Download Action Potentials in Earthworms

Experiment AN-4: Action Potentials in Earthworms
Background
In the resting cell, the permeability of the membrane to potassium (PK) is greater than its permeability
to sodium (PNa). Stimulation, like synaptic activity coming from other nerve cells, can depolarize
(make less negative) the cell membrane. Sodium channels in the cell membrane are sensitive to
membrane depolarization and they respond by opening, which increases the membrane’s permeability
to sodium. If the depolarization reaches or exceeds a certain level (threshold), an action potential is
produced. Action potentials develop because of a regenerative, positive feedback cycle. As the cell’s
permeability to sodium increases, sodium conductance increases, and increased sodium conductance
leads to greater depolarization of the membrane. As depolarization increases, sodium permeability
increases again, and more voltage-sensitive channels open. With more channels open, sodium
conductance and membrane depolarization increase until the membrane potential reaches the
equilibrium potential for sodium.
But, before the equilibrium potential for sodium is reached, two other events occur: the voltagesensitive sodium channels close soon after they open, and the voltage-sensitive potassium channels
open. With these channels open, potassium ions leave the cell and cause the membrane to repolarize
(hyperpolarize) towards its resting level. This process of membrane hyperpolarization closes the
voltage-sensitive potassium channels and re-primes the sodium channels so that they are ready to open
once more. This is called the refractory period.
Propagation of the action potential from the site of initiation to other locations along the nerve cell is
caused by the positive charges in the cell leaking to an adjacent (unstimulated) region and depolarizing
that region enough to create an action potential there. In this way, the signal moves from one region of
the axon to the adjacent one, and ultimately to the end of the axon. Some axons are myelinated; the
axon is covered with a series of Schwann cells, a type of glial cell which electrically insulates the axon.
The spaces between adjacent Schwann cells are called the nodes of Ranvier, and they are the only
regions along the axon where the membrane is exposed to the extracellular fluid. The myelin insulation
prevents the currents associated with action potentials from leaking out of the membrane until they
reach a node. So, action potentials take place only at the nodes in myelinated cells.
In this laboratory you will record action potentials from the ventral nerve cord of an annelid, the
earthworm Lumbricus terrestris. The ventral nerve cord of some invertebrates is a structure analogous
to the dorsal nerve cord of vertebrates. The ventral nerve cord of an earthworm contains three giant
neurons. The cord has one medial giant neuron with a lateral giant neuron on either side of the medial
neuron. Because of their size, the three giant neurons in the earthworm nerve cord can generate action
potentials with conduction velocities that permit the worms to have a fast escape reflex. When a large
stimulus is delivered to the nerve cord, the neurons respond and action potentials from the medial and
lateral neurons are seen.
You will examine certain principles associated with neuronal activity:
•
Neuron viability—determining the viability of the neurons in the nerve cord by observing
action potentials from the medial and lateral giant neurons.
•
Thresholds of neurons—determining the stimulus amplitudes needed to generate action
potentials from the medial neurons.
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•
Conduction velocity—measuring the speed at which an action potential propagates down the
medial neuron.
•
Effects of temperature—observing how cooling affects the conduction velocity of the medial
neuron.
•
Stimulus strength and duration—observing how the stimulus amplitude needed to generate an
action potential is related to the duration of the stimulus pulse.
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Experiment AN-4: Action Potentials in Earthworms
Equipment Required
PC or Mac Computer
IXTA data acquisition unit
USB cable
IXTA power supply
iWire-B3G input cable
NBC-402 Nerve Bath Chamber
C-BNC-P2 Stimulator Cable - BNC to Dual pin jack stimulating cable
C-ISO-FP5 Recording Cable - 5 Lead Pin jack recording cable
Room-Temp & Chilled Ringer's solution
Bottles containing 10% ethanol solution
IXTA Setup
1. Place the IXTA on the bench, close to the computer.
2. Check Figure T-1-1 in the Tutorial chapter for the location of the USB port and the power
socket on the IXTA.
3. Check Figure T-1-2 in the Tutorial chapter for a picture of the IXTA power supply.
4. Use the USB cable to connect the computer to the USB port on the rear panel of the IXTA.
5. Plug the power supply for the IXTA into the electrical outlet. Insert the plug on the end of the
power supply cable into the labeled socket on the rear of the IXTA. Use the power switch to
turn on the unit. Confirm that the red power light is on.
Start the Software
1. Click on the LabScribe shortcut on the computer’s desktop to open the program. If a shortcut is
not available, click on the Windows Start menu, move the cursor to All Programs and then to
the listing for iWorx. Select LabScribe from the iWorx submenu. The LabScribe Main window
will appear as the program opens.
