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http://dx.doi.org/10924-977X/& 2015 E

nCorrespondence tNeuroscience, Unive43123 Parma, Italy.

E-mail address: a

Please cite this arinhibitor URB694 in

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Antidepressant-like activityand cardioprotective effects of fatty acidamide hydrolase inhibitor URB694 in sociallystressed Wistar Kyoto rats

Luca Carnevalia, Federica Vacondiob, Stefano Rossic,Sergio Callegarid, Emilio Macchic, Gilberto Spadonie,Annalida Bedinie, Silvia Rivarab, Marco Morb, Andrea Sgoifoa,n

aDepartment of Neuroscience, University of Parma, ItalybDepartment of Pharmacy, University of Parma, ItalycDepartment of Life Sciences, University of Parma, ItalydDivision of Cardiology, Vaio Hospital, Fidenza (Parma), ItalyeDepartment of Biomolecular Sciences, University of Urbino “Carlo Bo”, Italy

Received 4 February 2015; received in revised form 15 June 2015; accepted 14 July 2015

KEYWORDSArrhythmia;Depression;Endocannabinoid sys-tem;Heart rate variability;Stress

0.1016/j.euroneurlsevier B.V. and E

o: Stress Physiologrsity of Parma, PaTel.: +39 0521 905ndrea.sgoifo@unip

ticle as: Carnevalisocially stressed

AbstractIn humans, depression is often triggered by prolonged exposure to psychosocial stressors and isoften associated with cardiovascular comorbidity. Mounting evidence suggests a role forendocannabinoid signaling in the regulation of both emotional behavior and cardiovascularfunction. Here, we examined cardiac activity in a rodent model of social stress-induceddepression and investigated whether pharmacological inhibition of the enzyme fatty acid amidehydrolase (FAAH), which terminates signaling of the endocannabinoid anandamide, exertsantidepressant-like and cardioprotective effects. Male Wistar Kyoto rats were exposed to fiveweeks of repeated social stress or control procedure. Starting from the third week, theyreceived daily administration of the selective FAAH inhibitor URB694 (0.1 mg/kg, i.p.) orvehicle. Cardiac electrical activity was recorded by radiotelemetry. Repeated social stresstriggered biological and behavioral changes that mirror symptoms of human depression, such as(i) reductions in body weight gain and sucrose solution preference, (ii) hyperactivity of thehypothalamic-pituitary-adrenocortical axis, and (iii) increased immobility in the forced swimtest. Moreover, stressed rats showed (i) alterations in heart rate daily rhythm and cardiacautonomic neural regulation, (ii) a larger incidence of spontaneous arrhythmias, and (iii) signs

o.2015.07.015CNP. All rights reserved.

y Laboratory, Department ofrco Area delle Scienze 11/a,625; fax: +39 0521 905673.r.it (A. Sgoifo).

, L., et al., Antidepressant-like activity and cardioprotective effects of fatty acid amide hydrolase.... European Neuropsychopharmacology (2015), http://dx.doi.org/10.1016/j.euroneuro.2015.07.015

L. Carnevali et al.2

Please cite this article as: Carnevalinhibitor URB694 in socially stressed

of cardiac hypertrophy. Daily treatment with URB694 (i) increased central and peripheralanandamide levels, (ii) corrected stress-induced alterations of biological and behavioralparameters, and (iii) protected the heart against the adverse effects of social stress. Repeatedsocial stress in Wistar Kyoto rats reproduces aspects of human depression/cardiovascularcomorbidity. Pharmacological enhancement of anandamide signaling might be a promisingstrategy for the treatment of these comorbid conditions.& 2015 Elsevier B.V. and ECNP. All rights reserved.

1. Introduction

Extensive evidence suggests that depression is a robust andindependent predictor of cardiovascular disease incidenceand progression (Van der Kooy et al., 2007). While a varietyof mechanisms – both genetic and behavioral – have beenproposed to explain this association, one specific pathophy-siological mechanism through which depression is thought toincrease cardiac risk is a dysregulation of the autonomicneural control of cardiac function. For example, depressivepatients have been found to display a predominance ofsympathoadrenergic activation and/or reduced parasympa-thetic modulation, as evidenced by increases in resting-state heart rate (HR) and decreases in its variability (HRV)(Udupa et al., 2007), both predictive of adverse cardiovas-cular events (Carney et al., 2005). However, other studieshave reported contradictory findings, leading to significantdebate and discussion as to whether depression per se isassociated with reduced HRV or, rather, autonomic dysfunc-tion is a consequence of pharmacological antidepressanttreatment (Licht et al., 2010; Kemp et al., 2014).

A common precipitating factor for the onset and progres-sion of both depressive and cardiovascular disorders invulnerable individuals is represented by prolonged orrepeated exposure to stressors of psychosocial nature(Albus, 2010). In preclinical settings, the resident-intruderparadigm is considered a relevant rat model of social stressthat relies on robust ethological prerequisites to meetconstruct and etiological validity for the human condition(Sgoifo et al., 2014). Rats repeatedly exposed to this socialstress paradigm show behavioral and biological changes thatmirror human depression (Becker et al., 2008; Wood et al.,2012; Carnevali et al., 2013). Importantly, such symptomshave been associated with long-lasting alterations in theautonomic neural modulation of HR (Wood et al., 2012) andpro-arrhythmic remodeling of electrical and structuralproperties of the myocardium (Carnevali et al., 2013).Therefore, this stress paradigm appears to be a usefulexperimental approach for studying the shared pathophy-siology and neural substrates that link depression andcardiovascular disease, and may be used to identify noveltherapeutic strategies for treating this comorbidity.

