ACTION POTENTIAL - IONIC BASIS AND RECORDING

1. IONIC BASIS AND RECORDING OF ACTION POTENTIAL Dr.Anu Priya.J . 11-12-2013 1

2. Introduction History Resting membrane potential Graded potential Action potential Ionic basis Types Recording Applied aspects 11-12-2013 2

3. Introduction Nerve and muscle are excitable tissues Can undergo rapid changes in their membrane potentials Change their resting potentials into electrical signals that aid in cellular communication These signaling events are mediated by ion channels 11-12-2013 3

4. 11-12-2013 4 Since the 18 th  century, when Galvani introduced the concept of "animal electricity", electric potentials have been observed and recorded in different nerves and muscles. History

5. 11-12-2013 5 Illustration of Italian physician Luigi Galvani's experiments, in which he applied electricity to frogs legs; from his book De Viribus Electricitatis in Motu Musculari (1792).

6. History 1963- A. L. Hodgkin and A. F. Huxley - Nobel prize in Physiology or Medicine- study of sodium and potassium channels – voltage clamp method Sir John Carew Eccles-shared-work on synapse 11-12-2013 6

7. 11-12-2013 7 The patch clamp technique - Erwin Neher and Bert Sakmann  - Nobel Prize in Physiology or Medicine in 1991 Record the currents of single ion channels for the first time, proving their involvement in fundamental cell processes such as action potential conduction. History

8. Sir A. F. Huxley passed away on 30 May 2012 – age 94 years 11-12-2013 8 History

9. 11-12-2013 9 It is the potential difference existing across the cell membrane at rest Interior of the cell is negatively charged in relation to the exterior State of polarisation Resting Membrane Potential

10. Resting Membrane Potential RMP is maintained by: Natural concentration gradient Selective permeability of cell membrane Impermeable anions Sodium-potassium ATPase pump 11-12-2013 10

11. Neurons have a selectively permeable membrane During resting conditions membrane is: permeable to potassium (K + ) (channels are open) impermeable to sodium (Na + ) (channels are closed) Diffusion force pushes K + out (concentration gradient) This creates a positively charged extra-cellular space. Electrostatic force pushes K + in Thus, there is a ‘dynamic equilibrium’ with zero net movement of ions. The resting membrane potential is negative Resting Membrane Potential 11-12-2013 11

12. Graded potential Subthreshold stimuli cause sensory receptors to depolarize and produce a voltage called a generator potential(Receptor Potential) Does not obey all or none law Graded response it is not propagated Summation No refractory period Duration(5-10 ms) 11-12-2013 12

13. Graded potential & Action potential 11-12-2013 13

14. Action potential An action potential is a rapid change in the membrane potential in response to a threshold stimulus followed by a return to the resting membrane potential. The size and shape of action potentials differ considerably from one excitable tissue to another. An action potential is propagated with the same shape and size along the whole length of a cell. The action potential is the basis of the signal-carrying ability of nerve cells. Voltage-dependent ion channel proteins in the plasma membrane are responsible for action potentials. 11-12-2013 14

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17. Hodgkin cycle 11-12-2013 17

18. Graded potential & Action potential 11-12-2013 18

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22. Role of other ions Impermeable Anions Calcium ions 11-12-2013 22

23. Properties Voltage inactivation Refractory period All or none law Propagative depolarization and repolarization No summation 11-12-2013 23

24. 11-12-2013 24 Mammalian axons less than 20 μ m diameter Squid-giant cells-largest axon in neck region-about 1 mm diameter Recording of action potential

25. 11-12-2013 25 Requirements of instrument used : It should be capable of responding extremely rapidly The potential changes which are in millivolts has to be amplified before being recorded Recording of action potential

26. 11-12-2013 26 The instruments used are: Microelectrodes Electronic amplifiers Cathode ray oscilloscope (CRO) Recording of action potential

27. Microelectrodes 11-12-2013 27 Micropipette – tip size less than 1 mm diameter Filled with strong electrolyte solution- KCl Resistance – 1 billion Ω The tip of the micropipette is passed through the cell membrane of the nerve fibre Indifferent electrode – in extracellular fluid Connected to cathode ray oscilloscope through amplifier

28. Electronic amplifier 11-12-2013 28 magnify the potential changes of the tissue to be recorded on the oscilloscope screen

29. Cathode ray oscilloscope 11-12-2013 29 Rapid and instantaneous recording of electrical events of living tissues Parts Glass tube Cathode Fluorescent screen Two sets ( horizontal and vertical ) electrically charged plates

