Volume 15 - 2021 | https://doi.org/10.3389/fncel.2021.786597 This article is part of the Research TopicGPCR and G Protein-Mediated Signalling Events in the Nervous SystemView all 9 articles There is evidence of ghrelinergic-cannabinoidergic interactions in the central nervous system (CNS) that may impact on the plasticity of reward circuits The aim of this article was to look for molecular and/or functional interactions between cannabinoid CB1 and ghrelin GHS-R1a receptors In a heterologous system and using the bioluminescence resonance energy transfer technique we show that human versions of cannabinoid CB1 and ghrelin GHS-R1a receptors may form macromolecular complexes Such receptor heteromers have particular properties in terms of CB1/Gi-mediated signaling and in terms of GHS-R1a-Gq-mediated signaling just co-expression of CB1R and GHS-R1a led to impairment of cannabinoid signaling cannabinoids led to an increase in ghrelin-derived calcium mobilization that was stronger at low concentrations of the CB1 receptor agonist arachidonyl-2’-chloroethylamide (ACEA) The expression of CB1-GHS-R1a receptor complexes in striatal neurons was confirmed by in situ proximity ligation imaging assays Upregulation of CB1-GHS-R1a- receptor complexes was found in striatal neurons from siblings of pregnant female mice on a high-fat diet the expression was upregulated after treatment of neurons with ghrelin (200 nM) or with ACEA (100 nM) These results help to better understand the complexities underlying the functional interactions of neuromodulators in the reward areas of the brain and YIL 781 hydrochloride were purchased from Tocris Bioscience (Bristol Concentrated (10 mM) stock solutions prepared in DMSO or ethanol were stored at −20°C aliquots of concentrated solutions of compounds were thawed and conveniently diluted in the appropriate experimental solution C57BL/6J female mice were used for the experiments All animals were subjected to a 12 h/12 h light/ dark cycle in a temperature- and humidity-controlled room and were allowed free access to water and standard laboratory chow C57BL/6J mice were randomly assigned to a high-fat diet (HFD; 60% kcal from fat; catalog no USA) or standard diet (STD; 10% kcal from fat; catalog no Primary striatal neurons were obtained from fetuses of mothers on STD or HFD diets Pregnant animals were killed by cervical dislocation during the light phase All animal procedures were performed in agreement with European guidelines (2010/63/EU) and approved by the University of Barcelona Ethical Committee which reports to the regional Government (Protocol #9659; Generalitat de Catalunya Human embryonic kidney HEK-293T (lot 612968) cells were acquired from the American Type Culture Collection (ATCC) They were amplified and frozen in liquid nitrogen in several aliquots Cells from each aliquot were used until passage 19 HEK-293T cells were grown in Dulbecco’s modified Eagle’s medium (DMEM; Gibco United Kingdom) supplemented with 2 mM L-glutamine MEM Non-Essential Amino Acid Solution (1/100) and 5% (v/v) heat-inactivated Fetal Bovine Serum (FBS; all supplements were from Invitrogen Cells were maintained in a humid atmosphere of 5% CO2 at 37°C Cells were transiently transfected with the corresponding cDNAs using the PEI (PolyEthylenImine, Sigma-Aldrich, St. Louis, MO, USA) method as previously described (Carriba et al., 2008; Hradsky et al., 2011; Navarro et al., 2012) growth medium was replaced by a complete medium To prepare primary striatal neurons, brains from fetuses of pregnant mice were removed (gestational age: 17 days). Neurons were isolated as described in Hradsky et al. (2013) and plated at a confluence of 40,000 cells/0.32 cm2 digested for 20 min at 37°C with 0.25% trypsin Trypsinization was stopped by adding an equal volume of culture medium (Dulbecco’s modified Eagle medium-F-12 nutrient mixture Cells were brought to a single cell suspension by repeated pipetting followed by passage through a 100 μm-pore mesh 200× g) cells were resuspended in supplemented DMEM and seeded at a density of 3.5 × 105 cells/ml the medium was replaced by neurobasal medium supplemented with 2 mM L-glutamine Neuronal cultures were used for assays after 15 days of culture the percentage of neurons in the cultures was >90% and D1R cloned in pcDNA3.1 were amplified without their stop codons using sense and antisense primers