BJP British Journal of Pharmacology DOI:10.1111/bph.13052 www.brjpharmacol.org REVIEW Correspondence Autophagy: an emerging therapeutic target in vascular diseases Cécile Vindis, Inserm, UMR-1048, Institute of Metabolic and Cardiovascular Diseases, F-31432 Toulouse, France E-mail: cecile.vindis@inserm.fr Received July 2014 Revised 27 November 2014 Accepted December 2014 Cécile Vindis1,2 Inserm, UMR-1048, Institute of Metabolic and Cardiovascular Diseases, Toulouse, France, and University of Toulouse III, Toulouse, France Autophagy is a cellular catabolic process responsible for the destruction of long-lived proteins and organelles via lysosome-dependent pathway This process is of great importance in maintaining cellular homeostasis, and deregulated autophagy has been implicated in the pathogenesis of a wide range of diseases A growing body of evidence suggests that autophagy can be activated in vascular disorders such as atherosclerosis Autophagy occurs under basal conditions and mediates homeostatic functions in cells but in the setting of pathological states up-regulated autophagy can exert both protective and detrimental functions Therefore, the precise role of autophagy and its relationship with the progression of the disease need to be clarified This review highlights recent findings regarding autophagy activity in vascular cells and its potential contribution to vascular disorders with a focus on atherogenesis Finally, whether the manipulation of autophagy represents a new therapeutic approach to treat or prevent vascular diseases is also discussed Abbreviations 3-MA, 3-methyladenine; 4-HNE, 4-hydroxynonenal; 7-KC, 7-ketocholesterol; AGE, advanced glycation end product; AMPK, AMP-activated protein kinase; ATG, AuTophaGy-related genes; CVD, cardiovascular disease; EC, endothelial cell; ER, endoplasmic reticulum; MAP1-LC3, microtubule-associated protein light chain 3; mTOR, mammalian target of rapamycin; oxidized LDL, oxidized low-density lipoprotein; ROS, reactive oxygen species; SMCs, smooth muscle cells; VSMC, vascular SMC © 2014 The British Pharmacological Society British Journal of Pharmacology (2015) 172 2167–2178 2167 BJP C Vindis Tables of Links TARGETS LIGANDS Catalytic receptorsa Enzymesc 4-hydroxynonenal (4-HNE) Metformin CD40 AMPK Acrolein Methylglyoxal (MGO) Integrin, αM subunit (CD11b) ATG4B Carbamazepine Osteopontin (OPN) RTP Type C (CD45) Cathepsin D CD40L PDGF-BB TLR7 ERK Chemerin Phosphatidylethanolamine VEGFR-2 JNK Dexamethasone Phosphatidylserine Transportersb JNK Everolimus Rac1 ABCA1 p38MAPK Huntingtin Rapamycin ULK1 Imiquimod Simvastatin IL-1β TNF-α IFN-γ VCAM-1 Lithium Valproic acid Lysophosphatidylcholine von Willebrand factor These Tables list key protein targets and ligands in this article which are hyperlinked to corresponding entries in http:// www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Pawson et al., 2014) and are permanently archived in the Concise Guide to PHARMACOLOGY 2013/14 (a,b,cAlexander et al., 2013a,b,c) Introduction Autophagy is a ‘housekeeping’ subcellular process for lysosome-mediated turnover of damaged proteins and organelles first discovered by Christian De Duve in 1963 (De Duve, 1963) Autophagy is ubiquitous in eukaryotic cells, being highly conserved from yeast to human Three major forms of autophagy have been described: macroautophagy, microautophagy and chaperone-mediated autophagy Of these, the most prevalent and common form is macroautophagy This review will focus on macroautophagy, hereafter referred to as autophagy In this process, the cytoplasmic structures targeted for destruction are sequestered within double-membrane vesicles called autophagosomes and delivered to the lysosome by fusion for breakdown and possible recycling of the resulting macromolecules Although autophagy is generally considered to be nonspecific, other intracellular components have been suggested to be selectively targeted by autophagy Under specific conditions, mitochondria, endoplasmic reticulum (ER), peroxisomes, ribosomes, lipid droplets and bacterial pathogens could be sequestered and degraded by autophagosomes (He and Klionsky, 2009; Dong and Czaja, 2011; Youle and Narendra, 2011; Huang and Brumell, 2014) Under physiological conditions, autophagy has an essential homeostatic role by releasing nutrients from macromolecules and by eliminating unwanted constituents from the cell Autophagy can also be stimulated by stressful conditions including starvation; the degradation of cytoplasmic materials generates amino acids and fatty acids that are used to produce ATP for promoting cell survival (Levine and Yuan, 2005) Besides acting as a cell protector, autophagy participates in embryonic development (Cecconi and Levine, 2008), differentia2168 British Journal of Pharmacology (2015) 172 2167–2178 tion (Mizushima and Komatsu, 2011), longevity (Rubinsztein et al., 2011) and immunity (Ma et al., 2013) However, autophagy dysfunction is correlated with diverse pathologies, such as neurodegeneration, cancer, infection and ageing, and also with vascular disorders, including myocardial ischaemia and reperfusion, cardiomyopathy/heart failure, and atherosclerosis (Boya et al., 2013) Despite remarkable progress in this domain, the regulation and functional significance of autophagy in human diseases are still not well defined and, depending on the context, autophagy may act as both a protective and detrimental process In this review the current knowledge on the role of autophagy in vascular diseases, with a focus on atherosclerosis, is discussed and the therapeutic potential of manipulating autophagy as a treatment for vascular disorders addressed The molecular machinery of autophagy The details of the autophagic machinery have been already extensively described in several recent reviews (Feng et al., 2014) Therefore, only the major components of the autophagy machinery for understanding the basic concept of autophagy will be described here (Figure 1) The process of autophagy consists of four sequential steps ending with the degradation of cytosolic ‘cargo’ in lysosomes: initiation and nucleation of phagophore (isolation membrane), expansion of autophagosomes, maturation of autophagosomes into autolysosomes, and the execution of autophagy (final degradation) Autophagy is tightly regulated by more than 30 highly conserved genes called ATG (AuTophaGy-related Autophagy in vascular diseases Figure Overview of the autophagy machinery Once activated, autophagy proceeds through four sequential steps, each step requiring specific regulatory proteins and complexes Autophagy stimuli lead to the formation of two important complexes, Atg1/ULK1 and PI3K III/ Beclin1, which are necessary for the initiation/nucleation step During this step, phagophore structures are formed from plasma or organellar membranes, the double-lipid bilayer expands and wraps cytoplasmic materials yielding a closed multi-lamellar organelle termed autophagosome Two ubiquitin-like conjugation systems are part of the elongation and maturation steps One system involves the covalent conjugation of Atg12 to Atg5 with the help of the E1-like enzyme Atg7 and the E2-like enzyme Atg10 The Atg12–Atg5 conjugate in turn associates non-covalently with Atg16 The presence of Atg16 is required for the localization of Atg5 and Atg12 to the phagophore The second system involves the conjugation of phosphatidylethanolamine to LC3/Atg8 by the sequential action of Atg4, Atg7 and Atg3 Lipid conjugation leads to the conversion of the soluble form of LC3-I to the autophagosome-associated form LC3-II The autophagosome undergoes fusion with a late endosome or lysosome, to create an autolysosome, in which sequestered materials are degraded by lysosomal enzymes BJP genes) that were initially characterized in Saccharomyces cerevisiae (Tsukada and Ohsumi, 1993; Thumm et al., 1994; Harding et al., 1996; Klionsky et al., 2003), followed by the discovery of their mammalian orthologues (Mizushima et al., 2011) Once activated, autophagy begins with the formation of the phagophore (a precursor of autophagosomes), the origin of which is a subject of considerable debate Several recent data suggest a multi-membrane source model for the biogenesis of autophagosome in mammalian cells: the ER (Axe et al., 2008; Hayashi-Nishino et al., 2009; Yla-Anttila et al., 2009a), the outer membrane of the mitochondrion (Hailey et al., 2010), the ER-mitochondrial junction (Hamasaki et al., 2013), clathrin-coated vesicles from the plasma membrane (Ravikumar et al., 2010; Moreau et al., 2011), early endosomes (Longatti et al., 2012) and vesicles budding from the ER and Golgi (Hamasaki et al., 2003; Zoppino et al., 2010; Guo et al., 2012) In a very recent study, Ge et al (2013) identified the ER-Golgi intermediate compartment as the most efficient membrane substrate for the biogenesis of the phagophore, thus integrating these two putative sources Two major essential complexes regulate the recruitment of specific proteins into newly forming autophagosomal membranes The first one requires the class III PI3K Vps 34 which recruits the autophagy-specific proteins (Atg17, Atg13) in the region of phagophore formation This macromolecular complex can also contain Beclin1 (the mammalian orthologue of yeast Atg6), p150 Vsp15 (p150), Atg14 or Ambra1 The second complex involved in the early steps of autophagy involves ULK1 (also called Atg1) which interacts with Atg5, Atg12, Atg16, Atg13 and the focal adhesion kinase family-interacting protein of 200 kD (FIP200) The elongation of membranes for the formation of the autophagosome requires two ubiquitin-like conjugating systems The Atg12Atg5-Atg16L system : Atg12 is conjugated to Atg5 by Atg7 which is similar to an E1 ubiquitin-activating enzyme and Atg10 is similar to an E2 ubiquitin-conjugating enzyme Then the conjugated Atg12–Atg5 complex interacts with Atg16L and this complex associates with phagophores localized to the outer membrane of nascent autophagosomes, but it dissociates before the autophagosome is formed The second ubiquitin-like reactions involve the microtubule-associated protein light chain (MAP1-LC3/Atg8/LC3), the cytosolic form of LC3 LC3-I is generated by the cleavage of pro-LC3 by ATG4B LC3-I is then conjugated to the lipid phosphatidylethanolamine by Atg7 and Atg3 to form LC3-II (Ravikumar et al., 2010) Since LC3-II is specifically associated with autophagosomes, the level of LC3-II is correlated with the number of autophagosomes and is considered as an indicator of autophagosome formation (Tanida et al., 2008) The mature autophagosomes traffic along microtubules to endosomes or lysosomes using the dynein-dynactin complex, the fusion of autophagosomes with endosomes/lysosomes appears to be mediated by an endosomal sorting complex required for transport, soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), GTPase Rab7 proteins and with the lysosomal-associated membrane proteins, LAMP-1 and LAMP-2 In the final step of the autophagy process, the encapsulated ‘cargo’ is degraded by lysosomal proteases and released (Mizushima, 2007) Therefore, each step between autophagic processes should be tightly regulated for efficient autophagic degradation British Journal of Pharmacology (2015) 172 2167–2178 2169 BJP C Vindis Autophagy in atherosclerosis Despite recent advances in medical and interventional therapies, cardiovascular diseases (CVDs) continue to be the leading cause of death worldwide Atherosclerosis is, by far, the main cause of most CVDs It is a progressive, complex disease often associated with the ageing process and recognized risk factors such as