Adult rats were given antidepressant drugs orally. Fluoxetine, but not moclobemide, venlafaxine, tianeptine or desipramine, increased total glucocorticoid receptor (GR) mRNA in the hippocampus after 4 weeks. Further examination revealed that GR mRNA containing the brain-specific exon 1(7) was increased across all hippocampal subregions. In contrast, expression of the major exon 1(10) and another brain-specific exon 1(5)-containing GR mRNAs were unchanged. Tissue-specific first exon usage may contribute to the differential regulation of GR by fluoxetine in brain subregions.

In aging humans and rodents, inter-individual differences in cognitive function have been ascribed to variations in long-term glucocorticoid exposure. 11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1) regenerates the active glucocorticoid cortisol from circulating inert cortisone, thus amplifying intracellular glucocorticoid levels in some tissues. We show that 11β-HSD1, but not 11β-HSD2, mRNA is expressed in the human hippocampus, frontal cortex, and cerebellum. In two randomized, double-blind, placebo-controlled crossover studies, administration of the 11β-HSD inhibitor carbenoxolone (100 mg three times per day) improved verbal fluency ( P < 0.01) after 4 weeks in 10 healthy elderly men (aged 55-75 y) and improved verbal memory ( P < 0.01) after 6 weeks in 12 patients with type 2 diabetes (52-70 y). Although carbenoxolone has been reported to enhance hepatic insulin sensitivity in short-term studies, there were no changes in glycemic control or serum lipid profile, nor was plasma cortisol altered. 11β-HSD1 inhibition may be a new approach to prevent/ameliorate cognitive decline.

A bstract : Glucocorticoids affect a wide range of processes in the brain, altering neurotransmission, electrophysiological activity, metabolism, cell division, and death. These actions are mediated by corticosteroid receptors (glucocorticoid and mineralocorticoid) that modify transcriptional activity of target genes. The amount of steroid available to activate these receptors is not only dependent on the circulating levels but also on pre‐receptor metabolism of glucocorticoids occurring intracellularly. This metabolism is carried out by the enzymes 11β‐hydroxysteroid dehydrogenases (11β‐HSDs). There are two distinct isozymes, the products of distantly related genes. 11β‐HSD type 2 inactivates glucocorticoids to its inert 11‐keto derivative, while 11α‐HSD type 1 elevates intracellular glucocorticoid levels by regenerating active glucocorticoids from circulating 11‐dehydrocorticosterone or cortisone. This review highlights the important and very different roles the two enzymes play in the brain, outlining recent results obtained from studying mice with a targeted gene deletion in the 11β‐HSD1 or 11β‐HSD2 genes.

Increasing evidence links chronically elevated glucocorticoid levels and cognitive impairments in a subpopulation of aged rodents and humans. Antidepressant drugs improve hypothalamic-pituitary-adrenal axis feedback regulation and reduce plasma glucocorticoid levels. Decreasing the cumulative lifetime exposure to glucocorticoid excess by long-term exposure to antidepressants may prevent the emergence of cognitive impairments in aged rats. To test this hypothesis, we treated middle-aged male Lister hooded rats (16 months) with amitriptyline until they were 24 months of age, and their cognitive function was assessed in the water maze. Performance in the spatial learning task declined significantly with aging (p < 0.01), with 33% of aged controls showing poorer (<2.5 SD) probe test performance than young controls. Amitriptyline treatment from midlife preserved water maze performance with aging (p < 0.01 compared with aged controls) and significantly (p < 0.01) reduced the proportion of poor performers (7%). Measures of anxiety-related behaviors in the elevated plus-maze were significantly (p < 0.05) decreased in the aged rats after amitriptyline. Furthermore, evening plasma corticosterone levels were reduced (30% decrease; p < 0.01 compared with aged controls) after 6 months of amitriptyline. These data suggest that long-term treatment with amitriptyline decreases the prevalence of cognitive impairment in aged rats and that this may, in part, be a consequence of reduced plasma corticosterone levels and reduced anxiety.

The brain serotonin (5-HT) system interacts closely with the hypothalamic-pituitary-adrenal axis. We examined the effects of stress on hippocampal 5-HT7 receptor and corticosteroid receptor (mineralocorticoid receptor (MR) and glucocorticoid receptor (GR)) mRNA expression measured by in situ hybridisation histochemistry. Acute restraint stress increased 5-HT7 receptor mRNA in CA2 and CA3 hippocampal subregions (32% increase) and had a small effect on GR but not MR mRNA (19% fall in GR in CA1). In contrast, chronic stress (1 week of variable stressors) had little effect on hippocampal 5-HT7 receptor mRNA (9% rise in CA3) but decreased MR mRNA (e.g. 34% decrease in CA2) and GR mRNA expression selectively in the dentate gyrus (26% decrease). The rise in 5-HT7 receptor mRNA expression following restraint stress parallels our previously reported increase in expression after inhibiting the synthesis of adrenal steroids. These data suggest that acute but not chronic stress regulates 5-HT7 receptor mRNA expression in a manner that is likely to be independent of its glucocorticoid actions.

