Methode de patch clamp

To achieve the inside-out configuration the patch pipette is attached to the cell membrane and is then retracted to break off a patch of membrane Figure 3. In this case the cytosolic surface of the membrane is exposed.

This is often used to investigate single channel activity with the advantage that the medium that is exposed to the intracellular surface can be modified. If the aim is to study the influence of extracellular cues such as neurotransmitters, the outside-out configuration Figure 3 should be chosen. In this case the pipette is retracted during the whole-cell configuration, causing a rupture and rearrangement of the membrane.

In this configuration the extracellular surface is exposed and thus extracellular cues can easily be applied. The ion channel of interest can also be isolated and expressed heterogeneously in a common cell line e.

Depending on the sample, either an inverted cultured cells or an upright fixed stage microscope for slices with a stable platform is needed. If cells in acute slices are investigated, an infrared DIC is recommendable to visualize the membrane. The microscope should be placed on an anti-vibration table because any movement could be fatal to the seal between the pipette and the membrane. A micromanipulator is needed to move the pipette precisely. Very fine pipettes are formed by heating and pulling small glass or quartz capillary tubes.

The tip of the pipette is heat-polished in a microforge to gain a high-resistance seal onto the membrane. The pipette is filled with a solution that resembles either the extracellular solution or the cytoplasm, depending on the recording mode. The pipette is mounted on a micromanipulator to permit precise movements towards the cell membrane. For conductance of the current a chlorided silver wire is used.

The bath electrode, which is also a chlorided silver wire, sets zero current value. A differential amplifier with a low-noise transistor is connected to a computer for data acquisition and digitization. Specific software can be purchased to control the amplifier and analyze the data. An oscilloscope can alternatively be used to monitor the currents.

If desired, a perfusion system can be added to the setup. Substances can either be applied via a perfusion pencil or by using a POC perfusion open and closed chamber. Patch-clamp experiments are used to approach a huge variety of physiological questions, not only in neuroscience. During the last two decades patch-clamp recordings have also become more important for the investigation of ion channels in non-excitable cells. It is also a very important method in medical research, since many diseases are related to a malfunction of definite ion channels.

In pharmacological research, automated patch-clamping is used to screen potent substances for ion channel modifications. The membrane current and changes in fluorescence are recorded simultaneously. Similar experiments can be performed with pH- or Cl - -sensitive dyes.

The video shows the preparation of mouse brain slices using a vibrating microtome. It depicts the whole workflow beginning with the dissection using a stereo microscope, the embedding in low gelling temperature agarose and the slicing itself. In this video the workflow for performing electrophysiological measurements on acute brain slices using a fixed stage microscope.

The video shows an exemplary workflow for performing electrophysiology on fluorescent cultured cells using an inverted microscope. It starts with the preparation of the patch pipettes and ends with the actual measurement on the microscope.

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November 09, Tutorial. A glass pipette containing electrolyte solution is tightly sealed onto the cell membrane and thus isolates a membrane patch electrically.

Currents fluxing through the channels in this patch hence flow into the pipette and can be recorded by an electrode that is connected to a highly sensitive differential amplifier. In the voltage-clamp configuration, a current is injected into the cell via a negative feedback loop to compensate changes in membrane potential. Recording this current allows conclusions about the membrane conductance.

Courtesy of Dr. Whole-cell: By applying another brief but strong suction, the cell membrane is ruptured and the pipette gains access to the cytoplasm. Inside-out: In the cell-attached mode, the pipette is retracted and the patch is separated from the rest of the membrane and exposed to air.

The cytosolic surface of the membrane is exposed. Outside-out: In the whole-cell mode, the pipette is retracted resulting in two small pieces of membrane that reconnect and form a small vesicular structure with the cytosolic side facing the pipette solution.

Adjust osmolarity and pH values. Prepare cells or brain slices — Prepare cultured cells, isolated neurons, brain slices, or whole animals. Pull and polish the pipette — Prepare the recording electrode. Pull the glass capillary tube and polish pipette tip. Set up the perfusion system — Set up the perfusion system and data acquisition software. Ensure that the system is shielded. Patching a cell — Use the manipulator to touch the cell membrane with the pipette.

Ensure a high resistance electrical seal is formed. Signal acquisition and amplification — The signal will be amplified. For best results, ensure you are using the correct type of amplifier for your research.

