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Nanosecond pulsed electric fields (nsPEFs) induce changes in the plasma membrane (PM), including PM permeabilization (termed nanoporation), allowing free passage of ions into the cell and, in certain cases, cell death. Recent studies from our laboratory show that the composition of the PM is a critical determinant of PM nanoporation. Thus, we hypothesized that the biological response to nsPEF exposure could be influenced by lipid microdomains, including caveolae, which are specialized invaginations of the PM that are enriched in cholesterol and contain aggregates of important cell signaling proteins, such as caveolin-1 (Cav1). Caveolae play a significant role in cellular signal transduction, including control of calcium influx and cell death by interaction of Cav1 with regulatory signaling proteins. Present results show that depletion of Cav1 increased the influx of calcium, while Cav1 overexpression produced the opposite effect. Additionally, Cav1 is known to bind and sequester important cell signaling proteins within caveolae, rendering the binding partners inactive. Imaging of the PM after nsPEF exposure showed localized depletion of PM Cav1 and results of co-immunoprecipitation studies showed dissociation of two critical Cav1 binding partners (transient receptor potential cation channel subfamily C1 (TRPC1) and inositol trisphosphate receptor (IP3R)) after exposure to nsPEFs. Release of TRPC1 and IP3R from Cav1 would activate downstream signaling cascades, including store-operated calcium entry, which could explain the influx in calcium after nsPEF exposure. Results of the current study establish a significant relationship between Cav1 and the activation of cell signaling pathways in response to nsPEFs.

Exposures to low intensity radiofrequency electromagnetic fields (RF-EMFs, 100 kHz to 6 GHz) have been associated with both positive and negative effects on cognitive behavior. To elucidate the mechanism of RF-EMF interaction, a few studies have examined its impact on neuronal activity and synaptic plasticity. However, there is still a need for additional basic research that verify the reported effects and further our understanding of the underlying mechanisms of RF-EMFs on the neuronal system. The present study investigated changes in neuronal activity and synaptic transmission following a 60-min exposure to 3.0 GHz RF-EMF at a low dose (specific absorption rate (SAR) < 1 W/kg). We showed that RF-EMF exposure decreased the amplitude of action potential (AP), depolarized neuronal resting membrane potential (MP), and increased neuronal excitability and synaptic transmission in cultured primary hippocampal neurons (PHNs). The results show that RF-EMF exposure can alter neuronal activity and highlight that more investigations should be performed to fully explore the RF-EMF effects and mechanisms.

Exposures to radiofrequency electromagnetic fields (RF-EMFs, 100 kHz to 6 GHz) have been associated with both positive and negative effects on cognitive behavior. To elucidate the mechanism of RF-EMF interaction, a few studies have examined its impact on neuronal activity and synaptic plasticity. However, there is still a need for additional basic research that further our understanding of the underlying mechanisms of RF-EMFs on the neuronal system. The present study investigated changes in neuronal activity and synaptic transmission following a 60-min exposure to 3.0 GHz RF-EMF at a low dose (specific absorption rate (SAR) 1 W/kg). We showed that RF-EMF exposure decreased the amplitude of action potential (AP), depolarized neuronal resting membrane potential (MP), and increased neuronal excitability and synaptic transmission in cultured primary hippocampal neurons (PHNs). The results show that RF-EMF exposure can alter neuronal activity and highlight that more investigations should be performed to fully explore the RF-EMF effects and mechanisms.

Nanosecond electric pulses have been shown to open nanopores in the cell plasma membrane by fluorescent imaging of calcium uptake and fluorescent dyes, including propidium (Pr) iodide and YO-PRO-1 (YP1). Recently, we demonstrated that nsEPs also induce the phosphoinositide intracellular signaling cascade by phosphatidylinositol-4,5-bisphosphate (PIP2) depletion resulting in physiological responses similar to those observed following stimulation of Gq11-coupled receptors. In this paper, we explore the role of receptor- and store-operated calcium entry (ROCE/SOCE) mechanisms in the observed response of cells to nsEP. We show that addition of the ROCE/SOCE and transient receptor potential channel (TRPC) blocker gadolinium (Gd3+, 300 μM) slows PIP2 depletion following 1 and 20 nsEP exposures. Lipid rafts, regions of the plasma membrane rich in PIP2 and TRPC, are also disrupted by nsEP exposure suggesting that ROCE/SOCE mechanisms are likely impacted. Reducing the expression of stromal interaction molecule 1 (STIM1) protein, a key protein in ROCE and SOCE, in cells exposure to nsEP resulted in a reduction in induced intracellular calcium rise. Additionally, after exposure to 1 and 20 nsEPs (16.2 kV/cm, 5 Hz), intracellular calcium rises were significantly reduced by the addition of GD3+ and SKF-96365 (1-[2-(4-methoxyphenyl)-2-[3-(4-methoxyphenyl) propoxy] ethyl-1H-imidazole hydrochloride, 100 μM), a blocker of STIM1 interaction. However, using similar nsEP exposure parameters, SKF-96365 was less effective at reducing YP1 uptake compared to Gd3+. Thus, it is possible that SKF-96365 could block STIM1 interactions within the cell, while Gd3+ could acts on TRPC/nanopores from outside of the cell. Our results present evidence of nsEP induces ROCE and SOCE mechanisms and demonstrate that YP1 and Ca2+ cannot be used solely as markers of nsEP-induced nanoporation.

High-energy, short-duration electric pulses (EPs) are known to be effective in neuromodulation, but the biological mechanisms underlying this effect remain unclear. Recently, we discovered that nanosecond electric pulses (nsEPs) could initiate the phosphatidylinositol

Nanosecond pulsed electric fields (nsPEFs) induce changes in the plasma membrane (PM), including PM permeabilization (termed nanoporation), allowing free passage of ions into the cell and, in certain cases, cell death. Recent studies from our laboratory show that the composition of the PM is a critical determinant of PM nanoporation. Thus, we hypothesized that the biological response to nsPEF exposure could be influenced by lipid microdomains, including caveolae, which are specialized invaginations of the PM that are enriched in cholesterol and contain aggregates of important cell signaling proteins, such as caveolin-1 (Cav1). Caveolae play a significant role in cellular signal transduction, including control of calcium influx and cell death by interaction of Cav1 with regulatory signaling proteins. Present results show that depletion of Cav1 increased the influx of calcium, while Cav1 overexpression produced the opposite effect. Additionally, Cav1 is known to bind and sequester important cell signaling proteins within caveolae, rendering the binding partners inactive. Imaging of the PM after nsPEF exposure showed localized depletion of PM Cav1 and results of co-immunoprecipitation studies showed dissociation of two critical Cav1 binding partners (transient receptor potential cation channel subfamily C1 (TRPC

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.

