General Physiology and Excitable Tissue – General Medicine
The virtual neuromuscular junction (NMJ)
The Virtual NMJ is a simulation of an experiment recording the electrical potentials associated
with neuromuscular transmission at the skeletal neuromuscular junction.
Electrophysiological measurements provide a more direct insight into the mechanisms of
neuromuscular transmission than the measurement of muscle tension. Bernard Katz's studies of the
endplate potential, for instance, lead to the discovery of the quantal nature of transmitter release and
further use of the techniques have revealed the precise mechanism of action of many of the drugs that
affect neuromuscular transmission.
The simulation allows you to observe the muscle action potential (AP) and endplate potentials
(EPPs) evoked by either nerve stimulation or by direct current stimulation of the muscle fibre. The
effects of a variety of drugs and of changes to ionic composition of the extracellular solution on the AP
and EPPs can be studied.
The preparation
The hemidiaphragm muscle with attached phrenic nerve
is dissected from a rat and pinned out in flat dish.
Oxygenated (95% O2, 5% CO2) Kreb's solution is
superfused in a constant stream over the preparation. The
dish is placed on a microscope stage and a fine (tip
diameter < 0.1 m) glass micropipette electrode inserted
in the endplate region of a muscle fibre.
The electrode is connected via a microelectrode
amplifier to an oscilloscope, displaying the intracellular
membrane potential of the muscle fibre.
Computer simulation NMJ
To start the simulation, select the icon:
Double click it or find the program in the
menu Start –> všechny programy –> NMJ Simulation.
Program control:
Basic control of the program is in the menu bar at the top of the window:
File:
 New Experiment – Start experiment with a new rat.
 Load Experiment – Load saved experiment.
 Save Experiment – Save current experiment.
 Print – Print out actual window.
 Exit – Leave the program.
View:
 Zoom In – Display magnification. Open the dialog box Display Magnification, where you can
(using catch and drag) set a rectangle, which will be shown in the main window.
 Zoom Out – Display reduction. Display in the main window will be reset to initial magnitude.
1
General Physiology and Excitable Tissue – General Medicine
Drugs:
Drugs can be applied in various doses and in any combination during the experiment. To add a
drug to the preparation (or change its concentration), select a drug from the Drugs menu; enter the
required concentration (expressed in mol per liter (M) and corresponds to the final concentration in the
solution); click the OK button and then the Do Sweep button.
When you set concentration of the drug to 0 M, it will be eliminated from the solution. To
remove all applied drugs select Drugs – Remove All Drugs.
Ions:
In the window Set Ion Concentration you can
change concentrations of some ions in the solutions.
External is the concentration in the organ bath, Internal
is the concentration in the intracellular solution, V.Rev is
the equilibrium potential of a given ion. To set
physiological concentration of the ions click on Reset to
Normal.
Calcium is responsible for acetylcholine release
from the nerve endings. Calcium influx enables moving
of vesicles with acetylcholine to the cell membrane and
their exocytosis. Leakage and entry of calcium ions from
and to the muscle cell is very limited. Thus muscle
contraction is almost independent on extracellular
concentration of calcium. Contraction can be affected
2
General Physiology and Excitable Tissue – General Medicine
only after long-lasting changes in calcium concentration.
Magnesium competitively blocks the calcium entry to the nerve cell. Thus magnesium affects
acetylcholine release. High concentration of magnesium blocks the neuromuscular transmission.
Experiment control:





