Network activation triggers a significant energy metabolism increase in both neurons and astrocytes. Questions of the primary neuronal energy substrate (e.g., glucose vs. lactate) as well as the relative contributions of glycolysis and oxidative phosphorylation and their cellular origin (neurons vs. astrocytes) are still a matter of debates. Using simultaneous measurements of electrophysiological and metabolic parameters during synaptic stimulation in hippocampal slices from mature mice, we show that neurons and astrocytes use both glycolysis and oxidative phosphorylation to meet their energy demands. Supplementation or replacement of glucose in artificial cerebrospinal fluid (ACSF) with pyruvate or lactate strongly modifies parameters related to network activity-triggered energy metabolism. These effects are not induced by changes in ATP content, pH i , [Ca 2 þ ] i or accumulation of reactive oxygen species. Our results suggest that during network activation, a significant fraction of NAD(P)H response (its overshoot phase) corresponds to glycolysis and the changes in cytosolic NAD(P)H and mitochondrial FAD are coupled. Our data do not support the hypothesis of a preferential utilization of astrocyte-released lactate by neurons during network activation in slices-instead, we show that during such activity glucose is an effective energy substrate for both neurons and astrocytes. Keywords: astrocytes; energy metabolism; glycolysis; lactate; network activity; neurons INTRODUCTION High cellular energy demands during network activation are met by upregulation of cytosolic glycolysis and mitochondrial oxidative phosphorylation. Mitochondrial metabolism provides most of the ATP but glycolysis is also enhanced and may contribute to the energy production. Journal of Cerebral Blood1-4 For elucidation of the cellular basis of neuroenergetics, measurements of metabolic signals including the oxygen utilization and NAD(P)H/FAD autofluorescence provide valuable information for connecting energy metabolism with neuronal activity. NADH (reduced form) is fluorescent when excited with UV light whereas NAD þ is not, leading to a decrease in observed fluorescence as a result of NADH oxidation. In contrast, FAD (oxidized form) is fluorescent, so the oxidation of FADH 2 to FAD causes an increase in fluorescence. The fluorescence of NADH cannot be separated from that of NADPH and their emission is measured in concert (NAD(P)H). NAD(P)H fluorescence represents a 'mixed' signal since this cofactor can be produced by both glycolysis and mitochondria, whereas FAD fluorescence is entirely mitochondrial. 5,6 Measurements of these parameters in combination with electrophysiological recordings have been used in many studies to monitor the energy status during neuronal activity in brain tissues.Typically, NAD(P)H transients induced by synaptic stimulation have a characteristic biphasic waveform: the initial short dip is followed by a long-lasting overshoot. While there exists a common

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