Influences of acute ethanol exposure on locomotor activities of zebrafish larvae under different illumination

Alcohol xxx (2015) 1e11

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Influences of acute ethanol exposure on locomotor activities of zebrafish larvae under different illumination Ning Guo a, *,1, Jia Lin b,1, Xiaolan Peng b, Haojun Chen a, Yinglan Zhang b, Xiuyun Liu b, Qiang Li b, ** a

Center for Chinese Medical Therapy and Systems Biology, Shanghai University of Traditional Chinese Medicine, 1200 Cailun Road, Shanghai 201203, China Translational Medical Center for Development and Disease, Shanghai Key Laboratory of Birth Defect, Institute of Pediatrics, Children’s Hospital of Fudan University, 399 Wanyuan Road, Shanghai 201102, China b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 16 March 2015 Received in revised form 18 August 2015 Accepted 18 August 2015

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 lightedark 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. Ó 2015 Elsevier Inc. All rights reserved.

Keywords: Ethanol Zebrafish Larvae Locomotor Light Neurotransmitter

Introduction Ethanol is a small molecule that is soluble in both aqueous and lipid environments, which render it permeable to biological membranes, including the bloodebrain barrier. The behavioral manifestations of acute ethanol administration in human include psycho-stimulation and euphoria at low and moderate doses, sedation at heavy doses, and death in extreme cases. It has been reported that ethanol has extensive influences on the functions of the central nervous system, ranging from motor ability, perception, and higher cognitive functions, such as learning and memory (Ewing, Mills, Bisgrove, & McManus, 1984; White, Matthews, & Best, 2000). * Corresponding author. Tel.: þ86 21 5132 2748; fax: þ86 21 5132 2642. ** Corresponding author. E-mail address: [email protected] (N. Guo). 1 Equal contribution. http://dx.doi.org/10.1016/j.alcohol.2015.08.003 0741-8329/Ó 2015 Elsevier Inc. All rights reserved.

The zebrafish is an aquatic vertebrate model organism. Although the nervous system of zebrafish is much simpler than that of the mammals, it possesses similar molecular pathways that are involved in the etiology of neurological disorders (Kily et al., 2008; Lockwood, Bjerke, Kobayashi, & Guo, 2004). Therefore, besides its popularity in genetic and developmental studies (Ackermann & Paw, 2003; Udvadia & Linney, 2003), the zebrafish has also been widely employed in neuro-pharmacological studies in order to elucidate the effects of neuroactive drugs on the nervous system (Ellis & Soanes, 2012; Kokel et al., 2010; van der Ven et al., 2005). It has been reported that, as early as 4e5 days post-fertilization (dpf), zebrafish larvae develop the ability to swim, and exhibit a broad spectrum of behaviors, such as hunting, avoidance, scototaxis, and thigmotaxis (Colwill & Creton, 2011a, b; Schnörr, Steenbergen, Richardson, & Champagne, 2012). Therefore, zebrafish larvae present unique opportunities to study the neurological activities of pharmaceuticals at very early developmental stages. Due to the small sizes of zebrafish larvae, larval behavior studies are

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usually carried out in multi-well plate formats; thus, locomotor activity has been extensively studied as the most straightforward and applicable parameter in investigations on the behavioral influences of environmental factors and neuroactive drugs. Zebrafish larvae demonstrate behavioral responses to acute ethanol treatment that are similar to rodents. Locomotor activities of zebrafish larvae displayed inverted U-shaped responses under the influence of ethanol. Increased locomotor activities, as measured by swimming speed or swimming distances, were observed in zebrafish larvae treated with ethanol at low concentrations, whereas, further increases in ethanol concentrations resulted in decreases in locomotor activities of the larvae (Ikeda et al., 2013; Lockwood et al., 2004; Puttonen, Sundvik, Rozov, Chen, & Panula, 2013). Mice receiving acute ethanol treatment at various doses demonstrated similar characteristic inverted Ushaped locomotor responses as observed in zebrafish. Low doses of ethanol treatment resulted in increases in locomotor activities, whereas high doses of ethanol treatment exerted inhibitory effects on locomotor activities (Aragon, Pesold, & Amit, 1992; Correa, Miquel, & Aragon, 2000; Phillips, Huson, Gwiazdon, BurkhartKasch, & Shen, 1995; Viana, Almeida-Santos, Aguiar, & Moreira, 2013). Transition in illumination from light to dark usually evokes an initial startle response in zebrafish larvae, followed by increased locomotor activities during the dark phase compared with the precedent light phase (Emran, Rihel, & Dowling, 2008; MacPhail et al., 2009). Acute ethanol treatments were reported to modulate the responses of zebrafish larvae to light changes (Irons, MacPhail, Hunter, & Padilla, 2010; MacPhail et al., 2009). Wild-type zebrafish larvae with unspecified genetic background at 6 dpf demonstrated increased locomotor activities during both the light and the dark phase when treated with 1% and 2% ethanol compared with the control group without ethanol treatment. Ethanol treatment at 4% resulted in complete inhibition of the locomotor activities of the zebrafish larvae, as well as the responses to light changes (Irons et al., 2010; MacPhail et al., 2009). In a different study using AB strain wild-type zebrafish larvae at 6 dpf, ethanol treatment at 1% resulted in increased locomotor activities during both light and dark conditions, whereas, across all illumination conditions, the larvae receiving 2% ethanol treatment demonstrated constant levels of locomotor activities that were comparable to the control group during the light condition (de Esch et al., 2012). In order to fully utilize the advantages of zebrafish larvae, behavior studies with zebrafish larvae are usually carried out between 5 and 7 dpf. AB is a well-characterized inbred wild-type zebrafish strain with a well-defined genetic background, which is widely employed to investigate the behavioral impacts of various neuroactive drugs. However, the effects of ethanol treatment on the locomotor activities of AB strain zebrafish larvae at 5e7 dpf have not been extensively examined. Therefore, the current study was designed to establish the profile of the locomotor activities of AB strain zebrafish larvae at both 5 and 7 dpf under the influence of ethanol in different illumination conditions, and to explore the possibility of using zebrafish larvae as model organisms to study the neural activities of ethanol and the related molecular mechanisms. The changes in neurotransmitter levels in response to ethanol treatments were also examined to shed light on the potential neurological mechanisms that are involved in ethanol intoxication.

