Identification of a Locus Coeruleus-amygdala Angiotensinergic Circuit: Implications for Stress-related Cardiovascular Diseases

Abstract

Background: The locus coeruleus (LC) is a significant noradrenergic nucleus in the brain sensitive to afferent interoceptive signals. It responds to behavioral challenges by increasing noradrenaline release through ascending projections to the hypothalamus, thalamus, cortex, and amygdala. The brain renin-angiotensin system (RAS) plays a role in stress-related cardiovascular diseases, and previous research has identified angiotensin II (Ang II) and its receptors in the LC. The current study aimed to gain a deeper understanding of the function of the Ang II type 1 receptor (AT1R) in the LC, particularly its involvement in transmitting interoceptive cardiovascular signals by regulating LC activity. 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. 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. Cre-inducible tracing with AT1R-cre mice was applied for circuit anterograde analysis and in vivo chemogenetics were used for behavior testing and analysis. Results: The majority of AT1R-eGFP+ neurons (94%) in the LC were found to be co-localized with TH. By using anterograde labeling with cre-inducible GFP virus (AAV-DIO-GFP) in AT1R-Cre mice, it was revealed that the AT1R+ neurons in the LC predominantly project to the amygdala and extended amygdala regions. Only a few or no projections to other brain areas known to receive LC inputs were observed. Within the amygdala, the AT1R+ nerve terminals were specifically limited to the medial division of the central amygdala (CeM) and the basomedial amygdala (BMA). Subsequently, AT1R was deleted from LC by injecting Cre virus into AT1R-Flox mice. Although the general anxiety level remained unchanged, restraint stress-induced anxiety was attenuated following LC AT1R deletion as shown by increased open arm distance in the elevated plus maze (EPM) test (21.7±6.6mm AAV-GFP vs. 98.1±27.5mm AAV-Cre, p<0.05, n=10). Moreover, the study employed Cre-dependent inhibitory and excitatory designer receptors exclusively activated by designer drug (hM4Di and hM3Dq DREADD) with Clozapine-n-oxide (CNO) to selectively silence and activate the AT1R+ neurons in the LC. The inhibition of AT1R+ neurons decreased the baseline anxiety level in mice (36.6±8.6mm Saline vs. 153.4±38.9mm CNO, p<0.01, n=8, EPM open arm distance), while activation of AT1R+ neurons increased baseline anxiety level (963.1±223.1mm Saline vs. 408.5±92mm CNO, p<0.01, n=10, EPM open arm distance). Additionally, the acoustic startle response, which is associated with anxiety levels, was reduced after silencing the LC AT1R+ neurons (324±39.2mV Saline vs. 168.8±54.1mV CNO, p<0.05, n=8). Conclusion: These findings suggest that a specific group of angiotensinergic LC neurons sends projections to the amygdala, highlighting the potential significance of AT1R in mediating noradrenergic activation between the LC and amygdala. Future investigations will explore the response of LC AT1R+ neurons to stress-induced cardiovascular stimuli and afferent interoceptive LC inputs, which may offer novel insights, and new therapeutic direction for the link between stress disorders and cardiovascular disease risk. NIH 1R01HL137103-01A1. This is the full abstract presented at the American Physiology Summit 2024 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.