AC1 and AC8 as Mediators of the Dendritic Response to Ethanol in a Model of FASD

描述(由申请人提供)：这项建议的总体目标是在胎儿酒精谱系障碍(FASD)模型中确定乙醇对神经元形态造成有害影响的潜在机制，从而支持NIAAA增加对与酒精使用有关的生物学功能的了解的使命。突触发生(脑生长突发期)发生在人类妊娠的最后三个月，是发育中的大脑中树突生长和棘突发育的关键窗口。树突棘是神经元之间进行突触交流的主要结构。在此期间接触乙醇对发育中的胎儿大脑有不利影响，导致学习障碍、行为异常和精神发育迟缓，称为FASD。BDNF已被证明通过与TrkB受体相互作用来调节树突状细胞生长和脊椎发育。在新生儿大脑的不同区域，BDNF的表达也被证明受到乙醇的调节。双胞胎研究表明，胎儿基因型影响FASD的严重程度，尽管涉及的基因(S)尚未确定。由于研究表明缺乏腺苷环化酶1和8的成年小鼠对乙醇的镇静作用有更高的敏感性，并且缺乏这些ACS的新生小鼠的纹状体对乙醇诱导的神经细胞死亡比WT小鼠更敏感，我们认为ACS可能在新生儿乙醇暴露的后遗症中发挥作用。这一建议将检验这一假说，即钙离子刺激的腺苷环化酶(AC1和AC8)通过调节BDNF信号来调节树突状细胞对乙醇的反应。该假说将用于验证下列目标：1)确定乙醇暴露对野生型(WT)和钙离子刺激的AC基因敲除(DKO)小鼠发育中纹状体神经元形态的影响；2)分离钙离子刺激的AC突触前和突触后在调节树突复杂性和脊柱形成中的作用；3)确定乙醇暴露后新生鼠纹状体内钙离子刺激的ACS对BDNF调节的影响。在目标1中，将用乙醇处理P5-7幼鼠(WT和DKO)，并在P14和P30对纹状体神经元进行胞体大小、树突长度、树突分支和棘密度的分析。在目标2中，来自WT和DKO小鼠的皮质纹状体共培养将被用来确定突触前和突触后在乙醇诱导的树突树枝和棘突形成中的作用。在目标3中，将检测乙醇处理的新生小鼠(P5-7)突触体组分中BDNF的mRNA和蛋白以及TrkB蛋白的表达，并将使用纹状体神经元的共聚焦成像来识别突触前和突触后钙离子刺激的ACS在调节BDNF表达中的作用。在四个主要领域的使用培训：原代神经元培养技术(皮质纹状体共培养)、免疫组织化学技术/共聚焦分析、二线荧光染料成像和神经元形态分析的使用为发起人和申请者提供了令人兴奋的研究机会，极大地提高了申请者未来取得科学成功的潜力。 公共卫生相关性：人类妊娠的第三个三个月是大脑发育的关键时期。在此期间饮酒可导致胎儿神经毒性，导致行为异常、学习障碍、智力低下、抑郁和称为胎儿酒精谱系障碍(FASD)的精神病。本研究旨在研究腺苷环化酶在调节新生儿酒精暴露后树突状细胞生长、脊柱发育和BDNF表达中的作用，从而确定FASD儿童的治疗靶点。

DESCRIPTION (provided by applicant): The overall objective of this proposal is to define the mechanisms underlying the deleterious effects caused by ethanol on neuronal morphology in a model of Fetal Alcohol Spectrum Disorder (FASD), thereby upholding the mission of the NIAAA to increase the understanding of biological functions relating to alcohol use. The synaptogenesis (brain growth spurt) period occurs during the last trimester of human gestation and is a critical window for dendritic growth and spine development in the developing brain. Dendritic spines are the primary structures used in synaptic communication between neurons. Exposure to ethanol during this period has been shown to have detrimental effects on the developing fetal brain, leading to learning disorders, behavioral abnormalities, and mental retardation, termed FASD. BDNF has been shown to regulate dendritic growth and spine development through its interaction with the trkB receptor. BDNF expression has also been shown to be modulated by ethanol in various regions of the neonatal brain. Fetal genotype has been shown to affect the severity of FASD as shown by twin studies, although the gene(s) involved have yet to be identified. Since studies have shown adult mice lacking the adenylyl cyclases 1 and 8 have an increased susceptibility to the sedative effects of ethanol, and the striatum of neonatal mice lacking these ACs are more sensitive to EtOH induced neuronal cell death than WT mice, we believe the ACs could play a role in the sequelae of neonatal EtOH exposure. This proposal will test the hypothesis that the Ca2+stimulated adenylyl cyclases (AC1 and AC8) regulate the dendritic response to ethanol through regulation of BDNF signaling. This hypothesis will be tested using the following Aims: 1) Determine the effects of ethanol exposure on neuronal morphology in the developing striatum from wild type (WT) and Ca2+stimulated AC knockout (DKO) mice; 2) Dissociate the pre- and postsynaptic roles of the Ca2+stimulated ACs in the regulation of dendritic complexity and spine formation; 3) Determine the effects of the Ca2+stimulated ACs on BDNF regulation in the neonatal striatum following ethanol exposure. In Aim 1, P5-7 pups (WT and DKO) will be treated with ethanol and striatal neurons will be analyzed at P14 and P30 for soma size, dendritic length, dendritic branching, and spine density. In Aim 2, corticostriatal co-cultures from WT and DKO mice will be used to determine the pre- and postsynaptic roles of the Ca2+stimulated ACs in ethanol-induced effects on dendritic arborization and spine formation. In Aim 3, BDNF mRNA and protein as well as trkB protein will be measured in synaptosomal fractions from ethanol-treated neonatal mice (P5-7) and confocal imaging of striatal neurons will be used to identify the pre- and postsynaptic roles of the Ca2+stimulated ACs in regulating BDNF expression. Training in the use of four main areas: primary neuronal culture techniques (corticostriatal co-cultures), immunohistochemical techniques/confocal analyses, use of diolistic fluorescent dye imaging and neuronal morphology analyses represents exciting opportunities of research for both the sponsor and applicant, greatly enhancing the applicant's potential for future scientific success. PUBLIC HEALTH RELEVANCE: The third trimester of gestation in humans is a critical time for brain development. Consumption of alcohol during this time can result in fetal neurotoxicity leading to behavioral abnormalities, learning disorders, mental retardation, depression, and psychosis termed fetal alcohol spectrum disorder (FASD). This proposal is designed to study the role of the adenylyl cyclases in the regulation of dendritic growth, spine development, and BDNF expression after neonatal alcohol exposure, which may lead to identification of therapeutic targets for children with FASD.