Advertisement
Browse Subject Areas
?

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

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

  • Pia Steensland,

    Affiliations Ernest Gallo Clinic and Research Center, University of California San Francisco, Emeryville, California, United States of America, Department of Clinical Neuroscience, Karolinska Institutet, Stockholm, Sweden

  • Jeffrey A. Simms,

    Affiliation Ernest Gallo Clinic and Research Center, University of California San Francisco, Emeryville, California, United States of America

  • Carsten K. Nielsen,

    Affiliation Ernest Gallo Clinic and Research Center, University of California San Francisco, Emeryville, California, United States of America

  • Joan Holgate,

    Affiliation Ernest Gallo Clinic and Research Center, University of California San Francisco, Emeryville, California, United States of America

  • Jade J. Bito-Onon,

    Affiliation Ernest Gallo Clinic and Research Center, University of California San Francisco, Emeryville, California, United States of America

  • Selena E. Bartlett

    selenab@gallo.ucsf.edu

    Affiliation Ernest Gallo Clinic and Research Center, University of California San Francisco, Emeryville, California, United States of America

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

  • Pia Steensland, 
  • Jeffrey A. Simms, 
  • Carsten K. Nielsen, 
  • Joan Holgate, 
  • Jade J. Bito-Onon, 
  • Selena E. Bartlett
PLOS
x

Abstract

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.

Introduction

Obesity-related pathology is an alarming public health problem worldwide. Homeostatic control systems precisely regulate body weight and adiposity in a restrictive food environment [1]. However, non-homeostatic factors, such as palatability and motivation, override these systems when a sedentary lifestyle is combined with accessibility of palatable and calorically dense foods or natural reinforcers [1]. In fact, the current obesity epidemic is suggested to be partly driven by over-consumption of natural reinforcers such as sugar [2][6].

Uncontrolled over-consumption of natural reinforcers share characteristics with drug addiction. For example, stimuli no longer ‘liked’ are still intensely ‘wanted’ [7], [8]. Additionally, there is overlap between brain regions regulating seeking and self-administration of substances of abuse and those regulating motivational and reinforcing aspects of foraging and intake of natural reinforcers [9][15]. For example, natural reinforcers, including sucrose, activate neurons in the ventral tegmental area (VTA). Conversely, VTA lesions selectively reduce sucrose consumption [16]. Furthermore, sucrose consumption increases dopamine release in the nucleus accumbens [17], a brain area exhibiting opiate-like activation following excessive sugar intake [18]. Finally, recent neuroimaging studies have discovered neuroadaptations in obese individuals that mimic those in cocaine addicted individuals [14], [19], [20].

The common molecular substrates underling the motivation to consume natural reinforcers and drugs of abuse are largely unknown. However, the endogenous opioid system is one possible candidate [15]. An interaction between sugar consumption and the opioid system is supported by cross-tolerance [21], [22] and cross-dependence [23][25] between sucrose consumption and opiates. In addition, both sugar and ethanol intake is decreased by the opioid antagonist naltrexone [26][28].

Recently it has been suggested that the neurokinin 1 (NK1)-receptor system, and its endogenous ligand substance P (SP), interacts with the opioid receptor systems to regulate reward related behaviors. For example, the NK1- and µ-opioid (MOR)-receptors are widely co-expressed in brain regions involved in reward, for example the amygdala, hypothalamus, and nucleus accumbens [29][33]. Furthermore, NK1-receptor knock-out mice are insensitive to the rewarding properties of morphine [34], [35]. NK1-receptor knock-out mice fail to develop a preference for morphine, using the conditioned place preference paradigm, and self-administer morphine at lower levels than wild-type controls [35]. Additionally, recent series of preclinical and clinical experiments identified the NK1-receptor as a novel therapeutic target for alcohol use disorders (AUDs) [36]. The preclinical studies showed that NK1-receptor knockout mice decreases voluntary ethanol consumption and increased sensitivity to sedative effects of ethanol [36]. In the clinical setting, the NK1-receptor antagonist LY686017 suppressed alcohol craving and improved overall well-being in recently detoxified AUD patients [36]. The role of NK1-receptor system in AUDs is further supported by a study showing that polymorphisms of the NK1-receptor are significantly associated with the development of AUDs in Caucasian individuals [37].

The involvement of the opioid system in rewarding properties of both sucrose and drugs of abuse, together with the interaction between the opioid and the NK1-receptor system in reward related behaviors led us to the hypothesis that the NK1-receptor system may play a role in appetitive behaviors. To further elucidate the role of the NK1-receptor system in the regulation of consumption of natural reinforcers and ethanol, we evaluated the efficacy of a clinically safe and selective NK1-receptor antagonist, ezlopitant (CJ-11,974) [38], [39] to decrease sucrose and ethanol consumption and seeking. Ezlopitant has previously been investigated in clinical trials as a potential therapy for pain, chemotherapy-induced emesis and irritable bowel syndrome [40], [41]. The present study gives further support to the hypothesis that the NK1-receptor system might be a novel therapeutic target for addictive disorders.

Results

The NK1-receptor antagonist ezlopitant decreases both sucrose and ethanol operant-self administration

The effect of the NK1-receptor antagonist, ezlopitant on sucrose and ethanol operant self-administration was evaluated in Long-Evans rats that had a stable level of ethanol or sucrose responding on a FR3 schedule. Ezlopitant (2, 5 or 10 mg/kg i.p.) or vehicle was administered 30 minutes before the start of the operant self-administration session. Ezlopitant treatment had an overall main effect on the number of presses on the active lever during operant self-administration of 5% sucrose [F(3,15) = 21.9, P<0.001] and post hoc analysis revealed that all doses of ezlopitant attenuated the number of active lever presses for 5% sucrose (Figure 1A). In addition, ezlopitant treatment had an overall main effect on the number of presses on the active lever during operant self-administration of 10% ethanol [F(3,14) = 5.5, P<0.01]. Post hoc analysis revealed that the highest dose of ezlopitant (10 mg/kg) significantly inhibited operant self-administration of 10% ethanol compared with vehicle (Figure 1B). There was no overall main effect on the number of presses on the inactive lever in the ethanol or the sucrose group [sucrose: F(3,15) = 1.4, non-significant (n.s.); ethanol: F(3,14) = 2.3, n.s., data not shown].

thumbnail
Figure 1. The NK1-receptor antagonist ezlopitant significantly inhibits both sucrose and ethanol operant self-administration in rats.

Ezlopitant (2, 5, 10 mg/kg i.p.) dose dependently decreases self-administration of 5% sucrose (A). Only the highest dose of ezlopitant (10 mg/kg i.p.) decreases self-administration of 10% ethanol (B). All values are expressed as the mean ± SEM number of presses on the active lever. **P<0.01, ***P<0.001 compared to vehicle.

https://doi.org/10.1371/journal.pone.0012527.g001

Ezlopitant inhibits progressive ratio responding for sucrose, but not ethanol

The effect of ezlopitant on the incentive motivation to respond for sucrose and ethanol was evaluated in Long-Evans rats that had achieved a stable level of ethanol or sucrose responding on a FR3 schedule. Ezlopitant (2, 5 or 10 mg/kg i.p.) or vehicle was administered 30 minutes before the start of the PR test. Ezlopitant treatment had an overall main effect on the breakpoint during PR tests for the 5% sucrose group [F(3,13) = 5.9, P<0.01] and post hoc analysis revealed that the 5 and 10mg/kg doses of ezlopitant attenuated the breakpoint for 5% sucrose (Figure 2A). In contrast, ezlopitant treatment had no overall main effect the breakpoint during PR tests for the 10% ethanol group [F(4,13) = 1.5, n.s] (Figure 2B). One sucrose animal was excluded from analysis because of low breakpoint responding following vehicle treatment and one ethanol animal was excluded from analysis due to lack of responding during regular FR3 sessions.

thumbnail
Figure 2. The NK1-receptor antagonist ezlopitant significantly decreases the breakpoint for sucrose but not ethanol operant self-administration in rats.

