The authors acknowledge Lichun Zhang for helping to establish the method and the Fluorescence Microscopy Service Unit, Carl von Ossietzky University of Oldenburg, for the use of the imaging facilities. This research was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany&#39;s Excellence Strategy -EXC 2177/1.

<ol>
	<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=Noise-induced+and+age-related+hearing+loss:+new+perspectives+and+potential+therapies+[version+1;+peer+review.">Noise-induced and age-related hearing loss: new perspectives and potential therapies [version 1; peer review.</a> F1000Research. 6 (927), (2017).</li><li>Heeringa, A. N., Koeppl, 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+aging+cochlea:+Towards+unraveling+the+functional+contributions+of+strial+dysfunction+and+synaptopathy.">The aging cochlea: Towards unraveling the functional contributions of strial dysfunction and synaptopathy.</a> Hearing. 376, 111-124 (2019).</li><li>Liberman, L. D., Wang, H., 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=Opposing+gradients+of+ribbon+size+and+AMPA+receptor+expression+underlie+sensitivity+differences+among+cochlear-nerve/hair-cell+synapses.">Opposing gradients of ribbon size and AMPA receptor expression underlie sensitivity differences among cochlear-nerve/hair-cell synapses.</a> The Journal of Neuroscience. 31 (3), 801-808 (2011).</li><li>Khimich, 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=Hair+cell+synaptic+ribbons+are+essential+for+synchronous+auditory+signalling.">Hair cell synaptic ribbons are essential for synchronous auditory signalling.</a> Nature. 434 (7035), 889-894 (2005).</li><li>Pangr&#353;i&#269;, T., 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=Hearing+requires+otoferlin-dependent+efficient+replenishment+of+synaptic+vesicles+in+hair+cells.">Hearing requires otoferlin-dependent efficient replenishment of synaptic vesicles in hair cells.</a> Nature Neuroscience. 13 (7), 869-876 (2010).</li><li>Meyer, A. C., 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=Tuning+of+synapse+number,+structure+and+function+in+the+cochlea.">Tuning of synapse number, structure and function in the cochlea.</a> Nature Neuroscience. 12 (4), 444-453 (2009).</li><li>Zhang, L., Engler, S., Koepcke, L., Steenken, F., Koeppl, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concurrent+gradients+of+ribbon+volume+and+AMPA-receptor+patch+volume+in+cochlear+afferent+synapses+on+gerbil+inner+hair+cells.">Concurrent gradients of ribbon volume and AMPA-receptor patch volume in cochlear afferent synapses on gerbil inner hair cells.</a> Hearing Research. 364, 81-89 (2018).</li><li>Steenken, F., 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=Age-related+decline+in+cochlear+ribbon+synapses+and+its+relation+to+different+metrics+of+auditory-nerve+activity.">Age-related decline in cochlear ribbon synapses and its relation to different metrics of auditory-nerve activity.</a> Neurobiology of Aging. 108, 133-145 (2021).</li><li>Merchan-Perez, A., 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=Ultrastructural+differences+among+afferent+synapses+on+cochlear+hair+cells:+Correlations+with+spontaneous+discharge+rate.">Ultrastructural differences among afferent synapses on cochlear hair cells: Correlations with spontaneous discharge rate.</a> Journal of Comparative Neurology. 371 (2), 208-221 (1996).</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>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> The Journal of Neuroscience. 33 (47), 18409-18424 (2013).</li><li>Yin, Y., Liberman, L. D., Maison, S. F., 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=Olivocochlear+innervation+maintains+the+normal+modiolar-pillar+and+habenular-cuticular+gradients+in+cochlear+synaptic+morphology.">Olivocochlear innervation maintains the normal modiolar-pillar and habenular-cuticular gradients in cochlear synaptic morphology.</a> Journal of the Association for Research in Otolaryngology. 15 (4), 571-583 (2014).</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 (1), 25056 (2016).</li><li>Reijntjes, D. O. J., K&#246;ppl, C., Pyott, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Volume+gradients+in+inner+hair+cell-auditory+nerve+fiber+pre-+and+postsynaptic+proteins+differ+across+mouse+strains.">Volume gradients in inner hair cell-auditory nerve fiber pre- and postsynaptic proteins differ across mouse strains.</a> Hearing Research. 390, 107933 (2020).</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=Single-neuron+labeling+in+the+cat+auditory+nerve.">Single-neuron labeling in the cat auditory nerve.</a> Science. 216 (4551), 1239-1241 (1982).</li><li>Bourien, J., 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=Contribution+of+auditory+nerve+fibers+to+compound+action+potential+of+the+auditory+nerve.">Contribution of auditory nerve fibers to compound action potential of the auditory nerve.</a> Journal of Neurophysiology. 112 (5), 1025-1039 (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> The Journal of Neuroscience. 33 (34), 13686-13694 (2013).</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>Batrel, C., 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=Mass+potentials+recorded+at+the+round+window+enable+the+detection+of+low+spontaneous+rate+fibers+in+gerbil+auditory+nerve.">Mass potentials recorded at the round window enable the detection of low spontaneous rate fibers in gerbil auditory nerve.</a> PLoS ONE. 12 (1), 0169890 (2017).</li><li>Jeffers, P. W. C., Bourien, J., Diuba, A., Puel, J. -. L., 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=Noise-induced+hearing+loss+in+gerbil:+Round+window+assays+of+synapse+loss.">Noise-induced hearing loss in gerbil: Round window assays of synapse loss.</a> Frontiers in Cellular Neuroscience. 15, 699978 (2021).</li><li>Gleich, O., Semmler, P., Strutz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+auditory+thresholds+and+loss+of+ribbon+synapses+at+inner+hair+cells+in+aged+gerbils.">Behavioral auditory thresholds and loss of ribbon synapses at inner hair cells in aged gerbils.</a> Experimental Gerontology. 84, 61-70 (2016).</li><li>Cheal, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+gerbil:+A+unique+model+for+research+on+aging.">The gerbil: A unique model for research on aging.</a> Experimental Aging Research. 12 (1), 3-21 (1986).</li><li>Gates, G. 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The authors have no conflicts of interest to declare.

