- To measure conduction velocity in a human reflex arc, using the Achilles tendon as the initiator of a reflex and contraction of the gastrocnemius muscle as the response (extracellular recording).
- To measure the threshold, conduction velocity, and refractory period of the sciatic nerve of a frog nerve and Earthworm giant nerve fiber by stimulating the nerve and measuring the response through external recording electrodes (extracellular recording).
- To measure the conduction velocity of the human sciatic nerve through external recording electrodes (extracellular recording).
Lab 9 Overview
I. Hypotheses about nerve conduction speed in different taxa
II. Test hypotheses about nerve conduction speed
- a. Human reflex arc
- b. Frog sciatic nerve
- c. Earthworm giant nerve fiber
III. Data analysis and presentation
- a. Graph means±SD comparing the human, frog,and earthworm conduction velocities
- b. Conduct an ANOVA comparing the human, frog and earthworm conduction velocities
Conduction Velocity in Nerves: Background
A neuron is a cell that is specialized for the transmission of nervous impulses. The axon is the part of the neuron that conducts impulses; the axon is usually a long outgrowth, or process, that carries impulses away from the cell body of a neuron toward target cells.
A nerve impulse, also called an action potential, is the signal that is transmitted along an axon that enables nerve cells to communicate and to activate many different systems in an organism. An Action potential may originate in the brain and result in a deliberate movement or they may be involved in a reflex arc that is independent of the brain. An action potential may be transmitted to a muscle cell, causing muscle contraction.
Neurons have the property of being able to generate action potentials. The action potential is caused by a change in the neuron membrane permeability. This change in permeability results in a change in distribution of ions across the membrane. The change in distribution of ions leads to a change in electrical charge (potential) across the membrane. Changes in electrical potential can be experimentally detected as the action potential passes along the axon of the neuron.
Changes in electrical potential of the axon can be detected and displayed on a recording device in the laboratory by one of two basic methods:
1. Intracellular Recording: Two electrodes are placed on either side of the membrane of the neuron, one inside the cell and one outside. As the ions move into and out of the cell a change in potential difference is recorded between the electrodes. This technique is performed on large, isolated neurons.
2. Extracellular Recording: A pair of electrodes is placed on the outside of the neuron. As the action potential passes along the neuron, a change in potential between the electrodes may be measured and recorded as a biphasic AP. This method does not measure ion flow but the net difference in potential as the action potential passes first one electrode and then the other electrode. This method has the distinct advantage that it can be used to record the passage of an action potential (as in a muscle) from the surface of the body and is also used to record action potentials from whole nerves (in contrast to having to puncture individual neurons).
In today's laboratory you will be using extracellular recording: In the frog and human you will be recording from a nerve, which is a bundle of neurons each with its own threshold, rather than from a single neuron. In the Earthworm, you will be recording from giant axons. You will not only visualize the action potential but also determine the speed at which the action potential travels along a nerve in each of these organisms.
Powerlab will act as a digital 2-channel oscilloscope. Time will be recorded on the X axis and voltage on the Y. Time and sensitivity can be adjusted on each channel. A useful feature of PowerLab is that the operator can initiate a sweep of the screen (i.e. the computer starts sampling). This is known as the TRIGGER. The trigger allows you to capture the time period immediately after an event. It is possible to "trigger" the computer, to begin to collect data (to sweep), at the same time as the stimulus is applied. Thus a record of the stimulating event and the time when the Action Potential appears (latency) can be measured. In this application of the trigger, the computer is set to generate a single sweep upon stimulation of the human achilles tendon as well as the frog nerve. Time can be measured on the X axis. You will be using Channel 1, where the trigger will be displayed, and Channel 3, where the responses will be recorded. The PowerLab set up is slightly different for the Earthworm giant axon recording, but the theory is similar.
The conduction velocity of the action potential is determined by measuring the distance traveled (length of the nerve in m) and dividing by the time (sec) taken to complete the reflex arc, also called the latency.
Conduction velocity = distance (m)/time (sec).
- Measurement of distance is relatively straightforward. It can be done using a ruler or a tape measure.
- The measurement of time is more complicated. Action potentials travel very quickly; therefore, the times to be measured are very small and require more sophisticated instrumentation. The computer with PowerLab, like the oscilloscope, is ideally suited to measure events that happen in a very short amount of time.
The conduction velocity of a particular neuron is correlated with nerve diameter and myelination. Myelin, a lipid-rich substance, acts like insulation to increase the conduction velocity of vertebrate neurons. Invertebrates lack myelinated neurons, and conduction velocity of their action potentials increases primarily as the result of increased axon diameter. Many invertebrates have specialized "giant" axons, like the earthworm, that conduct action potentials very rapidly.
