Direct targeting of hippocampal neurons for apoptosis by glucocorticoids is reversible by mineralocorticoid receptor activation

An important question arising from previous observations in vivo is whether glucocorticoids can directly influence neuronal survival in the hippocampus. To this end, a primary postnatal hippocampal culture system containing mature neurons and expressing both glucocorticoid (GR) and mineralocorticoid (MR) receptors was developed. Results show that the GR agonist dexamethasone (DEX) targets neurons (microtubule-associated protein 2-positive cells) for death through apoptosis. GR-mediated cell death was counteracted by the MR agonist aldosterone (ALDO). Antagonism of MR with spironolactone ([7alpha-(acetylthio)-3-oxo-17alpha-pregn-4-ene-21 carbolactone] (SPIRO)) causes a dose-dependent increase in neuronal apoptosis in the absence of DEX, indicating that nanomolar levels of corticosterone present in the culture medium, which are sufficient to activate MR, can mask the apoptotic response to DEX. Indeed, both SPIRO and another MR antagonist, oxprenoate potassium ((7alpha,17alpha)-17-hydroxy-3-oxo-7-propylpregn-4-ene-21-carboxylic acid, potassium salt (RU28318)), accentuated DEX-induced apoptosis. These results demonstrate that GRs can act directly to induce hippocampal neuronal death and that demonstration of their full apoptotic potency depends on abolition of survival-promoting actions mediated by MR.     

The transcriptional response to chronic stress and glucocorticoid receptor blockade in the hippocampal dentate gyrus

The dentate gyrus (DG) of the hippocampus plays a crucial role in learning and memory. This subregion is unique in its ability to generate new neurons throughout life and integrate these new neurons into the hippocampal circuitry. Neurogenesis has further been implicated in hippocampal plasticity and depression. Exposure to chronic stress affects DG function and morphology and suppresses neurogenesis and long-term potentiation (LTP) with consequences for cognition. Previous studies demonstrated that glucocorticoid receptor (GR) blockade by a brief treatment with the GR antagonist mifepristone (RU486) rapidly reverses the stress and glucocorticoid effects on neurogenesis. The molecular pathways underlying both the stress-induced effects and the RU486 effects on the DG are, however, largely unknown. The aim of this study was therefore (1) to investigate by microarray analysis which genes and pathways in the DG are sensitive to chronic stress and (2) to investigate to what extent blockade of GR can normalize these stress-induced effects on DG gene expression. Chronic stress exposure affected the expression of 90 genes in the DG (P < 0.01), with an overrepresentation of genes involved in brain development and morphogenesis and synaptic transmission. RU486 treatment of stressed animals affected expression of 107 genes; however, mostly different genes than those responding to stress. Interestingly, we found CREBBP to be normalized by RU486 treatment to levels observed in control animals, suggesting that CREB-signaling may play a central role in mediating the chronic stress effects on neurogenesis, LTP and calcium currents. The identified genetic pathways provide insight into the stress-induced adaptive plasticity of the hippocampal DG that is so central in learning and memory and will direct future studies on the functional outcome and modulation of these stress effects.     

The effect of chronic fluoxetine treatment on brain corticosteroid receptor mRNA expression and spatial memory in young and aged rats

As rats age, a subgroup will show spatial memory impairments, along with decreased corticosteroid receptors (MR and/or GR) in the hippocampus and a hyperactive hypothalamic-pituitary-adrenal axis. In previous work, we have shown that amitriptyline treatment increases hippocampal MR mRNA and improves spatial memory in young rats but had no effect in aged rats. Here, we examine the effect of 1-month treatment with the selective 5-HT re-uptake inhibitor, fluoxetine (10 mg/kg, p.o.) on hippocampal corticosteroid receptor mRNA and spatial memory in young 4-month-old and aged 24-month-old rats. Aged rats were impaired in spatial memory compared to young controls. MR mRNA expression was reduced with ageing in all hippocampal subfields except CA4 (35% decrease in dentate gyrus (DG) and CA2, P<0.05) and GR mRNA was decreased selectively in CA1 (17% decrease, P<0.05). Fluoxetine treatment increased GR mRNA in the hippocampus of young rats (24 and 46% increase in DG and CA3, respectively, P<0.01) but had no effect on hippocampal MR mRNA expression. In contrast, in aged rats, fluoxetine treatment increased hippocampal MR mRNA selectively in CA2 (43% increase, P<0.05), but had no effect on hippocampal GR mRNA. Fluoxetine treatment did not alter watermaze performance in either young or aged rats. It appears that increased hippocampal MR (at least in the CA2 region) which may underlie the enhancement in memory processing in young rats, is insufficient to improve memory in already cognitively impaired aged rats.     

