Biomedical subjects

E R De Kloet

Publications and source records attributed to E R De Kloet.

At least 19 recordsLinked to original sources

PubMed2006

The dynamic pattern of glucocorticoid receptor-mediated transcriptional responses in neuronal PC12 cells.

The aim of the current study was (i) to examine the overlap in the pattern of glucocorticoid receptor (GR)-mediated transcriptional responses between different neuronal substrates and (ii) to assess the nature of these responses by differentiating between primary and downstream GR-responsive genes. For this purpose, nerve growth factor-differentiated catecholaminergic PC12 cells were used in which endogenous GRs were activated briefly with a high dose of corticosterone followed by gene expression profiling 1 and 3 h afterwards using Affymetrix GeneChips. The results revealed a strikingly similar temporal pattern to that which was reported previously in hippocampus, with only down-regulated genes 1 h after GR activation and the majority of genes up-regulated 3 h after GR activation. Real-time quantatitive PCR of transcripts in cycloheximide-treated cells showed that all five GR-responsive genes selected from the 1-h time point were primary responsive, whereas all four GR-responsive genes selected from the 3-h time point were downstream responsive. At the level of individual genes, the overlap with the previously generated hippocampal data sets was small, illustrating the cell-type specifity of GR-mediated genomic responses. Finally, we identified a number of interesting genes, such as SWI/SNF, synaptosomal-associated protein 25 and certain Rab proteins which may play a role in the effects of glucocorticoids on catecholaminergic neuronal functioning.

Animals↗

PubMed2006

Acute activation of hippocampal glucocorticoid receptors results in different waves of gene expression throughout time.

Several aspects of hippocampal cell function are influenced by adrenal-secreted glucocorticoids in a delayed, genomic fashion. Previously, we used Serial Analysis of Gene Expression to identify glucocorticoid receptor (GR)-induced transcriptional changes in the hippocampus at a fixed time point. However, because changes in mRNA levels are transient and most likely precede the effects on hippocampal cell function, the aim of the current study was to assess the transcriptional changes in a broader time window by generating a time curve of GR-mediated gene expression changes. Therefore, we used rat hippocampal slices obtained from adrenalectomised rats, substituted in vivo with low corticosterone pellets, predominantly occupying the hippocampal mineralocorticoid receptors. To activate GR, slices were treated in vitro with a high (100 nM) dose of corticosterone and gene expression was profiled 1, 3 and 5 h after GR-activation. Using Affymetrix GeneChips, a striking pattern with different waves of gene expression was observed, shifting from exclusively down-regulated genes 1 h after GR-activation to both up and down regulated genes 3 h after GR-activation. After 5 h, the response was almost back to baseline. Additionally, real-time quantitative polymerase chain reaction was used for validation of a selection of responsive genes including genes involved in neurotransmission and synaptic plasticity such as the corticotropin releasing hormone receptor 1, monoamine oxidase A, LIMK1 and calmodulin 2. This permitted confirmation of GR-responsiveness of 15 out of 18 selected genes. In conclusion, direct activation of GR in hippocampal slices results in transient changes in gene expression. The pattern in which gene expression was modulated suggests that the fast genomic effects of glucocorticoids may be realised via transrepression, preceding a later wave of transactivation. Furthermore, we identified a number of interesting candidate genes which may underlie the glucocorticoid-mediated effects on hippocampal cell function.

Animals↗

PubMed2004

Localization of mRNA expression of P-glycoprotein at the blood-brain barrier and in the hippocampus.

The multidrug resistance (mdr) P-glycoprotein is an energy-dependent efflux transporter that protects the brain against a wide variety of neurotoxic compounds. This transmembrane protein is a well-known functional component of the blood-brain barrier and might be present in other brain cells as well. We have developed a riboprobe against the murine mdr1 mRNA recognizing both isoforms of the rodent mdr1 gene to determine the exact localization of P-glycoprotein expression. We have also studied the effects of treatment with a known inducer of P-glycoprotein expression. In situ mRNA hybridization demonstrates that mdr1 mRNA is present in the endothelial cells of brain capillaries throughout the rat brain, indicating that P-glycoprotein is expressed at the endothelial cells forming the blood-brain barrier. Surprisingly, specific mdr1 mRNA expression was also found in neuronal layers of hippocampal fields, particularly in the granule cells of the dentate gyrus. Kainic acid treatment decreased the expression levels of mdr1 mRNA in the dentate gyrus 6 and 24 h after treatment. Our data indicate that P-glycoprotein is expressed by endothelial cells and possibly dentate gyrus neurons The functional role of P-glycoprotein at dentate gyrus neurons is presently unknown.

