Pharmacological Blockade of Cannabinoid CB1 Receptors in Diet-Induced Obesity Regulates Mitochondrial Dihydrolipoamide Dehydrogenase in Muscle

Funding: This work was supported by CIBERobn (CB06/03/1008), Ministerio de Economía y Competitividad (MINECO) (PG: BFU2012-33334), Instituto de Salud Carlos III (ISCIII), MINECO, co-funded by UE-ERDF program (JS: CP12/03109), Red de Trastornos Adictivos (FRF: RD12/0028/0001, PG: RD12/0028/0004, JM: RD12/0028/0013), The Basque Country Government (PG: BCG IT764-13), Consejería de Economía, Innovación y Ciencia, Junta de Andalucía, UE-ERDF (FRF: CTS-8221, JM: CVI-6656), Consejería de Salud, Junta de Andalucía, UE-ERDF (FRF: SAS111224), and University of the Basque Country UPV/EHU (PG: UFI11/41). JS, FJP and AS hold “Miguel Servet” research contracts from the National System of Health, ISCIII, UE-ERDF (CP12/03109, CP14/00212, and CP14/00173 respectively)Peer reviewedPublisher PD

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

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

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 at symmetric synapses in mouse dentate ML.

<p>Preembedding immunogold method for electron microscopy (<b>A-C</b>). In TRPV1-WT (<b>A, B</b>), silver-intensified gold particles (arrowheads) are mostly distributed in dendritic (den) profiles but not in inhibitory synaptic terminals (ter). Importantly, TRPV1 immunolabeling is virtually abolished in the ML of TRPV1-KO mice (<b>C</b>). Scale bars: 0.5 μm. <b>D:</b> Percentages of TRPV1 positive dendritic profiles from the total analyzed dendritic sections with symmetric synapses (arrows in <b>A-C</b>) in the dentate ML of TRPV1-WT (dendrites: 32.89 ± 5.04%) and TRPV1-KO (dendrites: 4.76 ± 1.75%). Values in <b>D</b> mean ± SEM. <i>***P<0</i>.<i>0001</i>.</p

Immunolocalization of TRPV1 at dendrites in mouse dentate ML.

<p>Preembedding immunogold method for electron microscopy (<b>A-D</b>). TRPV1 immunolocalization (arrowheads) at dendritic (den) membranes receiving symmetric synapses (arrows) from axon terminals (ter) in the inner 1/3 (<b>A, B</b>) and outer 2/3 ML (<b>C, D</b>). Scale bars: 0.5 μm. <b>E:</b> Proportion of TRPV1 positive dendritic sections with symmetric synapses in the inner 1/3 (81.4%) and outer 2/3 (18.6%) of the dentate ML. <b>F:</b> Density of TRPV1 immunoparticles per area (part/μm²) in dendritic domains of the inner 1/3 (3.72 ± 0.85%) and outer 2/3 ML (1.95 ± 1.02%). Values mean ± SEM (<i>***P<0</i>.<i>0001</i>).</p

Endocannabinoid LTD in Accumbal D1 Neurons Mediates Reward-Seeking Behavior

International audienceThe nucleus accumbens (NAc) plays a key role in drug-related behavior and natural reward learning. Synaptic plasticity in dopamine D1 and D2 receptor medium spiny neurons (MSNs) of the NAc and the endogenous cannabinoid (eCB) system have been implicated in reward seeking. However, the precise molecular and physiological basis of reward-seeking behavior remains unknown. We found that the specific deletion of metabotropic glutamate receptor 5 (mGluR5) in D1-expressing MSNs (D1miRmGluR5 mice) abolishes eCB-mediated long-term depression (LTD) and prevents the expression of drug (cocaine and ethanol), natural reward (saccharin), and brain-stimulation-seeking behavior. In vivo enhancement of 2-arachidonoylglycerol (2-AG) eCB signaling within the NAc core restores both eCB-LTD and reward-seeking behavior in D1miRmGluR5 mice. The data suggest a model where the eCB and glutamatergic systems of the NAc act in concert to mediate reward-seeking responses

Endocannabinoid long-term depression revealed at medial perforant path excitatory synapses in the dentate gyrus

The endocannabinoid system modulates synaptic plasticity in the hippocampus, but a link between long-term synaptic plasticity and the type 1 cannabinoid (CB1) receptor at medial perforant path (MPP) synapses remains elusive. Here, immuno-electron microscopy in adult mice showed that similar to 26% of the excitatory synaptic terminals in the middle 1/3 of the dentate molecular layer (DML) contained CB1 receptors, and field excitatory postsynaptic potentials evoked by MPP stimulation were inhibited by CB1 receptor activation. In addition, MPP stimulation at 10 Hz for 10 min triggered CB, receptor-dependent excitatory long-term depression (eCB-eLTD) at MPP synapses of wild-type mice but not on CB1-knockout mice. This eCB-eLTD was group I mGluR-dependent, required intracellular calcium influx and 2-arachydonoyl-glycerol (2-AG) synthesis but did not depend on N-methyl-d-aspartate (NMDA) receptors. Overall, these results point to a functional role for CB1 receptors with eCB-eLTD at DML MPP synapses and further involve these receptors in memory processing within the adult brain.We thank all members of P. Grandes laboratory for their helpful comments, suggestions, and discussions during the performance of this study. The authors thank Giovanni Marsicano (INSERM, U1215 Neurocentre Magendie, Endocannabinoids and Neuroadaptation, Bordeaux, France. University de Bordeaux, France), Beat Lutz (Institute of Physiological Chemistry and German Resilience Center, University Medical Center of the Johannes Gutenberg University Mainz, Germany) and Susana Mato (Achucarro Basque Center for Neuroscience, Science Park of the UPV/EHU, Leioa, Vizcaya, Spain) for providing the CB1 receptor knock-out mice. This work was supported by MINECO/FEDER, UE (grant number SAF2015-65034-R to PG); The Basque Government (grant number BCG IT764-13 to PG); Red de Trastornos Adictivos, Instituto de Salud Carlos III (ISC-III) and European Regional Development Funds-European Union (ERDF-EU; grant RD16/0017/0012 to PG); PhD contract from MINECO (BES-2013-065057 to SP); Vanier Canada Graduate Scholarship (NSERC to CJF)

AM251 regulates the protein expressions of glucose/pyruvate-metabolizing enzymes.

<p>AM251 effects on the protein expressions of GPI, TPI, Eno3, PKM1, LDHa and Glo1 in the abdominal muscle of STD, HFD and HCD-fed rats (<b>A-F</b>). Representative 2D polyacrylamide gels showing the intensity of the identified spots (<b>G</b>). Bonferroni’s test (<i>n</i> = 6): *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001 <i>vs</i>. STD-vehicle; ^#<i>P</i><0.05, ^##<i>P</i><0.01, ^###<i>P</i><0.001 <i>vs</i>. HFD-vehicle; ^$<i>P</i><0.05, ^{$ $$}<i>P</i><0.001 <i>vs</i>. HCD-vehicle.</p