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Abstract:
The purpose of this experiment was to examine how the compound action potentials propagate down the sciatic nerve of a frog based on varying stimuli. Before the nerve could be tested it needed to be extracted from the thigh of the frog and then submerged in saline. Several varying stimuli were used and recorded by a software package. The second experiment requires a constant stimulus with varying reference pin locations. From this the velocity of the action potential propagated can be determined.
The experiment revealed that as the intensity of the stimulus increased so did the intensity of the compound action potential (CAP).
Introduction:
As taught to us during class different stimuli are going to have different effects on the force the muscle generates. The muscle first had to be extracted. Dissection involved the use of scalpels, scissors, and tweezers to cut and remove both the skin and the muscle. While removing the This experiment will allow us to analyze how the action potentials generated by a nerve from a frog changes in response to differences in amplitude of a stimulus from a voltage source.
By analyzing the changes in action potential, we will be able to determine whether there is a certain trend in the action potentials and whether or not the nerve will reach a saturation point at which there will no longer be a change in the value of the action potential. Also, we will analyze the change in speed of the propagation of the action potential due to alterations in amplitudes of the stimuli. We predict this speed of propagation to be between 0 and 80 meters per second.
Methods:
At the beginning of this lab, a frog was soaked in a paralyzing agent and then pithed. This is done to not create any discomfort for the frog and euthanize them in the quickest and least painful way possible. After the animal is pithed, the dissection can begin. First, the skin of the animal must be removed off the legs. Simply cut around the torso and “de-pants” the frog by pulling the skin down the legs exposing the muscles. After this, the sciatic nerve must be found by separating the two muscles in the upper leg. The sciatic nerve is white and a vein is running along-side it. To extract it, tie a knot around it right above the knee and trace it up the torso into the central nervous system.
When the section is large enough, another knot should be tied around the nerve. The nerve should then be cut above the knots so they can be utilized to help remove the section. The nerve will have lots of braches which must be removed to properly extract the nerve. The biggest section possible should be extracted to properly span the testing tray. After extracted soak the nerve in saline and properly connect the leads to their proper places on the testing tray, configuration shown in Figure 1 below. After connections are made, the nerve should be set on the tray making sure that it spans from the first to the last connector. This is vital for proper results. Set your pulse width to .05 and set stimulus to only fire when the button is pressed. Make sure to keep the nerve moist on the tray so the cells in the nerve do not die.
Figure 1: Configuration for Testing Tray
The first experiment that should be done is to vary the stimulus intensity creating different action potentials and the second is to test the velocity that the nerve propagates action potentials. This can be done first running a certain stimulus intensity through nerve like previous and then moving the +R connector down one pin. Then send the same stimulus through the nerve and measure the distance between pin to aid in determining the velocity. V=(distance between pins)/(time difference between two same intenity samples)
Results:
Table 1: The amplitude of the stimulus and the change in the intensity of the compound action potential. Amplitude (V)∆ CAP Intensity (mV)
0.050.134
0.10.317
0.150.505
0.20.738
0.251.569
0.35.04
0.355.167
Table 1 shows the amplitude of the stimulus applied in the first column. The second column is the results of the change in the intensity of the compound action potential. A quick observation of the table reveals that as the stimulus is increased the larger the intensity of the CAP. When the amplitude reaches .3V saturation is revealed. It was also noted that as the amplitude of the stimulus increased the CAP got wider and its duration increased.
The velocity of the action potential propagating through the nerve was determined by finding the difference of the peaks with the same stimuli of 1 volt but with different reference nodes. This is shown in the equation below:
V=X/∆t=(4*〖10〗^(-3) m)/((.75-.5)*〖10〗^(-3) s)=(4 m)/(.25 s)=16 m/s
No further conduction velocities could be evaluated because of nerve death.
Discussion:
The CAP is the sum of all individual fiber action potentials of the given nerve. As the strength of the stimulus increases the, more fibers are recruited. Addition of fibers, produce more action potentials to produce a CAP with a larger curve. This is why the CAP intensity grew larger as the stimulus was strengthened. Once the stimulus strength reached a certain level saturation occurred. This could have occurred for two reasons. One reason is that all the fibers in the nerve have been excited and are conducting action potentials. The second reason could be because the software IWORX has a gain of 1000, which means the intensity cannot go above 5 mv.
The results show that the highest CAP intensity reached 5.167 mv this could be the IWORX attempting to limit the voltage. Simple action potential is an all or none phenomenon, which means that there are no grades of response. Either the action potential occurs or it does not. Either a stimulus is higher than threshold in a single fiber and action potential occurs, or it is too low and there is no action potential. Compound action potential considers the response of an entire nerve. The entire nerve consists of many fibers of different types: small, large diameter, absence or presence of myelin, each of which have different thresholds. So, given a particular stimulus some fibers may produce action potentials whereas some may not. Hence there is a gradation in response depending upon the number of fibers excited which produces the compound action potential.
Figure1: Sample response of nerves CAP
The source of the stimulus artifact as shown if figure 1, is from the input. The artifact is results from the virtually instantaneous spread of stimulating electrodes to the recording electrodes. This part doesn’t involve the fiber just yet, that’s why it’s faster than the CAP. The conduction velocity of 16 m/s can be most accurately identified as the response from the large amount of Aδ fibers being excited in the frog nerve. According to Wikipedia, this is reasonable given that conduction velocities of Aδ fiber range from 3-30 m/s.
While the results are reasonable we cannot be sure if the data is entirely correct. Only one trial for conduction velocity was done. Accurate data requires multiple trials. Also the errors could have affected the data. An error could be from the anesthetic used in the experiment. The anesthetic injected in the frog could have blocked action potentials from being produced. A second source of error could be the duration of the experiment. As the experiment went on; nerve death was occurring. The deterioration of the nerve could have decreased the amount of action potentials being produced.
Conclusions:
Overall the lab was a success. The relationship of stimulus intensity and compound action potential were examined. The lab revealed that as the strength of the stimulus increased; the larger the compound action potential. For future labs a less potent anesthetic, should be used to ensure action potentials were being propagated. More experience in dissecting, would extend the life of the nerve; extending the duration of the experiment allowing us examine the different conduction velocities of the nerve. We could also switch the input leads to see how the conduction velocities of the nerve compare in opposite directions.
