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Research Report

Genetic variance contributes to dopamine receptorantagonist-induced inhibition of sucrose intake in inbred andoutbred mouse strains

Cheryl T. Dyma,c, Alexander Pinhasa, Magdalena Robaka,Anthony Sclafanib,c,d, Richard J. Bodnara,c,⁎aDepartment of Psychology, Queens College, The Graduate Center, City University of New York, Flushing, NY 11367, USAbDepartment of Psychology, Brooklyn College, The Graduate Center, City University of New York, Brooklyn, NY 11210, USAcDepartment of Neuropsychology, Brain and Behavior, Doctoral Sub-Programs, The Graduate Center, City University of New York, New York,NY 10016, USAdDepartment of Cognition, Brain and Behavior, Doctoral Sub-Programs, The Graduate Center, City University of New York, New York, NY10016, USA

A R T I C L E I N F O

⁎ Corresponding author.Department of PsychoUSA. Fax: +1 718 997 3257.

E-mail address: richard.bodnar@qc.cuny.e

0006-8993/$ – see front matter © 2008 Elsevidoi:10.1016/j.brainres.2008.12.042

A B S T R A C T

Article history:Accepted 15 December 2008Available online 25 December 2008

Preference and intake of sucrose varies across inbred and outbred strains of mice.Pharmacological analyses revealed that the greatest sensitivity to naltrexone-inducedinhibition of sucrose (10%) intake was observed in C57BL10/J and C57BL/6J strains, whereas129P3/J, SWR/J and SJL/J strains displayed far less sensitivity to naltrexone-inducedinhibition of sucrose intake. Given that dopamine D1 (SCH23390) and D2 (raclopride)receptor antagonism potently reduce sucrose intake in outbred rat and mouse strains, thepresent study examined the possibility of genetic variance in the dose-dependent(50–1600 nmol/kg) and time-dependent (5–120 min) effects of these antagonists uponsucrose (10%) intake in the eight inbred (BALB/cJ, C3H/HeJ, C57BL/6J, C57BL/10J, DBA/2J, SJL/J,SWR/J and 129P3/J) and one outbred (CD-1) mouse strains previously tested with naltrexone.SCH23390 significantly reduced sucrose intake across all five doses in 129P3/J and SJL/Jmice,across four doses in C57BL/6J and BALB/cJ mice, across three doses in DBA/2J, SWR/J, C3H/HeJand C57BL/10J mice, but only at the two highest doses in CD-1 mice. SCH23390 was 2–3-foldmore potent in inhibiting sucrose intake in 129P3/J and SJL/J mice relative to CD-1 mice. Incontrast, only the highest equimolar 1600 nmol/kg dose of raclopride significantly reducedsucrose intake in the BALB/cJ, C3H/HeJ, C57BL/6J, C57BL/10J, DBA/2J, SJL/J and 129P3/J, butnot the SWR/J and CD-1 strains. The present and previous data demonstrate specific anddifferential patterns of genetic variability in inhibition of sucrose intake by dopamine andopioid antagonists, suggesting that distinct neurochemical mechanisms control sucroseintake across different mouse strains.

© 2008 Elsevier B.V. All rights reserved.

Keywords:Dopamine D1 receptorDopamine D2 receptorSCH23390RacloprideSweet intake

logy, Queens College, The Graduate Center, City University of NewYork, Flushing, NY 11367,

du (R.J. Bodnar).

er B.V. All rights reserved.

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1. Introduction

The precise role for dopamine in reward has been a consistentsource of debate since the proposal of the “anhedoniahypothesis” over a quarter-century ago (e.g., Wise, 1982;Wise et al., 1978), and continues unabated in recent reviews(e.g., Baldo and Kelley, 2007; Barbano and Cador, 2007;Berridge, 2007; Salamone et al., 2007). In place of theanhedonia hypothesis, Berridge (Berridge, 1996; Berridge andRobinson, 1998) proposed a role for brain dopamine in‘wanting’, that is, the attribution of incentive salience toreward-related stimuli. An alternative role for brain opioidswas proposed in ‘liking’, that is, the hedonic impact of reward.Differential involvement for opioids and dopamine werefound for reinforcement processes related to food and waterintake (e.g., Agmo et al., 1993, 1995). Sucrose and saccharinintake were significantly reduced by systemic pretreatmentwith opioid (e.g., Apfelbaum and Mandenoff, 1981; Cooper,1983; Levine et al., 1982; Lynch and Libby, 1983) and dopamineD1 and D2 (e.g., Bello and Hajnal, 2006; Muscat and Willner,1989; Tyrka et al., 1992; but see (Muscat et al., 1991; Phillips etal., 1991) antagonists in rats. Using the sham-feeding proce-dure (Weingarten and Watson, 1982) in which ingested fluiddrains out of an open gastric fistula thereby minimizingpostingestive nutritive effects (Sclafani and Nissenbaum,1985), sugar intake is also reduced following pretreatmentwith opioid (Kirkham and Cooper, 1988a,b; Rockwood andReid, 1982) and dopamine D1 and D2 (Geary and Smith, 1985;Schneider et al., 1990) antagonists. However, opioid anddopamine antagonists differed in their effects upon sugar-conditioned flavor preferences. Whereas the opioid antago-nist, naltrexone failed to reduce sucrose-conditioned flavorpreferences in sham-feeding rats (Yu et al., 1999), fructose-conditioned flavor preferences in real-feeding rats (Bakeret al., 2004) or sucrose-conditioned flavor–nutrient prefer-ences induced by intragastric sucrose (Azzara et al., 2000),dopamine D1 and/or D2 receptor antagonism significantlyreduced sucrose-conditioned and fructose-conditioned flavorpreferences in these conditioningparadigms (Azzaraet al., 2001;Baker et al., 2003; Hsiao and Smith, 1995; Yu et al., 2000a,b).

