David J. Kennaway

Salivary Melatonin as a Circadian Phase Marker: Validation and Comparison to Plasma Melatonin

There are many situations in which it would be useful to know the phase state of the biological clock. It is recognized that measurement of melatonin levels can provide this information, but traditionally blood has been used for the analysis, and there are many problems in extending the measurements into the home or field situations. The aim of this study was to develop and validate a salivary melatonin radioimmunoassay and to compare results obtained against a plasma assay for determining the onset of melatonin secretion. The assay developed was sensitive (4.3 pM) and required only 200 μl of sample. A rhythm in melatonin was detected in saliva, peaking at approximately 120 pM or 30% of the plasma levels. Using an objective criterion for determining the onset of secretion (mean ± 2 standard deviations of three daytime samples), the time of onset was shown to exhibit low intraindividual variability (coefficient of variation = 1.5%-4.3%). The time of onset determined using saliva was significantly correlated with the plasma onset (r = .70, p < .05). The onsets determined were 22:30 h ± 22 min for the saliva and 21:50 h ± 16 min for plasma for 17 subjects. Similarly, the acrophases of the saliva and plasma melatonin rhythms were significantly correlated. Neither posture alone nor changes in posture affected the calculation of the onset of melatonin secretion using the saliva approach. Very high saliva flow rates induced by citric acid resulted in lower melatonin concentrations compared to the gentle chewing on parafin film. These results firmly establish the use of salivary melatonin measurements for phase typing of the melatonin rhythm in humans.

Differential effects of light wavelength in phase advancing the melatonin rhythm

Abstract:  Shorter wavelength light has been shown to be more effective than longer wavelengths in suppressing nocturnal melatonin and phase delaying the melatonin rhythm. In the present study, different wavelengths of light were evaluated for their capacity to phase advance the saliva melatonin rhythm. Two long wavelengths, 595 nm (amber) and 660 nm (red) and three shorter wavelengths, 470 nm (blue), 497 nm (blue/green), and 525 nm (green) were compared with a no‐light control condition. Light was administered via a portable light source comprising two light‐emitting diodes per eye, with the irradiance of each diode set at 65 μW/cm2. Forty‐two volunteers participated in up to six conditions resulting in 15 per condition. For the active light conditions, a 2‐hr light pulse was administered from 06:00 hr on two consecutive mornings. Half‐hourly saliva samples were collected on the evening prior to the first light pulse and the evening following the second light pulse. The time of melatonin onset was calculated for each night and the difference was calculated as a measure of phase advance. The shorter wavelengths of 470, 495 and 525 nm showed the greatest melatonin onset advances ranging from approximately 40–65 min while the longer wavelengths produced no significant phase advance. These results strengthen earlier findings that the human circadian system is more sensitive to the short wavelengths of light than the longer wavelengths.

Urinary 6-sulfatoxymelatonin excretion and aging: New results and a critical review of the literature

Abstract: The apparent age‐related decline in melatonin production has been thought to continue in a secular manner across the lifespan. While it is clear that melatonin levels in children and adolescents are elevated compared to older individuals, the question of whether there is a sudden or gradual change has not been adequately addressed. In this study, we report the excretion of the melatonin metabolite, 6‐sulfatoxymelatonin in 253 subjects aged between 21 and 82 yr. The correlation with age was significant (r= ‐ 0.24; P < 0.05). When the data was analysed by ANOVA using 5‐yr age spans, there was a significant effect of age, but post hoc analysis indicated that after 25 yr of age there was no significant decline in excretion of the metabolite. Thus, although the oldest subjects excreted 36% less melatonin metabolite than the youngest, the decrease occurred at a very early age. In the second part of the study, we re‐evaluated the data from seven previous studies that measured plasma melatonin levels or metabolite excretion across a wide range of ages and 11 studies comparing young versus older subjects. Statistical analysis by ANOVA again suggested that the changes in melatonin occurring with age were essentially complete before 30 yr of age. The youngest subjects produced at the most twice the amount of melatonin as the oldest subjects. Finally, we evaluated the mean plasma melatonin levels in 144 groups of normal subjects reported in 137 separate publications with respect to age. Again, whereas there was a significant correlation with age, ANOVA showed that there was no difference between groups after 35 yr of age, and the oldest groups had levels that were only 43% of the youngest groups. We conclude that melatonin production is lower in older people, but that the change occurs very early in life, around 20–30 yr of age.

