Effects of general anesthesia on behavioral circadian rhythms and clock-gene expression in the suprachiasmatic nucleus in rats.

The suprachiasmatic nucleus (SCN) of the hypothalamus is a nucleus that regulates circadian rhythms through the cyclic expression of clock genes. It has been suggested that circadian-rhythm-related, adverse postoperative events, including sleep disturbances and delirium, are partly caused by anesthesia-induced disruption of clock-gene expression. We examined the effects of multiple general anesthetics on the expression cycle of Period2 (Per2), one of the clock genes that regulate circadian rhythms in the SCN, and on the behavioral rhythms of animals. Rats were treated with sevoflurane, propofol, and dexmedetomidine for 4 h. The expression of Per2 in SCN was analyzed using in situ hybridization, and the behavioral rhythm before and after anesthesia was analyzed. Per2 expression in the SCN decreased significantly immediately after anesthesia in all groups compared with corresponding control groups. However, Per2 returned to normal levels within 24 h, and there was no phase change in the gene expression cycle or behavioral rhythm. This study suggests that acute suppression of Per2 expression may be a general phenomenon induced by general anesthesia, but that the molecular mechanism of the body clock is resilient to disturbances to some extent.

Expression analysis of neuropeptide FF receptors on neuroendocrine-related neurons in the rat brain using highly sensitive in situ hybridization.

RF-amide peptides, a family of peptides characterized by a common carboxy-terminal Arg-Phe-NH2 motif, play various physiological roles in the brain including the modulation of neuroendocrine signaling. Neuropeptide FF (NPFF) receptors exhibit a high affinity for all RF-amide peptides, which suggests that the neurons expressing these NPFF receptors may have multiple functions in the brain. However, the distribution of the neurons expressing NPFF receptors in the rat brain remains poorly understood. This study aimed to determine the detailed histological distribution of mRNA that encodes the neuropeptide FF receptors (Npffr1 and Npffr2) in the rat brain using in situ hybridization. Neurons with strong Npffr1 expression were observed in the lateral septal nucleus and several hypothalamic areas related to neuroendocrine functions, including the paraventricular nucleus (PVN) and arcuate nucleus, whereas Npffr2-expressing neurons were observed mainly in brain regions involved in somatosensory pathways, such as several subnuclei of the thalamus. Npffr1 expression was observed in 70% of corticotropin-releasing hormone neurons, but in only a small population of oxytocin and vasopressin neurons in the PVN. Npffr1 expression was also observed in the dopaminergic neurons in the periventricular nucleus and the dorsal arcuate nucleus, and in the kisspeptin neurons in the anteroventral periventricular nucleus. These results suggest that NPFFR1-mediated signaling may be involved in neuroendocrine functions, such as in reproduction and stress response. In conjunction with a detailed histological map of NPFFRs, this study provides useful data for future neuroendocrine research.

Region-specific changes in brain kisspeptin receptor expression during estrogen depletion and the estrous cycle.

Kisspeptin acts as a potent neuropeptide regulator of reproduction through modulation of the hypothalamic-pituitary-gonadal axis. Previous studies revealed sex differences in brain expression patterns as well as regulation of expression by estrogen. Alternatively, sex differences and estrogen regulation of the kisspeptin receptor (encoded by Kiss1r) have not been examined at cellular resolution. In the current study, we examined whether Kiss1r mRNA expression also exhibits estrogen sensitivity and sex-dependent differences using in situ hybridization. We compared Kiss1r mRNA expression between ovariectomized (OVX) rats and estradiol (E2)-replenished OVX rats to examine estrogen sensitivity, and compared expression between gonadally intact male rats and female rats in diestrus or proestrus to examine sex differences. In OVX rats, E2 replenishment significantly reduced Kiss1r expression specifically in the hypothalamic arcuate nucleus (ARC). A difference in Kiss1r expression was also observed between diestrus and proestrus rats in the hypothalamic paraventricular nucleus (PVN), but not in the ARC. Thus, estrogen appears to have region- and context-specific effects on Kiss1r expression. However, immunostaining revealed minimal colocalization of estrogen receptor alpha (ERα) in Kiss1r-expressing neuronal populations of ARC and PVN, indicating indirect or ERα-independent regulation of Kiss1r expression. Surprisingly, unlike the kisspeptin ligand, no sexual dimorphisms were observed in either the brain distribution of Kiss1r expression or in the number of Kiss1r-expressing neurons within enriched brain nuclei. The current study reveals marked differences in regulation between kisspeptin and kisspeptin receptor, and provides an essential foundation for further study of kisspeptin signaling and function in reproduction.

