Sex-dependent effects of angiotensin type 2 receptor expressing medial prefrontal cortex (mPFC) interneurons in fear extinction learning

Figures

• 

  Figure 1.. AT2R/td-Tomato+ mPFC expression in male and female mice. A. Generation of the AT2R-cre/td-Tomato reporter mouse and experimental approach to test mPFC AT2R distribution with immunohistochemistry (IHC). B. AT2R+ cells are equally expressed in the prelimbic (PL) and infralimbic (IL) regions of the mPFC of male (n=3) and female (n=3) mice (t(4)=0.90, p=0.42). C. Representative coronal sections showing laminar distribution of AT2R+ neurons in layers 2/3, 5, and 6 of the mPFC of male and female mice. D. Approach for assessing distribution of AT2R+ neurons through the depth of the mPFC. E. Rostral/caudal depth of mPFC had no impact on distribution of AT2R+ neurons in male (early mPFC, 175.0 ± 20.0 cells/mm2, mid mPFC, 222.6 ± 53.7 cells/mm2, late mPFC, 224.4 ± 75.2 cells/mm2, F(2,6)=0.26, p=0.78) or female (early mPFC, 136.2 ± 70.4 cells/mm2, mid mPFC, 121.4 ± 37.7 cells/mm2, late mPFC, 155.7 ± 70.0 cells/mm2 F(2,6)=0.08, p=0.93) brains. F. Representative coronal sections showing laminar distribution of AT2R+ neurons throughout rostral/caudal mPFC. All scale bars indicate 200μm.

• 

  Figure 2.. Low expression of AT2R/td-Tomato+ on excitatory mPFC neurons. A. Experimental approach for co-staining of AT2R-tdTomato+ cells with TBR1 neurons in the mPFC (n=3/group). B. Sex does not significantly affect AT2R colocalization with TBR1 in the PL and IL of the mPFC (t(4)=1.34, p=0.25). C-D. Left, representative coronal section from the male (C) and female (D) mPFC. AT2R-tdTomato and TBR1 signals are colocalized (indicated with arrows). Right, Percentage of AT2R+ neurons colocalized with TBR1 in the mPFC of male (C) and female (D) mice. E. Representative coronal sections from male (left) and female (right) mPFC showing laminar distribution of AT2R/TBR1 colocalizations (indicated with X).

• 

  Figure 3.. Characterization of AT2R/td-Tomato+ in mPFC cell types. A. Experimental approach to test mPFC-AT2R+ cell type in the mPFC of male and female mice (n=3/group). B-E. Sex has no effect on AT2R colocalization with PV interneurons (B, male: 5.5% ± 2.5% colocalized, female: 6.9% ± 2.6% colocalized, t(4)=0.41, p=0.70 ), nNos interneurons (C, male: 5.5% ± 3.3% colocalized, female: 3.5% ± 1.2% colocalized, t(4)=0.58, p=0.59) ), CB interneurons (D, male: 17.0% ± 4.0% colocalized, female: 19.6% ± 5.6% colocalized, t(4)=0.38, p=0.72 ), or CR interneurons (E, male: 6.1% ± 1.9% colocalized, female: 6.0% ± 1.7% colocalized, t(4)=0.03, p=0.98 ). PL, prelimbic cortex of the mPFC; IL, infralimbic cortex of the mPFC; Som, somatostatin; PV, parvalbumin; nNos, neuronal nitric oxide synthase; CB, calbindin; CR, calretinin. Colocalization indicated with arrows.

• 

  Figure 4.. High expression of AT2R/td-Tomato+ on mPFC somatostatin interneurons in female mice. A. Experimental approach for co-staining of AT2R-tdTomato+ cells with somatostatin neurons in the mPFC (n=3/group). B. Females have significantly greater AT2R/somatostatin colocalizations in the PL and IL of the mPFC (t(4)=3.52, p=0.02). C-D. Left, representative coronal section from the male (C) and female (D) mPFC. AT2R-tdTomato and somatostatin signals are colocalized (indicated with arrows). Right, Percentage of AT2R+ neurons colocalized with somatostatin in the mPFC of male (C) and female (D) mice. E. Representative coronal sections from male (left) and female (right) mPFC showing laminar distribution of AT2R/somatostatin colocalizations (indicated with X).

• 

  Figure 5.. AT2R cre/lox mPFC deletion delays extinction learning in females, but not males. A. Experimental protocol for the auditory cue-dependent fear extinction test. B. Left, strategy for injection of AAV-cre or AAV-GFP into the mPFC of male AT2R-flox mice and representative injection image in the mPFC. Right, injection of AAV-cre into the mPFC successfully decreased expression of AT2R in males (GFP 1.35 ± 0.38 fold change, Cre 0.40 ± 0.11 fold change, t(17)=2.54, p=0.02 ). C. Left, strategy for injection of AAV-cre or AAV-GFP into the mPFC of female AT2R-flox mice and representative injection image in the mPFC. Right, injection of AAV-cre into the mPFC successfully decreased expression of AT2R in females (GFP 1.20 ± 0.28 fold change, Cre 0.32 ± 0.07 fold change, t(18)=3.28, p<0.01 ). D. AT2R deletion from the male mPFC does not impact fear acquisition or extinction learning across three testing days in males (F(1,17)=0.09, p=0.76) E. In females, AT2R deletion from the mPFC significantly inhibits extinction learning (F(1,20)=5.35, p=0.03). n=9–12/group.

• 

  Figure 6.. Effects of AT2R cre/lox mPFC deletion on behavioral assays of locomotion and exploration in males and females. A,E. Representative locomotor traces of GFP- and Cre-injected males and females in the open field (OF) test. A-H. AT2R deletion has no impact on locomotion (B, t(15)=0.97, p=0.35 F t(17)=0.07, p=0.95, total distance traveled) or generalized anxiety (C t(15)=1.49, p=0.16, G t(17)=0.57, p=0.58, center entries; D, t(15)=1.47, p=0.16, H t(17)=1.11, p=0.29, time in center) in males or females. I,M. Representative locomotor traces of GFP- and Cre-injected males and females in the elevated plus maze (EPM) test. I-P. AT2R deletion has no impact on exploratory behavior (J, total arm entries t(15)=1.84, p=0.09 ) or generalized anxiety in males (K, open arm entries t(15)=0.45, p=0.66 ; L t(15)=0.29, p=0.78, time in open arm). mPFC AT2R deletion increases some aspects of exploratory behavior in females (N, total arm entries, t(17)=1.14, p=0.27; O, open arm entries, t(17)=2.64, p=0.02; P, open arm time, t(17)=1.38, p=0.19. OF, open field; EPM, elevated plus maze. n=8–10/group.

• 

  Figure 7.. Proposed circuit for mPFC AT2R+ mechanism. 1. Activation of AT2R on somatostatin interneurons in the PL and IL of the mPFC decreases the activity of these interneurons. 2. This decreases inhibition of glutamatergic projection neurons, increasing their firing to downstream regions. 3. Because of preferential somatostatin inhibition on brainstem (L2/3), amygdala (L5), and thalamic (L6) projecting excitatory neurons, mPFC top-down regulation is increased to these regions. 4. We hypothesize that activation of mPFC AT2Rs, via increased top-down regulation, will decrease behavioral (freezing, startle) and cardioautonomic (heart rate, blood pressure) measures of fear in a sex-dependent manner.