The Journal of Neuroscience, November 1, 2006, ():
Fusion of Microglia with Pyramidal Neurons after Retroviral Infection
J. Neurosci. Ackman et al.
26: 11413
Supplemental data
Files in this Data Supplement:
- supplemental material
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Supplementary Figure 1. eGFP retroviral labeling of SVZ/RMS in wildtype and flathead. GFP+ cells in the SVZ/RMS surrounding the lateral ventricle in wildtype and flathead rat after retrovirus injection at P11. Note the lack of cells with a bipolar, migratory morphology in the rostral extension of the SVZ in flathead. Vimentin staining (VIM, blue) labels the ependymal cells surrounding the lateral ventricle. Scale bars: 50 µm.
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Supplementary Figure 2. Retrovirally labeled neurons in the postnatal rat telencephalon. a, A GFP labeled layer VI neuron in frontal neocortex 3 days after virus injection. The neuron is positive for the neuronal labels, NeuN (blue) and anti-rat pyramidal cell antigen (RT-PYR, red). b, A labeled neuron in the striatum (arrow) 3 days after eGFP retrovirus injection. Note the two other retrovirally labeled cell types nearby (arrowheads). c, A labeled neuron in layer CA1 of hippocampus (arrow) from a P17 rat 3 days after retrovirus injection. Note the abundant labeling of cells along the posterior periventricular space dorsal to CA1, including two secondary cells that appear closely apposed to the GFP+ dendrite of the CA1 neuron (inset). DAPI is blue, Fluorescent Nissl in red. d, Two GFP+ neurons in layer VI of frontal neocortex (arrows) 2 days after stereotaxic injection. The GFP+ processes of the cells are vimentin negative (VIM, red) as is a nearby cell with morphology of a microglia (arrowhead). Scale bars: a, left panel, 40 µm; a, right panel, 20 µm; b, 20 µm; c, 40 µm; c, inset, 20 µm; d, 40 µm.
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Supplementary Figure 3. eGFP retrovirus does not transduce non-dividing cells. Primary neural cultures from neonatal wildtype or flathead rat were cultured either in the presence eGFP retrovirus alone (Virus control), or in the presence of the anti-mitotic drug Ara-C and then eGFP retrovirus (Ara-C + virus). GFP labeled cells were found only in virus control cultures that did not receive anti-mitotic treatment for both wildtype and flathead. Scale bar: 50 µm.
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Supplementary Figure 4. Labeled neurons in vitro that are not associated with secondary GFP+ cells contain secondary nuclei in the neuronal cell body. a, A GFP+ neuron with two nuclei 14 d. after addition of eGFP retrovirus to the culture. Notice that the GFP+ neuron is colabeled with MAP-2 (magenta) which reveals the presence of two nuclei in the soma as evidenced by two regions with no MAP-2 staining separated by a band of MAP-2+ staining (right panel). Separation of nuclei was confirmed by serial optical sectioning. b, A GFP+ neuron with two nuclei at14 d.s. Notice the GFP+ neuron has a second small nuclei within the cytoplasm (red, middle panel) and is colabeled with MAP-2 (blue, right panel). Scale bars: a, left panel, 50 µm; a, middle, right panels, 20 µm; b, 10 µm.
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Supplementary Figure 5. Fusion between non-retrovirally transduced neurons and microglia in vitro. a, Fusion between a GFP-/MAP-2+ neuron and a GFP- secondary cell (DAPI, blue, inset, arrows) from a culture that had received GFP retrovirus. b, Fusion between a GFP-/IB4+ microglia and a neuron (IB4+ filling of neuronal processes, arrows). c, Fusion between a GFP-/IB4+ microglia and a GFP-/MAP-2+ neuron (arrows). IB4 in red, MAP-2 in blue, overlay on phase contrast image. Notice the extensive IB4+ labeling of the MAP-2+ fused-neuron but not of nearby MAP-2+ neurons. d, Fusion between a IB4+ microglia and a neuron from a culture that had received GFP retrovirus (arrows). Notice the reactivity of the fused neuron’s processes and microglia for IB4. e, Fusion between a IB4+ microglia and neurons from a culture that had received GFP retrovirus (arrows). Notice that the only two reactive microglia with the characteristic elongated morphology of fused-type cells are the microglia fused to IB4+ neurons. Scale bars: a, 50 µm; a inset, 25 µm; b, 40 µm; c, 20 µm; d, 40 µm; e, 40 µm.
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Supplementary Figure 6. Cells fused to neurons incorporate BrdU. a, A GFP negative neuron that is MAP-2+ (red) and has a BrdU+ (blue) secondary cell fused to a dendrite (arrows). b, A GFP+ neuron that is MAP-2+ and has a GFP+/BrdU+ secondary cell fused to a dendrite (arrows). Scale bars: a, 40 µm; b, 20 µm.