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.

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.

High-energy, short-duration electric pulses (EPs) are known to be effective in neuromodulation, but the biological mechanisms underlying this effect remain unclear. Recently, we discovered that nanosecond electric pulses (nsEPs) could initiate the phosphatidylinositol4,5-bisphosphate (PIP2) depletion in non-excitable cells identical to agonist-induced activation of the Gq11 coupled receptors. PIP2 is the precursor for multiple intracellular second messengers critically involved in the regulation of intracellular Ca2+ homeostasis and plasma membrane (PM) ion channels responsible for the control of neuronal excitability. In this paper we demonstrate a novel finding that five day in vitro (DIV5) primary hippocampal neurons (PHNs) undergo significantly higher PIP2 depletion after 7.5 kV/cm 600 ns EP exposure than DIV1 PHNs and day 1–5 (D1-D5) non-excitable Chinese hamster ovarian cells with muscarinic receptor 1 (CHO-hM1). Despite the age of development, the stronger 15 kV/cm 600 ns or longer 7.5 kV/cm 12 µs EP initiated profound PIP2 depletion in all cells studied, outlining damage of the cellular PM and electroporation. Therefore, the intrinsic properties of PHNs in concert with nanoporation explain the stronger neuronal response to nsEP at lower intensity exposures. PIP2 reduction in neurons could be a primary biological mechanism responsible for the stimulation or inhibition of neuronal tissues.

Previously, it was demonstrated that nanometer-sized pores (nanopores) are formed in outer cellular membranes after exposure to nanosecond electric pulses (nsEPs). We reported that plasma membrane nanoporation affects phospholipids of the cell membrane, culminating in cascading phosphoinositide phosphatidylinositol-4,5-bisphosphate (PIP2) intracellular signaling. In the current study, we show that nsEPs initiated electric field (EF) dose-dependent PIP2 hydrolysis and/or depletion from the plasma membrane. This process was confirmed using fluorescent optical probes of PIP2 hydrolysis: PLCδ-PH-EGFP and GFP-C1-PKCγ-C1a. The 50% maximum response occurs with a single 600ns pulse achieving an effective dose (ED50) of EF~8kV/cm within our model cell system. At 16.2kV/cm, the ED50 for the pulse width was 484ns. Reduction of the pulse width or EF amplitude gradually reduced the observed effect, but twenty 60ns 16.2kV/cm pulses produced an effect similar to a single 600ns pulse of the same amplitude. Propidium iodide (PI) uptake after the nsEP exposure confirmed a strong relationship between EF-induced plasma membrane impact and PIP2 depletion. These results have expanded our current knowledge of nsEPs dependent cell physiological effects, and serve as a basis for model development of new exposure standards, providing novel tools for drug independent stimulation and approaches to differential modulation of key cellular functions.

Nanosecond electric pulses (nsEP's) are a well-studied phenomena in biophysics that cause substantial alterations to cellular membrane dynamics, internal biochemistry, and cytoskeletal structure, and induce apoptotic and necrotic cell death. While several studies have attempted to measure the effects of multiple nanosecond pulses, the effect of pulse repetition rate (PRR) has received little attention, especially at frequencies greater than 100 Hz. In this study, uptake of Propidium Iodide, FM 1–43, and YO-PRO-1 fluorescent dyes in CHO-K1 cells was monitored across a wide range of PRRs (5 Hz–500 KHz) using a laser-scanning confocal microscope in order to better understand how high frequency repetition rates impact induced biophysical changes. We show that frequency trends depend on the identity of the dye under study, which could implicate transmembrane protein channels in the uptake response due to their chemical selectivity. Finally, YO-PRO-1 fluorescence was monitored in the presence of Gadolinium (Gd3+), Ruthenium Red, and in calcium-free solution to elucidate a mechanism for its unique frequency trend.

Pulsed infrared (IR) laser energy has been shown to modulate neurological activity through both stimulation and inhibition of action potentials. While the mechanism(s) behind this phenomenon is (are) not completely understood, certain hypotheses suggest that the rise in temperature from IR exposure could activate temperature- or pressure-sensitive ion channels or create pores in the cellular outer membrane, allowing an influx of typically plasma-membrane-impermeant ions. Studies using fluorescent intensity-based calcium ion ([Formula: see text]) sensitive dyes show changes in [Formula: see text] levels after various IR stimulation parameters, which suggests that [Formula: see text] may originate from the external solution. However, activation of intracellular signaling pathways has also been demonstrated, indicating a more complex mechanism of increasing intracellular [Formula: see text] concentration. We quantified the [Formula: see text] mobilization in terms of influx from the external solution and efflux from intracellular organelles using Fura-2 and a high-speed ratiometric imaging system that rapidly alternates the dye excitation wavelengths. Using nonexcitable Chinese hamster ovarian ([Formula: see text]) cells and neuroblastoma-glioma (NG108) cells, we demonstrate that intracellular [Formula: see text] receptors play an important role in the IR-induced [Formula: see text], with the [Formula: see text] response augmented by ryanodine receptors in excitable cells.

Pulsed infrared light can excite action potentials in neurons; yet, the fundamental mechanism underlying this phenomenon is unknown. Previous work has observed a rise in intracellular calcium concentration following infrared exposure, but the source of the calcium and mechanism of release is unknown. Here, we used fluorescence lifetime imaging of Oregon Green BAPTA-1 to study intracellular calcium dynamics in primary rat hippocampal neurons in response to infrared light exposure. The fluorescence lifetime of Oregon Green BAPTA-1 is longer when bound to calcium, and allows robust measurement of intracellular free calcium concentrations. First, a fluorescence lifetime calcium calibration curve for Oregon Green BAPTA-1 was determined in solutions. The normalized amplitude of the short and long lifetimes was calibrated to calcium concentration. Then, neurons were incubated in Oregon Green BAPTA-1 and exposed to pulses of infrared light (0-1 J/cm2; 0-5 ms; 1869 nm). Fluorescence lifetime images were acquired prior to, during, and after the infrared exposure. Fluorescence lifetime images, 64x64 pixels, were acquired at 12 or 24 ms for frame rates of 83 and 42 Hz, respectively. Accurate α1 approximations were achieved in images with low photon counts by computing an α1 index value from the relative probability of the observed decay events. Results show infrared light exposure increases intracellular calcium in neurons. Altogether, this study demonstrates accurate fluorescence lifetime component analysis from low-photon count data for improved imaging speed.

