Neurotoxicology and Teratology 27 (2005) 365 – 384 www.elsevier.com/locate/neutera
NBTS 2005 Abstracts NBTS 1-7/BTS 1-7 Zebrafish Models of Neurobehavioral Toxicity Scalzo FM1, Levin ED2 (Bard College, Annandale, NY1 and Duke University Medical Center, Durham, NC2) The joint Neurobehavioral Teratology Society and Behavioral Toxicology Society symposium is entitled bZebrafish Models of Neurobehavioral ToxicityQ. Zebrafish models have much to offer for determining molecular mechanisms of neurobehavioral toxicology and teratology. These techniques have the potential to facilitate rapid advances in the understanding of normal neurobehavioral development and the mechanisms by which toxins and chemicals can perturb developmental processes. In addition zebrafish can provide a system for high throughput screening of compounds so that a starting point can be made for the thousands of compounds and mixtures that have yet to be characterized. This symposium will highlight some recent developments in approaches to studying neurodevelopmental processes in zebrafish and present data from neurobehavioral toxicology studies. Dr. Elwood Linney of the Duke University Medical Center will give an overview of the use of zebrafish in neurobehavioral toxicology. Dr. James Fadool of Florida State University will discuss the processes of retinal and visual development in zebrafish. Dr. Kathi Lefebvre of NOAA will discuss the behavioral effects and mechanistic action of algal toxins. Dr. John Ramsdell of NOAA will discuss the characterization of developmental toxicity of algal toxins using microinjection techniques. Dr. Frank Scalzo of Bard College will discuss stress effects in zebrafish. Dr. Edward Levin of the Duke University Medical Center will discuss the sensitivity of zebrafish to pharmacological and toxicological challenge in choice behavior studies. This joint symposium will provide the most recent developments in applying behavioral and molecular approaches to studying the mechanisms of abnormal neurobehavioral development.
NBTS 1/BTS 1 Zebrafish as a neurotoxicological and neurobehavioral model Linney E (Duke University Medical Center, Durham, NC) 0892-0362/$ - see front matter D 2005 Published by Elsevier Inc. doi:10.1016/j.ntt.2005.03.004
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NBTS 2/BTS 2 Molecular analysis of the zebrafish visual system Fadool JM (Florida State University) See BTS abstract listings for full text, unavailable at press time.
NBTS 3/BTS 3 Zebrafish: A tool for studying behavioral effects and mechanistic action of algal toxins Lefebvre K (National Oceanic and Atmospheric Administration, Seattle, WA) See BTS abstract listings for full text, unavailable at press time.
NBTS 4/BTS 4 Characterization of developmental toxicity of algal toxins after microinjection of fish embryos Ramsdell J (National Oceanic and Atmospheric Administration, Charleston, SC) See BTS abstract listings for full text, unavailable at press time.
NBTS 5/BTS 5 Neurobehavioral effects of stressors in embryonic zebrafish Scalzo FM (Psychology Program, Bard College, Annandale-onHudson, NY) See BTS abstract listings for full text, unavailable at press time.
NBTS 6/BTS 6 Zebrafish choice behavior: Sensitivity to pharmacological and toxicological challenge Levin ED (Duke University Medical Center, Durham, NC) See BTS abstract listings for full text, unavailable at press time.
NBTS 7 Imaging the Ontogeny of Self-Regulatory Control Peterson BS (Department of Psychiatry, Columbia University, and the New York State Psychiatry Institute, New York, New York (Introduced by D. Dow-Edwards)) Improvement in self-regulatory control largely defines the ontogeny of human development. Studying the neural basis of self-regulatory control has implications for understanding disease mechanisms that cut across multiple diagnostic domains. We will present findings from several anatomical and functional Magnetic Resonance Imaging (MRI) studies, comprising several hundred children and adults, that are helping to identify the neural bases of self-regulatory capacities in health and illness. Functional MRI studies of self-regulatory capacities include the Stroop and Simon interference tasks, anatomical studies include cortical and subcortical brain regions, and examples of pathological development are drawn from our studies of Tourette Syndrome and Bipolar Disorder. Findings from these studies suggest that increasing functional capacity of frontostriatal circuits are important for the development of increasing selfregulatory control, particularly during adolescence and early adulthood. Disturbances in the anatomical and functional integrity of these circuits seem to contribute to the development of serious psychopathologies in children and adolescents, and failure to develop appropriate compensatory responses likely determines adult outcome. Supported by NIH grants MH36197, MH068318, and DA017820.
NBTS 8 Binge alcohol exposure during adolescence produces behavioral, electrophysiological and biochemical adaptations in hippocampus Matthews DB, Silvers JM, Tokunaga S, Morrow AL (Department of Psychology, University of Memphis, Memphis Tennessee and University of North Carolina, Chapel Hill North Carolina (Introduced by Diana Dow-Edwards)) Alcohol use by adolescents is a profound, yet understudied, problem in the United States. Estimates suggest that over 2 million adolescent engage in binge alcohol use, yet the
underlying neurobiological consequences of this use is not clear. Male rats were injected with high dose alcohol, 5.0 g/ kg, every other day from postnatal (PN) day 30 to PN 50 and were then alcohol-free from PN 50 to PN 62. We found that animals develop tolerance to ethanol-induced hypnosis and metabolic tolerance that was long lasting. In addition, tolerance to ethanol induced spatial memory impairments were found at PN 50 but were absent at PN 62. Interestingly, tolerance to ethanol-induced inhibition of hippocampal pyramidal neurons was found at PN 50 but not at PN 62 mimicking the behavioral effect. Furthermore, tolerance to ethanol induced allopregnanolone levels in the hippocampus was found at PN 50 but levels were increased at PN 62. Finally, adaptations to hippocampal GABAA receptor peptide levels were found, and these adaptations were brain region and time specific. These data suggest that binge alcohol exposure during adolescents produces profound changes in hippocampal neurobiology. Supported by NIAAA.
NBTS 9 Cannabinoid/psychostimulant interactions in adolescent vs adult rats Izenwasser S (Dept. of Psychiatry and Behavioral Sciences, Univ. Miami School of Medicine, Miami, Florida (Introduced by: D. Dow-Edwards)) Studies show that a significant number of adolescents use cigarettes, and more than half of the teens who smoke daily also use illicit drugs. We have shown that chronic treatment with nicotine produces sensitization to its locomotor-activating effects in female and adult rats but not male adolescent rats. Thus, it appears that there are different adaptations in response to chronic nicotine in adolescent than adult rats. It is not known whether nicotine use alters the subsequent effects of cannabinoid and stimulant drugs. Thus we have been studying the effects of repeated nicotine administration on the behavioral effects of subsequently administered cocaine, amphetamine, methylphenidate, and cannabinoids. In adolescent male rats, there is a marked sensitization to cocaine, amphetamine, and methylphenidate-stimulated locomotor activity after treatment with nicotine, and this is not evident in adult rats or in female adolescent rats. In addition, there is an increase in cocaine self-administration in adult rats that were treated as adolescents with nicotine, but not in rats that were treated with nicotine during adulthood. Thus, it appears that the time during development of nicotine administration is the determining factor in the subsequent long-term adaptations that occur. Adolescent males pretreated with nicotine were also sensitized to the locomotor-decreasing effects of the cannabinoid agonist CP 55,940, whereas the adolescent females and adult males and females were not. This effect was blocked by co-administration of nicotine with the cannabinoid antagonist AM 251. Similarly, administration of AM
251 blocked the sensitization to cocaine seen subsequent to nicotine administration in adolescent male rats. We have found also that treatment with cannabinoid agonists leads to increased behavioral effects of cocaine in adolescent males. Thus, it appears that administration of nicotine or cannabinoids to adolescent males alters the response to cocaine or amphetamine long-term. In addition, it appears that the endogenous cannabinoid system is involved in the interaction between nicotine and cocaine in adolescent males, and that endogenous cannabinoids might play a more important role in regulating the effects of drugs in the adolescent male brain than in female or adult brains. Supported by the National Institute on Drug Abuse.
NBTS 10 Repeated exposure to low dose methylphenidate during adolescence enhances spatial working memory Dow-Edwards D, Lenderman M, Grullon L, Jackson A, Stephenson S (Department of Physiology/Pharmacology, SUNY Downstate, Brooklyn, New York) Methylphenidate (MPD; e.g., Ritalin) is a psychostimulant commonly administered to patients with ADHD. However, there are few pre-clinical studies of chronic clinically relevant MPD exposure during adolescence. In this study we wanted to see if various doses of MPD, administered to young rats of both genders would alter performance on the radial 8-arm maze (RAM) and other behavioral tests. The rats were housed in a reverse light– dark cycle and all procedures were performed with lights off. Rats received one daily dose of either 0, 1, or 3 mg/kg of MPD beginning on PND 22 by intragastric intubation. Forty minutes later they were tested on the RAM bated with chocolate chips or Noyes sugar pellets. Results from cohorts 1 and 2 (using chocolate chips) show that rats treated with 1 mg/kg of MPD performed significantly better on the RAM than rats in both the 3 mg/kg and control groups. Results from cohorts 3 and 4 (using the less-motivating sugar pellets) show that MPD at either 1 or 3 mg/kg improves performance but only after criterion is achieved and the maze is shifted to a new location. These data suggest that MPD improves spatial working memory in adolescence but that the effective dose depends on the motivation of the subject and the conditions of the test. In addition, MPD clearly has some lasting effects on responses to other psychostimulants and in other cognitive tests in adulthood. Supported by R21MH066852.
NBTS 11/BTS 7 Assessment of cognitive function: Bridging the gap from preclinical animal studies to the human condition Snyder PJ (Pfizer, Groton, CT)
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NBTS 12 Use of identical behavioral tasks in children and laboratory animals for studying chemical effects on a variety of cognitive functions Paule MG (Behavioral Toxicology Laboratories, Division of Neurotoxicology, FDA’s National Center for Toxicological Research (NCTR), Jefferson, Arkansas) Data needed to make predictions about a chemical’s neurotoxicity are obtained from lab animals in controlled experiments. While the use of appropriate animal models is critical, it is also important to utilize relevant endpoints. Maintenance of endpoint continuity across species (including humans) allows for the determination of interspecies similarities and validation of animal models and aids in the extrapolation of data. A battery of positively-reinforced behavioral tasks, the NCTR Operant Test Battery (OTB), has been developed for the assessment of behaviors thought to model motivation, color and position discrimination, time estimation, short-term memory, and learning. Comparative data show that the OTB performance of welltrained monkeys is generally indistinguishable from that of children. Human OTB performance correlates significantly with IQ, thus demonstrating relevance. The monkey model is a good predictor of chemical effects in humans and, for some chemicals, effects in rats are similar to those for monkeys and, thus, (presumably) humans. Tools like the OTB allow interspecies comparisons and provide the means for studying the effects of chemical agents in a variety of animal models using relevant endpoints. Use of the same or similar instruments in clinical settings could provide longitudinal data—within the same patients—on the adverse effects of chemical exposures, side effects and/ or efficacy of drug therapies or other interventions, and disease progression or remission.
