Investigation of Seizure-Susceptibility in a Drosophila melanogaster Model of Human Epilepsy with Optogenetic Stimulation.

We examined seizure-susceptibility in a Drosophila model of human epilepsy using optogenetic stimulation of ReaChR (red-activatable channelrhodopsin). Photostimulation of the seizure-sensitive mutant parabss1 causes behavioral paralysis that resembles paralysis caused by mechanical stimulation, in many aspects. Electrophysiology shows that photostimulation evokes abnormal seizure-like neuronal firing in parabss1 followed by a quiescent period resembling synaptic failure and apparently responsible for paralysis. The pattern of neuronal activity concludes with seizure-like activity just prior to recovery. We tentatively identify the mushroom body as one apparent locus of optogenetic seizure initiation. The α/ß lobes may be primarily responsible for mushroom body seizure induction.

Seizure Suppression by High Temperature via cAMP Modulation in Drosophila.

Bang-sensitive (BS) Drosophila mutants display characteristic seizure-like activity (SLA) and paralysis after mechanical shock . After high-frequency electrical stimulation (HFS) of the brain, they generate robust seizures at very low threshold voltage. Here we report an important phenomenon, which effectively suppresses SLA in BS mutants. High temperature causes seizure suppression in all BS mutants (parabss1, eas, sda) examined in this study. This effect is fully reversible and flies show complete recovery from BS paralysis once the temperature effect is nullified. High temperature induces an increase in seizure threshold after a brief pulse of heat shock (HS). By genetic screening, we identified the involvement of cAMP in the suppression of seizures by high temperature. We propose that HS induces adenylyl cyclase which in turn increases cAMP concentration which eventually suppresses seizures in mutant flies. In summary, we describe an unusual phenomenon, where high temperature can suppress SLA in flies by modulating cAMP concentration.

Mutations of the Calcium Channel Gene cacophony Suppress Seizures in Drosophila.

Bang sensitive (BS) Drosophila mutants display characteristic seizure-like phenotypes resembling, in some aspects, those of human seizure disorders such as epilepsy. The BS mutant parabss1, caused by a gain-of-function mutation of the voltage-gated Na+ channel gene, is extremely seizure-sensitive with phenotypes that have proven difficult to ameliorate by anti-epileptic drug feeding or by seizure-suppressor mutation. It has been presented as a model for intractable human epilepsy. Here we show that cacophony (cacTS2), a mutation of the Drosophila presynaptic Ca++ channel α1 subunit gene, is a particularly potent seizure-suppressor mutation, reverting seizure-like phenotypes for parabss1 and other BS mutants. Seizure-like phenotypes for parabss1 may be suppressed by as much as 90% in double mutant combinations with cacTS2. Unexpectedly, we find that parabss1 also reciprocally suppresses cacTS2 seizure-like phenotypes. The cacTS2 mutant displays these seizure-like behaviors and spontaneous high-frequency action potential firing transiently after exposure to high temperature. We find that this seizure-like behavior in cacTS2 is ameliorated by 85% in double mutant combinations with parabss1.

Disruption of Endocytosis with the Dynamin Mutant shibirets1 Suppresses Seizures in Drosophila.

One challenge in modern medicine is to control epilepsies that do not respond to currently available medications. Since seizures consist of coordinated and high-frequency neural activity, our goal was to disrupt neurotransmission with a synaptic transmission mutant and evaluate its ability to suppress seizures. We found that the mutant shibire, encoding dynamin, suppresses seizure-like activity in multiple seizure-sensitive Drosophila genotypes, one of which resembles human intractable epilepsy in several aspects. Because of the requirement of dynamin in endocytosis, increased temperature in the shi(ts1) mutant causes impairment of synaptic vesicle recycling and is associated with suppression of the seizure-like activity. Additionally, we identified the giant fiber neuron as critical in the seizure circuit and sufficient to suppress seizures. Overall, our results implicate mutant dynamin as an effective seizure suppressor, suggesting that targeting or limiting the availability of synaptic vesicles could be an effective and general method of controlling epilepsy disorders.

Drosophila sodium channel mutations: Contributions to seizure-susceptibility.

This paper reviews Drosophila voltage-gated Na(+) channel mutations encoded by the para (paralytic) gene and their contributions to seizure disorders in the fly. Numerous mutations cause seizure-sensitivity, for example, para(bss1), with phenotypes that resemble human intractable epilepsy in some aspects. Seizure phenotypes are also seen with human GEFS+ spectrum mutations that have been knocked into the Drosophila para gene, para(GEFS+) and para(DS) alleles. Other para mutations, para(ST76) and para(JS) act as seizure-suppressor mutations reverting seizure phenotypes in other mutants. Seizure-like phenotypes are observed from mutations and other conditions that cause a persistent Na(+) current through either changes in mRNA splicing or protein structure.

Modeling glial contributions to seizures and epileptogenesis: cation-chloride cotransporters in Drosophila melanogaster.

