Experiment AN-3: Neuromuscular Studies
Background
The purpose of this experiment is to demonstrate some of the electrical properties of a muscle and its
motor neurons. Nerve and muscle action potentials will be recorded from nerve-muscle preparation.
The conduction time and synaptic delay of the neuromuscular unit will be determined. The effect of
stimulus frequency, as well as the effects of specific chemical agents upon the muscle and nerve and
the neuromuscular junction, will also be measured.
The Motor Unit
Skeletal muscles are innervated by motor neurons whose cell bodies are located in the ventral horn of
the gray matter of the spinal cord. Although each skeletal muscle fiber is innervated by only one motor
neuron, each motor neuron innervates more than one muscle fiber. A motor neuron and the population
of muscle fibers it innervates form the basic functional unit of the neuromuscular system, the motor
unit. Each motor unit in a vertebrate behaves in an all or none fashion; when the motor neuron fires, the
innervated muscle fibers contract maximally and in unison. The number of muscle fibers innervated by
a single neuron varies from three to several hundred depending on the function of the muscle. Motor
units that control fine movement, such as those of the ocular muscles, are innervated by a large number
of neurons and contain very few muscle fibers. On the other hand, large muscles that do not require a
fine degree of control, such as the gastrocnemius, have several hundred muscle fibers in a motor unit.
The junction of a nerve axon terminal and another cell (neuron or muscle) is known as a synapse. The
synapse between a motor neuron and a muscle fiber is known as the neuromuscular junction. In 98% of
all muscle fibers, there is only one junction, and this synapse is located in the center of the muscle fiber.
The muscle action potential begins at this point and spreads to either end of the muscle fiber. This
spreading of the potential from the center allows for the nearly coincidental contraction of all
sarcomeres in the muscle. Since the units are contracting together, and not separately, the development
of muscle tension is more efficient. Usually muscle fibers from adjacent motor units interlock, with
small bundles of one motor unit lying among similar bundles in a second unit. This interaction also
increases the amount of coincident contraction.
The degree of muscle contraction is primarily a function of the number of motor units that are
activated. Motor units have different stimulus threshold values depending on the cell and fiber
composition of each unit. As the strength of the stimulus is increased, it surpasses the threshold
voltages of a greater number of motor units in the muscle. As more motor units are excited, the strength
of the muscle contraction increases to a maximum level, where all fibers are excited.
The Neuromuscular Junction
The neuromuscular junction of vertebrates has been intensely studied as a model of general synaptic
function because its size and accessibility are greater than synapses within the central nervous system
(Figure AN-3-B1). This synapse is a critical point in communication between the neural and muscular
systems.
Animal Nerve – Neuromuscular Studies – Background
AN-3-1
Figure AN-3-B1: The Neuromuscular Junction.
The distal end of the axon branches into many presynaptic terminals so that each muscle fiber has at
least one junction with the motor neuron. The terminals innervate the muscle at a specialized region of
the muscle (or postsynaptic) membrane known as the end-plate. The space between the terminal and
the end-plate is called the synaptic cleft. It is 20 to 30 nanometers wide and contains the basal lamina,
which is a thin layer of connective tissue through which extracellular fluids diffuse.
Synaptic Conduction
Motor neurons use acetylcholine (ACh) to transmit nerve impulses across the synapse to the muscle
fibers. The presynaptic terminals have a high concentration of membrane-bound synaptic vesicles
containing ACh. These vesicles fuse with the terminal membrane to release ACh into the synaptic cleft
(Figure AN-3-B2). Although the mechanism is not completely understood, calcium (Ca++) is known to
be necessary in this process. When a nerve action potential depolarizes the presynaptic terminal, Ca++
channels are opened causing an influx of Ca++ into the terminal. Once inside the terminal, Ca++ aids
with the fusion of synaptic vesicles to the terminal membrane. The time consumed during the processes
which occur after the arrival of the presynaptic action potential and before the elicitation of the muscle
action potential is known as the synaptic delay. The fusion of vesicles to the membrane and the
subsequent release of ACh occur about 0.3 msec after the arrival of the nerve action potential, which
accounts for most of the synaptic delay.
Animal Nerve – Neuromuscular Studies – Background
AN-3-2
Figure AN-3-B2: Disposition of Acetylcholine at the neuromuscular junction.
Once released, acetylcholine rapidly diffuses (in 0.01 msec) across the synaptic cleft and binds with
specific receptors on the muscle end-plate. This process greatly increases the permeability of the
muscle membrane to positive ions. The concentration and potential gradients of this membrane permit
Na+ to enter the muscle fiber, depolarizing the membrane. The muscle membrane potential rises in the
area of the end plate, creating a local end-plate potential, which further increases the membrane's
permeability to Na+. When the end-plate potential reaches its threshold value, a muscle action potential
is generated. If the muscle action potential is large enough, it will cause muscle fibers to contract.
Within 1 msec of its release from the presynaptic terminal, much of the excessive amount of
acetylcholine has already diffused out of the synaptic cleft and no longer has a chance of acting on the
postsynaptic membrane. The remaining molecules are hydrolyzed to choline and acetate by the enzyme
acetylcholinesterase, which is in the matrix of the basal lamina. The hydrolysis can occur before or
after acetylcholine binds to the receptor sites since the binding is freely reversible. The short period of
time that some acetylcholine molecules remain in contact with the muscle fiber membrane is almost
always sufficient to excite a muscle action potential.
After the hydrolysis of acetylcholine, choline is actively transported back into the axon terminal by a
pump in the presynaptic membrane. Inside the terminal, choline is acetylated by choline acetyl
transferase, and the resynthesized acetylcholine is stored in presynaptic vesicles to be used again.
Excitation-Contraction Coupling
Once a nerve action potential has been transmitted across the synaptic cleft, and the end-plate potential
has risen above its threshold value, a muscle action potential is initiated. This action potential elicits an
electrical current which spreads along the muscle fiber and its transverse tubules (T tubules), which
penetrate all the way through the muscle fiber. The action potentials in the T tubules release calcium
from the adjacent sarcoplasmic reticulum. When the calcium diffuses to the myofibrils, chemical
changes occur in the muscle fiber and contraction begins. This overall process for controlling muscle
contraction is called excitation-contraction coupling.
