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Review

Epilepsy research methods update: Understanding the causes of epileptic seizures and identifying new treatments using non-mammalian model organisms Vincent T. Cunliffe a,*, Richard A. Baines b, Carlo N.G. Giachello b, Wei-Hsiang Lin b, Alan Morgan c, Markus Reuber d, Claire Russell e, Matthew C. Walker f, Robin S.B. Williams g a

Bateson Centre, Department of Biomedical Science, University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, United Kingdom Faculty of Life Sciences, University of Manchester, AV Hill Building, Oxford Road, Manchester M13 9PT, United Kingdom c Department of Molecular and Cellular Physiology, Institute of Translational Medicine, University of Liverpool, Crown Street, Liverpool L69 3BX, United Kingdom d Academic Neurology Unit, University of Sheffield, Royal Hallamshire Hospital, Glossop Road, Sheffield S10 2JF, United Kingdom e Department of Comparative Biomedical Sciences, Royal Veterinary College, Royal College Street, London NW1 0TU, United Kingdom f Department of Clinical and Experimental Epilepsy, Institute of Neurology, University College London, Queen Square, London WC1N 3BG, United Kingdom g School of Biological Sciences, Royal Holloway College, University of London, Egham Hill, Egham, Surrey TW20 0EX, United Kingdom b

A R T I C L E I N F O

A B S T R A C T

Article history: Received 8 September 2014 Accepted 23 September 2014

This narrative review is intended to introduce clinicians treating epilepsy and researchers familiar with mammalian models of epilepsy to experimentally tractable, non-mammalian research models used in epilepsy research, ranging from unicellular eukaryotes to more complex multicellular organisms. The review focuses on four model organisms: the social amoeba Dictyostelium discoideum, the roundworm Caenorhabditis elegans, the fruit fly Drosophila melanogaster and the zebrafish Danio rerio. We consider recent discoveries made with each model organism and discuss the importance of these advances for the understanding and treatment of epilepsy in humans. The relative ease with which mutations in genes of interest can be produced and studied quickly and cheaply in these organisms, together with their anatomical and physiological simplicity in comparison to mammalian species, are major advantages when researchers are trying to unravel complex disease mechanisms. The short generation times of most of these model organisms also mean that they lend themselves particularly conveniently to the investigation of drug effects or epileptogenic processes across the lifecourse. ß 2014 British Epilepsy Association. Published by Elsevier Ltd. All rights reserved.

Keywords: Epilepsy Epileptogenesis Ictogenesis Anti epileptic drugs Non-mammalian Animal research

1. Introduction Epilepsy is characterised by recurrent unprovoked epileptic seizures. The ILAE definition of an epileptic seizure is a transient occurrence of signs and/or symptoms due to abnormal excessive or synchronous neuronal activity in the brain.1 Whilst most epileptic seizures do not seem to cause significant lasting damage, repeated

* Corresponding author. Tel.: +44 114 222 2389. E-mail addresses: v.t.cunliffe@sheffield.ac.uk (V.T. Cunliffe), [email protected] (R.A. Baines), [email protected] (A. Morgan), m.reuber@sheffield.ac.uk (M. Reuber), [email protected] (C. Russell), [email protected] (M.C. Walker), [email protected] (Robin S.B. Williams).

or prolonged seizures can cause persistent functional and morphological change. Around 0.7% of the general population suffer from epilepsy, and although many patients respond satisfactorily to the available anti-epileptic drugs (AEDs), around a third of people with epilepsy do not respond to currently available treatments.2 There is, therefore, a significant unmet need both for better understanding of the disease process and for new strategies that enable cost-efficient discovery of new anti-epileptic drugs. Human genetic studies indicate that defects in a wide variety of molecular and cellular mechanisms facilitating normal nervous system development and maintaining neural homeostasis underlie ictogenetic and epileptogenetic processes.3 For example, mutations in genes that encode subunits of neurotransmitter receptors and ion channels may be ictogenetic and epileptogenic, confirming roles for the synaptic and the intrinsic excitability machinery in seizure processes.4 In addition, epileptogenic

http://dx.doi.org/10.1016/j.seizure.2014.09.018 1059-1311/ß 2014 British Epilepsy Association. Published by Elsevier Ltd. All rights reserved.

Please cite this article in press as: Cunliffe VT, et al. Epilepsy research methods update: Understanding the causes of epileptic seizures and identifying new treatments using non-mammalian model organisms. Seizure: Eur J Epilepsy (2014), http://dx.doi.org/10.1016/ j.seizure.2014.09.018

