DA and receptors blockade increases the number of striatal TH⁺ neurons.

<p>Mice received a single i.p. injection with DA receptor ligands or with a selective nicotinic acetylcholine α4β2 receptor antagonist dihydro-β-erythroidine (DHβE) at PND4 and were killed at PND8. SKF = SKF38393 (10 mg/kg); SCH = SCH23390 (0.1 mg/kg); Q = quinpirole (0.1 mg/kg); RAC = raclopride (1 mg/kg); DHβE = dihydro-β-erythroidine (3.2 mg/kg). (E) PND4 mice subjected to striatal DA depletion by treatment with the TH inhibitor αMpT (150 mg/kg, i.p., twice, with 24 h of interval) were treated with quinpirole (0.1 mg/kg); (n = 6) or SKF38393 (10 mg/kg); (n = 6). Values are means+S.E.M. of 12 (A,C), 17 (B) or 6 (D,E) mice for group. In (A), *p<0.05 (One-way ANOVA+Bonferroni's test) <i>vs.</i> all other values; in (B), *p<0.05 (One-way ANOVA+Bonferroni's test) <i>vs.</i> values obtained in mice treated with saline or quinpirole alone; in (C,D,E), *p<0.05 (Student's t test) <i>vs.</i> values obtained in mice treated with saline.</p

DA depletion increases the number of intrinsic TH⁺ neurons.

<p>DA levels and the number of TH⁺ neurons in the striatum of mice treated with αMpT (150 mg/kg, i.p.; injected twice with 24 h of interval at PND4 and PND5), and killed 24 h (PND6) or 72 h (PND8) later are shown in (D) and (E). Values are means+S.E.M. of 10 mice for group. *p<0.05 (Student's test) <i>vs.</i> saline-treated mice. Correlation analysis between DA levels and the number of TH⁺ neurons in shown in (F) (r² = 0.65; p<0.05). Empty circles = mice treated with saline and killed at PND6; filled circles = mice treated with αMpT and killed at PND6; empty squares = mice treated with saline and killed at PND8; filled squares = mice treated with αMpT and killed at PND8.</p

DA depletion changes the spatial distribution of striatal TH⁺ neurons.

<p>The distribution profile of TH⁺ neurons in the striatum of mice treated with saline or in striatum of mice treated with saline or αMpT at PND4 (2 injections 24 h apart) and killed at PND6 is shown in (A). Representative images of neurons and fibers stained for TH are shown below the graph. The figure shows the triple vectors used for distance determination. Segments connecting the cell body of TH⁺ neurons to the central border and the two peripheral borders of the clusters are indicated). Note that most of the TH⁺ neurons are placed at shorter distance from the clusters of DA fibers in mice treated with αMpT. Double fluorescence immunostaining for TH and GAD, ChAT, Ki-67, and BrdU in mice treated with saline or αMpT as above is shown in (B), (C), (D), and (E), respectively.</p

Phenotypic characterization of intrinsic TH⁺ neurons.

<p>Double fluorescence staining for TH and DAT, or AADC and for TH and BrdU are shown in (A) and in (B), respectively. Co-localization was examined by densitometric analysis of red and green fluorescence in a selected region corresponding to the horizontal line in the right panels. The coincidence of the fluorescence peaks is indicative of a high level of co-localization.</p

Striatal TH⁺ neurons express D2 and D4 receptors.

<p>(A) Double fluorescence staining for TH and D1, D2 or D4 receptors in the striatum of mice at PND4 and PND8 is shown. Co-localization was examined by densitometric analysis of red and green fluorescence in a selected region corresponding to the horizontal line in the right panels. The coincidence of the fluorescence peaks is indicative of a high level of co-localization. (B) Immunoreactivity for D1 and D2 dopamine receptors in striatal sections of adult wild-type and D1 or D2 receptor knockout mice, respectively.</p

ELISA analysis of GDNF expression in the striatum of mice after treatment with saline, LY379268 (3 mg/kg, i.p.), MPTP (30 mg/kg, i.p.), or MPTP+LY379268.

