Investigating lactate dynamics in brain tissue is challenging, partly because in vivo data at cellular resolution are not available. We monitored lactate in cortical astrocytes and neurons of mice using the genetically encoded FRET sensor Laconic in combination with two-photon microscopy. An intravenous lactate injection rapidly increased the Laconic signal in both astrocytes and neurons, demonstrating high lactate permeability across tissue. The signal increase was significantly smaller in astrocytes, pointing to higher basal lactate levels in these cells, confirmed by a one-point calibration protocol. Trans-acceleration of the monocarboxylate transporter with pyruvate was able to reduce intracellular lactate in astrocytes but not in neurons. Collectively, these data provide in vivo evidence for a lactate gradient from astrocytes to neurons. This gradient is a prerequisite for a carrier-mediated lactate flux from astrocytes to neurons and thus supports the astrocyte-neuron lactate shuttle model, in which astrocyte-derived lactate acts as an energy substrate for neurons.
Synaptic activity is followed within seconds by a local surge in lactate concentration, a phenomenon that underlies functional magnetic resonance imaging and whose causal mechanisms are unclear, partly because of the limited spatiotemporal resolution of standard measurement techniques. Using a novel Förster resonance energy transfer-based method that allows real-time measurement of the glycolytic rate in single cells, we have studied mouse astrocytes in search for the mechanisms responsible for the lactate surge. Consistent with previous measurements with isotopic 2-deoxyglucose, glutamate was observed to stimulate glycolysis in cultured astrocytes, but the response appeared only after a lag period of several minutes. Na ϩ overloads elicited by engagement of the Na ϩ -glutamate cotransporter with D-aspartate or application of the Na ϩ ionophore gramicidin also failed to stimulate glycolysis in the short term. In marked contrast, K ϩ stimulated astrocytic glycolysis by fourfold within seconds, an effect that was observed at low millimolar concentrations and was also present in organotypic hippocampal slices. After removal of the agonists, the stimulation by K ϩ ended immediately but the stimulation by glutamate persisted unabated for Ͼ20 min. Both stimulations required an active Na ϩ /K ϩ ATPase pump. By showing that small rises in extracellular K ϩ mediate short-term, reversible modulation of astrocytic glycolysis and that glutamate plays a long-term effect and leaves a metabolic trace, these results support the view that astrocytes contribute to the lactate surge that accompanies synaptic activity and underscore the role of these cells in neurometabolic and neurovascular coupling.
The glycolytic rate is sensitive to physiological activity, hormones, stress, aging, and malignant transformation. Standard techniques to measure the glycolytic rate are based on radioactive isotopes, are not able to resolve single cells and have poor temporal resolution, limitations that hamper the study of energy metabolism in the brain and other organs. A new method is described in this article, which makes use of a recently developed FRET glucose nanosensor to measure the rate of glycolysis in single cells with high temporal resolution. Used in cultured astrocytes, the method showed for the first time that glycolysis can be activated within seconds by a combination of glutamate and K+, supporting a role for astrocytes in neurometabolic and neurovascular coupling in the brain. It was also possible to make a direct comparison of metabolism in neurons and astrocytes lying in close proximity, paving the way to a high-resolution characterization of brain energy metabolism. Single-cell glycolytic rates were also measured in fibroblasts, adipocytes, myoblasts, and tumor cells, showing higher rates for undifferentiated cells and significant metabolic heterogeneity within cell types. This method should facilitate the investigation of tissue metabolism at the single-cell level and is readily adaptable for high-throughput analysis.
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