Ezlopitant (5 and 10 mg/kg i.p.) decreases the breakpoint for 5% sucrose using a PR schedule (A), but had no effect on the breakpoint for 10% ethanol (B). All values are expressed as the mean ± SEM breakpoint. **P<0.01, *P<0.05 compared to vehicle.

https://doi.org/10.1371/journal.pone.0012527.g002

Ezlopitant selectively inhibits voluntary consumption of both sucrose and ethanol

Ezlopitant treatment had an overall main effect on voluntary consumption of 5% sucrose (g/kg) at both time points [6hrs; F(3,11) = 7.8, P<0.001; 24hrs: F(3,11) = 7.1, P<0.001]. Post hoc analysis showed that all doses of ezlopitant significantly decreased sucrose intake compared to vehicle at the 6 hour time point (Figure 3A). At the 24 hour time point, the two highest doses of ezlopitant significantly decreased sucrose intake compared to vehicle (5 mg/kg: P<0.05 and 10 mg/kg: P<0.001; data not shown). There was an overall main effect on the preference for sucrose over water at the 6 hour, but not the 24 hour time point [6hrs: F(3,11) = 4.0, P<0.05; F(3,11) = 2.1, n.s., data not shown]. Post hoc analysis of the 6 hour preference data revealed a significant decrease following treatment with the highest dose of ezlopitant (10 mg/kg) compared to vehicle (P<0.05, data not shown).

thumbnail
Figure 3. The NK1-receptor antagonist ezlopitant significantly and selectively decreases voluntary intake of both sucrose and ethanol.

Ezlopitant decreased consumption of 5% sucrose (A), whereas only the highest dose of ezlopitant (10 mg/kg) decreased consumption of 20% ethanol (B). All values are expressed as the mean intake (ethanol and sucrose: g/kg/6 hrs) ± SEM. *P<0.05, **P<0.01, ***P<0.001 compared to vehicle.

https://doi.org/10.1371/journal.pone.0012527.g003

In the high ethanol consumption model (intermittent-access-20%-ethanol), there was an overall main effect on the ethanol consumption (g/kg) 6 and 24 hours after the administration of ezlopitant [6hr: F(3,10) = 8.2, P<0.001; 24hrs: F(3,10) = 5.9, P<0.01). Post hoc analysis revealed that the highest dose of ezlopitant (10 mg/kg) significantly reduced the ethanol consumption compared to vehicle at both time points (6hrs: Figure 3B, 24hrs: data not shown). In addition, there was an overall main effect on the preference for ethanol over water at both time points [6hrs: F(3.10) = 6.9, P<0.01; 24hrs: F(3.10) = 4.2, P<0.05], however, post hoc analysis revealed a significant difference between the highest dose of ezlopitant (10 mg/kg) and vehicle only at the 6 hour time point (P<0.01, data not shown).

The ability of ezlopitant to decrease sucrose and ethanol consumption was specific for each of the solutions as treatment with ezlopitant had no overall main effect on water consumption in either the sucrose or ethanol group at any time point [sucrose group: 6hrs: F(3,10) = 1.4, n.s., Table 1, 24hrs: F(3,10) = 1.8, n.s. data not shown; ethanol group: 6hrs: F(3,11) = 1.3, n.s., Table 1, 24hrs: F(3,11) = 1.0, n.s., data not shown].

thumbnail
Table 1. Ezlopitant treatment had no effect on the water intake in rats that were given intermittent-access to 5% sucrose and 20% ethanol, respectively, in a two-bottle choice setting.

https://doi.org/10.1371/journal.pone.0012527.t001

The NK1-receptor antagonist ezlopitant decreases voluntary sucrose consumption in C57BL/6 mice

In the DID-model of sucrose consumption, the ezlopitant treatment had an overall main effect on the sucrose intake in C57BL/6 mice [F(3,39) = 4.2, P = 0.01]. Post hoc analysis showed that the highest dose of ezlopitant (15 mg/kg) significantly decreased the voluntary sucrose intake compared to vehicle (Figure 4A). Furthermore, there was an overall main effect on the preference for sucrose over water [F(3,39) = 4.8, P<0.01]. Post hoc analysis revealed that the 15 mg/kg dose decreased the preference for sucrose over water compared to vehicle (P>0.01, data not shown). The decrease in sucrose intake following ezlopitant treatment was specific since there was no overall main effect on the water intake [F(3,39) = 1.1, n.s., data not shown].

thumbnail
Figure 4. The NK1-receptor antagonist ezlopitant significantly decreases voluntary intake of sucrose using the drinking in the dark model in mice.

The highest dose of ezlopitant (15 mg/kg) decreased consumption of 5% sucrose (A). However, ezlopitant treatment failed to induce a significant decrease in ethanol consumption compared to vehicle (B). All values are expressed as the mean intake (g/kg/2 hrs) ± SEM. **P<0.01, compared to vehicle.

https://doi.org/10.1371/journal.pone.0012527.g004

In the DID-model of ethanol consumption, there was a trend, but no significant overall main effect on the ethanol consumption (g/kg/2hrs) after ezlopitant administration in C57BL/6 mice [F(3,38) = 2.6, P = 0.06, n.s, Figure 4B]. Subsequently, there was no overall main effect on the preference for ethanol over water following ezlopitant treatment [F(3,38) = 2.2, n.s., data not shown]. Furthermore, there was no overall main effect on the water intake following ezlopitant treatment [F(3,38) = 2.3, n.s., data not shown] in the C57BL/6 mice.

Ezlopitant does not affect locomotor activity

To examine the possibility that ezlopitant inhibited operant self-administration and consumption of ethanol and sucrose through a general effect on locomotor behavior, we administrated ezlopitant or vehicle to two different groups of Long Evans rats. Following habituation to the locomotor activity boxes, the NK1-receptor antagonist (10 mg/kg) or vehicle was administered and the ambulatory distance traveled was recorded for 60 minutes. The NK1-receptor antagonist treatment induced no significant effect on locomotor activity (ambulatory distance travelled) compared to vehicle (P = 0.92, n.s., Figure 5).

thumbnail
Figure 5. The NK1-receptor antagonist ezlopitant has no significant effect on general locomotor activity in rats.

Rats administered ezlopitant (10 mg/kg) did not display differences in ambulatory distances traveled compared to vehicle-treated rats. The values are expressed as the mean (± SEM) distance traveled (cm) per 3 minute-period over the 60 minute test-period.

https://doi.org/10.1371/journal.pone.0012527.g005

Ezlopitant selectively inhibits voluntary consumption of the non-caloric sweetener saccharin

To evaluate if the marked ezlopitant-induced decrease in sucrose compared to ethanol intake was dependent on the high caloric value of the sucrose solution, we tested the effect of the compound on a 0.2% saccharin solution with zero caloric value. Ezlopitant treatment had an overall main effect on voluntary consumption of 0.2% saccharin (g/kg) at both time points [6hrs: F(3,9) = 7.6, P<0.001; 24hrs: F(3,9) = 6.5, P<0.01]. Post hoc analysis showed that the two highest doses of ezlopitant (5 and 10 mg/kg) significantly decreased saccharin intake compared to vehicle at the 6 hour time point (Figure 6). At the 24 hour time point, only the highest ezlopitant dose significantly decreased saccharin intake compared to vehicle (P<0.01; data not shown).

thumbnail
Figure 6. The NK1-receptor antagonist ezlopitant significantly decreases voluntary intake of non-caloric saccharin solution.

