Genetic analysis of the corticosterone response to ethanol in BXD recombinant inbred mice.

The genetic control over the corticosterone response to ethanol (EtOH) and its possible relationship to other EtOH-related traits was examined using BXD recombinant inbred (RI) strains derived from an F2 cross of C57BL/6J (B6) and DBA/2J (D2) progenitor strains. Quantitative trait locus (QTL) analysis of corticosterone levels 1 hr following EtOH suggested the influence of a single major gene on this trait. Two loci were predicted to account for 47% of the genetic variance in plasma corticosterone levels 6 hr following EtOH, whereas 3 loci were predicted to account for 78% of the genetic variance in corticosterone levels 7 hr following EtOH. Markers associated with corticosterone levels 7 hr following EtOH and corrected corticosterone levels 6 hr post-EtOH overlapped with ones found to influence acute and chronic EtOH withdrawal severity, suggesting some degree of common genetic determination between these traits. Overall these results indicate that gene action significantly influences stress responsiveness and suggest possible chromosomal locations of these genes.

Corticosteroids enhance convulsion susceptibility via central mineralocorticoid receptors.

Recently, interest in the roles of central nervous system mineralocorticoid receptors (MR) and glucocorticoid receptors (GR) has increased. In vitro results have implicated MR in the enhancing effects of corticosteroids and GR in the suppressing effects of corticosteroids on hippocampal excitability. Although indirect evidence exists suggesting that opposing actions of central MR and GR occur in vivo, direct evidence from studies employing receptor agonists and antagonists is only beginning to emerge. Work in our laboratory suggests that increased corticosterone levels are associated with increased severity of ethanol, pentobarbital, and diazepam withdrawal. Further work with chemical convulsants suggests that MR mediate excitatory effects of corticosteroids on convulsion susceptibility. The circadian rhythm in convulsion susceptibility varies with the circadian rhythm of plasma corticosterone levels and MR binding. The types of convulsions affected by manipulations of MR activity are believed to be of limbic origin, suggesting that limbic convulsions may be alleviated by the use of specific MR antagonists. In addition, because MR are substantially bound at rest and maximally occupied during the circadian peak in corticosteroid levels and during stressor exposure, these receptors are implicated in the maintenance of and in changes in the arousal state of animals.

Sensitivity of the circadian rhythm of kainic acid-induced convulsion susceptibility to manipulations of corticosterone levels and mineralocorticoid receptor binding.

Increases in corticosterone levels have been associated with enhanced susceptibility and decreases in corticosterone levels have been associated with decreased susceptibility to convulsions in mice. The proconvulsant effects of corticosterone are believed to be mediated by central mineralocorticoid receptors (MR). Both convulsion susceptibility and plasma corticosteroid levels display circadian rhythmicity. When corticosterone levels are at their lowest, hippocampal MR binding is submaximal, whereas when corticosterone levels are at their circadian peak, hippocampal MR binding is maximal. In the present experiments the relationship between circadian rhythms of susceptibility to kainic acid-induced convulsions and plasma corticosterone levels was investigated. In addition, the effects of exogenously administered corticosterone and the MR antagonist spironolactone were examined at times of different convulsion susceptibility. In general, lower plasma corticosterone levels were associated with decreased convulsion susceptibility and higher plasma corticosterone levels were associated with greater convulsion susceptibility. Corticosterone, administered when endogenous levels were low, had a proconvulsant effect. Spironolactone, administered when corticosterone levels were higher and hippocampal MR were presumably maximally occupied, had an anticonvulsant effect. These results indicate that the circadian rhythm in susceptibility to kainic acid-induced convulsions is sensitive to manipulations of corticosterone levels and MR binding. Degree of central MR occupancy may, in part, mediate convulsion susceptibility in humans as well as laboratory animals.

Mineralocorticoid receptors mediate the enhancing effects of corticosterone on convulsion susceptibility in mice.

