Materials and methods
Animals and experimental design
Adult (4-5 month old) male CD1 mice were recruited in the experiments.
All animals were housed under 12/12 light–dark cycle with free access
to food and water. All experimental procedures were carried out in
accordance with EC Directive 86/609/EEC for animal experiments and
approved by the Institutional Animal Care and Use Committee at the
Sechenov Institute of Evolutionary Physiology and Biochemistry (#
12-2/2021).
Mice were divided into three experimental groups:
1) Group-housed (control) animals (GH group, n=10) which were housed in
standard vivarium cages, 5 animals per cage;
2) Mice exposed to hindlimb unloading (HU group, n=10). Each mouse was
housed in the individual cage and suspended by the tail through a swivel
attached to a metal rod at the top of the cage. The suspension angle was
adjusted to approximately 30°, so that the hindlimbs did not contact
with the floor but the animal was able to move freely around the cage
with use of the forelimbs (Morey-Holton & Globus, 2002).
3) Socially isolated mice (SI group, n=10) which were housed in the
individual cages similar to those for HU group, but without tail
suspension and any other movement restrictions.
The duration of the experiment consisted 3 days. All animals were
sacrificed on the following day.
Sample preparation
5 mice from each group were deeply anesthetized with i.p. injection of
Zoletil/Xylazine mixture (60 mg/kg+10 mg/kg; Virbac, France) and then
perfused transcardially with cold phosphate buffered saline (PBS)
followed by 4% PFA in 0.1 M PBS (pH 7.4). After perfusion all mice were
decapitated, the brains were removed, postfixed in 4% PFA (+4°C, 5
days), incubated in 20% sucrose/PBS for cryoprotection (+4°C, 3 days),
then frozen, and stored at - 80°C for further immunohistochemical assay.
The other 5 animals from each group were decapitated, each dorsal
striatum was dissected and homogenized separately for further Western
blot analysis.
Immunohistochemistry
After standard preliminary procedures cut sections were incubated
overnight with primary antibodies (Table 1). Further the sections were
washed in PBS and incubated for 1 hour with biotinylated anti-rabbit or
anti-mouse secondary antibodies (Tabl. 1) followed by one-hour
incubation with streptavidin-peroxidase complex (1:500, Supelco,
#S2438). The peroxidase reaction was revealed in the buffer containing
0.05% 3,3′-diaminobenzidine (DAB, Sigma-Aldrich, #D5637) and hydrogen
peroxide (0.01%).
Evaluation of sections. The frozen brains were sectioned
coronally at 10 µm using Leica cryostat. Every 15thsection was selected for analysis of TH, phospho(Ser31)-TH or
phospho(Ser40)-TH in the SNc, and TH, phospho(Ser31)-TH or
phospho(Ser40)-TH, D1R, D2R, or phospho(Ser133)-CREB in the dorsal
striatum. In total, five sections of the studied zone per each
immunostaining were analyzed for each animal. The sections were
processed under standardized conditions in every experiment, i.e.
control and experimental groups in each experiment were collected,
fixed, and processed for analysis simultaneously. To examine the
specificity of staining, we performed the negative control (the same
protocol without primary antibodies) that demonstrated no staining.
Analysis of immunostaining in the SNc and dorsal striatum was performed
with use of taken images by a 20x/0.5 objective on Zeiss Axio Imager A1
microscope (Carl Zeiss Microscopy GmbH).
Content of immunopositive substances was estimated as the optical
density normalized with background with use of ImageJ software (version
6.0). Calibration was done according ImageJ recommendation
(https://imagej.nih.gov/ij/docs/examples/calibration/). Each
analyzed image was converted into 8-bit grayscale image and optical
density of ROI was estimated. Optical density of the background was
evaluated at the same image in non-immunoreactive brain tissue field.
Western blot analysis
Each striatum was homogenized in lysis buffer (20 mM Tris pH 7.5, 1%
Triton-X100, 100 mM NaCl, 1 mM EDTA, 1 mM EGTA) with protease inhibitors
(Sigma-Aldrich, #P8340) and phosphatase inhibitors (Roche, #04 906 837
001) using tissue grinder at 4°C. Insoluble materials were removed by
centrifugation. Total protein content in samples was determined by Lowry
assay with bovine serum albumin (BSA) as a standard. The supernatant was
mixed in ratio 2:1 with 3x loading buffer (0.2 M Tris-HCl pH 6.7, 6%
sodium dodecyl sulfate, 15% glycerol, 0.003% bromophenol blue, and
10% β-mercaptoethanol) and incubated for 10 min at 96°C. Equal amounts
of samples (10 μg protein per line) were loaded for electrophoresis.
Proteins were separated on 10% or 8% polyacrylamide gel and then
transferred to nitrocellulose membrane (Santa Cruz Biotechnology,
#sc-3718). The membranes were incubated in 5% non-fat milk or 3% BSA
in Tris buffered saline with Tween (TBST) for 1 h and then incubated
overnight with primary antibodies (Table 1). Then the membranes were
washed in TBST and incubated with secondary antibodies (Table 1) for 1
hour at room temperature. Specific protein bands were visualized by
chemiluminescent reaction produced by SuperSignal™ West Dura Extended
Duration Substrate (ThermoFisher Scientific, #34075) with use of
ChemiDoc MP Imaging System (#12003154, Bio-Rad Laboratories Inc.,
Hercules, CA, USA).
Densitometric analysis of protein content was performed using
ImageJ software (version 6.0).
Phosphorylation of TH (Ser31, Ser40), ERK1/2 (Thr202/Tyr204), Akt
(Thr308), and GSK3β (Ser9) was estimated as ratios pTH/TH,
pERK1/2/ERK1/2, pAkt/Akt, and pGSK3β/GSK3β. Phosphorylation of CAMKII
(Thr286) was estimated by normalizing to actin. Overall phosphorylated
substrates of PKA were analyzed on the full blot, also with normalizing
to actin. Phosphorylation of specific PKA target, Ser157 of VASP
(vasodilator-stimulated phosphoprotein), was estimated as ratio
pVASP/VASP.
Statistical analysis
All data of Western-blot and imunohistochemical assays were processed
statistically by non-parametric Kruskal-Wallis test with post hoc Dunn’s
test using GraphPad 7 Software. All results were presented as median ±
interquartile range. Differences were regarded as significant at
p<0.05.