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The use and abuse of cannabis can be associated with significant pathophysiology, however, it remains unclear whether (1) acute administration of Δ-9-tetrahydrocannabinol (THC) during early adulthood alters the cannabinoid type 1 (CB1 ) receptor localization and expression in cells of the brain, and (2) THC produces structural brain changes. Here we use electron microscopy and a highly sensitive pre-embedding immunogold method to examine CB1 receptors in the hippocampus cornu ammonis subfield 1 (CA1) 30 min after male mice were exposed to a single THC injection (5 mg/kg). The findings show that acute exposure to THC can significantly decrease the percentage of CB1 receptor immunopositive terminals making symmetric synapses, mitochondria, and astrocytes. The percentage of CB1 receptor-labeled terminals forming asymmetric synapses was unaffected. Lastly, CB1 receptor expression was significantly lower at terminals of symmetric and asymmetric synapses as well as in mitochondria. Structurally, CA1 dendrites were significantly larger, and contained more spines and mitochondria following acute THC administration. The area of the dendritic spines, synaptic terminals, mitochondria, and astrocytes decreased significantly following acute THC exposure. Altogether, these results indicate that even a single THC exposure can have a significant impact on CB1 receptor expression, and can alter CA1 ultrastructure, within 30 min of drug exposure. These changes may contribute to the behavioral alterations experienced by young individuals shortly after cannabis intoxication.

[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 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 Government

Type 1 cannabinoid (CB1 ) receptors are widely distributed in the brain. Their physiological roles depend on their distribution pattern, which differs remarkably among cell types. Hence, subcellular compartments with little but functionally relevant CB1 receptors can be overlooked, fostering an incomplete mapping. To overcome this, knockin mice with cell-type-specific rescue of CB1 receptors have emerged as excellent tools for investigating CB1 receptors' cell-type-specific localization and sufficient functional role with no bias. However, to know whether these rescue mice maintain endogenous CB1 receptor expression level, detailed anatomical studies are necessary. The subcellular distribution of hippocampal CB1 receptors of rescue mice that express the gene exclusively in dorsal telencephalic glutamatergic neurons (Glu-CB1 -RS) or GABAergic neurons (GABA-CB1 -RS) was studied by immunoelectron microscopy. Results were compared with conditional CB1 receptor knockout lines. As expected, CB1 immunoparticles appeared at presynaptic plasmalemma, making asymmetric and symmetric synapses. In the hippocampal CA1 stratum radiatum, the values of the CB1 receptor-immunopositive excitatory and inhibitory synapses were Glu-CB1 -RS, 21.89% (glutamatergic terminals); 2.38% (GABAergic terminals); GABA-CB1 -RS, 1.92% (glutamatergic terminals); 77.92% (GABAergic terminals). The proportion of CB1 receptor-immunopositive excitatory and inhibitory synapses in the inner one-third of the dentate molecular layer was Glu-CB1 -RS, 53.19% (glutamatergic terminals); 2.30% (GABAergic terminals); GABA-CB1 -RS, 3.19% (glutamatergic terminals); 85.07% (GABAergic terminals). Taken together, Glu-CB1 -RS and GABA-CB1 -RS mice show the usual CB1 receptor distribution and expression in hippocampal cell types with specific rescue of the receptor, thus being ideal for in-depth anatomical and functional investigations of the endocannabinoid system. J. Comp. Neurol. 525:302-318, 2017. © 2016 Wiley Periodicals, Inc.

Activation of type 1 cannabinoid (CB(1)) 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 CB(1) receptors in the brain. In situ hybridization and immunohistochemical techniques have been crucial for defining the CB(1) receptor expression and localization at the cellular level. Nevertheless, more advanced methods are needed to reveal the precise topography of CB(1) 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 CB(1) receptors in the brain. Herein, we describe a single pre-embedding immunogold method for electron microscopy based on the use of specific CB(1) 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 CB(1) 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 CB(1) receptors in glutamatergic synapses, astrocytes, neuronal and astrocytic mitochondria in the brain.

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).

