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Extramural Research

2006 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
EPA Project Officer: Fields, Nigel
Project Period: November 1, 2001 through October 31, 2006
Project Period Covered by this Report: November 1, 2005 through October 31, 2006
RFA: Centers for Children's Environmental Health and Disease Prevention Research (2001)
Research Category: Children's Health , Health Effects

Description:

Objective:

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 is followed by a period of retardation where some skills fail to develop or do so well behind schedule, a period of regression 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 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 has been 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. Within each of these categories, subjects are evaluated for toxicant-induced deficits in social, cognitive, and/or motor skills.

Because this model was to focus 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 will be spontaneous locomotion, active avoidance responding, and water maze activity (exposed platform). Finally, intrusive stereotypic and self-injurious behaviors that 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, we added other strains, most recently concentrating on engrailed-2 knockout mice (see below).

For each strain, we identify the postnatal days during which each behavior first appears. Next, the disruptive effects of low dose exposure to toxicants on the ontogenic development of each behavior are 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 regression as 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.

The objectives 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 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:

Pharmacotherapy for and Neurochemical Basis of Self-Injurious Behavior

The first milestone achieved was insight into pharmacotherapy for and underlying mechanisms of the intrusive self-injurious behaviors. These behaviors are among the most difficult for parents to cope with. We demonstrated that self-injurious behaviors are caused by activation of both dopaminergic and serotonergic systems in the striatum and further and that drugs that reduce these behaviors reverse their neurochemical bases.
A New Animal Model of Autism: Prenatal and Postnatal Treatment With NaVal

Rationale. As noted above, we were not satisfied with the available animal models of autism and, therefore, elected to develop a new approach where toxicant or genetic-induced alterations in neurobehavioral maturation are categorized as retardations, regressions, or intrusions. Prenatal exposure to NaVal first 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.”

Methods and Results. As noted in our 2005 Annual Report, the NaVal did disrupt the maturation of a number of cognitive and motor skills in BALB/c mice in a manner consistent with autism. These data were included in a paper submitted to The Journal of Autism and Developmental Disorders, which now is in press.

Postnatal Treatment With MeHg

Rationale. We next 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. We used essentially the same approach as described above, and these data are now in preparation for publication. Briefly, we observed the following.

Mid-Air Righting (MeHg-Induced Retardation). Treatment with MeHg every other day between postnatal day (PND) 3 and PND 15 resulted in a significant reduction (retardation) in the ability to mid-air right on days PND 14-19 (p < 0.005). This effect was somewhat dose-dependent with mice receiving the highest dose showing virtually no improvement in performance through PND 19.

Negative Geotaxis (MeHg-Induced Regression). Mice receiving the MeHg through day PND 15 were able to perform the negative geotaxis response with the same latency as control-treated mice. On PND 16 and PND 17, however, the treated mice showed a significant loss (consistent with regression) of this negative geotaxis response manifest as an increased latency (p < 0.05).

Novel Object Approach (MeHg-Induced Hyper-Responsiveness). Mice receiving the postnatal MeHg treatment were tested on days PND 20-24 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 PND 25, 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-like intrusion.

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. These data indicate that the normal social behaviors are considerably reduced by the MeHg treatment and, in this place, intrusive aggressive attack occurs.

Vitamin E is Protective Against MeHg and Naval-Induced Behavioral Deficits

In our first attempt to protect mice against the deleterious effects of early toxicant exposure, we elected to pretreat subjects with an antioxidant, vitamin E, followed by our treatments with MeHg and valproic acid.The toxicant-induced regression was replicated fully in mice receiving the antioxidant vehicle pretreatment, but vitamin E pretreatment completely protected mice against the MeHg and the NaVal-induced regression in mid-air righting. This effect is quite important; in future studies we intend to replicate it using a second antioxidant and, further, to carefully evaluate the degree to which our biomarker of oxidative stress reflects the regression itself as well as the protection by antioxidants.

Oxidative Stress in Autistic Children

Dr. Xue Ming (a pediatric neurologist on sabbatical from 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 was also 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. This work has been accepted for publication in Prostaglandins, Leukotrienes, and Essential Fatty Acids.

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

NaVal- and Amphetamine-Induced Increase in Brain Isoprostane. 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 subcutaneously 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) as 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 toxicant-treated mice.

Neurobehavioral Deficits in Engrailed-2 Knockout Mice

Perhaps the most important aspect of our new model is that it allows us to study the effects of genetic alterations on sensitivity to toxicants. We have now used our model to evaluate the neurobehavioral effects of deletion of engrailed-2 in mice. This gene has been linked to autism, and we found our model to be remarkably sensitive to the genetic alteration. This work is detailed in a manuscript submitted to Nature Medicine.

Future Activities:

The investigators did not report any future activities.


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 Carlson KM, Wagner GC. Effects of phencyclidine on schedule-controlled responding following neurotoxic lesions of the striatum. Life Sciences 2005;77(4):372-385. R829391 (2004)
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  • Abstract from PubMed
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  • Abstract: ScienceDirect-Abstract
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  • Journal Article Halladay AK, Wagner GC, Sekowski A, Rothman RB, Baumann MH, Fisher H. Alterations in alcohol consumption, withdrawal seizures, and monoamine transmission in rats treated with phentermine and 5-hydroxy-L-tryptophan. Synapse 2006;59(5):277-289. R829391 (2005)
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    R829391C003 (2006)
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  • Journal Article Ming X, Stein TP, Brimacombe M, Johnson WG, Lambert GH, Wagner GC. Increased excretion of a lipid peroxidation biomarker in autism. Prostaglandins, Leukotrienes and Essential Fatty Acids 2005;73(5):379-384. R829391 (2004)
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    R829391C003 (2006)
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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, environmental toxicant, environmental health hazard, children's environmental health, growth & development

    Progress and Final Reports:
    Original Abstract
    2004 Progress Report
    2005 Progress Report


    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

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    The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.

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