Glucose metabolism links astroglial mitochondria to cannabinoid effects.

Astrocytes take up glucose from the bloodstream to provide energy to the brain, thereby allowing neuronal activity and behavioural responses1-5. By contrast, astrocytes are under neuronal control through specific neurotransmitter receptors5-7. However, whether the activation of astroglial receptors can directly regulate cellular glucose metabolism to eventually modulate behavioural responses is unclear. Here we show that activation of mouse astroglial type-1 cannabinoid receptors associated with mitochondrial membranes (mtCB1) hampers the metabolism of glucose and the production of lactate in the brain, resulting in altered neuronal functions and, in turn, impaired behavioural responses in social interaction assays. Specifically, activation of astroglial mtCB1 receptors reduces the phosphorylation of the mitochondrial complex I subunit NDUFS4, which decreases the stability and activity of complex I. This leads to a reduction in the generation of reactive oxygen species by astrocytes and affects the glycolytic production of lactate through the hypoxia-inducible factor 1 pathway, eventually resulting in neuronal redox stress and impairment of behavioural responses in social interaction assays. Genetic and pharmacological correction of each of these effects abolishes the effect of cannabinoid treatment on the observed behaviour. These findings suggest that mtCB1 receptor signalling can directly regulate astroglial glucose metabolism to fine-tune neuronal activity and behaviour in mice.

Differential Subcellular Distribution and Release Dynamics of Cotransmitted Cholinergic and GABAergic Synaptic Inputs Modify Dopaminergic Neuronal Excitability.

We identified three types of monosynaptic cholinergic inputs spatially arranged onto medial substantia nigra dopaminergic neurons in male and female mice: cotransmitted acetylcholine (ACh)/GABA, GABA-only, and ACh only. There was a predominant GABA-only conductance along lateral dendrites and soma-centered ACh/GABA cotransmission. In response to repeated stimulation, the GABA conductance found on lateral dendrites decremented less than the proximally located GABA conductance, and was more effective at inhibiting action potentials. While soma-localized ACh/GABA cotransmission showed depression of the GABA component with repeated stimulation, ACh-mediated nicotinic responses were largely maintained. We investigated whether this differential change in inhibitory/excitatory inputs leads to altered neuronal excitability. We found that a depolarizing current or glutamate preceded by cotransmitted ACh/GABA was more effective in eliciting an action potential compared with current, glutamate, or ACh/GABA alone. This enhanced excitability was abolished with nicotinic receptor inhibitors, and modulated by T- and L-type calcium channels, thus establishing that activity of multiple classes of ion channels integrates to shape neuronal excitability.SIGNIFICANCE STATEMENT Our laboratory has previously discovered a population of substantia nigra dopaminegic neurons (DA) that receive cotransmitted ACh and GABA. This study used subcellular optogenetic stimulation of cholinergic presynaptic terminals to map the functional ACh and GABA synaptic inputs across the somatodendritic extent of substantia nigra DA neurons. We determined spatially clustered GABA-only inputs on the lateral dendrites while cotransmitted ACh and GABA clustered close to the soma. We have shown that the action of GABA and ACh in cotransmission spatially clustered near the soma play a critical role in enhancing glutamate-mediated neuronal excitability through the activation of T- and L-type voltage-gated calcium channels.

Altered glial expression of the cannabinoid 1 receptor in the subiculum of a mouse model of Alzheimer's disease.

