Assessment of nicotine release from nicotine-loaded chitosan nanoparticles dry powder inhaler formulations via locomotor activity of C57BL/6 mice.

PURPOSE: This study aimed to assess the activity of controlled release nicotine from dry powder inhaler formulation via locomotor activity of C57BL/6 mice. METHODS: To achieve this we built a nose-only inhalation device for pulmonary administration of nicotine to mice and determined the optimal operational parameters. We used the locomotor activity test to compare the effects of the inhaled nicotine hydrogen tartrate-loaded chitosan nanoparticles (NHT-CS) with NHT in C57BL/6 mice. The minimum inhaled dose of NHT-CS required to alter locomotor activity was compared with inhaled and subcutaneously (s.c) injected NHT. Finally, histological examination of lung tissues was performed to ensure inhalation of NHT-CS did not cause lung damage. RESULTS: We found a flow rate of 0.9 L/min and an exposure time of 5 min achieved optimal delivery of nicotine. A minimum of 0.88 mg inhaled of NHT-CS or 0.59 mg inhaled of NHT was required to alter locomotor activity similarly to injection of 0.5 mg/kg nicotine, suggesting the reformulation process did not alter the activity of NHT-CS. No differences between untreated and NHT-CS treated lung tissue upon histological examination were observed. CONCLUSIONS: The results indicated the inhaled NHT-CS is a viable preclinical option for developing novel inhalation formulations as a potential anti-smoking therapeutic.

Modulation of serotonin and noradrenaline in the BLA by pindolol reduces long-term ethanol intake.

Repeated cycles of binge-like alcohol consumption and abstinence change the activity of several neurotransmitter systems. Some of these changes are consolidated following prolonged alcohol use and are thought to play an important role in the development of dependence. We have previously shown that systemic administration of the dual beta-adrenergic antagonist and 5-HT1A/1B partial agonist pindolol selectively reduces long-term but not short-term binge-like consumption of ethanol and alters excitatory postsynaptic currents in basolateral amygdala (BLA) principal neurons. The aim of this study was to investigate the effects of pindolol microinfusions in the BLA on long-term ethanol intake using the drinking-in-the-dark paradigm in mice. We also microinfused RU24969 (5-HT1A/1B receptor partial agonist) and CGP12177 (ß1/2 adrenergic antagonist) following long-term ethanol intake and determined the densities of 5-HT1A/1B receptors and ß1/2 adrenergic in the BLA following short-term (4 weeks) and long-term ethanol (12 weeks) consumption. We show that intra-BLA infusion of pindolol (1000 pmol/0.5 µl), RU24969 (0.3 and 3 pmol/0.5 µl) and CGP12177 (500 pmol/0.5 µl) produce robust decreases in long-term ethanol consumption. Additionally, we identified reduced ß1/2 adrenergic receptor expression and no change in 5-HT1A/1B receptor density in the BLA of long-term ethanol-consuming mice. Collectively, our data highlight the effects of pindolol on voluntary, binge-like ethanol consumption behavior following long-term intake.

Sex Specific Alterations in α4*Nicotinic Receptor Expression in the Nucleus Accumbens.

Background: The mechanisms leading from traumatic stress to social, emotional and cognitive impairment and the development of mental illnesses are still undetermined and consequently there remains a critical need to develop therapies for preventing the adverse consequences of traumatic stress. Research indicates nicotinic acetylcholine receptors containing α4 subunits (α4*nAChRs) are both impacted by stress and capable of modulating the stress response. In this study, we investigated whether varenicline, a partial α4β2*nAChR agonist which reduces nicotine, alcohol and sucrose consumption, can reduce stress, a driving factor in substance use disorders. We also examined the effect of stress on nucleus accumbens (NAc) α4*nAChR expression. Methods: Transgenic mice with fluorescent tags attached to α4*nAChRs were administered varenicline and/or yohimbine (a pharmacological stressor) and plasma corticosterone and NAc α4*nAChR expression were measured. A separated group of mice were exposed to maternal separation (MS) during post-natal day (P) 2⁻14, then restraint stressed (30 min) at six weeks of age. Body weight, anxiety-like behaviours (elevated plus maze), plasma corticosterone and NAc α4*nAChR levels were measured. Results: Varenicline attenuated yohimbine-induced plasma corticosterone increases with no effect on NAc α4*nAChR expression. MS reduced unrestrained plasma corticosterone levels in both sexes. In females, MS increased body weight and NAc α4*nAChR expression, whereas, in males, MS and restraint caused a greater change in anxiety-like behaviours and plasma corticosterone levels. Restraint altered NAc α4*nAChR expression in both male and female MS mice. Conclusions: The effects of stress on NAc α4*nAChR are sex-dependent. While varenicline attenuated acute stress-induced rises in corticosterone levels, future studies are required to determine whether varenicline is effective for relieving the effects of stress.

