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. 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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 antibody against otoferlin, with OHCs appearing much fainter than IHCs.
Perfusion may be carried out using two different setups. 1) A gravity-fed drip-line system in which a single bottle feeding a commercial drip line is suspended on a pulley wheel approximately 1.5 m above the animal. Fluids are introduced successively after lowering the bottle, which is open at the top. 2) A system using a digital variable-speed peristaltic pump, with a thin, long tube open at one end to take in the fluid and a needle that can be easily attached at the other end. Both work equally well, and the subtle differences will not be elaborated upon here. The authors specifically recommend, however, a down draft-style workbench, with the animal on a perforated platform and the fumes drawn off below. This enables good access to the surgical area without compromising the exhaust function (unlike working in an open fume cabinet).
Proper fixation of the tissue is of critical importance, since otherwise the sensory epithelium will detach and disintegrate during dissection. In the gerbil, more prolonged exposure to the fixative is necessary than commonly used (e.g., for mice17,27,13 or guinea pigs28). The preferred method for gerbils is rapid extraction of the cochlea after the animal&#39;s death and immersion-fixation for at least 1.5 days. If cardiovascular perfusion is preferred, it is crucial that fixation sets in within a few minutes and proceeds well. Since it can be difficult to ultimately rate the quality of fixation during perfusion, it is recommended to routinely postfix the cochleae as described.
It is important to comply with the washing steps, whereby the last washing step (step 2.7) is the most important one. If not adequately washed, the tissue is sticky and adheres to the dissection instruments, which makes the dissection difficult. It is also recommended to use an autofluorescence quencher in cochleae harvested from aged gerbils to reduce nonspecific fluorescence. Autofluorescence might originate from lipofuscin, which is common in tissue from aged animals29,30,31, and appears to be broadly excited as well as broadly emitting. When excited with a wavelength in the UV -spectrum (&#955; = 364 nm), lipofuscin has a broad emission range (&#955; = 400-700 nm, with a maximum at ~&#955; = 568 nm)32. In human myocardial tissue, lipofuscin is visible with an excitation of &#955; = 555 nm and emission of &#955; = 605 nm33. Similarly, in the IHCs of gerbils, the authors noticed an increase in nonspecific fluorescence most prominently in the channel used for the AF568 antibody, which suggests autofluorescence from lipofuscin granules. A general recommendation when working with tissue from animals is thus to use the excitation bandwidth around 550-600 nm for the least critical immunolabel.
The measurement of total cochlear length and the correct identification of specific cochlear positions is only possible if its entire length is preserved. If parts of the sensory epithelium are lost in dissection, it is recommended to still mount the remaining part or even only the spiral ganglion piece, and keep detailed notes. It is then usually possible to estimate the missing section with reasonable accuracy. If the apical portions of the cochlea are completely preserved, but the basal end is missing, specific cochlear locations on the apical part might be definable by calculating the positions based on the median cochlear length within the used gerbil population because the deviation from the real value is small.
An important limitation of the synapse volume quantification is that the resulting absolute volumes are not comparable across different confocal stacks. The critical step that determines the resulting volumes of synaptic elements is the choice of the intensity threshold for initial detection. However, the choice of this intensity threshold is made subjectively by the user in the current protocol and many others3,27,34. Furthermore, the brightness of the immunofluorescence in the confocal image depends on many factors, which are very difficult to standardize across specimens, such as tissue thickness, orientation of tissue relative to the optical path, duration of laser exposure, and precise confocal and deconvolution settings. All these caveats are exacerbated if rare material is acquired over a considerable length of time, as is typical for aging gerbils. Together, this means that biases are very difficult to exclude in pooled data, and the best-case scenario is a dataset with high variance. It is thus recommended to routinely normalize synaptic volumes to the respective median volume of the individual confocal stack, as introduced by Liberman et al.3. The obvious downside is that synaptic volumes are then only comparable within a given image stack, e.g., between different locations on the IHC but not between different cochlear locations or specimen ages.
The dual labeling of pre- and postsynaptic structures and the requirement of co-localization allows for a more reliable estimate of the number of functional synapses than using either label alone. If only one label is feasible, it is recommended to use the presynaptic anti-CtBP2, coupled to AF488 or fluorescent tags with similar wavelength specifics, for two reasons. First, orphaned ribbons, that is, CtBP2-labeled elements without a co-localized postsynaptic label, appear to be rare17, and the authors confirmed this for aged gerbils. Thus, the error introduced by not being able to verify co-localization with a postsynaptic partner is small. It should be noted, however, that this becomes a more significant issue when examining noise-exposed ears (e.g.,28). Second, in cases with high fluorescence signal of unclear origin, the noise typically affected the channel using an excitation wavelength around 568 nm to the greatest extent (example in Figure 2B, representing the GluA2 label). At least part of this noise may, in tissue from aged animals, be lipofuscin autofluorescence. Thus, to maximize the chance for a clean, single anti-CtBP2 label, it is advisable to avoid this wavelength channel. Finally, it was shown that, with proper cool and dark storage conditions, the immunolabeling used here is stable over long periods of time, up to at least 2.5 years (Figure 3B), which makes the re-evaluation of valuable tissue, such as from aged gerbils, possible.
The quantification of IHC afferent synapse number has become an important metric for evaluating the state of the peripheral auditory system in many contexts, among them age-related hearing loss. There are various protocols published now (e.g., gerbils21, mice17,13, guinea pigs10,28, and humans35). The method outlined here is, in its details, specific to young adult and aged gerbils but some general recommendations have been derived that may be of use in other species too.