The primers harbored either unique BamHI and KpnI sites for CB1R and HindIII and BamHI sites for GHS-R1a and D1R The fragments were subcloned to be in frame with an enhanced yellow fluorescent protein (pEYFP-N1; Clontech Germany) and the Renilla luciferase protein (Rluc; pRluc-N1; PerkinElmer MA) on the C-terminal end of the receptor to produce CB1R-YFP HEK-293T cells transfected with cDNAs for CB1R-YFP and GHS-R1a-Rluc were fixed in 4% paraformaldehyde for 15 min and then washed twice with PBS containing 20 mM glycine before permeabilization with the same buffer containing 0.2% Triton X-100 (5 min incubation) The samples were treated for 1 h with blocking solution (PBS containing 1% bovine serum albumin) and labeled with a mouse anti-Rluc (1/100; MAB4400 USA) as primary antibody and subsequently treated with Cy3-conjugated anti-mouse IgG (1/200; 715-166-150; Jackson ImmunoResearch) as the secondary antibody (1 h each) The samples were washed several times and mounted with 30% Mowiol (Calbiochem Samples were observed under a Zeiss 880 confocal microscope (Carl Zeiss HEK-293T cells growing in 6-well plates were transiently cotransfected with a constant amount of cDNA encoding for GHS-R1a fused to Renilla luciferase (GHS-R1a-Rluc) and with increasing amounts of cDNA corresponding to CB1 receptor fused to the yellow fluorescent protein (CB1R-YFP) cells were cotransfected with a constant amount of cDNA encoding for D1R-Rluc and with increasing amounts of cDNA for CB1R-YFP Forty-eight hours post-transfection cells were washed twice in quick succession with HBSS (137 mM NaCl; 5 mM KCl; 0.34 mM Na2HPO4; 0.44 mM KH2PO4; 1.26 mM CaCl2; 0.4 mM MgSO4; 0.5 mM MgCl2; and 10 mM HEPES pH 7.4) supplemented with 0.1% glucose (w/v) detached by gently pipetting and resuspended in the same buffer To have an estimation of the number of cells per plate protein concentration was determined using a Bradford assay kit (Bio-Rad Germany) with bovine serum albumin dilutions for standardization cells were distributed (20 μg protein) in 96-well microplates (black plates with a transparent bottom; Porvair Fluorescence was read using a Mithras LB 940 (Berthold Germany) equipped with a high-energy xenon flash lamp using a 10-nm bandwidth excitation and emission filters at 485 and 530 nm YFP-fluorescence expression was determined as the fluorescence of the sample minus the fluorescence of cells expressing only protein-Rluc the equivalent of 20 μg of cell suspension was distributed in 96-well microplates (white plates; Porvair) and 5 μM coelenterazine H was added (PJK GMBH the readings were collected using a Mithras LB 940 (Berthold which allowed the integration of the signals detected in the short-wavelength filter at 485 nm (440–500 nm) and the long-wavelength filter at 530 nm (510–590 nm) luminescence readings were collected 10 min after 5 μM coelenterazine H addition The net BRET is defined as [(long-wavelength emission)/(short-wavelength emission)]-Cf where Cf corresponds to [(long-wavelength emission)/(short-wavelength emission)] for the Rluc construct expressed alone in the same experiment The BRET curves were fitted assuming a single phase by a non-linear regression equation using the GraphPad Prism software (San Diego BRET values are given as milli-BRET units (mBU: 1000 × net BRET) HEK-293T cells transfected with the cDNAs for CB1R (1 μg) and/or GHS-R1a (1.5 μg) and neuronal primary cultures were plated in 6-well plates Two hours before initiating the experiment the cell-culture medium was replaced by the non-supplemented DMEM medium resuspended in the non-supplemented DMEM medium containing 50 μM zardaverine and plated in 384-well microplates (2,500 cells/well) Cells were pretreated (15 min) with the corresponding antagonists (1 μM rimonabant for CB1R and 1 μM YIL 781 for GHS-R1a) or vehicle and stimulated with agonists (1 nM and 1 μM ACEA for CB1R or 200 nM ghrelin for GHS-R1a; 15 min) before the addition of 0.5 μM FK or vehicle the reaction was stopped by the addition of the Eu-cAMP tracer and the ULight-conjugated anti-cAMP monoclonal antibody prepared in the “cAMP detection buffer” (PerkinElmer) Homogeneous time-resolved fluorescence energy transfer (HTRF) measures were performed after 60 min incubation at RT using the Lance Ultra cAMP kit (PerkinElmer Fluorescence at 665 nm was analyzed on a PHERAstar Flagship microplate reader