Effects of adrenal steroid manipulations and repeated restraint stress on dynorphin mRNA levels and excitatory amino acid receptor binding in hippocampus

Adrenal steroid and stress effects were determined in hippocampus on levels of dynorphin (DYN) mRNA, expressed in dentate gyrus, and excitatory amino acid receptors, measured in Ammon's horn and dentate gyrus. Adrenalectomy (ADX) decreased DYN mRNA levels in dentate gyrus and replacement with aldosterone (ALDO), a specific type I adrenal steroid receptor agonist, prevented the decrease. Ru28362, a specific type II receptor agonist, had no effect. Likewise, kainate receptor binding to the stratum lucidum and hilus region of dorsal hippocampus was decreased after ADX and this decrease was prevented by ALDO but not by Ru28362 treatment. Similar though smaller effects were found for CNQX binding to AMPA receptors but only in the dentate gyrus molecular or infra- and supragranular layers. Although corticosterone (CORT) treatment of intact rats (40 mg/kg for 3 weeks) elevated DYN mRNA levels in dentate gyrus, up to 14 days of daily restraint stress (1 or 6 h/day) had no significant effect. Neither CORT treatment nor repeated restraint stress altered NMDA and non-NMDA glutamate receptors in hippocampus. The results of this study showing ADX-induced decreases of DYN mRNA and CNQX binding in dentate gyrus and decreased kainate binding in mossy fiber terminal regions are consistent with morphological evidence showing that adrenal steroids maintain normal integrity and structure of dentate gyrus neurons and do so via type I adrenal steroid receptors. These same parameters are apparently not sensitive to chronic restraint stress although the effects of other stressors must be examined.     

Quantification of type I and II adrenal steroid receptors in neuronal, lymphoid and pituitary tissues

Circulating lymphocytes are frequently used to study glucocorticoid receptor (GR) regulation in various clinical disease states, such as depression. Since little is known about the relationship between lymphoid and neuronal GR, type II adrenal steroid receptors (i.e., GR) were quantitated in neuronal (hippocampus, frontal cortex, hypothalamus), lymphoid (circulating lymphocytes, spleen, thymus) as well as pituitary tissues of adrenal-intact and 1 day adrenalectomized (ADX) rats using the selective type II receptor ligand, [3H]RU 28362. Specific, high affinity (dissociation constant = 0.2-0.3 nM) type II receptors were present in all tissues examined with the density in 1 day ADX rats being thymus greater than frontal cortex = spleen greater than hippocampus = pituitary greater than hypothalamus greater than lymphocytes. Adrenal intact rats had fewer type II receptors in frontal cortex, hippocampus and spleen as compared to 1 day ADX rats. Dose-response competition studies using [3H]RU 28362 and various unlabelled steroids revealed a binding profile indicative of a type II receptor with the potency being RU 28362 greater than triamcinolone acetonide greater than dexamethasone = corticosterone much greater than aldosterone in both whole brain and spleen soluble fractions. In contrast to the high concentration of type II receptors in the various tissues, the density of type I (i.e., mineralocorticoid) receptors was very low or nondetectable in the same tissues of 1 day ADX rats with the notable exception of the hippocampus where there were approximately comparable levels of both receptors. These results document the widespread distribution of type II adrenal steroid receptors in neuronal and lymphoid tissues which are similar in affinity and steroid specificity.     

Corticosterone regulation of Type I and Type II adrenal steroid receptors in brain, pituitary, and immune tissue

Type I and Type II adrenal steroid receptor levels were compared in the brain, pituitary and immune system of adrenalectomized rats in the presence or absence of several replacement doses of corticosterone. Six days of adrenalectomy produced an up-regulation of Type II adrenal steroid receptors in the brain and spleen. The lowest replacement dose of corticosterone (equivalent to resting levels of this hormone) blocked this Type II receptor up-regulation, while higher replacement doses of corticosterone were associated with widespread Type I and Type II adrenal steroid receptor down-regulation. However, the dose of corticosterone required for receptor down-regulation varied between tissues. Specifically, hippocampal receptors were most sensitive to corticosterone, whereas pituitary receptors were the least sensitive. All tissues examined, except the pituitary, exhibited a down-regulation of Type II receptors with a high corticosterone replacement dose which approximated acute stress levels of this hormone. In summary, physiologically relevant concentrations of corticosterone were capable of down-regulating Type I and Type II adrenal steroid receptors in multiple brain areas and peripheral immune tissues, including peripheral blood mononuclear cells. In contrast, adrenal steroid receptor levels in the pituitary were relatively insensitive to regulation by corticosterone.     

