Experiment 2: Physiology of Nerve (Section E)

Laboratory Report
Maria Ronalee Dones, Vergel Jigs Enriquez, Annielle Cyreen Figueroa, Kym Gonzales

Department of Biological Sciences

College of Science, University of Santo Tomas
España, Manila, Philippines

Abstract various specified parts that functions in

The nerve is a specialized cell that receiving and transmitting signals namely
receives and transmits electric signals axons and dendrites. Dendrites transport
throughout the body. Voltage gated channels electrical signals to the cell body whereas
allow the passage an electric signal called an axons bring signals away from the soma.
action potential. In this experiment, the Neurons are classified in two ways; according
threshold voltage, compound action to number of its extensions and based on the
potential, and conduction velocity of the direction of transmission of information.
nerve were determined. Results are as There are three types of neurons according to
follows: threshold voltage of the nerve is the number of extensions. First, is a neuron
50mV while the maximum CAP is that is composed of a short extension divided
3.350mV;the absolute refractory period of the into two branches with one branch
examined nerve is 3.75mses in which the functioning as an axon and the second branch
relative refractory period immediately as a dendrite. This type of neuron is called
follows and lastly, nerve conduction velocity pseudounipolar neurons. Second, is a neuron
is 3.33 m/s. that contains several dendrites radiating from
the soma together with a single long axon.
I. Introduction This neuron is termed as multipolar neurons.
A neuron is a specialized cell that The third and last type of neuron according to
forms the basic unit of the nervous system. It the amount of extensions are the bipolar
is responsible in receiving and transmitting neurons. This neuron has two extensions
messages in a form of electrochemical exhibiting similar lengths. The second
impulses (Morgan & Bloom, 2006). It has classification of neurons are based on the
direction of information transmission. Under
this type are sensory neurons, motor neurons
and interneurons. Sensory neurons receive
signals from sensory organs and transmit
these signals through the axon into the central
nervous system. On the other hand, motor
neurons conduct signals/information from the
cortex into the spinal cord and eventually to
the muscles/muscle cells. Finally,
interneurons connect several neurons in the
central nervous system.
According to Paxinos & Stone (2012),
neurons receive impulse and transmits it to
the following neuron thus indicating that
nerve impulses function as a chain reaction. A
neuron is polarized first before it becomes
stimulated. In the polarization state (resting
membrane potential), the neuron has a
positive electrical charge outside of the
membrane due to the excess of sodium ions
and a negative electrical charge inside the
membrane because of the potassium ions
present. According to Levitan & Kaczmarek
(2015), at depolarization, the Na ions goes
into the cell. After depolarization,
repolarization occurs when the K ions move
departs the cell. Further K ions would move
out of the cell hyperpolarizing the membrane.
After hyperpolarization, the K ions and Na
ions will go back to its original state
exhibiting again the resting membrane
potential.
This sequence demonstrates the action
potential stimulation. Action potentials are
significant electrical signal that allows
communication among neurons used by the
nervous system in relaying information
(Palmer & Stuart, 2006). It is produced by the
opening and closing of voltage-gated sodium
channels and voltage-gated potassium
channels (Caldwell, 2009). Transfer of
information is generated as action potentials
move along axons. Every nerve is composed
of numerous axons that are parallel to one
another. When the stimulation given to a
nerve is large, more axons respond and a
compound action potential is produced. It is
the summation of all the axons responding to
the stimulus but it is limited because of the
restricted number of axons per neuron.
During stimulation, the Na channels are
opened and inactivated. This inactivation of
voltage-gated sodium channels for 1-2
milliseconds prevents the entry of sodium
ions. Inactivation will continue and during
this time, action potentials are not generated
and cannot pass through. This is called the
absolute refractory period. Potassium
channels are responsible in helping the
membrane to go back to its resting membrane
potential. After the absolute refractory period,
Na channels that have recovered from the
previous stimulation, s stronger stimulus is
then applied to generate a second action
potential. This period requires a stronger
stimulus in order to generate an action
potential and is called relative refractory
period. Action potentials travel through
axons quickly in order to produce a quick
response. Axons are covered with myelin
