NBTS 2003 Abstracts

Neurotoxicology and Teratology 25 (2003) 381 – 397 www.elsevier.com/locate/neutera

NBTS 2003 Abstracts NBTS 1 –7/BTS 8– 14 Developing zebrafish models for neurobehavioral toxicology Levin1 ED, Scalzo2 FM (Duke University Medical Center, Durham, NC1 and Bard College, Annandale, NY 2)

Balice-Gordon R (University of Pennsylvania, Philadelphia, PA) See BTS abstract listings for full text, unavailable at press time.

The Joint Behavioral Toxicology Society and Neurobehavioral Teratology Society symposium is entitled ‘‘Developing zebrafish models for neurobehavioral toxicology.’’ Zebrafish is an emerging model of choice for the study of the molecular basis of neurodevelopment. Their clear chorion and rapid development allow access to observe critical events in neurodevelopment including molecular signaling and neural proliferation, differentiation, and migration. Recently, behavioral tests to determine the functional capacities of zebrafish have opened the way for using this alternative species in neurobehavioral teratology and toxicology. This symposium will highlight some of the opportunities offered by the zebrafish model and present data from behavioral toxicology and teratology studies. Dr. Rita Balice-Gordon of the Univ. of Pennsylvania will speak about mechanisms of synaptogenesis in zebrafish. Dr. Patrick Page-McCaw of the Univ. of Calif., San Francisco will discuss the assessment of cognitive function in newly hatched zebrafish. Dr. Frank Scalzo of Bard College will speak about NMDA receptor mechanisms and neurobehavioral development in zebrafish. Dr. Joseph Bilotta of Western Kentucky Univ. will present a zebrafish model of fetal alcohol syndrome. Dr. Kathi Lefebvre of the NOAA will discuss impairments in zebrafish mechanosensory function caused by algal toxin exposure. Dr. Edward Levin of the Duke Univ. Med. Ctr. will compare the nature of the insecticide chlorpyrifos-induced neurobehavioral impairments in zebrafish and rat models. Dr. Michael J. Carvan of the Univ. of Wisconsin-Milwaukee will speak about the use of the zebrafish for mechanistic studies of neurotoxicity. This joint symposium will provide the latest methodological and mechanistic information concerning the use of this rapidly rising model of neurodevelopment.

NBTS 2/BTS 9 Assessment of cognitive function in newly hatched zebrafish Page-McCaw P (University of California-San Francisco, San Francisco, CA)

NBTS 1/BTS 8 Mechanisms of synaptogenesis in zebrafish

See BTS abstract listings for full text, unavailable at press time.

doi:10.1016/S0892-0362(03)00043-6

See BTS abstract listings for full text, unavailable at press time.

NBTS 3/BTS 10 NMDA receptor mechanisms and neurobehavioral development in zebrafish Scalzo F (Bard College, Annandale, NY) See BTS abstract listings for full text, unavailable at press time.

NBTS 4/BTS 11 A zebrafish model of fetal alcohol syndrome Bilotta J (Western Kentucky University, Bowling Green, KY) See BTS abstract listings for full text, unavailable at press time.

NBTS 5/BTS 12 Impairments in zebrafish mechanosensory function caused by algal toxin exposure Lefebvre K [Environmental Conservation Division, Northwest Fisheries Science Center, National Marine Fisheries Service, National Oceanic and Atmospheric Administration (NOAA), Seattle, WA]

382

Abstracts / Neurotoxicology and Teratology 25 (2003) 381–397

NBTS 6/BTS 13 A comparison of the nature of the insecticide chlorpyrifos-induced neurobehavioral impairments in zebrafish and rat models Levin ED (Duke University Medical Center, Durham, NC) See BTS abstract listings for full text, unavailable at press time. NBTS 7/BTS 14 The use of the zebrafish for mechanistic studies of neurotoxicity Carvan MJ (University of Wisconsin-Milwaukee, Milwaukee, WI) See BTS abstract listings for full text, unavailable at press time.

* Charles River Distinguished Speaker NBTS 8/BTS 15 A behavioral analysis of hippocampal function based on subregional, attribute, and process specificity Kesner RP (University of Utah, Salt Lake City, UT) See BTS abstract listings for full text, unavailable at press time.

NBTS 9 The Neurobehavioral Teratology Society online: 2002 – 2003 Livezey GT (Neuroscience, University of Minnesota, Minneapolis, MN) The Neurobehavioral Teratology Society has ‘‘unveiled’’ its new web site (http://www.NBTS.org), as of September. This was a great and highly successful effort, directed by our Public Affairs Chair, Jack Lipton. This modernized site includes many features not previously provided, including an online abstract submission form, and its own email addresses for our officers. The old site remains at http:// NBTS.bsbe.umn.edu as a backup for all data and for those few whose access is limited by older equipment and/or software. An analysis of all society online activities (websites and email) traffic for June 2002 – June 2003 and for calendar year 2002 will be presented at the meeting. For all of 2002, web traffic is up on the old site with 19,000 pages viewed by 4000 visitors. Our new site appears to be rapidly eclipsing the old site with 2000 page views a month through December. Data from our old site indicate that our website use is primarily society business conducted mostly from our places of business. Data from

our new site suggest a greater proportion of user referrals from search engines, indicating a much ‘‘higher public profile.’’ Our web sites are resources provided to better serve our society and its members, as well as to provide a public outreach venue. I thank all those who assisted me in responding to specific requests. I have sent or received over 2000 emails in the year 2002, while conducting society business as treasurer or webmaster. We are truly an ‘‘online’’ society in the new millenium.

NBTS 10 Periadolescent nicotine exposure alters dopamine function in adult C57 mice Kelley BM, Barb JR, Nipe G, Bell LM (Department of Psychology, Bridgewater College, Bridgewater, VA) Previous research in our lab has shown that adolescent nicotine exposure dose-dependently decreases cocaine reward in fully mature subjects (similar results also noted with ethanol). The reinforcing effects of nicotine, like most abused drugs, appear to be mediated in part by stimulation of dopamine receptors. Our studies suggest that adolescent nicotine exposure may permanently decrease the reinforcing effects of abused drugs through changes in dopamine receptor function. Behavioral pharmacological methods were used to evaluate dopamine D1, D2, and D3 receptors in adult mice exposed to nicotine (3.0 mg/kg sc, M– F, b.i.d.) or saline during adolescent development (postnatal days 25– 60). Prior to drug testing, subjects had a 20-day drug-free, time-off period. After acclimation to the testing apparatus (5 days/saline), the locomotor effects of three dopamine agonists (D1, SKF 38393; D2, quinpirole; D3, 7-OH-DPAT) were measured (30 min, cm traveled). All drug tests were proceeded and followed by saline control tests. After completing an ascending dose –response curve (four doses tested), subjects received the highest drug dose for five consecutive days to assess the development of tolerance or sensitization. The results indicate significant differences between the adolescent nicotine group and the control group across D1, D2, and D3 testing; however, the most robust findings were noted across D2 and D3 testing.

NBTS 11 An exploratory study examining adolescent methylphenidate exposure on alcohol sensitivity in adult C57 mice Barb JR, Hessberger N, Ports S, Woodring K, Kelley BM (Department of Psychology, Bridgewater College, Bridgewater, VA) Despite the alarming use and abuse of methylphenidate among children and adolescence, there has been virtually

Abstracts / Neurotoxicology and Teratology 25 (2003) 381–397

no research on its long-term impact on drug reward/ sensitivity. The present study was designed to examine the effect of adolescent methylphenidate exposure on ethanol sensitivity in adulthood. Sensitivity to ethanol was examined by testing motor activity of adult mice that were exposed to saline or methylphenidate (20.0 mg/kg sc, M– F, b.i.d.), during adolescent development (postnatal days 25– 60). Following a 3-week drug-free period, the subjects were acclimated to the testing chamber (4 days, saline injection). Thereafter, motor activity was measured (every 5 min for 30 min, cm traveled) after injections of ethanol (4%, 8%, 12%, 16%, and 20%, intraperitoneal). Sessions with saline injections were conducted before and after each ethanol test. Subsequently, in order to assess the development of tolerance subjects were tested with the highest concentration of ethanol for five consecutive days. Significant differences between methylphenidate-treated and control subjects were not observed at the lowest doses (4%, 8%, 12%, and 16%). However, at the 20% ethanol concentration, a statistically significant difference was found, in which the methylphenidate group showed reduced sensitivity and during repeated administration, an increased tolerance to ethanol’s motor-depressing effects was also noted.

NBTS 12 Pediatric lead poisoning: the clinical presentation in individual children with low blood lead levels (Introduced by A. Rabe) Lidsky TI*, Rosen* JF, Heaney* A, Schneider* JS (Institute for Basic Research, Staten Island, NY; The Children’s Hospital at Montefiore, Bronx, NY; Thomas Jefferson University Medical School, Philadelphia PA) Several studies have demonstrated that low blood lead levels have deleterious effects on cognitive functioning and academic skills. However, because these studies presented averaged results from large groups of children, lead’s effects in the individual child were obscured. The present study presents the results of neuropsychological evaluations of 18 children with peak blood lead levels ranging from 7 to 15 mg/dl. Considered as a group, mean IQ was in the lower part of the average range and performance in tests of memory, attention, and executive functioning in the low average range thereby giving the impression that lead poisoning caused a generalized lowering of intellectual functioning. Detailed consideration of each case in isolation, however, indicated that lead’s effects in the individual child were quite different. Each child showed areas of complete normalcy with test results in specific cognitive domains in the average or above average range. However, coupled with this adequate performance were test results in other cognitive domains that reflected severe impairment. This pattern of test

383

results in the individual is characteristic of brain injury rather than of a nonspecific dampening of intellectual functioning. This same pattern of findings was seen in children with blood lead levels below or equal to 10 mg/dl (n = 5), and with blood lead levels from 11 to 15 mg/dl (n = 13).

