), (2017).</li><li>Takeda, S., Mannstr&#246;m, P., Dash-Wagh, S., Yoshida, T., Ulfendahl, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+Aging+and+Noise+Exposure+on+Auditory+Brainstem+Responses+and+Number+of+Presynaptic+Ribbons+in+Inner+Hair+Cells+of+C57BL/6J+Mice.">Effects of Aging and Noise Exposure on Auditory Brainstem Responses and Number of Presynaptic Ribbons in Inner Hair Cells of C57BL/6J Mice.</a> Neurophysiology. 49 (5), 316-326 (2017).</li><li>Mehraei, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Auditory+brainstem+response+latency+in+noise+as+a+marker+of+cochlear+synaptopathy.">Auditory brainstem response latency in noise as a marker of cochlear synaptopathy.</a> Journal of Neuroscience. 36 (13), 3755-3764 (2016).</li></ol>

The authors have no conflicts of interest to disclose.

Since cochlear synaptopathy was first characterized in adult mice with a temporary threshold shift (TTS) induced by 8&#8210;16 kHz octave band noise at 100 dB SPL for 2 h31, researchers have increasingly investigated the effects of synaptopathy in various mammals, including monkeys and humans32,33. In addition to noise exposure, several other conditions have been associated with cochlear synaptopathy (e.g., aging, the use of ototoxic drugs...

Frequencies in the range of approximately 20&#8210;20,000 Hz can be perceived as auditory stimuli by humans. Human hearing is normally most sensitive near 1,000 Hz, where average sound pressure level is 20 &#956;Pa in young adults (i.e., 0 decibels of sound pressure level [dB SPL]). In some pathological conditions, hearing loss is restricted to specific frequencies. For example, in the early stages of noise-induced hearing loss (NIHL), a &#8220;notch&#8221; (i.e., hearing threshold elevation) can be observed in the audiogram at 4 kHz1. Along the mammalian cochlear partition, its gradations of stiffness and mass produce an exponential frequency map, with high-frequency sound detection at the base of the cochlea and low-frequency detection at the apex2. Indeed, there is a cochlear place-frequency map along the basilar membrane, leading to what is known as tonotopic organization2,3. Each given place on the basilar membrane has the highest sensitivity to only one particular sound frequency, which is usually termed the characteristic frequency3,4, although responses to other frequencies can also be observed.
To date, various mouse models have been employed to investigate normal function, pathological processes, and therapeutic efficacy in the auditory system. Precise knowledge of physiological parameters in the mouse cochlea is a prerequisite for such studies of hearing loss. The mouse cochlea is anatomically divided into apical, middle, and basal turns, which correspond to different frequency regions. By labeling auditory nerve afferents at the cochlear nucleus to analyze their corresponding peripheral innervation sites in the cochlea, M&#252;ller et al. succeeded in establishing the cochlear place-frequency map in the normal mouse in vivo5. In the interval of 7.2&#8211;61.8 kHz, which corresponds to positions between 90% and 10% of the full length of the basilar membrane, the mouse cochlear place-frequency map can be described by a simple linear regression function, suggesting a relation between the normalized distance from the cochlear base and the logarithm of the characteristic frequency5. In laboratory mice, the place-frequency map can be used to explore the relationship between hearing thresholds within specific frequency ranges and cochleograms showing the numbers of missing hair cells in relative regions along the basilar membrane6. Importantly, the place-frequency map provides a positioning system for the investigation of minimal structural damage, such as damage to the ribbon synapses of hair cells at specific cochlear frequency locations in mice with peripheral auditory trauma7,8.
In the mammalian cochlea, ribbon synapses are comprised of a presynaptic ribbon, an electron-dense projection that tethers a halo of release-ready synaptic vesicles containing glutamate within the IHC, and a postsynaptic density on the nerve terminal of the SGN with glutamate receptors9. During cochlear sound transduction, deflection of the hair cell bundle results in IHC depolarization, which leads to glutamate release from IHCs onto the postsynaptic afferent terminals, thereby activating the auditory pathway. Activation of this pathway leads to the transformation of sound-induced mechanical signals into a rate code in the SGN10. Indeed, the IHC ribbon synapse is highly specialized for indefatigable sound transmission at rates of hundreds of Hertz with high temporal precision, and is of critical importance for presynaptic mechanisms of sound encoding. Previous studies have revealed that ribbon synapses vary greatly in size and number at different frequency regions in the adult mouse cochlea11,12, likely reflecting structural adaptation to the particular sound coding for survival needs. Recently, experimental animal studies have demonstrated that cochlear synaptopathy contributes to multiple forms of hearing impairments, including noise-induced hearing loss, age-related hearing loss, and hereditary hearing loss13,14. Thus, methods for identifying correlated changes in synaptic number, structure, and function at specific frequency regions have been increasingly employed in studies of auditory development and inner ear disease, using models generated via experimental manipulation of genetic or environmental variables15,16,17.
In the current report, we present a protocol for analyzing the synaptic number, structure, and function at a specific frequency region of the basilar membrane in adult mice. Cochlear frequency localization is performed using a given place-frequency map in combination with a cochleogram. The normal morphological characteristics of cochlear ribbon synapses are evaluated via presynaptic and postsynaptic immunostaining. The functional status of cochlear ribbon synapses is determined based on the suprathreshold amplitudes of ABR wave I. With minor alterations, this protocol can be used to examine physiological or pathological conditions in other animal models, including rats, guinea pigs, and gerbils.

