Naloxone increases conditioned fear responses during social buffering in male rats.

Social buffering is the phenomenon in which the presence of an affiliative conspecific mitigates stress responses. We previously demonstrated that social buffering completely ameliorates conditioned fear responses in rats. However, the neuromodulators involved in social buffering are poorly understood. Given that opioids, dopamine, oxytocin and vasopressin play an important role in affiliative behaviour, here, we assessed the effects of the most well-known antagonists, naloxone (opioid receptor antagonist), haloperidol (dopamine D2 receptor antagonist), atosiban (oxytocin receptor antagonist) and SR49059 (vasopressin V1a receptor antagonist), on social buffering. In Experiment 1, fear-conditioned male subjects were intraperitoneally administered one of the four antagonists 25 min prior to exposure to a conditioned stimulus with an unfamiliar non-conditioned rat. Naloxone, but not the other three antagonists, increased freezing and decreased walking and investigation as compared with saline administration. In Experiment 2, identical naloxone administration did not affect locomotor activity, anxiety-like behaviour or freezing in an open-field test. In Experiment 3, after confirming that the same naloxone administration again increased conditioned fear responses, as done in Experiment 1, we measured Fos expression in 16 brain regions. Compared with saline, naloxone increased Fos expression in the paraventricular nucleus of the hypothalamus and decreased Fos expression in the nucleus accumbens shell, anterior cingulate cortex and insular cortex and tended to decrease Fos expression in the nucleus accumbens core. Based on these results, we suggest that naloxone blocks social buffering of conditioned fear responses in male rats.

Kiss1-dependent and independent release of luteinizing hormone and testosterone in perinatal male rats.

Prenatal and postnatal biphasic increases in plasma testosterone levels derived from perinatal testes are considered critical for defeminizing/masculinizing the brain mechanism that regulates sexual behavior in male rats. Hypothalamic kisspeptin neurons are indispensable for stimulating GnRH and downstream gonadotropin, as well as the consequent testicular testosterone production/release in adult male rats. However, it is unclear whether kisspeptin is responsible for the increase in plasma testosterone levels in perinatal male rats. The present study aimed to investigate the role of Kiss1/kisspeptin in generating perinatal plasma LH and the consequent testosterone increase in male rats by comparing the plasma testosterone and LH profiles of wild-type (Kiss1+/+) and Kiss1 knockout (Kiss1-/-) male rats. A biphasic pattern of plasma testosterone levels, with peaks in the prenatal and postnatal periods, was found in both Kiss1+/+ and Kiss1-/- male rats. Postnatal plasma testosterone and LH levels were significantly lower in Kiss1-/- male rats than in Kiss1+/+ male rats, whereas the levels in the prenatal embryonic period were comparable between the genotypes. Exogenous kisspeptin challenge significantly increased plasma testosterone and LH levels and the number of c-Fos-immunoreactive GnRH neurons in neonatal Kiss1-/- and Kiss1+/+ male rats. Kiss1 and Gpr54 (kisspeptin receptor gene) were found in the testes of neonatal rats, but kisspeptin treatment failed to stimulate testosterone release in the cultured testes of both genotypes. These findings suggest that postnatal, but not prenatal, testosterone increase in male rats is mainly induced by central kisspeptin-dependent stimulation of GnRH and consequent LH release.

Reduction of arcuate kappa-opioid receptor-expressing cells increased luteinizing hormone pulse frequency in female rats.

The brain mechanism responsible for the pulsatile secretion of gonadotropin-releasing hormone (GnRH) is important for maintaining reproductive function in mammals. Accumulating evidence suggests that kisspeptin/neurokinin B/dynorphin A (KNDy) neurons in the hypothalamic arcuate nucleus (ARC) play a critical role in the regulation of pulsatile GnRH and subsequent gonadotropin secretion. Dynorphin A (Dyn) and its receptor, kappa-opioid receptor (KOR, encoded by Oprk1), have been shown to be involved in the suppression of pulsatile GnRH/luteinizing hormone (LH) release. On the other hand, it is still unclear whether the inhibitory Dyn signaling affects KNDy neurons or KOR-expressing non-KNDy cells in the ARC or other brain regions. We therefore aimed to clarify the role of ARC-specific Dyn-KOR signaling in the regulation of pulsatile GnRH/LH release by the ARC specific cell deletion of KOR-expressing cells using Dyn-conjugated-saporin (Dyn-SAP). Estrogen-primed ovariectomized female rats were administered Dyn-SAP to the ARC. In situ hybridization of Oprk1 showed that ARC Dyn-SAP administration significantly decreased the number of Oprk1-expressing cells in the ARC, but not in the ventromedial hypothalamic nucleus and paraventricular nucleus. The frequency of LH pulses significantly increased in animals bearing the ARC Dyn-SAP administration. The number of Kiss1-expressing cells in the ARC was not affected by ARC Dyn-SAP treatment. Dyn-KOR signaling within the ARC seems to mediate the suppression of the frequency of pulsatile GnRH/LH release, and ARC non-KNDy KOR neurons may be involved in the mechanism modulating GnRH/LH pulse generation.

