Graphs and charts in Amphibian experiemnts in physiology

1. (A) PROPERTIES OF HEART: AUTOMATICITY, RHYTHMICITY, EXTRA-SYSTOLE AND
COMPENSATORY PAUSE
AIM: To study the properties of cardiac muscle in a beating frog’s heart
1.Automaticity and Rhythmicity
2.Extra-systole and Compensatory pause
REQUISITES: Kymograph (the instrument which records movement on a moving surface), heart
trough, Simple heart lever, Ringer solution, Thread, Bipolar electrodes or Thin loop of tinsel wire
for stimulating the ventricle and Bent pin (small pin to hook the apex of heart)
PROCEDURE: Set up the stimulator at single stimulus. Bipolar electrodes are used for stimulating
the heart muscle. Care should be taken that stimulation of heart with electrodes does not
displace the preparation; alternatively thin loop of tinsel wire can be used for stimulation of the
heart. Arrange the recording apparatus as required for recording normal cardiogram. Expose the
heart of the pithed frog. Pass the bent pin through the apex. Mount the frog on the board and
attach the heart to the heart lever. Speed of the drum should be 2.5 mm/sec.

2. 1) AUTOMATICITY AND RHYTHMICITY: The capability to contract in the absence of neural control is
called automaticity. Cardiac rhythmicity is the spontaneous depolarization and repolarization
event that occurs in a repetitive and regular manner. Normal cardiogram is recorded on slow
moving drum. You will observe that the heart beats spontaneously and in a regular fashion,
indicating the automaticity and rhythmicity.
2) EXTRA-SYSTOLE AND COMPENSATORY PAUSE: Normally the ventricle contracts as a result of
impulse generated from the pacemaker. However, if an artificial extra stimulus is given, it responds
by contracting prematurely. This is known as extrasystole or ventricular premature contraction.
The pause following the extrasystole is known as compensatory pause. Set up the experiment as
for recording a normal cardiogram. Apply electrode on the ventricle of heart. A few normal beats
are recorded. Stimulate the ventricle during different phases of the cardiac cycle (a) during systole,
(b) during early diastole, (c) during late diastole and record the effects.

4. OBSERVATIONS: It will be observed that there is no effect on normal heartbeat when
heart is stimulated during systole or early diastole. Stimulating the heart in late
diastole produces an extra systole (ES), i.e., heart muscle responds by producing an
extra premature contraction which is followed by a compensatory pause (CP).
QUESTIONS:
1) Define the terms: Extra-systole and compensatory pause.
2) What is compensatory pause due to?
3) Give the physiological significance of compensatory pause.
4) What are the factors which predispose to extra-systole in humans?
5) How many extra-systoles per minute are considered as normal? When do they
become clinically significant?
6) When is the heart refractory? What is meant by absolute and relative refractory
periods?
7) What purpose is served by the long refractory period for the cardiac muscle?

5. (B) PROPERTIES OF HEART: AUTORHYTHMICITY
AIM: To study the property of Autorhythmicity of cardiac muscle in a beating frog’s heart after Stannius
ligature.
AUTORHYTMICITY: It is the ability of the heart to initiate its beat continuously and regularly without
external stimulation.
A normal heart beats with a perfect regular rhythm. The time interval between 2 consecutive beats is
exactly the same, due to regular discharge from sinus venosus (pacemaker) in a frog. However, if the
pacemaker fails to generate impulse, the other parts of the heart can takeover & generate impulse. This is
known as autorhythmicity.
STANNIUS LIGATURE: The conduction of impulse can be blocked partially or completely by mechanical
pressure such as by tying a thin thread knot at various sites. These are known as stannous ligatures.
PROCEDURE: Prepare the Frog as for recording of the normal heart beat. With the held-up of fine
pointed forceps, pass a wet thread between the bulbus arteriosus and the rest of the heart. Lift the apex
upwards and forward by means of the hook attached to it. Bring the two ends of the thread down and
around, so that it lies between the auricles and the sinus venosus and leave it loose at present.
Mount the frog on the board and attach the heart to the lever. Record a few heart beats on slow moving
drum (speed 2.5 mm/sec)