International audience; The retrieval of odor-related memories shapes animal behavior. The anterior piriform cortex (aPC) is the largest part of the olfactory cortex, and it plays important roles in olfactory processing and memory. However, it is still unclear whether specific cellular mechanisms in the aPC control olfactory memory, depending on the appetitive or aversive nature of the stimuli involved. Cannabinoid-type 1 (CB1) receptors are present in the aPC (aPC-CB1), but their potential impact on olfactory memory was never explored. Here, we used a combination of behavioral, genetic, anatomical, and electrophysiological approaches to characterize the functions of aPC-CB1 receptors in the regulation of appetitive and aversive olfactory memory. Pharmacological blockade or genetic deletion of aPC-CB1 receptors specifically impaired the retrieval of conditioned odor preference (COP). Interestingly, expression of conditioned odor aversion (COA) was unaffected by local CB1 receptor blockade, indicating that the role of aPC endocannabinoid signaling is selective for retrieval of appetitive memory. Anatomical investigations revealed that CB1 receptors are highly expressed on aPC GABAergic interneurons, and ex vivo electrophysiological recordings showed that their pharmacological activation reduces miniature inhibitory post-synaptic currents (mIPSCs) onto aPC semilunar (SL), but not pyramidal principal neurons. COP retrieval, but not COA, was associated with a specific CB1-receptor-dependent decrease of mIPSCs in SL cells. Altogether, these data indicate that aPC-CB1 receptor-dependent mechanisms physiologically control the retrieval of olfactory memory, depending on odor valence and engaging modulation of local inhibitory transmission.

Summary The 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., Graphical Abstract, Highlights • mGluR5-D1-CB1-induced eCB-LTD mediates drugs of abuse and natural reward seeking • eCB-LTD in D2-MSNs plays no important role in processing of reward-seeking responses • Loss of eCB-LTD is a consequence of higher MAGL activity and lower CB1R expression • Acute drug administration stops craving for alternative rewards on following days, Neuroscience; Behavioral Neuroscience; Molecular Neuroscience

Astroglial type‐1 cannabinoid (CB1) receptors are involved in synaptic transmission, plasticity and behavior by interfering with the so‐called tripartite synapse formed by pre‐ and post‐synaptic neuronal elements and surrounding astrocyte processes. However, little is known concerning the subcellular distribution of astroglial CB1 receptors. In particular, brain CB1 receptors are mostly localized at cells' plasmalemma, but recent evidence indicates their functional presence in mitochondrial membranes. Whether CB1 receptors are present in astroglial mitochondria has remained unknown. To investigate this issue, we included conditional knock‐out mice lacking astroglial CB1 receptor expression specifically in glial fibrillary acidic protein (GFAP)‐containing astrocytes (GFAP‐CB1‐KO mice) and also generated genetic rescue mice to re‐express CB1 receptors exclusively in astrocytes (GFAP‐CB1‐RS). To better identify astroglial structures by immunoelectron microscopy, global CB1 knock‐out (CB1‐KO) mice and wild‐type (CB1‐WT) littermates were intra‐hippocampally injected with an adeno‐associated virus expressing humanized renilla green fluorescent protein (hrGFP) under the control of human GFAP promoter to generate GFAPhrGFP‐CB1‐KO and ‐WT mice, respectively. Furthermore, double immunogold (for CB1) and immunoperoxidase (for GFAP or hrGFP) revealed that CB1 receptors are present in astroglial mitochondria from different hippocampal regions of CB1‐WT, GFAP‐CB1‐RS and GFAPhrGFP‐CB1‐WT mice. Only non‐specific gold particles were detected in mouse hippocampi lacking CB1 receptors. Altogether, we demonstrated the existence of a precise molecular architecture of the CB1 receptor in astrocytes that will have to be taken into account in evaluating the functional activity of cannabinergic signaling at the tripartite synapse.