Estrogen increases KISS1 expression in newly generated immortalized KISS1-expressing cell line derived from goat preoptic area.

Kisspeptin neurons located in the hypothalamic preoptic area (POA) are suggested to be responsible for the induction of the gonadotropin-releasing hormone (GnRH) surge and the following luteinizing hormone (LH) surge to regulate female mammals' ovulation. Accumulating evidence demonstrates that the preovulatory level of estrogen activates the POA kisspeptin neurons (estrogen positive feedback), which in turn induces a GnRH/LH surge. This study aimed to derive a cell line from goat POA kisspeptin neurons as an in vitro model to analyze the estrogen positive feedback mechanism in ruminants. Neuron-derived cell clones obtained by the immortalization of POA tissue from a female Shiba goat fetus were analyzed for the expression of kisspeptin (KISS1) and estrogen receptor α (ESR1) genes using quantitative real-time reverse transcription-polymerase chain reaction and three cell clones were selected as POA kisspeptin neuron cell line candidates. One cell line (GP64) out of the three clones showed significant increase in the KISS1 level by incubation with estradiol for 24 h, indicating that the GP64 cells mimic endogenous goat POA kisspeptin neurons. The GP64 cells showed immunoreactivities for kisspeptin and estrogen receptor α and retained a stable growth rate throughout three passages. Further, intracellular calcium levels in the GP64 cells were increased by the KCl challenge, indicating their neurosecretory ability. In conclusion, we generated a new KISS1-expressing cell line derived from goat POA. The current GP64 cell line could be a useful model to elucidate the estrogen positive feedback mechanism responsible for the GnRH/LH surge generation in ruminants.

Establishment of immortalised cell lines derived from female Shiba goat KNDy and GnRH neurones.

Kisspeptin plays a critical role in governing gonadotrophin-releasing hormone (GnRH)/gonadotrophin secretion and subsequent reproductive function in mammals. The hypothalamic arcuate nucleus (ARC) kisspeptin neurones, which co-express neurokinin B (NKB) and dynorphin A (Dyn) and are referred to as KNDy neurones, are considered to be involved in GnRH generation. The present study aimed to establish cell lines derived from goat KNDy and GnRH neurones. Primary-cultured cells of female Shiba goat foetal hypothalamic ARC and preoptic area (POA) tissues were immortalised with the infection of lentivirus containing the simian virus 40 large T-antigen gene. Clones of the immortalised cells were selected by the gene expression of a neuronal marker, and then the neurone-derived cell clones were further selected by the gene expression of KNDy or GnRH neurone markers. As a result, we obtained a KNDy neurone cell line (GA28) from the ARC, as well as two GnRH neurone cell lines (GP11 and GP31) from the POA. Immunocytochemistry revealed the expression of kisspeptin, NKB and Dyn in GA28 cells, as well as GnRH in GP11 and GP31 cells. GnRH secretion from GP11 and GP31 cells into the media was confirmed by an enzyme immunoassay. Moreover, kisspeptin challenge increased intracellular Ca2+ levels in subsets of both GP11 and GP31 cells. Kisspeptin mRNA expression in GA28 cells, which expressed the oestrogen receptor alpha gene, was significantly reduced by 17ß-oestradiol treatment. Furthermore, the transcriptional core promoter and repressive regions of the goat NKB gene were detected using GA28 cells. In conclusion, we have established goat KNDy and GnRH neurone cell lines that could be used to analyse molecular and cellular mechanisms regulating KNDy and GnRH neurones in vitro, facilitating the clarification of reproductive neuroendocrine mechanisms in ruminants.

Role of kisspeptin neurons as a GnRH surge generator: Comparative aspects in rodents and non-rodent mammals.

