Industrializing electrophysiology: HT automated patch clamp on SyncroPatch® 96 using instant frozen cells.

PubMed

Polonchuk, Liudmila

2014-01-01

Patch-clamping is a powerful technique for investigating the ion channel function and regulation. However, its low throughput hampered profiling of large compound series in early drug development. Fortunately, automation has revolutionized the area of experimental electrophysiology over the past decade. Whereas the first automated patch-clamp instruments using the planar patch-clamp technology demonstrated rather a moderate throughput, few second-generation automated platforms recently launched by various companies have significantly increased ability to form a high number of high-resistance seals. Among them is SyncroPatch(®) 96 (Nanion Technologies GmbH, Munich, Germany), a fully automated giga-seal patch-clamp system with the highest throughput on the market. By recording from up to 96 cells simultaneously, the SyncroPatch(®) 96 allows to substantially increase throughput without compromising data quality. This chapter describes features of the innovative automated electrophysiology system and protocols used for a successful transfer of the established hERG assay to this high-throughput automated platform.

Sperm Patch-Clamp

PubMed Central

Lishko, Polina; Clapham, David E.; Navarro, Betsy; Kirichok, Yuriy

2014-01-01

Sperm intracellular pH and calcium concentration ([Ca2+]i) are two central factors that control sperm activity within the female reproductive tract. As such, the ion channels of the sperm plasma membrane that alter intracellular sperm [Ca2+] and pH play important roles in sperm physiology and the process of fertilization. Indeed, sperm ion channels regulate sperm motility, control sperm chemotaxis toward the egg in some species, and may trigger the acrosome reaction. Until recently, our understanding of these important molecules was rudimentary due to the inability to patch-clamp spermatozoa and directly record the activity of these ion channels under voltage clamp. Recently, we overcame this technical barrier and developed a method for reproducible application of the patch-clamp technique to mouse and human spermatozoa. This chapter covers important aspects of application of the patch-clamp technique to spermatozoa, such as selection of the electrophysiological equipment, isolation of spermatozoa for patch-clamp experiments, formation of the gigaohm seal with spermatozoa, and transition into the whole-cell mode of recording. We also discuss potential pitfalls in application of the patch-clamp technique to flagellar ion channels. PMID:23522465

Micromolded PDMS planar electrode allows patch clamp electrical recordings from cells.

PubMed

Klemic, Kathryn G; Klemic, James F; Reed, Mark A; Sigworth, Fred J

2002-06-01

The patch clamp method measures membrane currents at very high resolution when a high-resistance 'gigaseal' is established between the glass microelectrode and the cell membrane (Pflugers Arch. 391 (1981) 85; Neuron 8 (1992) 605). Here we describe the first use of the silicone elastomer, poly(dimethylsiloxane) (PDMS), for patch clamp electrodes. PDMS is an attractive material for patch clamp recordings. It has low dielectric loss and can be micromolded (Annu. Rev. Mat. Sci. 28 (1998) 153) into a shape that mimics the tip of the glass micropipette. Also, the surface chemistry of PDMS may be altered to mimic the hydrophilic nature of glass (J. Appl. Polym. Sci. 14 (1970) 2499; Annu. Rev. Mat. Sci. 28 (1998) 153), thereby allowing a high-resistance seal to a cell membrane. We present a planar electrode geometry consisting of a PDMS partition with a small aperture sealed between electrode and bath chambers. We demonstrate that a planar PDMS patch electrode, after oxidation of the elastomeric surface, permits patch clamp recording on Xenopus oocytes. Our results indicate the potential for high-throughput patch clamp recording with a planar array of PDMS electrodes.

Patch-clamp amplifiers on a chip

PubMed Central

Weerakoon, Pujitha; Culurciello, Eugenio; Yang, Youshan; Santos-Sacchi, Joseph; Kindlmann, Peter J.; Sigworth, Fred J.

2010-01-01

We present the first, fully-integrated, two-channel implementation of a patch-clamp measurement system. With this “PatchChip” two simultaneous whole-cell recordings can be obtained with rms noise of 8 pA in a 10 kHz bandwidth. The capacitance and series-resistance of the electrode can be compensated up to 10 pF and 100 MΩ respectively under computer control. Recordings of hERG and Nav 1.7 currents demonstrate the system's capabilities, which are on par with large, commercial patch-clamp instrumentation. By reducing patch-clamp amplifiers to a millimeter size micro-chip, this work paves the way to the realization of massively-parallel, high-throughput patch-clamp systems for drug screening and ion-channel research. The PatchChip is implemented in a 0.5 μm silicon-on-sapphire process; its size is 3 × 3 mm2 and the power consumption is 5 mW per channel with a 3.3 V power supply. PMID:20637803

Catch and Patch: A Pipette-Based Approach for Automating Patch Clamp That Enables Cell Selection and Fast Compound Application.

PubMed

Danker, Timm; Braun, Franziska; Silbernagl, Nikole; Guenther, Elke

2016-03-01

Manual patch clamp, the gold standard of electrophysiology, represents a powerful and versatile toolbox to stimulate, modulate, and record ion channel activity from membrane fragments and whole cells. The electrophysiological readout can be combined with fluorescent or optogenetic methods and allows for ultrafast solution exchanges using specialized microfluidic tools. A hallmark of manual patch clamp is the intentional selection of individual cells for recording, often an essential prerequisite to generate meaningful data. So far, available automation solutions rely on random cell usage in the closed environment of a chip and thus sacrifice much of this versatility by design. To parallelize and automate the traditional patch clamp technique while perpetuating the full versatility of the method, we developed an approach to automation, which is based on active cell handling and targeted electrode placement rather than on random processes. This is achieved through an automated pipette positioning system, which guides the tips of recording pipettes with micrometer precision to a microfluidic cell handling device. Using a patch pipette array mounted on a conventional micromanipulator, our automated patch clamp process mimics the original manual patch clamp as closely as possible, yet achieving a configuration where recordings are obtained from many patch electrodes in parallel. In addition, our implementation is extensible by design to allow the easy integration of specialized equipment such as ultrafast compound application tools. The resulting system offers fully automated patch clamp on purposely selected cells and combines high-quality gigaseal recordings with solution switching in the millisecond timescale.

Implementing Patch Clamp and Live Fluorescence Microscopy to Monitor Functional Properties of Freshly Isolated PKD Epithelium

PubMed Central

Pavlov, Tengis S.; Ilatovskaya, Daria V.; Palygin, Oleg; Levchenko, Vladislav; Pochynyuk, Oleh; Staruschenko, Alexander

2015-01-01

Cyst initiation and expansion during polycystic kidney disease is a complex process characterized by abnormalities in tubular cell proliferation, luminal fluid accumulation and extracellular matrix formation. Activity of ion channels and intracellular calcium signaling are key physiologic parameters which determine functions of tubular epithelium. We developed a method suitable for real-time observation of ion channels activity with patch-clamp technique and registration of intracellular Ca2+ level in epithelial monolayers freshly isolated from renal cysts. PCK rats, a genetic model of autosomal recessive polycystic kidney disease (ARPKD), were used here for ex vivo analysis of ion channels and calcium flux. Described here is a detailed step-by-step procedure designed to isolate cystic monolayers and non-dilated tubules from PCK or normal Sprague Dawley (SD) rats, and monitor single channel activity and intracellular Ca2+ dynamics. This method does not require enzymatic processing and allows analysis in a native setting of freshly isolated epithelial monolayer. Moreover, this technique is very sensitive to intracellular calcium changes and generates high resolution images for precise measurements. Finally, isolated cystic epithelium can be further used for staining with antibodies or dyes, preparation of primary cultures and purification for various biochemical assays. PMID:26381526

Murine epithelial cells: isolation and culture.

PubMed

Davidson, Donald J; Gray, Michael A; Kilanowski, Fiona M; Tarran, Robert; Randell, Scott H; Sheppard, David N; Argent, Barry E; Dorin, Julia R

2004-08-01

We describe an air-liquid interface primary culture method for murine tracheal epithelial cells on semi-permeable membranes, forming polarized epithelia with a high transepithelial resistance, differentiation to ciliated and secretory cells, and physiologically appropriate expression of key genes and ion channels. We also describe the isolation of primary murine nasal epithelial cells for patch-clamp analysis, generating polarised cells with physiologically appropriate distribution and ion channel expression. These methods enable more physiologically relevant analysis of murine airway epithelial cells in vitro and ex vivo, better utilisation of transgenic mouse models of human pulmonary diseases, and have been approved by the European Working Group on CFTR expression.

Effects of Tramadol on Substantia Gelatinosa Neurons in the Rat Spinal Cord: An In Vivo Patch-Clamp Analysis

PubMed Central

Yamasaki, Hiroyuki; Funai, Yusuke; Funao, Tomoharu; Mori, Takashi; Nishikawa, Kiyonobu

2015-01-01

Tramadol is thought to modulate synaptic transmissions in the spinal dorsal horn mainly by activating µ-opioid receptors and by inhibiting the reuptake of monoamines in the CNS. However, the precise mode of modulation remains unclear. We used an in vivo patch clamp technique in urethane-anesthetized rats to determine the antinociceptive mechanism of tramadol. In vivo whole-cell recordings of spontaneous inhibitory postsynaptic currents (sIPSCs) and spontaneous excitatory postsynaptic currents (sEPSCs) were made from substantia gelatinosa (SG) neurons (lamina II) at holding potentials of 0 mV and -70 mV, respectively. The effects of intravenous administration (0.5, 5, 15 mg/kg) of tramadol were evaluated. The effects of superfusion of tramadol on the surface of the spinal cord and of a tramadol metabolite (M1) were further analyzed. Intravenous administration of tramadol at doses >5 mg/kg decreased the sEPSCs and increased the sIPSCs in SG neurons. These effects were not observed following naloxone pretreatment. Tramadol superfusion at a clinically relevant concentration (10 µM) had no effect, but when administered at a very high concentration (100 µM), tramadol decreased sEPSCs, produced outward currents, and enhanced sIPSCs. The effects of M1 (1, 5 mg/kg intravenously) on sEPSCs and sIPSCs were similar to those of tramadol at a corresponding dose (5, 15 mg/kg). The present study demonstrated that systemically administered tramadol indirectly inhibited glutamatergic transmission, and enhanced GABAergic and glycinergic transmissions in SG neurons. These effects were mediated primarily by the activation of μ-opioid receptors. M1 may play a key role in the antinociceptive mechanisms of tramadol. PMID:25933213

Preparation of Drosophila central neurons for in situ patch clamping.

PubMed

Ryglewski, Stefanie; Duch, Carsten

2012-10-15

Short generation times and facile genetic techniques make the fruit fly Drosophila melanogaster an excellent genetic model in fundamental neuroscience research. Ion channels are the basis of all behavior since they mediate neuronal excitability. The first voltage gated ion channel cloned was the Drosophila voltage gated potassium channel Shaker(1,2). Toward understanding the role of ion channels and membrane excitability for nervous system function it is useful to combine powerful genetic tools available in Drosophila with in situ patch clamp recordings. For many years such recordings have been hampered by the small size of the Drosophila CNS. Furthermore, a robust sheath made of glia and collagen constituted obstacles for patch pipette access to central neurons. Removal of this sheath is a necessary precondition for patch clamp recordings from any neuron in the adult Drosophila CNS. In recent years scientists have been able to conduct in situ patch clamp recordings from neurons in the adult brain(3,4) and ventral nerve cord of embryonic(5,6), larval(7,8,9,10), and adult Drosophila(11,12,13,14). A stable giga-seal is the main precondition for a good patch and depends on clean contact of the patch pipette with the cell membrane to avoid leak currents. Therefore, for whole cell in situ patch clamp recordings from adult Drosophila neurons must be cleaned thoroughly. In the first step, the ganglionic sheath has to be treated enzymatically and mechanically removed to make the target cells accessible. In the second step, the cell membrane has to be polished so that no layer of glia, collagen or other material may disturb giga-seal formation. This article describes how to prepare an identified central neuron in the Drosophila ventral nerve cord, the flight motoneuron 5 (MN5(15)), for somatic whole cell patch clamp recordings. Identification and visibility of the neuron is achieved by targeted expression of GFP in MN5. We do not aim to explain the patch clamp technique itself.

HTS techniques for patch clamp-based ion channel screening - advances and economy.

PubMed

Farre, Cecilia; Fertig, Niels

2012-06-01

Ten years ago, the first publication appeared showing patch clamp recordings performed on a planar glass chip instead of using a conventional patch clamp pipette. "Going planar" proved to revolutionize ion channel drug screening as we know it, by allowing high quality measurements of ion channels and their effectors at a higher throughput and at the same time de-skilling the highly laborious technique. Over the years, platforms evolved in response to user requirements regarding experimental features, data handling plus storage, and suitable target diversity. This article gives a snapshot image of patch clamp-based ion channel screening with focus on platforms developed to meet requirements of high-throughput screening environments. The commercially available platforms are described, along with their benefits and drawbacks in ion channel drug screening. Automated patch clamp (APC) platforms allow faster investigation of a larger number of ion channel active compounds or cell clones than previously possible. Since patch clamp is the only method allowing direct, real-time measurements of ion channel activity, APC holds the promise of picking up high quality leads, where they otherwise would have been overseen using indirect methods. In addition, drug candidate safety profiling can be performed earlier in the drug discovery process, avoiding late-phase compound withdrawal due to safety liability issues, which is highly costly and inefficient.

Integration of autopatching with automated pipette and cell detection in vitro

PubMed Central

Wu (吴秋雨), Qiuyu; Kolb, Ilya; Callahan, Brendan M.; Su, Zhaolun; Stoy, William; Kodandaramaiah, Suhasa B.; Neve, Rachael; Zeng, Hongkui; Boyden, Edward S.; Forest, Craig R.

2016-01-01

Patch clamp is the main technique for measuring electrical properties of individual cells. Since its discovery in 1976 by Neher and Sakmann, patch clamp has been instrumental in broadening our understanding of the fundamental properties of ion channels and synapses in neurons. The conventional patch-clamp method requires manual, precise positioning of a glass micropipette against the cell membrane of a visually identified target neuron. Subsequently, a tight “gigaseal” connection between the pipette and the cell membrane is established, and suction is applied to establish the whole cell patch configuration to perform electrophysiological recordings. This procedure is repeated manually for each individual cell, making it labor intensive and time consuming. In this article we describe the development of a new automatic patch-clamp system for brain slices, which integrates all steps of the patch-clamp process: image acquisition through a microscope, computer vision-based identification of a patch pipette and fluorescently labeled neurons, micromanipulator control, and automated patching. We validated our system in brain slices from wild-type and transgenic mice expressing channelrhodopsin 2 under the Thy1 promoter (line 18) or injected with a herpes simplex virus-expressing archaerhodopsin, ArchT. Our computer vision-based algorithm makes the fluorescent cell detection and targeting user independent. Compared with manual patching, our system is superior in both success rate and average trial duration. It provides more reliable trial-to-trial control of the patching process and improves reproducibility of experiments. PMID:27385800

Giga-seal formation alters properties of sodium channels of human myoballs.

PubMed

Fahlke, C; Rüdel, R

1992-03-01

The influence of giga-seal formation on the properties of the Na+ channels within the covered membrane patch was investigated with a whole-cell pipette and a patch pipette applied to the same cell. Current kinetics, current/voltage relation and channel densities were determined in three combinations: (i) voltage-clamping and current recording with the whole-cell pipette, (ii) voltage-clamping with the whole-cell pipette and current recording with the patch pipette and, (iii) voltage-clamping and current recording with the patch pipette. The Hodgkin-Huxley (1952) parameters tau m and tau h were smaller for the patch currents than for the whole cell, and the h infinity curve was shifted in the negative direction. The channel density was of the order of 10 times smaller. All effects were independent of the extracellular Ca2+ concentration. The capacitive current generated in the patch by the whole-cell Na+ current and its effect on the transmembrane voltage of the patch were evaluated. The kinetic parameters of the Na+ channels in the patch did not depend on whether the voltage was clamped with the whole-cell pipette or the patch pipette. Thus, the results are not due to spurious voltage.

Digital PCR to determine the number of transcripts from single neurons after patch-clamp recording.

PubMed

Faragó, Nóra; Kocsis, Ágnes K; Lovas, Sándor; Molnár, Gábor; Boldog, Eszter; Rózsa, Márton; Szemenyei, Viktor; Vámos, Enikő; Nagy, Lajos I; Tamás, Gábor; Puskás, László G

2013-06-01

Whole-cell patch-clamp recording enables detection of electrophysiological signals from single neurons as well as harvesting of perisomatic RNA through the patch pipette for subsequent gene expression analysis. Amplification and profiling of RNA with traditional quantitative real-time PCR (qRT-PCR) do not provide exact quantitation due to experimental variation caused by the limited amount of nucleic acid in a single cell. Here we describe a protocol for quantifying mRNA or miRNA expression in individual neurons after patch-clamp recording using high-density nanocapillary digital PCR (dPCR). Expression of a known cell-type dependent marker gene (gabrd), as well as oxidative-stress related induction of hspb1 and hmox1 expression, was quantified in individual neurogliaform and pyramidal cells, respectively. The miRNA mir-132, which plays a role in neurodevelopment, was found to be equally expressed in three different types of neurons. The accuracy and sensitivity of this method were further validated using synthetic spike-in templates and by detecting genes with very low levels of expression.

An optogenetics- and imaging-assisted simultaneous multiple patch-clamp recording system for decoding complex neural circuits

PubMed Central

Wang, Guangfu; Wyskiel, Daniel R; Yang, Weiguo; Wang, Yiqing; Milbern, Lana C; Lalanne, Txomin; Jiang, Xiaolong; Shen, Ying; Sun, Qian-Quan; Zhu, J Julius

2015-01-01

Deciphering neuronal circuitry is central to understanding brain function and dysfunction, yet it remains a daunting task. To facilitate the dissection of neuronal circuits, a process requiring functional analysis of synaptic connections and morphological identification of interconnected neurons, we present here a method for stable simultaneous octuple patch-clamp recordings. This method allows physiological analysis of synaptic interconnections among 4–8 simultaneously recorded neurons and/or 10–30 sequentially recorded neurons, and it allows anatomical identification of >85% of recorded interneurons and >99% of recorded principal neurons. We describe how to apply the method to rodent tissue slices; however, it can be used on other model organisms. We also describe the latest refinements and optimizations of mechanics, electronics, optics and software programs that are central to the realization of a combined single- and two-photon microscopy–based, optogenetics- and imaging-assisted, stable, simultaneous quadruple–viguple patch-clamp recording system. Setting up the system, from the beginning of instrument assembly and software installation to full operation, can be completed in 3–4 d. PMID:25654757

The Nano-Patch-Clamp Array: Microfabricated Glass Chips for High-Throughput Electrophysiology

NASA Astrophysics Data System (ADS)

Fertig, Niels

2003-03-01

Electrophysiology (i.e. patch clamping) remains the gold standard for pharmacological testing of putative ion channel active drugs (ICADs), but suffers from low throughput. A new ion channel screening technology based on microfabricated glass chip devices will be presented. The glass chips contain very fine apertures, which are used for whole-cell voltage clamp recordings as well as single channel recordings from mammalian cell lines. Chips containing multiple patch clamp wells will be used in a first bench-top device, which will allow perfusion and electrical readout of each well. This scalable technology will allow for automated, rapid and parallel screening on ion channel drug targets.

Outside-out "sniffer-patch" clamp technique for in situ measures of neurotransmitter release.

PubMed

Muller-Chrétien, Emilie

2014-01-01

The mechanism underlying neurotransmitter release is a critical research domain for the understanding of neuronal network function; however, few techniques are available for the direct detection and measurement of neurotransmitter release. To date, the sniffer-patch clamp technique is mainly used to investigate these mechanisms from individual cultured cells. In this study, we propose to adapt the sniffer-patch clamp technique to in situ detection of neurosecretion. Using outside-out patches from donor cells as specific biosensors plunged in acute cerebral slices, this technique allows for proper detection and quantification of neurotransmitter release at the level of the neuronal network.

Lipid-glass adhesion in giga-sealed patch-clamped membranes.

PubMed

Opsahl, L R; Webb, W W

1994-01-01

Adhesion between patch-clamped lipid membranes and glass micropipettes is measured by high contrast video imaging of the mechanical response to the application of suction pressure across the patch. The free patch of membrane reversibly alters both its contact angle and radius of curvature on pressure changes. The assumption that an adhesive force between the membrane and the pipette can sustain normal tension up to a maximum Ta at the edge of the free patch accounts for the observed mechanical responses. When the normal component of the pressure-induced membrane tension exceeds Ta membrane at the contact point between the free patch and the lipid-glass interface is pulled away from the pipette wall, resulting in a decreased radius of curvature for the patch and an increased contact angle. Measurements of the membrane radius of curvature as a function of the suction pressure and pipette radius determine line adhesion tensions Ta which range from 0.5 to 4.0 dyn/cm. Similar behavior of patch-clamped cell membranes implies similar adhesion mechanics.

A hydrophilic polymer based microfluidic system with planar patch clamp electrode array for electrophysiological measurement from cells.

PubMed

Xu, Baojian; Ye, WeiWei; Zhang, Yu; Shi, JingYu; Chan, ChunYu; Yao, XiaoQiang; Yang, Mo

2014-03-15

This paper presents a microfluidic planar patch clamp system based on a hydrophilic polymer poly(ethylene glycol) diacrylate (PEGDA) for whole cell current recording. The whole chip is fabricated by UV-assisted molding method for both microfluidic channel structure and planar electrode partition. This hydrophilic patch clamp chip has demonstrated a relatively high gigaseal success rate of 44% without surface modification compared with PDMS based patch clamp devices. This chip also shows a capability of rapid intracellular and extracellular solution exchange with high stability of gigaseals. The capillary flow kinetic experiments demonstrate that the flow rates of PEGDA microfluidic channels are around two orders of magnitude greater than those for PDMS-glass channels with the same channel dimensions. This hydrophilic polymer based patch clamp chips have significant advantages over current PDMS elastomer based systems such as no need for surface modification, much higher success rate of cell gigaseals and rapid solution exchange with stable cell gigaseals. Our results indicate the potential of these devices to serve as useful tools for pharmaceutical screening and biosensing tasks. © 2013 Elsevier B.V. All rights reserved.

Cocaine acute "binge" administration results in altered thalamocortical interactions in mice.

PubMed

Urbano, Francisco J; Bisagno, Verónica; Wikinski, Silvia I; Uchitel, Osvaldo D; Llinás, Rodolfo R

2009-10-15

Abnormalities in both thalamic and cortical areas have been reported in human cocaine addicts with noninvasive functional magnetic resonance imaging. Given the substantial involvement of the thalamocortical system in sensory processing and perception, we defined electrophysiology-based protocols to attempt a characterization of cocaine effects on thalamocortical circuits. Thalamocortical function was studied in vivo and in vitro in mice after cocaine "binge" administration. In vivo awake electroencephalography (EEG) was implemented in mice injected with saline, 1 hour or 24 hours after the last cocaine "binge" injection. In vitro current- and voltage-clamp whole-cell patch-clamp recordings were performed from slices including thalamic relay ventrobasal (VB) neurons. In vivo EEG recordings after cocaine "binge" administration showed a significant increment, compared with saline, in low frequencies while observing no changes in high-frequency gamma activity. In vitro patch recordings from VB neurons after cocaine "binge" administration showed low threshold spikes activation at more negative membrane potentials and increments in both I(h) and low voltage activated T-type calcium currents. Also, a 10-mV negative shift on threshold activation level of T-type current and a remarkable increment in both frequency and amplitudes of gamma-aminobutyric acid-A-mediated minis were observed. Our data indicate that thalamocortical dysfunctions observed in cocaine abusers might be due to two distinct but additive events: 1) increased low frequency oscillatory thalamocortical activity, and 2) overinhibition of VB neurons that can abnormally "lock" the whole thalamocortical system at low frequencies.

A laser microsurgical method of cell wall removal allows detection of large-conductance ion channels in the guard cell plasma membrane

NASA Technical Reports Server (NTRS)

Miedema, H.; Henriksen, G. H.; Assmann, S. M.; Evans, M. L. (Principal Investigator)

1999-01-01

Application of patch clamp techniques to higher-plant cells has been subject to the limitation that the requisite contact of the patch electrode with the cell membrane necessitates prior enzymatic removal of the plant cell wall. Because the wall is an integral component of plant cells, and because cell-wall-degrading enzymes can disrupt membrane properties, such enzymatic treatments may alter ion channel behavior. We compared ion channel activity in enzymatically isolated protoplasts of Vicia faba guard cells with that found in membranes exposed by a laser microsurgical technique in which only a tiny portion of the cell wall is removed while the rest of the cell remains intact within its tissue environment. "Laser-assisted" patch clamping reveals a new category of high-conductance (130 to 361 pS) ion channels not previously reported in patch clamp studies on plant plasma membranes. These data indicate that ion channels are present in plant membranes that are not detected by conventional patch clamp techniques involving the production of individual plant protoplasts isolated from their tissue environment by enzymatic digestion of the cell wall. Given the large conductances of the channels revealed by laser-assisted patch clamping, we hypothesize that these channels play a significant role in the regulation of ion content and electrical signalling in guard cells.

In vivo experimental study on laser welded ICG-loaded chitosan patches for vessel repair

NASA Astrophysics Data System (ADS)

Rossi, Francesca; Matteini, Paolo; Esposito, Giuseppe; Albanese, Alessio; Puca, Alfredo; Maira, Giulio; Rossi, Giacomo; Pini, Roberto

2011-03-01

Laser welding of microvessels provides several advantages over conventional suturing techniques: surgical times reduction, vascular healing process improvement, tissue damage reduction. We present the first application of biopolymeric patches in an in vivo laser assisted procedure for vessel repair. The study was performed in 20 New Zealand rabbits. After anesthesia, a 3-cm segment of the right common carotid artery was exposed and clamped proximally and distally. A linear lesion 3 mm in length was carried out. We used a diode laser emitting at 810 nm and equipped with a 300 μm diameter optical fiber. To close the cut, ICG-loaded chitosan films were prepared: chitosan is characterized by biodegradability, biocompatibility, antimicrobial, haemostatic and wound healing-promoting activity. ICG is an organic chromophore commonly used in the laser welding procedures to mediate the photothermal conversion at the basis of the welding effect. The membranes were used to wrap the whole length of the cut, and then they were welded in the correct position by delivering single laser spots to induce local patch/tissue adhesion. The result is an immediate closure of the wound, with no bleeding at clamps release. The animals were observed during follow-up and sacrificed after 2, 7, 30 and 90 days. All the repaired vessels were patent, no bleeding signs were documented. The carotid samples underwent histological examinations. The advantages of the proposed technique are: simplification of the surgical procedure and shortening of the operative time; good strength of the vessel repair; decreased foreign-body reaction, reduced inflammatory response and improved vascular healing process.

QPatch: the past, present and future of automated patch clamp.

PubMed

Mathes, Chris

2006-04-01

The QPatch 16 significantly increases throughput for gigaseal patch clamp experiments, making direct measurements in ion channel drug discovery and safety testing feasible. Released to the market in the Autumn of 2004 by Sophion Bioscience, the QPatch originated from work done at NeuroSearch (Denmark) in the early days of automated patch clamp. Today, the QPatch provides many unique features. For example, only the QPatch includes an automated cell preparation station making several hours of unattended operation possible. The 16-channel electrode array, called the QPlate, includes glass-coated microfluidic channels for less compound absorption and, hence, more accurate IC(50) values. The microfluidic pathways also allow for very small amounts of compound used for each experiment ( approximately 5 microl per addition). Only the QPatch has four independent pipetting heads for more efficient liquid handling (especially for ligand-gated ion channel experiments). Patch clamp recordings with the QPatch match the high quality of conventional patch clamp and in some cases the results are even better. For example, only the QPatch includes 100% series resistance compensation for the elimination of false positives due to voltage errors. Finally, the modular QPatch 16 was designed with more channels in mind. The upgrade pathway to 48-channels (the QPatch HT) will be discussed.

A miniaturized planar patch-clamp system for transportable use.

PubMed

Boussaoud, Adrien; Fonteille, Isabelle; Collier, Guilhem; Kermarrec, Frédérique; Vermont, Fabien; Tresallet, Eric; De Waard, Michel; Arnoult, Christophe; Picollet-D'hahan, Nathalie

2012-02-15

In the last decade, planar patch-clamp (PPC) has emerged as an innovative technology allowing parallel recordings of cellular electrophysiological activity on planar substrates. If PPC is widely adopted by the pharmaceutical sector, it remains poorly extended to other areas (i.e. environment and safety organizations) probably because of the large, expensive and non-easily transportable format of those commercial equipments. The present work describes for the first time a new compact and transportable planar patch-clamp system (named Toxint'patch or TIP, for Toxin detection with integrated patch-clamp) focusing on environmental matters and meant to be used in coastal laboratories, for direct on-site monitoring of the seawater and shellfish quality. The TIP system incorporates silicon chips tailored to monitor cellular ionic currents from cultured cells stably expressing a phycotoxin molecular target. The functionality of this novel briefcase-sized PPC system is described in terms of fluidic control, electronic performances with amplifying and filtering boards and of user interface for data acquisition and control implemented on a computer. Copyright © 2011 Elsevier B.V. All rights reserved.

Slow oscillation of membrane currents mediated by glutamatergic inputs of rat somatosensory cortical neurons: in vivo patch-clamp analysis.

PubMed

Doi, Atsushi; Mizuno, Masaharu; Katafuchi, Toshihiko; Furue, Hidemasa; Koga, Kohei; Yoshimura, Megumu

2007-11-01

Using in vivo patch-clamp technique, the slow oscillation of membrane currents was characterized by its synaptic nature, correlation with electroencephalogram (EEG) and responses to different anesthetic agents, in primary somatosensory cortex (SI) neurons in urethane-anesthetized rats. In more than 90% of the SI neurons, the slow oscillation of the inward currents (0.1-2.5 Hz) with the duration of several hundreds of a millisecond was observed at the holding membrane potential of -70 mV. The reversal potential of the inward currents was approximately 0 mV and was suppressed by application of an alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor antagonist. In most cases (> 90%) the inward current was synchronized with positive wave of the surface EEG recorded from ipsilateral and even contralateral cortical regions. The frequency as well as duration of the slow oscillation decreased by a volatile anesthetic agent, isoflurane (1.5-5.0%), and excitatory postsynaptic currents (EPSCs) were almost abolished at the highest concentration. Intraperitoneal injection of pentobarbital (25 mg/kg) also decreased the frequency of the slow oscillation without affecting short EPSCs. When gamma-aminobutyric acid A (GABA(A)) receptors were activated by local microinjection of muscimol (3 x 10(-3) m, 1-10 microL) into the thalamus, the frequency of the slow oscillation markedly decreased, but was not abolished completely. These findings suggest that the slow oscillation of the inward currents is generated by the summation of glutamatergic EPSCs, and affected by isoflurane and pentobarbital differently. In addition, GABAergic system in the thalamus can affect the frequency, but is not essentially implicated in the genesis of the slow oscillation.

Rediscovering sperm ion channels with the patch-clamp technique

PubMed Central

Kirichok, Yuriy; Lishko, Polina V.

2011-01-01

Upon ejaculation, mammalian spermatozoa have to undergo a sequence of physiological transformations within the female reproductive tract that will allow them to reach and fertilize the egg. These include initiation of motility, hyperactivation of motility and perhaps chemotaxis toward the egg, and culminate in the acrosome reaction that permits sperm to penetrate the protective vestments of the egg. These physiological responses are triggered through the activation of sperm ion channels that cause elevations of sperm intracellular pH and Ca2+ in response to certain cues within the female reproductive tract. Despite their key role in sperm physiology and their absolute requirement for the process of fertilization, sperm ion channels remain poorly understood due to the extreme difficulty in application of the patch-clamp technique to spermatozoa. This review covers the topic of sperm ion channels in the following order: first, we discuss how the intracellular Ca2+ and pH signaling mediated by sperm ion channels controls sperm behavior during the process of fertilization. Then, we briefly cover the history of the methodology to study sperm ion channels, which culminated in the recent development of a reproducible whole-cell patch-clamp technique for mouse and human cells. We further discuss the main approaches used to patch-clamp mature mouse and human spermatozoa. Finally, we focus on the newly discovered sperm ion channels CatSper, KSper (Slo3) and HSper (Hv1), identified by the sperm patch-clamp technique. We conclude that the patch-clamp technique has markedly improved and shifted our understanding of the sperm ion channels, in addition to revealing significant species-specific differences in these channels. This method is critical for identification of the molecular mechanisms that control sperm behavior within the female reproductive tract and make fertilization possible. PMID:21642646

A novel way to go whole-cell in patch-clamp experiments.

PubMed

Inayat, Samsoon; Zhao, Yan; Cantrell, Donal R; Dikin, Dmitryi; Pinto, Lawrence H; Troy, John B

2010-11-01

With a conventional patch-clamp electrode, an Ag/AgCl wire sits stationary inside the pipette. To move from the gigaseal cell-attached configuration to whole-cell recording, suction is applied inside the pipette. We have designed and developed a novel Pushpen patch-clamp electrode, in which a W wire insulated and wound with Ag/AgCl wire can move linearly inside the pipette. The W wire has a conical tip, which can protrude from the pipette tip like a push pen, a procedure we call the Pushpen Operation. We use the Pushpen operation to impale the cell membrane in cell-attached configuration to go whole-cell without disruption of the gigaseal. We successfully recorded whole-cell currents from chinese hamster ovarian cells expressing influenza A virus protein A/M2, after obtaining whole-cell configuration with the Pushpen operation. This novel method of achieving whole-cell configuration may have a higher success rate than is the case with the conventional patch clamp technique.

Xenon inhibits excitatory but not inhibitory transmission in rat spinal cord dorsal horn neurons

PubMed Central

2010-01-01

Background The molecular targets for the promising gaseous anaesthetic xenon are still under investigation. Most studies identify N-methyl-D-aspartate (NMDA) receptors as the primary molecular target for xenon, but the role of α-amino-3-hydroxy-5-methyl-4-isoxazole-4-propionic acid (AMPA) receptors is less clear. In this study we evaluated the effect of xenon on excitatory and inhibitory synaptic transmission in the superficial dorsal horn of the spinal cord using in vitro patch-clamp recordings from rat spinal cord slices. We further evaluated the effects of xenon on innocuous and noxious stimuli using in vivo patch-clamp method. Results In vitro, xenon decreased the amplitude and area under the curve of currents induced by exogenous NMDA and AMPA and inhibited dorsal root stimulation-evoked excitatory postsynaptic currents. Xenon decreased the amplitude, but not the frequency, of miniature excitatory postsynaptic currents. There was no discernible effect on miniature or evoked inhibitory postsynaptic currents or on the current induced by inhibitory neurotransmitters. In vivo, xenon inhibited responses to tactile and painful stimuli even in the presence of NMDA receptor antagonist. Conclusions Xenon inhibits glutamatergic excitatory transmission in the superficial dorsal horn via a postsynaptic mechanism. There is no substantial effect on inhibitory synaptic transmission at the concentration we used. The blunting of excitation in the dorsal horn lamina II neurons could underlie the analgesic effect of xenon. PMID:20444263

Perforated Patch-clamp Recording of Mouse Olfactory Sensory Neurons in Intact Neuroepithelium: Functional Analysis of Neurons Expressing an Identified Odorant Receptor

PubMed Central

Jarriault, David; Grosmaitre, Xavier

2015-01-01

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of olfactory-driven behaviors and their modulation. These coding properties depend heavily on the initial interaction between odor molecules and the olfactory receptor (OR) expressed in the OSNs. The identity, specificity and ligand spectrum of the expressed OR are critical. The probability to find the ligand of the OR expressed in an OSN chosen randomly within the epithelium is very low. To address this challenge, this protocol uses genetically tagged mice expressing the fluorescent protein GFP under the control of the promoter of defined ORs. OSNs are located in a tight and organized epithelium lining the nasal cavity, with neighboring cells influencing their maturation and function. Here we describe a method to isolate an intact olfactory epithelium and record through patch-clamp recordings the properties of OSNs expressing defined odorant receptors. The protocol allows one to characterize OSN membrane properties while keeping the influence of the neighboring tissue. Analysis of patch-clamp results yields a precise quantification of ligand/OR interactions, transduction pathways and pharmacology, OSNs' coding properties and their modulation at the membrane level.  PMID:26275097

Correlation of open cell-attached and excised patch clamp techniques.

PubMed

Filipovic, D; Hayslett, J P

1995-11-01

The excised patch clamp configuration provides a unique technique for some types of single channel analyses, but maintenance of stable, long-lasting preparations may be confounded by rundown and/or rapid loss of seal. Studies were performed on the amiloride-sensitive Na+ channel, located on the apical surface of A6 cells, to determine whether the nystatin-induced open cell-attached patch could serve as an alternative configuration. Compared to excised inside-out patches, stable preparations were achieved more readily with the open cell-attached patch (9% vs. 56% of attempts). In both preparations, the current voltage (I-V) relation was linear, current amplitudes were equal at opposite equivalent clamped voltages, and Erev was zero in symmetrical Na+ solutions, indicating similar Na+ activities on the cytosolic and external surfaces of the patch. Moreover, there was no evidence that nystatin altered channel activity in the patch because slope conductance (3-4pS) and Erev (75 mV), when the bath was perfused with a high K:low Na solution (ENa = 80 mV), were nearly equal in both patch configurations. Our results therefore indicate that the nystatin-induced open cell-attached patch can serve as an alternative approach to the excised inside-out patch when experiments require modulation of univalent ions in the cytosol.

Planar patch clamp: advances in electrophysiology.

PubMed

Brüggemann, Andrea; Farre, Cecilia; Haarmann, Claudia; Haythornthwaite, Ali; Kreir, Mohamed; Stoelzle, Sonja; George, Michael; Fertig, Niels

2008-01-01

Ion channels have gained increased interest as therapeutic targets over recent years, since a growing number of human and animal diseases have been attributed to defects in ion channel function. Potassium channels are the largest and most diverse family of ion channels. Pharmaceutical agents such as Glibenclamide, an inhibitor of K(ATP) channel activity which promotes insulin release, have been successfully sold on the market for many years. So far, only a small group of the known ion channels have been addressed as potential drug targets. The functional testing of drugs on these ion channels has always been the bottleneck in the development of these types of pharmaceutical compounds.New generations of automated patch clamp screening platforms allow a higher throughput for drug testing and widen this bottleneck. Due to their planar chip design not only is a higher throughput achieved, but new applications have also become possible. One of the advantages of planar patch clamp is the possibility of perfusing the intracellular side of the membrane during a patch clamp experiment in the whole-cell configuration. Furthermore, the extracellular membrane remains accessible for compound application during the experiment.Internal perfusion can be used not only for patch clamp experiments with cell membranes, but also for those with artificial lipid bilayers. In this chapter we describe how internal perfusion can be applied to potassium channels expressed in Jurkat cells, and to Gramicidin channels reconstituted in a lipid bilayer.

Dendrimer-assisted patch-clamp sizing of nuclear pores

PubMed Central

Bustamante, J.O.; Michelette, E.R.F.; Geibel, J.P.; Hanover, J.A.; McDonnell, T.J.; Dean, D.A.

2015-01-01

Macromolecular translocation (MMT) across the nuclear envelope (NE) occurs exclusively through the nuclear pore complex (NPC). Therefore, the diameter of the NPC aqueous/electrolytic channel (NPCC) is important for cellular structure and function. The NPCC diameter was previously determined to be ≅10 nm with electron microscopy (EM) using the translocation of colloidal gold particles. Here we present patch-clamp and fluorescence microscopy data from adult cardiomyocyte nuclei that demonstrate the use of patch-clamp for assessing NPCC diameter. Fluorescence microscopy with B-phycoerythrin (BPE, 240 kDa) conjugated to a nuclear localization signal (NLS) demonstrated that these nuclei were competent for NPC-mediated MMT (NPC-MMT). Furthermore, when exposed to an appropriate cell lysate, the nuclei expressed enhanced green fluorescence protein (EGFP) after 5–10 h of incubation with the plasmid for this protein (pEGFP, 3.1 MDa). Nucleus-attached patch-clamp showed that colloidal gold particles were not useful probes; they modified NPCC gating. As a result of this finding, we searched for an inert class of particles that could be used without irreversibly affecting NPCC gating and found that fluorescently labeled Star-burst dendrimers, a distinct class of polymers, were useful. Our patch-clamp and fluorescence microscopy data with calibrated dendrimers indicate that the cardiomyocyte NPCC diameter varies between 8 and 9 nm. These studies open a new direction in the investigation of live, continuous NPC dynamics under physiological conditions. PMID:10784359

In vivo robotics: the automation of neuroscience and other intact-system biological fields

PubMed Central

Kodandaramaiah, Suhasa B.; Boyden, Edward S.; Forest, Craig R.

2013-01-01

Robotic and automation technologies have played a huge role in in vitro biological science, having proved critical for scientific endeavors such as genome sequencing and high-throughput screening. Robotic and automation strategies are beginning to play a greater role in in vivo and in situ sciences, especially when it comes to the difficult in vivo experiments required for understanding the neural mechanisms of behavior and disease. In this perspective, we discuss the prospects for robotics and automation to impact neuroscientific and intact-system biology fields. We discuss how robotic innovations might be created to open up new frontiers in basic and applied neuroscience, and present a concrete example with our recent automation of in vivo whole cell patch clamp electrophysiology of neurons in the living mouse brain. PMID:23841584

Integrated multiple patch-clamp array chip via lateral cell trapping junctions

NASA Astrophysics Data System (ADS)

Seo, J.; Ionescu-Zanetti, C.; Diamond, J.; Lal, R.; Lee, L. P.

2004-03-01

We present an integrated multiple patch-clamp array chip by utilizing lateral cell trapping junctions. The intersectional design of a microfluidic network provides multiple cell addressing and manipulation sites for efficient electrophysiological measurements at a number of patch sites. The patch pores consist of openings in the sidewall of a main fluidic channel, and a membrane patch is drawn into a smaller horizontal channel. This device geometry not only minimizes capacitive coupling between the cell reservoir and the patch channel, but also allows simultaneous optical and electrical measurements of ion channel proteins. Evidence of the hydrodynamic placement of mammalian cells at the patch sites as well as measurements of patch sealing resistance is presented. Device fabrication is based on micromolding of polydimethylsiloxane, thus allowing inexpensive mass production of disposable high-throughput biochips.

Diffusion-convection effects on drug distribution at the cell membrane level in a patch-clamp setup.

PubMed

Baran, Irina; Iftime, Adrian; Popescu, Anca

2010-01-01

We present a model-based method for estimating the effective concentration of the active drug applied by a pressure pulse to an individual cell in a patch-clamp setup, which could be of practical use in the analysis of ligand-induced whole-cell currents recorded in patch-clamp experiments. Our modelling results outline several important factors which may be involved in the high variability of the electric response of the cells, and indicate that with a pressure pulse duration of 1s and diameter of the perfusion tip of 600 μm, elevated amounts of drug can accumulate locally between the pipette tip and the cell. Hence, the effective agonist concentration at the cell membrane level can be consistently higher than the initial concentration inside the perfusion tubes. We performed finite-difference and finite-element simulations to investigate the diffusion/convection effects on the agonist distribution on the cell membrane. Our model can explain the delay between the commencement of acetylcholine application and the onset of the whole-cell current that we recorded on human rhabdomyosarcoma TE671 cells, and reproduce quantitatively the decrease of signal latency with the concentration of agonist in the pipette. Results also show that not only the geometry of the bath chamber and pipette tip, but also the transport parameters of the diffusive and convective phenomena in the bath solution are determinant for the amplitude and kinetics of the recorded currents and have to be accounted for when analyzing patch-clamp data. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

The slow inward calcium current is responsible for a part of the contraction of patch-clamped rat myoballs.

PubMed

Rivet, M; Cognard, C; Raymond, G

1989-01-01

The slow inward calcium current and the contractile response were simultaneously recorded in voltage clamped (whole cell patch clamp recording) rat myoballs in primary culture. The shape of the contraction(T)/potential(V) relationship and the application of the inorganic calcium channel blocker cadmium (1.5 mM), which suppresses a part of the contractile activity, demonstrate the existence of two components of contraction. One of them is related to the slow calcium current.

Laser microsurgery of higher plant cell walls permits patch-clamp access

NASA Technical Reports Server (NTRS)

Henriksen, G. H.; Taylor, A. R.; Brownlee, C.; Assmann, S. M.; Evans, M. L. (Principal Investigator)

1996-01-01

Plasma membranes of guard cells in epidermal peels of Vicia faba and Commelina communis can be made accessible to a patch-clamp pipet by removing a small portion (1-3 micrometers in diameter) of the guard cell wall using a microbeam of ultraviolet light generated by a nitrogen laser. Using this laser microsurgical technique, we have measured channel activity across plasma membranes of V. faba guard cells in both cell-attached and isolated patch configurations. Measurements made in the inside-out patch configuration revealed two distinct K(+)-selective channels. Major advantages of the laser microsurgical technique include the avoidance of enzymatic protoplast isolation, the ability to study cell types that have been difficult to isolate as protoplasts or for which enzymatic isolation protocols result in protoplasts not amenable to patch-clamp studies, the maintenance of positional information in single-channel measurements, reduced disruption of cell-wall-mediated signaling pathways, and the ability to investigate intercellular signaling through studies of cells remaining situated within tissue.

Odorant responses of olfactory sensory neurons expressing the odorant receptor MOR23: A patch clamp analysis in gene-targeted mice

PubMed Central

Grosmaitre, Xavier; Vassalli, Anne; Mombaerts, Peter; Shepherd, Gordon M.; Ma, Minghong

2006-01-01

A glomerulus in the mammalian olfactory bulb receives axonal inputs from olfactory sensory neurons (OSNs) that express the same odorant receptor (OR). Glomeruli are generally thought to represent functional units of olfactory coding, but there are no data on the electrophysiological properties of OSNs that express the same endogenous OR. Here, using patch clamp recordings in an intact epithelial preparation, we directly measured the transduction currents and receptor potentials from the dendritic knobs of mouse OSNs that express the odorant receptor MOR23 along with the green fluorescent protein. All of the 53 cells examined responded to lyral, a known ligand for MOR23. There were profound differences in response kinetics, particularly in the deactivation phase. The cells were very sensitive to lyral, with some cells responding to as little as 10 nM. The dynamic range was unexpectedly broad, with threshold and saturation in individual cells often covering three log units of lyral concentration. The potential causes and biological significance of this cellular heterogeneity are discussed. Patch clamp recording from OSNs that express a defined OR provides a powerful approach to investigate the sensory inputs to individual glomeruli. PMID:16446455

Odorant responses of olfactory sensory neurons expressing the odorant receptor MOR23: a patch clamp analysis in gene-targeted mice.

PubMed

Grosmaitre, Xavier; Vassalli, Anne; Mombaerts, Peter; Shepherd, Gordon M; Ma, Minghong

2006-02-07

A glomerulus in the mammalian olfactory bulb receives axonal inputs from olfactory sensory neurons (OSNs) that express the same odorant receptor (OR). Glomeruli are generally thought to represent functional units of olfactory coding, but there are no data on the electrophysiological properties of OSNs that express the same endogenous OR. Here, using patch clamp recordings in an intact epithelial preparation, we directly measured the transduction currents and receptor potentials from the dendritic knobs of mouse OSNs that express the odorant receptor MOR23 along with the green fluorescent protein. All of the 53 cells examined responded to lyral, a known ligand for MOR23. There were profound differences in response kinetics, particularly in the deactivation phase. The cells were very sensitive to lyral, with some cells responding to as little as 10 nM. The dynamic range was unexpectedly broad, with threshold and saturation in individual cells often covering three log units of lyral concentration. The potential causes and biological significance of this cellular heterogeneity are discussed. Patch clamp recording from OSNs that express a defined OR provides a powerful approach to investigate the sensory inputs to individual glomeruli.

Characterization of GABAA receptor ligands with automated patch-clamp using human neurons derived from pluripotent stem cells

PubMed Central

Yuan, Nina Y.; Poe, Michael M.; Witzigmann, Christopher; Cook, James M.; Stafford, Douglas; Arnold, Leggy A.

2016-01-01

Introduction Automated patch clamp is a recent but widely used technology to assess pre-clinical drug safety. With the availability of human neurons derived from pluripotent stem cells, this technology can be extended to determine CNS effects of drug candidates, especially those acting on the GABAA receptor. Methods iCell Neurons (Cellular Dynamics International, A Fujifilm Company) were cultured for ten days and analyzed by patch clamp in the presence of agonist GABA or in combination with positive allosteric GABAA receptor modulators. Both efficacy and affinity were determined. In addition, mRNA of GABAA receptor subunits were quantified by qRT-PCR. Results We have shown that iCell Neurons are compatible with the IonFlux microfluidic system of the automated patch clamp instrument. Resistance ranging from 15-25 MΩ was achieved for each trap channel of patch clamped cells in a 96-well plate format. GABA induced a robust change of current with an EC50 of 0.43 μM. Positive GABAA receptor modulators diazepam, HZ166, and CW-04-020 exhibited EC50 values of 0.42 μM, 1.56 μM, and 0.23 μM, respectively. The α2/α3/α5 selective compound HZ166-induced the highest potentiation (efficacy) of 810% of the current induced by 100 nM GABA. Quantification of GABAA receptor mRNA in iCell Neurons revealed high levels of α5 and β3 subunits and low levels of α1, which is similar to the configuration in human neonatal brain. Discussion iCell Neurons represent a new cellular model to characterize GABAergic compounds using automated patch clamp. These cells have excellent representation of cellular GABAA receptor distribution that enable determination of total small molecule efficacy and affinity as measured by cell membrane current change. PMID:27544543

Novel 384-well population patch clamp electrophysiology assays for Ca2+-activated K+ channels.

