Whole transcriptome analysis in offspring whose fathers were exposed to a developmental insult: a novel avian model.

Although the effects of paternal exposure to insults on the offspring received limited attention in the past, it is currently gaining interest especially after understanding the mechanisms which may mediate such exposure effects. In the current study, the well-controlled avian model (Fayoumi) was utilized to investigate the effects of paternal exposure to the developmental insult, chlorpyrifos on the offspring's gene expression via mRNA and small RNA sequencing. Numerous mRNA gene expression changes were detected in the offspring after paternal exposure to the developmental insult, especially in genes related to neurogenesis, learning and memory. qPCR analysis of several genes, that were significantly changed in mRNA sequencing, confirmed the results obtained in mRNA sequencing. On the other hand, small RNA sequencing did not identify significant microRNA genes expression changes in the offspring after paternal exposure to the developmental insult. The effects of the paternal exposure were more pronounced in the female offspring compared to the male offspring. The results identified expression alterations in major genes (some of which were pertinent to the functional changes observed in other forms of early developmental exposure) after paternal insult exposure and provided a direction for future studies involving the most affected genes.

Parental Preconception and Pre-Hatch Exposure to a Developmental Insult Alters Offspring's Gene Expression and Epigenetic Regulations: An Avian Model.

Parental exposure to insults was initially considered safe if stopped before conception. In the present investigation, paternal or maternal preconception exposure to the neuroteratogen chlorpyrifos was investigated in a well-controlled avian model (Fayoumi) and compared to pre-hatch exposure focusing on molecular alterations. The investigation included the analysis of several neurogenesis, neurotransmission, epigenetic and microRNA genes. A significant decrease in the vesicular acetylcholine transporter (SLC18A3) expression was detected in the female offspring in the three investigated models: paternal (57.7%, p < 0.05), maternal (36%, p < 0.05) and pre-hatch (35.6%, p < 0.05). Paternal exposure to chlorpyrifos also led to a significant increase in brain-derived neurotrophic factor (BDNF) gene expression mainly in the female offspring (27.6%, p < 0.005), while its targeting microRNA, miR-10a, was similarly decreased in both female (50.5%, p < 0.05) and male (56%, p < 0.05) offspring. Doublecortin's (DCX) targeting microRNA, miR-29a, was decreased in the offspring after maternal preconception exposure to chlorpyrifos (39.8%, p < 0.05). Finally, pre-hatch exposure to chlorpyrifos led to a significant increase in protein kinase C beta (PKCß; 44.1%, p < 0.05), methyl-CpG-binding domain protein 2 (MBD2; 44%, p < 0.01) and 3 (MBD3; 33%, p < 0.05) genes expression in the offspring. Although extensive studies are required to establish a mechanism-phenotype relationship, it should be noted that the current investigation does not include phenotype assessment in the offspring.

Reversal of prenatal heroin-induced alterations in hippocampal gene expression via transplantation of mesenchymal stem cells during adulthood.

Neurobehavioral teratology is the study of typically subtle neurobehavioral birth defects. Our previously described mouse model demonstrated septohippocampal cholinergic innervation-related molecular and behavioral deficits after prenatal exposure to heroin. Since the alterations are below malformation level, they are likely to represent consequences of regulatory processes, feasibly gene expression. Consequently, in the present study pregnant mice were injected with heroin on gestation days 9-18 and were transplanted with mesenchymal stem cells (MSC) on postnatal day (PD) 105. The hippocampi of the offspring were analyzed on PD120 for the expression of the pertinent genes. Heroin induced global gender-dependent statistically significant changes in the expression of several genes. Significant Treatment X Sex interaction occurred in D1 and SOX2 genes (p < 0.01). Transplantation of MSC reversed the prenatal heroin-induced alterations in approximately 80% of the genes. The reversal index (RI), shifting the score of the heroin-exposed offspring by transplantation back toward the control level, was 0.61 ± 0.10 for the difference from RI = 0 (p < 0.001), confirming the validity of the effect of the neuroteratogens across variations among different genes. The present study suggests that neurobehavioral defects induced by prenatal heroin exposure are likely to be a consequence of regulatory changes. This study on prenatal exposure to insults with subsequent MSC therapy provides a model for elucidating the mechanisms of both the neuroteratogenicity and the therapy, steps that are critical for progress toward therapeutic applications.