Short infrared laser pulses have many physiological effects on cells including the ability to stimulate action potentials in neurons. Here we show that short infrared laser pulses can also reversibly block action potentials. Primary rat hippocampal neurons were transfected with the Optopatch2 plasmid, which contains both a blue-light activated channel rhodopsin (CheRiff) and a red-light fluorescent membrane voltage reporter (QuasAr2). This optogenetic platform allows robust stimulation and recording of action potential activity in neurons in a non-contact, low noise manner. For all experiments, QuasAr2 was imaged continuously on a wide-field fluorescent microscope using a Krypton laser (647 nm) as the excitation source and an EMCCD camera operating at 1000 Hz to collect emitted fluorescence. A co-aligned Argon laser (488 nm, 5 ms at 10Hz) provided activation light for CheRiff. A 200 mm fiber delivered infrared light locally to the target neuron. Reversible action potential block in neurons was observed following a short infrared laser pulse (0.26-0.96 J/cm2; 1.37-5.01 ms; 1869 nm), with the block persisting for more than 1 s with exposures greater than 0.69 J/cm2. Action potential block was sustained for 30 s with the short infrared laser pulsed at 1-7 Hz. Full recovery of neuronal activity was observed 5-30s post-infrared exposure. These results indicate that optogenetics provides a robust platform for the study of action potential block and that short infrared laser pulses can be used for non-contact, reversible action potential block.

Nanosecond pulsed electric fields (nsPEFs) perturb membranes of cultured mammalian cells in a dose-dependent manner with different types of cells exhibiting characteristic survivability. Adherent cells appear more robust than non-adherent cells during whole-cell exposure. We hypothesize that cellular elasticity based upon the actin cytoskeleton is a contributing parameter, and the alteration of a cell's actin cortex will significantly affect viability upon nsPEF exposure. Chinese hamster ovary (CHO) cells that are (a) untreated, (b) treated with latrunculin A to inhibit actin polymerization, or (c) exposed to nsPEFs have been probed using atomic force microscopy (AFM) force-indentations. Exposure to 50 or 100 pulses of 10 ns duration and 150 kV/cm in a single dosage approximately lowers average CHO cell elastic modulus by half, whereas latrunculin lowers it more than 75%. Latrunculin pre-treatment disrupts the actin cortex enough that it negates cumulative damage by equally fractionated (i.e., two rounds of 50 pulses each, separated by 10 min) dosages of nsPEFs as seen in untreated and dimethyl sulfoxide (DMSO)-treated cells with propidium uptake, phosphatidylserine externalization, and 24 h viability according to MTT and CellTiter Glo assays. These results suggest a correlation among cell stiffness, cytoskeletal integrity, and susceptibility to recurrent exposures to nsPEFs, which emphasizes a mechanobiological underpinning of nsPEF bioeffects.

Exposure to nanosecond pulsed electrical fields (nsPEFs) results in a myriad of observable effects in mammalian cells. While these effects are often attributed to the direct permeabilization of both the plasma and organelle membranes, the underlying mechanism(s) are not well understood. We hypothesize that nsPEF-induced membrane disturbance will initiate complex intracellular lipid signaling pathways, which ultimately lead to the observed multifarious effects. In this article, we show activation of one of these pathways--phosphoinositide signaling cascade. Here we demonstrate that nsPEF initiates phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) hydrolysis or depletion from the plasma membrane, accumulation of inositol-1,4,5-trisphosphate (IP3) in the cytoplasm and increase of diacylglycerol (DAG) on the inner surface of the plasma membrane. All of these events are initiated by a single 16.2 kV/cm, 600 ns pulse exposure. To further this claim, we show that the nsPEF-induced activation mirrors the response of M1-acetylcholine Gq/11-coupled metabotropic receptor (hM1). This demonstration of PIP2 hydrolysis by nsPEF exposure is an important step toward understanding the mechanisms underlying this unique stimulus for activation of lipid signaling pathways and is critical for determining the potential for nsPEFs to modulate mammalian cell functions.

Hypothermic machine perfusion (HMP) for the preservation of kidneys, recovered from extended criteria organ donors (ECDs), presents the opportunity for assessing ex vivo parameters that may have value in predicting postimplantation organ viability. Organ perfusion and vascular resistance are the parameters most frequently cited as the basis for the decision to use or discard a donor kidney. The limitation of these measures is emphasized by the observation that a significant percentage of ECD kidneys with poor perfusion parameters can provide life-sustaining function after transplantation. It has been suggested that whole organ oxygen consumption (OC) during oxygenated HMP may better reflect the proportion of viable tissue in the organ and more reliably predict posttransplant organ function. Our study correlates renal OC and renal vascular resistance (RVR) during oxygenated HMP with postpreservation glomerular filtration rates (GFRs) in rodent kidneys after 24 hours of oxygenated HMP. Kidneys from adult rodents were preserved for 24 hours using oxygenated HMP and static cold storage (SCS). During oxygenated HMP preservation, organ OC, renal organ flow rates, and RVR were serially measured. After the preservation period, organs were mounted onto a Langendorff device for warming to normal body temperature and measurement of GFR. Oxygen consumption and RVR during HMP were correlated with postpreservation GFR. Oxygen consumption during oxygenated HMP was significantly correlated (r2 = 0.871; p < 0.05) with postpreservation GFR, suggesting that higher OC predicts better postpreservation GFR. In contrast, RVR was poorly correlated with postpreservation GFR (r2 = 0.258; p = 0.199). Glomerular filtration rate in SCS kidneys was 0.002 ± 0.003 ml/min/g. We demonstrate that measurement of organ OC during oxygenated HMP may have significant value in predicting postpreservation organ function.

