Moxd1 Is a Marker for Sexual Dimorphism in the Medial Preoptic Area, Bed Nucleus of the Stria Terminalis and Medial Amygdala

Abstract

The brain shows various sex differences in its structures. Various mammalian species exhibit sex differences in the sexually dimorphic nucleus of the preoptic area (SDN-POA) and parts of the extended amygdala such as the principal nucleus of the bed nucleus of the stria terminalis (BNSTpr) and posterodorsal part of the medial amygdala (MePD). The SDN-POA and BNSTpr are male-biased sexually dimorphic nuclei, and characterized by the expression of calbindin D-28K (calbindin 1). However, calbindin-immunoreactive cells are not restricted to the SDN-POA, but widely distributed outside of the SDN-POA. To find genes that are more specific to sexually dimorphic nuclei, we selected candidate genes by searching the Allen brain atlas and examined the detailed expressions of the candidate genes using in situ hybridization. We found that the strong expression of monooxygenase DBH-like 1 (Moxd1) was restricted to the SDN-POA, BNSTpr and MePD. The numbers of Moxd1-positive cells in the SDN-POA, BNSTpr and MePD in male mice were larger than those in female mice. Most of the Moxd1-positive cells in the SDN-POA and BNSTpr expressed calbindin. Neonatal castration of male mice reduced the number of Moxd1-positive cells in the SDN-POA, whereas gonadectomy in adulthood did not change the expression of the Moxd1 gene in the SDN-POA in both sexes. These results suggest that the Moxd1 gene is a suitable marker for sexual dimorphic nuclei in the POA, BNST and amygdala, which enables us to manipulate sexually dimorphic neurons to examine their roles in sex-biased physiology and behaviors.

Keywords: BNST; MPOA; Moxd1; SDN; amygdala; calbindin; masculinization; molecular marker.

Figure 1

Representative fluorescent images of in situ hybridization for Moxd1 mRNA in the MPOA and adjacent areas. (A–D) Coronal sections of male mice along the anterior-posterior axis. (E–H) Coronal sections of female mice along the anterior-posterior axis. BNSTpr, principal nucleus of the bed nucleus of the stria terminalis; MPOA, medial preoptic area; SDN, sexually dimorphic nucleus of the preoptic area; 3v, third ventricle. Scale bars: 200 μm.

Figure 2

Sex and gonadal steroid dependent differences in Moxd1-positive cells in the SDN-POA. Representative images of intact male (A), castrated (CAS) male (B), neonatally castrated male (C), intact female (D) and ovariectomized (OVX) female (E) mice. Dashed lines indicate the boundaries of the SDN-POA. Scale bars: 50 μm. (F) Difference in the number of Moxd1-positive cells. Significant differences were found between experimental groups indicated by a and b. The number in parenthesis of each bar indicates the number of mice. (P < 0.05, Welch’s t-test with Holm’s multiple correlation).

Figure 3

Comparison of the distribution of Moxd1-positive cells with calbindin-immunoreactive cells in the MPOA of male mice. (A–C) Representative images of Moxd1 (magenta) and calbindin (green) at low magnification. Scale bar: 100 μm. (D–F) High magnification images indicated by rectangles in (A–C). Scale bar: 50 μm. The inset shows a high magnification view of a double-labeled cell indicated by small rectangle. Scale bar: 5 μm. (G) Orthogonal view of Moxd1- and calbindin-positive cells. Sacle bar: 10 μm. ACN, anterior commissural nucleus; MPNm, medial preoptic nucleus, medial part; vMPOA, ventral part of the MPOA. (H–K) Representative images of calbindin (magenta), oxytocin-neurophysin I (blue) and Trh (green). Arrowhead indicates colocalization of calbindin and oxytocin (blue), or calbindin and Trh (magenta). (L–O) Representative images of Moxd1 (magenta), oxytocin-neurophysin I (blue) and Trh (green). Scale bars: 20 μm.

Figure 4

Sexually dimorphic expression of Moxd1 mRNA in the BNSTpr and MePD. Representative images in the BNSTpr of intact male (A), neonatally castrated male (B) and female (C) mice. (D) Difference in the Moxd1 mRNA-positive area in the BNSTpr. Representative images of the MePD of male (E), neonatally castrated male (F) and female (G) mice. (H) Difference in the Moxd1 mRNA-positive area in the MePD. *Significant difference from intact male mice (P < 0.05, n = 4 for each, Welch’s t-test with Holm’s correction). BNSTpr, principal nucleus of the bed nucleus of the stria terminalis; MePD, posterodorsal part of the medial amygdala; LV, lateral ventricle; fx, fornix; st, stria terminalis; opt, optic tract. Scale bars: 200 μm.

Figure 5

Comparison of the distribution of Moxd1-positive cells with calbindin-immunoreactive cells in the BNSTpr and MePD of male mice. (A–C) Representative images of Moxd1 (magenta) and calbindin (green) in the BNSTpr (dashed line). (D–F) Representative images of Moxd1 (magenta) and calbindin (green) in the MePD (dashed line). Scale bars: 200 μm. The inset shows a high magnification view indicated by small rectangle. Arrowhead indicates the overlap of the Moxd1-positive cells and the calbindin-immunoreactive cells. Scale bars: 10 μm.

Figure 6

Overlap of the Moxd1-positive cells and calbindin-immunoreactive cells in the amygdala nuclei and adjacent areas. (A) Moxd1, (B) calbindin, (C) merged image. Note that the MePD was located in the more posterior section. BLA, basolateral amygdala; BMA, basomedial amygdala; CEA, central amygdala; COA, cortical amygdala; CP, caudoputamen; CTX, cerebral cortex; EPI, endopiriform nucleus; LA, lateral amygdala; MEA, medial amygdala; PIR, piriform cortex; opt, optic tract. Scale bars: 0.5 mm. (D–I) High magnification images of double-labeled cells indicated by small rectangles and arrowheads in (C). (D) LA, (E) BLA, (F) BMA, (G) MEA, (H) COA, (I) PIR. Scale bars: 10 μm.

Figure 7

Comparison of the distribution of Moxd1-positive cells with calbindin-immunoreactive cells in the dorsal tenia tecta (DTT), cerebral cortex (CTX) and cerebellum. (A–C) Representative images of Moxd1 (magenta) and calbindin (green) in the dorsal tenia tecta. (D–F) Representative images of Moxd1 (magenta) and calbindin (green) in the CTX. (G–I) Representative images of Moxd1 (magenta) and calbindin (green) in the cerebellum. Arrowhead indicates the overlap of the Moxd1-positive cells and the calbindin-immunoreactive cells. Arrow indicates exclusive Moxd1 expression in the subplate layer of the CTX. Scale bars: 200 μm. Each inset shows a high magnification view of a double-labeled cell indicated by small rectangle. Scale bar: 10 μm. DTT, dorsal tenia tecta; CC, corpus callosum.