Innate Attractiveness and Associative Learnability of Odors Can Be Dissociated in Larval Drosophila

We investigate olfactory associative learning in larval Drosophila. A reciprocal training design is used, such that one group of animals receives a reward in the presence of odor X but not in the presence of odor Y (Train: X+ // Y), whereas another group is trained reciprocally (Train: X // Y+). After training, differences in odor preference between these reciprocally trained groups in a choice test (Test: X -- Y) reflect associative learning. The current study, after showing which odor pairs can be used for such learning experiments, 1) introduces a one-odor version of such reciprocal paradigm that allows estimating the learnability of single odors. Regarding this reciprocal one-odor paradigm, we show that 2) paired presentations of an odor with a reward increase odor preference above baseline, whereas unpaired presentations of odor and reward decrease odor preference below baseline; this suggests that odors can become predictive either of reward or of reward absence. Furthermore, we show that 3) innate attractiveness and associative learnability can be dissociated. These data deepen our understanding of odor-reward learning in larval Drosophila on the behavioral level, and thus foster its neurogenetic analysis

Circadian Desynchrony Promotes Metabolic Disruption in a Mouse Model of Shiftwork

Shiftwork is associated with adverse metabolic pathophysiology, and the rising incidence of shiftwork in modern societies is thought to contribute to the worldwide increase in obesity and metabolic syndrome. The underlying mechanisms are largely unknown, but may involve direct physiological effects of nocturnal light exposure, or indirect consequences of perturbed endogenous circadian clocks. This study employs a two-week paradigm in mice to model the early molecular and physiological effects of shiftwork. Two weeks of timed sleep restriction has moderate effects on diurnal activity patterns, feeding behavior, and clock gene regulation in the circadian pacemaker of the suprachiasmatic nucleus. In contrast, microarray analyses reveal global disruption of diurnal liver transcriptome rhythms, enriched for pathways involved in glucose and lipid metabolism and correlating with first indications of altered metabolism. Although altered food timing itself is not sufficient to provoke these effects, stabilizing peripheral clocks by timed food access can restore molecular rhythms and metabolic function under sleep restriction conditions. This study suggests that peripheral circadian desynchrony marks an early event in the metabolic disruption associated with chronic shiftwork. Thus, strengthening the peripheral circadian system by minimizing food intake during night shifts may counteract the adverse physiological consequences frequently observed in human shift workers

Genetische Manipulation des zentralen Schrittmachers im suprachiasmatischen Nucleus

