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Developmental exposure to either polycyclic aromatic hydrocarbons (PAHs) or heavy metals has been shown to cause persisting and overlapping neurobehavioral effects in animal models. However, interactions between these compounds have not been well characterized, despite their co-occurrence in a variety of environmental media. In two companion studies, we examined the effects of developmental exposure to cadmium (Cd) with or without co-exposure to prototypic PAHs benzo[a]pyrene (BaP, Exp. 1) or fluoranthene (FA, Exp. 2) using a developing zebrafish model. Zebrafish embryos were exposed to Cd (0-0.3 μM), BaP (0-3 μM), FA (0-1.0 μM), or binary Cd-PAH mixtures from 5 to 122 h post fertilization (hpf). In Exp. 1, Cd and BaP produced independent effects on an array of outcomes and interacting effects on specific outcomes. Notably, Cd-induced deficits in dark-induced locomotor stimulation were attenuated by BaP co-exposure in the larval motility test and BaP-induced hyperactivity was attenuated by Cd co-exposure in the adolescent novel tank test. Likewise, in Exp. 2, Cd and FA produced both independent and interacting effects. FA-induced increases on adult post-tap activity in the tap startle test were attenuated by co-exposure with Cd. On the predator avoidance test, FA- and 0.3 μM Cd-induced hyperactivity effects were attenuated by their co-exposure. Taken together, these data indicate that while the effects of Cd and these representative PAHs on zebrafish behavior were largely independent of one another, binary mixtures can produce sub-additive effects for some neurobehavioral outcomes and at certain ages. This research emphasizes the need for detailed risk assessments of mixtures containing contaminants of differing classes, and for clarity on the mechanisms which allow cross-class toxicant interactions to occur., Competing Interests: Declaration of competing interest The authors have no conflicts of interests for this article., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Per- and polyfluoroalkyl substances (PFAS) are commonly used as surfactants and coatings for industrial processes and consumer products. These compounds have been increasingly detected in drinking water and human tissue, and concern over their potential effects on health and development is growing. However, relatively little data are available for their potential impacts on neurodevelopment and the degree to which different compounds within this class may differ from one another in their neurotoxicity. The present study examined the neurobehavioral toxicology of two representative compounds in a zebrafish model. Zebrafish embryos were exposed to 0.1-100uM perfluorooctanoic acid (PFOA) or 0.01-1.0uM perfluorooctanesulfonic acid (PFOS) from 5 to 122 h post-fertilization. These concentrations were below threshold for producing increased lethality or overt dysmorphologies, and PFOA was tolerated at a concentration 100× higher than PFOS. Fish were maintained to adulthood, with behavioral assessments at 6 days, 3 months (adolescence) and 8 months of age (adulthood). Both PFOA and PFOS caused behavioral changes in zebrafish, but PFOS and PFOS produced strikingly different phenotypes. PFOA was associated with increased larval motility in the dark (100uM), and enhanced diving responses in adolescence (100uM) but not adulthood. PFOS was associated with a reversed light-dark response in the larval motility test (0.1-1uM), whereby the fish were more active in the light than the dark. PFOS also caused time-dependent changes in locomotor activity in the novel tank test during adolescence (0.1-1.0uM) and an overall pattern of hypoactivity in adulthood at the lowest concentration (0.01uM). Additionally, the lowest concentration of PFOS (0.01uM) reduced acoustic startle magnitude in adolescence, but not adulthood. These data suggest that PFOS and PFOA both produce neurobehavioral toxicity, but these effects are quite distinct from one another., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Adolescence is a period of risk for beginning tobacco addiction. Differential neural response to nicotine in adolescents vs. adults may help explain the increased vulnerability to nicotine self-administration seen with adolescent onset. We indexed the effects of acute nicotine ditartrate (0.4 mg/kg, salt weight) administration on dopamine (DA) and serotonin (5HT) as well as the DA metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) in several brain regions (nucleus accumbens, striatum and frontal cortex) of 6-week old (adolescent) and 10-week old (young adult) Sprague-Dawley rats. When nicotine was administered DA concentrations in the accumbens were significantly higher in adults than in adolescents, whereas there was no age-related difference without nicotine. However neither age group showed a significant effect of nicotine vs. age-matched controls. DA turnover in the accumbens was significantly greater in adolescent females in response to nicotine, but adult females did not show this effect and neither did males of either age group. DA turnover in the striatum was significantly higher in adolescents than adults regardless of nicotine administration. In the frontal cortex, there was a more complex effect. Without nicotine, adult male rats had higher DA concentrations than adolescent males, whereas female rats did not differ from adolescent to adult ages. When given nicotine, the age effect was no longer seen in males. However, there was not a significant effect of nicotine vs. age-matched controls in either age group. No age or nicotine effects were seen in females. 5HT in the accumbens was significantly increased by nicotine administration in adults but not in adolescents. Altered neural responsivity of adolescents to nicotine-induced neural effects particularly in accumbens DA and 5HT may be related to the increased nicotine dose concentrations they self-administer., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Polycyclic aromatic hydrocarbons (PAH) are products of incomplete combustion which are ubiquitous pollutants and constituents of harmful mixtures such as tobacco smoke, petroleum and creosote. Animal studies have shown that these compounds exert developmental toxicity in multiple organ systems, including the nervous system. The relative persistence of or recovery from these effects across the lifespan remain poorly characterized. These studies tested for persistence of neurobehavioral effects in AB* zebrafish exposed 5-120 h post-fertilization to a typical PAH, benzo[a]pyrene (BAP). Study 1 evaluated the neurobehavioral effects of a wide concentration range of BAP (0.02-10 μM) exposures from 5 to 120 hpf during larval (6 days) and adult (6 months) stages of development, while study 2 evaluated neurobehavioral effects of BAP (0.3-3 μM) from 5 to 120 hpf across four stages of development: larval (6 days), adolescence (2.5 months), adulthood (8 months) and late adulthood (14 months). Embryonic BAP exposure caused minimal effects on larval motility, but did cause neurobehavioral changes at later points in life. Embryonic BAP exposure led to nonmonotonic effects on adolescent activity (0.3 μM hyperactive, Study 2), which attenuated with age, as well as startle responses (0.2 μM enhanced, Study 1) at 6 months of age. Similar startle changes were also detected in Study 2 (1.0 μM), though it was observed that the phenotype shifted from reduced pretap activity to enhanced posttap activity from 8 to 14 months of age. Changes in the avoidance (0.02-10 μM, Study 1) and approach (reduced, 0.3 μM, Study 2) of aversive/social cues were also detected, with the latter attenuating from 8 to 14 months of age. Fish from study 2 were maintained into aging (18 months) and evaluated for overall and tissue-specific oxygen consumption to determine whether metabolic processes in the brain and other target organs show altered function in late life based on embryonic PAH toxicity. BAP reduced whole animal oxygen consumption, and overall reductions in total basal, mitochondrial basal, and mitochondrial maximum respiration in target organs, including the brain, liver and heart. The present data show that embryonic BAP exposure can lead to neurobehavioral impairment across the life-span, but that these long-term risks differentially emerge or attenuate as development progresses., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Edward D. Levin reports financial support was provided by National Institute of Environmental Health Sciences. Edward D. levin reports a relationship with National Institute of Environmental Health Sciences that includes: funding grants. None, (Copyright © 2022 Elsevier Inc. All rights reserved.)

