Co-localization of brain corticosteroid receptors in the rat hippocampus.

New developments in corticosteroid receptor research enabled us to perform a highly detailed study on the neuroanatomical topography of MR and GR in the rat hippocampus. Receptor immunocytochemistry was used to map the distribution of GR protein with the help of a monoclonal antibody raised against the purified rat liver GR-hormone complex. Furthermore, in situ hybridization with 35S-labeled RNA probes, which were transcribed from cDNAs complementary to either a fragment of the rat brain MR gene or to the rat liver GR gene, was applied to investigate the localization of MR and GR mRNA in the limbic brain. The pyramidal neurons of cell field Ca1 and CA2 and the granular neurons of the dentate gyrus showed marked GR immunoreactivity (GRir) as well as intense labeling of GR mRNA. The radiolabeled density of GR mRNA in cell fields CA3 and CA4 was considerable less, whereas low-to-almost-undetectable levels of GRir could be observed in these regions. MR mRNA appeared to be evenly distributed over all cell fields of the hippocampus and the dentate gyrus. The topography of GRir, GR mRNA and MR mRNA was found to agree with the cellular distribution of MR and GR binding sites in the hippocampus. Moreover, the microanatomy of MR and GR in the hippocampus appeared to overlap. Our data strongly suggest that MR and GR are co-expressed in the majority of pyramidal and granular neurons of the hippocampal formation. This assumption is based on coherence in the detection of different aspects of the receptor cycle of MR and GR.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cocaine withdrawal causes delayed dysregulation of stress genes in the hippocampus

Relapse, even following an extended period of withdrawal, is a major challenge in substance abuse management. Delayed neurobiological effects of the drug during prolonged withdrawal likely contribute to sustained vulnerability to relapse. Stress is a major trigger of relapse, and the hippocampus regulates the magnitude and duration of stress responses. Recent work has implicated hippocampal plasticity in various aspects of substance abuse. We asked whether changes in stress regulatory mechanisms in the hippocampus may participate in the neuroadaptations that occur during prolonged withdrawal. We therefore examined changes in the rat stress system during the course of withdrawal from extended daily access (5-hours) of cocaine self-administration, an animal model of addiction. Tissue was collected at 1, 14 and 28 days of withdrawal. Plasma corticosterone levels were determined and corticosteroid receptors (GR, MR, MR/GR mRNA ratios) and expression of other stress-related molecules (HSP90AA1 and HSP90AB1 mRNA) were measured in hippocampal subfields using in situ hybridization. Results showed a delayed emergence of dysregulation of stress genes in the posterior hippocampus following 28 days of cocaine withdrawal. This included increased GR mRNA in DG and CA3, increased MR and HSP90AA1 mRNA in DG, and decreased MR/GR mRNA ratio in DG and CA1. Corticosterone levels progressively decreased during the course of withdrawal, were normalized following 28 days of withdrawal, and were correlated negatively with GR and positively with MR/GR mRNA ratio in DG. These results suggest a role for the posterior hippocampus in the neuroadaptations that occur during prolonged withdrawal, and point to a signaling partner of GR, HSP90AA1, as a novel dysregulated target during cocaine withdrawal. These delayed neurobiological effects of extended cocaine exposure likely contribute to sustained vulnerability to relapse.     

Corticosteroid receptor mRNA expression in the brains of patients with epilepsy

The effects of corticosteroids in the brain are mediated through the glucocorticoid receptor (GR) and the mineralocorticoid receptor (MR). We used a sensitive competitive RT-PCR assay to quantify the amounts of GR and MR mRNA in human brain tissue specimens from patients with focal epilepsies. GR and MR mRNAs were expressed at approximately the same levels in the temporal lobe, frontal lobe, and hippocampus as compared to tissues with high glucocorticoid/mineralocorticoid receptor expression (liver/kidney). GR and MR mRNA concentrations in the temporal lobe increased markedly during childhood and reached adult levels at puberty. GR and MR mRNA expression was significantly higher in the temporal lobe and frontal lobe cortex of women than in those of men. In women, MR and GR mRNA concentrations were markedly lower in hippocampal tissue than in frontal and temporal lobe cortex tissue. In conclusion, our data demonstrate sex- and site-dependent expression of corticosteroid receptor mRNA in the human brain.     

