# Application Note: Electrophysiology

## Introduction

Electrophysiology is a field of research that deals with the electrical properties of cells and biological tissues. In some cases, it is used to test for nervous or cardiac diseases and abnormalities. In many research applications probes are used to measure the electrical activity of individual cells, tissues, and whole specimens. Measuring the activity of cells at various membrane potentials can give valuable information about ion transport mechanisms and cellular communication. Varying ionic strength and membrane potential on cell populations can be used to study contractile movements of muscle cells, and diseases affecting the normal propagation of impulses. Using various stimulation techniques along with a selection of quality equipment and setup design can give rise to a wealth of applications in electrophysiology.

## Electrophysiology Principle

Researchers and clinicians use electrophysiology when studying the electrical properties of neural and muscle tissue. In the clinical laboratory, electroencephalograms are routinely performed as a test for neural disorders like epilepsy, brain tumor, stroke, encephalitis, and others by measuring the electrical activity of the brain through external electrodes. In the research laboratory, electrophysiology methods are used to measure the ion-channel activity of cell membranes in various electrical environments. Here, an extremely thin micropipette is used to make intimate contact with the cell membrane to study membrane potential.

Neurons and other cell types derive their electrical properties from their lipid bilayer and the ion concentrations inside and outside of the cell. The ion concentration differences create a membrane potential difference on either side of the membrane. The flow of ions across the membrane generates a current that can be measured using Ohm’s law, where the change in voltage (V) is related to the current (I) and membrane resistance (R).

$$\Delta V = IR = {1 \over \Delta G}$$

Equation 1: Ohm's law for potential difference calculation

The membrane potential at equilibrium is also determined through experimentation. The Nernst Equation examines the electrical potential of a cell at equilibrium affected by a single ion. The elucidation of the threshold voltage necessary to generate an action potential can be derived.

$$E = {RT \over zF} ln {[C]_0 \over [C]_1}$$

Equation 2: The Nernst Equation for membrane voltage at equilibrium

E is the potential (Nernst potential) for a given ion
R is the universal gas constant and is equal to 8.314 J/(K *mol)
T is the temperature in Kelvin
Z is the valence of the ionic species
[C]o is the concentration of the ionic species C in the extracellular fluid.
[C]i is the concentration of the ionic species C in the intracellular fluid.

Measurement of the membrane voltage effected by multi-ion channels is widely studied, as resting membrane potential is likely to be accomplished via two or more ions at a time. This can be calculated by the Goldman-Hodgkin-Kats equation.11

$$V_m = {RT \over F} { P_K [K^+]_0 + p_{Na} [NA^+]_0 + p_{Cl} [Cl^-]_0 \over P_K [K^+]_i + p_{Na} [NA^+]_i + p_{Cl} [Cl^-]_i }$$

Equation 3: The Goldman-Hodgkin-Kats Equation for the calculation of membrane potential for K+, Na+, and Cl- ions

pK is the membrane permeability for K+. PNA is the relative membrane permeability of Na+ pCl is the relative membrane permeability of Cl- [K+]o is the concentration of K+ in the extracellular fluid [K+]i is the concentration of K+ in the intracellular fluid [Na+]o is the concentration of Na+ in the extracellular fluid [Na+]i is the concentration of Na+ in the intracellular fluid [Cl-]o is the concentration of Cl- in the extracellular fluid [Cl-]i is the concentration of Cl- in the intracellular fluid

In general, electrophysiology can be divided into two types, intracellular electrophysiology and extracellular electrophysiology:

### Intracellular Electrophysiology

Intracellular electrophysiology involves obtaining electrical conductivity information from across a single cell or junction. Intercellular electrophysiology applications require extremely fine glass capillaries impregnated with electrodes to contact the cell membrane and interior matrix. Intimate contact is made between the capillary and the single cell of interest. This is accomplished using extremely thin glass pipettes and careful manipulation to gather membrane potential of the cell of interest.

