siRNA silencing of estrogen receptor-α expression specifically in medial preoptic area neurons abolishes maternal care in female mice

Abstract

The medial preoptic area has been shown to be intricately involved in many behaviors, including locomotion, sexual behavior, maternal care, and aggression. The gene encoding estrogen receptor-α (ERα) protein is expressed in preoptic area neurons, and a very dense immunoreactive field of ERα is found in the preoptic region. ERα knockout animals show deficits in maternal care and sexual behavior and fail to exhibit increases in these behaviors in response to systemic estradiol treatment. In the present study, we used viral-vector mediated RNA interference to silence ERα expression specifically in the preoptic area of female mice and measured a variety of behaviors, including social and sexual aggression, maternal care, and arousal activity. Suppression of ERα in the preoptic area almost completely abolished maternal care, significantly increasing the latency to pup retrieval and significantly reducing the time the moms spent nursing and licking the pups. Strikingly, maternal aggression toward a male intruder was not different between control and preoptic ERα-silenced mice, demonstrating the remarkably specific role of ERα in these neurons. Reduction of ERα expression in preoptic neurons significantly decreased sexual behavior in female mice and increased aggression toward both sexual partners and male intruders in a seminatural environment. Estrogen-dependent increases in arousal, measured by home cage activity, were not mediated by ERα expression in the preoptic neurons we targeted, as ERα-suppressed mice had increases similar to control mice. Thus, we have established that a specific gene in a specific group of neurons is required for a crucially important natural behavior.

Fig. 1.

Immunohistochemical evaluation of estrogen receptor-α (ERα) silencing in the medial preoptic region (MPA). Animals received bilateral MPA microinjections of either AAV shRNA LUC or AAV shRNA ERα. Only animals with greater than 80% reduction in ERα expression in the MPA were included in shRNA ERα group. (Left) AAV shRNA LUC MPA-injected mouse. (Right) AAV shRNA ERα MPA-injected mouse. DAB stained brain tissue: brown coloration (DAB), EGFP; Black nuclear staining (DAB with nickel), ERα. (Scale bar, 200 μm.)

Fig. 2.

MPA suppression of ERα significantly reduced maternal behaviors but not maternal aggression. (A) Pup care. (B) Latency to pup retrieval. (C) Aggressive behaviors toward male intruder. Values represent mean ± SEM (SEM). *P < 0.05, **P < 0.01.

Fig. 3.

Suppression of ERα in the MPA significantly decreased rejective and receptive behaviors in female mice, specifically by decreasing the number of kicking and boxing episodes. (A) All sexual behaviors: rejective, proceptive and receptive. (B) Different components of rejective behavior: kicking, rearing, boxing, and fleeing. Values represent mean ± SEM. *P < 0.05, **P < 0.01.

Fig. 4.

Suppression of ERα in the MPA diminished social investigation and aggressive behaviors in a SNE and decreased aggression under food competition conditions. (A) Social interactions among females in the SNE and toward male intruders. (B) Social interactions in the SNE under restricted food availability. Values represent mean ± SEM. *P < 0.05, **P < 0.01.

Fig. 5.

Suppression of ERα expression in the MPA did not prevent estradiol-mediated increases in home-cage activity. (A) Horizontal activity. (B) Total distance. (C) Vertical activity. All values summed across 12-h dark (black bar) and light periods (white bar). Values represent mean ± SEM. *P < 0.05, ***P < 0.001.