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Utilising Automated Electrophysiological Platforms in Epilepsy Research

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Patch Clamp Electrophysiology

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2188))

Abstract

Genetic mutations have long been implicated in epilepsy, particularly in genes that encode ion channels and neurotransmitter receptors. Among some of those identified are voltage-gated sodium, potassium and calcium channels, and ligand-gated gamma-aminobutyric acid (GABA), neuronal nicotinic acetylcholine (CHRN), and glutamate receptors, making them key therapeutic targets. In this chapter we discuss the use of automated electrophysiological technologies to examine the impact of gene defects in two potassium channels associated with different epilepsy syndromes. The hKCNC1 gene encodes the voltage-gated potassium channel hKV3.1, and mutations in this gene cause progressive myoclonus epilepsy (PME) and ataxia due to a potassium channel mutation (MEAK). The hKCNT1 gene encodes the weakly voltage-dependent sodium-activated potassium channel hKCNT1, and mutations in this gene cause a wide spectrum of seizure disorders, including severe autosomal dominant sleep-related hypermotor epilepsy (ADSHE) and epilepsy of infancy with migrating focal seizures (EIMFS), both conditions associated with drug-resistance. Importantly, both of these potassium channels play vital roles in regulating neuronal excitability. Since its discovery in the late nineteen seventies, the patch-clamp technique has been regarded as the bench-mark technology for exploring ion channel characteristics. In more recent times, innovations in automated patch-clamp technologies, of which there are many, are enabling the study of ion channels with much greater productivity that manual systems are capable of. Here we describe aspects of Nanion NPC-16 Patchliner, examining the effects of temperature on stably and transiently transfected mammalian cells, the latter of which for most automated systems on the market is quite challenging. Remarkable breakthroughs in the development of other automated electrophysiological technologies, such as multielectrode arrays that support extracellular signal recordings, provide additional features to examine network activity in the area of ion channel research, particularly epilepsy. Both of these automated technologies enable the acquisition of consistent, robust, and reproducible data. Numerous systems have been developed with very similar capabilities, however, not all the systems on the market are adapted to work with primary cells, particularly neurons that can be problematic. This chapter also showcases methods that demonstrate the versatility of Nanion NPC-16 Patchliner and the Multi Channel Systems (MCS) multielectrode array (MEA) assay for acutely dissociated murine primary cortical neurons, enabling the study of potassium channel mutations implicated in severe refractory epilepsies.

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References

  1. Steinlein OK, Mulley JC, Propping P et al (1995) A missense mutation in the neuronal nicotinic acetylcholine receptor alpha 4 subunit is associated with autosomal dominant nocturnal frontal lobe epilepsy. Nat Genet 11:201–203

    Article  CAS  Google Scholar 

  2. Helbig I, Heinzen EL, Mefford HC et al (2016) Primer part 1-the building blocks of epilepsy genetics. Epilepsia 57:861–868

    Article  Google Scholar 

  3. Perucca P (2018) Genetics of focal epilepsies: what do we know and where are we heading? Epilepsy Curr 18:356–362

    Article  PubMed Central  Google Scholar 

  4. Epi PMC (2015) A roadmap for precision medicine in the epilepsies. Lancet Neurol 14:1219–1228

    Article  Google Scholar 

  5. Barros F, Pardo LA, Dominguez P et al (2019) New structures and gating of voltage-dependent potassium (Kv) channels and their relatives: a multi-domain and dynamic question. Int J Mol Sci 20:E248

    Article  CAS  Google Scholar 

  6. Markham MR, Kaczmarek LK, Zakon HH (2013) A sodium-activated potassium channel supports high-frequency firing and reduces energetic costs during rapid modulations of action potential amplitude. J Neurophysiol 109:1713–1723

    Article  CAS  PubMed Central  Google Scholar 

  7. Rudy B, Mcbain CJ (2001) Kv3 channels: voltage-gated K+ channels designed for high-frequency repetitive firing. Trends Neurosci 24:517–526

    Article  CAS  Google Scholar 

  8. Sierra F, Comas V, Buno W et al (2005) Sodium-dependent plateau potentials in electrocytes of the electric fish Gymnotus carapo. J Comp Physiol A Neuroethol Sens Neural Behav Physiol 191:1–11

    Article  CAS  Google Scholar 

  9. Bhattacharjee A, Gan L, Kaczmarek LK (2002) Localization of the slack potassium channel in the rat central nervous system. J Comp Neurol 454:241–254

    Article  CAS  Google Scholar 

  10. Oliver KL, Franceschetti S, Milligan CJ et al (2017) Myoclonus epilepsy and ataxia due to KCNC1 mutation: analysis of 20 cases and K(+) channel properties. Ann Neurol 81:677–689

