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

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.)

Visualization by high resolution immunoelectron microscopy of the transient receptor potential vanilloid-1 at inhibitory synapses of the mouse dentate gyrus.

We have recently shown that the transient receptor potential vanilloid type 1 (TRPV1), a non-selective cation channel in the peripheral and central nervous system, is localized at postsynaptic sites of the excitatory perforant path synapses in the hippocampal dentate molecular layer (ML). In the present work, we have studied the distribution of TRPV1 at inhibitory synapses in the ML. With this aim, a preembedding immunogold method for high resolution electron microscopy was applied to mouse hippocampus. About 30% of the inhibitory synapses in the ML are TRPV1 immunopositive, which is mostly localized perisynaptically (∼60% of total immunoparticles) at postsynaptic dendritic membranes receiving symmetric synapses in the inner 1/3 of the layer. This TRPV1 pattern distribution is not observed in the ML of TRPV1 knock-out mice. These findings extend the knowledge of the subcellular localization of TRPV1 to inhibitory synapses of the dentate molecular layer where the channel, in addition to excitatory synapses, is present

Cannabinoid CB1 Receptors Are Localized in Striated Muscle Mitochondria and Regulate Mitochondrial Respiration

The cannabinoid type 1 (CB1) receptor is widely distributed in the brain and peripheral organs where it regulates cellular functions and metabolism. In the brain, CB1 is mainly localized on presynaptic axon terminals but is also found on mitochondria (mtCB1), where it regulates cellular respiration and energy production. Likewise, CB1 is localized on muscle mitochondria, but very little is known about it. The aim of this study was to further investigate in detail the distribution and functional role of mtCB1 in three different striated muscles. Immunoelectron microscopy for CB1 was used in skeletal muscles (gastrocnemius and rectus abdominis) and myocardium from wild-type and CB1-KO mice. Functional assessments were performed in mitochondria purified from the heart of the mice and the mitochondrial oxygen consumption upon application of different acute delta-9-tetrahidrocannabinol (Δ9-THC) concentrations (100 nM or 200 nM) was monitored. About 26% of the mitochondrial profiles in gastrocnemius, 22% in the rectus abdominis and 17% in the myocardium expressed CB1. Furthermore, the proportion of mtCB1 versus total CB1 immunoparticles was about 60% in the gastrocnemius, 55% in the rectus abdominis and 78% in the myocardium. Importantly, the CB1 immunolabeling pattern disappeared in muscles of CB1-KO mice. Functionally, acute 100 nM or 200 nM THC treatment specifically decreased mitochondria coupled respiration between 12% and 15% in wild-type isolated mitochondria of myocardial muscles but no significant difference was noticed between THC treated and vehicle in mitochondria isolated from CB1-KO heart. Furthermore, gene expression of key enzymes involved in pyruvate synthesis, tricarboxylic acid (TCA) cycle and mitochondrial respiratory chain was evaluated in the striated muscle of CB1-WT and CB1-KO. CB1-KO showed an increase in the gene expression of Eno3, Pkm2, and Pdha1, suggesting an increased production of pyruvate. In contrast, no significant difference was observed in the Sdha and Cox4i1 expression, between CB1-WT and CB1-KO. In conclusion, CB1 receptors in skeletal and myocardial muscles are predominantly localized in mitochondria. The activation of mtCB1 receptors may participate in the mitochondrial regulation of the oxidative activity probably through the relevant enzymes implicated in the pyruvate metabolism, a main substrate for TCA activity

Cannabinoid CB1 Receptors Are Localized in Striated Muscle Mitochondria and Regulate Mitochondrial Respiration.

