TO STUDY THE EFFECT OF DRUGS ON FROG’S RECTUS MUSCLE

INTRODUCTION
The rectus abdominus of the frog is a skeletal muscle used to demonstrate effects of nicotinic
drugs and neuromuscular blockers.
REQUIREMENTS

Animal : Frog – small to medium sized, about 50gm in weight

Drug and solutions : Acetylcholine 100 µg/ml
Physostigmine 300µg/ml
Tubocurarine 100 µg/ml
Frog Ringer solution
Apparatus / instruments : Rough cloth, pithing needle, dissection instruments, isolated organ
bath, students kymograph, simple writing lever, aerator, syringes, needles.
PROCEDURE
1. Check the settings of your isolated organ bath, Fill the inner organ bath with Frog
Ringer and mark the level of Ringer with thread. Balance the simple lever.
2. Pith a frog and fix on a frog board.

3. Pick and cut the skin in the midline and then laterally. Two recti abdominis muscles
are seen on each side of the midline from the base of the sternum to the pubis.
4. Cut through sternum just above the xiphisternum and dissect out the rectus of one side.
5. Tie a thread on both ends of the muscle. Tie one end to the aerator – cum tissue holder
and the other end to the lever.
6. Start aeration. Put a relaxing weight (0.5-1 gm) on long arm of the lever with thread
and relax the muscle for approx. 30-45 minutes.
7. Take a log dose response curve for Acetylcholine starting with lowest effective dose
i.e. 1,2,4 μg/ml till maximum response is obtained.

8. Each response is taken for 90 seconds with 5 minute interval as follows :

 o min : Raise weight and start kymograph
30 sec : Add Acetylcholine
2 min : Stop Kymograph, wash the rectus 3-4 times, and lower the weight
5 min. : Raise weight and start kymograph

9. Wash the preparation 3-4 times after each response

10.Add 0.1 ml of physostigmine to the organ bath. Wait for 5-6 minutes and then again
record responses with same log doses of acetylcholine.

11.In another preparation, add 0.1ml of tubocurarine. Wait for 5-6 minutes and repeat
response with the same doses of acetylcholine.

OBSERVATION
Using a scale, measure the response obtained with acetylcholine alone, after adding
physostigmine and after adding tubocurarine. Tabulate your findings and plot dose response
curves.
1.

Acetylcholine allowed to act for 90 Sec.

Presence of physostigmine 0.5 µg/ml for 3 min
BATH CAPACITY : 20 ml

1) Explain the graph.

The graph shows the effect of drug responses on isolated Frog rectus muscle . The drugs administered
in this experiment are Acetylcholine and Acetylcholine with Physostigmine.It is seen that Ach causes
muscle contraction and an addition of Physostigmine increases contraction.
2) Describe the mechanism of action of physostigmine?
Physostigmine is an anticholinesterases (antiChEs) agent which inhibit ChE, protect ACh from
hydrolysis— produce cholinergic effects in vivo and potentiate ACh both in vivo and in vitro. The
antiChEs react with the enzyme(Ach esterase) essentially in the same way as ACh. It is a reversible
carbamate that carbamylate the esteratic site of the enzyme. Hence the cholinergic actions of anti -
ChEs are due to amplification of endogenous Ach.
3) Mention the clinical uses of Anticholinesterases.
As miotic – in glaucoma , to reverse the effect of mydriatics after refraction testing , to prevent
formation of adhesions between iris and lens or iris and cornea
Myasthenia gravis
Postoperative paralytic ileus/urinary retention
Postoperative decurarization
Belladonna poisoning
Alzheimer’s disease
2.

Acetylcholine allowed to act for 90 sec.

d-TC (10 ug) allowed to act for 3 min.
BATH CAPACITY : 20 ml

1) Explain the graph.