With hippocampal slice electrophysiology, direct evidence that mineralocorticoid receptors (MR) mediate excitatory effects of corticosteroids on neuronal activity was found. The present experiments extended this hypothesis to a whole animal model. The effects of manipulations of MR binding on convulsions produced by pentylenetetrazol (PTZ), strychnine and kainic acid were examined. Moderate increases in plasma corticosterone levels resulted in enhanced susceptibility to several convulsion types; decreased levels attenuated the susceptibility. The proconvulsant effects of corticosterone were inhibited by the MR antagonist, spironolactone. Convulsions typically affected by these manipulations were PTZ-induced myoclonic jerk and face and forelimb clonus and kainic acid-induced convulsions. Other convulsion types, e.g., PTZ-induced running bouncing clonus and tonic hindlimb extension and strychnine-induced convulsions, were generally unaffected by these manipulations, which suggests that central MR effects are not global. The convulsions that were sensitive to MR manipulations are believed to originate in limbic structures. These results provide the first direct evidence for a role of central MRs in the modulation of central nervous system excitability in a whole animal. Furthermore, they suggest that central MR action may be important in disruptions of excitability, such as epilepsy, drug withdrawal syndromes and arousal states in healthy animals.

Corticosterone increases severity of acute withdrawal from ethanol, pentobarbital, and diazepam in mice.

It has been suggested that withdrawal from several subclasses of central nervous system (CNS) depressants involves common underlying mechanisms. For example, mice genetically selected for severe ethanol withdrawal convulsions (Withdrawal Seizure Prone or WSP) have also been found to express severe withdrawal following treatment with barbiturates and benzodiazepines. Corticosteroids appear to modulate severity of withdrawal from CNS depressants. Therefore, it was hypothesized that corticosterone would enhance withdrawal convulsions following acute ethanol, pentobarbital, and diazepam in WSP mice. Corticosterone (20 mg/kg) administered following each of these drugs significantly increased severity of handling-induced convulsions during withdrawal. Corticosterone did not affect pre-withdrawal convulsion scores or handling-induced convulsions of drug-naive mice. These results suggest that withdrawal convulsions following acute ethanol, pentobarbital, and diazepam are sensitive to modulation by corticosterone and they support the hypothesis that stress may increase drug withdrawal severity.

Type I corticosteroid receptors modulate PTZ-induced convulsions of withdrawal seizure prone mice.

Corticosteroids have been shown to modulate convulsion expression in humans and animals. It is hypothesized that type I corticosteroid receptors mediate the excitatory effects of corticosteroids in vivo based on low-dose efficacy of corticosterone, and differential effects of mineralocorticoids vs. glucocorticoids on convulsions. In the present experiments, the effects of altering corticosterone levels, and the role of the type I receptor in mediating these effects, were examined using pentylenetetrazol (PTZ)-induced convulsions in ethanol withdrawal seizure prone (WSP) mice. It was hypothesized that stimulation of type I receptors partially mediates the expression of tonic hindlimb extensor (THE) convulsions produced by PTZ. Aminoglutethimide, a steroid synthesis inhibitor, increased latencies to PTZ-induced THE. This anticonvulsant effect was reversed by corticosterone and the type I agonist, deoxycorticosterone (DOC), but not by the type II agonist, dexamethasone. Furthermore, two type I receptor antagonists, spironolactone and RU26752, increased latencies to PTZ-induced THE, suggesting that they have anticonvulsant action. In summary, the results of these experiments suggest that type I corticosteroid receptors are important for expression of PTZ-induced convulsions.

Dissociation of the effect of aminoglutethimide on corticosterone biosynthesis from ataxic and hypothermic effects in DBA and C57 mice.

Adrenalectomy is frequently used to deplete adrenocortical hormones in physiological and receptor-binding studies in animals. However, this procedure is irreversible, removes both the cortex and medulla, and produces many negative side effects such as hypotension and hypoglycemia. Aminoglutethimide is a steroid synthesis inhibitor which depletes adrenocortical hormones without these negative effects. However, aminoglutethimide itself has been shown to produce behavioral and physiological deficits. In the present experiments, dose-response relationships were determined for the effects of aminoglutethimide on corticosterone levels, motor coordination, and body temperature in C57 and DBA mice. Aminoglutethimide (5.4-54 mg/kg) inhibited the increase in plasma corticosterone concentrations normally observed in response to restraint stress. Only at higher doses (170-1,000 mg/kg) were rotarod performance and body temperature affected. The corticosterone response to restraint stress recovered fully between 12 and 24 h after aminoglutethimide. In the present study, doses of aminoglutethimide were found that temporarily inhibit stressed corticosterone release without producing motor deficits and temperature decreases. These results indicate that aminoglutethimide is a potential substitute for adrenalectomy in studies on the effects of removal of adrenocortical hormones.

