Individual differences and knockout in zebrafish reveal similar cognitive effects of BDNF between teleosts and mammals.

The remarkable similarities in cognitive performance between teleosts and mammals suggest that the underlying cognitive mechanisms might also be similar in these two groups. We tested this hypothesis by assessing the effects of the brain-derived neurotrophic factor (BDNF), which is critical for mammalian cognitive functioning, on fish's cognitive abilities. We found that individual differences in zebrafish's learning abilities were positively correlated with bdnf expression. Moreover, a CRISPR/Cas9 mutant zebrafish line that lacks the BDNF gene (bdnf-/-) showed remarkable learning deficits. Half of the mutants failed a colour discrimination task, whereas the remaining mutants learned the task slowly, taking three times longer than control bdnf+/+ zebrafish. The mutants also took twice as long to acquire a T-maze task compared to control zebrafish and showed difficulties exerting inhibitory control. An analysis of habituation learning revealed that cognitive impairment in mutants emerges early during development, but could be rescued with a synthetic BDNF agonist. Overall, our study indicates that BDNF has a similar activational effect on cognitive performance in zebrafish and in mammals, supporting the idea that its function is conserved in vertebrates.

Medaka as a model for seasonal plasticity: Photoperiod-mediated changes in behaviour, cognition, and hormones.

Teleosts display the highest level of brain plasticity of all vertebrates. Yet we still know little about how seasonality affects fish behaviour and the underlying cognitive mechanisms since the common neurobehavioral fish models are native to tropical environments where seasonal variation is absent or reduced. The medaka, Oryzias latipes, which inhabits temperate zone habitats, represents a promising model in this context given its large phenotypic changes associated with seasonality and the possibility to induce seasonal plasticity by only manipulating photoperiod. Here, we report the first extended investigation of seasonal plasticity in medaka behaviour and cognition, as well as the potential underlying molecular mechanisms. We compared medaka exposed to summer photoperiod (16 h light:8 h dark) with medaka exposed to winter photoperiod (8 h light:16 h dark), and detected substantial differences. Medaka were more active and less social in summer photoperiod conditions, two effects that emerged in the second half of an open-field and a sociability test, respectively, and might be at least in part related to habituation to the testing apparatus. Moreover, the cognitive phenotype was significantly affected: in the early response to a social stimulus, brain functional lateralisation shifted between the two hemispheres under the two photoperiod conditions, and inhibitory and discrimination learning performance were reduced in summer conditions. Finally, the expression of genes encoding key pituitary hormones, tshß and gh, and of the tshß regulatory transcription factor tef in the brain was increased in summer photoperiod conditions. This work reveals remarkable behavioural and cognitive phenotypic plasticity in response to photoperiod in medaka, and suggests a potential regulatory role for the same hormones involved in seasonal plasticity of other vertebrates.

Size-selective mortality induces evolutionary changes in group risk-taking behaviour and the circadian system in a fish.

Intensive and trait-selective mortality of fish and wildlife can cause evolutionary changes in a range of life-history and behavioural traits. These changes might in turn alter the circadian system due to co-evolutionary mechanisms or correlated selection responses both at behavioural and molecular levels, with knock-on effects on daily physiological processes and behavioural outputs. We examined the evolutionary impact of size-selective mortality on group risk-taking behaviour and the circadian system in a model fish species. We exposed zebrafish Danio rerio to either large or small size-selective harvesting relative to a control over five generations, followed by eight generations during which harvesting was halted to remove maternal effects. Size-selective mortality affected fine-scale timing of behaviours. In particular, small size-selective mortality, typical of specialized fisheries and gape-limited predators targeting smaller size classes, increased group risk-taking behaviuor during feeding and after simulated predator attacks. Moreover, small size-selective mortality increased early peaks of daily activity as well as extended self-feeding daily activity to the photophase compared to controls. By contrast large size-selective mortality, typical of most wild capture fisheries, only showed an almost significant effect of decreasing group risk-taking behaviour during the habituation phase and no clear changes in fine-scale timing of daily behavioural rhythms compared to controls. We also found changes in the molecular circadian core clockwork in response to both size-selective mortality treatments. These changes disappeared in the clock output pathway because both size-selected lines showed similar transcription profiles. This switch downstream to the molecular circadian core clockwork also resulted in similar overall behavioural rhythms (diurnal swimming and self-feeding in the last hours of darkness) independent of the underlying molecular clock. To conclude, our experimental harvest left an asymmetrical evolutionary legacy in group risk-taking behaviour and in fine-scale daily behavioural rhythms. Yet, the overall timing of activity showed evolutionary resistance probably maintained by a molecular switch. Our experimental findings suggest that size-selective mortality can have consequences for behaviour and physiological processes.

