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

Abstract

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.

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Data Availability

The datasets used and/or analyzed in the current study are available from the corresponding author upon reasonable request.

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Acknowledgements

We are grateful to Dr. Pavel Musienko for critical discussion of the data and the manuscript. Part of the analysis was done at Research Resource Center #441590 at Sechenov Institute of Evolutionary Physiology and Biochemistry.

Funding

This study was supported by grants from RFBR 20-015-00062 (MVG), 17-29-01029-ofi_m (BSS), and 17-29-01034-ofi_m (NSM) and by Russian Government program.

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ASB performed experiments, analyzed the data, and revising manuscript. SAT and NSM performed animal experiments. SDN and AAN performed immunohistochemical staining and data analysis. BSS designed the experiments and revised manuscript. MVG conceived the study, designed the experiments, and wrote the manuscript. All authors discussed and commented on the manuscript.

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Correspondence to Margarita V. Glazova.

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10571_2020_922_MOESM1_ESM.pdf

Online Resource 1. Neither hindlimb unloading nor dynamic foot stimulation altered GABA expression in the hippocampus. a-c – GAD67 immunostaining of the hippocampus of control rats (a), the rats of DFS group (b) and after 3-day HU (c). DGg – granular layer of the dentate gyrus; DGm – molecular layer of the dentate gyrus; h – the hilus. d, e – western blot analysis of GAD67 expression: d – representative images of western blots of GAD67 and GAPDH; e – analysis of GAD67 expression in the hippocampus by calculation of the ration between GAD67 and GAPDH. n=8 (number of animals) for each group. f – qRT-PCR analysis demonstrated that GAD65 mRNA and GAD67 mRNA levels in the hippocampus of DFS and HU rats were not changed. n=6 for each group. c – control, DFS – the rats with dynamic foot stimulation during 3-day hindlimb unloading; HU – 3-day hindlimb unloading. Data are shown as median with interquartile range. (PDF 4065 kb)

10571_2020_922_MOESM2_ESM.pdf

Online Resource 2. Ponceau S staining of western blot membranes. After protein transfer, the nitrocellulose membranes were stained with Ponceau S and optical density of the selected zones for SNAP-25 and VAMP-2 (a), and for syntaxin 1 (b) analysis were calculated for analysis of SNAP-25, VAMP-2 and for syntaxin 1 expression. (PDF 3301 kb)

10571_2020_922_MOESM3_ESM.pdf

Online Resource 3. PKA activity do not change after hindlimb unloading or dynamic foot stimulation. a, b – western blot analysis of pPKA substrate phosphorylation (a) with Ponceau S staining as a standard (b). c – the result of PKA substrate phosphorylation. c – control, DFS – the rats with dynamic foot stimulation during 3-day hindlimb unloading; HU – 3-day hindlimb unloading. Data are shown as median with interquartile range. (PDF 2522 kb)

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Berezovskaya, A.S., Tyganov, S.A., Nikolaeva, S.D. et al. Dynamic Foot Stimulations During Short-Term Hindlimb Unloading Prevent Dysregulation of the Neurotransmission in the Hippocampus of Rats. Cell Mol Neurobiol 41, 1549–1561 (2021). https://doi.org/10.1007/s10571-020-00922-2

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Keywords

  • Simulated microgravity
  • Hippocampus
  • VGLUT1/2
  • SNARE proteins
  • Akt
  • CREB