Adenosine Receptors and Ethanol Withdrawal

腺苷受体和乙醇戒断

基本信息

批准号：
7749649
负责人：
Tracy Renee Butler
金额：
$ 3.04万
依托单位：
UNIVERSITY OF KENTUCKY
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-09-01 至 2011-05-08
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7749649
关键词：
Adenosine Agonist Air Alcohol withdrawal syndrome Brain Calcium Calculi Cell Culture Techniques Cell Death Cell Survival Cells Chronic Confocal Microscopy Data Densitometry Ethanol Excision Excitatory Amino Acids Female Fluorescence Formalin Glutamates Hippocampal Formation Hippocampus (Brain)Hour Human Image Imagery Immunofluorescence Immunologic Immunohistochemistry In Vitro Knowledge Label Laboratories Measures Mediating Microscopy Modeling N-Methyl-D-Aspartate Receptors N-Methylaspartate NMDA receptor antagonist NR1 gene Neurons Nuclear Protein Nuclear Proteins Oranges Pharmaceutical Preparations Pharmacotherapy Polyamines Propidium Diiodide Purinergic P1 Receptors Rattus Reporting Rodent Sex Characteristics Slice Structure System Techniques Toxic effect Treatment Protocols Western Blotting Withdrawal Work alcohol effect alcohol exposure density dentate gyrus design follow-up in vivo male neurotoxicity neurotransmission novel polypeptide prevent receptor receptor density research study response sex

项目摘要

DESCRIPTION (provided by applicant): Previous studies suggest an innate sex difference in adenosine receptor density and/or function, which is mediated, at least in part, by NMDA receptors (Butler et al., 2008). The currently proposed studies will investigate effects of chronic ethanol (EtOH) exposure on adaptive changes in adenosine and NMDA receptor proteins, which may confer sensitivity to neurotoxicity during ethanol withdrawal (EWD) in a concentration- and/or sex-dependent manner. Male and female organotypic hippocampal slice cultures will be continuously exposed to EtOH (0, 25, or 50 mM) for ten days. Upon removal of EtOH, immunohistochemistry will be employed for the adenosine A1 and A2A receptor subtypes and the NMDA NR1 and NR2B subunits to quantify receptor density in the primary cell layers of the dentate gyrus, and the CA3 and CAI regions of the hippocampus. Western blotting will be conducted for an additional quantitative measure of changes in receptor proteins throughout the whole hippocampal formation in male and female EtOH-exposed cultures (0, 25, 50 mM). Additional studies will examine functional consequences of pharmacological antagonism of adenosine Al receptor antagonism during EWD. Cultures will be exposed to EtOH for ten days before twenty-four hour EWD. For the duration of EWD, cultures will be exposed to an Air antagonist; an Air antagonist + Air agonist; or an Air antagonist + NMDAr antagonist; and all cultures will be exposed to propidium iodide (PI) for labeling of dead/dying cells. Cultures will then be formalin-fixed for immunohistochemical labeling of mature neurons with neuronal nuclear protein (NeuN). Additional cultures will be exposed to the same drug treatments described, but will be exposed to Calcium (Ca2+) Orange at the end of twenty-four hour EWD to measure whether toxicity caused by A1r antagonism during EWD is correlated with potentiated Ca2+ influx. Slices will be visualized using fluorescent microscopy and data will be quantified using densitometry and confocal imaging. Correlational analyses will determine if the measures of cell viability (PI, NeuN, Ca2-*- orange) are related. These techniques allow for a comprehensive characterization of changes in the adenosinergic and NMDA receptor systems induced by EtOH exposure, and the functional consequences of those hypothesized adaptations during EWD. A significant amount of evidence suggests that the female brain of both humans and rodents is more sensitive to neurotoxicity in response to EtOH exposure and/or EWD. The current studies will contribute to our knowledge of EtOH- induced neuronal adaptations and potential sex differences in brain structure and/or function, and may contribute to the advent of novel pharmacotherapies to ameliorate EtOH and/or EWD-related toxicity.

描述（由申请人提供）：先前的研究表明，腺苷受体密度和/或功能的先天性别差异，至少部分由NMDA受体介导（Butler等，2008）。目前提出的研究将研究慢性乙醇（ETOH）暴露对腺苷和NMDA受体蛋白适应性变化的影响，这可能会以浓度和/或性别依赖性方式赋予乙醇戒断（EWD）期间对神经毒性的敏感性。雄性和女性器官海马切片培养物将连续暴露于ETOH（0、25或50 mm）中十天。去除ETOH后，将采用免疫组织化学用于腺苷A1和A2A受体亚型以及NMDA NR1和NR2B亚基来量化齿状回的原代细胞层中的受体密度，以及Hippocampus的CA3和CAI区域。将进行蛋白质印迹，以对男性和女性eTOH暴露培养物（0、25、50 mm）的整个海马形成中受体蛋白变化的额外定量衡量。其他研究将检查EWD期间腺苷Al受体拮抗作用的药理拮抗作用后果。培养物将在二十四小时EWD之前暴露于ETOH中十天。在EWD期间，培养物将暴露于空气对手。空气拮抗剂 +空气动力学家；或空气拮抗剂 + NMDAR拮抗剂；并且所有培养物将暴露于碘化丙啶（PI），以标记死亡/垂死细胞。然后，将培养物进行福音固定，以用神经元核蛋白（NEUN）对成熟神经元进行免疫组织化学标记。其他培养物将暴露于所述的相同药物治疗中，但在二十四小时EWD结束时将暴露于钙（Ca2+）橙色，以衡量EWD期间A1R拮抗作用引起的毒性是否与增强的Ca2+涌入相关。将使用荧光显微镜可视化切片，并将使用光密度仪和共聚焦成像对数据进行定量。相关分析将确定细胞活力（PI，Neun，Ca2 - * - 橙色）的度量是否相关。这些技术允许全面表征ETOH暴露引起的腺苷能和NMDA受体系统的变化，以及在EWD期间假设适应的功能后果。大量证据表明，对于EtOH暴露和/或EWD，人类和啮齿动物的女性大脑都对神经毒性更敏感。当前的研究将有助于我们对eTOH诱导的神经元适应性以及大脑结构和/或功能的潜在性别差异的了解，并可能有助于使新型药物疗法的出现以改善EtOH和/或EWD相关的毒性。