Yvan Touitou

Ethical and Methodological Standards for Laboratory and Medical Biological Rhythm Research

The main objectives of this article are to update the ethical standards for the conduct of human and animal biological rhythm research and recommend essential elements for quality chronobiological research information, which should be especially useful for new investigators of the rhythms of life. A secondary objective is to provide for those with an interest in the results of chronobiology investigations, but who might be unfamiliar with the field, an introduction to the basic methods and standards of biological rhythm research and time series data analysis. The journal and its editors endorse compliance of all investigators to the principles of the Declaration of Helsinki of the World Medical Association, which relate to the conduct of ethical research on human beings, and the Guide for the Care and Use of Laboratory Animals of the Institute for Laboratory Animal Research of the National Research Council, which relate to the conduct of ethical research on laboratory and other animals. The editors and the readers of the journal expect the authors of submitted manuscripts to have adhered to the ethical standards dictated by local, national, and international laws and regulations in the conduct of investigations and to be unbiased and accurate in reporting never‐before‐published research findings. Authors of scientific papers are required to disclose all potential conflicts of interest, particularly when the research is funded in part or in full by the medical and pharmaceutical industry, when the authors are stock‐holders of the company that manufactures or markets the products under study, or when the authors are a recent or current paid consultant to the involved company. It is the responsibility of the authors of submitted manuscripts to clearly present sufficient detail about the synchronizer schedule of the studied subjects (i.e., the sleep‐wake schedule, ambient light‐dark cycle, intensity and spectrum of ambient light exposure, seasons when the research was conducted, shift schedule in studies involving shift work, and menstrual cycle stage in studies involving young women). Rhythm analysis of time series data should be performed with the perspective that rhythms of different periods might be superimposed upon the observed temporal pattern of interest. A variety of different and complementary statistical procedures can be used for rhythm detection. Fitting a mathematical model to the time series data provides a better and more objective analysis of time series data than simple data inspection and narrative description, and if rhythmicity is documented by objective methods, its characterization is required by relevant parameters such as the rhythms period (tau), MESOR (time series average), amplitude (range of temporal variation), acrophase (time of peak value), and bathyphase (time of trough value). However, the assumptions underlying the time series modeling must be satisfied and applicable in each case, especially the assumption of sinusoidality in the case of cosinor analaysis, before it can be accepted as appropriate. An important aspect of the peer review of manuscripts submitted to Chronobiology International entails judgment of the conformity of research protocols and methods to the standards described in this article.

Nocturnal Excretion of 6-Sulphatoxymelatonin in Children and Adolescents with Autistic Disorder

BACKGROUND Many studies in autistic disorder report sleep problems and altered circadian rhythms, suggesting abnormalities in melatonin physiology. Additionally, melatonin, a pineal gland hormone produced from serotonin, is of special interest in autistic disorder given reported alterations in central and peripheral serotonin neurobiology. METHODS Nocturnal urinary excretion of 6-sulphatoxymelatonin was measured by radioimmunoassay in groups of children and adolescents with autistic disorder (n = 49) and normal control individuals (n = 88) matched on age, sex, and Tanner stage of puberty. RESULTS Nocturnal 6-sulphatoxymelatonin excretion rate was significantly and substantially lower in patients with autism than in normal controls (mean +/- SEM, .75 +/- .11 vs. 1.80 +/- .17 microg/hr, p =.0001), and was significantly negatively correlated with severity of autistic impairments in verbal communication and play (p < .05). CONCLUSIONS These findings indicate clearly that nocturnal production of melatonin is reduced in autism. Further research is warranted in order to understand the mechanisms underlying the lower melatonin production, to assess the impact of altered melatonin on the pathophysiology and behavioral expression of autistic disorder, and to determine the utility of melatonin administration in individuals with autism.

