EXPERIMENT 3

NEUROPHYSIOLOGY OF NERVE IMPULSES

(ACTIVITY 6-9)
Y. A. Azucena, J. A. Bacud, S. C. Baquiran, T. J. Bautista 4BIO5
Department of Biological Sciences, College of Science, University of Santo Tomas, Espaa
Avenue, Manila

Keywords: action potential,

conduction velocity, nerve
impulses, neurotransmitter
release

Abstract
The nervous system is composed of neurons that fire up
action potentials. These signals are produced by the
opening of voltage-gated channels on the membrane. The
effect of the intensity of the stimulus voltage on the
frequency of action potentials was tested. The factors
affecting conduction velocity of the signals and the effect of
different ions on the neurotransmitter release were also
tested. It was found that the increasing intensity of the
stimulus caused an increase in frequency of action
potentials. The greater diameter and presence of
myelination contributed to a faster conduction of action
potential and this nerve impulse continue to traverse the
neuron until it reaches a synaptic cleft where calcium ions
aid in releasing neurotransmitters so that the propagation of
the signal can continue to other neurons.

Introduction
The nervous system functions as the
signaling center of a whole organism. It is
composed of neurons that fire up electrical
signals, known as action potentials, to
respond to the stimulus from the
environment. Action potentials are produced
by the depolarization of neurons due to the
opening of voltage-gated channels on the
membrane. Voltage-gated sodium channels
are integral membrane proteins that allow
the passage of sodium ions inside the cell
causing the depolarization. Potassium ions,
on the other hand, are released out of the
cell through voltage-gated potassium
channels in order to reestablish the neuron
to its non-conducting, polarized state. The
fluctuation of the amount of ions inside the

cell affects the membrane potential, which is

measured in millivolts (mV).
The amplitude of an action potential
is constant, regardless of how strong the
stimuli. The conduction or propagation of
an electrical signal through the axon is
considered as an all-or-none event. The
signal is regenerated as it passes along the
axon to ensure undiminished amplitude.
The intensity of the stimulus is defined
through the frequency of action potentials
per second. The time needed to recover
from the previous stimulation is also
affected by stimulus intensity and these are
called refractory periods. The relative
refractory period is the time after the first
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action potential when a second action

potential can already be conducted if there
is an increase of stimulus intensity.
Conversely, an absolute refractory period is
time after the first action potential is
conducted when a second action potential
cannot be conducted even with increased
stimulus intensity.
An action potential traverses through
an axon at different velocities depending on
the size and presence of glial cells or
myelination. Glial cells or neuroglia are nonneuronal cells that support conduction of
electrical signals. Myelination is the
wrapping of certain glial cells around the
axon. The glial cells form myelin sheaths
around the axon that increase the velocity of
the propagation of signals. These sheaths
have gaps that separate them. These are
called
the
nodes
of
Ranvier.
Oligodendrocytes and Schwann cells are
the types of glial cells that wrap around the
central nervous system and peripheral
nervous system respectively.
The axon propagates the signal until
it reaches the axon terminal synapse so it
can relay the signal to another neurons
dendrites then cell body. Sensory neurons
communicate with the motor neuron through
interneurons. An action potential causes
the release of neurotransmitters to the
synaptic gap and binds to receptor proteins
of neurons to produce the appropriate
reaction to a stimulus through a cascade of
molecular events. Axon terminals are
branches at the end of axons. These
terminals are separated by synaptic gaps
where neurotransmitters from intracellular
vesicles are released to a region called the
chemical synapse. Calcium ions in the axon
terminal trigger the exocytosis of the
neurotransmitter-containing
synaptic
vesicles. The neurotransmitters diffuse and
bind to membrane receptor proteins,
causing
a
postsynaptic
potential.
Neurotransmitters can act as paracrine
agents that affect local targets, autocrine
agents that target other neurons, or

endocrine agents that travel long distances

through circulation.
The objective of the simulation is to
determine the effect of stimulus intensity the
frequency of action potentials, effect of
myelination and axon diameter on the
conduction velocity of action potentials, role
of calcium ions in the release of
neurotransmitters, and response of the
functional areas of neurons on varying
stimuli.

Methodology
Activity 6 The Action Potential: Coding
for Stimulus Intensity
An axon was placed in a nerve
chamber. The oscilloscope was used to
observe if the timing of stimuli is appropriate
and changes in the voltage along the axon.
A stimulator was used to set the voltage of
the stimulus and deliver electric signals for
axon depolarization through stimulation
wires (S). Voltage changes in the axon were
recorded using recording electrodes, which
are set 2 cm from the stimulation wires. The
Oscilloscope was set at 100 milliseconds
per division.
Activity 7 The Action Potential:
Conduction Velocity
The oscilloscope, stimulator, and
stimulator wires were also used in the
simulation. Three types of axon were placed
in the nerve chamber in order to test the
differences in their conducting velocity
(m/s). Two recording electrodes were used
(R1 and R2). R1 was still 2 cm from the
stimulation wires while R2 was 2 cm from
R1. The distance between R1 and R2 was
10 cm (0.1 m).

