(E-170) Direction-dependent effects of nanomagnetic forces on the distribution of calcium hotspots in cortical neurons

Introduction:: Cells utilize the elevation of calcium (Ca²⁺) ions to regulate various cellular processes. In neurons, this may include Ca²⁺-dependent processes like neurotransmitter release, modifying gene expression, growth cone tuning, cell migration, or spine-mediated learning and memory formation ^[1,2,3,4]. The dynamic regulation of various modes of influx and efflux plays a significant role in achieving the temporal and spatial segregation of calcium ions in the cytoplasm of neurons ^[5]. However, considering the high polarity of a neuron cell, there is currently limited knowledge about the local effects and distinction of force-induced Ca²⁺ influx in highly compartmentalized neuronal cells and networks. To study this, we focus on microdomains called “calcium hotspots.” Calcium hotspots are localized regions within the cell that show higher calcium ion concentrations than the surrounding cytoplasm ^[6]. By application of nanomagnetic forces, we studied the spatial impact of these forces on the dynamics of cytosolic calcium hotspot regions.


Materials and Methods:: Glass-bottom Petri dishes were used to seed cortical neurons at 5x10⁴ cells/ml. To facilitate cell adhesion, the Petri dishes were coated with Poly-D-Lysine. Before imaging, the cells were incubated for 13 days, with media changed every three days. On day-in-vitro (DIV) 12, each Petri dish was loaded with starch-NH₂ magnetic nanoparticles and incubated for 24 hours before imaging. On DIV 13, cells were stained with Calbryte 520AM calcium staining probe at a concentration of 1 uM and then incubated for two hours. Following the incubation, cells were washed three times to remove excess nanoparticles and calcium probe. The control set (cells with nanoparticles but no nano-magnetic force stimulation) was imaged using a Leica DMI-8 fluorescent microscope at 100X magnification. Imaging was conducted for 1.5 min with a 90 ms exposure time for calcium and a 200 ms exposure time for magnetic nanoparticles. Afterward, the same Petri dishes were exposed to magnetic forces and incubated for 5 minutes before imaging. For the analyses, we measured i) the total number of the hotspots in the cytosol and ii) the distance of the calcium hotspots from the center of the cells in two study groups.


Results, Conclusions, and Discussions:: We aimed to characterize the distribution of cytosolic hotspots (Fig. 1a1-1a2) in cells subjected to nanomagnetic forces in four distinct Petri dish regions (Fig. 1a3) compared to cells not exposed to nanomagnetic forces. A total of eight cells per quadrant were randomly selected for analysis, and the number and spatial distribution of hotspots were quantified (Fig. 1b-1e). The results showed no significant difference in the number of hotspots between cells exposed to nanomagnetic forces and those unexposed (Fig. 1b). The distance between hotspots and the center of the cells was also analyzed. We found that hotspots were homogeneously distributed within the cell body when no magnetic forces were applied (Fig. 1c). In contrast, cells exposed to nanomagnetic forces show a trending number of hotspots further away from the cell center, with a tendency to accumulate in the cellular quadrille in parallel to the quadrant where the nanomagnetic forces were oriented (Fig. 1d, 1e). This trend was consistent across all directions of the forces applied to each cell. Our findings suggest that nanomagnetic forces can influence the spatial distribution of cytosolic hotspots, which may affect cellular function and response to external stimuli.



Without nanomagnetic forces, the spatial distribution of calcium hotspots in primary cortical neurons seems to occur uniformly. However, the application of nanomagnetic forces showed a tendency to shift the distribution of calcium hotspots in a direction-dependent manner from the cell center relative to the force direction. These findings indicate that nanomagnetic forces may localize cytosolic calcium hotspots to a particular cell compartment. Further research is necessary to understand the underlying mechanisms of this observation.


Acknowledgements (Optional): : This work is supported by a National Science Foundation (NSF) CAREER award (A.K., Grant# CBET-1846271). AK declares co-foundership of NanoMagnetic Solutions, Inc. No financial support was received from NanoMagnetic Solutions, Inc. for this research study.


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