Figure 8. Neuronal clusters activated during specific social behaviors revealed by MERFISH.

(A) Enrichment in cFos-positive cells within each neuronal cluster observed in males or females after displaying a given social behavior. Red bars marked with asterisks are clusters with statistically significant enrichment in cFos-positive cells, as compared to the fraction of cFos-positive cells in all cells (binomial test; false-discovery rate < 5%). Error bars represent standard error of the mean (N = 3 – 5 replicates). We measured fewer slices in behaviorally stimulated animals than in naive animals (4 versus 12 slices per animal) (29), and only clusters in which at least 10 cells are present in two or more behavior replicates are depicted. (B) Expression distributions of selected marker genes and genes of interest for neuronal clusters enriched in cFos-positive cells in the tested social behaviors. Expression distributions are calculated as in Fig. 2. (C) Representative in situ hybridization images of 16-μm-thick sections from the preoptic region showing cFos expression in cells expressing markers of neuronal clusters activated during parenting, in virgin females, mothers, and fathers. Regions in blue dashed boxes are magnified and shown on the right. Red, green, and blue mark the listed genes and white (or yellow for I-2) indicates co-expression in the merged images. Clusters that cannot be distinguished by a combination of two marker genes plus their spatial location (I-27 and I-10) were not tested. (D) Venn diagrams summarizing the clusters that were activated during specific behaviors in different sexes or physiological states.