Giordano de Guglielmo

Chronic Nicotine Activates Stress/Reward-Related Brain Regions and Facilitates the Transition to Compulsive Alcohol Drinking

Alcohol and nicotine are the two most co-abused drugs in the world. Previous studies have shown that nicotine can increase alcohol drinking in nondependent rats, yet it is unknown whether nicotine facilitates the transition to alcohol dependence. We tested the hypothesis that chronic nicotine will speed up the escalation of alcohol drinking in rats and that this effect will be accompanied by activation of sparsely distributed neurons (neuronal ensembles) throughout the brain that are specifically recruited by the combination of nicotine and alcohol. Rats were trained to respond for alcohol and made dependent using chronic, intermittent exposure to alcohol vapor, while receiving daily nicotine (0.8 mg/kg) injections. Identification of neuronal ensembles was performed after the last operant session, using immunohistochemistry. Nicotine produced an early escalation of alcohol drinking associated with compulsive alcohol drinking in dependent, but not in nondependent rats (air exposed), as measured by increased progressive-ratio responding and increased responding despite adverse consequences. The combination of nicotine and alcohol produced the recruitment of discrete and phenotype-specific neuronal ensembles (∼4–13% of total neuronal population) in the nucleus accumbens core, dorsomedial prefrontal cortex, central nucleus of the amygdala, bed nucleus of stria terminalis, and posterior ventral tegmental area. Blockade of nicotinic receptors using mecamylamine (1 mg/kg) prevented both the behavioral and neuronal effects of nicotine in dependent rats. These results demonstrate that nicotine and activation of nicotinic receptors are critical factors in the development of alcohol dependence through the dysregulation of a set of interconnected neuronal ensembles throughout the brain.

Activation of PPARγ by Pioglitazone Potentiates the Effects of Naltrexone on Alcohol Drinking and Relapse in msP Rats

BACKGROUND Pioglitazone is a selective peroxisome proliferator-activated receptor γ (PPARγ) agonist used for the treatment of insulin resistance and type 2 diabetes. Previous studies conducted in our laboratory showed that activation of PPARγ by pioglitazone reduces alcohol drinking, stress-induced relapse, and alcohol withdrawal syndrome in rats. Pioglitazone was not able to prevent relapse elicited by alcohol cues. Conversely, the nonselective opioid antagonist naltrexone has been shown to reduce alcohol drinking and cue- but not stress-induced relapse in rodents. METHODS Based on these findings, this study was sought to determine the efficacy of pioglitazone and naltrexone combination on alcohol intake and relapse behavior. Genetically selected alcohol-preferring Marchigian Sardinian (msP) rats were used for the study. RESULTS Pioglitazone (10 and 30 mg/kg) and naltrexone (0.25 and 1.0 mg/kg) each individually reduced alcohol drinking in msP rats. The combination of the 2 drugs resulted in a more potent alcohol drinking reduction than single agents. Confirming previous studies, pioglitazone (10 and 30 mg/kg) significantly reduced relapse induced by the pharmacological stressor yohimbine (1.25 mg/kg) but not by cues predictive of alcohol availability. Conversely, naltrexone reduced reinstatement of drug seeking elicited by alcohol cues but not by yohimbine. CONCLUSIONS The drug combination was effective in reducing both relapse behaviors. These findings open new vistas in the use pioglitazone in combination with naltrexone for the treatment of alcoholism.

Restraint Stress Alters Nociceptin/Orphanin FQ and CRF Systems in the Rat Central Amygdala: Significance for Anxiety-Like Behaviors