a 14-h light: 10-h dark cycle (lights on at 8:00 AM, lights off at 10:00 PM). Eggs were obtained by natural spawning, and were raised in groups of 50 in an incubator at 28.5  C from birth to 7 dpf, which was staged according to a previously published method (Kimmel, Ballard, Kimmel, Ullmann, & Schilling, 1995). Eggs and larvae were kept under the same lighting schedule as adult zebrafish. All animal experimental procedures complied with local and international regulations. All protocols were approved by the institutional animal care committee. Drugs Ethanol (10009259, Sinopharm Chemical Reagent) working solutions were freshly made by serial dilutions to appropriate concentrations with the zebrafish system water before experiments. Behavior tests

Experimental procedures

All behavior tests were performed in a room with an ambient temperature of 28.5  C. The room was humidified to minimize the evaporation of the water in the testing wells. To ensure adequate swimming spaces for zebrafish larvae, behavior tests were carried out in 24-well plates. The inner diameter of each well was 18 mm, which is about 6 times the body length of a 7 dpf zebrafish larva. Behavior tests were carried out with the zebrafish larvae at both 5 dpf and 7 dpf. The larvae were obtained from group mating, and were randomly assigned to the 5 dpf group and the 7 dpf group. Since the replicate 24-well dilution series experiments were performed on 2 separate days, 2 batches of group mating were carried out. On each testing day, 4 plates were tested consecutively. All the experiments were performed between 10:00 AM and 6:00 PM. The experiments were arranged in a way that all concentration groups were equally presented in each 24-well plate to avoid any inter-treatment variations due to differences in experiment timing during the day. The zebrafish larvae were carefully transferred to a 24-well plate with one single larva in each well. Excess fluid was removed, and 500 mL of fresh zebrafish system water was loaded into each well immediately. Subsequently, 500 mL of ethanol working solution was quickly added into the wells; therefore, each well contained 1 mL liquid. The final ethanol concentrations tested were 0% (control), 0.1%, 0.25%, 0.5%, 1%, and 2% (v/v%). The substantial volume of the ethanol working solution ensured a good mixture of the liquid in the wells. In addition, the relatively small size of the well avoided the occurrence of strong currents that might agitate the larvae. The plate was then placed into a Zebrabox apparatus (ViewPoint Life Sciences) to video record the zebrafish larvae activities. The zebrafish larvae were first given a 50 min acclimation period with illumination at 110 Lx. Then three 15-min cycles (10 min illumination at 110 Lx followed by 5 min dark, i.e., illumination off) were delivered to examine the responses of the zebrafish larvae to changes in lighting conditions under the influence of ethanol. Therefore, each experiment session lasted 95 min, including the acclimation period, and ethanol was presented during the whole 95 min experiment session. The quantification of zebrafish larvae locomotion activities was achieved using the tracking mode of Zebralab software (ViewPoint Life Sciences) with recorded videos. The videos of zebrafish larvae were taken at the rate of 25 fps, and were pooled into 1 min time bins. The distance moved by the larvae in the whole well was acquired for the analysis of locomotor activities.

Zebrafish husbandry

Analysis of neurotransmitters and systemic ethanol level

AB strain wild-type zebrafish were maintained at 28.5  C according to standard protocols (Westerfield, 1995). Fish were kept on

A separate set of zebrafish larvae at 7 dpf were used for analysis. The larvae were treated with 0% (control), 0.5%, 1%, and 2% (v/v%)

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ethanol for 95 min. Immediately after the ethanol treatment, the larvae were quickly rinsed with fresh zebrafish system water for less than 15 s, to remove the residual ethanol solution on the skin, and were rapidly frozen with liquid nitrogen. The larvae were thawed on ice before decapitation. Due to the small size of the larvae, the entire heads instead of the brains were used for the quantification of neurotransmitter contents in the central nervous system. The remaining bodies were used for the quantification of systemic ethanol level. High-precision liquid chromatography (HPLC) was employed to examine the levels of norepinephrine (NE), dopamine (DA) and its metabolite 3, 4-dihydroxyphenylacetic acid (DOPAC), and serotonin (5-HT) and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) in the head. For HPLC analysis, 7 samples were acquired for each ethanol concentration group. Each sample contained heads of 40 zebrafish larvae. The samples were suspended in ice-cold PBS (20 mL/sample) and were homogenized 4 times (3 s each time) at 700 rpm on ice. Then the samples were centrifuged at 9600  g for 10 min at 4  C, and 1 mL of the supernatant from each sample was used for protein content quantification. Two mL of stabilizer (0.2 N perchloric acid) was added into each sample and centrifuged again at 9600  g for 10 min at 4  C. The supernatant was collected and stored at 80  C. HPLC analysis was carried out using an Agilent 1200 HPLC system (Agilent, USA) with Antec DECARD SDC electrochemical detection (Antec, Netherlands). The column used was an Agilent Eclipse XDB-C18 column (5 mM, 4.6 mm  150 mm). The levels of neurotransmitters were normalized to the protein content. A Headspace Gas Chromatography-Mass Spectrometer was used to quantify the systemic ethanol level in the remaining decapitated bodies of zebrafish larvae. Similar to HPLC analysis, 7 samples were acquired for each ethanol concentration group. Each sample contained bodies of 40 zebrafish larvae. The samples were processed the same way as the samples for HPLC analysis of neurotransmitters. Quantifications of ethanol contents were carried out with an Agilent G1888 automatic head space sampler (Agilent, USA) and an Agilent 7890A gas chromatography system (Agilent, USA) equipped with an Agilent 5975C mass spectrometer (Agilent, USA) and a HP-5MS capillary column (30 m long, 0.35 mm ID). The levels of ethanol content were normalized to the protein content of the respective sample. Data presentation and statistics analysis The data are expressed as mean  SEM. Statistical analyses were performed using GraphPad Prism software package. For Fig. 1AeE and Fig. 2AeE, repeated-measures two-way ANOVAs with time as the repeated variable were performed to determine the significant influences of time and ethanol treatment on locomotor activities of the zebrafish larvae. Bonferroni’s multiple comparisons test was employed to determine the significant differences between the control group and the ethanoltreated groups at each different time point. For Fig. 1F and Fig. 2F, repeated-measures two-way ANOVAs with illumination condition as the repeated variable were performed to determine the significant influences of illumination and ethanol treatment on locomotor activities of zebrafish larvae. Bonferroni’s multiple comparisons test was employed to determine the significant differences in locomotor activities of the zebrafish larvae between the light and dark conditions under the same ethanol treatment condition. For Fig. 6, a one-way ANOVA was used to determine the significant influences of ethanol treatment on the tissue levels of ethanol and the neurotransmitters in the groups treated with different concentrations of ethanol. Tukey’s multiple comparisons test was employed to explore the significant differences between individual groups.

