Neurobehavioral Teratogenicity Research Articles

Chapter 27 - Effects of prenatal alcohol and cannabis exposure on neurodevelopmental and cognitive disabilities

Substance use during pregnancy and the short- and long-term impacts of different substances on maternal, fetal, and longer-term health outcomes of individuals prenatally exposed have been the subject of much investigation. Alcohol has been recognized as harmful during pregnancy and has been clearly recognized as a neurobehavioral teratogen, and the pattern of effects has been termed fetal alcohol spectrum disorder. More recently, the effects of prenatal cannabis exposure have been vigorously explored as a priority research area following decriminalization/legalization of cannabis in Canada and the United States. As the data become more and more robust, we are learning that cannabis during pregnancy can have negative effects on maternal and fetal outcomes and on longer-term neurodevelopmental and cognitive functions.

Neurodevelopmental outcomes in infants exposed in utero to antipsychotics: a systematic review of published data.

The proportion of pregnancies exposed to either second-generation antipsychotics (SGAs) or first-generation antipsychotics (FGAs) varies between 0.3%-2% of all pregnancies, but, until now, little is known about the potential neurobehavioral teratogenicity of antipsychotics. Assessing this safety facet is the aim of this article. PubMed, Scopus, and Google Scholar were searched for eligible articles. PubMed (1954 to May 2016) was searched using several medical subject headings, variously combined. PubMed search results were also limited using the search filter for human studies published in English. Scopus and Google Scholar searches were filtered for article title (antipsychotics/neuroleptics, pregnancy). After excluding duplicates, 9,250 articles were identified and 29 met the following inclusion criteria: only articles that provided original/primary data on neurodevelopmental outcome in human offspring older than 4 months of age, independently of the study design, were selected for review. Indeed, some relevant neurodevelopmental milestones are achieved at this time. Length of study and neurodevelopmental assessment methodology did not influence the study selection. Unfortunately, published data on neurodevelopmental teratogenicity of SGAs mainly derive from case reports and small case-series studies. Even findings emerging from case-control and prospective/retrospective studies are of limited clinical relevance because of their small sample sizes. Limited data are also available on FGAs. Hence, we have to conclude that the long-term neurodevelopmental outcomes for children exposed in utero remain unclear. Low to very low quality evidence of retrieved data makes impossible to confirm or exclude potential long-lasting untoward effects on infant neurocognitive development associate with antenatal exposure to either SGAs or FGAs.

Developmental exposure to organophosphate flame retardants causes behavioral effects in larval and adult zebrafish

BackgroundOrganophosphate flame retardants (OPFRs) have grown in usage since concerns about the health effects of the previously used polybrominated flame retardants led to their being phased out. The potential for OPFRs to cause adverse health effects of their own is still unexamined. Because of their structural similarities to organophosphate pesticides, which have themselves been heavily researched and shown to be neurobehavioral teratogens, we investigated the possibility that developmental exposure to two OPFRs, triphenyl phosphate (TPHP), and tris(1,3-dichloroisopropyl)phosphate (TDCIPP) might lead to behavioral impairment across the lifespan, as has been observed with the organophosphate pesticide chlorpyrifos. MethodsZebrafish were exposed to 0.03 or 0.3μM of TPHP, TDCIPP, or chlorpyrifos from 0 to 5days post fertilization. Vehicle control consisted of 0.03% solution of DMSO. At 6days post fertilization, larvae were tested on a locomotor assay. Separate cohorts of 6day old larvae that were not tested on the larval assay were allowed to grow to adulthood. At 12weeks post fertilization, these adult zebrafish were tested on a battery of behavioral assays that included tests of novel environment exploration, startle habituation, social affiliation, and predator escape. ResultsDevelopmental exposure altered zebrafish behavior across the lifespan. Larval zebrafish exposed to the 0.03μM doses of chlorpyrifos or TDCIPP exhibited significant (p<0.05) hyperactivity in the locomotor assay. Organophosphate exposure significantly (p<0.05) altered the time course of adult zebrafish behavior in the novel environment, startle habituation, and social affiliation assays. Predator escape behavior was significantly (p<0.05) reduced in fish exposed to the 0.3μM dose of TDCIPP. Exposure also caused hyperactivity in adult fish, with fish exposed to the 0.3μM dose of TDCIPP exhibiting significantly (p<0.05) elevated locomotor behavior in the novel environment assay. DiscussionEarly developmental exposure to OPFRs produced behavioral impairment that persisted into adulthood. These findings support broader research investigating the role of organophosphate compounds, including the OPFRs used here, in developmental neurotoxicity.

