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BACKGROUND ____________________________________________________________________
As you have previously learned in Quantitative Physiology, Biology, and BME Lab, information and
stimuli are transmitted throughout body via electro-chemical signals. The electrical component of these
signals, called action potentials, are the result of ionic fluxes across the cell membrane, that propagates
along the tissue and eventually induces an electro-chemical reaction, e.g., neurotransmitter release that
is used to transfer information along neurons or to stimulate muscular contractions. In this module, you
will induce excitations in neurons and skeletal muscle using a set of extracellular electrodes. You will
also measure the propagation of action potentials along a neuronal bundle.
Adequate Stimulus for Excitation
Action potentials are the electrical signals
that carry control and sensory
information through muscle or nerve.
Action potentials are generated by ions
flowing through the cell membrane into
and out of the cell. The ions flow through
small gaps (called ion channels) in the cell
membrane that open and close based on
the transmembrane electric field strength
which depends on the transmembrane
potential. Ion channels are often specific
to certain ions, Na+ or K+ for example. As
Figure 1 Schematic representation of an action potential
the channels open and close, ions flow in
or out of the cell along the concentration gradient causing local changes in transmembrane potential.
This change in potential propagates along the tissues, causing the action potentials that travel along
neurons or stimulate skeletal muscle (Figure 1).
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Action potentials can be induced through externally applied electrical stimulation. An electrical stimulus
can increase tissues resting membrane potential to the threshold potential, leading to action potential
genesis and propagation. To effectively induce action potential genesis in excitable tissues, a stimulus
must have:
1. An abrupt onset,
2. Adequate duration
3. Sufficient intensity.
Typically a rectangular current pulse is used to excite tissues. An example of a sequence of stimuli is
shown in Figure 2. In this procedure your will apply rectangular electrical pulses of varying durations and
amplitudes to excitable tissues and monitor for contractions in response to the stimuli. You will use this
technique to characterize the electrical excitability of the tissues.
Rheobase current (b) is the minimum intensity required for stimulation with an infinitely
long-duration pulse. Rheobase is largely dependent on the type of electrode and the
location of the electrode relative to the tissue to be stimulated.
Chronaxie (c) is the duration when the current is twice the rheobase (I = 2b). The
relationship between chronaxie and rheobase is tissue dependent. The chronaxie is
commonly used as a safe level to ensure successful stimulation, e.g., tissue is stimulated at
twice the chronaxie duration and at 2b. The chronaxie also corresponds the duration at
which the least amount of energy is necessary to elicit a response; an important parameter
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It has been empirically determined that the strength-duration curve for excitable tissue is of the general
hyperbolic form displayed in Figure 3, where:
= (1 +
(1)
In this expression, I is the peak stimulus current; d is the stimulus duration; b is the rheobase, c is the
chronaxie.
For a rectangular pulse, the charge necessary to elicit an excitation response is equal to the product of
current and pulse duration:
= = ( + )
(2)
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between the intracellular and extracellular space. Using this representation, it becomes possible to
derive an expression for the excitation of excitable tissue. It is only necessary to increase the resting
membrane potential (RMP) of an excitable cell to slightly above a threshold potential (TP) for excitation
to occur, Pearce et al. derived a first-order differential equation for the current required for stimulation
[1]. Figure 4 illustrates the basic concept, which involves division of the current between the membrane
resistance and capacitance. As shown in Figure 5, the pulse of current causes the RMP to increase by an
amount V to the TP. The cell becomes excited and generates its action potential.
= +
(3)
where:
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A solution to the differential equation provides Eqn. (4), quantifying the amplitude of the threshold
current required at a particular duration, d.
=
(4)
where is the membrane-specific time constant ( = .) Notice that for long durations, that is d ,
the amplitude approaches the rheobase, aligning with the previous definition of rheobasic current.
