Manganese (Mn) Enhanced Magnetic Resonance Imaging (MEMRI) can be used for different applications such as tracing neuronal connections or functional imaging^1-3. However, mechanisms of Mn cell accumulation and Mn transport are still unclear. The aim of this study is to better understand these mechanisms by the use of MEMRI in a hippocampal organotypic slice culture. The accumulation of Mn in hippocampal cells in different conditions was studied. There were: (1) in competition with other metals that use divalent metal transporters (DMT1 family) such as Fe and Zn; (2) in competition with Ca and in presence of Ca channel antagonist (verapamil) or a β-1 adrenergic agonist inducing an increase of Ca channel activity (dobutamine).Animals: Postnatal day 6 Sprague Dawley rats were used for this study. Slice preparation and culture: Hippocampi were dissected and coronal sections (300 μm) were sliced in an artificial CSF (aCSF) at 4ºC ⁴. Slices were cultured at the interface medium/air on a membrane insert (incubation at 37 ºC, 5%CO2) for 7 or 8 days (D7, D8). Mn and drug incubation: On D7 or D8, Mn was added at 150 μM to the culture medium for 2h before MRI. Zn 1mM, Fe 1mM, Zn/Fe 1mM or Ca 3mM-6mM were added to the medium culture at the same time as Mn. Dobutamine 50 μM was added 2h before Mn (4h before MRI). MEMRI: 2h after Mn addition to the medium culture (with or without metal or drug), the membrane insert was cut around the slice, rinse, placed in a perfusion chamber and fixed with a plastic anchor. Slices were continuously perfused with aCSF at room temperature. T₁ weighted images (FLASH, TR=25ms, TE=4.01 ms, voxel size: 100 μm3) were acquired on an 11.7T MRI system (Bruker) using a surface/volume cross coil configuration. Data analysis: ROIs were manually drawn around the slice and also in 6 different areas using a rat brain atlas as a visual reference: dentate gyrus (DG), Cornu Ammonis (CA3 and CA1/2), Schaffer collaterals (SC), subiculum/enhorinal cortex (Sub/EC) and signal was normalized to the background noise (Fig.1). The signal to noise ratio (SNR) was computed for each ROI and subtracted from the SNR of control slices (without MRI; ΔSNR).MRI images revealed excellent contrast in presence of 150 µM Mn in the medium (Fig.1). We have shown that manipulation of metal transporters and Ca channels affect Mn cell accumulation (Fig.2/3). A decrease of Mn cell accumulation was observed in presence of an excess of Ca (Ca 3 mM: -54% and Ca 6 mM: -58%) as well as in the presence of verapamil (54%; Fig.2). In presence of dobutamine, there is an increase of Mn accumulation (+16%) with a specific increase in SC and Sub/EC (Fig.2). To finish, a decrease of Mn accumulation was observed in presence of Zn (-51%), Fe (-41%) and Zn/Fe (-48%). All of these decreases concerned all ROIs with a more significant decrease in CA1/2 and 3 (Fig.3).Our results suggest that 50% of Mn enters hippocampal cells via Ca channel in agreement with literature⁵. There is still a signal in presence of Ca channel antagonist meaning that Mn cellular uptake is also performed via other channel or transporters. In competition with Zn, Mn uptake decrease in Zn rich fibers area (CA3 and DG⁶). The decrease in Mn accumulation in presence of Zn and Fe is in aggrement with the literature showing a transport of Mn via DMT-1, transferrin and ZIP8^7-8. To finish, in our experiment dobutamine increase Mn uptake in agreement with literature using cardiac cells⁹. It is known that β-1 adrenergic receptors are rich in CA1 and in subiculum¹⁰. The stimulation of these areas probably induced an increase of Mn transport in CA2/3 and DG. More investigation will be done by studying drug impact on Mn tract tracing following a microinjection. In conclusion, MRI of hippocampal slice culture is an excellent way to study the cellular mechanisms related to MEMRI.