The overall goal of this procedure is to introduce a reliable method to investigate outer hair cell or OHC motile responses, including electro motility, slow motility, and bending at acoustic frequencies. This is accomplished by first harvesting temporal bones from Guinea pigs, mice, or other mammalian animals, and opening the bula to expose the cochlea. The second step is to immerse the cochlea in Lebovits L 15, removing the apical region of the bony shell, the spiral ligament, and the straw of vais, and removing the organ of Cort.
Following a five minute incubation of the organ of Cort in one milligram per milliliter collagenase, it is moved to the recording chamber using a 50 microliter Hamilton syringe, and the cells are dissociated by refluxing them through the needle. The recording chamber is then moved to the microscope for cell stimulation with an external alternating electrical field and video recording of the motile responses of isolated outer hair cells with a high-speed camera. Ultimately, outer hair, cell, electro motility, slow motility, and bending at acoustic frequencies can be evaluated by computer-assisted image analysis.
Begin this procedure by harvesting the temporal bones from Guinea pigs, mice, or other mammalian animal models. Then open the temporal bones using als nipper. In order to expose the cochlear, remove excess bone carefully keep the cochlear bony shell intact and immerse it in livi L 15.
Under the microscope, open the apical region of the cochlear. Remove the dryer, VASIs, and spiral ligament with the tip of a scalpel blade, a micro point pick or a fine tweezer. Remove the organ of corti from the cochlear modis with the tweezers.
Next place the cochlear in one milligram per milliliter, collagenase at room temperature for five minutes. If er hair cells from the basal turns of the cochlear are required, remove the bony shell covering the base of the cochlear with the pick and separate the spiral from the temporal bone using the scalpel blade Before removing the organ of Corte, transfer the organ of corti to the recording chamber using a 50 microliter Hamilton syringe. Then dissociate the cells by refluxing them through the needle.
Here is the diagram of the external alternating electric field generator and the links with the image capture system, the control circuitry, and software specially implemented at the engineering core of the House Ear Institute. The experimental setup used in this experiment consists of an Axia vert 1 3 5 TV inverted microscope with an alternative LED based illumination system and two electronic micro manipulators. There are three camera reports on the microscope, A PC controlled ultra high speed photo tron, fast cam X 10 24 PCI camera in the teleport, which has high resolution and captures images at high frequencies up to 100, 000 FPS.
The images provided by the high speed camera are directly observed in the PC monitor. An additional regular CCD camera in the ocular port is connected to a high resolution monitor. A conventional digital photo camera in the front port allows the capture of still frames the homemade software running on the same PC controls the trigger of the high speed camera, the illumination system, and the external alternating electric field mount.
The two electrodes, which consists of two 0.25 millimeter diameter silver wires with tip distance of 0.8 millimeters on an electric micro manipulator. Next, calibrate the electrical field using an external electrode. This electrode measures the electric potential at different points generating a map of the electrical field.
If a single isolated outer hair cell is placed between the tips of the electrodes with its longitudinal axis parallel to the applied alternating electrical field, it moves elongating and shortening at the same frequency as the electric field. For instance, shown here is a cell being stimulated at 50 hertz and recorded at 1000 frames per second. This next image shows a cell being stimulated at four kilohertz and recorded at 18, 000 frames per second.
If the cell is placed perpendicular to the field, a different type of outer hair cell response bending can be observed and investigated. This cell, for example, is bending when stimulated at 50 hertz with an electrical field perpendicular to its longer axis. Outer hair cells can show changes in shape in response to non-electrical stimulation.
This response is called slow motility and can also be investigated with this technique. Select different stimulations among the four different protocols. The first one is continuous single frequency.
Note that the stimulus mode, frequency, amplitude, and wave type can be selected, which are indicated by red circles with the homemade control software. The second stimulation protocol is bursted single frequency in which the length of the bursts and the gaps between the bursts are adjustable. The third one is linear sweep in which the initial and final frequencies are adjustable.
The fourth stimulation protocol is multi stimulation, which allows the combination of different stimulus modes. After selecting the corresponding parameters, the control software configures the system and allows the operator to initiate light synchronized video recording and cell stimulation by clicking a single button in the computer screen. In this movie, the isolated outer hair cells show changes in the length or curvature when they're being stimulated with an external alternating electric field that is parallel or transversal respectively to their longitudinal axis.
Outer hair cells responses are analyzed offline using pro analysts software's feature tracking function shown here is an outer hair cell stimulated at a frequency of 50 hertz and recorded at 1000 frames per second. The software provides the distance between two points frame by frame as shown here during cell shortening. The distance between points selected at the apex indicated in red as cuticular plate, and the base of the cell indicated in green as basal pole is smaller and it increases with cell elongation, the software does a frame by frame analysis of the changes in outer hair cell length.
The panel at the bottom of the image shows the trace of the movement. In this example, the total changing length is about 6.5 pixels. Here is an example to show that the cell edge can be detected and the area of the optical section can be automatically measured by the contour tracking function with pro analys software.
In this experiment, polystyrene microspheres are added to the bath solution randomly and firmly attached to the plasma membrane. Different microspheres can be selected simultaneously and the software can automatically track all of them frame by frame. In this way, the cells can be divided in sections, and the motility of each section can be evaluated independently.
By selecting the microspheres located on the lateral edges of the cell, image changes in the length of each segment and changes in the angle of one segment. In respect to the other, can also be independently evaluated. Once mastered, this technique can be done in three hours if it is performed properly.
Of course, additional time is necessary for offline analysis of the recorded movies. However, with adequate software, this analysis can be completed in roughly three more hours per movie. After watching this video, you should have a good understanding of how to investigate outer hair, cell motile responses, including electro motility, slow motility, and bending at acoustic frequencies.
