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Spatial cognitive abilities are fundamental to foraging animal species. In particular, being able to encode the location of an object in relation to another object (i.e., spatial relationships) is critical for successful foraging. Whether egocentric (i.e., viewer-dependent) or allocentric (i.e., dependent on external environment or cues) representations underlie these behaviours is still a highly debated question in vertebrates and invertebrates. Previous research shows that bees encode spatial information largely using egocentric information. However, no research has investigated this question in the context of relational similarity. To test this, a spatial matching task previously used with humans and great apes was adapted for use with wild-caught bumblebees. In a series of experiments, bees first experienced a rewarded object and then had to spontaneously (Experiment 1) find or learn (Experiments 2 and 3) to find a second one, based on the location of first one. The results showed that bumblebees predominantly exhibited an allocentric strategy in the three experiments. These findings suggest that egocentric representations alone might not be evolutionary ancestral and clearly indicate similarities between vertebrates and invertebrates when encoding spatial information.

Being able to abstract relations of similarity is considered one of the hallmarks of human cognition. While previous research has shown that other animals (e.g. primates) can attend to relational similarity, they struggle to focus on object similarity. This is in contrast with humans. And it is precisely the ability to attend to objects that it is argued to make relational reasoning uniquely human. What about invertebrates? Despite earlier studies indicating that bees are capable of learning abstract relationships (e.g. ‘same’ and ‘different’), no research has investigated whether bees can spontaneously attend to relational similarity and whether they can do so when relational matches compete with object matches. To test this, a spatial matching task (with and without competing object matches) previously used with children and great apes was adapted for use with wild-caught bumblebees. When object matches were not present, bumblebees spontaneously used relational similarity. Importantly, when competing object matches were present, bumblebees still focused on relations over objects. These findings indicate that the absence of object bias is also present in invertebrates and suggest that the relational gap between humans and other animals is due to their preference for relations over objects.

Zebrafish larvae are increasingly used for pharmacological research, but internal drug exposure is often not measured. Understanding pharmacokinetics is necessary for reliable translation of pharmacological results to higher vertebrates, including humans. Quantification of drug clearance and distribution requires measurements of blood concentrations. Additionally, measuring drug metabolites is of importance to understand clearance in this model organism mechanistically. We therefore mechanistically study and quantify pharmacokinetics in zebrafish larvae, and compare this to higher vertebrates, using paracetamol (acetaminophen) as paradigm compound. A method was developed to sample blood from zebrafish larvae at five days post fertilization. Blood concentrations of paracetamol and its major metabolites, paracetamol-glucuronide and paracetamol-sulphate, were measured. Blood concentration data were combined with measured amounts in larval homogenates and excreted amounts and simultaneously analysed through non-linear mixed effects modelling, quantifying absolute clearance and distribution volume. Blood sampling from zebrafish larvae was most successful from the posterior cardinal vein with median volume (interquartile range) of 1.12 (0.676-1.66) nL per blood sample. Samples were pooled (n=15-35) to reach measurable levels. Paracetamol blood concentrations at steady state were only 10% of the external paracetamol concentration. Paracetamol-sulphate was the major metabolite and its formation was quantified using a time-dependent metabolic formation rate. Absolute clearance and distribution volume correlated well to reported values in higher vertebrates, including humans. Based on blood concentrations and advanced data analysis, the mechanistic and quantitative understanding of paracetamol pharmacokinetics in zebrafish larvae has been established. This will improve the translational value of this vertebrate model organism in drug discovery and development.

Young children have difficulty predicting a future physiological state that conflicts with their current state. This finding is explained by the fact that children are biased by their current state (e.g., thirsty and desiring water) and thus have difficulty imagining themselves in a different state (e.g., not thirsty and desiring pretzels) “tomorrow,” for example. Another potential explanation that we explore here is that young children have difficulty understanding how physiological states, like thirst, fluctuate over time. We asked 3-, 4- and 5-year-olds (Experiment 1) and adults (Experiment 2) to predict what a thirsty Experimenter–who preferred crisps to water—would want (“water” or “crisps”) “right now” and “tomorrow.” Only adults correctly predicted someone else’s future desires when this person’s future and current desires conflicted. In contrast, both adults and children in the control groups (in which the Experimenter was not thirsty) had no difficulty predicting that the Experimenter would want crisps “right now” and “tomorrow.” Our findings suggest that children’s difficulty predicting future desires cannot solely be attributed to their being biased by their current state since the children in our study were, themselves, not thirsty. We discuss our results in the context of children’s difficulty understanding fluctuations in physiological states.

