Evidence that the Bed Nucleus of the Stria Terminalis Contributes to the Modulation of Hypophysiotropic Corticotropin-Releasing Factor Cell Responses to Systemic Interleukin-1β

Systemic infection activates the hypothalamic-pituitary-adrenal (HPA) axis, and brainstem catecholamine cells have been shown to contribute to this response. However, recent work also suggests an important role for the central amygdala (CeA). Because direct connections between the CeA and the hypothalamic apex of the HPA axis are minimal, the present study investigated whether the bed nucleus of the stria terminalis (BNST) might act as a relay between them. This was done by using an animal model of acute systemic infection involving intravascular delivery of the proinflammatory cytokine interleukin-1 (IL-1, 1 g/kg). Unilateral ibotenic acid lesions encompassing the ventral BNST significantly reduced both IL-1-induced increases in Fos immunoreactivity in corticotropin-releasing factor (CRF) cells of the hypothalamic paraventricular nucleus (PVN) and corresponding increases in adrenocorticotropic hormone (ACTH) secretion. Similar lesions had no effect on CRF cell responses to physical restraint, suggesting that the effects of BNST lesions were not due to a nonspecific effect on stress responses. In further studies, we examined the functional connections between PVN, BNST, and CeA by combining retrograde tracing with mapping of IL-1-induced increases in Fos in BNST and CeA cells. In the case of the BNST, these studies showed that systemic IL-1 administration recruits ventral BNST cells that project directly to the PVN. In the case of the CeA, the results obtained were consistent with an arrangement whereby lateral CeA cells recruited by systemic IL-1 could regulate the activity of medial CeA cells projecting directly to the BNST. In conclusion, the present findings are consistent with the hypothesis that the BNST acts as a relay between the CeA and PVN, thereby contributing to CeA modulation of hypophysiotropic CRF cell responses to systemic administration of IL-1.

Performance and endocrine responses of group housed weaner pigs exposed to the air quality of a commercial environment

The growth performance and endocrine responses of male weaner pigs (3 to 8 weeks of age) was evaluated in two different environments (clean and dirty) and housing (single or groups of 10 pigs/pen) conditions. The dirty environment contained significantly elevated ammonia, carbon dioxide and dust levels compared with the clean environment. Pigs grew faster and consumed more feed in the clean environment and this was associated with reduced plasma cortisol concentrations compared with pigs in the dirty environment. Pigs housed in groups in the dirty environment had increased β-endorphin and decreased IGF-I concentrations compared to group housed pigs in the clean environment. Feed conversion efficiency did not differ due to environment or group housing. Plasma concentration of cortisol, p-endorphin, IGF-I and IGF-II did not differ between single and group housed pigs. Activity of the hypothalamic-pituitary-adrenal (HPA) axis was greater in response to environmental conditions than group housing, and this was associated with reduced growth in weaner pigs. © 2004 Elsevier B.V. All rights reserved.

Prolactin and the expression of suppressor of cytokine signaling-3 in the sheep adrenal gland before birth

Prolactin and the expression of suppressor of cytokine signaling-3 in the sheep adrenal gland before birth. Am J Physiol Regul Integr Comp Physiol 291: R1399-R1405, 2006. First published June 29, 2006; doi: 10.1152/ajpregu.00252.2006.-The fetal pituitary-adrenal axis plays a key role in the fetal response to intrauterine stress and in the timing of parturition. The fetal sheep adrenal gland is relatively refractory to stimulation in midgestation (90-120 days) before the prepartum activation, which occurs around 135 days gestation (term = 147 +/- 3 days). The mechanisms underlying the switch from adrenal quiescence to activation are unclear. Therefore, we have investigated the expression of suppressor of cytokine signaling-3 (SOCS-3), a putative inhibitor of tissue growth in the fetal sheep adrenal between 50 and 145 days gestation and in the adrenal of the growth-restricted fetal sheep in late gestation. SOCS-3 is activated by a range of cytokines, including prolactin (PRL), and we have, therefore, determined whether PRL administered in vivo or in vitro stimulates SOCS-3 mRNA expression in the fetal adrenal in late gestation. There was a decrease (P < 0.005) in SOCS-3 expression in the fetal adrenal between 54 and 133 days and between 141 and 144 days gestation. Infusion of the dopaminergic agonist, bromocriptine, which suppressed fetal PRL concentrations but did not decrease adrenal SOCS-3 mRNA expression. PRL administration, however, significantly increased adrenal SOCS-3 mRNA expression (P < 0.05). Similarly, there was an increase (P < 0.05) in SOCS-3 mRNA expression in adrenocortical cells in vitro after exposure to PRL (50 ng/ml). Placental and fetal growth restriction had no effect on SOCS-3 expression in the adrenal during late gestation. In summary, the decrease in the expression of the inhibitor SOCS-3 after 133 days gestation may be permissive for a subsequent increase in fetal adrenal growth before birth. We conclude that factors other than PRL act to maintain adrenal SOCS-3 mRNA expression before 133 days gestation but that acute elevations of PRL can act to upregulate adrenal SOCS-3 expression in the sheep fetus during late gestation.

