Quantitative abilities in a reptile (Podarcis sicula).

The ability to identify the largest amount of prey available is fundamental for optimizing foraging behaviour in several species. To date, this cognitive skill has been observed in all vertebrate groups except reptiles. In this study we investigated the spontaneous ability of ruin lizards to select the larger amount of food items. In Experiment 1, lizards proved able to select the larger food item when presented with two alternatives differing in size (0.25, 0.50, 0.67 and 0.75 ratio). In Experiment 2 lizards presented with two groups of food items (1 versus 4, 2 versus 4, 2 versus 3 and 3 versus 4 items) were unable to select the larger group in any contrast. The lack of discrimination in the presence of multiple items represents an exception in numerical cognition studies, raising the question as to whether reptiles' quantitative abilities are different from those of other vertebrate groups.

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.

Non-visual numerical discrimination in a blind cavefish (Phreatichthys andruzzii).

Over a decade of comparative studies, researchers have found that rudimentary numerical abilities are widespread among vertebrates. While experiments in mammals and birds have employed a variety of stimuli (visual, auditory and tactile), all fish studies involved visual stimuli and it is unknown whether fish can process numbers in other sensory modalities. To fill this gap, we studied numerical abilities in Phreatichthys andruzzii, a blind cave-dwelling species that evolved in the phreatic layer of the Somalia desert. Fish were trained to receive a food reward to discriminate between two groups of objects placed in opposite positions of their home tank. In Experiment 1, subjects learned to discriminate between two and six objects, with stimuli not controlled for non-numerical continuous variables that co-vary with numbers, such as total area occupied by stimuli or density. In Experiment 2, the discrimination was two versus four, with half of the stimuli controlled for continuous quantities and half not controlled for continuous quantities. The subjects discriminated only the latter condition, indicating that they spontaneously used non-numerical information, as other vertebrates tested in similar experiments. In Experiments 3 and 4, cavefish trained from the beginning only with stimuli controlled for continuous quantities proved able to learn the discrimination of quantities based on the sole numerical information. However, their numerical acuity was lower than that reported in other teleost fish tested with visual stimuli.

The lizard celestial compass detects linearly polarized light in the blue.

The present study first examined whether ruin lizards, Podarcis sicula, are able to orientate using plane-polarized light produced by an LCD screen. Ruin lizards were trained and tested indoors, inside a hexagonal Morris water maze positioned under an LCD screen producing white polarized light with a single E-vector, which provided an axial cue. White polarized light did not include wavelengths in the UV. Lizards orientated correctly either when tested with E-vector parallel to the training axis or after 90 deg rotation of the E-vector direction, thus validating the apparatus. Further experiments examined whether there is a preferential region of the light spectrum to perceive the E-vector direction of polarized light. For this purpose, lizards reaching learning criteria under white polarized light were subdivided into four experimental groups. Each group was tested for orientation under a different spectrum of plane-polarized light (red, green, cyan and blue) with equalized photon flux density. Lizards tested under blue polarized light orientated correctly, whereas lizards tested under red polarized light were completely disoriented. Green polarized light was barely discernible by lizards, and thus insufficient for a correct functioning of their compass. When exposed to cyan polarized light, lizard orientation performances were optimal, indistinguishable from lizards detecting blue polarized light. Overall, the present results demonstrate that perception of linear polarization in the blue is necessary - and sufficient - for a proper functioning of the sky polarization compass of ruin lizards. This may be adaptively important, as detection of polarized light in the blue improves functioning of the polarization compass under cloudy skies, i.e. when the alternative celestial compass based on detection of the sun disk is rendered useless because the sun is obscured by clouds.

Orientation of lizards in a Morris water-maze: roles of the sun compass and the parietal eye.

The present study examined for the first time whether a Morris water-maze can be used to explore compass and other orientation mechanisms in the ruin lizard Podarcis sicula. In the open field, during sunny days, lizards were individually trained to swim from the center of the water maze onto a hidden platform (the goal), positioned at the periphery of the maze in a single compass direction. The goal was invisible because it was placed just beneath the water surface and the water was rendered opaque. The results showed that lizards learn to swim directly towards the hidden goal under the sun in the absence of visual feature cues. We further examined whether the observed orientation response would be due to lizards learning the spatial position of the goal relative to the sun's azimuth, i.e. to the use of a time-compensated sun compass. Lizards reaching learning criteria were subjected to 6 h clock-shift (fast or slow), and tested for goal orientation in the Morris water-maze. Results demonstrated that the learned orientation response is mediated by a time-compensated sun compass. Further investigations provided direct evidence that in ruin lizards an intact parietal eye is required to perform goal orientation under the sun inside a Morris water-maze, and that other brain photoreceptors, like the pineal or deep brain photoreceptors, are not involved in orientation.