Hippocampal glucocorticoid receptor expression in the tree shrew: Regulation by psychosocial conflict

Summary 1. This study was conducted to determine whether chronic psychosocial conflict alters the expression of glucocorticoid receptor (GR) mRNA in the hippocampus of male tree shrews (Tupaia belangeri).2. To generate probes for thein situ hybridization, the tree shrew GR gene was partly cloned. There was a 90% homology between the deduced amino acid sequence of the cloned tree shrew GR and that of the corresponding human GR sequence.³⁵S-Labeled riboprobes which had been transcribed from the tree shrew GR clone hybridized to pyramidal neurons in all subregions of the tree shrew hippocampal formation and to granule neurons in the dentate gyrus.3. Afterin situ hybridization, the expression of GR mRNA was semiquantitatively determined by counting silver grains over single neurons of the hippocampal formation of psychosocially stressed tree shrews and control animals. After 12 days of social conflict, the number of silver grains in the CA1 and CA3 pyramidal neurons of stressed animals was significantly lower than in controls. No statistically significant differences in mRNA expression were observed in the pyramidal neurons of the subiculum and in the granule neurons of the dentate gyrus.4. The present results suggest that psychosocial stress leads to a site-specific down-regulation of hippocampal GR via modification of mRNA expression.     

Regulation of Kainate Receptor Subunit mRNA by Stress and Corticosteroids in the Rat Hippocampus

Kainate receptors are a class of ionotropic glutamate receptors that have a role in the modulation of glutamate release and synaptic plasticity in the hippocampal formation. Previous studies have implicated corticosteroids in the regulation of these receptors and recent clinical work has shown that polymorphisms in kainate receptor subunit genes are associated with susceptibility to major depression and response to anti-depressant treatment. In the present study we sought to examine the effects of chronic stress and corticosteroid treatments upon the expression of the mRNA of kainate receptor subunits GluR5-7 and KA1-2. Our results show that, after 7 days, adrenalectomy results in increased expression of hippocampal KA1, GluR6 and GluR7 mRNAs, an effect which is reversed by treatment with corticosterone in the case of KA1 and GluR7 and by aldosterone treatment in the case of GluR6. 21 days of chronic restraint stress (CRS) elevated the expression of the KA1 subunit, but had no effect on the expression of the other subunits. Similarly, 21 days of treatment with a moderate dose of corticosterone also increased KA1 mRNA in the dentate gyrus, whereas a high corticosterone dose has no effect. Our results suggest an interaction between hippocampal kainate receptor composition and the hypothalamic-pituitary-adrenal (HPA) axis and show a selective chronic stress induced modulation of the KA1 subunit in the dentate gyrus and CA3 that has implications for stress-induced adaptive structural plasticity.     

Transient global ischemia in rat brain promotes different NMDA receptor regulation depending on the brain structure studied

The mRNA expression of the major subunits of N-methyl-d-aspartate receptors (NR1, NR2A and NR2B) following ischemia–reperfusion was studied in structures with different vulnerabilities to ischemic insult in the rat brain. The study was performed using quantitative real-time PCR on samples from 3-month-old male Sprague–Dawley rats after global transient forebrain ischemia followed by 48 h of reperfusion. Expression of NMDA receptor subunits mRNAs decreased significantly in all structures studied in the injured animals as compared to the sham-operated ones. The hippocampal subfields (CA1, CA3 and dentate gyrus) as well as the caudate-putamen, both reported to be highly ischemic-vulnerable structures, showed outstandingly lower mRNA levels of NMDA receptor subunits than the cerebral cortex, which is considered a more ischemic-resistant structure. The ratios of the mRNA levels of the different subunits were analyzed as a measure of the NMDA receptor expression pattern for each structure studied. Hippocampal areas showed changes in NMDA receptor expression after the insult, with significant decreases in the NR2A with respect to the NR1 and NR2B subunits. Thus, the NR1:NR2A:NR2B (1:1:2) ratios observed in the sham-operated animals became (2:1:4) in insulted animals. This modified expression pattern was similar in CA1, CA3 and the dentate gyrus, in spite of the different vulnerabilities reported for these hippocampal areas. In contrast, no significant differences in the expression pattern were observed in the caudate-putamen or cerebral cortex on comparing the sham-operated animals with the ischemia-reperfused rats. Our results support the notion that the regulation of NMDA receptor gene expression is dependent on the brain structure rather than on the higher or lower vulnerability of the area studied.     