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><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 &amp;amp; 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>&lt;strong&gt;Phosphate-buffered saline:&lt;/strong&gt;</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&amp;nbsp; 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. Tissue from an inferior perfusion could still be saved by opening holes into the cochlear apex and base and postfixing the cochleae by immersion in 4% PFA for 2 days.
Length determination of the whole cochlea was conducted according to M&#252;ller25 to evaluate IHCs and their synapses at specific cochlear positions, equivalent to distinct characteristic frequencies (Table 1). For this, the desired cochlear positions were calculated as percentages of basilar membrane length from the cochlear base. The median cochlear length of 13 cochleae from young adult gerbils was 11.3 mm (interquartile range: 11.02-11.52 mm). The median cochlear length of 24 cochleae from aged gerbils was 11.5 mm (interquartile range: 11.24-11.73 mm). There was no significant difference between the length of young adult and aged cochleae (Mann-Whitney U-test: U = -1.62, p = 0.105); the overall median was 11.48 mm. One may argue that this variation in the length of individual cochleae was small and using fixed length positions (in mm) is adequate. However, the place-frequency function is non-linear. Therefore, a deviation from the median length causes a larger error for basal cochlear locations than for relatively more apical cochlear locations. For example, for the cochlear location equivalent to a frequency of 1 kHz, calculated based on the median cochlear length (i.e., 11.48 mm), the corresponding frequency ranged from 1.16 kHz to 0.91 kHz for the shortest (10.36 mm) and the longest cochlea (12.28 mm) in this sample, respectively. For a basally located frequency (e.g., 32 kHz), the frequency range was 51.62 kHz to 23.97 kHz, for the shortest and longest cochlea, respectively. Therefore, it is advisable to calculate the individual locations on each cochlea, in particular when examining basal cochlear locations.
Figure 1 depicts maximum intensity z-dimension projections of cochleae from a young adult (10 months, Figure 1A) and an aged gerbil (38 months, Figure 1B), which are examples of ideal immunolabels. In the image depicting the young adult gerbil&#39;s cochlea, the myoVIIa-labeled IHCs, here shown in blue, are in sharp contrast to the black background. Therefore, individual IHCs are easily detectable. The pre- and postsynaptic structures are clearly visible as green and red elements, respectively. They are assumed to form a functional synapse whenever they are in close juxtaposition (Figure 1A&#39;,B&#39;). There were rarely unpaired presynaptic structures (orphaned ribbons) apparent in the cochleae of aged gerbils. The cochlea from the old gerbil (Figure 1B) was treated with the autofluorescence quencher. The IHC label appears weaker, although the laser power used in this specimen was three times higher than that used for the cochlea from the young adult gerbil. Nevertheless, the IHCs are still distinct from the background. The pre-and postsynaptic structures are clearly visible, and the laser power used for both those channels was similar or even below that for the young adult specimen. Tissue from aged animals typically showed significantly more nonspecific-looking fluorescent signal. Therefore, the main difference in the protocol for young adult and aged material is the treatment with an autofluorescence quencher. Note that this was carried out after the immunostaining with fluorescence-tagged antibodies and might, in principle, also affect the intended antibody label. However, the treatment effectively reduced extraneous fluorescence of nonspecific origin, while leaving sufficient signal of the specific label of the structures of interest (compare Figure 1B with Figure 2B). Preliminary results indicated, however, that, in the stria vascularis, extraneous fluorescence did not manifest in samples from aged gerbils.
