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Drug and Alcohol Dependence 132 (2013) 420– 426

Contents lists available at ScienceDirect

Drug and Alcohol Dependence

j ourna l ho me p age: www.elsev ier .com/ locate /drugalcdep

ifferential effects of methadone and buprenorphine on the responsef D2/D3 dopamine receptors in adolescent mice�

. William Barwatta, Rebecca S. Hofforda, Michael A. Emerya,b, M. L. Shawn Batesa,b,aul J. Wellmana,b, Shoshana Eitana,b,∗

Behavioral and Cellular Neuroscience, Department of Psychology, Texas A&M University, 4235 TAMU, College Station, TX 77843, USAInterdisciplinary Program in Neuroscience, Texas A&M Institute for Neuroscience (TAMIN), USA

r t i c l e i n f o

rticle history:eceived 14 February 2013eceived in revised form 19 June 2013ccepted 12 July 2013vailable online 9 August 2013

eywords:ddictionependencerugs of abuseaintenance treatmentpioidain management

a b s t r a c t

Background: There is a lack of studies that examine the effects of opioid maintenance drugs on the devel-oping adolescent brain, limiting the ability of physicians to conduct a science-based risk assessmenton the appropriateness of these treatments for that age group. Our recent observations indicate higherpotential risks in repeated exposure to morphine during adolescence, specifically to the D2/D3 dopaminereceptors’ signaling. Disturbances in dopaminergic signaling could have broader implications for long-term mental health. Thus, this study examined whether buprenorphine and methadone differentiallyalter the responses of the D2/D3 dopamine receptors in adolescents.Methods: Adolescent mice were orally administered buprenorphine (0.1–0.4 mg/kg), methadone(25–100 mg/kg), or saline once daily for 6 days. Two hours or three days later, the mice were testedfor their locomotor response to 10 mg/kg quinpirole, a D2/D3 dopamine receptor agonist.Results: Buprenorphine-treated adolescent mice did not significantly differ from control drug-naïve ani-mals in their response to quinpirole. However, an enhanced response was observed in methadone-treatedadolescent animals. This enhanced locomotion was significantly higher two hours following the final doseof methadone, as compared to three days afterwards.

Conclusions: This study suggests that exposure to various opioids carries differential probabilities ofaltering the highly sensitive neurochemistry of adolescent brains. Methadone exposure disturbs the D2-like receptor’s response, indicating a potential risk in administering methadone to adolescents (eitherfor the treatment of opioid dependency/abuse or for pain management). In contrast, buprenorphineappears to have a significantly lower effect on the behavioral sensitivity of D2/D3 dopamine receptors inadolescents.

. Introduction

Nonmedical use of opioids is the second most common form ofllicit drug use in the United States after marijuana (SAMHSA, 2009).n 2008, one in five teens abused a prescription pain medicationPATS, 2009), and this high prevalence has continued over the lastew years (Johnston et al., 2011). Thus, there is growing need to

mprove our knowledge on the consequences of adolescents’ opi-id use, as well as on the treatment options available for them.ur recent observations indicate that adolescent mice exposed to

� Supplementary material can be found by accessing the online version of thisaper. Please see Appendix A for more information.∗ Corresponding author at: Department of Psychology, Texas A&M University,

235 TAMU, College Station, TX 77843, USA. Tel.: +1 979 845 2508;ax: +1 979 845 4727.

E-mail address: seitan@tamu.edu (S. Eitan).

376-8716/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved.ttp://dx.doi.org/10.1016/j.drugalcdep.2013.07.016

© 2013 Elsevier Ireland Ltd. All rights reserved.

morphine subsequently exhibited a supersensitive response to aD2/D3 dopamine receptor agonist (Hofford et al., 2012). This effectof morphine was extremely pronounced in adolescents, but wasbarely observed in adults. These findings suggest that opioid useduring adolescence results in markedly robust disturbances of thedopaminergic signaling as compared to use during adulthood.

