Journal of Biomedical Science (2006) 13:797–811 797 DOI 10.1007/s11373-006-9102-x

Methamphetamine-induced behavioral sensitization in mice: alterations in l-opioid receptor

Chi-Tso Chiu, Tangeng Ma & Ing K. Ho* Department of Pharmacology and Toxicology, University of Mississippi Medical Center, 2500 North State Street, Jackson, MS, 39216, USA

Received 11 May 2006; accepted 21 June 2006 2006 National Science Council, Taipei

Key words: l-opioid receptor, behavioral sensitization, constitutive activity, DAMGO, G protein, methamphetamine, neuroadaptations

Summary We had previously demonstrated that opioid receptors contribute to the induction and expression of behavioral sensitization induced by repeated daily injection with 2.5 mg/kg of methamphetamine for 7 days. Using the same regimen, the present study investigated the alterations in l-opioid receptor during the induction (on days 2, 5, and 8) and expression (on days 11 and 21) periods of behavioral sensitization. Radioligand binding revealed that the maximal binding of l-opioid receptor was not changed on days 2 and 5, but down-regulated on day 8. After cessation of drug treatment, the maximal binding of l-opioid receptor gradually and time-dependently returned to normal level on day 11 and up-regulated on day 21. In contrast, no changes in d-andj-opioid receptors were detectable on any given day examined. The potency of DAMGO for [35S]-GTPcS coupling was enhanced on days 2, 5, 11, and 21. Moreover, 1 lM of nal- trexone or b-chlornaltrexamine significantly suppressed the basal [35S]-GTPcS coupling on days 2, 11, and 21. These findings indicate enhanced responsiveness and elevated constitutive activity of l-opioid receptor. In summary, our data clearly demonstrate that alterations in l-opioid receptor are involved in and may contribute to the sensitization to locomotor stimulating effect of methamphetamine.

Abbreviations: ANOVA – analysis of variance; AMPH – amphetamine; b-CNA – b-chlornaltrexamine; 2 4 5 2,5 DAMGO – [D-Ala , N-Me-Phe , Gly -ol]-enkephalin; DPDPE – [D-Pen ]-enkephalin; GDP – guanosine 5¢-diphosphate;[35S]-GTPcS – guanosine 5¢-O-(3-[35S]-thio) triphosphate; METH – methamphetamine; NAcc – nucleus accumbens; NAT – naltrexone; U69,593 – ( + )-(5a,7a,8b)-N-methyl-N-[7-(1-pyrrolid- inyl)-1-oxaspiro[4.5]dec-8-yl]-benzeneacetamide; VTA – ventral tegmental area

Introduction sensitization, which is characterized by progressive and enduring augmentation of the behavioral Continued use of psychostimulants such as effects in response to subsequent exposure to the amphetamine (AMPH), methamphetamine same dose of the drug [1, 2]. Behavioral sensitiza- (METH), and cocaine is typically associated with tion has been used as an animal model for studying the development of tolerance. However, repeated the development of craving in addicts and psycho- intermittent exposure to these drugs was found to sis that arises from repeated exposure to psycho- produce ‘‘reverse tolerance’’ known as behavioral stimulants [2, 3]. *To whom correspondence should be addressed. Fax +1-601- METH is a synthetic drug and chemically 984-1637, E-mail: [email protected] related to AMPH but has a higher potential for 798 abuse. The behavioral activating effects of AMPH although agonists of d- and l-opioid receptors are thought to depend primarily on its ability to exhibit rewarding property, it has been found increase dopamine release in the terminal regions that l- but not d-opioid receptors play a major of the mesolimbic dopamine system [4]. The role in modulating mesolimbic dopamine system ventral tegmental area (VTA) is the somatoden- [13–15]. Pharmacological evidence reveals that dritic region of the mesolimbic dopamine neurons, mesolimbic dopaminergic activity can be con- whose nerve terminals project primarily to the trolled by c-aminobutyric acid (GABA), a major nucleus accumbens (NAcc), an important struc- inhibitory afferent in this brain region [16]. In the ture for mediating spontaneous and pharmacolog- VTA, activation of l-opioid receptors located ically stimulated locomotor activity [5]. The possibly on both GABA afferent and interneurons mesolimbic dopamine system has been implicated is known to hyperpolarize these GABA-containing in the rewarding effects of drugs of abuse [6]. It is neurons and decrease GABA release [17, 18]. clear that neuroadaptations in the VTA and NAcc Therefore, the neuronal activity of mesolimbic are critical for the induction and expression of dopamine neurons is also indirectly regulated behavioral sensitization to psychostimulants, by l-opioid receptors in the VTA through GABA. respectively [7, 8]. In addition to anatomical Accordingly, activation of l-opioid receptors led differences, the processes of induction and expres- to increase in dopamine neuronal activity and sion of behavioral sensitization are also temporally subsequent dopamine release in the NAcc [18, 19], distinct. An abundance of neuroadaptive phenom- whereas blockade of the receptor resulted in ena observed during the establishment and expres- a significant decrease in baseline and AMPH- sion of behavioral sensitization was occurring in a induced dopamine levels in the NAcc [15, 19]. time-dependent fashion [9], suggesting different Acute administration with AMPH or METH is neuronal mechanisms were involved. known to increase the expression of opioid peptide Topographic overlaps between opioid and mRNA and neurotransmission of endorphin in the dopamine neurons were found in the VTA, sub- brain [20, 21], and mesolimbic structures such as stantia nigra, striatum, and limbic areas, suggest- VTA and NAcc receive b-endorphin containing ing that there are interactions between these two fibers [22]. It was found that the GABA contents systems [10]. Our previous study in mice showed of the midbrain were decreased 1 h after a single that, pretreatment with naltrexone (NAT), a non- administration of METH [23], suggesting the selective opioid receptor antagonist, attenuated the activation of l-opioid receptors in the VTA. induction and expression of METH-induced Therefore, repeated METH treatment during the behavioral sensitization [11]. When administered induction period of behavioral sensitization may prior to daily METH treatment, the inhibitory cause repeated or persistent activation of l-opioid effect of NAT on the induction of behavioral receptor. Moreover, activation of l-opioid recep- sensitization appeared to vanish after a longer tors in the VTA is known to mediate food intake period of abstinence. Moreover, when adminis- behavior [24]. It was found that not only the tered prior to METH challenge, the inhibitory feeding [24, 25] but also the dopamine releasing effect of NAT on the expression of behavioral [26] effects of l-opioid receptor were greatly sensitization was found to be more effective after a enhanced after repeated AMPH or METH treat- longer period of abstinence. These differential ment, suggesting functional alterations in l-opioid effects of NAT indicated potential alterations in receptors. Taken together with the findings opioid receptors after repeated METH treatment, above, l-opioid receptors seem to play a major which might be time-dependent and definitive for role in modulating or mediating the effects of expression of behavioral sensitization. psychostimulants in the mesolimbic dopamine Endogenous opioids have been suggested to system and susceptible to repeated psychostimu- play a key role in mediating the rewarding effects lant treatment. Therefore, the present study was of psychostimulants. It is well accepted that designed to extend our research by addressing the j-opioid receptors play a role in opposing the question whether alterations in l-opioid receptor behavioral consequences induced by repeated psy- are associated with the induction and expression of chostimulant administration [12]. In addition, METH-induced behavioral sensitization. 799