NBTS 13 Cognitive testing in infant monkeys: Responding to biomedical and public health concerns Golub MS (California National Primate Research Center, Davis, California) Studies in nonhuman primates are often sought when substantial evidence has developed in rodent models but generalization to humans is uncertain, or when substantial evidence is available in humans but causal interpretations are difficult. Sometimes these studies are intended to settle a controversy and are considered the bdefinitiveQ test. Topics
that have been explored include malnutrition (protein, protein calorie), nutritional supplements (PUFA, iron), environmental toxicants (lead, mercury, TCDD, PCBs, methanol), abused substances (marihuana, cocaine, alcohol) and therapeutic agents (ritalin, obstetric drugs). Gradually standardized testing methods that parallel human infant assessment tools have been developed. The similarities between human and nonhuman primates in brain development, cognitive ability and mother–infant interaction lead to the impression that monkeys are always the bbestQ model for determining potential harmful effects of an agent on cognitive development. However, considerable further progress is needed for monkey experiments to meet this expectation. Basic research in brain and cognitive development of various nonhuman primate species is extremely limited and contemporary imaging and genetic screening technologies have seldom been applied. Sequential targeted hypothesis testing strategies are rare. New approaches, such as coordinated human– nonhuman primate and rodent–nonhuman primate research programs, are also needed if individual experiments are going to be able to contribute to resolution of high profile biomedical and public health issues. Supported by HD39386 and RR00169.
NBTS 14 Path integration versus spatial learning: What does the Cincinnati water maze (CWM) measure? Vorhees CV, Pu C, Fukumura M, Williams MT (Div. of Neurology, Cincinnati Children’s Research Foundation and Univ. of Cincinnati, Cincinnati, Ohio) The CWM is used to measure effects on learning, but unlike the Morris water maze (MWM) that was designed to measure a specific construct (spatial mapping), the CWM was validated empirically. We hypothesize that the CWM measures path integration learning. In Exp-1 rats were given hippocampal lesions. Lesioned rats were impaired in both mazes, but in the CWM only on path B. In Exp-2, comparable lesions were made but one subgroup received path order AB and the other half BA in the CWM followed by the MWM. Lesioned rats were impaired on both mazes, but this time on paths A and B, regardless of test order indicating that spatial cues are used in the CWM. To determine the role of these cues, rats were tested under different lighting conditions. In dim light, rats took longer to learn the CWM, whereas there was no difference in the MWM. In complete darkness, on the other hand, rats learned the CWM, albeit more slowly, whereas in the MWM rats learned a non-spatial strategy, but never reached asymptotic performance. The CWM measures path integration learning when tested in darkness, but both path integration and spatial mapping are used when distal cues are adequately lighted. Support by NIH grants DA06733 and DA14269.
NBTS 15 Adaptation to change as an index of cognitive function Weiss B (Department of Environmental Medicine, University of Rochester School of Medicine and Dentistry, Rochester NY) Adaptation to variable situational contingencies is the key to survival. Animals must respond appropriately to subtle cues about their environment to do so. Because natural environments are dynamic, they must be capable of discriminating and adjusting their behavior to unstable, often erratic circumstances. Much of human behavior also involves choice in response to changing contingencies; we make judgments all the time about the likely outcomes of our behavior (contingencies). It reflects our stochastic world, which we often treat, so to speak, as a Bayesian universe. To model such a situation in animals, we turn to schedule-controlled operant behavior because it provides the ability to trace, over time, how behavior responds to shifting contingencies. Examples come from research on lead, mercury, methanol, dioxin, and other neurotoxic agents. The examples demonstrate how responses to shifting relationships between behavior and its consequences serve as sensitive indicators of neurobehavioral function.
NBTS 16 Cigarette’s impact on cognitive performance in young adults following control for pre-smoking abilities Fried PA, Gray R (Carleton University, Ottawa, Ontario, Canada) Although the IQ of smokers is reported lower than nonsmokers, attributing that finding to cigarette use is problematic as smokers and nonsmokers differ on a host of demographic factors that impact on cognitive performance. A further issue is the performance prior to the initiation of regular cigarette use. These concerns are addressed utilizing the Ottawa Prenatal Prospective Study (OPPS) cohort. Assessed since birth, born to mothers recruited from a predominantly low-risk, middle class population, this cohort, presently young adults were examined in the present study. Using longitudinal smoking (and other drug use) data, the relationship between cigarette use and measures of cognitive performance was evaluated. Without considering controls, cigarette use is negatively related, in a dose response fashion to overall IQ, aspects of attention, and auditory aspects of memory—similar to the prenatal cigarette effects found in this sample prior to the initiation of smoking. When only either premorbid performance or prenatal nicotine exposure is controlled, these effects are somewhat attenuated. However, when both premorbid performance and prenatal exposure combined with demographic variables are considered, the negative relationships between current tobacco use
and cognitive performance are no longer evident. These results highlight the importance of considering a full spectra of background variables in interpreting the consequences of cigarette smoking on cognitive performance. Supported by NIDA grants to PAF.
NBTS 17 Perinatal nicotine exposure affects nicotine-stimulated rubidium efflux Britton AF, Vann RE, Robinson SE (Department of Pharmacology and Toxicology, Virginia Commonwealth University, Richmond, Virginia) Maternal smoking is a risk factor associated with nicotine abuse. We studied the effect of perinatal nicotine exposure on the response to nicotine across adolescence in the rat. On day 7 of gestation pregnant Sprague–Dawley rats were implanted subcutaneously with AlzetR osmotic minipumps delivering nicotine (l-nicotine tartrate, 2 mg/kg free base/day) or 0.9% saline. Rats were exposed both in utero and in the early postnatal period. Within 24 h of birth, litters were culled to no more than 10 pups, keeping equal numbers of each sex when possible. On postnatal days (PDs) 28, 35, 49, and 63, nicotine-stimulated 86Rb+ efflux was measured in synaptosomes prepared from the frontal cortex, striatum, hippocampus and thalamus–midbrain, using the method of Marks et al. (J. Pharmacol. Exp. Ther. 264: 542, 1995). One female and one male of each age were tested per litter. Data were analyzed by three-way (treatment x sex x age) analysis of variance (ANOVA). A main effect of treatment was found in the striatum, a main effect of age was found in the hippocampus and thalamus–midbrain, and a significant interaction of treatment x age was found in the striatum, cortex, and hippocampus. There was no effect of sex. A peak in nicotine-stimulated 86Rb+ efflux occurred on PDs 49 and/ or 35 in the vehicle control rats Essentially, perinatal nicotine exposure flattened this peak in nicotine-stimulated 86Rb+ efflux. The mechanism behind the reduced nicotine-stimulated efflux in adolescent rats remains to be investigated. Supported in part by Virginia Youth Tobacco Project.
NBTS 18 Prenatal cocaine and/or nicotine alters life span in laboratory rats in a sex-dependent manner Sobrian SK, Clark Jr E, Ressman K, Marr L, and Gerald ST (Department of Pharmacology, Howard University College of Medicine, Washington, DC) Prenatal exposure of laboratory rats to a variety of substance of abuse can alter longevity and enhance other age-related degeneration. To evaluate the hypothesis that prenatal exposure to cocaine and/or nicotine would shorten
life span, Sprague–Dawley females were exposed on GD 821 to 1 of 8 treatments: 20 mg/kg [LC]; 40 mg/kg [HC] cocaine; 2.5 mg/kg [LN]; 5 mg/kg [HN] nicotine; 20 mg/kg cocaine and 5 mg/kg nicotine [LC/HN]; 40 mg/kg cocaine and 2.5 mg/kg nicotine [HC/LN]; Pair Fed: yoked to HC [PF]; 8) Saline: injection of 0.9% NaCl [SAL]. Kaplan– Meier survival curves were constructed for uncensored mortality data, which were analyzed by the Logrank Test and ANOVAs. In female offspring, gestational exposure to either LC or HN alone shortened life-span; combined in utero drug exposure nullified this effect. In contrast, while neither HC or LN alone influenced life span, the combination of these two reduced longevity. The pattern of drug effects differed in males. Only the low dose of either drug alone reduced life-span. Observed effects were unlikely the result of prenatal undernutrition in that comparable life-spans were seen in PF and SAL offspring of both sexes, with female showing slightly longer longevity than males. However, at both cocaine doses and in the HC/LN group, males outlived females. These results indicate that prenatal exposure to cocaine or nicotine, either alone or in combination, can shorten life-span in rat. The sexually dimorphic nature of the changes suggest gender differences in prenatal sensitivity to drugs of abuse. Supporter by NIH Grant S06GM08016.
NBTS 19 Assessment of selective attention and error reactivity in fmr1 knockout (KO) mice: A mouse model of Fragile X syndrome (FXS) Strupp BJ, Moon J, Beaudin A, Veros-Ky S, Driscoll LL, Crnic LS, Levit-Sky DA (Cornell Univ., Ithaca, NY, and Univ. Colorado Health Sci. Ctr., Denver, CO) This study assessed selective attention and reactivity to errors in the fmr1 KO mouse model of Fragile X syndrome (FXS). Control (n = 19) and fmr1 KO (n = 20) male mice were tested on a selective attention task. The onset time and location of a brief visual cue varied randomly across trials; an olfactory distractor was presented prior to the cue on 1/3 of the trials. During the first 3 sessions (c200 trials), the fmr1 KO mice performed more poorly than controls. Because (a) the impairment was most pronounced on trials without a distractor, and (b) the groups did not differ on a baseline task without distractors, it appears that the fmr1 KO mice experienced a more generalized disruption to the distractors than controls. The drop in performance produced by an error on the prior trial was greater for the fmr1 KO mice than for controls. The pattern of effects indicated impaired regulation of arousal (caused by distractors or an error) in the fmr1 KO mice which, in turn, disrupted attention and inhibitory control. These findings are consistent with the problems in arousal regulation, attention,
and inhibitory control seen in humans with FXS. Videotapes of performance revealed that distractor-induced grooming was greater for the fmr1 KO mice than controls. These findings provide important support for the validity of the fmr1 KO mouse as a model of FXS; many previous studies have encountered difficulty in demonstrating cognitive deficits in these mice. More generally, this task provides a novel tool for assessing distractibility in mice. Supported by NIH (HDO4024 and HDO47029).
