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Neural Mechanisms of Action of Drugs of Abuse and Natural Reinforcers, 2008: 89-105 ISBN: 978-81-308-0245-9 Editors: Milagros Méndez Ubach and Ricardo Mondragón-Ceballos

Non-dopaminergic modulation of the discriminative properties 5 of

David N. Velázquez-Martínez1, Florencio Miranda2 Hugo Sánchez Castillo1 and Gabriela Orozco Calderón1 1Departamento de Psicofisiología, Facultad de Psicología, Universidad Nacional Autónoma de México, México, D.F. 04510, México; 2Facultad de Estudios Superiores Iztacala, Universidad Nacional Autónoma de México, Av de los Barrios No.1, Los Reyes-Iztacala, Tlalnepantla, Estado de México, 54090 México

Abstract Psychostimulants induce a direct or indirect increase in (DA) synaptic and extrasynaptic levels in mesocorticolimbic areas affecting the reward and appetitive system of the brain. Despite their therapeutical use, dependency, addiction and abuse to psychostimulants had become a worldwide problem that limits their usage. The modulation of the

DA neurotransmission through non-DA mechanisms

Correspondence/Reprint request: Dr. David N. Velázquez-Martínez, Departamento de Psicofisiología, Facultad de Psicología, Universidad Nacional Autónoma de México, México, D.F. 04510, México E-mail: [email protected] 90 David N. Velázquez-Martínez et al. may be of help in the treatment of addiction. Since the reinforcing properties of drugs have a concomitant subjective effect, the direct study of the stimulus properties of the drug has a predictive value for the abuse liability of the drug. The main objective here is to review evidence of the modulation of the discriminative properties of cocaine and amphetamine by , γ-aminobutyric acid (GABA) and glutamate that may give us some insight for the treatment of addiction.

Introduction Psychostimulants induce excitation of the Central Nervous System (CNS) and a subjective experience of awareness and welfare [1]. Examples of natural psychostimulants are cocaine, nicotine, and caffeine, while synthetic ones are amphetamine (AMPH)-related drugs. AMPHs had been used to improve physical fitness, delay the onset of the sleep period, increase mental activity, reduce fatigue and have therapeutic use in obesity, narcolepsy, depression and attention deficit disorder [1]. However, tolerance and dependence is rapidly generated and dependency and addiction has become a world problem. Besides biological factors, social environment, subject’s personality and personal history are important in the determination of dependency and addiction. Therefore an effective treatment for addiction should include diverse factors and approaches, but basic knowledge of their actions is essential for therapeutics.

Neurobiological mechanisms of psychostimulants Psychostimulants induce a direct or indirect increase in dopamine (DA) synaptic and extrasynaptic levels in mesocorticolimbic areas affecting the reward and appetitive system of the brain [2,3]; the indirect stimulation of DA receptors is particularly seen in the nucleus accumbens (NAcc) [4]. Cocaine and AMPH are indirect monoamine with affinity for the reuptake transporters of DA (DAT), but are also able to inhibit the norepinephrine (NET) and serotonin (SERT) transporters increasing the extracellular levels of DA, NE and serotonin (5-HT) [5-8]. AMPH also blocks the transporters located in presynaptic vesicles inducing an increase in cytoplasmatic monoamine concentrations and being able to revert the membrane transport system [9-11]. Both natural reinforces (such as food, water, sex, among others) and psychostimulants increase extracellular levels of DA in NAcc. Other brain areas such as the hypothalamus, the frontal cortex, and the amygdala also contribute to the reward system, but the mesocorticolimbic pathway leading DA fibers from the ventral tegmental area (VTA) to the NAcc are of Non-DA modulation of amphetamine cue 91 particular importance [12]. Additionally, VTA neurons are regulated by a DA feedback loop from the NAcc and this loop is regulated, among several others, by 5-HT, γ-aminobutyric acid (GABA) and glutamate (Glu) projecting pathways [see 13]. There is no direct pharmacological treatment for addiction, since the use of DA antagonists is highly aversive in addicts, besides that their prolonged use leads to the development of parkinsonism [14]. It has been suggested that in order to diminish the reinforcement properties of psychostimulants an alternative strategy may be through non-DAergic afferents that may modulate the DA mesocorticolimbic pathway. The main objective here is to present evidence of the modulation of the discriminative properties of AMPH by 5-HT and Glu that may give us some insight for the treatment of addiction.

