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During the last 10 years, the conference on 'Steroids and Nervous System' held in Torino (Italy) has been an important international point of discussion for scientists involved in this exciting and expanding research field. The present review aims to recapitulate the main topics that have been presented through the various meetings. Two broad areas have been explored: the impact of gonadal hormones on brain circuits and behaviour, as well as the mechanism of action of neuroactive steroids. Relationships among steroids, brain and behaviour, the sexual differentiation of the brain and the impact of gonadal hormones, the interactions of exogenous steroidal molecules (endocrine disrupters) with neural circuits and behaviour, and how gonadal steroids modulate the behaviour of gonadotrophin-releasing hormone neurones, have been the topics of several lectures and symposia during this series of meetings. At the same time, many contributions have been dedicated to the biosynthetic pathways, the physiopathological relevance of neurosteroids, the demonstration of the cellular localisation of different enzymes involved in neurosteroidogenesis, the mechanisms by which steroids may exert some of their effects, both the classical and nonclassical actions of different steroids, the role of neuroactive steroids on neurodegeneration, neuroprotection, and the response of the neural tissue to injury. In these 10 years, this field has significantly advanced and neuroactive steroids have emerged as new potential therapeutic tools to counteract neurodegenerative events.

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.

In Japanese quail, the presence of aromatase (oestrogen synthase) in the dorsal horn of the spinal cord suggests that spinal sensory processes might be controlled by local actions of oestrogens. This is supported by the presence of oestrogen receptors and aromatase in the dorsal horn of the spinal cord in rats, and by the alteration of sensitivity by oestrogens in various mammalian species and also in canaries. We investigated whether oestrogens that are locally produced in the quail spinal cord can bind to specific receptors in the vicinity of their site of synthesis. We demonstrate the presence of numerous oestrogen receptor α-immunoreactive (ERα-ir) cell nuclei, predominantly in laminae II and, to a lesser extent, I and III of the dorsal horn of the spinal cord (i.e. in the area where aromatase was previously identified). ERα-ir cells were also seen in various parts of the intermediate zone (laminae V–VII). This presence of ERα-ir cells in the dorsal horn and intermediate zone fits in well with the distribution of ERα-ir cells in homologous areas in mammals, including rats. Only a few labelled cells were found in the ventral horn in the cervical, brachial, thoracic and first lumbar segments, but a conspicuous dense group of large ERα-ir cells was identified in lamina IX of the ventral horn in synsacral segments 8–10, which contain the motoneurones innervating the muscles of the cloacal gland. The presence of ERα-ir cells in lamina IX of these synsacral segments in quail contrasts with the finding that motoneurones innervating penile muscles in rats contain androgen, but not oestrogen receptors, and are influenced by androgens rather than by oestrogens. Together, these data suggest that spinal actions of oestrogens may modulate the sensory and motor systems that participate in reproduction, as well as other nonreproductive functions in quail.

In adult male and female Japanese quail, aromatase-immunoreactive cells were identified in the spinal dorsal horns from the upper cervical segments to the lower caudal area. These immunoreactive cells are located mostly in laminae I–III, with additional sparse cells being present in the medial part of lamina V and, at the cervical level exclusively, in lamina X around the central canal. Radioenzyme assays based on the measurement of tritiated water release confirmed the presence of substantial levels of aromatase activity throughout the rostrocaudal extent of the spinal cord. Contrary to what is observed in the brain, this enzyme activity and the number of aromatase-immunoreactive cells in five representative segments of the spinal cord are not different in sexually mature males or females and are not influenced in males by castration with or without testosterone treatment. The aromatase activity and the numbers of aromatase-immunoreactive cells per section are higher at the brachial and thoracic levels than in the cervical and lumbar segments. These experiments demonstrate for the first time the presence of local estrogen production in the spinal cord of a higher vertebrate. This production was localized in the sensory fields of the dorsal horn, where estrogen receptors have been identified previously in several avian and mammalian species, suggesting an implication of aromatase in the modulation of sensory (particularly nociceptive) processes.

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.

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.