Disruption, action potential generation

Generation of action potentials (APs)

The action potential is a transient depolarization and repolarization of the membrane that occurs in electrically excitable cells (neurons, cardiac cells, muscle). Due to unequal distribution of charged ions between the inside and outside of the cell, a voltage difference (potential) exists between the intracellular and extracellular sides of the cell membrane. At “resting” levels of activity, this membrane potential is about -70 millivolts. Typically, sodium ions are much higher outside of the cell while potassium ions are higher inside of the cell. The action potential is initiated by a depolarizing stimulus that results in the opening of voltage-gated sodium channels in the membrane. Once a few sodium channels open, this triggers local depolarization opening other nearby VGSC and induction of a rapid depolarization to positive potentials (e.g. +20 mV) of the membrane. This rapid depolarization is due to influx of positively charged sodium ions into the cell through the VGSC. Sodium channels rapidly “inactivate”, closing even if the membrane remains depolarized, limiting the amount of sodium entering the cell and stopping additional depolarization. The VGSC induced depolarization of the membrane causes voltage-gated potassium channels to open, which results in positively charged potassium ions exiting the cell to repolarize the membrane to its resting potential. In myelinated neurons, myelin introduces insulation around the axon that allows depolarization to spread further down the axon with great efficiency. Sodium channel expression is higher around specialized “nodes” in the myelin sheath, nodes of Ranvier. The high concentration of VGSCs at these nodes increase the probability that a sufficient number of VGSC will open on depolarization to reach the threshold for firing an action potential. In this manner the sequestering of VGSC at the node allows the electrical impulse to quickly jump from node to node along the length of the axon, increasing the speed of propagation of the action potential.

The process described above is highly conserved across electrically excitable cells and is described in a generic manner here. Due to the diversity of different neuron types and expression of different combinations of ion channels across those neuron types, differences in the shape and temporal patterns of APs are observed across different cells in the nervous system. The basic components of the action potential as described above are well conserved in neurons from invertebrates to vertebrates including mammals and humans.

For an easy-to-read summary description including figures of action potential generation and propagation, see e.g.

• https://openbooks.lib.msu.edu/neuroscience/chapter/action-potentials/
• https://pressbooks.umn.edu/sensationandperception/chapter/action-potentials/

The action potential is a cycle of membrane depolarization, hyperpolarization and return to the resting value. It is measured most directly using electrophysiological approaches, which allow measurements to be made on a time scale that is consistent with the speed at which these events occur (milliseconds). Other approaches allow for more indirect assessments of APs using optical and other measures. Typically, these optical approaches do not have the temporal resolution of electrophysiological measurements.

Electrophysiological Techniques for Measurements of Action Potentials

There are a wide variety of electrophysiological techniques that allow for action potential measurement. At their core, all of them allow the recording of changes in either membrane potential or currents flowing across the membrane, and all can do so with high temporal resolution (milliseconds) necessary to record APs. Different configurations each have inherent advantages and disadvantages and the selection of the appropriate technique depends on the specific questions to be addressed by an experiment. All these approaches make use of one or more electrodes to measure the electrical responses (changes in membrane voltage or current) in a cell or group of cells. The electrodes can be of various sizes and shapes, and may be placed inside the cell (intracellular recordings), on the cell (patch clamp recordings), or adjacent to the cell (extracellular recordings). Please, see Khadria, 2022 and Ogden, 1994 for further details.

In patch recording, in contrast to evaluating specific channel activity as described in KE1 for VGSC activation, to evaluate AP, the current clamp configuration is commonly employed. Also distinct from KE1, pharmacological manipulations are not applied so that all channel types can contribute to the AP response - AP requires both sodium and potassium ion flow. The action potential is reflected in a rapid fluctuation in voltage.

In the intracellular recording, sharp glass microelectrodes are inserted directly into the intracellular space of the neuron and membrane voltage is measured. Membrane voltage increases dramatically once a threshold depolarization is reached and an action potential is reflected in a short duration steep increase in voltage followed by a rapid fall. Compared to patch clamp, sharp electrode intracellular recording is more difficult to perform, but allows recordings to be obtained for much longer periods of time. They measure the synaptic signals of cells with a high signal-to-noise ratio.

Rather than on or in the cell, extracellular recordings show changes in the activity of several cells surrounding a microelectrode. Alterations to the position and size of this electrode will change the nature of the measurement. These are referred to as unit recordings where APs are characterized by high frequency of activity on msec timescale. Often, action potential signals from multiple cells are recorded on the same electrode and one cell distinguished from the other using a process called spike sorting. Spike sorting uses computer algorithms to analyze the waveforms of the electrical activity and separate them based on their temporal profile, amplitude and other characteristics.

Microelectrode arrays (MEAs) are a form of extracellular recording, consisting of chips that contain multiple electrodes, typically arranged in a small grid. Rather than recording from a single electrode, action potential signals can be recorded from multiple electrodes simultaneously. The number of electrodes ranges from tens to thousands depending on the spatial resolution of the array and type of data required by the experiment. Different types of arrays can be used for a wide variety of in vitro and in vivo applications. MEA recordings provide multiple parameters of electrical activity, with firing rate and bursting rates as the most common to characterize APs.  

Local field potentials are also extracellular recordings, but measure the synchronized electrical potential of a group of cells whose source may be difficult to determine. The signals from these cells will overlap and the recording will be a sum of all of the electrical activity. Commonly used in laminated structures with known anatomical inputs, stimulating electrodes are placed on the input presynaptic axonal field and electrical responses induced after a short synaptic delay represent neurotransmission from pre to postsynaptic neurons.

Optical measurements of action potential

A variety of optical techniques are used as indirect measurement of the action potential. These have the advantage of being higher throughput than electrophysiological approaches, but the disadvantage of having a slower temporal resolution. They are highly correlated with action potential generation, but are subject to some confounders and do not possess the temporal resolution of electrophysiological approaches. These include the use of Na+ or Ca++ sensitive dyes that fluoresce in response to the binding of one of these ions.  Single APs are not detected, but changes in bulk ion concentration over a finite period of time primarily reflect the firing rate of the cells.  Another optical technique uses dyes that are sensitive to changes in voltage or fluorescence resonance energy transfer (FRET) using specialized fluorophores that respond based on changes in membrane potential. Large changes in FRET fluorescence are indicative of changes in electrical activity. Under the correct conditions, FRET fluorescence can reflect changes in action potential generation.

For additional information on these techniques see Khadria, 2022 and Ogden, 1994.

Action potentials or nerve impulses are rapid and transient electrical activity that are propagated in the membrane of excitable such as neurons and muscle cells. The same principal mechanism exists in all cells and therefore is independent of sex. Action potentials are present from fetal stages on in vertebrates and they are also present in invertebrates.

Khadria A,2022. “Tools to Measure Membrane Potential of Neurons”. Biomedical Journal ,45 (5),749–62. https://doi.org/10.1016/j.bj.2022.05.007.

Ogden D(ed.) ,1994. Microelectrode techniques. The Plymouth Workshop Handbook. Cambridge, The Company of Biologists Ltd, 448pp.http://plymsea.ac.uk/id/eprint/7954/

OECD,2023 Initial Recommendations on Evaluation of Data from the Developmental Neurotoxicity (DNT) In-Vitro Testing Battery; Series on Testing and Assessment No. 377. Available at: https://one.oecd.org/document/ENV/CBC/MONO(2023)13/en/pdf