The alteration of the endocannabinoid tone usually associates with changes in the expression and/or function of the cannabinoid CB1 receptor. In Alzheimer's disease (AD), amyloid beta (Aß)-containing aggregates induce a chronic inflammatory response leading to reactivity of both microglia and astrocytes. However, how this glial response impacts on the glial CB1 receptor expression in the subiculum of a mouse model of AD, a brain region particularly affected by large accumulation of plaques and concomitant subcellular changes in microglia and astrocytes, is unknown. The CB1 receptor localization in both glial cells was investigated in the subiculum of male 5xFAD/CB2 EGFP/f/f (AD model) and CB2 EGFP/f/f mice by immuno-electron microscopy. The findings revealed that glial CB1 receptors suffer remarkable changes in the AD mouse. Thus, CB1 receptor expression increases in reactive microglia in 5xFAD/CB2 EGFP/f/f , but remains constant in astrocytes with CB1 receptor labeling rising proportionally to the perimeter of the reactive astrocytes. Not least, the CB1 receptor localization in microglial processes in the subiculum of controls and closely surrounding amyloid plaques and dystrophic neurites of the AD model, supports previous suggestions of the presence of the CB1 receptor in microglia. These findings on the correlation between glial reactivity and the CB1 receptor expression in microglial cells and astrocytes, contribute to the understanding of the role of the endocannabinoid system in the pathophysiology of Alzheimer's disease.

Omega-3 Recovers Cannabinoid 1 Receptor Expression in the Adult Mouse Brain after Adolescent Binge Drinking.

Adolescent binge drinking is a social problem with a long-lasting impact on cognitive functions. The cannabinoid type-1 (CB1) receptor of the endocannabinoid system (ECS) is involved in brain synaptic plasticity, cognition and behavior via receptor localization at specific subcellular compartments of the cortical, limbic and motor regions. Alcohol (EtOH) intake affects the ECS, CB1 and their functions. Evidence indicates that binge drinking during adolescence impairs memory via the abrogation of CB1-dependent synaptic plasticity in the hippocampus. However, the impact of EtOH consumption on global CB1 receptor expression in the adult brain is unknown. We studied this using optical density analysis throughout brain regions processed for light microscopy (LM) immunohistotochemistry. CB1 staining decreased significantly in the secondary motor cortex, cerebellum, cingulate cortex, amygdala and nucleus accumbens. Next, as omega-3 (n-3) polyunsaturated fatty acids (PUFAs) rescue synaptic plasticity and improve EtOH-impaired cognition, we investigated whether docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) had any effect on CB1 receptors. N-3 intake during EtOH abstinence restored CB1 immunostaining in the secondary motor cortex, cerebellum and amygdala, and ameliorated receptor density in the cingulate cortex. These results show that n-3 supplementation recovers CB1 receptor expression disrupted by EtOH in distinct brain regions involved in motor functions and cognition.

GLAST versus GFAP as astroglial marker for the subcellular study of cannabinoid CB1 receptors in astrocytes.

The cannabinoid CB1 receptor-mediated functions in astrocytes are highly dependent on the CB1 receptor distribution in these glial cells relative to neuronal sites, particularly at the nearby synapses under normal or pathological conditions. However, the portrait of the CB1 receptor distribution in astroglial compartments remains uncompleted because of the scarce CB1 receptor expression in these cells and the limited identification of astrocytes. The glial fibrillary acidic protein (GFAP) is commonly used as astroglial marker. However, because GFAP is a cytoskeleton protein mostly restricted to the astroglial cell bodies and their main branches, it seems not ideal for the localization of CB1 receptor distribution in astrocytes. Therefore, alternative markers to decipher the actual astroglial CB1 receptors are required. In this work, we have compared the glutamate aspartate transporter (GLAST) versus GFAP for the CB1 receptor localization in astrocytes. We found by immunoelectron microscopy that GLAST reveals almost three-fold astroglial area and four-fold astroglial membranes compared to GFAP. In addition, this better visualization of astrocytes was associated with the detection of 12% of the total CB1 receptor labeling in GLAST-positive astrocytes.

A cannabinoid link between mitochondria and memory.

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.

Adolescent ethanol intake alters cannabinoid type-1 receptor localization in astrocytes of the adult mouse hippocampus.

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.

Cognitive and neurobehavioral benefits of an enriched environment on young adult mice after chronic ethanol consumption during adolescence.

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.

