Together, these in vivo and in vitro studies demonstrate that EAAC1 antisense-treated rats develop epilepsy and limbic hyperexcitability and that this hyperexcitability may be attributable, in part, to a reduction in new GABA synthesis in the hippocampus. These studies also suggest that glutamate transporters in general, and EAAC1 specifically, have a role in synthesis and release of new neurotransmitter GABA in the hippocampus of normal naive rats.
The EEG and behavioral monitoring have shown that EAAC1 antisense-treated rats develop behavioral changes manifested as staring–freezing episodes, which occur simultaneously with EEG epileptiform changes. These changes are specific and maximal at the time of maximal EAAC1 knockdown. The phenotype of the rats in this study differs from the EAAC1-deficient mice reported by Peghini et al. (1997)
. EAAC1 null mice have a deficit of the transporter protein during ontological development allowing for compensatory responses, whereas antisense knockdown results in the loss of this transporter during adulthood. Noteworthy in EAAC1 null mice was the decrease in locomotor activity with episodes of locomotor arrest (Peghini et al., 1997
); however, simultaneous EEG recording to rule out seizures were not reported in their study. In the present study, extracellular field potential recordings from both thalamocortical and hippocampal slices of antisense-treated rats showed hyperexcitability. The hippocampal hyperexcitability was correlated with the EEG hyperexcitability. Based on this finding and because of the large decrease in GABA synthesis, whole-cell patch-clamp recordings of CA1 pyramidal neurons were performed. The data suggest that EAAC1 antisense-induced hyperexcitability may be attributed to a decrease in mIPSC amplitude, but not mIPSC frequency, in CA1 pyramidal neurons.
A balance of excitation and inhibition is essential for the maintenance of normal function in the brain. Golan et al. (1996)
showed that GABA concentration determines the efficacy of inhibition. In the present study, a decrease in total GABA was observed in the hippocampus of antisense-treated rats but not in sense controls. This decrease was not significant in other regions, although it was present in the thalamus also. Inhibiting GABA synthesis causes seizures, and some of the effects of anticonvulsants occur through interference with enzymes associated with GABA metabolism (Petroff et al., 1996a
High-affinity glutamate transporter subtypes have been found to be specifically localized to both neuronal and astroglial membranes. Under normal conditions, these proteins maintain low extracellular levels of glutamate. A series of studies suggest that the astroglial transporters, GLT-1 in particular, are primarily responsible for the synaptic inactivation of glutamate and for preventing excitotoxic injury (Bergles and Jahr, 1997
; Otis and Jahr, 1998
; Otis and Kavanaugh, 2000
). Nevertheless, EAAC1 is the predominant neuronal transporter present widely throughout the CNS dendrites and somata of large and small pyramidal neurons. However, its function in normal synaptic biology has eluded investigators. Molecular anatomic studies suggested that this protein was unexpectedly localized to presynaptic GABA terminals (Rothstein et al., 1994
; Conti et al., 1998
; He et al., 2000
). Subsequently, preliminary antisense knockdown studies suggested a relationship between this protein and tissue GABA levels (Rothstein et al., 1996
GABA is synthesized primarily from the α
-decarboxylation of glutamate by glutamate decarboxylase (Martin and Rimvall, 1993
). An alternate pathway for GABA synthesis via putrescine has been described previously (Seiler and Al-Therib, 1974
). Although the contribution of this pathway to GABA synthesis appears to be small in the mature rat brain [~1% of total GABA synthesis (Noto et al., 1986
)], putrescine has been shown to be a GABA precursor in the developmentally immature retina (Yamasaki et al., 1999
). GABA carbon, which is lost from GABAergic neurons, must be replenished from other cells because mature neurons do not possess the necessary enzymes for de novo
synthesis. Glutamine produced in astrocytes is a major precursor of GABA, although few quantitative studies of the precursors of GABA in vivo
have been reported. Neostriatal microinjections of methionine sulfoximine, an inhibitor of glutamine synthetase, resulted in only a ~50% reduction of GABA synthesis (Paulsen et al., 1988
), suggesting that a pathway(s) other than glutamine may also supply glutamate precursors for GABA synthesis in this brain region. The large decrease in GABA levels and GABA synthesis from extracellular [14
C]glutamate in the hippocampus after knockdown of EAAC1 indicate that direct transport of glutamate into GABAergic neurons can provide precursors for GABA synthesis. Furthermore, the electrophysiologic studies suggest that the loss of EAAC1 leads to decreased mI PSC s, consistent with decreased presynaptic release of GABA.
Together, these metabolic and electrophysiologic studies clearly document a relationship between the presynaptic glutamate transporter and the inhibitory transmitter GABA. In turn, these metabolic studies strongly suggest that the mechanism of epilepsy in the EAAC1 antisense knockdown rats is mediated by decreased GABA synthesis and, therefore, decreased C NS inhibition. The data suggest that, in normal rat brain, EAAC1 may have an important role in regulating GABA synthesis and release synthesis. Recently, GTR AP3–18 (for glutamate transporter-associated protein 3–18), an EAAC1 inhibitory modulator, was described (Lin et al., 2001
), suggesting that, under normal conditions, EAAC1 may be modulated, perhaps to regulate presynaptic GABA synthesis. Preliminary studies also show that increased GTR AP3–18, through inhibition of EAAC1 (like antisense), produces epilepsy (Sepkuty et al., 2001
). Overall, these studies suggest a novel interaction between excitatory amino acid transporters and an inhibitory amino acid neurotransmitter system. Furthermore, they raise new possibilities of manipulating GABA metabolism through direct or indirect modulation of EAAC1 (e.g., GTR AP3–18) and may provide novel therapeutic modalities for the treatment of epilepsy.