Search

Oxytocin administration has been reported to decrease consumption, withdrawal, and drug-seeking associated with several drugs of abuse and thus represents a promising pharmacological approach to treat drug addiction. We used an established rat model of alcohol dependence to investigate oxytocin's effects on dependence-induced alcohol drinking, enhanced motivation for alcohol, and altered GABAergic transmission in the central nucleus of the amygdala (CeA). Intraperitoneal oxytocin administration blocked escalated alcohol drinking and the enhanced motivation for alcohol in alcohol-dependent but not nondependent rats. Intranasal oxytocin delivery fully replicated these effects. Intraperitoneal administration had minor but significant effects of reducing locomotion and intake of non-alcoholic palatable solutions, whereas intranasal oxytocin administration did not. In dependent rats, intracerebroventricular administration of oxytocin or the oxytocin receptor agonist PF-06655075, which does not cross the blood-brain barrier (i.e., it would not diffuse to the periphery), but not systemic administration of PF-06655075 (i.e., it would not reach the brain), decreased alcohol drinking. Administration of a peripherally restricted oxytocin receptor antagonist did not reverse the effect of intranasal oxytocin on alcohol drinking. Ex vivo electrophysiological recordings from CeA neurons indicated that oxytocin decreases evoked GABA transmission in nondependent but not in dependent rats, whereas oxytocin decreased the amplitude of spontaneous GABAergic responses in both groups. Oxytocin blocked the facilitatory effects of acute alcohol on GABA release in the CeA of dependent but not nondependent rats. Together, these results provide converging evidence that oxytocin specifically and selectively blocks the enhanced motivation for alcohol drinking that develops in alcohol dependence likely via a central mechanism that may result from altered oxytocin effects on CeA GABA transmission in alcohol dependence. Neuroadaptations in endogenous oxytocin signaling may provide a mechanism to further our understanding of alcohol use disorder., Competing Interests: The authors have declared that no competing interests exist.

Opioid use disorder (OUD) is associated with the emergence of persistent negative emotional states during drug abstinence that drive compulsive drug taking and seeking. Functional magnetic resonance imaging (fMRI) in rats identified neurocircuits that were activated by stimuli that were previously paired with heroin withdrawal. The activation of amygdala and hypothalamic circuits was related to the degree of heroin dependence, supporting the significance of conditioned negative affect in sustaining compulsive-like heroin seeking and taking and providing neurobiological insights into the drivers of the current opioid crisis.

The peptide hormone acyl-ghrelin and its receptor, GHSR 1a , represent intriguing therapeutic targets due to their actions in metabolic homeostasis and reward activity. However, this pleotropic activity makes it difficult to intervene in this system without inducing unwanted effects. Thus, it is desirable to identify passive and active regulatory mechanisms that allow differentiation between functional domains. Anatomical restriction by the blood brain barrier represents one major passive regulatory mechanism. However, it is likely that the ghrelin system is subject to additional passive mechanisms that promote independent regulation of orexigenic behavior and reward processing. By applying acyl-ghrelin sequestering antibodies, it was determined that peripheral sequestration of acyl-ghrelin is sufficient to blunt weight gain, but not cocaine rewarding effects. However, both weight gain and reward-associated behaviors were shown to be blocked by direct antagonism of GHSR 1a . Overall, these data indicate that GHSR 1a effects on reward are independent from peripheral acyl-ghrelin binding, whereas centrally-mediated alteration of energy storage requires peripheral acyl-ghrelin binding. This demonstration of variable ligand-dependence amongst functionally-distinct GHSR 1a populations is used to generate a regulatory model for functional manipulation of specific effects when attempting to therapeutically target the ghrelin system.

Background/objectives: Ghrelin, a stomach-derived hormone implicated in numerous behaviors including feeding, reward, stress, and addictive behaviors, acts by binding to the growth hormone secretagogue receptor (GHSR). Here, we present the development, verification, and initial characterization of a novel GHSR knockout (KO) Wistar rat model created with CRISPR genome editing., Methods: Using CRISPR/Cas9, we developed a GHSR KO in a Wistar background. Loss of GHSR mRNA expression was histologically verified using RNAscope in wild-type (WT; n = 2) and KO (n = 2) rats. We tested the effects of intraperitoneal acyl-ghrelin administration on food consumption and plasma growth hormone (GH) concentrations in WT (n = 8) and KO (n = 8) rats. We also analyzed locomotion, food consumption, and body fat composition in these animals. Body weight was monitored from early development to adulthood., Results: The RNAscope analysis revealed an abundance of GHSR mRNA expression in the hypothalamus, midbrain, and hippocampus in WTs, and no observed probe binding in KOs. Ghrelin administration increased plasma GH levels (p = 0.0067) and food consumption (p = 0.0448) in WT rats but not KOs. KO rats consumed less food overall at basal conditions and weighed significantly less compared with WTs throughout development (p = 0.0001). Compared with WTs, KOs presented higher concentrations of brown adipose tissue (BAT; p = 0.0322)., Conclusions: We have verified GHSR deletion in our KO model using histological, physiological, neuroendocrinological, and behavioral measures. Our findings indicate that GHSR deletion in rats is not only associated with a lack of response to ghrelin, but also associated with decreases in daily food consumption and body growth, and increases in BAT. This GHSR KO Wistar rat model provides a novel tool for studying the role of the ghrelin system in obesity and in a wide range of medical and neuropsychiatric disorders.

Aims: The development of novel and more effective medications for alcohol use disorder (AUD) is an important unmet medical need. Drug repositioning or repurposing is an appealing strategy to bring new therapies to the clinic because it greatly reduces the overall costs of drug development and expedites the availability of treatments to those who need them. Probenecid, p-(di-n-propylsulfamyl)-benzoic acid, is a drug used clinically to treat hyperuricemia and gout due to its activity as an inhibitor of the kidneys' organic anion transporter that reclaims uric acid from urine. Probenecid also inhibits pannexin1 channels that are involved in purinergic neurotransmission and inflammation, which have been implicated in alcohol's effects and motivation for alcohol. Therefore, we tested the effects of probenecid on alcohol intake in rodents., Methods: We tested the effects of probenecid on operant oral alcohol self-administration in alcohol-dependent rats during acute withdrawal as well as in nondependent rats and in the drinking-in-the-dark (DID) paradigm of binge-like drinking in mice., Results: Probenecid reduced alcohol intake in both dependent and nondependent rats and in the DID paradigm in mice without affecting water or saccharin intake, indicating that probenecid's effect was selective for alcohol and not the result of a general reduction in reward., Conclusions: These results raise the possibility that pannexin1 is a novel therapeutic target for the treatment of AUD. The clinical use of probenecid has been found to be generally safe, suggesting that it can be a candidate for drug repositioning for the treatment of AUD., (© The Author(s) 2019. Medical Council on Alcohol and Oxford University Press. All rights reserved.)

Depression is highly prevalent worldwide, but its etiology is not fully understood. An overlooked possible contributor to the epidemic of depression is feeding styles, particularly at early age when the brain is intensely changing. We have previously reported that unlimited sucrose consumption during adolescence leads to enduring changes in brain reward function. Here, we tested the hypothesis that sucrose consumption during adolescence would lead to a 'depressive-like' phenotype. Adolescent male rats were given unlimited access to 5% sucrose in their home cages from postnatal day 30 to postnatal day 46 and their emotional behavior was subsequently examined at adulthood. Sucrose consumption during adolescence caused anhedonia, decreased motivation for saccharin, increased immobility in the forced swim test and exacerbated anxiety-like behavior. Additionally, sucrose consumption during adolescence decreased cell proliferation in the hippocampus in adulthood. Chronic treatment with imipramine (10 mg/kg) normalized behavior and restored cell proliferation in the hippocampus of adult rats with a history of sucrose consumption during adolescence. A similar sucrose consumption starting at adulthood only increases immobility in the forced swim test, suggesting that sucrose intake affects also adults' behavior but to a lesser degree. Overall, our findings reveal an unsuspected protracted effect of sucrose consumption on behavior and suggest that unlimited sucrose consumption during critical periods of brain development may play an important role in the etiology of reward-related disorders such as depression.

