Laura N. Cedillo, Florencio Miranda. Introduction

Transcription

1 Pharmacological Reports 2013, 65, ISSN Copyright 2013 by Institute of Pharmacology Polish Academy of Sciences Effects of co-administration of the GABA B receptor agonist baclofen and a positive allosteric modulator of the GABA B receptor, CGP7930, on the development and expression of amphetamineinduced locomotor sensitization in rats Laura N. Cedillo, Florencio Miranda FES Iztacala, National Autonomous University of México, Av. de los Barrios 1, Los Reyes Iztacala Tlalnepantla, Edo. de México 54090, México Correspondence: Florencio Miranda, fmirandah@yahoo.com Abstract: Background: Several of the behavioral effects of amphetamine (AMPH) are mediated by an increase in dopamine neurotransmission in the nucleus accumbens. However, evidence shows that -aminobutyric acid B (GABA B ) receptors are involved in the behavioral effects of psychostimulants, including AMPH. Here, we examined the effects of co-administration of the GABA B receptor agonist baclofen and a positive allosteric modulator of the GABA B receptor, CGP7930, on AMPH-induced locomotor sensitization. Methods: In a series of experiments, we examined whether baclofen (2.0, 3.0 and 4.0 mg/kg), CGP7930 (5.0, 10.0 and 20.0 mg/kg), or co-administration of CGP7930 (5.0, 10.0 and 20.0 mg/kg) with a lower dose of baclofen (2.0 mg/kg) could prevent the development and expression of locomotor sensitization produced by AMPH (1.0 mg/kg). Results: The results showed that baclofen treatment prevented both the development and expression of AMPH-induced locomotor sensitization in a dose-dependent manner. Furthermore, the positive allosteric modulator of the GABA B receptor, CGP7930, increased the effects of a lower dose of baclofen on AMPH-induced locomotor sensitization under both conditions. Conclusion: These data provide further evidence that GABA B receptor ligands may modulate psychostimulant-induced behaviors. Key words: amphetamine, sensitization, GABA B receptors Introduction Cocaine and amphetamine (AMPH) are indirect monoamine agonists that exhibit an affinity for dopamine (DA), norepinephrine (NE), and serotonin (5-HT) transporters, which are involved in neurotransmitter reuptake and vesicular storage systems [52]. Cocaine inhibits the reuptake of DA, NE, and 5-HT, thereby increasing the synaptic levels of these neurotransmitters. AMPH blocks the uptake of DA, NE, and 5-HT into synaptic vesicles, promoting an increase in the cytoplasmic concentrations of these monoamines. As the levels of cytoplasmic monoamines increase, they exit neurons via reversal of the direction of plasma membrane transporters, which leads to an increase in synaptic DA, NE, and 5-HT levels [1, 28, 52]. The mesolimbic DAergic system, particularly the projection from the ventral tegmental area (VTA) to the nucleus accumbens (NAcc), is an important locus for the production of the locomotor, reinforcement, reward, and discriminative stimulus effects of psy Pharmacological Reports, 2013, 65,

2 Effects of BCF and CGP on AMPH-induced locomotor sensitization Laura N. Cedillo and Florencio Miranda chostimulants [17, 20, 35]. The administration of AMPH or cocaine rapidly increases DAergic neurotransmission by interfering with the function of DA transporters, as described above. Consequently, psychostimulant administration produces an increase in DAergic signaling in the limbic areas [35, 36]. Recent evidence also suggests a potential role for -aminobutyric acid (GABA) neurotransmission in modulating some of the behavioral effects of psychostimulants. GABA B receptor agonists are reportedly effective in attenuating some of the behavioral effects of psychostimulants that may be related to the abuse of these drugs. For example, the selective GABA B receptor agonist baclofen (BCF) reduces the reinforcing effects of cocaine [49], nicotine [19], methamph [48], and AMPH [9]. Furthermore, BCF administration decreases the conditioned locomotion elicited by cues associated with cocaine [25]. Other GABA B receptor agonists also reduce psychostimulant-related behaviors. Brebner et al. [10] reported that the selective GABA B receptor agonist CPG44532 was effective in attenuating cocaine self-administration in rats subjected to a progressive ratio schedule. Although some findings have indicated that GABA B agonists, such as BCF, may be useful in the treatment of drug abuse, other results have suggested that the muscle-relaxant properties of BCF, in conjunction with its sedative and hypothermic effects, limit its widespread application as a therapeutic