The dorsal striatum is a key node for many neurobiological processes such as motor activity, cognitive functions, and affective processes. The proper functioning of striatal neurons relies critically on metabotropic receptors. Specifically, the main adenosine and endocannabinoid receptors present in the striatum, ie, adenosine A(2A) receptor (A(2A)R) and cannabinoid CB1 receptor (CB1R), are of pivotal importance in the control of neuronal excitability. Facilitatory and inhibitory functional interactions between striatal A(2A)R and CB1R have been reported, and evidence supports that this cross-talk may rely, at least in part, on the formation of A(2A)R-CB1R heteromeric complexes. However, the specific location and properties of these heteromers have remained largely unknown. Here, by using techniques that allowed a precise visualization of the heteromers in situ in combination with sophisticated genetically modified animal models, together with biochemical and pharmacological approaches, we provide a high-resolution expression map and a detailed functional characterization of A(2A)R-CB1R heteromers in the dorsal striatum. Specifically, our data unveil that the A(2A)R-CB1R heteromer (i) is essentially absent from corticostriatal projections and striatonigral neurons, and, instead, is largely present in striatopallidal neurons, (ii) displays a striking G protein-coupled signaling profile, where co-stimulation of both receptors leads to strongly reduced downstream signaling, and (iii) undergoes an unprecedented dysfunction in Huntington's disease, an archetypal disease that affects striatal neurons. Altogether, our findings may open a new conceptual framework to understand the role of coordinated adenosine-endocannabinoid signaling in the indirect striatal pathway, which may be relevant in motor function and neurodegenerative diseases. This work was supported by grants from the Spanish Ministry of Economy and Competitiveness (MINECO/FEDER; grant SAF2015-64945-R to MG; grant SAF-2014-54840-R to EIC and VC; grant SAF2015-65034-R to PG; grant SAF2015-67474-R to SG; grants SAF2014-55700-P and PCIN-2013-019-C03-03 to FC); Centro de Investigacion Biomedica en Red sobre Enfermedades Neurodegenerativas (CIBERNED; grant PI2013/05 to MG, PJM and EIC); Comunidad de Madrid (grant S2010/BMD-2308 to MG); Generalitat de Catalunya (grant 2014-SGR-1236 to EIC); 'La Marato de TV3' Foundation (grant 20140610 to EIC; grant 20152031 to FC); Agentschap voor Innovatie door Wetenschap en Technologie (grant SBO-140028 to FC); BBSRC DTP studentship (to PJM and LB); EPSRC (grant EP/M006379/1 to LAH); Deutsche Forschungsgemeinschaft (DFG; grant MO 1920/1-1 to KM; grant CRC-TRR 58 to BL); Institute of Health Carlos III from the Spanish Ministry of Economy and Competitiveness (grant PIE14/00034 to FC; grant PI10/00172 and funding from FEDER grants to MJC and JP); The Basque Government (grant IT764-13 to PG); University of the Basque Country UPV/EHU (grant UFI11/41 to PG); Red de Trastornos Adictivos-Institute of Health Carlos III (grant RD12/0028/0004 to PG). AC is supported by the Spanish Ministry of Economy and Competitiveness (Juan de la Cierva Program). MM is supported by the Spanish Ministry of Education, Culture and Sport (FPU Program). The authors declare no conflict of interest.

The transient receptor potential vanilloid type 1 (TRPV1) is a non-selective cation channel that plays an important role in pain perception and modulates neurotransmitter release and synaptic plasticity in the brain. TRPV1 function must lay on its anatomical distribution in the peripheral and central nervous system regions involved in the physiological roles of the channel. However, the anatomical localization of TRPV1 is well established in the periphery, but in the brain it is a matter of debate. While some studies support the presence of TRPV1 in several brain regions, recent evidences suggest a restricted distribution of the channel in the central nervous system. To investigate to what extent central TRPV1 function stands on a precise brain distribution of the channel, we examined the mouse hippocampal dentate molecular layer (ML) where TRPV1 mediates long-term synaptic plasticity. Using pre-embedding immunocytochemistry for high resolution electron microscopy, we show that TRPV1 immunoparticles are highly concentrated in postsynaptic dendritic spines to asymmetric perforant path synapses in the outer 2/3 of the ML. However, TRPV1 is poorly expressed at the excitatory hilar mossy cell synapses in the inner 1/3 of this layer. Importantly, the TRPV1 pattern distribution disappeared in the ML of TRPV1-knockout mice. Taken together, these findings support the notion of the presence of TRPV1 in a brain region where the channel has been shown to have a functional role, such as the perforant path synapses in the hippocampal dentate ML.

