Lab 9: Conduction Velocity of Nerves

BISC 111/113: Introductory Organismal Biology

 Introduction to Organismal Biology Lab  Calendar and Assignments

 Statistics and Graphing  Science Writing Guidelines

 Lab 1: Biodiversity  Lab 2: Population Growth  Labs 3-6: Plant Biology  Labs 7-9: Animal Biology  Lab 10: Laboratory Exam  Lab 11: Population Growth 2  Lab 12: Beetle Presentations

Objectives

 * 1) To measure conduction velocity in a human reflex arc, using the Achilles tendon as the iniator of a reflex and contraction of the gastrocnemius muscle as the response.
 * 2) To measure the conduction velocity of the sciatic nerve of a frog by stimulating the nerve and measuring the response through external recording electrodes.
 * 3) To measure the threshold, conduction velocity, and refractory period of the earthworm giant nerve fiber.

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 </dl> III. Measure earthworm threshold and refractory parameters <dl>IV. Data analysis and presentation <dd>a. Compare means of the three data sets (human, frog, earthworm) <dd>b. Conduct an ANOVA comparing the means <dd>c. Compare today's data with a broader data set </dl>

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. Action potentials 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: 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. 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: you will be recording from a nerve, which is a bundle of neurons, rather than from a single neuron. This method not only enables you to visualize the action potential but also allows you to determine the speed at which the action potential travels along a nerve.

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 immediately after the stimulus, when the response occurs, is generated. In this application of the trigger, the computer is set to generate a single sweep upon stimulation of the nerve. PowerLab sweeps the screen once and displays the data. It will not collect any more data until it is re-triggered. Time can be measured on the X axis.This week you will be using both Channel 1 where the trigger will be displayed and Channel 3 where the responses will be recorded.

Speed or velocity is determined by measuring the distance traveled and dividing by the time required to travel the distance (speed = distance/time). Therefore, in order to estimate the speed of conduction of an action potential, it is necessary to measure both the length of the nerve and the time that it takes the action potential to travel along the measured distance. The nerves of any animal have a range of conduction speeds, and the rapidity of reaction of a given function can be correlated with the conduction speed of the axons involved. 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 increases in axon diameter. Many invertebrates have specialized "giant" axons that conduct action potentials very rapidly.
 * 1) Measurement of the distance is relatively straightforward. It can be done using a ruler or a tape measure.
 * 2) 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.

<hr style="width: 725px; border: 4px solid #FF0000;"> Draft a data table in your lab notebook for nerve conduction velocities of human, frog, and earthworm. Include three time trials for each animal and record mean time (msec or sec), distance (m), and calculated speed of conduction (m/sec) for each trial. Enter your data on the class data sheet as well. <hr style="width: 725px; border: 4px solid #FF0000;">

Conduction Velocity in a Human Reflex Arc
To determine the speed of conduction in a mammalian nerve a reflex is initiated by tapping the Achilles tendon. This will cause the gastrocnemius (calf) muscles to contract. The distance that the action potential travels is measured. The time between the tapping of the tendon and the contraction of the muscle can be measured on the computer screen. Hence speed of conduction can be calculated.

The Reflex Arc: When the Achilles tendon is stretched by tapping with a hammer, a contraction occurs in the gastrocnemius muscle. A reflex arc is initiated by stretching the 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.

Experimental Procedure
 * 1) 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: To make an EMG RECORDING:
 * 1) Open the desk top file:
 * 2) Press start in the lower right
 * 3) Rotate then rest your foot and observe the trace.
 * 4) 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.
 * 5) Press stop and close out of the file.
 * 1) Open the file: “EMG test settings”.  If you cannot find this file on the desktop, ask your instructor.
 * 2) 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.
 * 3) 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. Continue recording until you have 3 representative EMGs.
 * 4) When you have a good set of 3 EMGs (see Fig. 9.2), press STOP in lower right, and measure the time with the cursor from the start of the stimulus (at zero) to the middle of the largest (or middle) peak. Repeat on different recordings and average three.
 * 5) Record data in your lab manual and on the spreadsheet provided by your instructor.
 * 6) 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.
 * 7) 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 gastrocnemius

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 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 compound action potential is biphasic in nature as it represents a change in potential as the action potentials pass over the two recording electrodes (see Fig. 9.3A).

Fig. 9.3. A, Diagram of a biphasic action otential recorded from the outside 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 Achilles tendon). In this lab, the frog will have been double pithed (both its brain and spinal cord have been destroyed). The skin will have been removed from the leg and the urostyle (part of the frog pelvis) will have been removed. To Dissect the Sciatic Nerve
 * 1) Gently separate the dorsal thigh muscles with your fingers and 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.
 * 2) Keep the nerve moist with Amphibian Ringers (a solution that contains ions in the same concentration found in the frog).
 * 3) 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.
 * 4) Gently raise the nerve by lifting the thread and dissect the nerve to its origin in the spinal cord. Take great care with this dissection especially in the pelvis area.  Keep the nerve moist with Ringers until ready to be placed in the nerve chamber.

