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internal organs and glands tend to be smaller in diameter ( m) than those that innervate
skeletal muscles ( m). Smaller diameter axons tend to conduct action potentials more slowly
than larger diameter axons (Figure 2). The thresholds, refractory periods, and the durations of
action potentials differ across types of axons. Interpretation of the properties of compound action
potential involves thinking of the sciatic nerve as a heterogenous population of nerve fibers.
2
Figure 1: Cross section of the sciatic nerve of a rabbit (Young,
1951). Each of these outlines is the darkly-stained layer of
myelin that surrounds a single myelinated nerve fiber. Fiber
diameters vary. Unmyelinated fibers, which are much smaller
than myelinated fibers, are not visible with the histological
method used to prepare the tissue for this picture.
40
Conduction velocity (m/s)
bullfrog
30
Figure 2: Conduction velocity versus fiber diameter for individ-
ual myelinated nerve fibers in the bullfrog sciatic nerve (Tasaki,
20
1953, adapted from Figure 65). The slope of the line indicates
that the best fitting constant of proportionality is 2 m/s per m.
10
The fiber diameter was measured near the insertion of the nerve
into the gastrocnemius muscle. Temperature was C.
0
0 5 10 15 20
Diameter (µm)
3
Widely spaced recording electrodes Closely spaced recording electrodes Closely spaced with neural block
$+ !v(t) −
$+ !v(t) −
$+ !v(t) −
" " "
i(t) i(t) i(t)
" " "
i(t) i(t) i(t)
#t #t #t
wave of negative potential
reaches "−" electrode
!v(t) !v(t) !v(t)
#t #t #t
wave of negative potential
reaches "+" electrode diphasic waveform monophasic waveform
Figure 3: Three schemes for measuring compound action potentials. In each scheme, a current %&('*) is
applied to two stimulus electrodes, and a voltage response +&('*) is measured across two recording electrodes
(upper panels). The response to a pulse of current consists of two, temporally separated components, if the
recording electrodes are widely spaced (left panels). A diphasic waveform results for more closely spaced
recording electrodes (center panels). A monophasic waveform results if a portion of the nerve between
the recording electrodes (shown as a dark band) is altered to block transmission of action potentials (right
panels).
the compound action potential is blocked between the two recording electrodes (right panel) so that
it does not reach the recording electrode, a monophasic compound action potential is recorded.
The monophasic compound action potential consists of one or more peaks of (negative) po-
tential that last on the order of a millisecond.1 The maximum amplitude varies with the recording
conditions but rarely exceeds a few millivolts. Multiple peaks might represent repeated action po-
tentials produced by the same fibers or alternatively, sub-populations of fibers that differ in their
propagation velocities and thus produce components with different delays.
The shape of the compound action potential depends on the population of excited fibers within
the nerve. If all of the fibers in the nerve have similar diameters, then the propagation velocities
for action potentials will be similar. The resulting monophasic compound action potential will
be brief. If the nerve contains a heterogeneous mix of fibers with different diameters (as in the
bullfrog sciatic nerve), then the monophasic compound action potential will become broader as it
propagates. If the distance between the stimulating and recording electrodes is sufficiently large,
the monophasic compound action potential may consist of several peaks — each corresponding to
a different subpopulation of fibers that conducts action potentials at a different velocity. Studying
the electrical properties of the different peaks has led to insights about the thresholds, refractory
periods, and velocities of propagation of action potentials in different subpopulations of fibers
(Patton, 1960; Ochs, 1965; Ruch et al., 1965).
1
The convention of plotting negative monophasic action potentials as upward deflections (which is not used in this
laboratory manual) is commonly used in physiological publications.
4
1.4 Protocol is Important
The written record of your experiment is the original record of your work and the basis for every-
thing that you conclude. Such records are often called protocol, and are usually kept in a special
notebook with sewn binding (not a loose-leaf notebook), whose pages were numbered when the
notebook was printed. Records are written in this protocol book in ink. The protocol book is the
permanent record of your experiment.
Protocol should be sufficiently detailed so that (1) the experiment could be repeated at some
later time, (2) results from different experiments can be compared, and (3) so that your procedures
can be reconstructed at a later time without relying on your memory. The first page for each
experiment should give the date and time, as well as a brief description of the purpose of the
experiment. All relevant procedures and observations should be entered in the protocol book and
the time should be indicated regularly.
