1. Recording membrane voltage in current-clamp mode from Carbone, Cicirata, Aicardi, EdiSES, 1° ed. (2009)  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?

2. It can be used to make sums, subtractions, integrals, derivatives or any other mathematical operation of the input signals Recording membrane potentials with operational amplifiers What is an operational amplifier? Is a solid-state amplifier with the following characteristics: With open circuit: high gain (A) = ∞ (≈ 2x10 5 ) high R in = ∞ (≈ 1x10 14  ) low R out = 0 (≈ 10  )

3. 1 st example - The voltage inverter 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 i a =0 (V i -  ) (  -V o ) R1R1 = + i a R2R2 At the blue junction: i 1 = i 2 + i a ViVi VoVo R1R1 = - R2R2 VoVo R2R2 ViVi R1R1 (inverting) The gain is A = - R2R2 R1R1 R in = R 1 R out = 0 

4. 2 nd example - The non-inverter but i = ViVi R2R2 (V o - V i ) = R 1 i Assuming i a = 0 and  = 0: (non-inverting) The gain is A = 1 + R1R1 R2R2 R in = ∞  R out = 0  V o = V i + R 1 ViVi R2R2 thus V o = 1 + V i R1R1 R2R2

5. 3 rd example - The unity-gain, buffer amplifier (the “voltage-follower”) It has the same configuration of the previous case except that: R 2 = ∞ and R 1 = 0 V o = 1 + V i R1R1 R2R2 The previous equation: becomes: VoVo ViVi = 1 It is the ideal “buffer amplifier” for coupling high-resistance microelectrodes (>100 M  ) with instruments which measure the voltage (oscilloscopes, computer interfaces, ….) The gain is A = +1 (unity) R in = ∞  R out = 0 

6. A single-electrode current-clamp amplifier

7. Current-clamp and voltage-clamp recordings for complete electrophysiological analysis  Under these conditions, the Ohm law: V m = R m I m can be simplified to: K = R m I m I m = I m  g m K RmRm  Action potential recordings in current-clamp (I m = 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 (V m = K)

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

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: I Na = g Na (V m – E Na ) I K = g K (V m – E K ) with E Na = +63 mV with E K = -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  -conotoxin GVIA: the N-type Ca 2+ 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 Ca 2+ 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, 1 st ed. Technical: The axon guide: A Guide to Electrophysiology & Biophysics Laboratory Techniques Down-load from: http://www.moleculardevices.com/pages/instruments/axon_guide.html