Ezlopitant (5 and 10 mg/kg) significantly decreased consumption of the non-caloric 0.2% saccharin solution compared to vehicle. All values are expressed as the mean saccharin intake (g/kg/6 hrs) ± SEM. *P<0.05, ***P<0.001 compared to vehicle.

https://doi.org/10.1371/journal.pone.0012527.g006

Ezlopitant does not decrease intake of water or a salty solution

To further evaluate ezlopitant's selectivity for sweet solutions, we tested the effect of the NK1-receptor antagonist on water and salty (NaCl) solution intake, respectively. There were no significant difference in water or 0.175% NaCl solution intake at any time point following ezlopitant (10 mg/kg) treatment compared to vehicle (water: 6hrs: P = 0.39, n.s. Figure 7A, 24hrs: P = 0.52, n.s. data not shown; NaCl: 6hrs: P = 0.88, n.s., Figure 7B, 24hrs: P = 0.41, n.s.data not shown).

thumbnail
Figure 7. The NK1-receptor antagonist ezlopitant has no significant effect on intake of water or salty solution.

Rats administered ezlopitant (10 mg/kg) did not decrease the intake of water (A) or a 0.175% NaCl solution (B) compared to vehicle. All values are expressed as the mean intake (ml/6 hrs) ± SEM.

https://doi.org/10.1371/journal.pone.0012527.g007

The NK1-receptor antagonist ezlopitant inhibits SP-mediated [35S]GTPγS-stimulated binding in rat brain membranes

SP produced a dose-dependent stimulation of [35S]GTPγS-binding in rat membranes prepared from the cerebral cortex of both water exposed (EC50 = 57±3.8 nM, Figure 8A) and sucrose exposed rats (EC50 = 750±31 nM, Figure 8B). Furthermore, when the SP-stimulated (1 µmol/L) [35S]GTPγS-binding was performed in the presence of ezlopitant (0.1 nmol/L–100 µmol/L) the binding was potently inhibited in both groups (water group, IC50 = 1.5±0.5 nM, Figure 8C: sucrose group, IC50 = 61±3.1 nM, Figure 8D).

thumbnail
Figure 8. The NK1-receptor antagonist ezlopitant inhibits NK1-receptor-stimulated [35S]GTPγS binding in the cerebral cortex of rats.

SP produced a dose dependent stimulation of [35S]GTPγS-binding in rat membranes from naive (A) and sucrose- trained rats (B). Ezlopitant potently inhibits SP-stimulated (1 µmol/L) [35S]GTPγS NK1-receptor binding in the cerebral cortex of naive rats (C) and of rats following long-term sucrose consumption (D). The values are expressed as mean ± SEM percentage increase in basal [35S]GTPγS binding.

https://doi.org/10.1371/journal.pone.0012527.g008

Discussion

Emerging evidence indicates that the NK1-receptor system is involved in reinforcing mechanisms of drugs of abuse including alcohol. The NK1-receptor has been identified as a possible therapeutic target for AUDs [36] and genetic studies indicate that the NK1-receptor is involved in the etiology of AUDs [37]. The present study gives further support for a role of the NK1-receptor system in appetitive behaviors, as the NK1-receptor antagonist, ezlopitant, inhibits SP binding in rat membranes and decreases both sucrose and ethanol self-administration without decreasing water or salty solution consumption or inhibiting general locomotor activity.

In the present study, the NK1-receptor antagonist ezlopitant is effective in attenuating both sucrose and ethanol intake in rodents. Somewhat surprisingly, ezlopitant more potently inhibited sucrose consumption when compared to ethanol in all drinking models used. While the addictive properties of ethanol are well established (for review see [10], [49]), evidence for food or sugar addiction has been largely based on anecdotal evidence. However, some people claim that they feel compelled to eat sweet foods, similar to how an alcoholic might crave alcohol [9] and it has recently been shown that natural reinforcers stimulate the same neural systems and reward mediating neurotransmitters (including dopamine, acetylcholine and opioids) as ethanol (for review see: [9], [10]). Furthermore, emerging evidence indicate that sucrose is addictive in rodents [9], [18], [23], [50]. Several stages of addiction, for example bingeing [51], withdrawal [9], [23], [52], craving [53][55] and cross-sensitization to amphetamine and cocaine [56], [57], can all be induced following intermittent excessive sugar intake. Finally, the reinforcing properties of sugar are supported by a recent study showing that sweetened solutions can surpass cocaine reward, even in drug-sensitized and -addicted rats [58].

Recently, the NK1-receptor antagonist LY686017 was shown to suppress spontaneous alcohol craving in AUD patients [36]. Additionally, the amygdala, which expresses high levels of NK1-receptors, plays a role in the motivational aspects of alcohol drinking behaviors [59]. Thus, LY686017 might attenuate craving by suppression of pathologically elevated activity in the amygdala [36]. Furthermore, MOR and NK1-receptors are co-expressed in amygdala neurons [60] which is an important area for the NK1-receptor's involvement in the motivational aspects of morphine reward [29]. The present results support this hypothesis. In the operant self-administration paradigm, the rats were trained on a fixed ratio (FR3) schedule to obtain an sucrose or ethanol reward and following establishment of stable baseline responding were challenged using a PR schedule. This paradigm is thus used as a measure of the motivation to seek the reward [61]. Ezlopitant was more effective in inhibiting sucrose and ethanol under FR3 operant conditions than in a two-bottle-choice setting where the reward is freely available. Importantly, the decreases in operant behaviors (on both fixed and PR schedules) were more dramatic in the sucrose-trained animals when compared to the ethanol-trained animals. Because, ezlopitant had no effect on general locomotor activity and NK1-receptor antagonists act as antidepressants in rodents [62], the possibility that the ezlopitant-induced decreased activity in the operant self-administration paradigm could be a result of decreased general activity or anhedonia is minimal. Furthermore, ezlopitant significantly attenuated intake of the non-caloric sweetener saccharin, but had no effect on water and salty solution consumption. These results indicate that NK1-receptors play an important role in appetitive responding for sweet solutions, regardless of caloric value, and may regulate the motivational aspects of consumption of ethanol and sweetened solutions.

The present study shows that ezlopitant treatment significantly decreases ethanol consumption in rats, supporting a study showing that NK1-receptor knock-out mice have markedly lower ethanol consumption than wild-type controls [36]. However, in contrast to the rat data, we found that ezlopitant-treatment failed to significantly decrease ethanol consumption compared to vehicle in C57BL/6 mice. One possible explanation is that higher doses of the NK1-receptor antagonist are needed to attenuate ethanol consumption in mice than rats. This is supported by a study indicating that a near-complete inactivation of NK1-receptors is needed to suppress ethanol consumption in mice [36]. Furthermore, in the present study, the lowest dose of ezlopitant (2 mg/kg) significantly decreased sucrose consumption in rats, whereas a markedly higher dose (15 mg/kg) was needed to decrease sucrose intake in mice. These results indicate a species difference in the sensitivity to the behavioral response following NK1-receptor antagonism.