2-Arachidonoylglycerol Reduces Proteoglycans and Enhances Remyelination in a Progressive Model of Demyelination.

The failure to undergo remyelination is a critical impediment to recovery in multiple sclerosis. Chondroitin sulfate proteoglycans (CSPGs) accumulate at demyelinating lesions creating a nonpermissive environment that impairs axon regeneration and remyelination. Here, we reveal a new role for 2-arachidonoylglycerol (2-AG), the major CNS endocannabinoid, in the modulation of CSPGs deposition in a progressive model of multiple sclerosis, the Theiler's murine encephalomyelitis virus-induced demyelinating disease. Treatment with a potent reversible inhibitor of the enzyme monoacylglycerol lipase, which accounts for 85% of the 2-AG degradation in the mouse CNS, modulates neuroinflammation and reduces CSPGs accumulation and astrogliosis around demyelinated lesions in the spinal cord of Theiler's murine encephalomyelitis virus-infected mice. Inhibition of 2-AG hydrolysis augments the number of mature oligodendrocytes and increases MBP, leading to remyelination and functional recovery of mice. Our findings establish a mechanism for 2-AG promotion of remyelination with implications in axonal repair in CNS demyelinating pathologies.SIGNIFICANCE STATEMENT The deposition of chondroitin sulfate proteoglycans contributes to the failure in remyelination associated with multiple sclerosis. Here we unveil a new role for 2-arachidonoylglycerol, the major CNS endocannabinoid, in the modulation of chondroitin sulfate proteoglycan accumulation in Theiler's murine encephalomyelitis virus-induced demyelinating disease. The treatment during the chronic phase with a potent reversible inhibitor of the enzyme monoacylglycerol lipase, which accounts for 85% of the 2-arachidonoylglycerol degradation in the mouse CNS, modulates neuroinflammation and reduces chondroitin sulfate proteoglycan deposition around demyelinated lesions in the spinal cord of Theiler's murine encephalomyelitis virus-infected mice. The increased 2-arachidonoylglycerol tone promotes remyelination in a model of progressive multiple sclerosis ameliorating motor dysfunction.

Localization of the cannabinoid type-1 receptor in subcellular astrocyte compartments of mutant mouse hippocampus.

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.

Subcellular localization of NAPE-PLD and DAGL-α in the ventromedial nucleus of the hypothalamus by a preembedding immunogold method.

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.

Olfactory bulb astrocytes link social transmission of stress to cognitive adaptation in male mice.

Emotions and behavior can be affected by social chemosignals from conspecifics. For instance, olfactory signals from stressed individuals induce stress-like physiological and synaptic changes in naïve partners. Direct stress also alters cognition, but the impact of socially transmitted stress on memory processes is currently unknown. Here we show that exposure to chemosignals produced by stressed individuals is sufficient to impair memory retrieval in unstressed male mice. This requires astrocyte control of information in the olfactory bulb mediated by mitochondria-associated CB1 receptors (mtCB1). Targeted genetic manipulations, in vivo Ca2+ imaging and behavioral analyses reveal that mtCB1-dependent control of mitochondrial Ca2+ dynamics is necessary to process olfactory information from stressed partners and to define their cognitive consequences. Thus, olfactory bulb astrocytes provide a link between social odors and their behavioral meaning.

Target cell-specific plasticity rules of NMDA receptor-mediated synaptic transmission in the hippocampus.