Different opioids are known to have unique molecular profilesand to differentially modulate the activity of various opioid recep-tors (Zhang et al., 1998; Patel et al., 2002; Arttamangkul et al.,2008). Thus, different opioids are likely to differentially modulatebrain neurochemistry. However, there is a lack of studies examin-ing the differential effects of opioids on the developing brains ofadolescents. Specifically, buprenorphine and methadone are twodrugs approved for maintenance treatment of opioid addiction in

adults, and also recently in adolescents (Kleber et al., 2006). For ado-lescents, a few clinical studies demonstrated improved retentionin treatment programs using these drugs (Bell and Mutch, 2006).However, the advantages and risks associated with using these

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rugs in this age group are controversial (Kleber et al., 2006; Simkinnd Grenoble, 2010). Various authorities thus instated restrictionsn the use of pharmacological maintenance treatments, especiallyethadone, for treating opioid-dependent adolescents (Fiellin,

008; Hillier, 2011). Additionally, methadone and buprenorphinere also used for pain management in children and adolescentsAnghelescu et al., 2011; Michel et al., 2011). Methadone is alsoecreationally used (misused/abused) by adolescents (Johnstont al., 2011). However, there are currently very limited studies thatirectly examine the differential effects of these opioids on theeveloping brains of adolescents, limiting the ability of physicianso conduct a science-based risk assessment on the effects of usinghese treatments in youths.

Given the mental health implications of an altered dopaminer-ic system, this study examined the effects of orally administeringuprenorphine (0.1, 0.2 and 0.4 mg/kg) and methadone (25, 50nd 100 mg/kg) for 6 consecutive days on altering the locomotoresponses to quinpirole, a D2/D3 dopamine receptor agonist. Addi-ionally, in order to determine the equivalence between the dosessed in this study and doses used for treating humans, plasma levelsf buprenorphine and methadone were measured following thesereatment regimens.

. Methods and materials

.1. Animals

All procedures were conducted in accordance with the National Institutes ofealth (NIH) Guide for the Care and Use of Laboratory Animals, and were approvedy the Texas A&M Institutional Animal Care and Use Committee. Adolescent male57BL/6 mice, purchased from Harlan Lab (Houston, TX), were housed 4 per cageith food and water ad lib. They were acclimated to the temperature-controlled

21 ± 2 ◦C) vivarium with a 12 h/12 h light/dark cycle (light on at 07:00) for approx-mately one week prior to treatment.

The choice for the age of the adolescent mice was based on studies by Spear andolleagues (reviewed in Spear, 2000). Accordingly, mice were purchased at post-atal day 22 (PND 22). They were acclimated to the vivarium until PND 28, whenethadone, buprenorphine, or saline injections began, and behavioral testing was

erformed on PND 33 or 36. Thus, in this study, mice were injected during whats considered the late phase of their prepubescent period, and were tested duringheir mid-adolescence/periadolescent period. The different experimental groups areummarized in Table 1 of the Supplementary materials.

.2. Methadone and buprenorphine treatment regimen

Adolescent mice (n = 12–29 per group) were administered buprenorphine (0.1,.2, or 0.4 mg/kg, 10 ml/kg), methadone (25, 50, or 100 mg/kg, 10 ml/kg) or saline10 ml/kg) once daily (8 a.m.) for six days via gavage. Drugs were purchased fromigma–Aldrich Chemicals (St. Louis, MO). These doses were selected to representlasma levels generated by the therapeutic doses used for maintenance treatment

n humans (Leavitt, 2003; Bell and Mutch, 2006; Moody et al., 2011). The selec-ion was based on the existing literature on the pharmacokinetics of these drugsn mice (Middaugh et al., 1983; Kalliokoski et al., 2011). In addition, plasma levelsf buprenorphine and methadone were examined to compare with the reportedlasma levels in human opioid addicts receiving buprenorphine or methadone foraintenance treatments.

.3. Locomotion testing

The assessment of activity was made in a set of 8 automated optical beam activ-ty monitors (Model RXYZCM-16; Accuscan Instruments, Columbus, OH, USA) asescribed in detail in (Wellman et al., 2009). Briefly, each monitor is housed within

40 cm × 40 cm × 30.5 cm acrylic cage. Activity monitors and cages were located in sound-proof room with a 40 dB white noise generator continuously operating. Aultiplexor-analyzer simultaneously tracks the interruption of beams from optical

eam activity monitors. The multiplexor-analyzer updates the animal’s position inhe acrylic cage every 10 ms using a 100% real-time conversion system. The gen-ral activity is obtained from the computerized integration of the data using totalistance traveled scores (in cm; Sanberg et al., 1987).