When performing stimulation experiments, it is unlikely that membrane capacitance and resistance are
known, thus time constant is determined to characterize the tissues of interest. Figure 6 is a logarithmic
plot of this current strength-duration curve for different values of . The important message in Figure 6
is that the rate of stimulus intensity increase for stimulation with decreasing duration, i.e., the slope of
the curve prior to the rheobase, is the same. This means that the shapes of the strength-duration curves
are essentially the same for all tissues. The differences in the curves for the different tissues relate to
the membrane time constant, which manifests itself by the duration at which the current starts to
increase above rheobase (b) as the stimulus duration is shortened. The significance of this fact is that,
for a given tissue, a constant charge must be delivered to achieve excitation; current and duration are
reciprocally related in delivering this charge. Typical values for membrane time constants are 2 ms for
cardiac muscle, 500 s for sensory receptors and about 10 s for motor nerves. Using the exponential
expression for I, the current required to stimulate tissue with a rectangular pulse is Qstim:
= =
(5)
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Figure 6. A log-log plot of the exponential form of the strength-duration curve for various .
Figure 7. Analytically derived strength-duration curves for current, charge, and energy.
Figure 7 shows the charge-duration curve for normalized duration in terms of d/. Comparison of the
empirically-derived curves (Figure 3) and the analytically-derived expressions (Figure 7) reveals several
striking similarities. The quantity bc, in the empirical expression is the zero-duration charge (Eqn (1)). It
is possible to determine the significance of bc by equating the empirical (Eqn. (1)) and analytical (Eqn. (4)
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expressions for stimulus current and solving the resultant expression for an infinitesimally short
duration stimulus:
= +
(6)
2
3
+ 2 2 +
2! 3!
=1
(7)
Therefore, if the chronaxie of an excitable tissue is (empirically) determined, then we can also determine
the time constant, which can subsequently be used to predict what stimuli are appropriate for eliciting a
muscle contraction. Note that the chronaxie (time constant) can also be determined from (a) the lowest
energy value that results in action potential genesis or (b) the point of intersection of the normalized
current and charge strength-duration curves (Figure 3).
In summary, determination of an excitable tissues strength-duration behavior is critical for ensuring
that electrical stimuli are administered with adequate duration and intensity. In this procedure, we will
empirically determine the current strength duration curve for frogs sciatic nerve and for skeletal
muscle. The resultant strength duration curves will also be used to identify important tissue parameters
such as the rheobase and chronaxie.
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6. Connect the negative (-) terminal to a small resistance (10 100 ). This test resistance (Rtest) will be
used to confirm there is current flow in your circuit and to accurately quantify the stimulation
current using Ohms law. Connect the other end of Rtest to the stimulating pin closer to the frogs
foot.
OSC
(Ch. 1)
Rtest
OSC
(Ch. 2)
Figure 9 Block diagram of connections for stimulation. The position of the stimulating electrodes will change in
subsequent experiments
7. Connect the outputs of the stimulator to Channel 1 of the oscilloscope. The stimulator behaves as a
constant current source: It will produce enough voltage to result in the desired current across a
load. The measurement of the stimulus output will display the voltage necessary to achieve the
prescribed stimulus (vstim).
8. Connect another channel from the oscilloscope across Rtest. Make sure that the ground (black) lead
from the probe is connected to the side of the resistor going into the black input on the stimulator.
How will you use the measurement of vtest to determine istim?
9. Set the oscilloscope triggering to Normal mode, and make a rising edge on Channel 1 the trigger.
Set the trigger level to just above the baseline. This will cause the oscilloscope to perform a fixed
acquisition each time the leading edge of the pulse is detected.
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10. With the stimulator TURNED OFF, program the stimulator as follows (see Figure 10):
a. Stim Mode: EXT SYNC (a pulse of pre-defined duration will execute only when the TEST
button is pressed)
b. AUDIO: ON a tone will play whenever the stimulator output is on
c. WIDTH: 300 ms sets the duration of the rectangular pulse
d. CURRENT: 0 mA sets the current intensity of the rectangular pulse
11. Gradually increase the intensity the intensity of the stimulus and examine the muscle for a twitch.
Document vc (to determine istim) and vstim.
12. Turn the stimulator off and decrease the duration to 200 ms. Reduce the intensity to 0, then
gradually increase the current intensity until a twitch is detected.
13. Repeat Step 12 for shorter durations until a twitch cannot be achieved with the maximum intensity
stimulation. Strive to obtain 8 10 durations and document the threshold vstim and istim at each
duration. Hint: You want to have a higher resolution of measurement at the knee of the strengthduration characteristic, i.e., the range of values where the intensities require to elicit stimuli increase
from the rheobase.
