Zebrafish Models of Epilepsy and Epileptic Seizures

Zebrafish Models of Epilepsy and Epileptic Seizures

Chapter 26 Zebrafish Models of Epilepsy and Epileptic Seizures Daniëlle Copmans, Aleksandra Siekierska and Peter A.M. de Witte KU Leuven, Leuven, Bel...

761KB Sizes 0 Downloads 116 Views

Chapter 26

Zebrafish Models of Epilepsy and Epileptic Seizures Daniëlle Copmans, Aleksandra Siekierska and Peter A.M. de Witte KU Leuven, Leuven, Belgium

GENERAL DESCRIPTION Biology of the Model Zebrafish (Danio rerio) is a tropical freshwater fish native to South Asia belonging to the Actinopterygii (fish with fins reinforced by spines) that represent the largest class of animals within the vertebrates. The interest in zebrafish as an in vivo nonmammalian vertebrate model for biomedical research, with an initial focus on developmental biology, has been rapidly increasing over the last 25 years (Wyatt et al., 2015). The major difference in the zebrafish development, in comparison to humans, encompasses the fertilization and the further embryogenesis that occurs ex utero. The crucial developmental processes are fully carried out during the first 3 days postfertilization (dpf), and include several steps (Kimmel et al., 1995). Zebrafish eggs undergo incomplete cleavage and, interestingly, the first division occurs around 40 min after fertilization, a process repeated every 15 min. Conversely, in humans this process is among the slowest, as it occurs only 24 hours postfertilization (hpf), with subsequent cleavages 12–24 h apart (Gilbert, 2000). Further, after 24 hpf, the pharyngula stage of the zebrafish embryo begins, and refers to the period when the growing organism possesses the classic vertebrate body plan. At 3 dpf morphogenesis is mostly completed and the larval stage begins. From 30 dpf (1 month) onwards, zebrafish are considered to be juveniles, and from 90 dpf (3 months of age) on they are adults (Kimmel et al., 1995; Westerfield, 2000). The laboratory zebrafish have a mean lifespan of 3.5 years, but can live up to 5.5 years (at least 50% longer than in commonly used mouse strains) (Gerhard et al., 2002). The breeding is most optimal between 7 and 18 months of age (Westerfield, 2000), which is why zebrafish are typically kept in the laboratory until 18–24 months. Sex determination in zebrafish is a complex process. It is primarily genetic; however, in contrast to mammals, it is Models of Seizures and Epilepsy. http://dx.doi.org/10.1016/B978-0-12-804066-9.00026-2 Copyright © 2017 Elsevier Inc. All rights reserved.

not based on the action of sex chromosomes, but rather on a mechanism of polygenic sex determination (PSD), that is, multiple autosomal factors have an effect on the direction of gonad differentiation (Liew and Orban, 2014). Interestingly, at 10–12 dpf the gonads begin to differentiate into ovaries, irrespective of the individual’s genotypic sex. Only at around 3–4 weeks post fertilization (wpf) the ovaries in future males undergo a transformation to testis (Uchida et al., 2002). As a result, research on zebrafish larvae is not sex-specific.

Zebrafish as a Model of Human Diseases Nowadays, zebrafish is widely recognized as an important organism for modeling human diseases. Particularly, in the past decade, it has emerged as a valuable model in the field of epilepsy and drug discovery. The main advantages that underlie the ever-increasing interest are outlined further. l

Zebrafish are vertebrates that are highly conserved with regard to the human reference genome. For instance, 69% of zebrafish protein-coding genes have at least one human orthologue, and 82% of the genes that are involved in human genetic disorders have at least one zebrafish orthologue (Howe et al., 2013). l Adult zebrafish are small (c. 3 cm long), robust, and hence easy to keep in the laboratory without the need for highly specialized equipment. Since they exhibit high fecundity, large amounts of eggs can be collected easily, on a daily basis, and in a cost-effective way (Zon and Peterson, 2005). l Zebrafish embryos and larvae develop ex utero very quickly, and consequently most organ systems are fully functional by 5 dpf. Moreover, zebrafish embryos are transparent until 24 hpf, and therefore can be monitored through the developmental stages under a simple dissection microscope (Kimmel et al., 1995; Westerfield, 2000).

369

370 PART | IV  Non-Mammalian In Vivo Models

l

Since larvae between 3–7 dpf have a body length of c. 3.5–4.5 mm, they can easily be arrayed in microtiter plates, enabling high-capacity testing of therapies for human diseases (Tamplin et al., 2012). Compounds can simply be added to the water and, in general, molecules that exhibit logP values above 1 are expected to have a good bioavailability in zebrafish larvae (Milan et al., 2003), probably by means of dermal and enteral absorption (Nishimura et al., 2015). l Zebrafish can be easily manipulated genetically using various technologies, such as morpholino (MO)-based gene knockdown (KD) (Nasevicius and Ekker, 2000), transgenesis, and genome editing using CRISPR/Cas9 methodology (Bedell et al., 2012). Moreover, the Zebrafish Mutation Project (ZMP) at the Sanger Institute has led to the creation of knockout alleles in proteincoding genes in the zebrafish genome, and more than 10,000 alleles are currently available (Kettleborough et al., 2013).

models that truly reflect the pathogenesis and characteristics of the different human epilepsies are required. Among current zebrafish epilepsy models, genetic models based on morphant and mutant animals have been reported. In this case, the epileptic syndrome is driven by a gene knockdown (using morpholino antisense oligomers), by a chemically induced mutation (as a result of a randomly acting mutagenic agent), or by a gene knockout (using genetic tools like CRISPR/Cas9) in the epilepsy gene of interest (Fig. 26.1). Importantly, when engineering and phenotypically investigating a genetic epilepsy model, one should take into account that zebra­fish possess paralogs for many human genes. For example, in case of scn1lab mutant zebrafish, homozygous but not heterozygous mutants mimic Dravet syndrome (DS), a severe epilepsy syndrome that starts within the first year of life, and is caused by a heterozygous SCN1A mutation in humans (Baraban et al., 2013). This discrepancy can be e­ xplained by the zebrafish paralog scn1laa that partially compensates the scn1lab mutation.

METHODS OF GENERATION

Zebrafish Seizure Models

General Aspects

To generate a chemically-induced zebrafish seizure model, zebrafish are exposed to different concentrations or doses of a known or novel convulsant drug. Often the drug is added to the swimming water (Baraban et al., 2005; Leclercq et al., 2015; Winter et al., 2008), this being most suitable for high-throughput purposes. Another route of drug administration can be injection, for example, into the yolk sac at the embryonal or larval stage (Tiedeken and Ramsdell, 2009), or intraperitoneally in case of adult fish (Alfaro et al., 2011). To generate a hyperthermia-induced zebrafish seizure model, zebrafish are exposed to different temperatures and rates of temperature elevation. This can be done by means of a controlled heating device, and should be precisely monitored in proximity of the zebrafish. The drug concentration/dose or hyperthermia condition that reproducibly induces seizures after a short latency time, and with low lethality and seizure-unrelated toxicity, is selected as the optimal one to complete phenotypical characterization of the model (see Methods of characterization).

The first zebrafish model in the field of epilepsy was reported in 2005 (Baraban et al., 2005). This chemically induced model relies on the convulsant pentylenetetrazole (PTZ), and remains popular up to this day for the rapid identification of anticonvulsant hits (Baxendale et al., 2012; Buenafe et al., 2013; Long et al., 2014). Ever since, a range of diverse zebrafish models has been developed that, similarly to rodent models (Loscher, 2011), can be classified as models of epileptic seizures and epilepsy models. In models of epileptic seizures, seizures are induced either chemically by a convulsant drug, or by hyperthermia that refers to an elevated body temperature and can occur in humans in case of fever or when taking a hot water bath. Zebrafish seizure models are ideally suited for antiseizure drug (ASD) discovery and to study ictogenesis, but do not capture the entire disease process. When studying epilepsy and screening for antiepileptogenic drugs (AED), epilepsy

FIGURE 26.1  Classification of current zebrafish models of epilepsy and epileptic seizures. Models of epileptic seizures can be induced chemically by a convulsant drug, or by hyperthermia. Epilepsy models can be grouped into genetic mutant models and genetic morphant models.

Zebrafish Models of Epilepsy and Epileptic Seizures Chapter | 26

Genetic Zebrafish Models of Epilepsy Morpholino-Based Gene Knockdown The majority of current genetic zebrafish epilepsy models were generated using MOs (Nasevicius and Ekker, 2000). MOs are short antisense morpholino oligonucleotides that are synthetically derived from DNA, but have a six-­ membered morpholine ring instead of the deoxyribose ring, and a nonionic phosphorodiamidate linkage instead of the anionic phosphodiester bond. They target the RNA of interest by complementary base pairing and sterically hinder translation or splicing, resulting in a full or partial gene knockdown. Due to the small size and neutral charge, MOs diffuse through the embryo after microinjection in the 1–2 cell-stage (Bedell et al., 2011; Bill et al., 2009). The low cost, high efficacy, and the immediate availability of morphants for experiments made MOs popular tools for direct functional investigation of genes in zebrafish (Ekker, 2000). However, in recent years, MOs were criticized for their capacity to induce off-target effects (Law and Sargent, 2014; Schulte-Merker and Stainier, 2014). Therefore, generation of a morpholino-based model requires its validation with the adequate controls for correct interpretation of obtained results. In brief, MO dosing should be standardized for optimal efficacy, without overt off-target effects, a p53 MO can be coinjected to overcome the common off-target effect of p53-dependent neural toxicity (Robu et al., 2007), and proper control MOs should be used. Phenotypic validation requires at least a second MO that targets an independent nonoverlapping sequence. The efficacy of splice-blocking MOs should be demonstrated by qPCR analysis of the target, whereas for translational-blocking, MOs efficacy can be shown by a western blot or immunohistochemical methods, if a specific antibody is available. Moreover, reversal of the morphant phenotype by an RNA rescue (i.e., coinjection of MO with wild type target gene mRNA, or Tol2 transient transgenesis for a long-term rescue) (Kawakami et al., 2000; Schubert et al., 2014) is the golden standard for validation of MO specificity. However, it might not always be possible if the target gene is too large or too complex to be cloned, or is not commercially available as a ready-touse clone. The definitive control for a morpholino knockdown is to correlate the morphant phenotype with the phenotype of a corresponding mutant (Eisen and Smith, 2008), as was the case for the scn1lab morphant and mutant epilepsy model (Baraban et al., 2013; Zhang et al., 2015). Another validation can be to inject a MO against a specific epilepsy gene into a null genetic background that should not lead to any abnormal phenotype (Stainier et al., 2015).