Mental time travel (MTT) is the ability that allows humans to mentally project themselves backwards in time to remember past events (i.e., episodic memory) or forwards in time to imagine future events (i.e., future thinking). Despite empirical evidence showing that animals might possess MTT abilities, some still claim that this ability is uniquely human. Recent debates have suggested that it is the temporal cognitive mechanism (i.e., ability to represent the sense of past and future) that makes MTT uniquely human. Advances in the field have been constrained by a lack of comparative data, methodological shortcomings that prevent meaningful comparisons, and a lack of clear conceptualizations of the temporal cognitive mechanism. Here I will present a comprehensive review into MTT in humans and animals-with a particular focus on great apes. I will examine three of the most prominent and influential theoretical models of human MTT. Drawing on these accounts, I suggest that a basic way of understanding time might be shared across species, however culture and language will play a critical role at shaping the way we elaborate mental representations about past and future events. This article is categorized under: Cognitive Biology > Evolutionary Roots of Cognition Psychology > Comparative Psychology.

Humans are constantly acquiring new information and skills. However, forgetting is also a common phenomenon in our lives. Understanding the lability of memories is critical to appreciate how they are formed as well as forgotten. Here we investigate the lability of chimpanzees' short-term memories and assess what factors cause forgetting in our closest relatives. In two experiments, chimpanzees were presented with a target task, which involved remembering a reward location, followed by the presentation of an interference task-requiring the recollection of a different reward location. The interference task could take place soon after the presentation of the target task or soon before the retrieval of the food locations. The results show that chimpanzees' memories for the location of a reward in a target task were compromised by the presentation of a different food location in an interference task. Critically, the temporal location of the interference task did not significantly affect chimpanzees' performance. These pattern of results were found for both Experiment 1-when the retention interval between the encoding and retrieval of the target task was 60 seconds- and Experiment 2-when the retention interval between the encoding and retrieval of the target task was 30 seconds. We argue that the temporal proximity of the to-be-remembered information and the interference item during encoding is the factor driving chimpanzees' performance in the present studies.

Hypertonicity increases urea transport, as well as the phosphorylation and membrane accumulation of UT-A1, the transporter responsible for urea permeability in the inner medullary collect duct (IMCD). Hypertonicity stimulates urea transport through PKC-mediated phosphorylation. To determine whether PKC phosphorylates UT-A1, eight potential PKC phosphorylation sites were individually replaced with alanine and subsequently transfected into LLC-PK1 cells. Of the single mutants, only ablation of the S494 site dampened induction of total UT-A1 phosphorylation by the PKC activator phorbol dibutyrate (PDBu). This result was confirmed using a newly generated antibody that specifically detected phosphorylation of UT-A1 at S494. Hypertonicity increased UT-A1 phosphorylation at S494. In contrast, activators of cAMP pathways (PKA and Epac) did not increase UT-A1 phosphorylation at S494. Activation of both PKC and PKA pathways increased plasma membrane accumulation of UT-A1, although activation of PKC alone did not do so. However, ablating the PKC site S494 decreased UT-A1 abundance in the plasma membrane. This suggests that the cAMP pathway promotes UT-A1 trafficking to the apical membrane where the PKC pathway can phosphorylate the transporter, resulting in increased UT-A1 retention at the apical membrane. In summary, activation of PKC increases the phosphorylation of UT-A1 at a specific residue, S494. Although there is no cross talk with the cAMP-signaling pathway, phosphorylation of S494 through PKC may enhance vasopressin-stimulated urea permeability by retaining UT-A1 in the plasma membrane.

Vasopressin signaling is critical for the regulation of urea transport in the inner medullary collecting duct (IMCD). Increased urea permeability is driven by a vasopressin-mediated elevation of cAMP that results in the direct phosphorylation of urea transporter (UT)-A1. The identification of cAMP-sensitive phosphorylation sites, Ser486 and Ser499, in the rat UT-A1 sequence was the first step in understanding the mechanism of vasopressin action on the phosphorylation-dependent modulation of urea transport. To investigate the significance of multisite phosphorylation of UT-A1 in response to elevated cAMP, we used highly specific and sensitive phosphosite antibodies to Ser486 and Ser499 to determine cAMP action at each phosphorylation site. We found that phosphorylation at both sites was rapid and sustained. Furthermore, the rate of phosphorylation of the two sites was similar in both mIMCD3 cells and rat inner medullary tissue. UT-A1 localized to the apical membrane in response to vasopressin was phosphorylated at Ser486 and Ser499. We confirmed that elevated cAMP resulted in increased phosphorylation of both sites by PKA but not through the vasopressin-sensitive exchange protein activated by cAMP pathway. These results elucidate the multisite phosphorylation of UT-A1 in response to cAMP, thus providing the beginning of understanding the intracellular factors underlying vasopressin stimulation of urea transport in the IMCD.