With the method outlined in this protocol, it is possible to immunolabel IHCs and synaptic structures in cochleae from young adult and aged gerbils, identify presumed functional synapses by co-localization of pre- and postsynaptic elements, allocate them to individual IHCs, and quantify their number, volume, and location.&#160;The antibodies used in this approach also labeled outer hair cells (OHCs; myoVIIa) and their presynaptic ribbons. Furthermore, a viable alternative for immunolabeling of both IHCs and OHCs is an an...

Age-related hearing loss is one of the world's most prevalent diseases that affects more than one-third of the world's population aged 65 years and older1. The underlying causes are still under debate and actively being investigated but may include the loss of the specialized synapses connecting inner hair cells (IHCs) with afferent auditory nerve fibers2. These ribbon synapses comprise a presynaptic structure that has vesicles filled with the neurotransmitter glutamate tethered to it, as well as postsynaptic &#945;-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate receptors3,4,5. In the gerbil, ~20 afferent auditory nerve fibers contact one IHC6,7,8. Fibers on the IHC facing the modiolus are opposed to large synaptic ribbons, while the fibers connecting on the pillar side of the IHC face small synaptic ribbons (i.e., in cats9, gerbils7, guinea pigs10, and mice3,11,12,13,14). Furthermore, in the gerbil, the size of the presynaptic ribbons and the postsynaptic glutamate patches are positively correlated7,14. Fibers that are opposed to large ribbons on the modiolar side of the IHC are small in caliber and have low spontaneous rates and high thresholds15. There is evidence that low spontaneous rate fibers are more vulnerable to noise exposure10 and ototoxic drugs16 than high-spontaneous low-threshold fibers, which are located on the pillar side of IHCs15. 
The loss of ribbon synapses is the earliest degenerative event in cochlear neural age-related hearing loss, while the loss of spiral ganglion cells and their afferent auditory nerve fibers lags behind17,18. Electrophysiological correlates include recordings of auditory brainstem responses17 and compound action potentials8; however, these do not reflect the subtleties of synapse loss, since low spontaneous rate fibers do not contribute to these measures16. More promising electrophysiological metrics are the mass potential-derived neural index19 and the peristimulus time response20. However, these are only reliable if the animal has no other cochlear pathologies, beyond auditory nerve fiber loss, that affect the activity of the remaining auditory nerve fibers8. Furthermore, behaviorally assessed thresholds in the gerbil were not correlated with synapse numbers21. Therefore, reliable quantification of surviving ribbon synapses and, thus, the number of functional auditory nerve fibers is only possible by direct examination of the cochlear tissue.
The Mongolian gerbil (Meriones unguiculatus) is a suitable animal model for studying age-related hearing loss. It has a short life span, has low-frequency hearing similar to humans, is easy to maintain, and shows similarities to human pathologies related to age-related hearing loss2,22,23,24. Gerbils are considered aged when they reach 36 months of age, which is near the end of their average life span22. Importantly, an age-related loss of ribbon synapses has been demonstrated in gerbils raised and aged in quiet environments8,21. 
Here, a protocol to immunolabel, dissect, and analyze cochleae from gerbils of different ages, from young adults to aged, is presented. Antibodies directed against components of the presynapse (CtBP2), postsynaptic glutamate receptor patches (GluA2), and IHCs (myoVIIa) are used. An autofluorescence quencher is applied that reduces the background in aged cochleae and leaves the fluorescence signal intact. Further, a description is given of how to dissect the cochlea to examine both the sensory epithelium and the stria vascularis. The cochlear length is measured to enable the selection of distinct cochlear locations that correspond to specific best frequencies25. Quantification of synapse numbers is carried out with the freely available software ImageJ26. Additional quantification of synapse volumes and locations within the individual HC is performed with software custom written in Matlab. This software is not made publicly available, as the authors lack the resources to provide professional documentation and support.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Albumin Fraction V biotin-free</td>
			<td>Carl Roth</td>
			<td>0163.2</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-CtBP2 (IgG1 monoclonal mouse)</td>
			<td>BD Biosciences, Eysins</td>
			<td>612044</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-GluA2 (IgG2a monoclonal mouse)</td>
			<td>Millipore</td>
			<td>MAB39</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse (IgG1)-AF 488</td>
			<td>Molecular Probes Inc.</td>
			<td>A21121</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-MyosinVIIa (IgG polyclonal rabbit)</td>
			<td>Proteus Biosciences</td>
			<td>25e6790</td>
