The overall goal of this procedure is to demonstrate how to expose and distinguish the phasic and tonic motor terminals with morphological staining and electrophysiological recording in the crayfish extensor muscle. This is accomplished by first pinching off the first or second walking leg from a mid-size crayfish, cutting out the cuticle and removing the flexor muscle in the meite segment. This exposes the extensor muscle and the nerve bundle.
The second step of the procedure is to stain the preparation with four ethyl amino sterile and methyl peritoneum iodide, or four D two asp to visualize the axons and nerve terminals. The third step of the procedure is selectively stimulating tonic and or phasic neurons, and simultaneously doing intracellular recordings of evoked EPSPs in the muscle fiber. The final step of the procedure is to use a focal electrode to record spontaneous and evoked qu events from the terminals.
Ultimately, results can be obtained that separate tonic and phasic terminals, morphologically and physiologically through immunofluorescence microscopy and electrophysiology. This method can help answer the key questions in the field of synaptic physiology in areas such as muscle fatigue, synaptic depression, and synaptic crosstalk. The experiment starts by picking up the crayfish carefully so as to avoid its claws.
Detach the leg from the body at the isia pod segment. A forceful pinch at the isia pod segment induces the crayfish to automize or naturally break off the first walking leg. The crayfish will stop bleeding and is placed back in its holding tank.
The crayfish is not sacrificed. Place the leg on a piece of tissue paper so the preparation can be turned easily while making cuts. Turn the leg around until the outside, which is the lateral side is facing up on the dissection plate.
This generally has the arched side facing up. Using a sharp razor blade attached to a scalpel blade breaker etch the cuticle within the meite segment by following the pattern shown in the figure. Be careful not to cut too far distally on the dorsal to ventral cut along the meite carpod joint.
This cut results in a window in the meite above the underlying leg muscles. Leave the cuticle in place for now. Place the preparation in a dissection dish containing crayfish physiological saline.
At this point, place a pin in the middle of the carpod segment and in the dorsal aspect of the ischia pod segment with fine tweezers, lift the window cut in the cuticle slightly from the distal end, and using the razor cut the flexor muscle fibers away from the cuticle. Continue cutting in a distal to proximal manner, and finally lift the window of the cuticle off. To expose the extensor muscle, the overlying flexor muscle must be removed.
Separate the tendon of the flexor muscle by displacing it from the flexor muscle with a pin. Then cut the tendon at the Mari poddy carpod joint. As shown, be very careful to pull the tendon away from the leg cavity before making the cut and to cut only the tendon and not the main leg nerve that is on the inner side of the tendon.
Pinch the tendon where it was cut with tweezers and pull the flexor muscle off of the leg by lifting it in a coddle direction To expose the nerve to the extensor muscle, cut the main leg nerve at the merepod carpod joint and carefully pull the main leg nerve back over the extensor muscle. The separation of the nerve to the extensor muscle from the main leg nerve can be enhanced by gently pulling the distal stump of the main leg nerve to the side of the preparation. When peeling the main leg nerve back over the extensor muscle, small branches of the inhibitory axon may need to be cut.
The nerve bundle can be visualized with methylene blue staining. However, methylene blue would not be used for electrophysiological recordings in this top image. Red arrows to mark the nerve track of interest.
The nerve bundle of interest is the larger bundle branching off the main leg nerve near the proximal end of where the merepod is. Note the axon branching and the two readily visible axons within the nerve. Now that the nerve and muscle are exposed, prepare the apparatus used for the experiment.
The intra and extracellular recordings are done with a standard head stage and amplifier. We use a model axon clamp two B amplifier and a one XLU head. Stage three electrodes are needed, one intracellular and two extracellular.
The two extracellular electrodes will be used to stimulate the nerve and to record synaptic evoked and spontaneous qual events. Fire polish the focal electrodes and bend them slightly over a heating element. After filling a focal electrode with saline, place it in the stimulating electrode holder and insert the stimulator wire so that it contacts the saline.
