Dexamethasone Does Not Prevent Seven-Day ADX-Induced Apoptosis in the Dentate Gyrus of the Rat Hippocampus

Long-term adrenalectomy (ADX) is known to result in apoptosis within the dentate gyrus of the rat hippocampus. While the underlying mechanism is still unclear, adrenal steroids appear to play a pivotal role in granule cell survival, as administration of the mineralocorticoid receptor (MR) agonists, corticosterone and aldosterone, to ADX rats results in protection against the ADX-induced effect. The consequence of administration of the glucocorticoid receptor (GR) agonists, dexamethasone and RU28362, however, is less clear, and either complete or only partial protection for the ADX animal has been reported. This study investigated further the role played by GR in the degenerative process. After establishing the characteristics of seven-day ADX-induced apoptosis in the young male Wistar rat, the effect of chronically-implanted, subcutaneous pellets containing various doses of dexamethasone and corticosterone, on ADX-induced apoptosis was studied. Both high and low doses of corticosterone were found to be protective. In contrast to some other studies, however, neither dose of dexamethasone had any obvious protective effect and rather seemed to increase apoptosis in dentate gyrus of intact animals. Intracerebroventricular infusion of dexamethasone for seven days was also found to be ineffective in preventing apoptosis, demonstrating that it is occupation of MR, rather than GR, which is crucial to dentate gyrus granule cell survival.     

Support for a bimodal role for type II adrenal steroid receptors in spatial memory.

We investigated the effects of acute adrenal steroid treatment on spatial memory using the Y-maze and employing adrenal steroid receptor antagonists and agonists. For receptor activation, adrenalectomized rats were injected 2 h prior to their first Y-maze trial with sesame oil (adrenalectomy or SHAM), stress levels of corticosterone, a Type I receptor agonist (aldosterone), or a Type II receptor agonist (RU362). For receptor inactivation, unoperated rats were injected with a Type I receptor antagonist (RU318), a Type II receptor antagonist (RU555), sesame oil, or not injected at all. The findings indicated that spatial memory was impaired when the Type II receptors were blocked (RU555) or highly occupied (corticosterone or RU362) and normal for the other treatment conditions. These data suggest that the Type II receptors may be responsible for the inverted U-shaped relationship between spatial memory and corticosterone levels reported by others.     

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

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

Androgens are neuroprotective in the dentate gyrus of adrenalectomized female rats

Neuroprotective effects of androgens have not been well-characterized, but there is evidence that 5 alpha-androstane-3 alpha, 17 beta-diol (3 alpha-diol) has anti-seizure effects. To further examine androgens' neuroprotective effects, testosterone (T), dihydrotestosterone (DHT), 3 alpha-diol (1.0 mg/kg SC daily), or sesame oil vehicle was administered to adrenalectomized or sham-operated, young, female Long Evans rats (N = 52). After seven days, animals were perfused and trunk blood was collected for radioimmunoassay of plasma corticosterone and androgens. No pyknotic cells were seen in the dentate of the sham-operated animals or those animals that had incomplete adrenalectomies (n = 20); however, cresyl violet and TUNEL stains revealed pyknotic cells in the granule layer of the dentate gyrus of adrenalectomized rats (n = 28). Testosterone, DHT, or 3 alpha-diol significantly reduced the number of pyknotic cells in the dentate gyrus compared to vehicle administered, adrenalectomized rats. Steroid-administered animals had levels of T, DHT, or 3 alpha-diol within physiological concentrations. These findings suggest that T, DHT, or 3 alpha-diol may have neuroprotective effects via a common mechanism of action.     

The relationship between adrenal steroids and enrichment-induced brain growth.

The question of whether brain growth brought about by environmental enrichment is mediated by the adrenal cortex has not been answered. Accordingly, young male rats were either adrenalectomized (ADX) and infused with a constant maintenance dose of corticosterone (2 mg.kg-1.day-1) or sham-operated and implanted with a blank infusion device. Half of each surgical group was maintained in either impoverished (IC) or enriched conditions (EC). After 30 days, changes in forebrain growth and thickness of various cortical and subcortical regions were determined for each group. Enrichment and ADX independently increased forebrain weight and thickened cortical tissue at about the same anatomical sites. However, combined treatments were additive, not interactive. EC-induced brain growth is mimicked but not mediated by adrenalectomy.     