We present a RNA deep sequencing (RNAseq) analysis of a comparison of the transcriptome responses to infection of zebrafish larvae with Staphylococcus epidermidis and Mycobacterium marinum bacteria. We show how our developed GeneTiles software can improve RNAseq analysis approaches by more confidently identifying a large set of markers upon infection with these bacteria. For analysis of RNAseq data currently, software programs such as Bowtie2 and Samtools are indispensable. However, these programs that are designed for a LINUX environment require some dedicated programming skills and have no options for visualisation of the resulting mapped sequence reads. Especially with large data sets, this makes the analysis time consuming and difficult for non-expert users. We have applied the GeneTiles software to the analysis of previously published and newly obtained RNAseq datasets of our zebrafish infection model, and we have shown the applicability of this approach also to published RNAseq datasets of other organisms by comparing our data with a published mammalian infection study. In addition, we have implemented the DEXSeq module in the GeneTiles software to identify genes, such as glucagon A, that are differentially spliced under infection conditions. In the analysis of our RNAseq data, this has led to the possibility to improve the size of data sets that could be efficiently compared without using problem-dedicated programs, leading to a quick identification of marker sets. Therefore, this approach will also be highly useful for transcriptome analyses of other organisms for which well-characterised genomes are available. Electronic supplementary material The online version of this article (doi:10.1007/s00251-014-0820-3) contains supplementary material, which is available to authorized users.

Providing adults with relevant information (knowledge that they will be tested at some future time) increases motivation to remember. Research has shown that it is more effective to have this information prior to, rather than after, an encoding phase. We investigated this effect in apes and children in the context of tool-use tasks. In Experiment 1 we presented chimpanzees, orangutans, and bonobos with two tool-use tasks and three different two-tool sets. We had two conditions: prospective (PP) and retrospective (RP). In the PP subjects were shown the task that they would have to solve before they were shown the tools with which they could solve it. In the RP this order was reversed. Apes remembered the location of the useful tool better in the PP than in the RP. In Experiment 2 we presented 3- and 4-year-olds with the same conditions. Both age groups remembered the location of the correct tool in the PP, but only the 4-year-olds did so in the RP. Thus providing apes and preschool children with relevant information prior to, rather than after, the encoding phase enhances memory. These results have important implications for the understanding of the evolution of memory in general, and encoding mechanisms in particular.

Item does not contain fulltext The increasing use of zebrafish larvae for biomedical research applications is resulting in versatile models for a variety of human diseases. These models exploit the optical transparency of zebrafish larvae and the availability of a large genetic tool box. Here we present detailed protocols for the robotic injection of zebrafish embryos at very high accuracy with a speed of up to 2000 embryos per hour. These protocols are benchmarked for several applications: (1) the injection of DNA for obtaining transgenic animals, (2) the injection of antisense morpholinos that can be used for gene knock-down, (3) the injection of microbes for studying infectious disease, and (4) the injection of human cancer cells as a model for tumor progression. We show examples of how the injected embryos can be screened at high-throughput level using fluorescence analysis. Our methods open up new avenues for the use of zebrafish larvae for large compound screens in the search for new medicines.

Zebrafish larvae (Danio rerio) are increasingly used to translate findings regarding drug efficacy and safety from in vitro-based assays to vertebrate species, including humans. However, the limited understanding of drug exposure in this species hampers its implementation in translational research. Using paracetamol as a paradigm compound, we present a novel method to characterize pharmacokinetic processes in zebrafish larvae, by combining sensitive bioanalytical methods and nonlinear mixed effects modeling. The developed method allowed quantification of paracetamol and its two major metabolites, paracetamol-sulfate and paracetamol-glucuronide in pooled samples of five lysed zebrafish larvae of 3 days post-fertilization. Paracetamol drug uptake was quantified to be 0.289 pmole/min and paracetamol clearance was quantified to be 1.7% of the total value of the larvae. With an average volume determined to be 0.290 μL, this yields an absolute clearance of 2.96 × 10(7) L/h, which scales reasonably well with clearance rates in higher vertebrates. The developed methodology will improve the success rate of drug screens in zebrafish larvae and the translation potential of findings, by allowing the establishment of accurate exposure profiles and thereby also the establishment of concentration-effect relationships.

Salmonella enterica serovar Typhimurium (S. typhimurium) bacteria cause an inflammatory and lethal infection in zebrafish embryos. To characterize the embryonic innate host response at the transcriptome level, we have extended and validated previous microarray data by Illumina next-generation sequencing analysis. We obtained 10 million sequence reads from control and Salmonella-infected zebrafish embryos using a tag-based sequencing method (DGE or Tag-Seq) and 15 million reads using whole transcript sequencing (RNA-Seq), which respectively mapped to circa 65% and 85% of 28,716 known Ensembl transcripts. Both sequencing methods showed a strong correlation of sequence read counts per transcript and an overlap of 241 transcripts differentially expressed in response to infection. A lower overlap of 165 transcripts was observed with previous microarray data. Based on the combined sequencing-based and microarray-based transcriptome data we compiled an annotated reference set of infection-responsive genes in zebrafish embryos, encoding transcription factors, signal transduction proteins, cytokines and chemokines, complement factors, proteins involved in apoptosis and proteolysis, proteins with anti-microbial activities, as well as many known or novel proteins not previously linked to the immune response. Furthermore, by comparison of the deep sequencing data of S. typhimurium infection in zebrafish embryos with previous deep sequencing data of Mycobacterium marinum infection in adult zebrafish we derived a common set of infection-responsive genes. This gene set consists of known and putative innate host defense genes that are expressed both in the absence and presence of a fully developed adaptive immune system and that provide a valuable reference for future studies of host-pathogen interactions using zebrafish infection models.

