How-To-Do-It
The Use of an Isolated Earthworm
Crop-Gizzard Preparation as a Model for
Teaching Smooth Muscle Physiology
Kevin G. Krajniak
Smooth visceral muscle is one of
the major types of muscle tissue that
students examine in physiology
courses. It is an integral part of many
organ systems in animals and has several unique characteristics that distinguish it from skeletal and cardiac muscle. It lacks striation, contracts spontaneously, maintains its contractility
when stretched, and is controlled by
neurotransmitters and hormones.
In the classical laboratory for demonstrating smooth muscle properties,
part of a mammalian digestive tract
like the rabbit ileum or rat jejunum is
generally used (Tharp 1997). However,
when these and other mammalian
smooth muscle tissues are used, they
must be handled carefully since contamination will prevent them from
contracting. Also, they must be kept
at the body temperature of the animal,
which is well above room temperature,
thus requiring a heated tissue chamber. Other disadvantages include that
these animals are costly, require special care, may need the approval of
the college or university committee
that oversees animal care and usage,
and may be unsuitable for use in high
school settings.
We have developed a comparable
laboratory exercise showing the properties of smooth muscle with the crop
and gizzard of the earthworm. In this
experiment the student removes the
combined crop-gizzard from the animal and places it in a tissue bath at
room temperature. Once the tissues
are connected to a force transducer,
Kevin G. Krajniak is an Associate Professor and Ryan W. Klohr is a graduate
student in the Department of Biological Sciences at Southern Illinois University Edwardsville, Edwardsville, IL
62026-1651; e-mail: kkrajni@siue.edu.
Ryan W. Klohr
the student can record spontaneous
contractions and effects of neurotransmitters like acetylcholine and serotonin. This experiment has none of the
disadvantages of the mammalian exercise. The animals can be purchased
from a bait shop for only a few dollars
a dozen, kept in the refrigerator until
needed, and used at room temperature. Furthermore, since they are invertebrates, no special authorization is
required.
Earthworm crop-gizzard smooth
muscles are similar to mammalian
intestinal smooth muscles. Both contract spontaneously in isolated tissue
baths (Ruegg 1971; Tuladhar et al.
1997; Ukena et al. 1995, 1996; Krajniak
& Klohr 1999). Both tissues are excited
by acetylcholine (Ruegg 1971; Anderson & Fange 1967; Krajniak & Klohr
1999). Also, serotonin modulates the
motility of both preparations, although
in the earthworm it is inhibitory, while
in the mammal it is excitatory (Tuladhar
et al. 1997; Krajniak & Klohr 1999).
Materials & Methods
This experiment can be performed
with several species of earthworms
including Lumbricus terrestris (Carolina
Biological Supply Company, Burlington, NC) and worms of unknown species commonly sold as giant Canadian
nightcrawlers at local bait shops. In
this paper data are shown from experiments using giant Canadian nightcrawlers. Worms should be kept in
their container at 10o C prior to the
experiment. If they will be kept for
longer than a week, the worms should
be fed Magic Worm food which can
be purchased from Carolina Biological
Supply Company (Burlington, NC). A
list of supplies needed to record mus-
cle contractions of the isolated cropgizzard is given in Table 1.
Contractions of isolated crop-gizzard are recorded as follows: The
worm should be anesthetized by placing it in a 5% solution of ethanol for
5 to 10 minutes. Once anesthetized it
is placed in a dissection tray dorsal
side up, and pins are placed at the
anterior and posterior ends. An incision is made with a pair of fine scissors
from the anterior end to the clitellum
and the body wall is pinned back. The
exposed crop and gizzard is removed
from the animal by cutting connections
distal to the esophagus anteriorly and
proximal to the intestine posteriorly
along with the septa that connect these
structures to the body wall (Figure 1).
The crop-gizzard is placed in a salinefilled petri dish and ligatures are tied
around both ends of the tissue. One
end is secured to a stationary rod,
while the other is connected to a forcedisplacement transducer (Grass FT-03)
coupled to a Grass P122 AC/DC
amplifier and RPS 212 regulated power
supply (Astromed, West Warwick, RI).
Then the tissue should be suspended
in a 40 ml tissue bath filled with saline
(Figure 2). A plastic 30-ml syringe
without the plunger can be used as a
chamber. The saline reservoir is connected to the needle port of the syringe
with rubber tubing and a clamp on
the tubing is used to prevent the flow
of saline into the chamber except during washes. When completely filled,
this chamber holds 40 ml. All recordings are viewed with and stored on
a Pentium computer using a Sable
Systems AD503 A/D converter card
connected to the amplifier and either
the DATACAN V or TLS software
(Sable Systems International, Henderson, NV). Recordings can also be made
with other equipment, like a Harvard
Apparatus Heart/Smooth muscle
SMOOTH MUSCLE PHYSIOLOGY 59
Table 1. Supplies needed by each group of students
Supply
Amount
Giant Canadian nightcrawler
Dissection tray
Pins
Fine scissors
Fine forceps
Cotton thread
Tissue chamber, a 30 ml syringe body
1 Liter aspirator bottle
Rubber tubing to connect the aspirator bottle to the tissue
chamber
Tubing clamp
Ring stand
Clamps to hold the tissue chamber and the stationary rod
in place
Rack and pinion adjustable clamp (Harvard Apparatus,
Holliston, MA) to hold the transducer onto the ring
stand
Stationary rod to hold the crop-gizzard, this can be made
from a piece of wire hanger
Force transducer
Amplifier/power supply
Computer 386 processor or better
Sable Systems AD503 A/D converter card
Sable Systems DATACAN V or TLS software
Aquarium air pump connected to 2 feet tubing and
syringe needle
1.5ml microcentrifuge tubes
100–1000 ml variable volume pipetteman
pH meter
Worm saline*
Stock solutions of 1012 Molar acetylcholine chloride and
serotonin creatinine sulfate (Sigma) solution in distilled
water
1
1
10
1
1
1 spool/class
1
1
2 feet
1
1
2
1
1
1
1
1
1
1
1
6
1
1 / class
2 liters
100 ml of each
* Composed of 14.26 g NaCl, 0.59 g KCl, 3.02 g NaHCO3, 1.104 g NaH2PO4, 0.444g
CaCl2, and 0.406 g MgCl2 in 2 liters of distilled water adjusted to a pH of 7.3 with
NaOH and HCl (Vassileva et al. 1982).
transducer and a chart recorder. In
this instance the crop-gizzard should
be connected to one end of the wooden
lever that passes through the transducer, and a small counter weight
should be added to the other end.
A similar setup can be made with a
kymograph by connecting the tissue
to the stylus that writes on the recording drum.
The saline used in the tissue bath
and all dilutions of the neurotransmitters are prepared according to the
information given in Table 1 and
adjusted to pH 7.3 (Vassileva et al.
1982). Artificial seawater diluted to
one-third strength may also be used
as saline. Serial dilutions of the neurotransmitter with concentrations from
1013 to 1018 M are prepared from a
1012 M stock. A 0.4 ml aliquot of each
dilution of neurotransmitter is added
to the bath beginning with the lowest
concentration. The concentration
reported is the final molar concentration in the bath which can be calcu-
lated by dividing the concentration of
the neurotransmitter added to the bath
by 100, since the 0.4 ml of neurotransmitter added is one hundredth of the
tissue bath volume of 40 ml. Each dose
of neurotransmitter is followed by a
saline wash once the maximal effect
has been recorded. Air should be bubbled continuously into the tissue bath
to ensure a supply of oxygen and mix
the neurotransmitters added during
the experiment. Serotonin creatinine
hydrochloride and acetylcholine chloride can be purchased from Sigma
Chemicals (Sigma Aldrich, St. Louis,
MO); all inorganic salts can be bought
from Fisher Scientific (Houston, TX);
artificial sea salts can be purchased
from a local pet store.
The isolated crop-gizzard shows a
complex pattern of contraction and
relaxation. In some instances the contractions are regular and the pattern
is easily identified, whereas in others
the recording may yield a trace in
which small sporadic contractions are
60 THE AMERICAN BIOLOGY TEACHER, VOLUME 63, NO. 1, JANUARY 2001
interspersed between large contractions. To analyze these types of traces
only the large contractions are used
and the small ones are ignored. This
analysis results in a dose-response
curve similar to one produced by
recording from a regularly contracting
preparation. All amplitudes should be
determined by measuring from the top
to the bottom of each wave form. The
Sable Systems records the amplitude
in volts; however the transducer measures force, so it is necessary to determine a conversion factor from volts
to grams of force. This is done by
hanging a 0.5-gram weight on the
transducer and measuring how many
volts are registered on the computer.
