THESIS
2004
xvii, 88 leaves : ill. (some col.) ; 30 cm
Abstract
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 ([Ca
2+]
o) have long been shown to be necessary for excitotoxicity, however, the relationship between [Ca
2+]
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...[
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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 ([Ca
2+]
o) have long been shown to be necessary for excitotoxicity, however, the relationship between [Ca
2+]
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 [Ca
2+]
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 (P
f) at a given [Ca
2+]
o, as derived from the Goldman-Hodgkin-Katz (GHK) equation on the basis of constant field assumptions. The P
f of NMDAR at 2.7 mM [Ca
2+]
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, P
f, 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 Ca
2+ 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 [Ca
2+]
o, but induced toxicity at elevated [Ca
2+]
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.
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