The induction of immediate-early gene (IEG) expression in brain nuclei in response to an experience is necessary for the formation of long-term memories. Additionally, the rapid dynamics of IEG induction and decay motivates the common use of IEG expression as markers for identification of neuronal assemblies (“ensembles”) encoding recent experience. However, major gaps remain in understanding the rules governing the distribution of IEGs within neuronal assemblies. Thus, the extent of correlation between coexpressed IEGs, the cell specificity of IEG expression, and the spatial distribution of IEG expression have not been comprehensively studied. To address these gaps, we utilized quantitative multiplexed single-molecule fluorescence in situ hybridization (smFISH) and measured the expression of IEGs (Arc, Egr2, and Nr4a1) within spiny projection neurons (SPNs) in the dorsal striatum of mice following acute exposure to cocaine. Exploring the relevance of our observations to other brain structures and stimuli, we also analyzed data from a study of single-cell RNA sequencing of mouse cortical neurons. We found that while IEG expression is graded, the expression of multiple IEGs is tightly correlated at the level of individual neurons. Interestingly, we observed that region-specific rules govern the induction of IEGs in SPN subtypes within striatal subdomains. We further observed that IEG-expressing assemblies form spatially defined clusters within which the extent of IEG expression correlates with cluster size. Together, our results suggest the existence of IEG-expressing neuronal “superensembles,” which are associated in spatial clusters and characterized by coherent and robust expression of multiple IEGs.

响应于经验，在脑核中诱导立即早期基因（IEG）表达对于形成长期记忆是必要的。另外，IEG诱导和衰变的快速动力学激发了IEG表达作为识别编码最近经验的神经元组装（“集合”）的标志物的普遍用途。然而，在理解控制IEG在神经元组装体中分布的规则方面仍然存在重大差距。因此，尚未全面研究共表达的IEG之间的相关程度，IEG表达的细胞特异性和IEG表达的空间分布。为了解决这些差距，我们利用定量多路复用单分子荧光原位杂交（smFISH）并测量了IEGs（Arc，Egr2和Nr4a1）急性暴露于可卡因后，小鼠背侧纹状体的棘突投射神经元（SPN）内）。探索我们的观察结果与其他大脑结构和刺激的相关性，我们还分析了来自小鼠皮质神经元单细胞RNA测序研究的数据。我们发现，虽然IEG表达是分级的，但多个IEG的表达在单个神经元的水平上紧密相关。有趣的是，我们观察到特定区域的规则控制纹状体亚域内SPN亚型中IEG的诱导。我们进一步观察到，表达IEG的程序集形成空间定义的簇，其中IEG表达的程度与簇大小相关。总之，我们的结果表明存在表达IEG的神经元“超团”，