Electrophysiological Techniques, Membrane Potential, and Electrophysiological Techniques

Electrophysiological Techniques, Membrane Potential, and Electrophysiological Techniques ...

Electrophysiology is a procedure for measuring the electrical activity or excitability of biological cells (be they muscle cells, neurons, or stem cells). This can be done at a single-cell level, or it can involve simultaneous measurements from hundreds or thousands of cells.

What is electrophysiology?

Membrane potential, action potential steps, and action potential graph

Potential for Membrane

- Action possibilities and diagram

Potential rated versus action potential

Equipment in electrophysiology

Intracellular recording of electrophysiological techniques

Patch clamp

Current-clamp

- Voltage doppelgang

Extracellular recording of electrophysiological techniques

- Recording of single units

Multi-unit recording

Multielectrode arrays (MEA)

Integral recording versus extracellular recording

Electrophysiology applications and examples in practice

Pre-clinical research

Drug discovery

Electrophysiology in Clinical Practice

This article focuses on single-cell electrophysiology and outlines the fundamentals of electrophysiological research by describing the ionic basis of action potentials and the various electrophysiological techniques used to investigate and understand excitable tissues and organs.

What is electrophysiology?

Electrophysiology is a research into the electrical properties of biological circuits in tissues, tissues, whole organs, and systems.

Regardless of Ohms law, any electronic circuit (such as a radio or a computer) includes a battery connected to components by copper wires, through which negatively charged electrons flow and perform, according to voltage (V) = current (I) x resistance (R).

ions are regulated by ion channelsenzymes that catalyze the passive flow of ions down electrochemical gradients in biology. However, the biology also has a few layers of complexity.

Scientists have worked at the intersection of physics and physiology to pioneer our understanding of what makes cells excitable.1

Over the last hundred years, advances in electrophysiology theory, techniques, and equipment have greatly enhanced our understanding of how the body works, including fundamental knowledge of the heart, the brain (and other organs), and improved diagnosis and treatment of disease.

In the early 1950s, Hodgkin and Huxley uncovered the ionic basis of the neuronal action potential, triggering an intense period of research in electrode design.4 In 1981, Sakmann and Neher patented glass micropipettes capable of measuring ion flow through single ion channels or across a single cell membrane. This technology further encnered the exploration of how excitable cells work.6 This technique enables scientists to study the fundamental mechanisms behind excitability and brain function.

Membrane potential, action potential steps, and action potential graph

The voltage or potential difference across the cell membrane is caused by the hydrophobic membrane separating charges, acting as a capacitor and resistor to the movement of charged ions across it.

Na+, K+, Ca2+, and Cl- are the predominant cellular ions that carry charge. These ions have different concentrations inside and outside the cell. So when ion channels open, the ions want to move towards a neutral balance.

Generally, Na+, Ca2+, and Cl- ions exist in greater concentrations extracellularly, and K+ ions in greater concentrations intracellularly, with the inside of the cell having a net negative charge. This creates an electrochemical gradient (or driving force) so that when an ion channel opens, the permeable ions flow down their electrochemical gradient carrying charge across the membrane, causing a voltage decrease. This can result in either depolarization or hyperpolarization of the cell membrane

Na+ and Ca2+ are positively charged, boosting their movement into the negatively charged cell, so their positive charge depolarizes the electrochemical potential. On the other hand, K+ ions are highly concentrated inside the cell, and their driving force is in the opposite direction, out of the cell. This is why hyperpolarization is called common.

Ions cannot freely diffuse through the hydrophobic lipid bilayer cell membrane, but they can move through the channels or pores set inside it that extend from the inside to the outside. Many of these channels are highly selective for only one species of ion and often gated, opening only in response to a fluctuation in membrane voltage (voltage-gated ion channels) or on the binding of a small molecule or ligand.

The flow of ions across the membrane sets its overall resistance (according to Ohms law). As the more channels open, the membrane resistance (Rm) will be small, and as the more channels open, the more ions will flow, giving ergo greater conductance and ergo greater resistance.

