Search

Polymeric nanogels as drug delivery systems offer great advantages, such as high encapsulation capacity and easily tailored formulations; however, data on biocompatibility are still limited. We synthesized N-isopropylacrylamide nanogels, with crosslinker content between 5 and 20 mol%, functionalized with different positively charged co-monomers, and investigated the in vivo toxicity in zebrafish. Our results show that the chemical structure of the basic unit impacts the toxicity profile depending on the degree of ionization and hydrogen bonding capability. When the degree of crosslinking of the polymer was altered, from 5 mol% to 20 mol%, the distribution of the positively charged monomer 2-tert-butylaminoethyl methacrylate was significantly altered, leading to higher surface charges for the more rigid nanogels (20 mol% crosslinker), which resulted in >80% survival rate (48 h, up to 0.5 mg/mL), while the more flexible polymers (5 mol% crosslinker) led to 0% survival rate (48 h, up to 0.5 mg/mL). These data show the importance of tailoring both chemical composition and rigidity of the formulation to minimize toxicity and demonstrate that using surface charge data to guide the design of nanogels for drug delivery may be insufficient.

RBFOX1is a highly pleiotropic gene that contributes to several psychiatric and neurodevelopmental disorders. Both rare and common variants inRBFOX1have been associated with several psychiatric conditions, but the mechanisms underlying the pleiotropic effects ofRBFOX1are not yet understood. Here we found that, in zebrafish,rbfox1is expressed in spinal cord, mid- and hindbrain during developmental stages. In adults, expression is restricted to specific areas of the brain, including telencephalic and diencephalic regions with an important role in receiving and processing sensory information and in directing behaviour. To investigate the effect ofrbfox1deficiency on behaviour, we usedrbfox1sa15940, arbfox1loss-of-function line. We found thatrbfox1sa15940mutants present hyperactivity, thigmotaxis, decreased freezing behaviour and altered social behaviour. We repeated these behavioural tests in a secondrbfox1loss-of-function line with a different genetic background,rbfox1del19, and found thatrbfox1deficiency affects behaviour similarly in this line, although there were some differences.rbfox1del19mutants present similar thigmotaxis, but stronger alterations in social behaviour and lower levels of hyperactivity thanrbfox1sa15940fish. Taken together, these results suggest thatrbfox1deficiency leads to multiple behavioural changes in zebrafish that might be modulated by environmental, epigenetic and genetic background effects, and that resemble phenotypic alterations present inRbfox1-deficient mice and in patients with different psychiatric conditions. Our study thus highlights the evolutionary conservation ofrbfox1function in behaviour and paves the way to further investigate the mechanisms underlyingrbfox1pleiotropy on the onset of neurodevelopmental and psychiatric disorders.

While psychotic experiences are core symptoms of mental health disorders like schizophrenia, they are also reported by 5–10% of the population. Both smoking behaviour and genetic risk for psychiatric disorders have been associated with psychotic experiences, but the interplay between these factors remains poorly understood. We tested whether smoking status, maternal smoking around birth, and number of packs smoked/year were associated with lifetime occurrence of three psychotic experiences phenotypes: delusions (n = 2067), hallucinations (n = 6689), and any psychotic experience (delusions or hallucinations; n = 7803) in 157,366 UK Biobank participants. We next calculated polygenic risk scores for schizophrenia (PRSSCZ), bipolar disorder (PRSBP), major depression (PRSDEP) and attention deficit hyperactivity disorder (PRSADHD) in 144,818 UK Biobank participants of European ancestry to assess whether association between smoking and psychotic experiences was attenuated after adjustment of diagnosis of psychiatric disorders and the PRSs. Finally, we investigated whether smoking exacerbates the effects of genetic predisposition on the psychotic phenotypes in gene-environment interaction models. Smoking status, maternal smoking, and number of packs smoked/year were associated with psychotic experiences (p −5). Except for packs smoked/year, effects were attenuated but remained significant after adjustment for diagnosis of psychiatric disorders and PRSs (p −3). Gene-environment interaction models showed the effects of PRSDEP and PRSADHD (but not PRSSCZ or PRSBP) on delusions (but not hallucinations) were significantly greater in current smokers compared to never smokers (p

Early life stress (ELS) is defined as a short or chronic period of trauma, environmental or social deprivation, which can affect different neurochemical and behavioral patterns during adulthood. Zebrafish (Danio rerio ) have been widely used as a model system to understand human neurodevelopmental disorders and display translationally relevant behavioral and stress‐regulating systems. In this study, we aimed to investigate the effects of moderate ELS by exposing young animals (6‐weeks postfertilization), for 3 consecutive days, to three stressors, and analyzing the impact of this on adult zebrafish behavior (16‐week postfertilization). The ELS impact in adults was assessed through analysis of performance on tests of unconditioned memory (free movement pattern Y‐maze test), exploratory and anxiety‐related task (novel tank diving test), and social cohesion (shoaling test). Here, we show for the first time that moderate ELS increases the number of alternations in turn‐direction compared to repetitions in the unconditioned Y‐maze task, suggesting increased working memory, but has no effect on shoal cohesion, locomotor profile, or anxiety‐like behavior. Overall, our data suggest that moderate ELS may be linked to adaptive flexibility which contributes to build “resilience” in adult zebrafish by improving working memory performance.

