Figure legends
Figure 1: Expression and phosphorylation of tyrosine hydroxylase (TH) in the substantia nigra pars compacta (SNc) of mice after 3-day hindlimb unloading (HU) and social isolation (SI). (A) Immunohistochemical analysis revealed significant decrease in TH expression in the SNc of HU mice (n=5) in comparison with SI (n=5), but not group-housed (GH, n=5) animals. (B) Estimation of phospho(Ser31)TH in the SNc of GH (n=5), HU (n=5), and SI (n=5) mice showed similar pattern of inter-group difference. (C) Content of phospho(Ser40)TH was decreased in HU group (n=5) compared with both GH (n=5) and SI (n=5) mice. Plots show optical density of immunopositive substance (arbitrary units, a.u.). Data are shown as median ± interquartile range. * – p < 0.05. (D-F) Representative images of TH (D), phospho(Ser31)TH (E), and phospho(Ser40)TH (F) in the SNc of GH, HU, and SI mice. Scale bars: 100 μm.
Figure 2: The effect of 3-day HU and social isolation (SI) on the expression and phosphorylation of TH in the dorsal striatum of mice. (A) Immunohistochemical assay showed reduced expression of TH in the dorsal striatum of HU mice (n=5) in comparison with SI (n=5), but not GH (n=5) mice. (B, C) Western blot analysis detected elevated striatal phosphorylation of TH at Ser31 (B) in HU mice (n=5) in comparison with SI (n=4), but not GH (n=5) group, while TH phosphorylation at Ser40 (C) was decreased in the striatum of both HU (n=5) and SI (n=5) mice compared to GH control (n=5). Plot A shows optical density of immunopositive substance (arbitrary units, a.u.); plots B, C demonstrate Western blot results in arbitrary units (a.u.). Data are shown as median ± interquartile range. * – p < 0.05. (D) Representative images of TH in the dorsal striatum. Scale bars: 100 μm. (E) Representative immunoblot images of total and phosphorylated TH in the dorsal striatum samples.
Figure 3: Immunohistochemical analysis of dopamine receptors D1 and D2 in the dorsal striatum of mice exposed to 3-day HU and SI. (A) Striatal expression of D1-receptors was significantly reduced in the HU mice (n=5) in comparison with both GH (n=5) and SI (n=5) mice. (B) D2-receptor expression was similar in all experimental groups. Plots show optical density of immunopositive substance (arbitrary units, a.u.). Data are shown as median ± interquartile range. * – p < 0.05. (C, D) Representative images of D1 (C) and D2 (D) receptors in the dorsal striatum. Scale bars: 100 μm.
Figure 4: The effect of 3-day HU and SI on the activity of dopamine-associated signaling pathways in the dorsal striatum of mice. Western blot analysis detected a decrease in phosphorylation of protein kinase A (PKA) targets including overall substrates (A) and Ser157 of VASP (B) in the striatum of HU mice (n=5) in comparison with both GH (n=4 or n=5) and SI (n=5) groups. Phosphorylation of ERK1/2 (C), Akt (D), GSK3β (E), and CAMKII (F) did not differ between GH (n=4 or n=5), HU (n=5), and SI (n=5) animals after 3-day experiment. Plots demonstrate Western blot results in arbitrary units (a.u.). Data are shown as median ± interquartile range. * – p < 0.05. (G) Representative immunoblots of phosphorylated PKA substrates, phosphorylated and total forms of VASP, ERK1/2, Akt and GSK3β, phospho-CAMKII, and actin.
Figure 5: 3-day HU, but not SI, decreases CREB phosphorylation in the dorsal striatum of mice. (A) Immunohistochemical assay showed decreased content of CREB phosphorylated at Ser133 in striatal cell nuclei of HU mice (n=5) in comparison with GH (n=5) and SI (n=5) animals. Data are shown as median ± interquartile range. * – p < 0.05. (B) Representative images of phospho-CREB in the dorsal striatum of GH, HU, and SI mice. Phospho(Ser133)CREB immunopositive cell nuclei are pointed by arrowheads. Scale bars: 100 μm.