Relationships between rapid changes in local aromatase activity and estradiol concentrations in male and female quail brain

Estradiol-17β (E₂) synthesized in the brain plays a critical role in the activation of sexual behavior in many vertebrate species. Because E₂ concentrations depend on aromatization of testosterone, changes in aromatase enzymatic activity (AA) are often utilized as a proxy to describe E₂ concentrations. Utilizing two types of stimuli (sexual interactions and acute restraint stress) that have been demonstrated to reliably alter AA within minutes in opposite directions (sexual interactions = decrease, stress = increase), we tested in Japanese quail whether rapid changes in AA are paralleled by changes in E₂ concentrations in discrete brain areas. In males, E₂ in the pooled medial preoptic nucleus/medial portion of the bed nucleus of the stria terminalis (POM/BST) positively correlated with AA following sexual interactions. However, following acute stress, E₂ decreased significantly (approximately 2-fold) in the male POM/BST despite a significant increase in AA. In females, AA positively correlated with E₂ in both the POM/BST and mediobasal hypothalamus supporting a role for local, as opposed to ovarian, production regulating brain E₂ concentrations. In addition, correlations of individual E₂ in POM/BST and measurements of female sexual behavior suggested a role for local E₂ synthesis in female receptivity. These data demonstrate that local E₂ in the male brain changes in response to stimuli on a time course suggestive of potential non-genomic effects on brain and behavior. Overall, this study highlights the complex mechanisms regulating local E₂ concentrations including rapid stimulus-driven changes in production and stress-induced changes in catabolism.     

Sexual arousal, is it for mammals only?

Sexual arousal has many dimensions and has consequently been defined in various ways. In humans, sexual arousal can be assessed based in part on verbal communication. In male non-human mammalian species, it has been argued that arousal can only be definitively inferred if the subject exhibits a penile erection in a sexual context. In non-mammalian species that lack an intromittent organ, as is the case for most avian species, the question of how to assess sexual arousal has not been thoroughly addressed. Based on studies performed in male Japanese quail, we argue that several behavioral or physiological characteristics provide suitable measures of sexual arousal in birds and probably also in other tetrapods. These indices include, the performance of appetitive sexual behavior in anticipation of copulation (although anticipation and arousal are not synonymous), the activation of specific brain area as identified by the detection of the expression of immediate early genes (fos, egr-1) or by 2-deoxygucose quantitative autoradiography, and above all, by the release of dopamine in the medial preoptic area as measured by in vivo dialysis. Based on these criteria, it is possible to assess in birds sexual arousal in its broadest sense but meeting the more restrictive definition of arousal proposed for male mammals (erection in an explicit sexual context) is and will probably remain impossible in birds until refinement of in vivo imaging techniques such fMRI allow us to match in different species, with and without an intromittent organ, the brain areas that are activated in the presence of specific stimuli.     

Behavioral responses to pups in males with different reproductive experiences are associated with changes in central OT,TH and OTR,D1R,D2R mRNA expression in mandarin voles

Male rodents behave differently toward pups because of different sexual and/or paternal experiences; however, the mechanisms underlying these responses are not well understood. Using socially monogamous mandarin voles (Microtus mandarinus) we investigated the behavioral responses of males with different reproductive experiences (virgin males, paired males and new fathers) to new born pups. Central levels of neuropeptide oxytocin (OT), tyrosine hydroxylase (TH), as well as oxytocin receptor (OTR), dopamine 1-type receptor (D1R) and dopamine 2-type receptor (D2R) mRNA expression in the nucleus accumbens and medial amygdala were also measured in these males. Our data showed that new fathers exhibited more approaching behavior and contained more OT-immunoreactive and TH-immunoreactive neurons. In addition to increased OTR mRNA expression in the nucleus accumbens and medial amygdala, new fathers had higher D1R and D2R mRNA expression in the nucleus accumbens, and less D1R and D2R mRNA expression in the medial amygdala than paired males. These results demonstrate that males with different reproductive experiences display different behavioral responses to pups and that these differences are associated with altered OT and dopamine, and their receptors in specific brain regions.     