agent in humans and as a tool for behavioral research [16, 26]. However, a novel alternative approach, allosteric modulation of GABA B receptors, has been suggested. Positive allosteric modulators of GABA B receptors display no intrinsic activity of their own but can interact synergistically with GABA B agonists, including BCF, to enhance their effects. One strategy for attempting to overcome the side effects of BCF is to use lower dosages to reduce unwanted effects of the drug, in combination with positive allosteric modulators of GABA B receptors, such as CGP7930 (CGP). Locomotor sensitization is the progressive and persistent enhancement of a behavioral response to a drug after repeated and intermittent administration. This phenomenon has been well characterized for psychostimulant and cannabinoid drugs [32, 60, 63] and is thought to reflect neuroadaptations that contribute to drug addiction and model some aspects of addictive behaviors, such as drug craving [51]. Therefore, locomotor sensitization may reflect neurobiological changes related to drug addiction and may be useful for studying DA-GABA interactions. The present study was designed to examine the effects of coadministration of the GABA B receptor agonist BCF and CGP, a positive allosteric modulator of the GABA B receptor, on the development and expression of AMPH-induced locomotor sensitization. Materials and Methods Animals A total of 400 male Wistar rats that were 120 days old and weighed g at the beginning of the experiments were obtained from the breeding colony of the FES-Iztacala-UNAM, México. They were individually housed in stainless steel cages with freely available food (Teklad LM485 Rat Diet by Harlan, México City, México) and water and were maintained under a 12 h light/dark cycle, with the lights turned on at 08:00 h. The room was maintained at a temperature of 21 ± 1 C. All experiments were conducted during the light phase (between 11:00 a.m. and 1:00 p.m.). Animal care and handling procedures were performed in accordance with the Official Mexican Norm (NOM- 062-ZOO-1999) entitled Technical Specifications for the Production, Care, and Use of Laboratory Animals and all procedures were approved by the local bioethics committee. Drugs The drugs used in this study were D-amphetamine sulfate, (±)-baclofen (Sigma-Aldrich, St. Louis, MO, USA), and CGP7930 (Tocris, Ballwin, MO, USA). (±)-Baclofen and D-AMPH were dissolved in water, and CGP7930 was dissolved in 2 drops (20 µl/drop) of ethanol and then in 1% Tween 80. All drugs were prepared fresh daily and were administered via intraperitoneal injection (1 ml/kg). Apparatus Locomotor activity was measured with an open-field activity monitoring system (ENV-515 model; Med Associates, St. Albans, VT. USA). Each Plexiglas cage ( cm) was equipped with 2 sets of 8 photobeams that were placed 2.5 cm above the surface of the floor on opposite walls to record x y am- Pharmacological Reports, 2013, 65,

3 bulatory movements. Photobeam interruptions were recorded and translated by software to yield the horizontal distance traveled (in cm), which was the dependent measure used for analysis. Experimental procedure The timeline of the general procedure is shown in Figure 1. Each day or session started with a 10 min period of habituation to the cages, followed by administration of drug(s) or vehicle (VEH). The rats were returned to the cages, and their locomotor activity was recorded for 1 h. On day 1, the rats were habituated to the open-field cages and injection procedures (habituation session). On day 2, locomotor activity after VEH administration (locomotor activity baseline) was evaluated. On days 3 7, the rats received pharmacological compounds and/or AMPH (for development of AMPH-induced locomotor sensitization; see Tab. 1). On days 8 9, the rats were left undisturbed (resting days). In the experiments examining the development of AMPH sensitization, the rats were injected with VEH and AMPH on days 10 and 11, respectively (test days), while in the experiments examining the expression of AMPH sensitization, the rats were injected with VEH on day 10 and with pharmacological compounds and/or AMPH on day 11 (see Tab. 1). In all experiments, the rats were injected with CGP and BCF 1 min before AMPH injection and immediately returned to the cages, where their locomotor activity was recorded for 1 h. Acute effects of BCF and CGP on locomotor activity The locomotor activity in groups of animals (n = 10 rats per group) was assessed once in response to each different dose of BCF (0.0, 2.0, 3.0, and 4.0 mg/kg) and