Chronic stress, metabolism, and metabolic syndrome.

The prevalence of obesity has rapidly escalated and now represents a major public health concern. Although genetic associations with obesity and related metabolic disorders such as diabetes and cardiovascular disease have been identified, together they account for a small proportion of the incidence of disease. Environmental influences such as chronic stress, behavioral and metabolic disturbances, dietary deficiency, and infection have now emerged as contributors to the development of metabolic disease. Although epidemiological data suggest strong associations between chronic stress exposure and metabolic disease, the etiological mechanisms responsible remain unclear. Mechanistic studies of the influence of chronic social stress are now being conducted in both rodent and nonhuman primate models, and phenotypic results are consistent with those in humans. The advantage of these models is that potential neural mechanisms may be examined and interventions to treat or prevent disease may be developed and tested. Further, circadian disruption and metabolic conditions such as diabetes mellitus could increase susceptibility to other stressors or serve as a stressor itself. Here, we review data from leading investigators discussing the interrelationship between chronic stress and development of metabolic disorders.

Lentivirus-mediated downregulation of hypothalamic insulin receptor expression.

Regulation of feeding behavior and energy balance are among the central effects of insulin. For example, intracerebroventricular administration of insulin decreases food intake and body weight, whereas antisense oligodeoxynucleotide downregulation of insulin receptors (IRs) produces hyperphagia. To further examine the role of IRs in the central actions of insulin, we designed an IR antisense lentiviral vector (LV-IRAS) and injected this vector into the third ventricle to selectively decrease IR expression in the rat hypothalamus. Three weeks after LV-IRAS administration, the expression of IRs in the hypothalamus was significantly decreased, whereas no changes were observed in hippocampal IR levels. LV-IRAS administration decreased insulin-stimulated phosphorylation of hypothalamic IRs and translocation of the insulin-sensitive glucose transporter GLUT4 in the hypothalamus; no changes in IR signaling were observed in the hippocampus of LV-IRAS-treated rats. Lentivirus-mediated downregulation of IR expression and signaling produced significant increases in body weight, as well as increases in fat mass that were selective for the subcutaneous compartment. Conversely, lean muscle mass and water mass were not affected in LV-IRAS-treated rats compared to rats treated with control virus. Changes in peripheral adiposity were associated with increases in basal hypothalamic leptin signaling in the absence of changes in leptin receptor expression in LV-IRAS rats. Collectively, these data illustrate the important functional relationships between hypothalamic insulin and leptin signaling in the regulation of body composition and provide insight into the mechanisms through which decreases in IR expression and signaling dysregulates leptin activity, thereby promoting increases in peripheral adiposity.

Mapping of RNA accessible sites for antisense experiments with oligonucleotide libraries.

Antisense experiments are often complicated by the lack of reliable methods for selecting effective antisense sequences. Chimeric oligodeoxynucleotide (ODN) libraries and ribonuclease H (RNase H) were used to identify regions on the 1253 nucleotide angiotensin type-1 receptor (AT1) mRNA that are accessible to hybridization with antisense ODNs. Phosphorothioate antisense ODNs targeted against accessible sites reduced AT1 receptor levels by at least 50% in cell culture. ODNs to 4 sites produced a 70% to 80% reduction. In contrast, most sequences targeted between accessible sites were ineffective. When injected into the brains of rats, ODNs targeted to accessible sites reduced AT1 (by 65%) but not AT2 receptor levels. Additionally, AT1 receptor function as measured by agonist-induced water intake, was significantly attenuated in these rats. ODNs directed between accessible sites were ineffective at suppressing water intake. RNA mapping can be applied to any RNA target to facilitate selection of multiple, active antisense sequences for cell culture and in vivo experiments.

Regional distribution of 11 beta-hydroxysteroid dehydrogenase in rat brain.

