Transcriptomic changes in retinal ganglion cell types associated with the disruption of cholinergic retinal waves

In the early stages of retinal development, a form of correlated activity known as retinal waves causes periodic depolarizations of immature retinal ganglion cells (RGCs). Retinal waves are crucial for refining visual maps in the brain's retinofugal targets and for the development of retinal circuits underlying feature detection, such as direction selectivity. Yet, how waves alter gene expression in immature RGCs is poorly understood, particularly at the level of the many distinct types of RGCs that underlie the retina's ability to encode diverse visual features. We performed single-cell RNA sequencing on RGCs isolated at the end of the first postnatal week from wild-type (WT) mice and ß2KO mice, which lack the ß2 subunit of the nicotinic acetylcholine receptor, leading to the disruption of cholinergic retinal waves. Statistical comparisons of RGC transcriptomes between the two conditions reveal a weak impact of retinal waves on RGC diversity, indicating that retinal waves do not influence the molecular programs that instruct RGC differentiation and maturation. Although wave-dependent gene expression changes are modest in the global sense, we identified ~238 genes that are significantly altered in select subsets of RGC types. We focused on one gene, Kcnk9, which encodes the two-pore domain leak channel potassium channel TASK3. Kcnk9, which is highly enriched in aRGCs, was strongly downregulated in ß2KO. We validated this result using in situ hybridization and performed whole-cell recording to demonstrate a significant decrease in the leak conductance in ß2KO. Our dataset provides a useful resource for identifying potential targets of spontaneous activity-dependent regulation of neurodevelopment in the retina. Overall design: RGCs from wild-type and ß2KO mice at postnatal day 7 (P7) were isolated using CD90-conjugated magnetic beads and analyzed using scRNA-seq.

视网膜发育早期，一种被称为视网膜波（retinal waves）的相关性电活动会诱导未成熟视网膜神经节细胞（retinal ganglion cells，RGCs）发生周期性去极化。视网膜波对于重塑大脑视网膜投射靶点的视觉图谱，以及完善负责特征检测（如方向选择性）的视网膜环路发育至关重要。然而，视网膜波如何调控未成熟RGC的基因表达仍不甚明确，尤其是在构成视网膜多样化视觉特征编码能力的多种不同RGC亚型层面。我们对出生后第一周末期分离的野生型（wild-type，WT）小鼠和β2KO小鼠的RGC开展了单细胞RNA测序（single-cell RNA sequencing，scRNA-seq），其中β2KO小鼠缺乏烟碱型乙酰胆碱受体的β2亚基，这会导致胆碱能视网膜波的破坏。对两种条件下RGC转录组的统计学比较显示，视网膜波对RGC多样性的影响较弱，表明视网膜波并未影响指导RGC分化与成熟的分子程序。尽管依赖视网膜波的基因表达变化在全局层面较为温和，但我们在特定RGC亚型亚群中共鉴定出约238个发生显著改变的基因。我们重点聚焦基因Kcnk9，其编码双孔域漏电流钾通道TASK3。在aRGC中高度富集的Kcnk9在β2KO小鼠中被显著下调。我们通过原位杂交验证了这一结果，并通过全细胞膜片钳记录证实β2KO小鼠的漏电流电导显著降低。本数据集为鉴定视网膜中依赖自发活动调控神经发育的潜在靶点提供了宝贵资源。整体实验设计：于出生后第7天（P7）从野生型和β2KO小鼠中分离RGC，采用CD90偶联磁珠进行分选，并通过scRNA-seq进行分析。