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Cation-Chloride Cotransporters and Seizure State Transitions in Drosophila


Seizure disorders, including the epilepsies, are debilitating misfortunes suffered by over 1% of the world's human population. Spontaneous recurrent seizures--paroxysmal events characterized by large groups of hyperexcited and hypersynchronized neurons--are hallmarks of epilepsy and destructive to the quality of life of many individuals. Although antiepileptic drug (AED) development is continuingly improving, available cures for attenuating and reducing the frequency of seizures are completely ineffective for a third of epileptics. Basic research concerning properties of seizures and their causes will yield critical insight required for the design of novel therapeutics. Such scientific advances may arise from experimentation using model organisms, with one such promising animal model being the fruit fly, Drosophila melanogaster.

Flies, like most other organisms with complex nervous systems given a sufficient stimulus, exhibit transitions between behavioral seizure states with a detectable underlying neural correlate. Conservation of nervous system genes and functions throughout evolution has made the fly a relevant model for human seizure disorders. In addition to such relevance, the advantages of the Drosophila system techniques enable finer dissection and identification of members in molecular pathways related to seizure-genesis, seizure-propagation, seizure-termination and recovery. For my thesis work I capitalized on the powerful tools available in Drosophila to attempt to tease apart the pleiotropic functions of cation-chloride cotransporters (CCCs) throughout the nervous system that are important for seizure manifestation. I also screened a collection of chromosomal deletions for their effect on behavioral paralysis after a seizure in hope of eventually identifying endogenous seizure-suppression and recovery mechanisms.

kcc mutants are more seizure-susceptible than wild-type flies. kcc is the highly conserved Drosophila ortholog of K+/Cl- cotransporter genes (KCCs) thought to be expressed in all animal cell types. I examined the spatial and temporal requirements for kcc loss-of-function to modify seizure-susceptibility in flies. Targeted RNAi of kcc in various sets of neurons is sufficient to induce severe seizure-sensitivity. Interestingly, kcc RNAi in glia was found to be particularly effective in causing seizure-sensitivity. kcc knockdown during development causes reduction in seizure induction threshold, lengthening of seizure refractory period, cell swelling and blood-brain barrier (BBB) degradation in adult flies. Results suggest that a threshold of K+/Cl- cotransport dysfunction in the nervous system during development, and concomitant malformation of the BBB, are major determinants of seizure-susceptibility in Drosophila.

Although many genes and synaptic/non-synaptic mechanisms linked with seizure-susceptibility have been described, relatively much less is known about how seizures stop, regardless of etiology. Theoretically, given the significant chance of seizure occurrence, there exist mechanisms for terminating, and recovering from, seizures. I hypothesized that defects in such mechanisms would affect post-seizure states such as the postictal or refractory state of flies. To identify such possible mechanisms, I screened over 150 chromosomal deletions for their affect on the duration of paralysis of the seizure-susceptible bang-sensitive (BS) mutant, parabss1. 5 deletions gave rise to significant lengthening of parabss1 mean recovery time (MRT). charlatan, chn, a gene encoding a NRSF/REST transcriptional repressor, was identified as the causative gene for one of these deletions. chn RNAi caused an increase in paralysis times and synaptic failure duration after a parabss1 seizure, but seizure induction threshold was not significantly altered. RNAi of chn lengthened MRT of another BS mutant, eas, suggesting that chn is a general seizure-enhancer. Although identification of chn as a general seizure-enhancer did not expose specific molecular mechanisms, the screen was a proof of principle for the postulate that genetic factors may impact different aspects of seizures, some of which have been overlooked during the history of seizure disorder research.

My thesis lays a foundation for better understanding the role of CCCs in nervous system disease and draws needed attention to the relatively neglected postictal state. Specific manipulation and marking of glia in Drosophila has implicated CCC functions in these cells as important factors in seizure-susceptibility before and after seizures. Seizure-sensitivity arising from CCC loss can be further examined with precision using manipulations and rescue experiments already possible with today's techniques. Extracellular ion regulations are critical factors affecting seizure-onset, and perhaps also, seizure-recovery and refractory states. Nervous system parameters unique to seizures might trigger specialized mechanisms involved in restoring normal conditions after seizures and rendering the reoccurrence of seizures less-likely for some time. Examining effects of well-controlled perturbations on all aspects of seizures is critical for solving problems presented by seizure disorders.

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