equipped with an HTRF optical module (BMG Lab Technologies To determine extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation HEK-293T transfected cells and primary striatal neurons were plated (50,000 cells/well) in transparent Deltalab 96-well plates and kept in the incubator for 15 days the medium was replaced by non-supplemented DMEM medium the cells were pre-treated at RT for 10 min with antagonists (1 μM rimonabant for CB1R and YIL 781 for GHS-R1a) or vehicle and stimulated for an additional 7 min with selective agonists (1 nM 1 μM ACEA for CB1R and 200 nM ghrelin for GHS-R1a) cells were washed twice with cold PBS before the addition of 30 μl/well “Ultra lysis buffer” -PerkinElmer- (15 min treatment) 10 μl of each supernatant was placed in white ProxiPlate 384-well plates and ERK1/2 phosphorylation was determined using an AlphaScreen®SureFire® kit (PerkinElmer) following the instructions of the supplier and using an EnSpire® Multimode Plate Reader (PerkinElmer The reference value (100%) was the value achieved in the absence of any treatment (basal) Agonist effects were given in percentage with respect to the basal value HEK-293T cells were transfected with the cDNAs for CB1R (1 μg) and/or GHS-R1a (1.5 μg) in the presence of 1 μg cDNA for the calmodulin-based calcium GCaMP6 sensor (Chen et al., 2013) using the PEI method cells were detached using Mg+2-free Locke’s buffer (pH 7.4; 154 mM NaCl 5.6 mM glucose and 5 mM HEPES) supplemented with 10 μM glycine 1,500 cells per well were plated in 96-well black cells were incubated for 10 min with the CB1R and GHS-R1a antagonists (1 μM rimonabant or 1 μM YIL 781) and subsequently stimulated with selective agonists (1 nM real-time 515 nm fluorescence emission due to calcium-ion complexed GCaMP6 was recorded on the EnSpire® Multimode Plate Reader (every 5 s Physical interaction between CB1R and GHS-R1a was detected using the Duolink in situ PLA detection Kit (OLink; Bioscience Sweden) following the instructions of the supplier Primary neurons were grown on glass coverslips washed with PBS containing 20 mM glycine to quench the aldehyde groups and permeabilized with the same buffer containing 0.05% Triton X-100 (20 min) After 1 h incubation at 37°C with the blocking solution in a pre-heated humidity chamber primary neurons were incubated overnight in the antibody diluent medium with a mixture of equal amounts of mouse anti-CB1R (1/100; sc-293419 USA) and rabbit anti-GHS-R1a (1/100; ab95250 United Kingdom) to detect CB1R-GHS-R1a complexes Neurons were processed using the PLA probes detecting primary antibodies (Duolink II PLA probe plus and Duolink II PLA probe minus) diluted in the antibody diluent solution (1:5) Ligation and amplification were done as indicated by the supplier Samples were mounted using the mounting medium with Hoechst (1/100; Sigma-Aldrich) to stain nuclei Samples were observed in a Zeiss 880 confocal microscope (Carl Zeiss Germany) equipped with an apochromatic 63× oil immersion objective (N.A 1.4) and a 405 nm and a 561 nm laser lines a stack of two channels (one per staining) and four Z stacks with a step size of 1 μm were acquired The number of neurons containing one or more red spots vs and the unpaired t-test was used to compare the values (red dots/cell) obtained Molecular interaction between GHS-R1a and CB1 receptors expressed in HEK-293T cells (A) Confocal microscopy images of HEK-293T cells transfected with cDNAs for GHS-R1a-Rluc (1.5 μg) and/or for CB1R-YFP (1 μg) GHS-R1a-Rluc (red) was identified by immunocytochemistry using an anti-Rluc antibody (Merck-Millipore The CB1R-YFP (green) was identified by the fluorescence due to YFP Cell nuclei were stained with Hoechst (blue) (B,C) BRET saturation experiments were performed using HEK-293T cells co-transfected with a constant amount of GHS-R1a-Rluc cDNA (1.5 μg) and increasing amounts of CB1R-YFP cDNA (0–2 μg) and treated with ACEA (100 nM) or vehicle HEK-293T cells were transfected with a constant amount of D1R-Rluc cDNA (1.5 μg) and increasing amounts of CB1R-YFP cDNA (0–2 μg) BRET data are expressed as the mean ± SEM of eight independent experiments performed in duplicates Functional characterization of GHS-R1a and CB1 receptors expressed in HEK-293T cells (A,B) HEK-293T cells transfected with cDNA encoding for either CB1R (1 μg; A) or GHS-R1a (1.5 μg; B) were pre-treated with selective