Sex-specific transcriptomic profiles of nicotine sensitization in rats inform the genetic basis of human smoking behavior. Sex-specific transcriptomic profiles of nicotine sensitization in rats inform the genetic basis of human smoking behavior

Tobacco cigarette smoking, with nicotine (NIC) as the addictive component, is a large risk factor for human mortality. In animals, repeated NIC exposure leads to sensitization (SST) and enhances self-administration (SA) of NIC. However, the molecular basis of SST and SA and their genetic relevance to smoking behavior are poorly understood. Using F1 progeny of inbred Envigo rats (F344/BN), we carried out a transcriptional profiling of NIC SST and SA in ventral tegmental area (VTA), nucleus accumbens core (Nac) and shell (Nash). We observed male-specific NIC SST and a parental effect of NIC with SA only present in paternal F344 crosses. Gene differential expression (DE) analysis revealed sex and brain region-specific transcriptomic signatures of SST and SA, with genes downregulated in male VTA associated with both SST and SA, while genes upregulated in male Nac were associated with SA. DE genes associated with SST and SA are enriched for those related to synaptic processes, myelin sheath, and tobacco use disorder or chemdependency. Interestingly, we found that for SST the downregulated genes in the male VTA tended to be upregulated in female VTA, and the overlapping genes were strongly enriched for smoking genome-wide association study (GWAS) risk variants, which may thus explain male-specific SST. To gain mechanistic insight on the observed parental effect of SA, we analyzed the allelic imbalance of expression (AIE) in reciprocally crossed F1 rats that exhibited differential tendency of SA and found widespread region-specific AIE. For the SA-inclined rats, the genes showing AIE bias towards paternal F344 alleles in Nac were strongly enriched for genes upregulated in Nac DE—the gene set most relevant to NIC SA—and also for GWAS risk variants of smoking initiation. These findings show a prominent role for sex and region-specific gene expression and imprinting in NIC SST and SA, and suggest a mechanistic link between genes underlying these processes and human NIC addiction. This study provides a resource for understanding the biology underlying the genetic findings on human smoking phenotypes. Overall design: F1 progeny were generated by crossing two inbred Envigo strains (Fischer-344 [F344] and Brown Norway [BN]). Initial cross (F1i) was performed by crossing male F344 and female BN. Reciprocal cross (F1r) was performed by crossing female F344 and male BN. The proven breeding pairs (F344/NHsd and BN/RijHsd) were purchased from Envigo. The breeding was carried out at the University of Chicago animal facility. For sensitization, F1 males (n=5-6/group) and females (n=7/group) were randomly assigned the to three groups and tested in early adulthood (~250 g). The first group received 0.1 mg/kg of NIC (base), the second group received 0.4 mg/kg of NIC (base) and the third group received SAL (1.0 ml/kg). F1 rats were daily administered either NIC or SAL for 4 days. On days 1 and 4, rats were placed in an open field immediately after the injection and their locomotion measured for 2 hours. Rats were then left undisturbed in their home cages for 2 weeks. On day 19, all F1 rats were administered one injection of NIC (0.4 mg/kg, IP, base), immediately placed in the open fields and their locomotion measured for two hours. For the SST transcriptomic analysis F1 males (n=16) and females (n=16) were divided in 2 groups. In the first group (n=8) rats were exposed to 0.4 mg/kg of NIC (base) every day for 4 days. In the second group (n=8) rats were exposed to SAL (1.0 ml/kg) every day for 4 days. Brain tissues (~20mg) from Nac, Nash and VTA regions were harvested on day 17.

以尼古丁（nicotine, NIC）为成瘾性成分的烟草吸烟行为，是人类死亡的重大风险因素。在动物模型中，反复暴露于尼古丁可引发敏化（sensitization, SST）并增强尼古丁自身给药（self-administration, SA）行为。然而，敏化与自身给药的分子基础，及其与吸烟行为的遗传关联仍知之甚少。本研究以近交系Envigo大鼠的F1后代（F344/BN）为研究对象，对腹侧被盖区（ventral tegmental area, VTA）、伏隔核核心区（nucleus accumbens core, Nac）及伏隔核壳区（nucleus accumbens shell, Nash）内尼古丁诱导的敏化与自身给药行为开展转录组分析。研究观察到雄性特异性的尼古丁敏化现象，且仅在父本为F344的杂交子代中存在尼古丁自身给药的亲本效应。差异表达（differential expression, DE）分析显示，敏化与自身给药行为存在性别与脑区特异性的转录组特征：雄性VTA中下调的基因同时与敏化和自身给药相关，而雄性Nac中上调的基因仅与自身给药相关。与敏化和自身给药相关的差异表达基因显著富集于突触过程、髓鞘结构、烟草使用障碍或药物依赖相关的基因集。有趣的是，本研究发现雄性VTA中下调的基因往往在雌性VTA中呈现上调趋势，且二者的重叠基因显著富集吸烟相关全基因组关联研究（genome-wide association study, GWAS）风险变异，这或可解释雄性特异性敏化现象。为探究上述自身给药亲本效应的潜在机制，本研究对具有不同自身给药倾向的互交F1大鼠开展了表达等位基因失衡（allelic imbalance of expression, AIE）分析，发现广泛存在的脑区特异性表达等位基因失衡现象。在具有自身给药倾向的大鼠中，Nac中偏向父本F344等位基因的表达等位基因失衡基因，显著富集于Nac差异表达分析中上调的基因（这是与尼古丁自身给药最相关的基因集），同时也富集于吸烟启动相关的GWAS风险变异。上述研究结果表明，性别与脑区特异性的基因表达及基因组印记在尼古丁敏化与自身给药行为中发挥重要作用，并暗示这些过程相关的基因与人类尼古丁成瘾存在机制关联。本研究为解析人类吸烟表型遗传发现背后的生物学机制提供了重要资源。实验整体设计：通过杂交两种近交系Envigo大鼠（Fischer-344 [F344]与Brown Norway [BN]）获得F1子代。正向杂交组（F1i）由雄性F344与雌性BN杂交得到，反向杂交组（F1r）由雌性F344与雄性BN杂交得到。经繁育验证的种鼠（F344/NHsd与BN/RijHsd）购自Envigo，饲养于芝加哥大学动物实验设施。敏化行为实验中，将F1雄性大鼠（每组n=5~6）与雌性大鼠（每组n=7）随机分为三组，于成年早期（体重约250g）开展测试：第一组注射0.1mg/kg尼古丁游离碱，第二组注射0.4mg/kg尼古丁游离碱，第三组注射生理盐水（SAL，1.0ml/kg）。每日对F1大鼠注射尼古丁或生理盐水，持续4天。在第1天与第4天，大鼠于注射后立即被放入开放场装置，记录2小时的活动度。随后将大鼠放回饲养笼，静置2周。第19天时，所有F1大鼠均注射0.4mg/kg尼古丁（腹腔注射，游离碱），立即放入开放场装置并记录2小时的活动度。敏化转录组分析中，将F1雄性大鼠（n=16）与雌性大鼠（n=16）分为两组：第一组（n=8）每日注射0.4mg/kg尼古丁游离碱，持续4天；第二组（n=8）每日注射生理盐水（1.0ml/kg），持续4天。于第17天采集Nac、Nash与VTA脑区的脑组织（约20mg）。