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Results Ethanol dose dependently modified locomotor activities of zebrafish larvae The swimming distances of zebrafish larvae within each 1-min time bin were first plotted with the progression of the experiment to illustrate the changes in locomotor activities (Fig. 1AeE, Fig. 2AeE). To better demonstrate the effects of different concentrations of ethanol on the locomotor activities of zebrafish larvae, different dosage groups were plotted with the control group, one at a time. For zebrafish larvae at 5 dpf, the non-treated control group demonstrated a minimal level of locomotor activities during the light phase. Abrupt and sustained increases in locomotor activities were observed when the illumination changed from light to dark. Across the three light/dark cycles, no obvious habituation was observed, as the level of locomotor activities in the subsequent light/dark cycles remained comparable to those of the first one (Fig. 1AeE, lines with black circles). Two-way ANOVA analysis discovered significant influences of ethanol treatment on the locomotor activities of zebrafish larvae (F [5,186] ¼ 45.9, p < 0.05). Ethanol treatment at low concentrations did not yield any observable changes in the locomotor activities either during the light phase or during the dark phase (Fig. 1A and B, line with open circles). Beginning at 0.5%, compared with the control group, although there was no change in the locomotor activities during the light phase, small but significant increments in the locomotor activities during the dark phase were observed (Fig. 1C). The increases in locomotor activities during the dark phase were further enhanced when the 5 dpf larvae were treated with 1% ethanol (Fig. 1D). When treated with 2% ethanol, the larvae at 5 dpf demonstrated dramatically elevated locomotor activities during the light phase compared with the non-treated control group. During the dark phases, significant differences between the 2% ethanoltreated group and the control group were only observed during the beginning of the dark phase, and the dark-induced hyper-locomotor activities were almost abolished in the 2% ethanol-treated group (Fig. 1E). To better analyze the effects of ethanol treatment on the locomotor activities elicited by 5 dpf zebrafish larvae under different illumination conditions, the average swimming distances per minute under different illumination conditions during the first light/dark cycle were plotted (Fig. 1F). Two-way ANOVA analysis discovered significant influences of illumination conditions (F [1,186] ¼ 853.8, p < 0.05), ethanol treatments (F[5,186] ¼ 33.74, p < 0.05), as well as the interaction between the two factors (F [5,186] ¼ 32.56, p < 0.05) on the locomotor activities of the zebrafish larvae. The change in illumination condition from light to dark resulted in significant increases, within each concentration group, in the locomotor activities in all the groups as determined by Bonferroni’s multiple comparison test (Fig. 1F, open bar vs. shaded bar). It is noticeable that, in the 2% ethanol-treated group, although a significant increase in the locomotor activities in the dark phase was observed, the differences in locomotor activities under different illumination conditions is greatly reduced, compared with all other groups. The zebrafish larvae at 7 dpf demonstrated similar patterns of locomotor activity changes as the 5 dpf larvae, except that the base level locomotor activity of the non-treated control group during the light phase was much higher than that of the 5 dpf larvae (Fig. 2AeE). Ethanol treatments exerted significant influences on the locomotor activities of the larvae as determined by two-way ANOVA (F[5,186] ¼ 37.00, p < 0.05). Briefly, low concentrations of ethanol (0.1% and 0.25%) did not yield any changes in the locomotor

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activities either during the light phase or during the dark phase (Fig. 2A and B). Intermediate concentrations of ethanol (0.5% and 1%) resulted in increased locomotor activities during the dark phase without affecting that of the light phase (Fig. 2C and D). High concentration of ethanol (2%) induced hyper-locomotor activities during both the light and the dark phase. In addition, the darkinduced hyper-locomotor activities were almost abolished in the 2% ethanol-treated group (Fig. 2E). When the average swimming distances per minute during the first light/dark cycle were analyzed, two-way ANOVA analysis discovered significant influences of illumination conditions (F [1,186] ¼ 462.1, p < 0.05), ethanol treatments (F[5,186] ¼ 14.35, p < 0.05), and the interaction between the two factors (F [5,186] ¼ 20.10, p < 0.05) on the locomotor activities of the zebrafish larvae. Compared with the light phase, significant increases in locomotor activities during the dark phase were observed in all ethanol-treated groups, except the 2% group, as determined by Bonferroni’s multiple comparison test (Fig. 2F, open bar vs. shaded bar). Locomotor responses of individual zebrafish larva to ethanol treatment and changes in lighting conditions To better illustrate the influences of light changes and ethanol treatments on the locomotor responses of individual zebrafish larva, scatter plots were used to demonstrate the correlations between the swimming distance of each larva during the light phase and during the dark phase (Fig. 3). Each data point in the graph represents a single zebrafish larva. For the zebrafish larvae at 5 dpf (Fig. 3A), the group receiving 2% ethanol treatment demonstrated a drastically different distribution pattern compared with all other groups. First, very low levels of locomotor activities during the light phase were observed in all groups, except the 2% ethanol-treated group. In the groups treated with ethanol at concentrations lower than 2%, most of the data points distributed along the y-axis; while in the 2% ethanol-treated group, all the data points were away from the y-axis. This is a clear indication of enhanced locomotor activities of the zebrafish larvae with 2% ethanol treatment during the light phase. In addition, all the data points in the groups treated with ethanol at concentrations lower than 2% distributed above the diagonal line in the graphs, which indicated hyper-locomotor activities during the dark phase compared with the light phase. For the 2% ethanol-treated group, the data points demonstrated a rather linear relationship with the diagonal line, which indicated the lack of responsiveness in locomotor activities to illumination changes under the influence of 2% ethanol. For the zebrafish larvae at 7 dpf (Fig. 3B), one prominent difference from the 5 dpf larvae is that, in the control group and the groups treated with ethanol at concentrations lower than 2%, instead of sitting on the y-axis, the majority of the data points were away from the y-axis, which indicated increased locomotor activities in the 7 dpf larvae during the light phase compared with the 5 dpf larvae. As most of the data points in the groups treated with

