Patch & Voltage Clamping

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 refined from the voltage clamp technique, however a blunt electrode is 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) between 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
• Single-electrode voltage clamp

Current Clamp
The current clamp is basically 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

The PC-ONE patch clamp amplifier from Dagan is particularly suited for the entry level market while the BVC-700 patch clamp amplifier from Dagan is most suited for patch clampers working with tissue slices and using current clamp patch pipettes. The PC-ONE and BVC-700 have suitable BNC connections for a PowerLab data acquisition system.

For more information on various applications please visit:

• Intracellular Recordings application page
• Evoked Potentials application page

Software:

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.

LabChart software is also suitable for evoked potential recordings that do NOT require waveform averaging or if more than two recording channels are required. The triggering option within LabChart is extremely useful to synchronize recordings with a stimulus, while the analog or digital outputs on the PowerLab may be used to control a third-party stimulator. The LabChart software can be used to identify the specific waveform components such as amplitude and latency.

LabChart Extensions are free for download from the website for existing LabChart users and they include:

• Event Manager (Win Only): Allows the user to monitor user defined events online using different criteria, and to perform a variety of user defined actions.
• Export Axon (Win Only): Allows the user to save LabChart files in the ABF (Axon binary format) which can be read by pClamp.
• Evoked Response (Mac Only): 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 (Win or 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.
• Read Scope (Mac Only): Read Scope is a LabChart extension which allows LabChart for Mac to read Scope for Mac files directly.
• Export Matlab(Win only): Allows LabChart for Windows files to be saved and exported as MATLAB compatible files. MATLAB is a flexible data analysis program available from The Mathworks Inc. (www.mathworks.com).
• Translate Binary (Win only): 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 only): Allows LabChart to save data as an EDF file, and to read EDF files. Translate EDF does not support the EDF+ format.
• Telegraph (Win only): 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.

Hardware:

Data Recording and Analysis
PowerLab data acquisition systems and LabChart/Scope software are ideal for intracellular recordings due to their ability to record at high sampling rates. The high frequency of neuronal firing rates greatly exceeds the frequency of most other physiological signals. Sampling rates of 40 kHz to 200 kHz are recommended for most intracellular recordings. They include:

• ML866 PowerLab 4/30 – 4 channels
• ML870 PowerLab 8/30 – 8 channels
• ML880 PowerLab 16/30 – 16 channels

Two Electrode Voltage Clamp System
ML870B72 Two Electrode Voltage Clamp System includes:

• ML870 PowerLab 8/30 (LabChart and Scope software)
• MLS260 LabChart Pro software
• MLA190 19" Rack Adapter
• TEV-200A Two Electrode Voltage Clamp (includes TEV-201, TEV-202 and VCM/BC-203 headstages)
• BUZZ Foot Pedal Buzz
• HB180 Pipette Holders (for 1.5 to 1.8 mm pipettes) (2x)

Voltage Clamp Preamplifiers and Accessories

• TEV-200A Two Electrode Voltage Clamp
• PC-ONE Patch/Whole Cell Clamp
• BVC-700A Whole Cell Clamp (Bridge & Voltage Clamp Amplifier)
• CELL CLAMP 2-V Two Electrode Clamp
• AM-2 Audio Monitor
• BSI-950/220 Biphasic Stimulus Isolator (BSI-950/220)
• BSI-950/115 Biphasic Stimulus Isolator (BSI-950/115)
• 8018 Headstage Extension Cable (universal 15 pin, 3m)
• TEV-201 Headstage (for TEV-200A)
• TEV-208 Series Resistance/Leakage Current Adapter Box
• PC-ONE-(10-80) PC-ONE Headstages
• BUZ-1 Universal Buzz Box
• BUZZ Foot Pedal Buzz
• HB120 Glass Pipette Holder (1.0 - 1.2 mm)
• HB180 Glass Pipette Holder (1.5 - 1.8 mm)
• PS-1 Power supply for 220-240V AC

Stimulators

• STG4004 4 Channel Stimulus Generator
• STG4008 8 Channel Stimulus Generator

Heat-evoked activation of the ion channel, TRPV4
A D Guler; H Lee; T Iida; I Shimizu; M Tominaga and M Caterina, Journal of Neuroscience, 6408-4614, 2002

Adenine nucleotide-induced activation of adenosine A2B receptors expressed in Xenopus laevis oocytes: Involvement of a rapid and localized adenosine formation by ectonucleotidases
I Matsuoka, S Ohkubo, J Kimura and Y Uezono, Molecular Pharmacology, 606-613, 2002