Drug discrimination as a behavioral model of interoceptive effects of psychostimulants During training in the Drug Discrimination paradigm, reinforcement is correlated to responding to one alternative if drug A is injected, but when a different drug or saline is given, reinforcement is correlated with emitting an alternative response. That is, the drug is able to induce an internal “drug- state” that the subject uses as an indicator of which response will be reinforced. If administered with a similar drug, we expect subjects will respond in a similar way to the training drug, based on the assumption that similar drugs induce similar drug-states [15]. After training, during generalization or a substitution test, a different drug is given, while in combination tests another drug is given in conjunction with the training drug [16,17]. In has been described that AMPH is able to induce stimulus control in both humans [18] and animals [19,20]. Usually rats are trained in a discrimination test between 1 mg/kg of d-AMPH and saline in a two-lever operant task (Fig. 1A, C) or using a Conditioned Taste Aversion (CTA) procedure (Fig. 1B, D). Figure 1A presents data from our group. At the beginning of training, rats emitted random responses to either lever under both d-AMPH or saline, but as training progressed, and by the end of training, they emitted responses only to the d-AMPH lever when injected with d-AMPH and responded to the saline-lever under saline sessions. After training, different doses of d-AMPH were evaluated in generalization sessions. As shown on Figure 1C, after 0.1 mg/kg of d-AMPH, animals responded as if saline was injected, but as dose increased (up to 3 mg/kg) the preference for the d-AMPH-correlated lever increased, showing that d-AMPH induced a dose-dependent stimulus control. 92 David N. Velázquez-Martínez et al.

Figure 1. A: Acquisition of d-amphetamine-saline discrimination using an operant procedure. Data are means ± SEM of 8-10 rats. A one-way ANOVA revealed significant differences (F (5,40)=48.409 p < 0.001) and the Duncan test confirmed significant (p < 0.01) differences for the last 5 sessions from each condition. C: Generalization test to different doses of amphetamine (Drug: D; Saline: S). A one- way ANOVA revealed significant differences (F(5,65)=75.878 p<0.001) and the Duncan test confirmed that performance after 0.1 and 0.3 mg/kg had significant (p<0.01) differences to drug-training sessions. B: Acquisition of d-amphetamine- saline discrimination using CTA procedure. A one-Way ANOVA revealed significant (F(5,35)=11.753 p<0.001) differences and the Duncan test confirmed that the last 3 sessions from each condition attained significant (p<0.01) differences. D: Results of substitution tests with amphetamine in the CTA procedure. A one-way ANOVA revealed significant (F(4,28)=12.566 p < 0.001) differences and the Duncan test confirmed significant (p<0.01) differences from 0.1 and 0.3 mg/kg performances to drug training sessions. Saccharin preference (discrimination index) was calculated according to the formula A/(A+B), where A is saccharin intake and B water intake. With this formula, a value close to 0 indicates a strong aversion to saccharin, while a value close to 1 indicates strong preference for saccharin.

An alternative method of training is the use the CTA procedure. Water deprived rats are given 1 mg/kg of d-AMPH, then given access to a saccharin solution and, immediately after access, they are injected with LiCl to induce toxicosis. On alternating days, the same rats have access to saccharin Non-DA modulation of amphetamine cue 93 solution followed by administration of saline. In a few sessions rats learn to discriminate their pharmacological state and began to avoid saccharin after drug injections but showed an increased preference for saccharin when injected with saline. It has been shown that diverse drugs such as phencyclidine [21,22], 5-HT agonists [23], [24], [25] and AMPH [20], among others, are able to induce stimulus control with the CTA procedure. Figure 1B shows data from our laboratory, on the acquisition of d-AMPH (1 mg/kg) versus saline discrimination using the CTA procedure [26]. d-AMPH is able to induce discriminative control over saccharin intake in a few trials. When the administration of d-AMPH preceded saccharin-LiCl pairing, the subjects decreased their saccharin intake, while saccharin intake increased after saline administration. Once rats learned this discrimination, different doses of d-AMPH were evaluated in generalization tests (Fig. 1C). It was observed that different doses of d-AMPH produced a dose-dependent stimulus control.