Regulation of flunitrazepam binding in the dorsal horn of the spinal cord by adrenalectomy and corticosteroids.

Adrenal corticosteroids and adrenalectomy (ADX) have opposing effects on benzodiazepine binding sites in brain regions. These treatments were employed to study [3H]flunitrazepam (FLU) binding in regions punched out from the rat spinal cord. We found that binding was higher in dorsal horn than in ventral horn, and minimal in white matter. Clonazepam and RO 15-1788 largely displaced [3H]FLU binding, whereas RO 5-4864 was weakly active. Four days post-ADX, binding increased exclusively in the dorsal horn, and this effect was reversed by administration of corticosterone (CORT), but not dexamethasone (DEX) or aldosterone (ALDO) given over 4 days. When endogenous CORT was increased by administration of cold stress to adrenal-intact rats, reduced benzodiazepine (BDZ) binding was also observed in the dorsal horn. When added in vitro, only ALDO and not CORT or DEX, inhibited [3H]FLU binding. It is suggested that steroids with affinity for the type I corticosteroid receptor (CORT, ALDO) decrease [3H]FLU binding to a neural-type BDZ receptor in the dorsal horn. Reduction of the inhibitory BDZ system may be physiologically important, and can partly explain the enhancement of excitatory synaptic transmission produced by corticosteroids at the level of the spinal cord.     

Adrenal steroid regulation of neuropeptide Y (NPY) mRNA: differences between dentate hilus and locus coeruleus and arcuate nucleus

Regulation by adrenal steroids of neuropeptide Y (NPY) mRNA was investigated in hilus of the dentate gyrus, arcuate nucleus of hypothalamus and locus coeruleus (LC) of the adult rat brain. Adrenalectomy (ADX) increased NPY mRNA in hilus but decreased NPY mRNA levels in arcuate nucleus and LC. Using a steroid replacement paradigm previously shown to discriminate between Type I and Type II adrenal steroid receptor mediated effects, it was shown that Type I receptor stimulation by aldosterone prevented the ADX-induced increase of NPY mRNA in hilus, whereas Type II receptor stimulation by Ru28362 prevented the ADX-induced decrease in NPY mRNA in arcuate nucleus and LC. The results for hilus are consistent with evidence for a role of Type I receptors in maintaining levels of a number of gene products associated with neurotransmission. The different regulation in hilus from that in arcuate and LC indicate, along with evidence for regulation of NPY expression by insulin, NGF and cyclic AMP and phorbol esters, that the adrenal steroid regulation of NPY gene expression is part of a complex set of regulatory mechanisms that depend on the brain region and cell type.     

Adrenal steroid type I and type II receptor binding: estimates of in vivo receptor number, occupancy, and activation with varying level of steroid

Adrenal steroid (AS) receptors differ from other steroid receptors in the inability of the activated form of the cytosolic receptor to exchange ligand in an in vitro binding assay. We extended this finding by demonstrating that AS receptors extracted from isolated brain nuclei also failed to exchange ligand. Taking advantage of this unique feature of AS receptors, we measured type I and type II AS binding level in rats with varying amounts of endogenous glucocorticoids or exogenous dexamethasone (DEX). We estimated the degree of receptor occupation/activation in various brain areas and the pituitary during basal glucocorticoid conditions and after acute stress. There was a variable proportion of type I receptors in the hippocampus which were unactivated during basal conditions (0-35%). The proportion of unactivated type I receptors increased (55-65%) after DEX treatment. The hippocampus was especially sensitive to the ability of low basal corticosterone (CORT) levels to activate both type I and type II receptors, whereas the pituitary was very insensitive, evidenced by a failure of acute stress levels of endogenous glucocorticoids to occupy/activate type II receptors in the pituitary. Comparison of estimates of the degree of in vivo hippocampal type I and type II receptor activation for the various treatment groups with estimates of in vitro type I and type II receptor occupation by steroid suggested that DEX was more efficient than CORT in producing or maintaining the activated form of the type II receptor in vivo, whereas CORT was more efficient than DEX in activating the type I receptor. These studies suggest that AS receptors in the brain, and especially the hippocampus, are more sensitive to circulating levels of glucocorticoids than the pituitary. There also may be a greater capacity for physiological variations in type I receptor occupation in vivo than had previously been suggested. Finally, discrepancies between CORT and DEX affinity in vitro for type I and type II sites and their in vivo potency may be accounted for by differences in the ability of these compounds to activate type I and type II AS receptors.     