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AN-3-3
Muscle Action Potentials
The initiation and conduction properties of muscle action potentials are almost the same as those of
nerve action potentials, except for some quantitative differences:
•
The resting membrane potential of skeletal muscle fibers is approximately - 90 mV, which is the
same as in large myelinated nerve fibers, but more negative than in most neurons.
•
The duration of skeletal muscle action potentials is up to 5 times longer than the duration of
those from large myelinated nerve fibers.
•
The velocity of conduction in skeletal muscle fibers is significantly lower than the velocity of
conduction in the nerve fibers that innervate these muscle fibers.
Pharmacology
The neuromuscular junction is susceptible to many drugs which may have one of several effects:
Acetylcholinesterase Inhibition
Physostigmine (eserine) and diisopropyl fluorophosphate (DFP) inhibit the hydrolytic enzyme,
acetylcholinesterase. Inhibition of acetylcholinesterase prevents degradation of acetylcholine. Due to
the continued presence of acetylcholine, the muscle fiber contracts and then becomes unresponsive to
further stimulation (contracted paralysis). For example, small quantities of DFP induce twitching of the
facial muscles and tongue, but larger amounts lead to death from suffocation (breathing muscles are
continuously depolarized).
Receptor Blockage
Curare and its synthetic analog gallamine triethiodide bind to the postsynaptic receptor site and make it
unavailable to acetylcholine. These two drugs are inhibitory because they have molecular structures
which mimic acetylcholine and are therefore capable of binding at the receptor sites. However, these
drugs are not capable of stimulating muscle fibers. An injection of curare causes progressive flaccid
paralysis of the skeletal musculature, starting with the muscles of the eyes, moving to the head and
neck, and finally to the limbs and breathing.
The effects of many drugs which inhibit acetylcholinesterase or block ACh receptors are reversible
with time.
Other Effects
Neuromuscular transmission can be prevented by blocking Na+ channels and thereby inhibiting nerve
action potentials (tetrodotoxin), inhibiting acetylcholine synthesis (hemicholinium), inhibiting
acetylcholine release (procaine, botulinus toxin), mimicking acetylcholine by depolarization of
postsynaptic membrane (succinylcholine, decamethonium), or blocking metabolic sources of energy
(cyanide).
Neuromuscular transmission is also affected by altering ionic concentrations. Synaptic blockage can be
caused by permanently depolarizing the postsynaptic membrane with a high concentration of K+. Due
to its regulatory function, a low concentration of extracellular Ca++ will inhibit ACh release. Since
magnesium (Mg++) is also a divalent ion, it is capable of competing with Ca++. Therefore, a high
concentration of Mg++ blocks Ca++ from doing its job and will inhibit ACh release.
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AN-3-4
Experiment AN-3: Neuromuscular Studies
Equipment Required
PC or Mac Computer
IXTA data acquisition unit
USB cable
IXTA power supply
NBC-401 or 402 Nerve Chamber
iWire-B3G input cable
C-ISO-P5 pin connector-pinjack lead wires (5)
Glass hooks
Pasteur pipettes and bulbs
C-BNC-P2 BNC-dual pinjack stimulator cable
Pinjack-male banana ground cable
Room-Temp & Chilled Amphibian Ringer's solution
Small amounts of reagent solutions
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.
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AN-3-5
4. Pull down the Settings menu again. Select the Neuromuscular Studies-LS2 settings file from
Animal Nerve.
5. After a short time, LabScribe will appear on the computer screen as configured by the
NeuromuscularStudies-LS2 settings.
6. For your information, the settings used to configure the recording and stimulator channels in the
LabScribe software and IXTA for this experiment 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.
7. 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)
Settings on the Stimulator Window of the Preferences Dialog that Configure the iWorx System
for Experiment AN-3.
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
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AN-3-6
NBC-401 or 402 Nerve/Muscle Bath Chamber Setup
Figure AN-3-S1: The NBC-402 nerve bath chamber. Note that the NBC-401 is approximately half this
size.
1. Locate the following items in the iWorx kit: NBC-401 or 402 nerve bath chamber (Figure AN3-S1) and C-BNC-P2 stimulator cable (Figure AN-3-S2)
Figure AN-3-S2: The C-BNC-P2 stimulator cable.
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AN-3-7
2. Also, locate the C-ISO-B3G recording cable (Figure AN-3-S3) and C-ISO-P5 nerve chamber
lead wires in the iWorx kit.
Figure AN-3-S3: The C-ISO-B3G recording cable.
3. Attach the BNC connector of the C-BNC-P2 stimulator cable to the stimulator 1 input of the
IXTA (Figure AN-3-S4). Place the sockets, at the other end of the stimulator cable, on the
closely-spaced electrodes at one end of the NBC-401 or 402 nerve bath chamber (Figure AN-3S5). The red socket goes on the positive stimulating electrode (+S), which is the electrode
closest to the end of the chamber. The black socket goes on the negative stimulating electrode (S), on the opposite side of the chamber to avoid the possibility of the short circuit between the
two stimulator cables.
4. Insert the connector on the end of the C-ISO-B3G biopotential cable into the iWire 1 input of
the IXTA.
5. Attach the red, black, white, brown, and green C-ISO-P5 nerve chamber lead wires to the
corresponding sockets on the lead pedestal of the C-ISO-B3G biopotential cable. Place the
sockets, at the other end of the lead wires, on the appropriate electrodes of the nerve bath:
•
The green (C) lead is attached to the electrode (G1) between the negative stimulating
electrode (-S) and the proximal recording electrode for the nerve (-N).
•
The black (-1) lead is attached to the proximal recording electrode for the nerve (-N), the
one closest to the ground electrode (G1).
•
The red (+1) lead is attached to the distal recording electrode for the nerve (+N), the one
between the proximal recording electrode for the nerve (-N) and the ground electrode on
the muscle (G2).
