Elsevier

Neuropharmacology

Volume 105, June 2016, Pages 114-123
Neuropharmacology

Increasing brain angiotensin converting enzyme 2 activity decreases anxiety-like behavior in male mice by activating central Mas receptors

https://doi.org/10.1016/j.neuropharm.2015.12.026Get rights and content

Highlights

  • Overexpression of Angiotensin Converting Enzyme (ACE2) is anxiolytic.

  • Activation of ACE2 in the brain using diminazene aceturate is anxiolytic.

  • Central antagonism of Mas Receptor (MasR) abolishes the anxiolytic effect of ACE2.

  • MasRs are highly expressed on GABAergic neurons in the basolateral amygdala (BLA).

  • ACE2 overexpression promotes GABAergic transmission in the BLA by acting on MasR.

Abstract

Over-activation of the brain renin-angiotensin system (RAS) has been implicated in the etiology of anxiety disorders. Angiotensin converting enzyme 2 (ACE2) inhibits RAS activity by converting angiotensin-II, the effector peptide of RAS, to angiotensin-(1–7), which activates the Mas receptor (MasR). Whether increasing brain ACE2 activity reduces anxiety by stimulating central MasR is unknown. To test the hypothesis that increasing brain ACE2 activity reduces anxiety-like behavior via central MasR stimulation, we generated male mice overexpressing ACE2 (ACE2 KI mice) and wild type littermate controls (WT). ACE2 KI mice explored the open arms of the elevated plus maze (EPM) significantly more than WT, suggesting increasing ACE2 activity is anxiolytic. Central delivery of diminazene aceturate, an ACE2 activator, to C57BL/6 mice also reduced anxiety-like behavior in the EPM, but centrally administering ACE2 KI mice A-779, a MasR antagonist, abolished their anxiolytic phenotype, suggesting that ACE2 reduces anxiety-like behavior by activating central MasR. To identify the brain circuits mediating these effects, we measured Fos, a marker of neuronal activation, subsequent to EPM exposure and found that ACE2 KI mice had decreased Fos in the bed nucleus of stria terminalis but had increased Fos in the basolateral amygdala (BLA). Within the BLA, we determined that ∼62% of GABAergic neurons contained MasR mRNA and expression of MasR mRNA was upregulated by ACE2 overexpression, suggesting that ACE2 may influence GABA neurotransmission within the BLA via MasR activation. Indeed, ACE2 overexpression was associated with increased frequency of spontaneous inhibitory postsynaptic currents (indicative of presynaptic release of GABA) onto BLA pyramidal neurons and central infusion of A-779 eliminated this effect. Collectively, these results suggest that ACE2 may reduce anxiety-like behavior by activating central MasR that facilitate GABA release onto pyramidal neurons within the BLA.

Introduction

Excessive uncontrollable anxiety is the most prevalent mental illness with ≈18% of adults in the U.S. being afflicted with some sort of anxiety disorder (Kessler et al., 2005). Despite therapeutic advancements, it is estimated that 40% of patients with anxiety disorders are resistant to treatment (Bystritsky, 2006). While medications such as benzodiazepines are effective at relieving anxiety, they are also associated with severe side effects such as sedation, memory impairments, tolerance and dependence (reviewed by (Uzun et al., 2010)). Accordingly, it is imperative to identify new medications that effectively relieve anxiety without deleterious side effects.

Patients with anxiety have increased risk for developing cardiovascular disease (Johannessen et al., 2006, Thurston et al., 2013) and a recent epidemiological study found that symptoms of posttraumatic stress disorder (PTSD), a type of anxiety disorder, are less severe in patients taking anti-hypertensive medications that target the renin-angiotensin-system (RAS) (Khoury et al., 2012). The effector peptide of the RAS is synthesized through a series of proteolytic cleavage events whereby renin converts angiotensinogen into angiotensin-I, which is cleaved by angiotensin converting enzyme (ACE) into angiotensin-II (Ang-II), which in turn, activates the angiotensin type 1a receptor (AT1aR). Several preclinical studies have found that stressful and anxiogenic stimuli promote Ang-II induced activation of AT1aRs but inhibiting this receptor or the synthesis of Ang-II dampens stress responsiveness and relieves fear and anxiety-like behavior in laboratory rats and mice (Castren and Saavedra, 1988, Krause et al., 2011b, Marvar et al., 2014, Saavedra et al., 2005). Taken together, these results suggest that over-activation of the ACE/Ang-II/AT1aR axis contributes to the etiology of anxiety disorders and therapies that limit its activation may be anxiolytic.

The relatively recent discovery of angiotensin converting enzyme 2 (ACE2) revealed a counter-regulatory limb of the RAS that opposes many of the deleterious consequences of AT1aR activation (reviewed by (Xu et al., 2011)). Angiotensin converting enzyme 2 metabolizes Ang-II into angiotensin 1–7 (Ang-(1–7)) which promotes cardio-protection by activating the Mas receptor (MasR) to cause vasodilation and decreased sympathetic nervous system activity (reviewed by (Xia and Lazartigues, 2010)). Emerging evidence suggests that this counter-regulatory limb of the RAS also influences physiological and behavioral responses to psychological stress. Genetic deletion of the MasR increases anxiety-like behavior in mice (Walther et al., 1998) but central administration of Ang-(1–7) blunts cardiovascular reactivity in response to psychogenic stress (Martins Lima et al., 2013) and attenuates anxiety-like behavior in rats (Bild and Ciobica, 2013, Kangussu et al., 2013). The relatively short half-life (≈10s) of Ang-(1–7) delivered in vivo in rats (Yamada et al., 1998) limits its therapeutic utility; however, ACE2 activity can be sustained pharmacologically in rats (Qi et al., 2013) and has the advantage of lowering Ang-II levels while also providing ligand for the MasR. Anti-hypertensive therapies have targeted the RAS for decades (Gavras et al., 1978), and consequently, it is established that interventions that suppress RAS activity are not associated with the deleterious side effects (e.g. sedation, dependence, memory impairment) that occur with some of the current anti-anxiety medications (Townsend, 2015). Therefore, ACE2 may be well-suited to serve as a therapeutic target to relieve anxiety; however, preclinical studies evaluating the effects of ACE2 on anxiety-like behavior have not been conducted.

