Abstracts

Rationale: To examine very early events that possibly underlie the development of temporal lobe epilepsy (TLE), the Li-pilocarpine rat model of TLE was employed to study changes in layer 5 neurons of the deep entorhinal cortex (EC). Methods: Status epilepticus (SE) was induced in male Wistar rats, 2-3 months old, by injection of lithium and pilocarpine, and confirmed by EEG recording. One hour after onset of SE, diazepam was injected. Acute horizontal brain slices were prepared 2 or 3 weeks post-seizure, and from control rats that had received only lithium. EC layer 5 (L5) neuron synaptic potentials were recorded with sharp intracellular microelectrodes. mRNA and protein levels of Cl^- transporters were determined by fluorescent in-situ hybridization (FISH) and immuno-cytochemistry. Results: GABA_Aergic synaptic potentials reversed at increasingly more depolarized levels, well beyond resting potential, during the two to three week period following SE in 80% of the EC L5 neurons studied. FISH data showed a concurrent progressive increase for NKCC1, a Cl^- inward transporter (234.6±31.2 % 2 wk and 454.4±33.5 % of control at 3 wk post SE, and a strong decrease in mRNA for KCC2, an active Cl^- extruder (81.0±8.9 % 2 wk and 44.3±4.7 % of control at 3 wk post SE). These changes were seen only in the deep EC, but not in L1-3, subiculum, dentate gyrus or perirhinal cortex. In EC L5, NKCC1 protein progressively increased from nearly undetectable to 300% and 800%, of control background fluorescence at 2 and 3 weeks post SE respectively. At 3 weeks post insult 79% of neurons studied were positive for the NKCC1 protein (control 3%). KCC2 protein was localized to neurons and decreased in quantitative agreement with the mRNA data. Concurrent with the above changes, there was an increase in the number of cells responding to single synaptic stimuli with depolarizing polysynaptic bursts that persisted even after block of GABA_A receptors by picrotoxin (100 µM). This suggests that disinhibition, rather than GABAergic excitation, is involved in burst formation. Three weeks post SE, inhibition of Cl^- transport by bumetanide (10 µM) reversibly restored more negative GABA_A reversal potentials and eliminated polysynaptic bursts. Conclusions: Gradual changes in Cl^- transporters NKCC1 and KCC2 following SE make the deep EC hyper-excitable and possibly prone to ending the latent period by triggering a spontaneous partial or generalized seizure.