Long-term potentiation and depression of NMDA receptor-mediated synaptic transmission (NMDAR LTP/LTD) can significantly impact synapse function and information transfer in several brain areas. However, the mechanisms that determine the direction of NMDAR plasticity are poorly understood. Here, using physiologically relevant patterns of presynaptic and postsynaptic burst activities, whole-cell patch clamp recordings, 2-photon laser calcium imaging in acute rat hippocampal slices and immunoelectron microscopy, we tested whether distinct calcium dynamics and group I metabotropic glutamate receptor (I-mGluR) subtypes control the sign of NMDAR plasticity. We found that postsynaptic calcium transients (CaTs) in response to hippocampal MF stimulation were significantly larger during the induction of NMDAR-LTP compared to NMDAR-LTD at the MF-to-CA3 pyramidal cell (MF-CA3) synapse. This difference was abolished by pharmacological blockade of mGluR5 and was significantly reduced by depletion of intracellular calcium stores, whereas blocking mGluR1 had no effect on these CaTs. In addition, we discovered that MF to hilar mossy cell (MF-MC) synapses, which share several structural and functional commonalities with MF-CA3 synapses, also undergoes NMDAR plasticity. To our surprise, however, we found that the postsynaptic distribution of I-mGluR subtypes at these two synapses differ, and the same induction protocol that induces NMDAR-LTD at MF-CA3 synapses, only triggered NMDAR-LTP at MF-MC synapses, despite a comparable calcium dynamics. Thus, postsynaptic calcium dynamics alone cannot predict the sign of NMDAR plasticity, indicating that both postsynaptic calcium rise and the relative contribution of I-mGluR subtypes likely determine the learning rules of NMDAR plasticity.

Up-regulation of CB1 cannabinoid receptors located at glutamatergic terminals in the medial prefrontal cortex of the obese Zucker rat.

The present study describes a detailed neuroanatomical distribution map of the cannabinoid type 1 (CB1) receptor, along with the biochemical characterization of the expression and functional coupling to their cognate G i/o proteins in the medial prefrontal cortex (mPCx) of the obese Zucker rats. The CB1 receptor density was higher in the prelimbic (PL) and infralimbic (IL) subregions of the mPCx of obese Zucker rats relative to their lean littermates which was associated with a higher percentage of CB1 receptor immunopositive excitatory presynaptic terminals in PL and IL. Also, a higher expression of CB1 receptors and WIN55,212-2-stimulated [35S]GTPγS binding was observed in the mPCx but not in the neocortex (NCx) and hippocampus of obese rats. Low-frequency stimulation in layers II/III of the mPCx induced CB1 receptor-dependent long-term synaptic plasticity in IL of area obese Zucker but not lean rats. Overall, the elevated 2-AG levels, up-regulation of CB1 receptors, and increased agonist-stimulated [35S]GTPγS binding strongly suggest that hyperactivity of the endocannabinoid signaling takes place at the glutamatergic terminals of the mPCx in the obese Zucker rat. These findings could endorse the importance of the CB1 receptors located in the mPCx in the development of obesity in Zucker rats.

Dopaminergic modulation of endocannabinoid-mediated plasticity at GABAergic synapses in the prefrontal cortex.

Similar to dopamine (DA), cannabinoids strongly influence prefrontal cortical functions, such as working memory, emotional learning, and sensory perception. Although endogenous cannabinoid receptors (CB(1)Rs) are abundantly expressed in the prefrontal cortex (PFC), very little is known about endocannabinoid (eCB) signaling in this brain region. Recent behavioral and electrophysiological evidence has suggested a functional interplay between the dopamine and cannabinoid receptor systems, although the cellular mechanisms underlying this interaction remain to be elucidated. We examined this issue by combining neuroanatomical and electrophysiological techniques in PFC of rats and mice (both genders). Using immunoelectron microscopy, we show that CB(1)Rs and dopamine type 2 receptors (D(2)Rs) colocalize at terminals of symmetrical, presumably GABAergic, synapses in the PFC. Indeed, activation of either receptor can suppress GABA release onto layer 5 pyramidal cells. Furthermore, coactivation of both receptors via repetitive afferent stimulation triggers eCB-mediated long-term depression of inhibitory transmission (I-LTD). This I-LTD is heterosynaptic in nature, requiring glutamate release to activate group I metabotropic glutamate receptors. D(2)Rs most likely facilitate eCB signaling at the presynaptic site as disrupting postsynaptic D(2)R signaling does not diminish I-LTD. Facilitation of eCB-LTD may be one mechanism by which DA modulates neuronal activity in the PFC and regulates PFC-mediated behavior in vivo.

