Rewarding Properties of Methylphenidate: Sensitization by Prior Exposure to the Drug and Effects of Dopamine D1- and D2-Receptor Antagonists

Rewarding Properties of Methylphenidate: Sensitization by Prior Exposure to the Drug and Effects of Dopamine D1- and D2-Receptor Antagonists

National Public Health Institute, Department of Mental Health and Alcohol Research, Drug Research Unit, Helsinki, Finland

Esa Meririnne, M.D., Drug Research Unit, Department of Mental Health and Alcohol Research, National Public Health Institute, Mannerheimintie 166, FIN-00300 Helsinki, Finland. E-mail: esa.meririnne{at}ktl.fi

Abstract

In drug addiction, a sensitization phenomenon has been postulated to play a critical role. The aim of our study was to evaluate whether sensitization occurs to the rewarding properties of methylphenidate, a psychostimulant drug known to possess abuse potential, as assessed with the biased conditioned place preference method in rats. In addition, since the brain dopaminergic system is considered to be important in drug-reward, the involvement of dopamine D1- and D2-receptors both in the rewarding properties of methylphenidate and in sensitization to these properties was assessed. Conditioning with methylphenidate at doses of 1.25 to 20 mg/kg increased preference for the paired environment, whereas a dose of 0.31 mg/kg was ineffective. However, following the 7-day sensitization treatment with methylphenidate (0.62–20 mg/kg), conditioning with a dose of 0.31 mg/kg resulted in an increased preference for the paired environment, i.e., the rewarding properties of methylphenidate appeared to be sensitized. Control experiments indicated that the enhancement of preference was not due to attenuation of sensitization treatment-induced withdrawal nor to tolerance to aversive properties of methylphenidate. When conditioned with methylphenidate, D1-antagonist SCH 23390 but not D2-antagonist raclopride prevented place preference. However, when coadministered with methylphenidate during the sensitization treatment, both SCH 23390 and raclopride prevented the development of sensitization. These data indicate that the rewarding properties of methylphenidate are sensitized by prior exposure to the drug and that both D1- and D2-receptors, the latter of which possibly more specifically, appear to be involved in the development of this sensitization.

Methylphenidate (MP) is a psychostimulant drug widely used for the treatment of attention deficit hyperactivity disorder and narcolepsy. In controlled use for the treatment of attention deficit hyperactivity disorder among children and adolescents, the abuse of MP is considered to be rare, but its illicit use has been recognized by several reports indicating that MP possesses abuse potential (Rappley, 1997).

In the brain, MP increases the extracellular levels of dopamine and norepinephrine in a manner similar to cocaine and amphetamine. The mechanism of action is thought to include dopamine reuptake inhibition, yet other mechanisms may also be involved (Nomikos et al., 1990). However, unlike the two other psychostimulants, MP does not affect the serotonergic system (Koe, 1976; Segal and Kuczenski, 1999), and hence it has been used as a tool in animal experiments for characterizing dopamine-behavior relationship without a serotonin effect (e.g., Segal and Kuczenski, 1999). MP's behavioral profile in rats, including induction of locomotor activity and stereotypes, resembles those of cocaine and amphetamine, and in behavioral tests assessing abuse potential MP has been shown to possess rewarding properties (Martin-Iverson et al., 1985; Mithani et al., 1986).

The repeated use of a psychostimulant may result in an augmented behavioral response to the subsequent administration of the drug, frequently referred to as sensitization. Determination of whether sensitization develops to the rewarding properties of drugs is critical in view of the postulated role of sensitization in the development of drug-craving and compulsive drug-seeking behavior (Robinson and Berridge, 1993). The repeated administration of cocaine and amphetamine has been shown to sensitize their rewarding properties (Lett, 1989;Shippenberg and Heidbreder, 1995; Le Pen et al., 1998; Pierre and Vezina, 1998), as well as locomotor activity (Kuribara and Uchihashi, 1993; Mattingly et al., 1994). Regarding MP, however, whether sensitization occurs to its rewarding properties has not been elucidated. When sensitization to its locomotor-stimulating effects is assessed, somewhat inconsistent results have been obtained. In some studies, locomotor activity has been reported to be sensitized (Shuster et al., 1982; Gaytan et al., 1997; McDougall et al., 1999), whereas in other studies no such effect was found (McNamara et al., 1993;Izenwasser et al., 1999). In fact, some authors have suggested that this lack of sensitization could be attributed to the inability of MP to affect the serotonergic system (Izenwasser et al., 1999).