sheaths that increases the rate of conduction
(conduction velocity). The diameter of an
axon is also a factor in determining the
conduction velocity. This exercise aims to
determine the threshold voltage, CAP, and
conduction velocity of a nerve.
II. Methodology
A. Measurement of Compound Action
Potential
Setup and calibration of equipment
In setting up and calibrating the
equipment, red and black alligator clips were
connected (0.5 apart) to the two metal rungs
on opposites of the MLTO12/B Nerve bath.
The red positive and black negative BNC
connector on Power Lab was thenconnected
to the positive and negative analog output
connector, respectively. Afterwards, red and
black lead from the first recording electrode
were connected to two of the metal rungs of
the MLLT012/B Nerve Bath while the 8-pin
pod connector to the pod port on Input 1 of
the Power Lab. Same procedure was done for
the second recording electrode, except the
alligator clips were placed further away from
the stimulus electrode.
Moistened filter paper strip with
Ringer’s solution was put over the wires in
the nerve bath to test its connectivity.
PowerLab was turned on and Lab Chart® (or
Scope 3.0) was launched followed by
Experiments Gallery to open CAP Set Chart.
From the Lab Chart application window,
Macro menu was selected and the Test
Connection button chart. This automatically
recorded the data for 1 second. A series of
stimulus pulses were seen from the monitor
(this indicates that connections are working
and ready to be use). After doing all of this, a
sciatic nerve was carefully isolated, without
grasping it with forceps, was laid across the
wire electrodes, making it sure that it is
connected with the active connections.
A.1. Threshold voltage and maximal CAP
amplitude
In this section nerve were given a
series of electrical stimuli, each with
increasing amplitude. Afterwards, threshold
voltage and voltage required for maximum
CAP amplitude for the nerve were calculated.
From the Chart window, Macro: Threshold
voltage was clicked. This automatically
stimulated the nerve and record it for 1.1 s.
Waveform cursor was used to measure the
CAP amplitude at each stimulus voltage. The
stimulus level were the first CAP was seen
and the maximum CAP amplitude was also
noted.
A.2. Refractory Period
In this part of experiment, PowerLab
stimulates the nerve with series of pulses with
a decreasing interval. In order to do this,
minimum stimulus voltage required to draw a
maximal CAP from the nerve (mV) were
determined. Next, from the LabChart
window, Macro: Refractory _mV was
selected, with the voltage nearest to the
minimum stimulus voltage. With a 15 data
blocks and 10 milliseconds duration, results
were recorded and analyzed based from the
following: Two CAP’s recorded in CAP1 in
each block of data recorded in Part 2 were
selected. Zoom window was opened and data
was examined using Waveform Cursor.
Amplitude for second CAP in table ## and
stimulus intervals was recorded. The relative
and absolute refractory period were
elucidated, by determining the stimulus
interval where the amplitude of the second
CAP first shows a decreased and stimulus
interval where the second CAP completely
disappears, respectively.
A.3. Nerve Conduction Velocity
A ruler was used to determine the distance
between the black negative leads of each the
two recording electrodes. From the Chart
window, Macro: Conduction Velocity was
selected; this records a block of data in two
channels for 10 milliseconds. The data were
then recorded and analyzed by calculating the
conduction velocity.
A selection was made which includes the first
CAP. From the Zoom window, Marker and
Waveform Cursor was used to determine the
time interval for the CAP to trave; between
the two recording electrodes. After that,
Channel 1 and channel 2 was selected and a
marker was placed on the first CAP peak and
the waveform cursor over the second CAP
peak, respectively. Time differential (△t) was
read and recorded. To get the conduction
velocity, measurements for the distance
between the two recording electrodes and the
formula found appendix 1 was used.
III. Results and Discussion
A.1. Determination of threshold voltage
and maximum Compound Action
Potential
The first part of the experiment aims
to determine the threshold voltage and
maximum CAP amplitude. As seen in Figure
1, the CAP amplitude failed to rise above
0mV when the stimulus amplitude is less than
50mV. It was only when 50mV was applied A.2. Determination of refractory period
that the CAP amplitude started to rise. The Table 1. CAP amplitude versus stimulus
CAP amplitude continued to rise as the intensity
stimulus amplitude was increased.
Stimulus Interval Amplitude of Second
The maximum CAP amplitude was (ms) CAP

acquired at 3.350mV when 170mV of 4 -0.050 mV

stimulus was applied. When the maximum
3.5 -0.150 mV
CAP amplitude was acquired, increasing the
stimulus amplitude no longer went over the 3.0 -0.225 mV

