Recording Tips

Instructor’s Supplement

Equipment

This section lists equipment required for extracellular and intracellular recording. Unless otherwise indicated, each student rig requires each item. Each instructor’s supplement refers to one of these equipment lists and notes anything additional that is required. The common set is required for both extracellular and intracellular recording. This section is not intended as a buying guide for assembling a teaching lab; the Crawdad web site (crawdad.cornell.edu) has sources and suggestions for inexpensive substitutes.

Common to Extracellular and Intracellular

• Computer with A/D and D/A (optional if oscilloscope present, but extremely desirable).
• Oscilloscope (optional if computer is present; storage optional but useful if no computer).
• Dissecting microscope (6 to 50× objective with 10 × eyepieces is a good range).
• Microscope light (fiber-optic with two arms is most flexible).
• Faraday cage (metal or wood frame with metal window screen on sides, top, and back; approximately 32w × 32h × 24d in inches). This may not be needed in a room with low electrical interference. Before building a Faraday cage, you can check your room by attempting a recording with and without grounded aluminum foil shielding the preparation.
• Steel plate approximately 32w × 24d × 3/8 inch thick (avoid stainless steel, which will not hold magnets).
• Dampers for steel plate. Four size 6 to size 10 rubber stoppers are sufficient in many cases. If the lab has vibration problems, you may need to use partially inflated bicycle tires to isolate the plate (8- to 10-inch diameter tires for a child’s bike work well). Intracellular recording requires better damping than does extracellular recording.
• BNC cables (2 to 3 feet long, about six per rig).
• BNC T-connectors (minimum three per rig).
• Alligator clip cables (2 to 3 feet long, minimum four per rig).
• Ground wire (chlorided silver wire or Ag-AgCl pellet).
• Station for chloriding silver wire, resoldering broken wires, and other repairs that may be needed during the lab (one for the entire lab). If you are only doing extracellular recording, chlorided silver is not required as a ground, so the chloriding station is not required.

Extracellular

• AC amplifier (capable of 1000× gain, 60 Hz notch filter desirable); see Land et al. (2001) for an inexpensive design.
• Coarse micromanipulator on magnetic base.
• Suction electrode (for construction details see Johnson et al., 2007).
• If using the Johnson et al. (2007) suction electrode design, a microelectrode puller is desirable. It need not be of the quality required to produce intracellular electrodes. We use 1.5 mm outer diameter, 1.0 mm inner diameter electrode glass or capillary tubes for extracellular electrodes. Puller settings are not critical. Capillary glass can also be pulled manually over an alcohol or gas flame.

Intracellular

• Microelectrode puller (one for the entire lab).
• DC amplifier with headstage and electrode holder.
• Fine micromanipulator on magnetic base.
• 10 ml beaker for electrode backfilling.
• Microelectrode glass. We use standard borosilicate glass tubing with a filament, outer diameter 1.0 mm, inner diameter 0.5 or 0.75 mm, because it is inexpensive and readily available. If your electrode puller is used in research and is set for another type of glass, use whatever glass is most convenient. Students may find it easier to thread the Ag-AgCl wire of an electrode holder into electrodes made with thinner wall (larger interior diameter) glass.
• Waste containers for disposal of used and broken electrodes. Each lab group should be provided with one of these. A coffee can with a hole cut in the lid will suffice.
• Syringe with long thin needle for electrode backfilling (one per two to three rigs is enough). Fiberglass ones are cheaper and don’t clog but are easily broken by students. Metal ones are more durable but more expensive and more likely to become clogged. Flush out the needle with distilled water after each lab period.
• 3 M KCl (224 g/l); 500 ml will last several semesters.
• 10 ml beakers for electrode filling.

Ground Wire

The ground wire completes the recording circuit in both extracellular and intracellular recordings. Accurate measurement of DC potentials requires that the ground be nonpolarizable. Otherwise, fluctuating potentials from an unstable metal-fluid junction will dominate the recording (Katz, 1966; Purves, 1981). For a ground, you can either use a commercially made Ag-AgCl pellet or make your own chlorided silver wire. Solder the pellet or wire to a cable with either an alligator clip (to clip to the Faraday cage) or a banana plug (to plug into the oscilloscope or amplifier chassis ground) at the other end.

To coat a silver wire with silver chloride, use one of the following methods, starting with 0.008- to 0.025-inch bare silver wire. First, remove any oil from the wire with ethanol or sand it lightly with 600 grit or finer sandpaper.

• An easy method that requires no apparatus is to soak the silver wire in chloride bleach for 15-30 min.
• For a fast result, attach the silver wire to the positive pole of a 5 to 15 V DC power source. Place the negative pole (any metal) in 3 M KCl and dip the silver wire into the KCl, leaving it until it becomes slightly brown (usually only a few seconds).
• For a fast result, attach the wire to the positive pole of a 5 to 15 V DC power source. Place the negative pole in chloride bleach and dip the silver wire into the bleach, leaving it until it becomes slightly brown (usually only a few seconds).
• The fastest method is dipping a silver wire into ferric trichloride solution, which is inexpensive and readily available for etching of printed circuit boards and craft projects.
• For the most long-lasting result, attach one silver wire to the positive pole of a 5 to 15 V DC power source and another silver wire to the negative pole. Place both wires in 3 M KCl and reverse the polarity of the power source every few minutes for about 20 min. This produces two chlorided wires.

The third method seems to produce the longest-lasting chloride coating, although the coating will wear off eventually in any case. When that happens, just repeat the procedure.