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mutations have been discovered in genes such as LGI1, ARX, EFHC1, STXBP1, PCDH19, TBC1D24, CHD2, SYNGAP1 and ALG13, which perform diverse roles in cellular processes such as neuronal morphogenesis, synapse formation, transcriptional regulation and intercellular signalling within the nervous system,4–6 further widening the range of molecular mechanisms specifically implicated in human epilepsy. In order to build on the insights provided by human molecular genetic analysis, model systems are required that allow the fundamental mechanisms underlying seizure phenotypes and therapeutic drug actions to be elucidated. It is not always necessary to recapitulate human seizure phenotypes in every respect and indeed, there are many advantages in using simple models to understand the fundamental biological processes that underlie seizure generation and mechanisms of drug action. Moreover, whilst the greater similarities between the nervous systems of different mammalian species may make rodent models particularly appropriate for determining likely treatment responses in humans, variation in the genetic background of rodent strains can lead to opposing or contradictory results, confounding experimental studies.7 Rodent models are also more expensive and require complicated, invasive procedures to analyse gene functions in seizure processes. In higher non-mammalian systems, such as zebrafish, the network mechanisms that generate seizures are likely to be similar to those that underlie seizures in mammals,8 and many other non-mammalian organisms exhibit phenotypes with seizure-like characteristics when the balance between excitatory and inhibitory neurotransmission within the CNS is perturbed. For some types of anti-epileptic drugs, the extensive evolutionary conservation of the sequences of their protein targets means that even unicellular eukaryotes can be employed for drug screening and mechanism-of-action studies. Thus, for all of these reasons, a wide variety of species that are biologically simpler than mammals has begun to be deployed in epilepsy research. 2. Anti-epileptic drug discovery and pharmacology using Dictyostelium discoideum The unicellular social amoeba Dictyostelium has been used as a simple model system in epilepsy research to elucidate the modes of action of existing anti-epileptic drugs and then to use this information to identify new compounds with improved specificity and efficacy. D. discoideum is listed by the US National Institute of Health as a biomedical model system9 and has been used in a range of biochemical and pharmacogenetics studies to define the action of therapeutic compounds in mammalian models.10–12 The key advantages of this model include: enabling the rapid analysis of drug effects on normal cell function and on synchronised development; the screening of mutant cell banks to identify the genetic basis of drug mechanisms; the analysis of isogenic cell lines containing ablated or over-expressed proteins to define a role of individual or multiple proteins as potential drug targets; and the use of multiple gram weights of isogenic cell lines for biochemical analysis. These features enable Dictyostelium to be employed as a valuable animal replacement model, with a range of methodological attributes not available in mammalian models in epilepsy research. Sodium valproate is the most commonly prescribed treatment for epilepsy worldwide,13 but until recently, very little was known about its mechanism of action as an AED.14 Recent studies showed that valproic acid regulated the turnover of phosphoinositides in Dictyostelium at concentrations similar to those found in humans during treatment for epilepsy.12,15 To investigate the molecular mechanism underlying this effect, cell lines containing ablated target proteins involved in phosphoinositide production and recycling were tested for sensitivity to valproate in an attempt

to isolate a mutant lacking the protein target for valproate. The results of these studies suggested that the molecular target for valproate in Dictyostelium is likely to be involved in either the incorporation or transfer of inositol within phosphoinositide species.15 A structure–activity-based screen of a range of valproate-like compounds identified a range of medium chain fatty acids that inhibited phosphoinositide production in Dictyostelium more effectively than valproate. These fatty acids included a major component of the medium chain triglyceride (MCT) ketogenic diet, decanoic acid.15 Excitingly, the level of this fatty acid is elevated in the plasma of children on the ketogenic diet16,17 suggesting a role for decanoic acid in seizure control. The molecular basis of the therapeutic effect of the MCT ketogenic diet remains unclear. To improve tolerability, it will be critical to identify compounds within the diet that help to control seizures but lack the side-effects and the necessity for the severe dietary restrictions engendered by the ketogenic diet. Considerable research has initially focused on one physiological effect of the diet – the increasing level of ketone body formation in patients. However, this has since been shown to correlate poorly with seizure control in animal models.18,19 An alternative effect of the diet is the accumulation of medium chain fatty acids in blood (in particular decanoic and octanoic acids). The identification of decanoic acid and a range of octanoic acid derivatives, with similar effects to valproate on phosphoinositide signalling in Dictyostelium, suggested a common mechanism of action for these compounds in this simple model, and raised the possibility that they might also be effective in seizure control. To investigate this possibility, decanoic and octanoic acids were assessed in a rat hippocampal slice (PTZ) model of seizures.15,20 A subset of the tested compounds exhibited strong seizure control activity, with significant improvement over valproate. Compounds with high activity also showed high structural specificity, so that, for example, 4-methyloctanoic, 4-ethyloctanoic, and 2-proplyoctanoic acid demonstrated strongly improved seizure control, compared to valproic acid, whereas octanoic acid and 3,7dimethyloctanoic acid were ineffective. Some of these novel active compounds were effective both in a low magnesium (drugresistant) model of seizure control and in an in vivo status epilepticus model.15 The recent advances in this work have demonstrated that seizure-like activity, induced in primary rat neurons or in ex vivo hippocampal slices by PTZ, or in vivo by kainic acid, causes a reduction in the key signalling phosphoinositide, PIP3, and this reduction is blocked in the presence of valproate.21 This mechanism, initially identified in Dictyostelium, provides a credible new explanation for the anti-epileptic effect of valproate in seizure control. Since both enhanced efficacy and safety characteristics must be considered when introducing new epilepsy treatments, the family of potent fatty acids was also examined for potential side effects, including teratogenicity.20 Valproate and related short chain fatty acids have been well documented to inhibit histone deacetylase (HDAC) activity,22,23 causing altered gene transcription during embryonic development, leading to embryonic malformations, which consequently limits their use in women of childbearing age due to the increased risk of birth defects.24,25 Assessment of HDAC inhibition by these potent fatty acids identified a subset of compounds that are less likely to cause birth defects through this mechanism, providing potentially safer treatment during pregnancy. The identification of phosphoinositide signalling as a target for anti-epileptic drug discovery has led to the discovery of novel fatty acids with enhanced activity, including decanoic acid, which is elevated in the plasma of patients on the MCT ketogenic diet. Translation of discoveries made in Dictyostelium to mammalian epilepsy models suggest that this family of novel compounds may