Selective blockade of the mineralocorticoid receptor impairs hypothalamic-pituitary-adrenal axis expression of habituation

The present study investigated the role of mineralocorticoid receptors (MR) and glucocorticoid receptors (GR) in the expression of habituation of the hypothalamic-pituitary-adrenal (HPA) axis response to stress. Male rats were restrained for 1 h per day for six consecutive days. On day 6, 1 h prior to restraint stress, both restraint-naive and repeatedly restrained rats were injected s.c. with either vehicle (propylene glycol) or one of three corticosteroid receptor antagonist treatments: selective MR antagonist (RU28318 or spironolactone), selective GR antagonist (RU40555), or both MR and GR antagonists combined (RU28318 + RU40555). Blood samples were collected for corticosterone measurement at the beginning of stress, during stress, and 1 h after stress termination. Repeated restraint stress produced significant habituation of corticosterone responses. Acute treatment with the combined MR and GR antagonists prevented the expression of habituation. When tested alone, the MR antagonist also blocked the expression of corticosterone-response habituation, whereas the GR antagonist had no effect. Neither the MR, nor the GR antagonists alone, significantly altered the corticosterone response to restraint in rats exposed to restraint for the first time. The final experiment examined the corticosterone response to a corticotropin releasing hormone (CRH, 3 microg/kg i.p.) challenge. Neither previous exposure to restraint or acute pretreatment with the combined MR and GR antagonists (RU28318 + RU40555) altered the corticosterone response to CRH challenge. This result indicates that the expression of habituation and its blockade by corticosteroid receptor antagonists is not a result of altered pituitary-adrenal response to CRH. Overall, this study suggests that MR plays an important role in constraining the HPA axis response to restraint stress in restraint-habituated rats. The dependence of the HPA axis on MR-mediated corticosteroid negative feedback during acute stress may be an important mechanism that helps maximize the expression of stress habituation and thereby minimize exposure of target tissues to corticosteroids in the context of repeated stress.     

Postnatal ontogeny of mineralocorticoid and glucocorticoid receptor gene expression in regions of the rat tel- and diencephalon.

In situ hybridization was used to study the neuroanatomical distribution of mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) gene expression during development in the rat. This study was based on incubation of adjacent sections of brains from 2-, 8-, 12-, 16-day-old and adult (3 months) rats with 35S-labelled cRNA probes. These probes are transcribed from 513 and 500 basepair cDNA fragments with little homology from rat brain MR and rat liver GR respectively. Different patterns of expression were found in the brain of MR and GR during ontogeny. At postnatal day (pnd) 2, a high density of labelled MR mRNA was found in all pyramidal (CA1-4) and granular (dentate gyrus) cell fields of the hippocampal structure, the anterior hippocampus and indusium griseum, and cortex layer II. Modest to high labelling of MR mRNA was observed in the subfornical organ and the anterior hypothalamus. A variety of other telencephalic regions anterior and posterior of bregma exhibited modest to weak intensity of labelled MR mRNA. The diencephalon virtually lacked labelled MR mRNA. At older postnatal ages including the adult age, this regional distribution of radiolabelled MR mRNA did not change. At pnd 2, abundant radiolabelled GR mRNA was found widespread over the tel- and diencephalon, with the highest density observed in cell field CA1 and CA2 of the hippocampus and the parvocellular division of the hypothalamic paraventricular nucleus. Modestly labelled GR mRNA was observed in various hypothalamic and thalamic nuclei, basal ganglia, the lateral septum and the amygdala. At older postnatal ages and in adulthood, the intensity of labelled GR mRNA became progressively stronger in the hippocampus. Moreover, we observed a trend towards a more condensed and narrow band of cell bodies in the hippocampus for both MR and GR mRNA during ontogeny. A semi-quantitative comparison of the intensity of both labelled mRNA's performed at each age revealed a significantly lower expression of GR than MR mRNA in the CA3 cell field at pnd 2. At pnd 8 and 12, the amount of GR mRNA was significantly lower in the dentate gyrus and the CA3, whereas in adulthood, less GR mRNA was measured in all pyramidal and granular cell fields. The present study demonstrates that MR and GR genes are expressed in early postnatal development in a pattern resembling that in adulthood. As is the case in the adult brain, there is more MR than GR mRNA in the hippocampus during ontogeny, especially in the CA3 cell field and the DG.     

