Transcriptomes of accumbens nuclei from mice submitted to a short-term cocaine-dependent conditioned place preference

Molecular basis of transition to addiction in vulnerable individuals is largely unknown. We hypothesized that human susceptibility genes can be identified on the basis of conserved molecular mechanisms in rodent brains. We used a short-term cocaine-dependent conditioned place preference (CPP) to identify genetic hallmarks of early steps of reward memory in basal ganglia including accumbens nucleus (NAc), globus pallidus (GP) and subthalamic nucleus (STN). Using genome-wide microarray analysis and CPP as a quantitative trait, we found that synaptic plasticity-related genes are deregulated in these three structures. A significant enrichment in bona fide transcripts involved in dendritic spine local translation was evidenced. mGluR5 is transcriptionally deregulated in Acc and GP of cocaine-treated animals. Grin3a that encodes a NMDA receptor subunit involved in Ca++ permeability is deregulated in NAc. Furthermore, Orexin/Hcrt transcript level is decreased in STN, a region known to be involved in discriminating addictive drugs and natural rewards. We also found that mGluR5 and Grin3a expression deregulation is sufficient to induce changes in synaptic plasticity-related genes. Altogether, these results suggest that a combined deregulation of mGluR5 and Grin3A pathway in NAc, mGluR5 in GP and orexin system in STN may generate an incentive memory contrasted between addictive drugs and natural rewards. Such pathways may include clusters of genes that are potential susceptibility genes for transition to addiction. Agilent Whole Mouse Genome oligomicroarrays (GEO accession no. GPL2872, Agilent Technologies, Palo Alto, CA) were used. They contain 60-mer DNA probes synthesized in situ in a 44k format. Of 44,290 spots, 2756 are controls. The remaining 41,534 spots represent 33,661 unique transcripts which correspond to 20,202 unique human genes. Five independent (four accumbens nuclei from mice treated with cocaine compared to four accumbens nuclei from mice treated with saline solution) measurements were carried out for each group of biological conditions using exchanged dye-labeled RNA targets (i.e., Cy3 and Cy5 dyeswapping experiments). Each hybridization was numerized hybridization by a GenePix 4000B Microarray Scanner and an Agilent G6525 Microarray Scanner.

易感个体向成瘾转化的分子基础目前尚未完全阐明。本研究基于啮齿类动物脑中保守的分子机制，提出假说：可通过该机制识别人类成瘾易感性基因。本研究采用短期可卡因依赖型条件性位置偏爱（Conditioned Place Preference, CPP）模型，对基底神经节（包括伏隔核（accumbens nucleus, NAc）、苍白球（globus pallidus, GP）与丘脑底核（subthalamic nucleus, STN））中奖赏记忆早期阶段的遗传特征进行筛选。以全基因组微阵列分析为技术手段、CPP作为数量性状，我们发现上述三个脑区中与突触可塑性（synaptic plasticity）相关的基因均存在表达失调现象。研究证实，参与树突棘局部翻译（dendritic spine local translation）的功能性转录本存在显著富集。代谢型谷氨酸受体5（metabotropic glutamate receptor 5, mGluR5）的转录水平在可卡因处理小鼠的NAc与GP中出现失调。编码参与钙离子通透的NMDA（N-methyl-D-aspartate）受体亚基的Grin3a基因在NAc中存在表达失调。此外，食欲素/下丘脑泌素（Orexin/Hcrt）的转录本水平在STN中出现下调，而STN是已知参与区分成瘾性药物与自然奖赏的脑区。本研究同时证实，mGluR5与Grin3a的表达失调足以引发突触可塑性相关基因的表达变化。综上，本研究结果提示：NAc中mGluR5与Grin3a通路、GP中mGluR5以及STN中食欲素系统的协同失调，可能诱发成瘾性药物与自然奖赏之间的奖赏动机记忆差异。上述通路所涉及的基因簇，或可成为成瘾转化过程中的潜在易感基因。本研究采用安捷伦全小鼠基因组寡核苷酸微阵列（Agilent Whole Mouse Genome oligomicroarrays，GEO登录号：GPL2872，安捷伦科技公司，帕洛阿尔托，加利福尼亚州）。该微阵列采用44k格式，包含原位合成的60聚体DNA探针。在总计44290个探针点中，2756个为对照探针，剩余41534个探针点对应33661个独特转录本，涵盖20202个人类独特基因。针对每组生物学条件，本研究采用染料标记的RNA靶标进行交换（即Cy3与Cy5染料交换实验），开展5次独立重复检测：可卡因处理组小鼠的4个NAc样本与生理盐水处理组小鼠的4个NAc样本进行对照比较。每次杂交的信号均通过GenePix 4000B微阵列扫描仪与安捷伦G6525微阵列扫描仪进行数字化定量分析。