Hypergravity induced disruption of cerebellar motor coordination

The cerebellum coordinates voluntary movements for balanced motor activity in a normal gravity condition. It remains unknown how hypergravity is associated with cerebellum-dependent motor behaviors and Purkinje cell’s activities. In order to investigate the relationship between gravity and cerebellar physiology, we measured AMPA-mediated fast currents and mGluR1-mediated slow currents of cerebellar Purkinje cells along with cerebellum-dependent behaviors such as the footprint and irregular ladder under a hypergravity condition. We found abnormal animal behaviors in the footprint and irregular ladder tests under hypergravity. They are correlated with decreased AMPA and mGluR1-mediated synaptic currents of Purkinje cells. These results indicate that gravity regulates the activity of Purkinje cells, thereby modulating cerebellum-dependent motor outputs.

the rats in the cage are exposed to 4 G hypergravity. A high-resolution video camera, which was placed inside the cage, was used to evaluate whether the mice could move freely, and access food and water. The rats were exposed to HG for 23 hours and took an hour rest time under normal gravity (1 G). The conditioning process was repeated for 4 weeks (Fig. 1a). Hypergravity exposure process is continued twenty-three hours a day with one hour resting for 4 weeks (Fig. 1b). The behavior tests followed by electrophysiology were conducted right after hypergravity exposure.
Slice preparation. All animal handling procedures were approved by the Institutional Animal Care and Use Committee of Inha University and Incheon National University. Animals were treated in accordance with the National Institutes of Health Guide for the care and use of laboratory animals, and the Animal Welfare Act (7 U.S.C. et seq.). Sprague-Dawley rat (postnatal age: 11 weeks) for brain slicing were deeply anesthetized with halothane. The Brains were quickly removed and placed into chilled (4 °C), oxygenated (5% CO 2 and 95% O 2 ) slicing medium containing (in mM): 110 Choline Chloride, 2.5 KCl, 1.2 NaH 2 PO 4 , 25 NaHCO 3 , 20 glucose, 7 MgCl 2 , 0.5 CaCl 2 . Sagittal slices (300 μm) were cut using vibratome. Rat Brain Slices were then transferred to a holding chamber containing oxygenated physiological saline made up of (in mM): 124 NaCl, 2.5 KCl, 1 NaH 2 PO 4 , 26.2 NaHCO 3 , 20 glucose, 1.3 MgCl 2 , 2.5 CaCl 2 . After at least 1 h recovery, individual slices were transferred to a recording chamber. Oxygenated physiological saline was continuously superfused at a rate of 1.5 ml/min at 32-33 °C temperature. electrophysiological recordings. Whole-cell patch recordings were obtained using an Axon instruments Axoclamp 700B Amplifier (Molecular Devices), and recording pipettes had tip resistances of 4-6 MΩ when filled with a solution containing (in mM): 135 Cesium methanesulfonate, 10 CsCl, 0.2 EGTA, 10 HEPES, 4 Na 2 -ATP, 0.4 Na-GTP. The pH and osmolality of intracellular solution were adjusted to 7.3 and 290 mOsm, respectively. Recordings were done in current-clamp configuration and cells were held at −65 mV: the resting membrane potential was in the range of −65 to −70 mV after whole-cell configuration. During recordings, an access resistance was continually monitored. Recordings were excluded if an input resistance changed by>15%. pClamp Version 10.2 software (Molecular Devices) was used for data acquisition. Whole-cell patch recordings were made on Purkinje neurons. Give 1 pulse (10 ms duration) and 5 pulses (50 ms duration, 100 Hz, 1 ms pulse width) stimulation. During the recordings, the slices were continuously remained at 32-33 °C temperature with ACSF that contained 10μM SR (TOCRIS), 2μM NBQX (TOCRIS).
Behavior test. In order to measure animals' capability of motor coordination under gravity shift, we forced control and HG-exposed rats to perform a footprint and irregular ladder test (see Fig. 2 for illustration of behavior tests). As for the footprint test, we made a long, foot-printable glass plate (with 10 cm width and 200 cm length) where the rats can walk along. Also, they were tested in 1 day, 1 week and 4 weeks so as to measure effects of HG over time. While rats were walking on the glass plate from the beginning to the end, we measured the lateral distance between front and hind paws in both control and HG groups with the different exposure times. The result was analyzed according to the distance pattern of footprint on the walking pad. As for the irregular ladder test, control and HG groups were allowed to walk on a zigzag patterned ladder which challenges rats to balance and walk straightly. While rats were climbing on the irregular ladder from the bottom to top, slipping numbers were counted in both control and HG rats with different exposure times. Data analysis. All electrophysiological data are treated and expressed numerically using Axon TM pCLAMP TM 11 Electrophysiology Data Acquisition & Analysis Software (Molecular Devices, San Jose, CA). The student' t-test in www.nature.com/scientificreports www.nature.com/scientificreports/ For the differences between HG and control group, P < 0.05 was interpreted statistically significant (*P < 0.05, **P < 0.005, ***P < 0.001). All Graphs were prepared in GraphPad ® Prism 7 (GraphPad Software Inc., La Jolla, CA) and final arrangement and labeling were carried out using Adobe illustrator CC 2019 (Adobe Inc., San Jose, CA). All data are illustrated as mean ± Standard Error of the Mean (SEM). n indicates the number of animals in behavior tests, and slices in EPG.
Next, we wondered whether the hypergravity-induced impairment of motor coordination is associated with altered intrinsic membrane properties of Purkinje cells. For recording, we selected Purkinje cells in the lobule 9 around the posterior vermis of the cerebellum related to gravity and balance of gravity and balance [27][28][29] . We measured cells' resting membrane potential (RMP), Input resistance (MΩ), depolarizing sec (mv), firing frequency (Hz) which are indicators of static membrane property of quiescent cells, signal holding capability, hyperpolarized-induced rebound action of signals, intrinsic firing behavior, respectively. We found there were no differences between the control and hypergravity group in resting membrane potential (Fig. 3b, Control: Then, we questioned whether cells' synaptic properties are related to hypergravity. Purkinje cells have synaptic inputs from parallel fibers which are largely shaped by both mGluR1-dependent slow currents and AMPAR-dependent fast currents. We examined mGluR1-mediated slow (I slow ) and AMPAR-mediated fast (I fast ) currents by stimulating parallel fibers. There was significant reduction of I slow amplitude over various intensities in hypergravity rats (Fig. 4a: Control 10   www.nature.com/scientificreports www.nature.com/scientificreports/ control and hypergravity groups. MAX I slow in the hypergravity group was smaller than that in the control group (Fig. 4a, Control: 261.93 ± 36.25, n = 7; HG: 61.18 ± 9.03, n = 9. **P = 0.0012). Also, MAX I fast in the hypergravity group was smaller than that in control rats (Fig. 4b, Control: 548.63 ± 131.73, n = 6; HG: 215.01 ± 37.68, n = 9. *P = 0.0126). This result suggests that hypergravity modulates mGluR1-and AMPAR-dependent currents of Purkinje cells, which likely affects motor coordination.

