Microglia actively regulate the number of functional synapses

Abstract

Microglia are the immunocompetent cells of the central nervous system. In the physiological setting, their highly motile processes continually survey the local brain parenchyma and transiently contact synaptic elements. Although recent work has shown that the interaction of microglia with synapses contributes to synaptic remodeling during development, the role of microglia in synaptic physiology is just starting to get explored. To assess this question, we employed an electrophysiological approach using two methods to manipulate microglia in culture: organotypic hippocampal brain slices in which microglia were depleted using clodronate liposomes, and cultured hippocampal neurons to which microglia were added. We show here that the frequency of excitatory postsynaptic current increases in microglia-depleted brain slices, consistent with a higher synaptic density, and that this enhancement ensures from the loss of microglia since it is reversed when the microglia are replenished. Conversely, the addition of microglia to neuronal cultures decreases synaptic activity and reduces the density of synapses, spine numbers, surface expression of AMPA receptor (GluA1), and levels of synaptic adhesion molecules. Taken together, our findings demonstrate that non-activated microglia acutely modulate synaptic activity by regulating the number of functional synapses in the central nervous system.

Figure 1. Clondronate ablates microglia in organotypic hippocampal brain slices.

Hippocampal slices were obtained from MacGreen mice and were treated with clodronate (5 µg/ml) or MIF (100 µg/ml) for 6 or 14 days and examined using confocal microscopy. A. Microglia could not be detected in clodronate-treated slices at DIV14, whereas microglia in MIF-treated slices appear similar in morphology to microglia in control slices. Scale bar, 20 µm. B. Western blot analysis of NeuN, Iba-1, and GFAP. Alpha-tubulin was used as a loading control.

Figure 2. Increased mEPSC frequency in microglia-depleted organotypic hippocampal brain slices.

Hippocampal organotypic brain slices obtained from MacGreen mice were treated with clodronate (5 µg/ml) or MIF (100 µg/ml) for 6 or 14 DIV. A. Firing patterns of CA1 pyramidal neurons in response to current injections for control and clodronate- or MIF-treated slices. B. CNQX, an AMPA receptor antagonist, blocks mEPSCs. Representative traces of mEPSC from control or clodronate- or MIF-treated OHBS at DIV14 in the absence or presence of 10 µM CNQX. C. Representative recording traces of mEPSCs from CA1 neurons in control, clodronate- or MIF-treated brain slices. D. Summary of mean mEPSC frequency and amplitude from control, clodronate-, or MIF-treated brain slices. Error bars represent mean ± SEM.

Figure 3. Replenishment of microglia reverses the effect of microglial depletion on organotypic brain slices.

A. Organotypic slices obtained from MacGreen mice were treated with clodronate for 6 DIV. Clodronate was washed away and then the slices were cultured for 8 DIV in the absence of clodronate with or without primary microglia labeled with miniruby (5 µg/ml, 8×10² cells in 3 µl). B–D. Images showing exogenously added microglia (Miniruby, red) to CA1 hippocampal slices treated with clodronate. MacGreen indicates endogenous microglia (green). Higher magnification of miniruby⁺ microglia in C. Side view of the z-stacking images for B. DAPI staining was used to visualize nuclei. Scale bars, 50 µm in B; 20 µm in C. E–G. Recording traces (E) and summaries of frequency (F) and amplitude (G) of sEPSCs and mEPSCs from CA1 neurons in control, clodronate-treated, and clodronate-treated with replenished microglia organotypic slices. Error bars represent mean ± SEM. H–J. Cellular debris does not affect sEPSC and mEPSC frequency or amplitude. H. Organotypic slices were incubated either without (control) or with (red line) added cellular debris (control+debris). I, J. Summary of sEPSC and mEPSC frequency (I) and amplitude (J) from control or cultures with added cellular debris at DIV14. Data are expressed as means ± SEM (n = 2 for each).

Figure 4. Addition of microglia to neurons reduces mEPSC frequency.

