Article

Acute ethanol induces behavioral changes and alters c-fos expression in specific brain regions, including the mammillary body, in zebrafish

Article

Acute ethanol induces behavioral changes and alters c-fos expression in specific brain regions, including the mammillary body, in zebrafish

If you want to read the PDF, try requesting it from the authors.

Abstract

Ethanol is one of the most commonly abused substances in the world, and ethanol abuse and dependence disorders represent major societal problems. However, appropriate treatment is lacking as we still do not fully understand the molecular bases of these disorders. The zebrafish is one of the model organisms utilized for studying such mechanisms. In this study, we examined the effects of acute ethanol administration on the behavior of zebrafish, and we also analyzed correlated gene expression changes using whole-mount in situ hybridization focusing on a number of genes associated with different neurotransmitter systems, stress response, and neuronal activity. We found ethanol treatment to result in hyperactivity and reduced shoal cohesion compared to control. Analysis of c-fos expression demonstrated altered activity patterns in certain brain regions, including intense activation of the mammillary body in zebrafish with acute ethanol treatment. We also found reduced level of gad1b expression in the cerebellum of ethanol treated fish compared to control. However, we could not detect significant changes in the expression level of other genes, including vglut2b, th, crh, hdc, avp, pomc, and galn in ethanol treated fish compared controls. Our results suggest that zebrafish is a promising animal model for the study of mechanisms underlying alcohol induced behavioral changes and alcohol related human disorders.

No full-text available

Request Full-text Paper PDF

To read the full-text of this research,
you can request a copy directly from the authors.

Article
Mepanipyrim, an anilinopyrimidine fungicide, has been extensively used to prevent fungal diseases in fruit culture. Currently, research on mepanipyrim-induced toxicity in organisms is still very scarce, especially visual developmental toxicity. Here, zebrafish larvae were employed to investigate mepanipyrim-induced visual developmental toxicity. Intense light and monochromatic light stimuli-evoked escape experiments were used to investigate vision-guided behaviors. Meanwhile, transcriptomic sequencing and real-time quantitative PCR assays were applied to assess the potential mechanisms of mepanipyrim-induced visual developmental toxicity and vision-guided behavioral alteration. Our results showed that mepanipyrim exposure could induce retinal impairment and vision-guided behavioral alteration in larval zebrafish. In addition, the grk1b gene of the phototransduction signaling pathway was found to be a potential aryl hydrocarbon receptor (AhR)-regulated gene. Mepanipyrim-induced visual developmental toxicity was potentially related to the AhR signaling pathway. Furthermore, mepanipyrim-induced behavioral alteration was guided by the visual function, and the effects of mepanipyrim on long and middle wavelength light-sensitive opsins may be the main cause of vision-guided behavioral alteration. Our results provide insights into understanding the relationship between visual development and vision-guided behaviors induced by mepanipyrim exposure.
Article
Full-text available
Social cognition and social behaviors are complex phenomena that involve numerous brain areas and underlying neurobiological mechanisms. Embryonic alcohol exposure may lead to the development of Fetal Alcohol Spectrum Disorder (FASD), a disorder that manifests with varying symptoms including abnormal social behavior and other cognitive deficits. Animal models have been utilized to mimic aspects of the disease and to study potential underlying mechanisms. The zebrafish is a relative newcomer in this field but has been suggested as an optimal compromise between system complexity and practical simplicity for modeling FASD. Importantly, due to external fertilization and development of the embryo outside the mother and subsequent lack of parental care, this species allows precise control of the timing and dose of alcohol delivery during embryonic development. Furthermore, the zebrafish is a highly social species and thus may be particularly appropriate for the analysis of embryonic alcohol-induced alterations in this context. Here, we provide a succinct review focusing on shoaling, a prominent form of social behavior, in zebrafish. We summarize what is known about its behavioral mechanisms and underlying neurobiological processes, and how it is altered by exposure to ethanol during embryonic development. Lastly, we briefly consider possible future directions of research that would help us better understand the relationship between the behavioral expression and molecular basis of embryonic ethanol-induced social deficits in fish and humans.
Article
Full-text available
Schizophrenia is a severe disorder characterized by positive, negative and cognitive symptoms, which are still not fully understood. The development of efficient antipsychotics requires animal models of a strong validity, therefore the aims of the article were to summarize the construct, face and predictive validity of schizophrenia models based on rodents and zebrafish, to compare the advantages and disadvantages of these models, and to propose future directions in schizophrenia modeling and indicate when it is reasonable to combine these models. The advantages of rodent models stem primarily from the high homology between rodent and human physiology, neurochemistry, brain morphology and circuitry. The advantages of zebrafish models stem in the high fecundity, fast development and transparency of the embryo. Disadvantages of both models originate in behavioral repertoires not allowing specific symptoms to be modeled, even when the models are combined. Especially modeling the verbal component of certain positive, negative and cognitive symptoms is currently impossible.
Article
Full-text available
Zebrafish ( Danio rerio ) are quickly becoming an important model organism in behavioural neuroscience and drug addiction research. Conditioned place preference studies show that drugs of abuse produce responses in zebrafish that are similar to mammalian animal models. Repeated administration of ethanol in zebrafish results in withdrawal-induced behavioural responses that vary with dose and exposure duration, requiring additional investigation. Here, we examine the effects of ethanol withdrawal on anxiety-like behaviours in adult zebrafish after a 21-day ethanol dosing schedule at either 0.4% or 0.8%. Anxiety-like behaviour was measured with the novel object approach test; this test involves placing a fish in a circular arena with a novel object in the centre and observing the amount of exploration of the object. We found increased anxiety-like behaviour during ethanol withdrawal. This study adds to the growing body of literature that validates the zebrafish as a model organism in the field of behavioural neuroscience and addiction.