Discussion
In our studies, hypergravity impairs motor coordination which is accompanied by reduced mGluR1 and AMPA medicated currents of Purkinje cells. Meanwhile, the intrinsic properties of Purkinje's cells are unaffected. The alteration of gravity negatively might influence neurotransmitter release in synapses of parallel fibers to Purkinje cells. Purkinje cells' activity largely is influenced by two factors: 1) Intrinsic properties and 2) synaptic properties. There is no alteration in action potential generation under hypergravity as the cell's intrinsic properties including firing frequency and amplitude are not altered. In contrast, HG can reduce mGluR-and AMPAR-mediated currents of Purkinje cells, which is caused by sluggish release of glutamate in the presynaptic terminals of parallel fibers and/or altered kinetics and/or sensitivity of both mGluRs and AMPARs in the postsynaptic Purkinje cells. A previous study showed HG alteres neurotransmitter reuptake by presynaptic transporters 30 . Nonetheless, we suggest that HG primarily affects postsynaptic mechanisms such as kinetics and/or expression of mGluRs and AMPARs. A further study is required to reveal whether presynaptic mechanisms are involved in the altered condition of gravity.
The central function of cerebellum is to build a dynamic prediction (e.g., an internal model) of the self-motion to ensure postural and perceptual stability 31,32 . The internal model allows cerebellum to maintain body movement against gravity. Gravity is a constant force that we experience through our entire lives. Thus, it is a neural mechanism shaped by balanced consequence of adaptation and stabilization through a learning process [33][34][35][36][37] . It is generally assumed according to a previous notion that the brain's internal model is required to enforcedly adapt to the zero-G environment in outer space such that active motion generates an erroneous mismatch after re-entry 38 . For example, astronauts could experience disturbances in their internal balance when launching to and returning from earth under HG and staying in the outer space under MG. The exposure of different gravity could drive them to unlearn and later relearn the earth environment like the jet lag. As the learning process is closely associated with neural plasticity, there can be a potential contribution of gravity of long term potentiation/ depression, a suggested cellular mechanism underlying learning, which is involved in Ca 2+ -permitting channels/ receptors including NMDARs and mGluRs. Interestingly, we found hypergravity-induced disruption of AMPA and mGluR1 currents can be a molecular mechanism of such a mismatch underlying unlearning and relearning www.nature.com/scientificreports www.nature.com/scientificreports/ processes. In our experimental paradigm, there is a potential to affect synaptic transmission due to the abnormal animal behavior in rotation which can restrict normal access of food, water and space. Thus, the altered cerebellar synaptic activities under HG can be comorbid with abnormal animal welfare. Although the similar condition to astronauts can be considered, a further study to investigate the compounding effects seems to be required.
In conclusion, the Purkinje cells of cerebellum play an important role in regulating balanced movements as they provide inhibitory tones to motor network. Purkinje cells might be required to increase the inhibitory interference for stabilizing circuit excitability at the beginning of hypergravity. Purkinje cell's synaptic activity can be decreased during long-term exposure of hypergravity as an adaptation mechanism. This negative correlation between cerebellum activity and gravity might provide important strategies of how to alleviate adverse symptoms when a gravity shift occurs in certain environments (e.g. trip to space and back to earth).