VPT 321: Veterinary Neuropharmacology (2+1) copy 1

EFFECT OF DRUGS ON NEURO MUSCULAR JUNCTIONS

Aim

• To demonstrate nicotinic cholinergic response at neuromuscular junction of skeletal muscle and to demonstrate anticholinesterase activity of Physostigmine/neostigmine potentiating Ach response. To demonstrate d-tubocurarine competitive blockade of Ach at neuromuscular junction.

Apparatus

• Organ bath, kymograph or Chart recorder or digital polygraph

Drugs

• Acetylcholine, Physostigmine, d-tubocurarine.

Procedure

• The frog rectus preparation is usually made from Rana temporaria, Rana pipiens or Rana esculenta. Frog weighing 20 g will do for the experiment. These can be kept for many weeks at 4 degree celcius without feeding. The frog is stunned, decapitated and the spinal cord is destroyed.
• The frog is pinned down on the dissecting board and the skin of the abdomen but not the abdomen itself is picked with the forceps and cut from the above the sternum down to the fork. It is then cut laterally to expose whole of the abdomen.
• Two rectus muscle will be plainly seen, running each side of the midline from the base of the sternum to the fork. The sternum is cut through just above the xyphisternum at its base, and the pair of muscles attached to it are dissected out and transferred to a dish containing frog-ringer solution at room temperature.
• The muscles are divided, unless they are very small and threads are attached to the top and bottom of each piece. One end is attached to the pin in the organ bath and the other end to the lever.
• The load on the lever should be 0.5 g to 1.0 g and adjust it so that it is just balanced and then add the load, a small piece of plasticine is suitable. The preparation is kept at room temperature in frog ringer solution through which air is blown.
• The capacity of the organ bath is 5-10 ml. A simple side way writing lever is suitable though a gimbal is better but is expensive. A vibrator may be attached to avoid sticking on to the writing paper.
• After the muscle has been made to contract, it will not relax rapidly when the drug has been washed out, and it must be stretched gently by increasing the load. The experiment is then carried with the drugs and the effects are recorded on the kymograph.