Article
Full-text available
Hypocretins/Orexins neuropeptides are known to regulate numerous physiological functions, such as energy homeostasis, food intake, sleep/wake cycle, arousal and wakefulness, in vertebrates. Previous studies on mice have revealed an intriguing orexins/endocannabinoids (ECs) signaling interaction at both structural and functional levels, with OX-A behaving as a strong enhancer of 2-arachydonoyl-glycerol (2-AG) biosynthesis. In this study, we describe, for the first time in the brain of zebrafish, the anatomical distribution and co-expression of orexin (OX-2R) and endocannabinoid (CB1R) receptors, suggesting a functional interaction. The immunohistochemical colocalization of these receptors by confocal imaging in the dorsal and ventral telencephalon, suprachiasmatic nucleus (SC), thalamus, hypothalamus, preoptic area (PO) and cerebellum, is reported. Moreover, biochemical quantification of 2-AG levels by LC-MS supports the occurrence of OX-A-induced 2-AG biosynthesis in the zebrafish brain after 3 h of OX-A intraperitoneal (i.p.; 3 pmol/g) or intracerebroventricular (i.c.v.; 0.3 pmol/g) injection. This effect is likely mediated by OX-2R as it is counteracted by i.p./i.c.v administration of OX-2R antagonist (SB334867, 10 pmol/g). This study provides compelling morphological and functional evidence of an OX-2R/CB1R signaling interaction in the brain of adult zebrafish, suggesting the use of this well-established vertebrate animal model for the study of complex and phylogenetically conserved physiological functions.
Article
Full-text available
Emotional responses, such as fear and anxiety, are fundamentally important behavioral phenomena with strong fitness components in most animal species. Anxiety-related disorders continue to represent a major unmet medical need in our society, mostly because we still do not fully understand the mechanisms of these diseases. Animal models may speed up discovery of these mechanisms. The zebrafish is a highly promising model organism in this field. Here, we report the identification of a chemokine-like gene family, samdori (sam), and present functional characterization of one of its members, sam2 We show exclusive mRNA expression of sam2 in the CNS, predominantly in the dorsal habenula, telencephalon, and hypothalamus. We found knockout (KO) zebrafish to exhibit altered anxiety-related responses in the tank, scototaxis and shoaling assays, and increased crh mRNA expression in their hypothalamus compared with wild-type fish. To investigate generalizability of our findings to mammals, we developed a Sam2 KO mouse and compared it to wild-type littermates. Consistent with zebrafish findings, homozygous KO mice exhibited signs of elevated anxiety. We also found bath application of purified SAM2 protein to increase inhibitory postsynaptic transmission onto CRH neurons of the paraventricular nucleus. Finally, we identified a human homolog of SAM2, and were able to refine a candidate gene region encompassing SAM2, among 21 annotated genes, which is associated with intellectual disability and autism spectrum disorder in the 12q14.1 deletion syndrome. Taken together, these results suggest a crucial and evolutionarily conserved role of sam2 in regulating mechanisms associated with anxiety.
Article
Full-text available
Scopolamine (hyoscine) is a muscarinic acetylcholine receptor antagonist that has traditionally been used to treat motion sickness in humans. However, studies investigating depressed and bipolar populations have found that scopolamine is also effective at reducing depression and anxiety symptoms. The potential anxiety-reducing (anxiolytic) effects of scopolamine could have great clinical implications for humans; however, rats and mice administered scopolamine showed increased anxiety in standard behavioural tests. This is in direct contrast to findings in humans, and complicates studies to elucidate the specific mechanisms of scopolamine action. The aim of this study was to assess the suitability of zebrafish as a model system to test anxiety-like compounds using scopolamine. Similar to humans, scopolamine acted as an anxiolytic in individual behavioural tests (novel approach test and novel tank diving test). The anxiolytic effect of scopolamine was dose dependent and biphasic, reaching maximum effect at 800 μM. Scopolamine (800 μM) also had an anxiolytic effect in a group behavioural test, as it significantly decreased their tendency to shoal. These results establish zebrafish as a model organism for studying the anxiolytic effects of scopolamine, its mechanisms of action and side effects.
Article
Full-text available
Modafinil (2-((diphenylmethyl)sulfinyl)acetamide), a selective dopamine and norepinephrine transporter inhibitor, is most commonly prescribed for narcolepsy but has gained recent interest for treating a variety of disorders. Zebrafish ( Danio rerio) are becoming a model of choice for pharmacological and behavioural research. To investigate the behavioural effects of modafinil on anxiety, we administered doses of 0, 2, 20, and 200 mg/L for 30 minutes then tested zebrafish in the novel approach test. In this test, the fish was placed into a circular arena with a novel object in the center and motion-tracking software was used to quantify the time the fish spent in the outer area of the arena (thigmotaxis zone), middle third of the arena (transition zone) and center of the arena, as well as total distance traveled, immobility and meandering. Modafinil caused a decrease in time spent in the thigmotaxis zone and increased time spent in the transition zone across all doses. Modafinil did not significantly alter the time spent in the center zone (near the novel object), the distance moved, meandering, or the duration of time spent immobile. We also validated this test as a measure of anxiety with the administration of ethanol (1%) which decreased time spent in the thigmotaxis zone and increased time spent in the transition zone. These results suggest that modafinil decreases anxiety-like behaviour in zebrafish.
Article
Full-text available
Background: Dysfunction of the glutamatergic system has been implicated in alcohol addiction; however, the molecular underpinnings of this phenomenon are still poorly understood. In the current study we have investigated the possible function of matrix metalloproteinase-9 (MMP-9) in alcohol addiction because this protein has recently emerged as an important regulator of excitatory synaptic plasticity. Methods: For long-term studies of alcohol drinking in mice we used IntelliCages. Dendritic spines were analyzed using Diolistic staining with DiI. Whole-cell patch clamp was used to assess silent synapses. Motivation for alcohol in human subjects was assessed on the basis of a Semi-Structured Assessment for the Genetics of Alcoholism interview. Results: Mice devoid of MMP-9 (MMP-9 knockout) drank as much alcohol as wild-type animals; however, they were impaired in alcohol seeking during the motivation test and withdrawal. The deficit could be rescued by overexpression of exogenous MMP-9 in the central nucleus of the amygdala (CeA). Furthermore, the impaired alcohol seeking was associated with structural alterations of dendritic spines in the CeA and, moreover, whole-cell patch clamp analysis of the basal amygdala to CeA projections showed that alcohol consumption and withdrawal were associated with generation of silent synapses. These plastic changes were impaired in MMP-9 knockout mice. Finally, C/T polymorphism of MMP-9 gene at position -1562, which upregulates MMP-9 expression, correlated with increased motivation for alcohol in alcoholics. Conclusions: In aggregate, our results indicate a novel mechanism of alcohol craving that involves MMP-9-dependent synaptic plasticity in CeA.
