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BBio 351: Principles of Anatomy & Physiology I
Fall 2015
Lab 6: Earthworm action potentials
created by Doug Wacker with materials provided by ADInstruments;
revised by Greg Crowther
Today’s objectives
 Make extracellular recordings of action potentials from anesthetized earthworms.
 Use these recordings to explore four core concepts of neurophysiology: threshold, “allor-none” responses, nerve conduction velocity, and refractory period.
Supplies/equipment
 LabChart 7.1 software
 PowerLab Data Acquisition Unit with Bio Amp
 Stimulator Cable (BNC to Alligator Clips)
 5 Lead Shielded Bio Amp Cable
 Shielded Lead Wires (3 Alligator Clips)
 Three silver wires, chlorided at one end
 Modeling clay or adhesive tape
 Petri dish
 Millimeter ruler
 Tissues
 Blunt probe
 Dissection tray with wax or pad
 Dissection pins
 Eyedropper or Pasteur pipette
 One earthworm (Lumbricus spp.), at least 60 mm in length
 10% ethanol in earthworm saline
Pre-lab assignment
By the start of your lab section (Tuesday at 2pm, Thursday at 2pm, or Friday at 8:45am) you
should (1) print or download this lab handout, read it over, and bring it to lab; and (2) complete
the pre-lab assignment posted to Canvas (which will include questions on the material below).
Background
A characteristic of the nervous system is that signals are conducted from place to place by
nerve impulses – action potentials that travel along a nerve fiber, or axon. All sensation,
thought, and movement is mediated by coded patterns of nerve impulses. The properties of
action potentials in single fibers are therefore of fundamental physiological importance.
Annelids, such as the common earthworm (Lumbricus spp.), and arthropods have a ventral
nerve cord that is analogous to the dorsal spinal cord of chordates. The earthworm possesses
a medial giant nerve fiber and two lateral giant nerve fibers (that lie on either side of the medial
one) that run the length of the animal. The earthworm also has a multitude of small nerves that
do not run the length of the animal (there are about 500-1000 neurons per segment, or about
100,000 neurons altogether). Each giant fiber is actually made up of multiple large cells (one
per segment) that are electrically coupled to each other through gap junctions. This results in
the rapid conduction of action potentials from cell to cell so that each giant fiber behaves almost
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as though it were a single axon. In addition, the lateral giant fibers are extensively linked to
each other by cross-connections, and therefore function as a single unit.
Figure 1. Earthworm
ventral nerve cord and
surrounding structures.
Figure 2.
Crosssection of an
earthworm,
with the
ventral nerve
cord
expanded for
display.
Historically, giant axons (from squid, especially) played an important part in the discovery of the
membrane mechanisms underlying the action potential. The role of these giant fibers, which
conduct action potentials far faster than small nerves, is to allow the animal to respond rapidly to
threatening situations. Therefore, they take part in a variety of “escape” behaviors.
Action potentials occur when specialized voltage-sensitive membrane sodium channels are
activated. The large increase in sodium permeability results in membrane depolarization (i.e.,
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Fall 2015
the charge difference between the inside and outside of the cell decreases). This is followed by
repolarization as the sodium permeability returns to its low baseline value and potassium
permeability is transiently increased. From the beginning of the action potential to the
restoration of the resting membrane potential, the neuron is incapable of producing another
action potential. This period is referred to as the refractory period, which can be divided into
two phases. Initially there is the absolute refractory period, where it is impossible to initiate a
second action potential. This is followed by the relative refractory period, where only a
stimulus of greater-than-normal intensity can elicit a response.
Action potentials are “all-or-none” events, meaning that they either occur or do not; there is no
such thing as a “half action potential.” Once an action potential begins, it propagates down the
length of the axon. There is no relation between stimulus strength and response
amplitude in a single axon. This is one advantage of using an earthworm model – you can
see the “all-or- none” response. In other animal models, a graded response appears, since the
recording electrodes pick up signals from multiple axons.
