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

描述（由申请人提供）：本提案的总体目标是确定乙醇对胎儿酒精谱系障碍（FASD）模型中神经元形态的有害影响的潜在机制，从而支持NIAAA的使命，即增加对与酒精使用有关的生物学功能的理解。突触发生（大脑生长突增）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脊柱发育的关键窗口期。树突棘是神经元间突触通讯的主要结构。在此期间暴露于乙醇已被证明对发育中的胎儿大脑有不利影响，导致学习障碍、行为异常和智力迟钝，称为FASD。BDNF已被证明通过与trkB受体的相互作用来调节树突生长和脊柱发育。BDNF的表达也被证明是由乙醇在新生儿大脑的不同区域调节。双胞胎研究表明，胎儿基因型影响FASD的严重程度，尽管涉及的基因尚未确定。由于研究表明缺乏腺苷酸环化酶1和8的成年小鼠对乙醇镇静作用的敏感性增加，并且缺乏这些ACs的新生小鼠的纹状体对EtOH诱导的神经元细胞死亡比WT小鼠更敏感，我们认为ACs可能在新生儿EtOH暴露的后遗症中发挥作用。该提案将验证Ca2+刺激的腺苷酸环化酶（AC1和AC8）通过调节BDNF信号传导调节树突对乙醇的反应的假设。这一假设将通过以下目的进行验证：1)确定乙醇暴露对野生型（WT）和Ca2+刺激AC敲除（DKO）小鼠发育纹状体神经元形态的影响；2)解离Ca2+刺激的ACs在调节树突复杂性和脊柱形成中的突触前和突触后作用；3)确定Ca2+刺激的ACs对乙醇暴露后新生儿纹状体BDNF调节的影响。在Aim 1中，P5-7幼崽（WT和DKO）将用乙醇处理，纹状体神经元将在P14和P30时分析体细胞大小、树突长度、树突分支和脊柱密度。在Aim 2中，WT和DKO小鼠皮质纹状体共培养将用于确定Ca2+刺激的ACs在乙醇诱导的树突树突和脊柱形成中的突触前和突触后作用。在Aim 3中，BDNF mRNA和蛋白以及trkB蛋白将在乙醇处理的新生小鼠（P5-7）的突触体部分中进行测量，纹状体神经元的共聚焦成像将用于鉴定Ca2+刺激的ACs在调节BDNF表达中的突触前和突触后作用。在使用四个主要领域的培训：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，使用diolistic荧光染料成像和神经元形态学分析对申办者和申请人来说都是令人兴奋的研究机会，极大地提高了申请人未来科学成功的潜力。

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.