Fig. 1. Locomotor activities of 5 dpf larvae in response to light changes under the influence of ethanol. AeE: The distances moved by zebrafish larvae in each 1 min time bin. Data are presented as mean  SEM, n ¼ 32 animals per group. Only error bars above the data points are visualized. To better visualize the data, in each panel, the

control group (filled circles) was plotted with a single ethanol concentration group (open circles). Therefore, the controls are the same in all the panels. The shaded parts in each panel represent the 5 min dark challenge phase, and the non-shaded parts in each panel represent the 10 min light phase. F: Average distanced moved by zebrafish larvae per min during the first light (opened bars)/dark (filled bars) cycle. Data are presented as mean  SEM, n ¼ 32 animals per group. Statistical icons: AeE: *p < 0.05, significantly different from the control group under the same lighting condition as determined by two-way ANOVA followed by Bonferroni’s multiple comparison test. F: *p < 0.05, significant difference between the light and dark conditions within the same ethanol concentration group as determined by two-way ANOVA followed by Bonferroni’s multiple comparison test.

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ethanol at concentrations lower than 2% are located above the diagonal line, the data points in the 2% ethanol-treated group again demonstrated a linear relationship with the diagonal line, which is similar to what was observed with the 5 dpf larvae. This is a clear indication of abolished locomotor responses to illumination changes under the influence of 2% ethanol. Activity changes immediately following a transition under the influence of ethanol In order to further elucidate the effects of ethanol on the locomotor activities of zebrafish larvae, instantaneous changes in the locomotor activities of zebrafish larvae during a light transition were analyzed. The swimming distances of zebrafish larvae 30 s before and 30 s after a light to dark or dark to light transition were plotted. For the control group zebrafish larvae at 5 dpf, a transient spike followed by a quick decay to a level that is higher than the pretransition level in locomotor activities was observed following the light to dark transition, whereas the dark to light transition caused a gradual decay in locomotor activities (Fig. 4, black lines). For the groups treated with ethanol at concentrations lower than 1%, the 5 dpf larvae demonstrated similar patterns of locomotor changes in response to light transitions (Fig. 4, top 3 panels, gray lines), as compared with the control group. For the group treated with 1% ethanol, the transition from light to dark resulted in an abrupt increase in locomotor activities followed by sustained increases in locomotor activities to a plateau level that is much higher than the control group (Fig. 4, left panel on the 4th row, gray line). The dark to light transition resulted in a gradual decrease in locomotor activities of the zebrafish larvae with 1% ethanol treatment from a level that is higher than the control group to a level that is comparable to the control group (Fig. 4, right panel on the 4th row, gray line). For the group treated with 2% ethanol, a delayed increase in locomotor activity followed by a gradual decay to the control level, which is about the same level as the larvae with 2% ethanol treatment displayed before the transition, was observed (Fig. 4, bottom left panel, gray line). The dark to light transition did not cause any change in the locomotor activities displayed by the larvae with 2% ethanol treatment (Fig. 4, bottom right panel, gray line). The zebrafish larvae at 7 dpf demonstrated similar patterns of instantaneous locomotor responses to transitions in illumination as the 5 dpf larvae (Fig. 5), although the base level locomotor activities of the 7 dpf larvae were higher than those of the 5 dpf larvae. Ethanol and neurotransmitter levels in AB zebrafish larvae at 7 dpf To understand the differences in the behaviors of zebrafish larvae under the influences of ethanol at different concentrations, the systemic levels of ethanol in 7 dpf zebrafish larvae in different ethanol-treated groups were analyzed with head-space gas chromatography (Fig. 6A). The measured body ethanol contents were further normalized with the protein content of the same respective

Fig. 2. Locomotor activities of 7 dpf larvae in response to light changes under the influence of ethanol. AeE: The distances moved by zebrafish larvae in each 1 min time bin. Data are presented as mean  SEM, n ¼ 32 animals per group. Only error bars above the data points are visualized. To better visualize the data, in each panel, the

control group (filled circles) was plotted with a single ethanol concentration group (open circles). Therefore, the controls are the same in all the panels. The shaded parts in each panel represent the 5 min dark challenge phase, and the non-shaded parts in each panel represent the 10 min light phase. F: Average distanced moved by zebrafish larvae per min during the first light (opened bars)/dark (filled bars) cycle. Data are presented as mean  SEM, n ¼ 32 animals per group. Statistical icons: AeE: *p < 0.05, significantly different from the control group under the same lighting condition as determined by two-way ANOVA followed by Bonferroni’s multiple comparison test. F: *p < 0.05, significant difference between the light and dark conditions within the same ethanol concentration group as determined by two-way ANOVA followed by Bonferroni’s multiple comparison test.

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Fig. 3. Influences of ethanol on locomotor responses of individual zebrafish larva: scatter plot. Data shown are a geometric representation of the comparisons between the swimming distances of each larva under light and dark conditions during the first light/dark cycle. Each datum point represents a single zebrafish larva. The average swimming distances (mm/ min) under light condition are along the x axis, and those under dark condition are along the y-axis. The diagonal line has a slope of 1, to visually demarcate two regions: data points above the line indicate hyper-locomotor activities under the dark condition, and those below the line indicate hypo-locomotor activities under the dark condition. A: AB strain zebrafish larvae at 5 dpf. B: AB strain zebrafish larvae at 7 dpf. The concentrations of ethanol treatments that the larvae received are marked on the top-center of each graph.

sample to control for the differences across samples due to sample preparation. Progressively increased systemic ethanol levels in zebrafish larvae were observed with increases in environmental ethanol concentration. The influences of environmental ethanol concentration were determined to be significant by one-way ANOVA (F[3,24] ¼ 23.01, p < 0.01). Tukey’s multiple comparisons test was carried out to compare the systemic ethanol levels between individual groups. Compared with the control group, the

systemic ethanol levels of the 1% and 2% ethanol-treated groups were significantly higher. Among the groups with ethanol treatments, the systemic ethanol levels of the 2% ethanol-treated group were significantly higher than those of the 0.5% and 1% ethanoltreated groups. Previous studies have reported interactions between ethanol and neurotransmitter systems involving NE, DA, 5-HT, etc. To explore the possible neurochemical mechanisms underlying the