Article
Full-text available
Although innate color preference of motile organisms may provide clues to behavioral biases, it has remained a longstanding question. In this study, we investigated innate color preference of zebrafish larvae. A cross maze with different color sleeves around each arm was used for the color preference test (R; red, G; green, B; blue, Y; yellow). The findings showed that 5 dpf zebrafish larvae preferred blue over other colors (B > R > G > Y). To study innate color recognition further, tyrosinase mutants were generated using CRISPR/Cas9 system. As a model for oculocutaneous albinism (OCA) and color vision impairment, tyrosinase mutants demonstrated diminished color sensation, indicated mainly by hypopigmentation of the retinal pigment epithelium (RPE). Due to its relative simplicity and ease, color preference screening using zebrafish larvae is suitable for highthroughput screening applications. This system may potentially be applied to the analysis of drug effects on larval behavior or the detection of sensory deficits in neurological disorder models, such as autism-related disorders, using mutant larvae generated by the CRISPR/Cas9 technique.
Article
Full-text available
Background: The telencephalon shows a remarkable structural diversity among vertebrates. In particular, the everted telencephalon of ray-finned fishes has a markedly different morphology compared to the evaginated telencephalon of all other vertebrates. This difference in development has hampered the comparison between different areas of the pallium of ray-finned fishes and the pallial nuclei of all other vertebrates. Various models of homology between pallial subdivisions in ray-finned fishes and the pallial nuclei in tetrapods have been proposed based on connectional, neurochemical, gene expression and functional data. However, no consensus has been reached so far. In recent years, the analysis of conserved developmental marker genes has assisted the identification of homologies for different parts of the telencephalon among several tetrapod species. Results: We have investigated the gene expression pattern of conserved marker genes in the adult zebrafish (Danio rerio) pallium to identify pallial subdivisions and their homology to pallial nuclei in tetrapods. Combinatorial expression analysis of ascl1a, eomesa, emx1, emx2, emx3, and Prox1 identifies four main divisions in the adult zebrafish pallium. Within these subdivisions, we propose that Dm is homologous to the pallial amygdala in tetrapods and that the dorsal subdivision of Dl is homologous to part of the hippocampal formation in mouse. We have complemented this analysis be examining the gene expression of emx1, emx2 and emx3 in the zebrafish larval brain. Conclusions: Based on our gene expression data, we propose a new model of subdivisions in the adult zebrafish pallium and their putative homologies to pallial nuclei in tetrapods. Pallial nuclei control sensory, motor, and cognitive functions, like memory, learning and emotion. The identification of pallial subdivisions in the adult zebrafish and their homologies to pallial nuclei in tetrapods will contribute to the use of the zebrafish system as a model for neurobiological research and human neurodegenerative diseases.
Article
Full-text available
Fetal Alcohol Spectrum Disorders (FASD) describes a wide array of ethanol-induced developmental defects, including craniofacial dysmorphology and cognitive impairments. It affects ∼1 in 100 children born in the United States each year. Due to the pleiotropic effects of ethanol, animal models have proven critical in characterizing the mechanisms of ethanol teratogenesis. In this review, we focus on the utility of zebrafish in characterizing ethanol-induced developmental defects. A growing number of laboratories have focused on using zebrafish to examine ethanol-induced defects in craniofacial, cardiac, ocular, and neural development, as well as cognitive and behavioral impairments. Growing evidence supports that genetic predisposition plays a role in these ethanol-induced defects, yet little is understood about these gene–ethanol interactions. With a high degree of genetic amenability, zebrafish is at the forefront of identifying and characterizing the gene–ethanol interactions that underlie FASD. Because of the conservation of gene function between zebrafish and humans, these studies will directly translate to studies of candidate genes in human populations and allow for better diagnosis and treatment of FASD.
Article
Full-text available
Larval zebrafish present unique opportunities to study the behavioral responses of a model organism to environmental challenges during early developmental stages. The purpose of the current study was to investigate the locomotor activities of AB strain zebrafish larvae at 5 and 7 days post-fertilization (dpf) in response to light changes under the influence of ethanol, and to explore potential neurological mechanisms that are involved in ethanol intoxication. AB strain zebrafish larvae at both 5 and 7 dpf were treated with ethanol at 0% (control), 0.1%, 0.25%, 0.5%, 1%, and 2% (v/v%). The locomotor activities of the larvae during alternating light-dark challenges, as well as the locomotor responses immediately following the light transitions, were investigated. The levels of various neurotransmitters were also measured in selected ethanol-treated groups. The larvae at 5 and 7 dpf demonstrated similar patterns of locomotor responses to ethanol treatment. Ethanol treatment at 1% increased the swimming distances of the zebrafish larvae in the dark periods, but had no effect on the swimming distances in the light periods. In contrast, ethanol treatment at 2% increased the swimming distances in the light periods, but did not potentiate the swimming activity in the dark periods, compared to controls. Differences in the levels of neurotransmitters that are involved in norepinephrine, dopamine, and serotonin pathways were also observed in groups with different ethanol treatments. These results indicated the behavioral studies concerning the ethanol effects on locomotor activities of zebrafish larvae could be carried out as early as 5 dpf. The 1% and 2% ethanol-treated zebrafish larvae modeled ethanol effects at different intoxication states, and the differences in neurotransmitter levels suggested the involvement of various neurotransmitter pathways in different ethanol intoxication states.
