The Effects of General Anaesthesia and Light on Behavioural Rhythms and GABAA Receptor Subunit Expression in the Mouse SCN.

General anaesthesia (GA) is known to affect the circadian clock. However, the mechanisms that underlie GA-induced shifting of the clock are less well understood. Activation of γ-aminobutyric acid (GABA)-type A receptors (GABAAR) in the suprachiasmatic nucleus (SCN) can phase shift the clock and thus GABA and its receptors represent a putative pathway via which GA exerts its effect on the clock. Here, we investigated the concurrent effects of the inhalational anaesthetic, isoflurane, and light, on mouse behavioural locomotor rhythms and on α1, ß3, and γ2 GABAAR subunit expression in the SCN of the mouse brain. Behavioural phase shifts elicited by exposure of mice to four hours of GA (2% isoflurane) and light (400 lux) (n = 60) were determined by recording running wheel activity rhythms in constant conditions (DD). Full phase response curves for the effects of GA + light on behavioural rhythms show that phase shifts persist in anaesthetized mice exposed to light. Daily variation was detected in all three GABAAR subunits in LD 12:12. The γ2 subunit expression was significantly increased following GA in DD (compared to light alone) at times of large behavioural phase delays. We conclude that the phase shifting effect of light on the mouse clock is not blocked by GA administration, and that γ2 may potentially be involved in the phase shifting effect of GA on the clock. Further analysis of GABAAR subunit expression in the SCN will be necessary to confirm its role.

General Anaesthesia Shifts the Murine Circadian Clock in a Time-Dependant Fashion.

Following general anaesthesia (GA), patients frequently experience sleep disruption and fatigue, which has been hypothesized to result at least in part by GA affecting the circadian clock. Here, we provide the first comprehensive time-dependent analysis of the effects of the commonly administered inhalational anaesthetic, isoflurane, on the murine circadian clock, by analysing its effects on (a) behavioural locomotor rhythms and (b) PER2::LUC expression in the suprachiasmatic nuclei (SCN) of the mouse brain. Behavioural phase shifts elicited by exposure of mice (n = 80) to six hours of GA (2% isoflurane) were determined by recording wheel-running rhythms in constant conditions (DD). Phase shifts in PER2::LUC expression were determined by recording bioluminescence in organotypic SCN slices (n = 38) prior to and following GA exposure (2% isoflurane). Full phase response curves for the effects of GA on behaviour and PER2::LUC rhythms were constructed, which show that the effects of GA are highly time-dependent. Shifts in SCN PER2 expression were much larger than those of behaviour (c. 0.7 h behaviour vs. 7.5 h PER2::LUC). We discuss the implications of this work for understanding how GA affects the clock, and how it may inform the development of chronotherapeutic strategies to reduce GA-induced phase-shifting in patients.

The Effect of General Anaesthesia on Circadian Rhythms in Behaviour and Clock Gene Expression of Drosophila melanogaster.

General anaesthesia (GA) is implicated as a cause of postoperative sleep disruption and fatigue with part of the disturbance being attributed to a shift of the circadian clock. In this study, Drosophila melanogaster was used as a model to determine how Isoflurane affects the circadian clock at the behavioural and molecular levels. We measured the response of the clock at both of these levels caused by different durations and different concentrations of Isoflurane at circadian time 4 (CT4). Once characterized, we held the duration and concentration constants (at 2% in air for 6 h) and calculated the phase responses over the entire circadian cycle in both activity and period expression. Phase advances in behaviour were observed during the subjective day, whereas phase delays were associated with subjective night time GA interventions. The corresponding pattern of gene expression preceded the behavioural pattern by approximately four hours. We discuss the implications of this effect for clinical and research practice.

The functional changes of the circadian system organization in aging.

The circadian clock drives periodic oscillations at different levels of an organism from genes to behavior. This timing system is highly conserved across species from insects to mammals and human beings. The question of how the circadian clock is involved in the aging process continues to attract more attention. We aim to characterize the detrimental impact of aging on the circadian clock organization. We review studies on different components of the circadian clock at the central and periperal levels, and their changes in aged rodents and humans, and the fruit fly Drosophila. Intracellular signaling, cellular activity and intercellular coupling in the central pacemaker have been found to decline with advancing age. Evidence of degradation of the molecular clockwork reflected by clock gene expression in both central and peripheral oscillators due to aging is inadequate. The findings on age-associated molecular and functional changes of peripheral clocks are mixed. We conclude that aging can affect the circadian clock organization at various levels, and the impairment of the central network may be a fundamental mechanism of circadian disruption seen in aged species.

