场景恐惧型学习记忆 (contextual fear conditioning, CFC) 是研究学习记忆的细胞和分子机制以及相关疾病的致病机理的重要行为学范式，具有鲁棒性高和时序性强等特征。恐惧记忆的形成依赖于神经细胞中分子水平上的精细调控，包括染色质等表观遗传学、基因的转录以及翻译等层面。大脑海马体是恐惧记忆形成的重要脑区，也是老年性痴呆症等神经系统疾病的重要罹患部位。既往对恐惧记忆形成过程的海马体基因动态变化特征已经被广泛研究，然而海马体细胞群体高度异质性，包括神经细胞、星形胶质细胞和小胶质细胞等细胞类型等，不同类型的细胞在恐惧记忆形成过程中基因转录和翻译水平的变化特征和这些变化在恐惧记忆中的贡献迄今未见报道。综合利用单细胞测序技术 (single-cell RNA-seq, scRNA-seq) 和细胞特异翻译组研究方法 (RiboTag，分离纯化组织中特定细胞类型中核糖体结合的mRNAs的方法)，本论文全面解析了小鼠海马体在恐惧记忆形成过程中单细胞和特异细胞水平转录组和相关mRNA进入核糖体翻译水平的时序性变化。我们的结果提示不同类型的海马神经元和胶质细胞，在恐惧记忆形成过程中有不同的基因转录和翻译水平的变化特征，神经元的特征变化远比胶质细胞复杂。小胶质细胞作为中枢神经系统中驻留的免疫类型的细胞，监测并维持着大脑中环境稳态，参与调控神经网络的功能。目前对于小胶质细胞是否参与调控恐惧记忆尚存争议，我们通过scRNA-seq和RiboTag方法深入分析小胶质细胞在恐惧记忆形成过程中的分子变化特征，并发现小胶质细胞中富集表达的B细胞受体 (BCR) 信号通路基因mRNAs，在恐惧记忆刺激后进入核糖体翻译水平显著上调。小胶质细胞特异性敲除Blnk (B cell linker，编码BCR信号通路衔接蛋白) 小鼠恐惧型学习记忆能力受损，而对小鼠的空间记忆以及物体识别记忆能力没有显著影响。进一步研究发现，小胶质细胞中特异性敲除Blnk引起恐惧记忆刺激后小胶质细胞激活，并且通过非自主性效应 (non-cell autonomous) 方式，降低神经元对恐惧记忆刺激的响应。综上，我们的研究初步揭示了海马体各个细胞类型在恐惧记忆形成过程中的分子水平特征性变化，提示小胶质细胞的BCR信号通路参与抑制恐惧记忆形成过程中小胶质细胞的激活，进而增加神经元对于恐惧记忆刺激的响应。
Contextual fear conditioning （CFC） is a strong robustness to timing variation behavioral paradigm for studying the cellular and molecular mechanisms of learning and memory, as well as the pathogenesis of related neurological diseases. Numerous studies have investigated the molecular mechanisms under the contextual fear memory formation and reveled that it depends on sophisticated regulatory mechanisms, including chromatin modulation, transcription, and translation levels. The hippocampus maintains contextual fear memory and also related to alzheimer‘s and other neurological diseases pathogenesis. The hippocampus molecular dynamic changes during fear memory formation were also well studied. However, hippocampal cell type compositations are highly heterogeneous, including neurons, astrocyte, and microglia, the dynamic transcriptome and translatome changes of these different cell types are largely unknown. Here we used scRNA-seq （single cell RNA-seq） and RiboTag method （isolating translating mRNAs from a specific cell population by immunoprecipitation certain ribosome subunit） to probe the single-cell gene expression landscapes and cell-type-specific temporally distinct ribosome-bound mRNA profiles during contextual fear memory formation. We found that neurons and glial cells altered transcriptional and translational signatures associated with the fear memory, and neurons are more complicated.Microglia are the resident immune cells in the central nervous system （CNS）, and surveillance of brain microenvironment to maintain homeostasis. Whether microglia involve in the fear memory formation is still controversial. Deciphering the molecular changes of microglia during fear memory formation, we found that microglial ribosome-bound B cell receptor （BCR） signaling pathway related mRNAs are up-regulated. Depletion of microglia specifically expressed Blnk impairs mouse fear memory but has little effects on spatial and object recognition memories. Further, we found that Blnk deficiency induces microglia activation and has non-cell-autonomous effects on hippocampal neuronal cells during fear memory formation. In conclusion, our study preliminarily revealed the hippocampal cell-type-specific molecular changes during contextual fear memory formation, and suggested that microglia BCR signaling pathway involved in microglia activation and neuronal function regulation during fear memory formation.