The exact mechanism by which the NK1 receptor antagonist, ezlopitant decreases sucrose and ethanol intake remains unclear. There is strong evidence that both sucrose and ethanol stimulate the brain reward system through endogenous opioids (see for example [10], [11], [13]). The effectiveness of ezlopitant in attenuating sucrose and ethanol consumption indicates there may also be an interaction between NK1-receptors and the endogenous opioid system in reward-related behaviors, but this possibility remains to be determined.

In conclusion, the present study demonstrates that the NK1-receptor antagonist, ezlopitant, decreases sucrose and ethanol consumption and seeking in three different drinking models, giving further support of an involvement of the NK1-receptor system in AUDs and other reward-related behaviors. Additionally, these results indicate that the NK1-receptor system may serve as a therapeutic target for obesity induced by over-consumption of natural reinforcers. A clinical study is possible as ezlopitant is known to be safe in human subjects [40], [41].

Methods

Animals and Housing

Male, Long Evans rats (Harlan Indianapolis, IN) and C57BL/6 mice (Charles River Laboratories, Wilmington, MA) were individually housed in ventilated, climate controlled Plexiglas cages. The animals were acclimatized to the individual housing conditions and handling before the start of the experiments. All animals in the two-bottle-choice experiments were maintained on a 12 hour reversed light-dark cycle (lights off at 10 am) and rats in the operant self-administration and locomotor experiments were maintained on a regular 12 hour light-dark cycle (lights on at 7 am). Food and water were available ad libitum, except during initial training in the operant self-administration paradigm, as described below.

Ethical Consideration

The experiments contained herein comply with the current laws of the United States of America. All procedures were pre-approved by the Gallo Center Institutional Animal Care and Use Committee and conducted in accordance with NIH guidelines for the Humane Care and Use of Laboratory Animals.

Operant Self-Administration Paradigm

Fixed Ratio Schedule.

One group of Long-Evans rats (233±2 g, n = 16) was trained to self-administer 5% sucrose, and one group of Long-Evans rats (251±4 g, n = 15) was trained to self-administer 10% ethanol using a modified sucrose fading procedure [42] in standard operant conditioning chambers (Coulbourn Instruments, Allentown, PA), as described elsewhere [27]. Both groups were kept on a fixed ratio 3 schedule of reinforcement (FR3; three active lever presses required for 0.1 ml reward, 10% v/v ethanol or 5% sucrose, respectively), daily (Monday through Friday) for 30 minutes for at least five months prior to ezlopitant testing. All rats received all four treatment doses (vehicle, 2, 5 and 10 mg/kg intraperitoneal, i.p.) and each injection was given seven days apart using a Latin square design. Thus, each rat served as its own control.

Progressive Ratio Schedule.

One group of Long-Evans rats (214±3 g, n = 15) was trained to self-administer 5% sucrose, and one group of Long-Evans rats (238±3 g, n = 15) was trained to self-administer 10% ethanol as described above. Following ∼6 weeks (∼30 sessions) of 30 minute FR3 sessions, both groups of animals were treated with ezlopitant (vehicle, 2, 5 and 10 mg/kg, i.p.) 30 minutes prior to a progressive ratio (PR) test. The PR ratio method is as described by [43]. Briefly, the PR session was initiated by presentation of a compound cue (extension of the levers, illumination of the stimulus light over the active lever, tone sounding, and illumination of a raised dipper cup filled with alcohol or sucrose). After the compound cue, responding proceeded under a PR schedule that was the same for alcohol and sucrose rats. The PR schedule of reinforcement was determined by the equation . Briefly, after the compound cue, rats could lick the dipper cup, press a lever, or do nothing. If rats licked first, a PR schedule of reinforcement of 1, 1, 2, 2, 3, 4, 5, 6, 7, 9, 10, 12, 13, 15, 17, 20, 22, 25, 28, 32, 36, 40, 45, 50, etc., ensued. If rats pressed first, a PR of 1, 2, 2, 3, 4, 5, 6, 7, 9, 10, 12, 13, 15, 17, 20, 22, 25, 28, 32, 36, 40, 45, 50, etc., ensued. If the rat chose to do nothing, a 20 s timeout period occurred, and the rat was re-cued with the compound cue for up to 20 iterations. For the few rats that required re-cue, one or two re-cues were sufficient to elicit a response. Breakpoint was defined as the number of presses contained in the last, successfully completed ratio in either a 1 hr session or after 15 min of non-responding, whichever came first. Null responses, where an animal completed the required number of lever presses but did not lick to receive the reinforcer, were not counted toward the breakpoint. Ezlopitant-PR tests were performed on Tuesdays and Fridays in a Latin square design with regular FR3 sessions performed the other three days a week. Thus, each rat served as its own control.

Two-Bottle-Choice Drinking Paradigms

All fluids were presented in 100-ml graduated glass (for rats) or 50-ml plastic cylinders (for mice), with stainless steel drinking spouts inserted through two grommets in front of the cage. Bottles were weighed 6 and 24 hours (for rats) and 2 hours (for mice) after the fluids were presented, and measurements were taken to the nearest 0.1 gram. The weight of each animal was measured daily in order to calculate the gram per kilogram (g/kg) ethanol, sucrose and saccharin intake, respectively. Ethanol and sucrose preference (%) was calculated as the grams of ethanol, or sucrose, consumed divided by the total fluid consumption (grams of ethanol or sucrose+grams of water).

Intermittent-Access-20%-Ethanol: High Ethanol Consumption Model.

The intermittent-access-20%-ethanol two-bottle-choice drinking paradigm does not require sucrose fading and water is always available ad libitum [44], [45]. On the Monday following the end of the housing acclimatization period, 12 Long Evans rats were given access to one bottle of 20% v/v ethanol and one bottle of water. After 24 hours the ethanol bottle was replaced with a second water bottle that was available for the next 24 hours. This pattern was repeated on Wednesdays and Fridays. The rats had unlimited access to water over the weekend after the 24 hour measurement was taken on Saturday morning. After stable baseline drinking levels of 20% ethanol for at least 12 weeks, the rats were administered ezlopitant. All rats received all four treatment doses (vehicle, 2, 5 and 10 mg/kg, i.p.) and each injection was given seven days apart using a Latin square design. Thus, each rat served as its own control.

Intermittent-Access-5%-Sucrose.

Long-Evans rats (263±5, n = 11) were given intermittent-access to 5% sucrose solution according to the same schedule as the intermittent-access-20%-ethanol model. When rats had maintained stable baseline drinking levels for 12 weeks, administration of ezlopitant began. All rats received all four treatments (vehicle, 2, 5 and 10 mg/kg i.p.) and each injection was given seven days apart using a Latin square design.

Intermittent-Access-0.2%-Saccharin.

Long-Evans rats (n = 10) were given intermittent-access to 0.2% saccharin solution according to the same schedule as the intermittent-access-20%-ethanol model. When rats had maintained stable baseline drinking levels for ∼5 weeks, administration of ezlopitant began. All rats received all four treatments (vehicle, 2, 5 and 10 mg/kg i.p.) and each injection was given seven days apart using a Latin square design.

Continuous-Access to Water.

The effect of ezlopitant on consumption of water and salty solution, respectively, was evaluated in the same group Long-Evans rats given intermittent-access to 0.2% saccharin. Following the last ezlopitant-saccharin test occasion, the saccharin was removed and the rats were given continuous-access to two bottles of water. Following a four-day washout period, ezlopitant (10 mg/kg, i.p.) or vehicle was given to the rats to evaluate the effect on water consumption. The treatment was repeated and reversed between animals 48hrs later. Thus, each animal served as its own control.

Intermittent-Access-0.175%-Sodium Chloride (NaCl) solution.

