Fig. 4: Hemoglobin enhances c-Fos expressing cells in the dorsal region of the VMH and PAG only in lactating mothers.

a Representative ISH images of the posterior region of the MeA from C57BL/6 male, virgin female, and lactating mothers pre-stimulated with control- or hemoglobin (Hb)-cotton swabs. c-Fos cRNA probe (red) was used in conjunction with nuclear DAPI staining (blue). Abbreviations: MeApd, MeA posterodorsal region; MeApv, MeA posteroventral region; D, dorsal; and V, ventral. n = 3 for male-control and Hb, virgin female-Hb, n = 4 for virgin female-control, n = 6 for mother-control, and n = 7 for mother-Hb. Scale bar, 100 µm. b The number of c-Fos-positive cells in each sub-region of the MeA stimulated with control buffer or Hb. Abbreviations: MeAa, MeA anterior region. For counting MeAa; n = 3 for male-control and Hb, virgin female-Hb, n = 4 for virgin female-control, n = 6 for mother-control, and n = 7 for mother-Hb. 6 (MeAa) and 8 (MeAp) sections from each animal were quantified. Error bars, S.E.M. (MeApd) p = 0.004, and (MeApv) virgin female; p = 0.057, and mother; p = 0.001 by the two-sided Wilcoxon rank-sum test. c The number of c-Fos-positive cells in the BNST and PMCo stimulated with control buffer or Hb. n = 3 for virgin female-Hb, n = 4 for male-control and Hb, virgin female-control, n = 6 for mother-control, and n = 7 for mother-Hb. 7 (BNST) and 5 (PMCo) sections from each animal were quantified. Error bars, S.E.M. d Representative ISH images of the VMH from males, virgin females, and lactating mothers, stimulated with control buffer or hemoglobin. c-Fos cRNA probe (red) was used in conjunction with nuclear DAPI staining (blue). Abbreviations: d, dorsal region; vl, ventrolateral region. n = 3 for virgin female-Hb, n = 4 for male-control and Hb, virgin female-control, n = 6 for mother-control, and n = 7 for mother-Hb. Scale bar, 100 µm. e Quantification of c-Fos-positive neurons in the VMH. The number of sections counted to determine the number of c-Fos-positive neurons in each brain area of each animal was 10. Error bars, S.E.M. n = 3 for virgin female-Hb, n = 4 for male-control and Hb, virgin female-control, n = 6 for mother-control, and n = 7 for mother-Hb. p = 0.034 by the two-sided Wilcoxon rank-sum test. f Number of c-Fos-positive cells in the MPA stimulated with control buffer or Hb. n = 3 for virgin female-Hb, n = 4 for male-control and Hb, virgin female-control, n = 6 for mother-control, and n = 7 for mother-Hb. 4 sections from each animal were quantified. Error bars, S.E.M. g Representative ISH images of the dorsal region of the PAG from lactating mothers stimulated with control buffer or hemoglobin. c-Fos cRNA probe (red) was used in conjunction with nuclear DAPI staining (blue). n = 5 for mother-control and Hb. Scale bar, 100 µm. h Quantification of c-Fos-expressing neurons in the PAGd. n = 3 for virgin female-Hb, n = 4 for male-control and Hb, virgin female-control, n = 5 for mother-control and Hb. 4 sections from each animal were quantified. Error bars, S.E.M. p = 0.007 by the two-sided Wilcoxon rank-sum test.