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Modulation of Electrical Stimulation of Isolated Adrenal Chromaffin Cells is Achieved by Changing the Interphase Interval in Bipolar Pulses with Phase Durations between 3 and 50 ns
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Deep brain stimulation (DBS) is an approved method to treat numerous neurological disorders including epilepsy, dystonia, and Parkinson’s disease. DBS involves implanting electrodes into specific regions of the brain to deliver electrical pulses to modulate neurological activity. Risks and potential side effects associated with this invasive method generally include infections, and hemorrhages. Non-invasive techniques such as transcranial magnetic stimulation (TMS) in which electromagnetic coils stimulate nerve cells via magnetic fields and transcranial direct current stimulation (tDCS) which stimulates brain tissue via constant current application between two electrodes are also in clinical use. However, these electrostimulation methods have either limited penetration depth or low spatial resolution. To achieve the combined benefits of invasive and non-invasive electrostimulation techniques, our group has been investigating the potential for a novel bioelectric stimulus, nanosecond duration electric pulses (NEPs). NEPs have the potential to achieve both deep and focused electrical stimulation non-invasively via antennas, and thus without the need for implanted electrodes. One way to achieve this remote electrostimulation is based on a recent finding of complete cancellation of biological effects of NEPs with the application of a second pulse of opposite polarity, a process termed bipolar pulse cancellation. The combination of the bipolar pulse cancellation effects and electric field superposition allows possible application in deep tissues by a cancellation of cancellation or CANCAN effect, leaving surrounding tissue unaffected by delivered pulses. CANCAN achieves a single biologically-effective unipolar NEP resulting in focused stimulation without the risks associated with invasive DBS. To study effects of NEPs and application of bipolar pulse cancellation we utilized bovine adrenal chromaffin cells in our studies. Chromaffin cells were chosen as a cell model for their similarities with sympathetic neurons in that they release the catecholamine epinephrine and norepinephrine (Epi and NEpi) via the Ca2+-dependent process of exocytosis. The major finding is that a single 5 ns electric pulse causes membrane depolarization and Ca2+ influx via voltage-gated calcium channels (VGCCs). Recently, we found that a single unipolar pulse that was only 2 ns in duration caused Ca2+ influx via VGCCs that can be totally abolished when another 2 ns unipolar pulse with opposite polarity is applied afterwards. The resulting pulse with two opposite phases one after another is called a bipolar pulse. The cancelling effect disappears as the interval between the two phases of the bipolar pulse increases beyond 30 ns. Furthermore, it was found that increasing the pulse duration to 150 ns caused only the portion of Ca2+ influx attributable to membrane permeabilization to be cancelled, not that due to VGCC activation. This clearly demonstrates that bipolar pulse cancellation effects have a narrow window where they are manifested. Thus, the goal of the present study was to further identify bipolar pulse exposure parameters that cancel increases in [Ca2+]i in chromaffin cells mediated by influx of Ca2+ via VGCCs. To achieve this goal, we employed a combination of experimental and theoretical calculations, such as Accelerated Membrane Discharge (AMD) model and 1) identified threshold voltage amplitudes for single unipolar pulses with durations ranging from 3 to 50 ns, 2) determined whether bipolar cancellation can be achieved throughout the range of these durations, and 3) assessed whether the minimal interphase interval required to cancel the bipolar cancellation changes with pulse duration. The goals were achieved by exposing single isolated bovine adrenal chromaffin cells to external electric fields delivered from bipolar pulsers generated by pulsers by FID GmbH and applied via handmade tungsten rod electrodes. Changes of [Ca2+]i were assessed through rise of fluorescence levels of Calcium Green-1 dye recorded on electron multiplying CCD iXon Ultra 897 camera with the help of Leica Application software. Analysis of the recorded traces was performed by a custom MATLAB application developed during the thesis. Our results demonstrate an exponential decrease in unipolar pulse threshold voltage required to elicit a response in the cells with the increase of pulse duration from 3 to 50 ns. Meanwhile, symmetrical bipolar cancellation at the threshold voltage can only be achieved for pulse durations ranging from 3 to 12 ns. By 25 ns the effects elicited by a unipolar pulse were only partially attenuated by a symmetrical bipolar pulse, and bipolar pulse cancellation was completely lost when the pulse duration was increased to 50 ns. Furthermore, by increasing the interphase interval to only 4 ns the bipolar cancellation was started to wane down. This cancellation effect was found to be inversely dependent on the pulse duration, where for a shorter duration pulse of 3 ns, an 8 ns interphase interval was enough to mostly cancel the bipolar cancellation, and for longer duration pulses of 12 ns, a 4 ns interphase interval was sufficient. We have also found that some cells would react to a bipolar stimulus with very a small initial increase in [Ca2+]i, usually leading to a full blown response that was delayed by a few seconds. One of the most plausible mechanisms to explain bipolar pulse cancellation is the AMD hypothesis, which was successfully used to model bipolar cancellation for 2 ns pulses with variable interphase intervals. AMD assumes that the cell membrane acts as a capacitor that is being charged and discharged by the application and interruption of the external electric field, respectively. Since bipolar pulses have both positive and negative phases, the cell membrane voltage would become positively charged during the positive cycle and then brought back to the resting state with the negative cycle resulting in a very short time the membrane stays at a critical voltage for electropermeabilization. The AMD model was able to correctly model bipolar cancellation of Ca2+ responses for symmetrical bipolar pulses with a duration of 3 to 12 ns, and attenuation of the effects of unipolar pulses for symmetrical bipolar pulses of 25 to 50 ns. However, AMD was not able to model Ca2+ responses when the interphase intervals were introduced. To address these discrepancies, we developed an expanded AMD model which utilizes recorded experimental pulses and allows varying the membrane charging constant to see if AMD could still explain cancellation in pulses with very small interphase intervals. When the membrane charging constant was changed for the second phase, AMD showed a significantly better fit for our experimental results. In summary, our results show the exponential relationship between unipolar pulse amplitude and duration for eliciting a cellular response. The results also show a window where bipolar pulse cancellation is manifested, and minimal interphase intervals required to abolish it with the change of pulse duration. This bipolar cancellation window is crucial for the future development of remote electrostimulation applications based on the CANCAN effect.