After the last ezlopitant-water test occasion, the rats were given intermittent-access to one bottle of 0.175% NaCl in water and one bottle of water. Following five days, ezlopitant (10 mg/kg, i.p.) or vehicle was given to the rats to evaluate the effect on salty solution consumption. The treatment was repeated and reversed between animals 48hrs later. Thus, each animal served as its own control.

Drinking in the Dark Model.

The “drinking in the dark” (DID) model of ethanol consumption (adapted from [46]) induces high levels of ethanol consumption in mice. In brief, male C57BL/6 mice (5–6 weeks of age and 22±0.5g) were given access to one bottle of 20% ethanol and one bottle of water during a two-hour-period (1pm-3pm), Monday to Friday in a reverse light/dark cycle room. Two bottles of water were available at all other times. The weight of each mouse was measured daily to calculate the g/kg ethanol intake. To habituate the mice to injections, one saline injection (10 ml/kg body weight) was given on two consecutive days, a week before the start of the treatment. Following stable baseline consumption during six-seven weeks (30–38 drinking sessions, 5.0±0.2 g/kg/2 hrs), the mice were randomly assigned to four different doses of ezlopitant (vehicle (n = 12), 5 (n = 8), 10 (n = 15) or 15 (n = 7) mg/kg i.p).

The DID method was also used as a model of sucrose consumption. Male C57BL/6 mice (5–6 weeks of age and 19±0.5g) were given access to 5% sucrose instead of ethanol and subjected to ezlopitant treatment and the same habituation schedule as described above (vehicle (n = 15), 5 (n = 10), 10 (n = 12) or 15 (n = 6) mg/kg i.p).

General Locomotor Activity

Locomotor studies were run in activity-monitoring chambers (40×40 cm) with horizontal photo beams (Med Associates, St Albans, VT). Horizontal locomotor activity was monitored at 100 ms throughout the sessions. The study was run in 4 daily 2-hour-sessions as described previously [47]. In brief, after habituation of boxes (Day 1) and injections (Day 2 and 3) testing was conducted on Day 4. Data from Day 3 was used to assign animals to one of two treatment groups (vehicle, or ezlopitant (10 mg/kg, i.p.), n = 6 per group). After 60 minutes, a single injection of the assigned treatment was given, subsequently, the session continued for an additional 60 minutes. Data was collected across the entire 2-hour-session and recorded as distance traveled in cm.

[35S]GTPγS Binding in Rat Membranes

Single drug dose-response curve of Substance P (SP)-stimulated (0.1 nmol/L–100 µmol/L) [35S]GTPγS-binding and SP-(1 µmol/L)-stimulated [35S]GTPγS-binding in the presence of ezlopitant (0.1 nmol/L–100 µmol/L) were performed in triplicate in membranes prepared from rat cerebral cortex as described previously [48]. The brain tissue was collected from water rats (n = 3) as well as from rats that had consumed 5% sucrose (n = 3) according to the intermittent-access schedule described above. [35S]GTPγS-stimulated binding was assessed with NXT TOPCOUNTER and expressed as a percentage increase in basal [35S]GTPγS-binding.

Drugs and Chemicals

[35S]-guanosine 5′-(γ-thio)triphosphate ([35S]-GTPγS) (250 µCi; 9.25 MBq) (Perkin-Elmer, Boston, Massachusetts) and SP (Sigma Aldrich, St. Louis, MO, USA), other chemicals used in binding assays supplied as described previously [48]. All solutions were prepared in filtered water from 95% (v/v) ethanol (Gold Shield Chemical Ac., Hayward, CA, USA), (w/v) sucrose (Fisher Scientific, NJ, USA), saccharin or NaCl (Sigma Aldrich, St. Louis, MO, USA), respectively. The NK1-receptor antagonist, ezlopitant (CJ-11,974) [(2S,3S-cis)-2-diphenylmethyl)-N-[(2-methoxy,5-isopropylphenyl)methyl]-1-azabicyclo-[2.2.2]octan-3-amine)], was generously provided by Pfizer Global Research and Development, Groton, CT, USA. The compound was prepared in saline immediately before each injection. All injections were given as an acute i.p. injection (1 ml/kg for rats and 10 ml/kg for mice), 30 minutes before bottles were presented or before the start of the operant self-administration session.

Statistics

Statistical analysis was performed using GraphPadPrism software. Data were analyzed by one way ANOVA (mouse data) or repeated measures ANOVA (rat data) followed by Newman-Keuls Post hoc analysis when a significant overall main effect was found (P<0.05). The locomotor, water- and NaCl-2-bottle-choice data were analyzed with Student's t-test. Data from in vitro functional binding assays were analyzed by non-linear regression with a sigmoidal curve with variable slope to determine EC50 and IC50 values.

Acknowledgments

We thank Rui Li, Hilary Garcia, Tal Oppenheimer, and Nathan Santos for excellent technical assistance with the behavioral paradigms. We thank Pfizer Inc for providing ezlopitant for these studies.

Author Contributions

Conceived and designed the experiments: JAS JKH SEB. Performed the experiments: PS JAS CKN JKH JJBO. Analyzed the data: PS JAS CKN JKH JJBO SEB. Wrote the paper: PS JAS CKN JKH JJBO SEB.

