Figure 4.

Developmental microglia-neuron somatic junctions are dynamic and depend on P2Y12R function

(A) Schematic depicting the experimental procedure.

(B) In vitro 2-photon imaging of mouse neocortex from P1 animals confirmed the presence of dynamic somatic junctions between microglia and developing neurons. The area in the white box is enlarged in the right panel, which is also shown in the 3D model below. The contact site marked with a white arrow in the 3D model is shown at z2, with image planes marked by z1 and z3 above and below the contact, respectively.

(C) The slice was scanned over 1 h; insets show representative time frames from a smaller area. Microglial processes (green, CX3CR1-GFP) establish somatic contacts on cell bodies of multiple developing neurons (red, Cal-590). White arrows point to contacts, some of which are re-connected multiple times by microglial processes.

(D) Schematic of the P2Y12R-inhibition experiment (E) and representative measurement of a “PSB” experiment. The calcium trace and coverage values, measured over the 30-min experiment, are superimposed, and red arrows show the respective temporal positions (t1–t6) of the insets of the measured cell. White arrows point to contacts.

(F) Statistical analysis confirmed that acute inhibition of microglial P2Y12Rs induced a rapid and robust decrease of microglial process coverage on developing neurons (median, 38% decrease from baseline; interquartile range, 0.36–0.92; n = 30 cells, 3 mice). Coverage in control is 1, and PSB effect is plotted as an increase or decrease compared with the control. Wilcoxon matched-pairs test; ^∗∗p < 0.001.

(G) Same as (E) but from a “vehicle” experiment.

(H) Statistical analysis confirmed that vehicle addition did not induce a significant change in microglial coverage (median, 1.06-fold increase over baseline; interquartile range, 0.75–1.66; n = 25 cells, 3 mice). Wilcoxon matched-pairs test.

Scale bars represent 10 μm.