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Experiment AN-2: Compound Action Potentials Animal Nerve Chapter
1
Experiment AN-2: Compound
Action Potentials
Background
As the previous experiment showed, the interior of a
cell is negatively charged with respect to the outside,
and the magnitude of the potential difference is usually
between 50 and 80 mV. Some cells, like nerves and
muscles, can transiently reverse their membrane poten-
tials. This event is called an action potential and takes
place in milliseconds. During this process, the
membrane potential goes from negative to positive and
back to negative, again.
In the resting cell, the permeability of the membrane to
potassium (P
K
) is greater than its permeability to sodium
(P
Na
). 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
membranes 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 cells
permeability to sodium increases, sodium conductance
increases, and increased sodium conductance leads to
greater depolarization of the membrane. As depolar-
ization 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 hyperpolar-
ization 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 initi-
ation to other locations along the nerve cell is caused by
the positive charges in the cell leaking to an adjacent
(un-stimulated) 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 electri-
cally 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 sciatic nerve of a frog. Each nerve contains
hundreds of axons with different diameters, thresholds,
and degrees of myelination. The large, myelinated axons
with the fastest conduction velocities are known as Type
A fibers, which are further subdivided into
, , , and
types. Type B fibers are also myelinated, but have
smaller diameters and slower conduction velocities.
Type C fibers are very small, unmyelinated axons. When
a large stimulus is delivered to the nerve, many axons
respond and the recorded potential is the summation of
all the axons firing. This potential is known as the
compound action potential (CAP).
In this experiment, students will examine certain
principles associated with nerve conduction: Compound action potentialobserving the one or more
populations of different fiber types. Stimulus-response/axon recruitmentobserving how the
nerve response changes with increased stimulus voltage. Conduction velocitymeasuring the speed at which action
potentials propagate down the axons. Effects of temperatureobserving how cooling affects the
nerve conduction velocity. Bidirectionalitydetermining whether axons conduct in both
directions. Refractorinessobserving how stimulus frequency affects
the amplitude of compound action potentials Strength-Durationobserving how the amplitude of a
stimulus required to stimulate axons is related to the duration
of the stimulus.
Equipment Required
PC Computer
IWX/214 data acquisition unit
USB cable
IWX/214 power supply
NBC-300 Nerve Chamber
C-AAMI-504 input cable
C-ISO-P5 AAMI pin connector-pinjack lead wires (3)
Glass hooks
Pasteur pipettes and bulbs
C-STIM-BNC-P2 BNC-dual pinjack stimulator cable
Double male banana-female BNC adapter
Pinjack-male banana ground cable
Room-Temp & Chilled Amphibian Ringer's solution Animal Nerve Chapter
IWX/214 Setup
1
Place the IWX/214 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 IWX/214.
3
Check Figure T-1-2 in the Tutorial chapter for a picture of
the IWX/214 power supply.
4
Use the USB cable to connect the computer to the USB port
on the rear panel of the IWX/214.
5
Plug the power supply for the IWX/214 into the electrical
outlet. Insert the plug on the end of the power supply cable
into the labeled socket on the rear of the IWX/214. 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 computers 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,
IPLMv4.iwxgrp. Select this group and click Open.
4
Pull down the Settings menu again. Select the Compound
Action Potential-LS2 settings file.
5
After a short time, LabScribe will appear on the computer
screen as configured by the Compound Action Potential-
LS2 settings.
6
For your information, the settings used to configure the
recording channels in the LabScribe software and IWX/214
for this experiment are listed in Table AN-2-1 on page 2.
NBC-300 Nerve Bath Chamber Setup
1
Locate the following items in the iWorx kit: NBC-300 nerve
bath chamber (Figure AN-2-1 on page 2); C-STIM-BNC-P2
stimulator cable (Figure AN-2-2 on page 3); Male double
banana-female BNC adapter (Figure AN-2-3 on page 3).
Figure AN-2-1: The NBC-300 nerve bath chamber.
Table AN-2-1: Settings on the Channel Window
of the Preferences Dialog that Configure the
iWorx System for Experiment AN-2.
Parameter
Units/Title
Setting
Mode/
Function
Acquisition Mode
Scope
Multiple
Sweeps
Number
1
Delay between Sweeps
Sec
0.000
Start
User
Stop
Sec
Timed - 0.015
Display Time
Sec
0.015
Speed
Samples/Sec
20000
Channel A1
Compound AP
S
3-10KHz
Channel S1
Stimulus
S
Record
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The settings used to configure the stimulator for this exper-
iment are listed in Table AN-2-2 on page 2. 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.
Table AN-2-2: Settings on the Stimulator
Window of the Preferences Dialog that Configure
the iWorx System for Experiment AN-2.
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
0.250
Pulses (#pulses)
Number
1
Pulse Width (W)
msec
0.1
Time Off (T Off)
msec
0.9
Time Off Amplitude
Volts
0
Holding Potential (HP)
Volts
0 Animal Nerve Chapter
3
Figure AN-2-2: The C-STIM-BNC-P2 stimulator cable.
Figure AN-2-3: The male double banana-female BNC
adapter.
2
Also, locate the C-AAMI-504 recording cable (Figure AN-2-4
on page 3) and C-ISO-P5 nerve chamber lead wires in the
iWorx kit.
Figure AN-2-4: The C-AAMI-504 recording cable with three C-ISO-P5
nerve chamber lead wires.
3
Plug the male double banana-female BNC adapter into the
positive (red) and negative (black) banana jacks of the IWX/
214 stimulator (Figure AN-2-5 on page 3). 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 (Figure AN-2-3 on page 3).
4
Attach the BNC connector of the C-STIM-BNC-P2 stimu-
lator cable to the adapter on the stimulator outputs (Figure
AN-2-5 on page 3). Place the sockets, at the other end of
the stimulator cable, on the closely-spaced electrodes at
one end of the NBC-300 nerve bath chamber (Figure AN-2-
6 on page 3). The red socket goes on the positive stimu-
lating electrode (+S), which is the electrode closest to the
end of the chamber. The black socket goes on the ne