CGP (0.0, 5.0, 10.0, and 20.0 mg/kg). DAYS H BL Development of sensitization Resting days Test Days V A Fig. 1. Schematic diagram illustrating the timeline of AMPH (1 mg/ kg)-induced locomotor sensitization. H habituation, BL baseline locomotor activity, V vehicle, A amphetamine Experiment 1: Effects of BCF and CGP coadministration on the development of AMPHinduced locomotor sensitization (see Tab. 1 for the schedule of drug treatment for Experiment 1 and Experiment 2) a) Effects of BCF on the development of AMPHinduced locomotor sensitization. On the days where the development of sensitization was examined, groups of rats (n = 10 rats per group) received one of the following treatments: VEH + VEH (group 2V), VEH + AMPH (1.0 mg/kg; group VA), BCF (2.0 mg/ kg) + AMPH (1.0 mg/kg; group B2A), BCF (3.0 mg/ kg) + AMPH (1.0 mg/kg; group B3A), or BCF (4.0 mg/kg) + AMPH (1.0 mg/kg; group B4A). b) Effects of CGP on the development of AMPHinduced locomotor sensitization. On the days where the development of sensitization was examined, groups of rats (n = 10 rats per group) received one of the following treatments: VEH + VEH (group 2V), VEH + AMPH (1.0 mg/kg; group VA), CGP (5.0 mg/ kg) + AMPH (1.0 mg/kg; group C5A), CGP (10.0 mg/ kg) + AMPH (1.0 mg/kg; group C10A), or CGP (20.0 mg/kg) + AMPH (1.0 mg/kg; group C20A). c) Effects of the co-administration of CGP and BCF on the development of AMPH-induced locomotor sensitization. On the days where the development of sensitization was examined, groups of rats (n = 10 rats per group) received one of the following treatments: VEH + VEH + VEH (group 3V), VEH + VEH + AMPH (1.0 mg/kg; group 2VA), VEH + BCF (2.0 mg/kg) + AMPH (1.0 mg/kg; group VB2A), CGP (5.0 mg/kg) + BCF (2.0 mg/kg) + AMPH (1.0 mg/kg; group C5B2A), CGP (10.0 mg/kg) + BCF (2.0 mg/kg) + AMPH (1.0 mg/kg; group C10B2A), or CGP (20.0 mg/kg) + BCF (2.0 mg/kg) + AMPH (1.0 mg/ kg; group C20B2A). Experiment 2: Effects of BCF and CGP coadministration on the expression of AMPHinduced locomotor sensitization a) Effects of BCF on the expression of AMPHinduced locomotor sensitization. On the days where the development of sensitization was examined (3 7), one group (n = 10 rats) received an injection of VEH + VEH, and the other groups of rats (n = 10 rats per group) received an injection of VEH + AMPH (1.0 mg/kg) (see Tab. 1 for details). On day 10, all groups were injected with VEH. On day 11, each group of rats received one of the following treat Pharmacological Reports, 2013, 65,

4 Effects of BCF and CGP on AMPH-induced locomotor sensitization Laura N. Cedillo and Florencio Miranda ments: VEH + AMPH (group V-VA), VEH + AMPH (1.0 mg/kg; group A-VA), BCF (2.0 mg/kg) + AMPH (1.0 mg/kg; group B2A), BCF (3.0 mg/kg) + AMPH (1.0 mg/kg; group B3A), or BCF (4.0 mg/kg) + AMPH (1.0 mg/ kg; group B4A). b) Effects of CGP on the expression of AMPHinduced locomotor sensitization. On the days where the development of sensitization was examined, one group (n = 10 rats) received an injection of VEH + VEH, and the other groups of rats (n = 10 rats per group) received Tab. 1. Experimental design of development and expression of AMPH-induced locomotor sensitization Name of groups Repeated treatment Challenge Days 3 to 7 Day 10 Day 11 Development of sensitization 2V VEH + VEH VEH AMPH(1.0) VA VEH + AMPH(1.0) VEH AMPH(1.0) B2A BCF(2.0) + AMPH(1.0) VEH AMPH(1.0) B3A BCF(3.0) + AMPH(1.0) VEH AMPH(1.0) B4A BCF(4.0) + AMPH(1.0) VEH AMPH(1.0) 2V VEH + VEH VEH AMPH(1.0) VA VEH + AMPH(1.0) VEH AMPH(1.0) C5A CGP(5.0) + AMPH(1.0) VEH AMPH(1.0) C10A CGP(10.0) + AMPH(1.0) VEH AMPH(1.0) C20A CGP(20.0) + AMPH(1.0) VEH AMPH(1.0) 3V VEH + VEH + VEH VEH AMPH(1.0) 2VA VEH + VEH + AMPH(1.0) VEH AMPH(1.0) VB2A VEH + BCF(2.0) + AMPH(1.0) VEH AMPH(1.0) C5B2A CGP(5.0) + BCF(2.0) + AMPH(1.0) VEH AMPH(1.0) C10B2A CGP(10.0) + BCF(2.0) + AMPH(1.0) VEH AMPH(1.0) C20B2A CGP(20.0) + BCF(2.0) + AMPH(1.0) VEH AMPH(1.0) Expression of sensitization V-VA VEH + VEH VEH VEH + AMPH(1.0) A-VA VEH + AMPH(1.0) VEH VEH + AMPH(1.0) B2A VEH + AMPH(1.0) VEH BCF(2.0) + AMPH(1.0) B3A VEH + AMPH(1.0) VEH BCF(3.0) + AMPH(1.0) B4A VEH + AMPH(1.0) VEH BCF(4.0) + AMPH(1.0) V-VA VEH + VEH VEH VEH + AMPH(1.0) A-VA VEH + AMPH(1.0) VEH VEH + AMPH(1.0) C5A VEH + AMPH(1.0) VEH CGP(5.0) + AMPH(1.0) C10A VEH + AMPH(1.0) VEH CGP(10.0) + AMPH(1.0) C20A VEH + AMPH(1.0) VEH CGP(20.0) + AMPH(1.0) 2VA VEH + VEH + VEH VEH VEH + VEH + AMPH(1.0) A-2VA VEH + VEH + AMPH(1.0) VEH VEH + VEH + AMPH(1.0) VB2A VEH + VEH + AMPH(1.0) VEH VEH + BCF(2.0) + AMPH(1.0) C5B2A VEH + VEH + AMPH(1.0) VEH CGP(5.0) +BCF(2.0) + AMPH(1.0) C10B2A VEH + VEH + AMPH(1.0) VEH CGP10.0) + BCF(2.0) + AMPH(1.0) C20B2A VEH + VEH + AMPH(1.0) VEH CGP(20.0) + BCF(2.0) + AMPH(1.0) VEH: appropiate vehicle; AMPH: amphetamine; in parentheses doses: mg/kg; ip Pharmacological Reports, 2013, 65,

