Neuronal Synapse Formation Induced by Microglia and Interleukin 10
Figure 1
Neuronal synapse formation induced by microglia.
(A) Microglia were prepared from mixed glia without astrocytes. The prepared microglia were stained with antibodies of CD11b/c (red) and GFAP (green). CD11b/c and GFAP are specific markers of microglia and astrocytes respectively. The numbers of cells stained with antibodies of CD11b/c (microglia) or GFAP (astrocytes) were counted, and the purity of microglia was analyzed. Scale bar, 10 μm.
(B) An RT-PCR assay was performed to determine the purity of microglia prepared from mixed glia. The expressions of Iba-1 (a specific marker of microglia) and GFAP (a specific marker of astrocytes) were analyzed.
(C) The density of dendritic spines was increased by application of microglia. Rat cultured hippocampal neurons were transfected at days in vitro (DIV) 7 with pSuper.neo-gfp, and then immunostained using anti-GFP antibodies at DIV 15 for analysis. Microglia plated on a porous cell culture insert were applied to hippocampal neurons of DIV 8. The density of dendritic spines increased when microglia was applied. Means±SEM. n=48 dendrites for no microglia, 48 for 0.25 × 105 microglia, 48 for 0.5 × 105 microglia, 48 for 1.0 × 105 microglia. **p<0.01 and ***p<0.001, by the Newman-Keuls multiple comparison test after application of one-way ANOVA, F=9.915, p<0.0001. Scale bar, 10 μm.
(D) The number of excitatory synapses was increased by application of microglia. The cultured hippocampal neurons were immunostained using antibodies of GFP (green) and vGLUT, the excitatory synaptic marker (red). The addition of microglia increased the number of vGLUT. Means±SEM. n=40 dendrites for no microglia, 40 for 0.25 × 105 microglia, 40 for 0.5 × 105 microglia, 40 for 1.0 × 105 microglia. ***p<0.001, by the Newman-Keuls multiple comparison test after application of one-way ANOVA, F=8.206, p<0.0001. Scale bar, 10 μm.
(E) The number of inhibitory synapses was increased by application of microglia. Antibodies of GFP (green) and vGAT, the inhibitory synaptic marker (red), were used for the immunostaining of hippocampal neurons. The addition of microglia increased the number of vGAT. Means±SEM. n=40 dendrites for no microglia, 40 for 0.25 × 105 microglia, 40 for 0.5 × 105 microglia, 40 for 1.0 × 105 microglia. *p<0.05 and ***p<0.001, by the Newman-Keuls multiple comparison test after application of one-way ANOVA, F=31.81, p<0.0001. Scale bar, 10 μm.