Please cite this article in press as: Cunliffe VT, et al. Epilepsy research methods update: Understanding the causes of epileptic seizures and identifying new treatments using non-mammalian model organisms. Seizure: Eur J Epilepsy (2014), http://dx.doi.org/10.1016/ j.seizure.2014.09.018

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include drugs that can achieve seizure control in drug-resistant epilepsy, and highlights the advantages of an innovative 3Rs approach to epilepsy research. 3. Caenorhabditis elegans models of epilepsy Caenorhabditis elegans is a non-parasitic, soil-dwelling nematode worm. It mainly exists in a self-fertilising, hermaphrodite form, although males are present at very low frequencies. Development from egg to the approximately 1 mm-long adult takes around 3 days, and adults live for around 3 weeks. From the pioneering studies of Brenner in the 1960s,26 C. elegans rapidly became one of the major model organisms used in biology. Indeed, the genome of C. elegans was the first metazoan genome to be fully sequenced, in 1998. One of the main reasons why Brenner chose C. elegans was that its simplicity could enable an understanding of how genes specify the development of the nervous system and determine behaviour; a goal that was unfeasible with established animal model organisms. Indeed, the C. elegans nervous system comprises just 302 neurons, out of a total of 959 cells in the adult hermaphrodite. It is unique among animals in that the positions and fates of all of its cells are known, and the 7000 synaptic connections made by its 302 neurons have been mapped by serial section electron microscopy. Despite this simplicity, worms display a surprisingly complex set of behaviours and are capable of learning. The neurotransmitters that control many of these behaviours are conserved from worm to human and over 40% of human disease genes have worm homologues. A number of other special attributes make C. elegans particularly attractive as a model. The animal is naturally transparent, and virtually any specific neuron type can be visualised in freely moving animals via cellspecific promoters driving GFP fluorescence. The ability to grow thousands of hermaphrodites on agar plates without the need for mating makes it particularly well suited to high-throughput chemical and genetic screening. The unique ability to perform genome-wide knock down of protein expression via RNA interference simply by feeding the worms transformed versions of their standard laboratory food source, Escherichia coli, is particularly powerful in this regard.27 Another advantage is that homozygous loss-of-function mutations in conserved genes required for neuronal function are usually viable, in contrast to other models such as Drosophila, thus facilitating experimental analysis. There are disadvantages of C. elegans, however, with regard to its utility as an epilepsy model. Clearly, the extreme simplicity of the nervous system makes it more difficult to directly compare to the human disease than with rodent models. In addition, epilepsy is ultimately defined by episodes of abnormal electrical activity in the brain, but electrophysiological techniques are extremely challenging to perform using C. elegans. Finally, there are some differences between neurons in worms and humans that may prevent certain lines of epilepsy research – for example, C. elegans neurons lack voltage-gated sodium channels.28 Despite these caveats, the nematode represents a promising model with potential to shed light on certain aspects of epilepsy. Whilst the number of published studies using C. elegans to gain epileptological insights is relatively small compared to the huge number of studies using this model organism to explore basic neurobiology29 or to describe mechanisms of neurodegeneration,30 the pace of epilepsy research with this organism is beginning to gather momentum. The first publications focused on the worm homologue of the human lis1 gene that, when mutated, causes the epilepsy-prone disease lissencephaly.31 By using a particular allele of C. elegans lis-1, Caldwell and colleagues were able to demonstrate that these mutant worms exhibited repetitive convulsions when treated with the seizure-inducing GABA antagonist, pentylenetetrazole (PTZ). Importantly, the lis-1