			<td></td>
		</tr>
		<tr>
			<td>Blade Holder &#38; Breaker - Flat Jaws</td>
			<td>Fine Science Tools</td>
			<td>10052-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Bonn Artery Scissors - Ball Tip</td>
			<td>Fine Science Tools</td>
			<td>14086-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslip thickness 1.5H, 24 x 60 mm</td>
			<td>Carl Roth</td>
			<td>LH26.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Surgical Blade</td>
			<td>Henry Schein</td>
			<td>0473</td>
			<td></td>
		</tr>
		<tr>
			<td>donkey anti-rabbit (IgG)-AF647</td>
			<td>Life Technologies-Molecular Probes</td>
			<td>A-31573</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5 - Fine Forceps</td>
			<td>Fine Science Tools</td>
			<td>11254-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5SF Forceps</td>
			<td>Fine Science Tools</td>
			<td>11252-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol, absolute 99.8%</td>
			<td>Fisher Scientific</td>
			<td>12468750</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid</td>
			<td>Carl Roth</td>
			<td>8040.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Excel</td>
			<td>Microsoft Corporation</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Feather Double Edge Blade</td>
			<td>PLANO</td>
			<td>112-9</td>
			<td></td>
		</tr>
		<tr>
			<td>G19 Cannula</td>
			<td>Henry Schein</td>
			<td>9003633</td>
			<td></td>
		</tr>
		<tr>
			<td>goat anti-mouse (IgG2a)-AF568</td>
			<td>Invitrogen</td>
			<td>A-21134</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Ratiopharm</td>
			<td>N68542.04</td>
			<td></td>
		</tr>
		<tr>
			<td>Huygens Essentials</td>
			<td>Scientific Volume Imaging</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>Fiji</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Immersol, Immersion oil 518F</td>
			<td>Carl Zeiss</td>
			<td>10539438</td>
			<td></td>
		</tr>
		<tr>
			<td>Intrafix Primeline Classic, 150 cm (mit Datamatrix Code auf der Sterilverpackung)</td>
			<td>Braun</td>
			<td>4062957E</td>
			<td></td>
		</tr>
		<tr>
			<td>ISM596D</td>
			<td>Ismatec</td>
			<td></td>
			<td>peristaltic pump</td>
		</tr>
		<tr>
			<td>KL 1600 LED</td>
			<td>Schott</td>
			<td>150.600</td>
			<td>light source for stereomicroscope</td>
		</tr>
		<tr>
			<td>Leica Application suite X</td>
			<td>Leica Microsystem CMS GmbH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Leica TCS SP8 system</td>
			<td>Leica Microsystem CMS GmbH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Matlab</td>
			<td>The Mathworks Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mayo Scissors Tungston Carbide ToghCut</td>
			<td>Fine Science Tools</td>
			<td>14512-17</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini-100 Orbital-Genie</td>
			<td>Scientific Industries</td>
			<td>SI-M100</td>
			<td>for use in cold environment</td>
		</tr>
		<tr>
			<td>Narcoren (pentobarbital)</td>
			<td>Boehringer Ingelheim Vetmedica GmbH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon Eclipse Ni-Ei</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NIS Elements</td>
			<td>Nikon Europe B.V.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Carl Roth</td>
			<td>0335.3</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish without vents</td>
			<td>Avantor VWR</td>
			<td>390-1375</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Disodium phosphate</td>
			<td>AppliChem</td>
			<td>A1046</td>
			<td></td>
		</tr>
		<tr>
			<td>Monopotassium phosphate</td>
			<td>Carl Roth</td>
			<td>3904.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Carl Roth</td>
			<td>6781.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Sigma Aldrich</td>
			<td>31434-M</td>
			<td></td>
		</tr>
		<tr>
			<td>Screw Cap Containers</td>
			<td>Sarstedt</td>
			<td>75.562.300</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>Carl Roth</td>
			<td>K305.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Student Adson Forceps</td>
			<td>Fine Science Tools</td>
			<td>91106-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Student Halsted-Mosquito Hemostat</td>
			<td>Fine Science Tools</td>
			<td>91308-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Superfrost Adhesion Microscope Slides</td>
			<td>Epredia</td>
			<td>J1800AMNZ</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton&#160; X</td>
			<td>Carl Roth</td>
			<td>3051.2</td>
			<td></td>
		</tr>
		<tr>
			<td>TrueBlack Lipofuscin Autofluorescence Quencher</td>
			<td>Biotium</td>
			<td>23007</td>
			<td></td>
		</tr>
		<tr>
			<td>Vannas Spring Scissors, 3mm</td>
			<td>Fine Science Tools</td>
			<td>15000-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Vectashield Antifade Mounting Medium</td>
			<td>Vector Laboratories</td>
			<td>H-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Vibrax VXR basic</td>
			<td>IKA</td>
			<td>0002819000</td>
			<td></td>
		</tr>
		<tr>
			<td>VX 7 Dish attachment for Vibrax VXR basic</td>
			<td>IKA</td>
			<td>953300</td>
			<td></td>
		</tr>
		<tr>
			<td>Wild TYP 355110 (Stereomicroscope)</td>
			<td>Wild Heerbrugg</td>
			<td></td>
			<td>not available anymore</td>
		</tr>
	</tbody>
</table>