In left lower part of window there are control elements of the experiment:
Record – By clicking the Do Sweep button one electrical impulse is applied.
Stimulus – The muscle can be stimulated in either of two ways. DIRECTLY – by injecting a
small current into the muscle fiber via the recording electrode. The amplitude and duration of the
stimulus current can be set within the simulation. INDIRECTLY - by stimulating the phrenic
nerve via platinum wire electrode. You can also turn the stimulation off.
Cursor – The box Cursor displays the time (T) and electrical potential (Vm) at the point, where
the cursor (gray vertical line on the screen) crosses the current trace. You can move the cursor
using mouse (method catch and drag).
Drugs/Ions – In this box concentrations of drugs and ions in the solution are shown
Playback – Using the Playback scroll bar you can redisplay previous records on the screen.
Beside the scroll bar there is the box with actual/total record number. The checkbox Display
Auto Erase is used it to view previous records (when unchecked).
Applying drugs
The following drugs can be applied to the preparation:
Tubocurarine
Non-depolarizing competitive cholinergic antagonist, reversible blocker of the nicotinic
receptors. After the block of the nicotinic receptors, the receptor channels could not operate and nerveevoked depolarization of the subsynaptic membrane decreases. If this depolarization is too small, the
action potential on the muscle fiber does not appear. Tubocurarine and acetylcholine bind on the
nicotinic receptors reversibly and competitively. Recommended lower dose is 0.4 μM.
Tetrodotoxin
A sodium channel blocker. Tetrodotoxin decreases or prevents depolarization of the nerve and
muscle. Recommended lower dose is 0.05 μM.
-Conotoxin
Blocker of sodium channels of muscle membrane. Recommended lower dose is 0.5 μM.
Neostigmine
Competitive inhibitor of acetylcholine esterase (AChE) that causes reversible blockade of this
enzyme. AChE decreases concentration of acetylcholine in the synaptic cleft and cleaves it into cholin
and acetic acid. AChE blockade prolongs acetylcholine effect on the subsynaptic membrane.
Recommended lower dose is 1 μM.
3,4 diaminopyridine
A potassium channel blocker, which prolongs action potential. Recommended lower dose is
1 μM.
Suxamethonium
Short-time depolarizing myorelaxant. It binds to nicotinic receptors, where it acts like longtermed agonist. Binding of suxamethonium causes prolonged depolarization of the surrounding
sarcolemma that prevents recovery of the sodium channels and thus generation of action potentials.
Recommended lower dose is 10 μM.
3
General Physiology and Excitable Tissue – General Medicine
PROTOCOL
Describe effect of each drug on the action potential during the direct and indirect stimulation and
explain yours observations. (c = concentration of the drug in the solution):
indirect stimulation
Tubocurarine
c=
Tetrodotoxin
c=
-Conotoxin
c=
Neostigmine
c=
3,4-diaminopyridine
c=
Suxamethonium
c=
4
direct stimulation
General Physiology and Excitable Tissue – General Medicine
What is the stimulation threshold?
Tubocurarine and suxamethonium are used as myorelaxants. What is the difference between the two
drugs in the effect on the electrical potential of the cell?
Which drug prevents the action potential of the muscle cell in the lowest concentration?
Which drug effect could be eliminated by neostigmine and why?
Concentration of which ion (Na+, K+, Cl-) affects the resting membrane potential the most profoundly and
why? What happens if the extracellular concentration of this ion is increased by 7mM?
What is the effect of high magnesium concentration (5mM) on neuromuscular junction, how the
hypermagnesemia could be compensated and why?
How the action potential will be changed by hyponatremia (105 mM) and why?
How the parameters of the direct stimulation will be changed, when extracellular concentration of
potassium decreases to 1 mM and why?
5
General Physiology and Excitable Tissue – General Medicine
Some Properties of the Neuromuscular Transmission in the Albino Rat
In this experiment we study the skeletal muscle physiology using in vivo stimulation of a nervemuscle preparation in a rat. The effects of stimuli of varying strength and frequency will be investigated,
as well as the phenomena of summation, tetanus, fatigue and the effect of neuromuscular blocking
agents.
Skeletal muscle is composed of muscle fibers, each of which is innervated by the motor axon
branch originating in the CNS. The transfer of the signal from nerve to muscle takes place at the motor
end-plate (neuromuscular or myoneural junction, Fig.1). Within the axoplasm of the nerve terminals
there are located numerous membrane-bound vesicles containing neurotransmitter acetylcholine (ACh).
The end-plate membrane is that part of the muscle cell membrane lying immediately beneath the axon
terminals. Here the membrane is formed into postjunctional folds at which many nicotinic ACh-receptors
are located. The basal lamina of the postjunctional membrane contains enzyme acetylcholinesterase that
hydrolyses ACh into choline and acetate.
Schwann cell cytoplasm
Mitochondrion of axon
Synaptic vesicles
(Ach supply)
Axolemma
Basal lamina
Sarcolemma
Sarcoplasm
Junctional folds
(location of ACh receptors)
Location of acetylcholinesterase
Mitochondria of muscle
Fig 1. Cross section of the motor nerve ending at the motor nerve end plate
Mechanical events in muscle (Fig.2)
Muscle Twitch
A single action potential of a motor axon leads to a single muscle action potential that propagates
along the length of each muscle fiber innervated by that axon terminal. This leads to a single brief
contraction of the muscle knows as the twitch.
Summation
When motor impulses arrive at closely spaced intervals, tension developed by the second impulse
adds to that already present from the first impulse
Tetanic contraction
A burst of closely spaced impulses generates the maximal force possible for the muscle fiber
(tetanic contraction, partial or full tetanus).
6
General Physiology and Excitable Tissue – General Medicine
Superposition
Twitch
Muscle contraction record
Gradation in Tension in a Muscle
Each muscle fiber can develop a degree of tension between the twitch and full tetanus, depending
on the temporal sequence of nerve impulses. An impulse in the motor axons recruits all fibers in the
motor unit (a motor unit is a single α-motor neuron and all of the corresponding muscle fibers it
innervates). Tension developed by the muscle as a whole reflects the number of motor units active at that
time.
The number of muscle fibres in a motor unit varies greatly from one muscle to another. Large
limb muscles have many muscle fibres in a motor unit, while small eye and finger muscles have
relatively few ones.
Unfused tetanus
Fused tetanus
Summation
Twitch
Muscle contraction record
Stimulation
Stimulation
Fig. 2. Muscle twitch, summation and tetanisation
Neuromuscular Junction Fatigue
A muscle stimulated through the nerve will no longer contract after repeated stimulation, but the
muscle itself still responds if the stimulating electrode is applied directly to it. These findings showing
that the motor nerve is still able to function after prolonged stimulation have led to the conclusion that
the "site of fatigue" is the myoneural junction.
Effect of Blocking Agents
Curariform drugs block the neuromuscular transmission by the competing with acetylcholine for
the N-receptors.
Procedure:
1. Anaesthetize a rat with an intraperitoneal injection of urethan (1.5g/1 kg of body weight).
Preparation:
2. The preparation used consists of the sciatic nerve and gastrocnemius muscle in situ.
3. With the animal lying on its side, make a skin incision parallel to the femur, along the lateral
surface of the thigh (length of the incision about 3 cm).
7
General Physiology and Excitable Tissue – General Medicine
4. Retract the skin. A groove will be seen posterior to the femur and running parallel to it. This
groove lies between two muscles, the biceps femoris and the vastus lateralis.
5. Deepening the groove by separation of the muscles exposes the sciatic nerve. It is a large nerve
and easily freed for a considerable distance. Pass a loop under it and close the wound with the
skin.
6. Holding the nerve by the thread, you are now in position to stimulate the nerve. Keep the
preparation warm and moist with Tyrode solution.
Properties of muscle
A muscle in situ can be stimulated with electrodes attached directly to the muscle itself or
indirectly through its nerve.
Muscle twitch.
With the apparatus properly adjusted stimulate a muscle directly. (Frequency of stimulation is 1 Hz,
voltage 5 V, duration of the stimulus 1 ms).
Summation.
Stimulate the sciatic nerve [do not touch the muscle] so rapidly that each stimulus comes immediately at
the end of relaxation from the previous one (1-7 Hz, 5 V, 0.1 ms). The strength of muscular contraction
increases progressively.
Tetanus.
Increase the frequency of the stimulation. The impulses flow to the muscle too frequently to allow
complete relaxation; the muscle is in partial tetanus. (Frequency of stimulation is 12-20 Hz).
Increase the frequency of stimulation (more than 20 Hz). No time is permitted for relaxation; the muscle
is in complete tetanus. Stimulate with this frequency only for a short time!
Muscle fatigue.
Stimulate the sciatic nerve with the frequency about 10 Hz and observe a course and an intensity of the
contraction. When the muscle contraction weakens, stimulate the muscle directly and explain differences
between results of both stimulations.
Action of blocking agents
1. Place the animal on the back, make the incision in the middle line on the neck and isolate a trachea.
2. Place a catheter for an artificial breathing into the trachea.
3. Administer Arduan (the blocker of nicotinic receptors) intraperitoneally (0.2 mg of Arduan/1 kg of
body weight; a solution contains 0.2 mg/ml).
4. Start carefully the artificial ventilation. Explain a necessity of the artificial ventilation after the
administration of Arduan.
5. After 10 minutes try to stimulate the nerve (1 Hz, 5 V, 0.1 ms).
6. Now stimulate the muscle itself (1 Hz, 5 V, 1 ms).
PROTOCOL
Neuromuscular junction fatigue - explain differences between results of both stimulations.
Action of blocking agents – explain possible differences in contraction force between direct and indirect
stimulation after Arduan application.
8
General Physiology and Excitable Tissue – General Medicine
Muscle twitch
Rat phrenic nerve – hemidiaphragm simulation
Introduction
The Virtual Rat is a simulation of the rat
phrenic nerve-hemidiaphragm preparation - a
robust in vitro preparation which has been widely
used in the study the actions of neuromuscular
blocking and reversal agents, and other drugs
which affect neuromuscular transmission.
The hemidiaphraghm is a large, focally
innervated, respiratory skeletal muscle, composed
of fast-type muscle fibres. Neurotransmission is
mediated by nicotinic cholinoceptors. Electrical
stimulation of the phrenic nerve evokes fast,
shorting-lasting muscle twitches.
The preparation
A rat (0.5 kg) is killed by anaesthetising it with 100% CO2 (a rapid and fairly painless
procedure). The rib cage is opened and the phrenic nerve and triangular-shaped hemidiaphragm is
removed and placed into an organ bath containing Kreb's solution, bubbled with 95%O2/5%CO2, and
maintained at 32C in a surrounding water bath. As shown in the diagram, the base of the muscle is
attached to a support and the tendon attached to a tension transducer. The nerve is looped through a
stimulation electrode and another electrode is placed directly upon the muscle. Drugs are applied directly
into the bath. When required, drugs can be washed out, by flushing it out with new Kreb's solution.
Computer simulation
To start the simulation, select the icon:
by double-clicking it or in the menu Start –>
At the beginning you could write your name.
všechny programy –> Twitch V2.1.8.
Program control:
Basic control of the program is in the menu bar at the top of the window.
File:
 New Rat – Start experiment with new rat, all settings will be reset.
 Load Rat – Load saved experiment.
 Save Rat – Save current experiment.
 Print – Print out actual window.
 Exit – Leave program.
Drugs:
To apply a drug, choose one from the list of drugs. The dose of the drug can be selected from the
select menu. Drugs can be applied in various doses and in any combination during experiment.
Concentration is expressed in mol per liter (M) and corresponds to just added (not total) concentration.
Total concentration of the drug appears below the graphical record of the contraction.
The drugs accumulate in solution. When you want to replace a working solution with a new
Krebs solution, select Wash. Effect of some drugs lasts sometimes even after washing out.
9
General Physiology and Excitable Tissue – General Medicine
Ions:
Concentrations of calcium and magnesium in solution can be changed in this menu. Calcium is
responsible for acetylcholine release from the nerve endings. Calcium influx enables moving of vesicles
with acetylcholine to cell membrane and their exocytosis. Leak and entry of calcium from and to the
muscle cell is very limited. Thus muscle contraction is almost independent on extracellular concentration
of calcium. Contraction can be affected only after long-lasting changes in calcium concentration.
Magnesium competitively blocks the calcium entry to the nerve cell. Thus magnesium affects the
amount of acetylcholine release. High concentration of magnesium blocks a neuromuscular transmission.
Wash:
When you change the solution in the bath, all drugs and ionic changes will be removed. You can
choose between normal Krebs solution and Krebs solution without calcium. Effect of some drugs lasts
sometimes even after washing out.
Experiment control:
In the left lower part of the window there are control elements of the experiment. They are
divided into two parts: Controls a Stimulator.
The experiment is started up by pushing the button Start. Button Stop disrupts the experiment,
which can continue after pushing Start button. After disruption of the experiment you can browse
through the whole record using scrollbar. The actual time and magnitude of the contraction in a point of
the mouse cursor are showed in the field below Start and Stop buttons .
The muscle can be stimulated in either of two ways. DIRECTLY – by injecting a small current
into the muscle fiber via the recording electrode. The amplitude and duration of the stimulus current can
be set within the simulation. INDIRECTLY - by stimulating the phrenic nerve via platinum wire
electrode. You can also turn off the stimulation.
10
General Physiology and Excitable Tissue – General Medicine
Applying drugs
The following drugs can be applied to the preparation:
Tubocurarine
Non-depolarizing competitive cholinergic antagonist, reversible blocker of the nicotinic
receptors. After the block of the nicotinic receptors, the sodium channels could not operate and nerve
evoked depolarization of the subsynaptic membrane decrease. If this depolarization is too small, the
action potential on the muscle fiber does not appear. Tubocurarine and acetylcholine bonds on the
nicotinic receptors are reversible and competitive (i.e. dependent on drugs concentration). Recommended
lower dose is 0.2 μM.
Neostigmine
Competitive inhibitor of acetylcholinesterase (ACHE), that causes reversible blockade of this
enzyme. ACHE decreases concentration of acetylcholine in the synaptic gap and dissociates it into cholin
and acetic acid. ACHE blockade prolongs acetylcholine effect on the subsynaptic membrane.
Recommended lower dose is 0.2 μM.
4-aminopyridin
Blocker of potassium channel, which prolongs action potential duration. Recommended lower
dose is 20 μM.
Atropine
Competitive antagonist of muscarin receptors.
Suxamethonium
Short-time depolarizing myorelaxant. It binds to nicotinic receptors, where it effects like longtermed agonist. This bond causes prolonged depolarization of the membrane that is way the action
potential does not appear. Recommended lower dose is 5 μM.
Tetrodotoxin
Blocker of sodium channels. Tetrodotoxin decreases or prevents depolarization of the nerve and
muscle. Recommended lower dose is 0.2 μM.
11
General Physiology and Excitable Tissue – General Medicine
PROTOCOL
Describe effect of each drug on the action potential during the direct and the indirect stimulation
and explain your observations. (c = concentration of drug in the solution):
Indirect stimulation
Direct stimulation
Tubocurarine
c=
Neostigmine
c=
4-aminopyridine
c=
Atropine
c=
Suxamethonium
c=
Tetrodotoxin
c=
Magnesium
c=
12
General Physiology and Excitable Tissue – General Medicine
contraction force (gms)
Plot the graph of the dependence of the contraction magnitude on the drug concentration in the solution
with tubocurarine (minimal concentration 0.5 μM and increment 0.2 μM) or with suxamethonium
(minimal concentration 5 μM and increment 2 μM). Maximum concentration used corresponds to the
dose at which the muscle does not response to stimulation. Use only indirect stimulation.
concentration (mol/l)
Which drugs and in which mechanism can compensate low level of calcium concentration (0.5 mM)?
(Wash the bath with Krebs solution without calcium and then add 0.5 mM of calcium.)
How and why you should compensate hypermagnesemia (6 mM) without affecting potassium channels or
acetylcholinesterase?
13
General Physiology and Excitable Tissue – General Medicine
How can be completely restored the contraction after application of tetrodotoxin (0,5 μM) or
suxamethonium (20 μM)?
What is the difference in muscle response between the direct and the indirect stimulation, when the bath
solution is without calcium? Explain it.
Biofeedback
Introduction
Recently, there has been a renewed interest in mind-body interactions. There are many processes in the
body in which you exert voluntary control. For example, if you want a glass of water, you move your
body and go through the motions to take out a glass, fill it with water, and drink. You are consciously
“aware” of the motions that are under your “voluntary” control. However, once you start to swallow the
drink of water, you are not usually “aware” of the regulatory processes which follow automatically (e.g.,
you secrete saliva, rhythmical constrictions in the esophagus move the water down to the stomach, the
stomach starts churning, etc).
These processes are regulated by the autonomic nervous system and do not require conscious control by
the cerebral cortex. Muscle movement to obtain a drink of water involves some voluntary controls
(wherein your brain and body interact in a “loop” between sensory reinforcement of movement and the
brain) but there is usually no loop of feedback between consciousness and the involuntary actions
regulated by the autonomic nervous system, e.g., actions of the gastrointestinal tract.
The autonomic nervous system has two regulatory divisions, which can affect the same organs or tissues
but exert contrasting effects:
 sympathetic division — short-term response to acute stress, “fight-or-flight” response
 parasympathetic division — daily routine maintenance of homeostasis
For example, both systems are constantly regulating heart rate, but when the parasympathetic system
dominates, the heart rate is lower than when the sympathetic nervous system dominates.
Essentially, biofeedback completes the loop between autonomic functions and conscious awareness.
Biofeedback training is a learning process whereby people exert conscious control over physiological
processes controlled by the autonomic nervous system. Biofeedback instruments unobtrusively monitor
physiological functions (e.g., heart rate) and provide feedback in real time. The equipment provides
feedback using a signal that changes with the monitored variable. The person can then use the signal to
enhance the desired response.
With biofeedback training, people have been able to regulate many processes: lower heart rate;
lower blood pressure, control headaches, and manage responses to stressful situations. For example,
biofeedback training has been shown to be effective for controlling hypertension or high blood pressure.
One training method consists of a “hand warming” response — that is, the biofeedback signal is
associated with the temperature of the hands. What does “hand warming” have to do with high blood
pressure? Well, blood pressure is a result of cardiac output (volume output of the heart per minute) and
peripheral resistance. Peripheral resistance is inversely proportional to the amount of blood flow to the
periphery. Because blood is warmed in the body, when blood flow to the skin increases, the skin is
warmer. Therefore, warmer hands signify increased blood flow that occurred when peripheral resistance
and blood pressure decreased.
Biofeedback training has also been used to teach stress management techniques. In physiological
terms, relaxation using biofeedback training teaches people to activate specific controls of the
14
General Physiology and Excitable Tissue – General Medicine
parasympathetic part of the autonomic nervous system, e.g., to lower heart rate. At the same time,
biofeedback can be used to decrease activity of the sympathetic nervous system.
The galvanic skin response (GSR) or electrodermal activity is one variable traditionally
associated with sympathetic nervous system activity. The galvanic skin response is affected by sweat
gland activity and skin responses on the palmar surface of the hand. Unlike the heart, the sweat glands
are only activated by sympathetic activity. If the sympathetic branch of the autonomic nervous system is
highly aroused, then sweat gland activity increases and consequently, so does GSR. Because of this
association, the GSR is traditionally used as an index of sympathetic activity. When a person is relaxed,
then the GSR should be low. GSR has been used as a measure of emotional and sympathetic response.
For biofeedback purposes, however, the relationship of GSR response as an index of sympathetic activity
is complex for many reasons. GSR responses are delayed 1-3 seconds. Responses are not consistent when
given the same stimulus level. External factors, e.g., temperature and humidity, affect GSR
measurements, adding to inconsistent results. Internal factors, e.g., medications, can change GSR
measurements. A final complication is that the neurotransmitter for palmar sweat glands is acetylcholine,
not norepinephrine as is common for most of the sympathetic nervous system.
In this lesson, the biofeedback signal will be plotted on the screen as a thermometer style bar
chart that will rise and fall with changes in heart rate and GSR, allowing the Subject to become conscious
of his/her heart rate and arousal (GSR). The Subject will try to change the reading(s) without physical
movements and should be able to see that heart rate and level of arousal are independent.
Experimental objectives
1) Introduce the concept of biofeedback training for relaxation.
2) Measure levels of arousal via heart rate and galvanic skin response (GSR).
Materials