A sequence of different classes of synthetic insecticides have been used over the past 70 years. Over this period, the widely-used organochlorines were eventually replaced by organophosphates, with dichlorodiphenyltrichloroethane (DDT) and chlorpyrifos (CPF) as the principal prototypes. Considerable research has characterized the risks of DDT and CPF individually, but little is known about the toxicology of transitioning from one class of insecticides to another, as has been commonplace for agricultural and pest control workers. This study used adult zebrafish to investigate neurobehavioral toxicity following 5-week chronic exposure to either DDT or CPF, to or their sequential exposure (DDT for 5 weeks followed by CPF for 5 weeks). At the end of the exposure period, a subset of fish were analyzed for brain cholinesterase activity. Behavioral effects were initially assessed one week following the end of the CPF exposure and again at 14 months of age using a behavioral test battery covering sensorimotor responses, anxiety-like functions, predator avoidance and social attraction. Adult insecticide exposures, individually or sequentially, were found to modulate multiple behavioral features, including startle responsivity, social approach, predator avoidance, locomotor activity and novel location recognition and avoidance. Locomotor activity and startle responsivity were each impacted to a greater degree by the sequential exposures than by individual compounds, with the latter being pronounced at the early (1-week post exposure) time point, but not 3-4 months later in aging. Social approach responses were similarly impaired by the sequential exposure as by CPF-alone at the aging time point. Fleeing responses in the predator test showed flee-enhancing effects of both compounds individually versus controls, and no additive impact of the two following sequential exposure. Each compound was also associated with changes in recognition or avoidance patterns in a novel place recognition task in late adulthood, but sequential exposures did not enhance these phenotypes. The potential for chemical x chemical interactions did not appear related to changes in CPF metabolism to the active oxon, as prior DDT exposure did not affect the cholinesterase inhibition resulting from CPF. This study shows that the effects of chronic adult insecticide exposures may be relevant to behavioral health initially and much later in life, and that the effects of sequential exposures may be unpredictable based on their constituent exposures., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Organophosphate (OP) compounds comprise one of the most widely used classes of insecticides worldwide. OPs have been shown to have negative human health impacts, particularly developmental neurotoxicity. However, neurotoxic impacts in later adulthood and during the aging process are relatively uncharacterized. The present study examined diazinon (DZN), an OP, to determine the neurobehavioral consequences, in addition to mitochondrial dysfunction on a macroscale (whole organism basal respiration) and on a microscale (whole organ mitochondrial respiration), using zebrafish (ZF) as a model. One group of 14-month-old adult ZF were exposed acutely as adults (0.4, 1.25, and 4.0 μM) for five days and tested as adults, and another group was exposed developmentally 5-120 h post-fertilization (70, 210, and 700 nM) and tested at larval, adolescent, adult, and aging life stages. ZF exposed acutely as adults did not display many significant neurobehavioral impacts or mitochondrial dysfunction. Conversely, the embryonically exposed ZF showed altered behavioral functions at each stage of life which emerged and attenuated as fish transitioned from each developmental stage to the next. Mitochondrial oxygen consumptions measurement results for developmentally DZN exposed ZF showed significant increases in the low and middle dose groups in organs such as the brain and testes. Overall, there is an indication that early developmental exposure to DZN had continuing adverse neurobehavioral and cellular consequences throughout their lives well into adulthood and aging periods., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Vitamin D receptor (VDR) signaling is important for optimal neurobehavioral development. Disruption of VDR signaling by environmental toxicants during early development might contribute to the etiology of behavioral dysfunction. In the current set of studies, we examined ten compounds known to affect VDR function in vitro for neurobehavioral effects in vivo in zebrafish. Zebrafish embryos were exposed to concentrations of the compounds in their water during the first 5 days post-fertilization. On day 5, the embryos were tested in an alternating light-dark locomotor assay using a computerized video tracking system. We found that most of the compounds produced significant changes in locomotor behavior in exposed zebrafish larvae, although the direction of the effect (i.e., hypo- or hyperactivity) and the sensitivity of the effect to changes in illumination condition varied across the compounds. The nature of the behavioral effects generally corresponded to the effects these compounds have been shown to exert on VDR. These studies lay a foundation for further investigation to determine whether behavioral dysfunction persists into adulthood and if so which behavioral functions are affected. Zebrafish can be useful for screening compounds identified in high throughput in vitro assays to provide an initial test for how those compounds would affect construction and behavioral function of a complex nervous system, helping to bridge the gap between in vitro neurotoxicity assays and mammalian models for risk assessment in humans., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Response variability across the lifespan is an important consideration in toxicology and risk assessment, and the toxic effects of drugs and chemicals during adolescence need more research. This paper summarizes a workshop presented in March 2019, at the Society of Toxicology Annual Meeting in Baltimore, Maryland, that brought together experts in research on drug dependence and toxicity related to nicotine, cannabis, cocaine, and other illicit drugs during adolescence. The goal of the workshop was to address the following issues: (1) Do the effects of adolescent exposure differ from the same exposure in adults? (2) Are there unique biological markers of adolescent brain development? If so, what are they and how reliable are they? (3) Since multiple factors influence substance use disorder, can we disentangle risk factors for abuse and/or toxicity? What are the underlying biological susceptibilities that lead to dependence and neurotoxicity? What are the social, psychosocial and environmental factors that contribute to abuse susceptibilities? This paper reviews drug policy and national trends in adolescent substance use; the public health consequences of e-cigarettes; rat models of adolescent-onset nicotine self-administration and persisting effects of gestational nicotine; sex-dependent effects of delta-9-tetrahydrocannabinol on adolescent brain-behavior relationships; and translational approaches for identifying adolescent risk factors for transition to drug dependence. There is strong evidence that drug exposure prior to adulthood has longer lasting effects on behavior and the underlying neural circuitry. These effects, which are sex-dependent and influenced by stress, may be candidates as predictors of adolescent vulnerability. A major challenge to determining if adolescents have a unique susceptibility to dependence is whether and to what extent the human data allow distinction between the increased risk due to biological immaturity, an underlying biological susceptibility to dependence, or psychosocial and environmental factors for substance dependence. Factors important to consider for development of animal models include the timing and pattern of exposure as it relates to adolescence; age of assessment, and direct comparison with similar effects following exposures to adults to demonstrate that these effects are unique to adolescence. Here we provide a roadmap for further research into what makes adolescent brain development unique., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Farmers are often chronically exposed to insecticides, which may present health risks including increased risk of neurobehavioral impairment during adulthood and across aging. Experimental animal studies complement epidemiological studies to help determine the cause-and-effect relationship between chronic adult insecticide exposure and behavioral dysfunction. With the zebrafish model, we examined short and long-term neurobehavioral effects of exposure to either an organochlorine insecticide, dichlorodiphenyltrichloroethane (DDT) or an organophosphate insecticide chlorpyrifos (CPF). Adult fish were exposed continuously for either two or 5 weeks (10-30 nM DDT, 0.3-3 μM CPF), with short- and long-term effects assessed at 1-week post-exposure and at 14 months of age respectively. The behavioral test battery included tests of locomotor activity, tap startle, social behavior, anxiety, predator avoidance and learning. Long-term effects on neurochemical indices of cholinergic function were also assessed. Two weeks of DDT exposure had only slight effects on locomotor activity, while a longer five-week exposure led to hypoactivity and increased anxiety-like diving responses and predator avoidance at 1-week post-exposure. When tested at 14 months of age, these fish showed hypoactivity and increased startle responses. Cholinergic function was not found to be significantly altered by DDT. The two-week CPF exposure led to reductions in anxiety-like diving and increases in shoaling responses at the 1-week time point, but these effects did not persist through 14 months of age. Nevertheless, there were persistent decrements in cholinergic presynaptic activity. A five-week CPF exposure led to long-term effects including locomotor hyperactivity and impaired predator avoidance at 14 months of age, although no effects were apparent at the 1-week time point. These studies documented neurobehavioral effects of adult exposure to chronic doses of either organochlorine or organophosphate pesticides that can be characterized in zebrafish. Zebrafish provide a low-cost model that has a variety of advantages for mechanistic studies and may be used to expand our understanding of neurobehavioral toxicity in adulthood, including the potential for such toxicity to influence behavior and development during aging., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Dr. Theodore Slotkin has received consultant income in the past three years from Pardieck Law (Seymour, IN), Gjording Fouser (Boise, ID), Thorsnes Bartolotta McGuire (San Diego, CA), Walgreen Co. (Deerfield, IL) and Cracken Law (Dallas, TX). The other authors have no conflicts to report., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Studies of intergenerational effects of parental chemical exposure have principally focused on maternal exposure, particularly for studies of adverse neurobehavioral consequences on the offspring. Maternal nicotine exposure has long been known to cause adverse neurobehavioral effects on the offspring. However, paternal toxicant exposure has also been found to cause neurobehavioral toxicity in their offspring. Recent work suggests that paternal nicotine exposure can have epigenetic effects, although it remains unclear whether such changes lead to neurobehavioral effects. In the current study, we investigated the effects of paternal nicotine exposure on neurobehavioral development of their offspring. Male Sprague-Dawley rats were exposed to 0 or 2 mg/kg/day nicotine (sc) for 56 consecutive days with two consecutive 2ML4 osmotic minipumps. Following treatment, these males were mated with drug-naïve female rats. Offspring of both sexes were tested in a behavioral battery to assess locomotion, emotional function and cognition. Paternal nicotine exposure did not impact offspring viability, health or growth. However, behavioral function of the offspring was significantly altered by paternal nicotine exposure. Male offspring with paternal nicotine exposure exhibited locomotor hyperactivity in the Figure-8 apparatus when tested during adolescence. When retested in adulthood and regardless of sex, offspring of the nicotine exposed father showed significantly reduced habituation of locomotor activity over the course of the session. Compared to controls, female offspring of nicotine-exposed fathers showed significantly reduced response latency in the radial arm maze test. In addition to locomotor hyperactivity, the offspring of nicotine-exposed fathers also showed significantly diminished habituation in the novel object recognition test. These results indicate that chronic paternal nicotine exposure can impact the behavior of offspring, producing locomotor hyperactivity and impaired habituation., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Developmental neurotoxicity of a wide variety of toxicants mediated via maternal exposure during gestation is very well established. In contrast, the impacts of paternal toxicant exposure on offspring neurobehavioral function are much less well studied. A vector for paternal toxicant exposure on development of his offspring has been identified. Sperm DNA can be imprinted by chemical exposures of the father. Most but not all of the epigenetic marks in sperm are reprogrammed after fertilization. The persisting epigenetic marks can lead to abnormal genetic expression in the offspring. We have found that paternal delta-9-tetrohydrocannabinol (THC) exposure in rats causes changes in methylation of sperm (Murphy et al., 2018). This is similar to cannabis-associated changes in sperm DNA methylation we found in human males who smoke cannabis (Murphy et al., 2018). In the current study we investigated the intergeneration effects of THC exposure of young adult male rats (0 or 2 mg/kg/day orally for 12 days) to the neurobehavioral development of their offspring. This paternal THC exposure was not found to significantly impact the clinical health of the offspring, including litter size, sex ratio, pup birth weight, survival and growth. However, it did cause a long-lasting significant impairment in attentional performance in the offspring relative to controls when they were tested in adulthood. There was also a significant increase in habituation of locomotor activity in the adult offspring of the males exposed to THC prior to mating. This study shows that premating paternal THC exposure even at a modest dose for a brief period can cause deleterious long-term behavioral effects in the offspring, notably significant impairment in an operant attention task. Further research should be conducted to determine the degree to which this type of risk is seen in humans and to investigate the mechanisms underlying these effects and possible treatments to ameliorate these long-term adverse behavioral consequences of paternal THC exposure., (Copyright © 2019. Published by Elsevier Inc.)