Materials and methods regimen of METH successfully induced long- lasting behavioral sensitization in mice (at least Materials 3 weeks after the cessation of the drug treatment) [11]. METH was freshly dissolved in saline before 3 2 4 5 [ H]-[D-Ala , N-Me-Phe , Gly -ol]-enkephalin use and injected at a volume of 10 ml/kg of body 3 3 2,5 ([ H]-DAMGO; 50 Ci/mmol), [ H]-[D-Pen ]- weight. enkephalin ([3H]-DPDPE; 45 Ci/mmol), [3H]-(5a, 7a,8b)-(+)-N-methyl-N-(7-[1-pyrrolidinyl]-1-ox Brain membrane preparations aspiro[4.5]dec-8-yl)-benzeneacetamide ([3H]-U69, 593; 39.7 Ci/mmol), and guanosine 5¢- O-(3-[35S]- To investigate the neuronal alterations induced by thio) triphosphate ([35S]-GTPcS; 1250 Ci/mmol), repeated METH treatment during the induction were purchased from Perkin Elmer Inc. (Boston, period (days 1–7), brain samples were obtained MA). Scintillation cocktail (Bio-safe II) was 24 h after the first, fourth, and last daily injections obtained from RPI (Mount Prospect, IL). The of saline or METH on days 2, 5, and 8, respec- assay reagent for protein content was purchased tively, for the purpose of preventing the possible from Bio-Rad (Richmond, CA). METH, nalox- interference produced by acute effect of the drug. one, NAT, b-chlornaltrexamine (b-CNA), DAM- For studies of the neuronal alterations associated GO, guanosine 5¢-diphosphate (GDP), GTPcS, with the expression of behavioral sensitization after and all other chemicals, were purchased from short-term and long-term periods of abstinence, Sigma-Aldrich (St. Louis, MO). brain samples were obtained on days 11 and 21, respectively. Mice received 7 days of repeated Animals METH treatment without behavioral testing were assumed to be sensitized with the same consider- Male NIH Swiss mice (Harlan, Indianapolis, IN), ation. l-Opioid receptor knockout and wild-type weighing 20–25 g at the beginning of the experi- mice were sacrificed without any treatment. Mice ment were used. Upon arrival, mice were housed in were sacrificed by decapitation and the brains were groups of four in animal colony room on a 12-h removed and frozen immediately in liquid nitrogen light–dark cycle, and at constant temperature for 14 s. Whole brains without cerebella were (22 ± 2 C). Before any treatment, mice were homogenized in 30 volumes of ice-cold homoge- maintained in the colony room for at least 7 days nizing buffer (50 mM Tris–HCl, 3 mM MgCl2, without disturbance. l-Opioid receptor knockout 1 mM EGTA, pH 7.4 at 4 C). The membrane mice used in this study were developed by Loh homogenates were incubated for 20 min at 37 C et al. [27] and maintained on a 1:1 hybrid genetic to remove endogenous ligand and washed twice background (C57/BL6 and 129/Ola) as described. with the same buffer by centrifugation (49,000 Â Food and water were available ad libitum. All g for 20 min at 4 C) and re-suspension. After the procedures for animal handling and experiments last centrifugation, the brain membranes were were approved by Institutional Animal Care Com- suspended in the homogenizing buffer containing mittee of the University of Mississippi Medical 0.32 M sucrose and stored at ) 80 C until use. Center, and performed in compliance with the The protein content was measured using reagent NIH Guide for the Care and Use of Laboratory purchased from Bio-Rad (Richmond, CA). Animals. Saturation analysis of radioligand binding Induction of behavioral sensitization in brain membranes

For induction of behavioral sensitization, each All binding experiments were performed in 50 mM mouse was injected i.p. with either 2.5 mg/kg of Tris–HCl buffer (pH 7.4) in a final volume of METH (sensitized group) or saline (control non- 0.5 ml containing 80–120 lg of membrane protein. sensitized group) once a day for seven consecutive For total binding, aliquots of membrane days. Mice were kept in animal colony room and homogenates were assayed in tubes containing received all injections in their home cages. Our 0.05–8 nM of [3H]-DAMGO, [3H]-DPDPE, or previous study demonstrated that this treatment [3H]-U69,593 for the labeling of l-, d-, or j-opioid 800 receptors, respectively. Non-specific binding was 4 ml of scintillation cocktail. Net [35S]-GTPcS determined in the presence of 10 lM of naloxone coupling values were calculated by subtracting under identical conditions. Tubes were incubated non-specific coupling values. Drug-stimulated for 90 min at 25 C and the reactions were changes in [35S]-GTPcS coupling values were terminated by rapid filtration through Whatman expressed as % of basal coupling values under GF/B filters in a cell harvester. Filters were washed identical conditions. All experiments were per- three times with 3 ml of ice-cold Tris–HCl buffer formed in triplicate using pooled brain membranes (50 mM, pH 7.7) and transferred into vials con- from three mice and the results were obtained taining 4 ml of scintillation cocktail. The radioac- from at least five independent experiments with tivity in the samples was determined by a liquid different groups of animals. scintillation analyzer (Packard Instrument, Meri- den, CT). Specific binding values were calculated Data analysis by subtracting non-specific binding from total binding values. All experiments were performed in The parameters (Kd and Bmax) of radioligand duplicate using pooled brain membranes from binding were obtained from non-linear regression three mice and the results were obtained from four analysis of saturation binding curve, using Graph- independent experiments with different groups of Pad Prism 3.02 (GraphPad Software, San Diego, 35 animals. CA). The EC50 of DAMGO for [ S]-GTPcS coupling was obtained from non-linear regression [35S]-GTPcS coupling in brain membranes analysis of sigmoidal concentration–response curve using the same computer software. The For investigating concentration effect of l-opioid effects of METH on alterations in opioid receptors receptor agonist-stimulated [35S]-GTPcS coupling, during the induction or expression periods of aliquots (15 lg of protein) of brain membrane behavioral sensitization were analyzed by two-way homogenates were assayed in the buffer (50 mM analysis of variance (ANOVA) followed by Bon- Tris–HCl, 3 mM MgCl2, 1 mM DTT, 1 mM ferroni post-tests, using SigmaStat 3.0 (SPSS EGTA, 100 mM NaCl, 0.1% BSA, pH 7.5) con- Science, Rochester, MN). Data are expressed as taining 100 lM GDP, 0.1 nM [35S]-GTPcS, and mean ± standard errors of the mean (S.E.M.). different concentrations (1 nM to 10 lM) of DAMGO in a final volume of 0.5 ml. Constitutive activity of l-opioid receptor was determined by Results adding 1 lM of NAT, a non-selective opioid receptor antagonist, or 1 lMofb-CNA, an Effects of METH on receptor binding during antagonist of l-opioid receptor with negative the induction period of behavioral sensitization intrinsic activity (inverse agonist) [28], into the assay buffer containing 10 lM GDP. With the To investigate whether repeated METH treatment concentration of 1 lM, both agents have been produces alteration in opioid receptors, we first reported to specifically suppress the constitutive determined the changes in l-opioid receptor bind- activity of l-opioid receptor in mice brain homo- ing during the induction period on days 2, 5, and genates [29]. Changes in constitutive activity were 8, as described in methods. Saturation analysis of revealed by changes in the effects of antagonists on radioligand binding revealed that repeated saline 35 [ S]-GTPcS coupling. Basal coupling was deter- treatment did not affect either the Bmax (the mined in the absence of agonist or antagonist and maximum number of binding sites, fmol/mg; day non-specific coupling was determined in the pres- 2, 323 ± 9; day 5, 313 ± 14; day 8, 327 ± 10) ence of 10 lM of unlabeled GTPcS. All prepara- or Kd (binding affinity, nM; day 2, 1.4 ± 0.2; day tions were incubated for 90 min at 25 C and 5, 1.1 ± 0.1; day 8, 1.3 ± 0.1) values of [3H]- reactions were terminated by rapid filtration DAMGO to l-opioid receptor binding. Similar Kd through Whatman GF/B filters in a cell harvester. values were also obtained in the brain from Filters were washed three times with 3 ml of ice- METH-pretreated mice across the days examined cold buffer (50 mM Tris–HCl, 50 mM NaCl, (day 2, 1.2 ± 0.1; day 5, 1.1 ± 0.1; day 8, pH 7.7) and transferred into vials containing 1.4 ± 0.2). However, a two-way ANOVA 801

