The overall goal of this procedure is to prepare the bullfrog's sacculus for both micro-mechanical and bio-physical studies. This method can help answer key questions in the field of sensory neuroscience. How do auditory and vestibular hair cells adapt?

What causes spontaneous oscillation of hair bundles and how do hair cells actively respond to mechanical stimuli. This technique offers easy access to hair cells that remain healthy for several hours. Because hair cells are fragile and mechanically sensitive, preservation of their integrity can prove difficult during these procedures.

Here we provide a visual demonstration of this method, so that others can implement proper tissue handling techniques and can achieve success in biophysical studies of the inner ear. Begin by placing the freshly decapitated bullfrog head under a sterile dissection microscope. The dashed box in this image shows the location of the inner ear.

Use a scalpel to make a mid line cut through the palatal tissue from the vomerine teeth to the most posterior extent of the tissue. Next, make horizontal cuts with the scalpel to sever and clear any muscle lying below the palatal tissue to reveal the posterior cartilage. After removing the muscle, observe the lollipop shape of the temporal cartilage that forms the boundary of the otic capsule.

Sever the columella at its point of contact with the otic capsule's cartilage. Then repeatedly make shallow horizontal cuts through the cartilage to shave off several thin layers. Avoid deep cuts to prevent damage to the inner ear organs.

Stop shaving once the otic capsule found within the lollipop structure of the temporal cartilage is open and the inner ear organs are visible, as shown in this image. Next, trim the posterior and lateral edges of the otic capsule, taking care not to damage the inner ear organs. Frequently flow saline over the inner ear organs to ensure that they remain submerged and hydrated throughout the dissection.

Now, locate the two circular openings in the temporal bone at the media connection of the cartilage to the mid line. Cut downward through the most media opening to rupture the temporal bone. Then make a second downward cut through the otic capsule at its postural, lateral edge.

Pry loose the piece of cartilage between this and the previous cut to provide access to the inner ear organs. This action severs the nearest semi-circular canal, which can then be used as a handle for further manipulations. Next, taking care not to touch the sacculus, sever the lagena and eighth cranial nerve at the level of Scarpa's ganglion.

While holding the ampular of the nearest semi-circular canal, gently rotate the inner ear and sever the remaining two semi-circular canals. Holding the nerve or semi-circular canal ampular, extract the inner ear from the head and place it in a dish filled with chilled oxygenated artificial perilymph. Removal of the inner ear, permits visualization of the passages within the temporal bone through which the semi-circular canals once passed.

The sacculus is identified by the large wide mass of otoconia and saccular nerve on the surface. Identify the sacculus and then trim the semi-circular canals to render the inner ear more maneuverable. Next, make gentle cuts around the perimeter of the perilymphatic cistern overlying the neural side of the sacculus.

Sever the small pillars of tissue that bridge the cistern's membrane to the neural side of the sacculus and remove the cistern. Next, remove the nerve to the utriculus lagena and lagena nerve. Then, while holding the saccular nerve, gently lift the sacculus and cut through the thin membrane of the otoconia sac, as otoconia spill out of the sac, free the sacculus by cutting around its perimeter.

Use scissors or forceps to carefully wipe away any otoconia remaining on the sacculus. Take care not to touch the sacculus during this process. Now trim any remaining otoconia sac membrane from the sacculus'edge to prevent membrane adherence to plastic and glass surfaces.

Finally, gently flow saline over the sacculus with the Pasteur pipette, to remove any remaining otoconia. The sacculus in isolation is showed in detail here. The dashed line outlines the location of the otolithic membrane and the underlying sensory macula containing hair cells.

Using the back end of a Pasteur pipette with a broken tip, transfer the isolated sacculus to a Petri-dish containing Proteus 24 Solution. Then incubate the tissue for 30 minutes at 22 degree celsius. Transfer each digested sacculus to an open face experimental chamber, and secure the tissue with magnetic pins.

Then use a fine eyelash, to remove the overlying otolithic membrane and reveal the saccular macula containing the hair cells. After isolating the sacculus, fill the mounting block with artificial perilymph. Then apply two spots of vacuum grease to one opening, and place perforated aluminum on top, forming a weak seal.

Then transfer the sacculus to the foil, and center it on top of the hole with the macula facing down and the nerve stump facing up. Use a piece of twisted tissue to draw saline from the opposite side of the mounting chamber, in order to form a tight seal between the sacculus and the mounting foil. Wick the saline surrounding the sacculus from the surface of the aluminum square.

Use a poly-tetrafluoroethylene applicator to rapidly apply cyanoacrylate glue along the boundary between the edge of the sacculus and the aluminum square. Ensure that the entire circumference of the sacculus is covered with glue. When applying the cyanoacrylate glue, use the applicator to guide the glue around the tissues'edge while avoiding direct contact.

Your goal is to achieve a complete seal to isolate the endolymphatic and perilymphatic compartments. Proceed expediently to prevent the glue from creeping over the back of the tissue. Place a drop of saline on top of the mounted tissue to cure the glue.

A thin film of glue may form on top of the drop of saline, if so, remove it with forceps. Carefully remove the foil from the mounting block. Flip the mounted tissue over so that the macula side of the sacculus faces upward and floated on an artificial perilymph filled Petri-dish.

Add a drop of Proteus 24 Solution on top of the macula and incubate for 30 minutes at 22 degree celsius. Following the incubation, fill the lower canal of the two chamber apparatus with perilymph, and place vacuum grease around the central chamber. Place the foil mounted sacculus on the lower chamber with its nerve facing the chamber surface.

Apply grease around the perimeter of the foil. Place the upper chamber of the preparation on the foil, taking care to form a complete seal with the vacuum grease. Fill the upper chamber with bubbled artificial endolymph, then gently remove the otolithic membrane with an eyelash, to reveal the saccular media containing the hair cells.

This image shows the result of hair bundle displacement across discrete steps. Bundle displacement was tracked using a dual photo diode system with simultaneous sharp microelectrode receptor potential measurement. A plot of the bundle's receptor potential is a function of its displacement reveals a non-linear relationship between the bundle's response and its position.

Each color represents a set of time series, corresponding to the same displacement pulse. When viewed from above, hair bundles in a two chamber preparation oscillates spontaneously. In the absence of iontophoretic release of gentamycin, a hair bundle displays symmetric oscillations.

Then, as the magnitude of current passed through an iontophoretic pipette filled with 500 millimeter gentamycin sulfate grows, the frequency of hair bundle excursion falls in a dose dependent manner and the bundle is offset towards its tall edge for longer periods of time. Once mastered, this technique can be performed in 75 minutes. Following this procedure, experimental methods such as electrophysiology and mechanical load clapping can be performed to explore the hair cells'active process.

This technique paved the way for researchers in the field of sensory neuroscience. To explore the mechanics of hearing and balance in vertebrates. After watching this video, you should have a good understanding of how to prepare the bullfrog sacculus for biophysical exploration of hair cell physiology.

Thank you for watching and good luck with your experiments.

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