Selective inhibition of monoacylglycerol lipase is associated with passive coping behavior and attenuation of stress-induced dopamine release in the medial prefrontal cortex.

The endocannabinoid system is involved in the regulation of the stress response, but the relative contribution of N-arachidonoylethanolamine (AEA) and 2-arachidonoylglycerol (2-AG) and their mechanisms have to be elucidated. In this study, we compared the effects of the pharmacological inhibition of the two major endocannabinoid-degrading enzymes [fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) for AEA and 2-AG, respectively] on stress-coping [forced swim test (FST) and tail suspension test (TST)] and anxiety-like [elevated-plus maze (EPM) and light-dark test (LDT)] behaviors in wild-type and FAAH knockout mice. In vivo microdialysis estimated the effects of FAAH and MAGL inhibition on dopamine (DA) and serotonin (5-HT) levels in the medial prefrontal cortex (mPFC) during an FST. Mice were treated with PF-3845 (FAAH inhibitor), JZL184 (MAGL inhibitor), JZL195 (dual FAAH/MAGL inhibitor) or vehicle. Our data showed that PF-3845 increased latency to immobility and decreased total immobility time in FST, but no effects were observed in TST compared with vehicle-treated wild-type mice. By contrast, JZL184 decreased latency and increased immobility in TST and FST. JZL195 in wild-type mice and JZL184 in FAAH knockout mice reproduced the same passive coping behaviors as JZL184 in wild-type mice in TST and FST. In the microdialysis experiment, FST was associated with increased DA and 5-HT levels in the mPFC. However, JZL184-treated wild-type mice displayed a significant attenuation of forced swim stress-induced DA release compared with vehicle-treated wild-type mice and PF-3845-treated wild-type mice. Finally, FAAH and/or MAGL inhibitors induced robust and consistent anxiolytic-like effects in EPM and LDT. These results suggested differences between FAAH and MAGL inhibition in stress-coping behaviors. Notably, MAGL inhibition induced a consistent avoidant coping behavior and attenuated the stress-induced mPFC DA response in FST. However, more investigation is needed to elucidate the functional association between DA and 2-AG signaling pathways, and the molecular mechanism in the regulation of passive coping strategies during inescapable stress.

Deficient endocannabinoid signaling in the central amygdala contributes to alcohol dependence-related anxiety-like behavior and excessive alcohol intake.

Negative emotional states that are associated with excessive alcohol intake, particularly anxiety-like states, have been linked to opponent processes in the central nucleus of the amygdala (CeA), affecting stress-related transmitters and monoamines. This study extends these observations to include endocannabinoid signaling in alcohol-dependent animals. Rats and mice were exposed to chronic intermittent alcohol with vapor inhalation or liquid diet to induce dependence. In vivo microdialysis was used to estimate interstitial concentrations of endocannabinoids [N-arachidonoylethanolamine (anandamide; AEA) and 2-arachidonoylglycerol (2-AG)] and amino acids (glutamate and GABA) in rat CeA. Additionally, we evaluated the inhibition of endocannabinoids clearance enzymes [monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase] on anxiety-like behavior and alcohol consumption in alcohol-dependent rats and mice. Results revealed that alcohol dependence produced decreases in baseline 2-AG dialysate levels and increases in baseline levels of glutamate and GABA. Acute alcohol abstinence induced an enhancement of these dependence-induced effects and the levels of 2-AG and GABA were restored upon alcohol re-exposure. Additional studies showed that the increased CeA 2-AG levels induced by restraint stress and alcohol self-administration were blunted in alcohol-dependent rats. Pharmacological studies in rats and mice showed that anxiety-like behavior and alcohol consumption were increased in alcohol-dependent animals, and these behavioral effects were attenuated mainly by MAGL inhibitors [MJN110 (10 and 20 mg/kg) in rats and JZL184 (1 and 3 mg/kg) in mice]. The present results suggest a key role for endocannabinoid signaling in motivational neuroadaptations during alcohol dependence, in which a deficiency in CeA 2-AG signaling in alcohol-dependent animals is linked to stress and excessive alcohol consumption.

Phosphorylation of calcium/calmodulin-dependent protein kinase II in the rat dorsal medial prefrontal cortex is associated with alcohol-induced cognitive inflexibility.

