Activity of dopamine-linked signaling pathways in the dorsal striatum
To estimate activity of downstream signaling cascades of dopamine receptors, we analyzed activity of protein kinase A (PKA), extracellular signal-regulated kinases 1 and 2 (ERK1/2), protein kinase B (Akt), glycogen synthase kinase 3β (GSK3β), and Ca2+/calmodulin-dependent protein kinase II (CAMKII) in the dorsal striatum. Western-blot analysis showed that phosphorylation of total PKA substrates was decreased in the dorsal striatum of HU mice in comparison with both GH and SI freely moving animals, while no difference between GH and SI animals was observed (Figure 4A, G). Phosphorylation of VASP at Ser157, specific PKA target (Howe et al. , 2005; Tejeda et al. , 2020), showed similar pattern of alterations (Figure 4B, G). Altogether, these data indicated decreased activity of PKA in HU mice. At the same time we did not reveal any changes in phosphorylation of ERK1/2 (Thr202/Tyr204) (Figure 4C, G), Akt (Thr308) (Figure 4D, G), GSK3beta (Ser9) (Figure 4E, G), and CAMKII (Thr268) (Figure 4F, G) indicating that 3-day HU and SI did not affect corresponding signaling pathways.
It is also known that activation of D1R and D2R is accompanied with increased phosphorylation of transcriptional factor CREB (Beaulieu & Gainetdinov, 2011). Immunohistochemical analysis showed decreased amount of active CREB phosphorylated at Ser133 in the cell nuclei of the dorsal striatum in mice exposed to HU as compared with both GH and SI groups (Figure 5). Again, GH and SI animals demonstrated no difference.