D1R expression at P14.

<p>D1R expression in the frontal cortex and caudate-putamen of postnatal day 14 (P14; A- D) mice. In each panel, the upper band shows D1R and the lower band shows β-actin, which was used as a loading control. The bar graphs in each panel represent D1R band intensity (mean ± SEM) normalized to intensity of the loading control (integrated density value; IDV). The names of the mouse lines (<i>Dyt1</i> KO, <i>Dyt1</i> KI) are indicated to the right. (*<i>p</i><0.05, n = 3 or 4).</p

D1R expression at P60.

<p>D1R expression in the frontal cortex, caudate-putamen and ventral midbrain of postnatal day 60 (P60; A-I) mice. In each panel, the upper band shows D1R and the lower band shows β-actin, which was used as a loading control. The bar graphs in each panel represent D1R band intensity (mean ± SEM) normalized to intensity of the loading control (integrated density value; IDV). The names of the mouse lines (<i>Dyt1</i> KO, <i>Dyt1</i> KI, hMT) are indicated to the right. (*<i>p</i><0.05, **<i>p</i><0.01; n = 3 or 4).</p

Gα(olf) and Gα(s) expression at P60.

<p>Gα(olf) and Gα(s) expression in the frontal cortex, caudate-putamen and ventral midbrain of Dyt1 KO, and, Dyt1 KI mice at postnatal day 60 (P60; A-F). In each panel, the upper band shows Gα(olf) (A-F), and the lower band shows β-actin, which was used as a loading control. The bar graphs in each panel represent Gα(olf) band intensity (mean ± SEM) normalized to intensity of loading control (integrated density value; IDV). The names of the mouse lines (<i>Dyt1</i> KO, <i>Dyt1</i> KI) are indicated to the right (**<i>p</i><0.01, ***<i>p</i><0.001; n = 3 or 4).</p

D2R expression at P60.

<p>D2R expression in the frontal cortex, caudate-putamen and ventral midbrain of postnatal day 60 (P60; A-I) mice. In each panel, the upper band shows D2R and the lower band shows β-actin, which was used as a loading control. The bar graphs in each panel represent D2R band intensity (mean ± SEM) normalized to intensity of loading control (integrated density value; IDV). The names of the mouse lines (<i>Dyt1</i> KO, <i>Dyt1</i> KI, hMT) are indicated to the right (*<i>p</i><0.05, **<i>p</i><0.01; n = 3 or 4).</p

A comparison of the magnitude of reductions in D1R and D2R protein expression.

<p>A comparison of the magnitude of reductions in D1R and D2R protein expression in the frontal cortex (FC) and caudate putamen (CP) of postnatal day 60 (P60) and postnatal day 14 (P14) Dyt1 KO (KO) and Dyt1 KI (KI) mice.</p><p>A comparison of the magnitude of reductions in D1R and D2R protein expression.</p

A summary of the changes in D1R, D2R and Gα(olf) expression at P14.

<p>A summary of the changes in D1R, D2R and Gα(olf) expression at postnatal day 14 (P14) in the frontal cortex (FC) and caudate-putamen (CP) in the Dyt1 KO (KO) and Dyt1 KI (KI) lines of mouse.</p><p><b>↓</b> indicates statistically significant reductions in expression compared to wild-type control mice.</p><p>—indicates no statistically significant difference.</p><p>A summary of the changes in D1R, D2R and Gα(olf) expression at P14.</p

Gα(s) and Gα(olf) expression at P60.

<p>Gα(s) and Gα(olf) expression in the frontal cortex, caudate-putamen and ventral midbrain of Dyt1 KO and Gnal KO mice at postnatal day 60 (P60; A-D). In each panel, the upper band shows Gα(s) (Fig 4A and 4B), and Gα(olf) (Fig 4C and 4D), and the lower band shows β-actin, which was used as a loading control. The bar graphs in each panel represent Gα(s), and Gα(olf) band intensity (mean ± SEM) normalized to intensity of loading control (integrated density value; IDV). The names of the mouse lines (<i>Dyt1</i> KO, and <i>Gnal</i> KO) are indicated to the right.</p

Voxel based morphometry in cervical dystonia.