Nothing should ever be erased. If an error is detected in some procedure or some reading, that
should be noted in the book. The original observation should under no circumstances be obliterated
so that it cannot be read. Perhaps you will subsequently find that the original observation was
correct and that the correction was in error. The general rule is to write down everything that is
done that may later become relevant. Scientists are rarely sorry that they wrote too much in the
protocol book — only that they wrote too little.
You do not have to purchase a special protocol book for this laboratory session. However,
we do ask that you take protocol during your experiment, as a step toward learning about effective
practices in experimental work. You will be asked to attach your protocol to your laboratory report,
and we will assess the protocol as part of the grading procedure.
Animal experiments are to be undertaken only with the purpose of advancing knowl-
edge. Consideration should be given to the appropriateness of experimental proce-
dures, species of animals used, and number of animals required.
Only animals that are lawfully acquired shall be used in this laboratory, and their
retention and use shall be in every case in compliance with federal, state, and local
laws and regulations, and in accordance with the NIH guide.
Animals in the laboratory must receive every consideration for their comfort; they
must be properly housed, fed, and their surroundings kept in sanitary condition.
Appropriate anesthetics must be used to eliminate sensibility to pain during all surgical
procedures.
5
2 Methods
This section describes the experimental methods. Additional information, including photographs
of the surgical procedures, is available at http://umech.mit.edu/6.021J/index.html.
A video-taped demonstration of the surgery will be shown during the first 20 minutes of your lab-
oratory session. Arrive on time so you can start the procedures immediately after viewing the the
tape.
This is a physiology experiment and experimental animals can carry disease. You may not eat
or drink in the laboratory, and you must wear rubber gloves when you handle the animals.
2.1 Anesthesia
A bullfrog (Rana catesbeiana) is anesthetized by immersing it for about 20 minutes in tap water
that contains MS222 (Ethyl 3-aminobenzoate, methanesulfonic acid), an anesthetic commonly
used for aquatic animals. The concentration of anesthetic is 0.1 % by weight. The frog should
become limp.
2.2 Solutions
Throughout the dissection and the course of the experiment, the frog sciatic nerve should be kept
moist in Ringer’s solution (Table 1). Ringer’s solution approximates the ionic composition of
the extracellular fluids of the frog, and a sciatic nerve bathed in Ringer’s solution can remain
electrically responsive for hours. KRinger’s and Sucrose solutions are also available for students
interested in investigating the effects of changes in ion concentration on electrical responses.
6
1 Never close the jaws of the scissors without visual confirmation that the nerve will not be
cut.
1 Keep the nerve wet with Ringer’s solution (not with blood) at all times during the dissection.
1 Avoid picking up the nerve with forceps: handling should always be done by gently lifting
the nerve on a blunt instrument or by an attached thread. Stretching can destroy the action
potential generating mechanism in the nerve fibers.
Detailed description. The sciatic nerve runs down the leg between the large thigh muscles (Fig-
ure 4), i.e., between the semimembranosus and both the vastus externus and internus muscles. It
branches several times and the peroneal branch is found deep between the two large calf muscles,
i.e., the peroneus and gastrocnemius muscles.
Vastus externus
muscle
Vastus internus
muscle
3
Semimembranosus
muscle
2
Gastocnemius
muscle
Peroneus muscle
Figure 4: Dorsal (back) view of frog musculature (Carolina Biological Supply Company, 1965, adapted
from Review Sheet 55).
1. The frog is the source of your experimental material. Weigh the frog and measure its length
from snout to vent. Record these as part of your record of assembling an experimental
system.
7
2. Decapitate the frog with scissors. With a pithing needle, destroy (i.e., mosh up) the brain and
the spinal cord. The frog’s muscles will twitch during these manipulations. Pithing greatly
reduces subsequent muscle contractions, thus simplifying the dissection.
3. Place the frog on its back in the dissecting tray. Grasp the thin wall of skin and muscle
that overlie the viscera of the lower abdomen with forceps and pull it up and away from
the viscera. Using dissecting scissors, make a horizontal incision in the thin wall, and cut
through the wall — first to the left and up the left side of the abdomen, then to the right and
up the right side of the abdomen. Lift the thin wall of skin and muscle up and cut it off to
expose the viscera.