The hypocretin/orexin (HCRT) neuropeptide system regulates feeding, arousal state, stress responses, and reward, especially under conditions of enhanced motivational relevance. In particular, HCRT neurotransmission facilitates drug-seeking behavior in circumstances that demand increased effort and/or motivation to take the drug. The present study used a shRNA-encoding adeno-associated viral vector to knockdown Hcrt expression throughout the dorsal hypothalamus in adult rats and determine the role of HCRT in cocaine self-administration. Chronic Hcrt silencing did not impact cocaine self-administration under short-access conditions, but robustly attenuated cocaine intake under extended access conditions, a model that mimics key features of compulsive cocaine taking. In addition, Hcrt silencing decreased motivation for both cocaine and a highly palatable food reward (i.e., sweetened condensed milk; SCM) under a progressive ratio schedule of reinforcement, but did not alter responding for SCM under a fixed ratio schedule. Importantly, Hcrt silencing did not affect food or water consumption, and had no consequence for general measures of arousal and stress reactivity. At the molecular level, chronic Hcrt knockdown reduced the number of neurons expressing dynorphin (DYN), and to a smaller extent melanin-concentrating hormone (MCH), in the dorsal hypothalamus. These original findings support the hypothesis that HCRT neurotransmission promotes operant responding for both drug and non-drug rewards, preferentially under conditions requiring a high degree of motivation. Furthermore, the current study provides compelling evidence for the involvement of the HCRT system in cocaine self-administration also under low-effort conditions in rats allowed extended access, possibly via functional interactions with DYN and MCH signaling.

Alcohol use disorder (AUD) is a common and chronic disorder with substantial effects on personal and public health. The underlying pathophysiology is poorly understood but strong evidence suggests significant roles of both genetic and epigenetic components. Given that alcohol affects many organ systems, we performed a cross-tissue and cross-phenotypic analysis of genome-wide methylomic variation in AUD using samples from 3 discovery, 4 replication, and 2 translational cohorts. We identified a differentially methylated region in the promoter of the proprotein convertase subtilisin/kexin 9 (PCSK9) gene that was associated with disease phenotypes. Biological validation showed that PCSK9 promoter methylation is conserved across tissues and positively correlated with expression. Replication in AUD datasets confirmed PCSK9 hypomethylation and a translational mouse model of AUD showed that alcohol exposure leads to PCSK9 downregulation. PCSK9 is primarily expressed in the liver and regulates low-density lipoprotein cholesterol (LDL-C). Our finding of alcohol-induced epigenetic regulation of PCSK9 represents one of the underlying mechanisms between the well-known effects of alcohol on lipid metabolism and cardiovascular risk, with light alcohol use generally being protective while chronic heavy use has detrimental health outcomes.

Neuroinflammation appears to contribute to neurotoxicity observed with heavy alcohol consumption. To assess whether chronic alcohol results in neuroinflammation we used PET and [ 11 C]PBR28, a ligand that binds to the 18-kDa translocator protein (TSPO), to compare participants with an alcohol use disorder (AUD: n = 19) with healthy controls (HC: n = 17), and alcohol-dependent (n = 9) with -nondependent rats (n = 10). Because TSPO is implicated in cholesterol's transport for steroidogenesis, we investigated whether plasma cholesterol levels influenced [ 11 C]PBR28 binding. [ 11 C]PBR28 binding did not differ between AUD and HC. However, when separating by TSPO genotype rs6971, we showed that medium-affinity binders AUD participants showed lower [ 11 C]PBR28 binding than HC in regions of interest (whole brain, gray and white matter, hippocampus, and thalamus), but no group differences were observed in high-affinity binders. Cholesterol levels inversely correlated with brain [ 11 C]PBR28 binding in combined groups, due to a correlation in AUD participants. In rodents, we observed no differences in brain [ 11 C]PBR28 uptake between alcohol-dependent and -nondependent rats. These findings, which are consistent with two previous [ 11 C]PBR28 PET studies, may indicate lower activation of microglia in AUD, whereas failure to observe alcohol effects in the rodent model indicate that species differences do not explain the discrepancy with prior rodent autoradiographic studies reporting increases in TSPO binding with chronic alcohol. However, reduced binding in AUD participants could also reflect competition from endogenous TSPO ligands such as cholesterol; and since the rs6971 polymorphism affects the cholesterol-binding domain of TSPO this could explain why differences were observed only in medium-affinity binders.

Aldosterone regulates electrolyte and fluid homeostasis through binding to the mineralocorticoid receptors (MRs). Previous work provides evidence for a role of aldosterone in alcohol use disorders (AUDs). We tested the hypothesis that high functional activity of the mineralocorticoid endocrine pathway contributes to vulnerability for AUDs. In Study 1, we investigated the relationship between plasma aldosterone levels, ethanol self-administration and the expression of CYP11B2 and MR (NR3C2) genes in the prefrontal cortex area (PFC) and central nucleus of the amygdala (CeA) in monkeys. Aldosterone significantly increased after 6- and 12-month ethanol self-administration. NR3C2 expression in the CeA was negatively correlated to average ethanol intake during the 12 months. In Study 2, we measured Nr3c2 mRNA levels in the PFC and CeA of dependent and nondependent rats and the correlates with ethanol drinking during acute withdrawal. Low Nr3c2 expression levels in the CeA were significantly associated with increased anxiety-like behavior and compulsive-like drinking in dependent rats. In Study 3, the relationship between plasma aldosterone levels, alcohol drinking and craving was investigated in alcohol-dependent patients. Non-abstinent patients had significantly higher aldosterone levels than abstinent patients. Aldosterone levels positively correlated with the number of drinks consumed, craving and anxiety scores. These findings support a relationship between ethanol drinking and the aldosterone/MR pathway in three different species.

Methamphetamine (METH) is a highly addictive drug, but no pharmacological treatment is yet available for METH use disorders. Similar to METH, the wake-promoting drug (R)-modafinil (R-MOD) binds to the dopamine transporter (DAT). Unlike METH, R-MOD is not a substrate for transport by DAT and has low abuse potential. We tested the hypothesis that the atypical DAT inhibitor R-MOD and compounds that are derived from modafinil would decrease METH intake by reducing the actions of METH at the DAT. We tested the effects of systemic injections of R-MOD and four novel modafinil-derived ligands with increased DAT affinity (JJC8-016, JJC8-088, JJC8-089, and JJC8-091) on intravenous (i.v.) METH self-administration in rats that were allowed short access (ShA; 1 h) or long access (LgA; 6 h) to the drug. ShA rats exhibited stable METH intake over sessions, whereas LgA rats exhibited an escalation of drug intake. R-MOD decreased METH self-administration in ShA and LgA rats (in the 1st hour only). JJC8-091 and JJC8-016 decreased METH self-administration in both ShA and LgA rats. JJC8-089 decreased METH self-administration in LgA rats only, whereas JJC8-088 had no effect on METH self-administration in either ShA or LgA rats. These findings support the potential of atypical DAT inhibitors for the treatment of METH use disorders and suggest several novel compounds as candidate drugs., (Published by Elsevier Ltd.)

