Studio di Mutazioni Geniche e Variazioni Copy-Number implicate nel deficit combinato degli Ormoni Ipofisari(CPHD)

La crescita normale di un individuo è il risultato dell’azione coordinata di molteplici ormoni e recettori codificati da geni e a tal proposito, discreto interesse è stato dato ai geni tipici dell’asse del GH. Tuttavia altri geni, più a monte di questi e responsabili dello sviluppo dell’ipofisi contribuiscono alla crescita normale o patologica. Alcuni geni studiati sono POU1F1, PROP1, LHX3, LHX4, HESX1, SOX3 e svariate loro mutazioni sono state identificate come causa di panipopituarismo (CPHD=Combined Pituitary Hormone Deficiency). In realtà la ricerca genetica non spiega ancora molte anomalie ipofisarie e molte mutazioni devono ancora essere identificate. Uno degli scopi del dottorato, svoltosi nel laboratorio di Genetica molecolare di Pediatria, è stata l’identificazione di mutazioni geniche da un gruppo di pazienti CPHD considerando in particolare i geni POU1F1, LHX3, SOX3, non ancora messi a punto presso il laboratorio. L’approccio sperimentale si è basato sulle seguenti fasi: prelievo delle informazioni di sequenza da GeneBank, progettazione di primers per amplificare le porzioni esoniche, messa a punto delle fasi della PCR e del sequenziamento, analisi della sequenza e confronto con le informazioni di sequenza depositate allo scopo di rintracciare eventuali mutazioni o varianti. La bassa percentuale di mutazioni in questi geni non ha permesso finora di rintracciare mutazioni nelle porzioni esoniche salvo che in un soggetto, nell’esone 6 di LHX3b (nuova mutazione, recessiva eterozigote, c.1248A>G implicata nella mutazione p.T377A della sequenza proteica). Un metodo di screening di questa mutazione impiegando l’enzima di restrizione SacII è stato usato, senza rilevare nessun altra occorrenza dell’allele mutato in 53 soggetti di controllo. Oltre alla messa a punto del sequenziamento e di alcune tecniche di analisi di singoli SNP o piccoli INDELs per i 3 geni, la ricerca svolta è stata orientata all’impiego di metodi di rilevamento di riarrangiamenti genetici comportanti ampie delezioni e/o variazioni del copy-number di esoni/interi geni detto MLPA (Multiplex Ligation-dependent Probe Amplification) e progettato da MRC-Holland. Il sequenziamento infatti non permette di rilevare tali alterazioni quando sono ampie ed in eterozigosi. Per esempio, in un’ampia delezione in eterozigosi, l’intervallo delimitato dai primers usati per la PCR può non includere totalmente la porzione interessata da delezione su un cromosoma cosicché la PCR ed il sequnziamento si basano solo sulle informazioni dell’altro cromosoma non deleto. Un vantaggio della tecnica MLPA, è l’analisi contemporanea di una quarantina di siti posti su svariati geni. Questa metodo tuttavia può essere affetto da un certo margine di errore spesso dipendente dalla qualità del DNA e dovrebbe essere affiancato e validato da altre tecniche più impegnativa dal punto di vista sperimentale ma più solide, per esempio la Real Time PCR detta anche PCR quantitativa (qPCR). In laboratorio, grazie all’MLPA si è verificata la condizione di delezione eterozigote di un paziente “storico” per il gene GH1 e la stessa mutazione è stata rilevata anche con la qPCR usando lo strumento Corbett Rotor Gene 6000 (Explera). Invece un’analisi solo con la qPCR di variazioni del copy-number (CNV) per SOX3 in pazienti maschili non ha ancora evidenziato anomalie. Entrambe le tecniche hanno aspetti interessanti, il miglior approccio al momento sembra un’analisi iniziale di pazienti con l’MLPA, seguita dalla verifica di un eventuale esito anomalo impiegando la real-time PCR.

