Role of locus coeruleus expressing angiotensin type 1 receptors (AT1R) neurons in fear learning and stress‐induced anxiety

Abstract

BackgroundThe renin‐angiotensin system (RAS) has been implicated in stress‐related disorders, however the central mechanisms responsible for this remains unknown. The locus coeruleus (LC), a major noradrenergic nucleus of the brain, plays a critical role in modulating anxiety‐like behaviors. The LC has also been previously shown to express angiotensinogen (AGT), the pre‐cursor for angiotensin, as well as strong expression of angiotensin II receptors, but its role in stress‐related disorders has not been examined. Using angiotensin II type 1 receptor (AT1R)‐eGFP and Cre mice combined with neuroanatomical and behavioral approaches, we examined the role of LC expressing AT1R in fear‐ and anxiety‐related behavior.MethodsRNAscope® in situ hybridization assay was used to analyze cellular mRNA expression of AGT in the LC while in vivo chemogenetics combined with behavioral testing in AT1R‐Cre mice mouse was used to examine the role of LC‐AT1R cells in fear memory and stress‐induced anxiety. Further, dual immunohistochemistry plus retrograde tract tracing was used to characterize LC AT1R‐eGFP+ cells and limbic circuit connections.ResultsAGT mRNA and AT1R‐eGFP+ immunoactivity was found localized to the LC. The majority of AT1R‐eGFP+neurons (94%) in LC were co‐localized with tyrosine hydroxylase, a marker for norepinephrine‐containing neurons. Retrograde labeling revealed that some of these AT1R‐eGFP+ neurons send projections to the basolateral amygdala (BLA), an important brain structure for modulating stress induced fear related anxiety responses. Next, the AT1R+ neurons in LC were silenced using Cre‐dependent inhibitory designer receptor exclusively activated by designer drug (hM4Di DREADD) expression followed by Clozapine‐n‐oxide (CNO) administration. Silencing the LC AT1R‐expressing neurons prior to fear extinction training impaired the extinction of learned fear as shown by increased percent freezing during the training (time × drug interaction, F (7, 70) = 3.219, p<0.01, n=6) (Fig 1). Furthermore, restraint stress‐induced anxiety behavior was attenuated by LC AT1R+ neuron inhibition, as shown by increased center entries (22.7 ± 7.3 Saline v.s. 50.5 ± 8.8 CNO, p<0.05, n=6) and % time in center (2.97 ± 1.14 Saline v.s. 8.6 ± 1.4 CNO, p<0.05, n=6) in the open field test, and increased open arm entries (5 ± 1.3 Saline v.s. 12 ± 1.1 CNO, p<0.05, n=6) in the elevated plus maze test (Fig. 2).ConclusionThese findings provide evidence for a novel angiotensinergic LC cell type and position the LC AT1R as a potential mediator of noradrenergic regulation in learned fear and stress‐induced anxiety. Future studies are needed to fully characterize the underlying neurocircuits and neuropeptide modulators that likely interact with the LC AT1R expressing neurons during stress‐related behaviors.