PowerLab: Physiology of the Nerve

T. Pineda, P. M. Santiago, J. R. Serrano, A. E. Sutingco and M. T. Tuazon

Keywords: Summary

The nervous system is made up of the brain andspinal cord,

together with the nerves to conduct impulses to and from the
CNS. In this experiment, the Compound Action Potential
(CAP) was determined using the PowerLab. The data
obtained yielded the following values: Threshold voltage=
0.3008mV; Maximum CAP potential= 3.3505; Conduction
velocity= 3.33 m/s.

important structural and functional ways

Introduction
In this experiment, we stimulated
the frog's sciatic nerve, activating a large
number of individual nerve fibres
simultaneously, and record the resulting
aggregate electrical activity. The
objective of this experiment is to
determine the compound action potential
(CAP) of nerves that are not exposed and
exposed to anesthetics. We also studied
the characteristics of the frog's sciatic
nerve CAP, and also to construct a
strength or duration curve of the nerve.
The sciatic nerve is the main
nerve trunk from the spinal cord to the
leg. It consists of a bundle of nerve
fibers, each of which is the axon of a
neuron, whose cell body is near the
spinal cord. Axons are long, cylindrical
processes that project from the cell body
of a neuron and that act as a conduit for
neural messages called action potentials.
Some axons conduct action potentials
toward the brain: they are called afferent
fibers. Others, called efferent fibers,
conduct action potentials away from the
brain. The sciatic nerve contains both
efferent and afferent fibers. Axons
within the sciatic nerve differ in
(Fig. I-1). Myelinated axons are
surrounded by myelin, which looks in
cross-section like tightly-packed,
concentric rings around the perimeter of
the axon. Other axons lack myelin.
Axons that innervate internal organs and
glands tend to be smaller in diameter
than those that innervate skeletal
muscles. Smaller diameter axons tend to
conduct action potentials more slowly
than larger diameter axons. The
thresholds, refractory periods, and the
durations of action potentials differ
across types of axons. Interpretation of
the properties of compound action
potential involves thinking of the sciatic
nerve as a heterogenous population of
nerve fibers.
Fig.I-1: Cross section of the sciatic
nerve of a frog consists of only a single
bundle of fibers, surroundede by the
perinium and loose epineurium
 The origin of the action potential
is based on some amazing properties of
the cell membrane. All cells develop a
membrane potential (Vm). A surface of the nerve. If the recording
microelectrode with a very small tip can
be carefully introduced into the cell
through its membrane. Much research
has been done to ascertain the origin of
the membrane potential (Vm).
Fundamentally, a cell develops a
membrane voltage due to the separation
of charged ions across the cell
membrane. Because of differences in
concentration and the selective
permeability (or conductance) of the
membrane, only certain ions can cross
the barrier. The separation of charge
across the membrane eventually
achieves “electro-chemical” equilibrium
and at this point the resting membrane
potential is established. While all cells
develop a resting membrane potential,
only nerve and muscle cells have the
ability to change it dramatically. If the
Vm changes very quickly in response to
particular stimuli, this property is called
excitability. The response is called an
action potential. As action potentials
propagate along an axon, they produce
electric potentials that can be recorded
from the surface of the nerve. When a
nerve bundle is stimulated, many axons
produce action potentials synchronously.
The resulting electric responses recorded
from the surface of the nerve are called
compound action potentials to
distinguish them from the action
potentials generated by individual axons.
Stimulus electrodes are applied to one
end of the nerve; recording electrodes
are located along the nerve. If the nerve
is stimulated with a current pulse of
sufficient amplitude, the action
potentials produced in the fibers
propagate toward the recording
electrodes.
The aggregate effect of the many
action potentials is an extracellular wave
of negative potential, moving along the
electrodes are widely spaced, the wave
of negative potential produces a negative
pulse in recorded voltage as it passes the
(+) recording electrode. At a later time,
the negative wave of extracellular
potential passes the (-) recording
electrode, where it contributes a positive
pulse to the recorded voltage. If the
electrodes are more closely spaced, the
negative and positive parts of the
recorded voltage merge, and the
resulting waveform is called a diphasic
compound action potential. The
propagation can be blocked by a number
of methods including mechanical
methods (pressure applied to the nerve
or crushing the nerve with forceps),
electrical methods (passing a blocking
level of current through the nerve), or
chemical methods (applying local
anesthetics to the nerve).
Materials and Methods
The PowerLab was utilized to
determine the physiology of the nerve.
The equipment was first setup and
calibrated. For the setup, the red and
black alligator clips from the stimulator
electrodes were connected to two of the
metal rungs on the opposite sides of
MLT012/B Nerve Bath. Next, the red
and black BNC connector from the
stimulator electrode was connected to
the positive and negative analog output
connector on the powerlab, respectively.
The red and black leads from the first
recording electrode were connected to
two of the metal rungs of the MLT012/B
