2.1.

Specimen Preparation

The care and use of the animals was approved by the Institutional Animal Care and Use Committee at Baylor College of Medicine. We used wild-type adult mice of mixed background that were $4\text{to}6\text{weeks}$ old. The mouse was anesthetized with a ketamine/xylazine mixture and decapitated. Both cochleae were harvested and placed into oxygenated extracellular solution. The extracellular solution was similar to perilymph, the fluid that bathes the basolateral surfaces of the hair cells. However, we increased the calcium concentration within it to evoke larger responses. The extracellular solution contained $142\mathrm{mM}$ of NaCl, $4\mathrm{mM}$ of KCl, $10\mathrm{mM}$ of glucose, $10\mathrm{mM}$ of HEPES, and $4\mathrm{mM}$ of $\mathrm{Ca}{\mathrm{Cl}}_2$ . The pH value of the solution was adjusted to between 7.35 and 7.40, and the osmolality of the solution was $\sim 305\mathrm{mOsm}∕\mathrm{kg}$ .

The cochlea was microdissected from the surrounding bone and tissue under a stereo microscope (Stemi-2000C, Zeiss). Figure 1 shows a schematic of the dissected cochlea, and Fig. 1 shows a schematic of a cross section of one turn of the cochlea. Figures 1 and 1 show a mouse cochlea during dissection as imaged through a charge-coupled device (CCD) camera connected to the trinocular port of the microscope. The stapes was left attached to the oval window and the round window was untouched. Then, the cochlea was glued upright into a custom-made plexiglass chamber using dental glue (Iso-Dent, Ellman International, Oceanside, New York). Particular care was taken to make sure the stapes and round window were not contacted by the glue, which might affect their ability to conduct sound. The bone overlying the region of the cochlear epithelium to be studied was then opened with a fine knife and pick. The spiral ligament was gently displaced laterally, and Reissner’s membrane was incised to expose the cochlear epithelium containing the hair cells. Typically about one quarter to one third of a cochlear turn was exposed.

Fig. 1

Dissection of the mouse cochlea. (a) Schematic of the cochlea after dissection. An opening overlying the basal turn of the cochlea (i.e., near the stapes and round window) is shown in this example. (b) Schematic of a cross section of one turn of the cochlea. (c) The entire temporal bone, which contains the cochlea, is shown submerged in extracellular solution after removal from the skull, as visualized through the dissecting microscope. The round window, oval window, and stapes can be seen. The scale bar is $1\mathrm{mm}$ . (d) The cochlea has been glued upright into a recording chamber, so that only the cochlea (white area to the left of the oval and round windows) is visible. Particular care needs to be taken to leave the round window, oval window, and stapes unencumbered by glue. The scale bar is $1\mathrm{mm}$ . (e) The apical turn of the cochlea has been opened. This permits visualization of the cochlear epithelium where the hair cells are located (curved dark area at tip of arrow). About one third to one half of a cochlea turn can be opened. The scale bar is $0.5\mathrm{mm}$ .

The entire dissection process could be completed typically within about $20\min$ after animal sacrifice. After the dissection, the calcium indicator Oregon Green BAPTA 488-AM ( $10\mu \mathrm{M}$ ; Invitrogen, Carlsbad, California) was applied to the preparation, and the chamber was placed in a dark box for $30\min$ with a constant flow of oxygen over it. The chamber was then moved to the microscope for imaging, residual dye was rinsed away, and constant perfusion with oxygenated extracellular solution was performed by a peristaltic pump. The pump speed was limited to make sure the flow of extracellular solution did not disturb the optical image.

2.2.

Imaging setup

Imaging was performed using a custom-built upright microscope (Fig. 2 ) based on a moveable objective microscope (MOM, Sutter Instruments, Novato, California). An objective lens turret (OT1, Thorlabs, Newton, New Jersey) was inserted to permit easy changing of the objective without losing the orientation of the sample. A CCD camera was incorporated into the setup to allow visualization of the specimen using transmitted light. We typically used a $4\times$ long-working distance objective (Plan Fluor, Nikon) to find the area of interest on the sample. The focal plane and choice of region to image could be varied by moving the objective lens, which was mounted on 3-D translation motor stages (MP285-3Z/M, Sutter Instruments). Then, we rotated in a $20\times$ , $0.95\text{-}\mathrm{NA}$ water immersion lens (XFLUOR $20\times$ water immersion lens, Olympus) objective, which was used for imaging.

