Activation of Angiotensin type 2 Receptor (AT 2 R) Contributes to Fear Memory

Background Previous clinical studies identify the renin‐angiotensin system (RAS) as a potential therapeutic target in post‐traumatic stress disorder (PTSD), however the mechanism(s) are unknown. We propose that brain angiotensin receptors contribute to modulating inhibitory and excitatory fear circuits important for the consolidation and extinction of fear memory in PTSD. Methods Using a transgenic brain angiotensin type 2‐receptor (AT 2 R) eGFP‐BAC reporter mouse combined with pharmacological and behavioral approaches the aim of the study was to examine the role of the brain AT 2 R in fear memory. Results We first assessed AT 2 R‐eGFP + immunoreactive cell types in the mouse brain, which were highly co‐localized with the neuronal specific marker NeuN, while there were no detectable levels of co‐localization in astrocytes (GFAP marker) or microglia (Iba‐1 marker). The level of AT 2 R‐eGFP + expressing neurons in the major subnuclei of the amygdala, an important structure in the consolidation and extinction of fear memory (Fig. 1), were then quantified. Using Image J analysis, the total numbers of eGFP + neurons were counted in one field/section in a total of 4 sections per mouse ( n = 2). Within the amygdala sub‐nuclei, AT 2 R‐eGFP + neurons were found to be predominately expressed in medial amygdala (MeA) (203.8 ± 39.4 cells/mm 2 ) and the medial division of central amygdala (CeM) (221.2 ± 15.3 cells/mm 2 ), while the basolateral amygdala (BLA) (11.7 ± 1.8 cells/mm 2 ) contained very few AT 2 R‐eGFP + neurons. Moreover, within the CeM, 96% of the AT 2 R‐eGFP + neurons expressed the GABAergic marker GAD1, while approximately 40% GABAergic cells expressed interneuron markers (parvalbumin, calrentinin and somatostatin). To examine the behavioral role of AT 2 R in fear memory, we used classic Pavlovian fear conditioning, pairing auditory cues with footshocks. Following the acquisition of fear, AT 2 R mRNA expression was significantly elevated (t(22) = 2.5; p<0.05) within the CeA but not following extinction learning. In separate studies, a bilateral intra‐CeA injection of C21 (0.06 ug/ul) was administered prior to fear acquisition testing and freezing behavior quantified. Compared to vehicle control, mice receiving C21 into the CeA displayed enhanced extinction by a decreased percentage of freezing (61.7% ± 5.4 vehicle v.s. 42.1% ± 5.7 C21 group, p <0.05, n=10–12) during repeated conditioned stimuli (30 second auditory tones). These behavioral results were independent general anxiety‐like measures as C21 compared to vehicle did not affect locomotor activity or center entries in the open field test. Conclusions Together, these data provide anatomical, functional and behavioral evidence for activation brain AT2R in the consolidation and extinction of conditioned fear. Further studies are required to determine the neurobiological mechanism(s), which may involve changes in cerebral vascular blood flow and/or modulation of neuronal excitability and plasticity. Support or Funding Information American Heart Association ‐ 15CSA24340001 This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .