BIOLOGY 109.

2 PHYSIOLOGY LABORATORY
DEPARTMENT OF BIOLOGICAL SCIENCES
Laboratory 6: Nervous Physiology Heart and Intestine in Normal and Modified Ringers
(Ca and Mg Concentration Modifications)
Overview
Cardiac and smooth muscle have special structural and physiological
properties related to their distinctive functions. They also have certain properties
in common with each other.
Any of the three types of muscle cells can be called myocytes. This term is
preferable to muscle fiber for smooth and cardiac muscle because these two types
of cells do not have the long fibrous shape of skeletal muscle cells. They are
relatively short, and in further contrast to skeletal muscle fibers, they have only
one nucleus. Cardiac myocytes are also called cardiocytes.
Cardiac muscle and smooth muscle are involuntary muscle tissues, not
usually subject to our conscious control. They receive no innervation from somatic
motor neurons, but cardiac muscle and some smooth muscle receive nerves from
the sympathetic and parasympathetic divisions of the autonomic nervous system

Stimulation
Equipment

System
under test

Measuring
Equipment

Cardiac muscle is limited to the heart, where its function is to pump blood.
Knowing that, we can predict the properties that it must have: (1) It must contract
with a regular rhythm; (2) it must function in sleep and wakefulness, without fail
and without need of conscious attention; (3) it must be highly resistant to fatigue;
(4) the cardiocytes of a given heart chamber must contract in unison so that the
chamber can effectively expel blood; and (5) each contraction must last long
enough to expel blood from the chamber. These functional properties are the key
to understanding how cardiac muscle differs structurally and physiologically from
skeletal muscle.
Smooth muscle differs from skeletal muscle in several ways. It is
named smooth because it has no striations, for a reason to be described shortly.
Its myocytes are relatively small, allowing for fine control of such tissues and
organs as a single hair, the iris of the eye, and the tiniest arteries; yet in the
pregnant uterus, the myocytes become quite large and produce the powerful
contractions of childbirth.
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Smooth muscle is not always innervated, but when it is, the nerve supply
is autonomic, like that of the heart. Autonomic nerve fibers do not form precisely
localized neuromuscular junctions with the myocytes. Rather, a nerve fiber has as
many as 20,000 periodic swellings called varicosities along its length. Each
varicosity contains synaptic vesicles from which it releases neurotransmitters
usually norepinephrine from the sympathetic fibers and acetylcholine from the
parasympathetic fibers. The myocyte has no motor end plate, but instead has
receptors for these neurotransmitters scattered over its surface. The varicosities
simply release a flood of neurotransmitter into the tissue, and each myocyte may
respond to more than one nerve fiber.
Whether innervated or not, smooth muscle responds to a wide variety of
stimuli and often without any electrical excitation of the sarcolemma. It is much
slower than skeletal and cardiac muscle to contract and relax, but it can remain
contracted for a long time without fatigue and with minimal energy expenditure.
Composition of Normal Frog Ringer's (2.5 mM KCl)
Compound
_____ mM
KCl
2.50
NaCl
115.00
Na2HPO4
4.00
NaH2PO4
0.85
CaCl2*
1.80
MgCl2
1.00
*Add after spinning to oxygenate.

g for 1L
0.19
6.72
0.57
0.12
0.26
0.20

To make a 1L solution of normal frog Ringer's, prepare as follows:

1. Add to 100mL distilled H2O all ingredients above except CaCl2.
2. Add more distilled H2O to 1L.
3. Spin to oxygenate.
4. Add CaCl2
5. Adjust pH to 7.4 with 1M NaOH (if needed).
For the LOW Ca, HIGH Mg solution used to create a partial synaptic block
during EPP measurements, do not add the CaCl2, but instead add 0.12g CaCl2 to
800mL of the solution and 0.37g MgCl2 to 200mL of the solution, then mix
together.
The primary form of the response is the voltage across a single muscle cell
membrane that you will measure. A signal can be recorded on an oscilloscope or
computer but for this activity, you will measure the response using an improvised
devise such as a transparent ruler under the petri plate with the muscle-nerve
preparation on solution.
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Concept Application
Regardless of how a myocyte is stimulated, however, the immediate
trigger for contraction is the same as in skeletal and cardiac musclecalcium ions.
In some cases, the Ca2+ comes from the sarcoplasmic reticulum (SR), as it does in
skeletal muscle. With a relatively sparse SR, however, smooth muscle usually gets
most of its Ca2+ from the extracellular fluid by way of gated calcium channels in
the sarcolemma. Compensating for the paucity of SR, the sarcolemma has
numerous little pockets called caveolae that increase the cell surface area and
number of calcium channels. Calcium is 10,000 times as concentrated in the ECF
as in the cytosol, so if these channels are opened, it diffuses quickly into the cell.
Because smooth muscle cells are relatively small, the incoming Ca2+ can quickly
reach all of the myofilaments.
Objectives:
1. _______________________________________________________________
_______________________________________________________________
2. _______________________________________________________________
_______________________________________________________________
3. _______________________________________________________________
_______________________________________________________________
4. _______________________________________________________________
_______________________________________________________________
Materials and Chemicals
Petri plates, glass rod
Dissecting set (with sharp fine scissors and scalpel blades, forceps, hook)
Surgical gloves, clean cotton, string
Live large toads (frogs are more preferred depending on the availability), 3 per
group
Dissecting pan
Normal Ringers (based on the given composition, p31)
Modified Ringers (based on the given composition, p31)
Pasteur pipettes
pH meter (pen type)
1N NaOH, 1N HCl
Methods:
DISSECTION AND ISOLATION OF THE HEART AND SMALL INTESTINE OF TOAD
1. Paralyze the toad by pithing. When already paralyzed, lift the skin using
the shearing forceps in the lower abdomen and cut open with sharp scalpel
blade to access the abdominal muscle sheet.
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2. Carefully make a slit along the midline of the linea alba and cut through
the mid-ventral part of the muscles without damaging the delicate internal
organs underneath the thin abdominal sheet. Continue the straight cut to
the thoracic part along the sternal cartilage to the clavicle areas (to the
leaft and to the right side to completely open the ventral side of the toad
(Figure 1).