immunolabeling and counting ribbon synapses in young adult and aged gerbil cochleae

The loss of ribbon synapses connecting inner hair cells and afferent auditory nerve fibers is assumed to be one cause of age-related hearing loss. The most common method for detecting the loss of ribbon synapses is immunolabeling because it allows for quantitative sampling from several tonotopic locations in an individual cochlea. However, the structures of interest are buried deep inside the bony cochlea. Gerbils are used as an animal model for age-related hearing loss. Here, routine protocols for fixation, immunolabeling gerbil cochlear whole mounts, confocal imaging, and quantifying ribbon synapse numbers and volumes are described. Furthermore, the particular challenges associated with obtaining good material from valuable aging individuals are highlighted. 
Gerbils are euthanized and either perfused cardiovascularly, or their tympanic bullae are carefully dissected out of the skull. The cochleae are opened at the apex and base and directly transferred to the fixative. Irrespective of the initial method, the cochleae are postfixed and subsequently decalcified. The tissue is then labeled with primary antibodies against pre- and postsynaptic structures and hair cells. Next, the cochleae are incubated with secondary fluorescence-tagged antibodies that are specific against their respective primary ones. The cochleae of aged gerbils are then treated with an autofluorescence quencher to reduce the typically substantial background fluorescence of older animals' tissues. 
Finally, cochleae are dissected into 6-11 segments. The entire cochlear length is reconstructed such that specific cochlear locations can be reliably determined between individuals. Confocal image stacks, acquired sequentially, help visualize hair cells and synapses at the chosen locations. The confocal stacks are deconvolved, and the synapses are either counted manually using ImageJ, or more extensive quantification of synaptic structures is carried out with image analysis procedures custom-written in Matlab.

