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 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: fmirandah@yahoo.com
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 GABABreceptor agonist baclofen and a positive allosteric modulator of the GABABreceptor, 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 GABABreceptor, 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 GABABreceptor ligands may modulate psychostimulant-induced behaviors.
Key words:
amphetamine, sensitization, GABABreceptors
Introduction
Cocaine and amphetamine (AMPH) are indirect monoamine agonists that exhibit an affinity for dopa- mine (DA), norepinephrine (NE), and serotonin (5-HT) transporters, which are involved in neurotransmitter re- uptake and vesicular storage systems [52]. Cocaine in- hibits the reuptake of DA, NE, and 5-HT, thereby in- creasing the synaptic levels of these neurotransmitters.
AMPH blocks the uptake of DA, NE, and 5-HT into
synaptic vesicles, promoting an increase in the cyto- plasmic concentrations of these monoamines. As the levels of cytoplasmic monoamines increase, they exit neurons via reversal of the direction of plasma mem- brane transporters, which leads to an increase in synap- tic 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, 11321143 ISSN 1734-1140
Copyright © 2013 by Institute of Pharmacology Polish Academy of Sciences
chostimulants [17, 20, 35]. The administration of AMPH or cocaine rapidly increases DAergic neuro- transmission by interfering with the function of DA transporters, as described above. Consequently, psy- chostimulant administration produces an increase in DAergic signaling in the limbic areas [35, 36].
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 effective in attenuating some of the behavioral effects of psychostimulants that may be related to the abuse of these drugs. For example, the selective GABABre- ceptor agonist baclofen (BCF) reduces the reinforcing effects of cocaine [49], nicotine [19], methAMPH [48], and AMPH [9]. Furthermore, BCF administra- tion decreases the conditioned locomotion elicited by cues associated with cocaine [25]. Other GABABre- ceptor agonists also reduce psychostimulant-related behaviors. Brebner et al. [10] reported that the selec- tive GABAB receptor agonist CPG44532 was effec- tive in attenuating cocaine self-administration in rats subjected to a progressive ratio schedule.
Although some findings have indicated that GABABagonists, such as BCF, may be useful in the treatment of drug abuse, other results have suggested that the muscle-relaxant properties of BCF, in con- junction 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 GABABreceptors, has been suggested.
Positive allosteric modulators of GABAB receptors display no intrinsic activity of their own but can inter- act synergistically with GABAB agonists, including BCF, to enhance their effects. One strategy for at- tempting 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 GABABreceptors, such as CGP7930 (CGP).
Locomotor sensitization is the progressive and per- sistent 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 contrib- ute to drug addiction and model some aspects of ad- dictive behaviors, such as drug craving [51]. There- fore, locomotor sensitization may reflect neurobio- logical changes related to drug addiction and may be
useful for studying DA-GABA interactions. The pres- ent study was designed to examine the effects of co- administration of the GABAB receptor agonist BCF and CGP, a positive allosteric modulator of the GABABreceptor, 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 20–250 g at the beginning of the experi- ments were obtained from the breeding colony of the FES-Iztacala-UNAM, México. They were individu- ally housed in stainless steel cages with freely avail- able 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.). Ani- mal 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 Ani- mals” 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 (40 × 40 × 30 cm) was equipped with 2 sets of 8 photobeams that were placed 2.5 cm above the sur- face of the floor on opposite walls to record x–y am-
bulatory movements. Photobeam interruptions were recorded and translated by software to yield the hori- zontal distance traveled (in cm), which was the de- pendent 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 (lo- comotor 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 examin- ing 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 exam- ining the expression of AMPH sensitization, the rats were injected with VEH on day 10 and with pharmacol- ogical 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 re- turned 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).
