Electrophysiological recordings from the pharynx*
Alan Cook, Christopher J. Franks and Lindy Holden-Dye §
School of Biological Sciences, University of Southampton, SO16 7PX, UK

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Table of Contents

1. Summary of available methods
2. Protocol 1. Extrapharyngeal recordings (EPGs)
2.1. Equipment
2.2. The EPG experiment
3. Protocol 2. Intracellular recordings from the terminal bulb
3.1. Equipment
3.2. The intracellular recording experiment
4. Protocol 3. Patch clamp recordings from the pharynx
4.1. Equipment
4.2. The patch clamp experiment
5. Acknowledgements
6. References

 1. Summary of available methods
Extracellular, intracellular, whole-cell and single-channel patch clamp recordings may all be made from the pharyngeal muscle. The type of recording made will be dictated by the information that is being sought. Extracellular recordings, termed electropharyngeograms or EPGs were pioneered by Leon Avery and co-workers (Avery et al., 1995) and provide information on parameters such as rate of pharyngeal pumping, duration of each pump (i.e. time between contraction and relaxation phases) and, by analysis of the shape of the EPG, the activity of neurones within the pharyngeal nervous system including the inhibitory neurone M3 and the pacemaker neurone MC (for details and examples see Raizen and Avery, 1994; Raizen et al., 1995). EPGs have the advantage of being relatively easy to record and yield information both on the activity of the muscle and the pharyngeal neurones. However, they give little reliable information on the magnitude of the pharyngeal potentials as the amplitude of the EPGs is very dependent on the positioning of the recording electrode and the properties of the seal with the worm's cuticle. In contrast, intracellular recordings are technically more challenging but are necessary if information on the resting membrane potential or shape of the pharyngeal action potential is required (e.g., Lee et al., 1997). Detailed analysis of the currents underlying the pharyngeal action potential may be obtained from whole-cell recording techniques. Although the pharyngeal muscle is not very amenable to voltage-clamp because of extensive electrical coupling between cells, nonetheless, it is possible to make a limited analysis of voltage-activated currents (Shtonda and Avery, 2005). Future progress on detailed analysis of the ionic currents in pharyngeal muscle may come from voltage-clamp studies on pharyngeal myoblasts in primary culture. Further information on the properties of single channels may also be obtained from patches excised from the pharynx, in either outside-out or inside-out configuration. The basic protocols for each of the electrophysiological techniques available for C. elegans pharynx are outlined below. For further background to the black art of microelectrode recording techniques consult Purves (1981).

2. Protocol 1. Extrapharyngeal recordings (EPGs)
2.1. Equipment
A relatively stable recording table is required. An air-table is the best option but a stone slab, isolated from the frame and supported on rubber bungs, is usually quite sufficient provided that the environment is not mechanically very noisy.

An inverted microscope is better than an upright microscope as it provides more room to manoeuvre the electrode. The lamp should have an external DC power supply to limit problems with electrical noise. An X/Y moveable stage helps with positioning the preparation. Top of the range optics are not necessary, and x4 and x10 objectives will suffice.

A micromanipulator is needed to hold the suction electrode. As these are extracellular recordings, very fine movement is not required and a fairly coarse manual manipulator with fine movement in just the Z axis will suffice. Various options are available for mounting the manipulator on the recording stage, depending on the microscope being used, but the important consideration is stability. One option is to place the entire recording set-up on a metal surface (if using an air table this is the usual surface) and to use a manipulator with a magnetic base.

Electrical noise is one of the most severe limitations of the EPG technique as the amplitude of the EPG signal is relatively small. The apparatus should be enclosed in a Faraday cage to limit noise and to allow for earthing.

