Abstracts

RATIONALE: Synchronized bursting in a system of pyramidal neurons requires excitatory synaptic strength sufficient to drive the neuronal network as well as a critical number of cells (Traub and Wong, J Neurophys 49:459-471, 1983; Staley et al., Nat Neurosci 1:201-9, 1998). This study examined the relation between the genesis of NMDA receptor-containing synapses and the development of epileptiform bursting in a network of cultured hippocampal neurons.
METHODS: Hippocampi were removed from E18 Sprague-Dawley rats. Glia and neurons were co-cultured on separate surfaces as per Banker and Goslin. Patch clamp recordings were obtained from pyramidal neurons at 3-18 days in culture in external solution with or without Mg++. Following recording, neurons were fixed and labelled for synaptophysin, a presynaptic marker protein, and the NMDA receptor subunit NR1.
RESULTS: No cells exhibited spontaneous epileptiform action potential bursting in normal Mg++ (6 mM). When exposed to nominally zero external Mg++, bursting correlated with days in culture. Bursting occurred in 21/25 cells greater than 14 DIC; 9-11 DIC, 12/31; 6-8 DIC, 6/40; less than 6 DIC, 0/5. Epileptiform bursting in cells exposed to zero Mg++ was not blocked by the glutamatergic AMPA-type receptor antagonist CNQX but was eliminated by the glutamatergic NMDA-type receptor antagonist D-APV. In voltage clamp mode in normal Mg++, excitatory postsynaptic currents (EPSCs) were infrequent in cells less than 9 DIC (10.6 +/- 6 per minute; EPSC frequency increased 6 fold in cells greater than 14 DIC (62.8 +/- 22.5). In cultures less than 9 days old, EPSCs were usually completely abolished by CNQX. In cultures greater than 14 DIC, CNQX blocked approximately 85% of EPSCs; the remainder were blocked by D-APV. EPSC frequency increased only marginally in zero Mg++ in cultures less than 9 DIC but increased 50-100% in older cultures. AMPA receptor and NMDA receptor mediated EPSCs had distinct kinetic profiles with AMPA EPSCs displaying both a faster 10-90% rise (1.2 +/- 0.3 vs 4.4 +/- 0.9 ms) and decay (monoexponential time constant 33.3 +/- 2.2 vs 64.1 +/- 3.7 ms). Immunolabelling of cultured neurons with antibodies to synaptophysin and the NMDA receptor subunit NR1 indicated that NMDA receptors did not co-localize with synaptophysin prior to approximately 7 DIC but showed extensive co-localization after 14 DIC.
CONCLUSIONS: The results suggest that NMDA receptors are crucial to providing the increased excitatory drive required for recurrent bursting in a system of cultured neurons. The increasing bursting potential in maturing cultures correlates with localization of NMDA receptors to synaptic sites.
Support: This work was supported by NINDS grants NS 37192 (PSM) and NS 02081 and NS 40337 (JK).