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allele used did not display the neuronal migration and architecture defects evident in lissencephaly, suggesting that alterations in GABAergic presynaptic vesicle function may underlie the convulsions in both worms and humans. Consistent with this notion, mutations in genes involved in GABA synthesis (unc-25, encoding glutamic acid decarboxylase), vesicular uptake (unc-47, encoding a synaptic vesicle GABA transporter) or responsiveness (unc-49, encoding a GABAA receptor), all resulted in phenotypically similar convulsions to those seen with the lis-1 allele.31 Interestingly, these convulsions manifest as repetitive anterior muscle contractions – so called ‘head-bobbing’, while the posterior part of the worm is paralysed. In contrast, PTZ treatment of worms with a mutation in calmodulin-dependent protein kinase (unc-43) causes full body convulsions, with contraction of both the head and the tail region of the animal. The GABA-specific anterior head-bobbing phenotype was subsequently used in an RNAi screen to identify additional components of the lis-1 pathway, revealing that several genes involved in neuronal migration can contribute to the convulsive phenotype.32 It is likely that cytoskeletal transport processes are important in this regard, as both microtubule- and actin-based mechanisms have been implicated due to the effect of dynein and Rac mutations, respectively.31,33 An alternative C. elegans epilepsy model emerged from studies of neuronal acetylcholine receptors. A gain-of-function mutation in the acr-2 gene, which encodes a subunit of the acetylcholine receptor, was found to cause spontaneous ‘shrinking’ convulsions in the absence of seizure-inducing agents.34 Interestingly, an identical mutation in the b1 subunit of the human muscle acetylcholine receptor causes myasthenia gravis due to creation of a hyperactive channel. Consistent with this, electrophysiological analysis of the C. elegans acr-2 mutant revealed increased cholinergic transmission accompanied by reduced GABAergic transmission.34 To shed light on how this imbalance in excitatory/inhibitory inputs leads to the convulsive phenotype, a forward genetic screen was conducted to identify mutations that suppress the acr-2 mutant seizure phenotype.35 These experiments revealed that mutation of gtl-2, encoding a member of the transient receptor potential of the melastatin (TRPM) channel family, virtually eliminated spontaneous convulsions. Interestingly, GTL-2 is not expressed in neurons, suggesting that control of systemic ion homeostasis by non-neuronal TRPM channels enables sophisticated regulation of neuronal network activity. This may in turn suggest potentially new therapeutic approaches for epilepsy. Finally, a C. elegans epilepsy model has recently been developed that avoids the need to use either PTZ or defined mutants. Instead, this model simply involves heating the worms to over 26 8C in solution, which results in repetitive unilateral contractions of the anterior part of the animal.36 Clearly, the relevance of such heatinduced phenotypes to human epilepsy is not as obvious as the PTZ- or mutation-based approaches. Nevertheless, convulsions were reduced by Baccoside A, a plant-derived compound with reported anti-epileptic properties, and by mutation of cca-1, the putative worm homologue of T-type calcium channels.35 This led to the suggestion that Baccoside A acts to prevent convulsions by inhibiting CCA-1 channel activity. The use of C. elegans as a model for epilepsy research is in its infancy. Nevertheless, the few published studies to date give a hint of the great potential of the model for combining chemical screening with genetics, both to uncover underlying disease mechanisms and to suggest novel therapeutics. The real power of the nematode model is the ability to do this in a high-throughput, genome-wide scale that is not feasible with most other model organisms. For example, genetic screens using RNAi or classical mutagenesis could be used to isolate genetic suppressors or enhancers of induced seizures in vivo, as has been done for the acr2 mutant described above.35 Similarly, genetic screens could be

Please cite this article in press as: Cunliffe VT, et al. Epilepsy research methods update: Understanding the causes of epileptic seizures and identifying new treatments using non-mammalian model organisms. Seizure: Eur J Epilepsy (2014), http://dx.doi.org/10.1016/ j.seizure.2014.09.018