Genome Editing Technologies The current trend is to develop stable genetic models of human diseases using recent advances in genome engineering technologies that enable fast, easy, and affordable targeted

371

editing of the genome in zebrafish. In recent years, genome editing techniques, such as zinc finger nucleases (ZFNs) (Doyon et al., 2008; Gupta et al., 2012; Meng et al., 2008) or transcription activator-like effector nucleases (TALENs) (Bedell et al., 2012; Huang et al., 2011; Sander et al., 2011) have been shown to work well in zebrafish. Most recently, a more straightforward method has been developed, based on the clustered regulatory interspaced short palindromic repeats (CRISPR) mechanism of bacterial defense against invading foreign exogenous DNA, caused by type II CRISPR-associated nuclease (Cas9) (Wiedenheft et al., 2012). A short guide RNA (gRNA) complexed to Cas9 endonuclease binds to its complementary DNA target sequence, and leads to specific cleavage by Cas9, causing specific double strand breaks in a DNA target (Sander and Joung, 2014). By redesigning the gRNA sequence, one can retarget the Cas9 nuclease to cleave virtually any DNA sequence (Cho et al., 2014; Mali et al., 2013). The most suitable target regions for mutagenesis are usually exons found toward the 5′ end of the gene that will most likely cause a strong loss-of-function, but optionally the strategy can also be tailored to the type of causative human mutations identified in specific genes in patients. Zebrafish embryos can be injected with gene specific gRNA directly into the blastomere of early one-cell stage embryos. Next, batches of positive mutant fish are raised to adulthood in order to screen CRISPR-injected founders for germline transmission of potential mutants. Adult F1 zebrafish are then fin-clipped and genotyped in order to identify the mutated carriers. Later on, if necessary, they can be incrossed with F1 adult fish carrying the desired lesion to identify homozygous mutants in F2, if viable and fertile. The CRISPR/Cas9 method has been successfully used in zebrafish to perform efficient and specific knockout of target proteins of interest (Chang et al., 2013; Hwang et al., 2013a,b; Jao et al., 2013). Of particular interest is the recent report of the first CRISPR-based zebrafish model of epilepsy, that is, the homozygous stxbp1b mutant that displays spontaneous electrographic seizures (Grone et al., 2016). Many more CRISPR-based epilepsy models are expected soon, as the availability of this straightforward genome editing technique is spurring the generation of mutant zebrafish lines for epilepsypredisposing genes. Finally, CRISPR efficacy can be investigated at the early stages of mutant line generation by using T7 endonucleases that recognize and cleave nonperfectly matched DNA (Vouillot et al., 2015). If needed, evaluation of specificity can be performed either by assessing if unrelated gRNAs that target different sites of the same gene of interest result in the same mutant phenotype, or by rescuing the mutant phenotype by the introduction of the wild type protein (e.g., by injecting wild type mRNA), or Tol2 transient transgenesis for a long-term rescue (Kawakami et al., 2000).

372 PART | IV  Non-Mammalian In Vivo Models

METHODS OF CHARACTERIZATION General Aspects Commonly, a zebrafish epilepsy or seizure model is typified by its seizure-like behavior. In case of clonus-like convulsions, seen in many zebrafish models, behavioral characterization shows an increased locomotion. Importantly, increased locomotion can also result from a peripheral sensory neuron response, observed, for example, when larvae are exposed to cinnamon oil or mustard oil (Cousin et al., 2014). Therefore, validation of seizure-like behavior by a nonbehavioral seizure or epilepsy biomarker is absolutely required. Ideally, this is done by studying an electrophysiological biomarker (Engel et al., 2013), for example, by examining brain activity for the presence of recurrent epileptiform-like discharges using local field potential (LFP) recordings. Alternatively, the upregulated expression of molecular biomarkers can also be monitored by real time qPCR or WISH (Baraban et al., 2005). Then, the validated model can be pharmacologically characterized with clinically relevant ASDs or therapies to evaluate its applicability for drug discovery (Fig. 26.2). Mostly, 7 dpf larvae have been phenotypically characterized. They offer a fully functional small vertebrate with a developed central nervous system (CNS) that fits into the well of a 96-well plate. They are particularly suitable for drug discovery, and for disease modeling. Conversely, adult animals placed individually in an observational tank have

also been studied (Mussulini et al., 2013; Wong et al., 2010). They cannot be used for high-throughput purposes, but can extend and complement larval models, as they possess a fully mature nervous and endocrine system, and more complex locomotor behavior.

Behavioral Characterization The epileptic behavioral phenotype of zebrafish larvae can be monitored using automated video-tracking devices (simultaneously recorded by a top view camera) that allow a quantitative analysis of locomotor activity. Examples of such commercially available systems for zebrafish research include ZebraLab (created by ViewPoint, France) or Ethovision (produced by Noldus IT, Netherlands). Individual larvae can be monitored per well of a 96-well plate (Afrikanova et al., 2013; Baraban et al., 2005; Dinday and Baraban, 2015; Orellana-Paucar et al., 2012) to measure: total larval movement, total distance traveled, velocity, freezing, and burst activity. Locomotor behaviors can be monitored either without an additional treatment (i.e., spontaneously occurring seizures), or with cotreatment of subthreshold doses of convulsant compounds (i.e., increased seizure sensitivity). In case larvae display a series of more subtle seizure behaviors (e.g., atonic seizures, myoclonic twitches, tremor, and absences) that are difficult to quantify with commercially available systems, a high-resolution video camera

FIGURE 26.2  Phenotypic characterization of zebrafish models of epilepsy and epileptic seizures. Standard phenotypic characterization of zebrafish models of epilepsy and epileptic seizures starts with visual or automated observation and quantification of seizure-like behavior, for example, convulsive movements, atonic periods, jerking, and spasm. Next, to validate the behavioral phenotype, the presence of nonbehavioral seizure or epilepsy biomarkers is investigated, for example, recurrent epileptiform-like discharges (electrophysiological biomarker), and/or expression of molecular biomarker(s), such as c-fos level in the brain (unpaired t-test, **P ≤ 0.01). Finally, the validated model is pharmacologically characterized with clinically relevant antiseizure drugs, antiepileptogenic drugs, or therapies to evaluate its applicability. Data shown from 7 dpf zebrafish larvae incubated with vehicle (VHC) or 20 mM pentylenetetrazole (PTZ).

Zebrafish Models of Epilepsy and Epileptic Seizures Chapter | 26

can be employed in combination with manual scoring based on visual observation (Baraban et al., 2005; Mahmood et al., 2013).

Electrophysiological Biomarker A golden standard in clinical diagnosis of epilepsy, and ideally an essential characteristic of any animal model of epileptic seizures and epilepsy, is the presence of abnormal electrical discharges in the CNS. Zebrafish equivalent to human EEGs are LFP recordings that measure the electric potential (signal) generated by the summed electrical current coming from the extracellular space around neurons (Buzsaki et al., 2012). A glass electrode, connected to a high-impedance amplifier, is placed in or on larva’s brain structures for invasive (Baraban et al., 2005) or noninvasive LFP recordings (Zdebik et al., 2013), respectively. This allows registering of neuronal activity, and subsequently quantification of epilepsy parameters, that is, seizure onset, duration, clustering, frequency, and amplitude. LFPs are typically performed from the optic tectum (Afrikanova et al., 2013; Leclercq et al., 2015; Schubert et al., 2014) or forebrain (Baraban et al., 2013; Grone et al., 2016; Zhang et al., 2015) of zebrafish larvae immobilized in 1.2%–2% low-melting point agarose. If desirable, the occurrence of movement artefacts can be abolished by paralyzing the larvae with D-tubocurarine or α-bungarotoxin (Grone et al., 2016; Zdebik et al., 2013). Electrophysiology techniques have also been described for the recording of electrographic seizures in adult zebra­ fish (Johnston et al., 2013; Pineda et al., 2011). Similarly to the larvae, LFPs can be measured using a single recording electrode placed in the optic tectum of the anaesthetized fish (Johnston et al., 2013). In the study of Pineda and colleagues, so-called cerebral field potential (CFP) recordings were measured across the forebrain by the invasive placement of two recording electrodes in the anaesthetized fish (Pineda et al., 2011).

Molecular Biomarkers A commonly used molecular biomarker for epilepsy in zebrafish is c-fos, an early protooncogene and marker of neuronal activation (Hoffman et al., 1993). For instance, in a PTZ-induced zebrafish seizure model, an increase in the expression of c-fos mRNA was observed that was particularly upregulated in the optic tectum and cerebellum (Baraban et al., 2005). Promising new biomarkers are the neuronal PAS domain-containing protein 4 (npas4) and sestrin 3 (sesn3) that have been evaluated in zebrafish. The Npas4 gene was identified in rodents as an important regulator of synaptic plasticity and memory. The expression of its zebrafish homolog, npas4a gene, was shown to be restricted to the forebrain

373

during embryonic development, where it is upregulated in response to neuronal activity. Furthermore, knockdown of npas4a resulted in forebrain-specific defects including increased apoptosis and misexpression of some specific forebrain marker genes (Klaric et al., 2014). Sestrin 3 was recently discovered as a regulator of convulsant gene network in human epileptic hippocampus (Johnson et al., 2015). sesn3 normally regulates the levels of intracellular reactive oxygen species, and was shown to be broadly expressed in the brain of zebrafish larvae. It was demonstrated that sesn3 modulates locomotor convulsive behavior and expression of c-fos in the brain of a PTZ-induced zebrafish seizure model, that is, sesn3 MO knockdown attenuated PTZ-induced locomotor hyperactivity, and transcriptional response of c-fos (Johnson et al., 2015). Finally, it is also possible to monitor globally seizureinduced changes in gene expression by performing wholetranscriptome analysis, as was already performed for the zebrafish mind bomb mutant (Hortopan et al., 2010) and scn1lab zebrafish mutant (Baraban et al., 2013).

CHARACTERISTICS AND DEFINING FEATURES Chemically-Induced Zebrafish Seizure Models An overview of published zebrafish models of chemically induced epileptic seizures is given in Table 26.1. For all convulsants listed, their ability to induce seizure-like behavior was demonstrated in zebrafish larvae and/or adults. With the exception of the study of Alfaro et al. (2011), who injected kainate in adults intraperitoneally, seizures were induced by water immersion of the drug. Seizures are acute, and are regularly described as recurrent clonus-like convulsions, proceeded by high-speed (circular) movements, and followed by a loss of posture. This is the case for the larval and adult PTZ seizure model (Baraban et al., 2005; Mussulini et al., 2013), the adult kainate model (Alfaro et al., 2011), and the larval allylgycine model (Leclercq et al., 2015). In addition, seizures have been described as spasms, twitching, jerking, or tremor. These include the larval domoic acid seizure model (Tiedeken and Ramsdell, 2009), the adult strychnine seizure model (Stewart et al., 2012), the adult picrotoxin model (Wong et al., 2010), the larval ginkgotoxin model (Lee et al., 2012), the larval pilocarpine model (Vermoesen et al., 2011), and the larval linopirdine and XE991 model (Chege et al., 2012). Moreover, the adult caffeine seizure model (Wong et al., 2010) and the adult 1,3,5-trinitroperhydro-1,3,5-triazine (RDX) model (Williams et al., 2012) also display episodes of freezing. The latency time and persistence of the induced seizures depends on the selected convulsant and the concentration/ dose used. In case of exposure of larvae to 15–20 mM PTZ,

Convulsant

Concentration

Developmental Stage

Functional Target or Function

Seizures

Nonbehavioral Seizure Biomarker

PTZ

15–20 mM

7 dpf

GABAA-R

Yes

Upregulated c-fos, epileptiform discharges

Baraban et al. (2005); Afrikanova et al. (2013)

