Electrophysiology is the study of the electrical properties of biological cells and tissues. The cell is the structural and functional unit of all known living Organisms It is the smallest unit of an organism that is classified as living and is often called It involves measurements of voltage change or electrical current flow on a wide variety of scales from single ion channel proteins to whole tissues like the heart. Electrical tension (or voltage after its SI unit, the Volt) is the difference of electrical potential between two points of an electrical Electric current is the flow (movement of Electric charge. The SI unit of electric current is the Ampere. Ion channels are pore-forming Proteins that help establish and control the small Voltage Gradient across the Plasma membrane of all living Proteins are large Organic compounds made of Amino acids arranged in a linear chain and joined together by Peptide bonds between the Carboxyl The heart is a muscular organ in all Vertebrates responsible for pumping Blood through the Blood vessels by repeated rhythmic In neuroscience, it includes measurements of the electrical activity of neurons, and particularly action potential activity. Neuroscience is a field devoted to the scientific study of the nervous system Neurons (ˈnjuːɹɒn also known as neurones and nerve cells) are responsive cells in the Nervous system that process and transmit information In Neurophysiology, the action potential is a self-regenerating Wave of Electrochemical activity that allows Nerve cells to carry a signal
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), 2) tracings on printed circuit boards, and 3) hollow tubes filled with an electrolyte, such as glass pippettes. 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 (micrometres) 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, at the same time such invasive setup reduces the life of the cell. Intracellular activity may also be observed using a specially formed (hollow) glass pipette. In this technique, the microscopic pipette tip is pressed against the cell membrane, to which it tightly adheres. The electrolyte within the pipette may be brought into fluid continuity with the cytoplasm by delivering a pulse of pressure to the electrolyte 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 ion channels 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 of the electrical activity of a single cell, and is termed single unit recording. Depending on the preparation and precise placement, an extracellular configuration may pick up the activity of several nearby cells simultaneously, and this is 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. A local field potential (LFP is a particular class of Electrophysiological signals which is related to the sum of all Dendritic synaptic activity 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.
Optical electrophysiological techniques were created by scientists and engineers to overcome one of the main limitations of classical techniques. Classical techniques allow observation of electrical activity at approximately a single point within a volume of tissue. Essentially, classical techniques singularize a distributed phenomenon. Interest in the spatial distribution of bioelectric activity prompted development of molecules capable of emitting light in response to their electrical or chemical environment. Examples are voltage sensitive dyes and fluoresceing proteins.
After introducing one or more such compounds into tissue via perfusion, injection or gene expression, the 1 or 2-dimensional distribution of electrical activity may be observed and recorded.
Many particular electrophysiological readings have specific names:
Intracellular recording involves measuring voltage and/or current across the membrane of a cell. The heart is a muscular organ in all Vertebrates responsible for pumping Blood through the Blood vessels by repeated rhythmic The brain is the center of the Nervous system in animals All Vertebrates and the majority of Invertebrates have a brain Electrocorticography (ECoG is the practice of using electrodes placed directly on the exposed surface of the Brain to record electrical activity from the Cerebral The cerebral cortex is a structure within the Brain that plays a key role in Memory, Attention, perceptual Awareness, Thought, Electromyography (EMG is a technique for evaluating and recording the activation signal of muscles Muscle (from Latin musculus, diminutive of mus "mouse" is contractile tissue of the body and is derived from the Eyes are organs that detect Light, and send signals along the Optic nerve to the visual areas of the brain Electroretinography measures the electrical responses of various cell types in the Retina, including the photoreceptors ( rods and cones) The vertebrate retina is a light sensitive part inside the inner layer of the Eye. Electroantennogram or EAG is a technique by which we measure the average output of the antenna to the Brain for a given Odor. Olfactory receptors expressed in the Cell membranes of Olfactory receptor neurons are responsible for the detection of Odor molecules To make an intracellular recording, the tip of a fine (sharp) microelectrode must be inserted inside the cell, so that the membrane potential can be measured. Membrane potential (or transmembrane potential) is the Voltage difference (or Electrical potential difference between the interior and exterior of a Typically, the resting membrane potential of a healthy cell will be -60 to -80 mV, and during an action potential the membrane potential might reach +40 mV. In 1963, Alan Lloyd Hodgkin and Andrew Fielding Huxley won the Nobel Prize in Physiology or Medicine for their contribution to understanding the mechanisms underlying the generation of action potentials in neurons. Sir Alan Lloyd Hodgkin, OM, KBE, FRS (5 February 1914 Banbury, Oxfordshire, England – 20 December 1998 Cambridge Sir Andrew