PubMed

John, Victoria H; Dale, Tim J; Hollands, Emma C; Chen, Mao Xiang; Partington, Leanne; Downie, David L; Meadows, Helen J; Trezise, Derek J

2007-02-01

Planar array electrophysiology techniques were applied to assays for modulators of recombinant hIK and hSK3 Ca2+-activated K+ channels. In CHO-hIK-expressing cells, under asymmetric K+ gradients, small-molecule channel activators evoked time- and voltage-independent currents characteristic of those previously described by classical patch clamp electrophysiology methods. In single-hole (cell) experiments, the large cell-to-cell heterogeneity in channel expression rendered it difficult to generate activator concentration-response curves. However, in population patch clamp mode, in which signals are averaged from up to 64 cells, well-to-well variation was substantially reduced such that concentration-response curves could be easily constructed. The absolute EC50 values and rank order of potency for a range of activators, including 1-EBIO and DC-EBIO, corresponded well with conventional patch clamp data. Activator responses of hIK and hSK3 channels could be fully and specifically blocked by the selective inhibitors TRAM-34 and apamin, with IC50 values of 0.31 microM and 3 nM, respectively. To demonstrate assay precision and robustness, a test set of 704 compounds was screened in a 384-well format of the hIK assay. All plates had Z' values greater than 0.6, and the statistical cutoff for activity was 8%. Eleven hits (1.6%) were identified from this set, in addition to the randomly spiked wells with known activators. Overall, our findings demonstrate that population patch clamp is a powerful and enabling method for screening Ca2+-activated K+ channels and provides significant advantages over single-cell electrophysiology (IonWorks(HT)) and other previously published approaches. Moreover, this work demonstrates for the 1st time the utility of population patch clamp for ion channel activator assays and for non-voltage-gated ion channels.

In vivo robotics: the automation of neuroscience and other intact-system biological fields.

PubMed

Kodandaramaiah, Suhasa B; Boyden, Edward S; Forest, Craig R

2013-12-01

Robotic and automation technologies have played a huge role in in vitro biological science, having proved critical for scientific endeavors such as genome sequencing and high-throughput screening. Robotic and automation strategies are beginning to play a greater role in in vivo and in situ sciences, especially when it comes to the difficult in vivo experiments required for understanding the neural mechanisms of behavior and disease. In this perspective, we discuss the prospects for robotics and automation to influence neuroscientific and intact-system biology fields. We discuss how robotic innovations might be created to open up new frontiers in basic and applied neuroscience and present a concrete example with our recent automation of in vivo whole-cell patch clamp electrophysiology of neurons in the living mouse brain. © 2013 New York Academy of Sciences.

Distributions-per-level: a means of testing level detectors and models of patch-clamp data.

PubMed

Schröder, I; Huth, T; Suitchmezian, V; Jarosik, J; Schnell, S; Hansen, U P

2004-01-01

Level or jump detectors generate the reconstructed time series from a noisy record of patch-clamp current. The reconstructed time series is used to create dwell-time histograms for the kinetic analysis of the Markov model of the investigated ion channel. It is shown here that some additional lines in the software of such a detector can provide a powerful new means of patch-clamp analysis. For each current level that can be recognized by the detector, an array is declared. The new software assigns every data point of the original time series to the array that belongs to the actual state of the detector. From the data sets in these arrays distributions-per-level are generated. Simulated and experimental time series analyzed by Hinkley detectors are used to demonstrate the benefits of these distributions-per-level. First, they can serve as a test of the reliability of jump and level detectors. Second, they can reveal beta distributions as resulting from fast gating that would usually be hidden in the overall amplitude histogram. Probably the most valuable feature is that the malfunctions of the Hinkley detectors turn out to depend on the Markov model of the ion channel. Thus, the errors revealed by the distributions-per-level can be used to distinguish between different putative Markov models of the measured time series.

Construction, Calibration, and Validation of a Simple Patch-Clamp Amplifier for Physiology Education

ERIC Educational Resources Information Center

Rouzrokh, Ali; Ebrahimi, Soltan Ahmed; Mahmoudian, Massoud

2009-01-01

A modular patch-clamp amplifier was constructed based on the Strickholm design, which was initially published in 1995. Various parts of the amplifier such as the power supply, input circuit, headstage, feedback circuit, output and nulling circuits were redesigned to use recent software advances and fabricated using the common lithographic printed…

Influence of Global and Local Membrane Curvature on Mechanosensitive Ion Channels: A Finite Element Approach

PubMed Central

Bavi, Omid; Cox, Charles D.; Vossoughi, Manouchehr; Naghdabadi, Reza; Jamali, Yousef; Martinac, Boris

2016-01-01

Mechanosensitive (MS) channels are ubiquitous molecular force sensors that respond to a number of different mechanical stimuli including tensile, compressive and shear stress. MS channels are also proposed to be molecular curvature sensors gating in response to bending in their local environment. One of the main mechanisms to functionally study these channels is the patch clamp technique. However, the patch of membrane surveyed using this methodology is far from physiological. Here we use continuum mechanics to probe the question of how curvature, in a standard patch clamp experiment, at different length scales (global and local) affects a model MS channel. Firstly, to increase the accuracy of the Laplace’s equation in tension estimation in a patch membrane and to be able to more precisely describe the transient phenomena happening during patch clamping, we propose a modified Laplace’s equation. Most importantly, we unambiguously show that the global curvature of a patch, which is visible under the microscope during patch clamp experiments, is of negligible energetic consequence for activation of an MS channel in a model membrane. However, the local curvature (RL < 50) and the direction of bending are able to cause considerable changes in the stress distribution through the thickness of the membrane. Not only does local bending, in the order of physiologically relevant curvatures, cause a substantial change in the pressure profile but it also significantly modifies the stress distribution in response to force application. Understanding these stress variations in regions of high local bending is essential for a complete understanding of the effects of curvature on MS channels. PMID:26861405

Searching for new leads to treat epilepsy. Target-based virtual screening for the discovery of anticonvulsant agents.

PubMed

Palestro, Pablo; Enrique, Nicolas; Goicoechea, Sofia; Villalba, María Luisa; Sabatier, Laureano Leonel; Martin, Pedro; Milesi, Veronica; Bruno-Blanch, Luis E; Gavernet, Luciana

2018-06-05

The purpose of this investigation is to contribute to the development of new anticonvulsant drugs to treat patients with refractory epilepsy. We applied a virtual screening protocol that involved the search into molecular databases of new compounds and known drugs to find small molecules that interact with the open conformation of the Nav1.2 pore. As the 3D structure of human Nav1.2 is not available, we first assembled 3D models of the target, in closed and open conformations. After the virtual screening, the resulting candidates were submitted to a second virtual filter, to find compounds with better chances of being effective for the treatment of P-glycoprotein (P-gp) mediated resistant epilepsy. Again, we built a model of the 3D structure of human P-gp and we validated the docking methodology selected to propose the best candidates, which were experimentally tested on Nav1.2 channels by patch clamp techniques and in vivo by MES-test. Patch clamp studies allowed us to corroborate that our candidates, drugs used for the treatment of other pathologies like Ciprofloxacin, Losartan and Valsartan, exhibit inhibitory effects on Nav1.2 channel activity. Additionally, a compound synthesized in our lab, N,N´-diphenethylsulfamide, interacts with the target and also triggers significant Na1.2 channel inhibitory action. Finally, in-vivo studies confirmed the anticonvulsant action of Valsartan, Ciprofloxacin and N.N´-diphenethylsulfamide.

Automated Patch-Clamp Methods for the hERG Cardiac Potassium Channel.

PubMed

Houtmann, Sylvie; Schombert, Brigitte; Sanson, Camille; Partiseti, Michel; Bohme, G Andrees

2017-01-01

The human Ether-a-go-go Related Gene (hERG) product has been identified as a central ion channel underlying both familial forms of elongated QT interval on the electrocardiogram and drug-induced elongation of the same QT segment. Indeed, reduced function of this potassium channel involved in the repolarization of the cardiac action potential can produce a type of life-threatening cardiac ventricular arrhythmias called Torsades de Pointes (TdP). Therefore, hERG inhibitory activity of newly synthetized molecules is a relevant structure-activity metric for compound prioritization and optimization in medicinal chemistry phases of drug discovery. Electrophysiology remains the gold standard for the functional assessment of ion channel pharmacology. The recent years have witnessed automatization and parallelization of the manual patch-clamp technique, allowing higher throughput screening on recombinant hERG channels. However, the multi-well plate format of automatized patch-clamp does not allow visual detection of potential micro-precipitation of poorly soluble compounds. In this chapter we describe bench procedures for the culture and preparation of hERG-expressing CHO cells for recording on an automated patch-clamp workstation. We also show that the sensitivity of the assay can be improved by adding a surfactant to the extracellular medium.

Novel KCNQ2 channel activators discovered using fluorescence-based and automated patch-clamp-based high-throughput screening techniques

PubMed Central

Yue, Jin-feng; Qiao, Guan-hua; Liu, Ni; Nan, Fa-jun; Gao, Zhao-bing

2016-01-01

Aim: To establish an improved, high-throughput screening techniques for identifying novel KCNQ2 channel activators. Methods: KCNQ2 channels were stably expressed in CHO cells (KCNQ2 cells). Thallium flux assay was used for primary screening, and 384-well automated patch-clamp IonWorks Barracuda was used for hit validation. Two validated activators were characterized using a conventional patch-clamp recording technique. Results: From a collection of 80 000 compounds, the primary screening revealed a total of 565 compounds that potentiated the fluorescence signals in thallium flux assay by more than 150%. When the 565 hits were examined in IonWorks Barracuda, 38 compounds significantly enhanced the outward currents recorded in KCNQ2 cells, and were confirmed as KCNQ2 activators. In the conventional patch-clamp recordings, two validated activators ZG1732 and ZG2083 enhanced KCNQ2 currents with EC50 values of 1.04±0.18 μmol/L and 1.37±0.06 μmol/L, respectively. Conclusion: The combination of thallium flux assay and IonWorks Barracuda assay is an efficient high-throughput screening (HTS) route for discovering KCNQ2 activators. PMID:26725738

Protein kinase C enhances the swelling-induced chloride current in human atrial myocytes.

PubMed

Li, Ye-Tao; Du, Xin-Ling

2016-06-01

Swelling-activated chloride currents (ICl.swell) are thought to play a role in several physiologic and pathophysiologic processes and thus represent a target for therapeutic approaches. However, the mechanism of ICl.swell regulation remains unclear. In this study, we used the whole-cell patch-clamp technique to examine the role of protein kinase C (PKC) in the regulation of ICl.swell in human atrial myocytes. Atrial myocytes were isolated from the right atrial appendages of patients undergoing coronary artery bypass and enzymatically dissociated. ICl.swell was evoked in hypotonic solution and recorded using the whole-cell patch-clamp technique. The PKC agonist phorbol dibutyrate (PDBu) enhanced ICl.swell in a concentration-dependent manner, which was reversed in isotonic solution and by a chloride current inhibitor, 9-anthracenecarboxylicacid. Furthermore, the PKC inhibitor bis-indolylmaleimide attenuated the effect and 4α-PDBu, an inactive PDBu analog, had no effect on ICl.swell. These results, obtained using the whole-cell patch-clamp technique, demonstrate the ability of PKC to activate ICl,swell in human atrial myocytes. This observation was consistent with a previous study using a single-channel patch-clamp technique, but differed from some findings in other species.

Ion channel electrophysiology via integrated planar patch-clamp chip with on-demand drug exchange.

PubMed

Chen, Chang-Yu; Tu, Ting-Yuan; Jong, De-Shien; Wo, Andrew M

2011-06-01

Planar patch clamp has revolutionized characterization of ion channel behavior in drug discovery primarily via advancement in high throughput. Lab use of planar technology, however, addresses different requirements and suffers from inflexibility to enable wide range of interrogation via a single cell. This work presents integration of planar patch clamp with microfluidics, achieving multiple solution exchanges for tailor-specific measurement and allowing rapid replacement of the cell-contacting aperture. Studies via endogenously expressed ion channels in HEK 293T cells were commenced to characterize the device. Results reveal the microfluidic concentration generator produces distinct solution/drug combination/concentrations on-demand. Volume-regulated chloride channel and voltage-gated potassium channels in HEK 293T cells immersed in generated solutions under various osmolarities or drug concentrations show unique channel signature under specific condition. Excitation and blockage of ion channels in a single cell was demonstrated via serial solution exchange. Robustness of the reversible bonding and ease of glass substrate replacement were proven via repeated usage of the integrated device. The present approach reveals the capability and flexibility of integrated microfluidic planar patch-clamp system for ion channel assays. Copyright © 2011 Wiley Periodicals, Inc.

Establishment of the Dual Whole Cell Recording Patch Clamp Configuration for the Measurement of Gap Junction Conductance.

PubMed

Veenstra, Richard D

2016-01-01

The development of the patch clamp technique has enabled investigators to directly measure gap junction conductance between isolated pairs of small cells with resolution to the single channel level. The dual patch clamp recording technique requires specialized equipment and the acquired skill to reliably establish gigaohm seals and the whole cell recording configuration with high efficiency. This chapter describes the equipment needed and methods required to achieve accurate measurement of macroscopic and single gap junction channel conductances. Inherent limitations with the dual whole cell recording technique and methods to correct for series access resistance errors are defined as well as basic procedures to determine the essential electrical parameters necessary to evaluate the accuracy of gap junction conductance measurements using this approach.

QPatch: the missing link between HTS and ion channel drug discovery.

PubMed

Mathes, Chris; Friis, Søren; Finley, Michael; Liu, Yi

2009-01-01

The conventional patch clamp has long been considered the best approach for studying ion channel function and pharmacology. However, its low throughput has been a major hurdle to overcome for ion channel drug discovery. The recent emergence of higher throughput, automated patch clamp technology begins to break this bottleneck by providing medicinal chemists with high-quality, information-rich data in a more timely fashion. As such, these technologies have the potential to bridge a critical missing link between high-throughput primary screening and meaningful ion channel drug discovery programs. One of these technologies, the QPatch automated patch clamp system developed by Sophion Bioscience, records whole-cell ion channel currents from 16 or 48 individual cells in a parallel fashion. Here, we review the general applicability of the QPatch to studying a wide variety of ion channel types (voltage-/ligand-gated cationic/anionic channels) in various expression systems. The success rate of gigaseals, formation of the whole-cell configuration and usable cells ranged from 40-80%, depending on a number of factors including the cell line used, ion channel expressed, assay development or optimization time and expression level in these studies. We present detailed analyses of the QPatch features and results in case studies in which secondary screening assays were successfully developed for a voltage-gated calcium channel and a ligand-gated TRP channel. The increase in throughput compared to conventional patch clamp with the same cells was approximately 10-fold. We conclude that the QPatch, combining high data quality and speed with user friendliness and suitability for a wide array of ion channels, resides on the cutting edge of automated patch clamp technology and plays a pivotal role in expediting ion channel drug discovery.

Automated multi-slice extracellular and patch-clamp experiments using the WinLTP data acquisition system with automated perfusion control

PubMed Central

Anderson, William W.; Fitzjohn, Stephen M.; Collingridge, Graham L.

2012-01-01

WinLTP is a data acquisition program for studying long-term potentiation (LTP) and other aspects of synaptic function. Earlier versions of WinLTP (J. Neurosci. Methods, 162:346–356, 2007) provided automated electrical stimulation and data acquisition capable of running nearly an entire synaptic plasticity experiment, with the primary exception that perfusion solutions had to be changed manually. This automated stimulation and acquisition was done by using ‘Sweep’, ‘Loop’ and ‘Delay’ events to build scripts using the ‘Protocol Builder’. However, this did not allow automatic changing of many solutions while running multiple slice experiments, or solution changing when this had to be performed rapidly and with accurate timing during patch-clamp experiments. We report here the addition of automated perfusion control to WinLTP. First, perfusion change between sweeps is enabled by adding the ‘Perfuse’ event to Protocol Builder scripting and is used in slice experiments. Second, fast perfusion changes during as well as between sweeps is enabled by using the Perfuse event in the protocol scripts to control changes between sweeps, and also by changing digital or analog output during a sweep and is used for single cell single-line perfusion patch-clamp experiments. The addition of stepper control of tube placement allows dual- or triple-line perfusion patch-clamp experiments for up to 48 solutions. The ability to automate perfusion changes and fully integrate them with the already automated stimulation and data acquisition goes a long way toward complete automation of multi-slice extracellularly recorded and single cell patch-clamp experiments. PMID:22524994

From Understanding Cellular Function to Novel Drug Discovery: The Role of Planar Patch-Clamp Array Chip Technology

PubMed Central

Py, Christophe; Martina, Marzia; Diaz-Quijada, Gerardo A.; Luk, Collin C.; Martinez, Dolores; Denhoff, Mike W.; Charrier, Anne; Comas, Tanya; Monette, Robert; Krantis, Anthony; Syed, Naweed I.; Mealing, Geoffrey A. R.

2011-01-01

All excitable cell functions rely upon ion channels that are embedded in their plasma membrane. Perturbations of ion channel structure or function result in pathologies ranging from cardiac dysfunction to neurodegenerative disorders. Consequently, to understand the functions of excitable cells and to remedy their pathophysiology, it is important to understand the ion channel functions under various experimental conditions – including exposure to novel drug targets. Glass pipette patch-clamp is the state of the art technique to monitor the intrinsic and synaptic properties of neurons. However, this technique is labor intensive and has low data throughput. Planar patch-clamp chips, integrated into automated systems, offer high throughputs but are limited to isolated cells from suspensions, thus limiting their use in modeling physiological function. These chips are therefore not most suitable for studies involving neuronal communication. Multielectrode arrays (MEAs), in contrast, have the ability to monitor network activity by measuring local field potentials from multiple extracellular sites, but specific ion channel activity is challenging to extract from these multiplexed signals. Here we describe a novel planar patch-clamp chip technology that enables the simultaneous high-resolution electrophysiological interrogation of individual neurons at multiple sites in synaptically connected neuronal networks, thereby combining the advantages of MEA and patch-clamp techniques. Each neuron can be probed through an aperture that connects to a dedicated subterranean microfluidic channel. Neurons growing in networks are aligned to the apertures by physisorbed or chemisorbed chemical cues. In this review, we describe the design and fabrication process of these chips, approaches to chemical patterning for cell placement, and present physiological data from cultured neuronal cells. PMID:22007170

From understanding cellular function to novel drug discovery: the role of planar patch-clamp array chip technology.

PubMed

Py, Christophe; Martina, Marzia; Diaz-Quijada, Gerardo A; Luk, Collin C; Martinez, Dolores; Denhoff, Mike W; Charrier, Anne; Comas, Tanya; Monette, Robert; Krantis, Anthony; Syed, Naweed I; Mealing, Geoffrey A R

2011-01-01

All excitable cell functions rely upon ion channels that are embedded in their plasma membrane. Perturbations of ion channel structure or function result in pathologies ranging from cardiac dysfunction to neurodegenerative disorders. Consequently, to understand the functions of excitable cells and to remedy their pathophysiology, it is important to understand the ion channel functions under various experimental conditions - including exposure to novel drug targets. Glass pipette patch-clamp is the state of the art technique to monitor the intrinsic and synaptic properties of neurons. However, this technique is labor intensive and has low data throughput. Planar patch-clamp chips, integrated into automated systems, offer high throughputs but are limited to isolated cells from suspensions, thus limiting their use in modeling physiological function. These chips are therefore not most suitable for studies involving neuronal communication. Multielectrode arrays (MEAs), in contrast, have the ability to monitor network activity by measuring local field potentials from multiple extracellular sites, but specific ion channel activity is challenging to extract from these multiplexed signals. Here we describe a novel planar patch-clamp chip technology that enables the simultaneous high-resolution electrophysiological interrogation of individual neurons at multiple sites in synaptically connected neuronal networks, thereby combining the advantages of MEA and patch-clamp techniques. Each neuron can be probed through an aperture that connects to a dedicated subterranean microfluidic channel. Neurons growing in networks are aligned to the apertures by physisorbed or chemisorbed chemical cues. In this review, we describe the design and fabrication process of these chips, approaches to chemical patterning for cell placement, and present physiological data from cultured neuronal cells.

A simple technique for transferring excised patches of membrane to different solutions for single channel measurements.

PubMed

Quartararo, N; Barry, P H

1987-12-01

A technical problem associated with the patch clamp technique has been the changing of solutions bathing the membrane patch. The simple technique described here solves this problem by means of a movable polythene sleeve placed on the shaft of the patch clamp pipette. The sleeve is initially placed so that the tip of the pipette is exposed. A gigaohm seal is formed using standard techniques. The patch is then excised and the sleeve is slipped down a few mm past the end of the tip of the pipette. When the pipette and sleeve is now removed from the solution, a small drop of solution covering the membrane patch is held in place at the end of the sleeve by surface tension. The pipette is then easily transferred to a different solution without passing the membrane patch through the air-water interface. The sleeve is then simply pulled back up the pipette shaft to expose the membrane patch to the new solution.

New VMD2 gene mutations identified in patients affected by Best vitelliform macular dystrophy

PubMed Central

Marchant, D; Yu, K; Bigot, K; Roche, O; Germain, A; Bonneau, D; Drouin‐Garraud, V; Schorderet, D F; Munier, F; Schmidt, D; Neindre, P Le; Marsac, C; Menasche, M; Dufier, J L; Fischmeister, R; Hartzell, C; Abitbol, M

2007-01-01

Purpose The mutations responsible for Best vitelliform macular dystrophy (BVMD) are found in a gene called VMD2. The VMD2 gene encodes a transmembrane protein named bestrophin‐1 (hBest1) which is a Ca2+‐sensitive chloride channel. This study was performed to identify disease‐specific mutations in 27 patients with BVMD. Because this disease is characterised by an alteration in Cl− channel function, patch clamp analysis was used to test the hypothesis that one of the VMD2 mutated variants causes the disease. Methods Direct sequencing analysis of the 11 VMD2 exons was performed to detect new abnormal sequences. The mutant of hBest1 was expressed in HEK‐293 cells and the associated Cl− current was examined using whole‐cell patch clamp analysis. Results Six new VMD2 mutations were identified, located exclusively in exons four, six and eight. One of these mutations (Q293H) was particularly severe. Patch clamp analysis of human embryonic kidney cells expressing the Q293H mutant showed that this mutant channel is non‐functional. Furthermore, the Q293H mutant inhibited the function of wild‐type bestrophin‐1 channels in a dominant negative manner. Conclusions This study provides further support for the idea that mutations in VMD2 are a necessary factor for Best disease. However, because variable expressivity of VMD2 was observed in a family with the Q293H mutation, it is also clear that a disease‐linked mutation in VMD2 is not sufficient to produce BVMD. The finding that the Q293H mutant does not form functional channels in the membrane could be explained either by disruption of channel conductance or gating mechanisms or by improper trafficking of the protein to the plasma membrane. PMID:17287362

Automated patch clamp on mESC-derived cardiomyocytes for cardiotoxicity prediction.

PubMed

Stoelzle, Sonja; Haythornthwaite, Alison; Kettenhofen, Ralf; Kolossov, Eugen; Bohlen, Heribert; George, Michael; Brüggemann, Andrea; Fertig, Niels

2011-09-01

Cardiovascular side effects are critical in drug development and have frequently led to late-stage project terminations or even drug withdrawal from the market. Physiologically relevant and predictive assays for cardiotoxicity are hence strongly demanded by the pharmaceutical industry. To identify a potential impact of test compounds on ventricular repolarization, typically a variety of ion channels in diverse heterologously expressing cells have to be investigated. Similar to primary cells, in vitro-generated stem cell-derived cardiomyocytes simultaneously express cardiac ion channels. Thus, they more accurately represent the native situation compared with cell lines overexpressing only a single type of ion channel. The aim of this study was to determine if stem cell-derived cardiomyocytes are suited for use in an automated patch clamp system. The authors show recordings of cardiac ion currents as well as action potential recordings in readily available stem cell-derived cardiomyocytes. Besides monitoring inhibitory effects of reference compounds on typical cardiac ion currents, the authors revealed for the first time drug-induced modulation of cardiac action potentials in an automated patch clamp system. The combination of an in vitro cardiac cell model with higher throughput patch clamp screening technology allows for a cost-effective cardiotoxicity prediction in a physiologically relevant cell system.

Properties of the Nucleo-Olivary Pathway: An In Vivo Whole-Cell Patch Clamp Study

PubMed Central

Bazzigaluppi, Paolo; Ruigrok, Tom; Saisan, Payam; De Zeeuw, Chris I.; de Jeu, Marcel

2012-01-01

The inferior olivary nucleus (IO) forms the gateway to the cerebellar cortex and receives feedback information from the cerebellar nuclei (CN), thereby occupying a central position in the olivo-cerebellar loop. Here, we investigated the feedback input from the CN to the IO in vivo in mice using the whole-cell patch-clamp technique. This approach allows us to study how the CN-feedback input is integrated with the activity of olivary neurons, while the olivo-cerebellar system and its connections are intact. Our results show how IO neurons respond to CN stimulation sequentially with: i) a short depolarization (EPSP), ii) a hyperpolarization (IPSP) and iii) a rebound depolarization. The latter two phenomena can also be evoked without the EPSPs. The IPSP is sensitive to a GABAA receptor blocker. The IPSP suppresses suprathreshold and subthreshold activity and is generated mainly by activation of the GABAA receptors. The rebound depolarization re-initiates and temporarily phase locks the subthreshold oscillations. Lack of electrotonical coupling does not affect the IPSP of individual olivary neurons, nor the sensitivity of its GABAA receptors to blockers. The GABAergic feedback input from the CN does not only temporarily block the transmission of signals through the IO, it also isolates neurons from the network by shunting the junction current and re-initiates the temporal pattern after a fixed time point. These data suggest that the IO not only functions as a cerebellar controlled gating device, but also operates as a pattern generator for controlling motor timing and/or learning. PMID:23029495

Robotic multi-well planar patch-clamp for native and primary mammalian cells

PubMed Central

Milligan, Carol J; Li, Jing; Sukumar, Piruthivi; Majeed, Yasser; Dallas, Mark L; English, Anne; Emery, Paul; Porter, Karen E; Smith, Andrew M; McFadzean, Ian; Beccano-Kelly, Dayne; Bahnasi, Yahya; Cheong, Alex; Naylor, Jacqueline; Zeng, Fanning; Liu, Xing; Gamper, Nikita; Jiang, Lin-Hua; Pearson, Hugh A; Peers, Chris; Robertson, Brian; Beech, David J

2009-01-01

Multi-well robotic planar patch-clamp has become common in drug development and safety programmes because it enables efficient and systematic testing of compounds against ion channels during voltage-clamp. It has not, however, been adopted significantly in other important areas of ion channel research, where conventional patch-clamp remains the favoured method. Here we show the wider potential of the multi-well approach with the capability for efficient intracellular solution exchange, describing protocols and success rates for recording from a range of native and primary mammalian cells derived from blood vessels, arthritic joints, and the immune and central nervous systems. The protocol involves preparing a suspension of single cells to be dispensed robotically into 4-8 microfluidic chambers each containing a glass chip with a small aperture. Under automated control, giga-seals and whole-cell access are achieved followed by pre-programmed routines of voltage paradigms and fast extracellular or intracellular solution exchange. Recording from 48 chambers usually takes 1-6 hr depending on the experimental design and yields 16-33 cell recordings. PMID:19197268

Tetrodotoxin-sensitive, voltage-dependent sodium currents in hair cells from the alligator cochlea.

PubMed

Evans, M G; Fuchs, P A

1987-10-01

We have used whole-cell patch clamp techniques to record from tall hair cells isolated from the apical half of the alligator cochlea. Some of these cells gave action potentials in response to depolarizing current injections. When the same cells were voltage clamped, large transient inward currents followed by smaller outward currents were seen in response to depolarizing steps. We studied the transient inward current after the outward current had been blocked by external tetraethylammonium (20 mM) or by replacing internal potassium with cesium. It was found to be a sodium current because it was abolished by either replacing external sodium with choline or by external application of tetrodotoxin (100 nM). The sodium current showed voltage-dependent activation and inactivation. Most of the spiking hair cells came from the apex of the cochlea, where they would be subject to low-frequency mechanical stimulation in vivo.

Comparison of Cell Expression Formats for the Characterization of GABAA Channels Using a Microfluidic Patch Clamp System

PubMed Central

Chen, Qin; Yim, Peter D.; Yuan, Nina; Johnson, Juliette; Cook, James M.; Smith, Steve; Ionescu-Zanetti, Cristian; Wang, Zhi-Jian; Arnold, Leggy A.

2012-01-01

Abstract Ensemble recording and microfluidic perfusion are recently introduced techniques aimed at removing the laborious nature and low recording success rates of manual patch clamp. Here, we present assay characteristics for these features integrated into one automated electrophysiology platform as applied to the study of GABAA channels. A variety of cell types and methods of GABAA channel expression were successfully studied (defined as IGABA>500 pA), including stably transfected human embryonic kidney (HEK) cells expressing α1β3γ2 GABAA channels, frozen ready-to-assay (RTA) HEK cells expressing α1β3γ2 or α3β3γ2 GABAA channels, transiently transfected HEK293T cells expressing α1β3γ2 GABAA channels, and immortalized cultures of human airway smooth muscle cells endogenously expressing GABAA channels. Current measurements were successfully studied in multiple cell types with multiple modes of channel expression in response to several classic GABAA channel agonists, antagonists, and allosteric modulators. We obtained success rates above 95% for transiently or stably transfected HEK cells and frozen RTA HEK cells expressing GABAA channels. Tissue-derived immortalized cultures of airway smooth muscle cells exhibited a slightly lower recording success rate of 75% using automated patch, which was much higher than the 5% success rate using manual patch clamp technique by the same research group. Responses to agonists, antagonists, and allosteric modulators compared well to previously reported manual patch results. The data demonstrate that both the biophysics and pharmacologic characterization of GABAA channels in a wide variety of cell formats can be performed using this automated patch clamp system. PMID:22574655

Flow characterization and patch clamp dose responses using jet microfluidics in a tubeless microfluidic device.

PubMed

Resto, Pedro J; Bhat, Abhishek; Stava, Eric; Lor, Chong; Merriam, Elliot; Diaz-Rivera, Ruben E; Pearce, Robert; Blick, Robert; Williams, Justin C

2017-11-01

Surface tension passive pumping is a way to actuate flow without the need for pumps, tubing or valves by using the pressure inside small drop to move liquid via a microfluidic channel. These types of tubeless devices have typically been used in cell biology. Herein we present the use of tubeless devices as a fluid exchange platform for patch clamp electrophysiology. Inertia from high-speed droplets and jets is used to create flow and perform on-the-fly mixing of solutions. These are then flowed over GABA transfected HEK cells under patch in order to perform a dose response analysis. TIRF imaging and electrical recordings are used to study the fluid exchange properties of the microfluidic device, resulting in 0-90% fluid exchange times of hundreds of milliseconds. COMSOL is used to model flow and fluid exchange within the device. Patch-clamping experiments show the ability to use high-speed passive pumping and its derivatives for studying peak dose responses, but not for studying ion channel kinetics. Our system results in fluid exchange times slower than when using a standard 12-barrel application system and is not as stable as traditional methods, but it offers a new platform with added functionality. Surface tension passive pumping and tubeless devices can be used in a limited fashion for electrophysiology. Users may obtain peak dose responses but the system, in its current form, is not capable of fluid exchange fast enough to study the kinetics of most ion channels. Copyright © 2017 Elsevier B.V. All rights reserved.

Patch-Clamp Recording from Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes: Improving Action Potential Characteristics through Dynamic Clamp

PubMed Central

Veerman, Christiaan C.; Zegers, Jan G.; Mengarelli, Isabella; Bezzina, Connie R.

2017-01-01

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) hold great promise for studying inherited cardiac arrhythmias and developing drug therapies to treat such arrhythmias. Unfortunately, until now, action potential (AP) measurements in hiPSC-CMs have been hampered by the virtual absence of the inward rectifier potassium current (IK1) in hiPSC-CMs, resulting in spontaneous activity and altered function of various depolarising and repolarising membrane currents. We assessed whether AP measurements in “ventricular-like” and “atrial-like” hiPSC-CMs could be improved through a simple, highly reproducible dynamic clamp approach to provide these cells with a substantial IK1 (computed in real time according to the actual membrane potential and injected through the patch-clamp pipette). APs were measured at 1 Hz using perforated patch-clamp methodology, both in control cells and in cells treated with all-trans retinoic acid (RA) during the differentiation process to increase the number of cells with atrial-like APs. RA-treated hiPSC-CMs displayed shorter APs than control hiPSC-CMs and this phenotype became more prominent upon addition of synthetic IK1 through dynamic clamp. Furthermore, the variability of several AP parameters decreased upon IK1 injection. Computer simulations with models of ventricular-like and atrial-like hiPSC-CMs demonstrated the importance of selecting an appropriate synthetic IK1. In conclusion, the dynamic clamp-based approach of IK1 injection has broad applicability for detailed AP measurements in hiPSC-CMs. PMID:28867785

Localized Patch Clamping of Plasma Membrane of a Polarized Plant Cell 1

PubMed Central

Taylor, Alison R.; Brownlee, Colin

1992-01-01

We used an ultraviolet laser to rupture a small region of cell wall of a polarized Fucus spiralis rhizoid cell and gained localized access to the plasma membrane at the growing apex. Careful control of cell turgor enabled a small portion of plasma membrane-bound cytoplasm to be exposed. Gigaohm seals allowing single-channel recordings were obtained with a high success rate using this method with conventional patch clamp techniques. ImagesFigure 1 PMID:16669092

Monitoring and quantifying the passive transport of molecules through patch-clamp suspended real and model cell membranes.

PubMed

Messina, Pierluca; Lemaître, Frédéric; Huet, François; Ngo, Kieu An; Vivier, Vincent; Labbé, Eric; Buriez, Olivier; Amatore, Christian

2014-03-17

Transport of active molecules across biological membranes is a central issue for the success of many pharmaceutical strategies. Herein, we combine the patch-clamp principle with amperometric detection for monitoring fluxes of redox-tagged molecular species across a suspended membrane patched from a macrophage. Solvent- and protein-free lipid bilayers (DPhPC, DOPC, DOPG) patched from single-wall GUV have been thoroughly investigated and the corresponding fluxes measurements quantified. The quality of the patches and their proper sealing were successfully characterized by electrochemical impedance spectroscopy. This procedure appears versatile and perfectly adequate to allow the investigation of transport and quantification of the transport properties through direct measurement of the coefficients of partition and diffusion of the compound in the membrane, thus offering insight on such important biological and pharmacological issues. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Control of cerebellar granule cell output by sensory-evoked Golgi cell inhibition

PubMed Central

Duguid, Ian; Branco, Tiago; Chadderton, Paul; Arlt, Charlotte; Powell, Kate; Häusser, Michael

2015-01-01

Classical feed-forward inhibition involves an excitation–inhibition sequence that enhances the temporal precision of neuronal responses by narrowing the window for synaptic integration. In the input layer of the cerebellum, feed-forward inhibition is thought to preserve the temporal fidelity of granule cell spikes during mossy fiber stimulation. Although this classical feed-forward inhibitory circuit has been demonstrated in vitro, the extent to which inhibition shapes granule cell sensory responses in vivo remains unresolved. Here we combined whole-cell patch-clamp recordings in vivo and dynamic clamp recordings in vitro to directly assess the impact of Golgi cell inhibition on sensory information transmission in the granule cell layer of the cerebellum. We show that the majority of granule cells in Crus II of the cerebrocerebellum receive sensory-evoked phasic and spillover inhibition prior to mossy fiber excitation. This preceding inhibition reduces granule cell excitability and sensory-evoked spike precision, but enhances sensory response reproducibility across the granule cell population. Our findings suggest that neighboring granule cells and Golgi cells can receive segregated and functionally distinct mossy fiber inputs, enabling Golgi cells to regulate the size and reproducibility of sensory responses. PMID:26432880

Microfabricated Patch Clamp Electrodes for Improved Ion Channel Protein Measurements

NASA Astrophysics Data System (ADS)

Klemic, James; Klemic, Kathryn; Reed, Mark; Sigworth, Frederick

2002-03-01

Ion channels are trans-membrane proteins that underlie many cell functions including hormone and neurotransmitter release, muscle contraction and cell signaling cascades. Ion channel proteins are commonly characterized via the patch clamp method in which an extruded glass tube containing ionic solution, manipulated by an expert technician, is brought into contact with a living cell to record ionic current through the cell membrane. Microfabricated planar patch electrodes, micromolded in the silicone elastomer poly-dimethylsiloxane (PDMS) from microlithographically patterned structures, have been developed that improve on this method. Microfabrication techniques allow arrays of patch electrodes to be fabricated, increasing the throughput of the measurement technique. Planar patch electrodes readily allow the automation of cell sealing, further increasing throughput. Microfabricated electrode arrays may be readily integrated with microfluidic structures to allow fast, in situ solution exchange. Miniaturization of the electrode geometry should increase both the signal to noise and the bandwidth of the measurement. Microfabricated patch electrode arrays have been fabricated and measurements have been taken.

ACQ4: an open-source software platform for data acquisition and analysis in neurophysiology research.

PubMed

Campagnola, Luke; Kratz, Megan B; Manis, Paul B

2014-01-01

The complexity of modern neurophysiology experiments requires specialized software to coordinate multiple acquisition devices and analyze the collected data. We have developed ACQ4, an open-source software platform for performing data acquisition and analysis in experimental neurophysiology. This software integrates the tasks of acquiring, managing, and analyzing experimental data. ACQ4 has been used primarily for standard patch-clamp electrophysiology, laser scanning photostimulation, multiphoton microscopy, intrinsic imaging, and calcium imaging. The system is highly modular, which facilitates the addition of new devices and functionality. The modules included with ACQ4 provide for rapid construction of acquisition protocols, live video display, and customizable analysis tools. Position-aware data collection allows automated construction of image mosaics and registration of images with 3-dimensional anatomical atlases. ACQ4 uses free and open-source tools including Python, NumPy/SciPy for numerical computation, PyQt for the user interface, and PyQtGraph for scientific graphics. Supported hardware includes cameras, patch clamp amplifiers, scanning mirrors, lasers, shutters, Pockels cells, motorized stages, and more. ACQ4 is available for download at http://www.acq4.org.

Patch-clamp, ion-sensing, and glutamate-sensing techniques to study glutamate transport in isolated retinal glial cells.

PubMed

Billups, B; Szatkowski, M; Rossi, D; Attwell, D

1998-01-01

We have described how a combination of electrical, ion-sensing, and glutamate-sensing techniques has advanced our understanding of glutamate uptake into isolated salamander retinal glial cells. The next steps in understanding glutamate transport will inevitably depend strongly on molecular biological methods, as described elsewhere in this book, but will also require more detailed study of transporters in their normal environment, perhaps by using patch-clamping or imaging techniques to study cells in situ.

Coexistence of CLCN1 and SCN4A mutations in one family suffering from myotonia.

PubMed

Maggi, Lorenzo; Ravaglia, Sabrina; Farinato, Alessandro; Brugnoni, Raffaella; Altamura, Concetta; Imbrici, Paola; Camerino, Diana Conte; Padovani, Alessandro; Mantegazza, Renato; Bernasconi, Pia; Desaphy, Jean-François; Filosto, Massimiliano

2017-12-01

Non-dystrophic myotonias are characterized by clinical overlap making it challenging to establish genotype-phenotype correlations. We report clinical and electrophysiological findings in a girl and her father concomitantly harbouring single heterozygous mutations in SCN4A and CLCN1 genes. Functional characterization of N1297S hNav1.4 mutant was performed by patch clamp. The patients displayed a mild phenotype, mostly resembling a sodium channel myotonia. The CLCN1 c.501C>G (p.F167L) mutation has been already described in recessive pedigrees, whereas the SCN4A c.3890A>G (p.N1297S) variation is novel. Patch clamp experiments showed impairment of fast and slow inactivation of the mutated Nav1.4 sodium channel. The present findings suggest that analysis of both SCN4A and CLCN1 genes should be considered in myotonic patients with atypical clinical and neurophysiological features.

Series resistance compensation for whole-cell patch-clamp studies using a membrane state estimator

PubMed Central

Sherman, AJ; Shrier, A; Cooper, E

1999-01-01

Whole-cell patch-clamp techniques are widely used to measure membrane currents from isolated cells. While suitable for a broad range of ionic currents, the series resistance (R(s)) of the recording pipette limits the bandwidth of the whole-cell configuration, making it difficult to measure rapid ionic currents. To increase bandwidth, it is necessary to compensate for R(s). Most methods of R(s) compensation become unstable at high bandwidth, making them hard to use. We describe a novel method of R(s) compensation that overcomes the stability limitations of standard designs. This method uses a state estimator, implemented with analog computation, to compute the membrane potential, V(m), which is then used in a feedback loop to implement a voltage clamp; we refer to this as state estimator R(s) compensation. To demonstrate the utility of this approach, we built an amplifier incorporating state estimator R(s) compensation. In benchtop tests, our amplifier showed significantly higher bandwidths and improved stability when compared with a commercially available amplifier. We demonstrated that state estimator R(s) compensation works well in practice by recording voltage-gated Na(+) currents under voltage-clamp conditions from dissociated neonatal rat sympathetic neurons. We conclude that state estimator R(s) compensation should make it easier to measure large rapid ionic currents with whole-cell patch-clamp techniques. PMID:10545359

High-throughput electrophysiological assays for voltage gated ion channels using SyncroPatch 768PE.

PubMed

Li, Tianbo; Lu, Gang; Chiang, Eugene Y; Chernov-Rogan, Tania; Grogan, Jane L; Chen, Jun

2017-01-01

Ion channels regulate a variety of physiological processes and represent an important class of drug target. Among the many methods of studying ion channel function, patch clamp electrophysiology is considered the gold standard by providing the ultimate precision and flexibility. However, its utility in ion channel drug discovery is impeded by low throughput. Additionally, characterization of endogenous ion channels in primary cells remains technical challenging. In recent years, many automated patch clamp (APC) platforms have been developed to overcome these challenges, albeit with varying throughput, data quality and success rate. In this study, we utilized SyncroPatch 768PE, one of the latest generation APC platforms which conducts parallel recording from two-384 modules with giga-seal data quality, to push these 2 boundaries. By optimizing various cell patching parameters and a two-step voltage protocol, we developed a high throughput APC assay for the voltage-gated sodium channel Nav1.7. By testing a group of Nav1.7 reference compounds' IC50, this assay was proved to be highly consistent with manual patch clamp (R > 0.9). In a pilot screening of 10,000 compounds, the success rate, defined by > 500 MΩ seal resistance and >500 pA peak current, was 79%. The assay was robust with daily throughput ~ 6,000 data points and Z' factor 0.72. Using the same platform, we also successfully recorded endogenous voltage-gated potassium channel Kv1.3 in primary T cells. Together, our data suggest that SyncroPatch 768PE provides a powerful platform for ion channel research and drug discovery.

Electrophysiological and Electrochemical Methods Development for the Detection of Biologically Active Chemical Agents

DTIC Science & Technology

1988-11-01

Bilayer ........................................... 14 5. Current-Voltage Curve for Gramacidin in a Lecithin -Sphingomyelin Patch Bilayer... lecithin (Avanti). 9 2. MATERIALS 2.1 Patch Microprobe Instrumentation. The basis of the microprobe system is an AxoPatch Patch- Clamping Amplifier System...histogram of 1024 events cut above 2 pA. Events sampled are thought to be from the same single gramacidin channel in a lecithin : sphingomyelin (5:1) patch

Small Molecules for Early Endosome-Specific Patch Clamping.

PubMed

Chen, Cheng-Chang; Butz, Elisabeth S; Chao, Yu-Kai; Grishchuk, Yulia; Becker, Lars; Heller, Stefan; Slaugenhaupt, Susan A; Biel, Martin; Wahl-Schott, Christian; Grimm, Christian

2017-07-20

To resolve the subcellular distribution of endolysosomal ion channels, we have established a novel experimental approach to selectively patch clamp Rab5 positive early endosomes (EE) versus Rab7/LAMP1-positive late endosomes/lysosomes (LE/LY). To functionally characterize ion channels in endolysosomal membranes with the patch-clamp technique, it is important to develop techniques to selectively enlarge the respective organelles. We found here that two small molecules, wortmannin and latrunculin B, enlarge Rab5-positive EE when combined but not Rab7-, LAMP1-, or Rab11 (RE)-positive vesicles. The two compounds act rapidly, specifically, and are readily applicable in contrast to genetic approaches or previously used compounds such as vacuolin, which enlarges EE, RE, and LE/LY. We apply this approach here to measure currents mediated by TRPML channels, in particular TRPML3, which we found to be functionally active in both EE and LE/LY in overexpressing cells as well as in endogenously expressing CD11b+ lung-tissue macrophages. Copyright © 2017 Elsevier Ltd. All rights reserved.

Flip the tip: an automated, high quality, cost-effective patch clamp screen.

PubMed

Lepple-Wienhues, Albrecht; Ferlinz, Klaus; Seeger, Achim; Schäfer, Arvid

2003-01-01

The race for creating an automated patch clamp has begun. Here, we present a novel technology to produce true gigaseals and whole cell preparations at a high rate. Suspended cells are flushed toward the tip of glass micropipettes. Seal, whole-cell break-in, and pipette/liquid handling are fully automated. Extremely stable seals and access resistance guarantee high recording quality. Data obtained from different cell types sealed inside pipettes show long-term stability, voltage clamp and seal quality, as well as block by compounds in the pM range. A flexible array of independent electrode positions minimizes consumables consumption at maximal throughput. Pulled micropipettes guarantee a proven gigaseal substrate with ultra clean and smooth surface at low cost.

Neurophysiological modification of CA1 pyramidal neurons in a transgenic mouse expressing a truncated form of disrupted-in-schizophrenia 1

PubMed Central

Booth, Clair A; Brown, Jonathan T; Randall, Andrew D

2014-01-01

A t(1;11) balanced chromosomal translocation transects the Disc1 gene in a large Scottish family and produces genome-wide linkage to schizophrenia and recurrent major depressive disorder. This study describes our in vitro investigations into neurophysiological function in hippocampal area CA1 of a transgenic mouse (DISC1tr) that expresses a truncated version of DISC1 designed to reproduce aspects of the genetic situation in the Scottish t(1;11) pedigree. We employed both patch-clamp and extracellular recording methods in vitro to compare intrinsic properties and synaptic function and plasticity between DISC1tr animals and wild-type littermates. Patch-clamp analysis of CA1 pyramidal neurons (CA1-PNs) revealed no genotype dependence in multiple subthreshold parameters, including resting potential, input resistance, hyperpolarization-activated ‘sag’ and resonance properties. Suprathreshold stimuli revealed no alteration to action potential (AP) waveform, although the initial rate of AP production was higher in DISC1tr mice. No difference was observed in afterhyperpolarizing potentials following trains of 5–25 APs at 50 Hz. Patch-clamp analysis of synaptic responses in the Schaffer collateral commissural (SC) pathway indicated no genotype-dependence of paired pulse facilitation, excitatory postsynaptic potential summation or AMPA/NMDA ratio. Extracellular recordings also revealed an absence of changes to SC synaptic responses and indicated input–output and short-term plasticity were also unaltered in the temporoammonic (TA) input. However, in DISC1tr mice theta burst-induced long-term potentiation was enhanced in the SC pathway but completely lost in the TA pathway. These data demonstrate that expressing a truncated form of DISC1 affects intrinsic properties of CA1-PNs and produces pathway-specific effects on long-term synaptic plasticity. PMID:24712988

Cellular mechanisms of desynchronizing effects of hypothermia in an in vitro epilepsy model.

PubMed

Motamedi, Gholam K; Gonzalez-Sulser, Alfredo; Dzakpasu, Rhonda; Vicini, Stefano

2012-01-01

Hypothermia can terminate epileptiform discharges in vitro and in vivo epilepsy models. Hypothermia is becoming a standard treatment for brain injury in infants with perinatal hypoxic ischemic encephalopathy, and it is gaining ground as a potential treatment in patients with drug resistant epilepsy. However, the exact mechanism of action of cooling the brain tissue is unclear. We have studied the 4-aminopyridine model of epilepsy in mice using single- and dual-patch clamp and perforated multi-electrode array recordings from the hippocampus and cortex. Cooling consistently terminated 4-aminopyridine induced epileptiform-like discharges in hippocampal neurons and increased input resistance that was not mimicked by transient receptor potential channel antagonists. Dual-patch clamp recordings showed significant synchrony between distant CA1 and CA3 pyramidal neurons, but less so between the pyramidal neurons and interneurons. In CA1 and CA3 neurons, hypothermia blocked rhythmic action potential discharges and disrupted their synchrony; however, in interneurons, hypothermia blocked rhythmic discharges without abolishing action potentials. In parallel, multi-electrode array recordings showed that synchronized discharges were disrupted by hypothermia, whereas multi-unit activity was unaffected. The differential effect of cooling on transmitting or secreting γ-aminobutyric acid interneurons might disrupt normal network synchrony, aborting the epileptiform discharges. Moreover, the persistence of action potential firing in interneurons would have additional antiepileptic effects through tonic γ-aminobutyric acid release.