Draft data tables in your lab notebook to record the Threshold voltage needed to elicit an action potential in the frog and earthworm (both medial and lateral Giant Axon). From the three timed trials record the time between stimulus artifact and action potential (latency in ms) and measure the distance as described in the manual. Calculate the conduction velocity in m/sec for the human sciatic nerve, the frog sciatic nerve, and the earthworm medial and lateral giant axons and enter your data on the class data sheet. Calculate the Average conduction velocity for each using the class data.
Conduction Velocity in a Human Reflex Arc
When the Achilles tendon is stretched after being tapped with a reflex hammer, the induced action potential is conducted up the leg to the spinal cord and back down where it causes the gastrocnemius (calf) muscle to contract. To determine the speed of conduction, the distance that the action potential travels is measure and the time between the tapping of the tendon and the contraction of the muscle is measured using PowerLab and ADinstruments software.
The Reflex Arc: A reflex arc is initiated by stretching a tendon, an action that stimulates stretch receptors in the muscle. Those stretch receptors respond by initiating an action potential in sensory neurons. The action potential travels through those sensory neurons to the spinal cord where they synapse directly with motor neurons. The excitation travels back to the gastrocnemius muscle where it causes contraction of the muscle. Thus the tendon that was initially stretched is returned to its original length through contraction, completing the reflex arc.
The function of this type of reflex arc is to maintain posture. Muscles are continually stretching and returning to their original length without the intervention of the brain. Note that this response is monosynaptic. The sensory neuron synapses directly with the motor neuron in the spinal cord; there is no interneuron involved.
The Electromyogram (EMG): is a recording of a muscle contraction that can be taken from the skin above a muscle. An action potential travels down a nerve, through a nerve/muscle junction and into a muscle. In the muscle the action potential spreads throughout the muscle causing contraction of the muscle fibers. The passage of the action potentials can be sensed by electrodes placed on the skin above the muscle, which when amplified (as in the ECG) can be displayed on a computer screen.
The Reflex Hammer: is a percussion hammer used to test reflexes. The hammer that you will use has been modified so that when it hits the tendon, the hammer closes a circuit and generates a small signal. This signal is used to trigger a sweep by the computer.
- Seat the subject on the edge of the lab bench so that her legs are hanging freely. Attach two pre-jelled electrodes to the body of the calf (gastrocnemius) muscle, a bit to the left or right of the midline. The two electrodes should be placed so their outer edges touch in a vertical line on the muscle (See figure below). A third ground electrode should be placed on the ankle bone. Attach the cables to the correct electrodes: green for ground (on the ankle bone) and black and white to the calf muscle.
Fig. 9.1. A, Diagram of a reflex arc in a human. When the stretch receptor is stimulated by the hammer, the action potential travels up the sensory fibers to the spinal cord and synapses on the motor fibers The action potential then travels back down the nerve to cause the muscle contraction we observe as a reflex. B, Two electrodes are placed on the calf, close to each other as shown. The third electrode should be placed on a bony surface, such as the knee cap or ankle.
To test the settings:
- Open the desk top file: muscle test settings
- Press start in the lower right
- Rotate then rest your foot and observe the trace.
- Try at least 5 other positions that activate and then rest your calf muscle. Notice the changes in the height and intensity of the trace with every movement.
- Press stop and close out of the file.
To make an EMG RECORDING:
- Open the file: “EMG test settings”. If you cannot find this file on the desktop, ask your instructor.
- Check the hammer: Press START in the lower right of the screen to begin testing. Hit the flat part of the hammer in the palm of your hand and check channel 1 for a signal.
- To collect an EMG: All group members need to be still during recording. The subject’s leg should be relaxed. Press START in the lower right of the screen. Hold the bottom of the subject’s foot and then tap the Achilles tendon of the subject with the hammer. Record multiple EMG’s by hitting the back of the hammer (green part) on Achilles tendon and observing the reflex in Ch. 3. Repeat until you have 3 representative EMGs.
- When you have a good set of 3 EMGs (see Fig. 9.2), measure the time with the cursor from the start of the stimulus (at zero) to the middle of the first peak. Repeat on different recordings and average three.
- Record data in your lab manual and on the spreadsheet provided by your instructor.
- Use the tape measure to measure the distance in centimeters from the subject’s Achilles tendon to the base of the palpable rib cage (which is the approximate level at which this AP enters the spinal cord) and then down to the gastrocnemius muscles where the first electrode is attached.
