¹³C Magnetic Resonance Spectroscopy (MRS) has shown to be a very promising tool to study brain metabolism in vivo. After administration of a ¹³C-labeled probe to a subject, the enrichment of the produced metabolites can be followed over time and metabolic fluxes can be assessed through a modeling approach. Indirect carbon spectroscopy (¹H-[¹³C] MRS) is a technique that allows measuring ¹³C-labelled metabolites with higher sensitivity but with reduced bandwidth resolution. Recently, metabolic fluxes including TCA cycle in a 60µL voxel of the mouse brain, including striatum and cortex, were assessed using ¹H-[¹³C] MRS upon infusion of [U-¹³C₆] glucose at 14.1 Tesla. The aim of the present study was to measure metabolism in a smaller voxel (16.5 µl) including mainly the dorsal hippocampus, as this structure is important for many brain functions and implicated in many diseases.Male ICR/CD1 mice (n=6, 12 weeks) were scanned under isoflurane anesthesia (1-2%) in a horizontal 14.1T/26cm Varian magnet (Agilent Inc., USA). A homemade ¹H surface coil in quadrature combined with a linear ¹³C coil was designed specifically for ¹H-[¹³C] MRS. For instance, ¹H coil was made close to mouse brain and ¹³C coil was just on top of ¹H coil. The volume of interest (2x5.5x1.5 mm³) including bilateral dorsal hippocampi was localized using a set of T₂-weighted FSE images. Field inhomogeneity was adjusted using FASTMAP to reach a water linewidth <25Hz¹. On the target VOI, localized indirect ¹H-[¹³C] detection was applied using the full signal intensity BISEP-SPECIAL sequence (TE/TR=2.8/4000ms) together with OVS and VAPOR water suppression^2,3. Dorsal hippocampus metabolism was evaluated upon infusion of 70% enriched 20% (w/v) [U-¹³C₆] glucose for 3 hours. Spectra were frequency corrected and summed (nt=64/10min time point) for LCModel quantification and total creatine as internal reference set at 8µmol/g. Non-edited ¹H MR spectra contain ¹H resonances coupled to both ¹²C and ¹³C and thus can be quantified with a standard basis set for neurochemical profiles of mouse hippocampus. The ¹³C-editing spectra were quantified using another simulated basis set as previously³. The fraction of isotope enrichment (FE) in lactate, glutamate (Glu), glutamine (Gln) and the sum of Glu and Gln (Glx) was estimated. Results were fitted to a one compartment model of glucose metabolism using MATLAB nonlinear regression methods, and TCA rate (V_TCA), transmitocondrial flux (V_x), neurotransmission rate (V_NT), dilution factor (K_dil) and a composite flux (V_gt) were estimated. Monte–Carlo simulation was used to evaluate the errors of all adjusted metabolic fluxes⁴.Based on T₂ images, bilateral dorsal hippocampi were clearly identified (Figure 1). Figure 2 showed the typical spectra obtained after 3 hours of [U-¹³C₆] glucose infusion with good GluC4, GlnC4, GlxC3, and LacC3 resolution. The averaged FE time courses of lactate C3, GluC4, GlnC4 and GlxC3 were measured and therefore used for modeling (Figure 3). Lactate C3 FE reached steady state shortly, was used as the input function for metabolic flux quantification and assumed to be a step function. The resulting fluxes were V_TCA: 0.79±0.10, V_x: 0.32±0.05, V_NT: 0.42±0.35, K_dil: 0.85±0.08, and V_gt: 0.23±0.03, which were similar to previous study², as shown in Figure 4.This study shows for the first time in vivo measurements of the metabolic fluxes in the dorsal hippocampus using indirect ¹H-[¹³C] MRS detection. Time resolution was similar as compared to previous study with 60µl volume, without decreasing metabolite linewidths or affecting considerably the SNR. We conclude that metabolism of the mouse dorsal hippocampus can be assessed in vivo, which is one of the most studied structures of the brain.