Genetics of GHRH, GHRH-receptor, GH and GH-receptor: its impact on pharmacogenetics

When a child is not following the normal, predicted growth curve, an evaluation for underlying illnesses and central nervous system abnormalities is required and, appropriate consideration should be given to genetic defects causing GH deficiency (GHD). Because Insulin-like-Growth Factor-I (IGF-I) plays a pivotal role, GHD could also be considered as a form of IGF-I deficiency (IGFD). Although IGFD can develop at any level of the GHRH-GH-IGF axis, a differentiation should be made between GHD (absent to low GH in circulation) and IGFD (normal to high GH in circulation). The main focus of this review is on the GH-gene, the various gene alterations and their possible impact on the pituitary gland. However, although transcription factors regulating the pituitary gland development may cause multiple pituitary hormone deficiency they may present initially as GHD. These defects are discussed in various different chapters within this book, whereas, the impact of alterations of the GHRH-, GHRH-receptor- --as well as the GH-receptor (GHR) gene--will be discussed here.

Isolated GH deficiency type II: knockdown of the harmful Delta3GH recovers wt-GH secretion in rat tumor pituitary cells

Isolated GH deficiency type II (IGHD II) is the autosomal dominant form of GHD. In the majority of the cases, this disorder is due to specific GH-1 gene mutations that lead to mRNA missplicing and subsequent loss of exon 3 sequences. When misspliced RNA is translated, it produces a toxic 17.5-kDa GH (Delta3GH) isoform that reduces the accumulation and secretion of wild-type-GH. At present, patients suffering from this type of disease are treated with daily injections of recombinant human GH in order to maintain normal growth. However, this type of replacement therapy does not prevent toxic effects of the Delta3GH mutant on the pituitary gland, which can eventually lead to other hormonal deficiencies. We developed a strategy involving Delta3GH isoform knockdown mediated by expression of a microRNA-30-adapted short hairpin RNA (shRNA) specifically targeting the Delta3GH mRNA of human (shRNAmir-Delta3). Rat pituitary tumor GC cells expressing Delta3GH upon doxycycline induction were transduced with shRNAmir-Delta3 lentiviral vectors, which significantly reduced Delta3GH protein levels and improved human wild-type-GH secretion in comparison with a shRNAmir targeting a scrambled sequence. No toxicity due to shRNAmir expression could be observed in cell proliferation assays. Confocal microscopy strongly suggested that shRNAmir-Delta3 enabled the recovery of GH granule storage and secretory capacity. These viral vectors have shown their ability to stably integrate, express shRNAmir, and rescue IGHD II phenotype in rat pituitary tumor GC cells, a methodology that opens new perspectives for the development of gene therapy to treat IGHD patients.

HYPERANDROGENISM DUE TO 3-BETA-HYDROXYSTEROID DEHYDROGENASE-DEFICIENCY WITH ACCESSORY ADRENOCORTICAL TISSUE - A HORMONAL AND METABOLIC EVALUATION

1. Adrenal ectopic tissue has been detected in the paragonadal region of normal women. In patients with congenital adrenal hyperplasia due to 21-hydroxylase (21-OH) deficiency, the manifestation of hyperplasia of paragonadal accessory adrenal tissue has been usually reported to occur in males. Probably, this is the first report of a female with 3beta-hydroxysteroid dehydrogenase (3beta-HSD) deficiency with ectopic adrenal tissue in ovaries. However, the occurrence of hyperplasia of adrenal rests among women with classical congenital adrenal hyperplasia may not be rare, especially among patients with a late diagnosis.2. We report a woman with 3beta-HSD deficiency whose definitive diagnosis was made late at 41 years of age immediately before surgery for the removal of a uterine myoma. During surgery, exploration of the abdominal cavity revealed the presence of bilateral accessory adrenal tissue in the ovaries and in the para-aortic region. The patient had extremely high levels of ACTH (137 pmol/l), DHEA (901.0 nmol/l), DHEA-S (55.9 mumol/l), androstenedione (70.2 nmol/l), testosterone (23.0 nmol/l) and 17alpha-hydroxypregnenolone (234.4 nmol/l) suggesting 3beta-HSD deficiency.3. In view of these elevated androgen levels, with an absolute predominance of DHEA and DHEA-S, we evaluated the effect of this hormonal profile on carbohydrate tolerance and insulin response to glucose ingestion.4. The patient presented normal glucose tolerance but her insulin response was lower than that of 14 normal women (area under the curve, 3beta-HSD = 17,680 vs 50,034 pmol/l for the control group over a period of 3 h after glucose ingestion).5. These results support recent data suggesting that patients with increased serum DHEA and DHEA-S levels do not present resistance to insulin.

