Effects of Prepubertal or Adult Site-Specific Knockdown of Estrogen Receptor β in the Medial Preoptic Area and Medial Amygdala on Social Behaviors in Male Mice

Abstract

Testosterone, after being converted to estradiol in the brain, acts on estrogen receptors (ERα and ERβ) and controls the expression of male-type social behavior. Previous studies in male mice have revealed that ERα expressed in the medial preoptic area (MPOA) and medial amygdala (MeA) are differently involved in the regulation of sexual and aggressive behaviors by testosterone action at the time of testing in adult and/or on brain masculinization process during pubertal period. However, a role played by ERβ in these brain regions still remains unclear. Here we examined the effects of site-specific knockdown of ERβ (βERKD) in the MPOA and MeA on male social behaviors with the use of adeno-associated viral mediated RNA interference methods in ICR/Jcl mice. Prepubertal βERKD in the MPOA revealed that continuous suppression of ERβ gene expression throughout the pubertal period and adulthood decreased aggressive but not sexual behavior tested as adults. Because βERKD in the MPOA only in adulthood did not affect either sexual or aggressive behaviors, it was concluded that pubertal ERβ in the MPOA might have an essential role for the full expression of aggressive behavior in adulthood. On the other hand, although neither prepubertal nor adult βERKD in the MeA had any effects on sexual and aggressive behavior, βERKD in adulthood disrupted sexual preference of receptive females over nonreceptive females. Collectively, these results suggest that ERβ in the MPOA and MeA are involved in the regulation of male sexual and aggressive behavior in a manner substantially different from that of ERα.

Keywords: aggressive behavior; estrogen receptor β; medial amygdale; medial preoptic area; sexual preference; site-specific knockdown.

Figure 1.

Experimental procedures of Experiment 1 (top), Experiment 2 (middle), and Experiment 3 (bottom). Ticks under the horizontal bar indicate 1 week. SEX, Sexual behavior; AGG, aggressive behavior.

Figure 2.

Effects of prepubertal silencing of ERβ in the MPOA on the expression of male sexual and aggressive behaviors in adulthood. A, There was no difference between the MPOA-Cont and MPOA-βERKD groups in the number of mounts (left), intromissions (middle), or latency to the first mount (right). B, The duration (left) and number (right) of aggressive bouts was significantly reduced in the MPOA-βERKD group compared with the MPOA-Cont group (*p< 0.05). All behavioral data in A and B are presented as mean + SEM. C, Histological diagrams depicting the placement of the injection needle tip for each mouse in the MPOA-Cont (open circles) and MPOA-βERKD (solid circles) groups (top), and representative photomicrographs of MPOA sections with single-immunohistochemical staining for GFP (bottom; at bregma −0.10). Scale bar, 100 µm. 3V, third ventricle. D, Representative photomicrographs of MPOA sections with single-immunohistochemical staining for ERβ (top; at bregma −0.22), and MPOA sections with double-immunostaining for GFP and ERβ (bottom). Number of ERβ-immunoreactive cells in the targeted site was reduced in the βERKD group compared with the control group. Scale bars: top, 100 µm; bottom, 20 µm. Bottom, Black arrowheads indicate ERβ and GFP double-immunoreactive cells and white arrowheads indicate immunoreactive cells only for GFP.

Figure 3.

Effects of prepubertal silencing of ERβ in the MeA on the expression of male sexual and aggressive behaviors in adulthood. A, There were no differences between the MeA-Cont and MeA-βERKD groups in the number of mounts (left), intromissions (middle), or latency to the first mount (right). B, There were no differences between the MeA-Cont and MeA-βERKD groups in the duration (left) or number (right) of aggressive bouts. All behavioral data in A and B are presented as mean + SEM. C, Histological diagrams depicting the placement of the injection needle tip for each mouse in the MeA-Cont (open circles) and MeA-βERKD (solid circles) groups (top), and representative photomicrographs of MeA sections with single-immunohistochemical staining for GFP (bottom ; at bregma −1.82). Scale bar, 200 µm. opt, optic tract. D, Representative photomicrographs of MeA sections with single-immunohistochemical staining for ERβ (top; at bregma −1.94), and MeA sections with double-immunostaining for GFP and ERβ (bottom). Number of ERβ-immunoreactive cells in the targeted site was greatly reduced in the βERKD group compared with the control group. Scale bars: top, 200 µm; bottom, 20 µm. Bottom, Black arrowheads indicate ERβ and GFP double-immunoreactive cells and white arrowheads indicate immunoreactive cells only for GFP.

Figure 4.

Effects of βERKD in the MPOA in adulthood on male sexual and aggressive behaviors. A, There was no difference between the MPOA-Cont and MPOA-βERKD groups in the number of mounts (left), intromissions (middle), or latency to the first mount (right). B, There was no difference between the MPOA-Cont and MPOA-βERKD groups in the duration (left) or number (right) of aggressive bouts. All behavioral data are presented as mean + SEM.

Figure 5.

Effects of βERKD in the MeA in adulthood on male sexual preference and sexual and aggressive behaviors. A, In PTFF tests, mice in the MeA-Cont group showed longer SI duration toward a receptive female (vs toward a nonreceptive female mouse) but mice in the MeA-βERKD group failed to do so (left). Both of the MeA-Cont and MeA-βERKD groups showed longer SI duration toward a receptive female in PTFM tests (vs toward a male mouse; right; *p< 0.05, **p< 0.01). B, There were no differences between the MeA-Cont and MeA-βERKD groups in the number of mounts (left), intromissions (middle), or latency to the first mount (right). C, There were no differences between the MeA-Cont and MeA-βERKD groups in the duration (left) or number (right) of aggressive bouts. All behavioral data are presented as mean + SEM.