The hypothalamic paraventricular nucleus as a central hub for the estrogenic modulation of neuroendocrine function and behavior.

Estradiol and hypothalamic paraventricular nucleus (PVN) help coordinate reproduction with body physiology, growth and metabolism. PVN integrates hormonal and neural signals originating in the periphery, generating an output mediated both by its long-distance neuronal projections, and by a variety of neurohormones produced by its magnocellular and parvocellular neurosecretory cells. Here we review the cyto-and chemo-architecture, the connectivity and function of PVN and the sex-specific regulation exerted by estradiol on PVN neurons and on the expression of neurotransmitters, neuromodulators, neuropeptides and neurohormones in PVN. Classical and non-classical estrogen receptors (ERs) are expressed in neuronal afferents to PVN and in specific PVN interneurons, projecting neurons, neurosecretory neurons and glial cells that are involved in the input-output integration and coordination of neurohormonal signals. Indeed, PVN ERs are known to modulate body homeostatic processes such as autonomic functions, stress response, reproduction, and metabolic control. Finally, the functional implications of the estrogenic modulation of the PVN for body homeostasis are discussed.

Study of the NR4A family gene expression in patients with multiple sclerosis treated with Fingolimod.

BACKGROUND AND PURPOSE: Fingolimod is a drug approved for treatment of relapsing-remitting multiple sclerosis (RRMS) that exerts its effects via sequestering lymphocytes within the lymph nodes. The drug, acting on the sphingosine-1-phosphate pathway, is involved in a plethora of processes and, to date, its mechanism of action is not completely understood. Recently, it has been demonstrated that Fingolimod increases the expression of transcription factor NR4A2 in murine brain. NR4A2 belongs to nuclear receptor family 4, group A (NR4A) along with NR4A1 and NR4A3. The role of NR4A2 in the pathogenesis of multiple sclerosis is already known and supported by its down-regulation observed in blood obtained from patients with RRMS compared with healthy controls (HCs). It is notable that NR4A2 impairment is reversed in patients with RRMS during pregnancy, which represents a transitory state of immune tolerance, associated with reduced disease activity. An inverse correlation between NR4A2 gene expression levels and clinical parameters indicates that more aggressive forms of the disease are characterized by lower levels of NR4A2. METHODS: Gene expression levels of NR4A in blood obtained from HCs, treatment-naive (T0) and Fingolimod-treated patients with RRMS were evaluated to determine their contribution to drug response. RESULTS: Gene expression levels of NR4A were down-regulated in T0 patients compared with HCs. Patients treated with Fingolimod for >2 years were characterized by higher levels of NR4A2 compared with the T0 group, approaching those of HCs. NR4A1 and NR4A3 levels were not altered. CONCLUSIONS: Involvement of the NR4A family in the pathogenesis of multiple sclerosis and a role of Fingolimod in the recovery from NR4A2 deficit can be hypothesized based on our data.

Steroids, stress and the gut microbiome-brain axis.

It is becoming well established that the gut microbiome has a profound impact on human health and disease. In this review, we explore how steroids can influence the gut microbiota and, in turn, how the gut microbiota can influence hormone levels. Within the context of the gut microbiome-brain axis, we discuss how perturbations in the gut microbiota can alter the stress axis and behaviour. In addition, human studies on the possible role of gut microbiota in depression and anxiety are examined. Finally, we present some of the challenges and important questions that need to be addressed by future research in this exciting new area at the intersection of steroids, stress, gut-brain axis and human health.

Early postnatal genistein administration permanently affects nitrergic and vasopressinergic systems in a sex-specific way.

Genistein (GEN) is a natural xenoestrogen (isoflavonoid) that may interfere with the development of estrogen-sensitive neural circuits. Due to the large and increasing use of soy-based formulas for babies (characterized by a high content of GEN), there are some concerns that this could result in an impairment of some estrogen-sensitive neural circuits and behaviors. In a previous study, we demonstrated that its oral administration to female mice during late pregnancy and early lactation induced a significant decrease of nitric oxide synthase-positive cells in the amygdala of their male offspring. In the present study, we have used a different experimental protocol mimicking, in mice, the direct precocious exposure to GEN. Mice pups of both sexes were fed either with oil, estradiol or GEN from birth to postnatal day 8. Nitric oxide synthase and vasopressin neural systems were analyzed in adult mice. Interestingly, we observed that GEN effect was time specific (when compared to our previous study), sex specific, and not always comparable to the effects of estradiol. This last observation suggests that GEN may act through different intracellular pathways. Present results indicate that the effect of natural xenoestrogens on the development of the brain may be highly variable: a plethora of neuronal circuits may be affected depending on sex, time of exposure, intracellular pathway involved, and target cells. This raises concern on the possible long-term effects of the use of soy-based formulas for babies, which may be currently underestimated.