4. Slide the viscera up to uncover the spinal column and emerging sciatic nerve. The nerve is
white and cylindrical and should be distinguished from tendons and the surface connective
tissue (fascia) of muscles, both of which are white but are usually flatter and shinier than
nerves. Cut off the portion of the frog above the spinal cord entrance of the sciatic nerve,
including the viscera.
5. Grab the spinal column with a pair of forceps and with another pair of forceps grab a fold of
skin on the frog and pull down to remove the skin from the legs. If this proves difficult, grab
the muscles on each side of the spinal column with a pair of forceps and with a third pair of
forceps grab a fold of skin on the frog and pull down to remove the skin.
6. Free the sciatic nerve from surrounding tissue by cutting the surrounding tissue parallel to the
nerve being careful not to cut the nerve. After the portion of the sciatic nerve in the abdomen
has been freed of other tissue, slide small curved forceps underneath the sciatic nerve near its
insertion into the spinal cord, attach surgical thread to forceps and pull through. Tie off one
of the sciatic nerves at the backbone where it leaves the spinal cord, leaving about 2 inches
of thread attached for manipulating the nerve. Cut the nerve between the knot of thread and
the spinal cord.
7. Free the portion of the nerve in the abdomen from all surrounding tissue from the part at-
tached to the thread down to the hip joint. Try to minimize bleeding when you dissect the
nerve. Often the smaller blood vessels can be crushed with a hemostat and then safely cut
without causing much bleeding. If you inadvertently cut a large vessel, clamp it shut with
a hemostat and wash away the blood with frog Ringer’s solution. Be sure to keep the nerve
wet at all times.
8. When the nerve is free, poke a hole through the frog’s back by spreading the musculature
apart and pull the thread gently through from the ventral (abdominal) to the dorsal (back)
side. Do not tug on the nerve.
9. Dissecting the nerve at the hip is tricky. Switch to the dorsal side (flip the frog over) and
expose the nerve in the thigh where it lies deep between the large thigh muscles. To do this,
pull apart the two large groups of thigh muscles and pin them down so that their inner sides
are exposed. Cut through the overlying membranes and uncover the nerve and the nearby
blood vessel. Dissect the nerve free near the hip joint. Once the nerve is visualized on both
sides of the hip joint dissect through the hip joint. Take your time here and cut parallel to
the nerve and detach the tissue from the nerve. Always make certain that the nerve is not
between the blades of the scissors when you cut. You will notice that the sciatic nerve has
many branches throughout its course. These may be cut at any time, although for best results
8
cut them when the entire nerve is free of surrounding tissues. Cutting a branch of the nerve
may cause muscle contractions.
10. Midway down the thigh the sciatic nerve branches in two. You want the branch that goes
above the knee and down to the foot (the peroneal nerve). Separate and pin the muscles
of the calf apart. Follow the nerve from the thigh to the knee. The dissection of the nerve
through the knee is similar to its dissection through the hip. Before freeing the nerve at the
knee, dissect the nerve in the calf. When you are sure you can visualize the nerve, cut the
tendon which covers the nerve at the knee. Hemostat and cut any small blood vessels that
cross over the nerve.
11. Free the nerve down the length of the calf to the ankle. Tie off the nerve at the ankle, leaving
about 2 inches of thread for mounting. Cut the nerve just beyond where it is tied, and cut
away any remaining branches.
12. Measure the length of the nerve without stretching it.
Stimulating electrodes
S3
Plexiglass cover
R1
S2
R2
R3
R4
R5
R6
R7
R8
R9
R10
R11
R12
Recording electrodes
Figure 5: The nerve chamber. There are 12 recording electrodes spaced 1 cm apart. There are 3 stimulating
electrodes spaced 0.5 cm apart.
holes in the ends of the chamber and extend (do not stretch) the nerve to its original length. Then
secure it in place with tape attached to the thread and chamber. A small drop of Ringer’s solution
should be laid at each electrode-nerve crossing to ensure electrical connection. Cover the bottom
of the chamber with Ringer’s solution. Be careful not to short-circuit the electrodes; do not allow
the Ringer’s solution to reach the level of the electrodes in the chamber. Squirt a few drops of
Ringer’s solution on the rim of the chamber and cover with a Plexiglass slab to provide an airtight
seal. This keeps the atmosphere in the chamber saturated with water and prevents the nerve from
9
drying out. The nerve will be surrounded by moist air which acts as an electric insulator. The
three closely-spaced electrodes are normally used for electrically stimulating the nerve, and the
remaining electrodes are normally used for recording.
