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. 2002 Aug 1;22(15):6372-9.
doi: 10.1523/JNEUROSCI.22-15-06372.2002.

A neuronal glutamate transporter contributes to neurotransmitter GABA synthesis and epilepsy

Jehuda P Sepkuty  1 , Akiva S Cohen, Christine Eccles, Azhar Rafiq, Kevin Behar, Raquelli Ganel, Douglas A Coulter, Jeffrey D Rothstein
Affiliations

A neuronal glutamate transporter contributes to neurotransmitter GABA synthesis and epilepsy

Jehuda P Sepkuty et al. J Neurosci. .

Abstract

The predominant neuronal glutamate transporter, EAAC1 (for excitatory amino acid carrier-1), is localized to the dendrites and somata of many neurons. Rare presynaptic localization is restricted to GABA terminals. Because glutamate is a precursor for GABA synthesis, we hypothesized that EAAC1 may play a role in regulating GABA synthesis and, thus, could cause epilepsy in rats when inactivated. Reduced expression of EAAC1 by antisense treatment led to behavioral abnormalities, including staring-freezing episodes and electrographic (EEG) seizures. Extracellular hippocampal and thalamocortical slice recordings showed excessive excitability in antisense-treated rats. Patch-clamp recordings of miniature IPSCs (mIPSCs) conducted in CA1 pyramidal neurons in slices from EAAC1 antisense-treated animals demonstrated a significant decrease in mIPSC amplitude, indicating decreased tonic inhibition. There was a 50% loss of hippocampal GABA levels associated with knockdown of EAAC1, and newly synthesized GABA from extracellular glutamate was significantly impaired by reduction of EAAC1 expression. EAAC1 may participate in normal GABA neurosynthesis and limbic hyperexcitability, whereas epilepsy can result from a disruption of the interaction between EAAC1 and GABA metabolism.

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Figures

Fig. 1.
Fig. 1.
Antisense EAAC1 treatment leads to loss of hippocampal EAAC1 expression and epilepsy. a, Immunoblots from hippocampal tissue of three antisense-treated rats (4, 5, 6) have reduced expression of EAAC1 by 60–70% compared with the hippocampus of three sense controls (1, 2,3). b, e, EEG of an awake ambulating adult rat 1 or 2 d after infusion of antisense (b) or sense (e). A normal mixture of frequencies without spikes was seen in both animals.c, f, Recordings of the same rats (as inb and e, respectively) on day 4 or 5. Runs of high-voltage polyspike discharges for ∼2.5 sec, with return to baseline background EEG, can be seen in the antisense-treated rat (c) but not in the sense-treated rat (f). d, g, On days 9 or 10, the maximal antisense effect was seen as prolonged continuous high-voltage spikes, spike and wave complexes, background slowing, and decreased mixture of frequencies in the antisense-treated rat (d) but not in the sense-treated rat (g). h, The mean number of spikes per minute on days 9 or 10 in antisense EAAC1 compared with three controls (GLT-1 antisense-, GLAST antisense-, and EAAC1 sense-treated animals) is significantly increased. *p < 0.005 between the study group and each of the control groups but not between the different controls; ANOVA with Fisher's PLSD for mean spikes per minute.
Fig. 2.
Fig. 2.
Thalamocortical and HEC slices of EAAC1 antisense-treated rats are hyperexcitable. a,b, Thalamocortical slice field potential recordings showing a 10 sec run of spontaneous bursting rhythmic spike discharges (a; + +) in antisense-treated tissue but not in the controls (b) in which occasional spontaneous spike discharges were seen (b; +) (a andb are recorded at 5 mm/sec paper speed).c, The mean duration of spike discharges (seconds per minute) in the antisense-treated thalamocortical slices was significantly increased compared with control (*p < 0.05).d, e, HEC slice field potential recordings showing the occurrence of very frequent SISs in the antisense-treated rat slice (d; + +) and much less frequent SISs (e; +) in the sense-treated rat slice (d and e are recorded at 1 mm/sec).f, Mean number of spikes per minute in the HEC slices of antisense-treated rats was increased.
Fig. 3.
Fig. 3.
The hyperexcitability of hippocampal slices of EAAC1 antisense-treated rats may be secondary to decreased inhibition in CA1 pyramidal neurons. a, Representative mIPSCs (average of 50) from EAAC1 sense (a1), EAAC1 antisense (a2), and GLAST antisense (a3) CA1 pyramidal neurons. b, Cumulative frequency amplitude (b1) and t90 decay time (b2) histograms for neurons in a.c, Histogram of mean mIPSC amplitudes (c1) and t90 decay times (c2) for the three populations. *p< 0.05 denotes significant differences between EAAC1 antisense-treated and both EAAC1 sense- and GLAST antisense-treated groups.AS, Antisense; S, sense.
Fig. 4.
Fig. 4.
Total GABA is decreased significantly in the hippocampus (Hipp) and nonsignificantly in the thalamus (Thal) of EAAC1 antisense-treated rats, suggesting a role for EAAC1 in GABA synthesis in normal rat hippocampi.a, Histograms of mean total GABA levels in different brain regions (Crblm) comparing antisense-treated rats (gray) to sense controls (black). Antisense EAAC1 treatment leads to a loss of hippocampal GABA significantly (*p < 0.05) and thalamic GABA nonsignificantly. b, THA significantly decreases the [14C]GABA-specific activity measured in hippocampal tissue of normal rats (*p < 0.05).c, The addition of the glutaminase inhibitor DON with glutamate to hippocampal tissue produces a nonsignificant increase in [14C]GABA-specific activity. d, Addition of DON and DHK with glutamate to hippocampal tissue produces a significant increase in [14C]GABA-specific activity (*p < 0.05).
Fig. 5.
Fig. 5.
[14C]GABA-specific activity and synthesis rate are reduced in EAAC1 antisense-treated rats.a, [14C]GABA-specific activity after incubation of hippocampal tissue with [14C]glutamate is significantly decreased in EAAC1 antisense-treated rats (*p < 0.05). b, [14C]GABA is significantly increased with addition of [14C]glutamate in EAAC1 sense-treated animals (*p < 0.05). c, [14C]GABA is not increased significantly with added [14C]-labeled glutamate in EAAC1 antisense-treated animals. d, The rate of [14C]GABA synthesis with added [14C]glutamate is five times faster in tissue of EAAC1 sense-treated rats compared with tissue of EAAC1 antisense-treated rats.
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