Abstracts

Rationale: Seizures are characterized by an increase in network activity. Glutamine, a direct metabolic precursor of glutamate has been implicated in maintaining glutamate mediated excitatory activity, however this role remains controversial. To determine whether limiting the availability of glutamate precursors can suppress network activity in hyperexcitable states, we have utilized field recording and glutamate biosensor imaging to assess changes in network activity in acutely disinhibited cortical slices in response to a series of pharmacological manipulations. In addition, we analyzed spontaneous activity of layer V pyramidal neurons by whole cell recordings to determine if the effects at synaptic level correlate with network activity. Methods: Coronally sliced brain from 3-8 week old rats were placed in an interface or submerged chamber superfused with oxygenated aCSF and stimulated with a concentric bipolar electrode at layer VI-white matter boundary at various intervals and recorded with glass micropipettes. Glutamate release was imaged by analyzing changes in FRET ratio of slices preincubated with a previously characterized glutamate biosensor. To disinhibit the cortex, GABA_A and GABA_B receptor antagonists Gabazine (10μM) and CGP54626 (10nM) were added to the perfusate. In addition, the following inhibitors were used; 5-10mM methionine sulfoximine (MSO), 20mM methylaminoisobutyric acid (MeAIB), 30mM aminoisobutyric acid (AIB) and 4mM aminooxyacetic acid (AOAA). Results: We found that evoked glutamate release (directly measured through biosensor imaging and indirectly by fEPSP epileptiform events) in disinhibited cortex was directly correlated to the inter stimulus interval, suggesting a time sensitive limiting factor. Addition of physiological concentration of glutamine did not significantly alter evoked responses, indicating that availability of glutamine in aCSF is not the limiting factor. In contrast, modulating the level of glutamine within the slice exhibited robust effects. Addition of astrocytic glutamine synthetase inhibitor MSO induced intermittent failures of epileptiform events. This was not replicated by MeAIB, an inhibitor of the neuronal system A transporters that have been implicated in glutamine uptake, but application of a more general glutamine transport inhibitor AIB lead to partial failures of epileptiform events. Furthermore application of AOAA to inhibit de novo synthesis of glutamate resulted in complete suppression of epileptiform activity. MSO, AIB and AOAA effects were rescued by addition of physiological concentrations of glutamine. Conclusions: Under intense excitatory network demand, astrocytic transfer of glutamine to neurons through non-system A (and possibly system A) mediated transport is an essential step in the maintenance of epileptiform activity in acutely disinhibited cortical slices. In the absence of astrocytic source of glutamine, exogenously applied glutamine can maintain network activity. Our results suggest that modulating the availability of glutamine may be a strategy worth considering for therapeutic intervention of epilepsies.