Serotonin 5-HT2c agonists mimic the effect of light pulses on circadian rhythms

The serotonin agonist quipazine has been shown to cause phase shifts in melatonin and activity rhythms and to induce c-fos in the suprachiasmatic nucleus of rats. In this study, in vivo pharmacological characterisation of the phase shifting properties of serotonin agonists has been performed, with a view to determining the receptor sub-types involved. Agonists for the 5-HT2a/2c receptors, (+/-)-1-(4-iodo-2,5-dimethoxyphenyl)-2-aminopropane hydrochloride (DOI, 0.1 mg/k), 1-(3-chlorophenyl)-piperazine HCl (mCPP, 2 mg/kg) and N-(3-trifluoromethylphenyl)-piperazine HCl (TFMPP, 2 mg/kg) injected at CT18 resulted in acute transient inhibition of melatonin production and delays in the onset of production on the following nights of 1.2+/-0.2, 1.7+/-0.3 and 1. 4+/-0.8 h respectively. Drugs specific for 5-HT1a/7 and 5-HT3 receptors failed to affect melatonin production. At a dose of 0.07 micromole/kg, the serotonin antagonist, ritanserin inhibited the DOI induced phase delay whereas ketanserin was ineffective at this dose, providing strong evidence that DOI was acting through 5-HT2c receptors. DOI (0.5 mg/kg) at CT18 provoked a phase delay in the core body temperature rhythm of similar magnitude to that following a light pulse. Administration of DOI but not agonists active at other receptor sites resulted in the appearance of c-Fos in the ventrolateral division of the suprachiasmatic nucleus (SCN) at CT18 but not at CT6. Ritanserin was more potent than ketanserin at inhibiting the DOI induced increase in c-Fos labelled cells in the SCN. When rats were pre-treated with metergoline (15 mg/kg), ritanserin (3 mg/kg) or LY 53,857 (3 mg/kg) prior to a 2 lx/ 1 min light pulse, none of the drugs significantly inhibited the responses to light. The results of these experiments indicate that serotonergic agonists active at the 5-HT2c receptor mimic the effects of light on 2 independent rhythms and activate SCN neurones in the rat.

Reproductive biology of female Bmal1 null mice.

The light/dark cycle and suprachiasmatic nucleus rhythmicity are known to have important influences on reproductive function of rodents. We studied reproductive function in female heterozygous and homozygous brain and muscle ARNT-like protein 1 (Bmal1, also known as Arntl) null mice, which lack central and peripheral cellular rhythms. Heterozygous Bmal1 mice developed normally and were fertile, with apparent normal pregnancy progression and litter size, although postnatal mortality up to weaning was high (1.1-1.3/litter). The genotype distribution was skewed with both heterozygous and null genotypes underrepresented (1.0:1.7:0.7; P<0.05), suggesting loss of a single Bmal1 allele may impact on postnatal survival. Homozygous Bmal1 null mice were 30% lighter at weaning, and while they grew at a similar rate to the wild-type mice, they never achieved a comparable body weight. They had delayed vaginal opening (4 days), disrupted estrus cyclicity, and reduced ovarian weight (30%). Bmal1 null mice had a 40% reduction in ductal length and a 43% reduction in ductal branches in the mammary gland. Surprisingly, the Bmal1 mice ovulated, but progesterone synthesis was reduced in conjunction with altered corpora lutea formation. Pregnancy failed prior to implantation presumably due to poor embryo development. While Bmal1 null ovaries responded to pregnant mare serum gonadotropin/human chorionic gonadotropin stimulation, ovulation rate was reduced, and the fertilized oocytes progressed poorly to blastocysts and failed to implant. The loss of Bmal1 gene expression resulted in a loss of rhythmicity of many genes in the ovary and downregulation of Star. In conclusion, it is clear that the profound infertility of Bmal1 null mice is multifactorial.

Melatonin and Circadian Rhythms

Melatonin synthesis and secretion by the pineal gland is under the control of the suprachiasmatic nucleus and consequently has a profound circadian rhythm. In this review we discuss some of the issues surrounding the measurement of melatonin rhythmicity in biological fluids and the factors that influence melatonin circadian rhythmicity, including light and drugs. We also review the role of melatonin rhythmicity in sleep timing and sleep initiation.

Reproductive performance in female ClockΔ19 mutant mice

The relationship between circadian rhythmicity and rodent reproductive cyclicity is well established, but the impact of disrupted clock gene function on reproduction has not been well established. The present study evaluated the reproductive performance of mice carrying the ClockΔ19 mutation that were either melatonin deficient (ClockΔ19/Δ19) or had the capacity to synthesise melatonin reinstated (ClockΔ19/Δ19+MEL). The ClockΔ19/Δ19 mice took 2–3 days longer to mate, and to subsequently deliver pups, than their control line. The melatonin-competent mutants had a smaller, but still significant (P < 0.05), delay. The ClockΔ19 mutation resulted in smaller median litter sizes compared with control lines (seven v. eight pups; P < 0.05), whereas melatonin proficiency reversed this difference. Survival to weaning was 84% and 80% for the ClockΔ19/Δ19 and ClockΔ19/Δ19+MEL lines, respectively, compared with 94–96% for the two control lines. The ClockΔ19/Δ19 mutants became behaviourally arrhythmic in constant darkness but, despite this, seven of seven became pregnant when paired with males after at least 14 days of constant darkness (five of seven within 4 days of pairing). In the ClockΔ19/Δ19+MEL mice, seven of 15 became arrhythmic in constant darkness but still became pregnant. The seven mice that free ran for at least 14 days in constant darkness with a period of 27.1 h also became pregnant. The present study has demonstrated that the ClockΔ19 mutation has significant, but subtle, effects on reproductive performance. The reintroduction of melatonin competency and/or other genes as a result of crosses with CBA mice reduced the impact of the mutation further. It would appear that redundancy in genes in the circadian system allows the reproductive cyclicity to persist in mice, albeit at a suboptimal level.