3 + 3 +
2 Signal Stimulus
Generator − Isolator − 6Two channel
Oscilloscope
Nerve
$+ 5
4
Differential Computer
− Amplifier
Trigger
of 100 A/V.
The output of the stimulus isolator drives the stimulating electrodes of the nerve chamber (Fig-
ures 5 and 6). The center electrode is the cathode. The two outside electrodes should be connected
together to serve as anodes. This stimulation configuration reduces the threshold current required
to stimulate the nerve and reduces the spread of the stimulus current along the nerve.
A technical problem with electric stimulation is that a portion of the stimulus current produces
a voltage input to the recording amplifier which is called a stimulus artifact which can obscure the
nerve response. The circuit topology shown in Figure 6 is intended to reduce the stimulus artifact.
However, the size of the stimulus artifact is also affected by the geometrical arrangement of the
stimulus and recording electrodes. The lengths of unshielded wires should generally be kept as
small as possible.
10
3 Repeat
S1 duration
3
S2 duration
3
S1
7volts 3
S2
7volts
3
S1 delay
3
S2 delay
Train period
Trigger Trigger
Figure 7: The stimulus consists of a periodic train of pulses whose repetition rate is controlled by changing
the Train Rate knob of the signal generator. Each period consists of two pulses (S1 and S2) whose delays (S1
delay and S2 delay) from the trigger pulse, amplitudes (S1 volts and S2 volts), and durations (S1 duration
and S2 duration) can each be controlled.
200 ms. Set the delay of the first pulse to be minimal and its duration to 100 s. Set the delay of
the second pulse to 2.5 ms and its duration to 200 s. Measure the amplitude and timing of the
stimulus with the oscilloscope. Make sure that the scope’s horizontal sweep vernier is set in the
calibrated position.
11
2.6 Data Acquisition and Processing
The laboratory computer (an IBM compatible) is interfaced to an analog-to-digital conversion
system that has two analog inputs: channel A and channel B. The analog-to-digital conversion
system is programmed to monitor channel B for the arrival of a synchronization pulse from the
stimulus generator. When a synchronization pulse is detected, periodic sampling of channel A,
which is connected to the output of the preamplifier, begins immediately. The sampling interval is
< <
20 s and the samples have 16 bit resolution within the range to volts. Sampling continues
for 15 ms to give a sequence of 750 samples.
to make a new directory named c:\frog\b2 and to make that directory the current working
directory. Then type
C:\FROG\B2> frog
12
+β Print Notes
Continuous Get Wave Avg 10 G = 100 Print Plot
++ +
5
–– –
__
Electrode Voltage (mv)
A7 –3.1
__
A6 –4.3
__
__
__
++ +
–5 t = 1.520
–– –
– 0 + 1 2
Time (msec)
3 4 – 5 +
Stored Waveforms Notes
Prev Page A0: 31-Aug-94 6:58 -- amplitude = 27 uA; duration = 0.1 ms
A1: 31-Aug-94 6:59 -- amplitude = 28 uA; duration = 0.1 ms
A2: 31-Aug-94 7:03 -- amplitude = 29 uA; duration = 0.1 ms
A3:
A4:
A5:
A6: 31-Aug-94 7:12 -- positive electrode = #3, negative electrode = #4 --- before crushing nerve
A7: 31-Aug-94 7:10 -- positive electrode = #3, negative electrode = #4 --- after crushing nerve
A8:
Next Page A9:
Figure 8: Appearance of monitor screen after the acquisition of data via the frog program. The waveforms
shown are from a bullfrog sciatic nerve in response to stimulus pulses presented with a repetition rate of
10/s, an amplitude of 28 A, and a duration of 100 s. One waveform (dashed line) was recorded before
— and the other (solid line) was recorded after — the nerve was crushed at a location between the two
recording electrodes.