Impact of estrogen replacement throughout childhood on growth, pituitary-gonadal axis and bone in a 46,XX patient with CYP19A1 deficiency

The adequate replacement dose of estrogens during infancy and childhood is still not known in girls. Aromatase deficiency offers an excellent model to study how much estrogens are needed during infancy, childhood and adulthood.

Isolated growth hormone deficiency type 2: from gene to therapy

Isolated growth hormone deficiency type-2 (IGHD-2), the autosomal-dominant form of GH deficiency, is mainly caused by specific splicing mutations in the human growth hormone (hGH) gene (GH-1). These mutations, occurring in and around exon 3, cause complete exon 3 skipping and produce a dominant-negative 17.5 kD GH isoform that reduces the accumulation and secretion of wild type-GH (wt-GH). At present, patients suffering from IGHD-2 are treated with daily injections of recombinant human GH (rhGH) in order to reach normal height. However, this type of replacement therapy, although effective in terms of growth, does not prevent toxic effects of the 17.5-kD mutant on the pituitary gland, which can eventually lead to other hormonal deficiencies. Considering a well-known correlation between the clinical severity observed in IGHD-2 patients and the increased expression of the 17.5-kD isoform, therapies that specifically target this isoform may be useful in patients with GH-1 splicing defects. This chapter focuses on molecular strategies that could represent future directions for IGHD-2 treatment.

Ovarian and uterine development and hormonal feedback mechanism in a 46 XX patient with CYP19A1 deficiency under low dose estrogen replacement.

Abstract Background: Aromatase deficiency may result in a complete block of estrogen synthesis because of the failure to convert androgens to estrogens. In females, this results in virilisation at birth, ovarian cysts in prepuberty and lack of pubertal development but virilisation, thereafter. Objective and methods: We studied the impact of oral 17β-estradiol treatment on ovarian and uterine development, and on LH/FSH and inhibin B during the long-term follow-up of a girl harboring compound heterozygote point mutations in the CYP19A1 gene. Results: In early childhood, low doses of oral 17β-estradiol were needed. During prepuberty treatment with slowly increasing doses of E2 resulted in normal uterine and almost normal development of ovarian volume, as well as number and size of follicles. Regarding hormonal feedback mechanisms, inhibin B levels were in the upper normal range during childhood and puberty. Low doses of estradiol did not suffice to achieve physiological gonadotropin levels in late prepuberty and puberty. However, when estradiol doses were further increased in late puberty levels of both FSH and LH declined with estradiol levels within normal range. Conclusion: Complete aromatase deficiency provides an excellent model of how ovarian and uterine development in relation to E2, LH, FSH and inhibin B feedback progresses from infancy to adolescence.