Expression of estrogen receptor-alpha and -beta mRNA in the brain of Japanese quail embryos

The present study was conducted to investigate the mRNA expression of the two estrogen receptor (ER) subtypes ERalpha and ERbeta in the brain of Japanese quail embryos. We found expression of both ERalpha and ERbeta mRNA in homogenate of whole head from 6-day-old embryos, and in brain homogenate from 9- and 12-day-old embryos using real-time PCR. In 9- and 12-day-old embryos the ERalpha expression was higher in females than in males. We used in situ hybridization to examine the localization of the ERs in sections from male and female brains on day 9 and day 17 of incubation. On day 9, ERbeta mRNA was detected in the developing medial preoptic nucleus (POM), in the medial part of the bed nucleus of the striae terminalis (BSTm), and in the tuberal region of the hypothalamus. ERalpha signal could not be detected in the POM, the BSTm or the tuberal region in 9-day-old embryos. In 17-day-old embryos, ERbeta was highly expressed in the preoptic area, the nucleus Taeniae of the Amygdala (TnA) and the BSTm. Expression of ERalpha mRNA was detected in parts of the preoptic area and in the telencephalic TnA. No ERalpha expression was found in the BSTm, an area known to be sexually dimorphic in adults. The high embryonic expression of ERbeta in brain areas linked to sexual behavior indicates that ERbeta plays a role in sexual differentiation of the Japanese quail brain.     

Peptide immunoreactive neurons in the amygdala and the bed nucleus of the stria terminalis project to the midbrain central gray in the rat.

The central nucleus of the amygdala, bed nucleus of the stria terminalis, and central gray are important components of the neural circuitry responsible for autonomic and behavioral responses to threatening or stressful stimuli. Neurons of the amygdala and bed nucleus of the stria terminalis that project to the midbrain central gray were tested for the presence of peptide immunoreactivity. To accomplish this aim, a combined immunohistochemical and retrograde tracing technique was used. Maximal retrograde labeling was observed in the amygdala and bed nucleus of the stria terminalis after injections of retrograde tracer into the caudal ventrolateral midbrain central gray. The majority of the retrogradely labeled neurons in the amygdala were located in the medial central nucleus, although many neurons were also observed in the lateral subdivision of the central nucleus. Most of the retrogradely labeled neurons in the BST were located in the ventral and posterior lateral subdivisions, although cells were also observed in most other subdivisions. Retrogradely labeled neurotensin, corticotropin releasing factor (CRF), and somatostatin neurons were mainly observed in the lateral central nucleus and the dorsal lateral BST. Retrogradely labeled substance P-immunoreactive cells were found in the medial central nucleus and the posterior and ventral lateral BST. Enkephalin-immunoreactive retrogradely labeled cells were not observed in the amygdala or bed nucleus of the stria terminalis. A few cells in the hypothalamus (paraventricular and lateral hypothalamic nuclei) that project to the central gray also contained CRF and neurotensin immunoreactivity. The results suggest the amygdala and the bed nucleus of the stria terminalis are a major forebrain source of CRF, neurotensin, somatostatin, and substance P terminals in the midbrain central gray.     

Increased estrogen receptor alpha immunoreactivity in the forebrain of sexually satiated rats

Estrogen receptor alpha (ERalpha) participates in the neuroendocrine regulation of male sexual behavior, primarily in brain areas located in the limbic system. Males of many species present a long-term inhibition of sexual behavior after several ejaculations, known as sexual satiety. It has been shown that androgen receptor density is reduced 24 h after a single ejaculation or mating to satiety, in the medial preoptic area, nucleus accumbens and ventromedial hypothalamus. The aim of this study was to analyze if the density of ERalpha was also modified 24 h after a single ejaculation or mating to satiety. Sexual satiety was associated with an increased ERalpha density in the anteromedial bed nucleus of the stria terminalis (BSTMA), ventrolateral septum (LSV), posterodorsal medial amygdala (MePD), medial preoptic area (MPA) and nucleus accumbens core (NAc). A single ejaculation was related to an increase in ERalpha density in the BSTMA and MePD. ERalpha density in the arcuate (Arc) and ventromedial hypothalamic nuclei (VMN), and serum estradiol levels remained unchanged 24 h after one ejaculation or mating to satiety. These data suggest a relationship between sexual activity and an increase in the expression of ERalpha in specific brain areas, independently of estradiol levels in systemic circulation.     