Normalization of disrupted clock gene expression in males with tetraplegia: a crossover randomized placebo-controlled trial of melatonin supplementation.

STUDY DESIGN: Crossover double blind, randomized placebo-controlled trial. OBJECTIVES: Circadian oscillators are located both in the brain and in peripheral organs. Melatonin, the main brain-derived hormone governing circadian variations, is highly associated with daylight patterns. However, in subjects with tetraplegia the melatonin levels are blunted. Here we studied peripheral oscillators in peripheral blood mononuclear cells (PBMCs) in males with tetraplegia by examining how exogenous melatonin may influence the expression of clock gene mRNAs. SETTING: Sunnaas Rehabilitation Hospital, Nesoddtangen, Norway. METHODS: Six males with tetraplegia received 2 mg of melatonin or placebo 4 days before the study period. We also included six able-bodied men sleeping or kept awake during the night. Plasma samples were collected four times during a 24-h period. The mRNA expression levels of the clock genes PER1, PER2, BMAL1, and REV-ERBα were quantified in PBMCs using quantitative RT-PCR. RESULTS: The mRNA expression levels of PER-1 and -2 and REV-ERBα were increased at 04:00 h compared with the able-bodied controls (p < 0.05). Melatonin supplementation changed mRNA peak-time toward the time of supplementation. CONCLUSIONS: Several peripheral clock genes displayed distorted expression levels in tetraplegia. Supplementation with melatonin changed the mRNA expression levels of these genes toward those observed among able-bodied. SPONSORSHIP: Financial support was provided from the Throne Holst Foundation, Sunnaas Rehabilitation hospital and the University of Ferrara (FAR2016).

A blind circadian clock in cavefish reveals that opsins mediate peripheral clock photoreception.

The circadian clock is synchronized with the day-night cycle primarily by light. Fish represent fascinating models for deciphering the light input pathway to the vertebrate clock since fish cell clocks are regulated by direct light exposure. Here we have performed a comparative, functional analysis of the circadian clock involving the zebrafish that is normally exposed to the day-night cycle and a cavefish species that has evolved in perpetual darkness. Our results reveal that the cavefish retains a food-entrainable clock that oscillates with an infradian period. Importantly, however, this clock is not regulated by light. This comparative study pinpoints the two extra-retinal photoreceptors Melanopsin (Opn4m2) and TMT-opsin as essential upstream elements of the peripheral clock light input pathway.

Cryptochrome expression in the eye of migratory birds depends on their migratory status.

Most passerine birds are nocturnal migrants. When kept in captivity during the migratory periods, these species show a migratory restlessness, or Zugunruhe. Recent studies on Sylvia warblers have shown that Zugunruhe is an excellent proxy of migratory disposition. Passerine birds can use the Earth's geomagnetic field as a compass to keep their course during their migratory flight. Among the candidate magnetoreceptive mechanisms are the cryptochromes, flavoproteins located in the retina that are supposed to perceive the magnetic field through a light-mediated process. Previous work has suggested that expression of Cryptochrome 1 (Cry1) is increased in migratory birds compared with non-migratory species. Here we tested the hypothesis that Cry1 expression depends on migratory status. Blackcaps Sylvia atricapilla were caught before fall migration and held in registration cages. When the birds were showing robust Zugunruhe, we applied a food deprivation protocol that simulates a long migratory flight. When the birds were refed after 2 days, their Zugunruhe decreased substantially, as is expected from birds that would interrupt migration for a refuelling stopover. We found that Cry1 expression was higher at night than during daytime in birds showing Zugunruhe, whereas in birds that underwent the fasting-and-refeeding protocol and reduced their levels of Zugunruhe, night Cry1 expression decreased to daytime levels. Our work shows that Cry1 expression is dependent on the presence of Zugunruhe and not on species-specific or seasonal factors, or on the birds being active versus inactive. These results support the hypothesis that cryptochromes underlie magnetoreceptive mechanisms in birds.