The cannabinoid type 1 (CB1) receptor is widely distributed in the brain and peripheral organs where it regulates cellular functions and metabolism. In the brain, CB1 is mainly localized on presynaptic axon terminals but is also found on mitochondria (mtCB1), where it regulates cellular respiration and energy production. Likewise, CB1 is localized on muscle mitochondria, but very little is known about it. The aim of this study was to further investigate in detail the distribution and functional role of mtCB1 in three different striated muscles. Immunoelectron microscopy for CB1 was used in skeletal muscles (gastrocnemius and rectus abdominis) and myocardium from wild-type and CB1 -KO mice. Functional assessments were performed in mitochondria purified from the heart of the mice and the mitochondrial oxygen consumption upon application of different acute delta-9-tetrahydrocannabinol (Δ9-THC) concentrations (100 nM or 200 nM) was monitored. About 26% of the mitochondrial profiles in gastrocnemius, 22% in the rectus abdominis and 17% in the myocardium expressed CB1. Furthermore, the proportion of mtCB1 versus total CB1 immunoparticles was about 60% in the gastrocnemius, 55% in the rectus abdominis and 78% in the myocardium. Importantly, the CB1 immunolabeling pattern disappeared in muscles of CB1 -KO mice. Functionally, acute 100 nM or 200 nM THC treatment specifically decreased mitochondria coupled respiration between 12 and 15% in wild-type isolated mitochondria of myocardial muscles but no significant difference was noticed between THC treated and vehicle in mitochondria isolated from CB1 -KO heart. Furthermore, gene expression of key enzymes involved in pyruvate synthesis, tricarboxylic acid (TCA) cycle and mitochondrial respiratory chain was evaluated in the striated muscle of CB1 -WT and CB1 -KO. CB1 -KO showed an increase in the gene expression of Eno3, Pkm2, and Pdha1, suggesting an increased production of pyruvate. In contrast, no significant difference was observed in the Sdha and Cox4i1 expression, between CB1 -WT and CB1 -KO. In conclusion, CB1 receptors in skeletal and myocardial muscles are predominantly localized in mitochondria. The activation of mtCB1 receptors may participate in the mitochondrial regulation of the oxidative activity probably through the relevant enzymes implicated in the pyruvate metabolism, a main substrate for TCA activity

Subcellular distribution of TRPV1 at symmetric synapses in WT mouse dentate ML.

<p>Preembedding immunogold method for electron microscopy (<b>A-C</b>). TRPV1 immunoparticles are distributed in dendritic (den) sections receiving symmetric synapses (white arrowheads) from axon terminals (ter). Note that the metal particles (black arrowheads) are localized on the membranes and inside the dendritic profiles. Scale bars: 0.5 μm. <b>D:</b> Distribution of TRPV1 in dendrites (membrane: 75.25 ± 5.56%; inside: 24.75 ± 5.56%). Values mean ± SEM. (<i>***P<0</i>.<i>0001)</i>. <b>E:</b> Distribution of TRPV1 immunoparticles relative to the edge of postsynaptic membranes of symmetric synapses. The edge was defined as 0 with the peri/extrasynaptic side to the right. Note that the closest perisynaptic region (0–60 nm bin) contains the highest TRPV1 labeling (59.49%).</p

Immunolocalization of TRPV1 in granule cell perikarya of the mouse dentate ML.

<p>A high accumulation of TRPV1 immunoparticles is observed in the granule cytoplasm (cyt) of TRPV1-WT mouse (<b>A, B</b>). <b>A, B:</b> Arrows point to symmetric synapses made by axon terminals (ter) on the plasmalemma of granule cells. <b>C:</b> Notice the lack of TRPV1 labeling in the somatic cytoplasm of TRPV1-KO mice, but unspecific immunoparticles remain in the nucleus (nuc) that practically disappear after omission of the primary antibody (<b>D</b>). Scale bars: 0.5 μm. <b>E:</b> Density of TRPV1 immunoparticles per area (part/μm²) in the somatic cytoplasm of granule cells (TRPV1-WT 8.91 ± 0.58; TRPV1-KO 0.61 ± 0.11). Values mean ± SEM (<i>***P<0</i>.<i>0001</i>).</p