Nanosecond pulsed electric fields (nsPEF) are high voltage (1-15 kV/cm) nanosecond energy waveforms that can impact cellular activity. On a physical level, a nsPEF generates transient membrane perturbations in the form of nanopores to allow cation influx resulting in localized membrane depolarization. On a physiological level, a nsPEF exposure can activate receptors and channels on the membrane as well as second messenger cascades, both of which results in subcellular modulation that lasts beyond the nsPEF duration. An ongoing challenge is to characterize the extent/sequence of physiological events induced by nsPEF exposure, and potential to interplay with physical effects induced by the pulse. In our laboratory, C2C12 mouse myoblast cells have been demonstrated to be a useful in vitro model, as it is feasible to differentiate these immortalized progenitors into terminally transformed myotubes. From previous efforts, we quantified YO-PRO -1 (YO-PRO-1) uptake as a measurement of membrane perturbation, and concluded that membrane damage is proportional to applied pulsed electric field voltage. To expand upon these findings, we evaluated to what extent YOPRO-1 uptake at the membrane is physical or physiological in nature. Interestingly, the P2X7 receptor complex has been extensively studied utilizing YO-PRO-1 uptake as marker of apoptotic activity. For this reason, we tested the role of P2X7 receptor complex activation to mediate YO-PRO-1 uptake during pulsed electric field exposure. By blocking the P2X7 receptor, we reduced nsPEF-induced YO-PRO-1 uptake by 31.57%. Our results demonstrate that the P2X7 receptor complex is a subcellular candidate responsible for YO-PRO-1 uptake upon nsPEF exposure in myotubes.
Nanosecond pulsed electric fields (nsPEF) are high voltage (1-15 kV/cm) nanosecond energy waveforms that can impact cellular activity. On a physical level, nsPEF generates transient membrane perturbations in the form of nanopores to allow cation influx resulting in localized membrane depolarization. On a physiological level, nsPEF exposure can activate second messenger cascades resulting in subcellular modulation that lasts beyond the nsPEF duration. An ongoing challenge is to characterize the physiological events induced by nsPEF exposure, and potential to interplay with physical effects induced by the pulse. In our laboratory, C2C12 immortalized mouse myoblast cells have been demonstrated to be a useful in vitro model, by differentiating these progenitors into terminally transformed myotubes. We are not only able to further investigate the fundamental subcellular mechanisms activated by pulsed electric fields, but monitor muscle contraction as a functional output. From our previous efforts, we quantified calcium-green uptake as a measurement of cellular calcium uptake across a sweep of applied pulsed electric field voltages. To extend on these findings, we evaluated calcium dynamics in the intracellular space of myotubes. Given that sarcoplasmic reticulum efflux is required for muscle contraction, we tested the physiological role of the ryanodine receptor during pulsed electric field exposure on myotubes. By blocking the Ryanodine receptor with a competitive antagonist, we reduced nsPEF -induced calcium dynamics activation by 58.36% in media with calcium. Our results are the first to demonstrate that the Ryanodine receptor complex is a subcellular candidate responsible for generating calcium responses upon nsPEF exposure in myotubes.
An infrared laser pulse (IRLP) can trigger the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2), presumably via initial activation of phospholipase C (PLC), and activate multiple intracellular signaling cascades. Two main hydrolysis products are important second messengers, lipid diacylglycerol (DAG), that can lead to PKC activation, and soluble inositol 1,4,5-triphosphate (IP3) that stimulates calcium release from intracellular stores. The mobilization of these messengers can modulate ion conductivity through channels, and coordinate cytoskeletal rearrangements that promotes or suppresses further downstream signaling. The downstream effect impacts membrane conductance, membrane potential and overall cellular excitability. As a result, a single IRLP with a 2-4 millisecond duration may initiate a cellular effect that lasts for seconds to minutes. From our approach, we evaluated the PIP2 phosphoinositide signaling cascade from immortalized cells that exhibited genetically encoded reporters for PIP2/IP3 and DAG. Upon IRLP exposure, we observed a PIP2 depletion, IP3 increase and DAG decrease in cytosol in motor neuron-like NG108 cells. Our data suggest that IRLP may induce second messenger systems at the membrane, and as a result modulate ionic signaling across the cell body.
