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					Teaching Experiment

Compound Action Potentials in the Frog Sciatic Nerve
In this experiment, you will measure compound action potentials (CAP’s) from an isolated frog sciatic nerve to illustrate the basic physiological properties of nerve impulses.

Written by staff of ADInstruments.

Background
The fundamental unit of the nervous system is the neuron. Neurons and other excitable cells produce action potentials when they receive electrical or chemical stimulation. The action potential occurs as a large-scale depolarization when positive ions such as sodium rapidly enter the neuron via specialized membrane channel proteins. Action potentials are “all-or-none” events. Once an action potential begins, it propagates down the length of the axon. When the action potential reaches the end of the axon, a neurotransmitter is typically released into the synapse. After an action potential occurs, the neuron must repolarize. During this time, called the refractory period, the neuron is incapable of producing another action potential. Measuring action potentials from single neurons requires highly specialized equipment. In this lab, you will record compound action potentials (CAP’s) from the isolated frog sciatic nerve. CAP’s represent the summed action potentials of the multitude of neurons that comprise a nerve.

Required Equipment
A computer system PowerLab with analog output Chart software, v5.0 or later MLT012/B Nerve Bath Frog Ringer’s solution Isolated frog sciatic nerve Forceps Eyedropper Aluminum foil Filter paper

Procedures
Setup and calibration of equipment
1) Connect the red and black alligator clips from the stimulator electrodes to two of the metal rungs on opposite sides of the MLT012/B Nerve Bath (Figure 1). The distance between the electrodes should be 0.5 cm. It is not necessary to connect the green (ground) alligator clip. 2) Connect the red (positive) BNC connector from the stimulator electrode to the positive (+) analog output connector on the PowerLab. Connect the black (negative) BNC connector from the stimulator electrode to the negative (–) analog output connector.

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3) Connect the red and black leads from the first recording electrode to two of the metal rungs of the MLT012/B Nerve Bath (Figure 2). Connect the 8-pin pod connector to the Pod port on Input 1 of the PowerLab. 4) Repeat step 3 for the second recording electrode, only place the alligator clips further away from the stimulus electrode (Figure 3). Attach the pod connector to the Pod port on Input 2 on the PowerLab. 5) Using an eyedropper or Pasteur pipette, fill the lower reservoir of the Nerve Bath with frog Ringer’s solution. Fluid in the lower reservoir must not come in contact with the metal electrode rungs. Note: OVERFILLING the Nerve Bath in this manner will cause a short circuit in your experiment. 6) Cut a strip of filter paper and lay it over the wires in the nerve bath (Figure 4) so that it touches both stimulating electrodes and both sets of recording electrodes. Moisten the paper strip with frog Ringer’s solution, and place the cover on the nerve bath. This arrangement will be used to test your connections. 7) Turn on the PowerLab and make sure it is connected to the USB port on your computer. 8) Launch Chart from your computer. From the Experiments Gallery locate and double-click on the file called CAP Set Chart. 9) From the Chart Application window, select the Macro menu and choose Test Connection. Chart will now automatically record data for one second. You should see a series of stimulus pulses recorded (Figure 5). If not, check to make sure that the alligator clips are secure and the filter paper is moist and draped over all the active wires in the Nerve Bath. Once the connections are tested and working, you are ready to begin the lab. 10) Obtain an isolated frog sciatic nerve from your instructor. Using forceps, lift the nerve out of its dish by grasping the thread tied to either end of the nerve. Note: DO NOT GRASP THE NERVE WITH FORCEPS! Doing so will damage the nerve. 11) Gently blot the nerve on a piece of tissue or filter paper to remove any excess Ringer’s solution.

Figure 1. Placement of the stimulus electrodes on the MLT012/B Nerve Bath.

Figure 2. Placement of first recording electrodes on Nerve Bath.

Figure 3. Placement of second recording electrodes on Nerve Bath.

Figure 4. MLT012/B Nerve Bath set up for connection test with filter paper and frog Ringer's.

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Figure 5. Chart output after performing Connection Test macro.

12) Remove the filter paper from the Nerve Bath. Lay the nerve across the wire electrodes, making sure it is in contact with each of the active connections (Figure 6). If your nerve is too short, adjust the position of the recording electrodes as necessary. Place the cover back on the Nerve Bath.

Figure 6. Diagram of the complete setup of the MLT012/B and PowerLab 4/20T for the frog sciatic nerve experiment. Make sure that the nerve is resting on all the electrode pairs.

Exercise 1: Determination of threshold voltage and maximal CAP amplitude
In this part of the experiment, you will give the nerve a series of electrical stimuli, each increasing in amplitude. You will then be able to calculate the threshold voltage for the nerve, as well as the voltage required for maximum CAP amplitude. 1) From the Chart window, select Macro: Threshold Voltage. 2) Chart will automatically stimulate the nerve and record for 1.1 seconds. 3) Fill in Table 1 in your Data Notebook. Follow the directions from the Analysis section below.

