Psychopharmacology DOI 10.1007/s00213-015-3917-y

ORIGINAL INVESTIGATION

2-AG promotes the expression of conditioned fear via cannabinoid receptor type 1 on GABAergic neurons Alvaro Llorente-Berzal 1,6 & Ana Luisa B. Terzian 2,3 & Vincenzo di Marzo 4 & Vincenzo Micale 2,5 & Maria Paz Viveros 1,6 & Carsten T. Wotjak 2

Received: 14 November 2014 / Accepted: 10 March 2015 # Springer-Verlag Berlin Heidelberg 2015

Abstract Rationale The contribution of two major endocannabinoids, 2-arachidonoylglycerol (2-AG) and anandamide (AEA), in the regulation of fear expression is still unknown. Objectives We analyzed the role of different players of the endocannabinoid system on the expression of a strong auditory-cued fear memory in male mice by pharmacological means. Results The cannabinoid receptor type 1 (CB1) antagonist SR141716 (3 mg/kg) caused an increase in conditioned freezing upon repeated tone presentation on three consecutive days. The cannabinoid receptor type 2 (CB2) antagonist AM630

Electronic supplementary material The online version of this article (doi:10.1007/s00213-015-3917-y) contains supplementary material, which is available to authorized users. * Carsten T. Wotjak [email protected] 1

Departamento de Fisiología (Fisiología Animal II), Facultad de Biología, Universidad Complutense de Madrid, C/ Jose Antonio Novais 12, 28040 Madrid, Spain

2

RG BNeuronal Plasticity^, Department of Stress Neurobiology and Neurogenetics, Max Planck Institute of Psychiatry, Kraepelinstr. 210, 80804 Munich, Germany

3

Graduate School of Systemic Neuroscience, Ludwig-MaximiliansUniversität, Munich, Germany

4

Endocannabinoid Research Group, Institute of Biomolecular Chemistry, C.N.R., Via Campi Flegrei 34, Comprensorio Olivetti, 80078 Pozzuoli, NA, Italy

5

CEITEC—Central European Institute of Technology, Masaryk University, Brno, Czech Republic

6

Instituto de Investigación Sanitaria del Hospital Clínico San Carlos (IdISSC), Madrid, Spain

(3 mg/kg), in contrast, had opposite effects during the first tone presentation, with no effects of the transient receptor potential vanilloid receptor type 1 (TRPV1) antagonist SB366791 (1 and 3 mg/kg). Administration of the CB2 agonist JWH133 (3 mg/kg) failed to affect the acute freezing response, whereas the CB1 agonist CP55,940 (50 μg/kg) augmented it. The endocannabinoid uptake inhibitor AM404 (3 mg/kg), but not VDM11 (3 mg/kg), reduced the acute freezing response. Its co-administration with SR141716 or SB366791 confirmed an involvement of CB1 and TRPV1. AEA degradation inhibition by URB597 (1 mg/kg) decreased, while 2-AG degradation inhibition by JZL184 (4 and 8 mg/kg) increased freezing response. As revealed in conditional CB1deficient mutants, CB1 on cortical glutamatergic neurons alleviates whereas CB1 on GABAergic neurons slightly enhances fear expression. Moreover, 2-AG fear-promoting effects depended on CB1 signaling in GABAergic neurons, while an involvement of glutamatergic neurons remained inconclusive due to the high freezing shown by vehicle-treated Glu-CB1-KO. Conclusions Our findings suggest that increased AEA levels mediate acute fear relief, whereas increased 2-AG levels promote the expression of conditioned fear primarily via CB1 on GABAergic neurons.

Keywords Fear extinction . TRPV1 . CB1 . CB2 . URB597 . JZL184 . AM404 . Anandamide

Introduction Post-traumatic stress disorder (PTSD) is a fear-related anxiety disorder caused by the exposure to extreme traumatic events (Holmes and Singewald 2013; Andero and Ressler 2012). Cognitive behavioral therapy (CBT) has become the best

