Abstracts

Rationale: Mutations in the voltage gated sodium channel gene SCN1A have been linked to a wide spectrum of epilepsy disorders including genetic epilepsy with febrile seizures plus (GEFS+) and Dravet Syndrome (DS). Individuals with these disorders, especially DS, often respond poorly to existing anti-epileptic drugs. In this study we have used Drosophila as a rapid and low cost model system to search for new therapies to treat these disorders. Our data demonstrate that two different fly knock-in lines carrying SCN1A epilepsy-causing mutations (GEFS+ K1270T and DS S1231R) exhibit heat-induced seizures. Consistent with disease symptoms in humans, the seizure phenotype is more severe in DS compared to GEFS+ flies. Using a simple behavioral assay we can rapidly screen for modifiers that suppress heat-induced seizures in the DS and/or GEFS+ flies. Our initial studies have focused on monoamine signaling since we found that seizure-sensitivity was altered in DS and GEFS+ flies in a genetic background (white eyes) that affects monoamine levels.Methods: Two-day old female DS and GEFS+ flies were fed for 3 days on standard cornmeal food containing 0, 5, 25 or 50 mM 5-hydroxytryptophan (5-HTP), the immediate serotonin precursor. To examine sensitivity to heat-induced seizures, flies were placed in vials and heated in a water bath for 2 minutes. The flies were tested at temperatures at which the seizure probability at 2 minutes is ~50% (DS at 34 C and GEFS+ flies at 38 C). The total time flies spent seizing were determined for each of the four different treatment groups and compared using one-way ANOVA with Bonferroni post hoc test. Whole cell recordings from GABAergic local neurons were performed in the isolated whole brain preparation.Results: In GEFS+ flies, 5-HTP increased the sensitivity to heat-induced seizure in a dose dependent manner (n>45 flies per concentration). The total seizing time in flies fed 50 mM 5-HTP increased ~3 fold over flies that did not have 5-HTP in the food (28 3 to 79 4 s; p<0.001, n>45 flies/concentration). In DS flies in contrast, 5-HTP reduced the seizing time in a dose dependent manner (n>60 flies per concentration). During the 120 s heating period, the total seizing time in DS flies treated with 50 mM 5-HTP was reduced by 65% compared to flies that did not receive 5-HTP (70 4 s to 24 6 s) (p<0.001). To evaluate cellular mechanisms of seizure suppression we recorded from GABAergic LNs. Our previous work showed that the spontaneous burst frequency in LNs is significantly reduced in DS knock-ins compared to the Control line. In DS flies treated with 5-HTP the spontaneous burst firing frequency showed partial recovery, increasing from 0.6 0.1Hz in untreated to 1.0 0.1Hz in 50 mM 5-HTP (t-test, p<0.05).Conclusions: These data suggest that the serotonin pathway regulates seizure phenotypes caused by SCN1A mutations, implicating it as an alternative therapeutic target for the treatment of certain epileptic disorders.