The activity and distribution of 11 beta-hydroxysteroid dehydrogenase in rat brain is described. Oxidation of corticosterone to 11-dehydrocorticosterone was significantly increased by NADP; the reverse reaction was increased by NADPH. Cortisol was a poor substrate. Both 11-dehydrogenase (11-DH) and 11-oxoreductase (11-OR) activities were found in brains of rats from 6 days to adult, and 11-DH and 11-OR activities were positively correlated with each other. Highest enzyme activities were found in pituitary, cerebellum, hippocampus, and cortex. Lower levels were found in the olfactory region, hypothalamus, brain stem, preoptic nucleus, and amygdala. Two antisera to 11-DH, designated 56-125 and 56-126, reacted with a 34K component corresponding in mass to rat liver 11-DH on Western blots. The dominant species of protein in all brain regions reacting with rat liver 11-DH antibody 56-125, was at 26K mol wt. Antiserum 56-126 did not cross-react with the 26K protein. The 26K component was not a 34K degradation product. In each region of the brain, Western blot analysis showed that the 26K band intensity was directly proportional to enzyme activity. However, the 26K protein was devoid of 11-DH activity. All 11-DH and 11-OR activities were associated with the 34K antigen. The data demonstrate the nonuniform distribution of 11-DH in brain tissue. They are consistent with the notion that 11-DH may confer upon brain the ability to control intracellular levels of active glucocorticoids and in this way mediate steroid function within the cell.

Reciprocal changes in plasma corticosterone and testosterone in stressed male rats maintained in a visible burrow system: evidence for a mediating role of testicular 11 beta-hydroxysteroid dehydrogenase.

The purpose of these studies was to investigate the possible role of rat Leydig cell 11 beta-hydroxysteroid dehydrogenase (11HSD) in mediating the inhibitory effects of corticosterone on testosterone production. In a unique communal environment, the visible burrow system, male Long-Evans rats spontaneously segregated into unstressed dominant and stressed subordinate social relationships. Subordinate animals had elevated plasma corticosterone and diminished circulating testosterone levels relative to the dominant animals. The categories of animals were distinguished by behavioral criteria: weight change, wounds received, offensive and defensive behavior, and freedom of movement. As a result of their persistently elevated corticosterone levels, subordinate animals had smaller thymi and larger adrenals and spleens than dominants. We have postulated that Leydig cells are protected against the inhibitory effects of glucocorticoids on testosterone secretion by the inactivating effects of 11HSD. High corticosterone and low 11HSD are predicted to suppress testosterone production, and normal or diminished corticosterone levels combined with normal or elevated 11HSD should permit undiminished testosterone production. Consistent with these predictions, the testes of subordinate animals contained significantly lower 11HSD activity than those of dominant animals. The 11HSD of livers of subordinate and dominant animals were statistically indistinguishable. The results of this study support the postulated role of 11HSD as a protector of Leydig cell function.

Regulation of glucocorticoid receptor and mineralocorticoid receptor messenger ribonucleic acids by selective agonists in the rat hippocampus.

Adrenal steroids can have prodigious effects on the structure, function, and survival of hippocampal neurons. In the rat hippocampus, the actions of adrenal steroids are mediated by two receptor types, the glucocorticoid receptor (GR) and the mineralocorticoid receptor (MR). Using in situ hybridization, we have examined the regulation of the messenger RNAs (mRNAs) encoding the glucocorticoid and mineralocorticoid receptors, by aldosterone, which acts selectively through MR, and by RU28362, which acts selectively through GR. Our results demonstrate that there is autoregulation of each receptor subtype, such that activation of GR regulates GR mRNA levels and MR activation regulates MR mRNA expression. In addition, there is evidence that aldosterone, acting through MR, can affect the expression of GR mRNA. The extent to which a specific agonist can produce a significant change in the expression of a particular steroid receptor mRNA varies between the different subfields of the hippocampus.

Adrenal steroid regulation of neurotrophic factor expression in the rat hippocampus.