Ovulation is an essential phenomenon for reproduction in mammalian females along with follicular growth. It is well established that gonadal function is controlled by the neuroendocrine system called the hypothalamus-pituitary-gonadal (HPG) axis. Gonadotropin-releasing hormone (GnRH) neurons, localized in the hypothalamus, had been considered to be the head in governing the HPG axis for a long time until the discovery of kisspeptin. In females, induction of ovulation and folliculogenesis has been linked to a surge mode and pulse mode of GnRH releases, respectively. The mechanisms of how the two modes of GnRH are differently regulated had long remained elusive. The discovery of kisspeptin neurons, distributed in two hypothalamic nuclei, such as the arcuate nucleus in the caudal hypothalamus and preoptic area or the anteroventral periventricular nucleus in the rostral hypothalamic regions, and analyses of the detailed functions of kisspeptin neurons have led marked progress on the understanding of different mechanisms regulating GnRH surges (ovulation) and GnRH pulses (folliculogenesis). The present review will focus on the role of kisspeptin neurons as the GnRH surge generator, including the sexual differentiation of the surge generation system and factors that regulate the surge generator. Comparative aspects between mammalian species are especially focused on.

Characterization of rodent constitutively active estrogen receptor α variants and their constitutive transactivation mechanisms.

Estrogen receptor α (ERα) mRNAs exhibit remarkable heterogeneity owing to complicated alternative splicing. Some encode C-terminally-truncated ERα proteins, which display ligand-independent transactivation or dominant-negative activity. We previously characterized C-terminally-truncated ERα mRNA variants with cryptic sequences in humans and mice, and demonstrated that helices in the ligand-binding domains (LBDs) of ERα variants contribute to ligand-independent transcriptional activity. However, existence of non-conventional coding exons and generation of constitutively active ERα variants have remained to be examined in rats. To comparatively analyze modular organization and splicing profiles of the ERα genes, the range of C-terminally-truncated ERα variants was explored in rats and mice using rapid amplification of cDNA ends and RT-PCR cloning. Furthermore, their functions were determined in transiently transfected cells using expression constructs and luciferase reporter assays. Multiple cryptic exons and C-terminally-truncated ERα variant mRNAs were identified in rats and mice. Naturally occurring and artificially truncated variants/constructs lacking helix 5 potentially exhibited gain-of-function in transfected cells. Although cryptic exons and splicing profiles were poorly conserved among humans, mice, and rats, constitutively active variants were generated from the ERα genes. Moreover, the primary mechanism of ligand-independent activation by C-terminally-truncated ERα variants is C-terminus to helix 5 deletion in the LBD. This comparative study documented the complexity of ERα genes, mRNAs, and proteins, and further determined the underlying structural basis of ligand-independent activation by C-terminally-truncated ERα variants.

Human C-terminally truncated ERα variants resulting from the use of alternative exons in the ligand-binding domain.

The nuclear receptor genes contain alternative internal and terminal exons, with alternative exon incorporation yielding mRNA variants that encode various receptor types, including some with C-terminal truncation that exhibit constitutive activation or dominant-negative transcriptional transactivation. However, C-terminally truncated estrogen receptor α (ERα) variants with alternative sequences have rarely been reported in humans. Therefore, we assessed human ERα genomic organization and alternative splicing profiles, and identified both alternative exons and C-terminally truncated ERα variants. These naturally occurring C-terminally truncated ERα proteins were localized in the nuclei of transfected cells. In addition, ERαi45c and ERαΔ5 variants exhibited constitutive transactivation of an estrogen responsive element-driven promoter in transfected cells. We manufactured expression vectors encoding artificially truncated ERα constructs and evaluated their transactivation abilities to establish mechanisms determining the constitutive activity and dominant-negative properties of truncated variants. Lack of the region encoded in exon 8 eliminated basal and ligand-induced transcriptional transactivation. The C-terminally truncated ERα variants/constructs containing the helices 5 in their ligand-binding domains did not exhibit constitutive transactivation. Furthermore, we demonstrated that truncation from C-termini to helices 5 in the variant ligand-binding domains was required for constitutive activation and found that the remnant regions of the ligand-binding domains and variant-specific sequences influenced transcriptional transactivation efficiency. In conclusion, we elucidated the structural and functional features of novel C-terminally truncated ERα variants and revealed the mechanisms underlying constitutive transactivation by C-terminally truncated nuclear receptor variants.

Puerperal and parental experiences alter rat preferences for pup odors via changes in the oxytocin system.

