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International audience; Systemic injection of a thymidine analogue such as bromodeoxyuridine (BrdU) in vertebrates is commonly used to detect and study cell production during development, adulthood, and pathology, particularly in studies of adult neurogenesis. Although researchers are applying this technique to multiple species in various physiological conditions, the rate of BrdU clearance from the serum remains unknown in most cases. Changes in this clearance rate as a function of the species, sex or endocrine condition could however profoundly affect the interpretation of the results. We describe a rapid, sensitive, but simple bioassay for post-injection detection and quantification of BrdU in serum. This procedure was shown to be suitable for determining the length of time a thymidine analogue remains in the bloodstream of one avian species and seems applicable to any vertebrate provided sufficiently large blood samples can be collected. This technique was used to demonstrate that, in canaries, BrdU injected at a dose of 100 mg/kg is no longer available for incorporation into DNA between 30 and 60 min post-injection, a delay shorter than anticipated based on the available literature. Preliminary data suggest a similar fast clearance in Japanese quail and mice.

It is increasingly recognized that estrogens produce rapid and transient effects at many neural sites ultimately impacting physiological and behavioral endpoints. The ability of estrogens to acutely regulate cellular processes implies that their concentration should also be rapidly fine-tuned. Accordingly, rapid changes in the catalytic activity of aromatase, the limiting enzyme for estrogen synthesis, have been identified that could serve as a regulatory mechanism of local estrogen concentrations. However, the precise anatomical localization, time-course, triggering stimuli and functional significance of these enzymatic changes in vivo are not well understood. To address these issues as to where, when and why aromatase activity (AA) rapidly changes after sexual interactions, AA was assayed in six populations of aromatase-expressing cells microdissected from the brain of male quail that experienced varying durations of visual exposure to or copulation with a female. Sexual interactions resulted in a rapid AA inhibition. This inhibition occurred in specific brain regions (including the medial preoptic nucleus), in a context-dependent fashion and time-scale suggestive of post-translational modifications of the enzyme. Interestingly, the enzymatic fluctuations occurring in the preoptic area followed rather than preceded copulation and were tied specifically to the female's presence. This pattern of enzymatic changes suggests that rapid estrogen effects are important during the motivational phase of the behavior to trigger physiological events essential to activate mate search and copulation.

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.

It has long been known that the avian brain is capable of light detection independently of the eyes. The photoreceptive molecule neuropsin (OPN5) was identified in mammalian and avian brains, and shown to respond to biologically relevant light wavelengths. Whether OPN5 is functionally involved in light detection is unknown. Daylength plays a critical role in regulating the neuroendocrine control of reproduction in birds. The presence of light during a 'photoinducible' phase of the circadian cycle, which occurs 12-16 h after dawn, results in marked changes in hypothalamic gene expression. These changes ultimately control gonadotropin release from the pituitary gland that, in turn, stimulates gonadal development. In this study, we first measured OPN5 expression in the mediobasal hypothalamus (MBH) in border canaries during the photoinducible period in relation to thyrotropin (TSH) β-subunit mRNA expression, which is implicated in the control of avian reproduction. Second, the knockdown of OPN5 via small interfering RNA antisense in the MBH revealed that there is an inhibitory input in the photoinduced regulation of TSHβ mRNA expression. Our data indicate that a decrease in OPN5 mRNA expression is associated with the facilitation in TSHβ mRNA expression in the MBH, a critical step for the light-induced increase in gonadal recrudescence. We hypothesise that the removal of an inhibitory input by OPN5 in birds may be a step that occurs during the photoinducible period. Given the distribution of OPN5 in the brain and periphery, this suggests a possible multifunctional role for light information in regulating other physiological processes.

Beside their genomic mode of action, estrogens also activate a variety of cellular signaling pathways through non-genomic mechanisms. Until recently, little was known regarding the functional significance of such actions in males and the mechanisms that control local estrogen concentration with a spatial and time resolution compatible with these non-genomic actions had rarely been examined. Here, we review evidence that estrogens rapidly modulate a variety of behaviors in male vertebrates. Then, we present in vitro work supporting the existence of a control mechanism of local brain estrogen synthesis by aromatase along with in vivo evidence that rapid changes in aromatase activity also occur in a region-specific manner in response to changes in the social or environmental context. Finally, we suggest that the brain estrogen provision may also play a significant role in females. Together these data bolster the hypothesis that brain-derived estrogens should be considered as neuromodulators.

D1 dopamine receptors were pharmacologically characterized and localized by quantitative autoradiography in the basal ganglia of male and female European starlings (Sturnus vulgaris). The D1 selective antagonist SCH 23390 was used to label this receptor subtype. Starlings are songbirds and possess a neural circuit implicated in the learning and production of song. This circuit includes a sexually dimorphic nucleus, area X, that is a subregion of the parolfactory lobe of the basal ganglia and is known from work on zebra finches to receive dopaminergic input from the area ventralis of Tsai. We focused our investigation on the D1-like receptor subtype because they are abundant in the basal ganglia. Competition studies indicate that a variety of dopaminergic ligands compete with [3H] SCH 23390 for the binding site in an order of potency characteristic of a D1-like receptor. Autoradiographic studies of the basal ganglia revealed high D1 receptor densities in the avian homologues of the caudate-putamen and relatively low-receptor densities were observed in the avian homologue of the globus pallidus. In male starlings, area X could be reliably discerned on the autoradiograms by the higher density of D1 receptors compared to the surrounding parolfactory lobe (LPO). This was also true for females, though nt as reliably as in males. When we compared the mean D1 receptor density in area X for males and females we did not find a significant sex difference. However, we also analyzed the data by comparing sex differences in the degree to which area X has a higher receptor density in comparison with the surrounding LPO. When we normalized D1 receptor density in area X relative to the LPO, we did find a significant sex difference. This sex difference in relative receptor density represents another neural sex difference in the song circuit that may mediate sex differences in the learning and production of song in starlings and other songbirds. 1994 John Wiley & Sons, Inc.

