Introduction:

Our tasks where divided into four parts. These were:

2
-Determine the value of threshold stimulus (+-0,1A/ cm ), at the stimulus
duration of 0,1 ms.
2
-Calculate 2/3 of the determined threshold stimulus (+-0,1A/ cm ).
-After the subthreshold stimulus acts upon the cell membrane, determine the
value of threshold value in 4 time intervals; 1 ms, 2 ms, 3 ms and 4 ms.
-Interpret found data.

In order to solve these tasks, we have to know the definitions of threshold

stimulus and sub threshold stimulus; the threshold stimulus is the minimal
stimulus which initiates an action potential, while the sub threshold stimulus is a
stimulus which is not strong enough to either be perceived or to trigger an action
potential.

Throughout the experiment, we used a software, Neuron, which is a computer

model of neurons, which simulates the reaction of real-life cells under several
different conditions, for instance when stimulus is applied to its membrane.

Methods:
Relevant to our experiment, was using of current clamp method in the Neuron
program, as the voltage clamp measures the ion current across cell membranes,
while current clamp measure electrical current, or as dr. Zlumanczuk said, the
current clamp method is about controlling stimulus and observing change of
membrane potential. The stimulus is applied by a stimulus electrode to the cell
body, recording from the axon. The other method is voltage clamp; where
membrane potential is controlled by holding it at a given level, observing the
current which flows across the cell membrane. Certain currents can be expected
at different membrane potentials.

A stimulus had to be applied, through a current clamp, to a cell which had a

membrane potential of -60 mV. As mentioned, the threshold value is the minimal
stimulus which triggers an action potential, so our assignment was to search for
the unknown threshold stimulus. This was done by plotting in different values of
stimulus applied to the cell membrane, until an action potential was seen in the
displayed graph.
The membrane potential is free to vary, and the amplifier records whatever
voltage the cell generates on its own, or as a result of stimulation. This technique
is used to study how a cell responds when electric current enters a cell; this is
important for instance to understand how neurons respond
to neurotransmitters which react by opening ion channels in the cell membrane.

After having found the threshold stimulus value, we determined the sub threshold
value, 2/3 multiplied by the threshold stimulus. The next part of our assignment
was to determine the threshold stimulus value, after the sub threshold stimulus
was applied to the cell membrane, in 4 time intervals. The threshold stimulus in
this case, is a second stimulus. In the FIRST STIMULUS part in the computer
program, we plotted in the sub threshold value, duration of stimulus of which was
0,1 ms. In the SECOND STIMULUS part, the duration of applied stimulus
remained the same, but the start time would be 1, 2, 3 or 4 ms. Also the applied
stimulus would vary, as we had to search until the correct value could be seen as
an action potential curve in the graph.

FIRST STIMULUS:

Sub-threshold Amplitude
value
Starting time

0.1 Duration

SECOND STIMULUS

Varies Amplitude
depending on
the given
time
Time interval Starting time

0.1 ms Duration

After determining the threshold stimuli of each time interval, we wanted to find
the amount of time it would take for the threshold value of the cell with two
stimuli applied, to become the same as the threshold value which was found in
the cell with only one stimulus applied to its membrane. This was done by writing
the first threshold value in the SECOND STIMULUS part, and varying the
starting time until the graph would display an action potential:

FIRST STIMULUS

Sub-threshold Amplitude
stimulus
Starting time

0.1ms Duration

SECOND STIMULUS

Threshold Amplitude
stimulus
Varied until Starting time
graph would
indicate an
action
potential
0.1 ms Duration

Result:

The value of threshold stimulus at the stimulus duration of 0,1ms was 63,15 A/
cm 2 .

The sub threshold stimulus which was 2/3 of the threshold stimulus was 42,1 A/
cm 2 .