The graph shows the effect of drug responses on Frog rectus muscle . The drugs administered in this
experiment are Acetylcholine and Acetylcholine with d-tubocurarine.
2) Describe the mechanism of action of d-TC?
d-Tubocurarine acts as a non-depolarizing competitive blocker at
nicotinic acetylcholine(Nm) receptors on the motor end plate of the neuromuscular junction and
decreases frequency of opening of sodium channel thus causing the relaxation of skeletal muscle.
d-Tubocurarine competes with at least an equal affinity to acetylcholine, and at the same position
on nicotinic receptors. It prevents ACh from binding and activating the receptor-channel complex .
3) Name 2 centrally acting skeletal muscle relaxants.
Diazepam , Baclofen
 STUDY OF EFFECTS OF MYDRIATICS ON RABBIT EYE
Aim:
To determine the effects of mydriatic on rabbit eye.

Materials Required:
Rabbit, scissor. torch, cotton, pupilometer, dropper, normal saline, mydriatic drug
Procedure:
The rabbit is held in one hand in a semi dark room and its eyelashes are trimmed (both
eyes), Left eye serves as control and right eye as test eye. The medial canthus is pressed with
one finger and with two other fingers a pouch is made for instillation of drug. Two drops of
normal saline are instilled with the help of a dropper in control (left) eye and two drops of the
drug are instilled in the test (right) eye.Four parameters are measured before instillation of
drug and every 5 minutes thereafter for 30 minutes these are
1) Size of Pupil
2) Light reflex
3) Corneal reflex
4) State of conjunctiva
 1) Size of pupil
It is measured with the help of a pupillometer. Increase in size of the pupil is known as
mydriasis and decrease in size is known as miosis. The size is measured in mm.

2) Light reflex
The light of a torch is shed on the pupil. Bring the light from behind the eye as rabbit may
close the eye if light is brought from the front. The contraction of the pupil on exposure to
light is noted The reaction is known as light reflex.
3) Corneal reflex
A piece of cotton is taken and is rolled in to a fine pointed swab. It is then touched on the
lateral limbus of the rabbit eye. The closure of the eye is noticed, which is called Corneal
reflex.

4) State of conjunctival vessels

The lower eyelid of the rabbit is retracted to expose the lower palpebral conjunctiva. The
vessels of the conjunctiva are observed and congestion if any is noted.
Observation :
The following observations were made after instillation of unknown eye drops into the rabbit
eye. The results are tabulated as follows:

Active Mydriatic Effect of Drugs on Rabbit Eye

Time (Min) 0 5 10 15 20 25 30
Parameters C T C T C T C T C T C T C T
1. Pupil size (mm) 6 6 6 6 6 7 6 7 6 7.5 6 7.5 6 8
2. Light reflex + + + + + + + + + + + + + +
3. Corneal reflex + + + + + + + + + + + + + +
4. State of pin pink pink pin pin pale pink pale pin pale pink pal pin Pale
k k k k e k
conjunctiva

C = Control T= Test
 Passive Mydriatic Effect of Drugs on Rabbit Eye

Time (Min) 0 5 10 15 20 25 30
Parameters C T C T C T C T C T C T C T
1. Pupil size (mm) 6 6 6 6 6 7 6 7 6 7.5 6 7.5 6 8
2. Light reflex - - - - - - - - - - - - - -
3. Corneal reflex + + + + + + + + + + + + + +
4. State of pin pink pink pin pin pale pink pale pin pale pink pal pin Pale
k k k k e k
conjunctiva

C = Control T= Test
Questions:
1) Identify the nature of the drug.
Active mydriatic(Phenylephrine) and passive mydriatic(Atropine).

2) Describe the mechanism of action of identified drug.

Mydriasis is caused by contraction of dilator pupillae(contains alpha1 receptor) and by relaxation of sphincter
pupillae (contains M3 receptor).

3) Mention the therapeutic uses of the unknown drug.

Fundoscopy , retinoscopy , preoperative in surgeries , to break adhesions between lens and iris , corneal ulcer ,
uveitis

4) Explain active and passive mydriasis.