Role of adrenal steroid mineralocorticoid and glucocorticoid receptors in long-term potentiation in the CA1 field of hippocampal slices

We previously demonstrated in the dentate gyrus (DG) of anesthetized and freely behaving rats that both acute as well as chronic administration of corticosterone produces a suppression in long-term potentiation (LTP). In subsequent studies we showed, again in the DG, that activation of the two types of adrenal steroid receptors (mineralocorticoid (MR) and glucocorticoid (GR)) produce biphasic effects on synaptic plasticity; activation of MR produces an enhancement while activation of GR produces a suppression in LTP. In a separate study, we further demonstrated in rats administered the specific GR agonist RU 28362 that high-frequency stimulation, which normally produces LTP, instead produced long-term depression (LTD) in these animals. In the present study we investigated the effects of MR and GR activation by adrenal steroids on synaptic plasticity of the hippocampal CA1 field, but we studied this ex vivo, in a slice preparation. The results indicate that, as in our studies in the DG, adrenal steroids produce biphasic effects: in ADX rats, aldosterone (a specific MR agonist) enhanced while RU 28362 suppressed synaptic plasticity. Unlike the in vivo preparation, however, rarely was LTD observed in the animals receiving RU 28362. Also, ADX itself did not produce noticeable effects on synaptic plasticity. The present results are in agreement with previous studies showing that elevations in corticosterone or an acute episode of experimentally induced stress in vivo causes a suppression in LTP in the hippocampal CA1 field, in vitro.     

Blockade of corticotropin‐releasing hormone receptor 1 attenuates early‐life stress‐induced synaptic abnormalities in the neonatal hippocampus

Adult individuals with early stressful experience exhibit impaired hippocampal neuronal morphology, synaptic plasticity and cognitive performance. While our knowledge on the persistent effects of early‐life stress on hippocampal structure and function and the underlying mechanisms has advanced over the recent years, the molecular basis of the immediate postnatal stress effects on hippocampal development remains to be investigated. Here, we reported that repeated blockade of corticotropin‐releasing hormone receptor 1 (CRHR1) ameliorated postnatal stress‐induced hippocampal synaptic abnormalities in neonatal mice. Following the stress exposure, pups with fragmented maternal care showed retarded dendritic outgrowth and spine formation in CA3 pyramidal neurons and reduced hippocampal levels of synapse‐related proteins. During the stress exposure, repeated blockade of glucocorticoid receptors (GRs) by daily administration of RU486 (100 µg g^?1) failed to attenuate postnatal stress‐evoked synaptic impairments. Conversely, daily administration of the CRHR1 antagonist antalarmin hydrochloride (20 µg g^?1) in stressed pups normalized hippocampal protein levels of synaptophysin, postsynaptic density‐95, nectin‐1, and nectin‐3, but not the N‐methyl‐d ‐aspartate receptor subunits NR1 and NR2A. Additionally, GR or CRHR1 antagonism attenuated postnatal stress‐induced endocrine alterations but not body growth retardation. Our data indicate that the CRH‐CRHR1 system modulates the deleterious effects of early‐life stress on dendritic development, spinogenesis, and synapse formation, and that early interventions of this system may prevent stress‐induced hippocampal maldevelopment. © 2014 Wiley Periodicals, Inc.     

