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

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

Environmental Enrichment Rescues Endocannabinoid-Dependent Synaptic Plasticity Lost in Young Adult Male Mice after Ethanol Exposure during Adolescence

Binge drinking (BD) is a serious health concern in adolescents as high ethanol (EtOH) consumption can have cognitive sequelae later in life. Remarkably, an enriched environment (EE) in adulthood significantly recovers memory in mice after adolescent BD, and the endocannabinoid, 2-arachydonoyl-glycerol (2-AG), rescues synaptic plasticity and memory impaired in adult rodents upon adolescent EtOH intake. However, the mechanisms by which EE improves memory are unknown. We investigated this in adolescent male C57BL/6J mice exposed to a drinking in the dark (DID) procedure four days per week for a duration of 4 weeks. After DID, the mice were nurtured under an EE for 2 weeks and were subjected to the Barnes Maze Test performed the last 5 days of withdrawal. The EE rescued memory and restored the EtOH-disrupted endocannabinoid (eCB)-dependent excitatory long-term depression at the dentate medial perforant path synapses (MPP-LTD). This recovery was dependent on both the cannabinoid CB1 receptor and group I metabotropic glutamate receptors (mGluRs) and required 2-AG. Also, the EE had a positive effect on mice exposed to water through the transient receptor potential vanilloid 1 (TRPV1) and anandamide (AEA)-dependent MPP long-term potentiation (MPP-LTP). Taken together, EE positively impacts different forms of excitatory synaptic plasticity in water- and EtOH-exposed brains.This research was funded by ISCIII (“RD16/0017/0012” to P.G.), co-funded by ERDF/ESF, “Investing in your future”; The Basque Government (IT1230-19 to P.G.); Ministry of Science and Innovation (PID2019-107548RB-I00 to P.G.); Ph.D. contract from MINECO (BES-2013-065057 to S.P.); Ph.D. contract from UPV/EHU (PIF 18/315 to L.L.), and Ph.D. contract from UPV/EHU (PIF 19/164 to M.S.)

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