2. On the Main window, pull down the Settings menu and select Load Group.
3. Locate the folder that contains the settings group, IPLMv4Complete.iwxgrp. Select this group
and click Open.
4. Pull down the Settings menu again. Select the Action Potential-Worm-LS2 settings file from
Animal Nerve.
5. After a short time, LabScribe will appear on the computer screen as configured by the Action
Potential-Worm-LS2 settings.
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6. For your information, the settings used to configure the recording channels in the LabScribe
software and IXTA for this experiment are are programmed on the Channel window of the
Preferences Dialog.
7. The settings used to configure the stimulator for this experiment are listed in Table AN-4-S1.
Both groups of settings are programmed on the Channel and Stimulator windows of the
Preferences Dialog, which can be viewed by selecting Preferences from the Edit menu on the
LabScribe Main window.
8. Once the settings file has been loaded, click the Experiment button on the toolbar to open any
of the following documents:
•
•
•
•
Appendix
Background
Labs
Setup (opens automatically)
Table AN-4-S1: Settings on the Stimulator Window of the Preferences Dialog that Configure the
iWorx System for Experiment AN-4.
Parameter
Units/Title Setting
Stimulus Mode
Pulse
Stimulator Start
With Recording
Time Resolution
msec
0.01
Toolbar Step Frequency
Hz
1
Toolbar Step Amplitude
Volts
0.01
Toolbar Step Time
Sec
0.0001
Delay
Sec
0.005
Amplitude (Amp)
Volt
0.200
Pulses (#pulses)
Number
3
Pulse Width (W)
msec
0.1
Time Off (T Off)
msec
10
Time Off Amplitude
Volts
0
Holding Potential (HP)
Volts
0
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Earthworm Recording Setup
1. Plug the BNC adapter of the C-BNC-P2 stimulator cable into the stimulator 1 input of the IXTA
stimulator (Figure AN-4-S2). The banana plug that goes into the negative (black) stimulator
output is identified by a tab, embossed with the letters GND (ground), on that side of the
adapter.
2. Locate the C-ISO-B3G recording cable (Figure AN-4-S1) in the iWorx kit and plug it into the
iWire 1input on the front of the IXTA (Figure AN-4-S2).
Figure AN-4-S1: The C-ISO-B3G recording cable.
Figure AN-4-S2: The C-ISO-B3G recording cable and C-BNC-P2 stimulator cables attached to the
IXTA.
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Figure AN-4-S3: NBC-402 Nerve bath chamber with the C-BNC-P2 and C-ISO-FP5 cables for
recording worm action potentials.
Electrical Noise
Electrical noise is the most common problem associated with the recording of bioelectric signals. It
radiates through the air and comes from electrical devices in the lab room or building: lights, power
outlets, computers, monitors, and the power supplies. Since the source of power for these devices is
60Hz alternating current (AC), this electrical noise appears as a distorted sine wave with a repeating
period of 16.7 milliseconds (msec).
There are two major sources of electrical noise: pickup and ground loops.
Pickup
Pickup is caused by electrical radiation that produces currents in the electrodes and wires leading to the
amplifiers in the recording system. Because the resistance in the electrodes is high, small currents
produce large voltages that may be greater than the biopotential being recorded. The major ways to
reduce pickup are:
•
Faraday Cage: Put a grounded, screened enclosure, known as a Faraday cage, around the
preparation and the electrodes. the enclosure separates the source of the radiation from
the electrodes. The person operating the equipment might also be a source of noise, and
he or she may need to be grounded.
•
Shielded Cables: Use shielded cables to carry the signals from the electrodes to the
amplifier and the recorder; this puts a protective ground around the wires carrying the
bioelectric signal.
•
Differential Recording: Record using both a positive and a negative recording electrode
placed on a nerve or neuron. The noise signals that are equal in magnitude, but opposite
in polarity, will cancel each other out and leave a flat baseline.
•
Short Cables: Use the shortest cables available to reduce the length of wiring exposed to
electrical noise.
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•
Direct Current Equipment: Use equipment, like preamplifiers and illuminators, that are
powered by batteries or direct current (DC) transformers.
•
Equipment Removal: Unplug or remove unused alternating current (AC) equipment
from the area.
Ground Loops
Ground loops are a troublesome source of electrical noise caused by the ground cable itself serving as
an antenna for the noise radiating in the room. Using a Faraday cage to shield the preparation and the
recording electrodes does not remove the electrical noise caused by ground loops. To avoid ground
loops, use the following techniques:
•
Ground Hub: Ground all the equipment around the preparation to a common grounding
point (hub). This includes all the items that are electrically powered or are made of
metal, like illuminators or microscopes. Use simple cables, like banana cords equipped
with alligator clips, to connect each device directly to the common grounding point. The
common ground point is connected to the ground of the recording device with a single
cable. The recording device is connected to the building ground.