Electrode Holders

There are a variety of holders for intracellular electrodes. The simplest type (depicted in our figures and videos, and elsewhere) is a rod with a groove into which the electrode is lightly clamped. A chlorided silver wire is inserted into the electrode and attached to the amplifier headstage. When using this type of holder, it is usually easiest to thread the electrode onto the wire and then slide it into the groove of the holder. Be sure that the electrode is straight in the groove before it is clamped down and that it is not clamped too tightly, breaking the glass. You will periodically need to rechloride the wire (see above), since the coating is easily scraped off by electrodes.

The other common type of electrode holder is a plastic cylinder with a hole for the electrode and a rubber gasket to keep it in place. The holder usually plugs directly into the amplifier headstage, which is held by the micromanipulator. This type of holder has either a chlorided silver wire protruding from the hole or a silver chloride pellet at the base of the hole. If a wire is involved, it will need to be rechlorided periodically. If a pellet is involved, the hole into which the electrode is inserted must be filled with KCl and this must be in contact with the KCl inside the electrode. In either case, take care that KCl and saline do not get into the socket of the amplifier headstage.

Grounding

As emphasized in the student manual, the best way to reduce noise is to ground anything in the Faraday cage that might act as an antenna. However, this need not be taken to extremes. It should be sufficient to connect the cage to the oscilloscope’s chassis ground, the steel plate to the cage, the microscope to the cage, and the ground wire of the saline bath to the cage. Connecting all equipment to a single ground point like this not only makes a neater setup, it avoids possible noise from ground loops. Manipulators are already grounded through their magnetic bases. You may need to ground the light guides from your light source, although this is seldom necessary. If the light is a source of noise, turning it off may not help because the noise could be due to capacitative coupling between the electrode and the light’s power cord. In that case, you will need to unplug the light while recording. If you are using a microscope with a built-in or attached light, you will need to turn it off or unplug it while recording.

Common Problems

This section covers general troubleshooting. Any problems specific to particular labs or preparations are dealt with in the lab supplements.

Noise in extracellular recording:

• Are cage, steel plate, microscope, etc., grounded?
• When reaching into the Faraday cage, ground yourself by touching the steel plate.
• Is the saline bath ground in place and connected to ground?
• Is the external wire of the suction electrode contacting the saline?
• Are the syringe and tubing inside the cage?
• Are amplifier filters set properly? Experiment with different settings.

No signal in extracellular recording:

• Is the amplifier turned on and set to Recording mode?
• Does saline inside the electrode reach the internal wire?
• Is the nerve firmly sucked into the electrode tip?
• Is the electrode tip completely clogged? If so, replace it.
• Is the amplifier saturated? Check by trying a lower gain. If so, work to eliminate noise.
• Are high- and low-pass filters set too close to each other? Try other settings.
• Are oscilloscope settings correct? Check the scale knob.

Noise in intracellular recording:

• Are cage, steel plate, microscope, etc., grounded?
• Is the saline bath ground in place and connected to ground?

Oscillation after action potential in intracellular recording:

• Is the 60 Hz notch filter turned on? It often causes ringing (Figure C.2). Intracellular amplifier output can’t be zeroed or resistance can’t be measured:
• Turn off capacitance compensation, current injection, and DC Balance or Transient knobs.
• Check the state of the electrode holder; AgCl may have worn off the silver wire or pellet. Replace the holder or rechloride the silver wire as described above for ground wires.
• Does KCl in the electrode reach the silver wire or pellet of the electrode holder?
• Is there a tiny bubble at the tip? If so, replace the electrode.

Resting potential can’t be seen in oscilloscope:

• Set oscilloscope input mode to DC.
• Check that the correct amplifier output (10×) is being used.

Students can’t see the electrode tip and keep breaking it:

• Adjust lighting so that some light comes from the side.
• Try coloring electrodes with waterproof ink (Video C.1i, Electrode Inking). If you can find this ink in a bottle, dip the electrodes instead. Dipping electrodes in India ink may work, but sometimes plugs the electrode tip.
• The electrode tip is very difficult to see when it is stationary. Moving it back and forth horizontally with the manipulator often helps, but be sure to keep it above the level of the preparation.

References

Most of the material above is covered by references given in the student Appendix C, Recording Tips. The references below provide additional information on the reasons for chloriding silver wires. Answers to many general questions concerning electrophysiological recording techniques can be found at http://en.wikipedia.org/wiki/Electrophysiology.

• Johnson BR, Hauptman SA, Bonow RH (2007). Construction of a simple suction electrode for extracellular recording and stimulation. J Undergrad Neurosci Educ 6:A21-26. [pdf]
• Katz B (1966). Nerve, Muscle, and Synapse (McGraw-Hill, New York), pp. 17-19.
• Land BR, Johnson BR, Wyttenbach RA, Hoy RR (2007). Tools for physiology labs: Inexpensive equipment for physiological stimulation. J Undergrad Neurosci Educ 3:A30-35. [pdf]
• Land BR, Wyttenbach RA, Johnson BR (2001). Tools for physiology labs: An inexpensive high-performance amplifier and electrode for extracellular recording. J Neurosci Methods 106:47-55. [doi]
• Purves RD (1981). Microelectrode Methods for Intracellular Recording and Ionophoresis (Academic Press, New York), pp. 50-51.
• Sherman-Gold R (2012). The Axon Guide: Electrophysiology and Biophysics Laboratory Techniques, 3rd Edition (Molecular Devices Corporation, Union City CA). [pdf]