Daniel D. Savage, Ph.D. - US grants

Long term learning disabilities and other behavioral deficits have been described in children who were exposed to ethanol prenatally. A disorganization of neuronal and glial elements observed in the brains of such children and in animal models of ethanol exposure in utero may explain these learning and behavioral deficits. The hippocampal formation, an area of the brain involved with learning and memory, is quite sensitive to the effects of ethanol both pre- and postnatally. Anatomic and behavioral studies of fetal alcohol animals suggest a functional deficit within the hippocampal formation of these animals. The neurochemical manifestation(s) of this proposed functional deficit are unknown. The experiments outlined in this proposal are designed to study neurotransmitter receptors and several other neurochemical parameters in the hippocampal formation of animals exposed to ethanol prenatally. Attention will be focused on the dentate granule cells, their efferent projection to the hippocampal CA3-4 pyramidal cells and the afferent inputs to dentate granule cells. Using radiohistochemical techniques, the number and distribution of glutamate, muscarinic cholinergic, serotonergic, noradrenergic, GABA, kappa-opiate and adenosine receptors will be examined in the hippocampal formation of fetal alcohol animals and compared to ad lib fed and pair fed controls. The disposition of zinc in the axonal terminals of dentate granule cells after an intraventricular injection of zinc65 will also be examined. Finally, three neurochemical parameters will be examined that are thought to be associated with the mechanisms of long term potentiation in the hippocampal formation, and possibly with learning. The experiments described in this proposal should provide considerable information regarding the effects of ethanol exposure in utero on the hippocampal formation.

There is increasing concern regarding the reliability of anticonvulsant drug screens currently in use and the possibility that these screens may have impeded progress towards identifying new and better classes of anticonvulsant agents. This concern has led to efforts to develop more reliable anticonvulsant drug screens. The objectives of this research proposal are to 1) characterize a relatively new and little studied animal model of sponstaneously occurring limbic and motor seizures and 2) initiate an assessment of the utility of this model as an anticonvulsant drug screen by evaluating the effects of conventional anticonvulsants on the incidence of spontaneously occurring seizures. This effort represents a significant departure from more traditional methods of assessing anticonvulsant potential which usually rely on an elicited stimulus, such as electrical current or convulsant drug, to study drug effects on seizures. Using a computer automated kindling and EEG monitoring system, male Sprague-Dawley rats will receive kindling stimulations until they display spontaneous limbic and motor seizures. (This stage is quite different from the so called "kindled" state described by most investigators; where the animal has a permanent enhanced susceptibility to stimulus-induced motor seizures, but rarely displays spontaneous seizure activity). First, the spontaneous seizure model will be characterized in some detail to establish a standardized protocol for producing spontaneously seizing rats. Once the protocol is established, the effect of six drugs on the incidence of spontaneous seizures will be examined. Using osmotic minipumps to ensure constant plasma drug levels, the frequency and intensity of spontaneous seizures will be determied in drug and vehicle controls for a period of two weeks after minipump implantation. The effect of drug on spontaneous seizures will be correlated with plasma drug levels taken at several intervals during the two week drug trial. Should the spontaneous seizure model respond to conventional anticonvulsants in a pattern similar to human epileptic responses, future proposals will extend this work to drugs currently in clinical trial and experimental drugs having a rational or theoretical basis for anticonvulsant potential.

There is increasing concern regarding the reliability of anticonvulsant drug screens currently in use and the possibility that these screens may have impeded progress towards identifying new and better classes of anticonvulsant agents. This concern has led to efforts to develop more reliable anticonvulsant drug screens. The objectives of this research proposal are to 1) characterize a relatively new and little studied animal model of sponstaneously occurring limbic and motor seizures and 2) initiate an assessment of the utility of this model as an anticonvulsant drug screen by evaluating the effects of conventional anticonvulsants on the incidence of spontaneously occurring seizures. This effort represents a significant departure from more traditional methods of assessing anticonvulsant potential which usually rely on an elicited stimulus, such as electrical current or convulsant drug, to study drug effects on seizures. Using a computer automated kindling and EEG monitoring system, male Sprague-Dawley rats will receive kindling stimulations until they display spontaneous limbic and motor seizures. (This stage is quite different from the so called "kindled" state described by most investigators; where the animal has a permanent enhanced susceptibility to stimulus-induced motor seizures, but rarely displays spontaneous seizure activity). First, the spontaneous seizure model will be characterized in some detail to establish a standardized protocol for producing spontaneously seizing rats. Once the protocol is established, the effect of six drugs on the incidence of spontaneous seizures will be examined. Using osmotic minipumps to ensure constant plasma drug levels, the frequency and intensity of spontaneous seizures will be determied in drug and vehicle controls for a period of two weeks after minipump implantation. The effect of drug on spontaneous seizures will be correlated with plasma drug levels taken at several intervals during the two week drug trial. Should the spontaneous seizure model respond to conventional anticonvulsants in a pattern similar to human epileptic responses, future proposals will extend this work to drugs currently in clinical trial and experimental drugs having a rational or theoretical basis for anticonvulsant potential.