Another advantage of the earthworm model is that its giant fibers can be stimulated by
electrodes placed on the body of the animal, and the responses can be recorded extracellularly
from the animal's surface. Since the small nerves do not run over extended distances, their
activity is not recorded. Today you will be recording extracellularly and measuring the charge
difference (electrical potential) between two recording electrodes. Your experimental setup will
be similar to that in Figure 3.
What you are recording here is the difference in charge between two extracellular electrodes. In
the absence of a stimulus, there is no difference. Following a stimulus, a wave of depolarization
passes down the nerve (Figure 3). As this wave reaches the first recording electrode, the
surface of the nerve underneath the electrode becomes negative relative to the nerve surface
underneath the more distal recording electrode. By convention, this difference is shown as a
positive deflection in the recording (b in Figure 3). When the action potential is affecting the
membrane under both recording electrodes simultaneously, the potential difference between the
two returns to zero (c in Figure 3). Then, when this wave reaches the second recording
electrode, the nerve surface underneath that electrode now becomes negative relative to the
more proximal electrode’s nerve surface. This results in a negative deflection in the recording
(d in Figure 3). Once the action potential has passed both recording electrodes, the potential
returns to zero (e in Figure 3). This experimental setup results in recordings of biphasic,
extracellular action potentials, which look quite different in shape than action potentials recorded
using an intracellular electrode. It is important to understand the difference between these two
types of recordings and the appearance of the resulting data. There can be some variation in
the shape of the extracellular action potentials, depending mainly on the distance between the
recording electrodes.
The further the electrodes are from the surface of the nerve, the smaller the amplitude of the
action potential will appear. Remember, intracellular action potentials are all-or-none, and
therefore amplitudes don’t really differ. Most of the loss of amplitude is due to internal
current flow within the worm’s body. The amplitude also depends on how easily current can
flow across the skin of the worm and over its surface. If the region between the recording
electrodes is very wet with Ringer's solution, the peak deflection may be as small as 20 µV.
This is because the current dissipates throughout the solution.
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Figure 3. Extracellular recording of an action potential conducted along the earthworm.
In most, if not all, nerve fibers, there is a preferred natural direction of conduction – away from
the soma – called orthodromic, but this is determined by the fiber's connections and not by the
membrane ionic mechanisms responsible for the action potential. Using stimulating electrodes,
scientists can induce an antidromic action potential that travels towards the soma. In the
earthworm, the median and lateral fibers conduct their naturally generated impulses in opposite
directions: stimulation of the median giant fiber results in rapid head withdrawal, whereas
stimulation of the lateral giant fibers results in rapid tail withdrawal.
Equipment setup
1. Make sure that the PowerLab is turned off and the USB cable is connected to the computer.
2. Connect the Stimulator Cable to the Output connectors on the front panel of the PowerLab.
Connect the black (negative) BNC to the (-) Output, and connect the red (positive) BNC to
the (+) Output (Figure 4).
Figure 4.
Equipment
setup. You’ll only
use the white,
black, and green
wires connected
to the Bio Amp.
3. Connect the 5 Lead Shielded Bio Amp Cable to the Bio Amp Connector on the front panel of
the PowerLab (Figure 4). The hardware needs to be connected before you open the
settings file.
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BBio 351: Principles of Anatomy & Physiology I
Fall 2015
4. Attach the Shielded Lead Wires with Alligator Clips to the Bio Amp Cable. Refer to Figure 4
for proper placement, but do not attach them to the earthworm. Follow the color scheme on
the Bio Amp Cable. You’ll only use the white, black, and green wires connected to the Bio
Amp.
5. Check that everything is connected correctly. Turn on the PowerLab.
Earthworm preparation
1. Obtain a large earthworm, and carefully rinse it under tap water to remove excess dirt.
2. Place the earthworm in a Petri dish containing 10% ethanol in earthworm saline. The saline
should cover the earthworm completely. Allow the earthworm to become fully anesthetized;
this procedure usually takes a full five minutes or longer.
