Manganese (Mn²⁺) Enhanced Magnetic Resonance Imaging (MEMRI) can be used for different applications such as tracing neuronal connections or functional imaging^1-3. There are known to be multiple pathways of Mn²⁺ cell entry, however, mechanisms of Mn²⁺ cell accumulation and Mn²⁺ transport are still unclear. Therefore, the aim of this study is to better understand these mechanisms by the use of MEMRI in an organotypic hippocampal slice culture (OHSC). Mn²⁺ cell uptake under different conditions was studied. They were: (1) in competition with Ca²⁺ and in presence of Ca channel antagonist (verapamil); (2) in competition with other metals that use divalent metal transporters (DMT1 family) such as Fe and Zn; (3) in presence of AMPA, NMDA and GABA receptor antagonists (NBQX, MK-801 and bicuculline respectively) and (4) in presence of TTX, a Na channel blocker.

Animals: Postnatal day six 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, MnCl₂ was added at different concentration to the culture medium and at different times before MRI (Fig.1). Zn, Fe, Zn/Fe 1mM, Ca 3mM-6mM, MK-801 and/or NBQX (1 μM), bicuculline (20 μM) or TTX (1 μM) were added to the medium culture at the same time as Mn (2h before MRI). Verapamil 40 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=4.65 ms, voxel size: 50 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 signal was normalized to the background noise. The signal to noise ratio (SNR) was computed for each ROI and subtracted from the SNR of control slices (without MRI; ΔSNR).

Figure 1 shows MRI images from slices incubated with 10, 25, 50, 100 and 150 μM of MnCl₂ after 1, 2, 4 and 24h incubation time. Most of the drugs necessitate 2h of incubation, we choose to work with the 2h incubation at a high (150 μM) and low (25 μM) [MnCl₂]. Manipulation of metal transporters and Ca²⁺ channels affect Mn²⁺ cell uptake (Fig.2/3). A higher impact on OHSC incubated with the high [MnCl₂] was observed in presence of Ca²⁺ 6mM and verapamil (61% and 54%, respectively compared to 41% and 30% for the low [MnCl₂]). Interestingly, antagonism of glutamate receptors induced a decrease of Mn²⁺ cell uptake only at the low [MnCl₂]. GABA antagonist did not change Mn²⁺ cell accumulation (Fig.4). TTX induced a decrease of Mn cell uptake at a low [MnCl₂] (-36%) and unexpectedly an increase at a high [MnCl₂] (+52%; Fig.5).