Acute administration of a dopamine D2/D3 receptor agonist alters behavioral and neural parameters in adult zebrafish.

The dopaminergic neurotransmitter system is implicated in several brain functions and behavioral processes. Alterations in it are associated with the pathogenesis of several human neurological disorders. Pharmacological agents that interact with the dopaminergic system allow the investigation of dopamine-mediated cellular and molecular responses and may elucidate the biological bases of such disorders. Zebrafish, a translationally relevant biomedical research organism, has been successfully employed in prior psychopharmacology studies. Here, we evaluated the effects of quinpirole (dopamine D2/D3 receptor agonist) in adult zebrafish on behavioral parameters, brain-derived neurotrophic factor (BDNF) and neurotransmitter levels. Zebrafish received intraperitoneal injections of 0.5, 1.0, or 2.0 mg/kg quinpirole or saline (control group) twice with an inter-injection interval of 48 h. All tests were performed 24 h after the second injection. After this acute quinpirole administration, zebrafish exhibited decreased locomotor activity, increased anxiety-like behaviors and memory impairment. However, quinpirole did not affect social and aggressive behavior. Quinpirole-treated fish exhibited stereotypic swimming, characterized by repetitive behavior followed by immobile episodes. Moreover, quinpirole treatment also decreased the number of BDNF-immunoreactive cells in the zebrafish brain. Analysis of neurotransmitter levels demonstrated a significant increase in glutamate and a decrease in serotonin, while no alterations were observed in dopamine. These findings demonstrate that dopaminergic signaling altered by quinpirole administration results in significant behavioral and neuroplastic changes in the central nervous system of zebrafish. Thus, we conclude that the use of quinpirole administration in adult zebrafish may be an appropriate tool for the analysis of mechanisms underlying neurological disorders related to the dopaminergic system.

Inflammatory Pain Alters Dopaminergic Modulation of Excitatory Synapses in the Anterior Cingulate Cortex of Mice.

Pain modulation of dopamine-producing nuclei is known to contribute to the affective component of chronic pain. However, pain modulation of pain-related cortical regions receiving dopaminergic inputs is understudied. The present study demonstrates that mice with chronic inflammatory injury of the hind paws develop persistent mechanical hypersensitivity and transient anxiety. Peripheral inflammation induced by injection of complete Freund's Adjuvant (CFA) induced potentiation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic receptor (AMPAR) currents with a presynaptic component in layer II/III of the ACC. After four days of inflammatory pain, the dopamine-mediated inhibition of AMPAR currents was significantly reduced in the ACC. Furthermore, dopamine enhanced presynaptic modulation of excitatory transmission, but only in mice with inflammatory pain. High-performance liquid chromatography (HPLC) analysis of dopamine tissue concentration revealed that dopamine neurotransmitter concentration in the ACC was reduced three days following CFA. Our results demonstrate that inflammatory pain induces activity-dependent changes in excitatory synaptic transmission and alters dopaminergic homeostasis in the ACC.

Age-dependent effects of embryonic ethanol exposure on anxiety-like behaviours in young zebrafish: A genotype comparison study.

Fetal Alcohol Spectrum Disorder (FASD) is characterized by a variety of morphological, behavioural and cognitive deficits, ranging from mild to severe. Numerous animal models, including the zebrafish, have been employed to better understand the onset, expression and progression of this disorder. Embryonic ethanol-induced deficits in learning and memory, anxiety, social responses and elevated alcohol self-administration have been successfully demonstrated in zebrafish. Studies in zebrafish have also shown the expression of these behavioural deficits depends upon the developmental stage of ethanol exposure, the age of observation, as well as the genotype (strain or population origin) of the tested zebrafish. Here, we investigate how the genotype and age of observation may influence embryonic ethanol-induced alterations in anxiety-like responses in zebrafish. Zebrafish embryos exposed to either 0% or 1% (vol/vol) ethanol at 24hpf were tested in an open tank at one of three stages: larval (6-8 days post fertilization (dpf)), mid-larval (16-18dpf), or juvenile (26-28dpf). Two genotypes were tested in this manner, ABNS (a quasi-inbred strain) and ABSK (a mix of AB, TU and TL strains). We found embryonic ethanol induced behavioural changes to significantly differ depending on the genotype and age of observation. For example, significant differences between control and ethanol exposed zebrafish in both genotypes were observed in juvenile zebrafish, but few significant treatment effects were observed in larval zebrafish. Additionally, ethanol appeared to alter anxiety-like behaviours in the ABNS genotype but did not have as robust of an effect on the ABSK genotype. Lastly, there were significant behavioural differences between unexposed (control) zebrafish of the two genotypes, suggesting baseline behavioural differences despite a common AB genetic origin.