A sense of non-symbolic numerical magnitudes is widespread in the animal kingdom and has been documented in adult zebrafish. Here, we investigated the ontogeny of this ability using a group size preference (GSP) task in juvenile zebrafish. Fish showed GSP from 21 days post-fertilization and reliably chose the larger group when presented with discriminations of between 1 versus 3, 2 versus 5 and 2 versus 3 conspecifics but not 2 versus 4 conspecifics. When the ratio between the number of conspecifics in each group was maintained at 1 : 2, fish could discriminate between 1 versus 2 individuals and 3 versus 6, but again, not when given a choice between 2 versus 4 individuals. These findings are in agreement with studies in other species, suggesting the systems involved in quantity representation do not operate separately from other cognitive mechanisms. Rather they suggest quantity processing in fishes may be the result of an interplay between attentional, cognitive and memory-related mechanisms as in humans and other animals. Our results emphasize the potential of the use of zebrafish to explore the genetic and neural processes underlying the ontogeny and function of number cognition.

Psychiatric disorders represent a significant burden in our societies. Despite the convincing evidence pointing at gene and gene-environment interaction contributions, the role of genetics in the aetiology of psychiatric disease is still poorly understood. Forward genetic screens in animal models have helped elucidate causal links. Here we discuss the application of mutagenesis-based forward genetic approaches in common animal model species: two invertebrates, nematodes (Caenorhabditis elegans) and fruit flies (Drosophila sp.); and two vertebrates, zebrafish (Danio rerio) and mice (Mus musculus), in relation to psychiatric disease. We also discuss the use of large scale genomic studies in human populations. Despite the advances using data from human populations, animal models coupled with next-generation sequencing strategies are still needed. Although with its own limitations, zebrafish possess characteristics that make them especially well-suited to forward genetic studies exploring the aetiology of psychiatric disorders.

Synthetic cannabinoids can cause acute adverse psychological effects, but the potential impact when exposure happens before birth is unknown. Use of synthetic cannabinoids during pregnancy may affect fetal brain development, and such effects could be moderated by the genetic makeup of an individual. Disrupted in schizophrenia 1 (DISC1) is a gene with important roles in neurodevelopment that has been associated with psychiatric disorders in pedigree analyses. Using zebrafish as a model, we investigated (1) the behavioral impact of developmental exposure to 3 mM 1-pentyl-3-(1-naphthoyl)-indole (JWH-018, a common psychoactive synthetic cannabinoid) and (2) whether disc1 moderates the effects of JWH-018. As altered anxiety responses are seen in several psychiatric disorders, we focused on zebrafish anxiety-like behavior. Zebrafish embryos were exposed to JWH-018 from one to six days post-fertilization. Anxiety-like behavior was assessed using forced light/dark and acoustic startle assays in larvae and novel tank diving in adults. Compared to controls, both acutely and developmentally exposed zebrafish larvae had impaired locomotion during the forced light/dark test, but anxiety levels and response to startle stimuli were unaltered. Adult zebrafish developmentally exposed to JWH-018 spent less time on the bottom of the tank, suggesting decreased anxiety. Loss-of-function in disc1 increased anxiety-like behavior in the tank diving assay but did not alter sensitivity to JWH-018. Results suggest developmental exposure to JWH-018 has a long-term behavioral impact in zebrafish, which is not moderated by disc1.

Non-symbolic number cognition based on an approximate sense of magnitude has been documented in zebrafish. Here we investigated for the first time its neural bases. Zebrafish were habituated to a set of three or nine small dots associated with food reward. During habituation trials, the dots changed in their individual size, position and density maintaining their numerousness and overall surface area. In the dishabituation test, zebrafish faced a change (i) in number (from three to nine or vice versa with the same overall surface), (ii) in shape (with the same overall surface and number), or (iii) in size (with the same shape and number); in a control group (iv) zebrafish faced the same familiar stimuli as during the habituation. Using qPCR to measure modulation of the expression of the immediate early genes c-fos and egr-1 and in-situ hybridization to count egr1-positive cells we found a specific and selective activation of the caudal part of the dorso-central (Dc) division of the zebrafish pallium upon change in numerosity. As pallial regions are implicated in number cognition in mammals and birds, these findings support the existence of an evolutionarily conserved mechanism for approximate magnitude and provide an avenue for exploring the underlying molecular correlates.