Quantity Discrimination in Trained Lizards (Podarcis sicula).

Quantitative abilities have been reported in many animal species. Two main methods have been extensively used: spontaneous choice tests and training procedures. A recent study showed that ruin lizards are capable of spontaneously discriminating between the surface area of two food items of different size, but failed when food was presented in sets of discrete items differing in number. In the present study, we used a training procedure to further investigate quantitative abilities in ruin lizards. Subjects were presented with two sets of yellow disks differing either in number (Experiment 1) or in area (Experiment 2) and were trained on different discriminations of increasing difficulty (1 vs. 4, 2 vs. 4, and 2 vs. 3). Results showed that lizards were more accurate in discriminating sets of discrete items differing in number than the area of two individual items, in contrast to what had earlier been observed in spontaneous choice tests. Although we cannot exclude other factors that affected the performance of ruin lizards, the poor accuracy here observed in both experiments might reflect a true limit in lizards' quantitative abilities.

Extra-Visual Systems in the Spatial Reorientation of Cavefish.

Disoriented humans and animals are able to reorient themselves using environmental geometry ("metric properties" and "sense") and local features, also relating geometric to non-geometric information. Here we investigated the presence of these reorientation spatial skills in two species of blind cavefish (Astyanax mexicanus and Phreatichthys andruzzii), in order to understand the possible role of extra-visual senses in similar spatial tasks. In a rectangular apparatus, with all homogeneous walls (geometric condition) or in presence of a tactilely different wall (feature condition), cavefish were required to reorient themselves after passive disorientation. We provided the first evidence that blind cavefish, using extra-visual systems, were able i) to use geometric cues, provided by the shape of the tank, in order to recognize two geometric equivalent corners on the diagonal, and ii) to integrate the geometric information with the salient cue (wall with a different surface structure), in order to recover a specific corner. These findings suggest the ecological salience of the environmental geometry for spatial orientation in animals and, despite the different niches of adaptation, a potential shared background for spatial navigation. The geometric spatial encoding seems to constitute a common cognitive tool needed when the environment poses similar requirements to living organisms.

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.

Temporal variations of coagulation factor VII activity in mice are influenced by lighting regime.

It was recently reported that the circadian clock machinery controls plasma levels of factor (F) VII, the serine protease triggering blood coagulation. Here, by exploiting the mouse model, this study showed that variations of photoperiod (i.e., winter or summer conditions or simulated chronic jetlag conditions) have a strong impact on plasma FVII activity levels. Under conditions mimicking summer or winter photoperiods, FVII activity showed a clear 24 h rhythmicity. Interestingly, mean daily FVII activity levels were significantly reduced in mice exposed to summer photoperiods. Behavioral activity rhythms under both photoperiods were synchronized to LD cycles, and the amount of activity per 24 h was comparable. The authors also investigated the influence of chronic jetlag (CJL) on the FVII activity rhythms, which can be easily mimicked in mice through continuous abrupt shifts in the lighting schedule. The exposure of mice to simulated CJL of either consecutive westward or consecutive westward and eastward flights for 15 days did not abolish the behavioral activity rhythms but was associated with a period significantly different from 24 h. Intriguingly, both types of CJL exerted a strong influence on FVII activity rhythms, which were virtually suppressed. Moreover, the mean daily FVII activity was significantly lower in the CJL than in the winter photoperiod condition. Taken together, these findings in mice provide novel insights into the modulation of FVII activity levels, which might have implications for human pathophysiology.

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.

The circadian system of ruin lizards: a seasonally changing neuroendocrine loop?

Previous studies have shown that the amplitude of daily melatonin production in cultured ruin lizard pineal organs explanted in the summer is significantly higher than that from organs explanted in the winter. To test whether seasonal photoperiodic changes are decoded autonomously by the pineal gland, pineals explanted in summer were cultured in vitro and exposed to changes between winter and summer photoperiods. The changes in photoperiod duration did not affect the daily profiles of in vitro melatonin production. The discrepancy between the present in vitro results and those from lizards exposed to winter or summer photoperiods before pineal explantation supports the view that circadian information entering the pineal gland via its innervation is involved in determining seasonal changes of melatonin production in ruin lizards. We further examined whether a central component of the circadian system of ruin lizards, specifically the retinae of the lateral eyes, expresses similar seasonal changes in function as does the pineal gland. We did not find any difference between summer and autumn-winter in the effectiveness of either bilateral retinalectomy or optic nerve lesion-at the level of the optic chiasm-in altering circadian locomotor behavior in constant conditions. Both surgical procedures mostly induced a shortening of the free-running period of the locomotor rhythm of similar magnitude in all seasons. Thus, the retinae do not appear to participate in the seasonal reorganization of the circadian system in ruin lizards.