Opioid misuse is at historically high levels in the United States, with inhalation (ie, smoking and vaping) being one of the most common routes of consumption. We developed and validated a novel preclinical model of opioid self-administration by inhalation that does not require surgery and reliably produces somatic and motivational signs of dependence. Rats were trained to perform an operant response (nosepoke) to receive 10 s of vaporized sufentanil, a potent opioid, in 2 h daily sessions. Rats readily and concentration-dependently self-administered vaporized sufentanil. Rats exhibited a significant increase in responding for sufentanil when given the preferential μ-opioid receptor inverse agonist naloxone, suggesting the participation of μ-opioid receptors in the reinforcing properties of sufentanil vapor. Serum sufentanil concentrations significantly correlated with the number of sufentanil vapor deliveries. Rats that were given long access (LgA; 12 h/day) but not short access (ShA; 1 h/day) to vaporized sufentanil escalated their drug intake over time and exhibited both naloxone-precipitated somatic signs of opioid withdrawal and spontaneous withdrawal-induced mechanical hypersensitivity. After 6 months of forced drug abstinence, LgA rats returned to pre-escalation baseline levels of responding for sufentanil and mechanical sensitivity. Upon subsequent re-escalation (ie, after the return to extended access to sufentanil vapor), LgA rats again developed naloxone-precipitated somatic signs of withdrawal and spontaneous withdrawal-induced mechanical hypersensitivity. These findings demonstrate that the operant sufentanil vapor self-administration model has both face and construct validity and therefore will be useful for investigating the neurobiological basis of opioid addiction.

The original version of this Article contained an error in the spelling of the author Scott Edwards, which was incorrectly given as Scott Edward. This has now been corrected in both the PDF and HTML versions of the Article.

Alcohol-use disorder (AUD) is the most prevalent substance-use disorder worldwide. There is substantial individual variability in alcohol drinking behaviors in the population, the neural circuit mechanisms of which remain elusive. Utilizing in vivo electrophysiological techniques, we find that low alcohol drinking (LAD) mice have dramatically higher ventral tegmental area (VTA) dopamine neuron firing and burst activity. Unexpectedly, VTA dopamine neuron activity in high alcohol drinking (HAD) mice does not differ from alcohol naive mice. Optogenetically enhancing VTA dopamine neuron burst activity in HAD mice decreases alcohol drinking behaviors. Circuit-specific recordings reveal that spontaneous activity of nucleus accumbens-projecting VTA (VTA-NAc) neurons is selectively higher in LAD mice. Specifically activating this projection is sufficient to reduce alcohol consumption in HAD mice. Furthermore, we uncover ionic and cellular mechanisms that suggest unique neuroadaptations between the alcohol drinking groups. Together, these data identify a neural circuit responsible for individual alcohol drinking behaviors.

Rationale: Drugs of abuse can alter circuit dynamics by modifying synaptic efficacy and/or the intrinsic membrane properties of neurons. The juxtacapsular subdivision of the bed nucleus of stria terminalis (jcBNST) has unique connectivity that positions it to integrate cortical and amygdala inputs and provide feed-forward inhibition to the central nucleus of the amygdala (CeA), among other regions. In this study, we investigated changes in the synaptic and intrinsic properties of neurons in the rat jcBNST during protracted withdrawal from morphine dependence using a combination of conventional electrophysiological methods and the dynamic clamp technique., Results: A history of opiate dependence induced a form of cell type-specific plasticity characterized by reduced inward rectification associated with more depolarized resting membrane potentials and increased membrane resistance. This cell type also showed a lower rheobase when stimulated with direct current (DC) pulses as well as a decreased firing threshold under simulated synaptic bombardment with the dynamic clamp. Morphine dependence also decreased excitatory postsynaptic potential amplification, suggesting the downregulation of the persistent Na + current (I NaP )., Conclusion: These findings show that a history of morphine dependence leads to persistent cell type-specific plasticity of the passive membrane properties of a jcBNST neuronal population, leading to an overall increased excitability of such neurons. By altering the activity of extended amygdala circuits where they are embedded, changes in the integration properties of jcBNST neurons may contribute to emotional dysregulation associated with drug dependence and withdrawal.

Alcohol dependence is linked to dysregulation of the hypothalamic-pituitary-adrenal axis. Here, we investigated effects of repeated ethanol intoxication-withdrawal cycles (using chronic intermittent ethanol vapor inhalation; CIE) and abstinence from CIE on peak and nadir plasma corticosterone (CORT) levels. Irritability- and anxiety-like behaviors as well as glucocorticoid receptors (GR) in the medial prefrontal cortex (mPFC) were assessed at various intervals (2h-28d) after cessation of CIE. Results show that peak CORT increased during CIE, transiently decreased during early abstinence (1-11d), and returned to pre-abstinence levels during protracted abstinence (17-27d). Acute withdrawal from CIE enhanced aggression- and anxiety-like behaviors. Early abstinence from CIE reduced anxiety-like behavior. mPFC-GR signaling (indexed by relative phosphorylation of GR at Ser211) was transiently decreased when measured at time points during early and protracted abstinence. Further, voluntary ethanol drinking in CIE (CIE-ED) and CIE-naïve (ED) rats, and effects of CIE-ED and ED on peak CORT levels and mPFC-GR were investigated during acute withdrawal (8h) and protracted abstinence (28d). CIE-ED and ED increased peak CORT during drinking. CIE-ED and ED decreased expression and signaling of mPFC-GR during acute withdrawal, an effect that was reversed by systemic mifepristone treatment. CIE-ED and ED demonstrate robust reinstatement of ethanol seeking during protracted abstinence and show increases in mPFC-GR expression. Collectively, the data demonstrate that acute withdrawal from CIE produces robust alterations in GR signaling, CORT and negative affect symptoms which could facilitate excessive drinking. The findings also show that CIE-ED and ED demonstrate enhanced relapse vulnerability triggered by ethanol cues and these changes are partially mediated by altered GR expression in the mPFC. Taken together, transition to alcohol dependence could be accompanied by alterations in mPFC stress-related pathways that may increase negative emotional symptoms and increase vulnerability to relapse., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

We investigated the effects of physical exercise (PE) on locomotor activity and anxiety-like behavior in Lewis (LEW) and Spontaneously Hypertensive Rats (SHR) male rats. Rats received either four weeks of forced training, 5days/week, on a treadmill (experiment 1) or were given 21days of free access to running wheels (experiment 2). We also tested the effects of social isolation (SI) (seven days of isolation - experiment 3) on behavior. In experiment 1, 20% of LEW rats and 63% of SHR rats completed the training protocol. PE significantly increased central and peripheral locomotion in the open field (OF) and entries into the open arms in the elevated plus-maze (EPM) in both strains. In experiment 2, the distance traveled by SHR rats on running wheels was significantly higher compared with LEW rats. PE on running wheels also increased the time spent in the center of the OF in SHR rats only. In experiment 3, SI decreased central and peripheral locomotion in the OF in both strains. In summary, forced PE on a treadmill reduced anxiety-like behavior and increased locomotion in male rats of both strains, whereas voluntary PE on running wheels decreased anxiety-like behavior in SHR rats only. SI decreased locomotion in both strains in the OF. This study suggests that spontaneous activity levels are genotype-dependent and the effects of PE depend on the type of exercise performed., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The gateway hypothesis posits that initial exposure to legal drugs promotes subsequent addiction to illicit drugs. However, epidemiological studies are correlational and cannot rule out the alternative hypothesis of shared addiction vulnerability to legal and illegal drugs. We tested the gateway hypothesis using established rat alcohol exposure procedures and cocaine self-administration and reinstatement (relapse) procedures. We gave Wistar or alcohol-preferring (P) rats intermittent access to water or 20% alcohol in their homecage for 7 weeks (three 24-h sessions/week). We also exposed Wistar rats to air or intoxicating alcohol levels in vapor chambers for 14-h/day for 7 weeks. We then tested the groups of rats for acquisition of cocaine self-administration using ascending cocaine doses (0.125, 0.25, 0.5, 1.0 mg/kg/infusion) followed by a dose-response curve after acquisition of cocaine self-administration. We then extinguished lever pressing and tested the rats for reinstatement of drug seeking induced by cocaine-paired cues and cocaine priming (0, 2.5, 5, 10 mg/kg, i.p.). Wistar rats consumed moderate amounts of alcohol (4.6 g/kg/24 h), P rats consumed higher amounts of alcohol (7.6 g/kg/24 h), and Wistar rats exposed to alcohol vapor had a mean blood alcohol concentration of 176.2 mg/dl during the last week of alcohol exposure. Alcohol pre-exposure had no effect on cocaine self-administration, extinction responding, and reinstatement of drug seeking. Pre-exposure to moderate, high, or intoxicating levels of alcohol had no effect on cocaine self-administration and relapse to cocaine seeking. Our data do not support the notion that alcohol is a gateway drug to cocaine.

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 GABA A 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 GABA A 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., (Copyright © 2017 the authors 0270-6474/17/371140-17$15.00/0.)

The transition from moderate to compulsive alcohol drinking is driven by increasingly dysfunctional reward and stress systems. We review behavioral and pharmacological studies of alcohol self-administration in rats that were mainly conducted within the framework of the alcohol vapor model of dependence. We discuss neurotransmitter systems that are implicated in alcohol drinking, with a focus on contrasting those neurotransmitter systems that drive behavior in the dependent vs . nondependent states. We hypothesize that the identification of systems that become increasingly dysfunctional in alcohol dependence will reveal possible targets for successful interventions to reduce the motivation that drives compulsive alcohol drinking. In our opinion, drugs that (1) normalize, rather than block, a hypofunctional reward system via restoration of the function of hypothalamic stress systems, and (2) desensitize extrahypothalamic stress systems have the potential to selectively and effectively curb compulsive alcohol drinking., Competing Interests: Conflict of interest The authors declare no conflicts of interest.

Although women appear to be more vulnerable to alcohol-induced pathophysiology than men, the neurobiological basis for sex differences is largely unknown, partially because most studies on alcohol drinking are conducted in male subjects only. The present study examined sex differences in alcohol consumption in two rat strains, Long Evans and Wistar, using multiple behavioral paradigms. The effects of the estrous cycle on alcohol consumption were monitored throughout the study. The results indicated that females drank more alcohol than males when given either continuous or intermittent access to alcohol (vs. water) in their home cages (voluntary drinking). Under operant conditions, no sex or strain differences were found in drinking prior to development of alcohol dependence. However, upon dependence induction by chronic, intermittent alcohol vapor exposure, Wistar rats of both sexes substantially escalated their alcohol intake compared with their nondependent drinking levels, whereas Long Evans rats only exhibited a moderate escalation of drinking. Under these conditions, the estrous cycle had no effect on alcohol drinking in any strain and drinking model. Thus, strain, sex, and drinking conditions interact to modulate nondependent and dependent alcohol drinking. The present results emphasize the importance of including sex and strain as biological variables in exploring individual differences in alcohol drinking and dependence., (Published by Elsevier Inc.)

In the past years, a significant volume of research has implicated the appetitive hormone ghrelin in the mechanisms underlying drug use and addiction. From a neuroscientific standpoint, ghrelin modulates both reward and stress pathways, two key drivers of substance use behaviors. Previous investigations support a connection between the ghrelin system and alcohol, stimulants, and tobacco use in both animals and humans, while the research on opioids and cannabis is scarce. In general, upregulation of the ghrelin system seems to enhance craving for drugs as well as substances use. On the other hand, acute and chronic exposure to drugs of abuse influences the ghrelin system at different levels. This chapter summarizes the literature on the relationship between the ghrelin system and substance-related behaviors. We also review recent work investigating the ghrelin system as a potential pharmacological target for treating substance use disorders and discuss the need for additional research., (2017 Published by Elsevier Inc.)

Unlabelled: 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., (Copyright © 2016 the authors 0270-6474/16/369446-08$15.00/0.)

Unlabelled: Cocaine exposure alters brain-derived neurotrophic factor (BDNF) expression in the brain. BDNF signaling through TrkB receptors differentially modulates cocaine self-administration, depending on the brain regions involved. In the present study, we determined how brain-wide inhibition of TrkB signaling affects cocaine intake, the motivation for the drug, and reinstatement of drug taking after extinction. To overcome the inability of TrkB ligands to cross the blood-brain barrier, the TrkB antagonist cyclotraxin-B was fused to the nontoxic transduction domain of the tat protein from human immunodeficiency virus type 1 (tat-cyclotraxin-B). Intravenous injection of tat-cyclotraxin-B dose-dependently reduced cocaine intake, motivation for cocaine (as measured under a progressive ratio schedule of reinforcement), and reinstatement of cocaine taking in rats allowed either short or long access to cocaine self-administration. In contrast, the treatment did not affect operant responding for a highly palatable sweet solution, demonstrating that the effects of tat-cyclotraxin-B are specific for cocaine reinforcement. Cocaine self-administration increased TrkB signaling and activated the downstream Akt pathway in the nucleus accumbens, and had opposite effects in the prefrontal cortex. Pretreatment with tat-cyclotraxin-B normalized protein levels in these two dopamine-innervated brain regions. Cocaine self-administration also increased TrkB signaling in the ventral tegmental area, where the dopaminergic projections originate, but pretreatment with tat-cyclotraxin-B did not alter this effect. Altogether, our data show that systemic administration of a brain-penetrant TrkB antagonist leads to brain region-specific effects and may be a potential pharmacological strategy for the treatment of cocaine addiction., Significance Statement: Brain-derived neurotrophic factor (BDNF) signaling through TrkB receptors plays a well established role in cocaine reinforcement. However, local manipulation of BDNF signaling yields divergent effects, depending on the brain region, thereby questioning the viability of systemic TrkB targeting for the treatment of cocaine use disorders. Our study provides first-time evidence that systemic administration of a brain-penetrant TrkB antagonist (tat-cyclotraxin-B) reduces several behavioral measures of cocaine dependence, without altering motor performance or reinforcement by a sweet palatable solution. In addition, although cocaine self-administration produced opposite effects on TrkB signaling in the nucleus accumbens and prefrontal cortex, tat-cyclotraxin-B administration normalized these cocaine-induced changes in both brain regions., (Copyright © 2016 the authors 0270-6474/16/368150-11$15.00/0.)

The identification of new and more effective treatments for alcohol abuse remains a priority. Alcohol intake activates glucocorticoids, which have a key role in alcohol's reinforcing properties. Glucocorticoid effects are modulated in part by the activity of 11β-hydroxysteroid dehydrogenases (11β-HSD) acting as pre-receptors. Here, we tested the effects on alcohol intake of the 11β-HSD inhibitor carbenoxolone (CBX, 18β-glycyrrhetinic acid 3β-O-hemisuccinate), which has been extensively used in the clinic for the treatment of gastritis and peptic ulcer and is active on both 11β-HSD1 and 11β-HSD2 isoforms. We observed that CBX reduces both baseline and excessive drinking in rats and mice. The CBX diastereomer 18α-glycyrrhetinic acid 3β-O-hemisuccinate (αCBX), which we found to be selective for 11β-HSD2, was also effective in reducing alcohol drinking in mice. Thus, 11β-HSD inhibitors may be a promising new class of candidate alcohol abuse medications, and existing 11β-HSD inhibitor drugs may be potentially re-purposed for alcohol abuse treatment.

