Pharmacological blockage of NOP receptors decreases ventral tegmental area dopamine neuronal activity through GABAB receptor-mediated mechanism.

The Nociceptin/Orphanin FQ (N/OFQ) peptide and its receptor NOP are highly expressed within several regions of the mesolimbic system, including the ventral tegmental area (VTA). Evidence indicates that the N/OFQ-NOP receptor system is involved in reward processing and historically it has been proposed that activation of NOP receptors attenuates the motivation for substances of abuse. However, recent findings demonstrated that drug self-administration and relapse to drug-seeking are also attenuated after administration of NOP receptor antagonists. Here, to shed light on the mechanisms through which NOP receptor blockers modulate these processes, we utilized ex vivo patch-clamp recordings to investigate the effect of the selective NOP receptor antagonist LY2817412 on VTA dopaminergic (DA) function in male rats. Results showed that, similar to the endogenous NOP receptor agonist N/OFQ, LY2817412 reduced the spontaneous basal firing discharge of VTA DA neurons. Consistently, we found that NOP receptors are expressed both in VTA DA and GABA cells and that LY2817412 slice perfusion increased GABA release onto VTA DA cells. Finally, in the attempt to dissect the role of postsynaptic and presynaptic NOP receptors, we tested the effect of N/OFQ and LY2817412 in the presence of GABA receptors blockers. Results showed that the effect of LY2817412 was abolished following pretreatment with GABABR, but not GABAAR, blockers. Conversely, inhibition of DA neuronal activity by N/OFQ was unaffected by blockade of GABA receptors. Altogether, these results suggest that both NOP receptor agonists and antagonists can decrease VTA DA neuronal activity, but through distinct mechanisms of action. The effect of NOP receptor antagonists occurs through a GABABR-mediated mechanism while NOP receptor agonists seem to act via a direct effect on VTA DA neurons.

Different mechanisms underlie compulsive alcohol self-administration in male and female rats.

BACKGROUND: Sex is an important factor in the progression and treatment of alcohol addiction, and therapeutic approaches may have to be tailored to potential sex differences. This highlights the importance of understanding sex differences in behaviors that reflect key elements of clinical alcohol addiction, such as continued use despite negative consequences ("compulsive use"). Studies in experimental animals can help provide an understanding of the role sex plays to influence these behaviors. METHODS: Large populations of genetically heterogeneous male and female Wistar rats were tested in an established model of compulsive alcohol self-administration, operationalized as alcohol responding despite contingent foot shock punishment. We also tested baseline (fixed ratio, unpunished) operant alcohol self-administration, motivation to self-administer alcohol (progressive ratio), and temporal discounting for alcohol reward. In search of predictors of compulsivity, animals were screened for novelty-induced place preference, anxiety-like behavior, pain sensitivity and corticosterone levels. The estrous cycle was monitored throughout the study. RESULTS: Unpunished self-administration of alcohol did not differ between males and females when alcohol intake was corrected for body weight. Overall, females showed higher levels of compulsive responding for alcohol. Compulsive response rates showed bimodal distributions in male but not in female rats when intermediate shock intensities were used (0.2 and 0.25 mA); at higher shock intensities, responding was uniformly suppressed in both males and females. We also found less steep discounting in females when alcohol was devalued by delaying its delivery. Males exhibited a stronger motivation to obtain alcohol under unpunished conditions, while females showed higher corticosterone levels at baseline. Factor analysis showed that an underlying dimension related to stress and pain predicted compulsivity in females, while compulsivity in males was predicted by a reward factor. We did not find differences in alcohol-related behaviors throughout the various stages of the estrous cycle. CONCLUSIONS: Our results suggest that mechanisms promoting compulsivity, a key feature of alcohol addiction, likely differ between males and females. This underscores the importance of considering sex as a biological variable in both preclinical and clinical research, and has potential treatment implications in alcohol addiction.

