An Amygdalar Local Oscillator Coordinates Cellular and Behavioral Rhythms

Purpose: scRNA-Seq and snRNA were performed to characterize mWake-expressing neurons in the lateral amygdala The lateral amygdalae were collected from 7-8 wk old male mWake(Cre/+) mice at ZT0-2 using a previously published protocol. Briefly, the coronal plane (350 um) containing the LA was sliced using a vibratome in bubbling aCSF. LA were dissected into Hibernate-A media with a 2% B-27 and GlutaMAX supplement (0.5 mM final concentration). Both LA from 2 mice were used for scRNA-Seq analysis, which was repeated with 2 additional mice. This procedure was also performed for snRNA-Seq analysis (in total, 8 mice were used).研究目的：本实验通过单细胞RNA测序（single-cell RNA sequencing, scRNA-Seq）与单细胞核RNA测序（single-nucleus RNA sequencing, snRNA-Seq）技术，对外侧杏仁核（lateral amygdala, LA）中表达mWake的神经元进行特征分析。实验取材自7-8周龄雄性mWake(Cre/+)小鼠，于光照周期时间（zeitgeber time, ZT）0-2时段采集其外侧杏仁核，操作流程遵循已发表的标准方案。简要操作步骤如下：将包含外侧杏仁核的冠状脑组织切片（厚度350 μm）置于充有混合气的人工脑脊液（artificial cerebrospinal fluid, aCSF）中，使用振动切片机进行切片；随后将外侧杏仁核组织解离至添加有2% B27添加剂及终浓度0.5 mM GlutaMAX补充剂的Hibernate-A培养基中。本实验使用2只小鼠的双侧外侧杏仁核开展scRNA-Seq分析，并额外使用2只小鼠重复该实验流程；单细胞核RNA测序亦采用相同操作流程，本次实验总计使用8只小鼠完成全部实验。