Another important feature of the lipid bilayer is its capacitance (membrane capacitance, Cm) which arises from the physical properties of the bilayer; an insulator who is constantly conducting solutions on each side. The net effect is that the ion flow through the ion channels must charge the membrane capacitor first before changing the membrane potential. Rm.Cm = Tau is a neuron that is known for its rapid response to the time interval over which synaptic potentials summate (see

Excitable cells typically have resting membrane potentials of up to -60 mV (negative inside when measured with respect to the outside of the cell). Skeletal muscle, cardiac muscle, and neurons express voltage-gated Na+channels and voltage-gated K+channels.

During an action potential, voltage-gated Na+channels open allowing for the rapid influx of Na+ ions and rapid depolarization of the cell membrane potential. However, this powerful depolarization opens the voltage-gated K+channels through which K+ ions flow in the opposite direction and reduces the voltage to around-60 mV. The duration and waveform of the action potential are determined by the type and density of the ions present in the excitable cell membrane, as shown in Figures 1

Figure 1: Potential for neuronal action.

1) Action potentials must be triggered by a small depolarization of the resting membrane potential (around -60 mV); this might be caused by a synaptic input. 3) This accelerates the influx of Na+ ions, which depolarize the membrane for +40 mV or beyond, causing an afterhyperpolarizing potential. 5) On a much slower timescale, membrane-bound Na+/K+ exchangers (sometimes called ion pumps) that exchange K+

Figure 2: Cardiac Action Potential

2) The rapid Na+ influx through voltage-gated Na+channels depolarizes the cell membrane. 2) Voltage-gated K+channels open, beginning repolarization. 3) Ca2+channels open, the influx of Ca2+ positive charge is balanced by an efflux of K+ positive charges, producing a plateau phase. 4) Ca2+channels close and K+channels remain open, repolarizing membrane potential to -90 mV.

Graded potential vs action potential

When the cell membrane is depolarized to the threshold for Na+channel activation, an action potential is initiated. In neurons, action potentials are vital to neuronal communication.

Graded potentials are sub-threshold changes in the membrane potential in response to stimuli or incoming innervation from other cells. These postsynaptic potentials may be excitatory (depolarizing) or inhibitory (hyperpolarizing) depending on the type of channels that have influenced the sub-threshold response. In fact, the magnitude of the input and its subsequent response, the distance from the stage of action potential initiation, and the biophysical properties of the membrane, such as its

Graded potentials in Figure 3

Electrophysiology equipment

Electrophysiologists measure small cellular currents in the order of pAnA. They therefore require sensitive equipment that can exclude vibration and electrical interference (noise) from the ambient environment. The standard rig setup required for in vitro and in vivo electrophysiology is described below.

Figure 4: Standard electrophysiology rig setup

1) An air table is designed to prevent physical vibrations, adding movement artifacts to the experimental recording.

2) A faraday cage is required to protect the equipment from electrical interference. It''s critical to obtain clear, beneficial electrophysiological recordings.

3) A microscope and a micromanipulator are required to position the microelectrode(s) depending on the experimental setup. For in vivo experiments, the user may remove the microscope to make room for different machines, such as treadmills.

4) A speaker is required to collect and amplify your electrodes'' acquired signals. This is connected to a digitizer to convert analog signals into digital signals.

5) Data acquisition and analysis software is required to complete the experiment, design and run protocols, and to obtain substantial results from the collected data.

De plus, the user may need to consider incorporateping medication delivery systems and temperature control devices.

Intracellular recording of electrophysiological techniques

The five most common patch-clamp methods are described in Figure 5. It involves constructing a series circuit with a cell or patch of cell membrane without bending the wall. Instead, a glass micropipette, which includes a silver/silver-chloride wire attached to a patch-clamp amplifier, forms a high resistance gigaohm seal between the patch and the glass in the mouth.

The whole-cell configuration is achieved by approaching the cell membrane with a capillary glass microelectrod that has been micro-forge heated and pulled so that the mouth diameter is 1 m, with a resistance of between 2 and 6 M, and pipette offset zeroed.

When the micropipette is filled with an internal pressure of up to 2 ml, it is applied to the electrode''s release, which allows the membrane and pipette to become contiguous, forming a loose patch, increasing resistance and decreasing the current amplitude observed in the 5 mV seal test. This normally reduces the observed seal test current to a flat line with two fast pipette capacitance transients at the start and end of the voltage step. These are removed by

The electrode is gently pulled out and up to the surface of the cell, removing the danger of the nucleus blocking the patch and increasing accessresistance. The cell is now held at a negative potential approximately equal to resting membrane potential (-60 mgV) and negative pressure is applied to rupture the membrane. This means that the excitability of the whole-cell membrane can now be measured and manipulated.