Chronic prenatal ethanol exposure alters expression of central and peripheral insulin signaling molecules in adult guinea pig offspring

Maternal ethanol consumption during pregnancy can produce a range of teratogenic outcomes in offspring. The mechanism of ethanol teratogenicity is multi-faceted, but may involve alterations in insulin and insulin-like growth factor (IGF) signaling pathways. These pathways are not only important for metabolism, but are also critically involved in neuronal survival and plasticity, and they can be altered by chronic prenatal ethanol exposure (CPEE). The objective of this study was to test the hypothesis that CPEE alters expression of insulin and IGF signaling molecules in the prefrontal cortex and liver of adult guinea pig offspring. Pregnant Dunkin-Hartley-strain guinea pigs received ethanol (4 g/kg maternal body weight/day) or isocaloric-sucrose/pair-feeding (nutritional control) throughout gestation. Fasting blood glucose concentration was measured in male and female offspring at postnatal day 150–200, followed by euthanasia, collection of prefrontal cortex and liver, and RNA extraction. IGF-1, IGF-1 receptor (IGF-1R), IGF-2, IGF-2 receptor (IGF-2R), insulin receptor substrate (IRS)-1, IRS-2, and insulin receptor (INSR) mRNA expression levels were measured in tissues using quantitative real-time PCR. The mean maternal blood ethanol concentration was 281 ± 15 mg/dL at 1 h after the second divided dose of ethanol on GD 57. CPEE resulted in increased liver weight in adult offspring, but produced no difference in fasting blood glucose concentration compared with nutritional control. In the liver, CPEE decreased mRNA expression of IGF-1, IGF-1R, and IGF-2, and increased IRS-2 mRNA expression in male offspring only compared with nutritional control. Female CPEE offspring had decreased INSR hepatic mRNA expression compared with male CPEE offspring. In the prefrontal cortex, IRS-2 mRNA expression was increased in CPEE offspring compared with nutritional control. The data demonstrate that CPEE alters both central and peripheral expression of insulin and IGF signaling molecules at the mRNA level, which may be related to metabolic dysregulation in adult offspring. Furthermore, altered insulin and IGF signaling may be a mechanism of ethanol neurobehavioral teratogenicity.

Risks of neurobehavioral teratogenicity associated with prenatal exposure to valproate monotherapy: a systematic review with regulatory repercussions

Beyond its formal indications (epilepsy, bipolar disorder, and migraine), valproate sodium (VPA) is widely used in a number of other clinical conditions. Recently, however, the U.S. Food and Drug Administration (FDA) issued a warning regarding a decrease in IQ scores in children prenatally exposed to the drug. For patients with migraine, the pregnancy labeling of VPA will be changed from Category "D" to "X." VPA products will remain in pregnancy category "D" for treating epilepsy and manic episodes associated with bipolar disorder. Thus, this article aims to assess (through a computerized Medline/PubMed search) the neurobehavioral teratogenicity of valproate monotherapy, in order to evaluate alternative regulatory decisions. Reviewed information suggests a detrimental impact of antenatal valproate exposure on the global child neurodevelopment. Affected areas include not just reduced IQ scores, but also behavioral problems and a potential increase in the risk for a future diagnosis of attention-deficit/hyperactivity disorder. An increased risk of developing autism-spectrum disorders has also been reported. Thus, in my opinion, VPA should be assigned definitively to the Category "X," independent of any considerations about its clinical indications, and should be strictly avoided during pregnancy, due to the demonstrated risk of both neurobehavioral and neurocognitive teratogenicity.