Figure 1. Opening the mid-ventral region of the frog (www.sci.utah.edu)

3.

4.

5.

6.

The Heart and the Influence of Ions in the Cardiac Activity

The heart is enveloped by a thin pericardium. Cut through carefully the
thin sac to expose the beating heart. Observe the normal heart activity and
record the heart rate.
Add drop by drop Normal Ringers into the open sac so that the heart is
flooded with the solution. Start counting the heartbeat. Record the heart
rate. Allow the heart to stay under this state for 3-5min.
Using clean cotton, blot out the Normal Ringers solution without totally
making the heart dehydrated. Then drop by drop, flood the heart with the
Modified Ringers solution. Observe the changes in heartbeat after about
1 min. Record the heart rate.
Replace the Modified Ringers by blotting out with clean cotton.
Immediately add Normal Ringers. Observe for 1 to 2 min and then record
the heart rate again.

The Small Intestine and the Influence of Ions in the Smooth Muscle
7. Expose the small intestine of the toad (Figure 2).

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Figure 2. The small intestine (arrow) of the frog

(www.biologycorner.com)
8. Expose the jejunum the small intestine from the terminal of the duodenum
by lifting with the use of the hook. Drop Normal Ringers solution to the
intestine and observe the activity. If the toad is well fed, the intestine will
be actively doing peristalsis.
9. Pat dry the intestine and start dropping Modified Ringers and leave the
solution in the intestinal region for about 3min.
10. Observe the changes in the activity of the small intestine. Note all
observable changes in the movements.
References:
Anatomy and Physiology, Kenneth S. Saladin, 6th ed. McGraw-Hill. 11.7: Cardiac
and Smooth Muscle.

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NAME:___________________________________ DATE:__________________
COURSE/SECTION:______________ INSTRUCTORS SIGNATURE:____________
Laboratory 6: Nervous Physiology Heart and Intestine in Normal and Modified Ringers
(Ca and Mg Concentration Modifications)
Scientific Exploration
The entire frog heart experiment will allow the students to learn the
dissection of the frog, measure the resting heart rate and study the chemical
sensitivity and determine the effect of modified ionic composition. Performing the
detailed pharmacological studies on the heart to test actions of Calcium and
Magnesium, and other ions in two different physiological solutions. Using your
good senses, make valid judgments concerning the changes in a heartbeat and not
the electrical activity as in an electrocardiogram (ECG).
On the other hand, motility in the small intestine, as in all parts of the
digestive tube, is controlled predominantly by excitatory and inhibitory signals
from the enteric nervous system. These local nervous signals are however
modulated by inputs from the central nervous system, and a number of
gastrointestinal hormones appear to affect intestinal motility to some degree.
Table 1. Determination of resting heart rate.
Beats/min Difference between first Calculated Heart Expected
Heart
and last beat (sec)
Rate (BPM)
Rate (BPM)

Table 2. Effect of ionic composition on heart rate.

Condition
Heart Rate (BPM)
Normal
Modified
Modified to Normal
Exploratory Questions:
1. Give the significance of the following to the cardiac and smooth muscles:
a. Normal Ringers solution composition: __________________________
____________________________________________________________
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____________________________________________________________
b. Modified Ringers solution composition: _________________________
____________________________________________________________
____________________________________________________________
2. Describe the actions of substances in the heart muscle and the intestine:
a. NaCl: _____________________________________________________
____________________________________________________________
____________________________________________________________
b. KCl: ______________________________________________________
____________________________________________________________
____________________________________________________________
c. CaCl2: _______________________________________________________
____________________________________________________________
____________________________________________________________
3. Place a photo of your set up in the box below. Label the parts.

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4. Provide the required parameters for the above set up:

a. Duration of application of solution to heart/intestine: _______/______
e. Interval of application of solution to heart/intestine: _______/_______
f. Tissue reaction (please check): heart (Positive____
Negative____)
intestine (Positive____
Negative____)
5. What was the heart/intestines reaction to:
a. Modified Ringers: (Heart) ____________________________________
(Intestine) ___________________________________________________
b. Placed back to Normal Ringers: (Heart) _________________________
____________________________________________________________
(Intestine) ___________________________________________________
____________________________________________________________
6. Explain/discuss your observations._________________________________
______________________________________________________________
______________________________________________________________
7. Place your documentation of the frog/frog muscle-nerve preparations
below:

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Discussion:
______________________________________________________________
__________________________________________________________________
__________________________________________________________________
__________________________________________________________________
__________________________________________________________________
__________________________________________________________________
__________________________________________________________________
__________________________________________________________________
__________________________________________________________________

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