PURPOSE

MEMRI can be used for several 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 transport are still unclear. Visualization of the Mn²⁺ subcellular distribution will advance our understanding of these mechanisms. Therefore, the aim of this study is to obtain maps of the subcellular distribution of Mn²⁺ using TEM and X-ray elemental mapping of specimens obtained from an organotypic hippocampal slice culture (OHSC).

METHODS

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 incubation: On D7 or D8, MnCl2 was added at 0, 10, 25, 50, 100 or 150 μM to the culture medium for 24h before the experiment (Fig.1). MEMRI and electrophysiology: the membrane insert was cut around the slice, rinsed, and placed in a perfusion chamber. Slices were continuously perfused with bubbled aCSF. For the MRI, T₁ weighted images (FLASH, TR=25 ms, TE=4.65 ms, voxel size: 50 μm³) were acquired on an 11.7T MRI system (Bruker) using a surface/volume cross coil configuration. The highest Mn²⁺ concentration giving good contrast was 100 μM and was chosen for the rest of the experiments. Cell viability after MnCl₂ incubation was evaluated by electrophysiology. The intrinsic excitability and membrane properties were measured by whole cell recording of the principal neurons of CA3. The slice viability was measured by input-output curves from field recordings along the Schaffer collaterals (Field Excitatory Post-Synaptic Potentials, fEPSP; Fig.2). TEM and X-ray Nanoprobe: OHSC were rapidly frozen, cryo-sectioned, freeze-dried and imaged⁵. Sections with abundant neuropil were selected. X-ray maps with 80-nm pixel size of Mn and K were obtained in the bionanoprobe at beamline 2-ID-D of the Advanced Photon Source at Argonne National Laboratory (Fig.3)

RESULTS

Figure 2 shows that the membrane properties of OHSC after incubation for 24h with 100 μM MnCl₂ are similar to those of the control. Similar membrane potentials (control -64.4 ± 1.0 mV, MnCl₂ -64.7 ± 1.0 mV, tTEST p = 0.6) and input resistances (control 204.4 ± 22.0 MΩ, MnCl₂ 159.7 ± 30.6 MΩ, tTEST p = 0.26) were recorded in both groups. Intrinsic excitability of the neurons was also unchanged, with control cells’ rheobase at 101.6 ± 17.6 pA and incubated OHSC at 94.7 ± 16.8 pA, tTEST p = 0.78. Synchrotron x-ray maps (Fig.3) show that Mn²⁺ accumulation is confined to structures with diameters of 330 ± 200 nm and an average concentration of 70 ± 30 mM. Mn²⁺ structures are mainly intracellular with some clusters apparently at synapses. Focal regions containing Mn²⁺ atoms are distributed in the neuropil at an areal density of 0.5 / µm² and at a concentration of 2 mM. No significant accumulation of Mn²⁺ was detected in mitochondria.

DISCUSSION

After incubation with 100 μM MnCl₂ for 24h, the OHSC exhibits normal cellular function. This is important for the analysis of the X-ray images. Mn²⁺ is mainly located in intracellular compartments. Interestingly, our data show frequent Mn²⁺ accumulations in synapses in agreement with the current Mn²⁺ transport theory describing Mn²⁺ synaptic transmission via glutamatergic vesicle^6,7. In contrast, our data show a rare presence of Mn²⁺ in mitochondria, in contrast to the literature⁸. Some clusters are in cell body cytoplasm but do not appear to be associated with any cellular organelles.

Acknowledgements

No acknowledgement found.

References

¹Lin and Koretsky, 1997, MRM, 38:378-388; ²Aoki et al., 2004, Neuroimage, 22:1046-1059; ³Daoust et al., 2014, Neuroimage, 96:133-42; ⁴Fuller and Dailey, 2007, CSH protocol; ⁵Pivovarova et al., 2002, J. Neurosci., 22:10653-61; ⁶Takeda et al., 2002, Neuroscience, 114:669-74; ⁷Lin and Koretsky, 1997, Magn. Reson. Med., 38:378-88; ⁸Morello et al., 2008, Neurotoxicology, 29:60-72.