Electrophysiology - World Championship in Medical Science


Electrophysiology 

Electrophysiology studies test the electrical activity of your heart to find where an arrhythmia (abnormal heartbeat) is coming from. These results can help you and your doctor decide whether you need medicine, a pacemaker, an implantable cardioverter defibrillator (ICD), cardiac ablation or surgery.

Electrophysiology (see the etymology of "electron"]; φύσιςphysis, "nature, origin"; and -λογία-logia) is the study of the electrical properties of biological cells and tissues. It involves measurements ofvoltage changes or electric current or manipulations on a wide variety of scales from single ion channel proteins to whole organs like the heart. In neuroscience, it includes measurements of the electrical activity of neurons, and, in particular,action potential activity. Recordings of large-scale electric signals from the nervous system, such aselectroencephalography, may also be referred to as electrophysiological recordings.[1] They are useful for electrodiagnosisand monitoring.


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 Dr. Kendre Govind Manikrao from India is conferred with World Championship-2018 in Pediatrics (Antiretroviral Therapy) World Cup


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Definition and scope

Classical electrophysiological techniques

Principal and mechanisms

Electrophysiology is the branch of physiology that pertains broadly to the flow of ions (ion current) in biological tissues and, in particular, to the electrical recording techniques that enable the measurement of this flow. Classical electrophysiology techniques involve placing electrodes into various preparations of biological tissue. The principal types of electrodes are:

  1. simple solid conductors, such as discs and needles (singles or arrays, often insulated except for the tip),
  2. tracings on printed circuit boards, also insulated except for the tip, and
  3. hollow tubes filled with an electrolyte, such as glass pipettes filled with potassium chloride solution or another electrolyte solution.

The principal preparations include:

  1. living organisms,
  2. excised tissue (acute or cultured),
  3. dissociated cells from excised tissue (acute or cultured),
  4. artificially grown cells or tissues, or
  5. hybrids of the above.

If an electrode is small enough (micrometers) in diameter, then the electrophysiologist may choose to insert the tip into a single cell. Such a configuration allows direct observation and recording of the intracellular electrical activity of a single cell. However, this invasive setup reduces the life of the cell and causes a leak of substances across the cell membrane. Intracellular activity may also be observed using a specially formed (hollow) glass pipette containing an electrolyte. In this technique, the microscopic pipette tip is pressed against the cell membrane, to which it tightly adheres by an interaction between glass and lipids of the cell membrane. The electrolyte within the pipette may be brought into fluid continuity with the cytoplasm by delivering a pulse of negative pressure to the pipette in order to rupture the small patch of membrane encircled by the pipette rim (whole-cell recording). Alternatively, ionic continuity may be established by "perforating" the patch by allowing exogenous pore-forming agent within the electrolyte to insert themselves into the membrane patch (perforated patch recording). Finally, the patch may be left intact (patch recording).

The electrophysiologist may choose not to insert the tip into a single cell. Instead, the electrode tip may be left in continuity with the extracellular space. If the tip is small enough, such a configuration may allow indirect observation and recording ofaction potentials from a single cell, termed single-unit recording. Depending on the preparation and precise placement, an extracellular configuration may pick up the activity of several nearby cells simultaneously, termed multi-unit recording.

As electrode size increases, the resolving power decreases. Larger electrodes are sensitive only to the net activity of many cells, termed local field potentials. Still larger electrodes, such as uninsulated needles and surface electrodes used by clinical and surgical neurophysiologists, are sensitive only to certain types of synchronous activity within populations of cells numbering in the millions.

Other classical electrophysiological techniques include single channel recording and amperometry.


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Voltage clamp


The current clamp technique records the membrane potential by injecting current into a cell through the recording electrode. Unlike in the voltage clamp mode, where the membrane potential is held at a level determined by the experimenter, in "current clamp" mode the membrane potential is free to vary, and the amplifier records whatever voltage the cell generates on its own or as a result of stimulation. This technique is used to study how a cell responds when electric current enters a cell; this is important for instance for understanding how neurons respond to neurotransmitters that act by opening membrane ion channels.


Most current-clamp amplifiers provide little or no amplification of the voltage changes recorded from the cell. The "amplifier" is actually an electrometer, sometimes referred to as a "unity gain amplifier"; its main purpose is to reduce the electrical load on the small signals (in the mV range) produced by cells so that they can be accurately recorded by low-impedanceelectronics. The amplifier increases the current behind the signal while decreasing the resistance over which that current passes. Consider this example based on Ohm's law: A voltage of 10 mV is generated by passing 10 nanoamperes of current across 1  of resistance. The electrometer changes this "high impedance signal" to a "low impedance signal" by using avoltage follower circuit. A voltage follower reads the voltage on the input (caused by a small current through a big resistor). It then instructs a parallel circuit that has a large current source behind it (the electrical mains) and adjusts the resistance of that parallel circuit to give the same output voltage, but across a lower resistance.



