Robert L. Spencer

The role of adrenocorticoids as modulators of immune function in health and disease: neural, endocrine and immune interactions.

Bruce S. McEwen , Christine A. Biron , Kenneth W. Brunson , Karen Bulloch , William H. Chambers , Firdaus S. Dhabhar , Ronald H. Goldfarb , Richard P. Kitson , Andrew H. Miller , Robert L. Spencer , Jay M. Weiss d a Laboratory of Neuroendocrinology, Rockefeller UniÕersity, 1230 York AÕenue, Box 165, New York, NY 10021, USA b DiÕision of Biology and Medicine, Brown UniÕersity, ProÕidence, RI, USA c Pittsburgh Cancer Institute, UniÕersity of Pittsburgh, School of Medicine, Pittsburgh, PA, USA d Department of Psychiatry and BehaÕioral Sciences, Emory UniÕersity School of Medicine, Atlanta, GA, USA

Sex differences in dendritic atrophy of CA3 pyramidal neurons in response to chronic restraint stress

The present study investigated the effects of 21 days of chronic restraint stress on neural and endocrine parameters in male and female rats. Consistent with previous results, repeated restraint stress induced apical dendritic atrophy (a decrease in the number of apical branch points and dendritic length) of the CA3c pyramidal neurons in male rats. In contrast, female rats did not show significant dendritic atrophy in the apical field in response to repeated restraint stress. Female rats did show a decrease in the number of branch points in the basal dendritic tree compared to male rats in response to repeated restraint stress. Baseline and stress levels of plasma corticosterone were higher in female rats compared to male rats. Females exhibited slightly longer increases in corticosterone levels throughout the 21 days of restraint stress than males, indicating that the male corticosterone response to stress exhibited greater habituation. Plasma corticosteroid-binding globulin levels of female rats were also higher than those of male rats throughout the experiment. There was no change in plasma corticosteroid-binding globulin levels in male rats during the restraint stress, while there was a decrease in plasma corticosteroid-binding globulin levels in female rats during the restraint stress. Plasma estradiol levels in female rats also decreased in response to the chronic stress. In view of the qualitatively different dendritic atrophy found in males and females in appears unlikely that sex differences in the corticosteroid-binding globulin and corticosterone response can account for these morphological differences.

Visible burrow system as a model of chronic social stress: behavioral and neuroendocrine correlates.

In mixed-sex rat groups maintained in visible burrow systems (VBS), consistent asymmetries in offensive and defensive behaviors of male dyads are associated with the development of dominance hierarchies. Subordinate males are characterized by particular wound patterns, severe weight loss, and a variety of behavioral changes, many of them isomorphic to target symptoms of clinical depression. In two VBS studies, subordinate males showed increased basal levels of plasma corticosterone (CORT), and increased adjusted adrenal and spleen weights compared to controls, and often, to dominants as well. Thymus weights and testosterone levels of subordinates were not reliably different in one study using highly aggressive males, but were reduced, along with testes weights, in a second study using unselected males. Glucocorticoid receptor binding levels in hippocampus, hypothalamus, and pituitary were not different, nor were aldosterone levels. When tested in a restraint stress procedure, subordinates had higher basal CORT levels, but about 40% of these animals showed a reduced, or absent, CORT response to restraint. These findings indicate that subordination may be reflected in high magnitude changes consistent with physiological indices of prolonged stress. Dominant rats of such groups may also show physiological changes suggesting stress, particularly when the groups are comprised of highly aggressive males only. The VBS colony model thus appears to enable rat groups to produce natural, stress-engendering, social interactions that constitute a particularly relevant model for investigating the behavioral, neural, and endocrine correlates of chronic stress.

Stress response, adrenal steroid receptor levels and corticosteroid-binding globulin levels--a comparison between Sprague-Dawley, Fischer 344 and Lewis rats.