To Record a Single Action Potential 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 How can the biphasic response increase in size when an action potential has "all or none" properties? This 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. Notice that as the stimulus voltage is increased, a point will be reach when the wave form of the action potential stops changing. At this point all the fibers in the nerve are being stimulated (Fig 9.4B). This is a maximal response. Record and save all your trials 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.
 * 1) Check the Stimulator settings to make sure they match the image/table above. Fill the plastic nerve chamber with amphibian Ringers. Gently place (or weave) the nerve over (through) the first five or six electrodes (of the electrodes that are close together) starting at the left hand side of the chamber (Fig. 9.4 (see instructor)).  The nerve end on the left side of the chamber should be the anterior (pelvic) end of the nerve.
 * 2) Remove the excess 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).
 * 3) Cover the nerve chamber with the plastic lid.
 * 4) Open the PowerLab Scope program using the “frog settings file” which has the following settings. If you do not see the icon, ask your instructor.
 * 1) On the power supply box, 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.
 * 2) Briefly press down on the stimulator toggle switch once. This acts as the trigger stimulus and its amplitude should be visible in the upper display window of SCOPE.  If you get no response, check your settings and your cables. Look for the response from the nerve in the bottom window.
 * 3) 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. Most likely all you will be changing is the voltage.
 * 4) 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.
 * 5) 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.  The biphasic response represents the passage of the action potentials across the recording electrodes (see Figs. 9.3A and 9.4).
 * 6) 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.

Fig. 9.4. 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). Please note the stimulus artifact if present (stimulus traveling in the fluid outside the nerve). B. In Scope: Display: Overlay. Gradually increasing the stimulus amplitude results in progressively taller action potentials. How can this be reconciled with the fact that APs are "all-or-nothing"?

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. 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 all the neurons are refractory to the second stimulus. When making similar recordings from a single neuron a relative refractory period is observed. This is described as when the second stimulus can’t initiate an AP but if the amplitude of the second stimulus is increased the nerve will fire. There is also the absolute refractory period, where no matter how large the second stimulus, it is not able to initiate a second response in the nerve. In the compound action potential the refractory demonstration that you have observed 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. Try increasing the intensity of the second stimulus in refractory period 2 to see if there are any changes. 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.
 * 1) Open: New File.
 * 2) Set the stimulator on 'TWIN' pulses with the Delay set at 7 msec to start.
 * 3) 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).
 * 4) Press start
 * 5) A second action potential will appear on the screen (Fig. 9.5A).
 * 6) 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.
 * 7) 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.
 * 8) Use Display: Overlay to see your data displayed as in Fig. 9.5B

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.

Experimental Set-up
 * 1) 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); this procedure usually takes five minutes.
 * 2) Place the earthworm dorsal side up on your dissecting tray, and pin it down at each end with two dissecting pins about 0.5 cm apart. Be careful not to stretch the earthworm too far, as this can damage the nerve cord.
 * 3) Connect the stimulator leads as shown in Fig. 9.6A. The negative lead (cathode, black) should be posterior to the positive lead (anode, red).
 * 4) Clip a chlorided silver wire to each recording lead (Fig. 9.6B). Mount the alligator clips to the dissection board. You may need to bend the silver wire slightly to ensure that the chlorided (blackened) part of the wire is in good contact with the skin of the earthworm.
 * 5) Measure the distance in mm between the cathode and the first recording electrode (R1) and record.This is the distance that the action potential will travel.
 * 6) 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. 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 Fig. 9.7. A. An action potential recorded from the median giant axon. B. Using the Marker and Waveform Cursor in Scope to calculate the refractory period.
 * 1) 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.
 * 2) Increase the output by 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.
 * 3) 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 3V, ask for assistance.
 * 4) 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.
 * 5) 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.
 * 6) Save your file to the desktop.

Recruitment of the lateral giant axons Fig. 9.8. A. 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.
 * 1) Open a new file.
 * 2) Set the stimulus amplitude to just below the threshold value you determined for the median fiber and click Start.
 * 3) Increase the stimulus in 0.05 V steps until you observe the threshold for the median giant fiber.
 * 4) 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.

Determination of the refractory period
 * 1) Open a new file.
 * 2) From the Setup menu, choose the Stimulator...command. Set the number of pulses to two, and the time interval to about 15 msec. Click OK to close the dialog box.
 * 3) Click Start. With a pulse delay of 10 ms or more, you should see two stimulus artifacts, each followed by an action potential. This response indicates that the median giant fiber fired for both stimuli (Fig 9.9).
 * 4) Click the Interval arrows in the Stim Panel to decrease the interval by 1 ms., waiting at least 2 seconds (one Scope sweep) before decreasing the interval.
 * 5) Eventually you should find that the response to the second stimulus disappears. Click Stop, and record the stimulus interval. This is the refractory period.

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.

Assignments
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.

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