Sensitivity of modified Biel-maze task, compared with Y-maze task, to measure spatial learning and memory deficits of ethanol teratogenicity in the guinea pig

Ethanol consumption during pregnancy can produce a variety of teratogenic effects in offspring, termed Fetal Alcohol Spectrum Disorders (FASD). The most debilitating and permanent consequence of chronic prenatal ethanol exposure (CPEE) is neurobehavioral teratogenicity, which often manifests as cognitive and behavioral impairments, including deficits in spatial learning and memory. This study tested the hypothesis that a modified dry-land version of the multi-choice Biel-maze task is more sensitive than the rewarded-alternation Y-maze task for the determination of spatial learning and memory deficits of ethanol teratogenicity. Pregnant guinea pigs received ethanol (4g/kg maternal body weight/day) or isocaloric-sucrose/pair-feeding (control) for 5days/week throughout gestation. CPEE resulted in ethanol neurobehavioral teratogenicity in offspring, as demonstrated by increased spontaneous locomotor activity at postnatal day (PD) 10 and decreased brain weight at euthanasia (PD 150–200). On PD 21, offspring were randomly assigned to one of two tasks to assess spatial learning and memory performance: a dry-land version of the Biel maze or a rewarded-alternation Y-maze. Animals were habituated to the environment of their assigned task and performance of each CPEE or control offspring was measured. In the modified Biel maze, CPEE and control offspring were not different for percent completed trials or time to complete a trial. However, CPEE offspring made more errors (reversals and entering dead ends) in the Biel maze, demonstrating impaired spatial learning and memory. In contrast, CPEE offspring did not have impaired performance of the rewarded-alternation Y-maze task. Therefore, the modified dry-land version of the Biel-maze task, which measures cognitive performance using a complex multi-choice design, is more sensitive in demonstrating CPEE-induced spatial learning and memory deficits compared with a simple, rewarded-alternation Y-maze task.

Prenatal Exposures Associated with Neurodevelopmental Delay and Disabilities

Neurobehavioral teratology refers to the study of the abnormal development of the structure and the behavioral functions of the central nervous system, which result from exposure to exogenous agents during prenatal development. The focus of this review is the effects of various prenatal exposures on human neurodevelopment. Studies that deal with the adverse effects of infectious agents (rubella, cytomegalovirus, and toxoplasma), teratogenic drugs (e.g., antiepileptic drugs such as phenytoin, valproate, and carbamazepine, coumarin derivatives, and retinoids), alcohol, and other substances of abuse will be reviewed. Additionally, prenatal exposure to industrial or environmental chemicals (e.g., lead, methylmercury, and polycarbonated biphenyls) as well as exposure of the embryo or fetus to high amounts of ionizing radiation will be addressed. Possible mechanisms of selected neurobehavioral teratogens will also be discussed.

The teratogenicity and behavioral teratogenicity of di(2-ethylhexyl) phthalate (DEHP) and di-butyl Phthalate (DBP) in a chick model

Phthalates are industrial chemicals widely used in consumer products, plastics and children toys, and the risk of exposure to phthalates, especially prenatal exposure, is a growing concern justifying the development of an animal model to better understand their effect. The present study was designed to evaluate the suitability of a chick model for phthalate DEHP teratogenicity and neurobehavioral teratogenicity, a model which is simple and devoid of potential confounding factors such as maternal toxicity, maternal-fetal unit and maternal-neonatal interactions; major findings were confirmed in the DBP study. Prehatch exposure to DEHP in doses ranging from 20 to 100mg/kg, reduced the percent hatching from 80% in control eggs to 65%, and increased late hatchings from 12.5% in control eggs to 29.4%. In addition it induced developmental defects characterized by an opening or weakening of abdominal muscles allowing internal organs to protrude externally with or without a sac, omphalocele or gastroschisis, respectively. The effect was dose dependent ranging from 8% with DEHP (20mg/kg) to 22% (100mg/kg). Similar treatment with DBP 100mg/kg has reduced percentage hatching to 57% and increased late hatching to 37.5%, with a 14% increase in gastroschisis. Biochemical evaluation revealed elevated levels of alkaline phosphatase, which reflects non-specific toxicity of DEHP at such a high dose. Behavioral evaluation using an imprinting test and locomotor activity on chicks pretreated with DEHP (100mg/kg) has shown an abolishment of imprinting performance from the control (0.65) preference ratio. DNA damage measurements of the metabolite 8-hydroxydeoxyguanosine (8-OH-dG) in blood samples showed an increase of 39.7% after prehatch exposure to phthalates. This was statistically significant for DEHP and indicates genetic toxicity, since part of the teratogenic activity is associated with oxidative stress and DNA damage.

