Drug screening in Scn1a zebrafish mutant identifies clemizole as a potential Dravet syndrome treatment.

Dravet syndrome is a catastrophic pediatric epilepsy with severe intellectual disability, impaired social development and persistent drug-resistant seizures. One of its primary monogenic causes are mutations in Nav1.1 (SCN1A), a voltage-gated sodium channel. Here we characterize zebrafish Nav1.1 (scn1Lab) mutants originally identified in a chemical mutagenesis screen. Mutants exhibit spontaneous abnormal electrographic activity, hyperactivity and convulsive behaviours. Although scn1Lab expression is reduced, microarray analysis is remarkable for the small fraction of differentially expressed genes (~3%) and lack of compensatory expression changes in other scn subunits. Ketogenic diet, diazepam, valproate, potassium bromide and stiripentol attenuate mutant seizure activity; seven other antiepileptic drugs have no effect. A phenotype-based screen of 320 compounds identifies a US Food and Drug Administration-approved compound (clemizole) that inhibits convulsive behaviours and electrographic seizures. This approach represents a new direction in modelling pediatric epilepsy and could be used to identify novel therapeutics for any monogenic epilepsy disorder.

A novel zebrafish model of hyperthermia-induced seizures reveals a role for TRPV4 channels and NMDA-type glutamate receptors.

Febrile seizures are the most common seizure type in children under the age of five, but mechanisms underlying seizure generation in vivo remain unclear. Animal models to address this issue primarily focus on immature rodents heated indirectly using a controlled water bath or air blower. Here we describe an in vivo model of hyperthermia-induced seizures in larval zebrafish aged 3 to 7 days post-fertilization (dpf). Bath controlled changes in temperature are rapid and reversible in this model. Acute electrographic seizures following transient hyperthermia showed age-dependence, strain independence, and absence of mortality. Electrographic seizures recorded in the larval zebrafish forebrain were blocked by adding antagonists to the transient receptor potential vanilloid (TRPV4) channel or N-methyl-d-aspartate (NMDA) glutamate receptor to the bathing medium. Application of GABA, GABA re-uptake inhibitors, or TRPV1 antagonist had no effect on hyperthermic seizures. Expression of vanilloid channel and glutamate receptor mRNA was confirmed by quantitative PCR analysis at each developmental stage in larval zebrafish. Taken together, our findings suggest a role of heat-activation of TRPV4 channels and enhanced NMDA receptor-mediated glutamatergic transmission in hyperthermia-induced seizures.

Expression and function of KCNQ channels in larval zebrafish.

Members of the K(v)7 family generate a subthreshold potassium current, termed M-current, that regulates the excitability of principal central neurons. Mutations in two members of this family, K(v)7.2 (KCNQ2) and K(v)7.3 (KCNQ3) are associated with a neurological disorder known as benign familial neonatal convulsion (BFNC). Despite their importance in normal and pathological brain function, developmental expression and function of these channels remains relatively unexplored. Here, we examined the temporal expression of K(v)7 channel subunits in zebrafish larvae using a real-time quantitative PCR approach. Spatial expression in the larval zebrafish brain was assessed using whole-mount in situ hybridization. The mRNA for three members of the K(v)7 family (KCNQ2, 3 and 5) is reported in zebrafish between two and seven days post-fertilization (dpf). Using electrophysiological techniques, we show that inhibitors of K(v)7 channels (linopirdine and XE991) induce burst discharge activity in immature zebrafish between 3 and 7 dpf. This abnormal electrical activity is blocked by a K(v)7 channel opener (retigabine) and was also shown to evoke convulsive behaviors in freely swimming zebrafish. Using morpholino oligonucleotides directed against KCNQ3, we confirmed a role for KCNQ channels in generation of electrical burst discharges. These results indicate that functional K(v)7 channels are expressed in the larval zebrafish nervous system and could play a direct role in generation of seizure activity.