NBTS 13 Effects of neonatal cocaine on the serotonin system and behavior in the developing rat Summavielle T *, Magalha˜es A*, Sousa L*, Tavares MA (Neurobehavior Unit, IBMC, University of Porto, Porto, Portugal) Serotonin (5-HT) plays an important role in the development of the CNS. In the rat, serotoninergic neurons are first detected on gestational day 13. However, their axons and somatodendrites continue to grow throughout the first weeks of postnatal life. Here, we examined the effects of neonatal cocaine exposure on the role of 5-HT in the locomotor activity by measuring the levels of 5-HT in the raphe nuclei of developing rats, concurrently with openfield observations. Male and female Wistar rats were given cocaine hydrochloride (15 mg/Kg body weight/ day) sc in two daily doses, from postnatal day 1 (PND1) to PND30. Controls were given 0.9% saline. Open-field activity was registered on the PND14, 21, and 30 in three 15-min sessions. Also on PND14, 21, and 30, raphe nuclei were processed for determination of 5-HT and 5-HIAA by HPLC-EC. Our data revealed an increase in global activity, rearing behavior, central ambulations and peripheral movements on PND14 and 21, for both male and female cocaine-exposed rats. On the PND30, there was a significant decrease in recorded activities. On PND14 and 21, the 5-HT levels were significantly higher ( P < .001) for the cocaine-exposed animals. This difference was no longer observed on PND30. No gender differences were detected. These findings are in accordance with previous works that described the role of increased levels of 5-HT in cocaine-induced behavioral dysfunction. Supported by FCT PSAU/P/SAU/8/96, BD/14742/97 and BD/20075/99.

NBTS 14 Postnatal cocaine exposure and rearing effects on openfield behavior Magalha˜es* A, Summavielle* T, Tavares MA, de SOUSA* L (Neurobehavior Unit, Institute for Molecular and Cellular Biology, University of Porto, Portugal) This study examined the effects of environmental enrichment (EE) on open-field behavior of rats exposed to

384

Abstracts / Neurotoxicology and Teratology 25 (2003) 381–397

cocaine during the first month of life. Wistar rats were used in four groups. Group 1: injected with cocaine hydrochloride (15 mg/Kg body weight/day) sc in two daily doses, from postnatal day (PND) 1 to PND30 and reared in EE; Group 2: pups injected with cocaine as previously described and reared in standard environment (SE); Group 3: pups saline-injected and reared in EE; Group 4: pups saline-injected and reared SE. Open-field behaviors (rearing, center, and peripheral activity) were registered in three sessions during 15 min on PND14, 21, and 30. On PND14 and 21, results showed that rats postnatally exposed to cocaine and housed in SE increased peripheral activity. At age of 30 day, cocaine increased the frequency of rearing behavior and center activity during Session 1. On PND14, rats exposed to cocaine and housed in an EE (Group 1) increased rearing behavior and frequency of center visits in all sessions and had more peripheral activity during second and third sessions. On PND21, Group 1 showed increased of rearing and center activity during the second and third sessions and at PND30 the activity center is higher. These results suggest that rats postnatally exposed to cocaine increased motor activity but not the exploratory behavior, they seem to fear to explore this adverse environment. However, EE apparently overcomes this fear tendency, increasing exploratory behavior in all open-field dimensions. Supported by Project POCTI/PSI/39491/2001, BD/ 20075/99, BD/14742/97.

NBTS 15 Attention and executive functioning in preschool-age children prenatally exposed to alcohol, cigarettes, cocaine, and marijuana Noland* JS, Singer LT, Short* EJ, Arendt RE, Minnes* S, Bearer* CF (Departments of Pediatrics and Psychology, Case Western Reserve University, Cleveland, OH) Attention and executive functioning was investigated in a cohort (n = 316) of 4-year-old children, whose mothers had used varying combinations of cocaine, cigarettes, alcohol, and/or marijuana during pregnancy. Employing postpartum maternal report and biological assay, children were assigned to overlapping prenatal cocaine-, cigarette-, alcohol-, and marijuana-exposed groups, and complementary control groups. The postnatal environmental assessment included measures of maternal intellectual, psychosocial functioning, current drug/alcohol use, and home environment. The children in the alcohol-exposed group had worse tapping inhibition performance than children in the no alcohol exposure group. With half of the attention tasks data analyzed, the children in the cigarette-exposed group had worse performance on a measure of sustained attention. Both effects persisted when potential confounding environmental variables, other drug variables and concurrent

verbal intelligence were controlled for. Supported by NIDA R01-07957, General Clinical Research Center Grant to LTS, NIDA F32-05904 to JSN, a Schubert Center Grant to EJS and R03-AA12618, PO ES11261-01 to CFB.

NBTS 16 Effects of age and prenatal cocaine (COC) and/or nicotine (NIC) exposure on cocaine-induced conditioned place preference Sobrian SK, Marr L, Ressman K (Howard University College of Medicine, Washington, DC) A longitudinal analysis of COC-induced conditioned place preference (CPP) was undertaken in male and female offspring at 70, 150, and 270 days of age that were exposed daily on GD 8 – 20 to one of eight treatment conditions: (1) 20 mg/k COC [LC]; (2) 40 mg/kg COC [HC]; (3) 2.5 mg/kg of NIC [LN ]; (4) 5 mg/kg NIC [HN]; (5) 20 mg/kg COC and 5 mg/kg NIC [LC/HN]; (6) 40 mg/kg COC and 2.5 mg/ kg NIC [HC/LN]; (7) Pair fed: yoked to HC [PF]; (8) Saline controls: injection of 0.9% NaCl [SAL]. CPP was tested in a three-chamber apparatus. Side preference was determined 1 week prior to testing; during conditioning trials, COC (20 mg/kg) was always paired with the nonpreferred side. At PND 70, only LN offspring exhibited a preference for the COC-paired compartment. By PND150, both LN and HC/ LN groups spent more time on the COC-conditioned side. However, at 9 months of age, a different profile emerged; COC preference was only evident in HC and HN offspring. While SAL controls showed COC-induced conditioning at each age, it was only seen in PF offspring at PND 70. Aversion to COC was not apparent at any age, as no groups spent more time in the SAL-paired compartment. These data suggest that prenatal exposure to high-dose COC and both doses of NIC enhance the reinforcing properties of COC in an age-dependent manner. The aversion to COC, previously reported in aging LC, LN, and HN offspring, appears to be a late occurring phenomenon. Supported by NIH Grant S06 GM 08016.

NBTS 17 Kainate-induced toxicity to the adult rat retina is exacerbated by methamphetamine Rodrigues LG1, Tavares* MA2, Wood JPM1, Schmidt K-G1, Osborne NN1 (Nuffield Laboratory of Ophthalmology, Oxford University, UK1 and Medical School of Porto, Portugal *’ 2) The purpose of these study was to determine whether an intraocular injection of metamphetamine (MA) affects the healthy and/or the kainate (KA)-treated retina. Five microliter of saline, KA (40 mM), MA (1 mM), or a combination of KA and MA were injected into the vitreous

Abstracts / Neurotoxicology and Teratology 25 (2003) 381–397

humour of rats. The electroretinograms (ERGs) of the rat retinas were recorded before the injection of the drugs and then again 2 and 4 days after the injections. Retina samples were collected 1 day later and processed for immunohistochemistry (IHC) or western-blotting (WB) analysis. ERG, IHC, and WB analysis all showed that MA did not affect the retina on its own, as the results were indistinguishable from the saline-treated retinas. KA clearly affected the retina with the b-wave amplitude of the ERG reduced with clear changes in the localisation of GFAP, TH, ChAT, NO, and calretinin immunoreactivities. WB analysis also showed a change in GFAP and calretinin contents. While the IHC and WB data did not suggest that MA exacerbated or blunted the effect of KA, this was not the case for the ERG. Here the reduction in the b-wave amplitude caused by KA was similar whether measured at 2 or 4 days after injections. However, when MA was added to the KA, the reduction of the b-wave amplitude was greater at 4 days then at 2 days. The ERG data suggest that MA may exacerbate the toxic effect of KA. Rodrigues LG was supported by PRAXIS XXI/BD/18508/98 (Portugal).

NBTS 18 Thermoregulatory and behavioral sequelae of neonatal ‘‘ecstasy’’ (MDMA) treatment in rats Piper* BJ, Safain* M, Meyer JS (Neuroscience and Behavior Program, University of Massachusetts, Amherst, MA) Many users of ‘‘ecstasy’’ (MDMA; 3,4-methylenedioxymethamphetamine) are women of reproductive age. The present study was designed to examine the long-term functional consequences of MDMA treatment in a rat model of human third-trimester exposure. On postnatal days (PND) 1 –4, Sprague – Dawley rats received twice daily subcutaneous injections of either 10 mg/kg MDMA or saline vehicle (injections separated by 4 hour). Beginning on PND 70, male offspring were evaluated for object recognition memory, anxiety in the social interaction test, and aggressive behavior. Littermates were tested for their thermoregulatory responses to a single challenge with 10 mg/kg MDMA ip. Preliminary results from the challenge study show that acute MDMA treatment produced the expected hyperthermic response in control offspring, whereas the neonatal MDMA-exposed animals exhibited a significant hypothermia instead. Initial results of the behavioral testing indicate that neonatal MDMA may impair nonspatial working memory in adulthood but has no effect on anxiety-like behavior in the social interaction paradigm (examination of aggressive behavior is in progress). Although additional animals need to be studied to confirm our preliminary findings, the results thus far suggest that developmental exposure to MDMA has enduring functional effects that persist at

385

least into young adulthood. Supported by a Faculty Research Grant from the University of Massachusetts.