13
Next Page and Prev Page buttons select the set of ten waveforms whose notes are displayed.
When a waveform is saved, the page of notes associated with that waveform is automatically
selected and the date and time-of-day are automatically entered on the note line for that waveform.
You may add text to the note or modify existing text by pressing the button to the left of the note.
This button activates an editing mode prompt. Press text keys to add text, <Backspace> to delete
text, or strike the Return key to exit the prompt.
Notes can be modified at any time. Simply select the appropriate page (using the Next Page
and Prev Page buttons) and click on the button to the left of the appropriate note.
Relation between notes and protocol records. Notes are not substitutes for appropriate entries
in your protocol book! Notes only provide information about one measurement. It is generally
impossible to understand the context of one measurement in isolation. The context, working hy-
potheses, test setup, and tentative conclusions should all be recorded in your protocol records along
with a record of waveform IDs.
Scales. Voltages displayed on the screen are scaled by a constant G. To make the displayed volt-
ages correspond to those at the amplifier’s input, use the G= button to set G to the gain of the
amplifier, which is nominally 100 (more exact values are indicated on each amplifier).
The maximum and minimum values of the scales on both the abscissa and ordinate can be
changed using the nearby +, ++, -, and -- buttons. Pressing + or ++ increases the number by
a small or large amount respectively. Pressing - or -- decreases the number by a small or large
amount respectively.
Selecting waveforms. The color-keyed buttons to the right of the plot can be used to select stored
waveforms for display. Pressing one of those buttons initiates a prompting sequence. Enter a letter
and a digit to select a stored waveform or strike the spacebar to erase the entry or strike <esc> to
abort the prompting sequence.
Using the cursor. To obtain numerical values from the plotted waveforms, move the cursor to
the graphical area of the display and press the mouse button. A vertical line will appear, and the
position of that line can be changed by moving the mouse left or right. The time selected by the
vertical line is indicated just under the last of the color-keyed buttons to the right of the plot (see
Figure 8). Values of the plotted waveforms are also indicated as numbers typed to the right of the
corresponding color-keyed button.
14
2.6.6 Hardcopy
Hardcopies of the graphical portion of the display can be obtained by pressing the Print Plot
button. This button produces an encapsulated PostScript file in the current directory and then
initiates a sequence of messages on the campus computer network that will ultimately produce a
sheet of paper on the laser printer located in the laboratory.
Hardcopies of the notes can be obtained by pressing the Print Notes button. This button
produces a text file named NOTES.TXT in the current directory and sends that file off to the laser
printer.
The encapsulated Postscript files and NOTES.TXT are not deleted when the printing is com-
plete. These files provide opportunities to print additional copies of the plots or to electronically
paste the plots into your laboratory report. The file for the first plot that you make is named
PLOT001.EPS, the second is named PLOT002.EPS, and so forth. Typesetting and electronic
cut-and-paste are NOT required: these facilities are made available only for those who wish to use
them.
15
2.6.9 Exiting the Program
The frog program can be terminated at any time by pressing the Exit key. Data sessions can be
resumed simply by restarting the program. No data are lost by exiting and restarting.
where comp-name represents the name of the computer (e.g. athena.dialup). The laborato-
ry computers support file transfers to other computers using ftp. To activate ftp type
16
3 Experiments
3.1 Basic Observations
The purpose of these observations is give you some experience with basic features of compound
action potentials, before you begin your project. Try to complete these three observations in less
than 1/2 hour.
Determine the threshold current for the compound action potential for pulses with 100 s
duration, repeating at a rate of 10/s. Use the two most distal recording electrodes.
Your method should be well defined, though it may have some arbitrary constraints. Start
with a weak stimulus and gradually increase the level. You should observe a CAP about
2 ms after the stimulus with a current of a few tens of A. Notice that the vertical axis gain
of your oscilloscope can influence your ability to detect a response. Is the compound action
potential an “all-or-none” response?
Cautions. Avoid large currents, which can damage the nerve. Make sure that the stimulus
isolator is set for “1 mA” and remember that this means that 100 A of current will be
delivered to the nerve for every volt from the Grass S88.
