The behavioral pharmacological profile of a drug in a pertinent species is necessary for evaluating quantitatively not only how the drug functions as a reinforcer but also how use or abuse of the drug affects other aspects of the subject's behavior. Ongoing studies on the direct behavioral effects of drugs include a number of different paradigms, including two lever, choice, drug-discrimination behavior, operant behavior maintained by food or by postponement of avoidance of electric shock, and in vivo microdialysis measurements of neurotransmitter levels in discrete brain areas. In previous studies we have used laboratory animals to characterize interactions of caffeine with the effects of other psychomotor stimulants under conditions of acute and chronic caffeine exposure. Under specific schedules of administration caffeine was found to potentiate motor-activating, discriminative-stimulus and reinforcing effects of other psychostimulants, like amphetamine, cocaine and nicotine. At the same time we have been interested in the mechanism of action underlying these potentiating effects of caffeine and in previous studies we first found that behaviorally relevant doses of caffeine, produced dopamine release in the shell of the nucleus accumbens, which is usually correlated with rewarding effects of psychotropic drugs. Although the main current hypothesis is that adenosine A2A receptor blockade is the main mechanism responsible for the central effects of caffeine, its dopamine-releasing properties were found to be dependent on blockade of adenosine A1 receptors. Therefore, we have been conducting behavioral experiments to reevaluate the mechanism of action of caffeine responsible for its motor-activating and discriminative-stimulus effects by comparing the effects of caffeine with the effects of selective adenosine A1 and A2A receptor antagonists. Our findings to date underscore the role of A1 receptors in the behavioral and subjective effects of caffeine. Thus, the pharmacological profile of an acute systemic administration of caffeine has been that of an A1 receptor antagonist plus a weak A2A receptor antagonist in our studies. A2A receptors did not mediate, but, instead, played a permissive role in the motor-activating effects of caffeine. A1 receptor blockade was also clearly involved in the discriminative-stimulus effects of a motor-activating dose of caffeine. On the other hand, A2A receptor blockade was not involved and, in fact, counteracted the A1 receptor-mediated discriminative-stimulus properties of caffeine. We have also analyzed the role of A1 and A2A receptors in the effects of chronic administration of caffeine (1 mg/ml in the drinking water for 14 days). Our results suggest that development of tolerance to the effects of A1 receptor blockade might be mostly responsible for the tolerance that develops to the motor-activating effects of caffeine and that the residual motor-activating effects of caffeine in tolerant individuals are mostly attributable to A2A receptor blockade. A close correlation to our behavioral findings was found in our recent in vivo midrodialysis findings. The systemic administration of motor-activating doses of an A2A receptor antagonist significantly decreased extracellular levels of dopamine and glutamate in the shell of the rat nucleus accumbens and counteracted both dopamine and glutamate release in the nucleus accumbens induced by the systemic administration of motor-activating doses of either the A1 receptor antagonist CPT or caffeine. Furthermore, chronic exposure to caffeine in the drinking water was associated with tolerance to the effects of the acute administration of the A1 receptor antagonist and caffeine, but not of the A2A receptor antagonist, on the extracellular levels of dopamine and glutamate in the shell of the nucleus accumbens. These biochemical data confirm: first, the existence of opposite tonic effects of adenosine on extracellular concentrations of dopamine and glutamate in the shell of the nucleus accumbens that are mediated by A1 and A2A receptors; second, that A1 receptors mediate the dopamine- and glutamate-releasing effects of motor-activating doses of caffeine; third, that, at motor-activating doses, caffeine is a preferential A1 receptor antagonist. The results also suggest that the development of tolerance to the dopamine-releasing effects of caffeine in the shell of the nucleus accumbens could explain its weak addictive properties and atypical psychostimulant profile. Finally, as previously reported for caffeine, A1 and A2A receptor antagonists significantly potentiated the discriminative-stimulus effects of methamphetamine and cocaine (shift of the dose-response curves for methamphetamine and cocaine to the lef). Furthermore, both A1 and A2A receptor antagonists produced significant generalization to the drug-training stimuli, i.e., they substituted for methamphetamine and cocaine in the stimulus-discrimination task. Nevertheless, the A1 receptor antagonist was more potent than the A2A receptor antagonist in producing generalization to both the methamphetamine- (three times) and cocaine- (two times) training stimuli. We are now planning to evaluate the effects of selective A1 and A2A receptor antagonists and of acute and chronic treatment with caffeine on the stimulating effects of amphetamine or cocaine on motor-activity and on extracellular concentrations of dopamine and glutamate in the shell of the nucleus accumbens. At a more molecular level, we have recently provided evidence for the existence of heteromeric receptor complexes between subtypes of adenosine, dopamine and glutamate receptors. In the case of interactions between adenosine A2A and dopamine D2 receptors, we were able to obtain results, not only at the levels of behavior, brain neurochemistry and cellular function, but also at the protein level, with the demonstration of true A2A-D2 receptor heterodimers. These hererodimers result from an epitope-epitope electrostatic interaction between a strech of adjacent basic residues in the third intracellular loop of the dopamine D2 receptor and two adjacent aspartic acid residues and a serine that can be constitutively phosphorylated in the carboxyl terminus of the A2A receptor. By means of mass spectrometry, we demonstrated that short peptides, corresponding to sequences of the D2 and A2A receptors, interact efficiently. The results obtained by mass spectrometry were confirmed by using different constructs of the receptors in biochemical pull-down assays. Also, the interaction between wild type A2A receptor and an Arg-rich peptide of the D2 receptor was displaced by two peptides corresponding to the two different sequences in the carboxyl terminus of the A2A receptor. These results are the first example of epitope-epitope electrostatic interaction underlying receptor heteromerization.