Human neural network activity reacts to gravity changes in vitro

Abstract

During spaceflight, humans experience a variety of physiological changes due to deviations from familiar earth conditions. Specifically, the lack of gravity is responsible for many effects observed in returning astronauts. These impairments can include structural as well as functional changes of the brain and a decline in cognitive performance. However, the underlying physiological mechanisms remain elusive. Alterations in neuronal activity play a central role in mental disorders and altered neuronal transmission may also lead to diminished human performance in space. Thus, understanding the influence of altered gravity at the cellular and network level is of high importance. Previous electrophysiological experiments using patch clamp techniques and calcium indicators have shown that neuronal activity is influenced by altered gravity. By using multi-electrode array (MEA) technology, we advanced the electrophysiological investigation covering single-cell to network level responses during exposure to decreased (micro-) or increased (hyper-) gravity conditions. We continuously recorded in real-time the spontaneous activity of human induced pluripotent stem cell (hiPSC)-derived neural networks in vitro. The MEA device was integrated into a custom-built environmental chamber to expose the system with neuronal cultures to up to 6 g of hypergravity on the Short-Arm Human Centrifuge at the DLR Cologne, Germany. The flexibility of the experimental hardware set-up facilitated additional MEA electrophysiology experiments under 4.7 s of high-quality microgravity (10^-6 to 10^-5 g) in the Bremen drop tower, Germany. Hypergravity led to significant changes in activity. During the microgravity phase, the mean action potential frequency across the neural networks was significantly enhanced, whereas different subgroups of neurons showed distinct behaviors, such as increased or decreased firing activity. Our data clearly demonstrate that gravity as an environmental stimulus triggers changes in neuronal activity. Neuronal networks especially reacted to acute changes in mechanical loading (hypergravity) or de-loading (microgravity). The current study clearly shows the gravity-dependent response of neuronal networks endorsing the importance of further investigations of neuronal activity and its adaptive responses to micro- and hypergravity. Our approach provided the basis for the identification of responsible mechanisms and the development of countermeasures with potential implications on manned space missions.

Keywords: drop tower; electrophysiology; human induced pluripotent stem cell (hiPSC)-derived neurons; hypergravity; iNGN; microgravity; multi-electrode array (MEA); neural network.

FIGURE 1

Environmental chamber for multi-electrode array (MEA) electrophysiology experiments on altered gravity platforms. (A) Exploded-view illustration of the experimental hardware depicting the main electrical and structural elements. (B) Temperature profile for one drop tower experiment session. The recording starts after the evacuation of the tower for a baseline recording of 15 min before the drop (red striped line). Purple and green lines depict the temperature at the two respective MEA chips. Other colored temperature lines are measured at different locations inside the pressure chamber.

FIGURE 2

Investigation of morphology and electrophysiological activity of human neural networks subjected to 6 g hypergravity on a centrifuge. (A) Overview of the different experiment phases and the sections included in the analysis with their respective durations. (B) Phase contrast microscopy images of an exemplary MEA chip pre- and post-exposure to hypergravity. (C) Zoom images of the areas marked with a red frame in (B). (D) Firing and (E) bursting rates of neuronal cultures subjected to 6 g hypergravity on a human centrifuge. Five MEAs were measured during six centrifuge runs. Statistical analysis included 144 and 44 units for firing and burst rate, respectively. Repeated measures one-way ANOVA and Tukey’s multiple comparisons test were used (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001). Dashed lines mark the first, second, and third quartile in the violin. (F–H) Density plots of the firing rates during ramp (F), hypergravity (G), and post-exposure (H) phases compared to the baseline.

FIGURE 3

Electrophysiological activity of human neural networks subjected to microgravity in a drop tower experiment. (A) Overview of the different experiment phases with respective g-levels and durations. A representative electrode trace and a raster plot of all neurons of one exemplary MEA chip is shown with the microgravity phase marked with a red frame. Red brackets mark the section excluding safety margins which was used for analysis. (B) Phase-contrast microscopy image of an exemplary MEA chip pre- and post-drop. A zoomed area (red frame) of the culture is shown below. (C) Violin plot for comparison of firing rates averaged during baseline recordings prior to the drop (grey, left), during the microgravity phase (orange), during the impact (light orange), and post-drop baseline (grey, right). Five MEAs from three independent preparations were measured during five drops. Statistical analysis included 63 and 19 units for firing and burst rate, respectively. Repeated measures one-way ANOVA and Tukey’s multiple comparisons test were used (ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001). Firing rates of individual units are marked with a black triangle in each condition. Dashed lines mark the first, second, and third quartile. (D) Violin plot for comparison of burst rates during all four phases. (E,F) Density plots of the firing rates during microgravity and impact phases compared to baseline pre- and post-drop. (G–I) Subgroups of neurons show a distinct change in firing rate between baseline and microgravity. Units with low (G), medium (H), and high (I) firing rates during baseline and their respective firing rate during microgravity (dots connected by a line).