Age-dependent expression of glucocorticoid- and mineralocorticoid receptors on neural precursor cell populations in the adult murine hippocampus

Steroid hormones are regulators of adult hippocampal neurogenesis and are central to hypotheses regarding adult neurogenesis in age-related and psychiatric disturbances associated with altered hippocampal plasticity--most notably dementias and major depression. Using immunohistochemistry, we examined the expression of glucocorticoid (GR) and mineralocorticoid (MR) receptors during adult hippocampal neurogenesis. In young mice only 27% of dividing cells in the subgranular zone expressed GR, whereas 4 weeks after division 87% had become positive for GR and MR. GR was expressed by 50% of the radial glia-like type-1 and type-2a progenitor cells, whereas MR was expressed only by mature calbindin-positive granule cells. Doublecortin-positive neuronal progenitor cells (type-2b) and early postmitotic calretinin-positive neurons were devoid of GR and MR expression. Fifty per cent of the intermediate type-3 cells showed GR expression, possibly reflecting cells terminating maturation. Thus, all subpopulations of dividing precursor cells showed an identical receptor profile (50% GR, no MR), except for type-2b cells, which expressed neither receptor. There was also no overlap between calretinin and GR early postnatally (P8) or after physical activity or exposure to an enriched environment, both of which are potent neurogenic stimuli. In contrast, in old age calretinin-positive young neurons became GR and MR positive, suggesting increased steroid sensitivity. Age also increased the expression of GR in type-1 and type-2a precursor cells. Other intermediates were so rare in old age that they could not be studied. This course and variability of receptor expression in aging might help to explain differential vulnerability of adult neural precursor cells to corticoid-mediated influences.     

Immunoreactivities and Levels of Mineralocorticoid and Glucocorticoid Receptors in the Hippocampal CA1 Region and Dentate Gyrus of Adult and Aged Dogs

Corticosteroids are important factors in the maintenance of homeostasis in the brain. They are regulated via the interaction with two corticosteroid receptor systems—the mineralocorticoid (MR) and glucocorticoid receptor (GR). In the present study, we observed age-related changes in serum cortisol levels, and immunoreactivities and protein levels of MR and GR in the hippocampal CA1 region and dentate gyrus. The serum cortisol levels were significantly high (about twofold) in the aged group compared to that in the adult group. In the adult dog (2–3 years old), MR and GR immunoreactivity was detected in neurons in the pyramidal layer of the CA1 region, and in the granular and multiform layers of the dentate gyrus. In the aged dog (10–12 years old), MR immunoreactivity in the CA1 region was significantly decreased, especially, in the dentate multiform layer. In contrast, GR immunoreactivity in the aged dog was slightly decreased in the CA1 region and dentate gyrus. In the Western blot analysis, MR protein level in the aged dog was significantly lower compared to that of the adult dog; GR protein level in the aged dog was not significantly decreased. This result indicates that the reduction of MR immunoreactivity and protein level in the hippocampus of the aged dog may be associated with neural dysfunction in the aged hippocampus.     

Glucocorticoid Receptors are Localized to Dendritic Spines and Influence Local Actin Signaling

Glucocorticoids affect learning and memory but the cellular mechanisms involved are poorly understood. The present studies tested if the stress-responsive glucocorticoid receptor (GR) is present and regulated within dendritic spines, and influences local signaling to the actin cytoskeleton. In hippocampal field CA1, 13?% of synapses contained GR-immunoreactivity. Three-dimensional reconstructions of CA1 dendrites showed that GR aggregates are present in both spine heads and necks. Consonant with evidence that GR?? mRNA associates with the translation regulator Fragile X Mental Retardation Protein (FMRP), spine GR levels were rapidly increased by group 1 mGluR activation and reduced in mice lacking FMRP. Treatment of cultured hippocampal slices with the GR agonist dexamethasone rapidly (15?C30?min) increased total levels of phosphorylated (p) Cofilin and extracellular signal-regulated kinase (ERK) 1/2, proteins that regulate actin polymerization and stability. Dexamethasone treatment of adult hippocampal slices also increased numbers of PSD95+ spines containing pERK1/2, but reduced numbers of pCofilin-immunoreactive spines. Dexamethasone-induced increases in synaptic pERK1/2 were blocked by the GR antagonist RU-486. These results demonstrate that GRs are present in hippocampal spines where they mediate acute glucocorticoid effects on local spine signaling. Through effects on these actin regulatory pathways, GRs are positioned to exert acute effects on synaptic plasticity.     