Long term learning disabilities have been described in children exposed to ethanol prenatally. The hippocampal formation, an area of the brain involved with memory consolidation, is quite sensitive to the effects of prenatal ethanol exposure both in humans and animal models of in utero ethanol exposure. Morphologic, neurochemical, electrophysiological and behavioral evidence indicate the presence of a functional deficit in the hippocampal formation of animals exposed to ethanol prenatally. At present, little is known about which hippocampal neurochemical parameters are altered or how these alterations might lead to a net decrease in hippocampal neurotransmission. The long-term objective of this project is to identify what prenatal ethanol-induced neurochemical alterations occur in hippocampal formation and what relationships may exist between these alterations and some electrophysiological and behavioral consequences of prenatal ethanol exposure. The specific aims of our competing renewal grant application are: 1.) We will examine the effect of prenatal ethanol exposure on the N- methyl-D-aspartate (NMDA) and quisqualate (QUIS) subtypes of hippocampal glutamate receptors, using receptor autoradiography techniques. We have previously reported reductions in total glutamate and kainate-sensitive glutamate binding in fetal alcohol rat hippocampal formation. The NMDA and QUIS studies will complete our radiohistochemical investigation of glutamate receptor binding in fetal alcohol rat hippocampal formation. 2.) We will examine the interaction between the NMDA receptor and the phencyclidine (PCP) receptor along with the allosteric regulation of these binding sites by glycine. Our preliminary results indicate a decrease in NMDA receptors in fetal alcohol hippocampus. This reduction could affect how the NMDA/PCP system activates a cation-selective channel thought to be play an important role in hippocampal neuronal plasticity. 3.) We will examine basal and QUIS receptor-mediated activation of phosphoinositide (Pl) hydrolysis in fetal alcohol hippocampal formation. The accumulation of radiolabelled inositol phosphate (3H-lP1) in hippocampal slices will be measured. Pl hydrolysis is an important step for signal amplification by some neurotransmitters such as glutamate. Examination of basal and QUIS-stimulated Pl hydrolysis will allow us to determine whether this receptor- transduction system is affected by fetal alcohol exposure. 4.) We will examine the effect of fetal ethanol exposure on protein kinase C (PKC) mediated phosphorylation of hippocampal F1 protein. A relatively selective phosphorylation of F1 protein has been associated with the generation of long term potentiation, an electrophysiological phenomenon thought to mediate the memory consolidation function of hippocampal formation. We will also examine the amount and distribution of 3H-phorbol dibutyrate binding to PKC in various brain regions of control and fetal alcohol rats using radiohistochemical techniques. 5.) We will examine the effect of fetal ethanol exposure on the number of dentate granule and hippocampal pyramidal neurons. One explanation for a reduction in the number of glutamate receptor binding sites and hippocampal mossy fiber zinc may be a decrease in the number of hippocampal formation neurons containing glutamate receptors and zinc. We will measure cell density in the stratum granulosum of dentate gyrus and stratum pyramidale of hippocampal CA3, CA1 and subiculum in control and fetal alcohol rats.

Much evidence now indicates that the b-adrenoceptor blockers induce proatherosclerotic serum lipid changes (increases in serum triglycerides, total cholesterol, and LDL cholesterol, and a reduction in HDL cholesterol) while alpha 1-adrenoceptor blockers produce an opposite antiatherosclerotic serum lip profile. The mechanisms involved in the dyslipidemic effects of the alpha- and beta-adrenoceptor blockers are poorly defined, but they most likely involve modification of the actions of circulating catecholamines on lipid metabolism. Alpha1- and Beta2-Adrenoceptor activation appears to influence hepatic fatty acid disposition and ketogenesis, but their physiological roles are unclear. The primary goal of this proposal is to investigate regulation of hepatic fatty acid metabolism and serum lipid by catecholamines and adrenoceptor blockers. The principal hypothesis of this study is that hepatic alpha1- adrenoceptor activation by catecholamines acts most importantly in inhibit fatty acid oxidation and promote triglyceride synthesis and secretion, and that beta-adrenoceptor stimulation produces the opposite effect. Previous in vitro liver studies of this subject have give widely conflicting results. Beta-Adrenoceptor activation is reported to modestly stimulate or have no effects on hepatic fatty acid oxidation, while alpha1-Adrenoceptor activation is reported to inhibit, have no effect or stimulate this process. These varying results may have been due to differences in study conditions and predominant use of mature male rate which have many hepatic alpha1-but only low number of Beta2-adrenoceptors. Female rats are a better animal model for such studies because like humans, their livers contain large numbers of both alpha1-and beta2- adrenoceptors. To test the primary hypothesis, adrenoceptor regulation of the metabolism of isotopic fatty acid in isolated hepatocytes from female rats will be investigated. Optimal conditions will be identified for eliciting Beta-adrenoceptor- mediated stimulation, and alpha1-adrenoceptor inhibition, of fatty acid oxidation in hepatocytes. The roles of the primary alpha1-adrenoceptor intracellular mediators, diacylglycerol (DAG), inositol trisphosphate (lP3) and Ca++, in alpha1-regulation of hepatocyte fatty acid oxidation will be characterize. Subsequent studies will determine if adrenoceptor effects on hepatic fatty acid disposition involve changes in fatty acid oxidation, esterification, or intracellular triglyceride lipolysis. In vivo experiments, epinephrine and other adrenergic agonists, and alpha1- and beta-adrenoceptor blockers, alone and in combination, will be administered chronically to rats, and levels of serum lipids will be determined. These experiments will characterize the influence of chronic hyperadrenergic states, such as occurs in stress, on atherosclerotic serum lipid changes. Results of these studies will provide important new information on the contributions of catecholamines and their receptor antagonists to atherosclerotic serum lipid changes.

