practical physiology

1. Introduction to amphibian
experiments
Annepaka eliya raju
Tutor
Mamata medical college
khammam

2. 1. GENERAL INTRODUCTION
То study the nerve-muscle physiology, frogs are the
preferred animals because of the following reasons

3. (1) Frogs are cold blooded animals. Therefore, their nerve
muscle preparation survival under average experimental
conditions is superior to that of warm-blooded animals.
(2) They are easily available round the year and create no
nuisance during storage or while handling (absolutely harmless).
3) Their tissues can survive for quite some time in
vitro (outside the body) without oxygenation and temperature
control. Their muscles can absorb oxygen directly from the
atmosphere.
(4) They are easy to handle during experiment and can easily be
disposed of afterwards.

4. Source of current
Electrical stimulation can be given either with a direct
current (DC) source and a pair of stimulating electrodes
(galvanic current) or by using an induction coil and the
electrodes (faradic current or induced current).
Wires; Usually copper or aluminum wires are used in
laboratories to carry electric current. A single thick wire is used to
supply direct current. The wires are insulated by cotton, silk or
enamel. Copper wires are usually used.
The wire should not be damaged or have cracks.

7. Keys
The key is a device used for completing or interrupting a circuit.
Two types of keys are used, the simple or tap key, and the short
circuiting key.
The tap key ; This is connected in the primary circuit in
series with the DC source. The key is pressed gently and released
to make and break the circuit.
The short circuiting key This is connected in
the secondary circuit in parallel to prevent accidental leakage of
current into the tissues.

8. InductoriumThe inductorium( induction coil)
The inductorium used is known as the Du Bois-Reymond
inductorium (introduced by Du Bois-Reymond in 1849).
This is a simple device for transforming direct current into
induced current.
It consists of two separate coils: the primary coil and the
secondary coil. The primary coil is fixed on a frame and
the secondary coil is a movable one that covers the
primary coil,

10. The primary coil
The primary coil consists of 300 turns of insulated thick copper
wire wound around a soft iron core. The primary coil, the direct
current source and the tap key are connected in series and this
constitutes the primary circuit.
The secondary coil The secondary coil is made of 5000
turns of very copper fine wire. The secondary coil slide on two
horizontal metal slide rods. On one of the slide rods, a scale is
marked (in cm) by which the distance between the coils can be
measured. The two terminals of the secondary coil are connected
to the stimulating electrodes from secondary circuit

11. The inductorium also has a built-in interruptor
(Neef's hammer) which works on the same principle
as that of an electric bell.it is present behind frame,
work of primary circuit

12. Stimulating electrodes
Electrodes used in biological
experiments differ depending on their
manufacture and use .
Stimulating electrodes are used for
delivering the electric stimulus to the
tissues. It consists of two copper wire
held together by a piece of perspex

13. Kymograph
Kymograph is the name given to any
instrument that records movements on a
moving surface. It consists of a metal gear
box to which a vertical rotating shaft is
connected. The shaft is powered by a
horizontal axis running through the metal case.
To this axis, on one side, a series of pulleys are
attached. A belt connects one of these pulleys
to one of the pulleys in the power shaft. A
cylinder (6" x 6''), also called drum, is fixed to
the shaft. The drum rotates with the shaft. A
gear switch on the left side of the kymograph

14. Electric kymograph: (1) Kymograph body containing electric motor and gear system, (2)
Leveling screw, (3) On-off switch for mains current, (4) Calibrated speed setting system, (5)
Clutch lever, (6) Electric contact block, (7) Spindle, (8) Dual electric contact arms, (9)
Cylinder, (10) Screw lift for cylinder, (11) Lever for fixing cylinder on the spindle

15. There are two horizontal contact arms that
project
from the lower end of the vertical
shaft.projecting strickers These contact
arms can be separated and fixed with various
angles between them. The tips of the arms,
when they revolve, make contact with a
spring at the left side of the top of the
kymograph.
There are two terminals for electrical
connection: one is attached to the insulated
spring and the other to the metal case of the
gear box. By means of these connecting

16. Muscle trough
The muscle trough is a perspex or plastic
chamber used to keep the muscle moist
and viable in Ringer's solution. A block
carrying the stimulating electrodes is fixed
on the side walls of the trough.
The writing lever is fixed on the other
side wall of the trough. from the base of
the muscle trough, a drainage pipe is
provided with a clamp that helps to drain
the Ringer's solution from the trough
whenever required.