Primary hippocampal neurons (DIV21) were cultured for 2 days either alone or in the presence of microglia obtained from MacGreen mice. A. Neurons were stained with anti-NeuN antibody and visualized with Alexa Fluor555-conjugated secondary antibody. Scale bar, 50 µm. B. mEPSC recordings from neurons in the presence or absence of microglia. C, D. Summary of mean mEPSC frequency (C) and amplitude (D) from neurons in the presence or absence of microglia.

Figure 5. TNF-α levels are below detection levels in control or clodronate- or MIF-treated organotypic slices or in co-cultures of neurons and microglia.

Media from control or clodronate- or MIF-treated organotypic slices at DIV6 and 14, and hippocampal neuronal cultures (1°neurons) in the absence (Neu) or presence (Neu+mic) of microglia for 2 days were collected. TNF-α levels were measured by ELISA. TNF-α levels in media collected from primary microglia exposed for 12 h to LPS (100 ng/ml) were used as a positive control.

Figure 6. Microglia alter synaptic density and GluA1 expression in hippocampal neurons.

A, B. Hippocampal slices were treated with clodronate (5 µg/ml) or MIF (100 µg/ml) for 6 or 14 DIV. A, GluA1 expression in the CA1 neuronal layer in DIV6 and DIV14 hippocampal organotypic brain slices. B, Lysates from the organotypic slices were immunoblotted and probed using antibodies against GluA1 and α-tubulin. Scale bar, 10 µm. C, D. Primary WT hippocampal neuronal cultures were cultured in the absence or presence of microglia for 2 days. C, Immunohistochemistry was performed using anti-GluA1 antibody under non-permeabilized staining conditions and visualized with Alexa Fluor488-conjugated secondary antibody. After subsequent permeabilization phalloidin was used to stain F-actin. Scale bar, 20 µm. D, Quantitative analysis of surface GluA1 along dendrites for neurons with or without microglia using the LSM 5 Image Browser (Zeiss). Data are presented as mean ±SEM and expressed as a percent of the neurons-only control sample. E–H. E, Hippocampal neurons with or without microglia were stained with PSD95 (green), synapsin I (blue), and phalloidin (red). The smaller boxes show magnified images. Arrows depict PSD95⁺ synapsin I⁻ puncta. Scale bars, 20 µm (upper panel); 5 µm (lower panel). Quantification of spine numbers (F), PSD95⁺ synapsin I⁺ puncta (G), and PSD95⁺synapsin I⁺ puncta in total PSD95⁺ puncta (H) in neurons cultured with or without microglia. Values are presented as mean ±SEM expressed as a percent of the neurons-only control sample.

Figure 7. Microglia near neurons in co-cultures engulf neuronal materials.

A. Hippocampal neurons 19 DIV were coincubated with microglia for 2 days, immunostained with CD11b (green), a marker of microglia, and GluA1 (red), and visualized with Alexa Fluor488 or 555-conjugated secondary antibodies. Scale bar, 20 µm. B. Images showing microglia (phalloidin, red) and neurons (obtained from β-actin-EGFP mice, green) in co-cultures. Arrows point to neuronal material within the microglia. Scale bars, 20 µm (left); 5 µm (right). C. Images showing Mac-2⁺ microglia (green), the lysosomal marker LAMP-1 (red), and presynaptic puncta immunostained for synapsin I (white) in co-culture. Scale bars, 20 m. D. Orthogonal views of the engulfed presynaptic material (white arrowheads in C).

Figure 8. Microglia decrease the levels of synaptic adhesion molecules.

Hippocampal neurons at 19 DIV were co-cultured with microglia for 2 days. A. Western blot analysis of the levels of N-cadherin, pan-γ-protocadherin, and SynCAM-1 in neurons in the absence or presence of microglia. α-tubulin was used as a loading control. B. Quantification was performed using the ImageJ software and normalized against α-tubulin (n = 4). *P<0.05 compared to neurons alone.