explanation for the neuroprotective effect of Mg2+. However, excitotoxic glutamate levels are predicted to remove the Mg2+ block of NMDA receptors via AMPA receptor-mediated depolarization. 1 Magnesium Preserves Neuronal Metabolism In this study we tested the hypothesis that MgSO4 pre-treatment protects against mitochondrial bioenergetic failure caused by excitotoxic glutamate exposure through NMDA receptor-independent mechanism. Bioenergetic function was evaluated by two key parameters: 1) the initial change in O2 consumption rate in response to glutamate and 2) the change in respiratory capacity following transient glutamate receptor stimulation. Respiratory capacity was defined as the maximum respiration measured in the presence of the uncoupler FCCP and excess exogenous substrate. Relative respiratory capacity was defined as maximum respiration normalized to the basal O2 consumption rate. Results suggest that MgSO4 pretreatment protects against bioenergetic changes due to chronic moderate glutamate receptor stimulation but not due to acute excitotoxic glutamate receptor stimulation, primarily by NMDA receptor-dependent mechanisms. However, MgSO4 preserved neuronal ATP levels even though it was unable to rescue the reduction in relative respiratory capacity caused by an excitotoxic concentration of glutamate. respiration and the complex III inhibitor antimycin A was used to 
inhibit O2 consumption by the mitochondrial electron transport chain. ATP quantification Neuronal ATP levels were analyzed using the ATP bioluminescent somatic cell assay kit for cellular ATP determination. Neurons were incubated with or without treatment in a CO2-free 37uC incubator for one hour. Glutamate or vehicle was then added and cells were incubated for an additional two hours. Neuronal ATP was extracted using the kit’s ATP releasing agent and cellular ATP content was then determined 1685439 by luminescence using a FLUOstar OPTIMA multimodal plate reader. Total ATP content was normalized to cellular protein. Statistics An unpaired Student’s t-test was used to compare the absolute OCRs of Type I and Type II neuronal preparations. Two-way analysis of variance was employed to evaluate statistical significance among groups, with treatment and experiment number as factors. Tukey’s post-hoc analysis was used to compare individual groups. P,0.05 was considered significant. Statistical analyses were carried out using SigmaPlot 12.0. Results in the text are given as mean 6 standard error. Materials and Methods Materials Cell culture supplies were purchased from Invitrogen. All other reagents were obtained from Sigma-Aldrich. Pyruvate was made fresh from powder and pHadjusted for each individual experiment. Other reagents were diluted from concentrated pH-adjusted stocks stored at 220uC. Preparation of primary neurons Ethics Statement: All procedures were approved by the University of Maryland Institutional Animal Care and Use Committee and were in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Primary E18 rat cortical neurons were prepared by trypsin dissociation and plated and maintained in V7 microplates at a density of 0.86105 11414653 to 16105 cells/well as described. Cytosine BCTC site arabinofuranoside was added at 4 days in vitro to inhibit glial proliferation and 2:7 fresh Neurobasal/B27 culture medium was added on DIV 6. Neurons were maintained at 37uC in a humidified atmosphere of 95% air/5% CO2 and were used for experiments at 1115 DIV. Astrocytic contam