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 D 1 and D 2 receptor antagonists and then tested in a light-dark locomotor assay. We found that both developmental TDCIPP exposure and acute dopamine D 1 and D 2 antagonism decreased locomotor activity separately. The OPFR and DA effects were not additive; rather, TDCIPP blunted further D 1 and D 2 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., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Sex differences in cognitive processing and function have been documented in human and animal studies. Females have been found to perform better than males on non-spatial memory tasks, while males tend to outperform females on spatial memory tasks. The neural mechanisms underlying these sexual dimorphisms are unclear. However, it is known that the cholinergic system is critically involved in memory processes, and there are notable differences between males and females in cholinergic system function and receptor expression. In particular, there are sex differences in the processing of information in the frontal cortex and hippocampus. In this study, we examined the roles of muscarinic and nicotinic acetylcholine receptors in the medial frontal cortex (MfC) and ventral hippocampus (VH) on spatial working memory in male and female rats. Local infusions of scopolamine (SCOP) and mecamylamine (MEC) (10, 20, 50 μg/side) were used to antagonize these receptors in each respective brain region during performance in the 16-arm radial arm maze. Infusions of SCOP into the VH caused a significant increase in memory errors in female rats, but had no significant effect on males, while infusions of MEC into the VH had no effect on either sex. Infusions of both SCOP (50 μg/side) and MEC (20 μg/side) into the MfC caused working memory impairments in both sexes. These results show that muscarinic acetylcholine receptors in the VH are differentially vulnerable to spatial memory impairments in females. Ventral hippocampal muscarinic acetylcholine receptors may play a key role in male-female differences in spatial memory., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Dopaminergic signaling has long been known to be a critical factor in nicotine addiction, as well as other drugs of abuse. Dopaminergic projections from the VTA to the nucleus accumbens and prefrontal cortex have been well established to be critical to the reinforcing effects of these drugs. However, other projections of dopamine neurons are likely to have significant roles in this process. Also, the relative contributions of D1 and D2 dopamine receptors in drug addiction and its treatment remain to be fully understood. In this study, we examined the effects of blocking D1 and D2 receptors in the nucleus accumbens shell (AcS), anterior cingulate cortex (ACC), and parietal association cortex (PtA) on nicotine self-administration in rats. Female Sprague-Dawley rats were fitted with jugular catheters and allowed to self-administer nicotine (0.03 mg/kg/infusion) on an FR1 schedule. Rats were fitted with bilateral infusion cannulae to allow infusion of D1 or D2 antagonists (SCH-23390 or haloperidol) into each targeted brain area. Acute local infusions of SCH-23390 (1-4 μg/side) into the AcS and PtA significantly reduced nicotine self-administration by up to 75%. SCH-23390 infusion into the ACC was less effective with only suggestive non-significant reductions of nicotine self-administration. Acute, local infusions of haloperidol (0.5-2 μg/side) in any of the brain regions targeted did not have significant effects on nicotine self-administration. These results demonstrate a more significant role for D1 receptor mechanisms in the process of nicotine reinforcement and help provide a more detailed neuroanatomic map of nicotine dependence in the brain., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Cognitive dysfunction has been found in epidemiological studies to be among the most sensitive impairments associated with developmental exposure to a variety of environmental contaminants from heavy metals to polyhalogenated hydrocarbons and pesticides. These chemicals have been also shown to impair cognitive function after developmental exposure in experimental animal models. The radial-arm maze (RAM) has proven to be a sensitive and reliable way to assess both learning and memory in a variety of species, most often in rats and mice. The RAM is a very adaptable test method that takes advantage of rodents' instinct to explore new places in the environment to forage. That is, rodents do not need to be trained to run through the maze; they will normally do this from the initial session of testing. Training with differential reinforcement for arm choices provides a more rigorous test of learning and memory. The RAM is quite adaptable for assessing various aspects of cognition. Although the RAM has been mostly used to assess spatial learning and memory, it can be configured to assess non-spatial memory as well. Both working and reference memory can be easily distinguished. The RAM can be run with both appetitive (food reinforced) and aversive (water escape) motivators. The RAM has been found to be sensitive to a wide variety of developmental toxicants including heavy metals such as mercury and pesticides such as chlorpyrifos. There is an extremely rich literature especially with rats showing the effects of many types of brain lesions and drug effects so that the participation of a wide variety of neural systems in RAM performance is known. These systems, notably the hippocampus and frontal cortex, and acetylcholine and glutamate neurotransmitter systems, are the same neural systems that have been shown in humans to be critical for learning and memory. This considerably aids the interpretation of neurobehavioral toxicity studies., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Polybrominated diphenyl ethers (PBDEs) are persistent organic pollutants that are widely detected in the environment, biota, and humans. In mammals, PBDEs can be oxidatively metabolized to form hydroxylated polybrominated diphenyl ethers (OH-BDEs). While studies have examined behavioral deficits or alterations induced by exposure to PBDEs in both rodents and fish, no study to date has explored behavioral effects from exposure to OH-BDEs, which have been shown to have greater endocrine disrupting potential compared to PBDEs. In the present study, zebrafish (Danio rerio) were exposed during embryonic and larval development (0-6 days post fertilization, dpf) to a PBDE metabolite, 6-hydroxy, 2,2',4,4' tetrabromodiphenyl ether (10-50 nM) and then examined for short and long-term behavioral effects. Exposed zebrafish tested as larvae (6 dpf) showed an altered swimming response to light-dark transitions, exhibiting hypoactivity in light periods compared to control fish. When fish exposed from 0-6 dpf were tested as juveniles (45 dpf), they showed an increased fear response and hyperactivity in response to tests of novel environment exploration and habituation learning. These results demonstrate that early life exposure to a PBDE metabolite can have immediate or later life (more than a month after exposure) effects on activity levels, habituation, and fear/anxiety., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Cigarette smoke has been associated with a number of pathologies; however, the mechanisms leading to developmental effects are yet to be fully understood. The zebrafish embryo is regarded as a 'bridge model'; however, not many studies examined its applicability to cigarette smoke toxicity. This study examined the effects of total particulate matter (TPM) from 3R4F reference cigarettes on the early development of zebrafish (Danio rerio). Zebrafish embryos were exposed to two concentrations of TPM (0.4 and 1.4 μg/mL equi-nicotine units) or nicotine at equivalent doses. The exposures began at 2h post-fertilization (hpf) and lasted until 96 hpf. Several physiological parameters were assessed during or after the exposure. We show that TPM increased mortality, delayed hatching, and increased the incidence of deformities in zebrafish. TPM exposure also increased the incidence of hemorrhage and disrupted the angiogenesis of the major vessels in the brain. Moreover, TPM exposure reduced the larval body length, decreased the heart rate, and reduced the metabolic rate. Biomarkers of xenobiotic metabolism and oxidative stress were also affected. TPM-exposed zebrafish also differed behaviorally: at 24 hpf the embryos had a higher frequency of spontaneous contractions and at 144 hpf the larvae displayed swimming hyperactivity. This study demonstrates that TPM disrupts several aspects of early development in zebrafish. The effects reported for TPM were not attributable to nicotine, since embryos treated with nicotine alone did not differ significantly from the control group. Collectively, our work illustrates the utility of zebrafish as an alternative model to evaluate the toxic effects of cigarette smoke constituents., (Copyright © 2015 Elsevier Inc. All rights reserved.)