hypercholesterolaemia, hypertension, diabetes and cigarette smoking Atherosclerosis involves the build-up of fibrous and fatty deposits called plaque inside the arteries It can affect all of the arteries, but particularly those that supply blood to the heart (coronaries), the neck arteries that supply blood to the brain (carotids), and the arteries that supply the legs (peripheral) (Lusis, 2000) The disease develops through several stages, ultimately ending with a complex plaque accumulated in the artery wall that impedes blood flow Acute clinical manifestations such as myocardial infarction or stroke are the result of rupture or ulceration of an ‘unstable’ atherosclerotic plaque A large number of studies involving analysis of angiographic data and histological assessment of ruptured plaques have indicated that the composition rather than plaque size or stenosis severity plays a critical role in plaque rupture and thrombosis (Falk et al., 1995) Therefore, today’s challenges are the early detection of rupture-prone or so-called vulnerable plaque and the development of strategies that achieve plaque stabilization Most of the advanced plaques are composed of a ‘fibrous cap’ consisting of vascular smooth muscle cells (VSMCs) and extracellular matrix that encloses a lipid- and macrophage-rich necrotic core For example, unstable plaques contain a higher portion of inflammatory cells and lipids, and a lower proportion of VSMC compared with stable lesions (Finn et al., 2010) Vulnerable plaques are also characterized by the accumulation of apoptotic cells and defective phagocytic clearance (efferocytosis), resulting in the lipid-filled necrotic core (Moore and Tabas, 2011) The mechanisms involved in plaque stability and plaque rupture are rather complex and the oxidizing and inflammatory environment generated by the presence of proatherogenic factors [low-density lipoprotein (LDL) and oxidized lipids, oxidative stress, cytokines] can trigger prosurvival and prodeath processes, which are concomitantly activated in cells The outcome (life vs death) depends on the balance between these pathways In addition to apoptosis, there is a growing body of evidence showing that autophagy occurs in developing atherosclerotic plaques (Martinet and De Meyer, 2009) However, in many cell settings, autophagy and apoptosis are often activated by the same stimuli, and share identical effectors and regulators (Codogno and Meijer, 2005; Maiuri et al., 2007) Thus, given the importance of the stage-specific consequences of apoptosis in atherosclerotic lesions and the intricate interplay between apoptosis and autophagy, there is no doubt that autophagy could play a crucial role in plaque progression Detection of autophagy in atherosclerotic lesions Strong evidence for the presence of autophagy features in atherosclerotic lesions is limited and its occurrence 2170 British Journal of Pharmacology (2015) 172 2167–2178 is probably not appreciated and underestimated Although detection guidelines have recently been established for monitoring autophagy in higher eukaryotes (Klionsky et al., 2012), the detection of autophagy in tissue is still difficult to evaluate due to technical limitations Transmission electron microscopy (TEM) is recognized as the most accurate method to assess autophagy in tissue allowing the visualization of double-membraned autophagic structures; however, this method is time consuming and not appropriate for daily routine (Yla-Anttila et al., 2009b) Martinet et al (2013) recently evaluated the feasibility and specificity of immunohistochemical assessment of macroautophagyrelated marker proteins such as LC3, Atg5, CTSD/cathepsin D, Beclin1 or p62/SQSTM1 From their study, they concluded that only LC3 detection is suitable for monitoring autophagy; nevertheless, its staining in the different organs tested (liver, heart, kidney and gut) required a high-quality isoform-specific antibody coupled to a signal amplification system and overexpression of LC3 (e.g by GFP-LC3 mice) Therefore, when genetic manipulation or other in vitro techniques are not feasible, TEM remains the gold standard method for in situ evaluation of macroautophagy in human tissue samples (Martinet et al., 2013) Several studies have reported that TEM analysis of dying VSMC of both human and cholesterol-fed rabbit atherosclerotic plaques exhibit certain features of autophagy, such as vacuolization, formation of myelin figures and the inclusion of cytoplasmic ubiquitin (Kockx et al., 1998; Martinet et al., 2004; Jia et al., 2006) A recent report that provided a complete ultrastructural documentation of the autophagic process in human atherosclerotic plaques definitively confirmed that all the major cell types [smooth muscle cells (SMCs), macrophages and endothelial cells (ECs)] found in the lesion may undergo autophagic activation (Perrotta, 2013) However, this analysis did not address whether these observations on human atherosclerotic plaques were at a lesion-specific stage (early vs more complicated plaques) The marker proteins of autophagy, such as LC3-II, SQSTM1/p62 and Beclin1, have also been detected by immunoblot and immunofluorescence microscopy analysis in human plaques (Martinet et al., 2007) and in mouse models of atherosclerosis (Martinet et al., 2007; Liao et al., 2012; Razani et al., 2012) Although murine models are currently the most extensively used for atherosclerosis studies, caution must be taken when extrapolating mechanisms to human disease because representative lesions in mice models often consist of lipid-laden intimal macrophages without a well-developed fibrous cap or necrosis, both seen in chronic human atherosclerosis Additionally, intraplaque haemorrhage (IPH) in human plaques, which is a significant factor in necrotic core expansion, is rarely observed in mice (Getz and Reardon, 2012) Autophagic stimuli in vascular cells Several in vitro studies have demonstrated that autophagy can be induced by various pro-atherogenic stimuli in vascular cells (Table 1) Autophagy in vascular diseases BJP Table Autophagic stimuli in vascular cells Stimuli Setting/cell type References TNF-α Human atherosclerotic plaque/SMC Jia et al., 2006 Chemerin Angiogenesis/EC Shen et al., 2013 Osteopontin Abdominal aortic aneurysm/SMC Zheng et al., 2012 PDGF Phenotype transition/SMC Salabei et al., 2013 Shh Neointimal artery lesion/SMC Li et al., 2012 Oxidized LDL Apoptotic cell phagocytosis/EC Muller et al., 2011a LOX-1 down-regulation/EC Nowicki et al., 2007 Mouse atherosclerotic lesion/SMC Ding et al., 2013 4-HNE/acrolein/POVPC Removal of aldehyde-modified protein/SMC Hill et al., 2008 7-KC Mouse atherosclerotic lesion/SMC He et al., 2013 Human atherosclerotic lesion/SMC Martinet et al., 2004 AGE Cell proliferation/SMC Hu et al., 2012 MGO Angiogenesis/EC Liu et al., 2012 Hypoxia Inhibition of cell proliferation/SMC Lee et al., 2011b; Ibe et al., 2013 MGO, methylglyoxal; POVPC, 1-palmitoyl-2-oxovaleroyl phosphatidylcholine Induction of autophagy by cytokines Inflammatory cytokines, such as INF-γ and TNF-α, and CD40CD40-L interactions can induce autophagy in particular settings or conversely suppress it (Levine and Yuan, 2005; Deretic, 2011; Levine et al., 2011; Maiuri et al., 2013) TNF-α, which is secreted by inflammatory cells and SMCs in atheromas, was shown to increase vacuolization and the expression of LC3-II and Beclin1 in SMCs isolated from human atherosclerotic plaques (Jia et al., 2006) Other cytokines such as osteopontin (OPN), a protein involved in vascular inflammation, are able to induce autophagosome formation, the up-regulation of LC3 protein and autophagy-related genes, leading to VSMC cell death in abdominal aortic aneurysms (Zheng et al., 2012) Since inhibition of the integrin/CD44 and p38 MAPK-signalling pathways prevented OPN-induced autophagy, the authors concluded that OPN stimulates autophagy directly through the integrin/CD44 and p38 MAPK-mediated pathways in SMCs Interestingly, the adipokine chemerin contributes to human aorta EC angiogenesis through the up-regulation of autophagic activity (Shen et al., 2013) Because chemerin is associated with obesity and metabolic syndrome, the potential role of chemerin-induced autophagy in the neovascularization of atherosclerotic lesions needs to be further explored Induction of autophagy by reactive lipids Reactive oxygen species (ROS) (Scherz-Shouval and Elazar, 2011; Lee et al., 2012), oxidized LDL and secondary products of the oxidative degradation of lipids have all been implicated in the activation of autophagy Treatment of vascular ECs (Nowicki et al., 2007; Muller et al., 2011a) and SMCs (Ding et al., 2013) with oxidized LDL triggers an increase in autophagy-related proteins and in autophagosome formation Interestingly, exposure of SMCs to modest amounts of highly oxidized LDL (10–40 μg·mL−1) enhances autophagy and apoptosis, whereas exposure to higher concentrations (≥60 μg·mL−1) induces high levels of apoptosis and impairs autophagy, indicating that the stress response evoked by autophagy becomes defective when a threshold of cell injury is reached The oxidative degradation of lipids in lipoproteins leads to the generation of bioactive lipid intermediates and peroxidation end products (Esterbauer et al., 1992) Reactive lipid species such as free aldehydes [e.g 4-hydroxynonenal (4-HNE), acrolein] and to a lesser extent lipid hydroperoxides (e.g 1-palmitoyl-2-oxovaleroyl phosphatidylcholine) cause a robust increase in LC3-II, and electron micrographs of 4-HNE-treated SMCs show extensive vacuolization, pinocytic body formation, crescent-shaped phagophores and multilamellar vesicles (Hill et al., 2008) Likewise, human SMCs and mice macrophages exposed to 7-ketocholesterol (7-KC), one of the major oxysterols present in atherosclerotic plaques, display signs of ubiquitination and features of the autophagy process (Martinet et al., 2004; Liao et al., 2012) Recently, He et al (2013) investigated the molecular mechanism by which 7-KC induced autophagy in human SMCs Their study demonstrated that 7-KC increases Nox4-mediated ROS production, which triggers autophagy in SMC by inhibiting ATG4B activity Induction of autophagy by advanced glycation end products (AGEs) and hypoxia Driven by hyperglycemia and oxidative stress, the formation of AGEs has a pathophysiological role in the development and progression of different oxidative-based diseases including diabetes, atherosclerosis and neurological disorders (Giacco and Brownlee, 2010) Their putative role in the induction of autophagy has been recently demonstrated in vascular cells AGE-promoted autophagy was shown to conBritish Journal of Pharmacology (2015) 172 2167–2178 2171 BJP C Vindis tribute to cell proliferation through ERK, JNK and p38 activation in rat aortic SMCs, thus suggesting that the AGEautophagy pathway can accelerate the development of atherosclerosis in diabetic patients (Hu et al., 2012) Angiogenesis impairments in diabetic peripheral vasculature contribute to the delayed wound healing, the exacerbated peripheral limb ischaemia and even cardiac mortality in diabetic patients Methylglyoxal, a highly reactive α-oxoaldehyde, reduces endothelial angiogenesis through peroxynitrite (ONOO−)-dependent and autophagy-mediated VEGFR-2 protein degradation, which may represent a mechanism for diabetes-impaired angiogenesis (Liu et al., 2012) Atherosclerotic plaques develop intraplaque neovascularization, which is a typical feature of hypoxic tissue (Sluimer et al., 2008), and mice deficient in the autophagic protein Beclin1 display a pro-angiogenic phenotype associated with hypoxia (Lee et al., 