(METH treatment  days of treatment) revealed 31 and 6.4 ± 0.5 vs. 346 ± 35 and 6.2 ± 0.5; significant effects for Bmax values (METH treat- day 5, 347 ± 33 and 6.0 ± 0.2 vs. 353 ± 38 ment: F[1, 18] = 5.48, p = 0.031; interaction: and 6.7 ± 0.6; day 8, 365 ± 48 and 6.6 ± 0.9 F[2, 18] = 4.23, p = 0.031). Compared with the vs. 338 ± 66 and 6.4 ± 1.1) and [3H]-U69,593 corresponding saline-pretreated control group, the (day 2, 70 ± 6 and 2.1 ± 0.2 vs. 81 ± 5 and 3 Bmax values of [ H]-DAMGO binding in the brain 2.6 ± 0.2; day 5, 65 ± 12 and 1.8 ± 0.3 vs. 87 from METH-pretreated mice were nearly un- ± 19 and 2.5 ± 0.5; day 8, 75 ± 3 and 2.2 ± changed on days 2 (315 ± 18; Figure 1a) and 5 0.2 vs. 72 ± 3 and 1.9 ± 0.1) binding were (316 ± 9; Figure 1b), but decreased significantly detectable in the brain from METH-pretreated with prolonged METH treatment on day 8 mice on any given day tested (Figure 1a–c). (277 ± 9; 85% of control, p < 0.05, Bonferroni post-test; Figure 1c). To further investigate poten- Effects of METH on receptor binding during the tial alterations in other types of opioid receptors, expression period of behavioral sensitization radioligands [3H]-DPDPE and [3H]-U69,593 were used for the labeling of d- and j-opioid receptors, Next, we determined whether alterations in l-opioid respectively. Compared with the corresponding receptor are associated with the expression of saline-pretreated group, no significant changes in METH-induced behavioral sensitization after 3 Bmax or Kd values for [ H]-DPDPE (saline vs. short- and long-term periods of abstinence on METH, Bmax and Kd, respectively: day 2, 372 ± days 11 and 21, respectively. During the expression

Figure 1. Effects of METH on receptor binding in brain membranes during the induction period. Saturation analyses of radioli- gand binding using [3H]-DAMGO, [3H]-DPDPE, or [3H]-U69,593 were performed in the brain from mice 24 h after the first, fourth, and last daily injections of saline or METH on days 2 (a), 5 (b), and 8 (c), respectively. Data are mean values (mean ± S.E.M.) of four independent experiments. 802 periods, no changes in the Bmax and Kd values of 6.7 ± 0.9; day 21, 349 ± 13 and 5.1 ± 0.4 vs. [3H]-DAMGO binding were observed in the brain 363 ± 24 and 4.8 ± 0.2) and [3H]-U69,593 (day from non-sensitized (saline-pretreated) mice (Bmax 11, 76 ± 5 and 2.1 ± 0.3 vs. 85 ± 9 and and Kd, respectively: day 11, 311 ± 7 and 2.2 ± 0.5; day 21, 75 ± 6 and 2.1 ± 0.3 vs. 1.3 ± 0.1; day 21, 317 ± 13 and 1.2 ± 0.2), 83 ± 6 and 2.5 ± 0.2) binding remained un- and similar Kd values were also obtained from changed in sensitized mice at these time points sensitized (METH-pretreated) mice (day 11, tested (Figure 2a, b). These results support the 1.2 ± 0.1; day 21, 1.3 ± 0.2). However, a two- hypothesis that alterations in l-opioid receptor way ANOVA (METH treatment  days of absti- are involved in METH-induced behavioral sensi- nence) revealed significant effects for Bmax values tization and indicate that l-opioid receptors are (METH treatment: F[1, 12] = 12.42, p = 0.004; the most sensitive to repeated METH treatment. days of abstinence: F[1, 12] = 5.52, p = 0.037). Therefore, we only focused on the alterations 3 The Bmax value of [ H]-DAMGO binding in the in l-opioid receptor in the following functional brain from sensitized mice after a short-term studies. period of abstinence was returned, from down- regulated on day 8, to normal level on day 11 Effects of METH on DAMGO-stimulated (334 ± 23; 111% of control, this value did not [35S]-GTPcS coupling during the induction period reach statistical significance; Figure 2a). After a longer period of abstinence on day 21, the Bmax Exchange of GDP for GTP is the first step in G value of [3H]-DAMGO binding in the brain from protein-coupled receptor signaling and l-opioid sensitized mice was up-regulated when compared receptors are negatively coupled to adenylyl with the corresponding non-sensitized control cyclase via Gi/Goa proteins. Thus, to further group (393 ± 10; 124% of control, p < 0.01, investigate whether repeated METH treatment Bonferroni post-test; Figure 2b). In contrast, the produces functional alterations in l-opioid recep- 3 Bmax or Kd values for [ H]-DPDPE (non-sensitized tor, different concentrations of l-opioid receptor vs. sensitized, Bmax and Kd, respectively: day 11, agonist DAMGO, ranging from 1 nM to 10 lM, 398 ± 17 and 6.1 ± 0.9 vs. 388 ± 29 and were used to determine the changes in potency

Figure 2. Effects of METH on receptor binding in brain membranes during the expression period. Saturation analyses of radioli- gand binding using [3H]-DAMGO, [3H]-DPDPE, or [3H]-U69,593 were performed in the brain from saline- or METH-pretreated mice after short-term and long-term periods of abstinence on days 11 (a) and 21 (b), respectively. Data are mean values (mean ± S.E.M.) of four independent experiments. 803

(EC50) and efficacy of the agonist for receptor activation. During the induction period investi- gated, addition of DAMGO into the brain mem- branes from saline-pretreated mice produced similar increase in [35S]-GTPcS coupling by 50% with no significant changes in the EC50 values (nM; day 2, 714 ± 90; day 5, 734 ± 132; day 8, 747 ± 51; Figure 3a–c). The lack of DAMGO effect on brain membranes from l-opioid receptor gene knockout mice confirmed that the increase in [35S]-GTPcS coupling was resulted from l-opioid receptor activation (Figure 4). Repeated METH treatment did not affect the maximal responses (efficacy) induced by DAMGO, whereas the con- centration–response curves for DAMGO shifted to the left on days 2 (Figure 3a) and 5 (Figure 3b), but did not after prolonged METH treatment on day 8 (Figure 3c). A two-way ANOVA (METH treat- ment  days of treatment) revealed significant effects for EC50 values of DAMGO (METH treat- ment: F[1, 30] = 22.70, p < 0.001; days of treat- ment: F[2, 30] = 3.55, p = 0.041). Compared with the corresponding saline-pretreated group, the EC50 values were significantly decreased in the brain from METH-treated mice on days 2 (262 ± 36; p < 0.01, Bonferroni post-test) and 5 (368 ± 84; p < 0.01, Bonferroni post-test), but nearly unchanged on day 8 (641 ± 40).