Sudden unexpected death in epilepsy (SUDEP) accounts for 15% of all deaths in people with epilepsy and 50% in refractory epilepsy. The underlying mechanisms are not well understood, but seizure-induced cardiac and respiratory arrests are involved. The cardiovascular and respiratory systems are subject to precise reflex regulation to ensure appropriate oxygen supply under a wide range of circumstances. Barosensory and chemosensory afferents project into the nucleus tractus solitarius (NTS), which relays systemic data to higher brain centers for integration of homeostatic responses in heart rate, peripheral resistance, respiration, and other autonomic reactions. Being the afferent autonomic gatekeeper, NTS plays a critical role in cardiovascular and respiratory regulation. In the course of studying the kainic acid model, we became aware of progressive neuronal loss in the NTS and noted SUDEP-like deaths in rats with frequent convulsions. Increased autonomic susceptibility with inhalation anesthetics was also observed, often seen after impairment of baroreceptor and chemoreceptor reflex loops. Seizure-induced neuron loss in NTS may play a role impairing the integrative functions of NTS resulting in poor homeostatic responses during seizures and leading to SUDEP.

Previously, it was demonstrated that nanometer-sized pores (nanopores) are formed in outer cellular membranes after exposure to nanosecond electric pulses (nsEPs). We reported that plasma membrane nanoporation affects phospholipids of the cell membrane, culminating in cascading phosphoinositide phosphatidylinositol

Cells exposed to nanosecond-pulsed electric fields (nsPEF) exhibit a wide variety of nonspecific effects, including blebbing, swelling, intracellular calcium bursts, apoptotic and necrotic cell death, formation of nanopores, and depletion of phosphatidylinositol 4,5-biphosphate (PIP2) to induce activation of the inositol trisphosphate/diacylglycerol pathway. While several studies have taken place in which multiple pulses were delivered to cells, the effect of pulse repetition rate (PRR) is not well understood. To better understand the effects of PRR, a laser scanning confocal microscope was used to observe CHO-K1 cells exposed to ten 600ns, 200V pulses at varying repetition rates (5Hz up to 500KHz) in the presence of either FM 1-43, YO-PRO-1, or Propidium Iodide (PI) fluorescent dyes, probes frequently used to indicate nanoporation or permeabilization of the plasma membrane. Dye uptake was monitored for 30 seconds after pulse application at a rate of 1 image/second. In addition, a single long pulse of equivalent energy (200V, 6 μs duration) was applied to test the hypothesis that very fast PRR will approximate the biological effects of a single long pulse of equal energy. Upon examination of the data, we found strong variation in the relationship between PRR and uptake in each of the three dyes. In particular, PI uptake showed little frequency dependence, FM 1-43 showed a strong inverse relationship between frequency and internal cell fluorescence, and YO-PRO-1 exhibited a “threshold” point of around 50 KHz, after which the inverse trend observed in FM 1-43 was seen to reverse itself. Further, a very high PRR of 500 KHz only approximated the biological effects of a single 6 μs pulse in cells stained with YO-PRO-1, suggesting that uptake of different dyes may proceed by different physical mechanisms.

Recently, infrared light has been shown to both stimulate and inhibit excitatory cells. However, studies of infrared light for excitatory cell inhibition have been constrained by the use of invasive and cumbersome electrodes for cell excitation and action potential recording. Here, we present an all optical experimental design for neuronal excitation, inhibition, and action potential detection. Primary rat neurons were transfected with plasmids containing the light sensitive ion channel CheRiff. CheRiff has a peak excitation around 450 nm, allowing excitation of transfected neurons with pulsed blue light. Additionally, primary neurons were transfected with QuasAr2, a fast and sensitive fluorescent voltage indicator. QuasAr2 is excited with yellow or red light and therefore does not spectrally overlap CheRiff, enabling imaging and action potential activation, simultaneously. Using an optic fiber, neurons were exposed to blue light sequentially to generate controlled action potentials. A second optic fiber delivered a single pulse of 1869nm light to the neuron causing inhibition of the evoked action potentials (by the blue light). When used in concert, these optical techniques enable electrode free neuron excitation, inhibition, and action potential recording, allowing research into neuronal behaviors with high spatial fidelity.

Exposure of cells to nanosecond pulsed electric fields (nsPEF) causes a rapid increase in intracellular calcium. The mechanism(s) responsible for this calcium burst remains unknown, but is hypothesized to be from direct influx through nanopores, the activation of specific ion channels, or direct disruption of organelles. It is likely, however, that several mechanisms are involved/activated, thereby resulting in a complex chain of events that are difficult to separate by slow imaging methods. In this letter, we describe a novel high-speed imaging system capable of determining the spatial location of calcium bursts within a single cell following nsPEF exposure. Preliminary data in rodent neuroblastoma cells are presented, demonstrating the ability of this system to track the location of calcium bursts in vitro within milliseconds of exposure. These data reveal that calcium ions enter the cell from the plasma membrane regions closest to the electrodes (poles), and that intracellular calcium release occurs in the absence of extracellular calcium. We believe that this novel technique will allow us to temporally and spatially separate various nsPEF-induced effects, leading to powerful insights into the mechanism(s) of interaction between electric fields and cellular membranes.

Permeabilization of cell membranes occurs upon exposure to a threshold absorbed dose (AD) of nanosecond pulsed electric fields (nsPEF). The ultimate, physiological bioeffect of this exposure depends on the type of cultured cell and environment, indicating that cell-specific pathways and structures are stimulated. Here we investigate 10 and 600 ns duration PEF effects on Chinese hamster ovary (CHO) cell nuclei, where our hypothesis is that pulse disruption of the nuclear envelope membrane leads to observed cell death and decreased viability 24 h post-exposure. To observe short-term responses to nsPEF exposure, CHO cells have been stably transfected with two fluorescently-labeled proteins known to be sequestered for cellular chromosomal function within the nucleus - histone-2b (H2B) and proliferating cell nuclear antigen (PCNA). H2B remains associated with chromatin after nsPEF exposure, whereas PCNA leaks out of nuclei permeabilized by a threshold AD of 10 and 600 ns PEF. A downturn in 24 h viability, measured by MTT assay, is observed at the number of pulses required to induce permeabilization of the nucleus.

