Experimental study design, treatments and group assignments.

<p>Experimental study design, treatments and group assignments.</p

Striatal coronal sections immunostained with TH and counterstained with Nissl showing the morphology of the striatal dopaminergic cells.

<p>Control monkey (A) vehicle-treated monkey (B) and L-Dopa-treated monkey (C). Scale bar, 100 µm (A-C) and 20 µm (A’–C’).</p

Double immunofluorescence images of the striatal TH-ir cells showing one TH-ir cell that also express dopa-decarboxilase (DDC) (A), absence of TH-ir cells immunolabeled with the dopamine and cyclic AMP-regulated phosphoprotein (DARPP-32) (B) TH-ir interneurons expressing calretinin (CR) (C) and glutamate decarboxylase (GAD67) (D).

<p>Scale bars 20 µm.</p

Triple immunofluorescence images showing the different 4 phenotypes of the TH-ir cells here described.

<p>A) TH-ir cell labeled with CR and GAD67, B) two TH-ir cells that do not express either CR or GAD67, C) TH-ir cell expressing GAD67 but not CR and D) two TH-ir cells expressing CR but not for GAD67. Scale bars 20 µm.</p

General characteristics of the animals and TH-ir cell counting in the striatum and substantia nigra.

*<p>: <i>P</i>≤0.05 compared to vehicle or L-Dopa with control group.</p><p>#: <i>P</i>≤0.05 compared to L-Dopa with vehicle group. The data show the mean and its corresponding SEM.</p

Graphic representation of the distribution of striatal TH-ir neurons in each experimental group.

<p>Note that L-Dopa group has the highest number of TH-ir cells, particularly in the more rostral areas. The delineation of territory boundaries based on anatomical assessments is also showed. Sensorimotor (SM), Associative (ASS) and Limbic (Lim) territories of the caudate (Cd), Putamen (Pu) and part of nucleus accumbens (Acc) are included. The distances from the anterior commissure (AC) are indicated.</p

The graphic represents the percentage of TH-ir cells that co-localizes with glutamate decarboxylase (TH+/GAD67+), calretinin (TH+/CR+) and Dopa- decarboxylase (TH+/DDC+).

<p>Note the significant reduction of the double TH/CR immunoreactive cells in the L-Dopa group with respect to control group. * = p<0.05 using Tukey test following one way ANOVA repeated measures. Error bars represent SEM.</p

Striatal TH-ir cell distribution.

<p>(A) Mean of density of TH- ir cells in the pre- commissural and post-commissural striatum of control (n = 3), vehicle (n = 4) and L-Dopa (n = 4) groups. Mann and Whitney U: * = p<0.05. (B) Mean density of TH-ir cells in the anterior and posterior parts of pre-commissural striatum of control (n = 3), vehicle (n = 4) and L-Dopa (n = 4) groups. Mann and Whitney U: * = p<0.05.</p

The graphic represents the results of triple immunofluorescence studies in the pre-commissural striatum of every experimental group and the percentage of TH-ir cells that co-localizes with glutamate decarboxylase (GAD67+) and calretinin (CR+).

<p>Note that phenotype TH+/GAD67+/CR- was significantly increased in the L-Dopa group with respect to control group (* = p<0.05 using Tukey test following one way ANOVA repeated measures). Error bars represent SEM.</p

Cell renewal and differentiation dynamics of progenitors in the striatum.

<p>Schematic representation of hypothetic dynamics of endogenous striatal progenitors and the effects of MPTP. <b>A.</b> For reference we represented on top, the results of fate mapping of Sox-2⁺ cells in the murine dentate gyrus showing that non-radial Sox-2⁺ progenitors have self-renew potential and give rise to both neurons and astrocytes <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066377#pone.0066377-Muotri1" target="_blank">[26]</a>. <b>B, C.</b> Similarly, we propose than in the normal situation, Sox-2⁺ striatal progenitors (Sox-2⁺/GFAP^−/CR^–) divide to generate new Sox-2⁺ progenitors. Moreover, some Sox-2⁺ progenitors give rise to astrocytes and (few) Sox-2⁺/CR⁺ neurons that after several maturation steps could switch off Sox-2 expression and give rise to different mature phenotypes (including TH⁺). Following MPTP administration the number of Sox-2⁺ progenitors increases while the number of CR⁺ cells that express Sox-2 is decreased. Possible mechanisms are A) MPTP favours gliogenesis from Sox2 progenitors at the expense of neurogenesis. B) MPTP (or lack of dopamine) accelerates maturation of Sox-2⁺/CR⁺ cells. C) Sox-2⁺/CR⁺ cells are susceptible to MPTP toxicity. Abbreviations: 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine: MPTP; calretinin: CR; tyrosine hydroxylase: TH.</p