The neuropeptide-Y Y5 receptor antagonist L-152,804 decreases alcohol self-administration in inbred alcohol-preferring (iP) rats

Alcohol 36 (2005) 179–186

The neuropeptide-Y Y5 receptor antagonist L-152,804 decreases alcohol self-administration in inbred alcohol-preferring (iP) rats Jason P. Schroeder, David H. Overstreet, Clyde W. Hodge* Department of Psychiatry and Bowles Center for Alcohol Studies, University of North Carolina at Chapel Hill, Thurston-Bowles Building, CB #7178, Chapel Hill, NC 27599, USA Received 3 June 2005; received in revised form 26 September 2005; accepted 3 October 2005

Abstract Neuropeptide-Y (NPY) is the most abundant and widely distributed peptide in the mammalian central nervous system and increases feeding behavior through actions at the Y5 receptor subtype. Recent pharmacological evidence indicates that NPY activity at this receptor subtype can modulate ethanol reinforcement. The purpose of this study was to determine if NPY Y5 receptor antagonism reduces ethanol self-administration and reinforcement in a rodent genetic animal model of alcoholism. Selectively inbred alcohol-preferring (iP) rats were trained to voluntarily consume ethanol (10% vol/vol) versus H2O in a 24-h two-bottle choice test. An additional group of iP rats was trained in operant ethanol self-administration to lever press on a fixed-ratio 1 schedule for ethanol (10% vol/vol) reinforcement. Following establishment of baseline intake or ethanol-reinforced responding, iP rats were injected with L-152,804 (0–20 mg/kg) prior to two-bottle or operant ethanol self-administration sessions. In the two-bottle choice test, L-152,804 (3 and 10 mg/kg, ip) significantly reduced ethanol intake (g/kg) at 4- and 6-h postinjection and had no effect on food intake. In the operant procedure, L-152,804 (10 and 20 mg/kg, ip) significantly reduced both the dosage of self-administered ethanol (g/kg/1-h) and the total number of ethanol-reinforced responses. No effect was observed on latency to the first response or the number of inactive lever presses. These results indicate that blockade of NPY Y5 receptor activity decreases both voluntary ethanol drinking and ethanol reinforcement in a rodent genetic animal model of alcoholism. For this reason, NPY Y5 receptor antagonists may be useful in medical management of alcohol abuse and alcoholism in the human population. Ó 2005 Elsevier Inc. All rights reserved. Keywords: Alcohol; Reinforcement; Operant self-administration; NPY Y5 receptors; Inbred alcohol-preferring rats; iP rats

1. Introduction Neuropeptide-Y (NPY) has been implicated in a variety of neurobiological functions (Wettstein et al., 1995) and is the most abundant and widely distributed peptide in the mammalian central nervous system (CNS) (Allen et al., 1983; Heilig & Widerlov, 1990). The actions of NPY are mediated by a family of G-protein-coupled receptors (Balasubramaniam, 1997; Dumont et al., 1993), which includes at least five subtypes: Y1, Y2, Y4, Y5, and Y6 (Blomqvist & Herzog, 1997). The Y1 (Larsen et al., 1993; Mikkelsen & Larsen, 1992), Y2 (Gustafson et al., 1997), and Y5 (Gerald et al., 1996) receptor subtypes are located in the brain tissue of rodents as demonstrated by hybridization. NPY Y5 receptors are present at significant levels in the paraventricular nucleus of the hypothalamus (PVN), arcuate nucleus,

* Corresponding author. Tel.: 11-919-843-4823; fax: 11-919-9665679. E-mail address: [email protected] (C.W. Hodge). 0741-8329/05/$ – see front matter Ó 2005 Elsevier Inc. All rights reserved. doi: 10.1016/j.alcohol.2005.10.001

