Rapid changes in brain aromatase activity in the female quail brain following expression of sexual behaviour.

In male quail, oestrogens produced in the brain (neuro-oestrogens) exert a dual action on male sexual behaviour: they increase sexual motivation within minutes via mechanisms activated at the membrane but facilitate sexual performance by slower, presumably nuclear-initiated, mechanisms. Recent work indicates that neuro-oestrogens are also implicated in the control of female sexual motivation despite the presence of high circulating concentrations of oestrogens of ovarian origin. Interestingly, aromatase activity (AA) in the male brain is regulated in time domains corresponding to the slow "genomic" and faster "nongenomic" modes of action of oestrogens. Furthermore, rapid changes in brain AA are observed in males after sexual interactions with a female. In the present study, we investigated whether similar rapid changes in brain AA are observed in females allowed to interact sexually with males. A significant decrease in AA was observed in the medial preoptic nucleus after interactions that lasted 2, 5 or 10 minutes, although this decrease was no longer significant after 15 minutes of interaction. In the bed nucleus of the stria terminalis, a progressive decline of average AA was observed between 2 and 15 minutes, although it never reached statistical significance. AA in this nucleus was, however, negatively correlated with the sexual receptivity of the female. These data indicate that sexual interactions affect brain AA in females as in males in an anatomically specific manner and suggest that rapid changes in brain oestrogens production could also modulate female sexual behaviour.

Rapid control of reproductive behaviour by locally synthesised oestrogens: focus on aromatase.

Oestrogens activate nucleus- and membrane-initiated signalling. Nucleus-initiated events control a wide array of physiological and behavioural responses. These effects generally take place within relatively long periods of time (several hours to days). By contrast, membrane-initiated signalling affects a multitude of cellular functions in a much shorter timeframe (seconds to minutes). However, much less is known about their functional significance. Furthermore, the origin of the oestrogens able to trigger these acute effects is rarely examined. Finally, these two distinct types of oestrogenic actions have often been studied independently such that we do not exactly know how they cooperate to control the same response. The present review presents a synthesis of recent work carried out in our laboratory that aimed to address these issues in the context of the study of male sexual behaviour in Japanese quail, which is a considered as a suitable species for tackling these issues. The first section presents data indicating that 17ß-oestradiol, or its membrane impermeable analogues, acutely enhances measures of male sexual motivation but does not affect copulatory behaviour. These effects depend on the activation of membrane-initiated events and local oestrogen production. The second part of this review discusses the regulation of brain oestrogen synthesis through post-translational modifications of the enzyme aromatase. Initially discovered in vitro, these rapid and reversible enzymatic modulations occur in vivo following variations in the social and environment context and therefore provide a mechanism of acute regulation of local oestrogen provision with a spatial and time resolution compatible with the rapid effects observed on male sexual behaviour. Finally, we discuss how these distinct modes of oestrogenic action (membrane- versus nucleus-initiated) acting in different time frames (short- versus long-term) interact to control different components (motivation versus performance) of the same behavioural response and improve reproductive fitness.

Gonadotropin-releasing hormone plasticity: a comparative perspective.

Gonadotropin-releasing hormone 1 (GnRH1) is a key regulator of the reproductive neuroendocrine system in vertebrates. Recent developments have suggested that GnRH1 neurons exhibit far greater plasticity at the cellular and molecular levels than previously thought. Furthermore, there is growing evidence that sub-populations of GnRH1 neurons in the preoptic area are highly responsive to specific environmental and hormonal conditions. In this paper we discuss findings that reveal large variation in GnRH1 mRNA and protein expression that are regulated by social cues, photoperiod, and hormonal feedback. We draw upon studies using histochemistry and immediate early genes (e.g., c-FOS/ZENK) to illustrate that specific groups of GnRH1 neurons are topographically organized. Based on data from diverse vertebrate species, we suggest that GnRH1 expression within individuals is temporally dynamic and this plasticity may be evolutionarily conserved. We suggest that the plasticity observed in other neuropeptide systems (i.e. kisspeptin) may have evolved in a similar manner.

Variation in gonadotrophin-releasing hormone-1 gene expression in the preoptic area predicts transitions in seasonal reproductive state.

