Anthony N. Oliveri

Zebrafish model systems for developmental neurobehavioral toxicology

Zebrafish offer many advantages that complement classic mammalian models for the study of normal development as well as for the teratogenic effects of exposure to hazardous compounds. The clear chorion and embryo of the zebrafish allow for continuous visualization of the anatomical changes associated with development, which, along with short maturation times and the capability of complex behavior, makes this model particularly useful for measuring changes to the developing nervous system. Moreover, the rich array of developmental, behavioral, and molecular benefits offered by the zebrafish have contributed to an increasing demand for the use of zebrafish in behavioral teratology. Essential for this endeavor has been the development of a battery of tests to evaluate a spectrum of behavior in zebrafish. Measures of sensorimotor plasticity, emotional function, cognition and social interaction have been used to characterize the persisting adverse effects of developmental exposure to a variety of chemicals including therapeutic drugs, drugs of abuse and environmental toxicants. In this review, we present and discuss such tests and data from a range of developmental neurobehavioral toxicology studies using zebrafish as a model. Zebrafish provide a key intermediate model between high throughput in vitro screens and the classic mammalian models as they have the accessibility of in vitro models and the complex functional capabilities of mammalian models.

Neurobehavioral impairments caused by developmental imidacloprid exposure in zebrafish

BACKGROUND Neonicotinoid insecticides are becoming more widely applied as organophosphate (OP) insecticides are decreasing in use. Because of their relative specificity to insect nicotinic receptors, they are thought to have reduced risk of neurotoxicity in vertebrates. However, there is scant published literature concerning the neurobehavioral effects of developmental exposure of vertebrates to neonicotinoids. METHODS Using zebrafish, we investigated the neurobehavioral effects of developmental exposure to imidacloprid, a prototypic neonicotinoid pesticide. Nicotine was also administered for comparison. Zebrafish were exposed via immersion in aqueous solutions containing 45 μM or 60 μM of imidacloprid or nicotine (or vehicle control) from 4h to 5d post fertilization. The functional effects of developmental exposure to both imidacloprid and nicotine were assessed in larvae using an activity assay and during adolescence and adulthood using a battery of neurobehavioral assays, including assessment of sensorimotor response and habituation in a tactile startle test, novel tank swimming, and shoaling behavior. RESULTS In larvae, developmental imidacloprid exposure at both doses significantly decreased swimming activity. The 5D strains of zebrafish were more sensitive to both nicotine and imidacloprid than the AB* strain. In adolescent and adult fish, developmental exposure to imidacloprid significantly decreased novel tank exploration and increased sensorimotor response to startle stimuli. While nicotine did not affect novel tank swimming, it increased sensorimotor response to startle stimuli at the low dose. No effects of either compound were found on shoaling behavior or habituation to a startling stimulus. DISCUSSION Early developmental exposure to imidacloprid has both early-life and persisting effects on neurobehavioral function in zebrafish. Its developmental neurotoxicity should be further investigated.

Developmental exposure to organophosphate flame retardants causes behavioral effects in larval and adult zebrafish.

BACKGROUND Organophosphate flame retardants (OPFRs) have grown in usage since concerns about the health effects of the previously used polybrominated flame retardants led to their being phased out. The potential for OPFRs to cause adverse health effects of their own is still unexamined. Because of their structural similarities to organophosphate pesticides, which have themselves been heavily researched and shown to be neurobehavioral teratogens, we investigated the possibility that developmental exposure to two OPFRs, triphenyl phosphate (TPHP), and tris(1,3-dichloroisopropyl)phosphate (TDCIPP) might lead to behavioral impairment across the lifespan, as has been observed with the organophosphate pesticide chlorpyrifos. METHODS Zebrafish were exposed to 0.03 or 0.3 μM of TPHP, TDCIPP, or chlorpyrifos from 0 to 5 days post fertilization. Vehicle control consisted of 0.03% solution of DMSO. At 6 days post fertilization, larvae were tested on a locomotor assay. Separate cohorts of 6 day old larvae that were not tested on the larval assay were allowed to grow to adulthood. At 12 weeks post fertilization, these adult zebrafish were tested on a battery of behavioral assays that included tests of novel environment exploration, startle habituation, social affiliation, and predator escape. RESULTS Developmental exposure altered zebrafish behavior across the lifespan. Larval zebrafish exposed to the 0.03 μM doses of chlorpyrifos or TDCIPP exhibited significant (p<0.05) hyperactivity in the locomotor assay. Organophosphate exposure significantly (p<0.05) altered the time course of adult zebrafish behavior in the novel environment, startle habituation, and social affiliation assays. Predator escape behavior was significantly (p<0.05) reduced in fish exposed to the 0.3 μM dose of TDCIPP. Exposure also caused hyperactivity in adult fish, with fish exposed to the 0.3 μM dose of TDCIPP exhibiting significantly (p<0.05) elevated locomotor behavior in the novel environment assay. DISCUSSION Early developmental exposure to OPFRs produced behavioral impairment that persisted into adulthood. These findings support broader research investigating the role of organophosphate compounds, including the OPFRs used here, in developmental neurotoxicity.

