Neurons in the Dorso-Central Division of Zebrafish Pallium Respond to Change in Visual Numerosity.

We found a region of the zebrafish pallium that shows selective activation upon change in the numerosity of visual stimuli. Zebrafish were habituated to sets of small dots that changed in individual size, position, and density, while maintaining their numerousness and overall surface. During dishabituation tests, zebrafish faced a change in number (with the same overall surface), in shape (with the same overall surface and number), or in size (with the same shape and number) of the dots, whereas, in a control group, zebrafish faced the same stimuli as during the habituation. Modulation of the expression of the immediate early genes c-fos and egr-1 and in situ hybridization revealed a selective activation of the caudal part of the dorso-central division of the zebrafish pallium upon change in numerosity. These findings support the existence of an evolutionarily conserved mechanism for approximate magnitude and provide an avenue for understanding its underlying molecular correlates.

Characterizing ontogeny of quantity discrimination in zebrafish.

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.

Moderate early life stress improves adult zebrafish (Danio rerio) working memory but does not affect social and anxiety-like responses.

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.

Developmental Exposure to Low Concentrations of Methylmercury Causes Increase in Anxiety-Related Behaviour and Locomotor Impairments in Zebrafish.

Methylmercury (MeHg) is a ubiquitous pollutant shown to cause developmental neurotoxicity, even at low levels. However, there is still a large gap in our understanding of the mechanisms linking early-life exposure to life-long behavioural impairments. Our aim was to characterise the short- and long-term effects of developmental exposure to low doses of MeHg on anxiety-related behaviours in zebrafish, and to test the involvement of neurological pathways related to stress-response. Zebrafish embryos were exposed to sub-acute doses of MeHg (0, 5, 10, 15, 30 nM) throughout embryo-development, and tested for anxiety-related behaviours and locomotor activity at larval (light/dark locomotor activity) and adult (novel tank and tap assays) life-stages. Exposure to all doses of MeHg caused increased anxiety-related responses; heightened response to the transition from light to dark in larvae, and a stronger dive response in adults. In addition, impairment in locomotor activity was observed in the higher doses in both larvae and adults. Finally, the expressions of several neural stress-response genes from the HPI-axis and dopaminergic system were found to be disrupted in both life-stages. Our results provide important insights into dose-dependent differences in exposure outcomes, the development of delayed effects over the life-time of exposed individuals and the potential mechanisms underlying these effects.

Moderate alcohol exposure during early brain development increases stimulus-response habits in adulthood.

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.

rbfox1 loss of function in zebrafish leads to dysregulation in bdnf/trkb2 and pac1a expression resulting in HPI axis hyperactivation, altered stress response and allostatic overload.

RBFOX1 regulates transcriptional networks linked to synaptic transmission and neurodevelopment. Mutations in the RBFOX1 gene are associated with psychiatric disorders but how RBFOX1 influences psychiatric disorder vulnerability remains unclear. Recent studies showed that RBFOX1 mediates the alternative splicing of PAC1, a critical HPA axis activator. Further, RBFOX1 dysfunction is linked to dysregulation of BDNF/TrkB, a pathway promoting neuroplasticity, neuronal survival, and stress resilience. Hence, RBFOX1 dysfunction may increase psychiatric disorder vulnerability via HPA axis dysregulation, leading to disrupted development and allostatic overload. To test this hypothesis, we generated a zebrafish rbfox1 loss of function (LoF) line and examined behavioural and molecular effects during development. In larvae and adults, rbfox1 LoF resulted in hyperactivity, impulsivity and hyperarousal, and alterations in proliferation, fertility and survival, traits associated with allostatic overload. In larvae, rbfox1 LoF disrupted expression of pac1a, bdnf, trkb2, and HPI axis genes. These latter were restored after chronic TrkB agonist/antagonist treatment. In adults, bdnf/trkb2 and HPI axes dysregulation was only seen following acute stress. Our findings revealed a strict interplay between RBFOX1 and BDNF/TrkB in stress resilience and suggest that RBFOX1 LoF predisposes to psychiatric diseases through HPA axis hyperactivation during development, impairing adaptation and heightening vulnerability to allostatic overload.

Neural Basis of Number Sense in Larval Zebrafish.

