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Effects of embryonic exposure to ethanol on zebrafish visual function
Neurotoxicology and Teratology 24 (2002) 759 – 766 www.elsevier.com/locate/neutera
Effects of embryonic exposure to ethanol on zebrafish visual function Joseph Bilottaa,*, Shannon Saszika,b, Carla M. Givina, Heather R. Hardestya, Sarah E. Sutherlanda,c a
Department of Psychology and Biotechnology Center, Western Kentucky University, 1 Big Red Way, Bowling Green, KY 42101, USA b College of Optometry, University of Houston, Houston, TX 77204, USA c Southern College of Optometry, Memphis, TN 38104, USA Received 11 February 2002; received in revised form 1 July 2002; accepted 28 August 2002
Abstract Across a variety of species, including humans, it has been shown that embryos exposed to ethanol display eye abnormalities as well as deficiencies in visual physiology and behavior. The purpose of this study was to examine the effects of embryonic exposure to ethanol on visual function in zebrafish. Visual function was assessed physiologically, via electroretinogram (ERG) recordings, and behaviorally, by measuring visual acuity with the optomotor response. Zebrafish larvae were exposed to 1.5% ethanol at various times during development, including the period of maximal eye development. The results show that ethanol effects on visual function were most pronounced when exposure occurred during eye development. ERG recordings from ethanol-exposed larvae differed from normal subjects both in shape of the response waveform and in visual thresholds under both light and dark adaptation; the differences were more pronounced under lower levels of adaptation. Also, ethanol-exposed larvae displayed lower visual acuity as determined from the optomotor response. These results indicate embryonic ethanol exposure affects visual function particularly when exposure occurs during eye development. In addition, these findings illustrate the usefulness of the zebrafish as a viable animal model for studying Fetal Alcohol Syndrome (FAS). D 2002 Elsevier Science Inc. All rights reserved. Keywords: Ethanol; Fetal Alcohol Syndrome; Zebrafish; Visual acuity; Electroretinogram
1. Introduction A number of studies across a variety of species have shown that visual problems are associated with prenatal exposure to ethanol. Aside from microphthalmia in humans [11,33,35,37] and mice [24], other physical eye problems in both humans and other species include reduced optic nerve size, optic nerve hypoplasia [19,37], retinal vein abnormalities [19,20,27,37], and an increase in retinal ganglion cell death [13,27]. Deficits in optical and retinal structures lead to problems in visual ability or function, such as strabismus [34] and visual acuity [34 – 36]. Although a number of studies have demonstrated that prenatal exposure to alcohol appears to produce deficits in visual acuity in children, few studies have examined the effects of prenatal exposure to alcohol on visual physiology. Katz and Fox [23] examined the effects of prenatal exposure to ethanol on rat electro-
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Corresponding author. Tel.: +1-270-745-6314; fax: +1-270-745-6934. E-mail address: [email protected] (J. Bilotta).
retinograms (ERGs). The ERG is a gross electrical potential generated by retinal neurons [14]. Katz and Fox [23] found that hooded rats, exposed to ethanol prenatally, displayed deficits under light and dark adaptation as well as a decreased amount of rhodopsin in the retina. Similar deficiencies in the ERG responses have been reported in children diagnosed with Fetal Alcohol Syndrome (FAS) [21]. Thus, prenatal exposure appears to affect visual function as well as eye structures. Recently, the zebrafish (Danio rerio) has become an important animal model in developmental neuroscience, including visual neuroscience [4]. Zebrafish have transparent eggs and the embryos are semitransparent. Thus, the stage of embryonic development for an individual embryo can be determined without interfering with development. In addition, some chemicals, such as ethanol, pass through the chorion [7] making it relatively easy to study the effects of ethanol on the development of this species. Several studies have shown that ethanol exposure can affect zebrafish eye development. For example, zebrafish embryos, exposed to 2.5% and 3% solutions of ethanol,
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developed cyclopia (fusion of the two eyes [7,30]). In addition, preliminary work in our laboratory has shown that embryonic exposure to ethanol can produce microphthalmia in zebrafish [3]. Thus, it is clear that zebrafish eye development is affected by exposure to ethanol and that these effects are similar to those found in other species, including humans. The purpose of this study was to investigate the effects of embryonic exposure to ethanol on both visual physiology and visual function or acuity in zebrafish. Visual physiology was examined by obtaining ERG responses across various dark and light backgrounds. Visual acuity was determined behaviorally by using the optomotor response. Zebrafish larvae were exposed to ethanol at different times in development, including the period of eye development, in order to study the impact of ethanol at various times in development on visual physiology and behavior.
2. Methods
vertical black and white stripes consisting of 0.125 cycles per degree of visual angle, was placed on the outer wheel and rotated around the subject. If the fish ‘detects’ the moving stripes, then it will swim with the stimulus; if the animal cannot detect the moving stripes, then the animal will not swim with the movement of the stripes. This spatial frequency stimulus was chosen because previous work has shown that this stimulus is near the visual acuity limit of normal 6 –9 days postfertilization (dpf) zebrafish larvae [2]. In other words, normal zebrafish reliably respond to this spatial frequency stimulus, but have difficulty detecting higher spatial frequencies. Overhead room lights (Sylvania, F40/D; about 100 mW/cm2) illuminated the chamber. Prior to testing, the fish was placed into the fish chamber and a 5-min baseline swimming measure (number of laps around the chamber) was obtained. Next, the visual stimulus was rotated in one direction and the number of laps the fish swam with and against the direction of the stimulus were recorded for 5 min. Fish were tested between 6 and 9 dpf.
