Estrogen-mediated coupling via gap junctions in the suprachiasmatic nucleus.

The circadian clock orchestrates many physiological and behavioural rhythms in mammals with 24-h periodicity, through a hierarchical organisation, with the central clock located in the suprachiasmatic nucleus (SCN) in the hypothalamus. The circuits of the SCN generate circadian rhythms with precision, relying on intrinsic coupling mechanisms, for example, neurotransmitters like arginine vasopressin (AVP), vasoactive intestinal peptide (VIP), neuronal gamma-aminobutyric acid (GABA) signalling and astrocytes connected by gap junctions composed of connexins (Cx). In female rodents, the presence of estrogen receptors (ERs) in the dorsal SCN suggests an influence of estrogen (E2) on the circuit timekeeping that could regulate circadian rhythm and coupling. To investigate this, we used SCN explants together with hypothalamic neurons and astrocytes. First, we showed that E2 stabilised the circadian amplitude in the SCN when rAVPs (receptor-associated vasopressin peptides) were inhibited. However, the phase delay induced by VIPAC2 (VIP receptors) inhibition remained unaffected by E2. We then showed that E2 exerted its effects in the SCN via ERß (estrogen receptor beta), resulting in increased expression of Cx36 and Cx43. Notably, specific inhibition of both connexins resulted in a significant reduction in circadian amplitude within the SCN. Remarkably, E2 restored the period with inhibited Cx36 but not with Cx43 inhibition. This implies that the network between astrocytes and neurons, responsible for coupling in the SCN, can be reinforced through E2. In conclusion, these findings provide new insights into how E2 regulates circadian rhythms ex vivo in an ERß-dependent manner, underscoring its crucial role in fortifying the SCN's rhythm.

The trophoblast clock controls transport across placenta in mice.

In mammals, 24-h rhythms of physiology and behavior are organized by a body-wide network of clock genes and proteins. Despite the well-known function of the adult circadian system, the roles of maternal, fetal and placental clocks during pregnancy are poorly defined. In the mature mouse placenta, the labyrinth zone (LZ) is of fetal origin and key for selective nutrient and waste exchange. Recently, clock gene expression has been detected in LZ and other fetal tissues; however, there is no evidence of a placental function controlled by the LZ clock. Here, we demonstrate that specifically the trophoblast layer of the LZ harbors an already functional clock by late gestation, able to regulate in a circadian manner the expression and activity of the xenobiotic efflux pump, ATP-binding cassette sub-family B member 1 (ABCB1), likely gating the fetal exposure to drugs from the maternal circulation to certain times of the day. As more than 300 endogenous and exogenous compounds are substrates of ABCB1, our results might have implications in choosing the maternal treatment time when aiming either maximal/minimal drug availability to the fetus/mother.

Astrocytes as essential time-keepers of the central pacemaker.

Since the early observations made by Santiago Ramon y Cajal more than a century ago till now, astrocytes have gradually gained protagonism as essential partners of neurons in building brain circuits that regulate complex behavior. In mammals, processes such as sleep-wake cycle, locomotor activity, cognition and memory consolidation, homeostatic and hedonic appetite and stress response (among others), are synchronized in 24-h rhythms by the circadian system. In such a way, physiology efficiently anticipates and adapts to daily recurring changes in the environment. The hypothalamic suprachiasmatic nucleus (SCN) is considered the central pacemaker, it has been traditionally described as a nucleus of around 10,000 neurons nearly all GABAergic able to be entrained by light and to convey time information through multiple neuronal and hormonal pathways. Only recently, this neuro-centered view was challenged by breakthrough discoveries implicating astrocytes as essential time-keepers. In the present review, we will describe the current view on the SCN circuit and discuss whether astrocytic functions described in other brain regions and state-of-the-art experimental approaches, could help explaining better those well- and not so well-known features of the central pacemaker.

Pro-apoptotic effects of low doses of dimethoate in rat brain.