Adult exposure to tributyltin affects hypothalamic neuropeptide Y, Y1 receptor distribution, and circulating leptin in mice.

Tributyltin (TBT), a pesticide used in antifouling paints, is toxic for aquatic invertebrates. In vertebrates, TBT may act in obesogen- inducing adipogenetic gene transcription for adipocyte differentiation. In a previous study, we demonstrated that acute administration of TBT induces c-fos expression in the arcuate nucleus. Therefore, in this study, we tested the hypothesis that adult exposure to TBT may alter a part of the nervous pathways controlling animal food intake. In particular, we investigated the expression of neuropeptide Y (NPY) immunoreactivity. This neuropeptide forms neural circuits dedicated to food assumption and its action is mediated by Y1 receptors that are widely expressed in the hypothalamic nuclei responsible for the regulation of food intake and energy homeostasis. To this purpose, TBT was orally administered at a dose of 0.025 mg/kg/day/body weight to adult animals [male and female C57BL/6 (Y1-LacZ transgenic mice] for 4 weeks. No differences were found in body weight and fat deposition, but we observed a significant increase in feed efficiency in TBT-treated male mice and a significant decrease in circulating leptin in both sexes. Computerized quantitative analysis of NPY immunoreactivity and Y1-related ß-galactosidase activity demonstrated a statistically significant reduction in NPY and Y1 transgene expression in the hypothalamic circuit controlling food intake of treated male mice in comparison with controls. In conclusion, the present results indicate that adult exposure to TBT is profoundly interfering with the nervous circuits involved in the stimulation of food intake.

Neurosteroidogenesis Today: Novel Targets for Neuroactive Steroid Synthesis and Action and Their Relevance for Translational Research.

Neuroactive steroids are endogenous neuromodulators synthesised in the brain that rapidly alter neuronal excitability by binding to membrane receptors, in addition to the regulation of gene expression via intracellular steroid receptors. Neuroactive steroids induce potent anxiolytic, antidepressant, anticonvulsant, sedative, analgesic and amnesic effects, mainly through interaction with the GABAA receptor. They also exert neuroprotective, neurotrophic and antiapoptotic effects in several animal models of neurodegenerative diseases. Neuroactive steroids regulate many physiological functions, such as the stress response, puberty, the ovarian cycle, pregnancy and reward. Their levels are altered in several neuropsychiatric and neurological diseases and both preclinical and clinical studies emphasise a therapeutic potential of neuroactive steroids for these diseases, whereby symptomatology ameliorates upon restoration of neuroactive steroid concentrations. However, direct administration of neuroactive steroids has several challenges, including pharmacokinetics, low bioavailability, addiction potential, safety and tolerability, which limit its therapeutic use. Therefore, modulation of neurosteroidogenesis to restore the altered endogenous neuroactive steroid tone may represent a better therapeutic approach. This review summarises recent approaches that target the neuroactive steroid biosynthetic pathway at different levels aiming to promote neurosteroidogenesis. These include modulation of neurosteroidogenesis through ligands of the translocator protein 18 kDa and the pregnane xenobiotic receptor, as well as targeting of specific neurosteroidogenic enzymes such as 17ß-hydroxysteroid dehydrogenase type 10 or P450 side chain cleavage. Enhanced neurosteroidogenesis through these targets may be beneficial not only for neurodegenerative diseases, such as Alzheimer's disease and age-related dementia, but also for neuropsychiatric diseases, including alcohol use disorders.

Testosterone and estradiol differentially affect cell proliferation in the subventricular zone of young adult gonadectomized male and female rats.

