Figure 1.
Loss of CSF-1R results in severe depletion of microglia.
A. Coronal 5 µm sections of brains of 3 week old (a–f), E16 (g, h) and D1 PP (i, j) mice. Wt (a, c, e, g, i) and Csf1r−/− (b, d, f, h, j) immunostained with anti-Iba1 antibody and counterstained with hematoxylin. Sections from the hippocampus (a, b, g, h), thalamus (c, d, i, j) and preoptic area of the hypothalamus (e,f). Bar 50 µm. B. Higher power micrographs of sections of the thalamus (a–d, g, h), Cortex (e,f) from Wt (a, c, e, g,) and Csf1r−/− mice (b, d, f, h,) immunostained with anti-Ibal (a–f) or anti-F4/80 (g, h) antibodies. Mice at 3 weeks (a–d) and 1 day (e–h) of age. Brown cells in b indicate a cluster of rounded immunostained cells found in Csf1r−/− mice (b) compared to the highly dendritic cells found in WT mice (a). c and d higher power of individual immunostained cells (arrows) showing the lack of dendritic processes in Iba1 positive cells in Csf1r−/− mice. a, b (Bar 20 µm), c, d, e, f, g, h (Bar 10 µm). C. Enumeration of Iba1 positive cells in a complete half section of the brains of D1 PP WT and Csf1r−/− (−/−) mice. N = 3/genotype. Significantly different p<0.001.
Table 1.
IBA-1 Positive cell numbers in the brains isolated from 3 week old WT and Csf1r null mutant mice.
Figure 2.
Absence of CSF-1R results in perturbed brain architecture.
A. Gross architecture of paraformaldehyde fixed brains from 3-week-old mice cut 0.5 mm anterior to the optic chiasm from three WT (1–3) and three Csf1r−/− (4–6) mice. Note the enlarged ventricles and thinned cortex in Csf1r−/− mice. B. 5 µm Coronal sections stained with Nissl stain. 1, 2, 3, 4, anterior olfactory nucleus. 5, 6, Cortex, thalamus and hippocampus. 7, 8, cerebellum. 1, 3, 5, 7, WT, 2, 4, 6, 8, Csf1r−/−. Scale bar 1 mm. C. 5 µm Coronal sections stained with Nissl stain of brains from WT and Csf1r−/− E16 mice. Bar = 500 µm. D. 5 µm Coronal section through the cortex, thalamus and hippocampus from day 1PP WT (1) and Csf1r−/− (2) mice. Scale bar = 1 mm. E. Close up of sections of the cortex from B5 and 6 with lines with double arrows indicating matched regions in which cell counts and measurements were made from WT and Csf1r−/−. Bar = 200 µm. F. Cortical thickness from regions indicated in E. N = 3 for each genotype. p = 0.0168 Student's t test.
Figure 3.
Myelination patterns suggest largely normal late development but with periventricular disruption in Csf1r−/− mice.
Coronal sections from three week old wild type (A,C,E,G,I) and Csf1r−/− (B,D,F,H,J) mouse brain taken from equivalent co-ordinates along the rostrocaudal axis and immunostained for myelin basic protein (MBP) by immunoperoxidase (brown) and Hematoxylin counterstained (blue). Abbreviations: amygdale (a); alveus hippocampus (ah); cerebral cortex (c); corpus callosum (cc); cerebral peduncle (cp); dorsal hippocampal commissure (dhc); external capsule (ec); hippocampus (h); internal capsule (ic); lateral ventricle (lv); posterior commissure (pc); striatum (s); thalamus (t). (Section in H torn centrally during processing.) Scale bars A–F = 500 µm G–J = 200 µm.
Figure 4.
Effects of the loss of the CSF-1R on cell populations in the cortex.
A. 5 µm sections of cortex (a–d, g,h) and hippocampus (e, f) immunostained with anti NeuN (a, b), anti-GFAP (c–f) or anti-Nogo A antibodies (g,h). c–h counterstained with Haematoxylin. Wild Type (a,c,e,g) and Csf1r−/− mice (b,d,f,h) Bar = 20 µm. B. Total cell counts (N = 3/genotype) determined as described in the materials and methods in the regions indicated in Figure 2B 5, 6 of all Nissl stained cells (a), NeuN positive cells (b), GFAP positive cells (c) and Nogo A positive cells (d). GFAP positive cells are increased in Csf1r−/− mice compared to WT (p = 0.022) while Nogo A positive ones are decreased (p = 0.0314).
Figure 5.
Olfactory Deficits in Csf1op/op mice.
A. Representative gross morphology of brains of two WT mice and two Csf1op/op (op/op) mice. Note the runting of the olfactory bulbs in Csf1op/op mice. B. Nissl stained 5 µm sections through the anterior region of olfactory bulbs of WT and Csf1op/op mice indicate normal morphology in mutant mice. Scale bar = 1 mm. C. Olfaction tests: Mice of genotype WT (n = 9) and Csf1op/op (n = 7) as indicated given a food finding task were challenged on the first night with chow, and on the subsequent evening with cheese. Left panel. Latency in seconds to locate the buried food. Right panel. Latency in seconds to initiate consumption subsequent to finding food. Significant differences between genotypes across food types p<0.03 in both panels.
Figure 6.
The MacGreen mouse reports CSF-1R expression in the brain and co-localized only with microglia markers.
A. Representative 8 µm sections through the hypothalamus of the MacGreen mouse in which GFP reports CSF-1R promoter activity (Panel A) were immunostained with anti-mouse CSF-1R antibody labeled with rhodamine (Panel B). Note GFP is largely cytoplasmic and nuclear while CSF-1R is a membrane protein and therefore labels dendritic processes. Co-localization studies were performed using confocal microscopy and they showed complete co-incidence of labeling (merged image, panel C). * Third Ventricle Bar = 20 µm. B. Representative coronal confocal images of co-localization studies of 8 µm brain sections from the MacGreen mouse in which EGFP is expressed from the Csf1r promoter and that marks CSF-1R expressing microglia. Staining for the rhodamine-conjugated antibodies indicated was performed as described in the Materials and methods. Left panels Csf1r-eGFP reporter showing GFP labeled cells, Middle panels antibody staining in red, Right panels merged images. Antibody staining does not co-localize with astrocyte (anti-GFAP, a–c), neuronal (anti-NeuN, d–f) or oligodendrocyte (anti-MBP, g–i) markers. In contrast, there is complete congruence with the microglial marker Iba1 (j–l), F480 (m–o). In some cases there appeared to be occasional green cells that were not labeled when viewed on single plane sections in panels g–k. However, upon optical sectioning these were found to co-localize with the immunostain showing that single plane images can be misleading because of differential cellular localization of GFP and the antibody epitope (Data not shown). A-i m,o cortex; j–l hippocampus. Bar = 20 µm.
Table 2.
Csf1r-GFP Positive Cells Co-localize with Microglial markers.