The tonic discharge of rat medial vestibular nucleus (MVN) neurons, and their responsiveness to GABA receptor agonists were investigated in slices prepared from aged rats (24 months old). Aged MVN neurons showed regular spontaneous activity similar to that seen in slices from young adults. However the inhibitory effects of the GABA(A) agonist muscimol on the spontaneous activity of aged MVN neurons were significantly greater than in young MVN neurons. Inhibitory responses to the GABA(B) agonist baclofen also tended to be greater in slices from aged animals, but this difference was not statistically significant. The regular discharge of aged MVN neurons at firing rates similar to those in young animals suggests that the intrinsic excitability of MVN cells is maintained with age. The up-regulation of GABA(A) receptor efficacy in aged MVN neurons may compensate for changes in inhibitory inputs from vestibular commissures and cerebellum that may occur with neuronal loss in the aged brain.

11β-hydroxysteroid dehydrogenase type 1 (11β-HSD-1) intracellularly regenerates active corticosterone from circulating inert 11-dehydrocorticosterone (11-DHC) in specific tissues. The hippocampus is a brain structure particularly vulnerable to glucocorticoid neurotoxicity with aging. In intact hippocampal cells in culture, 11β-HSD-1 acts as a functional 11β-reductase reactivating inert 11-DHC to corticosterone, thereby potentiating kainate neurotoxicity. We examined the functional significance of 11β-HSD-1 in the central nervous system by using knockout mice. Aged wild-type mice developed elevated plasma corticosterone levels that correlated with learning deficits in the watermaze. In contrast, despite elevated plasma corticosterone levels throughout life, this glucocorticoid-associated learning deficit was ameliorated in aged 11β-HSD-1 knockout mice, implicating lower intraneuronal corticosterone levels through lack of 11-DHC reactivation. Indeed, aged knockout mice showed significantly lower hippocampal tissue corticosterone levels than wild-type controls. These findings demonstrate that tissue corticosterone levels do not merely reflect plasma levels and appear to play a more important role in hippocampal functions than circulating blood levels. The data emphasize the crucial importance of local enzymes in determining intracellular glucocorticoid activity. Selective 11β-HSD-1 inhibitors may protect against hippocampal function decline with age.

Chronic treatment with antidepressant drugs (2 weeks or longer) increases corticosteroid receptor mRNA expression in the hippocampus and reduces hypothalamic-pituitary-adrenal axis activity in parallel with improving mood and neuroendocrine function. Earlier effects are less well documented. We examined the effects of short term (9 days) treatment with fluoxetine (10 mg/kg) and venlafaxine (10 mg/kg) on hippocampal mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) mRNA expression and spatial memory in adult rats. In situ hybridization histochemistry showed that the antidepressants decreased MR mRNA expression in all hippocampal subregions (e.g. 45% decrease in CA1 with venlafaxine, P<0.001), while GR mRNA expression was selectively reduced in the CA3 subregion. There was a trend for decreased plasma corticosterone levels following fluoxetine (50% fall, P=0.07) and venlafaxine (30% fall, P=0.18) but neither antidepressants affected spatial memory in the watermaze. Thus antidepressants can have complex and opposing actions on hippocampal corticosteroid receptor expression depending on the duration of treatment.

Glucocorticoid receptor (GR) gene expression is regulated in a complex tissue-specific manner, notably by early-life environmental events that program tissue GR levels. We have identified and characterized several new rat GR mRNAs. All encode a common protein, but differ in their 5'-leader sequences as a consequence of alternate splicing of, potentially, 11 different exon 1 sequences. Most are located in a 3-kb CpG island, upstream of exon 2, that exhibits substantial promoter activity in transfected cells. Ribonuclease (RNase) protection analysis demonstrated significant levels of six alternate exons 1 in vivo in rat, with differences between liver, hippocampus, and thymus reflecting tissue-specific differences in promoter activity. Two of the alternate exons 1 (exons 1(6) and 1(10)) were expressed in all tissues examined, together present in 77-87% of total GR mRNA. The remaining GR transcripts contained tissue-specific alternate first exons. Importantly, tissue-specific first exon usage was altered by perinatal environmental manipulations. Postnatal handling, which permanently increases GR in the hippocampus, causing attenuation of stress responses, selectively elevated GR mRNA containing the hippocampus-specific exon 1(7). Prenatal glucocorticoid exposure, which increases hepatic GR expression and produces adult hyperglycemia, decreased the proportion of hepatic GR mRNA containing the predominant exon 1(10), suggesting an increase in a minor exon 1 variant. Such tissue specificity of promoter usage allows differential GR regulation and programming.