Fig. 2

Schematic of the microscope system.

An ultrafast Ti:saphpire laser (Chameleon, Coherent, Palestine, Texas) was used as a near-infrared (NIR) two-photon excitation light source. The center wavelength used in these experiments was $800\mathrm{nm}$ and the pulse width at the laser output was $\sim 140\mathrm{fs}$ . After a series of steering mirrors and telescopes, the laser was adjusted to a suitable beam size and angularly scanned by a pair of $3\text{-}\mathrm{mm}$ -aperture galvometer-controlled mirrors (6200H, Cambridge Technology, Lexington, Massachusetts ). The scan mirrors were imaged onto the back focal plane of the objective lens by the scan and tube lenses. The laser was focused into sample through the imaging objective. The average laser power was manually attenuated by a half-wave plate and polarizer to about $30\text{to}40\mathrm{mW}$ (as measured after passing through the objective lens) to minimize damage to the sample and photobleaching of the indicator, while still providing an adequate signal-to-noise ratio.

The fluorescence light was collected by the same objective lens and reflected to the detector by a dichroic mirror (type ET670SP-2P8, Chroma Technology, Rockingham, Vermont ) located between the objective and tube lenses. A bandpass fluorescence filter (type $\mathrm{ET}525∕50\mathrm{nm}$ , Chroma Technology) before the detector blocked the residue reflected or scattered NIR excitation light and only let the green fluorescence pass through. The fluorescence light signal was detected by a photomultiplier tube (C7319, Hamamatsu) and digitalized by an A/D converter (PXI 6259, National Instrument, Austin, Texas ).

Open source software (ScanImage Version 3.1, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York)²³ written in Matlab (The Mathworks, Natick, Massachusetts) was used to control the devices and collected images. We intensively modified the software to work with the hardware we used and our experimental protocols. Experimental data images were saved in tagged image file format (TIFF).

2.3.

Stimulus

Experiments were performed using a piezoelectric actuator (PA8-12, Piezosystem, Jena, Germany ) to generate movements of the stapes as previously described.²⁴ Briefly, the piezoelectric actuator drives a long thin shaft (a $4\text{-}\mathrm{cm}$ -long tungsten wire, $0.005\mathrm{in.}$ diameter). The piezoelectric actuator and shaft were mounted on a 3-D manipulator (PCS-5000 series, Burleigh, EXFO, Mississauga, Ontario ) and the tip of the shaft was positioned to contact the stapes. During the collection of a series of images, the piezoelectric actuator produced sound stimuli that varied from $5\text{to}12\mathrm{kHz}$ . The stimulus was $15\mathrm{s}$ long, and consisted of $150\text{-}\mathrm{ms}$ blocks of sine waves repeated at a rate of $5\mathrm{Hz}$ . The magnitude of stapes motion had been previously measured (using a laser Doppler vibrometer) to be in the nanometer range, recreating a sound intensity of $\sim 80\text{-}\mathrm{dB}$ sound pressure level.²⁴ Figure 3 shows a schematic of the experiment setup. Figure 3 shows a microscopic picture of the stimulator tip in contact with the stapes.

Fig. 3

Schematic diagram of the experiment setup. The size of the cochlea is exaggerated for clarity. In this diagram, the opening in the cochlea is shown overlying the basal turn. (b) Image of the preparation taken through the experimental microscope with the tip of the piezoactuator in contact with the stapes. The opening of the cochlea was over the apical turn in this example. The scale bar is $1\mathrm{mm}$ .

3.1.

