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Angiotensin Type 1 Receptor Inhibition Enhances the Extinction of Fear Memory

  • Paul J. Marvar
    Correspondence
    Address correspondence to Paul J. Marvar, Ph.D., Emory University School of Medicine, Department of Psychiatry and Behavioral Sciences, Center for Behavioral Neuroscience and Psychiatric Disorders, 954 Gatewood Road Atlanta, GA 30329
    Affiliations
    Behavioral Neuroscience and Psychiatric Disorders, Department of Psychiatry and Behavioral Sciences, Emory University School of Medicine, Atlanta, Georgia

    Department of Physiology and Pharmacology, University of Bristol, Bristol, United Kingdom
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  • Jared Goodman
    Affiliations
    Behavioral Neuroscience and Psychiatric Disorders, Department of Psychiatry and Behavioral Sciences, Emory University School of Medicine, Atlanta, Georgia
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  • Sebastien Fuchs
    Affiliations
    Department of Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, California
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  • Dennis C. Choi
    Affiliations
    Behavioral Neuroscience and Psychiatric Disorders, Department of Psychiatry and Behavioral Sciences, Emory University School of Medicine, Atlanta, Georgia
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  • Sunayana Banerjee
    Affiliations
    Behavioral Neuroscience and Psychiatric Disorders, Department of Psychiatry and Behavioral Sciences, Emory University School of Medicine, Atlanta, Georgia
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  • Kerry J. Ressler
    Affiliations
    Behavioral Neuroscience and Psychiatric Disorders, Department of Psychiatry and Behavioral Sciences, Emory University School of Medicine, Atlanta, Georgia
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      Background

      The current effective treatment options for posttraumatic stress disorder (PTSD) are limited, and therefore the need to explore new treatment strategies is critical. Pharmacological inhibition of the renin-angiotensin system is a common approach to treat hypertension, and emerging evidence highlights the importance of this pathway in stress and anxiety. A recent clinical study from our laboratory provides evidence supporting a role for the renin-angiotensin system in the regulation of the stress response in patients diagnosed with PTSD.

      Methods

      With an animal model of PTSD and the selective angiotensin receptor type 1 (AT1) antagonist losartan, we investigated the acute and long-term effects of AT1 receptor inhibition on fear memory and baseline anxiety. After losartan treatment, we performed classical Pavlovian fear conditioning pairing auditory cues with footshocks and examined extinction behavior, gene expression changes in the brain, as well as neuroendocrine and cardiovascular responses.

      Results

      After cued fear conditioning, both acute and 2-week administration of losartan enhanced the consolidation of extinction memory but had no effect on fear acquisition, baseline anxiety, blood pressure, and neuroendocrine stress measures. Gene expression changes in the brain were also altered in mice treated with losartan for 2 weeks, in particular reduced amygdala AT1 receptor and bed nucleus of the stria terminalis c-Fos messenger RNA levels.

      Conclusions

      These data suggest that AT1 receptor antagonism enhances the extinction of fear memory and therefore might be a beneficial therapy for PTSD patients who have impairments in extinction of aversive memories.