NBTS 20 Prenatal folate deficiency in mice increases adult anxiety levels Berry KJ1, Ferguson SA1, Hansen DK1, Antony AC2,3, Wall KS1, White G1,4 (1National Center for Toxicological Research, 2Indiana University School of Medicine, 3Richard L. Roudebush Veterans Affairs Medical Center, 4Toxicological Pathology Associates) Since the fortification of food with folic acid in 1997, human serum folate levels have increased; however, there remain high incidences of folic acid deficiencies worldwide. Concerns focused on severe developmental folate deficiency and the resulting teratological effects. The effects of less severe folate deficiency on CNS functioning has not been fully investigated. Because low serum folate levels in adults are associated with mood disorders, the effects of developmental folate deficiencies on later anxiety were investigated. Here, female CD-1 mice were fed chow containing 400-, 600-, or 1200-nmol folic acid/ kg diet for 8 weeks prior to breeding and until gestational day 18, at which time all dams were placed on folatereplete chow. A tester, blind to dietary group, evaluated anxiety behavior in PND 60 male and female offspring using the elevated plus maze (5-min sessions). Mice in the 400 nmol folate/kg group spent less time in the open arms ( p b 0.05) and more time in the closed arms ( p b 0.05). A significant effect of dietary group on total activity indicated that mice in the 400 nmol folate/kg group were more active overall ( p b 0.05). These data suggest that specific micronutrient deficiencies during prenatal development can later manifest in adulthood as changes in behavior thought to reflect increased anxiety.
NBTS 21 Rodent strain differences in performance of operant tasks measuring time estimation and impulsivity Ferguson SA, Paule MG, Cada A, Fogle CM, Gray EP (Division of Neurotoxicology, NCTR/FDA, Jefferson, AR) The Spontaneously Hypertensive Rat (SHR) is proposed as a model of childhood Attention Deficit Hyperactivity
Disorder (ADHD). However, the degree of impulsivity and inattentiveness is inconsistent across studies. Few studies compare the SHR to other common strains, although there is controversy in the continued use of the Wistar–Kyoto (WKY) as the control strain. Here, food-restricted male and female SHR (n = 20), WKY (n = 21), and Sprague– Dawley (SD) (n = 22) rats began training at 21 weeks of age on one of two operant tasks: 1) Temporal response differentiation (TRD) in which a lever hold duration of 10–14 s was reinforced, or 2) Differential reinforcement of low rates (DRL) in which the withholding of a lever press for 10–14 s was reinforced. There were no strain differences in TRD acquisition; however, male SHRs required more sessions to acquire the DRL task than SD or WKY males (38 vs. 15–21, respectively). Relative to SDs, baseline performance for both tasks was worse in the SHRs and WKYs (TRD and DRL accuracy was decreased by c 14% and 9%, respectively) but was unaffected by sex. TRD response rate was somewhat higher for SHRs and significantly higher for WKYs relative to SDs. These results indicate that the SHR and WKY strains are more alike than they are dissimilar and argue that cognitive functioning in these two strains, particularly that related to timing abilities, is impaired relative to the SD.
NBTS 22 An evaluation and interpretation of neurodevelopmental endpoints for human health risk assessment—Introductory remarks Walls I1, Brimijoin S2 (1ILSI Risk Science Institute, Washington DC, 2Mayo Clinic, Rochester, MN) In 1991, the US EPA issued a standardized protocol for evaluation of developmental neurotoxicity (DNT) and OECD is working on a consensus protocol for inclusion in the OECD Testing Guidelines Program. The EPA protocol is comprised of tests for evidence of deficits in neurobehavioral function, including auditory startle habituation, motor activity, associative learning and memory, and neuropathologic examination, including simple morphometric analysis. Sufficient data have now been generated to support the development of a scientific consensus regarding the endpoints measured in the DNT and their interpretation for human health risk assessment. In 2004 the ILSI Risk Science Institute established an Expert Panel to assess the lessons learned to date from the implementation of standardized tests for developmental neurotoxicity in experimental animals, and the subsequent application of test results to human health risk assessment. Five manuscripts are being prepared, on application to public health protection, use of positive controls, data interpretation, understanding variability in the data and appropriate statistical techniques. Supported by ILSI Risk Science Institute and USEPA.
This is an abstract of a proposed presentation and does not necessarily reflect EPA policy.
NBTS 23 Application of developmental neurotoxicity testing to public health protection Fenner-Crisp P1, Adams J 2, Balbus J 3, Bellinger D4, Brimijoin S 5, Makris S 6, Marrs T 7, Ray D 8 (1Consultant, ILSI Risk Science Institute, Charlottesville, VA, 2University of Massachusetts, Boston, MA, 3Environmental Defense, Washington, DC, 4Harvard University, Boston, MA, 5Mayo Clinic, Rochester, MN, 6NCEA, ORD, USEPA, Washington, DC, 7UK Food Standards Agency, London, UK, 8University of Nottingham, Nottingham, UK) Improved understanding of the neurotoxic effects of chemicals has become a high public health and societal priority in the United States. The extent to which environmental exposures contribute to the wide range of neurodevelopmental disorders is unclear. The developmental neurotoxicity guideline (OPPTS 870.6300) is the tool currently available for testing the neurotoxic potential of environmental and other agents in laboratory animals. Its ability to predict effects in the human when test results are positive is examined and illustrated with case examples. Its role as a public health protective tool when test results are negative also is explored. New and emerging technologies which may serve in addition to, or in place of, currently-tested endpoints are described. Lastly, the endpoints measured in the existing guideline and those that may be measured in follow-up studies are evaluated for their contribution to defining the mode/ mechanism of neurotoxic action. Supported by ILSI Risk Science Institute and USEPA. This is an abstract of a proposed presentation and does not necessarily reflect EPA policy.
NBTS 24 Undertaking positive control studies as part of developmental neurotoxicity testing Crofton KM1, Foss JA2, Hass U3, Jensen K1, Levin ED4, Parker SL5 (1Neurotoxicology, NHEERL, ORD, USEPA, RTP, NC, 2CRDDS Argus Division, Horsham, PA, 3Danish Institute for Food and Veterinary Research, Soborg, Denmark, 4Department of Psychiatry and Behavioral Sciences, Duke University Medical Center, Durham, NC, 5OrbusMedical Technologies, Fort Lauderdale, FL) Developmental neurotoxicity testing involves functional and neuropathological assessments in offspring during and following maternal exposure. Positive control data are instrumental in evaluating laboratory proficiency
in detecting chemical-induced changes in measured endpoints. Positive control data are valuable in a weight-of-evidence approach to help determine the biological significance of results and provide confidence in negative results from DNT studies. This review provides a practical guide for the use and interpretation of positive control studies in developmental neurotoxicology. Guidance is given on the selection and use of positive controls. The advantages and disadvantages of positive control agents are discussed for the endpoints in developmental neurotoxicity studies. Design issues specific to positive control studies in developmental neurotoxicity are examined. 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 that generate data used in risk decisions. Supported by ILSI Risk Science Institute and USEPA. This is an abstract of a proposed presentation and does not necessarily reflect EPA policy.
NBTS 25 Identification and interpretation of treatment-related effects in developmental neurotoxicity (DNT) testing Tyl RW 1, Brimijoin S 2, Moretto A 3, Sheets L 4, Sobotka T 5, Mendez E 6 (1RTI, Research Triangle Park, NC., 2Mayo Clinic, Rochester, MN, 3University of Padua, Italy, 4Bayer CropScience, Stilwell KS, 5FDA CFSAN, Laurel MD,6US EPA, OPP, Arlington, VA) The reliable detection, measurement and interpretation of treatment-related DNT effects depend on appropriate study design and execution, using scientifically established methodologies, with appropriate controls to minimize confounding factors. Appropriate statistical approaches should be optimized in advance for the specific endpoints, analyzing effects across time and functional domains as far as possible. Biomarkers of exposure are useful to assess the bioavailability of toxicants to the dam and offspring in utero and after birth. bWeight of evidenceQ principles then aid assessment of the biological significance of statistically significant differences from concurrent controls. These effects should be interpreted in light of available information from historical controls, positive controls, maternal and offspring systemic toxicity, and other relevant toxicological data. This review provides a framework for the integration of all these types of information in the interpretation of DNT studies. Supported by the ILSI Risk Science Institute and the US EPA. This is an abstract of a proposed presentation and does not necessarily reflect EPA policy.
NBTS 26 Framework for determining normal variability for endpoints measured in a developmental neurotoxicity test Raffaele KC 1, Fisher JE 2, Hancock S 3, Hazelden KP4, Sobrian SK 5 (1HED, OPP, US EPA, Washington DC, 2DNDP, CDER, FDA, Rockville, MD, 3Health Canada, Ottawa, Canada, 4 Huntingdon Life Sciences, East Millstone, NJ, USA, 5 Howard University College of Medicine, Washington DC) With the implementation of the Food Quality Protection Act in 1996, more detailed evaluations of possible health effects of pesticides on developing organisms have been required. As a result, considerable developmental neurotoxicity (DNT) data have been generated on a variety of endpoints, including developmental changes in motor activity, auditory startle habituation, and various learning and memory parameters. One issue in interpreting these data is the level of variability for such parameters, as measured in this type of study: excessive variability can obscure treatment-related effects, or conversely, small but statistically significant changes could be viewed as treatment-related, when they might in fact be within the normal range. To aid laboratories in designing useful regulatory DNT studies, a framework for evaluating observed variability in study data has been developed. Elements of the framework include: characterization of variability in the dataset; identification of appropriate datasets for comparison; evaluation of similarities and differences in variability between these datasets, and of possible sources of the variability, including those related to test conduct and test design. A case study using auditory startle habituation data is presented, employing the proposed framework. Supported by the ILSI Risk Science Institute and USEPA. This abstract of a proposed presentation does not necessarily reflect EPA policy.
NBTS 27 Statistical issues and techniques appropriate for developmental neurotoxicity (DNT) testing Holson RR1, Freshwater LL2, Maurissen JP 3, Moser VC4, Phang W 5 (1New Mexico Tech, Socorro, NM, 2BioSTAT Consultants, Portage, MI, 3The Dow Chemical Company, Midland, MI, 4 NTD, NHEERL, USEPA, Research Triangle Park, NC, 5 OPP/OPPTS, USEPA, Washington, DC) Statistical issues (design and analysis) raised by the EPA DNT guideline are complex. A review of submitted datasets illustrated several inadequate approaches, including issues of Type I error control, and ignoring gender, time and litter allocation as factors in the analyses. Since the DNT study
includes numerous experimental procedures conducted at several ages, it is not unusual to have hundreds of significance tests at a = 0.05; this drastically increases the overall error rate. Two general approaches to control experiment-wise Type I inflation are proposed: 1) statistical/design considerations that help to reduce the number of p values (e.g., factorial designs, multivariate techniques, repeated-measure analyses, etc.); 2) adjustments to the a level, including newer approaches less conservative than a Bonferonni correction. The design of the DNT includes testing of both sexes, and gender must be included in the statistical analysis for the determination of sex-related differences. Statistical power is also addressed in the context of control of Type II errors, and including both sexes may increase power. One male and one female can be drawn from each litter (mixed ANOVA design), or each litter can contribute one male or one female (fixed ANOVA design); the latter model has greater power than the former. Some of the statistical approaches for analyses of DNT data will be described. The importance of a priori specifying the planned statistical analysis is discussed. Supported by the ILSI Risk Science Institute and US EPA. This is an abstract of a proposed presentation and does not necessarily reflect EPA policy.
