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Electrostimulation of Ca2+ influx into adrenal chromaffin cells by nanoelectropulses: The impact of increasing pulse duration from 3 to 50 ns on Ca2+ entry pathways
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Electrostimulation is commonly used in clinical applications to treat neurological diseases and disorders. It can be achieved invasively by implanted electrodes such as in deep brain stimulation, or non-invasively such as in transcranial direct current stimulation where electrodes are placed on the scalp. Here, we propose a novel electrical stimulus, high intensity nanosecond duration electric pulses (NEPs) for their capability to target deep areas in the body non-invasively, without the use of implanted electrodes. However, before developing neuromodulation technologies based on this novel stimulus, it is imperative to understand how NEPs interact with neural cell types and identify the range of NEP parameters that trigger cell excitation in the absence of adverse effects. In this study, we used isolated bovine adrenal chromaffin cells, a well-characterized neural-type cell, as our cell model to investigate how NEPs that are 3 to 50 ns in duration affect chromaffin cell excitability. We have previously shown that in bovine adrenal chromaffin cells loaded with a fluorescent Ca2+ indicator, a single 5 ns electric pulse, similar to the physiological stimulus, causes an immediate rise in intracellular Ca2+ level ([Ca2+]i) that is solely due to Ca2+ influx via voltage-gated Ca2+ channels (VGCCs). However, when the electric pulse duration is increased to 150 ns, it causes Ca2+ influx into cells by at least two pathways. One is VGCCs (60-70%), and another via a plasma membrane pathway(s) (30-40%) that is yet to be identified. Thus, the main goal of this study was to identify the pulse duration at which Ca2+ influx begins to involve a non-VGCC pathway. To achieve our goal, we used two variable duration pulse generators, one with a pulse duration ranging from 3 to 11 ns, and another with a pulse duration ranging from 12 to 100 ns. First, we found that applying NEPs ranging from 3 to 50 ns in duration at their respective threshold electric fields caused a rapid rise in [Ca2+]i that increased in duration and magnitude as pulse duration was increased. Two patterns of responses were observed. For pulse durations less than 11 ns, Ca2+ responses were mainly transient (i.e. returned to baseline within 30 s following pulse application) in the majority (~75%) of the cells. However, when the pulse duration was increased to 11 ns, the majority (~77%) of the cells exhibited longer-lived Ca2+ responses, and by 50 ns, 100% of the cells had longer-lived activity. To determine if Ca2+ release from intracellular stores contributed to the longer-lived Ca2+ responses, we conducted experiments in the absence of external Ca2+. We found that none of the cells exhibited Ca2+ responses without extracellular Ca2+ indicating that the Ca2+ responses observed were solely due to Ca2+ influx into cells. Therefore, to identify the pathway for Ca2+ entry, VGCCs were blocked with a cocktail of inhibitors containing 100 nM ω-agatoxin IVA, 20 nM ω-conotoxin GVIA, and 20 µM nitrendipine to block P/Q-, N-, and L-type VGCCs, respectively. We found that Ca2+ responses evoked by 3 and 5 ns pulses were fully abolished, while those evoked by 11 were significantly attenuated relative to the control. Increasing pulse duration to 50 ns reduced Ca2+ entry into cells by about 50%, indicating that 50% of the Ca2+ responses were due to a non-VGCC entry pathway. In the next series of experiments, cells were treated with Cd2+, an inorganic, non-selective blocker of VGCCs. Similar to what we have found with a 150 ns pulse, all Ca2+ responses evoked by 11 and 50 ns durations were completely eliminated, indicating that Cd2+ blocked all Ca2+ entry pathways into cells. Because bipolar NEPs are known to reduced unwanted cellular membrane effects, we next determined if changing the shape of the 11 ns pulse, which is the pulse duration at which the cell membrane starts to involve a non-VGCC Ca2+ entry pathway, would reduce/eliminate the additional Ca2+ entry pathway. We found that application of a 11 ns bipolar pulse in which a positive pulse was immediately followed by a negative pulse of opposite polarity abolished Ca2+ responses in about 70% of the cells, with the remaining 30% exhibiting transient Ca2+ responses. Interestingly, blocking VGCCs with a cocktail of blockers caused Ca2+ responses to be totally eliminated in all cells tested, indicating that, unlike a 11 ns unipolar pulse, a 11 ns bipolar pulse caused Ca2+ influx into cells that was solely due to VGCCs, thus eliminating the additional pathway of Ca2+ entry into cells. Lastly, we investigated the Na+ dependency on the Ca2+ responses when cells were subjected to pulses that were 3, 5, 11 and 50 ns in duration. For this investigation, NEPs were applied to cells in Na+-free medium where either TMA+ or NMDG+ was used as a substitute for Na+. In both TMA+- and NMDG+-containing solutions, we found that for pulse durations less than 11 ns, the pulse-induced rise in [Ca2+]i was almost fully abolished, indicating that Na+ was necessary to evoke the pulse-induced Ca2+ rise. For the longer duration pulses (i.e. > 11 ns), the pulse-induced rise in [Ca2+]i was significantly attenuated in Na+-free solution relative to the control. These results indicate that for pulses shorter than 11 ns, Na+ entry played a role in the mechanism by which NEPs evoked Ca2+ influx, whereas, for pulses longer than 11 ns, there remains an NEP-induced Ca2+ influx pathway that was not dependent on external Na+. Of note is that, similar to what we previously found with 5 ns pulses, Na+ entry did not occur via voltage-gated Na+ channels since the Na+ channel blocker tetrodotoxin (TTX) failed to block the NEP-induced rise in [Ca2+]i for all pulse durations. Taken together, these results indicate that a single ultrashort (< 11 ns) electric pulse causes Ca2+ influx solely via VGCCs in a manner involving Na+ influx, while longer-duration pulses cause Ca2+ influx not only via VGCCs but also via a non-VGCC pathway that is yet to be identified. These results highlight the sensitivity of excitable adrenal chromaffin cells to ultrashort pulse durations that differ by only tens of nanoseconds, which is important for the development of future technologies aimed at using NEPs for neuromodulation.