Signal digitization — The analog signal is then digitized so that the signal can be analyzed. Data acquisition and analysis — With pCLAMP 11 Software Suite, longer and more sophisticated protocols can be programmed for faster data analysis and precise measurements.

Learn about electrophysiology lab set-up. Learn more about patch clamping techniques, from single channel to whole cell to extracellular field-potential recording.

An action potential is a rapid rise and subsequent fall in voltage or membrane potential across a cellular membrane with a characteristic pattern. Examples of cells that signal via action potentials are neurons and muscle cells. Read more. Ion channels are involved in many cell pathways and understanding the function of ion channels in response to changes in membrane potential or the presence or absence of other molecules is important in order to understand exactly how ion channels participate in normal and abnormal biological processes such as cell differentiation and migration, disease states, and neuronal communications.

What is continuous single-electrode voltage clamp cSEVC? It is an electrophysiology patch-clamp method that passes a membrane voltage into a cell and measures the change in current as the voltage steps. Current-clamp is a method used to measure the resulting membrane potential voltage from an injection of current. To measure the membrane potential, the MultiClamp B and Axoclamp A both monitor voltage drop initiated by current injection along an in-series resistor. Current-clamp is commonly used to inject simulated, but realistic current waveforms into a cell, and monitor membrane effect.

This technique is ideal for the evaluation of important cellular events such as action potentials. The current or voltage signal acquired by the amplifier is an analog signal, but to perform data analysis needed for high resolution patch-clamp measurements, the analog signal must be converted into a digital one.

Positioned between the amplifier and the computer, the digitizer accomplishes this important task. Signal quality is extremely important and is impacted by the sampling frequency. Ion channels play a role in many diseases including hypertension, cardiac arrhythmias, gastrointestinal, immune and neuromuscular disorders, pathological pain, and cancer.

By understanding the exact role that ion channel play in a particular disease, researchers might be able to find a way to affect the ion channel in such a way as to alter the course of the disease. In discontinuous single-electrode voltage clamp dSEVC , the tasks of voltage recording and current passing are allocated to the same micropipette.

Electrophysiology is the field of research studying current or voltage changes across a cell membrane. Electrophysiology techniques are widely used across a diverse range of neuroscience and physiological applications; from understanding the behavior of single ion channels in a cell membrane, to whole-cell changes in the membrane potential of a cell, to larger scale changes in field potential within the brain slices in vitro or brain regions in vivo.

Read More. An ion channel is a group of proteins that form a pore across the lipid bilayer of a cell. Each channel is permeable to a specific ion examples: potassium, sodium, calcium, chloride. Patch-clamp is used to evaluate current or voltage in the membrane associated with ion channel activity via direct measurement in real time using ultra-sensitive amplifiers, high-quality data acquisition systems, and powerful software to evaluate the results. The micropipette contains a wire bathed in an electrolytic solution to conduct ions.

The whole-cell technique involves rupturing a patch of membrane with mild suction to provide low-resistance electrical access, allowing control of transmembrane voltage. Alternatively, investigators can pull a patch of membrane away from the cell and evaluate currents through single channels via the inside-out or outside-out patch-clamp technique.

Series resistance is the sum of all resistances between the amplifier and the inside of the cell using the whole-cell recording method. Due to Ohms Law, the larger this resistance, the greater the difference between the command level and the measured values.

This creates an error in actual voltage or current measurement potentially leading to inaccurate observations. To overcome this, the Molecular Devices amplifiers have built-in circuitry to improve the bandwidth of the recording by compensating the error introduced by the voltage or current drop across the series resistance. The patch-clamp technique involves a glass micropipette forming a tight gigaohm seal with the cell membrane.

If a single ion channel is within the patch, currents can be measured. The Axopatch B, with extremely low-noise profile, is ideal for this application, maximizing signal for the smallest conductance ion channels.

A guide to Electrophysiology and Biophysics Laboratory Techniques. The purpose of this guide is to serve as an information and data resource for electrophysiologists. It covers a broad scope of topics ranging from the biological basis of bioelectricity and a description of the basic experimental setup to a discussion of mechanisms of noise and data analysis. Download Guide. In an experiment using the voltage-clamp method, the investigator controls the membrane voltage in a cell and measures the transmembrane current required to maintain that voltage.

This voltage control is called a command voltage.