•
The brown, or clear, (-2) lead is attached to the negative recording electrode for the
muscle (-M), which is between the second ground electrode (G2) and the proximal
recording electrode for the muscle (-M)
•
The white (+2) lead is attached to the positive recording electrode for the muscle (+M),
which is next to the negative muscle recording electrode (-M).
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AN-3-8
•
A jumper wire with sockets or insulated alligator clips is attached between the ground
electrode on the head of the muscle (G2) and the other side of ground electrode on the
nerve (G1). This ground is needed to prevent the artifact from the compound action
potential of the nerve from being recorded through the recording electrodes on the
muscle.
Figure AN-3-S5: The female BNC to Dual Banana Adapter, stimulator cable, and C-AAMI cable with
five lead wires connected to the IXTA.
Figure AN-3-S6: The NBC-401 nerve bath chamber with stimulator and recording lead wires attached.
Notice that the stimulator leads are attached to the electrodes that are spaced more closely.
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AN-3-9
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 the nerve. 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.
Animal Nerve – Neuromuscular Studies – Background
AN-3-10
•
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 as described in the section
showing the setup of the NBC-401 or 402 nerve bath chamber.
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/muscle preparation in the NBC-401 or 402 nerve/muscle bath
chamber is grounded to the ground (C) input on the C-ISO-B3G recording cable that is
connected to the isolated inputs of the IXTA. In this experiment, the muscle is also grounded
through the electrode near the knee joint. Grounding the head of the muscle prevents the nerve
CAP from being recorded by the muscle recording electrodes, and the muscle CAP from being
recorded by the nerve recording electrodes. Attach the green C-ISO-P5 electrode lead wire to
ground (C) socket of the lead pedestal of the C-ISO-B3G recording cable.
•
Attach the pin jack on the other end of the green lead wire to the electrode between the
negative stimulating electrode and the negative recording electrode. The ground
electrode separates the stimulating electrodes from the recording electrodes to reduce the
amplitude of the stimulus artifact picked up by the recording electrodes.
Animal Nerve – Neuromuscular Studies – Background
AN-3-11
•
Connect the ground electrode on the muscle to the ground electrode on the nerve with a
simple alligator-alligator cable. Make sure the electrode used for grounding the muscle
is between the positive recording electrode on the nerve and the negative recording
electrode on the muscle.
2. If the recordings of the CAP have a lot of 60Hz noise while using the ground of the C-ISO-B3G
cable, connect the ground electrode of the NBC-401 or 402 nerve bath chamber to one of the
green banana jack connectors on the IXTA.
•
Attach an optional pin jack-male banana electrode lead wire.
•
Attach the pin jack on one end of this electrode lead wire to the electrode between the
negative stimulating electrode and the negative recording electrode.
•
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 compound action potential, move the nerve/muscle chamber 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 Nerve or Muscle
Compound 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.
The Dissection
1. Place a frog in ice water for 15 minutes. Double pith the frog as soon as it is removed from the
ice water.
2. Remove the skin from the legs by making an incision through the skin around the entire lower
abdomen. Cut the connections between the skin and the body—especially around the base of
the pelvic girdle. Use stout forceps to pull the skin off the frog in one piece (like a pair of
pants).
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AN-3-12
3. Place the frog in a dissection tray with its dorsal side up. Moisten the exposed limbs of the frog
with Ringer's solution every five minutes or so.
4. Separate the muscles of the upper leg to expose the sciatic nerve. Muscles are surrounded by
connective tissue called fascia, and the large medial and lateral muscles on the dorsal side of the
upper leg are joined to each other by a fusion of their fascia along a thin "white line”. Grab the
muscle groups on either side of the “white line” with a forceps, and firmly pull the muscle
groups apart. The fascia will tear.
5. Deflect the muscles away from each other to expose the cream-colored sciatic nerve lying deep
between the muscles. The sciatic nerve is covered with fascia, which also includes some blood
vessels.
6. Use a glass hook, made by flaming the tip of a Pasteur pipet, to separate the nerve from the
fascia and the vessels. If possible, avoid cutting the blood vessels. If bleeding does occur, rinse
away the blood with lots of Ringer’s solution. Free the nerve from the knee joint to the pelvis.
Use the glass hook to place a thread under the nerve. Keep the exposed nerve moist at all times
with Ringer's solution.
7. Carefully separate the muscles of the pelvis to expose the sciatic nerve. Remember to rinse any
blood away with Ringer’s solution. The sciatic nerve enters the abdomen of the frog through an
opening at the end of the urostyle, a bone that forms part of the pelvis.
8. Carefully expose the remainder of the nerve through an opening along the lateral side of the
urostyle. To avoid cutting the nerve, lift the end of the urostyle with forceps as you cut the
muscle away from the urostyle with a blunt scissors. Cut along the urostyle from its tip to the
vertebral column.
9. Deflect the muscle away from the urostyle to expose the sciatic nerve. Use a glass hook to
separate connective tissue from the nerve and to place a piece of thread under the nerve. Move
the thread as close to the vertebral column as possible. Ligate the nerve; the leg should jump as
the knot is tied tightly.
10. Cut the nerve between the knot and the vertebral column. Keep the exposed nerve moist at all
times with Ringer's solution.
11. Use the thread to lift the proximal end of the nerve from the abdomen of the frog. Do not pinch
or stretch the nerve. Remove any connective tissues, blood vessels, or nerve branches that may
still keep the nerve attached to the body of the frog.
12. Continue to lift and release the sciatic nerve from any tissue that still keeps it attached to the
pelvis or thigh. The nerve should be separated from connecting tissues to a point just above the
knee joint.
13. Lay the nerve over the muscles of the lower leg and bathe the nerve and muscles with Ringer’s
solution. This nerve and the muscles of the lower leg are the preparation that needs to be
preserved for this experiment.
14. Expose the femur bone by dissecting away the muscles of the upper leg as close to the knee
joint as possible without damaging the sciatic nerve. Use a stout pair of scissors to cut the femur
bone as close to the knee joint as possible. Rinse the preparation with Ringer’s solution to
moisten the tissue and rinse away any blood.