* Travel Award Recipient NBTS 19 Prenatal ± MDMA exposure reduces striatal and NAc monoamine metabolism and increases locomotor activity in P21 rats Koprich JB, Campbell* NG, Kanaan* NM, Lipton JW (Department of Neurological Sciences, Rush University, Chicago, IL, USA) Recent epidemiologic data suggest that pregnant women who use MDMA limit its use to their first trimester. However, current MDMA models have focused on perinatal exposure regimens believed to be analogous to human third trimester. The current study examined the consequences of prenatal MDMA exposure [15 mg/kg sc, b.i.d., embryonic days (E) 14– 20] on the behavior and striatal and NAc neurochemistry of juvenile rats (P21). HPLC analyses revealed statistically significant decreases in the monoamine metabolites: DOPAC, HVA, and 5-HIAA in striatum and a significant reduction (males only) in 5-HIAA and dopamine activity (HVA/DA, DOPAC/DA) in the NAc of MDMA exposed rats vs. controls. In addition, rats exposed to MDMA prenatally had a 35% increase in ambulatory activity in a 20-min novel cage exposure as compared to controls [ F(1,58) = 6.21, P=.016]. These data are the first reports of long-term neurochemical and behavioral alterations in a rodent model of prenatal MDMA exposure. More importantly these data are reminiscent of those found in the Wistar –Kyoto SHR Rat, an accepted animal model of AD/ HD in which juvenile animals demonstrate reduced striatal DA and NAc activity and increased behavioral activation. Further analyses are needed to elucidate the consequences of prenatal MDMA exposure on neural development and subsequent behavioral alterations. Supported by: R01DA12624.

NBTS 20 Short-term effects of P11 MDMA treatment on monoamines, ACTH, corticosterone and serotonin transporter in rats Vorhees1 CV, Schaefer *’ 1 TL, Ehrman *’ 1 LA, Sah *’ 1 R, Gudelsky *’ 2 GA, Williams1 MT (Cincinnati Children’s Research Foundation and University of Cincinnati College of Medicine, Cincinnati, OH1 and College of Pharmacy, University of Cincinnati, Cincinnati, OH2) We previously reported that MDMA administration from P11 –20 results in adult deficits in spatial and sequential learning and memory, whereas P1 – 10 administration does not. At the end of testing, MDMA rats showed small

386

Abstracts / Neurotoxicology and Teratology 25 (2003) 381–397

hippocampal NE increases, small frontal cortical and hippocampal decreases, and no change in frontal cortex DA. None of these changes correlated with the learning deficits. In this experiment, we determined whether P11 –20 MDMA might cause monoamine and glucocorticoid changes during the treatment interval. On P11, we administered MDMA (10 mg/kg) or saline to entire litters (eight litters/group) every 2 hours for a total of four doses. Striatum, hippocampus, thymus, and blood were collected from one M/F pair from each litter at 17, 24, 30, or 78 hours after the first dose. Increased ACTH was observed at 1 and 7 hours and increased corticosterone was observed from 1 to 24 hours. 5-HT was reduced in hippocampus from 1 to 78 hours with peak reduction at 24 hours (reduced by 53%). 5-HT was reduced in the striatum from 24 to 78 hours with the peak at 24 hours (reduced by 42%). DOPAC in striatum and 5HIAA in striatum and hippocampus were also reduced, whereas striatal DA was not affected. Recent preliminary analyses show reductions in hippocampal SERT activity as well. Support by NIH DA11902, DA14269 and ES07051.

NBTS 21 The influences of adolescents’ attitudes and peers on their initiation and cessation of cigarette smoking and marijuana use Porath* AJ, Fried PA (Department of Psychology, Carleton University, Ottawa, Ontario, Canada) The correlates of both the initiation and cessation of cigarette smoking and marijuana use are currently under investigation in one-hundred fifty-two 16 – 21-year-old adolescents for whom prenatal exposure to cigarettes and marijuana had been ascertained. The subjects, participants in the ongoing longitudinal Ottawa Prenatal Prospective Study, are from a low-risk, predominately middle-class sample. Participants reported their history and current use of a variety of substances (including cigarettes and marijuana), and provided a urine sample, which enabled the validity of this self-report data to be determined. The adolescents also reported their disapproval of others’ drug use, perceptions of friends’ disapproval of use, assessment of risk, perceptions of difficulty associated with acquiring drugs, and the proportion of their friends who have both tried and regularly use drugs. Previous study on the correlates of youth drug use initiation and cessation have identified the influences of one’s attitudes and peers as being critical factors. This study, however, significantly extends previous inquiry on these phenomena in that in addition to determining the influence of prenatal drug exposure on both the initiation and cessation of cigarette smoking and marijuana use, it will also assess the extent to which the influences of attitudes and peers on drug use and cessation differ between those youth who were prenatally exposed to nicotine and marijuana, and those who were not.

The possible existence of sex differences will also be investigated.

NBTS 22 Acute embryonic exposure to alcohol raises circulating corticosterone in adult mice Rabe A, Dumas R, Schuller-Levis* G, Park* E (NY State Institute for Basic Research in Developmental Disabilities, Staten Island, NY) Hyperresponsiveness to stress, as expressed by a high level of circulating corticosterone, is an established consequence in progeny of rats that have been exposed to alcohol for multiple days during pregnancy. There appears to be no information whether an acute embryonic exposure has similar consequences. We explored this with adult offspring of C57Bl/6J mice exposed to alcohol on E9 (plug = E1). Two intraperitoneal injections of 2.9 g/kg alcohol were given 4 hours apart. Independent groups of alcohol-exposed and saline-injected control progeny were studied at mean ages of 7 and 22 months, either after a 12-hour restraint or norestraint in the same environment. A blood sample from the retro-orbital plexus was analyzed for corticosterone. The results from the young adult alcohol-exposed mice were consistent with findings based on chronic gestational exposure to alcohol: they secreted corticosterone in response to restraint stress well in excess of control levels ( P < .008). In contrast, the old alcohol-exposed mice showed control levels of corticosterone response to restraint, but a tendency to elevated levels without restraint stress. Does the loss of their proclivity to hyperrespond to stress indicate recovery of normal function, or does it suggest deterioration of the hypothalamic-pituitary-adrenal axis?

NBTS 23 Prenatal IV cocaine: teratogenic effect on the locus coeruleus (LC)? Mactutus CF, Hasselrot* U, Welch MA, Snow DM, Strupp BJ, Booze RM (Dept. Psychol., Univ. S. Carolina; Columbia, SC; Anat. and Neurobiol. Univ. of Kentucky, Lexington, KY; Dept. Psychol., Cornell University, Ithaca, NY) Attentional dysfunction in school-aged children appears to be the most reliable neurobehavioral alteration consequent to maternal cocaine (COC) abuse during pregnancy. The LC, the primary source of noradrenergic (NE) neurons in the CNS, was examined as a potential basis for this dysfunction. A gestational exposure period design was employed. Long– Evans female rats (N = 64) implanted with an intraventricular access port, were bred and given saline or COC HCl (3 mg/ kg/ml) from GD8 –14 (1
/day), GD14 –21 (2
/day), or GD8 – 21 (1
/day—GD8 – 14, 2
/day—GD15 – 21). COC had no significant effect on maternal/litter parameters. At 21

Abstracts / Neurotoxicology and Teratology 25 (2003) 381–397

days of age, one male and one female of each litter were anesthetized, perfused with paraformaldehyde, and their brains were removed for neuroanatomical measurements of the LC. Preliminary analyses suggested an 8% reduction in volume of the LC in GD8 – 14 (but not GD14 – 21) COCexposed pups, which was larger than the characteristic sexually dimorphic (6%) difference in volume. Using the optical disector method, a 12% (F > M) gender difference in LC neuron number was noted in the GD8 – 14 group, in contrast to a < 1% gender difference for controls and a 4 – 6% M>F pattern for both groups that received COC during GD14 –21. Thus, the COC early group (which also displays alterations in LC neurite outgrowth) suggests a true teratogenic effect on NE neurons in female offspring. Supported by NIH: DA09160, DA11337, DA12719, and DA13965.

NBTS 24 The effects of prenatal cocaine exposure on infant visual recognition memory and speed of information processing Singer L, Eisengart* LJ, Minnes* S, Jey* A, Arendt R, Min* MO (Department of Pediatrics, Case Western Reserve University, Cleveland, OH) Studies have found impaired selective attention after prenatal cocaine exposure. Our longitudinal study of 177 cocaine exposed (CE) and 175 nonexposed (NCE) infants examined visual information processing, using measures of novelty preference and fixation duration. The Fagan Test of Infant Intelligence (FTII) measured percent novelty and mean visual fixation duration at 6 and 12 months (corrected ages). Infants were recruited at birth from an urban, low socioeconomic status, polydrug-exposed population with exposure status determined through maternal self-report and urine and meconium screens. Potential confounding variables, sociodemographic characteristics, maternal use of other drugs, and infant medical risk were controlled. Cocaine exposure predicted novelty preference, with CE infants having lower scores (CE mean = 57% vs. NCE 59% P < .02). At 6 months heavily CE infants were more likely to be ‘‘at risk’’ than NCE infants (38% vs. 24%, P < .04). There was a nonsignificant trend for fixation duration scores to be differentially affected, CE infants had marginally shorter looking times at 6 months (1.6 vs. 1.7 s, P < .06). Looking times decreased with age. Thus, CE infants had poorer visual recognition memory suggesting risk for learning problems. Cocaine exposure had a marginal association with faster information processing speed, which did not enhance cognitive performance.