Activity was recorded two hours or three days following the final treatmentose of methadone, buprenorphine, or saline. Mice were placed in the testing room0 min prior to the test. Baseline activity was recorded for 30 min. The mice werehen injected with quinpirole (10 mg/kg, 10 ml/kg, i.p.) or vehicle and recorded for20 min. Different mice were used on each testing day. The data for the vehicle

ependence 132 (2013) 420– 426 421

tests are presented in the Supplementary materials. The apparatus was cleanedthoroughly with ethanol followed by water and completely dried between tests.

The choice of quinpirole to assess the effects of buprenorphine and methadoneon the behavioral sensitivity of the D2/D3 dopamine receptors was based on pre-vious studies in both rats (Piepponen et al., 1996; Druhan et al., 2000) and mice(Hofford et al., 2012) that demonstrated locomotor hypersensitivity to quinpirolefollowing morphine administration. Moreover, in our previous study (Hofford et al.,2012) a range of quinpirole doses (starting at 0.01 mg/kg) were tested. Morphinedid not alter the suppressive response of quinpirole (i.e., the response at the pre-synaptic D2 receptors) for any of the doses examined. However, morphine did alterthe subsequent activating effect of quinpirole (i.e., the response at the postsynap-tic receptors). Studies focused on the presynaptic receptors specifically used lowerdoses of quinpirole as to only activate the presynaptic receptors. These lower doseswould not be suitable here since they do not activate the postsynaptic receptors.Therefore, in this study we used a dose of quinpirole that is established to affect thepostsynaptic receptors.

It is important to note that the dose used is well within the range found in the lit-erature. In mice, quinpirole doses up to 20 mg/kg were used by many studies and wasconsidered to have specific effects on the D2-like dopamine receptors (Marstelleret al., 2009). Quinpirole specificity for up to 32 mg/kg was also established by thelack of effect in CBA/J mice that are deficient in the expression of dopamine receptors(Shannon et al., 1991).

2.4. Plasma levels of buprenorphine and methadone

Mice (n = 8–9 per group) were administered 0.2 mg/kg buprenorphine or50 mg/kg methadone for six days, as described above. Two, six or 24 h after thefinal treatment dose they were anesthetized with pentobarbital (100 mg/kg, i.p.) andtheir blood collected via intra-cardiac puncture. Plasma was separated via centrifu-gation (15 min, 1000 × g, 4 ◦C) and stored at −80 ◦C. Buprenorphine and methadonelevels in the plasma were determined using ELISA Kits (Neogen Corporation, St.Joseph, MI).

2.5. Data analyses

For each mouse, the scores for the total distance traveled (in cm) during the120 min post-vehicle or post-quinpirole were normalized to the total distance trav-eled (in cm) during the 30 min baseline locomotion using the formula: [total distancetraveled post-vehicle or post-quinpirole/baseline total distance traveled] × 100.Then, data for the between-subject factors of treatment was analyzed for thenormalized total distance traveled scores (% from baseline) during the 120 min post-vehicle or post-quinpirole using the Univariate Analysis of Variance (ANOVA, SPSSStatistics 18, Somers, NY). Additional temporal analyses were also computed forbetween-group factors of treatment (buprenorphine, methadone, or saline) and forthe within-group factor of time (1–120 min post-injection period summed in 5 minintervals). For this analysis, each animal’s the score for the last 5 min interval prior tovehicle or quinpirole injections (i.e., baseline) was used to normalize the data. Posthoc contrasts between each treatment group were computed using Bonferroni’s posthoc procedure. Differences with p-values of less than 0.05 were deemed statisticallysignificant. Results are presented as mean ± SEM. The data for the vehicle tests arepresented in the Supplementary Materials.

3. Results

3.1. Experiment I: the effects of buprenorphine and methadone onthe response to quinpirole three days following the final treatment

3.1.1. Total distance traveled. The scores for the total distance trav-eled (% from baseline) during the 120 min post-quinpirole arepresented in Fig. 1 Analysis revealed significant differences in thelocomotor response to quinpirole between animals treated withthe various drugs (F(6, 117) = 4.38, p < 0.0001). Post hoc comparisonrevealed no differences in quinpirole-induced suppression of activ-ity level between saline-injected mice and mice treated with 0.1, 0.2and 0.4 mg/kg buprenorphine. In contrast, significant differenceswere observed between saline-injected mice and mice treated withmethadone (p < 0.05). Specifically, significantly less suppression ofactivity by quinpirole was observed in methadone-treated animalsas compared to the drug-naïve animals.

