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Published in final edited form as: Neurotoxicol Teratol. 2015 Jan 16;48:1–8. doi: 10.1016/j.ntt.2015.01.005

Long-term Behavioral Impairment Following Acute Embryonic Ethanol Exposure in Zebrafish

JM Bailey 1, AN Oliveri 2, C Zhang 4, JM Frazier 3, S Mackinnon 4, GJ Cole 3,4, ED Levin 1,2
PMCID: PMC4363207  NIHMSID: NIHMS656584  PMID: 25599606

Abstract

BACKGROUND

Developmental exposure to ethanol has long been known to cause persisting neurobehavioral impairment. However, the neural and behavioral mechanisms underlying these deficits and the importance of exposure timing are not well-characterized. Given the importance of timing and sequence in neurodevelopment it would be expected that alcohol intoxication at different developmental periods would result in distinct neurobehavioral consequences.

METHODS

Zebrafish embryos were exposed to ethanol (0%, 1%, 3%) at either 8-10 or 24-27 hours post-fertilization (hpf) then reared to adolescence and evaluated on several behavioral endpoints. Habituation to a repeated environmental stimulus and overall sensorimotor function were assessed using a tap startle test; measurements of anxiety and exploration behavior were made following introduction to a novel tank; and spatial discrimination learning was assessed using aversive control in a three-chambered apparatus. Overt signs of dysmorphogenesis were also scored (i.e. craniofacial malformations, including eye diameter and midbrain-hindbrain boundary morphology).

RESULTS

Ethanol treated fish were more active both at baseline and following a tap stimulus compared to the control fish and were hyperactive when placed in a novel tank. These effects were more prominent following exposure at 24-27 hpf than with the earlier exposure window, for both dose groups. Increases in physical malformation were only present in the 3% ethanol group; all malformed fish were excluded from behavioral testing.

DISCUSSION

These results suggest specific domains of behavior are affected following ethanol exposure, with some but not all of the tests revealing significant impairment. The behavioral phenotypes following distinct exposure windows described here can be used to help link cellular and molecular mechanisms of developmental ethanol exposure to functional neurobehavioral effects.

Keywords: Zebrafish, ethanol, alcohol, development, hyperactivity

Introduction

The widespread effects of ethanol exposure on the fetal central nervous system (CNS) are well known to culminate in a constellation of behavioral impairments, a syndrome seen in a wide range of species (see reviews Cole et al., 2012; Gerlai et al., 2000; Ungerer et al., 2013). Moreover, it appears that the timing of ethanol exposure can be an important determinant of the CNS structures that are primarily affected and therefore presumably determines the nature of functional impairment (Hamre & West, 1993). In humans, the cognitive and behavioral effects associated with developmental ethanol exposure are classified clinically as fetal alcohol syndrome (FAS), or more broadly, fetal alcohol spectrum disorder (FASD). Characteristic signs and symptoms of FAS are growth retardation, craniofacial malformations, and neurodevelopmental abnormalities including intellectual delay (Jones & Smith, 1973; Warren & Foudin, 2001). While many of the overt characteristics (e.g. facial malformations and CNS structure) of FAS can be recapitulated with early embryonic ethanol exposure in rodents (see Sulik et al., 1981 and Sulik, 2005) some of the subtle cognitive and behavioral characteristics have not been well characterized following developmentally distinct time points of ethanol exposure. However, zebrafish have been used to model some behavioral aspects of fetal alcohol exposure (Bilotta, 2003; Loucks & Ahlgren, 2012) and it has been shown that moderate concentrations of ethanol exposure from 24-27 hpf impairs social affiliation in adult zebrafish (Fernandes & Gerlai, 2009) and 0-6 dpf of exposure to ethanol decreases accuracy on a spatial discrimination task (Carvan et al., 2004).

Despite the emerging emphasis on exposure timing and the importance of episodic binge drinking as a risk during pregnancy, the general practice in animal models of fetal alcohol effects (FAE) is to expose the fetus or larvae chronically to ethanol, which often corresponds to an entire trimester of human gestation (or more) and cannot sustain high-dose exposures due to lethality (see Zhang et al., 2011 and Zhang et al., 2013). This model fails to reflect the binge nature of ethanol consumption by humans, in which relatively high doses of ethanol are consumed during a short span of time. Therefore, in addition to characterizing the functional impact of ethanol during distinct developmental windows, a more valid model of human ethanol consumption is achieved by mimicking episodes of maternal binge drinking.