BIOPAC disposable vinyl electrodes (EL503), 3 electrodes per Subject
Electrode lead (SS2L)
GSR transducer (SS3L)
GEL100 electrode gel
Computer system
Biopac Student Lab software v3.6.7 PC or v3.0.7 Mac or greater
BIOPAC acquisition unit (MP30)
BIOPAC wall transformer (AC100A)
BIOPAC serial cable (CBLSERA) or USB cable (USB1W) if using a USB port
Experimental Methods
Overview
 As you complete the Experimental Methods (Set Up, Calibration, and Recording) and the Analysis,
you may need to use the following tools and/or display options. The window display shown below is
only a reference sample — it does not represent any lesson specific data. The sample screen shows 3
channels of data and four channel measurement boxes, but your screen display may vary between
lessons and at different points within the same lesson.
15
General Physiology and Excitable Tissue – General Medicine
channel measurement boxes
(channel #
)
measurement type result)
marker
marker tools
channel boxes
(Data analysis mode only)
vertical scales
marker label
vertical (amplitude)
scroll bar
channel labels
selection tool
horizontal (time) scroll bar
horizontal scale
zoom tool
I-Beam cursor
 The symbols explained below are used throughout Experimental Methods and Analysis.
Key to Symbols

If you encounter a problem or need further explanation of a concept, refer to the
Orientation Chapter for more details.

