List of selected genes in the synapse category differentially expressed at Day 5 of motor skill learning.

<p>List of selected genes in the synapse category differentially expressed at Day 5 of motor skill learning.</p

Annotation clusters of genes up-regulated at Day 5 identified by DAVID.

<p>Annotation clusters of genes up-regulated at Day 5 identified by DAVID.</p

Rats used for microarray analysis exhibited motor behaviors similar to the behaviors of the whole rat group.

<p><i>A,</i> The quality of the reaching behavior of the 4 rats in the <i>12-day-Reach</i> group (red, mean±SE) showed much improvement over time with the percentage of successful trials with dropped pellets of Day 8 to 12 being clearly lower than that of Day 1 to 5 (*, p<0.05, ANOVA). Similarly, a decreasing trend of this measure of learning was also observed in the <i>5-day-Reach</i> group (green). In this figure we have normalized the learning curve of every rat to its maximum value to account for inter-individual variability in initial performance. <i>B,</i> The probability of successful pellet retrieval per reach for the rats in the <i>12-day-Reach</i> group over the Learned (red, mean±SE) and Not-Learned (blue) Slots. Similar to the behavioral trend of the whole rat group (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061496#pone-0061496-g002" target="_blank">Fig. 2D</a>), the learning curve for the Learned Slots exhibited a sigmoid time course, with performance starting to increase at Day 5.1±3.2 (t_10%-max, black dotted line, mean±SE), and with the probability values of Day 1 to 5 smaller than those of Day 8 to 12 (*, p<0.05, ANOVA). The success probability values for the <i>5-day-Reach</i> group (N = 4; green; all slots) were also not statistically different from the values for the <i>12-day-Reach</i> group over the first 5 days (p>0.05, ANOVA).</p

Differentially expressed genes were grouped into functional clusters related to the synapse, the fibroblast growth factor family, and other processes.

<p><i>A,</i> The genes up-regulated at 5 days were grouped into 13 significantly enriched annotation clusters (p<0.01) using the DAVID bioinformatics resources. These clusters were then further condensed into 5 categories depending on the biological relationships between the clusters' annotation terms. Each of the five circles shown here represents one gene category. The area of the circle is directly proportional to the number of genes in that category. The thickness of the line connecting any two circles is proportional to the degree of overlap between the connected categories, indicated by the percentage of genes in the smaller of the two that are also present in the other. <i>B</i>, To estimate the effect of tightening the criterion for selecting differentially expressed genes on the rate of false positives, we successively increased our fold-change threshold from 1.5, to 1.75, and then 2.0 fold, and examined, at each threshold, the proportion of genes categorized into functionally enriched clusters. The percentages of genes remaining in any clusters (dark grey bars) and in the synapse category (black bars) were relatively unchanged as the fold-change threshold was increased. Assuming that the “true” proportion of "clusterable" genes is itself independent of the fold-change threshold, this observation implies that the false positive rate would probably not be much lower even with a more stringent fold-change threshold. DEG, differentially expressed genes.</p

A nontrivial forelimb reach-and-grasp task for the rat.

<p>Adult rats were trained to perform either a forelimb reach-and-grasp task (<i>Reach</i> groups) or a sham version of the task (<i>Sham</i> groups) for either 5 days (<i>Reach</i>, N = 4; <i>Sham</i>, N = 4) or 12 days (<i>Reach</i>, N = 4; <i>Sham</i>, N = 4) before tissue harvest. <i>A</i>, Behavioral setup for the <i>Reach</i> groups. In each trial a food pellet was placed at one of six possible slots arranged in three rows at different distances from the animal (near, mid, and far slots) and two columns that were ipsilateral and contralateral to the animal's preferred limb, respectively. The task goal was to reach and grasp the pellet using the preferred limb with single or multiple reaches. A reach was defined to be any hand trajectory from the box to the shelf area that crossed the border between the box and shelf (indicated by the thick black horizontal line), and then back to the box by crossing the same line. <i>B</i>, Behavioral setup for the <i>Sham</i> groups. In each trial the pellet was placed in the slit. Because the pellet was easily accessible, the rat naturally retrieved the reward with its tongue without using its forelimb. In all groups, the animal was conditioned to initiate a new trial by turning down either the left (left-handed rats, dotted-line arrows) or right (right-handed rats, solid-line arrows) aisle from the central aisle.</p

Most of the modulated probes in the <i>Reach</i> groups were differentially expressed at 5, but not 12, days.