Contraction of dilator pupillae(contains alpha1 receptor) : active mydriasis

Relaxation of sphincter pupillae (contains M3 receptor).: passive mydriasis

 Experiment No. 2
STUDY OF EFECTS OF MIOTIC ON RABBIT EYE
Aim:
To determine the effect of miotic on rabbit eye.

Materials required:
Rabbit, scissor. torch, cotton, pupilometer, dropper, normal saline, miotic drug

Procedure:
The rabbit is held in one hand in a semi dark room and its eyelashes are trimmed (both eyes),
Left eye serves as control and right eye as test eye. The medial canthus is pressed with one
finger and with two other fingers a pouch is made for instillation of drug. Two drops of normal
saline are instilled with the help of a dropper in control (left) eye and two drops of the drug
are instilled in the test (right) eye. Four parameters are measured before instillation of drug
and every 5 minutes thereafter for 30 minutes these are
1. Size of Pupil
2. Light reflex
3. Corneal reflex
4. State of conjunctiva
 1) Size of pupil
It is measured with the help of a pupillometer. Increase in size of the pupil is known as
mydriasis and decrease in size is known as miosis. The size is measured in mm.
2) Light reflex
The light of a torch is shed on the pupil. Bring the light from behind the eye as rabbit may
close the eye if light is brought from the front. The contraction of the pupil on exposure to
light is noted The reaction is known as light reflex.
3) Corneal reflex
A piece of cotton is taken and is rolled in to a fine pointed swab. It is then touched on the
lateral limbus of the rabbit eye. The closure of the eye is noticed, which is called Corneal
reflex.
4) State of conjunctival vessels
The lower eyelid of the rabbit is retracted to expose the lower palpebral conjunctiva. The
vessels of the conjunctiva are observed and congestion if any is noted.
Observation :
The following observations were made after instillation of unknown eye drops into the rabbit
eye. The results are tabulated as follows:

Time (Min) 0 5 10 15 20 25 30
Parameters C T C T C T C T C T C T C T
1. Pupil size (mm) 6 6 6 6 6 5 6 5 6 4.5 6 4.5 6 4
2. Light reflex + + + + + + + + + + + + + +
3. Corneal reflex + + + + + + + + + + + + + +
4. State of pink pink pink pink pink red pink red pink red pink red pink Red
conjunctiva

C = Control T= Test

Questions:

1)Identify the nature of the drug.

Miotic(cholinergic) – Pilocarpine

2) Describe the mechanism of action of identified drug.

Miosis is caused by contraction of sphincter pupillae(contains M3 receptor) and by relaxation of dilator

pupillae (contains alpha1 receptor).

3) Mention the therapeutic uses of the unknown drug.

As miotic
Cataract
Myasthenia gravis
Belladonna poisoning
Alzheimer’s disease
Experiment no. 3
STUDY OF EFECTS OF ANAESTHETICS ON RABBIT EYE

Aim:
To determine the effect of anaesthetics on rabbit eye.

Materials required:
Rabbit, scissor. torch, cotton, pupilometer, dropper, normal saline, anaesthetic drug

Procedure:
The rabbit is held in one hand in a semi dark room and its eyelashes are trimmed (both eyes),
Left eye serves as control and right eye as test eye. The medial canthus is pressed with one
finger and with two other fingers a pouch is made for instillation of drug. Two drops of normal
saline are instilled with the help of a dropper in control (left) eye and two drops of the drug
are instilled in the test (right) eye. Four parameters are measured before instillation of drug
and every 5 minutes thereafter for 30 minutes these are
1. Size of Pupil
2. Light reflex
3. Corneal reflex
4. State of conjunctiva
 1)Size of pupil
It is measured with the help of a pupillometer. Increase in size of the pupil is known as
mydriasis and decrease in size is known as miosis. The size is measured in mm.
2)Light reflex
The light of a torch is shed on the pupil. Bring the light from behind the eye as rabbit may
close the eye if light is brought from the front. The contraction of the pupil on exposure to
light is noted The reaction is known as light reflex.
3) Corneal reflex
A piece of cotton is taken and is rolled in to a fine pointed swab. It is then touched on the
lateral limbus of the rabbit eye. The closure of the eye is noticed, which is called Corneal
reflex.
4)State of conjunctival vessels
The lower eyelid of the rabbit is retracted to expose the lower palpebral conjunctiva. The
vessels of the conjunctiva are observed and congestion if any is noted.
Observation :
The following observations were made after instillation of unknown eye drops into the rabbit
eye. The results are tabulated as follows