Abstract Neonatal estradiol injections masculinize both the singing behavior and some morphological aspects of the song control nuclei in female zebra finches (Taeniopygia guttata), but there is to date no physiological evidence that estrogens are responsible for the differentiation of song and the song control nuclei in male zebra finches during normal ontogeny. Injections of estrogen into young males demasculinizes their copulatory behavior, so that it is difficult to understand how the same endocrine environment could simultaneously masculinize song and demasculinize copulation. Attempts to block early estrogen action in males have resulted in a paradoxical hypermasculinization of several aspects of the song control nuclei. In the present experiment, we tried to evaluate the role played by estrogens during zebra finch differentiation by blocking their secretion with the non-steroidal aromatase inhibitor, R76713 or racemic vorozole. Zebra finches received at 2-3 days post-hatch one Silastic implant filled with R76713 or left empty as a control. Implants were left in place until about 45 days of age and birds were then gonadectomized. At the age of 105 days, all birds received a Silastic implant filled with T and, starting two weeks later, they were repeatedly tested for singing and copulatory behavior during a period of 3 weeks. They were then perfused and their brains were analyzed by histological methods to measure the volume of the song control nuclei, HVc, RA, area X and MAN. Treatment with R76713 significantly decreased (± 50%) the number of song bouts produced by the birds but did not affect the copulatory behavior of the males. The behavior of females was not changed by the aromatase inhibitor and, in particular, no increase in mounting behavior was seen in the treated females. The presence of a strong sex dimorphism in the size of all 4 song control nuclei was confirmed but no significant effect of R76713 on these measures could be detected. These data are therefore consistent with the idea that estrogens are implicated in the differentiation of singing behavior in the zebra finch but the small amplitude of the behavioral effect observed and the absence of morphological effect suggest that either other factors also play a major role at this level or that higher doses or longer treatments with the aromatase blocker should be tested.

Two goals of research on neural sex differences are to establish the behavioral function of such sex differences and to identify precisely what features differ between males and females. Comparative studies of sex differences in the volume of brain nuclei within the songbird vocal control circuit provide one way to address these goals. Informative comparisons can be either inter-specific or intra-specific. Inter-specific comparisons of species within the songbird suborder allow one to establish how species variation in the degree to which there is a sex difference in nuclear volume relates to species variation in the degree to which there is a sex difference in vocal behavior. Intraspecific comparisons of sex differences in nuclear volume involve the comparison of a variety of histochemical methods to define nuclei and describe a nucleus within a species. Sex differences in nuclear volume have now been measured for at least some song control nuclei in 10 different passerine species. In species with more complex male than female song, the volume of key song control nuclei is on average larger in males than in females. However, future studies will require more refined measures of vocal behavior and perceptual abilities to make more precise correlations between brain and behavior. In European starlings (Sturnus vulgaris), the volume of the vocal control nucleus, area X was found to be on average 1.95 times bigger in males than in females based on Nissl stained sections. Variation in neurotransmitter receptor density as determined by quantitative receptor autoradiography can also be used to define clearly the boundaries of a nucleus. When the boundaries of area X in male and female starlings were defined based on variation in muscarinic cholinergic and alpha 2-adrenergic receptor densities, volumetric estimates were obtained that are nearly identical to those obtained with the use of Nissl stains. Intra-specific comparisons of this sort extend our knowledge concerning the neurochemical basis of sex differences in nuclear volume. The wide application of this method would greatly increase our understanding of neural sex differences.

Specialized neural circuitry has evolved in groups of birds where vocal learning is known to occur, such as in the oscine suborder of the Passeriformes (song-birds) and in the order Psittaciformes (parrots). These specializations are most prominent in the telencephalon, while the midbrain and medullary portions of the vocal control circuit are generally similar in all orders of birds. Specializations in songbirds have at least four components: 1) a set of distinct and interconnected vocal control nuclei in the forebrain; 2) unique connections between the auditory system and these vocal control nuclei; 3) the occurrence of receptors for sex steroid hormones in a subset of the vocal control nuclei; and 4) unique patterns in the distribution of various markers of the major classes of neurotransmitters within the vocal control nuclei. In the order Psittaciformes, as exemplified by budgerigars (Melopsittacus undulatus), it appears that neural specializations generally similar to those described in songbirds have evolved independently. Although anatomical studies have found a system of interconnected forebrain regions in budgerigars that are roughly similar to those described in oscines, detailed connectivity studies of this forebrain system suggest that it is only superficially similar to that of songbirds, and budgerigars also have unique connections between the auditory and motor systems. Also, analyses of the distribution of markers of neurotransmitter function in the budgerigar brain reveal patterns different from those described in songbird vocal control systems. This work suggests that songbirds and parrots have evolved separate neural ‘solutions’ to solve the problem of vocal plasticity. There are differences between these 'solutions' but also similarities that may be the result of convergent evolution. Although vocal behavior is learned in both songbirds and parrots, it differs in many respects. By taking advantage of this 'natural' experiment one can gain insight into the hormonal and neural events that mediate these different forms of vocal plasticity.

Steroid receptor coactivators are necessary for efficient transcriptional regulation by ligand-bound nuclear receptors, including estrogen and androgen receptors. Steroid receptor coactivator-2 (SRC-2) modulates estrogen- and progesterone-dependent sexual behavior in female rats but its implication in the control of male sexual behavior has not been studied to our knowledge. We cloned and sequenced the complete quail SRC-2 transcript and showed by semi-quantitative PCR that SRC-2 expression is nearly ubiquitous, with high levels of expression in the kidney, cerebellum and diencephalon. Real-time quantitative PCR did not reveal any differences between intact males and females the medial preoptic nucleus (POM), optic lobes and cerebellum. We next investigated the physiological and behavioral role of this coactivator using in vivo antisense oligonucleotide techniques. Daily injections in the third ventricle at the level of the POM of locked nucleic acid antisense targeting SRC-2 significantly reduced the expression of testosterone-dependent male-typical copulatory behavior but no inhibition of one aspect of the appetitive sexual behavior was observed. The volume of POM, defined by aromatase-immunoreactive cells, was markedly decreased in animals treated with antisense as compared with controls. These results demonstrate that SRC-2 plays a prominent role in the control of steroid-dependent male sexual behavior and its associated neuroplasticity in Japanese quail.