Genetic Basis of Pituitary Adenoma Predisposition

Much of the global cancer research is focused on the most prevalent tumors; yet, less common tumor types warrant investigation, since A rare disorder is not necessarily an unimportant one . The present work discusses a rare tumor type, the benign adenomas of the pituitary gland, and presents the advances which, during the course of this thesis work, contributed to the elucidation of a fraction of their genetic background. Pituitary adenomas are benign neoplasms of the anterior pituitary lobe, accounting for approximately 15% of all intracranial tumors. Pituitary adenoma cells hypersecrete the hormones normally produced by the anterior pituitary tissue, such as growth hormone (GH) and prolactin (PRL). Despite their non-metastasizing nature, these adenomas can cause significant morbidity and have to be adequately treated; otherwise, they can compromise the patient s quality of life, due to conditions provoked by hormonal hypersecretion, such as acromegaly in the case of GH-secreting adenomas, or due to compressive effects to surrounding tissues. The vast majority of pituitary adenomas arise sporadically, whereas a small subset occur as component of familial endocrine-related tumor syndromes, such as Multiple Endocrine Neoplasia type 1 (MEN1) and Carney complex (CNC). MEN1 is caused by germline mutations in the MEN1 tumor suppressor gene (11q13), whereas the majority of CNC cases carry germline mutations in the PRKAR1A gene (17q24). Pituitary adenomas are also encountered in familial settings outside the context of MEN1 and CNC, but unlike in the latter syndromes, their genetic background until recently remained elusive. Evidence in previous literature supported the notion that a tumor suppressor gene on 11q13, residing very close to but still distinct from MEN1, causes genetic susceptibility to pituitary tumors. The aim of the study was to identify the genetic cause of a low penetrance form of Pituitary Adenoma Predisposition (PAP) in families from Northern Finland. The present work describes the methodological approach that led to the identification of aryl hydrocarbon receptor interacting protein (AIP) as the gene causing PAP. Combining chip-based technologies (SNP and gene expression arrays) with traditional gene mapping methods and genealogy data, we showed that germline AIP mutations cause PAP in familial and sporadic settings. PAP patients were diagnosed with mostly adenomas of the GH/PRL-secreting cell lineage. In Finland, two AIP mutations accounted for 16% of all patients diagnosed with GH-secreting adenomas, and for 40% of patients being younger than 35 years of age at diagnosis. AIP is suggested to act as a tumor suppressor gene, a notion supported by the nature of the identified mutations (most are truncating) and the biallelic inactivation of AIP in the tumors studied. AIP has been best characterized as a cytoplasmic interaction partner of aryl hydrocarbon receptor (AHR), also known as dioxin receptor, but it has other partners as well. The mechanisms that underlie AIP-mediated pituitary tumorigenesis are to date largely unknown and warrant further investigation. Because AIP was identified in the genetically homogeneous Finnish population, it was relevant to examine its contribution to PAP in other, more heterogeneous, populations. Analysis of pituitary adenoma patient series of various ethnic origins and differing clinical settings revealed germline AIP mutations in all cohorts studied, albeit with low frequencies (range 0.8-7.4%). Overall, PAP patients were typically diagnosed at a young age (range 8-41 years), mainly with GH-secreting adenomas, without strong family history of endocrine disease. Because many PAP patients did not display family history of pituitary adenomas, detection of the condition appeared challenging. AIP immunohistochemistry was tested as a molecular pre-screening tool on mutation-positive versus mutation-negative tumors, and proved to be a potentially useful predictor of PAP. Mutation screening of a large cohort of colorectal, breast, and prostate tumors did not reveal somatic AIP mutations. These tumors, apart from being the most prevalent among men and women worldwide, have been associated with acromegaly, particularly colorectal neoplasia. In this material, AIP did not appear to contribute to the pathogenesis of these common tumor types and other genes seem likely to play a role in such tumorigenesis. Finally, the contribution of AIP in pediatric onset pituitary adenomas was examined in a unique population-based cohort of sporadic pituitary adenoma patients from Italy. Germline AIP mutations may account for a subset of pediatric onset GH-secreting adenomas (in this study one of seven GH-secreting adenoma cases or 14.3%), and appear to be enriched among young (≤25 years old) patients. In summary, this work reveals a novel tumor susceptibility gene, namely AIP, which causes genetic predisposition to pituitary adenomas, in particular GH-secreting adenomas. Moreover, it provides molecular tools for identification of individuals predisposed for PAP. Further elaborate studies addressing the functional role of AIP in normal and tumor cells will hopefully expand our knowledge on endocrine neoplasia and reveal novel cellular mechanisms of pituitary tumorigenesis, including potential drug targets.

