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

描述(申请人提供)：这项研究的目标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)通路中脑源性神经营养因子的减少与高酒精摄入量有关。DA通路是一种大脑回路，与酒精的回报作用有关。这一假说的提出是因为我们的发现表明，与不饮酒(NP)大鼠相比，饮酒(P)大鼠伏隔核(NACC)中BDNF的先天缺陷。之前在杂合子BDNF()小鼠身上进行的研究表明了BDNF与饮酒之间的关系，但他们无法确定BDNF可能在调节酒精摄入量方面发挥作用的大脑区域(S)。此外，对动物进行基因改造以进行靶标验证的方法往往受到发育适应和遗传补偿的限制，这可能掩盖了明确表型的建立。近年来，RNA干扰(RNAi)已成为研究基因功能的重要工具。申请人已经产生了一种慢病毒载体，它能够传递和表达靶向BDNF mRNA的短发夹状RNA。在我们的初步研究中，该载体已被证明在体外和体内都能有效地沉默BDNF基因。为了确定RNAi介导的对VTA和/或NACC中BDNF表达的抑制是否增加了酒精摄入量，该慢病毒和几种对照物质将被双侧注入到Wistar大鼠的VTA或NACC中，Wistar大鼠是P/NP大鼠的原始来源。然后，通过两瓶自由选择的方法，比较不同治疗组之间的饮酒量和非酒精品尝偏好的影响。治疗后，还通过酶联免疫吸附试验和免疫组织化学方法评估BDNF在每个脑区蛋白水平的表达，以确定BDNF表达的减少是否与酒精摄入量的增加有关。这项研究将为BDNF在酒精摄取中的作用提供更直接的证据，并特别研究中脑边缘DA通路中BDNF的减少是否与酒精摄入量的增加有关。由于到目前为止还没有关于体内沉默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，将有利于包括酒精中毒在内的药物成瘾的研究人员。

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.