Flies carrying a kcc loss-of-function mutation are more seizure-susceptible than wild-type flies. The kcc gene is the highly conserved Drosophila melanogaster ortholog of K+/Cl- cotransporter genes thought to be expressed in all animal cell types. Here, we examined the spatial and temporal requirements for kcc loss-of-function to modify seizure-susceptibility in flies. Targeted RNA interference (RNAi) of kcc in various sets of neurons was sufficient to induce severe seizure-sensitivity. Interestingly, kcc RNAi in glia was particularly effective in causing seizure-sensitivity. Knockdown of kcc in glia or neurons during development caused a reduction in seizure induction threshold, cell swelling, and brain volume increase in 24-48 hour old adult flies. Third instar larval peripheral nerves were enlarged when kcc RNAi was expressed in neurons or glia. Results suggest that a threshold of K+/Cl- cotransport dysfunction in the nervous system during development is an important determinant of seizure-susceptibility in Drosophila. The findings presented are the first attributing a causative role for glial cation-chloride cotransporters in seizures and epileptogenesis. The importance of elucidating glial cell contributions to seizure disorders and the utility of Drosophila models is discussed.

Seizure-sensitivity in Drosophila is ameliorated by dorsal vessel injection of the antiepileptic drug valproate.

Drosophila is a powerful model organism that can be used for the development of new drugs directed against human disease. A limitation is the ability to deliver drugs for testing. We report on a novel delivery system for treating Drosophila neurological mutants, direct injection into the circulatory system. Using this method, we show that injection of the antiepileptic drug valproate can ameliorate seizure-sensitive phenotypes in several mutant genotypes in the bang-sensitive (BS) paralytic mutant class, sda, eas, and para(bss1). This drug-injection method is superior to drug-feeding methods that we have employed previously, presumably because it bypasses potent detoxification systems present in the fly. In addition, we find that utilizing blood-brain barrier mutations in the background may further improve the injection results under certain circumstances. We propose that this method of drug delivery is especially effective when using Drosophila to model human pathologies, especially neurological syndromes.

Drosophila as a model for intractable epilepsy: gilgamesh suppresses seizures in para(bss1) heterozygote flies.

Intractable epilepsies, that is, seizure disorders that do not respond to currently available therapies, are difficult, often tragic, neurological disorders. Na(+) channelopathies have been implicated in some intractable epilepsies, including Dravet syndrome (Dravet 1978), but little progress has been forthcoming in therapeutics. Here we examine a Drosophila model for intractable epilepsy, the Na(+) channel gain-of-function mutant para(bss1) that resembles Dravet syndrome in some aspects (parker et al. 2011a). In particular, we identify second-site mutations that interact with para(bss1), seizure enhancers, and seizure suppressors. We describe one seizure-enhancer mutation named charlatan (chn). The chn gene normally encodes an Neuron-Restrictive Silencer Factor/RE1-Silencing Transcription factor transcriptional repressor of neuronal-specific genes. We identify a second-site seizure-suppressor mutation, gilgamesh (gish), that reduces the severity of several seizure-like phenotypes of para(bss1)/+ heterozygotes. The gish gene normally encodes the Drosophila ortholog of casein kinase CK1g3, a member of the CK1 family of serine-threonine kinases. We suggest that CK1g3 is an unexpected but promising new target for seizure therapeutics.

Rescue of easily shocked mutant seizure sensitivity in Drosophila adults.

Genetic factors that influence seizure susceptibility can act transiently during the development of neural circuits or might be necessary for the proper functioning of existing circuits. We provide evidence that the Drosophila seizure-sensitive mutant easily shocked (eas) represents a neurological disorder in which abnormal functioning of existing neural circuits leads to seizure sensitivity. The eas(+) gene encodes for the protein Ethanolamine Kinase, involved in phospholipid biosynthesis. We show that induction of eas(+) in adult mutant flies rescues them from seizure sensitivity despite previously known developmental defects in brain morphology. Additionally, through cell-type-specific rescue, our results suggest a specific role for eas(+) in excitatory rather than inhibitory neural transmission. Overall, our findings emphasize an important role for proper phospholipid metabolism in normal brain function and suggest that certain classes of epilepsy syndromes could have the potential to be treated with gene therapy techniques.

Seizure and epilepsy: studies of seizure disorders in Drosophila.

Despite the frequency of seizure disorders in the human population, the genetic and physiological basis for these defects has been difficult to resolve. Although many genetic contributions to seizure susceptibility have been identified, these involve disparate biological processes, many of which are not neural specific. The large number and heterogeneous nature of the genes involved makes it difficult to understand the complex factors underlying the etiology of seizure disorders. Examining the effect known genetic mutations have on seizure susceptibility is one approach that may prove fruitful. This approach may be helpful in both understanding how different physiological processes affect seizure susceptibility and identifying novel therapeutic treatments. We review here factors contributing to seizure susceptibility in Drosophila, a genetically tractable system that provides a model for human seizure disorders. Seizure-like neuronal activities and behaviors in the fruit fly are described, as well as a set of mutations that exhibit features resembling some human epilepsies and render the fly sensitive to seizures. Especially interesting are descriptions of a novel class of mutations that are second-site mutations that act as seizure suppressors. These mutations revert epilepsy phenotypes back to the wild-type range of seizure susceptibility. The genes responsible for seizure suppression are cloned with the goal of identifying targets for lead compounds that may be developed into new antiepileptic drugs.