ATP Binding Cassette Transporter, Subfamily B↗

PubMed2003

Regulation of the developing hypothalamic-pituitary-adrenal axis in corticotropin releasing hormone receptor 1-deficient mice.

During postnatal development, mice undergo a so-called stress hyporesponsive period, which is characterized by low basal corticosterone levels and the inability of mild stressors to induce a corticosterone response. The stress hyporesponsiveness is in part regulated by maternal factors. Twenty-four hours of deprivation results in an activation of basal and stress-induced corticosterone and a down-regulation of corticotropin releasing hormone (CRH), mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) expression in the brain. It has been hypothesized that the CRH receptor 1 (CRHr1) may play an important regulatory role during development by mediating the effects of maternal deprivation. Using CRHr1-deficient mice we examined the role of this receptor on the maternal deprivation effects and in regulating the expression of hypothalamic-pituitary-adrenal axis-related genes. We could demonstrate that the CRHr1 is essential for the activation of the corticosterone response following maternal deprivation, most likely due to the lack of the receptor in the pituitary. Furthermore, we could show that the CRHr1 is regulating the expression of CRH and MRs. In contrast, effects of maternal deprivation during postnatal development on GRs are not mediated by this receptor.

Animals↗

PubMed2002

The role of the efflux transporter P-glycoprotein in brain penetration of prednisolone.

In the present study, we have investigated the role of the multidrug resistance (mdr) P-glycoprotein (Pgp) at the blood-brain barrier in hampering the access of the synthetic glucocorticoid, prednisolone. In vivo, a tracer dose of [(3)H]prednisolone poorly penetrated the brain of adrenalectomised wild-type mice, but the uptake was more than threefold enhanced in the absence of Pgp expression in mdr1a (-/-) mice. In vitro, in stably transfected LLC-PK1 monolayers the human MDR1 P-glycoprotein was able to transport prednisolone present at a micromolar concentration. A specific Pgp blocker, LY 335979, could block this polar transport of [(3)H]prednisolone. Human Pgp does not transport all steroids, as cortexolone was not transported at all and aldosterone was only weakly transported. The ability of Pgp to export the synthetic glucocorticoid, prednisolone, suggests that uptake of prednisolone in the human brain is impaired, leading to a discrepancy between central and peripheral actions. Furthermore, the ensuing imbalance in activation of the two types of brain corticosteroid receptors may have consequences for cognitive performance and mood.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2001

Intracerebroventricular administration of a glucocorticoid receptor antagonist enhances the cardiovascular responses to brief restraint stress.

Intracerebroventricular (i.c.v.) administration of the glucocorticoid receptor antagonist 17beta-hydroxy-11beta(4-dimethylamino-phenyl)17alpha-(1-propynyl)estra-4,9dien-3one (RU38486) in conscious rats slowly increased systolic blood pressure as assessed with the indirect tail cuff method. However, direct measurement of blood pressure in freely moving rats did not reveal changes in blood pressure after i.c.v. injection of this antagonist either in the light or in the dark phase. In the present study, the hypothesis is tested that aspects of the tail cuff procedure, involving heat (30 min, 32 degrees C) and brief restraint stress, are necessary conditions to detect the glucocorticoid receptor-mediated cardiovascular effect. Freely moving rats equipped with a telemetric transmitter to directly measure heart rate and blood pressure were injected i.c.v. with either the glucocorticoid receptor or the mineralocorticoid receptor antagonist and were either left undisturbed for 24 h, or were subjected to the tail cuff procedure at 1.5, 6.5 and 23.5 h after injection. Then after 30-min warming and during brief restraint, blood pressure and heart rate showed a rapid increase. The mineralocorticoid receptor antagonist administered i.c.v. did not affect these stress-induced increases in cardiovascular responses. The glucocorticoid receptor antagonist i.c.v. significantly increased the heart rate and pressor response at 24 h. In the undisturbed rats, neither basal heart rate nor blood pressure were affected by either antagonist during the circadian cycle. In conclusion, the blockade of central glucocorticoid receptor causes a long-lasting facilitation of the stress-induced pressor and heart rate response, which does not require a 2-week training to the condition of heat and stress.

