ination of neuronal cultures was less than 5% as determined by GFAP immunocytochemistry to identify astrocytes and NeuN counterstaining to identify neurons. Results Oxygen consumption by 1115 DIV rat cortical neurons was measured using the XF24 microplate-based respirometer following a 1 hr incubation in the presence or absence of MgSO4. All experiments were conducted in artificial cerebrospinal fluid containing a physiological concentration of Mg2+. Different neuronal preparations exhibited markedly different responses to excitotoxic glutamate either in the absence or presence of MgSO4 pre- and co-treatment. In the absence of added MgSO4, populations of neurons exhibiting a decrease in O2 consumption rate upon glutamate addition, or only a modest transient increase, were classified as “Type I”neurons. Examples of absolute and baseline-normalized OCR measurements from Type I cell populations are given in Fig. 1A and C, respectively. Populations of neurons exhibiting.50% OCR stimulation in response to glutamate under the same experimental conditions were classified as Type II neurons. It should be noted that the Type I or Type II classification refers to the behavior of a given neuron preparation, not to the behavior of individual neurons. In addition, a preparation of neurons could only be categorized after experiments, based on the response to glutamate receptor stimulation. We failed to observe any obvious morphological differences between Type I and Type II neuronal populations. Overall, glutamate did not significantly increase OCR 21821695 in Type I neurons whereas it increased OCR to a mean of,188% of the control rate in Type II neurons. However, when Type I neurons were pre-treated with MgSO4 glutamate significantly stimulated OCR to,136% 15722457 of the control rate measured in the absence of glutamate addition. MgSO4 also slightly but significantly enhanced the OCR stimulation of Type II neurons in response to glutamate receptor activation. Measurement of O2 consumption by XF24 microplatebased respirometry O2 consumption measurements were made using an XF24 Extracellular Flux Analyzer as previously described. Artificial cerebrospinal fluid assay medium consisted of 120 mM NaCl, 3.5 mM KCl, 1.3 mM CaCl2, 0.4 mM KH2PO4, 1 mM MgCl2, 15 mM glucose, 4 mg/ml fatty acid free bovine serum album, and 5 HEPES, pH 7.2. For low Ca2+ aCSF, 1.3 mM CaCl2 was replaced by 1.86 mM CaCl2 and 5 mM EGTA to yield,100 nM free Ca2+. Neurons were incubated with or 71939-50-9 without treatment in a CO2-free 37uC incubator for one hour prior to measurements. Treatments were maintained throughout the experiments and were as indicated in the figure legends. XF assays consisted of cycles of 3 min mix, 2 min wait, 2 min measurement and were performed as described at 37uC. The protonophore carbonyl cyanide 4- phenylhydrazone was added to measure uncoupled Magnesium Preserves Neuronal Metabolism Increasing the concentration of MgSO4 to 10 mM further improved stimulation of OCR by glutamate in Type I neurons. Both 3 and 10 mM MgSO4 improved relative respiratory capacity in Type I neurons but not in Type II neurons. Because elevating MgSO4 to 10 mM in humans is not therapeutically realistic, we chose to focus the remainder of our study on the effects of 3 mM MgSO4. A possible reason for the failure of Type I 
neurons to increase OCR in response to glutamate was that energy demand was already elevated compared to Type II neurons. A higher basal demand for mitochondrial ATP should be reflected