Paternal and/or maternal preconception-induced neurobehavioral teratogenicity in animal and human models.

Prenatal insult exposure effects on the offspring, have and are still considered the main interest of most teratological studies, while paternal and maternal preconception effects have received relatively little interest. Once thought to be a myth, paternal exposure to insults leading to numerous detrimental effects in the offspring, has been confirmed on several occasions and is gaining increased attention. These effects could be demonstrated molecularly, biochemically and/or behaviorally. Different epigenetic mechanisms have been proposed for these effects to occur, including DNA methylation, histone modification and sperm RNA transmission. Paternal insult exposure has been shown to cause several neurobehavioral and developmental defects in the offspring. Findings on parental insult exposure effects on the progeny will be discussed in this review, with the main focus being on neurobehavioral effects after parental preconceptional exposure. The exposure to the insults induced long-lasting, mostly marked, defects. A few pioneering, prevention and reversal studies were published. Interestingly, most studies were conducted on paternal exposure and, at the present state of this field, on animal models. Clinical translation remains the subsequent challenge.

Implementation of a six-around-one optical probe based on diffuse light spectroscopy for study of cerebral properties in a murine mouse model of autism spectrum disorder.

Light reflectance spectroscopy (LRS) is a multispectral technique, sensitive to the absorption and scattering properties of biological molecules in tissues. It is used as a noninvasive tool to extract quantitative physiological information from human tissues and organs. A near-infrared LRS based on a single optical probe was used to monitor changes in optical and hemodynamic parameters in a mouse model of autism. A murine model of autism induced by developmental exposure to valproic acid (VPA) was used. Since autism could be attributed to neuroanatomical changes, we hypothesize that these changes can be detected using the LRS because spectral properties depend on both molecular composition and structural changes. The fiber-optic probe in the setup consisted of seven small optical fibers: six fibers for illumination placed in a circular manner around a central single collection fiber. Overall, measurements demonstrate changes in diffused reflectance spectra, cerebral optical tissue properties (absorption and scattering), and chromophore levels. Furthermore, we were able to identify differences between male and female groups. Finally, the effectiveness of S-Adenosylmethionine as a drug therapy was studied and found to improve the hemodynamic outcome. For the first time, to the best of our knowledge, the LRS is utilized to study variations in brain parameters in the VPA autism model mice through an intact scalp.

Gender Related Changes in Gene Expression Induced by Valproic Acid in A Mouse Model of Autism and the Correction by S-adenosyl Methionine. Does It Explain the Gender Differences in Autistic Like Behavior?

In previous studies we produced autism like behavioral changes in mice by Valproic acid (VPA) with significant differences between genders. S-adenosine methionine (SAM) prevented the autism like behavior in both genders. The expression of 770 genes of pathways involved in neurophysiology and neuropathology was studied in the prefrontal cortex of 60 days old male and female mice using the NanoString nCounter. In females, VPA induced statistically significant changes in the expression of 146 genes; 71 genes were upregulated and 75 downregulated. In males, VPA changed the expression of only 19 genes, 16 were upregulated and 3 downregulated. Eight genes were similarly changed in both genders. When considering only the genes that were changed by at least 50%, VPA changed the expression of 15 genes in females and 3 in males. Only Nts was similarly downregulated in both genders. SAM normalized the expression of most changed genes in both genders. We presume that genes that are involved in autism like behavior in our model were similarly changed in both genders and corrected by SAM. The behavioral and other differences between genders may be related to genes that were differently affected by VPA in males and females and/or differently affected by SAM.

