Many ion channels and neurotransmitter receptors that are found at the terminals of auditory and vestibular afferent neurons are also found at their cell bodies. Cultures of isolated cell bodies can be studied in vitro to understand how these ion channels and receptors influence the responses of the neurons. The compact morphology of the cell bodies allows for high quality electrical recordings to characterize the voltage-dependent properties of the ion channels and neurotransmitter receptors.
Moreover, easy access to a wide variety of cell types allows for high throughput by a physical analysis of cell diversity. The procedures will be demonstrated by graduate student Katherine Regalado, along with postdoctoral fellows, Dr.Daniel Bronson and Nathaniel Nowak from my laboratory. To begin hold a glass pasture pipette to the flame of a Bunsen burner to prepare tritration pipettes for cell dissociation.
Once the glass tip begins to melt, stretch the glass out to the desired tip diameter. Pull one end of the pipette away from the flame to create a small bend. Place the bent pipette under a microscope.
Using a scoring tile, score and break the glass at the desired diameter. Pass the pipette tip over the top of the burner flame. This will quickly polish the rough edges near the tip.
Scoop out the brain of the euthanized mouse using closed surgical scissors. Sever and remove the cranial nerve to completely detach the brain from the skull. Starting at the back of the head, pull the clear membranous material off with forceps, and then cut the top of the skull out using surgical scissors.
Remove the excess tissue from the back of the head and neck to make the dissection area and otic capsule cleaner and easier to access. Transfer the tissue to a second Petri dish with fresh L-15 solution. Then locate the otic capsule and the auditory, superior, vestibular, and inferior vestibular nerves.
Separate the superior and inferior ganglia using small spring scissors and cut away the auditory nerve. Using a scalpel, gently shave off the bony ridge to weaken the bony area under which the nerve dives into the bony capsule. Carefully remove the debris with a scalpel, exposing the entire swollen portion of the ganglion.
Use the fine spring scissors to cut and separate the ganglion from the peripheral nerve branch that is diving toward the utricle. Remove the superior ganglion using fine forceps and transfer it to a 35-millimeter Petri dish with fresh L-15 solution. After preheating the enzyme solution, pour the enzyme mixture into a 35-millimeter Petri dish and place it in a 37 degrees Celsius incubator for 10 to 15 minutes.
Clean the ganglion using fine forceps and small spring scissors by removing bone, excess tissue, nerve fibers, and any other superfluous structures. Take care to minimize the removal of any ganglionic tissue. Transfer the cleaned ganglia into the preheated enzyme solution and place it back in the incubator for 10 to 40 minutes.
Then transfer the ganglia to the 35-millimeter Petri dish with fresh L-15 solution. After two to three minutes, transfer the ganglia to another 35-millimeter Petri dish filled with filtered culture media. Transfer approximately 150 microliters of filtered culture media onto a coated glass bottom dish.
Then, transfer the desired number of ganglia to the cover slip. Draw a small amount of culture media from the culture dish and rinse the tritration pipette with medium to prevent the tissue from sticking to the sides of the glass pipette. Triturate by gently and repeatedly passing the tissue through the pipette until the ganglia are sufficiently dissociated.
After five minutes, check under a light microscope to see if the cells have settled on the cover slip. Carefully place a culture dish into a 37-degree Celsius incubator for 12 to 24 hours. Next, locate the otic capsule after bisecting the head and extract it from the skull by chipping away at the edges using forceps.
The spiral portion of the otic capsule houses the cochlea with the organ of Corti. Chip away at the bone, overlying the cochlear turns with fine forceps parallel to the curve of the bone. Use small spring scissors to sever the modiolus at the base of the cochlea to free it from the rest of the otic capsule.
Cut the organ of Corti into two or three turns using small spring scissors such that it lays flat, and excise the modiolus from each turn. Remove the stria vascularis by pinching the stria at the base with fine forceps. Unwind around the spiral and up to the apex to peel it free from the organ of Corti.
Remove the spiral ganglion by cutting at the edge of where the spiral ganglion neuron fiber tracts project toward the hair cells and then clean the ganglion as shown earlier. After enzymatically treating the spiral ganglion, tritrate the spiral ganglion in the culture medium supplemented with N2 and B27. Place the culture dish containing the dissociated ganglia in a 37-degree Celsius, 5%carbon dioxide incubator for 12 to 24 hours.
Dip the tip of the pipette into the clean solution of the culture dish to fill it with a clean solution that does not contain amphotericin. Fill the back of the pipette with the internal solution containing amphotericin. Dip the tip of the pipette back into the clean solution of the culture dish while the amphotericin slowly enters the tip from the back of the pipette.
Next, finish filling the pipette with the amphotericin solution of to two-thirds of the pipette. Lower the pipette into the bath of the recording chamber and locate the pipette tip in the middle of the field of view. Ensure that the tip is free of air bubbles or other debris.
Position the pipette close to the membrane and land firmly on the center of the cell. Apply negative pressure or suction using a syringe or mouth pipette. Turn on the holding potential of minus 60 millivolts.
The seal resistance should increase until it passes a giga ohm. Once a giga ohm seal forms, release the negative pressure. Before tyrosine begins to work, the input resistance slowly decreases, and the current flowing in response to the five millivolts voltage step, progressively increases as the amphotericin enters the membrane.
The representative examples of whole cell currents evoked from a vestibular ganglion neuron are shown in this figure. The net inward sodium currents are identified here by their transient activation and inactivation. The net outward currents are carried largely by potassium ions and have much slower activation and inactivation kinetics than sodium currents.
The hyperpolarization activated cyclic nucleotide gated currents, or HCN currents, are activated by a family of long duration hyperpolarizing voltages. The stability of ion channel characterization in the perforated patch at different time points during recording is shown in this figure. The voltage dependent activation curves of HCN currents were measured in a perforated patch configuration.
The curve has a sigmoidal shape and was stable throughout a long recording. The relative instability of the ruptured patch mode is illustrated by non-overlapping sigmoidal curves from different time points. The activation curves of HCN currents during a ruptured patch configuration are shown here.
The firing patterns in five vestibular ganglion somara, five spiral ganglion neurons, and the corresponding current steps are shown here. This heterogeneity in firing patterns reflects a fundamental diversity in the composition of underlying sodium and potassium channels in both vestibular ganglion neurons and spiral ganglion neurons. To maximize cell survival during this procedure, avoid forming air bubbles, do not overwork the tissue, and allow cells to settle before placing samples into the incubator.
Perforated patch clamp enables the study of ion channels that are modulated by cytosolic second messengers, which is critical for investigating the effects of efferent neurotransmitters on ganglion neurons. Patch clamp recordings of these bipolar neurons have been instrumental in revealing how biophysical diversity contributes to functional diversity in sensory neurons.
