In situ electroporation of mammalian cells through SiO2 thin film capacitive microelectrodes.

Electroporation is a widely used non-viral technique for the delivery of molecules, including nucleic acids, into cells. Recently, electronic microsystems that miniaturize the electroporation machinery have been developed as a new tool for genetic manipulation of cells in vitro, by integrating metal microelectrodes in the culture substrate and enabling electroporation in-situ. We report that non-faradic SiO2 thin film-insulated microelectrodes can be used for reliable and spatially selective in-situ electroporation of mammalian cells. CHO-K1 and SH-SY5Y cell lines and primary neuronal cultures were electroporated by application of short and low amplitude voltage transients leading to cell electroporation by capacitive currents. We demonstrate reliable delivery of DNA plasmids and exogenous gene expression, accompanied by high spatial selectivity and cell viability, even with differentiated neurons. Finally, we show that SiO2 thin film-insulated microelectrodes support a double and serial transfection of the targeted cells.

Electromagnetic field affects the voltage-dependent potassium channel Kv1.3.

PURPOSE: Theoretical and experimental evidences support the hypothesis that Extremely Low-Frequency Electromagnetic Fields (ELF-EMF) can modulate voltage-gated channels. In this work we investigated the effect of ELF-EMF on Kv1.3, a member of the family of the voltage-gated potassium channels that is thought to be involved in key physiological functions, including the regulation of T-cells activation during the immune response. MATERIALS AND METHODS: Kv1.3 expressing CHO-K1 cells were exposed to a 20 Hz electromagnetic field at two different intensities: 268 µT and 902 µT. Kv1.3 potassium currents were recorded by whole-cell patch-clamp before, during and after field exposure. RESULTS: We found that the Kv1.3 current was increased significantly by the ELF-EMF in a subpopulation of CHO-K1 cells. The increase developed after a few seconds from the start of exposure, reached a steady-state and took several minutes to return to the baseline after field removal. CONCLUSIONS: These findings suggest that Kv1.3 may mediate interactions between ELF-EMF and living cells, disclosing new research opportunities on the molecular mechanisms with which electromagnetic fields affect physiological and pathological processes, including immunomodulation, inflammation and cancer.

Automated analysis of local field potentials evoked by mechanical whisker stimulation in rat barrel cortex.

Local field potentials (LFPs) recorded in the barrel cortex in rats and mice are important to investigate somatosensory systems, the final aim being to start to understand mechanisms of brain representation of sensory stimuli in humans. Parameters extracted from LFP of particular interest include spike timing and transmembrane current flow. Recent improvements in microelectrodes technology have enabled neuroscientists to acquire a great amount of LFP signals during the same experimental session, calling for the development of algorithms for their quantitative automatic analysis. In the present work, an algorithm based on Phillips-Tikhonov regularization is presented to automatically detect the main features (in terms of amplitude and latency) of LFP waveforms recorded after whisker stimulation in rat. The accuracy of the algorithm is first assessed in a Monte Carlo simulation mimicking the acquisition of LFP in three different conditions of SNR. Then, the algorithm is tested by analyzing a set of 100 LFP recorded in the primary somatosensory (S1) cortex, i.e., the region involved in the cortical representation of touch in mammals.

Stimulation of Ca²+ signals in neurons by electrically coupled electrolyte-oxide-semiconductor capacitors.

Electrolyte-oxide-semiconductor capacitors (EOSCs) are a class of microtransducers for extracellular electrical stimulation that have been successfully employed to activate voltage-dependent sodium channels at the neuronal soma to generate action potentials in vitro. In the present work, we report on their use to control Ca²+ signalling in cultured mammalian cells, including neurons. Evidence is provided that EOSC stimulation with voltage waveforms in the microsecond or nanosecond range activates two distinct Ca²+ pathways, either by triggering Ca²+ entry through the plasma membrane or its release from intracellular stores. Ca²+ signals were activated in non-neuronal and neuronal cell lines, CHO-K1 and SH-SY5Y. On this basis, stimulation was tailored to rat and bovine neurons to mimic physiological somatic Ca²+ transients evoked by glutamate. Being minimally invasive and easy to use, the new method represents a versatile complement to standard electrophysiology and imaging techniques for the investigation of Ca²+ signalling in dissociated primary neurons and cell lines.

Mechanical and electrophysiological properties of the sarcolemma of muscle fibers in two murine models of muscle dystrophy: col6a1-/- and mdx.

