Abstracts

Rationale: Dentate granule cells (DGCs) in the hippocampus serve as a gate against hyperexcitation through the limbic system. Studies in pilocarpine-induced animal model of epilepsy suggest that inhibition of dentate granule cells is compromised, EGABA is positively shifted during latent period compared to the control due to the decreased expression of a K+-Cl- cotransporter, KCC2. Action potentials can be evoked only when the perforant path, afferent fibers convey neuronal signals from entorhinal cortex to hippocampus, is stimulated. Calcium transients, often measured with fluorescent calcium indicators, represent increases in intracellular calcium concentration which coincide with the action potential events. Therefore, to identify the changes in excitability of individual DGCs during epileptogenesis, we have used multicellular, fast calcium imaging within a small circuit. Methods: Adult male rats (2 months old) were injected with pilocarpine (PILO) to induce status epilepticus (SE), and diazepham was given to terminate the SE after 1 hr. One week after PILO injection, horizontal hippocampal slices were prepared and stained with a fluorescent calcium indicator, Oregon Green BAPTA 1 AM for 1 hr at 40 °C in the custom-made incubation chamber and washed for at least 30 min in artificial cerebrospinal fluid (ACSF) at room temperature. Multicellular calcium imaging at the single cell resolution were performed on the Live Cell Sweptfield confocal microscope system (NIKON) equipped with Cascade 512 EM-CCD camera (Photometrics) at 50Hz frame rate for 1 min (excitation at 488 nm). Perforant path was stimulated with 4 pulses (100 Hz, 200 µs, 10 s interval at 500 µA) in ACSF. For control slices from age-matched naïve rats, 10 µM and 100 µM picrotoxin were perfused into the slice chamber to see its blocking effect onto GABAaR-mediated excitability. Results: In slices from PILO-injected animals, 359 of 547 DGCs (7 different regions of interest, and 5 slices from three animals) showed evoked calcium transients (64.8 ± 2.3%, mean ± SEM). In slices from naïve animals, 16 of 299 DGCs (4 different regions of interest, and 3 slices from three animals) showed evoked calcium transients in ACSF (6 ± 5%), and 139 of 299 DGCs in 100 µM picrotoxin (44.1 ± 4.2%). Conclusions: The difference in number of evoked DGCs between naïve and PILO-injected animals are significant (P<0.01). Our results suggest that impaired filtering function of DGCs during latent period after pilocarpine treatment is due to the lowered threshold of excitability of DGCs. This is the first report of fast, multicellular calcium imaging of hippocampal neurons from adult rodents.