Through the use of inbred mouse strains, genetic variationhas been observed for sucrose and saccharin intake (Bachma-nov et al., 1997, 2001; Blizard et al., 1999; Capeless andWhitney, 1995; Fuller, 1974; Inoue et al., 2004; Lewis et al.,2005; Lush, 1989; Nachman, 1959; Pelz et al., 1973; Pothionet al., 2004; Reed et al., 2004; Stockton and Whitney, 1974;Tordoff et al., 2002), accounting for 78% and 83% of the geneticvariation associated with consumption of 0.1% saccharin and3% sucrose, respectively (Ramirez and Fuller, 1976). Thegenetic variation in the response to saccharin and dilutesucrose solutions is largely explained by polymorphisms inthe Tas1r3 gene that encodes for the T1R3 sweet receptor(Reed et al., 2004). In studies examining pairs of strains, C57BL/6Jmice displayed greater intake of five (0.005–1 M) glucose andsucrose concentrations than 101Bag/R1 mice (Stockton andWhitney, 1974), of a 0.1% saccharin solution than DBA/2J mice(Fuller, 1974), and of low sucrose concentrations than 129P3/Jmice (Bachmanov et al., 1997, 2001; Sclafani, 2006a,c; Sclafaniand Glendinning, 2005; Tordoff et al., 2002). Examination of 12

mouse strains across a range of nine sucrose concentrationsrevealed profound genetic variation in the sensitivity andmagnitude of intake as well as alterations in correspondingchow intake (Lewis et al., 2005). Intakes of dilute (0.1%) but notconcentrated (10%) sucrose correlated with Tas1r3 polymor-phisms indicating that sweet taste sensitivity does notcompletely explain the consumption of calorically densesugar solutions (Inoue et al., 2004). Moreover, profound geneticvariance was also observed in naltrexone's ability to reduceintake of a 10% sucrose solution in eight inbred and oneoutbred mouse strains (Dym et al., 2007).

To examine potential genetic variance in the dopaminergicreceptor modulation of sucrose intake, the present studytested eight inbred (BALB/cJ, C3H/HeJ, C57BL/6J, C57BL/10J,DBA/2J, SJL/J, SWR/J and 129P3/J) and one outbred (CD-1)mouse strains for differences in the ability of systemicadministration of D1 (SCH23390) andD2 (raclopride) dopaminereceptor antagonists to dose-dependently (50–1600 nmol/kg)and time-dependently (5–120 min) decrease intake of a 10%sucrose solution.

2. Results

2.1. Strain differences in sucrose intake following vehiclebaseline injections

Evaluationof sucrose intake followingvehicle baseline injectionsrevealed significant differences among strains (F(8,88)=8.53,P<0.0001) and for the interaction between strains and testtimes (F(40,440)=8.83, P<0.0001). The rank-order of the cumula-tive 2 h baseline vehicle sucrose intake among the nine strainswas: SWR/J (1.7 ml), BALB/cJ (1.7 ml), 129P3/J (1.6 ml), CD1(1.4 ml), C3H/HeJ (1.4 ml), C57BL/10J (1.3 ml), SJL/J (1.2 ml), C57BL/6J (1.2 ml), DBA/2J (1.2 ml). Thus, to adjust for baselinedifferences in sucrose intake across strains, the effects ofSCH23390 and raclopride across doses and timeswere evaluatedwithin each strain aswell as an evaluation of the percent vehiclebaseline values across strains, doses and times.

2.2. Strain differences in dopamine antagonist-inducedinhibition of sucrose intake

2.2.1. SCH23390 effectsOverall significant differences in sucrose intake followingSCH23390 were observed among the nine mouse strains(F(8,86) =5.54, P<0.0001), among doses (F(5,430) =115.76,P<0.0001), across test times (F(5,430)=590.04, P<0.0001), andfor all two-way and three-way interactions (P<0.0001). Fig. 1displays the marked strain-specific differences in the dose-dependent and time-dependent ability of SCH23390 to sig-nificantly reduce sucrose intake. The 129P3/J mice displayedsignificant reductions across all five SCH23390 doses (Fig. 1I),whereas the C57BL/6J and SJL/J mice displayed significantreductions following the four highest SCH23390 doses (Figs. 1Cand G). The C3H/HeJ, C57BL/10J and SWR/J mice displayedsignificant reductions following the three highest SCH23390doses (Figs. 1B, D and H), whereas the BALB/cJ, DBA/2J and theoutbred CD-1 mice displayed significant reductions followingonly the two highest SCH23390 doses (Figs. 1A, E and F).

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Moreover, significant enhancements of sucrose intakeoccurred following the lowest SCH23390 dose in CD-1 (15–60 min, Fig. 1E), DBA/2J (5–30 min, Fig. 1F) and SWR/J (15 min,Fig. 1H) mice.

Significant differences in percent vehicle baseline values ofsucrose intake following SCH23390 were also observed amongstrains (F(8,91)=2.84, P<0.007), among doses (F(4,364)=97.55,P<0.0001), across times (F(5,455)=9.41, P<0.0001), and for alltwo-way and three-way interactions (P<0.0001). Table 1summarizes the SCH23390-induced changes in sucrose intakeas the percentage of baseline vehicle intake for each strain,and indicates those instances in which one strain producedgreater inhibitory effects relative to another strain. Tosummarize some of the major differences, 129P3/J micedisplayed significantly greater magnitudes of SCH23390-induced inhibition than BALB/cJ, CD-1 and SWR/J mice. SJL/Jand C57BL/6J mice also displayed significantly greater magni-tudes of inhibition than BALB/cJ, CD-1 and SWR/Jmice, but to alesser degree than the 129P3/J strain.