5 an injection of VEH + AMPH (1.0 mg/kg). On day 10, all groups were injected with VEH. On day 11, each group of rats received one of the following treatments: VEH + AMPH (group V-VA), VEH + AMPH (1.0 mg/kg; group A-VA), CGP (5.0 mg/kg) + AMPH (1.0 mg/kg; group C5A), CGP (10.0 mg/kg) + AMPH (1.0 mg/kg; group C10A), or CGP (20.0 mg/kg) + AMPH (1.0 mg/kg; group C20A). c) Effects of CGP and BCF co-administration on the expression of AMPH-induced locomotor sensitization. On the days where the development of sensitization was examined, one group (n = 10 rats) received an injection of VEH + VEH + VEH, and the other groups of rats (n = 10 rats per group) received an injection of VEH + VEH + AMPH (1.0 mg/kg). On day 10, all groups were injected with VEH. On day 11, each group of rats received one of the following treatments: VEH + VEH + AMPH (group 2VA), VEH + VEH + AMPH (1.0 mg/kg; group A-2VA), VEH + BCF (2.0 mg/kg) + AMPH (1.0 mg/kg; group VB2A), CGP (5.0 mg/kg) + BCF (2.0 mg/kg) + AMPH (1.0 mg/kg; group C5B2A), CGP (10.0 mg/kg) + BCF (2.0 mg/kg) + AMPH (1.0 mg/kg; group C10B2A), or CGP (20.0 mg/kg) + BCF (2.0 mg/kg) + AMPH (1.0 mg/kg; group C20B2A). Data analysis The results of the investigated measure (distance traveled, in cm) are expressed as the mean ± SEM. The data obtained during the development of locomotor sensitization were analyzed via two-way ANOVA for repeated measures, with the group as the first factor and the day as the second factor. The data obtained during the baseline and testing days were analyzed through one-way ANOVA. When the ANOVA results were significant, Tukey s test (p < 0.05) was used to perform a posteriori comparisons. Results Acute effects of BCF and CGP on locomotor activity The results of this experiment revealed that neither BCF [F (3, 39) = 0.896, p > 0.05] nor CGP [F (3, 39) = 0.892, p > 0.05] altered the locomotor activity of the rats. EXPERIMENT 1 a) Effects of BCF on the development of AMPHinduced locomotor sensitization. The data obtained from the baseline locomotor activity measurements were similar in all groups [F (4, 49) = 0.781, p > 0.05]. Repeated administration of AMPH resulted in the development of sensitization to locomotor activity. However, administration of BCF at doses of 3.0 and 4.0 mg/kg, but not at 2.0 mg/kg, attenuated the locomotor activity produced by AMPH treatment. Two-way ANOVA for repeated measures indicated significant effects of the group [F (4, 45) = , p < 0.05], day [F (4, 180) = 8.483, p < 0.05], and the group day interaction [F (16, 180) = 3.999, p < 0.05]. The results of the VEH test are shown in Figure 2A. Animals that were treated with either AMPH alone or AMPH in combination with BCF showed conditioned locomotion. Here, one-way ANOVA indicated a significant group effect [F (4, 49) = 3.58, p < 0.05], and Tukey s test revealed that all other groups were significantly different from the 2V group. The results of the administration of AMPH on day 11 are shown in Figure 2B. When BCF was administered for 5 days in combination with AMPH injection, it was observed that BCF reduced the AMPH-induced locomotor activity in a dose-dependent manner [F (4, 49) = 2.921, p < 0.05]. Tukey s test revealed that the VA group was different from the 2V group and that the B3A and B4A groups were different from the VA group. b) Effects of CGP on the development of AMPHinduced locomotor sensitization. The baseline locomotor activity was similar in all groups [F (4, 49) = 0.264, p > 0.05]. Repeated administration of AMPH induced the development of sensitization to locomotor activity. The AMPH-induced locomotor sensitization was not altered after the administration of CGP (a positive allosteric modulator of GABA B receptors) at different doses. Two-way ANOVA for repeated measures indicated significant effects of the group [F (4, 45) = , p < 0.05], day [F (4, 180) = 6.721, p < 0.05], and the group day interaction [F (16, 180) = 1.306, p < 0.05]. The results of the VEH test are shown in Figure 2C. Animals treated with either AMPH alone or AMPH in combination with CGP showed conditioned locomotion. One-way ANOVA indicated a significant group effect [F (4, 49) = 3.79, p < 0.05], and Tukey s test revealed that all other groups were different from the 2V group. The results of the administration of 1136 Pharmacological Reports, 2013, 65,

