Abstracts

Rationale: In vivo brain microdialysis studies of epilepsy patients demonstrate elevated basal glutamate levels in epileptogenic foci when compared to non-epileptogenic regions (Ann Neurol 2016; 80: 35-45). The hypothesized mechanism of action by which this occurs is through deficiency in glutamine synthetase, the enzyme that metabolizes glutamate to glutamine, as this enzyme has been shown to be deficient in epileptogenic foci (Lancet 2004; 363: 28-37).  The objective of this study was to determine if inhibition of glutamine synthetase results in increased basal glutamate levels that correspond with an increased frequency and severity of seizures.   Methods: Male Sprague Dawley rats were implanted with an osmotic pump infusing either phosphate buffered saline (PBS) or glutamine synthetase inhibitor, methionine sulfoximine (MSO), into the entorhinal cortex for a period of up to 28 days.  Guide cannula were also placed in the hippocampus proper both ipsilateral and contralateral to the infusion location. Screw electrodes were placed above neocortex.  Upon initiation of the experiment, both the MSO and PBS treated rats were further subdivided into two categories.  One received microdialysis two weeks after pump placement (acute), and the other received microdialysis approximately six months after pump placement (chronic). EEG monitoring occurred simultaneously with microdialysis sampling, and samples were collected in one hour increments for a period of up to seven days. Collected dialysate samples were analyzed for 26 different amino acids using the AccQ-Tag Ultra Derivitization Kit (Waters, Milford, Mass) followed by LC/MS.   Results: In the acute study, glutamate levels increased over time on the contralateral side of MSO treated rats, but not on the MSO treated ipsilateral side, or on either side of the PBS control.  Glutamate levels increased to a point where it became significantly higher on the MSO contralateral side compared to the ipsilateral side (p < 0.05), and also compared to the bilateral sides of the PBS control (p < 0.05).  Basal glutamate levels in MSO animals did not directly correlate to the occurrence of seizures.  Furthermore, some rats with high levels of basal glutamate did not seize, while other rats with low glutamate levels did seize.  In the chronic MSO group, seizures occurred to a lesser extent than the acute MSO group.  Glutamate levels in the chronic MSO group did not change over time, and did not differ from the PBS control group.  Conclusions: The results suggest that glutamine synthetase inhibition increases basal glutamate levels in the contralateral projection region of the area of glutamine synthetase inhibition, but not in the inhibited area itself.   Furthermore, basal glutamate levels did not correlate directly with the occurrence of seizures.  These findings are significant because they help to elucidate the effect of glutamine synthetase inhibition on basal glutamate levels, and the causal role of basal glutamate in seizures and epilepsy.    Funding: NINDS K08 NS058674, R01 NS070824, and NCATS UL1 RR024139.