Perforated Patch-Clamp Recordings of Inner Ear Sensory Neuron Cell Bodies

Protocol

1. Recording

NOTE: In this procedure, patch-clamp recordings from the isolated ganglion are typically performed 12-24 h after the plating. Other labs have reported results from neurons after much longer periods in culture.

1. Prepare the recording chamber
   1. Use the quick exchange recording chamber (or equivalent) that allows for patch-clamp recordings directly in the culture dish. Set up the chamber on the inverted microscope.
   2. Insert the glass-bottom culture dishes into the chamber.
   3. Feed the L-15 solution through a perfusion tubing system to the recording chamber. Stabilize the perfusion in-flow and out-flow in the chamber at a rate of 0.5-1 mL per minute.
2. Prepare the internal solution for loading electrodes
   1. Thaw an aliquot of the perforated-patch internal solution. Allocate 2.5 mL of the solution onto a 35 mm culture dish. Replace the lid on the culture dish and label as "Tip Dip". Set aside in a dust-free zone, as it is very important to keep this solution as clean as possible.
   2. Weigh 1 mg of amphotericin-B and add 20 µL of dimethyl sulfoxide (DMSO). Vortex and spin down the solution until all the amphotericin-B is in the solution. Add 10 µL of the DMSO/amphotericin-B solution to the remaining 2 mL of the defrosted perforated-patch internal solution. Withdraw and dispel the solution with a pipette two or three times to ensure that the DMSO/amphotericin-B has uniformly mixed into the internal solution. The solution will have a mild-yellowish tinge.
   3. Draw the amphotericin-B perforated-patch internal solution into a 3 mL syringe. Add a 34 G tip to the syringe. Wrap the syringe in aluminum foil and keep the syringe on ice.
      NOTE: This solution must be remade every 2 h to ensure the effectiveness of the amphotericin-B in perforating the cell membrane. Amphotericin-B is light sensitive, and it must be shielded from light using aluminum foil or other methods.
3. Patch-clamp recordings
   1. Visualize the neurons using a 10x or 20x objective. Adjust the illumination and optimize the optics to see the boundaries and shadows around the cell.
   2. Check the quality of the isolated neurons. Only attempt neurons with smooth and even surfaces that are not too strongly contrasted and have only small, dispersed craters. Also, make sure to avoid pairs of neurons, neurons surrounded by debris, or other cells.
   3. Under 100x magnification, identify a neuron or field of neurons to patch and line them up in the center of the field of view.
   4. Fill the tip of the pipette with a clean solution that does not contain amphotericin by dipping the tip (for ~20 s) into the clean solution that was placed in a culture dish. Capillary action will draw a small amount of solution into the tip.
      NOTE: Perform this step under a stereo dissection microscope, which allows for determining how well the tip holds the Tip Dip solution. If the solution does not hold for ~10-20 s, the shape of the electrode is not ideal. In this case, reconfigure the electrode-pulling program to produce a longer tip.
   5. Next, fill the back of the pipette with the internal solution containing amphotericin. The filament within the pipette will draw down the solution to meet the clean solution in the tip. Allow the filament to smoothly pull the solution down, and do not attempt to tap out bubbles, as this will force the amphotericin to the tip too fast.
   6. Use a syringe to draw out solution that may be at the very back of the electrode.
      NOTE: It is very important to work fast from this point to land and form a high-resistance seal on the desired cell.
   7. Insert the pipette into the pipette holder. Check that the pipette is snug in the holder to prevent pipette drift. If the pipette is not stable, switch out the sealing O-rings in the front and back of the pipette holder to minimize drift and maintain strong suction. Replace the pipette with one that is freshly filled.
   8. Lower the pipette into the bath of the recording chamber.
   9. Locate the pipette tip in the middle of the field of view. Ensure that the tip is free of air bubbles or other debris.
   10. Apply a voltage step (5 mV) in the membrane test to monitor the pipette resistance. Continue monitoring the input resistance through the entire process of forming a seal on the cell.
   11. Cancel the pipette offset potential, such that the pipette current reads zero in the membrane test mode of pClamp.
   12. Correct for the pipette capacitance using the fast capacitance compensation function on the patch-clamp amplifier (Cp fast dials in the Multiclamp Commander soft panel).
   13. Move the pipette down to the cell.
   14. Once the pipette is close enough to the neuron, switch to the higher magnification objective and send the image through a camera to a monitor (use a 40x objective, total 400x magnification). Adjust the pipette and re-center the neuron.
   15. Move the recording electrode close to the soma. Locate the center of the neuron on the monitor and position the recording electrode above the neuron.
   16. Approach the neuron from above, such that the electrode will land in the center of the spherical cell. Adjust the pipette offset to zero the constant DC potentials in the system.
   17. Position the pipette close to the membrane. Land firmly on the center of the cell.
       NOTE: When landing, a small dimpling on the surface of the neuron and a doubling/tripling of the input resistance will occur.
   18. Apply negative pressure (suction) using a syringe or mouth pipette. Turn on the holding potential of -60 mV. The seal resistance should increase until it passes a giga-ohm.
       NOTE: Work fast to reduce the time between filling an electrode and landing on a cell. There is a limited time before the amphotericin added to the perforated-patch internal solution reaches the electrode tip. Once the tip is contaminated with amphotericin, it is difficult to form high-resistance seals, and the resistance often plateaus to ~200 megaohms.
   19. Once a giga-ohm seal forms, release the negative pressure. As soon as the seal forms, apply fast-capacitance compensation to reduce the amplitude of the pipette capacitance transients in membrane test mode as much as possible.
   20. As amphotericin begins to work, watch as the input resistance slowly decreases and the current flowing in response to the 5 mV voltage step progressively increases as the amphotericin enters the membrane. A sudden decrease in series resistance instead of gradual stabilization suggests that spontaneous rupture has occurred.
   21. Estimate the whole-cell capacitance and the series resistance using the amplifier's capacitive transient nulling function. Document and monitor the series resistance at the beginning of, and regularly during, the experiment.
       NOTE: The series resistance can take anywhere from 5-20 min to stabilize, depending on the size of the electrode and how much clean solution is drawn into the pipette. Voltage-clamp and current-clamp modes can be used once the series resistance has stabilized below 30 megaohms. Swelling or shrinking of the neurons during recording is sometimes observed, but rarely when the osmolarity and pH of the solutions are correctly adjusted.