KCNQ1-5 (Kv7.1-7.5) subunits assemble to form a variety of functional K(+) channels in the nervous system, heart, and epithelia. KCNQ1 and KCNQ4 homomers and KCNQ2/3 heteromers yield large currents, whereas KCNQ2 and KCNQ3 homomers yield small currents. Since the unitary conductance of KCNQ3 is five- to 10-fold greater than that of KCNQ4 or KCNQ1, these differences are even more striking. To test for differential membrane protein expression, we performed biotinylation and total internal reflection fluorescence imaging assays; however, both revealed only small differences among the channels, leading us to investigate other mechanisms at work. We probed the molecular determinants governing macroscopic current amplitudes, with focus on the turret and pore-loop domains of KCNQ1 and KCNQ3. Elimination of the putative N289 glycosylation site in KCNQ1 reduced current density by approximately 56%. A chimera consisting of KCNQ3 with the turret domain (TD) of KCNQ1 increased current density by about threefold. Replacement of the proximal half of the TD in KCNQ3 with that of KCNQ1 increased current density by fivefold. A triple chimera containing the TD of KCNQ1 and the carboxy terminus of KCNQ4 yielded current density 10- or sixfold larger than wild-type KCNQ3 or KCNQ1, respectively, suggesting that the effects on current amplitudes of the TD and the carboxy-terminus are additive. Critical was the role of the intracellular TEA(+)-binding site. The KCNQ3 (A315T) swap increased current density by 10-fold, and the converse KCNQ1 (T311A) swap reduced it by 10-fold. KCNQ3 (A315S) also yielded greatly increased current amplitudes, whereas currents from mutant A315V channels were very small. The KCNQ3 (A315T) mutation increased the sensitivity of the channels to external Ba(2+) block by eight- to 28-fold, consistent with this mutation altering the structure of the selectivity filter. To investigate a structural hypothesis for the effects of these mutations, we performed homology modeling of the pore region of wild-type and mutant KCNQ3 channels, using KvAP as a template. The modeling suggests a critical stabilizing interaction between the pore helix and the selectivity filter that is absent in wild-type KCNQ3 and the A315V mutant, but present in the A315T and A315S mutants. We conclude that KCNQ3 homomers are well expressed at the plasma membrane, but that most wild-type channels are functionally silent, with rearrangements of the pore-loop architecture induced by the presence of a hydroxyl-containing residue at the 315 position "unlocking" the channels into a conductive conformation.

Neural M-type (KCNQ/Kv7) K+ channels control somatic excitability, bursting and neurotransmitter release throughout the nervous system. Their activity is regulated by multiple signalling pathways. In superior cervical ganglion sympathetic neurons, muscarinic M1, angiotensin II AT1, bradykinin B2 and purinergic P2Y agonists suppress M current (IM). Probes of PLC activity show agonists of all four receptors to induce robust PIP2 hydrolysis. We have grouped these receptors into two related modes of action. One mode involves depletion of phosphatidylinositol 4,5-bisphosphate (PIP2) in the membrane, whose interaction with the channels is thought necessary for their function. The other involves IP3-mediated intracellular Ca2+ signals that stimulate PIP2 synthesis, preventing its depletion, and suppress IM via calmodulin. Carbon-fibre amperometry can evaluate the effect of M channel activity on release of neurotransmitter. Consistent with the dominant role of M current in control of neuronal discharge, M channel openers, or blockers, reduced or augmented the evoked release of noradrenaline neurotransmitter from superior cervical ganglion (SCG) neurons, respectively. We seek to localize the subdomains on the channels critical to their regulation by PIP2. Based on single-channel recordings from chimeras between high-PIP2 affinity KCNQ3 and low-PIP2 affinity KCNQ4 channels, we focus on a 57-residue domain within the carboxy-terminus that is a possible PIP2 binding site. Homology modelling of this domain using the published structure of IRK1 channels as a template predicts a structure very similar to an analogous region in IRK1 channels, and shows a cluster of basic residues in the KCNQ2 domain to correspond to those implicated in PIP2 regulation of Kir channels. We discuss some important issues dealing with these topics.

Purinergic P2Y receptors are one of four types of Gq/11-coupled receptors in rat superior cervical ganglia (SCG) sympathetic neurons. In cultured SCG neurons, purinergic and bradykinin suppression ofIMwere similar in magnitude and somewhat less than that by muscarinic agonists. The effects of the P2Y receptor agonist UTP on neuronal excitability and discharge properties were studied. Under current clamp, UTP increased action potential (AP) firing in response to depolarizing current steps, depolarized the resting potential, decreased the threshold current required to fire an AP, and decreased spike-frequency adaptation. These effects were very similar to those resulting from bradykinin stimulation and not as profound as from muscarinic stimulation or full M-current blockade. We then examined the P2Y mechanism of action. Like bradykinin, but unlike muscarinic, purinergic stimulation induced rises in intracellular [Ca2+]i. Tests using expression of IP3“sponge” or IP3phosphatase constructs implicated IP3accumulation as necessary for purinergic suppression ofIM. Overexpression of wild-type or dominant-negative calmodulin (CaM) implicated Ca2+/CaM in the purinergic action. Both sets of results were similar to bradykinin, and opposite to muscarinic, suppression. We also examined modulation of Ca2+channels. As for bradykinin, purinergic stimulation did not suppressICa, unless neuronal calcium sensor-1 (NCS-1) activity was blocked by a dominant-negative NCS-1 construct. Our results indicate that P2Y receptors modulate M-type channels in SCG cells via IP3-mediated [Ca2+]isignals in concert with CaM and not by depletion of phosphatidylinositol-4, 5-biphosphate. We group purinergic P2Y and bradykinin B2receptors together as having a common mode of action.

The nucleus of the solitary tract (NTS) is the central site of termination of baroreceptor afferents. We hypothesize that changes occur in voltage-gated calcium channels (VGCCs) within NTS neurons as a consequence of hypertension. Whole-cell patch-clamp recordings were obtained from adult normotensive (109±2 mm Hg; n=6 from 6 sham-operated and 31 nonsurgically treated) and hypertensive (158±6 mm Hg; n=24) rats. In some experiments, 4-(4-[dihexadecylamino]styryl)-N-methylpyridinium iodide was applied to the aortic nerve to visualize NTS neurons receiving baroreceptor synaptic contacts. Ba 2+ currents (500 ms; −80 mV prepotential; 500 ms voltage steps in 5-mV increments to +15mV) peaked between −20 and −10 mV and were blocked by 100 μm of Cd 2+ . Peak VGCCs were not different comparing non-4-(4-[dihexadecylamino]styryl)-N-methylpyridinium iodide-labeled and 4-(4- [dihexadecylamino]styryl)-N-methylpyridinium iodide-labeled NTS neurons in hypertensive and normotensive rats. The peak VGCC was significantly greater in cells from hypertensive compared with normotensive rats for both non–DiA-labeled ( P =0.02) and DiA-labeled ( P =0.04) neurons. To separate high-voltage activated (HVA) and low-voltage activated (LVA) components of VGCCs, voltage ramps (−110 mV to +30 mV over 50 ms) were applied from a holding potential of −60 mV (LVA channels inactivated) and a holding potential of −100 mV (both LVA and HVA currents activated). HVA currents were subtracted from HVA+LVA currents to yield the LVA current. Peak LVA currents were not different between hypertensive (8.9±0.8 pA/pF) and normotensive (7.8±0.6 pA/pF) groups of NTS neurons ( P =0.27). These results demonstrate that 4 weeks of renal wrap hypertension induce an increase in Ca 2+ influx through HVA VGCCs in NTS neurons receiving arterial baroreceptor inputs.