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AN-3-13
15. Separate the lower leg and the nerve from the muscles and bone of the upper leg. Cut across the
muscles of the thigh and the femur bone as close to the knee joint as possible and without
damaging the nerve.
Warning: Moisten the exposed limbs of the frog with Ringer's solution every five minutes or so.
16. Either muscle of the lower leg can be used with the sciatic nerve in this experiment. The
gastrocnemius muscle can be used in larger nerve/muscle chambers, but the diffusion of drugs
through this muscle takes more time. The tibialis anterior muscle fits into smaller chambers and
the diffusion of drugs takes less time through this smaller muscle. In either case, the muscles
must be separated from each other.
17. Use a glass hook to separate the gastrocnemius muscle from the bone and other muscles of the
lower leg.
18. Use scissors to free the Achilles tendon from the connective tissue around the heel of the foot.
If the gastrocnemius muscle is used:
1. Firmly tie a thread around the Achilles tendon, leaving the ends of the thread long enough to
secure the muscle in the nerve/muscle chamber.
2. Cut the Achilles tendon as close to the bottom of the foot as possible, so the thread is still
attached to the gastrocnemius muscle.
3. Move the gastrocnemius muscle away from the rest of the lower leg. Cut the tibiofibula bone
and tibialis anterior muscle just below the knee to separate the rest of the lower leg from the
preparation. Avoid damaging the gastrocnemius muscle or the sciatic nerve. Rinse the
preparation with Ringer’s solution to moisten the tissue and rinse away any blood.
If the tibialis anterior muscle is used:
1. Firmly tie a thread around the ankle. Leave the ends of the thread long enough to secure the
muscle in the nerve/muscle chamber.
2. Cut the Achilles tendon as close to the bottom of the foot as possible. Move the gastrocnemius
muscle away from the rest of the lower leg. Cut the across the head of the gastrocnemius muscle
as close to the knee joint as possible to separate it from the rest of the lower leg. Avoid
damaging the tibialis anterior muscle or the sciatic nerve. Rinse the preparation with Ringer’s
solution to moisten the tissue and rinse away any blood.
Placement of Preparation the Chamber
1. Before moving the nerve/muscle preparation from the dissecting tray to the nerve/muscle
chamber, read the following directions.
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AN-3-14
2. Orient the chamber so that the nerve/muscle preparation can be easily transferred to the
nerve/muscle bath chamber. The proximal end of the nerve will be at the end of the chamber
where the electrodes are closer together, and the distal end of the muscle will be at the end of
the chamber where the electrodes are farther apart.
Warning: Do not use the thread on the nerve to move the preparation into the bath chamber. Putting
too much tension on the nerve could separate the connections between the nerve and the muscle and
prevent signals from being conducted from the nerve to the muscle.
3. Carefully lift the preparation from the dissecting tray and place it in the nerve/muscle chamber
using the thread on the end of the muscle and a forceps on the tissue above the knee joint.
Warning: Do not touch the nerve with the forceps.
4. To improve contact between the nerve and the electrodes:
•
Place the thread on the proximal end of the nerve under the outermost stimulating
(positive) electrode.
•
Gently pull the thread on the proximal end of the nerve to position the section of nerve
that is just inside the knot under the outermost stimulating electrode. Secure the thread
on the wall of the chamber with soft wax.
5. Carefully move the muscle to a position over its three recording electrodes using the same
technique described in Step 3. Do not stretch the nerve past its in situ length. Secure the thread
on the end of the muscle to the wall of the chamber with soft wax.
6. Fill the nerve/muscle bath chamber with Ringer's solution to immerse the preparation. Cover
the bath chamber with a microscope slide to prevent evaporation of the Ringer’s solution when
the preparation is bathing, or dehydration of the preparation when the Ringer’s solution has
been removed.
Warning: The nerve/muscle preparation used in this experiment is functional for a limited period of
time. If the nerve is bathed periodically in Ringer’s solution, it will work for about four hours. To
conserve time, complete all the exercises in the experiment before analyzing the data.
Animal Nerve – Neuromuscular Studies – Background
AN-3-15
Experiment AN-3: Neuromuscular Studies
Exercise 1: Maximal Muscle Response
Aim: To apply stimulus pulses of increasing amplitude to the proximal end of the nerve, and record the
compound action potentials evoked from the nerve and the muscle.
Procedure
1. 1 Click the Stimulator Preferences icon on the LabScribe toolbar (Figure AN-3-L1) to open the
stimulator control panel (Figure AN-3-L2) on the Main window.
Figure AN-3-L1: The LabScribe toolbar.
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 0.250 V; the number of pulses
(#pulses) to 1; and, the pulse width (W) to 0.1ms. The value for a stimulus parameter can be
changed by either of two methods: click on the arrow buttons to the right of the window that
displays the value of the parameter to increase or decrease the value; or, type the value of the
parameter in the window next to the label of the parameter. Click the Apply button to finalize
the change in any stimulus parameter.
Figure AN-3-L2: The stimulator control panel
3. Use a Pasteur pipette to lower the level of Ringer’s solution in the nerve bath chamber below
the nerve. Make sure that no part of the nerve or the thread holding the nerve in place is in
contact with the Ringer’s solution still in the chamber. If necessary, carefully blot any large
drops of solution from the recording electrodes and the nerve with the corner of a laboratory
wipe.
Note: The stimulus amplitude and width set for this exercise should be strong enough to cause a CAP
in a healthy nerve.
Animal Nerve – Neuromuscular Studies – Background
AN-3-16
4. Click the Record button to stimulate and record from the nerve. The recording stops
automatically after one sweep that is 30 milliseconds wide. Type 0.250V in the Mark box to the
right of the Mark button. Press the Enter key on the keyboard to attach this notation regarding
the stimulus amplitude to the recording. Click the AutoScale buttons on the upper margins of
the Nerve CAP, Muscle CAP, and Stimulus channels. The recording should be similar to the one
in Figure AN-3-L3.
