Paired Patch Clamp Recordings from Motor-neuron and Target Skeletal Muscle in Zebrafish

1. Preparation of Fish for Paired Recordings

1. Prior to beginning the procedure, prepare five key solutions, which include: Bath Solution, Bath Solution with Tricaine (MS222), Bath Solution with Formamide, Neuron Internal Solution and Muscle Internal Solution.
2. Next, collect a single larval zebrafish between the ages of 72 to 96 hours and place in Bath Solution with Tricaine. Within one minute of exposure to the anesthetic, the fish will not respond to touch.
3. Place the fish on top of a plastic dish and, using a stereomicroscope, decapitate with a double edge razor blade.
4. Transfer the fish to a glass recording chamber coated with sylgard and filled with Bath Solution. Fasten at each end by inserting electrolytically sharpened tungsten pins through the notochord.
5. Create a skin flap near the rostral end of the fish with a pin that will provide a adequate grip for a pair of fine forceps. Remove the overlying skin by gripping it near the rostral pin with a pair of fine forceps. Peel slowly in the caudal direction to remove the skin.
6. Treat the fish with 3 mls of Bath Solution with Formamide for 3-5 minutes to block muscle contractions that might interfere with recordings. Wash 5 times with normal Bath Solution. Remove part of the superficial layer of muscle over 5 to 6 segments by gently scrapping with the side of a tungsten pin.
7. Transfer the fish to an upright microscope located in a Faraday cage to shield electrical noise. The microscope is equipped with a fixed stage, long working distance 40x objective and zoom of .5 to 4x. The microscope is mounted on a motorized X-Y translation stage in order to move the preparation between nerve and muscle under high power. The use of differential interference contrast optics helps to visualize the neurons.
8. Under 0.5 x zoom, use a wide bore 15-micron pipette to remove overlying muscle and expose the spinal cord. Negative pressure is applied through a 3 cc disposable syringe and muscle cells are removed one by one. This procedure is repeated for 5 to 6 dorsal segments of skeletal muscle. The same wide bore pipette is used to also remove the upper two layers of ventral muscle to obtain target fast skeletal muscle.
9. This completes the preparation for paired recordings and the microscope zoom is increased to approximately 1.6x.

2. Paired Recordings of CaP Motor-neuron and Target Muscle Cells

1. The cleaned segments are scanned to locate the CaP neurons. Each hemi-segment of spinal cord contains one large, 10 micron diameter, CaP motor-neuron. This teardrop shaped soma is located superficially under the spinal dura near the caudal most end of the segmental boundary. A CaP neuron is chosen for recording and the ventral target muscle in the adjacent caudal segment is scanned to check integrity.
2. Two patch electrodes are positioned for recording, one for the neuron and a second for muscle. The upper electrode has a shallow taper for penetration of the tough spinal dura and tip opening of approximately 2 microns. The lower electrode has a steeper taper for low resistance voltage clamp recording of skeletal muscle
3. Position the neuron electrode at an angle of approximately 30 degrees to penetrate the spinal dura, about one half-segment rostral to the selected CaP neuron. Advance the electrode using an axial drive manipulator while applying a gentle continuous positive pressure of approximately 60 millimeters mercury. As the electrode breaks through the dura, the CaP neuron may be displaced by the perfusion from the electrode, requiring a readjustment of the focus.
4. When the electrode touches the CaP neuron soma positive pressure is released and usually results in immediate formation of a gigaohm seal. However, in some cases where a seal fails to form, it becomes necessary to apply a gentle negative pressure. After seal formation, the electrode potential is adjusted to -80mV and application of negative pressure ruptures the cell membrane. The CaP neuron is characterized by an input resistance of 140 to 180 Mega-ohms and an action potential that overshoots +40 millivolts. This is tested by injecting incrementally increasing depolarizing steps under current clamp until the action potential is elicited.
5. Next, the microscope is relocated to ventral muscle using the X-Y motorized translator under high power. A fast muscle cell is chosen from the target field.
6. The muscle electrode is advanced toward the target muscle cell under positive pressure, which when released, forms a gigaohm seal. The electrode potential is adjusted to -50 millivolts to inactivate sodium channels and under gentle suck the cell membrane is ruptured. A drop in resistance to 100 to 200 Mega-ohms and the sudden appearance of the large capacitive transients signals entry.
7. To test for paired recording, the motor-neuron is depolarized by incrementally larger injections of current until an action potential is elicited. At this point an endplate current should be elicited in muscle. Continued stimulation of the motorneuron at 1 Hertz results in endplate currents without failure. As the stimulus frequency is increased to 100 Hertz the endplate currents fluctuate in amplitude and undergo functional depletion within 10 seconds of stimulation.

3. Representative Results

Typically, the neuron recording is stable for up to one hour but the muscle cells deteriorate as reflected as an increase in the series resistance. When this occurs the experiment is either terminated or another muscle cell is patch clamped. All recordings should be completed within one hour.

Figure 1. Recordings of the motorneuron action potential (AP, top) and the associated muscle endplate current (EPC, bottom). The action potential for a CaP neuron should overshoot +40 mV and the EPC should rise within 300 μsec and decay along an exponential time course with a time constant under 1 msec.

Table 1. Solutions.