Nerve Bath. Afterwards, the 8-pin pod
connector was connected to the pod port
on input 1 of the PowerLab. For the
second recording electrode, the alligator
clips were paced further Way from the
stimulus electrode; and the pod
connector was attached to pod port on
input 2 on the PowerLab.
For the setup for calibration, the
lower reservoir of the Nerve Bath was
filled with frog Ringer's solution
carefully to avoid contact between the
solution and the metal electrode rungs
which can cause short circuit. A strip of
paper was laid over the wires in the
nerve bath so that it touches both
stimulating electrodes and recording
electrodes. The strip was moistened with
frog Ringer's solution, and the cover was
placed on the nerve bath.
As for the calibration, the
PowerLab was connected to the USB
port in the computer and was switched
on. LabChart was launched and the file
named CAP Set Chart was opened. The
Macro menu was selected and Test
Connection was performed. A series of
stimulus pulses were recorder; if there is
none, check the setup. After the
connections were tested confirmed to be
working properly, the experiment proper
was conducted.
An isolated frog sciatic nerve
was utilized for the experiment. The
nerve was lifted out of the dish by
grasping the thread tied to the nerve
using forceps. The nerve was blotted
with tissue paper to remove excess
Ringer's solution. The filter paper from
the nerve bath was removed and the
nerve was laid across the wire electrodes
making sure it is in contact with each of
the active connections. The cover was
placed on the nerve bath.
The first experiment was to
determine the threshold voltage and
maximal CAP. A series of electrical
stimuli, each with increasing amplitude,
were give to the nerve. The stimulus
range was between 20 to 400 mV, and
increases in 10 mV. The Macro:
Threshold Voltage was selected from the
Chart window to stimulate and record
the nerve for 1.1 seconds. The table was
filled and the data were analyzed using
Waveform Cursor in order to measure
CAP amplitude at each stimulus voltage.
The data were recorded and maximum
CAP altitude was recorded.
The second experiment is the
determination of the refractory period. In
this part of the experiment, the
PowerLab stimulated the nerve with a
series if pulses. The pulse interval
decreased in each block of data. Using
the data from the first experiment, the
minimum stimulus voltage required to
elicit maximal CAP was determined.
Next, Macro: Refractory mV was
selected and one of the four versions of
Refractory macro was chosen, that is, the
voltage that is nearest the intensity
determined beforehand. The chart then
recorded 15 data block with each block
having 10 milliseconds in duration. For
the analysis, the first two CAPS's
recorded in CAP1 were selected in each
block of data recorded in part 2. The
zoom window was opened and the data
trace was examined using Waveform
Cursor. The amplitude for the second
CAP was recorded. The relative
refractory period was determined as the
first decrease in the amplitude of the
second CAP. Lastly, the absolute
refractory period was determined as
complete disappearance of the second
CAP.
The last experiment is the
determination of nerve conduction
velocity. The first step was to measure in
centimeters the distance between the
black leads of each of the two recording
electrodes. The Macro: Conduction
Velocity was selected from the chart
window. A block of data in two channels 150 2.2239 350 2.9247
was recorded in the chart. For the 160 3.2751 360 2.8735
analysis, first, a selection of the first 170 3.3487 370 2.8495
CAP in both channels was made. The 180 3.3503 380 2.7503
time interval for the CAP to travel 190 3.3247 390 2.7759
between the two recording electrodes 200 3.2751 400 2.8495
was determined using the Marker and 210 3.2239 410 2.7759
WaveformCursor. Channel 1 was
selected and the marker was placed on At the start, with low initial
the first peak. Then, channel 2 was stimulus amplitude of 20 mV no
selected and waveform cursor was compound action potential (CAP) is
placed over the second CAP peak. The visible. This is apparent until the 4th
value for the time differential was read stimulus amplitude (50mV). However
and recorded in the data notebook. The when 60 mV was reached a sign of CAP
following formula was used to determine was apparent and the CAP amplitude
the conduction velocity: equal to 0.3008 mV indicates this. The
Conduction Velocity (m/sec) = CAP is seen as a deflection after the
Distance between electrodes(cm)
x stimulus artifact. Figure 1 is showing the
Time interval between CAP′s(ms)
1m 1000ms first sign of CAP which is also denoted
x as the threshold stimulus voltage,
100cm 1sec
which is where the first sign of CAP can
RESULTS AND DISCUSSION be discerned. The threshold stimulus
voltage signifies the voltage needed to
Table 1- CAP amplitude VS stimulus generate at least on action potential from
intensity; threshold stimulus voltage the sciatic nerve fiber. So at 60mV we
highlighted in yellow and maximum can see that the sciatic nerve is capable
CAP amplitude highlighted in orange. of conduction action potentials.
Stimulu Stimulu
CAP CAP
s s
amplitu amplitu
amplitu amplitu
de de
de de
(mV) (mV)
(mV) (mV)
20 0.0000 220 3.2239
30 0.0000 230 3.1759
40 0.0000 240 3.1743
50 0.0000 250 3.0991
60 0.3008 260 3.1503
70 0.9744 270 3.0751
80 1.8235 280 3.1247
90 2.2991 290 2.9503
100 2.6495 300 3.0239
110 2.9007 310 2.9503
120 3.0293 320 3.9503
130 3.1247 330 2.9263
140 3.2735 340 2.9007
Figure 1 - CAP channel graph at Figure 2 - CAP channel graph at
60mV showing the threshold stimulus 180mV showing the maximum CAP
voltage. amplitude.

