Objective Principle Requirements Experimental specifications (conditions) Preparation of ACh stock and standard solutions Preparation of frog ringer solution (PSS) Procedure Kymograph recording of contractions Observation table Calculation of magnification value (Mf) Graphical presentation of CRC/ DRC Result and interpretation

1. To record concentration response curve (CRC)/ dose
response curve (DRC) of acetyl choline (ACh) using
isolated rectus abdominis muscle preparation of frog
Mr. Vishal Balakrushna Jadhav
Assistant Professor (Pharmacology)
GES’s Sir Dr. M. S. Gosavi COPER, Nashik-5
Experiment No. 4
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2. Overview of Discussion
• Objective
• Principle
• Requirements
• Experimental specifications (conditions)
• Preparation of ACh stock and standard solutions
• Preparation of frog ringer solution (PSS)
• Procedure
• Kymograph recording of contractions
• Observation table
• Calculation of magnification value (Mf)
• Graphical presentation of CRC/ DRC
• Result and interpretation
2

3. Objective
To record concentration response curve (CRC)/ dose
response curve (DRC) of acetyl choline (ACh) using
isolated rectus abdominis muscle preparation of frog.
3

4. Dose response curve/ dose response relationship
4
When a drug is administered systemically, the dose-response
relationship has two components-
A) Dose-plasma concentration relationship, and
B) Plasma concentration-response relationship.
The dose-plasma concentration relationship is determined by
pharmacokinetic considerations.
Descriptions of dose-response relationship refer to the plasma
concentration-response relationship, which can be more easily
studied in vitro.
Generally, the intensity of response increases with increase in
dose (or more precisely concentration at the receptor) and
the dose-response curve is a rectangular hyperbola (as shown
in figure).
Principle

5. 5
If the dose is plotted on a logarithmic scale, the curve becomes
sigmoid and a linear relationship between log of dose and the
response is seen in the intermediate (30-70% response) zone.
Various advantages of plotting log dose-response curves (DRC)
are- a) A wide range of drug doses can be easily displayed on a
graph, and b) Comparison between agonists and study of
antagonists becomes easier.
Figure- Dose response and log (dose)-response curves

6. Dose (concentration) response curves demonstrate graded
response to drugs or agonists where an increase in response
is recorded with a subsequent increase in the dose or
concentration of the drug.
The DRC is sigmoid or S-shaped (as shown)-
A- The first part (25% of graph) of the graph has
poor discrimination between the doses indicate
poor relationship between dose and response
B- The middle portion of the curve shows greater
sensitivity to different concentrations, and the
responses to increasing concentrations are
linearly differentiated.
C- The last part of the curve (plateau) shows the
ceiling effect where no more increase in the
response is seen with further increase in the
dose.
6
A
B
C

7. Sometimes, cumulative DRCs are employed for the study. The
cumulative DRC is obtained by increasing the concentration of the
drug in the organ bath step by step without washing the preceding
doses. This technique is simple and less time consuming. It is
generally employed in preparation where the tissue is slowly
contracting and slowly relaxing. However, this method is not suited
for drugs which shows fade phenomenon.
When the doses are increased in geometric progression (logarithmic
intervals) and the response is plotted against logarithms of doses,
the relationship is called as log DRC. The logarithmic transformation
of doses offers some advantages such as-
 The linear portion of the sigmoid curve becomes more straight,
 Comparison of two dose response curves is much simpler,
 Large dose ranges can be plotted which is otherwise difficult in
dose response curves, and
 Error is distributed all through the graph independent of the doses.
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8. The position of DRC on the dose axis (X-axis) is the index of drug
potency which refers to the amount of drug needed to produce a
certain response. A DRC positioned rightward indicates lower
potency (as shown in figure).
The upper limit of DRC is the index of drug efficacy and refers to the
maximal response that can be elicited by the drug, e.g. morphine
produces a degree of analgesia not obtainable with any dose of
aspirin- morphine is more efficacious than aspirin. Efficacy is a more
important factor in the choice of a drug.
The terms 'drug potency' and 'drug efficacy' are used interchangeably,
but these are not synonymous and refer to different characteristics
of the drug.
The slope of the DRC is also important. A steep slope indicates that a
moderate increase in dose will markedly increase the response,
while a flat one implies that little increase in response will occur over
a wide dose range. Slope of the curve indicates error and reliability
(precision) of the bioassay. Steeper the slope, more precise is the
assay, and vice versa is also true. 8

9. 9
Figure- Illustration of drug potency and efficacy. Dose response
curves of four drugs producing the same qualitative effects. Note-
Drug B is less potent but equally efficacious as drug A, Drug C is
less potent and less efficacious than drug A, Drug D is more potent
than drug A, B & C, but less efficacious than drugs A & B, and
equally efficacious as drug C.

10. The rectus abdominis muscle, also known as the abdominal muscle, is a
paired muscle running vertically on each side of the anterior wall of
the human abdomen, as well as that of some other mammals.
There are two parallel muscles, separated by a midline band
of connective tissue called the linea alba.
It extends from the pubic symphysis, pubic crest and pubic
tubercle inferiorly, to the xiphoid process and costal cartilages of
ribs V to VII superiorly.
The proximal attachments are the pubic crest and the pubic symphysis.
It attaches distally at the costal cartilages of ribs 5-7 and the xiphoid
process of the sternum.
Functions It is an important postural muscle. It helps to flexing lumbar
spine. It plays an important role in breathing and helps to keep
internal organ intact
Rectus abdominis muscle
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11. Rectus abdominis muscle of the frog is the voluntary skeletal muscle
that produces a slower contraction in response to the acetylcholine
(ACh).
Isolated rectus abdominis preparation is an isolated preparation of
tissue which is widely used for study of ACh as it operates on
nicotinic ACh mechanism at neuromuscular junctions (interaction
occur at Nm subtype of receptor present on neuromuscular
junction).
Cholinergic drug like Ach stimulate nicotinic receptors and produces
skeletal muscle contraction due to opening of Na+ channel. The
response of these drugs on nicotinic receptors is block
neuromuscular blocking drug like d-tubocurarine (d-TC).
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12. Frog’s Rectus
Abdominis Muscle
12

