High resolution imaging to unveil the subcellular layout of the cannabinoid type-1 receptor in rodent models of brain disease

179 p.The present Thesis focuses on the description of new subcellular localizations of CB1 receptors in normal brain and the study of the CB1 receptor expression in certain pathophysiological states. Different histological techniques have been crucial in defining the CB1 receptor expression and localization at the cellular level. However, it is extremely difficult to identify the subcellular distribution of CB1 receptors in some cell-types due to its low expression level on those cells. Moreover, it remains a key question to know the pattern of the subcellular CB1 receptor expression and distribution under pathological states. The high resolution immunoelectron microscopy applied in this study has shown to be an excellent approach for the fine detection of CB1 receptors in the brain. In particular, the single pre-embedding immunogold method for electron microscopy based on the use of specific primary CB1 receptor antibodies and silver-intensified 1.4 nm gold-labeled Fab' fragments was used, as well as the combined pre-embedding immunogold and immunoperoxidase method that implied the additional use of biotinylated secondary antibodies and avidin-biotin complex for the simultaneous localization of CB1 receptors and protein markers of specific brain cells or synapses

Altered glial expression of the cannabinoid 1 receptor in the subiculum of a mouse model of Alzheimer's disease.

The alteration of the endocannabinoid tone usually associates with changes in the expression and/or function of the cannabinoid CB1 receptor. In Alzheimer's disease (AD), amyloid beta (Aβ)-containing aggregates induce a chronic inflammatory response leading to reactivity of both microglia and astrocytes. However, how this glial response impacts on the glial CB1 receptor expression in the subiculum of a mouse model of AD, a brain region particularly affected by large accumulation of plaques and concomitant subcellular changes in microglia and astrocytes, is unknown. The CB1 receptor localization in both glial cells was investigated in the subiculum of male 5xFAD/CB2EGFP/f/f (AD model) and CB2EGFP/f/f mice by immuno-electron microscopy. The findings revealed that glial CB1 receptors suffer remarkable changes in the AD mouse. Thus, CB1 receptor expression increases in reactive microglia in 5xFAD/CB2EGFP/f/f, but remains constant in astrocytes with CB1 receptor labeling rising proportionally to the perimeter of the reactive astrocytes. Not least, the CB1 receptor localization in microglial processes in the subiculum of controls and closely surrounding amyloid plaques and dystrophic neurites of the AD model, supports previous suggestions of the presence of the CB1 receptor in microglia. These findings on the correlation between glial reactivity and the CB1 receptor expression in microglial cells and astrocytes, contribute to the understanding of the role of the endocannabinoid system in the pathophysiology of Alzheimer's disease.post-print4763 K

Lack of the transient receptor potential vanilloid 1 shifts cannabinoid-dependent excitatory synaptic plasticity in the dentate gyrus of the mouse brain hippocampus

[EN] The transient receptor potential vanilloid 1 (TRPV1) participates in synaptic functions in the brain. In the dentate gyrus, post-synaptic TRPV1 in the granule cell (GC) dendritic spines mediates a type of long-term depression (LTD) of the excitatory medial perforant path (MPP) synapses independent of pre-synaptic cannabinoid CB1 receptors. As CB1 receptors also mediate LTD at these synapses, both CB1 and TRPV1 might be influencing the activity of each other acting from opposite synaptic sites. We tested this hypothesis in the MPP–GC synapses of mice lacking TRPV1 (TRPV1-/-). Unlike wild-type (WT) mice, low-frequency stimulation (10min at 10Hz) of TRPV1-/- MPP fibers elicited a form of long-term potentiation (LTP) that was dependent on (1) CB1 receptors, (2) the endocannabinoid 2-arachidonoylglycerol (2-AG), (3) rearrangement of actin filaments, and (4) nitric oxide signaling. These functional changes were associated with an increase in the maximum binding efficacy of guanosine-5′-O-(3-[35S]thiotriphosphate) ([35S]GTPgS) stimulated by the CB1 receptor agonist CP 55,940, and a significant decrease in receptor basal activation in the TRPV1-/- hippocampus. Finally, TRPV1-/- hippocampal synaptosomes showed an augmented level of the guanine nucleotide-binding (G) Gai1, Gai2, and Gai3 protein alpha subunits. Altogether, the lack of TRPV1 modifies CB1 receptor signaling in the dentate gyrus and causes the shift from CB1 receptor-mediated LTD to LTP at the MPP–GC synapses.This work was supported by the Basque Government (IT1230- 19, to PG); MINECO/FEDER, UE (SAF2015-65034-R, to PG); Ministry of Science and Innovation (PID2019-107548RBI00, to PG); Red de Trastornos Adictivos, Instituto de Salud Carlos III (ISC-III); and European Regional Development Funds-European Union (ERDF-EU, Investing in your future; RD16/0017/0012, to PG); MINECO CTQ2017-85686-R (Spanish Ministry of Economy and Competitiveness, to JS); JE-H is a Postdoctoral Researcher contracted with funds of Red de Trastornos Adictivos, Instituto de Salud Carlos III (ISC-III), and European Regional Development Funds-European Union (ERDF-EU, Investing in your future; RD16/0017/0012), and the Basque Government (IT1230-19). IB-D holds a Postdoctoral Orientation Period contract (BES-2016-076766, BES-C-2016-0051). SA has a Ph.D. contract granted by University of the Basque Country (PIF 16/251). ES-G is funded by Ikerbasque and MINECO (PGC2018- 093990-A-I00; MICIU/AEI/FEDER, UE)