The recording chamber can be custom made from Perspex, or various options are available commercially. An easy and disposable alternative is to pour Sylgard at a depth of 0.5 cm in a 9 cm Petri dish. Allow it to set and then cut the required shape for the recording chamber out of the set Sylgard, allowing for a volume of approximately 1ml. Use a glass slide as the base and attach the Sylgard chamber to it. If the preparation is to be used for pharmacological experiments then important considerations are the volume of the bath versus the speed of the perfusion system to allow for rapid drug application and washing. To provide a stable reference potential, it is advisable to use an Agar bridge. This consists of a fine-bore glass tube filled with 4% Agar in 3 M KCl. The tube is bent so that one end can be placed in the recording chamber and the other in a small vial containing 3 M KCl to which the headstage ground is connected via a silver-chloride coated silver electrode.

A high-input impedance amplifier is required to record the EPGs, an analog to digital converter and a computer with software for data acquisition and to allow for off-line analysis.

A micropipette puller is required to pull the pipettes but a top of the range model is not necessary. A Narashige vertical drop puller for patch pipettes will suffice. The choice of pipette glass is probably not critical. Thick-walled borosilicate glass, 1 mm outer diameter without a filament is suitable. A pipette holder with a side-arm is used to hold the electrode.

All these items are obtainable from standard suppliers for biosciences research.

2.2. The EPG experiment
Pick animals into Dent's saline (composition in mM; D-glucose 10 mM, HEPES 10, NaCl 140, KCl 6, CaCl2 3, MgCl2 1, pH 7.4 with NaOH). It is important to consider the physiological status of the animals if comparisons are to be made between different groups. Therefore the age and nutritional status should be controlled for. Place the animals in a polystyrene tissue culture dish. The animals stick a little to the bottom of the dish which slows locomotion and makes it easier to cut the heads. An alternative method is to briefly chill the worms to immobilize them. Cut them (with a razor blade fragment held in a blade-holder) at the level of pharyngeal-intestinal valve. A solution of 0.1% bovine serum albumin (BSA) in Dent's saline should be used to release dissected tissue from the bottom of the Petri dish and ensure that it does not stick in pipettes. Transfer cut heads to the recording chamber in approx 5μl of 0.1% BSA Dent's using a Gilson pipette. To prepare the electrode for recording, pull a low resistance micropipette large enough to fit the head of the animal but small enough to provide a snug fit, i.e. about 10 μm diameter. A good pipette will last for half a dozen recordings or more if care is taken not to break it. Back-fill the micropipette with Dent's saline with a micropipette filler and attach it to the pipette holder. This has a silver-chloride coated silver wire to act as an electrode. The pipette holder should also have a side arm to which a length of tubing and a large (50 ml or more) syringe with a three-way tap is attached to enable suction to be applied. It is important that this is sealed well so that the suction cannot leak. Earth the apparatus to the signal ground input on the recording amplifier. Switch on the recording amplifier and set the tip potential to 0 mV using the voltage offset. Check the noise level. The EPGs are relatively small in amplitude and therefore the technique cannot tolerate a noise level of much more than 0.2 mV. Bring the electrode near to the anterior tip of the head and apply suction to the electrode thus attaching the head to the electrode. Close off the three-way tap to maintain suction. This helps to keep the head on the electrode. The noise will increase as the resistance of the seal increases. It may therefore be necessary to trouble-shoot additional sources of electrical noise (turning off the microscope lamp may help). If a perfusion system is being used then it can be started at this point. The amplitude and shape of the EPG are very dependent on the resistance of the seal and the relative placement of the electrode and therefore it is difficult to make comparisons between individual animals unless one is careful to control for these factors. Recordings may be made in voltage recording (‘Bridge’) mode. Recordings in either voltage-clamp or current-clamp do not add any advantage and simply introduce more electrical noise. This may necessitate filtering which can markedly distort the shape of the signal. Drugs may be applied via the perfusate or by aspiration of the bathing solution in the recording chamber.

EPG recordings are very stable and may be maintained for up to one hour or more, enabling detailed analysis of the response of the pharynx to different pharmacological agents. More care is required when making comparisons between mutant animals due to the intrinsic variability of the EPG shape. One aspect of EPG recordings that remains relatively limiting is that the analysis is still most accurately done manually, by cursor measurements on the digitized records. An example of an EPG is shown in Figure 1. There is scope for new development here in the form of software for reliable automated analysis of EPG shape.