The brain dopaminergic system is considered to be involved in mediating the rewarding properties of psychostimulants. In addition to the rewarding properties, there is evidence to suggest the involvement of at least D1-receptors in the development of sensitization to these properties. For example, the facilitation of amphetamine self-administration by prior treatment has been prevented by the D1-antagonist SCH 23390 (SCH; Pierre and Vezina, 1998). Furthermore, SCH prevented the development of sensitization to cocaine-induced place preference, whereas the D2-antagonist raclopride (RAC) was ineffective (Shippenberg and Heidbreder, 1995). Regardless of the latter finding, the conceivable involvement of D2-receptors in development of sensitization to rewarding properties of other psychostimulants should not be ignored, since D2-antagonists have prevented development of sensitization to the locomotor-stimulating effects of at least amphetamine and methamphetamine (Kuribara and Uchihashi, 1993; Meng et al., 1998). With regard to MP in particular, the role of dopaminergic mechanisms both in its rewarding properties and in the development of sensitization to these properties is unclear. Although the dopaminergic system is apparently involved in several behavioral responses to MP, various dopaminergic manipulations have failed to affect its rewarding properties. For example, MP can induce place preference in rats with 6-hydroxydopamine-induced dopaminergic lesions, as well as in dopamine-transporter knockout mice (Martin-Iverson et al., 1985; Sora et al., 1998). Concerning dopamine receptors, only the involvement of D2-receptors has been assessed; however, the D2-antagonist haloperidol did not affect MP-induced place preference, except at a dose of 1 mg/kg, which was considered to be nonspecifically high (Martin-Iverson et al., 1985; Mithani et al., 1986).

In the present study, the aims were to evaluate whether sensitization occurs to the rewarding properties of MP as assessed in the conditioned place preference method and to evaluate the roles of D1- and D2-receptors, using the respective antagonists SCH and RAC, both in the rewarding properties of MP and in the development of sensitization to these properties.

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Materials and Methods

Animals

Adult male Han:Wistar rats weighing 200 to 300 g were used in the study. The rats were obtained from Harlan Nederland B.V. (Horst, The Netherlands), at least 1 week before the experiments, and they were housed two per Macrolon III-type cage (18 × 33 × 15 cm) in a temperature-controlled room (22 ± 2°C) with a light cycle of 12 h. The lights were on from 8:00 AM to 8:00 PM, during which time all experiments were conducted. The animals had free access to standard laboratory chow and tap water, except in the conditioned taste aversion test (see Conditioned Taste Aversion). The animal experiments were approved by the local institutional animals care and use committee and the chief veterinarian of the county administrative board, and they were conducted according to the “European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes”.

Drugs

MP hydrochloride (donated by Novartis Pharma AG, Basel, Switzerland), SCH hydrochloride (D 054, Sigma/RBI, Natick, MA), RAC l-tartrate (R 121, Sigma/RBI), yohimbine hydrochloride (Y 100, Sigma/RBI), and lithium hydrochloride (L 0505, Sigma, St. Louis, MO) were dissolved in saline (SAL; 0.9% NaCl). All the drugs were administered intraperitoneally at a volume of 1 ml/kg, except that RAC was given subcutaneously and in the conditioned taste aversion test MP, lithium, and SAL were given at a volume of 8 ml/kg. Drug doses were calculated as free base.

Conditioned Place Preference

Apparatus.

The conditioned place preference test was conducted in eight identical rectangular boxes (60 × 30 × 45 cm) made of polyvinylchloride. Each box was divided into two compartments of equal size by a separating wall with a guillotine door (8 × 6 cm). Both compartments were covered with loose-fitting transparent plastic lids. One compartment was black with a smooth floor and small drops of 10% acetic acid added in both back corners, and the other was white with wire mesh on the floor and no acetic acid added. Thus, the compartments differed in three modalities: visual, tactile, and olfactory. The boxes were placed in a quiet dim room with white noise present for masking external sounds.