The Absence of the Transient Receptor Potential Vanilloid 1 Directly Impacts on the Expression and Localization of the Endocannabinoid System in the Mouse Hippocampus

The transient receptor potential vanilloid 1 (TRPV1) is a non-selective ligand-gated cation channel involved in synaptic transmission, plasticity, and brain pathology. In the hippocampal dentate gyrus, TRPV1 localizes to dendritic spines and dendrites postsynaptic to excitatory synapses in the molecular layer (ML). At these same synapses, the cannabinoid CB1 receptor (CB1R) activated by exogenous and endogenous cannabinoids localizes to the presynaptic terminals. Hence, as both receptors are activated by endogenous anandamide, co-localize, and mediate long-term depression of the excitatory synaptic transmission at the medial perforant path (MPP) excitatory synapses though by different mechanisms, it is plausible that they might be exerting a reciprocal influence from their opposite synaptic sites. In this anatomical scenario, we tested whether the absence of TRPV1 affects the endocannabinoid system. The results obtained using biochemical techniques and immunoelectron microscopy in a mouse with the genetic deletion of TRPV1 show that the expression and localization of components of the endocannabinoid system, included CB1R, change upon the constitutive absence of TRPV1. Thus, the expression of fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) drastically increased in TRPV1(-/-) whole homogenates. Furthermore, CB1R and MAGL decreased and the cannabinoid receptor interacting protein 1a (CRIP1a) increased in TRPV1(-/-) synaptosomes. Also, CB1R positive excitatory terminals increased, the number of excitatory terminals decreased, and CB1R particles dropped significantly in inhibitory terminals in the dentate ML of TRPV1(-/-) mice. In the outer 2/3 ML of the TRPV1(-/-) mutants, the proportion of CB1R particles decreased in dendrites, and increased in excitatory terminals and astrocytes. In the inner 1/3 ML, the proportion of labeling increased in excitatory terminals, neuronal mitochondria, and dendrites. Altogether, these observations indicate the existence of compensatory changes in the endocannabinoid system upon TRPV1 removal, and endorse the importance of the potential functional adaptations derived from the lack of TRPV1 in the mouse brain.This work was supported by the Basque Government (IT123019, to PG); MINECO/FEDER, UE (SAF2015-65034-R, to PG); Ministry of Science and Innovation (PID2019-107548RBI00, to PG); Red de Trastornos Adictivos, Instituto de Salud Carlos III (ISC-III) and European Regional Development Funds-European Union (ERDF-EU, Investing in your future; RD16/0017/0012, to PG); MINECO CTQ2017-85686-R (Spanish Ministry of Economy and Competitiviness, to JS); JE-H is a Postdoctoral Researcher contracted with funds of Red de Trastornos Adictivos, Instituto de Salud Carlos III (ISC-III) and European Regional Development Funds-European Union (ERDF-EU, Investing in your future; RD16/0017/0012), and the Basque Government (IT1230-19); IB-DR holds a Postdoctoral contract (BES2016-076766, BES-C-2016-0051); AM is the recipient of a PhD contract granted by the Department of Education of the Basque Governmen

Ingurune aberastuak nerabezaroko gehiegizko alkohol kontsumoaren ondoriozko portaera kalteak berreskuratzen ditu C57BL/6J sagu helduetan