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used to pinpoint the mechanism of action of various currently prescribed anti-epileptic drugs whose in vivo targets are unclear. Alternatively, phenotype-based screening for compounds capable of preventing convulsions could potentially reveal novel therapeutics. The availability of automated video imaging systems capable of recording worm movement in solution in a highthroughput manner makes large-scale chemical-genetic screens of this type feasible.37 Thus, despite its limitations, the simple nematode worm has significant untapped potential to contribute to epilepsy research. 4. Genetic control of seizure susceptibility in Drosophila melanogaster There are several prominent features of the fruit fly, D. melanogaster, that make it an attractive experimental model for epilepsy research. The simplicity of its nervous system, rapid life cycle, fully sequenced genome, and ability to manipulate its genetics all facilitate the analysis of complex interactions between genes and behaviour. Moreover, the possibility of performing electrophysiological recordings at different stages, provides a relatively new tool to investigate alterations in neuronal activity during development and/or seizure in this model system.38 It has been estimated that nearly 75% of disease-related genes in humans have functional orthologues in flies, establishing Drosophila as a valuable model system for the study of numerous human diseases, including epilepsy.39 In wild-type Drosophila, high frequency electrical stimuli can trigger seizure activity that results in convulsive spasms which are reminiscent of the motor convulsions caused by epileptic seizures in humans. Mutations in Drosophila genes encoding a range of distinct types of proteins, both reduce the threshold for such electrically-induced seizure onset, and also render seizure onset sensitive to mechanical shocks, such as a tap of the culture vial or brief vortexing on a laboratory mixer.40,41 These so-called Bang-Sensitive mutations (BS mutations) affect genes that encode proteins such as the voltage-sensitive sodium channel a-subunit (paralytic), ethanolamine kinase (easily shocked), aminopeptidase N (slamdance), an RNA binding protein (couch potato), and a LIM-domain transcription factor (prickle). Seizure onset is usually characterised, in adult flies, by leg shaking, abdominal muscle contractions, wing-flapping and proboscis extensions, after which a period of paralysis typically ensues. This is often followed by tonic-clonic-like convulsive spasms. Significantly, this seizure-like behaviour can be reduced by prior ingestion of clinically-relevant AEDs.42,43 After a refractory period, during which no further seizures can be induced, flies recover and re-acquire bang-sensitivity. The BS phenotype has been used to identify second-site mutations that suppress this phenotype.41 Such mutations include alleles of the voltage-gated potassium channel Shaker, the gap junction channel shaking-B, the voltage-gated sodium channel paralytic, the zinc finger transcription factor escargot, and DNA Topoisomerase I. Taken together, the diversity of gene products whose functions are affected by BS or Seizure-Suppressor mutations reflects the complexity of the biology underlying seizure susceptibility in Drosophila. Interestingly, AEDs such as valproate, phenytoin, gabapentin and Sodium bromide cause appreciable, albeit variable suppression of BS phenotypes of the BS mutant easily shocked. In contrast, the AEDs carbamazepine, ethosuximide and vigabatrin, do not suppress the BS phenotype of this mutant.44 The specificity of these pharmacological effects, together with the varied functions of the gene products affected in Bang-Sensitive and Seizure-Suppressor mutants, indicate that seizure onset in flies is a physiologically complex process, further study of which is likely to yield new insights to human epilepsy. Moreover, despite the evolutionary

distance, the similarities in the epileptiform activities between flies and mammals, together with the similar responses of seizures in these organisms to AEDs, make BS mutants attractive models for epilepsy research. Recent studies of the BS mutant slamdance (sda) have yielded particularly interesting insights into the mechanisms of both seizures and anti-epileptic drug action. Electrical stimulation of the larval stage of the BS mutant sda results in extended bouts of paralysis. Electrophysiological analysis of the CNS in this mutant reveals increased persistent sodium current (INap) mediated by voltage-gated sodium (Nav) channels in motor neurons.38 INap is a key pharmacological target to control seizures in humans. This current, which is present in almost all neurons, arises from incomplete inactivation of Nav channels and acts to potentiate action potential firing.45,46 An increase in INap has been associated with both symptomatic46 and idiopathic epilepsy.47 However, the molecular mechanism underlying INap is not well understood. Progress in this regard has been complicated by extensive genetic redundancy: mammalian genomes encode at least nine NaVs. By contrast, the genome of the fruitfly has just one Nav channel homolog, encoded by paralytic.48,49 This relative simplicity facilitates a combination of genetic, pharmacological, electrophysiological and behavioural techniques to be used in the fly to understand the mechanistic basis of INap and its contribution to seizure. Prior exposure of sda seizure mutant larvae to typical antiepileptics (e.g. phenytoin or gabapentin) reduced both INap and paralysis following electroshock, suggesting an association between the two. Molecular analysis of the expressed Nav between wild-type and sda showed a number of changes to the coding sequence as a direct consequence of differential alternative splicing in the two differing genetic backgrounds.50 One change in particular, the choice to include mutually exclusive exons K or L, is linked to the generation of seizure (paralysis) phenotypes. In wild-type larvae, 70–80% of Nav channel transcripts contain exon L (located in homology domain IIIS3-4), while the remainder contain exon K (that differs from L by 16 residues). By contrast, the inclusion of L is increased to 100% in sda.51 Heterologous expression of K- or L-containing splice variants in Xenopus oocytes shows that these exons have a marked influence on the magnitude of the INap current expressed. When present, exon L results in channels with a significantly larger INap.50 Thus, a change in alternative splicing of Nav may promote seizure onset in the fly. Accordingly, prior ingestion of phenytoin, which is sufficient to rescue seizure, also reversed the increased inclusion of exon L in sda back to a wild-type level.51 Ingestion of proconvulsants (e.g. picrotoxin) by wild-type larvae is sufficient to induce a seizure phenotype. Electrophysiological analysis in such larvae revealed enhancement of synaptic activity in motor circuitry and, importantly, increased inclusion of exon L in expressed Nav transcripts. Conversely, seizure behaviour was reduced in sda larvae following feeding with GABA, an effect that is associated with both decreased synaptic activity and reduction of inclusion of exon L.51 Taken together, these observations indicate that the mechanism regulating the inclusion of exon L, at the expense of exon K, is activity-dependent. Moreover, increasing activity promotes inclusion of exon L, which results in larger INap which further promotes cell excitability.51 This positive feedback loop provides a possible mechanistic explanation of the clinical observation in which small-scale paroxysmal activity, if left untreated, can directly promote subsequent seizures.52 Pasilla (Ps), a KH domain-containing RNA binding protein, was shown to influence the splicing balance between exons K and L.53 The inclusion of exon K is significantly increased in a Ps loss-offunction mutant, indicating that the presence of Ps is necessary for the inclusion of exon L.50 Thus, treatments that reduce the activity

