Persistent environmental pollutants were examined for their interaction with the glucocorticoid hormone system. The focus was placed on interference with the glucocorticoid synthesis and the glucocorticoid-signalling pathway in various in vitro test systems. Several aryl methyl sulphones competitively inhibited CYP11B1 activity in mouse adrenocortical Y1 cells. The DDT metabolite, 3-methylsulphonyl-2,2’-bis(4-chlorophenyl)-1,1’-dichloroethene (3-MeSO2-DDE) had a higher affinity to the enzyme than the endogenous substrate, 11-deoxycorticosterone. In fact, 3-MeSO2-DDE (Ki 1.6 μM) was almost as potent as the drug metyrapone (Ki 0.8 μM), a well-known inhibitor of the enzyme. 3-MeSO2-DDE inhibited CYP11B1 activity in human adrenocortical H295R carcinoma cells, and at higher concentrations the CYP21 activity. The human H295R cell line seems to be a useful test system for studies of enzyme activities and could be used to screen endocrine disrupting chemicals interfering with the glucocorticoid hormone synthesis. Several chiral PCB methyl sulphones and the fungicide tolylfluanid proved to be antagonists to the glucocorticoid receptor (GR) in rat hepatoma cells and/or Chinese hamster ovary cells stable transformed with a human GR and a responsive reporter vector. The 4-methylsulphonyl-2,3,6,2’,4’,5’-hexachlorobiphenyl (4-MeSO2-CB149) enantiomers had similar antagonistic effect on the GR. Co-exposure of substances led to additive inhibitory effects on glucocorticoid-regulated protein synthesis in rat hepatoma cells. In general, 4-substituted but not 3-substituted methylsulphonyl-PCBs interacted with the glucocorticoid hormone system. In the environment, humans and wildlife are constantly exposed to a wide range of chemicals. Considering the effects of these substances via mechanisms of actions described in this thesis, interference of xenobiotics with the glucocorticoid hormone system deserves further attention. In conclusion, environmental pollutants can interact with the glucocorticoid hormone system in vitro, yet the effects of the tested substances on this hormone system remain to be established in vivo.

Following social defeat, male hamsters exhibit behavioral changes characterized by a breakdown of normal territorial aggression and an increase in submissive/defensive behaviors in the presence of a non-aggressive intruder (NAI). We have termed this phenomenon conditioned defeat (CD). By contrast, only a small subset of defeated females exhibit submissive/defensive behavior in the presence of a NAI. We hypothesized that fluctuations in gonadal hormones might contribute to differences in the display of submissive behavior in intact female hamsters. Following social defeat, proestrous females (higher endogenous estradiol) were more likely to display conditioned defeat compared with diestrous 1 (lower endogenous estradiol) females. This finding suggests that there is an estrous cycle-dependent fluctuation in the display of CD in female hamsters and suggests that increased estradiol might contribute to increased submissive behavior. We then demonstrated that ovariectomized females given estradiol prior to CD testing exhibited significantly higher submissive behavior in the presence of a NAI suggesting that estradiol increases the expression of CD in female hamsters. We have also shown that castrated males that were singly housed for four weeks displayed significantly more submissive behavior than did their intact counterparts. Interestingly, castrated and intact males that were singly housed for 10 days prior to behavioral testing displayed similar behavior during CD testing. Together these data suggest that androgens and isolation modulate the display of CD in male hamsters. Finally, we examined brain activation following CD testing in defeated males and females (in diestrus 1 and proestrus). Defeated male and proestrous females exhibited increased Fos activation in the dorsal lateral septum and hypothalamic paraventricular nucleus relative to defeated diestrous 1 females. Diestrous 1 females exhibited increased Fos expression in the lateral bed nucleus of the stria terminalis compared with both defeated groups. Collectively, these data suggest that gonadal hormones and duration of individual housing modulate the display of CD in female and male hamsters and that those animals which display CD exhibit differences in patterns of neuronal activation than do those that do not display CD.