      Key Words

      There is increasing evidence that posttraumatic stress disorder (PTSD), a debilitating fear- and stress-related psychiatric illness, is associated with cardiovascular disease and its major comorbidities (
      • Ahmadi N.
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      ). Despite these emerging clinical and epidemiologic studies, the mechanisms responsible for the association between cardiovascular disease risk and PTSD remain unclear. Moreover, at present the current effective treatment options for PTSD are limited, and therefore the need to explore new treatment strategies is critical for improving future prevention efforts.
      The renin-angiotensin system is essential for cardiovascular regulation, and drugs targeting this pathway, for example angiotensin receptor blockers (ARBs) and angiotensin-converting enzyme inhibitors (ACE-I), are common strategies for treating hypertension and cardiovascular-related diseases. However, there is increasing interest and evidence supporting the role of the renin-angiotensin pathway in stress-related and neurodegenerative pathologies independent of its cardiovascular effects (
      • Saavedra J.M.
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      ,
      • Gard P.R.
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      ). It is known that stressful situations can elicit an increase in plasma levels of renin, which catalyzes the formation of angiotensin, thus giving rise to elevated levels of circulating angiotensin II (
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      ). Animal studies have demonstrated that in response to immobilization and isolation stress, the angiotensin type 1 receptor (AT1) binding is increased in the paraventricular nucleus (PVN) of the hypothalamus and is reduced with ARBs (
      • Armando I.
      • Volpi S.
      • Aguilera G.
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      Angiotensin II AT1 receptor blockade prevents the hypothalamic corticotropin-releasing factor response to isolation stress.
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      • Bregonzio C.
      • Seltzer A.
      • Armando I.
      • Pavel J.
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      Angiotensin II AT(1) receptor blockade selectively enhances brain AT(2) receptor expression, and abolishes the cold-restraint stress-induced increase in tyrosine hydroxylase mRNA in the locus coeruleus of spontaneously hypertensive rats.
      ). More recently, lentiviral knockdown of the AT1 receptor in the subfornical organ of the brain has been shown to prevent the neuroendocrine response to restraint stress (
      • Krause E.G.
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      • Scott K.A.
      • Flak J.N.
      • Jones K.
      • Smeltzer M.D.
      • et al.
      Blood-borne angiotensin II acts in the brain to influence behavioral and endocrine responses to psychogenic stress.
      ). Evidence also suggests the use of ARBs to prevent stress-related brain pathologies (
      • Saavedra J.M.
      Angiotensin II AT(1) receptor blockers as treatments for inflammatory brain disorders.
      ,
      • Wright J.W.
      • Harding J.W.
      The angiotensin AT4 receptor subtype as a target for the treatment of memory dysfunction associated with Alzheimer’s disease.
      ,
      • Gard P.R.
      Angiotensin as a target for the treatment of Alzheimer’s disease, anxiety and depression.
      ), and several preclinical and clinical reports have described protective effects of ARBs on cognition and memory (
      • Amenta F.
      • Mignini F.
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      • Veglio F.
      Protective effect of anti-hypertensive treatment on cognitive function in essential hypertension: Analysis of published clinical data.
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      • Anderson C.
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      • Unger T.
      • et al.
      Renin-angiotensin system blockade and cognitive function in patients at high risk of cardiovascular disease: Analysis of data from the ONTARGET and TRANSCEND studies.
      ). In line with these studies, we recently completed a clinical retrospective observational study of over 500 traumatized patients and found a significant association between individuals taking ACE-I/ARB medication and decreased PTSD symptoms (
      • Khoury N.M.
      • Marvar P.J.
      • Gillespie C.F.
      • Wingo A.
      • Schwartz A.
      • Bradley B.
      • et al.
      The renin-angiotensin pathway in posttraumatic stress disorder: Angiotensin-converting enzyme inhibitors and angiotensin receptor blockers are associated with fewer traumatic stress symptoms.
      ). To further understand the mechanism responsible for these clinical observations, the aim of the present study was to examine the role of the renin-angiotensin system, through inhibition of the AT1 receptor in an animal model of PTSD-like symptoms. Angiotensin type 1 receptor blockade has also been shown to influence the hypothalamic–pituitary–adrenal (HPA) axis (
      • Armando I.
      • Volpi S.
      • Aguilera G.
      • Saavedra J.M.
      Angiotensin II AT1 receptor blockade prevents the hypothalamic corticotropin-releasing factor response to isolation stress.
      ,
      • Gard P.
      Strain differences in the anxiolytic effects of losartan in the mouse.
      ), which could impact fear memory; therefore we also sought to examine the acute HPA axis stress response.
      Although PTSD is a complex disorder that includes chronic development over time of hyperarousal, intrusive, and avoidance/numbing symptoms (
      • Breslau N.
      • Kessler R.C.
      • Chilcoat H.D.
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      • Davis G.C.
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      Trauma and posttraumatic stress disorder in the community: The 1996 Detroit Area Survey of Trauma.
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      ), its cardinal pathology is thought to relate, in part, to the unmitigated fear response at the time of the trauma and the inability to inhibit fear in the aftermath of trauma (
      • Jovanovic T.
      • Ressler K.J.
      How the neurocircuitry and genetics of fear inhibition may inform our understanding of PTSD.
      ). Therefore, we use classical Pavlovian conditioning in mouse models to directly address the mechanisms of fear acquisition, expression, inhibition, and extinction. Numerous examples have now demonstrated that methods that facilitate extinction in rodent models might have great translational validity to enhancing extinction in human fear-related disorders marked by deficits in extinction processing (
      • Rothbaum B.O.
      • Davis M.
      Applying learning principles to the treatment of post-trauma reactions.
      ,
      • Davis M.
      • Ressler K.
      • Rothbaum B.O.
      • Richardson R.
      Effects of D-cycloserine on extinction: Translation from preclinical to clinical work.
      ). In these studies we hypothesized, on the basis of our prior human observational findings (
      • Khoury N.M.
      • Marvar P.J.
      • Gillespie C.F.
      • Wingo A.
      • Schwartz A.
      • Bradley B.
      • et al.
      The renin-angiotensin pathway in posttraumatic stress disorder: Angiotensin-converting enzyme inhibitors and angiotensin receptor blockers are associated with fewer traumatic stress symptoms.
      ), that losartan, an AT1 receptor antagonist, would be associated with decreased fear consolidation and/or enhanced extinction of fear.

      Methods and Materials

      Animals

      All experiments were performed on adult (3–4 months old) wild-type C57BL/6J male mice from Jackson Laboratory (Bar Harbor, Maine). All procedures were approved by the Institutional Animal Care and Use Committee of Emory University and were in compliance with National Institutes of Health guidelines.

      Drugs

      We administered losartan (Sigma-Aldrich, St. Louis, Missouri, catalog no. 61188), a selective AT1 receptor antagonist, intraperitoneally (IP) at a dose of 1 mg/kg and 10 mg/kg in a vehicle of .9% isotonic sterile saline; the same vehicle was also used in control groups. In other experiments, losartan was subcutaneously infused via osmotic mini-pump (10 mg/kg/day) (Model 2002; Alzet, Cupertino, California) over a 2-week period. In experiments where losartan was given IP, mice received a single dose 40 min before the appropriate behavioral procedure (see Figure S1 in Supplement 1 for experimental design).

      Cardiovascular Measures

      Blood pressure was measured invasively with radiotelemetry to resolve minute-to-minute acute blood pressure changes in freely moving animals or noninvasively with the tail cuff method as previously described (
      • Guzik T.J.
      • Hoch N.E.
      • Brown K.A.
      • McCann L.A.
      • Rahman A.
      • Dikalov S.
      • et al.
      Role of the T cell in the genesis of angiotensin II induced hypertension and vascular dysfunction.
      ,
      • Marvar P.J.
      • Thabet S.R.
      • Guzik T.J.
      • Lob H.E.
      • McCann L.A.
      • Weyand C
      • et al.
      Central and peripheral mechanisms of T-lymphocyte activation and vascular inflammation produced by angiotensin II-induced hypertension.
      ,
      • Marvar P.J.
      • Vinh A.
      • Thabet S.
      • Lob H.E.
      • Geem D.
      • Ressler K.J.
      • Harrison D.G.
      T lymphocytes and vascular inflammation contribute to stress-dependent hypertension.
      ).

      Anxiety Measures

      The elevated-plus maze consisted of a platform with two walled, closed arms and two nonwalled, open arms connected by an open center. The mice were placed onto the center between the plus maze arms and were recorded exploring the plus maze for 5 min. For analysis, the percentage of time spent exploring the open arms was calculated by dividing the time spent in the open arms by the combined time spent in open and closed arm (
      • Pellow S.
      • Chopin P.
      • File S.E.
      • Briley M.
      Validation of open:closed arm entries in an elevated plus-maze as a measure of anxiety in the rat.
      ). The open field consisted of a circular arena (60 cm diameter) made of black Plexiglass with a wall 20 cm high. Mice were allowed to explore for 10 min. Activity data were obtained and analyzed with the Activity Software (Med Associates, St. Albans, Vermont).

