Extracellular Electrophysiology

What is Electrophysiology?

Neurons and muscle cells in the body produce electrical signals transmitted from cell to cell. Electrophysiology is the method of studying the electrical properties of these electrically active (or electrogenic) biological cells and tissues. It involves measurements of voltages or electrical currents either inside (intracellular) or outside (extracellular) the cells.

What is the origin of electrical potentials in the cell?

In the early 1950s, Hodgkin and Huxley revealed the ionic basis of the neuronal action potential. The cell membrane acts as a semi-permeable barrier separating the cytosol (the fluid inside cells) from the extracellular fluid (ECF).

The electrical potential inside and outside cells is mainly determined by four different ions: chloride (Cl^-), sodium (Na⁺), potassium (K⁺), and calcium (Ca²⁺). These ions are not distributed equally within and outside cells, resulting in electrical potentials. Generally, the Na⁺, Ca²⁺, and Cl^- ions concentrations are higher in the surrounding ECF outside cells. Meanwhile, K⁺ ions are more concentrated in the cytosol inside cells.

The electric potential difference across the cell membrane is a voltage, also called the membrane potential. The ease of molecules and ions moving across the cell membrane depends on their electric charge, molar mass, and the molecule's polarity. The cell membrane is highly permeable for small molecules without an electrical charge, such as O₂, CO₂, urea, alcohol, and glucose.

How Ions Move Across the Cell Membrane

The diffusion direction across the cell membrane only depends on the concentration gradient. Because of its internal hydrophobic structure, the cell membrane is a significant diffusion barrier for charged ions such as K⁺, Na⁺, Ca²⁺, and Cl^- ions. They can move past the cell membrane through two primary pathways—ion channels and pumps.

Ion channels are specialized, water-filled pores that allow ions to cross the cell membrane along concentration gradients. Ion channels are often gated. They open either in response to a change in membrane voltage (voltage-gated ion channels) or on the binding of a small molecule or ligand (ligand-gated ion channels). Importantly, ion channels are selective.

The Na/K ATPase pump actively transports these ions using energy against their concentration gradients. The purpose of this pump is to maintain the intracellular and extracellular sodium and potassium concentrations.

image retrieved from en.wikipedia.org/wiki/Membrane_potential

Resting Membrane Potential

At the equilibrium potential, the rate at which ions cross the membrane by concentration gradient is equal to the rate at which ions travel by an electrochemical gradient in the opposite direction, creating a dynamic steady state. For example, diffusion because of a concentration gradient drives K⁺ ions from cell interiors—where their concentration is higher—to the ECF, where they are less concentrated.

The outflow of K⁺ ions from the cell leaves a negative charge behind, producing an electric field that opposes the further discharge of K⁺ ions. The equilibrium potential for potassium equals the potential across the membrane when there is no net movement of K⁺ ions (i.e., dynamic steady state).

The equilibrium membrane potential can be calculated with the Goldman-Hodgkin-Katz equation, which is a version of the Nernst equation:

Vm:resting membrane potential
R:universal gas constant (8.314 J/(K⋅mol))
T:temperature in Kelvin (K = °C + 273.15)
F:Faraday's constant (96485 C/mol)
pk:membrane permeability for K+ 
pNa:relative membrane permeability for Na+
pCl:relative membrane permeability for Cl-
[K+]o:concentration of K+ in the extracellular fluid.
[K+]i:concentration of K+ in the intracellular fluid
[Na+]o:concentration of Na+ in the extracellular fluid
[Na+]i:concentration of Na+ in the intracellular  fluid
[Cl-]o:concentration of Cl- in the extracellular fluid
[Cl-]i:concentration of Cl- in the intracellular  fluid

The main difference between the Nernst equation and the Goldman-Hodgkin-Katz equation is the membrane permeability terms for each ion, p_K, p_Na, and p_Cl. The permeability for a given ion is directly proportional to the total number of open ion channels in the membrane for this specific ion.

Permeability values are usually reported as relative permeabilities, with p_K having the reference value of one (in most cells at rest, p_K > p_Na, p_Cl). Relative permeability values are unitless.

For a typical neuron at rest, pK: pNa: pCl = 1: 0.05: 0.45. At the peak of a neuronal action potential, the relative permeability values are p_K:p_Na:p_Cl = 1:12:0.45. Importantly, in a cell where only one type of ion can cross the membrane, the resting membrane potential will equal the equilibrium potential for that particular ion.

In a typical neuron, the concentrations of ions inside and outside the cell are the following:

[K ⁺ ] _o = 5mM [K ⁺ ] _i = 150mM Equilibrium Potential: E _K = -90mV

[Na ⁺ ] _o = 150mM [Na ⁺ ] _i = 15mM Equilibrium Potential: E _Na = +60mV

[Cl ^- ] _o = 120mM [Cl ^- ] _i = 10mM Equilibrium Potential: E _Cl = -70mV

Overall, the intracellular cytosol is negatively charged compared to the extracellular environment, hence the resting potential of a typical cell is typically between -50mV and -75mV.

