Expression profiles of PERIOD1, 2, and 3 in peripheral blood mononuclear cells from older subjects.

AIMS: Circadian clocks regulate daily rhythms of behavior and physiology such as the sleep-wake cycle and hormonal secretion. Numerous characteristics of the behavioral and physiological processes change with age. In this study, we evaluated the circadian clockwork in older people by measuring daily profiles of PERIOD (PER) gene expression in peripheral blood mononuclear cells (PBMCs). MAIN METHODS: Blood samples were collected from 6 healthy older subjects (mean age 62 years) at 2-h intervals over a 24-h period under a semi-constant routine condition where masking effects are minimized. PBMCs were isolated from whole blood and temporal mRNA expression profiles of PER1, PER2, and PER3 were determined by RT-PCR. Phases of the PER rhythms, and times of sleep onset and offset were determined using data from those subjects who showed significant 24-h rhythms. The values for the parameters were compared between the older subjects and 8 young control subjects (mean age 21 years). KEY FINDINGS: Prominent daily rhythms of PER1, PER2, and PER3 mRNA levels, advanced sleep-wake timing and advanced phases of PER rhythms were observed in the older subjects compared to the young controls. There was no significant age-related phase difference in PER1 or PER2 rhythm with respect to sleep timing; however, PER3 expression pattern was altered in the older subjects. SIGNIFICANCE: This preliminary study shows that human circadian clockwork in PBMCs remains intact at least until the presenile stage and suggests that the altered PER3 expression pattern may reflect decreased homeostatic sleep drive in older people.

Expression profiles of 10 circadian clock genes in human peripheral blood mononuclear cells.

The circadian clock system regulates daily rhythms of physiology and behavior. The mammalian master clock in the suprachiasmatic nuclei orchestrates these biological rhythms in peripheral tissues. Since blood is the most accessible tissue source, we sought to dissect the human circadian clock system by characterizing clock gene expression in human peripheral blood mononuclear cells (PBMCs) isolated from eight young, healthy subjects. By evaluating the temporal expression profiles of 10 circadian clock genes, we found that Period 1 (Per1), Per2, and Per3 are rhythmically expressed in human blood samples. Our results suggest that evaluating the rhythmic expression of human Per genes could reveal an individual's circadian phenotype.

Dissociation between objective psychomotor impairment and subjective sleepiness after diazepam administration in the aged people.

The aim of the present study was to clarify whether subjective sleepiness accurately reflects benzodiazepine-related decline in psychomotor function after taking benzodiazepines (BZPs) in aged people. Subjects were eight healthy, young (mean age, 19.8 years) and seven healthy, older (mean age, 60.9 years) men. Placebo and diazepam (DZP) were administered orally in a single-blind crossover manner to the young subjects (placebo, 5 mg DZP and 10 mg DZP) and to the older subjects (placebo and 5 mg DZP). Plasma drug concentration, choice reaction time (CRT) as an objective measure of psychomotor function, and the Stanford Sleepiness Scale (SSS) as a measure of subjective sleepiness were monitored every 20 min from 1000 until 1600 h, being the drug administered at 1200 h. Pharmacokinetic variables did not differ significantly between the two age groups. DZP at 10 mg in young subjects induced significant increases in both the CRT and SSS score. DZP at 5 mg induced no significant increase in SSS score in either age group but did induce a significant increase in CRT only in the older subjects that matched that in young subjects given 10 mg DZP. The older subjects suffered from dissociation between subjective sleepiness and objective psychomotor impairment under DZP treatment. Such individuals may underestimate the detrimental effects on brain function.

The 3111T/C polymorphism of hClock is associated with evening preference and delayed sleep timing in a Japanese population sample.

Sleep timing is influenced by the circadian system. Morningness-eveningness (ME) preference in humans is affected by the free-running period, which is determined by circadian clock-relevant genes. In this study, we investigated association between the 3111T/C polymorphism in the 3'-flanking region of hClock (Homo sapiens Clock homolog) and ME preference in 421 Japanese subjects. The Horne-Ostberg ME questionnaire (MEQ) scores showed normal distribution, with mean score of 51.2 +/- 1.4 (range, 25-73), and scores were positively correlated with sleep onset time (r = 0.541, P < 0.001) and wake time (r = 0.513, P < 0.001). MEQ scores were significantly lower in subjects with 3111C/C (n = 12) than in subjects with 3111T/C (n = 106, P < 0.001) or 3111T/T (n = 303, P < 0.001), suggesting a stronger eveningness preference in 3111C/C homozygotes. This group also showed significantly delayed sleep onset (P < 0.001), shorter sleep time (P < 0.001), and greater daytime sleepiness (P < 0.001) in comparison to parameters in the subjects with the 3111T allele. There was no significant difference in any of these parameters between the 3111C/T and 3111T/T genotypes. The influence of the 3111T/C polymorphism on ME preferences in Caucasian populations remains controversial. The present findings in a Japanese population sample, which should have a relatively low risk of population stratification effects, suggest the significance of the association of the 3111C/C allele of hClock with evening preference.