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., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Tobacco smoking is characterized by repeated self-administration of nicotine by placing the cigarette in the mouth. The repeated hand-to-mouth self-administration is essentially a consummatory act. We recently developed a paradigm in which rats lick one of two spouts to trigger intravenous (IV) delivery of nicotine, which combines a consummatory act with rapid delivery of nicotine to model the act of tobacco smoking. We have found that rats will lick hundreds of times per nicotine infusion. In the current study, using the operant licking nicotine self-administration model with young adult Sprague-Dawley rats (0.03mg/kg/infusion of nicotine), we tested the effect of antagonists of H1 histamine receptors pyrilamine, serotonin (5HT) type 2 receptors ketanserin and N-methyl-d-aspartate (NMDA) glutamate receptors with d-cycloserine as well as an agonist of 5HT2c receptors lorcaserin, in dose ranges that we have found in previous studies to significantly reduce IV nicotine self-administration with the operant lever press operand. The H1 antagonist pyrilamine significantly reduced operant licking for nicotine self-administration. Pyrilamine caused significant reductions in the operant licking paradigm at lower doses (10 and 20mg/kg) than those we previously observed to affect responding in the operant lever press paradigm. In contrast, the 5HT2A and C antagonist ketanserin did not show an effect of reducing nicotine self-administration in the same dose range we had found in a previous study to significantly reduce operant lever press nicotine self-administration. The 5HT2C agonist lorcaserin significantly decreased nicotine self-administration in the licking paradigm at the same dose threshold as with lever press responding. The NMDA glutamate partial agonist d-cycloserine did not produce any change in nicotine self-administration with the licking operand, in contrast to its effect on the classic lever-pressing task. The rat model incorporating consummatory aspects of tobacco addiction can provide distinct and potentially more relevant information concerning possible new avenues of treatment to combat tobacco addiction., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Although nicotine accounts for a great deal of the neurodevelopmental damage associated with maternal smoking or second-hand exposure, tobacco smoke contains thousands of potentially neurotoxic compounds. We used PC12 cells, a standard in vitro model of neurodifferentiation, to compare tobacco smoke extract (TSE) to nicotine, matching TSE exposure (with its inherent nicotine content) to parallel concentrations of nicotine, or to benzo[a]pyrene, a tobacco combustion product. TSE promoted the transition from cell replication to differentiation, resulting in fewer, but larger cells with greater neurite extension. TSE also biased differentiation into the dopaminergic versus the cholinergic phenotype, evidenced by an increase in tyrosine hydroxylase activity but not choline acetyltransferase. Nicotine likewise promoted differentiation at the expense of cell numbers, but its effect on growth and neurite extension was smaller than that of TSE; furthermore, nicotine did not promote the dopaminergic phenotype. Benzo[a]pyrene had effects opposite to those of TSE, retarding neurodifferentiation, which resulted in higher cell numbers, smaller cells, reduced neurite information, and impaired emergence of both dopaminergic and cholinergic phenotypes. Our studies show that the complex mixture of compounds in tobacco smoke exerts direct effects on neural cell replication and differentiation that resemble those of nicotine in some ways but not others, and most importantly, that are greater in magnitude than can be accounted for from just the nicotine content of TSE. Thus, fetal tobacco smoke exposure, including lower levels associated with second-hand smoke, could be more injurious than would be anticipated from measured levels of nicotine or its metabolites., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Glucocorticoids are the consensus treatment given in preterm labor and are also elevated by maternal stress; organophosphate exposures are virtually ubiquitous, so human developmental coexposures to these two agents are common. This study explores how prenatal dexamethasone exposure modifies the neurobehavioral teratology of chlorpyrifos, one of the most widely used organophosphates. We administered dexamethasone to pregnant rats on gestational days 17-19 at a standard therapeutic dose (0.2 mg/kg); offspring were then given chlorpyrifos on postnatal days 1-4, at a dose (1 mg/kg) that produces barely-detectable (<10%) inhibition of brain cholinesterase activity. Dexamethasone did not alter brain chlorpyrifos concentrations, nor did either agent alone or in combination affect brain thyroxine levels. Assessments were carried out from adolescence through adulthood encompassing T-maze alternation, Figure 8 maze (locomotor activity, habituation), novelty-suppressed feeding and novel object recognition tests. For behaviors where chlorpyrifos or dexamethasone individually had small effects, the dual exposure produced larger, significant effects that reflected additivity (locomotor activity, novelty-suppressed feeding, novel object recognition). Where the individual effects were in opposite directions or were restricted to only one agent, we found enhancement of chlorpyrifos' effects by prenatal dexamethasone (habituation). Finally, for behaviors where controls displayed a normal sex difference in performance, the combined treatment either eliminated or reversed the difference (locomotor activity, novel object recognition). Combined exposure to dexamethasone and chlorpyrifos results in a worsened neurobehavioral outcome, providing a proof-of-principle that prenatal glucocorticoids can create a subpopulation with enhanced vulnerability to environmental toxicants., (© 2013.)

Zebrafish provide a powerful model of the impacts of embryonic toxicant exposure on neural development that may result in long-term behavioral dysfunction. In this study, zebrafish embryos were treated with 1.5mM strychnine for short embryonic time windows to induce transient changes in inhibitory neural signaling, and were subsequently raised in untreated water until adulthood. PCR analysis showed indications that strychnine exposure altered expression of some genes related to glycinergic, GABAergic and glutamatergic neuronal synapses during embryonic development. In adulthood, treated fish showed significant changes in swimming speed and tank diving behavior compared to controls. Taken together, these data show that a short embryonic exposure to a neurotoxicant can alter development of neural synapses and lead to changes in adult behavior., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Developmental exposure of rats to the pesticide chlorpyrifos (CPF) causes persistent neurobehavioral impairment. In a parallel series of studies with zebrafish, we have also found persisting behavioral dysfunction after developmental CPF exposure. We have developed a battery of measures of zebrafish behavior, which are reliable and sensitive to toxicant-induced damage. This study determined the critical duration of developmental CPF exposure for causing persisting neurobehavioral effects. Tests of sensorimotor response (tap startle response and habituation), stress response (novel tank diving test) and learning (3-chamber tank spatial discrimination) were conducted with adult zebrafish after early developmental CPF exposure. The CPF exposure level was 100 ng/ml with durations of 0-1, 0-2, 0-3, 0-4 and 0-5 days after fertilization. Developmental CPF exposure had persisting behavioral effects in zebrafish tested as adults. In the tactile startle test, CPF exposed fish showed decreased habituation to startle and a trend toward increased overall startle response. In the novel tank exploration test, exposed fish showed decreased escape diving response and increased swimming activity. In the 3-chamber learning test, the 0-5 day CPF exposure group had a significantly lower learning rate. There was evidence for persisting declines in brain dopamine and norepinepherine levels after developmental CPF exposure. In all of the measures the clearest persistent effects were seen in fish exposed for the full duration of five days after fertilization. In a follow-up experiment there were some indications for persisting behavioral effects after exposure during only the later phase of this developmental window. This study demonstrated the selective long-term neurobehavioral alterations caused by exposure to CPF in zebrafish. The zebrafish model can facilitate the determination of the molecular mechanisms underlying long-term neurobehavioral impairment after developmental toxicant exposure., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Zebrafish are increasingly used for developmental neurotoxicity testing because early embryonic events are easy to visualize, exposures are done without affecting the mother and the rapid development of zebrafish allows for high throughput testing. We used zebrafish to examine how exposures to three different organophosphorus pesticides (chlorpyrifos, diazinon and parathion) over the first five days of embryonic and larval development of zebrafish affected their survival, acetylcholinesterase (AChE) activity and behavior. We show that at non-lethal, equimolar concentrations, chlorpyrifos (CPF) is more effective at equimolar concentrations than diazinon (DZN) and parathion (PA) in producing AChE inhibition. As concentrations of DZN and PA are raised, lethality occurs before they can produce the degree of AChE inhibition observed with CPF at 300 nM. Because of its availability outside the mother at the time of fertilization, zebrafish provides a complementary model for studying the neurotoxicity of very early developmental exposures., (Copyright © 2011 Elsevier Inc. All rights reserved.)