17. Levers
Different types of levers are used in experimental physiology for various
purposes. Commonly used levers are the
• writing simple lever,
• starling heart lever,
• isometric lever,
• after load lever
• frontal lever

18. The lever consists of a horizontal arm fulcrum
, which bears holes and notches for hanging
the weights. The lever is fixed to the side wall
of the muscle trough. The writ-ing point of
the lever is made up of a triangular piece of
photographic film.
An ink writing stylus can be fitted into the
writing lever which can write on a white
glazed paper instead of on a smoked drum.
There is a screw (afterload screw) near the
fulcrum of the lever that limits the downward
movement of the lever.

19. The starling heart lever
This is used for recording the cardiogram of a frog's
heart. This lever is more sensitive than the writing lever,
as it records the contractions of the heart, which is weaker
than contractions of the gastronemius muscle. It consists
of a frame with a light steel lever, with holes and
notches, supported by a fine adjustable nickel silver
spring.

22. Isometric lever
This consists of a holder that carries a steel tension
spring and a flat writing lever. It is used for recording
isometric contractions.
Myograph stand
This is a vertical rod fixed to a heavy and triangular
base . The muscle trough can be fitted to
the rod and can be moved up and down with the help
of a fitted screw. The rod can be turned on its axis
so that the writing point of the muscle lever can be
made to touch or be removed from the drum with out
disturbing other adjustments

24. Tuning fork
A tuning fork with a frequency of 100
vibrations per second (100 Hz) is used for measuring
different time intervals. To the end of one arm of the
tuning fork, a writing point is attached. The tuning fork
is set to vibrate and is then made to write on the fast
rotating drum to obtain a tracing. This tracing consists
of different waves. Each wave of the tracing (from crest
to the crest) measures 0.01 seconds (10 ms).

26. Smoking
Before smoking the drum, a piece of a glazed paper is
properly pasted on the drum. Then the drum is placed
on the horizontal arm of the smoking stand The burner is
put on and the drum is smoked
uniformly by rotating manually on the flame. A thin
and uniformly black smoking is aimed at.

27. Varnishing
Varnishing is done to fix the recording on the smoked
paper. Labelling of the recording is done before
varnishing. The paper is cut at the jointed portion and
taken out of the drum and dipped in the 2 per cent
solution of resin or methylated spirit. Then the paper
is clipped and hung till it is completely dry.

28. Composition of Ringer solution
Nacl : 0.6% (isotonic with frog plasma)
КСІ : 0.014% (maintains membrane potential)
CaCl, : 0.012% (maintains muscle excitability)
NaHCO, :0.02% (maintains pH)
NaH,PO, : 0.001% (maintains pH )
Dextrose : 0.1% (provides nutrition)

29. GASTRONEMIUS MUSCLE AND SCIATIC NERVE
PREPARATION OF FROG
APPARATUS
PITHING NEEDLE .
SCISSORS
FORECEPS
BONE CUTTER WOODENBOARD
DISSECTION BOARD .
A PIECE OF CLOTH .
BEAKER .
RINGERS SOLUTION
,GLASS RODS AND COTTON
WOOL

30. PROCEDURE
1. Stunning of Frog
Hold the frog by its hind limbs and give a single hard
blow on its head against the edge of the table. The
frog becomes unconscious before it realises what has
happened

31. 2. Pithing of Frog
Pithing of frog, i.e. destroying its brain and the
spinal cord.
(i) Hold the unconscious frog in the left hand and
ventroflex its head with help of the index finger
(ii) Feel for the depression in the mid-dorsal line which
joins with the posterior border of the tympanic
membranes. This is the point of the
junction of the skull with the vertebral column.
Through this depression, insert a pithing needle into
the cranial cavity. This is indicated by sudden loss of
resistance. Destroy the brain by the rotatory
movements of the needle.
(iii) Take out the needle and introduce it into the
vertebral canal and, in the similar fashion, destroy
the spinal cord.

32. 3. Dissection of Sciatic Nerve
(i) Cut the skin of the frog all around at the middle of the trunk, and strip
the skin off its hind limbs.
(ii) Lay the frog on its abdomen on a wooden dissecting board. Mobilise
the tip of urostyle by cutting through the muscles along its side. Lift it up
and carefully cut it away, taking care not to injure, the underlying sciatic
nerve.
(iii) Identify the sciatic plexus, isolate 1.5-2 cm long vertebral column
from where sciatic plexus originates, by cutting with a bone-cutter below
and above the exit of the plexus

33. Iv. Bisect the vertebral column by
inserting pointed end of the scissor into
the vertebral canal, taking care not to
injure the sciatic nerve.
v) Expose the sciatic nerve in thigh between
muscle masses. Gently isolate the nerve
from adjacent structures proximally up to
the sciatic plexus and distally up to knee
joint.