Nanosecond electrical pulse (nsEP) exposure activates signaling pathways, produces oxidative stress, stimulates hormone secretion, causes cell swelling and induces apoptotic and necrotic death. The underlying biophysical connection(s) between these diverse cellular reactions and nsEP has yet to be elucidated. Using global genetic analysis, we evaluated how two commonly studied cell types, U937 and Jurkat, respond to nsEP exposure. We hypothesized that by studying the genetic response of the cells following exposure, we would gain direct insight into the stresses experienced by the cell and in turn better understand the biophysical interaction taking place during the exposure. Using Ingenuity Systems software, we found genes associated with cell growth, movement and development to be significantly up-regulated in both cell types 4 h post exposure to nsEP. In agreement with our hypothesis, we also found that both cell lines exhibit significant biological changes consistent with mechanical stress induction. These results advance nsEP research by providing strong evidence that the interaction of nsEPs with cells involves mechanical stress.

Phosphatidylinositol4,5-biphosphate (PIP2) is a membrane phospholipid of particular importance in cell-signaling pathways. Hydrolysis of PIP2 releases inositol-1,4,5-triphosphate (IP3) from the membrane, activating IP3 receptors on the smooth endoplasmic reticulum (ER) and facilitating a release of intracellular calcium stores and activation of protein kinase C (PKC). Recent studies suggest that nanosecond pulsed electric fields (nsPEF) cause depletion of PIP2 in the cellular membrane, activating the IP3 signaling pathway. However, the exact mechanism(s) causing this observed depletion of PIP2 are unknown. Complicating the matter, nsPEF create nanopores in the plasma membrane, allowing calcium to enter the cell and thus causing an increase in intracellular calcium. While elevated intracellular calcium can cause activation of phospholipase C (PLC) (a known catalyst of PIP2 hydrolysis), PIP2 depletion has been shown to occur in the absence of both extracellular and intracellular calcium. These observations have led to the hypothesis that the high electric field itself may be playing a direct role in the hydrolysis of PIP2 from the plasma membrane. To support this hypothesis, we used edelfosine to block PLC and prevent activation of the IP3/DAG pathway in Chinese Hamster Ovarian (CHO) cells prior to applying nsPEF. Fluorescence microscopy was used to monitor intracellular calcium bursts during nsPEF, while MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) survivability assays were utilized to determine whether edelfosine improved cell survival during nsPEF exposure. This work is critical to refine the role of PIP2 in the cellular response to nsPEF, and also to determine the fundamental biological effects of high electric field exposures.

Cellular delivery of pulsed IR laser energy has been shown to stimulate action potentials in neurons. The mechanism for this stimulation is not completely understood. Certain hypotheses suggest the rise in temperature from IR exposure could activate temperature- or pressure-sensitive channels, or create pores in the cellular outer membrane. Studies using intensity-based Ca 2+- responsive dyes show changes in Ca 2+ levels after various IR stimulation parameters; however, determination of the origin of this signal proved difficult. An influx of larger, typically plasma-membrane-impermeant ions has been demonstrated, which suggests that Ca 2+ may originate from the external solution. However, activation of intracellular signaling pathways, possibly indicating a more complex role of increasing Ca 2+ concentration, has also been shown. By usingCa 2+ sensitive dye Fura-2 and a high-speed ratiometric imaging system that rapidly alternates the excitation wavelengths, we have quantified the Ca 2+ mobilization in terms of influx from the external solution and efflux from intracellular organelles. CHO-K1 cells, which lack voltage-gated Ca 2+ channels, and NG-108 neuroblastoma cells, which do not produce action potentials in an early undifferentiated state, are used to determine the origin of the Ca 2+ signals and investigate the role these mechanisms may play in IR neural stimulation.

Previously, it was demonstrated that small nanometer-sized pores (nanopores) are preferentially formed after exposure to nanosecond pulsed electric fields (nsPEF). We have reported that nanoporation of the plasma membrane directly affects the phospholipids of the cell membrane, ultimately culminating in phosphatidylinositol 4,5 - bisphosphate (PIP 2 ) intracellular signaling. PIP 2 , located within the internal layer of the plasma membrane, plays a critical role as a regulator of ion transport proteins, a source of second messenger compounds, and an anchor for cytoskeletal elements. In this proceeding, we present data that demonstrates that nsPEFs initiate electric field dose-dependent PIP 2 hydrolysis and/or depletion from the plasma membrane through the observation of the accumulation of inositol 1,4,5 -trisphosphate (IP3) in the cytoplasm and the increase of diacylglycerol (DAG) on the inner surface of the plasma membrane. The phosphoinositide signaling cascade presented here involves activation of phospholipase C (PLC) and protein kinase C (PKC), which are responsible for a multitude of biological effects after nsPEF exposure. These results expand our current knowledge of nsPEF induced physiological effects, and serve as a basis for development of novel tools for drug independent stimulation or modulation of different cellular functions.

