In vitro preparations are a powerful tool to explore the mechanisms and processes underlying epileptogenesis and ictogenesis. In this review, we critically review the numerous in vitro methodologies utilized in epilepsy research. We provide support for the inclusion of detailed descriptions of techniques, including often ignored parameters with unpredictable yet significant effects on study reproducibility and outcomes. In addition, we explore how recent developments in brain slice preparation relate to their use as models of epileptic activity.

Case Western Reserve University Cleveland Ohio U S A

Department of Developmental Epileptology Institute of Physiology Czech Academy of Sciences Prague Czech Republic

Department of Epilepsy Movement Disorders and Physiology Kyoto University Graduate School of Medicine Sakyo ku Kyoto Japan

Department of Neurology College of Medicine and Hospital National Cheng Kung University Tainan Taiwan

Department of Neuroscience Tufts University School of Medicine Boston Massachusetts U S A

Departments of Neurology and Pediatrics University of Virginia Charlottesville Virginia U S A

Division of Cell Biology Department of Human Biology Neuroscience Institute University of Cape Town Cape Town South Africa

Epilepsy and Experimental Neurophysiology Unit The Foundation of the Carlo Besta Neurological Institute Milan Italy

Flocel Inc Cleveland Ohio U S A

Inserm Institut de Neurosciences des Systemes UMRS 1106 Aix Marseille University Marseille France

Laboratory of Developmental Epilepsy Saul R Korey Department of Neurology Dominick P Purpura Department of Neuroscience Albert Einstein College of Medicine Einstein Montefiore Epilepsy Center Montefiore Medical Center Bronx New York U S A

Neuroscience Research Center Charité Universitätsmedizin Berlin Berlin Germany

Zobrazit více v PubMed

Heinemann U, Staley KJ. What is the clinical relevance of in vitro epileptiform activity? Adv Exp Med Biol. 2014;813:25–41. PubMed

Khalilov I, Esclapez M, Medina I, et al. A Novel In Vitro Preparation: the Intact Hippocampal Formation. Neuron. 1997;19:743–749. PubMed

de Curtis M, Paré D, Llinás RR. The electrophysiology of the olfactory-hippocampal circuit in the isolated and perfused adult mammalian brain in vitro. Hippocampus. 1991;1:341–54. PubMed

de Curtis M, Librizzi L, Uva L. The in vitro isolated whole guinea pig brain as a model to study epileptiform activity patterns. J Neurosci Methods. 2015 PubMed

Heinemann U. An overview of in vitro seizure models in acute and organotypic slices, Model. Seizures. 2006

Müller CJ, Gröticke I, Hoffmann K, et al. Differences in sensitivity to the convulsant pilocarpine in substrains and sublines of C57BL/6 mice. Genes Brain Behav. 2009;8:481–92. PubMed

McKhann G, Wenzel H, Robbins C, et al. Mouse strain differences in kainic acid sensitivity, seizure behavior, mortality, and hippocampal pathology. Neuroscience. 2003;122:551–561. PubMed

Giorgi FS, Galanopoulou AS, Moshé SL. Sex dimorphism in seizure-controlling networks. Neurobiol Dis. 2014;72(Pt B):144–52. PubMed PMC

Velíšek L, Velíšková J, Etgen A. Region-specific modulation of limbic seizure susceptibility by ovarian steroids. Brain Res. 1999 PubMed

Taubøll E, Sveberg L, Svalheim S. Interactions between hormones and epilepsy. Seizure. 2015;28:3–11. PubMed

Herzog AG, Harden CL, Liporace J, et al. Frequency of catamenial seizure exacerbation in women with localization-related epilepsy. Ann Neurol. 2004;56:431–4. PubMed

Velísková J. The role of estrogens in seizures and epilepsy: the bad guys or the good guys? Neuroscience. 2006;138:837–44. PubMed

Galanopoulou AS, Moshé SL. In search of epilepsy biomarkers in the immature brain: goals, challenges and strategies. Biomark Med. 2011;5:615–28. PubMed PMC

Khazipov R, Khalilov I, Tyzio R, et al. Developmental changes in GABAergic actions and seizure susceptibility in the rat hippocampus. Eur J Neurosci. 2004;19:590–600. PubMed