Number sense, the ability to discriminate the quantity of objects, is crucial for survival. To understand how neurons work together and develop to mediate number sense, we used two-photon fluorescence light sheet microscopy to capture the activity of individual neurons throughout the brain of larval Danio rerio, while displaying a visual number stimulus to the animal. We identified number-selective neurons as early as 3 days post-fertilization and found a proportional increase of neurons tuned to larger quantities after 3 days. We used machine learning to predict the stimulus from the neuronal activity and observed that the prediction accuracy improves with age. We further tested ethanol's effect on number sense and found a decrease in number-selective neurons in the forebrain, suggesting cognitive impairment. These findings are a significant step towards understanding neural circuits devoted to discrete magnitudes and our methodology to track single-neuron activity across the whole brain is broadly applicable to other fields in neuroscience.

Pleiotropic contribution of rbfox1 to psychiatric and neurodevelopmental phenotypes in two zebrafish models.

RBFOX1 is a highly pleiotropic gene that contributes to several psychiatric and neurodevelopmental disorders. Both rare and common variants in RBFOX1 have been associated with several psychiatric conditions, but the mechanisms underlying the pleiotropic effects of RBFOX1 are not yet understood. Here we found that, in zebrafish, rbfox1 is 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 contribution of rbfox1 to behaviour, we used rbfox1sa15940, a zebrafish mutant line with TL background. We found that rbfox1sa15940 mutants present hyperactivity, thigmotaxis, decreased freezing behaviour and altered social behaviour. We repeated these behavioural tests in a second rbfox1 mutant line with a different genetic background (TU), rbfox1del19, and found that rbfox1 deficiency affects behaviour similarly in this line, although there were some differences. rbfox1del19 mutants present similar thigmotaxis, but stronger alterations in social behaviour and lower levels of hyperactivity than rbfox1sa15940 fish. Taken together, these results suggest that mutations in rbfox1 lead to multiple behavioural changes in zebrafish that might be modulated by environmental, epigenetic and genetic background effects, and that resemble phenotypic alterations present in Rbfox1-deficient mice and in patients with different psychiatric conditions. Our study, thus, highlights the evolutionary conservation of rbfox1 function in behaviour and paves the way to further investigate the mechanisms underlying rbfox1 pleiotropy on the onset of neurodevelopmental and psychiatric disorders.

The Role of Crosslinker Content of Positively Charged NIPAM Nanogels on the In Vivo Toxicity in Zebrafish.

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.

baz1b loss-of-function in zebrafish produces phenotypic alterations consistent with the domestication syndrome.

BAZ1B is a ubiquitously expressed nuclear protein with roles in chromatin remodeling, DNA replication and repair, and transcription. Reduced BAZ1B expression disrupts neuronal and neural crest development. Variation in the activity of BAZ1B has been proposed to underly morphological and behavioral aspects of domestication through disruption of neural crest development. Knockdown of baz1b in Xenopus embryos and Baz1b loss-of-function (LoF) in mice leads to craniofacial defects consistent with this hypothesis. We generated baz1b LoF zebrafish using CRISPR/Cas9 gene editing to test the hypothesis that baz1b regulates behavioral phenotypes associated with domestication in addition to craniofacial features. Zebrafish with baz1b LoF show mild underdevelopment at larval stages and distinctive craniofacial features later in life. Mutant zebrafish show reduced anxiety-associated phenotypes and an altered ontogeny of social behaviors. Thus, in zebrafish, developmental deficits in baz1b recapitulate both morphological and behavioral phenotypes associated with the domestication syndrome in other species.

Pleiotropic contribution of rbfox1 to psychiatric and neurodevelopmental phenotypes in a zebrafish model.

RBFOX1 is a highly pleiotropic gene that contributes to several psychiatric and neurodevelopmental disorders. Both rare and common variants in RBFOX1 have been associated with several psychiatric conditions, but the mechanisms underlying the pleiotropic effects of RBFOX1 are not yet understood. Here we found that, in zebrafish, rbfox1 is 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 of rbfox1 deficiency on behaviour, we used rbfox1sa15940, a rbfox1 loss-of-function line. We found that rbfox1sa15940 mutants present hyperactivity, thigmotaxis, decreased freezing behaviour and altered social behaviour. We repeated these behavioural tests in a second rbfox1 loss-of-function line with a different genetic background, rbfox1del19, and found that rbfox1 deficiency affects behaviour similarly in this line, although there were some differences. rbfox1del19 mutants present similar thigmotaxis, but stronger alterations in social behaviour and lower levels of hyperactivity than rbfox1sa15940 fish. Taken together, these results suggest that rbfox1 deficiency leads to multiple behavioural changes in zebrafish that might be modulated by environmental, epigenetic and genetic background effects, and that resemble phenotypic alterations present in Rbfox1-deficient mice and in patients with different psychiatric conditions. Our study thus highlights the evolutionary conservation of rbfox1 function in behaviour and paves the way to further investigate the mechanisms underlying rbfox1 pleiotropy on the onset of neurodevelopmental and psychiatric disorders.