Increased apoptosis during neonatal brain development underlies the adult behavioral deficits seen in mice lacking a functional paternally expressed gene 3 (Peg3)

Inactivation of the maternally imprinted, paternally expressed gene 3 (Peg3) induces deficits in olfactory function, sexual and maternal behaviors, oxytocin neuron number, metabolic homeostasis and growth. Peg3 is expressed in a number of developing hypothalamic and basal forebrain structures and is a component of the P53 apoptosis pathway. Peg3 inactivation in neuronal cell culture lines inhibits P53 mediated apoptosis, which is important in the early postnatal development and sexual differentiation of the brain. In this study, we investigated the effect of inactivating the Peg3 gene on the incidence of caspase 3 positive cells (a marker of apoptosis) in 4‐ and 6‐day postpartum mouse brain. Inactivating the Peg3 gene resulted in an increase in the incidence of total forebrain caspase 3 positive cells at 4 and 6 days postpartum. Increases in specific neuroanatomical regions including the bed nucleus of the stria terminalis, nucleus accumbens, caudate putamen, medial pre‐optic area, arcuate nucleus, medial amygdala, anterior cortical and posteriodorsal amygdaloid nuclei, were also observed. In wild‐type mice, sex differences in the incidence of caspase 3 positive cells in the medial amygdala, bed nucleus of the stria terminalis, nucleus accumbens, arcuate nucleus and the M2 motor cortex, were also observed. This neural sex difference was ameliorated in the Peg‐3 mutant. These findings suggest that the neuronal and behavioral deficits seen in mice lacking a functional Peg3 gene are mediated by increases in the incidence of early neonatal apoptosis in neuroanatomical regions important for reproductive behavior, olfactory and pheromonal processing, thermoregulation and reward. © 2009 Wiley Periodicals, Inc. Develop Neurobiol, 2009     

Lesions to the Medial Preoptic Area Affect Singing in the Male European Starling (Sturnus vulgaris)

The aromatization of testosterone (T) in the medial preoptic nucleus (POM) is known to regulate male courtship and sexual behaviors expressed prior to, and in anticipation of, copulation. Singing in male European starlings is used to attract mates prior to physical sexual contact, suggesting that the POM might be involved. The present study was performed to examine the effects of lesions targeting the POM on singing and courtship behavior in reproductively active male starlings. A significant decrease in song output and the gathering of green nest materials was observed in males with lesions to the POM compared to males with damage to brain areas outside of the POM. Lesions did not affect a male's tendency to remain near a female or to occupy a nestbox, suggesting that the effects of POM lesions were specific to courtship behaviors. Behavioral differences were not related to testis mass or volume, and GnRH immunoreactivity was observed within the hypothalamus and median eminence for each male, suggesting that the effects of POM lesions were related specifically to POM involvement in song expression rather than to a disruption of the GnRH axis. These results suggest a general role for the POM in the expression of behaviors related to sexual arousal or anticipation, including song.     

Lesions to the medial preoptic area affect singing in the male European starling (Sturnus vulgaris)

The aromatization of testosterone (T) in the medial preoptic nucleus (POM) is known to regulate male courtship and sexual behaviors expressed prior to, and in anticipation of, copulation. Singing in male European starlings is used to attract mates prior to physical sexual contact, suggesting that the POM might be involved. The present study was performed to examine the effects of lesions targeting the POM on singing and courtship behavior in reproductively active male starlings. A significant decrease in song output and the gathering of green nest materials was observed in males with lesions to the POM compared to males with damage to brain areas outside of the POM. Lesions did not affect a male's tendency to remain near a female or to occupy a nestbox, suggesting that the effects of POM lesions were specific to courtship behaviors. Behavioral differences were not related to testis mass or volume, and GnRH immunoreactivity was observed within the hypothalamus and median eminence for each male, suggesting that the effects of POM lesions were related specifically to POM involvement in song expression rather than to a disruption of the GnRH axis. These results suggest a general role for the POM in the expression of behaviors related to sexual arousal or anticipation, including song.     