Figure AN-3-L3: The compound action potential (CAP) from the sciatic nerve is displayed on the
upper channel. The CAP from the tibialis anterior muscle is displayed on the middle channel. The time
between the cursor at the onset of the stimulus artifact and the cursor at the onset of the muscle action
potential is the muscle latency.
5. Change the stimulus amplitude (Amp) to 0.300V by clicking on the arrow buttons next to the
value for this parameter as displayed on the stimulator control panel. The value for the stimulus
amplitude (Amp) can also be typed into window next the label Amp. Click the Apply button to
finalize the change in the stimulus amplitude.
6. Click Record to stimulate the nerve with 0.300V. Type 0.300V in the Mark box to the right of
the Mark button. and press the Enter key to attach a comment to the recording.
7. Increase the stimulus amplitude (Amp) by an increment of 0.050V using one of the techniques
explained in Step 5. Remember to click the Apply button each time to finalize the change in the
stimulus amplitude. Record and mark the response of the nerve.
8. Repeat Step 7 until the maximum compound action potential from the muscle is produced.
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.
Animal Nerve – Neuromuscular Studies – Background
AN-3-17
10. Fill the nerve/muscle chamber with fresh Ringer's solution to prevent the nerve and muscle
from drying out.
11. Increase the stimulus amplitude by another 10% before proceeding to the next exercise. Click
the Apply button to finalize the change in the stimulus amplitude. This level of stimulation
ensures a supramaximal response from the nerve and muscle.
Exercise 2: Synaptic Delay
Aim: To measure the time taken to transmit a signal across the synapse from the end of the nerve to the
surface of the muscle fibers.
The time between the nerve action potential and the muscle action potential depends on three major
factors: the conduction time down the nerve; the synaptic delay; and, the conduction time of the muscle
action potential from the neuromuscular junction (NMJ) to the muscle recording electrodes. Assume
that the third factor is negligible. The synaptic delay can be determined by subtracting the conduction
time along the nerve from the time interval between the nerve and muscle action potentials.
Procedure
1. Make sure the stimulus amplitude (Amp) is set to the supramaximal voltage determined at the
end of Exercise 1. If any change was made to the stimulus parameters, click the Apply button to
finalize the changes.
2. If necessary, lower the level of Ringer’s solution in the nerve bath chamber below the nerve and
blot any drops of solution from the preparation as described in Exercise 1.
3. Click Record to stimulate the nerve with the supramaximal stimulus. Type <Supramaximal> V
in the Mark box and press the Enter key to attach a comment to the recording.
4. Select Save in the File menu.
5. Fill the nerve/muscle chamber with fresh Ringer's solution to prevent the nerve and muscle
from drying out.
Exercise 3: Facilitation of the Muscle Response
Aim: To demonstrate facilitation in a muscle by stimulating the nerve/muscle preparation with a
succession of pulses that occur more frequently. Facilitation is an increase in the amplitude of the submaximal muscle action potentials that occur when there is less time between identical stimulus pulses.
Procedure
1. Adjust the Display Time and Timed Stop of the sweep to allow more action potentials to be
displayed on the computer screen.
•
Open the Edit menu on the Main window and select Preferences.
•
On the Channels window of the Preferences Dialog, change the Display Time and the
Timed Stop to 0.300 sec.
•
Click the OK button at the bottom of the Preferences Dialog.
Animal Nerve – Neuromuscular Studies – Background
AN-3-18
2. Adjust the critical stimulus parameters to the values listed in Table AN-3-L1 using the same
techniques used in Exercise 1. The stimulus amplitude must be set to a voltage that will
generate compound action potentials in the muscle with amplitudes that are 50% to 70% of the
maximal muscle CAP. Click the Apply button to finalize the changes to these stimulus
parameters.
Table AN-3-L1: Stimulus Parameters Required for Measuring the Facilitation of the Muscle.
Stimulus Parameter
Value
Stimulus Amplitude (Amp)
Volts for 50-70% of Max Muscle CAP
Number of Pulses (#pulses)
5
Pulse Width (W)
0.1 msec
Time Between Pulses (T Off)
20 msec
Holding Potential (HP)
0
3. If necessary, lower the level of Ringer’s solution in the nerve bath chamber below the nerve and
blot any drops of solution from the preparation as described in Exercise 1.
4. Click Record to stimulate the nerve. Type 20 msec Interval 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.
5. Change the time interval between stimulus pulses (T Off) to 15 msec. Click the Apply button to
finalize this change.
6. Click Record to stimulate the nerve. Type 15 msec Interval in the Mark box. Press the Enter key
on the keyboard to attach this notation to the recording.
7. Repeat Steps 5 and 6 to record the amplitudes of the muscle compound action potentials in the
series as the time interval between the stimuli is decreased. Change the time interval between
pulses (T Off) to 10, 5, and 2 msec. Record and mark the recordings at each new time interval.
8. Select Save in the File menu.
9. Fill the nerve chamber with fresh Ringer's solution to prevent the nerve from drying out.
Warning: Do not test the response of the muscle more than two times at each interpulse time
interval. The muscle may fatigue with frequent stimulation and may never recover.
Warning: Wear gloves when handling any drug solution, any tissue treated with
drugs, or any other object that has come in contact with a drug solution. Some of
these drugs are very effective poisons.
Animal Nerve – Neuromuscular Studies – Background
AN-3-19
Exercise 4: Effects of Eserine
Aim: To measure the changes in the compound action potential of muscle whose neuromuscular
junction was exposed to the anticholinesterase, eserine.
Procedure
1. Use the same Display Time and Timed Stop that was used in Exercise 3.
2. Adjust the critical stimulus parameters to the values listed in Table AN-3-L2 using the same
techniques used in Exercise 1.
3. Click the Apply button to finalize the changes to these stimulus parameters.
Table AN-3-L2: Stimulus Parameters Required for Measuring the Effects of Eserine and other
Drugs on the Nerve/Muscle Preparation.