Sex plays an important role in the progression and treatment of alcohol addiction. While men show a higher prevalence of alcohol addiction, women are more susceptible to the adverse effects of excessive alcohol consumption. Additionally, women often rely on heavy drinking as a maladaptive coping mechanism to alleviate stress and anxiety, driven by negative affect. On the other hand, men are more likely to report heavy drinking and relapse in response to positive emotions and social influences. These sex-based differences underline the importance of understanding how vulnerability to alcohol addiction and its treatment varies in males and females.We used genetically heterogeneous rats to explore the behavioral traits that contribute to compulsivity, a key clinical feature of alcohol addiction. We found that motivation to self-administer alcohol was higher in males, while females showed higher compulsive alcohol self-administration. In males, motivation to self-administer alcohol showed a significant correlation with compulsivity, while in females compulsivity was predicted by higher basal corticosterone levels.These findings underlie the importance of sex-specific factors in compulsive alcohol self-administration, with potential prevention and treatment implications in alcohol addiction.

Activation of GABAB receptors in central amygdala attenuates activity of PKCδ + neurons and suppresses punishment-resistant alcohol self-administration in rats.

Alcohol use despite negative consequences is a core phenomenon of alcohol addiction. We recently used alcohol self-administration that is resistant to footshock punishment as a model of this behavior, and found that activity of PKCδ + GABAergic neurons in the central amygdala (CeA) is a determinant of individual susceptibility for punishment resistance. In the present study, we examined whether activation of GABAB receptors in CeA can attenuate the activity of PKCδ + neurons in this region, and whether this will result in suppression of punishment- resistant alcohol self-administration in the minority of rats that show this behavior. Systemic administration of the clinically approved GABAB agonist baclofen (1 and 3 mg/kg) dose- dependently reduced punishment-resistant alcohol self-administration. Bilateral microinjections of baclofen into CeA (64 ng in 0.3 µl/side) reduced the activity of PKCδ + neurons, as measured by Fos expression. This manipulation also selectively suppressed punished alcohol self-administration in punishment-resistant rats. Expression analysis indicated that virtually all CeA PKCδ + neurons express the GABAB receptor. Using in vitro electrophysiology, we found that baclofen induced hyperpolarization of CeA neurons, reducing their firing rate in response to depolarizing current injections. Together, our findings provide a potential mechanism that contributes to the clinical efficacy of baclofen in alcohol addiction. Therapeutic use of baclofen itself is limited by problems of tolerance and need for dose escalation. Our findings support a mechanistic rationale for developing novel, improved alcohol addiction medications that target GABAB receptors, and that lack these limitations, such as e.g., GABAB positive allosteric modulators (PAM:s).

Abstinence-Induced Nicotine Seeking Relays on a Persistent Hypoglutamatergic State within the Amygdalo-Striatal Neurocircuitry.

Nicotine robustly sustains smoking behavior by acting as a primary reinforcer and by enhancing the incentive salience of the nicotine-associated stimuli. The motivational effects produced by environmental cues associated with nicotine delivery can progressively manifest during abstinence resulting in reinstatement of nicotine seeking. However, how the activity in reward neuronal circuits is transformed during abstinence-induced nicotine seeking is not yet fully understood. In here we used a contingent nicotine and saline control self-administration model to disentangle the contribution of cue-elicited seeking responding for nicotine after drug abstinence in male Wistar rats. Using ex vivo electrophysiological recordings and a network analysis approach, we defined temporal and brain-region specific amygdalo-striatal glutamatergic alterations that occur during nicotine abstinence. The results from this study provide critical evidence indicating a persistent hypoglutamatergic state within the amygdalo-striatal neurocircuitry over protracted nicotine abstinence. During abstinence-induced nicotine seeking, electrophysiological recordings showed progressive neuroadaptations in dorsal and ventral striatum already at 14-d abstinence while neuroadaptations in subregions of the amygdala emerged only after 28-d abstinence. The observed neuroadaptations pointed to a brain network involving the amygdala and the dorsolateral striatum (DLS) to be implied in cue-induced reinstatement of nicotine seeking. Together these data suggest long-lasting neuroadaptations that might reflect neuroplastic changes responsible to abstinence-induced nicotine craving. Neurophysiological transformations were detected within a time window that allows therapeutic intervention advancing clinical development of preventive strategies in nicotine addiction.