As more adults take the stimulant medication methylphenidate to treat attention deficit hyperactivity disorder (ADHD) residual type, the risk arises with regard to exposure during early development if people taking the medication become pregnant. We studied the neurobehavioral effects of methylphenidate in zebrafish. Zebrafish offer cellular reporter systems, continuous visual access and molecular interventions such as morpholinos to help determine critical mechanisms underlying neurobehavioral teratogenicity. Previously, we had seen that persisting neurobehavioral impairment in zebrafish with developmental chlorpyrifos exposure was associated with disturbed dopamine systems. Because methylphenidate is an indirect dopamine agonist, it was thought that it might also cause persistent behavioral impairment after developmental exposure. Zebrafish embryos were exposed to the ADHD stimulant medication methylphenidate 0-5 days post fertilization (12.5-50mg/l). They were tested for long-term behavioral effects as adults. Methylphenidate exposure (50mg/l) caused significant increases in dopamine, norepinepherine and serotonin on day 6 but not day 30 after fertilization. In the novel tank diving test of predatory avoidance developmental methylphenidate (50mg/l) caused a significant reduction in the normal diving response. In the three-chamber spatial learning task early developmental methylphenidate (50mg/l) caused a significant impairment in choice accuracy. These data show that early developmental exposure of zebrafish to methylphenidate causes a long-term impairment in neurobehavioral plasticity. The identification of these functional deficits in zebrafish enables further studies with this model to determine how molecular and cellular mechanisms are disturbed to arrive at this compromised state., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Environmental silver exposures are increasing due to the use of silver nanoparticles, which exert antimicrobial actions by releasing Ag+, a suspected developmental neurotoxicant. We evaluated the long-term neurochemical and behavioral effects of embryonic Ag+ exposure in zebrafish at concentrations that had no overt effects on morphological development. Exposure to 0.03, 0.1 or 0.3 μM Ag+ during the first five days post-fertilization caused elevations in both dopamine and serotonin turnover in the adult zebrafish brain without affecting basal neurotransmitter levels. Consistent with these synaptic effects, Ag+-exposed fish showed a faster acquisition of avoidance behavior in a three-chamber test apparatus, without any change in response latency or overall swimming ability. Our results indicate that Ag+ is a developmental neurotoxicant that causes persistent neurobehavioral effects, reinforcing health concerns about Ag+ released from silver nanoparticles., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Chlorpyrifos (CPF) an organophosphate pesticide causes persisting behavioral dysfunction in rat models when exposure is during early development. In earlier work zebrafish were used as a complementary model to study mechanisms of CPF-induced neurotoxicity induced during early development. We found that developmental (first five days after fertilization) chlorpyrifos exposure significantly impaired learning in zebrafish. However, this testing was time and labor intensive. In the current study we tested the hypothesis that persisting effects of developmental chlorpyrifos could be detected with a brief automated assessment of startle response and that this behavioral index could be used to help determine the neurobehavioral mechanisms for persisting CPF effects. The swimming activity of adult zebrafish was assessed by a computerized video-tracking device after a sudden tap to the test arena. Ten consecutive trials (1/min) were run to determine startle response and its habituation. Additionally, habituation recovery trials were run at 8, 32 and 128 min after the end of the initial trial set. CPF-exposed fish showed a significantly (p<0.025) greater overall startle response during the 10-trial session compared to controls (group sizes: Control N=40, CPF N=24). During the initial recovery period (8 min) CPF-exposed fish showed a significantly (p<0.01) greater startle response compared to controls. To elucidate the contributions of nicotinic and muscarinic acetylcholine receptors to developmental CPF-mediated effects, the effects of developmental nicotine and pilocarpine exposure throughout the first five days after fertilization were determined. Developmental nicotine and pilocarpine exposure significantly increased startle response, though nicotine (group sizes: Control N=32, 15 mM N=12, 25 mM N=20) was much more potent than pilocarpine (group sizes: Control N=20, 100 microM N=16, 1000 microM N=12). Neither was as potent as CPF for developmental exposure increasing startle response in adulthood. Lastly, developmental CPF exposure decreased dopamine and serotonin levels and increased transmitter turnover in developing zebrafish larvae (N=4 batches of 50 embryos/treatment). Only the decline in dopamine concentrations persisted into adulthood (group sizes: Control N=14, CPF N=13). This study shows that a quick automated test of startle can detect persisting neurobehavioral impairments caused by developmental exposure to CPF. This may be helpful in screening for persisting neurobehavioral defects from a variety of toxicants., (Copyright 2009. Published by Elsevier Inc.)

The consequences of exposure to developmental neurotoxicants are influenced by environmental factors. In the present study, we examined the role of dietary fat intake. We administered parathion to neonatal rats and then evaluated whether a high-fat diet begun in adulthood could modulate the persistent effects on 5HT and DA systems. Neonatal rats received parathion on postnatal days 1-4 at 0.1 or 0.2 mg/kg/day, straddling the cholinesterase inhibition threshold. In adulthood, half the animals in each exposure group were given a high-fat diet for 8 weeks. We assessed 5HT and DA concentrations and turnover in brain regions containing their respective cell bodies and projections. In addition, we monitored 5HT1A and 5HT2 receptor binding and the concentration of 5HT presynaptic transporters. Neonatal parathion exposure evoked widespread increases in neurotransmitter turnover, indicative of presynaptic hyperactivity, further augmented by 5HT receptor upregulation. In control rats, consumption of a high-fat diet recapitulated many of the changes seen with neonatal parathion exposure; the effects represented convergent mechanisms, since the high-fat diet often obtunded further increases caused by parathion. Neonatal parathion exposure causes lasting hyperactivity of 5HT and DA systems accompanied by 5HT receptor upregulation, consistent with "miswiring" of neuronal projections. A high-fat diet obtunds the effect of parathion, in part by eliciting similar changes itself. Thus, dietary factors may produce similar synaptic changes as do developmental neurotoxicants, potentially contributing to the increasing incidence of neurodevelopmental disorders.

Nicotinic receptor decreases in the frontal cortex and hippocampus are important mediators of cognitive impairment in both schizophrenia and Alzheimer's disease. Drug treatments for these diseases should take into account the impacts of compromised brain function on drug response. This study investigated the impact of compromised nicotinic receptor activity in the frontal cortex in rats on memory function. Since both Alzheimer's disease and schizophrenia can involve psychosis, antipsychotic drugs are often given. The impacts of antipsychotic drugs on cognitive function have been found to be quite variable. It is the hypothesis of this and previous studies that the cognitive effects of antispychotic drugs on cognitive function depend on the integrity of brain systems involved in cognition. Previously in studies of the hippocampus, we found that chronic inhibition of beta2-containing nicotinic receptors with dihydro-beta-erythrodine (DHbetaE) impaired working memory and that this effect was attenuated by the antipsychotic drug clozapine. In contrast, chronic hippocampal alpha7 nicotinic receptor blockade with methyllycaconitine (MLA) potentiated the clozapine-induced memory impairment which is seen in rats without compromised nicotinic receptor activity. The current study determined medial frontal cortical alpha7 and beta2-containing nicotinic receptor involvement in memory and the interactions with antipsychotic drug therapy with clozapine. Chronic DHbetaE and MLA infusion effects and interactions with systemic clozapine were assessed in female rats tested for memory on the radial-arm maze. Antipsychotic drug interactions with chronic systemic nicotine were investigated because nicotinic procognitive treatment has been proposed. The same local infusion DHbetaE dose that impaired memory with hippocampal infusion did not impair memory when infused in the medial frontal cortex. Frontal DHbetaE infusion potentiated clozapine-induced memory impairment, whereas previously the memory impairment caused by hippocampal DHbetaE infusion was attenuated by clozapine. Frontal cortical MLA infusions at a dose that previously was found to potentiate the clozapine-induced memory impairment with hippocampal infusion had no significant effect when infused into the medial frontal cortex. The location and subtype of nicotinic receptor underactivity are critical determinates for clozapine effects on memory. Patients with hippocampal beta2-containing nicotinic receptor loss may be well treated with clozapine therapy, while those with frontal cortical beta2-containing receptor loss may have a potentiated memory impairment caused by clozapine.