The hypothalamus and the endocannabinoid system are important players in the regulation of energy homeostasis. In a previous study, we described the ultrastructural distribution of CB1 receptors in GABAergic and glutamatergic synaptic terminals of the dorsomedial region of the ventromedial nucleus of the hypothalamus (VMH). However, the specific localization of the enzymes responsible for the synthesis of the two main endocannabinoids in the hypothalamus is not known. The objective of this study was to investigate the precise subcellular distribution of N-arachidonoylphospatidylethanolamine phospholipase D (NAPE-PLD) and diacylglycerol lipase α (DAGL-α) in the dorsomedial VMH of wild-type mice by a high resolution immunogold electron microscopy technique. Knock-out mice for each enzyme were used to validate the specificity of the antibodies. NAPE-PLD was localized presynaptically and postsynaptically but showed a preferential distribution in dendrites. DAGL-α was mostly postsynaptic in dendrites and dendritic spines. These anatomical results contribute to a better understanding of the endocannabinoid modulation in the VMH nucleus. Furthermore, they support the idea that the dorsomedial VMH displays the necessary machinery for the endocannabinoid-mediated modulation of synaptic transmission of brain circuitries that regulate important hypothalamic functions such as feeding behaviors.

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.

The dorsal striatum is a key node for many neurobiological processes such as motor activity, cognitive functions, and affective processes. The proper functioning of striatal neurons relies critically on metabotropic receptors. Specifically, the main adenosine and endocannabinoid receptors present in the striatum, ie, adenosine A2A receptor (A2AR) and cannabinoid CB1 receptor (CB1R), are of pivotal importance in the control of neuronal excitability. Facilitatory and inhibitory functional interactions between striatal A2AR and CB1R have been reported, and evidence supports that this cross-talk may rely, at least in part, on the formation of A2AR-CB1R heteromeric complexes. However, the specific location and properties of these heteromers have remained largely unknown. Here, by using techniques that allowed a precise visualization of the heteromers in situ in combination with sophisticated genetically modified animal models, together with biochemical and pharmacological approaches, we provide a high-resolution expression map and a detailed functional characterization of A2AR-CB1R heteromers in the dorsal striatum. Specifically, our data unveil that the A2AR-CB1R heteromer (i) is essentially absent from corticostriatal projections and striatonigral neurons, and, instead, is largely present in striatopallidal neurons, (ii) displays a striking G protein-coupled signaling profile, where co-stimulation of both receptors leads to strongly reduced downstream signaling, and (iii) undergoes an unprecedented dysfunction in Huntington’s disease, an archetypal disease that affects striatal neurons. Altogether, our findings may open a new conceptual framework to understand the role of coordinated adenosine-endocannabinoid signaling in the indirect striatal pathway, which may be relevant in motor function and neurodegenerative diseases.

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.

1,2-diacylglycerol lipase alpha (DAGLα) is responsible for the biosynthesis and release of 2-arachidonoyl-glycerol (2-AG), the most abundant endocannabinoid in the brain. Although its expression has been detected in discrete regions, we showed here an integrated description of the distribution of DAGLα mRNA and protein in the rat forebrain using in situ hybridization histochemistry and immunohistochemistry. As novelty, we described the distribution of DAGLα protein expression in the olfactory system, the rostral migratory stream, neocortex, septum, thalamus, and hypothalamus. Similar DAGLα immunostaining pattern was also found in the brain of wild-type, but not of DAGLα knockout mice. Immunohistochemical data were correlated by the identification of DAGLα mRNA expression, for instance, in the somata of specific cells in olfactory structures, rostral migratory stream and neocortex, cells in some septal-basal-amygdaloid areas and the medial habenula, and magnocellular cells of the paraventricular hypothalamic nucleus. This widespread neuronal distribution of DAGLα is consistent with multiple roles for endocannabinoids in synaptic plasticity, including presynaptic inhibition of neurotransmitter release. We discuss our comparative analysis of the forebrain expression patterns of DAGLα and other components of the endocannabinoid signaling system, including the CB1 receptor, monoacylglyceride lipase (MAGL), and fatty acid amide hydrolase (FAAH), providing some insight into the potential physiological and behavioral roles of this system. © 2011 IBRO.