NBTS 28 Toxicity test validation, positive controls and proficiency: Are chemicals necessary? MAURISSEN JP a, MARABLE BRb (Neurotoxicology, aThe Dow Chemical Company, Midland, Michigan and bSyracuse Research Corporation, Syracuse, New York) The USEPA neurotoxicity guidelines require the use of positive control data in support of toxicology test data submitted for the registration or re-registration of selected test materials and emphasize the use of chemicals to accomplish this. These guidelines, though, propose different rationales for the use of chemicals as positive control agents. An attempt is made to re-evaluate the cardinal questions that should be asked before a particular test is used. They can be summarized as follows: 1) What does the test measure? 2) Is the performing laboratory proficient in the use of the test? 3) Do the complementary data submitted in support of the test material provide enough context for the interpretation of the biological significance of an effect? Even though the almost exclusive use of chemicals has been emphasized to provide support for a test material submission, consideration should be given to the use of experimental procedures instead of chemicals, if possible, to accomplish the intended goal in light of animal welfare considerations. Supported by The Dow Chemical Company.
NBTS 29 Prenatal cocaine exposure-induced changes in the expression of dopamine receptor-signaling genes in the mouse cerebral cortex He F*, Novikova SI*, Bai J*, Lidow MS (Department of Biomed. Sci., University of Maryland, Baltimore, MD) The dopamine system is a major target of cocaine (COC). We used microarray analysis to examine alterations in the expression of dopamine receptor (DR) signaling genes in the mouse cerebral cortex (CC) induced by prenatal COC exposure (20 mg/kg s.c., t.i.d., E8-E18). Changes in the fetal (E18) and adult (P 50) CC were examined. We found alterations in multiple key molecules involved in DR signaling in the CC of the fetal and adult mice, which were exposed to COC in utero. These include changes not only in the DRs themselves [upregulation of DR1B (DR5) and DR3 and down-regulation of DR2], but also changes in the expression of such proteins as receptor deactivation- and sequestration-associated molecules as b2-arrestin and dynamin-2 (also linking dopamine receptors to ERK and JNK3 pathways), the third messenger of dopamine signaling, DARPP-32 and its regulators, and the DR1-Gq-protein linker, calcyon (fetal CC only). Our data suggest that prenatal COC exposure likely leads to a permanent reduction in the DARPP-32 induced suppression of PP1, which may reduce DR1 dephosphorylation (a previously-described effect). In addition, COC is likely to increase deactivation and sequestration of DRs. Prenatal COC exposure may also lead to over-activation of ERK and JNK3 pathways. Finally, in the fetal CC, COC may increase dopamineinduced activation of inositol phosphate turnover. These findings indicate that prenatal COC exposure has a strong potential to disrupt DR-associated intracellular signaling in the fetal and adult CC. Supported by NIDA RO1 grant DA08057.
NBTS 30 Harmful effects of perinatal omega-3 fatty acid deficiency and excess on the neurodiagnostic auditory brainstem response (ABR): Preliminary results Church MW, Jen K-LC * (Department Ob/Gyn and Nutrition and Food Sci., Wayne State Univ., Detroit, Michigan) It is well known that perinatal omega-3 fatty acid (x3) deficiency can cause neurodevelopmental and growth impairments. It is less well known that perinatal x-3 excess can have similar effects. Since large N-3 doses will be given clinically to prolong gestation in pregnant women at risk for preterm delivery, it is important to know more about the beneficial and harmful effects of
such treatments. Our objective was to compare perinatal N-3 deficiency and excess on offspring outcome, using the ABR as a measure of brain development and auditory function. Three diets were used: Low N-3 (safflower oil, ~0% N-3 fatty acids), High N-3 (Menhaden oil, N-3/N-6 ratio~14.5) and Control N-3 (soybean oil, N-3/N-6 ratio~0.14). The dams were kept on their diets during pregnancy and lactation. One male and one female offspring/litter were ABR tested at the postnatal age of 24 days. To date, 4 of 22 (18%) Low N-3 offspring, 4 of 16 (25%) High N-3 offspring, and 0 of 11 (0%) Control N-3 offspring had abnormal ABRs. Seven of 8 abnormal ABRs were indicative of prolonged brainstem neural transmission times (z 2 SD above norms). One abnormal ABR was indicative of a 20 dB sensorineural hearing loss. The Low and High N-3 litters also showed reduced birth weights, increased postnatal mortality, and alopecia. These preliminary results suggest that perinatal N-3 deficiency and excess cause similarly harmful neurological and developmental effects. Supported by a Gerber Foundation grant.
NBTS 31 Intergenerational effects of chronic and intermittent cocaine exposure on maternal behavior in next generation non-lactating offspring McMurray MS, Haslup A, Mirza R, Jarrett T, Walker C, Rodrigues T, Hofler V, Johns JM (Department of Psychiatry, University of North Carolina, Chapel Hill, North Carolina) Induction of maternal behavior in juveniles and nonparturient adults by continuous exposure to pups has been well documented and determined to be highly similar to the maternal behavior of parturient females hormonally. Research in our lab has shown that cocaine disrupts the normal onset of maternal behavior in lactating rat dams and their daughters. To determine if pup-induced maternal behavior is affected by rearing or prenatal drug exposure similarly to maternal behavior in cross-fostered lactating rats, next generation male and female offspring were tested for pup-induced maternal behavior at 28 and 60 (male only) days of age. Results demonstrate that rats prenatally exposed to cocaine or reared by dams treated with cocaine respond to pups significantly less at both test periods. Oxytocin levels in the medial preoptic area, ventral tegmental area, amygdala, and hippocampus were measured the day after test completion to compare with previous findings of cocaine’s effects on oxytocin and maternal behavior. Results indicate an intergeneration effect of cocaine on maternal behavior. This work was supported by NIH grant DA13362-01, and the NIH Federal Child Neglect Consortium.
NBTS 32 Confounding effects of prepulse stimulus intensity and frequency type on prepulse inhibition in scopolamine treated rats Andrus AKa,b, Marable BRc, Dunbar GLb, Reilly MPb, Maurissen JPJ a,b ( aToxicology and Environmental Research and Consulting, The Dow Chemical Company, Midland, Michigan. bDepartment of Psychology, Central Michigan University, Mt. Pleasant, Michigan. cSyracuse Research Corporation, Syracuse, New York) Prepulse inhibition (PPI) of the auditory startle response (ASR) has been recommended for use in the USEPA Developmental Neurotoxicity guideline. The purpose of this study was to assess aspects of the prepulse stimulus that may modulate treatment effects. To accomplish this, PPI of the ASR peak amplitude was measured in rats, and the intensity and type of auditory prepulse (a 10-kHz tone vs. a white noise burst) were varied to observe the potential interaction of these parameters with scopolamine treatment. Percent of PPI increased with the prepulse intensity. Although scopolamine treatment attenuated PPI, no interaction between scopolamine and prepulse frequency type or intensity was observed. It was noted that prepulses of certain intensities were eliciting a startle response. The response to the prepulse was greater when the prepulse was a white noise burst vs. a tone of the same intensity. Further, the startle to the prepulse altered the amount of inhibition and thus confounded the overall measure of PPI at the higher intensity levels. The parametric manipulation of the prepulse had a larger effect on PPI than scopolamine treatment. Sufficient precaution is necessary when reviewing the PPI literature or planning PPI experiments, as the methods used may alter whether or not treatment effects are observed. Supported by The Dow Chemical Company.
NBTS 33 Iron (Fe) deprivation and infant behavior in monkeys; Prenatal vs postnatal effects during the first three months of life Golub MS, Hogrefe CE, Germann SL, Capitanio JP (California National Primate Research Center, Davis, California) Developmental iron (Fe) deficiency is associated with motor and cognitive impairment in human infants, but critical periods for producing this effect are not known. In this study, rhesus monkeys (Macaca mulatta) were fed Fe deficient diets either prenatally (dams via purified diet, 10 Ag Fe/g, n = 14) or postnatally (infants, via formula, 12 Ag Fe/g, n = 12) and compared to controls fed Fe-adequate diets (dam, 30 Ag Fe/g, infant 93 Ag Fe/g) (n = 12). Prenatal
deprivation led to anemia in dams and altered Fe status in neonates that recovered by 1 month of age. Postnatal deprivation did not produce anemia in infants. There were no effects on birth weight or postnatal weight gain. Head width was smaller in the prenatally deprived group. Early behavioral evaluations (neonatal assessment, novelty preference, fine motor tests, gross motor observations) were not generally affected by Fe deprivation. Later in the 3 month period, the postnatal group was impaired in reaching criterion for object constancy and demonstrated higher frequencies of inactive behavior (laying on the bottom of the cage) during weekly observation sessions. Prenatally deprived infants showed more behavior changes during observation sessions in the third month of life. Behavior rating scales by a blind observer at 3–4 months of age characterized the prenatal group as less bfearfulQ and postnatal group as more btenseQ than controls. Continuing evaluation of this cohort will include cognitive testing. These results may help identify the optimal time for correcting developmental Fe deficiency. Supported by HD39386.
NBTS 34 Multiple toxic effects of in utero exposure to DEHP Mcfadden HG, Sahay N, Pizzi WJ (Northeastern Illinois University, Chicago, Illinois) Di(2-ethylhexyl) phthalate (DEHP) is used as a plasticizer in polyvinyl chloride (PVC) plastics. DEHP does not bind with PVC, and migrates into the environment at a rate responsive to temperature, agitation, and characteristics of the substance in contact with PVC. Given the ubiquitous use of PVC in medicine and in food storage, there are many points of exposure to DEHP. Routes of exposure for humans include inhalation, ingestion, and through medical fluids such as saline or plasma. DEHP is a suspected endocrine disrupter and thus may have extensive effects following in utero exposure. Two studies were conducted in which DEHP was delivered daily by oral gavage in an olive oil vehicle to pregnant rats at doses from 250–1000 mg/kg bodyweight from GD 8-21. The first study was a dose-ranging study, while the second was a litter design study with a larger subject pool. Widespread organ toxicity in offspring was found involving the heart, kidneys, testes, and seminal vesicles. A sex difference in the toxicity profile was present, with males showing toxicity while females were generally less or not at all affected. A reproduction screen showed reproductive deficits in DEHP-exposed males (females not screened). DEHP-exposed offspring showed decreased locomotor activity relative to controls on a pivoting locomotion task at PND 7-11. All toxic effects occurred at the higher doses, raising the question of likely exposure levels in humans. The selective toxicity in male
offspring suggests an endocrine mechanism related to testicular development and testosterone production during critical periods of development.