Epigenetic Dysregulation in Alcohol-Associated Behaviors: Preclinical and Clinical Evidence.

Alcohol use disorder (AUD) is characterized by loss of control over intake and drinking despite harmful consequences. At a molecular level, AUD is associated with long-term neuroadaptations in key brain regions that are involved in reward processing and decision-making. Over the last decades, a great effort has been made to understand the neurobiological basis underlying AUD. Epigenetic mechanisms have emerged as an important mechanism in the regulation of long-term alcohol-induced gene expression changes. Here, we review the literature supporting a role for epigenetic processes in AUD. We particularly focused on the three most studied epigenetic mechanisms: DNA methylation, Histone modification and non-coding RNAs. Clinical studies indicate an association between AUD and DNA methylation both at the gene and global levels. Using behavioral paradigms that mimic some of the characteristics of AUD, preclinical studies demonstrate that changes in epigenetic mechanisms can functionally impact alcohol-associated behaviors. While many studies support a therapeutic potential for targeting epigenetic enzymes, more research is needed to fully understand their role in AUD. Identification of brain circuits underlying alcohol-associated behaviors has made major advances in recent years. However, there are very few studies that investigate how epigenetic mechanisms can affect these circuits or impact the neuronal ensembles that promote alcohol-associated behaviors. Studies that focus on the role of circuit-specific and cell-specific epigenetic changes for clinically relevant alcohol behaviors may provide new insights on the functional role of epigenetic processes in AUD.

An epigenetic mechanism for over-consolidation of fear memories.

Excessive fear is a hallmark of anxiety disorders, a major cause of disease burden worldwide. Substantial evidence supports a role of prefrontal cortex-amygdala circuits in the regulation of fear and anxiety, but the molecular mechanisms that regulate their activity remain poorly understood. Here, we show that downregulation of the histone methyltransferase PRDM2 in the dorsomedial prefrontal cortex enhances fear expression by modulating fear memory consolidation. We further show that Prdm2 knock-down (KD) in neurons that project from the dorsomedial prefrontal cortex to the basolateral amygdala (dmPFC-BLA) promotes increased fear expression. Prdm2 KD in the dmPFC-BLA circuit also resulted in increased expression of genes involved in synaptogenesis, suggesting that Prdm2 KD modulates consolidation of conditioned fear by modifying synaptic strength at dmPFC-BLA projection targets. Consistent with an enhanced synaptic efficacy, we found that dmPFC Prdm2 KD increased glutamatergic release probability in the BLA and increased the activity of BLA neurons in response to fear-associated cues. Together, our findings provide a new molecular mechanism for excessive fear responses, wherein PRDM2 modulates the dmPFC -BLA circuit through specific transcriptomic changes.

A Role for Neuropeptide S in Alcohol and Cocaine Seeking.

The neuropeptide S (NPS) is the endogenous ligand of the NPS receptor (NPSR). The NPSR is widely expressed in brain regions that process emotional and affective behavior. NPS possesses a unique physio-pharmacological profile, being anxiolytic and promoting arousal at the same time. Intracerebroventricular NPS decreased alcohol consumption in alcohol-preferring rats with no effect in non-preferring control animals. This outcome is most probably linked to the anxiolytic properties of NPS, since alcohol preference is often associated with high levels of basal anxiety and intense stress-reactivity. In addition, NPSR mRNA was overexpressed during ethanol withdrawal and the anxiolytic-like effects of NPS were increased in rodents with a history of alcohol dependence. In line with these preclinical findings, a polymorphism of the NPSR gene was associated with anxiety traits contributing to alcohol use disorders in humans. NPS also potentiated the reinstatement of cocaine and ethanol seeking induced by drug-paired environmental stimuli and the blockade of NPSR reduced reinstatement of cocaine-seeking. Altogether, the work conducted so far indicates the NPS/NPSR system as a potential target to develop new treatments for alcohol and cocaine abuse. An NPSR agonist would be indicated to help individuals to quit alcohol consumption and to alleviate withdrawal syndrome, while NPSR antagonists would be indicated to prevent relapse to alcohol- and cocaine-seeking behavior.