Long-term learning disabilities have been described in children exposed to ethanol in utero. The hippocampal formation, a brain region involved with memory consolidation, is quite sensitive to the effects of prenatal ethanol exposure both in humans and animal models of in utero ethanol exposure. We have observed deficits in hippocampal glutamate neurotransmission in rat offspring whose mothers consumed moderate quantities of ethanol throughout gestation. As the hippocampal glutamatergic system is thought to participate in memory formation, this defect may contribute to subtle learning deficits in offspring whose mothers consumed moderate amounts of ethanol during gestation. The objectives of this proposal are to obtain a better understanding of prenatal ethanol-induced hippocampal glutamate receptor deficits and to initiate studies of environmental factors that affect the expression of these teratogenic effects. The specific aims are to: 1. Examine the effects of prenatal ethanol exposure on neurochemical markers of glutamate neurotransmission and mossy fiber zinc in developing offspring. We will determine what neurochemical teratology is expressed during development, which alterations persist into young adulthood and whether the pattern of alterations suggest developmental delay or permanent deficits. 2. Examine the impact of rat dam age on prenatal ethanol exposure-induced neurochemical deficits. We will determine which rat dam age produces offspring with the greatest degree of neurochemical teratology. 3. Examine the effect of varied periods of prenatal ethanol exposure on hippocampal neurochemistry. We will determine the minimal number of days of prenatal ethanol exposure that produce: 1) the smallest statistically significant changes and 2) the maximal changes in each neurochemical parameter. 4. Examine the effect of maternal blood ethanol concentration (BEC) on hippocampal neurochemistry. We will determine the lowest maternal BEC that produces significant neurochemical alterations and whether maximal changes in these parameters occur within the BEC range studied. These studies will provide a clearer picture of developmental expression of neurochemical defects in offspring, the impact of maternal age, ethanol dose and timing during fetal development on the expression of these neurochemical defects and a more detailed understanding of the nature of these neurochemical alterations. Finally, this data will provide information to facilitate the design of future studies of other environmental and genetic factors along with clues that may lead to studies designed to directly test hypotheses about the mechanism(s) of prenatal ethanol action on developing hippocampal formation.

DESCRIPTION: (Adapted from the Investigator's Abstract) Learning disabilities are among the more subtle, yet most pervasive, of fetal alcohol exposure-related defects in children. These learning deficits may not manifest until a child is school-aged and can occur in the absence of other physical evidence of alcohol-related birth defects. Deficits in hippocampal glutamatergic neurotransmission and synaptic plasticity have been observed in rats whose mothers consumed moderate quantities of ethanol during gestation. As glutamate receptor-dependent synaptic plasticity is important in learning, these defects are one candidate mechanism for the learning disabilities observed in offspring whose mothers drank moderate quantities of ethanol during pregnancy. The long-term objectives of our research program are two-fold: First, to more clearly delineate the neurobiological and behavioral mechanisms of activity-dependent synaptic plasticity deficits caused by prenatal ethanol exposure. Then, once these teratologic effects are better characterized, develop and explore rational treatment strategies for overcoming fetal ethanol exposure-induced learning deficits. The working hypothesis for this project states that: Prenatal ethanol exposure decreases metabotropic glutamate receptor (mGluR5)-mediated potentiation of amino acid transmitter release in dentate gyrus slices. In this proposal, we will focus on the initial steps in the process, the coupling of the mGluR5, the G-proteins Galphaq/11 and phospholipase C-beta1 (PLC-beta1) in the production of inositol 1, 4, 5 trisphosphate (IP3). The proposed deficit may be a function of: 1) Decreased mGluR5-coupled, phospholipase C (PLC)-stimulated IP3 production, which in turn, may result from 2) decreased levels of mGluR5, Galphaq/11 or PLC-beta1 proteins or 3) alterations in agonist-mediated desensitization of mGluR5-stimulated IP3 production. These studies will provide important new information about the effects of prenatal ethanol exposure on mGluR5 regulation of transmitter release, a critical component of synaptic plasticity in the hippocampal formation. Further, these results could provide insights into whether agents that affect mGluR function could be used to treat synaptic plasticity deficits and, ultimately, learning deficits in prenatal ethanol-exposed offspring.

The long-term objectives of this project and the other four projects in the IRP grant are to understand the neurochemical and neuropsychological mechanisms of synaptic plasticity which are affected by prenatal exposure to alcohol and their relationship to cognitive/learning deficits in adulthood. Even before life begins, hundreds of thousands of Americans are affected permanently by exposure to alcohol through maternal drinking. Prominent among the adverse effects of exposure to even moderate levels of alcohol is cognitive/learning deficits. The specific aims of this project are to evaluate experimentally in adult rats the effects of fetal alcohol exposure on: 1. long-lasting synaptic enhancement (LTP) in two areas of cerebral cortex important for learning and memory in freely-moving rats, 2. important inhibitory synapses in areas of cerebral cortex involved in cognition and learning, and 3. measures of learning and retention in challenging behavioral tasks linked to LTP by prior work. The results of these experiments, together with those in this IRP grant package, will provide a deeper understanding of the mechanisms by which prenatal alcohol exposure can disrupt the neurochemical and physiological mechanisms of synaptic plasticity that underlie cognition/learning. Such information can show the way to preventing life-long cognitive and behavioral handicaps.

alcoholism /alcohol abuse prevention; behavioral /social science research tag; embryo /fetus toxicology; mass spectrometry; protein quantitation /detection; two dimensional gel electrophoresis