Social and environmental enrichment has different effects on ethanol and sucrose consumption in mice.

BACKGROUND: Factors leading to the harmful consumption of substances, like alcohol and sucrose, involve a complex interaction of genes and the environment. While we cannot control the genes we inherit, we can modify our environment. Understanding the role that social and environmental experiences play in alcohol and sucrose consumption is critical for developing preventative interventions and treatments for alcohol use disorders and obesity. METHODS: We used the drinking in the dark two-bottle choice (2BC) model of ethanol and sucrose consumption to compare male C57BL/6 mice housed in the IntelliCage (an automated device capable of simultaneously measuring behaviors of up to 16 mice living in an enriched social environment) with mice housed in standard isolated and social environments. RESULTS: Consistent with previous publications on ethanol-naïve and -experienced mice, social and environmental enrichment reduced ethanol preference. Isolated mice had the highest ethanol preference and IntelliCage mice the least, regardless of prior ethanol experience. In mice with no prior sucrose experience, the addition of social and environmental enrichment increased sucrose preference. However, moving isolated mice to enriched conditions did not affect sucrose preference in sucrose-experienced mice. CONCLUSIONS: The impact of social and environmental enrichment on ethanol consumption differs from sucrose consumption suggesting that interventions and treatments developed for alcohol use disorders may not be suitable for sucrose consumption disorders.

A Rat Drinking in the Dark Model for Studying Ethanol and Sucrose Consumption.

Background: The intermittent access 2-bottle choice (IA2BC) and drinking in the dark (DID) models were developed for studying rodent binge-like consumption. Traditionally, IA2BC was used with rats and DID with mice. Recently, IA2BC was adapted to study mouse ethanol consumption. However, it is unknown whether DID is suitable for rats or if one rat model is more advantageous than another for studying binge-like consumption. Methods: Male Wistar rats consumed 20% ethanol or 5% sucrose using IA2BC or DID for 12 weeks. IA2BC drinking sessions occurred on alternate days (Mondays-Fridays) and lasted 24 h, whereas DID sessions ran 4 h/day, 5 days/week (Monday-Friday). Average consumption/session, week and hour was measured. To explore DID model suitability for screening novel compounds for controlling ethanol and sucrose intake, varenicline (2 mg/kg) or vehicle was administered to DID rats. Results: IA2BC rats consume more ethanol/session and similar amounts of ethanol/week than DID rats. While, IA2BC rats consume more sucrose/session and week than DID rats. Although IA2BC rats had more ethanol and sucrose access time, DID rats had greater ethanol and sucrose intake/hour. Varenicline significantly reduced ethanol and sucrose consumption in DID rats, consistent with previously published IA2BC studies. Conclusions: Despite the shorter access time, the rat DID model induced higher initial intake and greater consumption/hour for both ethanol and sucrose. The shorter duration of DID sessions did not prevent detection of varenicline-induced reductions in ethanol or sucrose consumption, suggesting the DID model may be suitable for studying binge-like ethanol and sucrose consumption.

The antihypertensive drug pindolol attenuates long-term but not short-term binge-like ethanol consumption in mice.