thalamus, and amygdala, which suggests the presence of functional hypothalamic–limbic neural circuits (Gerald et al., 1996), and the highly conserved expression of the Y5 receptor between human and rat hypothalamus and limbic brain regions (Nichol et al., 1999) indicates a major neurobiological role for this receptor. Evidence has accumulated implicating the NPY Y5 receptor in feeding behavior. Research using NPY Y5 agonists (Gerald et al., 1996; Haynes et al., 1998; Hu et al., 1996; Kirby et al., 1995; Wyss et al., 1998), antagonists (Criscione et al., 1998), and antisense nucleotides (Flynn et al., 1999; Tang-Christensen et al., 1998) indicates that ingestive behavior appears to be at least partially mediated by NPY activity at the Y5 receptor. The role of NPY activity at the Y5 receptor in feeding behavior suggests that NPY function at this receptor may also modulate ethanol intake, because addictive behaviors may usurp homeostatic mechanisms, which evolved to regulate food intake (Samson & Hodge, 1996). Consistent with this hypothesis, we have shown that blockade of NPY Y5 receptors with the

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selective antagonist L-152,804 modulates the onset and maintenance of operant ethanol self-administration in C57BL/6J mice (Schroeder et al., 2003a). Additional research indicates that genetically selected alcohol-preferring (P) ratlines exhibit different levels of NPY expression in the brain and different levels of ethanol intake following NPY administration. The increased drinking of P rats may be related to NPYergic activity in this selectively bred ratline. A genetic linkage analysis on the F2 intercross progenies of P and nonpreferring (NP) rats revealed a chromosomal region containing a NPY precursor gene (Bice et al., 1998; Carr et al., 1998). In addition, P rats and high-alcohol-drinking rats have lower levels of NPY in the amygdala when compared to their low-drinking counterparts (Ehlers et al., 1998a; Hwang et al., 1999). Moreover, P rats demonstrate greater NPY expression in the arcuate nucleus and PVN than NP rats (Hwang et al., 1999). Finally, pharmacological data have shown that intracerebral ventricular infusion of NPY decreases ethanol intake in P but not in NP rats (Badia-Elder et al., 2001; Gilpin et al., 2003) or unselected Wistar rats (Badia-Elder et al., 2001; Slawecki et al., 2000). To further examine the role of NPY Y5 receptors in the mediation of ethanol self-administration, the present set of experiments was designed to test the influence of the Y5 antagonist L-152,804 on voluntary ethanol intake and on ethanol-reinforced responding using a genetic model of high alcohol intake, which is the P rat. The P ratline has been found to fulfill the requirements of an animal model of alcoholism (Lester & Freed, 1973). P rats voluntarily consume ethanol in quantities that produce significant blood alcohol concentrations (50–200 mg%), respond for the pharmacological rather than the sensory effects of ethanol, and develop tolerance and dependence through voluntary drinking (Kampov-Polevoy et al., 2000; Li et al., 1987; Murphy et al., 2002). Two separate groups of inbred alcohol-preferring rats (iP) were allowed to self-administer 10% (vol/vol) ethanol in either the 24-h two-bottle choice test or 1-h operant sessions. Following the establishment of baseline intake or ethanol-reinforced responding, rats were injected with the novel and selective nonpeptide NPY Y5 antagonist L-152,804 (Kanatani et al., 2000) to determine the role of this receptor subtype in the maintenance of operant ethanol self-administration experiments in a rodent genetic model of alcoholism.

2. Materials and methods 2.1. Animals Twenty-four male iP rats weighing 350–500 g at the beginning of training were used in the studies. This stock of iP rats was derived from breeders of the selected line of P rats originally provided in 1999 by Indiana University (courtesy of Dr. T.K. Li) and has been bred for an additional 10