In this regard, recent years have witnessed an increasinginterest in the role of the endocannabinoid (ECB) system inthe regulation of emotional behavior (Gorzalka and Hill,2011; McLaughlin et al., 2014) and cardiovascular function(Hiley, 2009). ECBs, which include anandamide (AEA) and2-arachidonoylglycerol, and their receptors are prevalentthroughout neuroanatomical structures and circuits that are

i, L., et al., Antidepressant-like a.... European Neuropsychopharma

implicated in depression, such as the prefrontal cortex,hippocampus, amygdala and striatum (Bisogno et al., 1999;Moldrich and Wenger, 2000), as well as throughout thecardiovascular system (Bonz et al., 2003). In the brain,ECBs have been shown to constrain the stress response,acting presynaptically on CB1 receptors and, mainly, mod-ulating hypothalamic-pituitary-adrenal (HPA) axis and sym-pathetic nervous system activity, via down-regulation ofexcitatory (e.g. glutamatergic) neurotransmission (Riebeand Wotjak, 2011; Crowe et al., 2014). AEA is primarilycatabolized in vivo by the enzyme fatty acid amide hydro-lase (FAAH), which also cleaves the noncannabinoid fattyacid ethanolamides oleoylethanolamide (OEA) and palmi-toylethanolamide (PEA) (McKinney and Cravatt, 2005).Preliminary studies have demonstrated that facilitationof AEA signaling via inhibition of FAAH activity exertsantidepressant-like activity in chronically stressed rats(Bortolato et al., 2007), and that activation of the ECBpathway with exogenous AEA improves cardiac resistance toarrhythmias in rats (Krylatov et al., 2001; Ugdyzhekovaet al., 2001). Taken together, these findings provide arationale for the preclinical assessment of the therapeuticpotential of FAAH inhibitors for the treatment of depression-cardiovascular comorbidity.

Based on these considerations, in this study we applied aprotocol of repeated social stress on animals with a predis-position to stress-related psychophysiopathology (i.e., theWistar Kyoto (WKY) rats) (O’Mahony et al., 2011) in orderto: (i) verify the establishment of behavioral and biologicalsigns of a depressive-like state; (ii) determine whether suchsymptoms were associated with changes in the autonomicmodulation of HR (as indexed by HRV analysis) and increa-sed incidence of spontaneous arrhythmias; (iii) evaluatewhether pharmacological treatment with a selective FAAHinhibitor (URB694) exerts antidepressant-like activity andcardioprotective effects.

2. Experimental procedures

2.1. Animals

Experiments were conducted on 3-month-old Wistar Kyoto male rats(Charles River, Italy). They were singly housed with a 12-h lightcycle (lights on at 19.00 h) in climate-controlled rooms. Food andwater were available ad libitum, unless otherwise specified. Addi-tional older Wild-type Groningen male rats were housed with anoviduct-ligated female partner and used as residents in theresident-intruder paradigm (see below for details). All procedureswere approved by the Veterinarian Animal Care and Use Committee

ctivity and cardioprotective effects of fatty acid amide hydrolasecology (2015), http://dx.doi.org/10.1016/j.euroneuro.2015.07.015

3Antidepressant-like activity and cardioprotective effects of fatty acid amide hydrolase inhibitor URB694 in socially

of Parma University, with animals cared for in accordance with theEuropean Community Council Directives (2010/63/UE).

2.2. Experimental design

Fig. 1 displays the timeline of all procedures. We used 32 rats inExperiment 1 and 24 rats in Experiment 2. In each experiment,after baseline determinations, rats were randomly assigned toeither a social stress (groups 1 and 3) or a control (groups 2 and4) condition. Starting from day 19, stressed (STR) and control (CTR)groups received daily injection of either vehicle (VEH) or theselective FAAH inhibitor URB694. Specific experimental proceduresare described in the following sections.

2.3. Drug treatment

URB694 belongs to the class of carbamate FAAH inhibitors thatirreversibly carbamoylate the nucleophile catalytic serine in FAAHactive site (Tarzia et al., 2006). URB694 is a second generationinhibitor with improved metabolic stability and selectivity for FAAH(Clapper et al., 2009). URB694 was prepared using an improvedmethod that led to higher yield and easier purification compared tothose originally reported (Tarzia et al., 2006), following a reactionscheme that had been set up for a similar compound (Fiorelli et al.,2013). Physicochemical properties were identical to those pre-viously described (Tarzia et al., 2006). A solution of URB694 wasfreshly prepared for administration as in Bortolato et al. (2007).Starting from day 19, CTR and STR rats received daily injections ofVEH (vol: 1 ml/kg) or URB694 (0.1 mg/kg, i.p.) (Fig. 1). The dosewas chosen based on the available literature data and our experi-ence with FAAH inhibitors (Clapper et al., 2009; Vacondio et al.,2011). All injections were made between 14.00 and 15.00 h.

2.4. Surgery

Radiotelemetric transmitters (TA11CTA-F40, Data Sciences International,St. Paul, MN, USA) for ECG and locomotor activity (LOC, expressed ascounts/minute, cpm) recordings were implanted in animals intended forExperiment 1 under tiletamine hydrochloride+zolazepam hydrochloride(Zoletil, 20 mg/kg, s.c.) anesthesia, according to Carnevali et al. (2014).Rats intended for Experiment 2 underwent a similar procedure withouttransmitter implantation (sham operation). Animals recovered fromsurgery for at least 10 days before the start of recordings (Fig. 1).

Fig. 1 Timeline of experimental procedures in stressed (STR) and(0.1 mg/kg) or vehicle (VEH) starting from the beginning of the thi

Please cite this article as: Carnevali, L., et al., Antidepressant-like ainhibitor URB694 in socially stressed.... European Neuropsychopharma

2.5. Social stress

Social stress was based on a classical “resident–intruder” paradigmand was conducted similar to Carnevali et al. (2013). Briefly, eachrat from the stress groups (“intruder”) was transferred to theresident's cage, with a wire mesh partition separating the rats for30 min. Subsequently, the partition was removed allowing physicalinteraction for 10 min. Social stress exposure resulted in intrudersubordination and defeat (i.e., when the intruder rat assumed asupine posture that was held for at least 5 s). STR rats were exposedto 20 social defeat episodes over a period of 5 weeks, with socialdefeat sessions occurring daily for 4 consecutive days every week(Fig. 1). In the same days, CTRs were placed in a novel cage behinda partition for 30 min followed by 10 min of free exploration. Allrats were returned to their home cages after each session, whichtook place between 9.00 and 12.00 h.

2.6. Body weight (BW) and sucrose preference test

BW and sucrose solution consumption preference were measuredweekly for the duration of the study (Fig. 1). Animals were weighedand subsequently food and water deprived for 15 h prior to thesucrose solution consumption test (Bortolato et al., 2007). Duringthe test, animals were given access to water and 2% sucrosesolution in premeasured bottles (Grippo et al., 2011). One hourlater, the two bottles were removed and weighed again and foodand water were placed back in the cage. Sucrose solution intakewas expressed as the relative percentage of the total liquid intake,and was taken as an operational index of hedonic behavior.