Reversal of neurobehavioral teratogenicity in animal models and human: Three decades of progress.

Early studies of behavioral teratology were mostly descriptive, fulfilling the necessary first requirement in a new field. The next obvious stage was put forward in the 80's as mechanism driven science enabled reversal of the teratogens-induced deficits. Three decades later a plethora of studies have been published demonstrating the success of the new direction. Complete and long-term (ostensibly permanent) reversal has been demonstrated in numerous animal models representing the realization of the ultimate goal of the field. Perhaps less sought after, but still significant, are the studies on recovery which needs consistent treatment for its persistence The studies reviewed here have been summarized in Tables 1 and 2. Clinically, the field is only in its incipient stage because of the paucity in translational findings for complete reversal or even complete alleviation. Human findings are emerging but in partial alleviation, noteworthy were the demonstration of FASD children who showed improvement after choline treatment while others showed no effect. Consequently, while further studies in an animal model on the mechanism by which the teratogen exerts its deleterious effects and the reversal procedure action are important, the main thrust of the research should now be translation of the animal model findings into a standard clinical routine. Indeed, first steps towards these goals are being made in children with various neurodevelopmental disorders via the application of a variety of rehabilitation programs by physiotherapists, occupational therapists and speech and language therapists, but the results are partial and may not be long-lasting.

S-adenosyl methionine prevents ASD like behaviors triggered by early postnatal valproic acid exposure in very young mice.

INTRODUCTION: A common animal model of ASD is the one induced by valproic acid (VPA), inducing epigenetic changes and oxidative stress. We studied the possible preventive effect of the methyl donor for epigenetic enzymatic reactions, S-adenosine methionine (SAM), on ASD like behavioral changes and on redox potential in the brain and liver in this model. METHODS: ICR albino mice were injected on postnatal day 4 with one dose of 300 mg/kg of VPA, with normal saline (controls) or with VPA and SAM that was given orally for 3 days at the dose of 30 mg/kg body weight. From day 50, we carried out neurobehavioral tests and assessment of the antioxidant status of the prefrontal cerebral cortex, liver assessing SOD and CAT activity, lipid peroxidation and the expression of antioxidant genes. RESULTS: Mice injected with VPA exhibited neurobehavioral deficits typical of ASD that were more prominent in males. Changes in the activity of SOD and CAT increased lipid peroxidation and changes in the expression of antioxidant genes were observed in the prefrontal cortex of VPA treated mice, more prominent in females, while ASD like behavior was more prominent in males. There were no changes in the redox potential of the liver. The co-administration of VPA and SAM alleviated most ASD like neurobehavioral symptoms and normalized the redox potential in the prefrontal cortex. CONCLUSIONS: Early postnatal VPA administration induces ASD like behavior that is more severe in males, while the redox status changes are more severe in females; SAM corrects both. VPA-induced ASD seems to result from epigenetic changes, while the redox status changes may be secondary.

Is post exposure prevention of teratogenic damage possible: Studies on diabetes, valproic acid, alcohol and anti folates in pregnancy: Animal studies with reflection to human.

We discuss the possibilities to prevent the post-exposure teratogenic effects of several teratogens: valproic acid (VPA), diabetes and alcohol. Co-administration of folic acid with VPA reduced the rate of Neural Tube Defects (NTD) and other anomalies in rodents, but apparently not in pregnant women. Antioxidants or the methyl donor S-adenosyl methionine prevented Autism Spectrum Disorder (ASD) like behavior in mice and rats. In vivo and in vitro studies demonstrated that antioxidants, arachidonic acid, myoinositol and nutritional agents may prevent diabetes-embryopathy. Prevention of alcohol-induced embryonic and fetal injuries and neurodevelopmental deficits was achieved by supplementation of zinc, choline, vasoactive intestinal proteins (VIP related peptides), antioxidants and folic acid. While the animal research described in this review is indicative of possible preventions of the different teratogenic effects, this is not yet the focus in human research. Future research should promote further knowledge where our current understanding is the vaguest, human prevention.

