Expression and activity of TH in the nigrostriatal system
Firstly, we analyzed expression and phosphorylation of TH – the rate-limiting enzyme of dopamine biosynthesis. It is abundantly expressed in bodies of dopaminergic neurons localized in the substantia nigra pars compacta and in their axon terminals innervating striatal SPNs (Klein et al. , 2018). Immunohistochemical analysis showed that both expression of TH (Figure 1A, D) and its phosphorylation at Ser31 (Figure 1B, E) were significantly decreased in the SNc of mice in HU group compared with SI, but not GH animals. As conditions in HU and SI groups differed only by presence of unloading, we supposed that observed changes were caused rather by HU than isolation. Phosphorylation of TH at Ser40 (the main site of TH activation) was decreased in HU mice in comparison with both GH and SI groups (Figure 1C, F). Normally loaded GH and SI animals demonstrated no difference for all these markers. Thus, our data indicated that antiorthostatic unloading was the main factor which caused down-regulation of TH and dopamine biosynthesis in the SNc dopaminergic neurons.
In the dorsal striatum, TH expression estimated by immunochemical assay was as well lower in HU group in comparison with SI group (Figure 2A, D), however, TH phosphorylation at Ser31 was, in opposite, higher (Figure 2B, E). Besides, neither HU nor SI mice differed statistically from GH control. TH phosphorylation at Ser40 was reduced both in the HU and SI group in comparison with GH animals (Figure 2C, E). These data let us to suppose that both HU and SI affected dopaminergic innervation of striatal neurons.