Rivera C, Voipio J, Kaila K. Two developmental switches in GABAergic signalling: the K+-Cl- cotransporter KCC2 and carbonic anhydrase CAVII. J Physiol. 2005;562:27–36. PubMed PMC

Ben-Ari Y. Excitatory actions of GABA during development: the nature of the nurture. Nat Rev Neurosci. 2002;3:728–739. PubMed

Langer M, Brandt C, Löscher W. Marked strain and substrain differences in induction of status epilepticus and subsequent development of neurodegeneration, epilepsy, and behavioral alterations in rats [corrected] Epilepsy Res. 2011;96:207–24. PubMed

Galanopoulou AS. Dissociated gender-specific effects of recurrent seizures on GABA signaling in CA1 pyramidal neurons: role of GABA(A) receptors. J Neurosci. 2008;28:1557–67. PubMed PMC

Koe AS, Salzberg MR, Morris MJ, et al. Early life maternal separation stress augmentation of limbic epileptogenesis: the role of corticosterone and HPA axis programming. Psychoneuroendocrinology. 2014;42:124–33. PubMed

Young D, Lawlor PA, Leone P, et al. Environmental enrichment inhibits spontaneous apoptosis, prevents seizures and is neuroprotective. Nat Med. 1999;5:448–53. PubMed

Clark J, Baldwin R, Bayne K. Guide for the care and use of laboratory animals. DC Inst Lab. 1996

Matos G, Andersen ML, do Valle AC, et al. The relationship between sleep and epilepsy: evidence from clinical trials and animal models. J Neurol Sci. 2010;295:1–7. PubMed

Quigg M, Clayburn H, Straume M, et al. Effects of Circadian Regulation and Rest-Activity State on Spontaneous Seizures in a Rat Model of Limbic Epilepsy. Epilepsia. 2000;41:502–509. PubMed

Schmitt LI, Sims RE, Dale N, et al. Wakefulness affects synaptic and network activity by increasing extracellular astrocyte-derived adenosine. 2012;32:4417–4425. PubMed PMC

During MJ, Spencer DD. Adenosine: A potential mediator of seizure arrest and postictal refractoriness. Ann Neurol. 1992;32:618–624. PubMed

Mareš P, Kubová H. GABAB, not GABAA receptors play a role in cortical postictal refractoriness. Neuropharmacology. 2015;88:99–102. PubMed

Wolfensohn S, Lloyd M. Handbook of Laboratory Animal Management and Welfare (Google eBook) John Wiley & Sons; 2013.

Li X, Pearce RA. Effects of Halothane on GABA A Receptor Kinetics : Evidence for Slowed Agonist Unbinding. 2000;20:899–907. PubMed PMC

Zschenderlein C, Gebhardt C, von Bohlen Und Halbach O, et al. Capsaicin-induced changes in LTP in the lateral amygdala are mediated by TRPV1. PLoS One. 2011;6:e16116. PubMed PMC

Wölfer J, Bantel C, Köhling R, et al. Electrophysiology in ischemic neocortical brain slices: species differences vs. influences of anaesthesia and preparation. Eur J Neurosci. 2006;23:1795–800. PubMed

Tétrault S, Chever O, Sik A, et al. Opening of the blood-brain barrier during isoflurane anaesthesia. Eur J Neurosci. 2008;28:1330–41. PubMed

Aghajanian GK, Rasmussen K. Intracellular studies in the facial nucleus illustrating a simple new method for obtaining viable motoneurons in adult rat brain slices. Synapse. 1989;3:331–8. PubMed

Ting JT, Daigle TL, Chen Q, et al. Patch-Clamp Methods and Protocols. 2014;1183:221–242. PubMed PMC

Moyer JR, Brown TH. Methods for whole-cell recording from visually preselected neurons of perirhinal cortex in brain slices from young and aging rats. J Neurosci Methods. 1998;86:35–54. PubMed

Bischofberger J, Engel D, Li L, et al. Patch-clamp recording from mossy fiber terminals in hippocampal slices. Nat Protoc. 2006;1:2075–81. PubMed

Mainen ZF, Maletic-Savatic M, Shi SH, et al. Two-photon imaging in living brain slices. Methods. 1999;18:231–9. 181. PubMed