In many seasonally reproducing animals, the experience of prolonged exposure to constant photoperiods results in the induction of a state of photorefractoriness, which is defined as a lack of responsiveness to a previously stimulatory photoperiod. The physiological and genetic processes that control photorefractoriness are not well understood; however, the hallmark of photorefractoriness is an endogenous change in the physiological response to a constant photoperiod. It is already known that preoptic area (POA) gnrh1 gene expression declines during the development of refractoriness to long-day stimulation in European starlings. We employed in situ hybridisation histochemistry to characterise changes in POA gnrh1 mRNA expression during the reinstatement of photosensitivity in female starlings. Photorefractory starlings moved to short days (8L:16D) increased optical density of gnrh1 expressing cells within 10 days. Exposure to 30 short days resulted in greater visible gnrh1 cell numbers, with no detectable change in measures of ovarian follicular volume and oviduct mass. We subsequently examined the extent of gnrh1 expression in response to photostimulation after incremental periods on short day lengths. A significant long-day-induced increase in both gnrh1 expression and ovarian and oviduct mass occurred only after at least 30 short days. These findings demonstrate that the recovery of photorefractoriness involves an increase in gnrh1 mRNA expression and expands upon our previous knowledge that the development of photosensitivity is associated with an increase in both the precursor proGnRH1-GAP and GnRH1 peptides in the POA. Importantly, the change in the brain sensitivity occurs well before such changes can be detected via variation in ovarian activity.

Seasonal and individual variation in singing behavior correlates with α2-noradrenergic receptor density in brain regions implicated in song, sexual, and social behavior.

In seasonally breeding male songbirds, both the function of song and the stimuli that elicit singing behavior change seasonally. The catecholamine norepinephrine (NE) modulates attention and arousal across behavioral states, yet the role of NE in seasonally-appropriate vocal communication has not been well-studied. The present study explored the possibility that seasonal changes in alpha 2-noradrenergic receptors (α(2)-R) within song control regions and brain regions implicated in sexual arousal and social behavior contribute to seasonal changes in song behavior in male European starlings (Sturnus vulgaris). We quantified singing behavior in aviary housed males under spring breeding season conditions and fall conditions. α(2)-R were identified with the selective ligand [(3)H]RX821002 using autoradiographic methods. The densities of α(2)-R in song control regions (HVC and the robust nucleus of the arcopallium [RA]) and the lateral septum (LS) were lower in Spring Condition males. α(2)-R densities in the caudal portion of the medial preoptic nucleus (POM) related negatively to singing behavior. Testosterone concentrations were highest in Spring Condition males and correlated with α(2)-R in LS and POM. Results link persistent seasonal alterations in the structure or function of male song to seasonal changes in NE α(2)-Rs in HVC, RA, and LS. Individual differences in α(2)-R in the POM may in part explain individual differences in song production irrespective of the context in which a male is singing, perhaps through NE modification of male sexual arousal.

Differential effects of central injections of D1 and D2 receptor agonists and antagonists on male sexual behavior in Japanese quail.

A key brain site in the control of male sexual behavior is the medial pre-optic area (mPOA) where dopamine stimulates both D1 and D2 receptor subtypes. Research completed to date in Japanese quail has only utilized systemic injections and therefore much is unknown about the specific role played by dopamine in the brain and mPOA in particular. The present study investigated the role of D1 and D2 receptors on male sexual behavior by examining how intracerebroventricular injections and microinjections into the mPOA of D1 and D2 agonists and antagonists influenced appetitive and consummatory aspects of sexual behavior in male quail. Experiments 1 and 2 investigated the effects of intracerebroventricular injections at three doses of D1 or D2 agonists and antagonists. The results indicated that D1 receptors facilitated consummatory male sexual behavior, whereas D2 receptors inhibited both appetitive and consummatory behaviors. Experiment 3 examined the effects of the same compounds specifically injected in the mPOA and showed that, in this region, both receptors stimulated male sexual behaviors. Together, these data indicated that the stimulatory action of dopamine in the mPOA may require a combined activation of D1 and D2 receptors. Finally, the regulation of male sexual behavior by centrally infused dopaminergic compounds in a species lacking an intromittent organ suggested that dopamine action on male sexual behavior does not simply reflect the modulation of genital reflexes due to general arousal, but relates to the central control of sexual motivation. Together, these data support the claim that dopamine specifically regulates male sexual behavior.

Is sexual motivational state linked to dopamine release in the medial preoptic area?