Alles Leben dieser Erde ist dem täglich wiederkehrenden Wechsel von Tag und Nacht ausgesetzt. Um sich diesen Ryhtmen anzupassen, haben fast alle Lebewesen, vom Bakterium bis zum Menschen, zirkadiane Uhren entwickelt. Mittels solcher interner Zeitmesser können Lebewesen diese Rhythmen antizipieren und ihre Physiologie optimal anpassen. Der erste Teil dieser Arbeit beschreibt die Herstellung und Validierung eines neuen transgenen Mausmodels, in dem die zentrale zirkadiane Uhr ausgeschaltet wurde. Es ist allgemein anerkannt, dass das zirkadiane System von Säugern hierarchisch organisiert ist, mit einem zentralen Schrittmacher im Nucleus Suprachiasmaticus (SCN) des Hypothalamus und untergeordneten peripheren Uhren. Dieses Modell beruht auf SCN-Läsionsstudien, die zeigen, dass in Abwesenheit des SCN alle peripheren Uhren arrhythmisch werden. Der Nachteil von Läsionen ist, dass gleichermaßen afferente und efferente Verbindungen durchtrennt werden. In dieser Arbeit wurde daher ein genetisches Modell genutzt. Eine SCN-Cre-Treiberlinie (Syt10Cre) wurde hergestellt, in der gezeigt werden konnte, dass Cre im Großteil der SCN-Zellen aktiv ist, nicht aber in peripheren Geweben mit Ausnahme des Testis. Zur funktionellen Charakterisierung wurde mittels der Syt10Cre-Linie das essentielle Uhrengen Bmal1 deletiert (Bmal1fl/fl). Die Laufradaktivität von Syt10Cre Bmal1fl-Mäusen wurde unter Licht-Dunkel (LD) Bedingungen und in konstanter Dunkelheit (DD) untersucht. Es konnte ein Dosis-abhängiger Phänotyp gefunden werden: je weniger Zellen im SCN noch BMAL1-Protein exprimierten, desto weniger rhythmisch war die Laufradaktivität. Syt10Cre/Cre Bmal1fl/--Tiere wurden in konstanter Dunkelheit komplett arrhythmisch, und zeigten denselben Phänotyp wie Mäuse, in denen das Bmal1 Gen in allen Zellen deletiert ist (Bmal1-/-), was die Effizienz der genetischen Manipulation bestätigt. Die Syt10Cre-Treiberlinie wird hilfreich sein, um die Komplexität des zirkadianen Systems von Säugern zu untersuchen. Im zweiten Teil der Arbeit wurde die hierarchische Organisation des zirkadianen Systems an Hand des beschriebenen Syt10Cre/Cre Bmal1fl/--Mausmodels untersucht. Im Gegensatz zu den Ergebnissen der oben beschriebenen SCN-Läsionsstudien wurde gefunden, dass Syt10Cre/Cre Bmal1fl/--Mäuse unter LD-Bedingungen rhythmische Aktivität zeigen. Es konnte nachgewiesen werden, dass diese rhythmische Aktivität direkt Licht- und nicht Uhren-gesteuert ist. Außerdem wurde auch auf molekularer Ebene gezeigt, dass die Uhr im SCN komplett defekt ist. Trotz nicht funktioneller SCN-Uhr wurden unter LD-Bedingungen Glukokortikoide rhythmisch von der Nebenniere sezerniert. Dieser Glukokortikoidrhythmus war im Vergleich zu dem von Wildtyp-Tieren allerdings deutlich gedämpft. Daraus lässt sich ableiten, dass die Uhr im SCN für die rhythmische Glukokortikoidsekretion zwar nicht notwendig ist, aber an der Regulierung der Amplitude des Rhythmus beteiligt ist. Entlässt man diese Tiere in konstante Dunkelheit schwächt sich der Glukokortikoidrhythmus immer weiter ab, was darauf hinweist, dass in konstanter Dunkelheit die Uhr im SCN notwendig ist, um einen Glukokortikoidrhythmus aufrechtzuerhalten. Des Weiteren wurden unter LD-Bedingungen Uhrengene in peripheren Organen von Syt10Cre/Cre Bmal1fl/- Mäusen gemessen. Überraschend war, dass periphere Uhren in Syt10Cre/Cre Bmal1fl/- Mäusen in LD rhythmisch waren, was darauf hindeutet, dass der SCN als Gewebe zwar notwendig ist um periphere Uhren zu synchronisieren, die SCN-Uhr per se aber nicht. Sobald die Tiere in konstante Dunkelheit entlassen wurden, konnte eine deutliche Dämpfung der Rhythmen in peripheren Uhren gemessen werden. Einige Uhrengene waren sogar komplett arrhythmisch exprimiert, was die hierarchische Struktur des zirkadianen Systems unter konstanten Bedingungen bestätigt. Im dritten Teil dieser Arbeit werden die ersten Schritte zur Entwicklung eines kombinatorischen Cre-Systems beschrieben. Dieses kombinatorische Cre-System soll sehr spezifische genetische Manipulationen im Mausmodell ermöglichen. Die Idee basiert auf der Komplementierung zweier Cre-Teilproteine, die einzeln nicht aktiv sind. Sind sie aber in einer Zelle co-exprimiert, wird funktionelles Cre-Protein wiederhergestellt. Indem man beide Teile unter der Kontrolle verschiedener, aber überlappender Promotoren exprimiert, kann man ein deutlich spezifischeres Cre-Expressionsmuster erreichen, als es bisher möglich war. Ziel ist es, die funktionelle Anatomie des SCNs zu untersuchen, indem man die sogenannte „Core“-Region des SCN genetisch läsioniert. Es wird sehr interessant sein, die Effekte einer solchen genetischen SCN-Core Läsion auf das zirkadiane System zu untersuchen. Dieses Projekt wird dazu beitragen, die funktionelle Anatomie dieses komplexen Nukleus besser zu verstehen. Der vierte Teil dieser Arbeit beschäftigt sich mit der Rolle der zirkadianen Uhr als Vermittler zwischen verschiedenen physiologischen Systemen. Es ist bekannt, dass die zirkadiane Uhr und die metabolische Regulation eng miteinander verknüpft sind. Außerdem ist eine kürzere Schlafdauer mit metabolischen Dysfunktionen assoziiert, wie zum Beispiel einer erhöhten Wahrscheinlichkeit von Fettleibigkeit. Eine Möglichkeit ist, dass die metabolischen Effekte von Schlafentzug durch die zirkadiane Uhr vermittelt werden. Daher war die Hypothese dieser Studie, dass in Abwesenheit einer funktionellen zirkadianen Uhr die metabolischen Auswirkungen von Schlafentzug reduziert sind. Um dies zu testen, wurden transgene Mäuse untersucht, die keine funktionelle Uhr besitzen (Per1/2-Doppelmutanten). Es konnte gezeigt werden, dass Schlafentzug in Per1/2-Doppelmutanten tatsächlich weniger metabolische Auswirkungen hat, was nahelegt, dass die zirkadiane Uhr an der Vermittlung dieser Effekte beteiligt ist. Diese Studie gibt erste Hinweise, dass die zirkadiane Uhr möglicherweise ein wichtiger Integrator für Schlaf und Metabolismus ist. Außerdem ist sie klinisch relevant, da