Effects of METH on DAMGO-stimulated [35S]-GTPcS coupling during the expression period

Next, DAMGO-induced [35S]-GTPcS coupling were performed in brain membranes from mice on days 11 and 21 to investigate whether func- tional alterations in l-opioid receptor are associ- ated with the expression of behavioral sensitization after short-term and long-term peri- ods of abstinence, respectively. During the expres- sion periods, addition of DAMGO into the brain Figure 3. Effects of METH on DAMGO-stimulated membranes from non-sensitized mice produced [35S]-GTPcS coupling in brain membranes during the induc- similar increase in [35S]-GTPcS coupling with no tion period. [35S]-GTPcS coupling concentration–response significant changes in the EC values (day 11, curves for DAMGO were obtained in the brain from mice 50 24 h after the first, fourth, and last daily injections of saline 719 ± 50; day 21, 728 ± 23; Figure 5a, b). or METH on days 2 (a), 5 (b), and 8 (c), respectively. Data Similar maximal responses induced by DAMGO are expressed as % of basal level (mean ± S.E.M.) under were also observed in brain membranes from identical conditions of six independent experiments. sensitized mice; nevertheless, a two-way ANOVA (METH treatment  days of abstinence) of abstinence: F[1, 20] = 10.61, p = 0.004; revealed significant effects for EC50 values (METH interaction: F[1, 20] = 12.13, p = 0.002). Com- treatment: F[1, 20] = 44.62, p < 0.001; days pared with the corresponding non-sensitized 804

35 group, the EC50 value of DAMGO for [ S]- GTPcS coupling was decreased modestly in sensi- tized mice after a short-term period of abstinence on day 11 (597 ± 28; p < 0.05, Bonferroni post- test; Figure 5a). With the concentration–response curve shifting to the left, the EC50 value was decreased further in sensitized mice after a longer period of abstinence on day 21 (341 ± 46; p < 0.01, Bonferroni post-test; Figure5b).

Effects of METH on the constitutive activity of l-opioid receptor during the induction period Figure 4. DAMGO-stimulated [35S]-GTPcS coupling in wild-type and l-opioid receptor knockout mice. [35S]-GTPcS coupling concentration–response curves for DAMGO were Many G protein-coupled receptors, includ- obtained in the brain from wild-type and l-opioid receptor ing l-opioid receptor, exhibit constitutive activity knockout mice without any treatment. Data are expressed as which produces spontaneous regulation of G % of basal level (mean ± S.E.M.) under identical conditions proteins and effectors in the absence of activation of three independent experiments. by agonists [28, 30]. Recent in vitro [28, 30] and in vivo [29] studies found that chronic activation of l-opioid receptor by morphine treatment in- creased such agonist-independent activity. By suppressing the elevated constitutive activity, the property of NAT was found from neutral antag- onist in untreated cells, to turn into inverse agonist (antagonist with negative intrinsic activity) in cells after chronic morphine treatment. Moreover, as an irreversible antagonist, b-CNA was reported to serve as an inverse agonist and specifically sup- press the constitutive activity of l-opioid receptor [28]. To investigate whether repeated METH treat- ment affects the constitutive activity of l-opioid receptor, the effects of these antagonists on basal [35S]-GTPcS coupling were determined. Addition of NAT (1 lM) into brain mem- branes from saline-pretreated mice slightly in- creased [35S]-GTPcS coupling (open bars; Figure 6a). A two-way ANOVA (METH treat- ment  days of treatment) revealed significant effects for NAT-induced [35S]-GTPcS coupling (METH treatment: F[1, 30] = 7.45, p = 0.001). Compared with the corresponding saline-pretreat- ed group, NAT significantly suppressed the basal [35S]-GTPcS coupling in brain membranes from mice received a single METH injection on day 2

35 (p < 0.01, Bonferroni post-test). The NAT-in- Figure 5. Effects of METH on DAMGO-stimulated [ S]- 35 GTPcS coupling in brain membranes during the expression duced decrease in basal [ S]-GTPcS coupling period. [35S]-GTPcS coupling concentration-response curves indicates inverse agonist activity of the antagonist for DAMGO were obtained in the brain from saline- or and suggests the presence of constitutive activity. METH-pretreated mice after short-term and long-term peri- Although NAT also appeared to suppress the ods of abstinence on days 11(a) and 21 (b), respectively. Data 35 are expressed as % of basal level (mean ± S.E.M.) under basal [ S]-GTPcS coupling in METH-pretreated identical conditions of six independent experiments. 805 mice on days 5 and 8, they were not statistically constitutive activity of l-opioid receptor after a significant (closed bars; Figure 6a). single METH treatment. To further identify whether this elevated con- stitutive activity was resulted from l-opioid recep- Effects of METH on the constitutive activity tor, the effects of b-CNA on basal [35S]-GTPcS of l-opioid receptor during the expression period coupling were determined. Addition of b-CNA (1 lM) produced similar agonist-like effects in Next, the effects of 1 lM of NAT and b-CNA on saline-pretreated mice as did by NAT (open bars; basal [35S]-GTPcS coupling were determined to Figure 6b). The lack of inhibitory effect of b-CNA investigate whether alterations in constitutive activ- on basal [35S]-GTPcS coupling indicates that no ity of l-opioid receptor are associated with the constitutive activity of l-opioid receptor was expression of behavioral sensitization. In brain detectable in saline-pretreated mice under membranes from non-sensitized mice on days 11 the experimental conditions used. However, a and 21 (open bars in figures), both agents produced two-way ANOVA (METH treatment  days of similar agonist-like effects on [35S]-GTPcS coupling treatment) revealed significant effects for b-CNA- (Figure 7a, b), indicating that no constitutive induced [35S]-GTPcS coupling (METH treatment: activity was detectable. In contrast, two-way ANO- F[1, 30] = 6.38, p = 0.017). Consistent with the VA (METH treatment  days of abstinence) results produced by NAT, b-CNA significantly revealed significant effects for NAT- (METH treat- suppressed the basal [35S]-GTPcS coupling in ment: F[1, 16] = 18.331, p < 0.001) and b-CNA- METH-pretreated mice on day 2 (p < 0.05, Bon- induced [35S]-GTPcS coupling (METH treatment: ferroni post-test), but not on days 5 and 8 (closed F[1, 16] = 12.15, p = 0.003). Compared with the bars; Figure 6b). The b-CNA-induced decrease in corresponding non-sensitized group, the [35S]- basal [35S]-GTPcS coupling confirms enhanced

