C. Viglietti-Panzica

A sexually dimorphic nucleus in the quail preoptic area

The cytoarchitectural analysis of the preoptic-anterior hypothalamic region of the Japanese quail reveals a sexual dimorphism in the total volume of the medial preoptic nucleus (significantly larger in males than in females). Different nuclei of the region (dorsal preopticus, suprachiasmaticus) do not show any statistically significant difference. The sex-related difference is more consistent comparing the distribution of dark volume. This last is due to a larger number of cells containing high amount of Nissls substance in male than in female. Present findings represent the first example of sexual dimorphism in the avian hypothalamus.

Sexual differentiation and hormonal control of the sexually dimorphic medial preoptic nucleus in the quail.

We recently identified a sexually dimorphic nucleus in the preoptic region of the Japanese quail, the medial preoptic nucleus (POM), which is significantly larger in males than in females. In the present study, we investigated the hormonal control of this morphological neuroanatomical difference and the possible relationships between the sexual dimorphism in POM volume and in copulatory behavior. Treatments which are known to affect sexual behavior were thus applied to different groups of birds and the POM volume was then measured. In one experiment, male and female quails were either gonadectomized, gonadectomized and treated with testosterone or left intact. The larger size of the POM in males was confirmed and treatments significantly affected the nucleus size which was decreased by gonadectomy and restored by testosterone treatment in both sexes to a level similar to that seen in intact males. In two other experiments, eggs were injected with estradiol benzoate on day 9 of incubation and the POM volume was measured in adulthood either in intact birds or in gonadectomized birds receiving a replacement therapy with testosterone. Despite the fact that estradiol benzoate treatment completely suppressed copulatory behavior, it did not affect the volume of the POM or slightly increased it. These data thus show that the POM volume is controlled by testosterone levels in adulthood and could thus be an interesting model for the study of the effects of steroids on the brain.

Anatomical and neurochemical definition of the nucleus of the stria terminalis in Japanese quail (Coturnix japonica).

This study in birds provides anatomical, immunohistochemical, and hodological data on a prosencephalic region in which the nomenclature is still a matter of discussion. In quail, this region is located just dorsal to the anterior commissure and extends from the level of the medial part of the preoptic area at its most rostral end to the caudal aspects of the nucleus preopticus medialis. At this caudal level, it reaches its maximal elongation and extends from the ventral tip of the lateral ventricles to the dorsolateral aspects of the paraventricular nucleus. This area contains aromatase‐immunoreactive cells and a sexually dimorphic population of small, vasotocinergic neurons. The Nissl staining of adjacent sections revealed the presence of a cluster of intensely stained cells outlining the same region delineated by the vasotocin‐immunoreactive structures. Cytoarchitectonic, immunohistochemical, and in situ hybridization data support the notion that this area is similar and is probably homologous to the medial part of the nucleus of the stria terminalis of the mammalian brain. The present data provide a clear definition of this nucleus in quail: They show for the first time the presence of sexually dimorphic vasotocinergic neurons in this region of the quail brain and provide the first detailed description of this region in an avian species. J. Comp. Neurol. 396:141–157, 1998.

Afferent and Efferent Connections of the Sexually Dimorphic Medial Preoptic Nucleus of the Male Quail Revealed by in Vitro Transport of Dii

The medial preoptic nucleus of the Japanese quail is a testosterone-sensitive structure that is involved in the control of male copulatory behavior. The full understanding of the role played by this nucleus in the control of reproduction requires the identification of its afferent and efferent connections. In order to identify neural circuits involved in the control of the medial preoptic nucleus, we used the lipophilic fluorescent tracer DiI implanted in aldheyde-fixed tissue. Different strategies of brain dissection and different implantation sites were used to establish and confirm afferent and efferent connections of the nucleus. Anterograde projections reached the tuberal hypothalamus, the area ventralis of Tsai, and the substantia grisea centralis. Dense networks of fluorescent fibers were also seen in several hypothalamic nuclei, such as the anterior medialis hypothalami, the paraventricularis magnocellularis, and the ventromedialis hypothalami. A major projection in the dorsal direction was also observed from the medial preoptic nucleus toward the nucleus septalis lateralis and medialis. Afferents to the nucleus were seen from all these regions. Implantation of DiI into the substantia grisea centralis also revealed massive bidirectional connections with a large number of more caudal mesencephalic and pontine structures. The substantia grisea centralis therefore appears to be an important center connecting anterior levels of the brain to brain-stem nuclei that may be involved in the control of male copulatory behavior.

