CEBU DOCTORS’ UNIVERSITY

COLLEGE OF MEDICINE
DEPARTMENT OF PHYSIOLOGY
LEVEL I BLOCK III MODULE NERVOUS SYSTEM

LABORATORY EXPERIMENT AND ANSWER SHEET

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NAME: PEREZ, Marie Robin Frances A. DATE: November 4, 2021

Section and Group Number: Section A, Group 7

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EXPERIMENT NO. 4

FROG NERVE (PowerLab)

Introduction

In this laboratory you will measure compound action potentials (CAPS) from an isolated frog sciatic nerve
to explore the basic physiological properties of nerve impulses.

Learning Objectives

By the end of today’s laboratory you will be able to:

 Describe the nature of compound nerve action potentials.

 Explain how increasing stimulus strength results in increased magnitude of the CAP up to some
maximum.
 Know how the absolute and relative refractory periods of a nerve can be demonstrated and
understand the physiology significance periods.
 Determine nerve conduction velocity.

(Get the procedures from the server)

1. Exercise I
- to determine the threshold voltage and maximal CAP amplitude.
2. Exercise 2
- to determine the refractory period of the nerve
3. Exercise 3
- to determine the conduction velocity of the nerve.
- Pre-recorded results will be provided
- Students analyze, interpret the results
- Students make conclusion(s)
An early cathode ray tube. The paper in which this was first shown provides the first description of the
accurate measurement of compound nerve action potentials. This was made possible by the availability
of a suitable cathode ray tube. For their pioneering work in this field, Erlanger (1874-1965) and gasser
(1883-1963) were jointly awarded the Nobel Prize in Physiology or Medicine in 1944.

Nerve Bath Set up

Connect the stimulating electrodes to the nerve bath as shown.

The stimulating electrode cable has two BNC connectors at one end that plug into the PowerLab Output
connectors. It connects to the nerve bath at the end where the coils are closer together.
Connect the first set of recording electrodes to the nerve bath as shown.

The recording electrode cable has a single connector at the plug end.

Connect the second set of recording electrodes to the nerve bath as shown.
 In order to test the equipment, position a piece of most filter paper across all the electrodes as shown.

PowerLab Setup

Connect the positive and negative BNC connectors of the stimulating electrode leads to the analog
Outputs on the PowerLab.

Connect the first recording electrodes to inputs 1 on the PowerLab, and the second set to Input 2.

To stimulating
electrodes

To first recording
electrodes To second Recording
electrodes
Testing your stage

Note that the Labtutor panel is displaying data using Record Overlay.

Procedure

1. Click start. Labtutor will automatically stimulate and record data for one second.

2. You should see a series of stimulus pulses recorded in the Test channel that line up with the
Stimuli shown in the stimulus channel. 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 laboratory

Dissection

If you are required to dissect and mount your frog nerve, use the ‘Previous’and ‘Next’ buttons below to
navigate through the dissection instructions.

Gross Dissection

1. Obtain a double-pithed frog from your instructor. Secure the animal ventral side up to a
dissecting board using straight pins.

2. Place the frog in a dissection pan, and keep the animal moist at all times with Frog Ringer’s
solution.

3. Using a scalpel or sharp scissors, cut the skin of frog around its abdomen.

4. Peel the skin down and off the legs of the frog.

5. Keep the tissue moist at all time with frog Ringer’s solution.
 Cut skin around
Abdomen and grasp
frog here

Pull skin down and off

the animal

Exposing the Sciatic Nerve

1. Grasp the urostyle with forceps and cut it free; you should be able to observe the nerve plexus
below it. Be careful not to damage the nerve plexus.

2. Using a glass hook, locate and lift the sciatic nerve free from the associated fascia and the sciatic
artery. You may need to use blunt dissection techniques.

3. Cut the nerve from the spinal cord and reflect the nerve back onto the animal’s leg.

4. Tie a piece of thread around the free end of the nerve so that it can be handled gently.
Removing the Nerve

1. Sever the leg of the frog from the abdomen

2. Using forceps and the glass hook, continue to dissect the nerve away from the leg.
3. Sever the nerve from the gastrocnemius (calf) muscle.
Nerve Bath

1. Place the nerve across the electrodes of the nerve bath as shown.
2. Make sure that it’s in contact with the stimulating electrodes and both sets of recording
electrodes.
3. Keep the nerve moist at all times with the frog Ringer’s solution.
Exercise 1

In this exercise, you will determine the threshold voltage and maximal CAP amplitude.

