The central hypothesis of this project focuses on protective strategies for enhancing neuronal metabolism during and after short-term hypoglycemia. Several related mechanisms will be studied using physiological and mitochondrial imaging techniques in acute hippocampal slices, following hypoglycemia. In particular, we will directly assess neuronal-glial metabolic inter-relationships by analyzing whether the """"""""lactate shuttle"""""""" is altered as a function of age. We will also directly measure the presence or absence of mitochondrial permeability transition as a function of age. Because slice metabolism varies as a function of slice oxygenation, age of the tissue and slice conditions (i.e., interface versus submerged slice conditions), direct oxygen tension measurements will be performed in the tissue using a Clark-style oxygen microelectrode at the same depth as the electrical recordings. The oxygen tension monitoring will ensure that metabolic substrate provision is controlled appropriately within the slice. Neuronal-glial metabolic interactions will be analyzed using glial poisoning with fluoroacetate to estimate direct neuronal contribution to glycolysis and aerobic metabolism, by substituting lactate and pyruvate. Pyruvate will be assessed as a potential treatment for preventing hypoglycemic damage. Occurrence and prevention of mitochondrial permeability transition with these stresses will also be assessed, using cyclosporin A and newer analogs such as N-Me-VaI-CSA. Analysis of these mechanisms which lead to enhanced susceptibility to neuronal damage following hypoglycemia are likely to lead to enhanced treatment. Mechanisms of neuronal injury are expected to vary depending on the duration and severity of the hypoglycemia, and the age of the animal from which hippocampal slices were harvested. These in vitro slice models of hypoglycemia will contribute greatly to the understanding of neuronal metabolism, and particularly the metabolic interactions between neurons and glia as a function of lifespan.
