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Developmental bisphenol A (BPA) exposure has been implicated in adverse behavior and learning deficits. The mode of action underlying these effects is unclear. The zebrafish model was employed to investigate the neurobehavioral effects of developmental bisphenol A (BPA) exposure. The objectives of this study were to identify whether low-dose, developmental BPA exposure affects larval zebrafish locomotor behavior and whether learning deficits occur in adults exposed during development. Two control compounds, 17β-estradiol (an estrogen receptor ligand) and GSK4716 (a synthetic estrogen related receptor gamma ligand), were included. Larval toxicity assays were used to determine appropriate BPA, 17β-estradiol, and GSK4716 concentrations for behavior testing. BPA tissue uptake was analyzed using HPLC and lower doses were extrapolated using a linear regression analysis. Larval behavior tests were conducted using a ViewPoint Zebrabox. Adult learning tests were conducted using a custom-built T-maze. BPA exposure to ≤30 μM was nonteratogenic in zebrafish. Neurodevelopmental BPA exposure to 0.01, 0.1, or 1 μM led to larval hyperactivity or learning deficits in adult zebrafish. Exposure to 0.1 μM 17β-estradiol or GSK4716 also led to larval hyperactivity. This study demonstrates the efficacy of using the larval zebrafish model for studying the neurobehavioral effects of low-dose developmental BPA exposure.

Developmental exposures to methylmercury (MeHg) have life-long behavioral effects. Many micronutrients, including selenium, are involved in cellular defenses against oxidative stress and may reduce the severity of MeHg-induced deficits. Zebrafish embryos (<4 h post fertilization, hpf) were exposed to combinations of 0.0-0.30 microM MeHg and/or selenomethionine (SeMet) until 24 hpf then placed in clean medium. Fish were tested as adults under low light conditions ( approximately 60 microW/m(2)) for visual responses to a rotating black bar. Dose-dependent responses to MeHg exposure were evident (ANOVA, P<0.001) as evidenced by reduced responsiveness, whereas SeMet did not induce deficits except at 0.3 microM. Ratios of SeMet:MeHg of 1:1 or 1:3 resulted in responses that were indistinguishable from controls (ANOVA, P<0.001). No gross histopathologies were observed (H&E stain) in the retina or optic tectum at any MeHg concentration. Whole-cell, voltage-gated, depolarization-elicited outward K(+) currents of bipolar cells in intact retina of slices adult zebrafish were recorded and outward K(+) current amplitude was larger in bipolar cells of MeHg-treated fish. This was due to the intense response of cells expressing the delayed rectifying I(K) current; cells expressing the transient I(A) current displayed a slight trend for smaller amplitude among MeHg-treated fish. Developmental co-exposure to SeMet reduced but did not eliminate the increase in the MeHg-induced I(K) response, however, I(A) responses increased significantly over MeHg-treated fish to match control levels. Electrophysiological deficits parallel behavioral patterns in MeHg-treated fish, i.e., initial reactions to the rotating bar were followed by periods of inactivity and then a resumption of responses.

BackgroundMethylmercury (MeHg) is a known neurotoxic agent, but the mechanisms by which MeHg may act on reproductive pathways are relatively unknown. Several studies have indicated potential changes in hormone levels as well as declines in vertebrates with increasing dietary MeHg exposure.ObjectivesThe purpose of this study was to identify alterations in gene expression associated with MeHg exposure, specifically those associated with previously observed changes in reproduction and reproductive biomarkers. Fathead minnows, Pimephales promelas, were fed one of three diets that were similar to documented concentrations of MeHg in the diets of wild invertivorous and piscivorous fish. We used a commercial macroarray in conjunction with quantitative polymerase chain reaction to examine gene expression in fish in relation to exposure to these environmentally relevant doses of MeHg.ResultsExpression of genes commonly associated with endocrine disruption was altered with Hg exposure. Specifically, we observed a marked up-regulation in vitellogenin mRNA in individual Hg-exposed males and a significant decline in vitellogenin gene expression in female fish with increasing Hg concentrations. Other genes identified by the macroarray experiment included those associated with egg fertilization and development, sugar metabolism, apoptosis, and electron transport. We also observed differences in expression patterns between male and female fish not related to genes specifically associated with reproduction, indicating a potential physiological difference in the reaction of males and females to MeHg.ConclusionGene expression data may provide insight into the mechanisms by which MeHg affects reproduction in fish and indicate how MeHg differs in its effect from other heavy metals and endocrine-disrupting compounds.

Methylmercury (MeHg) is a ubiquitous environmental pollutant and has been shown to affect learning in vertebrates following relatively low exposures. Zebrafish were used to model long-term learning deficits after developmental MeHg exposure. Selenomethionine (SeMet) co-exposure was used to evaluate its role in neuroprotection. Embryos were exposed from 2-24 hours post fertilization to (1) MeHg without SeMet, (2) SeMet without MeHg and (3) in combination of MeHg and SeMet. In case (1), the levels of MeHg were 0.00, 0.01, 0.03, 0.06, 0.10, 0.30 µM. In case (2), the levels of SeMet were 0.00. 0.03, 0.06, 0.10, 0.30 µM. In case (3), co-exposure levels of (MeHg, SeMet) were (0.03, 0.03), (0.03, 0.06), (0.03, 0.10), (0.03, 0.30), (0.10, 0.03), (0.10, 0.06), (0.10, 0.10), (0.10, 0.30) µM. Learning functions were tested in individual adults, four months after developmental exposure using a spatial alternation paradigm with food delivery on alternating sides of the aquarium. Low levels of MeHg (<0.1 µM) exposure delayed learning in treated fish; fish exposed to higher MeHg levels were unable to learn the task; SeMet co-exposure did not prevent this deficit. These data are consistent with findings in laboratory rodents. The dorsal and lateral telencephalon are the primary brain regions in fish involved in spatial learning and memory. Adult telencephalon cell body density decreased significantly at all MeHg exposures >0.01 µM MeHg. SeMet co-exposure ameliorated but did not prevent changes in telencephalon cell body density. In summary, MeHg affected both learning and brain structure, but SeMet only partially reversed the latter.Descriptors developmental exposure; learning; mercury; selenium; spatial alternation; zebrafish

