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.

Intermittent ethanol exposure during adolescence impairs cannabinoid type 1 receptor-dependent long-term depression and recognition memory in adult mice.

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 (CB1) 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, CB1 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 CB1 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 CB1 receptor distribution and proportion of immunopositive excitatory synaptic terminals in the medial DGML. Furthermore, there was decreased binding of [35S]guanosine-5*-O-(3-thiotriphosphate) ([35S] 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 CB1 receptor expression, eCB-eLTD, and reduced recognition memory, but that these functional deficits can be restored by treatments that increase endogenous 2-arachidonoylglycerol.

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

The endocannabinoid system modulates synaptic plasticity in the hippocampus, but a link between long-term synaptic plasticity and the type 1 cannabinoid (CB1) receptor at medial perforant path (MPP) synapses remains elusive. Here, immuno-electron microscopy in adult mice showed that ∼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 CB1 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.

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.

Long-Term Effects of Intermittent Adolescent Alcohol Exposure in Male and Female Rats.

Alcohol is a serious public health concern that has a differential impact on individuals depending upon age and sex. Patterns of alcohol consumption have recently changed: heavy episodic drinking-known as binge-drinking-has become most popular among the youth. Herein, we aimed to investigate the consequences of intermittent adolescent alcohol consumption in male and female animals. Thus, Wistar rats were given free access to ethanol (20% in drinking water) or tap water for 2-h sessions during 3 days, and for an additional 4-h session on the 4th day; every week during adolescence, from postnatal day (pnd) 28-52. During this period, animals consumed a moderate amount of alcohol despite blood ethanol concentration (BEC) did not achieve binge-drinking levels. No withdrawal signs were observed: no changes were observed regarding anxiety-like responses in the elevated plus-maze or plasma corticosterone levels (pnd 53-54). In the novel object recognition (NOR) test (pnd 63), a significant deficit in recognition memory was observed in both male and female rats. Western Blot analyses resulted in an increase in the expression of synaptophysin in the frontal cortex (FC) of male and female animals, together with a decrease in the expression of the CB2R in the same brain region. In addition, adolescent alcohol induced, exclusively among females, a decrease in several markers of dopaminergic and serotonergic neurotransmission, in which epigenetic mechanisms, i.e., histone acetylation, might be involved. Taken together, further research is still needed to specifically correlate sex-specific brain and behavioral consequences of adolescent alcohol exposure.

Early maternal deprivation enhances voluntary alcohol intake induced by exposure to stressful events later in life.

In the present study, we aimed to assess the impact of early life stress, in the form of early maternal deprivation (MD, 24 h on postnatal day, pnd, 9), on voluntary alcohol intake in adolescent male and female Wistar rats. During adolescence, from pnd 28 to pnd 50, voluntary ethanol intake (20%, v/v) was investigated using the two-bottle free choice paradigm. To better understand the relationship between stress and alcohol consumption, voluntary alcohol intake was also evaluated following additional stressful events later in life, that is, a week of alcohol cessation and a week of alcohol cessation combined with exposure to restraint stress. Female animals consumed more alcohol than males only after a second episode of alcohol cessation combined with restraint stress. MD did not affect baseline voluntary alcohol intake but increased voluntary alcohol intake after stress exposure, indicating that MD may render animals more vulnerable to the effects of stress on alcohol intake. During adolescence, when animals had free access to alcohol, MD animals showed lower body weight gain but a higher growth rate than control animals. Moreover, the higher growth rate was accompanied by a decrease in food intake, suggesting an altered metabolic regulation in MD animals that may interact with alcohol intake.

Consequences of early life stress on the expression of endocannabinoid-related genes in the rat brain.

The endocannabinoid system is involved in several physiological and pathological states including anxiety, depression, addiction and other neuropsychiatric disorders. Evidence from human and rodent studies suggests that exposure to early life stress may increase the risk of psychopathology later in life. Indeed, maternal deprivation (MD) (24 h at postnatal day 9) in rats induces behavioural alterations associated with depressive-like and psychotic-like symptoms, as well as important changes in the endocannabinoid system. As most neuropsychiatric disorders first appear at adolescence, and show remarkable sexual dimorphisms in their prevalence and severity, in the present study, we analysed the gene expression of the main components of the brain cannabinoid system in adolescent (postnatal day 46) Wistar male and female rats reared under standard conditions or exposed to MD. For this, we analysed, by real-time quantitative PCR, the expression of genes encoding for CB1 and CB2 receptors, TRPV1 and GPR55 (Cnr1, Cnr2a, Cnr2b, Trpv1, and Gpr55), for the major enzymes of synthesis, N-acyl phosphatidyl-ethanolamine phospholipase D (NAPE-PLD) and diacylglycerol lipase (DAGL) (Nape-pld, Dagla and Daglb), and degradation, fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) (Faah, Magl and Cox-2), in specific brain regions, that is, the frontal cortex, ventral and dorsal striatum, dorsal hippocampus and amygdala. In males, MD increased the genetic expression of all the genes studied within the frontal cortex, whereas in females such an increase was observed only in the hippocampus. In conclusion, the endocannabinoid system is sensitive to early life stress at the gene expression level in a sex-dependent and region-dependent manner, and these changes are already evident in the adolescent brain.