How to Make the Different Patch-clamp Configurations in Figure 5

In the whole-cell configuration, it is possible to investigate the electrical properties of the cell in current-clamp and voltage-clamp modes.

The user observes a known current amplitude in the inside of the cell through their setup and observes the change in cell excitability in response to these current injections. This technique is useful because it can mimic physiological situations, like a synaptic input. In Figure 6 A, you can see how one cell responds to increasing step current injections from -20 to +20 pA in 10 pA steps, such that +10 pA of current injection was sufficient to allow the cell to threshold and fire action

Figure 6: A retrospective examination of mouse AgRP neurons in a brain slice preparation. Credit: Branco et al. 2016,7 reproduced under the Creative Commons Attribution 4.0 International license.

The membrane potential is measured at user-defined voltages in voltage-clamp mode. The feedback amplifier and headstage are able to activate the clamp, which allows the feedback amplifier and headstage to produce current to hold the cell''s membrane potential at different command voltages set by the user.

Ion channels open at different command voltages, indicating membrane resistance changes as ionic currents flow across the membrane. This instantaneously, the feedback amplifier compensates for this by injecting the reciprocal current to maintain the cell at the command voltage, giving a read out of this injected current as the membrane current.

The TEA ratio for cellular ionic properties is significant in that it is useful to research the conductances observed in cell membranes and those that influence cell excitability.2 This technique was performed in conjunction with pharmacological blockers to evaluate the total K+ conductances. In Figure 7, neurons in the medial nucleus of the trapezoid body (MNTB) and the anterior superior olive (LSO) in the mouse auditory brainstem were studied by a voltage clamp in the

Figure 7: K+current currents measured in auditory brainstem.Current traces from the MNTB (C) and LSO (D) demonstrate the amplitude of the peak and sustained current recorded at each command voltage. Credit: Choudhury et al. 2020, reproduced under the Creative Commons Attribution 4.0 international license.

Extracellular recording of electrophysiological techniques

In vivo, you may place wire electrodes or silicon probes directly into the subject, or, in vitro, layering primary cells, cultured cells, or tissue slices over electrodes.

Electrodes are generally thin in diameter and slide down into the tissue adjacent to the interest cells. This technique is beneficial in awake and behaving subjects, thus allowing them to gain insight into the neuronal response that motivates behaviors.

  • Single electrodes - one electrode, one recording site
  • Tetrodes - four electrodes bundled together, enables better cell sorting
  • Multielectrode arrays (MEAs) - arrays of multiple recording electrodes, records spiking across a greater area of tissue

The electrodes are able to record electronic field potentials produced by spiking or firing neurons. These waveforms are often called local field potentials and are composed of the spiking of several cells adjacent to the electrode. The number of neurons that an electrode or silicon probe may listen to is dependent on the electrode''s impedance and the number of recording sites (channels).

When doing in vivo electrophysiology experiments, researchers must prepare their subjects carefully. Using a micromanipulator, they can detect mechanical stress and avoid causing a bleed in the brain. Experimenters can then compare their position to published brain atlas coordinates, such as the Allen Brain atlas.

Experimenters working with transgenic animal subjects expressing channelrhodopsin in cell types of interest may combine in vivo electrophysiology with optogenetic stimulation by using a light source to irradiate their neurons of interest.9,10

After recording, use of spike sorting algorithms and software allows users to sort the local field potential into individual units or neurons.11 This step is crucial as the data recorded during in vitro recordings can be quite difficult.

Figure 8: A digital interface and the amplifier allow for recording, monitoring, and control of the experiment. The microelectrode is lowered into the tissue by a micromanipulator. The microdrive allows for fine positioning of the microelectrode once it is in the correct area. The operator also allows for recording, monitoring, analysis, and control of the experiment.

Recent advances in electrode design have led to the development of the Neuopixels probe.12 These probes utilize complementary metal-oxide semiconductor (CMOS) technology to allow simultaneous recording from tens of thousands of neurons.13 This number is expected to increase as advancements continue to minimize the size of the technology.