<p><i>A</i>, Isolating differentially expressed probes related to motor skill learning. The Venn diagram shows the degree of intersection between the set of modulated probes (Kruskal-Wallis test; p<0.05) in the <i>0-day</i> vs <i>5-day-Reach</i> vs <i>12-day-Reach</i> comparison (light grey circle), and that in the <i>0-day</i> vs <i>5-day-Sham</i> vs <i>12-day-Sham</i> comparison (dark grey circle). The 1184 probes specific to the <i>Sham</i> groups probably reflect gene expression changes that occurred as a result of the execution of the task contingency, the passage of time, and other factors, while the 719 modulated probes specific to the <i>Reach</i> groups are most likely specifically related to processes underlying motor skill learning. <i>B</i>, Dynamics of differential gene expression from 5 to 12 days for the modulated probes specific to the <i>Reach</i> groups. For every modulated probe, the extent of differential expression at 5 and 12 days (Δ5day, x-axis; and Δ12day, y-axis) were calculated by subtracting the mean expression of the <i>Sham</i> group of each day from that of the <i>Reach</i> group. A scatter plot of the 719 data points shown here revealed two distinct groups of probes clustering around the positive and negative Δ5day-axes with relatively small amplitudes along the Δ12day-axis, suggesting that most of the modulated probes were differentially expressed at 5 days but not at 12 days. <i>C</i>, Dynamic types of differential expression for the <i>Reach</i>-specific probes. Each modulated probe was grouped into one of the four dynamic types, depending whether |Δ5day| was larger (bars 1 and 2) or smaller (bars 3 and 4) than |Δ12day|, and whether the day with the larger magnitude of differential expression was an up- (bars 1 and 3) or down-regulation (bars 2 and 4). Most of the probes were grouped into the first and second types indicating a regulation at 5 days. The black bar indicates the number of genes up-regulated at Day 5 categorized into gene clusters in subsequent DAVID functional analysis. <i>D</i>, Dynamics of differential gene expression from 5 to 12 days for the modulated probes specific to the <i>Sham</i> groups. Unlike the <i>Reach</i>-specific genes shown in <i>B</i>, Δ5day and Δ12day for these probes showed a positive, significant correlation (p<0.05). It is therefore possible that these <i>Sham</i>-specific modulations are driven by a process with a dynamics very different from that underlying forelimb skill learning. <i>E</i>, Dynamic types of differential expression for the <i>Sham</i>-specific probes. Notice that in the set of probes up-regulated at Day 5, the number of probes categorized into functional clusters by DAVID (black bar) was much smaller than that for the <i>Reach</i>-specific probes, shown in <i>C</i>.</p

The <i>Sham</i> animals might be more motivated to perform the pellet retrieval task then the <i>Reach</i> animals.

<p>We used the average trial duration as an indicator of the animals' motivation to perform the pellet retrieval task. For both the <i>5-day</i> groups (<i>A</i>) and <i>12-day</i> groups (<i>B</i>), trial durations for the <i>Sham</i> animals (dotted line; mean±SD; N = 4) tended to be lower than those for the <i>Reach</i> animals (solid line; N = 4) even though these differences were not statistically significant for all days except two of the days in the <i>12-day</i> groups (*, p<0.05). We speculate that the <i>Sham</i> animals might be more motivated or excited to perform the task than the <i>Reach</i> animals because successful pellet retrieval was almost guaranteed for the <i>Sham</i> groups. This difference in the level of motivation could be an explanation for why we observed many differentially expressed probes specific to the <i>Sham</i> groups.</p

Microarray data were validated using qPCR.

<p><i>A</i>, Gene expression fold changes indicated by microarray and qPCR. Eighteen genes (listed on the figure's left) were selected from the synapse, FGF, and unclustered categories for qPCR validation. Array and qPCR fold change values were calculated for each gene for the comparisons, <i>5-day-Reach</i> versus <i>0-day, 5-day-Reach</i> versus <i>5-day-Sham</i>, and <i>5-day-Reach</i> versus <i>12-day-Reach</i>, resulting into a total of 18×3 = 54 comparisons. The extent of agreement between the direction of fold change values derived from the two methods was assessed by the ratio of qPCR-fold change to the array-fold change. A positive ratio indicates an agreement, and a negative ratio, a disagreement. The heat map on the left shows this ratio of each of the 54 comparisons using a color map with green showing the highest (positive) value, and red, the lowest (negative) value. Four genes (<i>Prkci</i>, <i>Rab11fip4</i>, <i>Ctnnd1</i>, and <i>Cask</i>) showed a disagreement in at least one of the three comparisons. These four genes showing mismatches between the array- and qPCR-fold change directions (*) were also the ones whose qPCR-▵C_T values of the <i>5-day-Reach</i> group were not significantly different from those of the other groups (p>0.05), and whose qPCR-▵C_T values did not correlate well with the microarray expression intensities (p>0.05). To the right of the heat map is a table indicating to which functional categories each gene belonged (S, synapse; D, development; I, intracellular signaling; T, transforming growth factor β receptor activity; F, fibroblast growth factor family; U, unclustered). <i>B</i>, For some of the genes, we observed an excellent correlation between the sample qPCR ▵C_T values and the sample microarray gene expression intensities. They included <i>Fgf2</i> (top panel; <i>r</i> = 0.64. p<0.01) and <i>Adcy1</i> (bottom panel; <i>r</i> = 0.73, p<0.01). In both graphs here, the qPCR C_T values were normalized with respect to those of the <i>Ywhaz</i> gene.</p

