Long-term integration of the transplanted neurons.

<p>Confocal microscopy images extracted from xyz-tile acquisitions showing GFP+ neuron implantation throughout the hippocampus 24 weeks post-transplantation. <b>a</b>) shows beads at the injection site carrying GFP+ neurons which are projecting their processes in the host hippocampus, <b>b</b>) shows neurons in Or -oriens layer of the hippocampus sending out processes through the radiatum layer, and <b>c</b>) shows cells in the stratum lucidum of the CA3. Brain slices were stained with CD11b a marker for microglia cells (<b>d</b>), and CD68 a marker for macrophages (<b>e</b>). Confocal microscopy images 4 xy frames extracted from xyz-tile acquisitions showing glass bead cluster were projected in z. Increase in microglia cells and macrophages was associated with the presence of GFP+ cells without processes (arrows). Beads without cells were free of microglia and macrophages, suggesting that these cells were there to clear non-integrated GFP+ neurons. All scale bars  =  100 µm.</p

Development and manipulation of neurons supported on silica beads.

<p>Confocal microscopy z series are projected on the xy scanning plane. E18 hippocampal neurons cultured seeded at 4k cells/cm² on 125 µm (<b>a-c</b>), and on 45 µm (<b>d-f</b>) PLL coated beads shown at DIV 4. Cells were fixed and stained with a neuron specific alphãtubulin antibody (green), and with an axon specific smi-312 antibody (red). Neurons were polarized in both preparations independently of bead radius of curvature. The number of neurons per bead is proportional to bead surface area, as 45 µm beads carried on average one cell, and 125 µm beads carried about 10 cells. (<b>g</b>) Bright field image of neurons seeded at 100k cells/cm² on 45 µm beads at DIV 4. (<b>h</b>) Cells were fixed and stained with a neuron specific Tuj-1 antibody (red), and the nuclear marker DAPI (blue). Twenty-one of the twenty-five cells on this bead are Tuj-1 positive. At this high density, cells in direct contact with the bead surface wrap their processes around the beads (highlighted in red) while the others sit on this layer (highlighted in blue) as illustrated in the color-coded picture (<b>i</b>). All Scale bars  =  50 µm.</p

Influence of the injection position on the distribution of the implanted GFP-neurons.

<p><b>a)</b> Schematic representation of the different hippocampus sub-regions. CA1-field SLu- stratum lucidum, Rad- radiatum layer of the hippocampus, PoDG- polymorph layer of the dentate gyrus, GrDG- granular layer of the dentate gyrus, MoDG- molecular layer of the dentate gyrus, LMol- lacunosum moleculare layer of the hippocampus, Py - pyramidal cell layer of the hippocampus, and Or -oriens layer of the hippocampus. Confocal microscopy images extracted from xyz-tile acquisitions showing GFP+ neuron implantation through out the hippocampus: <b>b</b>) shows the radiatum layer, <b>c</b>) the stratum lucidum of the CA3, <b>d</b>) part of the dentate gyrus. <b>f</b>) Fraction of the total GFP+ cells found in each region for injections in the CA3 (blue) and in the DG (red). Error bars represent the standard deviations for series of 10 animals. Scale bars  =  100 µm.</p

Functional integration of transplanted neurons.

<p><b>a</b>) Live confocal imaging of calcium response in a hippocampal slice containing a transplanted LiGluR6 neuron expressing GFP <b>b</b>) and labeled with a calcium indicator, Rhod-2 <b>c</b>). Panel <b>d</b>) shows an overlay of both channels. Scale bar  = 100 µm. LiGluR6 cell was stimulated by short exposure to 390nm light for a short period of time and we recorded the calcium response of the surrounding neurons. <b>a</b>) shows calcium variation of individual cells (single pixel) after binning (3x3) and subtraction of the fluorescence background. Response was color-coded using a rainbow scale. Corresponding fluorescence intensity changes during UV stimulation are shown in panel <b>e</b>). All neurons in the slice responded to the stimulation indicating that the transplanted cell has made functional connections with the surrounding neurons. For 6 neurons distributed above (labeled <i>a b c</i>) and below (labeled <i>1 2 3</i>) the transplanted cell we calculated ΔF/F for seven UV stimulations <b>f</b>). ΔF/F of the LiGluR6 neuron remains around 30% (+/−2.5%). (a<i>bc</i>) neurons have, in average, higher ΔF/F than the stimulated neurons with significant variations from one exposure to the next, while (1 2 3) neurons have, in average, a smaller ΔF/F.</p

Transplanted neurons in the adult rat hippocampus.

<p>DIV 5 GFP-neurons were injected unilaterally into the right hippocampus of 6 weeks old rats using 45 µm bead carriers. <b>a</b>) Schematic representation showing the injection location in the dentate gyrus (DG), and in the CA3 region (CA3). After a week the animals were sacrificed and their brains were sliced and immuno-stained with GFP antibody (green) and with Nissl (red) a nuclear cell marker. <b>b</b>) Fluorescence microscopy image of a brain slice taken at the injection site (scale bar  =  1 mm). Fields extracted from a XYZ-tile scan of the hippocampus, −3.7 mm A/P from the bregma, showing the extent of the transplanted neuron implantation in <b>c</b>) the CA3 stratum lucidum layer. <b>d</b>) Cross-section of a bead carrying two GFP+ neurons sending processes into the hippocampus in a 150 µm thick slice. <b>e</b>) A rare GFP+ neuron found in the brain section after dissociation from 2D support prior to injection. Scale bar  =  50 µm. Anterio-posterior GFP+ neuron distribution for injections made at [AP] = −3.5 in the CA3 (blue) and in the DG (red). <b>f</b>) shows the average number of GFP+ neuron (N_GFP-cell), and <b>g</b>) shows the average number of GFP-neuron (N_GFP-cell) per mm³. Error bars represent the standard deviations for series of 10 animals.</p