Lab Experiment 2: Measuring Action Potentials (nerve impulses) from a Reflex
Response
Objectives:
1. Understand the components of a reflex response
2. Record the time and intensity of a nerve impulse from the patellar
(knee-jerk) and Achilles’ reflex
3. Calculate the speed of an action potential
4. Determine if muscle fatigue is related to a slower action potential
5. Investigate what factors can affect the time or intensity of the
patellar reflex
Introduction
A neuron is a cell that is specialized for the transmission of electrical impulses.
When appropriately stimulated, a neuron propagates a wave of electrical activity (action
potentials) along its axon, which can be communicated to another neuron, muscle cell, or
endocrine cell. Action potentials can originate from several sources, including
voluntary processes (thought, response to physical stimuli) as well as involuntary
processes (monitoring rate of heartbeat, digestion).
As we have discussed, an action potential is caused by a sudden change in the
neuron’s membrane potential. This change is the result of movement of ions across the
membrane, so an action potential can be thought of as a difference in electrical charge
across the membrane. This can be detected experimentally using equipment that is able
to measure a charge difference in the extracellular fluid as a result of a passing action
potential.
In the laboratory, this can be done by a couple of methods:
1. Two electrodes are placed on either side of the membrane of the neuron, one
inside the cell and one outside. As the ions move into and out of the cell a change
in potential difference is recorded between the electrodes. It is from this type of
experiment that the information on ion movement during and action potential has
been obtained. Most of the diagrams of action potentials that are illustrated in
textbooks originate from this type of experiment. This technique is typically
performed on large or isolated neurons.
2. A pair of electrodes is placed on the outside of a neuron. As the action potential
passes along the neuron, the electrodes detect the charge difference. This is a
result of the net difference in potential as the action potential passes first one
electrode and then the other electrode. This method does not measure ion flow
directly, but a localized change in extracellular charge. This method has the
distinct advantage that it can be used to record the passage of an action potential
(as in a muscle) from the surface of the body. We will employ this method in
today’s lab.
In today’s experiment, you will determine the speed at which the action potential travels
along a nerve. Specifically, you will measure the activity of two neurons: one afferent
sensory neuron (that responds to stretch stimuli when a reflex hammer is used) and one
efferent motor neuron (inducing the minor muscle contraction that is the “reflex”).
The activity of these neuronal pathways coordinate what is called a simple reflex
arc. In every muscle of the body, there are fibers that monitor the degree of stretch of that
muscle. Attached to these fibers are sensory neurons that respond to stretch. Thus, when
a tendon is “stretched” by the strike of a reflex hammer, the afferent sensory neurons in
the muscle are activated and send signals to the spinal column, which coordinates two
efferent pathways: one that causes contraction of the muscle involved in the reflex and
one that inhibits contraction of the “opposite” muscle (a process called reciprocal
inhibition)(see Fig 1 below, showing the patellar reflex, reproduced from your textbook).
Note that in this figure it appears as though only one afferent and efferent neuron are
involved, but in reality several of both may be activated by the reflex.
Most reflexes are more complicated, involving several neural connections; for
example, those involved in maintaining posture. In these reflexes, several types of
proprioceptors (sensory neurons that monitor body position) relay information to the
spinal cord or brain stem, which coordinate tonic muscle contractions to allow standing
or sitting without falling over.
To determine the speed of conduction of a nerve impulse, a reflex is initiated by
tapping with the reflex hammer. The time between the tapping of the tendon and the
contraction of the muscle is recorded on the BioPac, and, by measuring the distance that
the action potential has traveled, the speed of conduction can be calculated (in
meters/second). To measure the nerve impulse time, the BioPac instrument does two
things: first, the recording is actually measuring the change in ion concentration (i.e.,
charge) that occurs when the muscle contracts (as we will discuss later, muscle
contraction involves membrane depolarization just like in a neuron). This measurement,
called electromyography (EMG), can be monitored by electrodes placed on the skin.
Since the muscle contraction is very small (only a few muscle fibers) and the reading is
taken from the surface of the skin, significant amplification of the signal is necessary; this
is the second part of the BioPac measurement, where the background “noise” of ion
movement in the ECF must be filtered out. Thus, it is important that during the
measurements you stay as still as possible to avoid background activity.
Nerve impulse velocity will be calculated and compared from both the patellar
reflex and the Achilles reflex, In addition to calculating nerve impulse velocity, you will
perform several experiments to investigate the properties of the reflex arc. These include
measurements of the reflex response during mental and physical distraction, during
competing reflex stimulation, and whether muscle fatigue influences nerve impulse
velocity.
Procedure:
1. Your BioPac workstation should be connected to a reflex hammer (Ch 1) and an
electrode assembly (Ch2). Open the BioPac PRO software. To access the
particular experiment, go to File  Open. When the Open window appears, at
the top drop down menu, enable “graph template”. On the left, highlight
“desktop”, and then choose Lab2_action potentials  open. A new window will
open. You are now ready to perform the experiment.
You will notice two divisions in the window. The top one will record the
hammer strike, and the lower will record the action potential intensity (via muscle
contraction).
Two other helpful points: first, each time you click “start”, recording
begins, and this is noted by the appearance of a diamond shape in the grey area
above the hammer window. This will allow you to keep track of separate
recordings. Second, two features that will help adjust the tracings: to get the
amplitude of the tracings back to the original display, from the display menu,
click “autoscale waveforms”; to get the horizontal portion of the tracings back to
the original display, from the display menu, choose “autoscale horizontal”.