PTZ

5–15 mM

Adult

GABAA-R

Yes

Upregulated c-fos, epileptiform discharges

Wong et al. (2010); Pineda et al. (2011); Stewart et al. (2012); Mussulini et al. (2013)

Domoic acid

360 µM

7 dpf

AMPA-R, Kainate-R

Yes

NI

Tiedeken and Ramsdell (2007, 2009)

4-Aminopyridine

450 µM

7 dpf

GLUT-R, K-channel

Yes

Epileptiform discharges

Baraban et al. (2007); Winter et al. (2008)

Maprotiline

14 µM

7 dpf

Histamine, mACh-R

Hyperactivity

NI

Winter et al. (2008)

Bemegride

220 µM

7 dpf

GABAA-R

Yes

NI

Winter et al. (2008)

Bicuculline

900 µM

7 dpf

GABAA-R

Yes

NI

Winter et al. (2008)

Physostigmine

200–500 µM

5 and 7 dpf

Cholinesterase

Yes

Epileptiform discharges

Winter et al. (2008); Kim et al. (2010)

Amoxapine

14 µM

7 dpf

GABAA-R, Ca

Yes

NI

Winter et al. (2008)

Enoxacin

450 µM

7 dpf

GABAA-R

Yes

NI

Winter et al. (2008)

Semicarbazide

900 µM

7 dpf

Amine oxidase

Yes

NI

Winter et al. (2008)

Aminophylline

450 µM

7 dpf

Adenosine

Yes

NI

Winter et al. (2008)

Acetylsalicylic acid

1.8 mM

7 dpf

Hypoglycemia, metabolic acidosis

Hyperactivity

NI

Winter et al. (2008)

Picrotoxin

30 µM

7 dpf

GABAA-R

Yes

NI

Winter et al. (2008)

Picrotoxin

170 µM

Adult

GABAA-R

Yes

NI

Wong et al. (2010)

Strychnine

References

3.5 µM

7 dpf

Glycine-R

Yes

NI

Winter et al. (2008)

Strychnine

15 µM

Adult

Glycine-R

Yes

NI

Stewart et al. (2012)

Caffeine

1.3 mM

Adult

Adenosine-R A1 and A2A

Yes

NI

Wong et al. (2010)

Pilocarpine

30–60 mM

3 and 7 dpf

mACh-R

Yes

Upregulated c-fos

Vermoesen et al. (2011); Lopes et al. (2016)

Kainate

6–8 mg/kg

Adult

Kainate-R

Yes

NI

Alfaro et al. (2011)

Kainate

50–500 µM

5, 7, and 15 dpf

Kainate-R

Hyper- and hypoactivity

Epileptiform discharges

Kim et al. (2010); Menezes et al. (2014)

Ginkgotoxin

0.2–1 mM

3 and 5 dpf

GABA synthesis

Yes

NI

Lee et al. (2012)

RDX

1 mM

Adult

GABAA-R

Yes

Upregulated c-fos

Williams et al. (2012)

Linopirdine

100 µM

3–7 dpf

Kv7 channels

Yes

Epileptiform discharges

Chege et al. (2012)

XE991

100 µM

3–7 dpf

Kv7 channels

Yes

Epileptiform discharges

Chege et al. (2012)

Allylglycine

300 mM

7 dpf

GAD enzyme

Yes

Epileptiform discharges

Leclercq et al. (2015)

Overview of zebrafish models of chemically induced epileptic seizures. PTZ, Pentylenetetrazole; RDX, Royal Demolition Explosive; 1,3,5-trinitroperhydro-1,3,5-triazine; dpf, days postfertilization; GAD, glutamic acid decarboxylase; NI, not investigated.

374 PART | IV  Non-Mammalian In Vivo Models

TABLE 26.1 Zebrafish Models of Chemically Induced Epileptic Seizures

Zebrafish Models of Epilepsy and Epileptic Seizures Chapter | 26

seizures occur within few minutes, and persist for hours until the zebrafish dies. Therefore, zebrafish are transferred to drug-free medium or sacrificed after the experiment. Finally, seizure-like behavior can be limited to altered behavior in absence of more specific traits, for example, the larval acetylsalicylic acid model and the maprotiline model that display hyperactivity (Winter et al., 2008), and the larval kainate model that shows a stage-dependent shift from hypo- to hyperactivity. (Menezes et al., 2014) Given the different terms used to describe seizures in zebrafish, and the difficulty to observe subtle events, it is not easy to correlate these behaviors with mammalian seizure types. In general, whole-body contractions seem to be interpreted as clonus-like convulsions and loss of posture or freezing as tonus-like events. Remarkably, only few chemical seizure models were investigated for the presence of a nonbehavioral seizure biomarker, that is, recurrent epileptiform discharges or significant upregulation of c-fos expression. This validation was only described for PTZ, 4-aminopyridine, physostigmine, pilocarpine, kainate, RDX, linopirdine, XE991, and allylglycine (Baraban et al., 2005, 2007; Chege et al., 2012; Kim et al., 2010; Leclercq et al., 2015; Lopes et al., 2016; Williams et al., 2012). Finally, only few studies focused on the long-term phenotype of zebrafish exposed to a convulsant (Menezes et al., 2014; Tiedeken et al., 2005). For instance, the study of Tiedeken and coworkers reported the occurrence of spontaneous seizures in 2 dpf embryos and 5 dpf larvae that were injected with domoic acid at the 128- to 512-cell stage (Tiedeken et al., 2005). Their data suggest a comparable sensitivity of zebrafish and rodents to the developmental toxicity of domoic acid, and is to our knowledge the first exploration of a chronic zebrafish seizure model.

Hyperthermia-Induced Zebrafish Seizure Models Up to now, only one zebrafish model of hyperthermia-induced seizures has been characterized (Hunt et al., 2012). Hunt and colleagues measured epileptiform discharges from the forebrain of agar-immobilized 3–7 dpf larvae that were exposed to an increase in bath temperature. The agar temperature rose from ±22 to ±33.5°C in 4–5 min, and after that the temperature slowly returned to baseline. The first epileptiform discharge was observed at a temperature of 25.5°C, and was a polyspiking, large-amplitude, longduration event. Discharges occurred closely thereafter, and progressed into high-frequent, small-amplitude, short-duration events. These acute electrographic seizures occurred for 2–6 min and were age-dependent with a peak at 5 dpf. Moreover, seizures were reproducible, did not show strain differences, and were not lethal, thereby capturing the key characteristics of mammalian febrile seizures.

375

In comparison to rodent models, this zebrafish model can be easily used to study hyperthermia-induced seizures in the developing brain. This was also demonstrated by the functional investigation of TRPV4 channels and NMDA-type glutamate receptors. The authors did not mention a long-term phenotypical investigation of the hyperthermia-exposed larvae. Behaviorally, seizures were not investigated, as the larvae were paralyzed to obtain stable electrographic recordings. Within zebrafish epilepsy models, an increased susceptibility to hyperthermia-induced seizures has been observed for the ocrl1 mutant model (Ramirez et al., 2012) and the stx1b morphant model (Schubert et al., 2014). Furthermore, hyperthermia-induced abnormalities were found in the scn1lab morphant model (Zhang et al., 2015) (Table 26.2).

Genetic Zebrafish Models of Epilepsy An overview of published zebrafish epilepsy models is given in Table 26.2. Although some epilepsy-relevant mutants were found in forward genetics screens using various mutagenesis methods (Baraban et al., 2013; Hortopan et al., 2010; McKeown et al., 2012; Ramirez et al., 2012), and one ZFNs-based model (Hoffman et al., 2016) and one CRISPR/Cas9-based mutant were studied (Grone et al., 2016), the majority of current genetic zebrafish epilepsy models were generated using MOs (Nasevicius and Ekker, 2000). An exponential increase in the number of genetic epilepsy models is to be expected given the successful use of CRISPR/Cas9 genome editing. Taken together, the results show that genetic stable mutant and morphant zebrafish are an excellent model for clinically-relevant epilepsies, as they recapitulate key features of the human condition. In most cases, seizure-like behavior was described and confirmed by the presence of spontaneous recurrent epileptiform discharges. Seizures are often described as recurrent high-speed movements or hyperactivity, accompanied by whole-body convulsions or jerking. This is the case for the mind bomb mutant (Hortopan et al., 2010), the lgi1a morphant (Teng et al., 2010), the scn1lab mutant (Baraban et al., 2013), and the scn1lab morphant (Zhang et al., 2015). Also, the kcnj10a morphant showed high-speed movements, but associated with ataxia, and loss of posture (Mahmood et al., 2013; Zdebik et al., 2013). Other descriptions of seizure-like behavior are more subtle, like the ocrl1 mutant that displayed occasional twitching and rigor (Ramirez et al., 2012), the chd2 morphant that showed frequent whirlpool-like swimming, whole-body trembling and twitching of the pectoral fin and jaw (Suls et al., 2013), and the stx1b morphant that had repetitive pectoral fin fluttering, increased orofacial movements, and myoclonus-like jerks (Schubert et al., 2014). No spontaneous seizures were described for the lgi1b (Teng et al., 2011), the pk1a morphant (Mei et al., 2013),

Human Gene

Functional Target or Function

Seizures

Nonbehavioral Epilepsy Biomarker

References

Retroviral insertional mutagenesis

KIAA1323

E3 ubiquitin ligase

Yes

Epileptiform discharges, TA

Hortopan et al. (2010)

lgi1a morphant

MOs

LGI1

Synapse transmission

Yes, also sensitized to PTZ

Upregulated c-fos

Teng et al. (2010, 2011)

lgi1b morphant

MOs

LGI1

Synapse transmission

Not observed, but sensitized to PTZ

Not observed, but sensitized to PTZ-induced upregulated c-fos

Teng et al. (2011)

kcnq3 morphant

MOs

KCNQ3

Kv7.3 channel

NI

Epileptiform discharges

Chege et al. (2012)

tnt mutant

ENU mutagenesis

SLC1A2

Glutamate transporter GLT-1

Not observed, but exaggerated body bends and paralysis

Prolonged bursts of motor neurons

McKeown et al. (2012)

ocrl1 mutant

Retroviral insertional mutagenesis

OCRL1

Phosphoinositide 5-phosphatase OCRL1

Yes

Sensitized to heat-induced epileptiform discharges

Ramirez et al. (2012)

pk1a morphant

MOs

PK1

Neuronal migration, axonal outgrowth

NI, but sensitized to PTZ

NI

Mei et al. (2013)

scn1lab mutant

ENU mutagenesis

SCN1A

Nav1.1 channel

Yes

Epileptiform discharges, TA

Baraban et al. (2013)

kcnj10a morphant

MOs

KCNJ10

Kv channel

Yes

Epileptiform discharges

Mahmood et al. (2013); Zdebik et al. (2013)

chd2 morphant

MOs

CHD2

Gene transcription modification

Yes

Epileptiform discharges

Suls et al. (2013)

stx1b morphant

MOs

STX1B

GABA and glutamate release

Yes

Epileptiform discharges

Schubert et al. (2014)

cnnm2a and cnnm2b morphant

MOs

CNNM2

Cyclin M2

Not observed, but increased embryonic contractions

NI

Arjona et al. (2014)

scn1lab morphant

MOs

SCN1A

Nav1.1 channel

Yes

Epileptiform discharges

Zhang et al. (2015)

cntnap2ab mutant

ZFNs

CNTNAP2

Contactin associated protein-like 2

NI, but sensitized to PTZ

NI

Hoffman et al. (2016)

stxbp1b mutant

CRISPR/Cas9

STXBP1

Neurotransmitter release

Not observed

Epileptiform discharges

Grone et al. (2016)

Overexpression of mutant fhf1b1

Tol2 transient ­transgenesis

FHF1

Nav channel

NI

Epileptiform discharges

Siekierska et al. (2016)

Model

Genetic Tool

mind bomb mutant

Overview of genetic zebrafish epilepsy models. MOs, morpholinos; ENU, N-ethyl-N-nitrosourea; ZFNs, zinc finger nucleases; Kv channel, voltage-gated potassium channel; Nav channel, voltage-gated sodium channel; TA, transcriptome analysis was done; NI, not investigated.