Alkondon M, Pereira EFR, Cartes WS, et al. Choline is a Selective Agonist of α7 Nicotinic Acetylcholine Receptors in the Rat Brain Neurons. Eur J Neurosci. 1997;9:2734–2742. PubMed

Tanaka Y, Tanaka Y, Furuta T, et al. The effects of cutting solutions on the viability of GABAergic interneurons in cerebral cortical slices of adult mice. J Neurosci Methods. 2008;171:118–25. PubMed

Ye JH, Zhang J, Xiao C, et al. Patch-clamp studies in the CNS illustrate a simple new method for obtaining viable neurons in rat brain slices: glycerol replacement of NaCl protects CNS neurons. J Neurosci Methods. 2006;158:251–9. PubMed

Dugué GP, Dumoulin A, Triller A, et al. Target-dependent use of co-released inhibitory transmitters at central synapses. J Neurosci. 2005;25:6490–6498. PubMed PMC

Zhao S, Ting JT, Atallah HE, et al. Cell type–specific channelrhodopsin-2 transgenic mice for optogenetic dissection of neural circuitry function. Nat Methods. 2011;8:745–752. PubMed PMC

Davie JT, Kole MHP, Letzkus JJ, et al. Dendritic patch-clamp recording. Nat Protoc. 2006;1:1235–47. PubMed PMC

Douglas RJ, Martin KAC. Inhibition in cortical circuits. Curr Biol. 2009;19:R398–402. PubMed

Dreier J, Heinemann U. Regional and time dependent variations of low Mg2+ induced epileptiform activity in rat temporal cortex slices. Exp Brain Res. 1991;3:581–596. PubMed

Kuenzi FM, Fitzjohn SM, Morton RA, et al. Reduced long-term potentiation in hippocampal slices prepared using sucrose-based artificial cerebrospinal fluid. J Neurosci Methods. 2000;100:117–122. PubMed

Dzhala V, Valeeva G, Glykys J, et al. Traumatic Alterations in GABA Signaling Disrupt Hippocampal Network Activity in the Developing Brain. J Neurosci. 2012;32:4017–4031. PubMed PMC

Capron B, Sindic C, Godaux E, et al. The characteristics of LTP induced in hippocampal slices are dependent on slice-recovery conditions. Learn Mem. 2006;13:271–7. PubMed PMC

Gähwiler BH, Capogna M, Debanne D, et al. Organotypic slice cultures: a technique has come of age. Trends Neurosci. 1997;20:471–7. PubMed

Debanne D, Guerineau NC, Gahwiler BH, et al. Physiology and pharmacology of unitary synaptic connections between pairs of cells in areas CA3 and CA1 of rat hippocampal slice cultures. J Neurophysiol. 1995;73:1282–1294. PubMed

Routbort M, Bausch S, McNamara J. Seizures, cell death, and mossy fiber sprouting in kainic acid-treated organotypic hippocampal cultures. Neuroscience. 1999;94:755–765. PubMed

Dyhrfjeld-Johnsen J, Berdichevsky Y, Swiercz W, et al. Interictal spikes precede ictal discharges in an organotypic hippocampal slice culture model of epileptogenesis. J Clin Neurophysiol. 2010;27:418–24. PubMed PMC

Stoppini L, Buchs P-A, Muller D. A simple method for organotypic cultures of nervous tissue. J Neurosci Meth. 1991;37:173–182. PubMed

De Simoni A, Yu LMY. Preparation of organotypic hippocampal slice cultures: interface method. Nat Protoc. 2006;1:1439–45. PubMed

Pomper JK, Graulich J, Kovacs R, et al. High oxygen tension leads to acute cell death in organotypic hippocampal slice cultures. Dev Brain Res. 2001;126:109–116. PubMed

Zhang M, Ladas TP, Qiu C, et al. Propagation of Epileptiform Activity Can Be Independent of Synaptic Transmission, Gap Junctions, or Diffusion and Is Consistent with Electrical Field Transmission. J Neurosci. 2014;34 PubMed PMC

Jackson J, Amilhon B, Goutagny R, et al. Reversal of theta rhythm flow through intact hippocampal circuits. Nat Neurosci. 2014;17:1362–70. PubMed

Jirsa VK, Stacey WC, Quilichini PP, et al. On the nature of seizure dynamics. Brain. 2014;137:2210–30. PubMed PMC

