317 - Glycolytic and redox control of neuronal excitability in vitro

Abstract

Metabolic control of epilepsy is recognized in part due to the efficacy of ketogenic diets, which provide alternate fuels rather than glucose for neuronal activity. It is documented that glycolytic rates are acutely increased during epileptic seizures and inhibition of glycolysis with 2-deoxyglucose (2-DG) has anti-seizure effects. Work from our laboratory has shown that seizure activity increases the steady-state levels of reactive oxygen species (ROS) production and causes mitochondrial dysfunction. Here we determined the relationship between glycolysis, ROS production and neuronal excitability. Mixed rat primary cortical cultures treated with 4-Aminopyridine (4-AP), a potassium channel blocker, showed an increase in a) Extracellular Acidification Rate (ECAR) levels (measure of glycolytic rate in the Seahorse XF analyzer), b) ROS production, measured by Amplex Red and c) increased neuronal excitability, assessed by a multiple electrode array system (Axion Biosystems). Pre-treatment of mixed primary cortical cultures with compounds like 2-DG, Bromopyruvic acid (3BP), palmitate - an anaplerotic substrate (along with glycogenic substrate limitation), nicotinamide riboside (NR) – an NAD+ precursor, oxaloacetate (OA) - a TCA cycle intermediate, and mTOR inhibitor rapamycin decreased ECAR rates and neuronal hyperexcitability induced by 4-AP. Pre-treatment with a reduced B-nicotinamide adenine dinucleotide (NADH), Thiazolidinediones – an acute specific inhibitor of the mitochondrial pyruvate carrier, PI3K inhibitor wortmannin decreased 4-AP induced increase in glycolysis, without significantly altering neuronal hyperexcitability. Lastly, 2-DG also reversed ROS production produced by 4-AP. Taken together, these results suggest that glycolysis contributes to ROS production, which in turn increases neuronal hyperexcitability.