<table border="0" cellpadding="0" cellspacing="0" style="width:828px;" width="828">
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		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ketamine hydrochloride</td>
			<td style="width:304px;">Gutian Pharmaceutical Co., Ltd., Fujian, China</td>
			<td style="width:141px;">H35020148</td>
			<td style="width:167px;">100mg/kg</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Xylazine hydrochloride</td>
			<td>Sigma-Aldrich, St. Louis, MO, USA</td>
			<td>X-1251</td>
			<td>10mg/kg</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TDT physiology apparatus</td>
			<td>Tucker-Davis Technologies, Alachua, FL, USA</td>
			<td colspan="2">Auditory Physiology System III</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SigGen/BioSig software</td>
			<td>Tucker-Davis Technologies, Alachua, FL, USA</td>
			<td colspan="2">Auditory Physiology System III</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Electric Pad</td>
			<td>Pet Fun</td>
			<td>11072931136</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dumont forceps 3#</td>
			<td>Fine Science Tools, North Vancouver, B.C., Canada</td>
			<td>0203-3-PO</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dumont forceps 5#</td>
			<td>Fine Science Tools, North Vancouver, B.C., Canada</td>
			<td>0209-5-PO</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stereo dissection microscope</td>
			<td>Nikon Corp., Tokyo, Japan</td>
			<td>SMZ1270</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Goat serum</td>
			<td>ZSGB-BIO, Beijing,China</td>
			<td>ZLI-9021</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-glutamate receptor 2, extracellular, clone 6C4</td>
			<td>Millipore Corp., Billerica, MA, USA</td>
			<td>MAB397</td>
			<td>mouse&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Purified Mouse Anti-CtBP2</td>
			<td>BD Biosciences, Billerica, MA, USA</td>
			<td>612044</td>
			<td>mouse&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 568 goat anti-mouse IgG1antibody</td>
			<td>Thermo Fisher Scientific Inc., Waltham, MA, USA</td>
			<td>A21124</td>
			<td>goat</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 488 goat anti-mouse IgG2a antibody</td>
			<td>Thermo Fisher Scientific Inc., Waltham, MA, USA</td>
			<td>A21131</td>
			<td>goat</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mounting medium containing DAPI</td>
			<td>ZSGB-BIO, Beijing,China</td>
			<td>ZLI-9557</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Confocal fluorescent microscopy</td>
			<td>Leica Microsystems, Wetzlar, Germany</td>
			<td>TCS SP8 II</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Image Pro Plus software</td>
			<td>Media Cybernetics, Bethesda, MD, USA</td>
			<td>version 6.0</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Professional diagnostic pocket otoscope</td>
			<td>Lude Medical Apparatus and Instruments Trade Co., Ltd., Shanghai,China</td>
			<td>HS-OT10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Needle electrode</td>
			<td>Friendship Medical Electronics Co., Ltd., Xi&#39;an,China</td>
			<td>1029</td>
			<td>20 mm, 28 G</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Closed-field speaker</td>
			<td>Tucker-Davis Technologies, Alachua, FL, USA</td>
			<td>CF1</td>
			<td></td>
		</tr>
	</tbody>
</table>

morphological and functional evaluation of ribbon synapses at specific frequency regions of the mouse cochlea