Figure 7. Effects of antagonists on basal [35S]-GTPcS cou- Figure 6. Effects of antagonists on basal [35S]-GTPcS cou- pling in brain membranes during the expression period. Ef- pling in brain membranes during the induction period. Effects fects of NAT (a) and b-CNA (b) were determined in the of NAT (a) and b-CNA (b) were determined in the brain brain from saline- or METH-pretreated mice after short-term from mice 24 h after the first, fourth, and last daily injections and long-term periods of abstinence on days 11 and 21, of saline or METH on days 2, 5, and 8, respectively. Data are respectively. Data are expressed as % of basal level (mean ± expressed as % of basal level (mean ± S.E.M.) under identi- S.E.M.) under identical conditions (n = 5). *p < 0.05, cal conditions (n = 6). *p < 0.05, **p < 0.01, compared with **p < 0.01, compared with response of corresponding saline- response of corresponding saline-pretreated group, according pretreated group, according to Bonferroni post-tests after a to Bonferroni post-tests after a two-way ANOVA (METH two-way ANOVA (METH treatment  days of abstinence). treatment  days of treatment). 806

GTPcS coupling in brain membranes from sensi- mediated, at least in part, through dopamine D2 tized mice (closed bars in figures) on day 11 were receptors. Continuous infusion of dopamine D2 but significantly suppressed by NAT (p < 0.01, Bon- not D1 receptor agonist increased the expression ferroni post-test; Figure 7a) or b-CNA (p < 0.05, of l- but not d-opioid receptors [35]. Conversely, Bonferroni post-test; Figure 7b), suggesting the chronic administration of dopamine D2 receptor presence of constitutive activity of l-opioid recep- antagonists decreased l-opioid receptor binding in tor. The [35S]-GTPcS coupling remained to be the brain [36]. Furthermore, AMPH- or METH- suppressed by NAT or b-CNA in sensitized mice induced behavioral sensitization was found to after a longer period of abstinence on day 21 associate with enhanced phosphorylation of cAMP (p < 0.05, Bonferroni post-test). These results indi- response element binding protein (CREB) and cate that elevated constitutive activity of l-opioid expression of transcription factors such as Fos receptor was associated with the expression of and Jun family proteins [37, 38]. These transcrip- METH-induced behavioral sensitization. tion factors are components of the AP-1 complex and the consensus binding sites of this complex are known to be located in the promoter region of Discussion the l-opioid receptor gene [39]. The finding that AP-1 binding activity was enhanced in various As described in previous section, acute adminis- brain regions after repeated METH treatment [37] tration of AMPH or METH increased endogenous supports the idea that METH-induced alteration in opioid contents in the brain and suggested the expression of l-opioid receptor might be resulted activation of l-opioid receptors by these endoge- from the induction of these transcription factors nous opioids. Therefore, it is reasonable to assume following dopamine receptor activation. that repeated or prolonged activation of l-opioid Chronic repeated administration of cocaine receptor by these endogenous opioids may ulti- resulted in up-regulation of l- and j-, but not mately lead to receptor down-regulation, a conse- d-opioid receptors in rats [33]. Moreover, a tran- quence resembles to those of morphine-induced sient elevated expression of d-opioid receptor tolerance and dependence [31]. In agreement with mRNA was found in the VTA after repeated this prediction, we demonstrated that the maximal AMPH treatment [34]. Unlike those findings, no binding of l-opioid receptor was not modified changes in Bmax or Kd values of d-andj-opioid during the early stage of the induction period but receptors were detectable in the present study. The was down-regulated with prolonged treatment of discrepancies may have resulted from differences in the drug. This observation is consistent with subjects, drugs, or experimental conditions. What- previous findings in rats that acute d-AMPH had ever the differences, it must however be noted that no effect on the expression of l-opioid receptor the effects of repeated METH treatment may not mRNA, whereas five intermittent injections of the globally affect the expression of opioid receptors in drug resulted in a significant down-regulation of the brain, the radioligand binding method using the receptor mRNA levels in the brain [32]. whole brain samples therefore may be insufficient to After the drug treatment was stopped, in con- reveal the region-specific changes in expression of trast, the maximal binding of l-opioid receptor was opioid receptors. Although the potential alterations gradually recovered, from down-regulation on day in receptors other than l-opioid receptors cannot 8, returning to normal levels on day 11, and up- be excluded, the present results not only indicate regulation on day 21. Significant up-regulation that l-opioid receptors are the most sensitive to of l-opioid receptor was also found in some brain repeated METH treatment, but also support the regions following chronic intermittent cocaine hypothesis that l-opioid receptors are involved in administration [33]. A study in rats identified that METH-induced behavioral sensitization. the expression of AMPH-induced behavioral sen- Decrease in the EC50 values of agonist for sitization was associated with up-regulation [35S]-GTPcS coupling represents increase in the of l-opioid receptor mRNA in the VTA [34]. potency of the agonist as well as the sensitivity Evidence in literature suggests that the mechanism of receptor to agonist stimulation. Administration leads to up-regulation of l-opioid receptor after of METH is known to cause overexpression of repeated psychostimulant treatment might be neuronal nitric oxide synthase in the mouse brain 807

