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

All procedures were carried out in accordance with the NRC/ILAR Guide for the Care and Use of Laboratory Animals (8th Edition). The study protocol was approved by the Institutional Animal Care and Use Committee of Capital Medical University, Beijing, China.

1. Animal Selection

1. For all experiments, use adult C57BL/6J male mice (8 weeks old) as the animal model.
   NOTE: C57BL/6J mice carrying a splice variant of the Cdh23 exhibit accelerated senescence in the auditory system, reflected as a 40% loss of ribbon synapses at the basal turn of the cochlea and a 10% loss at the middle turn by 6 months of age, followed by a rapid increase of this loss in the whole cochlea with age^18,19. Thus, we advise caution when using C57BL/6J mice older than 6 months for auditory research. Other strains of mice can be used depending on specific experimental aims.
2. Inspect the mice using a professional diagnostic pocket otoscope to rule out outer or middle ear pathologies prior to hearing assessments. Potential signs may include fluid or pus in the external auditory canal, redness and swelling in local tissue, and tympanic membrane perforation.
   NOTE: Although this is rarely encountered, once identified, mice with outer or middle ear diseases should be excluded.

2. Hearing Assessment

1. Anesthetize mice using an intraperitoneal injection of a mixture of ketamine hydrochloride (100 mg/kg) and xylazine hydrochloride (10 mg/kg). Judge the depth of anesthesia via painful stimuli (e.g., toe-pinch reflex).
   NOTE: When the toe-pinch reflex is completely absent, the animal has reached an adequate depth of anesthesia for auditory testing. If more time is required for bilateral ABR recordings, administer a lower dosage (one-fifth the original dosage) of anesthetics to restore the original anesthetic plane. Take care to avoid anesthetic overdose, as this may lead to death in mice.
2. Maintain the anesthetized animal’s body temperature at 37.5 °C using a thermoregulating heating pad. Place the anesthetized animal in an electrically and acoustically shielded room to avoid interference throughout the hearing test.
   NOTE: Maintain the physiological temperature during the entire procedure until the animal is totally awake, in order to prevent death caused by post-anesthesia hypothermia.
3. Place subdermal needle electrodes (20 mm, 28 G) at the vertex of the skull (recording electrode), in the ipsilateral parotid region below the pinna of measured ear (reference electrode), and in the contralateral parotid region (ground electrode), with a depth of 3 mm under the skin of the mouse head, respectively²⁰.
4. Use a closed-field speaker to perform acoustic stimulation via a 2 cm plastic tube with a cone-shaped tip. Fit the tip into the external ear canal²¹.
   NOTE: Ensure that the electrical impedance in the recording and reference electrodes is less than 3 kOhm (usually 1 kOhm). If the impedance is high, change the insertion site of the electrode, clean the electrode with alcohol, or replace the electrode to avoid alterations in ABR wave amplitude.
5. For ABR recording, generate tone pips (3 ms duration, 1 ms rise/fall times, at a rate of 21.1/s, frequency: 4–48 kHz) and present them at decreasing SPLs from 90 to 10 dB in 5‒10 dB SPL steps²⁰. During this step, the responses are amplified (10,000 times), filtered (0.1–3 kHz), and averaged (1,024 samples/stimulus level).
   NOTE: ABRs are collected for each stimulus level in 10 dB steps, with additional 5 dB steps near the threshold.
6. At each frequency, determine the ABR threshold, which refers to the minimal SPL resulting in a reliable ABR recording with one or more distinguishable waves that can be clearly identified by visual inspection (Figure 1).
   NOTE: It is usually necessary to repeat the process for low SPLs around the threshold to ensure the consistency of the waveforms. The response threshold is the lowest stimulus level at which the waveform can be observed, when a decrease of 5 dB would lead to the disappearance of the waveform.