Please cite this article in press as: Cunliffe VT, et al. Epilepsy research methods update: Understanding the causes of epileptic seizures and identifying new treatments using non-mammalian model organisms. Seizure: Eur J Epilepsy (2014), http://dx.doi.org/10.1016/ j.seizure.2014.09.018

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of Ps should, theoretically, be antiepileptic. To test this possibility, Ps levels were reduced in the sda mutant background. Knockdown of Ps, either by loss of one gene copy (ps+/ ) or expression of psspecific RNAi, was sufficient to diminish both the increased inclusion of exon L and seizure severity attributable to the sda mutation.51 Homologues of Ps in mammals are the neurooncological ventral antigen 1 and 2 splice factors (Nova-1 and Nova-2, respectively). The genomic region in and around exons K and L contain several putative [T/C]CA[T/C] Nova-recognition motifs.53,54 Indeed, comparative analyses concluded that the RNA regulatory maps between Ps and Nova-1/2 are highly conserved between flies and mammals, suggesting that splicing of mammalian Navs might be regulated by Nova proteins.54–56 Moreover, Nova-1/2 are reportedly involved in pilocarpine-induced epilepsy in the rat model, where pilocarpine promotes the appearance of spontaneous recurrent seizures after 1–4 weeks. Sodium current recordings from CA1 pyramidal cells in such animals showed an associated 1.5-fold increase in INap and a switch to burst firing within 1 week.46 Alternative splicing of exon 5 in mammalian Navs (e.g. human Nav1.1) has been shown to be sufficient to modulate inactivation and alter the magnitude of INap of heterologously expressed channels.57 Exon 5 (its variants being 5N and 5A) occupies a congruent region of the channel (homology domain I, S3–S4) to exons K or L (domain III, S3–S4 that includes the voltagesensor) indicative of conservation of function. Importantly, Novarecognition motifs flank exon 5 suggesting that this splicing event, which may influence the magnitude of INap, is regulated by Nova splicing factors. Because of this, an analysis of the Nova proteins, and identification of compounds that affect their activity, may have important consequences for the treatment of human epilepsy. 5. Zebrafish models of epileptic seizures Over the last fifteen years, the zebrafish has emerged as a powerful model vertebrate for the molecular genetic and pharmacological analysis of development, physiology and disease.58 Its forward genetic tractability stems from its relatively short generation time, the large size of clutches of offspring produced by breeding adults, the efficiency of germline mutagenesis, and the ease of maintaining large numbers of adults in a relatively small space. Moreover, the capability to perform large scale forward genetic screens is now matched by the availability of an array of reverse genetic techniques which allow mutations to be introduced into specific genes of interest,59–61 as well as antisense methods for inhibiting the expression of specific genes.62 The accessibility and transparency of its externally developing embryos and larvae also facilitate the visualisation of gene expression patterns in live organisms using fluorescent reporter transgenes. Importantly, the range of molecular tools available for these high resolution live imaging studies has recently been expanded to include genetically-encoded fluorescent calciumindicator GCaMP proteins, which can reveal the spatio-temporal activities of excitable cells such as neurons and muscle, in intact, living zebrafish embryos and larvae.63–65 The ease with which chemicals can be added to the water in which zebrafish develop and swim readily facilitates pharmacological interventions. The recent development of high-speed video tracking systems allow larval and adult swimming behaviours to be recorded, measured and analysed in detail. Thus, combining such pharmacological and behavioural techniques with imaging technology, mutations and transgenes offers the possibility of elucidating the neural mechanisms that regulate a remarkable range of normal and pathological behavioural phenotypes, including fear, aggression, sociality, reproductive dominance and learning, as well as anxiety, addiction, ADHD and epilepsy.66,67 The small size of the embryonic and larval stages, combined with the many advantages listed above,

