Pharmacological Reports Copyright © 2013 2013, 65, 11321143 by Institute of Pharmacology ISSN 1734-1140 Polish Academy of Sciences
Effects of co-administration of the GABAB receptor agonist baclofen and a positive allosteric modulator of the GABAB receptor, CGP7930, on the development and expression of amphetamine- induced 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, e-mail: [email protected]
Abstract: Background: Several of the behavioral effects of amphetamine (AMPH) are mediated by an increase in dopamine neurotransmis- sion in the nucleus accumbens. However, evidence shows that g-aminobutyric acid B (GABAB) receptors are involved in the behav- ioral effects of psychostimulants, including AMPH. Here, we examined the effects of co-administration of the GABAB receptor agonist baclofen and a positive allosteric modulator of the GABAB 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 develop- ment 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 GABAB receptor, CGP7930, in- creased the effects of a lower dose of baclofen on AMPH-induced locomotor sensitization under both conditions. Conclusion: These data provide further evidence that GABAB receptor ligands may modulate psychostimulant-induced behaviors.
Key words: amphetamine, sensitization, GABAB receptors
Introduction synaptic vesicles, promoting an increase in the cyto- plasmic concentrations of these monoamines. As the levels of cytoplasmic monoamines increase, they exit Cocaine and amphetamine (AMPH) are indirect neurons via reversal of the direction of plasma mem- monoamine agonists that exhibit an affinity for dopa- brane transporters, which leads to an increase in synap- mine (DA), norepinephrine (NE), and serotonin (5-HT) tic DA, NE, and 5-HT levels [1, 28, 52]. transporters, which are involved in neurotransmitter re- The mesolimbic DAergic system, particularly the uptake and vesicular storage systems [52]. Cocaine in- projection from the ventral tegmental area (VTA) to hibits the reuptake of DA, NE, and 5-HT, thereby in- the nucleus accumbens (NAcc), is an important locus creasing the synaptic levels of these neurotransmitters. for the production of the locomotor, reinforcement, AMPH blocks the uptake of DA, NE, and 5-HT into reward, and discriminative stimulus effects of psy-
1132 Pharmacological Reports, 2013, 65, 11321143 Effects of BCF and CGP on AMPH-induced locomotor sensitization Laura N. Cedillo and Florencio Miranda
chostimulants [17, 20, 35]. The administration of useful for studying DA-GABA interactions. The pres- AMPH or cocaine rapidly increases DAergic neuro- ent study was designed to examine the effects of co- transmission by interfering with the function of DA administration of the GABAB receptor agonist BCF transporters, as described above. Consequently, psy- and CGP, a positive allosteric modulator of the chostimulant administration produces an increase in GABAB receptor, on the development and expression DAergic signaling in the limbic areas [35, 36]. of AMPH-induced locomotor sensitization. Recent evidence also suggests a potential role for g-aminobutyric acid (GABA) neurotransmission in modulating some of the behavioral effects of psycho- stimulants. GABAB receptor agonists are reportedly Materials and Methods effective in attenuating some of the behavioral effects of psychostimulants that may be related to the abuse Animals of these drugs. For example, the selective GABAB re- ceptor agonist baclofen (BCF) reduces the reinforcing A total of 400 male Wistar rats that were 120 days old effects of cocaine [49], nicotine [19], methAMPH and weighed 20–250 g at the beginning of the experi- [48], and AMPH [9]. Furthermore, BCF administra- ments were obtained from the breeding colony of the tion decreases the conditioned locomotion elicited by FES-Iztacala-UNAM, México. They were individu- cues associated with cocaine [25]. Other GABAB re- ally housed in stainless steel cages with freely avail- ceptor agonists also reduce psychostimulant-related able food (Teklad LM485 Rat Diet by Harlan, México behaviors. Brebner et al. [10] reported that the selec- City, México) and water and were maintained under tive GABAB receptor agonist CPG44532 was effec- a 12 h light/dark cycle, with the lights turned on at tive in attenuating cocaine self-administration in rats 08:00 h. The room was maintained at a temperature of subjected to a progressive ratio schedule. 