3. Cochlear Tissue Processing

1. After ABR recording, euthanize the anesthetized mice via cervical dislocation, decapitate them, expose the bulla from the ventral side, and open with sharp scissors to gain access to the cochlea.
2. Using a fine forceps, remove the temporal bones, sever the stapes artery, remove the stapes from the oval window, and rupture the round window membrane. Make a small hole at the apex of the cochlea by gently rotating the tip of a needle (13 mm, 27 G).
3. Fix the isolated bones with 4% (wt/vol) paraformaldehyde in 0.1 M phosphate-buffered saline (PBS, pH 7.4) overnight at 4 °C. Using a fine-tipped pipette, gently flush fixative through the perilymphatic spaces via application to the oval or round windows (as an inlet) and the opening at the apex (as an outlet).
   NOTE: Some proteins require a short duration of fixation to avoid destruction of their epitopes for immunolabeling. In such cases, incubate bones in 4% paraformaldehyde at room temperature (RT) for 2 h, depending on the manufacturer’s instructions for immunohistochemistry. Fixation can also be performed via cardiac perfusion to remove the cochlear blood, avoiding background noise due to non-specific staining at later stages, particularly in mouse models of cochlear synaptopathy.
4. Rinse the bones three times for 5 min with 0.1 M cold PBS to remove residual paraformaldehyde. Decalcify the bones with 10% ethylenediaminetetraacetic acid (EDTA) either at RT for 4 h or at 4 °C for 24 h via gentle shaking in a horizontal shaker at 20 rpm. EDTA can be refreshed midway.
   NOTE: Decalcification times are subject to the concentration of EDTA and users’ preferences. Decalcified tissue should maintain a certain degree of toughness, which facilitates the manipulation of isolating cochlear whole-mounts at later steps. Temporal bones can be decalcified in 10% EDTA with rotation, allowing researchers to leave the laboratory following ABR tests and fixation experiments. The decalcification time is flexible within the range of 20 to 30 h at 4 °C.
5. Transfer one decalcified temporal bone from EDTA to 0.1 M PBS. Use #3, #5 Dumont forceps and the 27 G needle to dissect the apical, middle and basal cochlear regions in turn and then dissect the cochlea out of the bone under a stereo dissection microscope (as previously described²²). Make a series of small cuts along the spiral ligament using a razor blade, and remove the tectorial membrane and Reissner’s membrane (Figure 2).
   NOTE: As long as the dissected cochlea is intact, this process can be modified according to the individual operator's usual protocol.
6. Further dissect the remaining auditory epithelium including the spiral limbus into individual cochlear turns (apex, middle, and base with hook region) for whole-mount preparations.
7. Under a 40x oil objective of a light microscope, measure the basilar membrane length with a 250 μm scale placed in the eyepiece, which can be adjusted along the stereocilia of the IHCs.
8. Calculate the length of each cochlear turn by adding all segment lengths (250 μm per segment), and concomitantly obtain the total length of the basilar membrane by summing the lengths of each turn.
9. Convert the total length of the basilar membrane including the hook region into a percentage based on distance from the cochlear apex (0% refers to the cochlear apex, 100% to the cochlear base).
10. Convert this distance into the cochlear characteristic frequency using a logarithmic function (d(%) = 1 – 156.5 + 82.5 × log(f), with a slope of 1.25 mm/octave of frequency, where d is the normalized distance from the cochlear apex in percent, f is the frequency in kHz), as previously described^5,6. Thus frequency range in a corresponding area of the basilar membrane on each cochlear turn can be acquired.

4. Immunofluorescence Staining

1. After dissection, place each cochlear turn in a separate 2.5 mL centrifuge tube and incubate cochlear turns in 10% goat serum/PBS/0.1% Triton X-100 for 1 h at RT on a rotator.
2. Remove the above blocking/permeabilization solution from each tube using a 200 μL pipette tip under a dissection microscope and incubate the specimens with primary antibodies diluted in 5% goat serum/PBS/0.1% Triton X-100 overnight at 4 °C on a rotator.
   NOTE: For immunolabeling of cochlear synaptic ribbons, use the presynaptic marker mouse anti-carboxyl-terminal binding protein 2 IgG1 (CtBP2, labeling the B domain of the RIBEYE scaffolding protein, 1:400) and the postsynaptic marker mouse anti-glutamate receptor 2 IgG2a (GluR2, labeling a subunit of the AMPA receptor, 1:200)²³.
3. Rinse three times for 5 min with 0.1 M cold PBS to remove residual primary antibodies and incubate the specimens with secondary antibodies diluted in 5% goat serum/PBS/0.1% Triton X-100 at RT for 2‒3 h in darkness on a rotator.
   NOTE: Prepare appropriate secondary antibody mixtures using goat anti-mouse Alexa Fluor 568 (IgG1, 1:500) and goat anti-mouse Alexa Fluor 488 (IgG2a, 1:500), which are complementary to the primary antibodies used in step 4.2. To improve labeling efficiency for synaptic ribbons, we recommend selecting specific secondary antibodies. Some labs extend incubation with secondary antibodies to increase GluR2 immunolabeling²⁴.
4. Rinse three times for 5 min with 0.1 M PBS to remove residual secondary antibodies and transfer the specimens from 2.5 mL centrifuge tubes to 35 mm plates containing 0.1 M PBS.
5. Place a drop of mounting medium containing 4’,6-diamidino-2-phenylindole (DAPI) onto the slide and transfer the specimens from PBS to the mounting medium. Place one edge of a coverslip on the slide and release to let the coverslip fall gently.
   NOTE: To ensure that the hair cells face upward and that no folding or twisting of cochlear specimens occurs during the procedure, mount cochlear specimens under a stereo dissection microscope.
6. Place the slides in a slide box at 4 °C overnight to let slides dry and then image under a laser confocal microscope.