Finally after increasing the E.2 Determination of refractory

stimulus amplitude to 180mV we can see period
that it is at it’s maximum CAP
potential (Figure 2). Afterwards we can
observe a decline in the magnitude of the
CAP amplitude despite raising its
stimulus amplitude. The maximum CAP
potential, which is at 3.3503mV, is the
point at which a further increase in
stimulus voltage generates further
increase in the CAP amplitude. The size
of the CAP will no longer increase since
all the A-alpha fibers, which make up
the nerve, are excited and are conducting Figure 3 - Refractory period
action potentials.
Once the action potential starts,
voltage-gated sodium channels open and
at the peak of the action potential, these
channels will be inactive. After this
point, the channels are not able to close
right away; hence, a “recovery” period
takes place, the refractory period. An
absolute refractory period is defined as
an interval in which a second action
potential is not produced regardless of
increasing the intensity of stimulus. The conduction velocity can be
During this period, depolarization and easily calculated by knowing both the
repolarization takes place. An influx of distance the action potential travels or
sodium (Na+) ions and positive charge between CAP1 (𝑑1 ) and CAP2 (𝑑2 ) the
inside the membrance will take place amount of time it takes. Velocity has the
during depolarization and otherwise units of distance per time or m/s.
during repolarization, which would
entail increase of K+ ions and negative For the example given in the
charge inside the membrane. On the table below, the given value for the
other hand, relative refractory period is distance between recording electrodes is
defined as an interval in which a second (0.1m) but since the table above is in cm,
action potential may be produced if the 0.1 is converted to 10cm. The time
intensity of stimulus is increased. interval between CAP1 and CAP2 is
Hyperpolarization takes place during this 0.03s but since the table above is in ms,
period, which prepares the membrane for 0.03 is converted to 30ms. Then using
other upcoming electrical signals. the formula:
Voltage-gated sodium channels will be
closed during the course of Conduction Velocity (m/sec) =
hyperpolarization. In addition, if the Distance between electrodes(cm)
x
stimulus is kept constant and the interval Time interval between CAP′s(ms)
1m 1000ms
decreases, the threshold voltage for the x
100cm 1sec
second action potential will be higher
than the first. However, if a stimulus is
constant and the interval increase, then a
second action potential will occur. Distance 10 cm
between
E.3 Calculating the Conduction recording
Velocity electrodes
Time 30 ms
The variation in conduction interval
velocity in the various fibres in the nerve between
is an important factor in determining the CAP1 and
shape of the Compound Action CAP2
Potential, since the conduction velocity Conduction 3.33 m/s
of each fibre determines the latency of Velocity
its contribution to the CAP. Conduction
velocity is systematically related to fibre Substituting the values, the final
diameter in fibres of a given type. Conduction Velocity is 3.33m/s.

In summary, to calculate the conduction

velocity by the Difference Method, we
must take latency measurements using
two different recording positions. The
conduction distance is the distance
between the first recording electrode for
each position, and the conduction time is
the difference between the latencies at
the first recording electrode for each
position.
References:
Characteristics of the Compound Action
Potential. (n.d.). Retrieved February 22,
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http://www.medicine.mcgill.ca/physio/vl
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Goodman, B. P., Harper, C. M., & Boon,
A. J. (2009). Prolonged compound
muscle action potential duration
in critical illness myopathy. Muscle &
Nerve Muscle Nerve, 40(6), 1040-1042.
Remington, L. A., & Remington, L. A.
(2012). Clinical anatomy and physiology
of the visual system. St. Louis, MO:
Elsevier/Butterworth Heinemann.
Retrieved from:
http://www.medicine.mcgill.ca/physio/vl
ab/cap/methods.htm
DeLisa, J. A., & Mackenzie, K. (1982).
Manual of nerve conduction velocity
techniques. New York: Raven Press.
Retrieved from:
http://retina.anatomy.upenn.edu/~rob/lan
ce/conduction_velocity.html
Neuronal Action Potential – Important
features of the neuronal action
potential. (2014). Retrieved from
http://www.physiologyweb.com/l
ecture_notes/neuronal_action_po
tential/neuronal_action_potential
_refractory_periods.html
Compound Action Potential – Refractory
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http://www.medicine.mcgill.ca/p
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