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Shining a Light on the Future of Electrophysiology
Article

Shining a Light on the Future of Electrophysiology

Shining a Light on the Future of Electrophysiology
Article

Shining a Light on the Future of Electrophysiology

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Electrophysiology is the study of the electrical properties of tissues and cells. Studies can be at the level of whole organs, such as the heart, down to single ion channel proteins in the membranes of brain cells.

This article describes how electrophysiological techniques are used in academic research and drug discovery settings. It also considers what the future for its use may look like when we consider the emerging optical reporters of cellular excitability and imaging technologies that are currently in development. The article draws on the expertise of scientists working at the coal-face with these techniques to answer the question, ‘what is the future of electrophysiology?’

Amphibious advances

Bioelectricity has been explored for hundreds of years since the late 1700’s when the Italian scientist, Galvani, used electrodes to stimulate frog nerves inducing leg muscle contraction.1 In the 1950’s, scientists worked out that the electrical signals, or action potentials, carried by nerve cells are caused by the fast movement of ions across the cell’s membrane through protein channels, called ion channels.2

The patch breakthrough

Scientists then began investigating ion flow in more detail. In the 1980’s, German scientists, Erwin Neher and Bert Sakmann developed a technique in which they placed their electrodes inside hollow glass micropipettes. This set-up enabled them to move their small pipette up to a brain cell before sucking a tiny patch of the cell membrane into the mouth of the pipette. They could then directly measure ion channels opening and closing in these patches, and found they could control the open probability of these channels by injecting current to clamp the voltage across the membrane. This gave rise to the patch-clamp technique. When they applied more suction and ruptured the patch, they found they could measure the electrical activity of the whole cell. And the revolutionary technique, ‘whole-cell patch clamp electrophysiology’ was born.3 

See also: How to set up a patch-clamp rig

Combining electrophysiology with new techniques: improving insight

The practical approach of patch-clamping has not changed much since its inception. However, the kit used has improved and scientists are capitalizing on advances in optical and genetic techniques to do more elaborate experiments by incorporating new technologies with their patch-clamp investigations.4

Dr Christian Wilms, Application Specialist for Scientifica, a company that specializes in developing electrophysiological and imaging solutions, explains, “Neuroscientists now regularly combine multi-photon laser imaging with optogenetics and electrophysiology to probe deeper into the workings of single neurons and whole circuits in the brain.”

These techniques can be used to explore the electrical properties of cells in monolayer or 3D cultures in a dish, up to freely moving animals, providing incredible scientific insight.

Optogenetics is the targeted expression of light-activated ion channels, called channel rhodopsins, in cells. In excitable cells, activation of the channels by light enables manipulation of their activity, depending on the channel rhodopsin expressed. Different channel rhodopsins exist that can either depolarize and excite cells5 or hyperpolarise and silence them.6

Read more: Optogenetics, controlling cells with light

Multiphoton laser scanning microscopes can be targeted to excite cells that lay deeper in the tissue, and an optogenetic tool box exists for this.7

See also: Recent advances in multiphoton imaging

Whilst advanced, patch-clamp electrophysiology in combination with optogenetics is a low-throughput approach not immediately amenable to the drug discovery environment. For example, in a mouse brain, operators can only patch a small number of cells at a time, or implant electrodes with up to 64 channels to explore brain cell activity. It also requires significant capital investment in specialist equipment and highly trained staff. And, it is an inherently variable and invasive technique.

Improving Throughput

However, electrophysiology is still needed by the drug discovery industry especially in screening for compounds that interact with ion channels. A recent survey of outsourcing trends of electrophysiological testing to specialist contract research organizations revealed there is increasing demand for electrophysiological assays.8

Dr Andrew Southan, Head of Commercial Operations at Metrion Biosciences, a Contract Research Organization specializing in ion channel assays, explains, “During drug development you need to be able to test thousands of compounds against ion channel targets, both for efficacy and for safety, by confirming the compounds don’t affect cardiac action potentials.”


Adding, “Single cell electrophysiology is the gold-standard assay for investigating ion channel physiology. Improvements in automated patch clamp systems enable rapid high-throughput electrophysiological screening that can test upwards of 600 compounds per hour.” 


High-throughput electrophysiology is achieved by plating cells in 384 well plates and using automated patch clamp systems to carry out the screens.  