An avian model for ascertaining the mechanisms of organophosphate neuroteratogenicity and its therapy with mesenchymal stem cell transplantation.

INTRODUCTION: A fast and simple model which uses animals lower on the evolutionary scale is beneficial for progress in neuroteratological research. Here, we established this novel model and applied it in the study of the detrimental effects of pre-hatch exposure to chlorpyrifos on neurogenesis and several neurotransmitter systems in the chick and their reversal, using mesenchymal stem cell (MSC) transplantation. METHODS: Chicken eggs were injected with the organophosphate chlorpyrifos, 10mg/kg eggs - a dose below the threshold for dysmorphology - on incubation days (ID) 0 and 5 and subsequently the embryos were subjected to intravenous transplantation of MSC on ID 13. RESULTS: After hatching (day 1) the expression of the neurogenesis-related genes DCX (also confirmed by immunohistochemistry), BDNF, MAP 2, FGF 2, SOX 2 and VEGF in the lateral striatum area was decreased in the exposed group (p<0.005). Among the studied neurotransmitter systems (serotonergic, dopaminergic and cholinergic), increased gene expression was demonstrated for tyrosine hydroxylase (TH) and tryptophan hydroxylase (TPH) with a corresponding decrease in serotonin receptor 1A (5HTR1A) (p<0.05); no changes in gene expression of choline transporter, PKC beta and D2 were found following chlorpyrifos exposure. CONCLUSION: Transplantation of MSC reversed all the neurogenic and serotonergic alterations (p<0.01). The study of chick embryo exposure to insults with subsequent MSC therapy provides a fast and simple model for elucidating the mechanisms of both the neuroteratogenicity and the therapy, steps that are critical for progress toward therapeutic applications.

Mesenchymal stem cells can prevent alterations in behavior and neurogenesis induced by Aß25-35 administration.

Mesenchymal stem cells (MSCs) are known to enhance neurogenesis in the dentate gyrus, as well as to modulate immune cell activity and inflammation. Easily obtained and expanded from the bone marrow and other tissues, MSCs have been proposed as candidates for stem cell therapy in various neurodegenerartive diseases. In the present study, we sought to explore these therapeutic properties of MSC on Aß25-35-induced pathology when coadministered together. Apparently, coadministration of MSC prevented mild cognitive deficits observed following Aß administration alone, by promoting microglial activation and rapid clearance of injected Aß aggregates. Surprisingly, increased hippocampal neurogenesis was observed in the Aß-injected animals and was normal in MSC-coadministered animals just as in control animals. The observed increase in neurogenesis can be explained as a compensating mechanism responsible for the mild and temporary cognitive deficits observed in the Morris water maze assay in Aß-injected animals. Interestingly, MSC engrafted not only to the hippocampus but were also detected in the choroid plexus. We thus conclude that MSC may act in multiple pathways to protect the CNS from Aß pathology, while neurogenesis is a possible compensating mechanism; it is not always activated by MSC, which in turn may interact with local immune cells to regulate Aß accumulation.

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 100 mg/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 (20 mg/kg) to 22% (100 mg/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 (100 mg/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.

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.

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 5-7 days 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 (5 days), 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 24 h, 6 days and 14 days, 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.

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 (alpha, beta, gamma) 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 beta and gamma 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.

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).

A mechanism-based complementary screening approach for the amelioration and reversal of neurobehavioral teratogenicity.

