Department of Neurophysiology, National Institute of Neuroscience, NCNP, 4-1-1 Ogawa-Higashi, Kodaira, Tokyo 187-8502, Japan.
Neuroscience. 2011 Oct 13;193:249-58. doi: 10.1016/j.neuroscience.2011.06.080. Epub 2011 Jul 18.
Viral vector-mediated gene transfer has become increasingly valuable for primate brain research, in particular for application of genetic methods (e.g. optogenetics) to study neuronal circuit functions. Neuronal cell tropisms and infection patterns are viable options for obtaining viral vector-mediated transgene delivery that is selective for particular neuronal pathways. For example, several types of viral vectors can infect axon terminals (retrograde infections), which enables targeted transgene delivery to neurons that directly project to a particular viral injection region. Although recent studies in rodents have demonstrated that adeno-associated virus serotype 8 (AAV8) and 9 (AAV9) efficiently transduce neurons, the tropisms and infection patterns remain poorly understood in primate brains. Here, we constructed recombinant AAV8 or AAV9, which expressed an enhanced green fluorescent protein (EGFP) gene driven by a ubiquitous promoter (AAV8-EGFP and AAV9-EGFP, respectively), and stereotaxically injected it into several brain regions in marmosets and macaque monkeys. Immunohistochemical analyses revealed almost exclusive colocalization of EGFP fluorescence via AAV9-mediated gene transfer with a neuron-specific marker, indicating endogenous neuronal tropism of AAV9, which was consistent with our previous results utilizing AAV8. Injections of either AAV8-EGFP or AAV9-EGFP into the marmoset striatum resulted in EGFP expression in local striatal neurons as a result of local infection, as well as expression in dopaminergic neurons of the substantia nigra via retrograde transport along nigrostriatal axonal projections. Retrograde infections were also observed in the frontal cortex and thalamus, which are known to have direct projections to the striatum. These local and retrograde gene transfers were further demonstrated in the geniculocortical pathway of the marmoset visual system. These findings indicate promising capabilities of AAV8 and AAV9 to deliver molecular tools into a range of primate neural systems in pathway-specific manners through their neuronal tropisms and infection patterns.
病毒载体介导的基因转移在灵长类动物脑研究中变得越来越有价值，特别是在应用遗传方法（如光遗传学）研究神经元回路功能方面。神经元细胞趋向性和感染模式是获得选择性特定神经元通路的病毒载体介导转基因传递的可行选择。例如，几种类型的病毒载体可以感染轴突末梢（逆行感染），从而能够将转基因递送到直接投射到特定病毒注射区域的神经元。尽管最近在啮齿动物中的研究表明，腺相关病毒血清型 8（AAV8）和 9（AAV9）有效地转导神经元，但在灵长类动物大脑中，其趋向性和感染模式仍知之甚少。在这里，我们构建了表达增强型绿色荧光蛋白（EGFP）基因的重组 AAV8 或 AAV9，该基因由组成型启动子驱动（分别为 AAV8-EGFP 和 AAV9-EGFP），并通过立体定向注射到狨猴和猕猴的几个脑区。免疫组织化学分析显示，通过 AAV9 介导的基因转移，EGFP 荧光几乎完全与神经元特异性标志物共定位，表明 AAV9 的内源性神经元趋向性与我们之前利用 AAV8 的结果一致。将 AAV8-EGFP 或 AAV9-EGFP 注射到狨猴纹状体中，导致局部感染导致局部纹状体神经元表达 EGFP，以及通过黑质纹状体轴突投射逆行运输导致黑质多巴胺神经元表达 EGFP。在前脑皮层和丘脑也观察到逆行感染，已知它们与纹状体有直接投射。这些局部和逆行基因转移在狨猴视觉系统的视放射通路中得到了进一步证明。这些发现表明 AAV8 和 AAV9 具有通过其神经元趋向性和感染模式以特定途径将分子工具递送到一系列灵长类动物神经系统的有前途的能力。
