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The alteration of the endocannabinoid tone usually associates with changes in the expression and/or function of the cannabinoid CB

Activation of cannabinoid 1 receptors (CB1 R) modulates multiple behaviours, including exploration, motor coordination and response to psychostimulants. It is known that CB1 R expressed by either excitatory or inhibitory neurons mediates different behavioural responses to CB1 R activation, yet the involvement of CB1 R expressed by medium spiny neurons (MSNs), the neuronal subpopulation that expresses the highest level of CB1 R in the CNS, remains unknown. We report a new genetically modified mouse line that expresses functional CB1 R in MSN on a CB1 R knockout (KO) background (CB1 R(MSN) mice). The absence of cannabimimetic responses measured in CB1 R KO mice was not rescued in CB1 R(MSN) mice, nor was decreased spontaneous locomotion, impaired instrumental behaviour or reduced amphetamine-triggered hyperlocomotion measured in CB1 R KO mice. Significantly, reduced novel environment exploration of an open field and absence of amphetamine sensitization (AS) measured in CB1 R KO mice were fully rescued in CB1 R(MSN) mice. Impaired motor coordination in CB1 R KO mice measured on the Rotarod was partially rescued in CB1 R(MSN) mice. Thus, CB1 R expressed by MSN control exploration, motor coordination, and AS. Our study demonstrates a new functional roles for cell specific CB1 R expression and their causal link in the control of specific behaviors.

Omega-3 fatty acids (FA) are essential long-chain polyunsaturated FA, amongst others, α-linolenic acid (ALA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). The main food source of omega-3 is the oily fish which is found in salmon, anchovy or tuna. A diet enriched with omega-3 is known to favour healthy states by promoting molecular and functional changes during brain damage recovery, membranes fluidity, energy metabolism regulation, release of signalling molecules or gene expression. Likewise, the activation of signalling pathways by omega-3 improves neural transmission and plasticity and decreases oxidative stress and inflammation in cells, particularly in neurons. Therefore, omega-3 supplements have been used to prevent or treat many human disorders. This review is intended to provide the stateof- the art of omega-3 as a natural component with beneficial therapeutic properties in cardiovascular and neurodegenerative diseases (Alzheimer and Parkinson), cancer, alcoholism and overweight. Lastly, some insights into the potential benefits of omega-3 supplementation to dodge or treat some other diseases in the future are also considered.; Kate luzeko omega-3 mantenugaiak, azido α-linolenikoa (ALA), azido eikosapentaenoikoa (EPA) eta azido dokosahexaenoikoa (DHA), dietaren bitartez bereganatzen diren gantz-azido (GA) poliasegabeak dira. Propietate antioxidatzaileak barne hartzen dituzten hiru osagai horien elikagai-iturri nagusia arrain koipetsua (izokina, antxoa, hegalaburra…) eta horretatik eratorritako arrain-olioa dira batez ere. Omega-3 GA osagarriaz aberastutako dietak aldaketa molekular zein funtzional mesedegarriak eragiten ditu garunaren garapen prozesuan, zenbait garun lesioren berreskurapenean parte hartzen. Gehigarri horrek mintz zelularraren fluidotasuna areagotzen du, eta metabolismoaren erregulazioan parte hartzen du, seinaleztapen molekulen askapena sustatuz eta gene espresioan eraginez. Bi ekintza horien bidez seinaleztapen bideak aktibatzen dira, eta ondorioz garun plastikotasuna eta transmisio sinaptikoa suspertu. Areago, omega-3 GAk zeluletan oro har, eta neuronetan bereziki, oxidazio-estresak eta hanturak eragindako kalteak murriztu ditzake. Horregatik guztiagatik, omega-3 osagarria hainbat patologietan prebentzioan edo tratamenduan erabili da. Berrikuspen honek laburbiltzen ditu kate luzeko omega-3 GAetan aberastutako tratamenduak bihotz hodietako gaixotasunetan, minbizian, neuroendekapenezko gaixotasunetan (Alzheimer eta Parkinson), alkoholismoan eta gainpisuan, oinarrizko ikerkuntzan eta ikerketa klinikoan frogatu eta egiaztatu diren aurrerapen terapeutiko berriak; eta etorkizunera begira beste hainbat gaixotasuni aurrea hartzeko edo haiek tratatzeko potentzialtasun handiko eta albo ondoriorik gabeko osagarri ez-inbaditzaile aproposa izan daitekeela iradokitzen du.