2.2.2. Raclopride effectsOverall significant differences in sucrose intake followingraclopride were observed among strains (F(8,84) = 5.67,P<0.0001), among doses (F(5,420)=20.87, P<0.0001), acrosstimes (F(5,420)=699.57, P<0.0001), and for all two-way andthree-way interactions (P<0.0001), except between strains anddoses (F(40,420)=1.24, n.s). Fig. 2 displays the markedlylesser ability of raclopride to dose-dependently and time-dependently reduce sucrose intake in terms of the magnitudeof the antagonist-induced inhibition. Seven strains (inbredBALB/cJ, C3H/HeJ, C57BL/6J, C57BL/10J, DBA2/J, SJL/J and 129P3/J: Figs. 2A–D, F, I) displayed raclopride-induced reductions insucrose intake only at the highest 1600 nmol/kg dose, and twostrains (inbred SWR/J and outbred CD-1: Figs. 2E and H) failedto display any raclopride-induced reductions in sucroseintake. Indeed, raclopride significantly increased sucroseintake in DBA/2J mice following the 50 (15–90 min) and 400(5–30 min) nmol/kg doses (Fig. 2F), in C3H/HeJ mice followingthe 50 (60–90 min) nmol/kg dose (Fig. 2B), in SWR/J micefollowing the 400 (60–120 min) nmol/kg dose (Fig. 2H), and inoutbred CD-1 mice following the 200 (15–120 min) nmol/kgdose (Fig. 2E). Analysis of the percent of vehicle baseline valuesof sucrose intake following raclopride also failed to show anygenetic variability (data not shown).

2.3. Strain differences in the ID50 of SCH23390-inducedinhibition of sucrose intake

Table 2 summarizes the ID50 values for the ability of SCH23390to inhibit sucrose intake across the 120 min time course. The129P3/J mice consistently displayed the lowest ID50 valuesacross the first 90 min of the 2 h time course, and displayed2–3.5-fold greater potency in SCH23390-induced inhibition ofsucrose intake relative to CD-1mice in the first hour after drug

Fig. 1 – Alterations (mean,±SEM) in sucrose intake following thein nine mouse strains. Individual analyses of variance for each sbetween doses and times for all of the nine strains. Significant (Pfollowing specific drug doses relative to corresponding vehicle in

administration. In further agreement with the strain diffe-rences in SCH23390-induced inhibition in the percent ofbaseline sucrose intake, the SJL/J strain displayed the secondhighest degree of potency in inhibition of sucrose intakeduring the first 60 min of the time course.

2.4. Heritability estimates of SCH23390- andraclopride-induced inhibition of sucrose intake

Table 3 summarizes the narrow-sense heritability estimatesfor the five effective SCH23390 and one effective raclopridedose at each time point using the percent of vehicle baselinesucrose intake. Whereas moderate (h2=0.40–0.52) heritabilityestimates were obtained for baseline vehicle sucrose intakeacross the first 30 min of the time course (data not shown),relatively meager heritability estimates were typicallyobtained across the time course for the different effectiveSCH23390 (h2=0.05–0.32) and raclopride (h2=0.10–0.22) dosesacross test times.

2.5. Sucrose intake and T1R3 sweet taste receptor status

Four inbred mouse strains (C57BL/6J, C57BL/10J, SJL/J and SWR/J) had the sweet-sensitive form of the T1R3 receptor while fourstrains (BALB/CJ, C3H/HeJ, DBA/2J and 129P3/J) had the sweet-sub-sensitive form of the receptor (Reed et al., 2004). Evalua-tion of the percent vehicle sucrose intake scores at 30min onlyrevealed significant differences (F(1,78) = 6.61, P<0.012)between sensitive (25.3%) and sub-sensitive (12.3%) tasterstrains following the 1600 nmol/kg dose of SCH23390. Diffe-rences between sensitive (90.8%) and sub-sensitive (71%)taster strains in inhibition of sucrose intake following the200 nmol/kg dose of SCH23390 approached, but did achievesignificance (F=3.24, P=0.076). Differences between the twogroups in inhibition of sucrose intake failed to occur followingthe 50 (F=0.28, ns; sensitive: 111%; sub-sensitive: 104%), 400(F=0.13, ns; sensitive: 61%; sub-sensitive: 64%) and 800(F=1.81, ns; sensitive: 46.7%; sub-sensitive: 34.3%) nmol/kgdoses of SCH23390, and the 1600 nmol/kg dose of raclopride(F=0.46, ns; sensitive: 82.1%; sub-sensitive: 75%). Thus, thesweet-sensitive strains show less suppression in sucroseintake following the highest SCH23390 dose than sweet-sub-sensitive strains.

3. Discussion

Marked murine strain differences were observed in themagnitude, time course and potency of the dopamine D1(SCH23390), but not the D2 (raclopride), receptor antagonists toinhibit intake of a 10% sucrose solution. Whereas SCH23390produced a heterogeneous response pattern as a function ofmouse strain that differed across time course, magnitude anddose, raclopride's ability to reduce sucrose intake across

five doses of the D1 dopamine receptor antagonist, SCH23390train revealed significant main dose effects and interactions<0.05) increases (#) and decreases (*) in sucrose intaketake are denoted.

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Table 1 – SCH23390 inhibition of sucrose (10%) intake across strains, doses and test times as a measure of percent of vehiclebaseline intake

Strain 5 min 15 min 30 min 60 min 90 min 120 min

A: SCH 50 nmol/kgBALB/cJ 167%abcgh 160%abh 129% 110% 108% 94%C3H/HeJ 80%e 100%e 88%e 90% 100% 86%C57BL/6J 125%d 100% 100% 100% 91% 83%C57BL/10J 133% 140% 100% 113% 91% 85%CD-1 180%abcgh 186%abch 167%abch 145%ab 131% 129%DBA/2J 160%abcfg 180%abch 183%abch 138%abc 130% 117%SJL/J 120% 75% 78% 80% 73% 100%SWR/J 150%h 163%ab 140%a 125%ab 107% 106%129P3/J 100% 89% 80% 67% 64% 63%

B: SCH 200 nmol/kgBALB/cJ 133%abceg 100%abeh 86% 80% 85% 76%C3H/HeJ 60% 57% 63% 70% 109%ae 114%ade