21 ± 1°C. All experiments were conducted during the Although some findings have indicated that light phase (between 11:00 a.m. and 1:00 p.m.). Ani- GABAB agonists, such as BCF, may be useful in the mal care and handling procedures were performed in treatment of drug abuse, other results have suggested accordance with the Official Mexican Norm (NOM- that the muscle-relaxant properties of BCF, in con- 062-ZOO-1999) entitled “Technical Specifications junction with its sedative and hypothermic effects, for the Production, Care, and Use of Laboratory Ani- limit its widespread application as a therapeutic agent mals” and all procedures were approved by the local in humans and as a tool for behavioral research [16, bioethics committee. 26]. However, a novel alternative approach, allosteric modulation of GABAB receptors, has been suggested. Drugs Positive allosteric modulators of GABAB receptors display no intrinsic activity of their own but can inter- The drugs used in this study were D-amphetamine act synergistically with GABAB agonists, including sulfate, (±)-baclofen (Sigma-Aldrich, St. Louis, MO, BCF, to enhance their effects. One strategy for at- USA), and CGP7930 (Tocris, Ballwin, MO, USA). tempting to overcome the side effects of BCF is to use (±)-Baclofen and D-AMPH were dissolved in water, lower dosages to reduce unwanted effects of the drug, and CGP7930 was dissolved in 2 drops (20 µl/drop) in combination with positive allosteric modulators of of ethanol and then in 1% Tween 80. All drugs were GABAB receptors, such as CGP7930 (CGP). prepared fresh daily and were administered via Locomotor sensitization is the progressive and per- intraperitoneal injection (1 ml/kg). sistent enhancement of a behavioral response to a drug after repeated and intermittent administration. Apparatus This phenomenon has been well characterized for psychostimulant and cannabinoid drugs [32, 60, 63] Locomotor activity was measured with an open-field and is thought to reflect neuroadaptations that contrib- activity monitoring system (ENV-515 model; Med ute to drug addiction and model some aspects of ad- Associates, St. Albans, VT. USA). Each Plexiglas dictive behaviors, such as drug craving [51]. There- cage (40 × 40 × 30 cm) was equipped with 2 sets of 8 fore, locomotor sensitization may reflect neurobio- photobeams that were placed 2.5 cm above the sur- logical changes related to drug addiction and may be face of the floor on opposite walls to record x–y am-
Pharmacological Reports, 2013, 65, 11321143 1133 bulatory movements. Photobeam interruptions were Experiment 1: Effects of BCF and CGP co- recorded and translated by software to yield the hori- administration on the development of AMPH- zontal distance traveled (in cm), which was the de- induced locomotor sensitization (see Tab. 1 for the schedule of drug treatment for Experiment 1 pendent measure used for analysis. and Experiment 2)
Experimental procedure a) Effects of BCF on the development of AMPH- induced locomotor sensitization. On the days where The timeline of the general procedure is shown in Figure the development of sensitization was examined, 1. Each day or session started with a 10 min period of groups of rats (n = 10 rats per group) received one of habituation to the cages, followed by administration of the following treatments: VEH + VEH (group 2V), drug(s) or vehicle (VEH). The rats were returned to the VEH + AMPH (1.0 mg/kg; group VA), BCF (2.0 mg/ cages, and their locomotor activity was recorded for 1 h. kg) + AMPH (1.0 mg/kg; group B2A), BCF (3.0 mg/ On day 1, the rats were habituated to the open-field kg) + AMPH (1.0 mg/kg; group B3A), or BCF cages and injection procedures (habituation session). On (4.0 mg/kg) + AMPH (1.0 mg/kg; group B4A). day 2, locomotor activity after VEH administration (lo- b) Effects of CGP on the development of AMPH- comotor activity baseline) was evaluated. On days 3–7, induced locomotor sensitization. On the days where the rats received pharmacological compounds and/or the development of sensitization was examined, AMPH (for development of AMPH-induced locomotor groups of rats (n = 10 rats per group) received one of sensitization; see Tab. 1). On days 8–9, the rats were