The endocannabinoid system is involved in multiple physiological functions including reward. Cannabinoids potently control the activity of midbrain dopamine cells, but the contribution of cortical projections in this phenomenon is unclear. We show that the bed nucleus of the stria terminalis (BNST) efficient relays cortical excitation to dopamine neurons of the ventral tegmental area (VTA). Anatomical andin vivoelectrophysiological evidence demonstrate that excitatory projections arising exclusively from the infralimbic cortex converge on BNST neurons, which in turn project to and excite >80% VTA dopamine cells. At the ultrastructural level, cannabinoid type 1 receptors are detected within the BNST on axon terminals arising from the infralimbic cortex. We found that intra-BNST infusion of a cannabinoid agonist inhibits the firing of dopamine cells evoked by stimulation of the infralimbic cortex. Our data identify a new neuronal substrate for the actions of cannabinoids in the reward pathway.

Deep cerebellar dentate nuclei are in a key position to control motor planning as a result of an integration of cerebropontine inputs and hemispheric Purkinje neurons signals, and their influence through synaptic outputs onto extracerebellar hubs. GABAergic dentate neurons exhibit broader action potentials and slower afterhyperpolarization than non-GABAergic (presumably glutamatergic) neurons. Specific potassium channels may be involved in these distinct firing profiles, particularly, Kv3.1 and Kv3.3 subunits which rapidly activate at relatively positive potentials to support the generation of fast action potentials. To investigate the subcellular localization of Kv3.1b and Kv3.3 in GAD− and GAD+ dentate neurons of glutamic acid decarboxylase 67–green fluorescent protein (GAD67-GFP) knock-in mice a preembedding immunocytochemical method for electron microscopy was used. Kv3.1b and Kv3.3 were in membranes of cell somata, dendrites, axons and synaptic terminals of both GAD− and GAD+ dentate neurons. The vast majority of GAD− somatodendritic membrane segments domains labeled for Kv3.1b and Kv3.3 (96.1% and 84.7%, respectively) whereas 56.2% and 69.8% of GAD− axonal membrane segments were immunopositive for these subunits. Furthermore, density of Kv3.1b immunoparticles was much higher in GAD− somatodendritic than axonal domains. As to GAD+ neurons, only 70.6% and 50% of somatodendritic membrane segments, and 53.3% and 59.5% of axonal membranes exhibited Kv3.1b and Kv3.3 labeling, respectively. In contrast to GAD− cells, GAD+ cells exhibited a higher density labeling for both Kv3 subunits at their axonal than at their somatodendritic membranes. Taken together, Kv3.1b and Kv3.3 potassium subunits are expressed in both GAD− and GAD+ cells, albeit at different densities and distribution. They likely contribute to the distinct biophysical properties of both GAD− and GAD+ neurons in the dentate nucleus.

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 (similar to 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. P. Grandes' laboratory is provided by Ministerio de Economia y Competitividad (BFU2012-33334); Basque Government grant IT764-13; University of the Basque Country (UFI11/41) and by Red de Trastornos Adictivos, RETICS, Instituto de Salud Carlos III, grant number: RD07/0001/2001, RD12/0028/0004.

The existence of a neuronal-glial signalling through the activation of neurotransmitter receptors expressed in glia is well-documented. In excitatory synapses, glutamate released from presynaptic terminals activates not only postsynaptic receptors, but also ionotropic and metabotropic glutamate receptors localized in the glia ensheathing the synapses. The medial nucleus of the trapezoid body of the auditory system is involved in the localization of sounds in the space. In this nucleus, the large excitatory synaptic terminals formed by the calyces of Held on the principal globular cell bodies are wrapped by astrocytic processes. Since these synapses are functional from early postnatal days, glia receiving excitatory synaptic signals from the calyces may participate in modulating the maturation and development of the system. Groups I and II of metabotropic glutamate receptors (mGluRs) have been localized in glial cells in different brain regions. To investigate whether group II mGluRs are present in the medial nucleus of the trapezoid body, we have studied the pattern of expression of mGluR2/3 in the developing and mature nucleus by means of immunocytochemichal methods. The most remarkable finding was the switch in the occurrence of mGluR2/3 from glial to neuronal compartments. Thus, a preferential localization of mGluR2/3 immunoreactivity was observed in astrocytic processes surrounding the calyces of Held during the early postnatal development. In contrast, the main feature in adult rats was the presence of the group II mGluRs in presynaptic calyces of Held and postsynaptic principal globular cells. From these observations we suggest a role for group II mGluRs in neuronal-glial signalling in the calyx of Held-principal globular neuron synapses. Activation of these receptors might be relevant to the maturation and modulation of synaptic transmission in the medial nucleus of the trapezoid body.

