Claudia Castagna

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.

Differential effects of D1 and D2 dopamine-receptor agonists and antagonists on appetitive and consummatory aspects of male sexual behavior in Japanese quail.

Pharmacological studies in Japanese quail based on behavioral tests with a variety of dopaminergic compounds suggest that the activation of D2 dopamine receptors inhibits, and the activation of D1 dopamine receptors enhances, appetitive and consummatory components of male sexual behavior. This hypothesis was tested by studying the behavioral effects of specific D1 and D2 dopaminergic-receptor agonists and antagonists in castrated male Japanese quail chronically treated with exogenous testosterone (subcutaneous Silastic implants). The effects of 5 compounds were tested: 1 D1 (SKF38393) and 2 D2 (PPHT and quinpirole) agonists, and 1 D1 (SCH23390) and 1 D2 (Spiperone) antagonist. All compounds were tested at a low and a high dose (0.1 and 1 mg/kg, respectively, for all drugs, except spiperone where the doses were 2 and 10 mg/kg). A consistent effect of all drugs on consummatory sexual behavior was observed: it was stimulated by the D1 agonist and the D2 antagonist, but inhibited by the D1 antagonist and the D2 agonists. Far fewer effects of the treatments were detected on the measures of appetitive behavior. Measures of appetitive behavior were decreased by the 2 D2 agonists, but not affected by the other treatments. These data suggest that male copulatory behavior in quail is stimulated by dopamine acting on D1 receptors, but inhibited by activation of the D2 receptor subtype. The partial dissociation observed between the effects of the same treatments on appetitive and consummatory aspects of sexual behavior also suggests that these 2 behavioral systems may be controlled by the action of dopamine on different neuronal systems.

Aromatase inhibition blocks the activation and sexual differentiation of appetitive male sexual behavior in Japanese quail

Two experiments investigated the role of estrogens in the activation and sexual differentiation of appetitive sexual behavior (ASB) in Japanese quail (Coturnix japonica) as measured by a learned social proximity response. Injection of the aromatase inhibitor R767 13 in castrated, testosterone (T)-treated male quail completely suppressed ASB, confirming that, like consummatory sexual behavior, ASB is mediated by T aromatization. ASB is not observed in female quail, even if they are treated with T as adults. The role of embryonic estrogens in the sexual differentiation of ASB was tested by blocking estrogen synthesis in ovo. Control male and T-treated female quail deprived of estrogens during embryonic life learned the social proximity response used to assess ASB, whereas control female quail did not, despite the presence of high T. Thus, ASB is demasculinized by the action of embryonic estrogens during ontogeny as is consummatory behavior.

Testosterone effects on the neuronal ultrastructure in the medial preoptic nucleus of male Japanese quail.

Dorsolateral neurons of the medial preoptic nucleus (POM) of male Japanese quail are sensitive to the plasma levels of testosterone: their volume and optical density in Nissl-stained sections increase in castrated birds treated with testosterone. The present study was performed on castrated male quail treated or not with Silastic implants filled with testosterone to describe the ultrastructural variations induced by testosterone in these neurons. Gonadally intact male birds were included as controls. The ultrastructure of neurons, taken from the dorsolateral portion of the POM, was dramatically affected by the endocrine manipulations. Quantitative evaluations demonstrated a significant decrease in castrated birds of the rough endoplasmic reticulum (RER), of free polyribosomes, of Golgi complexes, and of dense bodies; these changes paralleled the decrease in cell size. The cell size and the percentage of volume occupied by the intracellular organelles in castrated birds treated with testosterone were comparable to values observed in controls. These ultrastructural changes are similar to those observed in neuronal targets for other gonadal hormones, supporting the idea that testosterone stimulates the development of cytoplasmic structures involved in protein synthesis and secretion. In addition, exposure to testosterone affects the synaptic inputs to POM. These ultrastructural changes are presumably related to the physiological effects (e.g., activation of male sexual behavior) exerted by testosterone on this preoptic region.

Effects of testosterone on the synaptology of the medial preoptic nucleus of male Japanese quail

The medial preoptic nucleus (POM) of male Japanese quail is a sexually dimorphic testosterone-dependent structure that plays a key role in the activation of male sexual behavior. Both the total volume of the nucleus and the size of the dorsolateral neurons are decreased in gonadectomized males. Immunocytochemical studies have revealed a complex pattern of innervation: immunopositive fibers for several neuropeptides and neurotransmitters have been detected in the POM; some of them (e.g. vasotocin-immunoreactive fibers) are sexually dimorphic and testosterone-dependent To understand the anatomical bases of these testosterone-dependent neurochemical changes, we performed an ultrastructural study of the POM neuropil in intact sexually mature, gonadectomized, or testosterone-treated gonadectomized males. A complex synaptic organization of the POM neuropil was observed in intact male quail reflecting the heterogeneity of the neurotransmitters and neuropeptides present in this nucleus. Changes in this organization were observed after the endocrine manipulations. The number of axosomatic synapses per cell body decreased after gonadectomy and was restored to the level observed in the intact group after the administration of testosterone. By contrast, no significant change was observed in the density of axodendritic and axospinal synapses after hormonal manipulations which suggests that the total number of synapses in the nucleus should be affected by testosterone (constant density in a changing total volume). The cross-sectional area of synaptic boutons was also decreased by castration and restored to intact level by testosterone. The action of testosterone on the activation of male copulatory behavior in gonadectomized birds is hence paralleled by an extensive rearrangement of neuropil in the POM.

Protein S100 immunoreactivity in glial cells and neurons of the Japanese quail brain.