After the sub threshold stimulus acted upon the cell membrane, the threshold
value in 4 time intervals were as follow:

FIRST STIMULUS:

42,1 A/ Amplitude
cm 2
Starting time

0.1ms Duration

SECOND STIMULUS

Varies Amplitude
depending on
the given
time
1/2/3/4ms Starting time

0.1 ms Duration

These are the results according to the given time intervals:

Time: Threshold value:
1 ms 2
30,22 A/ cm
2 ms 2
47,83 A/ cm
3 ms 2
68,07 A/ cm
4 ms 2
82,91 A/ cm

After determining the threshold stimuli of each time interval, we wanted to find
the amount of time it would take for the threshold value of the cell with two
stimuli applied, to become the same as the threshold value which was found in
the cell with only one stimulus applied to its membrane. This was done by writing
2
the first threshold value, 63,15 A/ cm , in the SECOND STIMULUS part, and
varying the starting time until the graph would display an action potential. These
are the results:

FIRST STIMULUS

42,1 A/ Amplitude
2
cm
Starting time

0.1ms Duration

SECOND STIMULUS

63,15 A/ Amplitude
cm 2
27,70ms Starting time

0.1 ms Duration
Time: Threshold value:
27,70 ms 2
63,15 A/ cm

Discussion:
Threshold stimulus is as mentioned above the minimal stimulus that trigger an
action potential. The membrane potential increases. When the channels open,
they allow an influx of sodium ions, which changes the electrochemical gradient,
which in turn produces a further rise in the membrane potential. So the lesser
negative it is inside the cell, the lower threshold value will be necessary in order
to trigger an action potential.

In the first task we found the threshold stimulus on a cell with the membrane
2
potential of -60mV. The result was 63,15 A/ cm . After this we determined the
2
sub threshold value, 42,1 A/ cm , which is a stimulus too low to trigger an
action potential.

What would happen if the cell was less negative; for instance a cell with a
membrane potential of -50 mV?
2
Our hypothesis is that the threshold value would be less than 63,15 A/ cm .
This would be cause by the less negative membrane potential, which would use
less stimuli for triggering of an action potential.

On the third part of the assignment, the graph shows that the threshold stimulus
is higher the longer the time interval is during a limited time. What is seen then is
that the threshold stimulus will decrease after a certain time. We know that for a
short time after an action potential occurs, it is impossible to evoke a second
action potential. This period is referred to as the absolute refractory period.
This period is followed by the relative refractory period, in which an action
potential can be evoked, but only by a larger stimulus than was required to evoke
the first action potential. Relative refractory period occurs during the
hyperpolarization. During the hyperpolarization the potassium channels are still
open due to the slowly closure of their gates. The cell will then be more negative
than it was during its resting state. We know that the more negative the
environment is inside the cell, the higher the threshold stimulus will be.
2
We firstly applied a stimulus of 42,1 A/ cm , which is the sub threshold
stimulus. Therefore it will not trigger an action potential. In order to induce an
action potential the second stimulus is applied after 1ms, and the threshold value
2
is 30,22 A/ cm , after which the threshold value will increase in a limiting time,
2
before it will start decrease and return to 15 A/ cm . The increasing of
threshold value can be seen in the table above.
From the information obtained we can conclude with the fact that the threshold
stimulus is acting on the cell during the relative refractory period.

Why does the threshold value increase as time increases and then return to
2
63,15 A/ cm ?

The hyperpolarization curve becomes more negative than in the resting state,
after which it returns to the resting state. The more out flux of potassium, the
more negative the interior of the cell is, and the more stimuli is required to
induce an action potential. While the potassium channels close slowly, less
potassium ions will be inside the cell, thereby the inside of the cell will be less
negative, hence less stimulus will be required for an initiation of an action
potential.

We calculated that it takes 27,70ms for the threshold stimulus to become the
same as the stimulus needed for triggering an action potential when the cell was
2
applied with only one stimulus at its resting state (63,15 A/ cm ).

2 2
What happens if the stimulus is increased from 63,15 A/ cm to 100 A/ cm ?
2
Since 100 A/ cm is above the threshold stimulus, it would cause the action
potential to be generated in less than 27,70ms, or in other words, before the end
of the relative refractory period. The increased stimulus will increase the rate of
conduction above 27,70ms.

When two stimuli where applied to the cell membrane, what would happen if the
first stimulus acting upon the cell was changes to a lower or a higher value?
Since we now know that the first stimulus affects the second stimulus, we know
that decreasing the value of the first stimulus will decrease the threshold value of
the second stimulus, and vice versa.