C57BL/6J 75%a 71% 67% 70% 73% 75%C57BL/10J 100% 120%abceh 100%abceh 100% 100% 92%CD-1 140%abcegh 143%abeh 111%abceh 100% 108% 129%DBA/2J 40% 60% 67% 50% 60% 58%SJL/J 60% 50% 44% 80% 91% 100%SWR/J 117%abceg 113%abh 90% 92%e 113%ae 112%a

129P3/J 71% 56% 60% 58% 64% 75%

C: SCH 400 nmol/kgBALB/cJ 67%b 80%abf 71% 70% 77% 76%C3H/HeJ 60%b 57% 50% 50% 73% 79%C57BL/6J 75% 57% 56% 60% 73% 75%C57BL/10J 67%b 60% 57% 63% 64% 69%CD-1 80%b 71%ab 78% 73% 85% 100%DBA/2J 60%b 80%b 67% 88% 90% 92%SJL/J 40% 38% 33% 50% 64% 83%SWR/J 50% 38% 40% 50% 67% 82%129P3/J 43% 44% 40% 50% 50% 69%

D: SCH 800 nmol/kgBALB/cJ 33% 40% 29% 50% 62% 65%C3H/HeJ 40% 29% 38% 30% 55% 79%e

C57BL/6J 25% 29% 22% 30% 45% 50%C57BL/10J 33% 20% 29% 38% 55% 54%CD-1 40% 29% 22% 36% 31% 64%DBA/2J 20% 20% 17% 25% 40% 42%SJL/J 40% 38% 33% 40% 55% 67%SWR/J 50% 38% 40% 50%ace 80%ei 94%ce

129P3/J 29% 22% 20% 25% 43% 56%

E: SCH 1600 nmol/kgBALB/cJ 0% 0% 0% 10% 23% 18%C3H/HeJ 20% 14% 25% 20% 36% 43%C57BL/6J 25% 14% 11% 10% 45%ade 50%e

C57BL/10J 0% 20% 14% 38%e 36% 38%CD-1 40%abcdefgh 43%acdefg 33%acdeg 36%e 38% 50%e

DBA/2J 0% 0% 0% 0% 10% 17%SJL/J 20% 25% 22% 20% 36% 67%ae

SWR/J 17% 13% 20% 25% 33% 47%129P3/J 14% 11% 10% 8% 21% 31%

NOTE: Significantly (P<.05) less %Vehicle inhibition than 129P3/J(a), SJL/J (b), C57BL/6J (c), BALB/cJ (d), DBA/2J (e), SWR/J (f), C57BL/10J (g), C3H/HeJ(h) or CD1 (i).

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strains was largely limited to effects following the 1600 nmol/kg dose for the BALB/cJ, C3H/HeJ, C57BL/6J, C57BL/10J, DBA/2J,SJL/J and 129P3/J, but not for the inbred SWR/J and outbredCD-1 strains. Further, heritability estimates in the ability of

particular strains to display raclopride-induced inhibition ofsucrose intake at the highest dose tended to be quite low(h2=0.10–0.22). Finally, strains that possessed the sweet-sensitive (C57BL/6J, C57BL/10J, SJL/J and SWR/J) and the

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sweet-sub-sensitive (BALB/CJ, C3H/HeJ, DBA/2J and 129P3/J)form of the T1R3 receptor (Reed et al., 2004) failed to differ inthe degree of inhibition of sucrose intake following the highestdose of raclopride. These antagonist effects are in agreementwith previously-observed significant dopamine antagonist-induced reductions in sucrose intake in sham-feeding rats(Geary and Smith, 1985; Hsiao and Smith, 1995; Schneider etal., 1986, 1990; Yu et al., 2000a,b) and in rat pups inindependent ingestion tests (Tyrka et al., 1992), as well as infructose intake in real-feeding rats (Baker et al., 2003). Thepresent study also indicated that low and moderate doses ofraclopride significantly increased sucrose intake in somemouse strains (C3H/HeJ, DBA/2J, SWR/J, and CD-1). These latterantagonist-induced facilitatory effects upon sucrose intakehave also been observed previously in rats (Muscat et al., 1991;Muscat and Willner, 1989; Phillips et al., 1991). Thus, thesedata thereby suggest that raclopride is quite ineffective inreducing sucrose intake in mice, and that any interpretationregarding genetic variability in D2 antagonist-induced sup-pression of sucrose intake is limited by the overall ineffec-tiveness of this response.

In contrast, genetic variability in SCH23390-induced inhibi-tion of sucrose intake was clearly demonstrated in twoindependent measures in the present study: magnitude ofeffect, and sensitivity to lower doses of the antagonist.SCH23390 significantly reduced sucrose intake across all fivedoses (50–1600 nmol/kg) in 129P3/J and SJL/J mice, across fourdoses (200–1600 nmol/kg) in C57BL/6J and BALB/cJmice, acrossthree doses (400–1600 nmol/kg) in SWR/J, C3H/HeJ and C57BL/10J mice, across three different doses (200, 800, 1600 nmol/kg)in DBA/2J mice, but only at the two highest doses (800,1600 nmol/kg) in CD-1 mice. A similar pattern of SCH23390-induced inhibitory actions were observed in assessing antago-nist ID50 potency across strains with the 129P3/J and SJL/Jinbred strains showing the greatest sensitivity consistentlyacross the time course. A 2–3-fold difference was observedbetween themost (129P3/J) and least (CD-1) sensitive strains inthe first hour following SCH23390 treatment. The strain-specific inhibition of SCH23390 sometimes occurred at differ-ent doses and time pointmeasurement intervals of the drug. Itis unclear from the present data as to whether different anddistinct multiple genetic mechanisms are responsible forindividual SCH23390 dose and time effects, or whether asingle genetic mechanism explains all effects. Interestingly,however, there also appeared to be some degree of geneticvariance in SCH23390-induced inhibition of sucrose intake inevaluating strains with the sweet-sensitive (C57BL/6J, C57BL/10J, SJL/J and SWR/J) and the sweet-sub-sensitive (BALB/CJ,C3H/HeJ, DBA/2J and 129P3/J) form of the T1R3 receptor (Reedet al., 2004) with significant differences following the1600 nmol/kg dose for the sensitive (25.3%) and sub-sensitive(12.3%) taster strains, and approaching significance followingthe 200 nmol/kg dose for the sensitive (90.8%) and sub-sensitive (71%) taster strains. Thus, the sweet-sensitive strainsappear to display less suppression in sucrose intake followingSCH23390 than sweet-sub-sensitive strains; potential implica-tions of these results will be discussed subsequently. Whereasa number of these previous criteria appear to support geneticvariance of the percent of vehicle intake in SCH23390-inducedinhibition of sucrose intake, narrow-sense heritability esti-