Repeated cycles of alcohol [ethanol (EtOH)] intoxication and withdrawal dysregulate excitatory glutamatergic systems in the brain and induce neuroadaptations in the medial prefrontal cortex (mPFC) that contribute to cognitive dysfunction. The mPFC is composed of subdivisions that are functionally distinct, with dorsal regions facilitating drug-cue associations and ventral regions modulating new learning in the absence of drug. A key modulator of glutamatergic activity is the holoenzyme calcium/calmodulin-dependent protein kinase II (CaMKII) that phosphorylates ionotropic glutamate receptors. Here, we examined the hypothesis that abstinence from chronic intermittent EtOH (CIE) exposure dysregulates CaMKII activity in the mPFC to impair cognitive flexibility. We used an operant model of strategy set shifting in male Long-Evans rats demonstrating reduced susceptibility to trial omissions during performance in a visual cue-guided task versus albino strains. Relative to naïve controls, rats experiencing approximately 10 days of abstinence from CIE vapor exposure demonstrated impaired performance during a procedural shift from visual cue to spatial location discrimination. Phosphorylation of CaMKII subtype α was upregulated in the dorsal, but not ventral mPFC of CIE-exposed rats, and was positively correlated with perseverative-like responding during the set shift. The findings suggest that abstinence from CIE exposure induces an undercurrent of kinase activity (e.g. CaMKII), which may promote aberrant glutamatergic responses in select regions of the mPFC. Given the role of the mPFC in modulating executive control of behavior, we propose that increased CaMKII subtype α activity reflects a dysregulated 'top-down' circuit that interferes with adaptive behavioral performance under changing environmental demands.

Diacylglycerol lipase disinhibits VTA dopamine neurons during chronic nicotine exposure.

Chronic nicotine exposure (CNE) alters synaptic transmission in the ventral tegmental area (VTA) in a manner that enhances dopaminergic signaling and promotes nicotine use. The present experiments identify a correlation between enhanced production of the endogenous cannabinoid 2-arachidonoylglycerol (2-AG) and diminished release of the inhibitory neurotransmitter GABA in the VTA following CNE. To study the functional role of on-demand 2-AG signaling in GABAergic synapses, we used 1,2,3-triazole urea compounds to selectively inhibit 2-AG biosynthesis by diacylglycerol lipase (DAGL). The potency and selectivity of these inhibitors were established in rats in vitro (rat brain proteome), ex vivo (brain slices), and in vivo (intracerebroventricular administration) using activity-based protein profiling and targeted metabolomics analyses. Inhibition of DAGL (2-AG biosynthesis) rescues nicotine-induced VTA GABA signaling following CNE. Conversely, enhancement of 2-AG signaling in naïve rats by inhibiting 2-AG degradation recapitulates the loss of nicotine-induced GABA signaling evident following CNE. DAGL inhibition reduces nicotine self-administration without disrupting operant responding for a nondrug reinforcer or motor activity. Collectively, these findings provide a detailed characterization of selective inhibitors of rat brain DAGL and demonstrate that excessive 2-AG signaling contributes to a loss of inhibitory GABAergic constraint of VTA excitability following CNE.

Persistent effects of chronic Δ9-THC exposure on motor impulsivity in rats.

RATIONALE: In humans, long-term marijuana use is associated with impaired impulse control and attentional capacity, though it has been difficult to distinguish pre-existing cognitive deficits from possible consequences of prolonged marijuana exposure. OBJECTIVE: To evaluate the effects of long-term exposure to Δ9-Tetrahydrocannabinol (Δ9-THC), the primary psychoactive constituent in marijuana, on indices of impulse control and attentional capacity using the rat 5-Choice Serial Reaction Time Task (5-CSRTT). METHODS: Ten 14-day cycles of Δ9-THC dosing and 5-CSRTT testing were employed, each comprised of 5-day Δ9-THC dosing (0.3 or 3 mg/kg b.i.d.) and 5-CSRTT testing during the 9 days of drug abstinence. Subsequent 5-CSRTT testing continued during 5 weeks of protracted abstinence. RESULTS: Dose-dependent increases in motor impulsivity (premature responses) and behavioral disinhibition (perseverative responses) emerged following 5 cycles of Δ9-THC exposure that persisted for the remaining dosing and testing cycles. Δ9-THC-related disruptions in motor impulsivity and behavioral inhibition were most pronounced during cognitively challenging 5-CSRTT sessions incorporating varying novel inter-trial intervals (ITIs), and these disruptions persisted for at least 5 weeks of Δ9-THC abstinence. Δ9-THC-related impairments in attentional capacity (response accuracy) were also evident during variable ITI challenge tests, though these attentional disruptions abated within 3 weeks of Δ9-THC abstinence. CONCLUSIONS: These observations demonstrate that long-term intermittent exposure to clinically meaningful Δ9-THC doses induces persistent impairments in impulse control and attentional function. If present in humans, these disruptions may impact academic and professional performance.

Increased impulsivity in rats as a result of repeated cycles of alcohol intoxication and abstinence.