If either a chart recorder or a kymograph is used the recording will show
how many millimeters the tissue has
moved. To calibrate these devices the
weight must be suspended from the
transducer lever or kymograph stylus
and the distance moved by the recording instrument should be noted. Contraction rate is determined by counting
the time interval between contraction
peaks which is minutes per contraction; the inverse of this number is
contractions per minute. In each
instance, 5 to 10 contractions are measured to establish a mean number.
To visualize effects of neurotransmitters it is useful to construct a log
dose-response curve. Since the baseline
of the contractions may shift during
the experiment, we record mean
amplitude and contraction rate before
and after the addition of each neurotransmitter concentration. Then the
percent response for each concentration is calculated using the following formula:
% Response 4
post-pre
2 100
pre
where post is the post-neurotransmitter value and pre is the pre-neurotransmitter value.
The log dose-response curve is created by plotting percent response
against log of the neurotransmitter
concentration. Log concentration can
be calculated by taking the log of the
molar concentration, e.g. a concentration of 1015 M would be 15 on
the graph since log 1015 is 15. From
this curve the threshold concentration
for each neurotransmitter can be
determined.
Another property of smooth muscle
the students should observe is the
effect of increased tension on the contraction patterns of the smooth muscle.
This is accomplished by increasing the
distance between the tissue and the
transducer.
Figure 1. Diagram showing the location of the crop-gizzard in the dissected earthworm. The crop-gizzard is located in the anterior portion of the worm. The ligatures
are tied to the region where the crop meets the esophagus and to the gizzard where
it comes in contact with the intestine. The septa that attach the crop-gizzard to the
body wall must also be severed.
Results
At room temperature the crop-gizzard contracts spontaneously for several hours (Figures 3 & 4). When there
is an increase in tension or stretch, the
tissues respond with a compensatory
relaxation and then resume the previous rhythmic pattern (Figure 5). Acetylcholine causes a concentration
dependent increase in contraction rate
and a biphasic change in contraction
amplitude (F ig ur e 3 ). Lo g d os eresponse curves of these responses are
shown in Figure 6. The increase in
contraction rate of the crop-gizzard
has a threshold between 1017 and 1016
M (Figure 6a). The increase in contraction amplitude has a threshold
between 1018 and 1017 M (Figure 6b).
Serotonin causes a concentration
dependent decrease in both contraction rate and amplitude (Figure 4). Log
dose-response curves are shown for the
pooled data in Figure 7. The decrease
in contraction rate has a threshold of
10 1 7 and 10 1 6 M (Figure 7a). The
decrease in amplitude has a threshold
between 1018 and 1017 M (Figure 7b).
Discussion
The muscle tissue of the earthworm
crop-gizzard reacts just like that of
mammalian smooth muscle. It responds
to increased stretch by relaxation and
a return to normal rhythmic contractions. The pharmacological results in
giant Canadian nightcrawlers are similar to those found in the earthworms
Lumbricus terrestris (Krajniak & Klohr
1999) and Eisenia foetida (Ukena et al.
1995, 1996). Acetylcholine and serotonin modulate the spontaneous contractions as they do in the isolated mam-
malian intestine (Ruegg 1971; Tuladhar
et al. 1997). Thus the giant Canadian
nightcrawler is a good model to demonstrate neurohormonal control of
smooth muscle.
This laboratory exercise usually takes
one three-hour session. The students
first assess the effects of stretch on the
preparation. Then they perform the
experiment with either acetylcholine
or serotonin. In the second half of the
session the students use the computers
to analyze their data and create log
dose-response curves. Once the students have generated the numbers for
their individual experiments they
share them with the other groups to
generate pooled data curves. They then
plot the log dose-response curves for
both their own data and the pooled
data. In their laboratory report they
are asked to explain the results and
compare them to the responses of
smooth muscles in other species of
annelids and mammals. If the single
animal data and pooled data curves
are different they are also asked to
explain the variation in the data.
This laboratory exercise teaches students several different principles of
physiology. It demonstrates that digestive tract smooth muscle contractions
are spontaneous, continue during an
increase in tension, and can be modulated by neurotransmitters like serotonin and acetylcholine. It also familiarizes students with the log doseresponse curve which defines the basic
interactions between a drug or neurotransmitter and its receptor.