Genetic deletion or pharmacological blockade of nociceptin/orphanin FQ receptors in the ventral tegmental area attenuates nicotine-motivated behaviour.

BACKGROUND AND PURPOSE: The nociceptin/orphanin FQ (N/OFQ)-nociceptin opioid-like peptide (NOP) receptor system is widely distributed in the brain and pharmacological activation of this system revealed therapeutic potential in animal models of substance use disorder. Studies also showed that genetic deletion or pharmacological blockade of NOP receptors confer resistance to the development of alcohol abuse. Here, we have used a genetic and pharmacological approach to evaluate the therapeutic potential of NOP antagonism in smoking cessation. EXPERIMENTAL APPROACH: Constitutive NOP receptor knockout rats (NOP-/- ) and their wild-type counterparts (NOP+/+ ) were tested over a range of behaviours to characterize their motivation for nicotine. We next explored the effects of systemic administration of the NOP receptor antagonist LY2817412 (1.0 & 3.0 mg·kg-1 ) on nicotine self-administration. NOP receptor blockade was further evaluated at the brain circuitry level, by microinjecting LY2817412 (3.0 & 6.0 µg·µl-1 ) into the ventral tegmental area (VTA), nucleus accumbens (NAc) and central amygdala (CeA). KEY RESULTS: Genetic NOP receptor deletion resulted in decreased nicotine intake, decreased motivation to self-administer and attenuation of cue-induced nicotine reinstatement. LY2817412 reduced nicotine intake in NOP+/+ but not in NOP-/- rats, confirming that its effect is mediated by inhibition of NOP transmission. Finally, injection of LY2817412 into the VTA but not into the NAc or CeA decreased nicotine self-administration. CONCLUSIONS AND IMPLICATIONS: These findings indicate that inhibition of NOP transmission attenuates the motivation for nicotine through mechanisms involving the VTA and suggest that NOP receptor antagonism may represent a potential treatment for smoking cessation.

A neural substrate of compulsive alcohol use.

Alcohol intake remains controlled in a majority of users but becomes "compulsive," i.e., continues despite adverse consequences, in a minority who develop alcohol addiction. Here, using a footshock-punished alcohol self-administration procedure, we screened a large population of outbred rats to identify those showing compulsivity operationalized as punishment-resistant self-administration. Using unsupervised clustering, we found that this behavior emerged as a stable trait in a subpopulation of rats and was associated with activity of a brain network that included central nucleus of the amygdala (CeA). Activity of PKCδ+ inhibitory neurons in the lateral subdivision of CeA (CeL) accounted for ~75% of variance in punishment-resistant alcohol taking. Activity-dependent tagging, followed by chemogenetic inhibition of neurons activated during punishment-resistant self-administration, suppressed alcohol taking, as did a virally mediated shRNA knockdown of PKCδ in CeA. These findings identify a previously unknown mechanism for a core element of alcohol addiction and point to a novel candidate therapeutic target.

Neurobiology of alcohol seeking behavior.