Vasotocinergic innervation of sexually dimorphic medial preoptic nucleus of the male Japanese quail: influence of testosterone

The distribution of vasotocin (VT)-immunoreactive (IR) fibers was described in the preoptic and septal regions of the male quail brain. The density of VT-IR fibers was measured in the sexually dimorphic preoptic nucleus (POM) and lateral septum (SL) of adult male quail (Coturnix japonica) by means of quantitative image analysis. Experimental manipulations of the hormonal environment in the peripubertal period influenced this distribution. In both regions, the VT immunoreactivity was reduced or absent when males were castrated. The immunoreactivity was restored to its original level in castrated males by Silastic implants of testosterone. These changes were anatomically specific as evidenced by the fact that the density of VT fibers did not vary in the hypothalamo-neurohypohysial tract as a function of the endocrine condition of the subjects. No change was also observed in the number of VT-IR cells in the periventricular region close to the POM. Previously published data show that VT or its mammalian homolog, vasopressin are implicated in the control of a wide range of instinctive behaviors. The steroid-dependent VT afferents to the POM, a key area controlling male copulatory behavior in quail could therefore be involved in the control of the sexual behavior in this species. The outputs of the POM which contains steroid-receptors could therefore be modulated by steroids in two different ways: directly through the steroid receptors it contains and indirectly through its steroid-sensitive peptidergic afferents.

Immunocytochemical studies on the LHRH system of the japanese quail: influence by photoperiod and aspects of sexual differentiation

SummaryImmunocytochemistry was used to determine if photoperiod and/or sex have any effect on the pattern of the luteinizing hormone-releasing hormone (LHRH) system in the brain of the Japanese quail. Immunopositive perikarya were found within three major areas of the brain: the rostral paraolfactory lobe, the preoptic, and the septal region. A quantitative analysis of LHRH cell numbers was performed on male and female quail after two photoperiodic treatments: sexually mature birds exposed to 24 weeks of 20 h light: 4 h darkness (20L∶4D), and birds with a regressed reproductive system (induced by transfer from a photoregime of 20L∶4D to 25 short days of 8L∶16D). Two-way analysis of variance showed that short-day males display significantly (p < 0.05) more immunopositive perikarya (607 + 134) than long-day males (291 + 114), short-day females (293 + 103) or long-day females (330 + 92). The density of LHRH-immunoreactive nerve fibres and the intensity of the immunostaining in the median eminence were always greater in long-day sexually mature quail (male and female) than in animals exposed to 25 days of 8L∶16D. These results demonstrate that the LHRH system of the quail is influenced by photoperiod and mirrors sexual differentiation.

Steroid-induced plasticity in the sexually dimorphic vasotocinergic innervation of the avian brain: behavioral implications.

Vasotocin (VT, the antidiuretic hormone of birds) is synthesized by diencephalic magnocellular neurons projecting to the neurohypophysis. In addition, in male quail and in other oscine and non-oscine birds, a sexually dimorphic group of VT-immunoreactive (ir) parvocellular neurons is located in a region homologous to the mammalian nucleus of the stria terminalis, pars medialis (BSTm) and in the medial preoptic nucleus (POM). These cells are not visible in females. VT-ir fibers are present in many diencephalic and extradiencephalic locations. Quantitative morphometric analyses demonstrate that, in quail, these elements are expressed in a sexually dimorphic manner (males>females) in regions involved in the control of different aspects of reproduction: i.e., the POM (copulatory behavior), the lateral septum (secretion of gonadotropin-releasing hormone [GnRH]), the nucleus intercollicularis (control of vocalizations), and the locus coeruleus (the main noradrenergic center of the avian brain). In many of these regions, VT-ir fibers are closely related to aromatase-ir, GnRH-ir, or estrogen receptor-expressing neurons. This dimorphism has an organizational nature: administration of estradiol-benzoate to quail embryos (a treatment that abolishes male sexual behavior) results in a dramatic decrease of the VT-immunoreactivity in all sexually dimorphic regions of the male quail brain. Conversely, the inhibition of estradiol (E2) synthesis during embryonic life (a treatment that stimulates the expression of male copulatory behavior in adult testosterone (T)-treated females) results in a male-like distribution of VT-ir cells and fibers. Castration markedly decreases the immunoreactivity in both the VT-immunopositive elements of the BSTm and the innervation of the SL and POM, whereas T-replacement therapy restores the VT immunoreactivity to a level typical of intact birds. These changes reflect modifications of VT mRNA concentrations (and probably synthesis) as demonstrated by in situ hybridization and they are paralleled by similar changes in male copulatory behavior (absent in castrated male quail, fully expressed in CX+T males). The aromatization of T into estradiol (E2) also controls VT expression and, in parallel limits the activation of male sexual behavior by T. In castrated male quail, the restoration by T of the VT immunoreactivity in POM, BSTm and lateral septum could be fully mimicked by a treatment with E2, but the androgen 5alpha-dihydrotestosterone (DHT) had absolutely no effect on the VT immunoreactivity in these conditions. At the doses used in this study, DHT also did not synergize with E2 to enhance the density of VT immunoreactive structures. Systemic or i.c.v. injections of VT markedly inhibit the expression of all aspects of male sexual behavior. VT, presumably, does not simply represent one step in the biochemical cascade of events that is induced by T in the brain and leads to the expression of male sexual behavior. Androgens and estrogens presumably affect reproductive behavior both directly, by acting on steroid-sensitive neurons in the preoptic area, and indirectly, by modulating peptidergic (specifically vasotocinergic) inputs to this and other areas. The respective contribution of these two types of actions and their interaction deserves further analysis.