The small freshwater zebrafish (Danio rerio) has emerged as a powerful tool for uncovering neural mechanisms of numerous syndromes and diseases because of the relative ease of using genetic and molecular tools in this species, coupled with highly conserved neural architecture and the capacity for complex behavior. The primary goal of this study was to characterize the behavioral effects of early (gastrulation) and late (organogenesis) developmental exposure to moderate-to-high doses of ethanol in zebrafish. Such data should facilitate further characterization of cellular and behavioral mechanisms that underlie FAS. To this end, the present design utilized a zebrafish model to investigate the persistent neurobehavioral deficits that result from short-term ethanol exposure during early development.

Methods

Animals

Zebrafish (Danio Rerio) from the AB strain (progenitors obtained from the Zebrafish International Resource Center) were bred in-house and generated all embryos used in the present study. All fish were housed in automatic fish housing systems at 28.5°C while at North Carolina Central University (NCCU) (Aquaneering, San Diego, CA, USA) and while at Duke University (Aquatic Habitats, Apopka, FL, USA). Fish were group housed with <20 fish per 3L tank on the multi-tank flow-through system and maintained on a 14 hr light,10 hr dark cycle 7 days per week. Ethanol exposure was carried out at NCCU and fish were subsequently transferred to Duke University for behavioral assessment, approximately 20 fish per ethanol exposure condition were reared for behavioral testing, fish with physical malformations (described below) were excluded from behavioral analyses. All behavioral testing was conducted between the hours of 12:00 and 5:00 pm, which was during the light phase. All fish were fed twice daily, artemia (Brine Shrimp Direct, Ogden, Utah) in the morning and dry flake fish food (TetraMin®, Tropical Flakes, Melle, Germany) in the evening; evening feeding was always withheld until the completion of behavioral testing. Each behavioral test is described in detail below, tests were conducted within subject on separate days with each fish experiencing each test only once, although the tap startle and spatial discrimination procedures are each made up of 10 intra-session trials. The protocols used were approved by the Institutional Animal Care and Use Committees at Duke University and North Carolina Central University in accordance with NIH and United States regulations.

Ethanol Treatment of Zebrafish Embryos

All breeding, embryo harvesting and ethanol exposures were conducted at North Carolina Central University, Durham, NC, USA, in the Cole laboratory. Zebrafish embryos in fish water containing a 1:500 dilution of 0.1% methylene blue (to prevent fungal infection) were exposed to 1.0% and 3.0% ethanol from 8 to 10 hpf or 24 to 27 hpf. Ethanol was diluted with fish water to its final concentration, and at the selected developmental stage for ethanol treatment embryos were placed in fresh fish water containing ethanol. At the end of the exposure period, fish water containing ethanol was removed, embryos were washed once with fresh fish water and then transferred to fresh fish water and placed on an aquarium rack system for rearing. At approximately 2 months of age (which corresponds to the late fry juvenile stage of development) all fish were transferred to the Levin laboratory at Duke University, Durham, NC, USA for approximately one-week acclimation prior to behavioral testing.

Brain and Facial Malformation Scoring

Eye size was measured at 2 dpf as previously described (Zhang et al., 2011; 2014), and involved measuring the longest axis along the eye, and calculated against a standard 50 μm ruler under the same magnification. For 2 dpf eye measurements, we designated a diameter of less than 240 μm as the “small eye” phenotype, since all untreated eyes were at least 250 μm in diameter. Malformation of the midbrain-hindbrain boundary (MHB) was assessed visually based on absence of the defined border between the midbrain and hindbrain. The presence of the MHB was defined as the presence of 3 or 4 ridges (tectal and cerebellar boundaries) perpendicular to the anterior-posterior (AP) axis of the CNS at the midbrain-hindbrain junction. Absence of this defined border was scored as a disruption of MHB development and has been previously described (see Zhang et al, 2014).

