Impairment of synaptic plasticity and novel object recognition in the hypergravity-exposed rats

The gravity is necessary for living organisms to operate various biological events including hippocampus-related functions of learning and memory. Until now, it remains inconclusive how altered gravity is associated with hippocampal functions. It is mainly due to the difficulties in generating an animal model experiencing altered gravity. Here, we demonstrate the effects of hypergravity on hippocampus-related functions using an animal behavior and electrophysiology with our hypergravity animal model. The hypergravity (4G, 4 weeks) group showed impaired synaptic efficacy and long-term potentiation in CA1 neurons of the hippocampus along with the poor performance of a novel object recognition task. Our studies suggest that altered gravity affects hippocampus-related cognitive functions, presumably through structural and functional adaptation to various conditions of gravity shift.

. Previous studies summarizing the physiological effects of altered gravity on hippocampus-related functions from genetic to molecular, behavioral, and electrophysiological levels.

Gravity
Exposure duration Age condition Strain Effect of gravity on the hippocampus References HG 1.85G 1 h for 5 days 7-9 weeks CD1 mice Upregulation of expression level in synaptic plasticity-related gene (proSAAS, neuroblastoma, thymosin beta-10, inhibin beta E) 46 Damage on discriminating a new spatial arrangement 47 2G 14 days 8 weeks C57BL/6J male mice Decreased brain-derived neurotrophic factor (BDNF) in the ventral hippocampus·Increased 5-HT receptor 1B in the ventral hippocampus 48 14 days 7 weeks Wistar male rats Impaired spatial memory (radial eight arms maze) The same serum cortisol level with the control Upregulation of insulin like growth factor binding protein 2 49 3,4G 14 days 150-180 g Wistar rats Impaired spatial learning task until 5 days, but no change after 5 days (radial arm maze) 50 14 days -Rats No change of Input / output relationships and Long-term potentiation 51 21 days 8 weeks C57BL/6J male mice Impaired spatial learning performance (water maze) 45 24, 48 h 8-9 weeks C57BL/6J male mice No detrimental effect on basal neurotransmission Increased LTP and phosphorylated AMPAR, but no change of L-LTP and phosphorylated CREB 44

MG
Tail-suspension 7 days 6-8 weeks BALB/c mice Major loss of proteins (tubulin, β-Synuclein) 41 28 days 8 weeks SD rats The decline of learning and memory (Morris water maze) Increased GluR1, GluR5, and glutamate whereas decreased 5-HT, dopamine, GABA, and epinephrine 39 Hindlimb www.nature.com/scientificreports/ length × 60 cm height) to be habituated to environmental factors including the place of objects. The two objects were placed in a diagonal position about 5 cm away from the white wall (Fig. 2a). The exploratory movement of the rat during the phase was recorded with a video camera installed at the top of the apparatus. In completing the first phase, the rat and familiar objects (F 1 , F 2 ) were removed from the apparatus for 6 h. In the second phase, the test phase, the rat explored a third copy of the familiar object (F 3 ) and a novel object (N) in the apparatus. Rats used to explore the novel object (N) more than the familiar one (F 3 ). To eliminate a certain variation by emotional instability, rats with excessive freezing behavior more than 60% (> 360 s) out of the whole period of test phase (10 mins) were excluded from analysis (Exclusion : 1G-3 out of 10 rats; HG 1day , HG 4weeks -each 1 out of 5 rats). Object exploration was defined when the nose of the rats directed towards the object at a distance below 2 cm and measured by the discrimination index which indicates the difference of time spent between a novel (T N ) and familiar object (T F3 ). It was calculated with the total amount of time spent with both objects in the test phase [Discrimination Index = (T N -T F3 )/(T N + TF 3 )].
Brain slice preparation. Age matched conditioned (HG group) and unconditioned (1G group) rats were deeply anesthetized with 2% isoflurane. Motionless rats were decapitated by a guillotine, and the brain was isolated quickly. The isolated brain was transferred to the ice-cold dissection buffer containing the following ingredients (in mM): 25 glucose, 75 sucrose, 87 NaCl, 2. In vitro field recording. Slices were transferred to a recording chamber where aCSF flowed (31 ± 0.5 °C; 1-2 ml/min). A slice harp anchored the slices in order to stabilize the recording position. A bipolar stimulating electrode was placed on the SC to evoke field excitatory postsynaptic potential (fEPSP) which were recorded on the stratum radiatum (SR) of CA1 by glass electrodes filled with aCSF. All responses were acquired using Axon Digidata Data analysis. All electrophysiological data were presented numerically using Axon pCLAMP11 Electrophysiology Data Acquisition and Analysis Software (Molecular Devices, San Jose, CA). The difference in behavior assessment was measured by One-way analysis of variance (ANOVA) of the Bonferroni post-hoc test. Two-way ANOVA was used to assess statistical significance for the differences between HG and 1G groups in EPG. Every statistical process was performed on SPSS Statics 25 (IBM, Armonk, NY). At least p < 0.05 was interpreted statistically significant (*p < 0.05, **p < 0.01, ***p < 0.001). All Graphs were prepared by GraphPad Prism 7 (GraphPad Software Inc., La Jolla, CA, USA) and final arrangement and labeling were carried out using Adobe Illustrator CC 2019 (Adobe Inc., San Jose, CA, USA). All data are presented in mean ± standard error of the mean (SEM). N indicates the number of animals in the NOR test and slices in EPG.