left the following treatments: VEH + VEH (group 2V), undisturbed (resting days). In the experiments examin- VEH + AMPH (1.0 mg/kg; group VA), CGP (5.0 mg/ ing the development of AMPH sensitization, the rats kg) + AMPH (1.0 mg/kg; group C5A), CGP (10.0 mg/ were injected with VEH and AMPH on days 10 and 11, kg) + AMPH (1.0 mg/kg; group C10A), or CGP respectively (test days), while in the experiments exam- (20.0 mg/kg) + AMPH (1.0 mg/kg; group C20A). ining the expression of AMPH sensitization, the rats c) Effects of the co-administration of CGP and were injected with VEH on day 10 and with pharmacol- BCF on the development of AMPH-induced locomo- ogical compounds and/or AMPH on day 11 (see Tab. 1). tor sensitization. On the days where the development In all experiments, the rats were injected with CGP and of sensitization was examined, groups of rats (n = 10 BCF 1 min before AMPH injection and immediately re- rats per group) received one of the following treat- turned to the cages, where their locomotor activity was ments: VEH + VEH + VEH (group 3V), VEH + VEH recorded for 1 h. + AMPH (1.0 mg/kg; group 2VA), VEH + BCF (2.0 mg/kg) + AMPH (1.0 mg/kg; group VB2A), CGP Acute effects of BCF and CGP on locomotor activity (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) The locomotor activity in groups of animals (n = 10 + AMPH (1.0 mg/kg; group C10B2A), or CGP rats per group) was assessed once in response to each (20.0 mg/kg) + BCF (2.0 mg/kg) + AMPH (1.0 mg/ different dose of BCF (0.0, 2.0, 3.0, and 4.0 mg/kg) kg; group C20B2A). and CGP (0.0, 5.0, 10.0, and 20.0 mg/kg).
Experiment 2: Effects of BCF and CGP co- administration on the expression of AMPH- induced locomotor sensitization Development of Resting Test sensitization days Days H BL V A a) Effects of BCF on the expression of AMPH- induced locomotor sensitization. On the days where DAYS 1 2 3 4 5 6 7 8 9 10 11 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 Fig. 1. Schematic diagram illustrating the timeline of AMPH (1 mg/ kg)-induced locomotor sensitization. H – habituation, BL – baseline groups were injected with VEH. On day 11, each locomotor activity, V – vehicle, A – amphetamine group of rats received one of the following treat-
1134 Pharmacological Reports, 2013, 65, 11321143 Effects of BCF and CGP on AMPH-induced locomotor sensitization Laura N. Cedillo and Florencio Miranda
ments: VEH + AMPH (group V-VA), VEH + AMPH b) Effects of CGP on the expression of AMPH- (1.0 mg/kg; group A-VA), BCF (2.0 mg/kg) + AMPH induced locomotor sensitization. On the days where the (1.0 mg/kg; group B2A), BCF (3.0 mg/kg) + AMPH development of sensitization was examined, one group (1.0 mg/kg; group B3A), or BCF (4.0 mg/kg) + (n = 10 rats) received an injection of VEH + VEH, and AMPH (1.0 mg/ kg; group B4A). 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 Repeated treatment Challenge of groups 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, 11321143 1135 an injection of VEH + AMPH (1.0 mg/kg). On day EXPERIMENT 1 10, all groups were injected with VEH. On day 11, each group of rats received one of the following treat- a) Effects of BCF on the development of AMPH- ments: VEH + AMPH (group V-VA), VEH + AMPH induced locomotor sensitization. (1.0 mg/kg; group A-VA), CGP (5.0 mg/kg) + AMPH The data obtained from the baseline locomotor (1.0 mg/kg; group C5A), CGP (10.0 mg/kg) + AMPH activity measurements were similar in all groups (1.0 mg/kg; group C10A), or CGP (20.0 mg/kg) + [F (4, 49) = 0.781, p > 0.05]. Repeated administration AMPH (1.0 mg/kg; group C20A). of AMPH resulted in the development of sensitization c) Effects of CGP and BCF co-administration on to locomotor activity. However, administration of the expression of AMPH-induced locomotor sensiti- BCF at doses of 3.0 and 4.0 mg/kg, but not at zation. On the days where the development of sensiti- 2.0 mg/kg, attenuated the locomotor activity produced zation was examined, one group (n = 10 rats) received by AMPH treatment. Two-way ANOVA for repeated an injection of VEH + VEH + VEH, and the other measures indicated significant effects of the group groups of rats (n = 10 rats per group) received an in- [F (4, 45) = 25.516, p < 0.05], day [F (4, 180) = 8.483, jection of VEH + VEH + AMPH (1.0 mg/kg). On day p < 0.05], and the group × day interaction [F (16, 180) 10, all groups were injected with VEH. On day 11, = 3.999, p < 0.05]. The results of the VEH test are each group of rats received one of the following treat- shown in Figure 2A. Animals that were treated with ments: VEH + VEH + AMPH (group 2VA), VEH either AMPH alone or AMPH in combination with + VEH + AMPH (1.0 mg/kg; group A-2VA), VEH BCF showed conditioned locomotion. Here, one-way + BCF (2.0 mg/kg) + AMPH (1.0 mg/kg; group ANOVAindicated a significant group effect [F (4, 49) VB2A), CGP (5.0 mg/kg) + BCF (2.0 mg/kg) = 3.58, p < 0.05], and Tukey’s test revealed that all + AMPH (1.0 mg/kg; group C5B2A), CGP (10.0 other groups were significantly different from the 2V mg/kg) + BCF (2.0 mg/kg) + AMPH (1.0 mg/kg; group. The results of the administration of AMPH on group C10B2A), or CGP (20.0 mg/kg) + BCF (2.0 day 11 are shown in Figure 2B. When BCF was mg/kg) + AMPH (1.0 mg/kg; group C20B2A). administered for 5 days in combination with AMPH injection, it was observed that BCF reduced the Data analysis AMPH-induced locomotor activity in a dose-depend- ent manner [F (4, 49) = 2.921, p < 0.05]. Tukey’s test The results of the investigated measure (distance trav- revealed that the VA group was different from the 2V eled, in cm) are expressed as the mean ± SEM. The group and that the B3A and B4A groups were differ- data obtained during the development of locomotor ent from the VAgroup. sensitization were analyzed via two-way ANOVA for b) Effects of CGP on the development of AMPH- repeated measures, with the group as the first factor induced locomotor sensitization. The baseline locomo- and the day as the second factor. The data obtained tor activity was similar in all groups [F (4, 49) = 0.264, during the baseline and testing days were analyzed p > 0.05]. Repeated administration of AMPH induced through one-way ANOVA. When the ANOVA results the development of sensitization to locomotor activity. were significant, Tukey’s test (p < 0.05) was used to The AMPH-induced locomotor sensitization was not perform a posteriori comparisons. altered after the administration of CGP (a positive al- losteric modulator of GABAB receptors) at different doses. Two-way ANOVA for repeated measures indi- cated significant effects of the group [F (4, 45) = 34.367, p < 0.05], day [F (4, 180) = 6.721, p < 0.05], Results and the group × day interaction [F (16, 180) = 1.306, p < 0.05]. The results of the VEH test are shown in Acute effects of BCF and CGP on locomotor Figure 2C. Animals treated with either AMPH alone or activity AMPH in combination with CGP showed conditioned locomotion. One-way ANOVA indicated a significant The results of this experiment revealed that neither BCF group effect [F (4, 49) = 3.79, p < 0.05], and Tukey’s [F (3, 39) = 0.896, p > 0.05] nor CGP [F (3, 39) = 0.892, test revealed that all other groups were different from p > 0.05] altered the locomotor activity of the rats. the 2V group. The results of the administration of
1136 Pharmacological Reports, 2013, 65, 11321143 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 one- way 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 locomotor sensitization was affected by the admini- was administered for 5 days in combination with stration of the combination of CGP at different dos- AMPH injection, it was found that CGP did not re- ages and a lower dose of BCF. Two-way ANOVA for duce the AMPH-induced locomotor activity [F (4, 49) repeated measures indicated significant effects of the = 5.313, p < 0.05]. Tukey’s test revealed that the VA group [F (5, 54) = 26.98, p < 0.05], day [F (4, 216) = group was different from the 2V group. 11.002, p < 0.05], and the group × day interaction c) Effects of CGP and BCF co-administration on [F (20, 216) = 3.74, p < 0.05], and Tukey’s test re- the development of AMPH-induced locomotor sensi- vealed that the C20B2A group was different than the tization. The baseline locomotor activity was similar 2VA group. The results of the VEH test are shown in in all groups [F (5, 59) = 1.30, p > 0.05]. Repeated ad- Figure 2E. Animals treated with either AMPH alone ministration of AMPH resulted in the development of or AMPH in combination with CGP and BCF exhib- sensitization to locomotor activity. AMPH-induced ited conditioned locomotion. One-way ANOVA indi-
