Abstracts

Rationale: The high fat, low carbohydrate ketogenic diet is a remarkably effective treatment for epilepsy, though its mechanism is far from understood. One of the primary metabolic consequences of for patients on the ketogenic diet is a rise in concentration of ketone bodies in the blood and CSF. Previously, we have shown that in vitro application of ketone bodies leads to a dampening of neuronal excitability in mouse brain slices. This effect is due to the opening of ATP-sensitive potassium (KATP) channels as it is eliminated via pharmacological block or in a genetic knock-out of Kir 6.2, the pore forming region of the KATP channel. To confirm that the ketone body effect is not due to the involvement of mitochondrial KATP (mitoKATP) channels, we sought independent evidence that these channels do indeed persist in the Kir 6.2 knock-out mice. Specifically, we investigated one of the phenomena associated with mitoKATP, ROS production in response to the application of diazoxide, a classical KATP channel opener.Methods: We utilized acute brain slices from P13-16 BL6 (wild-type) or Kir6.2 knock-out mice. Whole-cell patch clamp recordings were made from GABAergic neurons of the Substantia Nigra pars reticula in acute slices bathed in ACSF (12mM Glucose and bubbled with 95%O2 and 5%CO2). The recording pipette contained 20 µM of the a novel cell-impermeant reactive oxygen species (ROS) sensitive dye, reduced dichlorofluorescein – cysteic acid (H2DCF-CA). Images were acquired every 30 s to reduce photobleaching and the concentration of ROS in the cell was represented by the accumulation of the fluorescent, oxidized form of the dye. Following 10 minutes of baseline ROS detection, either diazoxide (200 µM) or the physiological ketone body R-β-hydroxybutyrate (2 mM) were added and the ROS signal was recorded for an additional 40 minutes.Results: We found that a phenomenon attributable to mitoKATP, an ROS burst due to the KATP channel opener diazoxide, is present in neurons both in wild-type and the Kir6.2 channel knock-out. Furthermore, using both H2DCF-CA and the genetically encoded ROS sensor HyPer, we found that ketone body metabolism itself produces an acute increase in reactive oxygen species.Conclusions: The persistence of mitoKATP channels in the Kir6.2 knock-out mouse strengthens the evidence that the ketogenic diet may dampen neuronal excitability via the opening of surface membrane KATP channels. Additionally, an ROS increase due to ketone body metabolism, in combination with the known neuronal activity-dependent ROS production, may have important implications in the anti-epileptic effect of the ketogenic diet.