Behavioral Testing

Novel tank dive task

The novel tank paradigm capitalizes on a species-specific pattern of responding to a novel environment in which zebrafish initially dwell on the tank floor before gradually exploring, occasioning the water surface after several minutes undisturbed in a novel environment (Bencan and Levin, 2008, Bencan et al., 2009, Levin, 2011 and Levin et al., 2007). This initial “dive response” is an effective strategy for predatory avoidance inasmuch as it removes the possibility of being consumed from below; the later exploration is presumably food-seeking/foraging behavior as zebrafish feed along the water column, including from the water's surface. Therefore, any deviation from this dive/explore response pattern is considered maladaptive.

Zebrafish were individually tested in 1.5-liter plastic tanks filled with 1350 ml of tank water, the surface of the water was approximately 10 cm from the tank floor. The trapezoid shaped tanks were 22.9 cm along the bottom and 27.9 cm along the top. The diagonal side of the tank was 15.9 cm and the opposite vertical side was 15.2 cm, the same design as used previously (see Levin et al., 2007). Behavior was tracked in real-time using EthoVision tracking software (Noldus Information and Technology, Wageningen, The Netherlands) which calculated distance from the tank floor and total distance traveled. Each trial was five-minutes in duration and commenced immediately after a fish was placed in the novel tank. The video signal was transmitted through a Samsung camcorder that was positioned approximately 88 cm away from the testing tanks.

Tap startle and habituation test

Twenty-four hours after completion of the dive test, animals were tested for sensorimotor function and habituation learning using a tap startle (Levin et al., 2007; Sledge et al., 2011). The startle test apparatus consisted of flat white 20.4 cm × 38.1 cm surface with white 12.7 cm × 15.2 cm frontal and rear blocking barriers attached. On the flat surface there were arranged eight identical 5.1 cm × 7.6 cm clear cylindrical arenas made of Plexiglas in a 2 × 4 array. Each of the eight arenas was filled with 30 ml of tank water. The apparatus was positioned between two white opaque barriers, which faced each other and projected a bare white screen. Mechanical solenoids were positioned beneath each arena. A Samsung camcorder was located approximately 71 cm above the apparatus. The solenoids were used to administer taps to the bottom of the cylindrical arena under control of the computer. Zebrafish were taken from the holding tanks and each one was placed into one of eight cylindrical arenas. Zebrafish were allowed to acclimate in the arena for 10 min prior to testing then solenoids tapped the testing arenas at 1 min intervals for 10 consecutive trials. As each session handles eight zebrafish, exposure group was counterbalanced across all arenas and across all sessions. EthoVision tracking software was used to calculate total distance traveled 5 s before and 5 s after delivery of the tap stimulus.

Spatial Discrimination Learning

A three-chamber tank was developed in our laboratory to assess spatial learning and memory in zebrafish (Arthur & Levin, 2001, Eddins et al., 2009, Levin & Cerutti, 2008, Levin & Chen, 2004, Levin et al., 2003 and Levin et al., 2006). The apparatus is made from a cylindrical pipe cut in half length-wise and divided into three chambers, the central start chamber and two choice chambers located to the right and left of the central alley. The central starting alley provides access to each side chamber when movable door-openings are adjusted (i.e. rotatable plastic partitions were movable along Plexiglas rods through both sides of the apparatus). Three thick horizontal black stripes along one wall of the chamber served as a visual cue to provide an axis of right–left orientation, so that the fish may discriminate right from left. Prior to training, five preference assessment trials were used to establish a side bias. After the preference assessment, 10 test trials were initiated in which the fish were trained to select their non-preferred side as determined by the preference assessment. At the start of each trial, zebrafish were individually placed in the central alley. After 60 s, the partitions to both of the side chambers were simultaneously opened. After correct choices, the partition was closed and drawn a maximum distance from the fish, creating the largest possible tank space for the fish. After incorrect choices, the partition between the start chamber and the end wall was drawn to provide a space one cm wide for 30 seconds. Choice accuracy and response latency were recorded for each trial.