Type I and type II corticosteroid receptor gene expression in the rat: effect of adrenalectomy and dexamethasone administration

We have used 32P-labeled cRNA probes directed against Type I (mineralocorticoid, high affinity glucocorticoid) and Type II (classical glucocorticoid) receptor mRNA to screen various tissues, and have investigated the effect of adrenalectomy (ADX) and dexamethasone (DM) administration on their levels in hippocampus. Both Northern blot and S1 nuclease analysis showed Type I mRNA to be high in hippocampus, colon, and heart; low in liver; and undetectable in thymus. Type II mRNA was high in liver, thymus, and brain; and low in testis and parotid. A transient increase in both hippocampal Type I and Type II mRNA was noted at 1-3 days post ADX. DM similarly elicited a rise in hippocampal Type I mRNA at 2-4 days after ADX, but prevented the ADX-induced increment in Type II mRNA. In contrast to the transient increase in Type I receptor mRNA levels, hippocampal levels of Type I receptors measured by [3H]aldosterone binding were constant 1-16 days post ADX. DM administration caused a doubling in Type I receptor levels over 4 days, with plateau levels at 4-16 days; previously, DM has been shown to lower Type II receptor levels in the hippocampus.(ABSTRACT TRUNCATED AT 250 WORDS)     

The responses of rat liver glucocorticoid receptors and genes for tyrosine aminotransferase, alpha-2-macroglobulin and gamma-fibrinogen to adrenalectomy-, dexamethasone- and inflammation-induced changes in the levels of glucocorticoids and proinflammatory cytokines

The responses of liver glucocorticoid receptor (GR) and genes coding for a glucocorticoid-inducible tyrosine aminotransferase (TAT) and two acute-phase proteins (APP) [alpha2-macroglobulin (alpha2-M) and gamma-fibrinogen (Fb)] to changes in glucocorticoid (GC) and proinflammatory (AP) cytokine contents have been examined in rats after single or combined treatments with turpentine oil, dexamethasone (Dex) and adrenalectomy. Activation of two APP genes in turpentine-induced inflammation was accompanied by an increase in the level of GR mRNA and a preferential translocation of GR-GC complexes to the nucleoplasm, while the expression of TAT remained unaltered. Dex alone caused a decrease in the levels of GR and Fb mRNAs, activation of TAT and alpha2-M genes, a decrease in the affinity of hormone binding sites and redistribution of translocated GR-Dex complexes within the nuclei. Inflammation potentiated the effect which Dex alone exerted on the GR content and the number of GR binding sites but counteracted its influence on the affinity of GR binding sites and nuclear distribution of GR-Dex complexes. Adrenalectomy promoted a fall in TAT mRNA, no changes in the GR and Fb mRNA, a decrease in the affinity of GR hormone binding sites and redistribution of GR-hormone complexes within the nuclei. The AP cytokines released in response to inflammation exerted a counteracting effect on the adrenalectomy-induced changes in the affinity of hormone binding sites and nuclear distribution of GR-hormone complexes. They potentiated a fall of TAT mRNA but promoted full expression of the Fb gene. These results argue strongly for the influence of AP cytokines on the functional state of the GR and GC signaling pathways.     

