Ca2 signaling from synaptic NMDA receptors is known to enhance de

Ca2 signaling from synaptic NMDA receptors is known to enhance dendritic out growth and synaptic delivery of NMDA receptors occurs within hours of synaptic activation. This scenario predicts an increase in the total NMDA receptor pool consistent with the slightly higher Ca2 response to bath applied NMDA in cultures exposed to overnight AP bursting. The sprouting and or growth of new NMDA receptor con taining synapses would also strengthen the relative contri bution of synaptic receptors to the response to bath applied NMDA. This would help counteract the extrasyn aptic NMDA receptor mediated toxic effects and facilitate neuroprotection in cultures treated with overnight AP bursting.

Regardless of any alteration in surface expression or distribution of NMDA receptors, the oppo sition Inhibitors,Modulators,Libraries of synaptic activity to NMDA receptor mediated death converges at a level downstream of the receptor, through opposing effects on CREB function and target gene activation. Conclusion We have developed and validated a technique for the iso lation Inhibitors,Modulators,Libraries and quantitative functional assessment of the extra synaptic NMDA receptor pool in cultured hippocampal neurons participating in neuronal networks. With this method we have shown that prolonged periods of AP bursting, which protects neurons from subsequent toxic insults, causes little change in the function of the extrasy naptic NMDA receptor pool, a receptor population Inhibitors,Modulators,Libraries linked to neuron death. Methods Hippocampal Cell Culture Hippocampal neurons from new born Sprague Dawley rats were prepared as described except that growth media was supplemented with B27 3% rat serum and 1 mM glutamine.

Neurons were plated onto 12 mm glass coverslips or plas tic 4 well dishes at a density between 400 and 600 cells Inhibitors,Modulators,Libraries per mm2. All stimulations and recordings were done after a culturing period of 10 to 12 days during which hippoc ampal neurons develop a rich network of processes, express functional NMDA type and AMPA kainate type glutamate receptors, and form synaptic contacts. The induction of network bursting and the cell death assay Bursts of AP firing throughout the neuronal network was induced by treatment of the neurons with 50 M bicucul line. Bicuculline was dissolved in DMSO which did not exceed a final concentration Inhibitors,Modulators,Libraries of 0. 05%. Cells with or without 16 h bicuculline pretreatment were subjected to 20 M NMDA for 10 min at 37 C to induce cell death. After washout of NMDA cells were incubated for a further 5 h at 37 C before fixation with paraformaldehyde and stained with Hoechst 33528. Cell death was evalu ated at a light microscope with 40�� mag nification by counting condensed nuclei in 20 fields of view for every condition in each experiment. Pictures of representative areas were taken with a CCD camera.

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