Edson X. Albuquerque - US grants

toxin; neurotransmitters;

DESCRIPTION: (Adapted from the applicant's abstract) This proposal addresses basic questions concerning the electrophysiological and molecular pharmacological mechanisms of the recently identified and essentially uncharacterized heterogeneous populations of nicotinic acetylcholine receptors (AChRs) located in various regions of the brain. The precise knowledge of the functional properties of such an elusive receptor is of major significance for the understanding of the physiology of the brain under normal conditions and in disease states. The novel neurotoxin (+) anatoxin-a (AnTX) and its synthetic analogs including the (-) enantiomer have proven essential to uncover the structural requirements for agonism at the muscle AChR. Furthermore, by virtue of its potency and specificity, (+)AnTX has enabled the recording of AChR currents in the central nervous system (CNS) and to reveal a great degree of heterogeneity of the AChRs, both within the same cell and among different cell types. Thus, the new long-term goal of this project is to investigate the fundamental functional and pharmacological properties of the AChR at different stages of development and under conditions of chronic exposure to selected drugs. In addition to ACh and the AnTX analogs, other selected weak agonists with similar specificity will be used to investigate AChR responses in the CNS. The noncompetitive antagonists that have been documented for the peripheral AChR will now be studied in the CNS, including a novel series of acridine araphane analogs which function as sensitive "rulers" to define the antagonist sites on the AChR. The effects of these selective toxins will also be studied at the N-methyl-D-aspartate receptor because of the great deal of structural and functional homology that has been observed with peripheral and central AChR. These studies are essential for the understanding of the interrelationships of homologous ligand-gated channels and related disease states such as Alzheimer's dementia. The proposed experiments will utilize a variety of electrophysiological techniques, including a new fast drug perfusion and withdrawal system developed in this laboratory, coupled with ligand binding, fluorescence labelling, and kinetic studies of receptors, and morphological studies of acutely dissociated and tissue cultured neuronal cells to reveal the basic mechanisms underlying the function of these different forms of AChR.

The ability of lead (Pb2+) to impair mental activity and neurodevelopment underscores its role as a pervasive environmental threat. Recently, we have observed that this cation is able to inhibit whole cell and single channel currents activated by N-methyl-D-aspartate (NMDA) in rat cultured hippocampal neurons in a concentration-dependent but voltage-independent manner, without significantly altering currents induced by either quisqualate or kainate. In view of the involvement of NMDA receptors in important physiological processes such as memory and learning, a detailed evaluation of the effect of Pb2+ on these receptors is vital. Furthermore, it is known that the impairment of neurodevelopment and cognitive processes induced by this element is much more severe in children than in adults. Indeed, our preliminary work showed that the effects of Pb2+ on NMDA receptors seem to be much more dramatic at early stages of neuronal development. Thus, several fundamental questions are raised: 1) How does Pb2+ affect the different conductance states of NMDA receptor channels at various stages of neuronal development in culture? 2) What is the site of action of Pb2+ on NMDA receptors? 3) At what age of culture of hippocampal neurons are the NMDA receptors most sensitive to the Pb2+? 4) Do glycine, Zn2+, Ca2+ and Mg2+ affect the actions of Pb2+ on NMDA receptors? 5) How does cell maturation influence the effects of Pb2+ on NMDA receptors? 6) What are the chronic effects of Pb2+ on cell development and NMDA receptor function in hippocampal neurons? To provide the answers to these questions, this project is aimed at studying the electrophysiological responses of cultured rat hippocampal neurons to NMDA in the absence of Pb2+ (control condition), and after acute as well as chronic exposure of the cell culture preparation to different concentrations of the cation. Recordings of whole cell and single channel currents activated by different agonists will be performed on the cultured neurons using standard patch clamp techniques. In addition, a new fast drug perfusion and withdrawal system developed in our laboratory will enable us to make a quantitative kinetic analysis of these currents. The elucidation of the actions of Pb2+ on NMDA receptors should constitute the first and most important step in an attempt to design a rational treatment of its neurotoxic effects in the central nervous system (CNS). As a secondary aim of the proposal, we would like to evaluate the interactions of Pb2+ with neuronal nicotinic acetylcholine receptors (AChR), since there is compelling evidence that in addition to NMDA receptors, AChRs may also play a significant role in cognitive processes. We have already demonstrated that low concentrations of Pb2+ similar to those that block NMDA receptors, markedly depress whole cell currents activated by anatoxin-a and ACh, well known agonists of the AChR. The studies described in this proposal will allow us to determine some of the molecular mechanisms that underlie the complex effects of Pb2+ in the CNS.