Telomere length reflects cellular ageing. Increased telomere shortening in leukocytes is associated with a range of neurodegenerative and cardiovascular diseases, the onset and progression of which may be mediated by behavioural traits such as anxiety and stress reactivity. However, the effects of the hypothalamus-pituitary-adrenal axis stress response are shown to be tissue specific. As such, leukocyte telomere length may not give an accurate measure of the relationship between stress-reactivity and telomere length in disease relevant tissues. To test the hypothesis that stress-reactivity contributes to age-related telomere shortening in a tissue specific manner, we examined the correlation between telomere length in heart and brain tissue and stress-reactivity in a population of young (6-9 month) and ageing (18 month) zebrafish. Stress-reactivity was assessed by tank diving, a zebrafish version of the rodent open-field test, and through gene expression. Telomere length was assessed using quantitative polymerase chain reaction. We show that ageing zebrafish have shorter telomeres in both heart and brain. Telomere length is inversely related to stress-reactivity in heart but not brain of ageing individuals. These data support the hypotheses that an anxious predisposition contributes to telomere shortening in heart tissue, and by extension age-related heart disease, and that stress-reactivity contributes to age-related telomere shortening in a tissue-specific manner.

An interactive effect between nicotine and 3,4 methylenedioxymethamphetamine (MDMA) has been reported but the mechanism underlying such interaction is not completely understood. This study used zebrafish to explore gene expression changes associated with altered sensitivity to the rewarding effects of MDMA following 2 week exposure to nicotine and 2-60 days of nicotine withdrawal. Reward responses to MDMA were assessed using a conditioned place preference (CPP) paradigm and gene expression was evaluated using quantitative real time PCR of mRNA from whole brain samples from drug-treated and control adult zebrafish. Zebrafish pre-exposed for 2 weeks to nicotine showed increased conditioned place preference in response to low dose, 0.1 mg/kg, MDMA compared to un-exposed fish at 2, 7, 30 and 60 days withdrawal. Pre-exposure to nicotine for two weeks induced a significant increase of c-Fos and vasopressin receptor expression but a decrease of D(3) dopaminergic and oxytocin receptor expression at 2 days of withdrawal. C-Fos mRNA increased also at 7, 30, 60 days of withdrawal. Nicotine pre-exposed zebrafish submitted to MDMA-induced CPP, showed an increase in expression of p35 at day 2, α4 at day 30, vasopressin at day 7, and D(3) dopaminergic receptor at day 7, 30, and 60. These gene alterations could account for the altered sensitivity to the rewarding effects of MDMA in nicotine pre-exposed fish, suggesting that zebrafish have an altered ability to modulate behaviour as a function of reward during nicotine withdrawal.

Synthetic cannabinoids can cause acute adverse psychological effects, but the potential impact when exposure happens before birth is unknown. Use of synthetic cannabinoids during pregnancy may affect fetal brain development, and such effects could be moderated by the genetic makeup of an individual. Disrupted in schizophrenia 1 (DISC1) is a gene with important roles in neurodevelopment which has been associated with psychiatric disorders in pedigree analyses. Using zebrafish as a model, we investigated (1) the behavioral impact of developmental exposure to JWH-018 (a common psychoactive synthetic cannabinoid) and (2) whether disc1 moderates the effects of JWH-018. As altered anxiety responses are seen in a several psychiatric disorders, we focused on zebrafish anxiety-like behavior. Zebrafish embryos were exposed to JWH-018 from one to six days post-fertilization. Anxiety-like behavior was assessed using forced light/dark and acoustic startle assays in larvae, and novel tank diving in adults. Compared to controls, developmentally exposed zebrafish larvae had impaired locomotion during the forced light/dark test, but anxiety levels and response to startle stimuli was unaltered. Adult zebrafish developmentally exposed to JWH-018 spent less time on the bottom of the tank, suggesting decreased anxiety. Loss-of-function in disc1 increased anxiety but did not alter sensitivity to JWH-018. Results suggest developmental exposure to JWH-018 has behavioral impact in zebrafish, which is not moderated by disc1.

Evidence has shown that a variety of vertebrates, including fish, can discriminate collections of visual items on the basis of their numerousness using an evolutionarily conserved system for approximating numerical magnitude (the so-called Approximate Number System, ANS). Here we combine a habituation/dishabituation behavioural task with molecular biology assays to start investigating the neural bases of the ANS in zebrafish. Separate groups of zebrafish underwent a habituation phase with a set of 3 or 9 small red dots, associated with a food reward. The dots changed in size, position and density from trial to trial but maintained their numerousness, and the overall areas of the stimuli was kept constant. During the subsequent dishabituation test, zebrafish faced a change (i) in number (from 3 to 9 or vice versa with the same overall surface), or (ii) in shape (with the same overall surface and number), or (iii) in size (with the same shape and number). A control group of zebrafish was shown the same stimuli as during the habituation. RT-qPCR revealed that the telencephalon and thalamus were characterized by the most consistent modulation of the expression of the immediate early genes c-fos and egr-1 upon change in numerousness; in contrast, the retina and optic tectum responded mainly to changes in stimulus size.