    Article  CAS  Google Scholar 

  11. Oyrer J, Maljevic S, Scheffer IE et al (2018) Ion channels in genetic epilepsy: from genes and mechanisms to disease-targeted therapies. Pharmacol Rev 70:142–173

    Article  CAS  PubMed Central  Google Scholar 

  12. Milligan CJ, Li M, Gazina EV et al (2014) KCNT1 gain of function in 2 epilepsy phenotypes is reversed by quinidine. Ann Neurol 75:581–590

    Article  CAS  PubMed Central  Google Scholar 

  13. Hamill OP, Marty A, Neher E et al (1981) Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch 391:85–100

    Article  CAS  Google Scholar 

  14. Neher E, Sakmann B, Steinbach JH (1978) The extracellular patch clamp: a method for resolving currents through individual open channels in biological membranes. Pflugers Arch 375:219–228

    Article  CAS  Google Scholar 

  15. Bell DC, Dallas ML (2018) Using automated patch clamp electrophysiology platforms in pain-related ion channel research: insights from industry and academia. Br J Pharmacol 175:2312–2321

    Article  CAS  Google Scholar 

  16. Milligan CJ, Li J, Sukumar P et al (2009) Robotic multiwell planar patch-clamp for native and primary mammalian cells. Nat Protoc 4:244–255

    Article  CAS  PubMed Central  Google Scholar 

  17. Petty SJ, Milligan CJ, Todaro M et al (2016) The antiepileptic medications carbamazepine and phenytoin inhibit native sodium currents in murine osteoblasts. Epilepsia 57:1398–1405

    Article  CAS  Google Scholar 

  18. Alexander SP, Catterall WA, Kelly E et al (2015) The concise guide to PHARMACOLOGY 2015/16: voltage-gated ion channels. Br J Pharmacol 172:5904–5941

    Article  CAS  PubMed Central  Google Scholar 

  19. Droge MH, Gross GW, Hightower MH et al (1986) Multielectrode analysis of coordinated, multisite, rhythmic bursting in cultured CNS monolayer networks. J Neurosci 6:1583–1592

    Article  CAS  PubMed Central  Google Scholar 

  20. Gross GW, Rieske E, Kreutzberg GW et al (1977) A new fixed-array multi-microelectrode system designed for long-term monitoring of extracellular single unit neuronal activity in vitro. Neurosci Lett 6:101–105

    Article  CAS  Google Scholar 

  21. Thomas CA Jr, Springer PA, Loeb GE et al (1972) A miniature microelectrode array to monitor the bioelectric activity of cultured cells. Exp Cell Res 74:61–66

    Article  Google Scholar 

  22. Pine J (2006) A history of MEA development. In: Advances in network electrophysiology. Springer, New York, pp 3–23

    Chapter  Google Scholar 

  23. Banach K, Halbach MD, Hu P et al (2003) Development of electrical activity in cardiac myocyte aggregates derived from mouse embryonic stem cells. Am J Physiol Heart Circ Physiol 284:H2114–H2123

    Article  CAS  Google Scholar 

  24. Sala L, Ward-Van Oostwaard D, Tertoolen LGJ et al (2017) Electrophysiological analysis of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) using multi-electrode arrays (MEAs). J Vis Exp 123:1–15

    Google Scholar 

  25. Tertoolen LGJ, Braam SR, Van Meer BJ et al (2018) Interpretation of field potentials measured on a multi electrode array in pharmacological toxicity screening on primary and human pluripotent stem cell-derived cardiomyocytes. Biochem Biophys Res Commun 497:1135–1141

    Article  CAS  PubMed Central  Google Scholar 

  26. Berdondini L, Massobrio P, Chiappalone M et al (2009) Extracellular recordings from locally dense microelectrode arrays coupled to dissociated cortical cultures. J Neurosci Methods 177:386–396

    Article  CAS  Google Scholar 

  27. Jimbo Y, Kawana A, Parodi P et al (2000) The dynamics of a neuronal culture of dissociated cortical neurons of neonatal rats. Biol Cybern 83:1–20

    Article  CAS  Google Scholar 

  28. Mcsweeney KM, Gussow AB, Bradrick SS et al (2016) Inhibition of microRNA 128 promotes excitability of cultured cortical neuronal networks. Genome Res 26:1411–1416

    Article  PubMed Central  Google Scholar 

  29. Mendis GDC, Berecki G, Morrisroe E et al (2019) Discovering the pharmacodynamics of conolidine and cannabidiol using a cultured neuronal network based workflow. Sci Rep 9:121

    Article  CAS  PubMed Central  Google Scholar 

  30. Bettencourt LM, Stephens GJ, Ham MI et al (2007) Functional structure of cortical neuronal networks grown in vitro. Phys Rev E Stat Nonlinear Soft Matter Phys 75:021915