Adrenal steroids and neurotrophic factors are important modulators of neuronal plasticity, function, and survival in the rat hippocampus. Adrenal steroids act through two receptor subtypes, the glucocorticoid receptor (GR) and the mineralocorticoid receptor, and activation of each receptor subtype has distinct biochemical and physiological consequences. Adrenal steroids may exert their effects on neuronal structure and function through the regulation of expression of neurotrophic and growth-associated factors. We have examined adrenal steroid regulation of the neurotrophins brain-derived neurotrophic factor, neurotrophin-3, and basic fibroblast growth factor, as well as the growth associated protein GAP-43, through activation of GR or mineralocorticoid receptor with selective agonists. Our findings indicated that in CA2 pyramidal cells, adrenalectomy resulted in decreases in the levels of basic fibroblast growth factor and neurotrophin-3 messenger RNA, which were prevented by activation of mineralocorticoid but not glucocorticoid receptors. Adrenalectomy-induced increases in GAP-43 and brain-derived neurotrophic factor messenger RNA levels could be blocked by activation of glucocorticoid receptors in CA1, but not in CA3, pyramidal cells. Thus the extent to which adrenal steroids regulate hippocampal neurotrophic and growth-associated factors, appears to be dependent both on the adrenal steroid receptor subtype activated and on the hippocampal subregion examined.

Serotonin receptor binding in a colony model of chronic social stress.

Male rats housed in mixed-sex groups quickly established dominance hierarchies in which subordinates appeared severely stressed. Subordinate rats had elevated basal corticosterone (CORT) levels relative to dominants and individually housed controls. Several subordinates had blunted CORT responses to a novel stressor, leading to the classification of subordinates as either stress-responsive or nonresponsive. Binding to 5-HT1A receptors was reduced in stress-responsive subordinates compared to controls throughout hippocampus and dentate gyrus. Decreased binding was observed in nonresponsive subordinates only in CA3 of hippocampus. In addition, 5-HT1A binding was decreased in CA1, CA3, and CA4 in dominants compared to controls. Binding to 5-HT2 receptors was increased in parietal cortex in both responsive and nonresponsive subordinates compared to controls. No changes were observed in binding to 5-HT1B receptors. These results are discussed in the context of regulation of the serotonergic system by stress and glucocorticoids and possible relevance to the pathophysiology of depression.

Repeated exposure to social stress has long-term effects on indirect markers of dopaminergic activity in brain regions associated with motivated behavior.

The visible burrow system (VBS) is a chronic social stress paradigm in which a dominance hierarchy forms among male rats housed with females. Males in the VBS undergo behavioral and physiological changes thought to be manifestations of chronic social stress. Since it is unclear whether chronic social stress affects motivation and reward behavior, brain areas related to these regions were examined. Long-term effects of a single or repeated VBS exposure on mesolimbic subregions were investigated by exposing rats to the VBS either once (one cycle of VBS housing and recovery) or repeatedly (three cycles). Behavior in the VBS was observed and rats were classified as dominants or subordinates. Subordinates were further sub-classified on the basis of stress hormone (corticosterone) response to an acute stressor (i.e. restraint stress). Normal responders were categorized as stress-responsive subordinates (SRS) and animals with a blunted hypothalamic-pituitary-adrenal axis response were designated as non-responsive subordinates (NRS). Controls males were pair-housed with a single female during VBS periods and alone during recovery. Lowered enkephalin-mRNA levels were observed in the nucleus accumbens (Acb) after single VBS exposure in SRS and repeated VBS exposure both subordinate groups (i.e. SRS + NRS) compared with controls. Decreased dopamine transporter density was detected after single VBS exposure in the dorsolateral caudate putamen (DLCPu) of NRS and after repeated VBS exposure in the Acb of NRS compared with controls. Dopamine D2 receptor density was elevated after single VBS exposure in the Acb of both subordinate groups (SRS + NRS) and after repeated VBS exposure in the DLCPu, dorsomedial CPu, and Acb of NRS compared with controls. No changes in dopamine D1 receptor binding were observed in any group. These results suggest that long-term changes in dopamine activity in mesolimbic structures persist after repeated exposures to chronic social stress and may provide insight into the neurochemical basis of depressive illness and subsequent comorbidity with drug abuse vulnerability.

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.

Chronic social stress produces reductions in available splenic type II corticosteroid receptor binding and plasma corticosteroid binding globulin levels.

