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.

COX-2 Inhibition Antagonizes Intra-Accumbens 2-Arachidonoylglycerol-Mediated Reduction in Ethanol Self-Administration in Rats.

BACKGROUND: Ethanol (EtOH) self-administration is particularly sensitive to the modulation of CB1 signaling in the nucleus accumbens (NAc) shell, and EtOH consumption increases extracellular levels of the endogenous cannabinoid CB1 receptor agonist 2-arachidonoyl glycerol (2-AG) in this brain region. Stimulation of CB1 receptor with agonists increases EtOH consumption, suggesting that EtOH-induced increases in 2-AG might sustain motivation for EtOH intake. METHODS: In order to further explore this hypothesis, we analyzed the alterations in operant EtOH self-administration induced by intra-NAc shell infusions of 2-AG itself, the CB1 inverse agonist SR141716A, the 2-AG clearance inhibitor URB602, anandamide, and the cyclooxygenase-2 (COX-2) inhibitor nimesulide. RESULTS: Surprisingly, self-administration of 10% EtOH was dose-dependently reduced by either intra-NAc shell SR141716A or 2-AG infusions. Similar effects were found by intra-NAc shell infusions of URB602, suggesting again a role for accumbal 2-AG on the modulation of EtOH intake. Intra-NAc shell anandamide did not alter EtOH self-administration, pointing to a specific role for 2-AG in the modulation of EtOH self-administration. Finally, the inhibitory effect of intra-NAc shell 2-AG on EtOH intake was significantly reversed by pretreatment with nimesulide, suggesting that oxidative metabolites of 2-AG might mediate these inhibitory effects on operant self-administration. CONCLUSIONS: We propose that 2-AG signaling in the NAc exerts an inhibitory influence on EtOH consumption through a non-CB1 receptor mechanism involving the COX-2 pathway.

Microglia Control Escalation of Drinking in Alcohol-Dependent Mice: Genomic and Synaptic Drivers.

BACKGROUND: Microglia, the primary immune cells of the brain, are implicated in alcohol use disorder. However, it is not known if microglial activation contributes to the transition from alcohol use to alcohol use disorder or is a consequence of alcohol intake. METHODS: We investigated the role of microglia in a mouse model of alcohol dependence using a colony stimulating factor 1 receptor inhibitor (PLX5622) to deplete microglia and a chronic intermittent ethanol vapor two-bottle choice drinking procedure. Additionally, we examined anxiety-like behavior during withdrawal. We then analyzed synaptic neuroadaptations in the central nucleus of the amygdala (CeA) and gene expression changes in the medial prefrontal cortex and CeA from the same animals used for behavioral studies. RESULTS: PLX5622 prevented escalations in voluntary alcohol intake and decreased anxiety-like behavior associated with alcohol dependence. PLX5622 also reversed expression changes in inflammatory-related genes and glutamatergic and GABAergic (gamma-aminobutyric acidergic) genes in the medial prefrontal cortex and CeA. At the cellular level in these animals, microglia depletion reduced inhibitory GABAA and excitatory glutamate receptor-mediated synaptic transmission in the CeA, supporting the hypothesis that microglia regulate dependence-induced changes in neuronal function. CONCLUSIONS: Our multifaceted approach is the first to link microglia to the molecular, cellular, and behavioral changes associated with the development of alcohol dependence, suggesting that microglia may also be critical for the development and progression of alcohol use disorder.

Increased IL-6 expression in astrocytes is associated with emotionality, alterations in central amygdala GABAergic transmission, and excitability during alcohol withdrawal.