The data collected in the associated step needs to be recorded in the Data Report (in the
section indicated by the alpha character). You can record the data individually by hand or
choose Edit > Journal > Paste measurements to paste the data to your journal for future
reference.

Most markers and labels are automatic. Markers appear at the top of the window as
inverted triangles. This symbol is used to indicate that you need to insert a marker and key
in a marker label similar to the text in quotes. You can insert and label the marker during
or after acquisition. On a Mac, press “ESC” and on a PC, press “F9.”
 Each section is presented in a two-column format, as described below.
FAST TRACK Steps
Detailed Explanation of Steps
This side of the lesson (left, shaded
This side of the lesson contains more detailed information to
column) is the “FAST TRACK”
clarify the steps and/or concepts in the FAST TRACK, and may
through the lesson, which contains a
include reference diagrams, illustrations, and screen shots.
basic explanation of each step.
A.
SET UP
FAST TRACK Set Up
1. Turn the computer ON.
2. Make sure the BIOPAC MP30 unit is
turned OFF.
Detailed Explanation of Steps
The desktop should appear on the monitor. If it does not
appear, ask the laboratory instructor for assistance. 

16
General Physiology and Excitable Tissue – General Medicine
3. Plug the equipment in as follows:
GSR transducer (SS3L) — CH 1
Electrode lead set (SS2L) —CH 2
BIOPAC MP30 unit
SS2L
Electrode Lead Set
SS3L
GSR
Transducer
Fig. 1
4. Turn the MP30 Data Acquisition Unit
ON.
5. Select a Subject (who has not
exercised within the last hour).
6. Place three electrodes on the Subject
(Fig. 2).

Subject must not have done any rigorous exercise within the
last hour.
one on right forearm
(just above wrist)
one on inside right leg
(just above ankle bone)
one on inside left leg
(just above ankle bone)
Set Up continues…
Fig. 2
 Place one electrode on the medial surface of the right
leg, just above the ankle bone
 Place one electrode on the medial surface of the left
leg, just above the ankle bone
 Place one electrode on the right anterior forearm at the
wrist (same side of arm as the palm of hand).
Note: For optimal electrode adhesion, the electrodes should
be placed on the skin at least 5 minutes before the start of
the Calibration procedure. 
17
General Physiology and Excitable Tissue – General Medicine
7. Attach the electrode lead (SS2L) to
the Subject following the color code
(Fig. 3).
 When the electrode cable is
connected properly, the LEAD II
electrode configuration will be
established.
right forearm
WHITE lead
right leg
BLACK lead
(ground)
8. Fill the electrode cavities with
(GEL100) gel and then attach the
GSR transducer (SS3L) to the
Subject’s fingertip (Fig. 4).
Note — Electrodes should be in
place for at least five
minutes prior to calibration
or recording.
Set Up continues…
left leg
RED lead
Fig. 3
Each of the pinch connectors on the end of the electrode
cable needs to be attached to a specific electrode. The
electrode cables are each a different color, and you should
follow Fig. 3 to ensure that you connect each cable to the
proper electrode.
The pinch connectors work like a small clothespin, but will
only latch onto the nipple of the electrode from one side of
the connector.
You need to fill both the electrode cavities with electrode gel
before attaching to the fingertips.
HINT
For a good signal to be picked up, it helps if
the subjects have a little sweat on their hands
(not a lot, but enough so that their hands are
not completely smooth or cold). If subjects
wash their hands just prior to the recording or if they have
been sitting in a cold room, then they must do something to
activate the sweat glands before beginning calibration or
recording. If subjects begin with colder hands, the scale will
be diminished and the signal will be easily saturated once
they “warm up” during the lesson.
18
General Physiology and Excitable Tissue – General Medicine
Sensors attach to
bottom of fingertips
Velcro straps
wrap around
fingers
9.
10.
11.
12.
Start the Biopac Student Lab Program
Choose Lesson 14 (L14-Biofbk-1).
Type in your filename.
Click OK.
Fig. .4
The SS3L is typically placed on the index and middle fingers of
the left hand, with the sensor on the bottom of the fingertips
(the part without the fingernail). Wrap the Velcro tape around
the finger so the transducer fits snugly (Fig. .4).


Use a unique identifier. 
This ends the Set Up procedure.
END OF SET UP
B.
CALIBRATION
The Calibration procedure establishes the hardware’s internal parameters (such as gain, offset, and
scaling) and is critical for optimum performance. Pay close attention to the Calibration procedure.
FAST TRACK Calibration
Detailed Explanation of Calibration Steps
1. Subject should sit in a relaxed state,
breathing normally.
2. Click on Calibrate (then YES, then
When you click on Calibrate, two dialog boxes will pop-up to
make sure you are ready for the calibration procedure. Click
OK).
on YES for the first box and click on OK for the second box.
You should see three tracings on the screen: the top is the GSR
recording, the middle is ECG, and the lower is ECG rate.
A beep should occur four seconds into the recording.
3. After the “beep,” inhale and exhale
The Subject should take one deep inhale and exhale, and then
deeply once.
return to normal breathing. The program needs to see some
variation in the GSR and BPM data.
Note: The Subject should inhale and exhale without tensing
the chest muscles too much, as this may cause excessive
EMG artifact.
The Calibration will run for 10 seconds and then stop
4. Wait for the Calibration to stop.
automatically, so let it run its course.
19
General Physiology and Excitable Tissue – General Medicine
5. Check your calibration data.
 If similar, proceed to the Data
Recording section.
 If different, Redo Calibration.
END OF CALIBRATION
At the end of the 10-sec calibration recording, your screen
should resemble Fig. 5.
Figure 5
Both recording channels should show some fluctuation. If
there is not any fluctuation, then it is possible that a transducer
is not connected properly and you must redo calibration.
If the lower recording of ECG rate shows large fluctuations
(20 BPM or more) during the interval from 4-10 seconds, then
it is possible that excessive EMG artifact during inhale and
exhale caused loss of BPM tracking and you should redo the
calibration. And have the Subject breathe less deeply.
If you did not hear a beep during the calibration, then you
should make sure your computer volume is turned up and redo
calibration. Redo by clicking on the Redo Calibration button
and repeating the entire calibration sequence.
20
General Physiology and Excitable Tissue – General Medicine
C. RECORDING LESSON DATA
1. Prepare for the recording.
In order to work efficiently, read this entire section so you will
know what to do for each recording segment.
This lesson requires that the Subject concentrate on the monitor
display of Heart Rate and Arousal (GSR) and try to change the
reading(s) without physical movement. Heart rate and level of
arousal (GSR) are independent, so the Subject may be able to
change one or the other or both.
Check the last line of the journal and note the total amount of
time available for the recording. 
When you begin recording, your heart rate and your GSR (Fig.6)
will be plotted in a thermometer style bar chart.
Faster
GSR
Arousal
Level
Heart
Rate
Slower
2. Click on Record.
3. For the first 90 seconds of the
recording, Subject should try to relax
so that both bars move down.
Recording continues…
Higher
Lower
Fig.6 Heart rate and Arousal (GSR)
The Heart Rate will be displayed to the left and will increase
(rise) as the heart rate increases. Arousal (GSR) will be
displayed on the right, and will increase (rise) as the body
resistance decreases, indicating a higher level of arousal.
The upper and lower range of each reading was set with your
calibration data. Both readings should begin with values in
approximately the center of the display. If your reading for either
bar begins beyond the mid-range, you may want to consider
redoing calibration.
The bar chart display only works while data is being recorded.
The display will change to a standard data plot when recording is
complete.
The recording will begin and will run continuously until you
click on the STOP button or the time indicated runs out.
Hints for achieving relaxation:
a) Relax your posture.
b) Breathe in and out very slowly.
c) Imagine yourself at a warm, relaxing seashore.
d) Smile.
e) Close your eyes.
21
General Physiology and Excitable Tissue – General Medicine
4. After 90 seconds, Subject should try to
move the bars up by increasing arousal
and Recorder should insert marker.
 insert marker at 90 seconds
(No marker label needed.)
5. Click on Stop twice.
6. Review the data on the screen.
 If correct, go to Step 7.
 If incorrect, click on Redo.
7. Click on Done.
To move the bars up, you need to become more aroused.
Recorder should insert a marker 90 seconds into the recording
to indicate that the Subject is now attempting arousal versus
relaxation. To insert Markers:
Mac = Esc key
PC = F9 key.
Markers can also be entered after the data is recorded. 
Hints for achieving arousal:
a) Think of a stressful situation.
b) Have the Director clap behind you.
c) Make a scowl with your face.
d) Hold your breath.
The first time you click on Stop, the data window containing the
Stop button will be activated.
When you click on Stop the second time, the bar chart data
display will be hidden and the data window will change to a
standard three-channel recording with GSR, Raw ECG and ECG
Rate. This data represents the entire recording.
If all went well, your data should look similar to Fig. 7 and you
can proceed to Step 7.
Note: The recording will vary greatly from person to person,
and it is hard to generalize about the “results.” Hopefully, you
were able to manipulate your physiological responses to some
degree. This may take some practice, as many people are not
used to thinking that they have some control over things like
their heart rate and nervous system.
Fig. 7 Sample data
The data would be incorrect if:
a) The BPM data fluctuates excessively, indicating that an
electrode may have “peeled” up.
b) The GSR signal goes outside of its plotting region,
indicating that one of the GSR transducers may not have
had good contact.
If incorrect, you should redo the recording by clicking on
“Redo” and repeating Steps 2-6. Note that once you press Redo,
the data you have just recorded will be erased.
A pop-up window with four options will appear. Make your
choice, and continue as directed.
If choosing the “Record from another subject” option:
a) Attach the electrodes and transducer to the new Subject
per Set Up Steps 5 and 6, and continue the entire lesson
from Set Up Step 9.
b) Each the new Subject will need to use a unique file
name.
22
General Physiology and Excitable Tissue – General Medicine
8. Remove the electrodes and transducers
and clean the GSR transducer’s gel
cavities.
END OF RECORDING
D. DATA ANALYSIS
FAST TRACK Data Analysis
1. Enter the Review Saved Data mode.
Note Channel Number(CH)
designation:
Channel
Displays
CH 41
heart rate
CH 42
GSR
2. Turn OFF Channel 2, Raw ECG.
Remove the electrode cable pinch connectors, and peel off the
electrodes. Throw out the electrodes (BIOPAC electrodes are not
reusable). Wash the electrode gel residue from the skin, using
soap and water. The electrodes may leave a slight ring on the
skin for a few hours. This is normal, and does not indicate that
anything is wrong.
Remove the GSR transducers and clean out the electrode gel
cavity using alcohol.
Detailed Explanation of Data Analysis Steps
Enter the Review Saved Data mode.
The data window should come up the same as Fig. 8.
Fig 8
To turn a channel OFF, click on the channel number box and
hold down the following key:
Mac: “option” key
PC: “Ctrl” key 
Fig. 9 Channel 2 turned off
3. Setup your display window for optimal The first data segment is the data between Time 0 and the first
viewing of the first data segment.
marker. The following tools help you adjust the data window:

