8 research outputs found
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
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
<i>In situ</i> hybridization of sagittal sections at basal ganglia level showing the expression of mRNA encoding GDNF (A) or NGF (B).
<p>Autoradiogram showing GDNF expression in the striatum of saline-treated
mice or LY379268 (0.25 mg/kg, i.p.)-treated mice (A). The inserts show
representative GDNF mRNA labeled cells (black grains) with increased
levels of labeling in LY379268-treated mice. Autoradiogram showing NGF
expression of saline-treated mice or LY379268 (0.25 mg/kg, i.p.)-treated
mice (B). Dose-response curve of GDNF mRNA levels in the striatum of
mice treated with saline or LY379268 (0.1, 0.25, 1, 3 or 4 mg/kg, i.p)
(C) and time-course of GDNF mRNA levels in the striatum of mice after a
single injection of LY379268 (0.25 mg/kg, i.p.) (D); values are
means±S.E.M
(n = 4–5, animals per group;
three independent experiments). Striatal GDNF mRNA levels in mice
treated with saline, LY379268 (0.25 mg/kg, i.p), LY341495 (1 mg/kg, i.p)
or LY379268+LY341495 (E); value are means±S.E.M
(n = 4, animals per group; three
independent experiments). *<i>p</i><0.05;
**<i>p</i><0.01 (One-way
ANOVA+Fisher's PLSD) vs. control mice. Scale bar:
A–B = 4 mm. Str, striatum;
Ctx, cortex; Hipp, hippocampus.</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
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
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
Immunohistochemical analysis of TH in the pars compacta of substantia nigra of mice injected with a single i.p. dose of 30 mg/kg of MPTP, alone or combined with LY379268 (0.25 or 3 mg/kg in a single i.p. injection, 30 min prior to MPTP injection or 0.25 mg/kg/7 days once a day, i.p.).
<p>Scale bar = 100 µm.
Stereological TH-positive cell counts are also shown. Values
(means+S.E.M.) were calculated from 7–8 mice per
group (10 sections - 10 µm thick, cut every 100 µm,
per animal were used for the calculation of the density of TH-positive
neurons in the pars compacta of the substantia nigra).
*<i>p</i><0.05 (One-way
ANOVA+Fisher's PLSD) vs. mice treated with MPTP
alone.</p
Western blot analysis of striatal GDNF expression in wild-type, <i>mGlu2<sup>−/−</sup></i> or <i>mGlu3<sup>−/−</sup></i> mice in basal conditions (A) and after treatment with LY379268, 3 mg/kg, i.p. (B).
<p>Animals were killed 24 h later. Densitometric data of GDNF are shown and
are the mean+S.E.M. of 3 animals performed two
times.*<i>p</i><0.05 (One-way
ANOVA+Fisher's PLSD) vs. saline-treated mice.</p