Fielding Huxley, OM, FRS (born 22 November 1917, Hampstead, London) is an English physiologist Their experiments involved intracellular recordings from the giant axon of Atlantic squid (Loligo pealei), and were among the first applications of the "voltage clamp" technique. The squid giant axon is the very large (up to 1 mm in diameter typically around 0 Today, most microelectrodes used for intracellular recording are glass micropipettes, with a tip diameter of < 1 micrometre, and a resistance of several megaohms. The micropipettes are filled with a solution that has a similar ionic composition to the intracellular fluid of the cell. A chlorided silver wire inserted in to the pipet connects the electrolyte electrically to the amplifier and signal processing circuit. The voltage measured by the electrode is compared to the voltage of a reference electrode, usually a silver-silver chloride wire in contact with the extracellular fluid around the cell. In general, the smaller the electrode tip, the higher its electrical resistance, so an electrode is a compromise between size (small enough to penetrate a single cell with minimum damage to the cell) and resistance (low enough so that small neuronal signals can be discerned from thermal noise in the electrode tip). Electrical resistance is a ratio of the degree to which an object opposes an Electric current through it measured in Ohms Its reciprocal quantity is
The voltage clamp technique allows an experimenter to "clamp" the cell potential at a chosen value. The Membrane potential, or better Membrane Voltage, is the difference of Electric potentials between two Aqueous solutions separated by a ( This makes it possible to measure how much ionic current crosses a cell's membrane at any given voltage. This is important because many of the ion channels in the membrane of a neuron are voltage gated ion channels, which open only when the membrane voltage is within a certain range. Ion channels are pore-forming Proteins that help establish and control the small Voltage Gradient across the Plasma membrane of all living Voltage-gated ion channels are a class of transmembrane Ion channels that are activated by changes in electrical Potential difference near the channel these Voltage clamp measurements of current are made possible by the near-simultaneous digital subtraction of transient capacitive currents that pass as the recording electrode and cell membrane are charged to alter the cell's potential. (See main article on voltage clamp. The voltage clamp is used by electrophysiologists to measure the Ion currents across a neuronal membrane while holding the membrane )
The current clamp technique records the membrane potential by injecting current into a cell through the recording electrode. Membrane potential (or transmembrane potential) is the Voltage difference (or Electrical potential difference between the interior and exterior of a 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 electrical current enters a cell; this is important for instance for understanding how neurons respond to neurotransmitters that act by opening membrane ion channels. See Chemical synapse for an introduction to concepts and terminology used in this article Ion channels are pore-forming Proteins that help establish and control the small Voltage Gradient across the Plasma membrane of all living
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 job is to change the nature of small signals (in the mV range) produced by cells so that they can be accurately recorded by low-impedance electronics. An electrometer is an electrical instrument for measuring Electric charge or electrical Potential difference. Electrical impedance, or simply impedance, describes a measure of opposition to a sinusoidal Alternating current (AC 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 MΩ of resistance. The ampere, in practice often shortened to amp, (symbol A is a unit of Electric current, or amount of Electric charge per second The ohm (symbol Ω) is the SI unit of Electrical impedance or in the Direct current case Electrical resistance, The electrometer changes this "high impedance signal" to a "low impedance signal" by using a voltage follower circuit. A buffer amplifier (sometimes simply called a buffer) is one that provides Electrical impedance transformation from one circuit to another A voltage follower reads the voltage on the input (caused by a small current through a big resistor). |- align = "center"| |width = "25"| | |- align = "center"| || Potentiometer |- align = "center"| | | |- align = "center"| 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.
This technique was developed by Erwin Neher and Bert Sakmann who received the Nobel Prize in 1991. The patch clamp technique is a Laboratory technique in Electrophysiology that allows the study of single or multiple Ion channels in cells Erwin Neher (born March 20, 1944 in Landsberg am Lech, Bavaria) is a German biophysicist. Bert Sakmann (born June 12, 1942) is a German cell Physiologist. 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 withdraw the electrode from the cell, pulling the patch of membrane away from the rest of the cell. This approach enables the membrane properties of the patch to be analysed pharmacologically.
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 micropipets (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 electrlolyte in the pipete. The resistance of the electrode in 10s or 100s of MΩ in this case. The ohm (symbol Ω) is the SI unit of Electrical impedance or in the Direct current case Electrical resistance, 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. Lucifer yellow is a fluorescent dye used in Cell biology. For common usage it is compounded with Carbohydrazide (CH and prepared as a Lithium salt The dyes are injected by applying a positive or negative, DC or pulsed voltage to the electrodes depending on the polarity of the dye.
