2-ns Electrostimulation of Ca\u3csup\u3e2+\u3c/sup\u3e 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

Quantitatively characterizing automotive interior surfaces using an optical TIR-based texture sensor

This paper presents the development of an optical total internal reflection (TIR) based surface texture sensor. Through combination of both contact and non-contact optical surface profiling mechanisms, the new sensor can be efficiently used for quantitative characterization of surface texture properties including texture pattern, roughness, and even mechanical property like softness, etc. We have used this sensor to measure and analyze texture properties of extensive automotive interior leather sample surfaces. The results demonstrate that the sensor can effectively assist the interior designer to quantify and classify essential texture features of automobile interior surfaces. Ongoing work is integrating the sensor into a scientific surface texture evaluation system for the purpose of improving automobile interior design. © 2009 IEEE.Link_to_subscribed_fulltex

High signal-to-noise imaging of spontaneous and 5 ns electric pulse-evoked Ca2+ signals in GCaMP6f-expressing adrenal chromaffin cells isolated from transgenic mice.

In studies exploring the potential for nanosecond duration electric pulses to serve as a novel modality for neuromodulation, we found that a 5 ns pulse triggers an immediate rise in [Ca2+]i in isolated bovine adrenal chromaffin cells. To facilitate ongoing efforts to understand underlying mechanisms and to work toward carrying out investigations in cells in situ, we describe the suitability and advantages of using isolated murine adrenal chromaffin cells expressing, in a Cre-dependent manner, the genetically-encoded Ca2+indicator GCaMP6f. Initial experiments confirmed that Ca2+ responses evoked by a 5 ns pulse were similar between fluorescent Ca2+ indicator-loaded murine and bovine chromaffin cells, thereby establishing that 5 ns-elicited excitation of chromaffin cells occurs reproducibly across species. In GCaMP6f-expressing murine chromaffin cells, spontaneous Ca2+ activity as well as nicotinic receptor agonist- and 5 ns evoked-Ca2+ responses consistently displayed similar kinetic characteristics as those in dye-loaded cells but with two-twentyfold greater amplitudes and without photobleaching. The high signal-to-noise ratio of evoked Ca2+ responses as well as spontaneous Ca2+ activity was observed in cells derived from Sox10-Cre, conditional GCaMP6f mice or TH-Cre, conditional GCaMP6f mice, although the number of cells expressing GCaMP6f at sufficiently high levels for achieving high signal-to-noise ratios was greater in Sox10-Cre mice. As in bovine cells, Ca2+ responses elicited in murine GCaMP6f-expressing cells by a 5 ns pulse were mediated by the activation of voltage-gated Ca2+ channels but not tetrodotoxin-sensitive voltage-gated Na+ channels. We conclude that genetically targeting GCaMP6f expression to murine chromaffin cells represents a sensitive and valuable approach to investigate spontaneous, receptor agonist- and nanosecond electric pulse-induced Ca2+ responses in vitro. This approach will also facilitate future studies investigating the effects of ultrashort electric pulses on cells in ex vivo slices of adrenal gland, which will lay the foundation for using nanosecond electric pulses to stimulate neurosecretion in vivo

Comparison of spontaneous fluctuations in [Ca²⁺]_i in Calcium Green-1 loaded and GCaMP6f-expressing ACC.

Representative fluorescence traces of spontaneous Ca2+ activity in (A) Calcium Green-loaded ACC, (B) ACC derived from Sox10-GCaMP6f mice or (C) from TH-GCaMP6f mice. In (A), traces are from ACC in different dishes from the same mouse. In (B) and (C), traces are from ACC from 3 different mice from different litters. In (A), photobleaching is evident by the drop in fluorescence intensity below F/F0 = 1.</p

Effect of blocking VGCC on Ca²⁺ responses evoked by a 5 ns pulse.

Individual cell responses to a 5 ns, 8 MV/m pulse (arrow), together with the averaged response ± SEM (red line), in the absence (A; n = 4, c = 13) or presence of 200 μM Cd2+ (B; n = 2, c = 10) or presence of a cocktail of VGCC blockers consisting of 3 μM ω-CTX GVIA, 2 μM ω-Aga IVA, 1 μM SNX-482, 3 μM nifedipine (C; n = 2, c = 15).</p

Immunohistochemical detection of SGC in mouse adrenal gland.

Adrenal gland cross sections from wt mice were stained with antibodies against TH to mark ACC and S100 to mark SGC. Low-magnification images (left column) demonstrate that immunoreactivity for each of these cell-specific markers is observed in the adrenal medulla (M) but not cortex (C). High-magnification images (right two columns) show distinct immunohistochemical staining patterns for each of these markers as well as nuclear counterstaining with Hoechst 33342. (TIF)</p

Comparison of 5 ns—evoked Ca²⁺ responses in ACC derived from <i>wt</i> and <i>Sox10-GCaMP6f</i> mice.

Comparison of 5 ns—evoked Ca2+ responses in ACC derived from wt and Sox10-GCaMP6f mice.</p

Comparison of Ca²⁺ responses to DMPP in <i>wt</i> and GCaMP-expressing ACC derived from <i>Sox10-GCaMP6f</i> mice.

(A) Ca2+ responses to 50 μM DMPP (arrows) in dye-loaded wt ACC (n = 2, c = 17) and (B) in GCaMP6f-expressing ACC (n = 3, c = 21). Traces show individual cell responses together with the averaged response (red lines). In (A), photobleaching is evident by the drop in fluorescence intensity below F/F0 = 1.</p

Effect of different GCaMP6f expression levels on DMPP-induced Ca²⁺ responses in <i>TH-GCaMP6f</i>-expressing ACC.

Representative fluorescence traces of Ca2+ responses evoked by 50 μM DMPP (arrows) in cells exhibiting (A) a “high” (n = 3, c = 13) or (B) “low” (n = 3, c = 8) level of GCaMP6f expression. In (B), photobleaching is evident by the drop in fluorescence intensity below F/F0 = 1.</p

Immunohistochemical expression of GCaMP6f in adrenal gland of <i>Sox10-GCaMP6f</i> and <i>TH-GCaMP6f</i> mice.

Adrenal gland cross sections Sox10-GCaMP6f mice (upper row) or TH-GCaMP6f mice (lower row) were stained with antibodies against GFP to mark GCaMP6f-expressing cells (left column, green) and TH to mark ACC (right column, red). Note the greater correlation between GFP and TH in Sox10-GCaMP6f versus TH-GCaMP6f mice, which results from greater recombination efficiency of GCaMP6f expression in ACC in Sox10-GCaMP6f mice. (TIF)</p