RNAi-medicated inhibition of BDNF expression and alcohol-drinking behavior

RNAi 抑制 BDNF 表达和饮酒行为

• 批准号: 7177105
• 负责人: QINGSHAN YAN
• 金额: $ 7.75万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-01-15 至 2008-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7177105
• 关键词: Address; Aftercare; Alcohol consumption; Alcoholism; Alcohols; Animal Model; Animals; Area; Brain; Brain region; Brain-Derived Neurotrophic Factor; Data; Dependency; Development; Dopamine; Drug Addiction; Enzyme-Linked Immunosorbent Assay; Financial compensation; Gene Expression; Gene Silencing; Genes; Genetic; Genetically Modified Animals; Goals; Growth Factor; Hippocampus (Brain); Human; Hyperphagia; Immunohistochemistry; Impairment; In Vitro; Indiana; Infusion procedures; Lentivirus Vector; Link; Masks; Mediating; Messenger RNA; Methods; Mus; Neuraxis; Neuromodulator; Neurons; Neuropeptides; Neurotransmitters; Nucleus Accumbens; Numbers; Pathway interactions; Peptides; Phenotype; Play; Prosencephalon; Proteins; RNA Interference; Rattus; Regulation; Reporting; Research Personnel; Rewards; Role; Seizures; Serotonin; Subfamily lentivirinae; System; Tissues; Tryptophan 5-monooxygenase; Tyrosine 3-Monooxygenase; Universities; Validation; Ventral Tegmental Area; Wistar Rats; alcohol effect; alcohol reward; dopaminergic neuron; drinking behavior; fetal; gene function; in vivo; mesolimbic system; neuronal survival; null mutation; preference; progenitor; research study; small hairpin RNA; tool; vector

DESCRIPTION (provided by applicant): The goal of the study is to link brain-derived neurotrophic factor (BDNF) to alcohol-drinking behavior. It is hypothesized that decreased BDNF in the mesolimbic dopamine (DA) pathway, a brain circuit that has been implicated in alcohol's rewarding effects, is associated with high alcohol intakes. This hypothesis is prompted by our finding showing innate deficiencies of BDNF in the nucleus accumbens (NACC) of alcohol-preferring (P) rats compared with alcohol-nonpreferring (NP) rats. Previous studies performed in heterozygous BDNF () mice have shown the relationship between BDNF and alcohol consumption, but they cannot identify brain area(s) in which BDNF may play a role in regulating alcohol intakes. In addition, the approach of genetically modifying animals for target validation is often limited by developmental adaptation and genetic compensation that may mask the establishment of a clear phenotype. Recently, RNA interference (RNAi) has become a valuable tool for investigating gene function. The applicant has generated a lentiviral vector that is capable of delivering and expressing short hairpin RNA that targets BDNF mRNA. This vector has been proved to be effective in silencing the BDNF gene both in vitro and in vivo in our preliminary study. To determine whether RNAi-mediated inhibitions of BDNF expression in the VTA and/or NACC increase alcohol intakes, this lentivirus and several control substances will be bilaterally infused into the VTA or the NACC of groups of the Wistar rat, the progenitor from which the P/NP rats were originally derived. Then, the effects of the infusion on alcohol consumptions and non-alcohol tastant preference are determined by a two-bottle free-choice method and compared among different treatment groups. BDNF expression at the protein level in each brain area after the treatments is also assessed via the enzyme-linked immunosorbent assay and immunohistochemistry to see whether reductions of BDNF expression are associated with increased alcohol intakes. This study would provide more direct evidence for the role of BDNF in alcohol intakes and specifically addresses whether reductions of BDNF in the mesolimbic DA pathway are associated with increased alcohol consumptions. Since there are no reports regarding silencing the BDNF gene in vivo to date, the establishment of the approach to inhibit BDNF expression, particularly in the mesolimbic DA system of intact animals, would benefit researchers in drug addiction including alcoholism.

描述（由申请人提供）：本研究的目的是将脑源性神经营养因子（BDNF）与饮酒行为联系起来。据推测，中脑边缘多巴胺（DA）通路中BDNF的减少与高酒精摄入量有关，多巴胺通路是一种与酒精奖励效应有关的脑回路。这一假说是由我们的发现提示，在神经核BDNF（NACC）的酒精偏好（P）大鼠与酒精非偏好（NP）大鼠相比，先天性缺陷。以前在杂合子BDNF（）小鼠中进行的研究已经显示了BDNF和酒精摄入之间的关系，但他们无法确定BDNF可能在调节酒精摄入方面发挥作用的脑区。此外，用于靶标验证的转基因动物的方法通常受到发育适应和遗传补偿的限制，这可能会掩盖明确表型的建立。近年来，RNA干扰技术已成为研究基因功能的重要工具。申请人已经产生了能够递送和表达靶向BDNF mRNA的短发夹RNA的慢病毒载体。我们的初步研究表明，该载体在体内外均能有效地沉默BDNF基因。为了确定RNAi介导的BDNF在VTA和/或NACC中表达的抑制是否增加酒精摄入，将该慢病毒和几种对照物质双侧输注到Wistar大鼠组的VTA或NACC中，Wistar大鼠是P/NP大鼠最初衍生的祖先。然后，通过两瓶自由选择法确定输液对酒精消耗和非酒精促味剂偏好的影响，并在不同治疗组之间进行比较。还通过酶联免疫吸附测定和免疫组织化学评估治疗后每个脑区中蛋白质水平的BDNF表达，以观察BDNF表达的减少是否与酒精摄入量增加相关。这项研究将为BDNF在酒精摄入中的作用提供更直接的证据，并具体说明中脑边缘DA通路中BDNF的减少是否与酒精摄入量增加有关。由于迄今为止还没有关于在体内沉默BDNF基因的报道，因此建立抑制BDNF表达的方法，特别是在完整动物的中脑边缘DA系统中，将使包括酒精中毒在内的药物成瘾的研究人员受益。