Fig. 5

Repopulation increases Cxcl13 expression in hippocampal microglia of male 3xTg mice. Schematic of experimental design (A, B). FACS-sorted cells were used as the input for scRNAseq (A). UMAP plots of control and PLX-repopulated groups illustrate clustering of cells (B). The inset shows the output of scMCA-based cell annotations. An overwhelming majority of sequenced cells were microglia (B). Proportions of different cell clusters out of all sequenced cells are depicted in C. Panel of genes identified through Seurat’s statistical framework or established literature is shown in D for annotation purposes. Red boxes highlight genes that are associated with their corresponding clusters. Increased Cxcl13 expression can be noted across the majority of repopulated microglia but in particular for PLX-mg and ARM/DAM clusters (E). Increasing tones of green denote increased Cxcl13 expression. ELISA on hippocampal lysates confirmed the upregulation of CXCL13 at the protein level (F). Representative immunofluorescent 10× images of in situ analysis of Cxcl13 expression (G, H). Scale bar represents 100 µm in G or 200 µm in H. The resultant yellow signal from green Cxcl13 in situ hybridization and red Iba1 immunohistochemistry suggests that all of the Cxcl13 transcripts we observed are associated with microglia (G). This representative image is from a PLX-repopulated sample. Cxcl13 staining showed strong trends towards increased expression in PLX-repopulated groups in Subiculum and CA1 (H–J), however did not change in the cortex (K). Student’s t-test (F, I–K), *p < 0.05. Data are presented as mean ± SEM