376 PART | IV  Non-Mammalian In Vivo Models

TABLE 26.2 Genetic Zebrafish Epilepsy Models

Zebrafish Models of Epilepsy and Epileptic Seizures Chapter | 26

and the cntnap2ab mutant (Hoffman et al., 2016); however, they did show an increased sensitivity for PTZ-induced seizures. The remaining models listed in Table 26.2 were not investigated for seizure traits, or seizures were not observed. For some of these models abnormal behavior was observed that could be associated with seizures (Arjona et al., 2014; McKeown et al., 2012). For others, the presence of a nonbehavioral biomarker was demonstrated, that is in the case of the kcnq3 morphant (Chege et al., 2012), the tnt mutant (McKeown et al., 2012), the stxbp1b mutant (Grone et al., 2016), and the overexpression model of mutant fhf1b1 (Siekierska et al., 2016).

RESPONSE TO ANTIEPILEPTIC DRUGS The convulsant models PTZ, ginkgotoxin, and allylglycine underwent a thorough pharmacological characterization by evaluating the anticonvulsant effects of clinically relevant ASDs (Afrikanova et al., 2013; Baraban et al., 2005; Berghmans et al., 2007; Leclercq et al., 2015; Lee et al., 2012). In brief, Baraban and colleagues showed for the larval PTZ seizure model that carbamazepine (CBZ) and ethosuximide (ETS) had little effect against induced epileptiform discharges, while phenytoin (PHT) and phenobarbital reduced the discharge amplitude, and valproic acid (VPA) and diazepam (DZP) strongly reduced epileptiform discharge (Baraban et al., 2005). This was confirmed by Afrikanova and coworkers, who observed a strong reduction of the number of ictal-like events and the total cumulative duration of epileptiform activity for PTZ exposed larvae that were treated with VPA or DZP (Afrikanova et al., 2013). The same observation was made for treatment with ETS or tiagabine. No significant effect against electrographical seizures was observed with CBZ, gabapentin (GBP), lamotrigine, levetiracetam (LEV), oxcarbazepine, PHT, primidone (PRD), topiramate (TPM), or zonisamide. Also, in the adult PTZ seizure model, VPA and DZP showed a significant reduction of behavioral seizures (Lee et al., 2010; Mussulini et al., 2013). The larval ginkgotoxin model showed a reduction in seizure behavior with PHT, GBP and PRD (Lee et al., 2012). Finally, several ASDs were evaluated in the larval allylglycine model. VPA, DZP, PHT, and TPM could significantly reduce the occurrence and cumulative duration of epileptiform discharges, while LEV did not. Importantly, Afrikanova and colleagues showed a good translation between pharmacological results of behavioral and electrophysiological investigation within the zebrafish model, and between the zebrafish and rodent model (Afrikanova et al., 2013; Leclercq et al., 2015). These studies demonstrate the utility of zebrafish seizure models for ASD discovery. To be complete, the convulsant models linopirdine and XE991 underwent a pharmacological evaluation of retigabine that suppressed both induced behavioral and electrographic seizures (Chege et al., 2012).

377

The pharmacological evaluation of seizures in the hyperthermia seizure model did not involve ASDs, and is therefore not further discussed in this section. With regard to genetic zebrafish epilepsy models, the scn1lab mutant and morphant model, and kcnj10a morphant model were pharmacologically characterized with clinically relevant ASDs (Baraban et al., 2013; Zdebik et al., 2013; Zhang et al., 2015). Epileptiform events of the scn1lab mutant model were reduced by VPA, DZP, potassium bromide, and stiripentol (STP), while acetazolamide and PHT had no effect. As expected, exposure to CBZ, ETS, and vigabatrin increased the seizure frequency. Again, a good translation was observed between pharmacological results of behavioral and electrophysiological investigation within the zebrafish model (Baraban et al., 2013). Also, in the scn1lab morphant model VPA treatment led to a strong reduction in epileptiform discharges (Zhang et al., 2015). Interestingly, the same observation was made for morphants exposed to fenfluramine, a promising new add-on therapeutic in the treatment of DS patients (Ceulemans et al., 2012). Both drugs were also effective in lowering the morphant’s behavioral hyperactivity. Other ASDs tested were STP, TPM, clobazam (CLB), and CBZ. As expected, CBZ had no effect or even increased locomotion, while STP, TPM, and CLB lowered the hyperactivity (Zhang et al., 2015). Finally, pentobarbitone, but not DZP, was reported to reduce significantly electrographic seizures in the kcnj10a morphant model (Zdebik et al., 2013).

USE IN THERAPY AND BIOMARKER DEVELOPMENT As mentioned earlier, zebrafish seizure and epilepsy models are particularly suitable for drug discovery and disease modeling. They can be used as a preliminary model, prior to validation in rodents, for the identification and initial evaluation of new therapies. Up to this day, especially the well-characterized larval PTZ seizure model and scn1lab mutant epilepsy model are commonly used for ASD discovery (Baraban et al., 2013; Baxendale et al., 2012; Buenafe et al., 2013; Challal et al., 2012; Dinday and Baraban, 2015; Long et al., 2014; Orellana-Paucar et al., 2012, 2013; Sourbron et al., 2016). In addition, by identifying genes differentially expressed in epilepsy mutants, one can gain insight into molecular pathways that may mediate these epileptic phenotypes, and get better understanding of how altered expression of certain genes can contribute to specific types of epilepsies and seizures. Hence, the fast generation of novel genetic zebrafish epilepsy models, and the characterization of seizure-induced changes in gene expression by wholetranscriptome analysis could accelerate the development of novel biomarkers.

378 PART | IV  Non-Mammalian In Vivo Models

LIMITATIONS Despite the key advantages, zebrafish also has its limitations in neuroscientific research that should be taken into account. First of all, the advantage of using zebrafish as a medium- to high-throughput model is limited to embryos and larvae, and to a lesser extent to juveniles. It does not apply to adults due to their large size (Delvecchio et al., 2011). Second, although at the genetic level zebrafish are highly similar to humans, they are evolutionarily more distinct than rodents (Stewart et al., 2014). These differences can lead to the so-called zebrafish annotation problem. Most commonly, the annotated activity of small molecules and/ or targets is based on mammalian studies, and although the assumption is made that it will be similar in zebrafish, it is not always the case (Rihel and Schier, 2012). Furthermore, not all brain regions are as developed as in mammals, for example, the cortex (Kalueff et al., 2014). However, this lower complexity can also be an advantage to unravel mechanisms of action. In addition, false positives can arise from the use of larvae at 5–7 dpf for drug discovery, because the blood–brain barrier (BBB) of zebrafish is only mature at 10 dpf (Fleming et al., 2013). Concerning drug discovery, compounds can be problematic to administer to zebrafish in certain cases; for example, water immersion of highly water-insoluble compounds or uptake of compounds with a low bioavailability. This can be anticipated by solubilizing the drug (Kalueff et al., 2014) or by applying different administration routes, for example, injection (into the yolk sac at the embryonal and larval stage, or intraperitoneally or subcutaneously in case of adult fish), or oral (with food, gavage: Collymore et al., 2013; or microgavage: Cocchiaro and Rawls, 2013). Nevertheless, it is likely that certain active compounds would not be identified in a screening setting due to water-insolubility, a possible source of false negative results. Finally, with regard to modeling of human disorders, zebrafish orthologues to single mammalian genes regularly come in paralog pairs that might have complementary functions. This is due to an additional whole-genome duplication in ancient fish ∼ 300 million years ago (Taylor et al., 2003). As a result, a mutagenic inactivation of one of the two genes could possibly lead to an unanticipated mild phenotype. The presence of paralogs therefore might necessitate a second hit to observe a full-blown phenotype in the exploration of a mutation that results in a loss-of-function.

MODEL OPTIMIZATION CONSIDERATIONS Model Characterization: General Aspects During the past decade it became clear that zebrafish epilepsy and seizure models need a thorough characterization, including pharmacological evaluation, before further interrogation

of molecular and cellular pathogenesis is possible, and before drug discovery work comes into the picture. Often, in zebrafish larvae, the difference between seizure-like behavior, hyperactivity, and abnormal behavior is not clearly distinct. Thus, as the observed phenotype can be unrelated to seizures, each model should be characterized not only for the presence of seizure-like behavior that may lead to hyperactivity, but also for at least one nonbehavioral seizure or epilepsy biomarker, that is, an electrophysiological biomarker or molecular biomarker. Further, subtle seizure behaviors, for example, wholebody trembling or twitching of the pectoral fin or jaw, are often only described or manually scored (Baraban et al., 2005; Mahmood et al., 2013; Schubert et al., 2014; Suls et al., 2013), largely reducing the throughput of a model. Therefore, high resolution imaging and processing would be a major asset to detect and quantify a full range of novel behavioral parameters that supplement the parameters used today, and together cover a large spectrum of seizure-related behaviors. Also, a thorough pharmacological evaluation should be done with clinically relevant ASDs to determine a model’s predictive and refractory potential (Afrikanova et al., 2013; Baraban et al., 2013; Leclercq et al., 2015; Lee et al., 2012; Zhang et al., 2015).

