Abstracts

Rationale: Seizures and status epilepticus (SE) in adults can be refractory to conventional AEDs. Pharmacoresistance is thought to result from changes in GABAergic inhibition which is based largely on insensitivity to diazepam. Effective GABAergic inhibition requires the maintenance of the chloride gradient, accomplished by the K+/Cl- co-transporter, KCC2. Seizure induced up-regulation of NKCC1, alterations in KCC2 and deficits in GABAergic inhibition have been observed in animal models of epilepsy and in epileptic tissue from humans with temporal lobe epilepsy. To understand the role of chloride homeostasis in seizure progression and the development of pharmacoresistance, we investigated the anticonvulsant potential of the NKCC1 antagonist, bumetanide, in vitro and in vivo.Methods: The anticonvulsant effects of bumetanide were assessed using in vivo EEG recordings of kainic acid induced seizures in 8-10 week old adult C57BL6 male mice and in vitro using the 0-Mg2+ model in acute brain slices. The ability of bumetanide to prevent the development of pharmacoresistance was determined by administering bumetanide in combinations with diazepam. Mice expressing channelrhodopsin specifically in GAD+ interneurons (ChR/GAD2) and mice lacking the GABAA receptor delta-subunit specifically in interneurons (Gabrd/GAD) were used for investigating whether bumetanide mediates its actions through the GABAergic network.Results: We show that bumetanide can effectively suppress seizures both in vivo and in vitro. Mice treated with bumetanide 30 minutes before administration of kainic acid showed reduced epileptiform activity in vivo. This effect was also observed in vitro with the reduction in duration of 0-Mg2+ induced seizure-like events (SLEs) by bumetanide. Interestingly, bumetanide reduced the duration of ictal discharges while increasing and sustaining persistent low-frequency activity. In addition, bumetanide can restore the efficacy of diazepam in suppressing seizures during status epilepticus. Together, our data suggest that bumetanide likely mediates is actions through the GABAergic network. To investigate whetherbumetanide maintains the inhibitory of actions of GABA throughout seizure progression and the development of status epilepticus, we utilized ChR/GAD2 mice to show that optogenetically driven GABAergic signaling can suppress seizures in the presence of bumetanide. Further, bumetanide was more effective at suppressing SLEs in mice with enhanced GABAergic drive onto principal neurons, strengthening our hypothesis that bumetanide likely mediates its anticonvulsant actions through the GABAergic inhibitory network.Conclusions: Our findings suggest that bumetanide treatment may preserve GABAergic inhibition and provide important preclinical support for of the use of bumetanide in combination with conventional AEDs.