Statistical Analysis

The data were analyzed for statistical significance using Superanova/Statview (SAS; Cary, NC, USA). The Type 1 error rate (α) was set at 0.05 for all tests. A mixed design repeated measures analysis of variance (ANOVA), with ethanol exposure as the between subjects factor and trial number or time as repeated-measure, was utilized. The Greenhouse-Geiser adjustment was used to control for possible deviations from sphericity in the dataset and log transformations (as noted) were applied when the distribution of the data was positively or negatively skewed. Dunnett's tests were used for post-hoc comparisons between control and treated groups. For the novel tank diving test, the mean distance from the floor (i.e. tank location) and swimming distance measured in centimeters per min were calculated. For the startle habituation test, the dependent measure was the distance traveled for the 5 s preceding and 5 s following the delivery of each of the ten taps that constituted a session. Log transforms were performed on distance traveled during the startle habituation test. Finally, for the 3-chamber spatial discrimination task, accuracy (percent correct) and latency to respond were calculated for each of 10 trials. For behavioral assays that spanned 10 trials (tap startle and spatial discrimination), data are presented as binned means of 2 trials, for a total of 5 data points.

Results

Novel Tank Diving

Ethanol-exposed fish were more active (i.e. traveled a greater overall distance) than control fish when placed in a novel tank environment (F(4,116)=4.08, p<0.005) (Fig. 1a). Dunnett's post-hoc comparisons of treated groups and controls revealed the 1% 24-27 hpf group and the 3% 8-10 and 24-27 hpf exposures caused significant hyperactivity (3% 8-10 hpf p<0.05; 1% 24-27 hpf p<0.01; 3% 24-27 hpf p<0.05). There was also a significant main effect of session time (F(4,464)=10.21, p<0.0005), where activity increased from the first to fifth minute of the session.

Figure 1.

Figure 1

Figure 1

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Panel A: Total distance traveled for each minute of a five-minute novel tank exploration trial. Panel B: Distance from the tank floor for each minute of a five-minute novel tank exploration trial. Each bar represents the mean distance for one minute (min 1-5), data are organized by exposure group. Error bars represent SEM.

The measure of location in the tank, i.e. distance from the bottom, was also significantly affected (F(4,116)=4.48, p<0.005) by ethanol treatment (see Fig. 1b). Dunnett's post hoc comparisons showed that the 24-27 hpf 3% ethanol exposure group deviated most (p<0.01) from the control group on tank location. The other treatment groups did not significantly differ from controls on this measure. The main effect of minute was also significant (F(4,464)=102.82, p<0.0005), indicating that fish increasingly explored the tank course of the five-minute session.

Startle Habituation

Developmental ethanol exposure caused overall hyperactivity among fish during the startle habituation test, as revealed by a significant main effect of treatment (F(4,108)=5.33, p<0.001) and Dunnett's post-hoc tests, which indicated that exposure during the 24-27 hpf period resulted in activity levels different from that of the control group (1% p<0.01, 3% p<0.05). There was a significant main effect of sample activity (i.e. 5 s pre- vs. post-tap) (F(1,108)=258.57, p<0.0005), with activity post-tap (1.36±0.01) being greater than pre-tap activity (1.02±0.02). There was also a significant main effect of tap bin (i.e. successive bins of 2 taps) (4,432)=3.91, p<0.005) and a significant sample x tap bin interaction (F(4,432)=16.91, p<0.0005). Follow-up tests of the simple main effects of tap bins separately during the pre- and post-tap activity samples revealed a significant main effect of tap bin during the post-tap sample (F(4,432)=23.22, p<0.0005) only (Fig. 2a), where the startle response decreased from 1.41±0.05 during the first tap bin (tap presentations 1-2) to 1.22±0.02 during the last bin (tap presentations 9-10). There was no effect of tap bin during the pre-tap sample (p>0.05, Fig. 2b). This effect highlights the specificity of habituation learning as it occurred only in the activity sample following (but not preceding) the tap delivery. There was no significant interaction of treatment x pre/post activity.

Figure 2.

Figure 2

Figure 2

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Panel A: Mean distance traveled in the 5 sec following the delivery of the tap stimulus. Panel B: Mean distance traveled in the 5 sec preceding the delivery of the tap stimulus. Each bar represents the mean distance traveled across a bin of two tap deliveries. Error bars represent SEM.