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Materials

Name	Company	Catalog Number	Comments
Amphotericin	Sigma-Aldrich	A4888-100MG	For perforated patch recordings.
ATP di-sodium	Sigma-Aldrich	A7699	Additive to internal solution
CaCl2	J.T. Baker	1311-01	Additive to internal solution
DMSO	Biotium	90082
EGTA	Sigma-Aldrich	E0396	Additive to internal solution
Electrode Puller	Narashige	PC-10
Ethanol	Decon Labs	2716	for cleaning head and around dissection bench
Filamented Borosilicate Capillaries for electrodes	Sutter Instruments	BF140-117-10
HEPES	Sigma-Aldrich	H3375-100G	for pH buffering all solutions in protocol
Hot plate / magnetic stirrers	VWR	76549-914
K2SO4, Potassium Sulfate	Sigma Aldrich	P9458-250G	Additive to internal solution
KCl	Sigma-Aldrich	P93333	Additive to internal solution
KOH (1 M)	Honeywell	319376-500ML	To bring internal solution to desired pH.
Low-profile-bath recording chamber for culture dishes	Warner Instruments	64-0236
MgCl2-Hexahydrate	Sigma-Aldrich	M1028	Additive to internal solution
microFil needle for filling micropipettes - 34 gauge	World Precision Instruments	MF34G
Microforge	Narashige	MF-90	For electrode polishing.
NaCl	Sigma-Aldrich	S7653	Additive to internal solution
NaOH (1 M)	Thomas Scientific	319511-500ML	for titration pH
Oxygen, Medical grade, with adequate regulator and tubing	USC Material Management	MEDOX200 (Identifier: 00015)	for dissolving into dissection and bath solutions
Poly-d-lysine coated glass bottomed culture dish	Mattek	P35GC-0-10-C	to plate neurons for culture
PES membrane filters , 0.2 micrometer	Nalgene	566-0020	for filtering solutions
PES membrane sterile syringe filters, 0.22 um, 30 mm	CELLTREAT	229747	for filtering solutions drawn into syringes
pH Meter	Mettler Toledo	Model S20
Reference Cell	World Precision Instruments	RC1T
Scientific Scale	Mettler Toledo	XS64
Volumetric flask, 1000 milliliter
Water, sterile u ltrapure, R>18.18 megaOhms cm (e.g., filtered by a Millipore-Sigma water purification system)	Millipore-Sigma	CDUFBI001