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Exercise 2: Determination of the refractory period
Before you perform this experiment, it is important that you complete the analysis section for Exercise 1 (Table 1). In this part of the experiment, the PowerLab will stimulate the nerve with a series of pulses. In each block of data, the pulse interval will decrease. You will be able to use this recording to determine the relative and absolute refractory periods of your nerve. 1) From your results in Table 1, determine the minimum stimulus voltage required to elicit a maximal CAP from your nerve. Indicate this voltage here: ____________ mV 2) From the Chart window, select Macro: Refractory __mV. Note: there are four versions of the Refractory macro. Each version uses a different stimulus voltage. Choose the voltage that is nearest the stimulus intensity that you wrote down in step one, above. 3) Chart will now record a series of 15 data blocks. Each block is 10 milliseconds in duration. 4) Fill in Table 2 in your Data Notebook. Follow the directions from the Analysis section below.

Exercise 3: Determination of nerve conduction velocity
In this part of the experiment, you will calculate the velocity of the CAP as it travels down the nerve. 1) Using a ruler, measure the distance in centimeters between the negative leads of each of the two recording electrodes. Record this value in Table 3. 2) From the Chart window, select Macro: Conduction Velocity. 3) Chart will record a block of data in two channels for 10 milliseconds. 4) Use the data from this recording to fill in Table 3. Follow the directions from the Analysis section below. 5) When you have finished making all the recordings, return the nerve to its dish of cold frog Ringer’s solution and return the nerve to your instructor.

Analysis
Determination of threshold voltage and maximum CAP amplitude
From your data trace in CAP1 (Channel 1) from Exercise 1, use the Waveform Cursor to measure CAP amplitude at each stimulus voltage. Record these data in Table 1 of your Data Notebook. The stimulus range is between 20 and 400 mV, and increases in 10 mV steps, as indicated in Table 1. Note the stimulus level where you first see a CAP. Record the maximum CAP amplitude.

Determination of refractory period
Select the first two CAP’s recorded in CAP1 in each of block of data recorded in Exercise 2. Open the Zoom window and examine the data trace using the Waveform Cursor. Record the amplitude for the second CAP in Table 2 of your Data Notebook. The stimulus intervals used in this part of the experiment are recorded in the order shown in Table 2. Determine the stimulus interval where the amplitude of the second CAP first shows a decrease. This is the relative refractory period. Determine the stimulus interval where the second CAP completely disappears. This is the absolute refractory period. Record both of these values in Table 2.
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Figure 7. Zoom window showing two pulses for determining refractory period.

Calculating conduction velocity
From your data in Exercise 3, make a selection in both channels that includes the first CAP. From the Zoom window, use the Marker and Waveform Cursor to determine the time interval for the CAP to travel between the two recording electrodes (figure 8). Overlay mode works best, but is not required. Select Channel 1 and place the marker on the first CAP peak. Select Channel 2 and place the waveform cursor over the second CAP peak. Read the value for time differential (t) from the top of the Zoom window. Record this value in Table 3 in your Data Notebook. Using your measurement for the distance between the two recording electrodes, make the following calculation and record your answer in Table 3.

 distance between electrodes (cm)   cm   100 1000 ms  Conduction velocity (m/sec) =         time interval between CAP' s (ms)   1 m   1 sec 



Figure 8. Zoom window in overlay mode showing analysis procedure for calculating conduction velocity.

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Data Notebook
Table 1. CAP amplitude versus stimulus intensity.
Stimulus amplitude (mV) 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 Threshold stimulus voltage: CAP amplitude (mV) Stimulus amplitude (mV) 220 230 240 250 260 270 280 290 300 310 320 330 340 350 360 370 380 390 400 410 CAP amplitude (mV)

mV

Maximum CAP amplitude:

mV

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Table 2. CAP amplitude versus stimulus interval.
Stimulus interval (ms) 4 3.5 3.0 2.5 2.0 1.9 1.8 1.7 1.6 1.5 1.4 1.3 1.2 1.0 Relative refractory period: Absolute refractory period: ms ms Amplitude of second CAP

Table 3. Calculation of conduction velocity
Distance between recording electrodes: Time interval between CAP1 and CAP2: Conduction velocity cm ms m/s

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Study Questions
Answer the following questions in complete sentences. 1) How does a CAP differ from a single action potential?

2)

What is the cause of the relative refractory period?

3)

Action potentials are said to be “all or none” responses. Why does the frog sciatic nerve give a graded response?

4)

Briefly describe the cellular events that occur during the refractory period. Hint: Discuss the mechanism of repolarization.

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