6 Effects of BCF and CGP on AMPH-induced locomotor sensitization Laura N. Cedillo and Florencio Miranda Fig. 2. Results for challenge days after the development of AMPH-induced locomotor sensitization. The bars represent the mean ± SEM from 10 rats. A, C and E represent the vehicle test. B, D and F represent the AMPH test. * p < 0.05, different from the 2V or 3V group based on oneway ANOVA followed by Tukey s post-hoc test. + p < 0.05, different from the VA or 2VA group based on one-way ANOVA followed by Tukey s post-hoc test. The name of the groups refers only the treatment received during the development of AMPH-induced locomotor sensitization (see Tab. 1 for further details) AMPH on day 11 are shown in Figure 2D. When CGP was administered for 5 days in combination with AMPH injection, it was found that CGP did not reduce the AMPH-induced locomotor activity [F (4, 49) = 5.313, p < 0.05]. Tukey s test revealed that the VA group was different from the 2V group. c) Effects of CGP and BCF co-administration on the development of AMPH-induced locomotor sensitization. The baseline locomotor activity was similar in all groups [F (5, 59) = 1.30, p > 0.05]. Repeated administration of AMPH resulted in the development of sensitization to locomotor activity. AMPH-induced locomotor sensitization was affected by the administration of the combination of CGP at different dosages and a lower dose of BCF. Two-way ANOVA for repeated measures indicated significant effects of the group [F (5, 54) = 26.98, p < 0.05], day [F (4, 216) = , p < 0.05], and the group day interaction [F (20, 216) = 3.74, p < 0.05], and Tukey s test revealed that the C20B2A group was different than the 2VA group. The results of the VEH test are shown in Figure 2E. Animals treated with either AMPH alone or AMPH in combination with CGP and BCF exhibited conditioned locomotion. One-way ANOVA indi- Pharmacological Reports, 2013, 65,

7 cated significant differences between groups [F (4, 49) = 3.37, p < 0.05]. Tukey s test revealed that all groups were different from the 3V group. The results of administration of AMPH on day 11 are shown in Figure 2F. After 5 days of AMPH administration in combination with CGP and BCF, it was observed that CGP increased the effects of BCF on AMPH-induced locomotor activity [F (5, 59) = 4.332, p < 0.05]. Tukey s test revealed that locomotor activity differed between the 2VA group and the 3V group and that the C20B2A group was different from the 2VA group. EXPERIMENT 2 a) Effects of BCF on the expression of AMPHinduced locomotor sensitization. The baseline locomotor activity on day 2 was similar in all groups [F (4, 49) = 1.370, p > 0.05]. Repeated administration of AMPH led to the development of sensitization to locomotor activity in all groups, except the V-VA group. Two-way ANOVA for repeated measures indicated significant effects of the group [F (4, 45) = , p < 0.05], day [F (4, 180) = 8.54, p < 0.05], and the group day interaction [F (16, 180) = 1.80, p < 0.05], and Tukey s test revealed that the V-VA group was different from all other groups. The results of the VEH test on day 10 are shown in Figure 3A. Animals treated with AMPH exhibited conditioned locomotion. One-way ANOVA indicated the existence of significant differences between groups [F (4, 49) = 5.16, p < 0.05], and Tukey s test revealed that all other groups were different from the V-VA group. The results of the administration of either AMPH or different doses of BCF and AMPH on day 11 are shown in Figure 3B. Administration of BCF led to a dose-dependent reduction in the expression of AMPH-induced locomotor activity [F (4, 49) = 5.05 p < 0.05]. Tukey s test revealed that the A-VA group was different than the V-VA group and that the B3A and B4A groups were different than the A-VA group. b) Effects of CGP on the expression of AMPHinduced locomotor sensitization. The baseline locomotor activity on day 2 was similar in all groups [F (4, 49) = 1.378, p > 0.05], and repeated administration of AMPH resulted in the development of sensitization to locomotor activity in all groups, except the V-VA group. Two-way ANOVA for repeated measures indicated a significant effect of the group [F (4, 45) = 27.74, p < 0.05] and day [F (4, 180) = 9.27, p < 0.05], but the group day interaction was not significant [F (16, 180) = 1.32, p > 0.05]. Tukey s test revealed that the V-VA group was different from all other groups. The results of the VEH test on day 10 are shown in Figure 3C. Animals treated with AMPH presented conditioned locomotion. One-way ANOVA indicated significant differences between