Zebrafish Shoaling, Its Behavioral and Neurobiological Mechanisms, and Its Alteration by Embryonic Alcohol Exposure: A Review.

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.

Developmental stage-dependent deficits induced by embryonic ethanol exposure in zebrafish: A neurochemical analysis.

FASD results from the developing fetus being exposed to alcohol, and is characterized by morphological, behavioural and cognitive deficits. However, the expression, severity and age of onset of these symptoms has been found to show variation. This variation may partly be due to the developmental stage at which alcohol reached the developing fetus. Previously, alcohol was shown to lead to significant concentration dependent behavioural as well as neurochemical changes detected in adult zebrafish when this substance was administered at 24 h post-fertilization (hpf) for 2 h. This alcohol exposure method arguably mimicked the milder, and more prevalent, forms of human FASD. However, whether the observed changes depended upon the developmental stage, i.e., the timing, of alcohol exposure has not been systematically analyzed. Here, we employ the same alcohol dosing regimen, where zebrafish eggs are immersed into 0% or 1% (vol/vol) alcohol for 2 h, but we perform the immersion at 5, 10, 16, 24, 36, or 48 hpf. We previously developed a sensitive HPLC method to quantify neurochemicals, and found levels of dopamine, serotonin and their metabolites DOPAC and 5-HIAA to be affected by embryonic alcohol treatment. Here, using the same method, we compare whole-brain levels of these neurochemicals in the embryonic alcohol exposed and control zebrafish at their age of 30 days post-fertilization (dpf). Consistent with previous reports, we found significant reduction of levels of dopamine, serotonin and their metabolites in the fish exposed to alcohol at 24 hpf. However, we also found significant dependency on the developmental stage at which alcohol was administered with particularly robust impairments when the exposure was at the early or middle of the developmental periods probed. Our results now demonstrate that one can detect functional abnormalities in the zebrafish brain induced by embryonic alcohol as early as 30 dpf and that the neurochemical deficits are dependent upon the developmental stage at which alcohol is administered.

The light-dark task in zebrafish confuses two distinct factors: Interaction between background shade and illumination level preference.

The light-dark preference task has been commonly used in rodents to screen for anxiogenic and anxiolytic drugs. However, recent adaptations of the light-dark preference test for zebrafish have produced inconsistent results. Several studies have reported that zebrafish exhibit a preference for light, while others have found a preference for black. We suggest the inconsistencies may be the result of confusing certain parameters of the test leading to improper interpretation. For example, researchers often use "light" interchangeably with "white" and "dark" with "black" when these are two distinct factors: level of illumination vs. background shade. In the current study, we use specifically designed preference tanks to investigate the influence of background shade (i.e. white vs. black) and level of illumination (i.e. light vs. dark) on preference and anxiety-like behaviour. Furthermore, we pharmacologically validate our results by quantifying the effects of ethanol, a drug with known anxiety-altering properties, on anxiety-like behaviours. Here we report that zebrafish's preference varies depending upon background shade and level of illumination. We also found that ethanol administration altered behavioural responses in an illumination- and background shade-dependent manner. Our findings reinforce the need to correctly differentiate between these factors when interpreting results obtained with this behavioural paradigm. Lastly, our results show that simple modifications to the experimental tank in which anxiety-related responses are measured can significantly alter behaviour of zebrafish, supporting the need for standardized testing procedures and/or for detailed description of experimental procedures and the apparatus.

Alcohol exposure during embryonic development: An opportunity to conduct systematic developmental time course analyses in zebrafish.

Ethanol affects numerous neurobiological processes depending upon the developmental stage at which it reaches the vertebrate embryo. Exposure time dependency may explain the variable severity and manifestation of life-long symptoms observed in fetal alcohol spectrum disorder (FASD) patients. Characterization of behavioural deficits will help us understand developmental stage-dependency and its underlying biological mechanisms. Here we highlight pioneering studies that model FASD using zebrafish, including those that demonstrated developmental stage-dependency of alcohol effects on some behaviours. We also succinctly review the more expansive mammalian literature, briefly discuss potential developmental stage dependent biological mechanisms alcohol alters, and review some of the disadvantages of mammalian systems versus the zebrafish. We stress that the temporal control of alcohol administration in the externally developing zebrafish gives unprecedented precision and is a major advantage of this species over other model organisms employed so far. We also emphasize that the zebrafish is well suited for high throughput screening and will allow systematic exploration of embryonic-stage dependent alcohol effects via mutagenesis and drug screens.

A Standardized Tank Design for the Light Dark Task in Zebrafish.

The light dark paradigm is a common behavioral test used to screen a variety of pharmacological agents, including anxiogenics and anxiolytics. Although most often used in rodents, the light dark task has recently been adapted for use in zebrafish. However, a number of inconsistent findings have been reported for this species. Some have found zebrafish to prefer black, while others report a preference for light. Careful analysis of light dark preference experiments using zebrafish reveals significant variation in testing tank design and test conditions, including lighting and substrate color. Additionally, in some experiments the designated dark side of the testing tank is completely covered, producing a "cave-like" environment which further confounds results. Lastly, authors commonly use the terms "light vs. dark" interchangeably with "white vs. black", when these are two separate factors that may influence preference: illumination level vs. background shade. To address these limitations, we designed testing tanks that differentiate illumination vs. background shade preference in zebrafish. This design allows for simple standardization of light dark testing apparatus in zebrafish, and facilitates more reliable comparison across studies.

Effect of social isolation on anxiety-related behaviors, cortisol, and monoamines in adult zebrafish.

Social isolation can be used to study behavioral, neural, and hormonal mechanisms that regulate interactions in social animals. Although isolation effects have been reported in social mammals and various fish species, systematic studies with isolated zebrafish are rare. Here, the authors examined behavior (social and nonsocial), physiological stress (whole-body cortisol levels), and neurochemicals (serotonin, dopamine, and their metabolites), following acute and chronic social isolation in adult zebrafish. To observe how isolated fish respond behaviorally to social stimuli, they exposed zebrafish to live conspecifics or animated images after acute (24 hr) or chronic (6 months) social isolation. The authors observed that isolation did not affect locomotor activity, but acute isolation had weak nonsignificant anxiogenic effects in adult zebrafish. They also found that all isolated fish responded to both live and animated social stimuli, and the stress hormone, cortisol was lower in chronically isolated fish. Finally, neurochemical analyses showed that serotonin levels increased when fish were exposed to social stimulus after acute isolation, but its metabolite 5HIAA decreased in response to social stimulus following both acute and chronic isolation. Levels of both dopamine and its metabolite DOPAC were also reduced in fish exposed to social stimulus after acute and chronic isolation. Overall, these results show that isolation in zebrafish is an effective tool to study fundamental mechanisms controlling social interaction at behavioral and physiological levels. (PsycINFO Database Record

Re-examining the factors affecting choice in the light-dark preference test in zebrafish.

The light-dark preference test has been extensively used to screen anxiolytic drugs and investigate mechanisms of anxiety with rodents. Recently, this task has been adapted to zebrafish, but a number of inconsistent findings have emerged. For example, some found zebrafish to avoid and others to prefer dark. Given the translational relevance of the zebrafish, its utility in high throughput drug screens, and that anxiety still represents a large unmet medical need, there is an urgent need to resolve these inconsistencies. We propose these inconsistencies are due to lack of distinction between two separate factors: background shade and level of illumination. Here, we systematically manipulated background shade (black vs. white) while keeping the illumination level constant (uniformly illuminated). We also manipulated the level of illumination (illuminated vs. not illuminated) while keeping the background constant (either uniformly black or white). We examined the time-course of numerous behavioural responses under these conditions, and found zebrafish to exhibit a significant preference for the black side of the tank within the first 3min of the test when the illumination level was constant. We found this response, along with other anxiety-like behaviours, to diminish over time. In contrast, we found zebrafish did not exhibit a preference for the unilluminated (dark) side of the tank when the background shade was kept constant. Our results demonstrate a dissociation between illumination level and background shade, shown by a preference for black to white, but not for dark to light, confirming the importance of differentiating these two distinct factors.

Zebrafish Are Able to Detect Ethanol in Their Environment.

