Our previous and other studies have demonstrated that moderate dosages of caffeine (15 and 20 mg/kg) induce locomotor sensitization. However, conditioned place preference was not reported with these dosages of caffeine. Instead, low dosage of caffeine (10 mg/kg), which is more in line with the amount normally ingested in beverages and food, can induce conditioned place preference but the locomotor sensitization has not been reported [2–6, 26]. In the present study, we showed that low dosage of caffeine (10 mg/kg) and low dosage of a selective adenosine A2A antagonist SCH58261 (2 mg/kg) elicited locomotor sensitization based on the observations that following chronic treatment with the test drugs and allowing for sufficient washout, acute challenge with the test drugs caused a larger response in the drug treated animals when compared to the vehicle-treated ones. Moreover, the expression of the sensitization was progressively enhanced when comparing the motor activity of the same animal on the first, 7th and 15th day following chronic treatment. Chronic treatment with a selective adenosine A1 antagonist DPCPX did not demonstrate locomotor sensitization. Our results suggest that chronic administration of low dosages of caffeine or SCH58261, which can induce CPP and behaviour sensitization, are able to elicit neuroadaptive changes similar to those observed with other psychostimulants. The behavioral sensitization of low dose of SCH58261 and the enhancement of acute caffeine-mediated response in SCH58261-sensitized mice strengthen our hypothesis that the effect of caffeine on behavioral reinforcing and sensitization may be mediated through adenosine A2A receptor.
Locomotor sensitization, proposed to reflect the increase of the wanting for drug reward, would result from an increase of the responsiveness of dopaminergic neurons to stimuli . Adenosine A2A receptors colocalized with dopamine D2 receptors in the medium-sized spiny GABAergic neurons are highly and selectively expressed in areas receiving a rich dopamine innervation, i.e., the dorsal and ventral striatum and tuberculum olfactorium [27–29]. Fenu and coworkers  have demonstrated that lower dose (10 mg/kg) but not higher dose (25 mg/kg) of caffeine and SCH58261 (3 mg/kg) can cross sensitized to a D2 dopamine agonist, bromocriptine. A strong antagonistic interaction between A2A and D2 receptors in the striatal projection neurons can explain the cross-sensitization between caffeine, or an A2A antagonist, and a D2 dopamine agonist. Activation of adenosine A2A receptors and dopamine D2 receptors produce the opposite response of increasing and decreasing the cAMP formation, respectively [31, 32]. This results in the opposite regulation of the activity of cAMP-dependent protein kinase involved in modulating the activity of numerous phosphoproteins and transcription factors, which control the expression of immediate early genes, such as c-fos and zif-268, leading to long-term adaptive responses [8, 10]. Consequently, antagonism of A2A receptors by caffeine and SCH58261 may directly facilitate the actions of D2 receptors on striatopallidal neurons. Therefore, it is reasonable to assume that chronic treatment with a selective A2A receptor antagonist, analogous to the chronic treatment with caffeine, can result in behavioral sensitization and cross-sensitization.
Our results also showed that chronic treatments with caffeine and SCH58261 increased the dopamine concentration and TH phosphorylation at Ser31 in the striatum in caffeine- and SCH58261-sensitized mice. Indeed, it has also been reported that 10 mg/kg of caffeine can reverse the catalepsy and decrease the activity produced by DA antagonists in rats [33, 34] and has effects on turning in unilateral 6-OHDA-lesioned rodents [35, 36]. Caffeine has been found to block the MPTP-induced decrease in the numbers of tyrosine hydroxylase-positive dopaminergic neurons in the striatum in mice . The dosage of 2 mg/kg SCH58261 can significantly improve the ability in an animal model of PD and enhance the therapeutic efficacy of L-DOPA . These observations indicated that in addition to mesolimbic dopaminergic pathway, caffeine in this dosage has effects on the nigrostriatal dopaminergic pathway, and is probably mediated by the adenosine A2A receptor. The effect of caffeine and SCH58261 on the neuroadaptation in the striatum, which is the target of mesolimbic and nigrostriatal dopaminergic pathways, may partially explain why they have behavioral sensitization, reinforcing and therapeutic effect in animal models of PD.
Most studies about caffeine and A2A antagonists focus on the neuroprotection against dopaminergic neurodegeneration in animal models of PD . In vivo, only two studies showed that chronic treatment with higher doses (25 and 50 mg/kg) of caffeine in rats significantly increased the DA in the striatum, whereas chronic lower dose of caffeine did not alter the DA content [22, 39]. Our previous studies showed that lower but not higher doses of caffeine can induce reinforcing and sensitization behavior. To reconcile the apparent discrepancy between the neuroadaptive and behavioral modifications, we chose the lower dosage of caffeine and demonstrated that chronic treatment with lower dose of caffeine (10 mg/kg) can increase the striatal DA in mice. Difference in the animal species and the use of internal standard (2, 3-dihydroxybutyric acid) for recovery of DA in the HPLC quantitation in our study may partially explain the discrepancy.
We also demonstrated that chronic treatment of caffeine and a selective A2A antagonist enhance the phosphorylation level of tyrosine hydroxylase at Ser31. Phosphorylation of TH is likely to be of physiological importance in maintaining catecholamine stores because TH is the rate-limiting enzyme in catecholamine biosynthesis and its activity is increased by phosphorylation . TH is phosphrylated at multiple sites. A recent study on intact bovine adrenal chromaffin cells has identified four phosphorylation sites on TH, at Ser8, Ser19, Ser31, and Ser40 . Treatment that increase Ser31 or Ser40 phosphorylation but not the others increase TH activity and catecholamine biosynthesis, and ERK-mediated phosphorylation of Ser31 play a role in dopaminergic related neurological disease . For example, chronic administration of morphine or cocaine increases phosphor-ERK immunoreactivity in the VTA , suggesting that dopamine biosynthesis may be elevated in this region. An earlier study has demonstrated that chronic treatment with caffeine (20 and 80 mg/kg for 9 days) increased the tyrosine hydroxylase mRNA levels in both the substantia nigra pars compacta and the ventral tegmental area .
In vitro, caffeine at mM concentrations can activate tyrosine hydroxylase in bovine chromaffin cells . Functional striatal hypodopaminergic activity was noted in mice with genetic deletion of adenosine A2A receptors . However, genetic deletion of adenosine A2A receptors results in persistent rather than transient and intermittent antagonism of the receptor and, in addition, in such study adenosine A2A receptors affected basal extracellular dopamine concentration but not total dopamine concentration in striatum. Our findings, together with previous studies, make it plausible that caffeine through adenosine A2A receptor-mediated phosphorylation of TH at Ser31, results in the dopaminergic neuroadaptations related to the treatment of PD and mechanism of drug dependence/addiction.
In conclusion, our study demonstrates that low dosages of caffeine and a selective adenosine A2A antagonist SCH58261 induce sensitization and cross-sensitization of locomotor activity, which are associated with elevated dopamine concentration and phosphorylation of TH at Ser31 in the striatum. Blockade of adenosine A2A receptor may play an important role in the striatal neuroadaptations observed in the caffeine- and SCH58261-sensitized mice.