Steroid hormones are important players to regulate adult neurogenesis in the dentate gyrus of the hippocampus, but their involvement in the regulation of the same phenomenon in the subventricular zone (SVZ) of the lateral ventricles is not completely understood. Here, in male rats, we tested the existence of activational effects of testosterone (T) on cell proliferation in the adult SVZ. To this aim, three groups of male rats: castrated, castrated and treated with T, and controls were treated with 5-bromo-2'-deoxyuridine (BrdU) and killed after 24h. The density of BrdU-labeled cells was significantly lower in castrated animals in comparison to the other two groups, thus supporting a direct correlation between SVZ proliferation and levels of circulating T. To clarify whether this effect is purely androgen-dependent, or mediated by the T metabolites, estradiol (E2) and dihydrotestosterone (DHT), we evaluated SVZ proliferation in castrated males treated with E2, DHT and E2+DHT, in comparison to T- and vehicle-treated animals, and sham-operated controls. The stereological analysis demonstrated that E2 and T, but not DHT, increase proliferation in the SVZ of adult male rats. Quantitative evaluation of cells expressing the endogenous marker of cell proliferation phosphorylated form of Histone H3 (PHH3), or the marker of highly dividing SVZ progenitors Mash1, indicated the effect of T/E2 is mostly restricted to SVZ proliferating progenitors. The same experimental protocol was repeated on ovariectomized female rats treated with E2 or T. In this case, no statistically significant difference was found among groups. Overall, our results clearly show that the gonadal hormones T and E2 represent important mediators of cell proliferation in the adult SVZ. Moreover, we show that such an effect is restricted to males, supporting adult neurogenesis in rats is a process differentially modulated in the two sexes.

Chronic unpredictable stress and long-term ovariectomy affect arginine-vasopressin expression in the paraventricular nucleus of adult female mice.

Arginine-Vasopressin (AVP) may regulate the hypothalamic-pituitary-adrenal axis (HPA) and its effects on depressive responses. In a recent study, we demonstrated that Chronic Unpredictable Stress (CUS) depressive effects are enhanced by long-term ovariectomy (a model of post-menopause). In the present study, we investigated the effects of long-term ovariectomy and CUS on AVP expression in different subdivision of the paraventricular nucleus (PVN) of female mice. Both long-term ovariectomy and CUS affect AVP immunoreactivity in some of the PVN subnuclei of adult female mice. In particular, significant changes on AVP immunoreactivity were observed in magnocellular subdivisions, the paraventricular lateral magnocellular (PaLM) and the paraventricular medial magnocellular (PaMM), the 2 subnuclei projecting to the neurohypophysis for the hormonal regulation of body homeostasis. AVP immunoreactivity was decreased in the PaLM by both the long-term deprivation of ovarian hormones and the CUS. In contrast, AVP immunoreactivity was increased in the PaMM by CUS, whereas it was decreased by ovariectomy. Therefore, present results suggest morphological and functional differences among the PVN's subnuclei and complex interactions among CUS, gonadal hormones and AVP immunoreactivity.

Androgen receptor deficiency alters the arginine-vasopressin sexually dimorphic system in Tfm rats.

In rodents as well as in many other mammalian and non-mammalian species, the arginine-vasopressin (AVP) system includes a parvocellular sexually dimorphic portion located within the bed nucleus of the stria terminalis (BST), the medial amygdaloid nucleus (MeA) and the lateral septum. In this system, males have more cells and denser projections than females, neurons show androgen and estrogen receptors, and gonadal hormones are required for the activation. However, the role of these hormones for the differentiation of the system is not clear. Previous studies performed on aromatase knockout mice suggested that estradiol is not necessary for the differentiation of the system, but it is important for its activation in adulthood. To elucidate the role of androgens on differentiation and functioning of AVP parvocellular system, we compared male and female rats with a non-functional mutation of androgen receptor (Tfm, testicular feminization mutation) to their control littermates. Our data show that the lack of a functional androgen receptor significantly decreases the expression of AVP immunoreactivity within the BST and MeA of male Tfm. Thus supporting the hypothesis that androgens, through the action of their receptor, should have a relevant role in the organization and modulation of the AVP parvocellular sexually dimorphic system.

Estrogenic regulation of NADPH-diaphorase in the supraoptic and paraventricular nuclei under acute osmotic stress.