Neurochemical Characterization of Neurons Expressing Estrogen Receptor ß in the Hypothalamic Nuclei of Rats Using in Situ Hybridization and Immunofluorescence.

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 ERß. However, the characteristics of ERß-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 ERß-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 ERß-expressing neurons identified in the current study can be very essential to understand the estrogen signaling via ERß.

Applicability of Anti-Human Estrogen Receptor ß Antibody PPZ0506 for the Immunodetection of Rodent Estrogen Receptor ß Proteins.

Several lines of controversial evidence concerning estrogen receptor ß (ERß) remain to be solved because of the unavailability of specific antibodies against ERß. The recent validation analysis identified a monoclonal antibody (PPZ0506) with sufficient specificity against human ERß. However, the specificity and cross-reactivity of PPZ0506 antibody against ERß proteins from laboratory animals have not been confirmed. In the present study, we aimed to validate the applicability of PPZ0506 to rodent studies. The antibody exhibited specific cross-reactivity against mouse and rat ERß proteins in immunoblot and immunocytochemical experiments using transfected cells. In immunohistochemistry for rat tissue sections, PPZ0506 showed immunoreactive signals in the ovary, prostate, and brain. These immunohistochemical profiles of rat ERß proteins in rat tissues accord well with its mRNA expression patterns. Although the antibody was reported to show the moderate signals in human testis, no immunoreactive signals were observed in rat testis. Subsequent RT-PCR analysis revealed that this species difference in ERß expression resulted from different expression profiles related to the alternative promoter usage between humans and rats. In conclusion, we confirmed applicability of PPZ0506 for rodent ERß studies, and our results provide a fundamental basis for further examination of ERß functions.

Recent Advances in High-sensitivity In Situ Hybridization and Costs and Benefits to Consider When Employing These Methods.

In situ hybridization (ISH), which visualizes nucleic acids in tissues and cells, is a powerful tool in histology and pathology. Over 50 years since its invention, multiple attempts have been made to increase the sensitivity and simplicity of these methods. Therefore, several highly sensitive in situ hybridization methods have been developed that offer researchers a wide range of options. When selecting these in situ hybridization variants, their signal-amplification principles and characteristics must be understood. In addition, from a practical point of view, a method with good monetary and time-cost performance must be chosen. This review introduces recent high-sensitivity in situ hybridization variants and presents their principles, characteristics, and costs.

Sex and interspecies differences in ESR2-expressing cell distributions in mouse and rat brains.

BACKGROUND: ESR2, a nuclear estrogen receptor also known as estrogen receptor ß, is expressed in the brain and contributes to the actions of estrogen in various physiological phenomena. However, its expression profiles in the brain have long been debated because of difficulties in detecting ESR2-expressing cells. In the present study, we aimed to determine the distribution of ESR2 in rodent brains, as well as its sex and interspecies differences, using immunohistochemical detection with a well-validated anti-ESR2 antibody (PPZ0506). METHODS: To determine the expression profiles of ESR2 protein in rodent brains, whole brain sections from mice and rats of both sexes were subjected to immunostaining for ESR2. In addition, to evaluate the effects of circulating estrogen on ESR2 expression profiles, ovariectomized female mice and rats were treated with low or high doses of estrogen, and the resulting numbers of ESR2-immunopositive cells were analyzed. Welch's t-test was used for comparisons between two groups for sex differences, and one-way analysis of variance followed by the Tukey-Kramer test were used for comparisons among multiple groups with different estrogen treatments. RESULTS: ESR2-immunopositive cells were observed in several subregions of mouse and rat brains, including the preoptic area, extended amygdala, hypothalamus, mesencephalon, and cerebral cortex. Their distribution profiles exhibited sex and interspecies differences. In addition, low-dose estrogen treatment in ovariectomized female mice and rats tended to increase the numbers of ESR2-immunopositive cells, whereas high-dose estrogen treatment tended to decrease these numbers. CONCLUSIONS: Immunohistochemistry using the well-validated PPZ0506 antibody revealed a more localized expression of ESR2 protein in rodent brains than has previously been reported. Furthermore, there were marked sex and interspecies differences in its distribution. Our histological analyses also revealed estrogen-dependent changes in ESR2 expression levels in female brains. These findings will be helpful for understanding the ESR2-mediated actions of estrogen in the brain.