Approximately 30% of human and mammalian populations develop cognitive impairments with ageing. Many of these impairments have been linked to dysfunction of the hippocampus, a well studied area of the medial‐temporal lobe, which is involved in episodic memory and control of the hypothalamo‐pituitary‐adrenal stress axis and, thus, of glucocorticoid secretion. This paper reviews the growing body of studies which explore a possible relationship between lifetime exposure to glucocorticoids and hippocampal impairment. There is now strong evidence which associates hypercortisolemia in aged men with later cognitive dysfunction and this complements a wealth of rodent and other human data. We conclude with a discussion of possible pharmacological and behavioural interventions.

The raphe–hippocampal 5-HT system plays a key role in the modulation of mood, memory and neuroendocrine responses. In the elderly, there is an increased incidence of disturbances of these functions. We examined the effects of ageing and of chronic antidepressant treatment upon 5-HT receptor subtype mRNA expression in the hippocampus and raphe of cognitively tested rats. Amitriptyline treatment decreased 5-HT1A receptor mRNA expression in the dorsal raphe nucleus of the aged rats (24% fall compared to saline treated controls, p<0.01) but not in the young rats. Neither age nor amitriptyline (10 mg/kg, i.p.) administration for 10 weeks altered 5-HT1A, 5-HT2A, 5-HT2C or 5-HT7 receptor mRNA expression in any hippocampal subregion. This suggests a difference in responsiveness to amitriptyline with ageing originating at the level of the raphe 5-HT1A autoreceptor gene expression.

The effects of ageing and of chronic antidepressant treatment upon 5-HT transporter sites ([3H]paroxetine binding) in the rat hippocampus was examined. [3H]paroxetine binding to transporter sites was decreased with ageing in the hippocampus of control rats (38% decrease in dentate gyrus and CA4). Amitriptyline (10 mg/kg, i.p.) had no significant effect on [3H]paroxetine binding in 10 months old rats, but increased binding sites in 24 months rats in all hippocampal subregions (greatest increase of 109% in CA1 compared to saline controls). These data indicate an age-related decrease in hippocampal serotonin transporter sites and upregulation of these sites following 10 weeks of amitriptyline. The observed increase in transporter sites following amitriptyline may contribute to the general lower effectiveness of tricyclic antidepressants with ageing.

Hippocampal mineralocorticoid receptors (MR) are proposed to mediate facilitation of cognition in the short-term. The acute central blockade of MR increases plasma corticosterone levels which itself can affect cognition thus complicating the interpretation of such studies. We therefore investigated the effects of chronic continuous central MR antagonism by spironolactone infused intracerebroventricularly (i.c.v.) (100 ng/h) via Alzet minipumps for 12 days. Rats given chronic i.c.v. infusion of spironolactone spent significantly less time (20% decrease, P < 0.05) within the platform quadrant during the watermaze probe test. Neither hippocampal MR nor glucocorticoid receptor mRNA expression were affected by spironolactone. Morning plasma corticosterone levels and adrenal and thymic weights were unchanged. Chronic central MR antagonism impairs spatial memory in the watermaze independent of changes in receptors or circulating glucocorticoid levels.

Both glucocorticoid excess and decreased serotoneergic (5-HT) transmission may cause depression. The recently cloned 5-HT6 and 5-HT7 receptors have high affinity for antidepressants. Here, we show that pharmacological adrenalectomy increases 5-HT6 and 5-HT7 receptor mRNA expression in specific hippocampal subfields, effects partly reversed by corticosterone replacement. Increased 5-HT6 and 5-HT7 receptor expression may provide a basis, in part, for the therapeutic actions of adrenal steroid synthesis inhibitors in resistant depression.

By inactivating potent glucocorticoid hormones (cortisol and corticosterone), 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) plays an important role in the placenta by controlling fetal exposure to maternal glucocorticoids, and in aldosterone target tissues by controlling ligand access to co-localized glucocorticoid and mineralocorticoid receptors. Amino acid sequence from homogeneous human placental 11β-HSD2 was used to isolate a 1897 bp cDNA encoding this enzyme (predicted Mr 44126; predicted pI 9.9). Transfection into mammalian (CHO) cells produces 11β-HSD2 activity which is NAD+-dependent, is without reductase activity, avidly metabolizes glucocorticoids (Km values for corticosterone, cortisol and dexamethasone of 12.4±1.5, 43.9±8.5 and 119±15 nM respectively) and is inhibited by glycyrrhetinic acid and carbenoxolone (IC50 values 10–20 nM). Rabbit antisera recognizing 11β-HSD2 have been raised to an 11β-HSD2-(370–383)-peptide–carrier conjugate. Recombinant 11β-HSD2, like native human placental 11β-HSD2, is detectable with affinity labelling and anti-11β-HSD2 antisera, and appears to require little post-translational processing for activity. 11β-HSD2 mRNA (~1.9 kb transcript) is expressed in placenta, aldosterone target tissues (kidney, parotid, colon and skin) and pancreas. In situ hybridization and immunohistochemistry localize abundant 11β-HSD2 expression to the distal nephron in human adult kidney and to the trophoblast in the placenta. 11β-HSD2 transcripts are expressed in fetal kidney (but not lung, liver or brain) at 21–26 weeks, suggesting that an 11β-HSD2 distribution resembling that in the adult is established by this stage in human development.