Examples of stacks that were suboptimally processed are shown in Figure 2. Figure 2A depicts the maximum-intensity z-projection of a stack from an old gerbil (36 months), where the IHCs, which were also either unnaturally bent or ripped apart, were not scanned in their entirety. As a result, only the apical and basal poles of IHCs are visible in this scan. It cannot be excluded that more synapses were located in the missing middle part of the IHCs and, therefore, a reliable analysis is not possible. Thus, it is crucial that all evaluated IHCs are scanned completely in the confocal stack. Figure 2B shows the maximum-intensity z-projection of a stack sampled in an aged gerbil (38 months). The fluorescence of unclear origin is high as the autofluorescence quencher was not used in this case. However, the extraneous fluorescence was largely confined to the (red) channel associated with the GluA2-label, which is quite typical. In such cases, it may still be possible to count functional synapses if the CtBP2-label of the ribbons is relatively clean and specific. Figure 2C depicts the maximum-intensity z-projection of a stack obtained from an aged gerbil (42 months). Here, the synapses cannot be allocated to individual IHCs as the IHCs were largely disintegrated; indeed, it is difficult to be sure how many IHCs are represented. In the example shown in Figure 3A, a mobile phone was used in close vicinity to the confocal microscope during the scan. Stripes are visible in the blue channel (IHC label). Fortunately, in this case, this did not affect the synaptic channels and affected only the upper part of the IHCs, thus, an analysis was still viable.
Figure 3 displays maximum-intensity z-projections of stacks taken from the same piece of cochlea from a young adult gerbil, acquired 3 months (Figure 3A,A&#39;) and 29.5 months (Figure 3B,B&#39;) after immunostaining. To remain comparable, the images were not taken from the exact same location (which might have suffered bleaching from the previous scan) but from the same cochlear piece. For the scan taken at the later time point, the laser power had to be increased ~2- to 3-fold. Nevertheless, the labeled structures are still clear. The signal-to-noise ratio had decreased, especially in the channels displaying postsynaptic and IHC structures, while the channel with the presynaptic label was less affected. Quantification of functional synapses per IHC is still possible.
Typical results for the analysis of synapse volume and position on the IHC are illustrated in Figure 4, using the confocal stack shown in Figure 1A. Individually defined IHCs are graphically displayed as grid reconstructions in different colors, with or without the functional synapses (defined by colocalization of pre- and postsynaptic labels) allocated to each. This display can be freely rotated in 3D to obtain various viewing angles (Figure 4A,B). IHCs can also be singled out for graphical display (Figure 4B). A large variety of data graphs, illustrating different aspects of the distribution of synaptic volumes within the IHC-centered coordinate system, is produced (examples in Figure 4C-E). The raw quantitative data for each IHC and each synaptic element are also available as spreadsheets which can then, for example, be combined across several confocal stacks for further statistical analysis.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63874/63874fig01.jpg" /> 
Figure 1: Examples of successfully processed cochleae. Maximum-intensity z-projections of confocal stacks from (A) a young adult gerbil (10 months), obtained at a cochlear location corresponding to 1 kHz, and (B)&#160;an aged gerbil (38 months, panel B), obtained at a cochlear location equivalent to 500 Hz. IHCs were stained with an antibody against myoVIIa (blue), presynaptic ribbons were labeled with anti-CtBP2 (green), and postsynaptic glutamate patches were labeled with anti-GluA2 (red). The cochlea of the aged gerbil was treated with an autofluorescence quencher after the immunostaining. For clarity, the IHCs&#39; outlines are indicated by dashed lines. Panels (A&#39;) and (B&#39;) display an enlargement of the areas outlined by the squares in the corresponding cochleae from panels (A) and (B). Yellow arrowheads point to functional synapses. The channels displaying the pre- and postsynaptic structures (but not the IHC channel) underwent deconvolution. Scale bars = 10 &#181;m (A,B), 1 &#181;m (A&#39;,B&#39;). Abbreviations: IHC = inner hair cell; myoVIIa = myosin VIIa; CtBP2 = C-terminal binding protein 2; GluA2 = ionotropic glutamate receptor. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63874/63874fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63874/63874fig02.jpg" /> 