Time (Min) 0 5 10 15 20 25 30
Parameters C T C T C T C T C T C T C T
1. Pupil size (mm) 6 6 6 6 6 6 6 6 6 6 6 6 6 6
2. Light reflex + + + + + + + + + + + + + +
3. Corneal reflex - - - - - - - - - - - - - -
4. State of Pink pink pink pink pink pale pink pale pink pale pink pale pink Pale
conjunctiva

Questions:
1) Identify the nature of the drug.

Local Anaesthetic LA - Lignocaine

2) Describe the mechanism of action of identified drug.

The LAs block nerve conduction by decreasing the entry of Na+ ions during upstroke of action
potential (AP). As the concentration of the LA is increased, the rate of rise of AP and maximum
depolarization decreases causing slowing of conduction. Finally, local depolarization fails to reach
the threshold potential and conduction block ensues.
Experiment no. 4
STUDY OF EFECTS OF COCAINE ON RABBIT EYE
Aim:
To determine the effect of cocaine on rabbit eye.

Materials required:
Rabbit, scissor. torch, cotton, pupilometer, dropper, normal saline, cocaine drug

Procedure:
The rabbit is held in one hand in a semi dark room and its eyelashes are trimmed (both eyes),
Left eye serves as control and right eye as test eye. The medial canthus is pressed with one
finger and with two other fingers a pouch is made for instillation of drug. Two drops of normal
saline are instilled with the help of a dropper in control (left) eye and two drops of the drug
are instilled in the test (right) eye. Four parameters are measured before instillation of drug
and every 5 minutes thereafter for 30 minutes these are
1. Size of Pupil
2. Light reflex
3. Corneal reflex
4. State of conjunctiva
 1)Size of pupil
It is measured with the help of a pupillometer. Increase in size of the pupil is known as
mydriasis and decrease in size is known as miosis. The size is measured in mm.
2)Light reflex
The light of a torch is shed on the pupil. Bring the light from behind the eye as rabbit may
close the eye if light is brought from the front. The contraction of the pupil on exposure to
light is noted The reaction is known as light reflex.
3) Corneal reflex
A piece of cotton is taken and is rolled in to a fine pointed swab. It is then touched on the
lateral limbus of the rabbit eye. The closure of the eye is noticed, which is called Corneal
reflex.
4)State of conjunctival vessels
The lower eyelid of the rabbit is retracted to expose the lower palpebral conjunctiva. The
vessels of the conjunctiva are observed and congestion if any is noted.
Observation :
The following observations were made after instillation of unknown eye drops into the rabbit
eye. The results are tabulated as follows

Time (Min) 0 5 10 15 20 25 30
Parameters C T C T C T C T C T C T C T
1. Pupil size (mm) 6 6 6 6 6 6 6 6.5 6 7 6 7.5 6 8
2. Light reflex + + + + + + + + + + + + + +
3. Corneal reflex - - - - - - - - - - - - - -
4. State of Pink pink pink pink pink pink pink red pink red pink red pink red
conjunctiva

1) Identify the nature of the drug.

Cocaine-Local anaesthetic (LA)

2) Describe the mechanism of action of identified drug.

In the periphery, it blocks uptake of NA and Adr into adrenergic nerve endings, resulting in higher
concentration of the transmitter around the receptors→ sympathomimetic effect, potentiation of
directly acting sympathomimetics and suppression of indirectly acting sympathomimetics. Local
vasoconstriction, tachycardia, rise in BP and mydriasis are the manifestations of its
sympathomimetic action. The only indication for cocaine is in ocular anaesthesia. It also causes
constriction of conjunctival vessels.