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. 2015 Feb 11;35(6):2417-22.
doi: 10.1523/JNEUROSCI.3279-14.2015.

Modulation of microglial process convergence toward neuronal dendrites by extracellular calcium

Ukpong B Eyo  1 , Nan Gu  2 , Srijisnu De  1 , Hailong Dong  3 , Jason R Richardson  4 , Long-Jun Wu  5
Affiliations

Modulation of microglial process convergence toward neuronal dendrites by extracellular calcium

Ukpong B Eyo et al. J Neurosci. .

Abstract

Extracellular calcium concentrations in the brain fluctuate during neuronal activities and may affect the behavior of brain cells. Microglia are highly dynamic immune cells of the brain. However, the effects of extracellular calcium concentrations on microglial dynamics have not been investigated. Here, we addressed this question in mouse brain slices and in vivo using two-photon microscopy. We serendipitously found that extracellular calcium reduction induced microglial processes to converge at distinct sites, a phenomenon we termed microglial process convergence (MPCs). Our studies revealed that MPCs target neuronal dendrites independent of neuronal action potential firing and is mediated by ATP release and microglial P2Y12 receptors. These results indicate that microglia monitor and interact with neurons during conditions of cerebral calcium reduction in the normal and diseased brain.

Keywords: P2Y12 receptor; calcium reduction; microglia; microglial process convergence; neuronal dendrites.

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Figures

Figure 1.
Figure 1.
Extracellular calcium reduction induces microglial process convergence in brain slices. A, Low-magnification two-photon z-stack image of GFP-expressing microglia in layer II/III of a cortical brain slice. B, C, Representative higher-magnification time-lapse images from boxed regions in (A) showing converging microglial processes during nominally free calcium conditions. Right most images are color-coded images before (red) and after (green) the process convergence where the focal points are indicated with white arrowheads. B′–C′, Images following the collapse of the converging microglial processes in B, C. D, Schematic of representative calcium switch experiments showing the location of convergent foci in the field-of-view. E, Quantitative data showing microglial process convergence in different extracellular calcium concentrations ([Ca2+]e); n = 30 slices at 0 mm, 19 slices at 1 mm and 27 slices at 2 mm. F, Velocity of extending processes during different calcium concentrations. G, Velocity of converging microglial processes during endogenous process convergence and exogenous ATP application through a pipette; **p < 0.01.
Figure 2.
Figure 2.
Microglial process convergence in vivo. A, Low-magnification two-photon z-stack image of GFP-expressing microglia in the intact cortex in vivo. B, C, Representative higher-magnification time lapse images from boxed regions in A showing converging microglial processes during calcium reduction induced by topical application of 2 mm EGTA. Rightmost images are color-coded images before (red) and after (green) the process convergence where the focal points are indicated with white arrowheads. D, Quantitative data showing process convergent events during calcium reduction in vivo (n = 8 mice under control and 4 mice under calcium-reduction conditions). E–G, Quantitative data showing responses between microglia in slices and in vivo during calcium reduction with respect to the duration of contact (E), the maximal distance from the focal point in which microglia respond (F), and the velocity of converging processes (G); ***p < 0.001.
Figure 3.
Figure 3.
Converging microglial processes target neuronal, but not astrocytic elements. A, Spontaneous calcium transients in representative astrocytes in hippocampal slices before and after incubation with FAC (10 mm; n = 10 representative cells). Inset, A single astrocyte with a calcium transient in control condition. B, C, Representative color-coded images (inset) of microglial dynamics (B, C) before (control, Ctl) and after incubation with 10 mm FAC (n = 4 slices). D, Quantitative data of microglial process convergence in 1–10 mm FAC (n = 7 slices in each condition). E, Low-magnification image of GFP-labeled microglia and SR101 stained astrocytes. F, G, Representative MPCs in boxed region in E with convergence events (white arrowheads). H, A representative image of an MPC in a double-transgenic (GFP microglia; YFP neuron) slice showing a process convergence event (white arrowhead) on a neuronal dendrite at high-magnification. I, Single z-stack images at 6 μm intervals of H. Yellow arrowheads point to the dendrite that is targeted by microglial processes.
Figure 4.
Figure 4.
Microglial process convergence requires microglial P2Y12 receptors. A, Schematic showing MPCs in the presence of ATP (1 mm; n = 5 slices) and in P2Y12 knock-out mouse slices (n = 8 slices) during calcium reduction. B, A sample initiating MPC that fails to be terminated upon application of ATP (asterisk indicates presumptive foci). C, D, MPC occurrence (C) and microglial density (D) in P2Y12 wild-type, heterozygote, and knock-out slices. E, F, Quantitative data showing effects of P2X7 antagonists (BBG; 10–20 μm; n = 5 slices), connexin blocker (CBX, 100 μm; n = 4 slices), and pannexin channel blocker (PB, 5 mm; n = 6 slices) on the occurrence of these events (D) and distance of responding microglia from the process convergence focal point (E); *p < 0.05, Student's t test; ***p < 0.001, ANOVA.
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