Aberrant expression of genes necessary for neuronal development and Notch signaling in an epileptic mind bomb zebrafish.

Mutation within an ubiquitin E3 ligase gene can lead to a failure in Notch signaling, excessive neurons, and depletion of neural progenitor cells in mind bomb mutants. Using mib(hi904) zebrafish, we reported seizures and a down-regulation of γ-aminobutyric acid (GABA) signaling pathway genes. A transcriptome analysis also identified differential expression pattern of genes related to Notch signaling and neurodevelopment. Here, we selected nine of these genes (her4.2, hes5, bhlhb5, hoxa5a, hoxb5b, dmbx1a, dbx1a, nxph1, and plxnd1) and performed a more thorough analysis of expression using conventional polymerase chain reaction, real-time polymerase chain reaction and in situ hybridization. Transgenic reporter fish (Gfap:GFP and Dlx5a-6a:GFP) were used to assess early brain morphology in vivo. Down-regulation of many of these genes was prominent throughout key structures of the developing mib(hi904) zebrafish brain including, but not limited to, the pallium, ventral thalamus, and optic tectum. Brain expression of Dlx5a-6a and Gfap was also reduced. In conclusion, these expression studies indicate a general down-regulation of Notch signaling genes necessary for proper brain development and suggest that these mutant fish could provide valuable insights into neurological conditions, such as Angelman syndrome, associated with ubiquitin E3 ligase mutation.

Spontaneous seizures and altered gene expression in GABA signaling pathways in a mind bomb mutant zebrafish.

Disruption of E3 ubiquitin ligase activity in immature zebrafish mind bomb mutants leads to a failure in Notch signaling, excessive numbers of neurons, and depletion of neural progenitor cells. This neurogenic phenotype is associated with defects in neural patterning and brain development. Because developmental brain abnormalities are recognized as an important feature of childhood neurological disorders such as epilepsy and autism, we determined whether zebrafish mutants with grossly abnormal brain structure exhibit spontaneous electrical activity that resembles the long-duration, high-amplitude multispike discharges reported in immature zebrafish exposed to convulsant drugs. Electrophysiological recordings from agar immobilized mind bomb mutants at 3 d postfertilization confirmed the occurrence of electrographic seizure activity; seizure-like behaviors were also noted during locomotion video tracking of freely behaving mutants. To identify genes differentially expressed in the mind bomb mutant and provide insight into molecular pathways that may mediate these epileptic phenotypes, a transcriptome analysis was performed using microarray. Interesting candidate genes were further analyzed using conventional reverse transcriptase-PCR and real-time quantitative PCR, as well as whole-mount in situ hybridization. Approximately 150 genes, some implicated in development, transcription, cell metabolism, and signal transduction, are differentially regulated, including downregulation of several genes necessary for GABA-mediated signaling. These findings identify a collection of gene transcripts that may be responsible for the abnormal electrical discharge and epileptic activities observed in a mind bomb zebrafish mutant. This work may have important implications for neurological and neurodevelopmental disorders associated with mutations in ubiquitin ligase activity.

Zebrafish as a model for studying genetic aspects of epilepsy.

Despite a long tradition of using rats and mice to model epilepsy, several aspects of rodent biology limit their use in large-scale genetic and therapeutic drug screening programs. Neuroscientists interested in vertebrate development and diseases have recently turned to zebrafish (Danio rerio) to overcome these limitations. Zebrafish can be studied at all stages of development and several methods are available for the manipulation of genes in zebrafish. In addition, developing zebrafish larvae can efficiently equilibrate drugs placed in the bathing medium. Taking advantage of these features and adapting electrophysiological recording methods to an agar-immobilized zebrafish preparation, we describe here our efforts to model seizure disorders in zebrafish. We also describe the initial results of a large-scale mutagenesis screen to identify gene mutation(s) that confer seizure resistance. Although the adaptation of zebrafish to epilepsy research is in its early stages, these studies highlight the rapid progress that can be made using this simple vertebrate species.