Histology and hormonal effect of Ghrelin on ovary of Benni (Barbus sharpeyi) in order to study sexual maturity, zygote and larval generation

Benni (Barbus sharpeyi) is valuable fish that Khuzastan fisheries office propagated it artificially in Susangerd Fish Propagation Center every year. Pituitary gland is used for this aim but female fish lost their fertilization power after 2-3 years, so in present research, new hormone, that is called Ghrelin. The aims of this research are histology, hormonal, zygote and larval generation studies and comparing the results with each other. Ghrelin is a multifunctional peptidyl hormone which increases GTH-II in fish, amphibian, and birds and mammalian so its effect on Benni sexual maturation was studied. Human Ghrelin (hGRL) was obtained from ANASPEC, Canada, with 28 amino acids. In the present study, three levels of ghrelin including 0 (sham treatments), 0.10 (treatment 1) and 0.15 μg/g (treatment 2) body wt and one level of pituitary gland 4000 μg/g (pituitary treatment) with two replications were used. 56 specimens were injected intraperitonealy and their ghrelin level was evaluated immediately after injection and after 24 h. Control fish(n=16) were just injected by physiological saline. For hormonal studies sham and experimental fish(n=40) were anesthetized with MS-222 at a concentration of 250 mg l-1, and blood samples were collected and kept at 4ْC, then spun to collect serum. Serum samples were stores at -20ْC until the RIA for CTH-II. For histology studies immediately after injection a piece of ovary was collected from control fish (Sham zero) after being anesthetized. The sampled ovaries were fixed in Buin solution and embedded in paraffin, and stained to Sections of 5–6 μm using haematoxylin and eosin. The ovarian samples were performed with a compound microscope. Histology and micrometry studies had done. The mature oocytes had given from mature fish, then weighted and the working fecundity were counted. The mature oocytes fertilized, the eggs were incubated and the percentage of fertilization was calculated. After 72h the eggs hatched and the percentage of hatch was counted. The percentage of hindrance was calculated after 6 days. Hormonal results indicate that ghrelin and pituitary increase significantly the GTH-II level in comparison to sham. Macroscopic observations (before taking ovary) showed that ovaries with green colored have couple oval structure located in the abdominal cavity. Microscopic studies of dissected ovaries indicated simultaneous growth of 127 oocytes with 6 stages. The type of the ovary is asynchronous. The results indicated that both of the ghrelin treatment increased the percentage of mature follicles followed by decrease of immature follicles. There were significant differences (P<0.05) between the number of mature and immature follicles. Average diameter of follicle in both of the ghrelin treatment was significantly (P<0.05) declined in the stages of the vitellogenesis when the result compared to the other treatment. Just treatment 1 and pituitary treatment can give mature oocytes. The fecundity of pituitary treatment significantly increase in comparision to ghrelin treatment (P<0.05). In food-restricted fish where endogenous ghrelin levels are known to be increased, a chronic administration of ghrelin induces overt negative effect in releasing mature oocytes. The percentage of fertilization was significantly increase (P<0.05) in ghrelin t. in comparison to pituitary t. and the percentage of hatch was significantly increase (P<0.05) in pituitary t. in comparison to ghrelin t. There was no significant difference (P>0.05) in terms of percentage of hindrance between treatments. In conclusion, the present study demonstrated that ghrelin has positive effect on the level of GTH-II, oocyte maturation, ovarian vitellogenesis and the number of mature follicles of Barbus sharpeyi ovary. Increasing of the mature follicles number reduces their average diameter, indicating stimulating effect of ghrelin in sexual maturation of Barbus sharpeyi.The ghrelin and pituitary treatment have equal chance in the post-stage of spawning.

