Abstract
Although there are now a number of antiepileptic drugs (AEDs) available, approximately one-third of epilepsy patients respond poorly to drug intervention. The reasons for this are complex, but are probably reflective of the increasing number of identified mutations that predispose individuals to this disease. Thus, there is a clear requirement for the development of novel treatments to address this unmet clinical need. The existence of gene mutations that mimic a seizure-like behaviour in the fruit fly, Drosophila melanogaster, offers the possibility to exploit the powerful genetics of this insect to identify novel cellular targets to facilitate design of more effective AEDs. In this study we use neuronal expression of GCaMP, a potent calcium reporter, to image neuronal activity using a non-invasive and rapid method. Expression in motoneurons in the isolated CNS of third instar larvae shows waves of calcium-activity that pass between segments of the ventral nerve cord. Time between calcium peaks, in the same neurons, between adjacent segments usually show a temporal separation of greater than 200 ms. Exposure to proconvulsants (picrotoxin or 4-aminopyridine) reduces separation to below 200 ms showing increased synchrony of activity across adjacent segments. Increased synchrony, characteristic of epilepsy, is similarly observed in genetic seizure mutants: bangsenseless1 (bss1) and paralyticK1270T (paraK1270T). Exposure of bss1 to clinically-used antiepileptic drugs (phenytoin or gabapentin) significantly reduces synchrony. In this study we use the measure of synchronicity to evaluate the effectiveness of known and novel anticonvulsive compounds (antipain, isethionate, etopiside rapamycin and dipyramidole) to reduce seizure-like CNS activity. We further show that such compounds also reduce the Drosophila voltage-gated persistent Na+ current (INaP) in an identified motoneuron (aCC). Our combined assays provide a rapid and reliable method to screen unknown compounds for potential to function as anticonvulsants.
Highlights
Epilepsy, which is the recurrence of spontaneous and seemingly unprovoked seizures, is a significant clinical disorder affecting ~1% of the global population
We show that imaging calcium-induced activity with GCaMP5 expression in motoneurons correlates well with action potential firing in these neurons, as measured by simultaneous loose-patch recordings
In the large majority of these studies validation of a linear relationship between GCaMP-signal and action potential firing has not been established. Prior to using this technique to image activity, we verified that the calcium signal we obtain from GCaMP expression in motoneurons of third instar isolated CNS mirror their electrical activity
Summary
Epilepsy, which is the recurrence of spontaneous and seemingly unprovoked seizures, is a significant clinical disorder affecting ~1% of the global population. Treatment is commonly through chronic administration of antiepileptic drugs (AEDs), many of which can have severe side-effects. Drug treatment has the severe limitation in that only about two-thirds of patients show reduction in frequency of seizure occurrence. No effective treatment is currently available [1]. Genetics is believed to be a primary cause of many types of epilepsy and the number of genetic loci considered to be contributory to seizure exceeds 500 [2]. Of these loci, mutations in neuronal sodium channels are considered to be the most common genetic cause of epilepsy in humans [3]. Identification of genetic mutations offers the possibility to identify novel targets to facilitate next-generation AED design
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