Response of ERalpha-IR and ERbeta-IR cells in the forebrain of female rats to mating stimuli

Sexual behavior in female rats depends on the action of estradiol on estrogen receptors (ERs) found in particular brain regions. While hormonal regulation of female sexual behavior requires ERalpha, the possible functions of ERbeta remain to be clarified. Mating stimulation has several behavioral and physiological consequences and induces Fos expression in many brain areas involved in the regulation of reproductive behavior and physiology. In addition, some cells in which mating induces Fos expression coexpress ERalpha. To determine whether cells in which Fos is induced by a particular mating stimulus coexpress ERalpha, ERbeta, or both, we used a triple-label immunofluorescent technique to visualize ERalpha-, ERbeta-, and mating-induced Fos-immunoreactivity (Fos-ir) in neurons in which mating stimulation reliably increases Fos expression. Ovariectomized, hormone-primed rats were either unmated, received 15 mounts, or received 15 intromissions. In the rostral medial preoptic area, Fos-ir was induced by mounts alone primarily in cells coexpressing ERalpha-ir, while Fos-ir was induced by intromissions mainly in cells coexpressing both ERalpha-ir and ERbeta-ir (ERalpha/ERbeta-ir). In the dorsal part of the posterodorsal medial amygdala, Fos-ir was induced by intromissions in cells coexpressing ERalpha-ir and ERalpha/ERbeta-ir. However, in the ventral part of the posterodorsal medial amygdala, Fos-ir was induced by intromissions primarily in cells coexpressing only ERbeta-ir. These data suggest that qualitatively different sexual stimuli may be integrated through distinct ER-containing circuits in the rostral medial preoptic area and posterodorsal medial amygdala. The diversity in coexpression of type of ER in cells in different brain areas after various mating stimuli suggests a role for both ERalpha and ERbeta in the integration of hormonal information and information related to mating stimuli.     

Secretin Activates Visceral Brain Regions in the Rat Including Areas Abnormal in Autism

1. The aim of this study was to determine whether central networks are involved in the presumptive behavioral and autonomic regulatory actions of secretin, a gut hormone that has been reported to have ameliorative effects in autistic children.2. Central neural responses monitored by regional c-fos gene expression were examined in response to intracerebroventricular secretin injection in awake, freely-moving Sprague-Dawley rats. Tissue sections were incubated in an antibody to the c-fosgene product, Fos, and processed immunohistochemically.3. Qualitative differences in Fos immunoreactivity in stress adaptation and visceral representation areas of the brain were observed between secretin- and vehicle-infused age-matched pairs (n = 4 pairs). Secretin-activated regions include the area postrema, dorsal motor nucleus, medial region of the nucleus of the solitary tract and its relay station in the lateral tegmentum, locus ceruleus, ventral periaqueductal gray, periventricular thalamic nucleus, paraventricular hypothalamus magnocellularis, medial and central amygdala, lateral septal complex as well as ependymal and subependymal nuclei lining the third ventricle. Specific areas of the cerebral cortex were heavily labeled in secretin-treated rats, as compared to controls: the medial bank of the anterior prefrontal cortex, orbitofrontal cortex, the piriform cortex, and the anterior olfactory nucleus. Secretin attenuated Fos immunoreactivity in the dorsal periaqueductal gray, intralaminar thalamus, medial parvicellular compartment of the hypothalamus, supraoptic nucleus of the hypothalamus, lateral amygdala, motor cortex, and the somatosensory and association areas of the parietal cortex.4. Secretin alters the activity of structures involved in behavioral conditioning of stress adaptation and visceral reflex reactions. This study predicts a possible cellular mechanism, activation of third ventricular ependymal and subependymal cells, as well as central regulatory actions of secretin. The physiological effects of secretin on behavioral, endocrine, autonomic and sensory neuronal activation patterns, together, contribute to central c-fos activation. Secretin alters the activity of structures involved in behavioral conditioning of stress adaptation and visceral reflex reactions. This study predicts a possible cellular mechanism, activation of third ventricular ependymal and subependymal cells, and central regulatory actions of secretin. The physiological effects of secretin on behavioral, endocrine, autonomic and sensory neuronal activation patterns, together, contribute to central c-fos activation. These findings mandate further investigation of secretin as a brain/gut stress regulatory hormone.     

