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For the nervous system to function, neurons must be able to send and receive signals. These signals are possible because each neuron has a charged cellular membrane (a voltage difference between the inside and the outside), and the charge of this membrane can change in response to neurotransmitter molecules released from other neurons and environmental stimuli. To understand how neurons communicate, one must first understand the basis of the baseline or "resting" membrane charge.

Neuronal Charged Membranes

The lipid bilayer membrane that surrounds a neuron is impermeable to charged molecules or ions. To enter or exit the neuron, ions must pass through special proteins called ion channels that span the membrane. Ion channels have different configurations: open, closed, and inactive, as illustrated in Figure 1. Some ion channels need to be activated in order to open and allow ions to pass into or out of the cell. These ion channels are sensitive to the environment and can change their shape accordingly. Ion channels that change their structure in response to voltage changes are called voltage-gated ion channels. Voltage-gated ion channels regulate the relative concentrations of different ions inside and outside the cell. The difference in total charge between the inside and outside of the cell is called the membrane potential.

This video discusses the basis of the resting membrane potential.

Resting Membrane Potential

A neuron at rest is negatively charged: the inside of a cell is approximately 70 millivolts more negative than the outside (−70 mV, note that this number varies by neuron type and by species). This voltage is called the resting membrane potential; it is caused by differences in the concentrations of ions inside and outside the cell. If the membrane were equally permeable to all ions, each type of ion would flow across the membrane and the system would reach equilibrium. Because ions cannot simply cross the membrane at will, there are different concentrations of several ions inside and outside the cell, as shown in Table 1.

The resting membrane potential is a result of different concentrations inside and outside the cell. The difference in the number of positively charged potassium ions (K⁺) inside and outside the cell dominates the resting membrane potential (Figure 2).

When the membrane is at rest, K⁺ ions accumulate inside the cell due to a net movement with the concentration gradient. The negative resting membrane potential is created and maintained by increasing the concentration of cations outside the cell (in the extracellular fluid) relative to inside the cell (in the cytoplasm). The negative charge within the cell is created by the cell membrane being more permeable to potassium ion movement than sodium ion movement. In neurons, potassium ions are maintained at high concentrations within the cell while sodium ions are maintained at high concentrations outside of the cell. The cell possesses potassium and sodium leakage channels that allow the two cations to diffuse down their concentration gradient.

However, the neurons have far more potassium leakage channels than sodium leakage channels. Therefore, potassium diffuses out of the cell at a much faster rate than sodium leaks in. Because more cations are leaving the cell than are entering, this causes the interior of the cell to be negatively charged relative to the outside of the cell. The actions of the sodium potassium pump help to maintain the resting potential, once established. Recall that sodium potassium pumps brings two K⁺ ions into the cell while removing three Na⁺ ions per ATP consumed. As more cations are expelled from the cell than taken in, the inside of the cell remains negatively charged relative to the extracellular fluid. It should be noted that calcium ions (Cl ⁻) tend to accumulate outside of the cell because they are repelled by negatively-charged proteins within the cytoplasm.

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For the nervous system to function, neurons must be able to send and receive signals. These signals are possible because each neuron has a charged cellular membrane (a voltage difference between the inside and the outside), and the charge of this membrane can change in response to neurotransmitter molecules released from other neurons and environmental stimuli.

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Neuronal electrophysiology (ephys) is the study of the electrical properties of biological cells and tissues in the nervous system. Changes in these properties allow the nervous system to perform its many functions, which are not only responsible for keeping us alive but also allow us to achieve higher levels of consciousness.

Ephys involves the measurements of these changes, usually voltage or current, on a variety of scales from single ion channels to the whole brain. Studying these properties has many implications beyond the basic understanding of how the brain works, including investigations of neurological disorders and the potential uses or effects of pharmaceutical compounds.

Plenty of potential

The electrical potentials across cell membranes (and their variations) is what makes some cells electrogenic. They are created by maintaining different concentrations of ions, such as sodium (Na⁺) and potassium (K⁺), between the inside of the cell (intracellular liquid) and the outside (extracellular liquid). The cell's membrane prevents the movement of ions across it. The only way they can diffuse across is be passing through specialised ion channels that can be open or closed.

When a neuron is at rest, the majority of Na+ channels are closed, but a few K⁺ channels are open. K⁺ ions are free to diffuse out of the cell down the concentration gradient while an electrical force, produced by K⁺ ions attracted to the negative cell interior pulls K⁺ ions into the cell. These forces will achieve an equilibrium where there is no net movement of K⁺ ions across the membrane. This is the equilibrium potential for K⁺. The resting membrane potential is the sum of the equilibrium potentials of all the ions and their relative permeability. In neurons, this leads to a -60 to -70 mV charge across the membrane.
This animation provides a great visual representation of how neurons create a resting membrane potential.
Action potentials occur in neurons after they are stimulated via a sensory or electrical input. This input may be hyperpolarising or depolarising. If a depolarising current reaches a certain threshold, then an action potential occurs. Action potentials are all-or-nothing events; the amplitude does not vary depending on the size of the preceding stimulus.
Action potentials firing in current clamp recording mode

This animation gives more details on the process of how action potentials occur.
The firing of a neuron is driven by the opening of increasing numbers of voltage-gated sodium channels and the entry of Na+ ions into the cell. After an action potential occurs, the cell membrane will rapidly repolarise to its resting membrane potential.