DESCRIPTION: The goal of this project is to understand the mechanisms by which lead (Pb2+) exerts neurotoxic effects in the central nervous system (CNS) particularly in the developing brain. N-methyl-D-aspartate (NMDA) receptor-ion channels (nAChRs) are very sensitive to inhibition by Pb2+. Whereas the inhibitory effect of Pb2+ on NMDA receptors is apparently due to its action on a Ca2+ site on the receptor, the mechanisms by which Pb2+ inhibits the activation of the alpha7-bearing nAChRs is still unknown and will be investigated in this proposal. Because NMDA receptors and alpha7-bearing nAChR are involved in memory and learning as well as in other forms of neuronal plasticity and development, it is very likely that inhibition of these receptors by Pb2+, particularly during early stages of neuronal maturation, could be associated with the severe learning disabilities caused by this heavy metal. Considering that distal dendritic development of the hippocampal neurons is particularly sensitive to the toxic effects of Pb2+, and that dendritic development(with formation and maturation of dendritic spines) is associated with learning and memory, the following question is raised: Is the expression of Pb2+-sensitive NMDA receptors and nAChRs restricted during development to particular neuronal regions, such as apical and basal dendrites, in distinct hippocampal areas (CA1, CA3, or dentate gyrus)? To answer this questions we will use state-of-the-art infrared microscopy (which allows visualization of axons, dendrites, and dendritic spines) combined with a computer-driven, robotic system of micromanipulators (which enables us to control the positions of patch electrodes and drug-delivery systems at precisely defined regions on the neuronal surfaces). Recordings can then be made of whole-cell and single-channel currents from neurons either visualized in or acutely dissociated from hippocampi of rats at different ages, and we will be able to map the distribution of Pb2+-sensitive nicotinic and NMDA receptors on cell bodies, dendrites, and dendritic spines of hippocampal neurons. Our preliminary studies also indicate that Pb2+ substantially increases spontaneous transmitter release from hippocampal neurons. Taking into account that NMDA receptors and alpha-BGT-sensitive nAChRs present on presynaptic terminals of CNS neurons can modulate the release of a number of neurotransmitters, it is critical to determine whether Pb2+ alters transmitter release by acting at these presynaptic receptors. Therefore, the effects of Pb2+ on spontaneous and evoked transmitter release will be investigated at the level of single synapses on hippocampal neurons, which will be acutely dissociated at various stages of development. We have developed a technique by which neurons can be acutely dissociated by mechanical means (without enzyme treatment) from hippocampi of rats at various ages. These neurons bear many synaptic terminals that are functional and can be electrically stimulated. Altogether these studies should provide the foundation for an understanding of the net effects of Pb2+ on receptor function, synaptic activation and maturation in the developing CNS.