NBTS 25 Passive avoidance learning of adult female rats exposed to cocaine during preweaning period

387

Torres-Reveron* A, Melnick SM, Dow-Edwards DL (Program in Neural and Behavioral Sciences, Physiology, Pharmacology, SUNY Downstate, Brooklyn, NY) Cocaine exposure during critical periods of brain development may result in learning deficits. To test this possibility, female rats received water or cocaine (50 mg/kg) during postnatal days (P) 11 – 20 and were tested for passive avoidance learning between P135– 152. On the first day of testing, rats were habituated to the chamber by receiving a 180-s trial initiated by illuminating the side where the animal was. The rat was able to freely move to the dark side of the chamber without receiving any shock. On the second day, rats received three 180-s trials. A footshock (0.5 mA) was delivered upon crossing to the dark side of the chamber and terminated when the animal returned to the illuminated side. The next day, rats received one trial to test for avoidance learning. Results indicated that female rats exposed to cocaine showed significantly less time to cross to the dark side during acquisition and also showed a delay in crossing back to the illuminated side compared to waterpretreated females. On the test day, cocaine-pretreated rats crossed to the dark side faster than the control group, suggesting a deficit in retention of passive avoidance. These data suggest that preweaning cocaine in females may have long-term effects on the neural circuits involved in passive avoidance learning. Supported by NIDA010990 and APA Neuroscience Minority Fellowship. Omnitech passive avoidance equipment was a donation of Eli Lilly.

NBTS 26 Preweaning cocaine: an examination of standard measures of anxiety Melnick SM, Dow-Edwards DL (Pharmacology/Physiology, SUNY Downstate Medical Center, Brooklyn, NY) Prenatal cocaine exposure has been shown to alter many neurobehavioral parameters including anxiety. The present study was designed to assess the role of preweaning cocaine administration, a model of 3rd trimester human exposure, on behavior in a variety of anxiety measures including the elevated plus maze, light/dark box and acoustic startle. During postnatal days (P) 11 – 20, male and female rats received either distilled water or 50 mg/kg cocaine daily. At P28, nondrugged animals were tested on the elevated plus maze. Cocaine-pretreated animals spent more time in and displayed a shorter latency to reach the open arm (less anxious behavior) compared to water-pretreated controls. When retested at P35, cocaine-pretreated rats spent less time beyond the rails (more anxious behavior). At both ages, indicators of exploratory behavior were decreased in cocaine-pretreated animals. However, cocaine-pretreated animals did not differ from controls either in time spent in the light area of the light/dark box at P30 or in decibels support-

388

Abstracts / Neurotoxicology and Teratology 25 (2003) 381–397

ing the half maximal startle response following a range of acoustic stimuli at P29. In addition, while open arm time assessed on the elevated plus maze at P66 was not different, cocaine-exposed males tended to display fewer open arm entries compared to other treatment groups. Therefore, preweaning cocaine exposure appears to interact with neural pathways involved in anxiety and/or exploratory activity but the effect is not a robust one. Supported by NIDA DA10990.

NBTS 27 Prenatal heroin exposure alters cholinergic receptor stimulated translocation and basal levels of the PKCbeta II isoform Yanai1,2 J, Yaniv*’ 1 SP (Ross Lab. for Neural Birth Defects, Dept. Anat. and Cell Biol., Hebrew U.-Hadassah Med. Sch., Jerusalem, Israel1 and Dept. Pharmacol. and Cancer Biol. Duke Med. Ctr., NC, USA2) Mice were exposed prenatally to heroin by injecting dams with 10 mg/kg daily on gestational days 9 –18. At adulthood, they showed behavioral deficits related to septohippocampal cholinergic synaptic function, concomitant with global cholinergic hyperactivity, including an increase in basal PKC activity, and a consequent desensitization of PKC to cholinergic input, both highly correlated with behavioral performance. Cholinergic grafting reversed the biochemical and behavioral deficits. Recent studies designed to pinpoint the changes in the behaviorally relevant PKC-gamma isoform showed a desensitization to cholinergic input, as attested to by the reduction in their carbachol-induced translocation. The current study showed a substantial decrease in basal levels of the behaviorally relevant PKC-betaII isoform in treated mice, both the phosphorylated (and therefore active) and the nonphosphorylated form as well as a reduction in their translocation to the cell membrane. There were no differences in the induction of the non-behaviorally related PKC-alpha. Supported by USPHS HD 40820 and Israeli Anti-Drug Authority.

NBTS 28 Juvenile animal studies: species selection and comparative organ system developmental schedules Hurtt M (Pfizer Global Research and Development, Groton, CT) Nonclinical animal studies are widely accepted as a critical component for assessing potential risk of a compound to ensure the safety of humans. Current testing guidelines, such as the assessment of developmental toxicity, fertility, and developmental neurotoxicity, do include the use of immature animals. Therefore, the need to conduct additional nonclinical juvenile animal studies may be necessary. There

are several factors that need to be considered in the selection of the test species for these juvenile animal studies. These factors include the existing toxicological data, comparative species developmental stage, likely route of human exposure, and technical feasibility. The presentation will briefly discuss these factors with a focus on reviewing speciesspecific developmental schedules for specific target organs. An understanding of species-specific development for the identified target organs from toxicological studies is critical in selection of animal models for juvenile toxicity studies. Species differences in development of key targets for the pulmonary, renal, skeletal, CNS, and reproductive systems will be presented.

NBTS 29 Nonclinical studies in juvenile animals: FDA perspective (Introduced by J.E. Fisher) Hastings* KL (Center for Drug Evaluation and Research, U.S. Food and Drug Administration, Rockville, MD) Studies in juvenile animals currently are not required to support clinical drug trials in pediatric patients, but are requested on a case-by-case basis. These studies are requested for a variety of reasons, but typically reflect concerns based on adverse effects that were observed in mature animals or in adult patients. Unfortunately, this practice is somewhat inconsistent with the idea that immature/developing biological systems can be more susceptible to harm and especially to irreversible adverse effects. The Center for Drug Evaluation and Research has written a guidance on juvenile animal studies. This document attempts to take into consideration what is known about developmental biology in formulating a reasonable approach to determining when juvenile animal studies are needed. Case studies were important in writing this guidance, and several examples will be discussed in this presentation.

NBTS 30 Current US EPA approach to assessing hazards of childhood exposures to pesticides Makris S (US EPA, Washington, DC) The US Environmental Protection Agency (EPA) conducts risk assessments for pesticides, toxic substances, and environmental pollutants, to address the potential for adverse outcomes in infants and children. Hazard and dose response assessments are the cornerstone of the risk assessment process. Information used in this process is typically derived from laboratory animal studies: a prenatal developmental toxicity study in two species, a two-generation reproduction study in rats, and for some chemicals a developmental neurotoxicity (DNT) study in rats. The reproduction and DNT studies include juvenile subjects that have been

Abstracts / Neurotoxicology and Teratology 25 (2003) 381–397

exposed to the test substance through critical pre- and postnatal stages of development. Common practice has assumed that (1) offspring exposure has occurred; (2) levels in offspring are similar to those in adults; and (3) exposures to young rodents are similar to potential exposures that infants and children might experience. Recognizing these assumptions may be inaccurate, EPA is making efforts to more directly characterize juvenile exposures. On a chemical-specific basis, adjustments to guideline protocols [e.g., alternative routes or periods of exposure and/or evaluation of additional endpoints (e.g., biomarkers of exposure/ effect)] have been used in studies that specifically address the immature animal. Such a chemical-specific approach to study design was taken by the EPA Office of Pesticide Programs for assessment of developmental neurotoxic potential and age-related sensitivity to cholinesterase inhibition for the organophosphorous pesticides. EPA encourages the expanded use of pharmacokinetic and pharmacodynamic information to characterize exposure and/or assist in designing targeted studies for the assessment of risks to infants and children.

NBTS 31 Design considerations in juvenile toxicity studies Buelke-Sam J (Toxicology Services, Greenfield, IN) Developmental testing approaches are evolving for both pharmaceutical and environmental agents as concerns for childhood exposures become more specific. Whether conducted for a class of compounds, such as the OP pesticides, or on a case-by-case basis for particular pharmaceuticals, many of these studies require designs that mesh both developmental and adult toxicity concerns. In addition to comparative age/organ system schedules, standard principles in developmental testing apply to the design and interpretation of juvenile studies: critical periods for exposure, critical periods for assessment, litter effects, function may be a more sensitive indicator of toxicity, etc. Standard components of acute and subchronic testing also apply: target organ assessment, defining systemic exposures, assessment of clinical and histopathologic endpoints, etc. Toxicity assessment is of greatest concern for those organs and systems that are not mature at birth in the human. Postnatal development continues as a complex ‘‘moving target,’’ not only for assessing target organ sensitivity, but also within other systems that impact developmental exposures, e.g., absorption, metabolism and renal excretion capabilities, etc. The design of these studies directly impacts the ability to identify treatment-related delayed/ accelerated development as well as specific toxicity: patterns of effect, within and across endpoints and ages, are key to interpreting results. Juvenile toxicity studies are expected to function as other current toxicological screening tools with observed high-dose effects assumed to be

389

predictive of an increased human risk. Overall assessment of risk for pharmaceuticals also includes the risk of not treating a childhood medical condition, as well as identifying risk of exposure.