generations (6–10 breeders per generation) at the University of North Carolina at Chapel Hill. The rats were housed individually in Plexiglas cages at the University of North Carolina at Chapel Hill vivarium. The animal colony room was maintained on a reversed 12-h light:12-h dark (12L:12D) cycle with the lights off at 07:00. All experimental procedures were conducted under institutional and NIH guidelines. 2.2. Two-bottle drinking procedure Oral alcohol and water intake were examined using a two-bottle choice protocol. Male iP rats (n 5 8) were allowed 1 week to acclimatize to individual housing conditions and handling. During this period water was the only fluid available. Then, rats were given a choice between ethanol (10% wt/vol) and water in the home cage. Following the establishment of baseline responding over a period of 3 weeks, L-152,804 (0, 3, 10 mg/kg, ip; pseudorandomized) was administered immediately prior to a 24-h access period at the beginning of the dark cycle. Injections were administered on nonsequential days (M–F, Monday–Friday) to allow at least 48 h for drug clearance. Fluid levels were recorded at 0, 2, 4, 6, and 24 h. Volume of ethanol consumed was converted to g/kg, and the alcohol preference was calculated. Additional male iP rats were used to determine the effects of L-152,804 injection on food intake at each of the time points examined in the previous two-bottle drinking study. In this control experiment L-152,804 (Vehicle or 3 mg/kg, ip) was administered immediately prior to a 24h access period at the beginning of the subjects’ dark cycle in two separate groups of iP rats (n 5 4). The food in each subject’s feeder bin was weighed at 0, 2, 4, 6, and 24 h to determine the effect of the most efficacious dose of L-152,804 (as measured by reductions in ethanol intake in the two-bottle paradigm) on food intake. 2.3. Ethanol self-administration apparatus Experimental sessions were conducted in Plexiglas chambers (27 3 37 3 2l cm) located in sound attenuating cubicles (MED Associates, model ENV 016M, Georgia, VT). Chambers were equipped with exhaust fans that masked external noise. The right wall of each chamber contained two response levers (MED Associates, model ENV-110M) and a liquid (0.1 ml) dipper (MED Associates, model ENV-202M). Responses on the right lever activated the dipper for 5 s, whereas responses on the left lever produced no programmed consequence. Chambers were interfaced to an IBM desktop computer that controlled experimental sessions and recorded data using commercially available software (MED Associates, MED-PC). 2.4. Ethanol self-administration procedure After 2 weeks of adaptation to laboratory housing conditions, fluid access was restricted to 1 h per day (for 2 days

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Ethanol solutions were prepared by mixing appropriate volumes of ethanol (95% wt/vol) and distilled water. Sucrose (wt/vol) solutions consisted of granulated cane sugar dissolved in distilled water. Ethanol, sucrose, and water solutions were presented at room temperature. The NPY Y5 antagonist L-152,804 (Tocris Cookson, Ellisville, MO) was suspended in (2-hydroxy)-b-cyclodextrin (45% wt/vol) in sterile deionized water by repeated vortexing and sonification.

2.6. Data analyses The volume of ethanol and water consumed by iP rats in the two-bottle method recorded to the nearest 0.1 ml, and the volume of ethanol consumed was converted to g/kg ethanol intake. In addition, percent ethanol preference was calculated as (ethanol ml/total fluid ml) 3 100. The principal parameters of ethanol-reinforced responding were the total number of ethanol-reinforced and inactive lever presses, volume of ethanol consumed (ml), response latency (i.e., temporal delay to the first ethanol response), and response termination (i.e., the time at which the last response occurred). Ethanol intake (g/kg) was estimated using each animal’s daily body weight and the volume of ethanol delivered per reinforcer (0.1 ml of 10% vol/vol ethanol). Drug dose effects were analyzed statistically by analysis of

3. Results 3.1. Effects of L-152,804 on two-bottle drinking behavior Fig. 1a illustrates the effects of L-152,804 on g/kg two-bottle ethanol intake. A two-way repeated measures ANOVA (injection 3 time) on cumulative g/kg ethanol intake revealed a significant interaction [F(4, 28) 5 5.44, p 5 .002] indicating that L-152,804 altered the dose of ethanol self-administered in a time-dependent manner. Student–Newman–Keuls analysis indicated that injection of the 3 and 10 mg/kg doses of L-152,804 significantly decreased ethanol intake as compared to vehicle levels at 4 and 6 h following injection. In addition, a significant effect of injection [F(2, 14) 5 6.82, p 5 .009] followed by post hoc Cumulative ethanol intake (mean g/kg +/- SEM)

2.5. Drugs

variance (ANOVA) followed by Student–Newman–Keuls comparisons using SigmaStat v. 2.0 (SPSS, Chicago, IL).