2.7. ECG and LOC data collection and analysis

ECG and LOC were sampled for 2 min every hour in baseline conditionsand at the end of each week of the stress protocol (Fig. 1). Dailyrhythms of HR, HRV indexes and LOC were calculated as previouslydescribed (Carnevali et al., 2014). Detailed procedures for HRV analysisare only briefly summarized here. Time- and frequency-domain analysisof HRV was conducted on multiple segments of continuous and stableECG signals. In the time-domain, we calculated the root mean squareof successive RR interval differences (RMSSD, ms), which reflects vagalinput to the heart (Ramaekers et al., 2002). In the frequency-domain(fast-Fourier transformation), we measured (i) the power of the low(LF; 0.2–0.75 Hz) (ms2) and the high (HF; 0.75–2.5 Hz) (ms2) frequency

control (CTR) rats that were treated daily with either URB694rd week of the experimental protocol.

ctivity and cardioprotective effects of fatty acid amide hydrolasecology (2015), http://dx.doi.org/10.1016/j.euroneuro.2015.07.015

L. Carnevali et al.4

bands, the latter reflecting respiratory-related vagal influences(Ramaekers et al., 2002; Reyes Del Paso et al., 2013), and (ii) the LFto HF ratio, which is taken as a synthetic measure of sympathovagalbalance (Ramaekers et al., 2002; Carnevali et al., 2014).

In addition, the occurrence of arrhythmic events was determinedand quantified off-line based on Surawicz and Knilans (2008) andCurtis et al. (2013).

2.8. Forced swim test

An adapted version of the forced swim test originally described byPorsolt et al. (1977) was used. 24 h after the last URB694/VEHinjection (Fig. 1), rats were forced to swim individually for 5 min ina Plexiglas cylinder (height: 40 cm, diameter: 30 cm) filled withwater (temperature: 2471 1C; depth: 30 cm). During the test, rats'behavior was videotaped. The overall time spent in (i) immobility(floating and making only those movements necessary to keep thehead above water), (ii) swimming (active swimming motions thatmoved the animal across the center or in circles within the centerof the cylinder), and (iii) climbing (attempts to climb the wall of thecylinder) was scored by a trained experimenter blind to animals'group. Immobility during the single session of the forced swimmingtest was used as an operational index of depressive behavior,according to previous tests of validity and reliability (Cryan et al.,2005; Calil and Marcondes, 2006).

2.9. Plasma collection and measurements at sacrifice

Tail vein blood was collected three times during the experimentalprotocol (Fig. 1) in heparinized tubes, 3 h after the onset of thedark phase. Blood was centrifuged (2600g; 4 1C; 10 min), and thesupernatant was collected and stored at �20 1C until analysis. 24 hafter the last URB694/VEH injection, animals were decapitatedunder anesthesia (see above). Brains were rapidly removed andprefrontal cortex, hippocampus and striatum were dissected overdry ice using a rat brain atlas (Paxinos and Watson, 1986) as a guide,snap-frozen in liquid nitrogen, and stored at �80 1C until analysis.Such brain regions were selected based on their involvement in theregulation of emotional behavior (prefrontal cortex, hippocampus)(Vertes, 2006) and in the integration of social information intocoding of social action and reward (striatum) (Báez-Mendoza andSchultz, 2013). The heart and the adrenal glands were alsoremoved, weighed and stored at �80 1C until analysis.

2.9.1. Corticosterone analysisPlasma was deproteinized by addition of two volumes of organicsolvent (ice-cold acetonitrile), containing the internal standarddexamethasone (structural analog of corticosterone, 75 nmol/L).After centrifugation (14000g, 10 min, 4 1C), the supernatant wasdirectly injected in the liquid chromatography/tandem mass spec-trometry system (HPLC/MS/MS) for quantification of corticosteronelevels, in accordance with previously published analytical methods(e.g., Plenis et al., 2011).

2.9.2. Analysis of fatty acid ethanolamides AEA, OEA, PEAFatty acid ethanolamides AEA, OEA and PEA were extracted from(i) plasma, and (ii) 10% w/v brain tissue and atrial and ventricularhomogenates by organic solvent (ice-cold acetonitrile) addition andquantified by HPLC/MS/MS. The HPLC/MS/MS analytical standardsAEA, OEA, PEA and the deuterated internal standards AEA-d4, PEA-d4, OEA-d4 were purchased from Cayman Chemicals (Ann Arbor,Michigan) as stock solutions in ethanol.

2.9.3. FAAH assayFor ex vivo determination of FAAH activity, frozen brain fractionswere thawed and homogenized in ice-cold Tris buffer (10 volumes,

Please cite this article as: Carnevali, L., et al., Antidepressant-like ainhibitor URB694 in socially stressed.... European Neuropsychopharma

50 mM, pH 7.5) containing 0.32 M sucrose. The homogenates werecentrifuged (1000g, 10 min, 4 1C) and total protein content wasquantified in the supernatant by Pierce BCA protein kit. FAAHactivity was measured at 37 1C for 30 min in 0.5 mL Tris buffer(50 mM, pH 7.5) containing fatty acid-free bovine serum albumin(BSA) (0.05%, w/v), 50 mg of protein from brain homogenates, 10 mManandamide and [3H]-anandamide (10000 disintegrations per min-ute) as described in Clapper et al. (2009). Briefly, the reactionswere stopped with 1 mL CHCl3/MeOH (1:1). After centrifugation(2000g, 10 min, 4 1C), [3H]-ethanolamine was measured in theaqueous phase by liquid scintillation counting. [3H]-anandamide(specific activity: 60 Ci/mmol), employed as a substrate for ex vivoFAAH assay, was purchased from American Radiolabeled Chemicals(ARC, St. Louis, Missouri).