Differences in adrenocortical secretory and gene expression responses to stimulation in vitro by ACTH or prolactin between high- and low-avoidance Hatano rats

Rats of the Hatano high-avoidance (HAA) and low-avoidance (LAA) strains have been genetically selected on the basis of their two-way active avoidance behavior, and have different endocrine responses to stress. The present study focused on the adrenal steroid hormone responses of the Hatano strains and identifies differences in regulation of the adrenal cortex in vitro of HAA and LAA rats. Although incubation with prolactin (PRL) and/or adrenocorticotrophic hormone (ACTH) resulted in a dose-dependent increase of corticosterone and progesterone release by adrenal cells from both HAA and LAA male rats, the responses were markedly increased for adrenal cells from LAA rats as compared with HAA rats. This finding suggested that adrenal glands of HAA rats are less sensitive to PRL and/or ACTH than adrenals from LAA rats. Several possible intra-adrenal regulators were investigated. The basal level of expression of steroidogenic acute regulatory protein (StAR) and the long form of the PRL receptor (PRLR-L) mRNAs was higher in adrenals of LAA rats. ACTH treatment of adrenal cells from HAA rats resulted in statistically significant increases in melanocortin receptor 2 (MC2R) mRNA expression, while neither ACTH nor PRL altered MC2R mRNA expression in adrenal cells of LAA rats. Conversely, the increase in PRLR-L mRNA expression induced by PRL was observed only in adrenal cells from LAA rats. Treatment of adrenal cells with PRL and/or ACTH increased the expression of StAR and CYP11A1 mRNAs for both Hatano strains. However, the induction of StAR mRNA expression was higher in LAA rats, but the CYP11A1 response was lower. These findings indicate that adrenal cells of the LAA strain have higher sensitivity to secretagogues than those of the HAA strain. These results suggest that PRL may also be important in stimulating secretion of adrenal steroid hormones.     

Accelerated cognitive aging in diabetic rats is prevented by lowering corticosterone levels

Diabetes and normal aging are both characterized by increases in levels of glucocorticoids. Because long-term exposure to elevated glucocorticoids can be detrimental to hippocampal function, we evaluated the performance of young diabetic rats in the 14-unit T-maze, a task that is sensitive to hippocampal deficits. To assess the contribution of diabetes-induced elevations in corticosterone levels, we examined maze learning in diabetic rats that had levels of corticosterone 'clamped' through adrenalectomy and low-dose corticosterone replacement. For comparison, we also tested a separate group of young and aged rats in the maze. Adrenally intact diabetic rats learned poorly in the 14-unit T-maze. Preventing the increases in corticosterone levels that accompanies the onset of experimental diabetes also prevented deficits in complex maze learning. The pattern of errors made by adrenally intact diabetic rats was similar to the pattern of errors made by aged rats, suggesting that the cognitive profiles of diabetic and aged rats share common features.     

Sex-dependent changes induced by prenatal stress in cortical and hippocampal morphology and behaviour in rats: an update

Recent prospective studies have shown that gestational stress in humans is more likely to cause cognitive and emotional problems in the offspring if it occurs during weeks 12-20 of pregnancy. There are also suggestions that such problems may be gender dependent. This review describes recent studies that found sex differences in the behaviour and brain morphology of rats stressed prenatally during the equivalent period of neuronal development in humans. Learning deficits are more prevalent in males and anxious behaviour in females but their appearance depends also on the timing and intensity of the stress and the age when the offspring were tested. Cognitive deficits and anxiety are linked to a sex-dependent reduction in neurogenesis and in measures of dendritic morphology in the prefrontal cortex and hippocampal formation. Maternal adrenalectomy prior to the stress prevents the anxiety in both sexes and learning deficits in males. Corticosterone administration to the dam to mimic levels induced by stress reinstates only the anxiety, indicating that it arises from foetal exposure to corticosterone from the maternal circulation. Learning deficits in males may result from a combination of a reduction in testosterone and in aromatase activity, together with the action of other adrenal hormones.     

Inhibitory avoidance deficit following short-term adrenalectomy in the rat: the role of adrenal catecholamines

Impaired retention of an inhibitory avoidance response was observed in rats subjected to adrenalectomy (ADX) up to 120 hr before the single learning trial. Corticosterone substitution failed to normalize this behavioral deficit. Rats ADX 240 hr prior to the learning trial showed a normalized behavior. Adrenomedullectomy (ADXM) 48 or 240 hr before learning caused a similar impairment as in short-term ADX rats. The 240-hr ADX rats subjected to corticosterone substitution showed the same behavioral deficit as short-term ADX rats or ADXM ones. Immediate postlearning subcutaneous injection of adrenaline in a dose range of 0.005-5.0 micrograms/kg or of noradrenaline (0.005-0.5 microgram/kg) to 48-hr ADX rats resulted in a dose-related improvement of later retention behavior. Higher doses of catecholamines were less or ineffective. Postlearning treatment of 48-hr ADXM rats with adrenaline (0.5-500 micrograms/kg) caused a similar pattern of behavioral changes. It is concluded that adrenal catecholamines play an important role in the modulation of consolidation of memory. In addition, the high circulating ACTH levels that follow long-term ADX may correct for the behaviorial deficit induced by the absence of adrenomedullary catecholamines.     