The demand for screening on these high-throughput systems is suggested to have increased in line with improvements in their functionality, such as giga-ohm seals and improved series resistance.8

Read more: Automated patch clamping trends

Illuminating another path to measuring cell excitability

Cell excitability can also be measured using voltage sensitive dyes which change their light emission spectra based on the electrical field that surrounds them. However, their usefulness is restricted because they cannot be easily targeted to specific cells. Overcoming this obstacle are genetically encoded voltage indicators (GEVI’s), the expression of which can be targeted to cells of interest. These GEVI’s fall into two categories: rhodopsin-based sensors, such as QuasAr1 and 29 and Archer1 and 210 and; sensors that are fusions between voltage sensitive domains and fluorescent proteins, such as VSFP-Butterfly11, ArcLight12 and ASAP1.13


 Professor Adam Cohen, Harvard University, describes how his lab have developed a genetically encoded channel rhodopsin and novel voltage sensor to optically stimulate and measure cell excitability.

The upside of GEVI’s is that they allow for higher throughput investigations of cell activity, as their expression can be targeted to specific cell types. It is also less invasive to express GEVI’s in excitable cells and observe their electrophysiological activity by imaging as opposed to patching them, making the process more physiological.

The downside of GEVI’s is that they are not as bright as other voltage reporters, and so recording from cells or processes in deep tissue is made difficult by unfavorable signal to noise ratios. Early GEVI’s were not able to distinguish single action potentials owing to their slow kinetics. However, newer, faster kinetic GEVI’s, such as ASAP113 can resolve fast events, such as action potentials, with high fidelity. Other challenges are the difficulty of calibrating the GEVI’s to get a true readout of membrane voltage. Also, imaging has technical limitations, such as the speed and resolving power of the optical sensors. 

Read more: Using GEVI’s to illuminate brain function

Optical Interrogation of electrophysiological properties

The development of advanced fluorescent calcium reporters like GCamp6, which can also be genetically encoded, has facilitated the optical recording of action potentials in neurons. Calcium entry is used as a proxy for membrane depolarization and increased excitability, and rapid binding and dissociation of calcium allows these reporters to track cellular excitability with high fidelity. The signal to noise ratio is much improved by these reporters, enabling imaging of calcium transients in small cellular components deep in tissue.14

Combining calcium imaging of GCamp6 with light-activation of expressed red-shifted channel rhodopsin C1V1, Dr Adam Packer, a Senior Research Associate at University College London, carried out a completely optical interrogation of neural circuits in vivo.15

As Adam explains, “We could simultaneously activate neurons and image their activity in vivo using 2-Photon optogenetic activation of cells with C1V1 and calcium imaging using GCamp6.”

Adding, “Using an optical approach meant we could, for the first time, select multiple cells of interest to stimulate while recording from those and hundreds of neurons nearby, simultaneously. This not only increased throughput but also enabled visualization of the impact of multiple cells on the surrounding local neural circuit."

See also: Increasing the 2-Photon field of view


Scaling-up throughput

As seen in Adam Cohen’s TED talk above, the Cohen group use a similar approach to Adam Packer, but they do it in cultured neurons with their developed channel rhodopsin activator CheRiff, and voltage sensor, QuasAr1.9

The video shows fast spreading membrane depolarizations through neural projections. It is not a difficult jump to imagine how this system could be used as a phenotypic screen to test the effects of compounds on the physiological properties of excitable cells.  


As Marc Rogers, Chief Scientific Officer of Metrion Biosciences explains, “Activating cells with light is desirable in a high throughput screening scenario. Not only can channel rhodopsins be used to stimulate cells, they can also be used to pace their activity.”

Adding, “This is an improvement on previous techniques to depolarize cells which required changing the extracellular solution to increase external potassium concentration.”


QState, a spin-out company from the labs of Harvard Professors, Adam Cohen and Kevin Eggan, is using their respective proprietary optogenetic and stem cell technologies to develop assays to aid in drug discovery for a host of neurological and cardiac diseases.


The future of electrophysiology remains bright

Optical techniques to measure and control the electrical activity of cells have advanced rapidly since their inception in 199716, creating the prospect of new assays for drug discovery. The incorporation of these technologies into cell-based assays and the development of automated high content systems, such as the OptoDyCE system, which enables fully optical cardiac electrophysiology17, is a growth area. In addition, the combination of these optical tools with stem cell technologies proffers an exciting new phase in the era of drug discovery.

However, despite the advances made in the optical approaches, the technology is still not ready to replace electrophysiology. As Adam Packer says, “You can’t do single channel recording with imaging techniques.”


And all interviewees agree, the insight provided by single cell electrophysiology remains invaluable. Both Adam Packer and Christian Wilms describe patch-clamp electrophysiology as the “ground-truth”, and Andy Southan confirmed that Metrion scientists still carry out confirmatory experiments in single cells on electrophysiology rigs.