The Persian Gulf Syndrome generally manifests as a set of nonspecific complaints with emphasis on central nervous system impairment. The purpose of this study was to determine if cognitive performance and middle cerebral artery blood flow velocity (MCABFV) were altered in symptomatic Gulf War veterans (sGWVs) and asymptomatic Gulf War veterans (aGWVs) by exposure to low levels of acetone. MCABFV was assessed in male aGWVs (n=8) and sGWVs (n=8) during cognitive challenges while breathing 1) clean air, 2) a clean air placebo, and 3) a mixture of air and 40 parts per million (ppm) acetone. Pulmonary function was also evaluated. Pulmonary function tests showed no statistical differences between aGWVs and sGWVs while breathing clean air or 40 ppm acetone in air. Cognitive performance was similar during the clean air, placebo, and acetone test conditions for sGWVs and aGWVs. Data pooled across test conditions for each group indicated a statistically significant (P

Background and Objective: A low cost laser imager was designed and fabricated for measurement of wound geometry. Methods: The accuracy of the imager was validated using reference depressions of known dimensions. Perimeter, area, and volume were compared to planimetric and packing techniques on simulated wound models. Results: Wound tracing and alginate measurement methods required approximately 20 times longer for the reference standards, and 11 times longer for the simulated wounds than with the laser scanning method (LSM). LSM consistently overestimated the reference perimeter by 0.73 ± 0.20 cm and the area by 0.98 ± 0.62 cm 2 . Volume estimates were not statistically different. The tracing method underestimated the perimeter by 0.34 ± 0.27 cm and the area by 1.07 ± 1.09 cm 2 . Volume measurements by the alginate method were not statistically different. The perimeters of the simulated wounds averaged 1.29 ± 0.27 cm greater using the LSM than obtained by the tracing method, and areas greater by 2.02 ± 1.30 cm 2 . Volume scans averaged 1.04 ± 0.61 cm 3 greater than by the alginate method.

The interaction between nsPEF-induced Ca(2+) release and nsPEF-induced phosphatidylinositol4,5-bisphosphate (PIP2) hydrolysis is not well understood. To better understand this interrelation we monitored intracellular calcium changes, in cells loaded with Calcium Green-1 AM, and generation of PIP2 hydrolysis byproducts (inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DAG)) in cells transfected with one of two fluorescent reporter genes: PLCδ-PH-EGFP or GFP-C1-PKCγ-C1a. The percentage fluorescence differences (ΔF %) after exposures were determined. Upon nsPEF impact, we found that in the absence of extracellular Ca(2+) the population of IP3 liberated during nsPEF exposure (ΔF 6%±3, n=22), is diminished compared to the response in the presence of calcium (ΔF 84%±15, n=20). The production of DAG in the absence of extracellular Ca(2+) (ΔF 29%±5, n=25), as well as in cells exposed to thapsigargin (ΔF 40%±12, n=15), was not statistically different from cells exposed in the presence of extracellular calcium (ΔF 22±6%, n=18). This finding suggests that the change in intracellular calcium concentration is not solely driving the observed response. Interestingly, the DAG produced in the absence of Ca(2+) is the strongest near the membrane regions facing the electrodes, whereas the presence of extracellular Ca(2+) leads to a whole cell response. The reported observations of Ca(2+) dynamics combined with IP3 and DAG production suggest that nsPEF may cause a direct effect on the phospholipids within the plasma membrane.

In cellular electrochemistry, ions respond to stimuli by constantly shuffling across cellular membranes to perform their physiological roles. This flow of ions, the electromotive force, leaves cells vulnerable to exogenous electromagnetic fields that can stimulate and/or modulate cellular activity. An irreparable link exists between changes in ionic concentration and the electric gradient of the cell (or its potential energy). Consequently, we can manipulate the physiology of the cell by altering its permeability to various ions, thereby modulating its electrical gradient. Only a few millivolts in excess of the resting membrane potential can stimulate a dramatic change in ion distribution within the cellular microenvironment. In excitable neural-type cells, electrical-stimulation-induced changes in membrane potential lead to the generation or inactivation of action potentials (AP). These AP trigger activities, such as nerve impulses in

Nanosecond pulsed electric fields (nsPEFs) are known to increase cell membrane permeability to small molecules in accordance with dosages. As previous work has focused on nsPEF exposures in whole cells, electrodeformation may contribute to this induced-permeabilization in addition to other biological mechanisms. Here, we hypothesize that cellular elasticity, based upon the cytoskeleton, affects nsPEF-induced decrease in cellular viability. Young’s moduli of various types of cells have been calculated from atomic force microscopy (AFM) force curve data, showing that CHO cells are stiffer than non-adherent U937 and Jurkat cells, which are more susceptible to nsPEF exposure. To distinguish any cytoskeletal foundation for these observations, various cytoskeletal reagents were applied. Inhibiting actin polymerization significantly decreased membrane integrity, as determined by relative propidium uptake and phosphatidylserine externalization, upon exposure at 150 kV/cm with 100 pulses of 10 ns pulse width. Exposure in the presence of other drugs resulted in insignificant changes in membrane integrity and 24-hour viability. However, Jurkat cells showed greater lethality than latrunculin-treated CHO cells of comparable elasticity. From these results, it is postulated that cellular elasticity rooted in actin-membrane interaction is only a minor contributor to the differing responses of adherent and non-adherent cells to nsPEF insults.

Simulation studies of neuromuscular incapacitation using high-intensity electric pulses (EPs) were previously reported. These studies hypothesized that a reversible action potential (AP) block can be achieved based on energy deposition causing neuronal electroporation. However, theoretical concepts were presented without elaboration of specific details on possible biological mechanisms. Recently, we discovered that nanosecond pulsed electric fields (nsPEF) could initiate phosphatidylinositol-4,5-bisphosphate (PIP2) depletion in non-excitable cells. PIP2 is the precursor for important second messengers and is a key modulator of the neuronal ion channels involved in AP generation. By using primary hippocampal neurons (PHN) and the PLCδ-PH-EGFP optical probe of PIP2 hydrolysis, we demonstrated that electric field (EF) exposure induced PIP2 depletion in the PHN, and defined EF exposure parameters necessary to safely elicit reversible effects without neuronal damage. Results show that five days after neuronal dissociation (D5), the pre-exposure level of the cytoplasmic PLCδ-PH-EGFP fluorescence is significantly higher in D5 neurons than in D1 neurons, likely due to higher levels of tonic inositol1,4,5-trisphosphate (IP3). Such biological sensitization caused D5 neurons to respond intensely following a single 7.5 kV/cm 600 ns EP, while the D1 neurons did not respond. Despite age of development, the stronger 15 kV/cm 600 ns or longer 7.5 kV/cm 12 µs EPs initiated profound PIP2 depletion in all neurons studied, outlining direct impact on the neuronal plasma membrane during electroporation. Accordingly, D1 neurons exposed to such EPs had significant post-exposure propidium iodide (PI) uptake. In the more sensitive D5 neurons, PIP2 recovery was achieved within 10 min after all 600 ns EPs exposures, but 12 µs EPs caused irreversible PIP2 depletion. Thus, nsPEF-induced PIP2 depletion in neurons could be the primary biological mechanism responsible for both stimulation and latent inhibition of neuronal tissues.