Chronic alcohol consumption disrupts glucocorticoid signaling at multiple physiological levels to interact with several disease-related processes associated with neuroendocrine and psychiatric disorders. Excessive alcohol use produces stress-related neuroadaptations at the level of the hypothalamic-pituitary-adrenal (HPA) axis as well as within central (extra-hypothalamic) neural circuitry, including the central amygdala (CeA) and prefrontal cortex (PFC). Altered glucocorticoid receptor (GR) signaling in these areas following excessive alcohol exposure is postulated to mediate the transition from recreational drinking to dependence, as well as the manifestation of a host of cognitive and neurological deficits. Specifically, a bidirectional regulation of stress systems by glucocorticoids leads to the development of an HPA axis tolerance and a concomitant sensitization of cortical and subcortical circuitries. A greater understanding of how hypothalamic and extra-hypothalamic glucocorticoid systems interact to mediate excessive drinking and related pathologies will lead to more effective therapeutic strategies for alcohol use disorder (AUD) and closely related comorbidities., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The combination of alcohol with an energy drink (ED) is believed to contribute to risky alcohol-drinking behaviors, such as binge drinking. However, the long-term effects on cognition and reward function that are caused by the repeated binge-like ingestion of alcohol and EDs are still poorly known. The present study examined the effects of a history of repeated exposure to alcohol and/or an ED on short-term memory and alcohol-seeking behavior. Male Wistar rats were given daily intragastric administration of alcohol (3.4g/kg) combined or not with an ED (10.71ml/kg) for 6 consecutive days. The rats were tested for locomotion 15min after the first intragastric treatment. Short-term memory was assessed in the novel object recognition and social discrimination tests 2-3days after the last intragastric administration. The rewarding effect of alcohol was tested 1-3weeks following the last intragastric administration in a conditioned place preference paradigm. The acute binge-like ingestion of alcohol decreased locomotor activity, whereas the combination of alcohol and an ED increased locomotion in the first minutes of assessment. Alcohol exposure produced cognitive deficits in both the object recognition and social discrimination tests, and adding the ED to the alcohol solution did not modify these effects. The combination of alcohol and the ED increased alcohol-induced conditioned place preference. Thus, a history of binge-like alcohol exposure combined with the ED caused subsequent cognitive deficits and increased alcohol seeking behavior, and such behavioral effects might contribute to the progression to alcohol abuse disorders., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Alcoholism, or alcohol use disorder, is a major public health concern that is a considerable risk factor for morbidity and disability; therefore, effective treatments are urgently needed. Here, we demonstrated that the glucocorticoid receptor (GR) antagonist mifepristone reduces alcohol intake in alcohol-dependent rats but not in nondependent animals. Both systemic delivery and direct administration into the central nucleus of the amygdala, a critical stress-related brain region, were sufficient to reduce alcohol consumption in dependent animals. We also tested the use of mifepristone in 56 alcohol-dependent human subjects as part of a double-blind clinical and laboratory-based study. Relative to placebo, individuals who received mifepristone (600 mg daily taken orally for 1 week) exhibited a substantial reduction in alcohol-cued craving in the laboratory, and naturalistic measures revealed reduced alcohol consumption during the 1-week treatment phase and 1-week post-treatment phase in mifepristone-treated individuals. Mifepristone was well tolerated and improved liver-function markers. Together, these results support further exploration of GR antagonism via mifepristone as a therapeutic strategy for alcoholism.

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., (Copyright © 2015 the authors 0270-6474/15/356241-13$15.00/0.)

Recent studies have suggested an association between alcoholism and DNA methylation, a mechanism that can mediate long-lasting changes in gene transcription. Here, we examined the contribution of DNA methylation to the long-term behavioral and molecular changes induced by a history of alcohol dependence. In search of mechanisms underlying persistent rather than acute dependence-induced neuroadaptations, we studied the role of DNA methylation regulating medial prefrontal cortex (mPFC) gene expression and alcohol-related behaviors in rats 3 weeks into abstinence following alcohol dependence. Postdependent rats showed escalated alcohol intake, which was associated with increased DNA methylation as well as decreased expression of genes encoding synaptic proteins involved in neurotransmitter release in the mPFC. Infusion of the DNA methyltransferase inhibitor RG108 prevented both escalation of alcohol consumption and dependence-induced downregulation of 4 of the 7 transcripts modified in postdependent rats. Specifically, RG108 treatment directly reversed both downregulation of synaptotagmin 2 (Syt2) gene expression and hypermethylation on CpG#5 of its first exon. Lentiviral inhibition of Syt2 expression in the mPFC increased aversion-resistant alcohol drinking, supporting a mechanistic role of Syt2 in compulsive-like behavior. Our findings identified a functional role of DNA methylation in alcohol dependence-like behavioral phenotypes and a candidate gene network that may mediate its effects. Together, these data provide novel evidence for DNA methyltransferases as potential therapeutic targets in alcoholism., (Copyright © 2015 the authors 0270-6474/15/356153-12$15.00/0.)

The hypocretin/orexin (HCRT) system has been associated with both positive and negative drug reinforcement, implicating HCRT receptor 1 (HCRT-R1) signaling in drug-related behaviors for all major drug classes, including opioids. However, to date there are limited studies investigating the role of HCRT receptor 2 (HCRT-R2) signaling in compulsive-like drug seeking. Escalation of drug intake with extended access has been suggested to model the transition from controlled drug use to compulsive-like drug seeking/taking. The current study examined the effects of a HCRT-R2 antagonist, NBI-80713, on heroin self-administration in rats allowed short- (1 h; ShA) or long- (12 h; LgA) access to intravenous heroin self-administration. Results indicate that systemically administered NBI-80713 dose-dependently decreased heroin self-administration in LgA, but not in ShA, animals. Quantitative PCR analyses showed an increase in Hcrtr2 mRNA levels in the central amygdala, a stress-related brain region, of LgA rats. These observations suggest a functional role for HCRT-R2 signaling in compulsive-like heroin self-administration associated with extended access and indicate HCRT-R2 antagonism as a potential pharmacological target for the treatment of heroin dependence.

Given that the κ opioid receptor (KOR) system has been implicated in psychostimulant abuse, we evaluated whether the selective KOR antagonist norbinaltorphimine dihydrochloride (nor-BNI) would attenuate the escalation of methamphetamine (METH) intake in an extended-access self-administration model. Systemic nor-BNI decreased the escalation of intake of long-access (LgA) but not short-access (ShA) self-administration. nor-BNI also decreased elevated progressive-ratio (PR) breakpoints in rats in the LgA condition and continued to decrease intake after 17 d of abstinence, demonstrating that the effects of a nor-BNI injection are long lasting. Rats with an ShA history showed an increase in prodynorphin immunoreactivity in both the nucleus accumbens (NAc) core and shell, but LgA animals showed a selective increase in the NAc shell. Other cohorts of rats received nor-BNI directly into the NAc shell or core and entered into ShA or LgA. nor-BNI infusion in the NAc shell, but not NAc core, attenuated escalation of intake and PR responding for METH in LgA rats. These data indicate that the development and/or expression of compulsive-like responding for METH under LgA conditions depends on activation of the KOR system in the NAc shell and suggest that the dynorphin-KOR system is a central component of the neuroplasticity associated with negative reinforcement systems that drive the dark side of addiction., (Copyright © 2015 the authors 0270-6474/15/354296-10$15.00/0.)

Opioids represent effective drugs for the relief of pain, yet chronic opioid use often leads to a state of increased sensitivity to pain that is exacerbated during withdrawal. A sensitization of pain-related negative affect has been hypothesized to closely interact with addiction mechanisms. Neuro-adaptive changes occur as a consequence of excessive opioid exposure, including a recruitment of corticotropin-releasing factor (CRF) and norepinephrine (NE) brain stress systems. To better understand the mechanisms underlying the transition to dependence, we determined the effects of functional antagonism within these two systems on hyperalgesia-like behavior during heroin withdrawal utilizing models of both acute and chronic dependence. We found that passive or self-administered heroin produced a significant mechanical hypersensitivity. During acute opioid dependence, systemic administration of the CRF1 receptor antagonist MPZP (20 mg/kg) alleviated withdrawal-induced mechanical hypersensitivity. In contrast, several functional adrenergic system antagonists (clonidine, prazosin, propranolol) failed to alter mechanical hypersensitivity in this state. We then determined the effects of chronic MPZP or clonidine treatment on extended access heroin self-administration and found that MPZP, but not clonidine, attenuated escalation of heroin intake, whereas both drugs alleviated chronic dependence-associated hyperalgesia. These findings suggest that an early potentiation of CRF signaling occurs following opioid exposure that begins to drive both opioid-induced hyperalgesia and eventually intake escalation., (© 2013 Society for the Study of Addiction.)