mates as a measure of genetic variance were quite meager(h2=0.05–0.32) across each of the time points and each of theSCH23390 doses that produced significant inhibition ofsucrose intake. A previous study (Dym et al., 2007) examiningopioid inhibition of sucrose intake indicated more consistentheritability estimates for effective naltrexone doses to pro-duce genetic variance in the inhibition of sucrose intake(h2=0.38–0.51). Thus, whereas measures of magnitude ofeffect, potency of effect and sensitive vs. sub-sensitive strainsrevealed genetic variance in SCH23390-induced inhibition ofsucrose intake, this interpretation should be tempered by themeager heritability data.

It is possible that these strain-specific effects of SCH23390could be due to other factors such as pharmacokinetic actions,actions at sites outside of the central nervous system, oractions upon activity or motor behavior. Although pharmaco-dynamic effects of SCH23390 acting at the D1 receptor wereinitially reported in rat studies (e.g., Briere et al., 1987; Hjorthand Carlsson, 1988; Reader et al., 1988; Schulz et al., 1985), thestrain-specific effects of SCH23390 could alternatively be dueto pharmacokinetics with SCH23390 acting as a poor D1antagonist in somemurine strains and a strong D1 antagonistin others. For instance, SCH23390 binding is significantlyreduced in homozygous mice for the recessive gene weaver(Pullara and Marshall, 1989, but see Ohta et al., 1989),dopamine D(1A) receptor knockout mice (Miyamoto et al.,2001; Montague et al., 2001), and diabetic mice (Saitoh et al.,1998), but is increased in Purkinje Cell Degeneration mutantmice (Delis et al., 2004). In contrast, SCH23390 binding isunchanged in hypoxicmice (Arregui et al., 1994), MPTP-treatedmice (Araki et al., 2001), methamphetamine-treatedmice (Yooet al., 2008), 6-hydroxydopamine and iron-deficient mice(Zhao et al., 2007), mu-opioid receptor knock-out mice (Tienet al., 2003), and cannabinoid CB1 receptor knockout mice(Houchi et al., 2005). In assessing whether inbred strainsdisplay similar or different SCH23390 binding characteristics,although restraint stress increased D1 receptor density in thenucleus accumbens of DBA/2 mice, while it reduced D1receptor density in the striatum of C57BL/6 mice (Cabib et al.,1998), no differences were observed in mesocorticolimbic andstriatal SCH23390 binding in normal DBA/2 and C57BL/6 mice(Erwin et al., 1993). BALB/c and DBA/1micemade aggressive byclonidine treatment displayed reduced SCH23390 binding inthe limbic forebrain, an effect not observed in clonidine-treated C57BL/6Jmice (Nikulina and Klimek, 1993). Kanes et al.(1993) did perhaps the most extensive strain survey inexamining D1 receptor binding with [3H]SCH23390, usingeight strains of which four (BALB/c, C3H/He, C57BL/6 andDBA/2) were evaluated in the present study. Although theyfailed to find any strain differences in [3H]SCH23390 uptakeinto the caudate or cerebellum, BALB/cJ mice displayed higher[3H]SCH23390 binding in the nucleus accumbens (19%), lateralcaudate–putamen (15%) and substantia nigra, pars reticulata(27%) than C57BL/6 mice with DBA/2 and C3H/He miceintermediate to the other strains. C3H/He mice displayedhigher [3H]SCH23390 binding in the dorsal caudate–putamen(23%) than C57BL/6 mice with DBA/2 and BALB/c miceintermediate to the other strains. Our behavioral dataindicated that C57BL/6J and C3H/HeJ inbred strains showedmoderate sensitivity, and BALB/cJ and DBA/2J inbred strains

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Table 2 – Linear regression values (ID50) of SCH23390-induced inhibition (nmol/kg) of sucrose intake among the inbred andoutbred mouse strains across the 120 min time course

Linear regression ID50

Strain 5 min 15 min 30 min 60 min 90 min 120 min

BALB/cJ 967 987 642 890 875 1165C3H/HeJ 628 733 622 763 1010 1465C57BL/6J 672 566 649 694 1478 1629C57BL/10J 830 888 735 998 1637 1288CD-1 1249 1183 1043 1059 1037 1300DBA/2J 713 896 751 681 814 1014SJL/J 521 493 414 729 945 1873SWR/J 1010 819 797 964 1604 1567129P3/J 505 410 321 513 650 1072Sensitivity ratio 2.5a 2.9a 3.2a 2.1a 2.5b 1.8 c

Note: The sensitivity ratio reflects the potency fold shift of SCH23390-induced inhibition of sucrose intake (ID50) between the least sensitivestrain at each time point relative to the most sensitive strain.a CD-1 vs 129P3/J.b C57BL/10J vs 129P3/J.c SJL/J vs DBA/2J.