Automated navigation of a glass micropipette on a high-density microelectrode array.

PubMed

Jing Lin; Obien, Marie Engelene J; Hierlemann, Andreas; Frey, Urs

2015-08-01

High-density microelectrode arrays (HDMEAs) provide the capability to monitor the extracellular electric potential of multiple neurons at subcellular resolution over extended periods of time. In contrast, patch clamp allows for intracellular, sub-threshold recordings from a single patched neuron for very limited time on the order of an hour. Therefore, it will be beneficial to combine HDMEA and patch clamp for simultaneous intra- and extracellular recording of neuronal activity. Previously, it has been shown that the HDMEA can be used to localize and steer a glass micropipette towards a target location without using an optical microscope [1]. Here, we present an automated system, implemented in LabVIEW, which automatically locates and moves the glass micropipette towards a user-defined target. The presented system constitutes a first step towards developing an automated system to navigate a pipette to patch a neuron in vitro.

Meclofenamic acid blocks the gap junction communication between the retinal pigment epithelial cells.

PubMed

Ning, N; Wen, Y; Li, Y; Li, J

2013-11-01

Nonsteroidal anti-inflammatory drugs (NSAIDs) are commonly used to manage the pain and inflammation. NSAIDs can cause serious side effects, including vision problems. However, the underlying mechanisms are still unclear. Therefore, we aimed to investigate the effect of meclofenamic acid (MFA) on retinal pigment epithelium (RPE). In our study, we applied image analysis and whole-cell patch clamp recording to directly measure the effect of MFA on the gap junctional coupling between RPE cells. Analysis of Lucifer yellow (LY) transfer revealed that the gap junction communication existed between RPE cells. Functional experiments using the whole-cell configuration of the patch clamp technique showed that a gap junction conductance also existed between this kind of cells. Importantly, MFA largely inhibited the gap junction conductance and induced the uncoupling of RPE cells. Other NSAIDs, like aspirin and flufenamic acid (FFA), had the same effect. The gap junction functionally existed in RPE cells, which can be blocked by MFA. These findings may explain, at least partially, the vision problems with certain clinically used NSAIDs.

Population patch clamp electrophysiology: a breakthrough technology for ion channel screening.

PubMed

Dale, Tim J; Townsend, Claire; Hollands, Emma C; Trezise, Derek J

2007-10-01

Population patch clamp (PPC) is a novel high throughput planar array electrophysiology technique that allows ionic currents to be recorded from populations of cells under voltage clamp. For the drug discovery pharmacologist, PPC promises greater speed and precision than existing methods for screening compounds at voltage-gated ion channel targets. Moreover, certain constitutively active or slow-ligand gated channels that have hitherto proved challenging to screen with planar array electrophysiology (e.g. SK/IK channels) are now more accessible. In this article we review early findings using PPC and provide a perspective on its likely impact on ion channel drug discovery. To support this, we include some new data on ion channel assay duplexing and on modulator assays, approaches that have thus far not been described.

Chronic cranial window with access port for repeated cellular manipulations, drug application, and electrophysiology

PubMed Central

Roome, Christopher J.; Kuhn, Bernd

2014-01-01

Chronic cranial windows have been instrumental in advancing optical studies in vivo, permitting long-term, high-resolution imaging in various brain regions. However, once a window is attached it is difficult to regain access to the brain under the window for cellular manipulations. Here we describe a simple device that combines long term in vivo optical imaging with direct brain access via glass or quartz pipettes and metal, glass, or quartz electrodes for cellular manipulations like dye or drug injections and electrophysiological stimulations or recordings while keeping the craniotomy sterile. Our device comprises a regular cranial window glass coverslip with a drilled access hole later sealed with biocompatible silicone. This chronic cranial window with access port is cheap, easy to manufacture, can be mounted just as the regular chronic cranial window, and is self-sealing after retraction of the pipette or electrode. We demonstrate that multiple injections can be performed through the silicone port by repetitively bolus loading calcium sensitive dye into mouse barrel cortex and recording spontaneous cellular activity over a period of weeks. As an example to the extent of its utility for electrophysiological recording, we describe how simple removal of the silicone seal can permit patch pipette access for whole-cell patch clamp recordings in vivo. During these chronic experiments we do not observe any infections under the window or impairment of animal health. PMID:25426027

Evaluation of Optogenetic Electrophysiology Tools in Human Stem Cell-Derived Cardiomyocytes.

PubMed

Björk, Susann; Ojala, Elina A; Nordström, Tommy; Ahola, Antti; Liljeström, Mikko; Hyttinen, Jari; Kankuri, Esko; Mervaala, Eero

2017-01-01

Current cardiac drug safety assessments focus on hERG channel block and QT prolongation for evaluating arrhythmic risks, whereas the optogenetic approach focuses on the action potential (AP) waveform generated by a monolayer of human cardiomyocytes beating synchronously, thus assessing the contribution of several ion channels on the overall drug effect. This novel tool provides arrhythmogenic sensitizing by light-induced pacing in combination with non-invasive, all-optical measurements of cardiomyocyte APs and will improve assessment of drug-induced electrophysiological aberrancies. With the help of patch clamp electrophysiology measurements, we aimed to investigate whether the optogenetic modifications alter human cardiomyocytes' electrophysiology and how well the optogenetic analyses perform against this gold standard. Patch clamp electrophysiology measurements of non-transduced stem cell-derived cardiomyocytes compared to cells expressing the commercially available optogenetic constructs Optopatch and CaViar revealed no significant changes in action potential duration (APD) parameters. Thus, inserting the optogenetic constructs into cardiomyocytes does not significantly affect the cardiomyocyte's electrophysiological properties. When comparing the two methods against each other (patch clamp vs. optogenetic imaging) we found no significant differences in APD parameters for the Optopatch transduced cells, whereas the CaViar transduced cells exhibited modest increases in APD-values measured with optogenetic imaging. Thus, to broaden the screen, we combined optogenetic measurements of membrane potential and calcium transients with contractile motion measured by video motion tracking. Furthermore, to assess how optogenetic measurements can predict changes in membrane potential, or early afterdepolarizations (EADs), cells were exposed to cumulating doses of E-4031, a hERG potassium channel blocker, and drug effects were measured at both spontaneous and paced beating rates (1, 2 Hz). Cumulating doses of E-4031 produced prolonged APDs, followed by EADs and drug-induced quiescence. These observations were corroborated by patch clamp and contractility measurements. Similar responses, although more modest were seen with the I Ks potassium channel blocker JNJ-303. In conclusion, optogenetic measurements of AP waveforms combined with optical pacing compare well with the patch clamp gold standard. Combined with video motion contractile measurements, optogenetic imaging provides an appealing alternative for electrophysiological screening of human cardiomyocyte responses in pharmacological efficacy and safety testings.

Evaluation of Optogenetic Electrophysiology Tools in Human Stem Cell-Derived Cardiomyocytes

PubMed Central

Björk, Susann; Ojala, Elina A.; Nordström, Tommy; Ahola, Antti; Liljeström, Mikko; Hyttinen, Jari; Kankuri, Esko; Mervaala, Eero

2017-01-01

Current cardiac drug safety assessments focus on hERG channel block and QT prolongation for evaluating arrhythmic risks, whereas the optogenetic approach focuses on the action potential (AP) waveform generated by a monolayer of human cardiomyocytes beating synchronously, thus assessing the contribution of several ion channels on the overall drug effect. This novel tool provides arrhythmogenic sensitizing by light-induced pacing in combination with non-invasive, all-optical measurements of cardiomyocyte APs and will improve assessment of drug-induced electrophysiological aberrancies. With the help of patch clamp electrophysiology measurements, we aimed to investigate whether the optogenetic modifications alter human cardiomyocytes' electrophysiology and how well the optogenetic analyses perform against this gold standard. Patch clamp electrophysiology measurements of non-transduced stem cell-derived cardiomyocytes compared to cells expressing the commercially available optogenetic constructs Optopatch and CaViar revealed no significant changes in action potential duration (APD) parameters. Thus, inserting the optogenetic constructs into cardiomyocytes does not significantly affect the cardiomyocyte's electrophysiological properties. When comparing the two methods against each other (patch clamp vs. optogenetic imaging) we found no significant differences in APD parameters for the Optopatch transduced cells, whereas the CaViar transduced cells exhibited modest increases in APD-values measured with optogenetic imaging. Thus, to broaden the screen, we combined optogenetic measurements of membrane potential and calcium transients with contractile motion measured by video motion tracking. Furthermore, to assess how optogenetic measurements can predict changes in membrane potential, or early afterdepolarizations (EADs), cells were exposed to cumulating doses of E-4031, a hERG potassium channel blocker, and drug effects were measured at both spontaneous and paced beating rates (1, 2 Hz). Cumulating doses of E-4031 produced prolonged APDs, followed by EADs and drug-induced quiescence. These observations were corroborated by patch clamp and contractility measurements. Similar responses, although more modest were seen with the IKs potassium channel blocker JNJ-303. In conclusion, optogenetic measurements of AP waveforms combined with optical pacing compare well with the patch clamp gold standard. Combined with video motion contractile measurements, optogenetic imaging provides an appealing alternative for electrophysiological screening of human cardiomyocyte responses in pharmacological efficacy and safety testings. PMID:29163220

A setup for combined multiphoton laser scanning microscopic and multi-electrode patch clamp experiments on brain slices

NASA Astrophysics Data System (ADS)

Helm, P. Johannes; Reppen, Trond; Heggelund, Paul

2009-02-01

Multi Photon Laser Scanning Microscopy (MPLSM) appears today as one of the most powerful experimental tools in cellular neurophysiology, notably in studies of the functional dynamics of signal processing in single neurons. Simultaneous recording of fluorescence signals at high spatial and temporal resolution and electric signals by means of multi electrode patch clamp techniques have provided new paths for the systematic investigation of neuronal mechanisms. In particular, this approach has opened for direct studies of dendritic signal processing in neurons. We report about a setup optimized for simultaneous electrophysiological multi electrode patch clamp and multi photon laser scanning fluorescence microscopic experiments on brain slices. The microscopic system is based on a modified commercially available confocal scanning laser microscope (CLSM). From a technical and operational point of view, two developments are important: Firstly, in order to reduce the workload for the experimentalist, who in general is forced to concentrate on controlling the electrophysiological parameters during the recordings, a system of shutters has been installed together with dedicated electronic modules protecting the photo detectors against destructive light levels caused by erroneous opening or closing of microscopic light paths by the experimentalist. Secondly, the standard detection unit has been improved by installing the photomultiplier tubes (PMT) in a Peltier cooled thermal box shielding the detector from both room temperature and distortions caused by external electromagnetic fields. The electrophysiological system is based on an industrial standard multi patch clamp unit ergonomically arranged around the microscope stage. The electrophysiological and scanning processes can be time coordinated by standard trigger electronics.

Automated Electrophysiology Makes the Pace for Cardiac Ion Channel Safety Screening

PubMed Central

Möller, Clemens; Witchel, Harry

2011-01-01

The field of automated patch-clamp electrophysiology has emerged from the tension between the pharmaceutical industry’s need for high-throughput compound screening versus its need to be conservative due to regulatory requirements. On the one hand, hERG channel screening was increasingly requested for new chemical entities, as the correlation between blockade of the ion channel coded by hERG and torsades de pointes cardiac arrhythmia gained increasing attention. On the other hand, manual patch-clamping, typically quoted as the “gold-standard” for understanding ion channel function and modulation, was far too slow (and, consequently, too expensive) for keeping pace with the numbers of compounds submitted for hERG channel investigations from pharmaceutical R&D departments. In consequence it became more common for some pharmaceutical companies to outsource safety pharmacological investigations, with a focus on hERG channel interactions. This outsourcing has allowed those pharmaceutical companies to build up operational flexibility and greater independence from internal resources, and allowed them to obtain access to the latest technological developments that emerged in automated patch-clamp electrophysiology – much of which arose in specialized biotech companies. Assays for nearly all major cardiac ion channels are now available by automated patch-clamping using heterologous expression systems, and recently, automated action potential recordings from stem-cell derived cardiomyocytes have been demonstrated. Today, most of the large pharmaceutical companies have acquired automated electrophysiology robots and have established various automated cardiac ion channel safety screening assays on these, in addition to outsourcing parts of their needs for safety screening. PMID:22131974

Obtaining spheroplasts of armored dinoflagellates and first single-channel recordings of their ion channels using patch-clamping.

PubMed

Pozdnyakov, Ilya; Matantseva, Olga; Negulyaev, Yuri; Skarlato, Sergei

2014-09-05

Ion channels are tightly involved in various aspects of cell physiology, including cell signaling, proliferation, motility, endo- and exo-cytosis. They may be involved in toxin production and release by marine dinoflagellates, as well as harmful algal bloom proliferation. So far, the patch-clamp technique, which is the most powerful method to study the activity of ion channels, has not been applied to dinoflagellate cells, due to their complex cellulose-containing cell coverings. In this paper, we describe a new approach to overcome this problem, based on the preparation of spheroplasts from armored bloom-forming dinoflagellate Prorocentrum minimum. We treated the cells of P. minimum with a cellulose synthesis inhibitor, 2,6-dichlorobenzonitrile (DCB), and found out that it could also induce ecdysis and arrest cell shape maintenance in these microalgae. Treatment with 100-250 µM DCB led to an acceptable 10% yield of P. minimum spheroplasts and was independent of the incubation time in the range of 1-5 days. We show that such spheroplasts are suitable for patch-clamping in the cell-attached mode and can form 1-10 GOhm patch contact with a glass micropipette, allowing recording of ion channel activity. The first single-channel recordings of dinoflagellate ion channels are presented.

Obtaining Spheroplasts of Armored Dinoflagellates and First Single-Channel Recordings of Their Ion Channels Using Patch-Clamping

PubMed Central

Pozdnyakov, Ilya; Matantseva, Olga; Negulyaev, Yuri; Skarlato, Sergei

2014-01-01

Ion channels are tightly involved in various aspects of cell physiology, including cell signaling, proliferation, motility, endo- and exo-cytosis. They may be involved in toxin production and release by marine dinoflagellates, as well as harmful algal bloom proliferation. So far, the patch-clamp technique, which is the most powerful method to study the activity of ion channels, has not been applied to dinoflagellate cells, due to their complex cellulose-containing cell coverings. In this paper, we describe a new approach to overcome this problem, based on the preparation of spheroplasts from armored bloom-forming dinoflagellate Prorocentrum minimum. We treated the cells of P. minimum with a cellulose synthesis inhibitor, 2,6-dichlorobenzonitrile (DCB), and found out that it could also induce ecdysis and arrest cell shape maintenance in these microalgae. Treatment with 100–250 µM DCB led to an acceptable 10% yield of P. minimum spheroplasts and was independent of the incubation time in the range of 1–5 days. We show that such spheroplasts are suitable for patch-clamping in the cell-attached mode and can form 1–10 GOhm patch contact with a glass micropipette, allowing recording of ion channel activity. The first single-channel recordings of dinoflagellate ion channels are presented. PMID:25199048

ACQ4: an open-source software platform for data acquisition and analysis in neurophysiology research

PubMed Central

Campagnola, Luke; Kratz, Megan B.; Manis, Paul B.

2014-01-01

The complexity of modern neurophysiology experiments requires specialized software to coordinate multiple acquisition devices and analyze the collected data. We have developed ACQ4, an open-source software platform for performing data acquisition and analysis in experimental neurophysiology. This software integrates the tasks of acquiring, managing, and analyzing experimental data. ACQ4 has been used primarily for standard patch-clamp electrophysiology, laser scanning photostimulation, multiphoton microscopy, intrinsic imaging, and calcium imaging. The system is highly modular, which facilitates the addition of new devices and functionality. The modules included with ACQ4 provide for rapid construction of acquisition protocols, live video display, and customizable analysis tools. Position-aware data collection allows automated construction of image mosaics and registration of images with 3-dimensional anatomical atlases. ACQ4 uses free and open-source tools including Python, NumPy/SciPy for numerical computation, PyQt for the user interface, and PyQtGraph for scientific graphics. Supported hardware includes cameras, patch clamp amplifiers, scanning mirrors, lasers, shutters, Pockels cells, motorized stages, and more. ACQ4 is available for download at http://www.acq4.org. PMID:24523692

Laser-assisted patch clamping: a methodology

NASA Technical Reports Server (NTRS)

Henriksen, G. H.; Assmann, S. M.; Evans, M. L. (Principal Investigator)

1997-01-01

Laser microsurgery can be used to perform both cell biological manipulations, such as targeted cell ablation, and molecular genetic manipulations, such as genetic transformation and chromosome dissection. In this report, we describe a laser microsurgical method that can be used either to ablate single cells or to ablate a small area (1-3 microns diameter) of the extracellular matrix. In plants and microorganisms, the extracellular matrix consists of the cell wall. While conventional patch clamping of these cells, as well as of many animal cells, requires enzymatic digestion of the extracellular matrix, we illustrate that laser microsurgery of a portion of the wall enables patch clamp access to the plasma membrane of higher plant cells remaining situated in their tissue environment. What follows is a detailed description of the construction and use of an economical laser microsurgery system, including procedures for single cell and targeted cell wall ablation. This methodology will be of interest to scientists wishing to perform cellular or subcellular ablation with a high degree of accuracy, or wishing to study how the extracellular matrix affects ion channel function.

Biological cell controllable patch-clamp microchip

NASA Astrophysics Data System (ADS)

Penmetsa, Siva; Nagrajan, Krithika; Gong, Zhongcheng; Mills, David; Que, Long

2010-12-01

A patch-clamp (PC) microchip with cell sorting and positioning functions is reported, which can avoid drawbacks of random cell selection or positioning for a PC microchip. The cell sorting and positioning are enabled by air bubble (AB) actuators. AB actuators are pneumatic actuators, in which air pressure is generated by microheaters within sealed microchambers. The sorting, positioning, and capturing of 3T3 cells by this type of microchip have been demonstrated. Using human breast cancer cells MDA-MB-231 as the model, experiments have been demonstrated by this microchip as a label-free technical platform for real-time monitoring of the cell viability.

Nanosecond laser pulse stimulation of spiral ganglion neurons and model cells.

PubMed

Rettenmaier, Alexander; Lenarz, Thomas; Reuter, Günter

2014-04-01

Optical stimulation of the inner ear has recently attracted attention, suggesting a higher frequency resolution compared to electrical cochlear implants due to its high spatial stimulation selectivity. Although the feasibility of the effect is shown in multiple in vivo experiments, the stimulation mechanism remains open to discussion. Here we investigate in single-cell measurements the reaction of spiral ganglion neurons and model cells to irradiation with a nanosecond-pulsed laser beam over a broad wavelength range from 420 nm up to 1950 nm using the patch clamp technique. Cell reactions were wavelength- and pulse-energy-dependent but too small to elicit action potentials in the investigated spiral ganglion neurons. As the applied radiant exposure was much higher than the reported threshold for in vivo experiments in the same laser regime, we conclude that in a stimulation paradigm with nanosecond-pulses, direct neuronal stimulation is not the main cause of optical cochlea stimulation.

The assessment of electrophysiological activity in human-induced pluripotent stem cell-derived cardiomyocytes exposed to dimethyl sulfoxide and ethanol by manual patch clamp and multi-electrode array system.

PubMed

Hyun, Soo-Wang; Kim, Bo-Ram; Hyun, Sung-Ae; Seo, Joung-Wook

2017-09-01

Recently, electrophysiological activity has been effectively measured in human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) to predict drug-induced arrhythmia. Dimethyl sulfoxide (DMSO) and ethanol have been used as diluting agents in many experiments. However, the maximum DMSO and ethanol concentrations that can be effectively used in the measurement of electrophysiological parameters in hiPSC-CMs-based patch clamp and multi-electrode array (MEA) have not been fully elucidated. We investigated the effects of varying concentrations of DMSO and ethanol used as diluting agents on several electrophysiological parameters in hiPSC-CMs using patch clamp and MEA. Both DMSO and ethanol at concentrations>1% in external solution resulted in osmolality >400mOsmol/kg, but pH was not affected by either agent. Neither DMSO nor ethanol led to cell death at the concentrations examined. However, resting membrane potential, action potential amplitude, action potential duration at 90% and 40%, and corrected field potential duration were decreased significantly at 1% ethanol concentration. DMSO at 1% also significantly decreased the sodium spike amplitude. In addition, the waveform of action potential and field potential was recorded as irregular at 3% concentrations of both DMSO and ethanol. Concentrations of up to 0.3% of either agent did not affect osmolality, pH, cell death, or electrophysiological parameters in hiPSC-CMs. Our findings suggest that 0.3% is the maximum concentration at which DMSO or ethanol should be used for dilution purposes in hiPSC-CMs-based patch clamp and MEA. Copyright © 2017 Elsevier Inc. All rights reserved.

Validation of a patch clamp screening protocol that simultaneously measures compound activity in multiple states of the voltage-gated sodium channel Nav1.2.

PubMed

Liu, Yi; Beck, Edward J; Flores, Christopher M

2011-12-01

Hyperactivity of voltage-gated sodium channels underlies, at least in part, a range of pathological states, including pain and epilepsy. Selective blockers of these channels may offer effective treatment of such disorders. Currently employed methods to screen for sodium channel blockers, however, are inadequate to rationally identify mechanistically diverse blockers, limiting the potential range of indications that may be treated by such agents. Here, we describe an improved patch clamp screening assay that increases the mechanistic diversity of sodium channel blockers being identified. Using QPatch HT, a medium-throughput, automated patch clamp system, we tested three common sodium channel blockers (phenytoin, lidocaine, and tetrodotoxin) with distinct mechanistic profiles at Nav1.2. The single-voltage protocol employed in this assay simultaneously measured the compound activity in multiple states, including the slow inactivated state, of the channel. A long compound incubation period (10 s) was introduced during channel inactivation to increase the probability of identifying "slow binders." As such, phenytoin, which preferentially binds with slow kinetics to the fast inactivated state, exhibited significantly higher potency than that obtained from a brief exposure (100 ms) used in typical assays. This assay also successfully detected the use-dependent block of tetrodotoxin, a well-documented property of this molecule yet unobserved in typical patch clamp protocols. These results indicate that the assay described here can increase the likelihood of identification and mechanistic diversity of sodium channel blockers from a primary screen. It can also be used to efficiently guide the in vitro optimization of leads that retain the desired mechanistic properties. © MARY ANN LIEBERT, INC.

Opto-current-clamp actuation of cortical neurons using a strategically designed channelrhodopsin.

PubMed

Wen, Lei; Wang, Hongxia; Tanimoto, Saki; Egawa, Ryo; Matsuzaka, Yoshiya; Mushiake, Hajime; Ishizuka, Toru; Yawo, Hiromu

2010-09-23

Optogenetic manipulation of a neuronal network enables one to reveal how high-order functions emerge in the central nervous system. One of the Chlamydomonas rhodopsins, channelrhodopsin-1 (ChR1), has several advantages over channelrhodopsin-2 (ChR2) in terms of the photocurrent kinetics. Improved temporal resolution would be expected by the optogenetics using the ChR1 variants with enhanced photocurrents. The photocurrent retardation of ChR1 was overcome by exchanging the sixth helix domain with its counterpart in ChR2 producing Channelrhodopsin-green receiver (ChRGR) with further reform of the molecule. When the ChRGR photocurrent was measured from the expressing HEK293 cells under whole-cell patch clamp, it was preferentially activated by green light and has fast kinetics with minimal desensitization. With its kinetic advantages the use of ChRGR would enable one to inject a current into a neuron by the time course as predicted by the intensity of the shedding light (opto-current clamp). The ChRGR was also expressed in the motor cortical neurons of a mouse using Sindbis pseudovirion vectors. When an oscillatory LED light signal was applied sweeping through frequencies, it robustly evoked action potentials synchronized to the oscillatory light at 5-10 Hz in layer 5 pyramidal cells in the cortical slice. The ChRGR-expressing neurons were also driven in vivo with monitoring local field potentials (LFPs) and the time-frequency energy distribution of the light-evoked response was investigated using wavelet analysis. The oscillatory light enhanced both the in-phase and out-phase responses of LFP at the preferential frequencies of 5-10 Hz. The spread of activity was evidenced by the fact that there were many c-Fos-immunoreactive neurons that were negative for ChRGR in a region of the motor cortex. The opto-current-clamp study suggests that the depolarization of a small number of neurons wakes up the motor cortical network over some critical point to the activated state.

Detergent Induction of HEK 293A Cell Membrane Permeability Measured under Quiescent and Superfusion Conditions Using Whole Cell Patch Clamp

PubMed Central

2015-01-01

Detergents have several biological applications but present cytotoxicity concerns, since they can solubilize cell membranes. Using the IonFlux 16, an ensemble whole cell planar patch clamp, we observed that anionic sodium dodecyl sulfate (SDS), cationic cetyltrimethylammonium bromide (CTAB), and cationic, fluorescent octadecyl rhodamine B (ORB) increased the membrane permeability of cells substantially within a second of exposure, under superfusion conditions. Increased permeability was irreversible for 15 min. At subsolubilizing detergent concentrations, patched cells showed increased membrane currents that reached a steady state and were intact when imaged using fluorescence microscopy. SDS solubilized cells at concentrations of 2 mM (2× CMC), while CTAB did not solubilize cells even at concentrations of 10 mM (1000× CMC). The relative activity for plasma membrane current induction was 1:20:14 for SDS, CTAB, and ORB, respectively. Under quiescent conditions, the relative ratio of lipid to detergent in cell membranes at the onset of membrane permeability was 1:7:5 for SDS, CTAB, and ORB, respectively. The partition constants (K) for SDS, CTAB, and ORB were 23000, 55000, and 39000 M–1, respectively. Combining the whole cell patch clamp data and XTT viability data, SDS ≤ 0.2 mM and CTAB and ORB ≤ 1 mM induced cell membrane permeability without causing acute toxicity. PMID:24548291

Enhanced Monitoring of Nanosecond Electric Pulse-Evoked Membrane Conductance Changes in Whole-Cell Patch Clamp Experiments.

PubMed

Yoon, Jihwan; Leblanc, Normand; Zaklit, Josette; Vernier, P Thomas; Chatterjee, Indira; Craviso, Gale L

2016-10-01

Patch clamp electrophysiology serves as a powerful method for studying changes in plasma membrane ion conductance induced by externally applied high-intensity nanosecond electric pulses (NEPs). This paper describes an enhanced monitoring technique that minimizes the length of time between pulse exposure and data recording in a patch-clamped excitable cell. Whole-cell membrane currents were continuously recorded up to 11 ms before and resumed 8 ms after delivery of a 5-ns, 6 MV/m pulse by a pair of tungsten rod electrodes to a patched adrenal chromaffin cell maintained at a holding potential of -70 mV. This timing was achieved by two sets of relay switches. One set was used to disconnect the patch pipette electrode from the pre-amplifier and connect it to a battery to maintain membrane potential at -70 mV, and also to disconnect the reference electrode from the amplifier. The other set was used to disconnect the electrodes from the pulse generator until the time of NEP/sham exposure. The sequence and timing of both sets of relays were computer-controlled. Using this procedure, we observed that a 5-ns pulse induced an instantaneous inward current that decayed exponentially over the course of several minutes, that a second pulse induced a similar response, and that the current was carried, at least in part, by Na + . This approach for characterizing ion conductance changes in an excitable cell in response to NEPs will yield information essential for assessing the potential use of NEP stimulation for therapeutic applications.

NUMERICAL SIMULATION OF NANOINDENTATION AND PATCH CLAMP EXPERIMENTS ON MECHANOSENSITIVE CHANNELS OF LARGE CONDUCTANCE IN ESCHERICHIA COLI

PubMed Central

Tang, Yuye; Chen, Xi; Yoo, Jejoong; Yethiraj, Arun; Cui, Qiang

2010-01-01

A hierarchical simulation framework that integrates information from all-atom simulations into a finite element model at the continuum level is established to study the mechanical response of a mechanosensitive channel of large conductance (MscL) in bacteria Escherichia Coli (E.coli) embedded in a vesicle formed by the dipalmitoylphosphatidycholine (DPPC) lipid bilayer. Sufficient structural details of the protein are built into the continuum model, with key parameters and material properties derived from molecular mechanics simulations. The multi-scale framework is used to analyze the gating of MscL when the lipid vesicle is subjective to nanoindentation and patch clamp experiments, and the detailed structural transitions of the protein are obtained explicitly as a function of external load; it is currently impossible to derive such information based solely on all-atom simulations. The gating pathways of E.coli-MscL qualitatively agree with results from previous patch clamp experiments. The gating mechanisms under complex indentation-induced deformation are also predicted. This versatile hierarchical multi-scale framework may be further extended to study the mechanical behaviors of cells and biomolecules, as well as to guide and stimulate biomechanics experiments. PMID:21874098

High-Content Electrophysiological Analysis of Human Pluripotent Stem Cell-Derived Cardiomyocytes (hPSC-CMs).

PubMed

Kong, Chi-Wing; Geng, Lin; Li, Ronald A

2018-01-01

Considerable interest has been raised to develop human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) as a model for drug discovery and cardiotoxicity screening. High-content electrophysiological analysis of currents generated by transmembrane cell surface ion channels has been pursued to complement such emerging applications. Here we describe practical procedures and considerations for accomplishing successful assays of hPSC-CMs using an automated planar patch-clamp system.

Giga-ohm seals on intracellular membranes: a technique for studying intracellular ion channels in intact cells.

PubMed

Jonas, E A; Knox, R J; Kaczmarek, L K

1997-07-01

A method is outlined for obtaining giga-ohm seals on intracellular membranes in intact cells. The technique employs a variant of the patch-clamp technique: a concentric electrode arrangement protects an inner patch pipette during penetration of the plasma membrane, after which a seal can be formed on an internal organelle membrane. Using this technique, successful recordings can be obtained with the same frequency as with conventional patch clamping. To localize the position of the pipette within cells, lipophilic fluorescent dyes are included in the pipette solution. These dyes stain the membrane of internal organelles during seal formation and can then be visualized by video-enhanced or confocal imaging. The method can detect channels activated by inositol trisphosphate, as well as other types of intracellular membrane ion channel activity, and should facilitate studies of internal membranes in intact neurons and other cell types.

Voltage clamp methods for the study of membrane currents and SR Ca2+ release in adult skeletal muscle fibres

PubMed Central

Hernández-Ochoa, Erick O.; Schneider, Martin F.

2012-01-01

Skeletal muscle excitation-contraction (E-C)1 coupling is a process composed of multiple sequential stages, by which an action potential triggers sarcoplasmic reticulum (SR)2 Ca2+ release and subsequent contractile activation. The various steps in the E-C coupling process in skeletal muscle can be studied using different techniques. The simultaneous recordings of sarcolemmal electrical signals and the accompanying elevation in myoplasmic Ca2+, due to depolarization-initiated SR Ca2+ release in skeletal muscle fibres, have been useful to obtain a better understanding of muscle function. In studying the origin and mechanism of voltage dependency of E-C coupling a variety of different techniques have been used to control the voltage in adult skeletal fibres. Pioneering work in muscles isolated from amphibians or crustaceans used microelectrodes or ‘high resistance gap’ techniques to manipulate the voltage in the muscle fibres. The development of the patch clamp technique and its variant, the whole-cell clamp configuration that facilitates the manipulation of the intracellular environment, allowed the use of the voltage clamp techniques in different cell types, including skeletal muscle fibres. The aim of this article is to present an historical perspective of the voltage clamp methods used to study skeletal muscle E-C coupling as well as to describe the current status of using the whole-cell patch clamp technique in studies in which the electrical and Ca2+ signalling properties of mouse skeletal muscle membranes are being investigated. PMID:22306655

VOLTAGE CLAMP BEHAVIOR OF IRON-NITRIC ACID SYSTEM AS COMPARED WITH THAT OF NERVE MEMBRANE

PubMed Central

Tasaki, I.; Bak, A. F.

1959-01-01

The current-voltage relation for the surface layer of an iron wire immersed in nitric acid was investigated by the voltage clamp technique. Comparing the phase of nitric acid to the axoplasm and the metallic phase to the external fluid medium for the nerve fiber, a striking analogy was found between the voltage clamp behavior of the iron-nitric acid system and that of the nerve membrane. The current voltage curve was found to consist of three parts: (a) a straight line representing the behavior of the resting (passive) membrane, (b) a straight line representing the fully excited (active) state, and (c) an intermediate zone connecting (a) and (b). It was shown that in the intermediate zone, the surface of iron consisted of a fully active patch (or patches) surrounded by a remaining resting area. The phenomenon corresponding to "repetitive firing of responses under voltage clamp" in the nerve membrane was demonstrated in the intermediate zone. The behavior of the cobalt electrode system was also investigated by the same technique. An attempt was made to interpret the phenomenon of initiation and abolition of an active potential on the basis of the thermodynamics of irreversible processes. PMID:13654740

Ionic requirements for membrane-glass adhesion and giga seal formation in patch-clamp recording.

PubMed

Priel, Avi; Gil, Ziv; Moy, Vincent T; Magleby, Karl L; Silberberg, Shai D

2007-06-01

Patch-clamp recording has revolutionized the study of ion channels, transporters, and the electrical activity of small cells. Vital to this method is formation of a tight seal between glass recording pipette and cell membrane. To better understand seal formation and improve practical application of this technique, we examine the effects of divalent ions, protons, ionic strength, and membrane proteins on adhesion of membrane to glass and on seal resistance using both patch-clamp recording and atomic force microscopy. We find that H(+), Ca(2+), and Mg(2+) increase adhesion force between glass and membrane (lipid and cellular), decrease the time required to form a tight seal, and increase seal resistance. In the absence of H(+) (10(-10) M) and divalent cations (<10(-8) M), adhesion forces are greatly reduced and tight seals are not formed. H(+) (10(-7) M) promotes seal formation in the absence of divalent cations. A positive correlation between adhesion force and seal formation indicates that high resistance seals are associated with increased adhesion between membrane and glass. A similar ionic dependence of the adhesion of lipid membranes and cell membranes to glass indicates that lipid membranes without proteins are sufficient for the action of ions on adhesion.

Automated and manual patch clamp data of human induced pluripotent stem cell-derived dopaminergic neurons.

PubMed

Franz, Denise; Olsen, Hervør Lykke; Klink, Oliver; Gimsa, Jan

2017-04-25

Human induced pluripotent stem cells can be differentiated into dopaminergic neurons (Dopa.4U). Dopa.4U neurons expressed voltage-gated Na V and K V channels and showed neuron-like spontaneous electrical activity. In automated patch clamp measurements with suspended Dopa.4U neurons, delayed rectifier K + current (delayed K V ) and rapidly inactivating A-type K + current (fast K V ) were identified. Examination of the fast K V current with inhibitors yielded IC 50 values of 0.4 mM (4-aminopyridine) and 0.1 mM (tetraethylammonium). In manual patch clamp measurements with adherent Dopa.4U neurons, fast K V current could not be detected, while the delayed K V current showed an IC 50 of 2 mM for 4-aminopyridine. The Na V channels in adherent and suspended Dopa.4U neurons showed IC 50 values for tetrodotoxin of 27 and 2.9 nM, respectively. GABA-induced currents that could be observed in adherent Dopa.4U neurons could not be detected in suspended cells. Application of current pulses induced action potentials in approx. 70 % of the cells. Our results proved the feasibility of automated electrophysiological characterization of neuronal cells.

A simple method for characterizing passive and active neuronal properties: application to striatal neurons.

PubMed

Lepora, Nathan F; Blomeley, Craig P; Hoyland, Darren; Bracci, Enrico; Overton, Paul G; Gurney, Kevin

2011-11-01

The study of active and passive neuronal dynamics usually relies on a sophisticated array of electrophysiological, staining and pharmacological techniques. We describe here a simple complementary method that recovers many findings of these more complex methods but relies only on a basic patch-clamp recording approach. Somatic short and long current pulses were applied in vitro to striatal medium spiny (MS) and fast spiking (FS) neurons from juvenile rats. The passive dynamics were quantified by fitting two-compartment models to the short current pulse data. Lumped conductances for the active dynamics were then found by compensating this fitted passive dynamics within the current-voltage relationship from the long current pulse data. These estimated passive and active properties were consistent with previous more complex estimations of the neuron properties, supporting the approach. Relationships within the MS and FS neuron types were also evident, including a graduation of MS neuron properties consistent with recent findings about D1 and D2 dopamine receptor expression. Application of the method to simulated neuron data supported the hypothesis that it gives reasonable estimates of membrane properties and gross morphology. Therefore detailed information about the biophysics can be gained from this simple approach, which is useful for both classification of neuron type and biophysical modelling. Furthermore, because these methods rely upon no manipulations to the cell other than patch clamping, they are ideally suited to in vivo electrophysiology. © 2011 The Authors. European Journal of Neuroscience © 2011 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.

Visual patch clamp recording of neurons in thick portions of the adult spinal cord.

PubMed

Munch, Anders Sonne; Smith, Morten; Moldovan, Mihai; Perrier, Jean-François

2010-07-15

The study of visually identified neurons in slice preparations from the central nervous system offers considerable advantages over in vivo preparations including high mechanical stability in the absence of anaesthesia and full control of the extracellular medium. However, because of their relative thinness, slices are not appropriate for investigating how individual neurons integrate synaptic inputs generated by large numbers of neurons. Here we took advantage of the exceptional resistance of the turtle to anoxia to make slices of increasing thicknesses (from 300 to 3000 microm) from the lumbar enlargement of the spinal cord. With a conventional upright microscope in which the light condenser was carefully adjusted, we could visualize neurons present at the surface of the slice and record them with the whole-cell patch clamp technique. We show that neurons present in the middle of the preparation remain alive and capable of generating action potentials. By stimulating the lateral funiculus we can evoke intense synaptic activity associated with large increases in conductance of the recorded neurons. The conductance increases substantially more in neurons recorded in thick slices suggesting that the size of the network recruited with the stimulation increases with the thickness of the slices. We also find that that the number of spontaneous excitatory postsynaptic currents (EPSCs) is higher in thick slices compared with thin slices while the number of spontaneous inhibitory postsynaptic currents (IPSCs) remains constant. These preliminary data suggest that inhibitory and excitatory synaptic connections are balanced locally while excitation dominates long-range connections in the spinal cord. Copyright 2010 Elsevier B.V. All rights reserved.

MUSCLEMOTION: A Versatile Open Software Tool to Quantify Cardiomyocyte and Cardiac Muscle Contraction In Vitro and In Vivo.

PubMed

Sala, Luca; van Meer, Berend J; Tertoolen, Leon G J; Bakkers, Jeroen; Bellin, Milena; Davis, Richard P; Denning, Chris; Dieben, Michel A E; Eschenhagen, Thomas; Giacomelli, Elisa; Grandela, Catarina; Hansen, Arne; Holman, Eduard R; Jongbloed, Monique R M; Kamel, Sarah M; Koopman, Charlotte D; Lachaud, Quentin; Mannhardt, Ingra; Mol, Mervyn P H; Mosqueira, Diogo; Orlova, Valeria V; Passier, Robert; Ribeiro, Marcelo C; Saleem, Umber; Smith, Godfrey L; Burton, Francis L; Mummery, Christine L

2018-02-02

There are several methods to measure cardiomyocyte and muscle contraction, but these require customized hardware, expensive apparatus, and advanced informatics or can only be used in single experimental models. Consequently, data and techniques have been difficult to reproduce across models and laboratories, analysis is time consuming, and only specialist researchers can quantify data. Here, we describe and validate an automated, open-source software tool (MUSCLEMOTION) adaptable for use with standard laboratory and clinical imaging equipment that enables quantitative analysis of normal cardiac contraction, disease phenotypes, and pharmacological responses. MUSCLEMOTION allowed rapid and easy measurement of movement from high-speed movies in (1) 1-dimensional in vitro models, such as isolated adult and human pluripotent stem cell-derived cardiomyocytes; (2) 2-dimensional in vitro models, such as beating cardiomyocyte monolayers or small clusters of human pluripotent stem cell-derived cardiomyocytes; (3) 3-dimensional multicellular in vitro or in vivo contractile tissues, such as cardiac "organoids," engineered heart tissues, and zebrafish and human hearts. MUSCLEMOTION was effective under different recording conditions (bright-field microscopy with simultaneous patch-clamp recording, phase contrast microscopy, and traction force microscopy). Outcomes were virtually identical to the current gold standards for contraction measurement, such as optical flow, post deflection, edge-detection systems, or manual analyses. Finally, we used the algorithm to quantify contraction in in vitro and in vivo arrhythmia models and to measure pharmacological responses. Using a single open-source method for processing video recordings, we obtained reliable pharmacological data and measures of cardiac disease phenotype in experimental cell, animal, and human models. © 2017 The Authors.

Expanding neurochemical investigations with multi-modal recording: simultaneous fast-scan cyclic voltammetry, iontophoresis, and patch clamp measurements.

PubMed

Kirkpatrick, D C; McKinney, C J; Manis, P B; Wightman, R M

2016-08-02

Multi-modal recording describes the simultaneous collection of information across distinct domains. Compared to isolated measurements, such studies can more easily determine relationships between varieties of phenomena. This is useful for neurochemical investigations which examine cellular activity in response to changes in the local chemical environment. In this study, we demonstrate a method to perform simultaneous patch clamp measurements with fast-scan cyclic voltammetry (FSCV) using optically isolated instrumentation. A model circuit simulating concurrent measurements was used to predict the electrical interference between instruments. No significant impact was anticipated between methods, and predictions were largely confirmed experimentally. One exception was due to capacitive coupling of the FSCV potential waveform into the patch clamp amplifier. However, capacitive transients measured in whole-cell current clamp recordings were well below the level of biological signals, which allowed the activity of cells to be easily determined. Next, the activity of medium spiny neurons (MSNs) was examined in the presence of an FSCV electrode to determine how the exogenous potential impacted nearby cells. The activities of both resting and active MSNs were unaffected by the FSCV waveform. Additionally, application of an iontophoretic current, used to locally deliver drugs and other neurochemicals, did not affect neighboring cells. Finally, MSN activity was monitored during iontophoretic delivery of glutamate, an excitatory neurotransmitter. Membrane depolarization and cell firing were observed concurrently with chemical changes around the cell resulting from delivery. In all, we show how combined electrophysiological and electrochemical measurements can relate information between domains and increase the power of neurochemical investigations.

High-speed pressure clamp.

PubMed

Besch, Stephen R; Suchyna, Thomas; Sachs, Frederick

2002-10-01

We built a high-speed, pneumatic pressure clamp to stimulate patch-clamped membranes mechanically. The key control element is a newly designed differential valve that uses a single, nickel-plated piezoelectric bending element to control both pressure and vacuum. To minimize response time, the valve body was designed with minimum dead volume. The result is improved response time and stability with a threefold decrease in actuation latency. Tight valve clearances minimize the steady-state air flow, permitting us to use small resonant-piston pumps to supply pressure and vacuum. To protect the valve from water contamination in the event of a broken pipette, an optical sensor detects water entering the valve and increases pressure rapidly to clear the system. The open-loop time constant for pressure is 2.5 ms for a 100-mmHg step, and the closed-loop settling time is 500-600 micros. Valve actuation latency is 120 micros. The system performance is illustrated for mechanically induced changes in patch capacitance.

Principal cells of the brainstem's interaural sound level detector are temporal differentiators rather than integrators.

PubMed

Franken, Tom P; Joris, Philip X; Smith, Philip H

2018-06-14

The brainstem's lateral superior olive (LSO) is thought to be crucial for localizing high-frequency sounds by coding interaural sound level differences (ILD). Its neurons weigh contralateral inhibition against ipsilateral excitation, making their firing rate a function of the azimuthal position of a sound source. Since the very first in vivo recordings, LSO principal neurons have been reported to give sustained and temporally integrating 'chopper' responses to sustained sounds. Neurons with transient responses were observed but largely ignored and even considered a sign of pathology. Using the Mongolian gerbil as a model system, we have obtained the first in vivo patch clamp recordings from labeled LSO neurons and find that principal LSO neurons, the most numerous projection neurons of this nucleus, only respond at sound onset and show fast membrane features suggesting an importance for timing. These results provide a new framework to interpret previously puzzling features of this circuit. © 2018, Franken et al.

An operational amplifier B1404UD1A-1 in the patch-clamp current-to-voltage converter.

PubMed

Korzun, A M; Rozinov, S V; Abashin, G I

1997-01-01

The applicability of the home-made operational amplifier B1404UD1A-1 in a patch-clamp current-to-voltage converter was analyzed. Its parameters (background noise, input bias current, and gain-bandwidth product) were estimated. Schematic solutions and practical recommendations for the use of this amplifier in a current-to-voltage converter were given. Based on the background noise and frequency parameters of the converter, we found that this device can be used for measuring ion channel currents with a high sensitivity and within a broad frequency range (0.055 pA, to 1 kHz; 0.4 pA, to 10 kHz). An example of the converter application in experiments is given.

Recording temperature affects the excitability of mouse superficial dorsal horn neurons, in vitro.

PubMed

Graham, B A; Brichta, A M; Callister, R J

2008-05-01

Superficial dorsal horn (SDH) neurons in laminae I-II of the spinal cord play an important role in processing noxious stimuli. These neurons represent a heterogeneous population and are divided into various categories according to their action potential (AP) discharge during depolarizing current injection. We recently developed an in vivo mouse preparation to examine functional aspects of nociceptive processing and AP discharge in SDH neurons and to extend investigation of pain mechanisms to the genetic level of analysis. Not surprisingly, some in vivo data obtained at body temperature (37 degrees C) differed from those generated at room temperature (22 degrees C) in spinal cord slices. In the current study we examine how temperature influences SDH neuron properties by making recordings at 22 and 32 degrees C in transverse spinal cord slices prepared from L3-L5 segments of adult mice (C57Bl/6). Patch-clamp recordings (KCH(3)SO(4) internal) were made from visualized SDH neurons. At elevated temperature all SDH neurons had reduced input resistance and smaller, briefer APs. Resting membrane potential and AP afterhyperpolarization amplitude were temperature sensitive only in subsets of the SDH population. Notably, elevated temperature increased the prevalence of neurons that did not discharge APs during current injection. These reluctant firing neurons expressed a rapid A-type potassium current, which is enhanced at higher temperatures and thus restrains AP discharge. When compared with previously published whole cell recordings obtained in vivo (37 degrees C) our results suggest that, on balance, in vitro data collected at elevated temperature more closely resemble data collected under in vivo conditions.

Dual patch voltage clamp study of low membrane resistance astrocytes in situ.

PubMed

Ma, Baofeng; Xu, Guangjin; Wang, Wei; Enyeart, John J; Zhou, Min

2014-03-17

Whole-cell patch clamp recording has been successfully used in identifying the voltage-dependent gating and conductance properties of ion channels in a variety of cells. However, this powerful technique is of limited value in studying low membrane resistance cells, such as astrocytes in situ, because of the inability to control or accurately measure the real amplitude of command voltages. To facilitate the study of ionic conductances of astrocytes, we have developed a dual patch recording method which permits membrane current and membrane potential to be simultaneously recorded from astrocytes in spite of their extraordinarily low membrane resistance. The utility of this technique is demonstrated by measuring the voltage-dependent activation of the inwardly rectifying K+ current abundantly expressed in astrocytes and multiple ionic events associated with astrocytic GABAA receptor activation. This protocol can be performed routinely in the study of astrocytes. This method will be valuable for identifying and characterizing the individual ion channels that orchestrate the electrical activity of low membrane resistance cells.

PREPARATION, IN VITRO AND IN VIVO CHARACTERIZATION OF HYDROPHOBIC PATCHES OF A HIGHLY WATER SOLUBLE DRUG FOR PROLONGED PLASMA HALF LIFE: EFFECT OF PERMEATION ENHANCERS.

