Slo2/KNa Channels in Drosophila Protect against Spontaneous and Induced Seizure-like Behavior Associated with an Increased Persistent Na+ Current.

Na+ sensitivity is a unique feature of Na+-activated K+ (KNa) channels, making them naturally suited to counter a sudden influx in Na+ ions. As such, it has long been suggested that KNa channels may serve a protective function against excessive excitation associated with neuronal injury and disease. This hypothesis, however, has remained largely untested. Here, we examine KNa channels encoded by the Drosophila Slo2 (dSlo2) gene in males and females. We show that dSlo2/KNa channels are selectively expressed in cholinergic neurons in the adult brain, as well as in glutamatergic motor neurons, where dampening excitation may function to inhibit global hyperactivity and seizure-like behavior. Indeed, we show that effects of feeding Drosophila a cholinergic agonist are exacerbated by the loss of dSlo2/KNa channels. Similar to mammalian Slo2/KNa channels, we show that dSlo2/KNa channels encode a TTX-sensitive K+ conductance, indicating that dSlo2/KNa channels can be activated by Na+ carried by voltage-dependent Na+ channels. We then tested the role of dSlo2/KNa channels in established genetic seizure models in which the voltage-dependent persistent Na+ current (INap) is elevated. We show that the absence of dSlo2/KNa channels increased susceptibility to mechanically induced seizure-like behavior. Similar results were observed in WT flies treated with veratridine, an enhancer of INap Finally, we show that loss of dSlo2/KNa channels in both genetic and pharmacologically primed seizure models resulted in the appearance of spontaneous seizures. Together, our results support a model in which dSlo2/KNa channels, activated by neuronal overexcitation, contribute to a protective threshold to suppress the induction of seizure-like activity.SIGNIFICANCE STATEMENT Slo2/KNa channels are unique in that they constitute a repolarizing K+ pore that is activated by the depolarizing Na+ ion, making them naturally suited to function as a protective "brake" against overexcitation and Na+ overload. Here, we test this hypothesis in vivo by examining how a null mutation of the Drosophila Slo2 (dSlo2)/KNa gene affects seizure-like behavior in genetic and pharmacological models of epilepsy. We show that indeed the loss of dSlo2/KNa channels results in increased incidence and severity of induced seizure behavior, as well as the appearance of spontaneous seizure activity. Our results advance our understanding of neuronal excitability and protective mechanisms that preserve normal physiology and the suppression of seizure susceptibility.

Flight and seizure motor patterns in Drosophila mutants: simultaneous acoustic and electrophysiological recordings of wing beats and flight muscle activity.

Abstract Tethered flies allow studies of biomechanics and electrophysiology of flight control. We performed microelectrode recordings of spikes in an indirect flight muscle (the dorsal longitudinal muscle, DLMa) coupled with acoustic analysis of wing beat frequency (WBF) via microphone signals. Simultaneous electrophysiological recording of direct and indirect flight muscles has been technically challenging; however, the WBF is thought to reflect in a one-to-one relationship with spiking activity in a subset of direct flight muscles, including muscle m1b. Therefore, our approach enables systematic mutational analysis for changes in temporal features of electrical activity of motor neurons innervating subsets of direct and indirect flight muscles. Here, we report the consequences of specific ion channel disruptions on the spiking activity of myogenic DLMs (firing at ∼5 Hz) and the corresponding WBF (∼200 Hz). We examined mutants of the genes enconding: 1) voltage-gated Ca(2+) channels (cacophony, cac), 2) Ca(2+)-activated K(+) channels (slowpoke, slo), and 3) voltage-gated K(+) channels (Shaker, Sh) and their auxiliary subunits (Hyperkinetic, Hk and quiver, qvr). We found flight initiation in response to an air puff was severely disrupted in both cac and slo mutants. However, once initiated, slo flight was largely unaltered, whereas cac displayed disrupted DLM firing rates and WBF. Sh, Hk, and qvr mutants were able to maintain normal DLM firing rates, despite increased WBF. Notably, defects in the auxiliary subunits encoded by Hk and qvr could lead to distinct consequences, that is, disrupted DLM firing rhythmicity, not observed in Sh. Our mutant analysis of direct and indirect flight muscle activities indicates that the two motor activity patterns may be independently modified by specific ion channel mutations, and that this approach can be extended to other dipteran species and additional motor programs, such as electroconvulsive stimulation-induced seizures.

Drosophila para bss Flies as a Screening Model for Traditional Medicine: Anticonvulsant Effects of Annona senegalensis.

Epilepsy is among the most common serious neurological disorders and affects around 50 million people worldwide, 80% of which live in developing countries. Despite the introduction of several new Anti-Epileptic Drugs (AEDs) in the last two decades, one third of treated patients have seizures refractory to pharmacotherapy. This highlights the need to develop new treatments with drugs targeting alternative seizure-induction mechanisms. Traditional medicine (TM) is used for the treatment of epilepsy in many developing countries and could constitute an affordable and accessible alternative to AEDs, but a lack of pre-clinical and clinical testing has so far prevented its wider acceptance worldwide. In this study we used Drosophila melanogaster paralytic bangsensitive (para bss ) mutants as a model for epileptic seizure screening and tested, for the first time, the anti-seizure effect of a non-commercial AED. We evaluated the effect of the African custard-apple, Annona senegalensis, which is commonly used as a TM for the treatment of epilepsy in rural Africa, and compared it with the classical AED phenytoin. Our results showed that a stem bark extract from A. senegalensis was significantly more effective than a leaf extract and similar to phenytoin in the prevention and control of seizure-like behavior. These results support that Drosophila constitutes a robust animal model for the screening of TM with potential value for the treatment of intractable epilepsy.

