Title: Imaging neuronal metabolism
Description: Epilepsy is an extremely common disorder and the most common neurological disorder in children. Although a majority of affected individuals are successfully treated with anti-convulsant drugs, there are many for whom medication is not effective. An alternative treatment is the ketogenic diet, a high fat/low carbohydrate and protein regimen that is remarkably effective at reducing and even eliminating seizures in these most difficult of cases. Despite many years of research, the exact mechanism of the diet has remained elusive. One possibility currently being explored is that the ketogenic diet works via an acute anticonvulsant action of ketone body metabolism on neuronal excitability. Previous work has demonstrated that when neurons from acute brain slices are exposed to an increase in ketone bodies similar to that seen in patients on the ketogenic diet, there is a depression in spontaneous neuronal electrical activity. This effect is due to opening of ATP-inhibited potassium channels (K ATP channels), membrane proteins that are known to couple metabolic state to cell excitability.

This dissertation works towards further testing the interaction between ketone bodies, K ATP channels, and neuronal excitability in three ways: (1) A novel, genetically-encoded fluorescent biosensor for measuring cellular energy level was engineered. The strategy used to develop the ATP sensor was to integrate a modified genetically encoded yellow fluorescent protein into a peptide loop of the bacterial ATP-binding protein, GlnK1. This purified protein reliably reported the ATP/ADP levels in vitro. When expressed in cultured mammalian cells, the probe responds to metabolic inhibition, although changes its pH sensitivity must be considered. (2) Two recently developed fluorescent probes to cellular reactive oxygen species (ROS) were characterized in neurons, and one of these probes, dihydrodichlorofluorescein-cysteic acid, H 2 DCF-CA, was used to investigate the molecular identity of a mitochondrial K ATP channel. (3) The effect of acute glycolytic inhibition and impact of K ATP channels on seizure susceptibility was tested in mice using the flurothyl seizure model. These tools will allow future work to begin to assay the complex relationship between metabolites, cellular energy, and neuronal activity.