2011a) Interestingly, in human cultured pulmonary vascular cells exposed to hypoxia, autophagy activation inhibits the hypoxic proliferation of these cells Moreover, hypoxia has been shown to activate autophagy through the metabolic sensor AMPK in human pulmonary SMCs and the suppression of AMPK expression prevents hypoxia-mediated autophagy and the induction of cell death (Ibe et al., 2013) Nevertheless, how hypoxia contributes to the induction of autophagy in atherosclerotic lesions remains to be determined Induction of autophagy by growth factors Vascular injury and chronic arterial diseases result in exposure of vascular SMCs to increased concentrations of growth factors As a consequence, SMCs develop a highly proliferative and synthetic phenotype Treatment of vascular SMCs with PDGF or sonic hedgehog (Shh) increases the expression of the synthetic phenotype markers and promotes autophagy, as assessed by LC3-II abundance, LC3 puncta formation and TEM (Li et al., 2012; Salabei et al., 2013) Autophagy mediated by PDGF or Shh is involved in the proliferation of SMCs and its pharmacological inhibition by 3-MA appears to prevent arterial restenosis However, the mechanisms involved in growth factor-promoted autophagy need to be further elucidated Functional role of autophagy in atherosclerosis: friend or foe? The functional role of autophagy in vascular diseases is currently under intense investigation and studies have characterized this process both in vitro and in vivo Given that increases in autophagy have been observed in various CVDs, a key unanswered question is whether autophagy is protective or harmful in vascular pathology Both beneficial and detrimental functions have beeen assigned to autophagy during atherosclerosis progression (Martinet and De Meyer, 2009) Recent data have shed light on the protective role of macrophage autophagy in the regulation of atherosclerotic plaque development Using ApoE-null mice, a wellestablished model to study atherogenesis, Razani et al (2012) showed that the autophagy markers p62/SQSTM1 and LC3 are mainly colocalized with plaque leukocytes (CD45 positive 2172 British Journal of Pharmacology (2015) 172 2167–2178 cells) and monocyte macrophages (CD11b, MOMA-2 positive cells) Interestingly, autophagy became defective in progressing atherosclerotic plaques from ApoE-null mice as assessed by the accumulation of the p62/SQSTM1 in atherosclerotic aortas Moreover, in ApoE-null mice completely lacking macrophage autophagy, enhanced plaque formation was observed and this led to macrophage inflammasome hyperactivation accompanied by increased IL-1β production The putative link between defective autophagy and activation of inflammasome could involve different mechanisms: (i) an increase in ROS production due to impaired mitophagy, since release of ROS from damaged mitochondria can activate inflammasome (Naik and Dixit, 2011; Nakahira et al., 2011), or (ii) the accumulation of dysfunctional lysosomes due to phagocytosed cholesterol crystals (Masters et al., 2011) However, recent data have shown that the induction of lysosomal biogenesis blunts the lysosomal dysfunction and inflammasome activation in macrophages isolated from atherosclerotic plaques, even in the absence of autophagy, thus supporting the involvement of additional mechanisms (Emanuel et al., 2014) Similarly, the group of Tabas has provided additional evidence for the protective role of macrophage autophagy (Liao et al., 2012) They explored how autophagy inhibition affects both apoptosis and phagocytic clearance (efferocytosis) in Atg5-deficient macrophages exposed to oxidative/ER stressors and in advanced atherosclerotic lesions They showed that defective macrophage autophagy led to increased apoptosis and oxidative stress in advanced lesional macrophages, promoted plaque necrosis and worsened efferocytosis in Atg5-deficient macrophage/LDLR-null mice The mechanism involved in defective efferocytosis of autophagy-inhibited apoptotic macrophages has not been fully elucidated, but the authors hypothesized that defective autophagy impairs the recognition and internalization of apoptotic cells by phagocytes perhaps by decreasing the expression of cell surface recognition molecules This makes sense since dying cells lacking the autophagy genes, Atg5 or Beclin1 in embryoid bodies, fail to express the ‘eat-me’ signal, phosphatidylserine (PS), and secrete lower levels of the ‘come-get-me’ signal, lysophosphatidylcholine (Qu et al., 2007) In support of these data, we found that vascular ECs silenced for Beclin1 and exposed to oxidized LDL exhibit less PS externalization and uptake by phagocytic macrophages (Muller et al., 2011a) Given the importance of efferocytosis in preventing plaque rupture, further investigations are necessary to establish why autophagy and efferocytosis fail during lesion progression Interestingly, the protective function of autophagy against atherosclerosis has been also linked with cholesterol metabolism and lipophagy Indeed, lipid droplets can be delivered to lysosomes through autophagy, thus facilitating the hydrolysis of cholesterol esters and subsequent ABCA1mediated cholesterol efflux (Ouimet et al., 2011) These findings were corroborated in Wip1-deficient mice The deletion of the Wip1 phosphatase, a known negative regulator of Atm-mTOR-dependent signalling, resulted in activated autophagy, suppression of macrophage conversion into foam cells and prevention of atherosclerotic plaque formation (Le Guezennec et al., 2012) The regulation of cholesterol efflux and autophagy via Wip1 may provide the basis to design Autophagy in vascular diseases novel therapeutic strategies for efficient cholesterol removal from foam cells, and thereby reduce lipid load in early atherosclerotic plaques Besides the protective role of macrophage autophagy in atherosclerotic plaque development, autophagy plays an important role in preserving vascular endothelial function by reducing oxidative stress and inflammation and increasing NO bioavailability (LaRocca et al., 2012) The activation of ECs by oxidized LDL with the subsequent increase in endothelial permeability occurs in the early stage of atherosclerosis Hence, the molecular mechanisms linking autophagy to endothelial dysfunction involve the degradation of oxidized LDL through the autophagic lysosome pathway as demonstrated by the colocalization of Dillabelled oxidized LDL with LC3 and LAMP-2 (Zhang et al., 2010) In addition, vascular ECs exposed to oxidized LDL undergo autophagy activation and phagocytic signal exposure through a common mechanism involving Beclin1 (Muller et al., 2011b) Therefore, it is conceivable that autophagy is actually anti-atherogenic, by favouring the processing of oxidized LDL and the clearance of pro-thrombotic apoptotic cells Interestingly, endothelial secretion of von Willebrand factor required for platelet adhesion to the injured vessel wall is altered in mice with an endothelial specific deletion of Atg7 although these animals have normal vessel architecture and capillary density (Torisu et al., 2013) In the context of IPH, autophagy may have a beneficial role against hemin-induced EC death by clearing the mitochondrial proteins modified by lipid peroxidation (Higdon et al., 2012) Overall, these observations suggest that modulating the autophagic flux may be a useful strategy for preventing thrombotic events The general consensus is that successful autophagy of damaged components protects plaque cells against oxidative stress and promotes cell survival Loss of SMCs contributes to the thinning of the fibrous cap which results in plaque destabilization and rupture (Clarke et al., 2006) Several reports have pointed to the beneficial role of SMC autophagy Martinet et al (2004; 2008) showed that aortic SMC death induced by low concentrations of statins was reduced by 7-KC-induced autophagy Similarly, a recent study demonstrated that the up-regulation of autophagy by 7-KC is protective and could be mediated by Nox4-induced ROS production (He et al., 2013) Inhibition of autophagy enhanced both cell apoptosis and necrosis; in contrast, the autophagy inducer rapamycin inhibited cell death of SMCs overloaded with an excess of free cholesterol (Xu et al., 2010) Furthermore, autophagy may be an important mechanism for the survival of vascular SMCs under conditions associated with excessive lipid peroxidation, since autophagy was shown to remove aldehyde-modified proteins, and inhibition of autophagy precipitates cell death in aldehyde-treated SMCs (Hill et al., 2008) Mechanistically, how autophagy suppresses SMC death programmes is not fully understood One possible mechanism could involve JNK-dependent ER stress activation, since the inhibition of ER stress with the chemical chaperone 4-phenylbutyric acid prevents JNK phosphorylation and autophagy (Haberzettl and Hill, 2013) In contrast, He et al (2013) demonstrated that 7-KC-triggered autophagy prevents SMC death by suppressing the ER stress-apoptosis BJP pathway, and the up-regulation of autophagy by rapamycin exhibited opposite effects However, these discrepancies could be explained by the nature of the stimuli 4-HNE, which is known to covalently modify proteins, has been found to promote the carbonylation of ER-sensor proteins such as protein disulfide isomerase, glucose-regulated protein 78; thereby causing unfolded protein response/ER stress and JNK activation (Haberzettl and Hill, 2013) Conversely, the inhibition of ER stress by 7-KC-induced autophagy could result from enhanced intracellular ROS, leading to ATG4B inhibition, thereby promoting autophagy; however, the molecular mechanisms underlying this process require further investigation (He et al., 2013) Another potential mechanism could involve the autophagic removal of damaged mitochondria (also called mitophagy), thus limiting the release of pro-apoptotic proteins such as cytochrome c As mentioned above, autophagy is predominantly considered as a protective mechanism in atherosclerosis; however, overwhelming stress and excessively stimulated autophagy may cause the autophagic death of SMCs (Levine and Yuan, 2005) leading to reduced collagen synthesis, thinning of the fibrous cap and ultimately to plaque destabilization Similarly, the autophagic death of ECs may increase vascular permeability and platelet aggregation, which enhance the risk of thrombosis and acute clinical events (Martinet and De Meyer, 2009) Interestingly, a novel role for autophagy in regulating VSMC phenotype has been recently uncovered Treatment of vascular SMCs with PDGF-BB which promotes the development of the synthetic vascular SMC phenotype is a robust inducer of autophagy as assessed by LC3-II abundance, LC3 puncta formation and electron microscopy (Salabei et al., 2013) Inhibition of autophagy blocked the degradation of contractile proteins and prevented the hyperproliferation and migration of SMCs, thus supporting the view that autophagy is required for PDGF-induced phenotype conversion and could have a detrimental role in the setting of restenosis However, future studies are necessary to identify the signalling pathway by which growth factors such as PDGF activate the autophagic programme Pharmacological modulation of autophagy in vascular diseases Pharmacological approaches to modulate autophagy have currently gained increasing attention in the treatment of CVDs Several drugs that have the potential to inhibit or stimulate autophagy have already been identified (Fleming et al., 2011), and now ongoing clinical trials are testing their association with cytotoxic drugs in a variety of cancers (Cheng et al., 