2005 Progress Report: Disruption of Ontogenic Development of Cognitive and Sensory Motor Skills

EPA Grant Number: R829391C003
Subproject: this is subproject number 003 , established and managed by the Center Director under grant R829391
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).

Center: CECEHDPR - University of Medicine and Dentistry of New Jersey Center for Childhood Neurotoxicology and Assessment
Center Director: Lambert, George H.
Title: Disruption of Ontogenic Development of Cognitive and Sensory Motor Skills
Investigators: Wagner, George
Institution: University of Medicine and Dentistry of New Jersey , University of Medicine and Dentistry of New Jersey
Current Institution: University of Medicine and Dentistry of New Jersey
EPA Project Officer: Louie, Nica
Project Period: November 1, 2001 through October 31, 2006
Project Period Covered by this Report: November 1, 2004 through October 31, 2005
RFA: Centers for Children's Environmental Health and Disease Prevention Research (2001) RFA Text |  Recipients Lists
Research Category: Children's Health , Health Effects , Health


One of the most striking features of many developmental disorders is that cognitive and sensory/motor development usually progress symptom-free for several months to years but then are followed by a period of retardationwhere some skills fail to develop or do so well behind schedule, or a period ofregression where some acquired skills are lost, or a period of intrusion where acquired skills are overshadowed by the appearance of behaviors aberrant in form or frequency. Importantly, the period when symptoms first begin to appear may represent the time when environmental toxicants have accumulated in the brain to critical levels and/or the deleterious effects of earlier exposure may become manifest through perturbation of normal ontogenic development of brain pathways. Furthermore, it is thought that certain individuals may be more sensitive to toxicants because of a genetic (perhaps immune-related) predisposition. Exposure during critical periods may disrupt neurobehavioral development by altering neural migration, circuitry, and/or synaptogenesis of brain areas required for expression of these behaviors.

Within this framework, the hypothesis that environmental toxicants are causally involved in developmental disorders is readily tested. That is, acute or chronic exposure should disrupt neurobehavioral development causing behavioral retardation, regression, and/or intrusions. These conditions can be identified only if normal developmental patterns are thoroughly understood. Traditionally, animal models of developmental disorders have not examined systematically each of these three possible scenarios but, instead, tend to focus on single aspects of neurobehavioral development. Accordingly, the objective of this research project is to develop a new paradigm for the study of toxicant-induced developmental disorders incorporating systematic assessment of retardation, regression and/or intrusions in the neurobehavioral development of mice.

The specific aims of this research project are to: (1) characterize the normal development of a wide range of behaviors thought to be dependent on maturation of cerebellum, hippocampus, and striatum in each strain of mice to be tested; (2) evaluate the effects of pre- and/or postnatal low-dose exposure to sodium valproate (NaVal) and methylmercury (MeHg) on these behaviors; (3) examine the brain regions for evidence of toxicant-induced damage in the context of disruption of neurobehavioral development; (4) evaluate the behavioral outcome in the context of this new paradigm in the form of the following question: did toxicant exposure result in retardation, regression, and/or intrusions in the neurobehavioral development of male or female mice of these strains; and (5) attempt to protect mice from the deleterious consequences of the toxicants with antioxidant pretreatment.

Progress Summary:

Because this model focuses on autism, three brain structures (cerebellum, hippocampus, and striatum) were selected because each has been shown to manifest pathological alteration upon autopsy of autistic individuals. For each of these brain areas, the development of neural circuitry and synapse formation in the context of the maturation of select behaviors shown to be dependent, at least in part, on these brain areas was examined. For the cerebellum, the behaviors included surface and mid-air righting reflex; for the hippocampus, the behaviors included water maze activity (hidden platform) and passive avoidance responding; and for the striatum, the behaviors include spontaneous locomotion, active avoidance responding, and water maze activity (exposed platform). Finally, intrusive stereotypic and self-injurious behaviors, which are stimulant-induced and mediated by the striatum, were included. The initial step was to characterize carefully the normal ontogeny of each of these behaviors in each strain of mice to be tested. We started with BALB/c mice, as our previous studies had shown this to be a sensitive strain. On the basis of preliminary data described below, we have added other strains overexpressing enzymes involved in the oxidative stress response following toxicant administration.