The use and abuse of alcohol (EtOH) is one of the world’s main health issues that strikingly impacts on our society, as heavy episodic drinking is becoming more and more common in the adolescence when the brain is particularly vulnerable to EtOH. However, molecular, anatomical, functional and behavioral alterations improve inyoung adult mice brains by an enriched environment (EE) exposure after adolescence EtOH consumption [21]. It remains unknown whether these beneficial effects are maintained over a long period of time after cessation of EtOH consumption. The aim of this study was to measure the long-term behavioral consequences of EtOH consumption and to explore the effects of EE in adulthood. For this goal, we treated C57BL/6J male mice with 20% EtOH or water during the 4 weeks of adolescence (p32-p56) followed by an abstinence period (p56-p90). Finally, they were exposed to EE for two weeks (p90-p104) and behavioral tests were conducted at their full adulthood: thigmotaxis for anxiety-like behaviour; novel object recognition test (NORT) for object recognition memory; novel object location test (NOLT) for location memory and beam walking balance test (BWBT) for motor coordination and balance. Object and spatial recognition memory were significantly lower in EtOH-treated mice. Also, motor coordination and balance were impaired after EtOH intake. Noticeably, memory and motor deficits reversed to control values after EE. In conclusion, we show that EE recovers the long-term behavioral and motor deficits after abusive EtOH consumption during adolescence. These results point to the beneficial effects EE have in EtOH addiction.; Alkohola (EtOH) munduan gehien kontsumitzen den substantzia psikoaktiboa da eta nerabezaroko alkoholaren kontsumo intentsiboa geroz eta ohikoagoa da. Adin tarte horretan burmuina garatzen ari da eta hainbat garun-atal zaurgarriagoak dira neurotoxikoen kalteen aurrean; hipokanpoa eta garuntxoa, esaterako. Ingurune aberastuak (IAk), aldaketa molekular, anatomiko zein funtzionalak eragiten ditu garunaren garapen prozesuan eta alkoholaren ondorioz helduaro goiztiarreko saguek galdutako portaera gaitasunen berreskurapena sustatzen du. Hala ere, IAk eragindako efektu mesedegarri horiek epe luzerago batean mantentzen diren aztertzeke dago. Ikerketa honen helburuak hurrengoak dira: nerabezaroko gehiegizko alkohol kontsumoak helduaroan eragiten dituen portaera aldaketak ikertzea eta parametro hauetan IAk izan ditzakeen onurak aztertzea. Horretarako, C57BL/6J sagu arrei nerabezaroko 4 astetan zehar (p32-p56) alkohol edo ur tratamendua eman zaie. Ondoren, helduaro goiztiarrean (p56-p90) animaliak abstinentzia egoeran mantendu dira eta helduaroan (p90-p104) saguen kumaldi erdia IAko baldintzetan jarri da 2 astez. Abstinentzia tarte horren azken egunetan portaera probak burutu dira: eremu irekiaren proba, antsietate maila neurtzeko; objektu berrien ezagutze proba, ezagutze oroimenerako; objektuen kokaleku berriaren ezagutze proba, oroimen espazialerako eta oreka proba, oreka eta koordinazio motorrerako. Alkohol taldeko saguek bereizketa indize baxuagoak erakutsi dituzte bai ezagutze oroimen proban baita oroimen espazialean ere, alkohol kontsumoaren ondoriozko narriadura kognitibo adierazgarria iradokiz. Antzeko emaitzak behatu dira oreka proban ere, non alkohol taldeko saguek (EtOH) oreka eta koordinazio motorra kaltetuta erakutsi duten. Interesgarriki, animaliak IAko baldintzapean jartzean objektuak eta kokalekuak bereizteko gaitasuna berreskuratzen dute eta oreka eta koordinazio maila hobetzen dute helduaroan, kontrol taldekoen (H2O) antzeko balioetaraino. IAk alkoholaren ondoriozko helduaroko efektu kaltegarriak leheneratzeko gaitasuna duela erakutsi du

High-resolution Immunoelectron Microscopy Techniques for Revealing Distinct Subcellular Type 1 Cannabinoid Receptor Domains in Brain

