Event Abstract

Plantar mechanical stimulation prevents neurochemical alterations in the hippocampus induced by stimulated microgravity

  • 1 Institute of Evolutionary Physiology and Biochemistry (RAS), Russia
  • 2 Institute of Biomedical Problems (RAS), Russia

Introduction. Fifty seven years have passed since the first manned flight into space, but our knowledge about alterations in the CNS is still limited. MRI studies demonstrated the long-term space flight significantly changed the brain morphology in astronauts resulting in decreased of gray matter and increased volume of the ventricles (Roberts et al., 2017). Moreover cognitive impairments were documented in astronauts after long-terms space missions (Casler and Cook, 1999). Obviously all these changings are mediated by neurochemical/molecular disturbances in the CNS and particular in the hippocampus, which controls memory and navigation by coordination of many inputs from forebrain and lower brain structures. In the hippocampus of rodents (in space and using hind-limb suspension model) it was demonstrated the alterations in the expression of glutamate receptors, neurotrophins and apoptotic proteins (Yasuhara et al., 2007; Santucci et al., 2012; Naumenko et al., 2015; Shang et al., 2017; Wang et al., 2017). In all these experiments biological materials were collected after 7 and more days in space or tail suspension. The initial stage of the brain alterations during exposure to unloading is still unexplored. Among the main causes of the brain function impairment during gravitational unloading the sensory deprivation is considered as the most important one. And withdrawal of the support afferentation under conditions of real or simulated microgravity is believed to trigger on the brain remodeling (Kozlovskaya et al., 1988). The roles of support afferentation are usually analyzed by means of the plantar mechanical stimulation during unloading (Layne et al., 1998; Kyparos et al., 2005). However the effects of plantar mechanical stimulation on functional activity of neurons and/or cognitive function have not been studied yet. In our study we analyzed effect of short-term stimulated microgravity (SM) and checked whether the repeated plantar mechanical stimulation (PMS) affects functional status of the hippocampal neurons. Materials and methods. 36 adult Wistar rats were recruited in the experiments. The rats were housed in individual cages at 12/12 light-dark cycle with free water and food access. All procedures were approved by the Biomedical Ethics Committee of the Institute of Biomedical Problems. The rats were divided for 3 groups: C – vivarium control, SM group - 3 days hind-limb suspension, PMS group – 4 hours repeated plantar mechanical stimulation each day during hind-limb suspension. The rats were anaesthetized and perfused with 4% formalin for immunohistochemical study (n=4 for each groups); or the hippocampi were dissected and homogenized for Western blot assay (n=8 for each groups). Used antibodies for immunostaining and Western blot: GAD67 (Millipore), VGLUT2 (Millipore), NR2B (Abcam), pCREB (Ser133; Millipore), Akt (Cell Signaling), pAkt (Ser473; Cell Signaling), GSK3b (Cell Signaling), pGSK3b (Ser9; Cell Signaling), GAPDH (Abcam). Statistical analysis was done by a nonparametric Kruskal-Wallis test. Results. Our data demonstrated that SM significantly reduced VGLUT2 immunostating in the hippocampus that was confirmed by immunoblotting. These data proposed a decrease in glutamate release, since it was shown that the expression of VGLUT2 correlates with glutamate secretion (Liguz-Lecznar and Skangiel-Kramska, 2007). In opposite PMS restored VGLU2 expression (Fig.1). Analysis of NMDA receptor subunit NR2b did not reveal any difference between all groups. Immunostaining for glutamate decarboxylase GAD67, which synthesize GABA, also did not show the differences between the groups. Analysis of Akt signaling demonstrated that in SM group phosphorylation of both Akt and GSK3b was significantly increased in the hippocampus (Fig. 2). We also observed increased number of pCREB immunopositive nuclei in the dentate gyrus and CA3 region of the hippocampus that revealed the activation of this transcription factor. Akt and CREB are the main factors that regulate the neuronal survival. It was demonstrated that short-term hindlimb suspension (1-3 days uploading) can cause a systemic stress (Morey-Holton and Globus, 2002). Exhausting of glutamatergic system in the hippocampus, probably caused by stress, may be a risk factor for cell damage, and the activation of Akt and CREB supposes the activation of neuroprotective mechanisms. On the other hand PMS during SM prevented damage of glutamatergic hippocampal system (Fig.1) and restored normal activity the members of Akt pathway and CREB (Fig.2). Conclusion. Repeated PMS during 3 days’ hindlimb uploading prevented damage of glutamatergic hippocampal system and restored normal activity of Akt/CREB signaling. Thus our data revealed that plantar mechanical stimulation protects hippocampus from the negative effects of SM. Figure legends Figure 1. Plantar mechanical stimulation protects glutamatergic system from negative effects of stimulated microgravity A-C - VGLUT2 immunostaining revealed a decreased VGLUT2 expression in the Dentate Gyrus of rats after 3 days hind-limb suspension (B) in comparison with control (A); in rats exposed to plantar mechanical stimulation (C) the VGLUT2 immunostaining intensity was the same with control. gDG - granular layer of the Dentate Gyrus; m - molecular layer of the Dentate Gyrus. Scale bar 200 mm. D - Western blot analysis of VGLUT2 expression in the hippocampus. Data are shown as mean +/- SEM. * - p<0.05 Figure 2. Plantar mechanical stimulation restores activity of Akt signaling pathway and CREB A, B - analysis of Akt (A) and GSK3b (B) activity were estimated by phosphorylation levels of Akt at Ser473 and GSK3b at Ser9 Data are shown as mean +/- SEM. * - p<0.05 C-D - pCREB immunostaining demonstrated an increased number of pCREB immunopositive nuclei in granular layer of the Dentate Gyrus and CA3 of rats after 3 days hind-limb suspension (D) in comparison with control (C); in rats exposed to plantar mechanical stimulation (E) the pCREB immunostaining intensity did not differ significant from the control. gDG - granular layer of the Dentate Gyrus; m - molecular layer of the Dentate Gyrus. Scale bar 200 mm.

Figure 1
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Acknowledgements

This study was supported by the RFBR 17-29-01034-ofi_m and by FASO of Russia (#АААА-А18-118012290371-3).

References

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Keywords: Hippocampus, Dentate Gyrus, Hind-limb suspension, VGLUT2, GAD67, Akt, Gsk3 beta, CREB

Conference: 39th ISGP Meeting & ESA Life Sciences Meeting, Noordwijk, Netherlands, 18 Jun - 22 Jun, 2018.

Presentation Type: Extended abstract

Topic: Neurosciences and psychology

Citation: Berezovskaya A, Tyganov S, Shenkman B and Glazova M (2019). Plantar mechanical stimulation prevents neurochemical alterations in the hippocampus induced by stimulated microgravity. Front. Physiol. Conference Abstract: 39th ISGP Meeting & ESA Life Sciences Meeting. doi: 10.3389/conf.fphys.2018.26.00021

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Received: 17 Jan 2019; Published Online: 16 Jan 2019.

* Correspondence: Dr. Margarita Glazova, Institute of Evolutionary Physiology and Biochemistry (RAS), Saint Petersburg, Russia, MGlazova@iephb.ru