Joan Holgate

Varenicline, an α4β2 nicotinic acetylcholine receptor partial agonist, selectively decreases ethanol consumption and seeking

Alcohol dependence is a disease that impacts millions of individuals worldwide. There has been some progress with pharmacotherapy for alcohol-dependent individuals; however, there remains a critical need for the development of novel and additional therapeutic approaches. Alcohol and nicotine are commonly abused together, and there is evidence that neuronal nicotinic acetylcholine receptors (nAChRs) play a role in both alcohol and nicotine dependence. Varenicline, a partial agonist at the α4β2 nAChRs, reduces nicotine intake and was recently approved as a smoking cessation aid. We have investigated the role of varenicline in the modulation of ethanol consumption and seeking using three different animal models of drinking. We show that acute administration of varenicline, in doses reported to reduce nicotine reward, selectively reduced ethanol but not sucrose seeking using an operant self-administration drinking paradigm and also decreased voluntary ethanol but not water consumption in animals chronically exposed to ethanol for 2 months before varenicline treatment. Furthermore, chronic varenicline administration decreased ethanol consumption, which did not result in a rebound increase in ethanol intake when the varenicline was no longer administered. The data suggest that the α4β2 nAChRs may play a role in ethanol-seeking behaviors in animals chronically exposed to ethanol. The selectivity of varenicline in decreasing ethanol consumption combined with its reported safety profile and mild side effects in humans suggest that varenicline may prove to be a treatment for alcohol dependence.

Partial agonists of the α3β4* neuronal nicotinic acetylcholine receptor reduce ethanol consumption and seeking in rats.

Alcohol use disorders (AUDs) impact millions of individuals and there remain few effective treatment strategies. Despite evidence that neuronal nicotinic acetylcholine receptors (nAChRs) have a role in AUDs, it has not been established which subtypes of the nAChR are involved. Recent human genetic association studies have implicated the gene cluster CHRNA3–CHRNA5–CHRNB4 encoding the α3, α5, and β4 subunits of the nAChR in susceptibility to develop nicotine and alcohol dependence; however, their role in ethanol-mediated behaviors is unknown due to the lack of suitable and selective research tools. To determine the role of the α3, and β4 subunits of the nAChR in ethanol self-administration, we developed and characterized high-affinity partial agonists at α3β4 nAChRs, CP-601932, and PF-4575180. Both CP-601932 and PF-4575180 selectively decrease ethanol but not sucrose consumption and operant self-administration following long-term exposure. We show that the functional potencies of CP-601932 and PF-4575180 at α3β4 nAChRs correlate with their unbound rat brain concentrations, suggesting that the effects on ethanol self-administration are mediated via interaction with α3β4 nAChRs. Also varenicline, an approved smoking cessation aid previously shown to decrease ethanol consumption and seeking in rats and mice, reduces ethanol intake at unbound brain concentrations that allow functional interactions with α3β4 nAChRs. Furthermore, the selective α4β2* nAChR antagonist, DHβE, did not reduce ethanol intake. Together, these data provide further support for the human genetic association studies, implicating CHRNA3 and CHRNB4 genes in ethanol-mediated behaviors. CP-601932 has been shown to be safe in humans and may represent a potential novel treatment for AUDs.

Varenicline, a partial agonist at neuronal nicotinic acetylcholine receptors, reduces nicotine-induced increases in 20% ethanol operant self-administration in Sprague-Dawley rats

Alcohol and nicotine use disorders are often treated as separate diseases, despite evidence that approximately 80–90% of alcohol dependent individuals are also heavy smokers. Both nicotine and ethanol have been shown to interact with neuronal nicotinic acetylcholine receptors (nAChRs), suggesting these receptors are a common biological target for the effects of nicotine and ethanol in the brain. There are few studies that have examined the effects of co‐administered nicotine and ethanol on the activity of nAChRs in rodents. In the present study, we show that Sprague‐Dawley rats, a strain often used for nicotine studies but not as often for voluntary ethanol intake studies, will consume 20% ethanol using both the intermittent‐access two‐bottle‐choice and operant self‐administration models without the need for sucrose fading. We show that nicotine (0.2 mg/kg and 0.8 mg/kg, s.c.) significantly increases operant 20% ethanol self‐administration and varenicline (2 mg/kg, s.c), a partial agonist at nAChRs, significantly decreases operant ethanol self‐administration and nicotine‐induced increases in ethanol self‐administration. This suggests that nAChRs play an important role in increasing ethanol self‐administration and that varenicline may be an efficacious treatment for alcohol and nicotine co‐dependencies.

