Methodologic issues in studying glucocorticoid-induced damage to neurons.
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Animals↗
Biomedical subjects
R Sapolsky
Publications and source records attributed to R Sapolsky.
Glucocorticoids mediate the stress-induced extracellular accumulation of glutamate.
The hippocampal damage caused by stress has been attributed to an increased glutamatergic tone brought about by secretion of glucocorticoids. Although exposure to stress has been shown to increase the outflow of glutamate, direct involvement of glucocorticoid in this phenomenon has not been examined. The present study demonstrates that adrenalectomy attenuates the stress-induced outflow of glutamate in the hippocampus and prefrontal cortex and that glucocorticoid replacement abolishes this attenuation.
Adrenalectomy↗
Stress exacerbates neuron loss and cytoskeletal pathology in the hippocampus.
Glucocorticoids (GCs), the adrenal steroids secreted during stress, endanger the hippocampus, compromising its ability to survive neurological insults. GCs probably do so by disrupting energetics in the hippocampus, thus impairing its ability to contain damaging fluxes of excitatory amino acids and calcium. Superficially, these observations suggest that stress itself should also exacerbate the toxicity of neurological insults. However, most studies have involved unphysiologic GC manipulations, limiting speculations about the endangering effects of stress. In this study, rats were infused with the excitotoxin kainic acid (KA) after either having been adrenalectomized and replaced with a range of physiologic concentrations of GCs, or having been stressed intermittently. We observed that within the CA3 region, increasing CORT concentrations exacerbated the KA-induced neuron loss, the extent of tau immunoreactivity, and of spectrin proteolysis. The transitions from low to high basal GC concentrations and from high basal to stress GC values were both associated with significant exacerbation of neuron loss and tau immunoreactivity; the extent of spectrin proteolysis was less sensitive to increments in GCs. As would be expected from these data, exposure to intermittent stress prior to KA infusion also exacerbated neuron loss, tau immunoreactivity, and spectrin proteolysis in CA3. Thus, physiological elevations of GCs, and stress itself, can exacerbate hippocampal neuron loss and the attendant degenerative markers following an excitotoxic insult. Of significance, seizure and hypoxia-ischemia provoke considerable GC stress responses, which may thus worsen the resultant damage. Furthermore, a number of neuropsychiatric disorders, as well as aging, are associated with elevated basal GC concentrations, which may endanger the hippocampus in the event of neurological insult.
Animals↗
Corticosterone is a preferable ligand for measuring rat brain corticosteroid receptors: competition by RU 28362 and RU 26752 for dexamethasone binding in rat hippocampal cytosol.
It is unclear whether in vitro corticosteroid receptor binding assays have used inappropriately high concentrations of synthetic corticosteroid competitors, thereby potentially introducing error into estimates of type I (mineralocorticoid) and type II (glucocorticoid) receptor binding. To determine more accurately the concentration of blockers necessary to discriminate between these two sites, we have derived Ki values for the competition of dexamethasone, RU 28362 and RU 26752 for [3H]corticosterone and [3H]dexamethasone binding in rat hippocampus. Non-specific binding of both radioligands was defined with unlabeled dexamethasone to exclude transcortin. The type II agonist RU 28362 competed for only a portion of [3H]corticosterone binding, exhibiting a Ki of 0.5 nM for this binding. In contrast, RU 28362 fully competed all binding of a saturating concentration of [3H]dexamethasone, even though [3H]dexamethasone also recognized type I receptors, defined as specific [3H]corticosterone binding in the presence of 80 nM RU 28362. RU 28362 competition for [3H]dexamethasone binding exhibited characteristics of a 2-site interaction, with Kis of 0.3 and 194 nM. The type I receptor antagonist RU 26752 competed less effectively for [3H]corticosterone and [3H]dexamethasone binding, but nonetheless competed fully within a 1000-fold concentration range. Even at a level less than 125 x its Ki for type I binding, RU 26752 still inhibited virtually all type II receptor binding by [3H]corticosterone. We conclude that type I and II receptors in rat brain are best distinguished using [3H]corticosterone as the labelling ligand, with cold RU 28362 and dexamethasone to eliminate binding to type II and transcortin sites, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
Androstanols↗
Augmented ACTH responses to stress in adrenalectomized rats replaced with constant, physiological levels of corticosterone are partially normalized by acute increases in corticosterone.
Adrenalectomized rats replaced with constant, physiological levels of corticosterone via a subcutaneous pellet (Pellet) have normal basal morning ACTH but exhibit enhanced and prolonged ACTH responses to stress vs. sham-operated (Sham) rats. It has not been determined if the lack of either stress-induced or circadian increases in corticosterone, both of which are missing in Pellet rats, may account for this enhanced response. To test the extent to which stress-associated increases in corticosterone alone can normalize stress-induced hypersecretion of ACTH, we approximated endogenous secretion by injecting additional corticosterone in Pellet rats via an indwelling subcutaneous cannula, 5 min before hypoxia stress (10% O2). A corticosterone dose of 666 micrograms/kg (Pellet+B), but not 333 micrograms/kg (Pellet+Low B), produced plasma corticosterone levels comparable to those in Shams and normalized stress-induced but not post-stress plasma ACTH. Administration of the type II corticosteroid receptor antagonist RU 38486 30 min before corticosterone reversed this inhibition. We conclude that enhanced ACTH responses to stress in Pellet rats result in large part from lack of type II receptor-mediated feedback inhibition by corticosterone increases during stress, although prior circadian increases in corticosterone may also be required.