Accumulating evidence from preclinical and clinical studies has implicated a role for the cytokine IL-6 in a variety of CNS diseases including anxiety-like and depressive-like behaviors, as well as alcohol use disorder. Here we use homozygous and heterozygous transgenic mice expressing elevated levels of IL-6 in the CNS due to increased astrocyte expression and non-transgenic littermates to examine a role for astrocyte-produced IL-6 in emotionality (response to novelty, anxiety-like, and depressive-like behaviors). Our results from homozygous IL-6 mice in a variety of behavioral tests (light/dark transfer, open field, digging, tail suspension, and forced swim tests) support a role for IL-6 in stress-coping behaviors. Ex vivo electrophysiological studies of neuronal excitability and inhibitory GABAergic synaptic transmission in the central nucleus of the amygdala (CeA) of the homozygous transgenic mice revealed increased inhibitory GABAergic signaling and increased excitability of CeA neurons, suggesting a role for astrocyte produced IL-6 in the amygdala in exploratory drive and depressive-like behavior. Furthermore, studies in the hippocampus of activation/expression of proteins associated with IL-6 signal transduction and inhibitory GABAergic mechanisms support a role for astrocyte produced IL-6 in depressive-like behaviors. Our studies indicate a complex and dose-dependent relationship between IL-6 and behavior and implicate IL-6 induced neuroadaptive changes in neuronal excitability and the inhibitory GABAergic system as important contributors to altered behavior associated with IL-6 expression in the CNS.

IL-1ß expression is increased and regulates GABA transmission following chronic ethanol in mouse central amygdala.

The interleukin-1 system (IL-1) is a prominent pro-inflammatory pathway responsible for the initiation and regulation of immune responses. Human genetic and preclinical studies suggest a critical role for IL-1ß signaling in ethanol drinking and dependence, but little is known about the effects of chronic ethanol on the IL-1 system in addiction-related brain regions such as the central amygdala (CeA). In this study, we generated naïve, non-dependent (Non-Dep) and dependent (Dep) male mice using a paradigm of chronic-intermittent ethanol vapor exposure interspersed with two-bottle choice to examine 1) the expression of IL-1ß, 2) the role of the IL-1 system on GABAergic transmission, and 3) the potential interaction with the acute effects of ethanol in the CeA. Immunohistochemistry with confocal microscopy was used to assess expression of IL-1ß in microglia and neurons in the CeA, and whole-cell patch clamp recordings were obtained from CeA neurons to measure the effects of IL-1ß (50 ng/ml) or the endogenous IL-1 receptor antagonist (IL-1ra; 100 ng/ml) on action potential-dependent spontaneous inhibitory postsynaptic currents (sIPSCs). Overall, we found that IL-1ß expression is significantly increased in microglia and neurons of Dep compared to Non-Dep and naïve mice, IL-1ß and IL-1ra bi-directionally modulate GABA transmission through both pre- and postsynaptic mechanisms in all three groups, and IL-1ß and IL-1ra do not alter the facilitation of GABA release induced by acute ethanol. These data suggest that while ethanol dependence induces a neuroimmune response in the CeA, as indicated by increased IL-1ß expression, this does not significantly alter the neuromodulatory role of IL-1ß on synaptic transmission.

Ethanol-induced alterations in endocannabinoids and relevant neurotransmitters in the nucleus accumbens of fatty acid amide hydrolase knockout mice.

Deletion of fatty acid amide hydrolase (FAAH), enzyme responsible for degrading endocannabinoids, increases alcohol consumption and preference. However, there is a lack of data on neurochemical events in mice exposed to alcohol in the absence of FAAH. Extracellular levels of endocannabinoids and relevant neurotransmitters were measured by in vivo microdialysis in the nucleus accumbens (NAc) of FAAH knockout (KO) and wild-type (WT) mice during an ethanol (EtOH; 2 g/kg, ip) challenge in EtOH-naive and repeated (r) EtOH-treated mice. In both genotypes, EtOH treatment caused no changes in baseline endocannabinoid levels, although FAAH KO mice displayed higher baseline N-arachidonoylethanolamine levels than WT mice. EtOH challenge caused a sustained increase in 2-arachidonoylglycerol (2-AG) levels in EtOH-naive WT mice but not in FAAH KO mice. In contrast, 2-AG levels were decreased following EtOH challenge in (r)EtOH-treated mice in both genotypes. Whereas (r)EtOH-treated mice showed higher baseline dopamine and serotonin levels than EtOH-naive mice in WT mice, these differences were attenuated in FAAH KO mice. Significant differences in baseline γ-aminobutyric acid (GABA) and glutamate levels by EtOH history were observed in WT mice but not in FAAH KO mice. Moreover, opposed effects on glutamate response were observed after EtOH challenge in EtOH-naive and (r)EtOH-treated FAAH KO mice. Finally, FAAH deletion failed to show EtOH-induced locomotion sensitivity. These data provide evidence of a potential influence of 2-AG in the neurochemical response to EtOH exposure in the NAc.