Vocal control nuclei in songbirds display seasonal changes in volume that are regulated by testosterone (T) and its androgenic (5α-dihydrotestosterone; DHT) or estrogenic (17β-estradiol; E(2)) metabolites. In male canaries, T regulates expression of the microtubule-associated protein doublecortin (DCX), a marker of neurogenesis. We examined the effect of T and its two metabolites alone or in combination on DCX expression in adult female canaries. Treatment with T or with DHT+E(2) increased HVC volume and neuron numbers as well as the total numbers of fusiform (migrating) and round (differentiating) DCX neurons in the nucleus but generally not in adjacent areas. DHT or E(2) alone did not increase these measures but increased the density of fusiform DCX cells per section. Similar results were observed in area X, although some effects did not reach significance, presumably because plasticity in X is mediated transsynaptically and follows HVC changes with some delay. There was no effect of any treatment on the total number of neurons in area X, and no change in DCX cell densities was detected in the lateral magnocellular nucleus of the anterior nidopallium, nor in other parts of the nidopallium. DHT and E(2) by themselves thus increase density of DCX cells migrating through HVC but are not sufficient in isolation to induce the recruitment of these newborn neurons in the nucleus. These effects are generally not observed in the rest of the nidopallium, implying that steroids only act on the attraction and recruitment of new neurons in HVC without having any major effects on their production at the ventricle wall.

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.

Songbirds show dramatic seasonal changes in their propensity to exhibit an endocrine response to environmental stimuli that regulate the timing of reproduction. There is a clear cycle in their ability to respond to changes in photoperiod but less is known about seasonal variation in their sensitivity to supplementary factors that augment the photo-induction of gonadal growth and fine tune the final onset of breeding. Such factors include behavioral interactions with a mate and the availability of a nest site. In birds, seasonal variations in the neural processing of photoperiod are mediated by substantial modifications in the availability of hypothalamic Gonadotropin-Releasing Hormone (GnRH)

Evoked potentials recorded from slices of pigeon hippocampus were reversibly attenuated by 2 mM kynurenic acid. High frequency stimulation (3 × 200 Hz for 1 sec, with 1 sec intervals) evoked a persistent increase in the evoked potential, lasting in a nondecremental form for at least 2 hr. This increase in the magnitude of the potential was not blocked by antagonists of the NMDA receptor (APV and MK-801). These data suggest that the synaptic facilitation observed in the pigeon hippocampus, which appears to be a form of long-term potentiation, does not depend on the activation of NMDA receptors. © 1993 Wiley-Liss, Inc.

The brain of adult homeothermic vertebrates exhibits a higher degree of morphological neuroplasticity than previously thought, and this plasticity is especially prominent in birds. In particular, incorporation of new neurons is widespread throughout the adult avian forebrain, and the volumes of specific nuclei vary seasonally in a prominent manner. We review here work on steroid-dependent plasticity in birds, based on two cases: the medial preoptic nucleus (POM) of Japanese quail in relation to male sexual behavior, and nucleus HVC in canaries, which regulates song behavior. In male quail, POM volume changes seasonally, and in castrated subjects testosterone almost doubles POM volume within 2 weeks. Significant volume increases are, however, already observable after 1 day. Steroid receptor coactivator-1 is part of the mechanism mediating these effects. Increases in POM volume reflect changes in cell size or spacing and dendritic branching, but are not associated with an increase in neuron number. In contrast, seasonal changes in HVC volume reflect incorporation of newborn neurons in addition to changes in cell size and spacing. These are induced by treatments with exogenous testosterone or its metabolites. Expression of doublecortin, a microtubule-associated protein, is increased by testosterone in the HVC but not in the adjacent nidopallium, suggesting that neuron production in the subventricular zone, the birthplace of newborn neurons, is not affected. Together, these data illustrate the high degree of plasticity that extends into adulthood and is characteristic of avian brain structures. Many questions still remain concerning the regulation and specific function of this plasticity.

In order to help elucidate how general the role of dopamine (DA) release in the medial preoptic area (mPOA) is for the activation of male sexual behavior in vertebrates, we recently developed an in vivo microdialysis procedure in the mPOA of Japanese quail. Using these techniques in the present experiment, the temporal pattern of DA release in relation to the pre-copulatory exposure to a female and to the expression of both appetitive and consummatory aspects of male sexual behavior was investigated. Extracellular samples from the mPOA of adult sexually-experienced male quail were collected every six minutes before, while viewing, while in physical contact with, and after exposure to a female. In the absence of a pre-copulatory rise in DA, males failed to copulate when the barrier separating them from the female was removed. In contrast, males that showed a substantial increase in mPOA DA during pre-copulatory interactions behind the barrier, copulated with females after its removal. However, there was no difference in DA during periods when the quail were copulating as compared to when the female was present but the males were not copulating. In addition, we show that pre-copulatory DA predicts future DA levels and copulatory behavior frequency. Furthermore, the size of the cloacal gland, an accurate indicator of testosterone action, is positively correlated with pre-copulatory DA. Taken together, these results provide further support for the hypothesis that DA action in the mPOA is specifically linked to sexual motivation as compared to copulatory behavior per se.

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.

In the years following the discovery of the song system, it was realized that this specialized circuit controlling learned vocalizations in songbirds (a) constitutes a specific target for sex steroid hormone action and expresses androgen and (for some nuclei) estrogen receptors, (b) exhibits a chemical neuroanatomical pattern consisting in a differential expression of various neuropeptides and neurotransmitters receptors as compared to surrounding structures and (c) shows pronounced seasonal variations in volume and physiology based, at least in the case of HVC, on a seasonal change in neuron recruitment and survival. During the past 30 years numerous studies have investigated how seasonal changes, transduced largely but not exclusively through changes in sex steroid concentrations, affect singing frequency and quality by modulating the structure and activity of the song control circuit. These studies showed that testosterone or its metabolite estradiol, control seasonal variation in singing quality by a direct action on song control nuclei. These studies also gave rise to the hypothesis that the probability of song production in response to a given stimulus (i.e. its motivation) is controlled through effects on the medial preoptic area and on catecholaminergic cell groups that project to song control nuclei. Selective pharmacological manipulations confirmed that the noradrenergic system indeed plays a role in the control of singing behavior. More experimental work is, however, needed to identify specific genes related to neurotransmission that are regulated by steroids in functionally defined brain areas to enhance different aspects of song behavior.