- Record length and then calculate and record the conduction velocity
Fig. 9.2. A sample of an EMG recorded on the computer using PowerLab. The trigger signal is on Input 1 (Ch 1) at time 0 and the EMG is on Ch 3. The double arrow indicates the time elapsed between the trigger signal and the gastrocnemius response, i.e. the time taken by the action potentials to propagate along the motor neurons of the sciatic nerve to the spinal cord and along the sensory neurons to the
Conduction Velocity in a Frog Sciatic Nerve
An action potential is initiated in the dissected sciatic nerve of a frog (Rana pipiens or Xenopus laevis) by a stimulator (a device for delivering precise electrical stimuli). The action potential travels along the nerve and is detected as it passes two external electrodes (according to method 2 described in the introduction) and the detected response is amplified and displayed on the computer screen. The trace on the computer of stimulus and response is triggered by the stimulus; time and distance are measured and the speed can then be calculated.
Compound Action Potential: A nerve is a collection of the axons of many neurons. The axons may have different thicknesses and hence their action potentials will have different sizes and speeds. When a nerve is stimulated, the action potential is recorded from the outside of the nerve and is known as a compound action potential, which in the vertebrate nerve is the result of many different action potentials added together. The compound action potential is derived from the action potentials passing first one electrode and then the other. The shape of the extracellular recording is biphasic in nature as it represents a change in potential as the compound action potentials pass over the two recording electrodes in succession (see Fig. 9.3A).
Fig. 9.3. A, Diagram of a biphasic action potential as an extracellular recording of a nerve. The stimulus is applied to the left end of the nerve. B, Dorsal view of exposed frog left hind limb and spinal column.
The Sciatic Nerve is the large nerve running from the spinal cord to the gastrocnemius muscle. It contains both sensory and motor neurons (it is the nerve that is stimulated when you stretch the Human Achilles tendon). In this lab, the frog will have been double pithed (both its brain and spinal cord have been destroyed). The skin was removed from the frog and the urostyle (part of the frog pelvis) may have been removed.
To Dissect the Sciatic Nerve
- Gently separate the dorsal thigh muscles with your fingers and use a blunt glass probe to reveal the white sciatic nerve and accompanying blood vessels (see Fig. 9.3B). Free the nerve from the surrounding tissue in the thigh using a blunt glass hook. Cut away muscle and connective tissue around the nerve as you hold the nerve out of the way. Try not to stretch the nerve and avoid touching the nerve with anything metal to avoid damaging the nerve.
- Keep the nerve moist with Amphibian Ringers (a solution that contains ions in the same concentration found in the frog).
- Tie a thread tightly around the knee end of the nerve. Then cut the nerve below the string and as close to the knee as possible.
- Gently raise the nerve by lifting the thread and then dissect the nerve to its origin in the spinal cord. Take great care with this dissection especially in the pelvic area. Keep the nerve moist with Ringers until ready to be placed in the nerve chamber.
To Record a Single Action Potential
- Check the Stimulator settings to make sure they match the image/table (below). Fill the plastic nerve chamber with amphibian Ringers. Gently place the nerve over the first five or six electrodes starting at the left hand side of the chamber (see instructor). The nerve end on the left side of the chamber should be the anterior end of the nerve. The anterior and posterior ends of the nerve are color coded with string. See your instructor if you are unsure of the color coding.
- Remove Ringers from the nerve chamber with a Pasteur pipette until none of the Ringers is touching the nerve. Also, avoid letting the threads dangle into the Ringers. It may help to place a small drop of mineral oil at each contact point between nerve and electrode (ask your instructor).
- Measure the distance from the stimulating electrode (wire 2) to the first recording electrode (wire 4).
- Cover the nerve chamber with the plastic lid.
- Open the PowerLab Scope program using the “frog settings file”. If you do not see the icon, ask your instructor. Be sure the stimulator (green box) matches the settings shown below.
During the experiment, record the stimulus amplitude and duration values on the page by adding comments (under commands menu – or apple/command K). Another option is to use the Display menu: Show overlay option to observe the changes with increasing voltage overlaid on one screen (see Fig. 9.8B).
To Record a Compound Action Potential
- On the power supply box (stimulator), start with stimulus duration of 0.1 msec. and the lowest voltage. On the computer, press the start button on the lower right of the screen. SCOPE is ready to record the signal and is now waiting for the trigger.
- Briefly press down on the stimulator toggle switch once. This acts as the trigger stimulus and the stimulus amplitude should be visible in the upper display window of SCOPE. If you get no signal, check your settings and your cables.
- Now slowly increase the stimulus voltage in 0.1 – 0.2 V intervals. Do not change the Delay or Duration values for this part of the experiment. All you will be changing is the voltage.