Synergistic action of corticosterone on kainic acid-induced electrophysiological alterations in the hippocampus

The present study investigates the effect of overexposure to high doses of the stress hormone corticosterone (CORT) on the electrophysiological changes produced in the hippocampus after local microinjection of KA. Extracellular recordings were performed in the CA1 area of mouse hippocampal slices prepared after a 7-day recovery period following KA microinfusion alone or combined with 3 days overexposure to CORT. The results showed that CORT shifts the KA response profile approximately 40-fold, since animals treated with a non-toxic dose of 0.01 μg KA and CORT exhibited epileptic activity and a shift on the paired-pulse response similar to that observed in animals treated with high doses of KA (0.4 μg). This synergistic action of CORT on the electrophysiological changes induced by KA was antagonized by the antiglucocorticoid RU486 whereas the antimineralocorticoid spironolactone was ineffective. These results suggest that CORT may play an important role in modulating the severity of KA-induced seizures in the hippocampal structure probably by GR-receptor mediated action.     

A randomized trial on mineralocorticoid receptor blockade in men: effects on stress responses, selective attention, and memory

Corticosteroids, released in high amounts after stress, exert their effects via two different receptors in the brain: glucocorticoid receptors (GRs) and mineralocorticoid receptors (MRs). GRs have a role in normalizing stress-induced effects and promoting consolidation, while MRs are thought to be important in determining the threshold for activation of the hypothalamic-pituitary-adrenal (HPA) axis. We investigated the effects of MR blockade on HPA axis responses to stress and stress-induced changes in cognitive function. In a double-blind, placebo-controlled study, 64 healthy young men received 400 mg of the MR antagonist spironolactone or placebo. After 1.5 h, they were exposed to either a Trier Social Stress Test or a non-stressful control task. Responses to stress were evaluated by hormonal, subjective, and physiological measurements. Afterwards, selective attention, working memory, and long-term memory performance were assessed. Spironolactone increased basal salivary cortisol levels as well as cortisol levels in response to stress. Furthermore, spironolactone significantly impaired selective attention, but only in the control group. The stress group receiving spironolactone showed impaired working memory performance. By contrast, long-term memory was enhanced in this group. These data support a role of MRs in the regulation of the HPA axis under basal conditions as well as in response to stress. The increased availability of cortisol after spironolactone treatment implies enhanced GR activation, which, in combination with MR blockade, presumably resulted in a decreased MR/GR activation ratio. This condition influences both selective attention and performance in various memory tasks.     

Distribution of glucocorticoid and mineralocorticoid receptor messenger RNA expression in human postmortem hippocampus

Corticosteroids bind to hippocampal glucocorticoid (GR) and mineralocorticoid (MR) receptors, thereby affecting behaviour and neurochemical transmission. Rat hippocampus has high levels of both receptors and their messenger RNAs (mRNA), but there is little information on receptors in human brain. We used in situ hybridization to determine the distribution of GR and MR mRNA expression in human hippocampus. Frozen sections of human postmortem hippocampus (5 patients, 58-88 years old, without cerebral pathology) were postfixed in paraformaldehyde and hybridized with 35S-UTP-labelled cRNA probes (transcribed in vitro from human cDNA subclones) under stringent conditions. Control included hybridization with sense probes and heterologous cRNA competition studies. GR mRNA was highly expressed in dentate gyrus, CA3 and CA4, but levels were significantly lower in CA1 and CA2. MR mRNA was also very highly expressed in hippocampus, with significantly higher levels in dentate gyrus and CA2, CA3 and CA4 than CA1. Controls confirmed the specificity of hybridization and there was little hybridization of sense probes. High GR and MR mRNA expression is found in both rat and human hippocampus but the subregional distributions clearly differ between the species.     