Recent studies suggest that microtubules (MTs) and tubulin proteins exhibit resonant frequencies in the radiofrequency (RF) range. We hypothesize that exposing neurons to externally applied RF waves tuned to an intrinsic resonant frequency of MTs or tubulin could disrupt the natural signaling occurring in and around them, leading to neurophysiological changes. To test this hypothesis, we assembled custom exposure systems that allow stable RF exposures of cell cultures in a controlled environment (37°C, 5% CO2, 95% humidity). We then exposed differentiated NG108-15 neuronal cells to RF waves tuned to selected resonance peaks for tubulin (91 MHz and 281 MHz) and for MTs (3.0 GHz) for 1 hr at a power density of 0.24 mW/cm2 (SAR = 0.012, 0.087, and 0.53 mW/kg, respectively). We used fluorescence imaging of endogenous MTs and current-clamp electrophysiology to investigate changes following RF exposures compared to sham. The results from the imaging data show a clear difference in the localization of fluorescent MTs between the sham and the RF exposed neuronal cells. The sham cells exhibited more fluorescence in the neurite projections, whereas the RF exposed cells showed a more diffuse pattern, with a stronger fluorescence in the cell body. The electrophysiological results showed that resting membrane potentials of the RF exposed neuronal cells were more depolarized than those of the sham cells. Consequently, we observed spontaneous action potentials in the RF exposed cells, which were not present in the sham cells. Overall, our results suggest that exposing neurons to MTs or tubulin resonant frequencies might affect MTs normal behavior, leading to neurophysiological changes. However, to confirm the specificity of resonant frequency effect and validate this idea, studies investigating exposures to nonresonant frequencies and additional tubulin and MTs resonant frequencies are warranted.
Infrared laser (IRL) exposure can induce a rapid temperature change (fast thermal gradient or FTG) that is able to stimulate or inhibit neurons and, thereby, modify neurological functions. Despite extensive research into this effect, the fundamental mechanism(s) underlying how FTG causes neurological stimulation or inhibition remains unclear. While it is hypothesized that IRL-induced FTG acts directly on the neuronal plasma membrane (PM), it is uncertain if the neurological effects observed in previous studies are mostly derived from presynaptic effects (i.e., modifications in action potential (AP) firing) or also from postsynaptic effects (i.e., alteration of the synaptic responses of the excitatory and inhibitory neuronal receptors). In the present study, we present an analysis of FTG-mediated changes in neuronal PM, AP firing rate, and miniature postsynaptic excitatory and inhibitory currents (mEPSCs and mIPSCs). Our results suggest FTG induces changes in both presynaptic and postsynaptic neurophysiological mechanisms. Specifically, we found that, after IRL pulse (IRLP)-induced FTG exposure, the amplitudes of APs are reduced, but the rate of APs are increased. In contrast, the quantities of both mEPSCs and mIPSCs are reduced, but the peak-to-peak frequency and peak amplitudes are increased. The results outlined in this study demonstrate the impact of FTG on neurons and neuronal network. This information is critical for understanding the complexity of the effects of FTG on neurological functions and for demonstrating how post-synaptic mechanisms might play a crucial role in neurological excitation or inhibition seen following IRL pulse exposure.
Irreversible electroporation therapy is utilized to remove cancerous tissues thru the delivery of rapid (250Hz) and high voltage (V) (1,500V/cm) electric pulses across microsecond durations. Clinical research demonstrated that bipolar (BP) high voltage microsecond pulses opposed to monophasic waveforms relieve muscle contraction during electroporation treatment. Our group along with others discovered that nanosecond electric pulses (nsEP) can activate second messenger cascades, induce cytoskeletal rearrangement, and depending on the nsEP duration and frequency, initiate apoptotic pathways. Of high interest across in vivo and in vitro applications, is how nsEP affects muscle physiology, and if nuances exist in comparison to longer duration electroporation applications. To this end, we exposed mature skeletal muscle cells to monopolar (MP) and BP nsEP stimulation across a wide range of electric field amplitudes (1-20 kV/cm). From live confocal microscopy, we simultaneously monitored intracellular calcium dynamics along with nsEP-induced muscle movement on a single cell level. In addition, we also evaluated membrane permeability with Yo-PRO-1 and Propidium Iodide (PI) across various nsEP parameters. The results from our findings suggest that skeletal muscle calcium dynamics, and nsEP-induced contraction exhibit exclusive responses to both MP and BP nsEP exposure. Overall the results suggest in vivo nsEP application may elicit unique physiology and field applications compared to longer pulse duration electroporation.
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