2.10. Statistical analysis

Two-way ANOVA for repeated measures with group as between-subject factor (4 levels) was applied for data obtained from: (i) BWand sucrose solution preference test with time as within-subjectfactor (6 levels: baseline; stress week 1, 2, 3, 4 and 5); (ii) ECG andLOC recordings separately for dark and light phases, with time aswithin-subject factor (6 levels: baseline; stress week 1, 2, 3, 4 and5); (iii) plasma determinations with time as within-subject factor (3levels: baseline; stress week 2 and 5). All other data were analyzedwith 2 (stress or control exposure)� 2 (URB694 or VEH treatment)factorial design ANOVAs. Follow-up analyses were conducted usingStudent's “t” tests, with a Bonferroni correction for multiplecomparisons for each outcome variable separately. Statisticalsignificance was set at po0.05.

3. Results

3.1. Body weight

BW changes are shown in Fig. 2A. Two-way ANOVA forrepeated measures yielded a significant effect of time(F=225.0, po0.01). Baseline BW was similar in the fourgroups. Starting from the end of the third week of the stressprotocol, BW was significantly lower in STR+VEH comparedto CTR+VEH rats (t=2.73, po0.05). This difference per-sisted until the end of the experiment (fifth week: t=3.6,po0.01). Chronic treatment with URB694 significantlyincreased BW gain in STR+URB rats compared to STR+VEH rats (fifth week: t=2.2, po0.05), whereas it didnot significantly modify BW gain in CTR+URB compared toCTR+VEH animals.

3.2. Sucrose solution preference

Changes in sucrose solution preference are depicted inFig. 2B. Two-way ANOVA for repeated measures yielded asignificant effect of group (F=10.8, po0.01). In baselineconditions, the four groups showed a similar preference forthe consumption of the sucrose solution. STR+VEH ratsshowed a reduction in sucrose solution preference com-pared to (i) CTR+VEH rats starting from the end of thesecond week (t=2.3, po0.05) until the end of the experi-ment (fifth week: t=4.8, po0.01), and (ii) baseline levelsat the end of the fourth (t=2.5, po0.05) and fifth (t=2.9,po0.05) weeks. STR+URB rats showed a significant reduc-tion in sucrose solution consumption preference comparedto CTR+URB rats at the end of the second week of the

ctivity and cardioprotective effects of fatty acid amide hydrolasecology (2015), http://dx.doi.org/10.1016/j.euroneuro.2015.07.015

Fig. 3 Time course of changes in plasma corticosterone levelsin baseline conditions and at the end of the second and fifthweek of the stress protocol. Data are reported as mean7SEMfor stressed (STR) and control (CTR) rats treated daily witheither URB694 (URB) or vehicle (VEH) (n=6 per group).Significant differences (Bonferroni test, p values are reportedin the text): n=versus CTR+VEH value; # =versus STR+URBvalue; †=versus baseline value.

Fig. 2 Time course of changes in body weight (panel A) andsucrose solution preference (panel B) in baseline conditions andduring the five weeks of repeated social stress. Data arereported as mean7SEM for stressed (STR) and control (CTR)rats treated daily with either URB694 (URB) or vehicle (VEH)(n=8 per group). Significant differences (Bonferroni test, pvalues are reported in the text): n=versus CTR+VEH value; #=versus STR+URB value; †=versus baseline value.

5Antidepressant-like activity and cardioprotective effects of fatty acid amide hydrolase inhibitor URB694 in socially

stress protocol (t=2.4, po0.05). Daily treatment withURB694 significantly increased sucrose solution preferenceup to baseline and control levels in STR+URB rats, whereasit did not significantly modify sucrose solution preference inCTR+URB animals compared to CTR+VEH counterparts.

3.3. HPA axis activity

Changes in plasma corticosterone levels are reported inFig. 3. Two-way ANOVA for repeated measures yielded asignificant effect of (i) time (F=24.2, po0.01), and (ii)group (F=4.6, po0.05). The four groups had similar base-line plasma corticosterone levels. Post-hoc analysesrevealed that at the end of the stress protocol, STR+VEHrats showed significantly higher plasma corticosteronelevels compared to (i) baseline levels (t=4.0, po0.01),

Please cite this article as: Carnevali, L., et al., Antidepressant-like ainhibitor URB694 in socially stressed.... European Neuropsychopharma

and (ii) CTR+VEH (t=2.6, po0.05) and STR+URB (t=3.2,po0.05) rats.

In addition, adrenal weight corrected for BW was sig-nificantly higher in STR+VEH rats compared to CTR+VEH(t=6.1, po0.01) and STR+URB (t=3.5, po0.01) rats(Table 1).

3.4. Forced swim test

Behavior during the forced swimming test is illustrated inFig. 4. STR+VEH rats exhibited a greater amount ofimmobility and a smaller amount of climbing behaviorcompared to CTR+VEH (immobility: t=3.0, po0.01; climb-ing: t=2.9, po0.05) and STR+URB (immobility: t=3.3,po0.01; climbing: t=2.9, po0.05) rats.

3.5. Cardiac activity

3.5.1. HR daily rhythmChanges in HR daily rhythm and its amplitude are shown inFig. 5. Two-way ANOVA for repeated measures yielded:(i) a significant effect of time for dark phase HR values(F=20.0, po0.01); (ii) a significant effect of group for darkphase HR values (F=3.4, po0.05) and HR rhythm amplitude(F=3.6, po0.05); (iii) a time� group interaction for darkphase HR values (F=3.4, po0.05). Follow-up analysesrevealed that during the dark phase of the fifth week,STR+VEH rats had significantly lower HR values comparedto baseline (t=3.2, po0.05) and CTR+VEH (t=2.9,po0.05) values, whereas no changes were observedbetween STR+URB and CTR+URB animals. In the sameperiod, HR rhythm amplitude resulted significantly reducedin STR+VEH rats compared to (i) the respective baselinevalue (t=3.5, po0.01), and (ii) CTR+VEH (t=3.9, po0.01)and STR+URB (t=3.8, po0.01) rats.

ctivity and cardioprotective effects of fatty acid amide hydrolasecology (2015), http://dx.doi.org/10.1016/j.euroneuro.2015.07.015

Table 1 Body weight, adrenal weight and heart weight in stressed (STR) and control (CTR) rats treated daily with eitherURB694 (URB) or vehicle (VEH) (n=6 per group).

