Experiment 3: REFLEX ACTION IN FROG

Objectives: In this experiment, you will 1) observe reflex actions in the frog
such as withdrawal reflex, corneal reflex, and righting reflex; 2) study the
effect of single-pithing or decerebration and double pithing on these reflexes;
and 3) learn some of the principal characteristics of reflexes such as reflex
time, synaptic delay, synaptic fatigue, and inhibition and coordination of
reflexes.
INTRODUCTION
 The neuron serves both as the receptor or detector of stimulus (internal or external
environmental change) and as a conductor of messages to the effector cells that executes
the response of the animal.
 This pathway generates simple automatic behavior called reflex or reflex action.
 The central nervous system (CNS) receives messages carried by sensory (afferent)
neurons and sends out messages carried by motor (efferent) neurons.
 It also has interneurons, processing neurons that can compare various sensory inputs
before they direct the actions to be performed.
 The reflexes of vertebrates are based on the reflex arc, a pathway which consists of
receptor or sense organ, sensory neuron, motor neuron, and effector organ. The knee jerk
reflex is an example.
 By way of inetrneurons, one reflex arc becomes connected to other reflex arcs.
 Somatic reflexes differ from visceral reflexes.
 Only one motor neuron is involved in a somatic reflex pathway while in a visceral reflex
pathway, there are two motor neurons involved.
 Somatic pathways reach the level of consciousness while visceral pathways do not.
PREPARATION
A. A normal frog
Note: In this experiment, you will be handling live frogs. One thing to always remember is that
there is a proper way of holding and carrying them. Make sure that the frog is held with its legs
extended. Do not allow it assume a flexed leg position in your hand; otherwise, it can push
against the palm of your hand and get away.
Another thing to remember is that you should not cause undue discomfort to your specimen.
Have several sheets of wet paper towels ready to cover the animal when it is not being observed.
Be sure to always wet your hands, too, before handling. This pointer also applies to the brain-
dead specimens.
A third thing to remember, especially with the live frogs, is to not startle or frighten the
specimen. Move slowly and speak quietly when observing it.
B. A spinal frog
A spinal frog is a single-pithed frog. Its brain has been destroyed but the spinal cord is still intact.
To single-pith:
1. Flex the head of the frog downward with the finger of the holding hand.
2. Thrust a sharp probe or stout needle into and through the foramen magnum (the
depression at the base of the skull) and, with the lateral movement of the probe or needle,
transect the brain. That the brain has been destroyed is indicated by the loss of some
reflexes, e.g., the lid and righting reflexes.
Touch the cornea of the frog. If the eyelid does not move, the brain ia already damaged. To check
for the loss of the righting reflex, place the single-pithed frog ventral side up on the counter. The
legs should be limp and the frog incapable of restoring a normal resting posture.
For a short period following decerebration, the animal normally passes through a state of "spinal
shock" characterized by either areflexia or hyperreflexia. The state is caused by the sudden loss
of higher center of control and should pass away within a few minutes.
C. A double-pithed frog
To produce a double-pithed frog:
1. After single-pithing, partly withdraw the needle or probe from the brain, then direct the tip
caudally.
2. Push the needle down the vertebral canal to destroy the spinal cord. The hind legs will first
straighten stiffly, then, will become completely flaccid and unresponsive to stimulation.
EXPERIMENT PROPER
Reflexes in Normal, Spinal, and Double-pithed Frogs
Make the following tests on normal, spinal, and double-pithed frogs. Observe the animals
carefully. Complete the chart of responses by recording all the responses or movements that you
observe. Include illustrations. In the case, especially of the normal frog, be detailed with your
descriptions. You can also apply the symbols (+) and (-) in the recording of observations of
responses.
1. Place the frog on the desk and observe its resting posture. Note position of the head, eyes,
forelegs, and hind legs. How are these positioned in the resting frog? What is the
configuration assumed by the body? Is the body parallel with the surface of the table or at an
angle? On the chart, make a drawing of the resting frog.
2. Observe the closing and opening of the nostrils or the raising and lowering of the floor of the
mouth. Frogs utilize buccal pumping to force air into their lungs. First, they gulp air and with
the elevation of the throat, at the same time, the closure of the nostrils, the air is swallowed.
Air is expelled with the compression of the sides of the abdomen and the opening of the
nostrils. What is the respiratory rate? There may be rapid oscillations of the throat between
ventilatory cycles. Do not count these since these serve mainly to flush the buccal cavity of
any residue of expired air in the mouth.
3. Test for maintenance of equilibrium.
Describe the motion and position of the head, eyes, forelegs, and hind legs while:
a) Frog is placed on its back on a frog board. Does the frog havr the ability to right itself?
How are the legs used when the animal rights itself? Illustrate the movements of the legs
and body while the animal is righting itself. How long did it take for the frog to right
itself?
b) Frog is ventral side down on the board that is being gently revolved in a horizontal plane.
c) Frog is ventral side down on a board that is being gently tilted so that the head is
elevated.
4. Drop the frog in a tank of water. What are the movements used in swimming?
In a spinal frog, swimming movements are frequently made although these may not enable
the animal to keep from going to the bottom. Place a floating board in the water close to the
frog. If the animal contacts the floating board, it may attempt to climb on the board. For a
brain-dead frog as well as the double-pithed one, place the forelegs on the board and describe
the climbing movement, if there is any. Turn the frog over and record the righting response in
water. Make simple sketches of the swimming, climbing, and righting movements.
5. Observe if there is any evidence of sight or hearing.
6. Test for muscular tension and the limb reflexes:
7. Gently prod the frog into jumping. Record the general form of the jumping behaviour. Does
the frog pull its legs while in the air or are the legs extended?
8. Test for pain response by pinching the toes. Did you observe a flexion- withdrawal reflex?
9. Dip the toes of the right foot into a beaker of cold water, then, gradually heat the water.
10. Dip the toes of the left foot into water at 80°C.
11. Turn the frog over and hold it upside down. Wet your fingers and stroke its belly. A male frog
may respond by clasping your fingers with its forelegs.
12. Test for the "wipe reflex" or "scratch reflex."
 ADDITIONAL NOTES, DATA, and OBSERVATIONS