Dexamethasone stimulates endothelin-1 gene expression in renal collecting duct cells

Aldosterone stimulates the endothelin-1 gene (Edn1) in renal collecting duct (CD) cells by a mechanism involving the mineralocorticoid receptor (MR) and the glucocorticoid receptor (GR). The goal of the present study was to determine if the synthetic glucocorticoid dexamethasone affected Edn1 gene expression and to characterize GR binding patterns to an element in the Edn1 promoter. Dexamethasone (1μM) induced a 4-fold increase in Edn1 mRNA in mIMCD-3 inner medullary CD cells. Similar results were obtained from cortical collecting duct-derived mpkCCD(c14) cells. RU486 inhibition of GR completely blocked dexamethasone action on Edn1. Similarly, 24h transfection of siRNA against GR reduced Edn1 expression by approximately 50%. However, blockade of MR with either spironolactone or siRNA had little effect on dexamethasone induction of Edn1. Cotransfection of MR and GR siRNAs together had no additive effect compared to GR-siRNA alone. The results indicate that dexamethasone acts on Edn1 exclusively through GR and not MR. DNA affinity purification studies revealed that either dexamethasone or aldosterone resulted in GR binding to the same hormone response element in the Edn1Edn1 promoter. The Edn1 hormone response element contains three important sequence segments. Mutational analysis revealed that one of these segments is particularly important for modulating MR and GR binding to the Edn1 hormone response element.     

Decreased Hippocampal Expression, but Not Functionality, of GABAB Receptors After Transient Cerebral Ischemia in Rats

Abstract : We have examined the effects of transient global ischemia on both the gene expression levels and the functionality of GABA_B receptors in rat brain, using antisense in situ hybridization and electrophysiological evaluations. At the level of gene expression, no significant change in GABA_B receptor expression was observed in any hippocampal subfield at either 6 or 12 h after challenge. At 24 h postchallenge, however, a significant decrease in GABA_B receptor expression was observed in both the CA1 and CA3 subfields, whereas no change was observed in the dentate granule cell layer. Although expression in both the vulnerable CA1 and less vulnerable CA3 subfields was diminished at this time postchallenge, there was no significant difference in the degree of the diminished expression between these subfields. At the functional level, the dose-dependent ability of baclofen (1-100 μ M ) to inhibit an evoked excitatory postsynaptic potential (f-EPSP) in the CA1 subfield was evaluated at 24 h postischemia, in comparison with the dose-response observed in sham-operated subjects. No significant differences were observed in the efficacy of GABA_B receptor-mediated inhibition of the elicited f-EPSP at any of the baclofen concentrations examined. These data demonstrate that although the mRNA expression levels for the GABA_B receptor are diminished in both vulnerable and less vulnerable neurons of Ammon's horn at 24 h following transient global ischemia, the functionality of the GABA_B receptor system is maintained at this time postchallenge.     

The role of development and adrenal steroids in the regulation of the mineralocorticoid receptor messenger RNA.

The ontogeny, adrenal-feedback regulation and regional distribution of the mineralocorticoid receptor (MR) mRNA were examined in the rat brain and kidney. In the kidney, MR mRNA levels in the adult were only 25-30% of the neonatal concentration. Adrenalectomy caused a 35% increase in total brain MR mRNA and a 94% increase in kidney MR mRNA levels. Examination of the regional distribution of the MR mRNA within the brain revealed that the hippocampus had the highest levels, and the mRNA abundance increased after adrenalectomy. The administration of dexamethasone to intact animals resulted in a significant reduction of MR mRNA in the kidney of neonatal rats but not in the brain. These data indicate that there are developmental changes in MR gene expression in kidney and that adrenal steroids can modulate MR gene expression in both the brain and kidney.     

Positive regulation of the glucocorticoid receptor in human T-cells sensitive to the cytolytic effects of glucocorticoids

Regulation of glucocorticoid receptor (GR) protein and mRNA were examined in the human leukemic T-cell line CEM-C7. Unlike other cells in which GR regulation has been examined, the growth of these cells is inhibited by glucocorticoids, leading to cell death. Treatment of glucocorticoid-sensitive CEM-C7 cells with 1 microM dexamethasone for 18 h resulted in an increase in both cytoplasmic and nuclear GR protein, as determined by immunoblotting with anti-human GR antisera. Analysis of GR mRNA levels by Northern blotting revealed a corresponding increase in mRNA in steroid-treated cells. An increase in GR mRNA was detectable after as little as 3 h of treatment with dexamethasone, and GR mRNA concentration continued to increase for at least 18 h, well before the onset of growth arrest or cell death. GR mRNA concentration was not altered after dexamethasone treatment of the glucocorticoid-resistant mutant cell line ICR27TK.3, which lacks functional GR. Thus, the increase in GR seen in glucocorticoid-sensitive cells is a GR-mediated response. These results are in sharp contrast to the down-regulation of GR reported in other cells and tissues, and suggest that regulation of the GR by its cognate ligand may be tissue-specific.     

