The overall goal of this procedure is to obtain simultaneous electrophysiological recordings of a motor neuron action potential and the associated synaptic response in target muscle. This is accomplished by first removing the skin on the top side of the fish. The second step of the procedure is to expose the spinal cord by removing all of the overlying dorsal muscle cells.
The third step of the procedure is to expose the target fast muscle by removing the upper layer of slow muscle. The final step of the procedure is to current clamp the coddle primary or cap neuron and voltage clamp the targets skeletal muscle. Ultimately, results can be obtained that show the synaptic responses in muscle that are associated with firing of motor neuron action potentials through simultaneous current clamp of the motor neuron and voltage clamp of the target muscle.
LaVar Super Fish offered a combination of advantages, facilitate paired motor neuron skeletal muscle, recording the small size of muscle permits, effective voltage clamp, and the stereotypical features of the cap motor neuron allow easy identification and accessing vi. Now, I'll step used through the procedures that are required to establish patch clamp of both neuron muscle Prior to beginning the procedure. Prepare five key solutions, which include Bath solution, bath solution with Trica bath solution with IDE neuron internal solution and muscle internal solution.
Next, collect a single larval zebra fish between the ages of 72 to 96 hours and place in bath solution with trica. Within one minute of exposure to the anesthetic, the fish will not respond. To touch place the fish on top of a plastic dish and using a stereo microscope decapitate with a double edge razor blade.
Transfer the fish to a glass recording chamber coated with sard and filled with bass solution. Create a skin flap near the rostral end of the fish with a pin that will provide an 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 coddle direction to remove the skin. Treat the fish with three milliliters of bath solution with form IDE for three to five minutes to block muscle contractions that might interfere with recordings. Wash five times with normal bath solution.
Remove part of the superficial layer of muscle over five to six segments by gently scraping with the side of a tungsten pin. 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, 40 x objective and zoom of 0.5 to four x.
The microscope is mounted on a motorized XY 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 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 three cc disposable syringe and a muscle cells are removed one by one. This procedure is repeated for five to six dorsal segments of skeletal muscle. The same wide boar pipette is used to also remove the upper two layers of ventral muscle to obtain target fast skeletal muscle.
This completes the preparation for paired recordings and the microscope zoom is increased to approximately 1.6 x. The clean 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 Jira, near the coddle most end of the segmental boundary. A cap neuron is chosen for recording and the associated coddle segment, ventral target muscle field is scanned to check integrity. 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 a tip opening of approximately two microns. The lower electrode has a steeper taper for low resistance voltage clamp recording of skeletal muscle. 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. When the electrode touches the cap neuron SOMA positive pressure is released and usually results in immediate formation of a giga 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 minus 80 millivolts 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 plus 40 millivolts.
This is tested by injecting incrementally increasing depolarizing steps under current clamp until the action potential is elicited. Next, the microscope is relocated to ventral muscle using the XY motorized translator. Under high power, a fast muscle cell is chosen from the target field.
The muscle electrode is advanced toward the target muscle cell under positive pressure, which when released forms a giga seal, the electrode potential is adjusted to minus 50 millivolts to inactivate sodium channels. And under gentle suction, the cell membrane is ruptured. A drop in resistance to 100 to 200 mega ohms and the sudden appearance of the large capacitive transient signals entry to test for pair.
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 motor neuron at one 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.
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.
Once mastered a pair, recording can be completed within one to one half hours. It's also possible to obtain one or two additional pairs in the neighboring segments. A typical day of recording will produce two to three pairs.
Paired motor neuron skeletal muscle recordings provide a unique opportunity to explore the fundamental workings of neuromuscular transmission. When used in conjunction with Motility mutant fish lines, it can pinpoint the source of neuromuscular dysfunction and provide new insights into the mechanisms underlying human diseases such as metastatic syndrome.
