Glutamate transporters function to control excitatory signaling by removal of glutamate from the synaptic cleft. Of this transporter family, low level expression of EAAC1/EAAT3 is primarily localized in a perisynaptic fashion on postsynaptic neurons. Unique to GABAergic neurons, EAAC1 is expressed presynpatically and functions to supply a primary source of glutamate for the synthesis of GABA via GAD65/GAD67. During excitotoxic insult, GABA response is enhanced to counter strong glutamate mediated signaling. Prior studies have noted diminished GABA synthesis in the presence of GABA synthesis and GABA stores increased susceptibility to seizure. The influence of enhanced EAAC1 expression has yet to be defined. While prior studies offered insight into the potential influence of EAAC1 on the GABA system, these studies lacked the ability to selectively evaluate the role of GABAergic EAAC1 in GABA synthesis and role of this glutamate transporter in seizure susceptibility.We first used an immortalized GABAergic cell line, HC2S2, to evaluate the influence of enhanced EAAC1 expression on GABA synthesis capacity. Preliminary studies show the over expression of EAAC1 in HC2S2 cells results in a 2 fold increase in GABA levels. Additionally, the over expression of GABAergic EAAC1 altered levels of glutamate, glutamine and aspartic acid. This presents the first data connecting the direct, cell type specific over expression of EAAC1 with enhanced GABA synthesis. We further examined the effect of EAAC1 modulation invivo.The rat GAD65 promoter sequence was used to selectively drive over expression and knockdown of GABAergic EAAC1 in adult rats. Three rAAV vectors were constructed containing flag tagged EAAC1, EAAC1 in the antisense (A/S) orientation, or a null cassette. Bilateral stereotaxic injections were performed into the hippocampi of adult male Sprague-Dawley rats with rAAV1/2-GAD65-flagEAAC1, rAAV1/2-GAD65-EAAC1 antisense (A/S), or rAAV1/2-GAD65-null. Thirty-five days post rAAV injection, injection of pilocarpine HCl was used to induce seizure activity. Subjects were monitored for progression to status epilepticus (SE) base on a modified Racine scale. The group receiving rAAV1/2-GAD65- EAAC1 A/S presented the initial seizure activity and progression to SE at a significantly increased rate when compared to the control group. The group receiving rAAV1/2-GAD65-flag-EAAC1 progressed to SE at a significantly slower rate when compared to the control group.This study presents the first evidence of altered EAAC1 expression in seizure susceptibility through modulation of protein expression specific to GABAergic neurons. This newly designed rAAV construct allows for selective targeting of GABAergic neurons, enabling the modulation of specific glutamate transporter subpopulations.Molecular Therapy (2006) 13, S344–S345; doi: 10.1016/j.ymthe.2006.08.983 [ABSTRACT FROM AUTHOR]