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.

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.

Precise localization of the voltage-gated potassium channel subunits Kv3.1b and Kv3.3 revealed in the molecular layer of the rat cerebellar cortex by a pre-embedding immunogold method.

A proper motor activity relies on a correct cerebellar function. The Kv3.1 and Kv3.3 voltage-gated potassium channels are key proteins involved in cerebellar function and dysfunction, as the lack of these causes severe motor deficits. Both channel subunits are coexpressed in granule cells and are rapidly activated at relatively positive potentials to support the generation of fast action potentials. However, the contribution of each subunit to the molecular architecture of the parallel fibers, the granule cell axons, is so far unknown. The goal of this study was to elucidate the relative distribution of Kv3.1b and Kv3.3 in specific compartments of the rat parallel fibers by using a pre-embedding immunocytochemical method for electron microscopy. Numerous Kv3.1b and Kv3.3 silver-intensified gold particles were associated with membranes of parallel fiber synaptic terminals and their intervaricose segments. Kv3.1b was found in about 85% of parallel fiber synaptic terminals and in about 47% of their intervaricose portions. However, only 28% of intervaricosities and 23% of parallel fiber presynaptic boutons were Kv3.3 immunopositive. The analysis also revealed that 54% of Purkinje cell dendritic spines localized Kv3.3. Although both potassium channel subunits share localization in the same presynaptic parallel fiber compartments, the present results with the method used indicate that there are a higher percentage of parallel fibers labeled for Kv3.1b than for Kv3.3, and that the labeling intensity for each subunit is higher in specific subcompartments analyzed than in others.

Deletion of the cannabinoid CB1 receptor impacts on the ultrastructure of the cerebellar parallel fiber-Purkinje cell synapses.

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.

GABAergic and cortical and subcortical glutamatergic axon terminals contain CB1 cannabinoid receptors in the ventromedial nucleus of the hypothalamus.

BACKGROUND: Type-1 cannabinoid receptors (CB(1)R) are enriched in the hypothalamus, particularly in the ventromedial hypothalamic nucleus (VMH) that participates in homeostatic and behavioral functions including food intake. Although CB(1)R activation modulates excitatory and inhibitory synaptic transmission in the brain, CB(1)R contribution to the molecular architecture of the excitatory and inhibitory synaptic terminals in the VMH is not known. Therefore, the aim of this study was to investigate the precise subcellular distribution of CB(1)R in the VMH to better understand the modulation exerted by the endocannabinoid system on the complex brain circuitries converging into this nucleus. METHODOLOGY/PRINCIPAL FINDINGS: Light and electron microscopy techniques were used to analyze CB(1)R distribution in the VMH of CB(1)R-WT, CB(1)R-KO and conditional mutant mice bearing a selective deletion of CB(1)R in cortical glutamatergic (Glu-CB(1)R-KO) or GABAergic neurons (GABA-CB(1)R-KO). At light microscopy, CB(1)R immunolabeling was observed in the VMH of CB(1)R-WT and Glu-CB(1)R-KO animals, being remarkably reduced in GABA-CB(1)R-KO mice. In the electron microscope, CB(1)R appeared in membranes of both glutamatergic and GABAergic terminals/preterminals. There was no significant difference in the percentage of CB(1)R immunopositive profiles and CB(1)R density in terminals making asymmetric or symmetric synapses in CB(1)R-WT mice. Furthermore, the proportion of CB(1)R immunopositive terminals/preterminals in CB(1)R-WT and Glu-CB(1)R-KO mice was reduced in GABA-CB(1)R-KO mutants. CB(1)R density was similar in all animal conditions. Finally, the percentage of CB(1)R labeled boutons making asymmetric synapses slightly decreased in Glu-CB(1)R-KO mutants relative to CB(1)R-WT mice, indicating that CB(1)R was distributed in cortical and subcortical excitatory synaptic terminals. CONCLUSIONS/SIGNIFICANCE: Our anatomical results support the idea that the VMH is a relevant hub candidate in the endocannabinoid-mediated modulation of the excitatory and inhibitory neurotransmission of cortical and subcortical pathways regulating essential hypothalamic functions for the individual's survival such as the feeding behavior.