Development of the Molecular Circadian Clock and Its Light Sensitivity in Drosophila Melanogaster.

The importance of the circadian clock for the control of behavior and physiology is well established but how and when it develops is not fully understood. Here the initial expression pattern of the key clock gene period was recorded in Drosophila from embryos in vivo, using transgenic luciferase reporters. PERIOD expression in the presumptive central-clock dorsal neurons started to oscillate in the embryo in constant darkness. In behavioral experiments, a single 12-h light pulse given during the embryonic stage synchronized adult activity rhythms, implying the early development of entrainment mechanisms. These findings suggest that the central clock is functional already during embryogenesis. In contrast to central brain expression, PERIOD in the peripheral cells or their precursors increased during the embryonic stage and peaked during the pupal stage without showing circadian oscillations. Its rhythmic expression only initiated in the adult. We conclude that cyclic expression of PERIOD in the central-clock neurons starts in the embryo, presumably in the dorsal neurons or their precursors. It is not until shortly after eclosion when cyclic and synchronized expression of PERIOD in peripheral tissues commences throughout the animal.

The Development and Decay of the Circadian Clock in Drosophila melanogaster.

The way in which the circadian clock mechanism develops and decays throughout life is interesting for a number of reasons and may give us insight into the process of aging itself. The Drosophila model has been proven invaluable for the study of the circadian clock and development and aging. Here we review the evidence for how the Drosophila clock develops and changes throughout life, and present a new conceptual model based on the results of our recent work. Firefly luciferase lines faithfully report the output of known clock genes at the central clock level in the brain and peripherally throughout the whole body. Our results show that the clock is functioning in embryogenesis far earlier than previously thought. This central clock in the fly remains robust throughout the life of the animal and only degrades immediately prior to death. However, at the peripheral (non-central oscillator level) the clock shows weakened output as the animal ages, suggesting the possibility of the breakdown in the cohesion of the circadian network.

Impact of anaesthesia on circadian rhythms and implications for laboratory experiments.

General anaesthesia is a widely used tool to enable surgery in animal experimentation. There is now convincing evidence that general anaesthesia can cause profound and strongly time-dependant shifts in circadian rhythms of behaviour (sleep-wake cycles), physiology (core body temperature, blood pressure, heart rate and hormone release) and cognitive parameters (learning and memory) in a range of species. These effects have the potential to confound laboratory experiments, and may lead to misinterpretation of results. Here, we summarise these effects and advise caution to those conducting laboratory experiments in which anaesthesia forms part of the protocol.

Clock gene expression and locomotor activity predict death in the last days of life in Drosophila melanogaster.

The importance of the circadian clock for the regulation of behaviour and physiology, and the molecular control of these rhythms by a set of clock genes are well defined. The circadian clock deteriorates with advancing age but the mechanism underlying is unclear. Here we recorded the expression of two key clock genes in young, middle-aged and old Drosophila using transgenic luciferase lines reporting period and timeless in vivo. We report a novel marker of imminent death in the expression of TIMELESS. In the days immediately preceding death TIMELESS expression increased to at least 150% of previous acrophase values (88.0% of n = 217) and lost circadian rhythmicity, which predicted death equally well in flies of different ages and under light and temperature cycles. We suggest this transient aberrant clock-gene expression is central to the mechanism of the disturbance in circadian behaviour before death (82.7% of n = 342). We also find that PERIOD expression in central-clock neurons remained robust with age, however PERIOD and TIMELESS in peripheral clocks showed a reduction in both expression level and rhythmicity. In conclusion, as flies age the molecular clock gradually declines at the peripheral level but continues to function at the central until days before death.

Chronotype and environmental light exposure in a student population.

In humans and most other species, changes in the intensity and duration of light provide a critical set of signals for the synchronisation of the circadian system to the astronomical day. The timing of activity within the 24 h day defines an individual's chronotype, i.e. morning, intermediate or evening type. The aim of this study was to investigate the associations between environmental light exposure, due to geographical location, on the chronotype of university students. Over 6 000 university students from cities in the Northern Hemisphere (Oxford, Munich and Groningen) and Southern Hemisphere (Perth, Melbourne and Auckland) completed the Munich ChronoType Questionnaire. In parallel, light measures (daily irradiance, timing of sunrise and sunset) were compiled from satellite or ground stations at each of these locations. Our data shows that later mid-sleep point on free days (corrected for oversleep on weekends MFSsc) is associated with (i) residing further from the equator, (ii) a later sunset, (iii) spending more time outside and (iv) waking from sleep significantly after sunrise. However, surprisingly, MSFsc did not correlate with daily light intensity at the different geographical locations. Although these findings appear to contradict earlier studies suggesting that in the wider population increased light exposure is associated with an earlier chronotype, our findings are derived exclusively from a student population aged between 17 and 26 years. We therefore suggest that the age and occupation of our population increase the likelihood that these individuals will experience relatively little light exposure in the morning whilst encountering more light exposure later in the day, when light has a delaying effect upon the circadian system.