3. Check whether the earthworm is fully anesthetized by poking it gently with a blunt probe. If
the earthworm does not react, it is fully anesthetized. If it does react, leave it in the Petri
dish a bit longer.
4. When ready, remove the earthworm from the ethanol and place it on the dissection tray. Pin
it to the tray using one pin on each end of the worm. Be careful not to stretch the
earthworm, as this can damage the nerve.
5. Insert a second dissecting pin toward the anterior end of the earthworm, 10-15 mm (use a
ruler) from the first pin. (The anterior end is the end nearest the clitellum, a smooth, nonsegmented band; see Figure 5.) Connect the stimulating electrodes as shown in the image.
The negative lead wire (cathode) should be posterior to the positive lead wire (anode).
Figure 5.
Earthworm
preparation.
Notice the
clitellum toward
the anterior end.
The stimulating
electrodes (red
and black) are
labeled “Anode”
and “Cathode”;
the recording
electrodes (white
and black)
correspond to R1
and R2.
6. To keep the worm anesthetized, apply a few drops of the ethanol/saline solution along the
length of the earthworm with the eyedropper. Soak up any excess fluid with the tissues. It
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BBio 351: Principles of Anatomy & Physiology I
Fall 2015
is not necessary to have a large puddle of solution, and too much may interfere with your
recording.
7. Place the three silver wires over the body of the worm, with the chlorided (blackened) ends
in contact with the body. Bend the wires such that they gently touch the body of the worm
without crushing it (Figure 5).
8. Connect the wires to the Alligator Clips of the recording electrodes. Fix the clips in position
using adhesive tape stuck to the dissection tray. Use Figure 5 as a guide. The Earth should
be closest to the stimulus pins, but its exact placement is not critical. Position the negative
recording lead (white or R1) anterior to the positive lead (black or R2) and approximately 10
mm apart. Getting these wires into the correct position and affixing the tape so they do not
move is perhaps the most challenging portion of the lab, so take the time needed to get it
right!
9. Make sure no wire is touching any other wire. Be careful when positioning the wires to
avoid damaging the worm. It may be easier to move the Alligator Clips than to move the
wire.
Exercise 1: Determining Threshold Voltage
In this exercise, you will record from the earthworm median giant axon to determine its
threshold voltage. The threshold voltage is defined as the minimum stimulus that produces an
action potential 50% of the time.
1. Launch the LabChart software and open the settings file “Earthworm AP Settings,” which
should be available in the LabChart Welcome Center, in the Experiments tab.
2. Start recording. LabChart is in Scope View and should display one 20 ms sweep every
time you click on Start. For each 20 ms sweep a stimulus pulse is generated through the
PowerLab Stimulator outputs.
3. Examine the recording. Something similar to Figure 6 should be recorded. The deflection
just after the start of the sweep is caused by spread of the stimulus current to the recording
electrodes. It is called a stimulus artifact. The stimulus artifact is often very large in
amplitude, and can exceed the range of the recording amplifier. Recovery from the artifact
should be rapid, however, so that the baseline is reached within one millisecond or so after
the stimulus pulse. NOTE: you may experience some electrical “noise” in the form of a large
ever-present wave in the background. This should not prevent you from conducting this
experiment; let your instructor know if you think you see such noise.
4. Open the Stimulator Panel by going to the Setup menu. A dialogue box should appear like
the one shown in Figure 7.
5. A Pulse Width of 0.2 ms and Max Repeat Rate of 1 Hz (or one pulse per second) have
been set for you. Do not adjust these unless directed to do so.
6. Set the Stimulator output by changing the Pulse Height in the Stimulator Panel (Figure 7).
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BBio 351: Principles of Anatomy & Physiology I
Fall 2015
Figure 6. Sub-threshold
stimulation creates a
stimulus artifact but no
response.
Figure 7.
Stimulator
panel.
7. Increase the Pulse Height in 0.2 V steps until a response from the median giant axon is
recorded (Figure 8). Remember, you’ll have to hit Start to record a response each time you
change your Pulse Height. It is a good idea to wait at least ten seconds between each
stimulation. Leave the Stimulator Panel open.