30. 11-12-2013 30 Cathode ray oscilloscope

31. Recording of action potential Patch clamp method Voltage clamp method 11-12-2013 31

32. Patch clamp method 11-12-2013 32

33. 11-12-2013 33 Voltage clamp method

34. Types Monophasic Biphasic Compound 11-12-2013 34

35. 11-12-2013 35 Biphasic action potential

36. Biphasic action potential 11-12-2013 36

37. 11-12-2013 37 Peripheral nerves in mammals are made up of many axons bound together in a fibrous envelope called the epineurium . Potential changes recorded extracellularly from such nerves therefore represent an algebraic summation of the all-or-none action potentials of many axons. The thresholds of the individual axons in the nerve and their distance from the stimulating electrodes vary. With subthreshold stimuli, none of the axons are stimulated and no response occurs. Compound action potential

38. 11-12-2013 38 When the stimuli are of threshold intensity, axons with low thresholds fire and a small potential change is observed. As the intensity of the stimulating current is increased, the axons with higher thresholds are also discharged. The electrical response increases proportionately until the stimulus is strong enough to excite all of the axons in the nerve. The stimulus that produces excitation of all the axons is the maximal stimulus, and application of greater, supramaximal stimuli produces no further increase in the size of the observed potential.

39. Compound action potential 11-12-2013 39

40. Applied aspects H ereditary spherocytosis (HS) Plasma membrane of red cells three times more permeable to Na + The level of Na + ,K + - ATPase elevated. When HS red blood cells have sufficient glucose to maintain normal ATP levels, they extrude Na + as rapidly as it diffuses into the cell cytosol . Hence the red blood cell volume is maintained. 11-12-2013 40

41. 11-12-2013 41 When HS erythrocytes are delayed in the venous sinuses of the spleen, where glucose and ATP are present at low levels, the intracellular ATP concentration falls. Therefore, Na + cannot be pumped out by the Na + ,K + - ATPase as rapidly as it enters. The red blood cells swell - osmotic effect of elevated intracellular Na + concentration. Spleen targets these swollen erythrocytes for destruction - anemia . Applied aspects

42. Tetrodotoxin (TTX)- a potent poison - specifically blocks the Na + channel- binds to the extracellular side of the sodium channel. Tetraethylammonium (TEA + ), another poison, blocks the K + channel when it is applied to the interior of the nerve fiber . Applied aspects 11-12-2013 42

43. 11-12-2013 43 The ovaries of certain species of puffer fish , also known as blowfish, contain TTX. Raw puffer fish - Japan. Applied aspects

44. Saxitoxin is another blocker of Na + channels that is produced by reddish- colored dinoflagellates that are responsible for so-called red tides. Applied aspects 11-12-2013 44

45. 11-12-2013 45 Shellfish eat the dinoflagellates and concentrate saxitoxin in their tissues. A person who eats these shellfish may experience life-threatening paralysis within 30 minutes after the meal Applied aspects

46. In an inherited disorder, called primary hyperkalemic paralysis, patients have episodes of painful spontaneous muscle contractions, followed by periods of paralysis of the affected muscles. Elevated levels of K + in the plasma and extracellular fluid. Some patients with this disorder have mutations of voltage-gated Na + channels that result in a decreased rate of voltage inactivation. This results in longer-lasting action potentials in skeletal muscle cells and increased K + efflux during each action potential. This can raise the extracellular levels of K + . Applied aspects 11-12-2013 46

47. 11-12-2013 47 The elevation of extracellular K + causes depolarization of skeletal muscle cells. Initially, the depolarization brings muscle cells closer to threshold, so that spontaneous action potentials and contractions are more likely. As depolarization of the cells becomes more marked, the cells accommodate because of the voltage-inactivated Na + channels. Consequently, the cells become unable to fire action potentials and are unable to contract in response to action potentials in their motor axons. Applied aspects

48. 11-12-2013 48 Low potentials recorded in neuropathy and spinal cord compression INJURY POTENTIAL   The difference in electrical potential between the injured and uninjured parts of a nerve or muscle – also called demarcation potential Applied aspects

49. Applied aspects 11-12-2013 49 TETANY Hypocalcemia – sodium channels activated by very little increase of membrane potential from resting state

50. THANK YOU 11-12-2013 50

51. References 11-12-2013 51 Guyton and Hall Textbook of Medical Physiology 12th edition Ganong's Review of Medical Physiology 23 rd edition Berne & Levy Physiology 6 th edition Boron and Boulpaep  Medical physiology 2 nd edition Basics of Medical physiology by Dr.Venkatesh.D 3 rd edition Textbook Of Medical Physiology  by Indu Khurana 1 st edition Internet references