For each strain, we identify the postnatal days during which each behavior first appears. Next, the disruptive effects of low-dose exposure to toxicants (drugs, metals) on the ontogenic development of each behavior were examined. The toxicants are administered either acutely or chronically and either pre- and/or postnatally. The prediction is that exposure will disrupt these behaviors in a dose-dependent fashion. Specifically, early exposure should cause a retardation in behavioral development, but exposure during or shortly after the maturation of the behavior should cause a regressionas evidenced by a plateau in normal development or a literal loss of some or all of these acquired behaviors. Finally, early toxicant exposure should render subjects more sensitive to intrusions of stereotypic and self-injurious behaviors following stimulant administration. On completion of behavioral testing, mice are subjected to histological and neurochemical examination of neural circuit maturation.

As noted, there is a wide spectrum of symptoms exhibited by autistic individuals and, therefore, in developing our mouse model we elected to test mice on a wide variety of tasks during the critical periods of maturation. The tasks were selected because they are known to be mediated, at least in part, by cerebellum, hippocampus, and striatum. In these preliminary studies mice were exposed to various neurotoxicants either prenatally or postnatally and the toxicant-induced deficits were categorized as retardations, regressions, or intrusions.

Prenatal Treatment With 600 mg/kg NaVal on Embryonic Day 13 (E13)

Rationale. Prenatal exposure to NaVal was used to model autism in mice because of case reports revealing that early exposure results in morphological abnormalities similar to that found in the autistic brain and because a high percentage of children exposed to NaVal in utero experienced developmental delays including deficits in language and communication and stereotypic and hyperexcitable behavior described as “fetal valproate syndrome.”

Body Weight. Pups born to pregnant mice receiving 600 mg/kg NaVal on E13 exhibited a significant increase in body weight from postnatal days P5-26 as they matured. In addition, there was a significant interaction of day and treatment with NaVal-treated pups having higher body weights than saline-treated controls (p< 0.01, data not shown).

Surface Righting (NaVal-Induced Retardation). Male and female pups receiving NaVal treatment in utero displayed longer latencies to surface right (i.e., roll onto their ventral surface when placed on their back) on days P5 and P6 as compared to control mice. This was interpreted as indicating that the NaVal treatment resulted in a retardation in the ability to perform this skill (p < 0.0001).

Mid-Air Righting (NaVal-Induced Retardation). When dropped from a height of 30 cm with ventral surface pointing up, mice normally rotate in mid-air, landing on a soft surface with all four paws. Their ability to perform this task normally matures around day P15. There was a significant overall effect of NaVal treatment on mid-air righting in males and females (p < 0.0001). Female pups treated with NaVal in utero were impaired in mid-air righting on P15, whereas males were impaired on P14, P15, and P16 (p < 0.0001). These data indicate that there was a long-lastingretardation following prenatal NaVal and that this effect was more evident in males than in females.

Hanging Wire (NaVal-Induced Retardation). Prenatal NaVal exposure significantly decreased latency-to-fall in hanging-wire-grip strength. Treated mice fell sooner than control mice, and the normal increased strength in male pups was not seen in the treated group (p < 0.0001). These data indicate that the prenatal NaVal treatment resulted in retardation in the maturation of this behavior.

Water Maze (NaVal-Induced Retardation). Preliminary investigation of the ontogeny of water maze performance in BALB/c mice revealed that, independent of what day training is initiated, a significant reduction in latency to escape to a platform hidden under opaque water does not emerge until around P23. On P23, saline-treated animals showed a significant improvement in ability to find the hidden platform. This was not the case for NaVal-treated pups, independent of sex. In fact, NaVal-treated pups did not show a significant improvement across days. On days P23 and P25, both male and female pups previously exposed to NaVal were significantly impaired in water maze performance; only males continued to be affected on P26 (p < 0.005) indicating a retardation in the maturation of water maze performance with males more affected more than females.