Corticotropin releasing factor (CRF) is the primary mediator of stress responses, and nociceptin/orphanin FQ (N/OFQ) plays an important role in the modulation of these stress responses. Thus, in this multidisciplinary study, we explored the relationship between the N/OFQ and the CRF systems in response to stress. Using in situ hybridization (ISH), we assessed the effect of body restraint stress on the gene expression of CRF and N/OFQ-related genes in various subdivisions of the amygdala, a critical brain structure involved in the modulation of stress response and anxiety-like behaviors. We found a selective upregulation of the NOP and downregulation of the CRF1 receptor transcripts in the CeA and in the BLA after body restraint. Thus, we performed intracellular electrophysiological recordings of GABAA-mediated IPSPs in the central nucleus of the amygdala (CeA) to explore functional interactions between CRF and N/OFQ systems in this brain region. Acute application of CRF significantly increased IPSPs in the CeA, and this enhancement was blocked by N/OFQ. Importantly, in stress-restraint rats, baseline CeA GABAergic responses were elevated and N/OFQ exerted a larger inhibition of IPSPs compared with unrestraint rats. The NOP antagonist [Nphe1]-nociceptin(1–13)NH2 increased the IPSP amplitudes in restraint rats but not in unrestraint rats, suggesting a functional recruitment of the N/OFQ system after acute stress. Finally, we evaluated the anxiety-like response in rats subjected to restraint stress and nonrestraint rats after N/OFQ microinjection into the CeA. Intra-CeA injections of N/OFQ significantly and selectively reduced anxiety-like behavior in restraint rats in the elevated plus maze. These combined results demonstrate that acute stress increases N/OFQ systems in the CeA and that N/OFQ has antistress properties.

A Transgenic Rat for Investigating the Anatomy and Function of Corticotrophin Releasing Factor Circuits

Corticotrophin-releasing factor (CRF) is a 41 amino acid neuropeptide that coordinates adaptive responses to stress. CRF projections from neurons in the central nucleus of the amygdala (CeA) to the brainstem are of particular interest for their role in motivated behavior. To directly examine the anatomy and function of CRF neurons, we generated a BAC transgenic Crh-Cre rat in which bacterial Cre recombinase is expressed from the Crh promoter. Using Cre-dependent reporters, we found that Cre expressing neurons in these rats are immunoreactive for CRF and are clustered in the lateral CeA (CeL) and the oval nucleus of the BNST. We detected major projections from CeA CRF neurons to parabrachial nuclei and the locus coeruleus, dorsal and ventral BNST, and more minor projections to lateral portions of the substantia nigra, ventral tegmental area, and lateral hypothalamus. Optogenetic stimulation of CeA CRF neurons evoked GABA-ergic responses in 11% of non-CRF neurons in the medial CeA (CeM) and 44% of non-CRF neurons in the CeL. Chemogenetic stimulation of CeA CRF neurons induced Fos in a similar proportion of non-CRF CeM neurons but a smaller proportion of non-CRF CeL neurons. The CRF1 receptor antagonist R121919 reduced this Fos induction by two-thirds in these regions. These results indicate that CeL CRF neurons provide both local inhibitory GABA and excitatory CRF signals to other CeA neurons, and demonstrate the value of the Crh-Cre rat as a tool for studying circuit function and physiology of CRF neurons.

Genetic and pharmacologic manipulation of TLR4 has minimal impact on ethanol consumption in rodents

Toll-like receptor 4 (TLR4) is a critical component of innate immune signaling and has been implicated in alcohol responses in preclinical and clinical models. Members of the Integrative Neuroscience Initiative on Alcoholism (INIA-Neuroimmune) consortium tested the hypothesis that TLR4 mediates excessive ethanol drinking using the following models: (1) Tlr4 knock-out (KO) rats, (2) selective knockdown of Tlr4 mRNA in mouse nucleus accumbens (NAc), and (3) injection of the TLR4 antagonist (+)-naloxone in mice. Lipopolysaccharide (LPS) decreased food/water intake and body weight in ethanol-naive and ethanol-trained wild-type (WT), but not Tlr4 KO rats. There were no consistent genotypic differences in two-bottle choice chronic ethanol intake or operant self-administration in rats before or after dependence. In mice, (+)-naloxone did not decrease drinking-in-the-dark and only modestly inhibited dependence-driven consumption at the highest dose. Tlr4 knockdown in mouse NAc did not decrease drinking in the two-bottle choice continuous or intermittent access tests. However, the latency to ethanol-induced loss of righting reflex increased and the duration decreased in KO versus WT rats. In rat central amygdala neurons, deletion of Tlr4 altered GABAA receptor function, but not GABA release. Although there were no genotype differences in acute ethanol effects before or after chronic intermittent ethanol exposure, genotype differences were observed after LPS exposure. Using different species and sexes, different methods to inhibit TLR4 signaling, and different ethanol consumption tests, our comprehensive studies indicate that TLR4 may play a role in ethanol-induced sedation and GABAA receptor function, but does not regulate excessive drinking directly and would not be an effective therapeutic target. SIGNIFICANCE STATEMENT Toll-like receptor 4 (TLR4) is a key mediator of innate immune signaling and has been implicated in alcohol responses in animal models and human alcoholics. Members of the Integrative Neuroscience Initiative on Alcoholism (INIA-Neuroimmune) consortium participated in the first comprehensive study across multiple laboratories to test the hypothesis that TLR4 regulates excessive alcohol consumption in different species and different models of chronic, dependence-driven, and binge-like drinking. Although TLR4 was not a critical determinant of excessive drinking, it was important in the acute sedative effects of alcohol. Current research efforts are directed at determining which neuroimmune pathways mediate excessive alcohol drinking and these findings will help to prioritize relevant pathways and potential therapeutic targets.