Procedure

1. In the Stimulator panel, check that the stimulus voltage is set to 10 mV.
2. Click Start. After a delay of 1 ms, LabTutor will stimulate the nerve and record for a total time
of 5 mins.
3. Now click the up arrow in the Stimulator panel Amplitude box. This increases the stimulus
voltage by 10 mV. Click Start.
4. Repeat step 3, increasing the voltage in 10 mV steps until you have at least three successive
responses that don’t increase in amplitude or you reach a stimulus voltage of 400 mV.

Analysis

1. Display the first record that shows an obvious CAP.

2. Select the entire waveform representing the CAP from peak to through. Be careful not to
include the stimulus artifact.
3. The Value panel will display the amplitude of the CAP. Drag the value to the cell in the CAP (mV)
column in the table, next to the appropriate stimulus (mV).
4. Repeat steps 1 & 3 for every subsequent record.
5. For all earlier records, enter a value of zero in the CAP (mV) column.

As you enter the data the Graph panel displays the relationship between the stimulus and the response.
Study Questions

1. How does a CAP differ from a single action potential?

A compound action potential is a collective response or summation of multiple action
potentials activated by more than one neuron. A single action potential represents the activity
of a single excitable neuron.
2. Action potentials are said to be all or none responses. Why does the frog sciatic nerve give
a grade response?
The frog sciatic nerve gave a graded response because it is made up of multiple nerve
fibers, creating a compound action potential when stimulated. This combined action potential
then results to an increased response than it would have been with a single nerve fiber.
Moreover, increasing the strength of the stimulus such as the experiment where it was
increased by increments of 10 mV, the CAP also increased, creating a graded response as a
result of the combined “all or none” responses of each individual fiber.
3. What was the smallest voltage required to produce the maximum (largest) CAP?
The smallest voltage required to produce the maximum (largest CAP) based on the graph
was 20 mV which resulted in a CAP of 1.44 mV. In the procedure, an actual maximum CAP was
not detected since the nerve was still responding, showing that there are still nerve fibers left
to stimulate.
4. Repeat steps 1 to 3 for every subsequent record.
5. For all earlier records, enter a value of zero in the CAP (mV) column.

Exercise 2

In this exercise, you will determine the refractory period of the nerve. You will deliver two stimuli to the
nerve, progressively decreasing the interval between them each time you stimulate.

Before you perform this experiment, you must have completed the analysis of Exercise

Procedure

1. In the Stimulator panel, set the stimulus voltage to the minimum voltage required to elicit a
maximal CAP in Exercise 1. Set the stimulus interval to 4.0 ms.
2. Click Start. LabTutor will stimulate the nerve twice at the interval that you have selected and
record for a total time of 10 ms.
3. Now Decrease the interval in the Stimulator panel to 3.5 ms and Click Start.
4. Repeat steps 2 and 3, decreasing the interval to 3.0 ms, the 2.5 ms, 2.0 ms, 1.9 ms, and then by
steps of 0.1 ms until the interval between the stimuli reaches 1.0 ms.

Analysis

1. Display the first record that shows an obvious second CAP.

2. Select the entire waveform representing the CAP from peak to though. Be careful not to include
the stimulus artifact.
 3. The Value panel will display the amplitude of the CAP. Drag the value to the cell in CAP (mV)
column in the table, next to the appropriate stimulus interval (ms.)
4. Repeat steps 1 to 3 for every subsequent record.

As you enter the data the Graph Panel displays the relationship between stimulus interval and the
magnitude of the 2nd CAP.

Note the stimulus interval at which detect a decrease in the magnitude of the 2nd CAP. This marks the end
of the relative refractory period.

Note the stimulus interval at which the 2nd CAP first disappears. This marks the end of the absolute
refractory period.