PubMed

Yaqoob, Ayesha; Ahmad, Mahmood; Mahmood, Asif; Sarfraz, Rai Muhammad

2016-11-01

Aim of present study was to develop metoprolol matrix patches using different enhancers. Combination of two hydrophobic polymers, ethyl cellulose and eudragit RL 100 (8 : 2) were used for preparation of unilaminated matrix patch. 10% w/w of isopropyl myristate (IPM), dimethyl sulfoxide (DMSO), span (20 (S20), Tween 20 (T20) and eucalyptus oil as enhancers and 40% of dibutyl phthalate as plasticizer were used. Prepared patches were evaluated for physical appearance, weight uniformity and thickness. FTIR studies were performed to assess compatibility among ingredients and developed formulation. Dissolution and permeation studies were performed to compare effects of enhancers. Surface morphology after release was examined by scanning electron microscopy. Selected formulation was subjected to in vivo studies by randomized crossover design in rabbits (n = 6) for pharmacokinetic comparison with oral solution administration. Physical evaluation revealed that translucent, flexible, non brittle patches of uniform weight and thickness were prepared. Release from patches followed Higuchi model. Mechanism of release was Fickian. Formulation containing IPM showed that release was by anomalous transport. Highest permeation flux was observed for formulation containing IPM with 2-fold enhancement in permeation. Permeation flux for patches was in order of formulation with no enhancer > IPM > T20 > S20 > DMSO = eucalyptus oil. Plasma concentration from in vivo studies exhibited sustained plasma levels of metoprolol after transdermal patch application in comparison to oral solution administration. Pharmacokinetic analysis of in vivo data elucidated that half life was increased 8 times when compared to oral administration, due to controlled release of drug for longer period of time. These findings suggested that hydrophobic transdermal patches of highly water soluble drug metoprolol were successfully prepared with 10% of IPM for sustained systemic delivery for prolonged half life.

Population patch-clamp electrophysiology analysis of recombinant GABAA alpha1beta3gamma2 channels expressed in HEK-293 cells.

PubMed

Hollands, Emma C; Dale, Tim J; Baxter, Andrew W; Meadows, Helen J; Powell, Andrew J; Clare, Jeff J; Trezise, Derek J

2009-08-01

Gamma-amino butyric acid (GABA)-activated Cl- channels are critical mediators of inhibitory postsynaptic potentials in the CNS. To date, rational design efforts to identify potent and selective GABA(A) subtype ligands have been hampered by the absence of suitable high-throughput screening approaches. The authors describe 384-well population patch-clamp (PPC) planar array electrophysiology methods for the study of GABA(A) receptor pharmacology. In HEK293 cells stably expressing human alpha1beta3gamma2 GABA(A) channels, GABA evoked outward currents at 0 mV of 1.05 +/- 0.08 nA, measured 8 s post GABA addition. The I(GABA) was linear and reversed close to the theoretical E(Cl) (-56 mV). Concentration-response curve analysis yielded a mean pEC(50) value of 5.4 and Hill slope of 1.5, and for a series of agonists, the rank order of potency was muscimol > GABA > isoguvacine. A range of known positive modulators, including diazepam and pentobarbital, produced concentration-dependent augmentation of the GABA EC( 20) response (1 microM). The competitive antagonists bicuculline and gabazine produced concentration-dependent, parallel, rightward displacement of GABA curves with pA(2) and slope values of 5.7 and 1.0 and 6.7 and 1.0, respectively. In contrast, picrotoxin (0.2-150 microM) depressed the maximal GABA response, implying a non-competitive antagonism. Overall, the pharmacology of human alpha1beta3gamma2 GABA(A) determined by PPC was highly similar to that obtained by conventional patch-clamp methods. In small-scale single-shot screens, Z' values of >0.5 were obtained in agonist, modulator, and antagonist formats with hit rates of 0% to 3%. The authors conclude that despite the inability of the method to resolve the peak agonist responses, PPC can rapidly and usefully quantify pharmacology for the alpha1beta3gamma2 GABA(A) isoform. These data suggest that PPC may be a valuable approach for a focused set and secondary screening of GABA(A) receptors and other slow ligand-gated ion channels.

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method.

PubMed

Ting, Jonathan T; Lee, Brian R; Chong, Peter; Soler-Llavina, Gilberto; Cobbs, Charles; Koch, Christof; Zeng, Hongkui; Lein, Ed

2018-02-26

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have validated the utility of this method for enhancing neuronal preservation and overall brain slice viability. The implementation of this technique by early adopters has facilitated detailed investigations into brain function using diverse experimental applications and spanning a wide range of animal ages, brain regions, and cell types. Steps are outlined for carrying out the protective recovery brain slice technique using an optimized NMDG artificial cerebrospinal fluid (aCSF) media formulation and enhanced procedure to reliably obtain healthy brain slices for patch clamp electrophysiology. With this updated approach, a substantial improvement is observed in the speed and reliability of gigaohm seal formation during targeted patch clamp recording experiments while maintaining excellent neuronal preservation, thereby facilitating challenging experimental applications. Representative results are provided from multi-neuron patch clamp recording experiments to assay synaptic connectivity in neocortical brain slices prepared from young adult transgenic mice and mature adult human neurosurgical specimens. Furthermore, the optimized NMDG protective recovery method of brain slicing is compatible with both juvenile and adult animals, thus resolving a limitation of the original methodology. In summary, a single media formulation and brain slicing procedure can be implemented across various species and ages to achieve excellent viability and tissue preservation.

Novel screening techniques for ion channel targeting drugs

PubMed Central

Obergrussberger, Alison; Stölzle-Feix, Sonja; Becker, Nadine; Brüggemann, Andrea; Fertig, Niels; Möller, Clemens

2015-01-01

Ion channels are integral membrane proteins that regulate the flux of ions across the cell membrane. They are involved in nearly all physiological processes, and malfunction of ion channels has been linked to many diseases. Until recently, high-throughput screening of ion channels was limited to indirect, e.g. fluorescence-based, readout technologies. In the past years, direct label-free biophysical readout technologies by means of electrophysiology have been developed. Planar patch-clamp electrophysiology provides a direct functional label-free readout of ion channel function in medium to high throughput. Further electrophysiology features, including temperature control and higher-throughput instruments, are continually being developed. Electrophysiological screening in a 384-well format has recently become possible. Advances in chip and microfluidic design, as well as in cell preparation and handling, have allowed challenging cell types to be studied by automated patch clamp. Assays measuring action potentials in stem cell-derived cardiomyocytes, relevant for cardiac safety screening, and neuronal cells, as well as a large number of different ion channels, including fast ligand-gated ion channels, have successfully been established by automated patch clamp. Impedance and multi-electrode array measurements are particularly suitable for studying cardiomyocytes and neuronal cells within their physiological network, and to address more complex physiological questions. This article discusses recent advances in electrophysiological technologies available for screening ion channel function and regulation. PMID:26556400

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

PubMed Central

Chong, Peter; Soler-Llavina, Gilberto; Cobbs, Charles; Koch, Christof; Zeng, Hongkui; Lein, Ed

2018-01-01

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have validated the utility of this method for enhancing neuronal preservation and overall brain slice viability. The implementation of this technique by early adopters has facilitated detailed investigations into brain function using diverse experimental applications and spanning a wide range of animal ages, brain regions, and cell types. Steps are outlined for carrying out the protective recovery brain slice technique using an optimized NMDG artificial cerebrospinal fluid (aCSF) media formulation and enhanced procedure to reliably obtain healthy brain slices for patch clamp electrophysiology. With this updated approach, a substantial improvement is observed in the speed and reliability of gigaohm seal formation during targeted patch clamp recording experiments while maintaining excellent neuronal preservation, thereby facilitating challenging experimental applications. Representative results are provided from multi-neuron patch clamp recording experiments to assay synaptic connectivity in neocortical brain slices prepared from young adult transgenic mice and mature adult human neurosurgical specimens. Furthermore, the optimized NMDG protective recovery method of brain slicing is compatible with both juvenile and adult animals, thus resolving a limitation of the original methodology. In summary, a single media formulation and brain slicing procedure can be implemented across various species and ages to achieve excellent viability and tissue preservation. PMID:29553547

Novel screening techniques for ion channel targeting drugs.

PubMed

Obergrussberger, Alison; Stölzle-Feix, Sonja; Becker, Nadine; Brüggemann, Andrea; Fertig, Niels; Möller, Clemens

2015-01-01

Ion channels are integral membrane proteins that regulate the flux of ions across the cell membrane. They are involved in nearly all physiological processes, and malfunction of ion channels has been linked to many diseases. Until recently, high-throughput screening of ion channels was limited to indirect, e.g. fluorescence-based, readout technologies. In the past years, direct label-free biophysical readout technologies by means of electrophysiology have been developed. Planar patch-clamp electrophysiology provides a direct functional label-free readout of ion channel function in medium to high throughput. Further electrophysiology features, including temperature control and higher-throughput instruments, are continually being developed. Electrophysiological screening in a 384-well format has recently become possible. Advances in chip and microfluidic design, as well as in cell preparation and handling, have allowed challenging cell types to be studied by automated patch clamp. Assays measuring action potentials in stem cell-derived cardiomyocytes, relevant for cardiac safety screening, and neuronal cells, as well as a large number of different ion channels, including fast ligand-gated ion channels, have successfully been established by automated patch clamp. Impedance and multi-electrode array measurements are particularly suitable for studying cardiomyocytes and neuronal cells within their physiological network, and to address more complex physiological questions. This article discusses recent advances in electrophysiological technologies available for screening ion channel function and regulation.

Creatine kinase is physically associated with the cardiac ATP-sensitive k+ channel in vivo

PubMed Central

Crawford, Russell M.; Ranki, Harri J.; Botting, Catherine H.; Budas, Grant R.; Jovanovic, Aleksandar

2007-01-01

Cardiac sarcolemmal ATP-sensitive K+ (KATP) channels, composed of Kir6.2 and SUR2A subunits, couple the metabolic status of cells with the membrane excitability. Based on previous functional studies, we have hypothesized that creatine kinase (CK) may be a part of the sarcolemmal KATP channel protein complex. The inside-out and whole cell patch clamp electrophysiology applied on guinea pig cardiomyocytes showed that substrates of CK regulate KATP channels activity. Following immunoprecipitation of guinea-pig cardiac membrane fraction with the anti-SUR2 antibody, Coomassie blue staining revealed, besides Kir6.2 and SUR2A, a polypeptide at ∼48 kDa. Western blotting analysis confirmed the nature of putative Kir6.2 and SUR2A, whereas matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis identified p48 kDa as a muscle form of CK. In addition, the CK activity was found in the anti-SUR2A immunoprecipitate and the cross reactivity between an anti-CK antibody and the anti-SUR2A immunoprecipitate was observed as well as vice verse. Further results obtained at the level of recombinant channel subunits demonstrated that CK is directly physically associated with the SUR2A, but not the Kir6.2, subunit. All together, these results suggest that the CK is associated with SUR2A subunit in vivo, which is an integral part of the sarcolemmal KATP channel protein complex. PMID:11729098

Identification of quaternary ammonium compounds as potent inhibitors of hERG potassium channels

PubMed Central

Xia, Menghang; Shahane, Sampada; Huang, Ruili; Titus, Steven A.; Shum, Enoch; Zhao, Yong; Southall, Noel; Zheng, Wei; Witt, Kristine L.; Tice, Raymond R.; Austin, Christopher P.

2011-01-01

The human ether-a-go-go-related gene (hERG) channel, a member of a family of voltage-gated potassium (K+) channels, plays a critical role in the repolarization of the cardiac action potential. The reduction of hERG channel activity as a result of adverse drug effects or genetic mutations may cause QT interval prolongation and potentially lead to acquired long QT syndrome. Thus, screening for hERG channel activity is important in drug development. Cardiotoxicity associated with the inhibition of hERG channels by environmental chemicals is also a public health concern. To assess the inhibitory effects of environmental chemicals on hERG channel function, we screened the National Toxicology Program (NTP) collection of 1408 compounds by measuring thallium influx into cells through hERG channels. Seventeen compounds with hERG channel inhibition were identified with IC50 potencies ranging from 0.26 to 22 μM. Twelve of these compounds were confirmed as hERG channel blockers in an automated whole cell patch clamp experiment. In addition, we investigated the structure-activity relationship of seven compounds belonging to the quaternary ammonium compound (QAC) series on hERG channel inhibition. Among four active QAC compounds, tetra-n-octylammonium bromide was the most potent with an IC50 value of 260 nM in the thallium influx assay and 80 nM in the patch clamp assay. The potency of this class of hERG channel inhibitors appears to depend on the number and length of their aliphatic side-chains surrounding the charged nitrogen. Profiling environmental compound libraries for hERG channel inhibition provides information useful in prioritizing these compounds for cardiotoxicity assessment in vivo. PMID:21362439

Optimizing Nanoelectrode Arrays for Scalable Intracellular Electrophysiology.

PubMed

Abbott, Jeffrey; Ye, Tianyang; Ham, Donhee; Park, Hongkun

2018-03-20

Electrode technology for electrophysiology has a long history of innovation, with some decisive steps including the development of the voltage-clamp measurement technique by Hodgkin and Huxley in the 1940s and the invention of the patch clamp electrode by Neher and Sakmann in the 1970s. The high-precision intracellular recording enabled by the patch clamp electrode has since been a gold standard in studying the fundamental cellular processes underlying the electrical activities of neurons and other excitable cells. One logical next step would then be to parallelize these intracellular electrodes, since simultaneous intracellular recording from a large number of cells will benefit the study of complex neuronal networks and will increase the throughput of electrophysiological screening from basic neurobiology laboratories to the pharmaceutical industry. Patch clamp electrodes, however, are not built for parallelization; as for now, only ∼10 patch measurements in parallel are possible. It has long been envisioned that nanoscale electrodes may help meet this challenge. First, nanoscale electrodes were shown to enable intracellular access. Second, because their size scale is within the normal reach of the standard top-down fabrication, the nanoelectrodes can be scaled into a large array for parallelization. Third, such a nanoelectrode array can be monolithically integrated with complementary metal-oxide semiconductor (CMOS) electronics to facilitate the large array operation and the recording of the signals from a massive number of cells. These are some of the central ideas that have motivated the research activity into nanoelectrode electrophysiology, and these past years have seen fruitful developments. This Account aims to synthesize these findings so as to provide a useful reference. Summing up from the recent studies, we will first elucidate the morphology and associated electrical properties of the interface between a nanoelectrode and a cellular membrane, clarifying how the nanoelectrode attains intracellular access. This understanding will be translated into a circuit model for the nanobio interface, which we will then use to lay out the strategies for improving the interface. The intracellular interface of the nanoelectrode is currently inferior to that of the patch clamp electrode; reaching this benchmark will be an exciting challenge that involves optimization of electrode geometries, materials, chemical modifications, electroporation protocols, and recording/stimulation electronics, as we describe in the Account. Another important theme of this Account, beyond the optimization of the individual nanoelectrode-cell interface, is the scalability of the nanoscale electrodes. We will discuss this theme using a recent development from our groups as an example, where an array of ca. 1000 nanoelectrode pixels fabricated on a CMOS integrated circuit chip performs parallel intracellular recording from a few hundreds of cardiomyocytes, which marks a new milestone in electrophysiology.

The Mechanosensitive Ca2+ Channel as a Central Regular of Prostate Tumor Cell Migration and Invasiveness

DTIC Science & Technology

2011-04-01

activation still needs to be determined (Strotmann et al. 2000). 7.2.4 The Use of MS Enzyme Inhibitors A further strategy for implicating potential MS...invasiveness and metastatic potential . 1.1 Use patch-clamp/pressure clamp techniques, confocal immunofluorescence, Westerns and surface biotinylation...9. Maroto, R. Kurosky, A. Hamill, O.P. Expression and function of canonical transient recptor potential channels in human prostate tumor cells

The Electrophysiological MEMS Device with Micro Channel Array for Cellular Network Analysis

NASA Astrophysics Data System (ADS)

Tonomura, Wataru; Kurashima, Toshiaki; Takayama, Yuzo; Moriguchi, Hiroyuki; Jimbo, Yasuhiko; Konishi, Satoshi

This paper describes a new type of MCA (Micro Channel Array) for simultaneous multipoint measurement of cellular network. Presented MCA employing the measurement principles of the patch-clamp technique is designed for advanced neural network analysis which has been studied by co-authors using 64ch MEA (Micro Electrode Arrays) system. First of all, sucking and clamping of cells through channels of developed MCA is expected to improve electrophysiological signal detections. Electrophysiological sensing electrodes integrated around individual channels of MCA by using MEMS (Micro Electro Mechanical System) technologies are electrically isolated for simultaneous multipoint measurement. In this study, we tested the developed MCA using the non-cultured rat's cerebral cortical slice and the hippocampal neurons. We could measure the spontaneous action potential of the slice simultaneously at multiple points and culture the neurons on developed MCA. Herein, we describe the experimental results together with the design and fabrication of the electrophysiological MEMS device with MCA for cellular network analysis.

Mucoadhesive buccal patches based on interpolymer complexes of chitosan–pectin for delivery of carvedilol

PubMed Central

Kaur, Amanpreet; Kaur, Gurpreet

2011-01-01

The study was designed to develop bioadhesive patches of carvedilol hydrochloride using chitosan (CH) and pectin (PE) interpolymer complexes and to systematically evaluate their in vitro and in vivo performances. Mucoadhesive buccal patches of carvedilol were prepared using solvent casting method. The physicochemical interaction between CH and PE was investigated by FTIR and DSC studies. The patches were evaluated for their physical characteristics like mass variation, content uniformity, folding endurance, ex vivo mucoadhesion strength, ex vivo mucoadhesion time, surface pH, in vitro drug release, in situ release study, and in vivo bioavailability study. The swelling index of the patches was found to be proportional to the PE concentration. The surface pH of all the formulated bioadhesive patches was found to lie between 6.2 and 7.2. The optimized bioadhesive patch (C1, CH:PE 20:80) showed bioadhesive strength of 22.10 ± 0.20 g, in vitro release of 98.73% and ex vivo mucoadhesion time of 451 min with in a period of 8 h. The optimized patch demonstrated good in vitro and in vivo results. The buccal delivery of carvedilol in rabbits showed a significant improvement in bioavailability of carvedilol from patches when compared to oral route. PMID:23960773

Mucoadhesive buccal patches based on interpolymer complexes of chitosan-pectin for delivery of carvedilol.

PubMed

Kaur, Amanpreet; Kaur, Gurpreet

2012-01-01

The study was designed to develop bioadhesive patches of carvedilol hydrochloride using chitosan (CH) and pectin (PE) interpolymer complexes and to systematically evaluate their in vitro and in vivo performances. Mucoadhesive buccal patches of carvedilol were prepared using solvent casting method. The physicochemical interaction between CH and PE was investigated by FTIR and DSC studies. The patches were evaluated for their physical characteristics like mass variation, content uniformity, folding endurance, ex vivo mucoadhesion strength, ex vivo mucoadhesion time, surface pH, in vitro drug release, in situ release study, and in vivo bioavailability study. The swelling index of the patches was found to be proportional to the PE concentration. The surface pH of all the formulated bioadhesive patches was found to lie between 6.2 and 7.2. The optimized bioadhesive patch (C1, CH:PE 20:80) showed bioadhesive strength of 22.10 ± 0.20 g, in vitro release of 98.73% and ex vivo mucoadhesion time of 451 min with in a period of 8 h. The optimized patch demonstrated good in vitro and in vivo results. The buccal delivery of carvedilol in rabbits showed a significant improvement in bioavailability of carvedilol from patches when compared to oral route.

Gut commensal microvesicles reproduce parent bacterial signals to host immune and enteric nervous systems.

PubMed

Al-Nedawi, Khalid; Mian, M Firoz; Hossain, Nazia; Karimi, Khalil; Mao, Yu-Kang; Forsythe, Paul; Min, Kevin K; Stanisz, Andrew M; Kunze, Wolfgang A; Bienenstock, John

2015-02-01

Ingestion of a commensal bacteria, Lactobacillus rhamnosus JB-1, has potent immunoregulatory effects, and changes nerve-dependent colon migrating motor complexes (MMCs), enteric nerve function, and behavior. How these alterations occur is unknown. JB-1 microvesicles (MVs) are enriched for heat shock protein components such as chaperonin 60 heat-shock protein isolated from Escherichia coli (GroEL) and reproduce regulatory and neuronal effects in vitro and in vivo. Ingested labeled MVs were detected in murine Peyer's patch (PP) dendritic cells (DCs) within 18 h. After 3 d, PP and mesenteric lymph node DCs assumed a regulatory phenotype and increased functional regulatory CD4(+)25(+)Foxp3+ T cells. JB-1, MVs, and GroEL similarly induced phenotypic change in cocultured DCs via multiple pathways including C-type lectin receptors specific intercellular adhesion molecule-3 grabbing non-integrin-related 1 and Dectin-1, as well as TLR-2 and -9. JB-1 and MVs also decreased the amplitude of neuronally dependent MMCs in an ex vivo model of peristalsis. Gut epithelial, but not direct neuronal application of, MVs, replicated functional effects of JB-1 on in situ patch-clamped enteric neurons. GroEL and anti-TLR-2 were without effect in this system, suggesting the importance of epithelium neuron signaling and discrimination between pathways for bacteria-neuron and -immune communication. Together these results offer a mechanistic explanation of how Gram-positive commensals and probiotics may influence the host's immune and nervous systems. © FASEB.

Structural basis of human PCNA sliding on DNA

NASA Astrophysics Data System (ADS)

de March, Matteo; Merino, Nekane; Barrera-Vilarmau, Susana; Crehuet, Ramon; Onesti, Silvia; Blanco, Francisco J.; de Biasio, Alfredo

2017-01-01

Sliding clamps encircle DNA and tether polymerases and other factors to the genomic template. However, the molecular mechanism of clamp sliding on DNA is unknown. Using crystallography, NMR and molecular dynamics simulations, here we show that the human clamp PCNA recognizes DNA through a double patch of basic residues within the ring channel, arranged in a right-hand spiral that matches the pitch of B-DNA. We propose that PCNA slides by tracking the DNA backbone via a `cogwheel' mechanism based on short-lived polar interactions, which keep the orientation of the clamp invariant relative to DNA. Mutation of residues at the PCNA-DNA interface has been shown to impair the initiation of DNA synthesis by polymerase δ (pol δ). Therefore, our findings suggest that a clamp correctly oriented on DNA is necessary for the assembly of a replication-competent PCNA-pol δ holoenzyme.

In vivo and in vitro functional characterization of Andersen's syndrome mutations.

PubMed

Bendahhou, Saïd; Fournier, Emmanuel; Sternberg, Damien; Bassez, Guillaume; Furby, Alain; Sereni, Carole; Donaldson, Matthew R; Larroque, Marie-Madeleine; Fontaine, Bertrand; Barhanin, Jacques

2005-06-15

The inward rectifier K(+) channel Kir2.1 carries all Andersen's syndrome mutations identified to date. Patients exhibit symptoms of periodic paralysis, cardiac dysrhythmia and multiple dysmorphic features. Here, we report the clinical manifestations found in three families with Andersen's syndrome. Molecular genetics analysis identified two novel missense mutations in the KCNJ2 gene leading to amino acid changes C154F and T309I of the Kir2.1 open reading frame. Patch clamp experiments showed that the two mutations produced a loss of channel function. When co-expressed with Kir2.1 wild-type (WT) channels, both mutations exerted a dominant-negative effect leading to a loss of the inward rectifying K(+) current. Confocal microscopy imaging in HEK293 cells is consistent with a co-assembly of the EGFP-fused mutant proteins with WT channels and proper traffick to the plasma membrane to produce silent channels alone or as hetero-tetramers with WT. Functional expression in C2C12 muscle cell line of newly as well as previously reported Andersen's syndrome mutations confirmed that these mutations act through a dominant-negative effect by altering channel gating or trafficking. Finally, in vivo electromyographic evaluation showed a decrease in muscle excitability in Andersen's syndrome patients. We hypothesize that Andersen's syndrome-associated mutations and hypokalaemic periodic paralysis-associated calcium channel mutations may lead to muscle membrane hypoexcitability via a common mechanism.

In vivo and in vitro functional characterization of Andersen's syndrome mutations

PubMed Central

Bendahhou, Saïd; Fournier, Emmanuel; Sternberg, Damien; Bassez, Guillaume; Furby, Alain; Sereni, Carole; Donaldson, Matthew R; Larroque, Marie-Madeleine; Fontaine, Bertrand; Barhanin, Jacques

2005-01-01

The inward rectifier K+ channel Kir2.1 carries all Andersen's syndrome mutations identified to date. Patients exhibit symptoms of periodic paralysis, cardiac dysrhythmia and multiple dysmorphic features. Here, we report the clinical manifestations found in three families with Andersen's syndrome. Molecular genetics analysis identified two novel missense mutations in the KCNJ2 gene leading to amino acid changes C154F and T309I of the Kir2.1 open reading frame. Patch clamp experiments showed that the two mutations produced a loss of channel function. When co-expressed with Kir2.1 wild-type (WT) channels, both mutations exerted a dominant-negative effect leading to a loss of the inward rectifying K+ current. Confocal microscopy imaging in HEK293 cells is consistent with a co-assembly of the EGFP-fused mutant proteins with WT channels and proper traffick to the plasma membrane to produce silent channels alone or as hetero-tetramers with WT. Functional expression in C2C12 muscle cell line of newly as well as previously reported Andersen's syndrome mutations confirmed that these mutations act through a dominant-negative effect by altering channel gating or trafficking. Finally, in vivo electromyographic evaluation showed a decrease in muscle excitability in Andersen's syndrome patients. We hypothesize that Andersen's syndrome-associated mutations and hypokalaemic periodic paralysis-associated calcium channel mutations may lead to muscle membrane hypoexcitability via a common mechanism. PMID:15831539

Properties of Single K+ and Cl− Channels in Asclepias tuberosa Protoplasts 1

PubMed Central

Schauf, Charles L.; Wilson, Kathryn J.

1987-01-01

Potassium and chloride channels were characterized in Asclepias tuberosa suspension cell derived protoplasts by patch voltage-clamp. Whole-cell currents and single channels in excised patches had linear instantaneous current-voltage relations, reversing at the Nernst potentials for K+ and Cl−, respectively. Whole cell K+ currents activated exponentially during step depolarizations, while voltage-dependent Cl− channels were activated by hyperpolarizations. Single K+ channel conductance was 40 ± 5 pS with a mean open time of 4.5 milliseconds at 100 millivolts. Potassium channels were blocked by Cs+ and tetraethylammonium, but were insensitive to 4-aminopyridine. Chloride channels had a single-channel conductance of 100 ± 17 picosiemens, mean open time of 8.8 milliseconds, and were blocked by Zn2+ and ethacrynic acid. Whole-cell Cl− currents were inhibited by abscisic acid, and were unaffected by indole-3-acetic acid and 2,4-dichlorophenoxyacetic acid. Since internal and external composition can be controlled, patch-clamped protoplasts are ideal systems for studying the role of ion channels in plant physiology and development. Images Fig. 5 PMID:16665712

Advances in the manufacturing, types, and applications of biosensors

NASA Astrophysics Data System (ADS)

Ravindra, Nuggehalli M.; Prodan, Camelia; Fnu, Shanmugamurthy; Padronl, Ivan; Sikha, Sushil K.

2007-12-01

In recent years, there have been significant technological advancements in the manufacturing, types, and applications of biosensors. Applications include clinical and non-clinical diagnostics for home, bio-defense, bio-remediation, environment, agriculture, and the food industry. Biosensors have progressed beyond the detection of biological threats such as anthrax and are finding use in a number of non-biological applications. Emerging biosensor technologies such as lab-on-a-chip have revolutionized the integration approaches for a very flexible, innovative, and user-friendly platform. An overview of the fundamentals, types, applications, and manufacturers, as well as the market trends of biosensors is presented here. Two case studies are discussed: one focused on a characterization technique—patch clamping and dielectric spectroscopy as a biological sensor—and the other about lithium phthalocyanine, a material that is being developed for in-vivo oxymetry.

High-Throughput Patch Clamp Screening in Human α6-Containing Nicotinic Acetylcholine Receptors

PubMed Central

Armstrong, Lucas C.; Kirsch, Glenn E.; Fedorov, Nikolai B.; Wu, Caiyun; Kuryshev, Yuri A.; Sewell, Abby L.; Liu, Zhiqi; Motter, Arianne L.; Leggett, Carmine S.; Orr, Michael S.

2017-01-01

Nicotine, the addictive component of tobacco products, is an agonist at nicotinic acetylcholine receptors (nAChRs) in the brain. The subtypes of nAChR are defined by their α- and β-subunit composition. The α6β2β3 nAChR subtype is expressed in terminals of dopaminergic neurons that project to the nucleus accumbens and striatum and modulate dopamine release in brain regions involved in nicotine addiction. Although subtype-dependent selectivity of nicotine is well documented, subtype-selective profiles of other tobacco product constituents are largely unknown and could be essential for understanding the addiction-related neurological effects of tobacco products. We describe the development and validation of a recombinant cell line expressing human α6/3β2β3V273S nAChR for screening and profiling assays in an automated patch clamp platform (IonWorks Barracuda). The cell line was pharmacologically characterized by subtype-selective and nonselective reference agonists, pore blockers, and competitive antagonists. Agonist and antagonist effects detected by the automated patch clamp approach were comparable to those obtained by conventional electrophysiological assays. A pilot screen of a library of Food and Drug Administration–approved drugs identified compounds, previously not known to modulate nAChRs, which selectively inhibited the α6/3β2β3V273S subtype. These assays provide new tools for screening and subtype-selective profiling of compounds that act at α6β2β3 nicotinic receptors. PMID:28298165

Positioning of the sensor cell on the sensing area using cell trapping pattern in incubation type planar patch clamp biosensor.

PubMed

Wang, Zhi-Hong; Takada, Noriko; Uno, Hidetaka; Ishizuka, Toru; Yawo, Hiromu; Urisu, Tsuneo

2012-08-01

Positioning the sensor cell on the micropore of the sensor chip and keeping it there during incubation are problematic tasks for incubation type planar patch clamp biosensors. To solve these problems, we formed on the Si sensor chip's surface a cell trapping pattern consisting of a lattice pattern with a round area 5 μm deep and with the micropore at the center of the round area. The surface of the sensor chip was coated with extra cellular matrix collagen IV, and HEK293 cells on which a chimera molecule of channel-rhodopsin-wide-receiver (ChR-WR) was expressed, were then seeded. We examined the effects of this cell trapping pattern on the biosensor's operation. In the case of a flat sensor chip without a cell trapping pattern, it took several days before the sensor cell covered the micropore and formed an almost confluent state. As a result, multi-cell layers easily formed and made channel current measurements impossible. On the other hand, the sensor chip with cell trapping pattern easily trapped cells in the round area, and formed the colony consisted of the cell monolayer covering the micropore. A laser (473 nm wavelength) induced channel current was observed from the whole cell arrangement formed using the nystatin perforation technique. The observed channel current characteristics matched measurements made by using a pipette patch clamp. Copyright © 2012 Elsevier B.V. All rights reserved.

Single-Molecule Patch-Clamp FRET Anisotropy Microscopy Studies of NMDA Receptor Ion Channel Activation and Deactivation under Agonist Ligand Binding in Living Cells.

PubMed

Sasmal, Dibyendu Kumar; Yadav, Rajeev; Lu, H Peter

2016-07-20

N-methyl-d-aspartate (NMDA) receptor ion channel is activated by the binding of two pairs of glycine and glutamate along with the application of action potential. Binding and unbinding of ligands changes its conformation that plays a critical role in the open-close activities of NMDA receptor. Conformation states and their dynamics due to ligand binding are extremely difficult to characterize either by conventional ensemble experiments or single-channel electrophysiology method. Here we report the development of a new correlated technical approach, single-molecule patch-clamp FRET anisotropy imaging and demonstrate by probing the dynamics of NMDA receptor ion channel and kinetics of glycine binding with its ligand binding domain. Experimentally determined kinetics of ligand binding with receptor is further verified by computational modeling. Single-channel patch-clamp and four-channel fluorescence measurement are recorded simultaneously to get correlation among electrical on and off states, optically determined conformational open and closed states by FRET, and binding-unbinding states of the glycine ligand by anisotropy measurement at the ligand binding domain of GluN1 subunit. This method has the ability to detect the intermediate states in addition to electrical on and off states. Based on our experimental results, we have proposed that NMDA receptor gating goes through at least one electrically intermediate off state, a desensitized state, when ligands remain bound at the ligand binding domain with the conformation similar to the fully open state.

An isolated Hda-clamp complex is functional in the regulatory inactivation of DnaA and DNA replication.

PubMed

Kawakami, Hironori; Su'etsugu, Masayuki; Katayama, Tsutomu

2006-10-01

In Escherichia coli, a complex consisting of Hda and the DNA-loaded clamp-subunit of the DNA polymerase III holoenzyme promotes hydrolysis of DnaA-ATP. The resultant ADP-DnaA is inactive for initiation of chromosomal DNA replication, thereby repressing excessive initiations. As the cellular content of the clamp is 10-100 times higher than that of Hda, most Hda molecules might be complexed with the clamp in vivo. Although Hda predominantly forms irregular aggregates when overexpressed, in the present study we found that co-overexpression of the clamp with Hda enhances Hda solubility dramatically and we efficiently isolated the Hda-clamp complex. A single molecule of the complex appears to consist of two Hda molecules and a single clamp. The complex is competent in DnaA-ATP hydrolysis and DNA replication in the presence of DNA and the clamp deficient subassembly of the DNA polymerase III holoenzyme (pol III*). These findings indicate that the clamp contained in the complex is loaded onto DNA through an interaction with the pol III* and that the Hda activity is preserved in these processes. The complex consisting of Hda and the DNA-unloaded clamp may play a specific role in a process proceeding to the DnaA-ATP hydrolysis in vivo.

Spontaneous Activity Drives Local Synaptic Plasticity In Vivo.

PubMed

Winnubst, Johan; Cheyne, Juliette E; Niculescu, Dragos; Lohmann, Christian

2015-07-15

Spontaneous activity fine-tunes neuronal connections in the developing brain. To explore the underlying synaptic plasticity mechanisms, we monitored naturally occurring changes in spontaneous activity at individual synapses with whole-cell patch-clamp recordings and simultaneous calcium imaging in the mouse visual cortex in vivo. Analyzing activity changes across large populations of synapses revealed a simple and efficient local plasticity rule: synapses that exhibit low synchronicity with nearby neighbors (<12 μm) become depressed in their transmission frequency. Asynchronous electrical stimulation of individual synapses in hippocampal slices showed that this is due to a decrease in synaptic transmission efficiency. Accordingly, experimentally increasing local synchronicity, by stimulating synapses in response to spontaneous activity at neighboring synapses, stabilized synaptic transmission. Finally, blockade of the high-affinity proBDNF receptor p75(NTR) prevented the depression of asynchronously stimulated synapses. Thus, spontaneous activity drives local synaptic plasticity at individual synapses in an "out-of-sync, lose-your-link" fashion through proBDNF/p75(NTR) signaling to refine neuronal connectivity. VIDEO ABSTRACT. Copyright © 2015 Elsevier Inc. All rights reserved.

Vps39 Interacts with Tom40 to Establish One of Two Functionally Distinct Vacuole-Mitochondria Contact Sites.

PubMed

González Montoro, Ayelén; Auffarth, Kathrin; Hönscher, Carina; Bohnert, Maria; Becker, Thomas; Warscheid, Bettina; Reggiori, Fulvio; van der Laan, Martin; Fröhlich, Florian; Ungermann, Christian

2018-06-04

The extensive subcellular network of membrane contact sites plays central roles in organelle biogenesis and communication, yet the precise contributions of the involved machineries remain largely enigmatic. The yeast vacuole forms a membrane contact site with mitochondria, called vacuolar and mitochondrial patch (vCLAMP). Formation of vCLAMPs involves the vacuolar Rab GTPase Ypt7 and the Ypt7-interacting Vps39 subunit of the HOPS tethering complex. Here, we uncover the general preprotein translocase of the outer membrane (TOM) subunit Tom40 as the direct binding partner of Vps39 on mitochondria. We identify Vps39 mutants defective in TOM binding, but functional for HOPS. Cells that cannot form vCLAMPs show reduced growth under stress conditions and impaired survival upon starvation. Unexpectedly, our mutant analysis revealed the existence of two functionally independent vacuole-mitochondria MCSs: one formed by the Ypt7-Vps39-Tom40 tether and a second one by Vps13-Mcp1, which is redundant with ER-mitochondrial contacts formed by ERMES. Copyright © 2018 Elsevier Inc. All rights reserved.

Single-neuron identification of chemical constituents, physiological changes, and metabolism using mass spectrometry.

PubMed

Zhu, Hongying; Zou, Guichang; Wang, Ning; Zhuang, Meihui; Xiong, Wei; Huang, Guangming

2017-03-07

The use of single-cell assays has emerged as a cutting-edge technique during the past decade. Although single-cell mass spectrometry (MS) has recently achieved remarkable results, deep biological insights have not yet been obtained, probably because of various technical issues, including the unavoidable use of matrices, the inability to maintain cell viability, low throughput because of sample pretreatment, and the lack of recordings of cell physiological activities from the same cell. In this study, we describe a patch clamp/MS-based platform that enables the sensitive, rapid, and in situ chemical profiling of single living neurons. This approach integrates modified patch clamp technique and modified MS measurements to directly collect and detect nanoliter-scale samples from the cytoplasm of single neurons in mice brain slices. Abundant possible cytoplasmic constituents were detected in a single neuron at a relatively fast rate, and over 50 metabolites were identified in this study. The advantages of direct, rapid, and in situ sampling and analysis enabled us to measure the biological activities of the cytoplasmic constituents in a single neuron, including comparing neuron types by cytoplasmic chemical constituents; observing changes in constituent concentrations as the physiological conditions, such as age, vary; and identifying the metabolic pathways of small molecules.

Finite element simulation of the gating mechanism of mechanosensitive ion channels

NASA Astrophysics Data System (ADS)

Bavi, Navid; Qin, Qinghua; Martinac, Boris

2013-08-01

In order to eliminate limitations of existing experimental or computational methods (such as patch-clamp technique or molecular dynamic analysis) a finite element (FE) model for multi length-scale and time-scale investigation on the gating mechanism of mechanosensitive (MS) ion channels has been established. Gating force value (from typical patch clamping values) needed to activate Prokaryotic MS ion channels was applied as tensional force to the FE model of the lipid bilayer. Making use of the FE results, we have discussed the effects of the geometrical and the material properties of the Escherichia coli MscL mechanosensitive ion channel opening in relation to the membrane's Young's modulus (which will vary depending on the cell type or cholesterol density in an artificial membrane surrounding the MscL ion channel). The FE model has shown that when the cell membrane stiffens the required channel activation force increases considerably. This is in agreement with experimental results taken from the literature. In addition, the present study quantifies the relationship between the membrane stress distribution around a `hole' for modeling purposes and the stress concentration in the place transmembrane proteins attached to the hole by applying an appropriate mesh refinement as well as well defining contact condition in these areas.

Single-neuron identification of chemical constituents, physiological changes, and metabolism using mass spectrometry

PubMed Central

Zhu, Hongying; Zou, Guichang; Wang, Ning; Zhuang, Meihui; Xiong, Wei; Huang, Guangming

2017-01-01

The use of single-cell assays has emerged as a cutting-edge technique during the past decade. Although single-cell mass spectrometry (MS) has recently achieved remarkable results, deep biological insights have not yet been obtained, probably because of various technical issues, including the unavoidable use of matrices, the inability to maintain cell viability, low throughput because of sample pretreatment, and the lack of recordings of cell physiological activities from the same cell. In this study, we describe a patch clamp/MS-based platform that enables the sensitive, rapid, and in situ chemical profiling of single living neurons. This approach integrates modified patch clamp technique and modified MS measurements to directly collect and detect nanoliter-scale samples from the cytoplasm of single neurons in mice brain slices. Abundant possible cytoplasmic constituents were detected in a single neuron at a relatively fast rate, and over 50 metabolites were identified in this study. The advantages of direct, rapid, and in situ sampling and analysis enabled us to measure the biological activities of the cytoplasmic constituents in a single neuron, including comparing neuron types by cytoplasmic chemical constituents; observing changes in constituent concentrations as the physiological conditions, such as age, vary; and identifying the metabolic pathways of small molecules. PMID:28223513

Simultaneous multi-patch-clamp and extracellular-array recordings: Single neuron reflects network activity

NASA Astrophysics Data System (ADS)

Vardi, Roni; Goldental, Amir; Sardi, Shira; Sheinin, Anton; Kanter, Ido

2016-11-01

The increasing number of recording electrodes enhances the capability of capturing the network’s cooperative activity, however, using too many monitors might alter the properties of the measured neural network and induce noise. Using a technique that merges simultaneous multi-patch-clamp and multi-electrode array recordings of neural networks in-vitro, we show that the membrane potential of a single neuron is a reliable and super-sensitive probe for monitoring such cooperative activities and their detailed rhythms. Specifically, the membrane potential and the spiking activity of a single neuron are either highly correlated or highly anti-correlated with the time-dependent macroscopic activity of the entire network. This surprising observation also sheds light on the cooperative origin of neuronal burst in cultured networks. Our findings present an alternative flexible approach to the technique based on a massive tiling of networks by large-scale arrays of electrodes to monitor their activity.

Simultaneous multi-patch-clamp and extracellular-array recordings: Single neuron reflects network activity.

PubMed

Vardi, Roni; Goldental, Amir; Sardi, Shira; Sheinin, Anton; Kanter, Ido

2016-11-08

The increasing number of recording electrodes enhances the capability of capturing the network's cooperative activity, however, using too many monitors might alter the properties of the measured neural network and induce noise. Using a technique that merges simultaneous multi-patch-clamp and multi-electrode array recordings of neural networks in-vitro, we show that the membrane potential of a single neuron is a reliable and super-sensitive probe for monitoring such cooperative activities and their detailed rhythms. Specifically, the membrane potential and the spiking activity of a single neuron are either highly correlated or highly anti-correlated with the time-dependent macroscopic activity of the entire network. This surprising observation also sheds light on the cooperative origin of neuronal burst in cultured networks. Our findings present an alternative flexible approach to the technique based on a massive tiling of networks by large-scale arrays of electrodes to monitor their activity.

Simultaneous multi-patch-clamp and extracellular-array recordings: Single neuron reflects network activity

PubMed Central

Vardi, Roni; Goldental, Amir; Sardi, Shira; Sheinin, Anton; Kanter, Ido

2016-01-01

The increasing number of recording electrodes enhances the capability of capturing the network’s cooperative activity, however, using too many monitors might alter the properties of the measured neural network and induce noise. Using a technique that merges simultaneous multi-patch-clamp and multi-electrode array recordings of neural networks in-vitro, we show that the membrane potential of a single neuron is a reliable and super-sensitive probe for monitoring such cooperative activities and their detailed rhythms. Specifically, the membrane potential and the spiking activity of a single neuron are either highly correlated or highly anti-correlated with the time-dependent macroscopic activity of the entire network. This surprising observation also sheds light on the cooperative origin of neuronal burst in cultured networks. Our findings present an alternative flexible approach to the technique based on a massive tiling of networks by large-scale arrays of electrodes to monitor their activity. PMID:27824075

Toward a New Gold Standard for Early Safety: Automated Temperature-Controlled hERG Test on the PatchLiner®

PubMed Central

Polonchuk, Liudmila

2012-01-01

The Patchliner® temperature-controlled automated patch clamp system was evaluated for testing drug effects on potassium currents through human ether-à-go-go related gene (hERG) channels expressed in Chinese hamster ovary cells at 35–37°C. IC50 values for a set of reference drugs were compared with those obtained using the conventional voltage clamp technique. The results showed good correlation between the data obtained using automated and conventional electrophysiology. Based on these results, the Patchliner® represents an innovative automated electrophysiology platform for conducting the hERG assay that substantially increases throughput and has the advantage of operating at physiological temperature. It allows fast, accurate, and direct assessment of channel function to identify potential proarrhythmic side effects and sets a new standard in ion channel research for drug safety testing. PMID:22303293

Trpm7 Protein Contributes to Intercellular Junction Formation in Mouse Urothelium*

PubMed Central

Watanabe, Masaki; Suzuki, Yoshiro; Uchida, Kunitoshi; Miyazaki, Naoyuki; Murata, Kazuyoshi; Matsumoto, Seiji; Kakizaki, Hidehiro; Tominaga, Makoto

2015-01-01

Trpm7 is a divalent cation-permeable channel that has been reported to be involved in magnesium homeostasis as well as cellular adhesion and migration. We generated urothelium-specific Trpm7 knock-out (KO) mice to reveal the function of Trpm7 in vivo. A Trpm7 KO was induced by tamoxifen and was confirmed by genomic PCR and immunohistochemistry. By using patch clamp recordings in primary urothelial cells, we observed that Mg2+-inhibitable cation currents as well as acid-inducible currents were significantly smaller in Trpm7 KO urothelial cells than in cells from control mice. Assessment of voiding behavior indicated a significantly smaller voided volume in Trpm7 KO mice (mean voided volume 0.28 ± 0.08 g in KO mice and 0.36 ± 0.04 g in control mice, p < 0.05, n = 6–8). Histological analysis showed partial but substantial edema in the submucosal layer of Trpm7 KO mice, most likely due to inflammation. The expression of proinflammatory cytokines TNF-α and IL-1β was significantly higher in Trpm7 KO bladders than in controls. In transmission electron microscopic analysis, immature intercellular junctions were observed in Trpm7 KO urothelium but not in control mice. These results suggest that Trpm7 is involved in the formation of intercellular junctions in mouse urothelium. Immature intercellular junctions in Trpm7 knock-out mice might lead to a disruption of barrier function resulting in inflammation and hypersensitive bladder afferent nerves that may affect voiding behavior in vivo. PMID:26504086

Trpm7 Protein Contributes to Intercellular Junction Formation in Mouse Urothelium.

PubMed

Watanabe, Masaki; Suzuki, Yoshiro; Uchida, Kunitoshi; Miyazaki, Naoyuki; Murata, Kazuyoshi; Matsumoto, Seiji; Kakizaki, Hidehiro; Tominaga, Makoto

2015-12-11

Trpm7 is a divalent cation-permeable channel that has been reported to be involved in magnesium homeostasis as well as cellular adhesion and migration. We generated urothelium-specific Trpm7 knock-out (KO) mice to reveal the function of Trpm7 in vivo. A Trpm7 KO was induced by tamoxifen and was confirmed by genomic PCR and immunohistochemistry. By using patch clamp recordings in primary urothelial cells, we observed that Mg(2+)-inhibitable cation currents as well as acid-inducible currents were significantly smaller in Trpm7 KO urothelial cells than in cells from control mice. Assessment of voiding behavior indicated a significantly smaller voided volume in Trpm7 KO mice (mean voided volume 0.28 ± 0.08 g in KO mice and 0.36 ± 0.04 g in control mice, p < 0.05, n = 6-8). Histological analysis showed partial but substantial edema in the submucosal layer of Trpm7 KO mice, most likely due to inflammation. The expression of proinflammatory cytokines TNF-α and IL-1β was significantly higher in Trpm7 KO bladders than in controls. In transmission electron microscopic analysis, immature intercellular junctions were observed in Trpm7 KO urothelium but not in control mice. These results suggest that Trpm7 is involved in the formation of intercellular junctions in mouse urothelium. Immature intercellular junctions in Trpm7 knock-out mice might lead to a disruption of barrier function resulting in inflammation and hypersensitive bladder afferent nerves that may affect voiding behavior in vivo. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

A comparison of 15 Hz sine on-line and off-line magnetic stimulation affecting the voltage-gated sodium channel currents of prefrontal cortex pyramidal neurons

NASA Astrophysics Data System (ADS)

Zheng, Yu; Dong, Lei; Gao, Yang; Dou, Jun-Rong; Li, Ze-yan

2016-10-01

Combined with the use of patch-clamp techniques, repetitive transcranial magnetic stimulation (rTMS) has proven to be a noninvasive neuromodulation tool that can inhibit or facilitate excitability of neurons after extensive research. The studies generally focused on the method: the neurons are first stimulated in an external standard magnetic exposure device, and then moved to the patch-clamp to record electrophysiological characteristics (off-line magnetic exposure). Despite its universality, real-time observation of the effects of magnetic stimulation on the neurons is more effective (on-line magnetic stimulation). In this study, we selected a standard exposure device for magnetic fields acting on mouse prefrontal cortex pyramidal neurons, and described a new method that a patch-clamp setup was modified to allow on-line magnetic stimulation. By comparing the off-line exposure and on-line stimulation of the same magnetic field intensity and frequency affecting the voltage-gated sodium channel currents, we succeeded in proving the feasibility of the new on-line stimulation device. We also demonstrated that the sodium channel currents of prefrontal cortex pyramidal neurons increased significantly under the 15 Hz sine 1 mT, and 2 mT off-line magnetic field exposure and under the 1 mT and 2 mT on-line magnetic stimulation, and the rate of acceleration was most significant on 2 mT on-line magnetic stimulation. This study described the development of a new on-line magnetic stimulator and successfully demonstrated its practicability for scientific stimulation of neurons.

Patch-clamp analysis of voltage-activated and chemically activated currents in the vomeronasal organ of Sternotherus odoratus (stinkpot/musk turtle)

PubMed Central

Fadool, D. A.; Wachowiak, M.; Brann, J. H.