Histocompatible Fischer 344 (F344) and Lewis (LEW) rats provide a comparative model for investigating the interactions between the nervous, endocrine and immune systems. The outbred Sprague-Dawley (SD) is the maternal strain for the inbred F344 and LEW strains. In this study we report large differences in the diurnal and stress corticosterone (CORT) profiles of these three genetically related strains: (1) F344 rats had significantly higher diurnal and stress CORT levels than SD and LEW rats; (2) in the morning, stress CORT levels of SD and F344 rats returned towards basal 1 h after cessation of the stressor, whereas stress CORT levels of LEW rats had not returned to basal by this time; and (3) in the evening, SD and F344 rats showed the expected evening rise in basal CORT levels, whereas LEW rats failed to show this rise. In light of the large differences in CORT levels, we expected to observe strain differences in absolute levels of Type I (mineralocorticoid) and Type II (glucocorticoid) adrenal steroid receptors in neural as well as immune tissue. However, we found no significant strain differences in levels of Type I receptors in the hippocampus, hypothalamus, pituitary, thymus, spleen and peripheral blood mononuclear cells. Similarly, we saw no significant strain differences in levels of Type II receptors in most of the tissues surveyed, with the notable exception that LEW rats showed higher Type II binding in the thymus, and SD rats showed small, but significantly higher Type II binding in the hippocampus. We also studied strain differences in levels of corticosteroid-binding globulin (CBG). F344 rats expressed significantly higher CBG levels than SD and LEW rats, in plasma, spleen and thymus. Future studies will investigate whether the substantial differences between strains in levels of CORT and CBG, in the context of few strain differences in post-adrenalectomy adrenal steroid receptor levels in neural and immune tissue, translate into differences in receptor occupancy/activation under resting conditions, or following stress.

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.

Adaptation to Prolonged or Repeated Stress – Comparison between Rat Strains Showing Intrinsic Differences in Reactivity to Acute Stress

Sprague-Dawley (SD), Fischer 344 (F344) and Lewis (LEW) rats are used in a wide variety of laboratory studies. Compared to SD and LEW rats, F344 rats show significantly greater activation of the hypothalamic-pituitary-adrenal (HPA) axis in response to acute stress, or to immunologic challenge. These differences in HPA axis responsivity have been the basis for numerous studies investigating strain differences in immunological and behavioral parameters. However, strain differences in the adaptation of the HPA axis response to prolonged stress, or to repeated stress, have not been investigated. This series of studies demonstrates that F344 rats maintain significantly higher ACTH and corticosterone levels than SD and LEW rats during a single prolonged stress session. Furthermore, F344 rats show virtually no habituation or adaptation of the corticosterone stress response during a single prolonged (4 h) stress session, or during stress sessions repeated over a period of 10 days. In contrast, SD and LEW rats show habituation both within and across stress sessions. Strain differences in HPA axis responsivity are also reflected in the significant adrenal hypertrophy observed in F344 rats (but not in SD or LEW rats) following repeated stress. These results show that strain differences in HPA axis responsivity, which are observed under conditions of acute stress, are further amplified during prolonged or repeated stress. These differences under prolonged or repeated stress conditions may consequently magnify the behavioral and immunological differences observed between strains under basal as well as challenged conditions.

Effects of aldosterone or RU28362 treatment on adrenalectomy-induced cell death in the dentate gyrus of the adult rat.

Previous studies have shown that granule cells of the adult dentate gyrus require adrenal steroids for their survival. In order to investigate whether activation of type I or type II adrenal steroid receptors can mediate granule cell survival, we have analyzed the density of pyknotic cells in the granule cell, CA1 and CA3 pyramidal cell layers in Nissl stained hippocampal sections from adult male rats which were either sham operated, adrenalectomized, or adrenalectomized and treated with aldosterone as a specific type I receptor agonist or RU28362 as a specific type II receptor agonist. Aldosterone treatment completely protected the dentate gyrus from adrenalectomy-induced cell death, while treatment with RU28362 resulted in only a partial protection against cell death in this region. These results indicate that type I adrenal steroid receptor activation is sufficient to protect against adrenalectomy-induced cell death.