Chronic corticosterone-induced deterioration in rat behaviour is not paralleled by changes in hippocampal NF-kappaB-activation

We investigated whether long-lasting stress induced by chronic glucocorticoid (GC) exposure affects activation of brain NF-kappaB and whether these changes are related to functional deterioration and structural changes in the rat hippocampus. Psychometric investigations were conducted using a holeboard test system in 28 one-year-old male Wistar rats. Thereafter, rats were divided into three groups for daily administration of 10 mg corticosterone (treatment) or sesame oil (placebo = sham control for effects of the vehicle) for 60 days. Additional control rats did not receive any treatment or handling until the end of the experiment. Behavioural and cognitive changes were tested again in the holeboard system. Rat body weights and corticosterone concentrations in plasma, hippocampus and urine were determined and adrenal glands were investigated histopathologically. Hippocampal concentrations of corticosterone, NF-kappaB and I-kappaBalpha were determined using RIA, EMSA and Western blotting techniques, respectively. Structural changes in rat hippocampus were measured using magnetic resonance imaging techniques. High peripheral corticosterone concentrations after chronic treatment led to significant reductions in rat body weight. Significant atrophy of both adrenal glands with marked histological deterioration was detected. Furthermore, an increase in hippocampal corticosterone concentrations was observed after chronic administration. Chronic corticosterone treatment also significantly altered behaviour and working and reference memory capacity without changing hippocampal structure. Daily injections of sesame oil in the placebo group, however, were also sufficient to reduce the pellet-finding time. However, neither in the corticosterone group nor in the placebo group were behavioural changes paralleled by significant changes in brain NF-kappaB activation and I-kappaBalpha expression. Thus, cognitive alterations in rats seen after chronic corticosterone exposure are not paralleled by hippocampal NF-kappaB modulation.     

Influence of Regularity of Exposure to Chronic Stress on the Pattern of Habituation of Pituitary-Adrenal Hormones, Prolactin and Glucose

The effect of regularity of exposure to two different chronic stressors (noise or immobilization (IMO)) on the pattern of habituation of pituitary-adrenal (PA) hormones, prolactin and glucose was evaluated in adult male rats. Animals were chronically subjected to either regular or irregular time schedule of noise (30 min/day) or IMO (2 h/day) for two weeks. The day after the last stress session the rats were killed without stress or after having been subjected to 30 min of the homotypic stressor. Whereas regular noise did not affect food intake, body weight gain or adrenal weight, irregular noise decreased body weight gain and induced a moderate adrenal hypertrophy. In addition, previous daily exposure to regular but not to irregular noise reduced both prolactin and corticosterone responses to acute noise. In contrast, glucose response to acute noise was reduced after both regular and irregular exposure to chronic noise. Either regular or irregular exposure to chronic IMO decreased food intake and body weight and increased adrenal weight to the same extent. Likewise, no influence of regularity of exposure to chronic IMO on corticosterone and prolactin responses to acute IMO was observed. However, habituation of the ACTH response to acute IMO was observed in rats subjected to chronic regular IMO, but not in rats subjected to chronic irregular IMO. Finally, acute IMO-induced hyperglycemia diminished to the same extent after regular and irregular IMO. From these results we can conclude that: first, the process of habituation of the PA axis to chronic stress is greatly dependent upon factors such as regularity of exposure to the stressor and stressor intensity, and second, the influence of regularity on the pattern of habituation to a repeated stressor is dependent on the physiological variable we are dealing with.     