The α5 Neuronal Nicotinic Acetylcholine Receptor Subunit Plays an Important Role in the Sedative Effects of Ethanol But Does Not Modulate Consumption in Mice

BACKGROUND Alcohol use disorders (AUDs) are a major public health problem, and the few treatment options available to those seeking treatment offer only modest success rates. There remains a need to identify novel targets for the treatment of AUDs. The neuronal nicotinic acetylcholine receptors (nAChRs) represent a potential therapeutic target in the brain, as recent human genetic studies have implicated gene variants in the α5 nAChR subunit as high risk factors for developing alcohol dependence. METHODS Here, we evaluate the role of the α5* nAChR for ethanol (EtOH)-mediated behaviors using male α5+/+ and α5-/- transgenic mice. We characterized the effect of hypnotic doses of EtOH and investigated drinking behavior using an adapted drinking-in-the-dark (DID) paradigm that has been shown to induce high EtOH consumption in mice. RESULTS We found the α5 subunit to be important in mediating the sedative effects of EtOH. The α5-/- mice showed slower recovery from EtOH-induced sleep, as measured by loss of righting reflex. Additionally, the α5-/- mice showed enhanced impairment to EtOH-induced ataxia. We found the initial sensitivity to EtOH and EtOH metabolism to be similar in both α5+/+ and α5-/- mice. Hence, the enhanced sedation is likely due to a difference in the acute tolerance of EtOH in α5-/- mice. However, the α5 subunit did not play a role in EtOH consumption for EtOH concentrations ranging from 5 to 30% using the DID paradigm. Additionally, varenicline was effective in reducing EtOH intake in α5-/- mice. CONCLUSIONS Together, our data suggest that the α5 nAChR subunit is important for the sedative effects of EtOH but does not play a role in EtOH consumption in male mice. Varenicline can be a treatment option even when there is loss of function of the α5 nAChR subunit.

The α5 subunit regulates the expression and function of α4*-containing neuronal nicotinic acetylcholine receptors in the ventral-tegmental area.

Human genetic association studies have shown gene variants in the α5 subunit of the neuronal nicotinic receptor (nAChR) influence both ethanol and nicotine dependence. The α5 subunit is an accessory subunit that facilitates α4* nAChRs assembly in vitro. However, it is unknown whether this occurs in the brain, as there are few research tools to adequately address this question. As the α4*-containing nAChRs are highly expressed in the ventral tegmental area (VTA) we assessed the molecular, functional and pharmacological roles of α5 in α4*-containing nAChRs in the VTA. We utilized transgenic mice α5+/+(α4YFP) and α5-/-(α4YFP) that allow the direct visualization and measurement of α4-YFP expression and the effect of the presence (α5+/+) and absence of α5 (-/-) subunit, as the antibodies for detecting the α4* subunits of the nAChR are not specific. We performed voltage clamp electrophysiological experiments to study baseline nicotinic currents in VTA dopaminergic neurons. We show that in the presence of the α5 subunit, the overall expression of α4 subunit is increased significantly by 60% in the VTA. Furthermore, the α5 subunit strengthens baseline nAChR currents, suggesting the increased expression of α4* nAChRs to be likely on the cell surface. While the presence of the α5 subunit blunts the desensitization of nAChRs following nicotine exposure, it does not alter the amount of ethanol potentiation of VTA dopaminergic neurons. Our data demonstrates a major regulatory role for the α5 subunit in both the maintenance of α4*-containing nAChRs expression and in modulating nicotinic currents in VTA dopaminergic neurons. Additionally, the α5α4* nAChR in VTA dopaminergic neurons regulates the effect of nicotine but not ethanol on currents. Together, the data suggest that the α5 subunit is critical for controlling the expression and functional role of a population of α4*-containing nAChRs in the VTA.

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.