1. Comparison of the Reflexes (Responses to Various Stimuli and Behaviour)

Double-Pithed
Reflex/Behavior Normal Frog Spinal Frog
Frog
1. Resting Posture Head facing The head was tilted The head was facing
forward. Both eyes to the left. The left down. Both left and
were widely open eye was partially right eyes were
and looking closed. The right eye closed. The
forward. It was in was open. The body appendages were in
normal sitting was tilted to the left full extension and
position. The floor in sitting position. total limpness. The
of the mouth was The floor of the floor of the mouth
moving in normal mouth was moving was not moving.
rhythm. in a slower rhythm.
2. Respiratory Rate 210/minute 50/min 23/min
3. Righting Reflex It immediately It remained on its No corrective action
a. Frog Is Turn Over corrected its body and dorsal recumbence. took place.
returned to the normal
position.
b. Frog Is Turn No movement was
Around On A observed.
Horizontal Plane
c. Frog Is Turned Till No movement was
Head Lies On observed.
Perpendicular Plane
4. It swam in a breast No reaction was No movement was
a) Swimming stroke fashion. It observed. observed.
Behaviour draws its hind legs
upward toward its
body then shoots its
legs out backward in
a straight line.
b) Righting No corrective action
Movement In Water took place.
c) Climbing No movement was
Behaviour observed.
5. The eyelid shuts The eyelid did not The eyelid did not
a) Blink/Protective upon touching. move. respond.
Eye Reflex
b) Auditory-Palpebral
Reflex
6.
a) Muscle Tonus
b) Limb Reflexes
c) Extensor Reflex
7. Flexor Reflex
8. Jumping Behaviour
9. Pain Response/Flexion-
Withdrawal Reflex
10. Response To Cold
Water
11. Response To Hot Water
12.
a) Clasp Reflex
 b) Quieting Reflex
13. Wipe Reflex
14. Spontaneous Activity

2. Reflex time in the spinal frog responding with the flexion-withdrawal Reflex to varying
concentrations of hydrochloric acid applied on its toe.