Autoscale horizontal
Horizontal(Time) Scroll Bar
Autoscale waveforms
Vertical (Amplitude) Scroll Bar
Zoom Tool
Zoom Previous
23
General Physiology and Excitable Tissue – General Medicine
4. Set up the measurement boxes as
follows:
Channel
Measurement
CH 41
value
CH 42
value
5. Measure the GSR and heart rate values
about 10 seconds into the recording.
A
6. Scroll to the point of maximal
relaxation.
The measurement boxes are above the marker region in the
data window. Each measurement has three sections: channel
number, measurement type, and result. The first two sections
are pull-down menus that are activated when you click on
them. Brief definition of measurements: 
value: displays the amplitude value for the channel at the
point selected by the I-beam cursor. If a single point is
selected, the value is for that point, if an area is selected, the
value is the endpoint of the selected area.
Note: The “selected area” is the area selected by the
I-beam tool (including the endpoints).
It takes a few heart rate cycles for the BPM data to be correct,
so measuring data 10 seconds into the record will be an
accurate baseline. Simply clicking the cursor at Time =10
seconds will update the value measurements.
Fig 10
Use the horizontal scroll bar to move through the data. Select a
point at the end of the first interval (before the marker)
representing the point of maximal relaxation.
Fig. 11
7. Take the measurements on this point as
needed.
A
8. Scroll to the point of maximal arousal. Select a point at the end of the second interval (after the marker)
representing the point of maximal arousal.
9. Take the measurements on this point as
needed.
A
10. Save or print the data file.
You may save the data to a floppy drive, save notes that are in
the journal, or print the data file. 

11. Exit the program.
END OF DATA ANALYSIS
24
General Physiology and Excitable Tissue – General Medicine
PROTOCOL
I. Data and Calculations
Subject Profile
Name
Age
Gender: Male / Female
Height
Weight
A.
Table 1
II.
Measurement
Heart Rate
(BPM)
CH. #
CH 41
value
GSR
(MHO)
CH 42
value
Baseline
(10 sec.)
Condition
Relaxation
Arousal
Questions
B. Based on the data from Table 1, did the effects of the parasympathetic nervous system change with
biofeedback? Explain the physiological mechanisms causing the results.
C. Describe a biofeedback program for “stress management.” Include details such as the physiological
variable(s) you would measure, the transducers needed, and your criterion for a successful training
program.
D. Name the branches of the autonomic nervous system and explain their function.
E. Define Biofeedback and explain in general terms how it works.
25
General Physiology and Excitable Tissue – General Medicine
F.
What change, if any, did your GSR recording show when you were aroused? Relaxed?
G. Why is GSR a useful measure for biofeedback training?
Determination of Blood Volume using T1824 (Evans Blue)
The volume of any fluid compartment in the body can be measured by administration of a
substance into that compartment, allowing it to spread throughout the compartment, and then by measuring
the extent to which the substance had become diluted. The concentration of the injected substance is
analyzed chemically, or by other means.
Ideal properties of a substance used for measurements of the body fluid volumes are:
1. It must be non-toxic.
2. It should diffuse quickly and uniformly.
3. It should be eliminated and metabolized slowly.
4. It should not have any influence on the distribution of water and other substances in the body.
5. It should be easy to determine.
A substance used for measuring blood volume must be capable of spreading throughout the blood
with ease, and it must remain in the circulatory system long enough for measurements to be made. The
plasma proteins remain in the circulatory system and any foreign substance that combines with them is
kept in the blood stream. A number of dyes, generally known as "vital dyes", have the ability to combine
with proteins.
The dye the most universally used for measuring the plasma volume is T-1824, also called Evans
blue. In making determinations of the plasma volume, a known quantity of the dye is injected, and it
immediately combines with the proteins and spreads throughout the circulatory system within
approximately five minutes. A sample of the blood is then taken and the red blood cells are removed from
the plasma by centrifugation. By spectrophotometric analysis, one can determine the exact quantity of the
dye in the sample of the plasma. From the quantity of the dye detected in each milliliter of the plasma and
the quantity of the dye injected, the plasma volume is calculated using this formula:
Volume in ml =
Quantity of dye injected
Concentration of dye per ml of plasma
This method does not measure the total blood volume because the dye does not enter the red blood
cells. However, the blood volume can be calculated from the plasma volume and the haematocrit using the
following formula:
26
General Physiology and Excitable Tissue – General Medicine
Blood volume = Plasma volume 
100
100 - Haematocrit
Procedure:
1. Anaesthetize the first adult rat, at first lightly with ether and then with urethan injected
intraperitoneally (1.5 g/kg of the body weight).
2. Put the anaesthetized rat on the operation table. Incise the skin on the neck along the midline and
reflect the skin laterally on each side. Make a vena jugularis externa preparation with surgical
forceps.
3. Aspirate the solution of the Evans blue into the insulin syringe in the amount proportional to the
rat's weight as indicated in the Table 1. Carefully inject the solution intravenously.
4. After 5 minutes decapitate the rat with scissors and collect its blood into a heparinized beaker.
5. Prepare the second rat in the same way (steps 1, 2)
6. Aspirate into the glass 2ml syringe the Evans blue solution in the amount indicated in the Table 1
and replenish the syringe up to the volume of 2 ml with hypertonic glucose (20%). Inject the
solution carefully intravenously into vena jugularis externa.
7. After 5 minutes decapitate the rat and collect its blood in the heparinized beaker in the same way
as in the first case (step 4)
8. Prepare the third rat in the same way (steps 1, 2)
9. After vena jugularis externa preparation inject the NaCl solution in the same amount as if it were
Evans blue according to the Table 1.
10. After 5 minutes decapitate the rat and collect its blood in the beaker in the same way as in step 4.
11. Take samples of the blood from the first and second rats into two glass capillaries, seal them on
the end and centrifuge them in the hematocrit centrifuge for 2 minutes at 16 000 r.p.m.
Determine the haematocrit value.
12. Transfer the blood from each of the three beakers into centrifuge test tubes and centrifuge them 5
minutes at 16 000 r.p.m. in the laboratory centrifuge.
13. Aspirate the plasmas with Pasteur's pipettes and transfer them to spectrophotometer cuvettes. The
plasma from the third rat serves as the blank. Read the absorbances of the first and second
samples against the blank on the spectrophotometer.
14. Determine the concentrations of the Evans blue using the calibration curve (Figure 1) and
calculate the plasma volume and the blood volume of the first and second rats according to the
formulas above.
15. Calculate the blood volume per kg of weight in both rats.
16. Compare both values.
17. Describe consequences of the injection of the hypertonic solution.
27
General Physiology and Excitable Tissue – General Medicine
Table 1.
1,1
1,0
0,9
Absorbance
0,8
0,7
0,6
0,5
0,4
0,3
0,2
0,1
0,0
0
1
2
3
4
5
6
7
8
9
10
11
Concentration (mg/l)
Absorbance calibration curve
28
12
13
14
15
16
General Physiology and Excitable Tissue – General Medicine
PROTOCOL
1.
Weight of the rat in kg
m1 =
2.
m2 =
Quantity of dye injected in mg
Q1 =
3.
Q2 =
Haematocrit
H1 =
4.
H2 =
Determination of Evans blue concentration (mg/l) using the calibration curve
C1 =
5.
C2 =
Plasma volume
V
P1

