Frequently Asked Questions

Q: I don’t see any living cells in my slice.

A: This is one of the most common problems in slice electrophysiology (See Troubleshooting: Slice Viability). The first thing to remember is that the ideal dissection, slicing, and incubation procedures vary considerably between brain regions, so find out what has already worked for other researchers slicing the same region. If you believe you are doing everything correctly, try cutting slices from younger animals, which are typically much easier to work with. Think critically about your procedure and remember the major causes of cell death in brain slices: pH, ischemic damage, excitotoxic damage, and mechanical damage. Consider also that a dead slice should have many cells that appear to be dead, whereas a slice with no cells may simply indicate an illumination or imaging problem. Finally: be persistent.

Q: I am not able to form a gigaohm seal

A: The most common cause is that the tip of the pipette is fouled. This can have several causes: 1. fingerprints / dust on pipette glass before pulling, 2. pipettes are too old (more than a day) or left uncovered too long, 3. the pipette contacted crystallized salt on the surface of the recording chamber water, 4. the pipette tip contacted brain tissue before it reached the cell due to insufficient pressure inside the pipette. 5. The bathing solution contains serum or bovine serum albumin. If you have to use a solution that contains these, make the seal first in a solution that does not contain proteins, then switch solutions.

Other reasons: 1. A dimple was not visible on the cell before releasing pressure (this often means that something else was compressed between the pipette and the target cell) 2. The cell is dead.

Q: I get a gigaohm seal very quickly, but the cell seems to be gone immediately after breaking in (indicated by very low input resistance or very high resting membrane potential).

A: The cell was probably dead before you patched it. Cells that look similar to this one are also likely to be dead; try changing your cell selection criteria.

Q: I can patch a cell, it looks healthy, but I lose it 10 minutes later.

A: This is often caused by a drifting pipette. Check to see that it has not moved more than about 10 μm from its location at the time of patching. If the pipette has moved, see the following question.

Another possibility is that vibrations transmitted to the pipette tip caused it to detach from the cell. This can be caused by vibrating equipment (unbalanced camera fans are a common culprit), poor isolation from ground vibrations, or the experimenter touching the setup. It is recommended to monitor the cell’s health frequently (or use an audible indicator of electrode potential) to increase the probability that you will discover the cause of a lost cell.

If this happens consistently, it is possible that your internal solution is poisoning the cell. If the cell becomes swollen or shriveled after patching, it is possible that the osmolarity of your internal solution is too low or too high. Try using a different aliquot of internal solution, or borrowing an aliquot of a different batch of internal solution from another experimenter to see if that helps the problem.

Q: My pipette tip is drifting! What do I do?

A: This is most commonly caused by over-tightening or under-tightening the electrode holder cap, which compresses and strains the O-ring holding the electrode. The O-ring relaxes slowly over time, causing the electrode to drift. Applying a small amount of grease to the O-ring can help release this strain before it becomes a problem. Also be sure that nothing is touching the pipette such as the edge of the recording chamber or the objective and that the pressure tube and headstage cable are properly secured to prevent transmission of any strain to the electrode holder. Another source of drift can be temperature changes in the vicinity of the headstage, or a malfunctioning manipulator.

Q: My patch pipettes keep clogging.

A: Clogged pipettes are a common but easily solvable problem. These are most commonly caused by either particulates suspended in the electrode solution or a dirty pipette filler. Electrode solutions should either be centrifuged at the beginning of the day or filtered immediately before filling the pipette, or both. We recommend against using most commercial pipette fillers, as they are difficult to clean. Instead make new fillers daily from plastic pipettes (see Section 4.3).

Q: My ACSF solution looks cloudy or has a precipitate.

A: This is often a sign that the pH of the solution is not in the right range (7.2-7.4). Check the pH. If the solution is being gassed with carbogen, check to be sure that the tank really contains 95% O2 and 5% CO2. One of the authors had an experience where the supplied tank did not have any CO2 in it, and this rapidly led to the death of the slices.

Q: My series resistance (or bridge balance) starts out OK, but increases over the course of the experiment.

A: Series resistance should be kept to a minimum (less than 15-20 MOhm) when possible. Increased series resistance is usually a sign that the tip of the pipette is clogging or the membrane is resealing. Sometimes a little pressure or suction will reopen the tip and allow access. It may also be a sign that the pipette is drifting away from the cell. In this case, visually check the pipette position and check the “My pipette tip is drifting!” FAQ. Increasing the diameter of your pipette tips can help avoid this problem.

Q: I can’t seem to compensate the amplifier in voltage clamp.

A: First, be sure that you understand the compensation procedure in the manufacturer’s manual, and practice the procedure on a model cell. Second, make sure that the electrodes are properly coated to reduce capacitance, and that the electrode series resistance is low (less than 5-10 MOhm). Make sure that the holding potential and the voltage step are in a linear range for the cell you are working with. The activation and deactivation of voltage-dependent channels can happen on the same time scale as the transients that you are trying to eliminate, making it difficult to properly adjust the compensation. Finally, remember that neurons with extensive dendrites do not appear to the clamp amplifier as a simple RC circuit with a single time constant, but have a large number of time constants. However, the amplifiers only are designed to provide proper compensation for a cell with a single time constant. In voltage clamp, you should be attempting to compensate the fastest time constant, and will not be able to correct for the slow components.

Q: There is a high frequency intermittent spiky noise on my recordings.

A: Turn off your cell phone and pager, or move them away from the rig. Make sure that the aspiration of solution from the chamber is not causing a charge separation (sometimes using a fine silver wire for the first 10-20 cm in the aspiration tube will help keep this from happening).