- 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 by stimulating the nerve and measuring the response through external recording electrodes (extracellular recording).
- To measure the threshold and conduction velocity of an earthworm giant axon through external recording electrodes.
- 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 black rubber part of the hammer in the palm of your hand and check channel 1 for a signal.
- To collect an EMG: The test subject should be seated and her legs and feet relaxed. Press START in the lower right of the screen. Gently lift the subject's toes to stretch the Achilles tendon on the back of her leg, and firmly rap the Achilles tendon of the subject with the black rubber part of the hammer. Record multiple EMG’s by hitting the black rubber part of the hammer on the 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 point of impact on the subject’s Achilles tendon to the approximate point at which the rib cage meets the spinal column (which is the approximate level at which this AP enters the spinal cord) and then down to the first electrode. Refer to PowerPoint slide provided by your instructor for a diagram of how to take this measurement.
- 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 peak of 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 anesthetized, sacrificed, and double pithed (both its brain and spinal cord will have been destroyed). You may need to remove the skin.
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.
Setting Up the Nerve Chamber
- Open the frog CAP file on the desktop. 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.
- Cover the nerve chamber with the plastic lid and make sure the nerve is still in contact with the wires after you close the lid. Connect the stimulating and recording electrodes as you see in the photos below. You will also have a copy of the photo in the blue binder on your bench. Check your electrode arrangement with your instructor before you proceed.
To Record a Compound Action Potential
- The file is set to start with a stimulation of 0.1 V. To stimulate the nerve at this initial setting, press the start button on the lower right of the screen.
- Now increase the stimulus voltage in 0.1 V intervals by clicking the up arrow under the word Amplitude at the top of screen. Do not change the Delay or Duration values for this part of the experiment. All you will be changing is the voltage. When you click the up arrow, the amplitude will increase by 0.01 V with each click, so you will need to click this arrow 9 times to reach 0.1V. Press start and observe the trace on the screen. Continue to increase the voltage in 0.1V increments.
- Eventually, at threshold, the compound action potential should begin to appear as a small positive deflection in the baseline.
- 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, note the voltage.
- Choose a voltage slightly below the maximum. Generate one action potential at this voltage. Measure the time with the cursor from the start of the stimulus 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 at this same voltage and record their latency values.
- The distance between the second stimulating electrode and the first recording electrode is 5 mm. Using this distance and your recorded latency values, calculate the conduction velocity 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. It is a good idea to save data frequently (under the File: save as menu) –in the lab course folder on the desktop). Be sure to include your animal and lab section in your file name.
Fig. 9.4. An example of compound action potentials at increasing stimuli from a frog's sciatic nerve shown in Scope: Display: Overlay. Gradually increasing the stimulus amplitude results in progressively taller action potentials. An initial response at 0.3V and a maximal response at 0.9V is shown in this example.
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 the frog refractory file. The amplitude is pre-set in this file at 0.5 V and will not change during this portion of the experiment.
- Check that the interval between the two stimuli pulses is set at 7 ms to start.
- Press start.
- Two action potentials will now appear (Fig. 9.5).
- Now decrease the interval between the two stimuli by 1 ms steps by clicking the down arrow by the word interval at the top of the screen. Each click of the down arrow will change the interval by 0.1 ms, so you will need to click the arrows 9 times to decrease the interval by 1ms. Press start. The second response moves toward the first with each decrease in interval. Continue to decrease the interval by 1ms and obtain the resulting action potentials at each interval. Note that once the interval is less than 1 ms, change the interval by just clicking once, decreasing the interval by 0.1 ms, until the second action potential disappears.
- Note the time when the second action potential decreases in size and the time when the second action potential disappears.
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. Multiple compound action potentials stimulated by twin pulses in a frog's sciatic nerve shown in Scope: Display: Show Overlay.
Conduction Threshold and Velocity 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 may take up to 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. .
- Place two stimulator pins about 2 cm below the clitellum. 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).
- See Fig. 9.6C before completing this step. The three recording electrodes (G, R1, R2) are chlorided silver wires. Gently insert them into the worm as shown in Fig. 9.6C in the order of G, R1, R2 in the central section of the body of the worm below the negative electrode. The pins can be placed fairly close together. Position the R2 electrode about 0.5 to 1 centimeter posterior to the R1 electrode.
- 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 PowerLab setup to record earthworm action potentials B. Earthworm anatomy and electrode placement on the earthworm
Determination of the threshold voltage for the medial and lateral axons, calculating , conduction velocity, and and observing recruitment of the lateral giant axon
- Open WormAP file on the computer desktop.
- Click start. Scope will display one sweep. 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.
- Repeat steps 2 and 3 increasing the Amplitude by 0.05 volts with each trial until you see a response from the median giant axon.
- When you see a response from the median giant axon (Fig. 9.7), record the threshold value. If you do not see a response and you are using a stimulus of more than 1V, ask for assistance.
- Keep increasing the stimulus until you observe a second response with a longer latent period (Fig. 9.7). Click Stop and record this threshold for the lateral giant fibers.
- Save your file to the desktop.
- To calculate conduction velocity, place the Marker at the start of the stimulus artifact and the Waveform Cursor on the action potential peak (Fig. 9.7). 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/ms which is easily converted to m/s.
Fig. 9.7. A recording from the earthworm that shows action potentials from the median and lateral giant fibers using the Marker and Waveform Cursor in Scope to calculate the latency.
10. Discard your file or place it in the folder for your lab section.
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.
Please take the post animal unit assessment: https://www.surveymonkey.com/s/2SRJ8WV .
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