Adrenalectomy↗
Polymerase chain reaction mapping of yeast GAL7 mRNA polyadenylation sites demonstrates that 3' end processing in vitro faithfully reproduces the 3' ends observed in vivo.
In general, synthetic RNA transcripts corresponding to the 3' ends of Saccharomyces cerevisiae genes appear to be accurately cleaved and polyadenylated in vitro under appropriate conditions in yeast cell extracts. Initially, however, the endpoints observed in vitro for the GAL7 gene failed to correlate adequately with those reported in vivo as derived from traditional S1 nuclease protection analyses. This led us to apply an independent method for analyzing mRNA 3' ends, using the polymerase chain reaction, with a first strand primer that incorporated a BamHI restriction site sequence near its 5' end, followed by (dT)17. This proved to be a sensitive and accurate means for determining precisely the major and minor polyadenylation sites of the GAL7 mRNA. Moreover, there was complete agreement between the sites identified with this technique when applied to cellular RNA and those generated in vitro by our 3' end mRNA processing reaction. This provides further support for the likelihood that processing in vitro faithfully reflects the endonucleolytic cleavage and polyadenylation events that occur within the living cell.
Base Sequence↗
The role of the hippocampus in feedback regulation of the hypothalamic-pituitary-adrenocortical axis.
There is considerable, although not entirely consistent, evidence that the hippocampus inhibits most aspects of HPA activity, including basal (circadian nadir) and circadian peak secretion as well as the onset and termination of responses to stress. Although much of the evidence for these effects rests only on the measurement of corticosteroids, recent lesion and implant studies indicate that the hippocampus regulates adrenocortical activity at the hypothalamic level, via the expression and secretion of ACTH secretagogues. Such inhibition results largely from the mediation of corticosteroid feedback, although more work is required to determine whether the hippocampus supplies a tonic inhibitory input in the absence of corticosteroids. It must be noted that the hippocampus is not the only feedback site in the adrenocortical system, since removal of its input only reduces, but does not abolish, the efficacy of corticosteroid inhibition, and since other elements of the axis appear eventually to compensate for deficits in feedback regulation. The importance of other feedback sites is further suggested not only by the presence of corticosteroid receptors in other parts of the brain and pituitary, but also by the improved prediction of CRF levels by combined hypothalamic and hippocampal receptor occupancy. The likelihood of feedback mediated by nonhippocampal sites underscores the need for future work to characterize hippocampal influence on HPA activity in the absence of changes in corticosteroid secretion. However, despite the fact that the hippocampus is not the only feedback site, it is distinguished from most potential feedback sites, including the hypothalamus and pituitary, by its high content of both type I and II corticosteroid receptors. The hippocampus is therefore capable of mediating inhibition over a wide range of steroid levels. The low end of this range is represented by corticosteroid inhibition of basal (circadian nadir) HPA activity. The apparent type I receptor specificity of this inhibition and the elevation of trough corticosteroid levels after hippocampal damage support a role for hippocampal type I receptors in regulating basal HPA activity. It is possible that basal activity is controlled in part through hippocampal inhibition of vasopressin, since the inhibition of portal blood vasopressin correlates with lower levels of hippocampal receptor occupancy, and the expression of vasopressin by some CRF neurons is sensitive to very low corticosteroid levels. At the high end of the physiological range, stress-induced or circadian peak corticosteroid secretion correlates strongly with occupancy of the lower affinity hippocampal type II receptors.(ABSTRACT TRUNCATED AT 400 WORDS)
Adrenal Cortex↗
Short term effects of glucocorticoids upon hippocampal ultrastructure.
This study evaluated hippocampal cytoarchitecture in male Sprague-Dawley (225-250 g) rats treated with corticosterone (CORT) and in unhandled, unstressed rats. CORT-treated animals received 20 mg/kg CORT in sesame oil/day x 3 days; each dose produces levels in the upper physiologic range, similar to those produced by major stressors. Using quantitative electron microscopy, sections from regions Ca3b, Ca1, and dentate gyrus were examined. Surface density and surface area of Golgi and the number of Golgi-stacks increased following CORT-treatment (p less than 0.05, ANOVA). Paralleling these changes surface area of rough endoplasmic reticulum and number of cisternae also increased in neuronal perikarya (p less than 0.001, p less than 0.003 respectively). In contrast, surface densities of mitochondria, multivesicular bodies, and lysosomes were unaffected by CORT treatment. These data suggest that both pyramidal and granule cells of hippocampus are subject to short term effects of high physiological levels of CORT, and that these effects may involve stimulation of protein biosynthesis.
