Abstracts

Temporal lobe epilepsy (TLE) is associated with a disruption in glutamate-glutamine cycle between neurons and glia. The effect of this disruption on synaptic functioning has not been well established. We tested the hypothesis that a disruption in glutamate synthesis from glutamine by phosphate-activated glutaminase (PAG) will alter GABAergic function by limiting the availability of glutamate for GABA synthesis by glutamic acid decarboxylase (GAD). We used a combination of electrophysiology and mass spectrometry to test this hypothesis. Field potential studies in hippocampal slices from young adult rats were used to assess the effects of diazo-oxo-norleucine (DON), a specific PAG inhibitor, on synaptic inhibition. A parallel series of experiments were done to assess the changes in the metabolite content of incubated slices and the rate of synthesis of glutamate, glutamine and GABA using sodium 2-13C-acetate. The levels of these compounds and the degree of isotopic labeling were measured using quantitative mass spectrometry. We found that DON was associated with profound excitation with the loss of paired pulse inhibition in the dentate granule cells studied using field potential recordings. These effects were first apparent as 1) an increase in the amplitude of the population spike such that there was a shift in the threshold stimulus intensity and 2) a decrease in paired pulse inhibition at 10 ms and 150 ms, corresponding roughly with the peaks of the fast and slow IPSP. There was a 43.6 [plusmn] 12.5% decrease in PPI at 1 hour following bath application of DON, n=4. In half of the slices tested, prolonged field bursts could be evoked in DON.
The responses to DON in the CA1 region were different from those seen in the dentate in two respects. First, while we saw a similar increase in the amplitude of the population spike and a reduction of paired pulse inhibition 1 hour following bath application of DON, we never saw the prolonged fields noted in the dentate. Second, the evoked response faded completely over the course of four hours such that a single population spike could not be evoked, even in slices where there were prolonged field bursts in the dentate.
In slices treated with DON in a static bath, we found the expected decrease in glutamate and GABA content and a slight increase in glutamine content. Surprisingly, the rate of GABA synthesis was not changed by the addition of DON. These data suggest that 1) there are differences in the dependence of GABA synthesis on PAG-generated glutamate between the dentate and CA1; 2) GABAergic function may be disrupted earlier that glutamatergic transmission when glutamate-glutamine cycling is impaired and 3) the levels of GABA in the whole slice may not correlate with functional GABAergic neurotransmission. (Supported by NIH grants RO1NS45792 and PO1139092)