Cellular changes in an in vitro neural circuit system under simulated microgravity

Physiological changes, some of which lead to neurological alterations and cognitive decline, have been reported to occur in space. To date, it has not been possible to identify the direct effect of microgravity alone on neural circuits in vitro. Therefore, this study aimed to elucidate the impact of simulated microgravity (sμG) on neural circuit dynamics using a microphysiological system (MPS). A unidirectional neural circuit MPS was engineered, and primary neurons from embryonic day 17 (E17) rat brains were extracted, seeded onto the system, and maintained under terrestrial conditions for two weeks to establish functional connectivity. Subsequently, cultures were exposed to either ground conditions or sμG using a rotating clinostat for an additional week. Neurons subjected to sμG exhibited a significant increase in oxidative stress and spontaneous Ca²⁺ activity, accompanied by a marked reduction in axonal density and synapsin-1 expression. Notably, sμG did not affect neuronal viability. Finally, transcriptomic analysis further revealed significant alterations in HSPA4 and SNCA expression, genes implicated in cellular stress responses and neurodegenerative pathology. This study represents the first practical application of a neural circuit MPS for physiological research. These findings underscore the utility of neural circuit MPSs as robust platforms for modeling the neurobiological consequences of microgravity and evaluating countermeasures to mitigate neural dysfunction in long-duration spaceflight. Statement of Significance: Long-term exposure to space environments, including microgravity and cosmic radiation, induces physiological changes, some leading to neurological impairments. However, the direct effects of microgravity on neural circuits remain unclear. Using a system that isolates microgravity, we demonstrate increased ROS generation, inhibited axon growth, altered synapse formation, and gene expression changes linked to neurodegenerative diseases. These findings highlight the potential risks of microgravity on neural function. MPS technologies, such as neural circuits on chips, are essential for space medicine and can provide platforms for drug testing to prevent space-induced cognitive decline. We anticipate that our technology will pave the way for examining the interaction between space environments and brain tissue at the cellular level in a practical and multifaceted manner.