5. Morphological Evaluation of Cochlear Ribbon Synapses

1. Image slides using a confocal microscope with three lasers—a 405 nm UV diode, a 488 nm argon laser, and a 561 nm diode-pumped solid-state (DPSS) laser to excite DAPI (Excitation spectrum 409–464 nm), Alexa Fluor 488 (Excitation spectrum 496–549 nm) and Alexa Fluor 568 (Excitation spectrum 573–631 nm), respectively.
2. Acquire confocal z-stacks over a distance of 8 μm from each cochlear turn using a 63x high-resolution oil immersion lens.
   ​NOTE: Once defined, all parameters for digitizing photomicrographs should be saved and applied uniformly to all slides.
3. For synaptic punctum counts, set the z-stacks (0.3 μm step size) to span the entire length of IHCs, thereby ensuring that all synaptic puncta can be imaged.
4. Merge the images containing puncta in a z-stack to obtain the z-axis projection, and import to the image-processing software.
5. Divide synaptic total counts in each z-stack at specific frequency regions by the number of IHCs (equal to the DAPI nuclear manual counts) to calculate the number of synaptic puncta for each IHC. At each specific frequency region, average all synaptic puncta in three images of different microscopic fields containing 9–11 IHCs.
   1. Outline the region of interests (ROIs) including the basolateral regions of each IHC using freehand selections button. Use the measure function for automatic quantification of puncta, and the watershed function to distinguish between closely adjacent spots.
   2. After each automated counting, perform visual inspections with manual corrections to ensure puncta counting reliable.
      NOTE: Experimenters should remain blinded as to whether the slide is from the apex, middle, or basal turn of the cochlea.
6. Visually assess synaptic structure and distribution, to manually isolate individual IHCs from their neighbors by the Pencil Tool (M) to better visualize the cytoskeletal architecture and synaptic localization.
7. To inspect the juxtaposition of presynaptic ribbons (CtBP2) and postsynaptic receptor patches (GluR2), extract the voxel space around ribbon by the Rectangular Marquee Tool and isolate individual ribbon by Crop. Through clicking Image > Image Size, acquire a thumbnail array of these miniature projections, which can then be used to identify paired synapses (appeared as closely juxtaposed pairs of CtBP2-positive and GluR2-positive puncta) versus orphan ribbons (lacking postsynaptic glutamate receptor patches) (Figure 3).
   NOTE: Normal cochlear synapses appear as combined immunolabeling of the presynaptic ribbon within the hair cell (anti-CtBP2) and the postsynaptic glutamate receptor patch on the auditory nerve terminal (anti-GluR2)²⁵. Some labs use confocal projections in conjunction with 3D modeling to quantify synaptic patch size or volume^26,27. Prior to significant loss of ribbon synapses, ribbons exhibiting changes in size or without paired glutamate receptor patches are likely indicative of synaptic dysfunction^27,28.

6. Functional Evaluation of Cochlear Ribbon Synapses

1. Collect all ABR waves for each frequency stimulus presented at an SPL of 90 dB for the analysis of suprathreshold ABR wave I amplitudes.
   NOTE: Neurophysiological and morphological studies have demonstrated that low spontaneous-rate, high-threshold fibers are especially vulnerable to aging and noise exposure^29,30. Although the simple loss of ribbon synapses cannot affect ABR thresholds, it commonly results in significant reductions in ABR wave I amplitudes, because these afferents including low spontaneous-rate, high-threshold fibers and high spontaneous-rate, low-threshold fibers contribute heavily to the summed activities of cochlear nerve fibers^28,29,31. A suprathreshold intensity of 90 dB SPL is selected here.
2. Determine peak-to-peak wave I amplitude using an offline analysis program (Figure 4). Each wave I in the ABR test consists of a starting positive (p) deflection and the subsequent negative (n) deflection. ABR wave I amplitude is defined as the difference in voltage between Ip (the positive peak of wave I) and In (the negative peak of wave I)²⁹.
   NOTE: In pathological conditions, cochlear synaptopathy can be determined based on the suprathreshold amplitudes of ABR wave I, which reflect the summed onset responses of SGNs evoked by sound. However, cochlear sensitivity that is not compromised due to OHC dysfunction is a prerequisite for this method.