14. Use the threshold intensities and durations to plot strength-duration curves for (a) current and (b)
charge, Q. Comment on these curves compared to the analytical predictions (Figure 7). Bonus: Use
your measurements to derive the energy (U) strength-duration curve.
15. Using the strength-duration curve(s) determine the rheobase and chronaxie for stimulation at the
gastrocnemius. Describe which curve you used (empirical or analytical model) and how you
calculated the parameters. Hint: Qualitative identification of the current strength-duration curve
horizontal asymptote is insufficient.
Procedure 2: Strength Duration Curve - Indirect Neuronal Stimulation
Muscles typically fire via action potentials that are transmitted from motor neurons. In this portion of
the procedure, the gastrocnemius muscle will be stimulated to contract via indirect stimulation of the
sciatic nerve in the frog quadriceps region.
1. BE SURE THAT THE STIMULATOR IS TURNED OFF
2. Disconnect the stimulating electrodes from the stimulator and remove the pins from the
gastrocnemius
3. On the opposite limb as the one used in the previous experiment position the bipolar electrode so
that it is in firm contact with the frogs thigh. It may be necessary to tie the electrode in place to
secure it.
4. Connect the bipolar electrode such that the red (+) terminal as nearer to the frogs head and the
black (-) at the electrode closer to the hindfoot
5. Repeat Steps 11 16 from the Strength Duration Curve: Direct Muscular Stimulation procedure.
6. The data from each group will be pooled and provide to you. Compare the rheobase and chronaxie
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values from the indirect motor nerve to the direct motor nerve.
Procedure 3: Strength Duration Curve - Direct Neuronal
Stimulation
In this and the next part of the procedure, muscle contractions
will be elicited through direct stimulation of the nerves that
control gastrocnemius. You will place electrodes proximate to
the spinal cord and sciatic nerve to induce the gastrocnemius
contraction
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(b)
(a)
Figure 13 Images of (a) the intact quadriceps region where the sciatic nerve and artery can be visualized and (b)
blunt dissection to expose the nerve
2. Expose the sciatic nerve in the thigh area of the skinned hindlimib. This can be accomplished by
blunt dissection of the surrounding musculature. The nerve will appear as a strip of white tissue
near the dark artery (see Figure 13).
3. Gently lift the nerve place small piece of sliced soft rubber/plastic tubing (sleeves). Be careful not to
damage the artery or the small vessels supplying the nerve.
4. Re-position the stimulating electrodes to the sleeve at the sciatic nerve.
5. Obtain a strength duration curve for direct simulation of the sciatic nerve
6. Plot the requisite strength duration curves and determine the chronaxie and rheobase for spinal
stimulation
Procedure 4: Effects of Nerve Block on Action Potential Propagation
1. Ensure that the stimulating electrode is positioned on the sciatic nerve at the level of the urostyle.
2. Slip a loose ligature on the nerve at a point inferior to the stimulating electrode. Be sure that the
ligature is long enough that its tightness may be adjusted without you having to make physical
contact with frog.
3. Apply stimuli to the nerve at a 1 Hz repetition rate and adjust the current intensity until twitches are
evident in the gastrocnemius.
4. Gradually tighten ligature. DO NOT MAKE CONTACT WITH THE FROG DURING TIGHTENING. Observe
the gastrocnemius twitches and note what occurs as the ligature tightens. Explain the effects of
tightening the ligature on AP propagation. Express your answer in terms of the cable model that you
learned in QPII. Hint: Consider the nerve as if it is a wire with a resistance that is dependent on the
tissues resistivity, length, and cross-sectional area.
Bonus: Determine Nerve Propagation Velocity
Nerve propagation, or conduction, velocity is an important parameter in the characterization of nervous
tissues (Remember all of the wonderful nerve characterization performed in Module II!) The
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propagation velocity can provide insight into the neuron type, firing rates, size, and relative health of
neurons within a nerve bundle. In this part of the procedure you will implement an experiment of your
own design to measure the NPV of the frog nerve. Hint: You essentially need to determine the time it
takes from the AP to travel from one location to another. Be sure to detail the procedure that you utilize
in your lab report. Here are a few procedural suggestions and reporting instructions:
You are welcome to conduct this procedure in coordination with the other direct nerve stimulation
experiments. Compare the NPV values from the frog to that of a human spinal cord.
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