recording membrane voltage in current-clamp mode from carbone, cicirata, aicardi, edises, 1° ed....
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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?
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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 )
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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
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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
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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
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A single-electrode current-clamp amplifier
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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)
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from Carbone, Cicirata, Aicardi, EdiSES (2009)
The voltage-clamp circuit (Cole & Curtis, 1948)
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The patch-clamp techniqueNeher & Sakmann (1981)
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Na+ and K+ currents at fixed voltages (Hodgkin & Huxley, 1952)
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Physiological and pharmacological separation of Na+ and K+ currents
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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
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The voltage dependence of Na+ and K+ conductances
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Tetrodotoxin (TTX): the classical Na+ channel blocker
A pufferfish containing TTX
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The -conotoxin GVIA: the N-type Ca2+ channel blocker
The conus geographus from Philippines
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Noxiustoxin (NTX): a blocker of voltage-gated K+
channels
Centruroides noxius (female from St. Rosa, México)
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The voltage-gated Na+, K+ and Ca2+ channels
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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