NBTS 35 Cocaine exposure limited to early pregnancy in rats produces lasting impairment in selective attention: Evidence from extra-dimensional shift (EDS) tasks Benedetto TL, Beaudin SA, Mactu-Tus CF, Levitsky DA, Booze RM, Straw-Derman M, Strupp BJ (Cornell Univ., Ithaca, NY, Univ. of S. Carolina, Columbia, SC) Previous research has demonstrated that prenatal cocaine exposure produces lasting deficits in attention and arousal regulation. In the present study, the timing and duration of cocaine exposure was varied in different groups of pregnant Long Evans dams. Specifically, cocaine was administered IV during E8-14, E15-21, or E8-21; control dams received IV saline. Following 8 months of behavioral testing, the offspring were euthanized and the density of several DA and NE receptors in specific brain regions was determined using autoradiography; these indices were correlated with the behavioral measures. This poster will present the results of a series of EDS tasks, in which the predictive dimension was shifted between olfactory, spatial, and visual domains. Preliminary analyses revealed that the rats exposed to cocaine only during E8-14 were significantly impaired relative to controls, specifically when the task involved very subtle predictive cues (visual) and salient nonpredictive cues (olfactory and spatial; p = 0.02). In contrast, prenatal cocaine exposure did not alter the rate of learning EDS tasks in which the predictive cues were salient and the irrelevant cues were subtle. The pattern of results supports earlier evidence that prenatal cocaine exposure produces lasting impairment in selective attention, and demonstrates this effect with a shorter duration of exposure than previously shown. Correlations between receptor density and performance measures will be discussed. Supported by NIH (DA13965, DA09160, DA014401).
NBTS 36 Adolescent nicotine exposure produces dose-dependent changes in cocaine sensitivity in adult rats Nolley EP, Kelley BM, Lyons MD, Wiley AR, Rowan JD (Department of Psychology, Bridgewater College, Bridgewater, Virginia) Recent findings suggest that adolescent nicotine exposure may permanently disrupt reward systems and drug sensitivity. Motor activity was used to evaluate
changes in cocaine sensitivity due to nicotine exposure during adolescence. Extending our previous work, this study used rats in place of mice. Testing was performed on adult male Sprague–Dawley rats exposed to nicotine (0.3, 1.0, and 3.0 mg/kg SC, M-F, b.i.d.) during adolescence. Prior to testing, subjects had a 19-day time-off period. After acclimating to the testing apparatus, the locomotor effects (30 min, cm traveled) of cocaine (5, 10, 20, 40 mg/kg, IP) were measured daily. Overall, subjects exposed to nicotine exhibited decreased sensitivity to cocaine as indicated by lower levels of motor activity. As in previous studies, an apparent dose-dependant relationship exists between the amount of nicotine received during development and adult cocaine sensitivity. Across the testing period, the control and 1.0 mg/kg dose groups exhibited heightened sensitization to cocaine’s locomotor effects compared to the low and high dose groups. While these results are somewhat different from previous findings in mice, the results demonstrate a doseresponse relationship between long-term, low-level adolescent nicotine exposure and changes in cocaine sensitivity in adulthood.
NBTS 37 Neuropsychological markers of ADHD risk in substance exposed and non-exposed infants Noland JS, Singer LT, Short EJ, Minnes S, Arendt RE, Simpson KS (Department of Psychology and Human Development, Vanderbilt Kennedy Center, Vanderbilt University, Nashville, Tennessee; Department of Pediatrics, Case Western Reserve University, Cleveland, OH) Response inhibition and working memory have demonstrated reliance on the neurological circuits by which neurotoxic exposures might interact with genetic predisposition for ADHD. Infant assessments of these processes are necessary for understanding the role of neuroteratology in ADHD. Look duration in the 6–12 month range may reflect response inhibition. In a longitudinal sample of poly-substance exposed children, infant look duration predicted unselective responses on a parallel search task at 4 years of age. In a new cohort of cigarette exposed infants, we have been developing assessments that separately tap working memory and response inhibition. The working memory task is a looking version of the delayed response task. The response inhibition tasks are 1) a reaching version of the delayed response task without the memory demands; and 2) an object retrieval task in which the target is hidden behind a clear barrier. Preliminarily, exposed infants are making faster and more impulsive responses and demonstrating less working memory. However, data collection is in the early stage.
NBTS 38 Developmental outcome of children prenatally exposed to carbamazepine Janulewicz P1, Adams J1, Dhillon R 2, Holmes LB2 (1Psychology, Univ. of Massachusetts Boston, 2Genetics and Teratology, Mass General Hospital, Boston, Massachusetts) Carbamazepine (CBZ; Tegretolk) has been associated with increased risks for neural tube defects, minor malformations involving craniofacial structures, digit and nail hypoplasia, reduced birthweight and head circumference, developmental delay, and reduced cognitive performance (Granstrom and Hiilesma, 1982; Jones et al. 1989; Lindhout et al. 1985; Ornoy and Cohen, 1996). Part of our ongoing research examines outcome following gestational monotherapy with CBZ (CBZ-E) in comparison to control children matched on age and maternal demographics. Herein we report on 35 CBZ-E and 35 matched control children recruited from a sample identified at birth in Boston hospitals, via referral from neurologists (serving mothers), or from other community resources. On WISCIII IQ measures, CBZ-E scored 8 or more points lower than controls on fullscale ( p = 0.001), verbal ( p = 0.001), and performance IQ ( p = 0.037). Across subtests from multiple cognitive domains, reductions in language-based processing scores more often represented an area of weakness. Our current results on 3 drugs suggest greater cognitive concerns for Phenobarbital and CBZ than for hydantoin monotherapy. Supported by Pfizer, Inc.
NBTS 39 Succimer chelation normalizes emotion regulation in lead-exposed rats: Evidence from an olfactory conditional association task Beaudin SA, Stangle DE1, Strawderman MS1, Smith D2, Levitsky DA1, Strupp BJ 1 (1Cornell University, Ithaca, NY, 2University of California, Santa Cruz, CA) This study evaluated a 3-week placebo-controlled trial of succimer chelation in rats exposed to Pb for the first 4 weeks of life. This presentation will focus on the results of a conditional olfactory discrimination task with surprising reward omission (SRO) on some correct trials. Pb exposure did not affect learning rate or asymptotic performance. However, the disruptive effect of committing an error on the previous trial was greater for the Pbexposed, placebo-treated rats than for controls, for both accuracy and response latency. Similarly, analysis of the latency to enter the testing alcove at trial onset revealed that these rats were more disrupted by the SRO procedure than controls. All three Pb effects indicate impaired
regulation of affect or emotion. In contrast, the succimer-treated Pb rats were indistinguishable from controls on all three measures, consistent with the finding that succimer also significantly speeded the removal of Pb from both blood and brain. Note, however, that some tasks and dependent measures administered in this study revealed greater benefit of succimer than others; some revealed no benefit. An additional important finding from this task, and from others in this study, is that succimer chelation produced lasting cognitive and affective dysfunction in rats that had not been exposed to Pb. These findings provide important new evidence that succimer chelation can significantly ameliorate the lasting dysfunction produced by early Pb exposure, but that there may be risks of continued succimer treatment after Pb has been removed from the body. Supported by NIH: ES07457, ES05950; DK07158.
NBTS 40 Prenatal cocaine exposure up-regulates caspase-3 in noradrenergic locus coeruleus neurons Dey S, Booze RM, Mactutus CF, Snow DM (Univ. of Kentucky, Lexington, KY, and USC, Columbia, SC) Prenatal exposure to cocaine induces neurological and behavioral alterations in children and increases the likelihood for attentional disorders. Previous studies from our groups have shown that prenatal cocaine exposure inhibits neurite initiation, elongation and branching of noradrenergic locus coeruleus (LC) neurons, and induces LC cell loss and decreased cell volume. Further, we showed that for LC neurons, cocaine induced a decrease in neurite outgrowth, but did not significantly affect outgrowth for dopaminergic, substantia nigra (SN) neurons, indicating specificity of cocaine for LC neurons. In the present study, we investigated putative mechanisms underlying these effects. We examined the regulation of the apoptotic pathway effector molecule, caspase-3 in LC and SN neurons, and correlated activity with cell differentiation. Fluorometric analyses to measure caspase-3 activity (Wallac Vector III Cytofluorometer) were performed using the caspase-3 substrate Ac-DEVD-AMC. Results showed that caspase-3 was activated at 1, 4 and 24 h in LC in response to treatment with cocaine (500 ng/ml), showing peak activity at 1 h. This upregulation occurred during robust stages of cell differentiation. However, significant caspase activity was not observed for SN. These data encourage further investigation of other upstream or downstream components involved in the apoptotic signaling cascade to provide potential targets for pharmacotherapeutic interventions for cocaine abused offspring. Support: DA12719, DA09160, DA013965 and DA014401.
NBTS 41 Cocaine exposure throughout gestation results in decreased sensitivity to cocaine in adulthood: Effects on timing behavior in rhesus monkeys Paule MG1,2, Chelonis JJ1,2,3, Gillam MP,1, Graham SA 2 (1Division of Neurotoxicology, National Center for Toxicological Research, Jefferson, Arkansas, 2Dept Pediatrics, Arkansas Children’s Hospital and 3U Arkansas Little Rock, Little Rock, Arkansas) Animals exposed to cocaine prenatally were challenged with several doses of cocaine as young adults. Treatment groups consisted of controls (n = 7) and animals that received cocaine throughout the whole of gestation (TGE; n = 5). TGE females were treated IM for several months, prior to and during mating, with doses of 1.0–3.0 mg/kg/injection, 3 / day, 5 days/week. After pregnancies were detected (ca. gestational day 30–40), doses were gradually escalated until birth as animals became tolerant to cocaine-induced appetite suppression. Maximum doses at birth ranged from 3.5 to 7.5 mg/kg/injection at term (~0.75–1.5 g/day, human equivalent). At birth, TGE offspring weighed significantly less and had smaller head circumferences than controls. At 6 years of age, cocaine dose–response curves were obtained: 0.3, 1.0 and 1.75 mg/kg were given IV prior to performance of a timing task for food treats. Subjects had to hold a response lever down for N 10 s but b 14 s. Endpoints included total holds and percent correct holds. Controls were significantly more sensitive to cocaine than TGE animals. Doses of 1.0 mg/kg greatly reduced responding in controls but had no effect in TGEs. These data demonstrate that exposure to cocaine throughout gestation can cause long-lasting (permanent?) alterations in sensitivity to subsequent exposures. Supported by NIDA grants 1 R03 DA12901-01 and 1 R01 DA13152-01 to JJC.