Previous studies on tool using have shown that presenting subjects with certain modifications in the experimental setup can substantially improve their performance. However, procedural modifications (e.g. trap table task) may not only remove task constraints but also simplify the problem conceptually. The goal of this study was to design a variation of the trap-table that was functionally equivalent to the trap-tube task. In this new task, the subjects had to decide where to insert the tool and in which direction the reward should be pushed. We also administered a trap-tube task that allowed animals to push or rake the reward with the tool to compare the subjects’ performance on both tasks. We used a larger sample of subjects than in previous studies and from all the four species of great apes (Gorilla gorilla, Pan troglodytes, Pan paniscus, and Pongo pygmaeus). The results showed that apes performed better in the trap-platform task than in the trap-tube task. Subjects solved the tube task faster than in previous studies and they also preferred to rake in rather than to push the reward out. There was no correlation in the level of performance between both tasks, and no indication of interspecies differences. These data are consistent with the idea that apes may possess some specific causal knowledge of traps but may lack the ability to establish analogical relations between functional equivalent tasks.

Contents: Age negatively affects reproductive success of broiler breeder flocks. One of the main hormones involved in the change of fertility with the age is testosterone (TT). The introduction of young males at later production stages of broiler flocks is known as spiking and is expected to reduce the fertility decline with age. The aim of this study was to determine the effect of injection of different levels of TT and the effect of spiking on reproductive fitness of broiler breeder flocks. Two experiments were conducted on Ross 308 breeder flocks for 10 weeks (41-50 weeks of age). The experiment 1 included a control and three treatments corresponding to three doses of TT (0.1, 0.2 and 0.3 cc), while the experiment 2 included a control and one treatment corresponding to spiking. The lower dose of TT (0.1 cc) and the spiking clearly slowed down the decline in hatchability. Therefore, either the external administration of TT to the males when the natural concentration of TT starts to decline or spiking the breeder flock with young roosters could contribute to improve reproductive success in later stages of the production cycle in broiler breeder flocks., Financial support by Rasht Branch, Islamic Azad University, grant number 4.5830 is gratefully acknowledged.

The translational value of zebrafish high-throughput screens can be improved when more knowledge is available on uptake characteristics of potential drugs. We investigated reference antibiotics and 15 preclinical compounds in a translational zebrafish-rodent screening system for tuberculosis. As a major advance, we have developed a new tool for testing drug uptake in the zebrafish model. This is important, because despite the many applications of assessing drug efficacy in zebrafish research, the current methods for measuring uptake using mass spectrometry do not take into account the possible adherence of drugs to the larval surface. Our approach combines nanoliter sampling from the yolk using a microneedle, followed by mass spectrometric analysis. To date, no single physicochemical property has been identified to accurately predict compound uptake; our method offers a great possibility to monitor how any novel compound behaves within the system. We have correlated the uptake data with high-throughput drug-screening data from Mycobacterium marinum -infected zebrafish larvae. As a result, we present an improved zebrafish larva drug-screening platform which offers new insights into drug efficacy and identifies potential false negatives and drugs that are effective in zebrafish and rodents. We demonstrate that this improved zebrafish drug-screening platform can complement conventional models of in vivo Mycobacterium tuberculosis -infected rodent assays. The detailed comparison of two vertebrate systems, fish and rodent, may give more predictive value for efficacy of drugs in humans.

One of the most contested areas in the field of animal cognition is non-human future-oriented cognition. We critically examine key underlying assumptions in the debate, which is mainly preoccupied with certain dichotomous positions, the most prevalent being whether or not ‘real’ future orientation is uniquely human. We argue that future orientation is a theoretical construct threatening to lead research astray. Cognitive operations occur in the present moment and can be influenced only by prior causation and the environment, at the same time that most appear directed towards future outcomes. Regarding the current debate, future orientation becomes a question of where on various continua cognition becomes ‘truly’ future-oriented. We question both the assumption that episodic cognition is the most important process in future-oriented cognition and the assumption that future-oriented cognition is uniquely human. We review the studies on future-oriented cognition in the great apes to find little doubt that our closest relatives possess such ability. We conclude by urging that future-oriented cognition not be viewed as expression of some select set of skills. Instead, research into future-oriented cognition should be approached more like research into social and physical cognition.

