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.