maximum CAP amplitude value. 2.5 -0.050 mV

2.0 0.000 mV

1.9 -0.05 mV

1.8 -0.02 mV

1.7 -1.50 mV

1.6 -2.50 mV

1.5 -2.50 mV
Nerves are governed by the all-or nothing law
wherein there is no median action potential. A 1.4 -0.025 mV
stimulus will either activate the nerve or it
1.3 -0.075 mV
will not. As seen in figure 1, the nerve only
1.2 -0.025 mV
began conducting action potentials above
50mV. More and more axons were activated 1.0 -0.200 mV
as the strength of the stimulus increased.
However, at 170 mV, all of the axons within Table 1 shows the results obtained in
the nerve were already activated. Thus CAP the said subsection of the experiment.
did not rise above 3.350mV even if the However, the results obtained failed to
stimulus amplitude was increased. Thus, the produce feasible and ideal data. Errors for
threshold voltage of the nerve is 50mV while this may include mishandling or damage to
the maximum CAP is 3.350mV. the nerve or in the usage of the apparatus.
Hence, to compensate for these results, it was
also done in PhysioEx 9.0.
Table 2. Results from PhysioEx
The threshold voltage
nerveused in PhysioEx 9.0 was set at 20mV.
With an interval of 250msec and 125msec, a
second action potential was
However, when the interval was lowered to
60msec, a second action potential was not
observed. It was only when the stimulus
voltage was raised to 30mV that a second
action potential was observed. When the
interval was further decreased to 30msec, it
needed 45mV stimulus to generate a second
potential. To determine the
refractory period of the nerve, the maximum
amount of stimulus, 60mV, was applied. A
60mV stimulus generated a second potential
when the intervals were lowered to 15msec
and 7.5msec. However, a 60mV stimulus
failed to produce a second potential at a
3.75msec interval.
The inactivation of the Na voltage
gated channels is observable when double
pulses are applied to a nerve. As the interval
is decreased, a stimulus higher than the
threshold voltage is needed to produce a
second action potential. A higher stimulus is
needed since most of the Na channels are still
recovering from the previous depolarization.
The absolute refractory period was
determined by applied the maximum amount
of stimulus voltage at a 3.75msec interval.
The absolute refractory period of the
of the examined nerve is 3.75msec. The nerve needs
more than 3.75msec for the Na channels to
return to a resting potential. The relative
produced. refractory period immediately follows the
absolute refractory period. In the relative
refractory period, a second action potential
can be produced at a higher stimulus.
A.3. Determination of nerve
conduction
Velocity
The conduction velocity can be easily
maximum
calculated by knowing both the distance the
action potential travels or between CAP1
(d_1) and CAP2 (d_2) the amount of time it
takes. Velocity has the units of distance per
time or m/s.
 In Table 3, the given value for the
distance between recording electrodes is
(0.1m) but since the table below is in cm, 0.1
is converted to 10cm. The time interval
between CAP1 and CAP2 is 0.03s but since
the table below is in ms, 0.03 is converted to
30ms. Then using the formula:
Compound Action Potential – Refractory
period.
(n.d.). Retrieved from
http://www.medicine.mcgill.ca/physio
/vlab/CAP/refract.htm
Goodman, B. P., Harper, C. M., & Boon, A. J.
(2009). Prolonged compound muscle
action potential duration in critical

Table 3. Calculation of Conduction Velocity illness myopathy. Muscle & Nerve

Muscle Nerve, 40(6), 1040-1042.

Neuronal Action Potential – Important

features
of the neuronal action potential
IV. Conclusion
(2014). Retrieved from
There are numerous factors to
http://www.physiologyweb.com/lectur
consider when studying nerve conduction.
e_notes/neuronal_action_potential/ne
First, the nerve must be stimulated about the
uronal_action_potential_refractory_pe
threshold stimulus to elicit an action
riods.html
potential. Second, the nerve needs time to
repolarize after stimulation. Third, both of the
former given factors greatly influence the
conduction velocity.

V. References
Characteristics of the Compound Action
Potential. (n.d.). Retrieved February
22, 2016, from
http://www.medicine.mcgill.ca/physio
/vlab/CAP/character.htm