Day and nighttime excretion of 6-sulphatoxymelatonin in adolescents and young adults with autistic disorder

BACKGROUND Several reports indicate that nocturnal production of melatonin is reduced in autism. Our objective was to examine whether melatonin production is decreased during the whole 24-h cycle, whether the melatonin circadian rhythm is inverted, and whether the reduction in melatonin production is related to the severity of autistic behavioral impairments. METHOD Day and nighttime urinary excretion of 6-sulphatoxymelatonin (6-SM) was examined during a 24-h period in post-pubertal individuals with autism (N=43) and typically developing controls (N=26) matched for age, sex and pubertal stage. RESULTS Low 6-SM excretion (mean ± SEM) was observed in autism, both at daytime (0.16 ± 0.03 vs. 0.36 ± 0.05 μg/h, p<0.01), nighttime (0.52 ± 0.07 vs. 1.14 ± 0.23 μg/h, p<0.05), and during 24h (8.26 ± 1.27 vs. 18.00 ± 3.43 μg/24-h collection, p<0.001). Intra-individual nighttime-daytime differences (delta values) in 6-SM excretion were smaller in individuals with autism than in controls (0.36 ± 0.07 vs. 0.79 ± 0.23 μg/h, p<0.05). Nocturnal excretion of 6-SM was negatively correlated with autism severity in the overall level of verbal language (Spearman ρ=-0.30, p<0.05), imitative social play (Spearman ρ=-0.42, p<0.05), and repetitive use of objects (Spearman ρ=-0.36, p<0.05). CONCLUSION A deficit in melatonin production is present both at daytime and at nighttime in individuals with autism, particularly in the most severely affected individuals. These results highlight interest in potential therapeutic uses of melatonin in autistic disorder, especially in individuals with severe autistic impairment and/or low urinary 6-SM excretion.

General Anesthetics Effects on Circadian Temporal Structure: An Update

Disruptions of circadian and biological rhythms as well as general anesthesia can induce sleep disorders, resulting in an increase in sleepiness and drowsiness and a decrease in vigilance. It has been previously shown that circadian time can influence the pharmacologic sensitivity and the duration of action of general anesthetics. Studies on interactions between general anesthesia and circadian rhythms are few, but all of them suggest an important role of general anesthetics on circadian rhythms. General anesthesia is a particular wake‐sleep state that could potentially alter circadian rhythms on the days following anesthesia. The aim of this review is to discuss the various effects of general anesthesia on animal and human circadian time structure. This topic is highly relevant to clinicians, especially those involved in that field of ambulatory practice responsible for post‐operative patient care, including patient recovery and fatigue.

Altered circadian patterns of salivary cortisol in low-functioning children and adolescents with autism

BACKGROUND Reports of higher stress responsivity, altered sleep-wake cycle and a melatonin deficit in autism have stimulated interest in the cortisol circadian rhythm in individuals with autism. METHODS The study was conducted on 55 low-functioning children and adolescents with autism (11.3 ± 4.1 years-old) and 32 typically developing controls (11.7 ± 4.9 years-old) matched for age, sex and puberty. Behavioral assessment was performed using the Autism Diagnostic Observation Schedule (ADOS). Salivary samples for measurement of cortisol were collected during a 24-h period (at least 0800 h-Day 1, 1600 h, 0800 h-Day 2 for 46 individuals with autism and 27 controls, and 0800 h-Day 1, 1100 h, 1600 h, 2400 h, 0800 h-Day 2 for 13 individuals with autism and 20 controls). Overnight (2000 h-0800 h) urinary cortisol excretion was also measured. RESULTS The autism group displayed significantly higher levels of salivary cortisol at all time-points, flatter daytime and nighttime slopes, higher 0800 h cortisol levels on Day 2 compared to Day 1, and greater variances of salivary and urinary cortisol. There was a significant relationship between salivary cortisol levels and impairments in social interaction and verbal language. Overnight urinary cortisol excretion was similar in the autism and control groups. CONCLUSION Anticipation of the stressful collection procedure appears to contribute to the higher 0800 h-Day 2 versus 0800 h-Day 1 salivary cortisol levels in autism. This sensitization to stressors might be as, or even more, important clinically than exposure to novelty in autism. The similar group means for overnight urinary cortisol excretion indicate that basal HPA axis functioning is unaltered in low-functioning autism. The elevated salivary cortisol levels observed in autism over the 24-h period in a repeated stressful condition, flattened diurnal cortisol patterns and the apparent effect of anticipation are consistent with prior findings in high trait anxiety.