- Eventually, at threshold, the compound action potential should begin to appear as a small deflection in the baseline (do not exceed 1 V). NOTE: Reverse the polarity of the recording electrodes (in Bio Amp ch 3), if necessary, so that the initial deflection of the displayed waveform is upward.
- Continue to gradually increase the voltage until you have a recording that looks like Fig. 9.4A. This is the threshold of the generation of action potentials in some of the neurons in the nerve (see Figs. 9.3A and 9.4).
- Continue to gradually increase the voltage (but never increase it above 1 V) until all the nerve fibers are responding and the amplitude of the compound action potential ceases to increase. As stronger voltages stimulate additional axons, the compound action potential will grow in amplitude.
- When you have a compound action potential whose amplitudes have reached their peak, press STOP and measure the time with the cursor from the start of the stimulus (not from zero) to the middle of the first peak of the biphasic response (see figure 9.4). Record this as the latency in your data table. Generate two more action potentials and record their latency values.
- Measure the distance in cm between the second stimulating electrode (#2 from the left side of the chamber) and the first recording electrode (#4 from the left side of the chamber). Calculate the conduction velocity along the nerve for all three trials and average your results to obtain one mean conduction velocity.
How can the response increase in amplitude when an action potential has "all or none" properties? This graded response phenomenon illustrates the differences in threshold that exists among the different sizes of fibers that make up the nerve. Remember, you are recording from a nerve, a large bundle of neurons, each with a different threshold. If the stimulus voltage is increased slowly and smoothly, you may observe discrete jumps in the amplitude of the compound action potential as different threshold classes of nerve fibers are “recruited”. As you increase the amplitude more neurons reach their threshold and contribute to the increase in size of the compound action potential. Eventually, as the stimulus voltage is increased, a point will be reached when the wave form of the action potential stops changing. At this point all the fibers in the nerve able to respond to the stimulus are being stimulated (Fig 9.4B). This is a maximal response.
Record and save all your trials on the desktop and annotate all the pages saved in PowerLab (page comment). It is a good idea to save data frequently (under the File: save as menu) –in the lab course folder on the desktop). Data pages in the saved file may be deleted later by selecting its number (lower edge) and typing Apple (Command) X. Be sure to include your animal and lab section in your file name.
Fig. 9.4A-B. A. Printout of a compound action potential recorded on the computer using PowerLab. The stimulator signal is on Channel 1(top) and the action potential is on Channel 3(bottom). Note the stimulus artifact (stimulus traveling in the fluid outside the nerve). B. In Scope: Display: Overlay. Gradually increasing the stimulus amplitude results in progressively taller action potentials.
To Measure the Refractory Period
When two stimuli are applied to the nerve in very quick succession, some or all of the neurons that make up the nerve are unable to respond to the second stimulus because the sodium channels are inactivated. They are refractory to the second stimulus.
- Open: New File.
- Set the stimulator on 'TWIN' pulses with the Delay set at 7 msec to start.
- Set the voltage and the duration of each pulse just high enough for production of the full action potential. Common settings for voltage range between 0.4 – 0.5 V and for duration range between 0.1 - 0.2 msec (if the nerve is very sensitive, it might be better to use 0.05msec).
- Press start
- A second action potential will appear on the screen (Fig. 9.5A).
- Now decrease the delay between the two stimuli by 1msec steps. The second response will seem to move toward the first. Record delay in ‘page comment’ each time.
- Record: Refractory period 1: the time when the second action potential decreases in size and refractory period 2: the time when the second action potential disappears.
- Use Display: Overlay to see your data displayed as in Fig. 9.5B
As you decrease the delay between the stimuli, the amplitude of the second action potential begins to decrease. This occurs because some of the fibers are refractory to the second stimulus. As you decrease the interval between pulses, the second action potential disappears because,eventually, all the neurons are refractory to the second stimulus.
In the compound action potential the refractory demonstration that you observe is due to different populations of neurons becoming refractory to the second stimulus. It is difficult to demonstrate the relative and absolute refractory periods in this recording situation. If no other students need to use your nerve, you can try increasing the intensity of the second stimulus in refractory period 2 to see if you can show a relative refractory period.
Fig. 9.5. A, Two compound action potentials stimulated by twin pulses - note that the second AP is smaller than the first one. B, Multiple compound action potentials stimulated by twin pulses in Scope: Display: Show Overlay.
Conduction Threshold, Velocity, and Refractory Period in Earthworm Nerves
NOTE: Parts of this procedure are modified from a protocol written by staff members of ADInstruments, and provided with purchase of the PowerLab instrumentation.
