Compound Action Potential Lab Report
Much like cells are the fundamental unit of a living being, neurons are the fundamental unit of the nervous system. These neurons work together with other excitable cells to produce action potentials when they receive electrical or chemical stimuli. Action potentials can be thought of as an “all-or-nothing” event and occur as a large-scale depolarization when sodium and other positive ions rapidly enter the neuron through membrane channel proteins. Once initiated, action potentials travel down the length of the axon and when it reaches the end a neurotransmitter is released into the synapse. Neurotransmitters work differently and some can act to inhibit the neurons while others activate them. After the action potential is complete, the neuron must repolarize and go through a refractory period. During this time the neuron is incapable of producing another action potential (Stillman, 2005). In this lab, compound action potentials (CAPs) will be measured from an isolated sciatic nerve from a cane toad, Bufo marinus. Compound action potentials are the total sum of action potentials from the multitude of neurons that compose a nerve (Stillman, 2005).
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Compound action potentials will be measured by stimulating the nerve with electrical impulses that increase in amplitude to determine the threshold voltage and maximal CAP amplitude.
The absolute and relative refractory periods will be found by stimulating the nerve with a series of pulses. The velocity of the compound action potential as it travels down the nerve will then be calculated to determine the nerve conduction velocity. Lastly, the temperature sensitivity of nerve conduction velocity will be tested by measuring the speed at which the compound action potential travels down the nerve at both hot and cold temperatures. It was hypothesized that threshold voltage, maximal CAP amplitude, refractory period, nerve conduction velocity and temperature sensitivity of nerve conduction velocity would be
determined.
Materials and Methods To test the sciatic nerve the nerve bath and PowerLab had to first be set up. Black and red alligator clips were were connected from the stimulator electrodes to two of the metal rungs on opposite sites of a nerve bath and the opposite ends were connected to PowerLab. Two more sets of black and red alligator clips were attached to metal rungs along one side of the nerve bath and to PowerLab. The lower reservoir of the nerve bath was filled with room temperature toad Ringer’s solution, taking care to make sure it did not come in contact with the metal electrode rungs. The connections were tested by placing a moist strip of filter paper over the wires and testing the connection through PowerLab (Stillman, 2005). Once the nerve bath and PowerLab were set up properly the sciatic nerve was removed from the double-pithed toad. This was done by cutting the skin around the abdomen and pulling it down over the legs. The urostyle was then cut free and the sciatic nerve was freed from the associated fascia and sciatic artery using a glass hook. The nerve was cut from the spinal cord and a piece of thread was tied around the end for easier handling. The nerve was further removed from the leg and severed at the gastrocnemius muscle. Ringer’s solution was used to rinse the nerve and filter paper was used to remove excess moisture. The nerve was laid across the wire electrodes of the nerve bath so that it was in contact with each of the active connections (Stillman, 2005). After the nerve was properly setup for testing, the nerve was given a series of increasing electrical stimuli. The nerve was stimulated using the “Threshold voltage” function of LabChart by automatically stimulating the nerve and recording for 1.1 seconds. This data was used to determine the threshold value voltage and the maximal CAP amplitude (Stillman, 2005). For the next part of the experiment LabChart stimulated the nerve with a series of pulses. In each block of data the pulse interval was decreased. The minimum stimulus voltage required to elicit a maximal CAP was found from the data of the last section of the experiment and used to adjust the voltage for this section. LabChart then recorded a series of 15 data blocks, each one 10 milliseconds in duration. The resulting data was used to determine the relative and refractory periods of the nerve (Stillman, 2005). For the third part of the experiment, the distance between the negative leads of each of the two recording electrodes was measure in centimeters. LabChart was then set to record a block of data in two channels for 10 milliseconds. This was used to calculate the velocity of the CAP as it travels down the nerve (Stillman, 2005). Lastly, the the effect of temperature velocity on nerve conduction was determined. The room temperature Ringer’s solution was removed from the nerve bath and cold Ringer’s solution was placed inside. LabChart was adjusted and set to record “Conduction Velocity”. This was repeated with warm Ringer’s solution (Stillman, 2005).
Results
To calculate the threshold voltage and maximum CAP amplitude data was taken over various increasing stimulus which ranged from 20mV to 410mV. To obtain a CAP the threshold stimulus voltage needed was reached at 180mV. The maximum CAP amplitude was observed at 8.36mV.