One-trial odour recognition learning and its underlying brain areas in the zebrafish.

Distinguishing familiar from novel stimuli is critical in many animals' activities, and procedures based on this ability are among the most exploited in translational research in rodents. However, recognition learning and the underlying brain substrates remain unclear outside a few mammalian species. Here, we investigated one-trial recognition learning for olfactory stimuli in a teleost fish using a behavioural and molecular approach. With our behavioural analysis, we found that zebrafish can learn to recognise a novel odour after a single encounter and then, discriminate between this odour and a different one provided that the molecular structure of the cues is relatively differentiated. Subsequently, by expression analysis of immediate early genes in the main brain areas, we found that the telencephalon was activated when zebrafish encountered a familiar odour, whereas the hypothalamus and the optic tectum were activated in response to the novel odour. Overall, this study provided evidence of single-trial spontaneous learning of novel odours in a teleost fish and the presence of multiple neural substrates involved in the process. These findings are promising for the development of zebrafish models to investigate cognitive functions.

Sporadic feeding regulates robust food entrainable circadian clocks in blind cavefish.

The circadian clock represents a key timing system entrained by various periodic signals that ensure synchronization with the environment. Many investigations have pointed to the existence of two distinct circadian oscillators: one regulated by the light-dark cycle and the other set by feeding time. Blind cavefish have evolved under extreme conditions where they completely lack light exposure and experience food deprivation. Here, we have investigated feeding regulated clocks in two cavefish species, the Somalian cavefish Phreatichthys andruzzii and the Mexican cavefish Astyanax mexicanus, in comparison with the surface-dwelling zebrafish Danio rerio. Our results reveal that feeding represents an extremely strong synchronizer for circadian locomotor rhythmicity in subterranean cavefish. Indeed, we showed that consuming just one meal every 4 days is sufficient to entrain circadian rhythmicity in both cavefish species, but not in zebrafish. These profound adaptations to an extreme environment provide insight into the connections between feeding and circadian clocks.

Encephalic photoreception and phototactic response in the troglobiont Somalian blind cavefish Phreatichthys andruzzii.

Many physiological and behavioural responses to changes in environmental lighting conditions are mediated by extraocular photoreceptors. Here we investigate encephalic photoreception in Phreatichthys andruzzii, a typical cave-dwelling fish showing an extreme phenotype with complete anophthalmy and a reduction in size of associated brain structures. We firstly identified two P. andruzzii photopigments, orthologues of rod opsin and exo-rod opsin. In vitro, both opsins serve as light-absorbing photopigments with λ(max) around 500 nm when reconstituted with an A(1) chromophore. When corrected for the summed absorption from the skin and skull, the spectral sensitivity profiles shifted to longer wavelengths (rod opsin: 521 nm; exo-rod opsin: 520 nm). We next explored the involvement of both opsins in the negative phototaxis reported for this species. A comparison of the spectral sensitivity of the photophobic response with the putative A(2) absorbance spectra corrected for skin/skull absorbance indicates that the A(2) versions of either or both of these pigments could explain the observed behavioural spectral sensitivity.

Loss of circadian rhythmicity in bdnf knockout zebrafish larvae.

Brain-derived neurotrophic factor (BDNF) plays a pivotal role in neuronal growth and differentiation, neuronal plasticity, learning, and memory. Using CRISPR/Cas9 technology, we generated a vital Bdnf null mutant line in zebrafish and carried out its molecular and behavioral characterization. Although no defects are evident on a morphological inspection, 66% of coding genes and 37% of microRNAs turned out to be differentially expressed in bdnf -/- compared with wild type sibling embryos. We deeply investigated the circadian clock pathway and confirmed changes in the rhythmic expression of clock (arntl1a, clock1a and clock2) and clock-controlled (aanat2) genes. The modulatory role of Bdnf on the zebrafish circadian clock was then validated by behavioral tests highlighting the absence of circadian activity rhythms in bdnf -/- larvae. The circadian behavior was partially rescued by pharmacological treatment. The bdnf -/- zebrafish line presented here is the first valuable and stable vertebrate model for the study of BDNF-related neurodevelopmental diseases.