Improvement in task goal achievement exhibited a sigmoid time course.

<p><i>A</i>, Eleven rats (rats 1–4 in <i>12-day-Reach</i> group) were trained for 12 days to reach and grasp pellets from six different locations in the workspace, including slots ipsilateral (ipsi) and contralateral (contra) to the rat's preferred side, at the Near, Mid, and Far levels (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061496#pone-0061496-g001" target="_blank">Fig. 1A</a>), respectively. The first measure of motor learning we used indicates the quality of reaching by quantifying the percentage of successful trials in which the pellet was dropped either to the inside of the slit or to the box's floor. Different rats showed different rates of improvement (blue, low rate of decrease; red, high rate) with 5 of the 11 rats showing a statistically significant decrease (*, Pearson's correlation coefficient<0, p<0.05). The top four rows correspond to data from rats included in the microarray analysis. <i>B,</i> For the 5 rats showing across-day improvement in movement quality, there was a clear decrease in the percentage of successful trials with dropped pellets over time (red, mean±SE). The percentage values of the first five days were greater than those of the last five days for these rats (*, p<0.05, ANOVA) but not for the other rats (blue). <i>C,</i> As a second measure of skill learning, we quantified the degree of task goal achievement across days. We examined whether there was across-day increase in the probability of successful retrieval per reach for each individual slot by linearly regressing the learning curve for each slot against time. Different rats exhibited performance improvement at different rates at different slots (blue, low improvement rate; red, high improvement rate) with different subsets of slots showing a significant increase in the success probability (*, Pearson's correlation coefficient>0, p<0.05; “Learned” slot). The top four rows correspond to data from rats included in the microarray analysis. <i>D,</i> The probability of successful retrieval per reach was calculated over the slots showing significant improvement (Learned Slots; red, mean±SE) and the other remaining slots showing no improvement (Not-Learned Slots; blue), respectively. The learning curve for the Learned Slots exhibited a sigmoid time course with the success probability starting to increase at Day 4.4±1.3 (t_10%-max, black dotted line; mean±SE). For the Learned Slots, the probability values of the first 5 days were also significantly lower than those of the last 5 days (*, p<0.05, ANOVA). <i>E,</i> The learning curve from the Learned Slots of every rat was regressed onto a sigmoid and an exponential function, respectively. When the regression R² values of the sigmoid fit were plotted against those of the exponential fit, all but one data point lay above the identify line (black dotted line) (green triangle, sigmoid>exponential; purple circle, exponential>sigmoid).</p

Successful pellet retrieval demanded strategic hand placement.

<p><i>A</i>, The frequency of use of different digits in successful trials for ipsilateral and contralateral slots. In the successful trials, immediately before grasping, each rat positioned its paw so that the pellet was directly under one of the digits, in between two adjacent digits, or under the palm of the hand. The graph shows the frequency of occurrence for the above possibilities (filled symbols: under a digit; unfilled symbols: between digits or under palm) within the successful trials for the ipsilateral (circles connected by solid line) and contralateral (squares connected by dotted line) slots (mean±SEM), across the animals in the <i>12-day-Reach</i> group (average over Day 1, 5, and 12). The two distributions were clearly separated with different means (Slot×Digit interaction, F(9,60) = 6.8, p<0.01) with the ipsilateral distribution peaking at the middle finger, and the contralateral, at the index finger. This suggests that grasping from these two different sets of slots involve the use of different digit sets. <i>B</i>, Strategies employed for slots at different distances from the rat. We calculated the rate of successful retrieval when, at the time of maximal forelimb extension, the pellet was directly under a finger (instead of between fingers) and was proximal in position to the PIP joint (dark grey bars), and when neither of these strategies was employed (light grey bars), over the near, mid, and far slots, respectively (mean±SEM). For all three slot sets, the success rate when the strategies were employed was clearly higher than when neither was employed (p<0.01).</p