References

  1. 1. Berthoud HR (2006) Homeostatic and non-homeostatic pathways involved in the control of food intake and energy balance. Obesity (Silver Spring) 14: Suppl 5197S–200S.HR Berthoud2006Homeostatic and non-homeostatic pathways involved in the control of food intake and energy balance.Obesity (Silver Spring)14Suppl 5197S200S
  2. 2. Blundell JE, Gillett A (2001) Control of food intake in the obese. Obes Res 9: Suppl 4263S–270S.JE BlundellA. Gillett2001Control of food intake in the obese.Obes Res9Suppl 4263S270S
  3. 3. Bray GA, York B, DeLany J (1992) A survey of the opinions of obesity experts on the causes and treatment of obesity. Am J Clin Nutr 55: 151S–154S.GA BrayB. YorkJ. DeLany1992A survey of the opinions of obesity experts on the causes and treatment of obesity.Am J Clin Nutr55151S154S
  4. 4. Hill JO, Peters JC (1998) Environmental contributions to the obesity epidemic. Science 280: 1371–4.JO HillJC Peters1998Environmental contributions to the obesity epidemic.Science28013714
  5. 5. Howard BV, Wylie-Rosett J (2002) Sugar and cardiovascular disease: A statement for healthcare professionals from the Committee on Nutrition of the Council on Nutrition, Physical Activity, and Metabolism of the American Heart Association. Circulation 106: 523–7.BV HowardJ. Wylie-Rosett2002Sugar and cardiovascular disease: A statement for healthcare professionals from the Committee on Nutrition of the Council on Nutrition, Physical Activity, and Metabolism of the American Heart Association.Circulation1065237
  6. 6. Ludwig DS, Peterson KE, Gortmaker SL (2001) Relation between consumption of sugar-sweetened drinks and childhood obesity: a prospective, observational analysis. Lancet 357: 505–8.DS LudwigKE PetersonSL Gortmaker2001Relation between consumption of sugar-sweetened drinks and childhood obesity: a prospective, observational analysis.Lancet3575058
  7. 7. Berridge KC (1996) Food reward: brain substrates of wanting and liking. Neurosci Biobehav Rev 20: 1–25.KC Berridge1996Food reward: brain substrates of wanting and liking.Neurosci Biobehav Rev20125
  8. 8. Berridge KC, Robinson TE (2003) Parsing reward. Trends Neurosci 26: 507–13.KC BerridgeTE Robinson2003Parsing reward.Trends Neurosci2650713
  9. 9. Avena NM, Rada P, Hoebel BG (2008) Evidence for sugar addiction: behavioral and neurochemical effects of intermittent, excessive sugar intake. Neurosci Biobehav Rev 32: 20–39.NM AvenaP. RadaBG Hoebel2008Evidence for sugar addiction: behavioral and neurochemical effects of intermittent, excessive sugar intake.Neurosci Biobehav Rev322039
  10. 10. Gianoulakis C (2009) Endogenous Opioids and Addiction to Alcohol and Other Drugs of Abuse. Curr Top Med Chem. C. Gianoulakis2009Endogenous Opioids and Addiction to Alcohol and Other Drugs of Abuse.Curr Top Med Chem
  11. 11. Mercer ME, Holder MD (1997) Food cravings, endogenous opioid peptides, and food intake: a review. Appetite 29: 325–52.ME MercerMD Holder1997Food cravings, endogenous opioid peptides, and food intake: a review.Appetite2932552
  12. 12. Shalev U, Yap J, Shaham Y (2001) Leptin attenuates acute food deprivation-induced relapse to heroin seeking. J Neurosci 21: RC129.U. ShalevJ. YapY. Shaham2001Leptin attenuates acute food deprivation-induced relapse to heroin seeking.J Neurosci21RC129
  13. 13. Tanda G, Di Chiara G (1998) A dopamine-mu1 opioid link in the rat ventral tegmentum shared by palatable food (Fonzies) and non-psychostimulant drugs of abuse. Eur J Neurosci 10: 1179–87.G. TandaG. Di Chiara1998A dopamine-mu1 opioid link in the rat ventral tegmentum shared by palatable food (Fonzies) and non-psychostimulant drugs of abuse.Eur J Neurosci10117987
  14. 14. Volkow ND, Wise RA (2005) How can drug addiction help us understand obesity? Nat Neurosci 8: 555–60.ND VolkowRA Wise2005How can drug addiction help us understand obesity?Nat Neurosci855560
  15. 15. Yeomans MR, Gray RW (2002) Opioid peptides and the control of human ingestive behaviour. Neurosci Biobehav Rev 26: 713–28.MR YeomansRW Gray2002Opioid peptides and the control of human ingestive behaviour.Neurosci Biobehav Rev2671328
  16. 16. Shimura T, Kamada Y, Yamamoto T (2002) Ventral tegmental lesions reduce overconsumption of normally preferred taste fluid in rats. Behav Brain Res 134: 123–30.T. ShimuraY. KamadaT. Yamamoto2002Ventral tegmental lesions reduce overconsumption of normally preferred taste fluid in rats.Behav Brain Res13412330
  17. 17. Hajnal A, Smith GP, Norgren R (2004) Oral sucrose stimulation increases accumbens dopamine in the rat. Am J Physiol Regul Integr Comp Physiol 286: R31–7.A. HajnalGP SmithR. Norgren2004Oral sucrose stimulation increases accumbens dopamine in the rat.Am J Physiol Regul Integr Comp Physiol286R317
  18. 18. Spangler R, Wittkowski KM, Goddard NL, Avena NM, Hoebel BG, et al. (2004) Opiate-like effects of sugar on gene expression in reward areas of the rat brain. Brain Res Mol Brain Res 124: 134–42.R. SpanglerKM WittkowskiNL GoddardNM AvenaBG Hoebel2004Opiate-like effects of sugar on gene expression in reward areas of the rat brain.Brain Res Mol Brain Res12413442
  19. 19. Wang GJ, Volkow ND, Thanos PK, Fowler JS (2004) : Similarity between obesity and drug addiction as assessed by neurofunctional imaging: a concept review. J Addict Dis 23: 39–53.GJ WangND VolkowPK ThanosJS Fowler2004: Similarity between obesity and drug addiction as assessed by neurofunctional imaging: a concept review.J Addict Dis233953
  20. 20. Wang GJ, Yang J, Volkow ND, Telang F, Ma Y, et al. (2006) Gastric stimulation in obese subjects activates the hippocampus and other regions involved in brain reward circuitry. Proc Natl Acad Sci U S A 103: 15641–5.GJ WangJ. YangND VolkowF. TelangY. Ma2006Gastric stimulation in obese subjects activates the hippocampus and other regions involved in brain reward circuitry.Proc Natl Acad Sci U S A103156415
  21. 21. d'Anci KE, Kanarek RB, Marks-Kaufman R (1996) Duration of sucrose availability differentially alters morphine-induced analgesia in rats. Pharmacol Biochem Behav 54: 693–7.KE d'AnciRB KanarekR. Marks-Kaufman1996Duration of sucrose availability differentially alters morphine-induced analgesia in rats.Pharmacol Biochem Behav546937
  22. 22. Lieblich I, Cohen E, Ganchrow JR, Blass EM, Bergmann F (1983) Morphine tolerance in genetically selected rats induced by chronically elevated saccharin intake. Science 221: 871–3.I. LieblichE. CohenJR GanchrowEM BlassF. Bergmann1983Morphine tolerance in genetically selected rats induced by chronically elevated saccharin intake.Science2218713
  23. 23. Colantuoni C, Rada P, McCarthy J, Patten C, Avena NM, et al. (2002) Evidence that intermittent, excessive sugar intake causes endogenous opioid dependence. Obes Res 10: 478–88.C. ColantuoniP. RadaJ. McCarthyC. PattenNM Avena2002Evidence that intermittent, excessive sugar intake causes endogenous opioid dependence.Obes Res1047888