Alcohol addiction is a chronic relapsing brain disease characterized by an impaired ability to stop or control alcohol use despite adverse consequences. A main challenge of addiction treatment is to prevent relapse, which occurs in more than >50% of newly abstinent patients with alcohol disorder within 3 months. In people suffering from alcohol addiction, stressful events, drug-associated cues and contexts, or re-exposure to a small amount of alcohol trigger a chain of behaviors that frequently culminates in relapse. In this review, we first present the preclinical models that were developed for the study of alcohol seeking behavior, namely the reinstatement model of alcohol relapse and compulsive alcohol seeking under a chained schedule of reinforcement. We then provide an overview of the neurobiological findings obtained using these animal models, focusing on the role of opioids systems, corticotropin-release hormone and neurokinins, followed by dopaminergic, glutamatergic, and GABAergic neurotransmissions in alcohol seeking behavior.

Stress-induced escalation of alcohol self-administration, anxiety-like behavior, and elevated amygdala Avp expression in a susceptible subpopulation of rats.

Comorbidity between alcohol use and anxiety disorders is associated with more severe symptoms and poorer treatment outcomes than either of the conditions alone. There is a well-known link between stress and the development of these disorders, with post-traumatic stress disorder as a prototypic example. Post-traumatic stress disorder can arise as a consequence of experiencing traumatic events firsthand and also after witnessing them. Here, we used a model of social defeat and witness stress in rats, to study shared mechanisms of stress-induced anxiety-like behavior and escalated alcohol self-administration. Similar to what is observed clinically, we found considerable individual differences in susceptibility and resilience to the stress. Both among defeated and witness rats, we found a subpopulation in which exposure was followed by emergence of increased anxiety-like behavior and escalation of alcohol self-administration. We then profiled gene expression in tissue from the amygdala, a key brain region in the regulation of stress, alcohol use, and anxiety disorders. When comparing "comorbid" and resilient socially defeated rats, we identified a strong upregulation of vasopressin and oxytocin, and this correlated positively with the magnitude of the alcohol self-administration and anxiety-like behavior. A similar trend was observed in comorbid witness rats. Together, our findings provide novel insights into molecular mechanisms underpinning the comorbidity of escalated alcohol self-administration and anxiety-like behavior.

Targeting the Opioid Receptors: A Promising Therapeutic Avenue for Treatment in "Heavy Drinking Smokers".

AIMS: Despite a general decline in tobacco use in the last decades, the prevalence of tobacco smoking in individuals with alcohol use disorder (AUD) remains substantial (45-50%). Importantly, the co-use of both substances potentiates the adverse effects, making it a significant public health problem. Substantial evidence suggests that AUD and Tobacco use disorder (TUD) may share common mechanisms. Targeting these mechanisms may therefore provide more effective therapy. Numerous studies describe a potential role of the endogenous opioid system in both AUD and TUD. Reviewing this literature, we aim to evaluate the efficacy of molecules that target the opioid system as promising therapeutic interventions for treating alcohol and tobacco co-use disorders. METHODS: We provide a synthesis of the current epidemiological knowledge of alcohol and tobacco co-use disorders. We evaluate clinical and preclinical research that focuses on the regulation of the endogenous opioid system in alcohol, nicotine, and their interactions. RESULTS: The epidemiological data confirm that smoking stimulates heavy drinking and facilitates alcohol craving. Pharmacological findings suggest that treatments that are efficacious in the dual addiction provide a beneficial treatment outcome in comorbid AUD and TUD. In this regard, MOP, DOP and NOP-receptor antagonists show promising results, while the findings prompt caution when considering KOP-receptor antagonists as a treatment option in alcohol and tobacco co-use disorders. CONCLUSIONS: Existing literature suggests a role of the opioid system in sustaining the high comorbidity rates of AUD and TUD. Molecules targeting opioid receptors may therefore represent promising therapeutic interventions in 'heavy drinking smokers.'

Dysregulation of the histone demethylase KDM6B in alcohol dependence is associated with epigenetic regulation of inflammatory signaling pathways.