Outer Hair Cell Calcium Imaging

When the preparation was ready for imaging, the recording chamber was transferred to the microscope and held fixed in place on a rigid column. First, the sample was visualized using transmitted light that originated from underneath the sample. We used a $4\times$ objective lens to position the tip of the stimulating probe against the top of the stapes (the capitulum). The angle of approach was within $25\mathrm{deg}$ of colinearity with the stapes to maximize the transmission of the vibrations of the piezoactuator to the fluids of the cochlea. We then found a region of the cochlear epithelium to image, and changed to the $20\times$ objective lens. Again, the sample was viewed using transmitted light to verify that the OHCs in the region to be imaged appeared organized and healthy. The microscope was then switched into two-photon mode by moving the flipper mirror to allow the laser to reach the sample.

Figure 4 shows typical transmitted light and Figs. 4 and 4 show fluorescence images of the cochlear epithelium. The quality of images depended on the intensity of the laser, the scanning resolution, and the number of averaged frames. While this calcium indicator is expected to partition relatively evenly among all cells, the OHCs always demonstrated the largest fluorescence intensity. This suggests that the indicator is either preferably taken up into OHCs, or that intracellular calcium concentration within OHCs is elevated. The latter is more likely, given the well-known fact that OHCs progressively deteriorate after animal sacrifice and are dead within about four hours. There was some variability in the brightness of different OHCs as well, presumably reflecting differences in their resting intracellular $\left[{\mathrm{Ca}}^{+2}\right]$ .

Fig. 4

Transmitted light view of the cochlear epithelium as imaged by the CCD camera. The one row of inner hair cells (IHCs) and three rows of outer hair cells (OHCs) can be seen. The pillar cells sit between the IHCs and OHCs. (b) A typical fluorescence image of the cochlear epithelium after staining with the calcium indicator Oregon Green BAPTA 488-AM. The OHCs demonstrate particularly high fluorescence intensity. The dotted line indicates the OHC region from which the total fluorescence was calculated. The scale bar is $15\mu \mathrm{m}$ . (c) By increasing the zoom factor, OHC stereociliary bundles can be noted. The scale bar is $5\mu \mathrm{m}$ . (a), (b), and (c) are from different samples.

Inner hair cells (IHCs) and some supporting cells, like pillar cells, also could be visualized, but their staining intensity was weaker. Increasing the scanning zoom factor permitted visualization of the V-shaped OHC stereociliary bundles, as in Fig. 4. We could not distinguish individual OHC stereocilia with this preparation. OHC stereocilia are $\sim 200\mathrm{nm}$ in diameter, which is beyond the resolution limit of the light wavelength we used. The staining intensity of the stereocilia was faint compared to that of the soma.

3.2.

Quantification of Outer Hair Cell Intracellular Calcium Concentration

To estimate the resting $\left[{\mathrm{Ca}}^{2+}\right]$ inside the OHCs, we measured the calcium indicator fluorescence within 16 OHCs using ImageJ software (NIH, Bethesda, Maryland). We selected individual OHCs from four cochleae before and after applying 0.1% Triton X-100 for $30\mathrm{s}$ . This procedure permeabilized the plasma membrane and allowed extracellular calcium to enter into the cell. The average increase in $\Delta F∕F$ was $115\pm 19\mathrm{\%}$ ( $\text{mean}\pm \mathrm{SEM}$ , $p<0.0001$ ), and varied depending on the initial fluorescence level of the OHC [Fig. 5 ]. Although cells that were brighter at rest demonstrated a lower increase in fluorescence after the application of Triton X-100 than those that were initially dimmer, all cells still demonstrated increases in $\Delta F∕F$ . This indicated that the calcium indicator was not saturated at the resting $\left[{\mathrm{Ca}}^{+2}\right]$ inside the OHCs.

Fig. 5

Change in fluorescence after membrane permeablization vs the initial OHC fluorescence. OHCs that were initially dimmer demonstrated a larger increase in their fluorescence as expected. Data were fitted with a line. (b) Change in fluorescence after membrane permeablization vs the initial OHC $\left[{\mathrm{Ca}}^{2+}\right]$ . Data were fitted with an exponential curve. Three data points at the extreme high range were $>2$ SD above the mean, and were excluded from the calculations of the average resting OHC $\left[{\mathrm{Ca}}^{2+}\right]$ .

