Current Opinion in Behavioral Sciences, Apr 1, 2015
Zebrafish behavioral phenotyping tools are becoming increasingly available.The diurnal nature of ... more Zebrafish behavioral phenotyping tools are becoming increasingly available.The diurnal nature of zebrafish allows the use of efficient visual stimuli.Both stimulus delivery and behavioral response quantification are automatable.Even lengthy and complex learning tasks, when automated, can run in parallel.High throughput behavioral screening is becoming a reality for zebrafish.The zebrafish is small, easy to keep, and breeds in large quantities, features that make it attractive for high throughput mutagenesis screening. It is translationally relevant due to evolutionary conservation of several of its characteristics, but particularly because of the high nucleotide sequence homology between zebrafish and human genes. Behavioral genetics also noticed this species. Although still very small, the number of behavioral paradigms designed for zebrafish is exponentially increasing and the marriage between behavioral sciences and genetics is becoming a reality for zebrafish. This paper reviews the latest developments of this rapidly expanding field with illustrative examples on novel behavioral paradigms drawn from the work of the author's laboratory along with a short discussion on modern forward genetic methods.
Techniques in the Behavioral and Neural Sciences, 1999
This chapter discusses the ethological approaches in behavioral neurogenetic research. The studie... more This chapter discusses the ethological approaches in behavioral neurogenetic research. The studies present in the chapter demonstrates that it is important to consider the ethological relevance of the tasks for the species in question and it is important to modify existing tests or design new ones, depending on the question asked and the genetic manipulation employed. It also illustrates that the importance of using ethologically-relevant tasks does not imply that there are no general features and common molecular mechanisms underlying behavioral phenomena, knowledge of the species-specific characteristics of behavior is essential for discovering these commonalities and generalizing the findings of animal research to human. The challenge is to design appropriate behavioral tests which tap into the natural memory capabilities of the species in question, and thereby make these tests sensitive enough to detect the genetic effects.
Fetal alcohol spectrum disorder (FASD) is a devastating disease of the brain caused by exposure t... more 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 vertebra...
Alcoholism: Clinical and Experimental Research, 2009
Zebrafish is becoming an important research tool for the analysis of brain function and behavior.... more Zebrafish is becoming an important research tool for the analysis of brain function and behavior. It has been proposed to model human alcoholism as well as fetal alcohol syndrome. Previous studies investigating the consequences of exposure to ethanol during early development of zebrafish employed robust dosing regimens (high ethanol concentration and long exposure) that may model a rare situation in the human clinic. These studies found major structural abnormalities developing in the exposed fish. Here we hope to avoid such gross changes and administer only low doses of ethanol (0.00, 0.25, 0.50, 0.75, 1.00 vol/vol %) at 24-hour postfertilization and for only a short period of time (for 2 hours). We analyze the behavior of exposed fish at adult stage using computerized stimulus presentation and automated videotracking response quantification. Despite the short ethanol exposure period and the modest concentrations, significant behavioral alterations were found: fish exposed to higher doses of ethanol swam at an increased distance from a computer-animated zebrafish shoal while their activity levels did not change. Although the interpretation of and the mechanisms underlying this finding will require further investigation, the results suggest that zebrafish will be an appropriate model organism for the analysis of the effects of moderate to mild prenatal ethanol exposure.
The function of the cannabinoid type 1 receptor (CB1-R) is poorly understood in zebrafish, and nu... more The function of the cannabinoid type 1 receptor (CB1-R) is poorly understood in zebrafish, and numerous inconsistent effects have been reported on it in the literature. The objective of the present study is to determine whether differences in the reported effects of CB1-R antagonism on anxiety-like behavioural responses, dopaminergic and serotonergic responses are due to concentration, context-dependent and/or population (genotype-related) effects. Two genetically distinct populations of zebrafish (AB and short fin (SF)) were treated with different concentrations of AM251 (0, 0.1, 1mg/L), and behavioural responses were quantified under two different contexts: one, following habituation and two, subsequently in a novel environment. The levels of dopamine, serotonin and their metabolites 3,4-dihydroxyindole acetic acid (DOPAC) and 5-hydroxyindoleacetic acid (5-HIAA) were quantified from whole-brain tissue. We demonstrate that a 60-min exposure to AM251 (0, 0.1, 1mg/L) does not alter b...