HORMONAL REPLACEMENT THERAPY IN FISH - HUMAN GROWTH-HORMONE GENE-FUNCTION IN HYPOPHYSECTOMIZED CARP

Transgenic common carp, Cyprinus carpio, produced by the microinjection of fertilized eggs with a linearized chimeric plasmid pMThGH, a human growth hormone (hGH) gene with a mouse metallothionein-I (MT) gene promoter in pBR322, were used to produce F1 and F2 transgenics. Following hypophysectomy of the transgenic F2 common carp, non-transgenic common carp and non-transgenic crucian carp, growth was monitored for up to 110 days. In addition, recombinant hGH was injected subcutaenously into a group of the non-transgenic crucian carp. Growth rate analyses indicated that (1) hypophysectomy of non-transgenic common carp and crucian carp results in the cessation of growth, (2) hGH administration can stimulate the growth of hypophysectomized crucian carp and (3) hypophysectomized hGH-transgenic common carp continue to grow in the absence of their own growth hormone, suggesting that the hGH-transgene is being expressed in tissues other than the pituitary.

Molecular basis for estrogen receptor alpha deficiency in BRCA1-linked breast cancer

Background BRCA1-mutant breast tumors are typically estrogen receptor alpha (ER alpha) negative, whereas most sporadic tumors express wild-type BRCA1 and are ER alpha positive. We examined a possible mechanism for the observed ER alpha-negative phenotype of BRCA1-mutant tumors.

Methods We used a breast cancer disease-specific microarray to identify transcripts that were differentially expressed between paraffin-embedded samples of 17 BRCA1-mutant and 14 sporadic breast tumors. We measured the mRNA levels of estrogen receptor 1 (ESR1) ( the gene encoding ER alpha), which was differentially expressed in the tumor samples, by quantitative polymerase chain reaction. Regulation of ESR1 mRNA and ER alpha protein expression was assessed in human breast cancer HCC1937 cells that were stably reconstituted with wild-type BRCA1 expression construct and in human breast cancer T47D and MCF-7 cells transiently transfected with BRCA1-specific short-interfering RNA ( siRNA). Chromatin immunoprecipitation assays were performed to determine if BRCA1 binds the ESR1 promoter and to identify other interacting proteins. Sensitivity to the antiestrogen drug fulvestrant was examined in T47D and MCF-7 cells transfected with BRCA1-specific siRNA. All statistical tests were two-sided.

Results Mean ESR1 gene expression was 5.4-fold lower in BRCA1-mutant tumors than in sporadic tumors ( 95% confidence interval [CI]=2.6-fold to 40.1-fold, P =.0019). The transcription factor Oct-1 recruited BRCA1 to the ESR1 promoter, and both BRCA1 and Oct-1 were required for ER alpha expression. BRCA1-depleted breast cancer cells expressing exogenous ER alpha were more sensitive to fulvestrant than BRCA1-depleted cells transfected with empty vector ( T47D cells, the mean concentration of fulvestrant that inhibited the growth of 40% of the cells [IC40] for empty vector versus ER alpha: > 10(-5) versus 8.0 x 10(-9) M [ 95% CI=3.1x10(-10) to 3.2 x 10(-6) M]; MCF-7 cells, mean IC40 for empty vector versus ER alpha : > 10(-5) versus 4.9 x 10(-8) M [ 95% CI=2.0 x 10(-9) to 3.9 x 10(-6) M]).

Conclusions BRCA1 alters the response of breast cancer cells to antiestrogen therapy by directly modulating ER alpha expression.