published_or_final_version / Medicine / Master / Doctor of Medicine

The Hypothalamic Pituitary Adrenal (HPA) axis is the coordinator of adaptive responses to physical and psychological stress. The central nervous system plays a key role in modulation of both basal and adaptive HPA axis functions. In fact, since long ago, animal studies have shown that acute and chronic exposure to glucocorticoids (a stress hormone released due to HPA axis activation, cortisol in humans) affects the function and the morphology of brain areas such as the hippocampus and the cingulate cortex. This thesis is based on novel neuroimaging methodologies used to investigate the interactions of psychological stress, cortisol and the brain. It consists of three functional studies and a morphometric one. In the first functional study we show that the hippocampus (where glucocorticoid receptors are most abundant) plays a role in initiation of an HPA axis stress response. In the second study, we provide evidence that besides hippocampus, the neural activity in the so-called "default mode network" (DMN), especially the anterior cingulate cortex (ACC), relates to interindividual variations in HPA axis response to psychological stress. In the third study we have investigated the cortisol-modulation of the DMN. Again, we provide evidence for a role of the ACC and the orbitofrontal cortex in negative feedback inhibition of the HPA axis activity. Finally, we show a morphological link between the ACC and the cortisol response to awakening which is an index of basal HPA axis activity. Overall, our findings confirm the critical role of the ACC and mesolimbic system in HPA axis regulation. These findings also draw attention to the interactions between functional subregions of the medial prefrontal cortex and states of HPA axis function prior to stress onset---suggesting an interplay of the monitoring and the executive planning roles of the medial prefrontal cortex in behavioral adaptation to stress. Beyond stress research, our findings offer a framework for combining neuroimaging and neuroendocrinology to better understand the interindividual variances in behavior, and perhaps to better identify subgroups at risk of psychological disorders.

No description available.

This project investigated postnatal development of the adrenocortical function in captive American kestrels (Falco sparverius) employing measurements of basal and stress-induced levels of corticosterone at specific developmental stages. Chicks aged 10-days exhibited partially functioning adrenocortical systems with baseline levels comparable to adults. The ability to respond to external stressors increased through postnatal development and by the age of 22 days, stress-induced maximal levels of corticosterone were indistinguishable from those of one-year old adults, and levels of 28-day old birds were significantly higher than these adults. In addition, baseline and maximum stress-induced levels of corticosterone at all ages were significantly higher in first-hatched chicks than all other siblings and these effects grew stronger through development. These results suggest that the brain-pituitary-adrenal axis in this semi-altricial species is (1) already partially developed in young chicks and (2) only becomes fully functional when behavioral and neuromuscular development is nearly complete. Furthermore, results from this study suggest that hatching asynchrony has an effect on this variation in stress-induced maximal levels of corticosterone during the latter half of postnatal development, with a higher degree of hatching asynchrony leading to larger disparity in adrenocortical function between first- and fourth-hatched chicks. This adrenocortical disparity resulting from female-mediated hatching asynchrony may potentially lead to both brood-reduction and brood survival under diametric food conditions, ensuring that the female's reproductive fitness is maximized in varying habitats. Variation of adrenocortical function among siblings may increase female efficiency in raising a brood of fit chicks, maximizing her reproductive success.

