Carsten K. Nielsen

The dual orexin/hypocretin receptor antagonist, almorexant, in the ventral tegmental area attenuates ethanol self-administration.

Recent studies have implicated the hypocretin/orexinergic system in reward-seeking behavior. Almorexant, a dual orexin/hypocretin R1 and R2 receptor antagonist, has proven effective in preclinical studies in promoting sleep in animal models and was in Phase III clinical trials for sleep disorders. The present study combines behavioral assays with in vitro biochemical and electrophysiological techniques to elucidate the role of almorexant in ethanol and sucrose intake. Using an operant self-administration paradigm, we demonstrate that systemic administration of almorexant decreased operant self-administration of both 20% ethanol and 5% sucrose. We further demonstrate that intra-ventral tegmental area (VTA) infusions, but not intra-substantia nigra infusions, of almorexant reduced ethanol self-administration. Extracellular recordings performed in VTA neurons revealed that orexin-A increased firing and this enhancement of firing was blocked by almorexant. The results demonstrate that orexin/hypocretin receptors in distinct brain regions regulate ethanol and sucrose mediated behaviors.

A Novel Delta Opioid Receptor Antagonist, SoRI-9409, Produces a Selective and Long-Lasting Decrease in Ethanol Consumption in Heavy-Drinking Rats

BACKGROUND Naltrexone, a compound with high affinity for the mu opioid receptor (MOP-R) reduces alcohol consumption. SoRI-9409 is a derivative of naltrexone that has highest affinity at delta opioid receptors (DOP-Rs). We have investigated the effects of SoRI-9409 on ethanol consumption to determine the consequences of altering the naltrexone compound to a form with increased efficacy at DOP-Rs. METHODS Effects of the opioid receptor antagonists, SoRI-9409 (0-30 mg/kg, IP), naltrexone (0-30 mg/kg, IP), or naltrindole (0-10 mg/kg, IP) on ethanol consumption was measured in high- and low-ethanol-consuming rats with two different drinking paradigms. SoRI-9409-, naltrexone-, and naltrindole-mediated inhibition of DOP-R-stimulated [(35)S]GTP gamma S binding was measured in brain membranes prepared from high-ethanol-consuming rats. The effects of SoRI-9409 on morphine-mediated analgesia, conditioned place preference, and anxiety were also examined. RESULTS In high- but not low-ethanol-consuming animals, SoRI-9409 is threefold more effective and selective at reducing ethanol consumption when compared with naltrexone or naltrindole for up to 24 hours. SoRI-9409 administered daily for 28 days continuously reduced ethanol consumption, and when the administration of SoRI-9409 was terminated, the amount of ethanol consumed remained lower compared with vehicle-treated animals. Furthermore, SoRI-9409 inhibits DOP-R-stimulated [(35)S]GTP gamma S binding in brain membranes of high-ethanol-consuming rats. CONCLUSIONS SoRI-9409 causes selective and long-lasting reductions of ethanol consumption. This suggests that compounds that have high affinity for DOP-Rs such as SoRI-9409 might be promising candidates for development as a novel therapeutic for the treatment of alcoholism.

δ-Opioid Receptor Function in the Dorsal Striatum Plays a Role in High Levels of Ethanol Consumption in Rats

Binge-like patterns of excessive drinking during young adulthood increase the propensity for alcohol use disorders (AUDs) later in adult life; however, the mechanisms that drive this are not completely understood. Previous studies showed that the δ-opioid peptide receptor (DOP-R) is dynamically regulated by exposure to ethanol and that the DOP-R plays a role in ethanol-mediated behaviors. The aim of this study was to determine the role of the DOP-R in high ethanol consumption from young adulthood through to late adulthood by measuring DOP-R-mediated [35S]GTPγS binding in brain membranes and DOP-R-mediated analgesia using a rat model of high ethanol consumption in Long Evans rats. We show that DOP-R activity in the dorsal striatum and DOP-R-mediated analgesia changes during development, being highest during early adulthood and reduced in late adulthood. Intermittent access to ethanol but not continuous ethanol or water from young adulthood leads to an increase in DOP-R activity in the dorsal striatum and DOP-R-mediated analgesia into late adulthood. Multiple microinfusions of naltrindole into the dorsal striatum or multiple systemic administration of naltrindole reduces ethanol consumption, and following termination of treatment, DOP-R activity in the dorsal striatum is attenuated. These findings suggest that DOP-R activity in the dorsal striatum plays a role in high levels of ethanol consumption and suggest that targeting the DOP-R is an alternative strategy for the treatment of AUDs.