Daily rhythm of salivary and serum urea concentration in sheep.

BACKGROUND: In domestic animals many biochemical and physiological processes exhibit daily rhythmicity. The aim of the present study was to investigate the rhythmic pattern of salivary and serum urea concentrations in sheep. METHODS: Six 3-year-old female sheep kept in the same environmental conditions were used. Sheep were sampled at 4 hour intervals for 48 consecutive hours starting at 08:00 of the first day and finishing at 04:00 of the second day. Blood samples were collected via intravenous cannulae inserted into the jugular vein; saliva samples were collected through a specific tube, the "Salivette". Salivary and serum urea concentrations were assayed by means of UV spectrophotometer. ANOVA was used to determine significant differences. The single Cosinor procedure was applied to the results showing significant differences over time. RESULTS: ANOVA showed a significant effect of time on salivary and serum urea concentrations. Serum and salivary urea peaked during the light phase. In the dark phase serum and salivary urea concentrations decreased, and the diurnal trough occurred at midnight. Cosinor analysis showed diurnal acrophases for salivary and serum urea concentrations. Daily mean levels were significantly higher in the serum than in the saliva. CONCLUSION: In sheep both salivary and serum urea concentrations showed daily fluctuations. Urea is synthesized in the liver and its production is strongly influenced by food intake. Future investigation should clarify whether daily urea rhythms in sheep are endogenous or are simply the result of the temporal administration of food.

Circadian rhythms in mouse blood coagulation.

The circadian clock, influencing many biological processes, has been demonstrated to modulate levels of specific coagulation factors, but its impact on the coagulation efficiency is unknown. In a mouse model, the authors evaluated the temporal variations in the initial rate of activated factor X (FXa) and thrombin generation. Upon coagulation activation through the FVIIa-TF pathway (extrinsic activation), both parameters showed rhythmic variations with a significant peak at ZT 12, the light-to-dark transition. In mice subjected to a 6-h delayed light-dark cycle, the peak was shifted as expected. These cyclic oscillations were also observed in constant darkness, thus demonstrating, for the first time, the existence of strong circadian rhythms of the initial rate of either FXa or thrombin generation activity levels. These circadian variations overlapped with those that have been recently described in factor VII (FVII) activity. The peak of FXa generation activity was simulated by the addition of purified human FVII, thus indicating that circadian variations in FVII activity are important determinants of the circadian rhythm of the procoagulant cascade efficiency. These findings help to elucidate the complex control on the coagulation process and might contribute in explaining the temporal variations in the frequency of cardiovascular events observed in humans.

Influence of exposure in ovo to different light wavelengths on the lateralization of social response in zebrafish larvae.

Exposure of the chick embryo to different wavelengths of light of the same intensity has shown that only certain wavelengths may be important in generating visual asymmetries. This study aimed to detect the possible influence of different wavelengths of light on development of asymmetry of social recognition in zebrafish larvae, tested using the fish's mirror image as the stimulus. From fertilization until day 10 post-hatching zebrafish were kept in five different lighting conditions: natural light/dark (LD) cycle, complete darkness (DD), and artificial LD cycles with 14 h of monochromatic light (red, green, or violet light) and 10 h of darkness (rLD 14:10, gLD 14:10, vLD 14:10, respectively). On day 10 after hatching, the zebrafish larvae were subjected to a mirror test. A preference for using the left eye to scrutinize their mirror image was apparent only in zebrafish larvae exposed to and reared under a natural LD cycle, and not following exposure to any of other lighting conditions. These results are discussed with reference to other evidence of brain lateralization.

Circadian expression of the clock gene Per2 is altered in the ruin lizard (Podarcis sicula) when temperature changes.

When exposed to the cold, the body temperature of the ruin lizard (Podarcis sicula), an ectothermic vertebrate, comes into equilibrium with that low environmental temperature. During this time, the behavioral output of the circadian clock, locomotor activity, disappears. We tested the activity of the circadian clockwork at low temperature (6 degrees C) by following the expression of one of its essential components, the Period2 (Per2) gene. Here we show that lizard Per2 (lPer2) expression, which is rhythmic and paralleling the behavioral rhythm of locomotor activity at higher temperature (29 degrees C), becomes constantly high at low temperature. When lizards are re-exposed to high temperature, rhythmic lPer2 expression is re-established after 2 days of adaptation and coincides with onset of locomotor activity. The alteration of the lPer2 expression pattern at low temperature indicates that the activity of the molecular feedback loop is modified under these conditions.