Down-regulation of central glucocorticoid receptor expression using antisense oligonucleotide technology

Endogenous glucocorticoids and serotonin have been implicated in the pathophysiology of depression, anxiety and schizophrenia. This thesis investigates the potential of downregulating expression of central Type II glucocorticoid receptors (GR) both in vitro and in vivo, with empirically-designed antisense oligodeoxynucleotides (ODN), to characterise GR modulation of 5-HT2A receptor expression using quantitative RT-PCR, Western blot analysis and radioligand binding. The functional consequence of GR downregulation is also determined by measuring 1-(2,5-dimethoxy 4-iodophenyl)-2-amino propane hydrochloride (DOI) mediated 5-HT2A receptor specific headshakes. Using a library of random antisense ODN probes, RNAse H accessibility mapping of T7-primed, in vitro transcribed GR mRNA revealed several potential cleavage sites and identified an optimally effect GR antisense ODN sequence of 21-mer length (GRAS5). In vitro efficacy studies using rat C6 glioma cells showed a 56% downregulation in GR mRNA levels and 80% downregulation in GR protein levels. In the same cells a 29% upregulation in 5-HT2A mRNA levels and 32% upregulation in 5-HT2A protein levels was revealed. This confirmed the optimal nature of the GRAS5 sequence to produce marked inhibition of GR gene expression, and also revealed GR modulation of the 50-HT2A receptor subtype in C6 glioma cells to be a tonic repression of receptor expression. The distribution of a fluorescently-labelled GRAS5 ODN was detected in diverse areas of the rat brain after single ICV administration, although this fluorescence signal was not sustained over a period of 5 days. However, fluorescently-labelled GRAS5 ODN, when formulated in polymer microspheres, showed diverse distribution in the brain which was maintained for 5 days following a single ICV administration. This produced no apparent neurotoxic effects on rat behaviour and hypothalamic-pituitary-adrenal (HPA) axis homeostasis. Furthermore, a single polymer microsphere injection ICV proved to be an effective means of delivering antisense ODNs and this was adopted for the in vivo efficacy studies. In vivo characterisation of GRAS5 revealed marked downregulation of GR mRNA in rat brain regions such as the frontal cortex (26%), hippocampus (35%), and hypothalamus (39%). Downregulation of GR protein was also revealed in frontal cortex (67%), hippocampus (76%), and hypothalamus (80%). In the same animals upregulation of 5-HT2A mRNA levels was shown in frontal cortex (13%), hippocampus (7%), and hypothalamus (5%) while upregulation in 5-HT2A protein levels was shown in frontal cortex (21 %). This upregulation in 5-HT2A receptor density as a result of antisense-mediated inhibition of GR was further confirmed by a 55% increase in DOl-mediated 5-HT2A receptor specific headshakes. These results demonstrate that GR is involved in tonic inhibitory regulation of 5-HT2A receptor expression and function in vivo, thus providing the potential to control 5-HT2A-linked disorders through corticosteroid manipulation. These experiments have therefore established an antisense approach which can be used to investigate pharmacological characteristics of receptors.