Cochlear inner hair cells (IHCs) transmit acoustic signals to spiral ganglion neurons (SGNs) through ribbon synapses. Several experimental studies have indicated that hair cell synapses may be the initial targets in sensorineural hearing loss (SNHL). Such studies have proposed the concept of cochlear &#34;synaptopathy&#34;, which refers to alterations in ribbon synapse number, structure, or function that result in abnormal synaptic transmission between IHCs and SGNs. While cochlear synaptopathy is irreversible, it does not affect the hearing threshold. In noise-induced experimental models, restricted damage to IHC synapses in select frequency regions is employed to identify the environmental factors that specifically cause synaptopathy, as well as the physiological consequences of disturbing this inner ear circuit. Here, we present a protocol for analyzing cochlear synaptic morphology and function at a specific frequency region in adult mice. In this protocol, cochlear localization of specific frequency regions is performed using place-frequency maps in conjunction with cochleogram data, following which the morphological characteristics of ribbon synapses are evaluated via synaptic immunostaining. The functional status of ribbon synapses is then determined based on the amplitudes of auditory brainstem response (ABR) wave I. The present report demonstrates that this approach can be used to deepen our understanding of the pathogenesis and mechanisms of synaptic dysfunction in the cochlea, which may aid in the development of novel therapeutic interventions.

ABR hearing tests were performed for 10 C57BL/6J mice (8 weeks of age) under anesthesia. ABRs were elicited using tone burst stimuli at 4, 8, 16, 32, and 48 kHz. The hearing threshold of each animal was visually detected by distinguishing at least one clear waveform in the ABR. All mice exhibited ABR thresholds in response to tone bursts, ranging between 25 and 70 dB SPL depending on the frequency of the stimulus. Our results indicated that the hearing threshold was lowest at 16 kHz (Figure 1</strong...

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Morphological and Functional Evaluation of Ribbon Synapses at Specific Frequency Regions of the Mouse Cochlea

This manuscript describes an experimental protocol for evaluating the morphological characteristics and functional status of ribbon synapses in normal mice. The present model is also suitable for noise-induced and age-related cochlear synaptopathy-restricted models. The correlative results of previous mouse studies are also discussed.

Cochlear inner hair cells (IHCs) transmit acoustic signals to spiral ganglion neurons (SGNs) through ribbon synapses. Several experimental studies have ...

