Associate Professor Montana State University Bozeman, Montana, United States
Introduction:: Implementing nano transduction technologies for neuromodulation has proven to be a valuable tool by generating local stimulation to neurons[1]. Primarily, nano transducers have focused on generating electrical gradients on the neuronal membrane to externally activate voltage-gated channels. Currently, the difficulty with this method is developing a brain-permeable primary signal that is strong enough to permit appropriate transduced electric energy to depolarize the membrane. Mechanical stimulation, conversely, has been shown to modulate neurons by driving an influx of calcium into the cytosol [2]. Pairing a magnetic nanoparticle (MNP) with an external magnetic field (nanomagnetic forces) has shown promising results in inducing calcium influx in primary cortical neurons[3]. The surface chemistry of the MNPs plays a crucial role in locating the nanoparticles, either at the cell membrane or within the cells. The observed calcium influx via nanomagnetic forces has been inconsistent between surface-bound and cell-uptaken MNPs, with the latter presenting less increase in calcium during force stimulation. Given that cell internal MNPs have better accessibility to neuronal processing, we think this inconsistency results from communication-dependent interactions. To test this hypothesis, we introduced starch-amine MNPs to high-density primary rat cortical neuron networks and tracked calcium signals to observe both activity and modulated influx levels before, during, and after nanomagnetic force modulation.
Materials and Methods:: E18 primary rat cortical neurons were dissociated, plated as high-density networks on poly-d-lysine coated 35 mm dishes, and incubated using a previous protocol [cite]. For MNP experiments, 100 nm Starch-Amine functionalized nano ferrite particles were incubated with the neurons from DIV 13 to DIV 14 (24h). On DIV 14, all cultures were subjected to a 1:1 ratio of media and Fluo-4AM with probenecid acid for 1 hour, gently washed with culture media, and allowed to stabilize for 30 minutes at standard incubation. The cultures were then placed in an Okolab on-stage incubator (37C) with a manual gas mixer (5% CO2, 95% air, saturated humidity) and given 5 minutes for stabilization. Then a 4-minute video was recorded (GFP FIM: 10%, 240 ms exposure, 4 Hz framerate) for a baseline; immediately following the video, a magnetic field was added, and a second video (modulation) was recorded with the same parameters. After the video concluded, the magnetic field was removed, and a third video (recovery) was recorded. Videos were segmented in ImageJ for all viable cells and analyzed in Matlab using in-house scripts (GitHub). Active cells were classified as cells with detected calcium influx events, while others were classified as inactive. We then quantified overall network activity using an activity index (AI). All statistical tests are Mann-Whitney U test (*U< 0.05, ** U< 0.01, *** U< 0.001, ****U< 0.0001).
Results, Conclusions, and Discussions:: To characterize the activity dependency of calcium modulation via nanomagnetic forces with starch-amine MNPs, we cultured primary rat neurons with high-density plating for two weeks to achieve active networks and incubated with the MNPs for 24 hours. Neuronal activity was then measured during a baseline period of 4 minutes as the average number of calcium influx events per minute (Fig 1a, b1-b2, c1-c2). No MNP cultures presented a baseline activity index (AI) of 1.39±1.12 events/min (Fig 1 d1). Furthermore, we observed two different AI during the baseline period for MNP-laden cultures, which we labeled low AI and high AI (1.25±1.93 and 4.18±4.50 events/min, Fig 1 d1). The no MNP cultures had 25.6±16.9% inactive cells, while the low and high AI cultures had 38.6±2.7% and 79.0±9.3% dormant cells. During modulation, no significant cumulative calcium influx was observed for either the No MNP cultures or MNP with low AI (Fig. 1 d2, e). However, there was a considerable influx (106.3±144.4 ΔF/F) for active neurons within the MNP-laden cultures. Similarly, we found no significant changes in activity rates during the modulation period for the no MNP. Still, we observed a significant decrease in calcium events for NMF cultures independent of the initial AI. We observed this decrease by minute 2 for the high AI cultures, while this trait did not present until minute 4 for the low AI cultures (Fig 1 d2). Finally, we tested if the neuronal calcium influx was dependent on the individual activity of the neuron during the baseline period (Fig. 1f). There was no significant correlation between increasing event rates and calcium influx, indicating the influx is not solely mediated by voltage-gated calcium channels. Altogether, this work shows that nanomagnetic force modulation with starch-amine magnetic nanoparticles of neuronal calcium depends on the base activity of the neuronal network, indicating a need further to characterize these cell internal MNP interactions with neuronal processing.
Acknowledgements (Optional): : This work is supported by a National Science Foundation (NSF) CAREER award (A.K., Grant# CBET-1846271). AK and CB declare inventorship on a provisional US patent application No. 63/446,770 (Prov #2) and co-foundership of NanoMagnetic Solutions, Inc. No financial support was received from NanoMagnetic Solutions, Inc. for this research study.
References (Optional): : [1] Li X, Xiong H, Rommelfanger N, Xu X, Youn J, Slesinger PA, Hong G, Qin Z. Nanotransducers for Wireless Neuromodulation. Matter. 2021 May 5;4(5):1484-1510. doi: 10.1016/j.matt.2021.02.012. PMID: 33997768; PMCID: PMC8117115.
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[3] Andy Tay, Anja Kunze, Coleman Murray, and Dino Di Carlo Induction of Calcium Influx in Cortical Neural Networks by Nanomagnetic Forces ACS Nano 2016 10 (2), 2331-2341, 10.1021/acsnano.5b07118