The identification of mechanisms and outcomes for neurobehavioral teratogenesis is critical to our ability to develop therapies to ameliorate or reverse the deleterious effects of exposure to developmental neurotoxicants. We established mechanistically-based complementary models for the study of cholinergic systems in the mouse and the chick, using both environmental neurotoxicants (chlorpyrifos, perfluoroalkyls) and drugs of abuse (heroin, nicotine, PCP). Behavioral evaluations were made using the Morris maze in the mouse, evaluating visuospatial memory related to hippocampal cholinergic systems, and imprinting in the chick, examining behavior dependent on cholinergic innervation of the IMHV. In both models we demonstrated the dependence of neurobehavioral deficits on impairment of cholinergic receptor-induced expression, and translocation of specific PKC isoforms. Understanding this mechanism, we were able to reverse both the synaptic and behavioral deficits with administration of neural progenitors. We discuss the prospects for clinical application of neural progenitor therapy, emphasizing protocols for reducing or eliminating immunologic rejection, as well as minimizing invasiveness of procedures through development of intravenous administration protocols.

Reversal of chlorpyrifos neurobehavioral teratogenicity in mice by nicotine administration and neural stem cell transplantation.

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 sarin in an avian model.

Nerve gas organophosphates like sarin are likely to be used in urban terrorism, leading to widespread exposures of pregnant women and young children. Here, we established a model for sarin neurobehavioral teratogenicity in the developing chick so as to explore the consequences of apparently subtoxic sarin exposure and the mechanisms underlying synaptic and behavioral deficits. Chicken eggs were injected with sarin (2, 6 and 12 microg/kg) on incubation days 2 and 6, treatments that did not decrease hatching and did not evoke dysmorphology. After hatching the chicks were tested for filial imprinting and neurochemical markers known to be critical for imprinting. Imprinting was reduced at 2 and 6 microg/kg but not at the highest dose. Acetylcholinesterase and choline acetyltransferase were unaffected but sarin reduced the concentration of the high-affinity choline transporter, the rate-limiting factor in acetylcholine utilization. The concentration of PKC isoforms was assessed in the imprinting-related intermediate part of the medial hyperstriatum ventrale, the region most closely associated with cholinergic function in imprinting behavior. Sarin reduced the concentration of all isoforms (alpha, beta, gamma) with a similar, biphasic dose-response curve to that seen for behavioral performance, a relationship noted in previous work with organophosphate pesticides. Our results indicate that otherwise subtoxic exposures to sarin produce neurodevelopmental deficits; since we utilized a chick model, which is devoid of maternal confounds that are present in mammalian development, the adverse effects of sarin are mediated directly in the developing organism.

Developmental neurotoxic effects of chlorpyrifos on acetylcholine and serotonin pathways in an avian model.

The developmental neurotoxicity of organophosphates such as chlorpyrifos (CPF) involves multiple mechanisms that ultimately compromise the function of specific neurotransmitter systems, notably acetylcholine (ACh) and serotonin (5-hydroxytryptamine, 5HT). Studies in mammalian models incorporate both direct effects on brain development and indirect effects mediated through maternal physiology and maternal/neonatal interactions. We examined the effects of CPF in an avian model, which does not share these potential confounds. Chick eggs were injected with CPF (10 or 20 mg/kg) on incubation days 2 and 6 and markers of ACh and 5HT systems were examined at hatching. The higher dose caused a reduction in cholinesterase activity but there was no consistent downregulation of m(2)-muscarinic ACh receptors as would have been expected from ACh hyperstimulation. Both doses evoked significant reductions in the presynaptic high-affinity choline transporter, the rate-limiting factor in ACh biosynthesis, as monitored by binding of hemicholinium-3. Choline acetyltransferase, a constitutive marker for ACh terminals, was unaffected. This suggests that CPF reduces ACh presynaptic activity rather than compromising the development of ACh projections per se. CPF exposure also reduced the expression of cerebrocortical 5HT(1A) receptors. These effects in the chick model recapitulate many of the actions of early gestational CPF exposure in rats, and thus suggest that CPF exerts direct actions on the immature brain to compromise the development of ACh and 5HT pathways.