Exposure Effects of Environmentally Relevant Concentrations of the Tricyclic Antidepressant Amitriptyline in Early Life Stage Zebrafish.

Antidepressants are one of the most globally prescribed classes of pharmaceuticals, and drug target conservation across phyla means that nontarget organisms may be at risk from the effects of exposure. Here, we address the knowledge gap for the effects of chronic exposure (28 days) to the tricyclic antidepressant amitriptyline (AMI) on fish, including for concentrations with environmental relevance, using zebrafish (Danio rerio) as our experimental model. AMI was found to bioconcentrate in zebrafish, was readily transformed to its major active metabolite nortriptyline, and induced a pharmacological effect (downregulation of the gene encoding the serotonin transporter; slc6a4a) at environmentally relevant concentrations (0.03 µg/L and above). Exposures to AMI at higher concentrations accelerated the hatch rate and reduced locomotor activity, the latter of which was abolished after a 14 day period of depuration. The lack of any response on the features of physiology and behavior we measured at concentrations found in the environment would indicate that AMI poses a relatively low level of risk to fish populations. The pseudopersistence and likely presence of multiple drugs acting via the same mechanism of action, however, together with a global trend for increased prescription rates, mean that this risk may be underestimated using current ecotoxicological assessment paradigms.

SRPK3 Is Essential for Cognitive and Ocular Development in Humans and Zebrafish, Explaining X-Linked Intellectual Disability.

OBJECTIVE: Intellectual disability is often the outcome of neurodevelopmental disorders and is characterized by significant impairments in intellectual and adaptive functioning. X-linked intellectual disability (XLID) is a subset of these disorders caused by genetic defects on the X chromosome, affecting about 2 out of 1,000 males. In syndromic form, it leads to a broad range of cognitive, behavioral, ocular, and physical disabilities. METHODS: Employing exome or genome sequencing, here we identified 4 missense variants (c.475C > G; p.H159D, c.1373C > A; p.T458N, and c.1585G > A; p.E529K, c.953C > T; p.S318L) and a putative truncating variant (c.1413_1414del; p.Y471*) in the SRPK3 gene in 9 XLID patients from 5 unrelated families. To validate SRPK3 as a novel XLID gene, we established a knockout (KO) model of the SRPK3 orthologue in zebrafish. RESULTS: The 8 patients ascertained postnatally shared common clinical features including intellectual disability, agenesis of the corpus callosum, abnormal eye movement, and ataxia. A ninth case, ascertained prenatally, had a complex structural brain phenotype. Together, these data indicate a pathological role of SRPK3 in neurodevelopmental disorders. In post-fertilization day 5 larvae (free swimming stage), KO zebrafish exhibited severe deficits in eye movement and swim bladder inflation, mimicking uncontrolled ocular movement and physical clumsiness observed in human patients. In adult KO zebrafish, cerebellar agenesis and behavioral abnormalities were observed, recapitulating human phenotypes of cerebellar atrophy and intellectual disability. INTERPRETATION: Overall, these results suggest a crucial role of SRPK3 in the pathogenesis of syndromic X-linked intellectual disability and provide new insights into brain development, cognitive and ocular dysfunction in both humans and zebrafish. ANN NEUROL 2024;96:914-931.

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.

A novel portable flip-phone based visual behaviour assay for zebrafish.

The optokinetic reflex (OKR) serves as a vital index for visual system development in early life, commonly observed within the first six months post-birth in humans. Zebrafish larvae offer a robust and convenient model for OKR studies due to their rapid development and manageable size. Existing OKR assays often involve cumbersome setups and offer limited portability. In this study, we present an innovative OKR assay that leverages the flexible screen of the Samsung Galaxy Z Flip to optimize setup and portability. We conducted paired slow-phase velocity measurements in 5-day post-fertilization (dpf) zebrafish larvae (n = 15), using both the novel flip-phone-based assay and a traditional liquid-crystal display (LCD) arena. Utilizing Bland-Altman plots, we assessed the agreement between the two methods. Both assays were efficacious in eliciting OKR, with eye movement analysis indicating high tracking precision in the flip-phone-based assay. No statistically significant difference was observed in slow-phase velocities between the two assays (p = 0.40). Our findings underscore the feasibility and non-inferiority of the flip-phone-based approach, offering streamlined assembly, enhanced portability, and the potential for cost-effective alternatives. This study contributes to the evolution of OKR assay methodologies, aligning them with emerging research paradigms.

adgrl3.1-deficient zebrafish show noradrenaline-mediated externalizing behaviors, and altered expression of externalizing disorder-candidate genes, suggesting functional targets for treatment.