Ivanov AI, Bernard C, Turner DA. Metabolic responses differentiate between interictal, ictal and persistent epileptiform activity in intact, immature hippocampus in vitro. Neurobiol Dis. 2015;75:1–14. PubMed PMC

Hájos N, Ellender TJ, Zemankovics R, et al. Maintaining network activity in submerged hippocampal slices: importance of oxygen supply. Eur J Neurosci. 2009;29:319–27. PubMed PMC

Morris G, Jiruska P, Jefferys JGR, et al. A New Approach of Modified Submerged Patch Clamp Recording Reveals Interneuronal Dynamics during Epileptiform Oscillations. Front Neurosci. 2016;10:519. PubMed PMC

Khalilov I, Holmes GL, Ben-Ari Y. In vitro formation of a secondary epileptogenic mirror focus by interhippocampal propagation of seizures. Nat Neurosci. 2003;6:1079–85. PubMed

Williamson A, Ferro M, Leleux P, et al. Localized Neuron Stimulation with Organic Electrochemical Transistors on Delaminating Depth Probes. Adv Mater. 2015 n/a-n/a. PubMed

Librizzi L, Pastori C, De Grazia U, et al. Rapid in vitro elimination of anesthetic doses of thiopental in the isolated guinea pig brain. Neurosci Lett. 2005;380:66–69. PubMed

Singer W, Lux HD. Extracellular potassium gradients and visual receptive fields in the cat striate cortex. Brain Res. 1975;96:378–383. PubMed

Mody I, Lambert JD, Heinemann U. Low extracellular magnesium induces epileptiform activity and spreading depression in rat hippocampal slices. J Neurophysiol. 1987;57:869–88. PubMed

Turner RW, Baimbridge KG, Miller JJ. Calcium-induced long-term potentiation in the hippocampus. Neuroscience. 1982;7:1411–1416. PubMed

Rheims S, Holmgren CD, Chazal G, et al. GABA action in immature neocortical neurons directly depends on the availability of ketone bodies. J Neurochem. 2009;110:1330–8. PubMed

Tyzio R, Allene C, Nardou R, et al. Depolarizing actions of GABA in immature neurons depend neither on ketone bodies nor on pyruvate. J Neurosci. 2011;31:34–45. PubMed PMC

Gruetter R, Ugurbil K, Seaquist ER. Steady-State Cerebral Glucose Concentrations and Transport in the Human Brain. J Neurochem. 2002;70:397–408. PubMed

Barros LF, Bittner CX, Loaiza A, et al. A quantitative overview of glucose dynamics in the gliovascular unit. Glia. 2007;55:1222–37. PubMed

Dudek FE, Obenaus A, Tasker JG. Osmolality-induced changes in extracellular volume alter epileptiform bursts independent of chemical synapses in the rat: importance of non-synaptic mechanisms in hippocampal epileptogenesis. Neurosci Lett. 1990;120:267–70. PubMed

Chesler M. Regulation and modulation of pH in the brain. Physiol Rev. 2003;83:1183–221. PubMed

Taira T, Smirnov S, Voipio J, et al. Intrinsic proton modulation of excitatory transmission in rat hippocampal slices. Neuroreport. 1993;4:93. PubMed

Pasternack M, Smirnov S, Kaila K. Proton Modulation of Functionally Distinct GABAA Receptors in Acutely Isolated Pyramidal Neurons of Rat Hippocampus. Neuropharmacology. 1996;35:1279–1288. PubMed

Dulla CG, Dobelis P, Pearson T, et al. Adenosine and ATP link PCO2 to cortical excitability via pH. Neuron. 2005;48:1011–23. PubMed PMC

Church J. A change from HCO3(-)-CO2- to hepes-buffered medium modifies membrane properties of rat CA1 pyramidal neurones in vitro. J Physiol. 1992;455:51–71. PubMed PMC

Hodgkin AL, Katz B. The effect of temperature on the electrical activity of the giant axon of the squid. J Physiol. 1949;109:240–249. PubMed PMC

Schuchmann S, Meierkord H, Stenkamp K, et al. Synaptic and nonsynaptic ictogenesis occurs at different temperatures in submerged and interface rat brain slices. J Neurophysiol. 2002;87:2929–2935. PubMed