References
Anderson, R. & Fange, R. (1967). Pharmacological receptors of an annelid
Lumbricus terrestris. Archives Internationales de Physiologie, de Biochimie et
de Biophysique, 75, 461–468.
Krajniak, K.G. & Klohr, R.W. (1999).
The effects of FMRFamide, serotonin,
and acetylcholine on the isolated
crop-gizzard of the earthworm, Lumbricus terrestris. Comparative Biochemistry and Physiology, 123A, 409–415.
Ruegg, J.C. (1971). Smooth muscle tone.
Physiological Reviews, 51, 201–248.
Tharp, G.D. (1997). Experiments in Physiology, 7th ed. Upper Saddle River, NJ:
Prentice Hall.
Tuladhar, B.R., Kaisar, M. & Naylor,
R.J. (1997). Evidence for a 5-HT3
receptor involvement in the facilitation of peristalsis on mucosal application of 5-HT in the guinea pig
isolated ileum. British Journal of Pharmacology, 122, 1174–1178.
Ukena, K., Oumi, T., Matsushima, O.,
Ikeda, T., Fujita, T., Minakata, H. &
SMOOTH MUSCLE PHYSIOLOGY 61
Figure 2. A diagram showing the apparatus used to record earthworm crop-gizzard contractions. The illustration shows the
crop-gizzard in the tissue bath. One end is tied to a support rod and the other is attached to the force-transducer. The tissue
bath is connected to a saline reservoir and an air line. The force-transducer is connected to a power source and an amplifier
which is linked to a recording computer.
62 THE AMERICAN BIOLOGY TEACHER, VOLUME 63, NO. 1, JANUARY 2001
Figure 3. The effects of different acetylcholine concentrations
on a single isolated crop-gizzard of a giant Canadian nightcrawler. The arrow at the top of the figure and the vertical
dashed lines indicate when the neurotransmitter was added;
the final molar concentration in the bath is indicated on the
left of each record.
Figure 4. Effects of increasing serotonin concentrations on a
single isolated crop-gizzard of a giant Canadian nightcrawler.
The arrow at the top of the figure and the vertical dashed
lines indicate when the neurotransmitter was added; the final
molar concentration in the bath is indicated on the left of
each record.
SMOOTH MUSCLE PHYSIOLOGY 63
Figure 5. The effect of stretch on the rhythmic contractions of the isolated crop-gizzard of a giant Canadian nightcrawler. The
dashed line indicates when the distance between the crop-gizzard and transducer was increased, resulting in a downward
deflection (increase in tension) and a compensatory relaxation (upward deflection).
Figure 6. Log dose-response curves showing the effects of
acetylcholine on the isolated crop-gizzard of giant Canadian
nightcrawlers. (a) Effects of acetylcholine on percent change
in contraction rate of the isolated crop-gizzard. (b) Effects of
acetylcholine on percent change in contraction amplitude of
the isolated crop-gizzard. In both graphs each point is the
average of five different preparations and the vertical bars
represent standard errors.
Figure 7. Log dose-response curves showing the effects of
serotonin on the isolated crop-gizzard of giant Canadian
nightcrawlers. (a) Effects of serotonin on percent change in
contraction rate of the isolated crop-gizzard. (b) Effects of
serotonin on percent change in contraction amplitude of the
isolated crop-gizzard. In both graphs each point is the average of five different preparations and the vertical bars represent standard errors.
64 THE AMERICAN BIOLOGY TEACHER, VOLUME 63, NO. 1, JANUARY 2001
Nomoto, K. (1995). Effects of annetocin, an oxytocin-related peptide isolated from the earthworm Eisenia
foetida, and some putative neurotransmitters on gut motility of the
earthworm. Journal of Experimental
Zoology, 272, 84–195.
Ukena K., Oumi T., Matsushima O.,
Takahashi T., Muneoka Y., Fujita T.,
Minakata H. & Nomoto K. (1996).
Inhibitory pentapeptides isolated
from the gut of the earthworm Eisenia foetida. Comparative Biochemistry
and Physiology, 114A, 245–249.
Vassileva, P.V., Stoyanov, I.N. & Vassileva, V.I. (1982) On the contractile
activity of the alimentary canal of
the earthworm (Lumbricus terrestris).
Comparative Biochemistry and Physiology, 71C, 127–129.
SMOOTH MUSCLE PHYSIOLOGY 65