Figure 2: Examples of suboptimally processed cochleae. Maximum-intensity z-projections of confocal stacks for which the processing was suboptimal, from gerbils that were (A)&#160;36 months and (B, C) 38 months old and from cochlear locations corresponding to 16 kHz, 8 kHz, and 32 kHz, respectively. Gerbils from which the cochleae in panels (A) and (C) were derived were transcardially perfused, and the cochlea from panel (C) was additionally postfixed for 3 days in 4% PFA. The cochlea from (B) was immersion-fixed in 4% PFA for 2 days. Cochleae from (A) and (C) were treated with the autofluorescence quencher. IHCs were stained with an antibody against myoVIIa (blue), presynaptic ribbons were labeled with anti-CtBP2 (green), and postsynaptic glutamate patches were labeled with anti-GluA2 (red). All channels were deconvolved. Brightness and contrast were further adjusted after the confocal scan. Scale bars = 10 &#181;m. Abbreviations: PFA = paraformaldehyde; IHC = inner hair cell; myoVIIa = myosin VIIa; CtBP2 = C-terminal binding protein 2; GluA2 = ionotropic glutamate receptor. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63874/63874fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63874/63874fig03.jpg" /> 
Figure 3: Stability of immunolabel after prolonged storage. Maximum-intensity z-projections of confocal stacks from a young adult gerbil (10 months), taken at the 16 kHz cochlear location, (A) 3 months after staining and (B) at a slightly more basal cochlear location on the same cochlear piece 29.5 months after staining. The cochlea was immersion-fixed in 4% PFA for 2 days. For clarity, (A&#39;) and (B&#39;) zoom in on only a few functional synapses from the areas indicated by squares in (A) and (B), respectively. IHCs were stained with an antibody against myoVIIa (blue), presynaptic ribbons were labeled with anti-CtBP2 (green), and postsynaptic glutamate patches were labeled with anti-GluA2 (red). The laser power for the IHC channels was 1.3% and 3%, for the presynaptic channels 0.4% and 1.1%, and for the postsynaptic channels 1.1% and 2% in (A) and (B), respectively. Note that in (A), blue stripes are visible in the upper part of the IHCs, which result from the use of a mobile phone in close vicinity to the confocal microscope. Scale bars = 10 &#181;m (A, B), 1 &#181;m (A&#39;, B&#39;). Abbreviations: PFA = paraformaldehyde; IHC = inner hair cell; myoVIIa = myosin VIIa; CtBP2 = C-terminal binding protein 2; GluA2 = ionotropic glutamate receptor. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63874/63874fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63874/63874fig04.jpg" /> 
Figure 4: Representative results of the quantification of synapse volume. (A) Ten IHCs shown as differently colored grid reconstructions based on the myoVIIa immunolabel. Their associated functional synapses, defined by colocalized CtBP2-(green) and GluA2-labeled elements (red), are displayed together with the IHCs, and also separately below, for clarity. Note that the angle of view was chosen to be perpendicular to the IHCs&#39; long axis and thus differs from the original confocal image (Figure 1A). (B) One of the IHCs singled out and shown rotated 90&#176;. The black plane was manually defined by the user and bisects the IHC along its pillar-modiolar axis. (C) Bubble plot showing the location of all functional synapses in this confocal stack, relative to the normalized three axes of their respective IHC. size of the symbols is proportional to the volume of the presynaptic element. Note that the angle of view was chosen to be similar to panel (B). (D) Boxplot of the normalized volumes of synaptic elements, separately for the presynaptic (left 2 boxes) and postsynaptic (right 2 boxes) partners of functional synapses and further separated according to their position in the modiolar or pillar half of their respective IHC (different colors). Boxes represent interquartile ranges, with the medians indicated by lines. Dashed whiskers indicate 1.5 times the interquartile range, and the crosses indicate outlying values beyond that. (E) Scatterplot of the normalized volumes of pre- vs. postsynaptic partners of functional synapses. Different symbols indicate the position in the modiolar or pillar half of their respective IHC. Abbreviations: IHC = inner hair cell; myoVIIa = myosin VIIa; CtBP2 = C-terminal binding protein 2; GluA2 = ionotropic glutamate receptor. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63874/63874fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: Distances from the apex that are equivalent to specific target frequencies in cochleae of different lengths. The equivalent best frequencies were calculated based on the equation given by M&#252;ller25. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63874/Table1_Steenken et al.xlsx" target="_blank">Please click here to download this Table.</a>