•
Simple Chain: Ground the devices to the common grounding point using the simplest
route that links the first device to the second device, the second device to the third
device, and so on. Start the chain at the device that is the farthest from the common
grounding point. End the chain by connecting the last device to the common grounding
point, which is connected to the ground of the recording device.
•
Free-Floating: In addition to using one of the grounding techniques described earlier,
plug all devices powered by alternating current (AC), like illuminators, amplifiers, and
recording units to power outlets using three-two prong adapters.
High Frequency Noise
High frequency noise can also be a problem when recording bioelectric potentials. This type of noise is
seen as the thickening of the recorded line. This noise contains many frequencies, and the amplitude of
the noise is proportional to the resistance of the electrode. Therefore, intracellular electrodes, with high
resistances, pick up a greater amplitude of high frequency noise than extracellular electrodes, with low
resistances.
Mechanical Noise
Mechanical noise, like vibrations from the ventilation system in the room, can cause the electrodes to
vibrate and produce voltage changes with each vibration cycle. To alleviate this problem, isolate the
platform holding the preparation with foam pads or bicycle inner tubes. Also, avoid bumping the table
when the recording electrodes are in place.
Grounding
The iWorx unit has two different connections for grounding:
•
One method uses the ground (C) input on the C-ISO-B3G recording cable.
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•
The other method uses either of the green (Ground or GND) banana jacks on the IXTA.
One jack is located on the front panel in the stimulator section; the other jack is located
on the back panel of the IXTA.
1. Try recording when the nerve cord is grounded to the ground (C) input on the C-ISO-B3G
recording cable that is connected to the isolated inputs of the IXTA.
•
Attach the needle electrode on the green C-ISO-N3 electrode lead wire to ground (C)
socket of the lead pedestal of the C-ISO-B3G recording cable.
•
Place this ground electrode along the midline between the stimulating electrodes and the
proximal recording electrode. With the ground electrode in this position, the amplitude
of the stimulus artifact picked up by the recording electrodes will be reduced.
2. If the action potential recording has a lot of 60Hz noise while using the ground of the C-ISOB3G cable, disconnect the green C-ISO-N3 electrode lead wire from the ground (C) socket of
the lead pedestal of the C-ISO-B3G recording cable.
•
Attach an optional alligator-male banana electrode lead wire.
•
Attach the alligator clip on one end of this lead wire to the ground electrode itself.
•
Attach the male banana plug on the other end of this lead wire to the green banana jack
on either the front or back panel of the iWorx unit.
Signal Improvement
1. To improve recordings of the action potential, move the preparation away from sources of 60Hz
noise. These sources include outlets, computers, monitors, lights, refrigerators, water baths, and
other AC powered devices.
2. If the recording still contains a great deal of electrical noise, apply the digital filtering function
to the data. Click on the add function button in the upper margin of the Action Potential
channel. Select Filter from the menu of computed functions.
3. In the Filter Setup Dialog window, the Filter Mode is set to the Hamming Window and the
Filter Order is set to 51, these are the default settings that should be used.
4. Set the Low Cutoff filter value to 65 and the High Cutoff filter value to 8000. The values for the
filter cutoffs can be set by:
•
Typing the values for the filter cutoffs in the boxes to the right of the names of the filter
cutoffs.
•
Clicking on the up or down arrows to the right of the boxes displaying the values of the
filter cutoffs.
•
Clicking on the margins of the colored area in the graphic display of the filter and
dragging the margins to the values required.
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Preparation of the Earthworm
1. Obtain an earthworm and rinse it off with tap or distilled water.
2. In a well ventilated area, place 10-25 ml of the 10% ethanol solution into a beaker.
3. Anesthetize the worm by placing it into the beaker. Remove the worm as soon as it stops
moving. This takes approximately 5 minutes, but may take a little longer. Note that too much
ethanol can kill the worm and too little will cause the worm to wriggle while attached to the
electrodes.
4. Dispose of the ethanol solution properly into a waste container. DO NOT REUSE the ethanol
for another worm.
5. Place the worm with its ventral side down on the NBC-402 Nerve Bath Chamber so that the
worm lays the length of the electrodes.
6. The anterior portion of the worm is marked by the clitellum (the thickened area). This end of
the worm will be at the stimulating end of the NBC-402 with the 3 pins.
