The overall goal of this procedure is to obtain a near normal preparation of the mouse vestibular sensory epithelium that can be used to study the fundamental cellular and subcellular processes of vestibular hair cells else. This is accomplished by first isolating the bony vestibular labyrinth from the cranial vault. The second step is to carefully scratch away at the bony labyrinth with fine forceps to expose the underlying membranous labyrinth before removing it completely from its bone casing.
Next, the vestibular sensory epithelium is exposed by cutting away the overlying membrane using fine iris scissors. The final step is to mount the semi intact sensory epithelium preparation on a glass bottomed chamber and stabilize it with a weighted nylon net. Ultimately, patch clamp electrophysiology and two photon microscopy are used to characterize the intrinsic membrane properties and calcium signaling profile respectively of each type of vestibular hair, cell, and or primary arine.
The major advantages of this technique over existing methods, including isolated hair cell preparations, is that we're able to maintain the arrangement of hair cells and their primary afferent neurons in as near normal conditions as possible. This means that we can make measurements in near physiological conditions and we can make these measurements simultaneously. Visual demonstration of this method is critical because the dissection is very difficult to learn, mainly because the vestibular apparatus is encased deep within the temporal bone of the skull and also because the sensory epithelium itself is very small and easily damaged during dissection.
Demonstrating the electroporation and the two photon procedures will be Stefano DeMarco, a colleague working with my laboratory After deeply anesthetizing and decapitating a C 57 black six mouse according to local institution and government guidelines and exposing the skull. Make a small incision in the skull at Lambda and cut along the sagittal suture. Ensure that the brain is not dragged by the shear blade.
During this step, apply ice cold A CSF to the brain to cool the tissue, and then carefully peel the parietal bones away laterally and the occipital bone posteriorly using shallow bend Pearson urs, once again, apply ice cold A CSF. Next, use a small stainless steel spatula to gently lift the brain from the surface of the middle and posterior cranial Fosse. The vestibular labyrinth is now clearly visible in the middle cranial fossa with the cochlea pointing.
An medially add ice cold A CSF and raju either side of the vestibular labyrinth. Then gently excise the vestibular labyrinth by gripping the anterior semi-circular canal and pulling laterally. Immerse the excised labyrinths in a dissecting dish containing ice cold.
Continuously gassed A CSF placed under a stereo microscope. This diagram shows the excised labyrinth in detail. Next, while working under the stereo microscope, grip the cochlea with fine forceps and hold the labyrinth to the bottom of the dish.
Use a second forceps to scratch away at the bone overlying the anterior semi-circular canal amp. Once a small hole in the bone is achieved, begin to flick the bone away from the amp. Be careful not to push the forceps through the bone as this can cause damage to the underlying membranous labyrinth and sensory epithelium.
Continue this procedure until both the anterior and adjacent horizontal semi-circular membranes, ducts and pul are exposed as shown here. Now using fine forceps, gently lift the pul and associated utricle away from the bony labyrinth and ensuring that the central region of the puli containing the sensory epithelium is not damaged. Transfer the triad containing the two pule and utricle into a Petri dish filled with lebovitz's L 15 culture medium.
Use a fiber optic to backlight the tissue to allow clear visualization of the Krista containing the sensory epithelium within the pul. Now use the fine iris scissors to carefully make an incision in the speckled roof of the amp. Take the incision as close to the edge of the sensory epithelium as possible without contacting it.
As shown here, ensure that there are no pieces of membrane overlying the sensory epithelium. This schematic shows the shape of the final preparation and placement of the recording Electrode transfer the isolated semi intact preparation to a small glass bottomed recording chamber filled with L 15 media way down the preparation using a grid of fine nylon fibers secured to a flattened U-shaped platinum wire. The tissue is now ready for electrophysiological recordings.
Perfuse the semi intact preparation with continuously oxygenated L 15 medium. Ensure that the media remains red to indicate a pH with oxygen of 7.3 to 7.4. Throughout the procedure, prepare recording pipettes from 1.5 millimeter, 1.19 millimeter internal diameter.
Borrow silicate glass using a two step protocol on a micro pipette puller to achieve a final impedance of three to four mega. Fill the shank of the pipette with three to four millimeters of potassium fluoride based internal solution. Then wrap the shank of the pipette two to three times and as far down to the tip as possible with a thin strip of param to insulate the electrode and reduce pipette capacitance.
Position the pipette over the sensory epithelium under low power five x magnification on an upright microscope. Switch to 40 x higher power magnification and visualize individual vestibular hair cells with an attached CCD camera. Use the micro manipulator to position the pipette on the membrane of a visualized hair cell.
Once a giga ohm seal is achieved, rupture the cell membrane with a small amount of negative pressure applied through a suction port on the pipette holder and make whole cell voltage clamp recordings using standard techniques for two photon microscopy. Keep the de deroofing semi intact. Preparation submerged and transfer onto a small piece of filter paper.
Ensure that the sensory epithelium is unobstructed from above. Place the filter paper and preparation onto the saline covered base platinum electrode of an electro por. Bring the top platinum electrode parallel with the base electrode and a distance of approximately two millimeters while being careful not to disrupt the orientation of the preparation.
When contact with the Oregon Green 4 88 bafta, one solution is made as seen in this image. Pass a brief current pulse across the preparation. Place a small dab of silicon oil on the bottom of a glass bottomed recording chamber filled with L 15 medium.
Then position the semi intact preparation onto it again, ensuring that the sensory epithelium is unobstructed from above. To ensure stability throughout imaging, replace the nylon grid over the preparation. Then using the two photon microscope, make optical recordings of spontaneous calcium activity in the sensory epithelium using standard imaging protocols.
This image shows whole cell currents recorded from a type one vestibular hair cell characterized by large collapsing tail currents indicated by the arrow here. Whole cell currents were recorded from a type two vestibular hair cell. Note the absence of tail currents.
This image shows whole cell recording from a calyx primary afferent characterized by transient sodium mediated currents as indicated by the arrow following depolarization from a negative resting potential of negative 120 millivolts. All cells were held at negative 60 millivolts. This figure shows IV relationships for type two hair cells recorded from young and older mice in response to increasing depolarization duration.
These histograms show the average conductance and an activation ratio of type two hair cells in young and old mice based on the 100 millisecond IV curves. These two photon micrographs showing BAFTA one Oregon green calcium activation in response to normal L 15 PERFU eight in the top panel or L 15 containing 100 millimolar potassium chloride in the bottom panel. The arrows indicate the same individual hair cell before and after the addition of potassium chloride.
This plot shows the calcium fluorescence profile of the individual cell indicated by the arrow in the previous image. This two photon micrograph of the sensory epithelium shows Oregon Green 4 88 bta one loading of individual hair cells. The inset shows the subcellular localization of calcium to the type one hair cell cytoplasm indicated by the arrow or the calyx primary afferent surrounding a type one hair cell indicated by the double arrows.
Once mastered, the dissection can take less than 10 minutes. After watching this video, you should have a good understanding of how to isolate the vestibular sensory epi epithelium, how to keep it intact, and how to maintain its viability for electrophysiological recordings and for two photon calcium imaging.
