Neuronal diversity of neuropeptide signaling, including galanin, in the mouse locus coeruleus

Purpose: The locus coeruleus (LC) is a small nucleus in the pons, with norepinephrine (NE) as its major transmitter. It plays key roles in wakefulness and is associated with disorders like depression. The study aims to analyze single-cell RNA-seq data to subdivide neurons in the LC and chart their neuropeptide, co-transmitter, and receptor profiles. It also seeks to build a precise map of galanin and its receptors in the mouse LC, and understand the role of neuropeptide expression and signaling. Results: The study identified 24 clusters in total, with 3 NE, 17 glutamate, and 5 GABA subtypes. NE neurons expressed at least 19 neuropeptide transcripts, mainly galanin (Gal) but not Npy, and >30 neuropeptide receptors. Among galanin receptors, Galr1 was expressed in ~19% of NE neurons, and highly expressed in GABA neurons surrounding the NE ensemble. Patch-clamp electrophysiology and cell-type-specific Ca2+-imaging using GCaMP6s revealed that a GalR1 agonist inhibits no more than ~35% of NE neurons, with the effect being direct and not relying on feed-forward GABA inhibition. Conclusions: The results define a role for the galanin system in NE functions and provide a conceptual framework for the action of many other peptides and their receptors. The study found that most NE neurons express mRNA transcripts for galanin, at least 19 neuropeptides (but not Npy), and >30 neuropeptide receptors, including Galr1 at low levels. Selective activation of GalR1 inhibited the spontaneous activity of a subset of NE neurons possibly through the activation of Kv currents. These findings offer new insights into the role of neuropeptide expression and signaling in mouse LC. The scRNA-seq experiment involved collecting cells from 3-4-week-old C57Bl/N mice of both sexes, dissociating the LC tissue, and fixing single cells in ice-cold methanol. cDNA libraries were prepared, and sequencing was performed using the 10x Genomics Chromium Single Cell Kit. Two independent samples were prepared and sequenced, resulting in a total of 2,578 cells. Data was analyzed using the Seurat pipeline, including quality control, filtering, and clustering.研究目的：蓝斑核（locus coeruleus, LC）是脑桥内的小型核团，以去甲肾上腺素（norepinephrine, NE）作为主要神经递质。该核团在觉醒调控中发挥关键作用，且与抑郁症等多种神经精神疾病密切相关。本研究旨在通过分析单细胞RNA测序（single-cell RNA-seq）数据，对蓝斑核内神经元进行分型，并绘制其神经肽、共递质及受体表达谱；同时构建小鼠蓝斑核内甘丙肽（galanin）及其受体的精准图谱，阐明神经肽表达与信号通路的生物学功能。研究结果：本研究共鉴定出24个细胞簇，包含3种去甲肾上腺素能神经元亚型、17种谷氨酸能神经元亚型与5种γ-氨基丁酸（GABA）能神经元亚型。去甲肾上腺素能神经元可表达至少19种神经肽转录本，其中以甘丙肽（Gal）为主，而非神经肽Y（Npy），同时表达超过30种神经肽受体。在甘丙肽受体家族中，Galr1在约19%的去甲肾上腺素能神经元中表达，且在环绕去甲肾上腺素能神经元集群的GABA能神经元中呈高表达。通过膜片钳电生理技术与基于GCaMP6s的细胞类型特异性钙成像实验发现，GalR1激动剂可抑制至多约35%的去甲肾上腺素能神经元，该抑制效应为直接作用，不依赖于前馈性GABA能抑制。研究结论：本研究明确了甘丙肽系统在去甲肾上腺素能神经元功能中的调控作用，为其他多种神经肽及其受体的作用机制研究提供了概念性框架。本研究发现，绝大多数去甲肾上腺素能神经元表达甘丙肽的mRNA转录本，以及至少19种神经肽（不含Npy）与超过30种神经肽受体，其中包括低水平表达的Galr1。选择性激活GalR1可通过激活Kv钾离子电流，抑制部分去甲肾上腺素能神经元的自发放电活动。上述发现为小鼠蓝斑核内神经肽的表达与信号通路功能研究提供了全新视角。本研究的单细胞RNA测序实验流程如下：采集3~4周龄、雌雄兼具的C57Bl/N小鼠组织样本，解离蓝斑核组织，将单细胞固定于冰预冷甲醇中；构建cDNA文库，采用10x Genomics Chromium单细胞试剂盒完成测序。共制备并测序2份独立样本，最终获得2578个有效细胞。数据分析采用Seurat分析流程，涵盖质量控制、数据过滤与细胞聚类等步骤。