NBTS 32 Dermal dosage administration of neonatal rat pups: mother –infant separation with occlusive dosing Barnett* JR, JF1, Learn* DB 1, York RG1, Hoberman AM1, Vedula* U 2, Osimitz* TG 3 (Argus Laboratories, A Division of Charles River Discovery and Development Services, Horsham, PA1, S.C. Johnson and Son, Inc., Racine, WI2 and Infoscientific, Inc., Charlottesville, VA3) Hazard identification studies for products intended to be applied dermally to young children should be conducted using dermal application of the test substance. Mother – infant separation (MIS) is often used to avoid test substance ingestion by the mother and resulting interference with the delivered dosage. In addition, an occlusive wrap is used to fix the applied dose in place as well as prevent oral ingestion of the test substance. Identification of test substance effects versus those associated with MIS and/or dosing procedures is critical for valid study interpretation. MIS and occlusive dermal dosing (6 hours daily, PNDs 9– 20) were used to apply dosages to F1 generation rat pups. Four groups were used: pups remaining with the dam (Group I); pups separated, but untreated (Group II); separated, treated with water and occluded (Group III); separated, treated with a test substance and occluded (Group IV). Daily measurements included body weights and clinical observations. On PND 21, a Detailed Clinical Observation (DCO) evaluation was performed on each pup. No adverse clinical signs were observed during the study. Body weight data analyses indicated (1) MIS alone had no effect on the pups (Group II v. I); (2) the occlusive procedure reduced body weight gain (Group III v. II); and (3) males receiving test substance showed a decrease in body weight gain during PND 18– 21, relative to those pups dosed with water (Group IV vs. III). The use of MIS with occlusive dermal dosing caused a reduction in body weight gain without adverse clinical responses.

NBTS 33 Factors affecting grip strength testing Marable BR, Andrus AK, Stebbins* KE, Maurissen JPJ (Toxicology and Environmental Research and Consulting, The Dow Chemical Company, Midland, MI) The rodent grip strength test is a putative measure of muscular strength and is part of the functional observational test battery to screen for neurobehavioral toxicity. Changes in grip strength have been interpreted as evidence of neurotoxicity. Despite its widespread use, questions remain about

390

Abstracts / Neurotoxicology and Teratology 25 (2003) 381–397

what the grip strength test truly measures. In this study, we tested potential grip strength confounders in female F344 rats, including operational parameters, disruption of sensory function (using doxorubicin), and changes in body weight. Operational parameters (sampling rate, grid support, trial angle, but not trial speed) had dramatic effects on grip strength data. Doxorubicin (10 mg/kg iv) caused decreases in forelimb and hindlimb grip strength (27% and 32%, respectively, compared to controls) that were correlated with degeneration of peripheral and central sensory components. Feed-restriction-induced loss of body weight (26% compared to controls) and muscle mass (20% compared to controls) reversibly decreased both forelimb and hindlimb grip strength (18% and 17%, respectively, compared to controls). Ignoring these confounding factors might lead to increased variability and data inconsistency within single studies and in historical control data sets. Furthermore, given that the grip strength test can be influenced by factors other than true muscular strength, use of the term grip performance is proposed to better reflect the apical nature of this test.

NBTS 34 Mercury levels in blood and brain of infant monkeys exposed to thimerosal Burbacher TM, Shen D, Liberato N, Grant K, Cernichiari E, Clarkson T (Departments of Environmental Health and Pharmacy, University of Washington, Seattle, WA and Department of Environmental Medicine, University of Rochester, Rochester, NY) Recently, questions have arisen about the safety of thimerosal, an ethylmercury containing preservative used in some infant vaccines. Current EPA guidelines for methylmercury exposure (primarily through fish in the diet) have been used to assess the public health risk to children receiving thimerosal-containing immunizations as infants. Depending on the exact vaccinations, schedule, and size of the infant, some children may receive ethylmercury (in the form of thimerosal) approaching or at the EPA guideline levels. The purpose of this study was to compare the distribution of total and inorganic mercury in newborn monkeys following thimerosal exposure (via injection) with newborns exposed to methylmercury (via oral gavage). Infant monkeys were exposed to thimerosal or methylmercury at birth and at 1, 2, and 3 weeks of age. Blood mercury levels were determined 2, 4 and 7 days after each exposure. Brain mercury levels were assessed 2, 4, 7 or 28 days after the last (3 week) exposure. Preliminary data indicate that the half-life of mercury in blood following thimerosal exposure is significantly lower than the half-life of mercury following methylmercury exposure. Brain levels of mercury were also significantly lower in the thimerosal group. Thus far, the data indicate that EPA guidelines for methylmercury exposure may not provide an accurate assessment of the public

health risk to children receiving thimerosal-containing immunizations. Supported by ES03745.

NBTS 35 Paraoxonase (PON1) polymorphisms and their significance for the developmental toxicity of organophosphates Cole* TB, Pettan-Brewer* C, Burbacher TM, Costa* LG, Furlong* CE (Departments of Environmental Health and Medicine, Division of Medical Genetics, University of Washington, Seattle, WA) Paraoxonase (PON1) is an HDL-associated enzyme involved in the metabolism of organophosphorous (OP) compounds, drugs, and oxidized lipids. PON1 activity is determined in part by a coding region SNP (Q192R) that affects its catalytic efficiency, and in part by differences in PON1 levels, which vary widely (>13-fold) among adults. PON1 levels are also developmentally regulated. Infants have very low PON1 levels and a variable onset of expression, reaching a plateau at 6 – 24 months. To assess the importance of the Q192R SNP for OP detoxication, a mouse model was used where human transgenes encoding PON1Q192 or PON1R192 were expressed in place of mouse PON1. Adult mice expressing hPON1R192 had similar PON1 levels as mice expressing hPON1Q192, but were significantly more resistant to dermal toxicity of chlorpyrifos oxon (CPO). The developmental onset of expression was similar for the hPON1Q192 and hPON1R192 transgenes. Thus, these mice could be used to assess the importance of the Q192R SNP for OP detoxication during development. PON1 knockout and wild-type mice are being used to determine the importance of PON1 for protecting against developmental toxicity associated with chronic CPO exposure. Endpoints for this study include cholinesterase levels, neuropathology, gene expression, and neurobehavioral assessment. Our findings indicate that children less than 2 years old, especially those homozygous for PON1Q192, would be particularly susceptible to OP toxicity. Supported by T32 AG00057, ES09601/EPA-R826886, ES09883, ES04696, ES07033.

NBTS 36 Postnatal behavioral functional evaluation of thalidomide in rabbits using eyeblink classical conditioning Denny KH, Barnett J, Hoberman* AM, Morseth* S, Stirling* D, Thomas* S, Teo S (Argus Laboratories, A Division of Charles River Discovery and Development Services, Horsham, PA; Milestone Biomedical Associates, Frederick, MD; Celgene Corporation, Warren, NJ) Current behavioral-functional evaluations in toxicology and teratology are performed in rodents. Testing of thalidomide

Abstracts / Neurotoxicology and Teratology 25 (2003) 381–397

(TH) required an alternate species for which there are few validated tests and/or control data. Evaluation of postnatal behavior in the rabbit is limited by behavioral and physiological characteristics that prohibit methods used in rodents. F1 pups from does dosed with 30, 150, and 500 mkd of (TH) from gestation day 18 through lactation day 28 were evaluated for postnatal behavior using an eyeblink classical conditioning test was validated and used as a surrogate of learning and memory. The test included two separate measurements (delay and trace) performed on randomly assigned animals (8/sex/dose). Each test session consisted of 60 trials in which a tone (the conditioned stimulus, CS) was paired with a corneal airpuff (the unconditioned stimulus, US). Each delay paradigm trial was designed to have the offset of the CS coincide with the onset of the US (an interstimulus interval). Each trace paradigm trial was designed to have the CS terminate with a ‘‘blank period’’ (400 ms without stimulus) before the onset of the US, for a total CS – US interval of 500 ms. Each paradigm continued for 5 consecutive days with a 2-day rest period followed by 3 days of consecutive testing for a total of 8 test days. Thalidomide in F0 does caused no statistically significant or biologically important differences in learning in F1 pups. A general tendency for increased learned responding occurred in the trace paradigm in the 150 mkd F1 generation rabbits. It is unclear if the increase was due to associative or nonassociative factors.

