Abstracts

Rationale: Control of seizures and epileptogenesis by modulation of metabolic processes such as glycolysis and mitochondrial metabolism is gaining renewed interest in mainstay epilepsy research. The importance of metabolic fuels in controlling seizures is highlighted by the anticonvulsant effect of decreasing glucose utilization via dietary therapies e.g. the ketogenic diet and 2-deoxyglucose. Implicit in these observations is that seizure activity or increased neuronal excitation increases glycolysis due to inherent mitochondrial dysfunction despite adequate oxygen availability i.e. induces the Warburg Effect. However, whether and how neuronal excitation increases glycolytic rates is poorly understood. We addressed this question by simultaneously measuring glycolytic flux and mitochondrial respiration in two model systems, primary cortical cultures and zebrafish embryos. Methods: Primary neuronal cultures (2 week-old) or zebrafish (4 days post-fertilization) were subjected to metabolic analysis by measuring real-time bioenergetics in an extracellular flux analyzer (XF Analyzer) which allows simultaneous measurement of glycolytic rates (extracellular acidification rates or ECAR) and mitochondrial respiration (oxygen consumption rates or OCR). Neuronal excitability was increased by treatment with two classical potassium channel blockers, 4-aminopyridine (4-AP) and tetraethylammine (TEA).Results: In primary cortical cultures 4-AP or TEA resulted in an instant 1.5 fold increase in basal and maximal glycolytic rates (n=15, p<0.01), which, in the case of 4-AP returned to control values after 4 hours. ECAR rates from TEA treated cultures are returned to control values at the 24 hour time-point. Mitochondrial was not altered after 4-AP treatment, but an immediate increase in OCR occurred after TEA treatment. 4-AP resulted in an overall 2-fold decrease in OCR after 4 hours (n=15, p<0.01), coinciding with the return of glycolysis to control rates and continued to decrease for the remainder of the time course. Likewise, OCR in the TEA treated cells decreased 2-fold at the 24 hour time-point (n=5, p<0.01). These experiments reveal a time course in which there is an initial increase in glycolysis that returns to baseline followed by an overall decrease in mitochondrial function. Further, we optimized assessment of bioenergetics using live zebrafish embryos. In zebrafish at 4 days post fertilization, 4-AP treatment of whole embryos produced a rapid 6-fold increase in ECAR (n=10, p<0.01) underscoring the utility of zebrafish as a model to study bioenergetics in response to seizure-like events or epilepsy mutations. Conclusions: The data suggests that increased neuronal excitation in cortical cultures results in immediate increase in glycolytic rates followed by a slow decline in mitochondrial respiration. The characterization of bioenergetic changes following neuronal excitability can lead to novel insights regarding the metabolic regulation of seizures and epileptogenesis.