Q
C
1
V
P2
1
C
Q
6.
Q
C
2
2
Evans blue concentration in the plasma (mg/l)
Quantity of the dye injected (mg)
Blood volume
V
B1
 V P1 
VP
H
7.

1
1 H
V
B2
 V P2 
1
1 H
Plasma volume
Haematocrit
The blood volume per kg of weight of the rat
V
m
B1
1

V
m
B2

2
Conclusion:
Compare the blood volumes in both animals and explain eventual differences.
29
General Physiology and Excitable Tissue – General Medicine
Electromyography – Muscle Fatigue
Introduction
Mechanical work, in the physical sense, refers
to the application of a force that results in the movement
of an object. Skeletal muscle performs mechanical work
when the muscle contracts and an object is moved, as in
lifting a weight. To lift a weight, your muscles must
exert a force great enough to overcome the weight. If
you exert less force, then the weight does not move
(Fig.1).
Fig. 1
Physiologically, skeletal muscle is stimulated to contract when the brain or spinal cord
activates motor units of the muscle.Motor units are defined as a motoneuron and all of the muscle
fibers that the motoneuron innervates. An action potential (AP) in a human motoneuron always
causes an action potential in all of the muscle fibers of the motor unit. As a matter of fact, humans
generally do not send just one AP at a time down a motoneuron. Instead, a train of APs is sent —
enough to induce tetany (the sustained fusion of individual muscle twitches) in the muscle fibers of
the motor unit. [A discussion of motor units and their control was presented in Lesson 1.]
Most human skeletal muscles are composed of hundreds of motor units (Fig. 2). When a skeletal
muscle is called on to perform mechanical work, the number of motor units in the muscle activated by the
brain is proportional to the amount of work to be done by the muscle; the greater the amount of work to
be done, the greater the number of motor units activated. Thus, more motor units are simultaneously
active when a skeletal muscle lifts 20 kilograms than when the same muscle lifts 5 kilograms.
Fig. 2.Example of Motor Units
The brain determines the number of active motor units required for a muscle to perform a given
task by utilizing sensory information from stretch receptors in the muscle and associated tendons and
joint capsules. For example, when lifting a bucket of water from the ground, the brain first activates
several motor units in the requisite skeletal muscles. If sensory information returning from the muscles
30
General Physiology and Excitable Tissue – General Medicine
indicates the muscles are contracting but not developing adequate power to lift the bucket, the brain
activates additional motor units until the sensory information indicates the bucket is being lifted. The
sequential activation of motor units to perform a designated task is called motor unit recruitment.
Once you have lifted a light object, the brain recruits approximately the same number of motor
units to keep the object up, but cycles between different motor units. The muscle fibers consume stored
energy available in the muscle, and generate a force by contracting. As the muscle fibers deplete this fuel
source, more energy must be created in order to continue contracting. By recruiting different motor units,
motor units can relax and replenish their fuel sources.
Skeletal muscles performing acute maximum work or chronic submaximum work of a repetitive
nature will eventually fatigue. Fatigue is defined as a decrease in the muscle’s ability to generate force
due to a reversible exhaustion of energy resources. If the muscle uses its energy sources faster than they
can be generated by cellular metabolism, fatigue occurs. During contraction, skeletal muscle cells convert
chemical energy into thermal and mechanical energy, and, in the process, produce chemical waste
products, mainly lactic acid, whose accumulation leads to acidosis. Some accumulated waste products
also stimulate pain receptors in surrounding connective tissue and induce cramping of skeletal muscle.
Pain and cramp of the working muscle are, in addition to a decrease in contraction force, characteristic
signs of the muscle fatigue. Normally the waste products are removed from the muscle by the circulatory
system as the blood brings nutrients to the muscle for energy transformation. If certain waste products
(metabolites) are not removed at an adequate rate, they will accumulate and chemically interfere with the
contractile process, thereby hastening the onset of fatigue.
The muscle work is divided into two basic groups – static work and dynamic work. The dynamic
work is characterized by a regular change of contraction and relaxation. During the dynamic work, the
blood flow in the working muscle varies, it decreases during contraction and increases during relaxation.
Thus the waste products are removed from the active muscle during relaxation. The static work is
characterized by an increase in muscle tone without a change in muscle length. The working muscle is
contracted and the blood flow is permanently decreased during the static work. This implies that the signs
of the muscle fatigue appear earlier in the static work then in the dynamic one.
In this lesson, you will examine motor unit recruitment and skeletal muscle fatigue by combining
electromyography with dynamometry.When a motor unit is activated, the component muscle fibers
generate and conduct their own electrical impulses, which cause the fibers to contract. Although the
electrical impulse generated and conducted by each fiber is very weak (less than 100 volts), many fibers
conducting simultaneously induce voltage differences in the overlying skin which are large enough to be
detected by a pair of surface electrodes.
The detection, amplification, and recording of changes in skin voltage produced by underlying
skeletal muscle contraction is called electromyography, and the recording thus obtained is called an
electromyogram (EMG). Power is defined as the amount of work done per unit of time. Dynamometry
means the measurement of power (dyno = power, meter = measure), and the graphic record derived from
the use of a dynamometer is called a dynagram. In this lesson, the power of contraction of clench
muscles will be determined by a hand dynamometer equipped with an electronic transducer for
recording.
The BIOPAC system will simultaneously record three bands of information:
1)
The force you exert on the transducer,
2)
The electrical signal produced by the muscle during contraction, and
3)
The integrated waveform, which is an indication of the activity levels of the muscle.
Experimental Objectives
1)
2)
To record the force produced by clench muscles, EMG, and integrated EMG when
inducing fatigue.
To compare the time to fatigue during static and dynamic work.
31
General Physiology and Excitable Tissue – General Medicine
Materials
 BIOPAC electrode lead set (SS2L)
 BIOPAC disposable vinyl electrodes (EL503), 6 electrodes per subject
 BIOPAC electrode gel (GEL1) and abrasive pad (ELPAD)
or
Skin cleanser or alcohol prep
 BIOPAC SS25 Hand Dynamometer
 BIOPAC Headphones (OUT1)
 Computer system
 Biopac Student Lab software v3.6.7 PC or v3.0.7 Mac or greater
 BIOPAC acquisition unit (MP30)
 BIOPAC wall transformer (AC100A)
 BIOPAC serial cable (CBLSERA) or USB cable (USB1W) if using a USB port.
Experimental Methods
Overview
 As you complete the Experimental Methods (Set Up, Calibration, and Recording) and the Analysis,
you may need to use the following tools and/or display options. The window display shown below is
only a reference sample — it does not represent any lesson specific data. The sample screen shows 3
channels of data and four channel measurement boxes, but your screen display may vary between
lessons and at different points within the same lesson.
channel measurement boxes
(channel #
)
measurement type result)
marker
marker tools
channel boxes
(Data analysis mode only)
vertical scales
marker label
vertical (amplitude)
scroll bar
channel labels
selection tool
horizontal (time) scroll bar
horizontal scale
zoom tool
I-Beam cursor
 The symbols explained below are used throughout Experimental Methods and Analysis.
Key to Symbols

If you encounter a problem or need further explanation of a concept, refer to the
Orientation Chapter for more details.