Psychopharmacology

treatment for PTSD (Graham and Milad 2011). It includes confrontation with trauma reminders (exposure therapy) (Dias et al. 2013; Kearns et al. 2012). This is particularly stressful to the patients and represents a significant challenge to compliance rates and therapeutic success. Thus, co-treatment with drugs, which may enhance fear extinction and/or suppress the acute negative affect, may promote exposure therapy (Bitencourt et al. 2013; Dias et al. 2013; Parsons and Ressler 2013). The search for optimal intervention strategies may benefit from basic research studying the mechanisms of acquisition, consolidation, and extinction of conditioned fear. In this task, animals learn to associate e.g. a tone with a punishment (e.g., electric foot shock). Subsequent reexposure to the tone leads to fear responses, which include freezing behavior. In the past decade, it became evident that the endocannabinoid system of the brain plays an essential role in extinction of conditioned fear (Marsicano et al. 2002). In fact, activation of cannabinoid receptor type 1 (CB1) by its endogenous ligands seems to play a dominant role in habituation-like processes leading to acute fear relief (Kamprath et al. 2006; Plendl and Wotjak 2010; for review, see Riebe et al. 2012). Importantly, this function of endocannabinoid signaling became evident for highly aversive fear memories (Kamprath et al. 2009), which led us to propose the critical range theory of endocannabinoid action (Moreira and Wotjak 2010; Hill et al. 2010). To d ay, w e kn o w a b ou t th e c o m p l e xi t y o f t h e endocannabinoid system, which includes a variety of receptors such as CB1 (Hillard 2014); cannabinoid receptor type 2 (CB2; Dhopeshwarkar and Mackie 2014) and transient receptor potential vanilloid receptor type 1 (TRPV1; Edwards et al. 2012); and several ligands such as 2-arachidonoylglycerol (2AG; Savinainen et al. 2012) and anandamide (AEA; GunduzCinar et al. 2013a), to name the two most prominent, and their synthesizing and degrading enzymes and, likely, specific transporters responsible for their uptake (Fowler 2013; for review, see Mechoulam and Parker 2013). Interestingly, AEA and 2-AG levels might be differentially affected by increasing shock intensities (Morena et al. 2014). CB1 receptors are presynaptically expressed by various different neuronal subpopulations (Marsicano and Lutz 1999) and also by glia cells (Han et al. 2012). Activation of CB1 leads to a decrease in neurotransmitter release. With the advent of conditional mouse genetics (Deussing 2013), it became possible to disentangle the behavioral impact of those different subpopulations. For instance, there is evidence that activation of CB1 receptors by a selective agonist may exert anxiogenic or anxiolytic effects, depending on whether they are expressed by GABAergic vs. cortical glutamatergic neurons (Rey et al. 2012). Similar opposing effects were reported for fear-related freezing behavior (Kamprath et al. 2009; Lafenêtre et al. 2009; Metna-Laurent et al. 2012). Furthermore, TRPV1 and CB1 seem to exert opposite effects on fear

and anxiety (Marsch et al. 2007; for review, see Moreira et al., Neuroscience 2012; Aguiar et al. 2014), and also CB2 receptors were reported to modulate anxiety-like behavior (Tambaro and Bortolato 2012). Given the multitude of endocannabinoid effects on fear and anxiety, pharmacological modulation of endocannabinoid signaling has been proposed as a valuable tool for the treatment of fear-related disorders (Parsons and Ressler 2013; Trezza and Campolongo 2013; Andero and Ressler 2012; Micale et al. 2013). Successful attempts in animal models included administration of selective agonists (Ganon-Elazar and Akirav 2013; Niyuhire et al. 2007; Pamplona et al. 2006) and pharmacological enhancement of AEA and/or 2-AG by inhibition of their major degrading enzymes and/or uptake processes (Trezza and Campolongo 2013). For instance, AM404 (i.e., an inhibitor of endocannabinoid uptake), and URB597 or AM3506 (i.e., inhibitors of fatty acid amide hydrolase (FAAH), the main degrading enzyme of AEA) enhanced extinction in several fear conditioning paradigms (Gunduz-Cinar et al. 2013b; Busquets-Garcia et al. 2011; Manwell et al. 2009; Bitencourt et al. 2008; Pamplona et al. 2008; Chhatwal et al. 2005). However, compared to the fear-alleviating effects of enhanced AEA signaling (Gunduz-Cinar et al. 2013b; Busquets-Garcia et al. 2011), surprisingly little is known about the specific involvement of 2-AG (Rea et al. 2014) and the inhibition of its degrading enzyme monoacylglycerol lipase (MAGL; Busquets-Garcia et al. 2011). To the best of our knowledge, there are no studies which directly compared the role of AEA vs. 2-AG in the expression of auditory-cued fear memories. Therefore, we decided to systematically modulate the various players of the endocannabinoid system and to analyze consequences on the expression of conditioned fear. This included three different strategies: (i) blockade of CB1, CB2, and TRPV1 to study their involvement in sustained fear responses; (ii) activation of CB1 and CB2 by selective agonists; and (iii) enhancement of AEA and/or 2-AG signaling by blocking degradation and/or uptake processes. Effects of repeated drug treatment on the expression of auditory-cued fear memories were opposed to general consequences on locomotor activity. Finally, we employed mice with conditional ablation of CB1 from GABAergic or cortical glutamatergic neurons to identify the neuronal subpopulation responsible for the fear-modulating effects of 2-AG.