La majorité des hyperplasies macronodulaires bilatérales des surrénales avec syndrome de Cushing ACTH-indépendant (AIMAH) est due à l’expression aberrante de divers récepteurs hormonaux au niveau du cortex surrénalien. Les gènes responsables des AIMAH familiales avec récepteurs aberrants n’ont pas été identifiés. Le but de ce projet est de les identifier. Une étude de liaison, visant à identifier la ou les régions du génome comprenant le ou les gènes pouvant être en cause dans les AIMAH familiales, a été réalisée en utilisant l’ADN des membres d’une famille (10 malades et 7 sains) originaire du Québec, atteinte d’AIMAH et syndrome de Cushing et caractérisée par l’expression des récepteurs β-adrénergique et V1-vasopressine. Diverses régions chromosomiques entre les personnes atteintes et non-atteintes de la famille ont été soulignées. Un total de 707453 SNPs a été obtenu, et après analyse statistique, 159 SNPs significatifs, pouvant être associés au phénotype, ont été mis en évidence entre les deux groupes. Il a été constaté que la majorité de ces SNPs se situaient sur les régions chromosomiques 1q32.1 et 16q12.2. Une étude du transcriptome a aussi été réalisée en utilisant l’ADN des tumeurs de deux patients de la famille, ainsi que l’ADN d'autres tumeurs surrénaliennes. Les analyses statistiques ont permis d’identifier 15 gènes susceptibles d’être reliés à la maladie (11 surexprimés et 4 sous-exprimés). En utilisant les données de ces deux études, nous avons ciblé six gènes du chromosome 1 (ATP2B4, PPP1R12B, SOX13, CACNA1S, ADORA1et PHLDA3), un du chromosome 16 (CHD9) et un du chromosome 13 (SPRY2), afin de rechercher la présence de mutations. Le séquençage n’a révélé aucun changement de nucléotide dans les gènes PPP1R12B et SOX13. Dans les gènes ATP2B4, CACNA1S, ADORA1et PHLDA3, le séquençage a révélé des changements de nucléotides n’entrainant soit pas de changement d’acide aminé soit un changement d’acide aminé jugé « non pertinent », du fait qu’il ne permettait pas de différencier les sujets sains des sujets atteints. Pour ce qui est de CHD9 et SPRY2, le séquençage a permis d’identifier des changements de nucléotides entrainant des changements d’acides aminés de façon plus fréquente chez les sujets atteints par rapport aux sujets sains. En conclusion, nos travaux nous ont donc permis d’identifier, par étude de liaison et par analyse du transcriptome, des gènes candidats qui pourraient être responsables de cette pathologie. Le séquençage de ces gènes candidats a révélé des mutations de CHD9 et SPRY2. Ces résultats s’avèrent prometteurs puisque ces deux gènes produisent des protéines impliquées dans le remodelage de la chromatine et dans la régulation de la signalisation des protéines kinases. Le phénotypage et le génotypage des patients atteints doivent être poursuivis pour vérification. / The majority of ACTH-independent macronodular adrenal hyperplasia with Cushing's syndrome (AIMAH) is due to the aberrant expression of various receptors in the adrenal cortex. The genes responsible for familial AIMAH with aberrant receptors have not been identified. The aim of this project is to characterize them. A linkage study to identify the region or regions of the genome comprising the gene or genes that may be involved in familial AIMAH was performed using DNA of family members (10 affected and 7 non affected) born in Quebec and harboring AIMAH and Cushing's syndrome, under the aberrant regulation of B-adrenergic and V1-vasopressin receptors. Various chromosomal regions between patients and non-affected family were highlighted. A total of 707,453 SNPs were obtained, and after statistical analysis, 159 significant SNPs, possibly associated with phenotype, were found between the two groups. It was found that the majority of these SNPs were located on chromosomal regions 1q32.1 and 16q12.2. A transcriptome analysis was conducted using DNA from tumours of two patients of the family, as well as DNA from other adrenal tumours; Statistical analysis identified 15 genes that may be linked to disease (11 up-regulated and 4 under-expressed). Using data from these two studies, we identified six genes on chromosome 1 (ATP2B4, PPP1R12B, SOX13, ADORA1, CACNA1S and PHLDA3), one on chromosome 16 (CHD9) and one on chromosome 13 (SPRY2), to investigate the presence of mutations. The sequencing revealed no nucleotide changes in gene PPP1R12B and SOX13. In ATP2B4, CACNA1S, ADORA1 and PHLDA3, the sequencing not revealed nucleotides changes leading to either amino acid changes or an amino acid changes considered “not-relevant”, because they do not differentiate healthy individuals from affected. The sequencing of CHD9 and SPRY2 identified nucleotide changes causing amino acid changes more frequently in patients compared to healthy subjects. In conclusion, our work has therefore identified by linkage analysis and DNA microarray candidate genes that can be responsible to this disease, and mutations in two of these genes, CHD9 and SPRY2. These results are promising because these genes produce proteins involved in chromatin remodeling and regulation of signaling protein kinases. Phenotyping and genotyping of patients should be pursued further.