NBTS 42 Analysis of the behavioral and neurochemical consequences of (F)3,4-methylenedioxymethamphetamine (MDMA or becstasyQ) Piper BJ, Fraiman JB, Owens CB, Meyer JS (Neuroscience and Behavior Program, Department of Psychology, University of Massachusetts, Amherst, MA) The recreational drug MDMA damages several markers of serotonin system integrity and alters functional behavior. However, it is not known whether MDMA induced serotonin neurotoxicity causes these behavioral consequences or if another mechanism is responsible. The objective of this investigation was to examine the relationship between the behavioral and neurochemical consequences of MDMA. Male, Sprague–Dawley rats (n = 67) received F MDMA
(4 10 mg/kg, s.c.) with or without citalopram (10 mg/kg) pretreatment. Core temperature, sexual response, and body weight were monitored during and immediately following drug treatments. A battery of assessments measuring motor, cognitive, exploratory, anxiety, social and aggressive behavior were also completed during a 10 week period after MDMA administration. MDMA altered motor activity, exploratory behavior in the object-recognition and holeboard tests, and social behavior in the social-interaction test. Citalopram pretreatment did not prevent most of the acute effects of MDMA (hyperthermia, weight loss) or the longterm (10 weeks) depletions in serotonin transporter binding. However, citalopram did block the MDMA induced decrease in aggressive behavior and the reduction in head dips in the hole-board. This study indicates that some, but not all, of the behavioral alterations are mediated by the effects of MDMA on the serotonin system. Possibly, nonserotonergic mechanisms contribute to the functional changes induced by MDMA. Supported by NIH Grant #T32 NS007490.
NBTS 43 Induction of the immediate-early gene Egr-1 by MDMA in neonatal rats Meyer JS1, Hsu A 2 (1Neuroscience and Behavior Program, University of Massachusetts, Amherst, Massachusetts, 2Neuroscience and Behavior Program, Mt. Holyoke College, South Hadley, Massachusetts) We recently reported that a single dose of MDMA (3,4-methylenedioxymethamphetamine) administered to rat pups on postnatal day (PD 4) stimulates c-fos expression in several brain areas, and that this effect is mediated at least partly by activation of D1 dopamine receptors (Meyer, Stavitsky, and Cipes, Society for Neuroscience, 2004). The present study examined the effects of MDMA on regional expression of another immediate-early gene, Egr-1 (also known as zif268 and Krox24). PD 4 male rats received a s.c. injection of either 1 mg/kg of the D1 antagonist SCH 23390 or saline vehicle, followed 30 min later by 20 mg/kg of MDMA or saline. The subjects were killed 2 h following the second injection, after which the brains were fixed, sectioned, and the sections were stained for Egr-1 immunoreactivity using standard methods. Relative numbers of stained cells were determined in the striatum and hippocampus, two areas previously shown to exhibit MDMA-induced c-fos expression at PD 4. In contrast to c-fos, there was measurable constitutive expression of Egr-1 in both the striatum and hippocampus. MDMA significantly increased this expression in both areas, but the effect of MDMA was blocked by the D1 antagonist only in the striatum. It is possible that Egr-1 induction by MDMA plays a role in
the increased apoptotic cell death we previously observed in neonatal rats treated repeatedly with this compound (Meyer et al., 2004).
NBTS 44 This is your (child’s) brain on drugs: In utero exposure to a cannabinoid agonist affects dendritic morphology of hippocampal CA1 neurons in the young rat Mervis RF1,2, Berberi N 3, Bachstetter A 2, Cassano T 4, Morgese MG4, Gaetani S 5, Cuomo V 5 (1Ctr for Aging and Brain Repair, Dept Neurosurgery., Univ. South Florida College of Medicine, Tampa, FL, 2 NeuroStructural Research Labs, Tampa, FL, 3Honors College, Univ of South Florida, Tampa, Fl., 4Univ Foggia, Italy, 5Univ La Sapienza Rome, Italy) Among women of reproductive age marijuana is the most widely used illegal drug. The impact of cannabis exposure on the developing brain is poorly understood. A specific cannabinoid receptor (CB1) is highly expressed in many brain regions, including the hippocampus. The synthetic CB1 agonist WIN 55,212-2 was administered daily to pregnant rats from gestational day 5 to 20 (0.5 mg/kg). This dose is equivalent to a moderate or low exposure of marijuana in humans and has no overt toxic effects. The behavioral consequences in 40 day-old (do) rats included hyperactive behavior and memory impairment. To assess the effects of this prenatal exposure on dendritic morphology of CA1 pyramids of the hippocampus, fixed brain tissue from WINexposed 40 do offspring (N = 6) and age-matched controls (N = 5) was Golgi stained. The basilar dendritic arbors of CA1s were quantified. In the prenatally WIN-exposed 40 do rats, there was a significant 12% increase in estimated total dendritic length and a highly significant increase in branching in the middle-third of the dendritic tree. This finding suggests that hippocampal circuitry was affected by the cannabinoid agonist; perhaps due to failure of normal developmental pruning back of the dendritic tree. Dysmorphic maturation of the hippocampus would be a factor underlying behavioral and cognitive changes seen in the young 40 day-old rats.
NBTS 45 Metallothionein expression and developmental exposure to mercury: Effects on learning in mice Levin ED, Eddins D, Petro A, Pollard N, Perraut C, Freedman JH (Dept. of Psychiatry, Nicholas School of Environ. and Earth Sci., Duke University, Durham, North Carolina) Metallothioneins are key components in the metabolism and detoxification of a variety of metals including mercury, which has been shown to cause neurocognitive impairment.
We have found that the deletion of two metallothionein genes (MT-1 and MT-2) in mice caused significant impairments on a win-shift task in the 8-arm radial maze. The metallothionein knockout mice were able to learn, but more slowly. In the next experiment, mercury, as mercuric chloride (HgCl2), was injected (sc) at concentrations of 0, 2 and 5 mg/kg on days 7, 14 and 21 after birth. As young adults the mice underwent training for spatial learning and memory on the 8-arm radial maze. The hypothesis that metallothionein knockout mice would be more sensitive to mercury-induced accuracy impairment on the radial-arm maze. Preliminary results support this hypothesis. Further work will determine the relationship of this sensitivity to enhanced depletion of frontal cortical dopamine. These results suggest that metallothioneins, through their roles in metal physiology or cellular protection, are involved in spatial learning and memory and affect vulnerability to developmental mercury neurotoxicity. Supported by the National Association for the Advancement of Autism Research (NAAR).
NBTS 46 Reversal of heroin neurobehavioral teratogenicity by grafting of neural progenitors Yanai J 1,2, Ben-Hur T3, Slotkin TA2, Katz S1 (1Ross Lab. for Neural Birth Defects, Dept. Anat. and Cell Biol., 3Dept. Neurol, Hadassah Hebrew U.-Med. Sch., Jerusalem, Israel, 2Dept. Pharmacol. and Cancer Biol. Duke Med. Ctr., NC, USA) A reversal of behavioral teratogenicity is dependent on the availability of a model where the mechanisms of the behavioral defect are established. We have developed a prenatal heroin model in mice, in which exposed offspring were deficient in septohippocampal cholinergic innervationrelated Morris maze behavior. The behavioral defects were associated with the hippocampal cholinergic receptorinduced translocation/activation of PKCc and could be reversed by manipulation of the regulating pathways, nicotine therapy, or by neural grafting. Here, we transplanted newborn mouse-derived multipotential neural precursor cells, expanded as neurospheres and tagged with BrdU into offspring that were exposed prenatally to heroin (10 mg/kg/ day SC, gestational days 9–18). The viability of the graft was confirmed with BrdU immunostaining. Prenatally exposed mice displayed impaired Morris maze performance, requiring more time than controls to reach the platform, concomitant with PKCc desensitization. While grafting neurospheres into normal mice had no effect on their performance, it reversed the deficits evoked by prenatal heroin exposure. We are currently studying the PKC isoforms in the brains of the exposed and transplanted offspring. Thus, our clinicalrelevant model is an appropriate platform for obtaining better understanding of behavioral defects induced by prenatal
heroin exposure and the role of stem cell transplantation in its treatment. Supported by USPHS HD 40820 and Israeli Anti-Drug Authority.
NBTS 47 Alterations in stress-associated behaviors and neuromarkers in adult rats after neonatal carrageenan injection of a hindpaw Lidow MS, Anseloni VCZ, He F, Novikova SI, Lidow IA (Dept. of Biomed. Sci., University of Maryland, Baltimore, MD) To examine whether adult animals, which as neonates were subjected to a short exposure to inflammation-inducing agents, exhibit alteration in their anxiety traits we developed a rat model of short lasting (b 2 days) early local inflammatory insult produced by a single injection of 0.25% carrageenan (CAR) into the plantar surface of a hindpaw. Here, we report that these animals also manifest a low anxiety trait associated with reduced emotional responsiveness to stress. This conclusion is based in the following observations: (a) rats in our model display reduced anxiety on an elevated plus-maze; (b) in the forced swim test, these rats exhibit behavioral characteristics associated with stronger ability for stress coping; and (c) these animals have reduced basal and stress-induced plasma levels of such stressrelated neuroendocrine markers as corticotropin-releasing factor, vasopressin, and adrenocorticotrophic hormone. To determine whether neonatal inflammatory insult induces elevation in maternal care, which may play a role in generating long-term behavioral alterations seen in our model, we examined maternal behavior for 3 days following CAR injection. Indeed, we observed a substantial increase in maternal attention to the pups at the time of inflammation, but this increase was not without its cost—a period of significant maternal neglect afterwards. Supported by NIH grantNS41384.
NBTS 48 Thyroid hormone insufficiency: Persistent deficits in brain structure and function Gilbert ME (Neurotoxicology Division, NHEERL, ORD, US EPA, RTP, North Carolina) Severe developmental hypothyroidism causes frank brain damage and mental retardation. Until recently, evaluation of more modest perturbations of the thyroid axis has been lacking. In this study we induced graded levels of hormone insufficiency in pregnant dams with the thyroid hormone synthesis blocker, propylthiouracil (PTU, 0, 3, 10 ppm in drinking water) from gestational day 6 and postnatal day 30. The effects on neurophysiology in hippocampus, brain
morphology, and behavior were evaluated in adult offspring following full recovery of hormone status. The data reveal that modest hormone reductions in dams and moderate reductions in pups are associated with perturbations in synaptic transmission in the dentate gyrus of hippocampus, structural abnormalities in the cortex, hippocampus, and corpus callosum, and learning deficits in a spatial and nonspatial hippocampal-dependent learning task. These findings corroborate recent human data indicating that even modest hormone insufficiencies can lead to permanent impairments in cognitive function. They also implicate perturbation of hippocampal function in the clinical manifestation of low level thyroid hormone insufficiency. Funded by USEPA, does not reflect US EPA policy).
