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 -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. 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 micropipettes, with a tip diameter of < 1 micrometre, and a resistance of several megohms. 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 into the pipette 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 chloride-coated silver 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). Maintaining healthy brain slices is pivotal for successful electrophysiological recordings. The preparation of these slices is commonly achieved with tools such as the Compresstome vibratome, ensuring optimal conditions for accurate and reliable recordings. Nevertheless, even with the highest standards of tissue handling,
slice preparation induces rapid and robust phenotype changes of the brain's major immune cells,
microglia, which must be taken into consideration when using this model.
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 ion channels, which open only when the membrane voltage is within a certain range. 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.
Current 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-
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 1
MΩ 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 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.
Patch-clamp recording uses a micropipette attached to the cell membrane to allow recording from a single ion channel. This 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 minimize 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 analyzed pharmacologically. Patch-clamp may also be combined with RNA sequencing in a technique known as
patch-seq by extracting the cellular contents following recording in order to characterize the electrophysiological properties relationship to gene expression and cell-type.
Sharp electrode recording 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 electrical resistance of the micropipette electrode is reduced by filling with 2-4M KCl, rather than a salt concentration which mimics the intracellular ionic concentrations as used in patch clamping. 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.
Microelectrode array recording While microelectrode arrays on semiconductor electronic chips, having thousands to hundreds of thousands of electrodes per array, served as a platform for long-term extracellular recording of in vitro neural cell cultures, their capability can also apply to intracellular recording of thousands of neural cells in
culture. ==Extracellular recording==