groups [F (4, 49) = 3.84, p < 0.05], and Tukey s test revealed that all groups were different from the V-VA group. The results obtained following the administration of AMPH or different doses of CGP and AMPH on day 11 (see Fig. 3D) showed that CGP does not affect the expression of AMPH-induced locomotor activity [F (4, 49) = 3.74, p < 0.05], and Tukey s test revealed that the A-VA group was different from the V-VA group. c) Effects of CGP and BCF co-administration on the expression of AMPH-induced locomotor sensitization. The baseline locomotor activity on day 2 was similar in all groups [F (5, 59) = 1.48, p > 0.05]. Repeated administration of AMPH led to the development of sensitization to locomotor activity in all groups, except the 2VA group. Two-way ANOVA for repeated measures indicated significant effects of the group [F (5, 54) = , p < 0.05] and day [F (5, 216) = 10.24, p < 0.05], but the group day interaction was not significant [F (14, 216) = 1.22, p > 0.05]. Tukey s test revealed that the 2VA group was different from all other groups. The results of the VEH test on day 10 are shown in Figure 3E. Animals treated with AMPH displayed conditioned locomotion. One-way ANOVA indicated significant differences between groups [F (4, 49) = 5.54, p < 0.05], and Tukey s test revealed that all other groups were different from the 2VA group. The results of the administration of different doses of CGP, BCF2.0, and AMPH on day 11 are shown in Figure 3F. Administration of CGP increased the effects of BCF on the expression of AMPH-induced locomotor activity [F (5, 59) = 2.21, p < 0.05]. Tukey s test revealed that the A-2VA group was different from the 2VA group and that the C20B2A group was different from the A-2VA group. Discussion The purpose of the present study was to examine the effects of the co-administration of the GABA B receptor agonist BCF with CGP, a positive allosteric modu Pharmacological Reports, 2013, 65,

8 Effects of BCF and CGP on AMPH-induced locomotor sensitization Laura N. Cedillo and Florencio Miranda Fig. 3. Results for challenge days during the expression of AMPH-induced locomotor sensitization. Bars represent the mean ± SEM from 10 rats. A, C and E represent the vehicle test. B, D and F represent the results obtained during treatment with pharmacological compounds and/or following AMPH injection. * p < 0.05, different from the V-VA or 2VA group based on one-way ANOVA followed by Tukey s post- hoc test. + p < 0.05, different from the A-VA or A-2VA group based on one-way ANOVA followed by Tukey s post-hoc test. The name of the groups refers only the treatment received during the expression of AMPH-induced locomotor sensitization (see Tab. 1 for further details) lator of GABA B receptors, on AMPH-induced locomotor sensitization. We found that AMPH increased locomotor activity and that BCF treatment resulted in prevention of both the development and the expression of AMPH-induced locomotor sensitization in a dose-dependent manner, whereas CGP had no effect on either the development or the expression of AMPH-induced locomotor activity. We also observed that administration of the positive allosteric modulator CGP increased the effects of a lower dose of BCF on AMPH-induced locomotor sensitization under both conditions. Additionally, an increase in locomotor activity was detected during the VEH test (day 10), which is interpreted as conditioned locomotion [58] and could mask the main effects of the drugs tested on day 11. However, although this possibility cannot be completely ruled out, we found that BCF and/or CGP reduced, rather than increased, AMPH- Pharmacological Reports, 2013, 65,

9 induced locomotor activity in a dose-dependent manner, in terms of both the development and expression of AMPH-induced locomotor sensitization. The behavioral results described above are consistent with those of previous studies demonstrating that the GABA B receptor agonist BCF attenuates AMPHinduced locomotor sensitization. For example, it has been reported previously that BCF prevents both the development [3] and expression [4] of sensitization to the locomotor effects of AMPH. BCF also attenuates the sensitization to the locomotor stimulant effects of cocaine [23], morphine [5, 24], and ethanol [13]. Furthermore, BCF attenuates psychostimulantrelated behaviors associated with drug addiction. BCF pre-treatment reduces cocaine self-administration in rats responding under fixed ratio schedules [11], progressive ratio schedules [2, 50], discrete trial schedules of reinforcement [10], and second-order schedules [18]. Additionally, BCF