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

描述（由申请人提供）：MicroRNAs (miRNAs)，小的（~22nt的非编码rna）作为基因表达的序列特异性转录后调节剂，通过降低靶mRNA水平和/或抑制蛋白质翻译。Schratt出版社。（2006）表明miRNA功能、神经元脊柱发育和至少一种可能在神经可塑性中发挥作用的生长因子之间存在联系。然而，对于成人大脑中的miRNA表达模式知之甚少，也不知道miRNA表达是否会在急性或反复用药后发生变化，也不知道是否有miRNA可能导致任何已知的信使RNA（或蛋白质）在药物使用（或滥用）后发生变化。我们假设miRNA表达改变在药物滥用的不同阶段是不同的，并且药物暴露会导致两组不同的啮齿动物中不同的miRNA表达模式（解剖和/或时间），这两组啮齿动物是根据寻求新奇的特征（高反应者（HR） vs低反应者（LR））选择性繁殖的，因为这两组啮齿动物在不同类型的挑战后基本上具有独特的基因表达模式。并显示出药物滥用倾向的个体差异。在Aim 1中，我们将使用激光捕获显微镜分离已知是可卡因直接靶点的特定大脑区域和/或在大规模miRNA分析之前经历药物诱导的突触可塑性变化。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。miRNA表达差异将通过miRNA qPCR验证，并通过miRNA原位杂交（ISH）方法分析表达的空间模式。在目标二中，我们将在HR和LR啮齿动物中使用可卡因致敏范式（药物暴露可以保持不变），在目标1中分离和监测的两个时间点（紧接可卡因和戒断一段时间后）检查可卡因对miRNA的调节。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）收集组织来检查miRNA调节。在Aim 4中，我们将使用拮抗剂来阻断基础或可卡因调节的HR/LR miRNA差异，以确定miRNA阻断是否会改变药物诱导的行为。总之，这些发现将阐明mirna是否有助于寻求药物滥用的初始倾向，或在诱导和/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.