Experiment 1: Effects of BCF and CGP co- administration on the development of AMPH- induced 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 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 (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 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 (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 locomo- tor sensitization. On the days where the development of sensitization was examined, groups of rats (n = 10 rats per group) received one of the following treat- ments: 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 co- administration on the expression of AMPH- induced locomotor sensitization
a) Effects of BCF on the expression of AMPH- induced 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-
1 2 3 4 5 6 7
Development of sensitization
Test Days Resting
days
A BL
8 9 10 11
H V
1 2 3 4 5 6 7
DAYS
Development of sensitization
Test Days Resting
days
A BL
8 9 10 11
H V
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
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 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, 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
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 treat- ments: 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 sensiti- zation. On the days where the development of sensiti- zation 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 in- jection 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 treat- ments: 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 trav- eled, 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 AMPH- induced 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) = 25.516, 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-depend- ent 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 differ- ent from the VA group.
b) Effects of CGP on the development of AMPH- induced locomotor sensitization. The baseline locomo- tor 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 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], 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
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 re- duce 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 sensi- tization. The baseline locomotor activity was similar in all groups [F (5, 59) = 1.30, p > 0.05]. Repeated ad- ministration of AMPH resulted in the development of sensitization to locomotor activity. AMPH-induced
locomotor sensitization was affected by the admini- stration of the combination of CGP at different dos- ages 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) = 11.002, p < 0.05], and the group × day interaction [F (20, 216) = 3.74, p < 0.05], and Tukey’s test re- vealed 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 exhib- ited conditioned locomotion. One-way ANOVA indi-
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-hoctest. The name of the groups refers only the treatment received during the development of AMPH-induced locomotor sensitization (see Tab. 1 for further details)
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 ad- ministration of AMPH on day 11 are shown in Figure 2F. After 5 days of AMPH administration in combina- tion with CGP and BCF, it was observed that CGP in- creased the effects of BCF on AMPH-induced loco- motor 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.
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 ad- ministration of AMPH on day 11 are shown in Figure 2F. After 5 days of AMPH administration in combina- tion with CGP and BCF, it was observed that CGP in- creased the effects of BCF on AMPH-induced loco- motor 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 AMPH- induced locomotor sensitization. The baseline loco- motor 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 indi- cated significant effects of the group [F (4, 45) = 16.356, 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 exis- tence 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 AMPH- 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- opment 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 administra- tion of AMPH or different doses of CGP and AMPH on day 11 (see Fig. 3D) showed that CGP does not af- fect the expression of AMPH-induced locomotor ac- tivity [F (4, 49) = 3.74, p < 0.05], and Tukey’s test re- vealed 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 sensiti- zation. The baseline locomotor activity on day 2 was similar in all groups [F (5, 59) = 1.48, p > 0.05]. Re- peated administration of AMPH led to the develop- ment 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) = 18.563, p < 0.05] and day [F (5, 216) = 10.24, p < 0.05], but the group × day in- teraction 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 locomo- tion. One-way ANOVA indicated significant differ- ences between groups [F (4, 49) = 5.54, p < 0.05], and Tukey’s test revealed that all other groups were differ- ent from the 2VA group. The results of the administra- tion 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 GABABrecep- tor agonist BCF with CGP, a positive allosteric modu-
lator of GABAB receptors, on AMPH-induced loco- motor sensitization. We found that AMPH increased locomotor activity and that BCF treatment resulted in prevention of both the development and the expres- sion 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 modula-
tor CGP increased the effects of a lower dose of BCF on AMPH-induced locomotor sensitization under both conditions. Additionally, an increase in locomo- tor 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-
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)
induced locomotor activity in a dose-dependent man- ner, in terms of both the development and expression of AMPH-induced locomotor sensitization.
The behavioral results described above are consis- tent with those of previous studies demonstrating that the GABABreceptor agonist BCF attenuates AMPH- induced 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 psychostimulant- related behaviors associated with drug addiction. BCF pre-treatment reduces cocaine self-administration in rats responding under fixed ratio schedules [11], pro- gressive ratio schedules [2, 50], discrete trial sched- ules of reinforcement [10], and second-order sched- ules [18]. Additionally, BCF decreases AMPH self- administration under a fixed ratio or progressive ratio schedule [9] and attenuates conditioned locomotion in response to cues associated with cocaine administra- tion [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 transmis- sion within the VTA. Several lines of evidence sup- port 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 locomo- tor, reinforcement, and reward effects of psy- chostimulants such as cocaine and AMPH [17, 31, 35], and this system plays an important role in both the development and expression of psychostimulant- induced locomotor sensitization [46]. Second, the VTA contains primary DAergic neurons, which re- lease DA in the NAcc and prefrontal cortex, and sec- ondary GABAergic interneurons, which reduce the firing rate of DAergic neurons in the VTA. In addi- tion, GABAergic neurons arising from the NAcc proj- ect to DAergic neurons within the VTA [29, 33]. This loop represents an important locus in the production of certain abuse-related behavioral effects of psy- chostimulants [35]. Third, anatomical evidence sug- gests that GABAB receptors are located within the VTA [8, 27, 29, 42]. Fourth, biochemical and behav- ioral studies have found that infusion of BCF into the
VTA decreases DA release in the NAcc [59, 62], which suggests that the activation of GABAB recep- tors located on the cell bodies of mesolimbic DAergic neurons is involved in the biochemical effects of BCF.