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

Universidad San Sebastian

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

DiOLISTIC Labeling of Neurons from Rodent and Non-human Primate Brain Slices

DiOLISTIC staining uses the gene gun to introduce fluorescent dyes, such as DiI, into neurons of brain slices (Gan et al., 2009; O'Brien and Lummis, 2007; Gan et al., 2000). Here we provide a detailed description of each step required together with exemplary images of good and bad outcomes that will help when setting up the technique. In our experience, a few steps proved critical for the successful application of DiOLISTICS. These considerations include the quality of the DiI-coated bullets, the extent of fixative exposure, and the concentration of detergent used in the incubation solutions. Tips and solutions for common problems are provided. This is a versatile labeling technique that can be applied to multiple animal species at a wide range of ages. Unlike other fluorescent labeling techniques that are limited to preparations from young animals or restricted to mice because they rely on the expression of a fluorescent transgene, DiOLISTIC labeling can be applied to animals of all ages, species and genotypes and it can be used in combination with immunostaining to identify a specific subpopulation of cells. Here we demonstrate the use of DiOLISTICS to label neurons in brain slices from adult mice and adult non-human primates with the purpose of quantifying dendrite branching and dendritic spine morphology.

We demonstrate the use of the gene gun to introduce fluorescent dyes, such as DiI, into neurons in brain slices from rodents and non-human primates of different ages. In this particular case, we use adult mice (3-6 months old) and adult cynomologus monkeys (9-15 years old). This technique, originally described by the laboratory of Dr. Lichtman (Gan et al., 2000), is well suited for the study of dendritic branching and dendritic spine morphology and can be combined with traditional immunostaining, if detergents are kept at a low concentration.

DiOLISTIC staining uses the gene gun to introduce fluorescent dyes, such as DiI, into neurons of brain slices (Gan et al., 2009; O'Brien and Lummis, 2007; ...

Quantifying Synapses: an Immunocytochemistry-based Assay to Quantify Synapse Number

One of the most important goals in neuroscience is to understand the molecular cues that instruct early stages of synapse formation. As such it has become imperative to develop objective approaches to quantify changes in synaptic connectivity. Starting from sample fixation, this protocol details how to quantify synapse number both in dissociated neuronal culture and in brain sections using immunocytochemistry. Using compartment-specific antibodies, we label presynaptic terminals as well as sites of postsynaptic specialization. We define synapses as points of colocalization between the signals generated by these markers. The number of these colocalizations is quantified using a plug in Puncta Analyzer (written by Bary Wark, available upon request, c.eroglu@cellbio.duke.edu) under the ImageJ analysis software platform. The synapse assay described in this protocol can be applied to any neural tissue or culture preparation for which you have selective pre- and postsynaptic markers. This synapse assay is a valuable tool that can be widely utilized in the study of synaptic development.

This protocol details how to quantify synapse number both in dissociated neuronal culture and in brain sections using immunocytochemistry. Using compartment-specific antibodies, we label presynaptic terminals as well as sites of postsynaptic specialization. We define synapses as points of colocalization between the signals generated by these markers.

One of the most important goals in neuroscience is to understand the molecular cues that instruct early stages of synapse formation. As such it has become ...

Postsynaptic Recordings at Afferent Dendrites Contacting Cochlear Inner Hair Cells: Monitoring Multivesicular Release at a Ribbon Synapse