We then calculated the $\left[{\mathrm{Ca}}^{2+}\right]$ within the cells using the equation per the instructions provided by the manufacturer of the indicator (Long-Wavelength Calcium Indicators Product Information, Invitrogen). This required knowing the fluorescence at rest, the fluorescence of the calcium saturated probe as determined by the permeabilization experiment, the background fluorescence, and the dissociation constant $\left(K_d\right)$ of the indicator dye $\left(170\mathrm{nM}\right)$ . From our sample, the OHC resting $\left[{\mathrm{Ca}}^{+2}\right]$ was found to vary from $52\text{to}875\mathrm{nM}$ . After excluding the three OHCs whose $\left[{\mathrm{Ca}}^{+2}\right]$ were $>2$ SD above the mean, the average OHC intracellular $\left[{\mathrm{Ca}}^{+2}\right]$ was $137\pm 21\mathrm{nM}$ $(\text{mean}\pm \mathrm{SEM})$ . Figure 5 demonstrates that the $\Delta F∕F$ after Triton X-100 application varied exponentially with the resting intracellular $\left[{\mathrm{Ca}}^{+2}\right]$ .

3.3.

Outer Hair Cell Fluorescence Change During Sound Stimulation

Our goal was to measure changes in intracellular calcium as an assessment of mechanotransduction. This was done by recording a series of 2-D image frames (100 images in $\sim 60\mathrm{s}$ ). No frame averaging was used for this process. During a portion of the recording, the sound stimulus was applied to the stapes.

Using ImageJ software (NIH, Bethesda, Maryland), we selected the OHC region of the epithelium and calculated the mean fluorescence intensity for each image frame. This value was plotted as a function of time. Due to photobleaching, the fluorescence intensity decreased during the time of data collection. This was accounted for by fitting the fluorescence data with a decreasing single exponential curve. The weight function of the fitting was adjusted to remove the effect of sound stimulation from the photobleaching background curve. For frames without any stimulus presented, the weighting was set to one, and for frames where there was a sound stimulus presented, the weighting was set to zero. Then, all the raw data were normalized by the fitted background curve.

Figures 6 and 6 show typical OHC calcium fluorescence intensity curves as a function of time (image frame count number). These examples were collected from the group of 24 OHCs within the apical turn of the cochlea (eight from each of the three rows) from Fig. 4. Figure 6 demonstrates the original data, the smoothed data, the fitted exponential, and background noise. The sound stimulus was presented between frames 27 through 58. The smoothing method was a moving window average with a nine-point span. In contrast to the fluorescence of the OHCs, the background noise was not affected by photobleaching or the sound stimulus. Figure 6 displays the normalized data $(\Delta F∕F)$ and the smoothed normalized data. An increase of the fluorescence intensity of up to 4% over baseline can be seen in the figure during the time of sound stimulation. This is consistent with an increase in intracellular calcium during sound transduction.

Fig. 6

Original data, smoothed data, and exponential fit, as a function of time (frame counts) from a group of 24 OHCs. The smoothing method was a moving window average with a nine-point span. The fit was a single exponential curve, excluding data during the time of the sound stimulus. Sound stimulation was presented between frames 27 and 58, as indicated by the rectangle. Background noise was not affected by photobleaching or the sound. (b) Original data and smoothed data after normalization to the exponential fit for the data shown in (a). (c) Representative response from a single OHC. (d) Average of responses from ten OHCs from six different cochleae. The noise floor threshold shown is 3 SD above average.

In addition to allowing the measurement of responses from groups of OHCs, this technique also permitted the measurement of calcium changes within single OHCs. Figure 6 demonstrates the $\Delta F∕F$ response of a single representative OHC. We then measured responses from six different cochleae, and averaged the responses of ten representative cells, as shown in Fig. 6. The noise floor threshold line is the mean noise floor plus three times the standard deviation, indicating that the response reaches statistical significance. This average change of $\sim 3\mathrm{\%}$ would correspond to a change in the intracellular $\left[{\mathrm{Ca}}^{+2}\right]$ of $\sim 4\text{to}5\mathrm{nM}$ .

As an additional control, we also tested six cochleae without sound stimulation, and no change in $\Delta F∕F$ was found after adjusting for photobleaching (data not shown).