The Absence of the Transient Receptor Potential Vanilloid 1 Directly Impacts on the Expression and Localization of the Endocannabinoid System in the Mouse Hippocampus.

The transient receptor potential vanilloid 1 (TRPV1) is a non-selective ligand-gated cation channel involved in synaptic transmission, plasticity, and brain pathology. In the hippocampal dentate gyrus, TRPV1 localizes to dendritic spines and dendrites postsynaptic to excitatory synapses in the molecular layer (ML). At these same synapses, the cannabinoid CB1 receptor (CB1R) activated by exogenous and endogenous cannabinoids localizes to the presynaptic terminals. Hence, as both receptors are activated by endogenous anandamide, co-localize, and mediate long-term depression of the excitatory synaptic transmission at the medial perforant path (MPP) excitatory synapses though by different mechanisms, it is plausible that they might be exerting a reciprocal influence from their opposite synaptic sites. In this anatomical scenario, we tested whether the absence of TRPV1 affects the endocannabinoid system. The results obtained using biochemical techniques and immunoelectron microscopy in a mouse with the genetic deletion of TRPV1 show that the expression and localization of components of the endocannabinoid system, included CB1R, change upon the constitutive absence of TRPV1. Thus, the expression of fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) drastically increased in TRPV1-/- whole homogenates. Furthermore, CB1R and MAGL decreased and the cannabinoid receptor interacting protein 1a (CRIP1a) increased in TRPV1-/- synaptosomes. Also, CB1R positive excitatory terminals increased, the number of excitatory terminals decreased, and CB1R particles dropped significantly in inhibitory terminals in the dentate ML of TRPV1-/- mice. In the outer 2/3 ML of the TRPV1-/- mutants, the proportion of CB1R particles decreased in dendrites, and increased in excitatory terminals and astrocytes. In the inner 1/3 ML, the proportion of labeling increased in excitatory terminals, neuronal mitochondria, and dendrites. Altogether, these observations indicate the existence of compensatory changes in the endocannabinoid system upon TRPV1 removal, and endorse the importance of the potential functional adaptations derived from the lack of TRPV1 in the mouse brain.

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.

Lack of the Transient Receptor Potential Vanilloid 1 Shifts Cannabinoid-Dependent Excitatory Synaptic Plasticity in the Dentate Gyrus of the Mouse Brain Hippocampus.

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 (10 min at 10 Hz) 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]GTPγS) 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) Gαi1, Gαi2, and Gαi3 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.

Acute Δ9-tetrahydrocannabinol prompts rapid changes in cannabinoid CB1 receptor immunolabeling and subcellular structure in CA1 hippocampus of young adult male mice.

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.

Subcellular specificity of cannabinoid effects in striatonigral circuits.

Recent advances in neuroscience have positioned brain circuits as key units in controlling behavior, implying that their positive or negative modulation necessarily leads to specific behavioral outcomes. However, emerging evidence suggests that the activation or inhibition of specific brain circuits can actually produce multimodal behavioral outcomes. This study shows that activation of a receptor at different subcellular locations in the same neuronal circuit can determine distinct behaviors. Pharmacological activation of type 1 cannabinoid (CB1) receptors in the striatonigral circuit elicits both antinociception and catalepsy in mice. The decrease in nociception depends on the activation of plasma membrane-residing CB1 receptors (pmCB1), leading to the inhibition of cytosolic PKA activity and substance P release. By contrast, mitochondrial-associated CB1 receptors (mtCB1) located at the same terminals mediate cannabinoid-induced catalepsy through the decrease in intra-mitochondrial PKA-dependent cellular respiration and synaptic transmission. Thus, subcellular-specific CB1 receptor signaling within striatonigral circuits determines multimodal control of behavior.