How does general anaesthesia affect the circadian clock?

Post-operative patients experience sleep disturbances. Animal studies demonstrate that general anaesthesia (GA) can disrupt circadian rhythms and cause changes in the molecular clock, indicating that anaesthesia contributes to post-operative circadian disruption. Here we review the effect of anaesthesia on the circadian clock and its rhythms in order to summarise current findings outline commonalities between studies and propose mechanisms by which effects may be mediated. KEY POINTS: 1) GA has strong effects on the main neurotransmitter systems linked with circadian control (Gamma aminobutyric acid/N-methyl-D-aspartate (GABA/NMDA)) and may act by interfering with light-entrainment of the clock. 2) Expression of the core clock gene per2 is inhibited by GA (possibly via a NMDA/glycogen synthase kinase 3ß (GSK3ß) pathway). 3) GA's effect on circadian rhythms appears greatest when administered during animals' active phases 4) GA may have different effects when administered under free-running and entrained conditions. 5) Anaesthesia may mimic the mechanism involved in adaptation of the clock to changes in daylength. There is agreement that GA can strongly affect the circadian clock. How anaesthesia-induced changes in the molecular clock lead to changes in behaviour remains unclear. The answer, and what it may mean for patients post-operatively, will rely on systematic studies at molecular, behavioural, and clinical levels using standardised protocols.

The innate immune cell response to bacterial infection in larval zebrafish is light-regulated.

The circadian clock, which evolved to help organisms harmonize physiological responses to external conditions (such as the light/dark cycle, LD), is emerging as an important regulator of the immune response to infection. Gaining a complete understanding of how the circadian clock influences the immune cell response requires animal models that permit direct observation of these processes within an intact host. Here, we investigated the use of larval zebrafish, a powerful live imaging system, as a new model to study the impact of a fundamental zeitgeber, light, on the innate immune cell response to infection. Larvae infected during the light phase of the LD cycle and in constant light condition (LL) demonstrated enhanced survival and bacterial clearance when compared with larvae infected during the dark phase of the LD cycle and in constant dark condition (DD). This increased survival was associated with elevated expression of the zebrafish orthologues of the mammalian pro-inflammatory cytokine genes, Tumour necrosis factor-α, Interleukin-8 and Interferon-γ, and increased neutrophil and macrophage recruitment. This study demonstrates for the first time that the larval zebrafish innate immune response to infection is enhanced during light exposure, suggesting that, similar to mammalian systems, the larval zebrafish response to infection is light-regulated.

The effects of the general anaesthetic isoflurane on the honey bee (Apis mellifera) circadian clock.

General anaesthesia administered during the day has previously been shown to phase shift the honey bee clock. We describe a phase response curve for honey bees (n=105) to six hour isoflurane anaesthesia. The honey bee isoflurane PRC is "weak" with a delay portion (maximum shift of -1.88 hours, circadian time 0 - 3) but no advance zone. The isoflurane-induced shifts observed here are in direct opposition to those of light. Furthermore, concurrent administration of light and isoflurane abolishes the shifts that occur with isoflurane alone. Light may thus provide a means of reducing isoflurane-induced phase shifts.

Way-finding in displaced clock-shifted bees proves bees use a cognitive map.

Mammals navigate by means of a metric cognitive map. Insects, most notably bees and ants, are also impressive navigators. The question whether they, too, have a metric cognitive map is important to cognitive science and neuroscience. Experimentally captured and displaced bees often depart from the release site in the compass direction they were bent on before their capture, even though this no longer heads them toward their goal. When they discover their error, however, the bees set off more or less directly toward their goal. This ability to orient toward a goal from an arbitrary point in the familiar environment is evidence that they have an integrated metric map of the experienced environment. We report a test of an alternative hypothesis, which is that all the bees have in memory is a collection of snapshots that enable them to recognize different landmarks and, associated with each such snapshot, a sun-compass-referenced home vector derived from dead reckoning done before and after previous visits to the landmark. We show that a large shift in the sun-compass rapidly induced by general anesthesia does not alter the accuracy or speed of the homeward-oriented flight made after the bees discover the error in their initial postrelease flight. This result rules out the sun-referenced home-vector hypothesis, further strengthening the now extensive evidence for a metric cognitive map in bees.