3.1.2. Temporal analysis. Additional temporal analyses were com-

puted using the distance traveled scores during each 5 min intervalof the 120 min post-quinpirole injection. The results for thebuprenorphine-treated animals are presented in Fig. 2A. Analy-sis revealed a main effect of time (F(23, 1633) = 13.37, p < 0.0001),

422 J.W. Barwatt et al. / Drug and Alcohol Dependence 132 (2013) 420– 426

Fig. 1. Mice (n = 13–29 per group) were orally administered buprenorphine (0.1, 0.2or 0.4 mg/kg) or methadone (25, 50, or 100 mg/kg) for 6 days. Three days later, theirresponse to 10 mg/kg quinpirole was tested. Results are presented as mean ± SEMof the total distance traveled scores (% from baseline) in the 120 min after admin-istration of quinpirole. * indicates a significant difference from saline-treated mice(p < 0.05, Bonferroni); § indicates a significant difference from saline-treated mice(p < 0.05, LSD).

Fig. 2. Mice (n = 13–29 per group) were orally administered buprenorphine (0.1, 0.2or 0.4 mg/kg; (A)) or methadone (25, 50, or 100 mg/kg; (B)) for 6 days. Three dayslater, their response to 10 mg/kg quinpirole was tested. Results are presented asmean ± SEM of the total distance traveled scores (% from baseline) for each 5 mininterval during the 30 min baseline and 120 min after administration of quinpi-role. Each time point represents the 5 min interval immediately preceding thattime. Quinpirole was administered at t = 0. Significant differences in the locomotorresponse to quinpirole between drug-naïve mice and mice treated with the variousdoses of methadone were observed in multiple 5 min interval periods during the120 min post-quinpirole (p < 0.05, Bonferroni). However, these post hoc contrastsare not individually identified to maintain the simplicity of the figure.

Fig. 3. Mice (n = 8–12 per group) were orally administered 0.2 mg/kg buprenorphineor 50 mg/kg methadone for 6 days. Two hours after the final dose, their response to10 mg/kg quinpirole was tested. Results are presented as mean ± SEM of the total

distance traveled scores (% from baseline) in the 120 min after administration ofquinpirole. * indicates a significant difference from saline-treated mice (p < 0.05,Bonferroni).

but no significant main effect of treatment (F(3, 71) = 1.68, p > 0.05,n.s.), and no significant interaction between treatment and time(F(69, 1633) = 0.87, p > 0.05, n.s.). Post hoc comparison revealedno differences in quinpirole-induced suppression of activity levelbetween drug-naïve mice and mice treated with the various dosesof buprenorphine.

The results for the temporal analysis post-quinpirole for themethadone-treated animals are presented in Fig. 2B. Analysisrevealed a main effect of time (F(23, 1702) = 21.37, p < 0.0001), amain effect of treatment (F(3, 74) = 3.41, p < 0.05), and a signifi-cant interaction between treatment and time (F(69, 1702) = 2.61,p < 0.0001). In contrast to buprenorphine, significant differences inthe locomotor response to quinpirole between drug-naïve mice andmice treated with the various doses of methadone were observed inmultiple 5 min interval periods during the 120 min post-quinpirole(p < 0.05).

3.2. Experiment II: the effects of buprenorphine and methadoneon the response to quinpirole two hours following the finaltreatment

3.2.1. Total distance traveled. The scores for the total distance trav-eled (% from baseline) during the 120 min post-quinpirole arepresented in Fig. 3. Analysis revealed significant differences in thelocomotor response to quinpirole between animals treated withthe various drugs (F(2, 28) = 18.53, p < 0.0001). Post hoc compar-ison revealed no differences in quinpirole-induced suppression

of activity level between drug-naïve mice (i.e., saline-injectedmice) and mice treated with 0.2 mg/kg buprenorphine. In contrast,a significant difference was observed between drug-naïve miceand mice treated with 50 mg/kg methadone (p < 0.05). Specifically,

J.W. Barwatt et al. / Drug and Alcohol Dependence 132 (2013) 420– 426 423

Fig. 4. Mice (n = 8–12 per group) were orally administered 0.2 mg/kg buprenorphineor 50 mg/kg methadone for 6 days. Two hours after the last dose, their response to10 mg/kg quinpirole was tested. Results are presented as mean ± SEM of the totaldistance traveled scores (% from baseline) for each 5 min interval during the 30 minbaseline and 120 min after administration of quinpirole. Each time point representsthe 5 min interval immediately preceding that time. Quinpirole was administeredasa