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showed lesser sensitivity to SCH23390-inhibition of sucroseintake. [3H]SCH23390 binding data on strains (e.g., 129P3/J andSJL/J) highly sensitive to SCH23390-induced inhibition ofsucrose intake are not available, so it may be premature todescribe these effects as D1-receptor specific, but moreappropriately to ascribe these effects as SCH23390-sensitiveor -subsensitive. A second caveat is that both D1 and D2antagonists were administered subcutaneously, and anysystemic effects, particularly for SCH23390, could be due toD1 receptor blockade in relevant sites outside of the centralnervous system. Relevant candidate sites displaying D1receptor binding as measured by SCH23390 include gastric(Glavin and Hall, 1995; Nomura et al., 1995) and intestinal(Fraga et al., 2004; Marmon et al., 1993) areas. Further studiesusing ventricular or intracerebral administration of D1antagonists are needed to confirm whether central sites ofaction are involved. A third and final caveat is to determinewhether the genetic variance in SCH23390-induced inhibitionof sucrose intake is an epiphenomenon of a generalized effecton activity and/or motor behavior. Although this factor wasnot directly studied in this paradigm, it is important to notethat the same animals in each strain were assessed for bothSCH23390-induced and raclopride-induced inhibition ofsucrose intake using equimolar concentrations of each drug.Whereas D1 antagonism produced both strain-specific anddose-dependent reductions in sucrose intake, raclopride onlyproduced reductions at the highest dose. Yet D1 antagonismwith SCH23390 and D2 antagonismwith related antagonists toraclopride produced equal reductions in hyperlocomotioninduced by caffeine, cocaine and amphetamine (Garrett andHoltzman, 1994; O'Neill and Shaw, 1999), albeit at doses higherthan those employed in the present study. Therefore, it doesnot appear that deficits in motor activity produced thedifferential strain-specific and dose-dependent reductions insucrose intake induced by SCH23390.

Fig. 2 – Alterations (mean,±SEM) in sucrose intake following thein ninemouse strains. Significant (P<0.05) increases (#) and decreto corresponding vehicle intake are denoted.

Genetic variability has also been previously observed in theability of the opioid antagonist naltrexone to suppress sucroseintake (Dym et al., 2007). Sucrose intake of SWR/Jmice failed todecrease significantly following a wide range (0.01–5mg/kg) ofnaltrexone doses. Naltrexone's maximal magnitude of inhibi-tory effects was small (35–40%, (ID50=7.5–10 mg/kg at 60 min))in 129P3/J and SJL/J mice, moderate (∼50%) in BALB/cJ, C3H/HeJ, CD-1 and DBA/2J mice, and profound (70–80%, (ID50=0.5–1.5 mg/kg at 60 min)) in C57BL/6J and C57BL/10J mice.Thus, there was a marked 37-fold difference across strains innaltrexone's effectiveness to reduce sucrose intake (Dymet al., 2007) which compares with the much smaller 2–3 folddifference observed with SCH23390 in the present study. Todirectly assess the relationship between the respectiveabilities of naltrexone and SCH23390 to inhibit sucrose intake,we derived the ID50 value of each antagonist for each of thenine common strains tested, and the correlation between theID50 values for each antagonist across the nine strains wascalculated. There was a weak, negative correlation betweenthe ID50 of naltrexone and SCH23390 to inhibit sucrose intake30 min following drug administration (r=−0.32; r2=0.11, ns).This particular data point produced the most consistentinhibition across drugs, doses and times. Fig. 3 illustratesdifferences in each drug's effects in the various strains. Forinstance, whereas the 129P3/J and SJL/J were among the leastresponsive strains to naltrexone, they were among the mostresponsive strains to SCH23390. In contrast, the C57BL/10J andC57BL/6J strains were the most responsive strains to naltrex-one, but were only moderately sensitive to SCH23390. Thedichotomy between dopaminergic (SCH23390) and opioid(naltrexone) antagonist modulation of sucrose intake hasbeen observed in other paradigms. Thus, although significantreductions in sucrose and saccharin intake have beenobserved in sham-feeding and real-feeding rats followingdopamine (SCH23390: e.g., Bello and Hajnal, 2006; Geary and

five doses of the D2 dopamine receptor antagonist, racloprideases (*) in sucrose intake following specific drug doses relative

Table 3 – Heritability estimates for percent baseline vehicle sucrose intake scores across strains for each SCH23390 dose ateach time point and for the effective raclopride dose

Heritability scores

Time → Condition ↓ 5 min 15 min 30 min 60 min 90 min 120 min

SCH2339050 nmol/kg .24 .25 .32 .17 .17 .15200 nmol/kg .22 .24 .18 .11 .19 .24400 nmol/kg .07 .15 .11 .06 .06 .05800 nmol/kg .05 .05 .06 .11 .15 .231600 nmol/kg .10 .15 .22 .15 .16 .18

Raclopride1600 nmol/kg .10 .22 .15 .16 .10 .12

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Smith, 1985; Muscat and Willner, 1989; Schneider et al., 1986,1990Tyrka et al., 1992) and opioid (naltrexone: e.g., Apfelbaumand Mandenoff, 1981; Cooper, 1983; Kirkham and Cooper,1988a,b; Levine et al., 1982; Lynch and Libby, 1983; Rockwoodand Reid, 1982) antagonists, these antagonists have differenteffects on the hedonic response to sweet taste (Barbano andCador, 2007; Berridge and Robinson, 1998) and flavor condition-ing effects of sugars (Azzara et al., 2000, 2001; Baker et al., 2003,2004; Hsiao and Smith, 1995; Yu et al., 1999, 2000a,b).