How is an action potential generated?

If the membrane potential becomes more positive than the resting potential, the membrane is said to be depolarized. The membrane is considered hyperpolarized if it becomes more negative than the resting potential.

Neurons

During an action potential (AP) in a neuron, voltage-gated sodium channels open, and Na+ ions diffuse through the cell membrane to the inside of the cell (due to concentration gradient). The threshold potential is the minimum membrane potential required to produce an AP (typically ≈ -55 mV).

A change in the membrane potential that does not reach the threshold potential does not generate an action potential (failed initiations). Importantly, once the threshold is reached, the triggered action potential is always of the same magnitude irrespective of the strength of the stimulus (“all-or-nothing”).

An influx of Na⁺ ions into the cell causes the fast depolarization of the cell membrane potential, which increases to about +40 mV. This depolarization causes voltage-gated sodium channels to close, voltage-gated potassium channels to open, and K⁺ ions flow from the higher concentration inside the cell to the lower concentration outside, i.e., in the opposite direction of the Na⁺ ions. This outflux of K ⁺ ions re-polarizes the membrane potential back to the resting potential of about -60 mV. Typically, repolarization overshoots the resting membrane potential, making the membrane potential more negative. This process is known as hyperpolarization.

While hyperpolarized, the neuron is in a refractory period, meaning it cannot generate subsequent action potentials. This period can be classified into absolute and relative refractory periods. The absolute refractory period occurs once the sodium channels close after an AP. Sodium channels then enter an inactive state during which they cannot be reopened, regardless of the membrane potential.

The relative refractory period occurs when sodium channels slowly come out of inactivation. During this time, the neuron can be excited with stimuli stronger than the one normally needed to initiate an AP. Early in the relative refractory period, the strength of the stimulus required is very high. Gradually, it becomes smaller throughout the relative refractory period as more sodium channels recover from the inactivation stage.

The refractory period lasts roughly 2 milliseconds. This period ensures that action potentials only move in one direction. So, the recently depolarized membrane section will not depolarize again during this time. The specific duration and waveform of the action potential are determined by the type and density of the ion channels present in the excitable cell membrane. This outflux of K ⁺ ions re-polarizes the membrane potential back to the resting potential of about -60 mV. Typically, repolarization overshoots the resting membrane potential, making the membrane potential more negative. This process is known as hyperpolarization.

Cardiac cells

Neuronal and cardiac action potentials share similarities. These include:

The depolarization of the cell membrane potential by a rapid influx of Na⁺ ions through voltage-gated sodium channels
The beginning repolarization of the cell membrane by opening voltage-gated potassium channels

However, in cardiac cells, voltage-gated calcium channels open, and Ca²⁺ ions flow from outside to the inside of the cell, thereby balancing the positive charge of K⁺ efflux. This process produces a plateau phase. Ca²⁺ channels then close while K⁺ ones remain open, repolarizing membrane potential to -90 mV.

Cardiac action potentials are about 100 times longer than neuronal ones. The time frame from the start of depolarization to the end of repolarization in a cardiac AP lasts 400 milliseconds. In contrast, a neuronal AP occurs in just 4 milliseconds.

Intracellular vs. Extracellular Electrophysiology

In pre-clinical research, where neurons and cardiac cells are used in 2D or 3D culture in vitro, their electrical signals provide essential information about cellular health and function. The electrical signals of these cells can be acquired either by intracellular or extracellular electrophysiology.

Intracellular Electrophysiology

Intracellular electrophysiology can be performed using patch-clamp, current-clamp, or voltage-clamp methods. In a patch-clamp technique, a small piece of the cell membrane containing at least one ion channel is isolated using a micropipette. In a voltage- or current-clamp method, a microelectrode is inserted inside the cell.

Advantages of Intracellular Electrophysiology

Using intracellular electrophysiology has several benefits. These include:

Relatively large signals (10s of mV)
High temporal resolution and signal-to-noise ratios
The ability to investigate individual ion channels
The capability to record sub-threshold membrane potentials

Disadvantages of Intracellular Electrophysiology

This technique also presents various drawbacks and limitations. This electrophysiology method is time-consuming, expensive, and usually limited to investigating single cells.

Methods of Intracellular Electrophysiology

The three most applied intracellular electrophysiology techniques are patch-clamp, current-clamp, and voltage-clamp.

Patch-Clamp

The patch-clamp technique was pioneered by Sakmann and Neher in the 1970s and 80s, and they shared the Nobel Prize for Physiology and Medicine in 1991 for their invention. In a patch-clamp experiment, a silver/silver chloride wire attached to a patch-clamp amplifier is placed inside a glass micropipette filled with ionic solution. This setup forms a high-resistance gigaohm seal between the membrane patch and the edge of the pipette. The different patch-clamp configurations are cell-attached, whole-cell, inside-out, and outside-out.

Current-Clamp

In a current-clamp method, the user injects a known current amplitude into the cell interiors and observes the change in cellular excitability. This technique is valuable because it can mimic physiological scenarios, like synaptic inputs.