Maternal smoking cessation and reduced academic and behavioral problems in offspring

BackgroundThere is some debate whether smoking during pregnancy causes or is only a risk factor for negative academic outcomes and increased risk of psychopathology in offspring. This study evaluated whether maternal smoking cessation would reduce the risk of adverse outcomes in school-aged children. MethodsWomen completed an online survey that included items about child scholastic performance and the Child Behavior Checklist (CBCL). Mothers were divided based on pre-pregnancy and pregnancy smoking status into: (1) Nonsmokers (N=320); (2) Women that smoked in the 3 months preceding and throughout pregnancy (Smokers, N=83); and (3) Smoking before, but not during pregnancy (Quitters, N=72). ResultsThe Smokers and Quitters groups each had lower education and incomes compared to Nonsmokers but were indistinguishable from each other on these measures. The offspring of Smokers were more likely (p<.05) to be behind their peers on standardized tests in math (27.8%) relative to both Nonsmokers (17.4%) and Quitters (13.0%) with similar findings for reading. Smokers reported more behavioral problems by their children in several areas including Hyperactivity and Impulsivity, Social problems, and Externalizing problems including Aggression and Rule-Breaking. Further, the children of Quitters had significantly fewer Attention and Externalizing problems than Smokers. These outcomes were observed even after accounting for the variance attributable to maternal education and several other potential confounds. ConclusionsTogether, these findings indicate that smoking cessation is associated with reduced risk of having children with academic and neuropsychological difficulties. These outcomes are discussed within the framework that nicotine may be a neurobehavioral teratogen.

Persistent behavioral impairment caused by embryonic methylphenidate exposure in zebrafish

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

Avian Models for Neurobehavioral Teratogenicity and Its Reversal by Stem Cell Transplantation

Avian Models for Neurobehavioral Teratogenicity and Its Reversal by Stem Cell Transplantation

Reversal of chlorpyrifos neurobehavioral teratogenicity in mice by allographic transplantation of adult subventricular zone‐derived neural stem cells

Neurobehavioral teratogenicity can be reversed with transplantation of neural stem cells. However, the usefulness of this therapy would be greatly enhanced by employing adult stem cells. In pursuit of this this goal, we developed a model that uses subventricular zone (SVZ) cells. HS/Ibg mice were exposed prenatally to chlorpyrifos on gestational days 9-18 (3 mg/kg/day, SC) in order to induce deficits in their performance in the Morris water maze test. Both the control and the exposed offspring were transplanted with SVZ cells (or vehicle) on postnatal day 35; this actually represents an allogenic transplantation, because the HS/Ibg strain is a heterogeneous stock. The transplanted cells were later observed in the host brain by DiI tracing, and their initial differentiation to cholinergic neurons and astrocytes was ascertained. On postnatal day 80, animals that had been exposed prenatally to chlorpyrifos displayed impaired Morris water maze performance, requiring more time to reach the platform. Transplantation of adult SVZ-derived neural stem cells (NSC) reversed the deficits. Applying autologous transplantation provides an important demonstration that the methodological obstacles of immunological rejection and the ethical concerns related to using embryonic stem cells may be successfully bypassed in developing stem cell therapies for neurodevelopmental disorders.

Prenatal SRI exposure: Where does neonatal behavioral syndrome end and neurobehavioral teratogenicity begin?

Prenatal SRI exposure: Where does neonatal behavioral syndrome end and neurobehavioral teratogenicity begin?

Reversal of chlorpyrifos neurobehavioral teratogenicity in mice by adult allographic subventricular zone-derived neural stem cell transplantation

Reversal of chlorpyrifos neurobehavioral teratogenicity in mice by adult allographic subventricular zone-derived neural stem cell transplantation