The Rewarding Properties of MP as Assessed by the Conditioned Place Preference Method.

The dose-response profile of MP in the place preference test was first assessed, and the highest dose that was clearly ineffective was chosen as a reference dose for the sensitization experiment. The biased place preference procedure used consisted of three phases:

1.: Preconditioning Phase (Days 1–3): The guillotine door of the box was open, and equal numbers of rats were placed in the black or white compartment and allowed to explore both compartments freely for 15 min (900 s). On the 3rd day, the times spent in both compartments were measured with a stopwatch. According to this preconditioning time, the rats were assigned to treatment groups with the less preferred compartment serving as the drug-paired compartment. If the preconditioning time for a rat was less than 200 s in either compartment, the rat was excluded from further testing. Further testing was discontinued in approximately 15% of the rats, following this criterion. In each treatment group, approximately half of the rats were assigned to the black compartment as the drug-paired compartment, while the other half were assigned to the white compartment as the drug-paired compartment (±1 rat when n ≤ 10; ± 2 rats when n > 10).
2.: Conditioning Phase (Days 4–6): During the conditioning phase, the rats experienced two conditioning sessions each day. In the first session, the rats received a SAL injection and were immediately confined to the SAL-paired compartment opposite the drug-paired compartment for 40 min. After an interval of at least 90 min, the second session of the day was begun; the rats received MP at different doses (0.31, 0.62, 1.25, 5, or 20 mg/kg) or SAL and were immediately confined to the drug-paired compartment for 40 min. The procedure of two conditioning sessions per day with this order of conditioning has been used previously to obtain results comparable with those obtained with more standard procedures (Calcagnetti and Schechter, 1992; Haile et al., 2001). During this phase the guillotine door was closed.
3.: Postconditioning Phase (Day 7): The guillotine door was opened, the rats placed in the drug-paired compartment, and the times they spent in the compartments measured for 15 min. The shifts in preference for the drug-paired compartment induced by conditioning (postconditioning time − preconditioning time) served as a measure of reward.

Sensitization to the Rewarding Properties of MP.

In the sensitization experiment the reference dose, which was the highest ineffective dose in the place preference test, was tested after a 7-day sensitization treatment with daily injections of MP at different doses to determine whether it would now induce place preference, i.e., whether prior exposure would sensitize the rewarding properties of MP. During the sensitization treatment, the rats received MP (0.31, 0.62, 1.25, 5, or 20 mg/kg) or SAL in their home cages once daily for 7 consecutive days. On the final day at least 3 h before the last sensitization injection, the place preference procedure as described above was commenced; the rats were allowed to freely explore the compartments of the box for 15 min. On 2 subsequent days (days 2–3 of the place preference test), the preconditioning phase was continued with no injections, and on the last day the preconditioning time of the rats was measured. During the conditioning phase (days 4–6), the rats received SAL in the SAL-paired compartment and MP (the reference dose of 0.31 mg/kg, chosen on the basis of the results in the MP-induced place preference experiments) or SAL in the drug-paired compartment. Thus, the interval between the sensitization and conditioning phases was 3 days, as there is evidence that, at least with cocaine injected once daily for 5 days, this may yield the greatest sensitization (Shippenberg and Heidbreder, 1995). Finally, on the last day (day 7) the postconditioning times were measured for 15 min.

Effects of D1- and D2-Receptor Blockade on the Rewarding Properties of MP.