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makes the zebrafish particularly attractive for high-throughput in vivo chemical screens for novel bioactive molecules in a high density, multi-well plate format. This combination of intrinsic biological features, together with the tools and technologies now available for the zebrafish, will enable powerful new approaches for investigating nervous system development, physiology and disease processes, as well as facilitating the discovery of new therapeutic drugs. Exposure of zebrafish embryos, larvae or adults to classical chemical convulsant agents, such as the GABAA receptor inhibitor Pentylenetetrazole (PTZ), rapidly induces vigorous locomotor convulsions, along with epileptiform electrical discharges in the brain that can be monitored by local field potential recordings from electrodes inserted into the tectum68 or resting on the skin.69 These convulsions are highly similar to those elicited by epileptic seizures in rodent brain slices and in human epilepsy patients, and the electrographic analysis of seizures in zebrafish larval stages reveals both large amplitude ictal discharges and small amplitude interictal discharges as components of the convulsant-induced neural activity.68,70 Similar studies in adults confirm that PTZ treatment induces bouts of hyperactivity, spasms, as well as circular and corkscrew swimming behaviour that is accompanied by a large increase in serum cortisol levels, consistent with the induction of stressful epileptic seizures.71 Moreover, an EEG technique has recently been developed for monitoring PTZ-induced seizures in intact adult zebrafish.72 Interestingly, administration of known anticonvulsants efficiently suppresses both convulsions and the epileptiform discharges in convulsant-treated embryos and larvae, further underscoring the similarity of the zebrafish epileptic seizures to those experienced by rodents and human patients.65,68,73,74 One of the hallmarks of seizure onset in mammals is the vigorous and rapid induction of a programme of immediate-early gene transcription in specific regions of the brain.75,76 A similar programme of rapidly induced gene transcription, which includes transcription factors such as cfos and npas4, is observed in the nervous system and skeletal muscle of PTZ-treated zebrafish embryos and larvae.65,68 The biological significance of these transcriptional changes remains incompletely understood, although expression of genes such as cfos is neuroprotective in rodents,77 and in cultured mammalian cells, npas4 promotes the formation of GABAergic inhibitory synapses and regulates the balance between excitatory and inhibitory activity within neural circuits,78,79 suggesting that at least some of these genes may play protective, homeostatic functions that limit the adverse, potentially excitotoxic effects of seizures. Seizure-induced gene expression can be readily detected in zebrafish embryos by whole-mount in situ hybridisation, which provides convenient markers for the onset of seizures that are particularly useful for high throughput chemical screens to identify compounds with anticonvulsant activities.65 The regulatory relationships and spatio-temporal in vivo expression patterns of many seizure-induced genes remains poorly characterised, and so further studies of these genes in zebrafish could enable their in vivo functional interrelationships to be determined relatively rapidly. Zebrafish models of specific epilepsies have potential for understanding disease mechanisms and identifying therapeutics. Inherited childhood epilepsy syndromes are often particularly intractable conditions. These rare syndromes have a major impact on the patients and their families but for many of these disorders, little is known about how the causative mutation results in epilepsy or the other disease symptoms. In the following sections we provide an overview of the zebrafish models of inherited epilepsies that have been generated to date. Late Infantile Neuronal Ceroid Lipofuscinosis (LINCL) is associated with progressive neurodegeneration in children, and is perhaps one of the most challenging types of inherited epilepsy

Please cite this article in press as: Cunliffe VT, et al. Epilepsy research methods update: Understanding the causes of epileptic seizures and identifying new treatments using non-mammalian model organisms. Seizure: Eur J Epilepsy (2014), http://dx.doi.org/10.1016/ j.seizure.2014.09.018

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syndrome to find a treatment for. Recently, a larval zebrafish model of LINCL was developed. Also known as CLN2 disease, LINCL is caused by loss-of-function mutations in the CLN2 gene encoding Tri-peptidyl peptidase 1 (TPP1),80 and interestingly, the majority of symptoms and pathologies found in LINCL are also present in the zebrafish tpp1 mutant.81 Thus, tpp1 homozygous mutant larvae at 72 hours post-fertilisation (hpf) exhibit much more frequent and prolonged bouts of movement than wild-type siblings. At later stages, tpp1 mutants become immobile and unresponsive to touch, making the detection of seizures more challenging. However, a non-invasive electroencephalography method has recently been used to demonstrate that epileptic seizure-like activity is readily detectable within the tpp1 mutant CNS (C. Russell, unpublished). Autosomal Dominant Partial Epilepsy with Auditory Features (ADPEAF) is caused by mutation of the LGI1 gene, of which there are two orthologues in zebrafish, lgi1a and lgi1b. Morpholinomediated knockdown of lgi1a increased basal locomotor activity and enhanced the convulsive movements elicited by exposure to PTZ,82 whereas morpholino-mediated knockdown of lgi1b did not increase basal locomotor activity of zebrafish larave, but did cause sensitisation to a PTZ-induced seizure-associated locomotor convulsive behaviour.83 Lowe Syndrome is an X-linked Oculocerebrorenal syndrome caused by mutation of a gene encoding OCRL1, a type II inositol polyphosphate 5-phosphatase involved in phosphoinositide metabolism, and characterised by multiple neurological abnormalities, including cystic lesions in the brain and seizures. ocrl1 homozygous mutant adult zebrafish develop gliosis and cystic brain lesions reminiscent of those in Lowe Syndrome patients, and mutant larvae exhibit temperature-sensitive electrographic seizure-like activity in the developing brain.84 The development of a larval zebrafish model for Epilepsy, Ataxia, Sensorineural deafness and Tubulopathy (EAST) syndrome,85 also known as Seizures, Sensorineural deafness, Ataxia, Mental retardation, and Electrolyte imbalance (SeSAME) syndrome,86 offers new hope for improved understanding of the pathogenetic mechanisms underlying this disorder. This syndrome is caused by mutations in an inwardly-rectifying potassium channel called KCNJ10 (previously known as Kir4.1), which most likely cause partial loss-of-function phenotypes. Recent experiments using an antisense morpholino to knock-down kcnj10 function in zebrafish revealed that morphant larvae exhibited excessive spontaneous tail movements at 30 hpf, and by 120 hpf, abnormally long bursts of locomotion during free-swimming that were followed by a loss of posture.87 Subsequent EEG recordings demonstrated that this zebrafish model responded positively to pentobarbitone but not diazepam,69 thus illustrating the relevance of these studies to understanding epilepsy mechanisms and identifying treatments. Mutations in genes encoding KQT-like potassium voltage-gated channel subfamily members KCNQ2 and KCNQ3 are associated with Benign Familial Neonatal Convulsions, which although not fatal, persist throughout life. Pharmacological inhibition of these channels elicited burst-like electrical discharges in the brain, as did microinjection of morpholinos targeted against the zebrafish orthologues of either KCNQ2 or KCNQ3.88 Burst-like discharges are also typical of the zebrafish Notch pathway mutant, mindbomb, which encodes an E3 Ubiqitin Ligase that is related to the gene affected in Angelman Syndrome, in which seizures are a characteristic feature.89 Functional deficits in voltage-gated sodium channels are implicated in many epilepsies, and mutations in the human sodium channel subunit gene SCN1A cause Dravet Syndrome, a severe, intractable form of childhood epilepsy. Recently, a loss-offunction recessive mutation in a zebrafish orthologue of SCN1A was described.90 The usefulness of this genetic model for anti-epileptic