Organizational Effects of Estrogens on Brain Vasotocin and Sexual Behavior in Quail

Reproductive behavior is sexually differentiated in quail: The male-typical copulatory behavior is never observed in females even after treatment with high doses of testosterone (T). This sex difference in behavioral responsiveness to T is organized during the embryonic period by the exposure of female embryo to estrogens. We showed recently that the sexually dimorphic medial preoptic nucleus (POM), a structure that plays a key role in the activation of male copulatory behavior, is innervated by a dense steroid-sensitive network of vasotocin-immunoreactive (VT-ir) fibers in male quail This innervation is almost completely absent in the female POM and is not induced by a chronic treatment with T, suggesting that this neurochemical difference could be organizational in nature. This idea was tested by injecting fertilized quail eggs of both sexes on day 9 of incubation with either estradiol benzoate (EB) (25 microg, a treatment that suppresses the capacity to show copulatory behavior in adulthood) or the aromatase inhibitor R76713 (10 microg, a treatment that makes adult females behaviorally responsive to T), or with the solvents as a control (C). At 3 weeks posthatch, all subjects were gonadectomized and later implanted with Silastic capsules filled with T. Two weeks later, all birds were perfused and brain sections were processed for VT immunocytochemistry. Despite the similarity of the adult endocrine conditions of the subjects (all were gonadectomized and treated with T Silastic implants providing the same plasma level of steroid to all subjects), major qualitative differences were observed in the density of VT-ir structures in the POM of the different groups. Dense immunoreactive structures (fibers and a few cells) were observed in the POM of C males but not females; EB males had completely lost this immunoreactivity (and lost the capacity to display copulatory behavior); and, conversely, R76713 females displayed a male-typical VT-ir system in the nucleus (and also high levels of copulatory behavior). Similar changes in immunoreactivity were seen in the nucleus of the stria terminalis and in the lateral septum (VT-ir fibers only in this case) but not in the magnocellular vasotocinergic system. These neurochemical changes closely parallel the effects of the embryonic treatments on male copulatory behavior. The vasotocinergic system of the POM can therefore be considered an accurate marker of the sexual differentiation of brain circuits mediating this behavior.

Vasotocinergic innervation of the septal region in the Japanese quail: sexual differences and the influence of testosterone

SummaryVasotocin (VT)-immunoreactive fibres were observed in the nuclei of the quail (Coturnix coturnix japonica) septal region. Their distribution in the nucleus septalis lateralis (SL) and the nucleus striae terminalis (nST) was sexually dimorphic: a dense network of immunoreactive fibres was seen in adult sexually stimulated males but not in females. Experimental manipulation of the hormonal environment influenced this distribution only in males. VT immunoreactivity was absent in SL and nST when male quail were exposed to a shortday photoperiod or castrated. The immunoreactivity was restored to its original level in castrated males by silastic implants of testosterone.

Changes in the Arginine‐Vasopressin Immunoreactive Systems in Male Mice Lacking a Functional Aromatase Gene

In male rodents, the arginine‐vasopressin‐immunoreactive (AVP‐ir) neurones of the bed nucleus of the stria terminalis (BNST) and medial amygdala are controlled by plasma testosterone levels (decreased after castration and restored by exogenous testosterone). AVP transcription in these nuclei is increased in adulthood by a synergistic action of the androgenic and oestrogenic metabolites of testosterone and, accordingly, androgen and oestrogen receptors are present in both BNST and medial amygdala. We used knockout mice lacking a functional aromatase enzyme (ArKO) to investigate the effects of a chronic depletion of oestrogens on the sexually dimorphic AVP system. Wild‐type (WT) and ArKO male mice were perfused 48 h after an i.c.v. colchicine injection and brain sections were then processed for AVP immunocytochemistry. A prominent decrease (but not a complete suppression) of AVP‐ir structures was observed in the BNST and medial amygdala of ArKO mice by comparison with the WT. Similarly, AVP‐ir fibres were reduced in the lateral septum of ArKO mice and but not in the medial preoptic area, a region where the AVP system is not sexually dimorphic in rats. No change was detected in the supraoptic and suprachiasmatic nuclei. However, a decrease in AVP‐ir cell numbers was however, detected in one subregion of the paraventricular nucleus. These data support the hypothesis that the steroid‐sensitive sexually dimorphic AVP system of the mouse forebrain is mainly under the control of aromatized metabolites of testosterone.