Adult male and female rats were housed for 2 weeks in a Visible Burrow System resulting in the development of strong dominant-subordinate relationships among the male rats. Neuroendocrine measures indicated that the subordinate rats, and to a lesser extent dominant rats, experienced chronic HPA axis hyperstimulation during the 2 week experience. This paper focuses on the consequences of this chronic social stress on cytosolic type II corticosteroid receptor binding in the spleen. In the first study, rats were adrenalectomized 18 h prior to sacrifice in order to measure total cellular receptor protein levels in each animal. In spite of the severity of the social stress, there was no decrease in splenic type II corticosteroid receptor binding levels in these short-term adrenalectomized animals. In the second study, rats were left adrenal-intact. Corticosteroid receptor levels in these adrenal-intact animals reflect the level of receptors (available receptors) that were unoccupied by endogenous hormone at the time of sacrifice. Both subordinate and dominant rats had fewer available splenic type II receptors than control rats, suggesting that a greater proportion of receptors in subordinate and dominant rats were occupied and activated by endogenous hormone at the time of sacrifice than in control rats. The differences in available receptor levels were not a function of total plasma corticosterone levels at the time of sacrifice (mean corticosterone levels were the same for control and subordinate rats). Instead, the differences in available receptor levels may have been a function of plasma corticosteroid binding globulin (CBG) levels which regulate free corticosterone levels. There was a large reduction in plasma CBG levels of subordinate (-70%) and dominant (-40%) rats relative to control rats, and there was a significant correlation between plasma CBG level and available type II receptors in the spleen. These results suggest that a decrease in CBG levels as a result of chronic social stress led to greater access of free corticosterone hormone to type II receptors in the spleen than is typically present in rats under basal or acute stress conditions. This result illustrates one mechanism by which chronic stress may have a greater impact than acute stress on splenic immune function.

Effects of chronic social stress on tyrosine hydroxylase mRNA and protein levels.

Males housed in mixed sex groups quickly form dominance hierarchies; subordinates can be further subdivided into stress responsive subordinates (SRS) and non-responsive subordinates (NRS) based on corticosterone responses to a novel stressor. Tyrosine hydroxylase (TH) mRNA levels measured with in situ hybridization were elevated in locus coeruleus (LC) of NRS compared to singly or pair-housed controls; NRS also had higher TH levels than dominants. TH protein levels determined by immunoautoradiography were also higher in LC of NRS and SRS versus pair-housed controls.

Immunocytochemical localization of 11 Beta-hydroxysteroid dehydrogenase in hippocampus and other brain regions of the rat.

The dehydrogenase form of 11 ß-hydroxysteroid dehydrogenase (11-DH) which catalyzes the oxidation of the biologically active steroid, corticosterone, to its inactive metabolite, 11-dehydrocorticosterone, is found in rat brain. The distribution and localization of 11-DH-like labeling in the rat brain was examined by immunocytochemistry. 11-DH-like immunostaining was found in all subfields of the hippocampus and in many other parts of the brain, including the preoptic area (POA), central nucleus of the amygdala, bed nucleus of the stria terminalis (NIST) and the cerebral cortex. Percentages of 11-DH-positive cells ranged from 10% in the POA and NIST to 50% to 60% in the hippocampus. When combined with neuronal or glial markers, 11-DH-like immunostaining was found to be predominantly localized within neurons, ranging from 10% or less glial labeling in hippocampus, amgydala and cortex to 22% glial labeling in the POA and NIST. Immunostaining was present in both the cytoplasmic and nuclear components of some cells in addition to their projections. In the kidney, 11-DH has been postulated to be a key component in a mechanism by which aldosterone gains access to renal Type I receptors despite the presence of much higher concentrations of glucocorticoids. The present data is consistent with a similar mechanism occurring in at least some parts of the brain, although the hippocampus appears to be an important exception because it does not appear to be differentially responsive to aldosterone in spite of its high 11-DH activity and immunoreactivity. However, the hippocampus is not implicated in neural control of salt appetite and fluid balance, whereas some of the other brain regions like the POA, NIST and amygdala are believed to be involved. Other aspects of 11-DH localization must therefore be examined in future studies, including the co-presence of mineraiocorticoid receptors and 11-DH in the same or adjacent cells and the possible significance of the relatively high glial localization of 11-DH immunoreactivity in the POA and NIST.