Dimethoate (DMT), a widely used Organophosphorous insecticide, was administered for 5 weeks (sub-chronic) at low dose (15 mg/kg b.w.) to male Wistar rats with the aim to simulate potential exposure to pesticide residues in food and water. The induction of cell death programs was investigated in two brain regions, cortex (Cx) and substantia nigra (SN), after the exposure period. We found that DMT increased cytochrome C (CytC) release from mitochondria, the Bax/Bcl-2 ratio, the activity of caspase-3 and calpains, in both brain regions compared to VEH injected ones. DMT treatment induced oxidative damage of lipids with a consequent enrichment in saturated over unsaturated fatty acids. However, the activity of mitochondrial respiratory complexes was not affected by DMT treatment. The activation of the pro-apoptotic pathway can be correlated with a decrease of TH-immunoreactive neurons in SN, comparable to the reduction observed in this cell population by aging. The results of this work contribute to understand the toxic mechanism of DMT and the possible etiological role that residues of this insecticide, might play in neurodegenerative diseases.

Mechanisms of Communication in the Mammalian Circadian Timing System.

24-hour rhythms in physiology and behaviour are organized by a body-wide network of endogenous circadian clocks. In mammals, a central pacemaker in the hypothalamic suprachiasmatic nucleus (SCN) integrates external light information to adapt cellular clocks in all tissues and organs to the external light-dark cycle. Together, central and peripheral clocks co-regulate physiological rhythms and functions. In this review, we outline the current knowledge about the routes of communication between the environment, the main pacemakers and the downstream clocks in the body, focusing on what we currently know and what we still need to understand about the communication mechanisms by which centrally and peripherally controlled timing signals coordinate physiological functions and behaviour. We highlight recent findings that shed new light on the internal organization and function of the SCN and neuroendocrine mechanisms mediating clock-to-clock coupling. These findings have implications for our understanding of circadian network entrainment and for potential manipulations of the circadian clock system in therapeutic settings.

Perinatal Programming of Circadian Clock-Stress Crosstalk.

An intact communication between circadian clocks and the stress system is important for maintaining physiological homeostasis under resting conditions and in response to external stimuli. There is accumulating evidence for a reciprocal interaction between both-from the systemic to the molecular level. Disruption of this interaction by external factors such as shiftwork, jetlag, or chronic stress increases the risk of developing metabolic, immune, or mood disorders. From experiments in rodents, we know that both systems maturate during the perinatal period. During that time, exogenous factors such as stress or alterations in the external photoperiod may critically affect-or program-physiological functions later in life. This developmental programming process has been attributed to maternal stress signals reaching the embryo, which lastingly change gene expression through the induction of epigenetic mechanisms. Despite the well-known function of the adult circadian system in temporal coordination of physiology and behavior, the role of maternal and embryonic circadian clocks during pregnancy and postnatal development is still poorly defined. A better understanding of the circadian-stress crosstalk at different periods of development may help to improve stress resistance and devise preventive and therapeutic strategies against chronic stress-associated disorders.

Short-Term High-Fat Diet Feeding Provides Hypothalamic but Not Hippocampal Protection against Acute Infection in Male Mice.

Obesity is associated with increased fever and sickness behavior in response to infection. The hypothalamic-pituitary-adrenal (HPA) axis plays a key role in the reaction to immune stimuli. Bacterial infection, or bacterial lipopolysaccharide (LPS), induces the expression of peripheral cytokines that stimulate the hypothalamus and the hippocampus and activate the HPA axis. In this study, we explored whether the hypothalamic and hippocampal responses to infection are altered during the development of diet-induced obesity. Male mice were exposed to a high-fat diet (HFD) or a low-fat diet (LFD) for 15 days. They were then administered a single intraperitoneal injection of bacterial LPS or vehicle and sacrificed 24 h later. LPS increased circulating levels of insulin and leptin, but only in LFD animals. LPS induced a significant decrease in hypothalamic corticotrophin-releasing hormone and glucocorticoid receptor mRNA levels in LFD animals but exerted the opposite effect in HFD-fed mice. LPS increased the hypothalamic expression of molecules involved in the leptin signaling pathway (SOCS3 and STAT3), nuclear factor-κB pathway members, inflammatory mediators (tumor necrosis factor-α and interleukin-6) and glial proliferation markers (Emr1 and CD68) in LFD animals. These effects were dampened in HFD-fed mice. In contrast, the hippocampal responses to LPS were largely insensitive to HFD. These results suggest that HFD feeding reduced the inflammatory response induced by LPS in the hypothalamus but not in the hippocampus.

Ontogeny of the circadian system: a multiscale process throughout development.

