DEPARTMENT OF PHYSIOLOGY

DOWUNIVERSITY OF HEALTH SCIENCES.

OBJECTIVE: TO RECORD SIMPLE MUSCLE TWICH (SMT)

LEARNING OBJECTIVES:

By the end of this experiment student should know:

  • How to pith the frog.
  • How to prepare nerve and muscle for further procedure.
  • How to record a graph of simple muscle twitch.

TO PITH THE FROG

REQUIREMENTS:

Frog and dissecting instruments.

THEORY:

Before using any animal for experimental purposes it should be made unconscious. To produce unconsciousness there are two methods:

Anesthesia:

The unconsciousness may be produced by using any anesthetic agent which may be used orally, intravenously or by inhalation of chloroform or ether, etc. but it is very costly and carries its own risk.

Pithing:

The unconsciousness may be produced by destroying the brain and spinal cord that is the CNS. This prevents the frog from perceiving any pain (the frog is clinically dead).

To study the nerve and muscle physiology of frog, it must be killed but tissues are kept alive. This can be done by pithing and destroying the CNS.

CLINICAL DEATH: The clinical death may be defined as loss of higher brain functions, irreversibly but its muscles and peripheral nerves are functional as long as the cells remain alive.

PROCEDURE:

  • Take the frog in the left hand and press the head downwards with the index finger so that the head makes a right angle with the trunk.
  • Place the nail of index finger of right hand in between the eyes and feel the OCCIPITOVERTIBRAL GROOVE by moving the nail backwards.
  • The nail dips into the groove. Press the nail so that it leaves a mark on the skin.
  • From this mark introduce the pithing needle into the CRANIAL CAVITY through the FORAMEN MAGNUM.
  • Pull the needle out in a rotating manner so that the BRAIN is completely destroyed.
  • This is known as SINGLE PITHING and the animal is called SPINAL ANIMAL.
  • Now introduce the needle into the VERTEBRAL COLUMN and pull it out in a rotating manner so that the SPINAL CORD is completely destroyed.
  • Complete unconsciousness is produced and the animal is termed as PITHED ANIMAL.
  • The muscles become flaccid.
  • Needle (fine probe) is inserted into aspot roughly given by the apex of anequilateral (0) whose base is formed by aline joining the two eyes.
  • Lay the pithed frog on frog board and test it for complete paralysis by touching the limbs with the pithing needle.
  • Any bleed occurring can be stopped by pressure with cotton.
  • This destroys spinal reflexes and frog’s legs straighten out.

Pithed Frog

PREPARATION OF NERVE AND MUSCLE OF FROG

OBJECTIVE:

The student should be able to

  • Prepare gastrocnemius sciatic preparation.
  • Explain the effects on nerve and muscle if not placed in Ringer's Solution.

REQUIREMENT:

Frog, dissecting instruments, Cotton, Ringer's solution, beaker, thread, dissectingboard, glass rod, Common pin.

PRINCIPLE:

The principle of this experiment is to obtain a separate independent muscle along with its nerve supply so that:

It may be stimulated and various effects observed.

THEORETICAL EXPLANATION:

From the frog following preparations of skeletal muscles along with their nerves may be obtained.

  1. Gastrocnemius and sciatic preparation.
  2. Hypoglossus with its hypoglossal nerve.
  3. Sartorious with its femoral nerve.

Among all of these, the Gastrocnemius sciatic preparation has been selected for experimental purposes due to following.

  1. It is a separate muscle and can be isolated very easily along with its independent nerve supply the sciatic preparation, Achilles tendon and the knee joint.
  2. It can be stimulated directly and indirectly through the nerve.
  3. Comparatively there is less chance of injury to the muscle and nerve.

Under proper conditions of isotonicity, nutrition and temperature etc. the life of nerve and muscle may be prolonged for several hours.

PROCEDURE:

Lay the pithed frog on the frog board. Cut the skin transversely all around the trunk and remove the skin from the two hind Limbs.

Open the abdominal cavity, remove the viscera, a white, shining nerve may be seen emerging from the last 3 vertebrae. Give a transverse cut just over the last 3 vertebrae and cut the two hind limbs into two pieces by giving a longitudinal cut very carefully so that the nerve is not damaged.

Remove all the muscles of the thigh completely and also cut the femur just near the knee joint so that a small portion of femur remains attached with the knee joint. The sciatic nerve is completely exposed and only a portion of the last 3 vertebrae remain attached with sciatic nerve.

Now separate the muscle with the help of a glass rod, tie a thread at Achilles tendon and cut it about 1 cm away from the knot. Now cut the tibia just near the knee joint so that a small portion of it remains attached with the knee joint. This is Nerve Muscle Preparation or Gastrocnemious Sciatic Preparation.