Voltage-Clamp

In a voltage-clamp configuration, the membrane potential is kept at user-specified voltages. Ion channels open at different command voltages, meaning membrane resistance changes as ionic currents flow across the membrane.

This setup does not provide a physiological measurement of cellular ionic properties. But it has given insights for investigating the conductances present in cell membranes and those that underlie cellular excitability.

Extracellular Electrophysiology

In extracellular electrophysiology, the cells' electrical signals are recorded using electrodes outside the cell. The primary advantages of an extracellular unit recording are:

The ease of obtaining recordings
Its capability to record over days and weeks
Its ability to simultaneously record signals with tens to hundreds of microelectrodes and measure collective oscillatory network dynamics.

Multielectrode Arrays (MEA)

Arrays of multiple electrodes in a dish can be used for extracellular recordings from cells in vitro or brain slices. MEA systems have been used in preclinical and drug discovery research for over 50 years.

The electrodes in these devices are arranged on the bottom of the dish in grids, like a chessboard. The cells are cultured above them, or the tissue slice is laid on top. An extracellular electrode measures cellular activity outside cells, known as local field potentials.

By recording from multiple electrodes simultaneously, researchers can investigate network dynamics within the tissue or between cells. They can also gather data from multiple cells at once, increasing the throughput of these experiments.

MEAs are typically made of glass, silicon, or plastic substrates with titanium or gold electrodes. The major disadvantages of these traditional MEAs are twofold. First, these MEAs consist of hard materials that are not physiologically relevant since the cellular environment in the body in vivo is soft. Second, traditional MEAs are rigid and do not allow the application of biomechanical cues. Both of these disadvantages contribute to the problem that cells on hard and rigid (traditional) MEAs do not behave in the same manner as cells inside the body.

BMSEED’s MEAs solve this problem. They are the only commercially available MEAs that use a soft and elastomeric silicone as the substrate with embedded stretchable electrodes that allow the combination of mechanical cues and electrophysiology. These stretchable MEAs (sMEAs) are consumables in the MEASSuRE platform.

60-Channel Glass MEA

The Advantages of the MEASSuRE System in Pre-Clinical Research

The materials used in electrophysiology experiments are notoriously fragile and delicate. So, researchers usually eliminate any motion in the sample, as this can damage the equipment and causes recording artifacts. For example, the glass micropipettes or small electrodes in intracellular electrophysiology break readily upon slight movement of the sample.

However, most cells in the body experience physical forces that cause mechanical deformation of the cells by tension or compression. These tensile and compressive forces affect the function of the cell by altering gene expression and cellular phenotype.

Over 9 out of 10 compounds that look promising in pre-clinical research ultimately fail clinical trials. Not taking mechanical forces on cells into account during preclinical research and drug development contributes to this high rate of failure.

Mechanical forces on cells can cause either physiological stretch or pathological stretch. Cells experience physiological stretch under natural conditions. For example, heart and lung cells expand and compress with each heartbeat and breath. The strains are typically less than 10%, and the stretch is usually slow (i.e., low strain rate). These mechanical forces are critical to the proper function of the cell.

Pathological stretch is a deformation beyond the healthy limit of the cell and causes trauma. A pathological stretch of the brain tissue is the root cause of most traumatic brain injuries and concussions. Traumatic brain injury is also a significant risk factor for neurodegenerative diseases such as Alzheimer’s.

The MEASSuRE system is the first and only research tool that combines three crucial modules for studying the effects of mechanical stretch on tissue electrophysiology. This device integrates:

● A cell-stretching apparatus to apply physiological and mechanical stretch

● A data-acquisition module for extracellular electrophysiology to assess the cell’s health, function, and maturity before and after stretching

● A live-cell imaging system to visualize cells and cellular processes during the injury

These three paradigms are applied concurrently and independently. The key to these unique capabilities of this electrophysiology, imaging, and mechanics module is the proprietary stretchable microelectrode array (sMEA). Currently, BMSEED is the only company offering stretchable microelectrodes for in vitro research applications.

This cutting-edge device can mimic physiological and pathological biomechanics in a controlled in-vitro environment. It also evaluates the condition, function, and maturity of the neurons and muscle cells (e.g., cardiac cells) before and after stretching.

The overall goal of the MEASSuRE system is to make cells in a dish (in vitro) behave more similarly to the same type of cells inside the body (in vivo). Doing so will enhance the accuracy of pre-clinical research and ultimately improve the success rate of clinical trials in developing treatments for complex diseases.

BMSEED’s 60- (up to 120-) Channel Electrophysiology Unit

Power Your Research With MEASSuRE

MEASSuRE is the only research tool that enables researchers to independently and simultaneously apply mechanical stretch, electrophysiology, and imaging using stretchable microelectrode arrays. Contact BMSEED to learn more about MEASSuRE.

At BMSEED, our mission is to drive innovation and make a positive impact on the biomedical research community. Learn more about us.