Absence of GDNF modulation by estradiol replacement in ovariectomized female mice.

<p>(A) Mouse experimental model. Ovariectomized (ovx) animals were separated in four groups and received either sham implant (O) or E₂ implant (Oi, E1 and E5) at surgery time. Three weeks later, mice received Vehicle (O and Oi), a single s.c. E₂ injection (E1), or five E₂ injections over 5 days (E5). Animals were sacrificed 24 hours after s.c. injection (O, Oi, E1) or 4–5 hours after the last injection (E5). (B) Vertical bar graph showing the mean ± SEM of plasma E₂ in O, Oi, E1 and E5 mice. This shows that the estrogen replacement protocol induced an increase in circulating E₂ in ovx mice. (C) Western blot analysis of phosphorylated Akt (pAkt) showing a representative blot (left), and pAkt / Akt ratio is shown with a vertical bar graph (right). The greatest difference is found between O and E1 (p = 0.053). (D) <i>Gdnf</i> mRNA expression, reported as <i>Gdnf1-2</i> relative to <i>bActin</i>, in the striatum of wild-type (<i>Gdnf</i>^<i>+/+</i>) and heterozygous mice (<i>Gdnf</i>^<i>+/-</i>). (E) GDNF protein content expressed as pg / mg total protein in <i>Gdnf</i>^<i>+/+</i> and <i>Gdnf</i>^<i>+/-</i> striatum. (F) Scatter plot diagram showing the positive correlation (ρ = 0.72, p = 0.002) between <i>Gdnf</i> mRNA (shown in D) and GDNF protein levels (shown in E) in <i>Gdnf</i>^<i>+/+</i> and <i>Gdnf</i>^<i>+/-</i> striata. This data shows that GDNF protein concentration measured by ELISA is consistent with <i>Gdnf</i> gene expression level. (G) QPCR analysis of <i>Gdnf</i> expression measured by <i>Gdnf1-2</i> relative to <i>Parv</i> mRNA (left graph) or <i>Hmbs</i> mRNA (right). (H) ELISA analysis of GDNF protein levels showing no difference between the experimental groups. All data are presented as the mean ± SEM. *, p < 0.05; **, p < 0.01; ***, p < 0.001. N is indicated at the bottom of each bar.</p

Estrogen receptor alpha (ERα) expression in the mouse striatum.

<p>(A) Photomicrographs show parvalbumin (Parv) (red) and ERα+ (green) staining in male and female coronal sections. Vertical bargraphs on the right indicate the mean percentage ± SEM of the Parv+ cells that are ERα+ (n = 3). (B) microphotograph illustrating ERα (red) expression by Darpp32+ medium spiny neurons (green) in a female brain. Note that ERα is almost exclusively expressed in Darpp32+ cells. (C) Quantification of the Darpp32 and ERα colocalization shows that 94.2 ± 1.9% of the Darpp32+ neurons are immuno-positive for ERα; and 95.1 ± 0.8% of striatal ERα is expressed in Darpp32+ neurons in male and female mice (n = 4, from 2 male and 2 female striata).</p

Induction of cFos by E₂ in medium spiny neurons (MSN).

<p>(A) Photomicrographs of the dorsal striatum showing progressive increase of cFos+ (red) expression in Darpp32+ (green) MSN in O, Oi, E1 and E5 groups. (B) Graph showing the percentage of cFos+ nuclei expressed by Darpp32+ MSNs. (C) Graph showing the mean number ± SEM of Darpp32+ cFos+ cells. (D) Graph showing the mean percentage ± SEM of Darpp32+ that are cFos+. All data are presented as the mean ± SEM. *, p < 0.05; **, p < 0.01. N is indicated at the bottom of each bar.</p

Induction of cFos by E₂ in striatum and cortex.

<p>(A) Low magnification photomicrographs of O and E1 female mouse coronal sections illustrate the increase of cFos+ nuclei in the brain under E₂ stimulation. Magnified views of striatum and cortex highlight the cFos staining difference between O and E1 mice. (B) Graphs showing the mean ± SEM of cFos+ nuclei in the striatum (left) and cortex (right) from O, Oi, E1 and E5 mice. (C) Correlation chart showing the positive correlation (ρ = 0.70, p = 0.004) between striatal and cortical number of cFos+ nuclei (shown in B). Note the gradual increase of cFos+ cells such as O < Oi < E1 ≤ E5. (D) Dual labeling for cFos (green) and Parv (red) showing the above mentioned increase of cFos+ nuclei in the striatum. Very few Parv+ cells are positive for cFos as indicated by the percentage of Parv+ cFos+ cells in the graph. n.s., not significant. *, p < 0.05; **, p < 0.01. N is indicated at the bottom of each bar.</p

Volcano plots illustrating differential gene expression in SNpc (A) & VTA (B) comparing late middle-aged (18months old) to young (2month old) mice.

<p>Differentially expressed genes were distributed in the top left and top right sections of each figure, corresponding <i>pFDR</i> <0.05 and fold change<−1.3 and <i>pFDR</i> <0.05 and fold change >1.3, respectively.</p

Validation of microarray results in SNpc by real time quantitative PCR.

<p>Relative log2 fold changes of mRNA were presented comparing late middle-aged (18months old) to young (2month old) mice. Positive numbers corresponded to up-regulated genes, whereas negative numbers indicated down-regulated genes. *, <i>p</i><0.05 comparing 18 to 2 months old mice using Student’s <i>t</i>-test. <i>n</i> = 4 replicates for each age group.</p

Representative immunohistochemical staining of GFAP in SNpc.

<p><b>A</b> and <b>B</b>, immuno-staining against GFAP antibody. <b>A'</b> and <b>B'</b>, immuno-fluorescent staining against TH antibody. <b>A''</b> and <b>B''</b>, merging of immuno-stainings against GFAP and TH antibodies, the later was represented with a pseudo-color. SNpc was highlighted in each panel. Inset, a higher magnification of SNpc for each picture. Scale bar, 200 µm.</p

Gene ontology analysis of age-dependent genes in SNpc and VTA comparing 18 to 2 months old mice.

<p>Gene ontology analysis of age-dependent genes in SNpc and VTA comparing 18 to 2 months old mice.</p

Gene ontology analysis of age-associated genes specific to SNpc.

<p>Gene ontology analysis of age-associated genes specific to SNpc.</p

SNpc and VTA dissection from mouse brain.

<p>Brain sections were labeled against tyrosine hydroxylase antibody without (<b>A</b>) and after (<b>B</b>) SNpc dissection, and without (<b>C</b>) and after (<b>D</b>) VTA dissection. Scale bar: 500 µm. <b>E</b>. Relative <i>Calb1</i> mRNA level. *, <i>p</i><0.05 comparing VTA to SNpc (<i>n</i> = 4 in each group).</p