Spatial Discrimination Learning

Developmental ethanol exposure did not significantly affect choice accuracy or response latency on the spatial discrimination task (all p's > 0.05). All fish acquired the spatial discrimination similarly, as indicated by the main effect of trial bin on accuracy (i.e. 5 bins of 2 consecutive trials each) (F(4,304)=14.01, p<0.0005) in which accuracy increased from 34.6±3.7% correct in the trial block 1-2 to 63.6±4.4% correct in the trial block 9-10 (Fig. 3). There was also a significant effect of trial bin (F(4,304)=3.47, p<0.01) on response latency, which varied from 4.0±0.4 s in trial block 1-2 to 6.8±1.0, 7.5±1.2, 5.4±0.8 and then 5.9±1.1 s in the subsequent two-trial blocks.

Figure 3.

Figure 3

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Mean accuracy (percent correct) is reported for each exposure condition during the spatial discrimination procedure, as a function of trial number. Error bars represent SEM.

Craniofacial Malformation and Survival

Our recent studies have assessed survival of embryos immediately following ethanol exposures, and then at 1 dpf and 7 dpf, and have shown that >90% of embryos survived to 7 dpf following either 1% or 3% ethanol exposure (Zhang et al, 2014). Similar survival rates were replicated here (>90%), with no observed difference in survival between control embryos and embryos exposed to either ethanol concentration (p>0.05). Similarly, eye diameter and MHB abnormalities were not detected in control, 1% 8-10 hpf, or 1% 24-27 hpf exposed fish. As MHB formation was assessed at 24 hpf, only embryos exposed to ethanol from 8-10 hpf were assessed for MHB malformations, with 30.9% (21/68) of 3% ethanol-exposed embryos exhibiting malformation. For eye diameter abnormalities, microphthalmia was detected in 20.6% (14/68) of the 3% 8-10 hpf group and in 42.4% (25/58) of the 3% 24-27 hpf group (see Fig. 4 for representative images of MHB and ophthalmic malformations).

Figure 4.

Figure 4

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Panels A-C: 1dpf; D-H: 2dpf. A, D: WT; B, E: 8-10 hpf 3%; G: 24-27hpf 1% and H: 24-27hpf 3%. Arrows indicate the MHB (A and B), which was disrupted in 21/68 of 8-10hpf 3% embryos (C). In 2dpf embryos, 14/68 of 8-10hpf 3% (F) and 25/58 of 24-27hpf 3% (H) showed small-eye phenotype, and the bar indicates 50 uM. P<0.01 for 1dpf MHB 3% 8-10hpf compared to control; 2dpf eye size in 3% 8-10hpf and 3% 24-27hpf compared to control. The ratio of disrupted MHB in control and 8-10hpf is 0/30. The ratio of small-eye in control, 8-10hpf 1% and 24-27hpf 1% is 0/30. We did not observe morphological abnormalities in the adult fish from the 3% 8-10hpf or the 24-27hpf group.

Discussion

The primary goal for the present study was to investigate the persisting neurobehavioral consequences of transient ethanol exposures (1% and 3%) early in neurodevelopment using a zebrafish model to mimic binge-like alcohol consumption by a pregnant woman. Therefore, this study was designed to test the specific hypothesis that brief exposure to ethanol during precisely defined periods of zebrafish embryogenesis would result in behavior change later in life. Here we report that brief embryonic exposure to ethanol produced later-life sensorimotor and exploration changes in adult zebrafish that depended upon the timing and dose of exposure, with the greatest manifestation of behavior change occurring with later exposure (24-27 hpf). Importantly, the test battery employed here was also able to identify insensitive domains of behavior (i.e. spatial learning and habituation learning) highlighting the specificity of ethanol exposure during these early developmental windows.