2013) Activators of autophagy (Table 2), for instance, rapamycin and its derivatives (everolimus) that trigger autophagy through the inhibition of mTOR (mammalian target of rapamycin), have been evaluated as potential plaque stabilizing drugs Local stent-based delivery of everolimus in atherosclerotic plaques from cholesterol-fed rabbits led to a striking reduction in macrophage content without altering SMCs (Verheye et al., 2007) In vitro studies showed that treatment of macrophages and SMCs with everolimus induced the inhibition of de novo protein synthesis in both British Journal of Pharmacology (2015) 172 2167–2178 2173 BJP C Vindis Table Pharmacological modulation of autophagy in the context of atherosclerosis Compound Pharmacological References Inducer Everolimus Macrolide, rapamycin derivative, mTOR inhibitor Verheye et al., 2007 Simvastatin Statin, HMG-CoA reductase inhibitor Wei et al., 2013 Imiquimod Imidazoquinoline, Toll-like receptor ligand De Meyer et al., 2012 z-VAD-fmk Fluoromethylketone, pan-caspase inhibitor Martinet et al., 2006 Valproic acid Carboxylic acid, myo-inositol-1-phosphate synthase inhibitor Dai et al., 2013 Trehalose Disaccharide, chemical chaperone LaRocca et al., 2012 Inhibitor Spautin-1 Fluorobenzylquinozaline, deubiquitinases USP10 and USP13 inhibitor Salabei et al., 2013 3-Methyladenine Purine derivative, class III PI3K inhibitor Salabei et al., 2013 Bafilomycin A1 Macrolide antibiotic, vacuolar-type H+-ATPase inhibitor Salabei et al., 2013 The pharmacological characteristics and mode of action of selected compounds that have been shown to modulate autophagy in the context of atherosclerosis are presented cell types by dephosphorylating the downstream mTOR target p70 S6 kinase The inhibition of translation promoted selective macrophage death and was characterized by bulk degradation of long-lived proteins, processing of LC3 and cytoplasmic vacuolization, which are all markers of autophagy The authors proposed that the macrophage selectivity is most likely due to the elevated metabolic activity of macrophages that makes them more sensitive to protein synthesis inhibitors than SMCs; however, protein translation inhibition can render SMCs less sensitive to cell death due to contractile-to-quiescent phenotype transition Hence, because macrophage efferocytosis and autophagy flux decreases as atherosclerosis progresses (Liao et al., 2012; Razani et al., 2012), the clearance of lesional macrophages in the vascular wall via everolimus-induced autophagy could be a promising strategy to promote stable plaque phenotype Although mTOR inhibitors have been shown to attenuate plaque progression in atherogenic models, they also enhance macrophage cholesterol efflux and reverse cholesterol transport A previous report demonstrated that sirolimus treatment for 12 weeks specifically reduces the cholesterol content of the aortic arch of ApoE-null mice compared with untreated mice (Basso et al., 2003) In support of the latter, two recent studies (Ouimet et al., 2011; Le Guezennec et al., 2012) have demonstrated that autophagy plays a role in the hydrolysis of stored cholesterol droplets in macrophages, thus facilitating cholesterol efflux Nevertheless, therapy with mTOR inhibitors is associated with side effects such as hypercholesterolaemia and hyperglycemia, which are not compatible with plaque stabilization (Martinet et al., 2014) Because statins lower plasma cholesterol and have been shown to induce autophagy via AMPK activation (Zhang et al., 2012) and/or Rac1-mTOR signalling (Wei et al., 2013), the combination of mTOR inhibitors with statin therapy would be beneficial to potentiate mTOR inhibitor-induced autophagy and to prevent unstable plaques Furthermore, hyperglycaemia could be manageable with the anti-diabetic drug metformin that lowers blood glucose levels but also triggers AMPK acti2174 British Journal of Pharmacology (2015) 172 2167–2178 vation through mTOR inhibition (Liao et al., 2011) Therefore, the development of a new generation of mTOR inhibitors with limited off-target effects would undeniably enhance their efficiency to treat vascular diseases Autophagy can also be modulated through mTORindependent pathways, albeit with different outcomes on plaque phenotype as described previously Macrophages express Toll-like receptors (TLRs) that recognize pathogens and eliminate intracellular pathogens by inducing autophagy Local administration of a TLR7 ligand imiquimod in atherosclerotic plaques of cholesterol-fed rabbits induced macrophage autophagy without affecting SMCs (De Meyer et al., 2012) Surprisingly, autophagy activation via imiquimod was detrimental because it was associated with cytokine release, up-regulation of VCAM-1, infiltration of T-cells and plaque progression The deleterious effect of imiquimod could be explained by its ability to activate NF-κB which could repress autophagy Although treatment with dexamethasone suppressed these pro-inflammatory effects in vivo, caution must be taken since TLR7 stimulation could play a role in promoting atherosclerosis by activating dentritic cells homing to atherosclerotic vessels (Doring et al., 2012; Macritchie et al., 2012) Several other drugs can induce autophagy by an mTOR-independent pathway, mainly by the regulation of inositol-1,4,5-triphosphate (IP3) levels, but whether these drugs affect macrophage cell fate or other cell types in the plaque is currently unknown Carbamazepine, valproic acid and lithium increase the intracellular clearance of misfolded protein accumulation through induction of autophagy by reducing the intracellular levels of IP3 (Williams et al., 2002; Sarkar et al., 2005) Interestingly, stimulation of autophagy by valproic acid decreases calcification by reducing matrix vesicle release in vascular SMCs (Dai et al., 2013) Additionally, using a cell-based screening method, several calcium channel blockers (CCBs) and antiarrhythmic drugs, such as verapamil, loperamide, amiodarone, nimodipine, nitrendipine, niguldipine and pimozide, have been identified as autophagy inducers by inhibiting Autophagy in vascular diseases intracellular levels of calcium (Fleming et al., 2011) Previous studies have shown that CCBs have anti-atherogenic effects beyond their BP-lowering effects Their pleiotropic actions in vascular cells involve, for instance, suppression of ROS and inflammation, inhibition of SMC proliferation and migration or activation of peroxisome proliferator-activated receptor gamma (PPAR-γ), but whether these effects are linked to the induction of autophagy has not presently been determined and certainly needs to be investigated further The pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-DL-Asp(Omethyl)-fluoromethylketone (z-VAD-fmk) has the ability to induce autophagy and necrotic cell death in macrophages and, indirectly, necrosis of vascular SMCs based mainly on the differential expression of receptor-interacting protein (Martinet et al., 2006) Consequently, a caspase inhibitor may have a detrimental effect due to stimulation of inflammatory responses and, indirectly, SMC necrosis Trehalose, a disaccharide present in many nonmammalian species (Sarkar et al., 2007), enhances the clearance of autophagy substrates such as mutant huntingtin and A53T α-synuclein, which are associated with Huntington’s disease and familial Parkinson’s disease Trehalose supplementation restores the expression of autophagy markers and rescues vascular endothelial function by increasing NO bioavailability, reducing oxidative stress and normalizing inflammatory cytokines in arteries of ageing mice (LaRocca et al., 2012) Advancing age is a major risk factor for CVD, therefore autophagy-enhancing strategies may have therapeutic efficacy for ameliorating age-associated arterial dysfunction A few studies have described a beneficial role of pharmacological inhibition of autophagy in vascular diseases Recently, Salabei et al (2013) revealed that autophagy plays a role in the contractile-to-synthetic VSMC phenotype transition induced by growth factors Autophagy inhibition by three pharmacological unrelated inhibitors, such as 3-methyladenine, spautin-1 or bafilomycin A1, stabilized the contractile phenotype The remarkable efficiency of spautin-1 in vitro suggests that it might be a useful therapeutic agent for preventing the phenotype switching and proliferation that occur in vascular injury, such as restenosis Conclusion and future challenges In conclusion, there is mounting evidence showing that autophagy plays a critical role in vascular diseases such as atherosclerosis Although many autophagic-specific genes and the basic molecular machinery of autophagy have now been well characterized, a first challenge is to identify more selective pharmacological compounds that target unique molecular effectors/regulators of autophagy to specifically modulate the process Similarly, it is also crucial to establish which of the four sequential autophagy steps should be preferentially targeted to develop a successful autophagy-based therapy Currently, the pharmacological modulation of autophagy by blocking mTOR function has shown beneficial effects on plaque phenotype An alternative approach to circumvent their side effects will be to explore compounds that control autophagy downstream of the mTOR complex, for instance, BJP the Beclin1 complex or the ubiquitin-like conjugation systems However, caution must be taken when enhancing autophagosome formation if impaired lysosome activity also takes place with the disease The consequences of the accumulation of autophagosomes in the cytosol could be detrimental for the cell A second challenge is to achieve a definite understanding of the autophagy process at all stages of the atherosclerotic lesion Indeed, the relevance of beneficial autophagy in the early stages and a dysfunctional autophagy observed in the late stages of mouse atherosclerotic models remains to be demonstrated in human clinical samples before we can consider targeting autophagy in the treatment of vascular diseases Moreover, the favourable effects of mTOR inhibitors on preventing the early stages of atherogenesis, such as monocyte recruitment, macrophage accumulation and SMC phenotypic modulation require further investigation to prove their effectiveness on the restoration of autophagy in advanced lesions Given the central role of macrophages in atherosclerotic plaque destabilization, the selective clearance of lesional macrophages in atherosclerotic plaques via drug-induced autophagy is a hopeful strategy However, chronic or excessive periods of autophagy can have detrimental consequences for the cell and ultimately lead to inflammation and cell death Therefore, a third challenge is how to accurately activate beneficial autophagy in a selective manner without inducing aberrant cell death or inflammation For instance, new attractive therapies based on cell specific-targeted nanoparticles and bioabsorbable drug-eluting scaffolds could be used to deliver relevant autophagy modulator compounds to atherosclerotic lesions with reduced side effects Overall, in view of the fundamental importance of autophagy in many cellular functions, the pharmacological modulation of autophagy undoubtedly represents a promising tool for the treatment of vascular diseases Acknowledgements C V is supported by INSERM and grants from La Fondation de France, La Fondation Coeur et Recherche, Association Française contre les Myopathies/Téléthon and La Fédération Française de Cardiologie Dr Frank Lezoualc’h is 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H1394–H1409 Hill BG, Haberzettl