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differential responses to ethanol treatment, the levels of NE, DA and its metabolite DOPAC, and 5-HT and its metabolite 5-HIAA were measured by HPLC (Fig. 6BeF) in the heads of 7 dpf zebrafish larvae after ethanol treatment at different concentrations (control, 0.5%, 1%, and 2%). The neurotransmitter contents were normalized to the protein contents of the same respective sample. For NE, all ethanoltreated groups demonstrated much lower levels of NE than the control group. One-way ANOVA discovered significant influences of ethanol treatments on the levels of NE (F[3,24] ¼ 5.921, p < 0.05, Fig. 6B). In addition, the 0.5% and 2% ethanol-treated groups were significantly different from the control group, as determined by Tukey’s multiple comparisons test. For DA, no significant influences of ethanol treatments were discovered by one-way ANOVA (F [3,24] ¼ 2.248, p ¼ 0.1086, Fig. 6C), and none of the differences between individual groups were statistically significant. Although the DA level was not significantly influenced by ethanol treatment, the level of its metabolite DOPAC was significantly affected by ethanol treatment (F[3,24] ¼ 3.039, p < 0.05, Fig. 6D). Significant differences in the DOPAC levels between the 1% and 0.5% ethanoltreated groups were discovered by Tukey’s multiple comparisons test. The influences of ethanol treatment on the levels of 5-HT and its metabolite 5-HIAA across all tested groups were similar. For both transmitters, the influences of ethanol treatments were significant as determined by one-way ANOVA (5-HT: F[3,24] ¼ 4.329, p < 0.05, Fig. 6E; 5-HIAA: F[3,24] ¼ 8.262, p < 0.05, Fig. 6F). The 1% ethanoltreated group demonstrated the highest levels in both transmitters, which were significantly different from all other groups. Discussion Ethanol treatment at 2% induced increases in locomotor activities during the light phase

Fig. 4. Locomotor activities of zebrafish larvae at 5 dpf immediately following transitions. Second-by-second swimming distances of zebrafish larvae 30 s prior to and 30 s following a light to dark or dark to light transition under the ethanol treatment at different concentrations are plotted. The shaded part in each panel represents the dark phase, and the non-shaded part in each panel represents the light phase. To better visualize the data, in each panel, the control group (black line) is plotted with a single

In the current study, the influences of ethanol treatment on the locomotor activities of zebrafish larvae under alternating light and dark conditions were examined. The zebrafish larvae at both 5 and 7 dpf demonstrated similar locomotor responses to ethanol treatment under light illumination. Across all the concentrations tested, ethanol treatment at concentrations lower than 2% did not yield significant changes in the locomotor activities as measured by swimming distances, whereas ethanol treatment at 2% markedly increased the locomotor activities of zebrafish larvae. Our finding is in accordance with previously published results. Swimming speed of AB strain zebrafish larvae at 7 dpf was significantly increased with acute ethanol treatment at 1%, 1.5%, and 2%. Treatment at higher ethanol concentrations, such as 4%, resulted in reduced swimming speed of zebrafish larvae compared with the non-treated control group (Lockwood et al., 2004). In a different study using Turku-strain wild-type zebrafish larvae at 7 dpf, acute ethanol treatment at 0.75% and 1.5% significantly enhanced the locomotor activities as measured by the swimming distances, while 3% ethanol treatment did not affect the swimming distances of the zebrafish larvae, compared with the non-treated control group (Puttonen et al., 2013). Increased swimming speed and swimming distances were observed when 6 dpf zebrafish larvae of Tubingen long-fin strain were treated with ethanol at the concentration of 300 mM, which is equivalent to 1.4%, whereas treatment at 100 mM did not affect either parameter (Ikeda et al., 2013). The variations in the active doses that induced hyper-locomotor activities across different

ethanol concentration group (gray line). Error bars are not displayed to better demonstrate the trend of changes in swimming distances following the transition. n ¼ 32 animals per group.

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studies may be the result of differences in the zebrafish strains, ages, and, more importantly, ethanol treatment regimens. Ethanol treatment at 0.5% and 1% enhanced the dark-induced hyperlocomotor activities during the dark phase without affecting the base level locomotor activities during the light phase It has been reported that zebrafish larvae demonstrated increases in locomotor activities when the illumination changed from light to dark (de Esch et al., 2012; Irons, Kelly, Hunter, Macphail, & Padilla, 2013; Irons et al., 2010; MacPhail et al., 2009; Vignet et al., 2013). Our findings are consistent with this phenomenon. The nontreated control group zebrafish larvae at both 5 and 7 dpf demonstrated marked increases in locomotor activities after the transition from light to dark. The effect of ethanol treatment on the dark-induced hyper-locomotor activities has also been reported. In one study that employed wild-type zebrafish larvae at 6 dpf with unspecified genetic background, ethanol treatments at 1% and 2% resulted in significant increases in the locomotor activities both in the light and the dark phase, compared with the non-treated control group. All groups (control, 1%, and 2%) demonstrated increased locomotor activities in the dark period compared with the light period (MacPhail et al., 2009). In a different study, the 6 dpf wild-type zebrafish larvae with unspecified genetic background demonstrated enhanced locomotor activities under the treatment of 1% and 2% ethanol during the light phase. During the dark phase, ethanol-induced increases in locomotor activities were only observed in the first and initial dark phase, and were absent from the following dark periods. The comparisons between the locomotor activities under different illuminations were not performed; however, dark-induced hyper-locomotor activities were obvious in both the control and the 1% ethanol-treated groups (Irons et al., 2010). AB strain wild-type zebrafish larvae at 6 dpf with ethanol treatment at 1% demonstrated increased locomotor activities during both light and dark conditions, whereas, across all illumination conditions, the larvae receiving 2% ethanol treatment demonstrated a constant level of locomotor activities that were comparable to the control group during the light condition (de Esch et al., 2012). In the current study, we have discovered a rather interesting phenomenon that was not reported by others. Ethanol treatment at 1% enhanced the dark-induced hyper-locomotor activities during the dark phase without affecting the base level locomotor activities during the light phase in both 5 and 7 dpf larvae. Ethanol treatment at 0.5% evoked similar locomotor responses in zebrafish larvae at 7 dpf, although to a much lesser extent. Ethanol treatment at 2% almost abolished the dark-induced hyperlocomotor activities

Fig. 5. Locomotor activities of zebrafish larvae at 7 dpf immediately following transitions. Second-by-second swimming distances of zebrafish larvae 30 s prior to and 30 s following a light to dark or dark to light transition under the ethanol treatment at different concentrations are plotted. The shaded part in each panel represents the dark phase, and the non-shaded part in each panel represents the light phase. To better visualize the data, in each panel, the control group (black line) is plotted with a single

Scatter plots were used to display the locomotor responses of individual zebrafish larva to changes in illumination conditions under the influences of ethanol (Fig. 3). The distribution patterns of the data points in the control group and the groups treated with ethanol at concentrations lower than 2% clearly indicated darkinduced hyper-locomotor activities in both the 5 and 7 dpf larvae. The rather linear distribution of the data points around the diagonal line demonstrated by the 5 and 7 dpf larvae under 2% ethanol

ethanol concentration group (gray line). Error bars are not displayed to better demonstrate the trend of changes in swimming distances following the transition. n ¼ 32 animals per group.

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Fig. 6. Influences of ethanol treatment on tissue ethanol and neurotransmitter levels. Systemic ethanol levels in the body (A), as well as levels of 5 neurotransmitters in the central nervous system NE (B), DA (C), DOPAC (D), 5-HT (E), and 5-HIAA (F) were measured. Data are presented as mean  SEM, n ¼ 7 samples per group. Each sample contained tissue from 40 zebrafish larvae. Statistic icons: *p < 0.05, significantly different from the control group as determined by one-way ANOVA followed by Tukey’s multiple comparisons test. #p < 0.05, significant difference between the bracketed ethanol-treated groups as determined by one-way ANOVA followed by Tukey’s multiple comparisons test.

treatment indicated that the locomotor responses of zebrafish larvae to illumination changes were diminished. This notion was further supported by our finding that the locomotor activities of 7 dpf larvae under 2% ethanol treatment during the dark phase did not significantly differ from that during the light phase (Fig. 2F). Although the locomotor activities of 5 dpf larvae under 2% ethanol treatment during the dark phase were significantly higher than that during the light phase, the difference was greatly reduced compared with other

groups (Fig. 1F). Impaired visual sensitivity could lead to the lack of responses to light changes. Previously published reports have demonstrated that early (before 3 dpf) and prolonged (lasting from 12 h to 3 days) exposure of zebrafish embryos to ethanol resulted in malformations and disrupted functions of the larval visual system (Bilotta, Saszik, Givin, Hardesty, & Sutherland, 2002; Matsui, Egana, Sponholtz, Adolph, & Dowling, 2006). However, whether acute ethanol treatment at 5 and 7 dpf for 90 min could affect the visual

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sensitivity of zebrafish larvae to illumination changes has not yet been investigated. Ethanol treatment at 1% and 2% altered the response immediately following the illumination transition It has been reported that, as early as 4 dpf, wild-type zebrafish larvae demonstrated startle responses to sudden transitions in illumination either from light to dark or from dark to light (Emran et al., 2008). Neuroactive drugs, such as pentylenetetrazole, aconitine, and 4-aminopyridine, were reported to modulate these illumination transition-induced startle responses in zebrafish larvae (Ellis, Seibert, & Soanes, 2012). A rather interesting observation is that, in both studies, the control group larvae without drug treatment always demonstrated greater startle responses following the transition from light to dark than those following the transition from dark to light. This phenomenon possibly indicated that the transition from light to dark was a more stressful condition than the transition from dark to light. Similar startle responses from light to dark were observed in the current study with control group zebrafish larvae at both 5 and 7 dpf (Figs. 4 and 5, left panels, black lines). However, the startle responses associated with dark to light transition were absent from our study (Figs. 4 and 5, right panels, black lines). This could possibly due to the differences in the genetic background of the zebrafish and the illumination transition paradigms employed in different studies. The distinct patterns of locomotor behaviors demonstrated by zebrafish larvae with ethanol treatment at 1% and 2% represented different ethanol intoxication states In the current study, ethanol treatment at 1% did not affect the locomotor activities of the zebrafish larvae under normal illumination, whereas the dark-induced hyper-locomotor activities were dramatically enhanced (Fig. 1D and F, Fig. 2D and F). On the other hand, 2% ethanol treatment induced significant increases in locomotor activities under the light, whereas the dark-induced hyperlocomotor activities were attenuated or even abolished (Fig. 1E and F, Fig. 2E and F). The contrast between the locomotor activities of the two groups of larvae with ethanol treatments at different concentrations provided evidence of different ethanol intoxication states. Zebrafish larvae with 1% ethanol treatment demonstrated characteristics of light ethanol intoxication. The behaviors of the subjects under normal conditions were not affected, but the responses to environmental stimuli were amplified. The 2% ethanol-treated group displayed characteristics of moderate ethanol intoxication, hyperactivity under normal conditions, and blunted responses to environmental stimuli. Ethanol treatment at different concentrations resulted in different systemic ethanol content in zebrafish bodies and affected levels of multiple neurotransmitters in the central nervous system Several studies have quantified the ethanol concentrations in zebrafish after acute ethanol treatments. The brain ethanol concentration was discovered to be at about 20% of the environmental ethanol concentration in adult AB strain zebrafish receiving ethanol treatments at 0.5% and 1% (v/v%) for 60 min (Tran, Chatterjee, & Gerlai, 2015). Adult AB strain zebrafish acutely treated with ethanol demonstrated progressively increased swimming distances with increases in environmental ethanol concentration. In a different study using adult zebrafish of a commercial strain with

