The adjustment of daily activity time (α) to the varying night length in nocturnal creatures was one of the functions originally attributed to a putative dual oscillator structure of circadian pacemakers in mammals. In two experimental approaches, we tested whether this ability is compromised in mice with functional deletions of one of the four circadian clock genes. First, we tested the capability of α compression by long days in mPer1(Brdm1) and mPer2(Brdm1) mutant mice. When exposed to a full L:D 18:6 photoperiod, wild-type and mPer1(Brdm1) mutant mice show compression followed by decompression of α in DD. mPer2(Brdm1) mutant mice did not compress their activity time. The interpretation of these data is, however, complicated by masking due to light. We, therefore, embarked on a second experiment, exploiting skeleton photoperiods. The skeleton photoperiod was changed stepwise from 0 to 24 h, and mCry1 and mCry2 knockout mice were now included in the design. We observed clear and systematic compression of α in wild-type and mCry1 and mCry2 knockout mice. mPer1(Brdm1) and mPer2(Brdm1) mice both poorly entrained to the skeleton photoperiod. The single mPer2(Brdm1) mutant mouse that did entrain did not show α compression. The results show that neither mCry1 nor mCry2 deletions compromise adjustment to day length, consistent with our earlier conclusions on period lengthening in constant light (Spoelstra & Daan, 2008). The mPer2(Brdm1) mutant behaves aberrantly and appears not to respond to the delaying action of light in the late subjective day.

Phase shifting of circadian systems by light has been attributed both to parametric effects on angular velocity elicited by a tonic response to the luminance level and to nonparametric instantaneous shifts induced by a phasic response to the dark-light (D>L) and light-dark (L>D) transitions. Claims of nonparametric responses are partly based on "step-PRCs," that is, phase response curves derived from such transitions. Step-PRCs in nocturnal mammals show mostly delays after lights-on and advances after lights-off, and therefore appear incompatible with phase delays generated by light around dusk and advances by light around dawn. We have pursued this paradox with 2 experimental protocols in mice. We first use the classic step-PRC protocol on wheel running activity, using the center of gravity as a phase marker to minimize the masking effects of light. The experiment was done for 3 different light intensities (1,10, and 100 lux). D > L transitions evoke mostly delays and L > D transitions show no clear tendency to either delay or advance. Overall there is little or no circadian modulation. A 2nd protocol aimed to avoid the problem of masking by assessing phase before and after the light stimuli, both in DD. Light stimuli consisted of either a slow light intensity increase over 48 h followed by abruptly switching off the light, or an abrupt switch on followed by a slow decrease toward total darkness during 48 h. If the abrupt transitions were responsible for phase shifting, we expected large differences between the 2 stimuli. Both light stimuli yielded similar PRCs characterized by delays only with circadian modulation. The results can be adequately explained by a model in which all PRCs evoked by steps result in fact from tonic responses to the light following a step-up or preceding a step-down. In this model only the response reduction of tonic velocity change after the 1st hour is taken into account. The data obtained in both experiments are thus compatible with tonic velocity responses. Contrary to standard interpretation of step-PRCs, non-parametric responses to the transitions are unlikely since they would predict delays in response to lights-off, advances in response to lights-on, while the opposite was found. Although such responses cannot be fully excluded, parsimony does not require invocation of a role for transitions, since all the data can readily be explained by tonic velocity (parametric) effects, which must exist because of the dependence of tau on light intensity.