Neuroendocrinology of Pyridoxine Deficiency

Veuruenducrim lri v j p .rim, deficienc:v. NEUROSCI BIOBEHAV REV 12(3/4) 189-193. 1988.- Dihydroxyphenylalanine decarboxvlase and 5-hydroxytryptophan decarboxvlase respectively have high and low affinities for pyridoxal phosphate. In the pyridoxinedeficient animal. hypothalamic serotonin content is significantly reduced without any change in catecholamine levels. Hypothalamic neurotransmitters affect the hvpothalamo-pituitary-end organ axes. Specifically, the decrease in hypothalamic serotonin in the pyridoxine-deficient rat results in tertiary hypothyroidism. In addition. pineal function is affected in deficient animals due to decreased synthesis of melatonin.

Hormonal regulation of the Menkes and Wilson copper-transporting ATPases in human placental Jeg-3 cells

Copper deficiency during pregnancy results in early embryonic death and foetal structural abnormalities including skeletal, pulmonary and cardiovascular defects. During pregnancy, copper is transported from the maternal circulation to the foetus by mechanisms which have not been clearly elucidated. Two coppertransporting ATPases, Menkes (ATP7A; MNK) and Wilson (ATP7B; WND), are expressed in the placenta and both are involved in placental copper transport, as copper accumulates in the placenta in both Menkes and Wilson disease. The regulatory mechanisms of MNKand WNDand their exact role in the placenta are unknown. Using a differentiated polarized Jeg-3 cell culture model of placental trophoblasts, MNK and WND were shown to be expressed within these cells. Distinct roles forMNKandWND are suggested on the basis of their opposing responses to insulin. Insulin and oestrogen increased both MNK mRNA and protein levels, altered the localization of MNK towards the basolateral membrane in a copper-independent manner, and increased the transport of copper across this membrane. In contrast, levels of WND were decreased in response to insulin, and the protein was located in a tight perinuclear region, with a corresponding decrease in copper efflux across the apical membrane. These results are consistent with a model of copper transport in the placenta in which MNK delivers copper to the foetus and WND returns excess copper to the maternal circulation. Insulin and oestrogen stimulate copper transport to the foetus by increasing the expression of MNK and reducing the expression of WND. These data show for the first time that MNK and WND are differentially regulated by the hormones insulin and oestrogen in human placental cells.

Investigating the Effects of Mifepristone (RU486) in the Behavioral, Hormonal and Central Responses to Acute Stress

Depression is associated with glucocorticoid hypersecretion, due to dysfunction of the hypothalamo-pituitary-adrenocorticol axis (HPA-axis). Because excess glucocorticoids are associated with depressive-like features in humans, glucocorticoid receptor antagonists are currently being tested for antidepressant efficacy in clinical trials. In the current study the hypothesis that mifepristone (RU486), a glucocorticoid receptor antagonist, would decrease the neuroendocrine and central HPA-axis responses to an acute stressor and attentuate depressive like behavior in an animal model of behavioral helplessness (forced swim test) was tested. Adult male rats were treated with 10 mglkg RU486 (subcutaneous) for five days and then exposed to a IO-minute forced swim test (FST), conducted in Plexiglas cylinders. FST sessions were videotaped for later analysis of behavioral immobility. Plasma ACTH and corticosterone CORT were measured at 15min and 90min after FST cessation. Animals were perfused and brains were collected for immunocytochemical assessment of c-Fos expression in the medial prefrontal cortex (mPFC), a brain region implicated in both depression and central control of the HPA axis. RU486 significantly decreased peak ACTH and CORT concentrations following FST exposure. In addition, glucocorticoid negative feedback was at1enuated in RU486-treated animals exposed to the FST. Exposure to FST alone induced c-FOS expression in the mPFC, as measured by the number of c-Fos positive neurons. Treatment with RU486 significantly increased the number of rnPFC c-Fos positive cell following FST exposure. The behavioral data obtained from FST paradigm, demonstrated that RU486 decreased immobility in the FST illustrating the potential efficacy of this drug as an antidepressant. Collectively these data suggest that RU486 dampens HPA-axis responses to stress, possibly by enhancing the excitability of stress-inhibitory neurons in the mPFC. This is particularly exciting, given the fact that this neural region is associated with decreased neural activity during depression in humans.