Genetic Approaches To Characterizing Drug Responses And Vulnerabilities: Mice

人和动物对滥用药物的行为、生理和毒理学反应存在很大的个体差异。在对药物的行为反应中，许多个体差异表现出实质性的遗传因素。转基因动物为实现几个相互关联的目标提供了手段：1)确定特定基因引入或中断的生化和行为后果；2)确定在人类研究中发现的过高或低表达候选基因的后果；3)阐明产生细胞类型特异性表达和跨突触基因调控的基因元件；4)研究不同基因组位置上变异之间的相互作用的影响；5)阐明体内单倍型特定的表达差异水平，6)使我们能够监测突触连接性及其通过给药和细胞黏附和其他重要脑分子中单倍型的变化而调节的情况，以及7)允许“去风险”或评估针对其表达被改变的基因的潜在药物疗法可能伴随的风险水平。在这一年里，奖励、强化和学习的兴趣机制导致了对这些系统的持续关注，因为我们已经为识别大脑中的连接差异奠定了许多基础。 在对候选基因敲除的持续研究中，由于它们编码药物靶标和/或在感兴趣的电路中表达，我们报道了关于这些敲除对a)可卡因适应性反应的不同影响的新数据，b)社会缺陷(MU/OPRM1敲除)c)抗抑郁和止痛药(OPRM1敲除)d)物质诱导的多巴胺释放(MU/OPRM1敲除)e)脑电路(DAT/SLC6A3敲除)和f)疼痛反应(DAT/SLC6A4，SERT/SLC6A4和MU/OPRM1敲除)。 我们通过对四个基因表达改变的小鼠的研究，推进了细胞黏附基因改变的影响的研究，我们的人类基因组广泛关联数据为这些基因提供了最一致的复制。我们已经提交了数据，在人类中我们有最强证据的两个基因CDH13、PTPRD和CSMD1纯合或杂合缺失的小鼠，降低了对10毫克/公斤可卡因剂量的环境的偏好。这些结果显示出特异性，因为可卡因保留了运动刺激，而且这些基因敲除的小鼠可以在Morris水迷宫和其他比较行为中表现出正常(或超常)的水平。初步数据支持在CDH13基因敲除中灭绝和恢复的幅度也有所降低的可能性。因此，这些动物提供了最好的小鼠确认全基因组关联的结果，这是复杂的、多基因的人类基因座造成的。有趣的是，这些小鼠在5毫克/公斤的可卡因剂量下也表现出更大的回报。这些数据与人类实验室对可卡因反应的个体差异以及对初始酒精剂量的反应的个体差异的研究结果一致。 我们已经开始寻找这些行为变化的神经和神经化学关联。在对CDH13基因敲除小鼠的初步研究中，我们发现了大脑皮层多巴胺水平和前额叶皮质多巴胺能纤维密度的变化。如果得到证实，这些结果将为选择性行为结果提供基础，这些结果伴随着CDH13在小鼠和人类中的表达变化。

There are large individual differences among humans and animals in behavioral, physiological and toxicological responses to drugs of abuse. Many of these individual differences in behavioral responses to drugs display substantial genetic components. Transgenic animals provide means for approaching several interrelated goals: 1)Ascertainment of biochemical and behavioral consequences of the introduction of or disruption of specific genes; 2)Ascertainment of the consequences of over- or under-expressing candidate genes identified in human studies; 3) Elucidation of gene elements yielding cell-type specific expression and trans-synaptic gene regulation; 4) Studying influences of interactions between variants at different genomic loci; 5)Elucidating haplotype-specific levels of expression differences in vivo, 6) allowing us to monitor synaptic connectivities and their modulation by drug administration and alterations in haplotypes in cell adhesion and other important brain molecules and 7) allowing "derisking" or assessment of the levels of risk likely to accompany potential pharmacotherapeutics that target the gene whose expression is modified. Interest mechanisms of reward, reinforcement and learning have led to continuing focus on these systems during this year, as we have established much of the groundwork for identification of wiring differences in brain. In continuing studies of knockouts of candidate genes that are interesting because they encode drug targets and/or are expressed in circuits of interest, we have reported novel data concerning the differential influences of these knockouts on a) adaptive responses to cocaine b) stress engendered by social defect (mu/OPRM1 knockouts)c) antidepressants and analgesics (OPRM1 knockouts) d) substance-induced dopamine release (mu/OPRM1 knockouts) e) brain circuitry (DAT/SLC6A3 knockouts) and f) pain responses (DAT/SLC6A4, SERT/SLC6A4 and mu/OPRM1 knockouts). We have moved forward with studies of the influences of cell adhesion gene alterations by studies of mice with altered expression of the four genes for which our human genome wide association datasests provide the most consistent replication. We have submitted data that mice with homozygous or heterozygous deletion of the two genes for which we have the strongest evidence in humans, CDH13,PTPRD and CSMD1, reduce preference for environments paired with 10 mg/kg cocaine doses. These results display specificity, since cocaine retains locomotor stimulation and since these knockout mice can perform at normal (or supranormal) levels in Morris water maze and other comparison behaviors. Initial data supports the possibility that the magnitude of extinction and reinstatement are also reduced in CDH13 knockouts. These animals thus provide one of the best mouse confirmations of a genome wide association result from a complex, polygenic human locus. Interestingly, these mice also display greater reward from 5 mg/kg cocaine doses. These data are convergent with results from human laboratory studies of individual differences in cocaine responses, and individual differences in responses to initial alcohol doses. We have begun to seek neural and neurochemical correlates of these behavioral changes. In initial studies of CDH13 knockout mice, we have identified alterations in levels of cerebral cortical dopamine and in densities of prefrontal cortical dopaminergic fibers. If confirmed, these results would provide a basis for the selective behavioral results that accompany altered expression of CDH13 in mice and in humans.