Pharmacological Reports, 2013, 65, 11321143 1137 cated significant differences between groups [F (4, 49) not significant [F (16, 180) = 1.32, p > 0.05]. Tukey’s = 3.37, p < 0.05]. Tukey’s test revealed that all groups test revealed that the V-VA group was different from were different from the 3V group. The results of ad- all other groups. The results of the VEH test on day ministration of AMPH on day 11 are shown in Figure 10 are shown in Figure 3C. Animals treated with 2F. After 5 days of AMPH administration in combina- AMPH presented conditioned locomotion. One-way tion with CGP and BCF, it was observed that CGP in- ANOVA indicated significant differences between creased the effects of BCF on AMPH-induced loco- groups [F (4, 49) = 3.84, p < 0.05], and Tukey’s test motor activity [F (5, 59) = 4.332, p < 0.05]. Tukey’s revealed that all groups were different from the V-VA test revealed that locomotor activity differed between group. The results obtained following the administra- the 2VAgroup and the 3V group and that the C20B2A tion of AMPH or different doses of CGP and AMPH group was different from the 2VAgroup. on day 11 (see Fig. 3D) showed that CGP does not af- fect the expression of AMPH-induced locomotor ac- EXPERIMENT 2 tivity [F (4, 49) = 3.74, p < 0.05], and Tukey’s test re- vealed that the A-VA group was different from the a) Effects of BCF on the expression of AMPH- V-VAgroup. induced locomotor sensitization. The baseline loco- c) Effects of CGP and BCF co-administration on motor activity on day 2 was similar in all groups the expression of AMPH-induced locomotor sensiti- [F (4, 49) = 1.370, p > 0.05]. Repeated administration zation. The baseline locomotor activity on day 2 was of AMPH led to the development of sensitization to similar in all groups [F (5, 59) = 1.48, p > 0.05]. Re- locomotor activity in all groups, except the V-VA peated administration of AMPH led to the develop- group. Two-way ANOVA for repeated measures indi- ment of sensitization to locomotor activity in all cated significant effects of the group [F (4, 45) = groups, except the 2VA group. Two-way ANOVA for 16.356, p < 0.05], day [F (4, 180) = 8.54, p < 0.05], repeated measures indicated significant effects of the and the group × day interaction [F (16, 180) = 1.80, p group [F (5, 54) = 18.563, p < 0.05] and day < 0.05], and Tukey’s test revealed that the V-VA [F (5, 216) = 10.24, p < 0.05], but the group × day in- group was different from all other groups. The results teraction was not significant [F (14, 216) = 1.22, p > of the VEH test on day 10 are shown in Figure 3A. 0.05]. Tukey’s test revealed that the 2VA group was Animals treated with AMPH exhibited conditioned different from all other groups. The results of the locomotion. One-way ANOVA indicated the exis- VEH test on day 10 are shown in Figure 3E. Animals tence of significant differences between groups treated with AMPH displayed conditioned locomo- [F (4, 49) = 5.16, p < 0.05], and Tukey’s test revealed tion. One-way ANOVA indicated significant differ- that all other groups were different from the V-VA ences between groups [F (4, 49) = 5.54, p < 0.05], and group. The results of the administration of either Tukey’s test revealed that all other groups were differ- AMPH or different doses of BCF and AMPH on day ent from the 2VAgroup. The results of the administra- 11 are shown in Figure 3B. Administration of BCF tion of different doses of CGP, BCF2.0, and AMPH led to a dose-dependent reduction in the expression of on day 11 are shown in Figure 3F. Administration of AMPH-induced locomotor activity [F (4, 49) = 5.05 p CGP increased the effects of BCF on the expression < 0.05]. Tukey’s test revealed that the A-VA group of AMPH-induced locomotor activity [F (5, 59) = was different than the V-VA group and that the B3A 2.21, p < 0.05]. Tukey’s test revealed that the A-2VA and B4A groups were different than the A-VAgroup. group was different from the 2VA group and that the b) Effects of CGP on the expression of AMPH- C20B2A group was different from the A-2VAgroup. induced locomotor sensitization. The baseline locomotor activity on day 2 was simi- lar in all groups [F (4, 49) = 1.378, p > 0.05], and re- peated administration of AMPH resulted in the devel- Discussion opment of sensitization to locomotor activity in all groups, except the V-VAgroup. Two-way ANOVAfor repeated measures indicated a significant effect of the The purpose of the present study was to examine the group [F (4, 45) = 27.74, p < 0.05] and day [F (4, 180) effects of the co-administration of the GABAB recep- = 9.27, p < 0.05], but the group × day interaction was tor agonist BCF with CGP, a positive allosteric modu-