In birds as in other vertebrates, estrogens produced in the brain by aromatization of testosterone have widespread effects on behavior. Research conducted with male Japanese quail demonstrates that effects of brain estrogens on all aspects of sexual behavior, including appetitive and consummatory components as well as learned aspects, can be divided into two main classes based on their time course. First, estrogens via binding to estrogen receptors regulate the transcription of a variety of genes involved primarily in neurotransmission. These neurochemical effects ultimately result in the activation of male copulatory behavior after a latency of a few days. Correlatively, testosterone and its aromatized metabolites increase the transcription of the aromatase mRNA, resulting in an increased concentration and activity of the enzyme that actually precedes behavioral activation. Second, recent studies with quail demonstrate that brain aromatase activity can also be modulated within minutes by phosphorylation processes regulated by changes in intracellular calcium concentration, such as those associated with glutamatergic neurotransmission. The rapid upregulations or downregulations of brain estrogen concentration (presumably resulting from these changes in aromatase activity) affect, by nongenomic mechanisms with relatively short latencies (frequency increases or decreases respectively within 10-15 min), the expression of male sexual behavior in quail and also in rodents. Brain estrogens thus affect behavior on different time scales by genomic and nongenomic mechanisms similar to those of a hormone or a neurotransmitter.

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.

The question of how sex differences in behavior among vertebrates emerge and are expressed has been the topic of intense study for over 50 years. Convergent evidence from birds and mammals, primarily rodents, has provided certain common principles while highlighting other species-specific properties. The importance of early hormonal effects on the developing brain to adult behavioral profile is pervasive throughout the vertebrate phyla and assures that brain sex phenotype will match gonadal phenotype. Variation in the magnitude of differences between males and females in sexual behavior, parenting and aggression are influenced by environmental and physiological parameters. Recent advances in the cellular and molecular mechanisms of steroid hormones in both organizing and activating neural circuits to control behavior reveal a wide variety of effector pathways and emphasize how much we have to learn.

Evidence has accumulated that the regulation of male sexual behavior by dopamine may not be the same in Japanese quail (and perhaps all birds) as it is in mammals. For example, the non-selective dopamine receptor agonist, apomorphine (APO) facilitates male sexual behavior in rats but inhibits it in quail. Although the general organization of the dopamine system is similar in birds and mammals, it is possible that the relative distribution and/or density of binding sites is different. We therefore compared the relative densities of D1-like and D2-like receptor subtypes in Japanese quail and rats, with the use of in vitro quantitative receptor autoradiography. Brain sections from 8 male rats and 8 male quail were labeled with [3H]SCH-23390 and [3H]Spiperone. In general we found a systematic species difference in the relative density of D1-like vs. D2-like receptors such that the D2/D1 ratio is higher in quail than in rats in areas well known to be important target sites for dopamine action such as striatal regions. We also uncovered significant differences in the relative density of D1-like and D2-like receptors in brain areas associated with sexual behavior, including the preoptic area, such that there was a greater D2/D1 ratio in quail as compared to rats. This difference may explain the variation in the behavioral effectiveness of APO in rats as compared to quail; with a higher relative density of D2-like receptors in quail, a similar dose of APO would be more likely to activate inhibitory processes in quail than in rats.

This chapter reviews the neural and endocrine bases of reproductive behaviors in birds with a particular focus on the role played by gonadal sex steroid hormones in the regulation of sexual behaviors. Studies of birds continue to be valuable for the elucidation of the neuroendocrine control of behavior. The avian brain also exhibits a remarkable degree of hormone-regulated neuroplasticity in associated neural circuits. Work on the neuroendocrine control of avian male sexual behavior has identified the key role played by estrogenic as well as androgenic metabolites of testosterone acting in the preoptic region to facilitate behavioral activation. Estrogenic metabolites of testosterone have relatively slow actions (i.e., days to weeks) that are often genomic in nature as well as relatively fast effects (e.g., minutes) that result from actions at the cell membrane. These different modes of action are associated with different temporal modes of the regulation of the enzyme aromatase (that converts testosterone to estradiol) in the brain. Female sexual behavior is activated by ovarian estrogens acting in the ventromedial nucleus of the hypothalamus. Parental care is hormonally primed in females by synergistic actions of sex steroids and prolactin while males tend to respond to cues provided by females.

Communication involves interactions between a sender and a receiver with the use of specialized signals. Different classes of hormones, such as steroid hormones, have profound effects on the probability that a signal will be produced via modulation of sensory inputs, central pattern generators, and effector organs. The perception of signals can result in marked changes in the endocrine physiology of the receiver, influencing whether the receiver will engage in subsequent appropriate behaviors. Hormone receptors have been localized to peripheral tissues and brain areas that mediate signal production and hormone-regulated gene expression has been identified in these areas related to signal production and processing.

Birdsong is a complex vocal signal that functions in a reproductive context to attract mates and defend territories. It is of interest to neuroscientists because it is a complex motor behavior that is learned via a developmental process and involves the precise guiding of motor patterns by auditory feedback. Song learning includes a sensory phase, when an auditory memory is formed, and this memory is used as a template for the later sensorimotor phase of development. There is substantial species variation in this learning process that may have been shaped by natural selection to optimize behavioral complexity and plasticity as needed to enhance reproductive success. Steroid hormones such as testosterone and its metabolite estradiol may promote song memory formation, and androgens close the sensorimotor phase and induce the crystallization of adult species-typical song.