  24. 24. Kanarek RB, Mathes WF, Heisler LK, Lima RP, Monfared LS (1997) Prior exposure to palatable solutions enhances the effects of naltrexone on food intake in rats. Pharmacol Biochem Behav 57: 377–81.RB KanarekWF MathesLK HeislerRP LimaLS Monfared1997Prior exposure to palatable solutions enhances the effects of naltrexone on food intake in rats.Pharmacol Biochem Behav5737781
  25. 25. Rudski JM, Billington CJ, Levine AS (1997) A sucrose-based maintenance diet increases sensitivity to appetite suppressant effects of naloxone. Pharmacol Biochem Behav 58: 679–82.JM RudskiCJ BillingtonAS Levine1997A sucrose-based maintenance diet increases sensitivity to appetite suppressant effects of naloxone.Pharmacol Biochem Behav5867982
  26. 26. Beczkowska IW, Bowen WD, Bodnar RJ (1992) Central opioid receptor subtype antagonists differentially alter sucrose and deprivation-induced water intake in rats. Brain Res 589: 291–301.IW BeczkowskaWD BowenRJ Bodnar1992Central opioid receptor subtype antagonists differentially alter sucrose and deprivation-induced water intake in rats.Brain Res589291301
  27. 27. Steensland P, Simms JA, Holgate J, Richards JK, Bartlett SE (2007) Varenicline, an {alpha}4beta2 nicotinic acetylcholine receptor partial agonist, selectively decreases ethanol consumption and seeking. Proc Natl Acad Sci U S A 104: 12518–23.P. SteenslandJA SimmsJ. HolgateJK RichardsSE Bartlett2007Varenicline, an {alpha}4beta2 nicotinic acetylcholine receptor partial agonist, selectively decreases ethanol consumption and seeking.Proc Natl Acad Sci U S A1041251823
  28. 28. Stromberg MF, Rukstalis MR, Mackler SA, Volpicelli JR, O'Brien CP (2002) A comparison of the effects of 6-beta naltrexol and naltrexone on the consumption of ethanol or sucrose using a limited-access procedure in rats. Pharmacol Biochem Behav 72: 483–90.MF StrombergMR RukstalisSA MacklerJR VolpicelliCP O'Brien2002A comparison of the effects of 6-beta naltrexol and naltrexone on the consumption of ethanol or sucrose using a limited-access procedure in rats.Pharmacol Biochem Behav7248390
  29. 29. Gadd CA, Murtra P, De Felipe C, Hunt SP (2003) Neurokinin-1 receptor-expressing neurons in the amygdala modulate morphine reward and anxiety behaviors in the mouse. J Neurosci 23: 8271–80.CA GaddP. MurtraC. De FelipeSP Hunt2003Neurokinin-1 receptor-expressing neurons in the amygdala modulate morphine reward and anxiety behaviors in the mouse.J Neurosci23827180
  30. 30. Jabourian M, Venance L, Bourgoin S, Ozon S, Perez S, et al. (2005) Functional mu opioid receptors are expressed in cholinergic interneurons of the rat dorsal striatum: territorial specificity and diurnal variation. Eur J Neurosci 21: 3301–9.M. JabourianL. VenanceS. BourgoinS. OzonS. Perez2005Functional mu opioid receptors are expressed in cholinergic interneurons of the rat dorsal striatum: territorial specificity and diurnal variation.Eur J Neurosci2133019
  31. 31. Nakaya Y, Kaneko T, Shigemoto R, Nakanishi S, Mizuno N (1994) Immunohistochemical localization of substance P receptor in the central nervous system of the adult rat. J Comp Neurol 347: 249–74.Y. NakayaT. KanekoR. ShigemotoS. NakanishiN. Mizuno1994Immunohistochemical localization of substance P receptor in the central nervous system of the adult rat.J Comp Neurol34724974
  32. 32. Pickel VM, Douglas J, Chan J, Gamp PD, Bunnett NW (2000) Neurokinin 1 receptor distribution in cholinergic neurons and targets of substance P terminals in the rat nucleus accumbens. J Comp Neurol 423: 500–11.VM PickelJ. DouglasJ. ChanPD GampNW Bunnett2000Neurokinin 1 receptor distribution in cholinergic neurons and targets of substance P terminals in the rat nucleus accumbens.J Comp Neurol42350011
  33. 33. Poulin JF, Chevalier B, Laforest S, Drolet G (2006) Enkephalinergic afferents of the centromedial amygdala in the rat. J Comp Neurol 496: 859–76.JF PoulinB. ChevalierS. LaforestG. Drolet2006Enkephalinergic afferents of the centromedial amygdala in the rat.J Comp Neurol49685976
  34. 34. Murtra P, Sheasby AM, Hunt SP, De Felipe C (2000) Rewarding effects of opiates are absent in mice lacking the receptor for substance P. Nature 405: 180–3.P. MurtraAM SheasbySP HuntC. De Felipe2000Rewarding effects of opiates are absent in mice lacking the receptor for substance P.Nature4051803
  35. 35. Ripley TL, Gadd CA, De Felipe C, Hunt SP, Stephens DN (2002) Lack of self-administration and behavioural sensitisation to morphine, but not cocaine, in mice lacking NK1 receptors. Neuropharmacology 43: 1258–68.TL RipleyCA GaddC. De FelipeSP HuntDN Stephens2002Lack of self-administration and behavioural sensitisation to morphine, but not cocaine, in mice lacking NK1 receptors.Neuropharmacology43125868
  36. 36. George DT, Gilman J, Hersh J, Thorsell A, Herion D, et al. (2008) Neurokinin 1 receptor antagonism as a possible therapy for alcoholism. Science 319: 1536–9.DT GeorgeJ. GilmanJ. HershA. ThorsellD. Herion2008Neurokinin 1 receptor antagonism as a possible therapy for alcoholism.Science31915369
  37. 37. Seneviratne C, Ait-Daoud N, Ma JZ, Chen G, Johnson BA, et al. (2009) Susceptibility locus in neurokinin-1 receptor gene associated with alcohol dependence. Neuropsychopharmacology 34: 2442–9.C. SeneviratneN. Ait-DaoudJZ MaG. ChenBA Johnson2009Susceptibility locus in neurokinin-1 receptor gene associated with alcohol dependence.Neuropsychopharmacology3424429
  38. 38. Lowe JA 3rd, Drozda SE, Snider RM, Longo KP, Zorn SH, et al. (1992) The discovery of (2S,3S)-cis-2-(diphenylmethyl)-N-[(2-methoxyphenyl)methyl]-1- azabicyclo[2.2.2]-octan-3-amine as a novel, nonpeptide substance P antagonisst. J Med Chem 35: 2591–600.JA Lowe 3rdSE DrozdaRM SniderKP LongoSH Zorn1992The discovery of (2S,3S)-cis-2-(diphenylmethyl)-N-[(2-methoxyphenyl)methyl]-1- azabicyclo[2.2.2]-octan-3-amine as a novel, nonpeptide substance P antagonisst.J Med Chem352591600
  39. 39. Tsuchiya M, Fujiwara Y, Kanai Y, Mizutani M, Shimada K, et al. (2002) Anti-emetic activity of the novel nonpeptide tachykinin NK1 receptor antagonist ezlopitant (CJ-11,974) against acute and delayed cisplatin-induced emesis in the ferret. Pharmacology 66: 144–52.M. TsuchiyaY. FujiwaraY. KanaiM. MizutaniK. Shimada2002Anti-emetic activity of the novel nonpeptide tachykinin NK1 receptor antagonist ezlopitant (CJ-11,974) against acute and delayed cisplatin-induced emesis in the ferret.Pharmacology6614452
  40. 40. Hesketh PJ, Gralla RJ, Webb RT, Ueno W, DelPrete S, et al. (1999) Randomized phase II study of the neurokinin 1 receptor antagonist CJ-11,974 in the control of cisplatin-induced emesis. J Clin Oncol 17: 338–43.PJ HeskethRJ GrallaRT WebbW. UenoS. DelPrete1999Randomized phase II study of the neurokinin 1 receptor antagonist CJ-11,974 in the control of cisplatin-induced emesis.J Clin Oncol1733843
  41. 41. Reed-Hagen AE, Tsuchiya M, Shimada K, Wentland JA, Obach RS (1999) Pharmacokinetics of ezlopitant, a novel non-peptidic neurokinin-1 receptor antagonist in preclinical species and metabolite kinetics of the pharmacologically active metabolites. Biopharm Drug Dispos 20: 429–39.AE Reed-HagenM. TsuchiyaK. ShimadaJA WentlandRS Obach1999Pharmacokinetics of ezlopitant, a novel non-peptidic neurokinin-1 receptor antagonist in preclinical species and metabolite kinetics of the pharmacologically active metabolites.Biopharm Drug Dispos2042939