Gz proteins are functionally coupled to dopamine D2-like receptors in vivo

The receptors that couple to the G protein Gz in vivo are still relatively unknown. In this study, we investigated the effects of various dopamine receptor agonists in a mouse deficient in the alpha subunit of Gz. The dopamine D1-like receptor agonist SKF38393 stimulated comparable locomotor activity in both wildtype mice and mice lacking Galphaz. In contrast, the dopamine D2-like receptor agonist quinpirole suppressed locomotor activity in both groups of mice, but this suppression was significantly smaller in Galphaz knockout mice. Consistent with these behavioural observations, quinpirole inhibition of dopamine release in the forebrain nucleus accumbens evoked by electrical stimulation of dopamine axons was significantly attenuated in mice lacking Galphaz. In addition, hypothermia and adrenocorticotropic hormone release resulting from activation of dopamine D2-like receptors were also significantly reduced in Galphaz knockout mice. However, adrenocorticotropic hormone secretion induced by corticotrophin releasing hormone and the serotonin 1A receptor agonist 8-hydroxy-dipropylamino-tetralin were similar between wildtype and Galphaz knockout mice. Western blot analysis showed that the expression levels of Galphai, Galphao, Galphas, Galphaq and Gbeta were the same in the brains of mice of both genotypes. Overall, our data suggest that Gz proteins are functionally coupled to dopamine D2-like receptors in vivo.

Mice deficient in the alpha subunit of Gz show changes in pre-pulse inhibition, anxiety and responses to 5-HT1A receptor stimulation, which are strongly dependent on the genetic background

RationaleGz, a member of the Gi G protein family, is involved in the coupling of dopaminergic and serotonergic receptors. In the present study, we investigated behaviour of mice deficient in the α subunit of Gz and focused on pre-pulse inhibition (PPI) and anxiety-like responses and the role of serotonin-1A (5-HT1A) receptors.Materials and methodsWe compared male and female wild-type and knock-out mice on either a C57Bl/6 or Balb/c background. We used automated startle boxes to assess startle and PPI and elevated plus maze to assess anxiety-like behaviours.ResultsBalb/c mice showed higher baseline PPI than C57Bl/6 mice, and there was no difference between the genotypes. The 5-HT1A receptor agonist, 8-hydroxy-di-propylaminotetralin (8-OH-DPAT), had no effect on PPI in C57Bl/6 mice but markedly increased PPI in Balb/c mice, with the effect being attenuated in Gαz knock-outs. On the elevated plus maze, there was little effect of the knock-out or 8-OH-DPAT in C57Bl/6 mice, whereas in Balb/c mice, Gαz knock-outs showed a phenotype of high levels of anxiety-like behaviour. 8-OH-DPAT was anxiogenic in Balb/c mice, but this effect was attenuated in Gαz knock-outs.Conclusions5-HT1A receptors couple to Gz. In a strictly background strain-dependent manner, Gαz knock-out mice display high levels of anxiety-like behaviour and are less sensitive to the action of 8-OH-DPAT. Balb/c mice show much more clear effects of the Gαz knock-out than C57Bl/6 mice, which are often considered the standard background strain for genetic modifications. Therefore, our results suggest caution when studying the behavioural effects of genetic modifications only in C57Bl/6 mice.

Neuronal Nicotinic Acetylcholine Receptor Modulators Reduce Sugar Intake.

Excess sugar consumption has been shown to contribute directly to weight gain, thus contributing to the growing worldwide obesity epidemic. Interestingly, increased sugar consumption has been shown to repeatedly elevate dopamine levels in the nucleus accumbens (NAc), in the mesolimbic reward pathway of the brain similar to many drugs of abuse. We report that varenicline, an FDA-approved nicotinic acetylcholine receptor (nAChR) partial agonist that modulates dopamine in the mesolimbic reward pathway of the brain, significantly reduces sucrose consumption, especially in a long-term consumption paradigm. Similar results were observed with other nAChR drugs, namely mecamylamine and cytisine. Furthermore, we show that long-term sucrose consumption increases α4β2 * and decreases α6β2* nAChRs in the nucleus accumbens, a key brain region associated with reward. Taken together, our results suggest that nAChR drugs such as varenicline may represent a novel treatment strategy for reducing sugar consumption.

Deletion of guanine nucleotide binding protein alpha-Z subunit in mice induces a gene dose dependent tolerance to morphine

The mechanism underlying the development of tolerance to morphine is still incompletely understood. Morphine binds to opioid receptors, which in turn activates downstream second messenger cascades through heterotrimeric guanine nucleotide binding proteins (G proteins). In this paper, we show that G(z), a member of the inhibitory G protein family, plays an important role in mediating the analgesic and lethality effects of morphine after tolerance development. We blocked signaling through the G(z) second messenger cascade by genetic ablation of the alpha subunit of the G protein in mice. The Galpha(z) knockout mouse develops significantly increased tolerance to morphine, which depends on Galpha(z) gene dosage. Further experiments demonstrate that the enhanced morphine tolerance is not caused by pharmacokinetic and behavioural learning mechanisms. The results suggest that G(z) signaling pathways are involved in transducing the analgesic and lethality effects of morphine following chronic morphine treatment.