BACKGROUND: Although there is clear evidence of genetic contributions to susceptibility to nicotine addiction, it has proved difficult to identify causal alleles and pathways from studies in humans. Mutagenesis in model species generates strong phenotypes not present in wildtype populations and can be used to identify biological mechanisms underlying quantifiable behaviours. We tested the hypothesis that a forward genetic screen for nicotine preference in zebrafish can predict loci and biological mechanisms influencing human smoking behaviour. METHODS: A population-based forward genetic screen of ethylnitrosurea- mutagenized zebrafish was used to identify lines of fish showing altered nicotine preference. Immunohistochemical, behavioral and quantitative PCR analyses were used to characterize mutant larvae. Focussed SNP analysis of the homologous human locus in cohorts from the UK and a Finnish Twin study assessed the predictive validity of the zebrafish data for human smoking behavior. RESULTS: We show nicotine preference is heritable in fish as in humans and identify loss-of-function mutations in the zebrafish Slit3 gene as associated with increased nicotine preference. Slit3 mutant larval fish showed altered sensitivity of habituation to acoustic startle to dopaminergic antagonists and increased Drd2 and Drd3 mRNA expression. Dopaminergic neuronal pathfinding was unaffected. Analysis of the SLIT3 locus in two independent human cohorts identified 2 genetic markers that predict level of cigarette consumption and likelihood of cessation. CONCLUSION: These findings suggest a role for SLIT3 signaling in development of dopaminergic pathways affecting behaviours associated with nicotine dependence and confirm the translational relevance of the zebrafish model in exploring complex human behaviors.

The larval zebrafish is inherently suitable for whole-brain single-cell resolution calcium imaging, making it an ideal model organism for identifying and dissecting the neural circuitry responsible for specific behaviors. Nonetheless, this capacity is restricted to very young animals. Classical and operant conditioning, the two main forms of associative learning, have been studied in a wide range of species for close to a century, but behavioral paradigms for zebrafish larvae have only begun to emerge in the past decade. In this chapter, we introduce these novel assays and describe some initial attempts to characterize the ontogeny of associative learning in larval zebrafish. Despite very modest performance levels, classical and operant conditioning have both been demonstrated as early as 1-week postfertilization, confirming their suitability for investigation with in vivo neuroimaging. Indeed, this approach has already yielded insights into their neurobiological substrates, with a particular focus on neurons in the cerebellum and habenula.

Mastering relational concepts and applying them to different contexts presupposes abstraction capacities and implies a high level of cognitive sophistication. One way to investigate extrapolative abilities is to assess cross-dimensional application of an abstract relational magnitude rule to new domains. Here we show that angelfish initially trained to choose either the shorter of two lines in a spatial task (line-length discrimination task) or the array with “fewer” items (numerical discrimination task) spontaneously transferred the learnt rule to novel stimuli belonging to the previously unseen dimension demonstrating knowledge of the abstract concept of “smaller”. Our finding challenges the idea that the ability to master abstract magnitude concepts across domains is unique to humans and suggests that the circuits involved in rule learning and magnitude processing might be evolutionary conserved.

Background Nicotine withdrawal syndrome is a major clinical problem. Animal models with sufficient predictive validity to support translation of pre-clinical findings to clinical research are lacking. Aims We evaluated the behavioural and neurochemical alterations in zebrafish induced by short- and long-term nicotine withdrawal. Methods Zebrafish were exposed to 1 mg/L nicotine for 2 weeks. Dependence was determined using behavioural analysis following mecamylamine-induced withdrawal, and brain nicotinic receptor binding studies. Separate groups of nicotine-exposed and control fish were assessed for anxiety-like behaviours, anhedonia and memory deficits following 2–60 days spontaneous withdrawal. Gene expression analysis using whole brain samples from nicotine-treated and control fish was performed at 7 and 60 days after the last drug exposure. Tyrosine hydroxylase (TH) immunoreactivity in pretectum was also analysed. Results Mecamylamine-precipitated withdrawal nicotine-exposed fish showed increased anxiety-like behaviour as evidenced by increased freezing and decreased exploration. 3H-Epibatidine labeled heteromeric nicotinic acethylcholine receptors (nAChR) significantly increased after 2 weeks of nicotine exposure while 125I-αBungarotoxin labeled homomeric nAChR remained unchanged. Spontaneous nicotine withdrawal elicited anxiety-like behaviour (increased bottom dwelling), reduced motivation in terms of no preference for the enriched side in a place preference test starting from Day 7 after withdrawal and a progressive decrease of memory attention (lowering discrimination index). Behavioural differences were associated with brain gene expression changes: nicotine withdrawn animals showed decreased expression of chrna 4 and chrna7 after 60 days, and of htr2a from 7 to 60 days.The expression of c-Fos was significantly increased at 7 days. Finally, Tyrosine hydroxylase (TH) immunoreactivity increased in dorsal parvocellular pretectal nucleus, but not in periventricular nucleus of posterior tuberculum nor in optic tectum, at 60 days after withdrawal. Conclusions Our findings show that nicotine withdrawal induced anxiety-like behaviour, cognitive alterations, gene expression changes and increase in pretectal TH expression, similar to those observed in humans and rodent models.

