Principle
Frog rectus abdominis muscle is voluntary muscle. At the neuromuscular junction, a nerve impulse liberates Ach from the nerve ending into the cleft between the muscle and nerve fiber. This Ach causes depolarization of the muscle fiber which in turn sets off a muscle action potential, and contraction of the muscle fiber. The muscle fiber of lower species like frog are multiply innervated and hence nerve stimulation causes persistent depolarization and a prolonged slow contraction of muscle. Local administration of Ach also produces similar effect. Frog rectus abdominis muscle contains nicotinic (N2) receptor. Ach acts as agonist.
Equipment required
1) Student kymograph
2) Student organ bath
3) Aeration tube
4) Aerator
5) Aeration tube holder
6) Frontal writing lever
7) Lever holder
8) Screw clip
9) Haemostatic forceps
10) Mariotte bottle
11) Rubber tubes
12) Tuberculine syringe
13) 26 no. needle
14) Scissor
15) Forceps.
Animal required
Frog.
Physiological solution required
Frog ringer solution.
Drug solution required
Ach 100µg/ml.
Procedure
1) Setup the assembly for the above experiment.
2) Pith the frog by passing a needle through occipito-atlantic junction between the brain and spinal cord. The stretching out of the limbs indicates that the pithing is proper.
3) Place the frog in a tray with the ventral side facing up.
4) Pick up the skin of the abdomen with the help of forceps and make proper incision to expose the abdomen.
5) Cut along the margin of the rectus abdominis muscle and then make a transverse cut through the sternum just above the base.
6) Free the rectus abdominis muscle from the interior abdominal vein.
7) Lift the muscle gently and divide the muscle longitudinally.
8) Tie a long thread on the upper side of the rectos muscle and a short thread on the lower side of the rectus abdominis muscle.
9) Transfer the muscle to a petri-dish containing frog ringer solution.
10) Tie the short thread to the hook of the aeration tube and place the rectus muscle in the inner organ bath containing frog ringer solution.
11) Tie the long thread to the frontal writing lever. The load on the lever should be 1 gm. The magnification should be between 5-7 times.
12) Stabilize the rectus muscle for 55 minutes period.
13) During the stabilization period replace the frog ringer solution in the inner organ bath at an interval of 5 minutes.
14) After stabilizing for a period of 55 minutes, switch on the kymograph and record the normal tracing for a period of 30 seconds. At the ends of 30 seconds period inject 0.1 ml of Ach solution to the inner organ bath and record the tracings for a period of 90 seconds (drug contact time). At the end of 90 seconds switch off the kymograph and give 3-4 washings of rectus muscle with frog ringer solution.
15) Inject 0.1 ml of Ach solution into the inner organ bath once again and record the response for 90 seconds.
16) If two equipotent responses are observed with similar doses of Ach, then record the responses of Ach with higher doses (0.2, 0.4, 0.8 and 1 ml).
17) After fixing the graph measure the height of contraction of the response produced by each dose of Ach and find out the dose which produces maximal response.
18) Plot a graph showing dose of Ach on X-axis and response on Y-axis.
19) Plot another graph showing log dose of Ach on X-axis and response on Y-axis.
20) Plot a third graph showing log molar concentration of Ach on X-axis and percentage on Y-axis
21) From the graph find out the EC50.
22) From the EC50 value finds out the pD2 value.
Observation and conclusions
1) Acetylcholine produces a dose-dependent contraction effect on frog rectus abdominis muscle.
2) A graph plot with a linear contraction scale shows non-sigmoid dose-response curve.
3) A graph plot with a logarithmic concentration scale shows a sigmoid dose-response curve.
4) pD2 value is useful in determination of affinity of a drug for a particular receptor.
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