Recruitment of a Neuronal Ensemble in the Central Nucleus of the Amygdala Is Required for Alcohol Dependence

Abstinence from alcohol is associated with the recruitment of neurons in the central nucleus of the amygdala (CeA) in nondependent rats that binge drink alcohol and in alcohol-dependent rats. However, whether the recruitment of this neuronal ensemble in the CeA is causally related to excessive alcohol drinking or if it represents a consequence of excessive drinking remains unknown. We tested the hypothesis that the recruitment of a neuronal ensemble in the CeA during abstinence is required for excessive alcohol drinking in nondependent rats that binge drink alcohol and in alcohol-dependent rats. We found that inactivation of the CeA neuronal ensemble during abstinence significantly decreased alcohol drinking in both groups. In nondependent rats, the decrease in alcohol intake was transient and returned to normal the day after the injection. In dependent rats, inactivation of the neuronal ensemble with Daun02 produced a long-term decrease in alcohol drinking. Moreover, we observed a significant reduction of somatic withdrawal signs in dependent animals that were injected with Daun02 in the CeA. These results indicate that the recruitment of a neuronal ensemble in the CeA during abstinence from alcohol is causally related to excessive alcohol drinking in alcohol-dependent rats, whereas a similar neuronal ensemble only partially contributed to alcohol-binge-like drinking in nondependent rats. These results identify a critical neurobiological mechanism that may be required for the transition to alcohol dependence, suggesting that focusing on the neuronal ensemble in the CeA may lead to a better understanding of the etiology of alcohol use disorders and improve medication development. SIGNIFICANCE STATEMENT Alcohol dependence recruits neurons in the central nucleus of the amygdala (CeA). Here, we found that inactivation of a specific dependence-induced neuronal ensemble in the CeA reversed excessive alcohol drinking and somatic signs of alcohol dependence in rats. These results identify a critical neurobiological mechanism that is required for alcohol dependence, suggesting that targeting dependence neuronal ensembles may lead to a better understanding of the etiology of alcohol use disorders, with implications for diagnosis, prevention, and treatment.

MT-7716, a potent NOP receptor agonist, preferentially reduces ethanol seeking and reinforcement in post-dependent rats

Dysregulation of the nociceptin (N/OFQ) system has been implicated in alcohol abuse and alcoholism, and growing evidence suggests that targeting this system may be beneficial for treating alcoholism. To further explore the treatment target potential of the N/OFQ system, the novel non‐peptide, small‐molecule N/OFQ (NOP) agonist MT‐7716, (R)‐2‐{3‐[1‐(Acenaphthen‐1‐yl)piperidin‐4‐yl]‐2‐oxo‐2,3‐dihydro‐1H‐benzimidazol‐1‐yl}‐N‐methylacetamide hydrochloride hydrate, was examined for its effects on ethanol self‐administration and stress‐induced reinstatement of alcohol seeking in non‐dependent and post‐dependent rats. Male Wistar rats were trained to self‐administer ethanol and then made ethanol dependent via repeated intragastric ethanol intubation. The effects of MT‐7716 (0.3 and 1 mg/kg; PO) on alcohol self‐administration were determined 2 weeks following dependence induction, when baseline self‐administration was restored. Effects of MT‐7716 on stress‐induced reinstatement were tested in separate cohorts of rats, 1 and 3 weeks post‐withdrawal. MT‐7716 reduced alcohol self‐administration and stress‐induced reinstatement of alcohol seeking in post‐dependent rats, but was ineffective in non‐dependent animals. Moreover, the prevention of stress‐induced reinstatement by MT‐7716 was more pronounced at 3 weeks post‐dependence. The results further confirm treatment target potential for the NOP receptor and identify non‐peptide NOP agonists as promising potential treatment drugs for alcohol abuse and relapse prevention. The findings also support dysregulation of the N/OFQ system as a factor in alcohol seeking and reinforcement.

