GIORNATE TECNICHE DELLA SCUOLA DI
DOTTORATO IN SCIENZE BIOLOGICHE E MEDICHE
Tecniche elettrofisiologiche: applicazioni in ricerca di base e clinica
Varese 6 luglio 2011
Antonio Peres
Tecniche di elettrofisiologia:
il Voltage-Clamp
The dawn of the clinical electrophysiology
Torpedo torpedo
Julius Caesar
Luigi Galvani 1737-1798
Julius Bernstein 1839-1917
Bernstein’s rheotome
The Bernstein’s hypothesis. In 1902
Julius Bernstein hypothesized that the
action potential could be due to a sudden
and transient loss of the ionic selectivity
of the membrane. According to this
hypothesis the resting cell membrane
was scarcely permeable and mainly to
potassium, while during the impulse a
non-selective permeability increase was
occurring. This phenomenon was called
the
“membrane breakdown”. It is
important to remember that Nernst’s
equation, describing the relation between
membrane permeability and electrical
potential, was developed only few years
earlier (1889)
Potenziale d’azione registrato da Bernstein;
esempio di registrazione extracellulare di
attività elettrica
The squid giant axon
A section of a squid nerve, showing in the
middle the giant axon surrounded by
hundreds of small-size axons
Stellate
ganglion
Giant
axon
Stellate ganglion
Intracellular recording of the action potential
Axon segment
Recording electrode
Amplifier
Reference electrode
stimulus
Hodgkin cycle (positive feedback)
Stimulus
1 – Membrane
depolarization
3 – Na+ enters the cell
carrying positive charges
2 – More Na+ channels open
Na+ permeability increases
Voltage-clamp
off
Voltage-clamp
off
Voltage-clamp
off
The idea of the voltage-clamp
(Cole & Marmont)
Kenneth S. Cole
1900-1984
George Marmont (searched with Google)
Voltage-clamp
The Na+ charges are
neutralized by the
externally provided current
Stimulus
1 – Membrane
depolarization
3 – Na+ enters the cell
carrying positive charges
2 – More Na+ channels open
Na+ permeability increases
Voltage-clamp
on
Voltage-clamp
on
Voltage-clamp
on
Voltage-clamp
on
Voltage-clamp
on
Voltage-clamp in the squid giant axon
(space clamp)
Eccles, Hodgkin & Huxley
Two-electrode voltage-clamp (TEVC)
Vm
V
GAT1
Vm= -40 mV
Voltage-clamp
on
A
Vcom
Vm
Im
comparatore
Vcom
Im
Vm
V
comparatore
Vcom
+
GABA
+
Voltage-clamp
on
Vm= -39 mV
A
Vcom
Vm
Im
Im
Vm
V
GABA
+ +
+
Voltage-clamp
on
+
Vm= -40 mV
A
Vcom
Vm
Im
comparatore
Vcom
Im
Vm
V
Voltage-clamp
on
Vm= -40 mV
A
Vcom
Vm
Im
comparatore
Vcom
Im
Patch Clamp
http://www.nature.com/nprot/journal/v1/n4/fig_tab/nprot.2006.266_F4.html
General components of a dual
patch clamp setup: (1) A/D
converter, (2) pulse generator,
(3) video monitor, (4)
oscilloscope, (5, 6) patch
clamp amplifiers, (7) computer
CPU containing acquisition and
analysis software, (8)
pneumatic table, (9) inverted
microscope, (10, 15) fine
position controllers for two
hydraulic manipulators, (11,
14) coarse controllers for two
hydraulic manipulators, (12,
13) two headstages for each
amplifier, (16) gravity perfusion
system, (17) Faraday cage,
(18, 19) syringes connecting to
the rear of patch pipette
holders, (20, 21) patch pipette
holders, (22) ground silver
wire, (23) perfusion system
outflow, (24, 25) patch
pipettes, (26) recording
chamber, (27) tube connected
to vacuum system, (28)
microscope stage adapter, (29)
microscope stage.
Main
configurations
Voltage step
applied between
pipette and bath
I
Pipette
in air
Pipette
in bath
Cell-attached
(gigaseal)
Whole
Cell
Gigaseal formation and rupture
Examples
A bacterial spheroplast patched
with a glass pipette.
http://www.conncad.com/gallery/patch_clamp.html
Hippocampal neurons
A piramidal
neuron
Single channel current
THE END