Department of Neuroscience, Baylor College of Medicine, BCM295, One Baylor Plaza, Houston, TX 77030, USA.
Eur J Neurosci. 2013 Apr;37(8):1203-20. doi: 10.1111/ejn.12126. Epub 2013 Jan 24.
The neonatal intraventricular injection of adeno-associated virus has been shown to transduce neurons widely throughout the brain, but its full potential for experimental neuroscience has not been adequately explored. We report a detailed analysis of the method's versatility with an emphasis on experimental applications where tools for genetic manipulation are currently lacking. Viral injection into the neonatal mouse brain is fast, easy, and accesses regions of the brain including the cerebellum and brainstem that have been difficult to target with other techniques such as electroporation. We show that viral transduction produces an inherently mosaic expression pattern that can be exploited by varying the titer to transduce isolated neurons or densely-packed populations. We demonstrate that the expression of virally-encoded proteins is active much sooner than previously believed, allowing genetic perturbation during critical periods of neuronal plasticity, but is also long-lasting and stable, allowing chronic studies of aging. We harness these features to visualise and manipulate neurons in the hindbrain that have been recalcitrant to approaches commonly applied in the cortex. We show that viral labeling aids the analysis of postnatal dendritic maturation in cerebellar Purkinje neurons by allowing individual cells to be readily distinguished, and then demonstrate that the same sparse labeling allows live in vivo imaging of mature Purkinje neurons at a resolution sufficient for complete analytical reconstruction. Given the rising availability of viral constructs, packaging services, and genetically modified animals, these techniques should facilitate a wide range of experiments into brain development, function, and degeneration.
腺相关病毒（adeno-associated virus）在新生期向脑室内注射，已被证明可广泛转导大脑中的神经元，但它在实验神经科学中的全部潜力尚未得到充分探索。我们报告了该方法的多功能性的详细分析，重点是在目前缺乏遗传操作工具的实验应用中。病毒注射到新生小鼠脑内快速、简单，并可访问包括小脑和脑干在内的脑区，这些脑区以前很难通过电穿孔等其他技术进行靶向。我们表明，病毒转导产生了固有马赛克表达模式，可以通过改变滴度来转导孤立的神经元或密集的神经元群体来加以利用。我们证明，病毒编码蛋白的表达比以前认为的更早开始活跃，从而允许在神经元可塑性的关键时期进行遗传干扰，但也具有长期稳定性，允许进行衰老的慢性研究。我们利用这些特征来可视化和操纵小脑后颅神经核中的神经元，这些神经元对通常在皮层中应用的方法具有顽固性。我们表明，病毒标记通过允许单个细胞易于区分，有助于分析小脑浦肯野神经元出生后的树突成熟，然后证明相同的稀疏标记允许对成熟的浦肯野神经元进行活体体内成像，其分辨率足以进行完整的分析重建。鉴于越来越多的病毒构建体、包装服务和基因修饰动物的出现，这些技术应该有助于广泛开展有关脑发育、功能和退化的实验。