The 24 h (circadian) timing system develops in mammals during the perinatal period. It carries out the essential task of anticipating daily recurring environmental changes to identify the best time of day for each molecular, cellular, and systemic process. Although significant knowledge has been acquired about the organization and function of the adult circadian system, relatively little is known about its ontogeny. During the perinatal period, the circadian system progressively gains functionality under the influence of the early environment. This review explores current evidence on the development of the circadian clock in mammals, highlighting the multilevel complexity of the process and the importance of gaining a better understanding of its underlying biology.

Sub-chronic exposure to the insecticide dimethoate induces a proinflammatory status and enhances the neuroinflammatory response to bacterial lypopolysaccharide in the hippocampus and striatum of male mice.

Dimethoate is an organophosphorus insecticide extensively used in horticulture. Previous studies have shown that the administration of dimethoate to male rats, at a very low dose and during a sub-chronic period, increases the oxidation of lipids and proteins, reduces the levels of antioxidants and impairs mitochondrial function in various brain regions. In this study, we have assessed in C57Bl/6 adult male mice, whether sub-chronic (5weeks) intoxication with a low dose of dimethoate (1.4mg/kg) affects the expression of inflammatory molecules and the reactivity of microglia in the hippocampus and striatum under basal conditions and after an immune challenge caused by the systemic administration of lipopolysaccharide. Dimethoate increased mRNA levels of tumor necrosis factor α (TNFα) and interleukin (IL) 6 in the hippocampus, and increased the proportion of Iba1 immunoreactive cells with reactive phenotype in dentate gyrus and striatum. Lipopolysaccharide caused a significant increase in the mRNA levels of IL1ß, TNFα, IL6 and interferon-γ-inducible protein 10, and a significant increase in the proportion of microglia with reactive phenotype in the hippocampus and the striatum. Some of the effects of lipopolysaccharide (proportion of Iba1 immunoreactive cells with reactive phenotype and IL6 mRNA levels) were amplified in the animals treated with dimethoate, but only in the striatum. These findings indicate that a sub-chronic period of administration of a low dose of dimethoate, comparable to the levels of the pesticide present as residues in food, causes a proinflammatory status in the brain and enhances the neuroinflammatory response to the lipopolysaccharide challenge with regional specificity.

Prenatal stress increases the expression of proinflammatory cytokines and exacerbates the inflammatory response to LPS in the hippocampal formation of adult male mice.

Early life experiences, such as prenatal stress, may result in permanent alterations in the function of the nervous and immune systems. In this study we have assessed whether prenatal stress affects the inflammatory response of the hippocampal formation of male mice to an inflammatory challenge during adulthood. Pregnant C57BL/6 mice were randomly assigned to stress (n=10) or non-stress (n=10) groups. Animals of the stress group were placed in plastic transparent cylinders and exposed to bright light for 3 sessions of 45min every day from gestational day 12 to parturition. Non-stressed pregnant mice were left undisturbed. At four months of age, non stressed and prenatally stressed male offspring were killed, 24h after the systemic administration of lipopolysaccharide (LPS) or vehicle. Under basal conditions, prenatally stressed animals showed increased expression of interleukin 1ß and tumor necrosis factor-α (TNF-α) in the hippocampus and an increased percentage of microglia cells with reactive morphology in CA1 compared to non-stressed males. Furthermore, prenatally stressed mice showed increased TNF-α immunoreactivity in CA1 and increased number of Iba-1 immunoreactive microglia and GFAP-immunoreactive astrocytes in the dentate gyrus after LPS administration. In contrast, LPS did not induce such changes in non-stressed animals. These findings indicate that prenatal stress induces a basal proinflammatory status in the hippocampal formation during adulthood that results in an enhanced activation of microglia and astrocytes in response to a proinflammatory insult.

Pesticide-induced decrease in rat testicular steroidogenesis is differentially prevented by lipoate and tocopherol.