  42. 42. Samson HH (1986) Initiation of ethanol reinforcement using a sucrose-substitution procedure in food- and water-sated rats. Alcohol Clin Exp Res 10: 436–42.HH Samson1986Initiation of ethanol reinforcement using a sucrose-substitution procedure in food- and water-sated rats.Alcohol Clin Exp Res1043642
  43. 43. Bowers MS, Hopf FW, Chou JK, Guillory AM, Chang SJ, et al. (2008) Nucleus accumbens AGS3 expression drives ethanol seeking through G betagamma. Proc Natl Acad Sci U S A 105: 12533–8.MS BowersFW HopfJK ChouAM GuillorySJ Chang2008Nucleus accumbens AGS3 expression drives ethanol seeking through G betagamma.Proc Natl Acad Sci U S A105125338
  44. 44. Simms JA, Steensland P, Medina B, Abernathy KE, Chandler LJ, et al. (2008) Intermittent access to 20% ethanol induces high ethanol consumption in Long-Evans and Wistar rats. Alcohol Clin Exp Res 32: 1816–23.JA SimmsP. SteenslandB. MedinaKE AbernathyLJ Chandler2008Intermittent access to 20% ethanol induces high ethanol consumption in Long-Evans and Wistar rats.Alcohol Clin Exp Res32181623
  45. 45. Wise RA (1973) Voluntary ethanol intake in rats following exposure to ethanol on various schedules. Psychopharmacologia 29: 203–10.RA Wise1973Voluntary ethanol intake in rats following exposure to ethanol on various schedules.Psychopharmacologia2920310
  46. 46. Rhodes JS, Best K, Belknap JK, Finn DA, Crabbe JC (2005) Evaluation of a simple model of ethanol drinking to intoxication in C57BL/6J mice. Physiol Behav 84: 53–63.JS RhodesK. BestJK BelknapDA FinnJC Crabbe2005Evaluation of a simple model of ethanol drinking to intoxication in C57BL/6J mice.Physiol Behav845363
  47. 47. Richards JK, Simms JA, Steensland P, Taha SA, Borgland SL, et al. (2008) Inhibition of orexin-1/hypocretin-1 receptors inhibits yohimbine-induced reinstatement of ethanol and sucrose seeking in Long-Evans rats. Psychopharmacology (Berl) 199: 109–17.JK RichardsJA SimmsP. SteenslandSA TahaSL Borgland2008Inhibition of orexin-1/hypocretin-1 receptors inhibits yohimbine-induced reinstatement of ethanol and sucrose seeking in Long-Evans rats.Psychopharmacology (Berl)19910917
  48. 48. Nielsen CK, Simms JA, Pierson HB, Li R, Saini SK, et al. (2008) A novel delta opioid receptor antagonist, SoRI-9409, produces a selective and long-lasting decrease in ethanol consumption in heavy-drinking rats. Biol Psychiatry 64: 974–81.CK NielsenJA SimmsHB PiersonR. LiSK Saini2008A novel delta opioid receptor antagonist, SoRI-9409, produces a selective and long-lasting decrease in ethanol consumption in heavy-drinking rats.Biol Psychiatry6497481
  49. 49. Heilig M, Egli M (2006) Pharmacological treatment of alcohol dependence: target symptoms and target mechanisms. Pharmacol Ther 111: 855–76.M. HeiligM. Egli2006Pharmacological treatment of alcohol dependence: target symptoms and target mechanisms.Pharmacol Ther11185576
  50. 50. Rada P, Avena NM, Hoebel BG (2005) Daily bingeing on sugar repeatedly releases dopamine in the accumbens shell. Neuroscience 134: 737–44.P. RadaNM AvenaBG Hoebel2005Daily bingeing on sugar repeatedly releases dopamine in the accumbens shell.Neuroscience13473744
  51. 51. Colantuoni C, Schwenker J, McCarthy J, Rada P, Ladenheim B, et al. (2001) Excessive sugar intake alters binding to dopamine and mu-opioid receptors in the brain. Neuroreport 12: 3549–52.C. ColantuoniJ. SchwenkerJ. McCarthyP. RadaB. Ladenheim2001Excessive sugar intake alters binding to dopamine and mu-opioid receptors in the brain.Neuroreport12354952
  52. 52. Galic MA, Persinger MA (2002) Voluminous sucrose consumption in female rats: increased “nippiness” during periods of sucrose removal and possible oestrus periodicity. Psychol Rep 90: 58–60.MA GalicMA Persinger2002Voluminous sucrose consumption in female rats: increased “nippiness” during periods of sucrose removal and possible oestrus periodicity.Psychol Rep905860
  53. 53. Avena NM, Long KA, Hoebel BG (2005) Sugar-dependent rats show enhanced responding for sugar after abstinence: evidence of a sugar deprivation effect. Physiol Behav 84: 359–62.NM AvenaKA LongBG Hoebel2005Sugar-dependent rats show enhanced responding for sugar after abstinence: evidence of a sugar deprivation effect.Physiol Behav8435962
  54. 54. Grimm JW, Fyall AM, Osincup DP (2005) Incubation of sucrose craving: effects of reduced training and sucrose pre-loading. Physiol Behav 84: 73–9.JW GrimmAM FyallDP Osincup2005Incubation of sucrose craving: effects of reduced training and sucrose pre-loading.Physiol Behav84739
  55. 55. Grimm JW, Osincup D, Wells B, Manaois M, Fyall A, et al. (2008) Environmental enrichment attenuates cue-induced reinstatement of sucrose seeking in rats. Behav Pharmacol 19: 777–85.JW GrimmD. OsincupB. WellsM. ManaoisA. Fyall2008Environmental enrichment attenuates cue-induced reinstatement of sucrose seeking in rats.Behav Pharmacol1977785
  56. 56. Avena NM, Hoebel BG (2003) A diet promoting sugar dependency causes behavioral cross-sensitization to a low dose of amphetamine. Neuroscience 122: 17–20.NM AvenaBG Hoebel2003A diet promoting sugar dependency causes behavioral cross-sensitization to a low dose of amphetamine.Neuroscience1221720
  57. 57. Gosnell BA (2005) Sucrose intake enhances behavioral sensitization produced by cocaine. Brain Res 1031: 194–201.BA Gosnell2005Sucrose intake enhances behavioral sensitization produced by cocaine.Brain Res1031194201
  58. 58. Lenoir M, Serre F, Cantin L, Ahmed SH (2007) Intense sweetness surpasses cocaine reward. PLoS One 2: e698.M. LenoirF. SerreL. CantinSH Ahmed2007Intense sweetness surpasses cocaine reward.PLoS One2e698
  59. 59. Heilig M, Koob GF (2007) A key role for corticotropin-releasing factor in alcohol dependence. Trends Neurosci 30: 399–406.M. HeiligGF Koob2007A key role for corticotropin-releasing factor in alcohol dependence.Trends Neurosci30399406
  60. 60. Yu YJ, Arttamangkul S, Evans CJ, Williams JT, von Zastrow M (2009) Neurokinin 1 receptors regulate morphine-induced endocytosis and desensitization of mu-opioid receptors in CNS neurons. J Neurosci 29: 222–33.YJ YuS. ArttamangkulCJ EvansJT WilliamsM. von Zastrow2009Neurokinin 1 receptors regulate morphine-induced endocytosis and desensitization of mu-opioid receptors in CNS neurons.J Neurosci2922233
  61. 61. Sanchis-Segura C, Spanagel R (2006) Behavioural assessment of drug reinforcement and addictive features in rodents: an overview. Addict Biol 11: 2–38.C. Sanchis-SeguraR. Spanagel2006Behavioural assessment of drug reinforcement and addictive features in rodents: an overview.Addict Biol11238
  62. 62. Dableh LJ, Yashpal K, Rochford J, Henry JL (2005) Antidepressant-like effects of neurokinin receptor antagonists in the forced swim test in the rat. Eur J Pharmacol 507: 99–105.LJ DablehK. YashpalJ. RochfordJL Henry2005Antidepressant-like effects of neurokinin receptor antagonists in the forced swim test in the rat.Eur J Pharmacol50799105