Patch Clamp Preliminaries

Electrode Glass

Many types of pipette glasses are available with differing electrical and thermal properties. An inexpensive glass which is suitable for most recording conditions is Drummond borosilicate 100 µl disposable micropipettes (Rochester Scientific Co., 15 Jet View Drive, Rochester, NY 14624). This pipette glass has relatively thin walls and must be cut to 95 mm length for the List EPC-7 electrode holder or 75 mm for the Axon Instruments electrode holder. Garner Glass Co. (Claremont, California, USA) sells pipette glass in a wide range of dimensions and made from a variety of of glass types. Before using your pipette glass, briefly fire polish each end to prevent wear on the teflon electrode holder. Then acid wash (boil) the glass in 80% nitric, 20% sulfuric acid for about 15 minutes. Be sure to acid wash under a hood. The acid wash removes trace contaminants and helps the electrodes fill more easily. Don't acid wash for too long or the glass will be etched in a manner which may affect the pipette tips. Rinse extensively in distilled water. Place the rinsed pipettes in a glass petri dish and set them on a hot plate at medium heat. They will be dry in about ten minutes.

Sylgard

Coating the pipette tip with Sylgard (Dow-Corning) will reduce the dielectric noise and help considerably to eliminate capacitance transients. While Sylgard is great for your recordings it can be difficult to work with. The stuff stains your clothes and sticks to everything. Make up a mixture of Sylgard with catalyst as described in the product literature. I make up about 5 mL at a time. Uusally there are lots of air bubbles in the mixture, which will increase noise. The air bubbles come out by spinning the mixture in a clinical centrifuge at low speed for a few minutes. Then you can keep it in the freezer and it will last for months (it hardens within hours at room temperature). Apply Sylgard to the taper of pulled pipettes, being careful not to come too close to the tip. I use a piece of unpulled electrode glass as an application tool with the ends fire-polished until they seal. You should slowly twist the pipette while you apply Sylgard so the glass will be evenly coated. Take advantage of the high viscosity of Sylgard to pull it toward the tip with the application tool. The Sylgard can then be hardened by drawing the pipette through the center of a coiled heating element. Since Sylgard flows away from heat make sure the tip is exposed to heat first, otherwise you will end up with lots of clogged pipettes. Another problem is that Sylgard can crack with too much heating, which will lead to increased noise in your recordings. Sylgard can be purchased from K.R. Anderson Co., Santa Clara, California (Tel 408-727-2800).

Chloriding Silver Wire

Are you still dipping your electrode wires in bleach? Or perhaps you chloride your wires by passing current through them in a Cl^- containing salt solution. Well people, its time to stop. This is THE BEST method for chloriding silver wire. It's fast and the chloride coating lasts for weeks. Weigh out about 3 g of silver chloride powder in a small ceramic crucible. Heat the silver chloride with a gas flame until it melts to a dark brown liquid. Now simply dip the end of your wire in the molten silver chloride. Result: a durable Ag/AgCl coating every time! Make sure you don't chloride the end that connects to your instrumentation since you need silver metal here to form the circuit.

Ground Electrode

The ground electrode should be made from the same saline as the recording pipette solution in order to minimize the junction potential. The junction potential results from the rate of diffusion of ions between different solutions. Keeping the ionic composition of the ground electrode solution the same as the recording pipette solution creates a reverse junction potential that cancels the junction potential of the recording pipette. Ground electrodes are typically made from large diameter (e.g., 2-4 mm i.d.) plastic or glass tubing filled with 1% agarose gel. A Ag/AgCl wire is inserted in the ground electrode to complete the circuit. At least a small concentration of Cl- must be present in the ground electrode solution to allow ionic exchange with the Ag/AgCl wire. The ground electrode resistance should be about 100 d. Celsius for for adequate grounding of the recording chamber.

Osmolarity

Most mammalian culture media and salt solutions have an osmolarity that ranges from about 290 - 338 mOsm. The osmolarity of many standard recording solutions range from 320 - 340 mOsm. I usually try to match the osmolarity of my recording and bath solutions to the osmolarity of the culture media, which is ~315 mOsm in most cases. Always check the osmolarity with an osmometer.