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.

Fatty acid amide hydrolase (FAAH) inactivation confers enhanced sensitivity to nicotine-induced dopamine release in the mouse nucleus accumbens.

Nicotine exerts its rewarding effects by promoting an increase in dopamine (DA) release in the nucleus accumbens (NAc), and this process is influenced by the endocannabinoid system. Fatty acid amide hydrolase (FAAH) is the main enzyme responsible for the degradation of the endocannabinoid anandamide and other non-cannabinoid N-acylethanolamines. Previous research has reported that both genetic deletion and pharmacological inhibition of FAAH enhance nicotine-induced conditioned place preference at low doses. We conducted a microdialysis study to characterize nicotine-induced changes in DA and serotonin (5-HT) levels in the NAc of FAAH knockout (KO) mice using a conditioned place preference-like paradigm with three nicotine doses (0.1, 1 and 10 mg/kg, s.c.). Additionally, the effects of the selective FAAH inhibitor PF-3845 (10 mg/kg, i.p.) were also examined. Our data indicated that compared with wild-type mice, genetic deletion of FAAH selectively enhanced the effect of low-dose nicotine on DA release (p < 0.001) and resulted in a strong post-nicotine elevation in DA levels (p < 0.01). However, there were no differences between the genotypes at higher doses. Furthermore, FAAH KO mice displayed a moderate enhancement of the effect of low-dose nicotine on NAc 5-HT release (p < 0.05), with no differences between the genotypes at higher doses. Compared with vehicle-pretreated mice, mice pretreated with PF-3845 displayed an enhancement of the effect of low-dose nicotine on NAc DA release (p < 0.001), which resulted in a sustained increase in DA levels (p < 0.05). Similar to FAAH KO mice, PF-3845-pretreated mice displayed a moderate enhancement of the effect of low-dose nicotine on NAc 5-HT release (p < 0.01). These observations in mice suggest that enhanced nicotine-induced NAc DA release might contribute to increased sensitivity to the conditioned rewarding effects of low-dose nicotine following FAAH inhibition, which has been previously reported. Future studies combining behavioral and neurochemical approaches are needed to elucidate the precise mechanism of these effects.

Effects of Withdrawal from Chronic Intermittent Ethanol Exposure on Sleep Characteristics of Female and Male Mice.

BACKGROUND: Sleep disruptions are an important consequence of alcohol use disorders. There is a dearth of preclinical studies examining sex differences in sleep patterns associated with ethanol (EtOH) dependence despite documented sex differences in alcohol-related behaviors and withdrawal symptoms. The purpose of this study was to investigate the effects of chronic intermittent EtOH on sleep characteristics in female and male mice. METHODS: Female and male C57BL6/J mice had access to EtOH/water 2-bottle choice (2BC) 2 h/d for 3 weeks followed by exposure to EtOH vapor (vapor-2BC) or air for 5 cycles of 4 days. An additional group never experienced EtOH (naïve). Mice were implanted with electroencephalographic (EEG) electrodes, and vigilance states were recorded across 24 hours on the fourth day of withdrawal. The amounts of wakefulness, slow-wave sleep (SWS), and rapid eye movement sleep were calculated, and spectral analysis was performed by fast Fourier transformation. RESULTS: Overall, vapor-2BC mice showed a decrease in the amount of SWS 4 days into withdrawal as well as a decrease in the power density of slow waves, indicating disruptions in both the amount and quality of sleep in EtOH-dependent mice. This was associated with a decrease in duration and an increase in number of SWS episodes in males and an increase in latency to sleep in females. CONCLUSIONS: Our results revealed overall deficits in sleep regulation in EtOH-dependent mice of both sexes. Female mice appeared to be more affected with regard to the triggering of sleep, while male mice appeared more sensitive to disruptions in the maintenance of sleep.