Diurnal differences and adrenal involvement in calmodulin stimulation of hippocampal adenylate cyclase activity.

Abstract 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 mug/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.

Dependence of adrenalectomy-induced sodium appetite on the action of angiotensin II in the brain of the rat.

Adrenalectomized rats express a robust sodium appetite that is accompanied by high levels of blood-borne angiotensin II and is caused by angiotensin II of cerebral origin. Blood-borne angiotensin II is elevated in rats consuming NaCl after adrenalectomy, and plasma angiotensin II concentrations are increased further when the animals cannot drink a NaCl solution. These phenomena are the result of the pathological removal of aldosterone, because replacement therapy returned both sodium intake and plasma angiotensin II concentrations to preadrenalectomy levels. The adrenalectomized rat's appetite for sodium is completely suppressed by interference with the central, but not the peripheral, action of angiotensin II. These data demonstrate that the mechanism of the sodium appetite of the adrenalectomized rat is a pathological instance of the angiotensin/aldosterone synergy that governs the sodium appetite of the adrenal-intact, sodium-depleted rat. Because aldosterone has been removed, angiotensin acts alone to produce the appetite. Furthermore, the data show that it is angiotensin II of central origin that is important for sodium appetite expression.

Salt appetite is enhanced by one prior episode of sodium depletion in the rat.

A single sodium depletion enhances the salt appetite that is expressed after a second and subsequent sodium depletions. The enhanced salt intake, as measured by a decrease in latency to drink and an increase in volume of 3% NaCl ingested, is not accounted for by an increased sodium loss. The enhanced salt intake occurs even when the interval between first and second depletion is as long as 4 months. The enhanced salt appetite does not depend on the drinking of salt after the animal's first sodium depletion and is specific for NaCl but not for KCl. Moreover, it can be produced without sodium depletion by the actions of the hormones aldosterone and angiotensin on the brain. These results suggest that angiotensin and aldosterone, which are released in response to sodium depletion, (a) increase renal sodium conservation, (b) evoke a salt appetite to restore the lost sodium, and (c) produce enduring changes in the brain that prepare it for more rapid and more vigorous expression of salt appetite in response to future sodium depletions. Thus the neural mechanisms that govern salt appetite are not only activated by the hormones of sodium conservation but appear also to be organized by them for a lifelong increase in avidity for salty substances.

Prior episodes of sodium depletion increase the need-free sodium intake of the rat.

Prior episodes of sodium depletion increase the daily 3% NaCl intake of rats. They ingest large volumes and continue to do so for as long as 3 months after recovery from sodium deficit while eating sodium-rich food and while plasma sodium concentration and renal function are normal. The increased daily intake of sodium is, therefore, need-free. There is a marked sex difference in the need-free intake of 3% NaCl. Female rats drink more salt than do male rats when they are sodium replete and depletion naive. Repeated depletions raise the need-free intakes of both sexes but the effect is greater in females. Plasma concentrations of angiotensin II and aldosterone, which are markedly elevated by each episode of sodium depletion, return to basal levels between and after depletions, and are not the cause of the chronically increased need-free salt intake of the multi-depleted rat. These results suggest that the persistent increase in daily 3% NaCl intake that occurs in the rat with a history of repeated sodium depletions is a permanent, nonpathological increase in avidity for the taste of salty substances that results in life-long overconsumption of salt.

Sex and sodium intake in the rat.

