Electrophysiology

Electrophysiology is the study of the electrical properties of biological cells and tissues. It involves measurements of voltage differences across cell membrane, and studies of how the flow of electrical current across membranes is regulated. In neuroscience, it includes measurements of the electrical activity of neurons, and particularly action potential activity.

Definition and scope
There are two major divisions of electrophysiology: intracellular recording and extracellular recording. Intracellular recordings are usually (but not always) made from cells "in vitro", whereas recordings of activity in living animals ("in vivo") are usually extracellular recordings. Extracellular recording includes single unit recording, field potential recording, single channel recording and amperometry. Intracellular recording techniques include voltage clamp and current clamp.

Many particular electrophysiological readings have specific names:
 * Electrocardiography - for the heart
 * Electroencephalography - for the brain
 * Electrocorticography - from the cerebral cortex
 * Electromyography - for the muscles
 * Electrooculography - for the eyes
 * Electroretinography - for the retina

Intracellular recording
Intracellular recording involves measuring voltage and/or current across the membrane of a cell. 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. Typically, the resting membrane potential of a healthy cell will be -60 to -70 mV, and during an action potential the membrane potential might reach +20 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. 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. Today, most microelectrodes used for intracellular recording are glass micropippetes, 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. 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 being small enough to penetrate a single cell with minimum damage to the cell, while having a low-enough resistance that small neuronal signals can be discerned from thermal noise in the electrode tip.

Voltage clamp
The voltage clamp technique allows an experimenter to "clamp" the cell potential at a chosen value. 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 channels -i.e. they are open only in a certain voltage range, and understanding how they work is important to understanding how neurons process information. 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)

"Current Clamp" describes recording the trans-membrane voltage with the ability to inject 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 electrical 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 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. 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 1M&Omega; of resistance. The electrometer changes this "high impedance signal" to a "low impedance signal" by using a voltage follower circuit. A voltage follower reads the voltage on the input (caused by a small current across 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.
The patch clamp technique was developed by Erwin Neher and Bert Sakmann who received the Nobel Prize in 1991. 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.

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 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. Hubel and Wiesel were awarded the Nobel Prize in Physiology or Medicine in 1981. If the electrode tip is slighly 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. 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.

Field potentials
Extracellular field potentials are local current sinks or sources that are generated by the collective activity of many cells. Usually a field potential is generated by the simultaneous activation of many neurons by synaptic transmission. 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. For more information, see neuronal field potentials.

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 neural and endocrine systems. Many monoamine neurotransmitters, e.g., norepinephrine (noradrenalin), dopamine, 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.