Entrainment may involve responses to dawn, to dusk, and to the light in between these transitions. Previous studies showed that the circadian system responds to only 2 light pulses, one at the beginning and one at the end of the day, in a similar way as to a full photoperiod, as long as the photoperiod is less than approximately 1/2 τ. The authors used a double 1-h light pulse protocol with different intervals of darkness in between (1, 2, 4, 7, 10, and 16 h) to study the phase responses of mice. The phase response curves obtained were compared to full light pulse PRCs of corresponding durations. Up to 6 hours, phase responses induced by double light pulses are virtually the same as by a corresponding full light pulse. The authors made a simple phase-only model to estimate the response reduction due to light exposure and response restoration due to dark exposure of the system. In this model, they assumed a 100% contribution of the first 1-h light pulse and fitted the reduction factor for the second light pulse to yield the best fit to the observations. The results suggest that after 1 h of light followed by less than 4 h of darkness, there is a considerable reduction in response to the second light pulse. Full response restoration requires more than 10 h of darkness. To investigate the influence of the duration of light on the response saturation, the authors performed a second series of experiments where the duration of the 2 light pulses was varied from 4 to 60 min each with a fixed duration of the stimulus (4 h). The response to 2 light pulses saturates when they are between 30 and 60 min long. In conclusion, double pulses replace single full light pulses of a corresponding duration of up to 6 h due to a response reduction during light, combined with response restoration during darkness. By the combined response reduction and response restoration, mice can maintain stable entrainment to the external LD cycle without being continuously exposed to it.

Recent theories on the function of arousals from torpor in hibernating mammals focus on the repair of the central nervous system from damage accumulating during prolonged hypothermia. In this framework, we investigated the synaptic ultrastructure in Layer 2 of the frontal cortex from hibernating European ground squirrels (Spermophilus citellus) sacrificed at four different phases in the torporarousal cycle. Using electron microscopy, we quantified synapse number and morphometric data on asymmetric axospinous synapses. Length, width, and surface area of postsynaptic densities (PSDs), and the synaptic apposition length of the analyzed synapse were measured. Five groups of animals were compared during entrance into torpor (Torpor Early, TE, n = 6), late torpor (Torpor Late, TL, n = 5), beginning of euthermic arousal episodes (Arousal Early, AE, n = 5), late in the euthermic arousal episode (Arousal Late, AL, n = 5), and during continuous euthermy in spring (EU, n = 6). The results showed that during torpor and at the beginning of arousals the PSD length and synaptic apposition length are significantly increased compared to synapses during late arousal and in spring conditions. In contrast, the width and surface area of the PSDs are decreased in torpor. At the beginning of an arousal the width of the PSD increases and gains maximum value in late arousals (AL), returning to spring (EU) values. No differences were found in total number of synapses during the torpor-arousal cycle. The results indicate reversible changes in ultrastructure of (asymmetric axospinous) synapses in the frontal cortex, which may be critical for the maintenance of cortical neuronal networks and for protection against potential deleterious effects of prolonged hypothermic phases of hibernation.

Bright light can influence human psychophysiology instantaneously by inducing endocrine (suppression of melatonin, increasing cortisol levels), other physiological changes (enhancement of core body temperature), and psychological changes (reduction of sleepiness, increase of alertness). Its broad range of action is reflected in the wide field of applications, ranging from optimizing a work environment to treating depressed patients. For optimally applying bright light and understanding its mechanism, it is crucial to know whether its effects depend on the time of day. In this paper, we report the effects of bright light given at two different times of day on psychological and physiological parameters. Twenty-four subjects participated in two experiments (n = 12 each). All subjects were nonsmoking, healthy young males (18-30 yr). In both experiments, subjects were exposed to either bright light (5,000 lux) or dim light <10 lux (control condition) either between 12:00 P.M. and 4:00 P.M. (experiment A) or between midnight and 4:00 A.M. (experiment B). Hourly measurements included salivary cortisol concentrations, electrocardiogram, sleepiness (Karolinska Sleepiness Scale), fatigue, and energy ratings (Visual Analog Scale). Core body temperature was measured continuously throughout the experiments. Bright light had a time-dependent effect on heart rate and core body temperature; i.e., bright light exposure at night, but not in daytime, increased heart rate and enhanced core body temperature. It had no significant effect at all on cortisol. The effect of bright light on the psychological variables was time independent, since nighttime and daytime bright light reduced sleepiness and fatigue significantly and similarly.