Zebrafish are becoming a valuable tool in the preclinical phase of drug discovery screenings as a whole animal model with high throughput screening possibilities. They can be used to bridge the gap between cell based assays at earlier stages and in vivo validation in mammalian models, reducing, in this way, the number of compounds passing through to testing on the much more expensive rodent models. In this light, in the present manuscript is described a new high throughput pipeline using zebrafish as in vivo model system for the study of Staphylococcus epidermidis and Mycobacterium marinum infection. This setup allows the generation and analysis of large number of synchronous embryos homogenously infected. Moreover the flexibility of the pipeline allows the user to easily implement other platforms to improve the resolution of the analysis when needed. The combination of the zebrafish together with innovative high throughput technologies opens the field of drug testing and discovery to new possibilities not only because of the strength of using a whole animal model but also because of the large number of transgenic lines available that can be used to decipher the mode of action of new compounds.

We have recently reported a mutation within the conserved immunoglobulin V-type domain of the predicted adhesion protein Mpzl3 (MIM 611707) in rough coat (rc) mice with severe skin abnormalities and progressive cyclic hair loss. In this study, we tested the hypothesis that the human orthologue MPZL3 on chromosome 11q23.3 is a candidate for similar symptoms in humans. The predicted conserved MPZL3 protein has two transmembrane motifs flanking an extracellular Ig-like domain. The R100Q rc mutation is within the Ig-domain recognition loop that has roles in T-cell receptors and cell adhesion. Results of the rc mouse study, 3D structure predictions, homology with Myelin Protein Zero and EVA1, comprehensive database analyses of polymorphisms and mutations within the human MPZL3 gene and its cell, tissue expression and immunostaining pattern indicate that homozygous or compound heterozygous mutations of MPZL3 might be involved in immune-mediated human hereditary disorders with hair loss.

Background MicroRNAs (miRNAs) have recently been shown to play important roles in development of the immune system and in fine-tuning of immune responses. Human miR-146 family members are known as inflammation-inducible miRNAs involved in negative feedback regulation of Toll-like receptor (TLR) signalling. Dysregulation of the miR-146 family has often been linked to inflammatory diseases and malignancies. This study reports on miR-146a and miR-146b as infection-inducible miRNAs in zebrafish, which has emerged as a model species for human disease. Results Using a custom-designed microarray platform for miRNA expression we found that both members of the zebrafish miR-146 family, miR-146a and miR-146b, were commonly induced by infection of zebrafish embryos with Salmonella typhimurium and by infection of adult fish with Mycobacterium marinum. The induction of these miRNAs was confirmed by Taqman miRNA assays. Subsequently, we used zebrafish embryos, in which adaptive immunity is not yet active, as an in vivo system to investigate the role of miR-146 in the innate immune response to S. typhimurium infection. Knockdown of traf6 and use of myd88 mutants demonstrated that the induction of miR-146a and miR-146b by S. typhimurium infection was affected by disruption of the MyD88-Traf6 pathway that mediates transduction of TLR signals and cytokine responses. In turn, knockdown of miR-146 itself had no major effects on the expression of known targets of MyD88-Traf6 signalling. Instead, RNA sequencing analysis showed that miR-146 knockdown led to an increased induction of six members of the apolipoprotein gene family in S. typhimurium-infected embryos. Conclusion Based on microarray analysis and Taqman miRNA assays we conclude that members of the miR-146 family, which is highly conserved between fish and human, are induced by bacterial infection in zebrafish in a MyD88 and Traf6 dependent manner. The combined knockdown of miR-146a and miR-146b in zebrafish embryos infected with S. typhimurium had no major effect on the expression of pro-inflammatory genes and transcription factors known to be downstream of the MyD88-Traf6 pathway. In contrast, apolipoprotein-mediated lipid transport emerged as an infection-inducible pathway under miR-146 knockdown conditions, suggesting a possible function of miR-146 in regulating lipid metabolism during inflammation.

Tool-using tasks that require subjects to overcome the obstacles to get a reward have been a major component of research investigating causal knowledge in primates. Much of the debate in this research has focused on whether subjects simply use certain stimulus features or instead use more functionally relevant information regarding the effect that certain features may have on a moving reward. Here, we presented two obstacle tasks, a trap platform and a barrier platform, to 22 great apes. Although perceptually similar, these two tasks contain two perceptually different but functionally equivalent obstacles: a trap and a barrier. In a pre-exposure phase, subjects either experienced an obstacle task or a task without any obstacle. In the transfer phase, all subjects were presented with an obstacle task, either the trap platform or the barrier platform. Our results show that those subjects who received an obstacle task prior to the second task performed better than those who first received a non-obstacle task. The type of obstacle task that subjects received first did not have any effect on their performance in the transfer phase. We suggest that apes possess some knowledge about the effects that obstacles have on slow-moving unsupported objects.