Pregabalin reduces cocaine self-administration and relapse to cocaine seeking in the rat

Pregabalin (Lyrica™) is a structural analog of γ‐aminobutyric acid (GABA) and is approved by the FDA for partial epilepsy, neuropathic pain and generalized anxiety disorders. Pregabalin also reduces excitatory neurotransmitter release and post‐synaptic excitability. Recently, we demonstrated that pregabalin reduced alcohol intake and prevented relapse to the alcohol seeking elicited by stress or environmental stimuli associated with alcohol availability. Here, we sought to extend these findings by examining the effect of pregabalin on cocaine self‐administration (0.25 mg/infusion) and on cocaine seeking elicited by both conditioned stimuli and stress, as generated by administration of yohimbine (1.25 mg/kg). The results showed that oral administration of pregabalin (0, 10 or 30 mg/kg) reduced self‐administration of cocaine over an extended period (6 hours), whereas it did not modify self‐administration of food. In cocaine reinstatement studies, pregabalin (10 and 30 mg/kg) abolished the cocaine seeking elicited by both the pharmacological stressor yohimbine and the cues predictive of cocaine availability. Overall, these results demonstrate that pregabalin may have potential in the treatment of some aspects of cocaine addiction.

Activation of Hypocretin-1/Orexin-A Neurons Projecting to the Bed Nucleus of the Stria Terminalis and Paraventricular Nucleus Is Critical for Reinstatement of Alcohol Seeking by Neuropeptide S

BACKGROUND Environmental conditioning is a major trigger for relapse in abstinent addicts. We showed that activation of the neuropeptide S (NPS) system exacerbates reinstatement vulnerability to cocaine and alcohol via stimulation of the hypocretin-1/orexin-A (Hcrt-1/Ox-A) system. METHODS Combining pharmacologic manipulations with immunohistochemistry techniques, we sought to determine how NPS and Hcrt-1/Ox-A systems interact to modulate reinstatement of alcohol seeking in rats. RESULTS Intrahypothalamic injection of NPS facilitated discriminative cue-induced reinstatement of alcohol seeking. This effect was blocked by the selective Hcrt-1/Ox-A antagonist SB334867 microinjected into the hypothalamic paraventricular nucleus (PVN) or into the bed nucleus of the stria terminalis (BNST) but not into the ventral tegmental area or the locus coeruleus. Combining double labeling and confocal microscopy analyses, we found that NPS-containing axons are in close apposition to hypothalamic Hcrt-1/Ox-A positive neurons, a significant proportion of which express NPS receptors, suggesting a direct interaction between the two systems. Retrograde tracing experiments showed that intra-PVN or intra-BNST red fluorobead unilateral injection labeled bilaterally Hcrt-1/Ox-A somata, suggesting that NPS could recruit two distinct neuronal pathways. Confirming this assumption, intra-BNST or PVN Hcrt-1/Ox-A injection enhanced alcohol seeking similarly to hypothalamic NPS injection but to a lesser degree. CONCLUSIONS Results suggest that the Hcrt-1/Ox-A neurocircuitry mediating the facilitation of cue-induced reinstatement by NPS involves structures critically involved in stress regulation such as the PVN and the BNST. These findings open to the tempting hypothesis of a role of the NPS system in modulating the interactions between stress and environmental conditioning factors in drug relapse.

Analgesic tolerance to morphine is regulated by PPARγ

Opioid drugs are potent analgesics. However, their chronic use leads to the rapid development of tolerance to their analgesic effects and subsequent increase of significant side effects, including drug dependence and addiction. Here, we investigated the role of PPARγ in the development of analgesic tolerance to morphine in mice.