To understand entrainment of circadian systems to different photoperiods in nature, it is important to know the effects of single light pulses of different durations on the free-running system. The authors studied the phase and period responses of laboratory mice (C57BL6J//OlaHsd) to single light pulses of 7 different durations (1, 3, 4, 6, 9, 12, and 18 h) given once per 11 days in otherwise constant darkness. Light-pulse duration affected both amplitude and shape of the phase response curve. Nine-hour light pulses yielded the maximal amplitude PRC. As in other systems, the circadian period slightly lengthened following delays and shortened following advances. The authors aimed to understand how different parts of the light signal contribute to the eventual phase shift. When PRCs were plotted using the onset, midpoint, and end of the pulse as a phase reference, they corresponded best with each other when using the mid-pulse. Using a simple phase-only model, the authors explored the possibility that light affects oscillator velocity strongly in the 1st hour and at reduced strength in later hours of the pulse due to photoreceptor adaptation. They fitted models based on the 1-h PRC to the data for all light pulses. The best overall correspondence between PRCs was obtained when the effect of light during all hours after the first was reduced by a factor of 0.22 relative to the 1st hour. For the predicted PRCs, the light action centered on average at 38% of the light pulse. This is close to the reference phase yielding best correspondence at 36% of the pulses. The result is thus compatible with an initial major contribution of the onset of the light pulse followed by a reduced effect of light responsible for the differences between PRCs for different duration pulses. The authors suggest that the mid-pulse is a better phase reference than lights-on to plot and compare PRCs of different light-pulse durations.

The mammalian retina contains both visual and circadian photoreceptors. In humans, nocturnal stimulation of the latter receptors leads to melatonin suppression, which might cause reduced nighttime sleepiness. Melatonin suppression is maximal when the nasal part of the retina is illuminated. Whether circadian phase shifting in humans is due to the same photoreceptors is not known. The authors explore whether phase shifts and melatonin suppression depend on the same retinal area. Twelve healthy subjects participated in a within-subjects design and received all of 3 light conditions—1) 10 lux of dim light on the whole retina, 2) 100 lux of ocular light on the nasal part of the retina, and 3) 100 lux of ocular light on the temporal part of the retina—on separate nights in random order. In all 3 conditions, pupils were dilated before and during light exposure. The protocol consisted of an adaptation night followed by a 23-h period of sustained wakefulness, during which a 4-h light pulse was presented at a time when maximal phase delays were expected. Nasal illumination resulted in an immediate suppression of melatonin but had no effect on subjective sleepiness or core body temperature (CBT). Nasal illumination delayed the subsequent melatonin rhythm by 78 min, which is significantly (p= 0.016) more than the delay drift in the dim-light condition (38 min), but had no detectable phase-shifting effect on the CBT rhythm. Temporal illumination suppressed melatonin less than the nasal illumination and had no effect on subjective sleepiness and CBT. Temporal illumination delayed neither the melatonin rhythm nor the CBT rhythm. The data show that the suppression of melatonin does not necessarily result in a reduction of subjective sleepiness and an elevation ofCBT. In addition, 100 lux of bright white light is strong enough to affect the photoreceptors responsible for the suppression of melatonin but not strong enough to have a significant effect on sleepiness and CBT. This may be due to the larger variability of the latter variables.

In this paper we examine the relationship between melatonin suppression and reduction of sleepiness through light by comparing three different data sets. In total 36 subjects participated in three studies and received 4 h of bright light either from midnight till 4:00 hours (experiments A and B) or from noon till 16:00 hours (experiment C). In experiment A (night-time light, partial illumination of the retina, pupil dilated) subjects were exposed to either 100 lx of ocular light on the temporal, 100 lx on the nasal part of the retina, or <10 lx of dim light on the whole retina. In experiments B (night-time light, whole retina, pupil not dilated) and C (daytime light, whole retina, pupil not dilated) subjects were exposed either to bright (5000 lx) or to dim light (<10 lx). Subjective sleepiness/fatigue and melatonin concentrations in saliva were assessed hourly in all three experiments. For experiment A, a significant suppression of melatonin due to nasal and temporal illumination of the retina was found, that was not accompanied by a detectable reduction of subjective sleepiness/fatigue. For experiment B we found a suppression of melatonin that was paralleled with a significant reduction in subjective sleepiness, but not in fatigue. During experiment C we found no melatonin suppression but a reduction of subjective sleepiness, but also no effect on fatigue. From these data we conclude that the effects of light on sleepiness/fatigue are not mediated by melatonin and that the influence of endogenous melatonin concentration on sleepiness/fatigue is restricted.