The experiments were conducted as described above with a modified injection schedule. During the conditioning phase, in the first session of the day, the rats were given two injections of SAL at an appropriate interval, and after the second injection they were immediately confined to the SAL-paired compartment. In the second session, the rats were injected first with either SCH (0.05, 0.1, or 0.2 mg/kg), RAC (0.2, 0.4, or 0.8 mg/kg), or SAL. After an appropriate interval, the rats were given a second injection of MP (5 mg/kg) or SAL, followed immediately by confinement in the drug-paired compartment. The intervals were 40 min for the SCH-treated groups and 10 min for the RAC-treated groups. According to previous studies, the doses of SCH and RAC with the intervals used should be behaviorally relevant (Ågmo et al., 1993; Mattingly et al., 1994;Hoffman and Donovan, 1995; Millan et al., 1998; Pierre and Vezina, 1998). Particularly, the possible affinity of SCH for serotonin-2-receptors appears to be insignificant at these doses (Bischoff et al., 1986). The dose of MP was chosen, based on results in the present and previous studies (Martin-Iverson et al., 1985; Mithani et al., 1986). When the effect of SCH and RAC alone was studied, the antagonists were assigned to the preferred compartment as the drug-paired compartment, since there is evidence that SCH may possess some aversive properties (Tzschentke, 1998). Furthermore, in separate experiments the effects of two higher doses of SCH (0.1 and 0.2 mg/kg) and RAC (0.4 and 0.8 mg/kg) alone were also associated with the less preferred compartment.

Effects of D1- and D2-Receptor Blockade on Sensitization to the Rewarding Properties of MP.

During the sensitization treatment, the rats were injected with SCH (0.1 or 0.2 mg/kg), RAC (0.2 or 0.4 mg/kg), or SAL. After an interval of 40 min for the SCH-treated groups and 10 min for the RAC-treated groups, MP (5 mg/kg) or SAL was administered. Otherwise, the experiments were conducted as described under Sensitization to the Rewarding Properties of MP.

Assessment of Withdrawal Symptoms

It is possible that place preference induced by MP following the 7-day sensitization treatment would reflect the alleviation of sensitization treatment-induced withdrawal symptoms rather than a true sensitization phenomenon. Therefore, to assess the magnitude of withdrawal symptoms, the rats were first injected with MP (5 or 20 mg/kg) or SAL once per day for 7 consecutive days, similar to the 7-day sensitization treatment in the place preference experiments, after which weight change (Planeta et al., 1994), food consumption (Kräuchi et al., 1984), anxiety-like behavior (Costall et al., 1989), and changes in motor activity (Pulvirenti and Koob, 1993) were measured.

Changes in Weight and Food Consumption.

Weight change and food consumption were measured daily for 3 days after the last injection. Daily weight change was calculated as the difference from the weight measured on the preceding day. Food consumption was assessed as the amount of food consumed per two rats, since the rats were housed in pairs, both of which always received the same treatment.

Anxiety-Like Behavior.

On the 3rd day after the last injection (corresponding to the beginning of the conditioning phase in the place preference experiments), anxiety-like behavior was measured by using the black-white box test modified from Timothy et al. (1999). The boxes used in the black-white box test were similar to those used in the place preference experiments, except that neither wire mesh was placed on the floor nor was acetic acid added. Instead, the black compartment was illuminated with a 20-W red bulb and the white compartment with a 10-W white bulb covered with a thin sheet of paper; both bulbs were placed approximately 25 cm above the box with a dark cardboard sheet between them, preventing light from illuminating the opposite compartment. Thus, the compartments differed only in the magnitude of luminousness. Each rat was tested once by placing it gently in the middle of the black compartment, facing away from the doorway. The following parameters were measured with a stopwatch for 8 min (480 s): time to enter the white compartment for the first time, total time spent in the white compartment, activity both in the black and white compartments (number of crosses over midline), and number of crosses between the black and white compartments. Prior to conducting the experiments with withdrawal-experiencing rats, the validity of the black-white box test used here was tested in a separate experiment by administering the known anxiogenic drug yohimbine (2.5 mg/kg) or SAL to rats 30 min before placing in the test box.

Motor Activity.

After the black-white box test, the motor activity of the rat was measured for 40 min in a test cage (transparent Macrolon III-type cage) by means of a computer-controlled photobeam activity system (San Diego Instruments, San Diego, CA). Both ambulatory and rearing activities were recorded as interruptions of photobeams at heights of 5 and 12.5 cm from the cage floor, respectively.