2.5

a) 2.0 1.5

* 1.0

* 0.5

*

*

0.0

2

4

6

Time (Hours)

Cumulative water intake (mean mls +/- SEM)

only) and iP rats were trained to lever press by autoshaping. Responses on the active lever were reinforced with sucrose (10% wt/vol) during two overnight sessions. Daily 1-h sessions were then conducted with sucrose (10% wt/vol). When sucrose response patterns stabilized, the rats were trained to self-administer ethanol (10% vol/vol) using a sucrose fading procedure. Briefly, ethanol was gradually added to the sucrose solution and then sucrose was faded out of the solution over a 1-month period until rats were self-administering 10% ethanol. After sucrose fading, the rats were allowed to self-administer ethanol (10% vol/vol) 5 days per week (M–F) for 2 months. Following this baseline period, the effects of the NPY Y5 antagonist L-152,804 (0, 3, 10, and 20 mg/kg; n 5 8) on the maintenance of operant ethanol self-administration were determined in a Latin-Square within-subjects design. Injections were administered on nonsequential days (M–F) to allow at least 48 h for drug clearance. Vehicle or drug was administered 2 h prior to the start of the 1-h session because results obtained from the two-bottle choice method in iP rats and operant ethanol self-administration in C57BL/6J mice (Schroeder et al., 2003a) indicated that L-152,804 had its greatest effect on ethanol intake 2 h after administration. In addition, previous research demonstrates that L152,804 levels in the brain are found at least 2 h following oral dosing (Kanatani et al., 2000).

181

6 Cyclodextrin L-152,804 (3 mg/kg) L-152,804 (10 mg/kg)

b) 5 4 3 2 1 0 2

4

6

Time (Hours)

Fig. 1. Effect of L-152,804 injection on cumulative g/kg ethanol intake (a) and cumulative ml of water consumed (b) from 6 h following L-152,804 injection. Data represent mean (6S.E.M.) of n 5 8 inbred alcohol-preferring rats. *Significantly different from cyclodextrin control injection.

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comparisons indicated that, collapsed across the 2-, 4-, and 6-h time points, iP rats drank significantly less after L152,804 injection than after vehicle administration. Fig. 1b illustrates the effects of L-152,804 on water intake in iP rats. A two-way repeated measures ANOVA (injection 3 time) on cumulative volume of water consumed from 0 to 6 h following injection did not reveal a significant interaction or a main effect of injection, indicating that L-152,804 did not alter water intake in a time-dependent manner or when collapsed across the time points measured. Finally, a two-way ANOVA (injection 3 time) on cumulative percent ethanol preference did not indicate a significant interaction, indicating that the effects of L-152,804 on percent ethanol preference were not dependent upon the timepoint following injection. A significant main effect of injection [F(2, 14) 5 9.48, p 5 .002] followed by Student– Newman–Keuls post hoc analysis indicated that, collapsed over the total 6 h following injection, L-152,804 decreased alcohol preference at both the 3 and 10 mg/kg doses. Fig. 2 illustrates the effects of L-152,804 injection g/kg ethanol intake (a), water (ml) intake (b), cumulative percent ethanol preference (c), and food intake (d) at 24 h following injection. Separate individual one-way repeated measure ANOVAs revealed that only g/kg ethanol intake differed significantly 24 h following injection of L-152,804 [F(2, 14) 5 5.33, p 5 .019]. Student–Newman–Keuls post hoc

analysis revealed that the 3 mg/kg dose of L-152,804 resulted in decreased cumulative g/kg ethanol intake at 24-h postinjection. Finally, injection of L-152,804 did not alter food consumption because a two-way mixed ANOVA on g/kg food intake did not reveal a significant effect of injection or an interaction between injection and time (Table 1), demonstrating that a dose of L-152,804, which significantly reduced ethanol intake in the two-bottle paradigm, had no significant effect on food intake. 3.2. Effects of L-152,804 on operant ethanol selfadministration Fig. 3a illustrates the effect of L-152,804 on operant ethanol self-administration as measured by g/kg ethanol intake. A one-way repeated measure ANOVA demonstrated a reduction in dose of self-administered ethanol [F(3, 21) 5 5.17, p 5 .008]. Student–Newman–Keuls post hoc analysis revealed that L-152,804 at 10 and 20 mg/kg reduced the estimated dose of self-administered ethanol below that which occurred following vehicle injection. Similarly, Fig. 3b illustrates the effects of L-152,804 on active and inactive lever responding. A two-way repeated measures ANOVA on operant responding (L-152,804 and lever selection) revealed a significant interaction between the factors [F(3, 21) 5 4.266, p 5 .017]. Analysis of the

