Impairment of reward-related learning by cholinergic cell ablation in the striatum

Abstract

The striatum in the basal ganglia-thalamocortical circuitry is a key neural substrate that is implicated in motor balance and procedural learning. The projection neurons in the striatum are dynamically modulated by nigrostriatal dopaminergic input and intrastriatal cholinergic input. The role of intrastriatal acetylcholine (ACh) in learning behaviors, however, remains to be fully clarified. In this investigation, we examine the involvement of intrastriatal ACh in different categories of learning by selectively ablating the striatal cholinergic neurons with use of immunotoxin-mediated cell targeting. We show that selective ablation of cholinergic neurons in the striatum impairs procedural learning in the tone-cued T-maze memory task. Spatial delayed alternation in the T-maze learning test is also impaired by cholinergic cell elimination. In contrast, the deficit in striatal ACh transmission has no effect on motor learning in the rota-rod test or spatial learning in the Morris water-maze test or on contextual- and tone-cued conditioning fear responses. We also report that cholinergic cell elimination adaptively up-regulates nicotinic ACh receptors not only within the striatum but also in the cerebral cortex and substantia nigra. The present investigation indicates that cholinergic modulation in the local striatal circuit plays a pivotal role in regulation of neural circuitry involving reward-related procedural learning and working memory.

Fig. 1.

Impairment of procedural learning in the tone-cued T-maze test. (A) The procedure of the tone-cued T-maze test is indicated. (B) The percentage of mice that passed the learning criterion in each session is indicated. The number of learned mice was significantly lower in cholinergic cell-eliminated mice than in IT-treated WT mice (n = 7 each; Wilcoxon-signed-ranks test, P < 0.05). (C) The number of successful sessions that exceeded the learning criterion in 10 sessions was averaged. Columns and error bars represent mean ± SEM, respectively. **, P < 0.01.

Fig. 2.

Spatial alternation test in the T-maze. (A) The number of sessions mice needed to reach the learning criterion in the spatial alternation task is indicated. Columns and error bars represent mean ± SEM, respectively. n = 15 each; statistically not different (P = 0.51). (B) The number of correct choices in the spatial delayed alternation test is indicated. The correct choice in delays of 30 and 90 s was more significantly reduced in IT-treated transgenic mice than in IT-treated WT mice (repeated-measure ANOVA, F_1,28 = 11.8, P < 0.01; **, P < 0.01; *, P < 0.05).

Fig. 3.

Behavioral analyses of different learning categories. (A) Motor learning test using a rota-rod. The points represent the mean rotation speed the animal reached with daily practices. The error bars represent mean ± SEM. IT-tg, n = 6; IT-wt, n = 7; statistically not different (repeated-measure ANOVA, F_1,15 = 0.25, P = 0.63). (B) The freezing time in contextual and tone-cued fear-conditioning tasks [n = 7 each; not statistically different in both fear responses (Student's t test, contextual, P = 0.40; tone-cued, P = 0.20)]. (C) IT-injected WT and transgenic mice (n = 9 each) improved performance in both standard water-maze task (repeated-measure ANOVA, IT-tg, F_8,64 = 9.48, P < 0.0001; IT-wt, F_8,64 = 13.5, P < 0.0001) and spatial reversal task (repeated-measure ANOVA, IT-tg, F_3,24 = 28.7, P < 0.0001; IT-wt, F_3,24 = 9.86, P = 0.0002). Escape latencies of both tests were not statistically different (repeated-measure ANOVA, standard, F_1,16 = 1.55, P = 0.23; reversal, F_1,16 = 0.64, P = 0.44). (D) The time spent in the trained quadrant was not statistically different (Student's t test: standard, P = 0.59; reversal, P = 0.18).

Fig. 4.

Exploratory hyperlocomotion of cholinergic cell-eliminated mice. The locomotion of cholinergic cell-eliminated mice was significantly higher than that of WT mice at the exploratory phase but not at habituated phase (IT-tg, n = 9; IT-wt, n = 7). Columns and error bars indicate mean ± SEM, respectively (Student's t test: **, P < 0.01).

Fig. 5.

Up-regulation of nAChRs in cholinergic cell-eliminated mice. (A) Expression levels of indicated proteins were determined by immunoblot analysis of homogenates obtained from the IT-injected (+) and uninjected (-) sides of the striatum of transgenic mice. Levels of ChAT and α4, β2, and α7 subunits of nAChRs were changed by cholinergic cell elimination, but no changes were observed for other proteins tested. (B–E) Levels of α4, β2, and α7 subunits of nAChRs and ChAT were quantified by immunoblot analysis of the indicated brain regions. Levels at the IT-injected side, relative to those of the IT-uninjected side, are indicated. Columns and error bars represent mean ± SEM, respectively. Each brain region was analyzed with at least five preparations (Student's t test: **, P < 0.01; *, P < 0.05).