Name: immunolabeling and counting ribbon synapses in young adult and aged gerbil cochleae
Uploaded: 2022-04-21T14:15:00
Description: Watch this Scientific Journal Video about Immunolabeling and Counting Ribbon Synapses in Young Adult and Aged Gerbil Cochleae at JoVE.com

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.

All protocols and procedures were approved by the relevant authorities of Lower Saxony, Germany, with permit numbers AZ 33.19-42502-04-15/1828 and 33.19-42502-04-15/1990. This protocol is for Mongolian gerbils (M. unguiculatus) of both sexes. Young adult refers to the age of 3-12 months, while gerbils are considered aged at 36 months and older. When not stated otherwise, buffers and solutions can be prepared and stored in the fridge for up to several months (4-8 &#176;C). Before use, ensure that the buffers and ...

<ol>
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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.

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	<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 ...

This protocol enables the evaluation of the synaptic integrity in the cochlea of the gerbil of all ages. This is important for fundamental research into hearing problems, in particular, due to aging. The protocol is relatively fast and yields quantitative data from several locations along the cochlea.Furthermore, it addresses a specific problem of aging tissue by using an autofluorescence quencher. Degeneration of synapses is the earliest sign of loss of function in the cochlea. Quantifying it is an important tool in research towards developing non-invasive diagnostic tools for synaptopathy.The cochlear dissection is the most crucial part that needs practice, so it is better to start with non-experimental cochli. Begin by removing the bullae. Cut the bone with scissors or crack the bone with forceps and remove it with forceps to reach the cochlea.Remove the malleus, incus, and semicircular canals. Carefully remove the bone covering the apex of the cochlea and scratch the cochlear bone with forceps to make small holes at the apex and in the basal turn or carefully poke holes in both locations. Immediately transfer the bullae into at least 50 milliliters of cold fixative.To cut the cochli in half, position a piece of razor blade longer than the coiled length of the cochlea into a blade holder. Then, place the cochlea in a Petri dish under a stereo microscope. Cut away excess tissue with a razor blade or with fine scissors.Hold the cochlea in place with fine forceps and cut in half along the modiolus. Prepare a 5%solution of the autofluorescence quencher by mixing it with 70%ethanol. Place the cochli in this solution and incubate it for one minute at room temperature on a shaker followed by washing thrice with one milliliter of PBS for five minutes.For the dissection of the cochlea, collect two fine forceps, super-fine forceps, vannas, spring scissors, a blade holder, a razor blade, and fill polystyrol Petri dish and its lid with PBS. Break pieces from the razor blade to obtain a cutting surface of two to four millimeters. Cut the cochlea in half as shown in the section for tissue preparation.Carefully fix the cochlear half with the forceps such that the cut edge is facing upwards. Use fine spring scissors to cut away the bone of the cochlea above the helicotrema covering the apex. Then, use scissors to isolate the apical turn by cutting through the modiolus and auditory nerve and scala vestibuli.Make two cuts on each side of the cochlear bone above, or if the stria vascularis is not needed for further evaluation, directly through the stria vascularis of the middle turn. Use the razor blade to separate the cochlear pieces. Alternatively, cut between the Organ of Corti and the stria vascularis.Leave the stria vascularis connected to the spiral ligament and remove the decalcified bone using forceps. Next, transfer the cochlear pieces into a Petri dish lid filled with PBS. Remove excess tissue on the pillar and modiolar sides such that the whole mounts will later lie on the slide as flat as possible.Large cochlear pieces, in particular, those originating from the basal turn, should be cut into two pieces. Carefully remove the tectorial membrane with super-fine forceps. Place the cochlear pieces onto a slide into a drop of mounting medium, ensuring that the Organ of Corti faces up.Shift the cochlear piece into the sagittal plane and look for invagination of the spiral limbus in close proximity to the inner hair cells. This invagination of the spiral limbus can be seen more clearly in the middle and basal cochlear pieces. Transfer the entire cochlea and the entire stria vascularis onto the microscope slide by repeating these steps.Cover slip the slide and add black nail polish around the edges for sealing. Dry in the dark and store them at four degree Celsius. Open a copy of the deconvolved stacks in ImageJ software loaded with BioVoxxel plugin.Split the channels by clicking on Image, then Color, and then Split Channels. Merge them by clicking on Image, then Color, and then Merge Channels to assign different colors. Convert the image to an RGB stack by clicking on Image, then go to Color and click Stack to RGB.Adjust the brightness and contrast by clicking on Image, Adjust, and then Brightness/Contrast. Use either auto or slider to ensure that the pre-and post-synaptic structures and the inner hair cell labels are distinct from the background. Label five inner hair cells with the text tool by clicking on the desired location within the stack.Next, go to Analyze, then Tools, and select ROI Manager. Check the tick box labels to label the points with a number. Zoom in on the inner hair cell of interest.Right click on the point tool to choose Multi-point mode. Scroll through the Z dimension and count the functional ribbon synapses, which are identified by a pre-synaptic ribbon in juxtaposition to a post-synaptic glutamate patch. After selecting all the structures of interest, click Add the ROI Manager, then click on the arbitrary name and choose Rename.With the hand tool, adjust the image to fully display the next inner hair cell. Change from Multi-point tool to Point tool to avoid adding more puncta to the previously stored data. Click on a structure of interest within the next inner hair cell and change to Multi-point tool.Continue adding the functional ribbon synapse in the ROI Manager until all the inner hair cells are evaluated. Click on a data set in the ROI Manager and then on Measure. Wait for a new window to appear that lists the measured data points and click on File and Save As to save this list as a spreadsheet.Save the image by activating Show All in the ROI Manager. Click on Flatten to permanently add the point labels to the stack and click on Agree to close by saving the changes. Z stack projections from the cochlea of a young gerbil aged 10 months show that the myosin 7A-labeled inner hair cells outlined here by white dashed lines were in sharp contrast to the background.The pre-synaptic ribbons were labeled with anti-CtBP2 two shown in green, while the post-synaptic glutamate patches were labeled with anti-GluA2 shown in red. The cochlea from an old gerbil aged 38 months was treated with the autofluorescence quencher and showed distinct inner hair cells with clearly visible pre-and post-synaptic structures. To test the stability of the immunolabeling, the same piece of cochlea from the young gerbil was imaged after three months and 29 1/2 months.In spite of the reduced signal-to-noise ratio, the functional synapses were distinct and quantifiable. Even if a cut fails, mount the remaining piece of tissue to later estimate the full cochlear length most accurately. The data from this protocol are particularly valuable if preceded by, and then individually correlated with, electrophysiological or psychophysical hearing tests.

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