Emerging data indicate that age-related brain changes alter seizure susceptibility, seizure-associated neurodegeneration, and responsiveness to AEDs. The present study assessed long-term animal survival in the Kainic Acid (KA) model along with in-vivo spontaneous seizure frequency, cellular hyperexcitability in CA1 in-vitro and in-vivo in subiculum, and responsiveness of in-vitro CA1 hyperexcitability to topiramate. Sprague-Dawley male rats were given KA to induce convulsive status epilepticus (KA-SE) at 2–3 months of age. The one-month mortality after KA-SE was 27%. One-month survivor rats had 37% sudden unexplained late mortality after KA-SE as compared to none in saline controls during their second year of life. In-vivo seizure frequency was examined prior to terminal experiments. The diurnal average seizure frequency in the KA-SE group at age 2 years was 1.06 ± 0.24 seizures/hour while no seizures were observed in the saline age-matched controls (p

Chronic exposure to intermittent hypoxia (CIH) has been used in animals to mimic the arterial hypoxemia that accompanies sleep apnea. Humans with sleep apnea and animals exposed to CIH have elevated blood pressures and augmented sympathetic nervous system responses to acute exposures to hypoxia. To test the hypothesis that exposure to CIH alters neurons within the nucleus of the solitary tract (NTS) that integrate arterial chemoreceptor afferent inputs, we measured whole cell currents induced by activation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and N-methyl-d-aspartate (NMDA) receptors in enzymatically dispersed NTS neurons from normoxic (NORM) and CIH-exposed rats (alternating cycles of 3 min at 10% O2 followed by 3 min at 21% O2 between 8 AM and 4 PM for 7 days). To identify NTS neurons receiving carotid body afferent inputs the anterograde tracer 4- (4-(dihexadecylamino)styryl- N-methylpyridinum iodide (DiA) was placed onto the carotid body 1 wk before exposure to CIH. AMPA dose-response curves had similar EC50 but maximal responses increased in neurons isolated from DiA-labeled CIH (20.1 ± 0.8 μM, n = 9) compared with NORM (6.0 ± 0.3 μM, n = 8) rats. NMDA dose-response curves also had similar EC50 but maximal responses decreased in CIH (8.4 ± 0.4 μM, n = 8) compared with NORM (19.4 ± 0.6 μM, n = 9) rats. These results suggest reciprocal changes in the number and/or conductance characteristics of AMPA and NMDA receptors. Enhanced responses to AMPA receptor activation could contribute to enhanced chemoreflex responses observed in animals exposed to CIH and humans with sleep apnea.

The inhibitory amino acid GABA is released within the nucleus of the solitary tract (NTS) during hypoxia and modulates the respiratory response to hypoxia. To determine if responses of NTS neurons to activation of GABA(A) receptors are altered following exposure to chronic hypoxia, GABA(A) receptor-evoked whole cell currents were measured in enzymatically dispersed NTS neurons from normoxic and chronic hypoxic rats. Chronic hypoxic rats were exposed to 10% O(2) for 9-12 days. Membrane capacitance was the same in neurons from normoxic (6.9+/-0.5 pF, n=16) and hypoxic (6.3+/-0.5 pF, n=15) rats. The EC(50) for peak GABA-evoked current density was significantly greater in neurons from hypoxic (21.7+/-2.2 microM) compared to normoxic rats (12.2+/-0.9 microM) (p

In anesthetized rats, increases in phrenic nerve amplitude and frequency during brief periods of hypoxia are followed by a reduction in phrenic nerve burst frequency [posthypoxia frequency decline (PHFD)]. We investigated the effects of chronic exposure to hypoxia on PHFD and on peripheral and central O2-sensing mechanisms. In Inactin-anesthetized (100 mg/kg) Sprague-Dawley rats, phrenic nerve discharge and arterial pressure responses to 10 s N2 inhalation were recorded after exposure to hypoxia (10 ± 0.5% O2) for 6-14 days. Compared with rats maintained at normoxia, PHFD was abolished in chronic hypoxic rats. Because of inhibition of PHFD, the increased phrenic burst frequency and amplitude after N2 inhalation persisted for 1.8-2.8 times longer in chronic hypoxic (70 s) compared with normoxic (25-40 s) rats ( P < 0.05). After acute bilateral carotid body denervation, N2 inhalation produced a short depression of phrenic nerve discharge in both chronic hypoxic and normoxic rats. However, the degree and duration of depression of phrenic nerve discharge was smaller in chronic hypoxic compared with normoxic rats ( P < 0.05). We conclude that after exposure to chronic hypoxia, a reduction in PHFD contributes to an increased duration of the acute hypoxic ventilatory response in anesthetized rats. Furthermore, after exposure to chronic hypoxia, the central network responsible for respiration is more resistant to the depressant effects of acute hypoxia in anesthetized rats.

Recognizing spatial relationships of neuro-degeneration in brains exposed to organic solvents is difficult when working from 2-dimensional serial slices. Recent advances in software have allowed the assembly of serial sections of stained tissue into a 3-dimensional (3D) representations. Appropriately chosen stains indicative of specific pathology can be highlighted and the 3D representation of its spatial distribution within the organ displayed. The purpose of this work was to develop a method for visualizing the spatial distribution of neuronal degeneration following organic solvent exposure. A cupric silver stain highly specific for degenerating neurons was used to identify neuronal degeneration in 83 serial histologic sections of brains of rodents exposed to toluene. Brain sections were scanned at 600 dpi using a grey-scale protocol. Scans were assembled into 3D images which were further processed into stereo pairs. Grey-scale scans were compared to the original sections in order to establish grey-scale ranges for healthy and damaged tissue and artifact staining. The respective categories then were assigned pseudo-colors to improve contrast.

Chronic wounds are a large part of daily medical practice, and are an increasing focus of research. As the number of modalities to treat chronic wounds increase, the validity of these modalities must be tested. A low cost laser imager was designed and fabricated for the measurement of wound geometry. The accuracy of the imager was validated using simulated wounds of known geometry. Perimeter, area, and volume measurements were compared with conventional measurement techniques on wound models simulated on plaster mockups of the lower leg and foot.