2.1. Subjects and rearing procedures 2.3. Physiological apparatus and procedures Zebrafish embryos (Danio rerio) bred inhouse [5] served as subjects. They were maintained at a water temperature of 28.5 C and a 14-h light on/10-h light off schedule. Fertilized embryos were placed into Petri dishes containing 20 ml of either distilled water or a 1.5% ethanol solution; the dishes were floated in 500 ml plastic containers that were floated in 10 gal aquarium tanks. Several different exposure durations were used: 6 –24, 12– 24, 24 –36, 48 – 60, and 60 – 72 h postfertilization (hpf). The 6 – 24 hpf exposure was used because it was shown to have significant effects on eye diameter and the presence of FAS characteristics in zebrafish [3]. The 12 –24 hpf exposure was used because this is the time period when the zebrafish eye develops; by 24 hpf, the eyecups are well-formed [31]. The 24– 36 hpf group represents a 12-h period just after eyecup development. The 48 –60 hpf period was chosen because it is around this time that the lens has formed and the optic nerve has reached the brain [9]. The last duration, 60 –72 hpf, was chosen because it is just prior to normal hatching; at this point, the optic nerve arbors have covered the optic tectum [10], and it marks the beginning of eye movements, including optokinetic nystagmus (OKN) [16]. Physical measurements such as eye diameter, as well as developmental staging, were obtained using an inverted microscope with phase contrast optics (World Precision Instruments, PIM). 2.2. Behavioral apparatus and procedures Zebrafish visual acuity was determined by measuring the optomotor response (for details, see Ref. [2]). The apparatus consisted of a small circular chamber (35-mm diameter) in which the fish is placed. A visual stimulus,
Details of the physiological apparatus and procedures are described elsewhere [22,29]. Briefly, visual stimuli were generated by using a two-channel optical system. One channel had a 150-W xenon arc lamp (Spectral Energy, Westwood, NJ, Model LH 150) light source that presented a broadband (white) test stimulus; the color temperature of this source was 9400 K. A 250-W tungsten – halogen bulb (Oriel, Stratford, CT, Model 6334) provided the broadband background irradiance and had a color temperature of 4500 K. The light sources were combined optically and focused onto one end of a liquid light guide (Oriel, Model 77556); the other end was placed in front of the subject’s eye. Neutral density filters placed in the light pathways controlled the irradiance of the two channels. Prior to the experiment, subjects were dark-adapted for at least one hour (zebrafish dark adaptation is complete by about 18 min [26]). Under dim red light, 6– 9 dpf subjects were anesthetized with a 0.01% dose of tricaine methanesulfonate [29] and placed on a piece of cotton, moistened with an anesthetic solution, located in a light – tight Faraday cage. Recording and reference electrodes were glass pipettes (tip diameter of 10 mm), filled with teleost saline solution that housed 36-gauge chlorided silver wires. The recording electrode was placed above the cornea on the subject’s eye; the reference electrode was placed on the body. Electrical signals were differentially amplified (bandpass of 0.1– 100 Hz) and recorded at a rate of 4 ms 1 by a computer. Once the subject was placed in the Faraday cage with the electrodes in position, the subject was dark-adapted for an additional 15 min. Following this time period, testing began. Each trial consisted of one 200 ms stimulus
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presentation; responses were recorded for an additional 2000 ms following stimulus termination. A baseline period of 50 ms was recorded prior to the stimulus presentation. Stimulus irradiance trials started below threshold and increased in 0.5 log unit steps until the response criterion was reached. To minimize possible light-adaptation effects, stimulus irradiances that produced responses beyond the linear portion of the response vs. log irradiance function were avoided [6]. Once an irradiance series was completed at a particular background, the background irradiance was increased. The subject adapted to the new background for at least 5 min and the procedures were repeated. Six background irradiances ranging from dark adaptation to 500 mW/cm2 were used. Increment threshold functions were derived by finding the stimulus irradiance that produced a criterion response for each background irradiance. Log threshold was defined as the log stimulus irradiance (mW/cm2) that produced the criterion response. Increment threshold functions were derived from the first voltage-positive ERG response (b-wave) measured from baseline or the first negative peak to the first positive peak [17]. Similar procedures have been used previously with zebrafish larvae [6,28,29]. 2.4. Statistical analyses A one-factor between-subjects analysis of variance (ANOVA) was used to determine whether there were significant differences in eye diameter across the six treatment groups (control, and those subjects exposed to 1.5% ethanol 6 – 24, 12 – 24, 24 – 36, 48 – 60, or 60 – 72 hpf). Similarly, a one-factor between-subjects ANOVA was calculated on the baseline optomotor response scores across the six treatment groups to determine whether there were differences in activity rates across the groups. Scheffe’s post hoc comparisons were made to determine where the specific significant differences occurred. A one-factor between-subjects analysis of covariance with the optomotor response to the moving visual stimulus as the dependent measure and the baseline response score as a covariate was calculated to determine whether there were differences in the optomotor response across the six treatment groups. A 2 (treatment: control vs. subjects exposed to 1.5% ethanol 6– 24 hpf ) 6 (background irradiance) mixed design ANOVA was calculated to determine whether there were any differences in ERG b-wave threshold between the two groups across the various background irradiances. Simple effects analyses were performed to determine where the significant differences occurred in the Treatment Background interaction. Finally, a 3 (control, subjects exposed to 1.5% ethanol 0 –12 or 12– 24 hpf ) 6 (background irradiance) mixed design ANOVA was used to determine whether there were differences in the ERG b-wave increment threshold functions across the different ethanol exposure conditions and background irradiances.
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3. Results 3.1. Visual acuity Visual acuity was assessed for larvae exposed to various ethanol conditions as well as control subjects. All subjects in the ethanol groups were exposed to 1.5% ethanol at various times during development. The majority of groups were exposed for 12 h; only one group was exposed longer (6– 24 hpf). The longer exposure condition was included since preliminary work demonstrated that it clearly affected zebrafish physical development [3]. Fig. 1 compares the eye diameter of the various groups measured at 3 dpf. A onefactor between-subjects ANOVA found significant effects on eye diameter across the groups [ F(5,122) = 29.27, P < .001]. Scheffe’s post hoc comparisons were made to determine where the significant differences occurred. The comparisons found that the subjects exposed to ethanol 6– 24 and 12 –24 hpf had significantly smaller eye diameters than all of the other groups, including the control group. Also, it was found that subjects exposed to ethanol 24 – 36 hpf had significantly smaller eye diameters than the 60 – 72 hpf group (all at P < .05). Thus, zebrafish eye diameter is affected by embryonic exposure to ethanol. Prior to examining the effects of ethanol on visual behavior using the optomotor response, it was important to ensure that there were no differences in swimming behavior across the groups. Since preliminary work has shown that exposure to ethanol could produce severe physical problems, such as an abnormally large body [3], only subjects that appeared to possess normal swimming
Fig. 1. Mean eye diameter of control subjects (n = 44) and subjects exposed to 1.5% ethanol at 6 – 24 (n = 10), 12 – 24 (n = 39), 24 – 36 (n = 10), 48 – 60 (n = 10), and 60 – 72 (n = 15) hpf. Subjects were examined at 3 dpf. Error bars represent ± 1 S.E.M. Groups designated with a single asterisk (*) indicate that these groups are significantly different from the other groups; groups designated with double asterisks (**) indicate that the two groups are significantly different from one another. All significance levels are P < .05.
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groups [ F(5,99) = 2.52, P < .05]. Scheffe’s post hoc comparisons revealed that the subjects exposed to ethanol 6– 24 and 12– 24 hpf had significantly lower responses than the control subjects. Also, these two groups had significantly lower responses than the subjects exposed to ethanol 48– 60 and 60– 72 hpf. Finally, subjects exposed to ethanol 24 – 36 hpf had significantly lower responses than those subjects exposed at 48 – 60 hpf. Fig. 2 shows the mean response scores for the various groups. The dotted line represents the average baseline response across the groups. Note that only the 6– 24 and 12 –24 hpf groups’ responses fall below the baseline response. Thus, ethanol exposure does have an effect on visual acuity; the effects are most pronounced when subjects are exposed to ethanol 6 –24 and 12– 24 hpf. 3.2. Retinal physiology Fig. 2. Mean optomotor response score of control subjects (n = 23) and subjects exposed to 1.5% ethanol at 6 – 24 (n = 17), 12 – 24 (n = 7), 24 – 36 (n = 16), 48 – 60 (n = 23), and 60 – 72 (n = 18) hpf. Subjects were examined between 6 and 9 dpf. Mean values are estimated marginal means from the covariance analysis. The dotted line shows the average baseline response score. Error bars represent ± 1 S.E.M. Groups designated with a single asterisk (*) indicate that these groups are significantly different from groups with a double asterisks (**); groups designated with the letter ‘a’ are significantly different from one another. All significance levels are P < .05.