### Extracellular Electrophysiology

Some applications require obtaining conductivity and impulse information from a group of neighboring cells or tissues in a field known as Extracellular Electrophysiology. One or more probes are used to stimulate the tissues or sample matrix whereby measurements of the electrical potential of the samples are collected while a high-speed movie is acquired. In many cases, the neurons or cells of interest are stimulated, generating a response that can be recorded and compared to a control site. Understanding the response recorded from cells, tissues, or whole animals has applications to study the human model in the treatment and understanding of disease.16

## Common Electrophysiology Techniques

### Patch Clamping

One commonly used technique in electrophysiology studies involves the attachment of a micropipette to a cell(s) of interest (Figure 1). The cell is punctured, and a small amount of suction is applied such that its membrane and cytoplasmic materials are partially sucked inside. This method is referred to as Patch-clamp.11 Patch clamping isolates a patch of the membrane from the external solution. The analysis of single cell membranes can give information about how the cell membrane responds when changes to ionic strength, voltage potential, or cell type are changed. Patch clamp analysis is often studied in Petri dishes or well-plates to minimize manipulation and increase the chances of cell viability. However, planar electrical stimulation geometries can also be used for ease of use. Additionally, several groups are using robotic targeting algorithms to improve the rate of specified cell targeting.2,9

### Voltage Clamping

Voltage clamping is performed by applying a sustained and consistent voltage to the sample to stimulate the membranes of excitable cells, initiating the ionic flow changes and muscle movements that would occur during or shortly after stimulation. These cells have a resting membrane potential and membrane resistance that must be measured and overcome before stimulation. Voltage clamping requires a voltage electrode for recording the transmembrane voltage and a current electrode for passing current through the membrane. A constant feedback loop of recording the membrane potential is generated, ensuring the cell remains at a constant potential. Once the desired potential has been stabilized, electrical potential measurements, as well as visual recordings are taken to determine the organelle functionality in the presence of various stimuli.11

### Current Clamping

The current clamping technique applies a known current through the specimen and measures how the membrane potential changes. Unlike voltage clamping which seeks to maintain the membrane potential at a given value, current clamping uses a single micropipette to apply the desired current and record how the membrane potential changes in response. By administering repetitive current pulses, the membrane resistance can be calculated using Ohm’s law.11

### Retinal Studies

The retina is widely studied in electrophysiology due to its selfcontained sensory circuitry and its usage as a model circuit system (Figure 2). Electroretinography involves the measurement of the retina’s electrical response to light of varying wavelengths and intensities using a filament electrode. Rods, cones, and other electrically active structures in the eye polarize and depolarize the field, causing measurable changes in the field. The resulting electroretinogram contains valleys and peaks representative of photoactivation, depolarization, and polarization due to ion flow in active cells in the field recording.

In the research laboratory, two-photon and multi-photon fluorescence microscopy is used to focus excited light on targeted receptors of the retina. Imaging using this technique is often done using a combination of infrared and visible light and is in conjunction with one or more additional methods.

### Calcium Imaging

Precise measurement and visualization of the action potential of a cell, tissue, or medium is an area of focus for many electrophysiologists. Calcium ions chelate with calcium indicators to create fluorescent molecules of varying lifetimes (Figure 3). These molecules are used as an indicator of calcium flux across the membrane. Calcium indicators can be synthesized chemically and added into the cell system, or can be synthesized by the cell via the transfection of genetically encoded fluorescent proteins. When performing calcium imaging with cells or tissues, several factors must be considered to maximize the amount of useful data collected.

First, an appropriate indicator and administration type must be carefully selected to minimize cell toxicity while maximizing transfection efficiency and enhance the chances of the desired response. The dissociation constant (Kd) and the expected quantum yield of the calcium indicator used can play a significant role in gathering enough fluorescent signal to quantify calcium concentration.

Lastly, calcium imaging would be extremely ineffective without the use of a very sensitive scientific camera coupled with the microscope used to visualize the sample. A careful check of the tested quantum efficiency of the sensor used in imaging against the expected emission wavelength of the calcium indicator used will provide great insight on whether that dye is suited for the type of experiment and cell type being used.

### Optogenetics

Optogenetics is a relatively new research technique that has gained a lot of popularity in electrophysiology communities due to its ability to target specific cells within a population. In optogenetics, cells are genetically modified to express exogenous proteins that are sensitive to light of particular wavelengths and intensities (Figure 4). The gene manipulations involve increasing the synthesis of photoactive proteins produced by the cell so that the cell membrane experiences a change in the polarization state of the membrane and thus a change in the ion permeability4.