Activation of type 1 cannabinoid (CB1) receptors by endogenous, exogenous (cannabis derivatives) or synthetic cannabinoids (i.e., CP 55.940, Win-2) has a wide variety of behavioral effects due to the presence of CB1 receptors in the brain. In situ hybridization and immunohistochemical techniques have been crucial for defining the CB1 receptor expression and localization at the cellular level. Nevertheless, more advanced methods are needed to reveal the precise topography of CB1 receptors in the brain, especially in unsuspected sites such as other cell types and organelles with low receptor expression (e.g., glutamatergic neurons, astrocytes, mitochondria). High-resolution immunoelectron microscopy provides a more precise detection method for the subcellular localization of CB1 receptors in the brain. Herein, we describe a single pre-embedding immunogold method for electron microscopy based on the use of specific CB1 receptor antibodies and silver-intensified 1.4 nm gold-labeled Fab' fragments, and a combined pre-embedding immunogold and immunoperoxidase method that employs biotinylated secondary antibodies and avidin-biotin-peroxidase complex for the simultaneous localization of CB1 receptors and protein markers of specific brain cells or synapses (e.g., GFAP, GLAST, IBA-1, PSD-95, gephyrin). In addition, a post-embedding immunogold method is also described and compared to the pre-embedding labeling procedure. These methods provide a relatively easy and useful approach for revealing the subcellular localization of low amounts of CB1 receptors in glutamatergic synapses, astrocytes, neuronal and astrocytic mitochondria in the brain

2-Arachidonoylglycerol Reduces Proteoglycans and Enhances Remyelination in a Progressive Model of Demyelination

The failure to undergo remyelination is a critical impediment to recovery in multiple sclerosis. Chondroitin sulfate proteoglycans (CSPGs) accumulate at demyelinating lesions creating a nonpermissive environment that impairs axon regeneration and remyelination. Here, we reveal a new role for 2-arachidonoylglycerol (2-AG), the major CNS endocannabinoid, in the modulation of CSPGs deposition in a progressive model of multiple sclerosis, the Theiler's murine encephalomyelitis virus-induced demyelinating disease. Treatment with a potent reversible inhibitor of the enzyme monoacylglycerol lipase, which accounts for 85% of the 2-AG degradation in the mouse CNS, modulates neuroinflammation and reduces CSPGs accumulation and astrogliosis around demyelinated lesions in the spinal cord of Theiler's murine encephalomyelitis virus-infected mice. Inhibition of 2-AG hydrolysis augments the number of mature oligodendrocytes and increases MBP, leading to remyelination and functional recovery of mice. Our findings establish a mechanism for 2-AG promotion of remyelination with implications in axonal repair in CNS demyelinating pathologies.This work was supported by Ministry of the Economy and Competition (MINECO) Grants SAF2013-42784-R and SAF2016-76449-R, Comunidad de Madrid Grant S2011/BMD-2308, and Red Española de Esclerosis Múltiple Grants RD12/0032/0008 and RD16/0015/0021 (sponsored by the Fondo de Investigación Sanitaria) to C.G., Basque Government Grant IT764-13, MINECO/FEDER Grant SAF2015-65034-R, and University of the Basque Country Grant UPV/EHU UFI11/41 to P.G., and MINECO Grant SAF2013-48271-C2-1-R to M.L.-R. None of the funding bodies played any role in the study design, data collection and analysis, the decision to publish, or the preparation of the manuscript. A.F. is a predoctoral fellow supported by MINECO Grant BES-2014-068459. We thank Laura Ramos for excellent technical support.Peer reviewe

Omega-3 Recovers Cannabinoid 1 Receptor Expression in the Adult Mouse Brain after Adolescent Binge Drinking

Adolescent binge drinking is a social problem with a long-lasting impact on cognitive functions. The cannabinoid type-1 (CB1) receptor of the endocannabinoid system (ECS) is involved in brain synaptic plasticity, cognition and behavior via receptor localization at specific subcellular compartments of the cortical, limbic and motor regions. Alcohol (EtOH) intake affects the ECS, CB1 and their functions. Evidence indicates that binge drinking during adolescence impairs memory via the abrogation of CB1-dependent synaptic plasticity in the hippocampus. However, the impact of EtOH consumption on global CB1 receptor expression in the adult brain is unknown. We studied this using optical density analysis throughout brain regions processed for light microscopy (LM) immunohistotochemistry. CB1 staining decreased significantly in the secondary motor cortex, cerebellum, cingulate cortex, amygdala and nucleus accumbens. Next, as omega-3 (n-3) polyunsaturated fatty acids (PUFAs) rescue synaptic plasticity and improve EtOH-impaired cognition, we investigated whether docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) had any effect on CB1 receptors. N-3 intake during EtOH abstinence restored CB1 immunostaining in the secondary motor cortex, cerebellum and amygdala, and ameliorated receptor density in the cingulate cortex. These results show that n-3 supplementation recovers CB1 receptor expression disrupted by EtOH in distinct brain regions involved in motor functions and cognition.This work has been supported by The Basque Government (IT1620-22); Red de Investigación en Atención Primaria de Adicciones (RIAPAd), Instituto de Salud Carlos III (RD21/0009/0006); Red de Trastornos Adictivos, Instituto de Salud Carlos III, European Regional Development Funds-European Union (ERDF-EU; RD16/0017/0012); and Ministry of Science and Innovation (PID2019-107548RB-I00). M.S. is in receipt of a PhD contract from The University of the Basque Country (PIF 19/164)

