Definitive Evidence for the Identification and Function of Renin-Expressing Cholinergic Neurons in the Nucleus Ambiguus

Background: The importance of the brain renin-angiotensin system (RAS) incardiovascular function is well accepted. However, not knowing the precise source ofrenin in the brain has been a limitation towards a complete understanding of how the brainRAS operates. Methods: Highly sensitive in situ hybridization techniques and conditionalknockout mice were used to address the location and function of renin in the brainstem.Results: We identified novel renin-expressing cholinergic neurons in the nucleusambiguus (NuAm), a major vagal cardioinhibitory center in the brainstem. The expressionof RAS genes was relatively abundant in the NuAm, implying that angiotensin II mightmediate an important regulatory role in this nucleus and other regions with neuralconnectivity to the NuAm. Then, we generated conditional knockout mice lacking theclassical renin isoform (Ren-aChAT-KO), specifically in cholinergic neurons. Ablation of Ren-a in cholinergic neurons abrogated renin expression in the NuAm. Moreover, studiesusing radiotelemetry, heart rate variability analyses, and pharmacological approachesrevealed that the parasympathetic nervous system is depressed in Ren-aChAT-KO maleswhile augmented in the Ren-aChAT-KO females. Subsequently, transcriptomic approacheswere used to infer putative genes and signaling pathways regulated by renin within theNuAm. Conclusions: This study revealed that renin in cholinergic neurons plays afundamental role in preserving autonomic balance and cardiovascular homeostasis in asex-dependent manner. These findings define the NuAm as an endogenous, local sourceof renin with biological function and serve as conclusive evidence for the presence andfunctionality of the brain RAS. Brains were collected from male and female WT and Ren-aChAT-KO mice. Brains were immediately harvested and frozen in 2-methyl butane on dry ice and stored in -80 C. Then, Palkovits technique was used to obtain bilateral NuAm brain punches samples as previously described.5 Briefly, the brains were embedded in Tissue-Tek O.C.T compound (Sakura) in a cryostat until AP: -7.2. Then, a 1.0 mm needle (Stoelting) was used to collect bilateral samples of the NuAm between AP: -7.2 to -6.5 mm coordinates caudal from bregma. The samples were maintained frozen until processing. Total RNA was extracted using Nucleospin RNA Plus XS kit (Takara). Total RNA was quantified using spectrophotometry (NanoDrop, Thermo Fisher Scientific) and confirmed using fluorometric methods (Qubit, Invitrogen). Samples were then submitted to Novogene (Beijing) for library preparation and RNA sequencing analysis. A total of 22 samples were submitted (WT male=6, Ren-aChAT-KO male=6, WT female=4, Ren-aChAT-KO female=6).背景：脑肾素-血管紧张素系统（renin-angiotensin system, RAS）在心血管功能中的重要性已得到广泛认可。然而，学界尚未明确脑中肾素的确切来源，这成为全面阐明脑RAS运作机制的一大阻碍。 方法：本研究采用高灵敏度原位杂交技术与条件性基因敲除小鼠模型，探究脑干中肾素的定位与功能。 结果：本研究在疑核（nucleus ambiguus, NuAm）——脑干内主要的迷走神经心血管抑制中枢——中发现了新型表达肾素的胆碱能神经元。RAS基因在疑核中表达相对丰富，提示血管紧张素Ⅱ可能在该核团及与疑核存在神经连接的其他脑区中发挥重要调控作用。随后，我们构建了仅在胆碱能神经元中缺失经典肾素亚型的条件性基因敲除小鼠（Ren-aChAT-KO）。胆碱能神经元中Ren-a的敲除完全消除了疑核内的肾素表达。此外，通过放射遥测技术、心率变异性分析与药理学手段开展的研究显示，雄性Ren-aChAT-KO小鼠的副交感神经系统受到抑制，而雌性Ren-aChAT-KO小鼠的副交感神经系统则被增强。后续，我们采用转录组学方法推断了疑核内受肾素调控的潜在基因与信号通路。 结论：本研究证实，胆碱能神经元中的肾素以性别依赖的方式，在维持自主神经平衡与心血管稳态中发挥核心作用。本研究结果明确疑核为具有生物学功能的内源性肾素局部来源，同时为脑RAS的存在与功能提供了确凿证据。 脑组织处理与测序：本研究采集了野生型（WT）与Ren-aChAT-KO雄性及雌性小鼠的脑组织。脑组织于取材后立即置于干冰预冷的2-甲基丁烷中速冻，并于-80℃条件下保存。随后采用Palkovits取材技术获取双侧疑核脑穿刺样本，具体操作参照既往文献5。简要步骤如下：将脑组织置于冰冻切片机中，用Tissue-Tek O.C.T包埋剂（Sakura）包埋，直至前囟（bregma）尾侧-7.2 mm的平面（AP: -7.2）；随后使用1.0 mm直径的穿刺针（Stoelting），在前囟尾侧AP: -7.2至-6.5 mm的坐标范围内采集双侧疑核样本。样本始终保持冷冻状态直至后续处理。总RNA采用Nucleospin RNA Plus XS试剂盒（Takara）提取，通过分光光度法（NanoDrop，Thermo Fisher Scientific）进行定量，并采用荧光法（Qubit，Invitrogen）验证RNA质量。随后将样本送至北京诺禾致源（Novogene）进行文库构建与RNA测序分析。本研究共提交22份样本（雄性野生型6份、雄性Ren-aChAT-KO 6份、雌性野生型4份、雌性Ren-aChAT-KO 6份）。