Glucocorticoid and mineralocorticoid receptor mRNA expression in squirrel monkey brain

Corticosteroids have been implicated in hippocampal atrophy in patients with severe psychiatric disorders, but little is known about receptor expression for corticosteroids in human or nonhuman primate brain. Both the glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) were surveyed in this study of squirrel monkey brain using in situ hybridization histochemistry. Regions of high GR mRNA levels included CA1 and CA2 of hippocampus, dentate gyrus, paraventricular hypothalamus, lateral geniculate, lateral>medial amygdala, and cerebellum. Western analysis confirmed that GR immunoreactivity in squirrel monkey brain tissue most likely reflects the alpha isoform. Regions of high MR mRNA levels included all hippocampal pyramidal cell fields, dentate gyrus granule cell layer, lateral septum, medial>lateral amygdala, and to a lesser extent, cerebellum. Low levels of MR were also expressed in caudate and putamen. Receptor expression for corticosteroids in deep brain structures and the hippocampal formation was similar to that previously reported in rodents, but GR and MR mRNA were expressed at higher levels in squirrel monkey cerebral cortex. GR expression was evident in all cortical layers, particularly the pyramidal cell-rich layers II/III and V. MR expression was restricted to the more superficial cortical layers, and was only moderately represented in layer V. Laminar patterns were apparent in all regions of cortex for GR expression in squirrel monkeys, but low MR mRNA levels were found in dorsomedial prefrontal cortex (PFC). Different subregional distributions and distinctive laminar patterns suggest specialized functions or coordinated interactions between GR and MR mediated functions in primate PFC.     

Medial septal cholinergic lesions increase hippocampal mineralocorticoid and glucocorticoid receptor messenger RNA expression.

Loss of the cholinergic innervation of the hippocampus and failure of central (presumably hippocampal) suppressive control of hypothalamic-pituitary-adrenal axis activity are important features of Alzheimer's dementia. We have examined the effects of electrolytic lesions of the medial septal cholinergic innervation on mineralocorticoid (MR) and glucocorticoid (GR) receptor mRNA expression in rat hippocampus using in situ hybridization histochemistry. Expression of both MR and GR mRNA was significantly increased in a subregions of the hippocampus, but not neocortex, with the greatest increase in the CA1 area for MR mRNA and dentate gyrus for GR mRNA. Since glucocorticoids potentiate the effects of neurotoxins in the hippocampus, the increased expression of receptors following loss of cholinergic inputs in Alzheimer's disease may increase hippocampal neuronal vulnerability.     

Brief neck restraint stress enhances long-term potentiation and suppresses long-term depression in the dentate gyrus of the mouse

We studied the effects of brief (10 min) neck restraint on long-term potentiation (LTP) and long-term depression (LTD) in mouse dentate gyrus (DG) slices. Brain slices were prepared immediately after neck restraint and LTP/LTD induction was attempted 3.5 h later. LTP enhancement and LTD suppression was observed in slices prepared from stressed animals. The corticosterone plasma concentration was elevated approx. fourfold just after the neck restraint session. To examine the role of glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) activation in mediating the effects of neck restraint on LTP and LTD, the animals were pretreated with either the specific GR antagonist RU38486, the specific MR antagonist spironolactone, the corticosterone synthesis inhibitor metyrapone, or vehicle Tween 80. Injection with Tween 80 and subsequent neck restraint did not influence LTP enhancement or LTD suppression, but the effects of neck restraint were prevented by RU38486, spironolactone and metyrapone pretreatment. These data indicate that acute stress caused by brief neck restraint enhances LTP and impairs LTD in the dentate gyrus and that these effects are mediated by activation of GRs and MRs.     