To assess whether alterations in the normal pattern of testosterone (T) secretion might be beneficial or detrimental, we studied a breeding population of darkeyed juncos in which we elevated T experimentally and measured its effect on potential correlates of fitness. We treated both free-living and captive males with implants that were either empty (C-males, controls) or packed with T (T-males, experimentals). Timing of implant varied and was designed to mimic natural peak breeding levels except that peaks were either prolonged or premature. We bled the birds at recapture and analyzed their plasma, and that of their female mates, for T and corticosterone (B). We also measured body mass and fat score in free-living T- and C-males. In the field, T-implants elevated T and kept it elevated for at least a month. Experimental males also had higher B than controls. In captives, the effect of the implants on plasma T was detectable within 24 hr. B in captive T-males was again higher than in captive C-males. In females, neither T nor B differed between mates of T- and C-males. T-males implanted in early spring lost more mass between implant and recapture in late spring than did controls and also had lower fat scores when recaptured. When implants were inserted in summer, treatment did not influence mass. Elevated T in early spring apparently hastened the transition from the winter to the breeding mode of fat storage. We suggest that prolonged elevation of testosterone might be selected against because of the association between T and B. Premature elevation of T might be costly because of the resultant loss of mass and fat reserves, which could lead to mortality when spring snowstorms prevent access to food.

Electrophysiological recordings from the hippocampus were taken using brain slices from 1-year-old, female, song sparrows (Melospiza melodia). The evoked responses were smaller and less stable as compared with those obtained from the mammalian hippocampus. They consisted of two spikes. The first spike had low calcium dependency and represented mainly fiber potential. The second spike demonstrated a clear calcium dependency proving its synaptic origin. Paired-pulse facilitation showed inhibition of the second response at latencies below 20 ms, facilitation at 30 ms and no changes above 30 ms. Train stimulation with 20 Hz (each train consisted of 1 s stimulation with 10-s intervals repeated 3 times) evoked a stable increase in the size of the evoked response lasting approximately 2 h. These data indicate that the avian hippocampus possesses several neurophysiological properties that typify the mammalian hippocampus including the long-term enhancement of the synaptic response following certain patterns of stimulation.

Estradiol facilitates the expression of male sexual behavior in Japanese quail within a few minutes. These rapid behavioral effects of estradiol could result from rapid changes in its local production in the preoptic area by aromatase, the enzyme converting testosterone into estradiol. Alternatively, aromatase activity may remain constant but fluctuations of local estradiol production could arise from rapid changes in the concentration of the enzymatic substrate, namely testosterone. Rapid increases of circulating testosterone levels have been observed in males of various species following social encounters. Surprisingly, in quail, the interaction with a female seems to result in a decrease in circulating testosterone levels. However, in that study conducted in quail, the samples were collected at intervals longer than the recently observed rapid effects of estradiol on sexual behavior. In the present study we investigated whether plasma testosterone concentrations fluctuate on a shorter time-frame. Eleven male were tested 5 min before and 5, 15 or 30 min after being allowed to have visual access to a female or to copulate with a female for 5 min. Both types of interactions resulted in a significant decline in circulating testosterone levels at latencies as short as 5 min. These data demonstrate that the decrease in testosterone levels is initiated shortly after sexual encounters. Because visual interactions with a female did not result in a rapid increase in testosterone concentrations, these findings rule out the possibility that a rapid rise in circulating testosterone levels participates in the rapid increase in brain estrogen synthesis and its facilitatory effects on copulatory behavior.

Dopaminergic and adrenergic receptors are G-protein-coupled receptors considered to be different based on their pharmacology and signaling pathways. Some receptor subtypes that are members of one family are actually closer in phylogenetic terms to some subtypes belonging to the other family, suggesting that the pharmacological specificity among these receptors from different families is not perfect. Indeed, evidence is accumulating that one amine can cross-talk with receptors belonging to the other system. However, most of these observations were collected in vitro using artificial cell models transfected with cloned receptors, so that the occurrence of this phenomenon in vivo as well as its distribution in the central nervous system is not known. In this study the pharmacological basis of possible in vivo interactions between dopamine and alpha(2)-adrenergic receptors was investigated in quail, zebra finches, and rats. Binding competitions showed that dopamine displaces the binding of the selective alpha(2)-adrenergic ligand, [(3)H]RX821002, in the brain of the three species with an affinity approximately 10-28-fold lower than that of norepinephrine. Dopamine also displaces with an affinity 3-fold lower than norepinephrine the binding of [(3)H]RX821002 to human alpha(h2A)-adrenergic receptors expressed in Sf9 cells. The anatomical distribution of this interaction was assessed in brain slices of quail and rat based on autoradiographic methods. Both norepinephrine and dopamine significantly displace [(3)H]RX821002 binding in all brain nuclei considered. Together, these data provide evidence for an interaction between the dopaminergic and noradrenergic systems in the vertebrate brain, albeit with species variations.

Sex differences in song behavior and in the neural system controlling song in songbirds are well documented but relatively little is known about sex differences in hearing. We recently demonstrated the existence of sex differences in auditory brainstem responses in a songbird species, the zebra finch (Taeniopygia guttata). Many sex differences are regulated by sex steroid hormone action either during ontogeny or in adulthood. As a first step to test the possible implication of sex steroids in the control of sex differences in the zebra finch auditory system, we evaluated via immunocytochemistry whether estrogens are produced and act in the zebra finch inner ear. Specifically we examined the distribution of aromatase, the enzyme converting testosterone into an estrogen, and of estrogen receptors of the alpha subtype (ERalpha) in adult zebra finch inner ears. The anatomy of the basilar papillae was visualized by fluorescein-phalloidin, which delineated the actin structure of hair cells and supporting cells at their apical surface. Whole mount preparations of basilar papillae stained by immunocytochemistry revealed in both males and females an abundant aromatase distribution in the cytoplasm of hair cells, while ERalpha was identified in the nuclei of hair cells and of underlying supporting cells. Double-labeled preparations confirmed the extensive co-localization of aromatase and ERalpha in the vast majority of the hair cells. These results are consistent with studies on non-avian species, suggesting a role for estrogens in auditory function. These findings are also consistent with the notion that estrogens may contribute to a sex difference in hearing. To our knowledge, this is the first demonstration of the presence of aromatase and of the co-localization of aromatase and ERalpha in the sensory epithelium of the inner ear in any animal model.