Externalizing disorders (ED) are a cause of concern for public health, and their high heritability makes genetic risk factors a priority for research. Adhesion G-Protein-Coupled Receptor L3 (ADGRL3) is strongly linked to several EDs, and loss-of-function models have shown the impacts of this gene on several core ED-related behaviors. For example, adgrl3.1-/- zebrafish show high levels of hyperactivity. However, our understanding of the mechanisms by which this gene influences behavior is incomplete. Here we characterized, for the first time, externalizing behavioral phenotypes of adgrl3.1-/- zebrafish and found them to be highly impulsive, show risk-taking in a novel environment, have attentional deficits, and show high levels of hyperactivity. All of these phenotypes were rescued by atomoxetine, demonstrating noradrenergic mediation of the externalizing effects of adgrl3.1. Transcriptomic analyses of the brains of adgrl3.1-/- vs. wild-type fish revealed several differentially expressed genes and enriched gene clusters that were independent of noradrenergic manipulation. This suggests new putative functional pathways underlying ED-related behaviors, and potential targets for the treatment of ED.

angptl4 gene expression as a marker of adaptive homeostatic response to social isolation across the lifespan in zebrafish.

Social isolation has detrimental health effects, but the underlying mechanisms are unclear. Here, we investigated the impact of 2 weeks of isolation on behavior and gene expression in the central nervous system at different life stages of zebrafish. Results showed that socially deprived young adult zebrafish experienced increased anxiety, accompanied by changes in gene expression. Most gene expression patterns returned to normal within 24 hours of reintroduction to a social environment, except angptl4, which was upregulated after reintroduction, suggesting an adaptive mechanism. Similarly, aging zebrafish displayed heightened anxiety and increased central nervous system expression of angptl4 during isolation, but effects were reversed upon reintroduction to a social group. The findings imply that angptl4 plays a homeostatic role in response to social isolation, which varies across the lifespan. The study emphasizes the importance of social interactions for psychological well-being and highlights the negative consequences of isolation, especially in older individuals. Further research may unravel how social isolation affects angptl4 expression and its developmental and aging effects.

Eye movement defects in KO zebrafish reveals SRPK3 as a causative gene for an X-linked intellectual disability.

Intellectual disability (ID) is a common neurodevelopmental disorder characterized by significantly impaired intellectual and adaptive functioning. X-linked ID (XLID) disorders, caused by defects in genes on the X chromosome, affect 1.7 out of 1,000 males. Employing exome sequencing, we identified three missense mutations (c.475C>G; p.H159D, c.1373C>A; p.T458N, and c.1585G>A; p.E529K) in the SRPK3 gene in seven XLID patients from three independent families. Clinical features common to the patients are intellectual disability, agenesis of the corpus callosum, abnormal smooth pursuit eye movement, and ataxia. SRPK proteins are known to be involved in mRNA processing and, recently, synaptic vesicle and neurotransmitter release. In order to validate SRPK3 as a novel XLID gene, we established a knockout (KO) model of the SRPK3 orthologue in zebrafish. In day 5 of larval stage, KO zebrafish showed significant defects in spontaneous eye movement and swim bladder inflation. In adult KO zebrafish, we found agenesis of cerebellar structures and impairments in social interaction. These results suggest an important role of SRPK3 in eye movements, which might reflect learning problems, intellectual disability, and other psychiatric disorders.

Novel non-stimulants rescue hyperactive phenotype in an adgrl3.1 mutant zebrafish model of ADHD.