This study aimed to analyse the sarcolemma of Col6a1-/- fibers in comparison with wild type and mdx fibers, taken as positive control in view of the known structural and functional alterations of their membranes. Structural and mechanical properties were studied in single muscle fibers prepared from FDB muscle using atomic force microscopy (AFM) and conventional electrophysiological techniques to measure ionic conductance and capacitance. While the sarcolemma topography was preserved in both types of dystrophic fibers, membrane elasticity was significantly reduced in Col6a1-/- and increased in mdx fibers. In the membrane of Col6a1-/- fibers ionic conductance was increased likely due to an increased leakage, whereas capacitance was reduced, and the action potential (ap) depolarization rate was reduced. The picture emerging from experiments on fibers in culture was consistent with that obtained on intact freshly dissected muscle. Mdx fibers in culture showed a reduction of both membrane conductance and capacitance. In contrast, in mdx intact FDB muscle resting conductance was increased while resting potential and ap depolarization rate were reduced, likely indicating the presence of a consistent population of severely altered fibers which disappear during the culture preparation.

Space and time-resolved gene expression experiments on cultured mammalian cells by a single-cell electroporation microarray.

Single-cell experiments represent the next frontier for biochemical and gene expression research. Although bulk-scale methods averaging populations of cells have been traditionally used to investigate cellular behavior, they mask individual cell features and can lead to misleading or insufficient biological results. We report on a single-cell electroporation microarray enabling the transfection of pre-selected individual cells at different sites within the same culture (space-resolved), at arbitrarily chosen time points and even sequentially to the same cells (time-resolved). Delivery of impermeant molecules by single-cell electroporation was first proven to be finely tunable by acting on the electroporation protocol and then optimized for transfection of nucleic acids into Chinese Hamster Ovary (CHO-K1) cells. We focused on DNA oligonucleotides (ODNs), short interfering RNAs (siRNAs), and DNA plasmid vectors, thus providing a versatile and easy-to-use platform for time-resolved gene expression experiments in single mammalian cells.

A potential role for the vanilloid receptor TRPV1 in the therapeutic effect of curcumin in dinitrobenzene sulphonic acid-induced colitis in mice.

A protective role of the transient potential vanilloid receptor 1 (TRPV1) in intestinal inflammation induced by dinitrobenzene sulphonic acid (DNBS) has been recently demonstrated. Curcumin, the major active component of turmeric, is also able to prevent and ameliorate the severity of the damage in DNBS-induced colitis. We evaluated the possibility that curcumin (45 mg kg(-1) day p.o. for 2 days before and 5 days after the induction of colitis) was able to reduce DNBS-induced colitis in mice, by acting as a TRPV1 agonist. Macroscopic damage score, histological damage score and colonic myeloperoxidase (MPO) activity were significantly lower (by 71%, 65% and 73%, respectively; P < 0.01), in animals treated with curcumin compared with untreated animals. Capsazepine (30 mg kg(-1), i.p.), a TRPV1 receptor antagonist, completely abolished the protective effects of curcumin. To extend these data in vitro, Xenopus oocytes expressing rat TRPV1 were examined. Capsaicin-evoked currents (3.3 micromol L(-1)) disappeared subsequent either to removal of the agonist or subsequent to the addition of capsazepine. However, curcumin (30 micromol L(-1)) was ineffective both as regard direct modification of cell membrane currents and as regard interference with capsaicin-mediated effects. As sensitization of the TRPV1 receptor by mediators of inflammation in damaged tissues has been shown previously, our results suggest that in inflamed, but not in normal tissue, curcumin can interact with the TRPV1 receptor to mediate its protective action in DNBS-induced colitis.

Dynamic localization and clustering of dendritic Kv2.1 voltage-dependent potassium channels in developing hippocampal neurons.

Dendritic excitability is modulated by the highly variable spatial and temporal expression pattern of voltage-dependent potassium channels. Somatodendritic Kv2.1 channels contribute a major component of delayed rectifier potassium current in cultured hippocampal neurons, where Kv2.1 is localized to large clusters on the soma and proximal dendrites. Here we found that dramatic differences exist in the clustering of endogenous Kv2.1 in cultured rat hippocampal GABAergic interneurons and glutamatergic pyramidal neurons. Studies on neurons developing in culture revealed that while a similar sequence of Kv2.1 localization and clustering occurred in both cell types, the process was temporally delayed in pyramidal cells. Localization and clustering of recombinant green fluorescent protein-tagged Kv2.1 occurred by the same sequence of events, and imaging of GFP-Kv2.1 clustering in living neurons revealed dynamic fusion events that underlie cluster formation. Overexpression of GFP-Kv2.1 accelerated the clustering program in pyramidal neurons such that the observed differences in Kv2.1 clustering in pyramidal neurons and interneurons were eliminated. Confocal imaging showed a preferential association of Kv2.1 with the basal membrane in cultured neurons, and electrophysiological recordings from neurons cultured on transistors revealed that Kv2.1 contributed the bulk of a previously described adherens junction delayed rectifier potassium conductance. Finally, Kv2.1 clusters were found spatially associated with ryanodine receptor intracellular Ca(2+) ([Ca(2+)](i)) release channels. These findings reveal a stepwise assembly of Kv2.1 potassium channels into membrane clusters during development, and an association of these clusters with Ca(2+) signaling apparatus. Together these data suggest that the restricted localization of Kv2.1 may play an important role in the previously observed contribution of this potassium channel in regulating dendritic [Ca(2+)](i) transients.

