Figure 1: An Imaris surface-rendered image of GFP-labeled microglia (green) surrounded by nuclei of other cells in the mouse hippocampus (blue).
When the brain is injured, specialized phagocytic microglial cells engulf and remove cellular debris. However, the role of these cells in uninjured brains is not clear. Researchers led by Dr. Cornelius Gross from the European Molecular Biology Laboratory (EMBL) in Italy studied microglia in uninjured developing mouse brains to find out if these cells help monitor and maintain synapses.
“Studies on microglial function so far were mainly performed in pathological conditions. The idea that microglia can also exert an active role in the intact developing brain is a relatively new concept,” said Dr. Rosa Paolicelli, who was part of the research team. A better understanding of microglia-mediated synaptic pruning might reveal more about how the brain and immune system interact and how neuronal connections form in the brain.
With confocal, simulated emission depletion (STED), and electron microscopy the researchers observed microglia in developing mouse brains. They used GFP to label microglial processes and immunohistochemistry was used to mark the presence of PSD95, a protein found at the post-synaptic density of dendritic spines. The confocal images showed that some PSD95 puncta colocalized with GFP. This observation was confirmed with STED microscopy, which resolved PSD95 puncta as small as ~ 80 nm.
Figure 2: Surface rendering of GFP presence located exclusively in the microglia in the hippocampal CA1 section
The researchers acquired serial optical sections (0.4 μm Z-step size) of hippocampal stratum radiatum with confocal microscopy and then used Imaris software to create 3-D reconstructions. The reconstructions showed that, in some cases, the PSD95 puncta that colocalized with GFP were surrounded by GFP-labeled microglial cytoplasm. Dr. Paolicelli said that the 3-D reconstruction of microglial cell processes containing synaptic material helped support their idea that the microglia engulfed dendritic spines and provided information that was complementary to the 2-D colocalization analysis.
Figure 3: The terminal processes of microglia (green) contain synaptic material (red), showing in this surface-rendered image that the microglia engulfed the synapses. The red particles are positive for PSD95, a protein located at the post-synaptic density of dendritic spines.
Collectively, the microscopy images taken with the three techniques showed that microglia engulf and eliminate synapses during development. The researchers also found that the developing brains of mice without CX3CR1 gene encoding a chemokine receptor expressed by microglia in the brain – had less microglia and delayed synaptic pruning, which resulted in extra dendritic spines and immature synapses. In other words, microglia are critical for setting up the right connectivity in the brain. “These findings shed light on a new important role for microglia in synaptic pruning, refining neural connections and circuit maturation,” Dr. Paolicelli said.
The researchers say that genetic variation in expression of CX3CR1 together with environmental pathogens that impact microglia function might lead to an increased susceptibility to developmental disorders such as autism that are associated with alterations in the number of synapses in the brain. The researchers plan to investigate microglia in the healthy adult brain, where their role is essentially unknown.