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Results & Discussion

Activity 6

Figure 1.0 From left to right A) A fiber

large-diameter, heavily myelinated axon B)
B fiber medium-diameter, lightly
myelinated axon C) C fiber thin,
unmyelinated axon

Activity
8

Chemical
Synaptic
Transmission
and
Neurotransmitter
Release
In the simulation, an axon terminal
was utilized to test the effect of varying
calcium ion levels and magnesium ion on
the release of neurotransmitters. A
stimulator was used to deliver a low
stimulus intensity or high stimulus intensity
on the axon terminal. The axon terminal
was immersed in solutions that contained
normal calcium ion level, low calcium ion
level, magnesium ion, and a solution that
contained no calcium.
Activity 9 The Action Potential: Putting
It All Together
A large sensory neuron and a large
interneuron were impaled with four small
microelectrode probes. Hook electrodes
were used to record the changes in the
extracellular voltage along the axon. The
oscilloscope was also utilized to observe
voltage changes in the neuron and
interneuron. The stimulator was also used
to deliver low or high stimulus intensity.

A single stimulus for 0.5 milliseconds

at threshold voltage (20 mV) resulted to a
single spike or action potential. When the
duration of the stimulus was set at 500
milliseconds at the same voltage, multiple
action potentials were recorded. The
interspike interval (ISI) was used to
determine the frequency of the action
potential: 1 / ISI (sec). The interspike
interval was calculated by subtracting the
time in milliseconds of an action potential to
a previous action potential. When the
stimulus voltage was set 30 mV for 500
milliseconds, an increase in the frequency
of action potentials were noted. With a
duration of 500 millisceonds and a higher
stimulus voltage (45 mV), the action
potential frequency increased of the axon
increased. An increase in the stimulus
intensity also increases the action potential
frequency. After one action potential was
propagated, the axon has become fully
recovered from its absolute refractory period
and relative refractory period. The stimulus
voltage was still present to produce another
potential.
Activity 7
Conduction velocity was calculated
by dividing the distance the action potential
travels along axon by the amount of time it
takes to traverse the axon. A sensory
Pacinian corpuscle and visceral sensory
fiber were examples of A and B fibers
respectively. Olfactory sensory neurons or
free nerve endings were examples of C
fibers. In the simulation, the conduction
velocity was calculated by dividing the
distance between wires R1 and R2 (0.1 m)
by the time it took for an action potential to
travel from R1 to R2. The stimulus voltage
was set at 30 mV for the three fibers. The
oscilloscope time scale for A, B, and C
fibers were set at 1, 10, and 50 milliseconds
per division respectively. The timescale was
adjusted for B and C fibers because the
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total time displayed would have been too

short for an action potential to be seen at
R2. The results suggest that the amount of
myelination and diameter of the axon or
fiber itself affect the conduction velocity of
action potentials. A greater diameter and
myelination increases the conductance of
an action potential along the fiber. This is
why A fiber has the fastest conduction
velocity while C fiber has the slowest
conduction velocity among the three fibers
used in the simulation.
Activity 8
The number of neurotransmitters
contained within synaptic vesicles released
in the presence of action potential varies
with the amount of calcium present in the
extracellular environment of the axon and
with the intensity of the stimulus. In the
simulation, the axon exposed to the control
calcium yielded to the greatest number of
neurotransmitter released. Two synaptic
vesicles exocytosed from the axon terminal
when stimulated with low intensity while six
synaptic vesicles exocytosed from the axon
terminal when stimulated with high intensity.
The low calcium solution and the solution
with magnesium yielded the same number
of exocytosed synaptic vesicles. One
synaptic vesicle exocytosed at low-intensity
stimulus while three synaptic vesicles
exocytosed at high-intensity stimulus. The
absence of calcium ions in the extracellular
solution did not result in the release of
neurotransmitter from the axon terminal
even with the application of low-intensity
and high-intensity stimulus.
Calcium is a vital element in the
process of neurotransmitter release from
the axon terminal (Byrne, 2016). When the
action potential reaches the axon terminal,
voltage-gated Ca2+ channels open allowing
the rush of Ca2+ into the neuron terminal.
The exocytosis of synaptic vesicles is
calcium
dependent;
therefore,
neurotransmitter release is inhibited when
Ca2+ channels are blocked and when there
is no Ca2+ in the extracellular environment.