1138 Pharmacological Reports, 2013, 65, 11321143 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 GABAB receptors, on AMPH-induced loco- tor CGP increased the effects of a lower dose of BCF motor sensitization. We found that AMPH increased on AMPH-induced locomotor sensitization under locomotor activity and that BCF treatment resulted in both conditions. Additionally, an increase in locomo- prevention of both the development and the expres- tor activity was detected during the VEH test (day sion of AMPH-induced locomotor sensitization in 10), which is interpreted as conditioned locomotion a dose-dependent manner, whereas CGP had no effect [58] and could mask the main effects of the drugs on either the development or the expression of tested on day 11. However, although this possibility AMPH-induced locomotor activity. We also observed cannot be completely ruled out, we found that BCF that administration of the positive allosteric modula- and/or CGP reduced, rather than increased, AMPH-
Pharmacological Reports, 2013, 65, 11321143 1139 induced locomotor activity in a dose-dependent man- VTA decreases DA release in the NAcc [59, 62], ner, in terms of both the development and expression which suggests that the activation of GABAB recep- of AMPH-induced locomotor sensitization. tors located on the cell bodies of mesolimbic DAergic The behavioral results described above are consis- neurons is involved in the biochemical effects of BCF. tent with those of previous studies demonstrating that Furthermore, microinjection of BCF into the VTA re- the GABAB receptor agonist BCF attenuates AMPH- duces cocaine self-administration under fixed ratio induced locomotor sensitization. For example, it has [53] and progressive ratio schedules [12], and micro- been reported previously that BCF prevents both the injection of BCF was also found to reduce heroin development [3] and expression [4] of sensitization to self-administration [61] and AMPH-induced motor the locomotor effects of AMPH. BCF also attenuates activity [30]. Altogether, these data support the hy- the sensitization to the locomotor stimulant effects of pothesis that the activation of GABAB receptors on cocaine [23], morphine [5, 24], and ethanol [13]. the cell bodies of DAergic neurons in the VTA plays Furthermore, BCF attenuates psychostimulant- an important role in the suppressive effects of BCF on related behaviors associated with drug addiction. BCF both the development and the expression of AMPH- pre-treatment reduces cocaine self-administration in induced locomotor sensitization. rats responding under fixed ratio schedules [11], pro- In this study, it was also observed that a positive al- gressive ratio schedules [2, 50], discrete trial sched- losteric modulator of GABAB receptors, CGP, in- ules of reinforcement [10], and second-order sched- creased the effects of a lower dose of BCF on ules [18]. Additionally, BCF decreases AMPH self- AMPH-induced locomotor sensitization. It is worth administration under a fixed ratio or progressive ratio noting that CGP did not alter AMPH-induced locomo- schedule [9] and attenuates conditioned locomotion in tor sensitization at any of the tested dosages. These response to cues associated with cocaine administra- results are consistent with those of at least one previ- tion [25]. Moreover, BCF attenuates the behavioral ous study, in which the combination of a lower dose effects of ethanol [15], nicotine [44], and heroin [18], of BCF with a dose of CGP reduced ethanol self- and we found in a previous study that BCF reduces administration [39]. Furthermore, it has been reported the discriminative stimulus properties of AMPH [41]. that CGP injections lead to a reduction of ethanol in- The mechanism underlying the observed effects of take in ethanol-preferring rats [39, 43], of cocaine BCF on AMPH-induced locomotor sensitization may self-administration in rats responding under several involve