Neuroendocrine control of a dynamic communication signal

Multiple physiological systems regulate the electric communication signal of the weakly electric gymnotiform fish, Brachyhypopomus pinnicaudatus. Fish were injected with neuroendocrine probes which identified pharmacologically relevant serotonin (5-HT) receptors similar to the mammalian 5-HT1AR and 5-HT2AR. Peptide hormones of the hypothalamic-pituitary-adrenal/interrenal axis also augment the electric waveform. These results indicate that the central serotonergic system interacts with the hypothalamic-pituitary-interrenal system to regulate communication signals in this species. The same neuroendocrine probes were tested in females before and after introducing androgens to examine the relationship between sex steroid hormones, the serotonergic system, melanocortin peptides, and EOD modulations. Androgens caused an increase in female B. pinnicaudatus responsiveness to other pharmacological challenges, particularly to the melanocortin peptide adrenocorticotropic hormone (ACTH). A forced social challenge paradigm was administered to determine if androgens are responsible for controlling the signal modulations these fish exhibit when they encounter conspecifics. Males and females responded similarly to this social challenge construct, however introducing androgens caused implanted females to produce more exaggerated responses. These results confirm that androgens enhance an individual's capacity to produce an exaggerated response to challenge, however another unidentified factor appears to regulate sex-specific behaviors in this species. These results suggest that the rapid electric waveform modulations B. pinnicaudatus produces in response to conspecifics are situation-specific and controlled by activation of different serotonin receptor types and the subsequent effect on release of pituitary hormones.

Neuroendocrine control of a dynamic communication signal

Multiple physiological systems regulate the electric communication signal of the weakly electric gymnotiform fish, Brachyhypopomuspinnicaudatus. Fish were injected with neuroendocrine probes which identified pharmacologically relevant serotonin (5-HT) receptors similar to the mammalian 5-HT1AR and 5-HT2AR. Peptide hormones of the hypothalamic-pituitary-adrenal/interrenal axis also augment the electric waveform. These results indicate that the central serotonergic system interacts with the hypothalamic-pituitaryinterrenal system to regulate communication signals in this species. The same neuroendocrine probes were tested in females before and after introducing androgens to examine the relationship between sex steroid hormones, the serotonergic system, melanocortin peptides, and EOD modulations. Androgens caused an increase in female B. pinnicaudatus responsiveness to other pharmacological challenges, particularly to the melanocortin peptide adrenocorticotropic hormone (ACTH). A forced social challenge paradigm was administered to determine if androgens are responsible for controlling the signal modulations these fish exhibit when they encounter conspecifics. Males and females responded similarly to this social challenge construct, however introducing androgens caused implanted females to produce more exaggerated responses. These results confirm that androgens enhance an individual's capacity to produce an exaggerated response to challenge, however another unidentified factor appears to regulate sex-specific behaviors in this species. These results suggest that the rapid electric waveform modulations B. pinnicaudatus produces in response to conspecifics are situation-specific and controlled by activation of different serotonin receptor types and the subsequent effect on release of pituitary hormones.

Caractérisation du potentiel de champ locale dans le cortex préfrontal médian du rat durant le stress et la prise alimentaire