It is well established that nicotinic systems in the brain are critically involved in attentional processes in both animals and humans. The current study assessed the effects of a novel nicotinic alpha7 receptor partial agonist and 5-HT3 antagonist, R3487/MEM3454 (also referred to as R3487 or MEM 3454) on sustained attention in rats performing an operant visual signal detection task. The effects of R3487/MEM3454 were compared to those of the acetylcholinesterase inhibitor/nicotinic alpha7 allosteric positive modulator galanthamine. Adult female Sprague-Dawley rats were injected subcutaneously with R3487/MEM3454 (0.03, 0.1, 0.15, 0.3 and 0.6 mg/kg), galanthamine (0.25, 0.5, 1, 2 mg/kg) or vehicle 30 min before the attentional test. In the second study, the time-dependent effects of R3487/MEM3454 were assessed by injecting the compound (0.6 mg/kg, s.c.) at different pretreatment intervals (30, 60 or 90 min) before the start of the attentional task. Our results show a significant dose-effect for R3487/MEM3454 on percent hit accuracy performance without any significant alteration on percent correct rejection performance. In the time-dependent test, R3487/MEM3454 significantly increased the percent hit accuracy performance when animals were injected 60 min before the start of the attentional task. Administration of galanthamine failed to significantly increase percent hit accuracy performance and increasing the dose of galanthamine produced a decrease in percent correct rejection performance. The present findings with R3487/MEM3454 suggest that nicotinic alpha7 receptors and/or 5-HT3 receptors may play an important role in modulating sustained attention and that R3487/MEM3454 may have therapeutic potential in improving sustained attention in humans.

Neonatal exposures to organophosphates that are not acutely symptomatic or that produce little or no cholinesterase inhibition can nevertheless compromise the development and later function of critical neural pathways, including serotonin (5HT) systems that regulate emotional behaviors. We administered parathion to newborn rats on postnatal days (PN) 1-4 at doses spanning the threshold for detectable cholinesterase inhibition (0.1 mg/kg/day) and the first signs of loss of viability (0.2 mg/kg/day). In adolescence (PN30), young adulthood (PN60) and full adulthood (PN100), we measured radioligand binding to 5HT(1A) and 5HT(2) receptors, and to the 5HT transporter in the brain regions comprising all the major 5HT projections and 5HT cell bodies. Parathion caused a biphasic effect over later development with initial, widespread upregulation of 5HT(1A) receptors that peaked in the frontal/parietal cortex by PN60, followed by a diminution of that effect in most regions and emergence of deficits at PN100. There were smaller, but statistically significant changes in 5HT(2) receptors and the 5HT transporter. These findings stand in strong contrast to previous results with neonatal exposure to a different organophosphate, chlorpyrifos, which evoked parallel upregulation of all three 5HT synaptic proteins that persisted from adolescence through full adulthood and that targeted males much more than females. Our results support the view that the various organophosphates have disparate effects on 5HT systems, distinct from their shared property as cholinesterase inhibitors, and the targeting of 5HT function points toward the importance of studying the impact of these agents on 5HT-linked behaviors.

Developmental neurotoxicity testing involves functional and neurohistological assessments in offspring during and following maternal and/or neonatal exposure. Data from positive control studies are an integral component in developmental neurotoxicity risk assessments. Positive control data are crucial for evaluating a laboratory's capability to detect chemical-induced changes in measured endpoints. Positive control data are also valuable in a weight-of-evidence approach to help determine the biological significance of results and provide confidence in negative results from developmental neurotoxicity (DNT) studies. This review is a practical guide for the selection and use of positive control agents in developmental neurotoxicology. The advantages and disadvantages of various positive control agents are discussed for the endpoints in developmental neurotoxicity studies. Design issues specific to positive control studies in developmental neurotoxicity are considered and recommendations on how to interpret and report positive control data are made. Positive control studies should be conducted as an integral component of the incorporation and use of developmental neurotoxicity testing methods in laboratories that generate data used in risk decisions.

Blockade of NMDA glutamate receptors with dizocilpine (MK-801) has been shown to cause substantial cognitive deficits and has been used to model symptoms of schizophrenia. Nicotine or nicotinic agonists, in contrast, may enhance cognitive or attentional functions and be of therapeutic potential in schizophrenia. Nicotinic-glutamatergic interactions, therefore, may have important implications in cognitive functions and antipsychotic treatments. Clozapine, a widely used antipsychotic drug, has been shown in some studies to be effective in ameliorating the cognitive deficits associated with schizophrenia. However, there is some evidence to suggest that clozapine similar to haloperidol may impair sustained attention in rats. In this study, we sought to determine whether chronic nicotine or dizocilpine may modify the effects of acute clozapine on attentional parameters and whether the behavioral effects would correlate with nicotinic or NMDA receptor densities in discrete brain regions. Adult female rats trained on an operant visual signal detection task were given 4 weeks of nicotine (5 mg/kg/day), dizocilpine (0.15 mg/kg/day), the same doses of both nicotine and dizocilpine as a mixture, or saline by osmotic minipump. While on chronic treatment, rats received acute injections of various doses of clozapine (0, 0.625, 1.25, 2.5 mg/kg, sc) 10 min prior to tests on attentional tasks. The pumps were removed on day 28 and 24 h later the animals were sacrificed for measurements of receptor densities in specific brain regions. The percent correct hit as a measure of sustained attention was significantly impaired by clozapine in a dose-related manner. Neither chronic nicotine nor dizocilpine affected this measure on their own or modified the effects of clozapine. Both nicotine and dizocilpine affected the receptor bindings in a region specific manner and their combination further modified the effects of each other in selective regions. Attentional performance was inversely correlated with alpha-bungarotoxin binding in the frontal cortex only. In conclusion, the data suggest attentional impairments with clozapine alone and no modification of this effect with nicotine or dizocilpine. Moreover, cortical low affinity nicotinic receptors may have a role in attentional functions.

Metallothioneins are central for the metabolism and detoxification of transition metals. Exposure to mercury during early neurodevelopment is associated with neurocognitive impairment. Given the importance of metallothioneins in mercury detoxification, metallothioneins may be a protective factor against mercury-induced neurocognitive impairment. Deletion of the murine metallothionein-1 and metallothionein-2 genes causes choice accuracy impairments in the 8-arm radial maze. We hypothesize that deletions of metallothioneins genes will make metallothionein-null mice more vulnerable to mercury-induced cognitive impairment. We tested this hypothesis by exposing MT1/MT2-null and wild-type mice to developmental mercury (HgCl(2)) and evaluated the resultant effects on cognitive performance on the 8-arm radial maze. During the early phase of learning metallothionein-null mice were more susceptible to mercury-induced impairment compared to wildtype mice. Neurochemical analysis of the frontal cortex revealed that serotonin levels were higher in metallothionein-null mice compared to wild-type mice. This effect was independent of mercury exposure. However, dopamine levels in mercury-exposed metallothionein-null mice were lower compared to mercury-exposed wild-type mice. This work shows that deleting metallothioneins increase the vulnerability to developmental mercury-induced neurocognitive impairment. Metallothionein effects on monoamine transmitters may be related to this cognitive effect.

Background: Developmental neurotoxicity of organophosphorous insecticides (OPs) involves multiple mechanisms in addition to cholinesterase inhibition. We have found persisting effects of developmental chlorpyrifos (CPF) and diazinon (DZN) on cholinergic and serotonergic neurotransmitter systems and gene expression as well as behavioral function. Both molecular/neurochemical and behavioral effects of developmental OP exposure have been seen at doses below those which cause appreciable cholinesterase inhibition., Objectives: We sought to determine if developmental DZN exposure at doses which do not produce significant acetylcholinesterase inhibition cause persisting cognitive deficits., Methods: Rats were exposed to DZN on postnatal days 1-4 at doses (0.5 and 2 mg/kg/d) that span the threshold for cholinesterase inhibition. They were later examined with a cognitive battery tests similar to that used with CPF., Results: In the T-maze DZN caused significant hyperactivity in the initial trials of the session, but not later. In a longer assessment of locomotor activity no DZN-induced changes were seen over a 1-hour session. Prepulse inhibition was reduced by DZN exposure selectively in males vs. females; DZN eliminated the sex difference present in controls. In the radial maze, the lower but not higher DZN dose significantly impaired spatial learning. This type of nonmonotonic dose-effect function has previously been seen with CPF as well. The lower dose DZN group also showed significantly greater sensitivity to the memory-impairing effects of scopolamine a muscarinic acetylcholine antagonist., Conclusions: Neonatal DZN exposure below the threshold for appreciable cholinesterase inhibition caused persisting neurocognitive deficits in adulthood. The addition of some inhibition of AChE with a higher dose reversed the cognitive impairment. This non-monotonic dose-effect function has also been seen with neurochemical effects. Some of the DZN effects on cognition resemble those seen earlier for CPF, some differ. Our data suggest that DZN and CPF affect transmitter systems supporting memory function, differently, implying participation of mechanisms other than their common inhibition of cholinesterase.