Common earthworms have a giant fiber system consisting of a single median giant fiber and two lateral giant fibers. The two lateral fibers are linked by numerous cross-connections and function as a single axon.
- Place your earthworm in a Petri dish containing 10% ethanol in earthworm saline. Allow the earthworm to become fully anesthetized (i.e. until it stops moving even when probed); this procedure usually takes as long as 25 minutes.
- Place the earthworm dorsal (dark) side up on your dissecting tray. Place the head end (with the clitellum (see Fig 9.6A)) at the top of the tray (fig 9.6C). Be careful not to stretch the earthworm too far, as this can damage the nerve cord. Pin the worm down at each end and then place a third dissecting pin about 0.5cm posterior to the head-end pin.
- Connect the stimulator leads coming from the power lab to the dissecting pins as shown in Fig. 9.6C. The negative lead (cathode, black) should be posterior to the positive lead (anode, red).
- The three recording electrodes are chlorided silver wires. Lay these across the worm as shown in Fig. 9.6C, line them up in the order of G, R1, R2 across the central section of the body of the worm. Check that there is contact between each wire and the earthworm's skin.
- Measure the distance in mm between the cathode (black) and the first recording electrode (R1) and record this measurement. This is the distance that the action potential will travel in your recording.
- Periodically moisten the entire earthworm with the 10% ethanol/saline solution using an eyedropper. Blot excess saline from the worm with a paper tissue.
Fig. 9.6. A Earthworm anatomy and orientation B, PowerLab setup to record earthworm action potentials. C. Electrode placement on the earthworm; the chlorided ends of the silver wires must be in contact with the earthworm's skin.
Determination of the threshold voltage and conduction velocity
- Open WormAP file on the computer desktop.
- Adjust the amplitude of the Simulator by clicking the Amplitude Down Arrow in the Stim Panel (upper left) to 0.15v.
- Click start. Scope will display one 20 ms sweep every 2 seconds. The deflection just after the start of the sweep is caused by spread of part of the stimulus voltage to the recording electrodes. It is called the stimulus artifact.
- Increase the output by 0.05 volts by clicking the Amplitude Up arrow in the Stim Panel, waiting at least 2 seconds (one Scope sweep) before increasing the voltage.
- Continue to increase the Amplitude until you see a response from the median giant axon.
- When you see a response from the median giant axon (Fig. 9.7A), click the Stop button and record the threshold value. If you do not see a response and you are using a stimulus of more than 2V, ask for assistance.
- Save your file to the desktop.
- To calculate conduction velocity, place the Marker on the stimulus artifact peak and the Waveform Cursor on the action potential peak. Read the time difference in the upper part of the Scope window.
- Divide the (previously measured) distance between the stimulus and recording electrodes by the time difference between the peaks to determine conduction velocity in mm/msec which is easily converted to m/s.
Fig. 9.7. A-B. A An action potential recorded from the median giant axon. B. Using the Marker and Waveform Cursor in Scope to calculate the refractory period.
Recruitment of the lateral giant axon
- Open a new file.
- Set the stimulus amplitude to just below the threshold value you determined for the median fiber and click Start.
- Increase the stimulus in 0.05 V steps until you observe the threshold for the median giant fiber.
- Keep increasing the stimulus until you observe a second response with a longer latent period (Fig. 9.7B). Click Stop and record this threshold for the lateral giant fibers.
- Save your file to destop.
Fig. 9.8. A-B. The response of the median giant fiber to dual stimuli. The time between the stimuli is longer than the refractory period of the nerve. B. A recording from the earthworm that shows action potentials from the median and lateral giant fibers.
Data Analysis and Presentation
1. Use data from the whole class to compare the mean conduction velocities of the three nerves examined today. Based on the means, does there appear to be a difference in conductivity in the nerves of the human, frog and earthworm?
2. Conduct an ANOVA comparing the speed of conduction (m/s) of the nerves of human, frog, and earthworm. Is the difference significant at the 0.05 probability level?
3. Data collected in this lab can be compared to previously documented conduction rates of the nerves of a wide variety of vertebrate and invertebrate animals. Are your data consistent with this broader data set?
Fig. 9.9. Velocity of nerve impulse conduction as a function of fiber diameter in a variety of animals. Modified from Bullock and Horridge, 1965, Structure and function of the Nervous System of Invertebrates. W. H. Freeman and Company.
Material in this lab will be included in the lab practical in Lab 10. Make sure that you understand the calculations, statistical tests, graphing, and concepts covered.
Other Labs in This Section
Lab 7: Vertebrate AnatomyLab 8: Vertebrate Circulation and Respiration