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Recording membrane voltage in current-clamp mode

Recording membrane voltage in current-clamp mode. Recording resting potentials, neuronal firings (trains of APs), pacemaker activities, graduate potentials requires glass microelectrodes of high resistance (10-100 M )

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Recording membrane voltage in current-clamp mode

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  1. Recording membrane voltage in current-clamp mode • Recording resting potentials, neuronal firings (trains of APs), pacemaker activities, graduate potentials requires glass microelectrodes of high resistance (10-100 M) • The cell can also be hyperpolarized or depolarized to regulate the resting and to evoke APs by passing a constant or stepwise membrane current. The current electrode is usually low-ohmic (k-M) and does not necessarily penetrate the cell. • Measuring voltages and passing currents can be done with the same microelectrode How? from Carbone, Cicirata, Aicardi, EdiSES, 1° ed. (2009)

  2. Recording membrane potentials with operational amplifiers What is an operational amplifier? Is a solid-state amplifier with the following characteristics: Withopen circuit: high gain (A) = ∞ (≈ 2x105) high Rin= ∞ (≈ 1x1014  ) low Rout= 0 (≈ 10 ) It can be used to make sums, subtractions, integrals, derivatives or any other mathematical operation of the input signals

  3. 1st example - The voltage inverter At the bluejunction: i1 = i2 + ia Due to the high gain of the op. amplif., the blue point acts as a “virtual ground”. There is no current flowing behind:  = 0 and ia =0 R2 Vo (Vi - ) Vo Vi ( -Vo) = - = - = + ia R1 R2 Vi R1 R2 R1 R2 (inverting) The gainisA= - R1 Rin= R1 Rout= 0 

  4. 2nd example - The non-inverter Vi but i = Assuming ia= 0 and  = 0: R2 (Vo- Vi) = R1 i R1 R1 Vi The gainisA= 1 + Vo = 1 + Vi Vo = Vi + R1 R2 R2 R2 thus (non-inverting) Rin= ∞  Rout= 0 

  5. 3rd example - The unity-gain, buffer amplifier (the “voltage-follower”) It has the same configuration of the previous case except that: R2 = ∞ and R1 = 0 R1 Vo = 1 + Vi R2 The previous equation: Vo = 1 Vi becomes: The gainisA = +1 (unity) Rin= ∞  Rout= 0  It is the ideal “buffer amplifier” for coupling high-resistance microelectrodes (>100 M) with instruments which measure the voltage (oscilloscopes, computer interfaces, ….)

  6. A single-electrode current-clamp amplifier

  7. Current-clamp and voltage-clamp recordings for complete electrophysiological analysis • Action potential recordings in current-clamp (Im = 0) is optimal for recording neuronal activity without perturbing the cell • Data interpretation in terms of voltage-gated ion channels, however, is difficult since membrane voltage changes continuously with time • A good compromise is “clamping” the voltage to a fixed value and measure the current (Vm = K) • Under these conditions, the Ohm law: • Vm = Rm Im • can be simplified to: • K = Rm Im Im = Im gm K Rm

  8. The voltage-clamp circuit (Cole & Curtis, 1948) from Carbone, Cicirata, Aicardi, EdiSES (2009)

  9. The patch-clamp technique Neher & Sakmann (1981)

  10. Na+ and K+ currents at fixed voltages (Hodgkin & Huxley, 1952)

  11. Physiological and pharmacological separation of Na+ and K+ currents

  12. The voltage dependence of Na+ and K+ conductances To calculate the Na+ and K+ conductances we use the following equations: INa = gNa (Vm – ENa) with ENa= +63 mV IK = gK (Vm – EK) with EK = -102 mV

  13. The voltage dependence of Na+ and K+ conductances

  14. Tetrodotoxin (TTX): the classical Na+ channel blocker A pufferfish containing TTX

  15. The w-conotoxin GVIA: the N-type Ca2+ channel blocker The conus geographus from Philippines

  16. Noxiustoxin (NTX): a blocker of voltage-gated K+ channels Centruroides noxius (female from St. Rosa, México) 

  17. The voltage-gated Na+, K+ and Ca2+ channels

  18. Suggested readings: General: Chapters 1-3 in Purves et al. Neuroscience, Sinauer, 4° ed. Chapters 1-3 in Carbone et al. Fisiologia: dalle molecole ai sistemi integrati, EdiSES, 1st ed. Technical:The axon guide: A Guide to Electrophysiology & Biophysics Laboratory Techniques Down-load from: http://www.moleculardevices.com/pages/instruments/axon_guide.html

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