Hypothalamic pituitary adrenal function in the extremely low birth weight infant.

Extremely premature infants manifest clinical features suggestive of adrenal insufficiency. Yet, serum cortisol levels are similar in ill and well preterm infants in a setting where one would expect high stress levels in the ill infants. We investigated the hypothalamic-pituitary-adrenal axis in 17 extremely low birth weight stressed premature infants, mean birth weight 739 g, gestational age, 26.1 weeks, using ovine CRH (oCRH) and ACTH stimulation. oCRH (1 microgram/kg) was administered at 2-7 days of life (mean = 4.1). ACTH rose from a basal value 6.0 +/- 0.8 pmol/L (mean +/- SEM) to 9.6 +/- 1.8 pmol/L (P < 0.01) at 15 min and 9.5 +/- 1.7 pmol/L (P < 0.01) at 60 min. Basal cortisol rose from 349.3 +/- 58.1 nmol/L to 422.3 +/- 57.9 nmol/L (P < 0.01) at 15 min and 568.7 +/- 60.2 nmol/L (P < 0.01) at 60 min. Cortisol values remained significantly (P < 0.05) elevated 24 h after oCRH. An ACTH stimulation test performed 24 h after the oCRH test demonstrated a significant cortisol rise from 603.5 +/- 130.5 nmol/L to 882.7 +/- 136.6 nmol/L (P < 0.05) at 60 min. Plasma CRH immunoactivity was also measured before oCRH testing and was detectable in 10 of 15 infants. The mean CRH immunoactivity was 21.8 +/- 4.4 pmol/L in the infants, significantly higher than 8 adult male controls (P < 0.04). Our results show a normal pituitary response to ovine CRH and a normal adrenal response to ACTH. We hypothesize that cortisol levels are inappropriately low in some ill preterm infants because of the inability of the extremely premature brain to recognize the stress of the illness or because of inadequate hypothalamic secretion of CRH. The significance of the measurable plasma CRH in the first week of life is unknown.

Specific and nonspecific effects of ethanol vapor on plasma corticosterone in mice.

The intent of this study was to determine whether chronic ethanol (EtOH) vapor inhalation, with or without adjunct pyrazole (PYR) administration, was stressful in mice, as defined by increases in plasma corticosterone (CORT) concentration. Mice were randomly assigned to groups differentiated both on the basis of EtOH vapor exposure and the presence or absence of PYR administration. Blood samples for blood EtOH concentration (BEC) and plasma CORT concentration were obtained from mice after 72-96 hours of treatment. Mice were sacrificed after 96 hours of treatment and body and adrenal weight determined. BEC was significantly higher in PYR-treated animals and animals treated with the higher EtOH vapor concentration. Plasma CORT was elevated in proportion to BEC; however, other nonspecific stresses, in particular that of PYR administration, also elevated plasma CORT. Nonspecific stresses associated with this protocol may reduce the generality of these observations. Nevertheless, the high correlation between BEC and plasma CORT concentration in the PYR groups indicates that, with suitable control groups, the PYR-EtOH vapor inhalation approach is viable for studies concerned with EtOH effects on hypothalamic-anterior pituitary-adrenocortical function.

Circadian rhythm abnormalities in totally blind people: incidence and clinical significance.

When people are completely isolated from environmental time cues, their circadian rhythms free run with a nearly 24-h cycle, generated by an internal body clock. Free-running temperature, cortisol, and melatonin rhythms have also been described in totally blind people, even though they were living in normal society and had access to abundant time cues; thus an intact visual system may be essential for synchronization of the circadian system. However, because of the small numbers of subjects studied, the incidence and clinical significance of circadian rhythm abnormalities among the blind has remained uncertain. In this study, plasma melatonin (n = 20), cortisol (n = 4), and sleep propensity (n = 1) were measured in serial samples taken from totally blind subjects for 24 h. Most totally blind subjects had circadian rhythm abnormalities. In about half of the subjects, the rhythms were free-running. Some blind subjects suffered recurrent insomnia and daytime sleepiness that were maximal when the internal rhythms were out of phase with the preferred sleep times. The high incidence of abnormal circadian rhythms in blind people underscores the importance of the light-dark cycle as an important environmental synchronizer for the human circadian system.

