Teaching Experiment

Earthworm Smooth Muscle

In this experiment, contractions of the earthworm gut are measured in an organ bath with a
force transducer. The effect of neurotransmitters and ionic concentrations on contraction
strength and rate will be investigated.
Written by staff of ADInstruments
With acknowledgement to: Dr. Stuart I. Cromarty, Department of Natural Sciences, Assumption
College, Worcester, MA, USA.

Background
Smooth muscle is one of three muscle fiber types found in animals. Unlike skeletal and cardiac
muscle cells, smooth muscle cells are not striated, and have single nuclei. Smooth muscles are
typically under control of the autonomic nervous system, and do not contract voluntarily.
Smooth muscle contracts slowly, and does not exhibit the characteristic twitch seen in
skeletal muscle. In addition, smooth muscle is not prone to muscle fatigue, making it an ideal
component of sphincter muscles. Smooth muscle is found in the gastrointestinal tract of many
animals, and is responsible for peristaltic movements. Smooth muscle is also present in the
walls of arteries and arterioles, where it helps to regulate blood pressure and flow.
Smooth muscle contractions are affected by calcium and potassium ions. Calcium ion influx
into the smooth muscle cell initiates a contraction. Potassium ion concentration in the
extracellular medium affects the resting membrane potential of the cell, bringing it closer to or
farther away from its threshold voltage. Neurotransmitters affect different types of smooth
muscle differently, depending on the association of the smooth muscle with excitable cells. In
general, acetylcholine increases the muscle cells permeability to calcium, while epinephrine
decreases the cells permeability to calcium.
The earthworm (Lumbricus spp.) gut can be dissected and examined in vitro using an organ
bath and force transducer. This preparation is robust; it can remain active for several hours. In
this experiment, you will measure the rate and force of contractions in the in vitro earthworm
gut, and examine its response to changes in extracellular ion concentration and to the presence
of neurotransmitters.

Required Equipment
A computer system
PowerLab 4/25T
Chart 5.0 or later
Bridge Pod
Force Transducer
Ring stand with micropositioner and clamps
Organ Bath
250 ml beaker

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Dissection tools:
Glass finger bowl
15% Ethanol
Sharp scissors
Blunt probe
Dissection tray with wax or pad
Dissection pins
Eyedropper
Earthworm saline solution of the following types:
Normal saline (room temperature)
Cold saline (5-10C)
Warm saline (37C)
High Ca2+
High K+
Ca2+-free
Acetylcholine
Epinephrine

Procedures
A. Set up and calibration of equipment
1.

Securely mount the organ bath, micropositioner, and force transducer to the ring stand.
The force transducer should be mounted so that it is over the opening of the organ
chamber. Close the drain valve on the bottom of the organ bath.

2.

Turn on the PowerLab and make sure the USB cable is connected to your computer.

3.

Make sure the Bridge Pod is connected to the Pod Port on Input 1 of the PowerLab.

4.

Connect the force transducer cable to the back of the Bridge Pod.

5.

Launch Chart and open the settings file for this experiment EW Gut Settings.

6.

A new blank data file will open after a few moments. This file should have one channel,
labeled Force.

7.

Click the Force channel function pop-up menu and select Bridge Pod. The Bridge Pod
dialog box will open.

8.

Observe the signal in the dialog box. Zero this signal by turning the knob on the front of
the Bridge Pod (figure). If you cannot zero the Bridge Pod, contact your instructor for
assistance.

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B. Earthworm dissection
Refer to the diagrams in your earthworm dissection guide for assistance.

Anesthetization
1.

Fill a fingerbowl halfway with 15% ethanol.

2.

Obtain a large earthworm and rinse it under tap water to remove excess dirt.

3.

Place the earthworm in the fingerbowl for five minutes; the earthworm should stop
moving.

Dissection: exposing the gut

1.

Remove the earthworm from the ethanol and place it on a dissection tray.

2.

Pin the earthworm to the tray using one pin on either end of the worm.

3.

Moisten the earthworm with room temperature earthworm saline. You must keep the
worm moist at all times during the dissection.

4.

Locate the clitellum (copulatory organ); this structure is closest to the anterior of the
earthworm.

5.

Using fine pointed scissors, carefully make a shallow incision in the clitellum. Make this
incision to the side of the midline; this techniques will prevent you from cutting into the
gut. NOTE: It is essential not to cut deeply during the dissection; you will damage the gut
tissue. If you tear the gut, obtain another earthworm and start over.

6.

Continue cutting the skin of the earthworm towards the anterior end. It is best to use an
upward-pointing direction with the scissors. As you cut the skin, pin back the skin to
expose the gut.

7.

When you have successfully opened the earthworm, inspect the gut and moisten the
worm with earthworm saline. If the gut is not damaged, continue with the next step.

Dissection: removing the gut

1.

With the gut exposed, use a blunt probe to dissect away the septa that connect the
underside of the gut to the body wall. You only need to expose 3-5 cm of the anterior gut.

2.
3.

When the gut is loosened from the body wall, again check to make sure it is not torn.
Tie a 20 cm piece of strong thread to the gut anterior to the pharynx and another piece
3-5 cm posterior to the pharynx. You should have 10-15 cm of loose thread available after
you tie off the gut so that you can attach the gut to the organ bath and force transducer.

4.

Using scissors, carefully remove the gut section from the earthworm.

Mounting the gut in the organ bath

1.

Tie the posterior end of the earthworm gut to the mounting hook and mount the hook on
the ring stand so the gut is in the organ bath. The distal end of the intestine should be 1
cm from the glass hook. Be sure to hold on to the thread on the anterior end.

2.

Lower the force transducer on the micropositioner to its lowest position.

3.

Tie the thread on the anterior end of the gut (pharynx end) to the force transducer. The
gut should be slack.

