Animal Physiology Lab

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Lab Two. FROG SCIATIC NERVE: PROPERTIES OF PERIPHERAL NERVE TRUNKS

Familiarize yourself with the oscilloscope as instructed. Calibrate it using the built in 400mV square wave. In this section we begin by practicing with a wet thread.

1) Instrument Complex and Arrangement

Soak an 80mm cotton thread in Ringer's Solution. Now place the thread across all the electrodes in the moist nerve chamber. Adjust the controls of the stimulator until you obtain an easily visible and stable trace on the oscilloscope. Adjust the voltage, frequency and duration controls to become familiar with the system. Observe the effects of increasing stimulus amplitude on the size and shape of this artifact. Try changing electrode positions. Be sure you can recognize the stimulus artifact (seen on the gray oscilloscope), for later you will need to be able to distinguish it from physiological responses.

• What effect does changing the voltage and duration controls have upon the shape of the oscilloscope display?

• What characteristics of the trace are common to all conditions? These are artifacts produced by the stimulator and may intrude upon any response trace!

• When we later study nerves, they will be kept moist with Ringer's Solution. Why do we soak our thread in Ringer's and not simple salted or distilled water?

• Move the recording electrodes to different positions and note the effect on the waveform.

2) Technique for Excision of the Sciatic Nerve. Performed by the instructor.

Double pith a large frog. Lay the frog ventral side up. With forceps raise the skin at the midline of the body between the forelegs and with scissors snip the skin around the entire body just below the forelegs. Now with one hand tightly grasp one forepaw and with the other hand strip the skin down and off the hind legs with one quick pull. Wash the frog in Ringer's and lay it on its back. Cut through the abdominal muscles and carefully remove the abdominal organs. to expose the three roots of the sciatic nerve on either side of the vertebral column. Turn the animal over and carefully separate the muscles of the dorsal side of the thigh. to expose the nerve. Do not cut the nerve yet. Follow the nerve into hip area, carefully cutting away the musculature and expose the three roots discussed above. Tie a ligature around the nerve at the spine and cut the nerve. Gently lift the cut nerve up with the thread and free it from the connective tissue and hip bone. Do not touch the nerve with metal dissecting instruments. Use glass probes to move it around. Isolate the nerve to the knee and there, tie another ligature, and cut the nerve free of the animal. Do not allow the nerve to touch the skin, do not touch it with any metal instruments, and do not stretch it. Then, place the nerve in Ringer's solution until you are ready to deal with it. Excise both nerves.

Anerve can be used for hours at a time for up to three days if it is stored at 8-12 degrees C in Ringer's solution when not in use. When being used the nerve should be wetted frequently even when within the moist nerve chamber. When you have obtained the live nerve, place a free end across the stimulating electrodes and lead the attached thread out of the end-hole and prevent movement by "locking" it into position on the spring. Pour a few ml of Ringer's in the chamber. Wet down the nerve, knock off any large droplets on the nerve or electrodes, and finally cover the chamber with its lid.

3. Live Frog Sciatic Nerve

A. Deriving the characteristics of the action potential.

i. Set the grey oscilloscope time sweep at 1 msec/div, the voltage sweep at 5mV/div, ground switch OUT, Triggering source Left IN (you will have to adjust the level knob to get a stationary artifact) and the stimulator frequency between 10-15/sec.

ii. Set the duration at 1msec and voltage at its lowest value. Gradually increase the intensity of the stimulus until the trace which appears on the oscilloscope (hopefully!) reaches its maximum height. This should be the full action potential of the sciatic nerve. Note the stimulating voltage, and the voltage of the action potential. You can adjust the voltage sweep to make it more sensitive. Measure the stimulating voltages using the black oscilloscope.

• Does the nerve follow the all-or-none law? Why?

• What is the value of the full a.p. in m.v.?

• Is the stimulus artifact seen in the a.p.? Under what conditions? You may have to repeat your study of the thread.

• In what way is a short stimulus of high intensity the same as a long stimulus of low intensity? Relate this to the molecular process of depolarization.

B. The Strength (Intensity) - Duration Curve

Determine and plot the threshold voltage for stimuli of different durations: 0.01, 0.1, 0.2, 0.5, 0.75 1, 2, and 5 msec.

• Is the product of intensity vs duration a constant for each point on your curve?

• If you were doing research on some aspect of nerve physiology, how would you use the idea of a "strength-duration constant" to give more precision to your investigation?

C. Conduction Velocity

Devise a method for measuring the conduction velocity of the nerve.

• Describe this method

NOTE: First, carefully define velocity as it relates to distance and time, both in the nerve chamber and on the oscilloscope screen. Remember that the stimulus artifact is in a constant position, and can be used as a reference for when the stimulus was delivered.

Move the recording electrodes to another pair of jacks and determine the speed of conduction over a greater distance.

• How does your measured velocity compare to published determinations of myelinated and non-myelinated neurons?

D. The Refractory Period

Set up two stimulators. Using the maximal voltage and the triggering source left IN/level knob, set up a stationary AP on the left side of the screen using one of the stimulators. Frequency 15/sec, duration 0.2msec seems to work well here. Then, set the second stimulator at the same level. Turn the second one on and watch as the second impulse travels towards the first (remember that the first must be locked) You will have to play with the frequency of the second to get it to move slowly towards the first.

• At what distance (time) does the second AP start to decrease?

• When does it completely disappear? Discuss both of these times.

• What happens after the two APs merge on the screen, and why? Explain this in terms of the function of the oscilloscope. Explain refractoriness in terms of the ionic basis of nerve activity.