unspecified genetic background, acute 1% ethanol treatment for 60 min resulted in a brain ethanol level of about 5.5 mg per mg of wet tissue (Rosemberg et al., 2012). Behaviorally, the adult zebrafish with 1% ethanol treatment for 60 min demonstrated significantly reduced swimming distance than the non-treated control group. Zebrafish embryos receiving acute ethanol treatment at concentrations of 1%e5% from 8 to 10 hours post-fertilization (hpf) or 24e27 hpf demonstrated tissue ethanol contents ranging from 50 mM to 300 mM (Zhang et al., 2014). Acute ethanol treatments of AB and TU strain zebrafish embryos at concentrations up to 1% (v/v %) for 2 h at 24 hpf resulted in tissue ethanol contents that were approximately 8% of the environmental ethanol concentrations (Mahabir, Chatterjee, & Gerlai, 2014). So far, the correlation between the intracellular ethanol level in zebrafish and the plasma ethanol content in humans has not yet been established. Therefore, it is hard to relate the tissue ethanol content in zebrafish to the physiological plasma ethanol levels in humans. In the current study, the systemic ethanol levels in zebrafish bodies increased in correspondence to the increases in the environmental ethanol concentration (Fig. 6A). Although the measurements were rather artificial and lacked physiological relevance, the results demonstrated that ethanol treatment at different concentrations did result in different systemic alcohol content in zebrafish. Ethanol treatments were discovered to influence the levels of various neurotransmitters in zebrafish. Ethanol treatments at concentrations up to 0.5% in AB strain zebrafish larvae at 24 hpf for 2 h not only impaired the shoaling behavior of the zebrafish at 70 dpf, but also significantly reduced the levels of DA, DOPAC, 5-HT, and 5-HIAA in the brains of the 70 dpf zebrafish (Buske & Gerlai, 2011). Acute 1-h ethanol exposure at concentrations up to 1% in 90 dpf AB strain zebrafish resulted in increased levels of DA, DOPAC, 5-HT, and 5-HIAA in the brains of the zebrafish (Chatterjee & Gerlai, 2009). In our current study, the levels of different neurotransmitters in 7 dpf larvae after ethanol treatment were measured, in order to shed light on which neurological pathways are involved in ethanol intoxication. Due to the lack of behavioral responses of zebrafish larvae with ethanol treatments at low concentrations (0.1%, 0.25%), only the 0.5%, 1%, and 2% ethanol-treated groups were investigated, in addition to the control group. Ethanol treatment significantly reduced the anxiety level as indicated by the decreased NE level in all ethanol-treated groups, although a robust (near 50%) yet nonsignificant decrease was observed in the 1% ethanol-treated group (Fig. 6B). Whereas the control, 0.5%, and 2% ethanol-treated groups demonstrated similar levels in DA, 5-HT, and their metabolites DOPAC and 5-HIAA, respectively, the 1% ethanol-treated group demonstrated the highest levels of these transmitters (Fig. 6CeF). Interestingly, it has been reported that non-selective and D1-selective dopaminergic agonists (apomorphine and SKF-38393, respectively) were able to enhance the dark-induced hyper-locomotor activities in zebrafish larvae with unspecified genetic background at 6 dpf without affecting the base level locomotor activities during the light period (Irons et al., 2013), which is similar to our result with 1% ethanol treatment (Fig. 1D and F, Fig. 2D and F). Our results indicated the involvement of multiple neurological pathways in the action of alcohol, which is in accordance with previous notions that alcohol exerts extensive effects on the nervous system, although studies that are more detailed are needed to dissect the exact molecular mechanisms of alcohol intoxication. Conclusions In summary, we have investigated the effects of ethanol on the locomotor activities of zebrafish larvae at both 5 and 7 dpf under different illumination conditions. The larvae at 5 and 7 dpf

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demonstrated similar patterns of locomotor responses to ethanol treatment, which indicated the behavioral studies concerning the ethanol effects on locomotor activities of zebrafish larvae could be carried out as early as 5 dpf. Ethanol treatment at 1% potentiated the zebrafish larvae to dark-induced hyper-locomotor activities, whereas ethanol treatment at 2% not only increased the base level locomotor activities in the light but also abolished the locomotor responses of zebrafish larvae to light changes. 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. Acknowledgments This work was supported by Innovation Program of Shanghai Municipal Education Commission (2012Z10268036) and Open Project of Key Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders, Ministry of Education, P.R. China to Guo N; National Natural Science Foundation of China Grant (81271509), Shanghai Pujiang Program (11PJ1401800) from Science and Technology Commission of Shanghai Municipality and Open Project of Key Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders, Ministry of Education, P.R. China to Li Q. Conflict of interest: All the authors declare no conflicts of interest. References Ackermann, G. E., & Paw, B. H. (2003). Zebrafish: a genetic model for vertebrate organogenesis and human disorders. Frontiers in Bioscience, 8, d1227e1253. Aragon, C. M., Pesold, C. N., & Amit, Z. (1992). Ethanol-induced motor activity in normal and acatalasemic mice. Alcohol, 9, 207e211. Bilotta, J., Saszik, S., Givin, C. M., Hardesty, H. R., & Sutherland, S. E. (2002). Effects of embryonic exposure to ethanol on zebrafish visual function. Neurotoxicology and Teratology, 24, 759e766. Buske, C., & Gerlai, R. (2011). Early embryonic ethanol exposure impairs shoaling and the dopaminergic and serotoninergic systems in adult zebrafish. Neurotoxicology and Teratology, 33, 698e707. Chatterjee, D., & Gerlai, R. (2009). High precision liquid chromatography analysis of dopaminergic and serotoninergic responses to acute alcohol exposure in zebrafish. Behavioural Brain Research, 200, 208e213. Colwill, R. M., & Creton, R. (2011a). Imaging escape and avoidance behavior in zebrafish larvae. Reviews in the Neurosciences, 22, 63e73. Colwill, R. M., & Creton, R. (2011b). Locomotor behaviors in zebrafish (Danio rerio) larvae. Behavioural Processes, 86, 222e229. Correa, M., Miquel, M., & Aragon, C. M. (2000). Lead acetate potentiates brain catalase activity and enhances ethanol-induced locomotion in mice. Pharmacology, Biochemistry, and Behavior, 66, 137e142. Ellis, L. D., Seibert, J., & Soanes, K. H. (2012). Distinct models of induced hyperactivity in zebrafish larvae. Brain Research, 1449, 46e59. Ellis, L. D., & Soanes, K. H. (2012). A larval zebrafish model of bipolar disorder as a screening platform for neuro-therapeutics. Behavioural Brain Research, 233, 450e457. Emran, F., Rihel, J., & Dowling, J. E. (2008). A behavioral assay to measure responsiveness of zebrafish to changes in light intensities. Journal of Visualized Experiments (20). http://dx.doi.org/10.3791/923, pii: 923. de Esch, C., van der Linde, H., Slieker, R., Willemsen, R., Wolterbeek, A., Woutersen, R., et al. (2012). Locomotor activity assay in zebrafish larvae: influence of age, strain and ethanol. Neurotoxicology and Teratology, 34, 425e433.

