Abstracts

Molecular Knockdown of Neuronal Glutamate Transporter EAAC1 Produces Epilepsy by Reduction of New GABA Synthesis in Rat Hippocampus.

Abstract number : I.05
Submission category :
Year : 2000
Submission ID : 1138
Source : www.aesnet.org
Presentation date : 12/2/2000 12:00:00 AM
Published date : Dec 1, 2000, 06:00 AM

Authors :
Jehuda P Sepkuty, Kevin Behar, Jeffrey D Rothstein, Johns Hopkins Univ, Baltimore, MD; Yale Univ, New Haven, CT.

RATIONALE: Reduced expression of the neuronal glutamate transporter EAAC1 leads to behavioral abnormalities including staring episodes, electrographic seizures and limbic hyperexcitability (Neurosci. Abs. 618.3, 1996, and 584.7, 1997, AAN abs. P01.039, 1999). It also reduces inhibitory efficacy in rat CA1 neurons (Neurosci. Abs. 491.10, 1999). Because EAAC1 has been localized to presynaptic GABAergic neurons and glutamate is a precursor for GABA synthesis, we hypothesized that EAAC1 may play a role in regulating GABA synthesis and could be causing epilepsy when knocked down. METHODS: EAAC1 expression was knocked down by intraventricular administration of antisense oligonucleotides. The rats' hippocampi and thalami were dissected and homogenated in fresh oxygenated Krebs buffer. C14 glutamate was added to the tissue in the presence of diazonorleucine (DON) and GABA-T inhibitor (gabaculine). New synthesis of C14 GABA was measured, using precursor C14 glutamate by HPLC. RESULTS: We found that the mean C14 GABA specific activity calculated for hippocampal samples (n=7) from EAAC1 antisense oligonucleotide treated animals was significantly smaller (1.6, p<0.02 unpaired t-test) than that calculated for samples from sense oligonucleotide treated animals (4.0, n=6). No significant differences in C14 GABA specific activity was found in thalamic samples from the same animals. When incubating hippocampal samples of normal rats (n=2) with a potent general glutamate transporter inhibitor (THA) and in the presence of the same inhibitors (DON and gabaculine), we found that the mean C14 GABA specific activity calculated for these samples was significantly smaller (2.2, p<0.01 unpaired t-test) than that calculated for non incubated hippocampal and thalamic samples (6.1, n=6). CONCLUSIONS: This suggests that limbic hyperexcitability and epilepsy induced by EAAC1 knockdown may be due, in part, to a reduction in new GABA synthesis in the hippocampus. It also suggests that glutamate transporter EAAC1 has a role in synthesis of new GABA in normal rats. (NIH 33958).