Hill MW, Wong M, Amarakone A, et al. Rapid Cooling Aborts Seizure-Like Activity in Rodent Hippocampal-Entorhinal Slices. Epilepsia. 2000;41:1241–1248. PubMed

Tancredi V, D’Arcangelo G, Zona C, et al. Induction of Epileptiform Activity by Temperature Elevation in Hippocampal Slices from Young Rats: An In Vitro Model for Febrile Seizures? Epilepsia. 1992;33:228–234. PubMed

Croning MD, Haddad G. Comparison of brain slice chamber designs for investigations of oxygen deprivation in vitro. J Neurosci Methods. 1998;81:103–111. PubMed

Bortolotto ZA, Bashir ZI, Davies CH, et al. Studies on the role of metabotropic glutamate receptors in long-term potentiation: some methodological considerations. J Neurosci Methods. 1995;59:19–24. PubMed

Avsar E, Empson RM. Adenosine acting via A1 receptors, controls the transition to status epilepticus-like behaviour in an in vitro model of epilepsy. Neuropharmacology. 2004;47:427–37. PubMed

Madisen L, Zwingman TA, Sunkin SM, et al. A robust and high-throughput Cre reporting and characterization system for the whole mouse brain. Nat Neurosci. 2010;13:133–40. PubMed PMC

Hu H, Cavendish JZ, Agmon A. Not all that glitters is gold: off-target recombination in the somatostatin-IRES-Cre mouse line labels a subset of fast-spiking interneurons. Front Neural Circuits. 2013;7:195. PubMed PMC

Raimondo JV, Joyce B, Kay L, et al. A genetically-encoded chloride and pH sensor for dissociating ion dynamics in the nervous system. Front Cell Neurosci. 2013;7:202. PubMed PMC

Raimondo JV, Irkle A, Wefelmeyer W, et al. Genetically encoded proton sensors reveal activity-dependent pH changes in neurons. Front Mol Neurosci. 2012;5:68. PubMed PMC

Karus C, Mondragão MA, Ziemens D, et al. Astrocytes restrict discharge duration and neuronal sodium loads during recurrent network activity. Glia. 2015 n/a-n/a. PubMed

Raimondo JV, Tomes H, Irkle A, et al. Tight Coupling of Astrocyte pH Dynamics to Epileptiform Activity Revealed by Genetically Encoded pH Sensors. J Neurosci. 2016;36:7002–13. PubMed PMC

Marvin JS, Borghuis BG, Tian L, et al. An optimized fluorescent probe for visualizing glutamate neurotransmission. Nat Methods. 2013;10:162–70. PubMed PMC

Bittner CX, Valdebenito R, Ruminot I, et al. Fast and reversible stimulation of astrocytic glycolysis by K+ and a delayed and persistent effect of glutamate. J Neurosci. 2011;31:4709–13. PubMed PMC

Boyden ES, Zhang F, Bamberg E, et al. Millisecond-timescale, genetically targeted optical control of neural activity. Nat Neurosci. 2005;8:1263–8. PubMed

Ellender TJ, Raimondo JV, Irkle A, et al. Excitatory effects of parvalbumin-expressing interneurons maintain hippocampal epileptiform activity via synchronous afterdischarges. J Neurosci. 2014;34:15208–22. PubMed PMC

Tønnesen J, Sørensen AT, Deisseroth K, et al. Optogenetic control of epileptiform activity. Proc Natl Acad Sci U S A. 2009;106:12162–7. PubMed PMC

Raimondo JV, Kay L, Ellender TJ, et al. Optogenetic silencing strategies differ in their effects on inhibitory synaptic transmission. Nat Neurosci. 2012;15:1102–4. PubMed PMC

Alfonsa H, Merricks EM, Codadu NK, et al. The Contribution of Raised Intraneuronal Chloride to Epileptic Network Activity. J Neurosci. 2015;35:7715–7726. PubMed PMC

How do we use in vitro models to understand epileptiform and ictal activity? A report of the TASK1-WG4 group of the ILAE/AES Joint Translational Task Force

A companion to the preclinical common data elements and case report forms for rodent EEG studies. A report of the TASK3 EEG Working Group of the ILAE/AES Joint Translational Task Force