34. 4. Dissection of Gastrocnemius Muscle
(1) Identify, disect and cut the gastrocnemius tendon from its
attachment. Tie one end of the long thread around it.
(ii) Separate out the gastrocnemius muscle from the adjacent
muscles by cutting through the delicate fascia in between. Then
free the muscle from tibia.
(iii) With the help of a bone-cutter, cut the tibia close to knee joint,
and also cut femur near knee joint.
Nerve muscle preparation is ready. Keep it immersed
in Amphibian warm Ringer's solution, till needed for experiment.

36. Simple muscle twitch
When a muscle is stimulated with a
single induction shock ,it exhibits a
momentary twitch like a contraction
.this momentary contraction of the
muscle in response to electrical
stimulation is called simple muscle
twitch .the contraction recorded on a
moving kymograph is known as a

37. The contraction period is the period between the point of
onset of contraction to the point that corresponds to
the peak of contraction.
The relaxation period is the period from the peak of
contraction to the end of relaxation.
Principle;
When a muscle is stimulated with a single induction
shock, the muscle contracts.This lifts the lever to
record a curve on a revolving smoked drum.

38. Requirements
1. Dissection instruments
2. Kymograph
3. Muscle trough
4. Inductorium
5. Short circuiting key
6.Tap key
7. Ringer's solution
8. Smoked drum
9. Low-resistance wires
10. Electrodes
11.Tuning fork (100 Hz)
12.Thread
13. Hook and weights

39. Procedure
1. Arrange the primary and secondary circuits for
recording a muscle twitch
2. Include the kymograph in the primary circuit.
3. Select the fastest speed of the kymograph
4. Dissect a frog to obtain a sciatic nerve-gastrocnemius
preparation
5. Fix the nerve-muscle preparation in the muscle trough.
6. Pour Ringer's solution into the trough.
7. Hang a 10 g weight from the writing lever and keep the
muscle in the afterloaded position.
8. Adjust the writing lever in such a way that the lever
touches the drum lightly in the horizontal position.

40. 9. Adjust the secondary coil to obtain a contraction at
break stimulus only.
10. Set the drum in motion and record a baseline.
11. Press the tap key and release it as soon as you record
the first contraction.
12. Vibrate the tuning fork by hitting against the thigh
or against the hypothenar eminence of the hand
and record a time tracing below the simple muscle
curve.
13. Stop the drum .

41. 14. Rotate the drum manually so that the contact arm
touch the kymograph key.
15. Mark the point of stimulation by moving
writing lever over the smoked drum
16.Mark of beginning of contraction peak of contraction
and end of relaxation
17caluculate and record the duration of latent period and
contraction period and relaxation period

42. Sherrington-
Starling’s
revolving
drum.
W
Muscl
e
Myograph
QT stand
Afterload screw
L-shaped
Isotonic
lever
Smoked
cylinder
SMT
Bipolar
electrode
Load
Bifid
striker Contact foil
on ebonite
block
Sec.
coil
SSRD
()
Tap Key
Low
Voltage
Unit 3V
Primary
coil
Leads
Electrical
circuit
Shaft
Dubois-Raymond’s
Induction coil
Experimental setup to record
muscle responses to its nerve stimulation:

43. Simple MuscleTwitch
P.S.

44. 0.01
Sec.
Simple MuscleTwitch
W
After load screw
SMT
Smoked
cylinder
Time tracing
100 Hz
Shatter/
physiological curves
C
B
A
Base
line
D
A = Point of stimulation.
B = Point of onset of contraction.
C = Point of maximum contraction/point of height of contraction.
D = Point of end of relaxation.
H = Apparent height of contraction.
AB = Latent period = 0.01 Sec.
BC = Contraction period = 0.04 Sec.
CD = Relaxation period = 0.05 Sec.
AD =Total twitch period = 0.1Sec.
0.01
Sec.
H =
Apparent
height
of
contraction

45. Observation and result
Observe and study the simple muscle curve Express the
duration of LP, CP and RP in ms; and also calculate the CP and
RP as a percentage of the total muscle curve (CP+RP) duration

46. DISCUSSION
The latent period, contraction period and relaxation period
vary according to the type of muscle.
The latent period is due to:
Time taken for the stimulus to travel along the
nerve to the neuromuscular junction.
Time taken for the impulse to cross the
neuromuscular junction, to stimulate the muscle.
Time taken for the excitation-contraction coupling to
occur.
Time taken by the lever to overcome the inertia of
rest.
Time taken to overcome the viscous resistance of
the muscle.

