Neural-type Cell Research Articles

Ultrashort nanosecond electric pulses activate a conductance in bovine adrenal chromaffin cells that involves cation entry through TRPC and NALCN channels

In whole-cell voltage clamped bovine adrenal chromaffin cells maintained at a holding potential of −70 mV, a single 5 ns, 5 MV/m pulse elicited an inward current carried mainly by Na+ that displayed inward rectification and a reversal potential near −3 mV, a voltage consistent with a non-selective cation current. The broad-spectrum inhibitors of transient receptor potential (TRP) channels, La3+ (10 μM), Gd3+ (10 μM), SKF-96365 (50 μM) and 2-aminoethoxydiphenyl borane (2-APB; 100 μM), inhibited the current similarly by ∼72%, ∼83%, ∼68% and ∼76%, respectively. Depleting membrane cholesterol with methyl-β-cyclodextrin (MβCD; 1–6 mg/ml) or inhibiting phosphatidylinositol 4,5-bisphosphate (PIP2) synthesis with wortmannin (20 and 40 μM) produced a similar level of inhibition on the NEP-induced conductance as the broad spectrum TRP channel inhibitors. Moreover, no additive inhibitory effect was detected by combining MβCD (3 mg/ml), wortmannin (20 μM) and La3+ (10 μM), suggesting that each agent targeted different levels of the same pathway to exert a full effect. RT-PCR experiments revealed robust expression at the mRNA level of TRPC4, TRPC5 and TRPM7 channels for which specific blockers were available. Whereas the TRPM7 blocker FTY720 had no effect, the TRPC4/5 channel inhibitor M084 (20 μM) blocked the conductance by ∼50%, indicating that TRPC4 and/or TRPC5 channel(s) may be partially involved in mediating the NEP-induced current. CP-96345 (20 μM), a specific blocker of the sodium leak current channel (NALCN), also reduced the NEP-induced current. The inhibition was ∼30% and additive to that caused by the TRPC4/5 blocker M084. RT-PCR experiments confirmed the expression of this channel at the mRNA level. Taken as a whole, these data provide evidence that a large fraction of the current evoked by a 5 ns pulse in adrenal chromaffin cells may be carried by both TRPC4/5 channels and the NALCN channel. Understanding the biophysical properties of the NEP-elicited conductance in a neural-type cell will be extremely valuable for the future development of NEP stimulation approaches for neuromodulation.

2-ns Electrostimulation of Ca2+ Influx into Chromaffin Cells: Rapid Modulation by Field Reversal

Cellular effects of nanosecond-pulsed electric field exposures can be attenuated by an electric field reversal, a phenomenon called bipolar pulse cancellation. Our investigations of this phenomenon in neuroendocrine adrenal chromaffin cells show that a single 2-ns, 16 MV/m unipolar pulse elicited a rapid, transient rise in intracellular Ca2+ levels due to Ca2+ influx through voltage-gated calcium channels. The response was eliminated by a 2-ns bipolar pulse with positive and negative phases of equal duration and amplitude and fully restored (unipolar-equivalent response) when the delay between each phase of the bipolar pulse was 30 ns. Longer interphase intervals evoked Ca2+ responses that were greater in magnitude than those evoked by a unipolar pulse (stimulation). Cancellation was also observed when the amplitude of the second (negative) phase of the bipolar pulse was half that of the first (positive) phase but progressively lost as the amplitude of the second phase was incrementally increased above that of the first phase. When the amplitude of the second phase was twice that of the first phase, there was stimulation. By comparing the experimental results for each manipulation of the bipolar pulse waveform with analytical calculations of capacitive membrane charging/discharging, also known as accelerated membrane discharge mechanism, we show that the transition from cancellation to unipolar-equivalent stimulation broadly agrees with this model. Taken as a whole, our results demonstrate that electrostimulation of adrenal chromaffin cells with ultrashort pulses can be modulated with interphase intervals of tens of nanoseconds, a prediction of the accelerated membrane discharge mechanism not previously observed in other bipolar pulse cancellation studies. Such modulation of Ca2+ responses in a neural-type cell is promising for the potential use of nanosecond bipolar pulse technologies for remote electrostimulation applications for neuromodulation.

VLSI FLOATING RESISTORS FOR NEURAL TYPE CELL ARRAYS

Two novel CMOS circuit designs implementing floating resistors are introduced, using the structure of a two-transistor CMOS bilateral linear resistor in the first configuration and two two-transistor CMOS bilateral linear resistors and cascode current mirrors in the second configuration. Linearity is achieved through nonlinearity cancellation via current mirrors over an applied range of ±5V. PSpice simulation results using parameters of MOSIS transistors are presented to verify the theory. These floating resistors can be used for coupling weights in VLSI neural-type cell arrays.