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. Overall design: 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).