DESCRIPTION (Adapted from applicant's abstract): This is a continuation application which has undergone two revisions. It has changed study sections at the request of Dr. Albuquerque. The objective of this project is to identify the physiological functions of the various subtypes of neuronal nicotinic acetylcholine receptors (AChRs) present in the hippocampus (the brain area mainly involved in learning and memory) and the mechanisms by which allosteric ligands control their activity. The foundation for this application rests upon the results of the studies conducted since the inception of this grant, namely that (I) a7-bearing nAChRs-the predominant nAChR subtype on hippocampal neurons-are highly permeable to Ca2+, are selectively activated by choline, a by-product of acetylcholine hydrolysis, and mediate synaptic transmission in the CA1 field of hippocampal slices; (ii) both a7 and a4b2 nAChRs are present in CA1 interneurons of hippocampal slices, and activation of these receptors facilitates the action potential-dependent release of GABA; (iii) non-competitive agonists have been identified that potentiate the activity of various nAChR subtypes. Considering these findings, the following questions have been posed: (i) What are the roles of neuronal nAChRs in modulation of synaptic activity in the hippocampus as a whole? (ii) Are there changes in the expression of functional nAChRs in the hippocampus along with in vivo development? (iii) Can a specific nAChR subtype be associated with a distinct interneuron type? (iv) What are the roles of endogenous choline in the a7 nAChR-mediated synaptic transmission in the hippocampus? (v) Does the potency or efficacy of non-competitive agonists depend on the receptor subtype? (vi) Is the a7 nAChR-mediated synaptic transmission in the hippocampus controlled by endogenous non-competitive agonists? (vii) Can this nicotinic synaptic transmission be altered by compounds that are known to act as non-competitive agonists? To address these questions, state-of-the-art technology, including infrared-assisted videomicroscopy, the patch-clamp technique, and a computerized system of mircomanipulators will be applied to neurons in the CA1 and CA3 fields of hippocampal slices. The implications of these studies can be far reaching given that the new findings, in addition to providing new insights into the understanding of the involvement of neuronal nAChRs in synaptic function, particularly in the hippocampus, may lay the groundwork for the development of efficacious therapeutic approaches to address physiopathological conditions in which the nAChR function is impaired.

DESCRIPTION: The dysfunction and degeneration of the nicotinic cholinergic system in the brain are integral physiopathological indicators of one of the most socially impacting neurological disorders, Alzheimer's disease (AD). In AD, the permanent loss of cholinergic neurons and nicotinic receptors (nAChRs) in brain areas that process cognitive functions, particularly the hippocampus and the frontal cortex, correlates well with the decline in cognition and memory. To date, treatment of patients with AD relies heavily on the use of acetylcholinesterases. These drugs, by increasing function of the cholinergic system, partially reverse the symptoms of AD patients. Recently, clinical trials have shown that nicotinic agonists (including nicotine) and drugs that allosterically potentiate the activity of nAChRs are more effective for treatment of patients with AD. The mechanisms underlying the effectiveness of these drugs remain unknown, because there is very little information on how function and expression of neuronal nAChRs in the brain are regulated by cholinergic afferents. In addition, detailed analysis of regulation of nAChR expression and function in the hippocampus by septal cholinergic afferents has been limited by the lack of a viable biological preparation that closely resembles the nicotinic cholinoceptive hippocampal system in vivo. Our initial characterization of the nicotinic properties of hippocampal neurons in organotypic, hippocampal and septal-hippocampal cultures constitutes the mainstay of the present proposal, as it establishes the septal-hippocampal co-cultures as an excellent model for in vitro study of the influences of septal innervation on nAChR expression in the hippocampus. Thus, this proposal is designed to use convergent, multidisciplinary approaches to address the central hypothesis that septal innervation and nicotine dynamically modify the hippocampal cholinergic system. The first goal of this study is to use electrophysiology, confocal microscopy, ligand binding and immunocytochemistry to determine whether septal innervation alters the nicotinic properties of different types of hippocampal neurons during development in organotypic cultures. The second goal is to use electrophysiological assays, recombinant DNA technology and "knock-out" mice, which have a null mutation in the gene encoding alpha7 nicotinic receptors, to study nAChR targeting and to investigate the motifs in the nAChR subunits that account for final receptor targeting in hippocampal neurons. The final goal is to use electrophysiological, biochemical and molecular biological techniques to evaluate how nicotine affects alpha7 and alpha4beta2 nAChR expression in the hippocampus. The results of these studies will have far reaching implications in identifying cellular and molecular mechanisms regulating nAChR expression and function in the hippocampus and provide the basis for developing therapeutic agents targeting regions of the brain affected in AD.