The medial preoptic area (mPOA) is a key site for the dopaminergic enhancement of male sexual behavior. Dopamine release increases in the rat mPOA with mating, supporting the critical stimulatory role played by preoptic dopamine on male sexual behavior. However, it has been questioned whether dopamine is specifically related to the occurrence of male sexual behavior and not simply involved in general arousal. To address this question, we asked whether dopamine release in the mPOA is linked to the production of male sexual behavior in Japanese quail (Coturnix japonica), a species that exhibits a much shorter temporal pattern of copulation than rats and does not have an intermittent organ, resulting in a very different topography of their sexual response. Extracellular samples from the mPOA of adult sexually experienced male quail were collected every 6 min before, during, and after exposure to a female using in vivo microdialysis and analyzed using high-performance liquid chromatography with electrochemical detection. Extracellular dopamine significantly increased in the presence of a female and returned to baseline after removal of the female. However, quail that failed to copulate did not display this increased release. These findings indicate that it is not solely the presence of a female that drives dopamine release in males, but how a male responds to her. Furthermore, in quail that copulated, dopamine release did not change in samples collected during periods of no copulation. Together, these findings support the hypothesis that dopamine action in the mPOA is specifically linked to sexual motivation and not only to copulatory behavior or physical arousal.

Sex differences in the expression of sex steroid receptor mRNA in the quail brain.

In Japanese quail, males will readily exhibit the full sequence of male-typical sexual behaviors but females never show this response, even after ovariectomy and treatment with male-typical concentrations of exogenous testosterone. Testosterone aromatisation plays a key-limiting role in the activation of this behavior but the higher aromatase activity in the brain of males compared to females is not sufficient to explain the behavioural sex difference. The cellular and molecular bases of this prominent sex difference in the functional consequences of testosterone have not been identified so far. We hypothesised that the differential expression of sex steroid receptors in specific brain areas could mediate this behavioural sex difference. Therefore, using radioactive in situ hybridisation histochemistry, we quantified the expression of the mRNA coding for the androgen receptor (AR) and the oestrogen receptors (ER) of the alpha and beta subtypes. All three receptors were expressed in an anatomically discrete manner in various nuclei of the hypothalamus and limbic system and, at usually lower densities, in a few other brain areas. In both sexes, the intensity of the hybridisation signal for all steroid receptors was highest in the medial preoptic nucleus (POM), a major site of testosterone action that is related to the activation of male sexual behaviour. Although no sex difference in the optical density of the AR hybridisation signal could be found in POM, the area covered by AR mRNA was significantly larger in males than in females, indicating a higher overall degree of AR expression in this region in males. By contrast, females tended to have significantly higher levels of AR expression than males in the lateral septum. ERalpha was more densely expressed in females than males throughout the medial preoptic and hypothalamic areas (including the POM and the medio-basal hypothalamus), an area implicated in the control of female receptivity) and in the mesencephalic nucleus intercollicularis. ERbeta was more densely expressed in the medio-basal hypothalamus of females but a difference in the reverse direction (males > females) was observed in the nucleus taeniae of the amygdala. These data suggest that a differential expression of steroid receptors in specific brain areas could mediate at least certain aspects of the sex differences in behavioural responses to testosterone, although they do not appear to be sufficient to explain the complete lack of activation by testosterone of male-typical copulatory behaviour in females.

D1-like dopamine receptor density in nuclei involved in social behavior correlates with song in a context-dependent fashion in male European starlings.

Research in songbirds shows that singing behavior is regulated by both brain areas involved in vocal behavior as well as those involved in social behavior. Interestingly, the precise role of these regions in song can vary as a function of the social, environmental and breeding context. To date, little is known about the neurotransmitters underlying such context-dependent regulation of song. Dopamine (DA) modulates highly motivated, goal-directed behaviors (including sexually motivated song) and emerging data implicate DA in the context-dependent regulation of singing behavior. This study was performed to begin to examine whether differences in DA receptors may underlie, in part, context-dependent differences in song production. We used autoradiographic procedures to label D1-like and D2-like DA receptors to examine the relationship between DA receptor density and singing behavior in multiple contexts in male European starlings (Sturnus vulgaris). Within a breeding context (when testosterone (T) was high), D1-like receptor density in the medial preoptic nucleus (POM) and midbrain central gray (GCt) negatively correlated with song used to attract a female. Additionally in this context, D1-like receptor density in POM, GCt, medial bed nucleus of the stria terminalis (BSTm), and lateral septum (LS) negatively correlated with song likely used to defend a nest box. In contrast, in a non-breeding context (when T was low), D1-like receptor density in POM and LS positively correlated with song used to maintain social flocks. No relationships were identified between song in any context and D2-like receptor densities. Differences in the brain regions and directional relationships between D1-like receptor binding and song suggest that dopaminergic systems play a region and context-specific role in song. These data also suggest that individual variation in singing behavior may, in part, be explained by individual differences in D1-like receptor density in brain regions implicated in social behavior.