Background Songbirds hold great promise for biomedical, environmental and evolutionary research. A complete draft sequence of the zebra finch genome is imminent, yet a need remains for application of genomic resources within a research community traditionally focused on ethology and neurobiological methods. In response, we developed a core set of genomic tools and a novel collaborative strategy to probe gene expression in diverse songbird species and natural contexts. Results We end-sequenced cDNAs from zebra finch brain and incorporated additional sequences from community sources into a database of 86,784 high quality reads. These assembled into 31,658 non-redundant contigs and singletons, which we annotated via BLAST search of chicken and human databases. The results are publicly available in the ESTIMA:Songbird database. We produced a spotted cDNA microarray with 20,160 addresses representing 17,214 non-redundant products of an estimated 11,500–15,000 genes, validating it by analysis of immediate-early gene (zenk) gene activation following song exposure and by demonstrating effective cross hybridization to genomic DNAs of other songbird species in the Passerida Parvorder. Our assembly was also used in the design of the "Lund-zfa" Affymetrix array representing ~22,000 non-redundant sequences. When the two arrays were hybridized to cDNAs from the same set of male and female zebra finch brain samples, both arrays detected a common set of regulated transcripts with a Pearson correlation coefficient of 0.895. To stimulate use of these resources by the songbird research community and to maintain consistent technical standards, we devised a "Community Collaboration" mechanism whereby individual birdsong researchers develop experiments and provide tissues, but a single individual in the community is responsible for all RNA extractions, labelling and microarray hybridizations. Conclusion Immediately, these results set the foundation for a coordinated set of 25 planned experiments by 16 research groups probing fundamental links between genome, brain, evolution and behavior in songbirds. Energetic application of genomic resources to research using songbirds should help illuminate how complex neural and behavioral traits emerge and evolve.

International audience; In rats, expression of the immediate early gene, c-fos observed in the brain following male copulatory behavior relates mostly to the detection of olfactory information originating from the female and to somatosensory feedback from the penis. However, quail, like most birds, are generally considered to have a relatively poorly developed sense of smell. Furthermore, quail have no intromittent organ (e.g., penis). It is therefore intriguing that expression of male copulatory behavior induces in quail and rats a similar pattern of c-fos expression in the medial preoptic area (mPOA), bed nucleus of the stria terminalis (BSTM) and parts of the amygdala. We analyzed here by immunocytochemistry Fos expression in the mPOA/BSTM/amygdala of male quail that had been allowed to copulate with a female during standardized tests. Before these tests, some of the males had either their nostrils plugged, or their cloacal area anesthetized, or both. A control group was not exposed to females. These manipulations did not affect frequencies of male sexual behavior and all birds exposed to a female copulated normally. In the mPOA, the increased Fos expression induced by copulation was not affected by the cloacal gland anesthesia but was markedly reduced in subjects deprived of olfactory input. Both manipulations affected copulation-induced Fos expression in the BSTM. No change in Fos expression was observed in the amygdala. Thus immediate early gene expression in the mPOA and BSTM of quail is modulated at least in part by olfactory cues and/or somatosensory stimuli originating from the cloacal gland. Future work should specify the nature of these stimuli and their function in the expression of avian male sexual behavior. (C) 2008 Elsevier B.V. All rights reserved.

The question of how sex differences in behavior among vertebrates emerge and are expressed has been the topic of intense study for over 50 years. Convergent evidence from birds and mammals, primarily rodents, has provided certain common principles while highlighting other species-specific properties. The importance of early hormonal effects on the developing brain to adult behavioral profile is pervasive throughout the vertebrate phyla and assures that brain sex phenotype will match gonadal phenotype. Variation in the magnitude of differences between males and females in sexual behavior, parenting and aggression are influenced by environmental and physiological parameters. Recent advances in the cellular and molecular mechanisms of steroid hormones in both organizing and activating neural circuits to control behavior reveal a wide variety of effector pathways and emphasize how much we have to learn.

Investigations of the cellular and molecular mechanisms of physiology and behaviour have generally avoided attempts to explain individual differences. The goal has rather been to discover general processes. However, understanding the causes of individual variation in many phenomena of interest to avian eco-physiologists will require a consideration of such mechanisms. For example, in birds, changes in plasma concentrations of steroid hormones are important in the activation of social behaviours related to reproduction and aggression. Attempts to explain individual variation in these behaviours as a function of variation in plasma hormone concentrations have generally failed. Cellular variables related to the effectiveness of steroid hormone have been useful in some cases. Steroid hormone target sensitivity can be affected by variables such as metabolizing enzyme activity, hormone receptor expression as well as receptor cofactor expression. At present, no general theory has emerged that might provide a clear guidance when trying to explain individual variability in birds or in any other group of vertebrates. One strategy is to learn from studies of large units of intraspecific variation such as population or sex differences to provide ideas about variables that might be important in explaining individual variation. This approach along with the use of newly developed molecular genetic tools represents a promising avenue for avian eco-physiologists to pursue.

In temperate zone songbirds, such as canaries (Serinus canaria), seasonal variation in gonadal activity and behavior are associated with marked brain changes. These include gonadotropin-releasing hormone (GnRH) expression and the volume of brain areas controlling song production. Questions have been raised about the consistency of seasonal brain changes in canaries. Laboratory studies of the American singer strain raised doubts as to whether this strain exhibits a robust photoperiodic response along with changes in brain GnRH content, and studies of free-living canaries have failed to identify seasonal changes in volume of song control nuclei. We assessed differences in brain GnRH and the song control system associated with photoperiod-induced variation in reproductive state in Border canaries. We found that males and females maintained for 10 weeks on long days (14L:10D) regress their gonads, exhibit a decline in testosterone and initiate molt; a response consistent with the onset of absolute photorefractoriness (i.e., failed to respond to previously stimulating daylengths). All birds regained photosensitivity (i.e., exhibited gonadal response to stimulating daylengths) after experiencing short days (8L:16D) for 6 weeks. Furthermore, comparisons of birds in either a photosensitive, photostimulated, or photorefractory state revealed a marked increase in GnRH protein expression in the photosensitive and photostimulated birds over photorefractory birds. A similar variation was observed in the volume of key forebrain song nuclei. Thus, Border canaries demonstrate measurable neuroplasticity in response to photoperiodic manipulations. These data, along with previous work on other strains of canaries, indicate the presence of intra-specific variation in photoperiodically regulated neuroplasticity.