Zebrafish have become a popular animal model for studying the development of alcohol addiction. Several behavioral paradigms for studying alcohol addiction have been developed for zebrafish, including conditioned place preference, alcohol-induced tolerance, and withdrawal. However, alcohol choice preference tasks have not been established in zebrafish as of yet. The ability of zebrafish to detect alcohol in their environment is required in alcohol choice or preference tasks. To our knowledge, it is currently unknown whether zebrafish are able to detect alcohol in their environment immediately following bath immersion. In the current study, we analyzed the time course of alcohol-induced behavioral changes of zebrafish while being immersed in alcohol solution in a 1.5 L tank. We recorded each trial in high-definition and quantified behavioral responses using automated video tracking-based and manual observation-based methods to quantify temporal changes in alcohol-induced behaviors. As alcohol is known to require several minutes of bath immersion to reach the brain in zebrafish, we argued that behavioral responses before this time point would prove zebrafish's ability to detect this substance in the water. Our results show that a 60-min exposure to 1% alcohol alters behavioral responses in a time-dependent manner. Notably, alcohol exposure significantly increased absolute turn angle, decreased distance to bottom, and variance of distance to bottom within the first 3 min immediately following exposure, a response that occurred before alcohol could reach the brain of the subjects in measurable amounts. These results imply that zebrafish are able to detect alcohol in their environment immediately following immersion into the drug solution.

Time-dependent interacting effects of caffeine, diazepam, and ethanol on zebrafish behaviour.

Zebrafish have become a popular animal model for behavioural pharmacology due to their small size, rapid development, and amenability to high throughput behavioural drug screens. Furthermore, water-soluble compounds can be administered via immersion of the fish in the drug solution, which provides a non-invasive drug delivery method. Numerous studies have demonstrated stimulant effects of alcohol. Diazepam and caffeine, on the other hand have been found to have inhibitory effect on locomotor activity in zebrafish. However, the time-dependent changes induced by these psychoactive drugs are rarely reported, and potential drug interactions have not been examined in zebrafish, despite the translational relevance of this question. In the current study, we examine time- and dose-dependent changes in zebrafish following exposure to caffeine, diazepam, and ethanol quantifying four different behavioural parameters over a 30min recording session. We subsequently analyze potential drug-drug interactions by co-administering the three drugs in different combinations. Our time-course and dose-response analyses for each of the three drugs represent so far the most detailed studies available serving as a foundation for future psychopharmacology experiments with zebrafish. Furthermore, we report significant interactions between the three drugs corroborating findings obtained with rodent models as well as in humans, providing translational relevance for the zebrafish model.

Acute alcohol exposure increases tyrosine hydroxylase protein expression and dopamine synthesis in zebrafish.

Zebrafish have become a popular animal model for investigating the effects of alcohol on the brain and behaviour. Acute exposure to alcohol has been shown to alter dopaminergic signalling in zebrafish, but the underlying mechanisms have not been well defined. In the current study, we characterize the effects of alcohol on the zebrafish dopaminergic system by focusing on tyrosine hydroxylase, the rate-limiting enzyme in dopamine synthesis. Using western blot analysis, we demonstrate that a 60min exposure to 1% alcohol increases tyrosine hydroxylase protein expression in the zebrafish brain. Enzymatic activity assays confirmed that alcohol also increases tyrosine hydroxylase enzymatic activity, whereas HPLC analysis demonstrated increased levels of whole-brain dopamine and its metabolite DOPAC. In addition to activation of the dopaminergic system, behavioural analysis revealed accompanying increase of distance traveled following 1% alcohol exposure. These findings suggest that acute alcohol exposure elevates dopamine synthesis via increased tyrosine hydroxylase protein expression. Our results support the hypothesis that alcohol alters dopaminergic signalling in the zebrafish brain in a similar manner as compared to mammals.

Alcohol-induced behavioral changes in zebrafish: The role of dopamine D2-like receptors.

