L. Donald Keith

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.

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.

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.

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.

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.

Influence of ethanol dependence on regional brain content of β-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.

Ethanol dependence and the pituitary-adrenal axis in mice. I. Genotypic differences in hormone levels.

Resting pituitary levels of beta-endorphin-(beta-EP-IR), ACTH-(ACTH-IR), and alpha-MSH-(alpha-MSH-IR)-like immunoreactive material were found to differ among 16 inbred mouse strains. Hormone levels correlated genetically with severity of withdrawal from ethanol, which also differed among the strains. Ethanol dependence led to reduced pituitary beta-EP-IR in 4 of 5 strains studied. After 24 hr of withdrawal, 3 of those 4 showed elevated pituitary beta-EP-IR. These results are consistent with the hypothesis that genetically-determined difference in pituitary hormone functioning underlie some of the genetically-determined differences in ethanol withdrawal severity.

Ethanol dependence and the pituitary adrenal axis in mice II. Temporal analysis of dependence and withdrawal.

The effects of ethanol dependence and withdrawal on pituitary hormone content and corticosterone release were investigated in AKR/J male mice in a vapor chamber. Both chronic ethanol inhalation and ethanol withdrawal were associated with increased adenohypophyseal-adrenocortical activity. Operationally, ethanol exposure was a stressor. Both physical dependence and withdrawal led to increased secretion/synthesis ratios of peptide hormones. The temporal pattern of pituitary ACTH-IR content changes was different from that of beta-endorphin-IR and alpha-MSH-IR. Differences in the pattern of ACTH-IR and alpha-MSH-IR most probably represent lobe-specific differences in the response to chronic ethanol exposure and withdrawal.

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.

Rapid corticosterone pulses: Confirmation by an alleged disconfirmation?

Data from a paper reporting a failure to confirm our report of rapid corticosterone pulses and suggesting that our data were artifactual were reanalyzed and reevaluated. It is shown that these data actually appear to confirm our original report. Artifacts in their procedure tend to introduce smearing that minimizes the observed effect. In addition, their method of data presentation combined with the lack of appropriate data analysis apparently led to their failure to draw the proper conclusions.