Berridge's (Berridge, 1996; Berridge and Robinson, 1998)proposed involvement of brain dopamine in food ‘wanting’and brain opioid systems in food hedonics (‘liking’) providesone widely-discussed framework to explain the differentialeffects of dopamine and opioid drugs on food-motivatedbehavior and reward in general. Whereas dopamine andespecially D1 receptor antagonism decreases sucrose intakein rats (e.g., Barbano and Cador, 2007; Berridge and Robinson,1998) and indeed all mouse strains tested in the present study,the magnitude and potency of the effects were strain-specific.Dopamine D1 antagonism is also particularly effective inblocking the acquisition of flavor–nutrient conditioning ofsimple carbohydrates (Azzara et al., 2001), the acquisition offlavor–flavor conditioning of sucrose in sham-feeding rats (Yu

Fig. 3 – Scatterplot and best-fit regression line of ID50 ofnaltrexone-induced inhibition (Dym et al., 2007) andSCH23390-induced inhibition of sucrose intake after 30 minacross nine mouse strains. A weak, negative andnon-significant correlation between the ID50 of naltrexoneand SCH23390 to inhibit sucrose intake 30 min followingdrug administration (r=−0.32; r2=0.11, ns) was observed.

et al., 2000b) and of fructose in real-feeding rats (Baker et al.,2003), as well as the expression of flavor–flavor conditioning(Baker et al., 2003; Yu et al., 2000a,b). All of these studiesimplicate the D1 dopamine receptor in reward mediated bysimple carbohydrates. The mechanism by which this media-tion of reward acts is still unclear. The present and previous(Dym et al., 2007) study demonstrating differences in thepattern and effectiveness of dopaminergic and opioid anta-gonism of sucrose intake across different inbred mousestrains may provide yet another form of converging evidencefor separation of these two pharmacological systems,although our knowledge of the ability of a wide variety ofmouse strains to display these specific attributes of foodseeking is presently limited to the eight inbred strainsevaluated. Other studies of the 129P3/J and C57BL/6J inbredstrains provide some intriguing information relevant to thisissue. These two strains differ in their sweet taste sensitivitywith C57BL/6Jmice possessing the sweet-sensitive form of theTas1R3 receptor, and 129P3/J possessing the sweet-sub-sensitive form of the receptor (Reed et al., 2004). Consistentwith this finding, 129P3/J mice underconsumed a variety ofnatural and artificial sweeteners compared to C57BL/6J mice(Bachmanov et al., 2001; Reed et al., 2004). However, afterexperience with concentrated sugar solutions, the differencein the sucrose preferences of the two strains disappeared, aneffect attributed to the postingestive conditioning actions ofthe sugar (Sclafani, 2006b). Indeed 129P3/J mice showed aflavor conditioning response to intragastric sucrose infusionsthat was as strong, if not more so, as that displayed by C57BL/6J mice when the sweetness of the cue flavors were equatedfor the two strains (Sclafani and Glendinning, 2005). Further-more, sucrose-experienced 129P3/J mice licked more on aprogressive ratio schedule for sucrose rewards than did C57BL/6J mice (Sclafani, 2006a). The respective strong and weaksensitivity of 129P3/J mice to SCH23390- and naltrexone-induced inhibition of sucrose intake may reflect their greaterreliance on the postingestive conditioning of sweet rewardsthan the activity of their subsensitive sweet taste receptors.Supporting this interpretation are the findings that SCH23390,but not naltrexone, blocks the postingestive conditioningactions of sugars (Azzara et al., 2000, 2001). Further limitedsupport for this concept is the present finding that the highestdose of SCH23390 produced significantly greater inhibition ofsucrose in the sweet-sub-sensitive strains (BALB/cJ, C3H/HeJ,DBA/2J and 129P3/J) relative to the sweet-sensitive strains

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(C57BL/6J, C57BL/10J, SJL/J and SWR/J), and is consistent withBerridge's proposals (Berridge, 1996; Berridge and Robinson,1998) that brain dopamine may be less involved in ‘liking’relative to ‘wanting’. Therefore, such data suggest that inbredgenetic differences amongmouse strainsmay provide a viablemodel for differentiating among the multiple processesinvolved in food reward.

4. Experimental procedures

4.1. Subjects

Outbred (CD-1, Charles River Laboratories, Wilmington, MA;n=11) and eight strains of inbred BALB/cJ, C3H/HeJ, C57BL/6J,C57BL/10J, DBA/2J, SJL/J, SWR/J and 129P3/J (The JacksonLaboratory, Bar Harbor, ME) male mice (6 weeks of age) wereinitially acclimated to the Queens College vivarium for 1 weekin group (5 per cage) housing. Ten to twelve mice of eachinbred strain completed testing; variability was due to isolateddeaths of animals due to unrelated factors. The animals werethen housed individually in plastic cages (30×20×15 cm) withstainless steel tops throughout the entire study, and main-tained on a 12 h light/12 h dark cycle (lights off at 2000 h) at aconstant temperature of 22 °C. All animals were provided withchow (Lab Diet Mouse Chow 5015) and water ad libitumthroughout the experiment, except when experimental test-ing was conducted.

4.2. Testing apparatus

Accurate measurement (±0.2 ml gradations) of the sucrosesolution was insured by using a retrofitted testing sippertube consisting of a leur slip tip syringe (10 ml, 0.2 mlgradations, Pharmaseal Laboratories, Glendale, CA), siliconesealant (All-Glass Aquarium Co., Inc., Franklin, WI), a rubberstopper and a straight sipper tube (63 mm in length, 8 mmin width, Lab Products, Seaford, DE) (e.g., Dym et al., 2007).The apparatus was created by drilling a hole into the top ofthe syringe, inserting the stopper and sipper tube into thehole, and securing them with the sealant that alsoprevented leaking. The sucrose sipper tube was firmlysecured to the stainless steel top of the cage by a tautmetal spring (100 mm) with clips at each end that affixed tothe cage top so that the gradations and meniscus wereeasily visible.