Model Characterization: Local Field Potential Recordings With regard to the current implementation and analysis of LFP recordings, several improvements could be undertaken. Above all, the well-established LFP technique is invasive, that is, a sharp glass micropipette (1 µm opening) is placed inside deep brain structures. While commonly used, this practice causes severe brain trauma that can result in artifacts. Recently, an alternative noninvasive method was developed to measure neural activity from the surface of the brain using electrodes with large openings (10–20 µm) that sample much larger populations of neurons. Such noninvasive LFPs are recorded from the skin with a blunt electrolyte-filled glass pipette placed on the larva head, mimicking surface EEG technology in human (Zdebik et al., 2013). The advantage of this noninvasive technique is that it is less prone to artifacts related to trauma caused by needle impalement in invasive techniques, and also allows for stable long-term recordings of EEG activity in zebrafish. Next, the evaluation of epileptiform discharges from zebrafish brain recordings typically implies the quantification of the number of events, together with their duration, and sometimes the amplitudes, by visual inspection of each recording by an experienced researcher (Baraban et al., 2005; Leclercq et al., 2015). Such a scoring manner is fairly subjective, prone to observer’s bias and error, and very laborious. A computational detection algorithm of epileptiform activity

Zebrafish Models of Epilepsy and Epileptic Seizures Chapter | 26

would overcome this problem—as already done in mice (Bergstrom et al., 2013)—by automatically counting the events and providing information about the start and duration of the seizures, as well as the frequency of those events, a fact that might be of interest when studying epileptogenesis. Such a program would not only help to reduce the time of analysis of brain recordings but, more importantly, would provide a more objective and therefore more reproducible method for automated processing of batches of LFP or CFP recordings. Additionally, by using bioinformatics tools and mathematical software, it is possible to create time-frequency maps that display power spectra of signals over time, making it possible to track changes in amplitudes over a given period of time (Schubert et al., 2014). Finally, arbitrary thresholds are often used to distinguish different types of brain activity, that is, ictal-like, interictallike, and status epilepticus (SE)-like epileptiform events. As these are based solely on the observer’s interpretation, and lack accompanying behavioral data, such differentiations for quantification of brain activity can be omitted. Alternatively, a differentiation based on the type of electrical discharges, for example, polyspiking discharges, isolated spikes, and highfrequency oscillations, could be useful (Schubert et al., 2014).

Model Generation: Genetic Models of Epilepsy Other lessons learned from the recent past relate to the potential, but also limitations of the methods commonly used in zebrafish epilepsy modeling, and to the possibilities to

379

control and/or overcome them. The once so popular MOs witnessed criticism as a genetic engineering tool especially due to their propensity for inducing off-target effects (Schulte-Merker and Stainier, 2014). A recent comparison of published morpholino-induced with mutant phenotypes from the Sanger ZMP revealed substantial discrepancies between morphant and mutant phenotypes (Kok et al., 2015). Therefore, for future use of knockdown and knockout approaches, it is important to take into account the possibility of phenotypic inconsistencies. When standard timelines that are needed to generate and characterize morphant and mutant models are compared, it is obvious that MOs allow a more rapid functional investigation of the gene of interest (Fig. 26.3). This stems from the fact that, in most cases, CRISPR mutant fish require a stable F2 generation, and thus the need for raising F0 and F1 generations to the age of fertility (minimum 3-months old). Nevertheless, the added time spent to generate a mutant model is beneficial in the long run, as there is no need for repeated injections and, more importantly, the phenotypic characteristics of the mutant are likely to be more specific—though exceptions can be observed due to genetic compensation (Rossi et al., 2015). However, it can be expected that the use of MOs will persist (Blum et al., 2015), especially as a tool for a rapid and initial investigation of newly identified candidate epilepsypredisposing genes, possibly before the generation of the corresponding mutant. Furthermore, to increase the range of disease modeling applications, it is possible to generate stable zebrafish

FIGURE 26.3  Expected timelines for the development and phenotypic characterization of a morphant (MOs timeline) and mutant (CRISPR/ Cas9 timeline) model.

380 PART | IV  Non-Mammalian In Vivo Models

lines with tissue-specific, inheritable gene knockout. It was shown that the CRISPR/Cas9 knockout technology could be spatially controlled in the blood lineage (erythrocyte-specific gata1 promoter) and skeletal muscles (muscle-specific mylz2 promoter) in zebrafish embryos (Ablain et al., 2015). Specifically for epilepsy disorders, it would be possible to do a brain-specific knockout of a gene of interest employing brain-specific promoters, such as her4 (Yeo et al., 2007) or elav/HuC (Park et al., 2000). Such a tissue-restricted expression would enable more precise control of the gene knockout itself, as well as avoid possible nonspecific peripheral side effects or embryonic lethality associated with the global knockout of ubiquitously expressed genes that would complicate their function analysis in vivo.

Model Generation: Nongenetic Models of Epilepsy or Epileptogenesis Despite the development of various chemically- and hyperthermia-induced models of epileptic seizures, and multiple genetic epilepsy models, some useful rodent models of epilepsy or epileptogenesis do not have a zebrafish counterpart yet. For obvious reasons, a few models will be inherently difficult to establish in zebrafish, for example, among others, weight-drop injury and cortical impact models of traumatic brain injury (TBI) (Chandel et al., 2016). Some epilepsy and epileptogenesis models, however, seem meaningful to be explored in larval zebrafish. They include (1) the TBI model, for instance by lateral fluid-percussion (Chandel et al., 2016) in the brain using the same equipment as for microinjections, (2) kindling models (Loscher and Brandt, 2010) by repeated subconvulsive chemical stimulation, and (3) post-SE models induced by chemical stimulation, followed by spontaneous recurrent seizures after a latent period (Loscher, 2011). As the larvae undergoing such treatments can be easily modified genetically, these models seem interesting especially for the mechanistic exploration of biochemical pathways underlying epileptogenesis.

INSIGHT INTO HUMAN DISORDERS: WHAT DOES A MODEL MODEL? Since the first pioneering study on zebrafish as a new paradigm for seizure investigations (Baraban et al., 2005), several groups have embarked on engineering and exploring epilepsy-related models using different chemical and genetic approaches in zebrafish. As a consequence, zebrafish is rapidly progressing from an “emerging” (Baraban, 2007) toward an “established” epileptic seizure and epilepsy model. This does not in any way mean that zebrafish models are nowadays replacing rodent models, as the latter ones are still regarded as the standard model organisms for the

understanding of epilepsy and the testing of new medications. However, the similarity between human and zebra­ fish epilepsy-related genes and potential drug targets, as evidenced by recent investigations and discovery work, positions zebrafish inevitably in a prominent place with regard to preclinical research. Moreover, the possibility to combine large-scale testing of numerous compounds on an intact and human-relevant whole organism, in addition to the potential to rapidly investigate the function of new epilepsy genes in vivo, is unparalleled by any other organism. These characteristics are presently opening up new opportunities in epilepsy research, as zebrafish is increasingly used and likely will acquire the status of a prerodent model to guide, finetune, and narrow down gene- and discovery-related issues. Especially in this era where next-generation sequencing (NGS) is revolutionizing the speed with which pathogenic mutations in genes are discovered, zebrafish could play an important role. Indeed, more than 70 epilepsy-­associated genes have been currently identified, and likely many more are yet to come (EpiPM Consortium, 2015). In this way, zebrafish might help to pave the way toward precision medicine, that is, an emerging approach for individualized care, in a very efficient way (EpiPM Consortium, 2015). Recent examples of such translational research are the development of the chd2 and stx1b morphant zebrafish models, and the overexpression model of mutant fhf1b1 that allowed a rapid functional determination of a candidate epilepsy gene in vivo (Schubert et al., 2014; Siekierska et al., 2016; Suls et al., 2013). Such research is important in the short run for a better understanding of the disease, but can also aid to improve patient therapy in the future. As a matter of fact, especially by using genetic approaches, it is anticipated that the refractory background of the human disease can be introduced in zebrafish models, allowing for the screen-based discovery of efficacious medication. Actually, a first example of this hyphenated genetic/discovery approach is already available. The use of the scn1lab mutant zebrafish model that mimics DS, a severe treatmentresistant epilepsy syndrome that starts within the first year of life, already led to the discovery of clemizole as a potential treatment (Baraban et al., 2013). Besides, similar research reconfirmed the activity of fenfluramine (Dinday and ­Baraban, 2015; Zhang et al., 2015), a compound that, as an add-on treatment in a recent clinical study, led to seizurefreedom in 7 of 10 DS children (Ceulemans et al., 2012; Schoonjans et al., 2015).

CONCLUSIONS The first decade of zebrafish research in the field of epilepsy has passed, and has provided us with strong experimental evidence that supports the validity of zebrafish as an epilepsy and seizure model. Various models have been developed, and resulted already in increased insights in multiple

Zebrafish Models of Epilepsy and Epileptic Seizures Chapter | 26

epileptic syndromes, and in the first successful large-scale zebrafish-based ASD screens. It is apparent that well characterized models, such as the zebrafish PTZ seizure model and the scn1lab mutant, have found great applicability in the field. Moreover, with the availability of effective genetic engineering and genome editing tools, the systematic development of zebrafish genetic epilepsy models for newly identified epilepsy-predisposing genes is now possible, and is promising for the discovery of both personalized medicines and therapies against refractory seizures. Taken together, current knowledge is introducing the next decade as one to fully benefit from zebrafish as an established epilepsy and seizure model that should improve the search for novel therapies against seizures and ultimately epilepsy.

REFERENCES Ablain, J., Durand, E.M., Yang, S., Zhou, Y., Zon, L.I., 2015. A CRISPR/ Cas9 vector system for tissue-specific gene disruption in zebrafish. Dev. Cell. 32 (6), 756–764. Afrikanova, T., Serruys, A.S., Buenafe, O.E., Clinckers, R., Smolders, I., de Witte, P.A., Crawford, A.D., Esguerra, C.V., 2013. Validation of the zebrafish pentylenetetrazol seizure model: locomotor versus electrographic responses to antiepileptic drugs. PLoS One 8 (1), e54166. Alfaro, J.M., Ripoll-Gomez, J., Burgos, J.S., 2011. Kainate administered to adult zebrafish causes seizures similar to those in rodent models. Eur. J. Neurosci. 33 (7), 1252–1255. Arjona, F.J., de Baaij, J.H., Schlingmann, K.P., Lameris, A.L., van Wijk, E., Flik, G., Regele, S., Korenke, G.C., Neophytou, B., Rust, S., Reintjes, N., Konrad, M., Bindels, R.J., Hoenderop, J.G., 2014. CNNM2 mutations cause impaired brain development and seizures in patients with hypomagnesemia. PLoS Genet. 10 (4), e1004267. Baraban, S.C., 2007. Emerging epilepsy models: insights from mice, flies, worms and fish. Curr. Opin. Neurol. 20 (2), 164–168. Baraban, S.C., Dinday, M.T., Castro, P.A., Chege, S., Guyenet, S., T ­ aylor, M.R., 2007. A large-scale mutagenesis screen to identify seizure-­ resistant zebrafish. Epilepsia 48 (6), 1151–1157. Baraban, S.C., Dinday, M.T., Hortopan, G.A., 2013. Drug screening in Scn1a zebrafish mutant identifies clemizole as a potential Dravet syndrome treatment. Nat. Commun. 4, 2410. Baraban, S.C., Taylor, M.R., Castro, P.A., Baier, H., 2005. Pentylenetetrazole induced changes in zebrafish behavior, neural activity and c-fos expression. Neuroscience 131 (3), 759–768. Baxendale, S., Holdsworth, C.J., Meza Santoscoy, P.L., Harrison, M.R., Fox, J., Parkin, C.A., Ingham, P.W., Cunliffe, V.T., 2012. Identification of compounds with anti-convulsant properties in a zebrafish model of epileptic seizures. Dis. Model Mech. 5 (6), 773–784. Bedell, V.M., Wang, Y., Campbell, J.M., Poshusta, T.L., Starker, C.G., Krug, 2nd, R.G., Tan, W., Penheiter, S.G., Ma, A.C., Leung, A.Y., Fahrenkrug, S.C., Carlson, D.F., Voytas, D.F., Clark, K.J., Essner, J.J., Ekker, S.C., 2012. In vivo genome editing using a high-efficiency TALEN system. Nature 491 (7422), 114–118. Bedell, V.M., Westcot, S.E., Ekker, S.C., 2011. Lessons from morpholinobased screening in zebrafish. Brief Funct. Genomics 10 (4), 181–188. Berghmans, S., Hunt, J., Roach, A., Goldsmith, P., 2007. Zebrafish offer the potential for a primary screen to identify a wide variety of potential anticonvulsants. Epilepsy Res. 75 (1), 18–28.