Clevidipine compared with nitroglycerin for blood pressure control in coronary artery bypass grafting: a randomized double-blind study.

PURPOSE: We tested the hypothesis that clevidipine, a rapidly acting dihydropyridine calcium channel blocker, is not inferior to nitroglycerin (NTG) in controlling blood pressure before cardiopulmonary bypass (CPB) during coronary artery bypass grafting (CABG). METHODS: In this double-blind study from October 4, 2003 to April 26, 2004, 100 patients undergoing CABG with CPB were randomized at four centres to receive intravenous infusions of clevidipine (0.2-8 µg·kg(-1)·min(-1)) or NTG (0.4 µg·kg(-1)·min(-1) to a clinician-determined maximum dose rate) from induction of anesthesia through 12 hr postoperatively. The study drug was titrated in the pre-CPB period with the aim of maintaining mean arterial pressure (MAP) within ± 5 mmHg of a clinician-predetermined target. The primary endpoint was the area under the curve (AUC) for the total time each patient's MAP was outside the target range from drug initiation to the start of CPB, normalized per hour (AUCMAP-D). The predefined non-inferiority criterion for the primary endpoint was a 95% confidence interval (CI) upper limit no greater than 1.50 for the geometric means ratio between clevidipine and NTG. RESULTS: Total mean [standard deviation (SD)] dose pre-bypass was 4.5 (4.7) mg for clevidipine and 6.9 (5.4) mg for NTG (P < 0.05). The geometric mean AUCMAP-D for clevidipine was 283 mmHg·min·hr(-1) (n = 45) and for NTG was 292 mmHg·min·hr(-1) (n = 48); the geometric means ratio was 0.97 (95% CI 0.74 to 1.27). The geometric mean AUCMAP-D during aortic cannulation was 357.7 mmHg·min·hr(-1) for clevidipine compared with 190.5 mmHg·min·hr(-1) for NTG. Mean (SD) heart rate with clevidipine was 76.0 (13.8) beats·min(-1) compared with 81.5 (14.4) beats·min(-1) for NTG. There were no clinically important differences between groups in adverse events. CONCLUSION: During CABG, clevidipine was not inferior to NTG for blood pressure control pre-bypass.

Identifying advanced and delayed sleep phase disorders in the general population: a national survey of New Zealand adults.

The aim was to estimate the prevalence of, and identify independent risk factors for, Advanced (ASPD) and Delayed Sleep Phase Disorder (DSPD) among Maori (indigenous New Zealanders) and non-Maori adults using a self-report questionnaire. The Munich Chronotype Questionnaire was mailed to a stratified sample of 9100 adults (5100 Maori and 4000 non-Maori) aged 20-59 years randomly selected from the electoral rolls (54% response rate). Different definitions for ASPD and DSPD were developed using combinations of symptoms including self-reported bed and rising times, current chronotype, and a desire to change sleep schedule. Logistic regression models were used to model the likelihood of reporting ASPD or DSPD separately after adjusting for ethnicity (Maori versus non-Maori), sex (males versus females), age (in decades), socio-economic deprivation (NZDep2006 deciles) and employment status (unemployed, night work versus employed with no night work). The prevalence of ASPD ranged from 0.25% to 7.13% whereas the prevalence of DSPD was 1.51 to 8.90% depending on the definition used. The prevalence of ASPD was higher among men and increased with age. The prevalence of DSPD was higher among those living in more deprived areas and decreased with age. After controlling for ethnicity, gender, age, socio-economic deprivation and employment status, people with ASPD were more likely to report excessive daytime sleepiness, whereas those with DSPD were more likely to report poor or fair self-rated health. Reporting ASPD and DSPD were associated with self-reported night work. In this large sleep timing survey, we found no differences in the prevalence of self-identified ASPD and DSPD between Maori and non-Maori. This has implications for the development and provision of sleep health services and strategies for managing the significant impact of work patterns on sleep.

Brachial arterial temperature as an indicator of core temperature: proof of concept and potential applications.

