Cellular Energy Metabolism in Neurons - PROJECT SUMMARY / ABSTRACT The brain uses energy intensively, and neuronal activity drives rapid, large swings in energy demand, yet we have limited quantitative understanding of how neurons trigger energy resupply and buffer cellular energy state on the seconds-to-minutes timescale. Textbook pathways describe how ATP can be produced from glucose, oxygen, and alternative fuels, but they do not explain how metabolic flux is dynamically engaged in intact neurons, nor how energy state is stabilized when demand changes abruptly. Our laboratory has developed a panel of genetically encoded metabolic biosensors with fluorescence-lifetime readouts that enable quantitative, real-time imaging of metabolites in defined neuron types and subcellular compartments. Using these tools, we find that neuronal stimulation evokes coordinated metabolic transients with unexpected features, including pronounced activity-dependent dynamics of cytosolic citrate and inorganic phosphate (Pi), and delayed changes in ATP consistent with strong buffering by the phosphocreatine/creatine kinase (PCr/CK) system. While intracellular Ca2+ signals are a well-known consequence of neuronal activity, our data suggest that Ca2+-dependent mitochondrial engagement and Ca2+-linked citrate dynamics may shape metabolic responses, but are not necessarily required for the rapid activation of glucose metabolism. In this project, we will use controlled stimulation and biosensor imaging in acute mouse hippocampal slices to (i) quantify the timing and magnitude of candidate trigger signals and metabolic outputs, during and after neuronal activation; and (ii) test mechanistic hypotheses about candidate metabolic triggers (including Pi-, Ca2+-, and citrate-linked mechanisms), buffering of energy by PCr/CK, and mechanisms governing recovery after stimulation. We will then determine how triggering, buffering, and recovery dynamics differ across neurons with different activity patterns (dentate granule cells, CA1 pyramidal neurons, and CA1 PV+ fast-spiking interneurons), and across subcellular compartments in dentate granule cells (soma, dendrites, and mossy fiber boutons). Neuronal activity will be quantified routinely with RCaMP and calibrated to action potential firing using cell-attached recordings, enabling metabolic responses to be compared at matched activity levels. These studies will establish quantitative principles governing rapid neuronal energy mobilization and buffering, providing a foundation for understanding how these processes may fail in aging and neurodegenerative disease.