ability were tested in the optomotor apparatus. In addition, since hyperactivity has been shown to be an effect of prenatal exposure to ethanol in rats [8] and humans [32], there was some concern that there might be differences in motor activity across the groups. Therefore, in order to be sure that the subjects in all groups were similar in swimming ability, the baseline responses (i.e., number of laps swum while the visual stimulus was stationary) across the groups were compared. A one-factor between-subjects ANOVA was calculated on the response scores across the six different groups (control, and those subjects exposed to 1.5% ethanol 6– 24, 12 –24, 24– 36, 48 –60, or 60– 72 hpf). The results found no significant differences in baseline swimming across the groups [ F(5,100) = 1.42, P >.05]. However, it is interesting to note that the mean response score for subjects exposed to 6 –24 hpf was slightly higher (m = 3.00, s = 2.12) than the mean response score for controls (m = 2.12, s = 1.58). Thus, if there is a difference between controls and ethanol-exposed groups in motor activity, then the optomotor response is not sensitive enough to detect it. This may be because the optomotor response only measures activity around the chamber and not lateral movement within the chamber. To further ensure that swimming or activity rates across the groups did not influence the responses to the rotating 0.125 cycles per degree stimulus, baseline swimming was entered as a covariate in a one-factor between-subjects analysis of covariance with the response to the moving visual stimulus as the dependent measure. The results showed that there were significant differences across the
Fig. 3 shows representative ERG responses from individual 6 dpf zebrafish larvae. The background illumination was 50 mW/cm2. Stimulus duration was 200 ms and is
Fig. 3. Individual sample ERG waveforms from normal and ethanolexposed subjects. The background illumination consisted of a 50-mW/cm2 white light. The stimulus duration was 200 ms and is indicated by the raised horizontal line on the abscissa. The negative values associated with each ERG waveform represent the stimulus irradiance in log attenuation values where 0.0 corresponds to a stimulus irradiance of 30 mW/cm2. The peak of the b-wave is labeled in each panel. (A) ERG responses of a 6 dpf normal larvae. (B) ERG responses of a 6 dpf larvae exposed to 1.5% ethanol at 6 – 24 hpf. See text for details.
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Fig. 4. Individual sample b-wave response amplitude vs. log irradiance response functions obtained under a dark background (filled symbols) and a background irradiance of 0.5 mW/cm2 (open symbols). Circles represent the responses of an ethanol-exposed subject and squares represent the responses of a control subject. The negative values associated with each ERG waveform represent the stimulus irradiance in log attenuation values where 0.0 corresponds to a stimulus irradiance of 30 mW/cm2.
represented by the raised bar on the abscissa. The negative values associated with each ERG waveform represent the stimulus irradiance in log attenuation values. Fig. 3A shows ERG responses of a control subject. The responses are similar to those of normal zebrafish larvae at 6 dpf [29]. There is a voltage-positive response (b-wave) whose amplitude is dependent upon stimulus irradiance. As in other studies with zebrafish larvae, some subjects show a small voltage-negative (a-wave) response prior to the b-wave, while others do not [28,29]. Thus, in the current study, only the responses of the ERG b-wave were analyzed. Fig. 3B shows representative ERG responses to different stimulus irradiances of a larvae zebrafish exposed to 1.5% ethanol 6 –24 hpf. The ERG response from this subject differs from the response of the control subject both in shape and in responsiveness to light. The b-wave shape appears to be different from that of controls in that it is more sustained than the response of control subjects; that is, it takes longer to return to a baseline response than the b-wave of the control subjects. Also, the b-wave response is much less responsive to light in ethanol-exposed subjects than in controls. Compare the ERG b-wave amplitudes of the control and ethanol-exposed subjects to the same stimulus irradiance ( 2.0). Although it clearly was the case that the ERG response of the ethanol-exposed subjects was reduced at background irradiance of 50 mW/cm2, it was not the case under all background conditions. Fig. 4 shows representative b-wave response amplitude vs. stimulus irradiance functions for a control subject (squares) and an ethanol-exposed subject (circles) obtained under a dark background (filled symbols) and a background of 0.5 mW/cm2 (open symbols). The most
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striking finding is that under the higher background irradiance, the response-irradiance functions of the ethanolexposed subject were very similar to those of the control subject. On the other hand, there was a large difference between the response-irradiance functions between the two subjects under the dark background. Thus, embryonic exposure to ethanol appears to be producing larger deficits under lower levels of light adaptation. To further investigate this finding, Fig. 5 shows the increment threshold functions for control subjects and those subjects exposed to 1.5% ethanol 6 –24 hpf. The increment threshold functions were obtained from subjects between 6 and 9 dpf. Log threshold was defined as the stimulus irradiance required to produce a 20-mV b-wave response. The background irradiance labeled ‘‘D’’ represents a state of complete dark adaptation. Error bars represent ± one standard error of the mean (S.E.M.). The increment threshold function obtained from the control subjects is similar to that obtained from a previous study in zebrafish larvae [28]. Under dim levels of illumination, threshold is relatively low. As the background illumination is increased, the threshold value increases. However, the increment threshold function obtained from the ethanol-exposed subjects was very different from controls. Threshold values did not improve with lower background levels. In fact, threshold values were very high across all background levels. A 2 (treatment: control vs. ethanolexposure) 6 (background irradiance) mixed design ANOVA revealed a significant Treatment Background irradiance interaction [ F(5,30) = 6.67, P < .001]. Simple effects analyses were performed to determine where the significant differences occurred. These analyses found that the ethanol-exposed subjects had significantly higher thresh-
Fig. 5. Averaged increment threshold functions based on the ERG b-wave response at 6 – 9 dpf control (circles; n = 3) and larvae exposed to 1.5% ethanol at 6 – 24 hpf (squares; n = 5). Log threshold is defined as the reciprocal of the log stimulus irradiance (mW/cm2) that produced a 20-mV bwave response. Error bars represent ± 1 S.E.M.
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olds than controls at all background levels except at the log background irradiance of 3.7 (all values tested at P < .05). Thus, 1.5% ethanol exposure at 6 – 24 dpf has dramatic effects on the ERG increment threshold function, particularly at the lower background levels. To determine whether shorter ethanol exposure durations would have any impact on long-term development of the ERG, two additional ethanol-exposed groups were compared to normal subjects. One group of larvae was exposed to 1.5% ethanol at 0 – 12 hpf and the other group was exposed to 1.5% ethanol at 12 –24 hpf. Increment threshold functions were obtained from all subjects, including controls, at 22 dpf. In general, the two ethanol-exposed groups had increment threshold functions similar to that of the control subjects, although the function obtained from control subjects had slightly lower thresholds across all backgrounds. A 3 (early exposure, late exposure and control) 6 (background irradiance) mixed design ANOVA found only a significant main effect of background irradiance [ F(5,30) = 18.27, P < .005]. Since the Type of exposure Background irradiance interaction was not significant, it appears that the three increment threshold functions were similar.