decreases AMPH selfadministration under a fixed ratio or progressive ratio schedule [9] and attenuates conditioned locomotion in response to cues associated with cocaine administration [25]. Moreover, BCF attenuates the behavioral effects of ethanol [15], nicotine [44], and heroin [18], and we found in a previous study that BCF reduces the discriminative stimulus properties of AMPH [41]. The mechanism underlying the observed effects of BCF on AMPH-induced locomotor sensitization may involve GABAergic modulation of DAergic transmission within the VTA. Several lines of evidence support this notion. First, the mesolimbic DA system, particularly the projection from the VTA to the NAcc, is an important locus in the production of the locomotor, reinforcement, and reward effects of psychostimulants such as cocaine and AMPH [17, 31, 35], and this system plays an important role in both the development and expression of psychostimulantinduced locomotor sensitization [46]. Second, the VTA contains primary DAergic neurons, which release DA in the NAcc and prefrontal cortex, and secondary GABAergic interneurons, which reduce the firing rate of DAergic neurons in the VTA. In addition, GABAergic neurons arising from the NAcc project to DAergic neurons within the VTA [29, 33]. This loop represents an important locus in the production of certain abuse-related behavioral effects of psychostimulants [35]. Third, anatomical evidence suggests that GABA B receptors are located within the VTA [8, 27, 29, 42]. Fourth, biochemical and behavioral studies have found that infusion of BCF into the VTA decreases DA release in the NAcc [59, 62], which suggests that the activation of GABA B receptors located on the cell bodies of mesolimbic DAergic neurons is involved in the biochemical effects of BCF. Furthermore, microinjection of BCF into the VTA reduces cocaine self-administration under fixed ratio [53] and progressive ratio schedules [12], and microinjection of BCF was also found to reduce heroin self-administration [61] and AMPH-induced motor activity [30]. Altogether, these data support the hypothesis that the activation of GABA B receptors on the cell bodies of DAergic neurons in the VTA plays an important role in the suppressive effects of BCF on both the development and the expression of AMPHinduced locomotor sensitization. In this study, it was also observed that a positive allosteric modulator of GABA B receptors, CGP, increased the effects of a lower dose of BCF on AMPH-induced locomotor sensitization. It is worth noting that CGP did not alter AMPH-induced locomotor sensitization at any of the tested dosages. These results are consistent with those of at least one previous study, in which the combination of a lower dose of BCF with a dose of CGP reduced ethanol selfadministration [39]. Furthermore, it has been reported that CGP injections lead to a reduction of ethanol intake in ethanol-preferring rats [39, 43], of cocaine self-administration in rats responding under several schedules of reinforcement [22, 54], of cocaineseeking behavior [21], of nicotine-induced locomotor stimulation in mice [40], and of nicotine self-administration [45]. In addition, CGP treatment was shown to produce approximately 41% BCF-appropriate responses but also to enhance the discriminative stimulus effects of BCF in pigeons trained to discriminate BCF from saline [34]. The above findings together with the present data provide behavioral evidence that BCF and related compounds, such as CGP, may represent potential pharmacological treatments for drug addiction. Positive allosteric modulators of GABA B receptors, such as CGP, display no intrinsic activity of their own but can modulate the activity of GABA or GABA B agonists, such as BCF, while avoiding the possible adverse side effects of BCF administration. In contrast to the data cited above, recent studies have shown that the administration of GS39783 alone, which is another positive modulator of GABA B receptors, significantly attenuates the locomotor activity induced by a single cocaine treatment and reduces modest co Pharmacological Reports, 2013, 65,