Le stress joue un rôle important dans le maintien de la qualité de vie quotidienne. Une exposition à une situation stressante peut causer divers désordres neuropsychiatriques du cerveau qui sont associés avec des problèmes liés au sommeil, à la dépression, à des problèmes digestifs et à des troubles de l’alimentation. Les traitements de ces troubles liés au stress sont très coûteux à travers le monde. De nos jours, des considérations importantes ont été soulevées afin de trouver des moyens appropriés pour la prévention plutôt que de dépenser ultérieurement plus de budget sur les traitements. De cette façon, l’étude et l’expérimentation sur les animaux des troubles liés au stress sont l’un des moyens les plus fiables pour atteindre une compréhension plus profonde des problèmes liés au stress. Ce projet visait à révéler la modulation des potentiels de champ locaux (LFP) lors de la consommation de sucrose dans deux conditions englobant la condition de contrôle non-stressante et celle stressante d’un choc électrique aiguë à la patte dans le cortex préfrontal médian (CPFm) du cerveau de rat. Le CPFm est une structure importante dans la réponse au stress et à l’anxiété par l’interaction avec l’axe hypothalamique-pituitaire surrénale (HPA). Les résultats de ce projet ont révélé que la plupart des coups de langue se sont produits dans les 15 premières minutes de l’accès à une solution de sucrose autant pour la condition contrôle non-stressante que pour la condition stressante. En outre, le stress aigu d’un choc à la patte affecte de manière significative la consommation horaire de sucrose en diminuant le volume de la consommation. Les résultats ont également révélé une présence importante du rythme thêta dans le CPFm pendant la condition de base et pendant l’ingestion de sucrose dans les deux conditions. De plus, les résultats ont montré une diminution de puissance des bandes delta et thêta lors des initiations de léchage du sucrose. Ce projet conduit à des informations détaillées sur les propriétés électrophysiologiques du cortex infra-limbique (IL) du CPFm en réponse à l’exposition à des conditions de stress et de l’apport d’une solution de sucrose. Ce projet permet également de mieux comprendre les mécanismes neurophysiologiques des neurones du CPFm en réponse à l’exposition à une condition stressante suivie d’apport de sucrose. Ce projet a également permis de confirmer les effets anorexigènes du stress et suggèrent également que la synchronisation neuronale dans le cortex IL peut jouer un rôle dans le comportement de léchage et sa désynchronisation pendant le léchage après une exposition à des conditions stressantes.

The role of the glucocorticoids in developing resilience to stress and addiction

There is emerging evidence that individuals have the capacity to learn to be resilient by developing protective mechanisms that prevent them from the maladaptive effects of stress that can contribute to addiction.The emerging field of the neuroscience of resilience is beginning to uncover the circuits and molecules that protect against stress-related neuropsychiatric diseases, such as addiction. Glucocorticoids (GCs) are important regulators of basal and stress-related homeostasis in all higher organisms and influence a wide array of genes in almost every organ and tissue. GCs, therefore, are ideally situated to either promote or prevent adaptation to stress. In this review, we will focus on the role of GCs in the hypothalamic-pituitary adrenocortical axis and extra-hypothalamic regions in regulating basal and chronic stress responses. GCs interact with a large number of neurotransmitter and neuropeptide systems that are associated with the development of addiction. Additionally, the review will focus on the orexinergic and cholinergic pathways and highlight their role in stress and addiction. GCs play a key role in promoting the development of resilience or susceptibility and represent important pharmacotherapeutic targets that can reduce the impact of a maladapted stress system for the treatment of stress-induced addiction.

Orphan nuclear receptor subfamily NR4A their interplay with other nuclear receptors and functions in osteoblasts