Study Questions

1. Explain the difference between the relative and absolute refractory periods.
A relative refractory period is the period of time when repolarization is almost complete
(Na+ channels have been mostly closed, and the nerve fiber is near or at its resting membrane
potential. In this state, the nerve fiber is available for a second action potential to occur, but
only with stimuli that is much stronger than normal. On the other hand, an absolute refractory
period is the time between firing level is reached and until the repolarization is almost
complete (Na+ channels are mostly still inactivated). In this state, no stimulus would be able
to excite the nerve and elicit an action potential no matter how strong. This enables neurons
to have an uninterrupted period of recovery and to make sure it will eventually be ready for
another action potential.
 2. Briefly described the cellular events responsible for the refractory period. (Hint: Discuss the
mechanism of repolarization).
A refractory period is the time in which the neuron recovers its resting state after firing
an action potential. After maximum strength and peak of action potential is reached, there is
potassium or K+ efflux as a result of one K+ channel becoming completely open and closure of
Na+ channels, a stage in action potential known as repolarization. This continuous with a slow
but prolonged opening of voltage-gated Ca2+ channels, and finally closure of the Ca2+ channels
and delayed increase of K+ through many types of K+ channels. This returns the nerve to its
normal polarization where the intracellular space is much more negatively charged than the
extracellular environment.

Exercise 3

In this exercise, you will determine the conduction velocity of the nerve.

The upper channel shows the CAP obtained from the proximal recording electrode and the lower channel
shows the CAP obtained the distal recording electrode.

Procedure

1. In the Stimulator panel enter a voltage that is twice that which you used in Exercise
2. Click Start. LabTutor is set up so that two channels of data are recorded for 5 ms.

Analysis

Data from the two channels is overlaid in the LabTutor panel.

1. Using a ruler, measure the distance in millimeters between the black negative leads of each of
the two recording electrodes. Enter this value in the table.
2. Use the Marker and waveform cursor to determine the time interval for the CAP to travel
between the two recording electrodes. To do this, place the marker on the first CAP peak.
3. Next, switch between channels by clicking on the Channel Buttons. Place the waveform cursor
over the second CAP peak and click to transfer the ∆ time value to the Value panel.
4. Drag the value to the appropriate cell the table.

The table is set up so that the conduction velocity in m/s is now calculated for you from the equation:
distance (mm) between electrodes / time interval (s) between CAPS x 1000.

When you have finished making your recordings, return the nerve to its dish of cold frog Ringer’s solution
and return the nerve to your instructor if asked to do so.
Study Question

1. Based on your calculation for CAP conduction velocity, how long would it take the CAP to
travel the length of the sciatic nerve? Assume a total length of 10 cm.

Data from experiment:

Length = 30 mm
Time = 0.71 ms

𝑑𝑖𝑠𝑡𝑎𝑛𝑐𝑒
(𝐶𝐴𝑃) 𝑐𝑜𝑛𝑑𝑢𝑐𝑡𝑖𝑜𝑛 𝑣𝑒𝑙𝑜𝑐𝑖𝑡𝑦 =
𝑡𝑖𝑚𝑒

30𝑚𝑚 1𝑚 1000𝑚𝑠 𝑚
(𝐶𝐴𝑃) 𝑐𝑜𝑛𝑑𝑢𝑐𝑡𝑖𝑜𝑛 𝑣𝑒𝑙𝑜𝑐𝑖𝑡𝑦 = × 1000𝑚𝑚 × = 42.3
0.71 𝑚𝑠 1𝑠 𝑠

𝑑𝑖𝑠𝑡𝑎𝑛𝑐𝑒
𝑡𝑖𝑚𝑒 = (𝐶𝐴𝑃) 𝑐𝑜𝑛𝑑𝑢𝑐𝑡𝑖𝑜𝑛 𝑣𝑒𝑙𝑜𝑐𝑖𝑡𝑦

10 𝑐𝑚 1𝑚 0.1𝑠
𝑡𝑖𝑚𝑒 = 42.3 𝑚⁄ × = 42.3 = 0.0024 𝑜𝑟 0.002 𝑠
𝑠 100 𝑐𝑚

It would take 0.002 seconds for CAP to travel a sciatic nerve with a length of 10 cm.