Sequential tool use is defined as using a tool to obtain another non-food object which subsequently itself will serve as a tool to act upon a further ( sub) goal. Previous studies have shown that birds and great apes succeed in such tasks. However, the inclusion of a training phase for each of the sequential steps and the low cost associated with retrieving the longest tools limits the scope of the conclusions. The goal of the experiments presented here was, first to replicate a previous study on sequential tool use conducted on New Caledonian crows and, second, extend this work by increasing the cost of retrieving a tool in order to test tool selectivity of apes. In Experiment 1, we presented chimpanzees, orangutans and bonobos with an out-of-reach reward, two tools that were available but too short to reach the food and four out-of-reach tools differing in functionality. Similar to crows, apes spontaneously used up to 3 tools in sequence to get the reward and also showed a strong preference for the longest out-of reach tool independently of the distance of the food. In Experiment 2, we increased the cost of reaching for the longest out-of reach tool. Now apes used up to 5 tools in sequence to get the reward and became more selective in their choice of the longest tool as the costs of its retrieval increased. The findings of the studies presented here contribute to the growing body of comparative research on tool use. Postprint

Following encoding, memory remains temporarily vulnerable to disruption. Consolidation refers to offline time-dependent processes that continue after encoding and stabilize, transform or enhance the memory trace. Memory consolidation resulting from sleep has been reported for declarative and non-declarative memories in humans. We first investigated the temporal course of memory retrieval in chimpanzees, bonobos and orangutans. We found that the amount of retrieved information was time dependent: apes' performance degraded after 1 and 2 h, stabilized after 4 h, started to increase after 8 and 12 h and fully recovered after 24 h. Second, we show that although memories during wakefulness were highly vulnerable to interference from events similar to those witnessed during the original encoding event, an intervening period of sleep not only stabilized apes' memories into more permanent ones but also protected them against interference.

Background The zebrafish embryo is an important in vivo model to study the host innate immune response towards microbial infection. In most zebrafish infectious disease models, infection is achieved by micro-injection of bacteria into the embryo. Alternatively, Edwardsiella tarda, a natural fish pathogen, has been used to treat embryos by static immersion. In this study we used transcriptome profiling and quantitative RT-PCR to analyze the immune response induced by E. tarda immersion and injection. Results Mortality rates after static immersion of embryos in E. tarda suspension varied between 25-75%, while intravenous injection of bacteria resulted in 100% mortality. Quantitative RT-PCR analysis on the level of single embryos showed that expression of the proinflammatory marker genes il1b and mmp9 was induced only in some embryos that were exposed to E. tarda in the immersion system, whereas intravenous injection of E. tarda led to il1b and mmp9 induction in all embryos. In addition, microarray expression profiles of embryos subjected to immersion or injection showed little overlap. E. tarda-injected embryos displayed strong induction of inflammatory and defense genes and of regulatory genes of the immune response. E. tarda-immersed embryos showed transient induction of the cytochrome P450 gene cyp1a. This gene was also induced after immersion in Escherichia coli and Pseudomonas aeruginosa suspensions, but, in contrast, was not induced upon intravenous E. tarda injection. One of the rare common responses in the immersion and injection systems was induction of irg1l, a homolog of a murine immunoresponsive gene of unknown function. Conclusions Based on the differences in mortality rates between experiments and gene expression profiles of individual embryos we conclude that zebrafish embryos cannot be reproducibly infected by exposure to E. tarda in the immersion system. Induction of il1b and mmp9 was consistently observed in embryos that had been systemically infected by intravenous injection, while the early transcriptional induction of cyp1a and irg1l in the immersion system may reflect an epithelial or other tissue response towards cell membrane or other molecules that are shed or released by bacteria. Our microarray expression data provide a useful reference for future analysis of signal transduction pathways underlying the systemic innate immune response versus those underlying responses to external bacteria and secreted virulence factors and toxins.

The fin bases constitute the main portal of rhabdovirus entry into rainbow trout ( Oncorhynchus mykiss ), and replication in this first site strongly conditions the outcome of the infection. In this context, we studied the chemokine response elicited in this area in response to viral hemorrhagic septicemia virus (VHSV), a rhabdovirus. Among all the rainbow trout chemokine genes studied, only the transcription levels of CK10 and CK12 were significantly upregulated in response to VHSV. As the virus had previously been shown to elicit a much stronger chemokine response in internal organs, we compared the effect of VHSV on the gills, another mucosal site which does not constitute the main site of viral entry or rhabdoviral replication. In this case, a significantly stronger chemokine response was triggered, with CK1, CK3, CK9, and CK11 being upregulated in response to VHSV and CK10 and CK12 being down-modulated by the virus. We then conducted further experiments to understand how these different chemokine responses of mucosal tissues could correlate with their capacity to support VHSV replication. No viral replication was detected in the gills, while at the fin bases, only the skin and the muscle were actively supporting viral replication. Within the skin, viral replication took place in the dermis, while viral replication was blocked within epidermal cells at some point before protein translation. The different susceptibilities of the different skin layers to VHSV correlated with the effect that VHSV has on their capacity to secrete chemotactic factors. Altogether, these results suggest a VHSV interference mechanism on the early chemokine response at its active replication sites within mucosal tissues, a possible key process that may facilitate viral entry.