Magnesium (Mg2+), a divalent ion, blocks

the calcium channels and inhibits the
release of neurotransmitter when added to
the extracellular fluid by competing with the
calcium ions in attaching to the voltagegated calcium channel (Hardingham,
Bannister, Read, Fox, Hardingham, & Jack,
2006).
Activity 9
The resting membrane potential is
the same value (-70mV) in both the sensory
neuron and the interneuron because the
value of the resting membrane potential
does not depend on the type of neuron. In
the exercise, R1 and R3 are located on the
cell body of a sensory neuron and an
interneuron, respectively. R2 and R4 are on
the axon of a sensory neuron and an
interneuron, respectively. A very weak,
subthreshold stimulus was first applied to
the sensory neuron, which resulted to a
small, depolarizing response at R1, and no
response at R2, R3, and R4. There was no
response at R3 because the very weak
stimulus did not depolarize the axon of the
sensory neuron to threshold. Application of
a moderate intensity stimulus to the sensory
receptor resulted to a larger, depolarizing
response at R1. Graded receptor potentials
occurred at R1 and R3, while action
potentials occurred at R2 and R4. Lastly,
when a strong intensity stimulus was
applied to the sensory receptor, a large
depolarizing response occurred at R1 and
R3, and action potentials were generated at
R2 and R4.
In the simulation, the increasing
intensity of the stimulus generated
increasing action potentials on the axons,
ranging from 0 to 33.3 Hz. Recalling that
action potentials are an all-or-nothing event,
sensory neurons respond to their adequate
sensory stimuli and generate action
potentials in the axon only if the stimulus is
strong enough to reach threshold. The
higher the intensity of the stimulus applied,
the higher the frequency of the action
potentials generated.
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Figure 2.1 A small, depolarizing response at

R1, and no response at R2, R3, and R4
upon application of a very weak,
subthreshold stimulus.

Figure 2.3 A large, depolarizing response at

R1 and R3, and action potentials at R2 and
R4 upon application of a strong stimulus.

Figure 2.2 A larger, depolarizing response

at R1, graded receptor potentials at R1 and
R3, and action potentials at R2 and R4
upon application of a moderate stimulus.

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Table 1.0 Summary of Results for Activity 6

Stimulus
Voltage (mV)
20
20
30
45

Stimulus Duration
(msec)
0.5
500
500
500

ISI (msec)

Action Potential
Frequency (Hz)

10
10
10

100
100
100

Table 2.0 Summary of Results for Activity 7

Axon
Type
A
fiber
B
fiber
C
fiber

Stimulus
Voltage
(mV)

Distance
From R1
to R2
(m)

Heavy

30

Light
None

Myelination

Time Between APs at

R1 and R2
(msec)

(sec)

Conduction
Velocity
(m/sec)

0.1

0.002

50

30

0.1

10

0.01

10

30

0.1

100

0.1

Table 3.0 Summary of Results for Activity 8

Stimulus
Intensity
Low
Intensity
High
Intensity

Number of
synaptic vesicles
exocytosed in
Control Ca2+

Number of
synaptic vesicles
exocytosed in No
Ca2+

Number of
synaptic vesicles
exocytosed in
Low Ca2+

Number of
synaptic vesicles
exocytosed in
Mg2+

Table 4.0 Summary of Results for Activity 9

Sensory Neuron
Stimulus

None
Weak
Moderate
Strong

Membrane
Potential
(mV)
Receptor
-70
-60
-40
-25

AP
Frequency
(Hz) in Axon

Vesicles
Released
from Axon
Terminal

0
16.6
33.3

0
4
6

Interneuron
Membrane
Potential
AP
(mV)
Frequency
Receiving
(Hz) in Axon
End
-70
-70
0
-50
5
-40
10

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Conclusion

References

Based on the recorded data from the

simulations, an increase in the stimulus
intensity also increases the action
potential frequency. A fiber has the
greatest conduction velocity while C
fiber has the slowest conduction
velocity; this suggests that a greater
diameter and myelination increases the
conductance of an action potential along
the fiber. The exocytosis of synaptic
vesicles is dependent upon the
presence of calcium ions; therefore,
neurotransmitter release is inhibited
when Ca2+ channels are blocked and
when there is no Ca2+ in the
extracellular
environment.
Lastly,
sensory neurons were found to respond
to adequate sensory stimuli and
generate action potentials in the axon
only if the stimulus is strong enough to
reach threshold.

Byrne,

J.
(2016).
Chapter
5:
Mechanisms of Neurotransmitter
Release.
Retrieved
from
http://neuroscience.uth.tmc.edu/s
1/chapter05.html

Hardingham, N., Bannister, N., Read, J.,

Fox, K., Hardingham, G., & Jack,
J. (2006). Extracellular Calcium
Regulates Postsynaptic Efficacy
through Group 1 Metabotropic
Glutamate
Receptors.
The
Journal
of
Neuroscience.
Retrieved
from
http://www.jneurosci.org/content/
26/23/6337.full
Neuroglial Cells. (2001). Retrieved from
http://www.ncbi.nlm.nih.gov/book
s/NBK10869/
The nervous system. (2008). Retreived
from
http://lrrpublic.cli.det.nsw.edu.au/l
rrSecure/Sites/LRRView/7700/do
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l

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