Abstracts

Rationale: The epilepsies are characterized by an increase in network excitability. This suggests that dynamic regulation of the synthesis and release of the excitatory neurotransmitter glutamate will likely influence the abnormal activity that underlies these disorders. Recent molecular and pharmacological advances have refined our understanding of how glutamine, the immediate precursor of synaptically released glutamate, is metabolized by neurons. Using a combined pharmacological and physiological approach we investigated the role of glutamate recycling through glutamine in maintenance of epileptiform activity in acute brain slice preparations treated with GABA A and GABA B receptor inhibitors.Methods: Brains were removed from anesthetized rats and transferred into ice cold low NaCl slicing solution bubbled with 95%O2 /5% CO2 and 400 micron coronal slices were made using a vibratome. Slices were incubated in aCSF at 32 degrees for 1 hour prior to recording. The solution was continuously bubbled with 95% O2/5% CO2. Slices were placed in an interface recording chamber partially submerged in and superfused continuously (~2.0 ml/min) with ACSF equilibrated with 95% O2/5% CO2. Extracellular field potentials were recorded from Layer V of the neocortex by using glass micropipettes (~1 M ohm) filled with ACSF. A bipolar stimulating electrode was placed to stimulate the layer VI/white matter boundary. Slices were stimulated with varying intensities and at varying intervals in the absence and presence of glutamine or glutamate as well as inhibitors of the components of the glutamine-glutamate shuttle and metabolic pathways for de novo synthesis of glutamate.Results: MeAIB, a specific inhibitor of neuronal glutamine uptake through the system A transporters SNAT1 and SNAT2, partially inhibited epileptiform discharges in the disinhibited slice - responses were weakened but not abolished. Histidine, a general inhibitor of glutamine transport, however, completely blocked prolonged epileptiform field depolarizations. Inhibition of de novo synthesis of glutamate through anaplerosis in astrocytes by blocking amino acid transferase with AOAA also completely blocked the epileptiform discharges. This latter effect could be overcome by the addition of exogenous glutamine or glutamate.Conclusions: Maintenance of epileptiform discharges in the disinhibited slice requires a continuous source of neuronal glutamate. Exogneously applied glutamate or its immediate metabolic precursor glutamine can suffice, but in their absence, synaptically released glutamate is derived through de novo synthesis via anaplerosis in astrocytes. Transfer of the glutamate carbon backbone to neurons occurs via a glutamine intermediate that is taken up by neurons through system A dependent and system A independent transport processes. These findings indicate that glutamate metabolism, and in particular, the de novo synthesis of glutamate and/or transfer of glutamine between astrocytes and neurons should be considered for rational therapeutic intervention in the epilepsies.