We have previously demonstrated that the sub-chronic administration of low doses of Toc or α-Toc, glyphosate and zineb to rats (i.p. 1/250 LD50, three times a week for 5 weeks) provoked severe oxidative stress (OS) in testicles. These effects were also reflected in plasma. Lipoic acid (LA) and α-tocopherol are considered as antioxidants due to their ability to neutralize reactive oxygenated species (ROS) and reset endogenous antioxidant levels. To investigate the possible protective effect on reproductive function, LA and Toc (i.p. 25, 50 and 100mg/kg) were administered simultaneously with the pesticide mixture (PM) for 5 weeks. Both drugs prevented OS and the damage to proteins and lipids caused by PM in a dose-dependent manner. The PM-induced increase levels of prostaglandins E2 and F2α was completely restored by LA but not by Toc. Similarly, only LA was able to restore the inhibition of testosterone production, the decrease of 3ß- and 17ß-hydroxysteroid dehydrogenases activities, and the elevation of gonatropins (FSH and LH) levels produced by PM. Furthermore, LA was more efficient than Toc in normalizing the histological alterations produced by PM administration, suggesting that pesticides act though other mechanisms that generate oxidative stress. In our experimental model LA displayed a higher protective role against pesticide-induced damage than that observed by Toc administration. Our results suggest that LA administration is a promising therapeutic strategy for coping with disorders suspected to be caused by OS generators - such as pesticides - in male reproductive system.

Circadian glucocorticoids throughout development.

Glucocorticoids (GCs) are essential drivers of mammalian tissue growth and maturation during one of the most critical developmental windows, the perinatal period. The developing circadian clock is shaped by maternal GCs. GC deficits, excess, or exposure at the wrong time of day leads to persisting effects later in life. During adulthood, GCs are one of the main hormonal outputs of the circadian system, peaking at the beginning of the active phase (i.e., the morning in humans and the evening in nocturnal rodents) and contributing to the coordination of complex functions such as energy metabolism and behavior, across the day. Our article discusses the current knowledge on the development of the circadian system with a focus on the role of GC rhythm. We explore the bidirectional interaction between GCs and clocks at the molecular and systemic levels, discuss the evidence of GC influence on the master clock in the suprachiasmatic nuclei (SCN) of the hypothalamus during development and in the adult system.

Effect of Sublethal Copper Overload on Cholesterol De Novo Synthesis in Undifferentiated Neuronal Cells.

Although copper (Cu) is an essential trace metal for cells, it can induce harmful effects as it participates in the Fenton reaction. Involuntary exposure to Cu overload is much more common than expected and has been linked with neurodegeneration, particularly with Alzheimer's disease (AD) evidenced by a positive correlation between free Cu in plasma and the severity of the disease. It has been suggested that Cu imbalance alters cholesterol (Chol) homeostasis and that high membrane Chol promotes the amyloidogenic processing of the amyloid precursor protein (APP) secreting the ß-amyloid (Aß) peptide. Despite the wide knowledge on the effects of Cu in mature brain metabolism, the consequence of its overload on immature neurons remains unknown. Therefore, we used an undifferentiated human neuroblastoma cell line (SH-SY5Y) to analyze the effect of sublethal concentrations of Cu on 1- de novo Chol synthesis and membrane distribution; 2-APP levels in cells and its distribution in membrane rafts; 3-the levels of Aß in the culture medium. Our results demonstrated that Cu increases reactive oxygen species (ROS) and favors Chol de novo synthesis in both ROS-dependent and independent manners. Also, at least part of these effects was due to the activation of 3-hydroxy-3-methyl glutaryl CoA reductase (HMGCR). In addition, Cu increases the Chol/PL ratio in the cellular membranes, specifically Chol content in membrane rafts. We found no changes in total APP cell levels; however, its presence in membrane rafts increases with the consequent increase of Aß in the culture medium. We conclude that Cu overload favors Chol de novo synthesis in both ROS-dependent and independent manners, being at least in part, responsible for the high Chol levels found in the cell membrane and membrane rafts. These may promote the redistribution of APP into the rafts, favoring the amyloidogenic processing of this protein and increasing the levels of Aß.

Clock Genes Profiles as Diagnostic Tool in (Childhood) ADHD-A Pilot Study.

Attention deficit hyperactivity disorder (ADHD) is a very common disorder in children and adults. A connection with sleep disorders, and above all, disorders of the circadian rhythm are the subject of research and debate. The circadian system can be represented on different levels. There have been a variety of studies examining 24-h rhythms at the behavioral and endocrine level. At the molecular level, these rhythms are based on a series of feedback loops of core clock genes and proteins. In this paper, we compared the circadian rhythms at the behavioral, endocrine, and molecular levels between children with ADHD and age- and BMI-matched controls, complementing the previous data in adults. In a minimally invasive setting, sleep was assessed via a questionnaire, actigraphy was used to determine the motor activity and light exposure, saliva samples were taken to assess the 24-h profiles of cortisol and melatonin, and buccal mucosa swaps were taken to assess the expression of the clock genes BMAL1 and PER2. We found significant group differences in sleep onset and sleep duration, cortisol secretion profiles, and in the expression of both clock genes. Our data suggest that the analysis of circadian molecular rhythms may provide a new approach for diagnosing ADHD in children and adults.