Enhanced heat loss and age-related hypersensitivity to diazepam.

Whether elderly people suffer from age-related changes in pharmacokinetics and/or pharmacodynamics with administration of benzodiazepines is still a matter of controversy. We investigated the course of brain function and thermoregulation after oral administration of a standard benzodiazepine, diazepam (DZP), in 8 healthy young men (mean age, 19.8 years; range, 18 to 23 years) and 8 healthy middle-aged and older men (mean age, 60.9 years; range, 53 to 71 years). Placebo or DZP was administered in a single-blind crossover manner to the young men (placebo, 5-mg, 10-mg DZP) and to the older men (placebo, 5-mg DZP), and plasma DZP concentration, choice reaction time, proximal body temperature, and distal body temperature were monitored with high time resolution under a modified constant routine condition to exclude masking effects. Whereas there was no evidence of age-related alterations in pharmacokinetics between the 2 groups, the older subjects, in comparison to the young subjects, showed a more delayed choice reaction time in response to the same plasma DZP level, suggesting that hypersensitivity is related to increased age. DZP at 5 mg in the older subjects induced acute and transient hypothermia to the same degree as that induced by DZP at 10 mg in the young subjects. The distal-proximal body temperature gradient (difference between distal body temperature and proximal body temperature), an indicator of blood flow in distal skin regions, showed strong positive correlation with the delay in choice reaction time in both groups. These findings suggest that hypersensitivity to benzodiazepine in older persons may be due, at least in part, to age-related changes in thermoregulation, especially in the heat loss process.

Similar profiles in human period1 gene expression in peripheral mononuclear and polymorphonuclear cells.

Increasing amounts of data have indicated the physiological significance of circadian clock gene regulation in various peripheral cells. In the present study, we examined expression of the human homolog of period1 (hPer1) in peripheral mononuclear cells (MNCs) and polymorphonuclear neutrophils (PMNs) in seven healthy young male volunteers (mean age, 21.0 years; range, 19-24 years) under modified constant routine conditions. The expression of hPer1 as determined by real-time PCR with gene-specific hybriprobes in MNCs and PMNs showed significant daily variations with similar acrophases and peak transcription in the subjective morning. The acrophases in hPer1 expression rhythms in MNCs and PMNs were found to correlate positively with that of the serum melatonin secretion rhythms, which is a reliable phase marker of the suprachiasmatic nucleus (SCN), the circadian master clock. The present findings indicate that clock gene activity could be preserved across different peripheral blood cell types and support the assumption that peripheral clocks are entrained by the SCN.

Stability of sleep timing against the melatonin secretion rhythm with advancing age: clinical implications.

Aging is often associated with decreased ability of sleep maintenance. It has been hypothesized that the elderly experience a delayed timing of sleep period relative to the circadian phase of various sleep-promoting physiological functions, possibly causing decreased sleep propensity in the latter part of their nocturnal sleep. We evaluated the relationship between the sleep timing and circadian phase of melatonin secretion, which is known as a possible human sleep modulator as well as a stable marker of biological clock phase (BCP). Actigraph sleep recordings were performed, followed by the evaluation of melatonin phase under dim light in 42 healthy elderly volunteers (mean age, 68.8 yr; male/female ratio, 16/26) and 27 healthy young male volunteers (22.5 yr). Elderly subjects showed remarkable clock time advances in both the midpoint of BCP and sleep timing, with a significant decrease in sleep maintenance ability. However, they showed no significant age-related changes in the sleep timing against the midpoint of BCP, suggesting that early morning awakening in the elderly appeared in a BCP for which sleep propensity remained sufficient to sustain sleep. The present findings do not support the hitherto known hypothesis that age-related delay in the sleep timing against the BCP induces the deterioration in sleep maintenance in the elderly.

Two pedigrees of familial advanced sleep phase syndrome in Japan.