4. Discussion The results of the present study showed that ethanol exposure during development can influence zebrafish visual behavior as well as visual physiology. This study also found that the timing of the ethanol exposure could impact the severity with which ethanol affects visual function. The present study showed that ethanol exposure produced smaller eyes as well as a reduction in visual acuity measured behaviorally. These findings support findings from studies on a variety of species that prenatal ethanol (or alcohol) exposure can impact the visual system. For example, children who have been exposed to alcohol prenatally display a number of visual problems such as microphthalmia [33], retinal vein problems [33,34], reduced optic disk [19,36], and poor visual acuity [25,34,35,37]. Similar findings in animal studies have been reported, including microphthalmia and reduced optic nerve size in rodents [24,27], as well as increased ganglion cell death in rats [27] and chicks [13]. In addition, the present study showed that ethanol exposure can alter retinal physiology. Larvae that were exposed to ethanol had ERG waveforms that were abnormal in shape and increment threshold functions that differed from normals. In general, visual thresholds in larvae exposed to ethanol were significantly higher than those of controls. However, even though these effects occurred under both light- and dark-adapted conditions, the deficits were more pronounced under lower backgrounds of illumination. These results support previous work that showed that rat retinal physiology was altered by prenatal exposure to
ethanol. Katz and Fox [23] found differences in ERG increment threshold functions between normal and ethanol-exposed rats; the differences were more pronounced under lower levels of illumination. Therefore, the ERG results found in zebrafish appear to be similar to those found in other species, including humans [21]. It also should be mentioned that the ERG increment threshold results are consistent with the deficits found in the optomotor response obtained under similar background illumination (about 100 mW/cm2). These findings suggest that the site of ethanol’s action on retinal physiology and visual behavior may be the same, and that this action takes place early in the visual system. One of the most interesting findings of the present results is that the effects of ethanol on vision appear to be most severe during the time in development when the eye forms. The 12– 24 hpf ethanol exposure group was chosen because this is the time when the eye begins to form [31]. Thus, one would expect more visual problems when the embryo is exposed to ethanol during this time. This was found to be the case. Larvae exposed to ethanol 12 –24 hpf had significantly smaller eye diameters and responded significantly less to high spatial frequency visual stimuli than any other 12-h exposure group. They also had slightly lower visual thresholds measured by the ERG than controls. In fact, these subjects displayed more similar visual deficits to those exposed to ethanol for approximately 24 h (6– 24 hpf) than any other group. The 6– 24 hpf ethanol-exposed subjects displayed significantly higher visual thresholds, smaller eye diameters, and poorer visual acuity than controls and subjects exposed to ethanol for any 12-h period following the first 24 h. It is not surprising that the 6– 24 hpf ethanolexposed group would display visual deficits similar to those found in the 12– 24 hpf ethanol-exposed group, since the last portion of exposure in the first group was the same as the second group. However, what is interesting is that the 6 –24 hpf ethanol-exposed group also displayed other physical deficits, such as physical deformities resembling FAS [3]. The 12 –24 hpf ethanol-exposed group did not display these physical characteristics; the only deficits for these subjects appeared to be in visual processing. Finally, the only other group that displayed any visual problems was the group exposed to ethanol at 24 –36 hpf. They displayed a slight deficit in visual acuity as measured by the optomotor response. This is not surprising given that this ethanol exposure takes place while the optic nerve is working its way to the brain (in the case of the zebrafish, the optic tectum); by about 2 dpf, the optic nerve has reached the optic tectum [9]. Since the optomotor response most likely represents brain functioning, one would expect some brain deficits in visual processing. One possible reason for the deficiencies found in visual behavior and physiology in ethanol-exposed subjects may be that ethanol-exposed subjects had smaller eyes. Smaller eyes will affect the optics of the eye and possibly retinal physiology. For example, it has been shown that the
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amplitudes of the ERG b-wave component are generally smaller with larger eyes (i.e., longer axial length), such as in the case with humans with myopia [12]. However, this cannot be the explanation for the differences found in this study with retinal physiology, since the present study finds the opposite results; larvae exposed to ethanol had both smaller eye diameters and higher ERG b-wave increment thresholds. In addition, previous work has shown that the ERG waveform and the response characteristics are not dramatically different at early ages of larvae development [29], where eye diameter does increase with age. Regarding the effects of eye size on visual behavior, further work is required to address this issue since zebrafish visual behavior depends on the coordinated development of optical and retinal structures, and the extraocular muscles (see Ref. [16]). It should be mentioned that even though the results from the present study with zebrafish show effects of ethanol that are similar to other animal species, there is a large discrepancy between the doses of ethanol across species. In order to find the effects found in rodent species, the doses necessary to produce these effects on zebrafish embryos are much higher. The most likely explanation for the discrepancy is that the chorion of the zebrafish embryo does not allow ethanol to pass through freely. Research examining the permeability of the zebrafish chorion supports this notion. Harvey et al. [18] found that when zebrafish eggs were placed in a glycerol solution, only about 8% of the glycerol solution actually entered the embryonic tissue. Similar effects were found when eggs were placed into a solution of dimethyl sulfoxide (DMSO). About 3% of DMSO reached the embryonic tissue with an intact chorion. However, when the chorion was removed, the percentage of DMSO that reached the embryo increased dramatically. At present, there are no studies that have examined the permeability of the zebrafish chorion to ethanol. However, if one assumes that roughly 8% of the ethanol reaches the embryonic tissue, then a 1.5% ethanol (1500 mg/dl) solution has an ‘‘effective’’ solution of 120 mg/dl. This ethanol dose falls within the range of doses used to produce effects in rodent pups [15] and corresponds to blood alcohol levels found in women who have children with FAS (see Ref. [1]). In summary, the zebrafish appears to be a useful model for studying embryonic exposure to ethanol not only because ethanol appears to affect zebrafish in a manner similar to its effects on other species, but also because of the advantages the zebrafish brings as a developmental model in general [4]. Because so much is known about its embryonic development, precise manipulations can be performed targeting particular structures. In the present study, we have shown that exposing zebrafish larvae to ethanol during the period of eye development produces significant visual deficits, including physical (microphthalmia), physiological (ERG), and behavioral (optomotor response) problems. Similar studies, targeting other aspects of zebrafish embryonic development,
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could provide valuable insight on the exact nature of the effects of ethanol.
Acknowledgements This work was supported by a Western Kentucky Research Grant and a Summer Fellowship Grant. The authors would like to thank Timothy Lawrence and Elizabeth Lemerise for their work on this project, and Angela McDowell and Codye Hill for their helpful comments on this work.
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J. Bilotta et al. / Neurotoxicology and Teratology 24 (2002) 759–766 during prenatal and perinatal life—digital image analysis, Surv. Ophthalmol. 44 (Suppl. 1) (1999) S63 – S73. A. Hellstrom, Y. Chen, K. Stromland, Fundus morphology assessed by digital image analysis in children with fetal alcohol syndrome, J. Pediatr. Ophthalmol. Strabismus 34 (1997) 17 – 23. T.E. Hug, K.M. Fitzgerald, G.W. Cibis, Clinical and electroretinographic findings in fetal alcohol syndrome, J. Am. Assoc. Pediatr. Ophthalmol. Strabismus 4 (2000) 200 – 204. A. Hughes, S. Saszik, J. Bilotta, P.J. DeMarco Jr., W.F. Patterson II, Cone contributions to the photopic spectral sensitivity of the zebrafish ERG, Vis. Neurosci. 15 (1998) 1029 – 1037. L.M. Katz, D.A. Fox, Prenatal ethanol exposure alters scotopic and photopic components of adult rat electroretinograms, Invest. Ophthalmol. Vis. Sci. 32 (1991) 2861 – 2872. L.A. Kennedy, M.J. Elliott, Ocular changes in the mouse embryo following acute maternal ethanol intoxication, Int. J. Dev. Neurosci. 4 (1986) 311 – 317. D.D. Lewis, S.E. Woods, Fetal alcohol syndrome, Am. Fam. Phys. 50 (1994) 1025 – 1032. L. Li, J.E. Dowling, Zebrafish visual sensitivity is regulated by a circadian clock, Vis. Neurosci. 15 (1998) 851 – 857. M.D. Pinazo-Duran, J. Renau-Piqueras, C. Guerri, K. Stromland, Optic nerve hypoplasia in fetal alcohol syndrome: an update, Eur. J. Ophthalmol. 7 (1997) 262 – 270.