[unreadable] DESCRIPTION (provided by applicant): In the hippocampus, the center for integration of cognitive functions, nicotinic receptors (nAChRs) modulate synaptic transmission mediated by the major excitatory and inhibitory transmitters - glutamate and GABA, respectively. However, hippocampal function relies also on tonically activated glutamatergic and GABAergic receptors. In fact7 some benzodiazepines that are clinically used for treatment of epileptic seizures affect more selectively tonic (steady state) than phasic (synaptic) GABAergic activity. Yet, the effects of nicotinic ligands on tonic activities in the hippocampus are unknown. In a recent study, we have demonstrated that the tryptophan metabolite kynurenic acid (KYNA), which is largely produced and released by astrocytes in the brain, modulates nicotinic cholinergic activity in the rat hippocampus. At physiologically relevant concentrations, KYNA inhibits more potently a7 nAChRs than NMDA receptors (once believed to be the major targets for KYNA in the brain) and regulates a4B2 nAChR expression. Other lines of evidence also indicate that there might be functional cross-talk between KYNA and the nicotinic cholinergic system: (i) nicotine regulates endogenous levels of KYNA in the brain and (ii) changes in KYNA levels often parallel alterations in the nicotinic cholinergic system in such neurological disorders as schizophrenia, Alzheimer's and Parkinson's diseases (AD, PD). This proposal is designed to use convergent, multidisciplinary approaches, including electrophysiological, biochemical and molecular biological assays, to address the central hypothesis that in the hippocampus the magnitude of nAChR activity modulates the degree o tonic and phasic glutamatergic or GABAergic activities and is regulated by endogenous KYNA. The specific aims are: (i) to investigate the effects of nAChR ligands on tonic and phasic forms of excitation or inhibition in the hippocampus; (ii) to examine how interactions between KYNA and the nicotinic cholinergic system modulate the tonic or phasic forms of GABAergic and glutamatergic activities in the hippocampus, and (iii) to determine the site of action of KYNA on o7 nAChRs, analyzing the possibility that the site for KYNA and for the allosteric potentiating ligand galantamine (that has been recent/y introduced for treatment of AD patients) on the nAChRs is one and the same. Knockout mice with null mutations in the gene that encodes the a7 nAChR subunit or the enzyme critical for ICYNA synthesis in the brain, kynurenine aminotransferase 11 (KATII), recombinant a7 nAChRs (mutants and chimeras) and different preparations (acute slices and heterologous systems) will be used to address these fundamental issues. The results of these studies win be far reaching; they will lead to a more comprehensive understanding of the means by which neuronal activity is regulated by nicotinic ligands, and provide the mechanistic basis for future development of therapeutic strategies designed to compensate, via the kynurenine pathway, for deficits in nicotinic function in disorders such as AD, PD and schizophrenia.