NMDA receptors (NMDARs) are classically known as coincidence detectors for the induction of long-term synaptic plasticity and have been implicated in hippocampal CA3 cell-dependent spatial memory functions that likely rely on dynamic cellular ensemble encoding of space. The unique functional properties of both NMDARs and mossy fiber projections to CA3 pyramidal cells place mossy fiber NMDARs in a prime position to influence CA3 ensemble dynamics. By mimicking presynaptic and postsynaptic activity patterns observed in vivo, we found a burst timing-dependent pattern of activity that triggered bidirectional long-term NMDAR plasticity at mossy fiber-CA3 synapses in rat hippocampal slices. This form of plasticity imparts bimodal control of mossy fiber-driven CA3 burst firing and spike temporal fidelity. Moreover, we found that mossy fiber NMDARs mediate heterosynaptic metaplasticity between mossy fiber and associational-commissural synapses. Thus, bidirectional NMDAR plasticity at mossy fiber-CA3 synapses could substantially contribute to the formation, storage and recall of CA3 cell assembly patterns.

International audience; The reason why neurons synthesize more than one endocannabinoid (eCB) and how this is involved in the regulation of synaptic plasticity in a single neuron is not known. We found that 2-arachidonoylglycerol (2-AG) and anandamide mediate different forms of plasticity in the extended amygdala of rats. Dendritic L-type Ca(2+) channels and the subsequent release of 2-AG acting on presynaptic CB1 receptors triggered retrograde short-term depression. Long-term depression was mediated by postsynaptic mGluR5-dependent release of anandamide acting on postsynaptic TRPV1 receptors. In contrast, 2-AG/CB1R-mediated retrograde signaling mediated both forms of plasticity in the striatum. These data illustrate how the eCB system can function as a polymodal signal integrator to allow the diversification of synaptic plasticity in a single neuron.

11 p. Background: The bed nucleus of the stria terminalis (BNST) is involved in behaviors related to natural reward, drug addiction and stress. In spite of the emerging role of the endogenous cannabinoid (eCB) system in these behaviors, little is known about the anatomy and function of this system in the anterolateral BNST (alBNST). The aim of this study was to provide a detailed morphological characterization of the localization of the cannabinoid 1 (CB1) receptor a necessary step toward a better understanding of the physiological roles of the eCB system in this region of the brain. Methodology/Principal Findings: We have combined anatomical approaches at the confocal and electron microscopy level to ex-vivo electrophysiological techniques. Here, we report that CB1 is localized on presynaptic membranes of about 55% of immunopositive synaptic terminals for the vesicular glutamate transporter 1 (vGluT1), which contain abundant spherical, clear synaptic vesicles and make asymmetrical synapses with alBNST neurons. About 64% of vGluT1 immunonegative synaptic terminals show CB1 immunolabeling. Furthermore, 30% and 35% of presynaptic boutons localize CB1 in alBNST of conditional mutant mice lacking CB1 mainly from GABAergic neurons (GABA-CB1-KO mice) and mainly from cortical glutamatergic neurons (Glu-CB1-KO mice), respectively. Extracellular field recordings and whole cell patch clamp in the alBNST rat brain slice preparation revealed that activation of CB1 strongly inhibits excitatory and inhibitory synaptic transmission. Conclusions/Significance: This study supports the anterolateral BNST as a potential neuronal substrate of the effects of cannabinoids on stress-related behaviors. Dr. Pedro Grandes' laboratory is supported by The Basque Country Government grant GIC07/70-IT-432-07, by Red de Trastornos Adictivos (RETICS), Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación (MICINN), grant RD07/0001/2001 and MICINN grant SAF2009-07065. Nagore Puente is supported by a Basque Country University grant for PhD Researcher's Specialization. Leire Reguero is in receipt of a predoctoral fellowship from the Basque Country Government. Dr. Olivier J. Manzoni's laboratory is supported by INSERM, ANR Neurosciences “Neurologie et Psychiatrie ANR-06-NEURO-043-01” and Région Aquitaine. Dr. Giovanni Marsicano's laboratory is supported by AVENIR/INSERM (with the Fondation Bettencourt-Schueller), by ANR (ANR-06-NEURO-043-01), by European Foundation for the Study of Diabetes (EFSD), by the EU-FP7 (REPROBESITY, contract number HEALTH-F2-2008-223713) and European Commission Coordination Action ENINET (contract number LSHM-CT-2005-19063). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Activation of cannabinoid type-1 receptors (CB(1)) is universally recognized as a powerful endogenous orexigenic signal, but the detailed underlying neuronal mechanisms are not fully understood. Using combined genetic and pharmacological approaches in mice, we found that ventral striatal CB(1) receptors exerted a hypophagic action through inhibition of GABAergic transmission. Conversely, brain CB(1) receptors modulating excitatory transmission mediated the well-known orexigenic effects of cannabinoids.