The use of photoactivatable proteins to generate changes in ion channels has resulted in some fascinating findings for the field of neural microbiology and the study of disease. Often, the activation of the proteins causes a change in the membrane potential. The Deisseroth group used the cation-specific, light-sensitive protein Channelrhodopsin-2 in the presence of blue light and triggered a brief, but reliable spike in the polarization of the recorded membrane potential of mammalian cells4. In a separate study, Li et al. demonstrated that antagonistic control of neural functions could also be achieved by using two light-sensitive proteins, showing reliable control of neural functions10.

## Concerns of the Electrophysiologist

### Series Resistance

A tightly controlled electrophysiology laboratory monitors and aims to reduce resistance between the amplifier and the cell, tissue, or specimen used. It is of extreme importance that the researcher delivers the desired voltage to the sample to accurately maintain the desired membrane potential in voltage clamping. A monitoring electrode is often used to measure the current membrane potential of the sample and delivers an adjusted amount of current to the cell in the case of voltage clamping. It is critical that the user limits the resistance in the system so that an accurate account of the applied voltage is maintained.

### Noise Reduction

Many disturbances in the measurement of the desired signal in an electrophysiology experiment can cause noise in the data. Most noise falls into one of four categories: thermal noise, shot noise, dielectric noise, and excess noise, which are described in more detail in Table 1. Noise can originate from many sources in the electrophysiology experiment and must be carefully minimized to maintain quality data. The variations in signalling that result in noise are random and cannot be predicted, only minimized. Several resources are available that provide lists of materials for noise minimization with various electrophysiology experimental setups.11

Table 1: Electrophysiology noise sources and common remediation methods.11, 14
Noise type Description Commonly used remediation methods
Thermal noise Noise arising from the thermal agitation of charge carriers in a conductor.
• Use signal filters
• Anti-vibration tables or micromanipulators
• Electrical isolation of charge carriers
Dielectric noise Noise arising from loss of current through the capacitors of the system. Frequency-dependent.
• For solid dielectrics, use high-quality materials with a low dissipation factor (Quartz, Sapphire, some ceramics).
• Careful selection of equipment, such as patch-clamp headstages.
• Remove high-loss dielectrics from the area.
Shot noise Variations in the number of photons contacting the sensor as a function of the discrete nature of photons (amplified at low photon counts).
• Sensor cooling
• Pixel correction
• Averaging multiple images together
Excess noise Electrode Noise – Noise associated with resistance in the electrode. Contributes to dielectric noise and thermal noise.
• Select low-noise patch-clamp pipette materials.
• Use only debris-free micropipettes and apply positive pressure before cell contact.
Vibrational Noise – Noise in the data collection due to small vibrations in the system or environment.
• Use an anti-vibration table.
• Use quality micromanipulators.
Seal Noise - Noise as a result of poor contact with cell membrane surface.
• Use pipette with appropriate aperture.
• If done by hand, improve laboratory techniques.
• Use robotic patch-clamping apparatus.2

### Signal Conditioning

There are often unwanted signals that are recorded in the data collected. These frequencies contaminate the data, making the challenge of matching the changes in potential to cellular events even more challenging. Filters are often used to block out these unwanted signals and decrease the likelihood of contamination. Low pass-filters, the most commonly used filter type, are often used to remove the slight fluctuations in the membrane potential and will significantly improve the quality of frequency data collected from intracellular nerve cell recordings.

Several other filter types exist to maximize the amount of desired signal collected at the given frequency. Filters can be classified as active, digital, or passive with the latter being the least common. Active filters are most commonly used because of their ease of incorporation into an existing electrophysiology system and proven noise filtering capability. Digital filtering can be implemented in the imaging or computer software. Digital filters can be applied to perform the passband mentioned above, filtering, as well as transfer functions like Elliptic, Cauer, Bessel, and more.11

## Imaging in Electrophysiology

The ability to prove that the target specimen has generated the desired response is an essential part of data collection. In electrophysiology, several microscopy techniques and methods are used to locate cells of interest and image the cellular functions being generated. The user must first locate the cell or area of the tissue/specimen of interest with accuracy, which can be troublesome using traditional widefield microscopy techniques. The use of optical contrast enhancement techniques can be used to aid in this process, but may not be sufficient for visualizing the activity of voltage-gated ion channels. When such information is necessary, fluorescent imaging techniques can also be used. Each method is specific to the type of ion or channel being studied and must be carefully reviewed before experimentation.