NBTS 49 Consequences of bullying during puberty Newman ML, Delville Y (Department of Psychology, University of Texas at Austin) Approximately 30% of adolescents are bullied regularly, and this bullying has been associated with a range of psychological disorders. In the present research, we tested a set of predictions about the consequences of being bullied, based on animal models of subjugation and stress. Male Syrian hamsters subjugated in adulthood show increased stress and inhibited aggression. Male Syrian hamsters subjugated in adolescence recover more quickly from stress and show increased aggression. The developmental differences may be due to the gradual development of the stress response during puberty. Do these timing differences apply to humans bullied in early vs. late puberty? Our participants were 1483 college students who reported on their victimization history and responded to a set of scenarios involving frustration. Those bullied in early puberty showed more anger and impulsive responses. Those bullied in late puberty showed less anger and withdrawn responses. These data suggest that bullying alters emotional reactivity, with the specific consequences depending on when it occurs. These data also suggest that animal models of violence and subjugation are a good starting point for predictions about human behavior. Supported by NIH ES10385 and NIH MH65728-2.
NBTS 50 Effect of low-dose acrylamide exposure on preweaning behavior of Fisher 344 rats Garey J, Ferguson SA, Paule MG (Division of Neurotoxicology, National Center for Toxicological Research/FDA, Jefferson, Arkansas) Recent studies have shown that small amounts of acrylamide (ACR) occur in a host of common foodstuffs,
thus providing a low level of constant exposure to a large number of humans. In this study, relatively low doses of ACR (0, 0.1, 0.3, 1.0 and 5.0 mg/kg/day) were provided to pregnant Fisher 344 dams by gavage starting on gestation day 6. Dams were dosed daily until litters were born; pups were gavaged with the same dose as their dam on postnatal days (PNDs) 1–22. In data analyzed to date, no treatment effects (treatment vs. control; repeated measures ANOVA) have been observed for righting reflex (PNDs 4–7), forelimb hang time (PNDs 12–16), open field measures of activity (PNDs 19–20) or Rotarod latency to fall time (PNDs 21– 22). No significant treatment effect was found for negative geotaxis latency to turn (PNDs 8–10), although the 5.0 mg/ kg/day treatment group had the shortest latency to turn overall. These results are in agreement with those obtained for doses of V 5 mg/kg/day in a previous range-finding study. At these relatively low doses, ACR does not appear to produce readily detectable effects on any of these early behavioral development measures. Supported by ORISE and NTP 224-93-0001.
NBTS 51 A single administration of ketamine produces an inflammatory response in the developing rat brain Wright LKM 1 Twaddle N 2, Branham W 3, Wang C 1, Schmued LC 1, Patterson TA 1, Paule MG 1 (Divisions of 1Neurotoxicology and 2Biochemical Toxicology and 3Center for Functional Genomics, National Center for Toxicological Research, Jefferson, Arkansas) Repeated administration of ketamine, an N-methyl-daspartate (NMDA) receptor antagonist, produces neuronal apoptosis in neonatal rats. To determine whether a single dose of ketamine also produces apoptosis, 7-day-old rats were injected subcutaneously with water (vehicle) or 40 mg/ kg ketamine. Plasma levels of ketamine were monitored for 4 h after drug administration, degenerating neurons were stained with Fluoro-Jade C, and electron microscopy (EM) and cDNA microarrays were used to investigate the mode of neuronal degeneration. The highest ketamine plasma levels were observed 15 min after administration (24.2 F 2.3 AM). A single dose of ketamine did not increase the number of Fluoro-Jade C positive neurons in either the cingulate cortex or the hippocampus 24 h after treatment. However, EM revealed necrotic neurons in the frontal cortex 24 h after ketamine treatment, and microarray analysis identified 707 significant (fold change N 1.5 and p b 0.05) gene expression changes in the thalamus 2 h after treatment. These genes are associated with a number of cellular processes, including inflammation, oxidative stress, altered protein processing, and decreased mitochondrial function. Thus, a single administration of 40 mg/kg ketamine appears to produce an inflammatory response in the developing rat brain, rather than apoptosis.
NBTS 52 Neonatal hippocampal Tat injections: Effects on prepulse inhibition (PPI) Fitting S, Booze RM, Wu G, Mactutus CF (Beh. Neurosci. Prgm., USC, Columbia, SC) More than 700,000 children under 15 years of age are infected worldwide with HIV/AIDS every year (UNAIDS, 2004). Some estimates suggest that up to 90% of the HIV1 infected children display progressive encephalopathy, including impaired brain growth, attentional and cognitive deficits. The presence of HIV in brain is believed to be responsible for mediating the process indirectly through the viral toxins gp120 and Tat. This study was designed to characterize the potential deficits in sensory gating, as indexed by PPI, following intrahippocampal administration of Tat. On postnatal day 1, one male and one female pup of 14 Sprague–Dawley litters were bilaterally injected with 50 lg Tat or saline (1 ll volume), directly into the hippocampus. Following 4 weeks recovery, all animals were tested for PPI of the auditory startle response (ASR) (ISIs of 0, 8, 40, 80, 120, and 4000 ms, 6-trial blocks, Latin square design). Tat had no significant effect on body weight or growth of the offspring. However, Tat altered PPI and the pattern of alterations was different for males and females. For males, a rightward shift was evident in ISI interval for peak inhibition of the ASR (v 2 = 8.5, p b 0.05). For females, Tat altered response latency across PPI trials (8–120 ms), as indexed by trend analyses, indicating less modulation of PPI by ISI. In sum, neonatal intrahippocampal Tat injection adversely affects the preattentive process of sensory gating, as indexed by PPI. Supported by DA13137, DA014401, HD043680.
NBTS 53 Prenatal cocaine exposure: Dose-dependent increase in intentional tremor Brown LM, Booze RM, Strupp BJ, Snow DM, Mactutus CF, (Behav. Neurosci. Prgm. USC, Columbia, SC) Prenatal exposure to cocaine causes neurological and behavioral alterations in young children, increasing the likelihood for attentional disorders and motor-related abnormalities (Singer et al., 2000). We investigated developmental alterations in the cerebellum as responsible for the potential motor/tone/reflex abnormalities. Long– Evans female rats were implanted with intravenous (IV) catheters, impregnated and administered saline, a low dose of cocaine (3 mg/kg/ml, 1 /day GD 8-21), or a high dose of cocaine (3 mg/kg/ml, 1 /day GD8-14, 2 / day GD 14-21). An uncatheterized untreated control group was included. Cocaine did not affect maternal or litter parameters. At 14 days of age, one male and one female from each litter were randomly selected, injected
with harmaline and placed in a tremor monitor (San Diego Instruments, Inc.) for 512 s. When tested in a large chamber (10 15 cm), which permits greater exploratory activity, a linear prenatal cocaine dose–response relationship was found across groups; the control groups were not significantly different. In a small chamber (10 9 cm), the tremor response was not significantly different across groups. An increased response to harmaline reflects greater Purkinje cell activation, mediated via the climbing fiber pathway. The present data replicate our prior findings that there are functional alterations in the cerebellum consequent to prenatal IV cocaine exposure and now extend these observations to indicate there is a clear intentional component to the altered tremor response. Support: DA09160, DA012719, DA013965 and DA014401.
NBTS 54 Early postnatal exposure to DE-71 produces lasting effects on learning in a visual discrimination task Dufault C, Poles G, Driscoll LL (Dept. of Psychology, Colorado College, Colorado Springs, Colorado) The effects of early postnatal exposure to polybrominated diphenyl ethers (PBDEs) on learning in adulthood were examined in a rodent model. On postnatal days 6–12, male Long–Evans rats were administered daily oral intubations of either corn oil alone or the commercial PBDE mixture DE-71, 30 mg/kg of body weight, dissolved in corn oil. They were then tested as adults on an automated, five-choice visual discrimination task. On each trial, the animals were food rewarded for making a nosepoke into the illuminated port. PBDE-exposed rats required a greater number of trials than controls to reach criterion (80% correct for two out of three daily sessions). The deficit in performance was due to increased frequencies of inaccurate responding and omission errors (failures to respond within 30 s of cue onset), particularly after the first session of the task. In contrast, premature response rates and latency measures did not differ between the groups. These findings suggest that brief, early exposure to PBDEs impairs the ability to associate visual cues with reward, and that this impairment lasts long past the period of exposure. Supported by CC Research and Development and Venture Grants to LLD and GP and a Howard Hughes Undergraduate Research Program Grant to CD.
NBTS 55 Similarities in methylphenidate sensitivity and reward were observed in adolescent mice and adult mice exposed to nicotine during adolescence
Kelley BM, Nolley EP, Binns L, Clarke R, Wadsworth SH (Department of Psychology, Bridgewater College, Bridgewater, Virginia) Previous data suggests adolescent nicotine use permanently disrupts reward systems. Behavioral pharmacological methods were used to assess the effects of adolescent nicotine exposure on methylphenidate (MPD) sensitivity and reward from adolescence to adulthood. Testing was performed on adult mice exposed to nicotine (0.3 and 3.0 mg/kg, SC, M-F, b.i.d.) or saline during adolescence (PND 25–57). After a 28day drug-free, time-off period, the locomotor effects (30 min, 30 cm traveled) of MPD (5, 10, 20, and 40 mg/kg, IP) were measured. Thereafter, mice underwent condition–place– preference testing. MPD (20 mg/kg) was paired with the subject’s non-preferred side and saline with the preferred side. Conditioning sessions were conducted for 8 days with a drug-free post-test on the day following the final conditioning session. A follow-up study compared adolescent mice (PND 25–46) to adult mice (PND 77–98) using identical MPD testing procedures. Adult subjects exposed to nicotine during adolescence behaved remarkably similar to adolescent subjects receiving only MPD. Both nicotine exposed subjects and naRve adolescents exhibited increased response to MPD’s motor activating effects and a decreased response to MPD’s rewarding effects. Adolescent nicotine exposure retards the development of reward systems, which could result in increased vulnerability to substance abuse problems across adulthood.