      Cue Fear Conditioning and Extinction

      As previously performed in our laboratory, fear conditioning was conducted in nonrestrictive acrylic cylinders (SR-LAB startle response system; San Diego Instruments, San Diego, California), and extinction testing was performed 24 and 48 hours after fear conditioning (
      • Choi D.C.
      • Maguschak K.A.
      • Ye K.
      • Jang S.W.
      • Myers K.M.
      • Ressler K.J.
      Prelimbic cortical BDNF is required for memory of learned fear but not extinction or innate fear.
      ,
      • Heldt S.A.
      • Stanek L.
      • Chhatwal J.P.
      • Ressler K.J.
      Hippocampus-specific deletion of BDNF in adult mice impairs spatial memory and extinction of aversive memories.
      ). Please refer to Supplement 1 for detailed description.

      Restraint Stress

      Mice were individually restrained in a well-ventilated 50-mL conical tube for 30 min. Immediately after the restraint period they were sacrificed, and the brains were removed and frozen with powdered dry ice and stored at −80°C. Brains were then cut on a freezing microtome in sections of 40 μm at −18°C, and tissue punches were obtained for messenger RNA (mRNA) and quantitative polymerase chain reaction (PCR).

      Reverse Transcription and PCR Quantification

      Gene expression changes in the amygdala, prefrontal cortex (PFC), and bed nucleus of the stria terminalis (BNST) were detected by relative quantitative reverse transcription PCR (FAST 7500; Applied Biosystems, Foster City, California). Bilateral tissue punches were performed according to the Mouse Brain Atlas by Watson and Paxinos (
      • Paxinos G.
      • Franklin K.
      The Mouse Brain in Stereotaxic Coordinates.
      ). Total RNA was isolated with Qiagen RNeasy kit (Qiagen, Valencia, California). Please refer to Supplement 1 for detailed description.

      Immunohistochemistry

      Mice were perfused intracardially with ice-cold saline followed by 4% paraformaldehyde in .1 mol/L phosphate buffer (pH 7.4). The brains were removed and stored in the same fixative for 24 hours at 4°C and subsequently immersed in 30% sucrose at 4°C. To visualize the c-Fos protein, an immunohistochemical avidin-biotin-peroxidase staining procedure was used (Elite ABC Kit; Vector Laboratories, Burlingame, California). The immunostaining reaction was developed with the oxidase-diaminobenzidine-nickel method (Sigma-Aldrich). Induction of c-Fos protein was evaluated by automated image analysis with ImageJ software (National Institutes of Health, Bethesda, Maryland). Please refer to Supplement 1 for detailed description.

      Neuroendocrine Measures

      Plasma serum corticosterone levels were measured by radioimmunoassay by the Emory University Biomarkers Core. Blood was collected on ice with .1 mol/L ethylenediamine tetraacetate, and plasma was separated in a refrigerated centrifuge and stored at −70°C until analysis. Plasma renin activity was measured in heparinized plasma with a fluorescent enzymatic assay adapted from (
      • Wang G.T.
      • Chung C.C.
      • Holzman T.F.
      • Krafft G.A.
      A continuous fluorescence assay of renin activity.
      ). Please refer to Supplement 1 for detailed description.

      Data Presentation and Statistical Analysis

      Data in the manuscript are expressed as the mean ± SEM, and p values < .05 were considered statistically significant. Comparisons between more than 2 groups were made with analysis of variance (ANOVA) with Prism 6.0 (GraphPad Software Inc., La Jolla, California). When differences were observed, a Bonferroni post hoc test was employed to compare specific groups. When identical measurements were made over time, we employed one-way repeated measures ANOVA with a Bonferroni post hoc test. When 2 groups were compared we used an unpaired two-tailed Student t test.

      Results

      Effect of Single Administration of Losartan on Learned Fear

      To further understand the mechanism by which angiotensin II blockers reduce PTSD symptoms (
      • Khoury N.M.
      • Marvar P.J.
      • Gillespie C.F.
      • Wingo A.
      • Schwartz A.
      • Bradley B.
      • et al.
      The renin-angiotensin pathway in posttraumatic stress disorder: Angiotensin-converting enzyme inhibitors and angiotensin receptor blockers are associated with fewer traumatic stress symptoms.
      ), we examined the effects of the selective AT1 receptor antagonist losartan in an animal model of PTSD-like symptoms. As shown in Figure 1A, in the absence of drug, both groups exhibited normal acquisition of fear to the five tone-shock or conditioned stimulus (CS)–unconditioned stimulus pairings. Twenty-four hours later, we examined the effects of losartan on fear expression (also considered the extinction training session) to the presentation of 15 trials of CS cues in a different context. Groups were given either losartan (1 mg/kg or 10 mg/kg IP) or saline before fear expression/extinction training. During both extinction training at 1 mg/kg losartan and 24 hours later during extinction retention testing, in the absence of drug, there were no differences in freezing between groups (Figure 1B,C,D). Therefore in a separate group, a higher dose of losartan (10 mg/kg IP) was examined. As shown in Figure 1E, during extinction training/fear expression, total average freezing was similar between groups. However, 24 hours later in the absence of drug, total overall freezing was significantly reduced during extinction retention, an index of long-term fear memory (Figure 1F) (t48 = 2.9; p < .01). As determined by repeated-measures two-way ANOVA, there was a significant main effect for treatment in the losartan group (10 mg/kg IP), which exhibited significantly less freezing to CS presentation during extinction retention (F1,144 = 14.6; p < .01) (Figure 1G). Bonferroni post hoc analysis revealed significant reductions in freezing during the first and third blocks of five CS tone presentations (p < .05) (Figure 1G). Taken together, these data indicate that losartan does not affect fear expression but enhances retention of fear extinction, in a dose-dependent manner, suggesting that AT1 receptor antagonism might reduce fear memory through enhancing the consolidation of extinction learning.
      Figure thumbnail gr1
      Figure 1Angiotensin type 1 receptor inhibition enhances the extinction of learned fear. Before losartan treatment (predrug) acquired fear during cued fear conditioning with five tone-shock pairings in pre-vehicle (n = 10) and pre-losartan (n = 11) groups (A). Twenty-four hours after fear acquisition, losartan (1 mg/kg or 10 mg/kg intraperitoneal) was given before extinction training (B, E) n = 20–25/group). Twenty-four hours after extinction training and in the absence of drug, mice were tested for extinction retention of learned fear, expressed as total average freezing and in blocks of five conditioned stimulus (CS) tones (C, D, F, G); *p < .05.