This work was supported in part by the University of St Andrews’ QR block grant and the following grants awarded to CHB: NC3Rs G1000053; BBSRC BB/M007863; Leverhulme RPG-2016-143. CHB and VJB are Members of the Royal Society Industry Fellows’ College. As a model organism, zebrafish have much to offer neuroscientific research and they are increasingly being used in behavioral neuroscience, for example to study the genetics of learning and memory. As fish are often considered “less clever” than mammals, it is important to understand how they learn and to establish optimal testing conditions. In this study, we compared the efficacy of food reinforcement and social stimuli in supporting Pavlovian conditioning, Pavlovian-to-instrumental transfer, and acquisition of a two-alternative forced choice visual discrimination. Although equally effective in conditioning and in motivating discrimination learning, fish responded with shorter latencies when they were anticipating food but responded for a greater number of trials when anticipating the social stimulus. After learning, the reward was changed: food-reinforcement was replaced with the social stimulus and vice versa. Performance accuracy did not change, but response latency did: the group previously rewarded with food, but now rewarded with the social stimulus, showed a decrease in response vigor. This is a negative contrast effect, which is well established in rats, but was thought to be absent in fish because they lacked goal representation. Our results show that zebrafish, like rats, do have goal representations. Furthermore, we have shown that whereas food has greater incentive salience than social stimuli, fish become satiated rapidly, but motivation to seek social stimuli is sustained. We conclude that zebrafish are well motivated by a mixed economy of social stimuli and food. Postprint

Background Impulsivity is poorly planned, premature or inappropriate behaviour, and deficits in impulse control are linked to a number of neuropsychiatric disorders. There are a number of theories relating to the neural basis of impulsivity, but the precise molecular mechanisms of impulse control are not clearly understood. Motor impulsivity has been observed in adult zebrafish, but in order to increase throughput it would be beneficial if early markers could be identified in larvae. One such method was recently shown to have some potential efficacy in zebrafish larvae, relating to an ADHD morphant (lphn3.1) showing increases in motor ‘peaks’ during free swimming behaviour. Objective The aim of this study was to carry out a pharmacological evaluation of motor ‘peaks’ as a measure of motor impulsivity in larval zebrafish, as well as evaluate the behaviour of mutant lines that might be predicted to show variation in motor impulsivity. Method 6dpf zebrafish larvae were filmed for 90-seconds following incubation in amisulpride (a DRD2/3 antagonist), nicotine, apomorphine (a non-specific dopamine agonist), atomoxetine (a norepinephrine reuptake inhibitor), and methylphenidate (a combined dopamine/norepinephrine agonist). We also filmed 6dpf larvae homozygous and heterozygous for mutations in pink1, disc1, and ache. In all larvae, we analysed distance moved and motor ‘peaks’, calculated as number of 3-second bins in which the larva traveled > 10mm. Results Atomoxetine and methylphenidate, which reduce impulsivity in humans and other mammalian models, reduced motor peaks while having no effect on overall distance traveled. Apomorphine increased locomotor peaks in a bimodal manner. disc1 homozygous mutants showed lower peaks than wild-type siblings. Finally, although nicotine increased distance traveled, there were no effects on motor peaks. Conclusion The results suggest that analysis of motor peaks may be a promising method for the early detection of impulsivity-related phenotypes.

Speed–accuracy trade-offs are well studied in human decision making, but we are only beginning to understand how such trade-offs affect other animals. Similarly, it is poorly understood how consistent individual differences in decision making are influenced by their social context. Here we investigated whether zebrafish, Danio rerio, show individual consistency (‘personality’) in speed–accuracy trade-offs based on a colour discrimination task, and how pairs of fish with distinct personalities make consensus choices. The results showed that zebrafish exhibit between-individual speed–accuracy trade-offs: some fish made ‘careful’, slow but accurate decisions, while others made swift but less accurate choices. We also found that these decision-making strategies were constant over time: fish retained the same strategy for 3 days. When testing pairs of careful and fast-and-inaccurate individuals, the combined choice strategy was intermediate in speed, but statistically indistinguishable from the careful individual, whereas accuracy of the dyad decision was moderately higher than that of each individual when tested singly, although this was again not significantly different from the more careful individual. For the first time, our study thus demonstrates that two individuals influence one another's speed–accuracy trade-off in decision making.