The novel tank diving task was developed as a test of the basic competing drives in animals to evade predators while still exploring the environment (Levin et al., 2007). In its well-established capacity to measure anxiety-like phenotypes in fish, it is comparable to the frequently-used elevated plus maze for rodents. In the novel tank test, spending more time exploring the upper regions of the tank (where the fish may be more likely to be eaten from below) is seen as analogous to spending more time on the open arms of the elevated plus maze, and can be similarly interpreted as maladaptive, impulsive or anxiety-like behavior (see Campos et al., 2013 for a review on animal models of anxiety). Here, zebrafish exposed to both doses of ethanol during the later developmental period (24-27 hpf) spent significantly more time swimming near the top of the tank than did either the controls or the fish exposed during the earlier period (8-10 hpf). Additionally, both late-exposure dose groups and the highest dose of the early-exposure group swam a greater total distance over the duration of the test. Taken together, the 24-27 hpf groups consistently swam more and did so at higher tank locations than the control group. Presumably, a general hyperactivity may have led to a swim path more evenly distributed throughout the area of the tank and consequently the tank location measure may not entirely reflect increased exploratory and/or decreased predator avoidance drives.

The tap startle test is used as a measure of both sensorimotor function and habituation learning. Here, overall the zebrafish exposed to either ethanol dose during the late-exposure window exhibited increased locomotion during the tap test, which is consistent with the hyperactivity captured during the novel tank assay. All fish, however, habituated to repeated presentations of the startling stimuli, suggesting the ethanol exposures tested in this study do not interfere with habituation learning. Post-startle swim speeds of all fish were significantly elevated from their baseline speed, verifying a startle response to the tap stimulus and indicating that the fish here were not experiencing any profound sensorimotor deficits. Additionally, there were no manifestations of behavioral impairment captured on the spatial discrimination assay. All exposure groups acquired the discrimination similarly and did so with comparable response latencies.

Taken together, the data from these three assays indicate that early life ethanol exposure has specific effects on motoric and exploratory endpoints in the absence of significant changes in learning function (either habituation learning or spatial discrimination) and implicate different exposure windows for divergent patterns of behavioral effects. Moreover, the results reported here suggest that a common behavioral mechanism may underlie the phenotype of ethanol exposure generated by these exposures. The fact that the 24-27 hpf group allocated more swimming behavior to the upper region of the tank (a characteristically “exploratory” response), traveled a greater distance when placed in a novel tank and were more active both at baseline and following the delivery of the tap stimulus can be characterized as “generalized hyperactivity”. While it is possible to interpret hyperactivity a number of ways, it might be reasonable to hypothesize that hyperactivity on the assays reported here might be caused by a disrupted sensitivity to aversive stimuli in fish exposed to ethanol during development. The fish exposed to ethanol from 24-27 hpf were hyperactive compared to controls on the tap startle assay, which was most evident during the baseline measures (5 s preceding each tap) and more active than controls in the novel tank assay. In this way, a decreased sensitivity to aversive stimuli might account for a behavioral phenotype of hyperactivity as a novel environment typically has aversive properties as does the confinement to a small cylindrical arena (the response to both is more likely to be a dive or freeze response in control fish). Moreover, this interpretation is consistent with evidence from human and rodent studies, which indicate anxiolytic effects associated with ethanol exposure.

Moreover, an accompanying neurochemical hypothesis could involve glutamate systems upregulating (or overdeveloping) and GABA systems downregulating (or underdeveloping) during exposure to ethanol (a glutamatergic antagonist and GABAergic agonist) throughout the critical neurodevelopmental stages examined in this study. Should these developmental alterations persist into later life, it is possible that hyperactive glutamate systems and blunted GABA systems would result in an exaggerated motoric response to the stimuli employed here. Therefore, these effects might suggest that GABA and/or glutamate systems are more sensitive during the 24-27 hpf timeframe than the 8-10 hpf developmental window. The 24-27 hpf window more closely corresponds to notochord development, neurogenesis and somatogenesis which strengthens a hypothesis that this developmental window might be quite sensitive to alterations in the behavior measured here. Moreover, such GABA or glutamate mechanisms might reasonably be considered independent from the mechanisms that drive structural malformations and as such can occur in the absence of craniofacial changes. The hypothesis that GABA/glutamate dysfunction during early life might serve as the origin of hyperactivity later in life, which is offered here, might be consistent with the acute or chronic effects of adult ethanol administration that has been observed in zebrafish (Mathur & Guo, 2011; Maximino et al., 2011), rodents (for a review see Silberman et al., 2009) and humans (for a review see Plebani et al., 2012) in which anxiolytic effects, including enhanced exploration behavior, are reported.