381

Bergstrom, R.A., Choi, J.H., Manduca, A., Shin, H.S., Worrell, G.A., Howe, C.L., 2013. Automated identification of multiple seizure-related and interictal epileptiform event types in the EEG of mice. Sci. Rep. 3, 1483. Bill, B.R., Petzold, A.M., Clark, K.J., Schimmenti, L.A., Ekker, S.C., 2009. A primer for morpholino use in zebrafish. Zebrafish 6 (1), 69–77. Blum, M., De Robertis, E.M., Wallingford, J.B., Niehrs, C., 2015. Morpholinos: antisense and sensibility. Dev. Cell 35 (2), 145–149. Buenafe, O.E., Orellana-Paucar, A., Maes, J., Huang, H., Ying, X., De Borggraeve, W., Crawford, A.D., Luyten, W., Esguerra, C.V., de Witte, P., 2013. Tanshinone IIA exhibits anticonvulsant activity in zebrafish and mouse seizure models. ACS Chem. Neurosci. 4 (11), 1479–1487. Buzsaki, G., Anastassiou, C.A., Koch, C., 2012. The origin of extracellular fields and currents—EEG, ECoG, LFP and spikes. Nat. Rev. Neurosci. 13 (6), 407–420. Ceulemans, B., Boel, M., Leyssens, K., Van Rossem, C., Neels, P., Jorens, P.G., Lagae, L., 2012. Successful use of fenfluramine as an add-on treatment for Dravet syndrome. Epilepsia 53 (7), 1131–1139. Challal, S., Bohni, N., Buenafe, O.E., Esguerra, C.V., de Witte, P.A., Wolfender, J.L., Crawford, A.D., 2012. Zebrafish bioassay-guided microfractionation for the rapid in vivo identification of pharmacologically active natural products. Chimia (Aarau) 66 (4), 229–232. Chandel, S., Gupta, S.K., Medhi, B., 2016. Epileptogenesis following experimentally induced traumatic brain injury—a systematic review. Rev. Neurosci. 27 (3), 329–346. Chang, N., Sun, C., Gao, L., Zhu, D., Xu, X., Zhu, X., Xiong, J.W., Xi, J.J., 2013. Genome editing with RNA-guided Cas9 nuclease in zebrafish embryos. Cell Res. 23 (4), 465–472. Chege, S.W., Hortopan, G.A., Dinday, T.M., Baraban, S.C., 2012. Expression and function of KCNQ channels in larval zebrafish. Dev. Neurobiol. 72 (2), 186–198. Cho, S.W., Kim, S., Kim, Y., Kweon, J., Kim, H.S., Bae, S., Kim, J.S., 2014. Analysis of off-target effects of CRISPR/Cas-derived RNAguided endonucleases and nickases. Genome Res. 24 (1), 132–141. Cocchiaro, J.L., Rawls, J.F., 2013. Microgavage of zebrafish larvae. J. Vis. Exp. (72), e4434. Collymore, C., Rasmussen, S., Tolwani, R.J., 2013. Gavaging adult zebrafish. J. Vis. Exp. (78). Cousin, M.A., Ebbert, J.O., Wiinamaki, A.R., Urban, M.D., Argue, D.P., Ekker, S.C., Klee, E.W., 2014. Larval zebrafish model for FDA-approved drug repositioning for tobacco dependence treatment. PLoS One 9 (3), e90467. Delvecchio, C., Tiefenbach, J., Krause, H.M., 2011. The zebrafish: a powerful platform for in vivo, HTS drug discovery. Assay Drug Dev. Technol. 9 (4), 354–361. Dinday, M.T., Baraban, S.C., 2015. Large-scale phenotype-based antiepileptic drug screening in a zebrafish model of Dravet syndrome (1,2,3). eNeuro 2 (4), 1–19. Doyon, Y., McCammon, J.M., Miller, J.C., Faraji, F., Ngo, C., Katibah, G.E., Amora, R., Hocking, T.D., Zhang, L., Rebar, E.J., Gregory, P.D., Urnov, F.D., Amacher, S.L., 2008. Heritable targeted gene disruption in zebrafish using designed zinc-finger nucleases. Nat. Biotechnol. 26 (6), 702–708. Eisen, J.S., Smith, J.C., 2008. Controlling morpholino experiments: don’t stop making antisense. Development 135 (10), 1735–1743. Ekker, S.C., 2000. Morphants: a new systematic vertebrate functional genomics approach. Yeast 17 (4), 302–306. Engel, Jr., J., Pitkänen, A., Loeb, J.A., Dudek, F.E., Bertram, 3rd, E.H., Cole, A.J., Moshe, S.L., Wiebe, S., Jensen, F.E., Mody, I., Nehlig, A., Vezzani, A., 2013. Epilepsy biomarkers. Epilepsia 54 (Suppl 4), 61–69.

382 PART | IV  Non-Mammalian In Vivo Models

EpiPM Consortium, 2015. A roadmap for precision medicine in the epilepsies. Lancet Neurol. 14 (12), 1219–1228. Fleming, A., Diekmann, H., Goldsmith, P., 2013. Functional characterisation of the maturation of the blood–brain barrier in larval zebrafish. PLoS One 8 (10), e77548. Gerhard, G.S., Kauffman, E.J., Wang, X., Stewart, R., Moore, J.L., Kasales, C.J., Demidenko, E., Cheng, K.C., 2002. Life spans and senescent phenotypes in two strains of Zebrafish (Danio rerio). Exp. Gerontol. 37 (8–9), 1055–1068. Gilbert, S.F., 2000. Developmental biology, sixth ed. Sinauer Associates, Sunderland, MA. Grone, B.P., Marchese, M., Hamling, K.R., Kumar, M.G., Krasniak, C.S., Sicca, F., Santorelli, F.M., Patel, M., Baraban, S.C., 2016. Epilepsy, behavioral abnormalities, and physiological comorbidities in syntaxin-binding protein 1 (STXBP1) mutant zebrafish. PLoS One 11 (3), e0151148. Gupta, A., Christensen, R.G., Rayla, A.L., Lakshmanan, A., Stormo, G.D., Wolfe, S.A., 2012. An optimized two-finger archive for ZFN-mediated gene targeting. Nat. Methods 9 (6), 588–590. Hoffman, E.J., Turner, K.J., Fernandez, J.M., Cifuentes, D., Ghosh, M., Ijaz, S., Jain, R.A., Kubo, F., Bill, B.R., Baier, H., Granato, M., Barresi, M.J., Wilson, S.W., Rihel, J., State, M.W., Giraldez, A.J., 2016. Estrogens suppress a behavioral phenotype in zebrafish mutants of the autism risk gene, CNTNAP2. Neuron 89 (4), 725–733. Hoffman, G.E., Smith, M.S., Verbalis, J.G., 1993. c-fos and related immediate early gene products as markers of activity in neuroendocrine systems. Front. Neuroendocrinol. 14 (3), 173–213. Hortopan, G.A., Dinday, M.T., Baraban, S.C., 2010. Spontaneous seizures and altered gene expression in GABA signaling pathways in a mind bomb mutant zebrafish. J. Neurosci. 30 (41), 13718–13728. Howe, K., Clark, M.D., Torroja, C.F., Torrance, J., Berthelot, C., Muffato, M., Collins, J.E., Humphray, S., McLaren, K., Matthews, L., McLaren, S., Sealy, I., Caccamo, M., Churcher, C., Scott, C., Barrett, J.C., Koch, R., Rauch, G.J., White, S., Chow, W., Kilian, B., Quintais, L.T., Guerra-Assuncao, J.A., Zhou, Y., Gu, Y., Yen, J., Vogel, J.H., Eyre, T., Redmond, S., Banerjee, R., Chi, J., Fu, B., Langley, E., Maguire, S.F., Laird, G.K., Lloyd, D., Kenyon, E., Donaldson, S., Sehra, H., AlmeidaKing, J., Loveland, J., Trevanion, S., Jones, M., Quail, M., Willey, D., Hunt, A., Burton, J., Sims, S., McLay, K., Plumb, B., Davis, J., Clee, C., Oliver, K., Clark, R., Riddle, C., Elliot, D., Threadgold, G., Harden, G., Ware, D., Begum, S., Mortimore, B., Kerry, G., Heath, P., Phillimore, B., Tracey, A., Corby, N., Dunn, M., Johnson, C., Wood, J., Clark, S., Pelan, S., Griffiths, G., Smith, M., Glithero, R., Howden, P., Barker, N., Lloyd, C., Stevens, C., Harley, J., Holt, K., Panagiotidis, G., Lovell, J., Beasley, H., Henderson, C., Gordon, D., Auger, K., Wright, D., Collins, J., Raisen, C., Dyer, L., Leung, K., Robertson, L., Ambridge, K., Leongamornlert, D., McGuire, S., Gilderthorp, R., Griffiths, C., Manthravadi, D., Nichol, S., Barker, G., Whitehead, S., Kay, M., Brown, J., Murnane, C., Gray, E., Humphries, M., Sycamore, N., Barker, D., Saunders, D., Wallis, J., Babbage, A., Hammond, S., Mashreghi-Mohammadi, M., Barr, L., Martin, S., Wray, P., Ellington, A., Matthews, N., Ellwood, M., Woodmansey, R., Clark, G., Cooper, J., Tromans, A., Grafham, D., Skuce, C., Pandian, R., Andrews, R., Harrison, E., Kimberley, A., Garnett, J., Fosker, N., Hall, R., Garner, P., Kelly, D., Bird, C., Palmer, S., Gehring, I., Berger, A., Dooley, C.M., Ersan-Urun, Z., Eser, C., Geiger, H., Geisler, M., Karotki, L., Kirn, A., Konantz, J., Konantz, M., Oberlander, M., Rudolph-Geiger, S., Teucke, M., Lanz, C., Raddatz, G., Osoegawa, K., Zhu, B., Rapp, A., Widaa, S., Langford, C., Yang, F., Schuster, S.C., Carter, N.P., Harrow, J., Ning, Z., Herrero,