We examined the effect of glutamate transporter blockade at the calyx of Held synapse. In immature synapses [defined as postnatal day 8 (P8) to P10 rats], transporter blockade causes tonic activation of NMDA receptors and strong inhibition of the AMPA receptor-mediated EPSC amplitude. EPSC inhibition was blocked with a metabotropic glutamate receptor (mGluR) antagonist [1μmLY341495 (2S-2-amino-2-(1S,2S-2-carboxycycloprop-1-yl)-3-(xanth-9-yl)propanoic acid)], suggesting that elevated resting glutamate concentration specifically activates group II and group III mGluRs. Using mGluR subtype-specific agonists and antagonists, we determined that increased glutamate activates presynaptic mGluR2/3 and mGluR8 receptors but not mGluR4, although this receptor is present. Surprisingly, in older animals (P16–P18), transporter blockade had no effect on EPSC amplitude because of a developmental downregulation of group II/III mGluR activation in rats and mice. In contrast to other CNS synapses, we observed no effect of transporter blockade on EPSC decay kinetics, although expression of glutamate transporters was strong in nearby glial processes at both P9 and P17. Finally, using a low-affinity AMPA receptor antagonist (γ-d-glutamylglycine), we show that desensitization occurs at P8–P10 but is absent at P16–P18, even during trains of high-frequency (100–300 Hz) stimulation. We suggest that diffusion and transporter activation are insufficient to clear synaptically released glutamate at immature calyces, resulting in significant desensitization. Thus, mGluRs may be expressed in the immature calyx to help limit glutamate release. In the more mature calyx, there is a far smaller diffusional barrier attributable to the highly fenestrated synaptic terminal morphology, so AMPA receptor desensitization is avoided and mGluR-mediated inhibition is not necessary.

We investigated the mechanisms by which activation of group I metabotropic glutamate receptors (mGluRs) and CB1 cannabinoid receptors (CB1Rs) leads to inhibition of synaptic currents at the calyx of Held synapse in the medial nucleus of the trapezoid body (MNTB) of the rat auditory brainstem. In ∼50% of the MNTB neurons tested, activation of group I mGluRs by the specific agonist (s)-3,5-dihydroxyphenylglycine (DHPG) reversibly inhibited AMPA receptor- and NMDA receptor-mediated EPSCs to a similar extent and reduced paired-pulse depression, suggestive of an inhibition of glutamate release. Presynaptic voltage-clamp experiments revealed a reversible reduction of Ca2+currents by DHPG, with no significant modification of the presynaptic action potential waveform. Likewise, in ∼50% of the tested cells, the CB1 receptor agonist (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone (WIN) reversibly inhibited EPSCs, presynaptic Ca2+currents, and exocytosis. For a given cell, the amount of inhibition by DHPG correlated with that by WIN. Moreover, the inhibitory action of DHPG was blocked by the CB1R antagonistN-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251) and occluded by WIN, indicating that DHPG and WIN operate via a common pathway. The inhibition of EPSCs by DHPG, but not by WIN, was abolished after dialyzing 40 mmBAPTA into the postsynaptic cell, suggesting that DHPG activated postsynaptic mGluRs. Light and electron microscopy immunolabeling indicated a presynaptic expression of CB1Rs and postsynaptic localization of mGluR1a. Our data suggest that activation of postsynaptic mGluRs triggers the Ca2+-dependent release of endocannabinoids that activate CB1 receptors on the calyx terminal, which leads to a reduction of presynaptic Ca2+current and glutamate release.