2011-01-01

Summary The electrophysiological basis of chemical communication in the specialized olfactory division of the vomeronasal (VN) organ is poorly understood. In total, 198 patch-clamp recordings were made from 42 animals (Sternotherus odoratus, the stinkpot/musk turtle) to study the electrically and chemically activated properties of VN neurons. The introduction of tetramethylrhodamine-conjugated dextran into the VN orifice permitted good visualization of the vomeronasal neural epithelium prior to dissociating it into single neurons. Basic electrical properties of the neurons were measured (resting potential, −54.5±2.7 mV, N=11; input resistance, 6.7±1.4GΩ, N=25; capacitance, 4.2±0.3 pF, N=22; means ± S.E.M.). The voltage-gated K+ current inactivation rate was significantly slower in VN neurons from males than in those from females, and K+ currents in males were less sensitive (greater Ki) to tetraethylammonium. Vomeronasal neurons were held at a holding potential of −60 mV and tested for their response to five natural chemicals, female urine, male urine, female musk, male musk and catfish extract. Of the 90 VN neurons tested, 33 (34 %) responded to at least one of the five compounds. The peak amplitude of chemically evoked currents ranged from 4 to 180 pA, with two-thirds of responses less than 25 pA. Urine-evoked currents were of either polarity, whereas musk and catfish extract always elicited only inward currents. Urine applied to neurons harvested from female animals evoked currents that were 2–3 times larger than those elicited from male neurons. Musk-evoked inward currents were three times the magnitude of urine-or catfish-extract-evoked inward currents. The calculated breadth of responsiveness for neurons presented with this array of five chemicals indicated that the mean response spectrum of the VN neurons is narrow (H metric 0.11). This patch-clamp study indicates that VN neurons exhibit sexual dimorphism in function and specificity in response to complex natural chemicals. PMID:11815645

Patch-clamp analysis of voltage-activated and chemically activated currents in the vomeronasal organ of Sternotherus odoratus (stinkpot/musk turtle).

PubMed

Fadool, D A; Wachowiak, M; Brann, J H

2001-12-01

The electrophysiological basis of chemical communication in the specialized olfactory division of the vomeronasal (VN) organ is poorly understood. In total, 198 patch-clamp recordings were made from 42 animals (Sternotherus odoratus, the stinkpot/musk turtle) to study the electrically and chemically activated properties of VN neurons. The introduction of tetramethylrhodamine-conjugated dextran into the VN orifice permitted good visualization of the vomeronasal neural epithelium prior to dissociating it into single neurons. Basic electrical properties of the neurons were measured (resting potential, -54.5 +/- 2.7 mV, N=11; input resistance, 6.7 +/- 1.4 G Omega, N=25; capacitance, 4.2 +/- 0.3 pF, N=22; means +/- S.E.M.). The voltage-gated K(+) current inactivation rate was significantly slower in VN neurons from males than in those from females, and K(+) currents in males were less sensitive (greater K(i)) to tetraethylammonium. Vomeronasal neurons were held at a holding potential of -60 mV and tested for their response to five natural chemicals, female urine, male urine, female musk, male musk and catfish extract. Of the 90 VN neurons tested, 33 (34 %) responded to at least one of the five compounds. The peak amplitude of chemically evoked currents ranged from 4 to 180 pA, with two-thirds of responses less than 25 pA. Urine-evoked currents were of either polarity, whereas musk and catfish extract always elicited only inward currents. Urine applied to neurons harvested from female animals evoked currents that were 2-3 times larger than those elicited from male neurons. Musk-evoked inward currents were three times the magnitude of urine- or catfish-extract-evoked inward currents. The calculated breadth of responsiveness for neurons presented with this array of five chemicals indicated that the mean response spectrum of the VN neurons is narrow (H metric 0.11). This patch-clamp study indicates that VN neurons exhibit sexual dimorphism in function and specificity in response to complex natural chemicals.iol

Evaluation of a 125I-labelled benzazepinone derived voltage-gated sodium channel blocker for imaging with SPECT.

PubMed

Pérez-Medina, Carlos; Patel, Niral; Robson, Mathew; Badar, Adam; Lythgoe, Mark F; Årstad, Erik

2012-12-21

Voltage-gated sodium channels (VGSCs) are a family of transmembrane proteins that mediate fast neurotransmission, and are integral to sustain physiological conditions and higher cognitive functions. Imaging of VGSCs in vivo holds promise as a tool to elucidate operational functions in the brain and to aid the treatment of a wide range of neurological diseases. To assess the suitability of 1-benzazepin-2-one derived VGSC blockers for imaging, we have prepared a (125)I-labelled analogue of BNZA and evaluated the tracer in vivo. In an automated patch-clamp assay, a diastereomeric mixture of the non-radioactive compound blocked the Na(v)1.2 and Na(v)1.7 VGSC isoforms with IC(50) values of 4.1 ± 1.5 μM and 0.25 ± 0.07 μM, respectively. [(3)H]BTX displacement studies revealed a three-fold difference in affinity between the two diastereomers. Iodo-destannylation of a tin precursor with iodine-125 afforded the two diastereomerically pure tracers, which were used to assess binding to VGSCs in vivo by comparing their tissue distributions in mice. Whilst the results point to a lack of VGSC binding in vivo, SPECT imaging revealed highly localized uptake in the interscapular region, an area typically associated with brown adipose tissue, which in addition to high metabolic stability of the iodinated tracer, demonstrate the potential of 1-benzazepin-2-ones for in vivo imaging.

An anion channel in Arabidopsis hypocotyls activated by blue light

NASA Technical Reports Server (NTRS)

Cho, M. H.; Spalding, E. P.; Evans, M. L. (Principal Investigator)

1996-01-01

A rapid, transient depolarization of the plasma membrane in seedling stems is one of the earliest effects of blue light detected in plants. It appears to play a role in transducing blue light into inhibition of hypocotyl (stem) elongation, and perhaps other responses. The possibility that activation of a Cl- conductance is part of the depolarization mechanism was raised previously and addressed here. By patch clamping hypocotyl cells isolated from dark-grown (etiolated) Arabidopsis seedlings, blue light was found to activate an anion channel residing at the plasma membrane. An anion-channel blocker commonly known as NPPB 15-nitro-2-(3-phenylpropylamino)-benzoic acid] potently and reversibly blocked this anion channel. NPPB also blocked the blue-light-induced depolarization in vivo and decreased the inhibitory effect of blue light on hypocotyl elongation. These results indicate that activation of this anion channel plays a role in transducing blue light into growth inhibition.

Synthesis, in vitro and in vivo studies, and molecular modeling of N-alkylated dextromethorphan derivatives as non-competitive inhibitors of α3β4 nicotinic acetylcholine receptor.

PubMed

Jozwiak, Krzysztof; Targowska-Duda, Katarzyna M; Kaczor, Agnieszka A; Kozak, Joanna; Ligeza, Agnieszka; Szacon, Elzbieta; Wrobel, Tomasz M; Budzynska, Barbara; Biala, Grazyna; Fornal, Emilia; Poso, Antti; Wainer, Irving W; Matosiuk, Dariusz

2014-12-15

9 N-alkylated derivatives of dextromethorphan are synthesized and studied as non-competitive inhibitors of α3β4 nicotinic acetylcholine receptors (nAChRs). In vitro activity towards α3β4 nicotinic acetylcholine receptor is determined using a patch-clamp technique and is in the micromolar range. Homology modeling, molecular docking and molecular dynamics of ligand-receptor complexes in POPC membrane are used to find the mode of interactions of N-alkylated dextromethorphan derivatives with α3β4 nAChR. The compounds, similarly as dextromethorphan, interact with the middle portion of α3β4 nAChR ion channel. Finally, behavioral tests confirmed potential application of the studied compounds for the treatment of addiction. Copyright © 2014 Elsevier Ltd. All rights reserved.

Dendritic excitation–inhibition balance shapes cerebellar output during motor behaviour

PubMed Central

Jelitai, Marta; Puggioni, Paolo; Ishikawa, Taro; Rinaldi, Arianna; Duguid, Ian

2016-01-01

Feedforward excitatory and inhibitory circuits regulate cerebellar output, but how these circuits interact to shape the somatodendritic excitability of Purkinje cells during motor behaviour remains unresolved. Here we perform dendritic and somatic patch-clamp recordings in vivo combined with optogenetic silencing of interneurons to investigate how dendritic excitation and inhibition generates bidirectional (that is, increased or decreased) Purkinje cell output during self-paced locomotion. We find that granule cells generate a sustained depolarization of Purkinje cell dendrites during movement, which is counterbalanced by variable levels of feedforward inhibition from local interneurons. Subtle differences in the dendritic excitation–inhibition balance generate robust, bidirectional changes in simple spike (SSp) output. Disrupting this balance by selectively silencing molecular layer interneurons results in unidirectional firing rate changes, increased SSp regularity and disrupted locomotor behaviour. Our findings provide a mechanistic understanding of how feedforward excitatory and inhibitory circuits shape Purkinje cell output during motor behaviour. PMID:27976716

Light-evoked hyperpolarization and silencing of neurons by conjugated polymers.

PubMed

Feyen, Paul; Colombo, Elisabetta; Endeman, Duco; Nova, Mattia; Laudato, Lucia; Martino, Nicola; Antognazza, Maria Rosa; Lanzani, Guglielmo; Benfenati, Fabio; Ghezzi, Diego

2016-03-04

The ability to control and modulate the action potential firing in neurons represents a powerful tool for neuroscience research and clinical applications. While neuronal excitation has been achieved with many tools, including electrical and optical stimulation, hyperpolarization and neuronal inhibition are typically obtained through patch-clamp or optogenetic manipulations. Here we report the use of conjugated polymer films interfaced with neurons for inducing a light-mediated inhibition of their electrical activity. We show that prolonged illumination of the interface triggers a sustained hyperpolarization of the neuronal membrane that significantly reduces both spontaneous and evoked action potential firing. We demonstrate that the polymeric interface can be activated by either visible or infrared light and is capable of modulating neuronal activity in brain slices and explanted retinas. These findings prove the ability of conjugated polymers to tune neuronal firing and suggest their potential application for the in-vivo modulation of neuronal activity.

Light-evoked hyperpolarization and silencing of neurons by conjugated polymers

PubMed Central

Feyen, Paul; Colombo, Elisabetta; Endeman, Duco; Nova, Mattia; Laudato, Lucia; Martino, Nicola; Antognazza, Maria Rosa; Lanzani, Guglielmo; Benfenati, Fabio; Ghezzi, Diego

2016-01-01

The ability to control and modulate the action potential firing in neurons represents a powerful tool for neuroscience research and clinical applications. While neuronal excitation has been achieved with many tools, including electrical and optical stimulation, hyperpolarization and neuronal inhibition are typically obtained through patch-clamp or optogenetic manipulations. Here we report the use of conjugated polymer films interfaced with neurons for inducing a light-mediated inhibition of their electrical activity. We show that prolonged illumination of the interface triggers a sustained hyperpolarization of the neuronal membrane that significantly reduces both spontaneous and evoked action potential firing. We demonstrate that the polymeric interface can be activated by either visible or infrared light and is capable of modulating neuronal activity in brain slices and explanted retinas. These findings prove the ability of conjugated polymers to tune neuronal firing and suggest their potential application for the in-vivo modulation of neuronal activity. PMID:26940513

Noninvasive diode laser activation of transient receptor potential proteins and nociceptors

NASA Astrophysics Data System (ADS)

Jiang, Nan; Cooper, Brian Y.; Nemenov, Michael I.

2007-02-01

We investigated diode laser (980 nm) evoked activation of transient receptor potential proteins (TRPV1 and TRPV2). C and A-delta (Aδ) nociceptor families are primarily responsible for pain mediation in the peripheral nervous system. TRPV1 proteins have been associated with heat evoked pain in C fibers while Aδ fibers have been associated with TRPV2. Diode laser stimulation allows a margin of safety between non-invasive activation and damage 19, 22, 34. Laser pulses (20-50 ms, 0.1-10 W, 980 nm) were used to stimulate: A) in vitro: excised patches from HEK293 cells expressing TRPV1; B) in vitro: rat DRG nociceptors expressing either TRPV1 or TRPV2; and C) in vivo: C-fibers of the rat saphenous nerve (SN) trunk. Cell currents were recorded using standard patch clamp methods. The SN was also stimulated electrically with bipolar electrodes. Stimulation (20-50 ms) of HEK and DRG cells expressing TRPV1 was highly reproducible. Activation and peak currents were achieved at estimated peak temperatures of 55°C and 70°C. Threshold activation was also observed in DRG neurons expressing TRPV2. The conduction velocity for laser-activated saphenous nerve afferents was in the C fiber range (0.5-1 m/s). Electrically stimulated nerve contained stimulation artifacts and complex neural components with conduction velocities ranging from 0.3-30 m/s. Diode laser activation of TRPV1 protein is a reproducible and effective means to probe TRP activity in both in vivo and in vitro preparations

Characterization of Ryanodine Receptor Type 1 Single Channel Activity Using “On-Nucleus” Patch Clamp

PubMed Central

Wagner, Larry E.; Groom, Linda A.; Dirksen, Robert T.; Yule, David I.

2014-01-01

In this study, we provide the first description of the biophysical and pharmacological properties of ryanodine receptor type 1 (RyR1) expressed in a native membrane using the on-nucleus configuration of the patch clamp technique. A stable cell line expressing rabbit RyR1 was established (HEK-RyR1) using the FLP-in 293 cell system. In contrast to untransfected cells, RyR1 expression was readily demonstrated by immunoblotting and immunocytochemistry in HEK-RyR1 cells. In addition, the RyR1 agonists 4-CMC and caffeine activated Ca2+ release that was inhibited by high concentrations of ryanodine. On nucleus patch clamp was performed in nuclei prepared from HEK-RyR1 cells. Raising the [Ca2+] in the patch pipette resulted in the appearance of a large conductance cation channel with well resolved kinetics and the absence of prominent subconductance states. Current versus voltage relationships were ohmic and revealed a chord conductance of ~750 pS or 450 pS in symmetrical 250 mM KCl or CsCl, respectively. The channel activity was markedly enhanced by caffeine and exposure to ryanodine resulted in the appearance of a subconductance state with a conductance ~40 % of the full channel opening with a Po near unity. In total, these properties are entirely consistent with RyR1 channel activity. Exposure of RyR1 channels to cyclic ADP ribose (cADPr), nicotinic acid adenine dinucleotide phosphate (NAADP) or dantrolene did not alter the single channel activity stimulated by Ca2+, and thus, it is unlikely these molecules directly modulate RyR1 channel activity. In summary, we describe an experimental platform to monitor the single channel properties of RyR channels. We envision that this system will be influential in characterizing disease-associated RyR mutations and the molecular determinants of RyR channel modulation. PMID:24972488

Facilitated giga-seal formation with a just originated glass surface.

PubMed

Böhle, T; Benndorf, K

1994-07-01

A simple technique of tip preparation in patch pipettes is described, which facilitates giga-seal formation. The pipettes were fabricated from thick-walled borosilicate glass tubing (external diameter 2.0 mm, internal diameter 0.5 mm) and the tips could be repeatedly broken in the bath. The pipette resistance correspondingly fell in steps of 3-20 M omega from about 80 M omega to about 2 M omega (double concentrated Tyrode). Scanning electron microscopy showed that the tip obtained after breaking was fairly plain. These broken tips were especially appropriate for patch-clamping. In cardiac myocytes in 11 out of 26 patches with Na+ channel activity, giga-seals developed spontaneously, i.e. without suction. In these patches the amplitude of the mean current with depolarizing pulses to -40 mV was significantly higher in comparison with patches formed under negative pressure. It is concluded that spontaneously sealed patches are most likely of planar configuration and the Na+ channel activity exceeds that in suction-induced patches.

Differentiation induction of mouse embryonic stem cells into sinus node-like cells by suramin

PubMed Central

Wiese, Cornelia; Nikolova, Teodora; Zahanich, Ihor; Sulzbacher, Sabine; Fuchs, Joerg; Yamanaka, Satoshi; Graf, Eva; Ravens, Ursula; Boheler, Kenneth R.; Wobus, Anna M.

2015-01-01

Background Embryonic stem (ES) cells differentiate into cardiac phenotypes representing early pacemaker-, atrial-, ventricular-, and sinus node-like cells, however, ES-derived specification into sinus nodal cells is not yet known. By using the naphthylamine derivative of urea, suramin, we were able to follow the process of cardiac specialization into sinus node-like cells. Methods Differentiating mouse ES cells were treated with suramin (500 μM) from day 5 to 7 of embryoid body formation, and cells were analysed for their differentiation potential via morphological analysis, flow cytometry, RT-PCR, immunohistochemistry and patch clamp analysis. Results Application of suramin resulted in an increased number of cardiac cells, but inhibition of neuronal, skeletal muscle and definitive endoderm differentiation. Immediately after suramin treatment, a decreased mesendoderm differentiation was found. Brachyury, FGF10, Wnt8 and Wnt3a transcript levels were significantly down-regulated, followed by a decrease in mesoderm- and cardiac progenitor-specific markers BMP2, GATA4/5, Wnt11, Isl1, Nkx2.5 and Tbx5 immediately after removal of the substance. With continued differentiation, a significant up-regulation of Brachyury, FGF10 and GATA5 transcript levels was observed, whereas Nkx2.5, Isl1, Tbx5, BMP2 and Wnt11 levels were normalized to control levels. At advanced differentiation stages, sinus node-specific HCN4, Tbx2 and Tbx3 transcript levels were significantly up-regulated. Immunofluorescence and patch-clamp analysis confirmed the increased number of sinus node-like cells, and electrophysiological analysis revealed a lower number of atrial- and ventricular-like cardiomyocytes following suramin treatment. Conclusion We conclude that the interference of suramin with the cardiac differentiation process modified mesoderm- and cardiac-specific gene expression resulting in enhanced formation of sinus node-like cells. PMID:19775764

A patch clamp study on reconstituted calcium permeable channels of human sperm plasma membranes.

PubMed

Ma, X H; Shi, Y L

1999-10-01

Ionic flux is thought to be important in the initiating process of gamete interaction such as acrosome reaction. However, modern electrophysiological methods, intracellular recording and patch-clamping, are difficult to approach the ion channels in mammal sperm membrane of an intact sperm due to its small size. In this work, by reconstituting the channel protein into lipid bilayer, Ca2+ channels in human spermatozoa were investigated with voltage clamp technique. Membrane proteins isolated from human sperm of 12 healthy donors were incorporated into lipid bilayer via fusion. In a cis 50//trans 10 mmol/L CaCl2 solution system, two types of channel events with similar reversal potential near the value of a perfect Ca2+ electrode, and sensitive to nifedipine and verapamil, were observed. Their unit conductance was 40 and 25 pS respectively. Percentage of channel open time was not dependent to holding potential for the former. However, for the channels of 25 pS, the percentage increased when the holding potential was changed from -20 to 100 mV. Ca(2+)-permeable channels were also detected from the spermatozoon samples of two infertile donors. Abnormal open time of these channels indicates that there are some defects in the conformation of the channel protein of infertile sperm membrane.

How Technique Is Changing Science.

ERIC Educational Resources Information Center

Hall, Stephen

1992-01-01

The author describes specific examples of the use of technology in science such as fiberoptic spectroscopy to observe galaxies and conduct three-dimensional maps of the universe. Adduces the following examples of technology influencing scientific investigations: gene cloning, gene sequencing, radioimmunoassays, patch-clamping of neurons, scanning…

The Promise of Microelectrode Array Approaches for Toxicity Testing: Examples with Neuroactive Chemicals

EPA Science Inventory

While high-throughput patch clamping formats provide rapid characterization of chemical effects on ion channel function and kinetics, the limitations of such systems often include the need for channel by channel characterization, requirements for transfected, rather than primary ...

Renal sodium transport in renin-deficient Dahl salt-sensitive rats

PubMed Central

Pavlov, Tengis S; Levchenko, Vladislav; Ilatovskaya, Daria V; Moreno, Carol; Staruschenko, Alexander

2016-01-01

Objective: The Dahl salt-sensitive rat is a well-established model of salt-sensitive hypertension. The goal of this study was to assess the expression and activity of renal sodium channels and transporters in the renin-deficient salt-sensitive rat. Methods: Renin knockout (Ren−/−) rats created on the salt-sensitive rat background were used to investigate the role of renin in the regulation of ion transport in salt-sensitive hypertension. Western blotting and patch-clamp analyses were utilized to assess the expression level and activity of Na+ transporters. Results: It has been described previously that Ren−/− rats exhibit severe kidney underdevelopment, polyuria, and lower body weight and blood pressure compared to their wild-type littermates. Here we found that renin deficiency led to decreased expression of sodium-hydrogen antiporter (NHE3), the Na+/H+ exchanger involved in Na+ absorption in the proximal tubules, but did not affect the expression of Na-K-Cl cotransporter (NKCC2), the main transporter in the loop of Henle. In the distal nephron, the expression of sodium chloride cotransporter (NCC) was lower in Ren−/− rats. Single-channel patch clamp analysis detected decreased ENaC activity in Ren−/− rats which was mediated via changes in the channel open probability. Conclusion: These data illustrate that renin deficiency leads to significant dysregulation of ion transporters. PMID:27443990

Dynamic Clamp in Cardiac and Neuronal Systems Using RTXI

PubMed Central

Ortega, Francis A.; Butera, Robert J.; Christini, David J.; White, John A.; Dorval, Alan D.

2016-01-01

The injection of computer-simulated conductances through the dynamic clamp technique has allowed researchers to probe the intercellular and intracellular dynamics of cardiac and neuronal systems with great precision. By coupling computational models to biological systems, dynamic clamp has become a proven tool in electrophysiology with many applications, such as generating hybrid networks in neurons or simulating channelopathies in cardiomyocytes. While its applications are broad, the approach is straightforward: synthesizing traditional patch clamp, computational modeling, and closed-loop feedback control to simulate a cellular conductance. Here, we present two example applications: artificial blocking of the inward rectifier potassium current in a cardiomyocyte and coupling of a biological neuron to a virtual neuron through a virtual synapse. The design and implementation of the necessary software to administer these dynamic clamp experiments can be difficult. In this chapter, we provide an overview of designing and implementing a dynamic clamp experiment using the Real-Time eXperiment Interface (RTXI), an open- source software system tailored for real-time biological experiments. We present two ways to achieve this using RTXI’s modular format, through the creation of a custom user-made module and through existing modules found in RTXI’s online library. PMID:25023319

Microglial Morphology and Dynamic Behavior Is Regulated by Ionotropic Glutamatergic and GABAergic Neurotransmission

PubMed Central

Fontainhas, Aurora M.; Wang, Minhua; Liang, Katharine J.; Chen, Shan; Mettu, Pradeep; Damani, Mausam; Fariss, Robert N.; Li, Wei; Wong, Wai T.

2011-01-01

Purpose Microglia represent the primary resident immune cells in the CNS, and have been implicated in the pathology of neurodegenerative diseases. Under basal or “resting” conditions, microglia possess ramified morphologies and exhibit dynamic surveying movements in their processes. Despite the prominence of this phenomenon, the function and regulation of microglial morphology and dynamic behavior are incompletely understood. We investigate here whether and how neurotransmission regulates “resting” microglial morphology and behavior. Methods We employed an ex vivo mouse retinal explant system in which endogenous neurotransmission and dynamic microglial behavior are present. We utilized live-cell time-lapse confocal imaging to study the morphology and behavior of GFP-labeled retinal microglia in response to neurotransmitter agonists and antagonists. Patch clamp electrophysiology and immunohistochemical localization of glutamate receptors were also used to investigate direct-versus-indirect effects of neurotransmission by microglia. Results Retinal microglial morphology and dynamic behavior were not cell-autonomously regulated but are instead modulated by endogenous neurotransmission. Morphological parameters and process motility were differentially regulated by different modes of neurotransmission and were increased by ionotropic glutamatergic neurotransmission and decreased by ionotropic GABAergic neurotransmission. These neurotransmitter influences on retinal microglia were however unlikely to be directly mediated; local applications of neurotransmitters were unable to elicit electrical responses on microglia patch-clamp recordings and ionotropic glutamatergic receptors were not located on microglial cell bodies or processes by immunofluorescent labeling. Instead, these influences were mediated indirectly via extracellular ATP, released in response to glutamatergic neurotransmission through probenecid-sensitive pannexin hemichannels. Conclusions Our results demonstrate that neurotransmission plays an endogenous role in regulating the morphology and behavior of “resting” microglia in the retina. These findings illustrate a mode of constitutive signaling between the neural and immune compartments of the CNS through which immune cells may be regulated in concert with levels of neural activity. PMID:21283568

Simultaneous recording of electrical activity and the underlying ionic currents in NG108-15 cells cultured on gold substrate.

PubMed

Acosta-García, Ma Cristina; Morales-Reyes, Israel; Jiménez-Anguiano, Anabel; Batina, Nikola; Castellanos, N P; Godínez-Fernández, R

2018-02-01

This paper shows the simultaneous recording of electrical activity and the underlying ionic currents by using a gold substrate to culture NG108-15 cells. Cells grown on two different substrates (plastic Petri dishes and gold substrates) were characterized quantitatively through scanning electron microscopy (SEM) as well as qualitatively by optical and atomic force microscopy (AFM). No significant differences were observed between the surface area of cells cultured on gold substrates and Petri dishes, as indicated by measurements performed on SEM images. We also evaluated the electrophysiological compatibility of the cells through standard patch-clamp experiments by analyzing features such as the resting potential, membrane resistance, ionic currents, etc. Cells grown on both substrates showed no significant differences in their dependency on voltage, as well as in the magnitude of the Na+ and K+ current density; however, cells cultured on the gold substrate showed a lower membrane capacitance when compared to those grown on Petri dishes. By using two separate patch-clamp amplifiers, we were able to record the membrane current with the conventional patch-clamp technique and through the gold substrate simultaneously. Furthermore, the proposed technique allowed us to obtain simultaneous recordings of the electrical activity (such as action potentials firing) and the underlying membrane ionic currents. The excellent conductivity of gold makes it possible to overcome important difficulties found in conventional electrophysiological experiments such as those presented by the resistance of the electrolytic bath solution. We conclude that the technique here presented constitutes a solution to the problem of the simultaneous recording of electrical activity and the underlying ionic currents, which for decades, had been solved only partially.

Melatonin mediates vasodilation through both direct and indirect activation of BKCa channels.

PubMed

Zhao, T; Zhang, H; Jin, C; Qiu, F; Wu, Y; Shi, L

2017-10-01

Melatonin, synthesized primarily by the pineal gland, is a neuroendocrine hormone with high membrane permeability. The vascular effects of melatonin, including vasoconstriction and vasodilation, have been demonstrated in numerous studies. However, the mechanisms underlying these effects are not fully understood. Large-conductance Ca 2+ -activated K + (BK Ca ) channels are expressed broadly on smooth muscle cells and play an important role in vascular tone regulation. This study explored the mechanisms of myocyte BK Ca channels and endothelial factors underlying the action of melatonin on the mesenteric arteries (MAs). Vascular contractility and patch-clamp studies were performed on myocytes of MAs from Wistar rats. Melatonin induced significant vasodilation on MAs. In the presence of N ω -nitro-l-arginine methyl ester (l-NAME), a potent endothelial oxide synthase (eNOS) inhibitor, melatonin elicited concentration-dependent relaxation, with lowered pIC 50 The effect of melatonin was significantly attenuated in the presence of BK Ca channel blocker iberiotoxin or MT1/MT2 receptor antagonist luzindole in both (+) l-NAME and (-) l-NAME groups. In the (+) l-NAME group, iberiotoxin caused a parallel rightward shift of the melatonin concentration-relaxation curve, with pIC 50 lower than that of luzindole. Both inside-out and cell-attached patch-clamp recordings showed that melatonin significantly increased the open probability, mean open time and voltage sensitivity of BK Ca channels. In a cell-attached patch-clamp configuration, the melatonin-induced enhancement of BK Ca channel activity was significantly suppressed by luzindole. These findings indicate that in addition to the activation of eNOS, melatonin-induced vasorelaxation of MAs is partially attributable to its direct (passing through the cell membrane) and indirect (via MT1/MT2 receptors) activation of the BK Ca channels on mesenteric arterial myocytes. © 2017 Society for Endocrinology.

Signal presequences increase mitochondrial permeability and open the multiple conductance channel.

PubMed

Kushnareva, Y E; Campo, M L; Kinnally, K W; Sokolove, P M

1999-06-01

We have reported that the signal presequence of cytochrome oxidase subunit IV from Neurospora crassa increases the permeability of isolated rat liver mitochondria [P. M. Sokolove and K. W. Kinnally (1996) Arch. Biochem. Biophys. 336, 69] and regulates the behavior of the mutiple conductance channel (MCC) of yeast inner mitochondrial membrane [T. A. Lohret and K. W. Kinnally (1995) J. Biol. Chem. 270, 15950]. Here we examine in greater detail the action of a number of mitochondrial presequences from various sources and of several control peptides on the permeability of isolated rat liver mitochondria and on MCC activity monitored via patch-clamp techniques in both mammalian mitoplasts and a reconstituted yeast system. The data indicate that the ability to alter mitochondrial permeability is a property of most, but not all, signal peptides. Furthermore, it is clear that, although signal peptides are characterized by positive charge and the ability to form amphiphilic alpha helices, these two characteristics are not sufficient to guarantee mitochondrial effects. Finally, the results reveal a strong correlation between peptide effects on the permeability of isolated mitochondria and on MCC activity: peptides that induced swelling of mouse and rat mitochondria also activated the quiescent MCC of mouse mitoplasts and induced flickering of active MCC reconstituted from yeast mitochondrial membranes. Moreover, relative peptide efficacies were very similar for mitochondrial swelling and both types of patch-clamp experiments. We propose that patch-clamp recordings of MCC activity and the high-amplitude swelling induced by signal peptides reflect the opening of a single channel. Based on the selective responsiveness of that channel to signal peptides and the dependence of its opening in isolated mitochondria on membrane potential, we further suggest that the channel is involved in the mitochondrial protein import process. Copyright 1999 Academic Press.

Flying-patch patch-clamp study of G22E-MscL mutant under high hydrostatic pressure.

PubMed

Petrov, Evgeny; Rohde, Paul R; Martinac, Boris

2011-04-06

High hydrostatic pressure (HHP) present in natural environments impacts on cell membrane biophysical properties and protein quaternary structure. We have investigated the effect of high hydrostatic pressure on G22E-MscL, a spontaneously opening mutant of Escherichia coli MscL, the bacterial mechanosensitive channel of large conductance. Patch-clamp technique combined with a flying-patch device and hydraulic setup allowed the study of the effects of HHP up to 90 MPa (as near the bottom of the Marianas Trench) on the MscL mutant channel reconstituted into liposome membranes, in addition to recording in situ from the mutant channels expressed in E. coli giant spheroplasts. In general, against thermodynamic predictions, hydrostatic pressure in the range of 0.1-90 MPa increased channel open probability by favoring the open state of the channel. Furthermore, hydrostatic pressure affected the channel kinetics, as manifested by the propensity of the channel to gate at subconducting levels with an increase in pressure. We propose that the presence of water molecules around the hydrophobic gate of the G22E MscL channel induce hydration of the hydrophobic lock under HHP causing frequent channel openings and preventing the channel closure in the absence of membrane tension. Furthermore, our study indicates that HHP can be used as a valuable experimental approach toward better understanding of the gating mechanism in complex channels such as MscL. Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.

MATLAB implementation of a dynamic clamp with bandwidth >125 KHz capable of generating INa at 37°C

PubMed Central

Clausen, Chris; Valiunas, Virginijus; Brink, Peter R.; Cohen, Ira S.

2012-01-01

We describe the construction of a dynamic clamp with bandwidth >125 KHz that utilizes a high performance, yet low cost, standard home/office PC interfaced with a high-speed (16 bit) data acquisition module. High bandwidth is achieved by exploiting recently available software advances (code-generation technology, optimized real-time kernel). Dynamic-clamp programs are constructed using Simulink, a visual programming language. Blocks for computation of membrane currents are written in the high-level matlab language; no programming in C is required. The instrument can be used in single- or dual-cell configurations, with the capability to modify programs while experiments are in progress. We describe an algorithm for computing the fast transient Na+ current (INa) in real time, and test its accuracy and stability using rate constants appropriate for 37°C. We then construct a program capable of supplying three currents to a cell preparation: INa, the hyperpolarizing-activated inward pacemaker current (If), and an inward-rectifier K+ current (IK1). The program corrects for the IR drop due to electrode current flow, and also records all voltages and currents. We tested this program on dual patch-clamped HEK293 cells where the dynamic clamp controls a current-clamp amplifier and a voltage-clamp amplifier controls membrane potential, and current-clamped HEK293 cells where the dynamic clamp produces spontaneous pacing behavior exhibiting Na+ spikes in otherwise passive cells. PMID:23224681

PA-6 inhibits inward rectifier currents carried by V93I and D172N gain-of-function KIR2.1 channels, but increases channel protein expression.

PubMed

Ji, Yuan; Veldhuis, Marlieke G; Zandvoort, Jantien; Romunde, Fee L; Houtman, Marien J C; Duran, Karen; van Haaften, Gijs; Zangerl-Plessl, Eva-Maria; Takanari, Hiroki; Stary-Weinzinger, Anna; van der Heyden, Marcel A G

2017-07-15

The inward rectifier potassium current I K1 contributes to a stable resting membrane potential and phase 3 repolarization of the cardiac action potential. KCNJ2 gain-of-function mutations V93I and D172N associate with increased I K1 , short QT syndrome type 3 and congenital atrial fibrillation. Pentamidine-Analogue 6 (PA-6) is an efficient (IC 50  = 14 nM with inside-out patch clamp methodology) and specific I K1 inhibitor that interacts with the cytoplasmic pore region of the K IR 2.1 ion channel, encoded by KCNJ2. At 10 μM, PA-6 increases wild-type (WT) K IR 2.1 expression in HEK293T cells upon chronic treatment. We hypothesized that PA-6 will interact with and inhibit V93I and D172N K IR 2.1 channels, whereas impact on channel expression at the plasma membrane requires higher concentrations. Molecular modelling was performed with the human K IR 2.1 closed state homology model using FlexX. WT and mutant K IR 2.1 channels were expressed in HEK293 cells. Patch-clamp single cell electrophysiology measurements were performed in the whole cell and inside-out mode of the patch clamp method. K IR 2.1 expression level and localization were determined by western blot analysis and immunofluorescence microscopy, respectively. PA-6 docking in the V93I/D172N double mutant homology model of K IR 2.1 demonstrated that mutations and drug-binding site are >30 Å apart. PA-6 inhibited WT and V93I outward currents with similar potency (IC 50  = 35.5 and 43.6 nM at +50 mV for WT and V93I), whereas D172N currents were less sensitive (IC 50  = 128.9 nM at +50 mV) using inside-out patch-clamp electrophysiology. In whole cell mode, 1 μM of PA-6 inhibited outward I K1 at -50 mV by 28 ± 36%, 18 ± 20% and 10 ± 6%, for WT, V93I and D172N channels respectively. Western blot analysis demonstrated that PA-6 (5 μM, 24 h) increased K IR 2.1 expression levels of WT (6.3 ± 1.5 fold), and V93I (3.9 ± 0.9) and D172N (4.8 ± 2.0) mutants. Immunofluorescent microscopy demonstrated dose-dependent intracellular K IR 2.1 accumulation following chronic PA-6 application (24 h, 1 and 5 μM). 1) KCNJ2 gain-of-function mutations V93I and D172N in the K IR 2.1 ion channel do not impair PA-6 mediated inhibition of I K1 , 2) PA-6 elevates K IR 2.1 protein expression and induces intracellular K IR 2.1 accumulation, 3) PA-6 is a strong candidate for further preclinical evaluation in treatment of congenital SQT3 and AF.

Double patch clamp reveals that transient fusion (kiss-and-run) is a major mechanism of secretion in calf adrenal chromaffin cells: high calcium shifts the mechanism from kiss-and-run to complete fusion.

PubMed

Elhamdani, Abdeladim; Azizi, Fouad; Artalejo, Cristina R

2006-03-15

Transient fusion ("kiss-and-run") is accepted as a mode of transmitter release both in central neurons and neuroendocrine cells, but the prevalence of this mechanism compared with full fusion is still in doubt. Using a novel double patch-clamp method (whole cell/cell attached), permitting the recording of unitary capacitance events while stimulating under a variety of conditions including action potentials, we show that transient fusion is the predominant (>90%) mode of secretion in calf adrenal chromaffin cells. Raising intracellular Ca2+ concentration ([Ca]i) from 10 to 200 microM increases the incidence of full fusion events at the expense of transient fusion. Blocking rapid endocytosis that normally terminates transient fusion events also promotes full fusion events. Thus, [Ca]i controls the transition between transient and full fusion, each of which is coupled to different modes of endocytosis.

Phenytoin preferentially inhibits L-type calcium currents in whole-cell patch-clamped cardiac and skeletal muscle cells.

PubMed

Rivet, M; Bois, P; Cognard, C; Raymond, G

1990-10-01

The effect of the anticonvulsant diphenylhydantoin (phenytoin) was tested on the inward calcium currents of whole-cell patch-clamped cells from rat and human muscles and from frog atrium. A concentration of 10 microM phenytoin was required to obtain a threshold inhibitory effect and, even with high concentrations (100 microM), the inhibition was not complete. In skeletal muscle (rat and human cells in culture), phenytoin (30 microM) exerted a more potent effect on the high-threshold calcium current (ICa,L inhibition: 53 +/- 6% mean +/- SDn-1) rather than on the low-threshold one (ICa,T inhibition: 16 +/- 10%). Similar results were obtained on dissociated frog atrial cells. These data are to be contrasted with those previously reported on neuronal cells, where specific inhibition of ICa,T was reported. Thus, the action of phenytoin appears to be different in muscle and nerve so that phenytoin does not appear to be a specific inhibitor of ICa,T.

Photoelectrical Stimulation of Neuronal Cells by an Organic Semiconductor-Electrolyte Interface.

PubMed

Abdullaeva, Oliya S; Schulz, Matthias; Balzer, Frank; Parisi, Jürgen; Lützen, Arne; Dedek, Karin; Schiek, Manuela

2016-08-23

As a step toward the realization of neuroprosthetics for vision restoration, we follow an electrophysiological patch-clamp approach to study the fundamental photoelectrical stimulation mechanism of neuronal model cells by an organic semiconductor-electrolyte interface. Our photoactive layer consisting of an anilino-squaraine donor blended with a fullerene acceptor is supporting the growth of the neuronal model cell line (N2A cells) without an adhesion layer on it and is not impairing cell viability. The transient photocurrent signal upon illumination from the semiconductor-electrolyte layer is able to trigger a passive response of the neuronal cells under physiological conditions via a capacitive coupling mechanism. We study the dynamics of the capacitive transmembrane currents by patch-clamp recordings and compare them to the dynamics of the photocurrent signal and its spectral responsivity. Furthermore, we characterize the morphology of the semiconductor-electrolyte interface by atomic force microscopy and study the stability of the interface in dark and under illuminated conditions.

One-channel Cell-attached Patch-clamp Recording

PubMed Central

Maki, Bruce A.; Cummings, Kirstie A.; Paganelli, Meaghan A.; Murthy, Swetha E.; Popescu, Gabriela K.

2014-01-01

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate depends on properties intrinsic to each channel protein as well as the mechanism by which it is generated and controlled and represents an important area of current research. Information about the operational dynamics of ion channel proteins can be obtained by observing long stretches of current produced by a single molecule. Described here is a protocol for obtaining one-channel cell-attached patch-clamp current recordings for a ligand gated ion channel, the NMDA receptor, expressed heterologously in HEK293 cells or natively in cortical neurons. Also provided are instructions on how to adapt the method to other ion channels of interest by presenting the example of the mechano-sensitive channel PIEZO1. This method can provide data regarding the channel’s conductance properties and the temporal sequence of open-closed conformations that make up the channel’s activation mechanism, thus helping to understand their functions in health and disease. PMID:24961614

Combination of High-density Microelectrode Array and Patch Clamp Recordings to Enable Studies of Multisynaptic Integration.

PubMed

Jäckel, David; Bakkum, Douglas J; Russell, Thomas L; Müller, Jan; Radivojevic, Milos; Frey, Urs; Franke, Felix; Hierlemann, Andreas

2017-04-20

We present a novel, all-electric approach to record and to precisely control the activity of tens of individual presynaptic neurons. The method allows for parallel mapping of the efficacy of multiple synapses and of the resulting dynamics of postsynaptic neurons in a cortical culture. For the measurements, we combine an extracellular high-density microelectrode array, featuring 11'000 electrodes for extracellular recording and stimulation, with intracellular patch-clamp recording. We are able to identify the contributions of individual presynaptic neurons - including inhibitory and excitatory synaptic inputs - to postsynaptic potentials, which enables us to study dendritic integration. Since the electrical stimuli can be controlled at microsecond resolution, our method enables to evoke action potentials at tens of presynaptic cells in precisely orchestrated sequences of high reliability and minimum jitter. We demonstrate the potential of this method by evoking short- and long-term synaptic plasticity through manipulation of multiple synaptic inputs to a specific neuron.

The etomidate analog ET-26 HCl retains superior myocardial performance: Comparisons with etomidate in vivo and in vitro.

PubMed

Liu, Xingxing; Song, Haibo; Yang, Jun; Zhou, Cheng; Kang, Yi; Yang, Linghui; Liu, Jin; Zhang, Wensheng

2018-01-01

(R)-2-methoxyethyl1-(1-phenylethyl)-1H-imidazole-5-carboxylate hydrochloride (ET-26 HCl) is a novel etomidate analogue. The purpose of this study was to characterize whether ET-26 HCl could retain the superior myocardial performance of etomidate in vivo and in vitro. In vivo, the influence of ET-26 HCl and etomidate on the cardiac function of dogs was confirmed using echocardiography and electrocardiogram. In vitro, a Langendorff preparation was used to examine direct myocardial performance in isolated rat hearts, and a whole-cell patch-clamp technique was used to study effects on the human ether-a-go-go-related gene (hERG) channel. In vivo, after a single bolus administration of ET-26 HCl or etomidate, no significant difference in echocardiography and electrocardiogram parameters was observed. No arrhythmia occurred and no QT interval prolongation happened during the study period. In the in vitro Langendorff preparation, none of the cardiac parameters were abnormal, and the hERG recordings showed that ET-26 HCl and etomidate inhibited the tail current of the hERG in a concentration-dependent manner with an IC50 of 742.51 μM and 263.60 μM, respectively. In conclusion, through an in vivo experiment and a whole organ preparation, the current study found that ET-26 HCl can maintain a myocardial performance that is similar to that of etomidate. In addition, the electrophysiology study indicated that ET-26 HCl and etomidate inhibited the hERG at a supra-therapeutic concentration.

Selective binding and transcytosis of Ulex europaeus 1 lectin by mouse Peyer's patch M-cells in vivo.

PubMed

Clark, M A; Jepson, M A; Simmons, N L; Hirst, B H

1995-12-01

The in vivo interaction of the lectin Ulex europaeus agglutinin 1 with mouse Peyer's patch follicle-associated epithelial cells was studied in the mouse Peyer's patch gut loop model by immunofluorescence and electron microscopy. The lectin targets to mouse Peyer's patch M-cells and is rapidly endocytosed and transcytosed. These processes are accompanied by morphological changes in the M-cell microvilli and by redistribution of polymerised actin. The demonstration of selective binding and uptake of a lectin by intestinal M-cells in vivo suggests that M-cell-specific surface glycoconjugates might act as receptors for the selective adhesion/uptake of microorganisms.

Novel nootropic dipeptide Noopept increases inhibitory synaptic transmission in CA1 pyramidal cells.

PubMed

Kondratenko, Rodion V; Derevyagin, Vladimir I; Skrebitsky, Vladimir G

2010-05-31

Effects of newly synthesized nootropic and anxiolytic dipeptide Noopept on inhibitory synaptic transmission in hippocampal CA1 pyramidal cells were investigated using patch-clamp technique in whole-cell configuration. Bath application of Noopept (1 microM) significantly increased the frequency of spike-dependant spontaneous IPSCs whereas spike-independent mIPSCs remained unchanged. It was suggested that Noopept mediates its effect due to the activation of inhibitory interneurons terminating on CA1 pyramidal cells. Results of current clamp recording of inhibitory interneurons residing in stratum radiatum confirmed this suggestion. Copyright 2010 Elsevier Ireland Ltd. All rights reserved.

[Peptidergic modulation of the hippocampus synaptic activity].

PubMed

Skrebitskiĭ, V G; Kondratenko, R V; Povarov, I S; Dereviagin, V I

2011-11-01

Effects of two newly synthesized nootropic and anxiolytic dipeptides: Noopept and Selank on inhibitory synaptic transmission in hippocampal CA1 pyramidal cells were investigated using patch-clamp technique in whole-cell configuration. Bath application of Noopept (1 microM) or Selank (2 microM) significantly increased the frequency of spike-dependent spontaneous m1PSCs, whereas spike-independent mlPSCs remained unchanged. It was suggested that both peptides mediated their effect sue to activation of inhibitory interneurons terminating on CA1 pyramidal cells. Results of current clamp recording of inhibitory interneurons residing in stratum radiatum confirmed this suggestion, at least for Noonent.

The Electrophysiological Biosensor for Batch-Measurement of Cell Signals

NASA Astrophysics Data System (ADS)

Suzuki, Kengo; Tanabe, Masato; Ezaki, Takahiro; Konishi, Satoshi; Oka, Hiroaki; Ozaki, Nobuhiko

This paper presents the development of electrophysiological biosensor. The developed sensor allows a batch-measurement by detecting all signals from a large number of cells together. The developed sensor employs the same measurement principle as the patch-clamp technique. A single cell is sucked and clamped in a micro hole with detecting electrode. Detecting electrodes in arrayed micro holes are connected together for the batch-measurement of signals a large number of cell signals. Furthermore, an array of sensors for batch-measurement is designed to improve measurement-throughput to satisfy requirements for the drug screening application.

Formulation of unidirectional release buccal patches of carbamazepine and study of permeation through porcine buccal mucosa

PubMed Central

Govindasamy, Parthasarathy; Kesavan, Bhaskar Reddy; Narasimha, Jayaveera Korlakunta

2013-01-01

Objective To achieve transbuccal release of carbamazepine by loading in unidirectional release mucoadhesive buccal patches. Methods Buccal patches of carbamazepine with unidirectional drug release were prepared using hydroxypropyl methyl cellulose, polyvinyl alcohol, polyvinyl pyrrolidone and ethyl cellulose by solvent casting method. Water impermeable backing layer (Pidilite® Biaxially-oriented polypropylene film) of patches provided unidirectional drug release. They were evaluated for thickness, mass uniformity, surface pH and folding endurance. Six formulations FA2, FA8, FA10, FB1, FB14 and FB16 (folding endurance above 250) were evaluated further for swelling studies, ex vivo mucoadhesive strength, ex vivo mucoadhesion time, in vitro drug release, ex vivo permeation, accelerated stability studies and FTIR and XRD spectral studies. Results The ex vivo mucoadhesion time of patches ranged between 109 min (FA10) to 126 min (FB14). The ex vivo mucoadhesive force was in the range of 0.278 to 0.479 kg/m/s. The in vitro drug release studies revealed that formulation FA8 released 84% and FB16 released 99.01% of drug in 140 min. Conclusions The prepared unidirectional buccal patches of carbamazepine provided a maximum drug release within specified mucoadhesion period and it indicates a potential alternative drug delivery system for systemic delivery of carbamazepine. PMID:24093793

Transmucosal delivery of domperidone from bilayered buccal patches: in vitro, ex vivo and in vivo characterization.

PubMed

Palem, Chinna Reddy; Gannu, Ramesh; Doodipala, Narender; Yamsani, Vamshi Vishnu; Yamsani, Madhusudan Rao

2011-10-01

Bilayered mucoadhesive buccal patches for systemic administration of domperidone (DOM), a dopamine-receptor (D(2)) antagonist, were developed using hydroxy propyl methyl cellulose and PVPK30 as a primary layer and Eudragit RLPO and PEO as a secondary layer. Ex vivo drug permeation through porcine buccal membrane was performed. Bilayered buccal patches were developed by solvent casting technique and evaluated for in vitro drug release, moisture absorption, mechanical properties, surface pH, in vitro bioadhesion, in vivo residence time and ex vivo permeation of DOM through porcine buccal membrane from a bilayered buccal patch. Formulation DB4 was associated with 99.5% drug release with a higuchi model release profile and 53.9% of the drug had permeated in 6 h, with a flux of 0.492 mg/h/cm(2) through porcine buccal membrane. DB4 showed 5.58 N and 3.28 mJ peak detachment force and work of adhesion, respectively. The physicochemical interactions between DOM and the polymer were investigated by differential scanning calorimetry (DSC) and fourier transform infrared (FTIR) Spectroscopy. DSC and FTIR studies revealed no interaction between drug and polymer. Stability studies for optimized patch DB4 was carried out at 40°C/75% relative humidity. The formulations were found to be stable over a period of 3 months with respect to drug content, in vitro release and ex vivo permeation through porcine buccal membrane. The results indicate that suitable bilayered mucoadhesive buccal patches with desired permeability could be prepared.

Ion channel pharmacology under flow: automation via well-plate microfluidics.