Article
Full-text available
The zebrafish has become an increasingly popular animal model for investigating ethanol's actions in the brain and its effects on behavior. Acute exposure to ethanol in zebrafish has been shown to induce a dose-dependent increase of locomotor activity, to reduce fear- and anxiety-related behavioral responses, and to increase the levels of dopamine and its metabolite 3,4-dihydroxyphenylacetic acid (DOPAC). The objective of the present study was to investigate the role of dopamine D1 receptors (D1-R) in ethanol-induced locomotor activity in zebrafish. Zebrafish were pre-treated with SCH-23390 (0 or 1 mg/L bath concentration), a D1-R antagonist, and subsequently exposed to ethanol (0, 0.25, 0.5, 1.0 % v/v). To explore potential underlying mechanisms, we quantified levels of dopamine, DOPAC, serotonin, and 5-HIAA from whole-brain tissue using high-precision liquid chromatography. We found pre-treatment with the D1-R antagonist to attenuate locomotor activity independent of ethanol concentration. Furthermore, unlike ethanol, D1-R antagonism did not alter behavioral responses associated with fear and anxiety. Pre-treatment with SCH-23390 decreased levels of dopamine and DOPAC, but this effect was also independent of ethanol concentration. The D1-R antagonist also reduced serotonin and 5-hydroxyindole acetic acid (5-HIAA) levels. These results suggest a multifaceted and at least partially independent role of dopamine D1 receptors in ethanol-induced locomotor activity and anxiety-related responses as well as in the functioning of the dopaminergic and serotoninergic neurotransmitter systems in zebrafish.
Article
Full-text available
Natural environment imposes many challenges to animals, which have to use cognitive abilities to cope with and exploit it to enhance their fitness. Since zebrafish is a well-established model for cognitive studies and high-throughput screening for drugs and diseases that affect cognition, we tested their ability for ambient color preference and 3D objects discrimination to establish a protocol for memory evaluation. For the color preference test, zebrafish were observed in a multiple-chamber tank with different environmental color options. Zebrafish showed preference for blue and green, and avoided yellow and red. For the 3D objects discrimination, zebrafish were allowed to explore two equal objects and then observed in a one-trial test in which a new color, size, or shape of the object was presented. Zebrafish showed discrimination for color, shape, and color+shape combined, but not size. These results imply that zebrafish seem to use some categorical system to discriminate items, and distracters affect their ability for discrimination. The type of variables available (color and shape) may favor zebrafish objects perception and facilitate discrimination processing. We suggest that this easy and simple memory test could serve as a useful screening tool for cognitive dysfunction and neurotoxicological studies.
Article
Full-text available
Anxiety testing in zebrafish is often studied in combination with the application of pharmacological substances. In these studies, fish are routinely netted and transported between home aquaria and dosing tanks. In order to enhance the ease of compound administration, a novel method for transferring fish between tanks for drug administration was developed. Inserts that are designed for spawning were used to transfer groups of fish into the drug solution, allowing accurate dosing of all fish in the group. This increases the precision and efficiency of dosing, which becomes very important in long schedules of repeated drug administration. We implemented this procedure for use in a study examining the behavior of zebrafish in the light/dark test after administering ethanol with differing 21 day schedules. In fish exposed to daily-moderate amounts of alcohol there was a significant difference in location preference after 2 days of withdrawal when compared to the control group. However, a significant difference in location preference in a group exposed to weekly-binge administration was not observed. This protocol can be generalized for use with all types of compounds that are water-soluble and may be used in any situation when the behavior of fish during or after long schedules of drug administration is being examined. The light/dark test is also a valuable method of assessing withdrawal-induced changes in anxiety.
Article
Full-text available
Recent studies in zebrafish have shown that exposure to ethanol in tank water affects various behaviors, including locomotion, anxiety and aggression, and produces changes in brain neurotransmitters, such as serotonin and dopamine. Building on these investigations, the present study had two goals: first, to develop a method for inducing voluntary ethanol intake in individual zebrafish, which can be used as a model in future studies to examine how this behavior is affected by various manipulations, and second, to characterize the effects of this ethanol intake on different behaviors and the expression of hypothalamic orexigenic peptides, galanin (GAL) and orexin (OX), which are known in rodents to stimulate consumption of ethanol and alter behaviors associated with alcohol abuse. Thus, we first developed a new model of voluntary intake of ethanol in fish by presenting this ethanol mixed with gelatin, which they readily consume. Using this model, we found that individual zebrafish can be trained in a short period to consume stable levels of 10% or 20% ethanol (v/v) mixed with gelatin and that their intake of this ethanol-gelatin mixture leads to pharmacologically relevant blood ethanol concentrations which are strongly, positively correlated with the amount ingested. Intake of this ethanol-gelatin mixture increased locomotion, reduced anxiety, and stimulated aggressive behavior, while increasing expression of GAL and OX in specific hypothalamic areas. These findings, confirming results in rats, provide a method in zebrafish for investigating with forward genetics and pharmacological techniques the role of different brain mechanisms in controlling ethanol intake.
Article
Full-text available
Addiction and substance abuse are found ubiquitously throughout human society. In the United States, these disorders are responsible for amassing hundreds of billions of dollars in annual costs associated with healthcare, crime and lost productivity. Efficacious treatments remain few in number, the development of which will be facilitated by comprehension of environmental, genetic, pharmacological and neurobiological mechanisms implicated in the pathogenesis of addiction. Animal models such as the zebrafish (Danio rerio) have gained momentum within various domains of translational research, and as a model of complex brain disorders (e.g., drug abuse). Behavioral quantification within the conditioned place preference (CPP) paradigm serves as a measure of the rewarding qualities of a given substance. If the animal develops an increase in preference for the drug paired environment, it is inferred that the drug has positive-reinforcing properties. This paper discusses the utility of the zebrafish model in conjunction with the CPP paradigm and reports CPP behavior following acute exposure to 0.0%, 0.25%, 0.50%, and 1.00% alcohol, and 0mg/L, 50mg/L, 100mg/L and 150mg/L caffeine.
Article
Full-text available
The zebrafish (Danio rerio) is emerging as a new important species for studying mechanisms of brain function and dysfunction. Focusing on selected central nervous system (CNS) disorders (brain cancer, epilepsy, and anxiety) and using them as examples, we discuss the value of zebrafish models in translational neuroscience. We further evaluate the contribution of zebrafish to neuroimaging, circuit level, and drug discovery research. Outlining the role of zebrafish in modeling a wide range of human brain disorders, we also summarize recent applications and existing challenges in this field. Finally, we emphasize the potential of zebrafish models in behavioral phenomics and high-throughput genetic/small molecule screening, which is critical for CNS drug discovery and identifying novel candidate genes.
