Cocaine Regulation of miRNAs in Rats with Differing Vulnerability to Drug Abuse

描述（由申请人提供）：微小RNA（miRNA），小（~ 22 nt非编码RNA），通过降低靶mRNA水平和/或抑制蛋白质翻译，作为基因表达的序列特异性转录后调节因子发挥作用。Schratt等人（2006）已经显示了miRNA功能、神经元棘发育和至少一种可能在神经可塑性中起作用的生长因子的作用之间的联系。然而，关于成年人大脑中的miRNA表达模式，或者miRNA表达的变化是否发生在急性或重复使用药物之后，或者任何miRNA是否可能是任何已知的信使RNA（或蛋白质）变化的基础，这些变化已知在药物使用（或滥用）之后发生。我们假设，在药物滥用的不同阶段，miRNA表达的改变是不同的，药物暴露将导致不同的miRNA表达模式（解剖学和/或时间）在两个不同的啮齿动物群体中，（高应答者（HR）对低应答者（LR）），因为这两组啮齿动物具有独特的基因表达模式，经过各种类型的挑战，并显示出药物滥用倾向的个体差异。在目标1中，我们将使用激光捕获显微镜来分离已知是可卡因的直接靶点和/或在大规模miRNA分析之前经历药物诱导的突触可塑性变化的特定脑区域。我们将在基础条件下使用来自HR和LR啮齿动物的组织;存在基因表达差异的条件。将通过miRNA qPCR验证miRNA表达差异，并通过miRNA原位杂交（ISH）方法分析表达的空间模式。在目标2中，我们将在HR和LR啮齿动物中使用可卡因致敏范例（其中药物暴露可以保持恒定）来检查可卡因在两个时间点（可卡因后立即和禁欲一段时间后）对miRNA的调节，如目标1中所分离和监测的。在目标3中，我们将在HR和LR啮齿动物中使用可卡因自我给药程序，在两个时间点（急性和戒断）收集组织以检测miRNA调控。在目标4中，我们将使用西洛米来阻断基础或可卡因调节的HR/LR miRNA差异，以确定miRNA阻断是否改变药物诱导的行为。总之，这些发现将揭示miRNAs是否有助于寻求滥用药物的初始倾向，或者在诱导和/或维持可卡因成瘾中发挥重要作用。 公共卫生相关性：我们的项目评估了被称为microRNAs（miRNAs）的小但新颖的遗传元件，这些元件被认为对细胞产生的蛋白质的类型或数量具有广泛的影响。在这项提议中，我们计划研究在两种不同的啮齿动物品系中是否存在miRNA的变化，这两种啮齿动物在基础条件下寻求药物的倾向不同。此外，我们计划在每种啮齿动物中研究不同的miRNA水平在多个大脑区域中的变化，作为可卡因使用的函数，无论是急性还是在药物治疗停止后很长时间。这些发现将揭示miRNAs是否有助于寻求滥用药物的初始倾向，或者在诱导和/或维持可卡因成瘾中发挥重要作用。

DESCRIPTION (provided by applicant): MicroRNAs (miRNAs), small (~22nt non-coding RNAs) function as sequence-specific post-transcriptional regulators of gene expression, by reducing target mRNA levels and/or inhibiting protein translation. Schratt et.al. (2006) have shown a link between miRNA function, neuronal spine development and the action of at least one growth factor that may play a role in neuroplasticity. Yet, little is known about miRNA expression patterns in the adult brain, or whether changes in miRNA expression occur following either acute or repeated drug use or whether any miRNA might underlie any known messenger RNA (or protein) changes known to occur following drugs use (or abuse). We hypothesize that miRNA expression alterations will be distinct across various stages of substance abuse and that drug exposure will result in distinctive miRNA expression patterns (anatomical and/or temporal) in two distinct groups of rodents selectively-bred based upon the novelty seeking trait (High Responders (HR) vs Low Responders (LR)), as these two groups of rodents have unique patterns of gene expression basally, after various types of challenges, and display individual differences in the propensity for substance abuse. In Aim 1, we will use laser capture microscopy to isolate specific brain regions known to be direct targets of cocaine and/or undergo drug-induced changes in synaptic plasticity prior to broad-scale miRNA profiling. We will use tissues derived from both HR and LR rodents under basal conditions; conditions where gene expression differences exist. miRNA expression differences will be validated by miRNA qPCR and spatial patterns of expression analyzed by miRNA in situ hybridization (ISH) methods. In aim two, we will use a cocaine sensitization paradigm (where drug exposure can be held constant) in both HR and LR rodents to examine miRNA regulation by cocaine at two time points (immediately following cocaine and following a period of abstinence) isolated and monitored as in aim 1. In aim 3, we will use cocaine self-administration procedures in HR and LR rodents, collect tissues at two time points (acute and abstinence) to examine miRNA regulation. In Aim 4, we will use antagomirs to block basal or cocaine regulated HR/LR miRNA differences to determine if miRNA blockade alters drug-induced behaviors. Together, these findings will shed light on whether miRNAs contribute to the initial propensity to seek drugs of abuse or play an important role in the induction and/or maintenance of cocaine addiction. PUBLIC HEALTH RELEVANCE: Our project evaluates small but novel genetic elements called microRNAs (miRNAs) that are thought to have broad influences on the types or amounts of proteins cells produce. In this proposal, we plan to examine whether there are miRNA changes in two different rodent lines that vary in their propensity to seek drugs under basal conditions. Further, we plan to examine in each rodent line how different miRNA levels vary in multiple brain regions as a function of cocaine use both acutely as well as long after the drug treatment has ceased. These findings will shed light on whether miRNAs contribute to the initial propensity to seek drugs of abuse or play an important role in the induction and/or maintenance of cocaine addiction.