Although it is axiomatic that males and females differ in relation to many aspects of reproduction related to physiology, morphology and behaviour, relatively little is known about possible sex differences in the response to cues from the environment that control the timing of seasonal breeding. This review concerns the environmental regulation of seasonal reproduction in birds and how this process might differ between males and females. From an evolutionary perspective, the sexes can be expected to differ in the cues they use to time reproduction. Female reproductive fitness typically varies more as a function of fecundity selection, while male reproductive fitness varies more as a function sexual selection. Consequently, variation in the precision of the timing of egg laying is likely to have more serious fitness consequences for females than for males, while variation in the timing of recrudescence of the male testes and accompanying territory establishment and courtship are likely to have more serious fitness consequences for males. From the proximate perspective, sex differences in the control of reproduction could be regulated via the response to photoperiod or in the relative importance and action of supplementary factors (such as temperature, food supply, nesting sites and behavioural interactions) that adjust the timing of reproduction so that it is in step with local conditions. For example, there is clear evidence in several temperate zone avian species that females require both supplementary factors and long photoperiods in order for follicles to develop, while males can attain full gonadal size based on photoperiodic stimulation alone. The neuroendocrine basis of these sex differences is not well understood, though there are many candidate mechanisms in the brain as well as throughout the entire hypothalamo–pituitary–gonadal axis that might be important.

There is considerable functional evidence implicating norepinephrine in modulating activity in the vocal control circuit of songbirds. However, our knowledge of noradrenergic inputs to the song system is incomplete. In this study, cholera toxin subunit B (CTB) injections into area X revealed projections from the noradrenergic nuclei locus coeruleus and subcoeruleus, and injections of biotinylated dextran amines into these noradrenergic nuclei labeled fibers in area X. The nonreciprocity of this connection was demonstrated by the absence of retrogradely labeled cells in area X following injections of CTB into the locus coeruleus. Additionally, we found novel inputs to area X from the nidopallium and arcopallium, the mesencephalic central gray, and the dorsolateralis anterior (DLL) and posterior (DLP) lateralis in the thalamus. Area X can be clearly distinguished from the surrounding medial striatum based on cytoarchitectural and chemical neuroanatomical criteria. We show here that neuromodulatory inputs to area X however, exhibit a considerable degree of overlap with the surrounding area. This finding suggests that regional specificity in neuromodulator action is most likely afforded by a specialization in receptor density and enzyme distribution rather than projections from the synthesizing nuclei. Our results extend current knowledge about noradrenergic projections to specialized nuclei of the song control circuit and provide neuroanatomical evidence for the functional action of norepinephrine-modulating context-dependent ZENK expression in area X. Furthermore, the novel projections to area X from telencephalic and thalamic areas could be new and interesting nodes in the striatopallidothalamic loop spanning the songbird brain.

Several studies have suggested dissociations between neural circuits underlying the expression of appetitive (e.g., courtship behavior) and consummatory components (i.e., copulatory behavior) of vertebrate male sexual behavior. The medial preoptic area (mPOA) clearly controls the expression of male copulation but, according to a number of experiments, is not necessarily implicated in the expression of appetitive sexual behavior. In rats for example, lesions to the mPOA eliminate male-typical copulatory behavior but have more subtle or no obvious effects on measures of sexual motivation. Rats with such lesions still pursue and attempt to mount females. They also acquire and perform learned instrumental responses to gain access to females. However, recent lesions studies and measures of the expression of the immediate early gene c-fos demonstrate that, in quail, sub-regions of the mPOA, in particular of its sexually dimorphic component the medial preoptic nucleus, can be specifically linked with either the expression of appetitive or consummatory sexual behavior. In particular more rostral regions can be linked to appetitive components while more caudal regions are involved in consummatory behavior. This functional sub-region variation is associated with neurochemical and hodological specializations (i.e., differences in chemical phenotype of the cells or in their connectivity), especially those related to the actions of androgens in relation to the activation of male sexual behavior, that are also present in rodents and other species. It could thus reflect general principles about POA organization and function in the vertebrate brain.

In many vertebrate species the medial preoptic area projects to a premotor nucleus, the periaqueductal central gray (PAG). This connection plays an important role in the control of reproductive behavior. In male Japanese quail (Coturnix japonica) specifically, the medial preoptic nucleus (POM), where various types of sensory inputs converge, is a critical site for the activational action of testosterone on male sexual behavior. To activate male copulatory behavior, testosterone must be aromatized to estradiol within the POM and aromatase-immunoreactive cells in the POM are the main source of projections to the PAG. The POM–PAG connection is thus an important functional circuit integrating the sensory with premotor components of sexual behavior. Contrary to what is observed in males, testosterone does not activate male-typical copulatory behavior in females and we investigated here via retrograde tracing methods whether this behavioral sexual difference is associated with a sex difference in connectivity between POM and PAG. Fluorescent microspheres were injected in the PAG of male and female quail and retrogradely labeled fluorescent cells counted in four fields of the POM in sections that had been immunolabeled for aromatase. Males had more aromatase-immunoreactive neurons projecting to the PAG than females and this difference was most prominent in the caudolateral part of the nucleus that has been specifically implicated in the control of male copulatory behavior. These data therefore support the hypothesis that sex differences in POM–PAG connectivity are causally linked to the sex difference in the behavioral response to testosterone. J. Comp. Neurol. 500