<p>Voxel based morphometry demonstrated reduced gray matter local tissue volume in the posterior cingulate (A and B, blue voxels, shown at two significance thresholds, presented as family-wise uncorrected p-values), but no differences in the thalamus, in cervical dystonia (family-wise error corrected p = 0.9996). When the analysis was restricted to only those voxels in a thalamic mask (to minimize the loss of statistical power by multiple-comparisons correction; C, green voxels), no significant differences in local tissue volume were noted (p = 0.34). Significant voxels (A, B) and thalamic mask (C) overlie the mean gray matter structural image. Note that identical structural scans were used in VBM analyses and segmentation analyses (i.e., scans used in this figure were the same as those used for data in Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155302#pone.0155302.g002" target="_blank">2</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155302#pone.0155302.g003" target="_blank">3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155302#pone.0155302.g005" target="_blank">5</a>). VBM results were corrected using threshold-free cluster enhancement (TFCE). All axial and coronal views are from a single plane, indicated in MNI Talairach coordinates. Color bars at bottom indicate TFCE-corrected p-values for the images above. Abbreviations: pat = patients; ctrl = controls. R = Right hemisphere; L = Left hemisphere.</p

Regional automated and manual gross volume measures.

<p>A reduction in thalamic volume, not seen in other regions involved in the control of movement, was seen in both cervical dystonia and spasmodic dysphonia. Total volume (i.e., number of voxels in left plus right hemispheres) is shown for each region of interest (mean ± standard error of the mean). Given the large differences in volume between brain regions, the axis has been adjusted to focus on each cluster of values. Breaks in the y-axis are indicated by hashed horizontal bars. All p-values corrected for multiple comparisons (Bonferroni corrected significance threshold, p = 0.00625); * p≤0.0060; ** p = 0.00020. Abbreviation: BA6 = Brodmann Area 6; Thal auto = automated thalamic segmentation.</p

Relationship of clinical measures to gross thalamic volume in CD patients.

<p>The relationship between individual gross thalamic volume (manually segmented, in mm³) and clinical measures suggests that reduced gross thalamic volume is a risk factor for dystonia, and is not a secondary effect of dystonia symptoms. Data are shown for all variables, even if excluded from regression models, so raw data for CD and SD can be viewed and compared. Both CD patients and controls (A) showed declining volume with age. Patient:control gross thalamic volume showed a qualitative divergence with age between CD and controls, but the divergence was not statistically significant. (B) There was no relationship between volume and age in the SD cohort (for either SD or controls), presumably reflecting the smaller age range in this cohort. Likewise, there was no divergence of slopes with age between SD and controls. Age at CD onset (C) appeared to correlate with gross thalamic volume, but this was likely driven by the relationship between age at scan and age of onset; age at SD onset (D) was not correlated with volume. Gross thalamic volume did not correlate significantly with duration for either CD (E); this relationship was not evaluated statistically for SD due to collinearity with other variables, but the positive slope suggests no indication of a decline in volume with increasing duration (F). Thalamic volume also did not correlate with severity of dystonia for either CD or SD in the multiple regression model, as measured by the Tsui scale for CD (G) or the voice-related quality of life score for SD (H), although the SD relationship to severity showed a trend toward significance (V-RQOL, p = 0.056), and appeared significant when evaluated post hoc as a single variable (p = 0.012). The asymmetry of muscles affected with cervical dystonia (as gauged by laterality of units of botulinum toxin injected) did not correlate with asymmetries in thalamic volume (I, p = 0.89). Note that for (C) and (D), thalamic volumes for control subjects are plotted vs. the age at onset for the matched patient, as control subjects do not have an age at onset. Volume for control subjects is included in C and D as a reference only (designated by Ω), to illustrate that patient:control differences persist (and in fact are more robust) when demographics (including age) are matched: with the exception of a single CD/control dyad, every patient showed lower volume than his/her matched control.</p