Epigenetic enzymes oversee long-term changes in gene expression by integrating genetic and environmental cues. While there are hundreds of enzymes that control histone and DNA modifications, their potential roles in substance abuse and alcohol dependence remain underexplored. A few recent studies have suggested that epigenetic processes could underlie transcriptomic and behavioral hallmarks of alcohol addiction. In the present study, we sought to identify epigenetic enzymes in the brain that are dysregulated during protracted abstinence as a consequence of chronic and intermittent alcohol exposure. Through quantitative mRNA expression analysis of over 100 epigenetic enzymes, we identified 11 that are significantly altered in alcohol-dependent rats compared with controls. Follow-up studies of one of these enzymes, the histone demethylase KDM6B, showed that this enzyme exhibits region-specific dysregulation in the prefrontal cortex and nucleus accumbens of alcohol-dependent rats. KDM6B was also upregulated in the human alcoholic brain. Upregulation of KDM6B protein in alcohol-dependent rats was accompanied by a decrease of trimethylation levels at histone H3, lysine 27 (H3K27me3), consistent with the known demethylase specificity of KDM6B. Subsequent epigenetic (chromatin immunoprecipitation [ChIP]-sequencing) analysis showed that alcohol-induced changes in H3K27me3 were significantly enriched at genes in the IL-6 signaling pathway, consistent with the well-characterized role of KDM6B in modulation of inflammatory responses. Knockdown of KDM6B in cultured microglial cells diminished IL-6 induction in response to an inflammatory stimulus. Our findings implicate a novel KDM6B-mediated epigenetic signaling pathway integrated with inflammatory signaling pathways that are known to underlie the development of alcohol addiction.

Downregulation of Synaptotagmin 1 in the Prelimbic Cortex Drives Alcohol-Associated Behaviors in Rats.

BACKGROUND: Alcohol addiction is characterized by persistent neuroadaptations in brain structures involved in motivation, emotion, and decision making, including the medial prefrontal cortex, the nucleus accumbens, and the amygdala. We previously reported that induction of alcohol dependence was associated with long-term changes in the expression of genes involved in neurotransmitter release. Specifically, Syt1, which plays a key role in neurotransmitter release and neuronal functions, was downregulated. Here, we therefore examined the role of Syt1 in alcohol-associated behaviors in rats. METHODS: We evaluated the effect of Syt1 downregulation using an adeno-associated virus (AAV) containing a short hairpin RNA against Syt1. Cre-dependent Syt1 was also used in combination with an rAAV2 retro-Cre virus to assess circuit-specific effects of Syt1 knockdown (KD). RESULTS: Alcohol-induced downregulation of Syt1 is specific to the prelimbic cortex (PL), and KD of Syt1 in the PL resulted in escalated alcohol consumption, increased motivation to consume alcohol, and increased alcohol drinking despite negative consequences ("compulsivity"). Syt1 KD in the PL altered the excitation/inhibition balance in the basolateral amygdala, while the nucleus accumbens core was unaffected. Accordingly, a projection-specific Syt1 KD in the PL-basolateral amygdala projection was sufficient to increase compulsive alcohol drinking, while a KD of Syt1 restricted to PL-nucleus accumbens core projecting neurons had no effect on tested alcohol-related behaviors. CONCLUSIONS: Together, these data suggest that dysregulation of Syt1 is an important mechanism in long-term neuroadaptations observed after a history of alcohol dependence, and that Syt1 regulates alcohol-related behaviors in part by affecting a PL-basolateral amygdala brain circuit.

Further evidence for the involvement of the PPARγ system on alcohol intake and sensitivity in rodents.