Material and methods Animals Male C57BL/6NCrl (B6N) mice were purchased from Charles River (Bad Sulzfeld, Germany) at age of 6–7 weeks. Mice with conditional deletion of CB1 from cortical

Psychopharmacology

glutamatergic (CB1f/f,Nex-Cre; Glu-CB1-KO; Monory et al. 2006) or GABAergic neurons of the forebrain (CB1f/f,Dlx5/6Cre ; GABA-CB1-KO; Monory et al. 2006) were kept on a mixed genetic background with a predominant B6N contribution. They were generated, bred, and genotyped essentially as described (Metna-Laurent et al. 2012), from our local breeding stocks (MPI for Biochemistry, Martinsried, Germany), and they were transferred to our animal facility when they were 8–12 weeks old. Mice were singled housed under an inverse 12 h:12 h lightdark cycle (lights off 09:00 a.m.), under standard laboratory conditions with water and food ad libitum for at least 12 days before starting with the experiments. All experiments were carried out according to the European Community Council Directive 2010/63/EEC and approved by the local government of Upper Bavaria (55.2.1.54-2532-44-09 and 55.2.1.54-2532-141-12). Efforts were made to minimize animal suffering and to reduce the number of animals used. Drugs AM630, JWH133, SB366791, and JZL184 were purchased from Tocris (Bristol, UK), and AM404, CP55,940, and URB597 from Sigma-Aldrich (Steinheim, Germany); SR141716 and VDM11 were from Cayman Chemical (Ann Arbor, USA), and OMDM198 was kindly provided by Dr. Vincenzo Di Marzo. Doses and vehicles were chosen based on previous findings (Busquets-Garcia et al. 2011; Rey et al. 2012; Micale et al. 2013); in the case of OMDM198, we followed recommendations made by Dr. Vincenzo Di Marzo. Thus, AM404 (3 mg/kg), VDM11 (3 mg/kg), OMDM198 (1, 2.5, and 5 mg/kg), and CP55,940 (1 and 50 μg/kg) were dissolved in ethanol:Cremophor (Sigma-Aldrich; Steinheim, Germany):saline in a 1:1:18 concentration. For AM630 (3 mg/kg), JWH133 (3 mg/kg), SB366791 (1 and 3 mg/kg), and SR141716 (3 mg/kg), the vehicle was a mixture of 3 drops of Tween-80 (Sigma-Aldrich; Steinheim, Germany) in 10 ml 2.5 % dimethylsulfoxide (DMSO; Sigma-Aldrich; Steinheim, Germany) in saline. URB597 (1 mg/kg) and JZL184 (4 and 8 mg/kg) were dissolved in 15 % DMSO:4.25 % polyethylene glycol (Carl Roth; Karlsruhe, Germany):4.25 % Tween 80:76.5 % saline. All pharmacological treatments were administered intraperitoneally in an injection volume of 10 ml/kg. Fear conditioning At day 0 (d0), mice were placed into cubic-shaped conditioning chambers with metal walls and metal grid floor (Kamprath and Wotjak 2004). After 180 s, a 20-s sine wave tone (9 kHz, 80 dB) was presented, which co-terminated with a 2-s electric foot shock (shocker/scrambler: ENV-414, MED Associates). Except for the first experiment, the shock intensity was