7. Gently stretch the worm the length of the nerve bath chamber.
8. Locate the C-BNC-P2 Stimulator Cable (Figure AN-4-S3). Place the stimulator jacks on the
electrodes as shown in Figure AN-4-S4.
9. Firmly push the leads onto the pins of the chamber so that:
10. The red lead is on the outer most pin of the stimulating section of the chamber.
11. There is a blank/empty pin between the red and black lead.
12. The black lead is on the third pin.
13. Locate the C-ISO-FP5 recording lead wires with pin jacks in the collection of equipment
needed for this experiment (Figure AN-4-S3).
Figure AN-4-S4: The C-BNC-P2 Stimulator cable attached to the NBC-402.
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Figure AN-4-S5: C-ISO-FP5 recording lead wires with pin electrodes attached to the NBC-402 for
short path recording.
14. Attach the red, black, and green C-ISO-FP5 recording lead wires to the corresponding sockets
on the lead pedestal of the C-ISO-B3G biopotential cable (Figure AN-4-S1).
15. Attach the leads to the pins on the nerve bath chamber as shown in figure Figure AN-4-S5.
Firmly push the leads onto the pins so that:
•
The green/ground lead is on the first pin diagonally opposite the stimulating electrodes.
•
Leave one pin empty, then place the black (-1) lead on the 3rd pin.
•
Place the red (+1) lead on the 4th pin.
•
Leave one pin empty, then place the white (+2) lead on the 6th pin.
•
Place the brown (-2) lead on the 7th pin.
Note: The photo shows the white and brown leads set up for short path recording - this setup will be
changed during the Nerve Conduction Velocity exercise.
Figure AN-4-S7: The earthworm preparation showing the position of the recording and stimulating
electrodes
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Warning: The earthworm preparation used in this experiment is functional for a limited period of
time. To conserve time, complete all the exercises in the experiment before analyzing the data. Keep
an eye on the earthworm during the experiments. If the worm begins to move, remove the electrodes
and re-anesthetize it, or carefully drip the ethanol solution on it and quickly wick away the excess.
Dispose of all waste in properly labeled containers.
Warning: You WILL need to blot any liquid from between the electrodes of the nerve bath chamber
to prevent bridging. If bridging occurs, there will be no conduction of current and no reaction from
the specimen.
Appendix: Recipe for Earthworm Ringer’s Solution.
Concentration (mMolar)
Grams/Liter DI H20
Salt
75.0
Sodium Chloride
4.38
4.0
Potassium Chloride
0.30
2.0
Calcium Chloride
0.22
1.0
Magnesium Chloride
0.10
10.0
Tris
1.21
23.0
Sucrose
7.87
Adjust the pH of the solution to 7.4 with 6N HCl
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Experiment AN-4: Action Potentials in Earthworms
Exercise 1: Viability of the Neurons
Aim: To test the viability of the nerve cord by stimulating the nerve cord with a stimulus amplitude that
should exceed the threshold of the neurons.
Procedure
1. Click the Stimulator Preferences icon on the LabScribe toolbar (Figure AN-4-L1) to open the
stimulator control panel (Figure AN-4-L2) on the Main window.
Figure AN-4-L1: The LabScribe toolbar.
Figure AN-4-L2: The stimulator control panel
2. Check the values for the stimulus parameters that are listed in the stimulator control panel on
the Main window. The pulse amplitude (Amp) should be set to 3.0 V, the number of pulses
(#pulses) to 1, the pulse width (W) should be set to 0.1 msec, and TimeOff to 10 msec. Click on
the Apply button to insure that the values set for the stimulus parameters are the ones that are
used in exercise.
3. Click the Record button to stimulate and record from the nerve cord. The recording stops
automatically after one sweep. On the Action Potential channel, use the + button to the left of
the AutoScale button to zoom in on the action potential. Click the AutoScale button on the
upper margin of the Stimulus channel. The recording should be similar to the one in Figure AN4-L3.
Note: The stimulus amplitude and width set for this exercise should be strong enough to cause an AP in
a healthy neuron. If there is a single AP, the medial neuron with its lower threshold is the one being
stimulated. If two adjacent APs are seen, both the medial and lateral neurons are being stimulated.
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Figure AN-4-L3: The monophasic action potential shown on both channel 1 and channel 2 of the
earthworm action potential recording.
4. If two adjacent action potentials are seen in the recording, lower the stimulus amplitude until
only one AP is seen. Use the down arrow next to the pulse amplitude (Amp) window to lower
the stimulus amplitude. Click on the Apply button, before clicking on the Record button, to
finalize the change in the stimulus amplitude.