5-HT modulation of psychostimulants Observations that 5-HT neurons innervate the DA mesoaccumbal pathway [27,28] and additional biochemical, electrophysiological and behavioral evidence suggests that 5-HT receptors may modulate the neurochemical and reinforcing effects of psychostimulants increasing or decreasing their behavioral effects [29-31]. This modulation is complex, since it depends on the differential participation of some of the 16 different 5-HTR subtypes that mediates 5-HT neurotransmission [32]. Specifically, the 5-HT1A, 5-HT1B, 5-HT2A, 5-HT2C and 5-HT3 receptors may be involved [33-36]. Microdialysis studies have shown an increase in DA and 5-HT in the prefrontal cortex, the striatum and the NAcc after AMPH or cocaine, but this effect is reduced after administration of 5-HT1A [37], 5-HT2A [38] or 5-HT3 [39] drugs. Hyperlocomotion induced by AMPH or cocaine is also inhibited by 5-HT1A agonists [40,41], 5-HT2A [42,43] and 5-HT3 [44] drugs. AMPH or cocaine are able to induce conditioned place preference (CPP: pairing a specific environment with a rewarding event) [45]. However, CPP to cocaine does not occur in 5-HT1BR knockout (KO) mice [46] or in mice that overexpress 5-HT3 receptor [47]. Additionally, although it has been suggested that the addictive, reinforcing and discriminative effects of psychostimulants are related to an increase in DA neurotransmission in the NAcc, Rocha (2003) [48] has shown that DAT-KO mice are still able to autoadminister cocaine, but a combined deletion of DAT and SERT (or NET) eliminates autoadministration in KO- mice, suggesting the importance of 5-HT mechanisms in the reinforcing effects of psychostimulants. Our main purpose was to use the drug discrimination paradigm to study such modulation. 94 David N. Velázquez-Martínez et al.

Figure 2. Left panel shows the effects of the administration of 8-OH-DPAT to rats trained to discriminate d-amphetamine (1 mg/kg intraperitoneal: ip) from saline (Training dose of d-AMPH: C; Saline: V). Repeated measures ANOVA revealed significant (F(4,53)=63.309 p < 0.001) differences and the Duncan test confirmed significant (p < 0.01) differences of drug training condition to 0.1-1 mg/kg dose of 8-OH-DAPT. Right panel shows the effects of the combined administration of 8-OH-DPAT and d-amphetamine. Repeated measures ANOVA showed significant (F(3,40)=5.317 p < 0.05) differences (Duncan test paired contrasts: **, p < 0.05) from control performance. Data are means ± SEM of 8 subjects.