Endokannabinoide sistemaren parte-hartzea arratoien garuntxoaren jaio osteko garapenean

Endokannabinoide-sistema (EKS) zelulen arteko komunikazio-sistema fisiologiko neuromodulatzaile garrantzitsuenetako bat da, eta helduaroan beraren funtzio ezagunena neurotransmisoreen doikuntza da. Azken urteotako ikerketek, aldiz, funtzio garrantzitsu horrez gain, garapen-prozesuetan ere parte-hartze handia duela erakutsi dute. Izan ere, zenbait artikuluk burmuinaren arauzko enbrioi-garapenerako EKSren beharra agerian utzi dute. Enbrioi-aroko ikerketek muga metodologiko handiak dituzte, eta orain arteko ikerketa gehienak hazkuntza zelularretan egin dira. Halere, burmuineko garuntxoaren kortexean, EKSren adierazpena handia da, eta jaio osteko garapen-prozesu guztiak biltzen ditu: pikor-zelulen morfogenesia, hain zuzen ere. Lan honetan, ultraegitura mailan, EKSren zenbait osagairen kokapena aztertu da; pikor-neuronen garapenaren faseetan baita helduaroan ere. CB1 kannabinoideen hartzailea, eta 2-arakidonil-glizerol (2-AG) endokannabinoidea sintetizatzen eta degradatzen dituzten entzimak —diazilglizerol lipasa (DAGL-α) eta monoazilglizerol lipasa (MAGL), hurrenez hurren— batera ageri dira hazkuntza- eta migrazio-prozesuetan dauden zuntz paraleloen axoietan jaio osteko garapen goiztiarrean. Konkretuki, CB1 eta DAGL-α zuntz paraleloen mintzean kokatzen dira, eta MAGL, mintzean ez ezik, zitoplasman ere adierazten da. Jaio osteko 12. egunean, zuntz paraleloek Purkinje neuronen arantza dendritikoekin kontaktu sinaptikoak ezartzen dituztenean, CB1 eta MAGL zuntz horien bukaera sinaptikoan mantenduko dira. CB1 bukaera sinaptiko horien mintzean kokatzen da, eta MAGL, aldiz, mintzaren hurrentasunean ez ezik, zitoplasman ere adierazten da. DAGL-α, zuntz paraleloen bukaera sinaptikotik desagertuko da, eta alde postsinaptikoan adieraziko da —hots, Purkinje neuronen arantza den- dritikoetan—. EKSn aztertutako 3 osagai horiek batera ageri dira zuntz paraleloetan garapenean zehar, kontaktu sinaptikoak ezarri arte. Horrela, zuntz horiek helduaren ezaugarriak lortzen dituztenean, hartzailea eta degradazio-entzima zuntz paraleloen bukaera sinaptikoan mantenduko dira. Sintesi-entzima, aldiz, axoi-profil horietatik desagertuko da, eta elementu postsinaptikoan adierazten hasiko da —hots, Purkinje neuronen arantza dendritikoetan—. Modu horretan, aztertutako aldi bakoitzean, EKSko osagaien kokapen espezifikoa bistaratu zen. Lortutako emaitzek EKS moldakorra dela eta funtzio desberdinetara egoki daitekeela iradokitzen dute, bere jarduna modu desberdinetan betez. Testuinguru horretan, garuntxoaren garapenean EKSren osagaien kokapen zehatza eta izaera identifikatzea alderdi kritikoa da, haurdun dauden emakumeen eta baita nerabeen kalamu-kontsumoak garunean egitura, funtzio eta portaera mailan eragiten dituen aldaketak ulertzeko. Horrez gain, jakintza hori terapeutikoki erabil daiteke, endokannabinoideen manipulazioak aplikazio klinikoak izan baititzake jaio osteko garapenean eta haurtzaroan gertatzen diren nerbio-sistemako gaixotasunen tratamenduan.; The endocannabinoid system (ECS) is widely distributed throughout the or-ganism. This system influences brain development and suppresses neurotransmitter re-lease in the mature brain, thus contributing to brain circuit formation and neural com-munication. Because of methodological limitations, most investigations focused on the study of the embryo development have been carried out in cell culture. In the central nervous system, the ECS is highly expressed in the cerebellar cortex where it plays a crucial role in the postnatal development of the granule cells. However, little is known about how the precise localization of the components of the ECS takes place during the early postnatal development, which is needed for the correct wiring of the cerebellar circuits. In this study, we used high resolution immunoelectron microscopy to investi-gate the subcellular localization of the cannabinoid CB1 receptor and the main enzymes for the synthesis and degradation of the endocannabinoid 2-arachidonoyl-glycerol (2-AG), diacylglycerol lipase (DAGL-α) and monoacylglycerol lipase (MAGL), re-spectively, in the granule cells during the early postnatal development of the cerebel-lum. The CB1 receptor, DAGL-α and MAGL were localized at the granule cell axons, the parallel fibers, during granule cell migration and axon elongation occurring at early postnatal age. Moreover, CB1 receptor and DAGL-α immunoparticles were placed in the parallel fiber membranes, while MAGL labeling was at the parallel fiber mem-branes and cytoplasm. At postnatal day 12, when parallel fibers make already synapses with the Purkinje cell dendritic spines, the CB1 receptor exhibited the typical presynap-tic localization in parallel fiber terminal membranes, and MAGL was close to the membrane and in the cytoplasm of the parallel fiber terminals. However, DAGL-α was excluded from the parallel fiber terminals but it was expressed at postsynaptic sites. Al-together, the expression of CB1 receptors, DAGL-α, and MAGL in the granule cell parallel fibers starts at early stages of the postnatal development. Later on, the CB receptors and MAGL remain in the mature parallel fiber synapses, while DAGL-α disap-pears from them and switches to the postsynaptic dendritic spines of the Purkinje cells. These structural changes correlate with the functions in which the ECS is involved dur-ing postnatal development. Hence, the identification of the specific localization of the components of the ECS in the developing cerebellum is crucial for understanding the structural, functional and behavioral changes taking place in the brain by cannabis con-sume. Furthermore, this knowledge could be exploited therapeutically as the ECS ma-nipulation might have potential clinical applications in the treatment of brain diseases caused by abnormal development of the brain