Animals↗

PubMed2001

Hippocampal apoptosis in major depression is a minor event and absent from subareas at risk for glucocorticoid overexposure.

Glucocorticoid (GC) overexposure in animals has been implicated in hippocampal dysfunctioning and neuronal loss. In major depression, hypercortisolemia, hypothalamic-pituitary-adrenocortical-axis alterations, and reduced hippocampal volumes are commonly observed; hence, hippocampal neurodegeneration is also expected. To study possible GC-related pathology, we investigated hippocampal tissue of 15 major-depressed patients, 16 matched controls, and 9 steroid-treated patients, using in situ-end-labeling for DNA fragmentation and apoptosis, and heat-shock protein 70 and nuclear transcription factor kappaB immunocytochemistry for damage-related responses. No obvious massive cell loss was observed in any group. In 11 of 15 depressed patients, rare, but convincing apoptosis was found in entorhinal cortex, subiculum, dentate gyrus, CA1, and CA4. Also in three steroid-treated patients, apoptosis was found. Except for several steroid-treated patients, heat-shock protein 70 staining was generally absent, nor was nuclear transcription factor-kappaB activation found. The detection in 11 of 15 depressed patients, in three steroid-treated, and in one control patient, demonstrates for the first time that apoptosis is involved in steroid-related changes in the human hippocampus. However, in absence of major pyramidal loss, its rare occurrence, that notably was absent from areas at risk for GC damage such as CA3, indicates that apoptosis probably only contributes to a minor extent to the volume changes in depression.

Adolescent↗

PubMed2001

Blood pressure, heart rate, and behavioral responses to psychological "novelty" stress in freely moving rats.

We developed a new model of psychological "open-field" stress in freely moving rats. Blood pressure and heart rate of the rats were measured by radiotelemetry and behavior analyzed by video tracking software. Open-field exposure induced marked increases in blood pressure and heart rate. Repeated daily exposure induced pressor responses that were slightly higher on Day 4 when compared to Day 1. Pretreatment with the beta1-adrenoceptor antagonist atenolol inhibited the tachycardia whereas the ganglion blocker pentolinium inhibited the pressor response, indicating involvement of the sympathetic nervous system. Pretreatment with diazepam prevented the novelty stress-induced pressor response and reduced the tachycardia. These results show that the psychological stress of exposing rats to an open field induces marked cardiovascular effects that are mediated by sympathetic hyperactivity. This model is unique in that it focuses on psychological stress and allows concomitant measurement of blood pressure, heart rate, and behavior in freely moving rats.

Animals↗

PubMed2001

Chronic psychosocial stress differentially affects apoptosis in hippocampal subregions and cortex of the adult tree shrew.

We studied the effect of chronic psychosocial stress on cell death and volume changes in the tree shrew hippocampus. In situ end labelling (ISEL) identified low frequent but convincing apoptosis in many hippocampal subregions. Also in entorhinal cortex, apoptosis was found, generally at higher frequencies. After 28 days of chronic stress, apoptosis was significantly reduced in the CA1 stratum radiatum, whereas an increase was observed in the hilus (P < 0.04). With all subregions taken together, the hippocampus showed a decrease, whereas in the cortex, an increase in apoptosis was found after stress (P < 0.04). In a parallel and similar chronic stress study, post mortem morphometry of the same brain regions was performed, revealing mild decreases (7.6%) in entire hippocampal volume. We conclude that (i) low frequent apoptosis occurs throughout the adult tree shrew brain, and (ii) 28 days of chronic stress differentially affects its occurrence in distinct hippocampal subregions and entorhinal cortex. As previous stereological investigations failed to detect any loss in the principal neuronal layers, psychosocial stress, therefore, must affect other (structural) parameters like dendritic tree, interneurons, neurogenesis, or glia.

Animals↗

PubMed2000

Effects of flesinoxan on corticosteroid receptor expression in the rat hippocampus.