The primary report is really stress importance of accurately locating the specific frequency region of the basilar membrane in most models, where synapse numbers altered and function can be analyzed. The cochlear localization of the specific frequency regions is reliably performed using, place frequency maps in conjunction with cochleogram data. The microscope provides insight into several research studies.It supporting the notion that the cochlear neuropathy is the primary initial events specifically preceding hearing loss, tinnitus, and hyperacusis. After confirming a lack of response to toe pinch in an anesthetized eight week old adult C57 black 6J male mouse. Place the mouse on a 37 degree celsius heat pad in an electrically and acoustically shielded room.Next, place a subdermal 20 millimeter 28 gauge needle recording electrode, with the depth of three millimeters under the skin at the vertex of the skull. A reference electrode in the ipsilateral parotid region, below the pinna of measured ear and a ground electrode in the contralateral parotid region. Place a closed field speaker equipped with a two centimeter plastic tube with a cone shaped tip next to the mouse and fit the tip into the external ear canal.For an auditory brainstem response, or ABR recording, generate tone pips at decreasing sound pressure levels from 90 to 10 decibels, in five to 10 decibels sound pressure level steps. During this step, the responses are amplified, filtered, and averaged. At each frequency, determine the ABR threshold, which is the minimal sound pressure level that results in a reliable ABR recording with one or more distinguishable waves that can be clearly identified by visual inspection.After the ABR recording, expose the bulla from the ventral side and open the bulla with sharp scissors to gain access to the cochlea. Using fine forceps, remove the temporal bones and sever the stapes artery. Then remove the stapes from the oval window and rupture the round window membrane.Gently rotate the tip of a 13 millimeter 27 gauge needle to make a small hole at the apex of the cochlea, before using a fine tipped pipette to gently flush 4%paraformaldehyde through the window into the perilymphatic spaces. Next, rinse the bones three times for five minutes per wash with cold fresh 0.1 molar PBS per wash. Followed by decalcification of the bones with 10%EDTA for four hours at room temperature and 20 rotations per minute on a horizontal shaker.At the end of the incubation, transfer a decalcified temporal bone into fresh 0.1 molar PBS under a dissecting microscope. Use 3 and 5 Dumont forceps and a 27 gauge needle to dissect the apical, middle, and basal cochlea regions in turn. Use a razor blade to make a small series of cuts along the spiral ligament and remove tectorial membrane and Reissner's membrane.Further dissect the remaining auditory epithelium, including the spiral limbus, into individual cochlear turns for whole mount preparations. Then use the 40X oil objective of a light microscope to measure the basilar membrane length with 250 micrometer scale placed in the eye piece. It can be adjusted along the stereocilia of the inner hair cells.After dissection, place each cochlear turn into individual 2.5 milliliter centrifuge tubes. Block any nonspecific binding with 10%goat serum in PBS in 0.1%Triton X-100 for one hour at room temperature on a rotator. At the end of incubation, use 200 microliter pipette tip to remove the solution under a dissection microscope.Incubate the specimens with the primary antibodies of interest diluted in 5%goat serum and PBS in 0.1%Triton X-100 overnight at 4 degrees Celsius on a rotator. The next morning, rinse the tissue samples three times for five minutes with cold 0.1 molar PBS per wash to remove any residual primary antibody. Then label the specimens with the appropriate secondary antibodies diluted in 5%goat serum in PBS in 0.1%Triton X-100 for two to three hours at room temperature on the rotator, protected from light.At the end of incubation, wash the samples three times with 0.1 molar PBS as demonstrated and transfer the specimens into individual 35 millimeter plates containing 0.1 molar PBS. Place a drop of DAPI supplemented mounting medium onto the slide and transfer the specimens from PBS to the mounting medium. Place one edge of a cover slip onto each slide and release to let the cover slips fall gently.Then dry the slides in a slide box at four degrees Celsius overnight. To image the specimens, use a confocal microscope with the appropriate lasers and 63X high resolution oil immersion lens to acquire eight micrometer confocal Z stacks from each cochlear turn. For synaptic puncta counts, set the Z stacks with the 0.3 micrometer step size to span the entire length of inner hair cells, insuring all of the synaptic puncta can be imaged and merge the puncta containing images in a Z stack to obtain the Z access projection.Import the merged images into an appropriate image processing software program. Divide the synaptic total counts in each Z stack at specific frequency regions by the number of DAPI positive inner hair cells to calculate the number of synaptic puncta for each cell. At each specific frequency region, average all of the synaptic puncta in three images of different microscopic fields, containing nine to 11 inner hair cells.To better visualize the cytoskeletal architecture and synaptic localization, use the brush tool to visually assess the synaptic structure and distribution of the individual inner hair cells. To inspect the juxtaposition of presynaptic ribbons and postsynaptic receptor patches, use the rectangular marquee tool extract the voxel space around the ribbons and use image cutting to isolate the individual ribbons. Then click image and image size to acquire a thumbnail array of these miniature projections that can be used to identify paired synapses versus orphan ribbons.All ten of the mice in this representative experiment exhibited ABR thresholds in response to tone bursts ranging between 25 and 70 decibels per sound pressure level depending on the frequency of the stimulus. The hearing threshold for this experiment was lowest at 16 kilohertz corresponding to a approximately 43%of the distance from the cochlear apex, suggesting that the acoustic sensitivity is significantly reduced in other cochlear regions. Whole mounts of the auditory epithelium is dissected into three pieces and the lengths are measured and converted into their percent distance from the cochlear apex.The frequency location on the basilar membrane of each cochlear turn is calculated using logarithmic function. In normal ears of adult mice, immunostaining reveals juxtaposed pairs of synaptic ribbons and glutamate receptor patches. Studying the surface of the basolateral membrane of the inner hair cells, with eight to 20 pairs per cell.Although the vast majority of the puncta appear as juxtaposed pairs in normal ears, orphan ribbons are infrequently observed at high magnifications. The number of inner hair cell ribbon synapses is highest at the 16 kilohertz region and significantly decreases as the distance from this location increases. The most important thing to remember during this procedure is that one must guarantee the integrity of cochlear basal membrane.We need to stress accuracy until technical proficiency is reached. Following this procedure, the gene therapy can be performed to answer whether the cochlear neuropathy can be repaired by this measure. This measure is wider technique for exploring cochlear neuropathy, which if the primary initial events associate with having hearing loss, tinnitus, and hyperacusis.

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11.5K Views.  Capital Medical University. The primary report is really stress importance of accurately locating the specific frequency region of the basilar membrane in most models, where synapse numbers altered and function can be analyzed. The cochlear localization of the specific frequency regions is reliably performed using, place frequency maps in conjunction with cochleogram data. The microscope provides insight into several research studies.It supporting the notion that the cochlear neuropathy is the primary initial eve...