Chronic Phase Shifts of the Photoperiod throughout Pregnancy Programs Glucose Intolerance and Insulin Resistance in the Rat

Shift work during pregnancy is associated with an increased risk for preterm birth and low birth weight. However, the impact upon the long term health of the children is currently unknown. In this study, we used an animal model to determine the consequences of maternal shift work exposure on the health of the adult offspring. Pregnant rats were exposed to chronic phase shifts (CPS) in their photoperiod every 3–4 days throughout gestation and the first week after birth. Adult offspring were assessed for a range of metabolic, endocrine, circadian and neurobehavioural parameters. At 3 months of age, male pups exposed to the CPS schedule in utero had increased adiposity (+29%) and hyperleptinaemia (+99% at 0700h). By 12 months of age, both male and female rats displayed hyperleptinaemia (+26% and +41% respectively) and hyperinsulinaemia (+110% and +83% respectively). 12 month old female CPS rats displayed poor glucose tolerance (+18%) and increased insulin secretion (+29%) in response to an intraperitoneal glucose tolerance test. In CPS males the glucose response was unaltered, but the insulin response was reduced by 35%. The glucose response to an insulin tolerance test was decreased by 21% in CPS females but unaltered in males. Disruption of circadian rhythmicity during gestation resulted in gender dependent metabolic consequences for the adult offspring. These results highlight the need for a thorough analysis of shift work exposure in utero on the health of the adult offspring in humans.

Programming of the fetal suprachiasmatic nucleus and subsequent adult rhythmicity

The suprachiasmatic nucleus (SCN) is the site of the generation and entrainment of circadian rhythms. Similar to other structures, it develops throughout gestation but is still immature for some time after. This suggests that the SCN could be vulnerable to maternal influences, such as poor nutrition, stress and drugs, all of which can affect neuronal development. Evidence is accumulating that suggests that this is the case, with body size at birth influencing melatonin production in adult humans and maternal malnutrition, and stress affecting sleep in rodents. Interestingly, the maternal environment affects the phase of rhythms and the response of the circadian timing system to light pulses. The nature of these changes in adult rhythmicity is similar to those commonly associated with depression in humans. Thus, abnormal fetal programming might predispose adults to depressive illness.

Sleep, Wake and Phase Dependent Changes in Neurobehavioral Function under Forced Desynchrony

STUDY OBJECTIVES The homeostatic-circadian regulation of neurobehavioral functioning is not well understood in that the role of sleep dose in relation to prior wake and circadian phase remains largely unexplored. The aim of the present study was to examine the neurobehavioral impact of sleep dose at different combinations of prior wake and circadian phase. DESIGN A between-participant design involving 2 forced desynchrony protocols varying in sleep dose. Both protocols comprised 7 repetitions of a 28-h sleep/wake cycle. The sleep dose in a standard protocol was 9.33 h per 28-h day and 4.67 h in a sleep-restricted protocol. SETTING A time-isolation laboratory at the Centre for Sleep Research, the University of South Australia. PARTICIPANTS A total of 27 young healthy males participated in the study with 13 in the standard protocol (age 22.5 ± 2.2 y) and 14 in the sleep-restricted protocol (age 21.8 ± 3.8 y). INTERVENTIONS Wake periods during both protocols were approximately 4 h delayed each 28-h day relative to the circadian system, allowing performance testing at different combinations of prior wake and circadian phase. The manipulation in sleep dose between the 2 protocols, therefore, allowed the impact of sleep dose on neurobehavioral performance to be examined at various combinations of prior wake and circadian phase. MEASUREMENTS AND RESULTS Neurobehavioral function was assessed using the psychomotor vigilance task (PVT). There was a sleep dose × circadian phase interaction effect on PVT performance such that sleep restriction resulted in slower and more variable response times, predominantly during the biological night. This interaction was not altered by prior wakefulness, as indicated by a nonsignificant sleep dose × circadian phase × prior wake interaction. CONCLUSIONS The performance consequence of sleep restriction in our study was prominent during the biological night, even when the prior wake duration was short, and this performance consequence was in forms of waking state instability. This result is likely due to acute homeostatic sleep pressure remaining high despite the sleep episode.