We report on results from an Internet survey of sleeping habits in a Dutch population using the Munich Chronotype Questionnaire (MCTQ), supplemented with the Horne-Ostberg Morningness-Eveningness Questionnaire (MEQ). The MCTQ was completed by 5,055 responders, of which 2,481 also completed the MEQ. MEQ score correlated well with the MCTQ assessment of time of mid-sleep on free days (MSF; r = - 0.73) and on workdays (MSW; r = - 0.61). MEQ was more strongly correlated with MSF (50% of sleep time) than with sleep onset (0%), rise time (100%), or with any other percentile (10 to 40, 60% to 90%) of sleep on free days. The study shows that chronotype (based on MSF as measured by the MCTQ) strongly correlates with morningness-eveningness (as measured by the MEQ). However, the MCTQ collects additional detailed information on sleep-wake behavior under natural conditions.

Anna Wirz-Justice,* Serge Daan,†,1 Simon Folkard,† Alfred Lewy,§ Reimer Lund,|| and Jurgen Zulley¶ *Centre for Chronobiology, University Psychiatric Clinics, Basel, Switzerland; †Centre for Life Sciences, University of Groningen, Haren, the Netherlands; †Laboratoire d’Anthropologie Appliquee, UPRES Ergonomie, Universite Rene Descartes–Paris V, Paris, France; §Sleep and Mood Disorders Laboratory, Department of Psychiatry, Oregon Health & Science University, Portland; ||Munich, Germany; ¶Sleep Disorders Center, University and Psychiatric Clinic Regensburg, Regensburg, Germany

The oscillating properties of the mammalian circadian system are generated by a molecular mechanism that involves the clock genes mPer1, mPer2, mCry1, and mCry2. Entrainment of the circadian system and its property to follow daylength are mediated by light input, but the precise mechanisms are still under debate. Light induced phase shifts of the circadian system are accompanied by induction of the c-Fos gene in the suprachiasmatic nucleus (SCN, the circadian pacemaker). Here we examine the role of the mammalian Per and Cry genes by means of c-Fos expression in genetically modified mice after entrainment to a light-dark cycle and in continuous dim light. Under continuous dim light, light induced c-Fos expression in mCry1-/and mCry2-/mice is about half that of wild type controls, and in mPer2Brdm1 mice, but not in mPer1Brdm1 mice, it is almost absent. Results after entrainment differed from continuous dim light in both mCry strains, but not in the mPer strains. Quantification of melanopsin containing ganglion cells and rod outer segment length in retinae of these mice revealed no differences that can be related to observed differences in light induced c-Fos in the SCN. This suggests that observed variation in light response between these genotypes is not driven by retinal photoreceptor density, but is likely to be an intrinsic property of the SCN. The SCN specific function in light response regulation of the analysed mCry and mPer genes is discussed in the frame work of the dual oscillator hypothesis (Evening Morning oscillator).

Female birds of several species have control over the production of daughters and sons. However, most studies failed to find a relationship between egg size and sex. This is intriguing as adjustment of egg size would constitute a powerful tool for the female to meet different resource demands of the sexes, particularly in size dimorphic species. Our results show that, within clutches of black-headed gulls (Larus ridibundus) the proportion of males was positively associated with egg mass. This applied for all three laying positions, independently of the absolute egg mass. There was a significant relationship between the distribution of the sexes over the laying sequence and the egg mass change. When egg mass decreased over the sequence, first-laid eggs were male biased and last-laid eggs female biased, and vice versa. The potential adaptive value of this allocation strategy is evaluated with regard to male sensitivity to egg quality and competitive differences between the sexes.