6. FIRST STANNIUS LIGATURE: Tighten the knot around the sinus venosus at a point which
is marked by the white crescentic line. This is the first stannous ligature. If the ligature
is correctly applied, the auricles and ventricle will stop beating after one or two beats,
the sinus continuing to beat at the previous rate. Stop the drum when the sinus beat
has been recorded.
After a lapse of a period of 5-30 minutes, the auricles and ventricle begin to beat again,
the auricles contracting first followed by the ventricle. Compare the rhythm and rate
with that of sinus.
SECOND STANNIUS LIGATURE: Tie a ligature around the auriculo-ventricular junction
and record the effects of this ligature. Ventricle will stop beating and after a lapse of
sometime, it starts beating rhythmically independent of auricular rhythm. This is
known as idioventricular rhythm. Compare the rhythm and rate with that of the
auricles.

8. QUESTIONS:
1)Where do you apply the first stannous ligature and why?
2)Why cannot these ligatures be applied to the mammalian heart

9. (C)PROPERTIES OF HEART: ALL-OR-NONE LAW; STAIRCASE PHENOMENON
AIM: To study the properties of cardiac muscle on quiescent heart (non-beating heart)
1)All-or-none law
2)Staircase phenomenon
• According to all or none law, when a stimulus is applied, whatever may be the strength, the
whole cardiac muscle gives maximum response or it does not give any response at all.
• If the heart is stimulated repeatedly with the interval between the consecutive stimuli not
less than 10 sec, the 1st
few contractions progressively increase in amplitude. This is known
as staircase phenomenon.
PROCEDURE: Tie the first stannius ligature around the sino-auricular junction. Stimulate the
quiescent heart with sub-threshold stimuli. And record the effect. Again, stimulate the heart
with a threshold and supra-threshold stimuli strength and observe the effect. It will be seen
that increasing the stimulus strength beyond the threshold value does not produce any change
in the amplitude of the contraction. There must be a time lapse of at least 25-30 seconds
between the two successive stimuli.

10. Figure represents All or None law
Figure represents Staircase Phenomenon (Beneficial effect)

11. Tie the first stannius ligature. Stimulate the quiescent heart with minimal threshold
stimuli in quick succession, allowing just enough time for the heart to contract and
relax in between two successive stimuli. Record the first 5 or 6 contractions on a slow-
moving drum. It will be seen that the amplitude of contraction keeps on increasing
with successive stimuli, because of beneficial effect.
QUESTIONS:
1)What is staircase effect due to?
2)Why was it necessary to wait about half a minute between the stimuli for
demonstration all or none law?

12. EFFECT OF VAGAL STIMULATION ON HEART
AIM: To show the effect of stimulation of vagosympathetic trunk and white crescentic
line on frog heart.
PRINCIPLE: Stimulation of the vagosympathetic trunk results in cardiac inhibition.
However, if the stimulation continues, the heart recovers from the inhibitory effect of
the vagus. This is demonstrated by recording the effect of vagosympathetic stimulation
of the heart on a moving drum.
White crescentic line (WCL): It is located at the junction between sinus venosus and the
atria.The ganglia, called BIDDER’S and REMAK’S are situated in the crescentic area. WCL
is constituted of cells of post ganglionic parasympathetic neurons.
REQUISITES: Student’s stimulator, kymograph, frog board, simple heart lever, electrodes,
pen marker, amphibian Ringer solution, dissection instruments, etc.

13. PROCEDURE: Fix a pithed frog to the frog board and open the chest to expose the
heart. Prolong the incision upwards to the lower jaw. Cut through the thin platysma
myoides and remove the threads of tissues running from the angle of the jaw. This will
expose the thin petro-hyoid muscle. This muscle runs from the base of the skull and is
inserted into the posterior cornu of the hyoid bone. Cut the brachial nerve. The
glossopharyngeal (lateral) and the hypoglossal (medial) nerves run forwards, crossing
the petro-hyoid muscle. Along the lower border of the petro-hyoid muscle there lies
a neurovascular bundle consisting of the laryngeal nerve, carotid-artery and the
vogues, from above downwards. For further identification, the area is cleared of the
tissues. A glistening tendon of levator scapulae inferioris can be seen in the depths
and neurovascular bundle runs across the tendon. The bundle is separated by a glass
rod. The nerve is kept moist with Ringer solution and is handled with great care. A
thread is passed beneath the trunk along-with the carotid artery. The dissection is
done on both sides.