Zebrafish express enzymes that metabolize ethanol in a manner comparable to that of mammals, incl... more Zebrafish express enzymes that metabolize ethanol in a manner comparable to that of mammals, including humans. We previously demonstrated that acute ethanol exposure increases alcohol dehydrogenase (ADH) activity in an inverted U-shaped dose-dependent manner. It was hypothesized that the biphasic dose-response was due to the increased activity of a high-affinity ADH isoform following exposure to low concentrations of ethanol and increased activity of a low-affinity ADH isoform following exposure to higher concentrations of ethanol. To test this hypothesis, we exposed zebrafish to different concentrations of ethanol (0%, 0.25%, 0.5%, and 1.0% v/v) for 30 min and measured the total ADH activity in the zebrafish liver. However, we also repeated this enzyme activity assay using a low concentration of the substrate (ethanol) to determine the activity of high-affinity ADH isoforms. We found that total ADH activity in response to ethanol induces an inverted U-shaped dose-response similar t...
Alcohol dependence and alcohol abuse represent major unmet medical needs. The zebrafish is consid... more Alcohol dependence and alcohol abuse represent major unmet medical needs. The zebrafish is considered to be a promising vertebrate species with which the effects of alcohol on brain function and behavior and the mechanisms underlying these effects may be studied. Alcohol is known to induce alterations in motor function as well as fear and anxiety. Here we present a recently developed fear paradigm in which we employ an animated (moving) image of a bird silhouette. We measure the effect of acute alcohol administration (dose range employed: 0.00 -0.75 vol/vol percentage, bath exposure for 60 minutes) on the behavioral responses of zebrafish. We test these responses during a pre-stimulus, stimulus and post-stimulus period of the task using both a videotracking and an observation based quantification method. The fear inducing stimulus was found to decrease the distance of the zebrafish from the bottom of the tank, to increase number of erratic movements, and to increase the number of jumps in alcohol exposed fish (versus control fish). Alcohol attenuated these fear responses in a dose dependent manner. In addition, alcohol decreased general activity at the highest dose, an effect that was independent of the presentation of the stimulus. We discuss the similarities and differences between observation and video-tracking based results and conclude that fear paradigms will be useful in revealing alcohol induced functional changes in the brain of zebrafish.
The ability to quantify, i.e. to estimate quantity, may provide evolutionary advantages in some c... more The ability to quantify, i.e. to estimate quantity, may provide evolutionary advantages in some contexts and has been demonstrated in a variety of animal species. In a prior study, we showed that angelfish (Pterophyllum scalare) were able to discriminate between groups (shoals) in which a large number of conspecifics swam preferring to join the larger of the two. Our results implied that angelfish can compare relative shoal sizes likely on the basis of some quantitative attributes of the shoal. Here, also using a binary preference test, we examined whether angelfish are able to discriminate between shoals of small numbers of conspecifics, and if so whether their performance reveals a comparable underlying mechanism to that proposed for discrimination of small quantities in human and non-human animals, namely the possible precursor of the ability to count. Our results demonstrate that fish reliably chose 4 versus 1, 3 versus 1, 2 versus 1 and 3 versus 2 individuals, but were at chance performance level when having to choose between 4 versus 3, 5 versus 4 and 6 versus 5. Findings also reveal that the density of the fish in the stimulus shoals did not significantly affect the performance of experimental angelfish. These results are compatible with the hypothesis of the existence of an object-file mechanism to discriminate small quantities in vertebrates and provide evidence for spontaneous discrimination of up to three elements in angelfish, a similar limit to that found in human and non-human animals. The findings add to the growing body of data, suggesting that the mechanisms underlying discrimination between different quantities of items may be shared across different taxa and have an evolutionary ancient origin.
Immediate early genes (IEGs) are transcription factors whose own transcription is initiated rapid... more Immediate early genes (IEGs) are transcription factors whose own transcription is initiated rapidly, for example, in the brain in response to environmental stimuli. c-fos is an IEG often used as a marker of neuronal activation. c-fos mRNA expression has started to be quantified and localized in the zebrafish brain following environmental manipulations but analysis of the expression of c-fos protein in the zebrafish brain has rarely been attempted. Here, we describe an immunofluorescence staining method for quantifying c-fos protein expression in different regions of the zebrafish brain. In addition, we expose zebrafish to caffeine, a positive control for c-fos activation in the brain. To confirm cell nucleus specific binding of the c-fos antibody, we counterstained brain sections with the nuclear fluorescent stain DAPI. Furthermore, we describe a method for reducing background autofluorescence often observed in zebrafish brain tissue. Our analysis showed that exposure to caffeine in...