The phase-resetting properties of the circadian system in mice with a functional deletion in mCry1, mCry2, mPer1, or mPer2 were studied in 2 experiments. In experiment 1, mCry1(-/-) and mCry2(-/-) mice as well as mPer1(Brdm1) and mPer2(Brdm1) mutant mice were exposed to 15-min light pulses during the 1st cycle following entrainment, either early (external time [ExT] 20) or late (ExT 4) in the subjective night. In experiment 2, a full PRC was measured for all these strains by exposure to light pulses of the same duration and intensity in free-running conditions in constant darkness. Directly after entrainment (experiment 1), mPer1(Brdm1) animals did not show significant phase advances by a light pulse in the late subjective night (ExT 4), as in the study by Albrecht et al. In the same experiment, mPer2(Brdm1) mice became arrhythmic too frequently to reliably measure their phase responses. Mice with a targeted gene disruption in mCry1 or mCry2 showed increased phase delays compared to wild type after exposure to a light pulse in the early subjective night (ExT 20). Otherwise, phase shifts were not significantly affected. In free run (experiment 2), all genotypes did show phase advances and phase delays. The mPer2(Brdm1) mutant PRC was above the mPer1(Brdm1) mutant and wild-type PRC (i.e., less delayed and more advanced) at most circadian phases. The mPer1(Brdm1) mutant PRC was not distinguishable from the wildtype PRC. The mCry2(-/-) mice showed much smaller phase delays than did mCry1(-/-) mice in the subjective evening (delay phase). In general, mPer2(Brdm1) mutant mice were more accelerated by light compared to mPer1(Brdm1) and wildtype control mice, whereas mCry1(-/-) mice were more delayed by light than were mCry2(-/-) mice.

Late swallows flew over the silent black crowd gathered on a Bavarian hil ls ide. They were paying their respects to a great ornithologist. With the passing of Ebo Gwinner last September, the study of biological rhythms suddenly lost one of its eminent scientists. Gwinner contributed to our field in a unique manner. Almost single-handedly, he created and consistently pursued the study of circannual rhythms. Ted Pengelley and he detected free-running circannual rhythms simultaneously and independently in the 1960s—Pengelley in squirrel hibernation, Gwinner in songbirds. Ever since, Ebo’s life was dedicated to the understanding of temporal organization in birds. He studied endogenous annual programs, their entrainment by photoperiodic and other cues, the control of annual long-distance migration and the adaptive nature of annual phenomena, as well as avian circadian clocks. Ebo was both a keen field naturalist and a thorough experimentalist. He had a vast knowledge of birds across the globe. He knew their natural habits and habitats. His deep interest and insight provided the necessary basis for successfully keeping, breeding, and experimenting with individual birds in captivity over many years in an unprecedented manner. Unusual institutional support and facilities are required for studying such slow and long processes, in which some experiments can last up to 10 years. The Max Planck Society provided the stable long-term environment for research hardly feasible in more ephemeral university settings. Gwinner first did his PhD thesis on the social behavior of ravens, guided by Gustav Kramer and Konrad Lorenz of the Max Planck Institute of Behavioral Physiology. He then started in 1964 as a postdoc in Jürgen Aschoff ’s department of the same institute in Andechs. This remained his home base from which he ventured abroad to work with leading scientists such as Don Farner in Seattle and Colin Pittendrigh in Stanford, to undertake field projects in Zaire, in Kenya, in Tanzania, in Kazakhstan, in the Galapagos, and so forth. The Arctic was to be his next target. Eventually, in 1991, he became director of the institute, recently transformed under his leadership into the new Institute of Ornithology. To his students, Ebo was an inspiring and warmly engaged mentor, who set an example as a dedicated and hardworking scientist. Many graduate students worked under his guidance in a plethora of fields ranging from biological rhythms to migration, behavioral endocrinology, and behavioral ecology. He infused them with his enthusiasm for science, nature, and birds; taught them thorough experimentation; and patiently guided them during manuscript and talk preparations. Despite his heavy administrative