PubMed

Spencer, C Ian; Li, Nianzhen; Chen, Qin; Johnson, Juliette; Nevill, Tanner; Kammonen, Juha; Ionescu-Zanetti, Cristian

2012-08-01

Automated patch clamping addresses the need for high-throughput screening of chemical entities that alter ion channel function. As a result, there is considerable utility in the pharmaceutical screening arena for novel platforms that can produce relevant data both rapidly and consistently. Here we present results that were obtained with an innovative microfluidic automated patch clamp system utilizing a well-plate that eliminates the necessity of internal robotic liquid handling. Continuous recording from cell ensembles, rapid solution switching, and a bench-top footprint enable a number of assay formats previously inaccessible to automated systems. An electro-pneumatic interface was employed to drive the laminar flow of solutions in a microfluidic network that delivered cells in suspension to ensemble recording sites. Whole-cell voltage clamp was applied to linear arrays of 20 cells in parallel utilizing a 64-channel voltage clamp amplifier. A number of unique assays requiring sequential compound applications separated by a second or less, such as rapid determination of the agonist EC(50) for a ligand-gated ion channel or the kinetics of desensitization recovery, are enabled by the system. In addition, the system was validated via electrophysiological characterizations of both voltage-gated and ligand-gated ion channel targets: hK(V)2.1 and human Ether-à-go-go-related gene potassium channels, hNa(V)1.7 and 1.8 sodium channels, and (α1) hGABA(A) and (α1) human nicotinic acetylcholine receptor receptors. Our results show that the voltage dependence, kinetics, and interactions of these channels with pharmacological agents were matched to reference data. The results from these IonFlux™ experiments demonstrate that the system provides high-throughput automated electrophysiology with enhanced reliability and consistency, in a user-friendly format.

Phenylquinoxalinone CFTR activator as potential prosecretory therapy for constipation

PubMed Central

CIL, ONUR; PHUAN, PUAY-WAH; SON, JUNG-HO; ZHU, JIE S.; KU, COLTON K.; TABIB, NILOUFAR AKHAVAN; TEUTHORN, ANDREW P.; FERRERA, LORETTA; ZACHOS, NICHOLAS C.; LIN, RUXIAN; GALIETTA, LUIS J. V.; DONOWITZ, MARK; KURTH, MARK J.; VERKMAN, ALAN S.

2017-01-01

Constipation is a common condition for which current treatments can have limited efficacy. By high-throughput screening, we recently identified a phenylquinoxalinone activator of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel that stimulated intestinal fluid secretion and normalized stool output in a mouse model of opioid-induced constipation. Here, we report phenylquinoxalinone structure-activity analysis, mechanism of action, animal efficacy data in acute and chronic models of constipation, and functional data in ex vivo primary cultured human enterocytes. Structure-activity analysis was done on 175 phenylquinoxalinone analogs, including 15 synthesized compounds. The most potent compound, CFTRact-J027, activated CFTR with EC50 ~ 200 nM, with patch-clamp analysis showing a linear CFTR current-voltage relationship with direct CFTR activation. CFTRact-J027 corrected reduced stool output and hydration in a mouse model of acute constipation produced by scopolamine and in a chronically constipated mouse strain (C3H/HeJ). Direct comparison with the approved prosecretory drugs lubiprostone and linaclotide showed substantially greater intestinal fluid secretion with CFTRact-J027, as well as greater efficacy in a constipation model. As evidence to support efficacy in human constipation, CFTRact-J027 increased transepithelial fluid transport in enteroids generated from normal human small intestine. Also, CFTRact-J027 was rapidly metabolized in vitro in human hepatic microsomes, suggesting minimal systemic exposure upon oral administration. These data establish structure-activity and mechanistic data for phenylquinoxalinone CFTR activators, and support their potential efficacy in human constipation. PMID:27815136

Functional regeneration of the ex-vivo reconstructed mesocorticolimbic dopaminergic system.

PubMed

Dossi, Elena; Heine, Claudia; Servettini, Ilenio; Gullo, Francesca; Sygnecka, Katja; Franke, Heike; Illes, Peter; Wanke, Enzo

2013-12-01

CNS reparative-medicine therapeutic strategies need answers on the putative recapitulation of the basic rules leading to mammalian CNS development. To achieve this aim, we focus on the regeneration of functional connections in the mesocorticolimbic dopaminergic system. We used organotypic slice cocultures of ventral tegmental area/substantia nigra (VTA/SN) and prefrontal cortex (PFC) on a multielectrode array (MEA) platform to record spikes and local field potentials. The spontaneously growing synaptically based bidirectional bursting activity was followed from 2 to 28 days in vitro (DIV). A statistical analysis of excitatory and inhibitory neurons properties of the physiological firing activity demonstrated a remarkable, exponentially increasing maturation with a time constant of about 5-7 DIV. Immunohistochemistry demonstrated that the ratio of excitatory/inhibitory neurons (3:1) was in line with the functional results obtained. Exemplary pharmacology suggested that GABAA receptors were able to exert phasic and tonic inhibition typical of an adulthood network. Moreover, dopamine D2 receptor inactivation was equally inhibitory both on the spontaneous neuronal activity recorded by MEA and on patch-clamp electrophysiology in PFC pyramidal neurons. These results demonstrate that axon growth cones reach synaptic targets up to full functionality and that organotypic cocultures of the VTA/SN-PFC perfectly model their newly born dopaminergic, glutamatergic and GABAergic neuronal circuitries.

Antigenic sites on the HN domain of botulinum neurotoxin A stimulate protective antibody responses against active toxin.

PubMed

Ayyar, B Vijayalakshmi; Tajhya, Rajeev B; Beeton, Christine; Atassi, M Zouhair

2015-10-28

Botulinum neurotoxins (BoNTs) are the most toxic substances known. BoNT intoxicates cells in a highly programmed fashion initiated by binding to the cell surface, internalization and enzymatic cleavage of substrate, thus, inhibiting synaptic exocytosis. Over the past two decades, immunological significance of BoNT/A C-terminal heavy chain (HC) and light chain (LC) domains were investigated extensively leading to important findings. In the current work, we explored the significance of BoNT/A heavy chain N-terminal (HN) region as a vaccine candidate. Mice were immunized with recombinant HN519-845 generating antibodies (Abs) that were found to be protective against lethal dose of BoNT/A. Immuno-dominant regions of HN519-845 were identified and individually investigated for antibody response along with synthetic peptides within those regions, using in vivo protection assays against BoNT/A. Results were confirmed by patch-clamp analysis where anti-HN antibodies were studied for the ability to block toxin-induced channel formation. This data strongly indicated that HN519-593 is an important region in generating protective antibodies and should be valuable in a vaccine design. These results are the first to describe and dissect the protective activity of the BoNT/A HN domain.

Ethanol Alters Alveolar Fluid Balance via Nadph Oxidase (NOX) Signaling to Epithelial Sodium Channels (ENaC) in the Lung

PubMed Central

Downs, Charles A.; Kreiner, Lisa H.; Eaton, Amity F.; Johnson, Nicholle M.; Brown, Lou Ann

2013-01-01

Chronic alcohol consumption is associated with increased incidence of ICU-related morbidity and mortality, primarily from acute respiratory distress syndrome (ARDS). However, the mechanisms involved are unknown. One explanation is that alcohol regulates epithelial sodium channels (ENaC) via oxidant signaling to promote a pro- injury environment. We used small rodent models to mimic acute and chronic alcohol consumption and tested the hypothesis that ethanol (EtOH) would affect lung fluid clearance by up-regulating ENaC activity in the lung. Fluorescence labeling of rat lung slices and in vivo mouse lung revealed an increase in ROS production in response to acute EtOH exposure. Using western blots and fluorescein-5-maleimide labeling, we conclude that EtOH exposure modifies cysteines of α-ENaC while data from single channel patch clamp analysis confirm that 0.16% EtOH increased ENaC activity in rat alveolar cells. In vivo lung fluid clearance demonstrated a latent increase in fluid clearance in mice receiving EtOH diet. Ethanol mice given a tracheal instillation of LPS demonstrated early lung fluid clearance compared to caloric control mice and C57Bl/6 mice. Standard biochemical techniques reveal that chronic EtOH consumption resulted in greater protein expression of the catalytic gp91phox subunit and the obligate Rac1 protein. Collectively these data suggest that chronic EtOH consumption may lead to altered regulation of ENaC, contributing to a ‘pro-injury’ environment in the alcohol lung. PMID:23382956

Interpretation of field potentials measured on a multi electrode array in pharmacological toxicity screening on primary and human pluripotent stem cell-derived cardiomyocytes.

PubMed

Tertoolen, L G J; Braam, S R; van Meer, B J; Passier, R; Mummery, C L

2018-03-18

Multi electrode arrays (MEAs) are increasingly used to detect external field potentials in electrically active cells. Recently, in combination with cardiomyocytes derived from human (induced) pluripotent stem cells they have started to become a preferred tool to examine newly developed drugs for potential cardiac toxicity in pre-clinical safety pharmacology. The most important risk parameter is proarrhythmic activity in cardiomyocytes which can cause sudden cardiac death. Whilst MEAs can provide medium- to high- throughput noninvasive assay platform, the translation of a field potential to cardiac action potential (normally measured by low-throughput patch clamp) is complex so that accurate assessment of drug risk to the heart is in practice still challenging. To address this, we used computational simulation to study the theoretical relationship between aspects of the field potential and the underlying cardiac action potential. We then validated the model in both primary mouse- and human pluripotent (embryonic) stem cell-derived cardiomyocytes showing that field potentials measured in MEAs could be converted to action potentials that were essentially identical to those determined directly by electrophysiological patch clamp. The method significantly increased the amount of information that could be extracted from MEA measurements and thus combined the advantages of medium/high throughput with more informative readouts. We believe that this will benefit the analysis of drug toxicity screening of cardiomyocytes using in time and accuracy. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

TPC2 polymorphisms associated with a hair pigmentation phenotype in humans result in gain of channel function by independent mechanisms.

PubMed

Chao, Yu-Kai; Schludi, Verena; Chen, Cheng-Chang; Butz, Elisabeth; Nguyen, O N Phuong; Müller, Martin; Krüger, Jens; Kammerbauer, Claudia; Ben-Johny, Manu; Vollmar, Angelika M; Berking, Carola; Biel, Martin; Wahl-Schott, Christian A; Grimm, Christian

2017-10-10

Two-pore channels (TPCs) are endolysosomal cation channels. Two members exist in humans, TPC1 and TPC2. Functional roles associated with the ubiquitously expressed TPCs include VEGF-induced neoangiogenesis, LDL-cholesterol trafficking and degradation, physical endurance under fasting conditions, autophagy regulation, the acrosome reaction in sperm, cancer cell migration, and intracellular trafficking of pathogens such as Ebola virus or bacterial toxins (e.g., cholera toxin). In a genome-wide association study for variants associated with human pigmentation characteristics two coding variants of TPC2, rs35264875 (encoding M484L) and rs3829241 (encoding G734E), have been found to be associated with a shift from brown to blond hair color. In two recent follow-up studies a role for TPC2 in pigmentation has been further confirmed. However, these human polymorphic variants have not been functionally characterized until now. The development of endolysosomal patch-clamp techniques has made it possible to investigate directly ion channel activities and characteristics in isolated endolysosomal organelles. We applied this technique here to scrutinize channel characteristics of the polymorphic TPC2 variants in direct comparison with WT. We found that both polymorphisms lead to a gain of channel function by independent mechanisms. We next conducted a clinical study with more than 100 blond- and brown/black-haired individuals. We performed a genotype/phenotype analysis and subsequently isolated fibroblasts from WT and polymorphic variant carriers for endolysosomal patch-clamp experimentation to confirm key in vitro findings.

Astrocyte-derived adenosine is central to the hypnogenic effect of glucose

PubMed Central

Scharbarg, Emeric; Daenens, Marion; Lemaître, Frédéric; Geoffroy, Hélène; Guille-Collignon, Manon; Gallopin, Thierry; Rancillac, Armelle

2016-01-01

Sleep has been hypothesised to maintain a close relationship with metabolism. Here we focus on the brain structure that triggers slow-wave sleep, the ventrolateral preoptic nucleus (VLPO), to explore the cellular and molecular signalling pathways recruited by an increase in glucose concentration. We used infrared videomicroscopy on ex vivo brain slices to establish that glucose induces vasodilations specifically in the VLPO via the astrocytic release of adenosine. Real-time detection by in situ purine biosensors further revealed that the adenosine level doubles in response to glucose, and triples during the wakefulness period. Finally, patch-clamp recordings uncovered the depolarizing effect of adenosine and its A2A receptor agonist, CGS-21680, on sleep-promoting VLPO neurons. Altogether, our results provide new insights into the metabolically driven release of adenosine. We hypothesise that adenosine adjusts the local energy supply to local neuronal activity in response to glucose. This pathway could contribute to sleep-wake transition and sleep intensity. PMID:26755200

Effects of nitric oxide on neuromuscular properties of developing zebrafish embryos.

PubMed

Jay, Michael; Bradley, Sophie; McDearmid, Jonathan Robert

2014-01-01

Nitric oxide is a bioactive signalling molecule that is known to affect a wide range of neurodevelopmental processes. However, its functional relevance to neuromuscular development is not fully understood. Here we have examined developmental roles of nitric oxide during formation and maturation of neuromuscular contacts in zebrafish. Using histochemical approaches we show that elevating nitric oxide levels reduces the number of neuromuscular synapses within the axial swimming muscles whilst inhibition of nitric oxide biosynthesis has the opposite effect. We further show that nitric oxide signalling does not change synapse density, suggesting that the observed effects are a consequence of previously reported changes in motor axon branch formation. Moreover, we have used in vivo patch clamp electrophysiology to examine the effects of nitric oxide on physiological maturation of zebrafish neuromuscular junctions. We show that developmental exposure to nitric oxide affects the kinetics of spontaneous miniature end plate currents and impacts the neuromuscular drive for locomotion. Taken together, our findings implicate nitrergic signalling in the regulation of zebrafish neuromuscular development and locomotor maturation.

Nucleus accumbens controls wakefulness by a subpopulation of neurons expressing dopamine D1 receptors.

PubMed

Luo, Yan-Jia; Li, Ya-Dong; Wang, Lu; Yang, Su-Rong; Yuan, Xiang-Shan; Wang, Juan; Cherasse, Yoan; Lazarus, Michael; Chen, Jiang-Fan; Qu, Wei-Min; Huang, Zhi-Li

2018-04-20

Nucleus accumbens (NAc) is involved in behaviors that depend on heightened wakefulness, but its impact on arousal remains unclear. Here, we demonstrate that NAc dopamine D 1 receptor (D 1 R)-expressing neurons are essential for behavioral arousal. Using in vivo fiber photometry in mice, we find arousal-dependent increases in population activity of NAc D 1 R neurons. Optogenetic activation of NAc D 1 R neurons induces immediate transitions from non-rapid eye movement sleep to wakefulness, and chemogenetic stimulation prolongs arousal, with decreased food intake. Patch-clamp, tracing, immunohistochemistry, and electron microscopy reveal that NAc D 1 R neurons project to the midbrain and lateral hypothalamus, and might disinhibit midbrain dopamine neurons and lateral hypothalamus orexin neurons. Photoactivation of terminals in the midbrain and lateral hypothalamus is sufficient to induce wakefulness. Silencing of NAc D 1 R neurons suppresses arousal, with increased nest-building behaviors. Collectively, our data indicate that NAc D 1 R neuron circuits are essential for the induction and maintenance of wakefulness.

Scalable electrophysiology in intact small animals with nanoscale suspended electrode arrays

NASA Astrophysics Data System (ADS)

Gonzales, Daniel L.; Badhiwala, Krishna N.; Vercosa, Daniel G.; Avants, Benjamin W.; Liu, Zheng; Zhong, Weiwei; Robinson, Jacob T.

2017-07-01

Electrical measurements from large populations of animals would help reveal fundamental properties of the nervous system and neurological diseases. Small invertebrates are ideal for these large-scale studies; however, patch-clamp electrophysiology in microscopic animals typically requires invasive dissections and is low-throughput. To overcome these limitations, we present nano-SPEARs: suspended electrodes integrated into a scalable microfluidic device. Using this technology, we have made the first extracellular recordings of body-wall muscle electrophysiology inside an intact roundworm, Caenorhabditis elegans. We can also use nano-SPEARs to record from multiple animals in parallel and even from other species, such as Hydra littoralis. Furthermore, we use nano-SPEARs to establish the first electrophysiological phenotypes for C. elegans models for amyotrophic lateral sclerosis and Parkinson's disease, and show a partial rescue of the Parkinson's phenotype through drug treatment. These results demonstrate that nano-SPEARs provide the core technology for microchips that enable scalable, in vivo studies of neurobiology and neurological diseases.

Reconstruction and Simulation of Neocortical Microcircuitry.

PubMed

Markram, Henry; Muller, Eilif; Ramaswamy, Srikanth; Reimann, Michael W; Abdellah, Marwan; Sanchez, Carlos Aguado; Ailamaki, Anastasia; Alonso-Nanclares, Lidia; Antille, Nicolas; Arsever, Selim; Kahou, Guy Antoine Atenekeng; Berger, Thomas K; Bilgili, Ahmet; Buncic, Nenad; Chalimourda, Athanassia; Chindemi, Giuseppe; Courcol, Jean-Denis; Delalondre, Fabien; Delattre, Vincent; Druckmann, Shaul; Dumusc, Raphael; Dynes, James; Eilemann, Stefan; Gal, Eyal; Gevaert, Michael Emiel; Ghobril, Jean-Pierre; Gidon, Albert; Graham, Joe W; Gupta, Anirudh; Haenel, Valentin; Hay, Etay; Heinis, Thomas; Hernando, Juan B; Hines, Michael; Kanari, Lida; Keller, Daniel; Kenyon, John; Khazen, Georges; Kim, Yihwa; King, James G; Kisvarday, Zoltan; Kumbhar, Pramod; Lasserre, Sébastien; Le Bé, Jean-Vincent; Magalhães, Bruno R C; Merchán-Pérez, Angel; Meystre, Julie; Morrice, Benjamin Roy; Muller, Jeffrey; Muñoz-Céspedes, Alberto; Muralidhar, Shruti; Muthurasa, Keerthan; Nachbaur, Daniel; Newton, Taylor H; Nolte, Max; Ovcharenko, Aleksandr; Palacios, Juan; Pastor, Luis; Perin, Rodrigo; Ranjan, Rajnish; Riachi, Imad; Rodríguez, José-Rodrigo; Riquelme, Juan Luis; Rössert, Christian; Sfyrakis, Konstantinos; Shi, Ying; Shillcock, Julian C; Silberberg, Gilad; Silva, Ricardo; Tauheed, Farhan; Telefont, Martin; Toledo-Rodriguez, Maria; Tränkler, Thomas; Van Geit, Werner; Díaz, Jafet Villafranca; Walker, Richard; Wang, Yun; Zaninetta, Stefano M; DeFelipe, Javier; Hill, Sean L; Segev, Idan; Schürmann, Felix

2015-10-08

We present a first-draft digital reconstruction of the microcircuitry of somatosensory cortex of juvenile rat. The reconstruction uses cellular and synaptic organizing principles to algorithmically reconstruct detailed anatomy and physiology from sparse experimental data. An objective anatomical method defines a neocortical volume of 0.29 ± 0.01 mm(3) containing ~31,000 neurons, and patch-clamp studies identify 55 layer-specific morphological and 207 morpho-electrical neuron subtypes. When digitally reconstructed neurons are positioned in the volume and synapse formation is restricted to biological bouton densities and numbers of synapses per connection, their overlapping arbors form ~8 million connections with ~37 million synapses. Simulations reproduce an array of in vitro and in vivo experiments without parameter tuning. Additionally, we find a spectrum of network states with a sharp transition from synchronous to asynchronous activity, modulated by physiological mechanisms. The spectrum of network states, dynamically reconfigured around this transition, supports diverse information processing strategies. VIDEO ABSTRACT. Copyright © 2015 Elsevier Inc. All rights reserved.

Mucoadhesive Fenretinide Patches for Site-specific Chemoprevention of Oral Cancer: Enhancement of Oral Mucosal Permeation of Fenretinide by Co-incorporation of Propylene Glycol and Menthol

PubMed Central

Wu, Xiao; Desai, Kashappa-Goud H.; Mallery, Susan R.; Holpuch, Andrew S.; Phelps, Maynard P.; Schwendeman, Steven P.

2012-01-01

The objective of this study was to enhance oral mucosal permeation of fenretinide by co-incorporation of propylene glycol (PG) and menthol in fenretinide/Eudragit® RL PO mucoadhesive patches. Fenretinide is an extremely hydrophobic chemopreventive compound with poor tissue permeability. Co-incorporation of 5-10 wt% PG (mean Js = 16-23 μg cm−2 h−1; 158-171 μg fenretinide/g tissue) or 1-10 wt% PG + 5 wt% menthol (mean Js = 18-40 μg cm−2 h−1; 172-241 μg fenretinide/g tissue) in fenretinide/Eudragit® RL PO patches led to significant ex vivo fenretinide permeation enhancement (p < 0.001). Addition of PG above 2.5 wt% in the patch resulted in significant cellular swelling in the buccal mucosal tissues. These alterations were ameliorated by combining both enhancers and reducing PG level. After buccal administration of patches in rabbits, in vivo permeation of fenretinide across the oral mucosa was greater (~43 μg fenretinide/g tissue) from patches that contained optimized permeation enhancer content (2.5 wt% PG + 5 wt% menthol) relative to permeation obtained from enhancer-free patch (~ 17 μg fenretinide/g tissue) (p < 0.001). In vitro and in vivo release of fenretinide from patch was not significantly increased by co-incorporation of permeation enhancers, indicating that mass transfer across the tissue, and not the patch, largely determined the permeation rate control in vivo. As a result of its improved permeation and its lack of deleterious local effects, the mucoadhesive fenretinide patch co-incorporated with 2.5 wt% PG + 5 wt% menthol represents an important step in the further preclinical evaluation of oral site-specific chemoprevention strategies with fenretinide. PMID:22280430

Determination of rheogenic ion transport in rat proximal colon in vivo.

PubMed

Haag, K; Lübcke, R; Knauf, H; Berger, E; Gerok, W

1985-01-01

A direct clamping technique is demonstrated, which allows monitoring of rapid changes of the short-circuit current (Isc) and the specific transepithelial resistance (Rm) as well as measurement of ion fluxes under short-circuit conditions in vivo. Due to the cylindrical symmetry of the colon the intraluminal electrode was devised as a centrally fixed silver rod, by which radial current injection was achieved. The geometrical arrangement of the electrodes guaranteed zero potential difference (PD) along the whole axis of the colon segment. The Isc was determined to 3.3 +/- 0.7 mueq h-1 cm-2 and Rm equal to 121 +/- 5 omega cm2. These data obtained by direct short-circuiting agree well with our earlier Rm and Isc data based on cable analysis, where the Isc was calculated from the open-circuit PD and Rm. This is considered as evidence for the reliability of the two independent in vivo techniques. Their validity was confirmed by the expected effects of drugs acting on rheogenic ion transport. Both the indirect (via Rm) as well as the direct Isc determination may be used alternatively as required; one may serve to match the other. For larger tubular structures like the rat colon the direct clamping should be preferred as the standard procedure for the Isc determination in vivo.

In vivo cardiac glucose metabolism in the high-fat fed mouse: Comparison of euglycemic–hyperinsulinemic clamp derived measures of glucose uptake with a dynamic metabolomic flux profiling approach

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kowalski, Greg M., E-mail: greg.kowalski@deakin.edu.au; De Souza, David P.; Risis, Steve

Rationale: Cardiac metabolism is thought to be altered in insulin resistance and type 2 diabetes (T2D). Our understanding of the regulation of cardiac substrate metabolism and insulin sensitivity has largely been derived from ex vivo preparations which are not subject to the same metabolic regulation as in the intact heart in vivo. Studies are therefore required to examine in vivo cardiac glucose metabolism under physiologically relevant conditions. Objective: To determine the temporal pattern of the development of cardiac insulin resistance and to compare with dynamic approaches to interrogate cardiac glucose and intermediary metabolism in vivo. Methods and results: Studies were conducted to determine themore » evolution of cardiac insulin resistance in C57Bl/6 mice fed a high-fat diet (HFD) for between 1 and 16 weeks. Dynamic in vivo cardiac glucose metabolism was determined following oral administration of [U-{sup 13}C] glucose. Hearts were collected after 15 and 60 min and flux profiling was determined by measuring {sup 13}C mass isotopomers in glycolytic and tricarboxylic acid (TCA) cycle intermediates. Cardiac insulin resistance, determined by euglycemic–hyperinsulinemic clamp, was evident after 3 weeks of HFD. Despite the presence of insulin resistance, in vivo cardiac glucose metabolism following oral glucose administration was not compromised in HFD mice. This contrasts our recent findings in skeletal muscle, where TCA cycle activity was reduced in mice fed a HFD. Similar to our report in muscle, glucose derived pyruvate entry into the TCA cycle in the heart was almost exclusively via pyruvate dehydrogenase, with pyruvate carboxylase mediated anaplerosis being negligible after oral glucose administration. Conclusions: Under experimental conditions which closely mimic the postprandial state, the insulin resistant mouse heart retains the ability to stimulate glucose metabolism. - Highlights: • Insulin clamp was used to determine the evolution of cardiac insulin resistance. • Clamp measures were compared to a dynamic metabolomics approach. • The clamp revealed the presence of cardiac insulin resistance after 3 weeks of HFD. • Cardiac glucose metabolism was not affected by HFD during an oral glucose challenge.« less

Drug Delivery and Transport into the Central Circulation: An Example of Zero-Order In vivo Absorption of Rotigotine from a Transdermal Patch Formulation.

PubMed

Cawello, Willi; Braun, Marina; Andreas, Jens-Otto

2018-01-13

Pharmacokinetic studies using deconvolution methods and non-compartmental analysis to model clinical absorption of drugs are not well represented in the literature. The purpose of this research was (1) to define the system of equations for description of rotigotine (a dopamine receptor agonist delivered via a transdermal patch) absorption based on a pharmacokinetic model and (2) to describe the kinetics of rotigotine disposition after single and multiple dosing. The kinetics of drug disposition was evaluated based on rotigotine plasma concentration data from three phase 1 trials. In two trials, rotigotine was administered via a single patch over 24 h in healthy subjects. In a third trial, rotigotine was administered once daily over 1 month in subjects with early-stage Parkinson's disease (PD). A pharmacokinetic model utilizing deconvolution methods was developed to describe the relationship between drug release from the patch and plasma concentrations. Plasma-concentration over time profiles were modeled based on a one-compartment model with a time lag, a zero-order input (describing a constant absorption via skin into central circulation) and first-order elimination. Corresponding mathematical models for single- and multiple-dose administration were developed. After single-dose administration of rotigotine patches (using 2, 4 or 8 mg/day) in healthy subjects, a constant in vivo absorption was present after a minor time lag (2-3 h). On days 27 and 30 of the multiple-dose study in patients with PD, absorption was constant during patch-on periods and resembled zero-order kinetics. Deconvolution based on rotigotine pharmacokinetic profiles after single- or multiple-dose administration of the once-daily patch demonstrated that in vivo absorption of rotigotine showed constant input through the skin into the central circulation (resembling zero-order kinetics). Continuous absorption through the skin is a basis for stable drug exposure.

Functional Evaluation of Biological Neurotoxins in Networked Cultures of Stem Cell-derived Central Nervous System Neurons

DTIC Science & Technology

2015-02-05

botulism or tetanus , whole-cell patch clamp electrophysiology was used to quantify spontaneous miniature excitory post-synaptic currents (mEPSCs) in...ESNs exposed to tetanus neurotoxin (TeNT) or botulinum neurotoxin (BoNT) serotypes / A-/G. In all cases, ESNs exhibited near-complete loss of synaptic

Evaluation of diel patterns of relative changes in cell turgor of tomato plants using leaf patch clamp pressure probes.

PubMed

Lee, Kang M; Driever, Steven M; Heuvelink, Ep; Rüger, Simon; Zimmermann, Ulrich; de Gelder, Arie; Marcelis, Leo F M

2012-12-01

Relative changes in cell turgor of leaves of well-watered tomato plants were evaluated using the leaf patch clamp pressure probe (LPCP) under dynamic greenhouse climate conditions. LPCP changes, a measure for relative changes in cell turgor, were monitored at three different heights of transpiring and non-transpiring leaves of tomato plants on sunny and cloudy days simultaneously with whole plant water uptake. Clear diel patterns were observed for relative changes of cell turgor of both transpiring and non-transpiring leaves, which were stronger on sunny days than on cloudy days. A clear effect of canopy height was also observed. Non-transpiring leaves showed relative changes in cell turgor that closely followed plant water uptake throughout the day. However, in the afternoon the relative changes of cell turgor of the transpiring leaves displayed a delayed response in comparison to plant water uptake. Subsequent recovery of cell turgor loss of transpiring leaves during the following night appeared insufficient, as the pre-dawn turgescent state similar to the previous night was not attained. Copyright © Physiologia Plantarum 2012.

Patch-clamp recordings of rat neurons from acute brain slices of the somatosensory cortex during magnetic stimulation

PubMed Central

Pashut, Tamar; Magidov, Dafna; Ben-Porat, Hana; Wolfus, Shuki; Friedman, Alex; Perel, Eli; Lavidor, Michal; Bar-Gad, Izhar; Yeshurun, Yosef; Korngreen, Alon

2014-01-01

Although transcranial magnetic stimulation (TMS) is a popular tool for both basic research and clinical applications, its actions on nerve cells are only partially understood. We have previously predicted, using compartmental modeling, that magnetic stimulation of central nervous system neurons depolarized the soma followed by initiation of an action potential in the initial segment of the axon. The simulations also predict that neurons with low current threshold are more susceptible to magnetic stimulation. Here we tested these theoretical predictions by combining in vitro patch-clamp recordings from rat brain slices with magnetic stimulation and compartmental modeling. In agreement with the modeling, our recordings demonstrate the dependence of magnetic stimulation-triggered action potentials on the type and state of the neuron and its orientation within the magnetic field. Our results suggest that the observed effects of TMS are deeply rooted in the biophysical properties of single neurons in the central nervous system and provide a framework both for interpreting existing TMS data and developing new simulation-based tools and therapies. PMID:24917788

Multimodal vibration damping of a plate by piezoelectric coupling to its analogous electrical network

NASA Astrophysics Data System (ADS)

Lossouarn, B.; Deü, J.-F.; Aucejo, M.; Cunefare, K. A.

2016-11-01

Multimodal damping can be achieved by coupling a mechanical structure to an electrical network exhibiting similar modal properties. Focusing on a plate, a new topology for such an electrical analogue is found from a finite difference approximation of the Kirchhoff-Love theory and the use of the direct electromechanical analogy. Discrete models based on element dynamic stiffness matrices are proposed to simulate square plate unit cells coupled to their electrical analogues through two-dimensional piezoelectric transducers. A setup made of a clamped plate covered with an array of piezoelectric patches is built in order to validate the control strategy and the numerical models. The analogous electrical network is implemented with passive components as inductors, transformers and the inherent capacitance of the piezoelectric patches. The effect of the piezoelectric coupling on the dynamics of the clamped plate is significant as it creates the equivalent of a multimodal tuned mass damping. An adequate tuning of the network then yields a broadband vibration reduction. In the end, the use of an analogous electrical network appears as an efficient solution for the multimodal control of a plate.

Prototype for Automatable, Dielectrophoretically-Accessed Intracellular Membrane–Potential Measurements by Metal Electrodes

PubMed Central

Sukhorukov, Vladimir L.; Zimmermann, Dirk

2013-01-01

Abstract Functional access to membrane proteins, for example, ion channels, of individual cells is an important prerequisite in drug discovery studies. The highly sophisticated patch-clamp method is widely used for electrogenic membrane proteins, but is demanding for the operator, and its automation remains challenging. The dielectrophoretically-accessed, intracellular membrane–potential measurement (DAIMM) method is a new technique showing high potential for automation of electrophysiological data recording in the whole-cell configuration. A cell suspension is brought between a mm-scaled planar electrode and a μm-scaled tip electrode, placed opposite to each other. Due to the asymmetric electrode configuration, the application of alternating electric fields (1–5 MHz) provokes a dielectrophoretic force acting on the target cell. As a consequence, the cell is accelerated and pierced by the tip electrode, hence functioning as the internal (working) electrode. We used the light-gated cation channel Channelrhodopsin-2 as a reporter protein expressed in HEK293 cells to characterize the DAIMM method in comparison with the patch-clamp technique. PMID:22994967

Synergistic Inhibition of Delayed Rectifier K+ and Voltage-Gated Na+ Currents by Artemisinin in Pituitary Tumor (GH3) Cells.

PubMed

So, Edmund Cheung; Wu, Sheng-Nan; Wu, Ping-Ching; Chen, Hui-Zhen; Yang, Chia-Jung

2017-01-01

Artemisinin (ART) is an anti-malarial agent reported to influence endocrine function. Effects of ART on ionic currents and action potentials (APs) in pituitary tumor (GH3) cells were evaluated by patch clamp techniques. ART inhibited the amplitude of delayed-rectifier K+ current (IK(DR)) in response to membrane depolarization and accelerated the process of current inactivation. It exerted an inhibitory effect on IK(DR) with an IC50 value of 11.2 µM and enhanced IK(DR) inactivation with a KD value of 14.7 µM. The steady-state inactivation curve of IK(DR) was shifted to hyperpolarization by 10 mV. Pretreatment of chlorotoxin (1 µM) or iloprost (100 nM) did not alter the magnitude of ART-induced inhibition of IK(DR) in GH3 cells. ART also decreased the peak amplitude of voltage-gated Na+ current (INa) with a concentration-dependent slowing in inactivation rate. Application of KMUP-1, an inhibitor of late INa, was effective at reversing ART-induced prolongation in inactivation time constant of INa. Under current-clamp recordings, ART alone reduced the amplitude of APs and prolonged the duration of APs. Under ART exposure, the inhibitory actions on both IK(DR) and INa could be a potential mechanisms through which this drug influences membrane excitability of endocrine or neuroendocrine cells appearing in vivo. © 2017 The Author(s). Published by S. Karger AG, Basel.

A Nanoscale Interface Promoting Molecular and Functional Differentiation of Neural Cells

NASA Astrophysics Data System (ADS)

Posati, Tamara; Pistone, Assunta; Saracino, Emanuela; Formaggio, Francesco; Mola, Maria Grazia; Troni, Elisabetta; Sagnella, Anna; Nocchetti, Morena; Barbalinardo, Marianna; Valle, Francesco; Bonetti, Simone; Caprini, Marco; Nicchia, Grazia Paola; Zamboni, Roberto; Muccini, Michele; Benfenati, Valentina

2016-08-01

Potassium channels and aquaporins expressed by astrocytes are key players in the maintenance of cerebral homeostasis and in brain pathophysiologies. One major challenge in the study of astrocyte membrane channels in vitro, is that their expression pattern does not resemble the one observed in vivo. Nanostructured interfaces represent a significant resource to control the cellular behaviour and functionalities at micro and nanoscale as well as to generate novel and more reliable models to study astrocytes in vitro. However, the potential of nanotechnologies in the manipulation of astrocytes ion channels and aquaporins has never been previously reported. Hydrotalcite-like compounds (HTlc) are layered materials with increasing potential as biocompatible nanoscale interface. Here, we evaluate the effect of the interaction of HTlc nanoparticles films with primary rat neocortical astrocytes. We show that HTlc films are biocompatible and do not promote gliotic reaction, while favouring astrocytes differentiation by induction of F-actin fibre alignment and vinculin polarization. Western Blot, Immunofluorescence and patch-clamp revealed that differentiation was accompanied by molecular and functional up-regulation of both inward rectifying potassium channel Kir 4.1 and aquaporin 4, AQP4. The reported results pave the way to engineering novel in vitro models to study astrocytes in a in vivo like condition.

Diadenosine tetraphosphate-induced inhibition of ATP-sensitive K+ channels in patches excised from ventricular myocytes.

PubMed Central

Jovanovic, A.; Terzic, A.

1996-01-01

Diadenosine 5',5''-P1,P4-tetraphosphate (Ap4A) has been termed 'alarmone' due to its role in intracellular signaling during metabolic stress. It is not known whether Ap4A could modulate ATP-sensitive K+ (KATP) channels, a family of channels regulated by the metabolic status of a cell. We applied the single-channel patch-clamp technique to measure the effect of Ap4A on KATP channels. When applied to the intracellular side of patches, excised from guinea-pig ventricular myocytes, Ap4A inhibited KATP channel activity, in a reversible and concentration-dependent (half-maximal concentration approximately 17 microM) manner. We conclude that Ap4A, a naturally occurring diadenosine polyphosphate, is actually an inhibitor of the myocardial KATP channel. PMID:8789372

Leaf patch clamp pressure probe measurements on olive leaves in a nearly turgorless state.

PubMed

Ehrenberger, W; Rüger, S; Rodríguez-Domínguez, C M; Díaz-Espejo, A; Fernández, J E; Moreno, J; Zimmermann, D; Sukhorukov, V L; Zimmermann, U

2012-07-01

The non-invasive leaf patch clamp pressure (LPCP) probe measures the attenuated pressure of a leaf patch, P(p) , in response to an externally applied magnetic force. P(p) is inversely coupled with leaf turgor pressure, P(c) , i.e. at high P(c) values the P(p) values are small and at low P(c) values the P(p) values are high. This relationship between P(c) and P(p) could also be verified for 2-m tall olive trees under laboratory conditions using the cell turgor pressure probe. When the laboratory plants were subjected to severe water stress (P(c) dropped below ca. 50 kPa), P(p) curves show reverse diurnal changes, i.e. during the light regime (high transpiration) a minimum P(p) value, and during darkness a peak P(p) value is recorded. This reversal of the P(p) curves was completely reversible. Upon watering, the original diurnal P(p) changes were re-established within 2-3 days. Olive trees in the field showed a similar turnover of the shape of the P(p) curves upon drought, despite pronounced fluctuations in microclimate. The reversal of the P(p) curves is most likely due to accumulation of air in the leaves. This assumption was supported with cross-sections through leaves subjected to prolonged drought. In contrast to well-watered leaves, microscopic inspection of leaves exhibiting inverse diurnal P(p) curves revealed large air-filled areas in parenchyma tissue. Significantly larger amounts of air could also be extracted from water-stressed leaves than from well-watered leaves using the cell turgor pressure probe. Furthermore, theoretical analysis of the experimental P(p) curves shows that the propagation of pressure through the nearly turgorless leaf must be exclusively dictated by air. Equations are derived that provide valuable information about the water status of olive leaves close to zero P(c) . © 2012 German Botanical Society and The Royal Botanical Society of the Netherlands.

Nonlinear finite element modeling of vibration control of plane rod-type structural members with integrated piezoelectric patches

NASA Astrophysics Data System (ADS)

Chróścielewski, Jacek; Schmidt, Rüdiger; Eremeyev, Victor A.

2018-05-01

This paper addresses modeling and finite element analysis of the transient large-amplitude vibration response of thin rod-type structures (e.g., plane curved beams, arches, ring shells) and its control by integrated piezoelectric layers. A geometrically nonlinear finite beam element for the analysis of piezolaminated structures is developed that is based on the Bernoulli hypothesis and the assumptions of small strains and finite rotations of the normal. The finite element model can be applied to static, stability, and transient analysis of smart structures consisting of a master structure and integrated piezoelectric actuator layers or patches attached to the upper and lower surfaces. Two problems are studied extensively: (i) FE analyses of a clamped semicircular ring shell that has been used as a benchmark problem for linear vibration control in several recent papers are critically reviewed and extended to account for the effects of structural nonlinearity and (ii) a smart circular arch subjected to a hydrostatic pressure load is investigated statically and dynamically in order to study the shift of bifurcation and limit points, eigenfrequencies, and eigenvectors, as well as vibration control for loading conditions which may lead to dynamic loss of stability.

Automatic tracking of cells for video microscopy in patch clamp experiments

PubMed Central

2014-01-01

Background Visualisation of neurons labeled with fluorescent proteins or compounds generally require exposure to intense light for a relatively long period of time, often leading to bleaching of the fluorescent probe and photodamage of the tissue. Here we created a technique to drastically shorten light exposure and improve the targeting of fluorescent labeled cells that is specially useful for patch-clamp recordings. We applied image tracking and mask overlay to reduce the time of fluorescence exposure and minimise mistakes when identifying neurons. Methods Neurons are first identified according to visual criteria (e.g. fluorescence protein expression, shape, viability etc.) and a transmission microscopy image Differential Interference Contrast (DIC) or Dodt contrast containing the cell used as a reference for the tracking algorithm. A fluorescence image can also be acquired later to be used as a mask (that can be overlaid on the target during live transmission video). As patch-clamp experiments require translating the microscope stage, we used pattern matching to track reference neurons in order to move the fluorescence mask to match the new position of the objective in relation to the sample. For the image processing we used the Open Source Computer Vision (OpenCV) library, including the Speeded-Up Robust Features (SURF) for tracking cells. The dataset of images (n = 720) was analyzed under normal conditions of acquisition and with influence of noise (defocusing and brightness). Results We validated the method in dissociated neuronal cultures and fresh brain slices expressing Enhanced Yellow Fluorescent Protein (eYFP) or Tandem Dimer Tomato (tdTomato) proteins, which considerably decreased the exposure to fluorescence excitation, thereby minimising photodamage. We also show that the neuron tracking can be used in differential interference contrast or Dodt contrast microscopy. Conclusion The techniques of digital image processing used in this work are an important addition to the set of microscopy tools used in modern electrophysiology, specially in experiments with neuron cultures and brain slices. PMID:24946774

Automatic tracking of cells for video microscopy in patch clamp experiments.

PubMed

Peixoto, Helton M; Munguba, Hermany; Cruz, Rossana M S; Guerreiro, Ana M G; Leao, Richardson N

2014-06-20

Visualisation of neurons labeled with fluorescent proteins or compounds generally require exposure to intense light for a relatively long period of time, often leading to bleaching of the fluorescent probe and photodamage of the tissue. Here we created a technique to drastically shorten light exposure and improve the targeting of fluorescent labeled cells that is specially useful for patch-clamp recordings. We applied image tracking and mask overlay to reduce the time of fluorescence exposure and minimise mistakes when identifying neurons. Neurons are first identified according to visual criteria (e.g. fluorescence protein expression, shape, viability etc.) and a transmission microscopy image Differential Interference Contrast (DIC) or Dodt contrast containing the cell used as a reference for the tracking algorithm. A fluorescence image can also be acquired later to be used as a mask (that can be overlaid on the target during live transmission video). As patch-clamp experiments require translating the microscope stage, we used pattern matching to track reference neurons in order to move the fluorescence mask to match the new position of the objective in relation to the sample. For the image processing we used the Open Source Computer Vision (OpenCV) library, including the Speeded-Up Robust Features (SURF) for tracking cells. The dataset of images (n = 720) was analyzed under normal conditions of acquisition and with influence of noise (defocusing and brightness). We validated the method in dissociated neuronal cultures and fresh brain slices expressing Enhanced Yellow Fluorescent Protein (eYFP) or Tandem Dimer Tomato (tdTomato) proteins, which considerably decreased the exposure to fluorescence excitation, thereby minimising photodamage. We also show that the neuron tracking can be used in differential interference contrast or Dodt contrast microscopy. The techniques of digital image processing used in this work are an important addition to the set of microscopy tools used in modern electrophysiology, specially in experiments with neuron cultures and brain slices.

Piezoresistive cantilever force-clamp system

DOE Office of Scientific and Technical Information (OSTI.GOV)

Park, Sung-Jin; Petzold, Bryan C.; Pruitt, Beth L.

2011-04-15

We present a microelectromechanical device-based tool, namely, a force-clamp system that sets or ''clamps'' the scaled force and can apply designed loading profiles (e.g., constant, sinusoidal) of a desired magnitude. The system implements a piezoresistive cantilever as a force sensor and the built-in capacitive sensor of a piezoelectric actuator as a displacement sensor, such that sample indentation depth can be directly calculated from the force and displacement signals. A programmable real-time controller operating at 100 kHz feedback calculates the driving voltage of the actuator. The system has two distinct modes: a force-clamp mode that controls the force applied to amore » sample and a displacement-clamp mode that controls the moving distance of the actuator. We demonstrate that the system has a large dynamic range (sub-nN up to tens of {mu}N force and nm up to tens of {mu}m displacement) in both air and water, and excellent dynamic response (fast response time, <2 ms and large bandwidth, 1 Hz up to 1 kHz). In addition, the system has been specifically designed to be integrated with other instruments such as a microscope with patch-clamp electronics. We demonstrate the capabilities of the system by using it to calibrate the stiffness and sensitivity of an electrostatic actuator and to measure the mechanics of a living, freely moving Caenorhabditis elegans nematode.« less

An Ultrasonic Clamp for Bloodless Partial Nephrectomy

NASA Astrophysics Data System (ADS)

Lafon, Cyril; Bouchoux, Guillaume; Murat, François Joseph; Birer, Alain; Theillère, Yves; Chapelon, Jean Yves; Cathignol, Dominique

2007-05-01

Maximum conservation of the kidney is preferable through partial nephrectomy for patients at risk of disease recurrence of renal cancers. Haemostatic tools are needed in order to achieve bloodless surgery and reduce post surgery morbidity. Two piezo-ceramic transducers operating at a frequency of 4 MHz were mounted on each arm of a clamp. When used for coagulation purposes, two transducers situated on opposite arms of the clamp were driven simultaneously. Heat delivery was optimized as each transducers mirrored back to targeted tissues the wave generated by the opposite transducer. Real-time treatment monitoring with an echo-based technique was also envisaged with this clamp. Therapy was periodically interrupted so one transducer could generate a pulse. The echo returning from the opposite transducer was treated. Coagulation necroses were obtained in vitro on substantial thicknesses (23-38mm) of pig liver over exposure durations ranging from 30s to 130s, and with acoustic intensities of less than 15W/cm2 per transducer. Both kidneys of two pigs were treated in vivo with the clamp (14.5W/cm2 for 90s), and the partial nephrectomies performed proved to be bloodless. In vitro and in vivo, wide transfixing lesions corresponded to an echo energy decrease superior to -10dB and parabolic form of the time of flight versus treatment time. In conclusion, this ultrasound clamp has proven to be an excellent mean for achieving monitored haemostasis in kidney.

KV7 Channel Pharmacological Activation by the Novel Activator ML213: Role for Heteromeric KV7.4/KV7.5 Channels in Guinea Pig Detrusor Smooth Muscle Function.

PubMed

Provence, Aaron; Angoli, Damiano; Petkov, Georgi V

2018-01-01

Voltage-gated K V 7 channels (K V 7.1 to K V 7.5) are important regulators of the cell membrane potential in detrusor smooth muscle (DSM) of the urinary bladder. This study sought to further the current knowledge of K V 7 channel function at the molecular, cellular, and tissue levels in combination with pharmacological tools. We used isometric DSM tension recordings, ratiometric fluorescence Ca 2+ imaging, amphotericin-B perforated patch-clamp electrophysiology, and in situ proximity ligation assay (PLA) in combination with the novel compound N -(2,4,6-trimethylphenyl)-bicyclo[2.2.1]heptane-2-carboxamide (ML213), an activator of K V 7.2, K V 7.4, and K V 7.5 channels, to examine their physiologic roles in guinea pig DSM function. ML213 caused a concentration-dependent (0.1-30 µ M) inhibition of spontaneous phasic contractions in DSM isolated strips; effects blocked by the K V 7 channel inhibitor XE991 (10 µ M). ML213 (0.1-30 µ M) also reduced pharmacologically induced and nerve-evoked contractions in DSM strips. Consistently, ML213 (10 µ M) decreased global intracellular Ca 2+ concentrations in Fura-2-loaded DSM isolated strips. Perforated patch-clamp electrophysiology revealed that ML213 (10 µ M) caused an increase in the amplitude of whole-cell K V 7 currents. Further, in current-clamp mode of the perforated patch clamp, ML213 hyperpolarized DSM cell membrane potential in a manner reversible by washout or XE991 (10 µ M), consistent with ML213 activation of K V 7 channel currents. Preapplication of XE991 (10 µ M) not only depolarized the DSM cells, but also blocked ML213-induced hyperpolarization, confirming ML213 selectivity for K V 7 channel subtypes. In situ PLA revealed colocalization and expression of heteromeric K V 7.4/K V 7.5 channels in DSM isolated cells. These combined results suggest that ML213-sensitive K V 7.4- and K V 7.5-containing channels are essential regulators of DSM excitability and contractility. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.