Schlafentzug und Fettleibigkeit essentielle Probleme unserer industrialisierten Gesellschaft sind. Ein besseres Verständnis der zugrunde liegenden Mechanismen kann zur Entwicklung neuer pharmakologischer Ansätze beitragen.Circadian timing systems evolved in most organisms to adapt to the daily alternation of light and dark and the accompanying changes of environmental parameters such as food availability and predator occurrence. Circadian clocks allow organisms to anticipate these changes, which in turn increases their evolutionary fitness. The first part of this thesis describes the generation and validation of a new genetic mouse model of central clock disruption. It is assumed, that the mammalian circadian timing system is hierarchically organized with a master pacemaker in the suprachiasmatic nucleus (SCN) of the hypothalamus and subordinated clocks in peripheral tissues. This view is based on SCN ablation studies showing that in the absence of the SCN all peripheral clocks become arrhythmic. An obvious flaw of SCN ablations is the concomitant scission of SCN afferents and efferents. Here we use a genetic model of SCN clock disruption. We generated a SCN Cre driver mouse line (Syt10Cre) and demonstrated strong Cre activity in the majority of SCN cells, but none in peripheral tissues with the exception of the testis. The Syt10Cre driver line was functionally validated by deleting a conditional allele of the essential clock gene Bmal1 (Bmal1fl/fl). Syt10Cre Bmal1fl mice were analyzed for their wheel running behavior in light dark (LD) and constant darkness (DD) conditions. We found a dose-dependent phenotype ranging from minor changes in period and amplitude in Syt10Cre/+ Bmal1fl/fl to markedly reduced rhythmicity in Syt10Cre/+ Bmal1fl/- mice to a complete loss of circadian rhythmicity in Syt10Cre/Cre Bmal1fl/- mice. The behavioral phenotype of Syt10Cre/Cre Bmal1fl/- mice was indistinguishable from the phenotype of Bmal1-/- mice, confirming the efficiency of SCN disruption. Furthermore the behavioral phenotype of Syt10Cre Bmal1fl mice was inversely correlated with the number of BMAL1 positive cells in the SCN. The Syt10Cre line will be a helpful tool to investigate the complexity of the mammalian circadian network. In the second part of this thesis Syt10Cre/Cre Bmal1fl/- mice are used to investigate the hierarchical structure of the mammalian circadian timing system. A major difference to the above-mentioned SCN ablation studies is that Syt10Cre/Cre Bmal1fl/- mice still show rhythmic wheel running behavior in LD conditions. We showed that this behavioral synchronization to the LD cycle is directly light- and not clock-driven. The disruption of the SCN clock on the molecular level was confirmed by in situ hybridization. Despite the absence of a functional SCN clock, corticosterone is rhythmic in Syt10Cre/Cre Bmal1fl/- mice in LD. However the amplitude of this rhythm is markedly reduced, indicating that although the SCN clock is dispensable for rhythmic release of corticosterone in LD, the amplitude of this rhythm is regulated by the SCN clock. Upon placing the mutant animals into DD, corticosterone rhythms dampen, indicating that in DD the SCN clock is essential for maintenance of corticosterone rhythmicity. Furthermore we analyze clock gene expression rhythms in peripheral tissues of Syt10Cre/Cre Bmal1fl/- mice in LD and DD. To our surprise, peripheral clocks continue to oscillate in Syt10Cre/Cre Bmal1fl/- mice in LD. This indicates that a functional clock in the SCN is not necessary for synchronized rhythmicity of peripheral clocks, but that light can directly synchronize peripheral clocks. Upon release into DD, however, clock gene rhythms in peripheral organs dampen. Thus in constant conditions the SCN clock is necessary to synchronize peripheral clocks. The third part of this thesis describes the first attempts towards the development of a combinatorial Cre system, which will allow very specific targeting of brain nuclei. The idea is based on complementation of two parts of the Cre enzyme, which individually are not active. If co-expressed in one cell, however, functional Cre is reconstituted. Expression of the two Cre parts under the control of different but overlapping promoters will markedly improve the specificity of the conventional Cre/loxP system. We aimed at investigating the functional anatomy of the SCN by targeting the SCN-core region which is believed to be the input region of the SCN. This SCN-core Cre line will then be used to set very specific genetic lesions to this SCN sub-region. It will be very interesting to analyze the effects of this SCN-core ablation on circadian output, which will help to elucidate the functional anatomy of this complex brain nucleus. The fourth part of this thesis addresses the role of the circadian clock in integrating different physiological systems. It has been shown that the circadian clock and metabolic regulation are tightly linked. In addition, sleep curtailment (which is often experienced during shift work) is associated with negative effects on metabolic homeostasis. This prompted us to analyze whether the clock might mediate the negative effects of sleep disturbance on metabolic regulation. Our hypothesis was that in the absence of a functional clock metabolic effects of sleep disruption should be less pronounced. We were able to show that arrhythmic Per1/2 mutants which lack a functional circadian clock indeed show less metabolic alterations after sleep disturbance, indicating that the circadian clock is involved in regulating this response. This study suggests that the circadian clock integrates sleep and metabolic regulation. Thus our work may help to understand the effects of sleep curtailment on obesity, which are major problems of industrialized societies. Deepening the understanding of the underlying mechanisms could contribute to the development of new pharmaceutical approaches for these disorders