Furthermore, microinjection of BCF into the VTA re- duces cocaine self-administration under fixed ratio [53] and progressive ratio schedules [12], and micro- injection of BCF was also found to reduce heroin self-administration [61] and AMPH-induced motor activity [30]. Altogether, these data support the hy- pothesis that the activation of GABAB 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 AMPH- induced locomotor sensitization.
In this study, it was also observed that a positive al- losteric modulator of GABAB receptors, CGP, in- creased the effects of a lower dose of BCF on AMPH-induced locomotor sensitization. It is worth noting that CGP did not alter AMPH-induced locomo- tor sensitization at any of the tested dosages. These results are consistent with those of at least one previ- ous study, in which the combination of a lower dose of BCF with a dose of CGP reduced ethanol self- administration [39]. Furthermore, it has been reported that CGP injections lead to a reduction of ethanol in- take in ethanol-preferring rats [39, 43], of cocaine self-administration in rats responding under several schedules of reinforcement [22, 54], of cocaine- seeking behavior [21], of nicotine-induced locomotor stimulation in mice [40], and of nicotine self-adminis- tration [45]. In addition, CGP treatment was shown to produce approximately 41% BCF-appropriate re- sponses but also to enhance the discriminative stimu- lus 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. Posi- tive allosteric modulators of GABABreceptors, such as CGP, display no intrinsic activity of their own but can modulate the activity of GABA or GABABago- nists, such as BCF, while avoiding the possible ad- verse side effects of BCF administration. In contrast to the data cited above, recent studies have shown that the administration of GS39783 alone, which is an- other positive modulator of GABABreceptors, signifi- cantly attenuates the locomotor activity induced by a single cocaine treatment and reduces modest co-
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 ethanol- induced locomotor sensitization [37].
It is important to note that the GABABreceptor is a G protein-coupled heterodimer composed of GABAB1 and GABAB2subunits, which have different functions [7]. The GABAB1subunit is essential for ligand bind- ing, while the GABAB2 subunit provides the G pro- tein-coupling mechanism and incorporates an alloste- ric modulatory site [47]. It has been suggested that the G protein-coupling mechanism of the GABAB2sub- unit involves an interaction with the extracellular do- main of the GABAB1subunit, 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 in- hibitory 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 ef- fect 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 re- leased onto these neurons is too low to activate the GABABreceptors. Positive modulators of GABABre- ceptors, such as CGP, are devoid of intrinsic activity, and their actions are dependent on the presence of en- dogenous GABA or other GABABagonists [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 GABABreceptors. The in- hibitory control of GABA over the VTA DAergic neu- rons can be enhanced in the presence of a positive al- losteric modulator of GABABreceptors, 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 syn- ergistically with GABA or with a GABABagonist, de- spite displaying no intrinsic activity of its own.
In conclusion, the results of the present study dem- onstrated that the GABABreceptor agonist BCF pre- vented both the development and the expression of AMPH-induced locomotor sensitization in a dose-
dependent manner. Furthermore, CGP, a positive al- losteric modulator of GABABreceptors, increased the effects of a lower dose of BCF on AMPH-induced lo- comotor sensitization under both conditions. These data provide further evidence that GABAB receptor ligands may modulate psychostimulant-induced be- haviors and that positive allosteric modulators of GABAB receptors may present pharmacological po- tential when combined with the use of conventional GABABagonists, such as BCF.
Acknowledgments:
This study was supported by grant 60872 from CONACyT (México) and a doctoral fellowship awarded to the first author (CONACyT, México).
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Received: June 22, 2012; in the revised form: April 5, 2013;
accepted: May 13, 2013.