      No Blood Pressure or Anxiety-Like Effects After Acute Administration of Losartan

      One possible hypothesis is that the aforementioned effects on extinction consolidation were due simply to compensatory changes after acutely lowered blood pressure or anxiety level. A dose of 10 mg/kg is commonly used in rodents, and when the dose is given acutely or chronically, some studies show reductions in baseline blood pressure (
      • Xia H.
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      • Hickman P.J.
      • Lazartigues E.
      Angiotensin II type 1 receptor-mediated reduction of angiotensin-converting enzyme 2 activity in the brain impairs baroreflex function in hypertensive mice.
      ,
      • Soltis E.E.
      • Jewell A.L.
      • Dwoskin L.P.
      • Cassis L.A.
      Acute and chronic effects of losartan (DuP 753) on blood pressure and vascular reactivity in normotensive rats.
      ,
      • Collister J.P.
      • Soucheray S.L.
      • Osborn J.W.
      Chronic hypotensive effects of losartan are not dependent on the actions of angiotensin II at AT 2 receptors.
      ), whereas others show no effect (
      • Senador D.
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      • Irigoyen M.C.
      • Morris M.
      • Elased K.M.
      Cardiovascular and autonomic phenotype of db/db diabetic mice.
      ,
      • Srinivasan J.
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      • Suresh B.
      • Ramanathan M.
      Effect of losartan and enalapril on cognitive deficit caused by Goldblatt induced hypertension.
      ,
      • Wong P.C.
      • Price W.A.
      • Chiu A.T.
      • Duncia J.V.
      • Carini D.J.
      • Wexler R.R.
      • et al.
      Nonpeptide angiotensin II receptor antagonists. VIII. Characterization of functional antagonism displayed by DuP 753, an orally active antihypertensive agent.
      ). In the current study, administration of losartan at a dose that enhanced extinction retention (10 mg/kg IP) had no effect on baseline blood pressure (Figure 2A).
      Figure thumbnail gr2
      Figure 2Acute administration of losartan (10 mg/kg, intraperitoneal) does not affect baseline blood pressure or anxiety measures. Minute analysis (every other minute) of blood pressure measured by radiotelemetry after acute administration of saline (n = 4) or losartan (10 mg/kg, intraperitoneal n = 4) (A). Arrows represent time point of injection and start extinction training testing at minute 40 after injection of drug or vehicle. Distance travelled during open-field testing (B) and percent time in open arms of elevated plus maze test of vehicle (C) (n = 6–8/group).
      In some animal studies, losartan has been previously shown to have anxiolytic effects (
      • Gard P.
      Strain differences in the anxiolytic effects of losartan in the mouse.
      ); therefore, to determine whether the dose of losartan used in the present study affects baseline levels of generalized anxiety-like behavior, animals were tested in the elevated plus maze. We found no differences in anxiety-like behavior between losartan- and saline-treated groups as measured by distance traveled and time spent in open arms in the elevated plus maze (Figure 2B,C). Overall, these data suggest that acute administration of losartan at 10 mg/kg IP, although enhancing extinction retention, does not affect baseline levels of blood pressure or measures of anxiety-like behavior in these animals.