ADHD is a highly prevalent neurodevelopmental disorder. The first-line therapeutic for ADHD, methylphenidate, can cause serious side effects including weight loss, insomnia, and hypertension. Therefore, the development of non-stimulant-based therapeutics has been prioritized. However, many of these also cause other effects, most notably somnolence. Here, we have used a uniquely powerful genetic model and unbiased drug screen to identify novel ADHD non-stimulant therapeutics. We first found that adgrl3.1 null (adgrl3.1-/-) zebrafish larvae showed a robust hyperactive phenotype. Although the hyperactivity was rescued by three ADHD non-stimulant therapeutics, all interfered significantly with sleep. Second, we used wild-type zebrafish larvae to characterize a simple behavioral phenotype generated by atomoxetine and screened the 1200 compound Prestwick Chemical Library® for a matching behavioral profile resulting in 67 hits. These hits were re-assayed in the adgrl3.1-/-. Using the previously identified non-stimulants as a positive control, we identified four compounds that matched the effect of atomoxetine: aceclofenac, amlodipine, doxazosin, and moxonidine. We additionally demonstrated cognitive effects of moxonidine in mice using a T-maze spontaneous alternation task. Moxonidine, has high affinity for imidazoline 1 receptors. We, therefore, assayed a pure imidazoline 1 agonist, LNP599, which generated an effect closely matching other non-stimulant ADHD therapeutics suggesting a role for this receptor system in ADHD. In summary, we introduce a genetic model of ADHD in zebrafish and identify five putative therapeutics. The findings offer a novel tool for understanding the neural circuits of ADHD, suggest a novel mechanism for its etiology, and identify novel therapeutics.

Zebrafish Optokinetic Reflex: Minimal Reporting Guidelines and Recommendations.

Optokinetic reflex (OKR) assays in zebrafish models are a valuable tool for studying a diverse range of ophthalmological and neurological conditions. Despite its increasing popularity in recent years, there are no clear reporting guidelines for the assay. Following reporting guidelines in research enhances reproducibility, reduces bias, and mitigates underreporting and poor methodologies in published works. To better understand optimal reporting standards for an OKR assay in zebrafish, we performed a systematic literature review exploring the animal, environmental, and technical factors that should be considered. Using search criteria from three online databases, a total of 109 research papers were selected for review. Multiple crucial factors were identified, including larval characteristics, sample size, fixing method, OKR set-up, distance of stimulus, detailed stimulus parameters, eye recording, and eye movement analysis. The outcome of the literature analysis highlighted the insufficient information provided in past research papers and the lack of a systematic way to present the parameters related to each of the experimental factors. To circumvent any future errors and champion robust transparent research, we have created the zebrafish optokinetic (ZOK) reflex minimal reporting guideline.

Transcriptomic underpinnings of high and low mirror aggression zebrafish behaviours.

BACKGROUND: Aggression is an adaptive behaviour that animals use to protect offspring, defend themselves and obtain resources. Zebrafish, like many other animals, are not able to recognize themselves in the mirror and typically respond to their own reflection with aggression. However, mirror aggression is not an all-or-nothing phenomenon, with some individuals displaying high levels of aggression against their mirror image, while others show none at all. In the current work, we have investigated the genetic basis of mirror aggression by using a classic forward genetics approach - selective breeding for high and low mirror aggression zebrafish (HAZ and LAZ). RESULTS: We characterized AB wild-type zebrafish for their response to the mirror image. Both aggressive and non-aggressive fish were inbred over several generations. We found that HAZ were on average more aggressive than the corresponding LAZ across generations and that the most aggressive adult HAZ were less anxious than the least aggressive adult LAZ after prolonged selective breeding. RNAseq analysis of these fish revealed that hundreds of protein-encoding genes with important diverse biological functions such as arsenic metabolism (as3mt), cell migration (arl4ab), immune system activity (ptgr1), actin cytoskeletal remodelling (wdr1), corticogenesis (dgcr2), protein dephosphorylation (ublcp1), sialic acid metabolism (st6galnac3) and ketone body metabolism (aacs) were differentially expressed between HAZ and LAZ, suggesting a strong genetic contribution to this phenotype. DAVID pathway analysis showed that a number of diverse pathways are enriched in HAZ over LAZ including pathways related to immune function, oxidation-reduction processes and cell signalling. In addition, weighted gene co-expression network analysis (WGCNA) identified 12 modules of highly correlated genes that were significantly associated with aggression duration and/or experimental group. CONCLUSIONS: The current study shows that selective breeding based of the mirror aggression phenotype induces strong, heritable changes in behaviour and gene expression within the brain of zebrafish suggesting a strong genetic basis for this behaviour. Our transcriptomic analysis of fish selectively bred for high and low levels of mirror aggression revealed specific transcriptomic signatures induced by selective breeding and mirror aggression and thus provides a large and novel resource of candidate genes for future study.

Modelling ADHD-Like Phenotypes in Zebrafish.