[unreadable] DESCRIPTION (provided by applicant): The objective of this new research project is to investigate whether cognition-enhancing agents are effective in reversing fetal ethanol-induced deficits in hippocampal synaptic plasticity and learning using a rat model of moderate prenatal ethanol exposure. Within the time and budgetary constraints of the R21 grant funding mechanism, we will limit this initial "proof of concept" investigation to an assessment of the effects of a single cognition enhancing agent on the performance of fetal ethanol-exposed offspring in two behavioral paradigms sensitive to moderate prenatal ethanol exposure. Subsequently, we will examine the effects of this agent on two measures of hippocampal synaptic plasticity, the physiologic mechanisms thought to subserve learning, which are also sensitive to moderate prenatal ethanol exposure. The working hypothesis for this project is that the cognition-enhancing histamine H3 receptor antagonist ABT-239 will diminish fetal ethanol-induced learning deficits by enhancing hippocampal synaptic plasticity in fetal ethanol-exposed offspring. We will examine the effects of five different doses of ABT-239 on fetal ethanolinduced deficits in contextual fear-conditioned learning (Aim 1A) and spatial navigation deficits in the Morris Water Task (Aim 1B). Then, we will conduct dose-response studies of the effects of ABT-239 on fetal ethanolinduced deficits in long-term potentiation in vivo (Aim 2A) and activity-dependent potentiation of electrically evoked amino acid release (Aim 2B) in the dentate gyrus of dorsal hippocampal formation. Based on our hypothesis, we predict that ABT-239 will improve performance on the learning tasks in a manner that corresponds to improvements in one or both measures of hippocampal synaptic plasticity. The long-term objectives of this research program are to determine whether this experimental approach can provide the pharmacologic rationale for initiating clinical trials of such agents for treating cognitive deficits associated with FASD and whether this rodent model of moderate prenatal ethanol exposure has utility for assessing the therapeutic potential of a variety of cognition-enhancing agents. [unreadable] [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The New Mexico Alcohol Research Center (NMARC) is a comprehensive, multidisciplinary program focused on fetal alcohol-related behavioral deficits. NMARC's prevailing philosophy is that significant progress towards the dual goals of better diagnoses and inventions for the behavioral deficits associated with Fetal Alcohol Spectrum Disorders (FASD) requires a well-coordinated effort integrating basic research advances that elucidate the mechanistic consequences of fetal ethanol exposure with combined neuropsychological and functional neuroimaging studies in human subjects with FASD. A research center organization that maximizes the coordination and communication across lines of investigation provides the best long-term prospect for overcoming the ongoing challenges of diagnosing fetal alcohol-induced behavioral deficits and devising more effective interventions to ameliorate these deficits. The NMARC is a composite of established fetal alcohol research investigators with a history of collaborative research interactions complemented by the addition of outstanding investigators from other fields whose expertise and contributions can synergize the center's research environment and facilitate progress towards achieving NMARC's strategic objectives. The strategic objectives of the NMARC program are to: 1) Advance our understanding of the teratogenic consequences of fetal ethanol exposure on the neurobiologic mechanisms that negatively impact behavior, both in rodents and humans, 2) develop more effective approaches for diagnosing subjects with FASD, through the use of combined neurobehavioral and functional neuroimaging assessments, as well as through the prospect of developing more sensitive and reliable biomarkers capable of detecting functional brain damage earlier in life, and 3) develop more effective interventions for fetal alcohol-related behavioral deficits. More efficacious interventions may require a combination of neurobehavioral, educational and pharmacotherapeutic approaches to ameliorate the often subtle, but long-lasting impact of these deficits on affected offspring. This NMARC P20 application consists of seven components including two cores (Administrative and Pilot Projects) and five R03-level Developmental Research components. Each of the three preclinical research components, the two clinical projects and the two pilot projects represent novel experimental approaches that will address at least one of the three strategic objectives of mechanisms, diagnoses or interventions. Likewise, a pipeline of future pilot project ideas described in the Pilot Project Core ensures continued pursuit of the NMARC's strategic objectives over the five year P20 project period. Oversight of the NMARC operation will be provided by the Administrative Core and the NMARC Executive Committee. Assessment of and advocacy for the NMARC's achievements along with guidance in operational matters will be provided by an Internal Advisory Committee consisting of five senior administrators in the UNM's Health Science Center, Main Campus and The MIND Institute. An External Advisory Committee of three internationally renowned fetal alcohol researchers will advise the NMARC Executive Committee and component investigators and monitor NMARC's progress towards the achievement of its specific aims and strategic objectives.

The Pilot Project Core of the New Mexico Alcohol Research Center (NMARC) will provide a critical opportunity to develop new basic, clinical and translational fetal alcohol research projects that complement and extend the NMARC's research program portfolio. Selected projects must fit within the context of the Center's three strategic objectives as well as build on the overall integration, synergies, and sustainability of the Center's research activities. Indeed, these elements are essential if NMARC is to achieve its five-year goal of becoming an important national research center worthy of P50-level support. As with the Developmental Projects (NMARC Components 2-6), the goal of each pilot project is to obtain sufficient preliminary data to develop competitive RO1 or R21 grant applications for submission to the NIAAA. The Pilot Project Core will support two relatively small pilot projects during the first year. Pilot Project 7A, directed by Dr. Kevin Caldwell (Neurosciences), will initiate an examination the interaction between genetic variation and prenatal ethanol exposure on the predisposition for developing Post-Traumatic Stress Disorderlike symptoms. Pilot Project 7B, directed by Dr. Julia Stephen (Radiology), will examine sensory integration responses in 6 to 22 month-old normal and FASD children using a unique "babySQUID" MEG system housed at the MIND Institute. The Center Director and NMARC Executive Committee will monitor progress of all pilot projects supported by the core. Pilot project progress will be reviewed tri-annually by the Internal Advisory Group and annually by the External Advisory Committee. The Administrative Core and NMARC Executive Committee will assist pilot project Principle Investigators in the preparation and submission of NIAAA grants to extend support for their research beyond the pilot project funding. During the mid-summer of the first year of the NMARC project period, a call for additional Developmental Project and Pilot Project proposals will be advertised within the scientific community at UNM. In September, these proposals will be evaluated by the NMARC Executive Committee for scientific merit and relevance to the strategic objectives of the Center. One or two pilot projects recommended for funding by the Executive Committee will be reviewed for approval at the Annual Meeting with the External Advisory Committee in October. Funding for newly approved projects would begin at the start of the second project year. In Years 02 through 04, a similar process will be followed to select multiple new Developmental Projects and Pilot Projects. An essential element in the long-term success of the Pilot Project Core is the assurance that there are other meritorious projects worthy of NMARC support in the "pipeline". A "Future Pilot Projects" section is presented to provide evidence that there are already a large number of other potential projects, some of which involve other faculty (designated at "Other Significant Contributors" in the NMARC), that would be considered for NMARC support as pilot project funds become available.

0-11 years old; Absolute ethanol; Address; Alcohol, Ethyl; Alcohols; Amino Acids; Ammon Horn; Animal Model; Animal Models and Related Studies; Behavioral; Behavioral Paradigm; Chemical Class, Alcohol; Chemosensitization; Chemosensitization/Potentiation; Child; Child Youth; Children (0-21); Classification; Clinical Research; Clinical Study; Clinical Trials; Clinical Trials, Unspecified; Cognition; Cognitive Disturbance; Cognitive Impairment; Cognitive decline; Cognitive deficits; Cognitive function abnormal; Common Rat Strains; Cornu Ammonis; Dentate Fascia; Dentate Gyrus; Development; Disturbance in cognition; Dorsal; Dose; ETOH; Ethanol; FASD; Fascia Dentata; Fetal Alcohol Exposure; Fetal Alcohol Spectrum Disorder; Fetal ETOH Exposure; Fetal Ethanol Exposure; Funding Mechanisms; Gestation; Grain Alcohol; Gyrus Dentatus; H3 Receptors; Hippocampal Formation; Hippocampus; Hippocampus (Brain); Histamine H3 Receptors; Human, Child; Hydrogen Oxide; Impaired cognition; In Utero Alcohol Exposure; In Utero ETOH Exposure; In Utero Ethanol Exposure; Intervention; Intervention Strategies; Investigation; Knowledge; Learning; Learning Disabilities; Learning disability; Long-Term Potentiation; Mammals, Rats; Measures; Methylcarbinol; Modeling; Mothers; Neurologic; Neurological; Numbers; Patients; Performance; Physiologic; Physiological; Potentiation; Predictive Value; Pregnancy; Prenatal Alcohol Exposure; Prenatal ETOH Exposure; Prenatal Ethanol Exposure; Programs (PT); Programs [Publication Type]; R01 Mechanism; R01 Program; RPG; Rat; Rattus; Receptors, Histamine H3; Research; Research Grants; Research Project Grants; Research Projects; Research Projects, R-Series; Rodent Model; Staging; Structure of dentate gyrus; Synaptic plasticity; Systematics; Testing; Therapeutic; Therapeutic Agents; Thinking; Thinking, function; Time; Treatment Efficacy; Water; Work; alcohol exposed; alcohol exposure; alcohol-exposed pregnancy; aminoacid; base; children; clinical investigation; cognitive dysfunction; cognitive loss; cognitively impaired; concept; conditioned fear; dentate gyrus; ethanol exposed; ethanol exposure; exposed to alcohol; exposed to alcohol prenatally; exposure to alcohol; fear conditioning; fetal; gestation ETOH exposure; gestation alcohol exposure; gestation ethanol exposure; hippocampal; improved; in vivo; interventional strategy; model organism; pregnancy ETOH exposure; pregnancy alcohol exposure; pregnancy ethanol exposure; prenatally alcohol exposed; prenatally exposed to alcohol; programs; response; spatial navigation; therapeutic efficacy; therapeutically effective; way finding; wayfinding; youngster

The NMARC Administrative Core will provide the scientific leadership and administrative support for all center-related activities. Under the supervision of the Center Director, the NMARC Program Coordinator will: 1) Assist component investigators and their staffs with routine fiscal and administrative matters, 2) coordinate all NMARC-related activities including the Executive Committee, Internal Advisory Committee and External Advisory Committee meetings 3) make all logistical arrangements for NMARC-supported seminar peakers and the Annual NMARC Symposium and 4) maintain the NMARC website and assist in publishing an electronic quarterly NMARC newsletter and a printed NMARC Annual Report. The Center Director and the NMARC Executive Committee, representing the preclinical and clinical areas of research endeavor, will be responsible for the NMARC's progress towards achieving the Center's specific aims. In addition, the Executive Committee will: 1) Monitor the progress of NMARC's research components, 2) oversee the Center's dissemination and outreach activities,3) review and recommend new projects for funding by the Pilot Project Core and 4) be responsible for developing plans for other center-based services in preparation for a subsequent P50 application, including core facilities for: a) Animal breeding and behavioral testing, b) human subject recruitment, c) statistics, and d) information management and data sharing. An Internal Advisory Committee composed of senior administrative leadership from the Health Sciences Center, UNM Main Campus, and The MIND Institute will monitor Center's progress, along with providing guidance in operational and regulatory matters. Collectively, the administrative leadership of the three organizational units has committed approximately $275,000 over five years to support the NMARC P20 effort. This support includes: 1) funding to support future pilot projects, 2) graduate research assistants, 3) waived costs for MRI scans and 4) wet bench laboratory space renovation costs. An External Advisory Committee of three internationally renowned fetal alcohol research scientists will meet annually with NMARC investigators and the Internal Advisory Committee to provide oversight of the Center's progress towards achievement of its specific aims and strategic objectives, and review the NMARC Annual Report to the NIAAA.

DESCRIPTION (provided by applicant): Learning disabilities are the most common behavioral deficit observed in children with Fetal Alcohol Spectrum Disorder. The development of effective pharmacotherapeutic interventions for these disabilities requires a clearer understanding of the neurobiologic bases of fetal ethanol-induced learning deficits and subsequently, the identification of therapeutic agents whose mechanisms of action would be predicted to have clinical utility. We have observed that the histamine H3 receptor antagonist ABT-239 ameliorates fetal ethanol-induced deficits in dentate gyrus long-term potentiation (LTP) and learning. We have also observed increased H3 receptor-effector coupling in dentate gyrus of fetal ethanol offspring. Given that presynaptic H3 receptors inhibit glutamate release;our results suggest that fetal ethanol exposure increases H3 receptor-mediated inhibition of glutamate release, and that ABT-239 reduces this heightened inhibitory influence. We hypothesize that: Fetal ethanol exposure elevates presynaptic histamine H3 receptor function in dentate gyrus. This heightened inhibitory influence reduces glutamate release at the perforant path - dentate granule cell synapse which, in turn, contributes to deficits in LTP and learned behaviors sensitive to functional damage in the dentate gyrus. To test this hypothesis, our specific aims will examine: 1: The binding of [3H]-A349821, a selective H3 receptor antagonist, in dentate gyrus to quantitate H3 receptor density in control and fetal alcohol offspring (Aim 1A). We will also use the selective H3 receptor agonist methimepip to conduct methimepip-displacement of [3H]-A349821 binding studies to determine the proportion of high- and low-affinity agonist binding sites of the H3 receptor (Aim 1B) and examine H3 receptor-effector coupling by measuring methimepip-stimulated [35S]-GTPgS binding (Aim 1C). 2: The effects of methimepip and ABT-239 on glutamate and GABA release by measuring paired-pulse plasticity and miniature postsynaptic currents along with the effects of these agents on LTP at perforant path dentate granule cell synapses in control and fetal ethanol-exposed offspring. 3: The effects of methimepip and ABT-239 on paired-pulse plasticity and LTP at the perforant path - granule cell synapse in vivo in control and fetal ethanol-exposed offspring. 4: The effects of methimepip on one-trial contextual fear conditioning (Aim 4A) and spatial navigation (Aim 4B) and the effects of methimepip and ABT-239 on a spatial pattern-separation variant of the radial arm maze (Aim 4C) in control and fetal ethanol-exposed offspring. We anticipate that these studies will provide important new insights on the impact of fetal ethanol exposure on histaminergic modulation of glutamatergic neurotransmission and synaptic plasticity in the dentate gyrus. In addition, the results could provide a preclinical pharmacologic rationale for considering drugs that act as H3 receptor antagonists as putative therapeutic agents for the treatment of learning deficits in humans with FASD. PUBLIC HEALTH RELEVANCE: Despite public awareness campaigns warning of the dangers of drinking during pregnancy, it is estimated that between 2% and 5% of children born in the United States each year have fetal alcohol-associated functional brain damage that will lead to learning deficits over time. Currently, there are no evidence-based clinically useful pharmacotherapeutic interventions for these deficits. The long-term objective of our research program is understand the neurobiologic bases of fetal ethanol-induced learning deficits and subsequently, to identify therapeutic agents whose mechanisms of action would be predicted to have clinical utility in treating fetal alcohol-associated learning disabilities.

DESCRIPTION (provided by applicant): NMARC is a NIAAA-designated Developmental Alcohol Research P20 Center at the UNM Health Sciences Center. The center is comprised of teams of preclinical and clinical scientists with a history of collaborative research interactions whose expertise and contributions have synergized the center's research environment and is facilitating progress towards the achievement of NMARC's three strategic objectives. These objectives are to: 1) Advance our understanding of the teratogenic consequences of fetal alcohol exposure that cause functional brain damage leading to adverse neurobehavioral outcomes. 2) Develop more effective approaches for diagnosing individuals with FASD through the establishment of more sensitive and reliable alcohol biomarkers of alcohol exposure coupled with the use of novel neurobehavioral and functional neuroimaging assessments that can detect functional brain damage earlier in life. 3) Develop more effective neurobehavioral, educational and pharmacotherapeutic interventions tor fetal alcohol-related behavioral deficits. NMARC's prevailing philosophy is that a research center organized to maximize the coordination, communication and synergistic integration across multiple lines of preclinical and clinical investigation provides the best long-term prospect of achieving significant progress towards the dual clinical goals of better diagnosis and more effective interventions for FASD. This P50 grant application contains five research components, each consisting of teams of investigators whose project addresses one or more of NMARC's three strategic objectives. Three core components support the center's research program: 1) A Pilot Project Core with one clinical and two preclinical projects led by investigators new to the FASD research field. 2) A Science Core that supports all NMARC investigators and serves as a forum for catalyzing collaboration and peer group mentoring. 3) An Administrative Core that will provide scientific and administrative leadership for the entire NMARC program along with administrative support and budgetary oversight of the center programs. The Administrative Core will also be responsible for ensuring progress toward achieving the center's goals of building research capacity through the recruitment of other UNM faculty and new faculty hires, and enhancing our knowledge dissemination capabilities. Assessment of NMARC's progress and achievements will be the responsibility of the Steering Committee working in conjunction with an external Program Advisory Committee of seven internationally-renowned FASD scientists, who will advise and assess NMARC's research activities and progress towards the achievement of the center's goals and strategic objectives.

NMARC's Administrative Core is responsible for providing scientific and administrative leadership for the entire center program along with the administrative support and budgetary oversight of all NMARC-related activities. Operational supervision of the NMARC is provided by a three-tiered system of management: 1) The Administrative Core, 2) the NMARC Steering Committee whose collective knowledge and experience represents the breadth of NMARC activities and, 3) the NMARC Program Advisory Committee (PAC) composed of seven internationally-renowned FASD research investigators. The specific aims of the Core are to: 1) Provide administrative leadership and support for the NMARC. All NMARC investigators receive routine administrative support. The Core also arranges all NMARC-related meetings, manages logistical arrangements for NMARC seminar visitors, the Annual Symposium and the Annual Meeting with the PAC. The Steering Committee meets monthly to review progress on the research components and resource allocation issues. The Steering Committee also meets with the PAC during the Annual NMARC meeting to discuss each research component's progress, the operation and coordination of the Administrative, Scientific and Pilot Project cores and the center's overall progress towards the achievement of the specific aims of the center as a whole. 2) Provide scientific leadership and direction to NMARC's investigators, working to help them achieve the aims of their individual research projects while advancing the specific aims of the center as a whole. The Core will facilitate research collaborative interaction and communication through the monthly Scientific Core meetings with component investigators, working to increase the integration of preclinical and clinical research projects and the interactions with NMARC's core facilities. The Core will also work to build NMARC's research capacity through oversight of a pilot project program, continued efforts to recruit new faculty investigators to NMARC, building research collaborations with other institutions and encouraging the training of future FASD investigators. 3) Manage all issues related to resource allocation within the NMARC and strive to maximize the synergistic utilization of research resources within the center. The Core will coordinate and facilitate NMARC investigator access to other resources within the university. 4) Enhance our knowledge dissemination capabilities through our NMARC Seminar Series, an Annual NMARC Symposium, a NMARC Webpage and expanding our community outreach activities. Assessment of NMARC's progress towards the achievement of these aims and the strategic objectives will be the responsibility of the Steering Committee working in conjunction with the Program Advisory Committee.

PROJECT SUMMARY ABSTRACT Learning disabilities are the most common behavioral deficit observed in children with Fetal Alcohol Spectrum Disorder (FASD). Currently, there are no established rationally-based clinically-effective pharmacotherapeutic interventions for these and related behavioral deficits. However, using a well-established rat model of FASD, we have observed that the histamine H3 receptor inverse agonist ABT-239 ameliorates prenatal alcohol exposure (PAE)-induced deficits in dentate gyrus long-term potentiation (LTP) and retention of memory. We have also observed increased H3 receptor-effector coupling and a heightened H3 receptor-mediated inhibition of glutamate release in the dentate gyrus of PAE rats. Our results suggest that PAE increases H3 receptor-mediated inhibition of glutamate release and that ABT-239 reduces this heightened inhibitory influence. These observations provide preclinical rationale for examining the efficacy of H3 receptor inverse agonists on treating learning and memory deficits in children with FASD. The working hypothesis for the preclinical UH2 phase of this proposal is that SAR152954, another histamine H3 receptor inverse agonist, will ameliorate PAE-induced behavioral and synaptic plasticity deficits by reversing PAE-induced decreases in activity-dependent potentiation of glutamate release. We will first examine the effects of four different doses of SAR152954 on PAE-induced deficits in one- trial contextual fear conditioning (Aim 1A) and the retention of spatial memory using the Morris Water Maze (Aim 1B), two behaviors quite sensitive to PAE-induced functional damage of the dentate gyrus. The milestone objective of Aim 1 will be to identify the optimal test dose (OTD) of SAR152954 that reverses PAE-induced memory deficits. Subsequently, we will examine whether the OTD dose of SAR152954 reverses PAE-induced deficits in dentate gyrus LTP (Aim 2A) and putative deficits in activity-dependent potentiation of glutamate levels in dentate gyrus (Aim 2B). The milestone objective of Aim 2 will be to demonstrate the amelioration of these neurophysiological deficits as the mechanistic cornerstone of a preclinical rationale for advancing H3 receptor inverse agonists to clinical trial. Assuming we achieve the preclinical milestones, we will submit a one-year request for funding to develop a clinical trial plan. A tentative draft of plans has been developed by our clinical investigator team. First, a Phase Ib trial is proposed using three drug doses in adolescents with FASD. In addition to acquiring pharmacokinetic and safety profile data, the primary Phase Ib milestone objective will be to identify the highest safe dose of the agent producing significant improvements in combined behavioral and neurophysiologic responses, as measured using hdEEG/magnetoencephalography. Subsequently, in a Phase IIa clinical trial, we would assess the therapeutic efficacy of the optimal drug dose using a double-blind crossover design that employs the same pharmacodynamic assessments used in the Phase Ib trial. Assuming these UH3 milestones are achieved, we anticipate a subsequent, more extensive trial to assess drug efficacy in a multisite clinical trial.

PROJECT SUMMARY Learning disabilities are the most common behavioral deficit observed in patients with Fetal Alcohol Spectrum Disorder (FASD). Currently, there are no established clinically effective pharmacotherapeutic interventions for these behavioral problems. Using a well-established rat model of FASD, we have reported that the histamine H3 receptor inverse agonist / antagonist ABT-239 ameliorates prenatal alcohol exposure (PAE)-induced deficits in synaptic plasticity and learning. We have also observed increased H3 receptor-effector coupling and a heightened H3 receptor-mediated inhibition of the probability of glutamate release in dentate gyrus of PAE rats. Collectively, these results suggest that PAE increases H3 receptor-mediated inhibition of glutamate release and that ABT-239 reduces this heightened inhibitory influence. One parsimonious explanation for why PAE rats are more sensitive to H3 receptor agents is a PAE-induced increase in the expression of the rH3A isoform of histamine H3 receptors relative to the rH3C isoform. This putative shift would confer greater sensitivity to the inhibitory effects of histamine H3 receptor agonists in PAE rats. The principal objectives of this NMARC research component application are to: 1) Examine whether PAE increases the expression of rH3A relative to rH3C in entorhinal cortical neurons projecting to the dentate gyrus. 2) More thoroughly establish the consequences of a PAE-induced elevation in H3 receptor-effector coupling and the effects of second H3 receptor inverse agonist / antagonist namely, SAR152954, on H3 receptor-mediated inhibition of glutamatergic neurotransmission in dentate gyrus. In Specific Aim 1A, we will employ in situ hybridization and qRT-PCR approaches to first confirm whether PAE increases the rH3A/rH3C mRNA expression ratio. In Aim 1B, we will use a [35S]-GTP?S binding assay in histological sections to conduct a more detailed examination of the effects of PAE on H3 receptor- effector coupling and assess the differential sensitivity of PAE rats to SAR152954 relative to controls. In Specific Aim 2A, we will conduct in vitro slice physiology studies to examine: 1) The impact of PAE on the effects of the H3 receptor agonist methimepip on fEPSP responses and pair-pulse plasticity in dentate gyrus, under baseline and after theta burst stimulation conditions. 2) The effects of SAR152954 alone and in the presence of methimepip on paired-pulse ratio and long-term potentiation. In Aim 2B, we will examine the impact of PAE on fEPSP and PPR before and after high frequency stimulations in awake freely moving rats. We predict that SAR152954 will ameliorate the heightened H3 receptor mediated inhibition of synaptic plasticity in PAE rats at concentrations/doses that do not impair synaptic transmission in control rats. These studies address two of the three strategic objectives of NMARC, namely advancing our understanding of the neurobiological consequences of PAE, as well as working towards establishing the mechanistic basis for novel interventions to treat of PAE- induced deficits in synaptic plasticity and memory. These studies could provide additional preclinical rational for considering drugs such as SAR152954 for clinical trials in patients with FASD. 1

PROJECT SUMMARY ? ADMINISTRATIVE CORE NMARC's Administrative Core is responsible for providing scientific and administrative leadership for the entire center program along with the administrative support and budgetary oversight of all NMARC-related activities. Operational supervision of the NMARC is provided by a three-tiered system of management: 1) The Admin Core Executive Committee. 2) The NMARC Steering Committee, composed of four directors and senior researchers from UNM and the MIND Research Network. 3) The NMARC Program Advisory Committee (PAC) composed of seven internationally renowned FASD research investigators. The Admin Core has four specific aims: Aim 1) Provide administrative leadership and support for the NMARC. All NMARC investigators receive routine administrative support. The Admin Core also arranges all NMARC-related meetings, manages logistical arrangements for NMARC seminar visitors, the Annual FASD Day Symposium, and the Annual NMARC Meeting with the PAC. The Steering Committee meets twice a year to review progress on the research components and discuss any resource allocation issues. The Steering Committee also meets with the PAC during the Annual NMARC meeting to discuss each research component's progress, the operation and coordination of the Administrative and Pilot Project cores, and the center's overall progress towards the achievement of the specific aims of the center as a whole. Aim 2) Provide scientific leadership and direction to NMARC's investigators, working to help them achieve the specific aims of their individual projects while advancing the specific aims of the center as a whole. The Admin Core will facilitate collaborative research interaction and communication through the monthly NMARC Investigator Group meetings with all NMARC-affiliated investigators, working to increase the integration of preclinical and clinical research projects. The Admin Core will also work to build NMARC's research capacity through oversight of the pilot project program, continued efforts to recruit new faculty investigators to NMARC, building research collaborations with other institutions and encouraging the training of future FASD investigators. Aim 3) Manage all issues related to resource allocation within the NMARC and strive to maximize the synergistic utilization of research resources within the center. The Admin Core will coordinate and facilitate NMARC investigator access to other resources within the university. Aim 4) Enhance our knowledge dissemination capabilities through our NMARC Seminar Series, the Annual FASD Day Symposium, a NMARC Webpage, and expanding our community outreach activities. Assessment of NMARC's progress towards the achievement of these aims and the strategic objectives is the responsibility of the Steering Committee working in conjunction with the PAC.