Nurr1, NGFI-B and Nor1 (NR4A2, NR4A1 and NR4A3, respectively) belong to the NR4A subfamily of nuclear receptors. The NR4A receptors are orphan nuclear receptors which means that activating or repressing ligands for these receptors have not been found. NR4A expression is rapidly induced in response to various stimuli including growth factors and the parathyroid hormone (PTH). The studies concerning the NR4A receptors in the central nervous system have demonstrated that they have a major role in the development and function of the dopaminergic neurons of the midbrain and in regulating hypothalamus-pituitary-adrenal-axis. However, the peripheral functions of the NR4A family are largely unknown. Cultured mouse primary osteoblasts, a preosteoblastic cell line and several osteoblastic cell lines were used to investigate the role of NR4A receptors in osteoblasts. NR4A receptors were shown to directly bind to and activate the promoter of the osteopontin gene (OPN) in osteoblastic cells, thus regulating its expression. OPN is a major bone matrix protein expressed throughout the differentiation of preosteoblastic cells into osteoblasts. The activation of the OPN promoter was shown to be dependent on the activation function-1 located in the N-terminal part of Nurr1 and to occur in both monomeric and RXR heterodimeric forms of NR4A receptors. Furthermore, PTH was shown to upregulate OPN expression through the NR4A family. It was also demonstrated that the fibroblast growth factor-8b (FGF-8b) induces the expression of NR4A receptors in osteoblasts as immediate early genes. This induction involved phosphatidylinositol-3 kinase, protein kinase C, and mitogen activated protein kinase, which are all major pathways of FGF signalling. Nurr1 and NGFI-B were shown to induce the proliferation of preosteoblastic cells and to reduce their apoptosis. FGF-8b was shown to stimulate the proliferation of osteoblastic cells through the NR4A receptors. These results suggest that NR4A receptors have a role both in the differentiation of osteoblasts and in the proliferation and apoptosis of preosteoblast. The NR4A receptors were found to bind to the same response element on OPN as the members of the NR3B family of orphan receptors do. Mutual repression was observed between the NR4A receptors and the NR3B receptors. This repression was shown to be dependent on the DNA-binding domains of both receptor families, but to result neither from the competition of DNA binding nor from the competition for coactivators. As the repression was dependent on the relative expression levels of the NR4As and NR3Bs, it seems likely that the ratio of the receptors mediates their activity on their response elements. Rapid induction of the NR4As in response to various stimuli and differential expression of the NR3Bs can effectively control the gene activation by the NR4A receptors. NR4A receptors can bind DNA as monomers, and Nurr1 and NGFI-B can form permissive heterodimers with the retinoid X receptor (RXR). Permissive heterodimers can be activated with RXR agonists, unlike non-permissive heterodimers, which are formed by RXR and retinoic acid receptor or thyroid hormone receptor (RAR and TR, respectively). Non-permissive heterodimers can only be activated by the agonists of the heterodimerizing partner. The mechanisms behind differential response to RXR agonists have remained unresolved. As there are no activating or repressing ligands for the NR4A receptors, it would be important to find out, how they are regulated. Permissiviness of Nurr1/RXR heterodimers was linked to the N-terminal part of Nurr1 ligand-binding domain. This region has previously been shown to mediate the interaction between NRs and corepressors. Non-permissive RAR and TR, permissive Nurr1 and NGFI-B, and RXR were overexpressed with corepressors silencing mediator for retinoic acid and thyroid hormone receptors (SMRT), and with nuclear receptor corepressor in several cell lines. Nurr1 and NGFI-B were found to be repressed by SMRT. The interaction of RXR heterodimers with corepressors was weak in permissive heterodimers and much stronger in non-permissive heterodimers. Non-permissive heterodimers also released corepressors only in response to the agonist of the heterodimeric partner of RXR. In the permissive Nurr1/RXR heterodimer, however, SMRT was released following the treatment with RXR agonists. Corepressor release in response to ligands was found to differentiate permissive heterodimers from non-permissive ones. Corepressors were thus connected to the regulation of NR4A functions. In summary, the studies presented here linked the NR4A family of orphan nuclear receptors to the regulation of osteoblasts. Nurr1 and NGFI-B were found to control the proliferation and apoptosis of preosteoblasts. The studies also demonstrated that cross-talk with the NR3B receptors controls the activity of these orphan receptors. The results clarified the mechanism of permissiviness of RXR-heterodimers. New information was obtained on the regulation and functions of NR4A receptors, for which the ligands are unknown.

Prenatal exposure to maternal depression, neonatal methylation of human glucocorticoid receptor gene (NR3C1) and infant cortisol stress responses

In animal models, variations in early maternal care are associated with differences in hypothalamic-pituitary-adrenal(HPA) stress response in the offspring, mediated via changes in the epigenetic regulation of glucocorticoid receptor (GR) gene (Nr3c1) expression.

Amniotic fluid and umbilical cord plasma corticotropin-releasing factor (CRF), CRF-binding protein, adrenocorticotropin, and cortisol concentrations in intraamniotic infection and inflammation at term