The use of multiple species to model complex human psychiatric disorders, such as ADHD, can give important insights into conserved evolutionary patterns underlying multidomain behaviors (e.g., locomotion, attention, and impulsivity). Here we discuss the advantages and challenges in modelling ADHD-like phenotypes in zebrafish (Danio rerio), a vertebrate species that has been widely used in neuroscience and behavior research. Moreover, multiple behavioral tasks can be used to model the core symptoms of ADHD and its comorbidities. We present a critical review of current ADHD studies in zebrafish, and how this species might be used to accelerate the discovery of new drug treatments for this disorder.

Deficiency of the ywhaz gene, involved in neurodevelopmental disorders, alters brain activity and behaviour in zebrafish.

Genetic variants in YWHAZ contribute to psychiatric disorders such as autism spectrum disorder and schizophrenia, and have been related to an impaired neurodevelopment in humans and mice. Here, we have used zebrafish to investigate the mechanisms by which YWHAZ contributes to neurodevelopmental disorders. We observed that ywhaz expression was pan-neuronal during developmental stages and restricted to Purkinje cells in the adult cerebellum, cells that are described to be reduced in number and size in autistic patients. We then performed whole-brain imaging in wild-type and ywhaz CRISPR/Cas9 knockout (KO) larvae and found altered neuronal activity and connectivity in the hindbrain. Adult ywhaz KO fish display decreased levels of monoamines in the hindbrain and freeze when exposed to novel stimuli, a phenotype that can be reversed with drugs that target monoamine neurotransmission. These findings suggest an important role for ywhaz in establishing neuronal connectivity during development and modulating both neurotransmission and behaviour in adults.

Using zebrafish (Danio rerio) models to understand the critical role of social interactions in mental health and wellbeing.

Social behavior represents a beneficial interaction between conspecifics that is critical for maintaining health and wellbeing. Dysfunctional or poor social interaction are associated with increased risk of physical (e.g., vascular) and psychiatric disorders (e.g., anxiety, depression, and substance abuse). Although the impact of negative and positive social interactions is well-studied, their underlying mechanisms remain poorly understood. Zebrafish have well-characterized social behavior phenotypes, high genetic homology with humans, relative experimental simplicity and the potential for high-throughput screens. Here, we discuss the use of zebrafish as a candidate model organism for studying the fundamental mechanisms underlying social interactions, as well as potential impacts of social isolation on human health and wellbeing. Overall, the growing utility of zebrafish models may improve our understanding of how the presence and absence of social interactions can differentially modulate various molecular and physiological biomarkers, as well as a wide range of other behaviors.

The potential for adverse effects in fish exposed to antidepressants in the aquatic environment.

Antidepressants are one of the most commonly prescribed pharmaceutical classes for the treatment of psychiatric conditions. They act via modulation of brain monoaminergic signaling systems (predominantly serotonergic, adrenergic, dopaminergic) that show a high degree of structural conservation across diverse animal phyla. A reasonable assumption, therefore, is that exposed fish and other aquatic wildlife may be affected by antidepressants released into the natural environment. Indeed, there are substantial data reported for exposure effects in fish, albeit most are reported for exposure concentrations exceeding those occurring in natural environments. From a critical analysis of the available evidence for effects in fish, risk quotients (RQs) were derived from laboratory-based studies for a selection of antidepressants most commonly detected in the aquatic environment. We conclude that the likelihood for effects in fish on standard measured end points used in risk assessment (i.e., excluding effects on behavior) is low for levels of exposure occurring in the natural environment. Nevertheless, some effects on behavior have been reported for environmentally relevant exposures, and antidepressants can bioaccumulate in fish tissues. Limitations in the datasets used to calculate RQs revealed important gaps in which future research should be directed to more accurately assess the risks posed by antidepressants to fish. Developing greater certainty surrounding risk of antidepressants to fish requires more attention directed toward effects on behaviors relating to individual fitness, the employment of environmentally realistic exposure levels, on chronic exposure scenarios, and on mixtures analyses, especially given the wide range of similarly acting compounds released into the environment.

Chip Protein U-Box Domain Truncation Affects Purkinje Neuron Morphology and Leads to Behavioral Changes in Zebrafish.