The afferent synapse between the inner hair cell (IHC) and the auditory nerve fiber provides an electrophysiologically accessible site for recording the postsynaptic activity of a single ribbon synapse 1-4. Ribbon synapses of sensory cells release neurotransmitter continuously, the rate of which is modulated in response to graded changes in IHC membrane potential 5. Ribbon synapses have been shown to operate by multivesicular release, where multiple vesicles can be released simultaneously to evoke excitatory postsynaptic currents (EPSCs) of varying amplitudes 1, 4, 6-11. Neither the role of the presynaptic ribbon, nor the mechanism underlying multivesicular release is currently well understood.
The IHC is innervated by 10-20 auditory nerve fibers, and every fiber contacts the IHC with a unmyelinated single ending to form a single ribbon synapse. The small size of the afferent boutons contacting IHCs (approximately 1 &mu;m in diameter) enables recordings with exceptional temporal resolution to be made. Furthermore, the technique can be adapted to record from both pre- and postsynaptic cells simultaneously, allowing the transfer function at the synapse to be studied directly 2. This method therefore provides a means by which fundamental aspects of neurotransmission can be studied, from multivesicular release to the elusive function of the ribbon in sensory cells.

Whole-cell patch-clamp recordings from auditory nerve fiber dendrites at the inner hair cell ribbon synapse in the mammalian cochlea.

The afferent synapse between the inner hair cell (IHC) and the auditory nerve fiber provides an electrophysiologically accessible site for recording the ...

Immunohistochemistry and Multiple Labeling with Antibodies from the Same Host Species to Study Adult Hippocampal Neurogenesis

Adult neurogenesis is a highly regulated, multi-stage process in which new neurons are generated from an activated neural stem cell via increasingly committed intermediate progenitor subtypes. Each of these subtypes expresses a set of specific molecular markers that, together with specific morphological criteria, can be used for their identification. Typically, immunofluorescent techniques are applied involving subtype-specific antibodies in combination with exo- or endogenous proliferation markers. We herein describe immunolabeling methods for the detection and quantification of all stages of adult hippocampal neurogenesis. These comprise the application of thymidine analogs, transcardial perfusion, tissue processing, heat-induced epitope retrieval, ABC immunohistochemistry, multiple indirect immunofluorescence, confocal microscopy and cell quantification. Furthermore we present&#160;a sequential multiple immunofluorescence protocol which circumvents problems usually arising from the need of using primary antibodies raised in the same host species. It allows an accurate identification of all hippocampal progenitor subtypes together with a proliferation marker within a single section. These techniques are a powerful tool to study the regulation of different progenitor subtypes in parallel, their involvement in brain pathologies and their role in specific brain functions.

This video article illustrates a comprehensive protocol to detect and quantify all stages of adult hippocampal neurogenesis within the same tissue section. We elaborated a method to overcome the limitations of indirect multiple immunofluorescence that arise when suitable antibodies from different host species are unavailable.

Adult neurogenesis is a highly regulated, multi-stage process in which new neurons are generated from an activated neural stem cell via increasingly ...

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 ...

Whole Mount Dissection and Immunofluorescence of the Adult Mouse Cochlea

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a spiral shape and have a tonotopic gradient for sound detection. The mouse cochlea is approximately 6 mm long and often divided into three turns (apex, middle, and base) for analysis. To investigate cell loss, cell division, or mosaic gene expression, the whole mount or surface preparation of the cochlea is useful. This dissection method allows visualization of all cells within the organ of Corti when combined with immunostaining and confocal microscopy to image cells at different planes in the z-axis. Multiple optical cross-sections can also be obtained from these z-stack images. In addition, the whole mount dissection method can be used for scanning electron microscopy, although a different fixation method is needed. Here, we present a method to isolate the organ of Corti as three intact cochlear turns (apex, middle, and base). This method can be used for mice ranging from one week of age through adulthood and differs from the technique used for neonatal samples where calcification of the cochlea is incomplete. A slightly modified version can be used for dissection of the rat cochlea. We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies.

We present a method to isolate the adult organ of Corti as three intact cochlear turns (apex, middle, and base). We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies. Together these techniques allow the study of hair cells, supporting cells, and other cell types found in the cochlea.

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a ...

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 ...

