Bryan Hummel
Vert.
Physiology 99
September
21,1999
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.