6

4

Water intake (mls/24 hours)

Ethanol intake (g/kg/24 hours)

20

a)

*

2

0

b) 15

10

5

0

0

3

0

10

L-152,804 mg/kg mg/kg Food consumed (grams/24 hours)

Alcohol preference (%/24 hrs)

100

c) 80

60

40

20

0

0

3

10

L-152,804 mg/kg

3

10

L-152,804 mg/kg 30

d) 25 20 15 10 5 0

0

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10

L-152,804 mg/kg

Fig. 2. Effect of L-152,804 injection on 24-h ethanol (g/kg) intake (a), water (volume) intake (b), alcohol (percent) preference (c), and food (grams) intake (d). Data represent mean (6S.E.M.) of n 5 8 inbred alcohol-preferring rats. *Significantly different from cyclodextrin control injection.

J.P. Schroeder et al. / Alcohol 36 (2005) 179–186 Table 1 Injection of L-152,804 (3 mg/kg) does not alter food intake in inbred male alcohol-preferring rats L-152,804 (mg/kg)

Time (h) postinjection 2

4

6

24

0 3

7.4 (61.2) 7.4 (61.2)

12.8 (63.1) 11.5 (63.7)

18.9 (64.1) 15.4 (64.8)

44.7 (611) 42.3 (69.0)

Data represent mean mg/kg food intake (6S.E.M.), n 5 4. S.E.M. 5 standard error of the mean.

Ethanol intake (Mean g/kg +/- SEM)

simple main effects of L-152,804 injection on ethanolreinforced responses [F(3, 21) 5 4.42, p 5 .018] followed by Student–Newman–Keuls post hoc tests revealed that the 10 and 20 mg/kg doses reduced responses on the active lever. Analysis of the simple main effects of L-152,804 on inactive lever presses revealed no effect of Y5 antagonism on this behavior. A two-way repeated measures ANOVA on response onset (L-152,804 and lever selection) indicated a significant effect of lever selection [F(1, 7) 5 29.96, p ! .001]

a) 0.6

* 0.4

*

0.2

0.0

0

3

10

20

L-152,804 mg/kg

Lever responses (Mean +/- SEM)

50

Active lever responses Inactive lever responses

b) 40

* *

30

20

10

0

0

3

10

20

L-152,804 mg/kg Fig. 3. Effect of L-152,804 injection on g/kg ethanol intake (a) and active and inactive lever presses in operant ethanol self-administration (b) over the 60-min session. Data represent mean (6S.E.M.) of n 5 8 inbred alcohol-preferring rats. *Significantly different from cyclodextrin control injections (Student–Newman–Keuls, p ! .05).

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because subjects responded more quickly on the active lever than on the inactive lever. No interaction between injection and lever press selection was found, however, indicating that L-152,804 did not alter the onset of active or inactive lever responding. A one-way ANOVA on active lever response termination revealed a trend toward early termination of operant responding for ethanol in iP rats following injection of L-152,804 [F(3, 21) 5 2.86, p 5 .061].