In birds and mammals, aromatase activity in the preoptic-hypothalamic region (HPOA) is usually higher in males than in females. It is, however, not known whether the enzymatic sex difference reflects the differential activation of aromatase transcription or some other control mechanism. Although sex differences in aromatase activity are clearly documented in the HPOA of Japanese quail (Coturnix japonica), only minimal or even no differences at all were observed in the number of aromatase-immunoreactive (ARO-ir) cells in the medial preoptic nucleus (POM) and in the medial part of the bed nucleus striae terminalis (BSTM). We investigated by in situ hybridization the distribution and possible sex differences in aromatase mRNA expression in the brain of sexually active adult quail. The distribution of aromatase mRNA matched very closely the results of previous immunocytochemical studies with the densest signal being observed in the POM, BSTM and in the mediobasal hypothalamus (MBH). Additional weaker signals were detected in the rostral forebrain, arcopallium and mesencephalic regions. No sex difference in the optical density of the hybridization signal could be found in the POM and MBH but the area covered by mRNA was larger in males than in females, indicating a higher overall expression in males. In contrast, in the BSTM, similar areas were covered by the aromatase expression in both sexes but the density of the signal was higher in females than in males. The physiological control of aromatase is thus neuroanatomically specific and with regard to sex differences, these controls are at least partially different if one compares the level of transcription, translation and activity of the enzyme. These results also indirectly suggest that the sex difference in aromatase enzyme activity that is present in the quail HPOA largely results from differentiated controls of enzymatic activity rather than differences in enzyme concentration.

The protein doublecortin (DCX) is expressed in post-mitotic migrating and differentiating neurons in the developing vertebrate brain and, as a part of the microtubule machinery, is required for neuronal migration. DCX expression is generally maximal during embryonic and early post-natal life but decreases markedly and almost disappears in older animals in parallel with the major decrease or cessation of neurogenesis. In several seasonally breeding songbird species such as canaries, the volume of several song control nuclei in the brain varies seasonally such that the largest nuclei are observed in the late spring and early summer. This variation is based on changes in cell size, dendritic branching, and, in nucleus HVC, on the incorporation of neurons newly born in adulthood. Because songbirds maintain an active neurogenesis and neuronal incorporation in their telencephalon throughout their lives, we investigated here the distribution of DCX-immunoreactive (ir) structures in the brain of adult male canaries. Densely stained DCX-ir cells were found exclusively in parts of the telencephalon that are known to incorporate new neurons in adulthood, in particular the nidopallium. Within this brain region, the boundaries of the song control nucleus HVC could be clearly distinguished from surrounding structures by a higher density of DCX-ir structures. In most telencephalic areas, about two thirds of these cells displayed a uni- or bipolar fusiform morphology suggesting they were migrating neurons. The rest of the DCX-ir cells in the telencephalon were larger and had a round multipolar morphology. No such staining was found in the rest of the brain. The broad expression of DCX specifically in adult brain structures that exhibit the characteristic of active incorporation of new neurons suggests that DCX plays a key role in the migration of new neurons in the brain of adult songbirds as it presumably does during ontogeny.

The contribution of social factors to seasonal plasticity in singing behavior and forebrain nuclei controlling song, and their interplay with gonadal steroid hormones are still poorly understood. In many songbird species, testosterone (T) enhances singing behavior but elevated plasma T concentrations are not absolutely required for singing to occur. Singing is generally produced either to defend a territory or to attract a mate and it is therefore not surprising that singing rate can be influenced by the sex and behavior of the social partner. We investigated, based on two independent experiments, the effect of the presence of a male or female partner on the rate of song produced by male canaries. In the first experiment, song rate was measured in dyads composed of one male and one female (M-F) or two males (M-M). Birds were implanted with T-filled Silastic capsules or with empty capsules as control. The number of complete song bouts produced by all males was recorded during 240 min on week 1, 2, 4, and 8 after implantation. On the day following each recording session, brains from approximately one-fourth of the birds were collected and the volumes of the song control nuclei HVC and RA were measured. T increased the singing rate and volume of HVC and RA but these effects were affected by the social context. Singing rates were higher in the M-M than in the M-F dyads. Also, in the M-M dyads a dominance-subordination relationship soon became established and dominant males sang at higher rates than subordinates in T-treated but not in control pairs. The differences in song production were not reflected in the size of the song control nuclei: HVC was larger in M-F than in M-M males and within the M-M dyads, no difference in HVC or RA size could be detected between dominant and subordinate males. At the individual level, the song rate with was positively correlated with RA and to a lower degree HVC volume, but this relationship was observed only in M-M dyads, specifically in dominant males. A second experiment, carried out with castrated males that were all treated with T and exposed either to another T-treated castrate or to an estradiol-implanted female, confirmed that song rate was higher in the M-M than in the M-F condition and that HVC volume was larger in heterosexual than in same-sex dyads. The effects of T on singing rate and on the volume of the song control nuclei are thus modulated by the social environment, including the presence/absence of a potential mate and dominance status among males. 2006 Wiley Periodicals, Inc. J Neurobiol, 2006

Steroid receptors such as the androgen and estrogen receptors require the presence of several proteins, known as coactivators, to enhance the transcription of target genes. The first goal of the present study was to define the role of SRC-1 on the steroid-dependent expression of the aromatase protein and its activity in male Japanese quail. The second goal was to analyze the rapid plasticity of the POM following antisense treatment interruption. We confirm here that the inhibition of SRC-1 expression by daily intracerebroventricular injections of locked nucleic acid antisense oligonucleotides in the third ventricle at the level of the preoptic area-hypothalamus (HPOA) significantly reduces testosterone-dependent male sexual behavior. In the first experiment, aromatase protein expression in HPOA was inhibited in SRC-1-depleted males but the enzymatic activity remained at the level measured in controls. We observed in the second experiment a recovery of the behavioral response to testosterone treatment after interruption of the antisense injection. However, several morphological characteristics of the POM were not different between the control group, the antisense-treated birds and antisense-treated birds in which treatment had been discontinued 3 days earlier. Antisense was also less effective in knocking-down SRC-1 in the present experiments as compared to our previous study. An analysis of this variation in the degree of knock-down of SRC-1 expression suggests dissociation among different aspects of steroid action on brain and behavior presumably resulting from the differential sensitivity of behavioral and neurochemical responses to the activation by testosterone and/or its estrogenic metabolites.