Stimulus Parameter
Value
Stimulus Amplitude (Amp)
0.100 V
Number of Pulses (#pulses)
1
Pulse Width (W)
0.1 msec
Time Between Pulses (T Off)
0.9 msec
Holding Potential (HP)
0
3. If necessary, lower the level of Ringer’s solution in the nerve bath chamber below the nerve and
blot any drops of solution from the preparation as described in Exercise 1.
4. Click Record to stimulate the nerve. Type 0.100V 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.
5. Change the stimulus amplitude (Amp) to 0.200V. Click the Apply button to finalize this change.
6. Click Record to stimulate the nerve. Type 0.200V in the Mark box. Press the Enter key on the
keyboard to attach this notation to the recording.
7. Repeat Steps 5 and 6 to record the amplitudes of the compound action potentials on the nerve
and muscle up to a maximal response from the muscle. Change the stimulus amplitude by
increments of 0.100V. Record and mark the recordings for each new stimulus amplitude.
8. After recording compound action potentials generated by stimulus pulses of different
amplitudes, put five drops of the eserine solution (0.01 mg/ml in Ringer's) on the surface of the
muscle. Put another five drops of eserine solution on the muscle at one minute intervals for a
total of 30 drops.
9. Reset the stimulus amplitude (Amp) to 0.100V. Click the Apply button to finalize this change.
10. Click Record to stimulate the nerve. Type 0.100V-Eserine in the Mark box to the right of the
Animal Nerve – Neuromuscular Studies – Background
AN-3-20
Mark button. Press the Enter key on the keyboard to attach this notation to the recording.
11. Change the stimulus amplitude (Amp) to 0.200V. Click the Apply button to finalize this change.
12. Click Record to stimulate the nerve. Type 0.200V-Eserine in the Mark box. Press the Enter key
on the keyboard to attach this notation to the recording.
13. Repeat Steps 11 and 12 to record the amplitudes of the compound action potentials from the
nerve and muscle. Use the same stimulus amplitudes as the ones applied to the untreated
nerve/muscle preparation. If there is a significant difference between muscle compound action
potentials recorded before and after the eserine treatment, proceed to Step 15.
14. If there is no significant difference between muscle compound action potentials recorded before
and after the eserine treatment:
•
Repeat the application of eserine in the same manner as it was applied in Step 8.
•
Test the response of the muscle using the procedures used in Steps 9 through 13.
•
If there is a difference between the muscle action potentials recorded before the first
eserine treatment and after the second eserine treatment, proceed to Step 15.
•
If there is no difference between the muscle action potentials recorded before the first
eserine treatment and after the second eserine treatment, apply a third treatment of
eserine. Test the response of the muscle after the third treatment of eserine using the
procedures used in Steps 9 through 13. Proceed to Step 15, whether there is a change in
the compound action potentials of the muscle or not.
15. Reverse the effects of eserine on the muscle:
•
Remove the Ringer’s solution containing eserine from the bottom of the bath chamber.
•
Rinse the inside of the bath chamber and the surface of the muscle and nerve with fresh
Ringer’s solution.
•
Submerge the nerve/muscle preparation in fresh Ringer's solution for five minutes,
changing the Ringer’s solution every minute.
16. Lower the level of Ringer’s solution in the nerve bath chamber below the nerve and blot any
drops of solution from the preparation as described in Exercise 1.
17. Record the compound action potentials from the preparation using the same procedures used in
Steps 9 through 13. Mark each recording to indicate the stimulus voltage used while testing the
reversibility of the effects of eserine. If the muscle compound muscle action potentials have
returned to normal, proceed to the next exercise.
18. If the muscle compound muscle action potentials have not returned to their pretreatment levels:
•
Submerge the nerve/muscle preparation in fresh Ringer’s solution for another five
minutes, changing the Ringer’s solution every minute.
•
Retest the response of the muscle using the same procedures used in Steps 9 through 13.
•
If the muscle compound muscle action potentials have returned to normal, proceed to the
next exercise.
•
If the muscle compound muscle action potentials have not returned to normal, continue
Animal Nerve – Neuromuscular Studies – Background
AN-3-21
rinsing the preparation with fresh Ringer’s solution until the muscle response recovers.
If the muscle has not fully recovered after 30 minutes of rinsing, consult your instructor.
19. Select Save in the File menu.
20. Fill the nerve/muscle chamber with fresh Ringer's solution to prevent the nerve and muscle
from drying out.
Exercise 5: Effects of Other Agents
Aim: To test the effects of other reagents on the compound action potentials of the nerve and the
muscle in the preparation. Each group will be assigned one of the following substances to test: curare,
atropine, acetylcholine (high concentration), nicotine, dantrolene, magnesium (high concentration), and
calcium (high concentration). Each reagent is dissolved in Ringer’s solution.
Procedure
1. Begin the study of the reagent that your group is assigned only after the effects of eserine have
been reversed, or if the response of the muscle is measurable and stable.
2. Use the same procedures employed in Exercise 4 to test the effects of your assigned reagent on
the compound action potentials of the nerve and the muscle.
3. Select Save in the File menu.
4. Follow the directions provided by your instructor for the cleaning of the equipment and the
disposal of tissue and reagents.
Data Analysis
Exercise 2-Synaptic Delay
1. Use the Sweep Selection bar at the bottom of the Main window (Figure AN-3-L4) to display the
sweep recorded in Exercise 2. Click on the tab on the selection bar for that sweep and the sweep
will appear on the Main window.
Figure AN-3-L4: The Sweep Selection bar showing the tab for Sweep 7 highlighted.
2. Transfer the sweep to the Analysis window. Click on the Analysis window icon in the toolbar or
select Analysis from the Windows menu to transfer the sweep from the Main window to the
Analysis window.
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AN-3-22
3. 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.
4. Maximize the height of the trace on both channels by clicking on the arrow to the left of the title
of each channel to open the channel menu. Select Scale from the menu and AutoScale from the
Scale submenu to increase the height of the data on that channel.
5. Once the cursors are placed in the correct positions for determining the latency of the nerve, the
time (T2-T1) 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.
6. 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 latency of the nerve.