Many agents that influence serotonergic neurotransmission modulate expression of hippocampal corticosteroid receptors. We have studied the effect of the specific 5-hydroxytryptamine, 5-HT(1A), receptor agonist flesinoxan on mRNA for glucocorticoid and mineralocorticoid receptors in the hippocampus and dorsal raphe nucleus. Since some responses to 5-HT(1A) receptor stimulation show a strong desensitization, we studied the effect of a single and repeated injections of flesinoxan. Because of the close interrelationship between the serotonergic system and the hypothalamo-pituitary-adrenal axis, we also studied the possible involvement of corticosterone as a mediator of the effects of flesinoxan. We found that a single injection of flesinoxan (3 and 10 mg/kg subcutaneously, s.c.) after 3 h leads to a downregulation of glucocorticoid receptor mRNA in the hippocampus (dentate gyrus and CA1 areas) and dorsal raphe nucleus. This effect does not desensitize after a second treatment over 2 days. Mineralocorticoid receptor mRNA expression remained unaltered. The decrease in hippocampal glucocorticoid receptor mRNA expression occurs independently of circulating corticosterone since flesinoxan reduced glucocorticoid receptor mRNA in the hippocampus of adrenalectomized rats with or without corticosterone replacement. These data indicate that the 5-HT(1A) receptor agonist flesinoxan alters glucocorticoid receptor expression via a direct pathway independently of corticosterone and argues for an intrinsic effect selective for hippocampal glucocorticoid receptor mRNA.

Animals↗

PubMed2000

The amount of free corticosterone is increased during lipopolysaccharide-induced fever.

The relation between lipopolysaccharide (LPS)-induced fever and bioavailability of corticosterone (B) was examined in male Wistar rats. Animals were injected with LPS (2.5 mg/kg i.p.) or saline and core temperature and heart rate were monitored continuously using a biotelemetry system. Blood samples were withdrawn from freely moving rats via jugular catheters for estimation of total and free plasma B. LPS induced a long-lasting increase (24-48 h) in core temperature and B secretion and a short-lasting increase (90 min) in heart rate. LPS-induced fever was accompanied by a significant increase in the free/total B ratio. In contrast, an acute injection of B, which resulted in circulating B levels similar to those found after LPS, did not affect the free/total B ratio. The important role of LPS-induced fever in the hormone secretion pattern and the equilibrium between free and total B was further demonstrated in an in vitro study showing that an increase in the temperature by 3 degrees C elevated the free B fraction and the free/total B ratio of plasma samples with concentrations of B in the physiological range (5-40 microg/dl). Taken together, these findings indicate that during LPS-induced fever there is an increase in the amount of biologically available B. Exposure of glucocorticoid-sensitive targets to elevated levels of free B could contribute to the restoration of homeostasis that is disturbed during inflammation.

Animals↗

PubMed2000

Maternal deprivation affects behaviour from youth to senescence: amplification of individual differences in spatial learning and memory in senescent Brown Norway rats.

Previous studies have shown that deprivation of the infant rat from maternal care has pronounced effects on the stress system during ontogeny. Here we test the hypothesis that 24 h of maternal deprivation at postnatal day 3 will cause persistent changes in behaviour. Spatial learning and memory of male Brown Norway rats deprived as infants were observed in the Morris water maze at 3, 12, 24 and 30-32 months of age (young, adult, aged, senescent). Their nondeprived mother-reared littermates served as controls. (i) With increasing age, water maze performance declined in deprived and nondeprived groups. However, once the task was learned the animals maintained their good performance during retest at later ages. (ii) Maternal deprivation delayed acquisition until adulthood and caused at every age a higher degree of persistent behaviour as judged from the performance of deprived rats' free swim trials and reversal trials. (iii) At senescence the mean performance in the water maze did not differ between the groups. Instead, the individual performance was strikingly different within each group. Senescent deprived rats were either nonimpaired or impaired with only a few animals showing an intermediate performance. Thus, a large group of animals ( approximately 40%) ages successfully as they are resistant to the effect of maternal deprivation. In contrast, the majority of the control animals displayed intermediate performance. Taken together, maternal deprivation has life-long consequences for behaviour and culminates at senescence in amplification of individual differences in learning ability rather than in a generalized deterioration of cognitive functions.

Aging↗

PubMed2000

Altered glucocorticoid regulation of the immune response in the chronic fatigue syndrome.