Adolescence is the life stage when tobacco addiction typically begins. Adolescent neurobehavioral development may be altered by nicotine self-administration in a way that persistently potentiates addiction. Previously, we showed that female adolescent rats self-administer more nicotine than do adults and that the increased nicotine intake then persists through the transition to adulthood [E.D. Levin, A. Rezvani, D. Montoya, J. Rose, H. Swartzwelder, Adolescent-onset nicotine self-administration modeled in female rats, Psychopharmacology 169 (2003) 141-149.]. In the current study, male Sprague-Dawley rats were given access to nicotine via the standard operant IV self-administration procedure (nicotine bitartrate dose of 0.03 mg/kg/infusion). One group of male rats started during adolescence the other group started in young adulthood. After the end of the four-week period of self-administration brain regions of the rats were assessed for alpha4beta2 nicotinic receptor binding. We found that male rats, like females, show higher nicotine self-administration when starting during adolescence as compared to starting in adulthood (p<0.001). Indeed, the effect in adolescent males was even greater than that in females, with more than triple the rate of nicotine self-administration vs. the adult-onset group during the first 2 weeks. The adolescent onset nicotine-self-administering rats also had significantly greater high affinity nicotinic receptor binding in the midbrain and the striatum, whereas hippocampal binding did not differ between the age groups. Striatal values significantly correlated with nicotine self-administration during the first 2 weeks in the adult-onset group but not the adolescent-onset rats, suggesting that the differences in self-administration may depend in part on underlying disparities in synaptic responses to nicotine. After the initial 2 weeks, nicotine self-administration in male rats declined toward adult-like levels, as the adolescent rats approached adulthood. This study showed that adolescent male rats self-administer significantly more nicotine than do male adult rats, but that adolescent-onset nicotine self-administration in male rats declines over weeks of continued use to approach adult-onset levels. In a previous study, we found that female rats also show greater nicotine self-administration with adolescent onset vs. adult onset, but that the females continued higher rates of self-administration into adulthood. Our results thus reinforce the concept that the adolescent brain is unusually receptive to the effects of nicotine in a manner that reinforces the potential for addiction.

Domoic acid (DA) is a marine biotoxin, produced by the diatom Pseudo-nitzchia spp., which has been shown to cause cognitive impairment in adults who are exposed via contaminated seafood. The neurobehavioral consequences of developmental exposure are much less well understood. In a previous study, we showed that a single prenatal exposure to DA in rats at mid-gestation caused neurobehavioral changes that persist into adulthood including increased susceptibility to the benchmark amnestic drug scopolamine. In the current study, we examined the lasting neurobehavioral consequences of DA exposure on the first day of postnatal life, a time in rats marking the completion of the major phase of neuroproliferation and corresponding to week 24 of human gestation. The effects of DA exposure at doses from 0.025-0.1 mg/kg (s.c.) twice per day on each of postnatal days 1 and 2 were compared with vehicle-treated controls and rats treated by the same protocol with 1 mg/kg of kainic acid. Following kainic acid exposure, a sex-selective effect was seen with females but not males showing a significant slowing of response latency in the radial-arm maze. The high DA dose of 0.1 mg/kg was quite toxic causing lethality in all of the offspring exposed and this group was excluded from further analysis. When the offspring in the 0.05 mg/kg DA dose group were tested, significant hypoactivity in the Figure-8 maze was observed during adolescence. No significant DA effects were seen in response latency or choice accuracy on the radial-arm maze during either acquisition or with challenge of the amnestic drug scopolamine. Early postnatal DA exposure in the rat can be lethal and sublethal exposure can cause neurobehavioral effects manifest in modest hypoactivity during the adolescent period. However, the sublethal persistent neurobehavioral toxicity appears to be less pervasive than reported effects following DA administered mid-gestation.

Although antipsychotic drugs are therapeutically effective in attenuating the hallmark symptoms of schizophrenia, these improvements do not return most patients to normative standards of cognitive function. Thus, complementary drug treatment may be needed to treat the attentional deficits of schizophrenia as well as to counteract the potential attentional impairments caused by some antipsychotic drugs. Nicotine, a drug commonly self-administered by a great majority of individuals with schizophrenia, has been shown to significantly improve cognitive function in some studies. The current study was conducted to determine the interactive effects of the atypical antipsychotic drugs clozapine and risperidone with chronic nicotine administration on attentional performance. Adult female Sprague-Dawley rats (N=35) were trained to perform an attentional task using an operant visual signal detection task. After training, rats were infused with a dose of 5 mg/kg/day (s.c.) nicotine base (n=18) or saline (n=17) for 28 consecutive days via osmotic pump. In Exp. 1, while being administered chronic nicotine or saline, rats were given acute doses of clozapine (0, 0.625, 1.25 and 2.5 mg/kg, s.c.) and were tested for attentional function. In Exp. 2, while on chronic nicotine or saline, other rats were challenged with acute doses of risperidone (0, 0.025, 0.05 and 0.1 mg/kg, s.c.) and were tested for attentional function. Results showed that acute administration of clozapine caused a significant dose-dependent impairment in choice accuracy (percent hit) in animals treated with chronic saline. Chronic nicotine treatment itself lowered accuracy, but attenuated further declines with acute clozapine treatment. Acute administration of risperidone at high dose significantly reduced performance (percent correct rejection) in chronically saline-treated rats, but in a similar fashion as in Exp. 1, chronic nicotine lowered accuracy but attenuated further impairment with acute risperidone. In summary, atypical antipsychotic drugs clozapine and risperidone significantly impaired choice accuracy in the visual signal detection task. Clozapine was more detrimental than risperidone but the adverse effects of both clozapine and risperidone on attentional performance were masked in rats chronically treated with nicotine.

Clozapine is an effective atypical antipsychotic drug used to treat schizophrenia. It has the advantage of producing fewer extrapyramidal motor side effects than typical antipsychotic drugs such as haloperidol. Schizophrenia involves more than the hallmark symptom of psychosis. Substantial cognitive impairment is also seen. Effective drug treatments against the cognitive impairment of schizophrenia need to be developed. The current study was conducted to determine the effects of clozapine on working memory in the rat neonatal hippocampal lesion model of schizophrenia, which includes symptoms of cognitive impairment. Infant Sprague-Dawley rats were given ibotenic acid lesions of the hippocampus on day 7 of age (using the day of birth as day 0). Controls were given vehicle infusions. In adulthood, the rats were trained on the 8-arm radial maze using the win-shift procedure. After 6 sessions of training, the lesioned rats and their controls were administered repeated injections of saline or clozapine (2.5 mg/kg) for the next 12 sessions of training. The females had significant radial-arm maze choice accuracy impairments caused by either clozapine or the hippocampal lesion, but the combination of the two treatments had no additive effect. The males showed a different pattern of effects. Intact males did not show a significant clozapine-induced impairment, whereas males with hippocampal lesions did show significant clozapine-induced impairment although hippocampal lesions by themselves did not significantly impair male choice accuracy. These data show that clozapine can cause memory impairment and it potentiates rather than reverses hippocampal lesion-induced deficits. There are critical sex-related differences in these effects.

To investigate the behavioral effects of prenatal exposure to the marine toxin domoic acid, pregnant female rats were injected subcutaneously with 0, 0.3, 0.6, or 1.2 mg/kg of domoic acid on gestational day 13. The offspring were then run through a behavioral testing battery to determine the developmental effects of the toxin on spontaneous alternation in the T-maze, on locomotor activity in the Figure-8 maze, and on working memory in the 8-arm radial maze. In the T-maze, no significant domoic acid induced differences were seen on spontaneous alternation, but there were significant domoic acid effects on latency. Prenatal domoic acid exposure caused a dose-related increase in response latency in the second spontaneous alternation test. There was also a significant domoic acid effect seen in the 1-h long Figure-8 maze test. Locomotor activity measured in the Figure-8 maze detected a persisting effect of the 1.2 mg/kg domoic acid dose, which significantly increased the rate of habituation over the activity test session. This was characterized by higher initial activity followed by greater decline in activity. In the radial-arm maze the control vehicle treated rats showed the normal sex-related difference in spatial learning and memory with males outperforming females. Developmental domoic acid exposure decreased this effect such that the normal sex difference in spatial memory was not seen with the 1.2 mg/kg domoic acid dose. The rats of both sexes with a history of prenatal domoic acid exposure showed increased susceptibility to the amnestic effects of the muscarinic acetylcholine scopolamine, suggesting that they had less functional reserve with which to solve the radial-arm maze memory task. This study demonstrates persisting neurobehavioral effects of acute prenatal exposure to domoic acid at doses that do not cause overt clinical signs of toxicity.