Zebrafish have become a popular animal model for behavioral neuroscience with an increasing numbe... more Zebrafish have become a popular animal model for behavioral neuroscience with an increasing number of studies examining the effects of pharmacological compounds targeting the brain. Exposure to MK-801, a non-competitive N-methyl-d-aspartate receptor antagonist has been shown to increase locomotor activity in zebrafish. However, others have failed to replicate this finding as several contradicting studies report no changes in locomotor activity following exposure to similar doses. In the current study we reconcile these behavioral reports by demonstrating that zebrafish do not exhibit changes in locomotor activity during exposure to non-sedative doses of MK-801. Interestingly, zebrafish do exhibit significant increases in locomotion if pre-treated with MK-801 followed by subsequent testing in a novel environment, which suggests the effects of MK-801 are context-dependent. In addition, we examine the potential role of the dopaminergic system in mediating MK-801's locomotor stimulant effect by quantifying the levels of dopamine and its metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) in the brains of zebrafish following a 30min exposure to 10μM of MK-801 (the dose found to induce the largest increase in locomotor activity). Our findings indicate that the MK-801-induced increase in locomotor activity is not accompanied by changes in whole-brain levels of dopamine or DOPAC. Overall, our results suggest that MK-801's context-dependent locomotor stimulant effect may be independent of whole-brain dopaminergic activation.
The popularity of the zebrafish has been growing in behavioral brain research. Previously utilize... more The popularity of the zebrafish has been growing in behavioral brain research. Previously utilized mainly in developmental biology and genetics, the zebrafish has turned out to possess a complex behavioral repertoire. For example, it is a highly social species, and individuals form tight groups, a behavior called shoaling. Social isolation induced changes in brain function and behavior have been demonstrated in a variety of laboratory organisms. However, despite its highly social nature, the zebrafish has rarely been utilized in this research area. Here, we investigate the effects of chronic social isolation (lasting 90 days) on locomotor activity and anxiety-related behaviors in an open tank. We also examine the effect of chronic social isolation on levels of whole-brain serotonin and dopamine and their metabolites. We found that long-term social deprivation surprisingly decreased anxiety-related behavious during open-tank testing but had no effect on locomotor activity. We also fo...
Zebrafish naturally form social groups called shoals. Previously, we have shown that submerging z... more Zebrafish naturally form social groups called shoals. Previously, we have shown that submerging zebrafish eggs into low concentrations of alcohol (0.00, 0.25, 0.50, 0.75 and 1.00 vol/vol% external bath concentration) during development (24h post-fertilization) for two hours resulted in impaired shoaling response in seven month old young adult zebrafish. Here we investigate whether this embryonic alcohol exposure induced behavioural deficit persists to older age. Zebrafish embryos were exposed either to fresh system water (control) or to 1% alcohol for two hours, 24h after fertilization, and were raised in a high-density tank system. Social behaviour was tested by presenting the experimental fish with a computer animated group of zebrafish images, while automated tracking software measured their behaviour. Control fish were found to respond strongly to animated conspecific images by reducing their distanceand remaining close to the images during image presentation, embryonic alcohol treated fish did not. Our results suggest that the impaired shoaling response of the alcohol exposed fish was not due to altered motor function or visual perception, but likely to a central nervous system alteration affecting social behaviour itself. We found the effects of embryonic alcohol exposure on social behaviour not to diminish with age, a result that demonstrates the deleterious and potentially life-long consequences of exposure to even small amount of alcohol during embryonic development in vertebrates.
Single gene manipulation allows one to create mutant mammalian organisms that may be useful for g... more Single gene manipulation allows one to create mutant mammalian organisms that may be useful for genetic dissection of mechanisms underlying brain function and behavior. Alterations in a mutant organism may be directly related to the mutation or due to compensatory mechanisms. Analysis of these responses may reveal molecular organization of the brain. However, the results of several recent molecular neurobiology studies are difficult to interpret since they are flawed by the problems resulting from genetic linkage of and variability in background genes. The behavioral analysis of mutant mice may also be uninterpretable if one ignores species-specific characteristics of the experimental animals.
Journal of comparative psychology (Washington, D.C. : 1983), 2015
The ability to discriminate between sets of items differing in quantity has shown a growing inter... more The ability to discriminate between sets of items differing in quantity has shown a growing interest in comparative studies as a diversity of animal species exhibit such quantitative competence. Previous studies with angelfish (Pterophyllum scalare) have demonstrated that this species is capable of spontaneously discriminating between fully visible groups (shoals) of conspecifics of different numerical size. In the present study, we investigated quantity discrimination in angelfish adopting a new procedure that we expected to make the task more difficult for the fish. During a pretest period, angelfish were allowed to fully see shoals of conspecifics of different numerical size, subsequently all fish but 1 in each stimulus shoal were hidden behind opaque barriers. Thus, during testing, experimental fish had to rely on their working memory, which implies a certain level of mental representation of the quantities or numbers discriminated. Angelfish chose the larger shoal with similar ...