EXCEED THE SPACE PROVIDED. The nerve agents soman, sarin and VX are organophosphorus compounds (OPs) chemically related to, but far more toxic than OP insecticides. Most of their acute toxicity results from the irreversible inhibition of acetylcholinesterase (AChE), the enzyme that inactivates the endogenous neurotransmitter acetylcholine. The limitations of available therapies against OP poisoning are well recognized, and more effective antidotes have to be developed. In this project, we will test the central hypothesis that an antidotal therapy composed of galantamine, a drug presently approved for treatment of Alzheimer's disease (AD), with or without the muscarinic antagonist atropine can counteract the immediate and delayed toxicity of nerve agents in guinea pigs of both sexes at different ages. We have evidence that the combination of galantamine and atropine, administered before or after an acute exposure to lethal doses of nerve agents or insecticides, effectively and safely counteracts their toxicity in peripubertal male guinea pigs. Although little is known regarding their neurophysiology, guinea pigs are considered the best non-primate model to predict the effectiveness of antidotes against OP intoxication in humans. In aim 1, we will determine the age and sex dependencies of the acute toxicity of soman, sarin, and VX, and optimize the antidotal therapy consisting of galantamine, with or without atropine, for male and female guinea pigs at three ages, i.e.neonatal, peripubertal, and adult. The effectiveness of the optimized therapy to prevent immediate and/or delayed toxic effects of the nerve agents will then be examined in neuronal function and brain integrity. In aim 2, we will investigate the effects of the therapy on the temporal relationship between changes in synaptic function (electrophysiological studies) and alterations in morphometry (non-invasive MRI studies) and neuronal viability (histopathological analysis) in the brains of guinea pigs acutely exposed to nerve agents. In collaboration with USAMRICD researchers, we will further examine the effectiveness of the antidotal therapy to maintain normal diaphragm and cognitive behavior in guinea pigs of both sexes exposed to nerve agents at different ages. In aim 3, we will derive pharmacokinetic parameters needed for subsequent clinical studies of the safety of the proposed therapy for human use. Still within this aim, we will determine the relevance of galantamine-induced reversible inhibition of AChE in distinct compartments to the effectiveness of the therapy. Particular emphasis will be given to the notion that galantamine, acting as a selective inhibitor of blood AChE, will facilitate the clearance of the nerve agents. In aim 4, we will determine, at whole animal level, how novel molecular mechanisms contribute to the toxicity of nerve agents and the effectiveness of galantamine/atropine. The results of these studies will be far reaching as they will provide the foundation to expedite the development of safe antidotes to be used by the first responders and the general population in the event of an exposure to nerve agents.

DESCRIPTION (provide by applicant) This application is for continuation of a toxicology training program at University of Maryland (UM). The objective is to train graduate students and postdoctoral fellows in neurotoxicology, cell injury and carcinogenesis, molecular epidemiology and aquatic toxicology, with special emphasis on mechanisms and application of cutting-edge technologies to toxicological research. This proposal brings together a cadre of well funded, highly productive faculty mentors from different sites within the UM System, including the School of Medicine (SOM) and the Chesapeake Biological Laboratory. Mentors form a cohesive program designed to prepare trainees for research careers in toxicology and environmental health sciences. One of the major strengths is the exposure of the students to translational aspects of basic research in toxicology. Chemical risk assessments and the development of countermeasures to treat and/or prevent disease states induced by toxicants depends on the determination of the mechanisms by which such toxicants act, on the identification of targets for therapeutic intervention, and on the characterization of new assays, models, tools, and technologies for toxicity testing. Thus, mentors have been selected for their common interest in basic, clinical and ecological research applied to such environmental toxicants as metals, phytoestrogens, dioxins, and insecticides, and interests span from basic to clinical and environmental research. This selection provides trainees with a unique opportunity to gain experience in fields that play a major role in determining the mechanism of action and the effects of environmental toxicants in susceptible subsets of the population, including sensitive stages of development. The graduate training in toxicology combines resources of the UM system-wide Program in Toxicology and the SOM Pharmacology and Experimental Therapeutics Departmrnt (toxicology track), and includes didactic course work, laboratory rotations, seminars, research and participation in national meetings. The postdoctoral program includes training in state-of-the-art toxicology research, teaching experience, and participation in seminars and national meetings. The breadth of the program is sufficient to allow emphasis in four primary areas: The Neurotoxicology Track provides training in molecular mechanisms of neurodegeneration due to environmental toxicants and the development of chemo- and gene therapy modalities. The Cell Injury and Carcinogenesis Track provides training in the action of free radicals, calcium homeostasis, mechanisms of lead and dioxin toxicities, estrogen biosynthesis and carcinogenesis. The Molecular EpidemiologyTrack provides training in identification of biomarkers of exposure and susceptibility, and in epidemiological methods for identifying associations between environmental chemical exposures and diseases. The Aquatic Toxicology Track provides training in pathobiology, immunotoxicology and molecular mechanisms of toxicity of heavy metals, dioxin and other pollutants.