Cloning of gonadotropin-releasing hormone I complementary DNAs in songbirds facilitates dissection of mechanisms mediating seasonal changes in reproduction.

Temperate zone animals exhibit seasonal variation in reproductive physiology. In most cases, seasonal changes in reproductive states are regulated by changes in GnRH1 secretion, rather than synthesis, from the preoptic area (POA)/anterior hypothalamus. An important exception occurs in some songbirds that become photorefractory to the stimulatory effects of long days and show profound decreases in brain GnRH1 protein content. Whether this decline reflects changes in gene expression is unknown because of past failures to measure GNRH1 mRNA levels, due in large part to the absence of available GNRH1 gene sequence in this taxon. Here, we report the first cloning of GNRH1 cDNAs in two songbirds: European starlings and zebra finches. Consistent with the size of the prepro-hormone in other avian and non-avian species, the open-reading frames predict proteins of 91 and 92 amino acids, respectively. Whereas the decapeptide in both species is perfectly conserved with chicken GnRH1, the amino acid identity in the signal peptide and GNRH associated peptide subdomains are significantly less well conserved. At the nucleotide level, the starling and zebra finch coding sequences are approximately 88% identical to each other but only approximately 70% identical to chicken GNRH1. In situ hybridization using radiolabeled cRNA probes demonstrated GNRH1 mRNA expression primarily in the POA, consistent with previous studies on the distribution of the GnRH1-immunoreactive cell bodies. Furthermore, we provide evidence for photoperiod-dependent regulation of GNRH1 mRNA in male starlings. Declines in GNRH1 mRNA levels occur in parallel with testicular involution. Thus, photorefractoriness is associated with decreases in GNRH1 gene expression in the medial POA.

Expression of reelin, its receptors and its intracellular signaling protein, Disabled1 in the canary brain: relationships with the song control system.

Songbirds produce learned vocalizations that are controlled by a specialized network of neural structures, the song control system. Several nuclei in this song control system demonstrate a marked degree of adult seasonal plasticity. Nucleus volume varies seasonally based on changes in cell size or spacing, and in the case of nucleus HVC and area X on the incorporation of new neurons. Reelin, a large glycoprotein defective in reeler mice, is assumed to determine the final location of migrating neurons in the developing brain. In mammals, reelin is also expressed in the adult brain but its functions are less well characterized. We investigated the relationships between the expression of reelin and/or its receptors and the dramatic seasonal plasticity in the canary (Serinus canaria) brain. We detected a broad distribution of the reelin protein, its mRNA and the mRNAs encoding for the reelin receptors (VLDLR and ApoER2) as well as for its intracellular signaling protein, Disabled1. These different mRNAs and proteins did not display the same neuroanatomical distribution and were not clearly associated, in an exclusive manner, with telencephalic brain areas that incorporate new neurons in adulthood. Song control nuclei were associated with a particular specialized expression of reelin and its mRNA, with the reelin signal being either denser or lighter in the song nucleus than in the surrounding tissue. The density of reelin-immunoreactive structures did not seem to be affected by 4 weeks of treatment with exogenous testosterone. These observations do not provide conclusive evidence that reelin plays a prominent role in the positioning of new neurons in the adult canary brain but call for additional work on this protein analyzing its expression comparatively during development and in adulthood with a better temporal resolution at critical points in the reproductive cycle when brain plasticity is known to occur.

Plasticity in the expression of the steroid receptor coactivator 1 in the Japanese quail brain: effect of sex, testosterone, stress and time of the day.

