已经证明，中脑边缘（mesolimbic）多巴胺通路是药物成瘾的解剖学基础，但目前该通路形成的机制不清。本项目旨在揭示mesolimbic多巴胺通路形成的调控机制，探究药物成瘾的起源性分子。在预实验中，我们创新性的采用Ribotag和Cre-loxp重组技术免疫共沉淀了mesolimbic通路形成期多巴胺亚类神经元转录本。经lncRNA表达谱分析，发现差异lncRNAs作用axon通路。其中MRUC007VKO靶向semaphorin5A。并且MA上调二者表达，初步显示与药物成瘾相关。下一步，拟分析MRUC007VKO对semaphorin5A及轴突生长的调控作用；采用胚胎脑内基因干扰获得lncRNAs及靶基因控制mesolimbic通路发育形成的可靠证据；通过模型鼠证实lncRNAs及靶基因与药物成瘾的相关性和干预性，有望找出药物成瘾的特异性分子靶点。

Previous studies have already demonstarted that mesolimbic dopaminergic pathway is the anatomical basis for drug addiction. However, the mechanisms of the formation of mesolimbic projection are unclear. Our proposal will explore the molecular mechanisms of mesolimbic projection formation and the origin of drug addiction. It’s a great challenge for isolation the transcripts of specific subtye dopaminergic neurons in mesolimbic projection.In our preliminary study, using Ribotag methology and Cre-loxp recombination technique, we labeled these neurons with Adeno-Cre virus injection to make tdtomato fluorescent protein expression, and to make Rpl22 protein expression taged with hemagglutinin (HA). We then immunoprecipitaed the HA associated transcripts from mesolimbic dopaminergic neurons. Subsequently, long non-coding RNA (lncRNA) microarry was employed to analyze lncRNA and mRNA expression profiles (lncRNA microarry contains mRNA probes). Bioinformatics analysis showed that differential expression lncRNAs targeting axon guidance pathway in which lncRNA-MRUC007VKO targeting semaphorin5A. In particular, methamphetamine increased both MRUC007VKO and semaphorin5A expression levels, which was closely related with addiction. The following of this proposal will perform the first experiment to analyze the function of MRUC007VKO and semaphorin5A by gene over-expression and knock-down in cultured dopaminergic neurons, as well as to know how lncRNA regulates semaphorin 5A was determined. The second experiment will confirm the regulation of MRUC007VKO and semaphorin5A in mesolimbic projection with in utero electroporation experiment. The final experiment will determine the expressions of MRUC007VKO and semaphorin5A and to investigate the possible intervention to methamphetamine abuse. These studies will provide important new insights into the molecular mechanisms of mesolimbic projection and should facilitate the understanding of reward and the development of new therapeutic strategies for drug abuse.