BW (g) AW (mg) AW/BW (mg/g) HW (g) HW/BW (g/g)

STR+VEH 327718a 3472b 0.10470.002a,b 1.1370.04 0.0034770.00011a,b

CTR+VEH 390712 2572 0.06570.004 1.2470.04 0.0031770.00005STR+URB 356710 2473 0.06970.009 1.1270.03 0.0031570.00003CTR+URB 35774 2572 0.07170.006 1.1370.04 0.0031770.00008

Data are reported as mean7SEM. BW=body weight; AW= adrenal weight; HW= heart weight. Significant differences (Bonferroni test,p values are reported in the text).

a=versus CTR+VEH value.b=versus STR+URB value.

Fig. 4 Behavior during the forced swim test in stressed (STR)and control (CTR) rats treated daily with either URB694 (URB)or vehicle (VEH) (n=8 per group). Data are reported asmean7SEM. Significant differences (Bonferroni test, p valuesare reported in the text): n=versus CTR+VEH value; # =versusSTR+URB value.

L. Carnevali et al.6

3.5.2. HRV parametersHRV parameters during the dark phases of the baselineperiod and the fifth week of the stress protocol areillustrated in Fig. 5. Two-way ANOVA for repeated measuresyielded a significant effect of time (F=5.0, po0.05) and atime� group interaction (F=4.5 po0.05) for dark phaseLF/HF values. Follow-up analyses revealed that STR+VEHrats showed significantly lower LF to HF ratio during thefifth week compared to (i) the respective baseline value(t=3.6, po0.01), and (ii) CTR+VEH rats (t=3.0, po0.05),whereas no changes were observed between STR+URB andCTR+URB animals (Fig. 5). There were no significantdifferences in RMSSD and HF values among the groups;therefore, no follow-up tests were performed.

3.5.3. Vulnerability to cardiac arrhythmiasExamples of the most common forms of arrhythmias foundin this study are reported in Fig. 6A. Arrhythmia vulner-ability was almost completely absent in baseline conditions(total incidence of arrhythmias: CTR+VEH=0.3870.25,STR+VEH=0.5670.37, CTR+URB=1.0370.58, CTR+VEH=0.6670.56). At the end of the fifth week of thestress protocol, the total incidence of arrhythmias was

Please cite this article as: Carnevali, L., et al., Antidepressant-like ainhibitor URB694 in socially stressed.... European Neuropsychopharma

significantly higher in STR+VEH rats compared to CTR+VEH (t=3.8, po0.01) and STR+URB (t=3.6, po0.01) rats(Fig. 6B). This was mainly due to a significantly higherincidence of sinus pauses in STR+VEH rats (t=3.2, po0.01vs. CTR+VEH; t=2.5, po0.05 vs. STR+URB) (Fig. 6B).

3.6. Heart weight

Heart weight corrected for BW was significantly heavier inSTR+VEH rats compared to CTR+VEH (t=2.4, po0.05) andSTR+URB (t=2.8, po0.05) rats (Table 1).

3.7. LOC activity

Two-way ANOVA for repeated measures yielded a significanteffect of time (F=4.8, po0.05) and a time� group inter-action (F=4.7 po0.05) for dark phase LOC values. Followup analyses revealed that during the dark phase of the fifthweek, STR+VEH rats showed significantly lower LOC valuescompared to (i) CTR+VEH (t=2.2, po0.05) rats, and (ii) therespective baseline value (t=2.5, po0.05).

3.8. FAAH activity

FAAH activity levels in the brain are depicted in Fig. 7.There were no significant effects of stress exposure on FAAHactivity levels in the prefrontal cortex, hippocampus andstriatum. Daily treatment with URB694 significantlydecreased FAAH activity in the (i) prefrontal cortex(F=299.7, po0.01), (ii) hippocampus (F=226.9, po0.01),and (iii) striatum (F=209.7, po0.01). Results of post-hocanalyses are reported in Fig. 7.

3.9. Fatty acid ethanolamide levels

3.9.1. Brain levelsBrain AEA, OEA and PEA levels at the end of the experimentalprotocol are illustrated in Fig. 7. There were no significanteffects of stress exposure on AEA, OEA and PEA levels in theprefrontal cortex, hippocampus and striatum. Daily treatmentwith URB694 significantly increased (i) AEA levels in theprefrontal cortex (F=30.2, po0.01), hippocampus (F=28.9,po0.01) and striatum (F=10.4, po0.01), (ii) OEA levels in theprefrontal cortex (F=61.7, po0.01), hippocampus (F=25.4,po0.01) and striatum (F=8.2, po0.05), and (iii) PEA levels in

ctivity and cardioprotective effects of fatty acid amide hydrolasecology (2015), http://dx.doi.org/10.1016/j.euroneuro.2015.07.015

Fig. 5 Top panels: time course of changes in (i) heart rate (HR) rhythm during the 12-h light (L) and 12-h dark (D) phases of thedaily cycle (left), and (ii) HR daily rhythm amplitude (i.e., the difference between average light and dark phase values) (right).Middle and bottom panels: heart rate variability data collected during the dark phases of the basal period and at the end of the fifthweek of the stress protocol. Data are reported as mean7SEM for stressed (STR) and control (CTR) rats treated daily with eitherURB694 (URB) or vehicle (VEH) (n=8 per group). Significant differences (Bonferroni test, p values are reported in the text): n=versusCTR+VEH value; # =versus STR+URB value; †=versus baseline value.

7Antidepressant-like activity and cardioprotective effects of fatty acid amide hydrolase inhibitor URB694 in socially

the prefrontal cortex (F=10.9, po0.01) and hippocampus(F=25.4, po0.01). Results of post-hoc analyses are reportedin Fig. 7.

Please cite this article as: Carnevali, L., et al., Antidepressant-like ainhibitor URB694 in socially stressed.... European Neuropsychopharma

3.9.2. Plasma levelsChanges in plasma AEA, OEA and PEA levels are reported inTable 2. Two-way ANOVAs for repeated measured yielded: (i) a

ctivity and cardioprotective effects of fatty acid amide hydrolasecology (2015), http://dx.doi.org/10.1016/j.euroneuro.2015.07.015

Fig. 6 Panel A: Examples of the arrhythmic events found in this study from a representative STR+VEH rat. Panel B: Incidence ofarrhythmias during the fifth week of the stress protocol in stressed (STR) and control (CTR) rats treated daily with either URB694(URB) or vehicle (VEH) (n=8 per group) (abbreviations: AEB= atrial escape beat; AV=atrioventricular; JEB=junctional escape beat;PVC=premature ventricular complex). Data are reported as mean7SEM. Significant differences (Bonferroni test, p values arereported in the text): n=versus CTR+VEH value;# =versus STR+URB value.

