Selective inactivation of adenosine A(2A) receptors in striatal neurons enhances working memory and reversal learning

Abstract

The adenosine A(2A) receptor (A(2A)R) is highly enriched in the striatum where it is uniquely positioned to integrate dopaminergic, glutamatergic, and other signals to modulate cognition. Although previous studies support the hypothesis that A(2A)R inactivation can be pro-cognitive, analyses of A(2A)R's effects on cognitive functions have been restricted to a small subset of cognitive domains. Furthermore, the relative contribution of A(2A)Rs in distinct brain regions remains largely unknown. Here, we studied the regulation of multiple memory processes by brain region-specific populations of A(2A)Rs. Specifically, we evaluated the cognitive impacts of conditional A(2A)R deletion restricted to either the entire forebrain (i.e., cerebral cortex, hippocampus, and striatum, fb-A(2A)R KO) or to striatum alone (st-A(2A)R KO) in recognition memory, working memory, reference memory, and reversal learning. This comprehensive, comparative analysis showed for the first time that depletion of A(2A)R-dependent signaling in either the entire forebrain or striatum alone is associated with two specific phenotypes indicative of cognitive flexibility-enhanced working memory and enhanced reversal learning. These selective pro-cognitive phenotypes seemed largely attributed to inactivation of striatal A(2A)Rs as they were captured by A(2A)R deletion restricted to striatal neurons. Neither spatial reference memory acquisition nor spatial recognition memory were grossly affected, and no evidence for compensatory changes in striatal or cortical D(1), D(2), or A(1) receptor expression was found. This study provides the first direct demonstration that targeting striatal A(2A)Rs may be an effective, novel strategy to facilitate cognitive flexibility under normal and pathologic conditions.

Figure 1.

Forebrain- or striatum-specific A_2AR KO is selective. (A) X-gal staining for brain regions of Cre expression demonstrates strong staining throughout the entire forebrain (i.e., cortex, hippocampus, and striatum) in Camk2a-cre(+)Rosa26^flox/flox mice (upper panel). X-gal staining also shows strong staining throughout the entire striatum (that is largely absent in the cortex or the hippocampus) in Dlx5/6-cre(+)Rosa26^flox/flox mice (lower panel). (B) Postnatal developmental time course of Cre recombination and deletion of the “floxed” A_2AR allele. Representative PCR analysis of genomic DNA isolated from (1) the striatum, (2) the cortex, and (3) the cerebellum of fb-A_2AR KO mice at the various developmental stages (P15, P23, and P35) demonstrates a forebrain-specific pattern of A_2AR deletion in the striatum and the cortex, but not in the cerebellum beginning around P23 (upper panel). Similar PCR analysis of (1) the striatum, (2) the cortex, and (3) the hippocampus from st-A_2AR KO mice at different developmental stages indicates a striatum-specific pattern of A_2AR deletion in the striatum, but not in the cortex or the hippocampus as early as P5 (lower panel). (C) ³H-ZM241385 (selective A_2AR antagonist) radioligand binding to quantify Cre-mediated loss of A_2AR expression in the striatum, the cortex, the hippocampus, and the olfactory bulb in fb-A_2AR KO mice and st-A_2AR KO mice. ³H-ZM241385 binding is reduced in all forebrain regions examined in fb-A_2AR KO mice (upper panel), but only in the striatum of st-A_2AR KO mice (lower panel). n = 5–7 per group. Mean ± SEM are plotted. *P = 0.06, **P < 0.01, ***P < 0.0001, KO vs. WT.

Figure 2.

Forebrain- and striatum-specific A_2AR KO is without compensatory changes in A₁, D₁, or D₂ receptor levels. (A,B) Quantitative analysis of ³H-DPCPX (selective A₁R antagonist), ³H-SCH23390 (selective D₁R antagonist), and ³H-raclopride (selective D₂R antagonist) in total membranes from the striatum and/or cortex of fb-A_2AR KO and fb-WT mice (n = 3–6 per group) (A), and of st-A_2AR KO and st-WT mice (n = 5–8 per group) (B). No differences in radioligand binding densities were demonstrated for these receptors (one-way ANOVA, P's > 0.13). Mean ± SEM are plotted.

Figure 3.

Fb-A_2AR KO and st-A_2AR KO mice show normal spatial recognition memory in the Y-maze. To evaluate memory for distant spatial cues, mice were first allowed 5 min to explore the start and familiar arms of the maze (sample phase) and then returned to the maze after a delay period (2 min, 30 min, 3.5 h, and then 1 d) and given 3 min to explore these same two arms plus an additional, novel arm (test phase). Preference for the novel arm, expressed as the percentage of time spent in the novel arm [(time in novel arm/time in all arms) × 100%], during each test phase was used to index spatial recognition memory. (A) Fb-A_2AR KO and fb-WT mice showed a similar preference for the novel arm, which progressively weakened at a comparable rate with increasing retention demands. (B) St-A_2AR KO and st-WT mice also showed a strong and comparable preference for the novel arm that gradually declined at a similar rate toward chance performance upon increasing the delay interval. Values depicted are mean ± SEM. Chance level = 33.33%, dashed line.

Figure 4.