2. Subject #1: attach pre-gelled electrodes for both reflex experiments. Before
applying electrodes, gently abrade the skin with the abrasion pad. This will
remove dead skin cells, allowing better conductance. Use pre-wrap or tape to
wrap secure the electrodes.
a. For the ankle reflex experiment
i. Place two electrodes on the inside of the gastrocnemius (calf)
muscle, approximately 5 in apart
ii. Place a ground electrode just inside the ankle on the same leg
b. For the patellar reflex experiment
i. Place two electrodes on the lower part of the quadriceps muscle,
approximately 5 in apart
ii. Place a ground electrode on the knee of the same leg
NOTE: wait 5 minutes before starting any recordings
3. Ankle jerk reflex experiment:
a. rest the shin on the lab stool, such that the foot is off the end. With the
reflex hammer, the other lab partner should strike the Achilles tendon to
evoke the reflex (foot jerking backwards), noting the place where the
tendon was struck.
b. Hookup the leads: red (+) lead to electrode closest to knee; white (-) lead
to electrode closest to tendon, and black (ground) to ankle electrode
c. Click “start” to begin recording.
d. Strike the Achilles tendon such that the strike results in a measurable
EMG reading. Repeat 9 more times so you have ten total readings. Click
“stop” when finished recording.
e. To determine the time and intensity of each impulse:
i. Use the magnifying glass icon (lower right corner) to highlight
each of the hammer strike recordings. You may need to do this a
couple of times to see a good separation of the hammer and EMG
recordings.
ii. Click the waveform cursor (middle icon, lower right) and highlight
the area from where the hammer strike trace starts to rise to where
the EMG trace begins. Record the Delta T value (shown near the
top of the window, right hand side, just below all the menu icons)
in your lab notebook, and calculate an average.
iii. To measure the intensity (amplitude) of the EMG trace: to the right
of the Delta T box, you’ll see another labeled “none”. Click on it,
and choose “Max” from the dropdown menu. Now, with the
waveform cursor, highlight the most positive portion of the EMG
trace. Record the mV value in your lab notebook, and calculate an
average.
f. Use the supplied tape measure to determine the general length of the
neural pathway: measure from your Achilles tendon to the small of your
back (where the lumbar region of the spinal cord is) and then from there to
the first electrode. Record this distance in your lab notebook. Calculate
the velocity of the nerve impulse in meters/sec.
g. Determine the effect of muscle fatigue on nerve conduction velocity
i. Remove the leads from the electrodes (but keep the electrodes
attached).
ii. Perform foot-raises by repeatedly moving to your tip-toes until you
can feel your calf muscle tiring.
iii. Repeat steps b-f above. Does muscle fatigue impact conduction
velocity and/or impulse intensity?
4. Repeat #2 and #3 above for the second subject.
5. Patellar reflex experiment
a. Subject should sit on lab stool or bench, with leg hanging off. Use the
reflex hammer to strike the knee just below the kneecap (the patellar
tendon) to induce the reflex, where the leg will jerk forward slightly. Note
the place where the hammer strikes.
b. Attach leads: red (+) to middle electrode; white (-) to electrode closest to
waist, and black (ground) to knee electrode
c. Click “start” and record 10 reflexes with clearly distinguishable EMG
tracings. This may take more than ten actual hammer strikes. Click
“stop” when finished recording.
d. Record the time and amplitude of each nerve impulse as you did for the
Achilles reflex tracings. Record the values in your lab notebook and
calculate averages.
e. Calculate the velocity of the impulse (meters/sec) by measuring the
distance from the patellar tendon to the lower back, and back to the first
electrode. Is there any difference in conduction velocity between the
Achilles and patellar reflex?
Note: Due to time constraints, the following experiments should be conducted on just one
person from each group.
6. Patellar reflex with Jendrassik maneuver.
a. The Jendrassik maneuver (named after the person who described it)
involves the effect of physical distraction on a simple reflex arc. To
demonstrate this, the subject measures a patellar reflex while gripping
both hands together across the chest, while using maximum force to
attempt to pull them apart. Develop a hypothesis as to what effect this
action will have on the time of the patellar reflex.
b. Repeat the patellar reflex experiment while performing the Jendrassik
maneuver. Record ten distinguishable EMG tracings, and determine the
time and amplitude for each and calculate an average. Does the time
and/or intensity change during physical distraction?
7. Patellar reflex with mental distraction
a. Make a prediction what, if any, effect mental distraction will have on the
patellar reflex. Prepare ten addition problems with 3-digit numbers (i.e.,
243 + 585).
b. Repeat the patellar reflex experiment, recording ten distinguishable EMG
traces while asking the subject to solve each addition problem in the
shortest time possible (without pen and paper). Determine the time and
amplitude of each EMG tracing and calculate an average. Does mental
distraction influence the time or intensity of the patellar reflex?
8. Voluntary knee jerk reflex
a. In this exercise, you will record the time and amplitude of the knee jerk
under voluntary control. Hypothesize whether there should be any
difference between the time and intensity of the muscle contracting under
voluntary and involuntary control.
b. Have the subject close his/her eyes. Record ten tracings by having the
subject voluntarily jerk his/her knee upon hearing the hammer strike the
table. Record time and amplitudes, and calculate averages. Is there any
difference from the involuntary reflex?