L. Carnevali et al.8

significant effect of time for AEA (F=6.9, po0.05), OEA(F=8.9, po0.01) and PEA (F=59.1, po0.01) levels; (ii) asignificant effect of group for AEA (F=5.9, po0.01), OEA(F=6.2, po0.01) and PEA (F=5.8, po0.01) levels; (iii) atime� group interaction for PEA levels (F=7.7, po0.01).Follow-up analysis revealed that at the end of the experi-mental protocol (week 5), STR+VEH and CTR+VEH rats hadsignificantly lower plasma levels of AEA, PEA and OEAcompared to (i) the respective baseline levels, and (ii) STR+URB and CTR+URB levels (p values are reported in thelegend of the figure). In addition, daily administration ofURB694 increased plasma levels of AEA in STR+URB and CTR+URB rats compared to pre-treatment (second week) levels (pvalues are reported in Table 2 legend).

3.9.3. Heart levelsIn atrial homogenates, there were no significant effects ofstress on AEA, OEA and PEA levels (Table 3). Daily treatmentwith URB694 significantly increased AEA (F=14.8, po0.01),OEA (F=29.9, po0.01) and PEA (F=42.8, po0.01) levels(Table 3). In ventricular homogenates, AEA levels weresignificantly increased by daily treatment with URB694(F=5.1, po0.05) and were not affected by stress exposure.Moreover, PEA levels were significantly reduced by stress

Please cite this article as: Carnevali, L., et al., Antidepressant-like ainhibitor URB694 in socially stressed.... European Neuropsychopharma

exposure (F=10.2, po0.01) and increased by daily treat-ment with URB694 (F=7.8, po0.05) (Table 3). Results ofpost-hoc analyses are reported in Table 3.

4. Discussion

The major and novel findings of the present study is that thepotent and selective FAAH inhibitor URB694 normalizes(i) biological and behavioral depressive-like symptoms,and (ii) cardiac alterations elicited by repeated social stressin WKY rats.

4.1. Depressive-like syndrome in socially stressedWKY rats

Chronic stressful life situations can increase the risk ofdeveloping depression in vulnerable individuals (Klengel andBinder, 2013). The WKY rat is an inbred strain that showsdepressive-related behaviors in response to acute stresssuch as the open field and the forced swim test (O’Mahonyet al., 2011). WKY rats have also an increased susceptibilityto chronic stress-induced dysregulation of the HPA axis(Malkesman et al., 2008), gastric ulceration (Paré andRedei, 1993) and gastrointestinal impairment (O’Malley

ctivity and cardioprotective effects of fatty acid amide hydrolasecology (2015), http://dx.doi.org/10.1016/j.euroneuro.2015.07.015

Fig. 7 Fatty acid amide hydrolase (FAAH) activity (panel A) and anandamide (AEA, panel B), oleoylethanolamide (OEA, panel C) andpalmitoylethanolamide (PEA, panel D) levels in the prefrontal cortex, hippocampus and striatum, 24 h after the last URB694/vehicleinjection. Data are reported as mean7SEM for stressed (STR) and control (CTR) rats treated daily with either URB694 (URB) orvehicle (VEH) (n=6 per group). FAAH activity was calculated as the relative % to CTR+VEH value. Significant differences (Bonferronitest, po0.05): n=versus respective VEH value.

9Antidepressant-like activity and cardioprotective effects of fatty acid amide hydrolase inhibitor URB694 in socially

et al., 2010). These traits mark WKY rats as a rodent modelwith a predisposition to stress-related psychophysiopathol-ogy, even though genetic and behavioral differences haveemerged between WKY lines obtained from different ven-dors (Browne et al., 2015). Based on these considerations,the application of a social stress paradigm to WKY rats is, inour view, a valid experimental approach to model beha-vioral and biological changes reminiscent of those observedin human depression.

Stressed WKY rats treated with vehicle displayed a long-term reduction in body weight growth, a common marker ofdepression in rats (Becker et al., 2008). Moreover, theyexhibited an increase in plasma corticosterone levels andadrenal gland weight. Of note, such changes were observedfour days after the last defeat, suggesting chronic HPA axishyperactivity, a well documented finding in depression(Carroll et al., 2007). In addition, the decrease in sucrosesolution preference in stressed animals may reflect theonset of an anhedonic state, a core symptom of depression(American Psychiatric Association, 2013). Finally, theirprolonged immobility in the forced swim test may beregarded as a relevant index of the depressive-like symptomof helplessness (Cryan et al., 2005). Taken together, thesefindings indicate that repeated social stress in WKY ratsinduced persistent changes in a variety of parametersrelevant to depression. In interpreting these results, it must

Please cite this article as: Carnevali, L., et al., Antidepressant-like ainhibitor URB694 in socially stressed.... European Neuropsychopharma

be noted that WKY rats appear to have heightened socialavoidance (Nam et al., 2014), which may make themespecially sensitive to the social stress paradigm used inthe present study.

4.2. Antidepressant-like activity of URB694

Previous studies have reported that chronic stress impairs CBsignaling, at either the ligands or the receptor levels(Rademacher et al., 2008; Reich et al., 2009). Here, we foundno differences in central levels of AEA and FAAH activitybetween stressed and control rats treated with vehicle,similarly to another study (Bortolato et al., 2007). Thesefindings seem to indicate that repeated stress exposure didnot compromise ECB neurotransmission in key neuroanatomicalbrain regions implicated in depression. A possible explanationis that control animals also presumably experienced certainamount of stress (e.g. novel cage test, daily injections).Indeed, at the end of the experimental protocol, plasma AEAlevels resulted significantly lower both in stressed and controlanimals treated with vehicle compared to the respectivebaseline levels. One limitation of this study is that we didnot determine the expression of CB receptors, which wouldhave provided a more complete picture on the effects of thissocial stress protocol on ECB system. As expected, daily

ctivity and cardioprotective effects of fatty acid amide hydrolasecology (2015), http://dx.doi.org/10.1016/j.euroneuro.2015.07.015

Table 2 Plasma levels of AEA, OEA and PEA in stressed (STR) and control (CTR) rats treated daily with either URB694 (URB)or vehicle (VEH) (n=6 per group), in basal condition and at the end of the 2nd and 5th week of the experimental protocol.