8. Each time you change the stimulus, click on Start and a new page will appear to the left of
the Scope View Window. This is the Page Explorer Window and allows for easy navigation
between ‘pages’, which represent iterations of a stimulus.
Note: If no response is seen with a stimulus of 3 V or greater, contact your instructor for
assistance.
Figure 8. Action potential
recorded from median
giant axon.
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BBio 351: Principles of Anatomy & Physiology I
Fall 2015
9. When the response first appears (see Figure 8), note the stimulus voltage as an estimate of
the threshold stimulus strength. Reduce the stimulus by 0.2 V, and click Start.
10. Begin to increase the stimulus by 0.05 V between each sweep. Continue adjusting the
stimulus strength upward and downward until you have defined the minimum stimulus that
usually elicits a response. This is your threshold voltage. Save your data, but do not close
the file. Record the final threshold value in Table 1 of the worksheet at the end of this lab
handout. Show your instructor your work before proceeding.
Exercise 2: Recruitment of the Lateral Giant Axons
In this exercise, you will attempt to record from the lateral giant axons. Because you are
stimulating externally, a significant voltage is required to obtain any response. Once you have
reached a threshold voltage for the median giant fiber, you will need to raise the voltage further
to stimulate the lateral giant fibers, which have higher stimulus thresholds. In addition to their
higher stimulus thresholds, the lateral fibers conduct more slowly than the median fiber. Their
action potential reaches the recording electrodes after a longer latent period and is seen as a
second, delayed peak in the recording.
Note: Remember to keep the earthworm anesthetized by dripping the earthworm saline in 10%
alcohol solution onto your animal periodically!
1. Set the Pulse Height to just below the threshold value you determined for the median giant
fiber. Start recording.
2. Increase the stimulus in 0.05 V steps until threshold for the median giant fiber is observed.
3. Keep increasing the stimulus by 0.05 V until a second response with a longer latent period is
observed (Figure 9). I may ask to see your traces before you leave, so if you are unsure
whether you are eliciting an action potential from the lateral giant axons, please ask for
assistance.
Figure 9. Action
potentials from
the median and
lateral giant
axons.
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BBio 351: Principles of Anatomy & Physiology I
Fall 2015
4. Record the stimulus threshold for the lateral giant fibers in Table 1 of the worksheet at the
end of this lab handout. Save your data, but do not close the file.
Note: In some specimens, the median and lateral fiber responses may not be separated – they
will appear to have the same threshold. Other worms may not show the second response,
possibly because the lateral giant fibers are damaged. Show your instructor your work before
proceeding.
Exercise 3: Determining Nerve Conduction Velocity
In this exercise, you will determine the nerve conduction velocity of the median giant fiber and
lateral giant fibers.
1. Adjust the Pulse Height so that action potentials from the median and lateral giant axons
should be seen. Click Start to record a single sweep or “page.”
2. Use the ruler to measure the distance between the cathode stimulating electrode and the R1
recording electrode. (See Figure 5 if you cannot remember what R1 is.) You will need this
value to calculate the nerve conduction velocity. Record this value in Table 2 of the
worksheet.
3. Examine the data from this exercise in the Scope View Window.
4. Place the Marker on the stimulus artifact peak of a representative trace, as shown in Figure
10. Then place the Waveform Cursor on the action potential peak from the median giant
axon. Read the time difference between the stimulus artifact and the action potential from
the Rate/Time display.
5. Fill out Table 2 of the worksheet at the end of this lab handout. As stated above, “distance”
is the distance between the cathode stimulating electrode and the R1 recording electrode.
Calculate a conduction velocity in mm/ms, then convert this value to m/s.
6. Repeat this procedure for the lateral giant axons. Show your instructor your work before
proceeding.
Exercise 4: Determining the Refractory Period
In this exercise, you will deliver pairs of stimuli to the nerve, progressively decreasing the
interval between the two stimuli. This information will be used to determine the refractory
period of the median giant fiber.
