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Figure 1. Example of an EPG recorded from a wild type (N2) pharynx. Note the separation of the positive going events corresponding to contraction of the corpus and terminal bulb, a number of “inhibitory potentials” correlating to activity of the M3 motorneurones. Negative going events are also to be observed which correspond to the relaxation of the corpus and terminal bulb respectively.
3. Protocol 2. Intracellular recordings from the terminal bulb
3.1. Equipment
The equipment is similar to that required for EPG recordings except that some of the items are required to a higher specification. First, a very stable surface is required and it is advisable to use an anti-vibration table. Two manipulators are required, one coarse and one fine. These should be mounted on opposite sides of the microscope. The microscope will also require better optics than for EPG recordings e.g., a x40 objective. A recording amplifier such as the Axoclamp 2B (Axon Instrument, USA) or similar is required. The properties of the intracellular electrode are the key to making successful recordings from the pharynx. It must be robust enough to penetrate the tough basement membrane without damaging the small muscle cells. This requires a short shank with a very sharp tip. It is also advisable to use hard glass e.g., quartz or aluminosilicate (1mm outer diameter, with filament). Ideally, the electrodes should be pulled on a high specification horizontal puller (e.g., Sutter P2000 laser puller) capable of reproducibly pulling hard glass. The electrodes should be pulled to a resistance of 80 to 100 MΩ resistance, filled with 4M KAcetate; 10mM KCl.
3.2. The intracellular recording experiment
Prepare cut heads and transfer them to the recording chamber as described for Protocol 1. Next pull a suction pipette, similar to, but with a slightly smaller tip opening than that used for the EPG recordings. The tip opening should fit over approximately one third of the terminal bulb. Place the suction electrode in an electrode holder (with a side-arm attached to a syringe via plastic tubing) and mount onto the coarse micromanipulator. Place the tip into the recording chamber and allow to back-fill with saline. Place the intracellular electrode in the pipette holder mounted in the fine manipulator on the opposite side of the microscope. This pipette will be used to hold the preparation in place and impart some structural rigidity to facilitate impalement of the terminal bulb muscle cell by the sharp electrode. Bring the tip of the suction pipette near to the terminal bulb of the pharynx and apply suction via the syringe so that the terminal bulb is held gently by the pipette against the bottom of the recording chamber. Then advance the intracellular recording electrode towards the opposite side of the terminal bulb until the tip dimples the muscle cell membrane without actually puncturing it. Intracellular access to the cell may be achieved by over-utilization of the capacitance compensation for the amplifier which causes the tip to cut through the membrane. (This ability is present on most amplifiers via a ‘buzz’ or ‘zap’ control). The resting membrane potential for wild type (N2) worms using standard Dent's saline outside is approximately −70 to −80mV. Every pharyngeal muscle pump coincides with an action potential that overshoots 0 mV by approximately 25 mV (Franks et al., 2002; Figure 2).
