Evaluation of prefrontal cortex expressing angiotensin type 2 receptors in fear-related behavior

Abstract

Background: Evidence suggests that the renin-angiotensin system (RAS), a regulator of blood pressure and fluid homeostasis, is a potential therapeutic target for PTSD. Brain angiotensin II receptors are expressed across corticolimbic circuits such as the medial prefrontal cortex (mPFC), however, their role and mechanism are largely unknown. Given the importance of the mPFC in fear-related behavior, our studies sought to examine the role of mPFC-expressing angiotensin type 2 receptor (AT2R) neurons in fear learning in male and female mice. We proposed that activity of mPFC-AT2Rs, via inhibition of glutamatergic transmission, facilitates the extinction of cued fear. Methods: Using an AT2R-eGFP BAC reporter mouse, immunohistochemistry, retrograde tracing, and whole-cell patch-clamp recording were performed for neuroanatomical and ex-vivo analysis. To understand the role of mPFC-AT2Rs in fear learning, we selectively deleted AT2R from mPFC neurons using an AAV-Cre or GFP virus in AT2R-flox male and female mice (n=9-12 per group). Following viral integration (3 weeks), Pavlovian auditory fear conditioning (5x tone/shock pairings, 30 CS extinction) was used to examine the extinction of learned fear. Anxiety-like behavior and locomotion were also examined using the open field and elevated plus maze behavioral tests. Results: The immunofluorescence microscopy results identified AT2R-eGFP+ cells as predominantly glutamatergic ( 88.1% Tbr1, 11.9% GAD) and highly expressed in the prelimbic ( 120.6±13.25 cells/mm2) and infralimbic ( 163.7±17.30 cells/mm2) cortices of the mPFC, while retrograde labeling demonstrated that some mPFC-AT2R-eGFP+ neurons project to the basolateral amygdala (BLA). Ex-vivo activation using slice electrophysiology in mPFC-AT2R-eGFP+ neurons decreased the distribution of both frequency ( D=0.19, p<0.01) and amplitude ( D=0.13, p<0.01) of spontaneous excitatory postsynaptic currents (sEPSC). Selective mPFC-AT2R deletion impaired extinction learning as measured by enhanced percent freezing (day 2, F(1, 719)=5.12, p=0.02), and this effect on extinction occurred only in female mice. Generalized anxiety measures (% time in open arm, M: GFP 14.42%, Cre 13.58%, p=0.78; F: GFP 14.85%, Cre 21.66%, p=0.19) and locomotion ( total distance, M: GFP 44.11m, Cre 56.00m, p=0.35; F: GFP 42.86m, Cre 42.39m, p=0.95) were not affected by mPFC-AT2R deletion in males or females. Conclusions: These findings provide novel evidence for mPFC-AT2R+ neurons as a potential sex-dependent corticolimbic regulator of conditioned fear. We propose that mPFC AT2Rs contribute to extinction in females by decreasing excitatory glutamatergic output from the mPFC onto the amygdala. To further evaluate these results, future studies will utilize in vivo chemogenetic approaches to examine the role of this circuit in the physiology and extinction of learned fear. NIH 1R01HL137103-01A1 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.