DESCRIPTION (provided by applicant): In the hippocampus-a major center for cognitive processing in the brain- a7* nicotinic receptors (nAChRs) modulates neuronal functions and viability. Reduced activity of these nAChRs impairs cognition in rats and mice, and has been proposed to contribute to the cognitive deficits observed in patients with schizophrenia and Alzheimer's disease (AD). In these disorders, decreased a7* nAChR activity is accompanied by increased brain levels of kynurenic acid (KYNA), an astrocyte-derived kynurenine metabolite that blocks a7* nAChRs and N-methyl-D-aspartate (NMDA) receptors J Neurosci 21:7463, 2001. Using mice with a null mutation in kynurenine aminotransferase II, an enzyme critical for brain KYNA synthesis, we obtained evidence to support the concept that normal KYNA levels maintain a tonic degree of inhibition of a7* nAChRs, but not NMDA receptors [J Neurosci 24:4635, 2004]. Recently, we also reported that the nAChR allosteric potentiating ligand galantamine, a drug used to treat mild-to-moderate AD, competitively antagonizes the effect of KYNA on a7* nAChRs [J Pharmacol Exp Ther 322:48, 2007]. The present study is designed to test the central hypothesis that elevated levels of glia-derived KYNA impair hippocampal synaptic function, disrupt hippocampal neuronal structures, and cause cognitive deficits via an inhibition of a7* nAChRs that can be prevented and/or reversed by galantamine. To address this hypothesis, we will use a complementary, multidisciplinary approach. Electrophysiological, molecular biological and biochemical studies to be performed on rat and human hippocampal slices will be complemented by in vivo biochemical and behavioral studies in rats. The experiments will rely on key pharmacological tools that cause selective fluctuations in brain KYNA levels. In aim 1, we will identify the effects of stimulation of neosynthesis of glia-derived KYNA on the activity/ expression of a7* nAChRs and other receptors in rat and human hippocampal neurons and astrocytes. In aim 2, we will examine the effects of acute and long-term stimulation of KYNA neosynthesis on hippocampal synaptic transmission and plasticity, neuronal structures, and cognitive functions. In aim 3, we will determine if galantamine prevents and/or reverses the effects of elevated levels of glia-derived KYNA on hippocampal a7* nAChRs, synaptic transmission and plasticity, neuronal integrity, and cognition. These translational studies will integrate data from the molecular to the behavioral level to provide a better understanding of the contribution of KYNA to the pathophysiology of catastrophic diseases such as AD and schizophrenia, and expedite the development of better treatments to improve cognitive functions known to be impaired in these diseases. PUBLIC HEALTH RELEVANCE: Although dysfunctions of the nicotinic cholinergic system in the hippocampus a brain region central to cognitive processing constitute a hallmark in catastrophic disorders such as Alzheimer's disease (AD) and schizophrenia, very little is known regarding potential mechanisms that make this system go awry. We have recently discovered that kynurenic acid, a metabolite whose levels are increased in the brain of patients with AD and schizophrenia, inhibits a subtype of nicotinic cholinergic receptors - the a7* receptors. As designed, the studies proposed herein will help establish a causal relationship between increased levels of kynurenic acid and decreased nicotinic functions in the hippocampus, and expedite the development of better treatments to improve the cognitive functions known to be impaired in these diseases.