Conditioned Taste Aversion

Instead of sensitization to the rewarding properties, it is also possible that after the sensitization treatment MP would induce place preference due to sensitization treatment-induced tolerance to the aversive properties of the reference dose (0.31 mg/kg) used in the place preference experiments. That is, without the sensitization treatment, conditioning with MP at the reference dose would not induce place preference because the rewarding and aversive properties have canceled each other out. Instead, following the sensitization treatment, the reference dose would induce place preference due to the development of tolerance to its aversive properties. Therefore, the aversive properties of MP at this dose were assessed in a conditioned taste aversion test (Nachman and Ashe, 1973). If the reference dose per se did not possess aversive properties, enhancement of place preference would probably not result from tolerance to, i.e., attenuation of, the aversive properties of the dose.

The rats were housed in pairs with laboratory chow freely available. During the experiment, daily drinking was restricted to two sessions: 1) Habituation/pairing/test session (15 min, from 8:15 AM to 8:30 AM): the rats were placed singly in separate Macrolon III-type test cages with one drinking bottle per cage, filled with 0.2% saccharin solution or water, except during the postconditioning phase (see below). Following this session, injections were given as appropriate, and fluid consumption was measured by weighing bottles before and after the session with the difference transformed into milliliters; 2) Free-drinking session (from 12:00 PM to 6:00 PM): the rats were allowed to drink water freely in their home cages with no injections or fluid measurements. Prior to beginning the experiment, the bottles were removed from the home cages at 6:00 PM the previous evening. During the preconditioning phase (days 1–3) in the habituation sessions, the bottle was filled with water. During this phase, no injections were given. The rats were assigned to treatment groups according to water consumption in the habituation session of the 3rd day. On the subsequent day, the 1st day of the conditioning phase (days 4–6) was begun; in the pairing session the bottle was filled with saccharin solution, and after the session the rat was injected with MP (0.31 mg/kg, the reference dose used in the place preference experiments), a known aversive drug lithium (8.2 mg/kg corresponding to approximately 51 mg/kg or 0.15 M of lithium chloride; Nachman and Ashe, 1973), or SAL. This saccharin-drug pairing was repeated for the next 2 days. During the following postconditioning phase (day 7) in the test session, two bottles, 1 filled with water and the other with saccharin solution, were placed in the test cages, and fluid consumption in both bottles was separately measured. The saccharin preference ratio in the test session was calculated with the following equation: saccharin solution consumption/(saccharin solution consumption + water consumption) and was taken as a measure of taste aversion.

Statistical Analysis

In the place preference experiments, the shift in preference for the drug-paired compartment induced by conditioning served as a measure of reward. Statistical analysis was performed using one-way ANCOVA (dependent factor: shift in preference; independent factor: drug doses; covariant: preconditioning time). In the assessment of withdrawal symptoms, weight change, food consumption, and motor activities were analyzed using ANOVA for repeated measures (dependent factor: weight change, food consumption, or motor activity counts; independent factor: drug doses; repeated measure: time), while the parameters of anxiety-like behavior in the black-white box test were assessed with one-way ANOVA (dependent factor: time to enter the white compartment for the first time, total time in the white compartment, line crossings in the white compartment, line crossings in the black compartment, or crossings between the compartments; independent factor: drug doses). In the conditioned taste aversion test, the saccharin preference ratio in the test session was taken as a measure of taste aversion, which was analyzed using the two-sample t test. Elsewhere, paired and two-sample t tests were used as appropriate. Post hoc comparisons were made using Bonferroni's test adjusted for appropriate number of comparisons. All data are expressed as mean ± S.E. unless otherwise stated.

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Results

The Rewarding Properties of MP as Assessed by the Conditioned Place Preference Method

As shown in Fig. 1, MP increased preference for the drug-paired compartment dose dependently. There was a statistically significant difference between the conditioning doses [F(5,43) = 7.292, p < 0.001, one-way ANCOVA (p = 0.093 for covariant)]. In the post hoc comparison, the doses of 1.25, 5, and 20 mg/kg differed from the SAL group. A dose of 0.62 mg/kg showed some tendency to increase preference, but a dose of 0.31 mg/kg was clearly ineffective. Therefore, this dose was chosen as the reference dose for the sensitization experiment.