Diurnal Differences and Adrenal Involvement in Calmodulin Stimulation of Hippocampal Adenylate Cyclase Activity

Calciam/calmodulin-dependent processes are altered by manipulations of the hypothalamic-pituitary-adrenal axis, and are associated with changes in synaptic efficacy in the hippocampus, such as long-term potentiation. Recent evidence indicates that there are diurnal variations in the threshold for long-term potentiation, as well as diverse effects of the adrenals and of adrenal steroids on electrical activity related to long-term potentiation. In order to probe possible mechanisms underlying these observations, we investigated the effects of the diurnal cycle, as well as adrenalectomy (ADX) and adrenal demedullation on adenylate cyclase activity. In hippocampal, but not cortical, membranes the adenylate cyclase response to calmodulin was higher during the beginning of the dark phase of the cycle, when endogenous corticosterone levels are high. Basal and forskolin-stimulated adenylate cyclase activity did not exhibit diurnal variation in either brain region. ADX (6 and 14 days) depressed the adenylate cyclase response to calmodulin in hippocampal membranes, and abolished the diurnal difference. ADX had smaller effects on this response in cortical membranes. ADX also attenuated basal and forskolin-stimulated adenylate cyclase activity, but these changes were less striking than effects on calmodulin-stimulated activity. Demedullation (14 days), generating corticosterone levels in the low physiological range, mirrored the effects of ADX on hippocampal adenylate cyclase activity. Corticosterone (20 to 25 μg/ml in the drinking water) did not consistently prevent ADX effects on adenylate cyclase activity. These results demonstrate that adrenal effects on adenylate case activity are regionally specific within the brain, and they suggest that other adrenal secretions besides glucocorticoids may be involved in the feedback of the diurnal rhythm on the hippocampus. Taken together with our recent finding that chronic stress or corticosterone injection selectively attenuated the adenylate cyclase response to calmodulin in cortical, but not hippocampal membranes our findings provide further support for a role of the pituitary-adrenal axis in modulating neural calmodulin-dependent adenylate cyclase activity.     

Hypophysectomy increases vasoactive intestinal peptide-stimulated cyclic AMP generation in the hippocampus of the rat

In investigating the feedback effects of circulating hormones on the brain, we showed previously that adrenalectomy (ADX) increases vasoactive intestinal peptide (VIP)-stimulated cAMP generation in slices from rat hippocampus, a brain structure with high levels of glucocorticoid receptors. This effect is reversed by replacement with glucocorticoids such as dexamethasone (DEX) and corticosterone (CORT). Here we report that, like ADX, hypophysectomy (HYPOX) also elevates VIP-stimulated cAMP generation, compared with sham-operated controls. Moreover, like glucocorticoid replacement, administration of ACTH to HYPOX rats causes a decrease in cAMP stimulation by VIP. Furthermore, ACTH had no effect when given to HYPOX + ADX rats, indicating that the effects of ACTH require the presence of adrenal steroid secretion. However, we find that ACTH may have a permissive role in this glucocorticoid effect because, in the absence of the pituitary, DEX treatment does not decrease VIP-stimulated cAMP levels in the hippocampus. In addition, hippocampal beta-adrenergic-stimulated cAMP accumulation was not suppressed by DEX treatment of HYPOX rats, which again is different from the effect of DEX treatment on ADX animals. These results are discussed in terms of possible synergism between pituitary hormones and steroid hormones in exerting feedback actions on brain function.     

Corticosteroid receptor plasticity and recovery of a deficient hippocampus-associated behavior after unilateral (dorsal) hippocampectomy