Motor Activity (NaVal-Induced Intrusion, Hyperactivity). Mice were placed in an open field activity chamber for a 10-minute test session. Each analysis consisted of a total activity count for the full 10-minute period and an analysis of each 2 minute session, with both treatment and sex as independent variables. Between day analysis examining total activity counts showed a significant increase in activity across days (p < 0.0001) as well as a significant sex difference, with females showing higher activity counts (p < 0.05). Within-days analysis on each specific 2-minute bout revealed animals only showed within-days habituation on P23. On P22, NaVal treatment resulted in consistently higher activity counts on bouts 2-4 (4-8 minutes) only, and a significant day by treatment interaction that approached significance (p = 0.06). These data indicate that the prenatal NaVal caused hyperactivity (intrusion) that was apparent only in a novel environment; both NaVal and control-treated mice did exhibit a habituation when retested in the same apparatus the next day.

Treatment With NaVal on P14

Rationale. In the following studies, mice born to untreated mothers received NaVal on day P14, a time critical for the maturation of a number of skills as described below.

Mid-Air Righting (NaVal-Induced Regression). There was a significant overall effect of NaVal treatment on mid-air righting following both the 200 mg/kg dose and the 400 mg/kg dose (p < 0.0001 for both doses). Both the 200 and 400 mg/kg dose produced significant deficits in mid-air righting on P15 and P16 (24-48 hours after treatment). Pups were able to mid-air right on P14, and this ability was lost (regression) following NaVal treatment (p < 0.0001 for both doses).

Water Maze (NaVal-Induced Retardation). Mice receiving 400 mg/kg NaVal on P14 showed a longer latency to reach the visible platform, a striatal-dependent task (p < 0.05). Mice receiving 200 mg/kg NaVal were not impaired, however, and were able to reach the platform in less than 30 seconds. When the platform was hidden (a hippocampal-dependent task), the saline-treated animals showed a significant improvement across days (p < 0.005). This analysis revealed a significant effect of treatment so that both NaVal doses produced a significant impairment (retardation) in escape latency on all 4 days of testing (p < 0.001).

Vitamin E Is Protective Against NaVal-Induced Behavioral Deficits

The mid-air righting procedure was repeated except that some NaVal-treated mice received subcutaneous (s.c.) vitamin E on P11-P14. The NaVal-induced regression was replicated fully in mice receiving the vehicle pretreatment, but vitamin E pretreatment completely protected mice against the NaVal-induced regression in mid-air righting. This effect is quite important; in the proposed studies we intend to replicate it using a second antioxidant, and further, to evaluate carefully the degree to which our biomarker of oxidative stress reflects the regression itself as well as the protection by antioxidants.

Postnatal Treatment With MeHg

Rationale. We chose to study an organic form of mercury because it is a toxicant widely distributed in the environment and has been implicated in the pathogenesis of autism. The toxic actions of MeHg depend on dose and age, with the developing brain being most sensitive to low dose exposure. MeHg does cross the placental barrier and, in humans exposed in utero to acute high doses, was shown to cause a retardation in cognitive and locomotor development along with numerous other neurological symptoms including seizures and cerebral palsy. Nonetheless, it is important to note that autism was not found to be associated with either pre- or neonatal exposure to high doses of organic mercury.

Mid-Air Righting (MeHg-Induced Retardation). Treatment with MeHg every other day between P3 and P15 resulted in a significant reduction (retardation) in the ability to mid-air right on days P14-P19 (p < 0.005). This effect was somewhat dose-dependent with mice receiving the highest dose showing virtually no improvement in performance through P19.

Negative Geotaxis (MeHg-Induced Regression). Mice receiving the MeHg through day P15 were able to perform the negative geotaxis response with the same latency as control-treated mice. On days P16 and P17, however, the treated mice showed a significant loss (consistent with regression) of this negative geotaxis response manifest as an increased latency (p < 0.05). Although the performance of the treated mice improved on days P18 and P19, they still were significantly slower than the control-treated mice (p < 0.005).