•
Transfer the names of the mathematical function used to determine the latency to the
Journal using the Add Title to Journal function in the Nerve CAP Channel pull-down
menu.
•
Transfer the values for the amplitude to the Journal using the Add Ch. Data to Journal
function in the Nerve CAP Channel pull-down menu.
7. Measure the latency of the nerve compound action potential:
•
Place one cursor on the onset of the stimulus artifact displayed on the Nerve CAP
channel.
•
Place the other cursor on the onset of the nerve compound action potential displayed on
the same channel.
•
The value for T2-T1 function on the Nerve CAP Channel is the latency of the nerve,
which is the time it takes the action potential to move from the negative stimulating
electrode to the negative nerve recording electrode.
•
Record the nerve latency in the Journal using the one of the techniques described in
Steps 5 or 6, and on Table AN-3-L3.
8. Measure the nerve conduction distance (in mm) between the negative stimulating electrode to
the negative nerve recording electrode. Record the nerve conduction distance in the Journal
using the one of the techniques described in Steps 5 or 6, and on the data table.
9. 9 Calculate the nerve conduction velocity (in millimeters per millisecond) of the nerve. Divide
the nerve conduction distance (in mm) that was measured in Step 9 by the nerve latency (in
msec) that was measured in Step 7.
For example, 10 mm/0.2 msec = 50 mm/msec
Record the nerve conduction velocity in the Journal, and on the data table.
Note:If the nerve and the nerve/muscle chamber are long enough that the nerve crosses three or more
electrodes that can be used for recording nerve action potentials, a more accurate method of
Animal Nerve – Neuromuscular Studies – Background
AN-3-23
determining the nerve conduction velocity can be utilized. This recording can be done monophasically
(without a positive nerve recording electrode) or biphasically (with both a positive and a negative
recording electrode). If the positive (second) nerve recording electrode is used, it should be placed on
electrode (F). Record the compound nerve action potential with the negative (first) nerve recording
electrode in position (D). Move this negative nerve recording electrode to a new position (E) and
record the compound nerve action potential, again. Measure the distance (in mm) between electrode
(D) and electrode (E). Divide this distance (D to E) by the time interval (in msec) between the peak of
the action potential recorded at (D) and the peak of the action potential recorded at (E).
10. Measure the muscle conduction distance (in mm) between the negative stimulating electrode
and the negative recording electrode on the muscle. Record this distance in the Journal, and on
the data table.
11. Calculate the conduction time (in msec) of the nerve compound action potential from the point
of stimulation of the nerve, which is the negative stimulating electrode, to the point of recording
on the muscle fibers, which is the negative recording electrode on the muscle. Divide the
muscle conduction distance (in mm) that was measured in Step 11 by the conduction velocity
(in mm/msec) of the nerve that was calculated in Step 10.
For example, 40 mm/(50 mm/msec) = 0.8 msec
Record the conduction time of the nerve in the Journal, and on the data table.
12. Measure the latency of the muscle. Place one cursor on the onset of the stimulus artifact
displayed on the Nerve CAP channel. Place the other cursor on the onset of the muscle
compound action potential displayed on the Muscle CAP channel. The value for T2-T1 function
on either the Nerve or Muscle CAP Channel is the latency of the muscle. Record the latency of
the muscle in the Journal, and on the data table.
13. Calculate synaptic delay. Subtract the nerve conduction time (stimulus electrode-NMJ) found in
Step 12 from the latency of the muscle found in Step 13. Record the synaptic delay in the
Journal, and on the data table.
14. Select Save in the File menu.
15. Click on the Main Window icon to return to that window.
Questions-Synaptic Delay
1. Is there a more accurate way to make this measurement?
2. What additional factors would be involved if the time between the nerve action potential and
muscle contraction were used instead of the time between the nerve and muscle action
potentials?
Animal Nerve – Neuromuscular Studies – Background
AN-3-24
Table AN-3-L3: Times, Velocities, and Distances Needed to Determine Synaptic Delay
Parameter
Value
Nerve Latency (msec)
Nerve Conduction Distance (mm)
Nerve Conduction Velocity (mm/msec)
Muscle Conduction Distance (mm)
Nerve Conduction Time, Stimulus to NMJ (msec)
Muscle Latency (msec)
Synaptic Delay (msec)
Exercise 3-Facilitation of the Muscle Response
1. Use the Sweep Selection bar at the bottom of the Main window to display the first sweep
recorded in Exercise 3. Click on the tab on the selection bar for that sweep and the sweep will
appear on the Main window. According to the design of the exercise, the five stimulus pulses
delivered to the nerve in this sweep were 20 msec apart.
2. Transfer the sweep to the Analysis window by clicking on the Analysis window icon in the
toolbar or selecting Analysis from the Windows menu.
3. Maximize the height of the traces on the Nerve and Muscle CAP Channels by clicking on the
arrow to the left of the title of each channel to open the channel menu. Select Scale from the
menu and AutoScale from the Scale submenu.
4. Measure the amplitude of the first muscle compound action potential in the series of five. Place
one cursor on the baseline of the recording on the Muscle CAP Channel in the section before
the first stimulus was delivered. Place the other cursor on the peak of the first muscle CAP. The
value for the V2-V1 function on the Muscle CAP Channel is the amplitude of this muscle
compound action potential.
5. Record the amplitude of the first muscle compound action potential in the Journal using the one
of the techniques described in data analysis section of Exercise 2, and on Table AN-3-L4.
6. Repeat Steps 4 and 5 on each of the other muscle compound action potentials in this sweep.
7. To display another sweep made with a different interval between stimulus pulses, click on the
tab for that sweep in the Sweep Selection bar at the bottom of the Analysis window. The sweep
that is selected will appear on the Analysis window.
Animal Nerve – Neuromuscular Studies – Background
AN-3-25
Table AN-3-L4: Amplitudes of Multiple Muscle Compound Action Potentials with Different
Stimulus Frequencies.