Arginine vasopressin (AVP) is a hypothalamic nonapeptide which regulates the hypothalamic-pituitary-adrenal axis response to stress by stimulating the secretion of adrenocorticotropin (ACTH) from corticotroph cells of the anterior pituitary. This effect is mediated by binding of AVP to the pituitary vasopressin receptor (V1bR). The V1bR belongs to the G protein-coupled receptor (GPCR) super family. Repeated stimulation of anterior pituitary cells with AVP has been shown to produce a loss of responsiveness to subsequent AVP stimulation. This phenomenon appears to be mediated by desensitisation of the V1bR, and may be due to phosphorylation of the receptor by G protein-coupled receptor kinase 5 (GRK5). The aim of this research was to establish and validate methods that would allow the role of GRK5 in the desensitisation of V1bR to AVP stimulation to be investigated. As no isoform specific inhibitors for GRK5 were available, HEK293 cells transiently transfected with the rat V1bR were used as a model system for this research. This allowed RNA interference (RNAi) to be used to knockdown GRK5 expression. The protocol for RNAi-mediated knockdown of GRK5 was established as part of this research. Protocols for Western blotting and qRT-PCR were also established to allow the RNAi-mediated knockdown of GRK5 protein and mRNA to be measured. Transfection of HEK293 cells with 10nM GRK5-targeting small interfering RNAs (siRNAs) reduced the expression of GRK5 protein to 53.4% ± 3.4% (mean ± SEM) of that seen in untreated control cells at 84 hours after transfection, while GRK5 mRNA levels were reduced to 28.7% ± 1.9% (mean ± SEM) of that of control cells 48 hours after transfection. An experimental protocol was designed in this research that would coordinate the RNAi-mediated knockdown of GRK5 with transient transfection of the HEK293 cells with the rV1bR. Since, activated V1bRs couple to Gq/11 and stimulate the production of inositol phosphates (IPs), the responsiveness of the V1bR can be determined by measuring the accumulation of [H³]-IPs in cells labelled with [H³]-myo-inositol. In the protocol designed, the effect of GRK5 knockdown on V1bR desensitisation is determined by stimulating HEK293 cells expressing the rV1bR (and previously transfected with GRK5-targeting siRNA) with 0nM or 100nM AVP for 0, 5, 15, 30 or 60 minutes, and comparing the accumulation if IPs over time with that of cells that are not transfected with GRK5-targeting siRNA. This protocol can be used in future to investigate the role of GRK5 in V1bR desensitisation, and may be adapted to determine if other GRK isoforms are involved in V1bR desensitisation.

No description available.

Maternal stress during the perinatal period has been linked to attention and behavioral problems and increased adrenocortical activity in children. Underlying this relationship is thought to be exposure to excessive glucocorticoids during development. The aim of this set of studies was to determine the effects of chronic maternal stress (CMS) during the perinatal period on behaviour and endocrine function in male and female guinea pig offspring at the juvenile and adult life stage. Environmental enrichment was investigated as a potential therapeutic tool to reverse changes induced by CMS. Pregnant guinea pigs were exposed to a sequence of stressors every other day over the second half of gestation until weaning on postnatal day 25. Offspring were tested for ambulatory activity, attention, cognitive function, sex-steroid levels and adrenocortical function. One cohort of animals were housed in an enriched environment, the remaining offspring were housed in standard conditions. A separate cohort was administered amphetamine (1 mg/kg) prior to behavioural testing to determine influence of CMS on dopaminergic function. Juvenile male and female offspring of mothers exposed to stress exhibited increased basal and decreased stress-induced salivary cortisol, and male offspring displayed reduced activity and a phase shift in their circadian rhythm of activity. Adult male offspring of CMS mothers exhibited increased activity in a novel environment and decreased activity in a familiar environment. Female adult offspring of CMS mothers exhibited reduced attention and increased activity in a novel environment. Enrichment acted independently of CMS, increasing plasma testosterone and attention in adult male offspring and reducing the adrenocortical response to stress and decreasing attention and activity in female offspring. Amphetamine decreased activity in a novel environment and increased activity in a familiar environment in both sexes, regardless of age or maternal treatment. Amphetamine improved attention in juvenile and adult males. The current studies demonstrated a strong effect of CMS on behaviour in juvenile and adult offspring. Enrichment was not effective for attenuating the effects of CMS. These studies clearly demonstrate behavioural changes as a result of CMS emerge over the lifetime of the offspring and have begun to uncover the underlying mechanisms of programming.