Estrogen receptors (ERs) α and ß are involved in the regulation of the nitrergic system in the supraoptic (SON) and paraventricular (PVN) nuclei under basal conditions. In this study we have assessed whether ERs are also involved in the modulation of the nitrergic system in the SON and PVN under acute systemic hypertonic conditions. Adult ovariectomized rats received a single injection of either estradiol, a selective ERα agonist, a selective ERß agonist, a selective ERα antagonist, a selective ERß antagonist or vehicle. Twenty-four hours later, animals received one i.p. injection of 1.5M NaCl to induce osmotic stress and were sacrificed after two additional hours. The number of NADPH-diaphorase-positive cells in the SON and PVN was determined. Their number in the SON was not affected by NaCl administration, whereas in the four PVN subdivisions it was decreased after NaCl administration. Estradiol and the ERα agonist prevented the action of NaCl in the four subdivisions of the PVN. In contrast, the inhibition of ERα enhanced the effect of NaCl, inducing a further decrease in the number of NADPH-diaphorase-positive cells. Moreover, the ERß agonist enhanced and the ERß antagonist blocked the effect of NaCl on the number of NADPH-diaphorase-positive neurons in the SON and in the medial magnocellular subdivision of the PVN. These findings indicate that estradiol regulates the nitrergic system in the SON and PVN under acute osmotic stress conditions, but the effects specifically depend on the anatomical subregions and different ERs.

Role of oestrogen receptors on the modulation of NADPH-diaphorase-positive cell number in supraoptic and paraventricular nuclei of ovariectomised female rats.

Modulation of the nitric oxide producing system (demonstrated via the NADPH-diaphorase histochemical reaction) by oestradiol has been established in several structures of the rat brain. The present study aimed to explore the possible regulation of NADPH-diaphorase activity by oestradiol in neurones of the supraoptic (SON) and paraventricular (PVN) nuclei and the role of oestrogen receptors (ERα and ERß) in this regulation. Adult ovariectomised rats were divided into six groups and injected either with vehicle or a single dose of oestradiol, a selective ERα agonist-PPT [4,4',4″-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol], a selective ERß agonist-DPN [2,3-bis(4-hydroxyphenyl)-propionitrile], a selective ERα antagonist-MPP [1,3-bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylethoxy)phenol]-1H-pyrazole dihydrochloride] or a selective ERß antagonist-PHTPP (4-[2-phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-3-yl]phenol). The number of NADPH-diaphorase positive elements in the SON and the PVN was modulated by both ERs but, depending on the nucleus, ERα and ERß ligands induced different effects. These results suggest that the regulation of nitrergic system by ERs may play a role in the control of oestrogen-dependent physiological mechanisms regulated by the SON and the PVN.

Implication of the VGF-derived peptide TLQP-21 in mouse acute and chronic stress responses.

The impact of stress is widely recognized in the etiology of multiple disorders. In particular, psychological stress may increase the risk of cardiovascular, metabolic, immune, and mood disorders. Several genes are considered potential candidates to account for the deleterious consequences of stress and recent data point to role of Vgf. VGF mRNA is abundantly expressed in the hypothalamus, where it has been involved in metabolism and energy homeostasis; more recently a link between VGF-derived peptides and mood disorders has been highlighted. The following experiments were performed to address the contribution of the VGF-system to stress induced changes in mice: the distribution of VGF immuno-reactivity in hypothalamic nuclei and its modulation by social stress; the role of VGF-derived peptide TLQP-21 in plasma catecholamine release induced by acute restraint stress (RS); the efficacy of chronic TLQP-21 in a mouse model of chronic subordination stress (CSS). VGF fibers were found in high density in arcuate, dorsomedial, and suprachiasmatic and, at lower density, in lateral, paraventricular, and ventromedial hypothalamic nuclei. Central administration of either 2 or 4 mM TLQP-21 acutely altered the biphasic serum epinephrine release and decreased norepinephrine serum levels in response to RS. Finally, 28-day of 40 µg/day TLQP-21 treatment increased CSS-induced social avoidance of an unfamiliar conspecific. Overall these data support a role for TLQP-21 in stress responses providing a promising starting point to further elucidate its role as a player in stress-related human pathologies.

Endocrine disrupters: a review of some sources, effects, and mechanisms of actions on behaviour and neuroendocrine systems.

