(K-427) Magnetic field strength effects in nanomagnetic force-based neuronal cell modulation.

Calcium is a common second messenger across many cell types throughout the body. In neurons, homeostatic calcium levels are crucial to proper cell function, synaptic transmission, and communication [1,2]. Most recently, we and others have shown that intracellular calcium dynamics can be modulated through mechanical stimulation using ultrasound, optical, or magnetic fields [3]. Using magnetic fields, we can control mechanical forces acting on magnetic nanoparticles (MNP) within a magnetic field, resulting in so-called nanomagnetic forces. With MNPs bound to the cell membrane, nanomagnetic forces (NMF) have been previously shown to cause an influx of calcium through mechano-sensitive N-type calcium channels [3]. Such calcium influx may alter neuronal communication, and we have seen trends that cell internalized MNPs, bound to subcellular structures and cargos, similarly modulate influx. Having studied the effects of NMF on calcium, we are interested in researching the electrophysiology of the cell while under NMF. By studying the electrophysiology of the cell, we expect to gain a clearer understanding of the effects of NMF on neuronal activity. To do so, we optimized a method to study how continuous low-strength NMF can modulate neuronal communication in primary rat cortical neurons.

E18 primary rat cortical neurons were dissociated using a standard protocol and plated onto 10 µm diameter microelectrode arrays (60MEA100/10iR-ITO, Multichannel Systems)[4]. MEAs were coated with poly-D-lysine and pre-treated (90% Neurobasal, 10% Horse serum). Neuronal cultures were plated dropwise to achieve high density ( >2000 neurons/mm²) and allowed to mature with standard incubation (37 C, 5% CO₂) and culture medium (96% Neurobasal+, 2% B27+, 1% Gluta-max, 1% Penicillin-Streptomycin). After 13 days in vitro (DIV), baseline recordings (4 minutes, 10 kHz, MEA 2100, Multichannel Systems) were performed at 0h, 2h, 4h, and 6h with standard incubation in-between. After 6h, magnetic nanoparticles (MNPs, 10 µg/mL, 100 nm, Starch-NH₂ shell, Micromod) were added with recordings at 8h, 10h, and 30h. After 24h MNP exposure (30h time point), excess magnetic nanoparticles were removed by 1X wash with culture medium, and MNP-laden cultures were subjected to either magnetic field A or magnetic field B, while no MNP cultures were left without magnetic fields. Magnetic fields and corresponding NMF were simulated in COMSOL Multiphysics via previously developed methods [5]. The magnetic fields on the MEAs were maintained continuously and recorded at 32h, 34h, 36h, and 38hh. Mean spike rates were obtained using an in-house MATLAB script. Briefly, microelectrode signals were filtered (300-4000 Hz), and spikes were detected using falling edge detection (4.25-4.5 std, consistent with the same microelectrode array). Mean spike rate calculations excluded electrodes with average spike rates less than 0.03 events/s or greater than 4 events/s across the 4-minutes to differentiate from excess noise.

In this study, we investigated the sensitivity of neuronal communication to nanomagnetic forces (NMF) by integrating two magnetic nanoparticles (MNP) assays with a microelectrode array (MEA). This setup allowed us to provide continuous MNP-transduced NMF over extended periods on cortical neuronal cultures. The selected assays, magnetic fields A and B, exerted forces between 10-100 fN and 1-10 fN across the MEA surface, respectively. We recorded three periods of activity: baseline, MNP uptake, and NMF. Baseline neuronal activity exhibited independent spikes/s (0.29±0.40) with no significant differences over 6 h (p >F: 0.68 >0.40). Upon MNP addition, minimal changes to spike rate were observed at 2 h (0.29±0.18), with a decrease at 4 h (0.17±0.18) sustained at 24 h (0.19±0.16). Subsequently, the application of nanomagnetic forces increased spiking. Initially, the response exhibited highly consistent spike rates independent of strong or weak NMF (strong - Mag A: 0.22±0.02 spikes/s; weak – Mag B: 0.13±0.01 spikes/s), indicating a consistent response across cultures. After an additional 2 h, a further increase in event rate was observed in the strong magnetic field but not in the weak (strong - Mag A: 0.22±0.02 spikes/s; weak – Mag B: 0.13±0.01 spikes/s). However, at hour 6, the weak magnetic field showed a significant increase in spike rate (0.54±0.34) before returning to values consistent with the initial spike rate at hour 8 (0.14±0.15). This data suggests that long-term nanomagnetic forces may contribute to an increase in neuronal firing, potentially mediated by an increase in intracellular calcium, a crucial factor in neuronal communication. Furthermore, our findings indicate that neuronal communication might still be sensitive but responded slower to nanomagnetic forces in the 1-10 fN range than forces in the 10-100 fN range. In conclusion, our study presents a long-term platform for electrophysiological recordings of neurons subjected to continuous nanomagnetic forces, providing valuable insights into the long-term impacts of nanomagnetic force exposure on neuronal cultures. These findings contribute to a better understanding of the dynamics of neuronal communication in response to nanomagnetic forces, which holds significant potential for various applications in neuroscience and biomedicine.

[1] Gleichmann, M.; Mattson, M. P. Neuronal calcium homeostasis and dysregulation.
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[2] Kawamoto, E. M.; Vivar, C.; Camandola, S. Physiology and pathology of calcium
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[3] Tay, A.; Kunze, A.; Murray, C.; Di Carlo, D. Induction of Calcium Influx in Cortical Neural Networks by Nanomagnetic Forces. ACS Nano 2016, 10, 2331–2341.

[4] Beck, C. L.; Hickman, C. J.; Kunze, A. Low-cost calcium fluorometry for long-term nanoparticle studies in living cells. Scientific Reports 2020, 10.

[5] Judge, D.; Kunze, A. Neural network growth under heterogenous magnetic gradient patterns. 2019 9th International IEEE/EMBS Conference on Neural Engineering (NER). 2019; pp 191–194.