Presenting Author The George Washington University
Background: The 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.
Methods: RNAscope® 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.
Results: AGT 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, plt;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, plt;0.05, n=6) and % time in center (2.97 ± 1.14 Saline v.s. 8.6 ± 1.4 CNO, plt;0.05, n=6) in the open field test, and increased open arm entries (5 ± 1.3 Saline v.s. 12 ± 1.1 CNO, plt;0.05, n=6) in the elevated plus maze test (Fig. 2).
Conclusion: These 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.
Fig 1. AT1R expressing neurons in the LC contribute to fear extinction (A) AGT mRNA and AT1R+ neurons were found in the LC. (B) Experimental protocol for Cre-dependent DREADD injection. (C) DREADD virus was injected into the LC of the AT1R-Cre mouse and AT1R-Cre-induced mCherry expression was detected after virus injection. (D) Auditory fear conditioning protocol. (E) Impaired extinction learning to conditioned fear after AT1R+ neuron silencing.; Fig 2. Chemogenetic silencing of the LC- AT1R+ neurons attenuate stress-induced anxiety. (A, E) Experimental protocol for open field and EPM test. (B-D) AT1R+ neuron silencing didn’t affect mice locomotor activity but blocked restraint stress-reduced anxiety in the open field test. (F-G) Inhibition of LC AT1R+ neurons didn’t affect mice locomotor activity but attenuated the restraint stress-reduced anxiety in the elevated plus maze test. EPM, elevated plus maze test.
