The cholinergic system’s essential involvement in both preclinical and clinical aspects of cognition processes has been proposed and reviewed extensively elsewhere. Terry and Buccafusco [1] provide the latest review on this subject. According to this review, all currently approved FDA drugs for the treatment of Alzheimer’s disease are cholinesterase inhibitors, which exert their efficacy apparently through stimulation of both muscarinic acetylcholine receptors (mAChRs) and nicotinic acetylcholine receptors (nAChRs). Gene targeting (knockout) technologies allow us to replace the gene of interest with one that is inactive, altered, or irrelevant [2]. In the case of a completed deletion, a gene is knocked out , and a mutant organism with a deficit in the gene product is generated. The lack of suitable embryonic stem (ES) cell lines prevents the application of these technologies into rats [3]. However, mice and humans are both mammals, and both species contain a similar number of genes that show a high degree of similarity [4,5]. Therefore, in rodents, mouse is a better species for employing the knockout approach compared with rat. In theory, knockout (KO) mice contain a targeted gene that is deleted; therefore, no product of the mutated gene is synthesized in the null mutants [6]. These mutant mouse lines, which have inactivating mutations of the individual genes, can be studied in a battery of physiological, pharmacological, behavioral, biochemical, and neurochemical tests to confirm or invalidate a hypothesis on the effects of specific proteins in the function of the brain. Müller [7] provided an excellent review article regarding targeted mouse mutants from vector design to phenotype analysis. The cited article provides detailed technical information on how to generate KO mice, including selection markers and screening strategies, potential problems and pitfalls, as well as construct design. Moreover, Bolivar et al. [8] have summarized behavioral profiles in all available knockout mice. They also provide updated information on available KO mice through their Web site, which is identified in their article. Additional information about Internet resources regarding transgenic rodent production is provided by Wells and Carter [3]. Because gene-targeting techniques enable us to analyze diverse aspects of gene function in whole animals, measurable phenotypes relevant to human pathology could be obtained in a good mouse model of human disease. However, for most diseases in the central nervous system (CNS), the coexistence of malfunctions in multiple subtypes of the same receptor or in multiple neurotransmitters typically contribute to a complex phenotype such as cognition. For example, Buccafusco and Terry [9] demonstrate that multiple CNS targets are needed to elicit beneficial effects on memory and cognition. Therefore, generating multiple mouse mutants that mimic all facets of a multifactorial disease would help us to address individual subsets of symptoms of the disease. In short, knockout technologies provide a powerful tool to reveal and refine treatment strategies for human disorders by building bridges between genetics and the pathogenesis of disease. Behavioral phenotypes discovered in the mutant can be used to evaluate (screen) the efficacy of potential new pharmacological therapies. For example, by evaluating the results of specific behavioral tests — including learning and memory tests in mAChR, nAChR, and acetylcholinesterase (AChE) KO mice — our knowledge of cognitional impairment would be broadened. This, in turn, will improve our ability to identify potential new drug therapies for the treatment of cognitional aspects of such diseases as Alzheimer’s disease, attention deficit hyperactivity disorder (ADHD), and schizophrenia. This chapter focuses on: Available cholinergic receptor KO mouse models, including muscarinic acetylcholine receptor (mAChR) KO mice, nicotinic acetylcholine receptor (nAChR) KO mice, and acetylcholinesterase (AChE) KO mice. Cognitional-related data for mAChR KO mice. Limitations on KO mouse models and future directions.
胆碱能系统在认知过程的临床前和临床方面的重要作用已在其他地方被广泛提出和综述。Terry和Buccafusco [1]提供了关于该主题的最新综述。根据该综述，目前美国食品药品监督管理局（FDA）批准的所有用于治疗阿尔茨海默病的药物都是胆碱酯酶抑制剂，它们显然通过刺激毒蕈碱型乙酰胆碱受体（mAChRs）和烟碱型乙酰胆碱受体（nAChRs）发挥其疗效。基因靶向（敲除）技术使我们能够用一个无活性、改变或不相关的基因替换感兴趣的基因[2]。在完全缺失的情况下，一个基因被敲除，产生一种基因产物有缺陷的突变生物体。缺乏合适的胚胎干细胞（ES）系阻碍了这些技术在大鼠中的应用[3]。然而，小鼠和人类都是哺乳动物，这两个物种都含有数量相似且相似度很高的基因[4,5]。因此，在啮齿动物中，与大鼠相比，小鼠是采用敲除方法的更好物种。理论上，敲除（KO）小鼠含有一个被删除的靶向基因；因此，在无效突变体中不会合成突变基因的产物[6]。这些具有单个基因失活突变的突变小鼠品系，可以通过一系列生理、药理、行为、生化和神经化学测试进行研究，以证实或否定关于特定蛋白质在脑功能中作用的假设。Müller [7]提供了一篇关于从载体设计到表型分析的靶向小鼠突变体的优秀综述文章。引用的文章提供了关于如何生成KO小鼠的详细技术信息，包括选择标记和筛选策略、潜在问题和陷阱以及构建体设计。此外,Bolivar等人[8]总结了所有可用敲除小鼠的行为特征。他们还通过其网站提供了关于可用KO小鼠的最新信息，该网站在他们的文章中被提及。Wells和Carter [3]提供了关于转基因啮齿动物生产的互联网资源的更多信息。因为基因靶向技术使我们能够在整体动物中分析基因功能的各个方面，所以在良好的人类疾病小鼠模型中可以获得与人类病理学相关的可测量表型。然而，对于中枢神经系统（CNS）中的大多数疾病，同一受体的多个亚型或多种神经递质中的功能障碍共存通常会导致复杂的表型，如认知。例如，Buccafusco和Terry [9]证明需要多个CNS靶点才能对记忆和认知产生有益影响。因此，生成模拟多因素疾病所有方面的多个小鼠突变体将有助于我们解决该疾病症状的各个子集。简而言之，敲除技术通过在遗传学和疾病发病机制之间架起桥梁，为揭示和完善人类疾病的治疗策略提供了一个强大的工具。在突变体中发现的行为表型可用于评估（筛选）潜在新药疗法的疗效。例如，通过评估特定行为测试的结果——包括mAChR、nAChR和乙酰胆碱酯酶（AChE）敲除小鼠的学习和记忆测试——我们对认知障碍的认识将得到拓宽。这反过来将提高我们识别潜在新药疗法的能力，以治疗诸如阿尔茨海默病、注意力缺陷多动障碍（ADHD）和精神分裂症等疾病的认知方面。本章重点关注：可用的胆碱能受体敲除小鼠模型，包括毒蕈碱型乙酰胆碱受体（mAChR）敲除小鼠、烟碱型乙酰胆碱受体（nAChR）敲除小鼠和乙酰胆碱酯酶（AChE）敲除小鼠。mAChR敲除小鼠的认知相关数据。敲除小鼠模型的局限性和未来方向。