Protein kinase-dependent oxidative regulation of the cardiac Na+–K+ pump: evidence from in vivo and in vitro modulation of cell signalling

PubMed Central

Galougahi, Keyvan Karimi; Liu, Chia-Chi; Garcia, Alvaro; Fry, Natasha A S; Hamilton, Elisha J; Rasmussen, Helge H; Figtree, Gemma A

2013-01-01

The widely reported stimulation of the cardiac Na+–K+ pump by protein kinase A (PKA) should oppose other effects of PKA to increase contractility of the normal heart. It should also reduce harmful raised myocyte Na+ levels in heart failure, yet blockade of the β1 adrenergic receptor (AR), coupled to PKA signalling, is beneficial. We treated rabbits with the β1 AR antagonist metoprolol to modulate PKA activity and studied cardiac myocytes ex vivo. Metoprolol increased electrogenic pump current (Ip) in voltage clamped myocytes and reduced glutathionylation of the β1 pump subunit, an oxidative modification causally related to pump inhibition. Activation of adenylyl cyclase with forskolin to enhance cAMP synthesis or inclusion of the catalytic subunit of PKA in patch pipette solutions abolished the increase in Ip in voltage clamped myocytes induced by treatment with metoprolol, supporting cAMP/PKA-mediated pump inhibition. Metoprolol reduced myocardial PKA and protein kinase C (PKC) activities, reduced coimmunoprecipitation of cytosolic p47phox and membranous p22phox NADPH oxidase subunits and reduced myocardial O2•−-sensitive dihydroethidium fluorescence. Treatment also enhanced coimmunoprecipitation of the β1 pump subunit with glutaredoxin 1 that catalyses de-glutathionylation. Since angiotensin II induces PKC-dependent activation of NADPH oxidase, we examined the effects of angiotensin-converting enzyme inhibition with captopril. This treatment had no effect on PKA activity but reduced the activity of PKC, reduced β1 subunit glutathionylation and increased Ip. The PKA-induced Na+–K+ pump inhibition we report should act with other mechanisms that enhance contractility of the normal heart but accentuate the harmful effects of raised cytosolic Na+ in the failing heart. This scheme is consistent with the efficacy of β1 AR blockade in the treatment of heart failure. PMID:23587884

Sound Rhythms Are Encoded by Postinhibitory Rebound Spiking in the Superior Paraolivary Nucleus

PubMed Central

Felix, Richard A.; Fridberger, Anders; Leijon, Sara; Berrebi, Albert S.; Magnusson, Anna K.

2013-01-01

The superior paraolivary nucleus (SPON) is a prominent structure in the auditory brainstem. In contrast to the principal superior olivary nuclei with identified roles in processing binaural sound localization cues, the role of the SPON in hearing is not well understood. A combined in vitro and in vivo approach was used to investigate the cellular properties of SPON neurons in the mouse. Patch-clamp recordings in brain slices revealed that brief and well timed postinhibitory rebound spiking, generated by the interaction of two subthreshold-activated ion currents, is a hallmark of SPON neurons. The Ih current determines the timing of the rebound, whereas the T-type Ca2+ current boosts the rebound to spike threshold. This precisely timed rebound spiking provides a physiological explanation for the sensitivity of SPON neurons to sinusoidally amplitude-modulated (SAM) tones in vivo, where peaks in the sound envelope drive inhibitory inputs and SPON neurons fire action potentials during the waveform troughs. Consistent with this notion, SPON neurons display intrinsic tuning to frequency-modulated sinusoidal currents (1–15Hz) in vitro and discharge with strong synchrony to SAMs with modulation frequencies between 1 and 20 Hz in vivo. The results of this study suggest that the SPON is particularly well suited to encode rhythmic sound patterns. Such temporal periodicity information is likely important for detection of communication cues, such as the acoustic envelopes of animal vocalizations and speech signals. PMID:21880918

Sound rhythms are encoded by postinhibitory rebound spiking in the superior paraolivary nucleus.

PubMed

Felix, Richard A; Fridberger, Anders; Leijon, Sara; Berrebi, Albert S; Magnusson, Anna K

2011-08-31

The superior paraolivary nucleus (SPON) is a prominent structure in the auditory brainstem. In contrast to the principal superior olivary nuclei with identified roles in processing binaural sound localization cues, the role of the SPON in hearing is not well understood. A combined in vitro and in vivo approach was used to investigate the cellular properties of SPON neurons in the mouse. Patch-clamp recordings in brain slices revealed that brief and well timed postinhibitory rebound spiking, generated by the interaction of two subthreshold-activated ion currents, is a hallmark of SPON neurons. The I(h) current determines the timing of the rebound, whereas the T-type Ca(2+) current boosts the rebound to spike threshold. This precisely timed rebound spiking provides a physiological explanation for the sensitivity of SPON neurons to sinusoidally amplitude-modulated (SAM) tones in vivo, where peaks in the sound envelope drive inhibitory inputs and SPON neurons fire action potentials during the waveform troughs. Consistent with this notion, SPON neurons display intrinsic tuning to frequency-modulated sinusoidal currents (1-15Hz) in vitro and discharge with strong synchrony to SAMs with modulation frequencies between 1 and 20 Hz in vivo. The results of this study suggest that the SPON is particularly well suited to encode rhythmic sound patterns. Such temporal periodicity information is likely important for detection of communication cues, such as the acoustic envelopes of animal vocalizations and speech signals.

Validation of simple indexes to assess insulin sensitivity during pregnancy in Wistar and Sprague-Dawley rats.

PubMed

Cacho, J; Sevillano, J; de Castro, J; Herrera, E; Ramos, M P

2008-11-01

Insulin resistance plays a role in the pathogenesis of diabetes, including gestational diabetes. The glucose clamp is considered the gold standard for determining in vivo insulin sensitivity, both in human and in animal models. However, the clamp is laborious, time consuming and, in animals, requires anesthesia and collection of multiple blood samples. In human studies, a number of simple indexes, derived from fasting glucose and insulin levels, have been obtained and validated against the glucose clamp. However, these indexes have not been validated in rats and their accuracy in predicting altered insulin sensitivity remains to be established. In the present study, we have evaluated whether indirect estimates based on fasting glucose and insulin levels are valid predictors of insulin sensitivity in nonpregnant and 20-day-pregnant Wistar and Sprague-Dawley rats. We have analyzed the homeostasis model assessment of insulin resistance (HOMA-IR), the quantitative insulin sensitivity check index (QUICKI), and the fasting glucose-to-insulin ratio (FGIR) by comparing them with the insulin sensitivity (SI(Clamp)) values obtained during the hyperinsulinemic-isoglycemic clamp. We have performed a calibration analysis to evaluate the ability of these indexes to accurately predict insulin sensitivity as determined by the reference glucose clamp. Finally, to assess the reliability of these indexes for the identification of animals with impaired insulin sensitivity, performance of the indexes was analyzed by receiver operating characteristic (ROC) curves in Wistar and Sprague-Dawley rats. We found that HOMA-IR, QUICKI, and FGIR correlated significantly with SI(Clamp), exhibited good sensitivity and specificity, accurately predicted SI(Clamp), and yielded lower insulin sensitivity in pregnant than in nonpregnant rats. Together, our data demonstrate that these indexes provide an easy and accurate measure of insulin sensitivity during pregnancy in the rat.

Phorbol ester impairs electrical excitation of rat pancreatic beta-cells through PKC-independent activation of KATP channels.

PubMed

Suga, S; Wu, J; Ogawa, Y; Takeo, T; Kanno, T; Wakui, M

2001-01-01

Phorbol 12-myristate 13-acetate (PMA) is often used as an activating phorbol ester of protein kinase C (PKC) to investigate the roles of the kinase in cellular functions. Accumulating lines of evidence indicate that in addition to activating PKC, PMA also produces some regulatory effects in a PKC-independent manner. In this study, we investigated the non-PKC effects of PMA on electrical excitability of rat pancreatic beta-cells by using patch-clamp techniques. In current-clamp recording, PMA (80 nM) reversibly inhibited 15 mM glucose-induced action potential spikes superimposed on a slow membrane depolarization and this inhibition can not be prevented by pre-treatment of the cell with a specific PKC inhibitor, bisindolylmaleimide (BIM, 1 microM). In the presence of a subthreshold concentration (5.5 mM) of glucose, PMA hyperpolarized beta-cells in a concentration-dependent manner (0.8-240 nM), even in the presence of BIM. Based on cell-attached single channel recordings, PMA increased ATP-sensitive K+ channel (KATP) activity. Based on inside-out patch-clamp recordings, PMA had little effect on KATP activity if no ATP was in the bath, while PMA restored KATP activity that was suppressed by 10 microM ATP in the bath. In voltage-clamp recording, PMA enhanced tolbutamide-sensitive membrane currents elicited by repetitive ramp pulses from -90 to -50 mV in a concentration-dependent manner, and this potentiation could not be prevented by pre-treatment of cell with BIM. 4alpha-phorbol 12,13-didecanoate (4alpha-PDD), a non-PKC-activating phorbol ester, mimicked the effect of PMA on both current-clamp and voltage-clamp recording configurations. With either 5.5 or 16.6 mM glucose in the extracellular solution, PMA (80 nM) increased insulin secretion from rat islets. However, in islets pretreated with BIM (1 microM), PMA did not increase, but rather reduced insulin secretion. In rat pancreatic beta-cells, PMA modulates insulin secretion through a mixed mechanism: increases insulin secretion by activation of PKC, and meanwhile decrease insulin secretion by impairing beta-cell excitability in a PKC-independent manner. The enhancement of KATP activity by reducing sensitivity of KATP to ATP seems to underlie the PMA-induced impairment of beta-cells electrical excitation in response to glucose stimulation.

Evaluation of Changes in Morphology and Function of Human Induced Pluripotent Stem Cell Derived Cardiomyocytes (HiPSC-CMs) Cultured on an Aligned-Nanofiber Cardiac Patch

PubMed Central

Khan, Mahmood; Xu, Yanyi; Hua, Serena; Johnson, Jed; Belevych, Andriy; Janssen, Paul M. L.; Gyorke, Sandor; Guan, Jianjun; Angelos, Mark G.

2015-01-01

Introduction Dilated cardiomyopathy is a major cause of progressive heart failure. Utilization of stem cell therapy offers a potential means of regenerating viable cardiac tissue. However, a major obstacle to stem cell therapy is the delivery and survival of implanted stem cells in the ischemic heart. To address this issue, we have developed a biomimetic aligned nanofibrous cardiac patch and characterized the alignment and function of human inducible pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) cultured on this cardiac patch. This hiPSC-CMs seeded patch was compared with hiPSC-CMs cultured on standard flat cell culture plates. Methods hiPSC-CMs were cultured on; 1) a highly aligned polylactide-co-glycolide (PLGA) nanofiber scaffold (~50 microns thick) and 2) on a standard flat culture plate. Scanning electron microscopy (SEM) was used to determine alignment of PLGA nanofibers and orientation of the cells on the respective surfaces. Analysis of gap junctions (Connexin-43) was performed by confocal imaging in both the groups. Calcium cycling and patch-clamp technique were performed to measure calcium transients and electrical coupling properties of cardiomyocytes. Results SEM demonstrated >90% alignment of the nanofibers in the patch which is similar to the extracellular matrix of decellularized rat myocardium. Confocal imaging of the cardiomyocytes demonstrated symmetrical alignment in the same direction on the aligned nanofiber patch in sharp contrast to the random appearance of cardiomyocytes cultured on a tissue culture plate. The hiPSC-CMs cultured on aligned nanofiber cardiac patches showed more efficient calcium cycling compared with cells cultured on standard flat surface culture plates. Quantification of mRNA with qRT-PCR confirmed that these cardiomyocytes expressed α-actinin, troponin-T and connexin-43 in-vitro. Conclusions Overall, our results demonstrated changes in morphology and function of human induced pluripotent derived cardiomyocytes cultured in an anisotropic environment created by an aligned nanofiber patch. In this environment, these cells better approximate normal cardiac tissue compared with cells cultured on flat surface and can serve as the basis for bioengineering of an implantable cardiac patch. PMID:25993466

Evaluation of Changes in Morphology and Function of Human Induced Pluripotent Stem Cell Derived Cardiomyocytes (HiPSC-CMs) Cultured on an Aligned-Nanofiber Cardiac Patch.

PubMed

Khan, Mahmood; Xu, Yanyi; Hua, Serena; Johnson, Jed; Belevych, Andriy; Janssen, Paul M L; Gyorke, Sandor; Guan, Jianjun; Angelos, Mark G

2015-01-01

Dilated cardiomyopathy is a major cause of progressive heart failure. Utilization of stem cell therapy offers a potential means of regenerating viable cardiac tissue. However, a major obstacle to stem cell therapy is the delivery and survival of implanted stem cells in the ischemic heart. To address this issue, we have developed a biomimetic aligned nanofibrous cardiac patch and characterized the alignment and function of human inducible pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) cultured on this cardiac patch. This hiPSC-CMs seeded patch was compared with hiPSC-CMs cultured on standard flat cell culture plates. hiPSC-CMs were cultured on; 1) a highly aligned polylactide-co-glycolide (PLGA) nanofiber scaffold (~50 microns thick) and 2) on a standard flat culture plate. Scanning electron microscopy (SEM) was used to determine alignment of PLGA nanofibers and orientation of the cells on the respective surfaces. Analysis of gap junctions (Connexin-43) was performed by confocal imaging in both the groups. Calcium cycling and patch-clamp technique were performed to measure calcium transients and electrical coupling properties of cardiomyocytes. SEM demonstrated >90% alignment of the nanofibers in the patch which is similar to the extracellular matrix of decellularized rat myocardium. Confocal imaging of the cardiomyocytes demonstrated symmetrical alignment in the same direction on the aligned nanofiber patch in sharp contrast to the random appearance of cardiomyocytes cultured on a tissue culture plate. The hiPSC-CMs cultured on aligned nanofiber cardiac patches showed more efficient calcium cycling compared with cells cultured on standard flat surface culture plates. Quantification of mRNA with qRT-PCR confirmed that these cardiomyocytes expressed α-actinin, troponin-T and connexin-43 in-vitro. Overall, our results demonstrated changes in morphology and function of human induced pluripotent derived cardiomyocytes cultured in an anisotropic environment created by an aligned nanofiber patch. In this environment, these cells better approximate normal cardiac tissue compared with cells cultured on flat surface and can serve as the basis for bioengineering of an implantable cardiac patch.

A proposed route to independent measurements of tight junction conductance at discrete cell junctions

PubMed Central

Zhou, Lushan; Zeng, Yuhan; Baker, Lane A; Hou, Jianghui

2015-01-01

Direct recording of tight junction permeability is of pivotal importance to many biologic fields. Previous approaches bear an intrinsic disadvantage due to the difficulty of separating tight junction conductance from nearby membrane conductance. Here, we propose the design of Double whole-cell Voltage Clamp - Ion Conductance Microscopy (DVC-ICM) based on previously demonstrated potentiometric scanning of local conductive pathways. As proposed, DVC-ICM utilizes two coordinated whole-cell patch-clamps to neutralize the apical membrane current during potentiometric scanning, which in models described here will profoundly enhance the specificity of tight junction recording. Several potential pitfalls are considered, evaluated and addressed with alternative countermeasures. PMID:26716077

Novel nitric oxide producing probiotic wound healing patch: preparation and in vivo analysis in a New Zealand white rabbit model of ischaemic and infected wounds.

PubMed

Jones, Mitchell; Ganopolsky, Jorge G; Labbé, Alain; Gilardino, Mirko; Wahl, Christopher; Martoni, Christopher; Prakash, Satya

2012-06-01

The treatment of chronic wounds poses a significant challenge for clinicians and patients alike. Here we report design and preclinical efficacy of a novel nitric oxide gas (gNO)-producing probiotic patch for wound healing. Specifically, a wound healing patch using lactic acid bacteria in an adhesive gas permeable membrane has been designed and investigated for treating ischaemic and infected full-thickness dermal wounds in a New Zealand white rabbit model for ischaemic wound healing. Kaplan-Meier survival curves showed increased wound closure with gNO-producing patch-treated wounds over 21 days of therapy (log-rank P = 0·0225 and Wilcoxon P = 0·0113). Cox proportional hazard regression showed that gNO-producing patch-treated wounds were 2·52 times more likely to close compared with control patches (hazard P = 0·0375, score P = 0·032 and likelihood ratio P = 0·0355), and histological analysis showed improved wound healing in gNO-producing patch-treated animals. This study may provide an effective, safe and less costly alternative for treating chronic wounds. © 2012 The Authors. © 2012 Blackwell Publishing Ltd and Medicalhelplines.com Inc.

Fluid shear stress enhances the cell volume decrease of osteoblast cells by increasing the expression of the ClC-3 chloride channel

PubMed Central

LIU, LI; CAI, SIYI; QIU, GUIXING; LIN, JIN

2016-01-01

ClC-3 is a volume-sensitive chloride channel that is responsible for cell volume adjustment and regulatory cell volume decrease (RVD). In order to evaluate the effects of fluid shear stress (FSS) stimulation on the osteoblast ClC-3 chloride channel, MC3T3-E1 cells were stimulated by FSS in the experimental group. Fluorescence quantitative polymerase chain reaction was used to detect changes in ClC-3 mRNA expression, the chloride ion fluorescent probe N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE) was used to detect the chloride channel activity, and whole-cell patch clamping was used to monitor the changes in the volume-sensitive chloride current activated by a hypotonic environment following mechanical stimulation. The results show that the expression of the osteoblast chloride channel ClC-3 was significantly higher in the FSS group compared with the control group. MQAE fluorescence intensity was significantly reduced in the FSS group compared to the control group, suggesting that mechanical stimulation increased chloride channel activity and increased the efflux of intracellular chloride ions. Image analysis of osteoblast volume changes showed that osteoblast RVD was enhanced by mechanical stimulation. Whole-cell patch clamping showed that the osteoblast volume-sensitive chloride current was larger in the stimulated group compared to the control group, suggesting that elevated ClC-3 chloride channel expression results in an increased volume-sensitive chloride current. In conclusion, FSS stimulation enhances the RVD of osteoblast cell by increasing the expression of the ClC-3 and enhancing the chloride channel activity. PMID:27073622

Redox regulation of epithelial sodium channels examined in alveolar type 1 and 2 cells patch-clamped in lung slice tissue.

PubMed

Helms, My N; Jain, Lucky; Self, Julie L; Eaton, Douglas C

2008-08-15

The alveolar surface of the lung is lined by alveolar type 1 (AT1) and type 2 (AT2) cells. Using single channel patch clamp analysis in lung slice preparations, we are able to uniquely study AT1 and AT2 cells separately from intact lung. We report for the first time the Na+ transport properties of type 2 cells accessed in live lung tissue (as we have done in type 1 cells). Type 2 cells in lung tissue slices express both highly selective cation and nonselective cation channels with average conductances of 8.8 +/- 3.2 and 22.5 +/- 6.3 picosiemens, respectively. Anion channels with 10-picosiemen conductance are also present in the apical membrane of type 2 cells. Our lung slice studies importantly verify the use of cultured cell model systems commonly used in lung epithelial sodium channel (ENaC) studies. Furthermore, we identify novel functional differences between the cells that make up the alveolar epithelium. One important difference is that exposure to the nitric oxide (NO) donor, PAPA-NONOate (1.5 microm), significantly decreases average ENaC NPo in type 2 cells (from 1.38 +/- 0.26 to 0.82 +/- 0.16; p < 0.05 and n = 18) but failed to alter ENaC activity in alveolar type 1 cells. Elevating endogenous superoxide (O2.) levels with Ethiolat, a superoxide dismutase inhibitor, prevented NO inhibition of ENaC activity in type 2 cells, supporting the novel hypothesis that O2. and NO signaling plays an important role in maintaining lung fluid balance.

Electrophysiological approach to determine kinetic parameters of sucrose uptake by single sieve elements or phloem parenchyma cells in intact Vicia faba plants.

PubMed

Hafke, Jens B; Höll, Sabina-Roxana; Kühn, Christina; van Bel, Aart J E

2013-01-01

Apart from cut aphid stylets in combination with electrophysiology, no attempts have been made thus far to measure in vivo sucrose-uptake properties of sieve elements. We investigated the kinetics of sucrose uptake by single sieve elements and phloem parenchyma cells in Vicia faba plants. To this end, microelectrodes were inserted into free-lying phloem cells in the main vein of the youngest fully-expanded leaf, half-way along the stem, in the transition zone between the autotrophic and heterotrophic part of the stem, and in the root axis. A top-to-bottom membrane potential gradient of sieve elements was observed along the stem (-130 mV to -110 mV), while the membrane potential of the phloem parenchyma cells was stable (approx. -100 mV). In roots, the membrane potential of sieve elements dropped abruptly to -55 mV. Bathing solutions having various sucrose concentrations were administered and sucrose/H(+)-induced depolarizations were recorded. Data analysis by non-linear least-square data fittings as well as by linear Eadie-Hofstee (EH) -transformations pointed at biphasic Michaelis-Menten kinetics (2 MM, EH: K m1 1.2-1.8 mM, K m2 6.6-9.0 mM) of sucrose uptake by sieve elements. However, Akaike's Information Criterion (AIC) favored single MM kinetics. Using single MM as the best-fitting model, K m values for sucrose uptake by sieve elements decreased along the plant axis from 1 to 7 mM. For phloem parenchyma cells, higher K m values (EH: K m1 10 mM, K m2 70 mM) as compared to sieve elements were found. In preliminary patch-clamp experiments with sieve-element protoplasts, small sucrose-coupled proton currents (-0.1 to -0.3 pA/pF) were detected in the whole-cell mode. In conclusion (a) K m values for sucrose uptake measured by electrophysiology are similar to those obtained with heterologous systems, (b) electrophysiology provides a useful tool for in situ determination of K m values, (c) As yet, it remains unclear if one or two uptake systems are involved in sucrose uptake by sieve elements, (d) Affinity for sucrose uptake by sieve elements exceeds by far that by phloem parenchyma cells, (e) Patch-clamp studies provide a feasible basis for quantification of sucrose uptake by single cells. The consequences of the findings for whole-plant carbohydrate partitioning are discussed.

Electrophysiological approach to determine kinetic parameters of sucrose uptake by single sieve elements or phloem parenchyma cells in intact Vicia faba plants

PubMed Central

Hafke, Jens B.; Höll, Sabina-Roxana; Kühn, Christina; van Bel, Aart J. E.

2013-01-01

Apart from cut aphid stylets in combination with electrophysiology, no attempts have been made thus far to measure in vivo sucrose-uptake properties of sieve elements. We investigated the kinetics of sucrose uptake by single sieve elements and phloem parenchyma cells in Vicia faba plants. To this end, microelectrodes were inserted into free-lying phloem cells in the main vein of the youngest fully-expanded leaf, half-way along the stem, in the transition zone between the autotrophic and heterotrophic part of the stem, and in the root axis. A top-to-bottom membrane potential gradient of sieve elements was observed along the stem (−130 mV to −110 mV), while the membrane potential of the phloem parenchyma cells was stable (approx. −100 mV). In roots, the membrane potential of sieve elements dropped abruptly to −55 mV. Bathing solutions having various sucrose concentrations were administered and sucrose/H+-induced depolarizations were recorded. Data analysis by non-linear least-square data fittings as well as by linear Eadie–Hofstee (EH) -transformations pointed at biphasic Michaelis–Menten kinetics (2 MM, EH: Km1 1.2–1.8 mM, Km2 6.6–9.0 mM) of sucrose uptake by sieve elements. However, Akaike's Information Criterion (AIC) favored single MM kinetics. Using single MM as the best-fitting model, Km values for sucrose uptake by sieve elements decreased along the plant axis from 1 to 7 mM. For phloem parenchyma cells, higher Km values (EH: Km1 10 mM, Km2 70 mM) as compared to sieve elements were found. In preliminary patch-clamp experiments with sieve-element protoplasts, small sucrose-coupled proton currents (−0.1 to −0.3 pA/pF) were detected in the whole-cell mode. In conclusion (a) Km values for sucrose uptake measured by electrophysiology are similar to those obtained with heterologous systems, (b) electrophysiology provides a useful tool for in situ determination of Km values, (c) As yet, it remains unclear if one or two uptake systems are involved in sucrose uptake by sieve elements, (d) Affinity for sucrose uptake by sieve elements exceeds by far that by phloem parenchyma cells, (e) Patch-clamp studies provide a feasible basis for quantification of sucrose uptake by single cells. The consequences of the findings for whole-plant carbohydrate partitioning are discussed. PMID:23914194

KATP channels in the nodose ganglia mediate the orexigenic actions of ghrelin

PubMed Central

Grabauskas, Gintautas; Wu, Xiaoyin; Lu, Yuanxu; Heldsinger, Andrea; Song, Il; Zhou, Shi-Yi; Owyang, Chung

2015-01-01

Abstract Ghrelin is the only known hunger signal derived from the peripheral tissues. Ghrelin overcomes the satiety signals evoked by anorexigenic molecules, such as cholecystokinin (CCK) and leptin, to stimulate feeding. The mechanisms by which ghrelin reduces the sensory signals evoked by anorexigenic hormones, which act via the vagus nerve to stimulate feeding, are unknown. Patch clamp recordings of isolated rat vagal neurons show that ghrelin hyperpolarizes neurons by activating K+ conductance. Administering a KATP channel antagonist or silencing Kir6.2, a major subunit of the KATP channel, abolished ghrelin inhibition in vitro and in vivo. Patch clamp studies show that ghrelin inhibits currents evoked by leptin and CCK-8, which operate through independent ionic channels. The inhibitory actions of ghrelin were abolished by treating the vagal ganglia neurons with pertussis toxin, as well as phosphatidylinositol 3-kinase (PI3K) or extracellular signal-regulated kinase 1 and 2 (Erk1/2) small interfering RNA. In vivo gene silencing of PI3K and Erk1/2 in the nodose ganglia prevented ghrelin inhibition of leptin- or CCK-8-evoked vagal firing. Feeding experiments showed that silencing Kir6.2 in the vagal ganglia abolished the orexigenic actions of ghrelin. These data indicate that ghrelin modulates vagal ganglia neuron excitability by activating KATP conductance via the growth hormone secretagogue receptor subtype 1a–Gαi–PI3K–Erk1/2–KATP pathway. The resulting hyperpolarization renders the neurons less responsive to signals evoked by anorexigenic hormones. This provides a mechanism to explain the actions of ghrelin with respect to overcoming anorexigenic signals that act via the vagal afferent pathways. Key points Ghrelin, a hunger signalling peptide derived from the peripheral tissues, overcomes the satiety signals evoked by anorexigenic molecules, such as cholecystokinin (CCK) and leptin, to stimulate feeding. Using in vivo and in vitro electrophysiological techniques, we show that ghrelin hyperpolarizes neurons and inhibits currents evoked by leptin and CCK-8. Administering a KATP channel antagonist or silencing Kir6.2, a major subunit of the KATP channel, abolished ghrelin inhibition. The inhibitory actions of ghrelin were also abolished by treating the vagal ganglia neurons with pertussis toxin, as well as phosphatidylinositol 3-kinase (PI3K) or extracellular signal-regulated kinase 1 and 2 (Erk1/2) small interfering RNA. Feeding experiments showed that silencing Kir6.2 in the vagal ganglia abolished the orexigenic actions of ghrelin. These data indicate that ghrelin modulates vagal ganglia neuron excitability by activating KATP conductance via the growth hormone secretagogue receptor subtype 1a–Gαi–PI3K–Erk1/2–KATP pathway. This provides a mechanism to explain the actions of ghrelin with respect to overcoming anorexigenic signals that act via the vagal afferent pathways. PMID:26174421

Design and in vivo evaluation of carvedilol buccal mucoadhesive patches.

PubMed

Thimmasetty, J; Pandey, G S; Babu, P R Sathesh

2008-07-01

The buccal region offers an attractive route of administration for systemic drug delivery. Carvedilol (dose, 3.125-25 mg) is beta-adrenergic antagonist. Its oral bioavailability is 25-35% because of first pass metabolism. Buccal absorption studies of a carvedilol solution in human volunteers showed 32.86% drug absorption. FTIR and UV spectroscopic methods revealed that there was no interaction between carvedilol and polymers. Carvedilol patches were prepared using HPMC, carbopol 934, eudragit RS 100, and ethylcellulose. The patches were evaluated for their thickness uniformity, folding endurance, weight uniformity, content uniformity, swelling behaviour, tensile strength, and surface pH. In vitro release studies were conducted for carvedilol-loaded patches in phosphate buffer (pH, 6.6) solution. Patches exhibited drug release in the range of 86.26 to 98.32% in 90 min. Data of in vitro release from patches were fit to different equations and kinetic models to explain release profiles. Kinetic models used were zero and first-order equations, Hixon-Crowell, Higuchi, and Korsmeyer-Peppas models. In vivo drug release studies in rabbits showed 90.85% of drug release from HPMC-carbopol patch while it was 74.63 to 88.02% within 90 min in human volunteers. Good correlation among in vitro release and in vivo release of carvedilol was observed.

Introduction to Solid Supported Membrane Based Electrophysiology

PubMed Central

Bazzone, Andre; Costa, Wagner Steuer; Braner, Markus; Călinescu, Octavian; Hatahet, Lina; Fendler, Klaus

2013-01-01

The electrophysiological method we present is based on a solid supported membrane (SSM) composed of an octadecanethiol layer chemisorbed on a gold coated sensor chip and a phosphatidylcholine monolayer on top. This assembly is mounted into a cuvette system containing the reference electrode, a chlorinated silver wire. After adsorption of membrane fragments or proteoliposomes containing the membrane protein of interest, a fast solution exchange is used to induce the transport activity of the membrane protein. In the single solution exchange protocol two solutions, one non-activating and one activating solution, are needed. The flow is controlled by pressurized air and a valve and tubing system within a faraday cage. The kinetics of the electrogenic transport activity is obtained via capacitive coupling between the SSM and the proteoliposomes or membrane fragments. The method, therefore, yields only transient currents. The peak current represents the stationary transport activity. The time dependent transporter currents can be reconstructed by circuit analysis. This method is especially suited for prokaryotic transporters or eukaryotic transporters from intracellular membranes, which cannot be investigated by patch clamp or voltage clamp methods. PMID:23711952

Introduction to solid supported membrane based electrophysiology.

PubMed

Bazzone, Andre; Costa, Wagner Steuer; Braner, Markus; Călinescu, Octavian; Hatahet, Lina; Fendler, Klaus

2013-05-11

The electrophysiological method we present is based on a solid supported membrane (SSM) composed of an octadecanethiol layer chemisorbed on a gold coated sensor chip and a phosphatidylcholine monolayer on top. This assembly is mounted into a cuvette system containing the reference electrode, a chlorinated silver wire. After adsorption of membrane fragments or proteoliposomes containing the membrane protein of interest, a fast solution exchange is used to induce the transport activity of the membrane protein. In the single solution exchange protocol two solutions, one non-activating and one activating solution, are needed. The flow is controlled by pressurized air and a valve and tubing system within a faraday cage. The kinetics of the electrogenic transport activity is obtained via capacitive coupling between the SSM and the proteoliposomes or membrane fragments. The method, therefore, yields only transient currents. The peak current represents the stationary transport activity. The time dependent transporter currents can be reconstructed by circuit analysis. This method is especially suited for prokaryotic transporters or eukaryotic transporters from intracellular membranes, which cannot be investigated by patch clamp or voltage clamp methods.

Potentiation of transient receptor potential V1 functions by the activation of metabotropic 5-HT receptors in rat primary sensory neurons

PubMed Central

Ohta, Toshio; Ikemi, Yuki; Murakami, Matsuka; Imagawa, Toshiaki; Otsuguro, Ken-ichi; Ito, Shigeo

2006-01-01

5-Hydroxytryptamine (5-HT) is one of the major chemical mediators released in injured and inflamed tissue and is capable of inducing hyperalgesia in vivo. However, the cellular mechanisms of 5-HT-induced hyperalgesia remain unclear. Transient receptor potential V1 (TRPV1) plays a pivotal role in nociceptive receptors. In the present study, we determined whether 5-HT changes TRPV1 functions in cultured dorsal root ganglion (DRG) neurons isolated from neonatal rats, using Ca2+ imaging and whole-cell patch-clamp techniques. In more than 70% of DRG neurons, 5-HT potentiated the increases of [Ca2+]i induced by capsaicin, protons and noxious heat. Capsaicin-induced current and depolarizing responses, and proton-induced currents were also augmented by 5-HT. RT-PCR analysis revealed the expression of 5-HT2A and 5-HT7 receptors in rat DRG neurons. Agonists for 5-HT2A and 5-HT7 receptors mimicked the potentiating effect of 5-HT, and their antagonists decreased it. In DRG ipsilateral to the complete Freund's adjuvant-injected inflammation side, expression levels of 5-HT2A and 5-HT7 mRNAs increased, and the potentiating effect of 5-HT was more prominent than in the contralateral control side. These results suggest that the PKC- and PKA-mediated signalling pathways are involved in the potentiating effect of 5-HT on TRPV1 functions through the activation of 5-HT2A and 5-HT7 receptors, respectively. Under inflammatory conditions, the increases of the biosynthesis of these 5-HT receptors may lead to further potentiation of TRPV1 functions, resulting in the generation of inflammatory hyperalgesia in vivo. PMID:16901936

Nonpsychotropic plant cannabinoids, cannabidivarin (CBDV) and cannabidiol (CBD), activate and desensitize transient receptor potential vanilloid 1 (TRPV1) channels in vitro: potential for the treatment of neuronal hyperexcitability.

PubMed

Iannotti, Fabio Arturo; Hill, Charlotte L; Leo, Antonio; Alhusaini, Ahlam; Soubrane, Camille; Mazzarella, Enrico; Russo, Emilio; Whalley, Benjamin J; Di Marzo, Vincenzo; Stephens, Gary J

2014-11-19

Epilepsy is the most common neurological disorder, with over 50 million people worldwide affected. Recent evidence suggests that the transient receptor potential cation channel subfamily V member 1 (TRPV1) may contribute to the onset and progression of some forms of epilepsy. Since the two nonpsychotropic cannabinoids cannabidivarin (CBDV) and cannabidiol (CBD) exert anticonvulsant activity in vivo and produce TRPV1-mediated intracellular calcium elevation in vitro, we evaluated the effects of these two compounds on TRPV1 channel activation and desensitization and in an in vitro model of epileptiform activity. Patch clamp analysis in transfected HEK293 cells demonstrated that CBD and CBDV dose-dependently activate and rapidly desensitize TRPV1, as well as TRP channels of subfamily V type 2 (TRPV2) and subfamily A type 1 (TRPA1). TRPV1 and TRPV2 transcripts were shown to be expressed in rat hippocampal tissue. When tested on epileptiform neuronal spike activity in hippocampal brain slices exposed to a Mg(2+)-free solution using multielectrode arrays (MEAs), CBDV reduced both epileptiform burst amplitude and duration. The prototypical TRPV1 agonist, capsaicin, produced similar, although not identical effects. Capsaicin, but not CBDV, effects on burst amplitude were reversed by IRTX, a selective TRPV1 antagonist. These data suggest that CBDV antiepileptiform effects in the Mg(2+)-free model are not uniquely mediated via activation of TRPV1. However, TRPV1 was strongly phosphorylated (and hence likely sensitized) in Mg(2+)-free solution-treated hippocampal tissue, and both capsaicin and CBDV caused TRPV1 dephosphorylation, consistent with TRPV1 desensitization. We propose that CBDV effects on TRP channels should be studied further in different in vitro and in vivo models of epilepsy.

Isolation and characterization of ventricular-like cells derived from NKX2-5eGFP/w and MLC2vmCherry/w double knock-in human pluripotent stem cells.

PubMed

Yamauchi, Kaori; Li, Junjun; Morikawa, Kumi; Liu, Li; Shirayoshi, Yasuaki; Nakatsuji, Norio; Elliott, David A; Hisatome, Ichiro; Suemori, Hirofumi

2018-01-01

Human pluripotent stem cell (hPSC)-derived cardiomyocytes (CMs) are a promising source for cell transplantation into the damaged heart, which has limited regenerative ability. Many methods have been developed to obtain large amounts of functional CMs from hPSCs for therapeutic applications. However, during the differentiation process, a mixed population of various cardiac cells, including ventricular, atrial, and pacemaker cells, is generated, which hampers the proper functional analysis and evaluation of cell properties. Here, we established NKX2-5 eGFP/w and MLC2v mCherry/w hPSC double knock-ins that allow for labeling, tracing, purification, and analysis of the development of ventricular cells from early to late stages. As with the endogenous transcriptional activities of these genes, MLC2v-mCherry expression following NKX2-5-eGFP expression was observed under previously established culture conditions, which mimic the in vivo cardiac developmental process. Patch-clamp and microelectrode array electrophysiological analyses showed that the NKX2-5 and MLC2v double-positive cells possess ventricular-like properties. The results demonstrate that the NKX2-5 eGFP/w and MLC2v mCherry/w hPSCs provide a powerful model system to capture region-specific cardiac differentiation from early to late stages. Our study would facilitate subtype-specific cardiac development and functional analysis using the hPSC-derived sources. Copyright © 2017 Elsevier Inc. All rights reserved.

Activation by intracellular GDP, metabolic inhibition and pinacidil of a glibenclamide-sensitive K-channel in smooth muscle cells of rat mesenteric artery.

PubMed Central

Zhang, H; Bolton, T B

1995-01-01

1. Single-channel recordings were made from cell-attached and isolated patches, and whole-cell currents were recorded under voltage clamp from single smooth muscle cells obtained by enzymic digestion of a small branch of the rat mesenteric artery. 2. In single voltage-clamped cells 1 mM uridine diphosphate (UDP) or guanidine diphosphate (GDP) added to the pipette solution, or pinacidil (100 microM) a K-channel opener (KCO) applied in the bathing solution, evoked an outward current of up to 100pA which was blocked by glibenclamide (10 microM). In single cells from which recordings were made by the 'perforated patch' (nystatin pipette) technique, metabolic inhibition by 1 mM NaCN and 10 mM 2-deoxy-glucose also evoked a similar glibenclamide-sensitive current. 3. Single K-channel activity was observed in cell-attached patches only infrequently unless the metabolism of the cell was inhibited, whereupon channel activity blocked by glibenclamide was seen; pinacidil applied to the cell evoked similar glibenclamide-sensitive channel activity. If the patch was pulled off the cell to form an isolated inside-out patch, similar glibenclamide-sensitive single-channel currents were observed in the presence of UDP and/or pinacidil to those seen in cell-attached mode; channel conductance was 20 pS (60:130 K-gradient) and openings showed no voltage-dependence and noisy inward currents, typical of the nucleoside diphosphate (NDP) activated K-channel (KNDP) seen previously in rabbit portal vein. 4. Formation of an isolated inside-out patch into an ATP-free solution did not increase the probability of channel opening which declined with time even when some single-channel activity had occurred in the cell-attached mode before detachment. However, application of 1 mM UDP or GDP, but not ATP, to inside-out patches evoked single-channel activity. Application of ATP-free solution to isolated patches, previously exposed to ATP and in which channel activity had been seen, did not evoke channel activity. 5. It is concluded that small conductance K-channels (KNDP) open in smooth muscle cells from this small artery in response to UDP or GDP acting from the inside, or pinacidil acting from the outside; the same channels open during inhibition of metabolism presumably mainly due to the rise in nucleoside diphosphates, but a fall in the ATP concentration on the inside of the channel did not by itself evoke channel activity.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:7735693

Synaptic dynamics and neuronal network connectivity are reflected in the distribution of times in Up states.

PubMed

Dao Duc, Khanh; Parutto, Pierre; Chen, Xiaowei; Epsztein, Jérôme; Konnerth, Arthur; Holcman, David

2015-01-01

The dynamics of neuronal networks connected by synaptic dynamics can sustain long periods of depolarization that can last for hundreds of milliseconds such as Up states recorded during sleep or anesthesia. Yet the underlying mechanism driving these periods remain unclear. We show here within a mean-field model that the residence time of the neuronal membrane potential in cortical Up states does not follow a Poissonian law, but presents several peaks. Furthermore, the present modeling approach allows extracting some information about the neuronal network connectivity from the time distribution histogram. Based on a synaptic-depression model, we find that these peaks, that can be observed in histograms of patch-clamp recordings are not artifacts of electrophysiological measurements, but rather are an inherent property of the network dynamics. Analysis of the equations reveals a stable focus located close to the unstable limit cycle, delimiting a region that defines the Up state. The model further shows that the peaks observed in the Up state time distribution are due to winding around the focus before escaping from the basin of attraction. Finally, we use in vivo recordings of intracellular membrane potential and we recover from the peak distribution, some information about the network connectivity. We conclude that it is possible to recover the network connectivity from the distribution of times that the neuronal membrane voltage spends in Up states.

PKD Phosphorylation as Novel Pathway of KV11.1 Regulation.

PubMed

Steffensen, Annette Buur; Bomholtz, Sofia Hammami; Andersen, Martin Nybo; Olsen, Jesper Velgaard; Mutsaers, Nancy; Lundegaard, Pia Rengtved; Lundby, Alicia; Schmitt, Nicole

2018-06-27

The voltage-gated potassium channel KV11.1 has been originally cloned from the brain and is expressed in a variety of tissues. The role of phosphorylation for channel function is a matter of debate. In this study, we aimed to elucidate the extent and role of protein kinase D mediated phosphorylation. We employed mass spectrometry, whole-cell patch clamp electrophysiology, confocal microscopy, site-directed mutagenesis, and western blotting. Using brain tissue from rat and mouse, we mapped several phosphorylated KV11.1 residues by LC-MS mass spectrometry and identified protein kinase D (PKD1) as possible regulatory kinase. Co-expression of KV11.1 with PKD1 reduced current amplitudes without altering protein levels or surface expression of the channel. Based on LC-MS results from in vivo and HEK293 cell experiments we chose four KV11.1 mutant candidates for further functional analysis. Ablation of the putative PKD phosphorylation site in the mutant S284A increased the maximal current indicating S284 as a main PKD target in KV11.1. Our data might help mitigating a long-standing controversy in the field regarding PKC regulation of KV11.1. We propose that PKD1 mediates the PKC effects on KV11.1 and we found that PKD targets S284 in the N-terminus of the channel. © 2018 The Author(s). Published by S. Karger AG, Basel.

Reduced high-frequency motor neuron firing, EMG fractionation, and gait variability in awake walking ALS mice

PubMed Central

Hadzipasic, Muhamed; Ni, Weiming; Nagy, Maria; Steenrod, Natalie; McGinley, Matthew J.; Kaushal, Adi; Thomas, Eleanor; McCormick, David A.

2016-01-01

Amyotrophic lateral sclerosis (ALS) is a lethal neurodegenerative disease prominently featuring motor neuron (MN) loss and paralysis. A recent study using whole-cell patch clamp recording of MNs in acute spinal cord slices from symptomatic adult ALS mice showed that the fastest firing MNs are preferentially lost. To measure the in vivo effects of such loss, awake symptomatic-stage ALS mice performing self-initiated walking on a wheel were studied. Both single-unit extracellular recordings within spinal cord MN pools for lower leg flexor and extensor muscles and the electromyograms (EMGs) of the corresponding muscles were recorded. In the ALS mice, we observed absent or truncated high-frequency firing of MNs at the appropriate time in the step cycle and step-to-step variability of the EMG, as well as flexor-extensor coactivation. In turn, kinematic analysis of walking showed step-to-step variability of gait. At the MN level, the higher frequencies absent from recordings from mutant mice corresponded with the upper range of frequencies observed for fast-firing MNs in earlier slice measurements. These results suggest that, in SOD1-linked ALS mice, symptoms are a product of abnormal MN firing due at least in part to loss of neurons that fire at high frequency, associated with altered EMG patterns and hindlimb kinematics during gait. PMID:27821773

Channel-mediated lactate release by K⁺-stimulated astrocytes.

PubMed

Sotelo-Hitschfeld, Tamara; Niemeyer, María I; Mächler, Philipp; Ruminot, Iván; Lerchundi, Rodrigo; Wyss, Matthias T; Stobart, Jillian; Fernández-Moncada, Ignacio; Valdebenito, Rocío; Garrido-Gerter, Pamela; Contreras-Baeza, Yasna; Schneider, Bernard L; Aebischer, Patrick; Lengacher, Sylvain; San Martín, Alejandro; Le Douce, Juliette; Bonvento, Gilles; Magistretti, Pierre J; Sepúlveda, Francisco V; Weber, Bruno; Barros, L Felipe

2015-03-11

Excitatory synaptic transmission is accompanied by a local surge in interstitial lactate that occurs despite adequate oxygen availability, a puzzling phenomenon termed aerobic glycolysis. In addition to its role as an energy substrate, recent studies have shown that lactate modulates neuronal excitability acting through various targets, including NMDA receptors and G-protein-coupled receptors specific for lactate, but little is known about the cellular and molecular mechanisms responsible for the increase in interstitial lactate. Using a panel of genetically encoded fluorescence nanosensors for energy metabolites, we show here that mouse astrocytes in culture, in cortical slices, and in vivo maintain a steady-state reservoir of lactate. The reservoir was released to the extracellular space immediately after exposure of astrocytes to a physiological rise in extracellular K(+) or cell depolarization. Cell-attached patch-clamp analysis of cultured astrocytes revealed a 37 pS lactate-permeable ion channel activated by cell depolarization. The channel was modulated by lactate itself, resulting in a positive feedback loop for lactate release. A rapid fall in intracellular lactate levels was also observed in cortical astrocytes of anesthetized mice in response to local field stimulation. The existence of an astrocytic lactate reservoir and its quick mobilization via an ion channel in response to a neuronal cue provides fresh support to lactate roles in neuronal fueling and in gliotransmission. Copyright © 2015 the authors 0270-6474/15/354168-11$15.00/0.

Effects of lacosamide and carbamazepine on human motor cortex excitability: a double-blind, placebo-controlled transcranial magnetic stimulation study.

PubMed

Lang, Nicolas; Rothkegel, Holger; Peckolt, Hannes; Deuschl, Günther

2013-11-01

Lacosamide (LCM) and carbamazepine (CBZ) are antiepileptic drugs both acting on neuronal voltage-gated sodium channels. Patch-clamp studies demonstrated significant differences in how LCM and CBZ affect neuronal membrane excitability. Despite valuable information patch-clamp studies provide, they also comprise some constraints. For example, little is known about effects of LCM on intracortical synaptic excitability. In contrast, transcranial magnetic stimulation (TMS) can describe drug-induced changes at the system level of the human cerebral cortex. The present study was designed to explore dose-depended effects of LCM and effects of CBZ on motor cortex excitability with TMS in a randomized, double-blind, placebo-controlled crossover trial in healthy human subjects. Subjects received 600 mg CBZ, 200 mg LCM, 400 mg LCM or placebo preceding TMS measurements. Compared to placebo, TMS motor thresholds were significantly increased after carbamazepine and lacosamide, with a trend for a dose dependent effect of lacosamide. Both, carbamazepine and lacosamide did not affect TMS parameters of intracortical synaptic excitability. TMS measurements suggest that lacosamide and carbamazepine predominantly act on neuronal membrane excitability. Copyright © 2013 British Epilepsy Association. Published by Elsevier Ltd. All rights reserved.

Patch clamp reveals powerful blockade of the mitochondrial permeability transition pore by the D2-receptor agonist pramipexole.

PubMed

Sayeed, Iqbal; Parvez, Suhel; Winkler-Stuck, Kirstin; Seitz, Gordon; Trieu, Isabelle; Wallesch, Claus-Werner; Schönfeld, Peter; Siemen, Detlef

2006-03-01

The dopamine-D2-agonist pramipexole (PPX) was tested for blocking mitochondrial permeability transition (PT) in order to give a possible explanation for its neuroprotective effect seen in PPX-treated Parkinson's disease patients. Patch-clamp techniques for studying single-channel currents in the inner mitochondrial membrane and large-amplitude swelling of energized mitochondria were used to study PPX action on the permeability transition pore (PTP), a key player in the mitochondrial route of the apoptotic cascade. Identity of the PTP was proven by measuring the concentration-response relation for cyclosporin A-blockade (IC50=26 nM). PPX inhibits the PTP reversibly with an IC50 of 500 nM, which is close to the values determined earlier as plasma concentrations after PPX medication in patients. Interaction of PPX with the PTP is further supported by demonstrating that it abolished Ca2+-triggered swelling in functionally intact mitochondria. Blockade of the PTP by PPX was attenuated by increasing concentrations of inorganic phosphate and by acidification. We suggest that PPX could exert part of its neuroprotective effect by inhibition of the PTP and thus, probably, blocking of the mitochondrial pathway of the apoptosis cascade.

Azadirachtin blocks the calcium channel and modulates the cholinergic miniature synaptic current in the central nervous system of Drosophila.

PubMed

Qiao, Jingda; Zou, Xiaolu; Lai, Duo; Yan, Ying; Wang, Qi; Li, Weicong; Deng, Shengwen; Xu, Hanhong; Gu, Huaiyu

2014-07-01

Azadirachtin is a botanical pesticide, which possesses conspicuous biological actions such as insecticidal, anthelmintic, antifeedancy, antimalarial effects as well as insect growth regulation. Deterrent for chemoreceptor functions appears to be the main mechanism involved in the potent biological actions of Azadirachtin, although the cytotoxicity and subtle changes to skeletal muscle physiology may also contribute to its insecticide responses. In order to discover the effects of Azadirachtin on the central nervous system (CNS), patch-clamp recording was applied to Drosophila melanogaster, which has been widely used in neurological research. Here, we describe the electrophysiological properties of a local neuron located in the suboesophageal ganglion region of D. melanogaster using the whole brain. The patch-clamp recordings suggested that Azadirachtin modulates the properties of cholinergic miniature excitatory postsynaptic current (mEPSC) and calcium currents, which play important roles in neural activity of the CNS. The frequency of mEPSC and the peak amplitude of the calcium currents significantly decreased after application of Azadirachtin. Our study indicates that Azadirachtin can interfere with the insect's CNS via inhibition of excitatory cholinergic transmission and partly blocking the calcium channel. © 2013 Society of Chemical Industry.