Background: A systems biology approach based on the assembly and interrogation of gene regulatory networks, or interactomes, was used to study neuroadaptation processes associated with the transition to alcohol dependence at the molecular level., Results: Using a rat model of dependent and non-dependent alcohol self-administration, we reverse engineered a global transcriptional regulatory network during protracted abstinence, a period when relapse rates are highest. We then interrogated the network to identify master regulator genes that mechanistically regulate brain region-specific signatures associated with dependent and non-dependent alcohol self-administration. Among these, the gene coding for the glucocorticoid receptor was independently identified as a master regulator in multiple brain regions, including the medial prefrontal cortex, nucleus accumbens, central nucleus of the amygdala, and ventral tegmental area, consistent with the view that brain reward and stress systems are dysregulated during protracted abstinence. Administration of the glucocorticoid antagonist mifepristone in either the nucleus accumbens or ventral tegmental area selectively decreased dependent, excessive, alcohol self-administration in rats but had no effect on non-dependent, moderate, alcohol self-administration., Conclusions: Our study suggests that assembly and analysis of regulatory networks is an effective strategy for the identification of key regulators of long-term neuroplastic changes within specific brain regions that play a functional role in alcohol dependence. More specifically, our results support a key role for regulatory networks downstream of the glucocorticoid receptor in excessive alcohol drinking during protracted alcohol abstinence.

The abuse of prescription opioids that are used for the treatment of chronic pain is a major public health concern, costing ∼$53.4 billion annually in lost wages, health-care costs, and criminal costs. Although opioids remain a first-line therapy for the treatment of severe chronic pain, practitioners remain cautious because of the potential for abuse and addiction. Opioids such as heroin are considered very rewarding and reinforcing, but direct and systematic comparisons of compulsive intake between commonly prescribed opioids and heroin in animal models have not yet been performed. In the present study, we evaluated the potential for compulsive-like drug seeking and taking, using intravenous self-administration of oxycodone, fentanyl, and buprenorphine in rats allowed long access sessions (12 h). We measured compulsive-like intake using an established escalation model and responding on a progressive ratio schedule of reinforcement. We compared the potential for compulsive-like self-administration of these prescription opioids and heroin, which has been previously established to induce increasing intake that models the transition to addiction in humans. We found that animals that self-administered oxycodone, fentanyl, or heroin, but not buprenorphine had similar profiles of escalation and increases in breakpoints. The use of extended access models of prescription opioid intake will help better understand the biological factors that underlie opioid dependence.

Electrophysiological data suggest a dual role of Y2 receptors (Y2 Rs) as autoreceptors regulating neuropeptide Y release and heteroceptors regulating gamma-aminobutyric acid release in the central amygdala (CeA). Here, we report that neither systemic (JNJ-31020028) nor intra-CeA (BIIE0246) Y2 R antagonism altered operant alcohol responding by alcohol-dependent or non-dependent rats. Conversely, BIIE0246 in the CeA reduced anxiety-like behavior in alcohol-dependent and alcohol-naïve rats. The finding that Y2 R antagonism reduces anxiety-like behavior but not alcohol drinking suggests that these two effects may occur via different functions of the Y2 R (e.g. autoreceptor versus heteroceptor function)., (© 2013 Society for the Study of Addiction.)

Rats emit 50kHz ultrasonic vocalizations (USVs) in situations of increased motivation, such as during the anticipation of palatable food or drugs of abuse. Whether the same holds true for the anticipation of alcohol intake remains unknown. Alcohol drinking in a nondependent state is thought to be mediated by its rewarding effects (positive reinforcement), whereas drinking in the dependent state is motivated by alcohol's stress-relieving effects (negative reinforcement). Here, we measured context-elicited 50kHz USVs in alcohol-dependent (alcohol vapor-exposed) and nondependent rats immediately before operant alcohol self-administration sessions. Dependent rats showed escalated levels of alcohol intake compared with nondependent rats. Overall, dependent and nondependent rats showed similar levels of anticipatory 50kHz USVs. However, the number of anticipatory USVs was positively correlated with alcohol intake in dependent rats but not nondependent rats. Additionally, dependent rats with higher alcohol intake displayed increased anticipatory 50kHz USVs compared with rats that had lower alcohol intake, whereas no difference was observed between rats with high and low alcohol intake in the nondependent group. Increased 50kHz USVs were specific for the anticipation of alcohol self-administration and did not generalize to a novel environment. These findings suggest that anticipatory 50kHz USVs may be an indicator of context-elicited negative reinforcement learning., (Copyright © 2014. Published by Elsevier B.V.)

Accumulating evidence demonstrates a functional role for the hippocampus in mediating relapse to cocaine-seeking behavior and extinction-induced inhibition of cocaine seeking, and dentate gyrus neurogenesis in the hippocampus may have a role. Here, we tested the hypothesis that disruption of normal hippocampal activity during extinction alters relapse to cocaine-seeking behavior as a function of dentate gyrus neurogenesis. Adult rats were trained to self-administer cocaine on a fixed-ratio schedule, followed by extinction and cocaine-primed reinstatement testing. Some rats received low-frequency stimulation (LFS; 2 Hz for 25 minutes) after each extinction session in the dorsal or ventral hippocampal formation. All rats received an injection of the mitotic marker 5-bromo-2'-deoxyuridine (BrdU) to label developing dentate gyrus neurons during self-administration, as well as before or after extinction and LFS. We found that LFS during extinction did not alter extinction behavior but enhanced cocaine-primed reinstatement. Cocaine self-administration reduced levels of 24-day-old BrdU cells and dentate gyrus neurogenesis, which was normalized by extinction. LFS during extinction prevented extinction-induced normalization of dentate gyrus neurogenesis and potentiated cocaine-induced reinstatement of drug seeking. LFS inhibition of extinction-induced neurogenesis was not due to enhanced cell death, revealed by quantification of activated caspase3-labeled cells. These data suggest that LFS during extinction disrupts hippocampal networking by disrupting neurogenesis and also strengthens relapse-like behaviors. Thus, newly born dentate gyrus neurons during withdrawal and extinction learning facilitate hippocampal networking that mediates extinction-induced inhibition of cocaine seeking and may play a key role in preventing relapse., (© 2012 The Authors, Addiction Biology © 2012 Society for the Study of Addiction.)

A history of stress produces increases in rodent relapse-like alcohol self-administration behavior and regional brain gene expression of phosphodiesterase 10A (PDE10A), a dual-specificity cyclic adenosine monophosphate/cyclic guanosine monophosphate-inhibiting enzyme. Here, we tested the hypothesis that administration of TP-10, a specific PDE10A inhibitor, would reduce alcohol self-administration in conditions predisposing to elevated self-administration. TP-10 administration dose-dependently (0.562, 1.0 mg/kg; subcutaneously) reduced relapse-like alcohol self-administration regardless of stress history enhancement of relapse-like behavior. TP-10 also reduced alcohol self-administration in genetically alcohol-preferring rats, as well as in alcohol-non-dependent and -dependent rats. Effective systemic TP-10 doses did not alter alcohol pharmacokinetics, significantly reduce motor activity or intrabout operant response speed, or promote a conditioned place aversion. TP-10 also reduced saccharin self-administration, suggesting a general role for PDE10A in the self-administration of reinforcing substances. PDE10A inhibition in the dorsolateral striatum, but not the nucleus accumbens, reduced alcohol self-administration. Taken together, the results implicate dorsolateral striatum PDE10A in facilitating alcohol intake and support further investigation of PDE10A systems in the pathophysiology and potential treatment of substance use disorders.