NBTS 37 Prenatal exposure to phenobarbital and behavior of adult mice offspring Christensen* HD, Gonzalez* CL, Rayburn WF (Dept. Pharmacology/Toxicology, University of Oklahoma, Oklahoma City, OK and Dept. Obstetrics and Gynecology, Univ. of New Mexico, Albuquerque, NM) Phenobarbital is an anticonvulsant with many therapeutic applications during pregnancy. The objective of this study was to assess the impact of prenatal exposure to a therapeutic dose of phenobarbital on behavior in adult mice offspring. C3H/He mice were randomly randomized to receive diet chow containing either phenobarbital (2.5 mg in 10-g chow) or a placebo for 1 week before mating and throughout gestation. This dose led to plasma concentrations that were therapeutic in humans and protective to maximal electroshock seizures. Adult offspring from eight litters of each group were evaluated. There were no significant differences between groups in duration of gestation, litter size, and birth weights. Fewer counts in a locomotor chamber were observed in phenobarbital offspring (524 vs. 688 for 60 min, P < .02). Impaired coordination of hindlimbs were observed in the phenobarbital offspring during the wire maneuver ( P < .001). The number of entries into the mirrored chamber was less after phenobarbital exposure (2.1 vs. 4.5, P < .05). These subtle effects on select

391

locomotor activity, hindlimb coordination, and responses to anxiety-provoking conditions require human correlation. Supported by Records Perinatal Research Fund.

NBTS 38 Residual neuropsychological sequelae of chlorine gas exposure Dilks LS, Matzenbacher DL (McNeese State University, Lake Charles, LA) According to the National Institute for Occupational and Health Safety, toxic levels of chlorine gas are reached at 1 part per 5000 (gas/air) and fatal levels are reached after a few breaths at 1000 parts per million. Immediate effects of chlorine exposure involve upper and lower respiratory distress. In one of the first studies examining neuropsychological effects of chlorine gas, Hilburn (2000) reported impaired recall and problems with concentration, balance, and following directions. In the current study, individuals exposed to toxic levels of chlorine gas from a ruptured pipeline in May of 1998 were administered five assessments of executive functioning, language, and adaptive functioning approximately 2.5 years postexposure. Participants included eight adults (seven males) with an average age of 45 years. Data indicated significant impairments in the areas of (1) attention and concentration, (2) speed of processing, (3) mental flexibility, (4) verbal and abstract processing, (5) insight, (6) speed of operations, (7) language processing, and (8) adaptive functioning. Premorbid status of these clients indicates gainful employment and no previous toxic exposure or deficiencies in the areas assessed. Given their occupational and educational histories, it is very unlikely they were functionally impaired prior to exposure. Given that our assessments were conducted more than 2 years postexposure, it appears neuropsychological impairments resulting from chlorine gas exposure are not transient but in fact may be permanent.

NBTS 39 Few behavioral alterations result from developmental cerebellar stunting induced by early postnatal alphadifluoromethylornithine (DFMO, eflornithine) treatment in rats Ferguson SA, Cada AM (Division of Neurotoxicology, National Center for Toxicological Research/FDA, Jefferson, AR) DFMO is used topically as a treatment for hirsutism and is under consideration as a chemopreventative agent and a treatment for malaria. Behavioral effects after developmental treatment have not been thoroughly described. Here, Sprague – Dawley rats were subcutaneously injected with 0 or 500 mg/kg DFMO (n = 16 litters/group) on postnatal days

392

Abstracts / Neurotoxicology and Teratology 25 (2003) 381–397

(PND) 5 – 12. Motor coordination (Rotarod, PND22– 25 and 80 – 82), play (PND32), dominance (water competition, PND54), long-term locomotor activity (PND57 –71), anxiety (light/dark box test, PND61 and elevated plus maze, PND79), acoustic startle (PND86) and prepulse inhibition (PND87) were assessed in males and females. DFMO-treated rats had shorter startle response latencies under both paradigms (acoustic startle and prepulse inhibition). Other tests indicated no significant effects of DFMO treatment. Adult siblings of these DFMO-treated rats exhibited a 10% decrease in cerebellar and 3% increase in brainstem weight (Ferguson et al., 2001). Despite this, the behaviors assessed here and the operant behaviors previously described (Ferguson et al., 2001) appeared remarkably normal. While more sensitive or challenging assessments may reveal DFMOinduced deficits, the comprehensive nature of the assessments used here suggests few alterations result from this paradigm of developmental DFMO treatment.

NBTS 40 Spatial working memory in female rhesus monkeys exposed to exogenous estrogen during puberty Golub MS, Hogrefe* CE, Germann SL (California National Primate Research Center, University of California, Davis, Davis, CA) Sex differentiation of spatial cognitive skills appears during adolescence. An automated (CANTAB) spatial working memory (SWM) test was used to assess the effects of pubertal treatment with exogenous estrogens on later brain function. Female rhesus monkeys received methoxychlor (MXC, 25 or 50 mg/kg/d) or diethylstilbestrol (DES, 0.5 mg/kg/d) orally from 24 to 36 months of age, 6 months before and after the anticipated age of menarche and were tested 6 months after discontinuation of dosing. A video monitor presented two, three, or four boxes that needed to be pressed in a nonrepeating sequence across trials with 3-s intertrial intervals to receive a sugar pellet. The MXC50 group was slower than controls learning the two-location sequence; which was required before three- and four-location sequences were added to the testing sessions. Both MXC50 and DES groups were delayed in improving performance with the three-location sequence after three 4session blocks ( P=.02). Only the MXC50 group failed to significantly improve performance across four 4-session blocks. Group differences were not significant on the more difficult four-location sequence. Further testing is needed to determine whether the performance impairment is specific to spatial tasks. Supported by USEPA STAR grant R827404.

* Travel Award Recipient NBTS 41 Postnatal exposure to fluoxetine (Prozac): enduring effects on reaction time and visual attention

Laroche* RB, Morgan RE (Psychology Department, Western Illinois University, Macomb, IL) Over the past two decades, the use of SSRIs to treat behavioral disorders in young children has grown rapidly. However, little evidence exists regarding the safety and efficacy of these drugs for use in children. Using a rodent model, the current study investigated whether postweaning exposure to a prototype SSRI, fluoxetine, influences performance on visual discrimination and attention tasks. Female Long –Evans rats (n = 24) were administered (via oral gavage) either fluoxetine (10 mg/kg) dissolved in apple juice or apple juice alone from PND25 to PND49 (25 days). After a 10-day washout period, Ss were trained to perform a simultaneous visual discrimination task to a criterion of 80% correct. Ss were then trained for 20 sessions on a visual attention task that consisted of varied stimulus delays (0,3,6, or 9 s) and cue durations (200, 400, or 700 ms). Animal’s ages ranged from 100 to 110 days by the completion of the study. Results indicated that fluoxetine-exposed animals responded more rapidly on both tasks (shorter response latencies) and they committed fewer omission errors. The groups did not differ on measures of impulsivity (commission errors), response accuracy, or motivation. These results indicate that pharmacological manipulation of the serotonergic system during early postnatal development can result in behaviorally relevant, long-term alterations in the central nervous system.

NBTS 42 Effects of satureja khuzestanica on reproduction potency of female rats Mortazavi* SHR, Ebrahimi* M, Salehnia* A, Abdollahi M (Department of Toxicology and Pharmacology, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran) This study has been carried out to evaluate the effects of satureja khuzestanica on reproduction in female rats. Infused solution of satureja khuzestanica was added in low dose (7.5 mg/ml) and high dose (15 mg/ml) 14 days before mating to female rats through drinking water. After 14 days of treatment, the female rats were mated with male rats. Mating was proved by smear test. In Day 18 of pregnancy before estimated normal delivery, the rats were anesthetized and cesarean surgery was done on them. The uterus is excised and examined, weighed and evaluated for the number of live, dead, and resorbed fetuses. Live fetuses were weighed and divided for evaluation of skeletal abnormalities and soft tissue anomalies. The results showed no statistical difference in external abnormalities between treated and control groups. Number of fortunate pregnancy and live offspring increased significantly in low-dose treated group. In comparison with control group, no fetus was observed in high-dose group. The results of this research

Abstracts / Neurotoxicology and Teratology 25 (2003) 381–397

show that product of herbal oral infusion in low doses increases the reproduction ability in female rat and inhibits it with high dose. Regarding components of satureja khuzestanica including carvacrol and thymol as known antioxidants many discussions can be made. Results of this study may be extrapolated to human viewing use of satureja khuzestanica as contraceptive or stimulant of reproduction. Further studies are needed to reach to that decision. Supported by Faculty of Pharmacy, Tehran University of Medical Sciences and The Khorraman.

NBTS 43 Different effects on behavioral development of general and CNS-specific knockout of the erbB4 receptor Golub MS, Lloyd KCK, Germann SL (Depts. of Internal Medicine and Anatomy, Physiology and Cell Biology, University of California, Davis, Davis, CA) ErbB4 is an important brain receptor for the neuregulin1 growth factor. Because deletion of both alleles leads to early embryonic death, knockout mice were created in which a single allele could be deleted in all cells at the blastocyst stage, or in neuronal precursors only (conditional knockout) at the embryonic stage. By breeding, a CNS-specific homozygous knockout could be created, along with the two different heterozygous knockouts. These mice were compared to wildtype (no gene deletion) littermates in a battery of behavioral tests. Mice with either allele deletion were delayed in early postnatal motor development, but the delay extended to the end of lactation for the blastocyst deletion. Blastocyst deletion also led to altered cue use in a Morris maze task. Mice with embryonic deletion displayed late lactation growth retardation, a higher level of activity as weanlings, and a lower level of spontaneous motor activity as adults. Mice with both deletions displayed primarily the embryo deletion phenotype. The data demonstrate the difficulty of interpreting genotype effects on early behavior of transgenic mice. Supported by a UCDavis Health System Award.