The distribution and roles of the cannabinoid CB

The cannabinoid CB1 receptor localizes to the glutamatergic parallel fiber (PF) terminals of the cerebellar granule cells and participates in synaptic plasticity, motor control and learning that are impaired in CB1 receptor knockout (CB 1 -KO) mice. However, whether ultrastructural changes at the PF-Purkinje cell (PC) synapses occur in CB 1 -KO remains unknown. We studied this in the vermis of the spinocerebellar lobule V and the vestibulocerebellar lobule X of CB 1 -KO and wild-type (CB 1 -WT) mice by electron microscopy. Lobule V, but not lobule X, of CB 1 -KO had significantly less and longer synapses than in CB 1 -WT. PF terminals were significantly larger in both lobules of CB 1 -KO with no changes in PC dendritic spines. The PF terminals in lobule V of CB 1 -KO contained less synaptic vesicles and lower vesicle density; by contrast, vesicle density in lobule X of CB 1 -KO remained unchangeable relative to CB 1 -WT. There were as many vesicles in lobule V of CB 1 -KO as in CB 1 -WT, but their distribution decreased drastically at 300 nm of the active zone. In lobule X of CB 1 -KO, less vesicles were found within 150 nm from the presynaptic membrane; however, no vesicles were at 450-600 nm of the active zone. A significant higher amount of synaptic vesicles close to the active zone in lobule V and X of CB 1 -KO was observed. In conclusion, the absence of CB1 receptors strikingly and distinctively impacts on the ultrastructural architecture of the PF-PC synapses located in cerebellar lobules that differ in vulnerability to damage and motor functions.

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.

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.).

Binge drinking (BD) is a common pattern of ethanol (EtOH) consumption by adolescents. The brain effects of the acute EtOH exposure are well-studied; however, the long-lasting cognitive and neurobehavioral consequences of BD during adolescence are only beginning to be elucidated. Environmental enrichment (EE) has long been known for its benefits on the brain and may serve as a potential supportive therapy following EtOH exposure. In this study, we hypothesized that EE may have potential benefits on the cognitive deficits associated with BD EtOH consumption. Four-week-old C57BL/6J male mice were exposed to EtOH following an intermittent 4-day drinking-in-the-dark procedure for 4 weeks. Then they were exposed to EE during EtOH withdrawal for 2 weeks followed by a behavioral battery of tests including novel object recognition, novel location, object-in-place, rotarod, beam walking balance, tail suspension, light-dark box and open field that were run during early adulthood. Young adult mice exposed to EE significantly recovered recognition, spatial and associative memory as well as motor coordination skills and balance that were significantly impaired after adolescent EtOH drinking with respect to controls. No significant permanent anxiety or depressive-like behaviors were observed. Taken together, an EE exerts positive effects on the long-term negative cognitive deficits as a result of EtOH consumption during adolescence.

Cannabinoid type-1 (CB1 ) receptors are widely distributed in the brain and play important roles in astrocyte function and the modulation of neuronal synaptic transmission and plasticity. However, it is currently unknown how CB1 receptor expression in astrocytes is affected by long-term exposure to stressors. Here we examined CB1 receptors in astrocytes of ethanol (EtOH)-exposed adolescent mice to determine its effect on CB1 receptor localization and density in adult brain. 4-8-week-old male mice were exposed to 20 percent EtOH over a period of 4 weeks, and receptor localization was examined after 4 weeks in the hippocampal CA1 stratum radiatum by pre-embedding immunoelectron microscopy. Our results revealed a significant reduction in CB1 receptor immunoparticles in astrocytic processes of EtOH-exposed mice when compared with controls (positive astrocyte elements: 21.50 ± 2.80 percent versus 37.22 ± 3.12 percent, respectively), as well as a reduction in particle density (0.24 ± 0.02 versus 0.35 ± 0.02 particles/μm). The majority of CB1 receptor metal particles were in the range of 400-1200 nm from synaptic terminals in both control and EtOH. Altogether, the decrease in the CB1 receptor expression in hippocampal astrocytes of adult mice exposed to EtOH during adolescence reveals a long lasting effect of EtOH on astrocytic CB1 receptors. This deficiency may also have negative consequences for synaptic function.

Binge drinking is a significant problem in adolescent populations, and because of the reciprocal interactions between ethanol (EtOH) consumption and the endocannabinoid (eCB) system, we sought to determine if adolescent EtOH intake altered the localization and function of the cannabinoid 1 (CB(1)) receptors in the adult brain. Adolescent mice were exposed to a 4-day-per week drinking in the dark (DID) procedure for a total of 4 weeks and then tested after a 2-week withdrawal period. Field excitatory postsynaptic potentials (fEPSPs), evoked by medial perforant path (MPP) stimulation in the dentate gyrus molecular layer (DGML), were significantly smaller. Furthermore, unlike control animals, CB(1) receptor activation did not depress fEPSPs in the EtOH-exposed animals. We also examined a form of excitatory long-term depression that is dependent on CB(1) receptors (eCB-eLTD) and found that it was completely lacking in the animals that consumed EtOH during adolescence. Histological analyses indicated that adolescent EtOH intake significantly reduced the CB(1) receptor distribution and proportion of immunopositive excitatory synaptic terminals in the medial DGML. Furthermore, there was decreased binding of [(35)S]guanosine-5*-O-(3-thiotriphosphate) ([(35)S] GTPγS) and the guanine nucleotide-binding (G) protein Gαi2 subunit in the EtOH-exposed animals. Associated with this, there was a significant increase in monoacylglycerol lipase (MAGL) mRNA and protein in the hippocampus of EtOH-exposed animals. Conversely, deficits in eCB-eLTD and recognition memory could be rescued by inhibiting MAGL with JZL184. These findings indicate that repeated exposure to EtOH during adolescence leads to long-term deficits in CB(1) receptor expression, eCB-eLTD, and reduced recognition memory, but that these functional deficits can be restored by treatments that increase endogenous 2-arachidonoylglycerol.

