General scientific context: Neuroenergetics, or understanding how the brain produces energy to maintain its functions, has attracted much attention recently. From the improvement of cognitive performances through lifestyle changes (e.g. exercise and nutrition) to novel neuroprotective strategies against neurodegenerative diseases, it appears that neuroenergetics is central for several and diverse aspects of neurobiology. More particularly, studying the cellular links between neuronal activity and energy homeostasis is of utmost importance to elucidate the mechanisms of energy supply dictated by costly neuronal activity. Focus: Nowadays, evidence demonstrates that lactate is efficiently used as an oxidative energy substrate in brain and some of the most convincing data on the specificity of its neuronal utilization were obtained using 13C-NMR spectroscopy, thus illustrating the power of the method for metabolic studies. However, most of the NMR studies were performed in vitro or ex vivo on perchloric acid extracts. Unfortunately, these procedures may introduce artifacts and have serious limitations. Our aim is to analyze brain metabolism of awake and stimulated rats using cutting edge NMR spectroscopy approaches and to demonstrate the intercellular metabolic cooperation between neurons and astrocytes during brain activation in vivo. Moreover, we hope to provide also some evidence that metabolic adaptations take place and are essential during the course of synaptic plasticity. Specific aims and Methodological approaches: The first original aim of this project will be to explore brain metabolism directly (i) on brain biopsies (using High Resolution at the Magic Angle Spinning -HRMAS- NMR spectroscopy), after perfusion of 13C-glucose and 13C-lactate on awake and unilateral-stimulated rats; and (ii) in vivo on unilateral-stimulated rats, using localized 1H-NMR spectroscopy, and 13C-NMR spectroscopy after injection of 13C-substrate. Neuronal stimulation will be obtained by right whisker stimulation, which leads to functional activity in the left barrel cortex. Each animal will be its own control since whisker stimulation will be performed on only one side. These two NMR approaches will allow us to perform both (i) isotopic steady state and (ii) dynamic metabolic studies and estimation of metabolic fluxes during brain activation. The second original aim of this project will be to work both on control rats and rats that will be downregulated for either the neuronal (MCT2) or the glial monocarboxylate transporters (MCT1 or MCT4). These genetically engineered rats will be obtained using an adenoviral vector approach. MCTs participate to the astrocyte-neuron lactate shuttle (ANLS), a highly cited intercellular metabolic exchange mechanism, as the critical transporters used to transfer astrocytic lactate to neurons during brain activation. However, this has never been demonstrated in vivo. A characterization of protein expression as well as protein remodeling linked to brain activation and synaptic plasticity (e.g. MCTs, Arc, zif268, GLAST, GLT1, etc) will be performed in parallel to metabolic studies. Results will give an overview (molecular and metabolic) on the intercellular cooperation that occurs between neurons and astrocytes during and following brain activation. Expected value of the proposed research: Finding ways to prevent or cure brain diseases is a primary goal of neuroscience research. Reaching it requires an ever-improving understanding of the brain’s normal functioning. For this reason, it is critically important to understand the specific mechanisms coupling metabolic and proteomic changes linked to brain activity. This project should give us further in vivo evidence of an astrocytic metabolic supply of lactate to neurons, through the MCTs, which is the missing piece of evidence that needs to be reported to validate the ANLS.