Expression of immediate early genes (IEGs) is critical for memory formation and has been widely used to identify the neural substrate of memory traces, termed memory engram cells. Functions of IEGs have been known to be different depending on their types. However, there is limited knowledge about the extent to which different types of IEGs are selectively or concurrently involved in the formation of memory engram. To address this question, we investigated the combinative expression of c-Fos, Arc, and Npas4 proteins using immunohistochemistry following aversive and rewarding experiences across subregions in the prefrontal cortex (PFC), basolateral amygdala (BLA), hippocampal dentate gyrus (DG), and retrosplenial cortex (RSC). Using an automated cell detection algorithm, we found that expression patterns of c-Fos, Npas4, and Arc varied across different brain areas, with a higher increase of IEG expressing cells in the PFC and posterior BLA than in the DG. The combinative expression patterns, along with their learning-induced changes, also differed across brain areas; the co-expression of IEGs increased in the PFC and BLA following learning whereas the increase was less pronounced in the DG and RSC. Furthermore, we demonstrate that different area-to-area functional connectivity networks were extracted by different IEGs. These findings provide insights into how different IEGs and their combinations identify engram cells, which will contribute to a deeper understanding of the functional significance of IEG-tagged memory engram cells.

即早基因（IEGs）的表达对于记忆形成至关重要，并被广泛用于识别记忆痕迹的神经底物，称为记忆印记细胞。不同类型的IEGs的功能已经知道是不同的。然而，关于不同类型IEGs在记忆印记形成过程中是否选择性参与或同时参与的知识仍然有限。为了解决这个问题，我们使用免疫组化技术，在前额皮层（PFC）、基底外侧杏仁核（BLA）、海马齿状回（DG）和后楔前叶皮质（RSC）的不同亚区中研究了c-Fos、Arc和Npas4蛋白的联合表达情况。使用自动细胞检测算法，我们发现c-Fos、Npas4和Arc的表达模式在不同脑区间存在差异，在PFC和BLA的后部区域IEGs表达细胞的数量增加更为显著，而DG则不如这些区域明显。IEGs的共表达模式及其学习诱导的变化也在不同脑区间有所不同；在PFC和BLA中，随着学习的发生，IEGs的共表达增加了，而在DG和RSC中这种变化不那么明显。此外，我们证明了由不同的IEGs提取的不同区与区之间的功能连接网络存在差异。这些发现提供了关于不同类型IEGs及其组合如何识别记忆印记细胞的见解，这将有助于更深入地理解IEG标记的记忆印记细胞的功能意义。