Basal week 2 week 5

AEA (nM) STR+VEH 0.9470.10 0.5370.05a 0.6170.07a

CTR+VEH 1.1870.18 0.7270.05a 0.6970.05a

STR+URB 0.9770.09 0.7670.12a 1.0270.09b,c

CTR+URB 1.0970.15 0.6970.06a 1.1270.08b,c

OEA (nM) STR+VEH 8.4670.33 7.7870.49 7.2070.43CTR+VEH 8.0870.77 8.9270.73 7.1970.35STR+URB 9.0370.78 8.2870.90 9.4170.42b

CTR+URB 12.1971.01 10.2070.48 9.7170.06PEA (nM) STR+VEH 10.8670.43 11.4770.35 4.9571.06a

CTR+VEH 10.9870.73 10.5070.94 5.3870.73a

STR+URB 11.8370.49 11.1870.92 8.5870.88b,a

CTR+URB 12.4370.82 10.6570.64 12.3270.68

Data are reported as mean7SEM. AEA=anandamide; OEA=oleoylethanolamide; PEA=palmitoylethanolamide. Significant differences(Bonferroni test).

a=po0.05 versus baseline value.b=po0.05 versus respective VEH value.c=po0.05 versus week 2 value.

Table 3 AEA, OEA and PEA levels in atrial and ven-tricular homogenates of stressed (STR) and control (CTR)rats treated daily with either URB694 (URB) or vehicle(VEH) (n=6 per group).

Atria Ventricles

AEA (pmol/g) STR+VEH 28.272.2 22.771.3CTR+VEH 23.172.5 33.674.4STR+URB 40.372.8a 34.774.0CTR+URB 31.072.6 38.474.2

OEA (pmol/g) STR+VEH 64.775.5 66.471.4CTR+VEH 57.478.1 79.275.2STR+URB 102.574.1a 86.672.8CTR+URB 100.0710.1a 74.376.6

PEA (pmol/g) STR+VEH 66.374.0 71.773.6CTR+VEH 56.278.7 87.577.8STR+URB 106.278.2a 100.977.6CTR+URB 178.5721.4a 172.0722.5a

Data are reported as mean7SEM. AEA=anandamide;OEA=oleoylethanolamide; PEA=palmitoylethanolamide.Significant differences (Bonferroni test).

a=po0.05 versus respective VEH value.

L. Carnevali et al.10

administration of the potent and selective FAAH inhibitorURB694 provoked a long-lasting reduction in FAAH activity inthe brain. This, in turn, resulted in an increase in AEA levelsonly in the prefrontal cortex of stressed rats, whereas controlrats showed higher AEA levels in all the selected brain areas.The differential effect of the FAAH inhibitor between stressedand control animals on brain levels of AEA, as well as of OEAand PEA, requires further scrutiny. Importantly, chronic treat-ment with URB694 normalized the effects of social stress onbody weight growth, sucrose solution preference and behaviorin the forced swim test. Of note, this drug regimen had noeffects on controls, suggesting that the FAAH inhibitor did notaffect normal biological processes and behavioral responses.Preclinical research regarding the role of the ECB system in theregulation of stress and emotional behavior creates a compel-ling argument that ECB signaling in the prefrontal cortex iscrucial for constraining activation of the neuroendocrine andbehavioral response to stress (Riebe and Wotjak, 2011;McLaughlin et al., 2014). For example, it has been shown thatCB1 activation in the prefrontal cortex suppresses glucocorti-coid secretion after cessation of a stressor (Hill et al., 2011).Accordingly, daily treatment with URB694 prevented thehypercorticosterolemia and adrenal hypertrophy observed instressed rats treated with vehicle. Therefore, our hypothesis isthat the beneficial effects of URB694 on the neuroendocrineand behavioral adverse consequences of repeated stressexposure may be due to the ability of FAAH inhibitors tomagnify endogenous AEA signaling at CB1 receptors, particu-larly in the prefrontal cortex (McLaughlin et al., 2014). This issupported by experimental evidence showing that the CB1antagonist rimonabant prevents the mood-enhancing actions ofa similar FAAH inhibitor (URB597) (Gobbi et al., 2005). Inter-estingly, a recent study reported that URB597 attenuated thehyperalgesia exhibited by WKY rats in response to noxiousstimuli, and that this effect was mediated by CB1 receptors inthe rostral ventromedial medulla (Rea et al., 2014). Theauthors suggested that the endocannabinoid system mayrepresent a useful and novel therapeutic approach for the

Please cite this article as: Carnevali, L., et al., Antidepressant-like ainhibitor URB694 in socially stressed.... European Neuropsychopharma

treatment of patients with pain that is co-morbid with stress-related psychiatric disorders (Rea et al., 2014). In our study,the lack of CB1 antagonist co-administration with URB694represents a major limitation. In addition, we cannot ruleout a possible involvement of PEA and OEA in the beneficialeffects of URB694, possibly via nuclear peroxisomeproliferator-activated receptors (Lo Verme et al., 2005).