Developmental exposure to a complex PAH mixture causes persistent behavioral effects in naive Fundulus heteroclitus (killifish) but not in a population of PAH-adapted killifish.

Acute exposures to some individual polycyclic aromatic hydrocarbons (PAHs) and complex PAH mixtures are known to cause cardiac malformations and edema in the developing fish embryo. However, the heart is not the only organ impacted by developmental PAH exposure. The developing brain is also affected, resulting in lasting behavioral dysfunction. While acute exposures to some PAHs are teratogenically lethal in fish, little is known about the later life consequences of early life, lower dose subteratogenic PAH exposures. We sought to determine and characterize the long-term behavioral consequences of subteratogenic developmental PAH mixture exposure in both naive killifish and PAH-adapted killifish using sediment pore water derived from the Atlantic Wood Industries Superfund Site. Killifish offspring were embryonically treated with two low-level PAH mixture dilutions of Elizabeth River sediment extract (ERSE) (TPAH 5.04 μg/L and 50.4 μg/L) at 24h post fertilization. Following exposure, killifish were raised to larval, juvenile, and adult life stages and subjected to a series of behavioral tests including: a locomotor activity test (4 days post-hatch), a sensorimotor response tap/habituation test (3 months post hatch), and a novel tank diving and exploration test (3months post hatch). Killifish were also monitored for survival at 1, 2, and 5 months over 5-month rearing period. Developmental PAH exposure caused short-term as well as persistent behavioral impairments in naive killifish. In contrast, the PAH-adapted killifish did not show behavioral alterations following PAH exposure. PAH mixture exposure caused increased mortality in reference killifish over time; yet, the PAH-adapted killifish, while demonstrating long-term rearing mortality, had no significant changes in mortality associated with ERSE exposure. This study demonstrated that early embryonic exposure to PAH-contaminated sediment pore water caused long-term locomotor and behavioral alterations in killifish, and that locomotor alterations could be observed in early larval stages. Additionally, our study highlights the resistance to behavioral alterations caused by low-level PAH mixture exposure in the adapted killifish population. Furthermore, this is the first longitudinal behavioral study to use killifish, an environmentally important estuarine teleost fish, and this testing framework can be used for future contaminant assessment.

Long-term behavioral impairment following acute embryonic ethanol exposure in zebrafish.

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 h 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.

Persistent behavioral effects following early life exposure to retinoic acid or valproic acid in zebrafish.

BACKGROUND Moderate to severe dysregulation in retinoid signaling during early development is associated with a constellation of physical malformations and/or neural tube defects, including spina bifida. It is thought that more subtle dysregulation of this system, which might be achievable via dietary (i.e. hypervitaminosis A) or pharmacological (i.e. valproic acid) exposure in humans, will manifest on behavioral domains including sociability, without overt physical abnormalities. METHODS During early life, zebrafish were exposed to low doses of two chemicals that disrupt retinoid signaling. From 0 to 5dpf, larvae were reared in aqueous solutions containing retinoic acid (0, 0.02, 0.2 or 2nM) or valproic acid (0, 0.5, 5.0 or 50μM). One cohort of zebrafish was assessed using a locomotor activity screen at 6-dpf; another was reared to adulthood and assessed using a neurobehavioral test battery (startle habituation, novel tank exploration, shoaling, and predator escape/avoidance). RESULTS There was no significant increase in the incidence of physical malformation among exposed fish compared to controls. Both retinoic acid and valproic acid exposures during development disrupted larval activity with persisting behavioral alterations later in life, primarily manifesting as decreased social affiliation. CONCLUSIONS Social behavior and some aspects of motor function were altered in exposed fish; the importance of examining emotional or psychological consequences of early life exposure to retinoid acting chemicals is discussed.