NBTS 44 Do rats have an IQ? Holson RR (Department of Psychology, New Mexico Tech, Socorro, NM) The concept of IQ continues to play an important, if controversial, role in human affairs. This concept is based upon factor analyses of human ‘‘cognitive’’ test performance, analyses, which have identified a single ‘‘g’’ or ‘‘general’’ factor in such performance. This g factor correlates almost perfectly with IQ test scores, and suggests that subjects who do well or badly on one subtest tend to do well or badly on all such tests. Whether this mathematical result

393

has biological meaning is, of course, quite another matter. Interestingly, there have been almost no published efforts to determine whether there is a similar g factor in rodent test performance, and hence whether rats too can be said to possess an ‘‘IQ.’’ I report here the results of four common behavioral tests (Barnes’ Maze, Radial 8-Arm Maze, Morris Maze and simple black – white Visual Discrimination) given to male rats from 36 litters after handling for 2 weeks. Principal component analysis suggested a first-order factor common to all but the Barnes Maze (in our hands this test was too easy, and hence had low reliability). However, this g factor seemed to be related to fear in the test setting NOT ‘‘cognitive’’ factors. Whether these findings suggest that rats lack a cognitive g factor, or simply that we are using the wrong tests, will be discussed.

NBTS 45 A teratologic study of prenatal nelfinavir in the rat Dow-Edwards DL, Melnick SM, Torres-Reveron A (Department of Physiology/ Pharmacology, Program of Neural and Behavioral Sciences, SUNY-Downstate, Brooklyn, NY) AIDS remains a significant health problem throughout the US, Europe and in particular in sub-Saharan Africa. In addition to traditional antiretroviral therapies such as AZT, newer therapies consist of a ‘‘cocktail’’ of AZT, 3TC and protease inhibitors such as nelfinavir. Since animal studies of the teratologic effects of prenatal nelfinavir have not been published, our group initiated a series of studies of prenatal nelfinavir (NFM) exposure in the rat. We examined the effects of 41 or 82 mg/kg/day NFM administered ig beginning on Day 1 of pregnancy (G1) in comparison to the vehicle (glycerol)-intubated control. On G20, dams were euthanized and the fetuses counted, weighed, and examined using the fresh visceral dissection technique. Brains were frozen for neurochemical analyses and the skeletons were preserved in alcohol for the alizarin-red method of visualizing bone. Preliminary data indicate that the 82 mg/kg dose of NFM decreased body weight of the fetuses compared to the other groups. There were no significant effects on litter size or male –female ratio. Examination of the viscera has shown that there were seven abnormalities in seven litters of the high-dose group, four in eight litters of the low-dose group and two in nine litters in the control group. Overall, the results suggest that prenatal nelfinavir at the highest dose tested has significant adverse effects on fetal development. Supported by NIH Grant #HD 35035.

NBTS 46 Neurobehavioural toxicity of in utero and lactational exposure to a chemical mixture based on blood levels in Canadian Arctic populations

394

Abstracts / Neurotoxicology and Teratology 25 (2003) 381–397

Bowers WJ, Nakai JS, Chu I, Wade M, Moir D, Gill S, Mueller R, Pulido O (Environmental Health Sciences Bureau, Health Canada, Ottawa, Canada) The current study examined the impact of in utero and lactational exposure of rodents to a mixture of environmental pollutants found in the blood of Canadian Arctic populations. The mixture contained methylmercury, 12 organochlorine pesticides, and 14 PCB congeners. Pregnant Sprague – Dawley rats were exposed to 0, 0.05, 0.5, or 5.0 mg/kg of the mixture or 15 mg/kg Aroclor 1254 from gestation day 1 to weaning at PND23. A 100-fold increase in mixture dose increased maternal blood Hg by 90-fold but offspring blood levels by 19-fold. Aroclor and the highest mixture dose decreased offspring weight gain 25% and delayed ear opening, accelerated eye opening and decreased grip strength at PND10 – 14. At PND21 only Aroclor reduced acoustic startle responding but the lowest mixture dose attenuated prepulse inhibition of startle responding, suggesting alterations in CNS processing of prepulse stimuli. Motor activity testing revealed increased locomotion in the high-dose group from PND17 to PND75 but decreased locomotion in the low dose group at PND17. At PND75, the lowest dose increased rearing in females but decreased it in males. The highest mixture dose also impaired visual discrimination learning, primarily in females. The results indicate that this chemical mixture based on human blood profiles affects neurobehavioural functioning but the nature of the effects varies with the dose, gender and the age of the animals.

* Travel Award Recipient NBTS 47 Gender-specific effects of 5-AZA-20-Deoxycytidine (5-AZA-CdR) in mice exposed during embryonic development (Introduced by S. Ferguson) Cisneros FJ *,1 BRANCH S *,2 (Division of Neurotoxicology, NCTR, AR1 and Department of Toxicology, NCSU, NC2) 5-AZA-CdR, a DNA demethylating agent, affects postnatal development and reproduction in mice exposed in utero. To determine the role of gender in these effects, pregnant CD-1 mice were administered 1 mg/kg ip of 5-AZA-CdR at gestation day (GD) 10. At GD17, a group of dams was killed and fetuses were collected to determine DNA methylation level. The remainders were allowed to give birth. At 3 months of age, reproductive capacity was studied. At 5 months old, mice were killed and serum was collected to determine glucose, corticosterone, and IGF-1 levels. Body weights were recorded at 21 days, 3 and 5 months of age. DNA methylation levels were higher in exposed than in control fetuses. Body weights of treated males and females were reduced at weaning compared to controls, but by 5 months of age, only the male body weight was reduced. Reproductive capacity of males was more affected than

females. To determine if this phenomenon was a behavior alteration, a male sexual behavior test was conducted. Exposed males exhibited low mating capacity and diminished sexual interest when exposed to a receptive female. Serum IGF-1 levels were lower in exposed males when compared to controls, but not in females. Levels of corticosterone and glucose were not altered. Our data suggest that the decreased serum IGF-1 levels associated with the treatment could be a key contributing factor to the observed growth retardation and altered male reproductive behavior in the in utero-exposed mice. Supported by NIH # ES08452.

NBTS 48 Ontogeny of timing ability in children and effects of stimulant medication on such in children with attention deficit hyperactivity disorder (ADHD) Paule MG, Baldwin* RL, Flake* RA, Blake* DJ, Edwards* MC, Field* CR, Meaux* JB, Chelonis JJ (National Center for Toxicological Research, Jefferson, AR and Arkansas Children’s Hospital, Little Rock, AR) An automated system was used in children (n = 720) aged 5 to 13 years to quantitate time estimation performance. The temporal response differentiation (TRD) task required subjects to hold a response lever down for at least 10 s but no more than 14 s. Nickels were delivered for each correct response. Evidence of timing ability (a peak in the percentage of lever holds occurring in the reinforced window of  30%) was noted in children as young as 6 years old, after which correct responding increased with age to  65% at age 13. There were no sex differences in TRD task performance. Children with higher IQ scores made more correct lever holds. The performance of young children with below average IQ was more variable than that of children with above average IQ. For children with ADHD (n = 17), TRD task performance improved significantly when they were tested within 2 hours of taking their prescribed dose of methylphenidate (MPH). Percent lever holds between 10 and 12 s increased; percent short (2 –4 s) lever holds decreased, percent burst responses (rapid lever presses) and response variability decreased. These data demonstrate that the TRD task provides useful measures in children and that MPH enhances timing precision in children with ADHD. Supported by NCTR and Arkansas Children’s Hospital.

NBTS 49 Succimer chelation significantly ameliorates the lasting cognitive and affective dysfunction produced by early lead (Pb) exposure in a rodent model Strupp BJ, Stangle* D, Strawder-Man* M, Smith D (Cornell University, Ithaca, NY; University of California at Santa Cruz, Santa Cruz, CA)

Abstracts / Neurotoxicology and Teratology 25 (2003) 381–397

The present study was designed to assess the efficacy of succimer chelation in alleviating the cognitive and affective changes produced by early lead exposure. A 2
3 factorial design was used, including three levels of Pb exposure and two succimer (SUCC) conditions (SUCC or vehicle). Pb exposure occurred for the first 30 days of life, followed by 21 days of SUCC or vehicle. Cognitive testing was then conducted for 6 – 8 months. Both Pb-exposed groups showed learning deficits, whereas rats in the higher exposure group also showed attentional dysfunction and heightened reactivity to committing an error on the prior trial. SUCC prevented some, but not all, of these Pb-induced alterations. Specifically, SUCC significantly ameliorated the learning impairment in both Pb groups but to a greater extent in the lower exposure group. In addition, the heightened reactivity to errors seen in the higher exposure group was completely normalized by SUCC. In contrast, SUCC did not significantly ameliorate the attentional dysfunction of the higher exposure group. Finally, this study provided evidence that SUCC treatment alone, in the absence of Pb exposure, is not benign. Rats treated with SUCC, but not Pb, showed deficits in learning and attention. These findings represent the first evidence that SUCC is effective in alleviating Pb-induced neurotoxicity but urge caution in continued use of the drug in the absence of elevated tissue Pb levels (after Pb levels have declined) or at very low exposure levels. Supported by NIH: ES07457, ES05950; DK07158.