Analysis of nuclear receptor action on the eukaryotic genome highlights the importance of coactivators on gene transcription. The steroid receptor coactivator-1 in particular is the focus of an intense research and physiological or behavioral studies have confirmed that it plays a major role in the modulation of steroid and thyroid receptors activity. However, little is known about the regulation of steroid receptor coactivator-1 expression the brain. The goal of this study was to determine the potential factors modulating steroid receptor coactivator-1 synthesis in Japanese quail by quantification of its mRNA with real time quantitative polymerase chain reaction and of the corresponding protein via Western blotting. Contrary to previously published results from our laboratory [Charlier TD, Lakaye B, Ball GF, Balthazart J (2002) The steroid receptor coactivator SRC-1 exhibits high expression in steroid-sensitive brain areas regulating reproductive behaviors in the quail brain. Neuroendocrinology 76:297-315], we found here that sexually mature females had a higher concentration of steroid receptor coactivator-1 in the preoptic area/hypothalamus compared with males. Steroid receptor coactivator-1 expression in the male preoptic area/hypothalamus was up-regulated by testosterone and tended to be decreased by stress. We also identified a significant correlation between the time of the day and the expression of the coactivator in the optic lobes, hippocampus, telencephalon and hindbrain but the pattern of changes in expression as a function of the time of the day varied from one brain area to another. Together, these data support the idea that steroid receptor coactivator-1 is not constitutively expressed but rather is finely regulated by steroids, stress and possibly other unidentified factors.

Neuroanatomical specificity in the expression of the immediate early gene c-fos following expression of appetitive and consummatory male sexual behaviour in Japanese quail.

We investigated the neural sites related to the occurrence of appetitive (ASB) and consummatory (CSB) aspects of male sexual behaviour in Japanese quail. Castrated males treated with testosterone were exposed for 5 min to one of four experimental conditions: (i) free interaction with a female (CSB group); (ii) expression of rhythmic cloacal sphincter movements in response to the visual presentation of a female (ASB-F group); (iii) or a male (ASB-M group), and (iv) handling as a control manipulation. Brains were collected 90 min after the start of behavioural tests and stained by immunocytochemistry for the FOS protein. An increase in FOS expression was observed throughout the rostro-caudal extent of the medial preoptic nucleus (POM) in CSB males, whereas the view of a female (ASB-F) induced an increased FOS expression in the rostral POM only. In the CSB group, there was also an increase in FOS expression in the bed nucleus striae terminalis, and both the CSB and ASB-F groups exhibited increased FOS expression in aspects of the ventro-lateral thalamus (VLT) related to visual processing. Moreover, both the CSB and ASB-M groups showed increased FOS expression in the lateral septum. These data provide additional support to the idea that there is a partial anatomical dissociation between structures involved in the control of both aspects of male sexual behaviour and independently provide data consistent with a previous lesion study that indicated that the rostral and caudal POM differentially control the expression of ASB and CSB in quail.

Rapid changes in production and behavioral action of estrogens.

It is well established that sex steroid hormones bind to nuclear receptors, which then act as transcription factors to control brain sexual differentiation and the activation of sexual behaviors. Estrogens locally produced in the brain exert their behavioral effects in this way but mounting evidence indicates that estrogens also can influence brain functioning more rapidly via non-genomic mechanisms. We recently reported that, in Japanese quail, the activity of preoptic estrogen synthase (aromatase) can be modulated quite rapidly (within minutes) by non-genomic mechanisms, including calcium-dependent phosphorylations. Behavioral studies further demonstrated that rapid changes in estrogen bioavailability, resulting either from a single injection of a high dose of estradiol or from the acute inhibition of aromatase activity, significantly affect the expression of both appetitive and consummatory aspects of male sexual behavior with latencies ranging between 15 and 30 min. Together these data indicate that the bioavailability of estrogens in the brain can change on different time-scales (long- and short-term) that match well with the genomic and non-genomic actions of this steroid and underlie two complementary mechanisms through which estrogens modulate behavior. Estrogens produced locally in the brain should therefore be considered not only as neuroactive steroids but they also display many (if not all) functional characteristics of neuromodulators and perhaps neurotransmitters.

Effects of calmodulin on aromatase activity in the preoptic area.