Although the brain is a major target organ of estrogens, the distribution of estrogen receptors in the brain is not fully understood. ESR2, also known as estrogen receptor ß, is an estrogen receptor subtype; its localization in the brain has long been controversial because it has traditionally been difficult to detect. In the present study, we analyzed the expression sites of ESR2 in mouse and rat brains using immunohistochemistry with a well-validated antibody, PPZ0506. The immunohistochemical analysis revealed a more localized expression of ESR2 protein in brain subregions than has previously been reported. Additionally, there were clear sex and interspecies differences in the distribution of this protein. We also observed changes in ESR2 expression in the female brain in response to circulating estrogen levels. Our results, which show the precise expression profiles of ESR2 protein in rodent brains, will be helpful for understanding the ESR2-mediated actions of estrogen.

Identification of Brain Regions Activated by Sevoflurane and Propofol and Regional Changes in Gene Expression.

General anesthetics have different efficacies and side effect incidences based on their mechanism of action. However, detailed comparative studies of anesthetics are incomplete. In this study, target brain regions and gene expression changes in these brain regions were determined for sevoflurane and propofol to understand the mechanisms that cause differences among anesthetics. Rats were anesthetized with sevoflurane or propofol for 1 hr, and brain regions with anesthesia-induced changes in neuronal activity were examined by immunohistochemistry and in situ hybridization for c-Fos. Among the identified target brain regions, gene expression analysis was performed in the habenula, the solitary nucleus and the medial vestibular nucleus from laser microdissected samples. Genes altered by sevoflurane and propofol were different and included genes involved in the incidence of postoperative nausea and vomiting and emergence agitation, such as Egr1 and Gad2. GO enrichment analysis showed that the altered genes tended to be evenly distributed in all functional category. The detailed profiles of target brain regions and induced gene expression changes of sevoflurane and propofol in this study will provide a basis for analyzing the effects of each anesthetic agent and the risk of adverse events.

Optimized Mouse-on-mouse Immunohistochemical Detection of Mouse ESR2 Proteins with PPZ0506 Monoclonal Antibody.

Despite the physiological significance of ESR2, a lack of well-validated detection systems for ESR2 proteins has hindered progress in ESR2 research. Thus, recent identification of a specific anti-human ESR2 monoclonal antibody (PPZ0506) and its specific cross-reactivity against mouse and rat ESR2 proteins heightened momenta toward development of appropriate immunohistochemical detection systems for rodent ESR2 proteins. Building upon our previous optimization of ESR2 immunohistochemical detection in rats using PPZ0506, in this study, we further aimed to optimize mouse-on-mouse immunohistochemical detection using PPZ0506. Our assessment of several staining conditions using paraffin-embedded ovary sections revealed that intense heat-induced antigen retrieval, appropriate blocking, and appropriate antibody dilutions were necessary for optimization of mouse-on-mouse immunohistochemistry. Subsequently, we applied the optimized immunostaining method to determine expression profiles of mouse ESR2 proteins in peripheral tissues and brain subregions. Our analyses revealed more localized distribution of mouse ESR2 proteins than previously assumed. Moreover, comparison of these results with those obtained in humans and rats using PPZ0506 revealed interspecies differences in ESR2 expression. We expect that our optimized methodology for immunohistochemical staining of mouse ESR2 proteins will help researchers to solve multiple lines of controversial evidence concerning ESR2 expression.

Modulation of synaptic plasticity by brain estrogen in the hippocampus.

The hippocampus is a center for learning and memory as well as a target of Alzheimer's disease in aged humans. Synaptic modulation by estrogen is essential to understand the molecular mechanisms of estrogen replacement therapy. Because the local synthesis of estrogen occurs in the hippocampus of both sexes, in addition to the estrogen supply from the gonads, its functions are attracting much attention. Hippocampal estrogen modulates memory-related synaptic plasticity not only slowly but also rapidly. Slow actions of 17ß-estradiol (17ß-E2) occur via classical nuclear receptors (ERα or ERß), while rapid E2 actions occur via synapse-localized ERα or ERß. Elevation or decrease of the E2 concentration changes rapidly the density and morphology of spines in CA1-CA3 neurons. ERα, but not ERß, drives this enhancement/suppression of spinogenesis. Kinase networks are involved downstream of ERα. The long-term depression but not the long-term potentiation is modulated rapidly by changes of E2 level. Determination of the E2 concentration in the hippocampus is enabled by mass-spectrometry in combination with derivatization methods. The E2 level in the hippocampus is as high as approx. 8 nM for the male and 0.5-2 nM for the female, which is much higher than that in circulation. Therefore, hippocampus-derived E2 plays a major role in modulation of synaptic plasticity. Many hippocampal slice experiments measure the restorative effects of E2 by supplementation of E2 to E2-depleted slices. Accordingly, isolated slice experiments can be used as in vitro models of in vivo estrogen replacement therapy for ovariectomized female animals with depleted circulating estrogen.