Unilateral ablation of the right dorsal hippocampus (HCX) produced changes in maximal corticosteroid binding capacity (B_max) in the contralateral hippocampal lobe of the rat with time. The mechanism by which this time course of changes was produced seemed to involve the pituitary-adrenal system, since a certain difference in corticosteroid receptor binding pattern was noted between chronic adrenalectomized (ADX) rats and rats which remained intact during postlesion survival. In the presence of endogenous adrenal hormones the HCX-induced changes in corticosteroid receptor binding relative to that observed in rats with the overlying neocortex ablated (control) were the following: (1) a 26% decrease at 5 days after HCX; (2) an increase the following 3 weeks with a maximum of 46% at 20 days postsurgery; and (3) recovery towards control values after longer survival times. After discrimination of corticosteroid binding into two corticosterone (CORT) binding receptor populations, e.g. glucocorticoid receptors (GR) and mineralocorticoid-like or CORT receptors (CR), the lesion-induced effect was more pronounced in GR than in CR. A 72% increase over controls was measured at 20 days postsurgery. In the absence of the adrenals, however, the B_max of corticosteroid binding was not decreased at 5 days after HCX. The relative increase in B_max reached a maximum of 39% over control levels at 30 days postsurgery and recovery towards control values after longer survival did not occur. The increase in corticosteroid receptor capacity after HCX, therefore, is transient in the presence of adrenocortical secretion and permanent in its absence. An attempt was made to relate the changes in corticosteroid receptor capacity to deficits in reversal learning behavior, since it is known that this type of behavior is associated with hippocampus function. Reversal learning behavior in a T-shaped maze was disturbed the first 2 weeks after HCX, but restored the following week. ADX seemed to facilitate recovery of the lesion-induced deficit. Bilateral removal of the hippocampus led to a more permanent deficiency in reversal learning behavior. It is concluded that: (1) unilateral HCX induces a period of considerable plasticity in corticosteroid receptors in particular of the GR type; (2) this receptor plasticity coincides with the recovery of a lesion-induced deficit in hippocampus-associated behavior; and (3) a role of adrenal hormones in regulation of neuronal and corticosteroid receptor plasticity cannot be excluded.     

Ultrastructural analysis of the hippocampus of adult rats after long-term adrenalectomy

Removal of adrenal steroids modulates various functions in the brain. However, adrenalectomy (ADX) induced cell death in the hippocampal formation of the adult rat is a recently described phenomenon. We undertook this ultrastructural study on long-term adrenalectomized (5 months) rats to investigate the mode of cell death in the hippocampus. Our results showed apoptotic changes in the hippocampus. In addition we have observed other types of degeneration in the hippocampal neurons. The novel finding in this study is that different morphological patterns of cell death were evident both in the dentate gyrus and in the pyramidal areas, which may reflect different stages of the same death process.     

Time- and region-dependent effect of adrenalectomy on neuropeptide gene expression in rat hippocampus and striatum

Seven days after removal of the adrenals in rats, the messenger RNA levels of preproenkephalin (ENK), preprodynorphin (DYN), cholecystokinin (CCK), and neuropeptide Y (NPY) were measured in hippocampus, striatum, and hypothalamus. Adrenalectomy (ADX) in the morning, when endogenous corticosterone levels were low, resulted 7 days later in a decrease of ENK mRNA and DYN mRNA levels in the hippocampus (41.3 ± 4.3 and 41.9 ± 5.7%, respectively) and in the striatum (32.1 ± 6.6 and 31.2 ± 12.9%, respectively), but no change was observed in the ENK mRNA content of the hypothalamus. When ADX was performed in the evening the opioid mRNA levels were not changed in these brain areas 7 days after ADX. Pretreatment with a single dose of corticosterone before surgery in the morning produced high corticosterone levels similar to those in the evening and prevented the decrease of ENK mRNA in the hippocampus. The decrease in hippocampal DYN mRNA and in striatal ENK mRNA and DYN mRNA persisted. CCK mRNA and NPY mRNA were not changed in any of the experimental groups in any of the three examined brain areas. This study demonstrates that ADX decreases opioid gene expression in the rat hippocampus and striatum. The effect of ADX on hippocampal ENK mRNA levels that persists for at least 7 days postsurgery is independent of the circulating corticosterone level at the time of surgery.     

Alteration of hippocampal cell proliferation in mice lacking the beta 2 subunit of the neuronal nicotinic acetylcholine receptor