Resveratrol opposite effects on rat tissue lipoperoxidation: pro-oxidant during day-time and antioxidant at night

Abstract We investigated the dosing-time dependency of acute resveratrol administration on lipoperoxidation level found in the heart, liver and kidney of male rats synchronized with a 12-h dark-light cycle. Resveratrol was administered by the i.p. route at the middle of the dark (6 h after dark onset, HADO) or light span (18 HADO) and thiobarbituric acid reactive species (TBARS) measured 4 h later at 10 and 22 HADO, respectively. Basal TBARS levels in the three organs were higher during the night span when compared to day span. Resveratrol effect on tissues TBARS was also dosing-time dependent. When administered during the dark phase, resveratrol decreased TBARS levels whereas at the light span, the polyphenol increased TBARS in the three organs. Resveratrol behaved as an antioxidant during the dark span and as a pro-oxidant during the light span. These data suggested a day/night rhythm in basal lipoperoxidation and in resveratrol antioxidant effect.

Presence of autism, hyperserotonemia, and severe expressive language impairment in Williams-Beuren syndrome

BackgroundDeletion of the Williams-Beuren syndrome (WBS) critical region (WBSCR), at 7q11.23, causes a developmental disorder commonly characterized by hypersociability and excessive talkativeness and often considered the opposite behavioral phenotype to autism. Duplication of the WBSCR leads to severe delay in expressive language. Gene–dosage effects on language development at 7q11.23 have been hypothesized.MethodsMolecular characterization of the WBSCR was performed by fluorescence in situ hybridization and high-resolution single-nucleotide polymorphism array in two individuals with severe autism enrolled in a genetic study of autism who showed typical WBS facial dysmorphism on systematic clinical genetic examination. The serotonin transporter promoter polymorphism (5-HTTLPR, locus SLC6A4) was genotyped. Platelet serotonin levels and urinary 6-sulfatoxymelatonin excretion were measured. Behavioral and cognitive phenotypes were examined.ResultsThe two patients had common WBSCR deletions between proximal and medial low copy repeat clusters, met diagnostic criteria for autism and displayed severe impairment in communication, including a total absence of expressive speech. Both patients carried the 5-HTTLPR ss genotype and exhibited platelet hyperserotonemia and low melatonin production.ConclusionsOur observations indicate that behaviors and neurochemical phenotypes typically associated with autism can occur in patients with common WBSCR deletions. The results raise intriguing questions about phenotypic heterogeneity in WBS and regarding genetic and/or environmental factors interacting with specific genes at 7q11.23 sensitive to dosage alterations that can influence the development of social communication skills. Thus, the influence of WBSCR genes on social communication expression might be dramatically modified by other genes, such as 5-HTTLPR, known to influence the severity of social communication impairments in autism, or by environmental factors, such as hyperserotonemia, given that hyperserotonemia is found in WBS associated with autism but not in WBS without autism. In this regard, WBS provides a potentially fruitful model with which to develop integrated genetic, cognitive, behavioral and neurochemical approaches to study genotype–phenotype correlations, possible gene–environment interactions and genetic background effects. The results underscore the importance of considering careful clinical and molecular genetic examination of individuals diagnosed with autism.