DESCRIPTION (provided by applicant): Organophosphorus (OP) pesticides are among the most heavily used pest control agents worldwide. In the US, malathion and chlorpyrifos account for more than 50% of the millions of pounds of OP pesticides used yearly. An ever growing body of epidemiological studies report that children whose mothers have been exposed to OP pesticides, including chlorpyrifos and malathion, during pregnancy present higher incidence of cognitive impairments, attention deficit/hyperactivity disorder, and autism spectrum disorders than non-exposed children. Although animal studies have confirmed that in-uterus or early postnatal exposure to chlorpyrifos compromises the brain development of rats and mice, the distinct temporal course of brain development and high levels of circulating carboxylesterases (enzymes that inactivate OP compounds) make these rodents inadequate models of human OP toxicity. In addition, even though concerns regarding the toxicity of OPs to the developing brain have been substantiated by their prompt permeation through the placenta, there have been no animal studies addressing the potential developmental toxicity of malathion. The present project is designed to test the central hypothesis that the developmental neurotoxicity of malathion and chlorpyrifos in guinea pigs - the best non-primate model of human OP intoxication - can be counteracted by galantamine and is largely accounted for by epigenetic mechanisms. Galantamine, a drug approved to treat Alzheimer's disease, is known to safely and effectively protect guinea pigs against the acute toxicity of OP compounds [PNAS 103: 13220, 2006]. Galantamine can also prevent the delayed cognitive dysfunctions that result from a single exposure of guinea pigs to sub-toxic doses of OPs. This project uses a multidisciplinary approach that involves electrophysiological, behavioral, magnetic resonance imaging and molecular biological assays to address three specific aims: (i) to identify, at various postnatal ages, neurological, structural, and neurobehavioral correlates of in-uterus exposure of guinea pigs to malathion or chlorpyrifos, (ii) to determine the effectiveness of postnatal treatment with galantamine to counteract the developmental toxicity of those pesticides, and (iii) to examine the contribution of epigenetic mechanisms to the developmental toxicity of the pesticides and the therapeutic effectiveness of galantamine. The overarching goal of this project is to provide fundamental and timely input for assessment and adequate treatment of the human developmental toxicity of malathion and chlorpyrifos. The results of this highly translational study of the potential health risks associated with exposure of the immature brain to pesticides will be far reaching as they will lay the groundwork necessary to advance research aimed at identifying novel therapeutic strategies to treat neurological diseases derived from in-uterus exposure to specific OP pesticides. PUBLIC HEALTH RELEVANCE: The overarching goal of this project is to provide fundamental and timely input for assessment of the human developmental toxicity of malathion and chlorpyrifos and identification of lead compounds for adequate treatment of neurological diseases that result from in-uterus exposure to these pesticides.

DESCRIPTION (provided by applicant): The sensitivity of the developing mammalian central nervous system (CNS) to organophosphorus (OP) nerve agents and the safety of antidotes such as atropine and pralidoxime, commonly used against acute OP toxicity, in pregnant women and the developing fetuses are hitherto unknown. Thus, during the 1995 terrorist attack with sarin in the Tokyo subway, pregnant women who sought medical attention after presenting mild signs of OP intoxication received no therapeutic treatment. The urgency of studies to investigate the developmental neurotoxicity of OP nerve agents is underscored by the recent report of the neurobehavioral teratogenicity of sarin in an avian model. In recent years, galantamine, a drug currently approved to treat Alzheimer's disease, emerged as an effective antidote against acute OP poisoning in neonatal, prepubertal, and adult guinea pigs. Treatment with galantamine was also shown to counteract the cognitive deficits and anxiety-like behavior seen months after a single exposure of prepubertal guinea pigs to 0.6-1.0xLD50 soman. The present project is, therefore, aimed at testing the central hypothesis that the developing mammalian CNS is exquisitely sensitive to the nerve agents sarin and soman and that galantamine or atropine will be effective medical countermeasures against the developmental neurotoxicity of these agents. The guinea pig will be the animal model of choice because: (i) brain development of guinea pigs closely resembles that of humans and non-human primates and (ii) like humans, guinea pigs have low levels of circulating carboxylesterases - the enzymes that metabolically inactivate OP compounds. The immediate goals of this project are: (i) to identify the effects of a prenatal exposure to soman or sarin on neurobehavior as well as functional, structural, and metabolic integrity of the brain of guinea pigs, and (ii) to assess the safety and effectiveness of galantamine or atropine, at doses that are compatible with human use, to counter the developmental toxicity of the nerve agents. Pregnant guinea pigs will receive, at a critical gestational period (GD 50-52), a subcutaneous injection of vehicle or a given dose (0.6x or 1.0xLD50) of soman or sarin. Subsequently (1 or 24 h later), the sows will be treated with saline (0.5 ml/kg, im) or a clinically relevant dose of galantamine (8 mg/kg, im) o atropine (0.5 mg/kg, im). On postnatal days 35-40 (prepuberty) and 120-125 (young adulthood), cognitive and emotional behavior of the animals will be analyzed. To examine the effects of the prenatal exposure to the nerve agents on electrical brain activity, electroencephalographic activity will be telemetrically monitored prior to the behavioral tests. Magnetic resonance imaging and spectroscopy will be used to identify deviations in brain structure and metabolism. The results of these translational, multidisciplinary studies will be far reaching as they will laythe groundwork necessary to advance research aimed at identifying safe and effective therapeutic strategies to treat even the most sensitive sector of the population in the event of a terrorist attack with sarin and soman. PUBLIC HEALTH RELEVANCE: The overarching goal of this project is to provide fundamental and timely input for assessment of the developmental toxicity of the nerve agents soman and sarin and identification of lead compounds for adequate treatment of neuropathologies that result from prenatal exposure to these agents.