1.5 mA, which was about five times the individual pain threshold of B6N mice (Siegmund et al. 2005; Siegmund and Wotjak 2007). After the shock presentation, mice remained in the chamber for another 60 s. Between sessions, the conditioning chamber was cleaned with 70 % ethanol. Mice were reexposed to the tone for 3 min at d1, d2, d3, and d10 after conditioning. This time, they were placed into a neutral test context with cylindrical shape and bedding. After 180 s, the tone was presented for 3 min, and after another 60 s, mice were returned to their home cages. In this neutral context, the bedding was renewed and scented with 1 % acetic acid. Conditioning and test contexts differed in multiple aspects, including shape, texture, and odor (Kamprath and Wotjak 2004), which kept generalized contextual fear at a minimum (Golub et al 2009). This enabled us to specifically study the involvement of endocannabinoids/endovanilloids in the expression of auditory-cued fear memory. The behavior of the animals was recorded and analyzed off-line by a trained observer (EVENTLOG software, designed by Robert Henderson, 1986). Open field test The open field test was performed in the TruScan Photo Beam Activity system (Coulbourn Instruments, Whitehall, PA, USA) as described previously (Jacob et al. 2009) under red light. Mice were placed into the center of a Plexiglas chamber (L26 cm×W26 cm×H38 cm) for 15 min. Horizontal locomotion and vertical exploration were automatically recorded by two photobeam sensor rings (2 and 5 cm above the floor; photobeams are spaced apart by 1.52 cm, providing a 0.73cm spatial resolution) which surrounded the Plexiglas chambers. Each test cage, including the sensor rings, was enclosed by opaque Plexiglas side walls (L47 cm × W47 cm × H38.5 cm). The floor was cleaned with soap and water and dried after each test session. Data were acquired with a sample rate of 4 Hz. The following measures were automatically calculated by TruScan Software Version 1.1 (Coulbourn Instruments): (i) movement time (s), (ii) distance moved in the horizontal plane (cm), and (iii) the number of vertical explorations (rearings). Experiments All experiments were performed during the active phase of the animals between 09:30 and 17:00 hours. The experimenter was blind to the treatment/genotype during both experiments and behavioral analyses. If not stated otherwise, drug treatment happened 30 min before reexposure to the tone at d1, d2, and d3 after conditioning, except for URB597 and JZL184 which were administered 120 min before in order to ensure adequate functionality (Micale et al. 2013). At d10, all mice were treated with vehicle. Treatment before exposure to the

Psychopharmacology

open field was the same as used at d1–d3. All experiments are described in the BResults^ section. Data analysis and statistics Freezing behavior was manually scored as described (Kamprath and Wotjak 2004), analyzed in 20-s bins, and expressed as a percentage of the observation intervals (i.e., 20 s). Open field behavior was analyzed in 3-min bins. Data are shown as mean±SEM. They were analyzed by two-way ANOVAs for repeated measures, with the main factors current intensity/drug and the repeated measure time interval/day, followed by Newman-Keuls post hoc test, if appropriate (Statistica 5, StatSoft). The sample sizes are depicted in the figures. Care was taken to use similar sample sizes for each experimental group of a given experiment. A p≤0.05 was considered statistically significant.

Results Conditioning with 1.5 mA promotes long-lasting freezing response to the tone To study the consequences of repeated drug administration on the expression of conditioned fear, we first established an animal model, which results in sustained freezing upon repeated tone presentations. For this reason, we conditioned three groups of B6N mice with a single tone-shock pairing (d0), whereby the intensity of the foot shock was 0.5, 0.75, or 1.5 mA, followed by repeated reexposure to tone (for 3 min each) at d1, d2, d3, and d10. To mimic the pharmacological interventions to come, we treated all animals with saline 30 min before each test session. As shown in Fig. 1a, conditioning with 1.5 mA caused the most prominent fear response to the tone at d1 [shock intensity: F2,28 =4.77, p=0.016; twoway ANOVAs (shock intensity, time interval) for repeated measures (time interval)], d2 (shock intensity: F2,28 =4.76, p=0.016), d3 (shock intensity×time interval: F16,224 =1.81, p=0.031), and, in tendency, d10 (shock intensity: F2,27 = 2.60, p=0.092; Fig. 1a). Therefore, we decided to use the highest shock intensity for the subsequent experiments because it induced the highest fear response and because we had previously seen that at this shock intensity there was an implication of the CB1 in fear extinction (Kamprath et al. 2009). Pharmacological blockade of CB1 and CB2, but not TRPV1, modulates the freezing response in opposite directions We next assessed whether endocannabinoid/endovanilloid signaling via CB1, CB2, and/or TRPV1 is involved in the

