Abstracts

Rationale: Dietary interventions such as the ketogenic diet (KD) have been proven to be effective in treating medically refractory epilepsy. However, the mechanisms underlying KD action remain unclear. Earlier studies have demonstrated that acetoacetate [ACA] and acetone are anticonvulsant in several in vivo models of induced seizures, but there is yet no in vitro evidence that ketone bodies can acutely influence neuronal hyperexcitability and/or synchrony [PMID: 10668691]. Whether beta-hydroxybutyrate (BHB) is anticonvulsant remains unclear as well. Methods: A novel photoconductive-stimulation (PCS) platform [PMID: 17406269] was used to study the chronic network effects of BHB in rat primary hippocampal cultures. PCS allows for the visualization and controlled - yet non-invasive stimulation - of a single neuron or hundreds of neurons in culture. This is accomplished by growing neuronal cultures on silicon chips and applying a beam of light to target cell(s) of interest while applying a voltage bias across the silicon wafer to induce a current, resulting in highly focused stimulation with minimal physiological manipulation. Two-week-old cultures were subsequently incubated for one week with either standard media or BHB at various concentrations (2mM, 5mM, and 10mM), then exposed to GFP-Fluor-4 which enabled real-time visualization of intracellular calcium levels associated with neuronal membrane firing. Video data were analyzed using custom software (MatLab) and excitation of the entire field (typically 500-1000 neurons) was quantified. Firing rates were calculated as the number of firing events of the neurons per minute. Results: We found that higher concentrations of BHB (5 & 10 mM; N =11 & 12, respectively) did not alter the average firing rate of the hippocampal neurons, whereas with 2 mM BHB, there was a significant (37%, p < 0.05) decrease compared to controls. The ratio of firing in the control vs. the 2mM cultures jumped from a baseline of 1 to 7.56, p=0.02 (a higher ratio correlates with a decrease in firing events per minute). Interestingly, 2mM BHB incubation also resulted in a 5-fold reduction in the number of active cells before PCS compared to during the stimulation period (60Hz at 5V, for 30 sec); N = 37; p<0.05). Conclusions: Our results indicate that chronic incubation of hippocampal cultures with BHB at a clinically relevant concentration leads to a significant reduction in the overall activity and excitability of neurons - effects that may contribute to an anticonvulsant effect in vivo, and may help explain in part the anticonvulsant mechanism of the KD. Our findings also suggest that a more rigorous examination of the molecular targets of BHB (and perhaps other ketone bodies) may provide novel avenues for the development of investigational compounds to treat medically refractory epilepsy.