Article
Full-text available
Alcohol abuse and alcoholism incur a heavy socioeconomic cost in many countries. Both genetic and environmental factors contribute to variation in the inebriating effects of alcohol and alcohol addiction among individuals within and across populations. From a genetics perspective, alcohol sensitivity is a quantitative trait determined by the cumulative effects of multiple segregating genes and their interactions with the environment. This review summarizes insights from model organisms as well as human populations that represent our current understanding of the genetic and genomic underpinnings that govern alcohol metabolism and the sedative and addictive effects of alcohol on the nervous system. Electronic supplementary material The online version of this article (doi:10.1007/s00438-013-0808-y) contains supplementary material, which is available to authorized users.
Article
Full-text available
The effects of ethanol exposure on Danio rerio have been studied from the perspectives of developmental biology and behavior. However, little is known about the effects of ethanol on the prey-predator relationship and chemical communication of predation risk. Here, we showed that visual contact with a predator triggers stress axis activation in zebrafish. We also observed a typical stress response in zebrafish receiving water from these conspecifics, indicating that these fish chemically communicate predation risk. Our work is the first to demonstrate how alcohol effects this prey-predator interaction. We showed for the first time that alcohol exposure completely blocks stress axis activation in both fish seeing the predator and in fish that come in indirect contact with a predator by receiving water from these conspecifics. Together with other research results and with the translational relevance of this fish species, our data points to zebrafish as a promising animal model to study human alcoholism.
Article
Full-text available
The zebrafish is gaining popularity in behavioral brain research. It may be a cost-effective tool with which we can improve our understanding of the biological and genetic mechanisms of human brain function and dysfunction. Some, myself and collaborators included, have argued that such translational relevance may be best achieved if one considers the ecology and species-specific characteristics of the study organism. In this review, I focus on our own studies investigating zebrafish fear responses, which may be utilized in analyzing the mechanisms of fear and anxiety, and which may be used for screening anxiolytic drugs. I review how zebrafish respond to their natural and synthetic alarm substance as well as to other fear-inducing stimuli, including sympatric and allopatric predatory fish, sympatric or allopatric harmless fish, moving (animated) images of predatory fish and moving images of abstract shapes. I discuss the behavioral responses these stimuli elicit, summarize the methods of the quantification of the behaviors, and speculate about their possible adaptive nature. Although we utilize complex visual stimuli and do not yet know what key features zebrafish may be sensitive to, our results, together with those published by others, imply that this simple vertebrate may have a bright future in behavioral brain research.
Article
Full-text available
Earlier studies in zebrafish have revealed that acutely given ethanol has a stimulatory effect on locomotion in fish larvae but the mechanism of this effect has not been revealed. We studied the effects of ethanol concentrations between 0.75 and 3.00% on 7-day-old larval zebrafish (Danio rerio) of the Turku strain. At 0.75-3% concentrations ethanol increased swimming speed during the first minute. At 3% the swimming speed decreased rapidly after the first minute, whereas at 0.75 and 1.5% a prolonged increase in swimming speed was seen. At the highest ethanol concentration dopamine levels decreased significantly after a 10-min treatment. We found that ethanol upregulates key genes involved in the biosynthesis of histamine (hdc) and dopamine (th1 and th2) following a short 10-min ethanol treatment, measured by qPCR. Using in situ hybridization and immunohistochemistry, we further discovered that the morphology of the histaminergic and dopaminergic neurons and networks in the larval zebrafish brain was unaffected by both the 10-min and a longer 30-min treatment. The results suggest that acute ethanol rapidly decreases dopamine levels, and activates both forms or th to replenish the dopamine stores within 30 min. The dynamic changes in histaminergic and dopaminergic system enzymes occurred in the same cells which normally express the transcripts. As both dopamine and histamine are known to be involved in the behavioral effects of ethanol and locomotor stimulation, these results suggest that rapid adaptations of these networks are associated with altered locomotor activity.
Article
Full-text available
Zebrafish have become a popular organism for the study of vertebrate gene function. The virtually transparent embryos of this species, and the ability to accelerate genetic studies by gene knockdown or overexpression, have led to the widespread use of zebrafish in the detailed investigation of vertebrate gene function and increasingly, the study of human genetic disease. However, for effective modelling of human genetic disease it is important to understand the extent to which zebrafish genes and gene structures are related to orthologous human genes. To examine this, we generated a high-quality sequence assembly of the zebrafish genome, made up of an overlapping set of completely sequenced large-insert clones that were ordered and oriented using a high-resolution high-density meiotic map. Detailed automatic and manual annotation provides evidence of more than 26,000 protein-coding genes, the largest gene set of any vertebrate so far sequenced. Comparison to the human reference genome shows that approximately 70% of human genes have at least one obvious zebrafish orthologue. In addition, the high quality of this genome assembly provides a clearer understanding of key genomic features such as a unique repeat content, a scarcity of pseudogenes, an enrichment of zebrafish-specific genes on chromosome 4 and chromosomal regions that influence sex determination.
Article
Full-text available
Previous research reports that acute alcohol exposure disrupts shoaling behavior in the zebrafish. The purpose of these studies is to better understand how acute alcohol exposure (0%, 0.125%, 0.25%, 0.5%, and 1.0%) alters zebrafish behavior. The effects of alcohol on aggressive behaviors in humans have been widely researched. Previous research from this lab has shown a bimodal effect of alcohol on shoaling behavior in zebrafish, with 0.5% and 2.0% (v/v) disrupting shoaling while 1.0% and 1.5% showing no direct effect. Because shoaling is a social behavior and is altered during acute alcohol exposure, aggressive behavior between fish should be addressed. In this series of experiments we explored alcohol's effects on aggressive behaviors. In order to address a possible role for alcohol induced aggression as it relates to shoaling we chose to examine the effects of acute alcohol exposure on zebrafish pairs. Fish were assessed during initial encounters occurring in our testing apparatus during acute alcohol exposure. Results show a change in biting as a function of all doses. Acute alcohol exposure (0.5%) also decreases overall occurrences of chasing and retreating but may increase the duration of each bout. Lastly in a separate experiment we looked at blood alcohol levels as a result of acute alcohol exposure. Acute alcohol exposure has been shown to modulate a variety of behaviors in adult zebrafish. Specifically, locomotor activity and shoal cohesion are two behaviors that can change when zebrafish are acutely exposed to alcohol (Gerlai, Lahav, Guo, & Rosenthal, 2000). Data collected with rodent, human and primate subjects suggest that induced alterations of motor function follow a characteristic inverted U-shaped function, with intermediate doses of ethanol enhancing locomotor activity and higher doses suppressing it (for reviews see Barr, Schwandt, Newman, & Higley, 2004; Fogarty & Vogel-Sprott, 2002; McBride & Li, 1998). The effects of alcohol on aggressive behaviors in humans have been widely researched. According to a meta-analysis by Bushman and Cooper entitled "Alcohol and Human Aggression" (1990), alcohol induces aggressive behavior in humans as much as any other social or non-social behavior. Acute alcohol exposure influences social shoaling behavior in zebrafish and has been shown to influence aggression in other species, including humans. Thus, it is possible that aggression may play a role in the disruption of social behavior seen in zebrafish under acute alcohol exposure, yet this relationship has not been investigated. In order to address this, we chose to examine the effects of acute alcohol exposure on aggression in zebrafish pairs.