Daily rhythms of serum leptin in ewes: effects of feeding, pregnancy and lactation.

The aim of the present study was to test whether serum concentrations of leptin in ewes vary with a daily rhythm. For this purpose, we examined 24 h serum leptin profiles of ewes exposed to natural photoperiodic conditions and subjected to two different feeding schedules (regular feeding and fasting). The results show for the first time the existence of daily rhythm of plasma leptin in regularly fed ewes, with a minimum during the light phase and a peak during the dark phase. Daily rhythms of serum leptin persisted after 50 h of fasting, although fasting shifted the peak of the rhythm to the beginning of the light phase and significantly reduced daily leptin production. To gain a better understanding of the role of leptin in the temporal organization of physiological events related to pregnancy and lactation, we measured serum leptin profiles throughout 24 h in ewes either during pregnancy or lactation. Daily leptin rhythms were found to persist during pregnancy and lactation, but both physiological conditions altered leptin concentrations. Maternal serum leptin concentration rose between early and mid pregnancy, then decreased in the late pregnancy and during lactation. Daily serum leptin concentration was significantly lower in nonpregnant, nonlactating ewes, compared either to lactating or to early pregnant ewes.

Toward a seasonal model of the circadian system: the case of Ruin lizards.

The pineal gland plays a role in the seasonal re-organization of the circadian system that occurs in Ruin lizard Podarcis sicula. In fact, either pinealectomy or melatonin implants induce an immediate transition from the circadian locomotor pattern typical of summer to the circadian locomotor pattern typical of spring or autumn. Furthermore, the degree of involvement of melatonin and the pineal in circadian organization changes dramatically with season: 1. The effects of pinealectomy, as well as those of melatonin implants on circadian locomotor rhythms are strong in summer and weak in the other seasons; 2. Daily melatonin injections entrain locomotor rhythms in summer, but not in other seasons; 3. A phase response curve for melatonin was generated in summer, but not in winter; 4. Daily injections of luzindole, an antagonist for melatonin receptors alter locomotor rhythms in summer, but not in other seasons; 5. A robust pineal-dependent circadian rhythm of circulating melatonin exists in summer, but not in spring or in autumn. This confirms that the central role of the pineal in determining circadian organization in summer and the marginal role of the pineal in either spring or autumn are respectively due to appearance (summer) or disappearance (spring, autumn) of a circadian melatonin signal in the circulation.

Daily and circadian rhythms of neurotransmitters and related compounds in the hypothalamic suprachiasmatic nuclei of a diurnal vertebrate.

By using immunocytochemistry we tested whether neurotransmitters, and enzymes specific to neurotransmitters synthesis are rhythmically expressed in the suprachiasmatic nuclei of the hypothalamus of Ruin lizards Podarcis sicula either kept in light-dark cycles or constant darkness. Within the suprachiasmatic nuclei, prominent 24 h rhythms under 12:12 light-dark cycles were found for vasoactive intestinal polypeptide (VIP) and for tyrosine hydroxylase (TH). Peaks of both VIP and TH fell in the light phase of the cycle. Rhythmic expression of TH persisted under constant temperature and darkness, demonstrating the existence of circadian rhythms of TH in the suprachiasmatic nuclei. No rhythmic expression of neurotransmitters and related compounds was found in the periventricular nuclei, the supraoptic nuclei, and the rest of the hypothalamus. Our data are the first demonstration of rhythmic expression of neurotransmitters and related compounds in the suprachiasmatic nuclei of a non-mammalian vertebrate. The demonstration of a diurnal peak of VIP in a diurnal reptile-vs. nocturnal peak of VIP typical of nocturnal mammals-provides new information for comparative studies on the circadian physiology of the suprachiasmatic nuclei across vertebrate classes and their adaptation strategies to different temporal niches.

Extraocular photoreception and circadian entrainment in nonmammalian vertebrates.

In mammals both the regulation of circadian rhythms and photoperiodic responses depend exclusively upon photic information provided by the lateral eyes; however, nonmammalian vertebrates can also rely on multiple extraocular photoreceptors to perform the same tasks. Extraocular photoreceptors include deep brain photoreceptors located in several distinct brain sites and the pineal complex, involving intracranial (pineal and parapineal) and extracranial (frontal organ and parietal eye) components. This review updates the research field of the most recent acquisitions concerning the roles of extraocular photoreceptors on circadian physiology and behavior, particularly photic entrainment and sun compass orientation.