Dexamethasone suppression of corticosteroid secretion: evaluation of the site of action by receptor measures and functional studies

A dose of dexamethasone was determined in rats (50 micrograms/kg s.c.) that suppressed the corticosterone response to restraint stress by 80%. Corticosteroid receptor occupancy estimates found that the 50 micrograms/kg s.c. dose of dexamethasone had no significant effect on available glucocorticoid receptor (GR) or mineralocorticoid receptor (MR) binding in brain regions (hypothalamus, hippocampus and cortex); on the other hand dexamethasone produced a selective and significant decrease in available GR in peripheral tissues (pituitary and spleen). Functional studies showed that the 50 micrograms/kg s.c. dose of dexamethasone completely blocked the effects of corticotropin-releasing hormone (CRH; 0.3-3.0 micrograms/kg i.p.) on corticosterone secretion, but did not inhibit the corticosterone response to an adrenocorticotropin hormone (ACTH; 2.5 I.U./kg i.p.) challenge. These studies indicate that this dose of dexamethasone exerts its inhibitory effects on the HPA axis primarily by acting at GR in the pituitary. The plasma dexamethasone levels produced by this dose of dexamethasone are similar to those present in humans the afternoon after an oral dexamethasone suppression test (DST), a time at which many depressed patients escape from dexamethasone suppression. These results support and extend other studies which suggest that the DST provides a direct test of the effects of increased GR activation in the pituitary on ACTH and cortisol secretion.

Habituation to repeated restraint stress is associated with lack of stress-induced c-fos expression in primary sensory processing areas of the rat brain

Rats repeatedly exposed to restraint show a reduced hypothalamic-pituitary-adrenal axis response upon restraint re-exposure. This hypothalamic-pituitary-adrenal axis response habituation to restraint does not generalize to other novel stressors and is associated with a decrease in stress-induced c-fos expression in a number of stress-reactive brain regions. We examined whether habituation to repeated restraint is also associated with adaptation of immediate early gene expression in brain regions that process and relay primary sensory information. These brain regions may not be expected to show gene expression adaptation to repeated restraint because of their necessary role in experience discrimination. Rats were divided into a repeated restraint group (five 1-hour daily restraint sessions) and an unstressed group (restraint naïve). On the sixth day rats from each group were either killed with no additional stress experience or at 15, 30 or 60 min during restraint. Immediate early gene expression (corticotrophin-releasing hormone heteronuclear RNA, c-fos mRNA, zif268 mRNA) was determined by in situ hybridization. A reduction in stress-induced hypothalamic-pituitary-adrenal axis hormone secretion (plasma corticosterone and adrenocorticotropic hormone) and immediate early gene expression levels in the paraventricular nucleus of the hypothalamus, the lateral septum and the orbital cortex was observed in repeated restraint as compared with restraint naïve animals. This reduction was already evident at 15 min of restraint. Unexpectedly, we also found in repeated restraint rats a reduction in restraint-induced c-fos expression in primary sensory-processing brain areas (primary somatosensory cortex, and ventroposteriomedial and dorsolateral geniculate nuclei of thalamus). The overall levels of hippocampal mineralocorticoid receptor heteronuclear RNA or glucocorticoid receptor mRNA were not decreased by repeated restraint, as may occur in response to severe chronic stress. We propose that repeated restraint leads to a systems-level adaptation whereby re-exposure to restraint elicits a rapid inhibitory modulation of primary sensory processing (i.e. sensory gating), thereby producing a widespread attenuation of the neural response to restraint.

Effects of chronic corticosterone ingestion on spatial memory performance and hippocampal serotonergic function

The effects of chronic ingestion of corticosterone (8 weeks via the drinking water, 400 micrograms/ml) on spatial memory performance and on monoamine levels in brain areas related to memory were investigated. Corticosterone treatment was associated with a long lasting (5 weeks post treatment) increase in 5-HT levels (44%) in the dentate gyrus of the hippocampus and decrease in 5-HT (50%) and NE (36%) levels in the frontal cortex. No effects were found in CA1, CA3 or in nucleus basalis. Performance of the rats on an 8-arm radial arm maze showed no overall effect of corticosterone treatment on trials to criterion or choice accuracy scores. However, three of the treated rats, who had consumed the most corticosterone during treatment, 12.5 +/- 0.3 mg/day, were impaired relative to all subjects. Thus, these results suggest that hippocampal serotonergic terminals show long lasting effects from corticosterone and may also be an early indicator of deleterious effects of glucocorticoids on hippocampal function. However, since only a small number of corticosterone-treated rats showed behavioral changes, future experiments are necessary to address the possibility that a higher level of corticosterone intake alters spatial memory as well as brain morphology and neurochemistry. Additional studies are also needed to determine whether such changes represent a threshold effect of the steroid or a dose-response function.