Alcoholism (alcohol dependence) is characterized by a compulsion to seek and ingest alcohol (ethanol), loss of control over intake, and the emergence of a negative emotional state during withdrawal. Animal models are critical in promoting our knowledge of the neurobiological mechanisms underlying alcohol dependence. Here, we review the studies involving operant alcohol self-administration in rat models of alcohol dependence and withdrawal with the focus on the alcohol vapor model. In 1996, the first articles were published reporting that rats made dependent on alcohol by exposure to alcohol vapors displayed increased operant alcohol self-administration during acute withdrawal compared with nondependent rats (i.e., not exposed to alcohol vapors). Since then, it has been repeatedly demonstrated that this model reliably produces physical and motivational symptoms of alcohol dependence. The functional roles of various systems implicated in stress and reward, including opioids, dopamine, corticotropin-releasing factor (CRF), glucocorticoids, neuropeptide Y (NPY), γ-aminobutyric acid (GABA), norepinephrine, and cannabinoids, have been investigated in the context of alcohol dependence. The combination of models of alcohol withdrawal and dependence with operant self-administration constitutes an excellent tool to investigate the neurobiology of alcoholism. In fact, this work has helped lay the groundwork for several ongoing clinical trials for alcohol dependence. Advantages and limitations of this model are discussed, with an emphasis on what future directions of great importance could be., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Rationale: Fifty kilohertz ultrasonic vocalizations (USVs) have been sometimes shown to reflect positive affective-like states in rats. Rewarding events, such as access to palatable food or drugs of abuse, increase the number of anticipatory 50-kHz USVs. However, little is known about the predictability of USVs, subtypes of USVs involved, and underlying neurobiological mechanisms., Objectives: We examined whether cue-induced anticipatory 50-kHz USVs predict palatable food intake and tested the effects of dopamine D1 and μ-opioid receptor antagonism on anticipatory USVs., Materials: Food-restricted rats received repeated sessions of a 2-min cue light immediately followed by a 5-min access to palatable food. Ultrasonic vocalizations were recorded during cue presentation. After 24 pairing sessions, the rats were pretreated with the D1 receptor antagonist SCH 23390 (5, 10, and 20 μg/kg) and μ-opioid receptor antagonist naltrexone (0.03, 0.06, 0.13, 0.25, 0.5, and 1 mg/kg) in a Latin-square design, and USVs were recorded during cue presentation., Results: Rats emitted 50-kHz USVs during cue presentation, and the number of USVs increased across sessions with robust and stable interindividual differences. Escalation in USVs was subtype-dependent, with nontrill calls significantly increasing over time. Palatable food intake was positively correlated with anticipatory 50-kHz USVs. Moreover, anticipatory USVs were dose-dependently prevented by antagonism of D1 and μ-opioid receptors., Conclusions: These findings demonstrate that anticipatory 50-kHz USVs represent a stable phenotype of increased motivation for food, and dopamine and opioid systems appear to mediate anticipatory 50-kHz USVs.

Drug addiction has been conceptualized as a chronically relapsing disorder of compulsive drug seeking and taking that progresses through three stages: binge/intoxication, withdrawal/negative affect, and preoccupation/anticipation. Drug addiction impacts multiple motivational mechanisms and can be conceptualized as a disorder that progresses from positive reinforcement (binge/intoxication stage) to negative reinforcement (withdrawal/negative affect stage). The construct of negative reinforcement is defined as drug taking that alleviates a negative emotional state. Our hypothesis is that the negative emotional state that drives such negative reinforcement is derived from dysregulation of key neurochemical elements involved in the brain stress systems within the frontal cortex, ventral striatum, and extended amygdala. Specific neurochemical elements in these structures include not only recruitment of the classic stress axis mediated by corticotropin-releasing factor (CRF) in the extended amygdala as previously hypothesized but also recruitment of dynorphin-κ opioid aversive systems in the ventral striatum and extended amygdala. Additionally, we hypothesized that these brain stress systems may be engaged in the frontal cortex early in the addiction process. Excessive drug taking engages activation of CRF not only in the extended amygdala, accompanied by anxiety-like states, but also in the medial prefrontal cortex, accompanied by deficits in executive function that may facilitate the transition to compulsive-like responding. Excessive activation of the nucleus accumbens via the release of mesocorticolimbic dopamine or activation of opioid receptors has long been hypothesized to subsequently activate the dynorphin-κ opioid system, which in turn can decrease dopaminergic activity in the mesocorticolimbic dopamine system. Blockade of the κ opioid system can also block anxiety-like and reward deficits associated with withdrawal from drugs of abuse and block the development of compulsive-like responding during extended access to drugs of abuse, suggesting another powerful brain stress/anti-reward system that contributes to compulsive drug seeking. Thus, brain stress response systems are hypothesized to be activated by acute excessive drug intake, to be sensitized during repeated withdrawal, to persist into protracted abstinence, and to contribute to the development and persistence of addiction. The recruitment of anti-reward systems provides a powerful neurochemical basis for the negative emotional states that are responsible for the dark side of addiction. This article is part of a Special Issue entitled 'NIDA 40th Anniversary Issue'., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The abuse of opioid drugs, both illicit and prescription, is a persistent problem in the United States, accounting for >1.2 million users who require treatment each year. Current treatments rely on suppressing immediate withdrawal symptoms and replacing illicit drug use with long-acting opiate drugs. However, the mechanisms that lead to preventing opiate dependence are still poorly understood. We hypothesized that κ opioid receptor (KOR) activation during chronic opioid intake contributes to negative affective states associated with withdrawal and the motivation to take increasing amounts of heroin. Using a 12 h long-access model of heroin self-administration, rats showed escalation of heroin intake over several weeks. This was prevented by a single high dose (30 mg/kg) of the long-acting KOR antagonist norbinaltorphimine (nor-BNI), paralleled by reduced motivation to respond for heroin on a progressive-ratio schedule of reinforcement, a measure of compulsive-like responding. Systemic nor-BNI also significantly decreased heroin withdrawal-associated anxiety-like behavior. Immunohistochemical analysis showed prodynorphin content increased in the nucleus accumbens core in all heroin-exposed rats, but selectively increased in the nucleus accumbens shell in long-access rats. Local infusion of nor-BNI (4 μg/side) into accumbens core altered the initial intake of heroin but not the rate of escalation, while local injection into accumbens shell selectively suppressed increases in heroin intake over time without altering initial intake. These data suggest that dynorphin activity in the nucleus accumbens mediates the increasing motivation for heroin taking and compulsive-like responding for heroin, suggesting that KOR antagonists may be promising targets for the treatment of opioid addiction.

Anxiety is one of the early symptoms of opioid withdrawal and contributes to continued drug use and relapse. The acoustic startle response (ASR) is a component of anxiety that has been shown to increase during opioid withdrawal in both humans and animals. We investigated the role of corticotropin-releasing factor (CRF) and norepinephrine (NE), two key mediators of the brain stress system, on acute heroin withdrawal-potentiated ASR. Rats injected with heroin (2 mg/kg s.c.) displayed an increased ASR when tested 4 h after heroin treatment. A similar increase in ASR was found in rats 10-20 h into withdrawal from extended access (12 h) to i.v. heroin self-administration, a model that captures several aspects of heroin addiction in humans. Both the α 2 adrenergic receptor agonist clonidine (10 μg/kg s.c.) and CRF1 receptor antagonist N,N-bis(2-methoxyethyl)-3-(4-methoxy-2-methylphenyl)-2,5-dimethyl-pyrazolo[1,5-a] pyrimidin-7-amine (MPZP; 20 mg/kg s.c.) blocked heroin withdrawal-potentiated startle. To investigate the relationship between CRF1 and α 2 adrenergic receptors in the potentiation of the ASR, we tested the effect of MPZP on yohimbine (1.25 mg/kg s.c.)-potentiated startle and clonidine on CRF (2 μg i.c.v.)-potentiated startle. Clonidine blocked CRF-potentiated startle, whereas MPZP partially attenuated but did not reverse yohimbine-potentiated startle, suggesting that CRF may drive NE release to potentiate startle. These results suggest that CRF1 and α 2 receptors play an important role in the heightened anxiety-like behaviour observed during acute withdrawal from heroin, possibly via CRF inducing the release of NE in stress-related brain regions.