In the rat, induction of maternal behavior depends on the parity of the female. For example, nulliparous (NP) females need longer exposure to pups than multiparous (MP) or lactating (L) females to exhibit similar maternal behavior. In this study, we investigated the role of brain oxytocin in the approaching behavior of these female rats. Olfactory preferences for pup odors were examined for 8 consecutive days. Each preference test was followed by direct overnight exposure to pups. On the 8th day, MP and L, but not NP females showed robust pup-odor preferences. After the behavioral test, half of the females were exposed to pups for 2 h, whereas the other half were not. The females were then sacrificed to analyze brain oxytocin (OXT) and vasopressin (AVP) activities by cFos immunohistochemistry and to quantify their receptor mRNA expression using real-time PCR. In the paraventricular nucleus (PVN), the percentage of cFos-positive OXT neurons was significantly larger in MP and L females than in NP females after pup exposure. No significant differences were found in cFos expression in OXT neurons of the supraoptic nucleus (SON) or in AVP neurons of either the PVN or SON. Expression of OXT receptor mRNA in the medial preoptic area and amygdala of the control groups was also higher in MP females than in NP females. Finally, we demonstrated that infusion of OXT into the lateral ventricle of NP females promoted preferences for pup odors. These results indicate that puerperal and parental experiences enhance the responsiveness of OXT neurons in the PVN to pup stimuli and establish olfactory preferences for these odors in a parity-dependent manner.

Estradiol rapidly modulates spinogenesis in hippocampal dentate gyrus: Involvement of kinase networks.

This article is part of a Special Issue "Estradiol and cognition". Estradiol (E2) is locally synthesized within the hippocampus and the gonads. Rapid modulation of hippocampal synaptic plasticity by E2 is essential for synaptic regulation. The molecular mechanisms of modulation through the synaptic estrogen receptor (ER) and its downstream signaling, however, are largely unknown in the dentate gyrus (DG). We investigated the E2-induced modulation of dendritic spines in male adult rat hippocampal slices by imaging Lucifer Yellow-injected DG granule cells. Treatments with 1 nM E2 increased the density of spines by approximately 1.4-fold within 2h. Spine head diameter analysis showed that the density of middle-head spines (0.4-0.5 µm) was significantly increased. The E2-induced spine density increase was suppressed by blocking Erk MAPK, PKA, PKC and LIMK. These suppressive effects by kinase inhibitors are not non-specific ones because the GSK-3ß antagonist did not inhibit E2-induced spine increase. The ER antagonist ICI 182,780 also blocked the E2-induced spine increase. Taken together, these results suggest that E2 rapidly increases the density of spines through kinase networks that are driven by synaptic ER.

Aging-induced changes in sex-steroidogenic enzymes and sex-steroid receptors in the cortex, hypothalamus and cerebellum.

We investigated age-induced changes in mRNA expression profiles of sex-steroidogenic enzymes and sex-steroid receptors in 3-, 12-, and 24-month-old male rat brain subregions [cerebral cortex (CC), hypothalamus (Hy) and cerebellum (CL)]. In many cases, the expression levels of mRNA decreased with age for androgen synthesis enzyme systems, including Cyp17a1, Hsd17b and Srd5a in the CC and CL, but not in the Hy. Estradiol synthase Cyp19a1 did not show age-induced decline in the Hy, and nearly no expression of Cyp19a1 was observed in the CC and CL over 3-24 m. Androgen receptor Ar increased in the Hy but decreased in the CC with age. Estrogen receptor Esr1 increased in the CC and Hy, and did not change in the CL with age. Esr2 did not change in the CC and Hy, but decreased in the CL with age. As a comparison, age-induced changes of brain-derived neurotrophic factor mRNA were also investigated.

Novel splicing events and post-transcriptional regulation of human estrogen receptor α E isoforms.

Expression of the estrogen receptor α (ERα) gene is subject to complex regulation. To elucidate the mechanisms of this regulation, the genomic organization and the physiological role of the multiple 5'-untranslated regions (5'-UTRs) must be determined. Here, we investigated the expression and splicing patterns of the human ERα E isoforms. We identified two novel untranslated exons, N1 and N2, in the 5'-region of the human ERα gene and multiple E isoform mRNA variants generated by alternative usage of non-coding internal exons. Expression of the N1-containing variants was observed only in the human breast adenocarcinoma cell line, MCF7, while the N2-containing variants were expressed in the adult liver and MCF7 cells. We examined post-transcriptional regulation of the variant mRNAs using luciferase reporter assays and quantitative PCR. The insertion of untranslated internal exons into the 5'-UTRs of the E isoforms reduced their translation efficiency, but barely influenced mRNA turnover. Our results indicate that the genomic organization of the human ERα gene and the splicing profiles of the human ERα E isoforms are more complicated than previously reported. Furthermore, the 5'-UTRs of the E isoforms post-transcriptionally control human ERα expression mainly through translational repression.