Cabergoline decreases alcohol drinking and seeking behaviors via glial cell line-derived neurotrophic factor.

BACKGROUND Cabergoline is an ergotamine derivative that increases the expression of glial cell line-derived neurotrophic factor (GDNF) in vitro. We recently showed that GDNF in the ventral tegmental area (VTA) reduces the motivation to consume alcohol. We therefore set out to determine whether cabergoline administration decreases alcohol-drinking and -seeking behaviors via GDNF. METHODS Reverse transcription polymerase chain reaction (RT-PCR) and Enzyme-Linked ImmunoSorbent Assay (ELISA) were used to measure GDNF levels. Western blot analysis was used for phosphorylation experiments. Operant self-administration in rats and a two-bottle choice procedure in mice were used to assess alcohol-drinking behaviors. Instrumental performance tested during extinction was used to measure alcohol-seeking behavior. The [35S]GTPgammaS binding assay was used to assess the expression and function of the dopamine D2 receptor (D2R). RESULTS We found that treatment of the dopaminergic-like cell line SH-SY5Y with cabergoline and systemic administration of cabergoline in rats resulted in an increase in GDNF level and in the activation of the GDNF pathway. Cabergoline treatment decreased alcohol-drinking and -seeking behaviors including relapse, and its action to reduce alcohol consumption was localized to the VTA. Finally, the increase in GDNF expression and the decrease in alcohol consumption by cabergoline were abolished in GDNF heterozygous knockout mice. CONCLUSIONS Together, these findings suggest that cabergoline-mediated upregulation of the GDNF pathway attenuates alcohol-drinking behaviors and relapse. Alcohol abuse and addiction are devastating and costly problems worldwide. This study puts forward the possibility that cabergoline might be an effective treatment for these disorders.

The delta opioid receptor antagonist, SoRI-9409, decreases yohimbine stress-induced reinstatement of ethanol-seeking.

A major problem in treating alcohol use disorders (AUDs) is the high rate of relapse due to stress and re‐exposure to cues or an environment previously associated with alcohol use. Stressors can induce relapse to alcohol‐seeking in humans or reinstatement in rodents. Delta opioid peptide receptors (DOP‐Rs) play a role in cue‐induced reinstatement of ethanol‐seeking; however, their role in stress‐induced reinstatement of ethanol‐seeking is not known. The objective of this study was to determine the role of DOP‐Rs in yohimbine‐stress‐induced reinstatement of ethanol‐seeking. Male, Long‐Evans rats were trained to self‐administer 10% ethanol in daily 30‐minute operant self‐administration sessions using a FR3 schedule of reinforcement, followed by extinction training. Once extinction criteria were met, we examined the effects of the DOP‐R antagonist, SoRI‐9409 (0–5 mg/kg, i.p.) on yohimbine (2 mg/kg, i.p.) stress‐induced reinstatement. Additionally, DOP‐R‐stimulated [35S]GTPγS binding was measured in brain membranes and plasma levels of corticosterone (CORT) were determined. Pre‐treatment with SoRI‐9409 decreased yohimbine stress‐induced reinstatement of ethanol‐seeking but did not affect yohimbine‐induced increases in plasma CORT levels. Additionally, yohimbine increased DOP‐R‐stimulated 35[S]GTPγS binding in brain membranes of ethanol‐trained rats, an effect that was inhibited by SoRI‐9409. This suggests that the DOP‐R plays an important role in yohimbine‐stress‐induced reinstatement of ethanol‐seeking behavior, and DOP‐R antagonists may be promising candidates for further development as a treatment for AUDs.

Intermittent access ethanol consumption dysregulates CRF function in the hypothalamus and is attenuated by the CRF-R1 antagonist, CP-376395

Corticotrophin‐releasing factor (CRF) is a mediator of stress responses and a key modulator of ethanol‐mediated behaviors. We report here that the CRF receptor 1 (CRF‐R1) antagonist, CP‐376395 reduces 20% ethanol consumption in animals trained to consume ethanol on an intermittent, but not a continuous, schedule. Furthermore, using [35S]GTPγS binding assays, we demonstrate that CRF‐mediated G‐protein signaling in the hypothalamus of the intermittent drinkers is decreased when compared to controls suggesting that the effects of CP‐376395 are mediated by extrahypothalamic mechanisms. The present study provides further support for the use of CRF‐R1 antagonists for the treatment of alcohol use disorders and suggests that ethanol consumption dysregulates CRF function in the hypothalamus.