[40]. As a second messenger, nitric oxide has been affinity of G proteins cannot be excluded. Further suggested to mediate METH-induced neurotoxicity investigations are required to clear this point. and behavioral sensitization [41]. It was found that The observation that inverse agonists decrease infusion with L-arginine for 8 h, a nitric oxide basal G protein coupling in membrane prepara- precursor, did not alter the Bmax or Kd values of tions is the most convincing evidence demonstrat- [3H]-DAMGO binding to the mouse brain mem- ing constitutive activation of l-opioid receptors. branes, whereas, it enhanced the efficacy and With the concentration of 1 lM, b-CNA has been potency of DAMGO for [35S]-GTPcS coupling reported to have negligible non-specific effects in [42]. These observations are similar to the present brain homogenates from l-opioid receptor gene study that they were induced by METH treatment. knockout mice, but significantly suppressed basal Although no significant differences in efficacy were [35S]-GTPcS coupling in those from genetically found, the enhanced potency of DAMGO on days 2 normal mice, indicating the presence of constitu- and 5 were not due to METH-induced increases tive activity of l-opioid receptors in vivo [29]. in l-opioid receptor binding, as the Bmax and Kd However, consistent with other research that failed values for [3H]-DAMGO binding were not signif- to demonstrate constitutive activity [47], b-CNA icantly changed on these days tested. Since nitric failed to demonstrate constitutive activity in oxide is known to regulate gene expression by saline-pretreated mice in the present study. The activating diverse signaling pathways [43], it is discrepancies may be resulted from differences in possible that the enhanced potency of DAMGO in subjects or experimental conditions such incuba- response to METH treatment was resulted from an tion temperature and incubation time used in these up-regulation of Gi/Goa proteins. In addition, studies. Nonetheless, by making comparisons nitric oxide has also been suggested to be involved between saline- and METH-pretreated groups, in opioid desensitization in the CNS [44]. Receptor the basal [35S]-GTPcS coupling was significantly desensitization was implicated as the mechanism suppressed by 1 lMofb-CNA on days 2, 11, and underlying opioid tolerance and dependence, and 21, indicating the elevated constitutive activity repeated administration of L-arginine was found to of l-opioid receptors. Moreover, increase in the mimic tolerance induced by repeated DAMGO constitutive activity of l-opioid receptors has been treatment [45]. As the reduction of total membrane found to increase the inhibitory effects of antag- receptors was reported to be the major mechanism onists such as naloxone and NAT on basal [35S]- for functional desensitization of the l-opioid recep- GTPcS coupling in brain homogenates [29]. Con- tor [46], the decrease in sensitivity of l-opioid sistent with the observation, our results showed receptor with prolonged METH treatment, from that NAT produced agonist-like effects in brain enhanced on day 2 to normal observed on day 8, membranes from saline-pretreated mice, but sig- might be due to down-regulation of the receptor, as nificantly suppressed the basal [35S]-GTPcS cou- the present results indicated. Although not mea- pling in those from METH-pretreated mice on sured, these findings support the hypothesis that days 2, 11, and 21. nitric oxide plays a role in mediating METH- The mechanism by which METH treatment induced functional alterations in l-opioid receptor. affects the constitutive activity of l-opioid recep- After cessation of repeated METH treatment, tor is currently unclear. Since acute administration the enhanced potency of DAMGO for [35S]- of AMPH or METH is known to increase the GTPcS coupling appeared to be parallel to the content of endogenous opioids, the effects of these 3 changes in the Bmax but not Kd values of [ H]- endogenous opioids acting presumably at l-opioid DAMGO binding during the expression period of receptors might bear some resemblance to the sensitization, whereas the changes only evident in effects of morphine. However, enhanced constitu- potency but not efficacy was unexpected. Due to tive activity of l-opioid receptor following mor- the experimental or methodological factors, tiny or phine treatment was found to associate with region-specific changes might not be revealed in reduced agonist-stimulated G protein activation the present study. Although the enhanced sensi- [29]. These results seem to be contradictory to the tivity of l-opioid receptor to agonist stimulation present study that the elevated constitutive may attribute to the up-regulation of the receptor, activities of the receptor were associated with the potential changes in expression or binding enhanced potency of DAMGO for [35S]-GTPcS 808 coupling. It should be mentioned that dopamine receptor contributes to the induction of behavioral receptors were found to exhibit constitutive activ- sensitization. ity as well [48, 49]. As an indirect dopamine The augmentation in dopamine transmission in receptor agonist, METH has profound effects on the NAcc underlies the expression of behavioral these and even other G protein-coupled receptors. sensitization [7], and dopamine D2 receptors play Therefore, in regard to constitutive activity an important role in the expression of sensitized of l-opioid receptor, the consequences after re- behaviors of AMPH or METH [58, 59]. Several peated METH treatment might be distinct from lines of evidence suggest that the locomotor effect those after morphine. A study using photolabeling of l-opioid receptor in the VTA is dependent on of Ga subunits demonstrated that individual G post-synaptic dopamine D2 receptor [60, 61]. In proteins activated by constitutively active l-opioid addition, tonic activation or the constitutive activ- receptors were different from those by agonist ity of l-opioid receptors in the VTA has been DAMGO [30]. Moreover, a study in mutant l- found to play a role in maintenance of basal opioid receptors which expressed elevated constit- dopamine levels in the NAcc [15, 19]. Therefore, utive activity also showed enhanced potency of alterations in the responsiveness of l-opioid recep- 35 DAMGO for [ S]-GTPcS coupling [50]. These tor may have profound effect on dopamine D2 findings may corroborate in part of our observa- receptor-mediated behavior. The elevated constit- tions. Without affecting the maximal binding of utive activity of l-opioid receptor may enhance or the receptor, the elevated constitutive activity at l- maintain the hyperreactivity of mesolimbic dopa- opioid receptors observed on day 2 might also mine neurons and thereby lower the threshold of result from an up-regulation of Gi/Goa proteins locomotor activity triggered by subsequent chal- coupling to the constitutively active receptors. lenge of METH. In fact, it has been suggested that Moreover, owing to a greater chance of encounter an increased expression of l-opioid receptor of a receptor in the active conformation with G mRNA in the VTA predisposes animals to a protein, it has been postulated that a high density sensitized response to psychostimulant challenge of receptors would result in a greater degree of [62]. Moreover, although the locomotor activity constitutive activity [51]. This provides a reason- induced by l-opioid receptor stimulation in the able explanation for our observations on days 11 NAcc is dopamine-independent [63], the expres- and 21 that the elevated constitutive activity at l- sion of l-opioid receptor in the NAcc is sensitive opioid receptors might simply result from the up- to the negative regulatory effect of dopamine D2 regulation of the newly synthesized receptors. receptor on enkephalin in this brain region [35]. The actions of psychostimulants in the VTA Furthermore, sensitization of the locomotor re- are critical for the induction of behavioral sensi- sponse to dopamine D2 receptor agonist induced tization [7]. AMPH and morphine were found to by lesion of the NAcc [5] may associate with an cross-sensitize the locomotor effect of each other’s enhanced behavioral response to locally [52, 53], and l-opioid receptor was found to play applied l-opioid receptor agonist [63]. Taken an important role in morphine-induced behavioral together with these findings, the long-lasting alter- sensitization [54]. Therefore, it is possible that ations in l-opioid receptor observed in the present morphine- and METH-induced behavioral sensi- study may be relevant to METH-induced changes tization share a common mechanism, activation in dopamine-dependent behavior and support the of l-opioid receptor. In fact, it has been identified hypothesis that l-opioid receptor contributes to that repeated activation of l-opioid receptor in the the expression of behavioral sensitization. This can VTA sensitized the mesoaccumbens dopamine be further supported by a parallel study from our neurons to l-opioid receptor stimulation in the laboratory that mice lacking l-opioid receptor VTA [55, 56] and the locomotor response to expressed significantly lower intensity of behav- AMPH [8, 57]. With prolonged METH treatment, ioral sensitization induced by METH than wild- the desensitization of l-opioid receptors observed type mice (Ma et al. unpublished data), even they in the present study not only reflects the conse- have been identified to possess compensatory up- quences of persistent activation of l-opioid recep- regulation of dopamine D2 receptor [64] and tor, but also supports the hypothesis that l-opioid showed enhanced locomotor response to apomor- phine [64] and AMPH [65]. 809