Adult hippocampal neurogenesis declines with age in parallel with decreased performance on a variety of hippocampal-dependent tasks. We measured the rate of cellular proliferation in the hippocampus of mice lacking the beta 2-subunit of the nicotinic acetylcholine receptor (beta 2-/- mice) at three ages: young adult (3 months old), fully adult (7-10 months old), and aged (22-24 months old). Consistent with previous studies, we observed an age-related decline in hippocampal proliferation in both groups. However, in fully adult beta 2-/- mice a 43% reduction of granule cell proliferation was detected compared to age-matched controls. This was accompanied by a significant decrease in dentate gyrus area/section and the length of the granule cell layer in beta 2-/- mice. These alterations were not the result of a change in plasma corticosterone levels or expression of the neurotrophic factor BDNF in the dentate gyrus, two known regulators of hippocampal cell proliferation. Similarly, there was no increase in gliosis, abnormal myelination, or apoptotic cell death in the beta 2-/- animals, although there was a significant shift in the location of apoptotic cells in the dentate gyrus indicative of a change in neuronal survival. These results suggest that the beta 2-subunit containing nicotinic acetylcholine receptors play an important role in regulating cell proliferation in the hippocampus and that endogenous acetylcholine may act to oppose the negative effects of normal aging and stress on cellular proliferation.     

Vasopressin V1 receptor in rat hippocampus is regulated by adrenocortical functions

Two subtypes of arginine vasopressin (AVP) receptors (V1 and V2) have been distinguished. In this study, we examined the characteristics of AVP binding in rat hippocampus and the effects of bilateral adrenalectomy and adrenal steroids on its [³H]AVP binding. [³H]AVP binding to rat liver and the hippocampal membranes was strongly inhibited by the V1 antagonist, OPC-21268. ADX resulted in a significant decrease in theB_max of AVP binding in the hippocampus. Chronic treatment with aldosterone and corticosterone restored the ADX-induced reduction, but treatment with dexamethasone did not. These results suggest that the AVP V1 receptor in the hippocampus is regulated by adrenocortical neuroregulatory function.     

Cannabinoid interactions with glucocorticoid receptors in rat hippocampus

Previous studies have found that chronic administration of delta9-tetrahydrocannabinol (THC), a psychoactive cannabinoid, can induce brain aging-like degenerative changes in hippocampal structures (e.g., pyramidal cell loss, glial reactivity). Normal aging changes in the hippocampus appear to be partly corticosteroid-dependent. Because THC is similar in molecular structure to corticosteroids (CORT), therefore, we have suggested that THC may act to induce pathology in the hippocampus through CORT receptors. The possibility of THC interactions with CORT receptors was tested more directly in the present studies. Binding of [3H]dexamethasone (DEX) to hippocampal cytosol, in vitro, was inhibited partially, but not completely, by 100-fold excess unlabeled THC and cannabidiol (CBD), a non-psychoactive cannabinoid. Even at 10,000-fold molar excess, moreover, THC could displace only 50% of radiolabeled DEX binding and CBD could inhibit only 22% of tracer binding. Scatchard plot analyses also pointed to a possible non-competitive site for cannabinoid interaction with glucocorticoid receptors. In addition, several studies utilizing the synthetic steroid RU-28362 indicated that THC interacts primarily with the type II class of glucocorticoid receptors. In a separate study, adrenalectomized rats were treated daily for 14 days with 5-10 mg/kg THC or vehicle, and examined 24 h later for [3H]CORT binding in hippocampal cytosol. In THC-treated animals, the Bmax for type II binding was reduced to a degree almost comparable to the down-regulation seen after chronic stress or high corticosteroid administration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Corticosterone modulation of neurotransmitter receptors in rat hippocampus: A quantitative autoradiographic study

The effect of adrenalectomy (ADX) and corticosterone (CORT) replacement on neurotransmitter receptors was studied in dorsal hippocampus of rat using quantitative autoradiography. ADX for one week causes an increase in [3H]5-HT binding to 5-HT1 receptors which is significant in the CA1 cell field. CORT treatment of ADX rats for 3-5 days results in localized reductions of [3H]5-HT binding including a partial reversal of the increase observed after ADX in CA1. CORT treatment of ADX animals also decreases binding of [3H]QNB to muscarinic receptors in the dorsal hippocampus, with a significant effect in an area designated as subiculum. No influence of CORT was detected on [3H]prazosin binding to alpha 1 adrenergic receptors in dorsal hippocampus. Possible mechanisms for hormone effects on neurotransmitter receptor levels are discussed.     