Sex differences in oxytocin receptor binding in forebrain regions: Correlations with social interest in brain region- and sex- specific ways

Social interest reflects the motivation to approach a conspecific for the assessment of social cues and is measured in rats by the amount of time spent investigating conspecifics. Virgin female rats show lower social interest towards unfamiliar juvenile conspecifics than virgin male rats. We hypothesized that the neuropeptide oxytocin (OT) may modulate sex differences in social interest because of the involvement of OT in pro-social behaviors. We determined whether there are sex differences in OT system parameters in the brain and whether these parameters would correlate with social interest. We also determined whether estrus phase or maternal experience would alter low social interest and whether this would correlate with changes in OT system parameters. Our results show that regardless of estrus phase, females have significantly lower OT receptor (OTR) binding densities than males in the majority of forebrain regions analyzed, including the nucleus accumbens, caudate putamen, lateral septum, bed nucleus of the stria terminalis, medial amygdala, and ventromedial hypothalamus. Interestingly, male social interest correlated positively with OTR binding densities in the medial amygdala, while female social interest correlated negatively with OTR binding densities in the central amygdala. Proestrus/estrus females showed similar social interest to non-estrus females despite increased OTR binding densities in several forebrain areas. Maternal experience had no immediate or long-lasting effects on social interest or OT brain parameters except for higher OTR binding in the medial amygdala in primiparous females. Together, these findings demonstrate that there are robust sex differences in OTR binding densities in multiple forebrain regions of rats and that OTR binding densities correlate with social interest in brain region- and sex-specific ways.     

Immediate early gene activity in song control nuclei and brain areas regulating motivation relates positively to singing behavior during, but not outside of, a breeding context

In some species, such as songbirds, much is known about how the brain regulates vocal learning, production, and perception. What remains a mystery is what regulates the motivation to communicate. European starlings (Sturnus vulgaris) sing throughout most of the year, but the social and environmental factors that motivate singing behavior differ seasonally. Male song is highly sexually motivated during, but not outside of, the breeding season. Brain areas outside the song control system, such as the medial preoptic nucleus (POM) and ventral tegmental area (VTA), have been implicated in regulating sexually motivated behaviors in birds, including song. The present study was designed to explore whether these regions, as well as three song control nuclei [area X, the high vocal center (HVC), and the robust nucleus of the arcopallium (RA)], might be involved differentially in song produced within compared to outside of a breeding context. We recorded the behavioral responses of breeding and nonbreeding condition male starlings to the introduction of a female conspecific. Males did not show context-dependent differences in the overall amount of song sung. However, immunocytochemistry for the protein product of the immediate early gene cFOS revealed a positive linear relationship between the total amount of songs sung and number of cFOS-labeled cells in POM, VTA, HVC, and RA for birds singing during, but not outside of, a breeding context. These results suggest that these regions differentially regulate male song production depending on reproductive context. Overall the data support the hypothesis that the POM and VTA interact with the song control system, specifically HVC and RA, to regulate sexually motivated vocal communication in songbirds.     

Japanese quail as a model system for studying the neuroendocrine control of reproductive and social behaviors

Japanese quail (Coturnix japonica; referred to simply as quail in this article) readily exhibit sexual behavior and related social behaviors in captive conditions and have therefore proven valuable for studies of how early social experience can shape adult mate preference and sexual behavior. Quail have also been used in sexual conditioning studies illustrating that natural stimuli predict successful reproduction via Pavlovian processes. In addition, they have proven to be a good model to study how variation in photoperiod regulates reproduction and how variation in gonadal steroid hormones controls sexual behavior. For example, studies have shown that testosterone activates male-typical behaviors after being metabolized into estrogenic and androgenic metabolites. A critical site of action for these metabolites is the medial preoptic nucleus (POM), which is larger in males than in females. The enzyme aromatase converts testosterone to estradiol and is enriched in the POM in a male-biased fashion. Quail studies were the first to show that this enzyme is regulated both relatively slowly via genomic actions of steroids and more quickly via phosphorylation. With this base of knowledge and the recent cloning of the entire genome of the closely related chicken, quail will be valuable for future studies connecting gene expression to sexual and social behaviors.     