      Effect of 2-Week Losartan Treatment on Learned Fear and Neuroendocrine Measures

      In our previous clinical study, most patients were on long-term treatment regimens of blood pressure lowering drugs, including ACE-I or ARBs (
      • Khoury N.M.
      • Marvar P.J.
      • Gillespie C.F.
      • Wingo A.
      • Schwartz A.
      • Bradley B.
      • et al.
      The renin-angiotensin pathway in posttraumatic stress disorder: Angiotensin-converting enzyme inhibitors and angiotensin receptor blockers are associated with fewer traumatic stress symptoms.
      ). Therefore, we next sought to examine the repeated effects of AT1 receptor antagonism on the extinction of fear. Two-week osmotic mini-pumps were implanted, and losartan (10 mg/kg/day) was subcutaneously infused, and on day 10, animals underwent Pavlovian fear conditioning. The dose of 10 mg/kg/day was chosen on the basis of our single IP administration study showing enhanced extinction retention at this bolus dose. As shown in Figure 3A, there was no difference in the acquisition of fear as determined by percent freezing in mice treated with losartan compared with vehicle. Furthermore, when we evaluated fear expression 24 hours later, despite the decreased trend, there was no significant difference in total average freezing or when expressed in blocks of 5 CS trials (Figure 3B,C). However, consistent with the effects of acute administration of losartan on extinction, mice treated for 2 weeks with losartan displayed enhanced extinction retention of fear memory when expressed over the total average freezing period (Figure 3D) (t35 = 2.4; p < .05). Repeated-measures ANOVA, during extinction retention testing, revealed a significant main effect of treatment in the losartan group, because they exhibited significantly less freezing to CS presentation (F1,108 = 8.8; p < .005) (Figure 3E). Bonferroni post hoc analysis revealed significant reductions in freezing during the second block of five CS tone presentations (Figure 3E) (p < .05). Similar to the acute administration, there were no differences between groups in generalized anxiety testing during the open field test or baseline blood pressure (Figure S2 in Supplement 1). These data provide additional evidence that long-term AT1 receptor inhibition is involved in fear memory, independent of blood pressure or measures of baseline anxiety.
      Figure thumbnail gr3
      Figure 3Chronic inhibition of angiotensin type 1 receptor enhances extinction of learned fear. After 2 weeks of losartan (10 mg/kg/day) (n = 17) or vehicle (n = 13) infusion, fear acquisition expressed as percent freezing during cued fear conditioning with five tone-shock pairings (A). Extinction training/fear expression was tested. Total average freezing within the session (B). Represented in blocks of 5 conditioned stimulus (CS) tones (C) 24 hours after fear acquisition. Extinction retention of learned fear, expressed as total average freezing and in blocks of 5 CS tones (D, E) (n = 18–20); *p < .05.
      The question of how AT1 inhibition affects fear memory and whether it is due to a primary central effect (brain specific) or a secondary peripheral action is unknown. Therefore, we next examined AT1 receptor expression and other stress and fear memory-related genes in brain nuclei important in the consolidation of learned fear, such as the amygdala, PFC, and BNST (
      • Mahan A.L.
      • Ressler K.J.
      Fear conditioning, synaptic plasticity and the amygdala: Implications for posttraumatic stress disorder.
      ). As shown in Figure 4A, after extinction training (24 hours after fear conditioning), 14-day losartan treatment significantly reduced amygdala AT1 receptor mRNA expression (t11 = 2.8; p < .05); however, no change was observed in the PFC, and a trend for reduced AT1 receptor mRNA was observed in the BNST (Figure 4B,C). We also examined additional genes that might be involved in the consolidation of fear memory in these brain regions. As shown in Figure 5A, losartan did not alter brain-derived neurotrophic factor (bdnf) mRNA expression in the BNST, a gene that we have previously shown to be important in fear learning (
      • Choi D.C.
      • Maguschak K.A.
      • Ye K.
      • Jang S.W.
      • Myers K.M.
      • Ressler K.J.
      Prelimbic cortical BDNF is required for memory of learned fear but not extinction or innate fear.
      ,
      • Heldt S.A.
      • Stanek L.
      • Chhatwal J.P.
      • Ressler K.J.
      Hippocampus-specific deletion of BDNF in adult mice impairs spatial memory and extinction of aversive memories.
      ,
      • Rattiner L.M.
      Brain-derived neurotrophic factor and tyrosine kinase receptor B involvement in amygdala-dependent fear conditioning.
      ,
      • Chhatwal J.P.
      • Stanek-Rattiner L.
      • Davis M.
      • Ressler K.J.
      Amygdala BDNF signaling is required for consolidation but not encoding of extinction.
      ,
      • Choi D.C.
      • Gourley S.L.
      • Ressler K.J.
      Prelimbic BDNF and TrkB signaling regulates consolidation of both appetitive and aversive emotional learning.
      ). The mRNA expression of corticotropin releasing hormone (crh), a gene involved in both fear and HPA stress activation in the BNST (
      • Sink K.S.
      • Walker D.L.
      • Freeman S.M.
      • Flandreau E.I.
      • Ressler K.J.
      • Davis M.
      Effects of continuously enhanced corticotropin releasing factor expression within the bed nucleus of the stria terminalis on conditioned and unconditioned anxiety.
      ), was also not affected (Figure 5B). However, c-fos mRNA levels were significantly reduced in the BNST compared with the vehicle group (Figure 5C) (t13 = 2.2; p < .05), whereas the expression levels of these genes were unchanged in the PFC after extinction training (Figure 5D,E,F). Taken together, these gene expression studies suggest that losartan has a central effect on AT1 receptor and c-fos mRNA levels that seem to be region-specific (Figure 4A).
      Figure thumbnail gr4
      Figure 4Angiotensin receptor type 1 (AT1) messenger RNA (mRNA) gene expression after extinction training in losartan-treated mice. The AT1 receptor mRNA levels in the amygdala (A), prefrontal cortex (B), and bed nucleus of the stria terminalis (C) of vehicle- (n = 7) or losartan-infused mice (n = 7). (D) Example of coronal brain section with black squares designating the surrounding regions of the amygdala isolated for reverse transcriptase quantitative polymerase chain reaction (reprinted from Paxinos and Franklin
      [
      • Paxinos G.
      • Franklin K.
      The Mouse Brain in Stereotaxic Coordinates.
      ]
      with permission from Elsevier, copyright 2006); *p < .05.
      Figure thumbnail gr5
      Figure 5Messenger RNA (mRNA) levels in the bed nucleus stria terminalis and prefrontal cortex of vehicle- or losartan-infused mice after extinction training in losartan-treated mice: quantitative polymerase chain reaction–determined quantitative levels (fold changes) of mRNAs encoding BNST (A, B, C) and prefrontal cortex (D, E, F) (n = 7–8); *p < .05. BDNF, brain-derived neurotrophic factor; CRH, corticotropin releasing hormone.
      Angiotensin receptor type 1 inhibition has been previously found to inhibit the neuroendocrine stress response mediated by the HPA (
      • Krause E.G.
      • de Kloet A.D.
      • Scott K.A.
      • Flak J.N.
      • Jones K.
      • Smeltzer M.D.
      • et al.
      Blood-borne angiotensin II acts in the brain to influence behavioral and endocrine responses to psychogenic stress.
      ), which could influence fear extinction. With a standard, well-validated HPA axis stressor, we first examined the effects of losartan on plasma corticosterone as well as renin, a measure of endogenous angiotensin II activity. After 30 min of restraint stress, significant elevations in corticosterone levels were observed, but these levels were unaltered by losartan (F1,35 = 193.4; p < .0001) (Figure 6A). Similarly plasma renin activity was elevated after restraint and unchanged by losartan (Figure 6B) (t17 = 2.1; p < .05). As an index of central HPA neuronal activation, we then examined c-Fos activation in the PVN. As shown in Figure 6C, losartan-treated animals exhibited reduced PVN c-Fos activation after restraint stress (t10 = 2.3; p < .05). These data suggest that chronic AT1 receptor inhibition does not prevent elevations in peripheral indices of the stress response but does influence central stress activation sites in the brain such as the PVN, which might influence other neuronal circuits (i.e., amygdala, PFC, BNST) involved in the extinction of fear.
      Figure thumbnail gr6
      Figure 6Effect of chronic losartan on central and peripheral indices of stress activation. Plasma corticosterone levels (A) and plasma renin activity (B) in mice treated with vehicle or losartan for 2 weeks after acute restraint stress (n =9–10/group). Induction of c-Fos protein quantified as number of positive cell counts/section of paraventricular nucleus (C) (n = 5–7/group). Representative images showing reduced c-Fos protein induction in losartan versus vehicle animals exposed to acute restraint stress (D, E). *p < .05.

