The Role of Estrogen Receptor β in the Dorsal Raphe Nucleus on the Expression of Female Sexual Behavior in C57BL/6J Mice

17β-Estradiol (E2) regulates the expression of female sexual behavior by acting through estrogen receptor (ER) α and β. Previously, we have shown that ERβ knockout female mice maintain high level of lordosis expression on the day after behavioral estrus when wild-type mice show a clear decline of the behavior, suggesting ERβ may be involved in inhibitory regulation of lordosis. However, it is not identified yet in which brain region(s) ERβ may mediate an inhibitory action of E2. In this study, we have focused on the dorsal raphe nucleus (DRN) that expresses ERβ in higher density than ERα. We site specifically knocked down ERβ in the DRN in ovariectomized mice with virally mediated RNA interference method. All mice were tested weekly for a total of 3 weeks for their lordosis expression against a stud male in two consecutive days: day 1 with the hormonal condition mimicking the day of behavioral estrus, and day 2 under the hormonal condition mimicking the day after behavioral estrus. We found that the level of lordosis expression in ERβ knockdown (βERKD) mice was not different from that of control mice on day 1. However, βERKD mice continuously showed elevated levels of lordosis behavior on day 2 tests, whereas control mice showed a clear decline of the behavior on day 2. These results suggest that the expression of ERβ in the DRN may be involved in the inhibitory regulation of sexual behavior on the day after behavioral estrus in cycling female mice

Long-Lasting Consequences of Neonatal Maternal Separation on Social Behaviors in Ovariectomized Female Mice

Maternal separation (MS) stress is known to induce long-lasting alterations in emotional and anxiety-related behaviors, but effects on social behaviors are not well defined. The present study examined MS effects on female social behaviors in the social investigation (SIT) and social preference (SPT) tests, in addition to non-social behaviors in the open-field (OFT) and light-dark transition (LDT) tests in C57BL/6J mice. All females were tested as ovariectomized to eliminate confounding effects of endogenous estrogen during behavioral testing. Daily MS (3 hr) from postnatal day 1 to 14 did not affect anxiety levels in LDT, but were elevated in OFT with modified behavioral responses to the novel environment. Furthermore, MS altered social investigative behaviors and preference patterns toward unfamiliar stimulus mice in SIT and short- and long-term SPT paradigms. In SIT, MS reduced social investigation duration and increased number of stretched approaches towards both female and male unfamiliar stimulus mice, suggesting increased social anxiety levels in MS females. Similarly, MS heightened levels of social anxiety during short-term SPT but no MS effect on social preference was found. On the other hand, MS females displayed a distinctive preference for female stimuli, unlike control females, when tested for long-term SPT over a prolonged period of 5 days. Evaluation of FosB expression in the paraventricular nucleus, medial and central amygdala following stimulus exposure demonstrated greater number of FosB immunopositive cells in all three brain regions in MS females compared to control females. These results suggest that MS females might differ in neuroendocrine responses toward unfamiliar female and male opponents, which may be associated with modifications in social behaviors found in the present study. Taken together, this study provides new evidence that early life stress modifies female social behaviors by highlighting alterations in behavioral responses to situations involving social as well as non-social novelty

Effects of MS on FosB immunoreactivity one hour following stimulus exposure in the Paraventricular Nucleus (PVN), Central Amygdala (CeA), and Medial Amygdala (MeA).

<p>Number of FosB immunopositive cells in the (A) PVN, (D) CeA, and (G) MeA of control and MS females not exposed to a stimulus mouse (basal), exposed to an unfamiliar female stimulus mouse, or exposed to an unfamiliar male stimulus mouse for 15 min. Representative images for FosB immunoreactivity in the PVN for (B) control and (C) MS female mice exposed to a male stimulus mouse, the CeA for (E) control and (F) MS females exposed to male stimuli, and the MeA for (H) control and (I) MS female mice exposed to female stimulus mice. All data are presented as mean + SEM. Scale bar, 100 µm. 3V, third ventricle; BLA, basolateral amygdala; OT, optic tract. *, <i>p</i><0.05; **, <i>p</i><0.01.</p

MS effects on anxiety-related behaviors in OFT and LDT.

<p>(A) The cumulative time spent in the center area, (B) total moving distance in the whole open field arena, and (C) number of rears within the whole open field arena, center area of the arena, and peripheral area of the arena during OFT were measured in control and MS female mice. No effect of MS was observed in the behavioral measurements of (D) time spent in the light compartment, (E) latency to enter the light compartment, and (F) number of transitions between compartments in female mice during LDT. All data are presented as mean + SEM. *, <i>p</i><0.05; **, <i>p</i><0.01 vs. control. C: control; MS: maternal separation.</p

MS-induced alterations in social preference during either ‘female vs. male,’ ‘female vs. empty,’ or ‘male vs. empty’ stimuli sets during long-term SPT.

<p>Total time spent in each tunnel connected to stimuli cages, indicated as length of horizontal bars, was measured in each stimuli paradigm during the dark phase in (A) control and (B) MS female mice. Female and male symbols on the left and right columns represent stimulus animals. Empty indicates the absence of a stimulus animal in the stimuli cage. Daily preference percentages during the 5 testing days within each stimuli paradigm in control and MS females are shown in C–E. (C) Percent of female stimuli preference in ‘female vs. male’ stimuli set, (D) percent of female stimuli preference in ‘female vs. empty cage’ stimuli set, and (E) percent of male stimuli preference in ‘male vs. empty cage’ stimuli set on each testing day. All data are presented as mean + SEM. MS: maternal separation.</p

MS effects on social preference for female and male stimuli mice during short-term SPT.

<p>MS females displayed reduced levels of total social investigation time towards both stimulus-containing cylinders in female mice. In addition, control females did not show preferences for either stimulus, whereas MS females tended to spend less time towards the male stimulus mouse. All data are presented as mean + SEM. *, <i>p</i><0.05. MS: maternal separation.</p

MS-induced reduction in social investigative behaviors toward unfamiliar female and male stimulus mice during SIT.

<p>(A) Cumulative social investigation duration, (B) total sniffing from corner duration, and (C) number of stretched approaches toward either a female or male stimulus-containing cylinder. All data are presented as mean + SEM. *, <i>p</i><0.05; **, <i>p</i><0.01.</p