Figure 3.

Microglial P2Y12Rs define somatic purinergic junctions on DCX+ developing neurons; these cells do not express Kv2.1 but contain LAMP1+ lysosomes in the vicinity of microglial contacts

(A–F) Correlated STORM super-resolution and CLSM images showing the abundant expression of the microglia-specific P2Y12Rs on microglial processes contacting DCX-+ cell bodies. DCX is shown in cyan, IBA1 in green, P2Y12R in yellow, and STORM localization points (LPs) for P2Y12R in red in the insers. Counting frames are marked with red lines in main panels and white grids in insets. Images are from E15 (A), P1 (B), P8 (C), P15 (D), P90 (E), and P90 P2Y12R knockout (F) mice. Note the complete absence of CLSM and STORM signals for P2Y12R in the knockout mice in (F). All IBA1+ microglial processes in contact with DCX+ neuronal cell bodies were expressing P2Y12Rs. In the VZ and SVZ of E15 brains n = 72; in the neocortex of P1 n = 60, P8 n = 85, and P15 n = 74; and in the dentate gyrus of P90 animals n = 93 contacts were tested (n = 2 mice in each age group, altogether n = 10 mice).

(G) Spatial analysis of super-resolution data shows the enrichment of P2Y12R labeling on the contacting side of microglial processes. Black dots represent data points, a blue bracket is the interquartile range, and median is shown by a red segment; n = 35 processes from 5 mice (Table S3). Kruskal-Wallis test followed by Tukey’s comparison; n.s., no significant difference; ^∗p < 0.05, ^∗∗∗p < 0.001.

(H) CLSM image showing the complete lack of Kv2.1 (yellow) expression in an E15 cortical plate (142 fully reconstructed DCX+ cells tested from two mice, zero Kv2.1+). DCX (blue), IBA1 (green); the ventricle (v) is delineated by a thin red line. Rectangular areas labeled with numbers are enlarged on the right.

(I) CLSM image showing robust expression of Kv2.1 (yellow) in the dentate granule cells of P90 mice, but DCX+ (blue) cells are completely devoid of Kv2.1 labeling (136 fully reconstructed DCX+ cells tested from two mice, zero Kv2.1+). The rectangular area is enlarged on the right. n, neuron; d, DCX+ cell.

(J and K) CLSM images showing examples of IBA1-labeled (green) microglial processes contacting the cell body of DCX+ postmitotic neurons (blue) with LAMP1+ puncta (red, white arrows) in the vicinity. The images are from the SVZ/VZ of E15 (J) and the DG of P90 mice (K).

(L) 69% of all contacts in E15 and 63% in P90 mice contained LAMP1+ vesicles within the DCX+ cell bodies in the close vicinity of microglial process contact. The red and green columns together show the number of measured contacts (49 for E15, 71 for P90, 2 mice for each group), and the red columns represent the number of contacts with LAMP1 labeling (34 for E15, 45 for P90).

Scale bars represent 2 μm in (A)–(F), 30 μm in (H) (5 μm in insets), 15 μm in (I) (5 μm in insets), 2 μm in (J), and 2 μm in (K). See also Figure S3.