recording membrane voltage in current-clamp mode from carbone, cicirata, aicardi, edises, 1° ed....

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?

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) = ∞ (≈ 2x105)

high Rin = ∞ (≈ 1x1014 )

low Rout = 0 (≈ 10 )

1st 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 ia =0

(Vi - ) ( -Vo) R1

= + ia R2

At the blue junction: i1 = i2 + ia

Vi Vo

R1 = -

R2 Vo R2

Vi = -

R1

(inverting) The gain is A = -

R2

R1

Rin = R1

Rout = 0

2nd example - The non-inverter

but i =Vi

R2

(Vo- Vi) = R1 i

Assuming ia= 0 and = 0:

(non-inverting) The gain is A = 1 +

R1

R2

Rin = ∞

Rout = 0

Vo = Vi + R1 Vi

R2 thus

Vo = 1 + Vi R1

R2

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

Vo = 1 + Vi R1

R2 The previous equation:

becomes:Vo

Vi = 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)

Rin = ∞

Rout = 0

A single-electrode current-clamp amplifier

Current-clamp and voltage-clamp recordings for complete

electrophysiological analysis

Under these conditions, the Ohm law:

Vm = Rm Im

can be simplified to:

K = Rm Im Im = Im gm

KRm

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)

from Carbone, Cicirata, Aicardi, EdiSES (2009)

The voltage-clamp circuit (Cole & Curtis, 1948)

The patch-clamp techniqueNeher & Sakmann (1981)

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

Physiological and pharmacological separation of Na+ and K+ currents

The voltage dependence of Na+ and K+ conductances

To calculate the Na+ and K+ conductances we use the following equations:

INa = gNa (Vm – ENa)

IK = gK (Vm – EK)

with ENa= +63 mV

with EK = -102 mV

The voltage dependence of Na+ and K+ conductances

Tetrodotoxin (TTX): the classical Na+ channel blocker

A pufferfish containing TTX

The -conotoxin GVIA: the N-type Ca2+ channel blocker

The conus geographus from Philippines   

Noxiustoxin (NTX): a blocker of voltage-gated K+

channels

Centruroides noxius (female from St. Rosa, México)   

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

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