Fb-A_2AR KO (female only) and st-A_2AR KO mice demonstrate normal visually guided escape behavior but improved spatial working memory. Mice were first pretrained in the water maze for up to two consecutive days (two trials per day) to swim directly to a visible platform for escape. Working memory was subsequently evaluated by examining over blocks of 4 d the improvement in escape latency (s) from trial 1 to 2 to reach a hidden platform whose location changed every day. (A) Both fb-A_2AR KO (left panel) and st-A_2AR KO (right panel) mice showed a comparable reduction in escape latency with each trial compared to fb-WT and st-WT mice, respectively. This indicates that A_2AR deletion did not alter motivation or sensory and motor capabilities required to successfully learn and execute an escape onto a visible platform. (B,C) Experiment set I: The forebrain cohort was tested over three blocks corresponding to a 20-sec, 10-min, or 15-min delay between trials. The striatal cohort was tested in a single block at the minimal delay of 20 sec. Among the female mice (B, left panel), clear evidence for a delay-dependent enhancement of working memory was observed in fb-A_2AR KO mice: these mice demonstrated a consistent and marked reduction in escape latency from trial 1 to 2 at all delays, whereas fb-WT mice only readily showed such improvement at the shorter delay, suggesting improved working memory capabilities in these female fb-A_2AR KO mice. In contrast, among the male mice (B, right panel), fb-A_2AR KO and fb-WT mice performed remarkably similarly, showing comparable improvements in escape latency from trial 1 to 2 at all delays. St-A_2AR KO mice, like female fb-A_2AR KO mice, readily showed improvement across the two trials, whereas st-WT mice did not (C). (D) Experiment set II: A separate striatal cohort was first trained for 4 d using a four-trial-per-day training protocol to facilitate learning and ensure mastery of the matching rule. Mice were then returned in the testing phase to a two-trial-per-day protocol as in Experiment set I and tested at 20-sec and 10-min delays. St-A_2AR KO and st-WT mice demonstrated comparable improvement from trial 1 to 2 during training, but as the task demands increased during testing, st-A_2AR KO mice again continued to out-perform st-WT mice, an effect that was not sex-dependent. Despite this lack of sex dependency in st-A_2AR KO mice, the data are further plotted split by sex in order to provide a parallel comparison with the enhanced working memory phenotype in fb-A_2AR KO mice, which did show a sex effect. Values depicted are mean ± SEM. *P < 0.05, ***P < 0.005, KO vs. WT. ^∧P < 0.02, ^∧∧∧P < 0.005, trial 1 vs. 2 in A_2AR KO mice; ^##P < 0.01, Genotype × Trials interaction.

Figure 5.

Reference memory performance is spared in fb-A_2AR KO and st-A_2AR KO mice. Mice were trained for 10 consecutive days (two trials per day) to acquire the fixed spatial location of a hidden escape platform. Following acquisition, two probe tests were conducted on days 11 (Probe test 1) and 13 (Probe test 2) during which the escape platform was removed from the pool and the spatial search pattern of mice was observed for 60 sec. (A) Fb-A_2AR KO mice, like fb-WT mice, demonstrated a steady decline in escape latency with each training day, showing highly comparable escape performance by the end of acquisition training (day 10). (B) Both fb-A_2AR KO and fb-WT mice showed a strong and similar search preference for the spatial location of the escape platform (i.e., target quadrant) in both probe tests. (C) Search accuracy defined by the number of annular crossings was comparable between fb-A_2AR KO and fb-WT mice. (D) Similarly, st-A_2AR KO and st-WT mice showed gradual performance improvement during acquisition, achieving near-identical performance at the end of training (day 10). (E) Both st-A_2AR KO and st-WT mice demonstrated an above-chance level search preference for the target quadrant in both probe tests; however, this preference was weaker in st-A_2AR KO mice. (F) St-A_2AR KO and st-WT mice did not significantly differ in the number of annular crossings, indicating comparable search accuracy in both probe tests. Values depicted are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 vs. chance level (=25%, dashed line). ^∧P < 0.05, st-A_2AR KO vs. st-WT.

Figure 6.

Faster reversal learning in fb-A_2AR KO and st-A_2AR KO mice. Reversal learning began once the mice had learned the fixed position of the hidden escape platform during acquisition. It lasted for 4 d (days 14–17) in the forebrain cohort and for 8 d (days 14–21) in the striatal cohort. During reversal, the location of the platform was shifted 180° to the opposite quadrant, and mice were required to learn the new escape location. Probe tests were conducted 24 h after a training session to evaluate the progress of reversal learning on day 18 (Probe test 3) in both cohorts, and additionally on days 20 and 22 (Probe tests 4 and 5) in the striatal cohort only. (A) Fb-A_2AR KO mice were less affected by the shift in platform location as indicated by faster escape latencies during the reversal phase. This effect appeared more pronounced with additional reversal training as demonstrated by a divergence in escape latencies. (B) Both fb-A_2AR KO and fb-WT mice exhibited a comparable search preference for the new target quadrant after 4 d of reversal training. (C) St-A_2AR KO mice also escaped more quickly throughout reversal learning compared to st-WT mice, indicating that they were less disrupted by the sudden change in platform location. (D) St-A_2AR KO mice also demonstrated a strong preference for the new target quadrant in all three probe tests. This preference was significantly greater in st-A_2AR KO mice during Probe test 3 compared to that in st-WT mice. A significant target preference was not observed in st-WT mice until the end of reversal training (i.e., Probe test 5). Values depicted are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.005 vs. chance level (=25%, dashed line). ^∧P < 0.05, st-A_2AR KO vs. st-WT.