PGC-1alpha is a transcriptional coactivator that controls energy homeostasis through regulation of glucose and oxidative metabolism. Both PGC-1alpha expression and oxidative capacity are decreased in skeletal muscle of patients and animals undergoing atrophy, suggesting that PGC-1alpha participates in the regulation of muscle mass. PGC-1alpha gene expression is controlled by calcium- and cAMP-sensitive pathways. However, the mechanism regulating PGC-1alpha in skeletal muscle during atrophy remains unclear. Therefore, we examined the mechanism responsible for decreased PGC-1alpha expression using a rodent streptozotocin (STZ) model of chronic diabetes and atrophy. After 21days, the levels of PGC-1alpha protein and mRNA were decreased. We examined the activation state of CREB, a potent activator of PGC-1alpha transcription, and found that phospho-CREB was paradoxically high in muscle of STZ-rats, suggesting that the cAMP pathway was not involved in PGC-1alpha regulation. In contrast, expression of calcineurin (Cn), a calcium-dependent phosphatase, was suppressed in the same muscles. PGC-1alpha expression is regulated by two Cn substrates, MEF2 and NFATc. Therefore, we examined MEF2 and NFATc activity in muscles from STZ-rats. Target genes MRF4 and MCIP1.4 mRNAs were both significantly reduced, consistent with reduced Cn signaling. Moreover, levels of MRF4, MCIP1.4, and PGC-1alpha were also decreased in muscles of CnAalpha-/- and CnAbeta-/- mice without diabetes indicating that decreased Cn signaling, rather than changes in other calcium- or cAMP-sensitive pathways, were responsible for decreased PGC-1alpha expression. These findings demonstrate that Cn activity is a major determinant of PGC-1alpha expression in skeletal muscle during diabetes and possibly other conditions associated with loss of muscle mass.

Episodic memory, as defined by Tulving, can be described in terms of behavioural elements (what, where and when information) but it is also accompained by an awareness of one’s past (chronesthesia) and a subjective conscious experience (autonoetic awareness). Recent experiments have shown that corvids and rodents recall the where, what and when of an event. This capability has been called episodic-like memory because it only fulfils the behavioural criteria for episodic memory. We tested seven chimpanzees, three orangutans and two bonobos of various ages by adapting two paradigms, originally developed by Clayton and colleagues to test scrub jays. In Experiment 1, subjects were fed preferred but perishable food (frozen juice) and less preferred but non-perishable food (grape). After the food items were hidden, subjects could choose one of them either after 5 min or 1 h. The frozen juice was still available after 5 min but melted after 1 h and became unobtainable. Apes chose the frozen juice significantly more after 5 min and the grape after 1 h. In Experiment 2, subjects faced two baiting events happening at different times, yet they formed an integrated memory for the location and time of the baiting event for particular food items. We also included a memory task that required no temporal encoding. Our results showed that apes remember in an integrated fashion what, where and when (i.e., how long ago) an event happened; that is, apes distinguished between different events in which the same food items were hidden in different places at different times. The temporal control of their choices was not dependent on the familiarity of the platforms where the food was hidden. Chimpanzees’ and bonobos’ performance in the temporal encoding task was age-dependent, following an inverted U-shaped distribution. The age had no effect on the performance of the subjects in the task that required no temporal encoding.

Novel high-throughput deep sequencing technology has dramatically changed the way that the functional complexity of transcriptomes can be studied. Here we report on the first use of this technology to gain insight into the wide range of transcriptional responses that are associated with an infectious disease process. Using Solexa/Illumina's digital gene expression (DGE) system, a tag-based transcriptome sequencing method, we investigated mycobacterium-induced transcriptome changes in a model vertebrate species, the zebrafish. We obtained a sequencing depth of over 5 million tags per sample with strong correlation between replicates. Tag mapping indicated that healthy and infected adult zebrafish express over 70% of all genes represented in transcript databases. Comparison of the data with a previous multi-platform microarray analysis showed that both types of technologies identified regulation of similar functional groups of genes. However, the unbiased nature of DGE analysis provided insights that microarray analysis could not have achieved. In particular, we show that DGE data sets are instrumental for verification of predicted gene models and allowed us to detect mycobacterium-regulated switching between different transcript isoforms. Moreover, genomic mapping of infection-induced DGE tags revealed novel transcript forms for which any previous EST-based evidence of expression was lacking. In conclusion, our deep sequencing analysis revealed in depth the high degree of transcriptional complexity of the host response to mycobacterial infection and resulted in the discovery and validation of new gene products with induced expression in infected individuals.

