The overall goal of this electrophysiological assay is to investigate the morphological and functional changes of cochlear hair cells during postnatal development. This method can help answer key questions in the audical assistant department field such as when do our hair cells gain their functions during the onset of hearing? The main advantage of this technique is that we are able to evaluate the function of individual out hair cells, isolated from the postnatal rat pups.
Demonstrating the procedure will be Wenlu Pan, Shasha Guo, and Nana Xu that were students from our lab. To begin this procedure, place the anesthetized animal in a polyethylene foam mold to immobilize the animal's body. Then, place the animal on an anti-vibration table in a sound-attenuating room.
Keep the animal's body temperature at 37.5 degrees Celsius with a heating pad. Next, wipe the head area with 70%ethanol. Make a one to two millimeter incision ventral-laterally to the external pinna to place the reference electrode or ground electrode.
Then, place a subdermal recording electrode over the skull vertex. Using the function generator, generate calibrated tone bursts of various frequencies and intensities. Deliver the sound stimuli through an electrostatic speaker located 10 centimeters away from the head of the animal.
To obtain the auditory brainstem responses, the sound-elicited potentials are filtered, amplified, and averaged using a multifunction processor. The 40 minute ABR recording is monitored online and stored for offline analysis. In this section, sterilize the animal's head by spraying with 70%ethanol.
Next, open the skull along the sagittal midline with scissors and remove the brain to expose the inner ear. Then, transfer the inner ear into a 35 millimeter petri dish filled with three milliliters of ice-cold Leibovitz's L-15 Medium. Under the dissection microscope, use fine forceps to remove the bony capsule of the cochlea.
After that, unwrap OC and SV associated from modiolus. By holding the basal portion of SV with forceps, separate OC from SV completely by unwinding slowly from base to apex. Subsequently, cut OC evenly into three pieces by using fine scissors.
All steps should be performed in ice-cold L-15 as fast as possible in order to minimize degradation and tissue deterioration. In this step, using a 200 microliter pipette tip, transfer the segments of OC onto a glass slide. Fix them with 100 microliters of 4%paraformaldehyde for at least four hours at four degrees Celsius.
Following that, wash the tissue by displacing the paraformaldehyde with 100 microliters of fresh PBS. Then, wash the tissue three times for 10 minutes each time. Afterward, incubate the tissue with 0.3%permeability agent in PBS for 30 minutes at room temperature.
After 30 minutes, discard the permeability agent and wash the tissue three times with PBS. Subsequently, block the tissue with 10%normal goat serum and PBS for one hour at room temperature. Incubate the tissue with anti-prestin C terminus antibody and blocking solution for two hours at room temperature or at four degrees Celsius overnight.
Then, wash the tissue three times with PBS for five minutes each time. Afterward, incubate the tissue with Alexa 488-conjugated secondary antibody in blocking solution for one hour at room temperature in the dark. Wash the tissue three times with PBS for five minutes each time in the dark.
Next, incubate the tissue with rhodamine phalloidin for 10 minutes at room temperature in the dark. Wash the tissue three times with PBS for five minutes in the dark. Then, mount the sample on a glass slide with mounting medium.
Image it with confocal microscopy using 405 nanometer, 488 nanometer, and 594 nanometer lasers. In this procedure, use the pipette puller and microforger. Make patch pipettes with a tip diameter of two to three microns.
Then, backfill the pipettes with intracellular solution. Using a 200 microliter pipette tip, transfer a piece of OC into a 35 millimeter petri dish. Digest the tissue with 100 microliters of enzymatic digestion medium for five minutes at room temperature.
Next, displace the enzymatic digestion medium with 100 microliters of L-15. Cut the tissue into small pieces using a microscalpel to isolate the hair cells. After gentle pipetting, transfer the cells into a homemade small plastic chamber filled with enzyme-free bath solution.
Then, place the chamber on the stage of an inverted microscope and find the healthy-appearing solitary OHCs. Subsequently, load the patch pipette into the head stage of a 700B amplifier. Position the patch pipette around the bottom of the outer hair cell.
Following that, whole cell patch clamp the OHC by sealing the lateral wall of the cell body. Apply light suction until the cell membrane is ruptured. Then, set the holding potential at 70 millivolts.
Cells with access resistance ranging from 10 to 17 megaohms and membrane resistance ranging from 100 to 500 megaohms are considered a successful whole cell configuration. Apply hyperpolarizing and depolarizing voltage to the cell to elicit whole cell currents. Measure the membrane capacitance of OHCs using a two sine-wave voltage stimulus protocol controlled by the patch clamp software.
Set the stimulate voltage range from 140 to 110 millivolts and store the data for offline analysis. After gentle digestion, the OHCs were isolated from the OC.Whole cell voltage clamp recordings were performed from OHCs acutely isolated from the rat cochlea. A representative example of the whole cell current recorded from an isolated P9 OHC in response to membrane potential changed from 140 to 107 millivolts is shown here.
This figure shows the non-linear membrane capacitance obtained from two OHCs at different ages. The capacitance voltage responses were fitted to the Boltzmann function. Once mastered, this technique can be done in two hours, if it is performed appropriately.
Following this procedure, other measures like Western blotting and the PCR can be performed in order to answer additional questions such as evaluating the expression level of some protein, such as prestin in out hair cells. After its development, this technique paved the way for researchers in the field of hair cells to explore electrophysiological properties in cochlear and the vestibular system. After watching this video, you should have a good understanding of how to investigate morphological, and the functional changes of cochlear hair cells during postnatal development.
