Amphibian instruments, circuits and nerve muscle preparation.pptx

Amphibian experiments

1. I NSTRUMENTS IN AMPHIBIAN EXPERIMENTS, ELECTRICAL CIRCUITS, FROG DISSECTION AND NERVE MUSCLE PREPARATION Dr Swati Bansal Senior Resident

2. I NSTRUMENTS

3. SIMPLE/ PRIMARY KEY Used to open or close the primary circuit

4. DU BOIS-REYMOND INDUCTION COIL Converts galvanic current into faradic current Principle : flow of current in a wire produces a magnetic field in the space around it which, in turn, induces a current in another coil nearby.

5. KYMOGRAPH Used for recording graphically the time course of events in tissues manifesting movement

6. SECONDARY KEY It is kept closed to p revents accidental passage of current to the tissues

7. ELECTRODES Stimulates the tissue

8. REVERSING KEY Shunts current from one electrode to another in experiments where 2 electrodes are used

9. TAPPING KEY Makes or breaks the circuit for a limited time

10. NEEF'S HAMMER Used to give repeated multiple stimuli 30-40 times/second

11. VARIABLE INTERRUPTER Interrupts the primary circuit at a wider range of frequency which is more precisely adjustable than Neef's hammer

12. MYOGRAPH BOARD WITH STAND Wooden board which is layered with wax Has corks to facilitate the pinning of tissues

13. LUCAS CHAMBER/ MUSCLE TROUGH Used in experiments where tissues are immersed in Ringer's solution To see effect of temperature, drugs

14. ELECTROMAGNETIC SIGNAL MARKER TUNING FORK TIME MARKING

15. ISOTONIC MUSCLE LEVER STARLING HEART LEVER

16. ELECTRICAL CIRCUITS

17. PRIMARY CIRCUIT SECONDARY CIRCUIT

18. ELECTRICAL CONNECTIONS CIRCUIT FOR SINGLE “MAKE” OR “BREAK” STIMULI Primary circuit- low voltage mains, simple key and primary coil connected in series Secondary circuit- secondary coil, short circuiting key and stimulating electrodes are connected in parallel with the primary coil.

19. TAKING KYMOGRAPH IN CIRCUIT Primary circuit- low voltage mains, simple key, primary coil and contact block of drum connected in series Secondary circuit- secondary coil, short circuiting key and stimulating electrodes are connected in parallel with the primary coil. The drum terminals are taken in the primary circuit when one wants to mark the point of stimulation or when two successive stimuli are needed.

20. TAKING NEEF’S HAMMER OR VARIABLE INTERRUPTER IN CIRCUIT Primary circuit- low voltage mains, simple key, Neef’s hammer of induction coil or variable interrupter and primary coil. Secondary circuit- secondary coil, short circuiting key and stimulating electrodes are connected in parallel with the primary coil. Used for getting repeated stimuli such as for producing tetanus or for stimulating the vagus nerve.

21. FROG DISSECTION AND NERVE MUSCLE PREPARATION

22. WHY FROG? Cold blooded animals and their nerve muscle preparation survival under experimental conditions is superior Tissues can survive outside body as it can absorb oxygen from atmosphere Easy availability round the year It is completely harmless Easy storage Easy to handle during experiment Easy to dispose off.

23. Dissection Of Gastrocnemius Nerve Muscle Preparation APPARATUS Pithing needle Scissors Forceps Bone cutter Wooden dissection board Amphibian Ringer’s solution Cotton wool

24. AMPHIBIAN RINGER’S SOLUTION Sodium chloride (0.6%)- isotonicity, generation of action potential Calcium chloride (0.012%)- excitability of living tissue Potassium chloride (0.014%)- resting membrane potential Sodium bicarbonate (0.02%)- optimal pH Dextrose (0.1%)- provide nutrition

25. PROCEDURE STUNNING Holding the frog gently and firmly give a sudden strong blow on its head by striking against the edge of the table. This renders the frog unconscious. Anesthesia not used as it may suppress the excitability of the nerve, neuromuscular junction or muscle

26. PITHING- purpose is to destroy the brain and spinal cord so that the animal neither feels pain nor there are any reflexes or voluntary movements during the dissection Hold the animal in the left hand and flex its head with the index finger. Ventro -flexing of the head helps to locate the triangular depression which marks the point where the pithing needle has to be inserted. Demarcate the point exactly in the center of the line joining the two tympanic membranes. There is a depression, where a sharp-pointed pithing needle should be pushed in. The pithing needle is pushed upward and rotated in all possible directions to completely destroy the brain.

