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.

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.

Identification of slit3 as a locus affecting nicotine preference in zebrafish and human smoking behaviour.

To facilitate smoking genetics research we determined whether a screen of mutagenized zebrafish for nicotine preference could predict loci affecting smoking behaviour. From 30 screened F3 sibling groups, where each was derived from an individual ethyl-nitrosurea mutagenized F0 fish, two showed increased or decreased nicotine preference. Out of 25 inactivating mutations carried by the F3 fish, one in the slit3 gene segregated with increased nicotine preference in heterozygous individuals. Focussed SNP analysis of the human SLIT3 locus in cohorts from UK (n=863) and Finland (n=1715) identified two variants associated with cigarette consumption and likelihood of cessation. Characterisation of slit3 mutant larvae and adult fish revealed decreased sensitivity to the dopaminergic and serotonergic antagonist amisulpride, known to affect startle reflex that is correlated with addiction in humans, and increased htr1aa mRNA expression in mutant larvae. No effect on neuronal pathfinding was detected. These findings reveal a role for SLIT3 in development of pathways affecting responses to nicotine in zebrafish and smoking in humans.

Developmental role of acetylcholinesterase in impulse control in zebrafish.

Cellular and molecular processes that mediate individual variability in impulsivity, a key behavioral component of many neuropsychiatric disorders, are poorly understood. Zebrafish heterozygous for a nonsense mutation in ache (ache (sb55/+)) showed lower levels of impulsivity in a 5-choice serial reaction time task (5-CSRTT) than wild type and ache(+∕+). Assessment of expression of cholinergic (nAChR), serotonergic (5-HT), and dopamine (DR) receptor mRNA in both adult and larval (9 dpf) ache (sb55/+) revealed significant downregulation of chrna2, chrna5, and drd2 mRNA in ache (sb55/+) larvae, but no differences in adults. Acute exposure to cholinergic agonist/antagonists had no effect on impulsivity, supporting the hypothesis that behavioral effects observed in adults were due to lasting impact of developmental alterations in cholinergic and dopaminergic signaling. This shows the cross-species role of cholinergic signaling during brain development in impulsivity, and suggests zebrafish may be a useful model for the role of cholinergic pathways as a target for therapeutic advances in addiction medicine.

Atomoxetine reduces anticipatory responding in a 5-choice serial reaction time task for adult zebrafish.

Deficits in impulse control are related to a number of psychiatric diagnoses, including attention deficit hyperactivity disorder, addiction, and pathological gambling. Despite increases in our knowledge about the underlying neurochemical and neuroanatomical correlates, understanding of the molecular and cellular mechanisms is less well established. Understanding these mechanisms is essential in order to move towards individualized treatment programs and increase efficacy of interventions. Zebrafish are a very useful vertebrate model for exploring molecular processes underlying disease owing to their small size and genetic tractability. Their utility in terms of behavioral neuroscience, however, hinges on the validation and publication of reliable assays with adequate translational relevance. Here, we report an initial pharmacological validation of a fully automated zebrafish version of the commonly used five-choice serial reaction time task using a variable interval pre-stimulus interval. We found that atomoxetine reduced anticipatory responses (0.6 mg/kg), whereas a high-dose (4 mg/kg) methylphenidate increased anticipatory responses and the number of trials completed in a session. On the basis of these results, we argue that similar neurochemical processes in fish as in mammals may control impulsivity, as operationally defined by anticipatory responses on a continuous performance task such as this, making zebrafish potentially a good model for exploring the molecular basis of impulse control disorders and for first-round drug screening.

The utility of zebrafish to study the mechanisms by which ethanol affects social behavior and anxiety during early brain development.

Exposure to moderate levels of ethanol during brain development has a number of effects on social behavior but the molecular mechanisms that mediate this are not well understood. Gaining a better understanding of these factors may help to develop therapeutic interventions in the future. Zebrafish offer a potentially useful model in this regard. Here, we introduce a zebrafish model of moderate prenatal ethanol exposure. Embryos were exposed to 20mM ethanol for seven days (48hpf-9dpf) and tested as adults for individual social behavior and shoaling. We also tested their basal anxiety with the novel tank diving test. We found that the ethanol-exposed fish displayed reductions in social approach and shoaling, and an increase in anxiety in the novel tank test. These behavioral differences corresponded to differences in hrt1aa, slc6a4 and oxtr expression. Namely, acute ethanol caused a spike in oxtr and ht1aa mRNA expression, which was followed by down-regulation at 7dpf, and an up-regulation in slc6a4 at 72hpf. This study confirms the utility of zebrafish as a model system for studying the molecular basis of developmental ethanol exposure. Furthermore, it proposes a putative developmental mechanism characterized by ethanol-induced OT inhibition leading to suppression of 5-HT and up-regulation of 5-HT1A, which leads, in turn, to possible homeostatic up-regulation of 5-HTT at 72hpf and subsequent imbalance of the 5-HT system.

Behavioural phenotyping of casper mutant and 1-pheny-2-thiourea treated adult zebrafish.

The ability to visualise neural circuits in zebrafish in vivo is one of the most useful aspects of this model organism in neuroscience. To maintain the transparency of embryos, however, drugs, such as 1-pheyl-2-thiourea (PTU) must be added, or researchers can use mutants that do not develop pigment (e.g., the casper). The behavioural characteristics of such strains, however, have not been documented. Here, we tested adult zebrafish from the casper line, as well as wild-type (Tübingen, TU) and wild-types treated as embryos with PTU on three commonly used behavioural endpoints in neuroscience: novel tank test (similar to open-field in rodents), conditioned place preference for nicotine, and social cohesion (using a new method of cluster analysis). We found no differences between the casper and the TU, but the adult TU treated with PTU as embryos showed a marked increase in anxiety during the novel tank test. These data suggest that where possible, labs interested in analysis of developmental processes involved in adult phenotypes should avoid the use of PTU in favour of transparent mutants, such as casper.

The role of zebrafish (Danio rerio) in dissecting the genetics and neural circuits of executive function.

Zebrafish have great potential to contribute to our understanding of behavioral genetics and thus to contribute to our understanding of the etiology of psychiatric disease. However, progress is dependent upon the rate at which behavioral assays addressing complex behavioral phenotypes are designed, reported and validated. Here we critically review existing behavioral assays with particular focus on the use of adult zebrafish to explore executive processes and phenotypes associated with human psychiatric disease. We outline the case for using zebrafish as models to study impulse control and attention, discussing the validity of applying extant rodent assays to zebrafish and evidence for the conservation of relevant neural circuits.

Treatment for tobacco dependence: a potential application for stratified medicine?

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.