In summary, the present study has examined sensitization of motor activity. Brain Res. Brain Res. the effects of repeated METH treatment and Rev. 16: 223–244, 1991. 8. Vezina P., D1 dopamine receptor activation is necessary for abstinence on l-opioid receptors in the brain of the induction of sensitization by amphetamine in the mice. Differences found between saline- and ventral tegmental area. J. Neurosci. 16: 2411–2420, 1996. METH-pretreated mice demonstrate that l-opioid 9. Zhang Y., Loonam T.M., Noailles P.A. and Angulo J.A., receptors are sensitive to repeated METH treat- Comparison of cocaine- and methamphetamine-evoked dopamine and glutamate overflow in somatodendritic and ment and alterations in l-opioid receptor are time- terminal field regions of the rat brain during acute, chronic, dependently associated with the induction and and early withdrawal conditions. Ann N.Y. Acad. Sci. 937: expression of METH-induced behavioral sensiti- 93–120, 2001. zation. Although the contributions of l-opioid 10. Sesack S.R. and Pickel V.M., Dual ultrastructural locali- zation of enkephalin and tyrosine hydroxylase immunore- receptor to the induction or expression of behav- activity in the rat ventral tegmental area: multiple ioral sensitization induced by METH require substrates for opiate–dopamine interactions. J. Neurosci. further investigation, these findings extend and 12: 1335–1350, 1992. 11. Chiu C.T., Ma T. and Ho I.K., Attenuation of metham- provide possible explanation for our previous phetamine-induced behavioral sensitization in mice by study that these time-dependent alterations systemic administration of naltrexone. Brain Res. Bull. in l-opioid receptor might be accountable for the 67: 100–109, 2005. differential effects of NAT on the induction and 12. Shippenberg T.S., Chefer V.I., Zapata A. and Heidbreder C.A, Modulation of the behavioral and neurochemical expression of behavioral sensitization. effects of psychostimulants by kappa-opioid receptor sys- tems. Ann. N.Y. Acad. Sci. 937: 50–73, 2001. 13. Devine D.P., Leone P., Pocock D. and Wise R.A., Acknowledgements Differential involvement of ventral tegmental mu, delta and kappa opioid receptors in modulation of basal meso- limbic dopamine release: in vivo microdialysis studies. This study was supported by research funds re- J. Pharmacol. Exp. Ther. 266: 1236–1246, 1993. ceived from the Center of Psychiatric Neurosci- 14. Jones D.N. and Holtzman S.G., Interaction between opioid ence at the University of Mississippi Medical antagonists and amphetamine: evidence for mediation by central delta opioid receptors. J. Pharmacol. Exp. Ther. Center which is supported by NIH Grant Num- 262: 638–645, 1992. ber P20 RR0l7701. 15. Schad C.A., Justice J.B. Jr. and Holtzman S.G., Differen- tial effects of delta- and mu-opioid receptor antagonists on the amphetamine-induced increase in extracellular dopa- mine in striatum and nucleus accumbens. J. Neurochem. References 67: 2292–2299, 1996. 16. Spanagel R. and Weiss F., The dopamine hypothesis of 1. Paulson P.E., Camp D.M. and Robinson T.E., Time course reward: past and current status. Trends Neurosci. 22: of transient behavioral depression and persistent behavioral 521–527, 1999. sensitization in relation to regional brain monoamine 17. Bergevin A., Girardot D., Bourque M.J. and Trudeau concentrations during amphetamine withdrawal in rats. L.E., Presynaptic mu-opioid receptors regulate a late step Psychopharmacology (Berlin) 103: 480–492, 1991. of the secretory process in rat ventral tegmental area 2. Robinson T.E. and Becker J.B., Enduring changes in brain GABAergic neurons. Neuropharmacology 42: 1065–1078, and behavior produced by chronic amphetamine adminis- 2002. tration: a review and evaluation of animal models of 18. Johnson S.W. and North R.A., Opioids excite dopa- amphetamine psychosis. Brain Res. 396: 157–198, 1986. mine neurons by hyperpolarization of local interneurons. 3. Itzhak Y. and Ali S.F., Behavioral consequences of J. Neurosci. 12: 483–488, 1992. methamphetamine-induced neurotoxicity in mice: relevance 19. Spanagel R., Herz A. and Shippenberg T.S., Opposing to the psychopathology of methamphetamine addiction. tonically active endogenous opioid systems modulate the Ann. N.Y. Acad. Sci. 965: 127–135, 2002. mesolimbic dopaminergic pathway. Proc. Natl. Acad. Sci. 4. Wise R.A. and Bozarth M.A., A psychomotor stimulant USA 89: 2046–2050, 1992. theory of addiction. Psychol. Rev. 94: 469–492, 1987. 20. Olive M.F., Koenig H.N., Nannini M.A. and Hodge C.W, 5. Clarke P.B., Jakubovic A. and Fibiger H.C., Anatomical Stimulation of endorphin neurotransmission in the nucleus analysis of the involvement of mesolimbocortical dopamine accumbens by ethanol, cocaine, and amphetamine. J. Neu- in the locomotor stimulant actions of d-amphetamine and rosci. 21: RC184, 2001. apomorphine. Psychopharmacology (Berlin) 96: 511–520, 21. Smith A.J. and McGinty J.F., Acute amphetamine or 1988. methamphetamine alters opioid peptide mRNA expression 6. Koob G.F., Drugs of abuse: anatomy, pharmacology and in rat striatum. Brain Res. Mol. Brain Res. 21: 359–362, function of reward pathways. Trends Pharmacol. Sci. 13: 1994. 177–184, 1992. 22. Mansour A., Khachaturian H., Lewis M.E., Akil H. and 7. Kalivas P.W. and Stewart J., Dopamine transmission in the Watson S.J., Anatomy of CNS opioid receptors. Trends initiation and expression of drug- and stress-induced Neurosci. 11: 308–314, 1988. 810