The zebrafish has been one of the primary study species utilized in developmental biology. Howeve... more The zebrafish has been one of the primary study species utilized in developmental biology. However, it is also gaining increasing amount of interest in other disciplines of biology including behavioral neuroscience; the numerous genetic tools developed and the large amount of genetic information accumulated for this species by now make it an excellent tool for the analysis of the mechanisms of complex central nervous system characteristics. Although several studies have investigated the biological and genetic underpinnings of associative learning (and memory), given the complexity of these phenomena, much remains to be discovered. In the past, the zebrafish has been employed particularly successfully in screening applications where a large number of mutations or drug effects had to be analyzed. Briefly, the practical simplicity and system complexity of the zebrafish may make this species an excellent tool also for the analysis of the mechanisms of associative learning. Screening, ho...
Zebrafish (zebra danio) are becoming increasingly popular in behavioral neuroscience and behavior... more Zebrafish (zebra danio) are becoming increasingly popular in behavioral neuroscience and behavior genetics. This small vertebrate may be utilized in modeling human brain disorders. One of the major neuropsychiatric conditions still not well understood is abnormally increased fear and anxiety. Zebrafish may be an appropriate organism with which these human diseases can be modeled and their biological mechanisms investigated. Predator induced anxiety paradigms have been suggested as useful methods in translational research. Shoaling fish, such as zebrafish, are known to respond to alarm substances with antipredatory or alarm reactions. However, these responses are not well characterized in zebrafish. In the current paper, we investigate the behavioral responses of zebrafish elicited by its alarm substance. Using observation-based as well as video-tracking aided behavior quantification methods we demonstrate significant alarm substance-induced behavioral changes that are independent of...
The zebrafish dopaminergic system is thought to be evolutionarily conserved and may be amenable t... more The zebrafish dopaminergic system is thought to be evolutionarily conserved and may be amenable to pharmacological manipulation using drugs developed for mammalian receptors. However, only few studies have examined the role of specific receptor subtypes in behaviour of adult zebrafish. The objectives of this study are to determine the translational relevance of the zebrafish and examine the psychopharmacology of specific dopamine receptors in this species. Using a behavioural pharmacological approach, we examine the effect of D1 and D2/3 receptor antagonisms on motor patterns of adult zebrafish during acute drug exposure and withdrawal. Acute exposure to SCH-23390 (D1 receptor antagonist) decreased total distance travelled in a dose-dependent manner. Exposure to amisulpride (D2/3 receptor antagonist) induced a biphasic dose-response in total distance travelled and in angular velocity. The results provide support for the existence of structurally and functionally conserved postsynaptic D1 and D2 receptors, as well as presynaptic D2 autoreceptors in the zebrafish brain. The behavioural effects of the employed antagonists did not persist following 30 min of withdrawal. The results suggest that zebrafish, a cheaper and simpler model organism compared to the rat and the mouse, may be an efficient translationally relevant tool for the analysis of the psychopharmacology of receptors of the vertebrate dopaminergic system.
Autism has been becoming the focus of attention as its apparently increasing prevalence is better... more Autism has been becoming the focus of attention as its apparently increasing prevalence is better appreciated. According to some estimates, the frequency of children with autistic spectrum disorder (ASD) can be as high as 1 in 150. The diagnosis can be made as early as 2 years of age, and autistic patients often have a normal life span. Thus, in terms of the number of "patient years," ASD represents a market that is as large as that of the biggest neurological indication, Alzheimer's disease. Despite the clear unmet medical need, no effective treatment is available. This may be because the mechanism of ASD is not understood. The aim of the present paper is to review recent advances in autism research and to discuss some of the most stressing problems mainly from a preclinical research standpoint. We hope to draw attention to the need to study this devastating disease that places an enormous burden on the society in general and the relatives and caregivers of autistic p...
Advances in molecular genetics and technology have led to the dawn of a new era for neuroscience:... more Advances in molecular genetics and technology have led to the dawn of a new era for neuroscience: manipulation of single genes now makes it possible to dissect the complexities of neurobiological phenotypes and to understand many of the intricacies of brain and behaviour, even in mammals. The phenotypical analysis of these mutant animals is complicated because the potential outcome of gene manipulation is difficult to predict. While behavioural analysis should form an integral part of any multidisciplinary research programme investigating the phenotypical effects of single genes on hippocampal function, it is crucial that the behavioural tests are designed and conducted appropriately. Approaches that take species-specific behavioural characteristics into account and use ethological methods could be the most useful for interpreting these behavioural findings and understanding the biological mechanisms of brain function.