[28] S. Saszik, J. Bilotta, Constant dark-rearing effects on visual adaptation of the zebrafish ERG, Int. J. Dev. Neurosci. 19 (2001) 611 – 619. [29] S. Saszik, J. Bilotta, C.M. Givin, ERG assessment of zebrafish retinal development, Vis. Neurosci. 16 (1999) 881 – 888. [30] E. Schmatolla, Retino-tectal course of optic nerves in cyclopic and synophthalmic zebrafish embryos, Anat. Rec. 180 (1975) 377 – 384. [31] E.A. Schmitt, J.E. Dowling, Early eye morphogenesis in the zebrafish, Brachydanio rerio, J. Comp. Neurol. 344 (1994) 532 – 542. [32] A.P. Streissguth, S. Landesman-Dwyer, J.C. Martin, D.W. Smith, Teratogenic effects of alcohol in humans and laboratory animals, Science 209 (1980) 353 – 361. [33] K. Stromland, Eyeground malformations in the fetal alcohol syndrome, Neuropediatrics 12 (1981) 97 – 98. [34] K. Stromland, Ocular abnormalities in the fetal alcohol syndrome, Acta Ophthalmol. Suppl. 171 (1985) 1 – 50. [35] K. Stromland, Ocular involvement in the fetal alcohol syndrome, Surv. Ophthalmol. 31 (1987) 277 – 284. [36] K. Stromland, A. Hellstrom, Fetal alcohol syndrome—an ophthalmological and socioeducational prospective study, Pediatrics 97 (1996) 845 – 850. [37] K. Stromland, K. Sundelin, Paediatric and ophthalmologic observations in offspring of alcohol abusing mothers, Acta Paediatr. Scand. 85 (1996) 1463 – 1468.
Effects of embryonic exposure to ethanol on zebrafish visual function Joseph Bilottaa,*, Shannon Saszika,b, Carla M. Givina, Heather R. Hardestya, Sarah E. Sutherlanda,c a
Department of Psychology and Biotechnology Center, Western Kentucky University, 1 Big Red Way, Bowling Green, KY 42101, USA b College of Optometry, University of Houston, Houston, TX 77204, USA c Southern College of Optometry, Memphis, TN 38104, USA Received 11 February 2002; received in revised form 1 July 2002; accepted 28 August 2002
Abstract Across a variety of species, including humans, it has been shown that embryos exposed to ethanol display eye abnormalities as well as deficiencies in visual physiology and behavior. The purpose of this study was to examine the effects of embryonic exposure to ethanol on visual function in zebrafish. Visual function was assessed physiologically, via electroretinogram (ERG) recordings, and behaviorally, by measuring visual acuity with the optomotor response. Zebrafish larvae were exposed to 1.5% ethanol at various times during development, including the period of maximal eye development. The results show that ethanol effects on visual function were most pronounced when exposure occurred during eye development. ERG recordings from ethanol-exposed larvae differed from normal subjects both in shape of the response waveform and in visual thresholds under both light and dark adaptation; the differences were more pronounced under lower levels of adaptation. Also, ethanol-exposed larvae displayed lower visual acuity as determined from the optomotor response. These results indicate embryonic ethanol exposure affects visual function particularly when exposure occurs during eye development. In addition, these findings illustrate the usefulness of the zebrafish as a viable animal model for studying Fetal Alcohol Syndrome (FAS). D 2002 Elsevier Science Inc. All rights reserved. Keywords: Ethanol; Fetal Alcohol Syndrome; Zebrafish; Visual acuity; Electroretinogram
1. Introduction A number of studies across a variety of species have shown that visual problems are associated with prenatal exposure to ethanol. Aside from microphthalmia in humans [11,33,35,37] and mice [24], other physical eye problems in both humans and other species include reduced optic nerve size, optic nerve hypoplasia [19,37], retinal vein abnormalities [19,20,27,37], and an increase in retinal ganglion cell death [13,27]. Deficits in optical and retinal structures lead to problems in visual ability or function, such as strabismus [34] and visual acuity [34 – 36]. Although a number of studies have demonstrated that prenatal exposure to alcohol appears to produce deficits in visual acuity in children, few studies have examined the effects of prenatal exposure to alcohol on visual physiology. Katz and Fox [23] examined the effects of prenatal exposure to ethanol on rat electro-
*
Corresponding author. Tel.: +1-270-745-6314; fax: +1-270-745-6934. E-mail address: [email protected] (J. Bilotta).
retinograms (ERGs). The ERG is a gross electrical potential generated by retinal neurons [14]. Katz and Fox [23] found that hooded rats, exposed to ethanol prenatally, displayed deficits under light and dark adaptation as well as a decreased amount of rhodopsin in the retina. Similar deficiencies in the ERG responses have been reported in children diagnosed with Fetal Alcohol Syndrome (FAS) [21]. Thus, prenatal exposure appears to affect visual function as well as eye structures. Recently, the zebrafish (Danio rerio) has become an important animal model in developmental neuroscience, including visual neuroscience [4]. Zebrafish have transparent eggs and the embryos are semitransparent. Thus, the stage of embryonic development for an individual embryo can be determined without interfering with development. In addition, some chemicals, such as ethanol, pass through the chorion [7] making it relatively easy to study the effects of ethanol on the development of this species. Several studies have shown that ethanol exposure can affect zebrafish eye development. For example, zebrafish embryos, exposed to 2.5% and 3% solutions of ethanol,
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developed cyclopia (fusion of the two eyes [7,30]). In addition, preliminary work in our laboratory has shown that embryonic exposure to ethanol can produce microphthalmia in zebrafish [3]. Thus, it is clear that zebrafish eye development is affected by exposure to ethanol and that these effects are similar to those found in other species, including humans. The purpose of this study was to investigate the effects of embryonic exposure to ethanol on both visual physiology and visual function or acuity in zebrafish. Visual physiology was examined by obtaining ERG responses across various dark and light backgrounds. Visual acuity was determined behaviorally by using the optomotor response. Zebrafish larvae were exposed to ethanol at different times in development, including the period of eye development, in order to study the impact of ethanol at various times in development on visual physiology and behavior.
2. Methods
vertical black and white stripes consisting of 0.125 cycles per degree of visual angle, was placed on the outer wheel and rotated around the subject. If the fish ‘detects’ the moving stripes, then it will swim with the stimulus; if the animal cannot detect the moving stripes, then the animal will not swim with the movement of the stripes. This spatial frequency stimulus was chosen because previous work has shown that this stimulus is near the visual acuity limit of normal 6 –9 days postfertilization (dpf) zebrafish larvae [2]. In other words, normal zebrafish reliably respond to this spatial frequency stimulus, but have difficulty detecting higher spatial frequencies. Overhead room lights (Sylvania, F40/D; about 100 mW/cm2) illuminated the chamber. Prior to testing, the fish was placed into the fish chamber and a 5-min baseline swimming measure (number of laps around the chamber) was obtained. Next, the visual stimulus was rotated in one direction and the number of laps the fish swam with and against the direction of the stimulus were recorded for 5 min. Fish were tested between 6 and 9 dpf.