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Figure 1

The rewarding properties of MP as assessed by the conditioned place preference method. The data shown represent time (mean ± S.E.) spent in the drug-paired compartment during the 15-min measurements before and after the conditioning phase (preconditioning and postconditioning times, respectively). During the conditioning phase, the effects of MP or SAL (referred to as conditioning drug) were associated with the drug-paired compartment in daily 40-min sessions for 3 consecutive days. Numbers within the Preconditioning time columns represent the number of rats in the group. **p < 0.01 and ***p < 0.001 when compared with the rats conditioned with SAL (Bonferroni's test).

Sensitization to the Rewarding Properties of MP

As evident in Fig. 2, after the 7-day sensitization treatment with SAL, conditioning with MP at a reference dose of 0.31 mg/kg caused no shift in preference. Instead, following the sensitization treatment with MP at different doses, conditioning with the reference dose resulted in a significant increase in preference for the drug-paired compartment, i.e., prior exposure to the drug appeared to sensitize the rewarding properties of MP. There was a statistically significant difference between the groups given SAL or MP at different doses during the sensitization treatment [F(5,65) = 4.360, p = 0.002, one-way ANCOVA (p = 0.03 for covariant)]. In the post hoc comparison, the doses of 0.62, 1.25, 5, and 20 mg/kg differed from the SAL group. When a separate group of rats was sensitized with MP at a dose of 20 mg/kg and conditioned with SAL, no shift in preference was observed in comparison with either the pre- and postconditioning times [t(5) = 0.045, p = 0.966, pairedt test], or to the group conditioned with SAL without sensitization (Fig. 2) [F(1,12) = 0.456,p = 0.512, one-way ANCOVA (p = 0.205 for covariant)].

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Figure 2

Sensitization to the rewarding properties of MP. The data shown represent time (mean ± S.E.) spent in the drug-paired compartment during the 15-min measurements before and after the conditioning phase (preconditioning and postconditioning times, respectively). Before the place preference test, during the 7-day sensitization treatment the rats received in their home cages daily one injection of MP or SAL (referred to as sensitization drug). After the interval of 3 days, during the conditioning phase, effects of MP at a dose of 0.31 mg/kg or SAL (referred to as conditioning drug) were associated with the drug-paired compartment in daily 40-min sessions for 3 consecutive days. Numbers within the Preconditioning time columns represent the number of rats in the group. *p < 0.05 and **p < 0.01 when compared with the rats sensitized with SAL and conditioned with MP at the dose of 0.31 mg/kg (Bonferroni's test).

In addition to increased preference, separate analysis revealed that after the sensitization treatment at a dose of 5 mg/kg, conditioning with the reference dose is able to induce a true place preference for the drug-paired compartment, i.e., during the postconditioning phase the rats spent more time in the drug- than in the SAL-paired compartment (Fig. 3). First, because in our biased method some rats showed a substantial aversion to the drug-paired compartment during the preconditioning phase, these rats may have spent less time in the drug- than in the saline-paired compartment during the postconditioning phase, despite a significantly increased shift in preference for the drug-paired compartment. Therefore, according to the preconditioning time (average of 375 s) the rats that showed only a mild initial aversion to the drug-paired compartment (preconditioning time > 375 s) were included in the analysis. Second, to gain statistical power, all rats receiving sensitization treatment with MP at a dose of 5 mg/kg alone or with SAL were pooled (see Materials and Methods). As shown in Fig. 3, after sensitization treatment with MP at a dose of 5 mg/kg and conditioning at the reference dose, the rats (preconditioning time = 414 ± 3 s) spent significantly more time in the drug- than in the SAL-paired compartment [t(20) = 4.724,p < 0.001, paired t test]. In contrast, after sensitization treatment with SAL and conditioning at the reference dose, the rats (preconditioning time = 407 ± 11 s) spent more time in the SAL- than in the drug-paired compartment [t(5) = −2.817, p < 0.037, paired t test]. Thus, after sensitization treatment with MP at a dose of 5 mg/kg, conditioning at the reference dose was able to induce a true place preference, at least in rats that had only a mild initial aversion to the drug-paired compartment.