Result
Impaired NOR in rats under HG. We wondered whether the gravity affects memory-related behavior according to an exposure time of HG. The NOR task was conducted with rats under normal gravity, HG 1day , and HG 4weeks in open field box ( Fig. 2a,b). Rats showed similar preference for each object regardless of HG in the familiarization phase ( Fig. 2c Reduced postsynaptic, but not presynaptic transmission under HG. In order to examine whether HG affects synaptic events in the hippocampal CA1 network, we tested the synaptic transmission of CA1 pyramidal cells in response to SC stimulation. The fiber volley (FV), the indicator of Ca 2+ influx into the presynaptic axon terminal, was considered as the input while the slope of postsynaptic fEPSPs, mostly AMPARs-mediated responses, was taken as the output. As predicted, the slope of fEPSPs increased as the amplitude of FVs increased in both groups (Fig. 3a,

Altered LTP in HG.
To test whether HG has an effect on synaptic plasticity, we examine short-term and long-term synaptic plasticity in both the 1G and HG groups. When SC was activated by the paired pulse with various intervals, differential paired pulse facilitation (PPF) was observed as the slope of the second fEPSPs (P2) over the first fEPSPs (P1) in both groups (Fig. 4a). There was no statistical significance between the two groups (1G-50 ms: 1.98 ± 0.23, n = 9 slices, 100 ms: 1.77 ± 0.17, n = 9 slices, 250 ms: 1.24 ± 0.07, n = 9 slices, 500 ms: 1. www.nature.com/scientificreports/

Discussion
It has been well studied that the vestibular system is greatly affected by altered gravity 21,22,[56][57][58] . The previous study implied that the vestibular organ was the main area influenced by HG in the brain 48 . Notably, the hippocampus, especially CA1, had electrophysiological and anatomical connections with the vestibular system [59][60][61][62][63][64][65][66] . For example, lesioned hippocampus aggravated HG-induced motion sickness 53 . The fact that HG has adverse effects on the brain triggered us to investigate a hippocampal function after the HG conditioning. Our current findings are as follows (1)  Differential effects of HG on pre-and post-synaptic neurons. Neurotransmitter release is determined by the incidence and pattern of action potentials, depolarization of nerve terminals, and release probability of vesicle machinery [67][68][69][70] . The previous study investigating the synaptosome in cerebral hemispheres showed that HG alters neurotransmitter release by modulation of neurotransmitter reuptake, indicating a role of HG in a presynaptic mechanism 71 . However, in our study, there was no significant difference of presynaptic FV amplitude over stimulation intensities and PPF representing presynaptic Ca 2+ influx and neurotransmitter releaseprobability, respectively. It demonstrates that HG does not alter the presynaptic activity at least in CA3-CA1 network. Instead, the strong reliance on postsynaptic AMPAR/NMDAR responses under HG condition depicts a postsynaptic mechanism. This finding is consistent with our early study that HG causes the impaired function of postsynaptic AMPAR and metabotropic glutamate receptors (mGluRs) subtype 1 in the cerebellum 72 .
A role of HG in cognitive behaviors and plasticity. Previous studies showed that HG causes various physiological changes, such as vestibular function 73 , signaling pathway in muscles 74 , and bone formation 75 which can lead to altered behaviors. As expected, our research group also observed that HG causes an abnormality of cerebellum-dependent motor coordination 72 . Now, our view is expanded to investigate a role of HG in cognitive behaviors with a memory test. HG-driven poor performance in NOR may be affected by a defect of memory function because NOR is dominantly dependent on the hippocampus [76][77][78][79][80] . Our result suggests that HG directly triggers the dysfunction of the hippocampus-dependent cognitive behavior. Given our and other results, HG could accompany multiple, parallel processing of various physiological systems such as HG to vestibular/motor behaviors and HG to cognitive behaviors. It is previously well known that NMDARs are deeply involved in neural plasticity and often behaviors 32,36,81-83 . A previous study described an increased LTP under short-term exposure of 4G (48 h) 44 . Meanwhile, we observed that the long-term, but not short-term, exposure of 4G (4 weeks) impairs a cognitive behavior followed by LTP deficit. Prior studies (various gravity levels, 3 weeks) revealed that HG-induced abnormality in various behaviors was sustained even after 15 days from centrifugation 45 . In our experimental condition, it seems to be worth testing how long the HG effect lasts.
Therapeutic strategy for HG. This study provides the scientific aspect of physiological effects by HG on hippocampus. Long-term gravity shift can cause the impairment of electrophysiological property in the hippocampus and the behavior in the NOR task, and it could be due to a defect of postsynaptic receptors. Therefore, HG-induced impairment may have the potential to be rescued by restoring the function of postsynaptic receptors. We have previously proposed transient potassium channels as a therapeutic target for various brain disorders. It is because the transient potassium channels are electrically counteracting channels to NMDARs and have clinical benefits of minimal interference in a normal synaptic transmission which can be impaired under www.nature.com/scientificreports/ the pharmacological modulation of NMDARs 84,85 . A pharmacological approach to enhance an NMDAR function can be further investigated under the condition of altered gravity.