<p>Animals were killed 1,2,3 or 7 days after treatments. Data of GDNF are the mean+S.E.M. of 8 animals. <i>p</i><0.05 (One-way ANOVA+Fisher's PLSD) vs. the corresponding groups of mice treated with saline (*) or with MPTP or LY379268 alone (#).</p

Double immunolabeling for GDNF and NeuN or GFAP in striatal cells showing the labelling of GDNF within neuronal cells (A).

<p>Arrows in the left panel, NeuN-positive cells containing GDNF mRNA black grains; arrow in the right panel, GDNF mRNA black grains, arrow head in the right panel, GFAP-positive cell. Immunohistochemical analysis of GDNF in the striatum of mice treated with a single injection of LY379268 (3 mg/kg, i.p.) and killed 24 h later (B). In both control mice and mice treated with LY379268, GDNF immunoreactivity is exclusively localized in neurons (note the absence of co-localization between GDNF and GFAP), and the extent of immunostaining increases after drug treatment. GDNF immunostaining in the striatum of mice treated 7 days before with MPTP, 20 mg/kg, i.p., x 3, two h apart (C). This treatment led to reactive gliosis in the striatum, as a result of the degeneration of nigro-striatal dopaminergic neurons. Under these conditions, GDNF immunostaining is localized both in neurons and reactive astrocytes. A single injection of LY379268 (3 mg/kg, i.p.) 7 days following MPTP injection did not enhance GDNF immunoreactivity in reactive astrocytes, but still enhanced immunoreactivity in neurons. Interestingly, the number of GDNF-positive reactive astrocytes is lower 24 h following LY379268 injection. Scale bar = 50 and 10 µm.</p

Quantitative real-time PCR analysis of GDNF mRNA in mouse striatum (A) and cortex (B) at 3, 6 or 12 h after systemic treatment with saline, LY379268 (3 mg/kg, i.p.), or LY379268 (3 mg/kg, i.p.)+LY341495 (1 mg/kg, i.p.).

<p>Values were normalized with respect to the amount of β-actin mRNA. Values are mean+S.E.M. of four determinations (each from triplicates). *<i>p</i><0.05 (One-way ANOVA+Fisher's PLSD) vs. saline-treated mice.</p

Basal GDNF levels in cultured mouse striatal neurons and in cultured astrocytes (A).

<p>Expression of phosphoERK1/2 and phospho-Akt in cultured striatal neurons treated with LY379268 (1 µM), LY341495 (1 µM) and LY379268+LY341495 for 15 min (B). Densitometric values are means+S.E.M. of 3–4 determinations. *p<0.05 (One-Way ANOVA+Fisher's PLSD) vs. basal values, #p<0.05 vs. LY379268 values. Treatment of cultured neurons with 1 µM LY379268 enhanced GDNF levels 24 h later (C), and it was abrogated by the co-application of the MEK inhibitor, PD98059, or the PI-3-K inhibitor, LY294002 (C). Application of LY379268 to astrocytes made “reactive” by several passages in culture and by the G5 supplement in the medium did not affect GDNF levels (D).</p

LY379268 fails to protects against MPTP toxicity in mice unilaterally implanted with anti-GDNF antibodies.

<p>Mice were implanted with a gelfoam (Spongostan) pre-soaked with saline alone (A,B) or a saline solution containing 5 µg of neutralizing anti-GDNF antibodies (A,B) in the left caudate nucleus. Stereological counts of TH-positive neurons in the substantia nigra pars compact in the implantation side (left) or contralateral side (right) in response to i.p. injection of saline, LY379268 (3 mg/kg), MPTP alone (30 mg/kg) or MPTP+LY379268 (injected 30 min prior to MPTP injection). Drugs were administered 24 h after the gelfoam implantation. Mice were killed 7 days after MPTP injection. Values (means+S.E.M.) were calculated from 6 mice per group. <i>p</i><0.05 (One-way ANOVA+Fisher's PLSD) vs. the corresponding values in mice treated with saline (*) or vs. the MPTP values of the right side (#).</p