Pharmacological analyses of learning and memory in zebrafish (Danio rerio)

Over the last decade, zebrafish (Danio rerio) have become valuable as a complementary model in behavioral pharmacology, opening a new avenue for understanding the relationships between drug action and behavior. This species offers a useful intermediate approach bridging the gap between in vitro studies and traditional mammalian models. Zebrafish offer great advantages of economy compared to their rodent counterparts, their complex brains and behavioral repertoire offer great translational potential relative to in vitro models. The development and validation of a variety of tests to measure behavior, including cognition, in zebrafish have set the stage for the use of this animal for behavioral pharmacology studies. This has led to research into the basic mechanisms of cognitive function as well as screening for potential cognition-improving drug therapies, among other lines of research. As with all models, zebrafish have limitations, which span pharmacokinetic challenges to difficulties quantifying behavior. The use, efficacy and limitations associated with a zebrafish model of cognitive function are discussed in this review, within the context of behavioral pharmacology.

Dopamine D1 and D2 receptor antagonism during development alters later behavior in zebrafish

&NA; This study sought to examine the long‐term behavioral impacts of dopamine D1 and D2 receptor antagonism during development in zebrafish (Danio rerio). Zebrafish embryos of both the AB* and 5D strains were exposed via immersion to either the D1 receptor antagonist SCH‐23,390 or the D2 receptor antagonist haloperidol, at either 0.5 or 1.5‐&mgr;M, from 5 h post‐fertilization to 5 days post‐fertilization. Aquarium water served as a control. Fish were then either tested as larvae on day 6 post‐fertilization on a light/dark locomotor assay, or were grown to adulthood and tested on a behavioral battery that included assays for novel environment exploration, startle habituation, social affiliation, and predator escape (AB* strain only). Overall, developmental exposure to dopamine D1 and D2 receptor antagonists caused clear effects in larval locomotor behavior, driving hyperactivity in dark phases and hypoactivity in light phases. Additionally, control fish from the two strains were significantly different from each other (p < 0.05) with the AB* fish being more active than SD during the dark periods of the test. In the adult behavioral battery, developmental exposure to 1.5‐&mgr;M of the D1 antagonist SCH‐23390 significantly reduced activity (p < 0.05) in the predator escape assay. Despite the fact that embryonic exposure to D1 and D2 receptor antagonists caused clear behavioral alterations in larval activity there were much more subtle effects persisting into adulthood. HighlightsZebrafish were exposed to dopamine antagonists during development.Exposures altered larval behavior in a light‐dark locomotor test.Adult locomotor behavior in a predator escape assay was also abnormal.Other adult behavioral assays did not appear affected.A second strain of fish was differentially affected in the larval assay.

Developmental exposure to an organophosphate flame retardant alters later behavioral responses to dopamine antagonism in zebrafish larvae

Human exposure to organophosphate flame retardants (OPFRs) is widespread, including pregnant women and young children with whom developmental neurotoxic risk is a concern. Given similarities of OPFRs to organophosphate (OP) pesticides, research into the possible neurotoxic impacts of developmental OPFR exposure has been growing. Building upon research implicating exposure to OP pesticides in dopaminergic (DA) dysfunction, we exposed developing zebrafish to the OPFR tris(1,3-dichloroisopropyl) phosphate (TDCIPP), during the first 5 days following fertilization. On day 6, larvae were challenged with acute administration of dopamine D1 and D2 receptor antagonists and then tested in a light-dark locomotor assay. We found that both developmental TDCIPP exposure and acute dopamine D1 and D2 antagonism decreased locomotor activity separately. The OPFR and DA effects were not additive; rather, TDCIPP blunted further D1 and D2 antagonist-induced decreases in activity. Our results suggest that TDCIPP exposure may be disrupting dopamine signaling. These findings support further research on the effects of OPFR exposure on the normal neurodevelopment of DA systems, whether these results might persist into adulthood, and whether they interact with OPFR effects on other neurotransmitter systems in producing the developmental neurobehavioral toxicity.