Decreased striatal Drd1 mRNA expression during the early, but not late, phase of motor skill learning.

<p>(A) Regional analysis of dopamine D1 receptor (Drd1) mRNA expression within sub-regions of the striatum of (top panel) early phase (EP) active controls (white bars) vs. EP trained rats (red bars), and of (bottom panel) late phase (LP) active controls (white bars) vs. LP trained rats (blue bars). Data in bar graphs show mean optical density (OD) values for Drd1 mRNA in the DMS, DLS, VLS, AcbC, and AcbSh. The results are presented as means ± SEM; <i>n</i> = 5 per group. Asterisks indicate where trained rats differ significantly (*<i>P</i> < 0.05 and **<i>P</i> < 0.01) from active control rats. (B) Representative autoradiographs showing the mRNA expression levels of Drd1 at the level of the striatum of active controls and trained rats during the early and late phases of motor skill learning. For more details, see Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140974#pone.0140974.g001" target="_blank">1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140974#pone.0140974.g002" target="_blank">2</a>.</p

Increased striatal DARPP-32 mRNA expression during the early, but not late, phase of motor skilled learning.

<p>(A) Regional analysis of dopamine- and cAMP-regulated neuronal phosphoprotein (DARPP-32) mRNA expression within sub-regions of the striatum of (top panel) early phase (EP) active controls (white bars) vs. EP trained rats (red bars), and of (bottom panel) late phase (LP) active controls (white bars) vs. LP trained rats (blue bars). Data in bar graphs show mean optical (OD) density values for DARPP-32 mRNA in the DMS, DLS, VLS, AcbC, and AcbSh. The results are presented as means ± SEM; <i>n</i> = 5 per group. Asterisks indicate where trained rats differ significantly (*<i>P</i> < 0.05 and **<i>P</i> < 0.01) from active control rats. (B) Representative autoradiographs showing the mRNA expression levels of Drd1 at the level of the striatum of active controls and trained rats during the early and late phases of motor skill learning. For more details, see Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140974#pone.0140974.g001" target="_blank">1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140974#pone.0140974.g002" target="_blank">2</a>.</p

Correlations between skilled-reaching performance during the late phase of motor skill learning and levels of either phospho-Thr75-DARPP-32 or phospho-Ser133-CREB.

<p>(A) Simple regression analysis indicating a significant correlation between success on the first reach attempt, in percent, during the late phase of motor skill learning and levels of phospho-Thr75-DARPP-32 or (B) phospho-Ser133-CREB. Filled circles represent values for individual rats from the late phase (LP) group. LP trained rats were divided into good and poor learners according to their individual learning curve profiles and endpoint performance. (C-D) The top row presents representative Western blot autoradiograms for phospho-Thr75-DARPP-32 and phospho-Ser133-CREB. (C) Data in bar graphs represent the levels of phospho-Thr75-DARPP-32 or (D) phospho-Ser133-CREB in controls (<i>n</i> = 6), good learners (<i>n</i> = 6), and poor learners (<i>n</i> = 4) expressed as means ± SEM. The asterisks indicate where poor or good learners differ significantly (*<i>P</i> < 0.05) from active controls.</p

Motor Skill Learning Is Associated with Phase-Dependent Modifications in the Striatal cAMP/PKA/DARPP-32 Signaling Pathway in Rodents

<div><p>Abundant evidence points to a key role of dopamine in motor skill learning, although the underlying cellular and molecular mechanisms are still poorly understood. Here, we used a skilled-reaching paradigm to first examine changes in the expression of the plasticity-related gene Arc to map activity in cortico-striatal circuitry during different phases of motor skill learning in young animals. In the early phase, Arc mRNA was significantly induced in the medial prefrontal cortex (mPFC), cingulate cortex, primary motor cortex, and striatum. In the late phase, expression of Arc did not change in most regions, except in the mPFC and dorsal striatum. In the second series of experiments, we studied the learning-induced changes in the phosphorylation state of dopamine and cAMP-regulated phosphoprotein, 32k Da (DARPP-32). Western blot analysis of the phosphorylation state of DARPP-32 and its downstream target cAMP response element-binding protein (CREB) in the striatum revealed that the early, but not late, phase of motor skill learning was associated with increased levels of phospho-Thr34-DARPP-32 and phospho-Ser133-CREB. Finally, we used the DARPP-32 knock-in mice with a point mutation in the Thr34 regulatory site (i.e., protein kinase A site) to test the significance of this pathway in motor skill learning. In accordance with our hypothesis, inhibition of DARPP-32 activity at the Thr34 regulatory site strongly attenuated the motor learning rate and skilled reaching performance of mice. These findings suggest that the cAMP/PKA/DARPP-32 signaling pathway is critically involved in the acquisition of novel motor skills, and also demonstrate a dynamic shift in the contribution of cortico-striatal circuitry during different phases of motor skill learning.</p></div

Deficits in skilled reaching behavior in DARPP-32 Thr34A mutant mice.

