Abstract
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.
Highlights
Estrogens exert actions through nuclear estrogen receptor α (ERα) and β (ERβ)
We aim to provide accurate and detailed data regarding the neurochemical characteristics of neurons expressing Esr2 mRNA in the hypothalamus of female and male rats using RNAscope in situ hybridization (ISH) for Esr2 mRNA combined with immunofluorescence against kisspeptin, arginine vasopressin (AVP), OXT, and corticotropin-releasing factor (CRF)
We identified Esr2 signals in the organum vasculosum laminae terminalis (OVLT), the ventrolateral hypothalamic tract, and in the ventrolateral parts of ventromedial hypothalamus (VMHvl) (Supplemental Figure S1A,B), even though the signals in these regions were not significantly strong
Summary
Estrogens exert actions through nuclear estrogen receptor α (ERα) and β (ERβ). In adulthood, ERα has been predominantly linked to reproductive neuroendocrine and sexual behaviors [1,2], whereas ERβ has been associated with many non-reproductive neurophysiological functions such as learning and memory [3,4], anxiety-related behaviors [5,6], and the stress response of the hypothalamic-pituitary-adrenal (HPA) axis [7]. To fully understand the involvement of estrogen receptors (ERs) in these functions, it is important to identify the neuroanatomical and neurochemical organization of neurons expressing ERs. To fully understand the involvement of estrogen receptors (ERs) in these functions, it is important to identify the neuroanatomical and neurochemical organization of neurons expressing ERs The distributions of both subtypes in the brain and their neurochemical phenotypes using in situ hybridization (ISH), immunohistochemistry, and PCR have been investigated in current literature [8,9,10] and many reports have successfully determined various characteristics of ERα-expressing neuronal populations [11,12]. Unlike ERα, ERβ’s respective protein distribution has been fairly controversial in some immunohistochemistry-based studies because of the absence of specific antibodies [13]. The reduction of immunoreactivity following ISH is problematic in further neurochemical characterization of ISH-based studies
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