NBTS 56 Blood and brain mercury levels in infant monkeys exposed to methylmercury or vaccines containing thimerosal Burbacher TMa,c,d, Shen DDb, Liberato NAa,c,d, Grant KSa,c,d, Cernichiari E e, Clarkson T e (Department of Environmental and Occupational Health Sciencesa, School of Public Health and Community Medicine, Departments of Pharmacy and Pharmaceuticsb, School of Pharmacy, Washington National Primate Research Centerc, and Center on Human Development and Disabilityd, University of Washington, Seattle, Washington, Department of Environmental Medicine, University of Rochester School of Medicinee, Rochester, New York) Thimerosal is a preservative that has been used in manufacturing vaccines since the 1930s. Reports have indicated that infants can receive ethylmercury (in the form of thimerosal) at or above the EPA guidelines for methylmercury exposure, depending on the exact vaccinations, schedule, and size of the infant. This study compared the systemic disposition and brain distribution of total and inorganic mercury in infant monkeys following thimerosal
exposure with infants exposed to methylmercury. Monkeys were exposed to methylmercury (via oral gavage) or vaccines containing thimerosal (via i.m. injection) at birth and 1, 2, and 3 weeks of age. Total blood mercury (Hg) levels were determined 2, 4 and 7 days after each exposure. Total and inorganic brain Hg levels were assessed 2, 4, 7 or 28 days after the last exposure. The initial and terminal half-life of Hg in blood following thimerosal exposure was 2.1 and 8.6 days, which are significantly shorter than the elimination half-life of Hg following MeHg exposure. Brain concentrations of total Hg were significantly lower by ~3-fold for the thimerosal-exposed infants, while the average brain-to-blood concentration ratio was slightly higher for the thimerosalexposed infants (3.5 F 1.0 vs. 2.5 F 0.6). A higher percentage of the total Hg in the brain was in the form of inorganic mercury for the thimerosal-exposed infants (34% vs. 7%). The current study indicates that methylmercury is not a suitable reference for risk assessment from exposure to thimerosal derived Hg. The significance of the higher inorganic Hg levels in the brain should be investigated, since this form of Hg is known to have a very long high-life, over 1 year. Knowledge of the toxicokinetics and developmental toxicity of thimerosal is needed to afford a meaningful interpretation of the developmental effects of thimerosalcontaining vaccines.
NBTS 57 Growth trajectories of children prenatally exposed to cocaine to age 6 years Kirchner HL, Minnes S, Ibrahim Y, Short E, Singer LT (Case Western Reserve University, Departments of Pediatrics, General Medical Sciences, and Psychology, Cleveland, Ohio) Long-term growth of infants exposed to cocaine is a growing concern, yet few longitudinal comparisons have been made. This study assessed the development of growth in cocaine exposed (CE: n = 218) and non-exposed children (NCE: n = 197) followed from birth. Groups did not differ on child gender and race, however differed on maternal age, marital status, number of prenatal visits, parity, and prenatal exposure of other drugs. Growth measures were obtained from 6 study visits (birth, 6, 12, 24, 48 months, 6 years), including height-(HAZ), weight-(WAZ), and head circumference-(HCZ)-for-age z-scores. Measures were converted to age–gender adjusted z-scores using the 2000 CDC reference population. Data were analyzed using random effects linear models to capture the growth trajectories controlling for other prenatal drug exposure and prenatal care. The sample was African American (80%), female (52%), and lower SES (98%). In the CE sample, mothers reported using 8 rocks/ week (median). Growth measures were available for z 4 visits from N90% of the children in each group. Across all visits CE were lighter than NCE. Around 12 months, the CE
group was taller then NCE, although not significantly. By 6 months of age both groups caught up to normal reference values for HAZ and WAZ. Only at birth did the CE group have significantly lower HAZ, WAZ, and HCZ scores. Data indicated that CE children demonstrated catch up growth.
NBTS 58 Preweaning cocaine alters acquisition of active avoidance in the adult male Sprague–Dawley rat Melnick SM1, Myrie JR2 , Dow-Edwards DL (Physiology/Pharmacology, SUNY Downstate Medical Center, Brooklyn, New York, 1SK Bio-Pharmaceuticals, Fairfield, New Jersey, 2Memorial Sloan Kettering Cancer Center, New York, New York) Cocaine exposure during development has been shown to alter many neurobehavioral parameters including learning. The present study examined the role of preweaning cocaine administration, a model of 3rd trimester human exposure, on the acquisition of active avoidance behavior. During postnatal days (P) 11–20, male and female rats received either dH20 or 50 mg/kg cocaine daily. At P94, non-drugged animals were tested in the active avoidance procedure for five consecutive days. Only cocaine-exposed males showed a reduced ability to escape while cocaine-exposed females showed performance within the control range. Preweaning cocaine administration significantly increased reaction time in males but significantly decreased reaction time in females. Thus, striatal neural pathways that may mediate active avoidance escape behavior may be altered in sexspecific ways. In addition, these data support a functional uncoupling of the D1-D2 receptors only in males treated with cocaine during development. Supported by NIDA DA10990).
NBTS 59 Prenatal intravenous (IV) cocaine: Cell loss in the locus coeruleus (LC) Mactutus CF, Hasselrot U, Fitting S, Adams S, Snow DM, Strupp BJ, Booze RM (Beh. Neurosci. Prgm. USC, Columbia, SC; Anat. and Neurobiol. UKY, Lexington, KY; Dept Psych., Cornell Univ., Ithaca, NY) Among the most reliable neurobehavioral alteration consequent to maternal cocaine (COC) abuse during pregnancy is the appearance of attentional dysfunction in school age children. As the LC plays a pivotal role in attentional processes and projects more widely than any other nucleus in the central nervous system, the LC was examined as a potential basis for this dysfunction. Both dose–response and gestational exposure period factors were included in the study
design. Nulliparous Long–Evans female rats (N = 64) were implanted with an IV access port, were bred and given saline or COC HCl (3 mg/kg/ml) 1 /day from GD8-21, 1 /day from GD8-14 and 2 /day from GD15-21), or 1 /day on GD13-15. COC had no significant effect on any standard maternal/litter parameters. At 21 days of age, one male and one female of each litter were anesthetized, perfused with paraformaldehyde, and their brains were removed for THimmunocytochemistry and cell counting of the LC using the optical dissector method. Preliminary analyses suggested no reduction in volume of the LC, but a 12–15% reduction in number of neurons as a function of dose of COC exposure. The GD13-15 COC exposure produced a reduction in LC volume and a 13% reduction in neuron number. Thus, not only is IV COC neurotoxic to the LC, but an early circumscribed exposure is sufficient to also cause cell loss, suggesting that low doses of COC are teratogenic to NE neurons. Support: DA09160, DA012719, DA013965 and DA014401.
NBTS 60 Correlations of brain glucose metabolic rates and behavior in adolescent rats exposed to prenatal cocaine and subsequent methylphenidate administration Torres-Reveron A, Dow-Edwards DL (Department of Physiology and Pharmacology and Program in Neural and Behavioral Science, SUNY Downstate, Brooklyn, New York) Prenatal cocaine (PC) exposure has been associated with deficits in arousal and attention. Since methylphenidate (MPD) is widely used to treat attention disorders, we wanted to determine whether PC exposure affects the local cerebral glucose utilization (LCGU) in response to MPD in adolescent rats of both genders. Pregnant dams received 60 mg/kg of cocaine or vehicle from gestational days 8–22 by intragastric intubation. The vehicle-intubated group was pair fed. On a single day between postnatal days 41–45, rats received a single injection of 10 mg/kg i.p. of MPD or saline. After 15 min, rats were injected with 2-deoxyglucose through the jugular vein and the quantified deoxyglucose method was performed. Pearson-product moment correlations were used to correlate locomotor and stereotyped behavior with LCGU in components of the nigrostriatal (NS) and mesolimbic (ML) systems. Results showed that behavior in the control females is not correlated with LCGU activity. Metabolism in a few structures of the NS system in females that received PC correlated with stereotyped behavior. In the control males, locomotor activity and stereotypy were correlated with metabolism in several structures of the NS system. In the PC males, LCGU in almost all of the components of the NS system correlated with locomotor activity. In conclusion, prenatal cocaine exposure produced gender-specific effects in the relation-
ship of brain metabolism and behavior in adolescent rats subsequently exposed to a psychostimulant. The NS system in male rats appears to be the most susceptible to the effects of developmental cocaine exposure since correlations in this system were more widespread compared to controls. Supported by APA_DNP to ATR and DA10990 to DDE.
NBTS 61 In utero cocaine exposure permanently alters behaviorally relevant brain circuits Stanwood GD, Thompson BL, Levitt P (Dept. Pharmacology and Vanderbilt Kennedy Center, Vanderbilt University, Nashville, Tennessee) Using low dose intravenous cocaine administration to pregnant rabbits, we have documented permanent alterations in dendritic morphology of cortical pyramidal cells, changes in gene expression in subtypes of cortical interneurons, and a loss of dopamine D1 receptor signaling. These abnormalities are elicited during a relatively brief period of embryonic development, comparable to the second trimester in humans, and occur only in cortical regions receiving dense dopaminergic input. Specific cognitive and motor behaviors are altered in adult offspring as a result. Similar changes in cortical circuitry can be observed in the brain of D1 receptor knockout mice. Thus, prenatal exposure to cocaine produces robust and long-lasting alterations in specific structural and functional properties of neurons of the cerebral cortex, and behavioral deficits reminiscent of the affected human population. These (mal)adaptive changes may be secondary to the loss of D1 receptor signaling, suggesting that restoration of this signaling system may be an key therapeutic target. Supported by DA11165, DA13792 and P30HD15052.
NBTS 62 Self-reported mental health symptoms of 9-year old prenatally cocaine-exposed children Singer LT, Minnes S, Linares T, Short E, Satayathum S, Kirchner HL (Case Western Reserve University, Departments of Pediatrics, Psychology, and General Medical Sciences, Cleveland, Ohio, United States) Prenatal cocaine exposure may negatively affect brain monoamine systems that regulate behavior. This study assessed self-reported mental health symptoms of cocaineexposed children and non-exposed children at 9 years of age. Children, 234, were administered the Dominic Interactive, a pictorial self-report of mental health symptoms across 7 domains (specific phobias, separation anxiety, generalized anxiety, depression, ODD, conduct and ADHD). Children with I.Q. b 70 were excluded.
Environmental and demographic covariates were assessed and entered into models if they were related to the outcome and associated with cocaine exposure. Cocaine exposure was associated with conduct disorder in girls only (adjusted OR = 10.3, 95% CI: 1.2, 90.4). Other drug and environmental effects were significant. Heavier marijuana exposure was associated with ODD ( p V 0.03). Better quality caregiving environment predicted lower
symptoms of generalized anxiety disorder ( p V 0.03), depression ( p V 0.03), conduct disorder ( p V 0.045), and ADHD ( p V 0.05). Cocaine-exposed children in adoptive/ foster care were less likely to report specific phobias ( p V 0.04). Results indicate specific effects of prenatal cocaine and marijuana exposure and quality of caregiving environment on children’s self-report of mental health symptoms.