2.1. Subjects and rearing procedures 2.3. Physiological apparatus and procedures Zebrafish embryos (Danio rerio) bred inhouse [5] served as subjects. They were maintained at a water temperature of 28.5 C and a 14-h light on/10-h light off schedule. Fertilized embryos were placed into Petri dishes containing 20 ml of either distilled water or a 1.5% ethanol solution; the dishes were floated in 500 ml plastic containers that were floated in 10 gal aquarium tanks. Several different exposure durations were used: 6 –24, 12– 24, 24 –36, 48 – 60, and 60 – 72 h postfertilization (hpf). The 6 – 24 hpf exposure was used because it was shown to have significant effects on eye diameter and the presence of FAS characteristics in zebrafish [3]. The 12 –24 hpf exposure was used because this is the time period when the zebrafish eye develops; by 24 hpf, the eyecups are well-formed [31]. The 24– 36 hpf group represents a 12-h period just after eyecup development. The 48 –60 hpf period was chosen because it is around this time that the lens has formed and the optic nerve has reached the brain [9]. The last duration, 60 –72 hpf, was chosen because it is just prior to normal hatching; at this point, the optic nerve arbors have covered the optic tectum [10], and it marks the beginning of eye movements, including optokinetic nystagmus (OKN) [16]. Physical measurements such as eye diameter, as well as developmental staging, were obtained using an inverted microscope with phase contrast optics (World Precision Instruments, PIM). 2.2. Behavioral apparatus and procedures Zebrafish visual acuity was determined by measuring the optomotor response (for details, see Ref. [2]). The apparatus consisted of a small circular chamber (35-mm diameter) in which the fish is placed. A visual stimulus,
Details of the physiological apparatus and procedures are described elsewhere [22,29]. Briefly, visual stimuli were generated by using a two-channel optical system. One channel had a 150-W xenon arc lamp (Spectral Energy, Westwood, NJ, Model LH 150) light source that presented a broadband (white) test stimulus; the color temperature of this source was 9400 K. A 250-W tungsten – halogen bulb (Oriel, Stratford, CT, Model 6334) provided the broadband background irradiance and had a color temperature of 4500 K. The light sources were combined optically and focused onto one end of a liquid light guide (Oriel, Model 77556); the other end was placed in front of the subject’s eye. Neutral density filters placed in the light pathways controlled the irradiance of the two channels. Prior to the experiment, subjects were dark-adapted for at least one hour (zebrafish dark adaptation is complete by about 18 min [26]). Under dim red light, 6– 9 dpf subjects were anesthetized with a 0.01% dose of tricaine methanesulfonate [29] and placed on a piece of cotton, moistened with an anesthetic solution, located in a light – tight Faraday cage. Recording and reference electrodes were glass pipettes (tip diameter of 10 mm), filled with teleost saline solution that housed 36-gauge chlorided silver wires. The recording electrode was placed above the cornea on the subject’s eye; the reference electrode was placed on the body. Electrical signals were differentially amplified (bandpass of 0.1– 100 Hz) and recorded at a rate of 4 ms 1 by a computer. Once the subject was placed in the Faraday cage with the electrodes in position, the subject was dark-adapted for an additional 15 min. Following this time period, testing began. Each trial consisted of one 200 ms stimulus
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presentation; responses were recorded for an additional 2000 ms following stimulus termination. A baseline period of 50 ms was recorded prior to the stimulus presentation. Stimulus irradiance trials started below threshold and increased in 0.5 log unit steps until the response criterion was reached. To minimize possible light-adaptation effects, stimulus irradiances that produced responses beyond the linear portion of the response vs. log irradiance function were avoided [6]. Once an irradiance series was completed at a particular background, the background irradiance was increased. The subject adapted to the new background for at least 5 min and the procedures were repeated. Six background irradiances ranging from dark adaptation to 500 mW/cm2 were used. Increment threshold functions were derived by finding the stimulus irradiance that produced a criterion response for each background irradiance. Log threshold was defined as the log stimulus irradiance (mW/cm2) that produced the criterion response. Increment threshold functions were derived from the first voltage-positive ERG response (b-wave) measured from baseline or the first negative peak to the first positive peak [17]. Similar procedures have been used previously with zebrafish larvae [6,28,29]. 2.4. Statistical analyses A one-factor between-subjects analysis of variance (ANOVA) was used to determine whether there were significant differences in eye diameter across the six treatment groups (control, and those subjects exposed to 1.5% ethanol 6 – 24, 12 – 24, 24 – 36, 48 – 60, or 60 – 72 hpf). Similarly, a one-factor between-subjects ANOVA was calculated on the baseline optomotor response scores across the six treatment groups to determine whether there were differences in activity rates across the groups. Scheffe’s post hoc comparisons were made to determine where the specific significant differences occurred. A one-factor between-subjects analysis of covariance with the optomotor response to the moving visual stimulus as the dependent measure and the baseline response score as a covariate was calculated to determine whether there were differences in the optomotor response across the six treatment groups. A 2 (treatment: control vs. subjects exposed to 1.5% ethanol 6– 24 hpf ) 6 (background irradiance) mixed design ANOVA was calculated to determine whether there were any differences in ERG b-wave threshold between the two groups across the various background irradiances. Simple effects analyses were performed to determine where the significant differences occurred in the Treatment Background interaction. Finally, a 3 (control, subjects exposed to 1.5% ethanol 0 –12 or 12– 24 hpf ) 6 (background irradiance) mixed design ANOVA was used to determine whether there were differences in the ERG b-wave increment threshold functions across the different ethanol exposure conditions and background irradiances.
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3. Results 3.1. Visual acuity Visual acuity was assessed for larvae exposed to various ethanol conditions as well as control subjects. All subjects in the ethanol groups were exposed to 1.5% ethanol at various times during development. The majority of groups were exposed for 12 h; only one group was exposed longer (6– 24 hpf). The longer exposure condition was included since preliminary work demonstrated that it clearly affected zebrafish physical development [3]. Fig. 1 compares the eye diameter of the various groups measured at 3 dpf. A onefactor between-subjects ANOVA found significant effects on eye diameter across the groups [ F(5,122) = 29.27, P < .001]. Scheffe’s post hoc comparisons were made to determine where the significant differences occurred. The comparisons found that the subjects exposed to ethanol 6– 24 and 12 –24 hpf had significantly smaller eye diameters than all of the other groups, including the control group. Also, it was found that subjects exposed to ethanol 24 – 36 hpf had significantly smaller eye diameters than the 60 – 72 hpf group (all at P < .05). Thus, zebrafish eye diameter is affected by embryonic exposure to ethanol. Prior to examining the effects of ethanol on visual behavior using the optomotor response, it was important to ensure that there were no differences in swimming behavior across the groups. Since preliminary work has shown that exposure to ethanol could produce severe physical problems, such as an abnormally large body [3], only subjects that appeared to possess normal swimming
Fig. 1. Mean eye diameter of control subjects (n = 44) and subjects exposed to 1.5% ethanol at 6 – 24 (n = 10), 12 – 24 (n = 39), 24 – 36 (n = 10), 48 – 60 (n = 10), and 60 – 72 (n = 15) hpf. Subjects were examined at 3 dpf. Error bars represent ± 1 S.E.M. Groups designated with a single asterisk (*) indicate that these groups are significantly different from the other groups; groups designated with double asterisks (**) indicate that the two groups are significantly different from one another. All significance levels are P < .05.