The ubiquitin ligase CHIP (C-terminus of Hsc70-interacting protein) is encoded by STUB1 and promotes ubiquitination of misfolded and damaged proteins. CHIP deficiency has been linked to several diseases, and mutations in the human STUB1 gene are associated with recessive and dominant forms of spinocerebellar ataxias (SCAR16/SCA48). Here, we examine the effects of impaired CHIP ubiquitin ligase activity in zebrafish (Danio rerio). We characterized the zebrafish stub1 gene and Chip protein, and generated and characterized a zebrafish mutant causing truncation of the Chip functional U-box domain. Zebrafish stub1 has a high degree of conservation with mammalian orthologs and was detected in a wide range of tissues in adult stages, with highest expression in brain, eggs, and testes. In the brain, stub1 mRNA was predominantly detected in the cerebellum, including the Purkinje cell layer and granular layer. Recombinant wild-type zebrafish Chip showed ubiquitin ligase activity highly comparable to human CHIP, while the mutant Chip protein showed impaired ubiquitination of the Hsc70 substrate and Chip itself. In contrast to SCAR16/SCA48 patients, no gross cerebellar atrophy was evident in mutant fish, however, these fish displayed reduced numbers and sizes of Purkinje cell bodies and abnormal organization of Purkinje cell dendrites. Mutant fish also had decreased total 26S proteasome activity in the brain and showed behavioral changes. In conclusion, truncation of the Chip U-box domain leads to impaired ubiquitin ligase activity and behavioral and anatomical changes in zebrafish, illustrating the potential of zebrafish to study STUB1-mediated diseases.

The impact of chronic unpredictable early-life stress (CUELS) on boldness and stress-reactivity: Differential effects of stress duration and context of testing.

Early-life stress (ELS) has been shown to result in a diverse array of long-lasting impacts; for example, increasing vulnerability to disease or building 'resilience' in adulthood. Previously, zebrafish (Danio rerio) have been used to understand the mechanisms by which ELS induces different behavioral phenotypes in adults, with alterations in both learning and anxiety observed in exposed individuals. Here, we subjected zebrafish larvae to chronic unpredictable early-life stress (CUELS) for 7 or 14 days, to investigate the impact on boldness towards a new environment and novel object, and stress-reactivity. We observed that 7 days of CUELS resulted in increased time spent in the top of a novel tank (indicating boldness) but did not alter approach to a novel object. Although CUELS did not affect stress-reactivity in terms of cortisol levels, decreased anxiety-like response to conspecific alarm substance (CAS) was observed in both ELS groups (7 and 14 days of CUELS). Therefore, for the first time, we observe a potential negative effect of CUELS by dampening the behavioral stress response following exposure to CAS. Overall, these data support the use of zebrafish as a translational model to study the broad range of ELS-induced permanent changes in behavior. It could also be used to investigate the mechanisms underlying both the positive and the negative effects of early-life adversity.

Cysteine Modification by Ebselen Reduces the Stability and Cellular Levels of 14-3-3 Proteins.

The 14-3-3 proteins constitute a family of adaptor proteins with many binding partners and biological functions, and they are considered promising drug targets in cancer and neuropsychiatry. By screening 1280 small-molecule drugs using differential scanning fluorimetry (DSF), we found 15 compounds that decreased the thermal stability of 14-3-3ζ Among these compounds, ebselen was identified as a covalent, destabilizing ligand of 14-3-3 isoforms ζ, ε, γ, and η Ebselen bonding decreased 14-3-3ζ binding to its partner Ser19-phosphorylated tyrosine hydroxylase. Characterization of site-directed mutants at cysteine residues in 14-3-3ζ (C25, C94, and C189) by DSF and mass spectroscopy revealed covalent modification by ebselen of all cysteines through a selenylsulfide bond. C25 appeared to be the preferential site of ebselen interaction in vitro, whereas modification of C94 was the main determinant for protein destabilization. At therapeutically relevant concentrations, ebselen and ebselen oxide caused decreased 14-3-3 levels in SH-SY5Y cells, accompanied with an increased degradation, most probably by the ubiquitin-dependent proteasome pathway. Moreover, ebselen-treated zebrafish displayed decreased brain 14-3-3 content, a freezing phenotype, and reduced mobility, resembling the effects of lithium, consistent with its proposed action as a safer lithium-mimetic drug. Ebselen has recently emerged as a promising drug candidate in several medical areas, such as cancer, neuropsychiatric disorders, and infectious diseases, including coronavirus disease 2019. Its pleiotropic actions are attributed to antioxidant effects and formation of selenosulfides with critical cysteine residues in proteins. Our work indicates that a destabilization of 14-3-3 may affect the protein interaction networks of this protein family, contributing to the therapeutic potential of ebselen. SIGNIFICANCE STATEMENT: There is currently great interest in the repurposing of established drugs for new indications and therapeutic targets. This study shows that ebselen, which is a promising drug candidate against cancer, bipolar disorder, and the viral infection coronavirus disease 2019, covalently bonds to cysteine residues in 14-3-3 adaptor proteins, triggering destabilization and increased degradation in cells and intact brain tissue when used in therapeutic concentrations, potentially explaining the behavioral, anti-inflammatory, and antineoplastic effects of this drug.