Differential regulation by dexamethasone of glucocorticoid receptor messenger RNA concentrations in neuronal cultures derived from fetal rat hypothalamus and cerebral cortex

1. Differential regulation, by dexamethasone, of glucocorticoid receptor gene expression was studied in three different neuronal cultures derived from hypothalamus amygdala, and cerebral cortex. 2. Cellular glucocorticoid receptor (GR) mRNA concentration was measured by hybridization using a 32P-labeled RNA probe complementary to a 2.2-kb fragment of the glucocorticoid receptor mRNA. Changes in the amount of GR mRNA were evaluated in relation to the content of beta-actin mRNA. 3. In cells derived from either hypothalamus or cerebral cortex, we observed a complex pattern of GR mRNA concentrations which were characterized by cyclic variations of GR mRNA content during continuous treatment with dexamethasone for up to 72 hr. 4. In contrast to cells derived from the hypothalamus where a persistent 30-40% reduction in GR mRNA levels was seen for up to a least 72 hr, we observed, in cells derived from the cerebral cortex, a sustained increased (1.4-fold) of the GR mRNA at this same time interval.     

Radiation-induced hyposuppression of the hypothalamic-pituitary-adrenal axis is associated with alterations of hippocampal corticosteroid receptor expression

Therapeutic brain irradiation in children can cause a progressive decline in cognitive functions through a diminished capability to learn and memorize. Because of the known involvement of the hippocampus in memory consolidation, this study was aimed at examining the late effects of gamma radiation on hypothalamic-pituitary-adrenal (HPA) axis activity and hippocampal corticosteroid receptor expression in an animal model of cranial radiotherapy. In the late-response phase, the basal and stress-induced corticosterone levels were not affected by radiation, but the suppression of glucocorticoid negative feedback by dexamethasone was attenuated in irradiated rats. Western blot analyses showed that exposure to radiation led to a decrease of cytosolic glucocorticoid receptor (GR) levels and a concomitant elevation of mineralocorticoid receptor (MR). The results obtained were complemented by those of RT-PCR, since the ratio of GR/MR mRNA was also decreased after radiation exposure. Dexamethasone appeared to be much less effective in shifting GR to the nuclear compartment in irradiated rats than in sham-irradiated animals. However, the expression of chaperones that aid GR intracellular trafficking, Hsp90 and Hsp70, remained unaffected. In conclusion, our data suggest that the hallmark of the late response to gamma radiation is a hyposuppressive state of the HPA axis that is associated with a decrease in both the GR/MR ratio and the nuclear accumulation of dexamethasone-activated GR in the hippocampus.     

Steroids and electrical activity in the brain

Corticosteroid hormones can enter the brain and bind to two receptor subtypes: the high affinity mineralocorticoid receptor (MR) and the glucocorticoid receptor (GR) with approximately 10-fold lower affinity. Under physiological conditions the degree of receptor occupation will range from a predominant MR occupation (at the beginning of the inactive period, under rest) to concurrent activation of MRs and GRs (at the circadian peak and after stress). With in vitro electrophysiological recording techniques we observed that neuronal excitability in the CA1 hippocampal field is under a long-term control of MR- and GR-mediated events. The predominant occupation of MRs is associated with a stable amino acid-carried synaptic transmission; calcium- and potassium-currents are small, as are the responses to biogenic amines. Occupation of GRs in addition to MRs results in a gradual failure of CA1 neurons to respond to repeated stimulation of amino acid-mediated input; ionic conductances and responses to biogenic amines are large. In general, electrical properties recorded when both MRs and GRs are unoccupied (i.e. after adrenalectomy) resemble the responses observed when both receptor types are activated. The corticosterone dependency of electrical properties is thus U-shaped. We conclude that MR occupation may be responsible for the maintenance of information processing in the CA1 field and the stability of the circuit. Additional activation of GRs will initially suppress synaptic activity, but may eventually result in an increased instability and even vulnerability of the neuronal networks.