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.

Solid state NMR is one of the most versatile characterization techniques used in materials science. The latest methodological developments (double and triple resonances, dipolar recoupling, J spectroscopy) and instrumental progress (ultra-high magnetic fields, ultra-fast MAS probes, gyrotrons for the generation of high power microwaves) allow us to obtain detailed information at the atomic level as well as connectivities in 3 dimensions. However, NMR suffers from an intrinsic sensitivity problem which makes its use unrealistic for the study of microscopic quantities of samples (typically < 100 micrograms). During the past years, remarkable efforts have been made in order to increase the signal-to-noise ratio of the NMR experiment (and consequently to decrease the corresponding experimental time): (i) hyperpolarized magnetic states (DNP, para-H2, hyperpolarized 129Xe), (ii) optimization of the NMR detector (micro-coils, cryogenic-NMR), (iii) novel signal treatments (non-usual schemes of sampling including Non Uniform Sampling), (iv) "exotic" detections of the NMR signal (mechanical and optical). The MicrogramNMR program is fully connected to this innovative challenge, namely the net increase of sensitivity in solid state NMR. The main goal of the project is perfectly defined: MicrogramNMR must popularize the study of solid samples for which the mass is roughly several tens of micrograms (using standard MAS probes with no hardware modification). The implemented methodology is based on the MACS approach (Magic Angle Coil Spinning), recently invented by D. Sakellariou and allowing gains in sensitivity of 10 (or 100 in time!). Durant the last years, C. Bonhomme and D. Sakellariou have obtained significant results on gold standards in solid state NMR by using MACS. Clearly, it is the right time to extend the MACS approach to much more complex materials for which the limitation of the mass is a critical issue in the fields of materials science and bio-nanocomposites (natural and synthetic samples). Another original goal of the project is to make the MACS approach "DNP-compatible", leading to unprecedented gains in sensitivity. In order to succeed in our tasks, we propose a compact project in terms of duration (24 months), with a limited size of funding (< 199 keuros) and manpower (a specialist in micro-mechanics, a post-doctoral fellow and 2 Master students). MicrogramNMR contains two main Tasks. The first Task is related to the optimization of micro-coils used in MACS in order to extend its application to NMR/EPR irradiation in DNP MAS. Two axes of research will be developed: (i) the generation of new coils with pure cylindrical geometry, (ii) the use of miniaturized dielectric resonators for DNP applications. The second Task of MicrogramNMR is related to the extension of MACS to complex topics in materials science for which the very small mass of the sample is a critical issue and a true challenge: (i) calcium phosphate germs at the surface of kidney stones called the Randall's plaques, in a very strong collaboration with physicians at the Tenon hospital, Paris, (ii) thin silica based mesoporous films labeled in 29Si and 17O. For the very first time, NMR experiments will be performed on a single thin film. The results will act as the first experimental proofs for the characterization of 2D nano-confinement effects on the oxide network, (iii) thin metallic oxide based mesoporous films. These films such as SrTiO3, BaxSr(1-x)TiO3, MgTa2O6 have many important applications in microelectronics. Here, the huge B1 fields delivered by the micro-coils will be of paramount importance for the study of "strong" quadrupolar nuclei such as 25Mg, 47/49Ti, 87Sr and 181Ta.

Nuclear Magnetic Resonance (NMR) has already proven to be a tremendous tool in –omics studies of Systems Biology, including the study of metabolome in biological systems known as metabolomics. Its major weakness is the low detection sensitivity that renders the analysis of microscopic quantities (submilligram) impractical, time consuming and often impossible. A cost-effective method is the use of micro(µ)-coils in NMR detections; however, implementing a NMR µcoil for heterogeneous samples such as tissues, cells and organisms is a challenging task. This is because of the necessity of spinning the sample at a 54.74° to the NMR magnetic field. This technique is known as Magic-Angle Spinning (MAS). The use of a spinning µcoil called High Resolution Magic Angle Coil Spinning (HRMACS) – developed by Alan Wong and his team at LSDRM marks the first successful µMAS NMR analyses of microscopic biospecimens, together with different biological expert-teams (INMG, ISA and CRMSB). Unfortunately HRMACS is greatly hindered by the impractical operation and the instability of µcoil. In 2015 without any financial supports, LSDRM has teamed up with a NMR-industry JEOL and developed an alternative technology, a standalone HRµMAS probe, specifically targeted to metabolomic applications. Unlike HRMACS, HRµMAS offers good metabolic analytical stability. However, further technological development and experimental validations are absolutely necessary in order for HRµMAS to have an impact in metabolomics. The aim of HRmicroMAS project is to implement and establish a truly convenient and accessible HRµMAS NMR-based application to the field of metabolomics. The project includes designing and constructing a stable HRµMAS probe, benchmarking and validating the HRµMAS NMR-based studies, and demonstrating its utilities with real applications. The success of the project will have a significant impact to the current NMR-platform for metabolomics, because it will be the first cornerstone of µMAS NMR-based metabolomics for microscopic specimens. The first half of the project will be dedicated to designing and building a reliable HRµMAS probe, and to benchmarking and validating the HRµMAS NMR experiments from sample-preparations to data acquisitions. The goal here is to develop a reliable HRµMAS NMR application that is convenient to everyone. The second half will carry out two independent pilot metabolomic studies with different teams whose are experts in metabolomic studies: (1) with the teams INMG and ISA, an metabolic investigation will be carried out on the heterogeneity of phenotype associated to aging within isogenic populations of Caenorhabditis elegans nematodes; and (2) with CRMSB, the metabolic profile of the brain energy metabolism in rat will be explored. These biological studies are designed to exploit and evaluate the different bioapplications of HRµMAS with different biological specimens, and will be an important milestone for HRmicroMAS, because it will demonstrate the wide utility of this innovative technology for NMR applications to in the field of metabolomics. HRmicroMAS fits well within the societal challenge #4 of “Life, Health and Well-Being” because it will be a vehicle that will lead to potential new discoveries and innovations in biological and medical science. And the strong collaborative nature in the multidisciplinary this project suits under the PRC financial instrument. It gathers µMAS experts (from LSDRM and JEOL), NMR spectroscopists (LSDRM and ISA) and biologists (INMG and CRMSB). This project also capitalizes a strong collaboration with a NMR industry, JEOL, whom will offer their involvements (probe design and construction) at zero-cost to the project. For this reason, HRmicroMAS can be considered a low-cost project (a requested fund of 304 k€) for a development of an innovative NMR application that could advance the analytical platform in metabolomics and open many new exciting venues.