Article
Full-text available
For the past 25 years NIH Image and ImageJ software have been pioneers as open tools for the analysis of scientific images. We discuss the origins, challenges and solutions of these two programs, and how their history can serve to advise and inform other software projects.
Article
Fetal Alcohol Spectrum Disorders (FASD) represent a large unmet medical need. Exposure of the developing human embryo to alcohol can lead to life-long suffering. Despite the well documented deleterious effects of alcohol on the developing fetus, pregnant women continue to drink alcohol, and FASD remains the leading cause of preventable mental retardation and other behavioral abnormalities. Particularly prevalent are the milder forms of the disease cluster, representing children who do not show obvious physical signs and who may be undiagnosed or misdiagnosed. To develop treatment and diagnostic tools, researchers have turned to animal models. The zebrafish is becoming one of the leading biomedical research organisms that may facilitate discovery of the biological mechanisms underlying this disease and the identification of biomarkers that may be used for diagnosis. Here we review the latest advances of this field, mostly focussing on the discoveries made in our own laboratory and others with zebrafish employed to analyze the effects of moderate to low level of exposure to alcohol. We argue that the zebrafish represents unique advantages, and adding information obtained with this species to the mix of other animal models will significantly increase translational relevance of animal biomedical research for the analysis of human FASD.
Article
Fluorescence in situ hybridization (FISH) has a wide spectrum of applications in current molecular cytogenetic and cancer research. This is a unique technique that can be used for chromosomal DNA analysis in all cell types, at all stages of the cell cycle, and at molecular resolution. Recent developments in microscopy and imaging systems have allowed quantification of digital FISH images (quantitative FISH or QFISH) and have provided a new way for molecular cytogenetic analysis at single-cell level. QFISH can be applied for studying chromosome imbalances in interphase nuclei or metaphase spreads, measuring relative DNA content at chromosomal loci and identifying parental origin of homologous chromosomes. Here, a QFISH protocol suitable for the majority of DNA probes using the popular US National Institute of Health developed ImageJ software is described.
Article
Zebrafish are increasingly used in neurobiological and behavioral studies. Possible stimuli to manipulate zebrafish behavior are being investigated. The presentation of colors appears to be one of the most used approaches, but there is much debate about the exact color preference. Here we have investigated the color preference in both larvae and adult zebrafish. We have studied the effects of wavelength, contrast, intensity, and location. Furthermore, we have tried to manipulate the preference with anxiolytic drugs (ethanol and buspirone). Our results show that both adults and larvae have a clear preference for blue zones while they avoid yellow-colored zones. Red and green zones have an intermediate preference. Part of the yellow aversion is caused by a fear for this color and can be diminished by the treatment with anxiolytic drugs. The location of the color appears to be of critical importance. Presentation of the color on the walls induces an approach response whereas presentation on the bottom induces an aversion. In conclusion, colors are important stimuli to manipulate zebrafish behavior and can be used in behavioral models. Standardization of the procedures and a clear description of the used methodologies are, however, essential.
Chapter
Alcohol addiction is a major unmet medical and economic issue for which very few efficacious pharmacological treatment options are currently available. The development and identification of new compounds and drugs to treat alcohol addiction is hampered by the high costs and low amenability of traditional laboratory rodents to high-throughput behavioral screens. The zebrafish represents an excellent compromise between systems complexity and practical simplicity by overcoming many limitations inherent in these rodent models. In this chapter, we review current advances in the behavioral and neurochemical characterization of ethanol-induced changes in zebrafish. We also discuss the basic principles and methods of and the most recent advances in using paradigms with which one can screen for compounds altering acute and chronic ethanol-induced effects in zebrafish.
Article
Fetal alcohol spectrum disorder (FASD) is a devastating disease of the brain caused by exposure to alcohol during prenatal development. Its prevalence exceeds 1%. The majority of FASD cases represent the milder forms of the disease which often remain undiagnosed, and even when diagnosed treatment options for the patient are limited due to lack of information about the mechanisms that underlie the disease. The zebrafish has been proposed as a model organism for exploring the mechanisms of FASD. Our laboratory has been studying the effects of low doses of alcohol during embryonic development in the zebrafish. This review discusses the methods of alcohol exposure, its effects on behavioral performance including social behavior and learning, and the potential underlying biological mechanisms in zebrafish. It is based upon a recent keynote address delivered by the author, and it focuses on findings obtained mainly in his own laboratory. It paints a promising future of this small vertebrate in FASD research. © 2015 Wiley Periodicals, Inc. Dev Psychobiol. © 2015 Wiley Periodicals, Inc.
Article
Acute alcohol intoxication causes cellular changes in the brain that last for hours, while chronic alcohol use induces widespread neuroadaptations in the nervous system that can last a lifetime. Chronic alcohol use and the progression into dependence involve the remodeling of synapses caused by changes in gene expression produced by alcohol. The progression of alcohol use, abuse, and dependence can be divided into stages, which include intoxication, withdrawal, and craving. Each stage is associated with specific changes in gene expression, cellular function, brain circuits, and ultimately behavior. What are the molecular mechanisms underlying the transition from recreational use (acute) to dependence (chronic)? What cellular adaptations result in drug memory retention, leading to the persistence of addictive behaviors, even after prolonged drug abstinence? Research into the neurobiology of alcoholism aims to answer these questions. This chapter will describe the molecular adaptations caused by alcohol use and dependence, and will outline key neurochemical participants in alcoholism at the molecular level, which are also potential targets for therapy.
