Abstracts

RATIONALE: Studies in the hippocampus suggest that synchronized bursting in a system of pyramidal neurons requires connectivity among a critical minimum of cells, reduced inhibitory efficacy, and sufficient excitatory synaptic strength to drive the network (Traub and Wong, J Neurophys 49:459-471, 1983; Staley et al., Nat Neurosci 1:201-9, 1998). This study presents an initial characterization of the conditions required to achieve epileptiform bursting in a system of cultured hippocampal neurons exposed to a magnesium-free environment. METHODS: Hippocampi were removed from E18 Sprague-Dawley rats. Glia and neurons were co-cultured on separate surfaces as per Banker and Goslin. Neurons were plated at two densities: 50,000 (low density-LD) or 200,000 (high density-HD) per 25 mm coverslip. Patch clamp recordings were obtained from pyramidal neurons at 3-18 days in culture in external solution (+) or (-) Mg2+ (6mM MgCl2). RESULTS: LD or HD cultures in normal Mg2+ did not exhibit epileptiform activity (EA - action potential bursts superimposed on 1-5 s depolarizing plateaus or continuous spiking activity) regardless of days in culture. Development of EA in cultures exposed to 0 Mg2+ solution was dependent on both cell density and days in culture. No LD culture developed EA regardless of culture duration or length of exposure to 0 Mg2+ (0/11). In HD cultures, EA did not develop at less than 8 days in culture (0/5), was more frequent at 8-10 days (6/10), and was always present at >11 days (7/7). EA correlated with the appearance of spontaneous postsynaptic potentials and synapse formation as determined from immunohistochemical and electron microcroscopy. In neurons displaying EA, the efficacy and potency of -aminobutyric acid in eliciting whole cell currents were reduced as compared with controls. CONCLUSIONS: The lack of EA in LD and young HD cultures support the hypothesis that establishment of synaptic connectivity among a sufficient number of neurons is necessary for the induction of burst discharges. A reduction in the strength of inhibitory synapses may also play a critical role in the generation of epileptiform activity.