The benefit of Neuropixels is the remarkable resolution provided by the many recording sites along the shank of the electrode. However, the drawback is that information can only be retrieved from neurons that are adjacent to the single shaft as it enters the brain tissue. This can be accomplished in a range of ways by combinating multiple electrodes in one recording session. However, there is a reasonable limitation to the number of electrodes that can be placed in the brain.

The CMOS-hosted in vivo multielectrode system (CHIME) provides an overview of hundreds of electrodes each day. These electrodes are made from a CMOS amplifier array.14 These devices are capable of monitoring neuronal activity over a wider spatial area.

Multiple electrodes in a dish may be used for higher-throughput extracellular recordings from cells in vitro, or from brain slices. MEA systems have been used in preclinical and drug discovery research for over 50 years.15

In circles, the electrodes are then cultured on top of them, or the tissue slice is laid on top. Molecular activity is measured extracellularly as local field potentials. Researchers can perform both recording from multiple electrodes simultaneously, thus increasing the output of these experiments.

With the advent of new technologies, such as CMOS arrays, the number of electrodes per dish has increased dramatically in terms of MEA design, increasing the number of sensors per dish. This increases the resolution scientists must monitor and record cellular activity.

Intracellular recording is the same as extracellular recording.

Electrophysiological investigation:

Intracellular

Extracellular

Level of recording:

Investigate individual cells

Record from multiple cells simultaneously

Preparation:

Can be in vivo or in vitro

Can be in vivo or in vitro

Possible configurations:

Whole-cell, cell-attached, loose-patch, single-channel, perforated patch, inside-out, and outside-out configurations

MEA, cardiac electrophysiology, high-throughput, automated electrophysiology

Throughput:

Medium to low

High

Experimental capacity:

Voltage-clamp, current-clamp and dynamic clamp possible

Normally only current-clamp configuration is possible. Although can be paired with electrical or optogenetic stimulation

Electrophysiological Investigation:

Intracellular

Extracellular

Recording level

Investigate individual cells

Record from multiple cells simultaneously

Preparation:

Can be in vivo or in vitro.

Can be in vivo or in vitro.

Possible configurations

Whole-cell, cell-attached, loose-patch, single-channel, perforated patch, inside-out, and outside-out configurations

MEA, cardiac electrophysiology, high-throughput, and automated electrophysiology

Throughput:

Medium to medium

High-quality footwear

Capacity experimentelle:

Voltage-clamp, current-clamp, and dynamic clamp are possible.

Normally, only the current-clamp configuration is possible. While it may be combined with electrical or optogenetic stimulation.

Electrophysiological Investigation:

Intracellular

Extracellular

Level of recording:

Investigate individual cells

Using multiple cell phones simultaneously, record

Preparation:

Can be in vivo or in vitro.

Can be in vivo or in vitro.

Possible configurations:

Whole-cell, cell-attached, loose-patch, single-channel, perforated patch, inside-out, and outside-out configurations

MEA, cardiac electrophysiology, high-throughput, and automated electrophysiology

Throughput:

Medium to low

High-End

Capacity experimentelle:

Voltage-clamp, current-clamp, and dynamic clamp are possible.

It is usually possible to use a current-clamp configuration only. Although it may be combined with electrical or optogenetic stimulation.

Electrophysiology in practice''s applications and examples

Electrophysiology is widely used in pre-clinical and academic research in in vitro and in vivo. These technologies have greatly contributed to our understanding of behavioral neuroscience, connectomics, neurophysiology, neuropharmacology, cardiology, and toxicology. In addition, electrophysiology remains the ground-truth assay when measuring neuronal activity.16

For compound screening or toxicological assays in cells, medium to high-throughput electrophysiology systems can automatically patch cells, while others may utilize electrode arrays to measure local field potentials from cells. Depending on the method, high-throughput electrophysiology systems may be used to measure cellular contraction, impedance, and other assays.17

Clinical electrophysiologists perform regular dose tests on patients to assist diagnosis and patient monitoring. These tests include 12 lead electrocardiograms (ECG), electroencephalogram (EEG), nerve conduction tests, and auditory testing.