5. On extracellular recordings of action potentials, a stimulus artifact may appear: The size of the
artifact depends on the width and amplitude of the stimulus pulse and the effectiveness of the
ground electrode separating the stimulating electrodes from the recording electrodes. If the end
of the stimulus artifact merges with the beginning of the action potential, move the proximal
(black) recording electrode away from the ground electrode and closer to the distal (red)
recording electrode. Record another action potential.
Note: You may find that these giant neurons “fatigue” with continued stimulation. If this happens, stop
stimulating for a few minutes to give the neurons time to recover, then try again.
6. If an action potential did not appear on the screen, check:
•
The wiring between the stimulator of the iWorx unit and the stimulating electrodes
•
The wiring between the isolated inputs of the iWorx unit and the recording electrodes.
•
The values for the stimulus parameters are shown on the stimulator control panel. These
values should match the ones listed in the table in the Setup document. If they do not
match, make the necessary adjustments. Click the Apply button on the stimulator control
panel to finalize the stimulus settings.
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7. Click the Record button to stimulate and record from the neuron. If the neuron does not respond
after the connections and the settings have been verified, increase the stimulus amplitude (Amp)
by 0.5 V. Remember to click the Apply button to finalize the change.
8. Click the Record button to stimulate and record from the neuron again. If no action potential is
generated, continue to increase the stimulus amplitude (Amp) in increments of 0.5V and record
from the nerve until the AP appears.
Warning: Do not exceed a stimulus amplitude of 4.0V without consulting your instructor.
9. Select Save As in the File menu, type a name for the file. Choose a destination on the computer
in which to save the file, like your lab group folder). Designate the file type as *.iwxdata. Click
on the Save button to save the data file.
Note: Remember to reanesthetize the earthworm as needed by dripping a small amount of 10% EtOH
on it. Quickly wick away the excess. If the earthworm is clearly dead, replace it with a new
anesthetized worm. Be careful not to over anesthetize your worm.
Exercise 2: Conduction Velocity
Aim: To measure the conduction velocity of the action potential in the medial neuron.
Procedure
1. Change the stimulus amplitude (Amp) to the lowest voltage that creates an action potential in
the medial neuron. Click the Apply button to finalize the change in the stimulus amplitude.
2. Make sure the recording electrodes are positioned as they were for Exercise 1.
3. Type Short Path in the Mark box to the right of the Mark button. Press the Enter key on the
keyboard to attach this notation to the recording. Click Record to stimulate the neuron.
4. Move the white and brown recording electrodes to pins 8 and 9 as shown in the Figure AN-4L4.
Figure AN-4-L4: C-ISO-FP5 recording lead wires with pin electrodes attached to the NBC-402 for
long path recording.
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5. Type Long Path in the Mark box to the right of the Mark button. Press the Enter key on the
keyboard to attach this notation to the recording. Click Record to stimulate the neuron.
6. Measure the distance between the two positions of the proximal (black and white) recording
electrodes that were used.
7. Select Save in the File menu.
8. This exercise can be repeated, moving the white and brown recording electrodes to pins farther
away from the red and black electrodes. Remember to measure the distance between the
proximal electrodes as stated in step 6.
9. Select Save in the File menu.
Exercise 3: Conduction Velocity and Temperature
Aim: To examine the effects of cooling the worm on the conduction velocity of the action potential in
the medial neuron.
Note: This part of the experiment must be done quickly since the earthworm will begin to warm as
soon as the cold paper towels are removed from the earthworm.
Procedure
1. Carefully cover the anesthetized worm with a few KimWipes or paper towels soaked in very
cold worm Ringer’s solution. Keep the earthworm covered for 2 minutes to get chilled.
2. Remove the paper towels and quickly measure the conduction velocity of the neuron using the
same procedures used in Exercise 3.
3. Remember to start with the electrodes in the Short Path position and move them to the Long
Path position.
4. Repeat Exercise 3 after the earthworm has warmed back up to room temperature.
Exercise 4: Refractory Period
Aim: To measure the effect of stimulus frequency on the occurrence of multiple action potentials in a
neuron.
After a neuron generates an action potential, it is unable to generate a second action potential until the
membrane recovers or the strength of the stimulus is increased. This time is known as the refractory
period. The refractory period can be divided into two sections: the absolute refractory period, which
occurs directly after the first action potential. During the absolute refractory period, the neuron is
unable to generate a second action potential even though the stimulus strength is set to a very high
level. The other section of the refractory period is known as the relative refractory period, and it occurs
after the neuron has been given additional time to recover. In the relative refractory period, the neuron
is able to generate a second action potential, but the strength of the stimulus needed to do this is greater
than the threshold stimulus of the neuron. As the neuron is given more time to recover, the strength of
the stimulus required to trigger the second action potential decreases. In fact, the end of the relative
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refractory period is defined as the time when the stimulus required to trigger the second action potential
is the same as the threshold stimulus that can trigger the first action potential in the pair.
Procedure
1. Adjust the critical stimulus parameters to the values listed in Table AN-4-L1 using the same
techniques used in earlier exercises. The stimulus amplitude must be set to the lowest voltage
that will generate an action potential in the medial neuron. Click the Apply button to finalize the
changes to these stimulus parameters.
2. Click Record to stimulate the neuron. Type 10msec in the Mark box to the right of the Mark
button. Press the Enter key on the keyboard to attach this notation to the recording.
3. Change the time interval between stimulus pulses (T Off) to 9 msec. Click the Apply button to
finalize this change.
4. Click Record to stimulate the neuron. Type 9msec in the Mark box. Press the Enter key on the
keyboard to attach this notation to the recording.
5. Repeat Steps 3 and 4 to record the effects of decreasing the time interval between the two
stimuli on the amplitude of the second action potential. Change the time interval between the
stimulus pulses (T Off) to 8, 7, 6, 5, 4, 3, 2, and 1 msec. Record the action potentials and mark
the recording for each new time interval.
6. Select Save in the File menu.
Table AN-4-L1: Stimulus Parameters Required for Measuring the Refractoriness of the Neuron.
Stimulus Parameter
Value
Stimulus Amplitude (Amp)
Threshold Level
Number of Pulses (#pulses)
2
Pulse Width (W)
0.1 msec
Time Between Pulses (T Off)
10 msec
Holding Potential (HP)
0
Exercise 5: Stimulus Strength and Duration
Aim: To demonstrate the inverse relationship between the stimulus amplitude and the stimulus duration
needed to generate action potentials.
Stimulus pulses change the flow (current) of ions through the neuronal membrane. In turn, changes in
ion currents lead to changes in the membrane potential of the neuron. If the change in the membrane
potential exceeds the critical level known as the threshold, the neuron generates an action potential. The
changes in ion currents are dependent on the stimulus amplitude and the stimulus duration. For
example, a stimulus pulse with a low amplitude and a long duration can stimulate a neuron just as
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easily as a stimulus pulse with a high amplitude and a short duration.
Procedure
1. Adjust the critical stimulus parameters to the values listed in Table AN-4-L2 using the same
techniques used in earlier exercises. Click the Apply button to finalize the changes to these
stimulus parameters.
2. Click Record to stimulate the neuron. Type 0.100 msec, and the stimulus amplitude in the Mark
box to the right of the Mark button. Press the Enter key on the keyboard to attach this notation
to the recording.
3. Change the stimulus duration to 0.010msec. Click the Apply button to finalize this change.
4. Click Record to stimulate the neuron. Type 0.010 msec and the stimulus amplitude in the Mark
box. Press the Enter key on the keyboard to attach this notation to the recording.
5. If an action potential is not detected when the stimulus duration is 0.010msec:
•
Increase the stimulus amplitude by an increment of 0.5 V, and click the Apply button.
•
Click Record to stimulate the neuron. Mark the recording with 0.01 msec and the current
stimulus amplitude.
•
Continue to raise the stimulus amplitude by 0.5 V and record until an action potential
appears. Mark the sweep with the stimulus duration, 0.010msec, and the value of the
stimulus amplitude that first caused an action potential at that stimulus duration.
6. Enter the stimulus amplitude that first causes an action potential at the duration of 0.010msec on
Table AN-4-L3.
Table AN-4-L2: Stimulus Parameters Required for Determining the Stimulus Strength-Duration
Relationship of the Neuron.
Stimulus Parameter
Value
Stimulus Amplitude (Amp)
Threshold Level
Number of Pulses (#pulses)
1
Pulse Width (W)
0.01 msec
Time Between Pulses (T Off)
0.9 msec
Holding Potential (HP)
0
7. Change the stimulus duration to 0.020msec. Click the Apply button to finalize this change.
8. Repeat Steps 4 and 5 for the stimulus duration of 0.020msec.
9. Repeat Steps 3, 4, and 5 for the following stimulus durations: 0.04, 0.08, 0.16, 0.32, 0.64, and
1.28msec.
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10. Select Save in the File menu.
Data Analysis
Exercise 1 - APs from Medial Neurons
1. Use the tabs in the Sweep Selection bar at the bottom of the Main window (Figure AN-4-L5) to
find the sweep displaying the action potential from the medial neuron caused by the stimulus
amplitude of 3 V.