### Contrast Enhancement in Imaging

The fine tip of the micropipette is used as a tool to pair the sample and electrode. The ability of the electrophysiologist to precisely manoeuvre the micropipette into the correct location on the specimen is of utmost importance to accurately collect data. However, this can present a challenge when selecting an imaging device and supporting materials.

As stated, micromanipulators are often used as the tool to fine-tune the location of the pipette tip and create a dimple on the surface of the cell or specimen. Validating the contact between the sample and pipette is often challenging since the cell membrane surface appears with very little contrast in traditional widefield microscopy techniques. Phase contrast displays an image that presents denser areas of the cell as being darker since there is a longer optical path length in those regions. This makes the corners and edges of the cell appear more in contrast with the surrounding areas, allowing for the better distinction between the membrane and extracellular matrix. Two main contrast improvement techniques are used in electrophysiology, differential interference contrast microscopy (DIC) and Dodt gradient contrast microscopy.

### Differential Interference Contrast Microscopy (DIC)

DIC imaging is commonly used in electrophysiology applications as a method to improve the contrast of the cell wall and outer structures while maintaining the ability to look for the micropipette tips. With DIC, features in the sample appear more prominent when there are gradients in the optical path length of light, such as corners and ridges inside and around the cell (Figure 5).

DIC is a powerful technique for studying live cells because it avoids the need for cell staining, preventing cytotoxic effects that may hinder natural cell functions. In the selection of an imaging device, the camera selected must be sensitive enough to detect the contrast differences created by using DIC. Additionally, the imaging sensor must have high responses across the visible wavelength range to detect the wide variety of voltage-sensitive and ion-sensitive dyes, which may be present in low concentrations. Lastly, because having the ability to capture rapidly changing cellular activity is extremely important when publishing and presenting findings, the camera selected for the electrophysiology lab must be fast enough to capture the rapidly changing cellular activity.

Another contrast enhancement technique commonly used to image unstained samples in thick tissues is Dodt gradient contrast microscopy. This method uses a gradient of light to improve the details of a sample. Dodt gradient contrast microscopy uses a tube to segment off a section of incoming light and sends it through a screen diffuser. The gradient light is collimated before interaction with the sample, which casts shadows of light on the edges of the sample. When several images are taken, shadows are cast along different areas of the field of view. The recombination of these images results in images with enhanced detail over any of the single images alone. This technique is popular because it requires very little setup and can be used with many existing microscopes.

### Fluorescence Microscopy

The use of fluorescence microscopy is used extensively in many areas of electrophysiology. Fluorescent probes and transgenic dyes are often used to image and measure the influx of ions in the study of ion channels, as well as visualize specific areas of the cell (Figure 6). Fluorescent labels can be used alone or with other dyes to display an image of two or more organelles overlaid over each other. Two-photon fluorescence microscopy is commonly used with dyes with emission wavelengths in different regions of the visible spectrum. Valuable spatial information about the structure of labelled features can be collected with this method. Using two-photon microscopy techniques, exciting advancements have been reported in the literature, proving its usefulness in the study of neural networks and differentiation13. There are also several groups using novel materials to improve the ease of microscopy imaging, such as the introduction of fluorescent pipettes to improve accuracy of single-cell patching.1,7

## Conclusion

Studying how the electrical environment of the cell can affect the flow of ions and thus, the functionality of cell, is studied widely for its applicability to disease prevention and remediation. Knowledge of electrophysiological techniques as well as the common complications can lead to higher quality data generation. Combining electrophysiology with other techniques such as optogenetics has contributed even further to the field of neuroscience and maintains the status of electrophysiology as a vital technique to our understanding of human biology.