Serotonin regulates type II corticosteroid receptor binding in hippocampal cell cultures

Previous work from our laboratory has shown that early postnatal handling of rat pups permanently increases hippocampal type II, but not type I, corticosteroid receptor binding. Handling also increases hippocampal 5-HT turnover, and the effect of handling on type II corticosteroid receptor binding is blocked by concurrent administration of the 5-HT2 receptor antagonist ketanserin. In view of these findings, the present studies examined the effects of 5-HT on type I ([3H]corticosterone) and type II ([3H]RU 28362) corticosteroid receptor binding in dispersed hippocampal cell cultures derived from animals killed at E19-20 in order to verify that 5-HT can act directly on hippocampal cells to alter corticosteroid receptor binding. Both type I and type II receptors were measurable in cultured hippocampal cells and the apparent affinity (Kd) for [3H]corticosterone (0.4 +/- 0.1 nM) and [3H]RU 28362 (0.8 +/- 0.1 nM) was similar to that from studies with intact animals. 5-HT increased type II, but not type I, corticosteroid receptor binding capacity in a dose-related manner, with the maximal effect (+188%) observed at 10 nM 5-HT and no change in the affinity of the receptor for [3H]RU 28362. The effect of 10 nM 5-HT on [3H]RU 28362 binding required a minimum of 4 d exposure and persisted for at least 7 d following the removal of 5-HT. The effect of 10 nM 5-HT on [3H]RU 28362 binding was completely blocked by the 5-HT2 receptor antagonists ketanserin and mianserin. There were no effects of the 5-HT1a antagonist, BMY 7378, or the 5-HT3 antagonist, MDL 72222.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hippocampal corticosterone receptors and novelty-induced behavioral activity: effect of kainic acid lesion in the hippocampus

Rats were injected bilaterally in the dorsal and ventral hippocampus with kainic acid (KA) or with artificial CSF and their behavior and brain corticosterone (B) receptor systems were studied. The hippocampal KA injection destroyed part of the pyramidal neurons and of the dentate gyrus neurons. These neurons contain a receptor system for B. At 2 weeks after the KA lesion this B receptor system displays an increase in apparent maximal binding capacity (Bmax) of approximately 25%. The compensatory increase in B receptor concentration is reflected in an increased uptake of [3H]B in cell nuclei of hippocampal slices incubated in vitro with saturating concentrations of the steroid. Administration of a tracer dose of [3H]B shows that labelled steroid can enter in vivo the cell nuclear compartment of the KA-lesioned lobe. The role of B was investigated on novelty-induced behavioral activities of KA-lesioned and sham-lesioned animals in a large open and a small closed field at 10 days after bilateral adrenalectomy (ADX) or sham-ADX which is 14 days after the (sham) lesion. B (300 micrograms/kg rat) was administered s.c. 1 h prior to the test. KA lesion resulted in an increase in exploratory activity and a reduction in grooming and immobility. After ADX the effect of KA on exploration was reduced in the 5 min open field and abolished in the 30 min closed field. ADX animals displayed more grooming behavior (closed-field). B replacement of ADX rats reinstated the exploratory hyperactivity of KA-lesioned rats. On some components of the behavior such as ambulation in open-field and locomotion in closed field, there was even a larger responsiveness to B in the KA-lesioned rats than in the control animals. It is concluded that (1) after KA lesion of receptor containing neurons, the remaining tissue displays a compensatory increase in number of B receptor sites; (2) B is required for full expression of exploratory activity of rats with or without KA lesions; (3) the KA-lesioned rats display a larger responsiveness to B; and (4) the increased number of B receptor sites may underlie the larger responsiveness to B.     

Modulation of presynaptic glucocorticoid receptors on glutamate release from rat hippocampal nerve terminals

In this study, we have examined the role of corticosterone (CORT) in the regulation of neuronal glutamate release using nerve terminals (synaptosomes) isolated from the rat hippocampus. Adult male Sprague‐Dawley rats received either a chronic systemic administration of CORT (daily 25 mg/kg in sesame oil, subcutaneously) or long‐term bilateral adrenalectomy (ADX) (3–4 weeks), and then the release of 4‐aminopyridine (4AP)‐evoked endogenous glutamate and the levels of glucocorticoid receptor (GR) expression from hippocampal nerve terminals were studied. Chronic administration of CORT resulted in a significant increase of 4AP‐evoked glutamate release from hippocampal nerve terminals, whereas ADX reduced 4AP‐evoked glutamate release. In addition, chronic administration of CORT and ADX induced a significant reduction and increase in GR expression in hippocampal synaptosomes, respectively, as detected by Western blots. Furthermore, acute treatment of CORT or dexamethasone facilitated 4AP‐evoked glutamate release from synaptosomes freshly isolated from naïve rat hippocampus and this effect can be significantly prevented by pretreatment of GR antagonist mifepristone, but not by mineralocorticoid receptor (MR) antagonist RU28318. Together, our results strongly support the presence of GRs on presynaptic nerve terminals in the rat hippocampus acting to facilitate the release of neuronal glutamate. Synapse 63:745–751, 2009. © 2009 Wiley‐Liss, Inc.     