The present study used genetic and pharmacological approaches in mice to test the hypothesis that increasing ACE2 in the brain reduces anxiety-like behavior by activating central MasRs. To identify brain circuits mediating the anxiolytic effects of ACE2, we assessed Fos induction within limbic regions subsequent to an anxiogenic stimulus. Experiments utilizing in situ hybridization and in vitro brain slice electrophysiology were conducted to reveal the neuronal phenotypes affected by MasR stimulation. Together, these studies suggest increasing brain ACE2 activity may be a novel therapuetic strategy for alleviating anxiety disorders.

Section snippets

Animals

All mice were male, 8–12 weeks-old at the initiation of the studies, individually-housed and given ad libitum access to pelleted rodent chow and water. All procedures were approved by the University of Florida Institutional Animal Care and Use Committee.

ACE2 KI mice robustly express ACE2 mRNA and have increased MasR mRNA in the amygdala

The hypothalami, amygdala, pituitaries and adrenals of ACE2 KI mice had significantly increased levels (P < 0.0001) of ACE2 mRNA (Fig. 1A) and significantly elevated (P < 0.0001) ACE2 activity (Fig. 1B) relative to WT littermates but ACE activity was similar between the groups (Fig. 1C). We measured mRNA levels for the AT1aR, AT2R, and MasR to determine whether increasing ACE2 influenced the expression of receptors for angiotensin-related peptides. Increasing ACE2 did not affect AT1aR and AT2R

Discussion

The goal of this study was to evaluate the effect of increased ACE2 on anxiety-like behavior in male mice. To this end, we determined that ubiquitous overexpression of ACE2 significantly reduced anxiety-like behavior assessed in the EPM. Increasing brain ACE2 activity with central administration of DIZE recapitulated the anxiolytic effects of ubiquitous ACE2 overexpression, demonstrating that pharmacologically up-regulating ACE2 activity in the brain is anxiolytic. Central administration of

Funding and disclosure

This study was supported by HL096830 (EGK), HL122494 (EGK), HL125805 (ADdK), HL116074 (ADdK), HL033610 (MKR), HL102033 (MKR and MJK), and HL 05921 (MKR and MJK).

Acknowledgments

No acknowledgements.

References (54)

  • R.C. Thurston et al.

    An anxious heart: anxiety and the onset of cardiovascular diseases

    Prog. Cardiovasc Dis.

    (2013)
  • Z. Tian et al.

    Estrogen receptor GPR30 exerts anxiolytic effects by maintaining the balance between GABAergic and glutamatergic transmission in the basolateral amygdala of ovariectomized mice after stress

    Psychoneuroendocrinology

    (2013)
  • T. Walther et al.

    Sustained long term potentiation and anxiety in mice lacking the Mas protooncogene

    J. Biol. Chem.

    (1998)
  • V.K. Yeragani et al.

    Diminished chaos of heart rate time series in patients with major depression

    Biol. Psychiatry

    (2002)
  • L.K. Becker et al.

    Immunofluorescence localization of the receptor Mas in cardiovascular-related areas of the rat brain

    Am. J. Physiol. Heart Circ. Physiol.

    (2007)
  • A. Bystritsky

    Treatment-resistant anxiety disorders

    Mol. Psychiatry

    (2006)
  • E. Castren et al.

    Repeated stress increases the density of angiotensin II binding sites in rat paraventricular nucleus and subfornical organ

    Endocrinology

    (1988)
  • P.J. Davern et al.

    Role of the sympathetic nervous system in Schlager genetically hypertensive mice

    Hypertension

    (2009)
  • M. Davis

    The role of the amygdala in fear and anxiety

    Annu. Rev. Neurosci.

    (1992)
  • M. Davis et al.

    Phasic vs sustained fear in rats and humans: role of the extended amygdala in fear vs anxiety

    Neuropsychopharmacology

    (2010)
  • A.D. de Kloet et al.

    Angiotensin type 1a receptors in the paraventricular nucleus of the hypothalamus protect against diet-induced obesity

    J. Neurosci.

    (2013)
  • A.D. de Kloet et al.

    Reporter mouse strain provides a novel look at angiotensin type-2 receptor distribution in the central nervous system, Brain Struct

    Funct

    (2014)
  • M.F. Doobay et al.

    Differential expression of neuronal ACE2 in transgenic mice with overexpression of the brain renin-angiotensin system

    Am. J. Physiol. Regul. Integr. Comp. Physiol.

    (2007)
  • S. Duvarci et al.

    Glucocorticoids enhance the excitability of principal basolateral amygdala neurons

    J. Neurosci.

    (2007)
  • A.J. Ferreira et al.

    Cardiovascular protection by angiotensin-converting enzyme 2: a new paradigm

    Future Cardiol.

    (2008)
  • K. Franklin et al.

    The Mouse Brain in Stereotaxic Coordinates

    (2008)
  • C.J. Frazier et al.

    Acute hypernatremia exerts an inhibitory oxytocinergic tone that is associated with anxiolytic mood in male rats

    Endocrinology

    (2013)
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