2. Click on a tab in the selection bar and the sweep associated with the tab will appear on the Main
window. On the Action Potential channel, use the + button to the left of the AutoScale button to
zoom in on the action potential as shown in Figure AN-4-L6.
3. Click the AutoScale button on the Stimulus channel to maximize the height of the stimulus.
4. Once the sweep is displayed on the Main window, transfer the sweep to the Analysis window
(Figure AN-4-L6) by clicking on the Analysis window icon in the toolbar or selecting Analysis
from the Windows menu.
5. Look at the Function Table that is above the uppermost channel displayed in the Analysis
window. The mathematical functions, V2-V1 and T2-T1, should appear in this table. The values
for V2-V1 and T2-T1 on each channel are seen in the table across the top margin of each
channel.
Table AN-4-L3: Stimulus Amplitudes that First Cause Action Potentials in the Medial Neuron at
Specified Stimulus Durations.
Stimulus Duration (ms)
Stimulus Amplitude (V)
0.100
0.010
0.020
0.040
0.080
0.160
0.320
0.640
1.280
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6. Once the cursors are placed in the correct positions for determining the amplitude of the action
potential, the amplitude (V2-V1) can be recorded in the on-line notebook of LabScribe by
typing its name and value directly into the Journal, or on a separate data table.
Figure AN-4-L5: The Sweep Selection bar showing the tab for Sweep 7 highlighted.
Figure AN-4-L6: Action potentials (AP) and stimulus pulse displayed in the Analysis window, with
cursors positioned to measure the amplitude of the AP.
7. The functions in the channel pull-down menus of the Analysis window can also be used to enter
the name and value of the parameter from the recording to the Journal. To use these functions:
•
Place the cursors at the locations used to measure the amplitude of the action potential.
•
Transfer the names of the mathematical functions used to determine the amplitude to the
Journal using the Add Title to Journal function in the Action Potential Channel pulldown menu.
•
Transfer the values for the amplitude to the Journal using the Add Ch. Data to Journal
function in the Action Potential Channel pull-down menu.
8. To measure the amplitude of the action potential, place one cursor on the baseline before the
stimulus artifact displayed on the Action Potential Channel. Place the other cursor on the peak
of the AP (Figure AN-4-L6). The value for the V2-V1 function on the Action Potential Channel
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is the amplitude of the action potential.
9. Record the amplitude of the action potential generated by 3V in the Journal using the one of the
techniques described in Steps 5 or 6, and on Table AN-4-L4.
Table AN-4-L4: Amplitude of Action Potentials Generated by Stimulus Pulses of Different
Amplitudes.
Action Potential Amplitude (mV)
Stimulus
StimulusAmp (V)
Medial Neuron Lateral Neuron
Initial Trial Stimulus
3
Threshold for Medial Neuron
Maximum for Medial Neuron
10. Use the tabs in the Sweep Selection bar at the bottom of the Analysis window to find the sweep
with the action potential from the medial neuron that was caused by the threshold stimulus
amplitude.
Note: Threshold amplitude may be higher or lower than the initial setting of 3 V.
11. To take measurements from a sweep displayed on the Analysis window, select its name from the
Sweep menu in the upper left margin of the data display window (Figure AN-4-L7).
12. Use the same techniques described earlier to measure and record the amplitude of the action
potential from the medial neuron caused by its threshold stimulus amplitude.
Figure AN-4-L7: The upper left corner of the Analysis window showing the Sweep menu used to select
the sweep from which the data is measured.
13. Select Save in the File menu.
14. Click on the Main Window icon to return to that window.
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Questions
1. When was threshold reached? Was it at 3 V, or higher, or lower?
2. Does the amplitude of the action potential in the medial neuron increase when the stimulus
amplitude is increased?
Exercise 2 - Conduction Velocity
1. Use the Sweep Selection bar at the bottom of the Main window to display the first sweep
needed to calculate the conduction velocity of the neuron. Click on the tab of that sweep and it
will appear on the Main window.
Note: Use the + button to maximize the action potential.
2. Transfer the sweep to the Analysis window using the Analysis window icon in the toolbar or the
Analysis listing on the Windows menu.
3. This sweep contains the APs from channels 1 and 2. Channel 1 represents the black and red
recording electrodes; channel 2 represents the white and brown recording electrodes.
4. Place one cursor on the peak of the AP shown in channel 1 and second cursor on the peak of the
AP in channel 2; measure T2-T1 (Figure AN-4-L8):
•
The value for T2-T1 is the time it takes the AP to move the distance between the two
positions of the recording electrodes used in this exercise.
•
Record the value for T2-T1 (in msec) in the Journal, and on Table AN-4-L5, for
temperature and direction labeled as Room-Normal.