Impulsivity is a risk factor for alcoholism, and long-term alcohol exposure may further impair impulse control in a manner that propels problematic alcohol use. The present study employed the rat 5-choice serial reaction time task (5-CSRTT) to measure behavioral inhibition and attentional capacity during abstinence from repeated 5-day cycles of alcohol liquid diet consumption. Task performance was not disrupted following the first cycle of alcohol exposure; however, evidence of impaired behavioral inhibition emerged following the third cycle of alcohol exposure. In comparison with controls, alcoholic rats exhibited deficits in inhibitory control during cognitively challenging 5-CSRTT tests employing variable intertrial interval (varITI). This behavioral disruption was not present during early abstinence (3 days) but was evident by 7 days of abstinence and persisted for at least 34 days. Interestingly, renewed alcohol consumption ameliorated these disruptions in impulse control, although deficient behavioral inhibition re-emerged during subsequent abstinence. Indices of increased impulsivity were no longer present in tests conducted after 49 days of abstinence. Alcohol-related impairments in impulse control were not evident in sessions employing highly familiar task parameters regardless of the abstinence period, and control experiments confirmed that performance deficits during the challenge sessions were unlikely to result from alcohol-related disruption in the adaptation to repeated varITI testing. Together, the current findings demonstrate that chronic intermittent alcohol consumption results in decreased behavioral inhibition in rats that is temporally similar to clinical observations of disrupted impulsive control in abstinent alcoholics performing tasks of behavioral inhibition.

The volitional nature of nicotine exposure alters anandamide and oleoylethanolamide levels in the ventral tegmental area.

Cannabinoid-1 receptors (CB(1)) have an important role in nicotine reward and their function is disrupted by chronic nicotine exposure, suggesting nicotine-induced alterations in endocannabinoid (eCB) signaling. However, the effects of nicotine on brain eCB levels have not been rigorously evaluated. Volitional intake of nicotine produces physiological and behavioral effects distinct from forced drug administration, although the mechanisms underlying these effects are not known. This study compared the effects of volitional nicotine self-administration (SA) and forced nicotine exposure (yoked administration (YA)) on levels of eCBs and related neuroactive lipids in the ventral tegmental area (VTA) and other brain regions. Brain lipid levels were indexed both by in vivo microdialysis in the VTA and lipid extractions from brain tissues. Nicotine SA, but not YA, reduced baseline VTA dialysate oleoylethanolamide (OEA) levels relative to nicotine-naïve controls, and increased anandamide (AEA) release during nicotine intake. In contrast, all nicotine exposure paradigms increased VTA dialysate 2-arachidonoyl glycerol (2-AG) levels. Thus, nicotine differentially modulates brain lipid (2-AG, AEA, and OEA) signaling, and these modulations are influenced by the volitional nature of the drug exposure. Corresponding bulk tissue analysis failed to identify these lipid changes. Nicotine exposure had no effect on fatty acid amide hydrolase activity in the VTA, suggesting that changes in AEA and OEA signaling result from alterations in their nicotine-induced biosynthesis. Both CB(1) (by AEA and 2-AG) and non-CB(1) (by OEA) targets can alter the excitability and activity of the dopaminergic neurons in the VTA. Collectively, these findings implicate disrupted lipid signaling in the motivational effects of nicotine.

Acquisition of and withdrawal from cocaine self-administration regulates 5-HT mRNA expression in rat striatum.

This study investigated how different stages of cocaine self-administration in rats affect the expression of two serotonin receptors in dorsal and ventral striatum, the 5-HT(1B) and 5-HT(6) subtypes, which have both been implicated in mediating some aspects of cocaine-related behaviors. In the first experiment, rats were trained to work for saccharin (oral) or cocaine (i.v.) reinforcers. We found that continuous access to cocaine for 23 days did not change the level of 5-HT(1B) mRNA expression compared to control animals receiving saccharin. However, a single cocaine session, given either by self-administration or non-contingently, increased 5-HT(1B) mRNA in dorsal striatum, whereas forced abstinence for two weeks after cocaine reduced 5-HT(1B) mRNA expression in the same subregion. 5-HT(6) mRNA was not changed by any of these treatments. A follow-up experiment investigated the effects of limited versus extended access to cocaine as well as forced abstinence, and we found that 14 days of forced abstinence significantly reduced 5-HT(1B) mRNA throughout the dorsal and ventral striatum compared to no withdrawal. These results suggest that the influence of 5-HT(1B) receptors in striatal projection neurons may be increased during cocaine acquisition and reduced after forced abstinence and may therefore be targets for pharmacological intervention in addiction.