Microglial P2Y12 is necessary for synaptic plasticity in mouse visual cortex

Microglia are the resident immune cells of the brain. Increasingly, they are recognized as important mediators of normal neurophysiology, particularly during early development. Here we demonstrate that microglia are critical for ocular dominance plasticity. During the visual critical period, closure of one eye elicits changes in the structure and function of connections underlying binocular responses of neurons in the visual cortex. We find that microglia respond to monocular deprivation during the critical period, altering their morphology, motility and phagocytic behaviour as well as interactions with synapses. To explore the underlying mechanism, we focused on the P2Y12 purinergic receptor, which is selectively expressed in non-activated microglia and mediates process motility during early injury responses. We find that disrupting this receptor alters the microglial response to monocular deprivation and abrogates ocular dominance plasticity. These results suggest that microglia actively contribute to experience-dependent plasticity in the adolescent brain.


Supplementary Figure 3. P2Y12 is highly expressed in cortical microglia
a-b. Confocal images of P2Y12 immunoreactivity in V1b of CXC3CR1 GFP/+ mice showing high expression on microglial processes. Images were taken using a 10x (a) and a 40x (b) objective. Square selection represents location of 40x image. Left panels show GFP fluorescence representing microglia, middle panels show P2Y12 immunoreactivity, and right panels represent the merge (GFP = green; P2Y12 = purple). c. High levels of colocalization between GFP and P2Y12 immunoreactivity in CXC3CR1 GFP/+ mice were observed in epifluorescence images.Images with circles denote in focus microglia counted for analysis. d. Graph showing quantification in three animals, where every microglia contained P2Y12 immunoreactivity. V1b: primary visual cortex binocular area. DHC: dorsal hippocampal commissure; V2L: secondary visual cortex lateral area. Scale bars = 100 µm (a); 50 µm (b); 50 µm (c).

Supplementary Figure 4. Changes in single eye responses after MD
The response from the ipsilateral and contralateral eye is plotted for the different conditions corresponding to the ocular dominance index (ODI) shifts presented in Fig. 5f. Notice that after 4D MD there is a reduced response to stimulation of the contralateral eye in control saline-treated mice but no change in the ipsilateral response. This reduction does not occur in P2Y12 KO mice. Graphs show mean +/-SEM.

Supplementary Figure 5. Confirmation of a lack of change in microglial motility in P2Y12 KO mice after 4 days of monocular deprivation
a. Images showing microglia imaged in vivo in P2Y12 KO mice. Traced processes in the boxed area are shown in insets in the upper right-hand corner of each panel. b. Quantification of traced processes shows no change in motility after deprivation in P2Y12 KO mice (Student's t-test, t(7) = 0.55 p=0.60). Scale bar = 20 µm. Graphs show mean +/-SEM.

Supplementary Figure 6. Interactions between microglia and synaptic and perisynaptic elements after MD
There was no effect of deprivation on microglial contacts with dendritic spines, axon terminals or astrocytic processes in either P2Y12 WT or KO mice (n=5, p=0.95, F(2,36) = 0.049; two-way ANOVA). Graphs show mean +/-SEM.

Supplementary Figure 7. Model of microglial actions during ODP
In non-deprived visual cortex, microglia are highly ramified and processes are motile. Within 12 hours after monocular deprivation, decreased activity in neurons leads to a rapid microglial hyper-ramification through an unknown mechanism. On a slower scale, ADP release from neurons or astrocytes activates P2Y12 on microglia, triggering microglial process targeting to depressed synapses, decreased process motility and phagocytosis of postsynaptic material.  Fig. 1b (top) and Fig. 1d (bottom)) after MD. Post hoc comparisons are made relative to the ND results from the same brain area. Notice that following 12 HR, 1D, 2D and 4D MD, significant hyper-ramification is observed between 20 and 32 m from the soma (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).