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Figure 3

MP-induced place preference after sensitization treatment in rats with mild initial aversion to the drug-paired compartment (preconditioning time > 375 s). The data shown represent time (mean ± S.E.) spent in the drug- and SAL-paired compartments during the 15-min measurement after the conditioning phase (postconditioning time). During the 7-day sensitization treatment before the place preference test, the rats daily received one injection of MP at a dose of 5 mg/kg or SAL (referred to as the sensitization drug) in their home cages. During the conditioning phase after a 3-day interval, the effect of MP at a dose of 0.31 mg/kg (referred to as the conditioning drug) was associated with the drug-paired compartment in daily 40-min sessions for 3 consecutive days. Numbers within the Time in the drug-paired compartment columns represent the number of rats in the group. *p < 0.05 and ***p< 0.001 when compared between the postconditioning times spent in the drug- and SAL-paired compartments (paired t test).

Effects of D1- and D2-Receptor Blockade on the Rewarding Properties of MP

The effects of SCH and RAC on MP-induced increase in preference for the drug-paired compartment are shown in Fig.4. MP-induced increase in preference was attenuated by SCH (Fig. 4A): there was a statistically significant difference between the groups conditioned with SAL or SCH at different doses in combination with MP (5 mg/kg) [F(3,40) = 4.751, p = 0.006, one-way ANCOVA (p = 0.386 for covariant)]. In the post hoc comparison, the dose of 0.2 mg/kg differed from the SAL group. Unlike SCH, RAC failed to affect MP-induced increase in preference (Fig. 4B) [F(3,28) = 0.467, p = 0.708, one-way ANCOVA (p = 0.002 for covariant)]. When conditioned alone in the preferred compartment, neither SCH nor RAC was able to induce shift in preference when tested with one-way ANCOVA (Fig. 4, C and D) [SCH-conditioned:F(3,19) = 0.119, p = 0.948 (p = 0.917 for covariant); RAC-conditioned:F(3,19) = 0.28, p = 0.839 (p = 0.01 for covariant)]. When conditioned alone in the less preferred compartment, SCH did not induce shift in preference (Fig. 4E) [F(2,17) = 1.364, p = 0.282, one-way ANCOVA (p = 0.376 for covariant)]. Instead, RAC increased preference for the drug-paired compartment (Fig. 4F) [F(2,24) = 3.469, p = 0.048, one-way ANCOVA (p = 0.186 for covariant)]. In the post hoc comparison, the dose of 0.8 mg/kg appeared to differ from the SAL group (p = 0.05, Bonferroni's test).

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Figure 4

Effects of dopamine D1-antagonist SCH (A) and D2-antagonist RAC (B) on the rewarding properties of MP; effects of SCH (C) and RAC (D) alone when associated with the preferred compartment; effects of SCH (E) and RAC (F) alone when associated with the less preferred compartment. The data shown represent time (mean ± S.E.) spent in the drug-paired compartment during the 15-min measurements before and after the conditioning phase (preconditioning and postconditioning times, respectively). During the conditioning phase, the effects of SCH, RAC, and SAL in combination with MP, or SCH and RAC alone [referred to as conditioning drug(s)] were associated with the drug-paired compartment in daily 40-min sessions for 3 consecutive days. Numbers within the Preconditioning time columns represent the number of rats in the group. **p < 0.01 and *p = 0.05 when compared with the rats conditioned with SAL alone, or SAL and MP at a dose of 5 mg/kg (Bonferroni's test).