Figure 1: Picture of the threshold voltage of compound action potentials of sciatic nerve. Series of increasing stimulus given to dissected sciatic nerve for 1.1 seconds. Stimulus from 20mV to 410mV.
Figure 2: Scatter plot of CAP amplitude vs Stimulus Voltage (mV). Graph showing the correlation between the stimulus voltage to obtain a CAP. Threshold voltage was around 180mV and maximum CAP was at 8.36mV.
To observe the refractory period a difference of the first and second CAP were determined and used to find the relative refractory period (where there is a first and second CAP are visible), and the absolute refractory period (where there is a first peak and no second peak).
Figure 3: Figure used to identify the refractory period between CAP’s. The distance between the first and second CAP show that there is a 4ms relative refractory period on left picture. On the right there is no CAP and this is where the absolute refractory period is.
Table 1. Conduction velocities for the sciatic nerve of Bufo marinus in cold, room temperature, and hot toad Ringer’s solution.
Conduction Velocity at 4⁰C
18.60 m/sec
Conduction Velocity at 22⁰C
29.63 m/sec
Conduction Velocity at 35⁰C
32.0 m/sec
Q10 effect: Q10= (R2/R1)10/(T2-T1)^ R1= 18.60 m/s at 4o C (T1) R2= 29.63 m/s at 22o C (T2) Q10= ((29.63 m/sec)/(18.60 m/sec))10/(22-4) = 1.295
Q10= (R2/R1)10/(T2-T1)^ R1= 29.63 m/s at 22o C (T1) R2= 32.0 m/s at 35o C (T2) Q10= ((32.0 m/sec)/(29.63 m/sec))10/(35-22) = 1.061
Conduction velocity increased with increasing temperature (Table 1). Q10 effect was greater when the temperature went from 4o C to 22o C than when the temperature rose from 22o C to 35oC.
Discussion
When the stimulus reaches a threshold potential, the nerve or muscle fiber will respond fully to the stimuli, but will not react at all if the stimulus is under threshold which is the “all or none response” (Withers, 1992). This all or none response pattern is shown clearly in these data of CAP amplitude vs stimulus intensity, since there was no CAP response to the stimulus until 180mV (see Figure 2).
The CAP differs from a single action potential in several ways. A single action potential is intracellular, and does not change with stimulus intensity above the threshold, where a CAP is an extracellular recording of many axons and is a graded response that does increase with intensity of the stimulation above the threshold (Withers, 1992).
To find the threshold voltage needed to elicit a compound action potential various readings were taken at different voltage stimulus. Voltage stimulus ranged from 20 to 410mV. The data recorded indicated that the threshold needed to elicit a compound action potential was 180mV. The maximum amplitude for the CAP was calculated at 8.36 mV. Once a CAP is elicited there is a period in which action potentials cannot be reinitiated or there is smaller response that does not reach an action potential. This was observed in figure 3, where there is a relative refractory period seen at 4ms on the leftmost picture and an absolute refractory period where there is no response after the first CAP, in the rightmost picture. It is therefore seen that there needs to be a certain time frame to repolarize the cells in order to create another CAP. If this time frame is not met the response is lower than a CAP or there is no response at all. The Q10 for increasing the frog Ringer’s solution from 4⁰C to 22 ⁰C was 1.295, while the Q10 from 22⁰C to 35⁰C was 1.061. If the rate of a reaction is independent of temperature, Q10 will be 1.0. If the rate of a reaction increases with temperature, Q10 will be more than 1.0 (Withers 1992). Therefore, if a temperature is more temperature dependent, the higher the Q10 will be (Withers 1992). For diffusion of ions in bulk solutions, Q10 is about 1.0 while for biochemical reactions and physiological rates, Q10 is generally 2-3 (Withers 1992). Since the Q10 values in this experiment were over 1.0, we can assume the conduction velocity of the frog sciatic nerve is temperature dependent (Withers 1992). Temperature dependent reactions are observed in other areas of a frog’s body as well. In a previous experiment involving temperature on the heart, cold Ringer’s solution slowed the heart rate while hot Ringer’s solution sped up the heart rate (Critchfield et al. 2014).