Chronic intermittent stress does not differentially alter brain corticosteroid receptor densities in rats prenatally exposed to ethanol.

Prenatal ethanol exposure produces hypothalamic-pituitary-adrenal (HPA) hyperresponsiveness to stressors. The present study tested the hypothesis that decreased corticosteroid receptor densities at HPA feedback sites may play a role in deficient feedback inhibition and the resultant HPA hyperresponsiveness that is observed following prenatal ethanol exposure. Brains of adult Sprague-Dawley rats from prenatal ethanol (E), pair-fed (PF) and ad libitum-fed control (C) treatment groups were examined for both mineralocorticoid receptor (MR; Type I) and glucocorticoid receptor (GR; Type II) densities using a cytosolic binding assay. Experiment 1 compared the effects of chronic intermittent stress (Stress Regimen I) and corticosterone (CORT) pellet implants on hippocampal corticosteroid receptor densities in control rats. Experiment 2 determined whether exposure to Stress Regimen I would differentially downregulate and whether adrenalectomy (ADX) would differentially upregulate hippocampal corticosteroid receptors in E compared with PF and C animals. Experiment 3 examined the effects of a modified chronic intermittent stress regimen (Stress Regimen II) on corticosteroid receptor densities at several HPA feedback sites (hippocampus, prefrontal cortex, hypothalamus, and anterior pituitary) in E compared with PF and C animals. CORT pellet implants significantly downregulated hippocampal GR and MR densities in control males and females. Exposure to Stress Regimen I produced downregulation of hippocampal GRs and MRs in males comparable with that produced with CORT pellet implants, and significant downregulation of hippocampal GRs in females across all prenatal treatment groups. This stress regimen also elevated basal plasma CORT levels without concurrent changes in plasma CBG levels, and increased relative adrenal weights in both males and females. In addition, upregulation of hippocampal GRs occurred at 7 days compared with 24 h following ADX in females that had previously been exposed to this stress regimen. Following exposure to Stress Regimen II, both the downregulation of hippocampal corticosteroid receptors and the increase in basal CORT levels in males and females appear to have been abolished by the changes in housing condition during the period of chronic stress. Importantly, prenatal ethanol exposure did not differentially alter GR or MR densities at any feedback site under non-stressed conditions. Exposure to Stress Regimen II, revealed subtle effects of prenatal treatments on hippocampal GRs however it is unlikely that these changes in corticosteroid receptor densities mediated the feedback inhibition deficits observed in E animals. Together, these data demonstrate that: (1) a relatively mild intermittent stress regimen can increase basal CORT levels and downregulate hippocampal corticosteroid receptor densities (2) a seemingly small change in housing conditions during stress appears to eliminate both receptor downregulation and increase in basal CORT levels and (3) decreased corticosteroid receptor densities at HPA feedback sites in the brain do not appear to underlie the HPA hyperresponsiveness observed in E animals.     

The Effects of Aging and Hormonal Manipulation on Amyloid Precursor Protein APP695 mRNA Expression in the Rat Hippocampus

In the rat hippocampus, neuronal morphology and survival are profoundly affected by adrenal steroids, and synaptic plasticity can be modulated by the ovarian sex steroids estrogen and progesterone, β-amyloid peptides, which accumulate in neuritic plaques and are derived from the amyloid precursor protein (APR), have been shown to be both trophic and toxic for hippocampal neurons. Of the various APR isoforms, APP695 is the predominant form found in rat brain and the APP695 mRNA is abundantly expressed in the hippocampus. In order to investigate the hypothesis that APR may serve as a mediator of the steroid effects, we have monitored the hippocampal expression of APP695 mRNA by in situ hybridization, with aging and with steroid manipulation. In aged female rats we observed a decrease in the level of APP695 mRNA relative to young female rats, while no such age difference was evident in male rats. Physiological, surgical and pharmacological manipulation of glucocorticoids appeared to have no effect on APP695 mRNA levels in the hippocampus. Treatment of young, ovariectomized female rats with estrogen and progesterone, resulted in an increase in hippocampal APP695 expression compared to untreated, ovariectomized controls.