Cochleae were either harvested after cardiovascular perfusion with fixative of the whole animal or rapidly dissected after euthanizing the animal and immersion-fixed. With the latter method, the IHCs stayed in place during dissection, whereas, in cases of unsuccessful perfusion and thus insufficiently fixed tissue, the sensory epithelium was often destroyed. Note that the authors encountered cases where fixation of the cochleae after transcardial perfusion was insufficient while fixation of the brain was still adequate. ...

Watch this Scientific Journal Video about Immunolabeling and Counting Ribbon Synapses in Young Adult and Aged Gerbil Cochleae at JoVE.com

Immunolabeling and Counting Ribbon Synapses in Young Adult and Aged Gerbil Cochleae

A protocol for processing young adult and aged gerbil cochleae by immunolabeling the afferent synaptic structures and hair cells, quenching autofluorescence in aged tissue, dissecting and estimating the length of the cochleae, and quantifying the synapses in image stacks obtained with confocal imaging is presented.

The loss of ribbon synapses connecting inner hair cells and afferent auditory nerve fibers is assumed to be one cause of age-related hearing loss. The ...

immunolabeling-counting-ribbon-synapses-young-adult-aged-gerbil

Research

JoVE Journal

Neuroscience

2.4K Views.  Carl von Ossietzky University Oldenburg. A protocol for processing young adult and aged gerbil cochleae by immunolabeling the afferent synaptic structures and hair cells, quenching autofluorescence in aged tissue, dissecting and estimating the length of the cochleae, and quantifying the synapses in image stacks obtained with confocal imaging is presented.

Video: Immunolabeling and Counting Ribbon Synapses in Young Adult and Aged Gerbil Cochleae

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect neuronal process outgrowth, shRNA or cDNA constructs can be introduced into primary neurons via chemical transfection or viral transduction. However, with primary cortical cells, a heterogeneous pool of cell types (glutamatergic neurons from different layers, inhibitory neurons, glial cells) are transfected using these methods. The use of in utero electroporation to introduce DNA constructs in the embryonic rodent cortex allows for certain subsets of cells to be targeted: while electroporation of early embryonic cortex targets deep layers of the cortex, electroporation at late embryonic timepoints targets more superficial layers. Further, differential placement of electrodes across the heads of individual embryos results in the targeting of dorsal-medial versus ventral-lateral regions of the cortex. Following electroporation, transfected cells can be dissected out, dissociated, and plated in vitro for quantitative analysis of neurite outgrowth. Here, we provide a step-by-step method to quantitatively measure neuronal process outgrowth in subsets of cortical cells.
The basic protocol for in utero electroporation has been described in detail in two other JoVE articles from the Kriegstein lab 1, 2. We will provide an overview of our protocol for in utero electroporation, focusing on the most important details, followed by a description of our protocol that applies in utero electroporation to the study of gene function in neuronal process outgrowth.

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In utero electroporation is a valuable method for transfecting neuronal progenitor cells in vivo. Depending upon the placement of the electrodes and the developmental timepoint of electroporation, certain subsets of cortical cells can be targeted. Targeted cells can then be analyzed in vivo or in vitro for effects of genetic alteration.

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect ...

Kinematics and Ground Reaction Force Determination: A Demonstration Quantifying Locomotor Abilities of Young Adult, Middle-aged, and Geriatric Rats

Behavior, in its broadest definition, can be defined as the motor manifestation of physiologic processes. As such, all behaviors manifest through the motor system. In the fields of neuroscience and orthopedics, locomotion is a commonly evaluated behavior for a variety of disease models. For example, locomotor recovery after traumatic injury to the nervous system is one of the most commonly evaluated behaviors 1-3. Though locomotion can be evaluated using a variety of endpoint measurements (e.g. time taken to complete a locomotor task, etc), semiquantitative kinematic measures (e.g. ordinal rating scales (e.g. Basso Beattie and Bresnahan locomotor (BBB) rating scale, etc)) and surrogate measures of behaviour (e.g. muscle force, nerve conduction velocity, etc), only kinetics (force measurements) and kinematics (measurements of body segments in space) provide a detailed description of the strategy by which an animal is able to locomote 1. Though not new, kinematic and kinetic measurements of locomoting rodents is now more readily accessible due to the availability of commercially available equipment designed for this purpose. Importantly, however, experimenters need to be very familiar with theory of biomechanical analyses and understand the benefits and limitations of these forms of analyses prior to embarking on what will become a relatively labor-intensive study. The present paper aims to describe a method for collecting kinematic and ground reaction force data using commercially available equipment. Details of equipment and apparatus set-up, pre-training of animals, inclusion and exclusion criteria of acceptable runs, and methods for collecting the data are described. We illustrate the utility of this behavioral analysis technique by describing the kinematics and kinetics of strain-matched young adult, middle-aged, and geriatric rats.