Some environmental contaminants interact with hormones and may exert adverse consequences as a result of their actions as endocrine disrupting chemicals (EDCs). Exposure in people is typically a result of contamination of the food chain, inhalation of contaminated house dust or occupational exposure. EDCs include pesticides and herbicides (such as dichlorodiphenyl trichloroethane or its metabolites), methoxychlor, biocides, heat stabilisers and chemical catalysts (such as tributyltin), plastic contaminants (e.g. bisphenol A), pharmaceuticals (i.e. diethylstilbestrol; 17α-ethinylestradiol) or dietary components (such as phytoestrogens). The goal of this review is to address the sources, effects and actions of EDCs, with an emphasis on topics discussed at the International Congress on Steroids and the Nervous System. EDCs may alter reproductively-relevant or nonreproductive, sexually-dimorphic behaviours. In addition, EDCs may have significant effects on neurodevelopmental processes, influencing the morphology of sexually-dimorphic cerebral circuits. Exposure to EDCs is more dangerous if it occurs during specific 'critical periods' of life, such as intrauterine, perinatal, juvenile or puberty periods, when organisms are more sensitive to hormonal disruption, compared to other periods. However, exposure to EDCs in adulthood can also alter physiology. Several EDCs are xenoestrogens, which can alter serum lipid concentrations or metabolism enzymes that are necessary for converting cholesterol to steroid hormones. This can ultimately alter the production of oestradiol and/or other steroids. Finally, many EDCs may have actions via (or independent of) classic actions at cognate steroid receptors. EDCs may have effects through numerous other substrates, such as the aryl hydrocarbon receptor, the peroxisome proliferator-activated receptor and the retinoid X receptor, signal transduction pathways, calcium influx and/or neurotransmitter receptors. Thus, EDCs, from varied sources, may have organisational effects during development and/or activational effects in adulthood that influence sexually-dimorphic, reproductively-relevant processes or other functions, by mimicking, antagonising or altering steroidal actions.

Milestones on Steroids and the Nervous System: 10 years of basic and translational research.

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.

Neuropeptides and enzymes are targets for the action of endocrine disrupting chemicals in the vertebrate brain.

Endocrine-disrupting chemicals (EDC) are molecules that interfere with endocrine signaling pathways and produce adverse consequences on animal and human physiology, such as infertility or behavioral alterations. Some EDC act through binding to androgen or/and estrogen receptors primarily operating through a genomic mechanism regulating gene expression. This mechanism of action may induce profound developmental adverse effects, and the major targets of the EDC action are the gene products, i.e., mRNAs inducing the synthesis of various peptidic molecules, which include neuropeptides and enzymes related to neurotransmitters syntheses. Available immunohistochemical data on some of the systems that are affected by EDC in lower and higher vertebrates are detailed in this review.

Neuroactive steroids: focus on human brain.

Studies in experimental animals have revealed important roles of neuroactive steroids in the control of central nervous system functions during physiological and pathological conditions, suggesting that they may represent good candidates for the development of neuroprotective strategies for neurodegenerative and psychiatric disorders. Even if the characterization of the roles played by neuroactive steroids in humans is still at the beginning, several data are already available showing that they may be synthesized within the human CNS. Among the different enzymes, a prominent role is dedicated to aromatase that synthesizes estradiol whose neuroprotective effects have been described in experimental animals. Neuroactive steroid levels are modified by neurodegenerative conditions (i.e. Alzheimer's and Parkinson's diseases, multiple sclerosis) or in other mental diseases (i.e. schizophrenia), and may have an important role in physiological conditions, as the reorganization of grey and white matter during human puberty and adolescence or as a consequence of emotional responses. The interaction of some neuroactive steroids (i.e., allopregnanolone and isopregnanolone) with GABA-A receptor is particularly important in mood disorders. The presumptive role of estradiol and progesterone in neuroprotection is here discussed by comparing contradictory data that have been collected in humans. In conclusion, the state of the art of our knowledge of the role of neuroactive steroids in the normal and pathological human brain suggests several lines of future therapeutic developments in the treatments of neurological, neurodegenerative and affective disorders. This article is part of a Special Issue entitled: Neuroactive Steroids: Focus on Human Brain.