BIOL 3810.506
Compound Action Potential of the Bullfrog Sciatic Nerve
TA: Vinoo Urity
Introduction
One of the primary and unique characteristics of action potential is that it can be “simultaneously elicited in every axion through electrical stimulation.” This scenario is referred to as CAP (compound action potential) which is artificial, collective nerve response. The action potential amplitude for every individual axon does not change alongside stimulus intensity, but increases alongside the CAP amplitude at higher stimulus voltage which includes other axons existing within the same nerve reaching their own threshold.
Materials and Methods
In this experiment, the sciatic nerve is removed from a frog to conduct a compound action potential experiment. PowerLab 500 hardware and the Lab Tutor software are used to test the nerve for oscillation and data.
I. Frog Sciatic Nerve Dissection
A bullfrog was captured and secured to a dissecting table using straight needle pins. The frog was dead and double-pithed and could not feel the pain during the procedure. The frog is successfully dissected and the nerve removed to be used in the experiment that followed.
II. Setup of Electrophysiology
Once the frog is successfully dissected using the appropriate procedures and the nerve retrieved, the experiment proceeded by setting up the nerve chamber and LabTutor.
Compound Action Potential Elicitation
The first exercise in the experiment used the sciatic nerve to identify the maximal and voltage CAP amplitude. In the LabTutor Stimulator panel, the stimulus voltage was originally set at 10Mv. After tapping Start button, the tool (LabTutor) had the nerve stimulated and gave 5ms. The stimulus voltage was then increased to another level to 15mV and then recorded. This was repeated two more times, increasing the voltage by 10mV and the three resultant responses were recorded.
Refractory Periods
The second exercise in the experiment involved sending two stimuli to the nerve, while decreasing the interval between them progressively every time to figure out the absolute refractory time. There was need to set the stimulus voltage at the least level which is needed to provide a maximal CAP in the initial exercise. A good reading was obtained at 200nV. The interval of the stimulus was placed at 4.0ms. After tapping star, the software (LabTutor) stimulated the nerve two times as the interval and recorded for 10ms time period. The interval was then decreased to 3.5ms in the stimulator panel then started. The procedure was repeated reducing the interval to 3.0ms followed by 2.5ms, 1.9ms, followed by 0.1ms steps until stimulus interval reached 1.0ms.
Conduction Velocity
The third exercise in the experiment was conducted to determine the nerve conduction velocity. A new voltage was then entered to the stimulator panel (twice as much as the voltage used in Exercise 2 above, which is 2(200mV). LabTutor was the run to record data for 5ms after tapping start button. The CAP was represented by the upper channel from the nearest electrode for recording and the lower channel represented the CAP from the far electrode. The distance between the electrodes was estimated through a measure of the distance between negative leads existing between the two electrodes in millimeters. The CAP time interval to move between the two electrodes was identified by estimating the distances between the further and nearest CAPs.
Results
Eliciting the Compound Action Potential
The sciatic nerve for the frog threshold stimulus Voltage was studies using 10Mv. With every increase of the voltage stimulus, a congruent increase was observed in the CAP amplitude. At 40mV stimulus voltage, the suprathreshold or the maximal stimulus was established at 1.96mV.
Refractory Periods
200mV was the stimulus voltage adopted in the second exercise in the experiment. The initial decrease, which was lower significantly, was detected at the 2nd CAP magnitude was observed at a stimulus interval of 3.5ms. The 2nd CAP stimulus interval first disappeared at 1.7ms. The connection to the stimulus intervals with the 2nd CAP in shown in figure 2. Figure 2 shows all the resulting amplitudes after testing the CAP.
Conduction velocity
In the conduction velocity exercise, (the third exercise), a 400mV stimulus voltage was used. The distance between the electrodes was 21mm. one CP was yielded at the nearest electrode taking about 1.1ms to reach the further end electrode. Using the following velocity formula and the obtained data :( V= Δd/Δt), the conduction velocity and the average fiber velocity, in the frog sciatic nerve was computed to be 18.9m/s. The used data summary for the computation of the velocity can be viewed in table 3. Table 3 shows CAPs amplitude elicited in the distal electrodes and proximal electrodes in regards to time.
Discussion
Compound Action Potential: Eliciting
Once every axon hits its threshold, the nerve assumes a fixed, all or nothing action potential. As the voltage got increased from 10mV, then to 20mV, to 30mV, etc., where progressively greater amplitudes were recorded in the CAPs. This suggested that at least one of the fibers existing within the nerve had met their thresholds previously. The initial voltage at 10mV voltage generated CAP amplitude of 8.12mV showing that at least one of the fibers attained threshold at that point. After gaining a 110mV stimulus voltage, the CAP amplitude did not increase again, showing that this was the maximal stimulus (the point where all the individual fibers thresholds within the nerve are achieved). The suprathreshold for the sciatic frog nerve of 110mV yielded a CAP amplitude not having the capability to reach an amplitude of more than 29.96mV. Finally, the nerves wanted a minimum voltage to elicit or trigger a compound action potential except that the amplitude of that compound action potential would not go up after attaining the maximal stimulus voltage.
Refractory Periods
While the time interval between the test stimuli and the conditioner becomes lower, the 2nd CAD indicated a significant reduction in amplitude at 2.5ms. A reduction in amplitude is significant relative to the refractory time because, at that time, only a small number of Na+ channels began to open again due to firing by the recent action potential. The 2.5ms intervals were hypothesized to form the true end of the relative refractory time since it was the shortest interval between the stimuli. The primary reduction could have been as a result of an error. When the distance between the stimuli hit 1.7ms, 2nd CAP was non-existent. This shows the end of the absolute refractory time, since after which, CAP can undergo propagation. The refractory time (absolute), which is the tie just after the firing of the action potential, the NA+ channels becomes deactivated, completely. This inactivation state renders the neuronormal membrane to get stimulated by any other stimulus. Since the axons existing within the nerve have different absolute and relative refractory periods, the refractory periods discovered mark the axon with the shortest relative and absolute refractory periods.
Conduction velocity
The bullfrog sciatic conduction velocity was estimated to be 18.9ms, which was just a little below the normal range for any other nerve fiber, which was estimated to be 50 to 60m/s. The stated results could be as a result of a conversion error that was initially in the program. In case the estimation in milliseconds was actually in seconds, the conduction velocity would have led to a lower level of estimated velocity.
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