A Timely Administration of Antenatal Steroids Is Highly Protective Against Intraventricular Hemorrhage: An Observational Multicenter Cohort Study of Very Low Birth Weight Infants.

Aim: The aim of the study is to evaluate the influence of the timing of antenatal steroids (ANSs) on neonatal outcome of very low birth weight infants (VLBWI) born before 30 weeks of gestation in the German Neonatal Network. Methods: The German Neonatal Network is a large population-based cohort study enrolling VLBWIs since 2009. We included 672 neonates, who were born between January 1, 2009 and December 31, 2019 in our analysis in 10 selected centers. Infants were divided into four subgroups based on the interval between the first steroid administration and preterm birth: (I) two doses of betamethasone, ANS-birth interval: >24 h to 7 days, n = 187, (II) only one dose of betamethasone, ANS-birth interval 0-24 h, n = 70, (III) two doses of betamethasone, ANS-birth interval >7 days, n = 177, and (IV) no antenatal steroids, n = 238. Descriptive statistics and logistic regression analyses were performed for the main neonatal outcome parameters. Group IV (no ANS) was used as a reference. Results: An ANS-birth interval of 24 h to 7 days after the first dose was associated with a reduced risk for intraventricular hemorrhage (OR 0.17; 95% CI 0.09-0.31, p < 0.001) and mechanical ventilation (OR 0.37; 95% CI 0.23-0.61, p < 0.001), whereas the group of infants that only received a single dose of steroids reflected a subgroup at high risk for adverse neonatal outcomes; an ANS-birth interval of >7 days was still associated with a lower risk for intraventricular hemorrhage (OR 0.43; 95% CI 0.25-0.72, p = 0.002) and the need for mechanical ventilation (OR 0.43; 95% CI 0.27-0.71, p = 0.001). Conclusion: Our observational data indicate that an ANS-birth interval of 24 h to 7 days is strongly associated with a reduced risk of intraventricular hemorrhage in VLBWIs. Further research is needed to improve the prediction of preterm birth in order to achieve a timely administration of antenatal steroids that may improve neonatal outcomes such as intraventricular hemorrhage.

Clinical parameters and biomarkers of oxidative stress in agricultural workers who applied copper-based pesticides.

Copper based-pesticides are widely used in agricultural practice throughout the world. We studied the (i) concentration of Cu and proteins involved in Cu homeostasis, (ii) plasma redox status, and (iii) biomarkers of exposure in Cu-based pesticide applicators in order to compare them with clinical biochemical tests. Thirty-one professional applicators and 32 control subjects were recruited. Oxidative stress biomarkers, ceruloplasmin (CRP), metallothioneins (MTs), copper, hematological parameters, and biochemical markers for pancreatic, hepatic and renal function were measured in plasma. Copper was increased in the exposed group compared to the control group concomitantly with TBARS, protein carbonyls, and nitrate+nitrite levels. In the exposed group, α-tocopherol and the FRAP assay were lower and LDH, transaminases, GGT, ALP, urea, creatinine, CRP and MTs were higher than in the control group. The relative leukocyte subclasses were also different between the two groups. Clinical chemistry tests did not surpass the upper reference limit. Our results suggest that the incorporation of oxidative stress biomarkers to biochemical/clinical tests should be considered for validation and included in the human health surveillance protocols.

Feto-Maternal Crosstalk in the Development of the Circadian Clock System.

The circadian (24 h) clock system adapts physiology and behavior to daily recurring changes in the environment. Compared to the extensive knowledge assembled over the last decades on the circadian system in adults, its regulation and function during development is still largely obscure. It has been shown that environmental factors, such as stress or alterations in photoperiod, disrupt maternal neuroendocrine homeostasis and program the offspring's circadian function. However, the process of circadian differentiation cannot be fully dependent on maternal rhythms alone, since circadian rhythms in offspring from mothers lacking a functional clock (due to SCN lesioning or genetic clock deletion) develop normally. This mini-review focuses on recent findings suggesting that the embryo/fetal molecular clock machinery is present and functional in several tissues early during gestation. It is entrained by maternal rhythmic signals crossing the placenta while itself controlling responsiveness to such external factors to certain times of the day. The elucidation of the molecular mechanisms through which maternal, placental and embryo/fetal clocks interact with each other, sense, integrate and coordinate signals from the early life environment is improving our understanding of how the circadian system emerges during development and how it affects physiological resilience against external perturbations during this critical time period.