1. From the Setup menu, select Stimulator Panel (Figure 7). The number of pulses has been
set to 2. You can change the Interpulse Interval by adjusting the frequency or “Max Repeat
Rate.” Set the Max Repeat Rate to 66.7 Hz to give an interpulse interval of 15 ms.
2. In the Stimulator Panel, set the Pulse Height to midway between the threshold for the
median giant nerve and lateral giant nerve. This should ensure that only the median fiber is
stimulated.
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BBio 351: Principles of Anatomy & Physiology I
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3. Start recording. Examine the recording. With an interpulse interval of ~5 ms or more, two
stimulus artifacts should be seen, each followed by an action potential. This indicates that
the median giant fiber fired for both stimuli (Figure 11). Since there are two responses, the
time between the stimulus pulses must be longer than the refractory period of the nerve.
Figure 10. Placement of the Marker (M) and Waveform Cursor (X) to record the latency of
the lateral giant nerve. Note: Only a single sweep or “page” is shown above. You can adjust
how many sweeps are shown at a time by moving the Overlay slider to the left (fewer
sweeps) or to the right (more sweeps).
Figure 11.
Response of the
median giant axon
to two stimuli.
4. Set the Max Repeat Rate to 71.4 Hz to give an interpulse interval of 14 ms. Start
recording.
5. Follow the table below to progressively decrease the interpulse interval by 1 ms. Start
recording, and then wait for at least 2 seconds before decreasing the interval again.
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BBio 351: Principles of Anatomy & Physiology I
frequency
(Hz)
interval
(ms)
Fall 2015
66.7
71.4
76.9
83.3
90.9
100
111.1
125
142.9
166.7
200
250
333.3
500
15
14
13
12
11
10
9
8
7
6
5
4
3
2
6. As you make the interpulse interval smaller and smaller, the response to the second
stimulus should eventually disappear.
7. The longest interpulse interval that does not permit a second response is the refractory
period. Record this in Table 3 of the worksheet at the end of this lab handout.
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BBio 351: Principles of Anatomy & Physiology I
Fall 2015
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BBio 351: Principles of Anatomy & Physiology I
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Worksheet for Lab 6: Earthworm Action Potentials
Table 1. Properties of the earthworm giant nerve fibers
Threshold
Voltage (µV)
Median Giant Fiber
Lateral Giant Fibers
Table 2. Nerve conduction velocity
Distance
(mm)
Median Giant Fiber
Time
(ms)
Conduction
Velocity (mm/ms)
Conduction
Velocity (m/s)
Lateral Giant Fibers
Table 3. Refractory period
Refractory
Period (ms)
Median Giant Fiber
Study Questions
1. Chemical versus electrical synapses. There are two basic types of signaling between two
neurons – chemical and electrical. Chemical signaling involves the intercellular movement of
neurotransmitters across the synaptic cleft. Electrical signaling involves gap junctions.
a. What are gap junctions?
b. Do the neurons of the earthworm’s giant fibers form chemical connections with each
other, or electrical connections with each other?
c. What advantage does this type of connection (from 1b) offer, relative to the other
type?
d. What DISadvantage does this type of connection (from 1b) have, relative to the other
type?
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BBio 351: Principles of Anatomy & Physiology I
Fall 2015
2. Conduction velocity.
a. What equation did you use to calculate conduction velocity in your table above?
b. Prior to collecting the data, which giant fibers (median or lateral) would you expect to
have a higher conduction velocity? Why?
c. Did your data meet this expectation?
d. Should conduction velocity vary with temperature? Why or why not?
3. Refractory periods.
a. What is refractoriness and when does it occur?
b. What are the two different types of refractory period associated with neuronal firing?
How do they differ?
c. Based on your measured refractory period, what is the maximum firing frequency of
the median giant neuron? How does this compare to the maximum firing frequencies
of other neurons? (Consult the Internet; cite any sources used.)
4. The introduction of this lab states, “There is no relation between stimulus strength and
response amplitude in a single axon.” What does this mean?
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