17
The following example projects are concerned with stimulus-response relations, spatial vari-
ations in the response, or effects of external agents. You may choose one of these or you can
construct an entirely new one. The descriptions given here are incomplete. Your plan should
be well-defined. Keep it simple so that you can complete the work, including some preliminary
plotting of results and thinking about interpretation within a one-hour lab session (dissecting the
nerve and doing the basic observations will take at least 2 hours). Your experimental manipulations
should avoid actions that may alter the nerve’s responsiveness over a long term.
18
Method: Straightforward.
1 Response components.
Motivation: Peripheral nerves are made up of nerve fibers of different diameters (Figures 1
and 2). Plots of numbers of fibers in particular diameter ranges have peaks indicating dis-
tinct size classes. These categories might have different response properties, e.g. different
thresholds, propagation velocities, refractory periods.
Hypothesis: Smaller nerve fibers have higher thresholds and lower propagation velocities
than larger fibers.
Method: Investigate the fine structure of the monophasic compound action potential. Is
it unimodal, or are there multiple peaks? Two peaks may represent populations of fibers
with different diameters. Determine whether each of the peaks has the same or a different
threshold.
1 Where is the CAP initiated?
Motivation: The electric stimulus is delivered to a region of the nerve defined by the stimulus
electrode locations. Does a propagated action potential begin at one or the other stimulus
electrode?
Hypothesis: The anode is the site where the CAP is initiated.
Methods: Measure the time of occurence of some feature of the response for different con-
figurations of the stimulus electrodes. For example, interchange the anode and cathode.
Alternatively, connect the output of the stimulus isolator to electrodes that are usually used
for recording (i.e., R1-R12).
19
Methods: Observe waveform of CAP at different recording locations and compare features
of the response (e.g. time of peak, height of peak, width of peak) to predictions based on
your hypothesis.
1 Superposition of responses from branches.
Motivation: Careful dissection of the nerve below the frog’s knee can yield two branches of
roughly equal size. These branches can then span over common recording electrodes and
thereby extend the length of many fibers in the recording chamber.
Hypothesis: The response recorded from the region where two branches are in contact with
the recording electrodes is the sum of the responses recorded with each branch alone.
Methods: Record with branch “a” alone, branch “b” alone, and both “a” and “b” on the
electrodes. Try to maintain the quality of contact of electrodes to nerve branches. Controls
(repeats after manipulation of branches) will be important.
20
When you choose your topic, remember that your experiment should take approximately one
hour to complete. Think through how many measurements you will need to make and how long it
will take to make each measurement. For example, it could take a long time — possibly hours —
to reverse the effects of a drug. Reversal time often depends on the size of the dose. Therefore, it
is generally prudent to investigate effects of small doses first.
Formulate a specific and testable hypothesis, and center your project on that hypothesis. Avoid
vague hypotheses, such as: “I would like to understand how temperature affects the compound
action potential.” Instead choose a more narrrowly focused hypothesis, such as: “Decreasing the
temperature of a nerve will decrease the conduction velocity of the compound action potential.”
This more-focused hypothesis is testable: it can be true or false. If you form a clear hypothesis,
you will be able to plan a logical set of measurements to test the hypothesis, and you will be able
to come to a clear conclusion when you write your report.
When you do your experiment, you may get unexpected results. For example, you may have
planned to measure temperature-induced changes in velocity of the compound action potential,
but you may also find large changes in amplitude. There may even be a temperature below which
there is no compound action potential at all. You should explore unexpected results and try to
understand their bases. Your aim should be not simply to reject or accept your hypothesis, but
to develop insight into the phenomena. For example, perhaps compound action potentials fail
to occur at low temperature because the threshold current is larger at low temperatures. If so, it
might be better to determine the threshold current for each new temperature and then measure the
conduction velocity when the stimulus current is equal to (for example) twice the threshold current.
Keep in mind that this is an experimental project. Your goal is to characterize what happens,
not why it is happening. Theoretical ideas about neural mechanisms will be the basis for your
second project (the Hodgkin-Huxley project).