Lead (Pb2+) exposure continues to be an important concern for fish populations. Research is required to assess the long-term behavioral effects of low-level concentrations of Pb2+ and the physiological mechanisms that control those behaviors. Newly fertilized zebrafish embryos (<2 hours post fertilization; hpf) were exposed to one of three concentrations of lead (as PbCl2): 0, 10, or 30 nM until 24 hpf. 1) Response to a mechanosensory stimulus. Individual larvae (168 hpf) were tested for response to a directional, mechanical stimulus. The tap frequency was adjusted to either 1 or 4 taps/sec. Startle response was recorded at 1000 fps. Larvae responded in a concentration-dependent pattern for latency to reaction, maximum turn velocity, time to reach Vmax and escape time. With increasing exposure concentrations, a larger number of larvae failed to respond to even the initial tap and, for those that did respond, ceased responding earlier than control larvae. These differences were more pronounced at a frequency of 4 taps/sec. 2) Response to a visual stimulus. Fish, exposed as embryos (2–24 hpf) to Pb2+ (0–10 uM) were tested as adults under low light conditions (~60 μW/m2) for visual responses to a rotating black bar. Visual responses were significantly degraded at Pb2+ concentrations of 30 nM. These data suggest that zebrafish are viable models for short- and long-term sensorimotor deficits induced by acute, low-level developmental Pb2+ exposures.

Zebrafish Danio rerio embryos were exposed to 0, 25, 50 or 75 ppb Hg 2þ from 0 to 24 h post-fertilization (hpf) then placed into Hg 2þ -free water. Inductively coupled plasma-mass spectrophotometer analysis of whole embryo Hg 2þ content at 24 hpf showed a positive correlation with exposure regime (Pearson's one-tailed, r 2 ¼ 0Á698, P < 0Á01); at 5 days posthatch (dph), whole larval Hg 2þ content was not detectable. Hg 2þ -induced behavioural deficits in larvae were, therefore, due to changes during embryogenesis and not to residual Hg 2þ in the larvae. At 5 dph, larvae were tested for responses to different frequencies but equal intensities of vibrational stimuli generated by a remotely controlled plastic hammer. Data were recorded by high-speed videography and computer-analysed for latency of response (ms), amplitude of the response as measured by maximum initial velocity [normalized as body (standard) lengths s ÿ1 ; V max ] and duration of behaviour from initial head movement to cessation of caudal tail movement (ms). A single mechanical stimulus resulted in behavioural outcomes that were related to embryonic Hg 2þ uptake. Response latency increased with exposure level and displayed an increase of Â1Á5-2Á5 over control values (ANOVA, P < 0Á01). The V max decreased with exposure level to a low of 71% of control at the highest Hg 2þ concentration (ANOVA, P < 0Á01). Duration of behaviour displayed a biphasic response pattern in which exposure to 0, 50 or 75 ppb Hg 2þ did not result in a significantly different response yet exposure to 25 ppb Hg 2þ caused a significantly longer time of active response (ANOVA, P < 0Á01). Repeated stimulation (1, 2 or 4 hits s ÿ1 ) resulted in a concentration-dependent increase in response failures. Regardless of stimulation frequency, larvae exposed to 0 or 25 ppb Hg 2þ as embryos maintained higher V max levels for longer intervals during the testing period than those exposed as embryos to either 50 or 75 ppb Hg 2þ .

Developmental bisphenol A (BPA) exposure is associated with adverse behavioral effects, although underlying modes of action remain unclear. Because BPA is a suspected xenoestrogen, the objective was to identify sex-based changes in adult zebrafish social behavior developmentally exposed to BPA (0.0, 0.1 or 1 μM) or one of two control compounds (0.1μM 17β-estradiol [E2], and 0.1 μM GSK4716, a synthetic estrogen-related receptor γ ligand). A test chamber was divided lengthwise so each arena held one fish unable to detect the presence of the other fish. A mirror was inserted at one end of each arena; baseline activity levels were determined without mirror. Arenas were divided into 3, computer-generated zones to represent different distances from mirror image. Circadian rhythm patterns were evaluated at 1–3 (= AM) and 5–8 (= PM) hr postprandial. Adult zebrafish were placed into arenas and monitored by digital camera for 5 min. Total distance traveled, % time spent at mirror image, and number of attacks on mirror image were quantified. E2, GSK4716, and all BPA treatments dampened male activity and altered male circadian activity patterns; there was no marked effect on female activity. BPA induced non-monotonic effects (response curve changes direction within range of concentrations examined) on male % time at mirror only in AM. All treatments produced increased % time at the mirror during PM. Male attacks on the mirror were reduced by BPA exposure only during AM. There were sex-specific effects of developmental BPA on social interactions and time-of-day of observation affected results.