Cochlear Surface Preparation in the Adult Mouse

Auditory processing in the cochlea depends on the integrity of the mechanosensory hair cells. Over a lifetime, hearing loss can be acquired from numerous etiologies such as exposure to excessive noise, the use of ototoxic medications, bacterial or viral ear infections, head injuries, and the aging process. Loss of sensory hair cells is a common pathological feature of the varieties of acquired hearing loss. Additionally, the inner hair cell synapse can be damaged by mild insults. Therefore, surface preparations of cochlear epithelia, in combination with immunolabeling techniques and confocal imagery, are a very useful tool for the investigation of cochlear pathologies, including losses of ribbon synapses and sensory hair cells, changes in protein levels in hair cells and supporting cells, hair cell regeneration, and determination of report gene expression (i.e., GFP) for verification of successful transduction and identification of transduced cell types. The cochlea, a bony spiral-shaped structure in the inner ear, holds the auditory sensory end organ, the organ of Corti (OC). Sensory hair cells and surrounding supporting cells in the OC are contained in the cochlear duct and rest on the basilar membrane, organized in a tonotopic fashion with high-frequency detection occurring in the base and low-frequency in the apex. With the availability of molecular and genetic information and the ability to manipulate genes by knockout and knock-in techniques, mice have been widely used in biological research, including in hearing science. However, the adult mouse cochlea is miniscule, and the cochlear epithelium is encapsulated in a bony labyrinth, making microdissection difficult. Although dissection techniques have been developed and used in many laboratories, this modified microdissection method using cell and tissue adhesive is easier and more convenient. It can be used in all types of adult mouse cochleae following decalcification.

This article presents a modified cochlear surface preparation method that requires decalcification and use of a cell and tissue adhesive to adhere the pieces of cochlear epithelia to 10 mm round cover slips for immunohistochemistry in adult mouse cochleae.

Auditory processing in the cochlea depends on the integrity of the mechanosensory hair cells. Over a lifetime, hearing loss can be acquired from numerous ...

Imaging the Aging Cochlea with Light-Sheet Fluorescence Microscopy

Deafness is the most common sensory impairment, affecting approximately 5% or 430 million people worldwide as per the World Health Organization1. Aging or presbycusis is a primary cause of sensorineural hearing loss and is characterized by damage to hair cells, spiral ganglion neurons (SGNs), and the stria vascularis. These structures reside within the cochlea, which has a complex, spiral-shaped anatomy of membranous tissues suspended in fluid and surrounded by bone. These properties make it technically difficult to investigate and quantify histopathological changes. To address this need, we developed a light-sheet microscope (TSLIM) that can image and digitize the whole cochlea to facilitate the study of structure-function relationships in the inner ear. Well-aligned serial sections of the whole cochlea result in a stack of images for three-dimensional (3D) volume rendering and segmentation of individual structures for 3D visualization and quantitative analysis (i.e., length, width, surface, volume, and number). Cochleae require minimal processing steps (fixation, decalcification, dehydration, staining, and optical clearing), all of which are compatible with subsequent high-resolution imaging by scanning and transmission electron microscopy. Since all the tissues are present in the stacks, each structure can be assessed individually or relative to other structures. In addition, since imaging uses fluorescent probes, immunohistochemistry and ligand binding can be used to identify specific structures and their 3D volume or distribution within the cochlea. Here we used TSLIM to examine cochleae from aged mice to quantify the loss of hair cells and spiral ganglion neurons. In addition, advanced analyses (e.g., cluster analysis) were used to visualize local reductions of spiral ganglion neurons in Rosenthal&#39;s canal along its 3D volume. These approaches demonstrate TSLIM microscopy&#39;s ability to quantify structure-function relationships within and between cochleae.

A light-sheet microscope was developed to image and digitize whole cochlea.

Deafness is the most common sensory impairment, affecting approximately 5% or 430 million people worldwide as per the World Health Organization1. Aging or ...

Immunolabeling of Neuronal Membrane Proteins in a Freeze-fractured Specimen of Mouse Brain Tissue

<a href='https://app-jove-com.remotexs.ntu.edu.sg/v/53853/combined-optogenetic-freeze-fracture-replica-immunolabeling-to'>Source: Sch&#246;nherr, S., et. al. Combined Optogenetic and Freeze-fracture Replica Immunolabeling to Examine Input-specific Arrangement of Glutamate Receptors in the Mouse Amygdala. J. Vis. Exp. (2016)</a>This video demonstrates the immunolabelling of freeze-fractured replicas of mouse brain tissue for electron microscopy. The fractured and coated replicas are digested to expose receptors. Primary and gold-tagged secondary antibodies are added to label specific neuronal membrane receptors. The replica is mounted on a grid and visualized under an electron microscope.

Source: Sch&#246;nherr, S., et. al. Combined Optogenetic and Freeze-fracture Replica Immunolabeling to Examine Input-specific Arrangement of Glutamate ...