The data collected in the associated step needs to be recorded in the Data Report (in the
section indicated by the alpha character). You can record the data individually by hand or
choose Edit > Journal > Paste measurements to paste the data to your journal for future
reference.

Most markers and labels are automatic. Markers appear at the top of the window as
inverted triangles. This symbol is used to indicate that you need to insert a marker and key
32
General Physiology and Excitable Tissue – General Medicine
in a marker label similar to the text in quotes. You can insert and label the marker during
or after acquisition. On a Mac, press “ESC” and on a PC, press “F9.”
 Each section is presented in a two-column format, as described below.
FAST TRACK Steps
Detailed Explanation of Steps
This side of the lesson (left, shaded
This side of the lesson contains more detailed information to
column) is the “FAST TRACK” through
clarify the steps and/or concepts in the FAST TRACK, and may
the lesson, which contains a basic
include reference diagrams, illustrations, and screen shots.
explanation of each step.
A.
SET UP
FAST TRACK SET UP
1. Turn your computer ON.
2. Make sure the BIOPAC MP30 unit is
OFF.
3. Plug the equipment in as follows:
Hand dynamometer (SS25L) — CH 1
Electrode lead (SS2L) — CH 3
Headphones (OUT1) — back of unit
Detailed Explanation of Steps for SET UP
The desktop should appear on the monitor. If it does not appear,
ask the laboratory instructor for assistance. 

Headphones (BIOPAC OUT1)
Plugs into back of MP30 unit
BIOPAC SS2L
plugs into CHannel 3
BIOPAC SS25L
plugs into CHannel 1
4. Turn ON the BIOPAC MP30 unit.
5. Attach three electrodes to the
forearm (Fig. 4).
Fig. 3 Equipment Setup

For the first recording segment, select the Subject’s dominant
forearm (generally the right forearm if the subject is righthanded, or the left forearm if the subject is left-handed) and
attach the electrodes onto the forearm as shown; this will be
Forearm 1.
Use the Subject’s “Other arm” for the second recording
segment; this will be Forearm 2.
Attach three electrodes to the forearm as shown in Fig. 4.
Set Up continues…
33
General Physiology and Excitable Tissue – General Medicine
Fig. 4 Electrode placement
Note: For optimal electrode adhesion, the electrodes should be
placed on the skin at least 5 minutes before the start of the
Calibration procedure.
6. Attach the electrode lead set
(SS2L) to the electrodes,
following the color code (Fig.
5).
IMPORTANT
Make sure the electrode lead
colors match Fig. 5.
White Lead
(-)
Red Lead
(+)
Black Lead
(Ground)
Fig. 5 Electrode lead attachment
Each of the pinch connectors on the end of the electrode cable
needs to be attached to a specific electrode. The electrode
cables are each a different color. Follow Fig. 5 to ensure that
you connect each cable to the proper electrode.
The pinch connectors work like a small clothespin, but will
only latch onto the nipple of the electrode from one side of the
connector.
34
General Physiology and Excitable Tissue – General Medicine