Context: Pregnant tissues express corticotropin-releasing factor (CRF), a peptide modulating fetal and placental ACTH and cortisol secretion. These actions are modulated by the locally expressed CRF-binding protein (CRF-BP). Objective: The objective of the study was to determine whether CRF, CRF-BP, ACTH, and cortisol concentrations change in amniotic fluid and umbilical cord plasma in the presence of intraamniotic infection/inflammation (IAI) in women with spontaneous labor at term. Design: This was a cross-sectional study. Setting: The study was conducted at a tertiary referral center for obstetric care. Patients: Patients included women in active labor at term with (n = 39) and without (controls; n = 78) IAI. Main Outcome Measures: Amniotic fluid and umbilical cord plasma concentrations of CRF, CRF-BP, ACTH, and cortisol measured by RIA and immunoradiometric assays were measured. Results: In patients with IAI, amniotic fluid CRF (0.97 +/- 0.18 ng/ml) and CRF-BP (33.06 +/- 5.54 nmol/liter) concentrations were significantly (P < 0.001) higher than in controls (CRF: 0.32 +/- 0.04 ng/ml; CRF-BP: 14.69 +/- 2.79 ml). The umbilical cord plasma CRF and CRF-BP concentrations were significantly (P < 0.001 for all) higher in women with IAI than in controls (CRF: 2.96 +/- 0.35 ng/ml vs. 0.38 +/- 0.18 ng/ml; CRF-BP: 152.12 +/- 5.94 nmol/liter vs. 106.9 +/- 5.97 nmol/liter). In contrast, amniotic fluid and umbilical cord plasma ACTH and cortisol concentrations did not differ between groups. Conclusions: Amniotic fluid and umbilical cord plasma CRF and CRF-BP concentrations are increased in women with spontaneous labor at term and IAI. CRF-BP may modulate CRF actions on ACTH and cortisol secretion, playing a pivotal role in limiting the inflammatory process and thus avoiding an overactivation of the fetal/placental hypothalamus-pituitary-adrenal axis at birth.

Relationship between 24-hour urinary-free cortisol excretion and salivary cortisol levels sampled from awakening to bedtime in healthy subjects

The utility of repeated salivary cortisol sampling as a substitute for 24-hour urinary-free cortisol (UFC) assessment was examined. Forty-four participants completed both 24-hour collections and 6 salivary collections at wake-up, 08:00, 12:00, 16:00, 20:00 and bedtime, during the same 24-hour period. The results demonstrated that mean, maximum, and amplitude (maximum minus minimum) for salivary cortisol all correlated positively with urinary cortisol, but the associations of these variables with urinary-free cortisol excretion were relatively small. Furthermore, a single salivary sample taken at wake-up was as good an indicator of overall cortisol production as the measures derived from multiple salivary samples. An examination of subject compliance indicated that many subjects failed to collect the timed salivary collections as instructed. The authors conclude that diurnal salivary cortisol sampling versus 24-hour urinary cortisol collections are likely to provide different information about ambient hypothalamic-pituitary-adrenal productivity, and therefore these measures should not be used interchangeably. In addition, subject compliance is a serious consideration in designing studies that employ home salivary collections. Published by Elsevier Science Inc.

Circulating insulin-like factor 3 (INSL3) in healthy and infertile women

STUDY QUESTION: How does insulin-like factor 3 (INSL3) concentration in blood vary across the menstrual cycle in women? SUMMARY ANSWER: INSL3 is secreted by the theca interna cells of growing antral follicles and is phasic in its expression. WHAT IS KNOWN ALREADY: The relaxin-like hormone INSL3 is known to be expressed in follicles of several mammal species, and was recently shown in cows to be specifically secreted into the bloodstream by growing antral follicles, corresponding to follicular waves. In males INSL3 is known to be acutely independent of the hormones of the hypothalamic-pituitary-gonadal axis, suggesting that in women INSL3 might be a novel biomarker for antral follicle recruitment and development. STUDY DESIGN, SIZE, DURATION: Two cohorts of women were studied. First, 18 healthy women of reproductive age were followed longitudinally for one and a half cycles, with blood sampling and hormone measurement every 2-3 days. A second cohort comprised a cross-sectional study of 909 women attending an infertility clinic, with a single blood sample taken at entry, together with other clinical and hormonal parameters. PARTICIPANTS/MATERIALS, SETTING, METHODS: Blood samples from both retrospective cohorts were analyzed for INSL3 using a highly sensitive time-resolved fluorescent immunoassay, and data were analyzed in comparison with other clinical and hormonal parameters. MAIN RESULT AND THE ROLE OF CHANCE: For young healthy women of reproductive age, we showed a phasic expression of INSL3 corresponding to antral follicle growth in both the follicular and luteal phases of the cycle, which was significantly (P < 0.05) elevated compared with that during menses. For women attending an infertility clinic, those with diagnosed polycystic ovarian syndrome indicated significantly (P < 0.0005) greater circulating INSL3 levels and those with low ovarian reserve showed significantly (P < 0.002) decreased INSL3 values. LIMITATIONS, REASONS FOR CAUTION: These were retrospective studies and the results were obtained from natural cycles only, with their inherent variability. WIDER IMPLICATIONS OF THE FINDINGS: We show for the first time that INSL3 in women does vary across the menstrual cycle, and appears to reflect the number of growing antral follicles recruited within both follicular and luteal phases. STUDY FUNDING/COMPETING INTEREST(S): The present retrospective study was largely supported by departmental funds. There were no competing interests.