When the stimulus reaches a threshold potential, the nerve or muscle fiber will respond fully to the stimuli, but will not react at all if the stimulus is under threshold which is the “all or none response” (Withers, 1992). This all or none response pattern is shown clearly in these data of CAP amplitude vs stimulus intensity, since there was no CAP response to the stimulus until 180mV (see Figure 2). The CAP differs from a single action potential in several ways. A single action potential is intracellular, and does not change with stimulus intensity above the threshold, where a CAP is an extracellular recording of many axons and is a graded response that does increase with intensity of the stimulation above the threshold.
Compound action potentials will be measured by stimulating the nerve with electrical impulses that increase in amplitude to determine the threshold voltage and maximal CAP amplitude.
The absolute and relative refractory periods will be found by stimulating the nerve with a series of pulses. The velocity of the compound action potential as it travels down the nerve will then be calculated to determine the nerve conduction velocity. Lastly, the temperature sensitivity of nerve conduction velocity will be tested by measuring the speed at which the compound action potential travels down the nerve at both hot and cold temperatures. It was hypothesized that threshold voltage, maximal CAP amplitude, refractory period, nerve conduction velocity and temperature sensitivity of nerve conduction velocity would be
determined.
Materials and Methods To test the sciatic nerve the nerve bath and PowerLab had to first be set up. Black and red alligator clips were were connected from the stimulator electrodes to two of the metal rungs on opposite sites of a nerve bath and the opposite ends were connected to PowerLab. Two more sets of black and red alligator clips were attached to metal rungs along one side of the nerve bath and to PowerLab. The lower reservoir of the nerve bath was filled with room temperature toad Ringer’s solution, taking care to make sure it did not come in contact with the metal electrode rungs. The connections were tested by placing a moist strip of filter paper over the wires and testing the connection through PowerLab (Stillman, 2005). Once the nerve bath and PowerLab were set up properly the sciatic nerve was removed from the double-pithed toad. This was done by cutting the skin around the abdomen and pulling it down over the legs. The urostyle was then cut free and the sciatic nerve was freed from the associated fascia and sciatic artery using a glass hook. The nerve was cut from the spinal cord and a piece of thread was tied around the end for easier handling. The nerve was further removed from the leg and severed at the gastrocnemius muscle. Ringer’s solution was used to rinse the nerve and filter paper was used to remove excess moisture. The nerve was laid across the wire electrodes of the nerve bath so that it was in contact with each of the active connections (Stillman, 2005). After the nerve was properly setup for testing, the nerve was given a series of increasing electrical stimuli. The nerve was stimulated using the “Threshold voltage” function of LabChart by automatically stimulating the nerve and recording for 1.1 seconds. This data was used to determine the threshold value voltage and the maximal CAP amplitude (Stillman, 2005). For the next part of the experiment LabChart stimulated the nerve with a series of pulses. In each block of data the pulse interval was decreased. The minimum stimulus voltage required to elicit a maximal CAP was found from the data of the last section of the experiment and used to adjust the voltage for this section. LabChart then recorded a series of 15 data blocks, each one 10 milliseconds in duration. The resulting data was used to determine the relative and refractory periods of the nerve (Stillman, 2005). For the third part of the experiment, the distance between the negative leads of each of the two recording electrodes was measure in centimeters. LabChart was then set to record a block of data in two channels for 10 milliseconds. This was used to calculate the velocity of the CAP as it travels down the nerve (Stillman, 2005). Lastly, the the effect of temperature velocity on nerve conduction was determined. The room temperature Ringer’s solution was removed from the nerve bath and cold Ringer’s solution was placed inside. LabChart was adjusted and set to record “Conduction Velocity”. This was repeated with warm Ringer’s solution (Stillman, 2005).
Results
To calculate the threshold voltage and maximum CAP amplitude data was taken over various increasing stimulus which ranged from 20mV to 410mV. To obtain a CAP the threshold stimulus voltage needed was reached at 180mV. The maximum CAP amplitude was observed at 8.36mV.
Figure 1: Picture of the threshold voltage of compound action potentials of sciatic nerve. Series of increasing stimulus given to dissected sciatic nerve for 1.1 seconds. Stimulus from 20mV to 410mV.
Figure 2: Scatter plot of CAP amplitude vs Stimulus Voltage (mV). Graph showing the correlation between the stimulus voltage to obtain a CAP. Threshold voltage was around 180mV and maximum CAP was at 8.36mV.
