Sex steroid action is critical to form sexually dimorphic nuclei, although it is not fully understood. We previously reported that masculinization of the principal nucleus of the bed nucleus of the stria terminalis (BNSTp), which is larger and has more neurons in males than in females, involves aromatized testosterone that acts via estrogen receptor-α (ERα), but not estrogen receptor-β (ERβ). Here, we examined sex steroid action on the formation of the anteroventral periventricular nucleus (AVPV) that is larger and has more neurons in females. Morphometrical analysis of transgenic mice lacking aromatase, ERα, or ERβ genes revealed that the volume and neuron number of the male AVPV were significantly increased by deletion of aromatase and ERα genes, but not the ERβ gene. We further examined the AVPV and BNSTp of androgen receptor knockout (ARKO) mice. The volume and neuron number of the male BNSTp were smaller in ARKO mice than those in wild-type mice, while no significant effect of ARKO was found on the AVPV and female BNSTp. We also examined aromatase, ERα, and AR mRNA levels in the AVPV and BNSTp of wild-type and ARKO mice on embryonic day (ED) 18 and postnatal day (PD) 4. AR mRNA in the BNSTp and AVPV of wild-type mice was not expressed on ED18 and emerged on PD4. In the AVPV, the aromatase mRNA level was higher on ED18, although the ERα mRNA level was higher on PD4 without any effect of AR gene deletion. Aromatase and ERα mRNA levels in the male BNSTp were significantly increased on PD4 by AR gene deletion. These results suggest that estradiol signaling via ERα during the perinatal period and testosterone signaling via AR during the postnatal period are required for masculinization of the BNSTp, whereas the former is sufficient to defeminize the AVPV.

Estrogens play an essential role in multiple physiological functions in the brain, including reproductive neuroendocrine, learning and memory, and anxiety-related behaviors. To determine these estrogen functions, many studies have tried to characterize neurons expressing estrogen receptors known as ERα and ERβ. However, the characteristics of ERβ-expressing neurons in the rat brain still remain poorly understood compared to that of ERα-expressing neurons. The main aim of this study is to determine the neurochemical characteristics of ERβ-expressing neurons in the rat hypothalamus using RNAscope in situ hybridization (ISH) combined with immunofluorescence. Strong Esr2 signals were observed especially in the anteroventral periventricular nucleus (AVPV), bed nucleus of stria terminalis, hypothalamic paraventricular nucleus (PVN), supraoptic nucleus, and medial amygdala, as previously reported. RNAscope ISH with immunofluorescence revealed that more than half of kisspeptin neurons in female AVPV expressed Esr2, whereas few kisspeptin neurons were found to co-express Esr2 in the arcuate nucleus. In the PVN, we observed a high ratio of Esr2 co-expression in arginine-vasopressin neurons and a low ratio in oxytocin and corticotropin-releasing factor neurons. The detailed neurochemical characteristics of ERβ-expressing neurons identified in the current study can be very essential to understand the estrogen signaling via ERβ.

Neurons expressing Calb1 that encodes calbindin D-28K (Calb), a calcium-binding protein, are significant components of the sexually dimorphic nucleus (SDN) of the preoptic area in mice. Calb is therefore used as a marker to study the SDN. The number of Calb neurons in the SDN is greater in males than in females, and this sex difference emerges before puberty. However, the timing of emergence of this difference and the role of Calb1 in the SDN remain unclear. In this study, we investigated when the sex difference in Calb neurons appears and whether Calb1 is required for organizing the SDN. Profiling the temporal changes in Calb neurons revealed that these neurons dramatically increased in the SDN during postnatal days 15 to 20 in males but not in females, resulting in a significant difference between sexes. However, the Calb1 mRNA in the SDN remained unchanged during the late postnatal period and did not differ between sexes. Calb1 knockdown (KD) was performed by injecting an adeno-associated virus vector into the preoptic area of neonatal mice. The analysis of postnatal Calb1-KD mice revealed that Calb1 KD reduced not only Calb1 expression but also the number of neurons comprising the SDN. These findings suggest that compared with the female SDN, more Calb neurons migrate to and are incorporated into the male SDN, thereby yielding the sex difference in the number of Calb neurons at the late postnatal period, and that Calb1 plays a significant role in clustering neurons in the SDN.

Stress contributes significantly to visceral pain hypersensitivity in irritable bowel syndrome (IBS). Although stress during critical developmental periods, such as chronic social defeat stress (CSDS) in adolescence or early-life adversities, like maternal separation, has been extensively researched and is known to increase visceral pain sensitivity, little is known about the effects of CSDS on visceral pain processing in full adulthood. This gap is important because IBS onset and exacerbation of symptoms frequently occur in adults experiencing chronic psychosocial stress. Using a CSDS protocol in adult mice, we examined changes in visceral pain thresholds, anxiety-like behavior, and neuroimmune plasticity, particularly focusing on microglia linked to pain and emotions. CSDS-exposed mice exhibited significantly reduced visceral pain thresholds and heightened visceral sensitivity, accompanied by adrenal gland hypertrophy, thymic atrophy, and elevated anxiety-like behavior. Morphological analysis revealed pronounced microglial process shrinkage and decreased branching within the anterior cingulate cortex (ACC) after CSDS, without considerable changes in traditional inflammation-associated mRNA levels. No microglial remodeling was detected in other regions. Microglial depletion with PLX3397 partially reduced visceral hypersensitivity, producing an intermediate phenotype, but did not improve anxiety-like behavior. Our findings suggest that microglia play a modulatory, but not exclusive role in stress-induced visceral pain. Regional differences indicate the ACC acts as a hub for chronic pain plasticity, governed by complex microglial and circuit-level mechanisms.

Sex differences are well-documented across many functions of the orexin system; however, whether such differences extend to pain processing has been examined far less thoroughly. Therefore, we investigated whether males and females differ in orexin system engagement during nociception, and whether any differences reflect structural organization or functional recruitment. Adult C57BL/6N mice of both sexes were used, with females tested during proestrus and metestrus. Formalin was injected into the hind paw, and c-Fos induction in lateral hypothalamic orexin neurons was quantified together with orexin A/B fiber density and orexin receptor OX1R/OX2R mRNA expression in the lumbar cord. Orexin A and B were fully colocalized, allowing orexin B to serve as a reliable marker. The total number of orexin-immunoreactive neurons did not differ between the sexes or across estrous stages, and spinal orexinergic innervation and OX1R/OX2R mRNA levels were comparable across groups. In contrast, formalin induced a significantly higher proportion of c-Fos-positive orexin neurons in males, indicating male-biased recruitment of orexin neurons during nociception, despite equivalent anatomical substrates. These findings suggest that sex differences in orexin involvement in nociception are not attributable to structural disparities in the number of orexin neurons, spinal projections, or receptor expression, but instead arise from sex- and context-dependent differences in the functional activation of orexin neurons in response to painful stimuli.