Modulation of discriminative effects of amphetamine by 5-HT1A receptors The highest densities of the 5-HT1A receptor in humans are located in the hippocampus and the neocortex, where receptors are postsynaptic to 5-HT neurons [49], but in the raphe nuclei, they are autoreceptors [50,51]. In animal studies, 5-HT1A agonists increase prefrontal DA release [51] and increase DA cell firing in the VTA [35,52]. Accordingly, the selective 5-HT1A , repinotan, increases the firing rate and burst firing of DA neurons in the VTA and DA release in the VTA and the medial prefrontal cortex (mPFC), while the 5-HT1A antagonist, N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-2- pyridinylcyclohexanecarboxamide maleate (WAY-100635), reversed the effects of repinotan in both areas [53]. Interestingly, application of repinotan in the mPFC increased extracellular DA at a 3 µM concentration, but reduced it at a higher concentration (30 µM) [53]. From their location and the observed modulation of firing activity and release we should expect that 5-HT1A receptor may be able to modulate the effects of d-AMPH. Figure 2 shows the interaction of d-AMPH with 8-hydroxy-2-dipropylaminotetralin (8-OH-DPAT), a 5-HT1A agonist. The left panel shows that the administration of 8-OH-DPAT did not Non-DA modulation of amphetamine cue 95 mimic the stimulus properties of d-AMPH, since the rats responded as if they received saline. The right panel shows the effects of the combined administration of 8-OH-DPAT and d-AMPH. Data should lie along the horizontal line at 0 difference if the administration of 8-OH-DAPT had no effect, but, as observed, 8-OH-DPAT increased or decreases the effects of low and high dosels of d-AMPH, respectively. In a different behavioral model, the prototypical 5-HT1A receptor agonist, 8-OH-DAPT, prevented -induced catalepsy in a dose-dependent manner [54]. This effect can be blocked by the 5-HT1A/1B antagonist [55] and by WAY-100635 [56]. The mechanism by which 5-HT1A agonists reduce catalepsy induced by DA antagonists or increased the discriminative properties of AMPH is not clear. Stimulation of 5-HT1A autoreceptors in the raphe may be important given that 8-OH-DPAT applied locally reduced neuroleptic-induced catalepsy [57]. A plausible mechanism for the anticataleptic effect involves a 5-HT-DA interaction, in which the stimulation of 5-HT1A autoreceptors reduces the activity of the 5-HT projections that inhibit DA nigrostriatal neurons through postsynaptic 5-HT2 receptors [58]. The observed decrease in the stimulus properties of AMPH by 8-OH-DPAT may be related to the stimulation of postsynaptic 5-HT1A receptors.

Modulation of discriminative effects of amphetamine by 5-HT2 receptors The 5-HT2A receptors are present on DA neurons of the VTA and the substantia nigra [59,60], as well as in the target regions of the DA pathways, including the prefrontal cortex and the NAcc [60,61]. They may modulate DA activity [62], since it has been described that the stimulation of 5-HT2A receptors influences DA release in the prefrontal cortex and the striatum [63,64]. It was also observed that the increase in DA release induced by cocaine or AMPH is prevented by 5-HT2A antagonists [38,65,66]. At a behavioral level, 5-HT2A antagonists have been shown to antagonize d-AMPH-induced hyperlocomotion [42,43] and to reverse its effect on latent inhibition [43]. In rats trained to discriminate cocaine (0.3 or 1 mg/kg) from saline, the 5-HT2A antagonists, and , did not generalize to cocaine when administered alone. Although, given in combination with cocaine, ketanserin attenuated the discriminative effects of cocaine in most subjects, while ritanserin attenuated the discriminative effects of cocaine in subjects trained at the higher dose [67]. Later it was confirmed that R-(+)-alpha-(2,3-dimethoxyphenyl)-1-[2-(4- fluorophenylethyl)]-4-piperidine methanol (MDL-100907) (0.05-1.6 mg/kg) or ketanserin (0.05-4 mg/kg) attenuated the stimulus effects of cocaine (5 mg/kg) [68]. Conflicting results have also been reported, since ketanserin (3 mg/kg) 96 David N. Velázquez-Martínez et al. produced a significant shift to the right of the cocaine dose-response curve, while different doses (1-10 mg/kg) of ketanserin did not blocked the discriminative- stimulus effects of the training dose of cocaine [69]. Another conflicting result is the observation that the stimulus properties of AMPH were not reverted by ketanserin [70] or MDL-100907 [43]. The latter was also unable to reverse the AMPH suppressant effect on overall response rate [71]. Figure 3 shows the interaction of d-AMPH with ketanserin. When given alone, ketanserin did not produce generalization in d-AMPH trained subjects (left panel of Fig. 3). When given in combination with d-AMPH, the low doses of ketanserin diminished the discriminative cue of medium doses of d-AMPH, while responses induced by higher doses of d-AMPH were unaffected by ketanserin (right panel of Fig. 3). The conflicting results may be related to the affinity and/or dose range used of the antagonist, as shown by our results. Interestingly, however, 5-HT2A antagonists alone have generally been found to have little effect on basal DA function [72-74], although there have been some contradictory reports [75,76]. It seems that 5-HT2A receptor stimulation has a facilitatory effect on DA release under certain conditions, but has little effect on basal DA activity [77], that may also be related to the differential effects observed at different doses of the antagonists.