Extending julius seizure, a bang-sensitive gene, as a model for studying epileptogenesis: Cold shock, and a new insertional mutation.

The bang-sensitive (BS) mutants of Drosophila are an important model for studying epilepsy. We recently identified a novel BS locus, julius seizure (jus), encoding a protein containing two transmembrane domains and an extracellular cysteine-rich loop. We also determined that jussda iso7.8, a previously identified BS mutation, is an allele of jus by recombination, deficiency mapping, complementation testing, and genetic rescue. RNAi knockdown revealed that jus expression is important in cholinergic neurons and that the critical stage of jus expression is the mid-pupa. Finally, we found that a functional, GFP-tagged genomic construct of jus is expressed mostly in axons of the neck connectives and of the thoracic abdominal ganglia. In this Extra View article, we show that a MiMiC GFP-tagged Jus is localized to the same nervous system regions as the GFP-tagged genomic construct, but its expression is mostly confined to cell bodies and it causes bang-sensitivity. The MiMiC GFP-tag lies in the extracellular loop while the genomic construct is tagged at the C-terminus. This suggests that the alternate position of the GFP tag may disrupt Jus protein function by altering its subcellular localization and/or stability. We also show that a small subset of jus-expressing neurons are responsible for the BS phenotype. Finally, extending the utility of the BS seizure model, we show that jus mutants exhibit cold-sensitive paralysis and are partially sensitive to strobe-induced seizures.

Shortened Lifespan and Other Age-Related Defects in Bang Sensitive Mutants of Drosophila melanogaster.

Mitochondrial diseases are complex disorders that exhibit their primary effects in energetically active tissues. Damage generated by mitochondria is also thought to be a key component of aging and age-related disease. An important model for mitochondrial dysfunction is the bang sensitive (bs) mutants in Drosophila melanogaster Although these mutants all show a striking seizure phenotype, several bs mutants have gene products that are involved with mitochondrial function, while others affect excitability another way. All of the bs mutants (parabss , eas, jus, ses B, tko are examined here) paralyze and seize upon challenge with a sensory stimulus, most notably mechanical stimulation. These and other excitability mutants have been linked to neurodegeneration with age. In addition to these phenotypes, we have found age-related defects for several of the bs strains. The mutants eas, ses B, and tko display shortened lifespan, an increased mean recovery time from seizure with age, and decreased climbing ability over lifespan as compared to isogenic CS or w1118 lines. Other mutants show a subset of these defects. The age-related phenotypes can be rescued by feeding melatonin, an antioxidant, in all the mutants except ses B The age-related defects do not appear to be correlated with the seizure phenotype. Inducing seizures on a daily basis did not exacerbate the phenotypes and treatment with antiepileptic drugs did not increase lifespan. The results suggest that the excitability phenotypes and the age-related phenotypes may be somewhat independent and that these phenotypes mutants may arise from impacts on different pathways.

julius seizure, a Drosophila Mutant, Defines a Neuronal Population Underlying Epileptogenesis.

Epilepsy is a neural disorder characterized by recurrent seizures. Bang-sensitive Drosophila represent an important model for studying epilepsy and neuronal excitability. Previous work identified the bang-sensitive gene slamdance (sda) as an allele of the aminopeptidase N gene. Here we show through extensive genetic analysis, including recombination frequency, deficiency mapping, transposon insertion complementation testing, RNA interference (RNAi), and genetic rescue that the gene responsible for the seizure sensitivity is julius seizure (jus), formerly CG14509, which encodes a novel transmembrane domain protein. We also describe more severe genetic alleles of jus RNAi-mediated knockdown of jus revealed that it is required only in neurons and not glia, and that partial bang-sensitivity is caused by knockdown in GABAergic or cholinergic but not glutamatergic neurons. RNAi knockdown of jus at the early pupal stages leads to strong seizures in adult animals, implicating that stage as critical for epileptogenesis. A C-terminal-tagged version of Jus was generated from a fosmid genomic clone. This fosmid fusion rescued the bang-sensitive phenotype and was expressed in the optic lobes and the subesophageal and thoracic abdominal ganglia. The protein was primarily localized in axons, especially in the neck connectives, extending into the thoracic abdominal ganglion.

The ketogenic diet metabolite beta-hydroxybutyrate (ß-HB) reduces incidence of seizure-like activity (SLA) in a Katp- and GABAb-dependent manner in a whole-animal Drosophila melanogaster model.

The high-fat, low-carbohydrate ketogenic diet (KD) is an effective clinical treatment for epilepsy in juveniles, especially for drug-resistant seizures. The KD results in elevated production of ketone bodies (KB's), such as beta-hydroxybutyrate (ß-HB), which are thought to have anticonvulsant properties; however, their exact mechanism of action is unknown. In vitro, KB effects on reducing neuronal firing rates are mediated in part by Katp channel activity and GABAb signaling. In order to study metabolic and pharmacological effects in a whole-animal model, we used the eas "bang-sensitive" (BS) mutant strain of Drosophila, which exhibits seizure-like activity (SLA) upon mechanical stimulation. Direct application of the KB ß-HB to food reduced BS SLA. Application either of tolbutamide, a Katp blocker, or of CGP-55845, a GABAb antagonist, concomitantly with ß-HB, partially reversed these KB effects on SLA, verifying a role for Katp channels and GABAb signaling in mediating the anticonvulsant effects of KB's and validating this whole-animal model of KD effects on seizure.