Tissue-specific dissociation of diurnal transcriptome rhythms during sleep restriction in mice

Study objectives: Shortened or mistimed sleep affects metabolic homeostasis, which may in part be mediated by dysregulation of endogenous circadian clocks. In this study, we assessed the contribution of sleep disruption to metabolic dysregulation by analysing diurnal transcriptome regulation in metabolic tissues of mice subjected to a sleep restriction (SR) paradigm. Methods: Male mice were subjected to 2 × 5 days of SR with enforced waking during the first 6 hours of the light phase. SR and control mice were sacrificed at different time points of the day and RNA preparations from the mediobasal hypothalamus (MBH), liver, and epididymal white adipose tissue (eWAT) were subjected to whole-genome microarray hybridization. Transcriptional rhythms were associated with changes in behavioral and physiological parameters such as sleep, body temperature, and food intake. Rhythm detection was performed with CircWave and transcription profiles were compared by 2-way analysis of variance and t-tests with Benjamini-Hochberg corrections. Results: Clock gene rhythms were blunted in all tissues, while transcriptome regulation was associated with either clock gene expression, sleep patterns, or food intake in a tissue-specific manner. Clock gene expression was associated with apoptosis pathways in the MBH and with tumor necrosis factor alpha signalling in liver. Food intake-associated genes included cilium movement genes in the MBH and lipid metabolism-associated transcripts in liver. Conclusions: In mice, repeated SR profoundly alters behavioral and molecular diurnal rhythms, disrupting essential signalling pathways in MBH, liver, and eWAT, which may underlie the metabolic and cognitive disturbances observed in sleep-restricted humans such as shift workers

Circadian Clock Genes <em>Per1</em> and <em>Per2</em> Regulate the Response of Metabolism-Associated Transcripts to Sleep Disruption

<div><p>Human and animal studies demonstrate that short sleep or poor sleep quality, e.g. in night shift workers, promote the development of obesity and diabetes. Effects of sleep disruption on glucose homeostasis and liver physiology are well documented. However, changes in adipokine levels after sleep disruption suggest that adipocytes might be another important peripheral target of sleep. Circadian clocks regulate metabolic homeostasis and clock disruption can result in obesity and the metabolic syndrome. The finding that sleep and clock disruption have very similar metabolic effects prompted us to ask whether the circadian clock machinery may mediate the metabolic consequences of sleep disruption. To test this we analyzed energy homeostasis and adipocyte transcriptome regulation in a mouse model of shift work, in which we prevented mice from sleeping during the first six hours of their normal inactive phase for five consecutive days (<em>timed sleep restriction</em> – TSR). We compared the effects of TSR between wild-type and <em>Per1/2</em> double mutant mice with the prediction that the absence of a circadian clock in <em>Per1/2</em> mutants would result in a blunted metabolic response to TSR. In wild-types, TSR induces significant transcriptional reprogramming of white adipose tissue, suggestive of increased lipogenesis, together with increased secretion of the adipokine leptin and increased food intake, hallmarks of obesity and associated leptin resistance. Some of these changes persist for at least one week after the end of TSR, indicating that even short episodes of sleep disruption can induce prolonged physiological impairments. In contrast, <em>Per1/2</em> deficient mice show blunted effects of TSR on food intake, leptin levels and adipose transcription. We conclude that the absence of a functional clock in <em>Per1/2</em> double mutants protects these mice from TSR-induced metabolic reprogramming, suggesting a role of the circadian timing system in regulating the physiological effects of sleep disruption.</p> </div

TSR-induced transcriptional reprogramming of WAT in wild-type mice.