4.3. Social stress-induced cardiac abnormalities

Stressed rats treated with vehicle showed a progressivedampening of the daily amplitude of HR rhythm, which was

ctivity and cardioprotective effects of fatty acid amide hydrolasecology (2015), http://dx.doi.org/10.1016/j.euroneuro.2015.07.015

11Antidepressant-like activity and cardioprotective effects of fatty acid amide hydrolase inhibitor URB694 in socially

mainly due to a reduction of HR values during the darkphase of the daily cycle. Such an effect may be ascribed tolower levels of somatomotor activity in these animals.However, we did not find a significant correlation betweenthe magnitudes of HR and LOC decreases. This suggests thatother factors likely concurred to determine stress-inducedbradycardia. Indeed, HRV analysis revealed that repeatedsocial stress induced a shift of the sympathovagal balancetowards parasympathetic prevalence (decreased LF to HFratio) in vehicle-treated rats, without an increase in theabsolute levels of vagal tone (RMSSD and HF indexes). TheseHRV data suggest that sympathetic influences on cardiacpacemaker activity were likely reduced in these animals,causing bradycardia. This was an unexpected phenomenon,as human depression is often associated with sympathetichyperactivity (Udupa et al., 2007; Kemp et al., 2012).However, in a recent study depressed patients have beenshown to exhibit signs of parasympathetic prevalence(decreased LF to HF ratio) during mental stress comparedto controls (Liang et al., 2014). The authors suggested thatthis finding may signal the accumulative effects of depres-sion on the cardiovascular system in real life stress situa-tions. Like psychosocial stress in humans, social stress inrats has been well characterized as engaging the sympa-thetic nervous system (Sgoifo et al., 1999). Therefore, ourhypothesis is that repeated episodes of social defeat mighthave determined a desensitization/downregulation of car-diac β-adrenoreceptors in vehicle-treated animals. Thiswould explain why cardiac sympathetic influences werereduced in these animals. Although these changes mightappear adaptive, they could represent the mechanismunderlying the increased incidence of spontaneous bradyar-rhythmias, particularly sinus pauses, observed in stressedrats. Sinus pauses, alternatively known as sinus arrest oratrial standstill, are due to cessation of the sinus nodeimpulse and result in absent PQRSTcomplexes (Surawicz andKnilans, 2008). These arrhythmic events are generallyattributed to conditions of excessive vagal modulation(Jordan and Mendel, 1995; Surawicz and Knilans, 2008),indicating that persistent vagal predominance may havepredisposed the heart of stressed rats to sinus nodedysfunction. However, we cannot exclude that arrhythmo-genesis was due to stress-induced alterations in the struc-tural characteristics (such as the reported cardiachypertrophy) and/or in the electrophysiological propertiesof the myocardium (Carnevali et al., 2013). It may beargued that the incidence and severity of the arrhythmicevents reported in this study were rather moderate. How-ever, it should be kept in mind that arrhythmia suscept-ibility was assessed in young, otherwise healthy rats duringundisturbed resting conditions.

4.4. Cardioprotective effects of URB694

Chronic treatment with URB694 normalized resting heartrate, restored the autonomic balance and preventedarrhythmia occurrence in stressed animals. The drug regi-men had no effects on controls, in line with the idea thatECBs do not mediate tonic control over the cardiovascularsystem in normal physiology (Hiley, 2009). ECB1 receptorsare present on pre-synaptic sympathetic nerve terminals

Please cite this article as: Carnevali, L., et al., Antidepressant-like ainhibitor URB694 in socially stressed.... European Neuropsychopharma

and in vitro studies report that their activation decreasessympathetic outflow by inhibition of noradrenaline release(Niederhoffer and Szabo, 1999). It is therefore tempting tospeculate that the cardioprotective effects of URB694 mightbe due to its ability to dampen stress-induced sympathetichyperactivity via enhancement of AEA signaling at thesereceptors. However, we cannot exclude that the effects ofthe FAAH inhibitor are mediated directly on the myocar-dium. Indeed, our data indicate that administration ofURB694 determined a general increase in AEA, OEA andPEA levels in heart homogenates. Moreover, preliminarystudies have shown that exogenous in situ application ofeither AEA or PEA confers cardiac protection (Lepicieret al., 2003; Underdown et al., 2005) and increases cardiacresistance to the arrhythmogenic effects of ischemia andreperfusion (Krylatov et al., 2001), with the underlyingmolecular and electrophysiological mechanisms that haveyet to be elucidated. Further investigation is required inorder to clarify whether URB694 exerts its anti-arrhythmiceffects directly on the myocardium by increasing thethreshold for arrhythmias and/or indirectly by restoringthe autonomic balance either at the central or peripherallevel. Whatever the mechanism prevails, our results suggestthat inhibition of FAAH activity improves cardiac function inWKY rats exposed to repeated social defeat stress.

5. Conclusion and perspectives

This study provides solid evidence that repeated socialstress induces depressive-like biological and behavioralsymptoms in WKY rats, which can be corrected by thepotent and selective FAAH inhibitor URB694. Intriguingly,this pharmacological approach appears to confer also car-dioprotection against the adverse consequences of socialstress in this animal model. The precise neurobiologicalpathways by which URB694 exerts its effects need to beaddressed in future studies. In interpreting our results, itmust also be acknowledged that WKY rats have been shownto have higher levels of brain FAAH compared to Wistar rats(Vinod et al., 2012) and therefore might be hyper-responsive to the treatment with the FAAH inhibitor usedin this study.

Nevertheless, our findings provide a strong basis forfurther investigation aimed at determining whether FAAHmight represent an effective therapeutic target for thetreatment of depression-cardiovascular comorbidity underchronic stress conditions.

Role of funding source

Funding for this study was provided by a grant from the Universityof Parma (FIL 2012); the University of Parma had no further role instudy design; in the collection, analysis and interpretation of data;in the writing of the report; and in the decision to submit the paperfor publication.

Contributors

Author Luca Carnevali wrote the protocol, performed the experi-ments, analyzed the data, interpreted the results and wrote thefirst draft of the manuscript. Authors Federica Vacondio and Stefano

ctivity and cardioprotective effects of fatty acid amide hydrolasecology (2015), http://dx.doi.org/10.1016/j.euroneuro.2015.07.015

L. Carnevali et al.12

Rossi analyzed the data and revised the manuscript for importantintellectual content. Authors Sergio Callegari and Emilio Macchiinterpreted the data and revised the manuscript for importantintellectual content. Authors Gilberto Spadoni and Annalinda Bedinisynthesized URB694 and revised the manuscript for importantintellectual content. Authors Silvia Rivara and Marco Mor designedthe study, interpreted the results and revised the manuscript forimportant intellectual content. Author Andrea Sgoifo designed thestudy, wrote the protocol, and revised the manuscript for importantintellectual content. All authors contributed to and have approvedthe final manuscript.

Conflict of interest

All authors declare that they have no conflicts of interest.

Acnowledgments

Funding for this study was provided by a grant from the Universityof Parma (FIL 2012).

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