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groups [ F(5,99) = 2.52, P < .05]. Scheffe’s post hoc comparisons revealed that the subjects exposed to ethanol 6– 24 and 12– 24 hpf had significantly lower responses than the control subjects. Also, these two groups had significantly lower responses than the subjects exposed to ethanol 48– 60 and 60– 72 hpf. Finally, subjects exposed to ethanol 24 – 36 hpf had significantly lower responses than those subjects exposed at 48 – 60 hpf. Fig. 2 shows the mean response scores for the various groups. The dotted line represents the average baseline response across the groups. Note that only the 6– 24 and 12 –24 hpf groups’ responses fall below the baseline response. Thus, ethanol exposure does have an effect on visual acuity; the effects are most pronounced when subjects are exposed to ethanol 6 –24 and 12– 24 hpf. 3.2. Retinal physiology Fig. 2. Mean optomotor response score of control subjects (n = 23) and subjects exposed to 1.5% ethanol at 6 – 24 (n = 17), 12 – 24 (n = 7), 24 – 36 (n = 16), 48 – 60 (n = 23), and 60 – 72 (n = 18) hpf. Subjects were examined between 6 and 9 dpf. Mean values are estimated marginal means from the covariance analysis. The dotted line shows the average baseline response score. Error bars represent ± 1 S.E.M. Groups designated with a single asterisk (*) indicate that these groups are significantly different from groups with a double asterisks (**); groups designated with the letter ‘a’ are significantly different from one another. All significance levels are P < .05.
ability were tested in the optomotor apparatus. In addition, since hyperactivity has been shown to be an effect of prenatal exposure to ethanol in rats [8] and humans [32], there was some concern that there might be differences in motor activity across the groups. Therefore, in order to be sure that the subjects in all groups were similar in swimming ability, the baseline responses (i.e., number of laps swum while the visual stimulus was stationary) across the groups were compared. A one-factor between-subjects ANOVA was calculated on the response scores across the six different groups (control, and those subjects exposed to 1.5% ethanol 6– 24, 12 –24, 24– 36, 48 –60, or 60– 72 hpf). The results found no significant differences in baseline swimming across the groups [ F(5,100) = 1.42, P >.05]. However, it is interesting to note that the mean response score for subjects exposed to 6 –24 hpf was slightly higher (m = 3.00, s = 2.12) than the mean response score for controls (m = 2.12, s = 1.58). Thus, if there is a difference between controls and ethanol-exposed groups in motor activity, then the optomotor response is not sensitive enough to detect it. This may be because the optomotor response only measures activity around the chamber and not lateral movement within the chamber. To further ensure that swimming or activity rates across the groups did not influence the responses to the rotating 0.125 cycles per degree stimulus, baseline swimming was entered as a covariate in a one-factor between-subjects analysis of covariance with the response to the moving visual stimulus as the dependent measure. The results showed that there were significant differences across the
Fig. 3 shows representative ERG responses from individual 6 dpf zebrafish larvae. The background illumination was 50 mW/cm2. Stimulus duration was 200 ms and is
Fig. 3. Individual sample ERG waveforms from normal and ethanolexposed subjects. The background illumination consisted of a 50-mW/cm2 white light. The stimulus duration was 200 ms and is indicated by the raised horizontal line on the abscissa. The negative values associated with each ERG waveform represent the stimulus irradiance in log attenuation values where 0.0 corresponds to a stimulus irradiance of 30 mW/cm2. The peak of the b-wave is labeled in each panel. (A) ERG responses of a 6 dpf normal larvae. (B) ERG responses of a 6 dpf larvae exposed to 1.5% ethanol at 6 – 24 hpf. See text for details.
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Fig. 4. Individual sample b-wave response amplitude vs. log irradiance response functions obtained under a dark background (filled symbols) and a background irradiance of 0.5 mW/cm2 (open symbols). Circles represent the responses of an ethanol-exposed subject and squares represent the responses of a control subject. The negative values associated with each ERG waveform represent the stimulus irradiance in log attenuation values where 0.0 corresponds to a stimulus irradiance of 30 mW/cm2.
represented by the raised bar on the abscissa. The negative values associated with each ERG waveform represent the stimulus irradiance in log attenuation values. Fig. 3A shows ERG responses of a control subject. The responses are similar to those of normal zebrafish larvae at 6 dpf [29]. There is a voltage-positive response (b-wave) whose amplitude is dependent upon stimulus irradiance. As in other studies with zebrafish larvae, some subjects show a small voltage-negative (a-wave) response prior to the b-wave, while others do not [28,29]. Thus, in the current study, only the responses of the ERG b-wave were analyzed. Fig. 3B shows representative ERG responses to different stimulus irradiances of a larvae zebrafish exposed to 1.5% ethanol 6 –24 hpf. The ERG response from this subject differs from the response of the control subject both in shape and in responsiveness to light. The b-wave shape appears to be different from that of controls in that it is more sustained than the response of control subjects; that is, it takes longer to return to a baseline response than the b-wave of the control subjects. Also, the b-wave response is much less responsive to light in ethanol-exposed subjects than in controls. Compare the ERG b-wave amplitudes of the control and ethanol-exposed subjects to the same stimulus irradiance ( 2.0). Although it clearly was the case that the ERG response of the ethanol-exposed subjects was reduced at background irradiance of 50 mW/cm2, it was not the case under all background conditions. Fig. 4 shows representative b-wave response amplitude vs. stimulus irradiance functions for a control subject (squares) and an ethanol-exposed subject (circles) obtained under a dark background (filled symbols) and a background of 0.5 mW/cm2 (open symbols). The most
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striking finding is that under the higher background irradiance, the response-irradiance functions of the ethanolexposed subject were very similar to those of the control subject. On the other hand, there was a large difference between the response-irradiance functions between the two subjects under the dark background. Thus, embryonic exposure to ethanol appears to be producing larger deficits under lower levels of light adaptation. To further investigate this finding, Fig. 5 shows the increment threshold functions for control subjects and those subjects exposed to 1.5% ethanol 6 –24 hpf. The increment threshold functions were obtained from subjects between 6 and 9 dpf. Log threshold was defined as the stimulus irradiance required to produce a 20-mV b-wave response. The background irradiance labeled ‘‘D’’ represents a state of complete dark adaptation. Error bars represent ± one standard error of the mean (S.E.M.). The increment threshold function obtained from the control subjects is similar to that obtained from a previous study in zebrafish larvae [28]. Under dim levels of illumination, threshold is relatively low. As the background illumination is increased, the threshold value increases. However, the increment threshold function obtained from the ethanol-exposed subjects was very different from controls. Threshold values did not improve with lower background levels. In fact, threshold values were very high across all background levels. A 2 (treatment: control vs. ethanolexposure) 6 (background irradiance) mixed design ANOVA revealed a significant Treatment Background irradiance interaction [ F(5,30) = 6.67, P < .001]. Simple effects analyses were performed to determine where the significant differences occurred. These analyses found that the ethanol-exposed subjects had significantly higher thresh-
Fig. 5. Averaged increment threshold functions based on the ERG b-wave response at 6 – 9 dpf control (circles; n = 3) and larvae exposed to 1.5% ethanol at 6 – 24 hpf (squares; n = 5). Log threshold is defined as the reciprocal of the log stimulus irradiance (mW/cm2) that produced a 20-mV bwave response. Error bars represent ± 1 S.E.M.