RATIONALE: The dopaminergic system has been proposed to mediate alcohol-induced locomotor activity, yet the mechanisms underlying this behavioral response remain poorly understood. OBJECTIVES: This study was conducted to investigate the role of dopamine D2-like receptors in mediating alcohol-induced behavioral responses. METHODS: In experiment 1, we examined the effects of high concentrations (0, 2.5, 5, 10 µM) of haloperidol on motor responses. In experiment 2, we examined the effects of low concentrations (0, 0.625, 1.25, 2.5 µM) of haloperidol on anxiety-like behavioral responses using the novel tank test. In experiment 3, we examined the effect of pre-treating zebrafish with different concentrations of haloperidol (0, 0.625, 2.5 µM) and subsequently exposing them to 0 or 1 % alcohol. RESULTS: In experiment 1, haloperidol induced an inverted U-shaped concentration-dependent increase in locomotor activity. In experiment 2, haloperidol (2.5 µM) reduced the absolute turn angle and freezing behavior in a new environment. In experiment 3, acute alcohol exposure significantly increased locomotor activity and decreased anxiety-like behavioral responses. Pre-treating zebrafish with the lower dose of haloperidol (0.625 µM) abolished the alcohol-induced locomotor activity, without altering anxiety-like behavioral responses. However, pre-treating with the higher dose of haloperidol (2.5 µM) abolished both alcohol-induced increase of locomotor activity and reduction of anxiety-like behavioral responses. CONCLUSION: The results suggest alcohol-induced locomotor hyperactivity in zebrafish is mediated via activation of dopamine D2-like receptors, whereas anxiety-like behavioral responses may only be altered by a high haloperidol concentration, at which dose the drug may affect receptors other than D2-R.

Concentration, population, and context-dependent effects of AM251 in zebrafish.

RATIONALE: 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. OBJECTIVE: 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. METHOD: 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. RESULTS: We demonstrate that a 60-min exposure to AM251 (0, 0.1, 1mg/L) does not alter behavioural performance following habituation in either populations. However, when subsequently transferred to a novel environment, zebrafish that were pre-treated with the highest dose of AM251 (1mg/L) exhibited increased anxiety-like behavioural responses including elevated absolute turn angle, freezing and bottom dwelling. We found that exposure to the highest dose of AM251 (1mg/L) for 60min increased serotonin in fish of both populations tested. In contrast, exposure to 0.1mg/L AM251 decreased, whereas to 1mg/L AM251 increased dopamine, DOPAC and 5-HIAA in fish of both populations. CONCLUSION: Our results demonstrate a genotype-independent effect of AM251 but imply that the inconsistent findings obtained after pharmacological blockade of CB1-Rs in zebrafish may be due to a combination of concentration- and environmental context-dependent effects.

Home tank water versus novel water differentially affect alcohol-induced locomotor activity and anxiety related behaviours in zebrafish.

The zebrafish may be uniquely well suited for studying alcohol's mechanisms of action in vivo, since alcohol can be administered via immersion in a non-invasive manner. Despite the robust behavioural effects of alcohol administration in mammals, studies reporting the locomotor stimulant and anxiolytic effects of alcohol in zebrafish have been inconsistent. In the current study, we examined whether differences in the type of water used for alcohol exposure and behavioural testing contribute to these inconsistencies. To answer this question, we exposed zebrafish to either home water from their housing tanks or novel water from an isolated reservoir (i.e. water lacking zebrafish chemosensory and olfactory cues) with 0% or 1% v/v alcohol for 30 min, a 2 × 2 between subject experimental designs. Behavioural responses were quantified throughout the 30-minute exposure session via a video tracking system. Although control zebrafish exposed to home water and novel water were virtually indistinguishable in their behavioural responses, alcohol's effect on locomotor activity and anxiety-like behavioural responses were dependent on the type of water used for testing. Alcohol exposure in home tank water produced a mild anxiolytic and locomotor stimulant effect, whereas alcohol exposure in novel water produced an anxiogenic effect without altering locomotor activity. These results represent a dissociation between alcohol's effects on locomotor and anxiety related responses, and also illustrate how environmental factors, in this case familiarity with the water, may interact with such effects. In light of these findings, we urge researchers to explicitly state the type of water used.

Ethanol-Induced ADH Activity in Zebrafish: Differential Concentration-Dependent Effects on High- Versus Low-Affinity ADH Enzymes.

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 to our previous study. Using a lower substrate level in our enzyme assay targeting high-affinity isozymes, we found a similar dose-response. However, the difference in activity between the high and low substrate assays (high substrate activity - low substrate activity), which provide an index of activity for low-affinity ADH isoforms, revealed no significant effect of ethanol exposure. Our results suggest that the inverted U-shaped dose-response for total ADH activity in response to ethanol is driven primarily by high-affinity isoforms of ADH.