Action potentials propagate along an axon to deliver electrical signals to other parts of the cell. The inward flow of current at one point on the axon spreads along the axon and depolarises adjacent sections of the membrane. Membrane patches need time to recover after firing before another action potential can occur. This limits the frequency of action potential firing but also ensures that it can only travel in one direction along the axon.

Intracellular and patch clamp recordings

To study these potentials, electrophysiologists make a variety of recordings.

Traditional intracellular recordings require the insertion of a microelectrode into the interior of the cell. Most electrodes come in the form of a glass micropipette with a <1 mm tip. These pipettes are filled with intracellular fluid solution (similar to the fluid in the cell). A silver chloride wire inside the pipette connects this solution to an amplifier that can record and process this signal.
(Source: Allen Institute for Brain Science)
The patch-clamp technique removes the need to impale the cell, and the pipette is placed on a 'patch' of membrane instead. Patch-clamp micropipettes often have a slightly larger diameter and are placed adjacent to a cell. Gentle suction draws a part of the membrane into the tip creating a situation known as cell-attached mode. Cell-attached mode can be used to measure the electrical properties of the ion channels in that specific patch of membrane (technically not a form of intracellular recording).
provides an excellent overview of the patch-clamp technique and its different variations.

This animation gives a visual description of how the voltage clamp method works.
The application of more suction can cause the patch to break leaving the electrode in contact with the whole-cell, hence the name whole-cell mode. This enables stable intracellular recordings to be made. There are other modes of patch clamp including the inside-out, outside-out, perforated and loose patches. Each of these modes enables the study of slightly different properties of the ion channels within the membrane.

The voltage clamp method allows electrophysiologists to measure the changes in ionic currents across cell membranes. It requires an amplifier that holds or 'clamps' the membrane voltage at a set level. It does this by iteratively measuring the membrane potential and altering the current to maintain the voltage at the desired value. The currents are recorded and reveal how changes in potential effect the cell, this is of particular interest because many of the ion channels in the membrane of a neuron open and close depending on the voltage.

The current clamp method allows a researcher to record the membrane potential while injecting positive or negative current into the cell. In current clamp mode, the voltage across the membrane can vary. Voltage changes in response to the administration of a drug or stimulus indicate differences to the ionic conductance of the membrane.

Extracellular recordings and microelectrode arrays

Intracellular recordings are great for measurements of the ionic conditions in a single cell. A different set of measurements can look at changes in ion concentrations in the extracellular fluid or a group of neurons.

Extracellular recordings show changes in the current or potential of several cells surrounding a microelectrode. Alterations to position and size of this electrode will change the nature of the measurement, depending on what properties are being investigated. By using two or more electrodes and a process called spike sorting, it is possible to work out the number of cells being recorded from and the activity occurring in each cell. Spike sorting uses computer algorithms to analyse the waveforms of the electrical activity from multiple electrodes and distinguish the activity of the individual neurons.
An example of an in vitro multielectrode array (Source: MultiChannel Systems)
Extracellular field potentials measure the electrical potential of a group of cells whose source is difficult to determine. The signals from these cells will overlap and the recording will be a sum of all of the electrical activity. These recordings are known as local field potentials.

Microelectrode arrays are chips that contain multiple electrodes through which neural signals can be recorded. They commonly have stimulating electrodes to deliver signals to a sample as well. The number of electrodes ranges from tens to thousands depending on the spatial resolution and amount of data required by the experiment. Different types of array can be used for a wide variety of in vitro and in vivo applications.

The present and the future

Currently, electrophysiology is being enhanced through its combination with techniques such as multiphoton microscopy and optogenetics.

One of the advantages of multiphoton microscopy is its ability to create detailed images of the brain deep within brain tissue, making it possible to see the morphology of a neuron at depths unattainable with standard fluorescence microscopy. By combining this information with detailed electrical activity, researchers can establish new insights into the structure and function of the cells.

Optogenetics enables researchers to excite or inhibit specific neurons with unprecedented temporal and spatial resolutions using light. By recording the electrical activity surrounding a neuron that has been stimulated or inhibited it is possible to establish a direct link between a neurons excitability and the consequences in associated cells.
Banner Image Credit: Psyrakis Dimitrios & Sodikdjon A. Kodirov
Further developments in optogenetics and microscopy will enable researchers to do more sophisticated experiments in different brain regions and help improve our knowledge of the nervous system.

One potential development in electrophysiology is the creation of more advanced voltage sensitive dyes. Current dyes can be used to measure electrical activity within cells without the need for a microelectrode. The advantages of voltage-sensitive dyes include the ability to measure from large populations of cells simultaneously. This enables visualisation and quantification of action potential initiation and propagation through a neural network. However, improvements in the way they function will allow further insights to be made. These improvements include improved quantification, less pharmacological effects and compatibility with two-photon imaging.

Neurons have been studied for millennia, but there is still so much to be discovered about the nervous system and the human brain. Electrophysiology is a great way to investigate neuronal activity on a variety of scales, and with more advanced techniques and technologies becoming available there are even more opportunities to make discoveries.

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Neuronal electrophysiology: the study of excitable cells

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Neuronal electrophysiology (ephys) is the study of the electrical properties of biological cells and tissues in the nervous system. Changes in these properties allow the nervous system to perform its many functions, which are not only responsible for keeping us alive but also allow us to achieve higher levels of consciousness.