Evaluation of Ribbon Synapses at Specific Frequency Regions of Mouse Cochlea

Begin with tubes containing pre-treated mouse cochlear turns with a permeabilizing agent and a blocking agent that prevent non-specific interactions.

Each turn detects different sound frequencies and contains hair cells that convert sound into electrical signals. These signals are transmitted via neurotransmitters in ribbon synapses to the auditory nerve.

Remove the solution and add primary antibodies that interact with proteins in hair and nerve cells at the ribbon synapses, then wash.

Incubate with secondary antibodies tagged with red and green fluorophores, which bind to their primary antibodies.

Wash to remove unbound secondary antibodies.

Place the specimen on a glass slide with a mounting medium containing nuclear dye to stain nuclei blue.

Mount the coverslip. Dry it, then use a confocal microscope to image each segment at various depths.

Ribbon synapses appear as closely positioned red and green puncta.

Count these functional puncta in each turn to correlate their response to sound frequency.

After dissection, place each cochlear turn into individual 2.5-milliliter centrifuge tubes. Block any nonspecific binding with 10% goat serum in PBS in 0.1% Triton X-100 for 1 hour at room temperature on a rotator.

At the end of the incubation, use a 200-microliter pipette tip to remove the solution under a dissection microscope. Incubate the specimens with the primary antibodies of interest diluted in 5% goat serum in PBS and 0.1% Triton X-100 overnight at 4 degrees Celsius on a rotator.

The next morning, rinse the tissue samples three times for 5 minutes with cold 0.1 molar PBS per wash to remove any residual primary antibody. Then, label the specimens with the appropriate secondary antibodies diluted in 5% goat serum in PBS and 0.1% Triton X-100 for two to three hours at room temperature on the rotator, protected from light.

At the end of the incubation, wash the samples three times with 0.1 molar PBS as demonstrated, and transfer the specimens into individual 35-millimeter plates containing 0.1 molar PBS. Place a drop of DAPI-supplemented mounting medium onto the slide, and transfer the specimens from PBS to the mounting medium. Place one edge of a coverslip onto each slide and release to let the coverslips fall gently. Then, dry the slides in a slide box at 4 degrees Celsius overnight.

To image the specimens, use a confocal microscope with the appropriate lasers and a 63x high-resolution oil immersion lens. To acquire 8-micrometer confocal z-stacks from each cochlea turn. For synaptic punctum counts, set the z-stacks with the 0.3-micrometer step size to span the entire length of inner hair cells, ensuring that all of the synaptic puncta can be imaged, and merge the puncta-containing images in a stack to obtain the z-axis projection.

Import the merged images to an appropriate image processing software program. Divide the synaptic total counts in each z-stack at specific frequency regions by the number of DAPI-positive inner hair cells to calculate the number of synaptic puncta for each cell. At each specific frequency region, average all of the synaptic puncta in 3 images of different microscopic fields containing 9 to 11 inner hair cells.

To better visualize the cytoskeletal architecture and synaptic localization, use the brush tool to visually assess the synaptic structure and distribution of the individual inner hair cells. To inspect the juxtaposition of presynaptic ribbons and postsynaptic receptor patches, use the rectangular marquee tool to extract the voxel space around the ribbons, and use image cutting to isolate the individual ribbons. Then, click Image and Image Size to acquire a thumbnail array of these miniature projections that can be used to identify paired synapses versus orphan ribbons.