Compound Action Potentials and the Sciatic Nerve

The northern leopard frog (Lithobates pipiens) was cooled in ice water for 15 minutes and then double pithed. To remove the skin from the legs, an incision was made around the entire lower abdomen. The skin was pulled off the frog much like pulling off a pair of pants. The frog was moistened with Ringer’s solution periodically. The muscles and urostyle were cut and pushed to the side to reveal a cream colored sciatic nerve. A piece of suture thread was tied to the proximal end of the nerve and it was cut superior to the knot. The nerve was dissected down shortly past the knee. The sciatic nerve was then ready for testing.

The sciatic nerve was placed onto the nerve bath chamber with a stimulator connected to a power supply and recording lead wires. To minimize electrical noise, the nerve bath chamber was set up in a grounded Faraday cage, the shortest shielded cables were used and personal cell phones and other surrounding electrical devices not necessary for the experiment were turned off. To test our hypotheses seven exercises were conducted. Throughout each exercise the nerve was periodically removed from the chamber and bathed in Ringer’s solution.

In the first exercise we briefly applied a stimulus at the proximal end of the nerve and recorded a compound action potential from the distal end as a test of the viability of the nerve. The pulse amplitude was set at 0.025 volts, number of pulses to one and pulse width at 0.1ms. The stimulus was increased by 0.05v until a moderate sized compound action potential (CAP) was recorded.

Exercise two quantified the relationship between amplitude of the stimulus and amplitude of the CAP. Stimulus amplitude was set at 0.00v to produce a flat line. The stimulus amplitude was increased to 0.05v. A compound action potential was not detected so we increased the stimulus amplitude by an increment of 0.05v until the threshold response of the nerve was detected at 0.5v. From there we continued to increase the stimulus amplitude in 0.05 increments until the maximal response of the nerve was observed.

Exercise three measured the velocity of action potential conduction. The stimulus amplitude was set to the lowest voltage that creates a maximal CAP, 0.65v also known as the short path. The lead wires were adjusted so that the recoding lead wire was farther from the ground electrode approximately 1.0cm. This was the long path.

Exercise four measured the effects of cooling on the velocity of action potential conduction. The sciatic nerve was bathed in chilled Ringer’s solution for several minutes then followed the same protocol as exercise three.

Exercise five measured the effect of stimulus frequency on the amplitude. The stimulus amplitude was set to 0.65v, number of pulses set to two, pulse width to 0.1 msec, time between pulses was 10 msec and holding potential set to 0. The time between intervals was then decreased by increments of 1 msec.

Exercise six determined the relationship between the stimulus amplitude and the stimulus duration needed to generate a compound action potential of defined amplitude. The initial critical stimulus parameters were set as the following: stimulus amplitude-0.100v, number of pulses-1, pulse width -0.1msec, time between pulses-0.9msec, holding potential-0. The stimulus amplitude was increased by increments of 0.100v until the compound action potential reaches a maximum level. The CAPs at each new stimulus amplitude were recorded. The criterion was determined from this data as the stimulus amplitude that delivered a mid-sized compound action potential. We used to criterion CAP as the set amplitude and then adjusted the stimulus durations to 5, 2, 1, 0.5, 0.2, 0.05 msec.

The final exercise used the same techniques in the previous exercises to determine if action potentials can travel in either direction and if there is a difference in the conduction velocity in one direction compared to the other.