Oestrogens derived from the neural aromatisation of testosterone play a key role in the activation of male sexual behaviour in many vertebrates. Besides their slow action on gene transcription mediated by the binding to nuclear receptors, oestrogens have now been recognised to have more rapid membrane-based effects on brain function. Rapid changes in aromatase activity, and hence in local oestrogen concentrations, could thus rapidly modulate behavioural responses. We previously demonstrated that calcium-dependent kinases are able to down-regulate aromatase activity after incubations of 10-15 min in phosphorylating conditions. In the present study, in quail hypothalamic homogenates, we show that Ca2+ or calmodulin alone can very rapidly change aromatase activity. Preincubation with 1 mM EGTA or with a monoclonal antibody raised against calmodulin immediately increased aromatase activity. The presence of calmodulin on aromatase purified by immunoprecipitation and electrophoresis was previously identified by western blot and two consensus binding sites for Ca2+-calmodulin are identified here on the deduced amino acid sequence of the quail brain aromatase. The rapid control of brain aromatase activity thus appears to include two mechanisms: (i) an immediate regulatory process that involves the Ca2+-calmodulin binding site and (ii) a somewhat slower phosphorylation by several protein kinases (PKC, PKA but also possibly Ca2+-calmodulin kinases) of the aromatase molecule.

Interactions between kinases and phosphatases in the rapid control of brain aromatase.

Aromatization of testosterone into oestradiol plays a key role in the activation of male sexual behaviour in many vertebrate species. Rapid changes in brain aromatase activity have recently been identified and the resulting changes in local oestrogen bioavailability could modulate fast behavioural responses to oestrogens. In quail hypothalamic homogenates, aromatase activity is down-regulated within minutes by calcium-dependent phosphorylations in the presence of ATP, MgCl2 and CaCl2 (ATP/Mg/Ca). Three kinases (protein kinases A and C and calmodulin kinase; PKA, PKC and CAMK) are potentially implicated in this process. If kinases decrease aromatase activity in a reversible manner, then it would be expected that the enzymatic activity would increase and/or return to baseline levels in the presence of phosphatases. We showed previously that 0.1 mM vanadate (a general inhibitor of protein phosphatases) significantly decreases aromatase activity but specific protein phosphatases that could up-regulate aromatase activity have not been identified to date. The reversibility of aromatase activity inhibition by phosphorylations was investigated in the present study using alkaline and acid phosphatase (Alk and Ac PPase). Unexpectedly, Alk PPase inhibited aromatase activity in a dose-dependent manner in the presence, as well as in the absence, of ATP/Mg/Ca. By contrast, Ac PPase completely blocked the inhibitory effects of ATP/Mg/Ca on aromatase activity, even if it moderately inhibited aromatase activity in the absence of ATP/Mg/Ca. However, the addition of Ac PPase was unable to restore aromatase activity after it had been inhibited by exposure to ATP/Mg/Ca. Taken together, these data suggest that, amongst the 15 potential consensus phosphorylation sites identified on the quail aromatase sequence, some must be constitutively phosphorylated for the enzyme to be active whereas phosphorylation of the others is involved in the rapid inhibition of aromatase activity by the competitive effects of protein kinases and phosphatases. Two out of these 15 putative phosphorylation sites occur in an environment corresponding to the consensus sites for PKC, PKA (and possibly a CAMK) and, in all probability, represent the sites whose phosphorylation rapidly blocks enzyme activity.

Rapid decreases in preoptic aromatase activity and brain monoamine concentrations after engaging in male sexual behavior.

In Japanese quail, as in rats, the expression of male sexual behavior over relatively long time periods (days to weeks) is dependent on the local production of estradiol in the preoptic area via the aromatization of testosterone. On a short-term basis (minutes to hours), central actions of dopamine as well as locally produced estrogens modulate behavioral expression. In rats, a view of and sexual interaction with a female increase dopamine release in the preoptic area. In quail, in vitro brain aromatase activity (AA) is rapidly modulated by calcium-dependent phosphorylations that are likely to occur in vivo as a result of changes in neurotransmitter activity. Furthermore, an acute estradiol injection rapidly stimulates copulation in quail, whereas a single injection of the aromatase inhibitor vorozole rapidly inhibits this behavior. We hypothesized that brain aromatase and dopaminergic activities are regulated in quail in association with the expression of male sexual behavior. Visual access as well as sexual interactions with a female produced a significant decrease in brain AA, which was maximal after 5 min. This expression of sexual behavior also resulted in a significant decrease in dopaminergic as well as serotonergic activity after 1 min, which returned to basal levels after 5 min. These results demonstrate for the first time that AA is rapidly modulated in vivo in parallel with changes in dopamine activity. Sexual interactions with the female decreased aromatase and dopamine activities. These data challenge established views about the causal relationships among dopamine, estrogen action, and male sexual behavior.

Sexual behavior activates the expression of the immediate early genes c-fos and Zenk (egr-1) in catecholaminergic neurons of male Japanese quail.