Novel Object Approach (MeHg-Induced Hyper-Responsiveness). Mice receiving the postnatal MeHg treatment were tested on days P20-24 in an open field apparatus with a single, novel object in a fixed location. The number of contacts each mouse made with this object was counted. On day P25, the location of that object was changed. Control mice noticed this change and significantly increased their number of object contacts for about 20 minutes; thereafter, their contacts with the object decreased back to baseline levels. The MeHg-treated mice also approached the object significantly more during the first 20 minutes of the session. Unlike the controls, however, the treated mice repetitively approached the object in its new location, making more and more approaches throughout the rest of the session. These data indicate that the treated mice were hyperresponsive to the location switch, a behavior that may be classified as an obsessive-likeintrusion.

Social Behavior (MeHg-Induced Intrusions). Male mice receiving the postnatal MeHg treatment were housed in individual cages until 90 days of age. An intruder male of the same age and treatment was then introduced into the home cage of these resident mice. Control residents approached the intruder with sniffing and grooming behavior. The treated mice exhibited significantly less sniffing and grooming behavior (p < 0.005) and significantly more attack behavior (p < 0.05). These data indicate that the normal social behaviors are considerably reduced by the MeHg treatment and, in this place,intrusive aggressive attack occurs.

Preliminary Evidence of Sensitization

Repeated Exposures (MeHg-induced sensitization). Mice received prenatal MeHg on days E10-14. They then were treated once again with a single exposure on P15; water maze testing was initiated on P23. It was found that the prenatal MeHg exposure primed the animal such that the second exposure on P15 caused a developmental retardation, with water maze escape latency significantly longer than in control mice or mice receiving any of the other treatment combinations including single MeHg exposure (p < 0.05).

Oxidative Stress in Autistic Children

Dr. Xue Ming (a pediatric neurologist on sabbatical from the New Jersey Medical School and working in Dr. Wagner’s laboratory) conducted a study of oxidative stress in autism. Urinary excretion of oxidative stress biomarkers, 8-OHdG and 8-isoprostane, were determined in 33 children with autism and 29 healthy children. Both biomarkers were normalized to urinary excretion of creatinine. 8-Isoprostane levels were significantly higher in children with autism (autism group: 32.92 + 1.98 ng creatinine-1M; control: 5.71 + 0.98 ng creatinine-1M, p < 0.01). There also was a trend of increased 8-OHdG urinary excretion in autistic subjects, but this did not reach statistical significance possibly because of the small number of subjects studied (autism: 13.73 + 1.03 ng creatinine-1M; control: 11.87 + 0.81 ng creatinine-1M, p = 0.08). These results showed that lipid peroxidation is increased in this cohort of autistic children.

Preliminary Study of Oxidative Stress Biomarker in Brains of Naval-Treated Mice

NaVal- and Amphetamine-Induced Increase in Brain Isoprostane. The objective of this proposal is to develop an animal model corresponding as closely as possible to the clinical research on autism. The critical observation that autistic individuals have higher urinary isoprostane levels prompted us to assess the effects of amphetamine, NaVal, and MeHg on brain lipid peroxidation. In our first study, mice received our standard doses of amphetamine or NaVal s.c and were sacrificed 90 minutes later. The striatal tissue was dissected and isoprostane levels were found to be elevated, though this effect failed to reach statistical significance with p < 0.06. Assay development will continue and we plan to add a second assay of lipid peroxidation (MDA) because we do not know whether isoprostane is the targeted lipid peroxidation products of NaVal or MeHg. The intent is to characterize the magnitude of the toxicant-induced lipid peroxidation at the time of exposure, linking this effect to the later and long-lasting toxicant-induced neurobehavioral deficits. It is predicted that the higher the degree of toxicant-induced oxidative stress, the higher the degree and duration of the neurobehavioral deficits. Furthermore, it is predicted that sensitized mice will show a greatly enhanced oxidative stress response, and finally, it is predicted that antioxidant treatment that protects mice against toxicant-induced neurobehavioral deficits will also protect these subjects against the toxicant-induced oxidative stress. In any case, this preliminary result suggests that oxidative stress is enhanced in the brain tissue of NaVal-treated mice.