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Fig. 5. Mice (n = 8–9 per group) were orally administered 0.2 mg/kg buprenor-phine or 50 mg/kg methadone for 6 days. Plasma levels of buprenorphine (A) and

t t = 0. In each 5 min interval period from t = 10 until the end of the testing period,ignificant differences were observed between drug-naïve and methadone-treatednimals in their locomotor response to quinpirole (p < 0.05, Bonferroni).

uinpirole significantly increased the activity levels of methadone-reated animals as compared to the suppression of activity observedn the drug-naïve animals.

.2.2. Temporal analysis. An additional temporal analysis was com-uted using the distance traveled scores during each 5 min intervalf the 120 min post-quinpirole injection. The results are presentedn Fig. 4. Analysis revealed a main effect of time (F(23, 644) = 13.36,

< 0.0001), a main effect of treatment (F(2, 28) = 23.06, p < 0.0001),nd a significant interaction between treatment and time (F(46,44) = 11.95, p < 0.0001). Post hoc comparison revealed no differ-nces in quinpirole-induced suppression of activity level betweenrug-naïve mice and mice treated with 0.2 mg/kg buprenorphine.

n contrast, significant differences was observed between drug-aïve mice and mice treated with 50 mg/kg methadone (p < 0.05).pecifically, from 10 min following quinpirole administration untilhe end of the testing period, significant differences were observedetween drug-naïve and methadone-treated animals in their loco-otor response to quinpirole for each of the 5 min interval periods.

.3. Plasma levels of buprenorphine and methadone

Six days of oral administration of 0.2 mg/kg buprenorphineielded a peak plasma concentration of 7.6 ± 0.8 ng/ml at 2 h afterhe last dose of buprenorphine (Fig. 5A). Six days of oral administra-ion of 50 mg/kg methadone yielded a peak plasma concentrationf 133 ± 28 ng/ml at 6 h after the last dose of methadone (Fig. 5B).

. Discussion

This study demonstrates that buprenorphine and methadoneifferentially modulate the locomotor response to a D2/D3opamine receptor agonist, quinpirole. Quinpirole reduced loco-otor activity in drug-naïve adolescent mice. This suppression

asted for the entire 120 min following quinpirole administration.his is consistent with the literature demonstrating that the sup-ressive effect of quinpirole on motor activity is predominant

n mice (Halberda et al., 1997). Buprenorphine-treated adoles-

ent mice did not significantly differ from drug-naïve animals inheir responses to quinpirole. However, in methadone-treated ado-escent mice, the initial suppression of locomotor activity wasollowed by enhanced locomotion.

methadone (B) were examined two, six or 24 h after the last administration. Resultsare presented as mean ± SEM.

Quinpirole is suggested to reduce locomotor activity bydecreasing dopamine release (Starke et al., 1989). This presynapticeffect is mediated by the short isoform of the D2 dopamine recep-tor (D2S; Mogenson and Wu, 1991; Usiello et al., 2000). In mice,the D2S receptor was sufficient for quinpirole’s suppressive effecton motor activity, and both the long isoform of the D2 dopaminereceptor (D2L; Wang et al., 2000) and the D3 dopamine receptor(Li et al., 2010a) were not necessary. In contrast, the motor acti-vating effect of quinpirole is thought to be mediated by D2L/D3postsynaptic receptors (Millan et al., 2004; Marsteller et al., 2009).The fact that suppression was initially observed in the methadone-treated animals suggests that the response of the presynapticD2 receptor is not altered, and that the main effect is on post-synaptic D2L/D3 dopamine receptors’ response. Additionally, theresponse to quinpirole of the methadone-treated mice examined2 h post-methadone administration greatly surpassed the activityof the vehicle-treated mice. Thus, the altered response of the D2/D3receptors is likely an effect at the postsynaptic receptors, given thata reduced suppressive effect at the presynaptic receptors could notentirely account for such an altered response.