Glucocorticoids and serotonin alter glucocorticoid receptor (GR) but not mineralocorticoid receptor (MR) mRNA levels in fetal mouse hippocampal neurons, in vitro

Studies utilizing rats and guinea pigs have demonstrated that the hypothalamo-pituitary-adrenal (HPA) axis can be programmed by glucocorticoids during fetal life. Such programming is believed to occur, at least partially, at the level of hippocampal glucocorticoid receptors (GR) and mineralocorticoid receptors (MR). Studies have also demonstrated that serotonin up regulates GR levels within the developing hippocampus. However, the cell type in which these changes take place has not been determined. We hypothesized that dexamethasone, corticosterone and serotonin exposure modify GR and MR mRNA levels in fetal mouse hippocampal cultures, and that these effects are confined to neurons. Cultures were derived from CD1 mouse fetuses on day 18 of gestation (n=8 dams). Fetal hippocampi were dissected, then mechanically and chemically dispersed. Cultures were exposed to dexamethasone, corticosterone or serotonin (1-100 nM) for 4 days. Levels of GR and MR mRNA were examined by in situ hybridization and high-resolution silver emulsion autoradiography. Four days exposure to dexamethasone or corticosterone (10 or 100 nM) decreased levels of GR mRNA within neurons. There was no significant change in MR mRNA in either experiment. Exposure to serotonin (100 nM) significantly increased expression of GR mRNA in hippocampal neurons. MR mRNA levels were unaffected by serotonin treatment. Dexamethasone, corticosterone or serotonin exposure did not alter expression of GR mRNA within glial cells. We conclude that synthetic and endogenous glucocorticoids, as well as serotonin, can influence neuronal levels of GR mRNA during hippocampal development. However, whether these effects are permanent remains to be determined.     

Blockade of glucocorticoid receptors rapidly restores hippocampal CA1 synaptic plasticity after exposure to chronic stress

Prolonged exposure to stressful events has been reported to inhibit the ability of hippocampal synapses to increase their synaptic efficacy. Here we tested if these effects could be prevented by blocking activation of glucocorticoid receptors during the last 4 days of the stress paradigm. In order to address this question, animals were exposed to 21 days of variable and inescapable stressors. Handled animals served as controls. During the last 4 days of the stress regime, animals were treated with the glucocorticoid receptor antagonist RU486. We found that 1 day after the last stressor, synaptic plasticity in the CA1 area of hippocampal slices is impaired in chronically stressed animals. Importantly, treating chronically stressed animals with RU486 for 4 days completely prevented this decrease in synaptic potentiation; RU486 treatment of handled controls did not affect potentiation. Treating hippocampal slices from control animals with high levels of corticosterone also impaired synaptic plasticity; this effect was similar for untreated and RU486-treated animals. Treating slices from chronically stressed animals with corticosterone did not further decrease synaptic plasticity. These data indicate that 4 days blockade of the glucocorticoid receptor, during a stress regime, is sufficient to fully restore synaptic plasticity.     

Distribution of corticosteroid receptors in the rhesus brain: relative absence of glucocorticoid receptors in the hippocampal formation.

Chronic stress has been associated with degenerative changes in the rodent and primate hippocampus, presumably mediated in part via neuronal glucocorticoid receptors (GRs). In the rat brain, GRs are widely distributed and are particularly dense in the hippocampus. The distribution of GRs in the primate brain, however, has not been fully characterized. In this study, we used in situ hybridization histochemistry and immunohistochemistry to map the distribution of GR mRNA and GR protein, respectively, in adult rhesus monkeys (Macaca mulatta). In contrast to its well established distribution in the rat brain, GR mRNA was only weakly detected in the dentate gyrus (DG) and Cornu Ammonis (CA) of the macaque hippocampus, whereas it was abundant in the pituitary (PIT), cerebellum (CBL), hypothalamic paraventricular nucleus (PVN), and, to a lesser extent, the neocortex. Immunohistochemical staining indicated a very low density of GR-like immunoreactive cells within the macaque hippocampal formation in contrast to the high density observed within the PVN, prefrontal and entorhinal cortices, and cerebellar cortex. Relative to the low level of GR, mineralocorticoid receptor (MR) mRNA and protein expression were abundant within the DG and CA of the rhesus monkey hippocampal formation. These results indicate that, in the primate, neocortical and hypothalamic areas may be more important targets for GR-mediated effects of glucocorticoids than the hippocampus. Alternatively, it is also possible that glucocorticoid effects are mediated through the MRs present in the hippocampal formation.