Translational relevance of forward genetic screens in animal models for the study of psychiatric disease.

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 etiology 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 etiology of psychiatric disorders.

Quantity as a Fish Views It: Behavior and Neurobiology.

An ability to estimate quantities, such as the number of conspecifics or the size of a predator, has been reported in vertebrates. Fish, in particular zebrafish, may be instrumental in advancing the understanding of magnitude cognition. We review here the behavioral studies that have described the ecological relevance of quantity estimation in fish and the current status of the research aimed at investigating the neurobiological bases of these abilities. By combining behavioral methods with molecular genetics and calcium imaging, the involvement of the retina and the optic tectum has been documented for the estimation of continuous quantities in the larval and adult zebrafish brain, and the contributions of the thalamus and the dorsal-central pallium for discrete magnitude estimation in the adult zebrafish brain. Evidence for basic circuitry can now be complemented and extended to research that make use of transgenic lines to deepen our understanding of quantity cognition at genetic and molecular levels.

Ankk1 Loss of Function Disrupts Dopaminergic Pathways in Zebrafish.

Ankyrin repeat and kinase domain containing 1 (ANKK1) is a member of the receptor-interacting protein serine/threonine kinase family, known to be involved in cell proliferation, differentiation and activation of transcription factors. Genetic variation within the ANKK1 locus is suggested to play a role in vulnerability to addictions. However, ANKK1 mechanism of action is still poorly understood. It has been suggested that ANKK1 may affect the development and/or functioning of dopaminergic pathways. To test this hypothesis, we generated a CRISPR-Cas9 loss of function ankk1 zebrafish line causing a 27 bp insertion that disrupts the ankk1 sequence introducing an early stop codon. We found that ankk1 transcript levels were significantly lower in ankk1 mutant (ankk127ins ) fish compared to their wild type (ankk1 +/+) siblings. In ankk1 +/+ adult zebrafish brain, ankk1 protein was detected in isocortex, hippocampus, basolateral amygdala, mesencephalon, and cerebellum, resembling the mammalian distribution pattern. In contrast, ankk1 protein was reduced in the brain of ankk127ins/27ins fish. Quantitative polymerase chain reaction analysis revealed an increase in expression of drd2b mRNA in ankk127ins at both larval and adult stages. In ankk1 +/+ adult zebrafish brain, drd2 protein was detected in cerebral cortex, cerebellum, hippocampus, and caudate homolog regions, resembling the pattern in humans. In contrast, drd2 expression was reduced in cortical regions of ankk127ins/27ins being predominantly found in the hindbrain. No differences in the number of cell bodies or axonal projections detected by anti-tyrosine hydroxylase immunostaining on 3 days post fertilization (dpf) larvae were found. Behavioral analysis revealed altered sensitivity to effects of both amisulpride and apomorphine on locomotion and startle habituation, consistent with a broad loss of both pre and post synaptic receptors. Ankk127ins mutants showed reduced sensitivity to the effect of the selective dopamine receptor antagonist amisulpride on locomotor responses to acoustic startle and were differentially sensitive to the effects of the non-selective dopamine agonist apomorphine on both locomotion and habituation. Taken together, our findings strengthen the hypothesis of a functional relationship between ANKK1 and DRD2, supporting a role for ANKK1 in the maintenance and/or functioning of dopaminergic pathways. Further work is needed to disentangle ANKK1's role at different developmental stages.

Zebrafish behavioural assays of translational relevance for the study of psychiatric disease.