      Discussion

      Posttraumatic stress disorder is a debilitating psychiatric illness with increasing prevalence and limited treatment options. Moreover, PTSD and other chronic stress and anxiety disorders eventually lead to pathologies, including cardiovascular disease and hypertension (
      • Edmondson D.
      • Cohen B.E.
      Posttraumatic stress disorder and cardiovascular disease.
      ,
      • Coughlin S.S.
      Post-traumatic stress disorder and cardiovascular disease.
      ). In the present study we demonstrate that losartan, a selective AT1 receptor antagonist and widely used anti-hypertensive drug, enhances extinction and reduces fear memory. These data illustrate a novel role of the AT1 receptor in an animal model of PTSD-like symptoms and suggest that this class of medications might be particularly useful for decreasing fear responses and enhancing the extinction of fear memories. These results also begin to provide a mechanistic understanding to support our previous clinical report suggesting that inhibition of the renin-angiotensin system might be beneficial for patients diagnosed with PTSD (
      • Khoury N.M.
      • Marvar P.J.
      • Gillespie C.F.
      • Wingo A.
      • Schwartz A.
      • Bradley B.
      • et al.
      The renin-angiotensin pathway in posttraumatic stress disorder: Angiotensin-converting enzyme inhibitors and angiotensin receptor blockers are associated with fewer traumatic stress symptoms.
      ).
      The renin-angiotensin system has long been known to play a key role in cardiovascular homeostasis. Angiotensin II, the main effector molecule, binds to its receptor subtypes, which include AT1, angiotensin type 2 receptor (AT2), and angiotensin type 4 receptor (
      • Wright J.W.
      • Harding J.W.
      Brain angiotensin receptor subtypes AT1, AT2, and AT4 and their functions.
      ). The AT1 receptor is widely expressed across many organs, including the heart, kidney, and vasculature, and throughout many brain regions (
      • Gard P.R.
      The role of angiotensin II in cognition and behaviour.
      ). The major systemic cardiovascular effects, including elevation in blood pressure, increased sympathetic activity, and fluid homeostasis as well as proliferative and hypertrophic effects, are mediated by AT1 signaling. Therefore, antagonists of this receptor are widely used to treat hypertension and cardiovascular-related diseases. Several animal studies have also shown that, independent of their beneficial effects on hypertension and cardiovascular related diseases, ARBs can improve stress-related symptoms (
      • Bregonzio C.
      • Seltzer A.
      • Armando I.
      • Pavel J.
      • Saavedra J.M.
      Angiotensin II AT(1) receptor blockade selectively enhances brain AT(2) receptor expression, and abolishes the cold-restraint stress-induced increase in tyrosine hydroxylase mRNA in the locus coeruleus of spontaneously hypertensive rats.
      ,
      • Armando I.
      • Carranza A.
      • Nishimura Y.
      • Hoe K.L.
      • Barontini M.
      • Terrón J.A.
      • et al.
      Peripheral administration of an angiotensin II AT(1) receptor antagonist decreases the hypothalamic-pituitary-adrenal response to isolation Stress.
      ,
      • Shekhar A.
      Angiotensin-II is a putative neurotransmitter in lactate-induced panic-like responses in rats with disruption of GABAergic inhibition in the dorsomedial hypothalamus.
      ). A recent excellent review highlights the beneficial effects of AT1 antagonists on brain disorders, and they are suggested as potential therapy for neurodegenerative diseases such as Alzheimer’s disease (
      • Saavedra J.M.
      Angiotensin II AT(1) receptor blockers as treatments for inflammatory brain disorders.
      ). In support of these data, we recently reported in a clinical population diagnosed with PTSD that individuals taking blood pressure medications that blocked the renin-angiotensin system had fewer PTSD symptoms. This effect was not observed with other blood pressure medications in the study (
      • Khoury N.M.
      • Marvar P.J.
      • Gillespie C.F.
      • Wingo A.
      • Schwartz A.
      • Bradley B.
      • et al.
      The renin-angiotensin pathway in posttraumatic stress disorder: Angiotensin-converting enzyme inhibitors and angiotensin receptor blockers are associated with fewer traumatic stress symptoms.
      ). Our current study extends these findings as we demonstrate a role for AT1 receptor inhibition in the extinction of fear memories.
      Patients with PTSD and other anxiety disorders are thought to have deficits in extinction of aversive memories (
      • Wessa M.
      • Flor H.
      Failure of extinction of fear responses in posttraumatic stress disorder: Evidence from second-order conditioning.
      ,
      • Blechert J.
      • Michael T.
      • Vriends N.
      • Margraf J.
      • Wilhelm F.H.
      Fear conditioning in posttraumatic stress disorder: Evidence for delayed extinction of autonomic, experiential, and behavioural responses.
      ,
      • Norrholm S.D.
      • Jovanovic T.
      • Olin I.W.
      • Sands L.A.
      • Karapanou I.
      • Bradley B.
      • Ressler K.J.
      Fear extinction in traumatized civilians with posttraumatic stress disorder: Relation to symptom severity.
      ,
      • Glover E.M.
      • Jovanovic T.
      • Mercer K.B.
      • Kerley K.
      • Bradley B.
      • Ressler K.J.
      • Norrholm S.D.
      Estrogen levels are associated with extinction deficits in women with posttraumatic stress disorder.
      ). Similarly, rodents with anxiety-like behavior or trauma exposure demonstrate a deficit in extinction of conditioned fear (
      • Andero R.
      • Ressler K.J.
      Fear extinction and BDNF: Translating animal models of PTSD to the clinic.
      ). In the current study, mice treated with losartan one time or over 2 weeks show less retention of fear memory or enhanced extinction of fear. Interestingly, these effects were independent of baseline blood pressure, anxiety, or locomotor activities. Losartan has been previously shown to have anxiolytic effects (
      • Srinivasan J.
      • Jayadev S.
      • Kumaran D.
      • Ahamed K.F.
      • Suresh B.
      • Ramanathan M.
      Effect of losartan and enalapril on cognitive deficit caused by Goldblatt induced hypertension.
      ,
      • Lopez L.H.
      • Caif F.
      • Garcia S.
      • Fraile M.
      • Landa A.I.
      • Baiardi G.
      • et al.
      Anxiolytic-like effect of losartan injected into amygdala of the acutely stressed rats.
      ); therefore it is possible that AT1 receptor antagonism could influence the level of fear acquired during fear training. However, at the doses we used, animals exposed to losartan for 2 weeks acquired fear similarly to control groups. These data are in line with other studies showing that losartan does not influence baseline anxiety levels in rodents when given acutely or chronically (