Current Opinion in Behavioral Sciences, Apr 1, 2015
Zebrafish behavioral phenotyping tools are becoming increasingly available.The diurnal nature of ... more Zebrafish behavioral phenotyping tools are becoming increasingly available.The diurnal nature of zebrafish allows the use of efficient visual stimuli.Both stimulus delivery and behavioral response quantification are automatable.Even lengthy and complex learning tasks, when automated, can run in parallel.High throughput behavioral screening is becoming a reality for zebrafish.The zebrafish is small, easy to keep, and breeds in large quantities, features that make it attractive for high throughput mutagenesis screening. It is translationally relevant due to evolutionary conservation of several of its characteristics, but particularly because of the high nucleotide sequence homology between zebrafish and human genes. Behavioral genetics also noticed this species. Although still very small, the number of behavioral paradigms designed for zebrafish is exponentially increasing and the marriage between behavioral sciences and genetics is becoming a reality for zebrafish. This paper reviews the latest developments of this rapidly expanding field with illustrative examples on novel behavioral paradigms drawn from the work of the author's laboratory along with a short discussion on modern forward genetic methods.
Techniques in the Behavioral and Neural Sciences, 1999
This chapter discusses the ethological approaches in behavioral neurogenetic research. The studie... more This chapter discusses the ethological approaches in behavioral neurogenetic research. The studies present in the chapter demonstrates that it is important to consider the ethological relevance of the tasks for the species in question and it is important to modify existing tests or design new ones, depending on the question asked and the genetic manipulation employed. It also illustrates that the importance of using ethologically-relevant tasks does not imply that there are no general features and common molecular mechanisms underlying behavioral phenomena, knowledge of the species-specific characteristics of behavior is essential for discovering these commonalities and generalizing the findings of animal research to human. The challenge is to design appropriate behavioral tests which tap into the natural memory capabilities of the species in question, and thereby make these tests sensitive enough to detect the genetic effects.
Fetal alcohol spectrum disorder (FASD) is a devastating disease of the brain caused by exposure t... more 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 vertebra...
Alcoholism: Clinical and Experimental Research, 2009
Zebrafish is becoming an important research tool for the analysis of brain function and behavior.... more Zebrafish is becoming an important research tool for the analysis of brain function and behavior. It has been proposed to model human alcoholism as well as fetal alcohol syndrome. Previous studies investigating the consequences of exposure to ethanol during early development of zebrafish employed robust dosing regimens (high ethanol concentration and long exposure) that may model a rare situation in the human clinic. These studies found major structural abnormalities developing in the exposed fish. Here we hope to avoid such gross changes and administer only low doses of ethanol (0.00, 0.25, 0.50, 0.75, 1.00 vol/vol %) at 24-hour postfertilization and for only a short period of time (for 2 hours). We analyze the behavior of exposed fish at adult stage using computerized stimulus presentation and automated videotracking response quantification. Despite the short ethanol exposure period and the modest concentrations, significant behavioral alterations were found: fish exposed to higher doses of ethanol swam at an increased distance from a computer-animated zebrafish shoal while their activity levels did not change. Although the interpretation of and the mechanisms underlying this finding will require further investigation, the results suggest that zebrafish will be an appropriate model organism for the analysis of the effects of moderate to mild prenatal ethanol exposure.
The function of the cannabinoid type 1 receptor (CB1-R) is poorly understood in zebrafish, and nu... more The function of the cannabinoid type 1 receptor (CB1-R) is poorly understood in zebrafish, and numerous inconsistent effects have been reported on it in the literature. The objective of the present study is to determine whether differences in the reported effects of CB1-R antagonism on anxiety-like behavioural responses, dopaminergic and serotonergic responses are due to concentration, context-dependent and/or population (genotype-related) effects. Two genetically distinct populations of zebrafish (AB and short fin (SF)) were treated with different concentrations of AM251 (0, 0.1, 1mg/L), and behavioural responses were quantified under two different contexts: one, following habituation and two, subsequently in a novel environment. The levels of dopamine, serotonin and their metabolites 3,4-dihydroxyindole acetic acid (DOPAC) and 5-hydroxyindoleacetic acid (5-HIAA) were quantified from whole-brain tissue. We demonstrate that a 60-min exposure to AM251 (0, 0.1, 1mg/L) does not alter b...