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olds than controls at all background levels except at the log background irradiance of 3.7 (all values tested at P < .05). Thus, 1.5% ethanol exposure at 6 – 24 dpf has dramatic effects on the ERG increment threshold function, particularly at the lower background levels. To determine whether shorter ethanol exposure durations would have any impact on long-term development of the ERG, two additional ethanol-exposed groups were compared to normal subjects. One group of larvae was exposed to 1.5% ethanol at 0 – 12 hpf and the other group was exposed to 1.5% ethanol at 12 –24 hpf. Increment threshold functions were obtained from all subjects, including controls, at 22 dpf. In general, the two ethanol-exposed groups had increment threshold functions similar to that of the control subjects, although the function obtained from control subjects had slightly lower thresholds across all backgrounds. A 3 (early exposure, late exposure and control) 6 (background irradiance) mixed design ANOVA found only a significant main effect of background irradiance [ F(5,30) = 18.27, P < .005]. Since the Type of exposure Background irradiance interaction was not significant, it appears that the three increment threshold functions were similar.
4. Discussion The results of the present study showed that ethanol exposure during development can influence zebrafish visual behavior as well as visual physiology. This study also found that the timing of the ethanol exposure could impact the severity with which ethanol affects visual function. The present study showed that ethanol exposure produced smaller eyes as well as a reduction in visual acuity measured behaviorally. These findings support findings from studies on a variety of species that prenatal ethanol (or alcohol) exposure can impact the visual system. For example, children who have been exposed to alcohol prenatally display a number of visual problems such as microphthalmia [33], retinal vein problems [33,34], reduced optic disk [19,36], and poor visual acuity [25,34,35,37]. Similar findings in animal studies have been reported, including microphthalmia and reduced optic nerve size in rodents [24,27], as well as increased ganglion cell death in rats [27] and chicks [13]. In addition, the present study showed that ethanol exposure can alter retinal physiology. Larvae that were exposed to ethanol had ERG waveforms that were abnormal in shape and increment threshold functions that differed from normals. In general, visual thresholds in larvae exposed to ethanol were significantly higher than those of controls. However, even though these effects occurred under both light- and dark-adapted conditions, the deficits were more pronounced under lower backgrounds of illumination. These results support previous work that showed that rat retinal physiology was altered by prenatal exposure to
ethanol. Katz and Fox [23] found differences in ERG increment threshold functions between normal and ethanol-exposed rats; the differences were more pronounced under lower levels of illumination. Therefore, the ERG results found in zebrafish appear to be similar to those found in other species, including humans [21]. It also should be mentioned that the ERG increment threshold results are consistent with the deficits found in the optomotor response obtained under similar background illumination (about 100 mW/cm2). These findings suggest that the site of ethanol’s action on retinal physiology and visual behavior may be the same, and that this action takes place early in the visual system. One of the most interesting findings of the present results is that the effects of ethanol on vision appear to be most severe during the time in development when the eye forms. The 12– 24 hpf ethanol exposure group was chosen because this is the time when the eye begins to form [31]. Thus, one would expect more visual problems when the embryo is exposed to ethanol during this time. This was found to be the case. Larvae exposed to ethanol 12 –24 hpf had significantly smaller eye diameters and responded significantly less to high spatial frequency visual stimuli than any other 12-h exposure group. They also had slightly lower visual thresholds measured by the ERG than controls. In fact, these subjects displayed more similar visual deficits to those exposed to ethanol for approximately 24 h (6– 24 hpf) than any other group. The 6– 24 hpf ethanol-exposed subjects displayed significantly higher visual thresholds, smaller eye diameters, and poorer visual acuity than controls and subjects exposed to ethanol for any 12-h period following the first 24 h. It is not surprising that the 6– 24 hpf ethanolexposed group would display visual deficits similar to those found in the 12– 24 hpf ethanol-exposed group, since the last portion of exposure in the first group was the same as the second group. However, what is interesting is that the 6 –24 hpf ethanol-exposed group also displayed other physical deficits, such as physical deformities resembling FAS [3]. The 12 –24 hpf ethanol-exposed group did not display these physical characteristics; the only deficits for these subjects appeared to be in visual processing. Finally, the only other group that displayed any visual problems was the group exposed to ethanol at 24 –36 hpf. They displayed a slight deficit in visual acuity as measured by the optomotor response. This is not surprising given that this ethanol exposure takes place while the optic nerve is working its way to the brain (in the case of the zebrafish, the optic tectum); by about 2 dpf, the optic nerve has reached the optic tectum [9]. Since the optomotor response most likely represents brain functioning, one would expect some brain deficits in visual processing. One possible reason for the deficiencies found in visual behavior and physiology in ethanol-exposed subjects may be that ethanol-exposed subjects had smaller eyes. Smaller eyes will affect the optics of the eye and possibly retinal physiology. For example, it has been shown that the
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amplitudes of the ERG b-wave component are generally smaller with larger eyes (i.e., longer axial length), such as in the case with humans with myopia [12]. However, this cannot be the explanation for the differences found in this study with retinal physiology, since the present study finds the opposite results; larvae exposed to ethanol had both smaller eye diameters and higher ERG b-wave increment thresholds. In addition, previous work has shown that the ERG waveform and the response characteristics are not dramatically different at early ages of larvae development [29], where eye diameter does increase with age. Regarding the effects of eye size on visual behavior, further work is required to address this issue since zebrafish visual behavior depends on the coordinated development of optical and retinal structures, and the extraocular muscles (see Ref. [16]). It should be mentioned that even though the results from the present study with zebrafish show effects of ethanol that are similar to other animal species, there is a large discrepancy between the doses of ethanol across species. In order to find the effects found in rodent species, the doses necessary to produce these effects on zebrafish embryos are much higher. The most likely explanation for the discrepancy is that the chorion of the zebrafish embryo does not allow ethanol to pass through freely. Research examining the permeability of the zebrafish chorion supports this notion. Harvey et al. [18] found that when zebrafish eggs were placed in a glycerol solution, only about 8% of the glycerol solution actually entered the embryonic tissue. Similar effects were found when eggs were placed into a solution of dimethyl sulfoxide (DMSO). About 3% of DMSO reached the embryonic tissue with an intact chorion. However, when the chorion was removed, the percentage of DMSO that reached the embryo increased dramatically. At present, there are no studies that have examined the permeability of the zebrafish chorion to ethanol. However, if one assumes that roughly 8% of the ethanol reaches the embryonic tissue, then a 1.5% ethanol (1500 mg/dl) solution has an ‘‘effective’’ solution of 120 mg/dl. This ethanol dose falls within the range of doses used to produce effects in rodent pups [15] and corresponds to blood alcohol levels found in women who have children with FAS (see Ref. [1]). In summary, the zebrafish appears to be a useful model for studying embryonic exposure to ethanol not only because ethanol appears to affect zebrafish in a manner similar to its effects on other species, but also because of the advantages the zebrafish brings as a developmental model in general [4]. Because so much is known about its embryonic development, precise manipulations can be performed targeting particular structures. In the present study, we have shown that exposing zebrafish larvae to ethanol during the period of eye development produces significant visual deficits, including physical (microphthalmia), physiological (ERG), and behavioral (optomotor response) problems. Similar studies, targeting other aspects of zebrafish embryonic development,
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could provide valuable insight on the exact nature of the effects of ethanol.
Acknowledgements This work was supported by a Western Kentucky Research Grant and a Summer Fellowship Grant. The authors would like to thank Timothy Lawrence and Elizabeth Lemerise for their work on this project, and Angela McDowell and Codye Hill for their helpful comments on this work.
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