Alcohol dependence is a debilitating disorder with current therapies displaying limited efficacy and/or compliance. Consequently, there is a critical need for improved pharmacotherapeutic strategies to manage alcohol use disorders (AUDs). Previous studies have shown that the development of alcohol dependence involves repeated cycles of binge-like ethanol intake and abstinence. Therefore, we used a model of binge-ethanol consumption (drinking-in-the-dark) in mice to test the effects of compounds known to modify the activity of neurotransmitters implicated in alcohol addiction. From this, we have identified the FDA-approved antihypertensive drug pindolol, as a potential candidate for the management of AUDs. We show that the efficacy of pindolol to reduce ethanol consumption is enhanced following long-term (12 weeks) binge-ethanol intake, compared with short-term (4 weeks) intake. Furthermore, pindolol had no effect on locomotor activity or consumption of the natural reward sucrose. Because pindolol acts as a dual beta-adrenergic antagonist and 5-HT1A/1B partial agonist, we examined its effect on spontaneous synaptic activity in the basolateral amygdala (BLA), a brain region densely innervated by serotonin and norepinephrine-containing fibres. Pindolol increased spontaneous excitatory post-synaptic current frequency of BLA principal neurons from long-term ethanol-consuming mice but not naïve mice. Additionally, this effect was blocked by the 5-HT1A/1B receptor antagonist methiothepin, suggesting that altered serotonergic activity in the BLA may contribute to the efficacy of pindolol to reduce ethanol intake following long-term exposure. Although further mechanistic investigations are required, this study demonstrates the potential of pindolol as a new treatment option for AUDs that can be fast-tracked into human clinical studies.

The effect of varenicline on binge-like ethanol consumption in mice is ß4 nicotinic acetylcholine receptor-independent.

BACKGROUND: Our laboratory has previously shown that the smoking-cessation agent varenicline, an agonist/partial agonist of α4ß2*, α3ß4*, α3ß2*, α6ß2* (* indicates the possibility of additional subunits) and α7 subunits of nicotinic acetylcholine receptors (nAChRs), reduces ethanol consumption in rats only after long-term exposure (12 weeks). As compounds having partial agonistic activity on α3ß4* nAChRs were shown to decrease ethanol consumption in rodents, we assessed here the involvement of the ß4 subunit in the effect of varenicline in the reduction of short- and long-term binge-like ethanol drinking in mice. METHODS: We used the well-validated drinking-in-the-dark (DID) paradigm to model chronic binge-like ethanol drinking in ß4-/- and ß4+/+ littermate mice and compare the effect of intraperitoneal injection of varenicline (2mg/kg) on ethanol intake following short- (4 weeks) or long-term (12 weeks) exposure. RESULTS: Drinking pattern and amounts of ethanol intake were similar in ß4-/- and ß4+/+ mice. Interestingly, our results showed that varenicline reduces ethanol consumption following short- and long-term ethanol exposure in the DID. Although the effect of varenicline on the reduction of ethanol consumption was slightly more pronounced in ß4-/- mice than their ß4+/+ littermates no significant differences were observed between genotypes. CONCLUSION: In mice, varenicline reduces binge-like ethanol consumption both after short- and long-term exposure in the DID and this effect is independent of ß4 nAChR subunit.

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 α4ß2 * and decreases α6ß2* 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.

Early Life Stress, Nicotinic Acetylcholine Receptors and Alcohol Use Disorders.

Stress is a major driving force in alcohol use disorders (AUDs). It influences how much one consumes, craving intensity and whether an abstinent individual will return to harmful alcohol consumption. We are most vulnerable to the effects of stress during early development, and exposure to multiple traumatic early life events dramatically increases the risk for AUDs. However, not everyone exposed to early life stress will develop an AUD. The mechanisms determining whether an individual's brain adapts and becomes resilient to the effects of stress or succumbs and is unable to cope with stress remain elusive. Emerging evidence suggests that neuroplastic changes in the nucleus accumbens (NAc) following early life stress underlie the development of AUDs. This review discusses the impact of early life stress on NAc structure and function, how these changes affect cholinergic signaling within the mesolimbic reward pathway and the role nicotinic acetylcholine receptors (nAChRs) play in this process. Understanding the neural pathways and mechanism determining stress resilience or susceptibility will improve our ability to identify individuals susceptible to developing AUDs, formulate cognitive interventions to prevent AUDs in susceptible individuals and to elucidate and enhance potential therapeutic targets, such as the nAChRs, for those struggling to overcome an AUD.

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 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.

Pharmacological consequence of the A118G µ opioid receptor polymorphism on morphine- and fentanyl-mediated modulation of Ca²âº channels in humanized mouse sensory neurons.