It is increasingly recognized that glucocortiocoids (GCs) can have subtle modulatory effects in immunoregulation rather than having generalized immunosuppressive effects. GCs suppress Th1 cells and cellular immunity, but may favor Th2 responses and humoral immunity. The chronic fatigue syndrome (CFS) appears to be associated with a disturbed HPA-axis. Moreover, CFS patients show several immunological changes suggestive of decreased cellular immunity. It is postulated herein that in CFS patients a decreased Th1/Th2 balance may be the result of selective effects of GC on the IL-10/IL-12 regulatory circuit.

Fatigue Syndrome, Chronic↗

PubMed2000

Differential expression and regional distribution of steroid receptor coactivators SRC-1 and SRC-2 in brain and pituitary.

Members of the p160 family of steroid receptor coactivator proteins mediate the stimulatory effects on gene transcription brought about by nuclear receptors, which comprise all steroid receptors. Using in situ hybridization we have examined the neuroanatomical distribution of the messenger RNAs (mRNAs) for two functionally distinct splice variants of Steroid Receptor Coactivator 1 (SRC-1/NCoA-1) and of Steroid Receptor Coactivator 2 (SRC-2/NCoA-2/GRIP-1/TIF-2). Transcripts encoding these coactivators show highly differential expression patterns. SRC-2 mRNA is expressed at very low levels in brain, but shows expression in the anterior pituitary. SRC-la and le mRNA are expressed in many brain areas, including hippocampus, amygdala, hypothalamus, basal ganglia, and isocortex. Striking differences between SRC-1a and le expression were observed in several brain nuclei. Relative levels of SRC-1a mRNA were much higher in anterior pituitary, and the arcuate, paraventricular and ventromedial nucleus of the hypothalamus, the locus coeruleus and the trigeminal motor nucleus, all important targets of steroid hormones in the brain. SRC-le mRNA showed modest elevation of relative expression in the caudal nucleus accumbens (shell), basolateral amygdala, and some thalamic nuclei. The differential and uneven neuroanatomical distribution of these coactivators may underlie diversity and cell-specificity of steroid receptor mediated signals in the brain.

Amygdala↗

PubMed2000

Corticosterone effects on BDNF expression in the hippocampus. Implications for memory formation.

The adrenal steroid corticosterone has profound effect on the structure and function of the hippocampus. Probably as a result of that, it modulates memory formation. In this review, the question is addressed if the corticosterone effects on memory processes are mediated by alterations in the expression of the neurotrophin Brain-Derived Neurotrophic Factor (BDNF) in the hippocampus. First, studies are described investigating the effect of corticosterone on BDNF expression in the rat hippocampus. It appears that corticosterone suppresses the BDNF expression at the mRNA and protein level in a subfield-specific way. Second, a model for the mechanism of action is proposed. In this model, activated mineralocorticoid and glucocorticoid receptors repress transcriptional activity of the BDNF promoter site-specifically via interaction with other transcription factors. Third, the implications for learning and memory are discussed. Studies show that during water maze training, corticosterone levels rise significantly, but the BDNF expression is not suppressed in any hippocampal subfield. Furthermore, high BDNF expression levels in specific subfields correlate with a good memory performance. Therefore, we suggest that the resistance of the hippocampal BDNF expression to suppression by corticosterone, as seen after water maze training, may contribute to an optimal memory performance.

Adrenalectomy↗

PubMed1999

Corticosterone effects on BDNF mRNA expression in the rat hippocampus during morris water maze training.

Corticosterone and Brain-Derived Neurotrophic Factor (BDNF) have both been shown to be involved in spatial memory formation in rats. In the present study we have investigated the effect of corticosterone on hippocampal BDNF mRNA expression after training in the Morris water maze in young adult Wistar rats. Therefore, we first studied BDNF mRNA levels in the hippocampus in relation to corticosterone levels at several time points after 4 training trials in the Morris water maze. Corticosterone levels were significantly increased after this procedure, and hippocampal BDNF mRNA levels only displayed a minor change: an increase in CA1 at 1 hr after training. However, in a previous study we observed dramatically decreased hippocampal BDNF mRNA levels in dentate gyrus and CA1 at 3 hr after injection of corticosterone. In order to analyze this discrepancy, we subsequently investigated if hippocampal BDNF mRNA expression is affected by corticosterone at 3 hr after water maze training. Therefore, we incorporated ADX animals and ADX animals which were injected with corticosterone in our study. ADX animals which were subjected to water maze training displayed similar hippocampal BDNF mRNA levels 3 hr after training compared to control ADX animals. Furthermore, ADX animals which were injected with corticosterone showed decreased BDNF mRNA levels in all hippocampal regions compared to control ADX animals. Water maze training did not alter this effect. Thus, the increased corticosterone levels during water maze training do not affect hippocampal BDNF mRNA expression, although exogenous corticosterone is effective under these conditions. Hence, our results suggest that in this situation BDNF is resistant to regulation by endogenous corticosterone, which may be important for learning and memory processes.