21
the response between R6 and R7. The intentional manipulation is the crushing between R7 and
R8, which should have no effect on the response between R6 and R7. Therefore, if crushing the
nerve also changes the response measured between R6 and R7, then the manipulation did more
than just affect the nerve at the site of the crush.
One variable that is not under the control of the experimenter is the passage of time. Each
observation that you make will happen at a different time, and it is important to know the extent
to which differences in responses simply reflect the passage of time. To assess this effect, one
can design a control in which a particular measurement is periodically repeated throughout the
experiment. For example, one could measure the diphasic response between R4 and R5 to a 10 A
pulse of current with a duration of 100 s at the beginning of the experiment and at 10 minute
intervals throughout the experiment. If the responses differ, then the passage of time is an important
factor that must be considered when the data are interpreted.
4.2 Proposal
After your laboratory session has been scheduled, you should meet with your partner to plan your
project and to write a proposal. The proposal should contain a brief statement of the hypothesis
you propose to test and the method that you will use to test it. Include a list of the experiments
you will perform and the measurements you plan to make in each experiment. Indicate how the
measurements will be used to help you test your hypothesis. The proposal should fit on a single
sheet of paper. A sample proposal is shown in Figure 9.
The proposal should be submitted before 5 pm on Monday, September 20, 1999. Electronic
submission via email2 to freeman@mit.edu is encouraged. The staff will review the proposals
and make suggestions. Acceptable proposals will be APPROVED; flawed proposals will be RE-
JECTED. Proposals that are not approved may be revised and resubmitted. All proposals must be
approved before 5 pm on Friday, September 24, 1999.
2
Please submit simple ascii text; please do not submit vendor-specific formats.
22
Proposal: Effects of Temperature on Compound Action
Potentials of the Frog Sciatic Nerve
Partner Names (E-mail): Partner One (pone@mit.edu) and Partner Two (ptwo@mit.edu)
Hypothesis: Decreasing temperature will decrease the velocity of the compound action potential
of the frog sciatic nerve.
Background: The speed of many chemical reactions tends to increase as temperature increas-
es. Therefore, it seems plausible that the speed of neural conduction might similarly depend on
temperature.
Procedure: We will obtain a 100 mL sample of Ringer’s solution on the day before our laboratory
session, and place the solution in a refrigerator to chill it to approximately 4 C. After performing
the basic observations described in the laboratory manual, we will rinse the nerve with chilled
Ringer’s solution and place 10 mL of chilled Ringer’s solution in the bottom of the nerve chamber.
We will place a small thermocouple in contact with the distal end of the nerve to monitor it’s
temperature. We will then record diphasic compound action potentials at three pairs of electrodes:
R1 and R2; R3 and R4; and R5 and R6. These measurements will be repeated once a minute for
10 minutes. We will record the temperature at the time that each measurement is made. The speed
of the compound action potential will be measured by dividing the distance between the first and
last pairs of electrodes (4 cm) by the difference between the times of the first negative peak of the
compound action potential measured on the first and last electrode pairs. Measurements using the
center pair of electrodes will be used to check consistency: if the speed measured from the first
pair to middle pair differs significantly from that measured from the first pair to last pair, we will
investigate the source of the difference.
We obtained a thermocouple by special arrangements that we made with the TA. We have also
tested the basic method by measuring the temperature of a piece of thread that was mounted in an
experimental chamber as though it were a nerve. We found that the temperature increased from
about 15 C to about 25 C over a time course of about 10 minutes. We therefore expect that we
have time to repeat this experiment three times to check the repeatability of our results.
23
4.3 Project Report
The project report should be concise. Do not repeat material that is easily referenced. For example,
there is no need to reproduce any figure from this laboratory manual or from the course text: simply
refer to it. Technical writing is necessarily directed at some particular intended audience. Write
the project report as though it were to be published in a journal that is read primarily by students
who have taken this subject. Thus, you can assume some working knowledge about the subject.
The project report should contain the following sections.
Cover Page. On the cover page include the title of the laboratory session, the authors’ names, your
laboratory subsection, the dates of the laboratory session(s), and the name of your partner (if
not a co-author).
Abstract. The abstract is a one paragraph ( O 100 words) summary of the report including the
question investigated, the methods used, and the principal results and conclusions. Your
intended audience should be able to understand the abstract without having to read any of
the report. This section should be written last.