Regulatory agencies recommend that centrally active drugs are tested for abuse potential before approval. Standard preclinical assessments are conducted in rats or non-human primates (NHPs). This study evaluated the ability of the zebrafish conditioned place preference (CPP) model to predict human abuse outcomes. Twenty-seven compounds from a variety of pharmacological classes were tested in zebrafish CPP, categorized as positive or negative, and analyzed using standard diagnostic tests of binary classification to determine the likelihood that zebrafish correctly predict robust positive signals in human subjective effects studies (+HSE) and/or Drug Enforcement Administration drug scheduling. Results were then compared with those generated for rat self-administration and CPP, as well as NHP self-administration, using this same set of compounds. The findings reveal that zebrafish concordance and sensitivity values were not significantly different from chance for both +HSE and scheduling. Although significant improvements in specificity and negative predictive values were observed for zebrafish relative to +HSE, specificity without sensitivity provides limited predictive value. Moreover, assessments in zebrafish provided no added value for predicting scheduling. By contrast, rat and NHP models generally possessed significantly improved concordance, sensitivity, and positive predictive values for both clinical measures. Although there may be predictive value with compounds from specific pharmacological classes (e.g., µ-opioid receptor agonists, psychostimulants) for zebrafish CPP, altogether these data highlight that using the current methodology, the zebrafish CPP model does not add value to the preclinical assessment of abuse potential.

Zebrafish are an important model species with unparalleled potential to advance understanding of the genetics and neurobiology of behaviour through genetic and pharmacological screening and mutant analysis. However, advances using this species have been limited by the lack of robust, standardised methodology and equipment suitable for assessing adult behaviour. Here we describe a simple, fully automated, computer based, operant system for measuring behaviour in juvenile and adult zebrafish and provide detailed protocols for appetitive and aversive assays to assess cognitive function in adult zebrafish. Applications include the study of cognition in zebrafish (and other similar sized fish species) and in zebrafish models of psychiatric and neurodegenerative diseases (e.g., Alzheimer’s disease, schizophrenia, Huntington’s disease, frontotemporal dementia), and characterisation of the role of select brain regions, neurotransmitter systems and genes in zebrafish. Further, the scalable nature of the system makes the protocols suitable for use in pharmacological and genetic screening programmes.

With lifespans rapidly increasing worldwide there has been a marked increase in age-related diseases—particularly those affecting cognition—that place a major socioeconomic burden on society. Despite this, much of what occurs during the aging process at a molecular level is poorly understood, facilitating the need for a greater understanding of the processes involved. In recent years, zebrafish have proved a useful model for the identification of genetic and cellular mechanisms affecting a variety of disease processes. Here we review the potential of zebrafish as a model for the study of cognitive ageing.

Exposure to alcohol during early central nervous system development has been shown variously to affect aspects of physiological and behavioural development. In extreme cases, this can extend to craniofacial defects, severe developmental delay and mental retardation. At more moderate levels, subtle differences in brain morphology and behaviour have been observed. One clear effect of developmental alcohol exposure is an increase in the propensity to develop alcoholism and other addictions. The mechanisms by which this occurs, however, are not currently understood. In this study, we tested the hypothesis that adult zebrafish chronically exposed to moderate levels of ethanol during early brain ontogenesis would show an increase in conditioned place preference for alcohol and an increased propensity towards habit formation, a key component of drug addiction in humans. We found support for both of these hypotheses and found that the exposed fish had changes in mRNA expression patterns for dopamine receptor, nicotinic acetylcholine receptor and μ-opioid receptor encoding genes. Collectively, these data show an explicit link between the increased proclivity for addiction and addiction-related behaviour following exposure to ethanol during early brain development and alterations in the neural circuits underlying habit learning.

Substantial evidence shows that the hypophyseal–pituitary–adrenal (HPA) axis and corticosteroids are involved in the process of addiction to a variety of agents, and the adrenal cortex has a key role. In general, plasma concentrations of cortisol (or corticosterone in rats or mice) increase on drug withdrawal in a manner that suggests correlation with the behavioural and symptomatic sequelae both in man and in experimental animals. Corticosteroid levels fall back to normal values in resumption of drug intake. The possible interactions between brain corticotrophin releasing hormone (CRH) and proopiomelanocortin (POMC) products and the systemic HPA, and additionally with the local CRH–POMC system in the adrenal gland itself, are complex. Nevertheless, the evidence increasingly suggests that all may be interlinked and that CRH in the brain and brain POMC products interact with the blood-borne HPA directly or indirectly. Corticosteroids themselves are known to affect mood profoundly and may themselves be addictive. Additionally, there is a heightened susceptibility for addicted subjects to relapse in conditions that are associated with change in HPA activity, such as in stress, or at different times of the day. Recent studies give compelling evidence that a significant part of the array of addictive symptoms is directly attributable to the secretory activity of the adrenal cortex and the actions of corticosteroids. Additionally, sex differences in addiction may also be attributable to adrenocortical function: in humans, males may be protected through higher secretion of DHEA (and DHEAS), and in rats, females may be more susceptible because of higher corticosterone secretion.