Transistor probes local potassium conductances in the adhesion region of cultured rat hippocampal neurons.

Adhesion interactions of neurons in a tissue may affect the ion conductance of the plasma membrane, inducing selective localization and modulation of channels. We studied the adhesion region of cultured neurons from rat hippocampus as a defined model where such effects could be observed electrophysiologically, taking advantage of extracellular recording by a transistor integrated in the substrate. We observed the K(+) current through the region of soma adhesion under voltage-clamp and compared it with the current through the whole cell. We found that the specific A-type conductance was depleted, even completely, in the region of adhesion, whereas the specific K-type conductance was enhanced up to a factor of 12. The electrophysiological approach opens a new way to investigate targeting of ion channels in the cell membrane as a function of adhesion processes.

On the mechanism of fatty acid-induced proton transport by mitochondrial uncoupling protein.

Uncoupling protein mediates electrophoretic transport of protons and anions across the inner membrane of brown adipose tissue mitochondria. The mechanism and site of proton transport, the mechanism by which fatty acids activate proton transport, and the relationship between fatty acids and anion transport are unknown. We used fluorescent probes to measure H+ and anion transport in vesicles reconstituted with purified uncoupling protein and carried out a comparative study of the effects of laurate and its close analogue, undecanesulfonate. Undecanesulfonate was transported by uncoupling protein with a Km value similar to that observed for laurate as it activated H+ transport. Both laurate and undecanesulfonate inhibited Cl- with competitive kinetics. Undecanesulfonate inhibited laurate-induced H+ transport with competitive kinetics. Undecanesulfonate and laurate differed in two important respects. (i) Laurate caused uncoupling protein-mediated H+ transport, whereas undecanesulfonate did not. (ii) Lauric acid was rapidly transported across the bilayer by nonionic diffusion, whereas undecanesulfonic was not. We infer that the role of uncoupling protein in H+ transport is to transport fatty acid anions and that fatty acids induce H+ transport because they can diffuse electroneutrally across the membrane. According to this hypothesis, uncoupling protein is a pure anion porter and does not transport protons; rather it is designed to enable fatty acids to behave as cycling protonophores.

Modulation of the mitochondrial permeability transition pore. Effect of protons and divalent cations.

We have studied the induction of the mitochondrial cyclosporin A-sensitive permeability transition pore (PTP) by the bifunctional SH group reagent phenylarsine oxide (PhAsO). Addition of nanomolar concentrations of the electroneutral H(+)-K+ ionophore nigericin to nonrespiring mitochondria in sucrose medium determines a dramatic increase of the time required for PTP induction by PhAsO, while no effect of nigericin is apparent in KCl medium. Using mitochondria loaded with the internal pH indicator 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein, we show that the effect of nigericin is mediated by the ionophore-induced acidification of matrix pH. Indeed, experimental manipulation of pHi by a number of treatments indicates that PTP induction is directly related to matrix pH, in that the PTP induction process becomes slower as pHi decreases at constant pHo. PTP induction by PhAsO in respiration-inhibited mitochondria is stimulated by Ca2+ and inhibited by a series of divalent cations. Since PhAsO induces the PTP even in the presence of excess EGTA and in the absence of respiration (Lenartowicz, E., Bernardi, P., and Azzone, G.F. (1991) J. Bioenerg. Biomembr. 23, 679-688), we have been able to study the Ca2+ dependence of the induction process. We show that the apparent Km for Ca2+ activation is about 10(-5) M and that Ca2+, cyclosporin A, and inhibitory Me2+ ions behave as if they were competing for the same binding site(s) on the pore. Since similar results are obtained from patch-clamp experiments on the mitochondrial megachannel (Szabó, I., Bernardi, P., and Zoratti, M. (1992) J. Biol. Chem. 267, 2940-2946), we suggest that (i) the PTP and the mitochondrial megachannel are the same molecular structures and (ii) the same factors affect both the process of pore induction and its open-closed orientation.