Genetic differences in hypothalamic-pituitary-adrenal axis responsiveness to acute ethanol and acute ethanol withdrawal.

There is a growing body of evidence suggesting that corticosteroids contribute to the increased neural excitability observed during ethanol withdrawal. In the present study, this was further investigated using mouse strains which differ in ethanol withdrawal severity. DBA/2 (DBA) mice were found to display more severe acute ethanol withdrawal seizures than C57BL/6 (C57) mice. Additionally, DBA mice showed a greater stress response than C57 mice, as measured by higher plasma concentrations of adrenocorticotropic hormone (ACTH) and corticosterone, to an acute dose of ethanol. Mimicking withdrawal plasma corticosterone levels by administering corticosterone to ethanol-naive mice resulted in increases in handling-induced convulsions in the range observed during withdrawal. There did not appear to be a strain difference in sensitivity to the excitatory effects of corticosterone. In summary, the greater stress response to ethanol by DBA mice may account, in part, for the more severe ethanol withdrawal syndrome of this strain.

Differential modulation by the stress axis of ethanol withdrawal seizure expression in WSP and WSR mice.

Withdrawal from both acute and chronic ethanol (EtOH) exposure is associated with increased neural excitability and increased activity of the hypothalamic-pituitary-adrenal axis. There is some evidence that glucocorticoids are necessary for EtOH withdrawal seizure expression. Lines of mice that were selected for severe (WSP) and minimal (WSR) EtOH withdrawal (as estimated from handling-induced convulsion scores) have been shown to differ in their stress response following an acute dose of EtOH. In this study we provide evidence that these lines of mice also differ in their sensitivity to the excitatory effects of glucocorticoids. EtOH withdrawal seizures of WSP mice were significantly increased by chronic and acute corticosterone treatment, whereas those of the WSR mice were unaffected. Neural excitability was decreased in the WSP mice when aminoglutethimide, a glucocorticoid synthesis blocker, was administered. Thus, it appears that genetic differences in EtOH withdrawal seizure severity may be due, in part, to differences in sensitivity to the excitatory effects of glucocorticoids.

Melatonin administration to blind people: phase advances and entrainment.

The purpose of this study was to test the phase-shifting and entraining effects of melatonin in human subjects. Five totally blind men were found in a previous study to have free-running endogenous melatonin rhythms. Their rhythms were remarkably stable, so that any deviation from the predicted phase was readily detectable. After determination of their free-running period and phase, they were given exogenous melatonin (5 mg) at bedtime (2200 hr) for 3 weeks, in a double-blind, placebo-controlled trial. The effects on the endogenous melatonin rhythm were assessed at intervals ranging from several days to 2 weeks. Exogenous administration of melatonin phase-advanced their endogenous melatonin rhythms. In three of the subjects, cortisol was shown to be phase-shifted in tandem with the melatonin rhythm. A sixth subject [one of the coauthors (JS)] was previously found to have free-running cortisol and temperature rhythms and was plagued by recurrent insomnia and daytime sleepiness. He had tried unsuccessfully to entrain his rhythms for over 10 years. After he took melatonin (7 mg at 2100 hr), his insomnia and sleepiness resolved. Determination of his endogenous melatonin rhythm after about a year of treatment demonstrated endogenous rhythms that appeared normally entrained. The treatment of blind people with free-running rhythms has many advantages for demonstrating chronobiological effects of hormones or drugs.

Diminished adrenocorticotropin response to insulin-induced hypoglycemia in nondepressed, actively drinking male alcoholics.