4. Discussion The present results suggest that NPY activity at Y5 receptors is required for full expression of ethanol’s reinforcing effects in selectively bred iP rats. This is consistent with a growing literature that implicates NPY receptors in regulating ethanol self-administration (Kelley et al., 2001; Schroeder et al., 2003a, 2003b; Thiele et al., 1998, 2000, 2002). More specifically, reductions in ethanol intake in iP rats produced by L-152,804 are consistent with the effects of this compound on the maintenance of operant ethanol self-administration in C57BL/6J mice (Schroeder et al., 2003a). The emergence of reductions in cumulative g/kg ethanol intake 2 h following injection in the two-bottle method is also consistent with previous research indicating that L-152,804 modulates ethanol intake in C57BL/6J mice from 2 to 5 h following administration (Schroeder et al., 2003a). In addition, the 2-h pretreatment time used in the operant procedure resulted in decreased operant ethanol self-administration throughout the entire 60-min session in iP rats, which is also consistent with the time course of L-152,804 effects on ethanol intake in mice. The most likely mechanism by which L-152,804 might have decreased g/kg ethanol intake in both behavioral models is by attenuation of its reinforcing properties. This was evidenced by reduced ethanol-reinforced responding in the absence of changes in response onset, which suggests that the NPY Y5 antagonist did not alter neurobiological processes that control the onset of ethanol responding but inhibited responding once it was initiated. The hypothesis that NPYergic function might mediate some of ethanol’s effects, including reinforcement, is suggested by the fact that NPY and ethanol have similar effects on event-related potentials recorded from cortex and amygdala (Ehlers et al., 1999), and that combined administration of the compounds produces additive effects (Ehlers et al., 1998b). Thus, the Y5 antagonist may have inhibited an effect of ethanol at this receptor. In further support of this hypothesis, coadministration of NPY enhances the sedative hypnotic effects of ethanol in P and NP rats (Gilpin et al., 2004a). This further suggests that the Y5 antagonist may have blocked a CNS pharmacological effect of ethanol, which may contribute to its reinforcing function. Finally, site-specific infusion of L-152,804 in the nucleus accumbens inhibits muscimol-stimulated increases in food intake (Stratford & Wirtshafter, 2004), which further supports the involvement

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of NPY Y5 receptors in reinforcement. Together these data suggest that the NPY Y5 antagonist L-152,804 may have reduced ethanol intake in iP rats by decreasing the reinforcing properties of ethanol. An alternative to the interpretation that L-152,804 reduced operant ethanol self-administration by attenuating the reinforcing properties of ethanol is that injection of the NPY Y5 antagonist may have reduced general locomotor activity, resulting in decreased ethanol-reinforced responding. Although this possibility remains, it appears unlikely for three main reasons. First, in the two-bottle drinking paradigm, iP rats consumed the same amount of water and food following L-152,804 injection as that following vehicle administration. If NPY Y5 antagonism decreased ethanol intake by inhibiting locomotor function, then one would expect reductions in water and/or intake following L-152,804 administration. Second, L-152,804 did not significantly alter the onset of responding on the ethanol-reinforced or inactive lever. This suggests that the ability to engage in motor activity was not altered. However, the possibility does remain that ability to engage in high-rate activity may have been altered. Third, previous research indicates that L-152,804 does not reduce locomotor activity when administered alone in rodents (Schroeder et al., 2003a) suggesting that the antagonist did not alter general activity. Another mechanism by which L-152,804 may have decreased ethanol intake is by the production of a negative affective state in the iP rats. Recent pharmacological evidence indicates that the NPY Y5 agonist ([cPP(1–7),NPY(19– 23),Ala(31),Aib(32),Gln(34)]hPP) is anxiolytic in the open-field and elevated plus maze tests in rats (Sorensen et al., 2004). This raises the possibility, that the NPY Y5 antagonist used in the present study may have produced anxiogenic effects. However, this hypothesis would also predict a reduction in water and food consumption in the two-bottle method, neither of which was observed. Given the role of NPY in feeding behavior, another alternative mechanism by which L-152,804 may have reduced intake in iP rats in the two behavioral models examined in the present experiment (Gerald et al., 1996; Haynes et al., 1998; Hu et al., 1996; Kirby et al., 1995; Wyss et al., 1998) could be that L-152,804 delayed ethanol-reinforced responding because of the caloric value of ethanol, which would not be reflected by a concomitant delay in water responding. This seems unlikely because L-152,804 reduces feeding that is increased by central administration of bPP, a Y4/Y5 receptor agonist, but not spontaneous feeding in satiated rats (Kanatani et al., 2000), and injection of L152,804 had no significant effect on food intake in the present study. A final mechanism by which L-152,804 may have decreased ethanol intake is that the NPY Y5 antagonist may have impaired recall for the association between contextual cues and the availability of ethanol because NPY has been found to enhance memory function (Flood & Morley,