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 generally called "single unit" recording. The action potentials recorded are very 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 animals are made in this way, and all recordings of single neurons in conscious animals. 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 primary visual cortex of the anesthetized cat, and showed how single neurons in this area respond to very specific features of a visual stimulus. David Hunter Hubel (born February 27, 1926) was co-recipient with Torsten Wiesel of the 1981 Nobel Prize in Physiology or Medicine, for their Torsten Nils Wiesel (b June 3, 1924) was a Swedish co-recipient with David H The term visual cortex refers to the primary visual cortex (also known as striate cortex or Hubel and Wiesel were awarded the Nobel Prize in Physiology or Medicine in 1981. 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 which 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 called spike sorting and is suitable in areas where there are identified types of cells with well defined spike characteristics. Spike sorting is a class of techniques used in the analysis of electrophysiological data If the electrode tip is bigger still, generally 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.
Extracellular field potentials are local current sinks or sources that are generated by the collective activity of many cells. The extracellular field potential is the Electrical potential produced by cells e Usually a field potential is generated by the simultaneous activation of many neurons by synaptic transmission. Neurotransmission (latin transmissio = passage crossing from transmitto = send let through also called synaptic transmission, is an electrical movement The diagram to the right shows hippocampal synaptic field potentials. At the right, the lower trace shows a negative wave that corresponds to a current sink caused by positive charges entering cells through postsynaptic glutamate receptors, while the upper trace shows a positive wave that is generated by the current that leaves the cell (at the cell body) to complete the circuit. Glutamate receptors are Transmembrane receptors located on Neuron membranes For more information, see local field potential. A local field potential (LFP is a particular class of Electrophysiological signals which is related to the sum of all Dendritic synaptic activity
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 neural and endocrine systems. Many monoamine neurotransmitters, e. See Chemical synapse for an introduction to concepts and terminology used in this article g. , norepinephrine (noradrenalin), dopamine, serotonin (5-HT), are oxidizable. Norepinephrine ( INN) (abbreviated norepi or NE) or noradrenaline ( BAN) (abbreviated NA or NAd) is a Dopamine is a Hormone and Neurotransmitter occurring in a wide variety of animals including both vertebrates and invertebrates Serotonin (ˌsɛrəˈtoʊnən ( 5-hydroxytryptamine, or 5-HT) is a Monoamine Neurotransmitter synthesized in serotonergic Neurons The method can also be used with cells that do not secrete oxidizable neurotransmitters by "loading" them with 5-HT or dopamine.
Planar patch clamp is a novel method developed for high throughput electrophysiology. Instead of positioning a pipette on an adherent cell, cell suspension is pipetted on a chip containing a microstructured aperture. The development of biochips is a major thrust of the rapidly growing Biotechnology industry which encompasses a very diverse range ofresearch efforts including Genomics
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: - it allows for integration of microfluidics, which enables automatic compound application for ion channel screening. Microfluidics deals with the behavior precise control and manipulation of Fluids that are geometrically constrained to a small typically sub-millimeter scale Ion channels are pore-forming Proteins that help establish and control the small Voltage Gradient across the Plasma membrane of all living - the system is accessible for optical or scanning probe techniques - perfusion of the intracellular side can be performed. Scanning probe microscopy (SPM is a branch of Microscopy that forms images of surfaces using a physical probe that scans the specimen In Physiology, perfusion is the process of nutritive delivery of Arterial Blood to a Capillary bed in the Biological tissue. Not to be confused with Intercellular, meaning "between cells"
The Bioelectric Recognition Assay (BERA) is a novel method for measuring changes in the membrane potential of cells immobilized in a gel matrix. Apart from the increased stability of the electrode-cell interface, immobilization preserves the viability and physiological functions of the cells. BERA is primary used in biosensor applications in order to assay analytes which can interact with the immobilized cells by changing the cell membrane potential. In this way, when a positive sample is added to the sensor, a characteristic, ‘signature-like’ change in electrical potential occurs. BERA has been used for the detection for human viruses (Hepatitis B and C viruses, herpes viruses) and veterinary disease agents (foot and mouth disease virus, prions, blue tongue virus) and plants (tobacco and cucumber viruses) in a highly specific, rapid (1-2 minutes), reproducible and cost-efficient fashion. The method has also been used for the detection of environmental toxins, such as herbicides and the determination of very low concentrations of superoxide anion in clinical samples. A recent advance in the evolution of the BERA technology was the development of a technique called Molecular Identification through Membrane Engineering (MIME). This technique allows for building cells with absolutely defined specificity against virtually any molecule of interest, by embedding thousand of artificial receptors into the cell membrane.