The emergence of cognitive deficits in a subgroup of aged rats is associated with increased hypothalamic-pituitary-adrenal axis activity, decreased hippocampal mineralocorticoid and/or glucocorticoid receptor gene expression and neuronal loss. Short-term treatment with antidepressant drugs in young rats increases hippocampal corticosteroid receptor gene expression. In this study, the effects of chronic antidepressant administration on hippocampal mineralocorticoid and glucocorticoid receptor gene expression and spatial memory in young and aged rats were investigated. Young (eight months) and old (22 +/- 1 months) Lister-hooded rats were ranked according to watermaze performance. Matched pairs of rats were treated with amitriptyline (10 mg/kg) or saline daily for nine weeks, then reassessed in the watermaze. Amitriptyline significantly improved spatial memory in the young rats (33% increase in transfer test time) and increased hippocampal mineralocorticoid, but not glucocorticoid receptor messenger RNA expression. By contrast, in aged rats, amitriptyline had no effect on spatial memory or hippocampal corticosteroid receptor gene expression, either in cognitively unimpaired or cognitively-impaired animals. In aged rats, basal plasma corticosterone levels, which were significantly higher than in young animals, correlated negatively with spatial memory, while hippocampal glucocorticoid receptor mRNA expression correlated negatively with plasma corticosterone levels and positively with spatial memory. Amitriptyline had no significant effect on basal morning plasma corticosterone levels in either young or aged rats, but significantly decreased evening corticosterone levels in aged rats. Our data support the notion that corticosterone exerts a concentration-dependent biphasic influence, via selective activation of hippocampal mineralocorticoid and glucocorticoid receptor, on spatial memory. Amitriptyline improves spatial memory in young rats and increases hippocampal mineralocorticoid receptor gene expression. The lack of amitriptyline effect on spatial memory in aged rats may reflect decreased plasticity of both the synaptic processes underlying spatial memory and the regulation of hippocampal mineralocorticoid/glucocorticoid receptor expression, with mineralocorticoid receptors fully occupied due to elevated basal plasma corticosterone levels (in part a consequence of inadequate glucocorticoid receptor function).

Both central serotonergic dysfunction and glucocorticoid hypersecretion have been separately implicated in the aetiology of affective disorders. The hippocampus highly expresses receptors for 5-hydroxytryptamine and glucocorticoids, and adrenalectomy alters the responsivity of hippocampal neurons to 5-hydroxytryptamine. The hippocampus thus represents a prime locus for interactions between the two systems. In this study we examined the effects of glucocorticoid manipulations on neuronal expression of messenger RNA encoding corticosteroid receptor and 5-hydroxytryptamine receptor subtypes in the hippocampus and 5-hydroxytryptamine1A messenger RNA expression in the dorsal raphe, in the rat. Interestingly, there was no effect of adrenalectomy on 5-hydroxytryptamine1A or 5-hydroxytryptamine2A receptor messenger RNA expression in the dorsal or ventral hippocampus at any time point measured. Furthermore, no changes in 5-hydroxytryptamine1A receptor gene expression were seen in the dorsal raphe (encoding autoreceptors) after adrenalectomy. However, 5-hydroxytryptamine2C (5-hydroxytryptamine1C) receptor messenger RNA expression was increased specifically in posterior CA1 and CA3 neurons following adrenalectomy, an effect that was reversed by glucocorticoid replacement. Following adrenalectomy, glucocorticoid and mineralocorticoid receptor messenger RNA expression increased in the dentate gyrus, CA1 and CA3 subfields of the hippocampus. These increases were apparent 6 h after adrenalectomy, were maintained at two days, but 14 days after adrenalectomy hippocampal glucocorticoid receptor and mineralocorticoid receptor gene expression had returned to control levels. These effects of adrenalectomy were abolished by dexamethasone, but not aldosterone administration, suggesting mediation by autoregulatory glucocorticoid receptors. Our results show that adrenalectomy only transiently increases corticosteroid receptor gene expression in the hippocampus, and selectively increases hippocampal 5-hydroxytryptamine2C receptor messenger RNA expression. The resulting change in 5-hydroxytryptamine2C receptor-mediated responses may produce the alterations in hippocampal neuronal activity in response to 5-hydroxytryptamine observed after adrenalectomy.