A hallmark of skeletal muscle atrophy is increased activities of several proteolytic systems, including caspase-3. We have previously shown that conditions involving insulin deficiency or insulin resistance increase both overall protein degradation and caspase-3-mediated actin cleavage. In the present experiments, we examined how insulin regulates caspase-3 activity in L6 myotubes. Reducing the serum concentration in the culture media from 2 to 0.5% overnight increased caspase-3 activity and actin cleavage. Addition of insulin to proteolytically active cells attenuated both responses within 4 h. Individually, inhibitors of either phosphatidylinositide 3-kinase (PI3K) or MEK1/2 partially blocked the insulin-induced reduction in caspase-3 activity; in combination, the inhibitors completely prevented insulin from attenuating caspase-3 activity. Insulin suppressed caspase-3 activity by a complex mechanism that included direct inhibition due to an increased interaction between caspase-3 and cellular inhibitor of apoptosis-1 and indirect inhibition via phosphorylation (i.e., inactivation) of the proapoptotic protein Bad, which participates in the intrinsic (i.e., mitochondrial) apoptosis activation cascade. Unlike other cell types, the phosphorylation of Bad Ser112 was mediated by the PI3K/Akt pathway rather than the MEK/ERK/ribosomal S6 protein kinase pathway. In summary, our findings indicate that insulin regulates caspase-3 activity by a multistep process that is unique to skeletal muscle, thus providing insights about the muscle-specific nature of the atrophy process.

International audience; Using a novel paradigm, Tayloret al.[1] recently investigated whether NewCaledonian crows make causal interventions in comparison to 24-month-oldchildren. They view a causal intervention as the ability, after having onlyobserved a correlation between cause and effect, to produce a novel behaviouralpattern to recreate the same outcome. They conclude that New Caledoniancrows cannot make causal interventions, whereas most children can. Theyalso question whether any previous work has provided evidence for causalinterventions in corvids. We argue that their conclusions are prematurebecause of both methodological and theoretical limitations to their study, andbecause their analysis of previous work is ambiguous.

Botulinum neurotoxin (BoNT) is a potent biological substance used to treat neuromuscular and pain disorders. Both BoNT type A and BoNT type E display high-affinity uptake into motor neurons and inhibit exocytosis through cleavage of the synaptosome-associated protein of 25 kDa (SNAP25). The therapeutic effects of BoNT/A last from 3 to 12 months, whereas the effects of BoNT/E last less than 4 weeks. Using confocal microscopy and site-specific mutagenesis, we have determined that the protease domain of BoNT/A light chain (BoNT/A-LC) localizes in a punctate manner to the plasma membrane, colocalizing with the cleaved product, SNAP25 197 . In contrast, the short-duration BoNT/E serotype is cytoplasmic. Mutations in the BoNT/A-LC have revealed sequences at the N terminus necessary for plasma membrane localization, and an active dileucine motif in the C terminus that is likely involved in trafficking and interaction with adaptor proteins. These data support sequence-specific signals as determinants of intracellular localization and as a basis for the different durations of action in these two BoNT serotypes.

Summary Determining the memory systems that support nonhuman animals' capacity to remember distant past events is currently the focus an intense research effort and a lively debate [1–3]. Comparative psychology has largely adopted Tulving's framework by focusing on whether animals remember what-where-when something happened (i.e., episodic-like memory) [4–6]. However, apes have also been reported to recall other episodic components [7] after single-trial exposures [8, 9]. Using a new experimental paradigm we show that chimpanzees and orangutans recalled a tool-finding event that happened four times 3 years earlier (experiment 1) and a tool-finding unique event that happened once 2 weeks earlier (experiment 2). Subjects were able to distinguish these events from other tool-finding events, which indicates binding of relevant temporal-spatial components. Like in human involuntary autobiographical memory, a cued, associative retrieval process triggered apes' memories: when presented with a particular setup, subjects instantaneously remembered not only where to search for the tools (experiment 1), but also the location of the tool seen only once (experiment 2). The complex nature of the events retrieved, the unexpected and fast retrieval, the long retention intervals involved, and the detection of binding strongly suggest that chimpanzees and orangutans' memories for past events mirror some of the features of human autobiographical memory.

Pregnant primiparous rats were exposed to low immobilization stress or ACTH injections (one unit) throughout pregnancy. We measured the following parameters in the female offsprings: puberty (vaginal opening and first oestrous), oestrous cycle and sexual behaviour. Compared to the controls, female sexual receptivity measured by means of lordosis and the lordosis quotient (LQ) increased in both the experimental groups, but the puberty parameters of the offspring did not alter.