J., Searle, S.M., Enright, A., Geisler, R., Plasterk, R.H., Lee, C., Westerfield, M., de Jong, P.J., Zon, L.I., Postlethwait, J.H., Nusslein-Volhard, C., Hubbard, T.J., Roest Crollius, H., Rogers, J., Stemple, D.L., 2013. The zebrafish reference genome sequence and its relationship to the human genome. Nature 496 (7446), 498–503. Huang, P., Xiao, A., Zhou, M., Zhu, Z., Lin, S., Zhang, B., 2011. Heritable gene targeting in zebrafish using customized TALENs. Nat. Biotechnol. 29 (8), 699–700. Hunt, R.F., Hortopan, G.A., Gillespie, A., Baraban, S.C., 2012. A novel zebrafish model of hyperthermia-induced seizures reveals a role for TRPV4 channels and NMDA-type glutamate receptors. Exp. Neurol. 237 (1), 199–206. Hwang, W.Y., Fu, Y., Reyon, D., Maeder, M.L., Kaini, P., Sander, J.D., Joung, J.K., Peterson, R.T., Yeh, J.R., 2013a. Heritable and precise zebrafish genome editing using a CRISPR-Cas system. PLoS One 8 (7), e68708. Hwang, W.Y., Fu, Y., Reyon, D., Maeder, M.L., Tsai, S.Q., Sander, J.D., Peterson, R.T., Yeh, J.R., Joung, J.K., 2013b. Efficient genome editing in zebrafish using a CRISPR-Cas system. Nat. Biotechnol. 31 (3), 227–229. Jao, L.E., Wente, S.R., Chen, W., 2013. Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system. Proc. Natl. Acad. Sci. USA 110 (34), 13904–13909. Johnson, M.R., Behmoaras, J., Bottolo, L., Krishnan, M.L., Pernhorst, K., Santoscoy, P.L., Rossetti, T., Speed, D., Srivastava, P.K., ChadeauHyam, M., Hajji, N., Dabrowska, A., Rotival, M., Razzaghi, B., Kovac, S., Wanisch, K., Grillo, F.W., Slaviero, A., Langley, S.R., Shkura, K., Roncon, P., De, T., Mattheisen, M., Niehusmann, P., O’Brien, T.J., Petrovski, S., von Lehe, M., Hoffmann, P., Eriksson, J., Coffey, A.J., Cichon, S., Walker, M., Simonato, M., Danis, B., Mazzuferi, M., Foerch, P., Schoch, S., De Paola, V., Kaminski, R.M., Cunliffe, V.T., Becker, A.J., Petretto, E., 2015. Systems genetics identifies Sestrin 3 as a regulator of a proconvulsant gene network in human epileptic hippocampus. Nat. Commun. 6, 6031. Johnston, L., Ball, R.E., Acuff, S., Gaudet, J., Sornborger, A., Lauderdale, J.D., 2013. Electrophysiological recording in the brain of intact adult zebrafish. J. Vis. Exp. 81, e51065. Kalueff, A.V., Echevarria, D.J., Stewart, A.M., 2014. Gaining translational momentum: more zebrafish models for neuroscience research. Prog. Neuropsychopharmacol. Biol. Psychiatry 55, 1–6. Kawakami, K., Shima, A., Kawakami, N., 2000. Identification of a functional transposase of the Tol2 element, an Ac-like element from the Japanese medaka fish, and its transposition in the zebrafish germ lineage. Proc. Natl. Acad. Sci. USA 97 (21), 11403–11408. Kettleborough, R.N., Busch-Nentwich, E.M., Harvey, S.A., Dooley, C.M., de Bruijn, E., van Eeden, F., Sealy, I., White, R.J., Herd, C., Nijman, I.J., Fenyes, F., Mehroke, S., Scahill, C., Gibbons, R., Wali, N., Carruthers, S., Hall, A., Yen, J., Cuppen, E., Stemple, D.L., 2013. A systematic genome-wide analysis of zebrafish protein-coding gene function. Nature 496 (7446), 494–497. Kim, Y.H., Lee, Y., Lee, K., Lee, T., Kim, Y.J., Lee, C.J., 2010. Reduced neuronal proliferation by proconvulsant drugs in the developing zebrafish brain. Neurotoxicol. Teratol. 32 (5), 551–557. Kimmel, C.B., Ballard, W.W., Kimmel, S.R., Ullmann, B., Schilling, T.F., 1995. Stages of embryonic development of the zebrafish. Dev. Dyn. 203 (3), 253–310. Klaric, T., Lardelli, M., Key, B., Koblar, S., Lewis, M., 2014. Activitydependent expression of neuronal PAS domain-containing protein 4 (npas4a) in the developing zebrafish brain. Front. Neuroanat. 8, 148.

Zebrafish Models of Epilepsy and Epileptic Seizures Chapter | 26

Kok, F.O., Shin, M., Ni, C.W., Gupta, A., Grosse, A.S., van Impel, A., Kirchmaier, B.C., Peterson-Maduro, J., Kourkoulis, G., Male, I., DeSantis, D.F., Sheppard-Tindell, S., Ebarasi, L., Betsholtz, C., SchulteMerker, S., Wolfe, S.A., Lawson, N.D., 2015. Reverse genetic screening reveals poor correlation between morpholino-induced and mutant phenotypes in zebrafish. Dev. Cell 32 (1), 97–108. Law, S.H., Sargent, T.D., 2014. The serine-threonine protein kinase PAK4 is dispensable in zebrafish: identification of a morpholino-generated pseudophenotype. PLoS One 9 (6), e100268. Leclercq, K., Afrikanova, T., Langlois, M., De Prins, A., Buenafe, O.E., Rospo, C.C., Van Eeckhaut, A., de Witte, P.A., Crawford, A.D., Smolders, I., Esguerra, C.V., Kaminski, R.M., 2015. Cross-species pharmacological characterization of the allylglycine seizure model in mice and larval zebrafish. Epilepsy Behav. 45, 53–63. Lee, G.H., Sung, S.Y., Chang, W.N., Kao, T.T., Du, H.C., Hsiao, T.H., Safo, M.K., Fu, T.F., 2012. Zebrafish larvae exposed to ginkgotoxin exhibit seizure-like behavior that is relieved by pyridoxal-5’-phosphate, GABA and anti-epileptic drugs. Dis. Model Mech. 5 (6), 785–795. Lee, Y., Kim, D., Kim, Y.H., Lee, H., Lee, C.J., 2010. Improvement of pentylenetetrazol-induced learning deficits by valproic acid in the adult zebrafish. Eur. J. Pharmacol. 643 (2–3), 225–231. Liew, W.C., Orban, L., 2014. Zebrafish sex: a complicated affair. Brief Funct. Genomics 13 (2), 172–187. Long, S.M., Liang, F.Y., Wu, Q., Lu, X.L., Yao, X.L., Li, S.C., Li, J., Su, H., Pang, J.Y., Pei, Z., 2014. Identification of marine neuroactive molecules in behaviour-based screens in the larval zebrafish. Mar. Drugs 12 (6), 3307–3322. Lopes, M.W., Sapio, M.R., Leal, R.B., Fricker, L.D., 2016. Knockdown of carboxypeptidase A6 in zebrafish larvae reduces response to seizureinducing drugs and causes changes in the level of mRNAs encoding signaling molecules. PLoS One 11 (4), e0152905. Loscher, W., 2011. Critical review of current animal models of seizures and epilepsy used in the discovery and development of new antiepileptic drugs. Seizure 20 (5), 359–368. Loscher, W., Brandt, C., 2010. Prevention or modification of epileptogenesis after brain insults: experimental approaches and translational research. Pharmacol. Rev. 62 (4), 668–700. Mahmood, F., Mozere, M., Zdebik, A.A., Stanescu, H.C., Tobin, J., Beales, P.L., Kleta, R., Bockenhauer, D., Russell, C., 2013. Generation and validation of a zebrafish model of EAST (epilepsy, ataxia, sensorineural deafness and tubulopathy) syndrome. Dis. Model Mech. 6 (3), 652–660. Mali, P., Aach, J., Stranges, P.B., Esvelt, K.M., Moosburner, M., Kosuri, S., Yang, L., Church, G.M., 2013. CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nat. Biotechnol. 31 (9), 833–838. McKeown, K.A., Moreno, R., Hall, V.L., Ribera, A.B., Downes, G.B., 2012. Disruption of Eaat2b, a glutamate transporter, results in abnormal motor behaviors in developing zebrafish. Dev. Biol. 362 (2), 162–171. Mei, X., Wu, S., Bassuk, A.G., Slusarski, D.C., 2013. Mechanisms of prickle1a function in zebrafish epilepsy and retinal neurogenesis. Dis. Model Mech. 6 (3), 679–688. Menezes, F.P., Rico, E.P., Da Silva, R.S., 2014. Tolerance to seizure induced by kainic acid is produced in a specific period of zebrafish development. Prog. Neuropsychopharmacol. Biol. Psychiatry 55, 109–112. Meng, X., Noyes, M.B., Zhu, L.J., Lawson, N.D., Wolfe, S.A., 2008. Targeted gene inactivation in zebrafish using engineered zinc-finger nucleases. Nat. Biotechnol. 26 (6), 695–701.