Neurodevelopmental effects of prenatal exposure to psychotropic medications

Until now, studies on the reproductive safety of psychotropics have typically assessed the risk of congenital malformations and perinatal complications associated with in utero exposure to such medications. However, little is known of their inherent potential neurobehavioral teratogenicity. The objective is to analyze available data from studies investigating developmental outcome of children exposed prenatally to psychotropics. A computerized Medline/PubMed/TOXNET/ENBASE search (1960-2010) was conducted using the following keywords: pregnancy, child/infant development/neurodevelopment, antidepressants, benzodiazepines, mood stabilizers, and antipsychotics. A separate search was also run to complete the safety profile of single specific medications. Resultant articles were cross-referenced for other relevant articles not identified in the initial search. A noncomputerized review of pertinent journals and textbooks was also performed. All studies published in English and reporting primary data on the developmental outcome of infants exposed in utero to psychotropics and born without malformations were collected. As regards antiepileptic drugs, only studies that provided data on specific medications approved for psychiatric practice use (carbamazepine, lamotrigine, and valproate) were considered. Data were extracted from 41 articles (38 identified electronically and 3 nonelectronically), which met the inclusion criteria. Despite reviewed studies showing relevant methodological limitations, concordant, albeit preliminary, information seems to exclude that prenatal exposure to both selective serotonin reuptake inhibitors and tricyclic antidepressants may interfere with the infants' psychological and cognitive development. Conversely, information on valproate strongly discourages its use in pregnant women. Moreover, although data on carbamazepine remain controversial, information on whole classes of drugs and single medications is either absent (second-generation antipsychotics) or too limited (first-generation antipsychotics, benzodiazepines, lithium, and lamotrigine) to inform the decision-making process. For all classes of psychotropics, new and/or further studies are warranted to answer definitively the urgent question about the impact of prenatal exposure to such medications on infant development.

An avian model for the reversal of neurobehavioral teratogenicity with neural stem cells

A fast and simple model which uses lower animals on the evolutionary scale is beneficial for developing procedures for the reversal of neurobehavioral teratogenicity with neural stem cells. Here, we established a procedure for the derivation of chick neural stem cells, establishing embryonic day (E) 10 as optimal for progression to neuronal phenotypes. Cells were obtained from the embryonic cerebral hemispheres and incubated for 5–7days in enriched medium containing epidermal growth factor (EGF) and basic fibroblast growth factor (FGF2) according to a procedure originally developed for mice. A small percentage of the cells survived, proliferated and formed nestin-positive neurospheres. After removal of the growth factors to allow differentiation (5days), 74% of the cells differentiated into all major lineages of the nervous system, including neurons (Beta III tubulin-positive, 54% of the total number of differentiated cells), astrocytes (GFAP-positive, 26%), and oligodendrocytes (O4-positive, 20%). These findings demonstrate that the cells were indeed neural stem cells. Next, the cells were transplanted in two allograft chick models; (1) direct cerebral transplantation to 24-h-old chicks, followed by post-transplantation cell tracking at 24h, 6days and 14days, and (2) intravenous transplantation to chick embryos on E13, followed by cell tracking on E19. With both methods, transplanted cells were found in the brain. The chick embryo provides a convenient, precisely-timed and unlimited supply of neural progenitors for therapy by transplantation, as well as constituting a fast and simple model in which to evaluate the ability of neural stem cell transplantation to repair neural damage, steps that are critical for progress toward therapeutic applications.

Nicotine-like excitatory effects of the new pesticide, neonicotinoids on rat cerebellar neurons

Identifying the mechanisms underlying the adverse effects of developmental neurotoxicants enables the design of therapies that can potentially reverse neurobehavioral deficits in adulthood. We administered chlorpyrifos (CPF), a model organophosphate pesticide to pregnant mice and identified visuospatial deficits in adult offspring using performance in the Morris maze. We then evaluated two strategies to reverse the effects, nicotine administration and transplantation of neural stem cells. Daily administration of nicotine prior to behavioral testing did not alter maze performance by itself, but completely reversed the deficits evoked by prenatal CPF exposure. Similarly, control animals grafted with neural stem cells in adolescence did not show any alterations in behavioral performance as adults, but the grafts completely reversed the effects of prenatal CPF treatment. This study thus provides a model for the development and application of both pharmacologic and cell-based therapies to offset the effects of neurobehavioral teratogens.