mRNA expression of corticotropin-releasing factor and urocortin 1 after restraint and foot shock together with alprazolam administration

Corticotropin-releasing factor (CRF) is expressed in the paraventricular nucleus of the hypothalamus (PVN), and act centrally to provoke stress-like autonomic and behavioral responses. Urocortins 1-3 are additional ligands to the CRF receptors 1 and 2. Ucn 1 neurons are primarily concentrated in the Edinger-Westphal (EW) nucleus and also have been associated with stress responses. It is also known that UCN 1 respond in different ways depending on the stressor presented. Benzodiazepines can act via the CRF peptidergic system and chronic administration of alprazolam does not interfere with CRF mRNA expression in the PVN, but significantly increase Ucn 1 mRNA expression in the EW. The aim of our study was to investigate the relationship between different stressor stimuli, foot shock (FS) and restraint (R), and the mRNA expression of CRF and Ucn 1 in the PVN and EW using alprazolam (A). We employed fos activation and in situ hybridization. Restraint group presented increased fos-ir and CRF mRNA expression in the PVN compared to FS group. The stress responses of R group were prevented by A. In the EW,fos-ir was higher in the FS group than in the R group, whereas Ucn 1 mRNA expression was higher in the R group than in the FS group. Alprazolam significantly increased fos-ir and Ucn 1 mRNA expression in both groups. Our results show that PVN and EW respond in different ways to the same stressors. Furthermore, EW of stressed animals replies in a complementary way comparing to PVN with the use of Alprazolam. (C) 2010 Elsevier Inc. All rights reserved.

Projections to the preoptic area from the paraventricular nucleus, acuate nucleus and the bed nucleus of the stria terminalis are unlikely to be involved in stress-induced suppression of GnRH secretion in sheep

Stress compromises reproductive function and the major physiological system activated during stress is the hypothalamo-pituitary-adrenal axis. Corticotrophin-releasing hormone and arginine vasopressin (AVP), which are produced in neurones of the paraventricular nucleus (PVN), drive the hypothalamo-pituitary-adrenal axis and are also implicated in the suppression of the reproductive axis. We used retrograde tracing and Fos labelling to map the projections from the PVN to the preoptic area (POA) where most gonadotrophin releasing hormone (GnRH) neurones are found. Fluorogold (FG) injections were made into the POA of gonadectomised male and female sheep (n = 5/sex), the animals were stressed and the brains recovered for histochemistry. All animals responded to stress with an increase in the number of Fos-labelled nuclei in the PVN. Few retrogradely labelled cells of the PVN were activated by stress. Dual labelling showed that very few FG-labelled cells also stained for corticotrophin-releasing hormone, none for AVP or enkephalin. Dual labelling for FG and Fos in the bed nucleus of the stria terminalis (BNST) and the arcuate nucleus showed that no FG-labelled cells in the BNST and only few in the ARC were activated by stress. No sex differences were observed in the activation of FG-labelled cells in any of the nuclei examined. We conclude that, although cells of the PVN, BNST and/or arcuate nucleus may affect reproduction via the GnRH cells of the POA, this is unlikely to involve direct input to the POA. If cells of these regions are involved in GnRH suppression during stress, this may occur via interneuronal pathways.