GABAergic modulation of DAergic transmis- schedules of reinforcement [22, 54], of cocaine- sion within the VTA. Several lines of evidence sup- seeking behavior [21], of nicotine-induced locomotor port this notion. First, the mesolimbic DA system, stimulation in mice [40], and of nicotine self-adminis- particularly the projection from the VTA to the NAcc, tration [45]. In addition, CGP treatment was shown to is an important locus in the production of the locomo- produce approximately 41% BCF-appropriate re- tor, reinforcement, and reward effects of psy- sponses but also to enhance the discriminative stimu- chostimulants such as cocaine and AMPH [17, 31, lus effects of BCF in pigeons trained to discriminate 35], and this system plays an important role in both BCF from saline [34]. the development and expression of psychostimulant- The above findings together with the present data induced locomotor sensitization [46]. Second, the provide behavioral evidence that BCF and related VTA contains primary DAergic neurons, which re- compounds, such as CGP, may represent potential lease DA in the NAcc and prefrontal cortex, and sec- pharmacological treatments for drug addiction. Posi- ondary GABAergic interneurons, which reduce the tive allosteric modulators of GABAB receptors, such firing rate of DAergic neurons in the VTA. In addi- as CGP, display no intrinsic activity of their own but tion, GABAergic neurons arising from the NAcc proj- can modulate the activity of GABA or GABAB ago- ect to DAergic neurons within the VTA [29, 33]. This nists, such as BCF, while avoiding the possible ad- loop represents an important locus in the production verse side effects of BCF administration. In contrast of certain abuse-related behavioral effects of psy- to the data cited above, recent studies have shown that chostimulants [35]. Third, anatomical evidence sug- the administration of GS39783 alone, which is an- gests that GABAB receptors are located within the other positive modulator of GABAB receptors, signifi- VTA [8, 27, 29, 42]. Fourth, biochemical and behav- cantly attenuates the locomotor activity induced by ioral studies have found that infusion of BCF into the a single cocaine treatment and reduces modest co-
1140 Pharmacological Reports, 2013, 65, 11321143 Effects of BCF and CGP on AMPH-induced locomotor sensitization Laura N. Cedillo and Florencio Miranda
caine-induced locomotor sensitization [38]. It has also dependent manner. Furthermore, CGP, a positive al- been reported that GS39783 reduces the locomotor losteric modulator of GABAB receptors, increased the activity arising from acute ethanol administration, effects of a lower dose of BCF on AMPH-induced lo- without any effects on ethanol-induced locomotor comotor sensitization under both conditions. These sensitization, whereas when GS39783 is administered data provide further evidence that GABAB receptor in conjunction with ethanol, it potentiates ethanol- ligands may modulate psychostimulant-induced be- induced locomotor sensitization [37]. haviors and that positive allosteric modulators of It is important to note that the GABAB receptor is GABAB receptors may present pharmacological po- a G protein-coupled heterodimer composed of GABAB1 tential when combined with the use of conventional and GABAB2 subunits, which have different functions GABAB agonists, such as BCF. [7]. The GABAB1 subunit is essential for ligand bind- ing, while the GABAB2 subunit provides the G pro- tein-coupling mechanism and incorporates an alloste- Acknowledgments: ric modulatory site [47]. It has been suggested that the This study was supported by grant 60872 from CONACyT (México) and a doctoral fellowship awarded to the first author (CONACyT, G protein-coupling mechanism of the GABAB2 sub- México). unit involves an interaction with the extracellular do- main of the GABAB1 subunit, and as a result of this interaction, agonist affinity and coupling efficacy are increased [6]. In line with this suggestion, it has been References: reported that CGP is effective in facilitating the in- hibitory effects of BCF on the spontaneous firing 1. Amara SG, Sonders MS: Neurotransmitter transporters rates of VTA DAergic neurons. CGP shifts the BCF as molecular targets for addictive drugs. 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