Figure 3. The left panel shows the effects of the administration of ketanserin to rats trained to discriminate d-amphetamine (1 mg/kg ip) from saline (training dose of d-AMPH: C; Saline: V). Repeated measures ANOVA revealed significant (F(4,57)=78.002 p<0.001) differences and the Duncan test confirmed that performance after all doses of ketanserin were different (p<0.01) from drug training condition. The right panel shows the effects of the combined administration of ketanserin and d-amphetamine. Repeated measures ANOVA showed significant (F(3,43)=5.456 p < 0.005) differences (Duncan test paired contrasts: **, p < 0.05) from control performance. Data are means ± SEM of 8 subjects. Non-DA modulation of amphetamine cue 97

Figure 4. The left panel shows the effects of the administration of mCPBG to rats trained to discriminate d-amphetamine (1 mg/kg ip) from saline (training dose of d-AMPH: C; Saline: V). Repeated measures ANOVA revealed significant (F([5,56)=79.053 p<0.001) differences and the Duncan test confirmed that performance after 0.1, 0.3 and 1 mg/kg doses of mCPBG were different (p<0.01) from drug training condition. The right panel shows the effects of the combined administration of mCBG and d-amphetamine or mCPBG+d-amphetamine+ MDL72222. Repeated measures ANOVA showed significant (F(3,37)=19.821, p < 0.001) differences (Duncan test paired contrasts: **, p < 0.05) from control performance. Data are means ± SEM of 8.

Modulation of discriminative effects of amphetamine by 5-HT3 receptors In previous studies it was found that the 5-HT3 antagonists or 5-HT3 antagonist 3-tropanyl-3,5-dichlorobenzoate (MDL72222) did not produce generalization in cocaine trained rats [78]. Also, neither tropisetron [78], 1-Methyl-N-(8-methyl-8-azabicyclo[3.2.1]-oct-3-yl)-1H-indazole-3- carboxamide maleate (LY-278,584) [79], [79], nor MDL72222 [78-80] were able to antagonize the stimulus effects of the training dose of cocaine (from 1 to 5 mg/kg). Moreover, it was also described that MDL72222 failed to alter the discriminative stimulus effects of methamphetamine [81]. Few studies had tested the effects of 5-HT3 agonists on the effects of psychostimulants. For example, it was found that in substitution tests, m-chlorophenilbiguanide (mCPBG, 2.5-20 mg/kg) showed partial substitution for the cocaine stimulus [36,82]. However, in combination tests conflicting results emerged, since some found that the combination of mCPBG with low doses of cocaine resulted in increased cocaine lever 98 David N. Velázquez-Martínez et al. selection [36], while others found that neither MDL72222 (10 mg/kg) or ondansetron attenuated the cocaine cue [82]. Figure 4 shows results from our labortory on the interaction of d-AMPH with mCPBG. We found that at high doses (10 mg/kg), mCPBG is able to induce partial generalization of the discriminative stimulus properties of d-AMPH. This effect has also been described for cocaine [36]. When the 3 mg/kg dose of mCPBG is given in combination with the 0.3 mg/kg dose of d-AMPH, it is able to decrease the selection of d-AMPH-lever, and this effect is reverted by MDL72222 (3 mg/kg). An earlier study found that none of the 5-HT3 antagonists tested, 3-tropanyl-3,5-dichlorobenzoate methyl quaternary ammonium MDL72222EF) (0.3-10 mg/kg), mesilate, tropisetron, or ondansetron, antagonized the effects of AMPH [83]. However, a recent study found that the 5-HT3 partial agonist N-(3-chlorophenyl)guanidine (MD-354) neither substituted for, nor antagonized, the AMPH stimulus, although its administration with some doses of AMPH shifted the AMPH dose-response curve to the left. Furthermore, MD-354 at doses of 0.1, 0.3 and 1 mg/kg, but not at doses of 0.01, 0.5, 1.5 or 3 mg/kg, administered in combination with the effective dose 50% (ED(50)) of AMPH (0.33 mg/kg) resulted in stimulus generalization. This patterning of effects is similar to our findings with mCPBG and d-AMPH. In addition, we showed that MDL72222 antagonized the effects of mCPBG, demonstrating that the effects are mediated through 5-HT3 receptors. It is concluded that, even though some 5-HT3 agonists lacks AMPH-like central stimulant actions on their own, they can, at certain doses, modulate the discriminative stimulus effects of AMPH [84].