Introduction. The introduction is a brief section (about 1 page) designed to motivate the reader.
Include background information on the problem, hypotheses to be tested, significance of the
work, etc. You may give citations to material in this laboratory manual or elsewhere. The
introduction should be directly relevant to your report; broad discussions of neurophysiology
or brain function are not needed.
Methods. Briefly describe any special methods that you used that are not in this laboratory de-
scription. Give details of calculations used to obtain the results. Do not repeat material that
is in this description; just refer to it.
Results. Describe the measurements (whether or not they fit with expectations) in the results sec-
tion. Generally, results can be communicated more efficiently and accurately with pictures
and graphs than with words alone. Describe those aspects that are important to your inter-
pretation, but omit interpretations from this section. A collection of printed graphs without a
written description of their relevance is unacceptable as a results section. Students frequently
err on the side of including a large number of graphs and little description of their relevance.
You need only include results that are relevant to your conclusions. Most of us have trouble
leaving things out. Ask yourself why a particular result is a necessary part of your story;
omit those that aren’t necessary.
Discussion. In the discussion section, assess the results for dependability and accuracy, and in-
terpret results in the light of other knowledge. The discussion section can include relevant
speculations and ideas for improving the experiment to test the hypotheses more rigorously.
Appendix. The appendix should include a copy of the protocol taken during the laboratory ses-
sion.
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4.4 Grade
The grade for the experimental project will be based on the proposal and on the project report using
the following considerations:
Proposal (10%). The proposal should reflect a carefully constructed plan. Thinking about what
you plan to do before entering the laboratory is crucial to collecting meaningful data when
you are there. Late proposals will receive no credit.
First draft & Critique (10%). Your first draft and your critique of a colleague’s first draft will be
graded primarily for completeness.
Protocol (10%). The protocol should provide enough information so that a reader can follow the
course of the experiment. Indeciphereable scribbles will receive no credit.
Report structure (10%). The sections of the report should be coherent.
Clarity/Conciseness (20%). A good report is easy to read. The content of each paragraph and
each graph should be clear. Everything included in the report should be there for a reason.
Points will be deducted for extraneous material. Reports should be less than 10 pages long,
unless there are good reasons for additional pages.
Conceptual correctness (20%). Are there clear errors? Are the results confused? Are the results
(which follow directly from the measurements) confused with the interpretations (which rely
on information other than what was observed)?
Insightfulness (20%). Insightfulness can be demonstrated by (1) proposing an experimental method
that can resolve some scientific issue, (2) carrying out experiments and/or analyses that lead
to clear conclusions, (3) preparing a report that demonstrates a clear understanding of the
strengths and weaknesses of your results and analyses. Simply performing one of the s-
tandard experiments and showing unmotivated measurements will receive 0 points. Clever
design of an experiment or imaginative analysis of the results will receive 20 points. Demon-
strating a clear understanding of your experiment, your analyses, and what can be concluded
is sufficient for 10 points.
DUE DATES ARE FIRM, AND THERE IS A SEVERE LATENESS PENALTY. The grade
for a late report will be multiplied by a lateness factor
PRQ S S
0UTWV - > 0UTWVYX
,
where is the number of hours late. The lateness factor is plotted in Figure 10. Notice that the
maximum grade for a report that is more than ONE DAY LATE is less than 50%.
References
Carolina Biological Supply Company (1965). Frog musculature.
Ochs, S. (1965). Elements of Neurophysiology. John Wiley & Sons, Inc., New York, NY.
25
1.0
Lateness factor L
0.5
[one day
0.0
1 10 100
Z
Time t past deadline (hours)
Patton, H. D. (1960). Special properties of nerve trunks and tracts. In Ruch, T. and Fulton, J. F.,
editors, Medical Physiology and Biophysics, pages 66–95. W. B. Saunders Company, Philadel-
phia, PA.
Ruch, T. C., Patton, H. D., Woodbury, J. W., and Towe, A. L. (1965). Neurophysiology. W. B.
Saunders Company, Philadelphia, PA.
Young, J. Z. (1951). Doubt and Certainty in Science. Oxford University Press, London.
26