Interval Between Stimulus Pulses
(msec)
Amplitude (mV) of Muscle Compound Action Potential in Series
1st
2nd
3rd
4th
5th
20
15
10
5
2
6. To take measurements from the second 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-3L5). Repeat Steps 4 and 5 to measure and record the amplitudes of the five muscle compound
action potentials in the sweep.
Figure AN-3-L5: The upper left corner of the Analysis window showing the Sweep menu used to select
the sweep to be measured.
9. Measure and record the amplitudes of the muscle compound action potentials in the three other
sweeps in Exercise 3. Follow the directions explained earlier in this data analysis section to
display, select, measure, and record data.
10. Select Save in the File menu.
11. Click on the Main Window icon to return to that window.
Questions-Facilitation of the Muscle Response
1. How does decreasing the interval between successive stimulus pulses affect the amplitude of the
compound muscle action potential?
2. What practical implication does this phenomena have for muscle function?
Animal Nerve – Neuromuscular Studies – Background
AN-3-26
Exercise 4-Effects of Eserine
1. Use the Sweep Selection bar at the bottom of the Main window to display the first sweep
recorded in Exercise 4. Click on the tab on the selection bar for that sweep and the sweep will
appear on the Main window. According to the design of the exercise, the nerve-muscle
preparation was bathed with normal Ringer’s solution and the stimulus pulse delivered to the
nerve in this sweep had an amplitude of 0.100V.
2. Transfer the sweep to the Analysis window by clicking on the Analysis window icon in the
toolbar or selecting Analysis from the Windows menu.
3. Maximize the height of the traces on the Nerve and Muscle CAP Channels by clicking on the
arrow to the left of the title of each channel to open the channel menu. Select Scale from the
menu and AutoScale from the Scale submenu.
4. Measure the amplitude of the muscle compound action potential. Place one cursor on the
baseline of the recording on the Muscle CAP Channel in the section before the stimulus was
delivered. Place the other cursor on the peak of the muscle CAP. The value for the V2-V1
function on the Muscle CAP Channel is the amplitude of this muscle compound action
potential.
5. Record the amplitude of the muscle compound action potential in the Journal using the one of
the techniques described in data analysis section of Exercise 2, and on Table AN-3-L5.
6. Measure the duration of the muscle compound action potential (CAP). Place one cursor on the
beginning of the muscle CAP displayed on the Muscle CAP Channel. Place the other cursor at
the end of the muscle CAP. The value for the T2-T1 function on the Muscle CAP Channel is the
duration of the muscle compound action potential in this sweep.
7. Record the duration of the muscle compound action potential in the Journal using the one of the
techniques described in data analysis section of Exercise 2, and on the data table.
8. To display another sweep made with a different stimulus amplitude, click on the tab for that
sweep in the Sweep Selection bar at the bottom of the Analysis window. The sweep that is
selected will appear on the Analysis window.
9. To take measurements from the second sweep displayed on the Analysis window, select its
name from the Sweep menu in the upper left margin of the data display window. Repeat Steps 4
through 7 to measure and record the amplitude and duration of the muscle compound action
potential in the sweep.
10. Measure and record the amplitudes and durations of the muscle compound action potentials in
the other sweeps made with different stimulus amplitudes in both treatments (Ringer’s solution
and Ringer’s solution with eserine). Use the same techniques explained earlier to display, select,
measure, and record the data.
11. Select Save in the File menu.
12. Click on the Main Window icon to return to that window.
Animal Nerve – Neuromuscular Studies – Background
AN-3-27
Table AN-3-L5: Amplitudes and Durations of Muscle Compound Action Potentials Before and
After Treatment with Eserine.
Normal Ringer’s
Stimulus Amplitude
(V)
Ringer’s with Eserine
Muscle
CAP
Muscle CAP Amp (V)
Muscle CAP Amp (V)
Period
(msec)
Muscle
CAP
Period
(msec)
0.100
0.200
0.300
0.400
0.500
Questions-Effects of Eserine
1. Does eserine have any effect on the excitability or threshold of the muscle?
2. What is the mechanism of action of eserine?
Exercise 5-Effects of Other Agents
1. Use the same techniques used in the analysis of Exercise 4 to take measurements from the data
in Exercise 5.
2. Use the same techniques used in Exercises 2, 3 and 4 to record the measurements from this
exercise in the Journal, and on Table AN-3-L6.
3. Select Save in the File menu.
Questions-Effects of Other Agents
1. Does the agent that you tested have any effect on the excitability or threshold of the muscle?
Those of the nerve?
2. What is the mechanism of action of your agent?
Animal Nerve – Neuromuscular Studies – Background
AN-3-28
Table AN-3-L6: Amplitudes and Durations of Muscle Compound Action Potentials Before and
After Treatment with Other Agents.
Normal Ringer’s
Stimulus Amplitude
(V)
Ringer’s with _______
Muscle
CAP
Muscle CAP Amp (V)
Muscle CAP Amp (V)
Period
(msec)
Muscle
CAP
Period
(msec)
0.100
0.200
0.300
0.400
0.500
Animal Nerve – Neuromuscular Studies – Background
AN-3-29
Experiment AN-3: Neuromuscular Studies
Appendix
Recipe for Amphibian Ringer’s Solution.
Concentration
(mMolar)
Grams/Liter
DI H20
Salt
111.0
Sodium Chloride
6.49
1.9
Potassium Chloride
0.142
1.06
Calcium Chloride∗2H2O
0.154
1.0
Tris
0.121
5.55
Glucose
1.00
Adjust the pH of the solution to 7.6 with 6N HCl
Concentrations of Reagents in Ringer’s that Alter Nerve and Muscle Activity.
Concentration
(mMolar)
Reagent
mg/ml in Ringer’s
0.036
Eserine
0.01
0.016
D-Tubocurarine Chloride
0.01
0.035
Atropine Sulfate
0.01
0.061
Acetylcholine Chloride
0.01
0.055
Nicotine Sulfate
0.01
10
Dantrolene
3.14
20
Magnesium Chloride∗6H2O
4.06
20
Calcium Chloride∗2H2O
2.94
Animal Nerve – Neuromuscular Studies – Background
AN-3-30