383

Milan, D.J., Peterson, T.A., Ruskin, J.N., Peterson, R.T., MacRae, C.A., 2003. Drugs that induce repolarization abnormalities cause bradycardia in zebrafish. Circulation 107 (10), 1355–1358. Mussulini, B.H., Leite, C.E., Zenki, K.C., Moro, L., Baggio, S., Rico, E.P., Rosemberg, D.B., Dias, R.D., Souza, T.M., Calcagnotto, M.E., Campos, M.M., Battastini, A.M., de Oliveira, D.L., 2013. Seizures induced by pentylenetetrazole in the adult zebrafish: a detailed behavioral characterization. PLoS One 8 (1), e54515. Nasevicius, A., Ekker, S.C., 2000. Effective targeted gene ‘knockdown’ in zebrafish. Nat. Genet. 26 (2), 216–220. Nishimura, Y., Murakami, S., Ashikawa, Y., Sasagawa, S., Umemoto, N., Shimada, Y., Tanaka, T., 2015. Zebrafish as a systems toxicology model for developmental neurotoxicity testing. Congenit. Anom. (Kyoto) 55 (1), 1–16. Orellana-Paucar, A.M., Afrikanova, T., Thomas, J., Aibuldinov, Y.K., Dehaen, W., de Witte, P.A., Esguerra, C.V., 2013. Insights from zebrafish and mouse models on the activity and safety of ar-turmerone as a potential drug candidate for the treatment of epilepsy. PLoS One 8 (12), e81634. Orellana-Paucar, A.M., Serruys, A.S., Afrikanova, T., Maes, J., De Borggraeve, W., Alen, J., Leon-Tamariz, F., Wilches-Arizabala, I.M., Crawford, A.D., de Witte, P.A., Esguerra, C.V., 2012. Anticonvulsant activity of bisabolene sesquiterpenoids of Curcuma longa in zebrafish and mouse seizure models. Epilepsy Behav. 24 (1), 14–22. Park, H.C., Kim, C.H., Bae, Y.K., Yeo, S.Y., Kim, S.H., Hong, S.K., Shin, J., Yoo, K.W., Hibi, M., Hirano, T., Miki, N., Chitnis, A.B., Huh, T.L., 2000. Analysis of upstream elements in the HuC promoter leads to the establishment of transgenic zebrafish with fluorescent neurons. Dev. Biol. 227 (2), 279–293. Pineda, R., Beattie, C.E., Hall, C.W., 2011. Recording the adult zebrafish cerebral field potential during pentylenetetrazole seizures. J. Neurosci. Methods 200 (1), 20–28. Ramirez, I.B., Pietka, G., Jones, D.R., Divecha, N., Alia, A., Baraban, S.C., Hurlstone, A.F., Lowe, M., 2012. Impaired neural development in a zebrafish model for Lowe syndrome. Hum. Mol. Genet. 21 (8), 1744–1759. Rihel, J., Schier, A.F., 2012. Behavioral screening for neuroactive drugs in zebrafish. Dev. Neurobiol. 72 (3), 373–385. Robu, M.E., Larson, J.D., Nasevicius, A., Beiraghi, S., Brenner, C., Farber, S.A., Ekker, S.C., 2007. p53 activation by knockdown technologies. PLoS Genet. 3 (5), e78. Rossi, A., Kontarakis, Z., Gerri, C., Nolte, H., Holper, S., Kruger, M., Stainier, D.Y., 2015. Genetic compensation induced by deleterious mutations but not gene knockdowns. Nature 524 (7564), 230–233. Sander, J.D., Cade, L., Khayter, C., Reyon, D., Peterson, R.T., Joung, J.K., Yeh, J.R., 2011. Targeted gene disruption in somatic zebrafish cells using engineered TALENs. Nat. Biotechnol. 29 (8), 697–698. Sander, J.D., Joung, J.K., 2014. CRISPR-Cas systems for editing, regulating and targeting genomes. Nat. Biotechnol. 32 (4), 347–355. Schoonjans, A.S., Lagae, L., Ceulemans, B., 2015. Low-dose fenfluramine in the treatment of neurologic disorders: experience in Dravet syndrome. Ther. Adv. Neurol. Disord. 8 (6), 328–338. Schubert, J., Siekierska, A., Langlois, M., May, P., Huneau, C., Becker, F., Muhle, H., Suls, A., Lemke, J.R., de Kovel, C.G., Thiele, H., Konrad, K., Kawalia, A., Toliat, M.R., Sander, T., Ruschendorf, F., Caliebe, A., Nagel, I., Kohl, B., Kecskes, A., Jacmin, M., Hardies, K., Weckhuysen, S., Riesch, E., Dorn, T., Brilstra, E.H., Baulac, S., Moller, R.S., Hjalgrim, H., Koeleman, B.P., Euro, E.R.E.S.C., Jurkat-Rott, K., Lehman-Horn, F., Roach, J.C., Glusman, G., Hood, L., Galas, D.J., Martin,

384 PART | IV  Non-Mammalian In Vivo Models

B., de Witte, P.A., Biskup, S., De Jonghe, P., Helbig, I., Balling, R., Nurnberg, P., Crawford, A.D., Esguerra, C.V., Weber, Y.G., Lerche, H., 2014. Mutations in STX1B, encoding a presynaptic protein, cause fever-associated epilepsy syndromes. Nat. Genet. 46 (12), 1327–1332. Schulte-Merker, S., Stainier, D.Y., 2014. Out with the old, in with the new: reassessing morpholino knockdowns in light of genome editing technology. Development 141 (16), 3103–3104. Siekierska, A., Isrie, M., Liu, Y., Scheldeman, C., Vanthillo, N., Lagae, L., de Witte, P.A., Van Esch, H., Goldfarb, M., Buyse, G.M., 2016. Gainof-function FHF1 mutation causes early-onset epileptic encephalopathy with cerebellar atrophy. Neurology 86 (23), 2162–2170. Sourbron, J., Schneider, H., Kecskes, A., Liu, Y., Buening, E.M., Lagae, L., Smolders, I., de Witte, P., 2016. Serotonergic modulation as effective treatment for Dravet syndrome in a zebrafish mutant model. ACS Chem. Neurosci. 7 (5), 588–598. Stainier, D.Y., Kontarakis, Z., Rossi, A., 2015. Making sense of anti-sense data. Dev. Cell 32 (1), 7–8. Stewart, A.M., Braubach, O., Spitsbergen, J., Gerlai, R., Kalueff, A.V., 2014. Zebrafish models for translational neuroscience research: from tank to bedside. Trends Neurosci. 37 (5), 264–278. Stewart, A.M., Desmond, D., Kyzar, E., Gaikwad, S., Roth, A., Riehl, R., Collins, C., Monnig, L., Green, J., Kalueff, A.V., 2012. Perspectives of zebrafish models of epilepsy: what, how and where next? Brain Res. Bull. 87 (2–3), 135–143. Suls, A., Jaehn, J.A., Kecskes, A., Weber, Y., Weckhuysen, S., Craiu, D.C., Siekierska, A., Djemie, T., Afrikanova, T., Gormley, P., von Spiczak, S., Kluger, G., Iliescu, C.M., Talvik, T., Talvik, I., Meral, C., Caglayan, H.S., Giraldez, B.G., Serratosa, J., Lemke, J.R., HoffmanZacharska, D., Szczepanik, E., Barisic, N., Komarek, V., Hjalgrim, H., Moller, R.S., Linnankivi, T., Dimova, P., Striano, P., Zara, F., Marini, C., Guerrini, R., Depienne, C., Baulac, S., Kuhlenbaumer, G., Crawford, A.D., Lehesjoki, A.E., de Witte, P.A., Palotie, A., Lerche, H., Esguerra, C.V., De Jonghe, P., Helbig, I., Euro, E.R.E.S.C., 2013. De novo loss-of-function mutations in CHD2 cause a fever-sensitive myoclonic epileptic encephalopathy sharing features with Dravet syndrome. Am. J. Hum. Genet. 93 (5), 967–975. Tamplin, O.J., White, R.M., Jing, L., Kaufman, C.K., Lacadie, S.A., Li, P., Taylor, A.M., Zon, L.I., 2012. Small molecule screening in zebrafish: swimming in potential drug therapies. Wiley Interdiscip. Rev. Dev. Biol. 1 (3), 459–468. Taylor, J.S., Braasch, I., Frickey, T., Meyer, A., Van de Peer, Y., 2003. Genome duplication, a trait shared by 22000 species of ray-finned fish. Genome Res. 13 (3), 382–390. Teng, Y., Xie, X., Walker, S., Rempala, G., Kozlowski, D.J., Mumm, J.S., Cowell, J.K., 2010. Knockdown of zebrafish Lgi1a results in abnormal development, brain defects and a seizure-like behavioral phenotype. Hum. Mol. Genet. 19 (22), 4409–4420. Teng, Y., Xie, X., Walker, S., Saxena, M., Kozlowski, D.J., Mumm, J.S., Cowell, J.K., 2011. Loss of zebrafish lgi1b leads to hydrocephalus and sensitization to pentylenetetrazol induced seizure-like behavior. PLoS One 6 (9), e24596. Tiedeken, J.A., Ramsdell, J.S., 2007. Embryonic exposure to domoic acid increases the susceptibility of zebrafish larvae to the chemical convulsant pentylenetetrazole. Environ. Health Perspect. 115 (11), 1547–1552.

Tiedeken, J.A., Ramsdell, J.S., 2009. DDT exposure of zebrafish embryos enhances seizure susceptibility: relationship to fetal p,p’-DDE burden and domoic acid exposure of California sea lions. Environ. Health Perspect. 117 (1), 68–73. Tiedeken, J.A., Ramsdell, J.S., Ramsdell, A.F., 2005. Developmental toxicity of domoic acid in zebrafish (Danio rerio). Neurotoxicol. Teratol. 27 (5), 711–717. Uchida, D., Yamashita, M., Kitano, T., Iguchi, T., 2002. Oocyte apoptosis during the transition from ovary-like tissue to testes during sex differentiation of juvenile zebrafish. J. Exp. Biol. 205 (Pt 6), 711–718. Vermoesen, K., Serruys, A.S., Loyens, E., Afrikanova, T., Massie, A., Schallier, A., Michotte, Y., Crawford, A.D., Esguerra, C.V., de Witte, P.A., Smolders, I., Clinckers, R., 2011. Assessment of the convulsant liability of antidepressants using zebrafish and mouse seizure models. Epilepsy Behav. 22 (3), 450–460. Vouillot, L., Thelie, A., Pollet, N., 2015. Comparison of T7E1 and surveyor mismatch cleavage assays to detect mutations triggered by engineered nucleases. G3 (Bethesda) 5 (3), 407–415. Westerfield, M., 2000. The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio), 4th edition University of Oregon Press, Eugene, OR. Wiedenheft, B., Sternberg, S.H., Doudna, J.A., 2012. RNA-guided genetic silencing systems in bacteria and archaea. Nature 482 (7385), 331–338. Williams, L.R., Wong, K., Stewart, A., Suciu, C., Gaikwad, S., Wu, N., Dileo, J., Grossman, L., Cachat, J., Hart, P., Kalueff, A.V., 2012. Behavioral and physiological effects of RDX on adult zebrafish. Comp. Biochem. Physiol. C Toxicol. Pharmacol. 155 (1), 33–38. Winter, M.J., Redfern, W.S., Hayfield, A.J., Owen, S.F., Valentin, J.P., Hutchinson, T.H., 2008. Validation of a larval zebrafish locomotor assay for assessing the seizure liability of early-stage development drugs. J. Pharmacol. Toxicol. Methods 57 (3), 176–187. Wong, K., Stewart, A., Gilder, T., Wu, N., Frank, K., Gaikwad, S., Suciu, C., Dileo, J., Utterback, E., Chang, K., Grossman, L., Cachat, J., Kalueff, A.V., 2010. Modeling seizure-related behavioral and endocrine phenotypes in adult zebrafish. Brain Res. 1348, 209–215. Wyatt, C., Bartoszek, E.M., Yaksi, E., 2015. Methods for studying the zebrafish brain: past, present and future. Eur. J. Neurosci. 42 (2), 1746–1763. Yeo, S.Y., Kim, M., Kim, H.S., Huh, T.L., Chitnis, A.B., 2007. Fluorescent protein expression driven by her4 regulatory elements reveals the spatiotemporal pattern of Notch signaling in the nervous system of zebrafish embryos. Dev. Biol. 301 (2), 555–567. Zdebik, A.A., Mahmood, F., Stanescu, H.C., Kleta, R., Bockenhauer, D., Russell, C., 2013. Epilepsy in kcnj10 morphant zebrafish assessed with a novel method for long-term EEG recordings. PLoS One 8 (11), e79765. Zhang, Y., Kecskes, A., Copmans, D., Langlois, M., Crawford, A.D., Ceulemans, B., Lagae, L., de Witte, P.A., Esguerra, C.V., 2015. Pharmacological characterization of an antisense knockdown zebrafish model of Dravet syndrome: inhibition of epileptic seizures by the serotonin agonist fenfluramine. PLoS One 10 (5), e0125898. Zon, L.I., Peterson, R.T., 2005. In vivo drug discovery in the zebrafish. Nat. Rev. Drug Discov. 4 (1), 35–44.