Up-regulation of CB1 Cannabinoid Receptors Located at Glutamatergic Terminals in the Medial Prefrontal Cortex of the Obese Zucker Rat.

The present study describes a detailed neuroanatomical distribution map of the cannabinoid type 1 (CB1) receptor, along with the biochemical characterization of the expression and functional coupling to their cognate G i/o proteins in the medial prefrontal cortex (mPCx) of the obese Zucker rats. The CB1 receptor density was higher in the prelimbic (PL) and infralimbic (IL) subregions of the mPCx of obese Zucker rats relative to their lean littermates which was associated with a higher percentage of CB1 receptor immunopositive excitatory presynaptic terminals in PL and IL. Also, a higher expression of CB1 receptors and WIN55,212-2-stimulated [35S]GTPγS binding was observed in the mPCx but not in the neocortex (NCx) and hippocampus of obese rats. Low-frequency stimulation in layers II/III of the mPCx induced CB1 receptor-dependent long-term synaptic plasticity in IL of area obese Zucker but not lean rats. Overall, the elevated 2-AG levels, up-regulation of CB1 receptors, and increased agonist-stimulated [35S]GTPγS binding strongly suggest that hyperactivity of the endocannabinoid signaling takes place at the glutamatergic terminals of the mPCx in the obese Zucker rat. These findings could endorse the importance of the CB1 receptors located in the mPCx in the development of obesity in Zucker rats.This work was supported by grants of the Basque Government (grants numbers IT1230-19 and IT1620-22 to PG), the Spanish Government (grant numbers CTQ2017-85686-R to MG and JS) and the Instituto de Salud Carlos III, Centro de Investigación Biomédica en Red de Salud Mental, CIBERSAM. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript