Patch & Voltage Clamping - Education

Overview:

Voltage Clamp

The voltage clamp technique is used to measure the ion currents across a neuronal membrane while holding the membrane voltage at a constant level. The neuronal membrane contains different kinds of ion channels including voltage gated ion channels. Manipulation of the membrane voltage using voltage clamping allows the current-voltage relationships of these ion channels to be studied.

The voltage clamp technique is commonly used to investigate ion channel properties in native Xenopus Oocytes. Two microelectrodes are placed within the Oocyte: one records the membrane voltage while the second passes a current to maintain the membrane at a constant command voltage. This data is then used to calculate the membrane resistance of the Oocyte according to Ohm’s Law (Voltage = Current x Resistance).

Current Clamp

The current clamp technique is used to measure biological voltages such as the action potential of an excitable cell with a microelectrode while keeping electrical current through the recording electrode constant. It can be used for both extracellular and intracellular recordings.

Patch Clamp

The patch clamping technique is a modified form of the voltage clamp technique, using a blunt electrode placed on the surface of the cell membrane. It is a common intracellular recording technique in electrophysiology that allows the study of individual ion channels in cells. It can be used to measure currents passing through individual ion channels or through whole cells. Suction is applied via the blunt electrode (glass pipette in classical patch clamp technique) to create a high resistance (gigaohm) seal between the electrode the electrode and cell surface. This seal isolates the ion channels within the patch from the rest of the membrane and allows the neurophysiologist to record only from these ion channels.

Method:

Voltage Clamp

Voltage clamping with sharp electrodes is a predecessor to the patch clamp method. The voltage clamp can be thought of as a current generator with two electrodes; a "voltage electrode" that records the membrane potential and a "current electrode" that passes current into the cell. The measured membrane potential voltage is amplified. A signal generator is used to set the voltage level or the command potential. The amplified membrane potential and the command potential feed into a feedback amplifier, which subtracts the amplified membrane potential from the command potential, magnifies any differences, and sends an output to the “current electrode”. Whenever the membrane potential varies from the command potential, this feedback circuit passes a current into the cell to reduce differences to zero. Therefore, the clamp circuit produces a current equal and opposite to the ionic current, which can be measured, giving an accurate reproduction of the currents flowing across the membrane. Voltage clamp techniques include:

Two-electrode voltage clamp (TEVC)
Single-electrode voltage clamp (SEVC)

The Two Electrode Voltage Clamp Recording System and Oocyte Clamp Workstation System are used for clamping large cells and cell structures such as Xenopus Oocytes and squid axons.

Epithelial Voltage Clamp Systems are available for studies of epithelial transport and the electrical properties of tissue or cell layers held in Ussing Chambers.

Current Clamp

The current clamp is a low impedance microelectrode and a ground electrode that are connected to a differential voltage amplifier. Noise and resistance are minimized in this circuit. The output from the amplifier over time is measured and analyzed. Voltage changes about 0.1 mV to 200 mV, which last a fraction of a millisecond are usually recorded.

Patch Clamping

Classical patch clamping uses a glass pipette, with an open tip diameter of about one micrometer, and is made such that the tip forms a smooth surfaced circle, rather than a sharp point. The interior of the pipette is filled with an appropriate solution. The composition of this solution may be altered or drugs may be added to study the ion channels under different conditions. A metal electrode is inserted in this solution from the other end of the glass pipette.

The open tip of the patch clamp electrode is pressed against a cell membrane and suction is applied to the inside of the electrode to pull the cell's membrane inside the tip of the electrode. The suction causes the cell to form a tight seal with the electrode (creating a "gigaohm seal” because the electrical resistance of that seal is > 1 gigaohm). The metal electrode conducts the electrical changes to an amplifier. The cell can then be voltage clamped (keeping the voltage constant) to observe changes in current or current clamped (keeping current constant) to observe changes in membrane voltage.

There are many types of patch clamping including:

On-Cell Patch/Cell-Attached Recording
Whole Cell Clamp
Inside-Out Patch
Outside-Out Patch

Several Patch Clamp Recording Systems with varying headstage sensitivities are available for measuring current levels passing through whole cells or individual ion channels using a blunt electrode.

For more information on various applications please visit:

Intracellular Recordings application page
Evoked Potentials application page

Software:

The LabChart Advantage:

(may require additional Modules and Extensions)

Sample up to 200,000 Hz in a single channel (up to 400,000 Hz aggregate) using the 35 series PowerLabs
Smoothing, Absolute Value, Integration and RMS Arithmetic calculations for quantification of neural activity
Spectrum feature for online or offline determination of the power and frequency components of recorded signals
Cyclic Measurements for calculating firing Frequency, Period, and action potential Count
Scope View for spike-triggered averaging and the overlay of evoked responses
Telegraph Extension for correct scale display of data after a hardware gain change
Peak Analysis Module for analysis of peak heights, half-widths, latency periods, population spike heights, rise times and areas
Provides the ability to record, display and analyze any high frequency signal that is time-locked to a stimulus
Generates stimuli of differing intensities and waveform structures (i.e. single-pulse, multiple pulse, simple ramps) and control an external stimulator using the analog output on the front of the PowerLab.

LabChart

LabChart software (for Windows and Macintosh) combines the familiar simplicity of a traditional strip chart recorder with the powerful analysis features of a digital acquisition system. LabChart software and a PowerLab data acquisition unit provide data integrity, easy selection of hardware settings, powerful online and offline analysis, procedure automation, seamless extraction of experimental data and flexible display options. Acquisition and analysis capabilities can be further increased with LabChart Extensions and LabChart Modules. LabChart Modules are available as part of LabChart Pro and LabChart Extensions are free for download from the website for existing LabChart users.

Useful LabChart Extensions

Event Manager (Win): Allows the user to monitor user defined events online using different criteria, and to perform a variety of user defined actions..
Evoked Response (Mac): This Extension analyzes physiological responses to a stimulus (evoked response experiments). A number of response parameters can be measured and logged to the Data Pad. This process can be automated to analyze a series of stimuli/response cycles. Physiological responses of neurons to stimuli can be measured include value, latency, peak height, half-width, latency to peak, slope, population spike height and population spike area.
Peak Parameters (Mac): Allows the user to determine a number of parameters for an individual peak. Parameters calculated include peak height, width, slope and various time parameters. It is useful for determining parameters of action potentials such as cardiac potentials, EPSP and IPSP.
Export MATLAB (Mac or Win): Allows LabChart for Windows files to be saved and exported as MATLAB compatible files.
Translate Axon (Win): Allows the user to save LabChart files in the ABF (Axon binary format) which can be read by pClamp
Translate Binary (Win): Translate Binary is a LabChart Extension which allows LabChart for Windows files to be saved and exported in a simple binary format. Translate Binary can import documents that have either been exported from LabChart or generated by another application.
Translate EDF (Win): Allows LabChart to save data as an EDF file, and to read EDF files. Translate EDF does not support the EDF+ format.
Telegraph (Win): Makes use of the gain-telegraph output from an electrophysiological amplifier to continue to display data at the correct scale after a gain change. The Telegraph Extension uses the gain telegraph voltage from the amplifier to automate the display of electrophysiological data in LabChart, so that the correct units and scale are used.

Peak Analysis Module

The Peak Analysis Module (Windows) automatically detects and analyses multiple, non-overlapping peaks in recorded waveforms.

Features and benefits include:

View automatically detected peaks with highlighted parameter markers, values and peak areas in the Peak Analysis View.
Log calculated peak parameters to a table. Easily export parameters from the table into other programs.
Select from the default analysis settings available for general and specific signal types.
Customizable detection and calculation settings to suit your application or waveform.

Scope View

By providing digital oscilloscope feature Scope View (LabChart Windows) facilitates recording and viewing consecutive sweeps of data. These sweeps, or pages, can be overlaid and averaged. The signal averaging functions are necessary to extract the evoked response from background noise. There are several powerful features in Scope View that enhance analysis and display including:

Create pages or sweeps from Comments in Chart View in addition to block mode and Event Mode.
Use the new 3D Depth and Saturation Overlay feature in the Scope Overlay Options to help distinguish overlaid traces.
Alter the active trace and overlay trace colors.
Change the display background to black for better contrast.

GLP and 21 CFR Part 11

For those researchers working within a laboratory requiring GLP and 21 CFR Part 11 compliance the GLP Client and GLP Server are available for use with LabChart (Windows only) and PowerLab data acquisition systems. For more information, visit the Good Laboratory Practice application page or contact your nearest ADInstruments representative.

Hardware:

PowerLab Data Acquisition Systems

The PowerLab is a high-performance data acquisition unit capable of recording at speeds of up to 400,000 samples per second continuously to disk (aggregate). PowerLab units are compatible with instruments, signal conditioners and transducers supplied by ADInstruments, as well as many other third-party companies. In addition to standard single-ended BNC inputs, 4 differential Pod ports are also available for direct connection of Pod signal conditioners and appropriate transducers. Research PowerLab units include:

PL3504 PowerLab 4/35 - 4 Channels
PL3508 PowerLab 8/35 – 8 Channels
PL3516 PowerLab 16/35 – 16 Channels

Research Systems

PL3508B75-V Two Electrode Voltage Clamp Recording System

PL3508 PowerLab 8/35 (includes LabChart software)
MLS260/7 LabChart Pro software
PLA190 19 Inch Rack Adapter
OC-725C-V Oocyte Clamp Amplifier, which includes:
- 7250V Voltage Headstage
- 7251I Bath Clamp Headstage
- 7259C Current Electrode Cable
- 725MC Oocyte Model Cell (includes rack mount hardware)
ESW-F15V E Series Electrode Holder (Str, Vent, Ag Wire, 1.5 mm)
E45W-F15VH E Series Electrode Holder (45°, Vent, Handle, Ag Wire, 1.5 mm)

PL3508B76-V Oocyte Clamp Workstation System

PL3508/P PowerLab 8/35
MLS260/7 LabChart Pro software
PLA190 19" Rack Adapter
TEV700-V Complete Oocyte Clamp Workstation
ESW-F15V E Series Electrode Holder (Str, Vent, Ag Wire, 1.5 mm)
E45W-F15VH E Series Electrode Holder (45°, Vent, Handle, Ag Wire, 1.5 mm)

PL3508B77-V Single Channel Epithelial Voltage Clamp System

PL3508/P PowerLab 8/35
MLS260/7 LabChart Pro software
PLA190 19” Rack Adapter
EC-800-V Epithelial Voltage Clamp (120V compliance)

PL3508B78-V Dual Channel Epithelial Voltage Clamp System

PL3508/P PowerLab 8/35
MLS260/7 LabChart Pro software
PLA190 19” Rack Adapter
EC-825A-V Dual Channel Epithelial Voltage Clamp (50V compliance)

PL3508B79/10-V Patch Clamp Recording System with 5101-10G Headstage

PL3508/P PowerLab 8/35
MLS260/7 LabChart Pro software
PLA190 19” Rack Adapter
PC-501A/10-V Patch Clamp with 5101-10G Headstage,
MC-10G Model Cell for 5101-10G Headstage
QSW-A15P Q Series Electrode Holder (Str, Port, Ag Wire, 1.5 mm)

PL3508B79/8-V Patch Clamp Recording System with 5101-100M Headstage

PL3508/P PowerLab 8/30
MLS260/7 LabChart Pro software
PLA190 19” Rack Adapter
PC-501A/8-V Patch Clamp with 5101-100M Headstage (100 MΩ)
MC-100M Model Cell for 5101-100M Headstage (100 MΩ)
QSW-A15P Q Series Electrode Holder (Str, Port, Ag Wire, 1.5 mm)

PL3508B79/9-V Patch Clamp Recording System with 5101-01G Headstage

PL3508/P PowerLab 8/30 with LabChart Pro* and Scope
MLS260/7 LabChart Pro software
PLA190 19” Rack Adapter
PC-501A/9-V Patch Clamp with 5101-01G Headstage (1 GΩ)
MC-01G Model Cell for 5101-01G Headstage (1 GΩ)
QSW-A15P Q Series Electrode Holder (Str, Port, Ag Wire, 1.5 mm)

Instruments

Ussing Chambers

For the complete range of Ussing chambers click here:

U9926/A Single Ussing Chamber w/Round Insert (O-Ring, 3.8 mm)
U9926/B Single Ussing Chamber w/Round Insert (5 pins, 3.8 mm)
U9926/C Single Ussing Chamber w/Slotted Insert (6 pins, 1.6x7 mm)
U9926/M Single Ussing Chamber w/Millicell Insert (12 mm)
U9926/S Single Ussing Chamber w/Snapwell Insert (12 mm)
U9926/T Single Ussing Chamber w/Transwell Insert (6.5 mm)
U2500/A Dual Ussing Chamber w/Round Insert (O-Ring, 3.8 mm)
U2500/B Dual Ussing Chamber w/Round Insert (5 pins, 3.8 mm)
U2500/C Dual Ussing Chamber w/Slotted Insert (6 pins, 1.6x7 mm)
U2500/M Dual Ussing Chamber w/Millicell Insert (12 mm)
U2500/S Dual Ussing Chamber w/Snapwell Insert (12 mm)
U2500/T Dual Ussing Chamber w/Transwell Insert (6.5 mm)

Amplifiers

Stimulators

Transducers

Headstages

Accessories

Ussing Chamber

Electrode Holders

For the complete range of electrode holders click here:

E45W-F10VH E Series Electrode Holder (45°, Vent, Handle, Ag Wire, 1.0 mm)
E45W-F12VH E Series Electrode Holder (45°, Vent, Handle, Ag Wire, 1.2 mm)
E45W-F15NH E Series Electrode Holder (45°, Handle, Ag Wire, 1.5 mm)
E45W-F15VH E Series Electrode Holder (45°, Vent, Handle, Ag Wire, 1.5 mm)
E45W-F20VH E Series Electrode Holder (45°, Vent, Handle, Ag Wire, 2.0 mm)
QSW-A10P Q Series Electrode Holder (Str, Port, Ag Wire, 1.0 mm)
QSW-A12P Q Series Electrode Holder (Str, Port, Ag Wire, 1.2 mm)
QSW-A15P Q Series Electrode Holder (Str, Port, Ag Wire, 1.5 mm)
QSW-A17P Q Series Electrode Holder (Str, Port, Ag Wire, 1.7 mm)
QSW-A20P Q Series Electrode Holder (Str, Port, Ag Wire, 2.0 mm)
QSW-B10P Q Series E Holder w/BNC (Str, Port, Ag Wire, 1.0 mm)
QSW-B12P Q Series E Holder w/BNC (Str, Port, Ag Wire, 1.2 mm)
QSW-B15P Q Series E Holder w/BNC (Str, Port, Ag Wire, 1.5 mm)
QSW-B20P Q Series E Holder w/BNC (Str, Port, Ag Wire, 2.0 mm)
ESW-F10N E Series Electrode Holder (Str, Ag Wire, 1.0 mm)
ESW-F10V E Series Electrode Holder (Str, Vent, Ag Wire, 1.0 mm)
ESW-F12N E Series Electrode Holder (Str, Ag Wire, 1.2 mm)
ESW-F12V E Series Electrode Holder (Str, Vent, Ag Wire, 1.2 mm)
ESW-F15N E Series Electrode Holder (Str, Ag Wire, 1.5 mm)
ESW-F15V E Series Electrode Holder (Str, Vent, Ag Wire, 1.5 mm)
ESW-F20N E Series Electrode Holder (Str, Ag Wire, 2.0 mm)
ESW-F20V E Series Electrode Holder (Str, Vent, Ag Wire, 2.0 mm)
ESW-M10P E Series Electrode Holder (Str, Port, Ag Wire, 1.0 mm)
ESW-M12P E Series Electrode Holder (Str, Port, Ag Wire, 1.2 mm)
ESW-M15P E Series Electrode Holder (Str, Port, Ag Wire, 1.5 mm)
ESW-M20P E Series Electrode Holder (Str, Port, Ag Wire, 2.0 mm)

Note: Selection is based on the outer diameter of user’s glass electrode

Telmisartan, an angiotensin II type 1 receptor antagonist, attenuates T-type Ca2+ channel expression in neonatal rat cardiomyocytes
Morishima M, Wang Y, Akiyoshi Y, Miyamoto S, Ono K, European Journal of Pharmacology, 105-112, 2009

Inhibition of the KCa3.1 channels by AMP-activated protein kinase in human airway epithelial cells
Klein H, Garneau L, Trinh N T N, Prive A, Dionne F, Goupil E, Thuringer D, Parent L, Brochiero E and Sauve R, American Journal of Physiology: Cell Physiology, C285–C295, 2009

4-Aminopyridine-sensitive outward currents in preBo¨ tzinger complex neurons influence respiratory rhythm generation in neonatal mice
J. A. Hayes,J. L. Mendenhall,B. R. Brush and C. A. Del Negro, Journal of Physiology, 1921 - 1936, 2008

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