14. The vagosympathetic trunk is placed on the pair of electrodes. The voltage knob of
the stimulator is adjusted to give a stimulus of 6-8 volts and the frequency regulator
turned to 20 or 30 cps. Record a few normal cardiograms in the usual manner and
then turn the stimulus mode selector knob of the stimulator to Pulses position, in
order to stimulate the vagosympathetic trunk. After recording the effect for a few
seconds, discontinue the stimulation and record the normal cardiogram again for a
few second. Then stimulate the white crescentic line located at the junction of the
sinus venous and the auricles and record the effect in a similar way.
To Record ‘Vagal Escape’ Phenomenon: When the vagus is stimulated continuously,
after some time, the heart escapes from the inhibitory action of vagus. To
demonstrate this ‘vagal escape’ phenomenon, the vagus is continuously stimulated
till the heart resumes its normal contractions. Repeat the observation by stimulating
the white crescentic line.

16. OBSERVATION: Study the effect of stimulation (of different strengths) of vagosympathetic trunk
on the cardiac activities. Note that at a lower strength of stimulus, there is a slowing down of
the heart, but the heart stops with a higher strength of stimulus. Also note that the heart beat
reappears when stimulation is continued for a longer duration. The heart rate of the vagal
escape is significantly less than the heart rate prior to the vagal stimulation (normal heart rate).
Vagal Inhibition: When the vagus nerve (vagosympathetic trunk) is stimulated in a frog, there is
inhibition of the heart. The heart rate and the force of contraction decrease. This is called vagal
inhibition. If the stimulus is strong, the heart stops. Cardiac inhibition occurs due to the release
of acetylcholine at the nerve endings. This increases the potassium conductance in the nodal
tissues by opening a special set of potassium channels.
Acetylcholine also decreases the concentration of cyclic AMP in the cells. The decrease in cyclic
AMP slows the opening of the calcium channels, which decreases the firing rate. A decrease in
calcium concentration in the myocardial cells decreases the force of contraction

17. Vagal Escape:
A strong vagal stimulation may abolish the spontaneous discharge of the heart for some
time and the heart stops temporarily. But the heart recovers automatically even after the
continuation of vagal stimulation. This is called vagal escape. The heart rate following vagal
escape is significantly lower than the normal heart rate because the escape rhythm is the
ventricular rhythm.
Causes of vagal escape
1.Idioventricular rhythm: When the heart stops due to vagal stimulation, the ventricle starts
generating the impulse, which is known as idioventricular rhythm. It is so called because the
cardiac rhythm is due to the pacemaker activity of the ventricular muscle, and the exact cause of
the ventricular pacemaking activity is not known. As the discharge rate of the ventricle is much
less, the idioventricular rhythm is significantly lower than the normal heartrate.
2.Depletion of acetylcholine: When the vagus nerve is stimulated continuously, the acetylcholine
released at the nerve ending is depleted after some time.Acetylcholine is degraded rapidly the
enzyme cholinesterase. Therefore, the effect of vagal stimulation on the heart is temporary.

18. PRECAUTIONS:
1.The vagosympathetic trunk should be identified by its anatomical landmark.
2.Before carrying out the experiment, vagosympathetic trunk should be confirmed by
stimulating the trunk and observing the slowness of the heart.
3.The effect of the vagal stimulation on the heart should be studied from a lower
strength to a higher strength of the stimuli.
4.After recording the vagal inhibition , the stimulation should be continued to study the
phenomenon of vagal escape.
5.The cardiogram should be recorded before, during and after stimulation of
vagosympathetic trunk.
6.The recording should be labeled properly to mark the start and termination of
stimulation.

19. QUESTIONS:
1. What are the differences between the stimulation of the left and the right vago-
sympathetic trunks?
2.Describe the type of fibers present in the vago-sympathetic trunk?
3.What is the mechanism of vagal and white crescentic line inhibitions?
4.What is vagal escape? What are its causes?
5.What is the importance of the white crescentic line?