Hyper-sensitivity to D2 and D3, but not D1, dopamine recep-tor agonists was demonstrated in adult rats undergoing morphinewithdrawal (Lee et al., 1987; Reddy et al., 1993; Piepponen et al.,1996; Druhan et al., 2000). We recently observed that this behav-ioral supersensitivity of the D2L/D3 postsynaptic receptor signalingfollowing morphine treatment is markedly enhanced in adoles-cents as compared to adults (Hofford et al., 2012). Thus, this studydemonstrates that, similar to morphine, methadone treatmentresults in a supersensitivity response of the D2L/D3 postsynapticreceptors in adolescents. Moreover, this enhanced effect in adoles-cents is likely a direct effect of repeated exposures to methadone,

rather than the effect of withdrawal, given that a larger effect wasobserved two hours following the final methadone dose as com-pared to three days later. Moreover, buprenorphine treatment didnot alter the behavioral response of D2L/D3 receptors. These results

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uggest that exposure to different opioids carry differential proba-ilities of altering the activity of the highly sensitive dopaminergicystem in adolescents.

Alteration of the D2/D3 dopamine receptors’ responses couldave long term implications for the abuse potential of opioids. The2 dopamine receptors are involved in the reinforcing propertiesf morphine (Elmer et al., 2002), and in mediating drug-seekingehaviors in mice (Dockstader et al., 2001) and rats (De Vriest al., 1999) undergoing morphine withdrawal. Specifically, the D2Leceptor was demonstrated to be essential for the rewarding prop-rties of morphine (Maldonado et al., 1997; Smith et al., 2002), andas demonstrated to mediate the aversive properties of morphineithdrawal (Smith et al., 2002). The D3 dopamine receptor wasemonstrated to play a role in opioid sensitization (Narita et al.,003; Li et al., 2010b) and reward (Ashby et al., 2003; Narita et al.,003). Additionally, disturbances in the signaling of the D2-likeopamine receptors are expected to have broader implications.ssociation was suggested between D2 receptor genes and thebuse of various illicit drugs, as well as between D2 receptor genesnd alcoholism (Blum et al., 1991; Nemoda et al., 2011). More-ver, the D2/D3 receptors were also suggested to be involved inhe pathophysiology of affective and psychotic disorders, and in theesponse to pharmacological treatments (Svestka, 2005; Gershont al., 2007; Seeman, 2011).

The adult therapeutic dosage of sublingual buprenorphineecommended by the American Psychiatric Association for main-enance treatment ranges from 2 to 32 mg, daily to 3 times a week.

range of 4–16 mg tablets is reported for the treatment of adoles-ents (Bell and Mutch, 2006). Buprenorphine plasma levels in maleatients maintained on 16 mg sublingual tablets was recorded toe roughly 40 ng × h/ml over a 24 h period, reaching a maximumlasma concentration of about 5 ng/ml 1.2 h after administrationMoody et al., 2011). Thus, this study examined doses of buprenor-hine that generated plasma levels in mice that are above thoseecorded in human patients receiving this drug for maintenancereatments.

A wide range of oral methadone doses are used in patients,ith the recommendations generally ranging from 20 to 100 mgaily. An average maintenance dose of approximately 50 mg wassed for clinical studies in adolescents (Bell and Mutch, 2006).ethadone metabolism, plasma levels, and clearance vary among

atients and are influenced by genetic factors and by interac-ions with other substances. Therapeutic methadone plasma levelsange between 100 and 1000 ng/ml, with studies demonstratinghat plasma levels over 200 ng/ml are usually necessary. A plasmaange of 400–500 ng/ml is recommended for optimal therapeu-ic efficiency (Leavitt, 2003). Thus, this study examined doses of

ethadone that generated plasma levels which are at the lownd of the range of plasma levels recommended for maintenancereatment in human patients. Thus, this study demonstrated that

ethadone – even at what might be viewed as suboptimal ther-peutic doses – alters the response of the D2L/D3 postsynapticeceptors in adolescents. However, buprenorphine – even at higheroses than used therapeutically – does not affect the activity at the2L/D3 receptors.

It is important to note that although efforts were made toesign a translation-oriented study, a limitation of this study ishat it is extremely difficult to absolutely compare all the aspectsf a ‘therapeutic dose’ across different species and ages. Dif-erent species require different ranges of doses to experiencehe therapeutic effects of drugs. Importantly, this study testshether treatment with these drugs carries differential odds of

nwanted (‘side’) effects. This study does not examine the ben-ficial effects of buprenorphine and methadone as maintenancerugs or for pain management – that had already been establishedrior to this study. Thus, we deemed it more clinically relevant to

ependence 132 (2013) 420– 426

examine the unwanted effects when administering doses at levelsthat are required for human therapy. Nonetheless, note that thedoses of methadone used in this study (which resulted in alteredD2/D3 responses) are in the range of doses used to reduce heroin-seeking in rats. Specifically, a 30 mg/kg/day s.c. administration ofmethadone was demonstrated to be effective in reducing heroin-seeking in rats (Leri et al., 2004). Such a dose roughly corresponds tothe 50 mg/kg/day oral methadone used in our study. Thus, the dosesused in our studies appear to be within the range that is effectivein reducing opioid-seeking in rodents.