<p>Endpoint measurements of reaching behavior are presented. (A) Success on the first reach attempt, in percentage. (B) Total success, in percentage. (A′ and B′) Bar graphs show the learning rate for each endpoint. The results are presented as means± SEM; n = 8 per group. Asterisks denote where DARPP-32 Thr34A mutant mice differ significantly (* <i>P</i> < 0.05, ** <i>P</i> < 0.01, *** <i>P</i> < 0.001) from wild type (WT) mice.</p

Enhanced phosphorylation of DARPP-32 and CREB in the striatum is associated with the early phase of motor skill learning.

<p>(A) Levels of phospho-Thr34-DARPP-32, (B) phospho-Thr75-DARPP-32, (C) phospho-Ser97-DARPP-32, (D) total DARPP-32, (E) phospho-Ser133-CREB, and (F) total CREB were evaluated in the striatum contralateral to the trained forelimb of active controls (white bars) and trained (red bars) rats after 3 days of motor skill learning (i.e., early phase) by means of Western blotting. The top row presents representative autoradiograms. The bottom graph is a summary of data represented as means ± SEM (<i>n</i> = 6–8 per group). Asterisks indicate where trained rats differ significantly (*<i>P</i> < 0.05) from active control rats. Heat Shock Protein 90 (HSP90) served as the loading control in A–F.</p

Arc mRNA expression is differentially induced in cortical regions during different phases of motor skill learning.

<p>(A) Regional analysis of Arc mRNA expression in various cortical regions of (top panel) early phase (EP) active controls (white bars) vs. EP trained rats (red bars), and of (bottom panel) late phase (LP) active controls (white bars) vs. LP trained rats (blue bars). Data in bar graphs show mean optical density (OD) values for Arc mRNA in M1, mPFC, and OFC. The results are presented as means ± SEM; <i>n</i> = 4–5 per group. Asterisks indicate where trained rats differ significantly (*<i>P</i> < 0.05) from active control rats. Abbreviations are as follows: ipsilateral (Ipsi) and contralateral (Contra). (B) Representative autoradiographs showing the mRNA expression levels of Arc at the level of the frontal cortex of active controls and trained rats during the early and late phases of motor skill learning. The pseudo-coloring indicates signal intensity ranging from low (black/purple) to high (yellow/white). For more details, see Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140974#pone.0140974.g001" target="_blank">1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140974#pone.0140974.g002" target="_blank">2</a>.</p

Schematic representation of the brain regions analyzed.

<p>Drawings illustrate the location where measures for gene expression studies were obtained. Coronal sections are marked in millimeters from bregma according to the Rat Brain atlas of Paxinos and Watson (1998). Labeled areas correspond to the following brain regions: medial prefrontal cortex (mPFC); orbitofrontal cortex (OFC); cingulate cortex (CG); primary motor cortex (M1); nucleus accumbens core (AcbC); nucleus accumbens shell (AcbSh); dorsal lateral striatum (DLS); dorsal medial striatum (DMS); ventrolateral striatum (VLS); paramedian lobule (PML).</p

Distance to decision boundary in relation to BW and IQ.

<p>In parts A & B the distance to decision boundary (i.e. vertical distance relative to horizontal yellow line) is displayed for both groups. In part A, all 143 subjects are shown while in part B only those with IQ data are represented. The color code in part A indicates birth weight of the individual while on the bottom it represents the full IQ scores (see respective color bars in middle). In part C the distance to decision boundary (horizontal axis) and the full IQ score (vertical axis) are plotted against each other for the group of subjects born preterm. The red dashed line indicates the linear fit to the data (R = –0.34, p < 0.0103).</p

Arc mRNA expression is differentially induced within sub-regions of the striatum during different phases of motor skill learning.

<p>(A) Regional analysis of Arc mRNA expression within sub-regions of the striatum of (top panel) early phase (EP) active controls (white bars) vs. EP trained rats (red bars), and of (bottom panel) late phase (LP) active controls (white bars) vs. LP trained rats (blue bars). Data in bar graphs show mean optical density (OD) values for Arc mRNA in DMS, DLS, VLS, AcbC, and AcbSh. The results are presented as means ± SEM; <i>n</i> = 5 per group. Asterisks indicate where trained rats differ significantly (*<i>P</i> < 0.05, **<i>P</i> < 0.01, and ***<i>P</i> < 0.001) from active control rats. (B) Representative autoradiographs showing the mRNA expression levels of Arc at the level of the striatum of active controls and trained rats during the early and late phases of motor skill learning. For more details, see Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140974#pone.0140974.g001" target="_blank">1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140974#pone.0140974.g002" target="_blank">2</a>.</p