Stimulus Reflex Time (sec)

(% HCI) Trial 1 Trial 2 Trial 3 Average
0.05 * * * *
0.1 * * * *
0.3 50 57 85 65
0.5 43 42 47 44
1.0 15 12 11 12.67
Procaine plus acid 16 8 12 12
* No response observed after 90 seconds.
3. Graph showing the relation of stimulus strength to reflex time.

70

60

50

40
Reflex Time (sec)
30

20

10

0
0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1

Concentration of Acis

4. Inhibition of the righting reflex and its explanation

5. Inhibition of the flexion-withdrawal reflex under varying conditions of the test *

REFLEX TIME
TRIAL 1 TRIAL 2 TRIAL 3 AVERAGE
Spinal Frog
Cerebral Hemispheres
removed by a cut at just the
back of the eyes
Cerebral Hemispheres
removed and leg stimulated
with feeble tetanising current
Cerebral Hemispheres
removed and optic lobes
stimulated with few crystals
of sodium chloride
Both cerebral hemispheres
and optic lobes removed

1. Explanation of central inhibition involved in the experiment.

2. Diagram of the components of the crossed-extensor reflex arc and the order of fatigability of
the parts starting with first to undergo synaptic fatigue designated as (#1).
3.
 SUMMARY
VPP3231 VETERINARY PHYSIOLOGY I LABORATORY
REPORT REFLEX ACTION IN A FROG NAME CIK
NASRIAH BINTI SAID MATRIC NO. 156721 IFFAH
NADZIRAH BINTI ABD RAZAK 157613 AFIQAH BINTI
ZAINURIN MUHAMAD FAIZAL BIN HAHLAN MUHAMAD
ALIF BIN ZAKARIA NURUL HAFIZAH BINTI ABU JAZID
MAJDI BIN AHMAD 158593 160107 158677 158322
159530 GROUP 10
EXPERIMENT 2: REFLEX ACTION IN A FROG. OBJECTIVE: 1) To determine strength of a
single threshold stimulus 2) To determine strength of a single maximal stimulus 3) To study the
effect of frequency of the stimulus MATERIALS: Scissors,glass probe,thread,Petri
dish,forceps,Pasteur pipette,Ringers solution. PROCDURE: (A) Normal Frog. 1. The frog was
left on the table. The position of the head, eyes, forelimb and hind limb was observed. 2. The
closing and opening of the nostrils and as well as the movement of the floor of the buccal cavity
was observed. The respiration rate of the frog was calculated. 3. Maintenance of equilibriuma)
The frog was left on the dorsal recumbence position and it¶s action was observed. b) The frog
was left on a piece of wood and the wood rotated horizontally at a slow rate. The movement and
position of the head, eyes, forelimb and hind limb was observed. c) The wood was tilted to the
front, back, left and right. The response of the frog was observed. 4. Muscular tension a) The
tension of the thigh and lower leg muscle was felt and palpated. b) When the frog is in a sitting
position, one of t he hind limb was pulled gently and the tension was felt. The limb was then
released after that c) The toes were pinched and any actions observed was recorded.
5. The frog is then put into a water tank and the frog¶s swimming action and style was
observed. (B) Spinal Frog 1. The brain of the frog was destroyed using a surgical needle. 2. The
above method 1-5 was repeated on the frog and any difference in observations was recorded.
(C) Double Pithed Frog 1. The spinal cord of the frog was destroyed using a surgical needle. 2.
The above method 1-5 was repeated on the frog and any difference in observations was
recorded. (II) Pithing of the frog 1. The frog is held with the left hand, with three fingers
positioned below the stomach, the forefinger above the head and the thumb on the top of the
vertebral column. 2. Using a scalpel, the skin from the area below the head to the forth vertebra
was removed. 3. The needle is inserted into the cranium through the occipital Atlantic joint, then
the foramen magnum and into the brain. 4. The needle is then retrieved back from the cranium
cavity until the foramen magnum where the needle is inserted into the spinal canal instead for
the destruction of the spinal cord. 5. When the spinal cord is destroyed, the hind limb would
seem tense, convulse violently and may urinate. (III) Preparation of the Gastrocnemius muscle-
Sciatic Nerve (A) Dissection of the Sciatic Nerve 1. The pithed frog if positioned with its stomach
facing the table. The skin around the urostyle region is lifted up and was cut all the way up until
the head. 2. The skin from the urostyle until the knee joint was cut. 3. The sciatic nerve was
observed when the urostyle was lifted up.
4. To prevent any damage to the nerve, the pelvic girdle that is connected to the vertebra
column was cut and removed. 5. The nerve was lifted up gently to prevent any damage. 6. The
sciatic nerve was cut from its origin until the knee joint and both nerve endings were tied with a
piece of thread. 7. To prevent dehydration, the sciatic nerve was constantly being moisturized
with Ringer¶s solution. (B) Dissection of the Gastrocnemius Muscle 1. The skin around one leg
was removed. 2. Using a pair of forceps, the skin was separated from the skin (just like
removing a pair of socks). 3. The gastrocnemius muscle was separated from the surrounding
muscle using a glass probe. 4. The Achilles tendon was cut at the distal end of the
gastrocnemius muscle. 5. Using a pair of scissors, the lower end of the limb was detached at
the knee joint region. 6. The proximal end of the limb was also detached. 7. Using a glass
probe, all the upper muscle of the hind limb that surrounds the femur was separated. 8. The
sciatic nerve at the proximal ending was cut. 9. All muscle from the femur was cut at the knee
joint region. 10. The Achilles tendon was tied to a piece of thread.
CONCLUSION Reflex action is fast reaction which does not involve brain of the animal but only
involve the spinal cord which the important for the reflex action to occur. Different temperature
and Ph has different effect on the muscle contraction.