antagonists 1 μM rimonabant -CB1R- or 1 μM YIL 781 -GHS-R1a- and subsequently treated with the selective agonists 1 μM) -CB1R- or ghrelin (200 nM) -GHS-R1a- cAMP levels after 0.5 μM forskolin (FK) stimulation were detected by the Lance Ultra cAMP kit Results are expressed in % respect to levels obtained upon FK stimulation (100%) The values are the mean ± SEM of 10 independent experiments performed in triplicates One-way ANOVA followed by Bonferroni’s multiple comparison post-hoc test were used for statistical analysis (C,D) HEK-293T cells expressing an engineered calcium sensor and CB1R (C) or GHS-R1a (D) were pre-treated with selective antagonists for 10 min followed by agonist stimulation Real-time traces of cytoplasmic Ca2+ levels detected by EnSpire® Multimode Plate Reader (PerkinElmer USA) over time are shown (six independent experiments) These data demonstrate that cannabinoids may regulate GHS-R1a function depending on the concentration and that GHS-R1a expression suppresses cannabinoid receptor-mediated events supposedly by the establishment of heteromeric complexes Functional characterization of the CB1-GHS-R1aHet expressed in HEK-293T cells (A–C) HEK-293T cells were transfected with cDNAs encoding for GHS-R1a (1.5 μg) and for CB1R (1 μg; A,C) or cDNAs encoding for GHS-R1a (1.5 μg) CB1R (1 μg) and the 6GCaMP calcium sensor (1 μg; B) and stimulated with selective agonists cAMP levels were analyzed by the Lance Ultra cAMP kit and results are expressed in % respect to levels obtained upon 0.5 μM FK stimulation (A; 100% Representative traces of intracellular Ca2+ responses over time are shown (eight independent experiments; B) ERK 1/2 phosphorylation was determined by an AlphaScreen®SureFire® kit (PerkinElmer) using an EnSpire® Multimode Plate Reader (PerkinElmer In cAMP accumulation and MAPK pathway signaling-related assays the values are the mean ± SEM of eight independent experiments performed in triplicates In the 90s different laboratories proved interactions between GPCRs to form heteromeric complexes These complexes can be detected by energy transfer techniques in heterologous expression systems or by proximity ligation assays (PLA) in natural sources either primary cultures or tissue sections An often-found property of a heteromer formed by two GPCRs is that the antagonist of one of the receptors not only blocks the signaling originated at the receptor but also the signaling originated at the partner receptor which is due to conformational changes transmitted from one receptor to the other may serve as a print to detect the heteromer Another possibility is that coactivation leads to a smaller effect than that obtained upon activating only one receptor of the complex; this phenomenon is known as negative crosstalk the antagonist of one receptor may restore the signaling via the partner receptor in the heteromer there was no cross-antagonism in GHS-R1a receptor/Gq-mediated signaling when the CB1R receptor was blocked by a selective antagonist neither cross-antagonism nor restoration of CB1R-Gi coupling was observed when addressing direct Gi- or direct Gq-induced outputs using selective antagonists Effect of antagonists in the functionality of the CB1-GHS-R1aHet HEK-293T cells were transfected with cDNAs encoding for GHS-R1a (1.5 μg) and for CB1R (1 μg; A) or with cDNAs encoding for GHS-R1a (1.5 μg) and the 6GCaMP calcium sensor (1 μg; B) 1 μM YIL 781 -for GHS-R1a- or 1 μM rimonabant -for CB1R- and subsequently stimulated with selective agonists 200 nM ghrelin -for GHS-R1a- or 100 nM ACEA -for CB1R- cAMP levels were analyzed by the Lance Ultra cAMP kit and results were expressed in % respect to levels obtained upon 0.5 μM FK stimulation (A; 100%) Representative real- time traces of cytoplasmic Ca2+ responses are shown (eight independent experiments; B) These results suggest that the proportion of CB1R-GHS-R1aHets in primary striatal cultures is relatively low and that CB1 and GHS-R1a receptors may be forming complexes with other GPCRs (see “Discussion” section) CB1R-GHS-R1aHet signaling in primary striatal neurons from C57BL/6J mice Primary striatal neurons obtained from C57BL/6J brain fetuses were pre-treated with selective antagonists 1 μM YIL 781 -for GHS-R1a- or 1 μM rimonabant -for CB1R- or vehicle and subsequently stimulated with selective agonists cAMP levels (A,B) were collected by the