The purpose of this study was to investigate the effects of the adenosine A 2A receptor antagonist istradefylline on cognitive deficits due to dysfunction of cortical dopaminergic transmission. The main findings are as follows: (1) intra-prefrontal cortex injection of 6-OHDA induced a significant reduction in dopamine levels in the PFC and working memory impairment assessed by the object recognition task and the delayed alternation task; (2) istradefylline, as well as donepezil and methamphetamine, improved the cognitive performance of PFC-lesioned rats and (3) istradefylline increased dopamine levels in the PFC in normal and PFC-lesioned rats. These findings suggest that istradefylline can improve impaired cognitive function associated with dopamine loss in the PFC of 6-OHDA lesioned rats.

The overall role of adenosine A 2A receptors in the control of cognitive function is not well understood. Pharmacological inactivation of A 2A receptors with selective antagonists has been reported to enhance short-term social memory and enhance long-term memory in passive avoidance (Prediger et al. 2005a; Prediger and Takahashi 2005; Kopf et al. 1999). Selective A 2A antagonists also improved the deficits in short-term social memory and spatial learning in spontaneously hypertensive rats and in aged rats (Takahashi et al. 2008; Prediger et al. 2005b, c). For working memory, the evidence is conflicting as selective A 2A antagonists did not reverse the scopolamine-induced reduction in spontaneous alternation in a mouse Y-maze task, but they did reverse MK-801-induced reduction in spontaneous alternation (Fraser et al. 1997). However, previous studies have demonstrated that overexpression of A 2A receptors in the forebrain causes impairment of working memory in an object recognition task, radial maze task and the Morris water maze, whereas genetic inactivation of A 2A receptors enhances working memory in the radial maze and Morris water maze with no effect on reference memory (Wei et al. 2011; Zhou et al. 2009; Giménez-Llort et al. 2007). Overall, these findings suggest that A 2A receptors negatively modulate cognitive function, especially working memory.

In this study, we used two different working memory tasks that evaluated prefrontal function to assess the effects of istradefylline in the PFC of 6-OHDA lesioned rat. The object recognition test is based on spontaneous exploratory behaviour of rats toward a novel object, and it is considered as a “pure working memory task” that has no reference memory component, such as rule learning, and positive or negative reinforcement (Ennaceur and Meliani 1992). This task has been reported to be sensitive to changes in dopaminergic dysfunction in the PFC but not to hippocampal lesions (Ventura et al. 2004; Mumby 2001; Ennaceur and Meliani 1992). Consistent with previous reports, dopaminergic lesions of the PFC significantly worsened the performance of rats in the object recognition task with a 3-min retention interval. Performance in this task might be affected partially by physical and emotional states such as locomotor activity and anxiety. However, locomotor activity and total exploration time of the novel object were not different in sham and PFC-lesioned rats, and no other abnormal behaviours were observed (data not shown). The decline of discrimination in PFC-lesioned rats is therefore suggested to be due to cognitive dysfunction.

Delayed alternation task in a T-maze is a standard method for testing spatial working memory that requires location awareness through visual cues around the maze (spatial recognition) and memory retention of the last arm visited (working memory). The former is mainly associated with hippocampal functions and the latter with PFC function (Sánchez-Santed et al. 1997). We showed here that dopaminergic lesions in the PFC induced by 6-OHDA injection decreased the correct responses in the delayed alternation task with 45-s intra-trial interval but not in the non-delayed task as previously reported (Izaki et al. 2001), indicating a working memory-related cognitive impairment.

Treatment with istradefylline significantly improved the performance of PFC-lesioned rats in the object recognition task, and its effects reached a plateau at 0.1–0.3 mg/kg. The decreased number of correct responses of PFC-lesioned rats was also reversed by 0.1 mg/kg istradefylline in the delayed alternation task. Istradefylline produces effects on motor function in experimental models of PD with an ED 50 of 0.05 and 0.26 mg/kg in CGS21680 (A 2A receptor agonist) and reserpine-induced catalepsy in mice, respectively (Shiozaki et al. 1999). Istradefylline also ameliorates motor deficits induced by the nigral injection of 6-OHDA in rats, at doses of more than 0.32 mg/kg (Mihara et al. 2007). So, the current results show that istradefylline is effective in reversing working memory impairment related to a loss of PFC dopaminergic input at the same dose levels that improve motor performance and this would be consistent with an effect on cognition occurring at doses used to treat PD in man.

The question then becomes how istradefylline improves cognition related to PFC deficits. The mesocortical dopaminergic pathway is critically involved in cognitive processing, including working memory, and reduced dopaminergic transmission in the PFC underlies age-related or stress-induced working memory deficits (Mizoguchi et al. 2009). These deficits could be reversed by the intra-PFC injection of a dopamine D-1 agonist drug. Importantly, increased levels of dopamine are found in the PFC during delayed alternation task in both primates and rodents (Floresco and Magyar 2006; Watanabe et al. 1997). All of these suggest that improved dopaminergic transmission in the PFC leads to improved cognition. Indeed, we showed that methamphetamine, a dopamine releasing agent and reuptake inhibitor, improves the cognitive impairment in PFC-lesioned rats. Since blockade of A 2A receptors facilitates dopaminergic transmission in the PFC but not in the nucleus accumbens (Acquas et al. 2002) or in the striatum (unpublished data), we investigated whether the effects of istradefylline were associated with alterations in PFC dopaminergic transmission. The prefrontal dopamine levels in PFC-lesioned rats declined to about 19 % of normal content and 42 % of normal extracellular concentrations. Oral administration of 0.1 mg/kg istradefylline significantly increased extracellular dopamine levels in the PFC in both normal and PFC-lesioned rats. The present findings support previous reports showing that a marked reduction of dopamine levels in PFC-lesioned rats is responsible for their reduced performance. This also strongly supports the concept that the improvement produced by istradefylline is mediated by the enhancement of PFC dopamine levels. This conclusion is supported by the effects of donepezil which also ameliorated cognitive deficits in PFC-lesioned rats and induced a significant improvement in the object recognition task. Previous studies have shown that cholinesterase inhibitors elevate cortical dopamine levels through both cholinergic dependent and independent pathways (Sherman et al. 2006; Liang and Tang 2006; Giacobini et al. 1996; Cuadra et al. 1994). The mechanisms underlying istradefylline’s enhancement of PFC dopamine release are not fully understood. A pharmacological study showing that local injection of the adenosine A 2A receptor agonist CGS21680 into the posterior cingulate cortex impairs social recognition behaviour in rats (Pereira et al. 2005) supports the importance of the cortical A 2A receptors in cognitive functions. This finding suggested that istradefylline might control dopamine release by blockade of cortical adenosine A 2A receptors. In addition, adenosine A 2A receptors in the striatum may exert their effect on cortical functions including working memory indirectly through a basal ganglia–cortical loop. Neural activity of the striatum is markedly affecting neuronal activity in cerebral cortex and alters working memory.