Zebrafish express enzymes that metabolize ethanol in a manner comparable to that of mammals, incl... more Zebrafish express enzymes that metabolize ethanol in a manner comparable to that of mammals, including humans. We previously demonstrated that acute ethanol exposure increases alcohol dehydrogenase (ADH) activity in an inverted U-shaped dose-dependent manner. It was hypothesized that the biphasic dose-response was due to the increased activity of a high-affinity ADH isoform following exposure to low concentrations of ethanol and increased activity of a low-affinity ADH isoform following exposure to higher concentrations of ethanol. To test this hypothesis, we exposed zebrafish to different concentrations of ethanol (0%, 0.25%, 0.5%, and 1.0% v/v) for 30 min and measured the total ADH activity in the zebrafish liver. However, we also repeated this enzyme activity assay using a low concentration of the substrate (ethanol) to determine the activity of high-affinity ADH isoforms. We found that total ADH activity in response to ethanol induces an inverted U-shaped dose-response similar t...
Alcohol dependence and alcohol abuse represent major unmet medical needs. The zebrafish is consid... more Alcohol dependence and alcohol abuse represent major unmet medical needs. The zebrafish is considered to be a promising vertebrate species with which the effects of alcohol on brain function and behavior and the mechanisms underlying these effects may be studied. Alcohol is known to induce alterations in motor function as well as fear and anxiety. Here we present a recently developed fear paradigm in which we employ an animated (moving) image of a bird silhouette. We measure the effect of acute alcohol administration (dose range employed: 0.00 -0.75 vol/vol percentage, bath exposure for 60 minutes) on the behavioral responses of zebrafish. We test these responses during a pre-stimulus, stimulus and post-stimulus period of the task using both a videotracking and an observation based quantification method. The fear inducing stimulus was found to decrease the distance of the zebrafish from the bottom of the tank, to increase number of erratic movements, and to increase the number of jumps in alcohol exposed fish (versus control fish). Alcohol attenuated these fear responses in a dose dependent manner. In addition, alcohol decreased general activity at the highest dose, an effect that was independent of the presentation of the stimulus. We discuss the similarities and differences between observation and video-tracking based results and conclude that fear paradigms will be useful in revealing alcohol induced functional changes in the brain of zebrafish.
The ability to quantify, i.e. to estimate quantity, may provide evolutionary advantages in some c... more The ability to quantify, i.e. to estimate quantity, may provide evolutionary advantages in some contexts and has been demonstrated in a variety of animal species. In a prior study, we showed that angelfish (Pterophyllum scalare) were able to discriminate between groups (shoals) in which a large number of conspecifics swam preferring to join the larger of the two. Our results implied that angelfish can compare relative shoal sizes likely on the basis of some quantitative attributes of the shoal. Here, also using a binary preference test, we examined whether angelfish are able to discriminate between shoals of small numbers of conspecifics, and if so whether their performance reveals a comparable underlying mechanism to that proposed for discrimination of small quantities in human and non-human animals, namely the possible precursor of the ability to count. Our results demonstrate that fish reliably chose 4 versus 1, 3 versus 1, 2 versus 1 and 3 versus 2 individuals, but were at chance performance level when having to choose between 4 versus 3, 5 versus 4 and 6 versus 5. Findings also reveal that the density of the fish in the stimulus shoals did not significantly affect the performance of experimental angelfish. These results are compatible with the hypothesis of the existence of an object-file mechanism to discriminate small quantities in vertebrates and provide evidence for spontaneous discrimination of up to three elements in angelfish, a similar limit to that found in human and non-human animals. The findings add to the growing body of data, suggesting that the mechanisms underlying discrimination between different quantities of items may be shared across different taxa and have an evolutionary ancient origin.
Immediate early genes (IEGs) are transcription factors whose own transcription is initiated rapid... more Immediate early genes (IEGs) are transcription factors whose own transcription is initiated rapidly, for example, in the brain in response to environmental stimuli. c-fos is an IEG often used as a marker of neuronal activation. c-fos mRNA expression has started to be quantified and localized in the zebrafish brain following environmental manipulations but analysis of the expression of c-fos protein in the zebrafish brain has rarely been attempted. Here, we describe an immunofluorescence staining method for quantifying c-fos protein expression in different regions of the zebrafish brain. In addition, we expose zebrafish to caffeine, a positive control for c-fos activation in the brain. To confirm cell nucleus specific binding of the c-fos antibody, we counterstained brain sections with the nuclear fluorescent stain DAPI. Furthermore, we describe a method for reducing background autofluorescence often observed in zebrafish brain tissue. Our analysis showed that exposure to caffeine in...
Zebrafish have become a popular animal model for behavioral neuroscience with an increasing numbe... more Zebrafish have become a popular animal model for behavioral neuroscience with an increasing number of studies examining the effects of pharmacological compounds targeting the brain. Exposure to MK-801, a non-competitive N-methyl-d-aspartate receptor antagonist has been shown to increase locomotor activity in zebrafish. However, others have failed to replicate this finding as several contradicting studies report no changes in locomotor activity following exposure to similar doses. In the current study we reconcile these behavioral reports by demonstrating that zebrafish do not exhibit changes in locomotor activity during exposure to non-sedative doses of MK-801. Interestingly, zebrafish do exhibit significant increases in locomotion if pre-treated with MK-801 followed by subsequent testing in a novel environment, which suggests the effects of MK-801 are context-dependent. In addition, we examine the potential role of the dopaminergic system in mediating MK-801's locomotor stimulant effect by quantifying the levels of dopamine and its metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) in the brains of zebrafish following a 30min exposure to 10μM of MK-801 (the dose found to induce the largest increase in locomotor activity). Our findings indicate that the MK-801-induced increase in locomotor activity is not accompanied by changes in whole-brain levels of dopamine or DOPAC. Overall, our results suggest that MK-801's context-dependent locomotor stimulant effect may be independent of whole-brain dopaminergic activation.
The popularity of the zebrafish has been growing in behavioral brain research. Previously utilize... more The popularity of the zebrafish has been growing in behavioral brain research. Previously utilized mainly in developmental biology and genetics, the zebrafish has turned out to possess a complex behavioral repertoire. For example, it is a highly social species, and individuals form tight groups, a behavior called shoaling. Social isolation induced changes in brain function and behavior have been demonstrated in a variety of laboratory organisms. However, despite its highly social nature, the zebrafish has rarely been utilized in this research area. Here, we investigate the effects of chronic social isolation (lasting 90 days) on locomotor activity and anxiety-related behaviors in an open tank. We also examine the effect of chronic social isolation on levels of whole-brain serotonin and dopamine and their metabolites. We found that long-term social deprivation surprisingly decreased anxiety-related behavious during open-tank testing but had no effect on locomotor activity. We also fo...
Zebrafish naturally form social groups called shoals. Previously, we have shown that submerging z... more Zebrafish naturally form social groups called shoals. Previously, we have shown that submerging zebrafish eggs into low concentrations of alcohol (0.00, 0.25, 0.50, 0.75 and 1.00 vol/vol% external bath concentration) during development (24h post-fertilization) for two hours resulted in impaired shoaling response in seven month old young adult zebrafish. Here we investigate whether this embryonic alcohol exposure induced behavioural deficit persists to older age. Zebrafish embryos were exposed either to fresh system water (control) or to 1% alcohol for two hours, 24h after fertilization, and were raised in a high-density tank system. Social behaviour was tested by presenting the experimental fish with a computer animated group of zebrafish images, while automated tracking software measured their behaviour. Control fish were found to respond strongly to animated conspecific images by reducing their distanceand remaining close to the images during image presentation, embryonic alcohol treated fish did not. Our results suggest that the impaired shoaling response of the alcohol exposed fish was not due to altered motor function or visual perception, but likely to a central nervous system alteration affecting social behaviour itself. We found the effects of embryonic alcohol exposure on social behaviour not to diminish with age, a result that demonstrates the deleterious and potentially life-long consequences of exposure to even small amount of alcohol during embryonic development in vertebrates.
Single gene manipulation allows one to create mutant mammalian organisms that may be useful for g... more Single gene manipulation allows one to create mutant mammalian organisms that may be useful for genetic dissection of mechanisms underlying brain function and behavior. Alterations in a mutant organism may be directly related to the mutation or due to compensatory mechanisms. Analysis of these responses may reveal molecular organization of the brain. However, the results of several recent molecular neurobiology studies are difficult to interpret since they are flawed by the problems resulting from genetic linkage of and variability in background genes. The behavioral analysis of mutant mice may also be uninterpretable if one ignores species-specific characteristics of the experimental animals.
Journal of comparative psychology (Washington, D.C. : 1983), 2015
The ability to discriminate between sets of items differing in quantity has shown a growing inter... more The ability to discriminate between sets of items differing in quantity has shown a growing interest in comparative studies as a diversity of animal species exhibit such quantitative competence. Previous studies with angelfish (Pterophyllum scalare) have demonstrated that this species is capable of spontaneously discriminating between fully visible groups (shoals) of conspecifics of different numerical size. In the present study, we investigated quantity discrimination in angelfish adopting a new procedure that we expected to make the task more difficult for the fish. During a pretest period, angelfish were allowed to fully see shoals of conspecifics of different numerical size, subsequently all fish but 1 in each stimulus shoal were hidden behind opaque barriers. Thus, during testing, experimental fish had to rely on their working memory, which implies a certain level of mental representation of the quantities or numbers discriminated. Angelfish chose the larger shoal with similar ...