Lance Ultra cAMP kit and results are expressed in % respect to levels obtained upon 0.5 μM FK stimulation (100% ERK1/2 phosphorylation (C,D) was analyzed using an AlphaScreen®SureFire® kit (PerkinElmer) and results are expressed in % respect to basal levels Values are the mean ± SEM of six independent experiments performed in triplicates One-way ANOVA followed by Bonferroni’s multiple comparison post-hoc tests were used for statistical analysis the expression of the CB1R-GHS-R1aHet was higher in the progeny of HFD mothers and may be regulated by ghrelin and Expression and function of CB1-GHS-R1aHets in primary striatal neurons isolated from fetuses of pregnant C57BL/6J female mice subjected to a standard (STD) or high-fat (HFD) diet (A) CB1-GHS-R1aHets were detected by the in situ proximity ligation assay (PLA) in primary striatal neurons isolated from the striatum of fetuses from STD or HFD pregnant mothers Experiments were performed in samples from six animals (B,C) The number of red dots/cell was quantified using Andy’s algorithm Fiji’s plug-in; nuclei were stained using Hoechst (blue) In (B) the number of dots/cell in HFD samples are compared with that in STD samples In (C) neurons obtained from fetuses from HFD mothers were stimulated with 200 nM ghrelin -for GHS-R1a- or 1 nM and 100 nM ACEA -for CB1R- and analyzed by PLA (C) The negative control was obtained omitting one of the primary antibodies One-way ANOVA followed by Bonferroni’s multiple comparison post-hoc test was used for statistical analysis on comparing to basal or to STD: ***p < 0.001 Bonferroni’s multiple comparison test showed significance in comparing results from ACEA treatments: ##p < 0.01 (C) the first aim of the present study was to address the possible interaction between the ghrelin receptor and the CB1R and to characterize the functional consequences of such interaction the previous and the present studies demonstrate that the GHS-R1a receptor may interact with either CB1 or CB2 receptors and that the resulting heteromers may occur in physiological environments allosteric interactions within CB1R-GHS-R1aHets and of CB2R-GHS-R1aHets lead to the blockade of cannabinoid receptor/Gi-mediated signaling The blockade occurs just by simple co-expression it does not require the activation of the GHS-R1a receptor cells expressing cannabinoid and ghrelin receptors heteromers on the cell surface would not be responsive to cannabinoids unless there is a pool of cell surface CB1Rs that are not interacting with the ghrelin receptor caution must be taken when trying to make general conclusions as the allosteric-cross interactions will occur in neurons expressing CB1R and GHS-R1a receptors and also CB1R-GHS-R1aHets; i.e. and a given neuron may express the CB1R-GHS-R1aHet plus other CB1R-containing heteromers (see GPCRs that interact with the CB1R; accessed on October 22 the benefits of cannabinoids acting on populations of striatal neurons expressing CB1R-GHS-R1aHet would be lost by the blockade exerted by the ghrelin receptor thus prevailing the effect of such cannabinoids on other systems such as the dopaminergic the CB1R-GHSR1aHet coactivated by ghrelin and cannabinoids provides a more robust calcium response Such bursts in the concentration of cytosolic calcium must be relevant since calcium regulates almost any event of neuronal physiology Our results suggest the potential for GHS-R1a receptor antagonists which could offer a double benefit: (i) reduce food intake and (ii) revert the detrimental effects of HFD on the functionality of the CB1R in striatal neurons It is quite likely that the CB1R-GHSR1aHet does occur in given subpopulations of neurons the next stage would be an accurate description of the regions and specific neurons where the two receptors are co-expressed The raw data supporting the conclusions of this article will be made available by the authors The animal study was reviewed and approved by University of Barcelona Ethical Committee All authors have read and agreed to the published version of the manuscript All authors contributed to the article and approved the submitted version This work was partially supported by the AARFD-17-503612 grant the US Alzheimer’s Association and by grants SAF2017-84117-R and PID2020-113430RB-I00 funded by Spanish MCIN/AEI/10.13039/501100011033 and by “ERDF A way of making Europe” by the “European