27. Thereafter, it is pushed downward into the vertebral canal. The pithing needle is rotated in all possible directions to destroy the spinal cord properly. When the spinal cord is destroyed and pithing is effectively complete then the limbs become flaccid, the animal neither feels pain nor does it move its limb during dissection and shows loss of conjunctival and corneal reflexes. Destroying the brain alone is not sufficient as the spinal reflexes may interfere with muscle contractions. Bring out the needle from the spial cord. The frog is ready for dissection.

28. DISSECTION Cut through the skin with scissors completely all around the trunk just below the forelimbs. Seize the skin in a duster and strip off the skin down to the toes. Place the frog on the myograph board on its abdomen. Pick up the tip of the urostyle with the forceps and cut the pelvic girdle on its either side , taking care not to injure the sciatic nerve.

29. Lift up the urostyle and cut 2cm long piece of vertebral column above and below the exit of sciatic nerves. This piece of vertebral column is retained to keep the nerve stretched over the stimulating electrodes. It also provides a rigid body to prevent nerve injury during accidental rough handling. Using a bone forceps, cut and divide this piece of vertebral column into two pieces. Lift each piece with forceps and snip away the nerves going to nearby tissues, taking care not to injure the sciatic nerves which can be seen disappearing into the thigh muscles. Expose the sciatic nerve in the thigh between the muscles and gently separate the nerve from the thigh muscle. Use a glass rod to handle the nerve, identify, dissect and cut the gastrocnemius tendon from its attachment. Tie a thread around the tendon.

30. Using any metal object will stimulate the nerve and cause the contraction of the muscle. Repeated contraction with metal objects will lead to muscle fatigue. Free the muscle from the tibia. Cut off the tibia-fibula below the knee joint and the femur close to the knee joint. Remove all redundant muscle other than the gastrocnemius. The knee joint is kept intact as it is required to fix the nerve muscle preparation to the myograph board. Moreover, the nerve may get injured in the process of cutting the knee joint. The nerve muscle preparation is now ready. Keep it immersed in frog’s Ringer’s solution till the commencement of the experiment.

31. PRECAUTIONS Ensure minimum injury to the nerve-muscle preparation. Nerve should never be stretched or repeatedly touched by a metal object. Do not let the nerve-muscle preparation to dry (keep moistening with amphibian Ringer’s solution). Do not use forceps or other metallic objects to handle the nerve-muscle preparation.

32. QUESTIONS 1. What is the advantage of using a frog for dissection? Cold blooded animals and their nerve muscle preparation survival under experimental conditions is superior Tissues can survive outside body as it can absorb oxygen from atmosphere Easy availability round the year It is completely harmless Easy storage Easy to handle during experiment Easy to dispose off.

33. 2. Why gastrocnemius is the muscle of choice? Gastrocnemius being a bulky muscle- Is easily identifiable Cannot be fatigued easily Gives good amplitude of contraction on being stimulated

34. 3. Why sciatic is the nerve of choice? it is a thick, shiny nerve and can be easily identified It can be easily mounted as it is the longest nerve of the body

35. 4. What is the composition of Ringer’s solution? Ans- Sodium chloride (0.6%)- isotonicity, generation of action potential Calcium chloride (0.012%)- excitability of living tissue Potassium chloride (0.014%)- resting membrane potential Sodium bicarbonate (0.02%)- optimal pH Dextrose (0.1%)- provide nutrition

36. 5. How will you pith the frog? Hold the animal in the left hand and flex its head with the index finger. Ventroflexing of the head helps to locate the triangular depression which marks the point where the pithing needle has to be inserted. Demarcate the point exactly in the center of the line joining the two tympanic membranes. There is a depression, where a sharp-pointed pithing needle should be pushed in. The pithing needle is pushed upward and rotated in all possible directions to completely destroy the brain. Thereafter, it is pushed downward into the vertebral canal. The pithing needle is rotated in all possible directions to destroy the spinal cord properly. When the spinal cord is destroyed and pithing is effectively complete then the limbs become flaccid, the animal neither feels pain nor does it move its limb during dissection and shows loss of conjunctival and corneal reflexes. Destroying the brain alone is not sufficient as the spinal reflexes may interfere with muscle contractions. Bring out the needle from the spial cord. The frog is ready for dissection.

37. 6. Why nerve muscle has to be dissected out with a part of bone? A piece of vertebral column is retained to keep the nerve stretched over the stimulating electrodes. It also provides a rigid body to prevent nerve injury during accidental rough handling. The knee joint is kept intact as it is required to fix the nerve muscle preparation to the myograph board. Moreover, the nerve may get injured in the process of cutting the knee joint.

38. 7. How will you identify and dissect out sciatic nerve? S ciatic nerve identified as a long, thick and shiny structure, can be seen disappearing into the thigh muscles. Expose the sciatic nerve in the thigh between the muscles and gently separate the nerve from the thigh muscle.

39. THANK YOU!