<p>A) Gene ontology analysis of microarray data of epididymal WAT at ZT18 on the last day of TSR compared to control WAT at ZT18. Only nodes (GO categories) with at least 10 regulated genes are shown. Significant overrepresentation of nodes is highlighted in red. B) Individual normalized log-transformed expression values for all genes involved in lipid metabolic pathways which are regulated at least 2 fold between control and TSR are plotted sorted for fold change. C) Individual normalized log-transformed expression values for all genes involved in glucose metabolic pathways which are regulated at least 2 fold between control and TSR are plotted ordered by fold change. D) Clock gene regulation by TSR in WAT at ZT18 sorted for fold change. Only clock genes with significant expression under control conditions are shown. With the exception of <i>Per2</i> and <i>Rora</i>, all clock genes were significantly regulated by TSR. Green represents low expression, red represents high expression. Sample sizes were 3 per group.</p

Sustained and wild-type-specific induction of lipogenic and glycolytic genes in WAT.

<p>A–J) Expression of glycolytic and lipogenic genes in epididymal WAT at ZT6 and at ZT18 in control conditions, on the last day of TSR and on the 7^th day of recovery for wild-type and <i>Per1/2</i> mutant animals. Expression values are normalized to the mean of the wild-type control group at ZT6. Data are shown as mean ± SEM and data for each ZT are statistically compared using two-way ANOVAs (details are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052983#pone.0052983.s005" target="_blank">Table S1</a>), followed by Bonferroni post-tests, comparing control vs. TSR and control vs. recovery for each genotype. * p<0.05 in post-test. Post-tests comparing genotypes for each condition (control, TSR and recovery) are shown in Suppl. table ST1. K) Schematic overview of WAT glycolysis and lipogenesis pathways. Transcripts which were found to be changed by TSR are highlighted in red. Sample sizes were 3–4 per group.</p

TSR alters diurnal activity profiles, but not food intake or plasma leptin levels in <i>Per1/2</i> mutants.

<p>A) Representative double plotted activity recording during five days of control, five days of TSR and five days of recovery. TSR (ZT0–6) is highlighted by a red rectangle. Light and dark phases are indicated by white and grey boxes, respectively. B) Mean diurnal activity profiles (n = 5) were generated by plotting the relative locomotor activity for every 30 min bin as percentage of total daily activity. Light and dark phases are indicated in white and grey, respectively. Data are plotted as mean ± SEM (dotted lines). C) Relative activity during the second half of the night (ZT 18–24). Activity is expressed relative to the average activity during the same time in the control week (in %). *: p<0.001 control vs. TSR, two-way ANOVA with Bonferroni post-test, see also Suppl. Table ST1. D) Food intake during one day of control, during the last day of TSR and during the 7^th day of recovery. Two-way ANOVA with Bonferroni post-test not significant, see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052983#pone.0052983.s005" target="_blank">Table S1</a>. E) Body weight gain per day of control, TSR and recovery. *: p<0.001 control vs. TSR, p<0.05 control vs. recovery, two-way ANOVA with Bonferroni post-test, see also Suppl. Table ST1. F) Plasma leptin levels at ZT18 measured on one day of control, the last day of TSR and the 7^th day of recovery. Two-way ANOVA with Bonferroni post-test, not significant, see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052983#pone.0052983.s005" target="_blank">Table S1</a>. All data are shown as mean ± SEM. Sample sizes were 5 per group for activity, 4–8 per group for food intake, 17–33 per group for body weight and 4–5 per group for leptin.</p

Effects of TSR on plasma metabolite levels in wild-types and <i>Per1/2</i> mutants.

<p>Plasma metabolites were measured at ZT6 and ZT18 in control conditions, on the last day of TSR and on the 7^th day of recovery for wild-type and <i>Per1/2</i> mutant animals. Data are shown as mean +/− SEM. Sample sizes were 3–5 per group.</p>*<p>: p<0.05 compared to control conditions in the same genotype,</p>#<p>: p<0.05 compared to wild-type in the same condition using 2-way ANOVA and Bonferroni post-tests. Statistical details are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052983#pone.0052983.s005" target="_blank">Table S1</a>. NEFAs: non-esterified fatty acids.</p