13. Requirements
Animal: Frog.
Physiological solution: Frog ringer solution.
Chemicals: Acetylcholine in different concentrations (1:10000 i.e.
100 μg/ml), Fixing solution.
Instruments: Sherrington recording drum , Student organ bath
Aerator, Insulin or tuberculin syringe to inject drugs in small
fractions, Dissecting board and various dissecting instruments.
Simple straw lever or frontal writing lever and stand, Pipette,
Stop watch etc.
Miscellaneous: Kymograph paper, plasticin, clips, and thread.
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14. Experimental specifications (conditions)
 Isolated tissue- Isolated Frog’s rectus abdominis preparation
 Drug- Acetylcholine (ACh) (Stock solution- 1 mg/ml) (Std. conc.-
1, 10 and 100 μg/ml)
 Physiological salt solution (PSS)- Frog ringer solution
 Time cycle- Total- 5 minutes, Base line- 30 seconds, Contact
time- 90 seconds, Washing period- 3 minutes
 Applied load/ tension- 1 g
 Bath capacity- 40 ml
 Bath temperature- Doesn’t matter
 Speed of rotation of drum- 0.25 mm/ second
 Magnification value (Mf) = d (F-W)/ d (F-T)
 Aeration- Normal air (1- 2 bubbles/ second)
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15. Preparation of ACh stock and standard solutions
Stock solution- 1 mg/ml or 1000 μg/ml
 Dissolve 1 mg of ACh in 1 ml of distilled water → 1000 μg/ml
Standard solutions
 Dilute 1 ml of stock solution up to 10 ml with distilled water
→ 100 μg/ml
 Dilute 1 ml of 100 μg/ml solution up to 10 ml with distilled water
→ 10 μg/ml
 Dilute 1 ml of 10 μg/ml solution up to 10 ml with distilled water
→ 1 μg/ml
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16. Preparation of frog ringer solution (PSS)
 Prepare 1 litre of frog ringer solution by dissolving NaCl (6.0 g),
KCl (0.14 g), NaHCO3 (0.2 g), and glucose (2 g) in distilled
water.
 CaCl2 (0.12 g) should be dissolved separately in distilled water
to avoid chances of precipitation of salt.
 Mix CaCl2 solution to the higher volume of PSS.
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17. Procedure
 Pith or stun the frog and lay it on its back on the frog-
dissecting board. Pin or tape the four limbs to the frog board.
 Remove the skin on the abdomen and expose the rectus
abdominis muscle
 Cut and prepare two rectus muscle preparations from each
frog. Tie a thread to the top and bottom of each muscle
preparation before detaching the muscle from the body of the
frog.
 Mount the preparation in up-right position in the organ bath
containing frog ringer solution under a tension of 1 g. There is
no need of maintaining bath temperature since it is an
amphibian tissue preparation. Bubble the organ bath with air.
 Relax the tissue for 45 minutes during which wash the tissue
with fresh volume of frog ringer for at least four times.
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18.  Record the contraction due to ACh using either simple side
way or frontal writing lever. Contact time of 90 seconds and 5
minutes of total time cycle may be followed for recording of
response of each dose of standard concentration.
 Record the responses with an increased doses of ACh std. till
the ceiling point or plateau stage is observed i.e. till the
maximum response is achieved if one gets same or slightly less
response with a higher concentration or dose.
 Properly label and fix the kymograph recording of responses
with the help of fixing solution.
 Measure the height of the response (cm/ mm) and plot a CRC/
DRC graphically.
 Put all the values in observation table (as shown).
 Describe the shape and various parts of CRC/ DRC and
interpret the result.
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19. Kymograph recording of contractions
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ACh Std.
(100 μg/ml)
0.1 0.2 0.4 0.8 1.6
Submaximal Dose
Ceiling Dose
Supramaximal Dose
Fig. Dose Response Curve (DRC) of ACh Std.

20. Observation table
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21. Observation table- Example
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Sr.
No.
Drug
(Conc.)
Dose
(ml)
Dose
(μg)
log
(dose)=
log (dose
in μg )
Response in Conc. of ACh in 40 ml
organ bath=
Dose (μg)/ Bath
Capacity (ml)
Height
(cm/mm)
%
1
ACh
(100 μg/ml )
0.1 10 1.0 0.3 16.67 0.25
2 0.2 20 1.3010 0.5 27.78 0.5
3 0.4 40 1.6021 1.4 77.78 1
4 0.8 80 1.9031 1.8 100.00 2
5 1.6 160 2.2041 1.8 100.00 4
0.00
20.00
40.00
60.00
80.00
100.00
120.00
0 0.5 1 1.5 2 2.5
%
RESPONSE
LOG (DOSE)
Log (DRC) of ACh Standard (100μg/ml)

22. Calculation of magnification value (Mf)
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Magnification value (Mf) = d (F-W)/ d (F-T)
Where-
d (F-W) → distance between fulcrum and stylus (writing tip)
d (F-T) → distance between fulcrum and point of attachment of
tissue

23. Graphical presentation of CRC/ DRC
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24. Result and interpretation
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The concentration response curve (CRC) or dose response
curve (DRC) of standard concentration of ACh by using isolated
frog’s rectus abdominis muscle preparation was recorded and
interpreted graphically as sigmoid or S-shape curve.
The magnification value was found to be-

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Thank You...