Constitutive Increases in Amygdalar Corticotropin-Releasing Factor and Fatty Acid Amide Hydrolase Drive an Anxious Phenotype.

BACKGROUND: Corticotropin-releasing factor (CRF) mediates anxiogenic responses by activating CRF type 1 (CRF1) receptors in limbic brain regions. Anxiety is further modulated by the endogenous cannabinoid (eCB) system that attenuates the synaptic effects of stress. In the amygdala, acute stress activates the enzymatic clearance of the eCB N-arachidonoylethanolamine via fatty acid amide hydrolase (FAAH), although it is unclear whether chronic dysregulation of CRF systems induces maladaptive changes in amygdalar eCB signaling. Here, we used genetically selected Marchigian Sardinian P (msP) rats carrying an innate overexpression of CRF1 receptors to study the role of constitutive upregulation in CRF systems on amygdalar eCB function and persistent anxiety-like effects. METHODS: We applied behavioral, pharmacological, and biochemical methods to broadly characterize anxiety-like behaviors and amygdalar eCB clearance enzymes in msP versus nonselected Wistar rats. Subsequent studies examined the influence of dysregulated CRF and FAAH systems in altering excitatory transmission in the central amygdala (CeA). RESULTS: msPs display an anxious phenotype accompanied by elevations in amygdalar FAAH activity and reduced dialysate N-arachidonoylethanolamine levels in the CeA. Elevations in CRF-CRF1 signaling dysregulate FAAH activity, and this genotypic difference is normalized with pharmacological blockade of CRF1 receptors. msPs also exhibit elevated baseline glutamatergic transmission in the CeA, and dysregulated CRF-FAAH facilitates stress-induced increases in glutamatergic activity. Treatment with an FAAH inhibitor relieves sensitized glutamatergic responses in msPs and attenuates the anxiety-like phenotype. CONCLUSIONS: Pathological anxiety and stress hypersensitivity are driven by constitutive increases in CRF1 signaling that dysregulate N-arachidonoylethanolamine signaling mechanisms and reduce neuronal inhibitory control of CeA glutamatergic synapses.

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.

Regional Influence of Cannabinoid CB1 Receptors in the Regulation of Ethanol Self-Administration by Wistar Rats.

Substantial evidence suggests a facilitatory influence of cannabinoid CB1 receptors in the modulation of ethanol consumption by rodents. Studies performed in rats selectively bred for high alcohol preference point to an involvement of CB1 receptors in the nucleus accumbens (NAC), ventral tegmental area (VTA) and medial prefrontal cortex (mPFC) in the modulation ethanol self-administration. However, the neural mechanisms through which CB1 receptors regulate ethanol intake in out-bred Wistar rats have not been investigated. The present study evaluated alterations in ethanol self-administration induced by localized infusions of the CB1 receptor antagonist SR141716A (0, 1 and 3 µg/side) into the NAC, anterior and posterior VTA and mPFC. Separate groups of Wistar rats were trained to operantly respond for an oral ethanol solution and prepared with bilateral injection cannulae aimed at each brain region. Results revealed significant decreases in ethanol intake following intra-NAC SR141716A administration, consistent with our prior observation of ethanol-induced increases extracellular 2-arachidonoyl glycerol (2-AG) in this brain region. We also observed a significant dose-dependent reduction in ethanol intake following SR141716A administration into the posterior, but not anterior VTA, consistent with evidence of a specific involvement of the posterior VTA in the regulation of ethanol intake. Ethanol consumption was unaltered following intra-mPFC SR141716A administration and ethanol self-administration did not induce robust changes in anandamide or 2-AG levels in mPFC microdialysates. These findings implicate an involvement of CB1 receptors in the NAC and posterior VTA, but not anterior VTA and mPFC in the regulation of ethanol self-administration behavior by outbred Wistar rats.

Attenuation of cue-induced heroin-seeking behavior by cannabinoid CB1 antagonist infusions into the nucleus accumbens core and prefrontal cortex, but not basolateral amygdala.

