CaMKIIα-Positive Interneurons Identified via a microRNA-Based Viral Gene Targeting Strategy
CaMKIIα-Positive Interneurons Identified via a microRNA-Based Viral Gene Targeting Strategy
复制标题
DOI：
10.1523/jneurosci.2570-19.2020
复制
发表时间：
2020-12-09
影响因子：
5.3
通讯作者：
Han, Xue
中科院分区：
文献类型：
--
作者：
Keaveney, Marianna K.;Rahsepar, Bahar;Han, Xue
单细胞分析揭示脑细胞基因表达谱的多样性日益增加。然而，传统的基于启动子的病毒基因表达技术无法捕获单细胞中不断增长的多样性。我们展示了一种新的病毒基因表达策略，使用 miRNA 引导的神经元标签 (mAGNET) 来靶向具有特定 miRNA 表达的细胞。我们设计了包含 CaMKII α 启动子和 microRNA-128 (miR-128) 结合位点的 mAGNET 病毒载体，并在雄性和雌性小鼠中标记了天然低表达 miR-128 的 CaMKII α(+) 细胞（Lm128C 细胞）。尽管CaMKII α传统上被认为是兴奋性神经元标记物，但我们的单细胞测序结果表明，Lm128C细胞是小清蛋白或生长抑素亚型的CaMKH α(+)抑制性神经元。对脑切片中 Lm128C 细胞生理特性的进一步评估表明，Lm128C 细胞表现出升高的膜兴奋性，其生物物理特性与快速放电的中间神经元非常相似，这与 miR-128 在调节控制膜兴奋性的基因网络中的先前转录组学发现一致。为了进一步证明这种新病毒表达策略的实用性，我们在运动皮层浅层的 Lm128C 细胞中表达 GCaMP6f，并在小鼠运动过程中进行体内钙成像。我们发现 Lm128C 细胞在运动过程中表现出比整体 CaMKII α(+) 细胞更高的钙事件率和更大的群体内相关性。总之，本文描述的基于 miRNA 的病毒基因靶向策略使我们能够标记稀疏的 CaMKII α(+) 中间神经元群体以进行功能研究，为研究大脑中基因表达与生理特性之间的关系提供新的能力。
Single-cell analysis is revealing increasing diversity in gene expression profiles among brain cells. Traditional promotor-based viral gene expression techniques, however, cannot capture the growing variety among single cells. We demonstrate a novel viral gene expression strategy to target cells with specific miRNA expression using miRNA-guided neuron tags (mAGNET). We designed mAGNET viral vectors containing a CaMKII alpha promoter and microRNA-128 (miR-128) binding sites, and labeled CaMKII alpha(+) cells with naturally low expression of miR-128 (Lm128C cells) in male and female mice. Although CaMKII alpha has traditionally been considered as an excitatory neuron marker, our single-cell sequencing results reveal that Lm128C cells are CaMKH alpha(+) inhibitory neurons of parvalbumin or somatostatin subtypes. Further evaluation of the physiological properties of Lm128C cell in brain slices showed that Lm128C cells exhibit elevated membrane excitability, with biophysical properties closely resembling those of fast-spiking interneurons, consistent with previous transcriptomic findings of miR-128 in regulating gene networks that govern membrane excitability. To further demonstrate the utility of this new viral expression strategy, we expressed GCaMP6f in Lm128C cells in the superficial layers of the motor cortex and performed in vivo calcium imaging in mice during locomotion. We found that Lm128C cells exhibit elevated calcium event rates and greater intrapopulation correlation than the overall CaMKII alpha(+) cells during movement. In summary, the miRNA-based viral gene targeting strategy described here allows us to label a sparse population of CaMKII alpha(+) interneurons for functional studies, providing new capabilities to investigate the relationship between gene expression and physiological properties in the brain.