Your Actions Are Getting On My Frogs Nerves.

Introduction: The sciatic nerve of the American bullfrog was used to extracellularly measure action potential movement down the nerve. An action potential is an all or none reversal of a membrane potential produced by regenerative inward current in excitable membranes (Eckert G-1). The sciatic nerve is a compound nerve consisting of several individual axons, so the actual extracellular action potential that is measured will be the sum of the action potentials of each individual axon. By using the distance between the recording electrodes and the stimulus, and by measuring the time it takes for the nerve impulse to get from the stimulus to the electrode, we can calculate the conduction velocity of the compound nerve. The effects of temperature and nerve damage on the speed of the action potential will also be tested.

Materials and Methods: An American bullfrog was dissected to remove one of the sciatic nerves between the spine and the “knee” joint. The nerve was placed across two electrodes in a nerve chamber and then submersed in Ringer’s solution. There was a separate stimulator which was touched to the nerve in order to stimulate it into producing an action potential. A computer was used to both record the size of the nerve impulse and the time it took for the pulse to reach the recording electrode.

Results: The bullfrog was dissected to remove one of the sciatic nerves between the spine and the “knee” joint being careful to avoid touching the nerve with any metal instruments. Before the nerve was removed from the frog, each end was tightly clamped shut with a silk suture. The nerve was placed in a nerve chamber across several electrodes and then submersed in Ringer’s solution. The ringer’s solution was removed from the nerve chamber before the testing began to prevent the solution from carrying the current instead of the nerve. There was a separate stimulator which was touched to the nerve in order to stimulate it into producing an action potential. The stimulation intensity was increased until the action potential reached a maximum peak. A computer was used to both record the size of the nerve impulse and the time it took for the pulse to reach the recording electrode. The polarities of the recording electrodes were reversed to see what effect it had on the biphasic action potential waveform. By using the distance between the recording electrodes and the stimulus, and by measuring the time it takes for the nerve impulse to get from the stimulus to the electrode, we can calculate the conduction velocity of the compound nerve. The same process of calculating the nerve impulse conduction velocity was repeated after the nerve was cooled to 4 degrees Celsius and again after it was heated to 37° C. The nerve was then actually reversed in the chamber to see if the action potentials can flow in both directions. The nerve was also subjected to TTX (tetrodotoxin, 4-toothed poison) and then crushed to observe the effect of trauma on the nerves ability to carry a charge. What was observed was that the conduction velocities at room temperature were 0.022m/s, 0.027m/s at 4°C and 0.021m/s at 37°C. Neither the TTX nor crushing the nerve had any effect.

Discussion: The action potential in a neuron are dependent on unequal distributions of Ions (mainly Na⁺ and K⁺), and the functioning of both chemical and electrical selective gated Ion channels. Once the cell membrane becomes depolarized to a certain threshold, the voltage gated sodium channels open and allow sodium to rush in which further depolarizes the membrane and causes the perpetuation of the action potential. At the highest state of depolarization, the potassium channels open and initiate the falling phase by allowing potassium to follow its concentration gradient and flow out of the cell. This causes hyper- polarization and eventually allows the cell to come back to its resting potential, ready to promote another action potential. Like many life processes, the conduction velocities of the action potentials were expected to slow when cooled and speed when heated, but this was not supported in this experiment. The sciatic nerve did exactly opposite of what was expected. The nerve-blocking agent TTX was expected to stop the action potential from forming because the sodium channels would be blocked and the wave of depolarization would not propagate. The tetraethylammonium, which blocks the potassium gates, was expected to severely reduce the cadence of the nerve impulses because of the long time period it would take the neuron to achieve its resting potential once stimulated. The frog nerve could be stimulated several more times after the addition of TTX with no significant change.

The crushing of the nerve was theoretically supposed to stop the action potential by not allowing the wave of depolarization to pass the point of damage, but this frog’s nerves were destined to defy the theory by acting normal even after being crushed multiple times by multiple people. Action potentials do propagate in both directions in the frog sciatic nerve. This was tested by physically reversing the nerve’s direction in the nerve bath and recording what happened. Typically in vivo, the action potential is unidirectional, but new research shows that a reduced version of the forward propagating action potential is actually sent in the direction from which it just came (Heinz Valtin 1996). The axon terminal of the sciatic nerve attaches to (synapses with) muscle fibers, so the neurotransmitter that it releases would be acetylcholine. Because the sciatic nerve is a compound nerve composed of several neurons with different destinations and importance, It would seem logical that they were not all the same size. Finally, the results from the electrodes were biphasic because the electrodes were measuring the difference between them. This means that as the action potential approaches the first electrode, they read the same charge. When the action potential reaches the first electrode, the first electrode is much more negative than the second. When the maximum of the action potential reaches the second, it is much more negative than the first. This accounts for the biphasic nature of the graphs. My final conclusion is that I am finished writing this lab report.

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