Receptor tyrosine kinases (RTKs) and integrins cooperate to stimulate cell migration and tumour metastasis. Here we report that an integrin influences signalling of an RTK, c-Met, from inside the cell, to promote anchorage-independent cell survival. Thus, c-Met and β1-integrin co-internalize and become progressively recruited on LC3B-positive ‘autophagy-related endomembranes' (ARE). In cells growing in suspension, β1-integrin promotes sustained c-Met-dependent ERK1/2 phosphorylation on ARE. This signalling is dependent on ATG5 and Beclin1 but not on ATG13, suggesting ARE belong to a non-canonical autophagy pathway. This β1-integrin-dependent c-Met-sustained signalling on ARE supports anchorage-independent cell survival and growth, tumorigenesis, invasion and lung colonization in vivo. RTK–integrin cooperation has been assumed to occur at the plasma membrane requiring integrin ‘inside-out' or ‘outside-in' signalling. Our results report a novel mode of integrin–RTK cooperation, which we term ‘inside-in signalling'. Targeting integrin signalling in addition to adhesion may have relevance for cancer therapy., Cooperative signalling between receptor tyrosine kinases (RTKs) and integrins is thought to occur at the cell surface. Here the authors show that β1 integrin influences signalling of an RTK, c-Met, from a novel intracellular compartment they call autophagy-related endomembranes.

In zebrafish developmentally exposed to ambient ethanol (20mM-50mM) 1-9 days post fertilization (dpf), the cortisol response to stress has been shown to be significantly attenuated in larvae, juveniles and 6 month old adults. These data are somewhat at variance with similar studies in mammals, which often show heightened stress responses. To test whether these cortisol data correlate with behavioural changes in treated animals, anxiety-like behaviour of zebrafish larvae (9dpf and 10dpf) and juveniles (23dpf) was tested in locomotor assays designed to this end. In open field tests treated animals were more exploratory, spending significantly less time at the periphery of the arena. Behavioural effects of developmental exposure to ethanol were sustained in 6-month-old adults, as judged by assessment of thigmotaxis, novel tank diving and scototaxis. Like larvae and juveniles, developmentally treated adults were generally more exploratory, and spent less time at the periphery of the arena in thigmotaxis tests, less time at the bottom of the tank in the novel tank diving tests, andless time in the dark area in scototaxis tests. The conclusion that ethanol-exposed animals showed less anxiety-like behaviour was validated by comparison with the effects of diazepam treatment, which in thigmotaxis and novel tank diving tests had similar effects to ethanol pretreatment. There is thus a possible link between the hypophyseal-pituitary-interrenal axis and the behavioural actions of developmental ethanol exposure. The mechanisms require further elucidation.

Zebrafish are an established and widely utilized developmental genetic model system, but limitations in developed behavioral assays have meant that their potential as a model in behavioral neuroscience has yet to be fully realized. Here, we describe the development of a novel operant behavioral assay to examine a variety of aspects of stimulus control in zebrafish using a 3 choice serial reaction time task (3 CSRTT). Fish were briefly exposed to three spatially distinct, but perceptually identical stimuli, presented in a random order after a fixed-time inter-trial interval (ITI). Entries to the correct response aperture either during the stimulus presentation, or within a brief limited hold period following presentation, were reinforced with illumination of the magazine light and delivery of a small food reward. Following training, premature responding was probed with a long-ITI session three times; once at baseline, once following a saline injection and once following an injection of a low dose of amphetamine (AMPH; 0.025 mg/kg). We predicted that if premature responding was related to impulsivity (as in rodents) it would be reduced following the AMPH injection. Results confirmed that zebrafish could learn to perform a complex operant task similar to tasks developed for rodents which are used to probe sustained attention and impulsivity, but the results from the AMPH trials were inconclusive. This study provides the foundations for development and further validation of this species as a model for some aspects of human attentional and impulse control disorders, such as substance abuse disorder.

Zebrafish are fast becoming one of the most widely used animal models in systems biology and developmental genetics, but their potential as models for behavioural neuroscience is only just beginning to be realised. This progress is dependent upon the rate at which behavioural assays for adult zebrafish are designed, reported and validated. As models for drug abuse and addiction, this species has been shown thus far to show high face validity, in that they show robust place preference for drug-related cues, as well as evidence for withdrawal, relapse and compulsive drug seeking. This review will outline the case for using zebrafish as models to study endophenotypes relating to drug addiction.

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal cancers largely due to disseminated disease at the time of presentation. Here, we investigated the role and mechanism of action of the metastasis-associated protein anterior gradient 2 (AGR2) in the pathogenesis of pancreatic cancer. AGR2 was induced in all sporadic and familial pancreatic intraepithelial precursor lesions (PanIN), PDACs, circulating tumor cells, and metastases studied. Confocal microscopy and flow cytometric analyses indicated that AGR2 localized to the endoplasmic reticulum (ER) and the external surface of tumor cells. Furthermore, induction of AGR2 in tumor cells regulated the expression of several ER chaperones (PDI, CALU, RCN1), proteins of the ubiquitin-proteasome degradation pathway (HIP2, PSMB2, PSMA3, PSMC3, and PSMB4), and lysosomal proteases [cathepsin B (CTSB) and cathepsin D (CTSD)], in addition to promoting the secretion of the precursor form pro-CTSD. Importantly, the invasiveness of pancreatic cancer cells was proportional to the level of AGR2 expression. Functional downstream targets of the proinvasive activity of AGR2 included CTSB and CTSD in vitro, and AGR2, CTSB, and CTSD were essential for the dissemination of pancreatic cancer cells in vivo. Taken together, the results suggest that AGR2 promotes dissemination of pancreatic cancer and that its cell surface targeting may permit new strategies for early detection as well as therapeutic management. Cancer Res; 71(22); 7091–102. ©2011 AACR.