Our preliminary data demonstrate that we have developed a neurobehavioral model of autism that is sensitive to the deleterious effects of NaVal and MeHg exposure. We demonstrated that cognitive, motor, and social behavioral deficits engendered by this model can be categorized as retardations, regressions, or intrusions. On the basis of human data demonstrating that autistic individuals have higher levels of oxidative stress biomarkers, we began evaluating the efficacy of vitamin E pretreatment, observing that this antioxidant effectively protected mice against NaVal-induced regression.

Future Activities:

We have developed a hypothesis that early exposure to a toxicant may prime and individual such that a second exposure to the same or different toxicant(s) may trigger a sensitized response leading to autistic regression. We plan to test this hypothesis examining the same brain regions for evidence of proinflammatory cytokine induction and oxidative stress responses following second exposures. We plan to continue our studies on the protective effects of antioxidants and to continue to develop our behavioral models as well, particularly in the realm of social behaviors.

Journal Articles on this Report : 3 Displayed | Download in RIS Format

Other subproject views: All 21 publications 9 publications in selected types All 9 journal articles
Other center views: All 86 publications 50 publications in selected types All 49 journal articles
Type Citation Sub Project Document Sources
Journal Article Brenz Verca MS, Bahi A, Boyer F, Wagner GC, Dreyer J-L. Distribution of α- and γ-synucleins in the adult rat brain and their modification by high-dose cocaine treatment. European Journal of Neuroscience 2003;18(7):1923-1938. R829391 (2004)
R829391 (2005)
R829391 (2006)
R829391C003 (2005)
  • Abstract from PubMed
  • Abstract: Wiley-Abstract
  • Journal Article Halladay AK, Tessarollo L, Zhou R, Wagner GC. Neurochemical and behavioral deficits consequent to expression of a dominant negative EphA5 receptor. Molecular Brain Research 2004;123(1-2):104-111. R829391 (2004)
    R829391 (2005)
    R829391 (2006)
    R829391C003 (2005)
  • Abstract from PubMed
  • Full-text: ScienceDirect-Full Text HTML
  • Other: ScienceDirect-Full Text PDF
  • Journal Article Kita T, Wagner GC, Nakashima T. Current research on methamphetamine-induced neurotoxicity: animal models of monoamine disruption. Journal of Pharmacological Sciences 2003;92(3):178-195. R829391 (2004)
    R829391 (2005)
    R829391 (2006)
    R829391C003 (2005)
  • Abstract from PubMed
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  • Supplemental Keywords:

    children’s health, disease and cumulative effects, ecological risk assessment, environmental chemistry, health risk assessment, risk assessments, susceptibility/sensitive population/genetic susceptibility, toxicology, genetic susceptibility, assessment of exposure, assessment technology, autism, behavioral assessment, behavioral deficits, childhood learning, children, developmental disorders, developmental effects, environmental health hazard, environmental toxicant, exposure assessment, gene-environment interaction, neurodevelopmental, neurological development, neuropathological damage, neurotoxic, neurotoxicity, outreach and education, public health,, RFA, Health, Scientific Discipline, Health Risk Assessment, Biochemistry, Children's Health, developmental neurotoxicity, biological response, motor development, neurodevelopmental toxicity, cognitive development, children's environmental health, environmental health hazard, environmental toxicant, growth & development

    Progress and Final Reports:

    Original Abstract
  • 2002
  • 2003
  • 2004 Progress Report
  • Final

  • Main Center Abstract and Reports:

    R829391    CECEHDPR - University of Medicine and Dentistry of New Jersey Center for Childhood Neurotoxicology and Assessment

    Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
    R829391C001 Neurotoxicant Effects on Cell Cycle Regulation of Neurogenesis
    R829391C002 Adhesion and Repulsion Molecules in Developmental Neurotoxic Injury
    R829391C003 Disruption of Ontogenic Development of Cognitive and Sensory Motor Skills
    R829391C004 Exposure Assessment and Intervention Project (EAIP)
    R829391C005 Clinical Sciences Project