10 Effects of BCF and CGP on AMPH-induced locomotor sensitization Laura N. Cedillo and Florencio Miranda caine-induced locomotor sensitization [38]. It has also been reported that GS39783 reduces the locomotor activity arising from acute ethanol administration, without any effects on ethanol-induced locomotor sensitization, whereas when GS39783 is administered in conjunction with ethanol, it potentiates ethanolinduced locomotor sensitization [37]. It is important to note that the GABA B receptor is a G protein-coupled heterodimer composed of GABA B1 and GABA B2 subunits, which have different functions [7]. The GABA B1 subunit is essential for ligand binding, while the GABA B2 subunit provides the G protein-coupling mechanism and incorporates an allosteric modulatory site [47]. It has been suggested that the G protein-coupling mechanism of the GABA B2 subunit involves an interaction with the extracellular domain of the GABA B1 subunit, and as a result of this interaction, agonist affinity and coupling efficacy are increased [6]. In line with this suggestion, it has been reported that CGP is effective in facilitating the inhibitory effects of BCF on the spontaneous firing rates of VTA DAergic neurons. CGP shifts the BCF concentration-response curve to the left but has no effect when administered alone [14]. According to the authors, the reason that CGP alone does not cause any change in the spontaneous firing rate of VTA DAergic neurons is most likely that the amount of GABA released onto these neurons is too low to activate the GABA B receptors. Positive modulators of GABA B receptors, such as CGP, are devoid of intrinsic activity, and their actions are dependent on the presence of endogenous GABA or other GABA B agonists [56, 57]. It is well known that DAergic neurons are tonically inhibited by GABAergic interneurons within the VTA [27] and that the VTA DAergic neurons receive inputs from GABAergic neurons that originate in the NAcc [55]. This circuit provides the endogenous levels of GABA required to activate GABA B receptors. The inhibitory control of GABA over the VTA DAergic neurons can be enhanced in the presence of a positive allosteric modulator of GABA B receptors, such as CGP. Therefore, these previous observations could explain the results of the current study and of some studies in which CGP was administered alone, if CGP acts synergistically with GABA or with a GABA B agonist, despite displaying no intrinsic activity of its own. In conclusion, the results of the present study demonstrated that the GABA B receptor agonist BCF prevented both the development and the expression of AMPH-induced locomotor sensitization in a dosedependent manner. Furthermore, CGP, a positive allosteric modulator of GABA B receptors, increased the effects of a lower dose of BCF on AMPH-induced locomotor sensitization under both conditions. These data provide further evidence that GABA B receptor ligands may modulate psychostimulant-induced behaviors and that positive allosteric modulators of GABA B receptors may present pharmacological potential when combined with the use of conventional GABA B agonists, such as BCF. Acknowledgments: This study was supported by grant from CONACyT (México) and a doctoral fellowship awarded to the first author (CONACyT, México). References: 1. Amara SG, Sonders MS: Neurotransmitter transporters as molecular targets for addictive drugs. Drug Alcohol Depend, 1998, 51, Arnold JM, Roberts DCS: A critique of fixed and progressive ratio schedules used to examine the neural substrates of drug reinforcement. Pharmacol Biochem Behav, 1997, 57, Bartoletti M, Gubellini C, Ricci F, Gaiardi M: Baclofen blocks the development of sensitization to the locomotor stimulant effect of amphetamine. Behav Pharmacol, 2005, 16, Bartoletti M, Gubellini C, Ricci F, Gaiardi M: The GABA B agonist baclofen blocks the expression of sensitisation to the locomotor stimulant effect of amphetamine. Behav Pharmacol, 2004, 15, Bartoletti M, Ricci F, Gaiardi MA: GABA B agonist reverses the behavioral sensitization to morphine in rats. Psychopharmacology, 2007, 192, Binet V, Brajon C, Le Corre L, Acher F, Pin JP, Prezeau L: The heptahelical domain of GABA B2 is activated directly by CGP7930, a positive allosteric modulator of the GABA B receptor. J Biol Chem, 2004, 279, Bowery NG: GABA B receptor: A site of therapeutic benefit. Curr Opin Pharmacol, 2006, 6, Bowery NG, Hudson AL, Price GW: GABA A and GABA B receptor site distribution in the rat central nervous system. Neuroscience, 1987, 20, Brebner K, Ahn S, Phillips AG: Attenuation of d-amphetamine self-administration by baclofen in the rat: behavioral and neurochemical correlates. Psychopharmacology, 2005, 177, Brebner K, Froestl W, Andrews M, Phelan R, Roberts DCS: The GABA B agonist CGP decreases cocaine self-administration in rats: demonstration using a progressive ratio and a discrete trials procedure. Neuropharmacology, 1999, 38, Pharmacological Reports, 2013, 65,

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