4.

Carefully raise the micropositioner by turning the adjustment knob until the gut is under
slight tension. NOTE: Be extremely careful not to overstretch the gut during this step.
The gut is very delicate and could tear easily.

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5.

Make sure the drain stopcock on the organ bath is closed. Fill the organ bath with
normal earthworm saline. The gut should now be completely submerged. If the gut is not
completely submerged, you may need to re-tie the lower thread to the hook.

Figure 1. The earthworm gut segment mounted in the organ chamber for recording. The ring stand and
force transducer are not shown.

Figure 2. Anatomy of the earthworm gut section.

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Exercise 1: Recording baseline data

1.

From the Chart view window, click Start to begin data recording.

2.

Observe your recording for five minutes. The contraction rate and strength should
increase and become regular as the anesthesia wears off.

3.

Make sure you have at least two minutes of consistent baseline data before
proceeding.

Figure 3. Typical recording of an earthworm gut segment under normal conditions.

Exercise 2: Effect of neurotransmitters on contraction rate and force

1.

Drain the normal earthworm saline from the bath by opening the stopcock valve and
draining the contents into a beaker placed underneath the bath.

2.

Replace the solution with saline containing acetylcholine. Pour the saline down the side
of the organ bath chamber to minimize disturbances to the gut tissue.

3.

Add a comment to your trace called acetylcholine, and record for five minutes.

4.

Drain the organ bath and replace it with epinephrine saline.

5.

Add a comment to your data called epinephrine.

6.

Record the response for five minutes.

7.

Drain the organ bath, and refill it with normal saline.

8.

Add a comment called wash and record for three minutes.

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Exercise 3: Effect of temperature on contraction rate

1.

Record the temperature of the normal saline in Table 2 of your Data Notebook.

2.

Drain the saline from the organ bath.

3.

Replace the normal saline with cold earthworm saline. Pour the saline down the side of
the organ bath chamber wall to minimize disturbance to your preparation.

4.

Add a comment to your data file called cold.

5.

Record the saline temperature in Table 2 of your Data Notebook.

6.

Record data for five minutes.

7.

Drain the cold saline and replace it with warm saline. Record the temperature of the
warm saline in Table 2 of your Data Notebook.

8.

Add a comment called warm to your data file and record for another five minutes.

9.

Drain the warm saline and replace it with normal (room temperature) earthworm saline.

10.

Add a comment called normal and record for three minutes.

Exercise 4: Effect of extracellular ions on contraction

1.

Drain the normal saline from the organ bath and replace it with saline marked high
Ca2+.

2.

Add a comment to your data called high calcium, and record for five minutes.

3.

Replace the high calcium saline with normal saline.

4.

Add a comment called wash and record for three minutes.

5.

Drain the normal saline and replace it with high K+ saline.

6.

Add a comment called high potassium and record for five minutes.

7.

Drain the organ bath and replace the solution with Ca2+-free saline.

8.

Add a comment called calcium free and record for five minutes.

Clean up
1.

When your experiment is complete, drain the organ bath and remove the earthworm gut
from the force transducer.

2.

Detach the gut from the glass hook.

3.

Discard the earthworm and gut according to your instructors instructions.

4.

Discard waste saline according to your instructors directions.

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Analysis
A. Determining contraction rate
1.

Determine the average rate of contraction in beats per minute (BPM) for each
experimental condition.

2.

Select data showing at least five contractions and click the Zoom View button.

3.

Place the Marker on the first contraction peak.

4.

Move the Waveform Cursor to the last contraction peak.

5.

Record the time differential from the Zoom View window.

Figure 4. Calculating contraction rate with the Marker and Waveform Cursor.

6.

Calculate the rate using the following equation:

Rate( BPM ) =

T (sec)
60
n 1

Where n= the total number of beats used in the calculation

7.

Calculate the contraction frequency for each condition.

Frequency (Hz) is calculated in the following manner:

f=

BPM
60

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B. Determining contraction force

1.

Use the Marker and Waveform Cursor to determine the average contraction force
during each condition.

2.

Select five or more contractions from the Chart view window and click the Zoom button
to open the Zoom window.

3.

From the Zoom view, place the marker on the waveform baseline.

Figure 5. Determining contraction force with the Marker and Waveform Cursor.

4.

Move the Waveform Cursor to the first peak and record the relative amplitude.

5.

Repeat this step for the rest of the peaks and calculate the average amplitude.

6.

Record the average amplitude for each condition in your Data Notebook.

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Data Notebook
Table 1. Effect of neurotransmitters on the earthworm gut

Condition

Average contraction
rate (BPM)

Contraction
frequency (Hz)

Average contraction
force (N)

Epinephrine 10-5M
Acetylcholine 10-5M

Table 2. Effect of temperature on the earthworm gut

Condition

Average contraction
rate (BPM)

Contraction
frequency (Hz)

Average contraction
force (N)

Normal Saline
(____)C
Cold Saline (_____)C
Warm Saline
(_____)C

Table 3. Effect of extracellular ion concentration on the earthworm gut

Condition

Average contraction
rate (BPM)

Contraction
frequency (Hz)

Average contraction
force (N)

High Ca2+
High K+
Ca2+-free

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Study Questions
1. Describe the response (contraction rate and contraction force) of the earthworm gut to the
following conditions:
a) Acetylcholine

b) Epinephrine

c) Cold

d) Warm

e) High Ca2+

f)

High K+

g) Ca2+- free

2.

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Which condition caused the largest increase in contraction rate?

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3.

Which condition caused the largest increase in contraction force?

4.

Which condition(s) caused a decrease in contraction rate and/or force?

5.

The contraction waveforms often appear to have two peaks. What is the physiological
basis for these two peaks?

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