      • Gard P.R.
      The role of angiotensin II in cognition and behaviour.
      ,
      • Braszko J.J.
      • Wincewicz D.
      • Jakubów P.
      Candesartan prevents impairment of recall caused by repeated stress in rats.
      ,
      • Braszko J.J.
      Valsartan abolishes most of the memory-improving effects of intracerebroventricular angiotensin II in rats.
      ) or on acquisition of an aversive memory (
      • Kulakowska A.
      • Karwowska W.
      • Wisniewski K.
      • Braszko J.J.
      Losartan influences behavioural effects of angiotensin II in rats.
      ). Moreover, these data demonstrate that AT1 receptor inhibition during fear conditioning enhances the extinction of an aversive memory and improves emotional learning, thus suggesting a role for endogenous angiotensin II in fear-related neurobiological processes.
      In many animal studies examining the role of angiotensin II and AT1 antagonism in learning and memory, inhibitory avoidance learning paradigms have been used, and these data have produced mixed results. For example, some have shown that angiotensin II improves avoidance memory (
      • Kulakowska A.
      • Karwowska W.
      • Wisniewski K.
      • Braszko J.J.
      Losartan influences behavioural effects of angiotensin II in rats.
      ,
      • Georgiev V.
      • Yonkov D.
      Participation of angiotensin II in learning and memory. I. Interaction of angiotensin II with saralasin.
      ,
      • Braszko J.J.
      • Wisniewski K.
      Effect of angiotensin II and saralasin on motor activity and the passive avoidance behavior of rats.
      ,
      • DeSouza F.A.M.
      • Sanchis-Segura C.
      • Fukada S.Y.
      • de Bortoli V.C.
      • Zangrossi H.
      • de Oliveira A.M.
      Intracerebroventricular effects of angiotensin II on a step-through passive avoidance task in rats.
      ), whereas others using similar learning paradigms have shown that angiotensin II impairs or has no effect on learning and memory (
      • Kulakowska A.
      • Karwowska W.
      • Wisniewski K.
      • Braszko J.J.
      Losartan influences behavioural effects of angiotensin II in rats.
      ,
      • Morgan J.M.
      Angiotensin injected into the neostriatum after learning disrupts retention performance.
      ,
      • Kerr D.S.
      • Bevilaqua L.R.M.
      • Bonini J.S.
      • Rossato J.I.
      • Köhler C.A.
      • Medina J.H.
      • et al.
      Angiotensin II blocks memory consolidation through an AT2 receptor-dependent mechanism.
      ,
      • Wright J.W.
      • Miller-Wing A.V.
      • Shaffer M.J.
      • Higginson C.
      • Wright D.E.
      • Hanesworth J.M.
      • Harding J.W.
      Angiotensin II(3-8) (ANG IV) hippocampal binding: Potential role in the facilitation of memory.
      ). Given that one of the main aims of the current study was to further understand the role of the renin-angiotensin system in the fear response, we used Pavlovian fear conditioning, a robust animal model for assessing memory in fear learning in both animals and humans. We demonstrate for the first time that inhibition of the AT1 receptor plays a role in the extinction of fear memory. In a comparison of these data with studies using inhibitory avoidance, our results would support studies suggesting that angiotensin II improves memory because, in the presence of the AT1 antagonist, memory retention as determined by freezing behavior was attenuated. Conversely, our results are in contrast to some studies using inhibitory avoidance, which show improvements in aversive memory after AT1 antagonism (
      • Barnes N.M.
      • Champaneria S.
      • Costall B.
      • Kelly M.E.
      • Murphy D.A.
      • Naylor R.J.
      Cognitive enhancing actions of DuP 753 detected in a mouse habituation paradigm.
      ,
      • Raghavendra V.
      • Chopra K.
      • Kulkarni S.K.
      Comparative studies on the memory-enhancing actions of captopril and losartan in mice using inhibitory shock avoidance paradigm.
      ,
      • Bonini J.S.
      • Bevilaqua L.R.
      • Zinn C.G.
      • Kerr D.S.
      • Medina J.H.
      • Izquierdo I.
      • Cammarota M.
      Angiotensin II disrupts inhibitory avoidance memory retrieval.
      ). These opposing results could be due to study differences in aversive learning paradigms, the dose of the AT1 antagonist and/or whether the drug was delivered via brain injection or systemically, and time of antagonist administration (i.e., before acquisition or retention).
      Studies have also shown that avoidance learning paradigms have produced inconsistent results with regard to the involvement of brain structures essential to the fear response such as the amygdala (
      • Tinsley M.R.
      The role of muscarinic and nicotinic cholinergic neurotransmission in aversive conditioning: Comparing Pavlovian fear conditioning and inhibitory avoidance.
      ). The amygdala is an integral part of the fear circuitry (
      • Jovanovic T.
      • Ressler K.J.
      How the neurocircuitry and genetics of fear inhibition may inform our understanding of PTSD.
      ), and key inputs to the amygdala from the medial PFC are thought to be required for the extinction of fear (
      • Likhtik E.
      • Pelletier J.G.
      • Paz R.
      • Paré D.
      Prefrontal control of the amygdala.
      ,
      • Likhtik E.
      • Popa D.
      • Apergis-Schoute J.
      • Fidacaro G.A.
      • Paré D.
      Amygdala intercalated neurons are required for expression of fear extinction.
      ). Immunohistological studies have revealed that brain AT1 receptors are expressed throughout regions involved in emotional learning, including the amygdala and hippocampus (
      • Marc Y.
      • Llorens-Cortes C.
      The role of the brain renin-angiotensin system in hypertension: Implications for new treatment.
      ,
      • Gonzalez A.D.
      • Wang G.
      • Waters E.M.
      • Gonzales K.L.
      • Speth R.C.
      • Van Kempen T.A.
      • et al.
      Distribution of angiotensin type 1a receptor-containing cells in the brains of bacterial artificial chromosome transgenic mice.
      ). In the current study, after extinction, losartan-treated animals showed decreased amygdala AT1 receptor mRNA expression as well as reduced c-fos mRNA levels in another key limbic structure involved in learned fear, the BNST (
      • Walker D.L.
      • Toufexis D.J.
      • Davis M.
      Role of the bed nucleus of the stria terminalis versus the amygdala in fear, stress, and anxiety.
      ,
      • Davis M.
      • Walker D.L.
      • Lee Y.