11

Ewing, J. A., Mills, K. C., Bisgrove, E. Z., & McManus, K. (1984). Selective attenuation of ethanol-induced performance impairment by naloxone in humans. Advances in Alcohol & Substance Abuse, 3, 47e59. Ikeda, H., Delargy, A. H., Yokogawa, T., Urban, J. M., Burgess, H. A., & Ono, F. (2013). Intrinsic properties of larval zebrafish neurons in ethanol. PLoS One, 8, e63318. Irons, T. D., Kelly, P. E., Hunter, D. L., Macphail, R. C., & Padilla, S. (2013). Acute administration of dopaminergic drugs has differential effects on locomotion in larval zebrafish. Pharmacology, Biochemistry, and Behavior, 103, 792e813. Irons, T. D., MacPhail, R. C., Hunter, D. L., & Padilla, S. (2010). Acute neuroactive drug exposures alter locomotor activity in larval zebrafish. Neurotoxicology and Teratology, 32, 84e90. Kily, L. J., Cowe, Y. C., Hussain, O., Patel, S., McElwaine, S., Cotter, F. E., et al. (2008). Gene expression changes in a zebrafish model of drug dependency suggest conservation of neuro-adaptation pathways. The Journal of Experimental Biology, 211(Pt 10), 1623e1634. Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B., & Schilling, T. F. (1995). Stages of embryonic development of the zebrafish. Developmental Dynamics, 203, 253e310. Kokel, D., Bryan, J., Laggner, C., White, R., Cheung, C. Y., Mateus, R., et al. (2010). Rapid behavior-based identification of neuroactive small molecules in the zebrafish. Nature Chemical Biology, 6, 231e237. Lockwood, B., Bjerke, S., Kobayashi, K., & Guo, S. (2004). Acute effects of alcohol on larval zebrafish: a genetic system for large-scale screening. Pharmacology, Biochemistry, and Behavior, 77, 647e654. MacPhail, R. C., Brooks, J., Hunter, D. L., Padnos, B., Irons, T. D., & Padilla, S. (2009). Locomotion in larval zebrafish: Influence of time of day, lighting and ethanol. Neurotoxicology, 30, 52e58. Mahabir, S., Chatterjee, D., & Gerlai, R. (2014). Strain dependent neurochemical changes induced by embryonic alcohol exposure in zebrafish. Neurotoxicology and Teratology, 41, 1e7. Matsui, J. I., Egana, A. L., Sponholtz, T. R., Adolph, A. R., & Dowling, J. E. (2006). Effects of ethanol on photoreceptors and visual function in developing zebrafish. Investigative Ophthalmology & Visual Science, 47, 4589e4597. Phillips, T. J., Huson, M., Gwiazdon, C., Burkhart-Kasch, S., & Shen, E. H. (1995). Effects of acute and repeated ethanol exposures on the locomotor activity of BXD recombinant inbred mice. Alcoholism: Clinical and Experimental Research, 19, 269e278. Puttonen, H. A., Sundvik, M., Rozov, S., Chen, Y. C., & Panula, P. (2013). Acute ethanol treatment upregulates Th1, Th2, and Hdc in larval zebrafish in stable networks. Frontiers in Neural Circuits, 7, 102. Rosemberg, D. B., Braga, M. M., Rico, E. P., Loss, C. M., Córdova, S. D., Mussulini, B. H., et al. (2012). Behavioral effects of taurine pretreatment in zebrafish acutely exposed to ethanol. Neuropharmacology, 63, 613e623. Schnörr, S. J., Steenbergen, P. J., Richardson, M. K., & Champagne, D. L. (2012). Measuring thigmotaxis in larval zebrafish. Behavioural Brain Research, 228, 367e374. Tran, S., Chatterjee, D., & Gerlai, R. (2015). An integrative analysis of ethanol tolerance and withdrawal in zebrafish (Danio rerio). Behavioural Brain Research, 276, 161e170. Udvadia, A. J., & Linney, E. (2003). Windows into development: historic, current, and future perspectives on transgenic zebrafish. Developmental Biology, 256, 1e17. van der Ven, K., De Wit, M., Keil, D., Moens, L., Van Leemput, K., Naudts, B., et al. (2005). Development and application of a brain-specific cDNA microarray for effect evaluation of neuro-active pharmaceuticals in zebrafish (Danio rerio). Comparative Biochemistry and Physiology. Part B, Biochemistry & Molecular Biology, 141, 408e417. Viana, T. G., Almeida-Santos, A. F., Aguiar, D. C., & Moreira, F. A. (2013). Effects of aripiprazole, an atypical antipsychotic, on the motor alterations induced by acute ethanol administration in mice. Basic & Clinical Pharmacology & Toxicology, 112, 319e324. Vignet, C., Bégout, M. L., Péan, S., Lyphout, L., Leguay, D., & Cousin, X. (2013). Systematic screening of behavioral responses in two zebrafish strains. Zebrafish, 10, 365e375. Westerfield, M. (1995). The zebrafish book: A guide for the laboratory use of zebrafish, Brachydanio rerio. Eugene, OR: University of Oregon Press. White, A. M., Matthews, D. B., & Best, P. J. (2000). Ethanol, memory, and hippocampal function: a review of recent findings. Hippocampus, 10, 88e93. Zhang, C., Frazier, J. M., Chen, H., Liu, Y., Lee, J. A., & Cole, G. J. (2014). Molecular and morphological changes in zebrafish following transient ethanol exposure during defined developmental stages. Neurotoxicology and Teratology, 44, 70e80.