Effects of D1- and D2-Receptor Blockade on Sensitization to the Rewarding Properties of MP

The effects of SCH and RAC on the development of sensitization to the rewarding properties of MP are shown in Fig.5. SCH prevented sensitization to the rewarding properties of MP (Fig. 5A): after the 7-day sensitization treatment with SAL combined with MP (5 mg/kg), conditioning with MP at a reference dose of 0.31 mg/kg increased preference for the drug-paired compartment, whereas after the sensitization treatment with SCH at different doses combined with MP (5 mg/kg), conditioning with the reference dose failed to do so. There was a statistically significant difference between the groups given SAL or SCH at different doses in combination with MP during the sensitization treatment [F(2,33) = 3.875, p = 0.031, one-way ANCOVA (p = 0.774 for covariant)]. In the post hoc comparison, the dose of 0.2 mg/kg differed from the SAL group. Similarly, RAC prevented sensitization to the rewarding properties of MP (Fig. 5B). There was a statistically significant difference between the groups given SAL or RAC at different doses in combination with MP during the sensitization treatment [F(2,29) = 7.189,p = 0.003, one-way ANCOVA (p = 0.046 for covariant)]. In the post hoc comparison, the dose of 0.4 mg/kg differed from the SAL group.

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Figure 5

Effects of dopamine D1-antagonist SCH (A) and D2-antagonists RAC (B) on sensitization to the rewarding properties of MP; effects of SCH (C) and RAC (D) alone. The data shown represent time (mean ± S.E.) spent in the drug-paired compartment during the 15-min measurements before and after the conditioning phase (preconditioning and postconditioning times, respectively). Before the place preference test, during the 7-day sensitization treatment the rats received daily injections of SCH, RAC, and SAL in combination with MP, or SCH and RAC alone [referred to as sensitization drug(s)]. After the 3-day interval, during the conditioning phase, effects of MP at a dose of 0.31 mg/kg or SAL (referred as conditioning drug) were associated with the drug-paired compartment in daily 40-min sessions for 3 consecutive days. Numbers within the Preconditioning time columns represent the number of rats in the group. *p < 0.05 and **p < 0.01 when compared with the rats sensitized with SAL and MP at a dose of 5 mg/kg (Bonferroni's test).

After sensitization with SCH at a dose of 0.2 mg/kg or RAC at a dose of 0.4 mg/kg alone, conditioning with SAL induced no shift in preference (Fig. 5, C and D) in comparison between the pre- and postconditioning times tested with the paired t test [SCH-sensitized:t(7) = 0.528, p = 0.614; RAC-sensitized: t(7) = 0.442, p = 0.672], or in comparison with the group conditioned with SAL without sensitization and tested with the one-way ANCOVA [SCH-sensitized:F(1,16) = 0.033, p = 0.857 (p = 0.155 for covariant); RAC-sensitized:F(1,16) = 0.096, p = 0.76 (p = 0.421 for covariant)]. Similarly, after sensitization with SCH or RAC alone, conditioning with MP at a dose of 0.31 mg/kg induced no shift in preference (Fig. 5, C and D) in comparison between the pre- and postconditioning times tested with the paired t test [SCH-sensitized: t(5) = −0.106, p = 0.92; RAC-sensitized: t(5) = 0.705, p = 0.513], or in comparison with the group sensitized with SAL and conditioned with MP at the dose of 0.31 mg/kg tested with the one-way ANCOVA [SCH-sensitized:F(1,15) = 0.062, p = 0.807 (p = 0.151 for covariant); RAC-sensitized:F(1,15) = 0.025, p = 0.877 (p = 0.492 for covariant)].

Withdrawal Symptoms

The results of the experiments assessing withdrawal symptoms following the 7-day treatment with MP at doses of 5 and 20 mg/kg are summarized in Fig. 6.

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Figure 6

The withdrawal symptoms weight change (A) (n = 8), food consumption (B) (n = 4), anxiety-like behavior (C) (n = 8), and changes in ambulatory activity (D) (n = 8) induced by 7-day treatment with daily injections of MP [5 mg/kg (●), 20 mg/kg (▴)], or SAL (■). Daily weight change and mean food consumption of two rats in the cage were measured for 3 consecutive days after the last injection. Anxiety-like behavior in the black-white box test and motor activity in an automated activity cage were assessed on the 3rd day after the last injection. All the data are represented as mean ± S.E. *p < 0.05 when compared with the SAL treatment (Bonferroni's test).