Astrocytes play a critical role in brain function, but their contribution during ethanol (EtOH) consumption remains largely understudied. In light of recent findings on the heterogeneity of astrocyte physiology and gene expression, an approach with the ability to identify subtypes and capture this heterogeneity is necessary. Here, we combined measurements of calcium signaling and gene expression to define EtOH-induced astrocyte subtypes. In the absence of a demonstrated EtOH receptor, EtOH is believed to have effects on the function of many receptors and downstream biological cascades that underlie calcium responsiveness. This mechanism of EtOH-induced calcium signaling is unknown and this study provides the first step in understanding the characteristics of cells displaying these observed responses. To characterize underlying astrocyte subtypes, we assessed the correlation between calcium signaling and astrocyte gene expression signature in response to EtOH. We found that various EtOH doses increased intracellular calcium levels in a subset of astrocytes, distinguishing three cellular response types and one nonresponsive subtype as categorized by response waveform properties. Furthermore, single-cell RNA-seq analysis of astrocytes from the different response types identified type-enriched discriminatory gene expression signatures. Combining single-cell calcium responses and gene expression analysis identified specific astrocyte subgroups among astrocyte populations defined by their response to EtOH. This result provides a basis for identifying the relationship between astrocyte susceptibility to EtOH and corresponding measurable markers of calcium signaling and gene expression, which will be useful to investigate potential subgroup-specific influences of astrocytes on the physiology and pathology of EtOH exposure in the brain.

星形胶质细胞在大脑功能中发挥着关键作用，但它们在乙醇 (EtOH) 消耗过程中的作用仍未得到充分研究。鉴于最近关于星形胶质细胞生理学和基因表达异质性的发现，有必要采用一种能够识别亚型并捕获这种异质性的方法。在这里，我们结合钙信号和基因表达的测量来定义 EtOH 诱导的星形胶质细胞亚型。在缺乏已证实的 EtOH 受体的情况下，据信 EtOH 对许多受体的功能和作为钙反应基础的下游生物级联反应有影响。这种 EtOH 诱导的钙信号传导机制尚不清楚，本研究为了解显示这些观察到的反应的细胞特征提供了第一步。为了表征潜在的星形胶质细胞亚型，我们评估了钙信号与星形胶质细胞基因表达特征之间的相关性，以响应 EtOH。我们发现各种 EtOH 剂量增加了星形胶质细胞子集中的细胞内钙水平，区分了三种细胞反应类型和一种按反应波形特性分类的无反应亚型。此外，来自不同反应类型的星形胶质细胞的单细胞 RNA-seq 分析确定了类型丰富的区分基因表达特征。结合单细胞钙反应和基因表达分析，确定了星形胶质细胞群中特定的星形胶质细胞亚群，这些亚群由它们对 EtOH 的反应定义。该结果为确定星形胶质细胞对 EtOH 的易感性与钙信号传导和基因表达的相应可测量标志物之间的关系提供了基础，