Sex differences and gonadal hormone regulation of brain cardiolipin, a key mitochondrial phospholipid.

Cardiolipin (CL) is a phospholipid that is almost exclusively located in the inner mitochondrial membrane of eukaryotic cells. As a result of its unique structure and distribution, CL establishes non-covalent bonds with a long list of proteins involved in ATP production, mitochondria biogenesis, mitophagy and apoptosis. Thus, the amount of CL, as well as its fatty acid composition and location, strongly impacts upon mitochondrial-dependent functions and therefore the metabolic homeostasis of different tissues. The brain is particularly sensitive to mitochondrial dysfunction as a result of its high metabolic demand. Several mitochondrial related-neurodegenerative disorders, as well as physiological ageing, show altered CL metabolism. Furthermore, mice lacking enzymes involved in CL synthesis show cognitive impairments. CL content and metabolism are regulated by gonadal hormones in the developing and adult brain. In neuronal cultures, oestradiol increases CL content, whereas adult ovariectomy decreases CL content and alters CL metabolism in the hippocampal mitochondria. Transient sex differences in brain CL metabolism have been detected during development. At birth, brain CL has a higher proportion of unsaturated fatty acids in the brain of male mice than in the brain of females. In addition, the expression of enzymes involved in CL de novo and recycling synthetic pathways is higher in males. Most of these sex differences are abolished by the neonatal androgenisation of females, suggesting a role for testosterone in the generation of sex differences in brain CL. The regulation of brain CL by gonadal hormones may be linked to their homeostatic and protective actions in neural cells, as well as the manifestation of sex differences in neurodegenerative disorders.

The Concept of Coupling in the Mammalian Circadian Clock Network.

The circadian clock network regulates daily rhythms in mammalian physiology and behavior to optimally adapt the organism to the 24-h day/night cycle. A central pacemaker, the hypothalamic suprachiasmatic nucleus (SCN), coordinates subordinate cellular oscillators in the brain, as well as in peripheral organs to align with each other and external time. Stability and coordination of this vast network of cellular oscillators is achieved through different levels of coupling. Although coupling at the molecular level and across the SCN is well established and believed to define its function as pacemaker structure, the notion of coupling in other tissues and across the whole system is less well understood. In this review, we describe the different levels of coupling in the mammalian circadian clock system - from molecules to the whole organism. We highlight recent advances in gaining knowledge of the complex organization and function of circadian network regulation and its significance for the generation of stable but plastic intrinsic 24-h rhythms.

Functional Divergence of Mammalian TFAP2a and TFAP2b Transcription Factors for Bidirectional Sleep Control.

Sleep is a conserved behavioral state. Invertebrates typically show quiet sleep, whereas in mammals, sleep consists of periods of nonrapid-eye-movement sleep (NREMS) and REM sleep (REMS). We previously found that the transcription factor AP-2 promotes sleep in Caenorhabditiselegans and Drosophila In mammals, several paralogous AP-2 transcription factors exist. Sleep-controlling genes are often conserved. However, little is known about how sleep genes evolved from controlling simpler types of sleep to govern complex mammalian sleep. Here, we studied the roles of Tfap2a and Tfap2b in sleep control in mice. Consistent with our results from C. elegans and Drosophila, the AP-2 transcription factors Tfap2a and Tfap2b also control sleep in mice. Surprisingly, however, the two AP-2 paralogs play contrary roles in sleep control. Tfap2a reduction of function causes stronger delta and theta power in both baseline and homeostasis analysis, thus indicating increased sleep quality, but did not affect sleep quantity. By contrast, Tfap2b reduction of function decreased NREM sleep time specifically during the dark phase, reduced NREMS and REMS power, and caused a weaker response to sleep deprivation. Consistent with the observed signatures of decreased sleep quality, stress resistance and memory were impaired in Tfap2b mutant animals. Also, the circadian period was slightly shortened. Taken together, AP-2 transcription factors control sleep behavior also in mice, but the role of the AP-2 genes functionally diversified to allow for a bidirectional control of sleep quality. Divergence of AP-2 transcription factors might perhaps have supported the evolution of more complex types of sleep.