Locomotion is often examined as a behavioural outcome in various models of disease in fields such as neuroscience and orthopedics. This video paper intends to describe a method for collecting ground reaction forces and kinematics from rats during unrestrained locomotion.

Behavior, in its broadest definition, can be defined as the motor manifestation of physiologic processes.  As such, all behaviors manifest through the ...

Targeted Training of Ultrasonic Vocalizations in Aged and Parkinsonian Rats

Voice deficits are a common complication of both Parkinson disease (PD) and aging; they can significantly diminish quality of life by impacting communication abilities. 1, 2 Targeted training (speech/voice therapy) can improve specific voice deficits,3, 4 although the underlying mechanisms of behavioral interventions are not well understood. Systematic investigation of voice deficits and therapy should consider many factors that are difficult to control in humans, such as age, home environment, age post-onset of disease, severity of disease, and medications. The method presented here uses an animal model of vocalization that allows for systematic study of how underlying sensorimotor mechanisms change with targeted voice training. The ultrasonic recording and analysis procedures outlined in this protocol are applicable to any investigation of rodent ultrasonic vocalizations.
The ultrasonic vocalizations of rodents are emerging as a valuable model to investigate the neural substrates of behavior.5-8 Both rodent and human vocalizations carry semiotic value and are produced by modifying an egressive airflow with a laryngeal constriction.9, 10 Thus, rodent vocalizations may be a useful model to study voice deficits in a sensorimotor context. Further, rat models allow us to study the neurobiological underpinnings of recovery from deficits with targeted training.
To model PD we use Long-Evans rats (Charles River Laboratories International, Inc.) and induce parkinsonism by a unilateral infusion of 7 &mu;g of 6-hydroxydopamine (6-OHDA) into the medial forebrain bundle which causes moderate to severe degeneration of presynaptic striatal neurons (for details see Ciucci, 2010).11, 12 For our aging model we use the Fischer 344/Brown Norway F1 (National Institute on Aging).
Our primary method for eliciting vocalizations is to expose sexually-experienced male rats to sexually receptive female rats. When the male becomes interested in the female, the female is removed and the male continues to vocalize. By rewarding complex vocalizations with food or water, both the number of complex vocalizations and the rate of vocalizations can be increased (Figure 1).
An ultrasonic microphone mounted above the male's home cage records the vocalizations. Recording begins after the female rat is removed to isolate the male calls. Vocalizations can be viewed in real time for training or recorded and analyzed offline. By recording and acoustically analyzing vocalizations before and after vocal training, the effects of disease and restoration of normal function with training can be assessed. This model also allows us to relate the observed behavioral (vocal) improvements to changes in the brain and neuromuscular system.

Voice disorders are debilitating in aging and Parkinson disease. The ultrasonic vocalizations of rats, also affected by these conditions, can be used to study these voice disorders, their neural substrates, and the nature of functional recovery with behavioral intervention.

Voice deficits are a common complication of both Parkinson disease (PD) and aging; they can significantly diminish quality of life by impacting ...

Cerebrovascular Casting of the Adult Mouse for 3D Imaging and Morphological Analysis