Circadian Disruption of Body Core Temperature and Rest–Activity Rhythms after General (Propofol) Anesthesia in Rats

Background:General anesthesia is commonly associated with sleep disorders, fatigue, drowsiness, and mood alterations in patients. The authors examined whether general (propofol) anesthesia can impact the circadian temporal structure by disturbing circadian rest–activity and body temperature rhythms under normal light–dark conditions (light–dark 12:12 h) in rats. Methods:A group of rats was anesthetized with propofol, and another was injected with 10% Intralipid, which was used as a control lipidic solution. The authors examined six groups of rats according to the Zeitgeber time of intraperitoneal administration (ZT6, ZT10, ZT16) and the substance injected (propofol or Intralipid). Results:On the day after anesthesia, propofol induced a significant 60- to 80-min phase advance of both rest–activity and body temperature rhythms. A significant 45- to 60-min phase advance of body temperature and a significant 20-min phase advance of rest–activity were still observed on the second day after anesthesia. The amplitudes of both rest–activity and body temperature rhythms were decreased on the first and second days after anesthesia. The 24-h mean rest–activity rhythm was decreased on the day after anesthesia, whereas the 24-h mean body temperature rhythm was not modified. Conclusion:The results demonstrate the disturbing effects of propofol anesthesia on the circadian time structure in rats under normal light conditions.

Plasma Corticosterone in Rats Is Specifically Increased at Recovery from Propofol Anesthesia without Concomitant Rise of Plasma ACTH

General anesthesia combined with surgery is commonly associated with post-operative stress-response in humans. Effects on the hypothalamic-pituitary-adrenal axis (HPA) during and after anesthesia are correlated with the magnitude of surgery and choice of anesthetics. The aim of our study in rats was to characterize the effects of general anesthesia without any surgery on HPA regulation of corticosterone and adrenocorticotropic hormone (ACTH) secretions. First, to assess whether the acute effects of general anesthesia on corticosterone concentration depend on time of day, rats were anesthetized with propofol at three different Zeitgeber times (ZT6, ZT10, and ZT16; with lights-on and -off at ZT0 and ZT12, respectively). Second, to determine the prolonged effects of general propofol anesthesia on daily corticosterone and ACTH concentrations, rats were anesthetized at ZT16 (4 h after lights-off) and euthanized either 1, 4, 12, 16, 20, or 24 h later. Third, the effects of propofol anesthesia on corticosterone and ACTH secretion were studied in rats instrumented with intracarotid cannulation. This permitted us to examine the individual patterns of corticosterone responses to propofol anesthesia as compared to their respective baseline corticosterone secretion. All of the results obtained showed that general propofol anesthesia, independent of the time-of-day of its administration, induces a significant increase of corticosterone secretion during the early recovery period without effect on ACTH secretion (i.e., no pituitary mediated stress-response). (Author correspondence: yvan.touitou@upmc.fr)

Responses of the Steroid Circadian System to Alcohol in Humans: Importance of the Time and Duration of Intake

Reports provide conflicting data about the effects of alcohol consumption on the hormonal system. Any study of these effects must control for a number of variables, including sex, alcohol status (alcoholic addiction vs. non-addiction), medical status (malnutrition, liver disease), and conditions of alcohol exposure, including an acute or continuous pattern of intake. The latter appears to be an especially critical factor in interpreting these effects. The authors therefore conducted a trial with a circadian design in which alcohol was administered repeatedly and regularly over a 26 h period for a total dose of 256 g. Because this protocol involves continuous alcohol administration, it is similar to administration among alcoholics and thus sheds new light on alcohols effect on hormone secretion. Using healthy volunteers rather than alcoholics, however, prevents any confounding due to liver disorders and nutritional deficiencies, and thus makes it possible to focus on the direct role of alcohol in hormonal modifications. In these conditions, the continuous administration of alcohol did not affect cortisol secretion, but serum testosterone levels were significantly higher at all time points during the alcohol session than at the corresponding time points during the control session. These data are not consistent with previously reported findings for the relation between alcohol and both cortisol and testosterone, because in the current experiment the action of ethanol on steroid secretion should involve the circadian clock more than the hormonal system itself.