Identification of a locus coeruleus-amygdala angiotensinergic circuit: Implications for anxiogenesis and stress-related cardiovascular disease
Authors: Yu Zhe , Marvar Paul 
Published: May 2023

Background: The brain renin-angiotensin system (RAS) mediates stress-related cardiovascular diseases. However, the synthetic pathways of angiotensin peptides in the brain, remains unclear. Angiotensin (Ang) II and its receptors have been previously identified in the locus coeruleus (LC), a major noradrenergic nucleus of the brain, that plays a critical role in modulating cardiovascular arousal and anxiety-like behaviors. The current study sought to further understand the function of Ang II and its type 1 receptor (AT1R) in this nucleus. Methods: Using AT1R-eGFP and AT1R-Cre mice combined with neuroanatomical tract-tracing, chemogenetic and behavioral approaches, we examined AT1R expressing neurons in the LC to assess their role in anxious behavior. Dual immunohistochemistry was used in AT1R-eGFP reporter mice to characterize LC-AT1R-eGFP+ cells by looking at the colocalization of noradrenergic neuron maker tyrosine hydroxylase (TH) and GFP. Efferent connectivity of the LC was verified by injecting GFP-expressing adeno-associated virus (AAV-GFP) into the LC of wildtype mice. Cre-inducible tracing with AT1R-cre mice was applied for circuit anterograde analysis and in vivo chemogenetics were used for anxiety behavior testing and analysis. Results: The majority of AT1R-eGFP+ neurons (94%) in the LC were co-localized with TH. Anterograde labeling with cre-inducible GFP virus (AAV-DIO-GFP) in AT1R-Cre mice revealed that the AT1R+ neurons in the LC predominantly send projections to the amygdala and extended amygdala regions, with very few or no projections to other brain areas known to receive LC inputs. Furthermore, within the amygdala, the AT1R+ nerve terminals were restricted to the medial division of the central amygdala (CeM) and the basomedial amygdala (BMA). Following these findings, we used Cre-dependent inhibitory designer receptor exclusively activated by designer drug (hM4Di DREADD) with Clozapine-n-oxide (CNO) to selectively silence the AT1R+ neurons in the LC prior to restraint stress. Restraint stress-induced anxiety behavior was attenuated by LC AT1R+ neuron inhibition, as shown by increased center entries (28.6±6.7 Saline vs. 53.6±6.6 CNO, p<0.05, n=8) and center distance (3.2± .8 Saline vs. 6.9±1.0 CNO, p<0.05, n=8) in the open field test, and increased open arm entries (4.8±0.9 Saline vs. 10.4±1.3 CNO, p<0.01, n=8) and % time in the open arm (4.1±1.3 Saline vs 13.0±2.3 CNO, p<0.01, n=8) in the elevated plus maze test. Conclusion: These findings suggest that an angiotensinoceptive LC neuron population projecting to the extended amygdala positions Ang II acting on the AT1R as a potential mediator of LC-amygdala noradrenergic activation. Future studies are needed to examine Ang II noradrenergic facilitation, a likely circuit mediating stress responses that are associated with cardiovascular disease. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.