47. Effect ofTemperature on Simple MuscleTwitch
A change in temperature in Ringer's
solution causes a change in muscle
contraction. With a change in temperature
there is a change in amplitude and the
duration of different periods of contraction
(latent, contraction and relaxation periods).
The changes are mainly due to change in
rate of conduction velocity in the nerve,
enzymatic and chemical activities in the
muscle and a change in the viscosity of the
muscle.

48. Requirements
1. Same as for ‘simple muscle twitch'
2. Cold Ringer's solution (10 °C)
3. Warm Ringer's solution (40 °C)
4. Centigrade thermometer

49. Procedure
1. Pith and dissect the frog to make the nerve-
muscle preparation.
2. Set up the nerve-muscle preparation for recording
the simple muscle curve.
3. Record a simple muscle curve on the revolving drum
with Ringer's solution in the muscle trough at room
temperature.
4. Drain the Ringer's solution from the muscle trough
and replace with warm Ringer's solution and wait for 1-
3 minutes to allow the muscle to warm up.
5. Using the same baseline, same point of stimulation
and same strength of stimulus, record the effect of
warm Ringer's solution on the simple muscle curve.
6. Note the temperature of the Ringer's solution and

50. 7. Pour the normal Ringer's solution and wait
for some time for the preparation to come
back to normal temperature. Pour cold
Ringer's solution into the muscle trough and
wait for 2–5 minutes to allow the muscle to
cool.
8. Using the same baseline, point of
stimulation and strength of stimulus, record
the effect of cold Ringer's on muscle
contraction.
9. Record a time tracing below the recordings
with the help of a tuning fork.
10. Calculate the latent period, contraction

51. 250C
370C
50C
500C Heat rigor
Cold rigor
P.S.
Effect of varying temperature 0n G.S. Preparation.

52. P.S.
Effect of varying temperature on frog’s G.S. Preparation.
Rigor: Rigidity/Stiffness/Shortening of muscle.
Heat rigor: Rigidity that develops in muscle when it is subjected to hot (>40 o C) temperature resulting
in coagulation of muscle proteins.
Cold rigor: Rigidity that develops in the muscle when it is exposed to cold(< 5o C)
temperature and it is due to inhibition of calcium pump and raising sarcoplasmic contraction of Ca++
and failure to delink the myosin head with binding sites over actin filament.
Rigor mortis: Rigidity that develops in the muscle some time after death.Time of onset: 2-4 hrs. due to
fall in conc. of ATP < 60% and Phospocreatine < 80%, that is necessary to lower sarcoplasmic Ca++ conc.
and delink cross linkage. Weans off: 24-36 hrs. after death due to autolysis of muscle protein.
Medico-legal application: Used to find out time since death.
Heat rigor
Base
line
Warm temp.
Room temp.
Cold temp.
>40o
C

53. Observation
Compare the recordings of the three
muscle curves
recorded at three different temperatures
of Ringer's
solution. Study the height and slope of
contraction,
and the duration of latent period,
contraction period
and relaxation period of each curve

54. Precautions
The precautions are the same as those for
‘simple muscle
twitch'. In addition to these:
strength of stimulation for all the recordings
should be kept constant.
2. The temperature of Ringer's solution should
not exceed 42°C. Note When the temperature
of the solution is 43 °C or more the muscle
proteins are denatured. Muscles remain in a
state of sustained contraction. This
phenomenon is known as heat rigor.

55. 3. The temperature of the cold solution should
not be less than 4 °C. Note When the
temperature of the solution is very low the
muscle proteins are coagulated. This prevents
muscle contraction. The temperature of the
solution should be
recorded just prior to or immediately after
muscle contraction.
The effects of the warm solution should be
recorded before recording the effects of cold
Ringer's because cold Ringer's inhibits all
enzymatic and metabolic activities of the
muscle. It may be difficult to revive muscle