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.

Glutamatergic Fate of Neural Progenitor Cells of Rats with Inherited Audiogenic Epilepsy.

Epilepsy is associated with aberrant neurogenesis in the hippocampus and may underlie the development of hereditary epilepsy. In the present study, we analyzed the differentiation fate of neural progenitor cells (NPC), which were isolated from the hippocampus of embryos of Krushinsky-Molodkina (KM) rats genetically prone to audiogenic epilepsy. NPCs from embryos of Wistar rats were used as the control. We found principal differences between Wistar and KM NPC in unstimulated controls: Wistar NPC culture contained both gamma-aminobutyric acid (GABA) and glutamatergic neurons; KM NPC culture was mainly represented by glutamatergic cells. The stimulation of glutamatergic differentiation of Wistar NPC resulted in a significant increase in glutamatergic cell number that was accompanied by the activation of protein kinase A. The stimulation of KM NPC led to a decrease in immature glutamatergic cell number and was associated with the activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and protein kinase B/ glycogen synthase kinase 3 beta (Akt/GSK3ß), which indicates the activation of glutamatergic cell maturation. These results suggest genetically programmed abnormalities in KM rats that determine the glutamatergic fate of NPC and contribute to the development of audiogenic epilepsy.