P, Ahmed Y, Srivastava S, Bhatnagar A (2008) Unsaturated lipid peroxidation-derived aldehydes activate autophagy in vascular smooth-muscle cells Biochem J 410: 525–534 Hu P, Lai D, Lu P, Gao J, He H (2012) ERK and Akt signaling pathways are involved in advanced glycation end product-induced autophagy in rat vascular smooth muscle cells Int J Mol Med 29: 613–618 C M Keenan et al 200 * 100 10–5 10–4 10–3 10–2 10–1 1.0 250 1500 *** * 1250 1000 750 500 250 C57Bl/6 CB1-/- CB2-/- Vehicle min AM841 WIN min AM841 WIN 0.1 mg·kg–1 0.3 mg·kg–1 1.0 mg·kg–1 3.0 mg·kg–1 D Colonic Bead Expulsion (s) Colonic Bead Expulsion (s) 1750 * 500 10 Vehicle AM841 mg·kg–1 *** *** ** *** 750 log [AM841] mg·kg–1 C *** 1000 * *** 1250 20 120 220 300 1500 20 120 220 *** *** 1750 20 120 220 400 *** *** *** 500 B 20 120 220 AM841 WIN 20 120 220 Colonic Bead Expulsion (% Control) A Colonic Bead Expulsion (s) BJP 1750 1500 *** *** 1250 1000 750 500 250 Vehicle AM251 AM630 mg·kg–1 mg·kg–1 AM841 0.1 mg·kg–1 Figure The effects of AM841 on colonic bead expulsion (A) AM841 and WIN55,212-2 dose-dependently reduced colonic transit Note that AM841 significantly slowed transit at doses as low as 0.1 mg·kg−1 n = 6–23 per group *P < 0.05, ***P < 0.001 compared with vehicle – one-way ANOVA with Sidak’s multiple comparison (B) The time-dependent effects of AM841 on colonic transit were compared with those of WIN55,212-2 Note that at the lower doses (0.1 mg·kg−1 AM841, 0.3 mg·kg−1 WIN55,212-2) WIN55,212-2 had no significant effect on colonic transit, whereas AM841 significantly reduced transit over nearly h n = 6–26 per group *P < 0.05, **P < 0.01, ***P < 0.001 compared with vehicle at the same time point – one-way ANOVA with Dunnett’s multiple comparison (C) The effects of AM841 (1 mg·kg−1) on colonic transit in wild-type (WT), CB1−/− mice and CB2−/− mice Note that mg·kg−1 AM841 had no effect in CB1−/− mice, whereas motility was reduced in both WT and CB2−/− mice to a similar extent n = 5–11 per group *P < 0.05, ***P < 0.001 compared with vehicle – t-test for each genotype of mice tested (D) The effects of CB1 (AM251) and CB2 (AM630) receptor antagonists on the actions of AM841 Pretreatment with AM251 completely blocked the effects of 0.1 mg·kg−1 AM841 at mg·kg−1 n = 6–21 per group ***P < 0.001 between groups indicated – one-way ANOVA with Sidak’s multiple comparison AM630 had no significant effect on the action of AM841 AM841 is a classical CB analogue with an isothiocyanate group at the last carbon of its dimethylheptyl chain (Picone et al., 2005) AM841 binds to the helix of the CB1 and CB2 receptor, through different binding motifs (Picone et al., 2005; Pei et al., 2008; Szymanski et al., 2011) For both receptors, the functional potencies of this ligand exceeded, by 20to 50-fold, that of structurally related non-covalent ana2414 British Journal of Pharmacology (2015) 172 2406–2418 logues, and so AM841 was designated as a ‘megagonist’ (Szymanski et al., 2011; Makriyannis, 2014) Building on our understanding of the chemical properties of AM841, we first undertook some in vitro electrophysiological and receptor trafficking studies to confirm that it behaved as expected in these biological assay systems Here, we found that AM841 behaved as a very potent (low nM) agonist which AM841 regulates GI motility Small Intestinal Transit (% Inhibition) A 80 60 40 20 Normal Transit Stress-Enhanced Transit –20 10–5 10–4 10–3 10–2 10–1 1.0 [AM841] mg·kg–1 B Small Intestinal Transit (% Control) 140 120 10 ** * ** 100 80 60 VEH AM841 VEH AM841 AM251 AM630 Non-Stressed Stressed AM841 C Faecal Pellet Output (% Control) 400 300 * * 200 100 VEH AM841 Non-Stressed VEH AM841 Stressed Figure The effects of AM841 on small intestinal transit and colonic bead expulsion in acutely stressed mice (A) AM841 dose-dependently reduced small intestinal transit in normal and stressed mice There was a slight leftward shift of the dose-response curve in the stressed mice n = 3–21 per group (B) The effects of AM841 (0.001 mg·kg−1) on small intestinal transit in non-stressed and stressed mice in the presence and absence of AM251 (5 mg·kg−1) and AM630 (5 mg·kg−1) Vehicle-treated stressed mice had a significantly increased small intestinal transit when compared with vehicle-treated non-stressed mice n = 3–24 per group *P < 0.05 – t-test Note that at this dose, AM841 had no significant effect in non-stressed mice, but significantly reduced transit in stressed animals **P < 0.01 compared with vehicle-treated stressed mice – two-way ANOVA with Sidak’s multiple comparison The effects of AM841 were blocked by AM251 but unaffected by AM630 **P < 0.01 compared to vehicletreated stressed mice – one-way ANOVA with Sidak’s multiple comparison (C) The effects of AM841 on stress-enhanced faecal pellet output Faecal pellet output was increased ∼3-fold by placing the animals in a novel environment Pretreatment with AM841 (0.01 mg·kg−1) normalized the stress-enhanced faecal pellet output n = 10–11 per group *P < 0.05 between groups as indicated – two-way ANOVA with Sidak’s multiple comparison BJP was not displaced from the receptor after binding, using the inverse agonist SR141716, in contrast to the classic ligand WIN 55,212-2, which was readily displaced Activation of CB1 receptors by agonists often leads to CB1 receptor internalization (Hsieh et al., 1999) Incubation of CB1 receptorexpressing HEK cells with AM841 caused a dose-dependent loss of cell surface CB1 receptor immunoreactivity If agonist application is brief and followed by antagonist, internalized CB1 receptors can be recycled back to the cell surface (Hsieh et al., 1999) Presumably, this occurs because after the antagonist displaces the agonist, the receptor is dephosphorylated and recycled back to the cell surface (Moore et al., 2007) The absence of receptor recycling following antagonist treatment suggests that AM841 remained covalently bound to the CB1 receptor, even after internalization When animals were injected with AM841, it was immediately apparent that their behaviour was similar to that of vehicle controls and no adverse events were observed following treatment This finding led us to investigate the brain penetration of AM841 and to conduct behavioural tests to assess if this ligand displays any classical CB actions, namely, hypothermia, analgesia and/or hypomotility In no case did we observe that AM841 exhibited the classical CNS actions of a CB1 receptor agonist and this we attribute to its demonstrated inability to access the brain in significant quantities One possible explanation for this observation is that the compound is a substrate for one or both of the multidrug resistance proteins (MRP1 and MRP2) as has been reported for the naturally occurring phenethyl isothiocyanate (Ji and Morris, 2005; Morris and Dave, 2014) Similar mechanisms have also recently been proposed for the exclusion of other peripherally restricted CB ligands (Pryce et al., 2014) However, these authors noted that the strains of mice we used have polymorphic CB drug pumps that lack functionality (Pryce et al., 2014) If this is the case, then other pumps or different exclusion mechanisms might exist for AM841 This has to be determined in future studies Having the properties of a peripherally restricted ligand makes AM841 a very interesting molecule for studies of the GI tract The endocannabinoid system regulates GI motility, but investigations of the actions of CBs in vivo are frequently confounded by central effects of these drugs Here, we have isolated the actions to the periphery and demonstrated that AM841 is an efficacious and highly potent agonist of CB1 receptors Indeed, these results demonstrate the remarkable ability of AM841 to slow down transit with an EC50 in the small intestine of around μg·kg−1 In the colon, the apparent potency was about 10-fold lower In contrast, the potency of WIN55,212-2 did not seem to change much between these gut regions, although in both cases it was far less potent It is not completely clear why this should be the case In the colon, we were able to use a lower dose of AM251 to block the effects of AM841 than were required to block small intestinal transit (2 mg·kg−1 vs mg·kg−1) These data suggest that in the colon, there is a lower effective receptor density on the enteric nerves than in the ileum However, that has yet to be determined experimentally Alternatively, the CB1 receptor in the colon might be constitutively desensitized or in a state of inactivation to a greater extent than the ileum because of a higher endocannabinoid tone in that region of the gut Tonically-released endocannabinoids are present throughout British Journal of Pharmacology (2015) 172 2406–2418 2415 BJP C M Keenan et al the GI tract and CB ‘tone’ has been reported in both the small and large intestine (Pertwee, 2001; Izzo and Sharkey, 2010; Storr et al., 2010), but the relative endocannabinoid tone between these regions of gut has never been compared As mentioned earlier, CB1 receptors are found on cholinergic nerves of the ENS and reduce the release of ACh (Duncan et al., 2005; Izzo and Sharkey, 2010) The proportion of cholinergic and non-cholinergic nerves in the ileum and colon differs along the mouse GI tract, with a much higher proportion being non-cholinergic in the colon (De Man et al., 2002; Mule et al., 2007; Baldassano et al., 2009; Bashashati et al., 2012) Whether this also somehow influences the results of these in vivo transit studies remains to be determined In the present study, we still observed an effect of AM841 on colonic propulsion h after injection in control mice Future investigations should examine the duration of action of covalent ligands, such as AM841, under both physiological and pathophysiological conditions and the potential consequences of irreversible agonism AM841 has been used previously as a high potency ligand to investigate the role of CB receptors in colitis (Fichna et al., 2014) Here, it was given by the same route, i.p., and was found to block the development of colitis, albeit with a slightly lower potency than was observed for colonic transit in the current study Interestingly, the effects of AM841 were found to be mediated by both CB1 and CB2 receptors in this pathophysiological condition (Fichna et al., 2014), whereas for GI motility under the conditions of our studies, AM841 acted only on CB1 receptors In this study, AM841 was compared with another peripherally restricted compound CB13 (Dziadulewicz et al., 2007), which was not able to block colitis following i.p administration CB13 [aka SAB378 (Dziadulewicz et al., 2007; Cluny et al., 2010a)] was shown by us to reduce motility via CB1 receptors to a similar extent as seen for AM841 in the current study (Cluny et al., 2010a) This suggests that the covalent nature or the potency of AM841 seems to confer some unique properties on AM841, as it is clearly peripherally-restricted and yet blocks colitis and inhibits motility Acute stress leads to an acceleration of small intestinal and colonic transit (Taché and Perdue, 2004) Under these conditions, we observed that AM841 was effective at slowing the accelerated transit and remarkably, a dose of AM841 without significant effect in normal animals now significantly slowed small intestinal transit and normalized colonic transit These findings occurred without an alteration in receptor mRNA expression in the GI tract Previous studies from our group showed in a similar paradigm that the endogenous lipid signalling molecule oleoylethanolamide was also able to reverse accelerated small intestinal motility with a higher potency than under physiological conditions (Cluny et al., 2009) The receptor mediating this effect was not identified, but was not the CB1 receptor Nevertheless, it is tempting to speculate that stress leads to an alteration in lipid/CB signalling in the GI tract as an adaptive mechanism to slow small intestinal motility Our findings of enhanced faecal pellet output are very similar to those previously reported by Million et al (2007) They showed that acute novel environment stress leads to enhanced faecal pellet output, the magnitude and duration of which are virtually identical to what we observed This effect 2416 British Journal of Pharmacology (2015) 172 2406–2418 is mediated by corticotrophin-releasing factor (CRF) signalling pathways and cholinergic neurons of the myenteric plexus Consistent with this model, we propose that AM841 acts on CB1 receptors on myenteric cholinergic neurons to reduce transmission and slow the gut From the results of our in vivo studies we cannot pinpoint the exact site of action, which could be through inhibition of presynaptic neurotransmitter release or prejunctional release or both Currently, there are very limited treatment options for functional GI disorders, in which motility is altered leading to severe symptoms (Camilleri, 2013) Cannabis is frequently used to relieve the symptoms of these disorders However, the unwanted psychotropic effects limit its usefulness as a therapeutic agent Here, we have discovered a class of molecule with potential beneficial actions on abnormally accelerated GI motility that lacks any central actions Because of its high potency, efficacy and duration of action, it seems well suited to further development as a therapeutic agent In summary, AM841 is a novel covalent CB agonist that behaved as an irreversible CB1 receptor ligand in vitro AM841 showed little brain penetration and so behaved as a peripherally restricted CB ligand AM841 potently reduced GI motility in vivo by acting at CB1 receptors in the small and large intestine When GI transit was accelerated under conditions of acute stress, AM841 was able to normalize it These data suggest that this novel CB ligand represents a new class of potential therapeutic agents for the treatment of GI disorders Acknowledgements Supported by the Canadian Institutes of Health Research (K A S.), National Institutes of Health (DA011322 and DA021696, K M.; DA09158, DA7215 and DA3801, A M.) and Deutsche Forschingsgemeinschaft (DFG STO645/9-1, M A S.) K A S is the Crohn’s and Colitis Foundation of Canada Chair in Inflammatory Bowel Disease Research Author contributions C M K., M A S., G A T., J T W., J W-M., A S., M R E., S P N., M B and H H performed the research C M K., M A S., K M., A M and K A S designed the research study G A T., M R E and S P N contributed essential research tools C M K., M A S., J T W., J W-M., A S., M B., K M and K A S analysed the data C M K., M A S., G A T., J T W., A S., S P N., M B., K M., A M and K A S wrote the paper All authors read, revised and approved the paper for publication M A S., K M., A M and K A S obtained funding for these studies Conflicts of interest The authors declare no conflicts of interest References Abalo R, Vera G, Lopez-Perez AE, Martinez-Villaluenga M, Martin-Fontelles MI (2012) The gastrointestinal pharmacology of cannabinoids: focus on motility Pharmacology 90: 1–10 AM841 regulates GI motility Adam JM, Clark JK, Davies K, Everett K, Fields R, Francis S et al (2012) Low brain 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Szarka LA, Burton D et al (2011) Pharmacogenetic trial of a cannabinoid agonist shows reduced fasting colonic motility in patients with nonconstipated irritable bowel syndrome Gastroenterology 141: 1638–1647, e1631–1637 Wong BS, Camilleri M, Eckert D, Carlson P, Ryks M, Burton D et al (2012) Randomized pharmacodynamic and pharmacogenetic trial of dronabinol effects on colon transit in irritable bowel syndrome-diarrhea Neurogastroenterol Motil 24: 358–e169 Yu XH, Cao CQ, Martino G, Puma C, Morinville A, St-Onge S et al (2010) A peripherally restricted cannabinoid receptor agonist produces robust anti-nociceptive effects in rodent models of inflammatory and neuropathic pain Pain 151: 337–344 BJP British Journal of Pharmacology DOI:10.1111/bph.13070 www.brjpharmacol.org RESEARCH PAPER Correspondence Relief learning is dependent on NMDA receptor activation in the nucleus accumbens Markus Fendt, Institute for Pharmacology and Toxicology, Leipziger Straße 44, D-39120 Magdeburg, Germany E-mail: markus.fendt@med.ovgu.de *Present address: Department of Anesthesiology, University Hospital Würzburg, Würzburg, Germany Received 17 October 2014 Revised Milad Mohammadi1,2* and Markus Fendt1,3 December 2014 21 December 2014 Accepted Institute for Pharmacology and Toxicology, 2Integrative Neuroscience Program, and 3Center of Behavioral Brain Sciences, Otto-von-Guericke University Magdeburg, Magdeburg, Germany BACKGROUND AND PURPOSE Recently, we demonstrated that the nucleus accumbens (NAC) is required for the acquisition and expression of relief memory The purpose of this study was to investigate the role of NMDA receptors within the NAC in relief learning EXPERIMENTAL APPROACH The NMDA receptor antagonist 2-amino-5-phosphonopentanoic acid (AP-5) was injected into the NAC The effects of these injections on the acquisition and expression of relief memory, as well as on the reactivity to aversive electric stimuli, were tested KEY RESULTS Intra-accumbal AP-5 injections blocked the acquisition but not the expression of relief memory Furthermore, reactivity to aversive electric stimuli was not affected by the AP-5 injections CONCLUSION AND IMPLICATION The present data indicate that NMDA-dependent plasticity within the NAC is crucial for the acquisition of relief memory Abbreviations AP-5, 2-amino-5-phosphonopentanoic acid; NAC, nucleus accumbens Tables of Links TARGETS LIGANDS GPCRsa Ligand-gated ion channelsb Dopamine receptors NMDA receptors Dopamine These Tables list key protein targets and ligands in this article which are hyperlinked to corresponding entries in http:// www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Pawson et al., 2014) and are permanently archived in the Concise Guide to PHARMACOLOGY 2013/14 (a,bAlexander et al., 2013a,b) Introduction Learning about relations between stimuli and events is essential for adaptive behaviour (McNally and Westbrook, 2006) A © 2015 The British Pharmacological Society well-investigated example for such learning is fear conditioning in which animals or humans learn that a particular cue predicts an aversive event (Fendt and Fanselow, 1999; LeDoux, 2012) This cue then becomes a conditioned fear British Journal of Pharmacology (2015) 172 2419–2426 2419 BJP M Mohammadi and M Fendt stimulus that prepares the brain and body for future environmental dangers Less well-known cues that are presented after an aversive event can also lead to associations As the to-belearned stimulus is presented in the moment of relief from the aversive event, this learning was called relief learning (Gerber et al., 2014) In contrast to conditioned fear stimuli that induce aversive behaviour (avoidance or startle potentiation), conditioned relief stimuli trigger appetitive-like behaviour such as approach behaviour or startle attenuation (Tanimoto et al., 2004; Andreatta et al., 2012) Initial research on relief learning was performed in fruit flies (Tanimoto et al., 2004; Yarali et al., 2008) but relief learning has also been demonstrated in humans and laboratory rodents (Andreatta et al., 2010; 2012; Mohammadi et al., 2014) Due to the appetitive-like nature of relief learning, it was suggested that the brain’s reward system is involved in relief learning Indeed, a human imaging study showed an activation of the nucleus accumbens (NAC), a crucial part of the reward system (Ikemoto, 2007), during expression of conditioned relief (Andreatta et al., 2012) Furthermore, temporary inactivation of the NAC in rats blocks both the acquisition and expression of relief memory (Andreatta et al., 2012; Mohammadi et al., 2014) Because the plasticity within the NAC is responsible for reward learning and responsereinforcement learning (Kelley, 2004; Miller and Marshall, 2005), these data suggest that the NAC is also the brain site of relief learning Most forms of associative learning are dependent on NMDA receptors (Maren, 2000; Martin et al., 2000; Chapman, 2001) In line with this general observation, it was repeatedly demonstrated that reward learning and responsereinforcement learning are mediated by NMDA receptors within the NAC (Kelley et al., 1997; Smith-Roe and Kelley, 2000; Di Ciano et al., 2001; Kelley, 2004) These findings suggest that relief learning may also be mediated by accumbal NDMA receptors The present study aims to address this hypothesis Therefore, rats received local injections of the NMDA receptor antagonist 2-amino-5-phosphonopentanoic acid (AP-5) into the NAC and were then submitted to relief conditioning One day later, conditioned relief was tested using the acoustic startle paradigm In a second experiment, it was tested if AP-5 injections affect the expression of conditioned relief and the reactivity to the electric stimuli The electric stimuli were used as an unconditioned stimulus (US) in the relief conditioning procedure Methods Animals and surgery Thirty-nine adult male Sprague Dawley rats aged between and months (250–350 g) at the time of the surgery were used in this experiment Rats were bred and reared at the local animal facility (original breeding stock: Taconic, Ry, Denmark) They were kept in groups of four to six animals per cage (Makrolon Type IV; Tecniplast, Hohenpeißenberg, Germany) in temperature- and humidity-controlled rooms (22 ± 2°C, 50 ± 10%) under a light : dark cycle of 12 h:12 h (lights 2420 British Journal of Pharmacology (2015) 172 2419–2426 on 6:00 h) and had free access to water and food All experiments and surgeries were performed during the light phase All studies involving animals are reported in accordance with the ARRIVE guidelines for reporting experiments involving animals (Kilkenny et al., 2010; McGrath et al., 2010) and were approved by the local ethical committee (Landesverwaltungsamt Sachsen-Anhalt, Az 42502-2-1172 UniMD) The animals were anaesthetized with isoflurane (Baxter Germany GmbH; Unterschleißheim, Germany) mixed with pure oxygen (5% isoflurane for induction, then 2.0–2.5%) The depth of anaesthesia was assessed by testing reflexes to a hind-paw pinch Then, the animals were fixed in a rodent stereotaxic apparatus The skull was exposed and stainless steel guide cannulas (custom-made; diameter: 0.7 mm, length: 8.0 mm) were bilaterally implanted aiming at the NAC: 1.2 mm rostral, ± 1.5 mm lateral and 7.4 mm ventral to the bregma (Paxinos and Watson, 1997) Cannulas were fixed with dental cement to the skull and three anchoring screws After the surgery, the animal was housed on its own and supervised for h and then returned to the colony After the surgery, there was a recovery period of 5–7 days Apparatus We used a startle system with eight chambers (35 cm × 35 cm × 35 cm; SR-LAB, San Diego Instruments, San Diego, CA, USA) Each chamber consisted of a stable platform holding a horizontal, cylindrical, transparent animal enclosure with an inner diameter of cm and an inner length of 16 cm Underneath the platform, a piezoelectric motion sensor was mounted for measuring animal movements The output signal of this sensor was digitalized with a sampling rate of kHz and sent to the computer Beginning at startle stimulus or electric stimulus onset, consecutive ms