5. Measure the conduction distance (in mm) between the two positions used for the proximal
(black and white) recording electrodes. Record this distance (in mm) in the Journal, and on
Table AN-4-L5, as the conduction distance for temperature and direction labeled as RoomNormal.
6. Calculate the conduction velocity (in meters per second). Divide the distance (in mm) between
the two positions of these electrodes by T2-T1, which is the time (in msec) between the peaks
of the two action potentials.
For example: 10 mm/0.2 msec = 50 mm/msec = 50 m/sec
7. Record the conduction velocity in the Journal, and on the data table for the temperature and
direction labeled as Room Temp.
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Figure AN-4-L8: Two sweeps used to determine the conduction velocity of the neuron.
8. Go back to main window to select another sweep at Room Temp when the electrodes may have
been moved farther apart. Follow the same directions in Steps 3-6 to calculate the Conduction
Velocity.
9. Select Save in the File menu.
Exercise 3 - Conduction Velocity at Low Temperature
1. Use the same techniques used in the analysis of Exercise 2 to make and record the
measurements needed to determine the conduction velocities of the neuron. Record the values
for these measurements in the Journal and on Table AN-4-L5.
2. Calculate and record the conduction velocity of the neuron at the cold temperature (Cold Temp).
3. Calculate and record the conduction velocity of the neuron at room temperature (Room-Post)
after the neuron cooled and warmed.
4. Select Save in the File menu.
Questions
1. Does the conduction velocity change when the earthworm is cooled?
2. What properties of the ion channels may change with temperature?
3. After warming back up to room temperature, was the conduction velocity the same as it was
prior to cooling? Why or why not?
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Table AN-4-L5: Conduction Velocities of a Neuron at Two Different Temperatures.
Temperature
Distance (mm) Time (msec) Conduction Velocity (m/sec)
Room Temp
Cold Temp
Room - Post
Exercise 4 - Refractory Period
1. Use the Sweep Selection bar at the bottom of the Main window to display the sweep with a pair
of stimulus pulses that are 10msec apart. Click on its tab to make it appear on the Main window.
2. Transfer the sweep to the Analysis window using the Analysis window icon in the toolbar or the
Analysis listing on the Windows menu.
3. Measure and record the amplitudes of the two action potentials using the techniques described
in the analysis of Exercise 2. Record the amplitudes of the first and second action potentials on
Table AN-4-L6.
Figure AN-4-L9: Pair of action potentials stimulated by a pair of pulses that are 5 msec apart. Note channel 2 is not displayed in this image.
4. To display the other sweeps where the times between the stimulus pulses are getting shorter,
click on the tabs for those sweeps in the Sweep Selection bar at the bottom of the Analysis
window. The sweeps that are selected will appear together on the Analysis window.
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5. To take measurements from another sweep displayed on the Analysis window, select its name
from the Sweep menu in the upper left margin of the data display window. Measure and record
the amplitudes of these action potentials as described in Step 3.
6. Select Save in the File menu.
Questions
1. Does the first AP in the pair have the same amplitude at all of the frequencies?
2. Does the second AP in the pair have the same amplitude at all of the frequencies?
3. At which frequency, or interpulse interval, does the second AP in the pair decline or disappear?
4. Is a neuron that generates a full-sized action potential with a stimulus of increased amplitude in
the absolute or relative portion of its refractory period?
Table AN-4-L6: Amplitudes of Action Potentials Occurring at Different Frequencies.
Amplitudes (mV)
Interpulse Interval (ms)
Effective Frequency (Hz)
First AP Second AP
10
100
9
111
8
125
7
143
6
167
5
200
4
250
3
333
2
500
1
1000
Exercise 5 - Stimulus Strength - Duration
1. Graph the stimulus amplitude needed to create an action potential as a function of the stimulus
duration. Data is in Table AN-4-L3. Place the values for the stimulus amplitudes on the Y-axis,
and the values for the stimulus durations on the X-axis.
2. The minimum stimulus amplitude which will elicit action potentials at an infinitely long
stimulus duration is a value known as the rheobase. Another value, chronaxie, is the stimulus
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duration where the stimulus amplitude is twice the value of the rheobase. Chronaxie values are
used as measures of the excitability of neurons. The most excitable neurons have the smallest
chronaxies. Using the graph created in Step 1, determine rheobase and chronaxie for the medial
neuron.
Questions
1. How does the excitability of your medial neuron compare to the medial neurons used by other
groups in your lab section?
2. Collect conduction velocity and chronaxie data from the other groups in your lab section. Is
there a relationship between conduction velocity and neuron excitability? If so, what is it?
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