COMPONENTS:

  1. ACHILLIS TENDON: It may be tied with the muscle lever (vertical arm) by means of a thread tied to it.
  2. SCIATIC NERVE: The muscle can be stimulated indirectly by placing the electrodes on the nerve and applying electrical stimuli to it.
  3. KNEE JOINT: From the knee joint the muscle may be fixed into the Lucas bath by means of a pin, inserting into the hole.
  4. GASTROCNEMIOUS MUSCLE: An artificial stimulus (i.e. electrical stimulus) is applied to the muscle to record its response.
  5. A PORTION OF LAST 3 VERTEBRAE: The sciatic nerve can be handled from this portion.

PRECAUTION:

  1. Keep the nerve and the muscle wet with Ringer's Solution or Normal Saline during its preparation and afterwards so that the nerve and the muscle are not dried and maintain their normal shape.
  2. Do not touch the nerve and the muscle with the hand or any other instrument. Use a glass rod for this purpose.

Avoid pulling, twisting and kinking of the nerve, so that it is not damaged.

TO RECORD SIMPLE MUSCLE TWICH (SMT)

OBJECTIVE:

The student should be able to:

  1. Fix the NMP in the muscle chamber.
  2. See the response of stimulus on nerve muscle preparation

REQUIREMENTS:-

Nerve muscle preparation, Lab tutor, Stand, Ringer’s solution or normal saline, Pins and dissecting instruments.

THEORY:-

The simple muscle twitch (S.M.T) may be defined as the response of the muscle to a single stimulus applied to a muscle, it give rise to a sudden contraction for a fraction of time.

S.M.T consists of three phases:

1. LATENT PERIOD:-

A phase following the point of contraction i.e. from point of stimulation (PS) to point of contraction (PC) during which no apparent changes take place in the muscle.

During this period the action potential is generated at PS in the nerve, this impulse propagates towards the neuromuscular junction, transmitted from the nerve to muscle fibers and prepares them for the contraction. The duration of L.P is about 0.01 second.

2. CONTRACTION PERIOD:-

A phase of shortening and contraction of muscle during which the actin and myosin filaments slides on each other and the muscle shortens in length. The duration of this period is about 0.04 seconds.

3.RELAXATION PERIOD:-

A phase of returning or relaxation during which the muscle returns to its original length. Its duration is about 0.05 seconds.

EQUIPMENT SETUP:

POWERLAB:

  1. Connect the Bridge Pod to Input 1 of the PowerLab.

2. Connect the positive and negative BNC connectors of the stimulating electrode leads to the analog Outputs on the PowerLab.

Force Transducer & Micropositioner

  1. Mount the micropositioner on to the Retort Stand.
  2. Securely mount the force transducer in the micropositioner mounting bracket.
  3. Plug the force transducer cable into the back of the Bridge Pod.

Muscle Holder

  1. Attach the Muscle Holder to the Retort Stand.
  2. Fix the muscle in the Muscle Holder by positioning the knee joint just below the constriction in the perspex molding, as shown in the figure.
  3. Connect the alligator clips from the stimulating electrodes to the inner two connectors at the top of the muscle holder.

Retort Stand

  1. Secure the thread attached to the achilles tendon of the muscle through the hole in the metal tab of the force transducer. Make sure that there is some slack in the thread.
  2. Raise the micropositioner using the adjustment knob so that the muscle is vertical but not under tension. The thread should not be loose, but should have some slack in it. Make sure that there is room to increase the height of the force transducer by at least 10 mm.
  3. Check that the stimulating electrodes on the muscle holder are in contact with the muscle
  4. Rotate the base of the muscle holder and insert an appropriately sized beaker to collect waste solution.
  5. Finally, rinse the muscle with Frog Ringer's solution to keep it moist.

PROCEDURE:-

•Make sure the muscle is moist and is in contact with the stimulating electrodes.

•In the Stimulator panel, check that the voltage is set to 50 mV.

•Click Start. The software is set up so that Lab Tutor will now record for 625 ms with a single stimulus being delivered after an initial delay of 50 ms.

•The contractile response of the muscle and the time at which the stimulus was delivered are shown in the LabTutor panel. LabTutor will also enter a comment showing the amplitude of stimulation.

•Continue to increase the stimulus amplitude in 50 mV steps, recording the responses until you have 3 successive stimuli that do not produce any further increase in the amplitude of the muscle contraction

RESULTS:

S.NO. / PHASES / STANDARD VALUES / OBSERVE VALUES
1. / Latent Period (L.P) / 0.01 sec.
2. / Contraction Period (C.P) / 0.04 sec.
3. / Relaxation Period (R.P) / 0.05 sec.
4. / Height of Contraction

PRECAUTIONS:

  1. Nerve and muscle preparation should remain wet with ringer’s solution.
  2. Nerve and muscle preparation should be placed properly on stand.
  3. Lab tutor is properly assembled.
  4. Electrode is placed over muscle fibers.