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RESULTS AND DISCUSSION

The basic unit for behavior is the reflex. Reflexes generally occur quickly and are the result
of excitation of sensory receptors, nerve fibers, integration by the Central Nervous System, transmission
of motor impulses and excitation of an effector organ or tissue.

a. Motor Activity

Through the course of the experiment, reflex action is verified via the use of normal, spinal and

and double-pithed frogs. In normal frog, the brain and spinal cord are still intact. In single-

pithed frogs, all connections between the brain and the spinal cord are broken, therefore it

eliminates any influence the brain could have on reflex activities. Lastly, in a double-pithed frog

all control by the central nervous system are eliminated including the brain and spinal cord.

Several actions are tested to compare the differences in the reflex of frog in the three states such

as the posture reflex, locomotion, righting reflex, swimming reflex, pain response and scratch

reaction. In a normal frog, the eyelids blink when touched and croaks as response to posture

reflex with a respiration rate of 210/minute, at this state the nostrils were observed to be used

during respiration; it reacts with dilute acetic acid as response to scratch reflex within

five seconds; other leg flexed as the remaining leg is pinched for withdrawal reflex; it also

jumped more frequently with a continuous and coordinated jumping movements. As it was

placed on its dorsal side, it makes a righting movement immediately for two seconds (fast

righting reflex) and swims immed/iately with a well-coordinated swimming movements. As the

normal frog was hanged, both legs flexed and extended as they reacted to pain immediately after

pinching.

In a spinal state, where single pit/hing was done, the frog remained motionless and lost muscle

tone; however the respiration rate lowered to 50/min. This can be explained as the frog was kept

moist when tested in swimming /reflex for they breathe primarily through the skin, not the lungs.