23. Kubota Y., Ito C., Sakurai E., Sakurai E., Watanabe T. 38. Turgeon S.M., Pollack A.E. and Fink J.S., Enhanced and Ohtsu H., Increased methamphetamine-induced loco- CREB phosphorylation and changes in c-Fos and FRA motor activity and behavioral sensitization in histamine- expression in striatum accompany amphetamine sensitiza- deficient mice. J. Neurochem. 83: 837–845, 2002. tion. Brain Res. 749: 120–126, 1997. 24. Badiani A., Leone P., Noel M.B. and Stewart J., Ventral 39. Law P.Y. and Loh H.H., Regulation of opioid receptor tegmental area opioid mechanisms and modulation of activities. J. Pharmacol. Exp. Ther. 289: 607–624, 1999. ingestive behavior. Brain Res. 670: 264–276, 1995. 40. Deng X. and Cadet J.L., Methamphetamine administration 25. Nencini P. and Stewart J., Chronic systemic administration causes overexpression of nNOS in the mouse striatum. of amphetamine increases food intake to morphine, but not Brain Res. 851: 254–257, 1999. to U50-488H, microinjected into the ventral tegmental area 41. Itzhak Y., Martin J.L. and Ali S.F., Comparison between in rats. Brain Res. 527: 254–258, 1990. the role of the neuronal and inducible nitric oxide synthase 26. Yokoo H., Yamada S., Yoshida M., Tanaka T., Mizoguchi in methamphetamine-induced neurotoxicity and sensitiza- K., Emoto H., Koga C., Ishii H., Ishikawa M. and tion. Ann. N.Y. Acad. Sci. 914: 104–111, 2000. Kurasaki N., Effect of opioid peptides on dopamine release 42. Heinzen E.L., Booth R.G. and Pollack G.M., Neuronal from nucleus accumbens after repeated treatment with nitric oxide modulates morphine antinociceptive tolerance methamphetamine. Eur. J. Pharmacol. 256: 335–338, 1994. by enhancing constitutive activity of the mu-opioid recep- 27. Loh H.H., Liu H.C., Cavalli A., Yang W., Chen Y.F. and tor. Biochem. Pharmacol. 69: 679–688, 2005. Wei L.N, mu Opioid receptor knockout in mice: effects on 43. Hemish J., Nakaya N., Mittal V. and Enikolopov G., ligand-induced analgesia and morphine lethality. Brain Nitric oxide activates diverse signaling pathways to regulate Res. Mol. Brain Res. 54: 321–326, 1998. gene expression. J. Biol. Chem. 278: 42321–42329, 2003. 28. Burford N.T., Wang D. and Sadee W., G-protein coupling 44. Torrecilla M., Pineda J. and Ugedo L., NO synthase of mu-opioid receptors (OP3): elevated basal signalling inhibitors reduce opioid desensitization in rat locus coeru- activity. Biochem. J. 348(Pt 3) 531–537, 2000. leus neurons in vitro. Neuroreport 12: 1601–1604, 2001. 29. Wang D., Raehal K.M., Lin E.T., Lowery J.J., Kieffer 45. Aley K.O. and Levine J.D., Dissociation of tolerance and B.L., Bilsky E.J. and Sadee W., Basal signaling activity of dependence for opioid peripheral antinociception in rats. J. mu opioid receptor in mouse brain: role in narcotic Neurosci. 17: 3907–3912, 1997. dependence. J. Pharmacol. Exp. Ther. 308: 512–520, 2004. 46. Pak Y., Kouvelas A., Scheideler M.A., Rasmussen J., 30. Liu J.G., Ruckle M.B. and Prather P.L., Constitutively O’Dowd B.F. and George S.R., Agonist-induced functional active mu-opioid receptors inhibit adenylyl cyclase activity desensitization of the mu-opioid receptor is mediated by in intact cells and activate G-proteins differently than the loss of membrane receptors rather than uncoupling from G agonist [D-Ala2,N-MePhe4,Gly-ol5]enkephalin. J. Biol. protein. Mol. Pharmacol. 50: 1214–1222, 1996. Chem. 276: 37779–37786, 2001. 47. Liu J.G. and Prather P.L., Chronic exposure to mu-opioid 31. Yoburn B.C., Billings B. and Duttaroy A., Opioid receptor agonists produces constitutive activation of mu-opioid regulation in mice. J. Pharmacol. Exp. Ther. 265: 314–320, receptors in direct proportion to the efficacy of the agonist 1993. used for pretreatment. Mol. Pharmacol. 60: 53–62, 2001. 32. Vecchiola A., Collyer P., Figueroa R., Labarca R., Bustos 48. Cai G., Gurdal H., Smith C., Wang H.Y. and Friedman E., G and Magendzo K., Differential regulation of mu-opioid Inverse agonist properties of dopaminergic antagonists at receptor mRNA in the nucleus accumbens shell and core the D(1A) dopamine receptor: uncoupling of the D(1A) accompanying amphetamine behavioral sensitization. dopamine receptor from G(s) protein. Mol. Pharmacol. 56: Brain Res. Mol. Brain Res. 69: 1–9, 1999. 989–996, 1999. 33. Unterwald E.M., Rubenfeld J.M. and Kreek M.J., Repeated 49. Nilsson C.L., Ekman A., Hellstrand M. and Eriksson E., cocaine administration upregulates kappa and mu, but not Inverse agonism at dopamine D2 receptors. Haloperidol- delta, opioid receptors. Neuroreport 5: 1613–1616, 1994. induced prolactin release from GH4C1 cells transfected 34. Magendzo K. and Bustos G., Expression of amphetamine- with the human D2 receptor is antagonized by R( ) )-n- induced behavioral sensitization after short- and long-term propylnorapomorphine, raclopride, and phenoxybenz- withdrawal periods: participation of mu- and delta-opioid amine. Neuropsychopharmacology 15: 53–61, 1996. receptors. Neuropsychopharmacology 28: 468–477, 2003. 50. Brillet K., Kieffer B.L. and Massotte D., Enhanced 35. Chen J.F., Aloyo V.J. and Weiss B., Continuous treatment spontaneous activity of the mu opioid receptor by cysteine with the D2 dopamine receptor agonist quinpirole mutations:characterization of a tool for inverse agonist decreases D2 dopamine receptors, D2 dopamine receptor screening. BMC Pharmacol. 3: 14, 2003. messenger RNA and proenkephalin messenger RNA, and 51. Leff P., The two-state model of receptor activation. Trends increases mu opioid receptors in mouse striatum. Neuro- Pharmacol. Sci. 16: 89–97, 1995. science 54: 669–680, 1993. 52. Vanderschuren L.J., De Vries T.J., Wardeh G., Hogen- 36. Chen J.F., Aloyo V.J., Qin Z.H. and Weiss B., Irreversible boom F.A and Schoffelmeer A.N., A single exposure to blockade of D2 dopamine receptors by fluphenazine-N- morphine induces long-lasting behavioural and neurochem- mustard increases D2 dopamine receptor mRNA and ical sensitization in rats. Eur. J. Neurosci. 14: 1533–1538, proenkephalin mRNA and decreases D1 dopamine recep- 2001. tor mRNA and mu and delta opioid receptors in rat 53. Vezina P. and Stewart J., Amphetamine administered to the striatum. Neurochem. Int. 25: 355–366, 1994. ventral tegmental area but not to the nucleus accumbens 37. Ishihara T., Akiyama K., Kashihara K., Ujike H., Ha- sensitizes rats to systemic morphine: lack of conditioned mamura T., Okada S. and Kuroda S., Activator protein-1 effects. Brain Res. 516: 99–106, 1990. binding activities in discrete regions of rat brain after acute 54. Vigano D., Rubino T., Di Chiara G., Ascari I., Massi P. and chronic administration of methamphetamine. J. Neu- and Parolaro D., Mu opioid receptor signaling in morphine rochem. 67: 708–716, 1996. sensitization. Neuroscience 117: 921–929, 2003. 811

55. Kalivas P.W. and Duffy P., Effect of acute and daily 61. Mori T., Baba J., Ichimaru Y. and Suzuki T., Effects of neurotensin and enkephalin treatments on extracellular rolipram, a selective inhibitor of phosphodiesterase 4, on dopamine in the nucleus accumbens. J. Neurosci. 10: 2940– hyperlocomotion induced by several abused drugs in mice. 2949, 1990. Jpn. J. Pharmacol. 83: 113–118, 2000. 56. Vezina P., Kalivas P.W. and Stewart J., Sensitization 62. Nikulina E.M., Covington H.E. III, Ganschow L., Ham- occurs to the locomotor effects of morphine and the specific mer R.P. Jr. and Miczek K.A., Long-term behavioral and mu opioid receptor agonist, DAGO, administered repeat- neuronal cross-sensitization to amphetamine induced by edly to the ventral tegmental area but not to the nucleus repeated brief social defeat stress: Fos in the ventral accumbens. Brain Res. 417: 51–58, 1987. tegmental area and amygdala. Neuroscience 123: 857–865, 57. Chen J.C., Liang K.W. and Huang E.Y., Differential effects 2004. of endomorphin-1 and )2 on amphetamine sensitization: 63. Churchill L. and Kalivas P.W., Dopamine depletion neurochemical and behavioral aspects. Synapse 39: 239– produces augmented behavioral responses to a mu-, but 248, 2001. not a delta-opioid receptor agonist in the nucleus accum- 58. Levy A.D., Kim J.J. and Ellison G.D., Chronic amphet- bens: lack of a role for receptor upregulation. Synapse 11: amine alters D-2 but not D-1 agonist-induced behavioral 47–57, 1992. responses in rats. Life Sci. 43: 1207–1213, 1988. 64. Tien L.T., Park Y., Fan L.W., Ma T., Loh H.H. and Ho 59. Ujike H., Akiyama K. and Otsuki S., D-2 but not D-1 I.K, Increased dopamine D2 receptor binding and dopamine agonists produce augmented behavioral response enhanced apomorphine-induced locomotor activity in in rats after subchronic treatment with methamphetamine mu-opioid receptor knockout mice. Brain Res. Bull. 61: or cocaine. Psychopharmacology (Berlin) 102: 459–464, 109–115, 2003. 1990. 65. Kas M.J., van den Bos R., Baars A.M., Lubbers M., 60. Cook C.D. and Beardsley P.M., The modulatory actions of Lesscher H.M., Hillebrand J.J., Schuller A.G., Pintar J.E. dopamine D2/3 agonists and antagonists on the locomotor- and Spruijt B.M., Mu-opioid receptor knockout mice show activating effects of morphine and caffeine in mice. Phar- diminished food-anticipatory activity. Eur. J. Neurosci. 20: macol. Biochem. Behav. 75: 363–371, 2003. 1624–1632, 2004.