drug discovery was exemplified through the identification of the FDA-approved, histamine antagonist Clemizole as a potent anticonvulsant, in a drug re-profiling screen carried out with the scn1Lab mutant.90 Such in vivo re-purposing screens of existing drug collections for new therapeutic applications may be a particularly cost-effective and rapid approach to drug discovery, which could complement more established, but costly, de novo lead identification strategies. Taken together, the recent progress in creating a range of new zebrafish models of human epilepsy is helping to advance understanding into the pathogenetic mechanisms underlying this group of devastating neurological disorders, and identify new candidate drugs. As more new mutations are reported in novel human epilepsy genes, the application of techniques for targeted mutagenesis of zebrafish genes, using TALENs and CRISPRs will provide additional models for use in epilepsy research.59,60 Moreover, recent studies showing that mutation of the zebrafish Notch pathway component mind bomb causes epileptic seizures in larvae implicate fundamental developmental mechanisms in epileptogenesis89 and raise the possibility that additional insights from fundamental developmental biology could also help to improve understanding of epilepsy in the future. 6. Summary A broad range of non-mammalian model laboratory organisms have been established as genetically and pharmacologically highly tractable in vivo systems for experimental studies of epilepsy. As discussed in this review, the biological relevance of each of these model systems to human epilepsy has been extensively validated and documented in the research literature. Many non-mammalian organisms were first used in experiments to elucidate developmental mechanisms. In contrast to mammals, these model organisms can be maintained relatively simply and cheaply in the laboratory, and the comparative ease with which mutations in genes of interest can be created, along with the availability of tools for monitoring cell behaviours, make these organisms very amenable to gene function analysis. Their tractability for largescale pharmacological screens also makes them particularly suitable for developing models of human diseases which can then be employed in drug discovery. The non-mammalian models discussed here have distinct strengths and limitations which complement those of mammalian models of epilepsy. Thus, whilst the social amoeba D. discoideum lacks a nervous system and consequently has no physiological equivalent of an epileptic seizure, the great simplicity of this organism has real advantages for elucidating biochemical pathways and the mechanism of drug action. By contrast, the roundworm C. elegans, fruit fly D. melanogaster and zebrafish Danio rerio have nervous systems that exhibit behaviours which are equivalent to epileptic seizures in humans, and their anatomical and physiological simplicity, taken together with their ease of maintenance at relatively low cost, help rather than hinder research efforts to understand disease mechanisms. As illustrated in this review, research using non-mammalian models has the potential to reduce the need for research using physiologically more complex species, but it will not replace all research using mammalian models in the foreseeable future. Nevertheless, the laboratory organisms discussed here are likely to play growing roles in helping to elucidate mechanisms of epileptogenesis and in identifying new classes of antiepileptic drugs. Conflict of interest statement The authors state that there are no known conflicts of interest associated with the publication of this article.

Please cite this article in press as: Cunliffe VT, et al. Epilepsy research methods update: Understanding the causes of epileptic seizures and identifying new treatments using non-mammalian model organisms. Seizure: Eur J Epilepsy (2014), http://dx.doi.org/10.1016/ j.seizure.2014.09.018

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Please cite this article in press as: Cunliffe VT, et al. Epilepsy research methods update: Understanding the causes of epileptic seizures and identifying new treatments using non-mammalian model organisms. Seizure: Eur J Epilepsy (2014), http://dx.doi.org/10.1016/ j.seizure.2014.09.018