Animals↗
Interleukin-1 stimulates the secretion of hypothalamic corticotropin-releasing factor.
There is now evidence that the immune system, during times of infectious challenge, can stimulate the secretion of glucocorticoids, the adrenal steroids that mediate important aspects of the response to stress. Specifically, secretion of interleukin-1 (IL-1), a monocyte lymphokine secreted after infection, appears at least in part responsible for this effect. Glucocorticoids are secreted in response to a neuroendocrine cascade involving, first, the brain, then the pituitary, and finally the adrenal gland. In this report, human IL-1 is shown to activate the adrenocortical axis at the level of the brain, stimulating the release of the controlling hormone corticotropin-releasing factor (CRF) from the hypothalamus. Infusion of IL-1 induced a significant secretion of CRF into the circulation exiting the hypothalamus, whereas immunoneutralization of CRF blocked the stimulatory effect of IL-1 on glucocorticoid secretion. IL-1 appeared to have no acute direct stimulatory effects on the pituitary or adrenal components of this system. Furthermore, IL-1 did not cause a nonspecific release of other hypothalamic hormones. Thus, the lymphokine acts in a specific manner to activate the adrenocortical axis at the level of the brain; this effect appears to be unrelated to the known pyrogenic effects of IL-1 within the hypothalamus.
Adrenal Cortex↗
Stress and glucocorticoids in aging.
This article has considered two themes that have permeated the gerontologic literature--namely, that aging is a time of decreased efficiency in responding to stress and that chronic stress can accelerate aspects of aging. Given the restricted framework of considering adrenocortical function (as a component of the stress response) and glucocorticoid over-exposure (as a component of chronic stress), there is considerable evidence for both of these ideas. The capacity of glucocorticoids to damage the rat hippocampus slowly over the life span and the glucocorticoid hypersecretion that seems to ensue during aging as a result of such hippocampal damage support these long-standing ideas. It should be noted that these two components interact with each other--excessive glucocorticoid secretion damages the hippocampus, and hippocampal damage produces excessive glucocorticoid secretion. This dysregulatory cascade appears to be a normal part of aging in the rat. The role of glucocorticoids in triggering programmed aging and death, while quite dramatic, is probably a phylogenetically rare event; it remains to be seen if the dysregulatory cascade of glucocorticoid excess in the rat is of relevance to aging in other species. Numerous published studies suggest that this cascade is not an obligatory aspect of normal human aging; rather, it appears to be a significant factor in the explanation of some features of pathologies associated with human aging.
Adrenal Cortex↗
Tuberculosis in wild olive baboons, Papio cynocephalus anubis (Lesson), in Kenya.
Several wild olive baboons from a single troop in the Masai Mara Game Reserve, Kenya were observed to be lethargic and emaciated. Five were trapped and tuberculin tested by intradermal inoculation of 0.1 cc (100 IU) mammalian old tuberculin in the upper eyelid. Two of the five showed positive reaction at 72 hr and were examined at necropsy. Gross lesions in both animals consisted of multiple nodules with caseation in the lung, spleen and tracheobronchial lymph nodes. There were multiple granulomas throughout the lung, spleen and the lymph nodes. Tissues were cultured on Lowenstein-Jensen media with and without pyruvic acid. Isolates were typed as Mycobacterium bovis.
Animals↗
Failure of beta-amyloid protein fragment 25-35 to cause hippocampal damage in the rat.
Considerable excitement has been generated as of late over reports that fragments of the amyloid precursor protein can be neurotoxic both in vivo and in vitro. In this brief report we study the neurotoxicity of the fragment corresponding to amino acids 25-35 of the beta-amyloid protein in the hippocampus in vivo. Under the conditions studied, we do not observe any evidence of consistent, dose-related damage above that seen with vehicle alone.
Amyloid beta-Peptides↗
Peripheral blood lymphocyte immunocompetence in wild African green monkeys (Cercopithecus aethiops) and the effects of capture and confinement.
Capture and prolonged confinement of wild African green monkeys (AGM, C. aethiops) in single housing have been shown to result in high morbidity and mortality. The present study was designed to analyse immune modulation associated with these procedures in AGMs. Four wild resting AGMs were stunned with a rifle shot as controls (group I). Seven newly captured monkeys (group II) and seven laboratory conditioned monkeys (group III) housed singly were anaesthetised with ketamine hydrochloride on day 18 and 45, and 180 and 210 post-capture, respectively. Heparinized blood (10 ml) was drawn from each animal and lymphocytes were cultured in six wells per sample. A triplicate subset was stimulated with 10 microliters Concanavalin A for 48 hours. The cells were pulsed with tritiated thymidine and harvested on filter discs 18 hours later. Lymphocyte stimulation indices (stimulated cell count-unstimulated cell count) were 3598 in group I. 3843 and 4395 in group II and 2809 and 2196 in group III, respectively. This indicates that AGMs confined to single housing for a prolonged period exhibit immune suppression. The results confirm our previous findings that the AGM is more susceptible to stress associated with immune suppression and subsequent high frequency of infectious diseases than other East African nonhuman primates confined to laboratory housing.
Animals↗