readings were recorded to obtain the magnitude of the animal’s response to the startle stimulus or electric stimulus (arbitrary units) The startle magnitude, average readout in the ‘startle response peak window’, was taken every 10–30 ms after startle stimulus onset For relief conditioning, aversive electric stimuli (US) and light stimuli (conditioned stimulus, CS) were used The light stimulus was produced by a 10 W bulb, had an intensity of ∼1000 lux and duration of s The electric stimuli were administered via a floor grid (six bars with mm diameter, distance: 10 mm), had an intensity of 0.4 mA and a duration of 0.5 s For the application of acoustic stimuli, a loudspeaker mounted on the ceiling of the box was used During all tests, a background noise with an intensity of 50 dB sound pressure level (SPL) was presented to mask environmental noises The acoustic startle stimulus was a noise burst with an intensity of 96 dB SPL for a duration of 40 ms For testing the reactivity to electric stimuli, stimulus intensities of 0.0, 0.1., 0.2, 0.3 and 0.4 mA were used As a readout for reactivity to electric stimuli, the summed readout of the piezoelectric motion sensor during the electric stimuli (500 ms) was used Behavioural protocol Experiment 1: effects of intra-NAC AP-5 injections on acquisition of relief memory Day (baseline session): animals (n = 31) were put in the chambers and after of acclimatization, 10 startle stimuli were delivered with an inter-trial interval of 30 s (see also Figure 1) Then, the animals were put back into Accumbal NMDA receptors mediate relief learning BJP Figure Behavioural protocols of the present study Upper panels depict the treatment schedule and the test sessions performed Lower panels give detailed information on the different test sessions their home cages Based on the mean startle amplitude of this session, the animals were distributed into groups with balanced mean baseline startle amplitude Day (training session): the animals were injected with either the vehicle or AP-5 (2.5 or nmol·0.3 μL−1, Sigma Aldrich, Munich, Germany) To this, the animals were gently held in place by hand to insert the injection cannulas (custom-made; diameter: 0.3 mm, 12 mm long), which were connected via a tube to a 10 μL syringe into the guide cannulas Then a volume of 0.3 μL was injected with a speed of 0.2 μL·min−1 using a microinjection pump (CMA/100, Microdialysis AB, Stockholm, Sweden) The cannulas were left in place for additional minute Ten minutes later, the animals were put into the startle chambers After of acclimatization, the relief conditioning protocol was performed Fifteen electric stimuli followed by a light stimulus (fixed inter-stimulus interval: s from onset electric stimulus to onset light stimulus) were delivered to the animals The intertrial interval (onset electric stimulus to onset next electric stimulus) was pseudo-randomized and varied between 30 and 100 s No startle stimuli were delivered to the animals during the conditioning session Day (retention test): the animals were put into the startle chamber Following of acclimatization, 10 startle stimuli were presented to habituate the animal, followed by 20 startle stimuli, 10 of them without the light CS and 10 of them upon presentation of the light CS (CS and startle stimuli co-terminated) The order of the trials with and without the light CS was pseudo-randomized Experiment 2: effects of intra-NAC AP-5 injections on expression of relief memory Day and 2: eight animals were used for this experiment The baseline and training session was identical to those of experiment except that no injections were performed before or during the relief conditioning session Day 3: half of the animals were injected with the vehicle and the other half with nmol·0.3 μL−1 AP-5 The animals were put into the chambers immediately after injection and the retention test was run Day 4: the animals were reconditioned with relief conditioning protocol from day Day 5: the test of day was repeated However, animals that received injections of saline on day were now injected with AP-5 and vice versa British Journal of Pharmacology (2015) 172 2419–2426 2421 BJP M Mohammadi and M Fendt Experiment 3: effects of intra-NAC AP-5 injections on locomotor reactivity to electric stimuli For this experiment, the animals of experiment were used One day after completion of experiment 2, the animals were injected with either saline or nmol·0.3 μL−1 AP-5 Then they were put into the startle chambers After an acclimatization time of min, five electric stimuli with increasing intensities (0.0, 0.1, 0.2, 0.3 and 0.4 mA) were administered with an inter-stimulus interval of 30 s One day later, the same procedure was repeated However, rats that received saline on the day before now received injections of AP-5 and vice versa We used doses of 2.5 and nmol AP-5·0.3 μL−1 These doses are based on the effective doses found in several published studies (Miserendino et al., 1990; Kelley et al., 1997; Schauz and Koch, 2000; Palencia and Ragozzino, 2004; Hsu and Packard, 2008) Histology At the end of the behavioural experiments, the animals were killed by CO2 The brains were removed and put into 30% sucrose and 4% formalin solution for fixation The brains were sectioned by a cryostat in 60 μm slices The slides were Nissl-stained with cresyl violet, examined with light microscopy and the injection sites (injection cannula trace) were located and marked on sections of a brain atlas (Paxinos and Watson, 1997) Data analysis For each animal, the mean response to the electric stimuli, the mean startle amplitudes with and without the light CS (peak amplitudes within the 100 ms after the startle stimulus onset) and their difference were calculated Because all data were normally distributed (D’Agostino and Pearson’s omnibus normality test), means and SEM were shown in the figures and parametric statistical tests were used for analysis (Prism 6.0, GraphPad Software Inc., La Jolla, CA, USA) A significance level of P < 0.05 was used for all tests Results Histology Histological analyses of the injection sites revealed that 35 animals received bilateral injections of saline or AP-5 into the NAC (ns = 7–11 per group) Most of the injection sites were localized in the core region of the NAC (Figure 2) Four animals were discarded because of misplaced injections Behaviour Experiment 1: effects of intra-NAC AP-5 injections on acquisition of relief memory In this experiment, saline or two different doses of AP-5 (2.5 or nmol·0.3 μL−1) were injected into the NAC immediately before the relief conditioning session (see Figure 1) On the following day, a retention test was performed without any injections Figure 3A depicts the mean startle magnitudes of startle alone and CS-startle trials of the retention test as well as their difference The data show that AP-5 injections into the NAC dose-dependently inhibit the acquisition of relief memory This is supported by an ANOVA with startle trial type as within-subject factor and treatment as between-subject factor There was a main effect of trial type 2422 British Journal of Pharmacology (2015) 172 2419–2426 Figure Reconstruction of the vehicle and AP-5 injection sites into the NAC on frontal brain sections (Paxinos and Watson, 1997) (A) Representative photomicrograph showing a brain slide with injection sites into the NAC (B) Injections before relief conditioning (acquisition) Black circles, saline; inverse triangles, 2.5 nmol AP-5; diamonds, nmol AP-5 (C) Injections (gray circles) before expression of relief memory and before testing on reactivity to electric stimuli Values represent the anterior distance to bregma (mm) according to Paxinos and Watson (1997) CPu, caudate putamen; Ctx, cortex; ec, external capsule; LSi, lateral septal nucleus (F1,22 = 8.90, P < 0.0001) and significant interaction between trial type and treatment (F2,22 = 4.58, P = 0.02) There were no main effects of treatment (F2,22 = 1.13, P = 0.34) indicating that the startle response itself was not affected by AP-5 injections Post hoc Sidak’s multiple comparison tests show significant trial type effects in the saline-injected group (t = 3.94, P < 0.01) but not in the two AP-5-injected groups (ts < 0.69, Ps > 0.05) This effect of intra-NAC AP-5 is further supported by an ANOVA on the difference scores (F2,22 = 4.50, P = 0.02) and post hoc Dunnett’s tests showing significant differences Accumbal NMDA receptors mediate relief learning BJP Figure Effects of intra-NAC injections on the acquisition and expression of relief memory Depicted is the startle magnitude (arbitrary units + SEM) without and with the presence of the relief CS (light stimulus), as well as the difference between these two trial types (A) AP-5 injected before conditioning dose-dependently blocks acquisition of relief memory *P < 0.05 comparison with nmol (Dunnett’s test) after significant effects in ANOVA (B) AP-5 injected before testing expression of relief memory has no effects between animals treated with saline and animals treated with the two AP-5 doses (Ps < 0.05) Experiment 2: effects of intra-NAC AP-5 injections on expression of relief memory Animals were relief conditioned without treatment On the following day, they received injections of saline and AP-5 (5 nmol·0.3 μL−1) into the NAC and they were tested for their relief memory There was apparently no effect of intra-NAC AP-5 injections on the expression of relief memory (Figure 3B) An ANOVA with startle trial type and treatment as within-subject factors revealed a significant effect of trial type (F1,6 = 10.79, P = 0.02) but neither of treatment (F1,6 = 0.88, P = 0.88) nor an interaction between trial type and treatment (F1,6 = 0.30, P = 0.61) Furthermore, post hoc Sidak’s multiple comparison tests showed significant trial type effects on both, saline- and AP-5-injected animals (ts > 2.97, Ps < 0.05) This is supported by a comparison of the difference scores showing no treatment effects (paired t-test: t = 0.59, P = 0.57) Notably, the testing order had no effect (F1,6 = 0.003, P = 0.96) Experiment 3: effects of intra-NAC AP-5 injections on locomotor reactivity to electric stimuli Animals were injected with either saline or AP-5 (5 nmol·0.3 μL−1) and then tested for their reactivity to electric stimuli with increasing intensities (0.0 to 0.4 mA) In Figure 4, the mean locomotor response of the animals during the 0.5 s duration of the electric stimuli is depicted Injections of AP-5 into the NAC did not affect the reactivity to electric stimuli An ANOVA with intensity and treatment as within-subject factors revealed a significant effect of intensity (F4,28 = 21.26, P < 0.001) but neither of treatment (F1,28 = 0.10, P = 0.76) nor an interaction between intensity and treatment (F4,28 = 0.21, P = 0.93) Discussion The present study investigated the role of accumbal NMDA receptors in the acquisition and expression of relief memory Figure Effects of intra-NAC injections on the locomotor reactivity to electric stimuli Shown is the mean locomotor activity (arbitrary units + SEM) during the electric stimuli (500 ms duration, different intensities) AP-5 injections did not affect the reactivity to electric stimuli Therefore, local injections of the NMDA receptor antagonist AP-5 were performed either directly before relief conditioning (acquisition) or before the retention test on conditioned relief (expression) Our data clearly show that accumbal NMDA receptor blockade before conditioning, but not before the retention test, prevented conditioned relief Furthermore, we demonstrated that NMDA receptor blockade does not affect the