Article
Mice display robust, stereotyped behaviours towards pups: virgin males typically attack pups, whereas virgin females and sexually experienced males and females display parental care. Here we show that virgin males genetically impaired in vomeronasal sensing do not attack pups and are parental. Furthermore, we uncover a subset of galanin-expressing neurons in the medial preoptic area (MPOA) that are specifically activated during male and female parenting, and a different subpopulation that is activated during mating. Genetic ablation of MPOA galanin neurons results in marked impairment of parental responses in males and females and affects male mating. Optogenetic activation of these neurons in virgin males suppresses inter-male and pup-directed aggression and induces pup grooming. Thus, MPOA galanin neurons emerge as an essential regulatory node of male and female parenting behaviour and other social responses. These results provide an entry point to a circuit-level dissection of parental behaviour and its modulation by social experience.
Article
The zebrafish strikes a good balance between system complexity and practical simplicity and as a result it is becoming increasingly frequently utilized in biomedical research as a translational tool. Numerous human brain disorders are associated with abnormal social behavior and the zebrafish has been suggested for modeling such disorders. To start this line of research, however, one may need to first thoroughly examine the laboratory organism, zebrafish, and its features, social behavior in this case. Proper methods need be developed to induce and quantify social behavior. These paradigms may be able to open a window to the brain and facilitate the understanding of the biological mechanisms of social behavior and its abnormalities. This review is based on an oral paper presented at the last Measuring Behavior Conference, and as such it is mainly focused on research conducted in my own laboratory. Tracing the temporal progression of our own work, it discusses questions including what shoaling is, how it can be induced and measured and how it can be utilized in the modeling of certain human brain disorders, for example, alcohol induced abnormalities.
Article
Ethanol (EtOH) and its metabolite, acetaldehyde (ALD), induce deleterious effects on central nervous system (CNS). Here we investigate the in vitro toxicity of EtOH and ALD (concentrations of 0.25%, 0.5%, and 1%) in zebrafish brain structures [telencephalon (TE), opticum tectum (OT), and cerebellum (CE)] by measuring the functionality of glutamate transporters, MTT reduction, and extracellular LDH activity. Both molecules decreased the activity of the Na(+)-dependent glutamate transporters in all brain structures. The strongest glutamate uptake inhibition after EtOH exposure was 58% (TE-1%), and after ALD, 91% (CE-1%). The results of MTT assay and LDH released demonstrated that the actions of EtOH and its metabolite are concentration and structure-dependent, in which ALD was more toxic than EtOH. In summary, our findings demonstrate a differential toxicity in vitro of EtOH and ALD in zebrafish brain structures, which can involve changes on glutamatergic parameters. We suggest that this species may be an interesting model for assessing the toxicological actions of alcohol and its metabolite in CNS.
Article
The zebrafish is increasingly utilized in the analysis of the effects of ethanol (alcohol) on brain function and behavior. We have shown significant population-dependent alcohol-induced changes in zebrafish behavior and have started to analyze alterations in dopaminergic and serotoninergic responses. Here, we analyze the effects of alcohol on levels of selected neurochemicals using a 2 × 3 (chronic × acute) between-subject alcohol exposure paradigm randomized for two zebrafish populations, AB and SF. Each fish first received the particular chronic treatment (0 or 0.5 vol/vol % alcohol) and subsequently the acute exposure (0, 0.5 or 1.0 % alcohol). We report changes in levels of dopamine, DOPAC, serotonin, 5HIAA, glutamate, GABA, aspartate, glycine and taurine as quantified from whole brain extracts using HPLC. We also analyze monoamine oxidase and tyrosine hydroxylase enzymatic activity. The results demonstrate that compared to SF, AB is more responsive to both acute alcohol exposure and acute alcohol withdrawal at the level of neurochemistry, a finding that correlates well with prior behavioral observations and one which suggests the involvement of genes in the observed alcohol effects. We discuss correlations between the current results and prior behavioral findings, and stress the importance of characterization of zebrafish strains for future behavior genetic and psychopharmacology studies.
Article
The zebrafish (Danio rerio) is rapidly becoming a popular model organism in pharmacogenetics and neuropharmacology. Both larval and adult zebrafish are currently used to increase our understanding of brain function, dysfunction, and their genetic and pharmacological modulation. Here we review the developing utility of zebrafish in the analysis of complex brain disorders (including, for example, depression, autism, psychoses, drug abuse and cognitive disorders), also covering zebrafish applications towards the goal of modeling major human neuropsychiatric and drug-induced syndromes. We argue that zebrafish models of complex brain disorders and drug-induced conditions have become a rapidly emerging critical field in translational neuropharmacology research.
Article
The zebrafish has been used in the analysis of the effects of drugs of abuse, including alcohol and nicotine. In the current study, we investigate the effects of these drugs on shoaling, group-forming behavior, in zebrafish, using a newly developed set of behavioral measures. We expose our fish acutely to 0.25, 0.50, 0.75 or 1.00% (vol/vol %) ethyl alcohol or 4 or 8mg/l nicotine by immersing the fish in the corresponding solutions. The behavior of the exposed fish is compared to controls in a large (91cm diameter) circular tank in which shoals of 8 subjects under the same treatment are allowed to swim freely. Several measures of shoaling are quantified including the nearest neighbour distance (NND), inter-individual distance (IID), swimming speed, polarization (a measure of the directional synchronization of the shoal), and the number and duration of excursions (departures from the shoal). Alcohol and nicotine were both found to exert significant effects on shoaling but impaired the behavior in different ways. For example, alcohol strongly disrupted polarization and only modestly reduced shoal cohesion, while nicotine had only a modest effect on polarization but robustly decreased shoal cohesion. Neither drug affected the number or the duration of excursions, but both reduced swimming speed. These results underscore the notion that using multiple measures of social behavior may allow one to characterize and distinguish different aspects of drug effects on behavior, which may facilitate discovery of novel drugs in drug screens and may also be utilized in the analysis of underlying mechanisms.