Osmolarity and Patch Configurations

For the whole-cell and outside-out patch configuration, seal resistances may be increased by using slightly hypoosmotic solutions in the recording pipette. Try a pipette solution of about 310 mOsm and a bath solution of about 325 mOsm. When switching from cell-attached to outside-out, use a smooth continuous movement of the recording pipette away from the cell. Patch excision should be completed after about 5 s. The speed of patch excision and solution osmolarity are critical variables for success in forming outside-out patches. Note: in forming outside-out patches from frog oocytes it will appear that the seal is lost when you break into the cell interior. This is because of large leakage currents through the oocyte membrane. Continue the patch excision process and chances are the gigaseal will reappear once you have completely excised the patch.

Filling the Recording Pipette

There are several things that can be done to optimize how you fill the recording pipette with solution. I store the recording electrode solution in 1.5 mL microcentrifuge tubes. Connect the recording pipette to a 10 mL syringe, place the tip in the solution (careful to keep it centered). Pull back on the plunger. If you hold the electrode so there is adequate backlight, you can actually see solution flow into the tip. Usually the tip appears blunted after about 10 s of filling. Next, remove the recording pipette from the syringe and proceed to backfill. Don't use a metal syringe needle. There's a real possibility that metal ions you don't want will be taken up into solution. This can be especially bad if you are trying to measure the kinetics of a channel that is blocked by metal ions. A number of labs fill their recording pipettes with tuberculin syringes in which the tip is melted to a fine taper with a gas flame. To make these, take a 1 mL tuberculin syringe and heat the center of the syringe above a gas flame. When it starts to bend, quickly move the syringe away from the flame and hold it over a trash can with the tip pointing down. Then, through the miracle of science, the syringe will start to elongate as the weight of the tip pulls it downward. The tip should fall into the trash and you can cut the syringe at the point where it is narrow enough to fit inside the recording electrode. Fill the recording electrode with as little solution as possible to keep the dielectric noise of the pipette to a minimum. Before putting the recording pipette into the holder, hold it with the tip pointed down and tap the side a few times to get out the air bubbles.

Measurement of Pipette Resistance

A standard pipette resistance range for single channel recording is 3 - 5 MOhm. Lower resistance pipettes seal with more difficulty, but the chances of patching a channel are improved. To measure the pipette resistance, first find the current. If the observed amplifier output is 1 V and the gain is 1 mV/pA, then I = 1 V/0.001 V x 10^-12 A = 1000 x 10^-12 A (or 1000 pA). Next apply Ohm's Law: R=V/I. For example, if the input voltage pulse is 5 mV, then

Measurement of Seal Resistance

Successful patching of single channels requires a seal resistance of at least 1 GOhm. You should expect seal resistances of about 30 - 60 GOhm when using a recording pipette solution such as 110 mM Ba. But be wary of seal resistances >100 GOhm. This may mean that you actually have a gigaclog! During seal formation the reduction in leakage currents reduces the closed channel noise and the patch can be clamped to various voltages without large voltage offsets. To measure the seal resistance, first find the current. If the observed amplifier output is 1 V and the gain is 1000 mV/pA, then I = 1 V/1 V x 10^-12 A = 1 x 10^-12 A or 1 pA. Next apply Ohm's Law. for example if the input voltage is 0.01 V, then

Direction of Current Flow

Current is defined as the flow of positive charges down a voltage gradient (electrons move in the opposite direction). The direction of currents flowing through ion channels is expressed relative to the cell interior. In whole-cell mode when the recording electrode has access to the cell interior, inward current is represented by a downward deflection on the oscilloscope as positive ions move from the cell exterior into the cell. This current is negative with respect to the cell as well as with respect to the recording electrode. However, in cell-attached mode things are quite different. The recording electrode does not have access to the cell interior. In this case, inward current is the movement of positive ions away from the recording electrode into the cell interior. The inward current is now positive with respect to the recording electrode and is represented by an upward deflection on the oscilloscope. In order to make things consistent, electrophysiologists invert the display of currents recorded in cell-attached mode. This way, current flow is always displayed relative to the inside of the cell: downward deflections represent negative inward currents and upward deflections represent positive outward currents. Consequently, as illustrated by the adjacent figure, all current-voltage relations will generally have a positive slope (allowing for some variability due to rectification and blocking processes).