Understanding the pathogenesis of the complex behavioural disorders that constitute psychiatric disease is a major challenge for biomedical research. Assays in rodents have contributed significantly to our understanding of the neural basis of behavioural disorders and continue to be one of the main focuses for the development of novel therapeutics. Now, owing to their genetic tractability and optical transparency (allowing in vivo imaging of circuit function) and the rapid expansion of genetic tools, zebrafish are becoming increasingly popular for behavioural genetic research. The increased development of behavioural assays in zebrafish raises the possibility of exploiting the advantages of this system to identify molecular mechanisms contributing to behavioural phenotypes associated with psychiatric disorders as well as potential therapeutics. This mini-review describes behavioural paradigms in zebrafish that can be used to address endophenotypes associated with psychiatric disease. The content reflects the interests of the author and covers tests of cognitive functions, response choice and inhibition, social interaction and executive function.

Behavioral Effects of Developmental Exposure to JWH-018 in Wild-Type and Disrupted in Schizophrenia 1 (disc1) Mutant Zebrafish.

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 µM 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.

Stress reactivity elicits a tissue-specific reduction in telomere length in aging zebrafish (Danio rerio).

Individual differences in personality are associated with variation in healthy aging. Health behaviours are often cited as the likely explanation for this association; however, an underlying biological mechanism may also exist. Accelerated leukocyte telomere shortening is implicated in multiple age-related diseases and is associated with chronic activation of the hypothalamus-pituitary-adrenal (HPA) axis, providing a link between stress-related personality differences and adverse health outcomes. However, the effects of the HPA axis are tissue specific. Thus, leukocyte telomere length may not accurately reflect telomere length in disease-relevant tissues. Here, we examined the correlation between stress reactivity and telomere length in heart and brain tissue in young (6-9 month) and aging (18 month) zebrafish. Stress reactivity was assessed by tank diving and through gene expression. Telomere length was assessed using quantitative PCR. We show that aging zebrafish have shorter telomeres in both heart and brain. Telomere length was inversely related to stress reactivity in heart but not brain of aging individuals. These data support the hypotheses that an anxious predisposition contributes to accelerated telomere shortening in heart tissue, which may have important implications for our understanding of age-related heart disease, and that stress reactivity contributes to age-related telomere shortening in a tissue-specific manner.

Increased Response to 3,4-Methylenedioxymethamphetamine (MDMA) Reward and Altered Gene Expression in Zebrafish During Short- and Long-Term Nicotine Withdrawal.

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 2 weeks induced a significant increase of c-Fos and vasopressin receptor expression but a decrease of D3 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 D3 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.

Effects of pollution on adolescent mental health: a systematic review protocol.

BACKGROUND: Whilst there is little uncertainty about the deleterious impact of pollution on human and planetary health, pollution's impact on adolescent mental health is less well understood. This is particularly true for young people in underdeveloped and developing world contexts, about whom research is generally lacking. Furthermore, although adolescent resilience continues to be a research priority, little attention has been paid to adolescent pathways of resilience in the face or aftermath of pollution exposure. The objective of this study will be to examine the associations between pollution and mental health in 10- to 24-year-olds (i.e. adolescents). METHODS: We designed and registered a study protocol for a systematic review of studies which link pollution and mental health in adolescents. We will include observational studies (e.g. cohort, case-control, time series analyses) that assess the associations between exposure to any form of pollution and the mental health of 10- to 24-year-olds. The primary outcome will be symptoms associated with neurodevelopmental disorders; disruptive, impulse-control, and conduct disorders; depressive disorders; anxiety disorders; substance disorders; and schizophrenia. No secondary outcomes will be considered. Literature searches will be conducted in multiple electronic databases (from inception onwards), including PubMed, MEDLINE, SCOPUS, Web of Science, CINAHL, PsycINFO, SciELO, ERIC, and Africa-Wide. Two investigators will independently screen all citations, full-text articles, and abstract data. The methodological quality (or bias) of included studies will be appraised using appropriate tools. We will provide a narrative synthesis of the evidence. DISCUSSION: This systematic review will evaluate the evidence on the associations between pollution and the mental health of 10- to 24-year-olds. Our findings will be of potential interest to multiple audiences (including adolescent patients/clients, their families, caregivers, healthcare professionals, scientists, and policy makers) and could be used to develop prevention and intervention strategies as well as focus future research. Results will be published in a peer-reviewed journal. SYSTEMATIC REVIEW REGISTRATION: PROSPERO CRD42020176664.

Conservation of mechanisms regulating emotional-like responses on spontaneous nicotine withdrawal in zebrafish and mammals.

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.