Specific activation of estrogen receptor alpha and beta enhances male sexual behavior and neuroplasticity in male Japanese quail

Two subtypes of estrogen receptors (ER), ERα and ERβ, have been identified in humans and numerous vertebrates, including the Japanese quail. We investigated in this species the specific role(s) of each receptor in the activation of male sexual behavior and the underlying estrogen-dependent neural plasticity. Castrated male Japanese quail received empty (CX) or testosterone-filled (T) implants or were daily injected with the ER general agonist diethylstilbestrol (DES), the ERα-specific agonist PPT, the ERβ-specific agonist DPN or the vehicle, propylene glycol. Three days after receiving the first treatment, subjects were alternatively tested for appetitive (rhythmic cloacal sphincter movements, RCSM) and consummatory aspects (copulatory behavior) of male sexual behavior. 24 hours after the last behavioral testing, brains were collected and analyzed for aromatase expression and vasotocinergic innervation in the medial preoptic nucleus. The expression of RCSM was activated by T and to a lesser extent by DES and PPT but not by the ERβagonist DPN. In parallel, T fully restored the complete sequence of copulation, DES was partially active and the specific activation of ERα or ERβ only resulted in a very low frequency of mount attempts in few subjects. T increased the volume of the medial preoptic nucleus as measured by the dense cluster of aromatase-immunoreactive cells and the density of the vasotocinergic innervation within this nucleus. DES had only a weak action on vasotocinergic fibers and the two specific ER agonists did not affect these neural responses. Simultaneous activation of both receptors or treatments with higher doses may be required to fully activate sexual behavior and the associated neurochemical events.     

Rapid action on neuroplasticity precedes behavioral activation by testosterone

Testosterone has been shown to increase the volume of steroid-sensitive brain nuclei in adulthood in several vertebrate species. In male Japanese quail the volume of the male-biased sexually dimorphic medial preoptic nucleus (POM), a key brain area for the control of male sexual behavior, is markedly increased by testosterone. Previous studies assessed this effect after a period of 8–14 days but the exact time course of these effects is unknown. We asked here whether testosterone-dependent POM plasticity could be observed at shorter latencies. Brains from castrated male quail were collected after 1, 2, 7 and 14 days of T treatment (CX+T) and compared to brains of untreated castrates (CX) collected after 1 or 14 days. POM volumes defined either by Nissl staining or by aromatase immunohistochemistry increased in a time-dependent fashion in CX+T subjects and almost doubled after 14 days of treatment with testosterone while no change was observed in CX birds. A significant increase in the average POM volume was detected after only one day of testosterone treatment. The optical density of Nissl and aromatase staining was also increased after one or two days of testosterone treatment. Activation of male copulatory behavior followed these morphological changes with a latency of approximately one day. This rapid neurochemical and neuroanatomical plasticity observed in the quail POM thus seems to limit the activation of male sexual behavior and offers an excellent model to analyze features of steroid-regulated brain structure and function that determine behavior expression.     

Gene c-Fos expression in brain of rats resistant and predisposed to emotional stress after intraperitoneal injection of the ACTH(4-10)analog--semax

The effect of the ACTH(4-10) analog Semax on immediate early gene c-Fos expression was studied in Wistar rats with high and low resistance to emotional stress under the usual conditions and during psychoemotional loading. Fos-immunoreactive cells in the were counted automatically with the help of a computer. It was shown that under the usual conditions the intraperitoneal Semax injection induced immediate early gene c-Fos expression in the lateral septal region in rats predisposed to emotional stress and in the paraventricular hypothalamus in rats of both groups. Preliminary Semax injection decreased the stress-induced c-Fos expression in the paraventricular hypothalamus and medial septum in rats predisposed to emotional stress and tended to reduce the number of stress-induced c-Fos-immunopositive cells in the lateral septum and basolateral amygdala in both groups of animals. The obtained data suggest that Semax differently affects the immediate early c-Fos gene expression in the brain of rats resistant and predisposed to emotional stress and this effect reflects the antistressor properties of the regulatory peptide.