Union” or by the “European Union Next Generation EU/PRTR” The research group of the University of Barcelona is considered of excellence (grup consolidat #2017 SGR 1497) by the Regional Catalonian Government which does not provide any specific funding for reagents or for payment of services or Open Access fees The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations Any 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This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) distribution or reproduction in other forums is permitted provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited in accordance with accepted academic practice distribution or reproduction is permitted which does not comply with these terms *Correspondence: Rafael Franco, cmZyYW5jbzEyM0BnbWFpbC5jb20=; Gemma Navarro, ZGltYXJ0dHNAaG90bWFpbC5jb20= † These authors have contributed equally to this work Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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Catalan citizens and voters in the 2017 referendum are giving their testimonies this week in Madrid’s Supreme Court They were called to the court by the defense of Jordi Sànchez and Quim Forn and told the court about their experiences of Spanish police aggression on the day of the vote described how police officers grabbed him by the testicles and threw him to the ground replied: “We were sent here to do this.” Following this Font says he was then punched in the face by a female police officer The day began with the testimony of Joan Porras a well-known political activist in Catalonia He is known for having visited the jailed Catalan leaders every day to wish them good night while they were imprisoned in the Lledoners penitentiary center His testimony was the first time he revealed his face to the public Porras said that in his voting station in Manresa Catalan police arrived to confiscate referendum material and ballot boxes The witness said they did so without causing any damage or injuring people Also testifying today were a number of citizens who voted in the town of Dosrius They spoke about what they saw when the Spanish police arrived to carry out their operations One witness described how the police began by throwing the women to the floor first out of all of the voters that were gathered in the polling station said she broke down in tears when a police officer threw her to the ground and grabbed her bag from her shoulder Martínez also told the court a police officer filmed her crying insulting her saying that she was acting “ridiculously,” and called her a "retard."  said she knew the referendum had been suspended but that said she believes “voting is the essence of democracy.” She also spoke about tension in her town leading up to the day of the vote pointing out that people were waking up to news of arrests of leaders many days and 42 of the Catalan trial have been reserved for 50 testimonies of Catalan voters Get the day's biggest stories right to your phone v1.1.0. Copyright © 2025. Powered by EBANTIC. All rights reserved. Catalan Mossos d'Esquadra police have carried out a raid against an Italian drug trafficking group based in Catalonia in the early hours of Tuesday morning Police expect to detain around 20 people from the group which smuggles hashish and marijuana internationally Law enforcement agents entered several sites across Catalonia The investigation is part of a joint effort between the Mossos' criminal investigation unit and the Italian Direzione Centrale Servizi Antidroga Police also carried out an operation against Neo-Nazi group Combat 18 on Tuesday morning.  The Mossos d'Esquadra arrested at least four people in a joint operation with Spain's National Police against the organization linked to white supremacism.  A man and a woman were arrested in Sentmenat (Vallès Occidental County), 30km north of Barcelona. Officers came across marijuana plants there and related equipment.  The other two arrests, also a man and a woman, took place in Mollerussa (Pla d'Urgell County), in western Catalonia.  A total of ten properties were searched in Catalonia, including in Prat de Llobregat (Baix Llobregat County), Navarcles (Bages) and Lloret de Mar (Selva).  Spain's National Police carried out related operations in Arganda del Rey (Madrid), Talavera de la Reina (Castilla–La Mancha), Lugo (Galicia) and Málaga (Andalusia).