RATIONALE: Peroxisome Proliferator Activator receptors (PPARs) are intracellular receptors that function as transcription factors, which regulate specific metabolic and inflammatory processes. PPARs are broadly distributed in the body and are also expressed in the central nervous system, especially in areas involved in addiction-related behavioral responses. Recent studies support a role of PPARs in alcoholism and pioglitazone: a PPARγ agonist used for treatment of type 2 diabetes showed efficacy in reducing alcohol drinking, stress-induced relapse, and alcohol withdrawal syndrome in rats. OBJECTIVES AND METHODS: In the current work, we tested the pharmacological effects of pioglitazone on binge-like alcohol consumption using an intermittent two-bottle choice paradigm in Wistar rats and on the "drinking in the dark" (DID) model in mice with selective deletion of PPARγ in neurons. RESULTS: Our data show that repeated administration of pioglitazone (10, 30 mg/kg) reduces high voluntary alcohol consumption in Wistar rats. Pre-treatment with the selective PPARγ antagonist GW9662 (5 mg/kg) completely prevented the effect of pioglitazone, demonstrating that its action is specifically mediated by activation of PPARγ. In line with this result, repeated administration of pioglitazone (30 mg/kg) attenuated binge alcohol consumption in PPARγ(+/+) mice. Whereas in PPARγ(-/-) mice, which exhibit reduced alcohol consumption, pioglitazone had no effect. Of note, PPARγ(-/-) mice exhibited lower patterns of alcohol drinking without showing difference in sucrose (control) intake. Interestingly, PPARγ(-/-) mice displayed a higher sensitivity to the sedative and ataxic effect of alcohol compared with their wild-type counterpart. CONCLUSIONS: Collectively, these data suggest that PPARγ agonists, and specifically pioglitazone, could be potential therapeutics for the treatment of binge alcohol drinking.

Nicotine increases alcohol self-administration in male rats via a µ-opioid mechanism within the mesolimbic pathway.

BACKGROUND AND PURPOSE: Alcohol and nicotine use disorders are commonly comorbid. Both alcohol and nicotine can activate opioid systems in reward-related brain regions, leading to adaptive changes in opioid signalling upon chronic exposure. The potential role of these adaptations for comorbidity is presently unknown. Here, we examined the contribution of µ and κ-opioid receptors to nicotine-induced escalation of alcohol self-administration in rats. EXPERIMENTAL APPROACH: Chronic nicotine was tested on alcohol self-administration and motivation to obtain alcohol. We then tested the effect of the κ antagonist CERC-501 and the preferential µ receptor antagonist naltrexone on basal and nicotine-escalated alcohol self-administration. To probe µ or κ receptor adaptations, receptor binding and G-protein coupling assays were performed in reward-related brain regions. Finally, dopaminergic activity in response to alcohol was examined, using phosphorylation of DARPP-32 in nucleus accumbens as a biomarker. KEY RESULTS: Nicotine robustly induced escalation of alcohol self-administration and motivation to obtain alcohol. This was blocked by naltrexone but not by CERC-501. Escalation of alcohol self-administration was associated with decreased DAMGO-stimulated µ receptor signalling in the ventral tegmental area (VTA) and decreased pDARPP-32 in the nucleus accumbens shell in response to alcohol. CONCLUSIONS AND IMPLICATIONS: Collectively, these results suggest that nicotine contributes to escalate alcohol self-administration through a dysregulation of µ receptor activity in the VTA. These data imply that targeting µ rather than κ receptors may be the preferred pharmacotherapeutic approach for the treatment of alcohol use disorder when nicotine use contributes to alcohol consumption.

Activation of PPARγ Attenuates the Expression of Physical and Affective Nicotine Withdrawal Symptoms through Mechanisms Involving Amygdala and Hippocampus Neurotransmission.