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Figure 2. An automated system for the correlation of electrical potentials and muscle movement in the C. elegans pharynx. Intracellular recordings from the terminal bulb were made in the manner described. The muscle was simultaneously imaged using a black and white CCD. The CCD supplied a standard PAL, interlaced video signal from which the vertical sync was extracted using a simple circuit developed in-house. The extraction circuit was placed in-line between the video camera and a PC-based frame grabber. It consisted of an LM1881 chip, used to read vertical sync data and pass it to a monostable oscillator (a 555 chip) set to generate one TTL pulse (Tag) for every two fields in the video feed. In an interlaced video signal, two fields corresponds to one full frame of video. Therefore, every frame in the video sequence had an associated tag which was then passed to the Digidata acquisition system to be recorded simultaneously with the electrophysiological events. A single command switch simultaneously controlled the video feed to the frame grabber and also the output of the tag signal to ensure that the first and last recorded video frames corresponded to the first and final tags in the sequence sent to the data acquisition system. Each video frame was automatically analysed using a simple image/signal correlation algorithm. The degree of muscle movement between video frames was calculated and matched back to the timing tags in the electrophysiological recordings. Briefly, a region of interest ROI was defined manually and then signal intensity for each pixel in the ROI was automatically calculated. The mean square difference (MSD) of ROIs in adjacent frames was calculated and used as a measure of muscle movement. As the timing tags were based on the vertical sync signal of the PAL video feed MSD calculations were 40 ms apart. The figure shows output from the full recording setup. A. Each frame of the video (iii) signal can be directly associated with a TTL timing tag (ii) recorded simultaneously with the electrophysiological recording of membrane potential (i). The lumen of the terminal bulb can be seen to gradually open and close in each successive frame during the action potential. One action potential corresponds to one motor cycle in the terminal bulb. B. In this recording each full action potential (i) is reflected in the MSD trace (ii). The mean latency of peak muscle contraction to peak action potential amplitude in this experiment was approximately 150 ms primarily reflecting the latency associated with excitation-contraction coupling in the pharyngeal muscle. Only six action potentials are shown in the trace, which spans 4.5 seconds. However, the number of action potentials that can be recorded with corresponding video and then processed, is limited only by the hard disk capacity and processing power of the PC used.
4. Protocol 3. Patch clamp recordings from the pharynx
4.1. Equipment
As for Protocol 2. Although a voltage-clamp amplifier such as the Axoclamp 2B may be used for these studies, a patch-clamp amplifier such as the Axopatch 200B is preferable.
4.2. The patch clamp experiment
A protocol for recording whole cell currents from the corpus region of the pharyngeal muscle has been described by Avery (Shtonda and Avery, 2005). Recordings may also be made from the terminal bulb region (Vinogradova et al., 2006). The methods are similar to patch clamp techniques described for other cells with the additional challenge, and limitation, that the geometry of the pharyngeal muscle makes it less amenable to this approach and voltage control will not be accurate. Accordingly, care must be taken not to over interpret data from such studies. For recordings from the terminal bulb, heads are transferred to the recording chamber, which contains a suitable external saline e.g., Dent's saline. A suction pipette (filled with Dent's saline) is attached to the cut edge of the cuticle and used to gently hold the preparation against the base of the recording chamber. The pharynx of C. elegans is surrounded by a basement membrane which will interfere with seal formation and obtaining whole cell access. Therefore, before one can begin to record from the muscle cells one needs remove the basement membrane by enzymatic digestion. A method modified from (Richmond and Jorgensen, 1999) is suitable. First, treat the preparation with a solution of 0.1% trypsin in Dent's saline for 30 seconds and then wash gently with Dent's saline to remove trypsin. Next, treat for 2–4 minutes with a solution containing (in Dent's): 0.23 mg ml-1 Protease Type XIV and 0.62 mg ml-1 Collagenase P. (The exact timing of this second treatment is crucial for success and will have to be determined by the operator). Finally, wash gently with Dent's to stop digestion. Patch electrodes for whole-cell recordings may be pulled from Haematokrit capillaries (resistance 3–5 MΩ) and filled with appropriate internal solutions (e.g., composition, in mM: 150 N-methyl-D-glucamine, 4 MgCl2, 5 HEPES, 0.25 CaCl2, 36 sucrose, 5 EGTA; pH 7.2). Single-channel recordings may be performed in outside-out and inside-out configuration using thick-walled electrodes (GC150-7.5 glass, Harvard Apparatus). The electrode tips need to be coated with beeswax (to reduce electrode capacitance) and fire-polished. Using these methods seal resistances in the range of 10–30 GΩ can be achieved. Data may be acquired with a patch-clamp amplifier in the standard manner. However, a special consideration for whole cell recordings from pharynx is that the input capacitance is very large and cannot be reliably and accurately compensated. Doubtless this is because of the highly folded surface of the muscle cell membrane and extensive electrical coupling between cells. An example of a whole cell recording is shown in Figure 3.