References

1. Piccolino M. Animal electricity and the birth of electrophysiology: Luigi Galvani''s legacy. Brain Res Bull. 1998;46(5):381-407. doi:10.1016/s0361-9230(98)00026-4

A quantitative description of membrane current and its potential implications for conduction and excitation in the nerve. J Physiol. 1952;117(4):500-544. doi:10.1113/jphysiol.1952.sp004764

4. Hausser M. The Hodgkin-Huxley theory of the action potential. Nat Neurosci. 2000;3,1165. doi:10.1038/81426

The discovery of potentials from motoneurones with an intracellular electrode, according to the authors. J Physiol. 1952;117(4):431-460. doi:10.1113/jphysiol.1952.sp004759

Hamill O, Marty A, Neher E, Sakmann B, and Sigworth F. Improved patch-clamp techniques for high-resolution current recording from cell-free membrane patches. Pflugers Archiv - Eur J Physiol. 1981;391(2):85-100. doi:10.1007/bf00656997

Carmeliet E. From Bernstein''s rheotome to Neher-Sakmanns patch electrode. The action potential. Physiol Rep. 2019;7(1):e13861. doi:10.14814/phy2.13861

7. Branco T, Tozer A, Magnus C, and others. Na v 1.7 in hypothalamic neurons has a near-perfect synaptic relationship, regulating body weight. Cell. 2016;165(7):1749-1761. doi:10.1016/j.cell.2016.05.019

8. Choudhury N, Linley D, Richardson A, and coll. Kv3.1 and Kv3.3 subunits are differentially linked to Kv3 channels and action potential repolarization in the principal neurons of the auditory brainstem. J Physiol. 2020;598(11):2199-2222. doi:10.1113/jp279668

A guide to optogenetically identified cortical inhibitory interneurons in in in vivo. J Vis Exp. 2014;(93):e51757. 2014 Nov 7. doi:10.13791/51757

10. Guerrero DKR, Donnett JG, Csicsvari, J., and Kovacs, K. A. Tetrode recording from the hippocampus of behaving mice coupled with a four-point-irradiation closed-loop optogenetics: a technique to test the importance of hippocampal SWR events to learning. Eneuro. 2018;5(4). doi:10.1523/ENEURO.0087-18.2018

11. Quiroga RQ (2012). Spike sorting. Curr Biol, 22(2), R45-R46. doi:10.1016/j.cub.2011.11.005

12. Jun J, Steinmetz N, Siegle J, and others. High-density neural activity recording using fully integrated silicon probes. Nature. 2017;551(7679):232-236. doi:10.1038/nature24636

13. Steinmetz N, Koch C, Harris K, and Carandini M. Challenges and Opportunities for a large-scale electrophysiology with Neuropixels probes. Curr Opin Neurobiol. 2018;50:92-100. doi:10.1016/j.conb.2018.01.009

CHIME: CMOS-hosted in vivo microelectrodes for massively scalable neuronal recordings. Front Neurosci. 2020;14. doi:10.3389/fnins.2020.00834

Biological application of microelectrode arrays in drug discovery and basic research, according to anal Bioanal Chem. 2003;377, 486495. doi:10.1007/s00216-003-2149-x

An introduction to patch clamp recording in Dallas M. and Bell D. (eds) Electrophysiology, Methods in Molecular Biology, 2021;2188. Humana, New York, NY. doi:10.1007/978-1-0716-0818-0_1.

17. Priest BT, Swensen AM, and McManus OB. Drug discovery in automated physiology. Curr Pharm Des. 2007;13(23):2325-2337. doi:10.12174/138161207781368701

18:476-475-1. Johnson-Smith, D.C. Clinical Electrophysiology: Electrotherapy and Electrophysiologic Testing. Philadelphia: Lippincott Williams & Wilkins; 2008. ISBN 13:9780781744843

About the authors

Adam Tozer, Dr.

Adam completed postdoctoral training in the Forsythe lab where his research focused on neural communication in an area of the brain vital for controlling the auditory environment. He then returned to the Heisler and subsequently Branco labs at the University of Cambridge, UK, and the MRC Laboratory of Molecular Biology, Cambridge UK. Adam is an avid science communicator and has moved away from the lab to full-time science communication and marketing to help promote the great work done in labs across the globe.

Prof. Ian Forsythe is an assistant professor.

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