Although changes in hypothalamic-pituitary-adrenal axis function have frequently been reported in alcoholics, the majority of studies have used recently detoxified subjects in whom abstinence phenomena and clinical depression may contribute to observed stress axis alterations. To isolate the primary effects of alcohol dependence on the stress axis, the ACTH and cortisol responses to insulin-induced hypoglycemia were measured in seven actively drinking male alcoholics recruited from the general public through a newspaper advertisement along with eight age-matched male controls. The alcoholic subjects met current American Psychiatric Association diagnostic criteria for alcohol dependence, were stably employed, and had no concurrent psychiatric disorders, cognitive impairment, or psychometric evidence of depression. While relatively young (30.0 yr; range, 22-48 yr), they had lengthy histories of alcohol-related problems (11.9 yr; range, 5-30 yr). Insulin administration resulted in similar nadirs in blood sugar in both alcoholic and control groups. However, the plasma ACTH response was markedly blunted in the alcoholics (P = 0.040, by Mann-Whitney U test). There was a nonsignificant trend toward increased cortisol levels in the alcoholic group. The findings suggest that altered hypothalamic-pituitary-adrenal axis function in alcoholics is a primary results of chronic ethanol exposure rather than a confounding effect of clinical depression or recent detoxification.

Influence of ethanol dependence on regional brain content of beta-endorphin in the mouse.

beta-Endorphin-like immunoreactivity (BE-LI) was measured in 7 brain regions of Swiss-Webster mice after 24, 48 and 72 h of exposure to ethanol vapor following a priming injection of ethanol and daily injections of pyrazole HCl to inhibit ethanol metabolism. Control mice in identical chambers received pyrazole injections but breathed air only. Ethanol dependence was confirmed by scoring additional groups of mice for handling-induced convulsions during withdrawal after each exposure duration. Measurement of anterior and neurointermediate (NIL) pituitary BE-LI, alpha-MSH and ACTH and plasma corticosterone confirmed earlier results showing NIL depletion of all 3 peptides at 24 h and increased plasma corticosterone concentrations at 72 h in ethanol-exposed mice. In brain extracts from ethanol-dependent mice, BE-LI was significantly reduced in the hypothalamus and midbrain with the greatest reduction occurring at 24 h. In forebrain, cerebral cortex, septum and hippocampus, pyrazole treatment significantly reduced BE-LI relative to an unhandled control group, and ethanol exposure tended to reverse this effect. HPLC of hypothalamic extracts revealed no differences in proportions of molecular forms of beta-endorphin-like peptides between 24 h control and ethanol-exposed groups. The predominant BE-LI peak in both groups co-eluted with opiate-active unmodified beta-endorphin. Ethanol dependence in mice is associated with regionally selective decreases in brain beta-endorphin concentration.

Stimulation of corticosterone secretion in vitro by brief ACTH exposure.

We examined the relationship between ACTH concentration and exposure duration on stimulation of corticosterone (B) secretion in vitro using perifused enzymatically dispersed rat adrenocortical cells. A modular perifusion apparatus was used that permitted evaluation of 20-24 cell chambers per experimental session. In expt 1, 20-1000 pg/ml concentrations of synthetic ACTH-(1-24) were presented to cells for 1 min. In expt 2, 100 pg ACTH-(1-24) was presented to adrenal cells in five dose-duration regimens ranging from 5 pg/min for 20 min to 100 pg/min for 1 min. Perifusal rate was 1 ml/min in all sessions. B was determined by radioimmunoassay. In expt 1 (constant-duration paradigm), 1-min presentation of ACTH-(1-24) produced log-linear dose-response effects across these concentrations (r = 0.926, P less than 0.01). In expt 2 (constant-mass paradigm), identical masses administered in different dose-duration regimens had different steroidogenic efficacies (P less than 0.02): low-dose long-duration regimens provoked greater total release than high-dose short-duration regimens. Overall, every dose-duration regimen was associated with stimulation of B secretion. These results indicate that very brief exposure to physiological concentrations of ACTH-(1-24) is a significant stimulus for corticosteroid secretion; variations in the dose-duration regimen over the physiological range modifies both the maximum rate of secretion and the duration of secretion, but not the response latency; and ACTH-(1-24) presentation mass is not the sole determinant of B secretion.

Ethanol-stimulated endorphin and corticotropin secretion in vitro.