sustained freezing response to the tone after conditioning with 1.5 mA. For this reason, we treated new groups of B6N mice with the TRPV1 antagonist SB366791 (1 and 3 mg/kg), the CB1 receptor antagonist/inverse agonist SR141716 (3 mg/kg), the CB2 receptor antagonist/inverse agonist AM630 (3 mg/kg), or the respective vehicle before reexposure to the tone at d1, d2, and d3 after conditioning. At d10, all mice received vehicle in order to estimate long-term effects of the pharmacological interventions. Treatment with SB366791 did not affect the freezing response (d1–d3, drug: F 2,27 < 1.04, p > 0.369; drug×time interval: F16,216 0.368; Fig. 1b); however, treatment with SR141716 further prolonged freezing, which could be statistically secured for d2 after conditioning (drug×time interval: F8,168 =2.29, p=0.023). There was also a long-lasting effect of treatment, which could be observed at d10 (drug: F1,20 =4.63, p=0.043; Fig. 1c). Treatment with AM630, in contrast, significantly reduced freezing at d1 after conditioning (drug×time interval: F8,168 =3.16, p=0.002), without any effect at the subsequent days (statistics not shown; Fig. 1d). Together, these data indicate that CB1 and CB2 receptors modulate the expression of conditioned fear in opposite directions, with no evidence for a direct involvement of tonic TRPV1 activity. The fear-promoting effects of CB2 became evident only during the first tone presentation; the fear-alleviating effects of CB1 could be observed only upon repeated tone exposure. An exogenous CB1 agonist further enhances conditioned fear responses, whereas a CB2 agonist shows no effects Treatment of B6N mice with a low dose of the CB1 agonist CP55,940 (1 μg/kg) had no effects on freezing. A higher dose (50 μg/kg), in contrast, further enhanced conditioned fear (Fig. 1e). This became evident at d2 (drug: F2,32 =3.54, p= 0.041) and d3 (drug: F2,32 =6.79, p=0.003) after conditioning. There were no lasting consequences of the treatment, since we failed to observe any group difference at d10, when all mice received vehicle before testing (drug: F2,31 =1.09, p=0.348). Administration of the CB2 agonist JWH133 (3 mg/kg; Fig. 1f) induced no significant effects (d1–d3, drug: F1,21 < 0.83, p>0.372; drug×time interval: F8,168 0.237). Interference with endocannabinoid uptake and/or degradation has bidirectional effects on conditioned fear responses To assess whether enhancement of endocannabinoid signaling may have similar fear-promoting effects as revealed for CP55,940, we tested various compounds with different levels of specificity in terms of enhancing AEA and/or 2-AG action. Treatment with VDM11 (3 mg/kg; Fig. 2a), an endocannabinoid uptake inhibitor with no direct action at

Psychopharmacology

Fig. 1 Pharmacological agonism and antagonism of CB1 increase, while CB2 blockade decreases, sustained freezing to the tone in response to a 1.5-mA fear conditioning, with no effects of CB2 activation and TRPV1 blockade. Mice were assigned to three groups, which underwent a single tone-shock pairing with 0.5, 0.75, or 1.5 mA (d0). All mice were reexposed to the tone for 3 min at d1, d2, d3, and d10 after conditioning. They were treated with a saline (i.p.) 30 min before tone presentation. Mice underwent a single tone-shock (1.5 mA) pairing at d0, followed by reexposure to the tone for 3 min at d1, d2, d3, and d10. At d1–d3, they were treated with b the TRPV1 receptor antagonist SB366791 (1 or 3 mg/kg), c the CB1 receptor antagonist/inverse agonist SR141716

(3 mg/kg), d the CB2 receptor antagonist AM630 (3 mg/kg), e the CB1 receptor agonist CP55,940 (1 or 50 μg/kg), f the CB2 receptor agonist JWH133 (3 mg/kg, all i.p.), or vehicle 30 min before tone presentation at d1–d3. At d10, all mice were treated with vehicle. Note that SR141716 and AM630 were tested in the same experiment and, therefore, share the same vehicle control. Data (mean±SEM, sample sizes in brackets) were analyzed and depicted in 20-s bins; x>y (e.g., III>I)—significant group differences (p0.580). The genotype differences reflect the slightly more pronounced decrease in locomotor activity in GABA-CB1KO compared to GABA-CB1-WT towards the end of the exposure. Therefore, the genotype differences in the fearpromoting effects of JZL184 (Fig. 4a) cannot be explained by general changes in exploratory behavior.

Fig. 4 The fear-promoting effects of JZL184 are completely abolished in GABA-CB1-KO and Glu-CB1-KO. a–c GABA-CB1-KO (n=18), d–f Glu-CB1-KO (n = 9), and their respective wild-type (WT) controls underwent a single tone-shock (1.5 mA) pairing at d0, followed by reexposure to the tone for 3 min at d1, d2, d3, and d10. They were treated with

vehicle or JZL184 (4 mg/kg, all i.p.) 2 h before tone presentation at d1– d3. At d10, all mice were treated with vehicle. Data were depicted in 20-s bins (a, b, d, e) or averaged over the total 3-min tone presentation (c, f). **p

2-AG promotes the expression of conditioned fear via cannabinoid receptor type 1 on GABAergic neurons.

The contribution of two major endocannabinoids, 2-arachidonoylglycerol (2-AG) and anandamide (AEA), in the regulation of fear expression is still unkn...
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