4.3. Sucrose intake procedure

All procedures were approved by the Queens College Institu-tional Animal Care and Use Committee. At the start of thetesting procedure at approximately 3–7 h into the light cycle,chow andwater were removed from the cage, and each animalwas given access to approximately 8 ml of 10% sucrose in thesipper tube for 2 h. Sucrose intake was measured by readingthe meniscus of the sucrose solutions along the gradationsafter 5, 15, 30, 60, 90, and 120 min, whereupon the sipper tubewas removed and food and water were returned. Each animalwas exposed to one sucrose session per day until a criterionminimum of 1 ml was consumed over three consecutive

exposures; this criterion was employed to avoid “floor effects”of antagonist treatment. Three to five sessions were needed toreach this criterion in tests conducted approximately threetimes each week. All mice in all strains met the above criteria,and thereupon underwent testing. Following this initial base-line treatment, the mice received a subcutaneous (sc) vehicle(0.3 ml distilled water/30 g body weight) injection, and 30 minthereafter, sucrose intake was measured over 2 h. Followingdetermination that each mouse met the minimal 1 mlcriterion sucrose intake following vehicle treatment, 2-hrsucrose intakes were measured 30-min after sc injection ofthe D1 (SCH23390) and D2 (raclopride) dopamine receptorantagonists (Sigma Chemical Co., St Louis, MO) at doses of 50,200, 400, 800 and 1600 nmol/kg. The drugs were mixed atconcentrations of 5, 20, 40, 80 and 160 nmol/ml andadministered at 10 ml/kg with a minimum 72 h intervalbetween doses. This dose range and post-injection intervalwere chosen on the basis of significant effects observed in rats(Azzara et al., 2001; Baker et al., 2003; Yu et al., 2000a,b).Subgroups of animals of each strain, matched for vehiclesucrose intake, received an ascending series of SCH23390doses followed by a descending series of raclopride doses, adescending series of SCH23390 doses followed by an ascen-ding series of raclopride doses, an ascending series ofraclopride doses followed by a descending series ofSCH23390 doses, and a descending series of raclopride dosesfollowed by an ascending series of SCH23390 doses. Orders ofdrug or specific dose presentation failed to produce anysignificant effects, insuring lack of order or carry-over effects.Following all dopamine antagonist treatments, sucrose intakewas reassessed following a second vehicle injection. Analysesof sucrose intake under baseline and the two vehicle treat-ments failed to reveal any differences, and therefore, thesedata were pooled as a representative baseline treatment foreach strain. Sample size differences among strains reflectedunrelated deaths of individual animals.

4.4. Statistics

Because there were no significant differences in sucrose intakefollowing the baseline and the two vehicle injections, a pooledvehicle score comprising these sessions was assessed for eachtime point for each animal in each strain. The first types ofanalyses assessed drug-induced changes in cumulative sucroseintake across strains using two three-way randomized blockanalyses of variancewith thenine strains as a between-subjectsvariable, the pooled vehicle and five SCH23390 or raclopridedoses as one within-subject variable, and the six test times asthe second within-subject variable. Bonferroni comparisons(P<0.05) evaluated significant drug effects within groups in thisand subsequent analyses. The second types of analysesevaluated individual strain-specific effects of SCH23390 orraclopride across the six dose conditions and six test timesupon cumulative sucrose intake using two-way repeated-measures analyses of variance. Because the mouse strainsdiffered in their 2 h baseline sucrose intakes, the third types ofanalyses evaluated between-strain differences by transformingthe intakes following drug treatments into percent intakes(intake/baseline intake×100), and performing three-way rando-mized-block analyses of variance with the nine strains as a

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between-subjects variable, the five SCH23390 or raclopridedoses as one within-subject variable and the six test times asthe second within-subject variable. The fourth type of analysesexaminedstrain-specific differences in thepotencyof SCH23390that produced significant dose-dependent inhibitory effectssuch that post-drug intake difference scores were calculated bysubtracting sucrose intake following each SCH23390 dosecondition from pooled baseline intake for each animal in eachstrain. Then linear regression analyseswere performed for eachtime point for each strain with the SCH23390 dose as theindependent variable and the difference scores for each mousein each strain as the dependent variable to determine the dosethatwould inhibit sucrose intakeby50%(ID50).A sensitivity ratioreflecting the potency fold shift of SCH23390-induced inhibitionof sucrose intake (ID50) was calculated by obtaining the quotientbetween the least sensitive strain at each time point relative tothe most sensitive strain. The fifth type of analyses examinedpotential differences in narrow-sense trait heritability betweenstrains for those drug doses (all SCH23390 doses and thehighestraclopride dose) that previously produced significant inhibitoryeffects. A better variable for estimating heritability of drugeffects would be relative sucrose intake (% of baseline) becauseof baseline differences in sucrose intake across strains. Herit-abilitywasdetermined by comparing thebetween-strain sumofsquares to the total sum of squares for each percent vehiclebaseline score for the five effective doses of SCH23390 and thehigh effective dose of raclopride at each time point after drugadministration. Because animals are isogenic (i.e., geneticallyidentical) within individual inbred strains, between-strainvariance (measured by the sum of squares between-subjectsscore) provides a measure of additive genetic (‘allelic’) variation(VA),whereaswithin-strainvariance (‘error variance’,measuredby the sum of squares error score) represents environmentalvariability (VE). Thus, using all eight inbred strains, an estimateof narrow-sense heritability (h2) for each trait was obtainedusing the formula: h2=VA/(VA+VE). Finally, a sixth type ofanalysis assessed whether differences in the magnitude ofdopamine D1 and D2 receptor antagonist inhibition varied as afunction of strains that possessed the sweet-sensitive form ofthe Tas1R3 receptor (C57BL/6J, C57BL/10J, SJL/J and SWR/J)relative to the sweet-sub-sensitive form (BALB/cJ, C3H/HeJ,DBA/2J and 129P3/J) (Reed et al., 2004). Two-way analyses ofvariance were performed on the percentage of vehicle baselineintake score at 30 min for the five doses of SCH23390 and thehigh dose of raclopride, all of which displayed significantinhibition of sucrose intake. The 30 min intake point waschosen because overall baseline intakes of the sweet-sensitiveand sweet-sub-sensitive strains were equal at this interval.

Acknowledgments

This research was supported in part by National Institute ofDiabetes and Digestive and Kidney Diseases Grant DK071761to AS and RJB; CD is a CUNYDoctoral Chancellor's Fellow, AP isa student in the CUNY Honors College at Queens College andMR was a high school student participating in the New YorkAcademy of Sciences Summer Research Training Program.Wethank three anonymous reviewers for their excellent sugges-tions concerning this manuscript.

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