1989b; Flood et al., 1987, 1989a). Although it is possible that NPY Y5 antagonism decreased operant responding by impairing the expression of operant behavior, evidence indicates that L-152,804 was more likely to have reduced operant ethanol self-administration by some other mechanism. First, L-152,804 injection did not alter response latencies on either the active or inactive levers. If L152,804 had impaired the representation of the active barpress ethanol contingency, then one would have expected to observe an increase in the latency to active bar-pressing behavior. Second, following injection of the effective doses of L-152,804 (10 and 20 mg/kg) bar-pressing behavior remained selective for the ethanol-paired lever. That is, even though the total number of ethanol-reinforced lever presses decreased as a function of L-152,804 dose, subjects were still able to remember which lever was associated with ethanol reinforcement. Together these internal data indicate that subjects were able to recall the association between responses on the active lever and ethanol presentation. The present data are consistent with evidence showing that administration of NPY increases (Gilpin et al., 2004b; Kelley et al., 2001) and of NPY antagonist decreases (Ehlers et al., 1998b; Schroeder et al., 2003a, 2003b; Sparta et al., 2004) alcohol self-administration and support the hypothesis that elevations of NPYergic function and ethanol intake are directly proportional. However, additional data indicate that NPYergic activity and ethanol intake are inversely related. Ventricular administration of high (microgram) concentrations of NPY has been found to decrease ethanol intake in P rats (Badia-Elder et al., 2001; Gilpin et al., 2003). In addition, intracerebral ventricular infusion of the NPY Y2 antagonist BIIE0246, which acts predominately at autoreceptors, effectively enhancing NPY function, also reduces ethanol self-administration (Rimondini et al., 2005; Thorsell et al., 2002). Similarly, evidence from transgenic and null mutant mice indicates that the function of NPY (Thiele et al., 1998) and the existence of Y1 receptors (Thiele et al., 2002) are inversely related to ethanol consumption. In addition, mutant mice lacking NPY Y5 receptors are more sensitive to the sedative effects of acute ethanol (Thiele et al., 2000), which suggests that NPY Y5 deletion makes subjects more sensitive to ethanol, a finding that argues against the hypothesis that L-152,804 injection reduces ethanol intake by attenuating the reinforcing properties of ethanol (Schroeder et al., 2003a, 2003b). Although the reason(s) for these discrepant findings are as yet unclear, one possible explanation is that the influence of NPY function at NPY receptors on ethanol intake is brain region dependent. Research indicating that NPY infusion increases ethanol intake has used intracranial infusion into the PVN (Gilpin et al., 2004b; Kelley et al., 2001), a fact that is in accordance with the orexigenic effects of NPY infusion into this brain region (Stanley & Leibowitz, 1984, 1985). In addition, the fact that NPY Y5 receptors are located primarily in the hypothalamus (Berglund et al., 2003) suggests that reductions in ethanol intake

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produced by peripheral injection of L-152,804 in iP rats and C57BL/6J mice (Schroeder et al., 2003a) could be the result of NPY Y5 antagonism in the hypothalamus. Despite the similar effects of NPY and its antagonists on ethanol intake, manipulations of NPYergic function may aid the amelioration of alcoholism. A Leu(7)-to-Pro(7) polymorphism in the signal peptide part of NPY is associated with higher average alcohol consumption in humans, suggesting a role of NPY in human alcohol-seeking behavior (Kauhanen et al., 2000). Combined with research in rodents on NPY and alcohol intake, this finding suggests that further examination of NPY function at its receptors in specific brain regions may yield novel therapeutics in the treatment of alcohol use and abuse in humans.

Acknowledgments This work was supported by grants AA09981 and AA011605 from the National Institute on Alcohol Abuse and Alcoholism to CWH and by The Bowles Center for Alcohol Studies at The University of North Carolina Chapel Hill.

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