Another important limitation of the study is that stable blood-concentration levels were not achieved in the adolescent miceeven after 6 days of methadone administration. Methadone wasnot detected in the blood 24 h after the last administration (i.e.,by the time of its next daily dose). This is most likely due to theshort half-life of methadone in mice (LeVier et al., 1995). In con-trast, buprenorphine was still detected in the blood even 24 h afterthe last administration. Thus, it is possible that the alteration ofD2/D3 dopamine receptors will occur when methadone is mis-used recreationally or used for pain management, but not whenused for maintenance therapy (e.g., when blood levels are keptstable for longer periods). Nonetheless, although methadone elim-ination from blood is relatively quick in mice, the elimination rateof methadone is significantly slower from their brains. Actually, theelimination half-life of methadone from mice brains was reportedto be almost identical to the plasma half-life measured clinicallyin humans (Kalvass et al., 2007). Thus, although stable blood lev-els were not achieved in this study, it is possible that stable brainlevels were still achieved, which would suggest that alterations inthe response of D2/D3 dopamine receptors might also occur duringmethadone maintenance therapy.

Lastly, the adolescent mice in this study were drug-naïve priorto the administration of buprenorphine or methadone. Cross-sensitization can develop between various opioids. Thus, it ispossible that the effects of buprenorphine and/or methadonewill differ in opioid-dependent adolescents (i.e., after chronicuse/abuse/exposure to opioids) as compared to drug-naïve ado-lescents. Thus, these findings call for further research to revealdifferences among other opioids in modulating the responses ofthe dopaminergic system, as well as potential cross-sensitizationbetween them and pharmacological maintenance treatments. Thisincludes opioids known to be recreationally used by adolescents aswell as opioids used for maintenance treatment or pain manage-ment in children and adolescents.

This study demonstrates behavioral supersensitivity in adoles-cent mice in response to a D2/D3 dopamine receptor agonist afterexposure to methadone, but not buprenorphine. Thus, this studysuggests that exposure to methadone during adolescence can resultin markedly robust disturbances of the responses of the D2-likereceptors. In contrast to methadone, buprenorphine appears tohave a significantly lower effect on the responses of the D2L/D3dopamine receptors in adolescents. Thus, these results also suggestthat exposure to different opioids carries differential probabilitiesof altering the highly sensitive neurochemistry of the adolescentbrain. This study focused only on examining adolescents given thehigher susceptibility of adolescents to the effects of opioids on thesensitivity of the D2/D3 dopamine receptors (Hofford et al., 2012).However, future studies should also examine the effects in adultsof methadone and buprenorphine on the responses of the D2/D3receptors.

Role of funding source

M.A.E. and M.L.S.B. are supported by the Heep fellowship in Neu-roscience awarded by the Texas A&M Institute for Neuroscience

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TAMIN). The funding source had no further role in the studyesign; in the collection, analysis and interpretation of data; in theriting of the report; or in the decision to submit the paper forublication.

ontributors

S.E. designed the study, supervised the performance of thexperiments, analyzed the data, and wrote the manuscript. J.W.B.nd R.S.H. performed the experiments and assisted with data anal-sis and editing the manuscript. M.A.E. and M.L.S.B. assisted witherforming the experiments and editing the manuscript. P.J.W.ssisted with the design of the study, statistical analysis and writ-ng of the manuscript. All authors contributed to and have approvedhe final manuscript.

onflict of interest

The authors have no conflicts of interest.

cknowledgements

We would like to thank Mr. Menachum M. Slodowitz for hisditorial assistance. M.A.E. and M.L.S.B. are supported by the Heepellowship in Neuroscience awarded by the Texas A&M Institute foreuroscience (TAMIN).

ppendix A. Supplementary data

Supplementary data associated with this article can beound, in the online version, at http://dx.doi.org/10.1016/.drugalcdep.2013.07.016.

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