BACKGROUND: The most common functional single nucleotide polymorphism of the human OPRM1 gene, A118G, has been shown to be associated with interindividual differences in opioid analgesic requirements, particularly with morphine, in patients with acute postoperative pain. The purpose of this study was to examine whether this polymorphism would modulate the morphine and fentanyl pharmacological profile of sensory neurons isolated from a humanized mouse model homozygous for either the 118A or 118G allele. METHODS: The coupling of wild-type and mutant µ opioid receptors to voltage-gated Ca channels after exposure to either ligand was examined by employing the whole cell variant of the patch-clamp technique in acutely dissociated trigeminal ganglion neurons. Morphine-mediated antinociception was measured in mice carrying either the 118AA or 118GG allele. RESULTS: The biophysical parameters (cell size, current density, and peak current amplitude potential) measured from both groups of sensory neurons were not significantly different. In 118GG neurons, morphine was approximately fivefold less potent and 26% less efficacious than that observed in 118AA neurons. On the other hand, the potency and efficacy of fentanyl were similar for both groups of neurons. Morphine-mediated analgesia in 118GG mice was significantly reduced compared with the 118AA mice. CONCLUSIONS: This study provides evidence to suggest that the diminished clinical effect observed with morphine in 118G carriers results from an alteration of the receptor's pharmacology in sensory neurons. In addition, the impaired analgesic response with morphine may explain why carriers of this receptor variant have an increased susceptibility to become addicted to opioids.

Induction of multiple reinstatements of ethanol- and sucrose-seeking behavior in Long-Evans rats by the α-2 adrenoreceptor antagonist yohimbine.

RATIONALE: Developing models to efficiently explore the mechanisms by which stress can mediate reinstatement of drug-seeking behavior is crucial to the development of new pharmacotherapies for alcohol use disorders. OBJECTIVES: We examined the effects of multiple reinstatement sessions using the pharmacological stressor, yohimbine, in ethanol- and sucrose-seeking rats in order to develop a more efficient model of stress-induced reinstatement. METHODS: Long-Evans rats were trained to self-administer 10% ethanol with a sucrose-fading procedure, 20% ethanol without a sucrose-fading procedure, or 5% sucrose in 30-min operant self-administration sessions, followed by extinction training. After reaching extinction criteria, the animals were tested once per week with yohimbine vehicle and yohimbine (2 mg/kg), respectively, 30 min prior to the reinstatement sessions or blood collection. Levels of reinstatement and plasma corticosterone (CORT) were determined each week for four consecutive weeks. RESULTS: Yohimbine induced reinstatement of ethanol- and sucrose-seeking in each of the 4 weeks. Interestingly, the magnitude of the reinstatement decreased for the 10% ethanol group after the first reinstatement session but remained stable for the 20% ethanol group trained without sucrose. Plasma CORT levels in response to injection of both vehicle and yohimbine were significantly higher in the ethanol-trained animals compared to sucrose controls. CONCLUSIONS: The stable reinstatement in the 20% ethanol group supports the use of this training procedure in studies using within-subject designs with multiple yohimbine reinstatement test sessions. Additionally, these results indicate that the hormonal response to stressors can be altered following extinction from self-administration of relatively modest amounts of ethanol.

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.

Partial agonists of the α3ß4* 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 ß4 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 ß4 subunits of the nAChR in ethanol self-administration, we developed and characterized high-affinity partial agonists at α3ß4 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 α3ß4 nAChRs correlate with their unbound rat brain concentrations, suggesting that the effects on ethanol self-administration are mediated via interaction with α3ß4 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 α3ß4 nAChRs. Furthermore, the selective α4ß2(*) nAChR antagonist, DHßE, 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.

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.

Mice deficient in the alpha subunit of G(z) show changes in pre-pulse inhibition, anxiety and responses to 5-HT(1A) receptor stimulation, which are strongly dependent on the genetic background.

RATIONALE: G(z), a member of the G(i) 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 alpha subunit of G(z) and focused on pre-pulse inhibition (PPI) and anxiety-like responses and the role of serotonin-1A (5-HT(1A)) receptors. MATERIALS AND METHODS: We 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. RESULTS: Balb/c mice showed higher baseline PPI than C57Bl/6 mice, and there was no difference between the genotypes. The 5-HT(1A) 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 Galpha(z) 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, Galpha(z) 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 Galpha(z) knock-outs. CONCLUSIONS: 5-HT(1A) receptors couple to G(z). In a strictly background strain-dependent manner, Galpha(z) 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 Galpha(z) 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.

Varenicline, an alpha4beta2 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 alpha4beta2 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 alpha4beta2 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.

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.