In mammals, sparse data illustrated the neuronal expression of S100 protein in central and peripheral nervous system. Similar studies have not been performed in other vertebrate species, in particular in birds. We provide here a detailed description of the distribution of the calcium-binding protein S100 in neuronal and glial elements in the central nervous system of an avian species, the Japanese quail (Coturnix japonica) largely used for neuroanatomical and functional studies. The distribution of S100-like immunoreactivity was analyzed by three different antisera: a polyclonal, against S100 protein, and two monoclonals, against the beta-subunit (S100beta) and the alpha-subunit (S100alpha) of this protein. All sera showed glial positive elements, which were more abundant in the brainstem than in the prosencephalon. Moreover, the polyclonal and the monoclonal antibodies against the beta-subunit evidenced a neuronal population with a wide distribution, variable morphology and staining intensity. In the telencephalon and diencephalon a few S100-positive neurons were observed in basal ganglia, nucleus paraventricularis hypothalami, nucleus rotundus and nucleus geniculatus lateralis, pars ventralis. In the mesencephalon and pons a wide S100-immunoreactive neuronal population was detected in several regions, including motor and sensory nuclei of most cranial nerves (i.e. oculomotoris, abducens, trigeminus, cochlearis, trochlearis and vestibularis nuclei). This distribution appears very similar to that previously described in the rat hindbrain by both immunocytochemistry and in situ hybridization, as well as to sparse observations on different vertebrates. Therefore, our results suggest that the distribution pattern of this protein (both in glial and in neuronal elements) is highly conserved throughout the phylogeny.

Sexual Dimorphism, Steroid-Induced Plasticity, and Behavioral Significance of the Vasotocinergic Innervation of the Avian Brain

This review focuses on the immunocytochemical demonstration of vasotocin (VT), the antidiuretic hormone in the avian brain. This peptide is synthetized by a well described system of diencephalic magnocellular neurons, but more recent studies have also demonstrated the presence of immunoreactive sexually dimorphic parvocellular groups or fibers in diencephalic and extradiencephalic regions of different avian species, including the Japanese quail. The main cluster has been detected in the male in a region considered as the avian homologue of the mammalian nucleus of the stria terminalis (nST). These cells are not visible in the female. This dimorphism has also been confirmed by in situ hybridization studies. Moreover, sexually dimorphic vasotocin-positive fibers are present in regions involved in the control of different aspects of reproduction, i.e. the nucleus preopticus medialis (copulatory behavior), the lateral septum (secretion of GnRH), and the nucleus intercollicularis (vocalization control). In the male, the vasotocin-immunoreactivity in these regions is strictly testosterone-dependent: castration, or exposure to a short-day photoperiod, decrease VT-immunoreactivity to female levels. Administration of estradiol-benzoate to embryos (a treatment that abolishes masculine sexual behavior) results in a dramatic decrease of the VT-immunoreactivity in all these brain regions of male quail. Behavioral experiments demonstrate that intracerebroventricular administration of vasotocin strongly inhibits male sexual behavior. These data suggest that in birds this peptide is not only the antidiuretic hormone, but also plays a central role in the control of diverse aspects of reproduction.

Post-natal development of the Reeler mouse cerebellum: An ultrastructural study

Reelin, an extracellular protein promoting neuronal migration in brain areas with a laminar architecture, is missing in the Reeler mouse (reelin(-/-)). Several studies indicate that the protein is also necessary for correct dendritic outgrowth and synapse formation in the adult forebrain. By transmission electron microscopy, we characterize the development and synaptic organization of the cerebellar cortex in Reeler mice and wild type control littermates at birth, postnatal day (P) 5, 7, 10 and 15. Ultrastructural analysis shows deep alterations in cortical architecture and mispositioning of the Purkinje neurons (Pns), which remain deeply embedded in a central cellular mass within the white matter, with highly immature features. Quantitative examination shows that Reeler mice display: (i) a lower density of granule cells and a higher density of Pns, from P10; (ii) a lower density of synaptic contacts between Pn dendrites and parallel or climbing fibers, from P5; (iii) a lower density of synaptic contacts between basket cells and Pns, from P5; and (iv) a lower density of mossy fiber rosettes, from P10. Our results demonstrate that Reelin profoundly affects the structure and synaptic connectivity of post-natal mouse cerebellum.

Ultrastructural characterization of the sexually dimorphic medial preoptic nucleus of male Japanese quail.

The medial preoptic nucleus is a sexually dimorphic structure whose cytoarchitecture, afferent and efferent connections, and functions have been previously described. No detailed ultrastructural study has, however, been perfomed to date. Here we describe the ultrastructural organization of this important preoptic structure of the male quail. Neuronal cell bodies of the medial preoptic nucleus generally show extensive development of protein-synthesis-related organelles (rough endoplasmic reticulum, polysomes), and of secretory structures (Golgi complexes, secretory vesicles, dense bodies). Previous morphometrical studies at the light-microscopical level have demonstrated the presence of a medial and a lateral neuronal population distinguished by the size of their cell bodies (the medial neurons are smaller than the lateral neurons). The present ultrastructural investigation confirms the difference in size, but no difference has been observed in the ultrastructural organization of the neurons. In both the medial and the lateral part, the nucleus is characterized by a large variety of cell bodies, including some that, on the basis of their ultrastructure, can be considered as putative peptidergic neurons. Close contacts are frequently observed between adjacent cell bodies that are normally arranged in clusters. Various types of synaptic endings are also present, suggesting a rich supply of nerve fibers. A few glial cells are scattered within the nucleus. In view of the crucial role of this region in regulating quail sexual behavior, the large heterogeneity of neurons and of afferent nervous fibers suggest that this region might have an important role in the integration of information arriving from different brain regions.