reactivity to electric stimuli Taken together, this indicates that relief learning depends on NMDA receptor activation within the NAC To measure conditioned relief, we used the acoustic startle paradigm In line with the data of our previous studies (Andreatta et al., 2012; Mohammadi et al., 2014), the relief CS robustly attenuated the startle magnitude under control conditions (injections of saline into the NAC) It is important to note that such an attenuation of the startle magnitude by a CS can only be observed if the CS has a positive valence This can be obtained not only by relief conditioning (CS presented shortly after an aversive US) but British Journal of Pharmacology (2015) 172 2419–2426 2423 BJP M Mohammadi and M Fendt also after safety conditioning (CS explicitly unpaired with an aversive US; e.g Mohammadi et al., 2014) and after ‘pleasure conditioning’ (CS precedes an appetitive US; e.g Schmid et al., 1995) In contrast, fear conditioning, in which the CS precedes an aversive US, induces a startle potentiation (Davis et al., 1993; Fendt and Koch, 2013) No modulation of the startle magnitude can be observed if the presentations of the CS and the US are randomized during the conditioning phase (i.e that by chance the CS and the US can also simultaneously appear) or if the CS is presented without any US during the conditioning phase (Davis and Astrachan, 1978; Andreatta et al., 2012) Previous studies from our group demonstrated that the NAC is crucial for the acquisition and expression of conditioned relief (Andreatta et al., 2012; Mohammadi et al., 2014) Importantly, the NAC is not involved in safety learning, that is the learning that a cue predicts the absence of the US (Josselyn et al., 2005; Mohammadi et al., 2014) In latter studies, safety learning was either induced by explicit unpairing of the US and the CS or by a conditioned inhibition procedure (cf Christianson et al., 2012) This demonstrates that there is a neural dissociation between relief learning and safety learning, strongly supporting the view that relief learning and safety learning represent distinct learning processes (Gerber et al., 2014) In humans and rats, we previously demonstrated a neural dissociation between fear learning and relief learning (Andreatta et al., 2012) The amygdala is crucial for fear learning but not for relief learning, whereas the NAC is crucial for relief learning but not for fear learning Taken together, the NAC is only involved in relief learning, not in fear or safety learning This strongly suggests that the observed effects after local injections into the NAC are specific to relief learning and not unspecific to any associative relationship between CS and US (as e.g in safety or fear learning) The present data now show an important role of accumbal NMDA receptors in relief learning Both of our AP-5 doses, 2.5 and nmol AP-5·0.3 μL−1, significantly blocked the acquisition of relief memory These doses are in line with effective doses found in the literature (e.g Kelley et al., 1997) However, it is important to note that the effects observed in the present study can also be explained by state- dependency, that is the AP-5 injections before relief conditioning induced a specific state which is necessary later to express conditioned relief However, such a state- dependency has not been observed yet for NMDA receptor blockade in associative learning (e.g Tzschentke and Schmidt, 1997; Bast et al., 2003) Therefore, we are confident that the observed AP-5 effects are based on a blockade of acquisition and not on state-dependency The NAC consists of different regions, the core and the shell regions (Zahm and Brog, 1992) Based on data from Kelley et al (1997), it is thought that the NAC core mediates the blockade of relief conditioning observed in this study This is supported by the fact that our injection sites were almost exclusively located within the core region (cf Figure 2) However, we injected a volume of 0.3 μL and such a volume may diffuse ca 0.5 μm (Martin, 1991) This means that we cannot exclude the possibility that the injected AP-5 also reaches neurons within the NAC shell In fact, it could be that the shell region of the NAC is more important for relief 2424 British Journal of Pharmacology (2015) 172 2419–2426 learning than the core region The shell region is the projection target of dopaminergic neurons within the ventral tegmental area which show an excitatory response to the offset of electric stimuli (Brischoux et al., 2009), that is in the moment of relief from the electric stimulus In our second experiment, the animals were relief conditioned without any treatment and AP-5 was then injected immediately before the retention test These injections clearly did not affect the expression of relief memory This demonstrates not only that accumbal NMDA receptors are not involved in the expression of relief memory but also that the sensory processing of the visual relief CS is not disturbed by AP-5 injections into the NAC The latter indicates that the blockade of relief learning observed in our first experiment is not due to AP-5 effects on the sensory processing of the relief CS However, a blockade of relief learning could also be explained by disturbed sensory processing of the US Therefore, we performed a third experiment in which we tested the effects of accumbal AP-5 injections on the locomotor reactivity to electric stimuli Clearly, AP-5 injections did not affect this reactivity indicating that the blockade of relief learning by AP-5 injections into the NAC is also not caused by disturbed US processing That means the sensory processing of both the CS and the US are not impaired after AP-5 injections into the NAC Therefore, the most obvious interpretation of the AP-5 effects in our first experiment is that AP-5 prevented the association between the CS and the US Thus, relief learning is based on NMDA receptor-dependent plasticity within the NAC, for example long-term potentiation (Schotanus and Chergui, 2008) Several studies have already demonstrated that accumbal NMDA receptors are involved in both appetitive Pavlovian and instrumental conditioning (Kelley et al., 1997; Smith-Roe and Kelley, 2000; Di Ciano et al., 2001; Dalley et al., 2005) In these learning processes, rewarding stimuli are used as an US, whereas in relief conditioning experiments, an aversive US is used However, as discussed earlier and in several publications before (Gerber et al., 2014; e.g Tanimoto et al., 2004), the timing of CS and US presentation is critical for the valence of the learned association If the CS precedes the US in Pavlovian conditioning (forward pairing), the CS will gain negative valence and will later induce behavioural signs of fear (summarized in Fendt and Fanselow, 1999; Davis, 2006; LeDoux, 2012) The underlying plasticity of this learning occurs in the amygdala (Maren, 2005; Pape and Pare, 2010) However, if the US precedes the CS (backward pairing), the CS will gain positive valence as the relief from an aversive stimulus can be considered as a reward In line with this idea, midbrain dopaminergic neurons were described that were phasically excited after the offset of aversive electric stimuli (Brischoux et al., 2009) These neurons were dopaminergic and located in the ventral region of the ventral tegmental area, a brain site which plays a key role in reward processing (Schultz, 1998; Wise, 2004) The ventral tegmental area projects to the NAC and is its main dopaminergic input (Fallon and Moore, 1978) For relief conditioning, it is not known whether accumbal dopamine is involved However, as it is involved in appetitive Pavlovian and instrumental conditioning, we suggest that this is also the case in relief learning We will address this hypothesis in future studies in our laboratory Furthermore, Accumbal NMDA receptors mediate relief learning appetitive conditioning depends on a coincident activation of NMDA and dopamine receptors within the NAC (Smith-Roe and Kelley, 2000; Di Ciano et al., 2001; Dalley et al., 2005) If accumbal dopamine is involved in relief learning, it is very probable that this coincident receptor activation is also the molecular mechanism underlying relief learning Taken together, the present results clearly demonstrate that acquisition of relief memory is dependent on NMDA receptors within the NAC These receptors not seem to be crucial for the expression of relief memory and the sensory processing of the CS and the US used in relief learning Future studies should investigate the role of accumbal dopamine in relief learning and whether accumbal dopamine receptors interact with NMDA receptors during the establishment of relief memory Acknowledgements The authors thank Dr Jorge Bergado-Acosta and Evelyn Kahl for their help in performing the experiments, Kathrin Freke for animal care and Timothy French for language editing This study was funded by the Deutsche Forschungsgemeinschaft (SFB779/B13) Author contributions M M and M F conceived and designed the experiments and wrote the manuscript M M performed the experiments and analysed the data Conflict of interest The authors have no conflict of interest to declare BJP Bast T, Zhang WN, Feldon J (2003) Dorsal hippocampus and classical fear conditioning to tone and context in rats: effects of local NMDA-receptor blockade and stimulation Hippocampus 13: 657–675 Brischoux F, Chakraborty S, Brierley DI, Ungless MA (2009) Phasic excitation of dopamine neurons in ventral VTA by noxious stimuli Proc Natl Acad Sci U S A 106: 4894–4899 Chapman PF (2001) The diversity of synaptic plasticity Nat Neurosci 4: 556–558 Christianson JP, Fernando ABP, Kazama AM, Jovanovic T, Ostroff LE, Sangha S (2012) Inhibition of fear by learned safety signals: a mini-symposium review J Neurosci 32: 14118–14124 Dalley JW, Lääne K, Theobald DEH, Armstrong HC, Corlett PR, Chudasama Y et al (2005) Time-limited modulation of appetitive Pavlovian memory by D1 and NMDA receptors in the nucleus accumbens Proc 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Brain Res 84: 99–107 Wise RA (2004) Dopamine, learning and motivation Nat Rev Neurosci 5: 483–494 Mohammadi M, Bergado-Acosta JR, Fendt M (2014) Relief learning is distinguished from safety learning by the requirement of the nucleus accumbens Behav Brain Res 272: 40–45 Yarali A, Niewalda T, Chen YC, Tanimoto H, Duerrnagel S, Gerber B (2008) Pain relief’ learning in fruit flies Anim Behav 76: 1173–1185 Palencia CA, Ragozzino ME (2004) The influence of NMDA receptors in the dorsomedial striatum on response reversal learning Neurobiol Learn Mem 82: 81–89 Zahm DS, Brog JS (1992) On the significance of subterritories in the ‘accumbens’ part of the rat ventral striatum Neuroscience 50: 751–767 2426 British Journal of Pharmacology (2015) 172 2419–2426 ISSN 0007-1188 (print) ISSN 1476-5381 (online) www.brjpharmacol.org BJP British Journal of Pharmacology www.bps.ac.uk Editor-in-Chief J.C (Ian) McGrath Glasgow, UK & Sydney, Australia Senior Editors Amrita Ahluwalia London, UK Richard Bond 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Colorimetric methods were 2182 British Journal of Pharmacology (2015) 172 21 792 2 09 commonly used The concentration of p-chloro-m-xylenol... Campbell et al., 198 8; 198 9a) However, Ahmed et al ( 198 8) reported high serum dihydrotestosterone levels after scrotal 2186 British Journal of Pharmacology (2015) 172 21 792 2 09 application and... Alza in 198 4 (US Patent 4,460,372) (Campbell and Chandrasekaran, 198 4) led to an unexpected drug delivery profile despite the presence of a British Journal of Pharmacology (2015) 172 21 792 2 09 2187