Adrenalectomy↗

PubMed1998

Flesinoxan treatment reduces 5-HT1A receptor mRNA in the dentate gyrus independently of high plasma corticosterone levels.

Flesinoxan acts as a full 5-HT1A receptor agonist and displays anxiolytic and anti-depressant properties. 5-HT1A receptor agonists, including flesinoxan, increase corticosterone (B) levels in the blood and reduces 5-HT1A receptor mRNA expression in the hippocampus. In this study, we examined whether the 5-HT1A receptor downregulation induced by flesinoxan involves corticosterone control of 5-HT1A receptor gene transcription. In experiment I, intact male Wistar rats (180-200 g) were treated with flesinoxan (1.0, 3.0 and 10 mg/kg bw, sc) or vehicle and decapitated 3 h later. Flesinoxan administration resulted in a significant, dose-dependent downregulation of 5-HT1A receptor mRNA in the dentate gyrus and dorsal raphe nucleus. In experiment II, rats were sham-operated and implanted with a cholesterol pellet (100 mg) or were adrenalectomized and implanted with a corticosterone pellet (20 mg corticosterone + 80 mg cholesterol). Flesinoxan injection also caused a dose-dependent decrease of 5-HT1A mRNA in the dentate gyrus of adrenalectomized animals with corticosterone replacement. There was no effect in the dorsal raphe nucleus. In experiment III, adrenalectomized and adrenalectomized + corticosterone rats were sc injected with flesinoxan (10 mg/kg bw) or vehicle, and flesinoxan appeared to downregulate 5-HT1A receptor expression in the dentate gyrus independently of corticosterone as well. No significant effects were observed in the dorsal raphe nucleus. It is concluded that flesinoxan reduces 5-HT1A receptor expression in the dentate gyrus both through homologous downregulation and a corticosterone-mediated effect on the serotonergic (5-HT) system.

Animals↗

PubMed1998

Glucocorticoid negative feedback on the HPA axis in five inbred rat strains.

The aim of the present work was to study the influence of altering glucocorticoid negative feedback on both basal activity of the hypothalamic-pituitary-adrenal (HPA) axis and its response to acute stress (tail shock) in five inbred rat strains known to differ in some depression-like behaviors: Brown Norway (BN), Fischer 344 (F344), Lewis (Lew), spontaneously hypertensive (SHR), and Wistar-Kyoto (WKY) rats. Two complementary approaches were used: 1) enhancement of negative feedback by administration of 0.05 and 0.2 mg/kg dexamethasone (Dex) and 2) attenuation of negative feedback by pharmacological adrenalectomy (PhADX). The results indicate that 1) Lew rats consistently show adrenocorticotropic hormone (ACTH) and corticosterone hyporesponsiveness to stress, 2) interstrain differences in the effect of Dex on the HPA axis were very weak and not related apparently to differences in the metabolism of the steroid, 3) the suppressive effect of the highest dose of Dex on basal corticosterone levels was lower in BN rats than in the other strains, and 4) after PhADX, an increase in ACTH levels was observed in response to acute stress in BN, F344, and WKY but not in Lew and SHR rats, suggesting possible interstrain differences in pituitary sensitivity to neural stimuli induced by stress. In summary, our results indicate that there are differences among the strains with regard to both 1) the suppressive effect of Dex on the HPA axis, BN rats showing a certain degree of resistance, and 2) the capability of PhADX rats to respond to acute stress, which suggests a defective release of ACTH in Lew and SHR rats. The biological meaning of these alterations of corticosteroid negative feedback among the five inbred strains studied remains to be established.

Adrenal Glands↗