Olanzapine is a widely used atypical antipsychotic drug. It is quite effective in reducing psychotic symptoms. However, the syndrome of schizophrenia encompasses more than psychosis. There is a pronounced cognitive impairment among other negative neurobehavioral symptoms. Classic antipsychotic drugs such as haloperidol do not alleviate cognitive impairment associated with schizophrenia and have been shown to exacerbate the dysfunction. The atypical antipsychotic drugs have a different profile of receptor actions and may have a different array of actions on cognitive function. The purpose of the current studies was to determine the effects of olanzapine on working memory function as measured by choice accuracy in the radial-arm maze. In determining the cognitive effects of any antipsychotic drug it is critical to determine its interactions with nicotinic manipulations since the great majority of people with schizophrenia smoke cigarettes and alterations in nicotinic receptors have been found in people with schizophrenia. Olanzapine caused a significant working memory impairment. Nicotine attenuated olanzapine-induced memory deficits. It may be the case that nicotinic co-treatment will be useful in addressing the cognitive impairment of schizophrenia.

Pre-pulse inhibition (PPI) is a phenomenon of neurobehavioral plasticity in which the motor response to a startling stimulus is inhibited by a preceding stimulus of a lower intensity. Most often this is tested in the auditory mode. PPI is impaired in a variety of clinical states, most notably schizophrenia. PPI is easily modeled in experimental animals and serves as a useful basis for determining the neural bases for behavioral plasticity. In the current study we examined the interactions of N-methyl-D-aspartate (NMDA) glutamate and nicotinic cholinergic receptor systems in the expression of PPI. Female Sprague-Dawley rats were tested for auditory PPI after s.c. injections of the NMDA antagonist dizocilpine (also known as MK-801), the prototypic nicotinic agonist nicotine or both. Vehicle (saline) injections served as the control. Nicotine (0.2-0.8 mg/kg) by itself caused a modest but significant dose-related improvement in PPI. Dizocilpine (25-100 microg/kg) caused a dramatic dose-related impairment in PPI. Interestingly, the low to moderate doses of nicotine potentiated the PPI impairment by dizocilpine. In a second experiment nicotine and dizocilpine interactions with the atypical antipsychotic drug clozapine were assessed. As in the first experiment, nicotine potentiated the adverse effects of dizocilpine on PPI. The combination of nicotine with clozapine effectively attenuated the PPI impairment caused by dizocilpine when neither alone was effective. Inasmuch as PPI is impaired in schizophrenia, its reversal by the antipsychotic drug clozapine may depend on co-administration of nicotine by smoking in the patients. Development of nicotinic-based co-treatments for schizophrenia may achieve this benefit of nicotine without the hazards of smoking. Sensory modulation deficit, which is a syndrome of sensory over-responsiveness may also benefit from such combination therapy.

Zebrafish are an emerging model for the study of the molecular mechanisms of brain function. To conduct studies of the neural bases of behavior in zebrafish, we must understand the behavioral function of zebrafish and how it is altered by perturbations of brain function. This study determined nicotine actions on memory function in zebrafish. With the methods that we have developed to assess memory in zebrafish using delayed spatial alternation (DSA), we determined the dose effect function of acute nicotine on memory function in zebrafish. As in rodents and primates, low nicotine doses improve memory in zebrafish, while high nicotine doses have diminished effect and can impair memory. This study shows that nicotine affects memory function in zebrafish much like in rats, mice, monkeys and humans. Now, zebrafish can be used to help understand the molecular mechanisms crucial to nicotine effects on memory.

Chlorpyrifos (CPF), a widely used organophosphate insecticide and potent acetylcholinesterase inhibitor, interferes with neurobehavioral development. Rat models have been key in demonstrating that developmental CPF exposure causes learning deficits and locomotor activity alterations, which persist into adulthood. Complementary nonmammalian models can be useful in determining the neurodevelopmental mechanisms underlying these persisting behavioral effects. Zebrafish (Danio rerio) with their clear chorion and extensive developmental information base provide an excellent model for assessment of molecular processes of toxicant-impacted neurodevelopment. We have developed methods for assessing spatial discrimination learning in adult zebrafish and have documented persisting effects of developmental CPF exposure on swimming activity and learning after low and high doses of CPF (10 and 100 ng/ml) administered to zebrafish embryos on Days 1-5 postfertilization (pf). In the current study, we developed methods for behavioral assessment of CPF exposure on swimming activity in newly hatched zebrafish. An equal area segmented annular grid (concentric circles divided into quadrants through the diameter) was made in a 16-mm diameter cylinder. The test area was placed on a heating device secured to an Olympus SZH10 dissecting scope stage. Zebrafish embryos were exposed to 10 ng/ml CPF, 100 ng/ml CPF, or vehicle control (25 microl/ml DMSO) (n=8-10/treatment group). Each treatment group was kept in a total volume of 25 ml of egg water (60 mg/ml Instant Ocean) including DMSO with or without CPF mixed to above dilutions in an incubator set at 28.5 degrees C. CPF dilutions or vehicle were changed daily with exposure ending on Day 5 pf. Testing of larval zebrafish was performed on Days 6 and 9 pf. The fish were placed in the test cylinder with 1.5 ml of egg H(2)O (28.5 degrees C). After a 2-min acclimation period, the swimming activity of the fish was measured for a 3-min testing session. The 100 ng/ml CPF dose caused significant slowing of swimming activity on Days 6 and 9 pf and had persisting effects of impairing spatial discrimination and decreasing response latency in adulthood. Developmental exposure to 10 ng/ml of CPF did not cause a significant change in locomotor activity during the period soon after hatching. CPF exposure during early development caused clear behavioral impairments detectable during the posthatching period. In a previous study, we found that early developmental CPF exposure caused behavioral alterations in zebrafish, which lasted throughout adulthood. The molecular mechanisms by which early developmental CPF exposure produces these behavioral impairments expressed in adulthood can now be studied in the zebrafish model.

The widely used organophosphate insecticide, chlorpyrifos (CPF), elicits neurobehavioral abnormalities after apparently subtoxic neonatal exposures. In the current study, we administered 1 or 5 mg/kg/day of CPF to pregnant rats on gestational days 9-12, the embryonic phase spanning formation and closure of the neural tube. Although there were no effects on growth or viability, offspring showed behavioral abnormalities when tested in adolescence and adulthood. In the CPF-exposed groups, locomotor hyperactivity was noted in early T-maze trials, and in the elevated plus-maze; alterations in the rate of habituation were also identified. Learning and memory were adversely affected, as assessed using the 16-arm radial maze. Although all CPF-exposed animals eventually learned the task, reference and working memory were impaired in the early training sessions. After training, rats in the CPF group did not show the characteristic amnestic effect of scopolamine, a muscarinic acetylcholine antagonist, suggesting that, unlike the situation in the control group, muscarinic pathways were not used to solve the maze. These results indicate that apparently subtoxic CPF exposure during neurulation adversely affects brain development, leading to behavioral anomalies that selectively include impairment of cholinergic circuits used in learning and memory. The resemblance of these findings to those of late gestational or neonatal CPF exposure indicates a prolonged window of vulnerability of brain development to CPF.

Azidothymidine (AZT) is administered to pregnant women with HIV to prevent the spread of infection to their fetuses. Since gestation is a period of critical neurodevelopment, it is important to determine the risk AZT exposure may pose to neurobehavioral function of the offspring. The current study focused on teratological risks of developmental AZT exposure to neurocognitive function. Male and female Swiss mice were administered AZT or vehicle (0, 100, or 200 mg/kg/day po given twice daily in equal amounts for 32 weeks before and during gestation). Adult male and female offspring (n = 10/sex/treatment group) underwent neurobehavioral testing focused on determining learning and memory capabilities in the radial-arm maze. AZT exposure did not cause significant deficits during radial-arm maze acquisition. No impairment was seen in asymptotic levels of choice accuracy indicative of working memory function. Attempts to unmask subtle learning impairments following developmental AZT by the introduction of behavioral challenges such as reduction of motivational state (food restriction either 4-6 h or 22-24 h) or imposition of intrasession delays of 1.5 min to 2.5 h were unsuccessful. With a 4-week intersession delay, a significant AZT Treatment x Delay effect was seen with a significantly greater decline seen in the controls as compared to the 100 mg/kg/day AZT group. Locomotor activity on the radial-arm maze was significantly affected by AZT treatment (100 mg/kg/day) during the acquisition phase, but not during the other test phases. No behavioral alterations were seen related to stress as measured by the elevated plus maze. Vestibulomotor functioning on the balance beam remained unaltered. Using an extended dosing regimen including dosing of both sires and dams, as well as placing a greater demand on reproductive system performance with three continuous breedings, this study detected only subtle neurobehavioral impairments in mice after prenatal AZT exposure at clinically relevant doses.