Temporal lobe epilepsy is the most prevalent seizure disorder in adults. Compromised inhibitory neurotransmitter function in the hippocampus contributes to the hyperexcitability generating this condition, but the underlying molecular mechanisms are unknown. Combining patch-clamp recording and single-cell mRNA amplification (aRNA) techniques in single dentate granule cells, we demonstrate that expression of GABA(A) receptor subunit mRNAs is substantially altered in neurons from epileptic rats. These changes in gene expression precede epilepsy onset by weeks and correlate with profound alterations in receptor function, indicating that aberrant GABA(A) receptor expression and function has an essential role in the process of epileptogenesis.

Alterations in GABAergic function associated with the development of temporal lobe epilepsy (TLE) were examined with the use of patch-clamp recording techniques in dentate granule (DG) and CA1 neurons acutely isolated from control and spontaneously epileptic rats in which TLE was elicited by pilocarpine injection 3-17 wk before use. The maximal efficacy of gamma-aminobutyric acid (GABA) in activating whole cell GABA currents increased significantly in epileptic DG neurons relative to controls. This efficacy increase was due to a 78% enhancement in the functional capacitance-normalized GABA(A) receptor (GABA(A)R) current density in epileptic DG neurons. Increased DG GABA(A)R current density was not accompanied by alterations in GABA potency (EC50). However, the maximal sensitivity of DG GABA-evoked currents to blockade by zinc increased 190% in epileptic neurons. Augmentation of epileptic DG neuron GABA-evoked currents by the broad-spectrum anticonvulsant benzodiazepine clonazepam (100 nM) was enhanced 114% relative to controls, whereas augmentation by the benzodiazepine, (BZ1)-selective agonist zolpidem (100 nM) was decreased by 66%. In contrast to DG neurons, maximal efficacy of GABA in activating GABA currents decreased in epileptic CA1 neurons relative to controls, due to a 52% decrease in functional capacitance-normalized GABA(A)R current density. This altered efficacy of GABA was accompanied by an increased GABA potency (GABA EC50 was 35.8 and 24.5 microM in control and epileptic neurons, respectively). Sensitivity of GABA-evoked currents to blockade by zinc was unchanged in epileptic CA1 neurons, whereas clonazepam (100 nM) augmentation of CA1 GABA-evoked currents decreased 81% relative to controls. These regionally distinct epilepsy-associated modifications in hippocampal GABAergic function may be due to discrete structural alterations in postsynaptic GABA(A)Rs accompanying epileptogenesis, could be therapeutically important, and undoubtedly could contribute to the enhanced limbic excitability underlying TLE.

Profound alterations in the function of GABA occur over the course of postnatal development. Changes in GABA A receptor expression are thought to contribute to these differences in GABAergic function, but how subunit changes correlate with receptor function in individual developing neurons has not been de®ned precisely. In the current study, we correlate expression of 14 different GABA A receptor subunit mRNAs with changes in the pharmacological properties of the receptor in individual hippocampal dentate granule cells over the course of postnatal development in rat. We demonstrate signi®cant developmental differences in GABA A receptor subunit mRNA expression, including greater than two-fold lower expression of a1-, a4-and g2-subunit mRNAs and 10-fold higher expression of a5-mRNA in immature compared with adult neurons. These differences correlate both with regional changes in subunit protein level and with alterations in GABA A receptor function in immature dentate granule cells, including two-fold higher blockade by zinc and three-fold lower augmentation by type-I benzodiazepine site modulators. Further, we ®nd an inverse correlation between changes in GABA A receptor zinc sensitivity and abundance of vesicular zinc in dentate gyrus during postnatal development. These ®ndings suggest that developmental differences in subunit expression contribute to alterations in GABA A receptor function during postnatal development.

Summary:Purpose: To define the changes in gene and protein expression of the neuronal glutamate transporter (EAAT3/ EAAC1) in a rat model of temporal lobe epilepsy as well as in human hippocampal and neocortical epilepsy.Methods: The expression of EAAT3/EAAC1 mRNA was measured by reverse Northern blotting in single dissociated hippocampal dentate granule cells from rats with pilocarpineinduced temporal lobe epilepsy (TLE) and age-matched controls, in dentate granule cells from hippocampal surgical specimens from patients with TLE, and in dysplastic neurons microdissected from human focal cortical dysplasia specimens. Immunolabeling of rat and human hippocampi and cortical dysplasia tissue with EAAT3/EAAC1 antibodies served to corroborate the mRNA expression analysis.Results: The expression of EAAT3/EAAC1 mRNA was increased by nearly threefold in dentate granule cells from rats with spontaneous seizures compared with dentate granule cells from control rats. EAAT3/EAAC1 mRNA levels also were high in human dentate granule cells from patients with TLE and were significantly elevated in dysplastic neurons in cortical dysplasia compared with nondysplastic neurons from postmortem control tissue. No difference in expression of another glutamate transporter, EAAT2/GLT-1, was observed. Immunolabeling demonstrated that EAAT3/EAAC1 protein expression was enhanced in dentate granule cells from both rats and humans with TLE as well as in dysplastic neurons from human cortical dysplasia tissue.Conclusions: Elevations of EAAT3/EAAC1 mRNA and protein levels are present in neurons from hippocampus and neocortex in both rats and humans with epilepsy. Upregulation of EAAT3/EAAC1 in hippocampal and neocortical epilepsy may be an important modulator of extracellular glutamate concentrations and may occur as a response to recurrent seizures in these cell types. Key Words: Glutamate transporter-EAAT3/ EAAC1-Epilepsy-Dentate-Dysplasia.Glutamate is the primary excitatory neurotransmitter in the mammalian central nervous system and has been implicated as a neurotoxic agent in several neurologic disorders including epilepsy, ischemia, and certain neurodegenerative diseases (1,2). Unbound extracellular glutamate is cleared primarily by sodium-dependent transport of glutamate into glia and neurons (for review, see 3,4), and glutamate transport is thought to be crucial for preventing accumulation of neurotoxic levels of extracellular glutamate. Multiple subtypes of sodiumdependent glutamate transporters have been identified pharmacologically, and four rat (GLAST, GLT-1, EAAC1, and EAAT4) and five human (EAAT1-5) transporters have been identified by molecular cloning (5). The excitatory amino acid transporters EAAT1 (GLAST) and EAAT2 (GLT-1) are expressed primarily in astroglial cells, whereas EAAT3 (EAAC1) and EAAT4 are enriched in neurons. EAAT3/EAAC1 is the most abundant neuronal transporter, and is selectively enriched in neurons of the hippocampus, cerebellum, and basal ganglia (6).A disturbance in glutamate-mediated excitatory neurotrans...

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