Vascular imaging is crucial in the clinical diagnosis and management of cerebrovascular diseases, such as brain arteriovenous malformations (BAVMs). Animal models are necessary for studying the etiopathology and potential therapies of cerebrovascular diseases. Imaging the vasculature in large animals is relatively easy. However, developing vessel imaging methods of murine brain disease models is desirable due to the cost and availability of genetically-modified mouse lines. Imaging the murine cerebral vascular tree is a challenge. In humans and larger animals, the gold standard for assessing the angioarchitecture at the macrovascular (conductance) level is x-ray catheter contrast-based angiography, a method not suited for small rodents.
In this article, we present a method of cerebrovascular casting that produces a durable skeleton of the entire vascular bed, including arteries, veins, and capillaries that may be analyzed using many different modalities. Complete casting of the microvessels of the mouse cerebrovasculature can be difficult; however, these challenges are addressed in this step-by-step protocol. Through intracardial perfusion of the vascular casting material, all vessels of the body are casted. The brain can then be removed and clarified using the organic solvent methyl salicylate. Three dimensional imaging of the brain blood vessels can be visualized simply and inexpensively with any conventional brightfield microscope or dissecting microscope. The casted cerebrovasculature can also be imaged and quantified using micro-computed tomography (micro-CT)1. In addition, after being imaged, the casted brain can be embedded in paraffin for histological analysis.
The benefit of this vascular casting method as compared to other techniques is its broad adaptation to various analytic tools, including brightfield microscopic analysis, CT scanning due to the radiopaque characteristic of the material, as well as histological and immunohistochemical analysis. This efficient use of tissue can save animal usage and reduce costs. We have recently demonstrated application of this method to visualize the irregular blood vessels in a mouse model of adult BAVM at a microscopic level2, and provide additional images of the malformed vessels imaged by micro-CT scan. Although this method has drawbacks and may not be ideal for all types of analyses, it is a simple, practical technique that can be easily learned and widely applied to vascular casting of blood vessels throughout the body.

In this article, we present a simple, practical technique for cerebrovascular casting that is easy to perform and can be utilized to image the vascular tree of the adult mouse brain.

Vascular imaging is crucial in the clinical diagnosis and management of cerebrovascular diseases, such as brain arteriovenous malformations (BAVMs). ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

Susceptibility to neuron cell death associated to neurodegeneration and ischemia are exceedingly increased in the aged brain but mechanisms responsible are badly known. Excitotoxicity, a process believed to contribute to neuron damage induced by both insults, is mediated by activation of glutamate receptors that promotes Ca2+ influx and mitochondrial Ca2+ overload. A substantial change in intracellular Ca2+ homeostasis or remodeling of intracellular Ca2+ homeostasis may favor neuron damage in old neurons. For investigating Ca2+ remodeling in aging we have used live cell imaging in long-term cultures of rat hippocampal neurons that resemble in some aspects aged neurons in vivo. For this end, hippocampal cells are, in first place, freshly dispersed from new born rat hippocampi and plated on poli-D-lysine coated, glass coverslips. Then cultures are kept in controlled media for several days or several weeks for investigating young and old neurons, respectively. Second, cultured neurons are loaded with fura2 and subjected to measurements of cytosolic Ca2+ concentration using digital fluorescence ratio imaging. Third, cultured neurons are transfected with plasmids expressing a tandem of low-affinity aequorin and GFP targeted to mitochondria. After 24 hr, aequorin inside cells is reconstituted with coelenterazine and neurons are subjected to bioluminescence imaging for monitoring of mitochondrial Ca2+ concentration. This three-step procedure allows the monitoring of cytosolic and mitochondrial Ca2+ responses to relevant stimuli as for example the glutamate receptor agonist NMDA and compare whether these and other responses are influenced by aging. This procedure may yield new insights as to how aging influence cytosolic and mitochondrial Ca2+ responses to selected stimuli as well as the testing of selected drugs aimed at preventing neuron cell death in age-related diseases.

Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

Intracellular Ca2+ remodeling in aging may contribute to excitotoxicity and neuron damage, processes mediated by Ca2+ overload. We aimed at investigating Ca2+ remodeling in the aging brain using fluorescence and bioluminescence imaging of cytosolic and mitochondrial Ca2+ in long-term cultures of rat hippocampal neurons, a model of neuronal aging.

Susceptibility to neuron cell death associated to neurodegeneration and ischemia are exceedingly increased in the aged brain but mechanisms responsible ...

High-Resolution Quantitative Immunogold Analysis of Membrane Receptors at Retinal Ribbon Synapses

Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA receptors (NMDARs) and AMPA receptors (AMPARs). Physiological data have indicated that glutamate receptors at RGCs are expressed not only in postsynaptic but also in perisynaptic or extrasynaptic membrane compartments. However, precise anatomical locations for glutamate receptors at RGC synapses have not been determined. Although a high-resolution quantitative analysis of glutamate receptors at central synapses is widely employed, this approach has had only limited success in the retina. We developed a postembedding immunogold method for analysis of membrane receptors, making it possible to estimate the number, density and variability of these receptors at retinal ribbon synapses. Here we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of retinal fixation, 2) freeze-substitution, 3) postembedding immunogold electron microscope (EM) immunocytochemistry and, 4) quantitative visualization of glutamate receptors at ribbon synapses.

