NMDA receptor mediated toxicity in primary neuronal cultures from rodent cerebral cortex

In various neurological disorders, excess glutamate over-stimulates N-methyl-D-aspartate receptors (NMDARs) leading to excess calcium influx and subsequent neuronal injury and death. NMDAR and extracellular calcium ([Ca2+]o) have long been shown to be necessary for excitotoxicity, however, the relationship between [Ca2+]o, NMDAR activity and NMDA-induced cytotoxicity is still unclear. Here, I used cultures of primary cortical neurons as a model system to investigate the relationships. The use of a specific antagonist, ifenprodil, revealed that NMDA-induced cytotoxicity in these cultures was mainly mediated by NR2B-containing NMDARs. Both ifenprodil and APV totally blocked NMDA-induced cytotoxicity. Consistently, NMDA-induced whole cell current was ~971.3 pA, while that supplemented with ifenprodil and APV were ~125.9 pA and ~14.0 pA, respectively. Parallel patch-clamping experiments and lactate dehydrogenase (LDH) cytotoxicity assays with a wide range of NMDA concentrations revealed that NMDA-induced cytotoxicity was directly correlated to NMDA-induced whole-cell currents in these cultures. A positive linear correlation was observed by plotting NMDA-induced cytotoxicity vs. NMDA-induced whole-cell currents. Performing LDH cytotoxicity assays, complete dose/response curves of the NMDA-induced cytotoxicity were obtained at different extracellular calcium concentrations (0.27 - 27 mM) and plotted vs. NMDA-induced whole-cell currents. Fitting curves using four parameter logistic equation showed that elevated [Ca2+]o, augmented NMDA-induced cytotoxicity by shifting the curves leftwards. By obtaining Hill slopes from the curves, this potentiation of NMDA toxic effect could be explained by the fractional calcium current (Pf) at a given [Ca2+]o, as derived from the Goldman-Hodgkin-Katz (GHK) equation on the basis of constant field assumptions. The Pf of NMDAR at 2.7 mM [Ca2+]o, which is ~21.5%, was calculated using GHK equation. These results support the notion that NMDA-induced excitotoxicity is limited by its intrinsic biophysical property, Pf, but not its subsequent intracellular neurotoxic signaling pathways near the NMDARs. Activating and blocking fluxes from other sources (non-NMDAR and VSCC) failed to significantly modulate NMDA-induced cytotoxicity. This result further supports the essential role of Ca2+ influx through NMDAR in neurotoxic process. To characterize the role of synaptic NMDARs in excitotoxicity, synaptic glutamate release was stimulated by high K+ exposure. High K+ exposure was not toxic at physiological [Ca2+]o, but induced toxicity at elevated [Ca2+]o. Comparing with toxicities induced by NMDA bath applications, NMDARs stimulated by high K+ exposure was found to be equivalent to that by ~10 μM NMDA. By using these numbers as reference, I calculated that synaptic NMDARs were contributing ~14.5% of NMDA-induced whole cell current