    Article  CAS  Google Scholar 

  31. Gullo F, Maffezzoli A, Dossi E et al (2009) Short-latency cross- and autocorrelation identify clusters of interacting cortical neurons recorded from multi-electrode array. J Neurosci Methods 181:186–198

    Article  Google Scholar 

  32. Grosser S, Queenan BN, Lalchandani RR et al (2014) Hilar somatostatin interneurons contribute to synchronized GABA activity in an in vitro epilepsy model. PLoS One 9:e86250

    Article  CAS  PubMed Central  Google Scholar 

  33. Gullo F, Manfredi I, Lecchi M et al (2014) Multi-electrode array study of neuronal cultures expressing nicotinic beta2-V287L subunits, linked to autosomal dominant nocturnal frontal lobe epilepsy. An in vitro model of spontaneous epilepsy. Front Neural Circuits 8:87

    Article  CAS  PubMed Central  Google Scholar 

  34. Ishii MN, Yamamoto K, Shoji M et al (2017) Human induced pluripotent stem cell (hiPSC)-derived neurons respond to convulsant drugs when co-cultured with hiPSC-derived astrocytes. Toxicology 389:130–138

    Article  CAS  Google Scholar 

  35. Odawara A, Katoh H, Matsuda N et al (2016) Physiological maturation and drug responses of human induced pluripotent stem cell-derived cortical neuronal networks in long-term culture. Sci Rep 6:26181

    Article  CAS  PubMed Central  Google Scholar 

  36. Odawara A, Matsuda N, Ishibashi Y et al (2018) Toxicological evaluation of convulsant and anticonvulsant drugs in human induced pluripotent stem cell-derived cortical neuronal networks using an MEA system. Sci Rep 8:10416

    Article  CAS  PubMed Central  Google Scholar 

  37. Maccione A, Gandolfo M, Massobrio P et al (2009) A novel algorithm for precise identification of spikes in extracellularly recorded neuronal signals. J Neurosci Methods 177:241–249

    Article  Google Scholar 

  38. Mendis GD, Morrisroe E, Petrou S et al (2016) Use of adaptive network burst detection methods for multielectrode array data and the generation of artificial spike patterns for method evaluation. J Neural Eng 13:026009

    Article  CAS  Google Scholar 

  39. Ferreira G, Yi J, Rios E et al (1997) Ion-dependent inactivation of barium current through L-type calcium channels. J Gen Physiol 109:449–461

    Article  CAS  PubMed Central  Google Scholar 

  40. Veselovskii NS, Fedulova SA (1986) Effect of replacing calcium ions with barium ions in studies of the inward currents of mammalian neurons. Neirofiziologiia 18:313–318

    CAS  PubMed  Google Scholar 

  41. Kostyuk PG, Krishtal OA, Pidoplichko VI (1975) Effect of internal fluoride and phosphate on membrane currents during intracellular dialysis of nerve cells. Nature 257:691–693

    Article  CAS  Google Scholar 

  42. Becker N, Stoelzle S, Gopel S et al (2013) Minimized cell usage for stem cell-derived and primary cells on an automated patch clamp system. J Pharmacol Toxicol Methods 68:82–87

    Article  CAS  Google Scholar 

  43. Richards KL, Milligan CJ, Richardson RJ et al (2018) Selective NaV1.1 activation rescues Dravet syndrome mice from seizures and premature death. Proc Natl Acad Sci U S A 115:E8077–E8085

    Article  CAS  PubMed Central  Google Scholar 

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Acknowledgments

The authors wish to thank Professor S Petrou, whose National Health and Medical Research Council (NHMRC) Project Grant (1106027) supported the research using mouse primary cortical neurons. The Florey Institute of Neuroscience and Mental Health is supported by Victorian State Government infrastructure funds.

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Authors and Affiliations

  1. Florey Institute of Neuroscience and Mental Health, Melbourne Brain Centre, Parkville, VIC, Australia

    Carol J. Milligan & Svenja Pachernegg

  2. Reproductive Development Group, Murdoch Children’s Research Institute, Parkville, VIC, Australia

    Svenja Pachernegg

Authors
  1. Carol J. Milligan
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  2. Svenja Pachernegg
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Corresponding author

Correspondence to Carol J. Milligan .

Editor information

Editors and Affiliations

  1. University of Reading, Reading, UK

    Mark Dallas

  2. Sophion Bioscience A/S, Ballerup, Denmark

    Damian Bell

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Milligan, C.J., Pachernegg, S. (2021). Utilising Automated Electrophysiological Platforms in Epilepsy Research. In: Dallas, M., Bell, D. (eds) Patch Clamp Electrophysiology. Methods in Molecular Biology, vol 2188. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0818-0_7

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  • DOI: https://doi.org/10.1007/978-1-0716-0818-0_7

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0817-3

  • Online ISBN: 978-1-0716-0818-0

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