Female rats drink more 3% NaCl solution than do males, both when they need sodium (need-induced sodium intake or sodium appetite) and when they do not (need-free sodium intake). The sexual dimorphism of sodium intake is a secondary sexual characteristic because after castration at 1 day of age male rats drank 3% NaCl in adulthood in a manner similar to that of females in both the need-free and need-induced state, and females given long-term, neonatal testosterone drank low, malelike volumes of 3% NaCl on a daily need-free basis, but their response to sodium depletion was unchanged. This sexual dimorphism of sodium intake seems to be governed by testosterone that has been converted in the brain to estrogen because treatment of Day 1 castrated females with a nonaromatizable androgen, dihydrotestosterone, did not change either their need-free or their need-induced 3% NaCl intake. Castration in adulthood of male and female rats did not change their sodium consumption. However, when castrated adults received testosterone, need-free intakes of NaCl were suppressed in both sexes, but the suppression of 3% NaCl intake occurred only while the steroid was present. Exogenous testosterone also lowered the need-induced sodium intake of adult castrated females. Thus, in castrated adults, need-free intake was actively suppressed by exogenous testosterone in both sexes, whereas need-induced intake of NaCl was suppressed only in females. These data indicate that sodium intake in the rat is a sexually dimorphic behavior that is organized neonatally and can be actively suppressed in adulthood by testosterone.

Differential inhibition of 11 beta-hydroxysteroid dehydrogenase by carbenoxolone in rat brain regions and peripheral tissues.

Carbenoxolone (CX), the succinyl ester of glycyrrhetinic acid, causes hypokalemia and hypernatremia. Its pharmacological effects are believed to be due to its inhibition of 11 beta-hydroxysteroid dehydrogenase (11-HSD). There was a marked inhibition of this enzyme in the liver, kidney, pituitary, hippocampus, hypothalamus and amygdala 1 h after intraperitoneal administration of CX (100 mg kg-1) to intact male rats. Intracerebral injection of CX (1.5 mg kg-1) into the 3rd ventricle inhibited the oxidation of corticosterone to 11-dehydrocorticosterone by 11-HSD in the pituitary and hippocampus and produced marked behavioral hyperactivity but had no effect in the liver or kidney. Lower amounts of CX (10-50 micrograms/rat) given intracerebroventricularly (i.c.v) were without significant effect on 11-HSD in the pituitary or amygdala 1 h after infusion but inhibited this enzyme differentially in the hippocampus and hypothalamus. Inhibition of 11-HSD activity in the hippocampus and hypothalamus was observed up to 6 h after i.c.v. administration of CX (50 micrograms/rat) together with some decrease in activity of this enzyme in the pituitary at 3 h. The findings that low doses of CX given i.c.v. can alter the activity of 11-HSD in specific brain regions without affecting its activity in peripheral tissues, and only marginally in the pituitary, provides a method to study the central role of this enzyme independently of systemic effects.

11beta-hydroxysteroid dehydrogenase functions reversibly as an oxidoreductase in the rat hippocampus in vivo.

The localization in the brain and metabolism of 3H-labeled corticosterone (B) and 11-dehydrocorticosterone (A) of high specific radioactivity was determined after stereotaxic injection into the hippocampus of anesthetized rats. [3H]B was cleared very rapidly with, on average, only about 7% being recovered after 5 min and 0.5% after 30 min. Most of this 3H-radioactivity was localized in the area surrounding the site of injection with little diffusion to adjacent areas. These findings make it possible to compare the short term metabolism of [3H]A and [3H]B in different lobes of the hippocampus in the same animal and establish their local equilibrium point in vivo. Under these conditions, about 5% conversion of each steroid to the other was observed in contrast to the situation in cultured hippocampal cells where 11beta-hydroxysteroid dehydrogenase (11-HSD) has been shown by others to act primarily as a reductase catalyzing the conversion of A to B. This method can also be used to study the effect of inhibitors such as 11alpha-hydroxyprogesterone, applied locally in the brain, on the metabolism of corticosteroids. The rate of conversion [3H]B or [3H]A to their dihydro- and tetrahydro-derivatives capable of modulating the GABAa receptor in the hippocampus was much lower than their interconversion. Thus, factors which influence the direction of the 11-HSD catalyzed reaction are important in regulating not only salt appetite and blood pressure but also the levels of neuroactive metabolites of corticosterone.