      Amygdala and bed nucleus of the stria terminalis: Differential roles in fear and anxiety measured with the acoustic startle reflex.
      ). The mechanism for this reduction in amygdala AT1 and BNST c-fos mRNA levels and whether it is a direct or indirect result of AT1 inhibition with systemic losartan is unclear. Although speculative, these data suggest that reduced AT1 receptor interaction might be contributing to the enhanced extinction; however, further AT1 receptor binding studies would be required and likely involve other brain angiotensin receptor subtypes such as AT2 and angiotensin type 4 receptor (
      • Wright J.W.
      • Harding J.W.
      The angiotensin AT4 receptor subtype as a target for the treatment of memory dysfunction associated with Alzheimer’s disease.
      ).
      Angiotensin type 1 receptor inhibition can also inhibit the central and peripheral neuroendocrine HPA stress response in rodents (
      • Armando I.
      • Volpi S.
      • Aguilera G.
      • Saavedra J.M.
      Angiotensin II AT1 receptor blockade prevents the hypothalamic corticotropin-releasing factor response to isolation stress.
      ,
      • Bregonzio C.
      • Seltzer A.
      • Armando I.
      • Pavel J.
      • Saavedra J.M.
      Angiotensin II AT(1) receptor blockade selectively enhances brain AT(2) receptor expression, and abolishes the cold-restraint stress-induced increase in tyrosine hydroxylase mRNA in the locus coeruleus of spontaneously hypertensive rats.
      ,
      • Armando I.
      • Carranza A.
      • Nishimura Y.
      • Hoe K.L.
      • Barontini M.
      • Terrón J.A.
      • et al.
      Peripheral administration of an angiotensin II AT(1) receptor antagonist decreases the hypothalamic-pituitary-adrenal response to isolation Stress.
      ), and therefore we speculated that these effects could influence learned fear. We demonstrate that, in response to restraint stress, mice treated with losartan for 14 days have decreased PVN c-fos activation, but surprisingly this treatment did not alter the downstream peripheral adrenal corticosterone response. This dissociation between central and peripheral indices of HPA activation might reflect the time frame chosen to analyze c-fos in our study or other neural input unrelated to the HPA response could be affecting c-fos activation, thus reflecting a limitation of this technique.
      Brain angiotensin II can also interfere with different neurotransmitters and hormones, such as norepinephrine, serotonin, vasopressin, and dopamine, which are all involved in memory consolidation (
      • Gard P.R.
      The role of angiotensin II in cognition and behaviour.
      ,
      • Braszko J.J.
      • Wisniewski K.
      Effect of angiotensin II and saralasin on motor activity and the passive avoidance behavior of rats.
      ). Moreover, angiotensin II has been found to modulate neurotrophic factors such as BDNF, a critical molecular mediator in learning and memory (
      • Szekeres M.
      • Nadasy G.L.
      • Turu G.
      • Supeki K.
      • Szidonya L.
      • Buday L.
      • et al.
      Angiotensin II-induced expression of brain-derived neurotrophic factor in human and rat adrenocortical cells.
      ). In our study, 14-day losartan treatment did not alter bdnf mRNA levels in the BNST or PFC after extinction training, although it is possible that amygdala bdnf might be involved in this pathway. In addition to bdnf, angiotensin II may activate other signaling pathways involved in fear memory that could indirectly influence AT1 or AT2 receptor expression and function. For example, Nostramo et al. (
      • Nostramo R.
      • Tillinger A.
      • Saavedra J.M.
      • Kumar A.
      • Pandey V.
      • Serova L.
      • et al.
      Regulation of angiotensin II type 2 receptor gene expression in the adrenal medulla by acute and repeated immobilization stress.
      ) recently showed in rat adrenal medulla cells in vitro that pituitary adenylate cyclase-activating polypeptide (PACAP), a key peptide in the cellular stress response, modulates the AT2 receptor. These authors suggested that, in response to stress, PACAP-triggered elevations in cyclic adenosine monophosphate in chromaffin cells of the adrenal medulla mediate a downregulation of AT2 receptor. Interestingly, in a clinical study, a single nucleotide polymorphism in the PACAP receptor gene as well as differential levels of circulating PACAP peptide have recently been linked to level of fear physiology and PTSD severity (
      • Ressler K.J.
      • Mercer K.B.
      • Bradley B.
      • Jovanovic T.
      • Mahan A.
      • Kerley K.
      • et al.
      Post-traumatic stress disorder is associated with PACAP and the PAC1 receptor.
      ).
      In summary, we show that inhibition of the AT1 receptor in mice enhances the extinction of fear memory, a process that is dysregulated in humans with PTSD. This was shown after both acute and repeated administration of losartan and was independent of effects on blood pressure, measures of anxiety, and fear acquisition. Furthermore, amygdala AT1 and c-fos BNST mRNA expression is reduced in losartan-treated animals after extinction training, which implies that downstream AT1 signaling events might be important in consolidation of extinction of fear. Importantly, these data also support the recent clinical observation that this class of medication might improve symptoms of PTSD (
      • Khoury N.M.
      • Marvar P.J.
      • Gillespie C.F.
      • Wingo A.
      • Schwartz A.
      • Bradley B.
      • et al.
      The renin-angiotensin pathway in posttraumatic stress disorder: Angiotensin-converting enzyme inhibitors and angiotensin receptor blockers are associated with fewer traumatic stress symptoms.
      ). Future questions for translating our current findings into potential novel therapies include identifying the appropriate timing and use of AT1 receptor antagonists as therapy after trauma exposure and determining whether circulating levels of angiotensin II are altered in patients diagnosed with PTSD and whether angiotensin receptor polymorphisms are present in these patients. Future studies are therefore aimed at further understanding these questions and the mechanism, because inhibition of this pathway might serve as a safe, powerful, and novel treatment for PTSD.
      This work was financially supported by The National Institutes of Health (K99 HL107675-01; R01 MH096764) and the Burroughs Wellcome Foundation. We thank the Emory University Biomarkers Yerkes Core supported by a National Primate Research Center Base Grant 2P51RR000165-51and Rodent Behavioral Cores for their contribution to this manuscript.
      All authors report no biomedical financial interests or potential conflicts of interest.

      Appendix A. Supporting information

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