On the contrary, black bears do not show such regular arousal Mepiroxol episodes. Therefore we were able to determine that tau phosphorylation is a general, hibernation-related phenomenon and that different natures of hibernation and/or divergent hibernation conditions do not result in a different tau phosphorylation signature. The three species were compared using a panel of phosphorylation-dependent tau antibodies that was applied to different brain regions. To analyse whether hibernation-state related tau phosphorylation is a specifically regulated process or passively driven by a brain temperature-dependent shift of kinase- and phosphatase activities, we assessed the phosphate net turnover of tau protein in different hibernation-states. Moreover, to elucidate the molecular mechanisms regulating tau phosphorylation in vivo, we determined the state-dependent activity profile of glycogen synthase Tulathromycin B kinase 3 beta, cyclin dependent kinase 5, stress-activated protein kinase/Jun-amino-terminal kinase and mitogen activated protein kinase/extracellular regulated protein kinase during hibernation. The hibernation-related tau phosphorylation was analysed immunohistochemically and by Western blot using different phosphosite-specific tau antibodies. A summary of the investigated tau phosphorylation sites and the applied antibodies is shown in Figure 1. Generally, tau protein is highly phosphorylated during torpor states and phosphorylation levels decrease after arousal as demonstrated in Figure 2 where the monoclonal antibody AT8 recognizing tau protein phosphorylated at S202/T205 was applied to analyse generation and distribution of phospho-tau during hibernation in the neocortex of Syrian hamsters. Euthermic animals are characterised by a complete absence of AT8 immunoreactivity whereas already four hours after entry into torpor a marked increase of tau phosphorylation was observed. During progression of torpor state immunolabelling showed a constant increase. Phosphorylated tau protein was first observed in the cell body and the adjacent part of the apical dendrite. However, after a more prolonged time in the state of torpor labelling extended further away from the cell body into the band of Bechterew. In late torpor, a particularly intense labelling was observed in the apical dendrites and cell bodies of many pyramidal cells in layer II and IV. Noteworthy, the fibres in the band of Bechterew at the upper lamina of layer II were also strongly labelled. A rapid decline in AT8-immunoreactivity was determined after induced arousal. For instance, as early as 1 hour after induction of arousal no phospho-tau was detectable in the cell bodies in layer II and in the band of Bechterew. Furthermore, there was no labelling except for some apical dendrites as short as 2.5 hours after induction of arousal. A complete reversal of AT8 immunoreactivity was observed 72 hours after induced arousal. Antibodies that detect specific phosphorylation sites on tau protein were used to characterise the regional tau phosphorylation pattern during the hibernation cycle by Western blot. In total, seven phosphosites were analysed in five or two different brain regions. Brain samples of five different hibernation-states were compared in arctic ground squirrels and Syrian hamsters. In black bears, euthermic animals were compared to hibernating animals. The data are summarised in the Figures 3, 4 and 5 and Tables 1 and 2. A number of differences regarding the degree and the kinetics of tau phosphorylation between species, phosphosites, brain regions and hibernation-states were observed. The analysis of results from arctic ground squirrels and Syrian hamsters revealed the most detailed findings since in contrast to black bears additional hibernation-states and brain regions were studied resulting in information about the kinetics of tau phosphorylation/dephosphorylation during torpor and arousal periods. A comprehensive summary of the results is listed in Table S1. All of the analysed tau phosphosites are affected during hibernation and showed increased degree of phosphorylation in torpor states.