Without the brain, perception of any sensory phenomena is impossible. This explained the reason

why when the eyelid was touched, the frog did not blink. It also jumped four times but was not

continuous. It did not respond to the righting reflex but the swimming reflex was observed
though slower compared to the normal state. A spinal frog cannot perceive pain. After a recovery

period however it will be able to resume its ability to carry on spinal reflex activity. The frog

may crouch, jump, scratch or even make noise, but it is not in pain. These actions are neural

reflexes controlled via motor senses in the spinal cord, not brain functions. While at the

decerebrated state, where the brain is removed, the frog does not blink but does have a slight

croak; does not react upon exposure to acetic acid; does still flex other leg as the remaining leg is

pinched; does make a slow righting movement and does still swim. Thus, it still has the

ability to respond to external stimuli. In decerebrated frog, brain stem is transected at the middle

to lower mesencephalic level, which blocks normal inhibitory signals from the higher control

centers of the brain to the pontile and vestibular muscle control nuclei resulting to involuntary

extension of extremities in response to external stimuli. The result is that the spinal cord motor

reflexes become very excitable and, therefore, easy to activate by even the slightest sensory input

signals to the cord.

In a double-pithed frog, the spinal cord is severed including the brain. Still, it managed to have

a respiration rate of 23/min but the breathing may be through the skin or called cutaneous respiration. At

this state the legs of the frog become completely limp due to flaccid paralysis of skeletal muscle. The frog

will never assume a crouching position again, and if pinched the frog will not respond after the spinal

cord is severed, the frog will not feel anything and there will not be a reflex withdrawal along with the

other reflexes. #n other words, spinal severing makes work easier because the muscle no longer reflexes.

Scratch reflex

Another is the scratch reflex of the frog in which dilute acetic acid is introduced. As itchiness occurs, it

proceeds with scratch reflex as to relieve cutaneous irritation. This is a form of defense mechanism when

parasites or irritants are present. Pain receptors are activated as acid is applied onto the skin. In addition to

this, acid is permeable to the frog skin that allows easy access of the acid to the frog’s internal system

resulting to a greater effect of the acid on the frog.

Withdrawal reflex

Upon conduction of stimuli either painful or harmful, there will be a counteracting flex

movement of a specific part of a body called withdrawal reflex. Pinching is a type of cutaneous

stimulation and its natural response is to withdraw that part of the body that is stimulated. Upon pinching,
the pain receptors made up of nerve endings in the skin transmit signal to the cerebellum which controls

motor function thus, causing a flex to the muscle affected by the stimulus. So, withdrawal reflex is

executed by pinching off the legs or toes of either side of the frog. In a normal frog, when left leg is

pinched, right leg flexed followed by the left leg. Theoretically, as expected, decerebrated frog responses

as what normal frog does; but not in the case of pithed frogs. What is responsible for the pain is the spinal

cord.

Righting reflex

Recovery to its normal position after orienting from different position of the body is called

righting reflex. In a normal frog, there is righting movement due to functional brain and spinal

cord observed while in decerebrated frog, a slow righting movement is observed. However, on

the pithed frogs, righting movements were not observed. Righting reflex is a mobilizing response

which requires a coordinated sensory input and motor output to be able to materialize. If the

somatic motor neurons are no longer transmitted to the sensory receptor organs, no action potenti

al will travel to the medulla oblongata and, hence, will not reach the effector organ which is the

muscles in the limbs.

Swimming reflex

Swimming reflex is the coordinated movement of the limbs to sustain its body afloat the water and to gain

movement. The normal frog was observed to immediately swim when it was placed in the water because

its brain and spinal cord are intact, and, thus, function the way they should be in order to maintain

equilibrium. In the case of single-pithed frog it still swims but accompanied with the movement was not

well"coordinated. %hile decerebrated and

double" pithed frogs still swim, but it should not swim since complete removal of the brain whichcontaine

d the nuclei necessary for the processing of any sensory information from the receptors is taken.