Glutamate neurotransmission involved in the discriminative properties of amphetamine As it was previously mentioned, the mesolimbic DA system is known to be an important mechanism for the production of locomotor, reinforcing, rewarding and discriminative stimulus effects of psychostimulants like cocaine and AMPH [2]. There is now evidence that Glu-DA interactions play a critical role in some behavioral effects of psychostimulants as AMPH and cocaine. It is known that KO mice lacking the mGlu receptor subtype 5 (mGluR5) do not self-administer cocaine [85]. On the other hand, the selective mGluR5 receptor antagonist, 6-methyl-2-(phenylethynyl)pyridine (MPEP), inhibits the stimulant effects of cocaine and AMPH in locomotor activity of mice [86]. We evaluated the effects of MPEP on the discriminative stimulus properties of d-AMPH using CTA as the drug discrimination procedure [26]. After rats learned d-AMPH-saline discrimination with the CTA procedure, d-AMPH was substituted by different doses of MPEP or a combination of Non-DA modulation of amphetamine cue 99

Figure 5. Saccharin preference (discrimination index) during generalization tests with MPEP (left panel) and d-amphetamine + pretreatment with MPEP (right panel; training dose of d-amphetamine: d-amph; Saline: V). Single administration of MPEP (F(4,36)=8.034 p < 0.01) or MPEP+d-amphetamine administration (F(4,36)=8.985 p < 0.001) produced significant differences in saccharin preference compared to the training dose of d-AMPH (Newman-Keuls test: *, p< 0.05 vs d-amphetamine). Data are means ± SEM of 10 rats.

MPEP+d-AMPH. The results show that MPEP did not substitute for d- AMPH (Fig. 5, left panel), while the pretreatment with MPEP reduced the discriminative signal of d-AMPH in a dose-dependent fashion (Fig. 5, right panel). These results demonstrated that MPEP reduces the discriminative signal of d-AMPH and confirm the attenuation of locomotor, reinforcing, rewarding and discriminative effects of psycostimulants as AMPH and cocaine by MPEP [86,87] and provide further evidence for a role of mGluR5 in modulating the behavioral effects of psychostimulants related to their abuse. It has been suggested that the mechanism underlying MPEP effects on psychostimulant-related behaviors could involve Glu-DA interactions in the NAcc. The NAcc receives Glu projections from the prefrontal cortex, the hippocampus and the amygdala and also receives DAergic projection from the VTA. Both DA and Glu terminals synapse on common neurons in the NAcc. In addition, mGluR5 are localized in the NAcc. Thus, it could be possible that blockade of mGluR5 by MPEP regulates psychostimulant- induced behaviors by interfering with DA neurotransmission, although more research is needed to reach a definitive conclusion.

GABA neurotransmission involved in the discriminative properties of amphetamine Recent evidence also suggests a potential role for GABA neurotransmission to modulate some behavioral effects of psychostimulants. 100 David N. Velázquez-Martínez et al.