NBTS 50 Utility of fetal movement coordination in assessing nervous system functioning after prenatal administration of the neurotoxin methylazoxymethanol (MAM) Kleven GA, Queral* L, Robinson* SR (Department of Psychology, University of Iowa, Iowa City, IA) It is now thought that some neuromotor disorders (e.g., cerebral palsy, ALS, Parkinson disease) may be the result of toxin exposure early in development. Consequently, methods to assess impairments in early nervous system functioning are of considerable interest to both researchers and clinicians. While such methods already have been developed for the postnatal period, few tools exist for the assessment of fetal nervous system functioning. Behavioral measures used in our lab have shown clear developmental patterns, suggesting they may be useful in detecting early assaults to the central nervous system. To test this hypothesis, pregnant rats were given an injection of the neurotoxin methylazoxymethanol (MAM) on E17 of gestation. On E20, 72 hours later, fetuses were prepared for in vivo testing, which consisted of the following behavioral observations: (a) spontaneous movement, (b) facial wiping in response to lemon infusion, and (c) response to an artificial nipple

395

presentation. Quantification of these three measures showed clear disruptions of movement coordination in the MAMtreated subjects, with no discernable differences from saline controls in weight or anatomical measures. These findings suggest that prenatal behavioral assessments of motor coordination, either spontaneous or evoked, may be useful in identifying neural insult during fetal development.

NBTS 51 Selection of a light source for fetal neurobehavior testing Rayburn* BB, Theele* D, Bolnick* J, Rayburn WF (Department of Obstetrics and Gynecology, University of New Mexico School of Medicine, Albuquerque, NM) In utero exposure to illicit drugs affects fetal behavior patterns. The fetal response to light stimulation has been proposed as a means to assess behavior testing, although a reliable light source has not been identified. The objective of the current investigation was to select the most intense light source that penetrates tissues while being safe for fetal behavioral testing. Two experiments were undertaken using a digital light meter (Extech 403125) to measure light intensity. First, the intensity of light through a filter was compared between the sun and four commercially available light sources. Secondly, penetration of light through the abdominal subcutaneous tissue (2.5 – 3.5-cm thickness) of six adult pigs was compared between these light sources. Light emitted from a halogen bulb was significantly more intense than from a photoflash, krypton bulb, or penlight. Light intensity of the halogen bulb was 41.3% that of the sun (median: 1320 vs. 3200 lx). Maximal intensity was gained with the light source being placed against the pig’s skin. The halogen bulb penetrated the tissues more than the photographic flash or krypton bulb (median: 650 vs. 260 or 15 lx). Light from the penlight did not penetrate the tissue. No thermal injury was observed for exposures less than 60 s. In summary, the halogen bulb was represented the best light source for comparative fetal neurobehavior testing. This external stimulus will be used to evaluate fetuses with recent maternal exposure to illicit substances.

NBTS 52 Thyroid hormone, brain development and the environment Zoeller RT (University of Massachusetts, Amherst, MA, USA) Thyroid hormone is essential for normal mammalian brain development. Historically, both clinical and experimental studies have focused on the effects of severe hypothyroidism. More recent clinical studies have focused on the neurological consequences of mild and/or transient thyroid

396

Abstracts / Neurotoxicology and Teratology 25 (2003) 381–397

hormone insufficiency. Experimental studies lag behind in focusing on the effects of mild thyroid hormone insufficiency, especially as it relates to fetal brain development. These issues are important for toxicological studies focused on assessing risk to human and wildlife populations of environmental chemicals that may affect thyroid hormone signaling. Toxicological studies commonly focus on endpoints of thyroid function; including circulating levels of thyroid hormones and thyrotropin, and thyroid histopathology. These studies are based on the implicit assumption that the hypothalamic-pituitary-thyroid axis is more sensitive to subtle thyroid hormone insufficiency than are other tissues, including the developing brain. The recent report of bisphenol A and two polyhalogenated derivatives binding to the thyroid hormone receptor and affecting gene expression represent a potential important case where a compound can interfere with thyroid hormone signaling without affecting endpoints of thyroid function. This example also suggests that chemicals may differentially influence thyroid hormone signaling mediated by the various receptor isoforms. Therefore, clinical and experimental studies are urgently needed to address very specific data gaps. These include an understanding of the clinical consequences of subtle thyroid hormone insufficiency occurring at different times during development. Moreover, we must identify developmental events in experimental systems that are affected by subtle thyroid hormone insufficiency, understanding the receptor isoforms mediating these events. The available genetic models and molecular tools will enhance these goals.

NBTS 53 Prenatal and perinatal thyroid hormone deficiencies: neuropsychological outcome Rovet J (The Hospital for Sick Children and University of Toronto, Toronto, Ontario, Canada) Thyroid hormone (TH) is essential for the developing nervous system. Early in gestation, the maternal thyroid is the fetal brain’s sole source of hormone while later as the fetal thyroid develops, it assumes an increasingly greater role to be fully independent by term. Because timing of need for TH varies among brain structures, different types of brain impairment and, correspondingly, different neurobehavioral deficits occur depending on exactly when the hormone is lacking. Several human conditions that provide distinct models of TH insufficiencies at different stages of development are maternal hypothyroidism, which reflects an early hypothyroidism; premature birth, which severs the maternal thyroid contribution in the third trimester and reflects a later-in-pregnancy TH insufficiency; and congenital hypothyroidism (CH), which involves a postnatal thyroid hormone insufficiency and, depending on etiology, a late third trimester

insufficiency as well. In my lab, we have been following infants and children with these conditions and have used neuropsychological and electrophysiological approaches to identify their particular deficits and to relate them to maternal or fetal TH levels at different stages of intrauterine or postnatal life. Two neural systems that appear to be especially vulnerable to TH insufficiency are the visual system and selective aspects of memory. This presentation will describe our recent findings on infants and children who lacked TH in utero or postnatally. We will report our findings on tasks of infant visual processing and attention and later childhood memory and will show that an early hypothyroidism is associated with deficits in visual contrast sensitivity and visual engagement, whereas a postnatal TH deficiency contributes to problems in episodic, spatial, and associative memory. Our findings suggest that in the human, retinal and thalamic development appear to need TH in early pregnancy, whereas the hippocampal formation appears to need TH postnatally.

NBTS 54 Evaluation of the thyroid and thyroid hormone levels in nonclinical studies in rats Christian MS (Argus International, Inc., Horsham, PA) Thyroid hormones regulate growth and development, cellular metabolism, use of oxygen and basal metabolic rate. Despite some modulation of metabolic rate by other hormones, e.g., testosterone, growth hormone and norepinephrine, T3 and T4 are the most important metabolic rate modulators. Thyroid hormone levels have been minimally evaluated to date in neonatal rodents at critical times of their development. This poses a problem for investigators because only minimal data are available for background use in determining the normalcy of the developmental pattern of the thyroid hormones under conditions of insult. Endpoints used for thyroid function assessment in neonatal and adult rats include thyroid weights, TSH, T3 and T4 levels and histopathology. In rodents, increased thyroid weights, decreased T3 and T4 serum levels, increased serum TSH levels, and sustained release of TSH and follicular cell hypertrophy/hyperplasia are hormonal and histopathological findings attributable to compounds altering thyroid function. Hypothyroidism early in the neonatal period (the first 2 weeks postnatal) affects both brain and reproductive endpoints in male and female rats. It has been shown to reduce levels of gonadotrophins and delay pubertal spermatogeneis in male rats and to block gonadotropin-induced first ovulation in immature female rats by decreasing FSH and LH serum concentrations. Inclusion of evaluations of TSH, T3 and T4 assays in multigeneration and developmental neurotoxicity protocols has resulted in new challenges for investigators. All data from our laboratory were based on use of an

Abstracts / Neurotoxicology and Teratology 25 (2003) 381–397

RIA kit. Suggestions for improving collection of such data also will be presented.

NBTS 55 Pesticides and thyroid status—observations in rats using the EPA Test Battery for Developmental Neurotoxicity Sheets LP (Bayer CropScience, Stilwell, KS/USA) The U.S. EPA Guideline for a Developmental Neurotoxicity (DNT) study (OPPTS 870.6300) includes a standardized battery of tests (e.g., neurobehavioral measures and neuropathology, with brain measurements) administered using the offspring at specific ages. Before testing a proprietary chemical, labs must verify the adequacy of technician training and validate their test procedures by establishing test norms and demonstrating competence in evaluating effects in neonatal animals perinatally exposed to chemicals, and by providing positive control data that demonstrate the suitability and sensitivity of their procedures. The antithyroid drug methimazole has been recommended for use as a positive control for such a developmental test battery (Comer and Norton, 1982). This paper discusses the design and results from a study with methimazole that was conducted in accordance with OPPTS 870.6300 and how those results contribute toward validation of the test procedures. This information is then used for comparison with the results from a DNT study with a herbicide that was tested due to indirect antithyroid activity in adult rats (acting via hepatic enzyme induction, which enhances the degradation

397

of serum T3 and T4). The results from that study establish that dietary exposure to this herbicide from GD6 through PD11 did not affect thyroid parameters (serum [T3/T4] or thyroid morphology); therefore, secondary antithyroid effects on behavior and brain morphology were not evident. This outcome supports an approach to investigate the primary effect of concern when the mechanism is known and then decide whether a DNT study is appropriate.

NBTS 56 Genomic approaches to the developing nervous system: thyroid hormone and the retina as a case study (Introduced by Mildred S. Christian) Harpavat* S, Cepko* C (Harvard Medical School, Boston, MA) Thyroid hormone—and lack of it—has a tremendous effect on the developing nervous system. However, the mechanism by which thyroid hormone mediates these effects remains poorly understood. We have approached this problem by focusing on a model neural tissue, the retina, and using a variety of ‘‘genomic’’ techniques. This talk will introduce these techniques and clarify the rationale of their use. It will also highlight how these techniques are illuminating thyroid hormone’s role in retinal development specifically and neural development generally.