7. Start the BIOPAC Student Lab
Program.
8. Choose lesson “L02-EMG-2” and click 
OK.
No two people can have the same filename, so use a unique
9. Type in a unique filename.
identifier, such as the subject’s nickname or student ID#. 
This ends the Set Up procedure.
10. Click OK.
END OF SET UP
B. CALIBRATION
The Calibration procedure establishes the hardware’s internal parameters (such as gain, offset, and
scaling) and is critical for optimum performance. Pay close attention to the Calibration procedure.
FAST TRACK CALIBRATION
Detailed Explanation of Steps for Calibration
A pop-up window will tell you to remove any grip force from
1. Click on Calibrate.
the hand dynamometer. To do so, set the hand dynamometer
down.
2. Set the hand dynamometer down and Remove your hands from the transducer to ensure there is no
force on the transducer. This establishes a zero-force calibration
click OK.
before you continue the calibration sequence.
3. Grasp the BIOPAC hand dynamometer For Forearm 1, clench with the hand of your dominant forearm.
with your hand as close to the dynagrip For Forearm 2, clench with your other hand.
Dynagrip crossbar
crossbar as possible without actually
touching the crossbar (Fig. 6).
IMPORTANT
You need to hold the
dynamometer in the same
position for all measurements
from each arm, so note your
hand position with respect to
the crossbar for the first
segment and try to repeat it for
the other segments.
4. Follow the instructions on the two
successive pop-up windows and click
on OK when ready for each.
5. Wait about two seconds, then clench
the hand dynamometer as hard as
possible, then release.
Hand close to
bracket but not
touching.
Fig. 6
Pop-up windows will guide you through the initial calibration
sequence. After you click OK on the third pop-up window, the
Calibration recording will begin.
The program needs a reading of your maximum clench to
perform an auto-calibration.
35
General Physiology and Excitable Tissue – General Medicine
6. Wait for Calibration to stop.
The Calibration procedure will last eight seconds and stop
automatically, so let it run its course.
7. Check the Calibration data.
At the end of the eight-second Calibration recording, the screen
should resemble Fig.7.
 If correct, proceed to the data
Recording Section.
 If incorrect, Redo Calibration.
Fig. 7
If your calibration recording did not begin with a zero baseline
(Subject clenched before waiting two seconds), you need to
repeat calibration to obtain a reading similar to Fig. 7.
END OF CALIBRATION
C. RECORDING LESSON DATA
FAST TRACK RECORDING
1. Prepare for the recording.
SEGMENT 1
2. Click on Record.
3. Clench the hand dynamometer with
your maximum force. Note this force
and try to maintain it.
4. When the maximum clench force
displayed on the screen has decreased
by more than 50%, click on Suspend.
5. Review the data on the screen.
 If correct, go Step 7.
Detailed Explanation of Steps for Recording Lesson Data
You will record two segments on each forearm:
a. Segment 1 records Fatigue during the static work.
b. Segment 2 records Fatigue during the dynamic work
In order to work efficiently, read this entire section so you will
know what to do before recording.
Check the last line of the journal and note the total amount of
time available for the recording. Stop each recording segment as
soon as possible so you don’t waste recording time (time is
memory) .
After you click on record, the screen will change to display only
the hand dynamometer channel, and a grid using your assigned
increment as a division scale will appear so that you can visually
review the force level. You will begin to record data.
Note the maximum clench force so you can determine when the
force has decreased by 50% (the maximum force may scroll out
of view). Try to maintain the maximum clench force (the
forearm will fatigue and the force will decrease).
The time to fatigue to 50% of maximal clench force will vary
greatly among individuals.
When you click on Suspend, the recording should halt, giving
you time to review the data for segment one.
If all went well, your data should look similar to Fig. 8.
36
General Physiology and Excitable Tissue – General Medicine
 If incorrect, go to Step 6.
6. Click on Redo if your data was
incorrect, and repeat Steps 3-5.
Fig. 8 Fatigue during the static work
Note that the peak found immediately following the start of
Segment 2 represents the maximal clench force. This example
shows the point of fatigue to 50% maximal clench force
captured on the same screen, but your maximum force may
scroll out of view. You may use the horizontal (time) scroll bar
to view your entire recording.
The data would be incorrect if:
a) You didn’t record to the point of 50% maximal clench
force.
b) The Suspend button was pressed prematurely.
c) The instructions were not followed.
Click “Redo” and have the Subject rest so the arm muscles
recover and the fatigue data will be meaningful. When ready
repeat Steps 3-5. Note that once you press Redo, the data you
have just recorded will be erased.
SEGMENT 2
7. Click on Resume.
A marker labeled “Continued clench at maximum force” will
automatically be inserted when you click Resume and the
recording will continue from the point it left off.
8. Repeatedly and periodically clench the Repeat a cycle of Clench-Release. One cycle should last about 1
hand dynamometer with your maximum second.
force and then relax.
9. When the maximum clench force
The time to fatigue to 50% of maximal clench force will vary
greatly among individuals.
displayed on the screen has decreased
by more than 50% of the initial value, When you click on Suspend, the recording should halt, giving
you time to review the data for segment two.
click on Suspend
10. Review the data on the screen.
If all went well, your data should look similar to Fig. 9.
 If correct, go to Step 13.
37
General Physiology and Excitable Tissue – General Medicine
11. If incorrect, go to Step 12.
12. Click on Redo if your data was
incorrect, and repeat Steps 8-10.
13. Click Forearm 2.
or, if you’ve recorded both arms,
Go to Step 16.
14. To record Forearm 2, attach electrodes
(per Set Up Step 5 and Step 6) to the
Subject’s other forearm.
15. Complete the entire Calibration section
and the Recording section up to this
point for the Forearm 2.
16. Click Done.
17. Remove the electrodes from your
forearm.
Fig. 9 Fatigue during the dynamic work
The data would be incorrect if:
a) You didn’t record to the point of 50% maximal clench force.
b) The Suspend button was pressed prematurely.
c) The instructions were not followed.
Click “Redo” and have the Subject rest so the arm muscles
recover and the fatigue data will be meaningful. When ready
repeat Steps 8-10. Note that once you press Redo, the data you
have just recorded will be erased.
When you click on Forearm 2, the program returns you to the
Calibration sequence.
Refer to the Set Up section for proper placement of electrodes
and electrode leads.
Complete the entire Calibration sequence (outlined in the
preceding section) and then follow the entire Recording
procedure.
A pop-up window with four options will appear. Make your
choice, and continue as directed.
If choosing the “Record from another subject” option:
c) Attach electrodes per Set Up Step 5 and continue the
entire lesson from Set Up Step 8.
d) Each person will need to use a unique file name.
Remove the electrode cable pinch connectors, and peel off the
electrodes. Throw out the electrodes (BIOPAC electrodes are
not reusable). Wash the electrode gel residue from the skin,
using soap and water. The electrodes may leave a slight ring on
the skin for a few hours, which is quite normal.
END OF RECORDING
38
General Physiology and Excitable Tissue – General Medicine
D. DATA ANALYSIS
FAST TRACK DATA ANALYSIS
Detailed Explanation of Data Analysis Steps
1. Enter the Review Saved Data mode and Enter Review Saved Data from the Lessons menu. 
choose the correct file.
For the first part of the analysis, choose the data file from the
Subject’s first arm (Forearm 1, saved with file name extension
“1-L02”).
For the second part of the analysis, choose the data file from the
subject’s second arm (Forearm 2, saved with file name extension
“2-L02”).
Note Channel Number (CH)
designations:
Channel Displays
CH 1
Force
CH 3
Raw EMG
CH 40
Integrated EMG
Fig. 10
2. Setup your display window for optimal The first data segment was the recording before the first marker.
viewing of the first data segment.
See Fig. 11.
The following tools help you adjust the data window: 
Autoscale horizontal Horizontal(Time) Scroll Bar
Autoscale waveforms Vertical (Amplitude) Scroll Bar
Zoom Tool
Zoom Previous
3. Set up the measurement boxes as
The following is a brief description of the specific
follows:
measurements not previously covered. 
Channel Measurement
value: displays the amplitude value for the channel at the
CH 1
value
point selected by the I-beam cursor. If a single point is
CH 40
delta T
selected, the value is for that point, if an area is selected,
the value is the endpoint of the selected area.
delta T: displays the amount of time in the selected
segment (the difference in time between the endpoints of
the selected area).
4. Using the I-Beam cursor, select a point
of maximal clench force immediately
following the start of Segment 1.
A
Fig. 11
The point selected should represent the maximal clench force at
39
General Physiology and Excitable Tissue – General Medicine
the start of Segment 1 (continuous maximal clench), as shown in
Fig. 11.
You will need this number to complete Step 7.
5. Calculate 50% of the maximum clench
force from Step 4.
A
6. Find the point of 50% maximum clench Make an eyeball approximation of the point that is 50% down
force by using the I-beam cursor and
from the maximal clench point. Then, use the I-beam cursor to
leave the cursor at this point.
click on points near this region, noting the value displayed in the
measurement box, until you are on a point within 5% of the
maximal clench force. Leave the cursor at this point.
7. Select the area from the point of 50%
One way to select the area is as follows:
clench force back to the point of
The cursor should be flashing on the point of 50% maximal
maximal clench force by using the Iclench force. Hold down the mouse button and drag to the left of
beam cursor and dragging. (Fig. 12).
this point until you reach the point of maximal clench force, then
Note the time to fatigue (CH 40 delta T) release the mouse button.
measurement.
A
8. Scroll to Segment 2 and set up your
display for optimal viewing.
Fig. 12 showing area max-50%
Segment 2 begins at the second append marker.
9. Set up the measurement boxes as
The following is a brief description of the specific
follows:
measurements not previously covered. 
Channel Measurement
max: finds the maximum amplitude value within the area
CH 1
max
selected by the I-Beam cursor (including the endpoints).
CH 40
delta T
10. Using the I-Beam cursor, select an area
of the first maximal clench force
immediately following the start of
Segment 2.
B
Fig. 13
The area selected should represent the maximal clench force at
40
General Physiology and Excitable Tissue – General Medicine
the start of Segment 2 (continuous maximal clench), as shown in
Fig. 13.
You will need this number to complete Step 13.
11. Calculate 50% of the maximum clench
force from Step 10.
B
12. Find the point of 50% maximum clench Make an eyeball approximation of the point that is 50% down
force by using the I-beam cursor and
from the maximal clench point. Then, use the I-beam cursor to
leave the cursor at this point.
click on points near this region, noting the value displayed in the
measurement box, until you are on a point within 5% of the
maximal clench force. Leave the cursor at this point.
13. Select the area from the point of 50%
One way to select the area is as follows:
clench force back to the point of
The cursor should be flashing on the point of 50% maximal
maximal clench force by using the Iclench force. Hold down the mouse button and drag to the left of
beam cursor and dragging. (Fig. 14).
this point until you reach the point of maximal clench force, then
Note the time to fatigue (CH 40 delta T) release the mouse button.
measurement.
B
Fig. 14 showing area max-50%
14. Repeat the entire Analysis section
starting with Step 1 with the data file
for Forearm 2.
15. Exit the program.

END OF DATA ANALYSIS
END OF LESSON 2
Complete the Lesson 2 Data Report that follows.
41
General Physiology and Excitable Tissue – General Medicine
PROTOCOL
I.
Data and Calculations
Subject Profile
Name
Age
Gender: Male / Female
Height
Weight
Dominant forearm: Right / Left
Muscle Fatigue
A. Complete Table 1 using Segment 1 data from each arm.
Table 1 Segment 1 data – Static work
Forearm 1 (Dominant)
Maximum
50% of max
Time to
Maximum
Clench Force
clench force
fatigue
Clench Force
calculate
CH 1 value
CH 40 delta T* CH 1 value
Forearm 2
50% of max
clench force
calculate
Time to
fatigue
CH 40 delta T*
Forearm 2
50% of max
clench force
calculate
Time to
fatigue
CH 40 delta T*
B. Complete Table 2 using Segment 2 data from each arm.
Table 2 Segment 2 data – Dynamic work
Forearm 1 (Dominant)
Maximum
50% of max
Time to
Maximum
Clench Force
clench force
Fatigue
Clench Force
calculate
CH 1 max
CH 40 delta T* CH 1 max
*Note: You do not need to indicate the delta T (time to fatigue) polarity. The polarity of the delta T
measurement reflects the direction the "I-beam" cursor was dragged to select the data. Data
selected left to right will have a positive ("+") polarity, while data selected right to left will have
a negative ("-") polarity.
II.
Questions
C. Compare the initial contraction force during the static and dynamic works.
Forearm 1 (dominant)
Forearm 2
D. Does the time to fatigue vary between the static and dynamic works? If yes, explain why.
Forearm 1 (dominant)
Forearm 2
42
General Physiology and Excitable Tissue – General Medicine
E. Define Fatigue
F. As you fatigue, the force exerted by your muscles decreases. What physiological
processes explain the decline in strength?
G. Define Motor unit
H. Define Motor unit recruitment
43