The Neurokinin 1 Receptor Antagonist, Ezlopitant, Reduces Appetitive Responding for Sucrose and Ethanol

Background The current obesity epidemic is thought to be partly driven by over-consumption of sugar-sweetened diets and soft drinks. Loss-of-control over eating and addiction to drugs of abuse share overlapping brain mechanisms including changes in motivational drive, such that stimuli that are often no longer ‘liked’ are still intensely ‘wanted’ [7], . The neurokinin 1 (NK1) receptor system has been implicated in both learned appetitive behaviors and addiction to alcohol and opioids; however, its role in natural reward seeking remains unknown. Methodology/Principal Findings We sought to determine whether the NK1-receptor system plays a role in the reinforcing properties of sucrose using a novel selective and clinically safe NK1-receptor antagonist, ezlopitant (CJ-11,974), in three animal models of sucrose consumption and seeking. Furthermore, we compared the effect of ezlopitant on ethanol consumption and seeking in rodents. The NK1-receptor antagonist, ezlopitant decreased appetitive responding for sucrose more potently than for ethanol using an operant self-administration protocol without affecting general locomotor activity. To further evaluate the selectivity of the NK1-receptor antagonist in decreasing consumption of sweetened solutions, we compared the effects of ezlopitant on water, saccharin-, and sodium chloride (NaCl) solution consumption. Ezlopitant decreased intake of saccharin but had no effect on water or salty solution consumption. Conclusions/Significance The present study indicates that the NK1-receptor may be a part of a common pathway regulating the self-administration, motivational and reinforcing aspects of sweetened solutions, regardless of caloric value, and those of substances of abuse. Additionally, these results indicate that the NK1-receptor system may serve as a therapeutic target for obesity induced by over-consumption of natural reinforcers.

Defining the role of corticotropin releasing factor binding protein in alcohol consumption

The corticotropin releasing factor (CRF) exerts its effects by acting on its receptors and on the binding protein (CRFBP), and has been implicated in alcohol use disorder (AUD). Therefore, identification of the exact contribution of each protein that mediates CRF effects is necessary to design effective therapeutic strategies for AUD. A series of in vitro/in vivo experiments across different species were performed to define the biological discrete role of CRFBP in AUD. First, to establish the CRFBP role in receptor signaling, we developed a novel chimeric cell-based assay and showed that CFRBP full length can stably be expressed on the plasma membrane. We discovered that only CRFBP(10 kD) fragment is able to potentiate CRF-intracellular Ca2+ release. We provide evidence that CRHBP gene loss increased ethanol consumption in mice. Then, we demonstrate that selective reduction of CRHBP expression in the center nucleus of the amygdala (CeA) decreases ethanol consumption in ethanol-dependent rats. CRFBP amygdalar downregulation, however, does not attenuate yohimbine-induced ethanol self-administration. This effect was associated with decreased hemodynamic brain activity in the CRFBP-downregulated CeA and increased hemodynamic activity in the caudate putamen during yohimbine administration. Finally, in alcohol-dependent patients, genetic variants related to the CRFBP(10 kD) fragment were associated with greater risk for alcoholism and anxiety, while other genetic variants were associated with reduced risk for anxiety. Taken together, our data provide evidence that CRFBP may possess both inhibitory and excitatory roles and may represent a novel pharmacological target for the treatment of AUD.

The Role of Delta Opioid Receptors in Ethanol Consumption and Seeking: Implications for New Treatments for Alcohol Use Disorders

There are few effective medications available for the treatment of alcohol use disorders (AUDs). To date, the opioid antagonist, naltrexone, is the most effective in reducing alcohol consumption in combination with behavioral therapy. Naltrexone has high affinity for the mu opioid peptide receptor (MOP-R) with moderate activity at the delta opioid peptide receptor (DOP-R) and kappa opioid peptide receptor (KOP-R). Preclinical studies suggest that the MOP-R plays a significant role in ethanol-mediated behaviors, however, there is evidence suggesting the DOP-R may be a better therapeutic target for treating AUDs. This chapter will review studies investigating the role of the opioid receptors in ethanol-mediated behaviors with the view of identifying improved therapeutics for the treatment of AUDs.