We analyzed the expression of the immediate early genes c-fos and Zenk (egr-1) in the brain of male quail that were gonadally intact (I) or castrated and treated (CX+T) or not (CX) with testosterone and had been exposed for 60 min either to a sexually mature female (F), or to an empty arena (EA) or were left in their home cage (HC). Alternate sections in the brains collected 90 min after the start of behavioral interactions were stained by immunocytochemistry for the proteins FOS or ZENK alone or in association with tyrosine hydroxylase (TH), a marker of catecholaminergic neurons. C-fos and Zenk expression was statistically increased in six brain areas of sexually active birds (I+F, CX+T+F) compared with controls (CX+F, CX+T+EA, CX+T+HC), i.e. the preoptic area, bed nucleus striae terminalis, arcopallium, nucleus intercollicularis, periaqueductal gray and the ventral tegmental area. Interestingly, c-fos and Zenk expression was high in the nucleus intercollicularis, a midbrain vocal control nucleus, of I+F and CX+T+F birds that displayed copulatory behavior but emitted few crows but not in the nucleus intercollicularis of CX+T+EA birds that crowed frequently. Increases in c-fos expression were observed in TH-immunoreactive cells in the periaqueductal gray and ventral tegmental area, but not in the substantia nigra, of I+F and CX+T+F birds indicating the activation of dopaminergic neurons during sexual behavior. Together, these data confirm the implication of the steroid-sensitive preoptic area and bed nucleus striae terminalis in the control of copulation and support the notion that dopamine is involved in its control.

Song activation by testosterone is associated with an increased catecholaminergic innervation of the song control system in female canaries.

In canaries, singing and a large number of morphological features of the neural system that mediates the learning, perception and production of song exhibit marked sex differences. Although these differences have been mainly attributed to sex-specific patterns of the action of testosterone and its metabolites, the mechanisms by which sex steroids regulate brain and behavior are far from being completely understood. Given that the density of immunoreactive catecholaminergic fibers that innervate telencephalic song nuclei in canaries is higher in males, which sing, than in females, which usually do not sing, we hypothesized that some of the effects induced by testosterone on song behavior are mediated through the action of the steroid on the catecholaminergic neurons which innervate the song control nuclei. Therefore, we investigated in female canaries the effects of a treatment with exogenous testosterone on song production, on the volume of song control nuclei, and on the catecholaminergic innervation of these nuclei as assessed by immunocytochemical visualization of tyrosine hydroxylase. Testosterone induced male-like singing in all females and increased by about 80% the volume of two telencephalic song control nuclei, the high vocal center (HVC) and the nucleus robustus archistriatalis (RA). Testosterone also significantly increased the fractional area covered by tyrosine hydroxylase-immunoreactive structures (fibers and varicosities) in most telencephalic song control nuclei (HVC, the lateral and medial parts of the magnocellular nucleus of the anterior neostriatum, the nucleus interfacialis, and to a lesser extent RA). By contrast, testosterone did not affect the catecholaminergic innervation of the telencephalic areas adjacent to HVC and RA. Together these data demonstrate that, in parallel to its effects on song behavior and on the morphology of the song control system, testosterone also regulates the catecholaminergic innervation of most telencephalic song control nuclei in canaries. The endocrine regulation of singing may thus involve the neuromodulatory action of specialized dopaminergic and/or noradrenergic projections onto several key parts of the song control system.

The neuroendocrinology of reproductive behavior in Japanese quail.

Sex steroid hormones such as testosterone have widespread effects on brain physiology and function but one of their best characterized effects arguably involves the activation of male sexual behavior. During the past 20 years we have investigated the testosterone control of male sexual behavior in an avian species, the Japanese quail (Coturnix japonica). We briefly review here the main features and advantages of this species relating to the investigation of fundamental questions in the field of behavioral neuroendocrinology, a field that studies inter-relationship among hormones, brain and behavior. Special attention is given to the intracellular metabolism of testosterone, in particular its aromatization into an estrogen, which plays a critical limiting role in the mediation of the behavioral effects of testosterone. Brain aromatase activity is controlled by steroids which increase the transcription of the enzyme, but afferent inputs that affect the intraneuronal concentrations of calcium also appear to have a pronounced effect on the enzyme activity through rapid changes in its phosphorylation status. The physiological significance of these slow genomic and rapid, presumably non-genomic, changes in brain aromatase activity are also briefly discussed.