The suprachiasmatic nucleus (SCN) is engaged in modulation of memory retention after (fear) conditioning, but it is unknown which pathways and neurotransmitter system(s) play a role in this action. Here we examine immunocytochemically whether muscarinic acetylcholine receptors (mAChRs), mediating cholinergic signal transduction in the SCN, are involved. For this purpose, mAChR immunoreactivity (mAChR‐ir) was studied in the SCN after various stages of passive shock avoidance (PSA) and active shock avoidance (ASA) training and, for ASA, at various posttraining time points. mAChR‐ir was significantly enhanced in SCN neurons as a result of the training procedure, and the number of mAChR‐positive glial cells in the SCN increased significantly. The increase in mAChR‐ir as a result of PSA and ASA training was not due to fear conditioning or the number of correct avoidances (in case of ASA training) but rather to behavioral arousal as a consequence of (brief) exposure to a novel environment (the test apparatus). This finding was confirmed by a cage‐change experiment in which the rats were allowed to stay in a novel cage for 15 min or 24 hr. Only the brief exposure to the fresh cage triggered alterations for SCN mAChRs 24 hr later. These results shed new light on a possible function of the cholinergic system in the SCN mediated by mAChRs in relation to modulation of memory processes and demonstrate that behavioral arousal during (the habituation stage of) a learning task is sufficient to alter the mAChR system in the SCN. © 2004 Wiley‐Liss, Inc.

Treatment of female zebra finches (Taeniopygia guttata) with 17-beta-estradiol leads to a female-biased sex ratio in their offspring at the age of independence [Horm. Behav. 35 (1999) 135]. It is unclear whether this is due to a bias of the primary sex ratio or to sex-specific survival. We replicated this experiment and found again a significantly higher total number of daughters than sons at independence in the estradiol-treated group. This was due to higher embryonic survival of daughters compared with sons in the estradiol-treated group and the reverse in the control group. There was no effect of the hormone treatment on the primary sex ratio. Treatment with 17-beta-estradiol led to a significantly shorter hatching time and to heavier offspring at day 7 after hatching. This weight was correlated with maternal plasma estradiol levels on the day of the first egg, which were significantly higher in the estradiol-treated group than in the control group. The results do not support the idea that maternal estradiol levels influence the primary sex ratio. They indicate that maternal estradiol differentially affects survival of sons and daughters via an influence on the embryonic environment, possibly enhancing offspring growth. beta 2004 Elsevier Inc. All rights reserved.

In this study we tested the hypothesis that in a passerine bird (great tit, Parus major) individuals differing for coping strategies differ in the magnitude of the adrenocortical response to social stress as well. Furthermore, we aimed at characterizing daily rhythms in corticosteroid release before and after social stress. We used 16 males from either of two lines bidirectionally selected for different coping strategies (fast and slow explorers). Social stress was induced by confrontation with an aggressive resident male. Corticosteroid metabolites were analyzed in feces collected at 90-min intervals from 900 to 1630 h on a baseline day, on the day of the social conflict, and on the following day. In both days and in both lines levels varied with time of day in a robust rhythm with a peak in the first sample of the morning and a trough at the end of the light phase. This rhythm correlates with activity (perch hopping). An overall increase in levels relative to baseline day was observed between 30 and 140 min after the challenge. Birds of the less aggressive and more cautious line (slow explorers) showed a trend for a higher response compared to birds of the more aggressive and bolder line (fast explorers), which showed almost no response. On the day after the challenge the birds of the slow line exhibited significantly reduced corticosteroid secretion, probably due to an increased negative feedback. The results provide evidence for a physiological basis of different coping strategies in birds, emerging in response to social stress and with a pattern similar to that in other vertebrates.