The cannabinoid CB

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.

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).

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.

Binge drinking (BD) is a common pattern of ethanol (EtOH) consumption by adolescents. The brain effects of the acute EtOH exposure are well-studied; however, the long-lasting cognitive and neurobehavioral consequences of BD during adolescence are only beginning to be elucidated. Environmental enrichment (EE) has long been known for its benefits on the brain and may serve as a potential supportive therapy following EtOH exposure. In this study, we hypothesized that EE may have potential benefits on the cognitive deficits associated with BD EtOH consumption. Four-week-old C57BL/6J male mice were exposed to EtOH following an intermittent 4-day drinking-in-the-dark procedure for 4 weeks. Then they were exposed to EE during EtOH withdrawal for 2 weeks followed by a behavioral battery of tests including novel object recognition, novel location, object-in-place, rotarod, beam walking balance, tail suspension, light-dark box and open field that were run during early adulthood. Young adult mice exposed to EE significantly recovered recognition, spatial and associative memory as well as motor coordination skills and balance that were significantly impaired after adolescent EtOH drinking with respect to controls. No significant permanent anxiety or depressive-like behaviors were observed. Taken together, an EE exerts positive effects on the long-term negative cognitive deficits as a result of EtOH consumption during adolescence.

Cannabinoid type-1 (CB

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.

Cellular activity in the brain depends on the high energetic support provided by mitochondria, the cell organelles which use energy sources to generate ATP. Acute cannabinoid intoxication induces amnesia in humans and animals, and the activation of type-1 cannabinoid receptors present at brain mitochondria membranes (mtCB1) can directly alter mitochondrial energetic activity. Although the pathological impact of chronic mitochondrial dysfunctions in the brain is well established, the involvement of acute modulation of mitochondrial activity in high brain functions, including learning and memory, is unknown. Here, we show that acute cannabinoid-induced memory impairment in mice requires activation of hippocampal mtCB1 receptors. Genetic exclusion of CB1 receptors from hippocampal mitochondria prevents cannabinoid-induced reduction of mitochondrial mobility, synaptic transmission and memory formation. mtCB1 receptors signal through intra-mitochondrial Gαi protein activation and consequent inhibition of soluble-adenylyl cyclase (sAC). The resulting inhibition of protein kinase A (PKA)-dependent phosphorylation of specific subunits of the mitochondrial electron transport system eventually leads to decreased cellular respiration. Hippocampal inhibition of sAC activity or manipulation of intra-mitochondrial PKA signalling or phosphorylation of the Complex I subunit NDUFS2 inhibit bioenergetic and amnesic effects of cannabinoids. Thus, the G protein-coupled mtCB1 receptors regulate memory processes via modulation of mitochondrial energy metabolism. By directly linking mitochondrial activity to memory formation, these data reveal that bioenergetic processes are primary acute regulators of cognitive functions.

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.

Type 1 cannabinoid (CB

The mature calyx of Held ending on principal neurons of the medial nucleus of the trapezoid body (MNTB) has very specialized morphological and molecular features that make it possible to transmit auditory signals with high fidelity. In a previous work we described an increased localization of the ionotropic α-amino-3-hydroxy-5-methyl-4 isoxazolepropionic acid (AMPA) glutamate receptor (GluA) subunits at postsynaptic sites of the calyx of Held-principal cell body synapses from postnatal development to adult. The aim of the present study was to investigate whether the pattern of the synaptic distribution of GluA2/3/4c and -4 in adult MNTB principal cell bodies correlated with preferential subcellular domains (stalks and swellings) of the calyx. We used a postembedding immunocytochemical method combined with specific antibodies to GluA2/3/4c and GluA4 subunits. We found that the density of GluA2/3/4c in calyceal swellings (19 ± 1.54 particles/μm) was higher than in stalks (10.93 ± 1.37 particles/μm); however, the differences for GluA4 were not statistically significant (swellings: 13.84 ± 1.39 particles/μm; stalks: 10.42 ± 1.24 particles/μm). Furthermore, GluA2/3/4c and GluA4 labeling co-localized to some extent in calyceal stalks and swellings. Taking these data together, the distribution pattern of GluA subunits in postsynaptic specializations are indicative of a spatial compartmentalization of AMPA subunits in mature calyx-principal neuron synapses that may support the temporally precise transmission required for sound localization in the auditory brainstem. J. Comp. Neurol. 518:163–174, 2010. © 2009 Wiley-Liss, Inc.

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.