Abstracts

We have shown that ketone bodies, both [beta]-hydroxybutyrate (BHB) and acetoacetate, at physiologic concentrations, have direct antiepileptic properties and that this action is mediated by enhanced activity at the GABA[sub]A[/sub] receptor. A possible mechanism of action for this effect is suggested by the negative shift in the reversal potential of the GABA[sub]A[/sub]- mediated IPSP (E[sub]GABA[/sub]) produced by BHB and was investigated further.
Hippocampi were prepared from 7 day old Wistar rats according to the method of Niesen and Ge (1999). Whole cell patch recordings were performed on CA1 neurons after 2-7 days [italic]in vitro[/italic] (DIV). The microelectrode contained (in mM): 140 KGluc, 10 HEPES, 0.1 CaCl[sub]2[/sub], 1.1 BAPTA, 2 Mg-ATP and 0.1 GTP. For these experiments, BHB was perfused onto brain slices at 0.1-1 mM to reflect known physiologic levels. GABA[sub]A[/sub]- mediated potentials were studied in the presence of CNQX (50 [mu]M) and APV (30 [mu]M) that blocked AMPA and NMDA receptor activity, respectively.
Perfusion of 0.1-1 mM BHB produced a 20-50% increase in the early IPSP amplitude recorded in CA1 neurons from cultured rat hippocampal slices. This effect was due to a 10 mV negative shift in the E[sub]GABA[/sub]. E[sub]GABA[/sub] is determined primarily by E[sub]Cl[/sub] since the GABA[sub]A[/sub] ionophore is five fold more permeable to Cl^- than to HCO^-[sub]3[/sub]. HCO^-[sub]3[/sub], however, tends to efflux from the cell and thus, competes with the hyperpolarizing effect of Cl^- influx.
To test the hypothesis that BHB alters anion flux through the GABA[sub]A[/sub] receptor, we replaced HCO^-[sub]3[/sub] in the perfusate with HEPES. Under these conditions, BHB did not potentiate the early IPSP. To prevent the generation of bicarbonate in these cells, 10 [mu]M acetazolamide, a known inhibitor of carbonic anhydrase, the enzyme that mediates the synthesis of HCO^-[sub]3[/sub], was added to the perfusate. BHB was again unable to increase the IPSP amplitude. In the third experiment, 1 mM amiloride which blocks Na⁺-H⁺ exchange, produced a negative shift in the E[sub]GABA[/sub] and prevented the action of BHB.
BHB is a potent agonist of GABA-mediated inhibition. By reducing the efflux of the HCO^-[sub]3[/sub] anion through the GABA ionophore, BHB produced a net increase in Cl^- influx, causing a larger hyperpolarizing response in these neurons.
[Supported by: The Charlie Foundation]