Neurobehavioral teratogenicity of perfluorinated alkyls in an avian model

Perfluorinated alkyls are widely-used agents that accumulate in ecosystems and organisms because of their slow rate of degradation. There is increasing concern that these agents may be developmental neurotoxicants and the present study was designed to develop an avian model for the neurobehavioral teratogenicity of perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS). Fertilized chicken eggs were injected with 5 or 10mg/kg of either compound on incubation day 0. On the day of hatching, imprinting behavior was impaired by both compounds. We then explored underlying mechanisms involving the targeting of protein kinase C (PKC) isoforms (α, β, γ) in the intermedial part of the hyperstriatum ventrale, the region most closely associated with imprinting. With PFOA exposure, cytosolic PKC concentrations were significantly elevated for all three isoforms; despite the overall increase in PKC expression, membrane-associated PKC was unaffected, indicating a defect in PKC translocation. In contrast, PFOS exposure evoked a significant decrease in cytosolic PKC, primarily for the β and γ isoforms, but again without a corresponding change in membrane-associated enzyme; this likely partial, compensatory increases in translocation to offset the net PKC deficiency. Our studies indicate that perfluorinated alkyls are indeed developmental neurotoxicants that affect posthatch cognitive performance but that the underlying synaptic mechanisms may differ substantially among the various members of this class of compounds, setting the stage for disparate outcomes later in life.

Apoptosis may involve in prenatally heroin exposed neurobehavioral teratogenicity?

Heroin abuse during pregnancy is a serious problem worldwide. Among all the illicit drugs, heroin is known as the most commonly abused opioid in the United States and China. Most women addicts are of child-bearing age. Heroin abuse during pregnancy, together with related factors like poor nutrition and inadequate maternal care, has been associated with adverse consequences including developmental delay of the offspring and their neurobehavioral teratogenicity. Researchers have done a lot of work to focus mainly on the variation of neurobehavior and its related factors such as the changes of neurotransmitters, receptors and involvement of the limited brain regions, but no one clearly and comprehensively explain the possible mechanism that may participate in the neurobehavioral teratogenicity induced by prenatal heroin exposure. Studies on animals have shown that heroin is a common neuroteratogen which can produce neurobehavioral defects. There must be some underlying mechanisms in the central nervous system which may take part in these defects. We hypothesized that the alterations in developmental apoptosis during embryogenesis could be one of the possible mechanisms which can cause neurobehavioral teratogenicity in prenatally heroin exposed offspring. Heroin is believed to pass through the placenta and blood-brain barrier much more rapidly than morphine due to the presence of acetyl groups and affects the developing brain. So far, it still remains obscure that whether the apoptosis in a particular brain region induced by heroin exposure in uterus is involved in neurobehavioral teratogenicity. Our hypothesis perhaps provides a more logical and possible explanation of the mechanism responsible for neurobehavioral teratogenicity caused by the prenatal heroin exposure during embryonic development. It can help to develop appropriate experimental animal models to understand the detailed mechanisms involved.

Survival, differentiation, and reversal of heroin neurobehavioral teratogenicity in mice by transplanted neural stem cells derived from embryonic stem cells

Cell therapies in animal models of neurobehavioral defects are normally derived from neural stem cells (NSC) of the developing cortex. However, the clinical feasibility of NSC therapies would be greatly improved by deriving transplanted cells and from a tissue culture source that is self-renewing, containing cells that potentially differentiate into the desired neuronal phenotypes. These cultures can be engineered to contain the appropriate factors to support their therapeutic action and likely evoke lesser immune reactions. In the current study, we employed our model of mice neurobehaviorally impaired via prenatal exposure to heroin, to test the therapeutic efficacy of NSC derived from murine embryonic stem cells culture (ESC). The culture contained elongated bipolar cells, 90% of which are positive for nestin, the intermediate filament protein found in neural precursors. After removal of growth factors, the NSC differentiated into neurons (34.0% +/- 3.8% NF-160 positive), including cholinergic cells (ChAT positive), oligodendrocytes (29.9% +/- 4.2% O(4)), and astrocytes (36.1% +/- 4.7% GFAP positive). Reverse transcriptase polymerase chain reaction (RT-PCR) analysis confirmed the immunocytochemical findings. Mice made deficient in Morris maze behavior by prenatal heroin exposure (10 mg/kg heroin s.c. on gestational days 9-18) were transplanted into the hippocampus region on postnatal day 35 with the ES culture-derived NSC (ES-NSC) labeled with dialkylcarbocyanine (Dil) cell tracker. Dil+ and NF160+ cells were detected in the hippocampal region (50% +/- 8% survival). The transplantation completely restored maze performance to normal; e.g., on day 3, transplantation improved the behavior from the deficient level of 11.9-sec latency to the control of 5.6-sec latency (44.5% improvement).