Chronic sleep deprivation markedly reduces coagulation factor VII expression.

Chronic sleep loss, a common feature of human life in industrialized countries, is associated to cardiovascular disorders. Variations in functional parameters of coagulation might contribute to explain this relationship. By exploiting the mouse model and a specifically designed protocol, we demonstrated that seven days of partial sleep deprivation significantly decreases (-30.5%) the thrombin generation potential in plasma evaluated upon extrinsic (TF/FVIIa pathway) but not intrinsic activation of coagulation. This variation was consistent with a decrease (-49.8%) in the plasma activity levels of factor VII (FVII), the crucial physiologicalal trigger of coagulation, which was even more pronounced at the liver mRNA level (-85.7%). The recovery in normal sleep conditions for three days completely restored thrombin generation and FVII activity in plasma. For the first time, we demonstrate that chronic sleep deprivation on its own reduces, in a reversible manner, the FVII expression levels, thus influencing the TF/FVIIa activation pathway efficiency.

Circadian Clock and OxInflammation: Functional Crosstalk in Cutaneous Homeostasis.

Circadian rhythms are biological oscillations that occur with an approximately 24 h period and optimize cellular homeostasis and responses to environmental stimuli. A growing collection of data suggests that chronic circadian disruption caused by novel lifestyle risk factors such as shift work, travel across time zones, or irregular sleep-wake cycles has long-term consequences for human health. Among the multiplicity of physiological systems hypothesized to have a role in the onset of pathologies in case of circadian disruption, there are redox-sensitive defensive pathways and inflammatory machinery. Due to its location and barrier physiological role, the skin is a prototypical tissue to study the influence of environmental insults induced OxInflammation disturbance and circadian system alteration. To better investigate the link among outdoor stressors, OxInflammation, and circadian system, we tested the differential responses of keratinocytes clock synchronized or desynchronized, in an in vitro inflammatory model exposed to O3. Being both NRF2 and NF-κB two key redox-sensitive transcription factors involved in cellular redox homeostasis and inflammation, we analyzed their activation and expression in challenged keratinocytes by O3. Our results suggest that a synchronized circadian clock not only facilitates the protective role of NRF2 in terms of a faster and more efficient defensive response against environmental insults but also moderates the cellular damage resulting from a condition of chronic inflammation. Our results bring new insights on the role of circadian clock in regulating the redox-inflammatory crosstalk influenced by O3 and possibly can be extrapolated to other pollutants able to affect the oxinflammatory cellular processes.

Evidence for circadian rhythms in human trophoblast cell line that persist in hypoxia.

Circadian clock governs daily rhythmicity of a number of physiological processes such as reproductive functions. The existence of circadian clocks in the placenta is not clearly established. In order to investigate whether human placenta may function as circadian oscillator, we utilized HTR-8/SVneo cells derived from human first-trimester trophoblast. In serum-shocked cells we found circadian expressions for the clock genes Per2 and Dec1 as well as for Dbp, a canonical clock-controlled gene. We obtained similar results for Vegf, a circadian output involved in the control of placental vasculogenesis and trophoblast functions. Interestingly, circadian oscillations persisted and even enhanced in cells experimentally rendered hypoxic with CoCl(2). These results could be explained since the hypoxic milieu of the first-trimester placenta is considered the optimal condition for normal placentation. These data collectively support a possible role for the differential rhythmic expression of Vegf, influenced by circadian clock, in the adjustment of placental vascularization and trophoblast functions to the specific requirements of the different gestational ages.

Feeding Entrainment of the Zebrafish Circadian Clock Is Regulated by the Glucocorticoid Receptor.