STUDY OBJECTIVES: To determine whether a known missense mutation (bp2106 A/G) in hPer2 (a human homolog of the Drosophila period gene) for familial advanced sleep phase syndrome in a Caucasian family is involved in Japanese familial advanced sleep phase syndrome pedigrees. MEASUREMENTS AND RESULTS: We identified 2 new Japanese families with advanced sleep phase syndrome, and a systematic survey was carried out in 28 relatives of theses 2 families. A total of 9 affected subjects were identified. The affected members showed significantly strong morningness tendencies compared with the unaffected members in various circadian parameters including the Horne-Ostberg Morningness-Eveningness Questionnaire score (77.3 +/- 4.8 vs 57.5 +/- 7.6, p < 0.001), average sleep-onset time (20:45 +/- 75 min vs 23:16 +/- 64 min, p < 0.02), and average wake time (4:55 +/- 38 min vs 6:13 +/- 25 min, p < 0.01), as well as saliva dim-light melatonin-onset time (20:15 +/- 21 min vs 22:25 +/- 65 min, p < 0.02). DNA samples were obtained from 7 affected and 7 unaffected subjects. None of the tested subjects possessed the missense mutation (bp2106 A/G) in hPer2. Furthermore, there is no significant linkage between affected subjects with hPer2 region by 2-point mapping and by direct sequencing of 23 exons of hPer2. CONCLUSION: These findings support the notion of genetic heterogeneity of familial advanced sleep phase syndrome cases in humans. The search for more familial advanced sleep phase syndrome cases and for loci other than hPer2 are necessary to further examine the roles of circadian-related genes in genetically determined human circadian rhythm disorders.

Heat loss, sleepiness, and impaired performance after diazepam administration in humans.

In spite of the accumulation of knowledge regarding the neuropharmacological action of benzodiazepines (Bz), the physiological process by which their sedative/hypnotic effects are induced remains poorly understood. We conducted a single-blind, crossover trial to evaluate the role of the thermoregulatory process in sleepiness and impaired psychomotor performance induced by a standard Bz, diazepam (DZP). Each of the eight healthy young male volunteers (mean age, 19.75 years; range, 18-23 years) was given a single oral dose of either 5 or 10 mg of DZP or placebo 12 h after his average sleep onset time. Changes in plasma DZP concentration, proximal body temperature (p-BT), distal body temperature (d-BT), subjective sleepiness measured by the Visual Analog Scale and Stanford Sleepiness Scale, and psychomotor performance measured by Choice Reaction Time were monitored under a modified constant routine condition in which various factors affecting thermoregulation, alertness, and psychomotor performances were strictly controlled. Orally administered DZP induced a significant transient decrease in p-BT and psychomotor performance as well as an increase in d-BT and subjective sleepiness. Distal-p-BT gradient (DPG; difference between d-BT and p-BT), which is an indicator of blood flow in distal skin regions, showed a strong positive correlation with the plasma DZP concentration, indicating that DZP in clinical doses promotes heat loss in a dose-dependent manner. The DPG also correlated positively with the magnitude of subjective sleepiness and impaired psychomotor performance. These findings indicate that the sedative/hypnotic effects of Bz could be due, at least in part, to changes in thermoregulation, especially in the process of heat loss, in humans.

Physical activity increases the dissociation between subjective sleepiness and objective performance levels during extended wakefulness in human.

The process of heat loss has been shown to be a key pathway regulating sleepiness in humans. The influence of physical exercise with its attending heat production on subjective sleepiness and performance levels during total sleep deprivation (SD) was assessed in eight healthy young volunteers (mean age 21.1 years). Each subject participated in a SD cross-over study in which sleepiness and performance levels were tested under exercise and non-exercise conditions. The exercise entailed 15 min walking/h (3.0 Kcal/kg per h caloric consumption). Physical exercise significantly alleviated subjective sleepiness depending on the magnitude of the core body temperature elevation. This indicates that suppressing heat loss could prevent progression of subjective sleepiness during the nighttime. We found a strong positive correlation between increased sleepiness and decreased performance levels in each of the two experimental sessions. However, ANCOVA revealed a significant difference in the slope of the regression lines representing two sessions, indicating less subjective sleepiness with physical exercise despite the same decrease in performance. The present findings alert us to the possibility that increased physical activity during extended wakefulness could increase the dissociation between subjective evaluation of sleepiness and actual brain function, resulting in increased risk of human error.