The patch-clamp technique

This technique was developed by Erwin Neher and Bert Sakmann who received the Nobel Prize in 1991.[3] Conventional intracellular recording involves impaling a cell with a fine electrode; patch-clamp recording takes a different approach. A patch-clamp microelectrode is a micropipette with a relatively large tip diameter. The microelectrode is placed next to a cell, and gentle suction is applied through the microelectrode to draw a piece of the cell membrane (the 'patch') into the microelectrode tip; the glass tip forms a high resistance 'seal' with the cell membrane. This configuration is the "cell-attached" mode, and it can be used for studying the activity of the ion channels that are present in the patch of membrane. If more suction is now applied, the small patch of membrane in the electrode tip can be displaced, leaving the electrode sealed to the rest of the cell. This "whole-cell" mode allows very stable intracellular recording. A disadvantage (compared to conventional intracellular recording with sharp electrodes) is that the intracellular fluid of the cell mixes with the solution inside the recording electrode, and so some important components of the intracellular fluid can be diluted. A variant of this technique, the "perforated patch" technique, tries to minimise these problems. Instead of applying suction to displace the membrane patch from the electrode tip, it is also possible to make small holes on the patch with pore-forming agents so that large molecules such as proteins can stay inside the cell and ions can pass through the holes freely. Also the patch of membrane can be pulled away from the rest of the cell. This approach enables the membrane properties of the patch to be analysed pharmacologically.



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Sharp electrode technique

In situations where one wants to record the potential inside the cell membrane with minimal effect on the ionic constitution of the intracellular fluid a sharp electrode can be used. These micropipettes (electrodes) are again like those for patch clamp pulled from glass capillaries, but the pore is much smaller so that there is very little ion exchange between the intracellular fluid and the electrolyte in the pipette. The resistance of the micropipette electrode is tens or hundreds of . Often the tip of the electrode is filled with various kinds of dyes like Lucifer yellow to fill the cells recorded from, for later confirmation of their morphology under a microscope. The dyes are injected by applying a positive or negative, DC or pulsed voltage to the electrodes depending on the polarity of the dye.



Extracellular recording

Single-unit recording

Main article: single-unit recording

An electrode introduced into the brain of a living animal will detect electrical activity that is generated by the neurons adjacent to the electrode tip. If the electrode is a microelectrode, with a tip size of about 1 micrometre, the electrode will usually detect the activity of at most one neuron. Recording in this way is in general called "single-unit" recording. The action potentials recorded are very much like the action potentials that are recorded intracellularly, but the signals are very much smaller (typically about 1 mV). Most recordings of the activity of single neurons in anesthetized and conscious animals are made in this way. Recordings of single neurons in living animals have provided important insights into how the brain processes information. For example, David Hubel and Torsten Wiesel recorded the activity of single neurons in the primaryvisual cortex of the anesthetized cat, and showed how single neurons in this area respond to very specific features of a visual stimulus.[4] Hubel and Wiesel were awarded the Nobel Prize in Physiology or Medicine in 1981.[5]


Multi-unit recording

If the electrode tip is slightly larger, then the electrode might record the activity generated by several neurons. This type of recording is often called "multi-unit recording", and is often used in conscious animals to record changes in the activity in a discrete brain area during normal activity. Recordings from one or more such electrodes that are closely spaced can be used to identify the number of cells around it as well as which of the spikes come from which cell. This process is calledspike sorting and is suitable in areas where there are identified types of cells with well defined spike characteristics. If the electrode tip is bigger still, in general the activity of individual neurons cannot be distinguished but the electrode will still be able to record a field potential generated by the activity of many cells.


Amperometry

Amperometry uses a carbon electrode to record changes in the chemical composition of the oxidized components of a biological solution. Oxidation and reduction is accomplished by changing the voltage at the active surface of the recording electrode in a process known as "scanning". Because certain brain chemicals lose or gain electrons at characteristic voltages, individual species can be identified. Amperometry has been used for studying exocytosis in the nervous and endocrine systems. Many monoamineneurotransmitters; e.g., norepinephrine (noradrenalin), dopamine, and serotonin (5-HT) are oxidizable. The method can also be used with cells that do not secrete oxidizable neurotransmitters by "loading" them with 5-HT or dopamine.



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Planar patch clamp

Planar patch clamp is a novel method developed for high throughput electrophysiology.[6] Instead of positioning a pipette on an adherent cell, cell suspension is pipetted on a chip containing a microstructured aperture.


A single cell is then positioned on the hole by suction and a tight connection (Gigaseal) is formed. The planar geometry offers a variety of advantages compared to the classical experiment:


Other methods

Solid-supported membrane (SSM)-based

With this electrophysiological approach, proteoliposomes, membrane vesicles, or membrane fragments containing the channel or transporter of interest are adsorbed to a lipid monolayer painted over a functionalized electrode. This electrode consists of a glass support, a chromium layer, a gold layer, and an octadecyl mercaptane monolayer. Because the painted membrane is supported by the electrode, it is called a solid-supported membrane. It is important to note that mechanical perturbations, which usually destroy a biological lipid membrane, do not influence the life-time of an SSM. The capacitiveelectrode (composed of the SSM and the absorbed vesicles) is so mechanically stable that solutions may be rapidly exchanged at its surface. This property allows the application of rapid substrate/ligand concentration jumps to investigate the electrogenic activity of the protein of interest, measured via capacitive coupling between the vesicles and the electrode.[7]


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Electrophysiology Studies (EPS) - American Heart Association






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