Although acute administration of ethanol in vivo results in increased plasma glucocorticoid concentration, it is unclear whether this effect is mediated by corticotropin (ACTH) from the anterior pituitary. Secretion of beta-endorphin-like (BE-IR) and corticotropin-like (ACTH-IR) immunoreactivity from perifused, dispersed mouse adenohypophyseal cells was used to evaluate the effect of 17 mM ethanol on secretion of pituitary peptides. Cells were also exposed to 10 nM synthetic corticotropin-releasing factor (CRF), 1 microM vasopressin, 54 mM KCl, 100 nM corticosterone, and calcium-free medium, separately and in combination. Secretion of BE-IR and ACTH-IR were markedly sensitive to low concentrations of ethanol. Exposure to 17 mM ethanol produced 3-fold stimulation of the rate of hormone release. This represented one-third to two-thirds that of the rate of maximum stimulation by CRF. Unlike CRF-stimulated secretion, ethanol-stimulated secretion was transient. Further, a second ethanol exposure 1 h after the first did not stimulate peptide secretion. Similar to CRF-stimulation, ethanol-stimulated peptide secretion required extracellular calcium and was inhibited by the glucocorticoid corticosterone. We suggest that this system is a useful model for investigation of the actions of low concentrations of ethanol at the cellular level.

Dynamics of thyrotropin-releasing hormone-induced thyrotropin and prolactin secretion by acutely dispersed rat adenohypophyseal cells. Evidence for 'all-or-none' secretion by heterogeneous secretory units, each with a specific response threshold.

We have examined the dynamics of thyrotropin-releasing hormone (TRH)-stimulated secretion of prolactin (PRL) and thyrotropin-stimulating hormone (TSH) using enzymatically dispersed rat adenohypophyseal cells suspended in a perfusion chamber with a volume of 0.2 ml to minimize mixing and dilution. One-min exposure to 3-300 nM TRH, the effective dose range, elicited immediate pulses of PRL and TSH secretion with dose-dependent amplitudes. At all TRH concentrations, following a brief burst of secretion lasting less than 1 min, release of both hormones declined precipitously. Increasing the duration of stimulation up to 30 min with half-maximal TRH concentrations did not alter the dynamics of the initial response and was ineffective in maintaining the initial amplitude of secretion. This phenomenon could not be attributed to exhaustion of readily releasable intracellular PRL and TSH, since an increment in TRH concentration elicited a second pulse of hormone secretion with temporal response characteristics identical to the first. The amplitude of the second pulse was dependent on both the initial concentration of TRH and the magnitude of the increment in TRH concentration. With a stepwise increase in TRH concentration during continuous perfusion, the sum of PRL or TSH secreted from all bursts of secretory activity approximated that achieved with a single exposure to the highest concentration of TRH employed. The high-amplitude secretory response to a given concentration of TRH was restored after an 8-min perfusion with medium alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification and secretion of gamma-endorphin and beta-endorphin-like peptides from the neurointermediate pituitary of the rat.

Gamma-endorphin (gamma E) and related peptides were chromatographically identified in extracts of neurointermediate (NIL) pituitary. When extracts of this tissue were purified and subjected to HPLC in order to identify gamma E-related peptides gamma E, des-tyrosine gamma E, des-enkephalin-gamma E, and alpha-N acetyl gamma E co-elutable peptides were evident. Perifusates of dispersed NIL tissue were also shown to contain beta E and gamma E-immunoreactivity, and the secretion of both was suppressed by exposure of the tissue to dopamine. This study provides evidence of the endogenous presence and secretion of pharmacologically active gamma E-related peptides by the intermediate lobe of the rat pituitary.

Potassium modulation of corticosterone secretion from perifused mouse adrenal cells.

Potassium has long been recognized as a significant modulator of aldosterone synthesis and secretion. Its role as a regulator of glucocorticoids is less clear. Using a perifusion of dispersed mouse adrenal cells, we found a dose-related effect of extracellular potassium (K+) on secretion of the major rodent glucocorticoid, corticosterone. The maximal elicitable responses was 33% of the maximal response to ACTH. An increase in K+ concentration enhanced the effect of ACTH, while K+-free medium depressed the response to ACTH. The temporal characteristics of the corticosterone response to K+ were similar to that of ACTH and consistent with a primary effect on biosynthesis rather than on discharge of a preformed pool of hormone. The results indicate that a significant ion-mediated mechanism modulates ACTH-stimulated glucocorticoid secretion and suggest that further studies of endogenous substances operative by ionic mechanisms might be warranted.