This is connected to a grass P 15 stimulation isolation unit, which is connected to an S 88 grass stimulator. During electrophysiological recordings, the vital dye for D two ASP is used to visualize the axons to stain the axons, exchange the bath saline with saline containing five micromolar of four D two as per dye for five minutes. After five minutes, refill the dish with the crayfish physiological saline, placing the preparation under a microscope and using a Forex objective.
And at 10 x eyepiece, move the stimulating electrode to the area of interest. The next step is to place the lumen of the macro patch stimulating electrode on either the phasic or the tonic axon. This photograph of a four dye two as dye stained preparation shows that the tonic axon is more brightly visible due to the increased mitochondrial content.
The tonic terminals show numerous varicosities and the phasic terminals are of a thinner nature under fluorescence microscopy. And the 20 x objective place the electrode on either the tonic or phasic axon. Here the electrode is being placed on the tonic axon.
Place the intracellular electrode into its micro manipulator, place the intracellular electrode into an extensor muscle cell. This will be used to record intracellular excitatory postsynaptic potentials or EPSPs. For recording qual EPSPs.
Place the second focal electrode into a micro manipulator. This is done by removing the past intracellular electrode and holder and replacing with the clean electrode holder. Use the microm manipulator to place the lumen of the second focal electrode directly over a synaptic varicosity.
Electrode and seal resistance are determined by passing test current pulses through the electrode. In our experiments, seal resistances ranged from 0.3 to one mega ohm and the electrode resistance range from 0.5 to one. Mega ohm seal resistance can be monitored throughout the recording.
When ready to begin the stimulation, turn on the scope 5.0 software and start stimulating the isolated axon. In the nerve bundle, the tonic axon is stimulated by the suction electrode and the EPSPs of the muscle fiber are recorded with a the intracellular or extracellular electrode. The EPSPs are recorded to a computer via a power lab forest interface.
Following stimulation of the tonic axon, the stimulating electrode can be moved to the phasic axon and the experiment repeated following nerve stimulation. The intracellular electrode records EPSPs generated in response to both tonic and phasic motor neuron activity. Synaptic facilitation of the tonic neuromuscular junctions depends on the frequency of nerve stimulation as shown here for successive trains of approximately 20 stimuli each at a frequency of 60 hertz.
Note the facilitation that occurs with multiple stimuli. This figure compares the amplitudes of the EPSPs generated by stimulation of the tonic nerve at frequencies of 20 hertz in blue, 40 hertz in green, and 60 hertz In red, note the market facilitation shown at higher frequencies of stimulation. Tonic terminals are low in synaptic efficacy, but show dramatic facilitated responses shown.
Here are the tonic EPSPs alone, and then the phasic responses are superimposed at the same scale. In contrast to the tonic terminals, the phasic terminals are high in qual efficacy, but show synaptic depression with high frequency stimulation. Synaptic depression at the phasic terminals is also related to frequency of stimulation as shown here.
A continuous five hertz stimulation over 30 minutes depresses the response at the neuromuscular junction. With higher stimulation frequency, the preparation will depress more rapidly with appropriate stimulation. The crayfish neuromuscular junction generates nons spiking EPSPs.
These EPSPs can be used to investigate the qual properties that make up the graded electrical signals in these muscles. Here, the qual synaptic responses of the tonic terminal are shown. Note that not all stimulation trials result in a qual event.
Direct counting of qual events is possible with low frequency stimulation for each evoked response. The number of qual events can be readily determined for the tonic terminals. By counting quantal events, the synaptic responses from the phasic motor neuron can be recorded in the same manner.
However, the evoked responses are multi qual in nature. Mean qual content is estimated by using the mean amplitude or area of the deflections along with the average peak amplitude or area of spontaneous events. A final reminder, be careful when removing the flexor muscle so as not to damage the nerve in waiting the extensor muscle.
That's it. Thanks for watching and good luck with your experiments.