The zebrafish has been one of the primary study species utilized in developmental biology. Howeve... more The zebrafish has been one of the primary study species utilized in developmental biology. However, it is also gaining increasing amount of interest in other disciplines of biology including behavioral neuroscience; the numerous genetic tools developed and the large amount of genetic information accumulated for this species by now make it an excellent tool for the analysis of the mechanisms of complex central nervous system characteristics. Although several studies have investigated the biological and genetic underpinnings of associative learning (and memory), given the complexity of these phenomena, much remains to be discovered. In the past, the zebrafish has been employed particularly successfully in screening applications where a large number of mutations or drug effects had to be analyzed. Briefly, the practical simplicity and system complexity of the zebrafish may make this species an excellent tool also for the analysis of the mechanisms of associative learning. Screening, ho...
Zebrafish (zebra danio) are becoming increasingly popular in behavioral neuroscience and behavior... more Zebrafish (zebra danio) are becoming increasingly popular in behavioral neuroscience and behavior genetics. This small vertebrate may be utilized in modeling human brain disorders. One of the major neuropsychiatric conditions still not well understood is abnormally increased fear and anxiety. Zebrafish may be an appropriate organism with which these human diseases can be modeled and their biological mechanisms investigated. Predator induced anxiety paradigms have been suggested as useful methods in translational research. Shoaling fish, such as zebrafish, are known to respond to alarm substances with antipredatory or alarm reactions. However, these responses are not well characterized in zebrafish. In the current paper, we investigate the behavioral responses of zebrafish elicited by its alarm substance. Using observation-based as well as video-tracking aided behavior quantification methods we demonstrate significant alarm substance-induced behavioral changes that are independent of...
The zebrafish dopaminergic system is thought to be evolutionarily conserved and may be amenable t... more The zebrafish dopaminergic system is thought to be evolutionarily conserved and may be amenable to pharmacological manipulation using drugs developed for mammalian receptors. However, only few studies have examined the role of specific receptor subtypes in behaviour of adult zebrafish. The objectives of this study are to determine the translational relevance of the zebrafish and examine the psychopharmacology of specific dopamine receptors in this species. Using a behavioural pharmacological approach, we examine the effect of D1 and D2/3 receptor antagonisms on motor patterns of adult zebrafish during acute drug exposure and withdrawal. Acute exposure to SCH-23390 (D1 receptor antagonist) decreased total distance travelled in a dose-dependent manner. Exposure to amisulpride (D2/3 receptor antagonist) induced a biphasic dose-response in total distance travelled and in angular velocity. The results provide support for the existence of structurally and functionally conserved postsynaptic D1 and D2 receptors, as well as presynaptic D2 autoreceptors in the zebrafish brain. The behavioural effects of the employed antagonists did not persist following 30 min of withdrawal. The results suggest that zebrafish, a cheaper and simpler model organism compared to the rat and the mouse, may be an efficient translationally relevant tool for the analysis of the psychopharmacology of receptors of the vertebrate dopaminergic system.
Autism has been becoming the focus of attention as its apparently increasing prevalence is better... more Autism has been becoming the focus of attention as its apparently increasing prevalence is better appreciated. According to some estimates, the frequency of children with autistic spectrum disorder (ASD) can be as high as 1 in 150. The diagnosis can be made as early as 2 years of age, and autistic patients often have a normal life span. Thus, in terms of the number of "patient years," ASD represents a market that is as large as that of the biggest neurological indication, Alzheimer's disease. Despite the clear unmet medical need, no effective treatment is available. This may be because the mechanism of ASD is not understood. The aim of the present paper is to review recent advances in autism research and to discuss some of the most stressing problems mainly from a preclinical research standpoint. We hope to draw attention to the need to study this devastating disease that places an enormous burden on the society in general and the relatives and caregivers of autistic p...
Advances in molecular genetics and technology have led to the dawn of a new era for neuroscience:... more Advances in molecular genetics and technology have led to the dawn of a new era for neuroscience: manipulation of single genes now makes it possible to dissect the complexities of neurobiological phenotypes and to understand many of the intricacies of brain and behaviour, even in mammals. The phenotypical analysis of these mutant animals is complicated because the potential outcome of gene manipulation is difficult to predict. While behavioural analysis should form an integral part of any multidisciplinary research programme investigating the phenotypical effects of single genes on hippocampal function, it is crucial that the behavioural tests are designed and conducted appropriately. Approaches that take species-specific behavioural characteristics into account and use ethological methods could be the most useful for interpreting these behavioural findings and understanding the biological mechanisms of brain function.
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Papers by Robert Gerlai