As with other drugs of abuse, heroin use is characterized by a high incidence of relapse following detoxification that can be triggered by exposure to conditioned stimuli previously associated with drug availability. Recent findings suggest that cannabinoid CB(1) receptors modulate the motivational properties of heroin-conditioned stimuli that induce relapse behavior. However, the neural substrates through which CB(1) receptors modulate cue-induced heroin seeking have not been elucidated. In this study, we evaluated alterations in cue-induced reinstatement of heroin-seeking behavior produced by infusions of the CB(1) receptor antagonist SR 141716A (0, 0.3 and 3 microg per side) delivered into the prefrontal cortex (PFC), nucleus accumbens (NAC), and basolateral amygdala (BLA) of rats. Results show that following extinction of operant behavior the presentation of a discriminative stimulus conditioned to heroin availability reinstated nonreinforced lever pressing to levels comparable to preextinction levels. Intra-PFC SR 141716A dose-dependently reduced cue-induced reinstatement of heroin seeking, with a significant reduction following the 3 microg per side dose. In the NAC, both SR 141716A doses induced a significant reduction in cue-induced reinstatement, with the highest dose completely blocking the effect of the cue. In contrast, intra-BLA SR 141716A did not alter cue-induced reinstatement of responding while systemic administration of this antagonist (3 mg/kg, i.p.) significantly blocked cue-induced reinstatement in all three-placement groups (BLA, PFC, and NAC). These findings provide new insights into the neural mechanisms through which CB(1) receptors modulate the motivational properties of heroin-associated cues inducing relapse.

Specific alterations of extracellular endocannabinoid levels in the nucleus accumbens by ethanol, heroin, and cocaine self-administration.

Ethanol and opiate self-administration are sensitive to manipulations of cannabinoid CB1 receptor function and, from this, a role for the endogenous cannabinoid system in the modulation of drug reward has been hypothesized. However, direct in vivo evidence of drug-induced alterations in brain endocannabinoid (eCB) formation has been lacking. To address this issue, we explored the effect of drug self-administration on interstitial eCB levels in the nucleus accumbens (NAc) shell using in vivo microdialysis. Ethanol, heroin, and cocaine were compared because the rewarding properties of ethanol and heroin are reduced by CB1 receptor inactivation, whereas cocaine reward is less sensitive to these manipulations. Ethanol self-administration significantly increased dialysate 2-arachidonoylglycerol (2-AG) levels with no concomitant change in dialysate anandamide (AEA) concentrations. Conversely, heroin self-administration significantly increased dialysate AEA levels, and induced a subtle but significant decrease in dialysate 2-AG levels. In each case, the relative change in dialysate eCB content was significantly correlated with the amount of drug consumed. In contrast, cocaine self-administration did not alter dialysate levels of either AEA or 2-AG. Local infusion of the CB1 antagonist SR 141716A into the NAc significantly reduced ethanol, but not cocaine, self-administration. Together with our previous observation that intra-NAc SR 141716A reduces heroin self-administration, these data provide novel in vivo support for an eCB involvement in the motivational properties of ethanol and heroin but not cocaine. Furthermore, the selective effects of ethanol and heroin on interstitial 2-AG and AEA provide new insight into the distinct neurochemical profiles produced by these two abused substances.

Biphasic alterations in serotonin-1B (5-HT1B) receptor function during abstinence from extended cocaine self-administration.

Alterations in 5-HT1B receptor function during cocaine abstinence were evaluated in rats given either limited- or extended access (LA and EA, respectively) to cocaine self-administration. The locomotor response to the 5-HT1B/1A agonist RU24969 was significantly reduced in cocaine-experienced animals relative to cocaine-naïve controls following 6 h of abstinence but became sensitized over the subsequent 14 days of abstinence. Both the early phase subsensitivity and later phase supersensivity to RU 24969-induced activity were greater in EA versus LA animals. Intra-nucleus accumbens administration of the 5-HT1B agonist CP 93, 129 produced significantly greater increases in dialysate dopamine levels in EA versus control animals following 14 days of abstinence. However, there was no difference between EA and cocaine-naïve control animals in the augmentation of cocaine-induced increases in nucleus accumbens DA produced by intra-VTA CP 93, 129. Collectively these findings demonstrate that 5-HT1B receptor function is persistently altered by cocaine self-administration.