Tobacco addiction is a leading preventable cause of death worldwide and places a heavy social and financial burden on society. Therefore, ways of helping people to overcome nicotine dependence are a key element of strategies aimed at improving public health. Current treatments are only partially effective and there is a need to develop more efficient approaches to help smokers to stop. There exists a substantial genetic variability in smoking behavior and the likelihood of cessation – tailoring treatment according to an individual’s genetic profile is now technologically feasible and could lead to more successful cessation attempts. Here we review studies of the genetic effects on smoking cessation in randomized controlled trials of pharmacological therapy and discuss the potential value of a personalized approach to help people stop smoking.

The zebrafish community has been steadily growing in the last 20 years in Europe. Given the federal structure of Europe, this increase in zebrafish research generated a need for a strategic forum to identify and discuss exciting new areas of research and funding opportunities as well as to address infrastructural and legal issues of experimentation, transport, and husbandry of zebrafish. To foster this exchange, the European Union (EU)-funded network EuFishBioMed (Cost Action BM0804) organized an international scientific meeting of zebrafish principal investigators in Padova, Italy, in March this year. More than 120 researchers from all over the globe presented their latest work in talks and posters. A number of workshops addressed future directions of research and infrastructural issues.

Although the zonation of the adrenal cortex has a clear functional role, the mechanisms that maintain it remain largely conjectural. The concept that an outer proliferative layer gives rise to cells that migrate inwards, adopting sequentially the zona glomerulosa, fasciculata and reticularis phenotypes, has yet to be explained mechanistically. In other tissues, Eph receptor (EphR)/ephrin signalling provides a mechanism for cellular orientation and migration patterns. Real-time PCR and other methods were used to determine the possible role of Eph/ephrin systems in the rat adrenal. mRNA coding for several members of the EphR family was detected, but out of these, EphA2 provided the closest parallel to zonal organisation. In situ hybridisation showed that EphA2 mRNA and EphA protein were predominantly located in the zona glomerulosa. Its transcription closely reflected expected changes in the glomerulosa phenotype, thus it was increased after a low-sodium diet, but decreased by pretreatment with the angiotensin-converting enzyme inhibitor, captopril. It was also decreased by ACTH treatment, but unaffected by betamethasone. mRNA coding for ephrin A1, the major ligand for the EphA receptors, was also detected in the rat adrenal, though changes evoked by the various pretreatments did not clearly reflect the expected changes in zonal function. Because the maintenance of cellular zonation requires clear positional signals within the adrenal cortex, these data support a role for Eph forward and reverse signalling in the maintenance of adrenocortical zonation.

Spinal cord injury often leads to permanent incapacity because long axons cannot regenerate in the CNS. Eph receptors inhibit axon extension through an effect on the actin cytoskeleton. We have previously reported that after injury EphA4 appears at high levels in stumps of corticospinal axons, while a cognate ligand, ephrinB2, is upregulated at the lesion site so as to confine the injured axons. In this study we have infused lesioned spinal cords with a peptide antagonist of EphA4. In treated animals the retrograde degeneration that normally follows corticospinal tract injury is absent. Rather, corticospinal tract axons sprout up to and into the lesion centre. In a behavioural test of corticospinal tract function, peptide treatment substantially improved recovery relative to controls. These results suggest that blocking EphA4 is likely to contribute to a future successful clinical treatment for spinal cord injury.

Strong evidence suggests that phospholipase Cγ1 (PLCγ1) is a suitable target to counteract tumourigenesis and metastasis dissemination. We recently identified a novel signalling pathway required for PLCγ1 activation which involves formation of a protein complex with 3-phosphoinositide-dependent protein kinase 1 (PDK1). In an effort to define novel strategies to inhibit PLCγ1-dependent signals we tested here whether a newly identified and highly specific PDK1 inhibitor, 2-O-benzyl-myo-inositol 1,3,4,5,6-pentakisphosphate (2-O-Bn-InsP5), could affect PDK1/PLCγ1 interaction and impair PLCγ1-dependent cellular functions in cancer cells. Here, we demonstrate that 2-O-Bn-InsP5 interacts specifically with the pleckstrin homology domain of PDK1 and impairs formation of a PDK1/PLCγ1 complex. 2-O-Bn-InsP5 is able to inhibit the epidermal growth factor-induced PLCγ1 phosphorylation and activity, ultimately resulting in impaired cancer cell migration and invasion. Importantly, we report that 2-O-Bn-InsP5 inhibits cancer cell dissemination in zebrafish xenotransplants. This work demonstrates that the PDK1/PLCγ1 complex is a potential therapeutic target to prevent metastasis and it identifies 2-O-Bn-InsP5 as a leading compound for development of anti-metastatic drugs.