Thus, it has been reported that GABAB receptor agonists are effective in attenuating some behavioral effects of psychostimulants related to its abuse. For example, the selective GABAB agonist baclofen reduces the reinforcing effects of cocaine [88], nicotine [89], methamphetamine [90] and AMPH [88]. Figure 6 shows the effect of d-AMPH with baclofen on d-AMPH discrimination in rats using CTA as the drug discrimination procedure [91]. After rats learned d-AMPH-saline discrimination, d-AMPH was substituted by different doses of baclofen (data not shown) or a combination of baclofen+d-AMPH. The results show that baclofen did not substitute for d- AMPH, but the pretreatment with baclofen reduced the discriminative signal of d-AMPH in a dose-dependent fashion (Fig. 6, left panel). To determine whether these effects result from the specific action of baclofen on GABAB receptors, we also evaluated the effects of the selective GABAB antagonist, 2-hydroxysaclofen, on the effects of baclofen on d-AMPH discriminative signal. As it can be noted in Figure 6 (right panel), the administration of 2-hydroxysaclofen produced a dose-dependent reduction of the effects of baclofen on d-AMPH induced discriminative signal. The results of the present study are in agreement with the attenuation of locomotor, reinforcing and rewarding effects of psychostimulants as AMPH and cocaine by GABAB agonists such as baclofen or (3-amino-2(S)-hydroxypropyl)methylphosphinic

Figure 6. Results of substitution tests with baclofen + d-amphetamine (left) and 2-hydroxysaclofen + baclofen + d-AMPH (right). Single administration of baclofen (F(3,27)=5.867 p < 0.01) and combined administration of 2-hydroxysaclofen + baclofen (F(3,27)=6.62 p < 0.05) produced significant differences in saccharin preferences compared to single d-AMPH treatment (Newman-Keuls test: *, p<0.05 vs 1 mg/kg AMPH alone). Data are means ± SEM of 10 rats. Non-DA modulation of amphetamine cue 101 acid (CPG 44532) [92-94]. Also, our results with 2-hydroxysaclofen are in accordance with the observation that pretreatment with the GABAB antagonist [[3-[1-(S)-[[3-cyclohexylmethyl)hydroxy phosphinyl]-2-(S) hydroxy propyl]amino]ethyl]benzoic acid (CGP56433A) attenuated the effects of baclofen on cocaine self-administration. The mechanism underlying baclofen effects on d- AMPH-induced discriminative signal could involve stimulation of GABAB receptors in the VTA. There is evidence that GABAB receptors are located postsynaptically in cell bodies of VTA neurons that project to the NAcc. Activation of these receptors decreases extracellular DA levels in the NAcc [95-97]. Thus, it is possible that baclofen administration can produce hyperpolarization of DA neurons in the VTA and inhibit DA release in the NAcc. Therefore, baclofen attenuates the AMPH-induced discriminative signal. This suggestion is supported by a previous report which demonstrated that microinjections of baclofen into the VTA reduced NAcc DA levels [98]. However, baclofen might also be acting on other sites such as the prefrontal cortex.

Conclusions As mentioned previously, it has been suggested that the reinforcing properties of psychostimulants are related to a facilitating effect of DA neurotransmission. Several neurotransmitter systems (including, but not limited to, NA, 5-HT, Glu, and GABA) may modulate the effects of the psychostimulants through their direct or indirect effects upon DAergic mechanisms. Here, we provide evidence that the stimulus properties of AMPH (established through an operant or CAS procedure) may be modulated by several 5-HT receptor subtypes. While the functional significance of 5-HT receptors has not been fully elucidated, data from several groups demonstrated that changes in 5-HT activity modulate the effects of psychostimulants under a variety of experimental conditions. One commonality among the studies with positive findings is that the effects of AMPH or cocaine are only partially modified by 5-HT agents, regardless of the direction of change. There is growing evidence that Glu and GABA may also modulate the effects of psychostimulants. Although the evidence is not conclusive, revised results open the possibility that in the near future some non-DAergic drugs may be useful in the treatment of addiction.

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