ABSTRACT Poisoning with organophosphorus (OP) pesticides during gestation is a life-threatening condition for both mothers and fetuses. Treatment relies heavily on the use of high doses of atropine to block muscarinic receptor overactivation by acetylcholine (ACh) build up due to OP-induced block of acetylcholinesterase (AChE). However, despite therapeutic intervention spontaneous miscarriages, infant and/or maternal deaths, and postnatal neurological complications (including seizures and cognitive deficits) can ensue. Although rarely taken into account, the non-selective inhibition of all muscarinic receptor (mAChR) subtypes by atropine may be an important determinant of these poor outcomes. Specifically, inhibition of presynaptic mAChRs (mostly M2), which are part of a negative feedback loop that limits ACh release from cholinergic neurons, can exacerbate the OP-induced cholinergic crisis. The pharmacological profile of R,S-trihexyphenidyl (THP), a drug that has been safely used during pregnancy and is approved for treatment dystonia and Parkinson?s disease, makes it an attractive candidate to treat gestational OP poisoning. In contrast to atropine, THP more selectively inhibits M1 and M3 than M2 mAChRs. In addition, THP inhibits as-of-yet unidentified subtypes of neuronal nicotinic ACh receptors (nAChRs). Overactivation of M1/M3 mAChRs and neuronal nAChRs in the placenta and myometrium, and in the cardiorespiratory and nervous systems can contribute to poor health outcomes following acute OP intoxication during pregnancy. Thus, this project will test the hypothesis that, in part by sparing M2 mAChRs and potentially by blocking nAChRs in addition to M1/M3 mAChRs, THP will be more potent and efficacious than atropine to treat gestational OP poisoning. The focus will be on chlorpyrifos (CPF), a widely used OP pesticide currently included in the U.S. Department of Homeland Security Chemical Threat Risk Assessment list of chemicals that may be deployed to poison large numbers of people in terrorist attacks. A multidisciplinary approach, a translationally relevant animal model (the guinea pig), and a placebo-controlled, randomized, blind design that minimizes experimental bias and maximizes scientific rigor will be used to address three aims. Aims 1 and 2 will establish the effectiveness of THP to save lives, reduce signs of acute toxicity, and prevent the development of neurological complications in mothers and fetuses gestationally exposed to a high dose of CPF. Aim 3 will shed light on the mechanisms that contribute to the toxicity of CPF and the antidotal effectiveness of THP. Successful completion of this project will lay the groundwork for the development of more effective antidotes to treat acute CPF intoxication during pregnancy. Identification of therapeutic interventions that can have a positive impact on the health outcomes of populations acutely intoxicated with OP pesticides lends support to the initiative of the World Health Organization to tackle the issue of acute OP pesticide intoxication, particularly in the developing world. In addition, it fulfills an unmet medical need for the effective treatment of victims of a deliberate attack with these pesticides.