Glucocorticoids (GCs) are steroid hormones mainly acting as key regulators of body homeostasis and stress responses. Their activities are primarily based on the binding to the GC receptor (GR), a member of the nuclear receptor family, that regulates tissue-specific sets of genes. GCs secretion follows a circadian rhythmicity with a peak linked to the animal's activity phase. In mammals, GCs are also implicated in feeding entrainment mechanisms as internal zeitgeber. Here, we investigated, by means of behavioural and molecular approaches, the circadian clock and its regulation by light and food in wild-type (WT) and null glucocorticoid receptor (gr-/-) zebrafish larvae, juveniles and adults. In both WT and gr-/- larvae and adults, behavioural activity and clock gene expression were entrained to the light-dark (LD) cycle and rhythmic in constant conditions. Differences in the pattern of clock genes' expression indicated a modulatory role of GCs. A significant role of Gr was detected in the feeding entrainment which was absent or markedly dampened in mutants. Furthermore, the expression of two clock-regulated genes involved in glucidic and lipidic metabolism was altered, highlighting the participation of GCs in metabolic processes also in fish. Taken together, our results confirmed the role of GC-mediated Gr signaling in the feeding entrainment in a non-mammalian species, the zebrafish.

Circadian clock as possible protective mechanism to pollution induced keratinocytes damage.

Ozone is among the most toxic environmental stressors to which we are continuously exposed. Due to its critical location, skin is one of the most susceptible tissues to oxidative stress damaging effect of ozone. An increasing collection of data suggests a significant role of circadian system in regulation of cellular response to oxidative stress. However, the molecular mechanism linking circadian clock and antioxidant pathway it is not completely understood. Here we investigated a possible protective role of entrained circadian clock to ozone induced damage in keratinocytes, the main cellular component of human epidermis. Our results showed that, clock-synchronized keratinocytes compared to arrhythmic ones exhibited a more efficient antioxidant response, attested by a faster activation of the master antioxidant regulatory factor NRF2. Moreover, analysis of clock gene expression profiles reveals a more rapid induction of the cardinal clock gene Bmal1 in entrained cells. Based on these findings, we suppose that an adequate coordination of circadian system and antioxidant pathway might be essential to maintain homeostasis in the skin. Alteration of metabolic pathways occurred in neurological diseases or in irregular schedule of life activity could negatively influence tissue gene expression programs and associated organ physiology via its effect on the circadian system.

Evolution shapes the responsiveness of the D-box enhancer element to light and reactive oxygen species in vertebrates.

The circadian clock is a highly conserved cell-autonomous mechanism that directs daily rhythms in most aspects of biology. Daily entrainment by environmental signals, notably light, is essential for its function. However, our understanding of the mechanisms and the evolution of photic entrainment remains incomplete. Fish represent attractive models for exploring how light regulates the circadian clock due to the direct light sensitivity of their peripheral clocks. Central to this property is the light induced expression of clock genes that is mediated by D-box enhancer elements. Here, using zebrafish cells, we reveal that the light responsive D-box enhancer serves as a nuclear target for reactive oxygen species (ROS). We demonstrate that exposure to short wavelengths of visible light triggers increases in ROS levels via NADPH oxidase activity. Elevated ROS activates the JNK and p38 MAP kinases and in turn, induces clock gene expression via the D-box. In blind cavefish and mammals, where peripheral clocks are no longer entrained by direct illumination, ROS levels are still increased upon light exposure. However, in these species ROS no longer induces D-box driven clock gene transcription. Thus, during evolution, alterations in ROS-responsive signal transduction pathways underlie fundamental changes in peripheral clock photoentrainment.

Modulation of DNA Repair Systems in Blind Cavefish during Evolution in Constant Darkness.

How the environment shapes the function and evolution of DNA repair systems is poorly understood. In a comparative study using zebrafish and the Somalian blind cavefish, Phreatichthys andruzzii, we reveal that during evolution for millions of years in continuous darkness, photoreactivation DNA repair function has been lost in P. andruzzii. We demonstrate that this loss results in part from loss-of-function mutations in pivotal DNA-repair genes. Specifically, C-terminal truncations in P. andruzzii DASH and 6-4 photolyase render these proteins predominantly cytoplasmic, with consequent loss in their functionality. In addition, we reveal a general absence of light-, UV-, and ROS-induced expression of P. andruzzii DNA-repair genes. This results from a loss of function of the D-box enhancer element, which coordinates and enhances DNA repair in response to sunlight. Our results point to P. andruzzii being the only species described, apart from placental mammals, that lacks the highly evolutionary conserved photoreactivation function. We predict that in the DNA repair systems of P. andruzzii, we may be witnessing the first stages in a process that previously occurred in the ancestors of placental mammals during the Mesozoic era.