Brief and precise time points of exposure were selected here for several reasons. First, this arrangement is better at capturing binge drinking behavior, which is more common in pregnant women than chronic, continuous drinking during pregnancy (especially during the first trimester) (de Chazeron et al., 2008; Ethen et al., 2008; Kesmodel, 2001; Kesmodel and Olsen, 2001; Sayal et al., 2009). Moreover, the chronic (6-24 hpf) exposures seen commonly in the zebrafish literature (e.g. Bilotta et al., 2004; Loucks et al., 2007; Kashyap et al., 2011; McCarthy et al., 2013) fail to model binge drinking and are instead approximately equivalent to drinking for the majority of the first trimester of pregnancy. Additionally, high-dose exposures are not possible using the chronic (6-24 hpf) exposure paradigm, as most of the exposed embryos will not survive past the larval stage of development (Zhang et al., 2011; Zhang et al., 2013) and some mechanistic studies also utilize a binge-like pattern of exposure to pin-point the neurotoxicity of ethanol (e.g. Flentke et al., 2014a; Flentke et al., 2014b).

Another key reason for the dosing regimen utilized here was a proof-of-concept study aimed at characterizing the behavioral phenotypes associated with exposure windows that are known to cause structural changes to the CNS, changes in gene expression and physical malformations (see Godin et al., 2010; Godin et al., 2011; Maier et al., 1997; Parnell et al., 2009; Van Maele–Fabry et al., 1995; Zhang et al., 2011; Zhang et al., 2013; Zhang et al., 2014). While animal studies have shown that the severity of morphological phenotypes of FAS clearly depends upon the precise embryonic stage during which exposure occurs as well as the alcohol dose, behavioral phenotypes have not been well-characterized following exposure at various developmental windows, but could further validate the use of this protocol in modeling FAS. Evidence from zebrafish studies has described gene expression and structural changes in the CNS that are differentially sensitive to ethanol exposure during different developmental windows. Specifically, the gastrulation/neurulation transition period (8-10 hpf) and early nervous system development (24-27 hpf) have been shown to be sensitive to low levels (1.5 and 2%) of transient ethanol (resulting in eye and brain malformations) when agrin or Shh gene expression has been partially reduced using morpholino oligonucleotides (MOs) that disrupt agrin or Shh function (Zhang et al., 2013). More is known about structural changes to the CNS following longer durations of developmental ethanol exposure, including for example microphthalmia and/or cyclopia (exposed 0–72 hpf) (Bilotta et al., 2004; Dlugos and Rabin, 2007; Santos-Ledo et al., 2011) and increased cell death in the hindbrain (Carvan et al., 2004; Loucks and Carvan, 2004).

Finally, both exposure windows selected here for zebrafish fall squarely within the first trimester of human gestation, but correspond to distinct periods of embryogenesis. The 8-10 hpf exposure corresponds to the gastrula phase of embryogenesis (corresponding to the transition from gastrulation to neurulation in zebrafish) which starts around the 16th day of gestation (16-20 dpf) in humans, or roughly during the third week of the first trimester. This exposure is equivalent to GD7-8 in mouse, a common exposure time for the study of FASD. The 24-27 hpf exposure corresponds to the pharyngula phase of embryogenesis (and is a key CNS developmental stage characterized by the formation of the 5-vesicle brain in zebrafish) which corresponds to somatogenesis and notochord development in humans, occurring between weeks 4 and 5 of the first trimester (see Kimmel et al., 1995 for a description of zebrafish embryogenesis). The 24-27 hpf exposure is similar to gestational day (GD) 9-10 in mouse and GD 28-30 in human.