An isoform of peroxisome proliferator-activated receptors (PPARs), PPARγ, is the receptor for the thiazolidinedione class of anti-diabetic medications including pioglitazone. Neuroanatomical data indicate PPARγ localization in brain areas involved in drug addiction. Preclinical and clinical data have shown that pioglitazone reduces alcohol and opioid self-administration, relapse to drug seeking, and plays a role in emotional responses. Here, we investigated the behavioral effect of PPARγ manipulation on nicotine withdrawal in male Wistar rats and in male mice with neuron-specific PPARγ deletion (PPARγ(-/-)) and their littermate wild-type (PPARγ(+/+)) controls. Real-time quantitative RT-PCR and RNAscope in situ hybridization assays were used for assessing the levels of expression and cell-type localization of PPARγ during nicotine withdrawal. Brain site-specific microinjections of the PPARγ agonist pioglitazone were performed to explore the role of this system on nicotine withdrawal at a neurocircuitry level. Results showed that activation of PPARγ by pioglitazone abolished the expression of somatic and affective nicotine withdrawal signs in rats and in (PPARγ(+/+)) mice. This effect was blocked by the PPARγ antagonist GW9662. During early withdrawal and protracted abstinence, the expression of PPARγ increased in GABAergic and glutamatergic cells of the amygdala and hippocampus, respectively. Hippocampal microinjections of pioglitazone reduced the expression of the physical signs of withdrawal, whereas excessive anxiety associated with protracted abstinence was prevented by pioglitazone microinjection into the amygdala. Our results demonstrate the implication of the neuronal PPARγ in nicotine withdrawal and indicates that activation of PPARγ may offer an interesting strategy for smoking cessation.SIGNIFICANCE STATEMENT Smoking cessation leads the occurrence of physical and affective withdrawal symptoms representing a major burden to quit tobacco use. Here, we show that activation of PPARγ prevents the expression of both somatic and affective signs of nicotine withdrawal. At molecular levels results show that PPARγ expression increases in GABAergic cells in the hippocampus and in GABA- and glutamate-positive cells in the basolateral amygdala. Hippocampal microinjections of pioglitazone reduce the insurgence of the physical withdrawal signs, whereas anxiety linked to protracted abstinence is attenuated by pioglitazone injected into the amygdala. Our results demonstrate the implication of neuronal PPARγ in nicotine withdrawal and suggest that PPARγ agonism may represent a promising treatment to aid smoking cessation.

Sub-dimensions of Alcohol Use Disorder in Alcohol Preferring and Non-preferring Rats, a Comparative Study.

Recent animal models of alcohol use disorder (AUD) are centered in capturing individual vulnerability differences in disease progression. Here, we used genetically selected Marchigian Sardinian alcohol-preferring (msP) and Wistars rats to apply a multidimensional model of AUD adapted from a previously described DSM-IV/DSM-5 multisymptomatic cocaine addiction model. As proof of concept, we hypothesized that msP rats, genetically selected for excessive drinking, would be more prone to develop dependence-like behavior compared to Wistars. Before exposure of animals to alcohol, we monitored basal anxiety in the elevated plus maze (EPM). Animals were then trained in prolonged operant alcohol self-administration, consisting of 30-min daily sessions for 60 days in total. Each session consisted of two 10-min periods of alcohol reinforcement separated by 10-min interval of non-reinforcement. Following training, we applied three criteria of individual vulnerability for AUD: (1) persistence of lever pressing for alcohol when it was not available; (2) motivation for alcohol in a progressive ratio (PR) schedule of reinforcement; and (3) resistance to punishment when alcohol delivery was anticipated by a foot-shock (0.3 mA). We obtained four groups corresponding to the number of criteria met (0-3 crit). Rats in the 0crit and 1crit groups were characterized as resilient, whereas rats in the 2crit and 3crit groups were characterized as prone to develop a dependent-like phenotype. As predicted, the 2-3crit groups were enriched with msP rats while the 0-1crit groups were enriched in Wistar rats. In further analysis, we calculated the global addiction score (GAS) per subject by the sum of the normalized score (z-score) of each criterion. Results showed GAS was highly correlated with animal distribution within the 3 criteria. Specifically, GAS was negative in the 0-1crit groups, and positive in the 2-3crit groups. A positive correlation between basal anxiety and quantity of alcohol intake was detected in msP rats but not Wistars. In conclusion, we demonstrated that the 0/3criteria model is a suitable approach to study individual differences in AUD and that msP rats, selected for excessive-alcohol drinking, show a higher propensity to develop AUD compared to non-preferring Wistars.