The postembedding immunogold method is one of the most effective ways to provide high-resolution analyses of the subcellular localization of specific molecules. Here we describe a protocol to quantitatively analyze glutamate receptors at retinal ribbon synapses.

Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA ...

Combined Optogenetic and Freeze-fracture Replica Immunolabeling to Examine Input-specific Arrangement of Glutamate Receptors in the Mouse Amygdala

Freeze-fracture electron microscopy has been a major technique in ultrastructural research for over 40 years. However, the lack of effective means to study the molecular composition of membranes produced a significant decline in its use. Recently, there has been a major revival in freeze-fracture electron microscopy thanks to the development of effective ways to reveal integral membrane proteins by immunogold labeling. One of these methods is known as detergent-solubilized Freeze-fracture Replica Immunolabeling (FRIL).
The combination of the FRIL technique with optogenetics allows a correlated analysis of the structural and functional properties of central synapses. Using this approach it is possible to identify and characterize both pre- and postsynaptic neurons by their respective expression of a tagged channelrhodopsin and specific molecular markers. The distinctive appearance of the postsynaptic membrane specialization of glutamatergic synapses further allows, upon labeling of ionotropic glutamate receptors, to quantify and analyze the intrasynaptic distribution of these receptors. Here, we give a step-by-step description of the procedures required to prepare paired replicas and how to immunolabel them. We will also discuss the caveats and limitations of the FRIL technique, in particular those associated with potential sampling biases. The high reproducibility and versatility of the FRIL technique, when combined with optogenetics, offers a very powerful approach for the characterization of different aspects of synaptic transmission at identified neuronal microcircuits in the brain.
Here, we provide an example how this approach was used to gain insights into structure-function relationships of excitatory synapses at neurons of the intercalated cell masses of the mouse amygdala. In particular, we have investigated the expression of ionotropic glutamate receptors at identified inputs originated from the thalamic posterior intralaminar and medial geniculate nuclei. These synapses were shown to relay sensory information relevant for fear learning and to undergo plastic changes upon fear conditioning.

This article illustrates how the expression of neurotransmitter receptors can be quantified and the pattern analyzed at synapses with identified pre and postsynaptic elements using a combination of viral transduction of optogenetic tools and the freeze-fracture replica immunolabeling technique.

Freeze-fracture electron microscopy has been a major technique in ultrastructural research for over 40 years. However, the lack of effective means to ...

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.

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 ...

Morphological and Functional Evaluation of Axons and their Synapses during Axon Death in Drosophila melanogaster

Axon degeneration is a shared feature in neurodegenerative disease and when nervous systems are challenged by mechanical or chemical forces. However, our understanding of the molecular mechanisms underlying axon degeneration remains limited. Injury-induced axon degeneration serves as a simple model to study how severed axons execute their own disassembly (axon death). Over recent years, an evolutionarily conserved axon death signaling cascade has been identified from flies to mammals, which is required for the separated axon to degenerate after injury. Conversely, attenuated axon death signaling results in morphological and functional preservation of severed axons and their synapses. Here, we present three simple and recently developed protocols that allow for the observation of axonal morphology, or axonal and synaptic function of severed axons that have been cut-off from the neuronal cell body, in the fruit fly Drosophila. Morphology can be observed in the wing, where a partial injury results in axon death side-by-side of uninjured control axons within the same nerve bundle. Alternatively, axonal morphology can also be observed in the brain, where the whole nerve bundle undergoes axon death triggered by antennal ablation. Functional preservation of severed axons and their synapses can be assessed by a simple optogenetic approach coupled with a post-synaptic grooming behavior. We present examples using a highwire loss-of-function mutation and by over-expressing dnmnat, both capable of delaying axon death for weeks to months. Importantly, these protocols can be used beyond injury; they facilitate the characterization of neuronal maintenance factors, axonal transport, and axonal mitochondria.

Morphological and Functional Evaluation of Axons and their Synapses during Axon Death in Drosophila melanogaster

Here, we provide protocols to perform three simple injury-induced axon degeneration (axon death) assays in Drosophila melanogaster to evaluate the morphological and functional preservation of severed axons and their synapses.

Axon degeneration is a shared feature in neurodegenerative disease and when nervous systems are challenged by mechanical or chemical forces. However, our ...