Therefore, it seems that for the foreseeable future, there will always be a place for electrophysiology rigs in academic and industrial research, and drug development.

References:

1. Verkhratsky, A. & Parpura, V. in 1–19 (2014). doi:10.1007/978-1-4939-1096-0_1

2. Hodgkin, A. L. & Huxley, A. F. A quantitative description of membrane current and its application to conduction and excitation in nerve. J. Physiol. 117, 500–44 (1952).

3. Hamill, O. P., Marty, A., Neher, E., Sakmann, B. & Sigworth, F. J. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch. 391, 85–100 (1981).

4. Scanziani, M. & Häusser, M. Electrophysiology in the age of light. Nature 461, 930–939 (2009).

5.        Boyden, E., Zhang, F., Bamberg, E., Nagel, G. and Deisseroth, K. (2005). Millisecond -timescale, genetically targeted optical control of neural activity. Nature Neuroscience, 8(9), pp.1263-1268.

6. Zhang, F., Wang, L., Brauner, M., Liewald, J., Kay, K., Watzke, N., Wood, P., Bamberg, E., Nagel, G., Gottschalk, A. and Deisseroth, K. (2007). Multimodal fast optical interrogation of neural circuitry. Nature, 446(7136), pp.633-639.

7. Prakash, R., Yizhar, O., Grewe, B., Ramakrishnan, C., Wang, N., Goshen, I., Packer, A., Peterka, D., Yuste, R., Schnitzer, M. and Deisseroth, K. (2012). Two-photon optogenetic toolbox for fast inhibition, excitation and bistable modulation. Nature Methods, 9(12), pp.1171-1179.

8. Comley, J. Outsourced Ion Channel Testing Trends 2016, HTStec Limited

9. Hochbaum, D., Zhao, Y., Farhi, S., Klapoetke, N., Werley, C., Kapoor, V., Zou, P., Kralj, J., Maclaurin, D., Smedemark-Margulies, N., Saulnier, J., Boulting, G., Straub, C., Cho, Y., Melkonian, M., Wong, G., Harrison, D., Murthy, V., Sabatini, B., Boyden, E., Campbell, R. and Cohen, A. (2014). All-optical electrophysiology in mammalian neurons using engineered microbial rhodopsins. Nature Methods, 11(8), pp.825-833.

10. Flytzanis, N., Bedbrook, C., Chiu, H., Engqvist, M., Xiao, C., Chan, K., Sternberg, P., Arnold, F. and Gradinaru, V. (2014). Archaerhodopsin variants with enhanced voltage-sensitive fluorescence in mammalian and Caenorhabditis elegans neurons. Nature Communications, 5, p.4894.

11. Akemann, W., Mutoh, H., Perron, A., Park, Y., Iwamoto, Y. and Knopfel, T. (2012). Imaging neural circuit dynamics with a voltage-sensitive fluorescent protein. Journal of Neurophysiology, 108(8), pp.2323-2337.

12. Jin, L., Han, Z., Platisa, J., Wooltorton, J., Cohen, L. and Pieribone, V. (2012). Single Action Potentials and Subthreshold Electrical Events Imaged in Neurons with a Fluorescent Protein Voltage Probe. Neuron, 75(5), pp.779-785.

13. St-Pierre, F., Marshall, J., Yang, Y., Gong, Y., Schnitzer, M. and Lin, M. (2014). High-fidelity optical reporting of neuronal electrical activity with an ultrafast fluorescent voltage sensor. Nature Neuroscience, 17(6), pp.884-889.

14. Chen, T., Wardill, T., Sun, Y., Pulver, S., Renninger, S., Baohan, A., Schreiter, E., Kerr, R., Orger, M., Jayaraman, V., Looger, L., Svoboda, K. and Kim, D. (2013). Ultrasensitive fluorescent proteins for imaging neuronal activity. Nature, 499(7458), pp.295-300.

15. Packer, A., Russell, L., Dalgleish, H. and Häusser, M. (2014). Simultaneous all-optical manipulation and recording of neural circuit activity with cellular resolution in vivo. Nature Methods, 12(2), pp.140-146.

16. Siegel, M. and Isacoff, E. (1997). A Genetically Encoded Optical Probe of Membrane Voltage. Neuron, 19(4), pp.735-741.

17. Klimas, A., Ambrosi, C., Yu, J., Williams, J., Bien, H. and Entcheva, E. (2016). OptoDyCE as an automated system for high-throughput all-optical dynamic cardiac electrophysiology. Nature Communications, 7, p.11542.

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Adam Tozer PhD
Adam Tozer PhD
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