Mutations in blind cavefish target the light-regulated circadian clock gene, period 2.

Light represents the principal signal driving circadian clock entrainment. However, how light influences the evolution of the clock remains poorly understood. The cavefish Phreatichthys andruzzii represents a fascinating model to explore how evolution under extreme aphotic conditions shapes the circadian clock, since in this species the clock is unresponsive to light. We have previously demonstrated that loss-of-function mutations targeting non-visual opsins contribute in part to this blind clock phenotype. Here, we have compared orthologs of two core clock genes that play a key role in photic entrainment, cry1a and per2, in both zebrafish and P. andruzzii. We encountered aberrantly spliced variants for the P. andruzzii per2 transcript. The most abundant transcript encodes a truncated protein lacking the C-terminal Cry binding domain and incorporating an intronic, transposon-derived coding sequence. We demonstrate that the transposon insertion leads to a predominantly cytoplasmic localization of the cavefish Per2 protein in contrast to the zebrafish ortholog which is distributed in both the nucleus and cytoplasm. Thus, it seems that during evolution in complete darkness, the photic entrainment pathway of the circadian clock has been subject to mutation at multiple levels, extending from opsin photoreceptors to nuclear effectors.

Influence of temperature on the liver circadian clock in the ruin lizard Podarcis sicula.

Reptiles represent an interesting animal model to investigate the influence of temperature on molecular circadian clocks. The ruin lizard Podarcis sicula lives in a continental climate and it is subjected to wide range of environmental temperatures during the course of the year. As consequence, ruin lizard daily activity pattern includes either the hibernation or periods of inactivity determined by hypothermia. Here we showed the rhythmic expression of two clock genes, lPer2 and lClock, in the liver of active lizards exposed to summer photo-thermoperiodic conditions. Interestingly, the exposition of lizards to hypothermic conditions, typical of winter season, induced a strong dampening of clock genes mRNA rhythmicity with a coincident decrease of levels. We also examined the qualitative and quantitative distribution of lPER2 and lCLOCK protein in different cellular compartments during the 24-h cycle. In the liver of active lizards both proteins showed a rhythmic expression profile in all cellular compartments. After 3 days at 6 degrees C, some temporal fluctuations of the lCLOCK and lPER2 are still detectable, although, with some marked modifications in respect to the values detected in the liver of active lizards. Besides demonstrating the influence of low temperature on the lizard liver circadian oscillators, present results could provide new essential information for comparative studies on the influence of temperature on the circadian system across vertebrate classes.

Daily and circadian rhythms of tissue factor pathway inhibitor and factor VII activity.

OBJECTIVE: Diurnal variations in levels of factor VII (FVII), FVIII, proteins C and S, antithrombin, plasminogen activator inhibitor-1, prothrombin fragment F1+2, and D-dimers in healthy humans point to the existence of circadian rhythms of coagulation factors. We sought for temporal fluctuations of tissue factor pathway inhibitor (TFPI) activity in human and mouse plasma. METHODS AND RESULTS: TFPI activity showed significant daily variations with highest levels in the morning in healthy men (+11%) and in mice at the light-to-dark transition (+63%), the beginning of the physically active period. Variations in FVII activity paralleled those in TFPI. In mice, the feeding schedule had a strong impact on these rhythms. Although restricted feeding and fasting shifted the peak of TFPI, the FVII peak disappeared. Investigation of temporal fluctuations in constant darkness indicated the existence of daily rhythms for TFPI and of true circadian rhythms for FVII. CONCLUSIONS: For the first time, we report, both in humans and mice, temporal variations in TFPI activity. The coherent variations in FVII and TFPI activity could interplay to maintain the coagulation equilibrium. The chronobiological patterns should be considered to analyze activity levels of these factors. Moreover, the mouse model could be exploited to investigate modifiers of coagulation rhythms potentially associated to morning peaks of cardiovascular events.