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.