The use of zebrafish in alcohol-related research has dramatically increased in recent decades and has resulted in mounting evidence that comparable FAS phenotypes can be generated in zebrafish as those in rodents and humans. Accordingly, there is a growing literature on the behavioral effects associated with alcohol exposure in zebrafish, which spans acute adult exposures and subchronic developmental exposures. Both the reinforcing efficacy of ethanol and the negative effects on cognitive endpoints have been evaluated. Adult zebrafish have been shown to prefer ethanol, as measured by a conditioned place preference arrangement (Mathur et al., 2011). In fact, many domains of behavioral function are clearly altered in ethanol-exposed zebrafish, much of which clearly corresponds to the well-characterized effects seen in rodents and humans. For instance, embryonic or larval exposure is associated with reduced shoaling behavior (Fernandes and Gerlai, 2009), altered sensory startle response (Carvan et al., 2004), decreased learning ability (Carvan et al., 2004), motoric changes and differences in exploration behavior (Lockwood et al., 2004). Following adult exposure similar changes in shoaling and motoric function have been documented (Echevarria et al., 2011; Gerlai et al., 2006; 2008; Wong et al., 2010). Moreover, a number of reports have noted reduced anxiety or fear responses in adults exposed to ethanol (Dlugos and Rabin, 2003; Mathur and Guo, 2011) which in some ways mimics the effects reported here following brief embryonic exposure. Importantly, as mentioned above, many of these phenotypes that are apparent in zebrafish are consistent with the behavioral profiles of humans and rodents exposed to early life ethanol and are likely interpretable within the context of changes in sensitivity to aversive stimuli (as is offered above).

It is difficult to make comparisons between the ethanol doses used here and a typical binge alcohol exposure in humans, because higher concentrations of ethanol are required in zebrafish to elicit the hallmark facial features of FAS. For example, 1% ethanol exposure in this study results in tissue ethanol levels that are approximately 50 mM (Zhang et al, 2014). This tissue ethanol level in humans would likely result in FAS, but produces no overt morphological abnormalities in zebrafish. The 3% binge exposure produces tissue ethanol levels of approximately 280-300 mM, which results in FAS in zebrafish. If the embryos used here had been dechorionated, the tissue ethanol levels would be reduced about 2.5-fold based on our previous studies (Zhang et al., 2013; Zhang et al., 2014), suggesting tissue ethanol levels of approximately 120 mM after a 3% binge exposure. This is substantially higher than binge alcohol levels in humans, but necessary to elicit FAS in zebrafish. Therefore, the doses selected here were chosen based on the known concentrations of ethanol that elicit FAS, while also taking into consideration levels that are below that which leads to FAS in zebrafish so that we could assess behavioral malformation in the absence of physical malformation. And finally, a note regarding the design of the present study. As mentioned in the methods section, the fish tested were juveniles. This age was selected as fish are large enough to be visualized by our tracking software and have been living in clean water for over one month. We would predict similar effects in older zebrafish, but this remains to be tested.

In conclusion, the data reported here provide support for the gastrulation/neurulation transition period (8–10 hpf) and early nervous system development (24–27 hpf), as being sensitive to transient ethanol exposure on functional endpoints later in life, with exposure during the later stage associated with the greatest dysfunction. These data add to the growing literature that utilizes zebrafish as a model for FAS, which has begun to show comparable phenotypes (i.e. physical and structural malformations) to those already described in the rodent and human literatures. However, there remain significant gaps in our understanding of how developmental exposure to ethanol modifies the life-long neurobehavioral developmental trajectory of vertebrates. The present study has tapped only a small subset of behavioral domains and cognitive capacities. Fortunately, numerous studies have developed assays for a number of other behavioral domains in zebrafish, including classical and operant arrangements, social tasks, fear/anxiety preparations, and others (Bailey et al., 2013; Tierney, 2011), and examining how ethanol exposure affects these behaviors would greatly deepen our understanding of the consequences of FAS. Similarly, the growth of genetic and molecular tools available for the zebrafish model will allow a much more detailed map of the pathway linking ethanol exposure to behavioral toxicity.

Supplementary Material

Highlights.

Zebrafish were exposed to 1 or 3% ethanol 8-10 or 24-27 hpf to evaluate long-term behavioral effects.

Ethanol exposure during the 24-27 hpf window caused locomotor hyperactivity during adolescence.

Behavioral effects were detected at doses that did not cause physical malformation.

Behavioral phenotypes of embryonic ethanol exposure are determined by window of exposure.

Acknowledgements

Research support from Duke University Superfund Research Center ES10356, NIAAA U54 AA019765 and the Duke-RJR Leon Golberg Toxicology Fellowship.

Footnotes

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