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iWorx Physiology Lab Experiment

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Experiment AN-4

Action Potentials in Earthworms

Note: The lab presented here is intended for evaluation

purposes only. iWorx users should refer to the User

Area on www.iworx.com for the most current versions of

labs and LabScribe2 Software.

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 voltage-

sensitive 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.

Animal Nerve – Earthworm Action Potentials – SetupIXTA AN-4-1

� 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.



Equipment Required

PC or Mac Computer

IXTA data acquisition unit

USB cable

IXTA power supply

C-ISO-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.


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


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.


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.


� 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.


� 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-ISO-

B3G 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.


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.


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


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) Salt Grams/Liter DI H20

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



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 AN-

4-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.


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.


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-4-

L4.

Figure AN-4-L4: C-ISO-FP5 recording lead wires with pin electrodes attached to the NBC-402 for

long path recording.


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.


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


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.


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


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.



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


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 pull-

down 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


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.

Stimulus StimulusAmp (V)

Action Potential Amplitude (mV)

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.


14. Click on the Main Window icon to return to that window.


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 Room-

Normal.

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.


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.


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.


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?


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.


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.


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.

Interpulse Interval (ms) Effective Frequency (Hz)

Amplitudes (mV)

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


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?


xermnt an-4 action potentials in earthworms · to sodium. if the depolarization reaches or exceeds...

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