To observe the refractory period a difference of the first and second CAP were determined and used to find the relative refractory period (where there is a first and second CAP are visible), and the absolute refractory period (where there is a first peak and no second peak).
Figure 3: Figure used to identify the refractory period between CAP’s. The distance between the first and second CAP show that there is a 4ms relative refractory period on left picture. On the right there is no CAP and this is where the absolute refractory period is.
Table 1. Conduction velocities for the sciatic nerve of Bufo marinus in cold, room temperature, and hot toad Ringer’s solution.
Conduction Velocity at 4⁰C
18.60 m/sec
Conduction Velocity at 22⁰C
29.63 m/sec
Conduction Velocity at 35⁰C
32.0 m/sec
Q10 effect: Q10= (R2/R1)10/(T2-T1)^ R1= 18.60 m/s at 4o C (T1) R2= 29.63 m/s at 22o C (T2) Q10= ((29.63 m/sec)/(18.60 m/sec))10/(22-4) = 1.295
Q10= (R2/R1)10/(T2-T1)^ R1= 29.63 m/s at 22o C (T1) R2= 32.0 m/s at 35o C (T2) Q10= ((32.0 m/sec)/(29.63 m/sec))10/(35-22) = 1.061
Conduction velocity increased with increasing temperature (Table 1). Q10 effect was greater when the temperature went from 4o C to 22o C than when the temperature rose from 22o C to 35oC.
Discussion
When the stimulus reaches a threshold potential, the nerve or muscle fiber will respond fully to the stimuli, but will not react at all if the stimulus is under threshold which is the “all or none response” (Withers, 1992). This all or none response pattern is shown clearly in these data of CAP amplitude vs stimulus intensity, since there was no CAP response to the stimulus until 180mV (see Figure 2).
The CAP differs from a single action potential in several ways. A single action potential is intracellular, and does not change with stimulus intensity above the threshold, where a CAP is an extracellular recording of many axons and is a graded response that does increase with intensity of the stimulation above the threshold (Withers, 1992).
To find the threshold voltage needed to elicit a compound action potential various readings were taken at different voltage stimulus. Voltage stimulus ranged from 20 to 410mV. The data recorded indicated that the threshold needed to elicit a compound action potential was 180mV. The maximum amplitude for the CAP was calculated at 8.36 mV. Once a CAP is elicited there is a period in which action potentials cannot be reinitiated or there is smaller response that does not reach an action potential. This was observed in figure 3, where there is a relative refractory period seen at 4ms on the leftmost picture and an absolute refractory period where there is no response after the first CAP, in the rightmost picture. It is therefore seen that there needs to be a certain time frame to repolarize the cells in order to create another CAP. If this time frame is not met the response is lower than a CAP or there is no response at all. The Q10 for increasing the frog Ringer’s solution from 4⁰C to 22 ⁰C was 1.295, while the Q10 from 22⁰C to 35⁰C was 1.061. If the rate of a reaction is independent of temperature, Q10 will be 1.0. If the rate of a reaction increases with temperature, Q10 will be more than 1.0 (Withers 1992). Therefore, if a temperature is more temperature dependent, the higher the Q10 will be (Withers 1992). For diffusion of ions in bulk solutions, Q10 is about 1.0 while for biochemical reactions and physiological rates, Q10 is generally 2-3 (Withers 1992). Since the Q10 values in this experiment were over 1.0, we can assume the conduction velocity of the frog sciatic nerve is temperature dependent (Withers 1992). Temperature dependent reactions are observed in other areas of a frog’s body as well. In a previous experiment involving temperature on the heart, cold Ringer’s solution slowed the heart rate while hot Ringer’s solution sped up the heart rate (Critchfield et al. 2014).
When the stimulus reaches a threshold potential, the nerve or muscle fiber will respond fully to the stimuli, but will not react at all if the stimulus is under threshold which is the “all or none response” (Withers, 1992). This all or none response pattern is shown clearly in these data of CAP amplitude vs stimulus intensity, since there was no CAP response to the stimulus until 180mV (see Figure 2). The CAP differs from a single action potential in several ways. A single action potential is intracellular, and does not change with stimulus intensity above the threshold, where a CAP is an extracellular recording of many axons and is a graded response that does increase with intensity of the stimulation above the threshold.