(A) Quantification of Fos+ cells per mm2 in brain areas of interest (oriented from top to bottom, rostral to caudal) in virgin males (attacking), mated males (parental), and mated females (parental) after pup exposure. One-way ANOVA followed by Tukey’s multiple comparison test reveals significant differences in the number of Fos+ cells identified between parental and infanticidal animals in medial bed nucleus of stria terminalis (BNSTm) (F2,8 = 12.47, p<0.0035), ventral lateral septum (LSv) (F2,8 = 14.31, p<0.0023), medial preoptic area (MPOA) (F2,8 = 8.536, p<0.0104), and PeFA (F2,8 = 12.02, p<0.0039) (virgin male n = 5; fathers n = 3; mothers n = 3). (B) Representative in situ hybridization images of Fos (red channel) expression in PeFA and MPOA after animal exposure to pups with counterstain DAPI (blue channel) (scale bar: 100 µm). Dotted lines in right and left denote the boundary of brain regions of interest (PeFA and MPOA, respectively), solid lines denote the fornix, arrowheads indicate location of third ventricle in the left panels, and solid lines denote the edge of the brain and third ventricle in the right panel. (C) Laser-capture microscopy strategy to identify markers of cells activated during infanticide (scale bar 100 µm). Tissue material corresponding to areas labeled by Fos (C) after pup-mediated attack, as well as oxytocin+ (Oxt) (O) and adjacent negative (N) area was laser-dissected and the corresponding transcripts characterized by microarray analysis. (D) Volcano plot showing the fold change (log base 2) against the p-value (log base 10) for all genes. Transcripts with greater than a twofold change in expression in the Fos+ compared to negative region and a p-value less than 0.001 are shown in red: vasopressin (Avp), thyrotropin-releasing hormone (Trh), galanin (Gal), Oxt, and Ucn3 (n = 3 biological replicates, each containing pooled tissue from six males). (E) Representative in situ hybridization showing colocalization of Ucn3 (red) and Fos (green) expression after infanticide (scale bar 100 µm), arrowheads indicate colocalization. (F) Plot depicting number (#) of neurons expressing Fos (green triangles), Ucn3 (red circles) and percent of cells expressing both Fos and Ucn3 (yellow bars) per brain section across Bregma coordinates (n = 6) indicate high colocalization in rostral PeFA. Overall representation of Ucn3+ neurons in the PeFA (2.74%) is indicated by the red line. (G) Representative in situ hybridization showing expression of Ucn3 (red) and Fos (green) after infanticide and other behaviors in the rostral PeFA, with colocalization (white arrowheads) only observed after infanticide (scale bar 100 µm). (H) Percentage of Ucn3+ cells colocalized with Fos after various behaviors (yellow bars; *significance) and number of Fos cells induced by various behaviors across sections of the rostral PeFA (green triangles; significance #) (n = 3–6/group). Kruskal–Wallis test followed by Dunn’s multiple comparisons test reveals significant differences in number of Fos+ cells in rostral PeFA between fresh bedding control exposure and pup attack in virgin males and virgin females, intermale aggression, and acute stress (#p<0.0001); yet, despite the observed widespread activation of PeFA cells during various behaviors, only pup-directed attacks by virgin males induced significant Fos expression in PeFAUcn3 neurons (*p<0.0002).