Chronic unpredictable early-life stress (CUELS) protocol: Early-life stress changes anxiety levels of adult zebrafish.

Early-life stress can lead to two different behavioral responses: (1) increased susceptibility to psychiatric disorders or (2) resilience. Here, we created a chronic unpredictable early-life stress (CUELS) protocol to assess the effects of early experiences in adult zebrafish. Animals were exposed to mild stressors twice a day and the duration was varied between groups (0, 1, 3, 7 and 14 days of stress). The stressor consisted of light/dark cycle changes; social isolation; overcrowding; water changes; water cooling; mechanical stirring; water heating; and immersion in shallow water. Behavior was assessed at young stages (21 days post-fertilization - open field analysis) and adulthood (4-months-old - novel tank diving test, light/dark task, shoaling, free movement pattern Y-maze and Pavlovian fear conditioning). Cortisol levels were assessed to evaluate the impact of CUELS in the HPI axis. Zebrafish exposed to 7 days of CUELS showed a decreased anxiety-like phenotype in two behavioral tasks, presenting increased time spent in top and decreased time spent in the dark area. Animals exposed to 14 days of CUELS showed an opposite anxious phenotype compared to 3 and 7 days of CUELS. No significant changes were observed in memory and cognition, social behavior and cortisol levels. In general, 7 days of CUELS protocol decreased anxiety in young and adult zebrafish, and could be used to understand the mechanisms underlying early-life experiences-derived alterations in neural circuits of anxiety.

Skin swabbing is a refined technique to collect DNA from model fish species.

Model fish species such as sticklebacks and zebrafish are frequently used in studies that require DNA to be collected from live animals. This is typically achieved by fin clipping, a procedure that is simple and reliable to perform but that can harm fish. An alternative procedure to sample DNA involves swabbing the skin to collect mucus and epithelial cells. Although swabbing appears to be less invasive than fin clipping, it still requires fish to be netted, held in air and handled-procedures that can cause stress. In this study we combine behavioural and physiological analyses to investigate changes in gene expression, behaviour and welfare after fin clipping and swabbing. Swabbing led to a smaller change in cortisol release and behaviour on the first day of analysis compared to fin clipping. It also led to less variability in data suggesting that fewer animals need to be measured after using this technique. However, swabbing triggered some longer term changes in zebrafish behaviour suggesting a delayed response to sample collection. Skin swabbing does not require the use of anaesthetics and triggers fewer changes in behaviour and physiology than fin clipping. It is therefore a more refined technique for DNA collection with the potential to improve fish health and welfare.

The zebrafish histamine H3 receptor modulates aggression, neural activity and forebrain functional connectivity.

AIM: Aggression is a behavioural trait characterized by the intention to harm others for offensive or defensive purposes. Neurotransmitters such as serotonin and dopamine are important mediators of aggression. However, the physiological role of the histaminergic system during this behaviour is currently unclear. Here, we aimed to better understand histaminergic signalling during aggression by characterizing the involvement of the histamine H3 receptor (Hrh3). METHODS: We have generated a novel zebrafish Hrh3 null mutant line using CRISPR-Cas9 genome engineering and investigated behavioural changes and alterations to neural activity using whole brain Ca2+ imaging in zebrafish larvae and ribosomal protein S6 (rpS6) immunohistochemistry in adults. RESULTS: We show that genetic inactivation of the histamine H3 receptor (Hrh3) reduces aggression in zebrafish, an effect that can be reproduced by pharmacological inhibition. In addition, hrh3-/- zebrafish show behavioural impairments consistent with heightened anxiety. Larval in vivo whole brain Ca2+ imaging reveals higher neuronal activity in the forebrain of mutants, but lower activity in specific hindbrain areas and changes in measures of functional connectivity between subregions. Adult hrh3-/- zebrafish display brain region-specific neural activity changes in response to aggression of both key regions of the social decision-making network, and the areas containing histaminergic neurons in the zebrafish brain. CONCLUSION: These results highlight the importance of zebrafish Hrh3 signalling for aggression and anxiety and uncover the brain areas involved. Targeting this receptor might be a potential novel therapeutic route for human conditions characterized by heightened aggression.