Acute predator stress impairs the consolidation and retrieval of hippocampus-dependent memory in male and female rats

We have studied the effects of an acute predator stress experience on spatial learning and memory in adult male and female Sprague-Dawley rats. All rats were trained to learn the location of a hidden escape platform in the radial-arm water maze (RAWM), a hippocampus-dependent spatial memory task. In the control (non-stress) condition, female rats were superior to the males in the accuracy and consistency of their spatial memory performance tested over multiple days of training. In the stress condition, rats were exposed to the cat for 30 min immediately before or after learning, or before the 24-h memory test. Predator stress dramatically increased corticosterone levels in males and females, with females exhibiting greater baseline and stress-evoked responses than males. Despite these sex differences in the overall magnitudes of corticosterone levels, there were significant sex-independent correlations involving basal and stress-evoked corticosterone levels, and memory performance. Most importantly, predator stress impaired short-term memory, as well as processes involved in memory consolidation and retrieval, in male and female rats. Overall, we have found that an intense, ethologically relevant stressor produced a largely equivalent impairment of memory in male and female rats, and sex-independent corticosterone-memory correlations. These findings may provide insight into commonalities in how traumatic stress affects the brain and memory in men and women.     

Administration of corticosterone after memory reactivation disrupts subsequent retrieval of a contextual conditioned fear memory: dependence upon training intensity

Reactivation of stabilized memories returns them to a labile state and causes them to undergo extinction or reconsolidation processes. Although it is well established that administration of glucocorticoids after training enhance consolidation of contextual fear memories, but their effects on post-retrieval processes are not known. In this study, we first asked whether administration of corticosterone after memory reactivation would modulate subsequent expression of memory in rats. Additionally, we examined whether this modulatory action would depend upon the strength of the memory. We also tested the effect of propranolol after memory reactivation. Adult male Wistar rats were trained in a fear conditioning system using moderate (0.4 mA) or high shock (1.5 mA) intensities. For reactivation, rats were returned to the chamber for 90 s 24h later. Immediately after reactivation, rats were injected with corticosterone (1, 3 or 10mg/kg) or vehicle. One, 7 and 14 days after memory reactivation, rats were returned to the context for 5 min, and freezing behavior was scored. The findings indicated that corticosterone when injected after memory reactivation had no significant effect on recall of a moderate memory, but it impaired recall of a strong memory at a dose of 3mg/kg. Propranolol (5mg/kg) given after the reactivation treatment produced a modest impairment that persisted over three test sessions. Further, the results showed that corticosterone, but not propranolol deficit was reversed by a reminder shock. These findings provide evidence that administration of glucocorticoids following memory reactivation reduces subsequent retrieval of strong, but not moderate, contextual conditioned fear memory likely via acceleration of memory extinction. On the other hand, propranolol-induced amnesia may result from blockade of reconsolidation process. Further studies are needed to determine the underlying mechanisms.     

Exploration and avoidance learning after ibotenic acid and radio frequency lesions in the rat amygdala

Open-field activity, avoidance behavior, and plasma corticosterone levels were studied after intraamygdala injections of 3.0 micrograms ibotenic acid (IBO) and radio-frequency (RF) lesions in the amygdala complex of male Wistar rats. The experiments were undertaken to evaluate the importance of amygdala neurons versus axons of passage in fear-motivated behavior. The IBO lesions led to increased open-field activity, but no impairments in active avoidance learning, nor changes in basal or experimental levels of plasma corticosterone. The RF lesions, on the other hand, led to an increase in experimental plasma corticosterone levels. In the one-way avoidance task the RF lesions, in contrast to the IBO lesions, led to significant impairments in the acquisition of the avoidance response. Although the long-term axon-sparing effect of IBO is questioned since cavities were detected in the affected areas 8 weeks after the injections, the differences in avoidance learning and in corticosterone levels between the RF and the IBO lesions indicate that the axons were functionally active at the time of testing (14-26 days postoperatively). The increase in open-field activity is attributed to the destruction of amygdala neurons and neurons in the overlying cortex, while an avoidance deficit seem to depend on the destruction of axons. On the basis of the behavioral results and the corticosterone data in these experiments, it is suggested that the behavioral changes are not attributable to a general reduction in the arousal of fear. However, since the IBO lesions did not affect the most medial parts of the amygdala complex including the central amygdala nucleus, the role of this nucleus in fear arousal has to be investigated further.     

Persistent neuroendocrine changes in multiple hormonal axes after a single or repeated stressor exposures

Many researchers have studied acute responses to stress in animals and how they are modified by prior stressor exposure, but relatively few have examined whether responses to stressors might last for prolonged periods of time. We have previously demonstrated that trough plasma corticosterone levels in rats are elevated for three to five days after single or repeated exposures to mild restraint and inescapable tailshock. The current study measured other aspects of the adrenal axis, and activity in other neuroendocrine systems, 24 hours after one or three consecutive exposures to the same stress paradigm. The data indicated persistent activation of the adrenal axis and prolactin levels, whereas the thyroid and reproductive hormone axes were inhibited after either one or three stress sessions. These changes are remarkable in that one would have expected acute responses to even intense stressors to have ended within hours after the end of the stressor. It will be important to understand the interactions among these responding neuroendocrine systems and to know how long such persistent changes last. Finally, it will be critical to understand the relative contributions of neuroendocrine and psychological factors in maintaining these persistent neuroendocrine changes after exposure to intense stressors.     