There is potential for heat loss and hypothermia during anesthesia and also for hyperthermia if heat conservation and active warming measures are not accurately titrated. Accurate temperature monitoring is particularly important in procedures in which the patient is actively cooled and then rewarmed such as during cardiopulmonary bypass surgery (CPB). We simultaneously measured core, nasopharyngeal, and brachial artery temperatures to investigate the last named as a potential peripheral temperature monitoring site. Ten patients undergoing hypothermic CPB were instrumented for simultaneous monitoring of temperatures in the pulmonary artery (PA), aortic arterial inflow (AI), nasopharynx (NP), and brachial artery (BA). Core temperature was defined as PA temperature before and after CPB and the AI temperature during CPB. Mean deviations of BA and NP temperatures from core temperature were calculated for three steady-state periods (before, during, and after CPB). Mean deviation of BA and NP temperatures from AI temperature was also calculated during active rewarming. A total of 1862 measurements were obtained and logged from eight patients. Mean BA and NP deviations from core temperature across the steady-state periods (before, during, and after CBP) were, respectively: .23 +/- .25, -.26 +/- .3, and -.09 +/- .05 degrees C (BA), and .11 +/- .19, -.1 +/- .47, and -.04 +/- .3 degrees C (NP). During steady-state periods, there was no evidence of a difference between the mean BA and NP deviation. During active rewarming, the mean difference between the BA and AI temperatures was .14 +/- .36 degrees C. During this period, NP temperature lagged behind AI and BA temperatures by up to 41 minutes and was up to 5.3 degres C lower than BA (mean difference between BA and NP temperatures was 1.22 +/- .58 degrees C). The BA temperature is an adequate surrogate for core temperature. It also accurately tracks the changing AI temperature during rewarming and is therefore potentially useful in detecting a hyperthermic perfusate, which might cause cerebral hyperthermia.

A honey bee (Apis mellifera) light phase response curve.

The authors report a phase response curve (PRC) for individual honey bees (Apis mellifera) to single 1-h light pulses (1000 lux) using an Aschoff Type 1 protocol (n = 134). The bee PRC is a weak (Type 1) PRC with a maximum advance of 1.5 h between circadian time (CT) 18 and 3 and a maximum delay of 1.5 h between CT 12 and 18. This is the first published honey bee light PRC and provides an important resource for chronobiologists and honey bee researchers. It may also have practical applications for what is an economically important species frequently transported across different time zones.

General anesthesia alters time perception by phase shifting the circadian clock.

Following general anesthesia, people are often confused about the time of day and experience sleep disruption and fatigue. It has been hypothesized that these symptoms may be caused by general anesthesia affecting the circadian clock. The circadian clock is fundamental to our well-being because it regulates almost all aspects of our daily biochemistry, physiology, and behavior. Here, we investigated the effects of the most common general anesthetic, isoflurane, on time perception and the circadian clock using the honeybee (Apis mellifera) as a model. A 6-h daytime anesthetic systematically altered the time-compensated sun compass orientation of the bees, with a mean anticlockwise shift in vanishing bearing of 87° in the Southern Hemisphere and a clockwise shift in flight direction of 58° in the Northern Hemisphere. Using the same 6-h anesthetic treatment, time-trained bees showed a delay in the start of foraging of 3.3 h, and whole-hive locomotor-activity rhythms were delayed by an average of 4.3 h. We show that these effects are all attributable to a phase delay in the core molecular clockwork. mRNA oscillations of the central clock genes cryptochrome-m and period were delayed by 4.9 and 4.3 h, respectively. However, this effect is dependent on the time of day of administration, as is common for clock effects, and nighttime anesthesia did not shift the clock. Taken together, our results suggest that general anesthesia during the day causes a persistent and marked shift of the clock effectively inducing "jet lag" and causing impaired time perception. Managing this effect in humans is likely to help expedite postoperative recovery.

Prevalence of causes of insomnia in primary care: a cross-sectional study.

BACKGROUND: As a result of a research interest in primary insomnia, the prevalence of other causes of insomnia in primary care must be ascertained. No source was found in the literature. It is also essential to know the epidemiology of the common causes of a condition to make an accurate diagnosis in primary care. AIM: To determine the prevalence of causes of insomnia in primary care, as part of a method of identifying patients with primary insomnia. DESIGN AND SETTING: Cross-sectional study in three general practices in Auckland, New Zealand. METHOD: Consecutive patients from the waiting room were asked to complete a nine-page questionnaire on possible causes of insomnia. RESULTS: In total, 1517 patients were approached and 955 completed the nine-page questionnaire (63%). Of the 41% (388) who reported difficulty with sleeping, primary insomnia occurred in 12% (45) of the population (95% confidence interval = 9% to 15%); 50% (195) had depression, 48% (185) had anxiety and 43% (165) had general (physical) health problems. Obstructive sleep apnoea occurred in 9% (34) and delayed sleep phase disorder in 2% (7). Only primary insomnia and delayed sleep phase disorder are mutually exclusive; the others can co-exist. CONCLUSION: This is the first description of the prevalence of causes of insomnia in primary care. It is hoped that the focus on primary insomnia will result in more behavioural treatments and lower the use of hypnotics in primary care; it should also assist in the appropriate detection and treatment of other causes of insomnia in primary care.