In search of stress: analysis of stress-related markers in mice after hindlimb unloading and social isolation.

OBJECTIVES: Hindlimb unloading (HU), widely used to simulate microgravity effects, is known to induce a stress response. However, as single-housed animals are usually used in such experiments, social isolation (SI) stress can affect experimental results. In the present study, we aimed to delineate stressful effects of 3-day HU and SI in mice. METHODS: Three animal groups, HU, SI, and group-housed (GH) control mice, were recruited. A comprehensive analysis of stress-related markers was performed using ELISA, western blotting, and immunohistochemistry. RESULTS: Our results showed that blood corticosterone and activity of glucocorticoid receptors and cAMP response element-binding protein (CREB) in the hippocampus of SI and HU animals did not differ from GH control. However, SI mice demonstrated upregulation of the hippocampal corticotropin-releasing hormone (CRH), inducible NO synthase (iNOS), vesicular glutamate transporter 1 (VGLUT1), and glutamate decarboxylases 65/67 (GAD65/67) along with activation of Fos-related antigen 1 (Fra-1) in the amygdala confirming the expression of stress. In HU mice, the same increase in GAD65/67 and Fra-1 indicated the contribution of SI. The special HU effect was expressed only in neurogenesis attenuation. DISCUSSION: Thus, our data indicated that 3-day HU could not be characterized as physiological stress, but SI stress contributed to the negative effects of HU.

Short-term hindlimb unloading negatively affects dopaminergic transmission in the nigrostriatal system of mice.

The nigrostriatal system composed of the dorsal striatum and the substantia nigra (SN) is highly involved in the control of motor behavior. Various extremal and pathological conditions as well as social isolation (SI) may cause an impairment of locomotor function; however, corresponding alterations in the nigrostriatal dopaminergic pathway are far from full understanding. Here, we analyzed the effect of 3-day hindlimb unloading (HU) and SI on the key players of dopamine transmission in the nigrostriatal system of CD1 mice. Three groups of mice were analyzed: group-housed (GH), SI, and HU animals. Our data showed a significant decrease in the expression and phosphorylation of tyrosine hydroxylase (TH) in the SN and dorsal striatum of HU mice that suggested attenuation of dopamine synthesis in response to HU. In the dorsal striatum of HU mice, the downregulation of TH expression was also observed indicating the effect of unloading; however, TH phosphorylation at Ser40 was mainly affected by SI pointing on an impact of isolation too. Expression of dopamine receptors D1 in the dorsal striatum of HU mice was increased suggesting a compensatory response, but the activity of downstream signaling pathways involving protein kinase A and cAMP response element-binding protein was inhibited. At the same time, SI alone did not affect expression of DA receptors and activity of downstream signaling in the dorsal striatum. Obtained data let us to conclude that HU was the main factor which impaired dopamine transmission in the nigrostriatal system but SI made some contribution to its negative effects.

Plantar Stimulations during 3-Day Hindlimb Unloading Prevent Loss of Neural Progenitors and Maintain ERK1/2 Activity in the Rat Hippocampus.

Adult neurogenesis is a flexible process that depends on the environment and correlates with cognitive functions. Cognitive functions are impaired by various factors including space flight conditions and reduced physical activity. Physically active life significantly improves both cognition and the hippocampal neurogenesis. Here, we analyzed how 3-day simulated microgravity caused by hindlimb unloading (HU) or dynamic foot stimulation (DFS) during HU can affect the hippocampal neurogenesis. Adult Wistar rats were recruited in the experiments. The results demonstrated a decrease in the number of doublecortine (DCX) positive neural progenitors, but proliferation in the subgranular zone of the dentate gyrus was not changed after 3-day HU. Analysis of the effects of DFS showed restoration of neural progenitor population in the subgranular zone of the dentate gyrus. Additionally, we analyzed activity of the cRaf/ERK1/2 pathway, which is one of the major players in the regulation of neuronal differentiation. The results demonstrated inhibition of cRaf/ERK1/2 signaling in the hippocampus of HU rats. In DFS rats, no changes in the activity of cRaf/ERK1/2 were observed. Thus, we demonstrated that the process of neurogenesis fading during HU begins with inhibition of the formation of immature neurons and associated ERK1/2 signaling activity, while DFS prevents the development of mentioned alterations.

Dynamic Foot Stimulations During Short-Term Hindlimb Unloading Prevent Dysregulation of the Neurotransmission in the Hippocampus of Rats.

Spaceflight and simulated microgravity both affect learning and memory, which are mostly controlled by the hippocampus. However, data about molecular alterations in the hippocampus in real or simulated microgravity conditions are limited. Adult Wistar rats were recruited in the experiments. Here we analyzed whether short-term simulated microgravity caused by 3-day hindlimb unloading (HU) will affect the glutamatergic and GABAergic systems of the hippocampus and how dynamic foot stimulation (DFS) to the plantar surface applied during HU can contribute in the regulation of hippocampus functioning. The results demonstrated a decreased expression of vesicular glutamate transporters 1 and 2 (VGLUT1/2) in the hippocampus after 3 days of HU, while glutamate decarboxylase 67 (GAD67) expression was not affected. HU also significantly induced Akt signaling and transcriptional factor CREB that are supposed to activate the neuroprotective mechanisms. On the other hand, DFS led to normalization of VGLUT1/2 expression and activity of Akt and CREB. Analysis of exocytosis proteins revealed the inhibition of SNAP-25, VAMP-2, and syntaxin 1 expression in DFS group proposing attenuation of excitatory neurotransmission. Thus, we revealed that short-term HU causes dysregulation of glutamatergic system of the hippocampus, but, at the same time, stimulates neuroprotective Akt-dependent mechanism. In addition, most importantly, we demonstrated positive effect of DFS on the hippocampus functioning that probably depends on the regulation of neurotransmitter exocytosis.