Electrical self-stimulation of dentate gyrus granule cells

Male albino rats implanted with a chronic stimulating electrode electrophysiologically guided into the granule cell layer of the dorsal dentate gyrus were tested in a self-stimulation paradigm. It was found that subjects self-administer electrical stimulation to the granule cells at low, steady rates. Consistent with the presence of opioid-like immunoreactivity in the granule cell mossy fiber pathway, intraperitoneal injection of the opiate antagonist naloxone diminishes granule cell self-stimulation in a dose-related fashion. The results suggest that a granule cell opioid synapse participates in expression of reward at this site.     

Adrenal gland responses to lipopolysaccharide after stress and ethanol administration in male rats

All forms of stress, including restraint stress (RS) and lipopolysaccharide (LPS) administration, activate the hypothalamic-pituitary-adrenal (HPA) axis. LPS binds to a recognition protein (CD14) and toll-like receptor 2/4 in different cells and tissues, including the adrenal gland, to induce the production of cytokines and cause upregulation of cyclooxygenase and nitric oxide synthase (NOS) enzymes. Acute ethanol exposure activates the HPA axis, but in some conditions prolonged administration can dampen this activation as well as decrease the inflammatory responses to LPS. Therefore, this study was designed to evaluate the adrenal response to a challenge dose of LPS (50 μg/kg) injected i.p., after submitting male rats to RS, twice a day (2 h each time) for 5 days and/or ethanol administration (3 g/kg) by gavage also for 5 days, twice daily. At the end of the experiment, plasma corticosterone concentrations and adrenal gland content of prostaglandin E (PGE) and NOS activity were measured as stress mediators. The results showed that repetitive ethanol administration attenuated the adrenal stress response to LPS challenge alone and after RS, by preventing the increase in plasma corticosterone concentrations and by decreasing the PGE content and NOS activity in the adrenal gland. Therefore, we conclude that moderate alcohol consumption could attenuate the effects of psychophysical stress and impair an inflammatory response.     

Glucocorticoids, the hippocampus, and behavior: interactive relation between task activation and steroid hormone binding specificity

A previous investigation established a modulatory effect of adrenal glucocorticoids on a behavior generally believed to require participation of the hippocampus for normal expression. For further delineation of whether the observed effects on appetitive extinction are consistent with a glucocorticoid action on hippocampus, adrenalectomized rats were administered either corticosterone or dexamethasone, a synthetic glucocorticoid that is accumulated much less intensively by the hippocampus than is corticosterone. While corticosterone normalized extinction, dexamethasone did not. In two other hippocampal-dependent behaviors, alternation and exploration, there was no glucocorticoid modulation of behavior, which may indicate that factors such as the level of activation of the type of learning determine whether a glucocorticoid effect will occur. These findings of a glucocorticoid action on one of three tasks that involve hippocampal function reinforce the notion that in addition to their well-studied actions in protein and carbohydrate metabolism, the adrenal glucocorticoids may also be subtle modulators of behavior.     

A facilitative role for corticosterone in the acquisition of a spatial task under moderate stress

Emotionally charged experiences alter memory storage via the activation of hormonal systems. Previously, we have shown that compared with rats trained for a massed spatial learning task in the water maze in warm water (25°C), animals that were trained in cold water (19°C) performed better and showed higher levels of the stress hormone corticosterone. Here, we examined whether manipulating the levels of corticosterone can determine the strength of spatial information acquisition and retention. Rats were injected with metyrapone (25, 50, and 75 mg/kg, i.p.) or with corticosterone (10 and 25 mg/kg, i.p.) and trained in a massed spatial task in either cold (19°C) or warm (25°C) water. We found that whereas animals injected with vehicle performed well in the spatial task in cold water (moderate stress), rats injected with the intermediate metyrapone dose showed impairment in performance. Moreover, whereas animals injected with vehicle on average did not perform well in warm water (mild stress), rats injected with the lower corticosterone dose showed improvement in performance in warm water. These two mirror experiments of corticosterone blockade and enhancement strongly suggest that corticosterone is instrumental in the acquisition and retention of the spatial learning task.