Heroin addiction, a chronic relapsing disorder characterized by excessive drug taking and seeking, requires constant psychotherapeutic and pharmacotherapeutic interventions to minimize the potential for further abuse. Vaccine strategies against many drugs of abuse are being developed that generate antibodies that bind drug in the bloodstream, preventing entry into the brain and nullifying psychoactivity. However, this strategy is complicated by heroin's rapid metabolism to 6-acetylmorphine and morphine. We recently developed a "dynamic" vaccine that creates antibodies against heroin and its psychoactive metabolites by presenting multihaptenic structures to the immune system that match heroin's metabolism. The current study presents evidence of effective and continuous sequestration of brain-permeable constituents of heroin in the bloodstream following vaccination. The result is efficient blockade of heroin activity in treated rats, preventing various features of drugs of abuse: heroin reward, drug-induced reinstatement of drug seeking, and reescalation of compulsive heroin self-administration following abstinence in dependent rats. The dynamic vaccine shows the capability to significantly devalue the reinforcing and motivating properties of heroin, even in subjects with a history of dependence. In addition, targeting a less brain-permeable downstream metabolite, morphine, is insufficient to prevent heroin-induced activity in these models, suggesting that heroin and 6-acetylmorphine are critical players in heroin's psychoactivity. Because the heroin vaccine does not target opioid receptors or common opioid pharmacotherapeutics, it can be used in conjunction with available treatment options. Thus, our vaccine represents a promising adjunct therapy for heroin addiction, providing continuous heroin antagonism, requiring minimal medical monitoring and patient compliance.

Genetic deletion of the neurokinin 1 receptor (NK1R) has been shown to decrease the reinforcing properties of opioids, but it is unknown whether pharmacological NK1R blockade has the same effect. Here, we examined the effect of L822429, a rat-specific NK1R antagonist, on the reinforcing properties of heroin in rats on short (1 h: ShA) or long (12 h: LgA) access to intravenous heroin self-administration. ShA produces heroin self-administration rates that are stable over time, whereas LgA leads to an escalation of heroin intake thought to model important dependence-related aspects of addiction. L822429 reduced heroin self-administration and the motivation to consume heroin, measured using a progressive-ratio schedule, in both ShA and LgA rats. L822429 also decreased anxiety-like behavior in both groups, measured on the elevated plus maze, but did not affect mechanical hypersensitivity observed in LgA rats. Expression of TacR1 (the gene encoding NK1R) was decreased in reward- and stress-related brain areas both in ShA and LgA rats compared with heroin-naïve rats, but did not differ between the two heroin-experienced groups. In contrast, passive exposure to heroin produced increases in TacR1 expression in the prefrontal cortex and nucleus accumbens. Taken together, these results show that pharmacological NK1R blockade attenuates heroin reinforcement. The observation that animals with ShA and LgA to heroin were similarly affected by L822429 indicates that the SP/NK1R system is not specifically involved in neuroadaptations that underlie escalation resulting from LgA self-administration. Instead, the NK1R antagonist appears to attenuate acute, positively reinforcing properties of heroin and may be useful as an adjunct to relapse prevention in detoxified opioid-dependent subjects.

Buprenorphine, a synthetic opioid that acts at both μ and κ opioid receptors, can decrease cocaine use in individuals with opioid addiction. However, the potent agonist action of buprenorphine at μ opioid receptors raises its potential for creating opioid dependence in non-opioid-dependent cocaine abusers. Here, we tested the hypothesis that a combination of buprenorphine and naltrexone (a potent μ opioid antagonist with weaker δ and κ antagonist properties) could block compulsive cocaine self-administration without producing opioid dependence. The effects of buprenorphine and various doses of naltrexone on cocaine self-administration were assessed in rats that self-administered cocaine under conditions of either short access (noncompulsive cocaine seeking) or extended access (compulsive cocaine seeking). Buprenorphine alone reproducibly decreased cocaine self-administration. Although this buprenorphine-alone effect was blocked in a dose-dependent manner by naltrexone in both the short-access and the extended-access groups, the combination of the lowest dose of naltrexone with buprenorphine blocked cocaine self-administration in the extended-access group but not in the short-access group. Rats given this low dose of naltrexone with buprenorphine did not exhibit the physical opioid withdrawal syndrome seen in rats treated with buprenorphine alone, and naltrexone at this dose did not block κ agonist-induced analgesia. The results suggest that the combination of buprenorphine and naltrexone at an appropriate dosage decreases compulsive cocaine self-administration with minimal liability to produce opioid dependence and may be useful as a treatment for cocaine addiction.

Alcoholism is characterized by a compulsion to seek and ingest alcohol, loss of control over intake, and the emergence of a negative emotional state during abstinence. We hypothesized that sustained activation of neuroendocrine stress systems (e.g., corticosteroid release via the hypothalamic-pituitary-adrenal axis) by alcohol intoxication and withdrawal and consequent alterations in glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) activation drive compulsive alcohol drinking. Our results showed that rats exposed to alcohol vapor to the point of dependence displayed increased alcohol intake, compulsive drinking measured by progressive-ratio responding, and persistent alcohol consumption despite punishment, assessed by adding quinine to the alcohol solution, compared with control rats that were not exposed to alcohol vapor. No group differences were observed in the self-administration of saccharin-sweetened water. Acute alcohol withdrawal was accompanied by downregulated GR mRNA in various stress/reward-related brain regions [i.e., prefrontal cortex, nucleus accumbens (NAc), and bed nucleus of the stria terminalis (BNST)], whereas protracted alcohol abstinence was accompanied by upregulated GR mRNA in the NAc core, ventral BNST, and central nucleus of the amygdala. No significant alterations in MR mRNA levels were found. Chronic GR antagonism with mifepristone (RU38486) prevented the escalation of alcohol intake and compulsive responding induced by chronic, intermittent alcohol vapor exposure. Chronic treatment with mifepristone also blocked escalated alcohol drinking and compulsive responding during protracted abstinence. Thus, the GR system appears to be involved in the development of alcohol dependence and may represent a potential pharmacological target for the treatment of alcoholism.

Animal models of drug dependence have described both reductions in brain reward processes and potentiation of stress-like (or anti-reward) mechanisms, including a recruitment of corticotropin-releasing factor (CRF) signaling. Accordingly, chronic exposure to opiates often leads to the development of mechanical hypersensitivity. We measured paw withdrawal thresholds (PWTs) in male Wistar rats allowed limited (short access group: ShA) or extended (long access group: LgA) access to heroin or cocaine self-administration, or in rats made dependent on ethanol via ethanol vapor exposure (ethanol-dependent group). In heroin self-administering animals, after transition to LgA conditions, thresholds were reduced to around 50% of levels observed at baseline, and were also significantly lower than thresholds measured in animals remaining on the ShA schedule. In contrast, thresholds in animals self-administering cocaine under either ShA (1 h) or LgA (6 h) conditions were unaltered. Similar to heroin LgA rats, ethanol-dependent rats also developed mechanical hypersensitivity after eight weeks of ethanol vapor exposure compared to non-dependent animals. Systemic administration of the CRF1R antagonist MPZP significantly alleviated the hypersensitivity observed in rats dependent on heroin or ethanol. The emergence of mechanical hypersensitivity with heroin and ethanol dependence may thus represent one critical drug-associated negative emotional state driving dependence on these substances. These results also suggest a recruitment of CRF-regulated nociceptive pathways associated with escalation of intake and dependence. A greater understanding of relationships between chronic drug exposure and pain-related states may provide insight into mechanisms underlying the transition to drug addiction, as well as reveal new treatment opportunities. This article is part of a Special Issue entitled 'Post-Traumatic Stress Disorder'., (Copyright © 2011 Elsevier Ltd. All rights reserved.)