Abstracts

Seizure activity is known to produce oxidative stress in neurons; conversely, recent studies have shown that exogenous oxidant administration results in neuronal hyperexcitability. Given recent data suggesting a neuroprotective role of ketone bodies, we asked whether [beta]-hydroxybutyrate (BHB) and acetoacetate (ACA) can attenuate or prevent hydrogen peroxide-induced hyperexcitability of layer V neocortical neurons, and whether these ketones can directly reduce reactive oxygen species (ROS) production in isolated mitochondria. Transverse 300 [mu]m brain slices from somatosensory cortex of normal 2-3 week-old Sprague-Dawley rats were acutely prepared, and whole-cell electrophysiological recordings were made of layer V pyramidal neurons under direct visualization with infrared microscopy and Nomarski optics. Hydrogen peroxide and ketone bodies were bath applied at 31[deg]C, and data were acquired with pCLAMP software and digitized for off-line analysis. For ROS assays, mitochondria were acutely isolated with standard subcellular fractionation techniques, and both basal and oligomycin-induced ROS production was measured using the indicator H[sub]2[/sub]DCFDA (dichlorofluorescein). Bath application of hydrogen peroxide (1.25 mM) elicited an initial hyperpolarization which was associated with a large decrease in input resistance (R[sub]N[/sub]), and upon washout, was followed by an irreversible depolarization (in 9 of 14 cells tested). When slices were pre-incubated with a cocktail of 1 mM BHB and 1 mM ACA for 30 minutes, subsequent administration of 1.25 mM hydrogen peroxide prevented the irreversible depolarization upon washout, but not the initial hyperpolarization (in 7 of 8 neurons tested). In additional control experiments, co-application of BHB and ACA (both 1 mM) to layer V pyramidal neurons decreased R[sub]N[/sub], but did not alter resting membrane potential or spike threshold. In mitochondrial experiments, both BHB and ACA directly reduced basal and oligomycin-induced ROS production (N=6/group, P[lt]0.05). The major ketone bodies BHB and ACA prevent the hyperexcitability of neocortical neurons exposed to acute oxidative stress. This effect could be mediated through a direct antioxidant effect of BHB and ACA at the mitochondrial level. Thus, the antioxidant action of ketone bodies may contribute in part to the anticonvulsant effect of the ketogenic diet. (Supported by NIH K02 NS 044846 (JMR) and VA Merit Review (WJS).)