## References

1. Andrásfalvy, B. K., Galiñanes, G. L., Huber, D., Barbic, M., Macklin, J. J., Susumu, K., Delehanty, J. B., Huston, A. L., Makara, J. K. & Medintz, I. L. (2014) Quantum dot–based multiphoton fluorescent pipettes for targeted neuronal electrophysiology. Nature Methods. 11, 1237–1241. doi: 10.1038/nmeth.3146
2. Annecchino, L. A., Morris, A. R., Copeland, C. S., Agabi, O. E., Chadderton, P. & Schultz, S. R. (2017) Robotic Automation of In Vivo Two-Photon Targeted Whole-Cell Patch-Clamp Electrophysiology. Neuron. 95, (5), pp1048–1055. doi: 10.1016/j.neuron.2017.08.018.
3. Borghuis, B. G., Tian, L., Xu, Y., Nikonov, S. S., Vardi, N., Zemelman, B. V. & Looger, L. L. (2011) Imaging Light Responses of Targeted Neuron Populations in the Rodent Retina. J Neurosci. Feb 23; 31(8): 2855–2867. doi: 10.1523/JNEUROSCI.6064-10.2011
4. Boyden, E. S., Zhang, F., Bamberg, E., Nagel, G. & Deisseroth, K. (2005) Millisecond-timescale, genetically targeted optical control of neural activity. Nat. Neurosci. 8, (9), pp.1263–1268. DOI: 10.1038/nn1525
5. Deisseroth, K. (2011) Optogenetics. Nature Methods. 8, 26–29. doi:10.1038/nmeth.f.324
6. Ding, S., Mattam, S. G. & Zhou F. M. (2011) Kv3-like potassium channels are required for sustained high-frequency firing in basal ganglia output neurons. J Neurophysiol. Feb;105(2):554-70. doi: 10.1152/jn.00707.2010
7. Gentet, L. J., Stuart, G. J. & Clements, J. D. (2000) Direct Measurement of Specific Membrane Capacitance in Neurons. Biophysical Journal. 79, (1) pp. 314-320. doi: 10.1016/S0006-3495(00)76293-X
8. Harris Lab (APIG). Image-Guided Electrophysiology. Janelia Research Campus. Accessed from https://www.janelia.org/lab/harris-lab-apig/research/activity-tools/image-guided-electrophysiology
9. Kushibiki, T., Okawa, S., Hirasawa, T. & Ishihara, M. (2014) Optogenetics: Novel Tools for Controlling Mammalian Cell Functions with Light. Int. J. Photoenergy. pp 1-10. http://dx.doi.org/10.1155/2014/895039
10. Li, X., Gutierrez, D. V., Hanson, M. G., Han, J., Mark, M. G., Chiel, H., Hegemann, P., Landmesser, L. T. & Herlitze, S. (2005) Fast noninvasive activation and inhibition of neural and network activity by vertebrate rhodopsin and green algae channelrhodopsin, Proc Natl Acad Sci USA. 102, (49), pp.17816–17821. DOI: 10.1073/pnas.0509030102
11. Molecular Devices. (2012) The Axon Guide: Electrophysiology and Biophysics Laboratory Techniques. Third Edition.
12. Ogden, D. & Stanfield, P. (1994). Patch clamp techniques for single channel and whole-cell recording in Microelectrode techniques: the Plymouth workshop handbook, Second Edition. pp 53–78.
13. Prè, P., Nestor, M. W., Sproul, A. A., Jacob, S., Koppensteiner, P., Chinchalongporn, V., Zimmer, M., Yamamoto, A., Noggle, S. A. & Arancio, O. (2014) A Time Course Analysis of the Electrophysiological Properties of Neurons Differentiated from Human Induced Pluripotent Stem Cells (iPSCs). PLOS One. Jul 29;9(7):e103418. doi: 10.1371/journal.pone.0103418
14. Suk, H. J., van Welie, I., Kodandaramaiah, S. B., Allen, B., Forest, C. R., Boyden, E. S. (2017) Closed-Loop Real-Time Imaging Enables Fully Automated Cell-Targeted Patch-Clamp Neural Recording In Vivo. Neuron. 95, (5), p1037–1047.e11. DOI: 10.1016/j.neuron.2017.09.012
15. van Wyk, M., Pielecka-Fortuna, J., Löwel, S. & Kleinlogel, S. (2015) Restoring the ON Switch in Blind Retinas: Opto-mGluR6, a Next-Generation, Cell-Tailored Optogenetic Tool. PLoS Biol. May 7;13(5):e1002143. doi: 10.1371/journal.pbio.1002143
16. Zilberstein, Y., Ewer, J. & Ayali, A. (2006) Neuromodulation of the locust frontal ganglion during the moult: a novel role for insect ecdysis peptides. J. Exp. Biol. 209, pp. 2911-2919. DOI: 10.1242/jeb.02339