Article
Background: Ethanol is a teratogen that affects numerous developmental processes in the nervous system, which includes development and survival of GABAergic and glutamatergic neurons. Possible molecular mechanisms accounting for ethanol's effects on nervous system development include perturbed fibroblast growth factor (Fgf) and Sonic hedgehog (Shh) signaling. In zebrafish, forebrain GABAergic neuron development is dependent on Fgf19 and Shh signaling. The present study was conducted to test the hypothesis that ethanol affects GABAergic and glutamatergic neuron development by disrupting Fgf, Shh, and agrin function. Methods: Zebrafish embryos were exposed to varying concentrations of ethanol during a range of developmental stages, in the absence or presence of morpholino oligonucleotides (MOs) that disrupt agrin or Shh function. In situ hybridization was used to analyze glutamic acid decarboxylase (GAD1) gene expression, as well as markers of glutamatergic neurons. Results: Acute ethanol exposure results in marked reduction in GAD1 gene expression in forebrain and hindbrain, and reduction of glutamatergic neuronal markers in hindbrain. Subthreshold ethanol exposure, combined with agrin or Shh MO treatment, produces a similar diminution in expression of markers for GABAergic and glutamatergic neurons. Consistent with the ethanol effects on Fgf and Shh pathways, Fgf19, Fgf8, or Shh mRNA overexpression rescues ethanol-induced decreases in GAD1 and Atonal1a gene expression. Conclusions: These studies demonstrate that GABAergic and glutamatergic neuron development in zebrafish forebrain or cerebellum is sensitive to ethanol exposure, and provides additional evidence that a signaling pathway involving agrin, Fgfs and Shh may be a critical target of ethanol exposure during zebrafish embryogenesis.
Article
Receiver biases towards specific sensory signals have been demonstrated in insects, birds and fish, both in the context of foraging and mate choice. In some cases, signals important in sexual selection appear to have evolved by exploiting a pre-existing bias in the sensory system. For instance, female preferences for male nuptial colouration may have arisen from selection on foraging practices. Using the zebrafish (Danio rerio), a species in which red is not a factor in mate choice, we tested for a foraging bias towards the colour red. We further investigated the plasticity of foraging biases by raising groups of fish on diets consisting solely of red, blue, green or white food. When we subsequently tested their colour preferences in a foraging context, each group responded most strongly to red, irrespective of the colour of food with which they had been conditioned. We also detected a significant effect of conditioning on colour preferences; fish responded more strongly to the colour that matched diet colour than to other colours. The observed receiver bias towards red may have evolved as an adaptive preference for carotenoid compounds in their diet. While the bias to red appears to be innate, our results indicate that learning is also important in shaping foraging biases.
Article
We describe a series of stages for development of the embryo of the zebrafish, Danio (Brachydanio) rerio. We define seven broad periods of embryogenesis—the zygote, cleavage, blastula, gastrula, segmentation, pharyngula, and hatching periods. These divisions highlight the changing spectrum of major developmental processes that occur during the first 3 days after fertilization, and we review some of what is known about morphogenesis and other significant events that occur during each of the periods. Stages subdivide the periods. Stages are named, not numbered as in most other series, providing for flexibility and continued evolution of the staging series as we learn more about development in this species. The stages, and their names, are based on morphological features, generally readily identified by examination of the live embryo with the dissecting stereomicroscope. The descriptions also fully utilize the optical transparancy of the live embryo, which provides for visibility of even very deep structures when the embryo is examined with the compound microscope and Nomarski interference contrast illumination. Photomicrographs and composite camera lucida line drawings characterize the stages pictorially. Other figures chart the development of distinctive characters used as staging aid signposts. ©1995 Wiley-Liss, Inc.
Article
The three-dimensional structure of flocks of dunlin, Calidris alpina, and starlings, Sturnus vulgaris, was studied while birds were in transit between feeding, loafing and roosting sites. A technique was developed that uses standard photogrammetric methods to determine the three-coordinate position of birds in flocks from stereoscopic pairs of simultaneously exposed photographs. A comparison of nearest neighbour distances indicates that dunlin have a tighter, more compact flock structure than do starlings (Fig. 2; Table 2). Analysis of interbird angles in both the vertical and horizontal planes indicates that each dunlin's nearest neighbour is most likely to be behind and below it. This spatial structure results in areas in which few nearest neighbours occur (e.g., immediately in front and below) (Fig. 3). Flight speeds during transit flights are also presented (Table 4). The spatial structure and behaviour of dunlin and starling flocks appear to be very similar to the structure and behaviour of schools of fish.
Article
Taurine (TAU) is an amino sulfonic acid that plays protective roles against neurochemical impairments induced by ethanol (EtOH). Mounting evidence shows the applicability of zebrafish for evaluating locomotor parameters and anxiety-like behavioral phenotypes after EtOH exposure in a large scale manner. In this study, we assess the effects of TAU pretreatment on the behavior of zebrafish in the open tank after acute 1% EtOH (v/v) exposure (20 and 60 min of duration) and on brain alcohol contents. The exposure for 20 min exerted significant anxiolytic effects, which were prevented by 42, 150, and 400 mg/L TAU. Conversely, the 60-min condition induced depressant/sedative effects, in which the changes on vertical activity were associated to modifications on the exploratory profile. Although all TAU concentrations kept locomotor parameters at basal levels, 150 mg/L TAU, did not prevent the impairment on vertical activity of EtOH[60]. Despite the higher brain EtOH content detected in the 60-min exposure, 42, 150, and 400 mg/L TAU attenuated the increase of alcohol content in EtOH[60] group. In conclusion, our data suggest that both protocols of acute EtOH exposure induce significant changes in the spatio-temporal behavior of zebrafish and that TAU may exert a preventive role by antagonizing the effects induced by EtOH possibly due to its neuromodulatory role and also by decreasing brain EtOH levels. The hormetic dose-response of TAU on vertical exploration suggests a complex interaction between TAU and EtOH in the central nervous system.