Mechanism of pain relief by low-power infrared irradiation: ATP is an IR-target molecule in nociceptive neurons.

PubMed

Yachnev, Igor L; Plakhova, Vera B; Podzorova, Svetlana A; Shelykh, Tatiana N; Rogachevsky, Ilya V; Krylov, Boris V

2012-01-01

Effects of infrared (IR) radiation generated by a low-power CO2-laser on the membrane of cultured dissociated nociceptive neurons of newborn rat spinal ganglia were investigated using the whole-cell patch-clamp method. Low-power IR radiation diminished the voltage sensitivity of activation gating machinery of slow sodium channels (Na(v)1.8). Ouabain known to block both transducer and pumping functions of Na+,K+-ATPase eliminated IR irradiation effects. The molecular mechanism of interaction of CO2-laser radiation with sensory membrane was proposed. The primary event of this interaction is the process of energy absorption by ATP molecules. The transfer of vibrational energy from Na+,K+- ATPase-bound and vibrationally excited ATP molecules to Na+,K+-ATPase activates this enzyme and converts it into a signal transducer. This effect leads to a decrease in the voltage sensitivity of Na(v)1.8 channels. The effect of IR-radiation was elucidated by the combined application of a very sensitive patch-clamp method and an optical facility with a controlled CO2-laser. As a result, the mechanism of interaction of non-thermal low-power IR radiation with the nociceptive neuron membrane is suggested.

Safinamide Differentially Modulates In Vivo Glutamate and GABA Release in the Rat Hippocampus and Basal Ganglia.

PubMed

Morari, Michele; Brugnoli, Alberto; Pisanò, Clarissa Anna; Novello, Salvatore; Caccia, Carla; Melloni, Elsa; Padoani, Gloria; Vailati, Silvia; Sardina, Marco

2018-02-01

Safinamide has been recently approved as an add-on to levodopa therapy for Parkinson disease. In addition to inhibiting monoamine oxidase type B, it blocks sodium channels and modulates glutamate (Glu) release in vitro. Since this property might contribute to the therapeutic action of the drug, we undertook the present study to investigate whether safinamide inhibits Glu release also in vivo and whether this effect is consistent across different brain areas and is selective for glutamatergic neurons. To this aim, in vivo microdialysis was used to monitor the spontaneous and veratridine-induced Glu and GABA release in the hippocampus and basal ganglia of naive, awake rats. Brain levels of safinamide were measured as well. To shed light on the mechanisms underlying the effect of safinamide, sodium currents were measured by patch-clamp recording in rat cortical neurons. Safinamide maximally inhibited the veratridine-induced Glu and GABA release in hippocampus at 15 mg/kg, which reached free brain concentrations of 1.89-1.37 µ M. This dose attenuated veratridine-stimulated Glu (but not GABA) release in subthalamic nucleus, globus pallidus, and substantia nigra reticulata, but not in striatum. Safinamide was ineffective on spontaneous neurotransmitter release. In vitro, safinamide inhibited sodium channels, showing a greater affinity at depolarized (IC 50 = 8 µ M) than at resting (IC 50 = 262 µ M) potentials. We conclude that safinamide inhibits in vivo Glu release from stimulated nerve terminals, likely via blockade of sodium channels at subpopulations of neurons with specific firing patterns. These data are consistent with the anticonvulsant and antiparkinsonian actions of safinamide and provide support for the nondopaminergic mechanism of its action. Copyright © 2018 The Author(s).

Multiple pathological events in exercised dystrophic mdx mice are targeted by pentoxifylline: outcome of a large array of in vivo and ex vivo tests.

PubMed

Burdi, Rosa; Rolland, Jean-François; Fraysse, Bodvael; Litvinova, Karina; Cozzoli, Anna; Giannuzzi, Viviana; Liantonio, Antonella; Camerino, Giulia Maria; Sblendorio, Valeriana; Capogrosso, Roberta Francesca; Palmieri, Beniamino; Andreetta, Francesca; Confalonieri, Paolo; De Benedictis, Leonarda; Montagnani, Monica; De Luca, Annamaria

2009-04-01

The phosphodiesterases inhibitor pentoxifylline gained attention for Duchenne muscular dystrophy therapy for its claimed anti-inflammatory, antioxidant, and antifibrotic action. A recent finding also showed that pentoxifylline counteracts the abnormal overactivity of a voltage-independent calcium channel in myofibers of dystrophic mdx mice. The possible link between workload, altered calcium homeostasis, and oxidative stress pushed toward a more detailed investigation. Thus a 4- to 8-wk treatment with pentoxifylline (50 mg x kg(-1) x day(-1) ip) was performed in mdx mice, undergoing or not a chronic exercise on treadmill. In vivo, the treatment partially increased forelimb strength and enhanced resistance to treadmill running in exercised animals. Ex vivo, pentoxifylline restored the mechanical threshold, an electrophysiological index of calcium homeostasis, and reduced resting cytosolic calcium in extensor digitorum longus muscle fibers. Mn quenching and patch-clamp technique confirmed that this effect was paralleled by a drug-induced reduction of membrane permeability to calcium. The treatment also significantly enhanced isometric tetanic tension in mdx diaphragm. The plasma levels of creatine kinase and reactive oxygen species were both significantly reduced in treated-exercised animals. Dihydroethidium staining, used as an indicator of reactive oxygen species production, showed that pentoxifylline significantly reduced the exercise-induced increase in fluorescence in the mdx tibialis anterior muscle. A significant decrease in connective tissue area and profibrotic cytokine transforming growth factor-beta(1) was solely found in tibialis anterior muscle. In both diaphragm and gastrocnemius muscle, a significant increase in neural cell adhesion molecule-positive area was instead observed. This data supports the interest toward pentoxifylline and allows insight in the level of cross talk between pathogenetic events in workloaded dystrophic muscle.

Ube3a loss increases excitability and blunts orientation tuning in the visual cortex of Angelman syndrome model mice.

PubMed

Wallace, Michael L; van Woerden, Geeske M; Elgersma, Ype; Smith, Spencer L; Philpot, Benjamin D

2017-07-01

Angelman syndrome (AS) is a neurodevelopmental disorder caused by loss of the maternally inherited allele of UBE3A Ube3a STOP/p+ mice recapitulate major features of AS in humans and allow conditional reinstatement of maternal Ube3a with the expression of Cre recombinase. We have recently shown that AS model mice exhibit reduced inhibitory drive onto layer (L)2/3 pyramidal neurons of visual cortex, which contributes to a synaptic excitatory/inhibitory imbalance. However, it remains unclear how this loss of inhibitory drive affects neural circuits in vivo. Here we examined visual cortical response properties in individual neurons to explore the consequences of Ube3a loss on intact cortical circuits and processing. Using in vivo patch-clamp electrophysiology, we measured the visually evoked responses to square-wave drifting gratings in L2/3 regular-spiking (RS) neurons in control mice, Ube3a -deficient mice, and mice in which Ube3a was conditionally reinstated in GABAergic neurons. We found that Ube3a -deficient mice exhibited enhanced pyramidal neuron excitability in vivo as well as weaker orientation tuning. These observations are the first to show alterations in cortical computation in an AS model, and they suggest a basis for cortical dysfunction in AS. NEW & NOTEWORTHY Angelman syndrome (AS) is a severe neurodevelopmental disorder caused by the loss of the gene UBE3A Using electrophysiological recording in vivo, we describe visual cortical dysfunctions in a mouse model of AS. Aberrant cellular properties in AS model mice could be improved by reinstating Ube3a in inhibitory neurons. These findings suggest that inhibitory neurons play a substantial role in the pathogenesis of AS. Copyright © 2017 the American Physiological Society.

Is GABA neurotransmission enhanced in auditory thalamus relative to inferior colliculus?

PubMed Central

Cai, Rui; Kalappa, Bopanna I.; Brozoski, Thomas J.; Ling, Lynne L.

2013-01-01

Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the central auditory system. Sensory thalamic structures show high levels of non-desensitizing extrasynaptic GABAA receptors (GABAARs) and a reduction in the redundancy of coded information. The present study compared the inhibitory potency of GABA acting at GABAARs between the inferior colliculus (IC) and the medial geniculate body (MGB) using quantitative in vivo, in vitro, and ex vivo experimental approaches. In vivo single unit studies compared the ability of half maximal inhibitory concentrations of GABA to inhibit sound-evoked temporal responses, and found that GABA was two to three times (P < 0.01) more potent at suppressing MGB single unit responses than IC unit responses. In vitro whole cell patch-clamp slice recordings were used to demonstrate that gaboxadol, a δ-subunit selective GABAAR agonist, was significantly more potent at evoking tonic inhibitory currents from MGB neurons than IC neurons (P < 0.01). These electrophysiological findings were supported by an in vitro receptor binding assay which used the picrotoxin analog [3H]TBOB to assess binding in the GABAAR chloride channel. MGB GABAARs had significantly greater total open chloride channel capacity relative to GABAARs in IC (P < 0.05) as shown by increased total [3H]TBOB binding. Finally, a comparative ex vivo measurement compared endogenous GABA levels and suggested a trend towards higher GABA concentrations in MGB than in IC. Collectively, these studies suggest that, per unit GABA, high affinity extrasynaptic and synaptic GABAARs confer a significant inhibitory GABAAR advantage to MGB neurons relative to IC neurons. This increased GABA sensitivity likely underpins the vital filtering role of auditory thalamus. PMID:24155003

Robust optical fiber patch-cords for in vivo optogenetic experiments in rats.

PubMed

Trujillo-Pisanty, Ivan; Sanio, Christian; Chaudhri, Nadia; Shizgal, Peter

2015-01-01

In vivo optogenetic experiments commonly employ long lengths of optical fiber to connect the light source (commonly a laser) to the optical fiber implants in the brain. Commercially available patch cords are expensive and break easily. Researchers have developed methods to build these cables in house for in vivo experiments with rodents [1-4]. However, the half-life of those patch cords is greatly reduced when they are used with behaving rats, which are strong enough to break the delicate cable tip and to bite through the optical fiber and furcation tubing. Based on [3] we have strengthened the patch-cord tip that connects to the optical implant, and we have incorporated multiple layers of shielding to produce more robust and resistant cladding. Here, we illustrate how to build these patch cords with FC or M3 connectors. However, the design can be adapted for use with other common optical-fiber connectors. We have saved time and money by using this design in our optical self-stimulation experiments with rats, which are commonly several months long and last four to eleven hours per session. The main advantages are: •Long half-life.•Resistant to moderate rodent bites.•Suitable for long in vivo optogenetic experiments with large rodents.

Simultaneous recording of the action potential and its whole-cell associated ion current on NG108-15 cells cultured over a MWCNT electrode

NASA Astrophysics Data System (ADS)

Morales-Reyes, I.; Seseña-Rubfiaro, A.; Acosta-García, M. C.; Batina, N.; Godínez-Fernández, R.

2016-08-01

It is well known that, in excitable cells, the dynamics of the ion currents (I i) is extremely important to determine both the magnitude and time course of an action potential (A p). To observe these two processes simultaneously, we cultured NG108-15 cells over a multi-walled carbon nanotubes electrode (MWCNTe) surface and arranged a two independent Patch Clamp system configuration (Bi-Patch Clamp). The first system was used in the voltage or current clamp mode, using a glass micropipette as an electrode. The second system was modified to connect the MWCNTe to virtual ground. While the A p was recorded through the micropipette electrode, the MWCNTe was used to measure the underlying whole-cell current. This configuration allowed us to record both the membrane voltage (V m) and the current changes simultaneously. Images acquired by atomic force microscopy (AFM) and scanning electron microscopy (SEM) indicate that cultured cells developed a complex network of neurites, which served to establish the necessary close contact and strong adhesion to the MWCNTe surface. These features were a key factor to obtain the recording of the whole-cell I i with a high signal to noise ratio (SNR). The experimental results were satisfactorily reproduced by a theoretical model developed to simulate the proposed system. Besides the contribution to a better understanding of the fundamental mechanisms involved in cell communication, the developed method could be useful in cell physiology studies, pharmacology and diseases diagnosis.

A clamp fixture with interdigital capacitive sensor for in situ evaluation of wire insulation

NASA Astrophysics Data System (ADS)

Sheldon, Robert T.; Bowler, Nicola

2014-02-01

An interdigital capacitive sensor has been designed and optimized for testing aircraft wires by applying a quasinumerical model developed and reported previously. The sensor consists of two patches of interdigitated electrodes, connected by a long signal bus strip, that are intended to conform to two sides of an insulated wire. The electrodes are deposited using photolithography upon a 25.4-μm-thick Kapton® polyimide film. The two electrode patches are attached to the two jaws of a plastic spring-loaded clamp, with each jaw having a milled groove designed such that the electrodes conform to the curved surface of the insulated wire. An SMA connector and cable connect between the electrodes on the clamp and an LCR meter. Segments of pristine M5086/2 aircraft wire, each 10 cm long, were immersed in fluids commonly found in aircraft environments, to cause accelerated chemical degradation. The effects of Jet A fuel, deicing fluid, hydraulic fluid, aircraft cleaner, isopropyl alcohol and distilled water were studied. The frequency-dependent capacitance and dissipation factor of one pristine wire segment and of those degraded in the six fluid environments were measured within the frequency range 100 Hz to 1 MHz. Significant changes in capacitance and dissipation factor were observed for all degraded wires, compared with results for the pristine sample, suggesting the feasibility of detecting insulation degradation in the field. The results were also consistent with those of a similar experiment performed on sheets of Nylon 6, the material that comprises the outermost layer of M5086/2 wire.

Real-time Experiment Interface for Biological Control Applications

PubMed Central

Lin, Risa J.; Bettencourt, Jonathan; White, John A.; Christini, David J.; Butera, Robert J.

2013-01-01

The Real-time Experiment Interface (RTXI) is a fast and versatile real-time biological experimentation system based on Real-Time Linux. RTXI is open source and free, can be used with an extensive range of experimentation hardware, and can be run on Linux or Windows computers (when using the Live CD). RTXI is currently used extensively for two experiment types: dynamic patch clamp and closed-loop stimulation pattern control in neural and cardiac single cell electrophysiology. RTXI includes standard plug-ins for implementing commonly used electrophysiology protocols with synchronized stimulation, event detection, and online analysis. These and other user-contributed plug-ins can be found on the website (http://www.rtxi.org). PMID:21096883

Hyperthyroidism impairs pancreatic beta cell adaptations to late pregnancy and maternal liporegulation in the rat.

PubMed

Holness, M J; Greenwood, G K; Smith, N D; Sugden, M C

2005-11-01

Hyperthyroidism modifies lipid dynamics (increased oxidation), impairs insulin action and can suppress insulin secretion. We therefore examined the impact of hyperthyroidism on the relationship between glucose-stimulated insulin secretion (GSIS) and insulin action, using late pregnancy as a model of physiological insulin resistance that is associated with compensatory insulin hypersecretion to maintain glucose tolerance. Our aim was to examine whether hyperthyroidism compromises the regulation of insulin secretion and the ability of insulin to modulate circulating lipid concentrations in late pregnancy. Hyperthyroidism was induced by tri-iodothyronine (T(3)) administration from day 17 to 19 of pregnancy. GSIS was assessed during an IVGTT and during hyperglycaemic clamps in vivo and in vitro, using step-up and -down islet perifusions. Hyperthyroidism in pregnancy elevated the glucose threshold for GSIS and impaired GSIS at low and high glucose concentrations in islet perifusions. In the intact animal, insulin secretion (after bolus glucose) was more rapidly curtailed following removal of the glucose stimulus to secretion. In contrast, GSIS was maintained during protracted hyperglycaemia (hyperglycaemic clamps) in the hyperthyroid pregnant state in vivo. Hyperthyroidism in vivo during late pregnancy blunts GSIS in subsequently isolated and perifused islets at low and high glucose concentrations. It also adversely affects GSIS under conditions of an acute glucose challenge in vivo. In contrast, GSIS is maintained during sustained hyperglycaemia in vivo, suggesting that in vivo factors can rescue GSIS. The ability of insulin to suppress systemic lipid levels during hyperglycaemic clamps was impaired. We therefore suggest that higher circulating lipids may preserve GSIS under conditions of sustained hyperglycaemia in the hyperthyroid pregnancy.

Cellular mechanisms underlying behavioral state-dependent bidirectional modulation of motor cortex output.

PubMed

Schiemann, Julia; Puggioni, Paolo; Dacre, Joshua; Pelko, Miha; Domanski, Aleksander; van Rossum, Mark C W; Duguid, Ian

2015-05-26

Neuronal activity in primary motor cortex (M1) correlates with behavioral state, but the cellular mechanisms underpinning behavioral state-dependent modulation of M1 output remain largely unresolved. Here, we performed in vivo patch-clamp recordings from layer 5B (L5B) pyramidal neurons in awake mice during quiet wakefulness and self-paced, voluntary movement. We show that L5B output neurons display bidirectional (i.e., enhanced or suppressed) firing rate changes during movement, mediated via two opposing subthreshold mechanisms: (1) a global decrease in membrane potential variability that reduced L5B firing rates (L5Bsuppressed neurons), and (2) a coincident noradrenaline-mediated increase in excitatory drive to a subpopulation of L5B neurons (L5Benhanced neurons) that elevated firing rates. Blocking noradrenergic receptors in forelimb M1 abolished the bidirectional modulation of M1 output during movement and selectively impaired contralateral forelimb motor coordination. Together, our results provide a mechanism for how noradrenergic neuromodulation and network-driven input changes bidirectionally modulate M1 output during motor behavior. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Diversity of bilateral synaptic assemblies for binaural computation in midbrain single neurons.

PubMed

He, Na; Kong, Lingzhi; Lin, Tao; Wang, Shaohui; Liu, Xiuping; Qi, Jiyao; Yan, Jun

2017-11-01

Binaural hearing confers many beneficial functions but our understanding of its underlying neural substrates is limited. This study examines the bilateral synaptic assemblies and binaural computation (or integration) in the central nucleus of the inferior colliculus (ICc) of the auditory midbrain, a key convergent center. Using in-vivo whole-cell patch-clamp, the excitatory and inhibitory postsynaptic potentials (EPSPs/IPSPs) of single ICc neurons to contralateral, ipsilateral and bilateral stimulation were recorded. According to the contralateral and ipsilateral EPSP/IPSP, 7 types of bilateral synaptic assemblies were identified. These include EPSP-EPSP (EE), E-IPSP (EI), E-no response (EO), II, IE, IO and complex-mode (CM) neurons. The CM neurons showed frequency- and/or amplitude-dependent EPSPs/IPSPs to contralateral or ipsilateral stimulation. Bilateral stimulation induced EPSPs/IPSPs that could be larger than (facilitation), similar to (ineffectiveness) or smaller than (suppression) those induced by contralateral stimulation. Our findings have allowed our group to characterize novel neural circuitry for binaural computation in the midbrain. Copyright © 2017 Elsevier B.V. All rights reserved.

Somatosensory neuron types identified by high-coverage single-cell RNA-sequencing and functional heterogeneity

PubMed Central

Li, Chang-Lin; Li, Kai-Cheng; Wu, Dan; Chen, Yan; Luo, Hao; Zhao, Jing-Rong; Wang, Sa-Shuang; Sun, Ming-Ming; Lu, Ying-Jin; Zhong, Yan-Qing; Hu, Xu-Ye; Hou, Rui; Zhou, Bei-Bei; Bao, Lan; Xiao, Hua-Sheng; Zhang, Xu

2016-01-01

Sensory neurons are distinguished by distinct signaling networks and receptive characteristics. Thus, sensory neuron types can be defined by linking transcriptome-based neuron typing with the sensory phenotypes. Here we classify somatosensory neurons of the mouse dorsal root ganglion (DRG) by high-coverage single-cell RNA-sequencing (10 950 ± 1 218 genes per neuron) and neuron size-based hierarchical clustering. Moreover, single DRG neurons responding to cutaneous stimuli are recorded using an in vivo whole-cell patch clamp technique and classified by neuron-type genetic markers. Small diameter DRG neurons are classified into one type of low-threshold mechanoreceptor and five types of mechanoheat nociceptors (MHNs). Each of the MHN types is further categorized into two subtypes. Large DRG neurons are categorized into four types, including neurexophilin 1-expressing MHNs and mechanical nociceptors (MNs) expressing BAI1-associated protein 2-like 1 (Baiap2l1). Mechanoreceptors expressing trafficking protein particle complex 3-like and Baiap2l1-marked MNs are subdivided into two subtypes each. These results provide a new system for cataloging somatosensory neurons and their transcriptome databases. PMID:26691752

Bumetanide enhances phenobarbital efficacy in a rat model of hypoxic neonatal seizures.

PubMed

Cleary, Ryan T; Sun, Hongyu; Huynh, Thanhthao; Manning, Simon M; Li, Yijun; Rotenberg, Alexander; Talos, Delia M; Kahle, Kristopher T; Jackson, Michele; Rakhade, Sanjay N; Berry, Gerard T; Berry, Gerard; Jensen, Frances E

2013-01-01

Neonatal seizures can be refractory to conventional anticonvulsants, and this may in part be due to a developmental increase in expression of the neuronal Na(+)-K(+)-2 Cl(-) cotransporter, NKCC1, and consequent paradoxical excitatory actions of GABAA receptors in the perinatal period. The most common cause of neonatal seizures is hypoxic encephalopathy, and here we show in an established model of neonatal hypoxia-induced seizures that the NKCC1 inhibitor, bumetanide, in combination with phenobarbital is significantly more effective than phenobarbital alone. A sensitive mass spectrometry assay revealed that bumetanide concentrations in serum and brain were dose-dependent, and the expression of NKCC1 protein transiently increased in cortex and hippocampus after hypoxic seizures. Importantly, the low doses of phenobarbital and bumetanide used in the study did not increase constitutive apoptosis, alone or in combination. Perforated patch clamp recordings from ex vivo hippocampal slices removed following seizures revealed that phenobarbital and bumetanide largely reversed seizure-induced changes in EGABA. Taken together, these data provide preclinical support for clinical trials of bumetanide in human neonates at risk for hypoxic encephalopathy and seizures.

Bumetanide Enhances Phenobarbital Efficacy in a Rat Model of Hypoxic Neonatal Seizures

PubMed Central

Cleary, Ryan T.; Sun, Hongyu; Huynh, Thanhthao; Manning, Simon M.; Li, Yijun; Rotenberg, Alexander; Talos, Delia M.; Kahle, Kristopher T.; Jackson, Michele; Rakhade, Sanjay N.; Berry, Gerard; Jensen, Frances E.

2013-01-01

Neonatal seizures can be refractory to conventional anticonvulsants, and this may in part be due to a developmental increase in expression of the neuronal Na+-K+-2 Cl− cotransporter, NKCC1, and consequent paradoxical excitatory actions of GABAA receptors in the perinatal period. The most common cause of neonatal seizures is hypoxic encephalopathy, and here we show in an established model of neonatal hypoxia-induced seizures that the NKCC1 inhibitor, bumetanide, in combination with phenobarbital is significantly more effective than phenobarbital alone. A sensitive mass spectrometry assay revealed that bumetanide concentrations in serum and brain were dose-dependent, and the expression of NKCC1 protein transiently increased in cortex and hippocampus after hypoxic seizures. Importantly, the low doses of phenobarbital and bumetanide used in the study did not increase constitutive apoptosis, alone or in combination. Perforated patch clamp recordings from ex vivo hippocampal slices removed following seizures revealed that phenobarbital and bumetanide largely reversed seizure-induced changes in EGABA. Taken together, these data provide preclinical support for clinical trials of bumetanide in human neonates at risk for hypoxic encephalopathy and seizures. PMID:23536761

Chloride channel blockers promote relaxation of TEA-induced contraction in airway smooth muscle.

PubMed

Yim, Peter D; Gallos, George; Perez-Zoghbi, Jose F; Trice, Jacquelyn; Zhang, Yi; Siviski, Matthew; Sonett, Joshua; Emala, Charles W

2013-01-01

Enhanced airway smooth muscle (ASM) contraction is an important component in the pathophysiology of asthma. We have shown that ligand gated chloride channels modulate ASM contractile tone during the maintenance phase of an induced contraction, however the role of chloride flux in depolarization-induced contraction remains incompletely understood. To better understand the role of chloride flux under these conditions, muscle force (human ASM, guinea pig ASM), peripheral small airway luminal area (rat ASM) and airway smooth muscle plasma membrane electrical potentials (human cultured ASM) were measured. We found ex vivo guinea pig airway rings, human ASM strips and small peripheral airways in rat lungs slices relaxed in response to niflumic acid following depolarization-induced contraction induced by K(+) channel blockade with tetraethylammonium chloride (TEA). In isolated human airway smooth muscle cells TEA induce depolarization as measured by a fluorescent indicator or whole cell patch clamp and this depolarization was reversed by niflumic acid. These findings demonstrate that ASM depolarization induced contraction is dependent on chloride channel activity. Targeting of chloride channels may be a novel approach to relax hypercontractile airway smooth muscle in bronchoconstrictive disorders.

Orexin-A and Endocannabinoid Activation of the Descending Antinociceptive Pathway Underlies Altered Pain Perception in Leptin Signaling Deficiency.

PubMed

Cristino, Luigia; Luongo, Livio; Imperatore, Roberta; Boccella, Serena; Becker, Thorsten; Morello, Giovanna; Piscitelli, Fabiana; Busetto, Giuseppe; Maione, Sabatino; Di Marzo, Vincenzo

2016-01-01

Pain perception can become altered in individuals with eating disorders and obesity for reasons that have not been fully elucidated. We show that leptin deficiency in ob/ob mice, or leptin insensitivity in the arcuate nucleus of the hypothalamus in mice with high-fat diet (HFD)-induced obesity, are accompanied by elevated orexin-A (OX-A) levels and orexin receptor-1 (OX1-R)-dependent elevation of the levels of the endocannabinoid, 2-arachidonoylglycerol (2-AG), in the ventrolateral periaqueductal gray (vlPAG). In ob/ob mice, these alterations result in the following: (i) increased excitability of OX1-R-expressing vlPAG output neurons and subsequent increased OFF and decreased ON cell activity in the rostral ventromedial medulla, as assessed by patch clamp and in vivo electrophysiology; and (ii) analgesia, in both healthy and neuropathic mice. In HFD mice, instead, analgesia is only unmasked following leptin receptor antagonism. We propose that OX-A/endocannabinoid cross talk in the descending antinociceptive pathway might partly underlie increased pain thresholds in conditions associated with impaired leptin signaling.

Design and in vivo evaluation of a patch delivery system for insulin based on thiolated polymers.

PubMed

Grabovac, Vjera; Föger, Florian; Bernkop-Schnürch, Andreas

2008-02-04

The aim of this study was to develop and evaluate a novel three-layered oral delivery system for insulin in vivo. The patch system consisted of a mucoadhesive layer, a water insoluble backing layer made of ethylcellulose and an enteric coating made of Eudragit. Drug release studies were performed in media mimicking stomach and intestinal fluids. For in vivo studies patch systems were administered orally to conscious non-diabetic rats. Orally administered insulin in aqueous solution was used as control. After the oral administration of the patch systems a decrease of glucose and increase of insulin blood levels were measured. The mucoadhesive layer, exhibiting a diameter of 2.5mm and a weight of 5mg, comprised polycarbophil-cysteine conjugate (49%), bovine insulin (26%), gluthatione (5%) and mannitol (20%). 74.8+/-4.8% of insulin was released from the delivery system over 6h. Six hours after administration of the patch system mean maximum decrease of blood glucose level of 31.6% of the initial value could be observed. Maximum insulin concentration in blood was 11.3+/-6.2ng/ml and was reached 6h after administration. The relative bioavailability of orally administered patch system versus subcutaneous injection was 2.2%. The results indicate that the patch system provides enhancement of intestinal absorption and thereby offers a promising strategy for peroral peptide delivery.

A PC-PU nanoparticle/PU/decellularized scaffold composite vascular patch: Synergistically optimized overall performance promotes endothelialization.

PubMed

Zhang, Jun; Liu, Cheng; Feng, Fuling; Wang, Dawei; Lu, Shuaishuai; Wei, Guo; Mo, Hong; Qiao, Tong

2017-12-01

Composite vascular patches have gained increasingly attention due to the limited availability of autologous patches (vascular graft materials made from the blood vessels of the same recipient), the lack of growth capability of nonautologous patches (vascular graft materials made from the blood vessels of a different donor) and the disadvantages of synthetic patches. In this study, we report a highly biocompatible phosphatidylcholine-polyurethane nanoparticle/polyurethane/decellularized scaffold composite vascular patch (PCVP). It was fabricated by a facile method - cosedimentation. Its in vitro blood and cell compatibility including hemolysis, plasma recalcification time, coagulation time, platelet adhesion and cytotoxicity was evaluated. The surface modified with phosphatidylcholine-polyurethane (PC-PU) nanoparticles exhibited the improved anticoagulation activity. The in vivo performance of the PCVP was investigated in a mouse model. The nanopatterned surface that resembled the concave-convex structure of the luminal surface of native blood vessels enhanced cell attachment, proliferation, migration and differentiation. The decellularized scaffold had the mechanical property similar to that of the targeted blood vessels, which could withstand in vivo dynamic blood pressure. The overall performance of the PCVP was synergistically optimized by each layer of the multilayer design. The patched artery remained patent and the formation of endothelial tissue - endothelialization was achieved 30days after the in vivo implantation in a mouse model. Copyright © 2017 Elsevier B.V. All rights reserved.

Piezoresistive cantilever force-clamp system

PubMed Central

Park, Sung-Jin; Petzold, Bryan C.; Goodman, Miriam B.; Pruitt, Beth L.

2011-01-01

We present a microelectromechanical device-based tool, namely, a force-clamp system that sets or “clamps” the scaled force and can apply designed loading profiles (e.g., constant, sinusoidal) of a desired magnitude. The system implements a piezoresistive cantilever as a force sensor and the built-in capacitive sensor of a piezoelectric actuator as a displacement sensor, such that sample indentation depth can be directly calculated from the force and displacement signals. A programmable real-time controller operating at 100 kHz feedback calculates the driving voltage of the actuator. The system has two distinct modes: a force-clamp mode that controls the force applied to a sample and a displacement-clamp mode that controls the moving distance of the actuator. We demonstrate that the system has a large dynamic range (sub-nN up to tens of μN force and nm up to tens of μm displacement) in both air and water, and excellent dynamic response (fast response time, <2 ms and large bandwidth, 1 Hz up to 1 kHz). In addition, the system has been specifically designed to be integrated with other instruments such as a microscope with patch-clamp electronics. We demonstrate the capabilities of the system by using it to calibrate the stiffness and sensitivity of an electrostatic actuator and to measure the mechanics of a living, freely moving Caenorhabditis elegans nematode. PMID:21529009

Enhancement of skin permeation of flurbiprofen via its transdermal patches using isopulegol decanoate (ISO-C10) as an absorption enhancer: pharmacokinetic and pharmacodynamic evaluation.

PubMed

Chen, Yang; Quan, Peng; Liu, Xiaochang; Guo, Wenjia; Song, Wenting; Cun, Dongmei; Wang, Zhongyan; Fang, Liang

2015-09-01

The study aimed to prepare a transdermal patch for flurbiprofen using isopulegol decanoate (ISO-C10) as a permeation enhancer, and to evaluate the in-vitro and in-vivo percutaneous permeation of the drug, as well as the pharmacodynamic efficacy of the formulation. The permeation experiments were conducted on rabbit skin, and the pharmacokinetic profiles and synovial fluid drug concentration were measured after in-vivo transdermal administration. A deconvolution approach was employed to analyse the correlation between the in-vitro and in-vivo drug permeation. The anti-inflammatory and analgesic effects were, respectively, assessed using the adjuvant arthritis model and the acetic acid induced pain model. ISO-C10 could increase the in-vitro permeation of flurbiprofen from 46.22 ± 5.65 μg/cm(2) to 101.07 ± 10.85 μg/cm(2) . The in-vivo absorption of the drug was also improved by the enhancer, and a good linear correlation was observed between the in-vitro and in-vivo drug permeation. Meanwhile, the ISO-C10 contained patches increased the drug disposition in synovial fluid and enhanced the pharmacodynamic efficacy of the formulation. ISO-C10 would be a promising permeation enhancer for improving the in-vitro and in-vivo delivery of flurbiprofen from its transdermal patches. © 2015 Royal Pharmaceutical Society.

(-)-Terpinen-4-ol changes intracellular Ca2+ handling and induces pacing disturbance in rat hearts.

PubMed

Gondim, Antonio Nei Santana; Lara, Aline; Santos-Miranda, Artur; Roman-Campos, Danilo; Lauton-Santos, Sandra; Menezes-Filho, José Evaldo Rodrigues; de Vasconcelos, Carla Maria Lins; Conde-Garcia, Eduardo Antonio; Guatimosim, Silvia; Cruz, Jader S

2017-07-15

(-)-Terpinen-4-ol is a naturally occurring plant monoterpene and has been shown to have a plethora of biological activities. The objective of this study was to investigate the effects of (-)-terpinen-4-ol on the rat heart, a key player in the control and maintenance of arterial blood pressure. The effects of (-)-terpinen-4-ol on the rat heart were investigated using isolated left atrium isometric force measurements, in vivo electrocardiogram (ECG) recordings, patch clamp technique, and confocal microscopy. It was observed that (-)-terpinen-4-ol reduced contraction force in an isolated left atrium at millimolar concentrations. Conversely, it induced a positive inotropic effect and extrasystoles at micromolar concentrations, suggesting that (-)-terpinen-4-ol may have arrhythmogenic activity on cardiac tissue. In anaesthetized animals, (-)-terpinen-4-ol also elicited rhythm disturbance, such as supraventricular tachycardia and atrioventricular block. To investigate the cellular mechanism underlying the dual effect of (-)-terpinen-4-ol on heart muscle, experiments were performed on isolated ventricular cardiomyocytes to determine the effect of (-)-terpinen-4-ol on L-type Ca 2+ currents, Ca 2+ sparks, and Ca 2+ transients. The arrhythmogenic activity of (-)-terpinen-4-ol in vitro and in vivo may be explained by its effect on intracellular Ca 2+ handling. Taken together, our data suggest that (-)-terpinen-4-ol has cardiac arrhythmogenic activity. Copyright © 2017 Elsevier B.V. All rights reserved.

Coronary arterial BK channel dysfunction exacerbates ischemia/reperfusion-induced myocardial injury in diabetic mice.

PubMed

Lu, Tong; Jiang, Bin; Wang, Xiao-Li; Lee, Hon-Chi

2016-09-01

The large conductance Ca(2+)-activated K(+) (BK) channels, abundantly expressed in coronary artery smooth muscle cells (SMCs), play a pivotal role in regulating coronary circulation. A large body of evidence indicates that coronary arterial BK channel function is diminished in both type 1 and type 2 diabetes. However, the consequence of coronary BK channel dysfunction in diabetes is not clear. We hypothesized that impaired coronary BK channel function exacerbates myocardial ischemia/reperfusion (I/R) injury in streptozotocin-induced diabetic mice. Combining patch-clamp techniques and cellular biological approaches, we found that diabetes facilitated the colocalization of angiotensin II (Ang II) type 1 receptors and BK channel α-subunits (BK-α), but not BK channel β1-subunits (BK-β1), in the caveolae of coronary SMCs. This caveolar compartmentation in vascular SMCs not only enhanced Ang II-mediated inhibition of BK-α but also produced a physical disassociation between BK-α and BK-β1, leading to increased infarct size in diabetic hearts. Most importantly, genetic ablation of caveolae integrity or pharmacological activation of coronary BK channels protected the cardiac function of diabetic mice from experimental I/R injury in both in vivo and ex vivo preparations. Our results demonstrate a vascular ionic mechanism underlying the poor outcome of myocardial injury in diabetes. Hence, activation of coronary BK channels may serve as a therapeutic target for cardiovascular complications of diabetes.

Rapid multislice T1 mapping of mouse myocardium: Application to quantification of manganese uptake in α-Dystrobrevin knockout mice.

PubMed

Jiang, Kai; Li, Wen; Li, Wei; Jiao, Sen; Castel, Laurie; Van Wagoner, David R; Yu, Xin

2015-11-01

The aim of this study was to develop a rapid, multislice cardiac T1 mapping method in mice and to apply the method to quantify manganese (Mn(2+)) uptake in a mouse model with altered Ca(2+) channel activity. An electrocardiography-triggered multislice saturation-recovery Look-Locker method was developed and validated both in vitro and in vivo. A two-dose study was performed to investigate the kinetics of T1 shortening, Mn(2+) relaxivity in myocardium, and the impact of Mn(2+) on cardiac function. The sensitivity of Mn(2+)-enhanced MRI in detecting subtle changes in altered Ca(2+) channel activity was evaluated in a mouse model with α-dystrobrevin knockout. Validation studies showed strong agreement between the current method and an established method. High Mn(2+) dose led to significantly accelerated T1 shortening. Heart rate decreased during Mn(2+) infusion, while ejection ratio increased slightly at the end of imaging protocol. No statistical difference in cardiac function was detected between the two dose groups. Mice with α-dystrobrevin knockout showed enhanced Mn(2+) uptake in vivo. In vitro patch-clamp study showed increased Ca(2+) channel activity. The saturation recovery method provides rapid T1 mapping in mouse hearts, which allowed sensitive detection of subtle changes in Mn(2+) uptake in α-dystrobrevin knockout mice. © 2014 Wiley Periodicals, Inc.

Intracortical Microstimulation (ICMS) Activates Motor Cortex Layer 5 Pyramidal Neurons Mainly Transsynaptically.

PubMed

Hussin, Ahmed T; Boychuk, Jeffery A; Brown, Andrew R; Pittman, Quentin J; Teskey, G Campbell

2015-01-01

Intracortical microstimulation (ICMS) is a technique used for a number of purposes including the derivation of cortical movement representations (motor maps). Its application can activate the output layer 5 of motor cortex and can result in the elicitation of body movements depending upon the stimulus parameters used. The extent to which pyramidal tract projection neurons of the motor cortex are activated transsynaptically or directly by ICMS remains an open question. Given this uncertainty in the mode of activation, we used a preparation that combined patch clamp whole-cell recordings from single layer 5 pyramidal neurons and extracellular ICMS in slices of motor cortex as well as a standard in vivo mapping technique to ask how ICMS activated motor cortex pyramidal neurons. We measured changes in synaptic spike threshold and spiking rate to ICMS in vitro and movement threshold in vivo in the presence or absence of specific pharmacological blockers of glutamatergic (AMPA, NMDA and Kainate) receptors and GABAA receptors. With major excitatory and inhibitory synaptic transmission blocked (with DNQX, APV and bicuculline methiodide), we observed a significant increase in the ICMS current intensity required to elicit a movement in vivo as well as to the first spike and an 85% reduction in spiking responses in vitro. Subsets of neurons were still responsive after the synaptic block, especially at higher current intensities, suggesting a modest direct activation. Taken together our data indicate a mainly synaptic mode of activation to ICMS in layer 5 of rat motor cortex. Copyright © 2015 Elsevier Inc. All rights reserved.

Quantifying Electrical Interactions between Cardiomyocytes and Other Cells in Micropatterned Cell Pairs

PubMed Central

Nguyen, Hung; Badie, Nima; McSpadden, Luke; Pedrotty, Dawn; Bursac, Nenad

2014-01-01

Micropatterning is a powerful technique to control cell shape and position on a culture substrate. In this chapter, we describe the method to reproducibly create large numbers of micropatterned heterotypic cell pairs with defined size, shape, and length of cell–cell contact. These cell pairs can be utilized in patch clamp recordings to quantify electrical interactions between cardiomyocytes and non-cardiomyocytes. PMID:25070342

Rod electrical coupling is controlled by a circadian clock and dopamine in mouse retina

PubMed Central

Jin, Nan Ge; Chuang, Alice Z; Masson, Philippe J; Ribelayga, Christophe P

2015-01-01

Key points Rod photoreceptors play a key role in vision in dim light; in the mammalian retina, although rods are anatomically connected or coupled by gap junctions, a type of electrical synapse, the functional importance and regulation of rod coupling has remained elusive. We have developed a new technique in the mouse: perforated patch-clamp recording of rod inner segments in isolated intact retinae maintained by superfusion. We find that rod electrical coupling is controlled by a circadian clock and dopamine, and is weak during the day and stronger at night. The results also indicate that the signal-to-noise ratio for a dim light response is increased at night because of coupling. Our observations will provide a framework for understanding the daily variations in human vision as well as the basis of specific retinal malfunctions. Abstract Rod single-photon responses are critical for vision in dim light. Electrical coupling via gap junction channels shapes the light response properties of vertebrate photoreceptors, but the regulation of rod coupling and its impact on the single-photon response have remained unclear. To directly address these questions, we developed a perforated patch-clamp recording technique and recorded from single rod inner segments in isolated intact neural mouse retinae, maintained by superfusion. Experiments were conducted at different times of the day or under constant environmental conditions, at different times across the circadian cycle. We show that rod electrical coupling is regulated by a circadian clock and dopamine, so that coupling is weak during the day and strong at night. Altogether, patch-clamp recordings of single-photon responses in mouse rods, tracer coupling, receptive field measurements and pharmacological manipulations of gap junction and dopamine receptor activity provide compelling evidence that rod coupling is modulated in a circadian manner. These data are consistent with computer modelling. At night, single-photon responses are smaller due to coupling, but the signal-to-noise ratio for a dim (multiphoton) light response is increased at night because of signal averaging between coupled rods. PMID:25616058

Antisense oligodeoxynucleotide inhibition of a swelling-activated cation channel in osteoblast-like osteosarcoma cells

NASA Technical Reports Server (NTRS)

Duncan, R. L.; Kizer, N.; Barry, E. L.; Friedman, P. A.; Hruska, K. A.

1996-01-01

By patch-clamp analysis, we have shown that chronic, intermittent mechanical strain (CMS) increases the activity of stretch-activated cation channels of osteoblast-like UMR-106.01 cells. CMS also produces a swelling-activated whole-cell conductance (Gm) regulated by varying strain levels. We questioned whether the swelling-activated conductance was produced by stretch-activated cation channel activity. We have identified a gene involved in the increase in conductance by using antisense oligodeoxynucleotides (ODN) derived from the alpha 1-subunit genes of calcium channels found in UMR-106.01 cells (alpha1S, alpha1C, and alpha1D). We demonstrate that alpha 1C antisense ODNs abolish the increase in Gm in response to hypotonic swelling following CMS. Antisense ODNs to alpha1S and alpha1D, sense ODNs to alpha1C, and sham permeabilization had no effect on the conductance increase. In addition, during cell-attached patch-clamp studies, antisense ODNs to alpha1c completely blocked the swelling-activated and stretch-activated nonselective cation channel response to strain. Antisense ODNs to alpha1S treatment produced no effect on either swelling-activated or stretch-activated cation channel activity. There were differences in the stretch-activated and swelling-activated cation channel activity, but whether they represent different channels could not be determined from our data. Our data indicate that the alpha1C gene product is involved in the Gm and the activation of the swelling-activated cation channels induced by CMS. The possibility that swelling-activated cation channel genes are members of the calcium channel superfamily exists, but if alpha1c is not the swelling-activated cation channel itself, then its expression is required for induction of swelling-activated cation channel activity by CMS.

Plasma Membrane Permeabilization by 60- and 600-ns Electric Pulses Is Determined by the Absorbed Dose

PubMed Central

Ibey, Bennett L.; Xiao, Shu; Schoenbach, Karl H.; Murphy, Michael R.; Pakhomov, Andrei G.

2008-01-01

We explored how the effect of plasma membrane permeabilization by nanosecond-duration electric pulses (nsEP) depends on the physical characteristics of exposure. The resting membrane resistance (Rm) and membrane potential (MP) were measured in cultured GH3 and CHO cells by conventional whole-cell patch-clamp technique. Intact cells were exposed to a single nsEP (60 or 600 ns duration, 0-22 kV/cm), followed by patch-clamp measurements after a 2-3 min delay. Consistent with earlier findings, nsEP caused long-lasting Rm decrease, accompanied by the loss of MP. The threshold for these effects was about 6 kV/cm for 60 ns pulses, and about 1 kV/cm for 600 ns pulses. Further analysis established that it was neither pulse duration nor the E-field amplitude per se, but the absorbed dose that determined the magnitude of the biological effect. In other words, exposure to nsEP at either pulse duration caused equal effects if the absorbed doses were equal. The threshold absorbed dose to produce plasma membrane effects in either GH3 or CHO cells at either pulse duration was found to be at or below 10 mJ/g. Despite being determined by the dose, the nsEP effect clearly is not thermal, as the maximum heating at the threshold dose is less than 0.01 °C. The use of the absorbed dose as a universal exposure metric may help to compare and quantify nsEP sensitivity of different cell types and of cells in different physiological conditions. The absorbed dose may also prove to be a more useful metric than the incident E-field in determining safety limits for high peak, lowaverage power EMF emissions. PMID:18839412

Role of gap junctions in the contractile response to agonists in the mesenteric artery of spontaneously hypertensive rats.

PubMed

Ma, Ke-Tao; Li, Xin-Zhi; Li, Li; Jiang, Xue-Wei; Chen, Xin-Yan; Liu, Wei-Dong; Zhao, Lei; Zhang, Zhong-Shuang; Si, Jun-Qiang

2014-02-01

To investigate the effects of hypertension on the changes in gap junctions between vascular smooth muscle cells (VSMCs) in the mesenteric artery (MA) of spontaneously hypertensive rats (SHRs). Whole-cell patch clamp, pressure myography, real-time quantitative reverse transcription PCR (qRT-PCR), western blot analysis and transmission electron microscopy were used to examine the differences in expression and function of the gap junction between MA VSMCs of SHR and control normotensive Wistar-Kyoto (WKY) rats. (1) Whole-cell patch clamp measurements showed that the membrane capacitance and conductance of in-situ MA VSMCs of SHR were significantly greater than those of WKY rats (P<0.05), suggesting enhanced gap junction coupling between MA VSMCs of SHR. (2) The administration of phenylephrine (PE) and KCl (an endothelium-independent vasoconstrictor) initiated more pronounced vasoconstriction in SHR versus WKY rats (P<0.05). Furthermore, 2-APB (a gap junction inhibitor) attenuated PE- and KCl-induced vasoconstriction, and the inhibitory effects of 2-APB were significantly greater in SHR (P<0.05). (3) The expression of connexin 45 (Cx45) mRNA and protein in the MA was greater in SHR versus WKY rats (P<0.05). The level of phosphorylated Cx43 was significantly higher in SHR versus WKY rats (P<0.05), although the expression of total Cx43 mRNA and protein in the MA was equivalent between SHR and WKY rats. Electron microscopy revealed that the gap junctions were significantly larger in SHR versus WKY rats. Increases in the expression of Cx45 and phosphorylation of Cx43 may contribute to the enhancement of communication across gap junctions between MA VSMCs of SHR, which may increase the contractile response to agonists.

A unique alkaline pH-regulated and fatty acid-activated tandem pore domain potassium channel (K2P) from a marine sponge

PubMed Central

Wells, Gregory D.; Tang, Qiong-Yao; Heler, Robert; Tompkins-MacDonald, Gabrielle J.; Pritchard, Erica N.; Leys, Sally P.; Logothetis, Diomedes E.; Boland, Linda M.

2012-01-01

SUMMARY A cDNA encoding a potassium channel of the two-pore domain family (K2P, KCNK) of leak channels was cloned from the marine sponge Amphimedon queenslandica. Phylogenetic analysis indicated that AquK2P cannot be placed into any of the established functional groups of mammalian K2P channels. We used the Xenopus oocyte expression system, a two-electrode voltage clamp and inside-out patch clamp electrophysiology to determine the physiological properties of AquK2P. In whole cells, non-inactivating, voltage-independent, outwardly rectifying K+ currents were generated by external application of micromolar concentrations of arachidonic acid (AA; EC50 ∼30 μmol l–1), when applied in an alkaline solution (≥pH 8.0). Prior activation of channels facilitated the pH-regulated, AA-dependent activation of AquK2P but external pH changes alone did not activate the channels. Unlike certain mammalian fatty-acid-activated K2P channels, the sponge K2P channel was not activated by temperature and was insensitive to osmotically induced membrane distortion. In inside-out patch recordings, alkalinization of the internal pH (pKa 8.18) activated the AquK2P channels independently of AA and also facilitated activation by internally applied AA. The gating of the sponge K2P channel suggests that voltage-independent outward rectification and sensitivity to pH and AA are ancient and fundamental properties of animal K2P channels. In addition, the membrane potential of some poriferan cells may be dynamically regulated by pH and AA. PMID:22723483

A unique alkaline pH-regulated and fatty acid-activated tandem pore domain potassium channel (K₂P) from a marine sponge.

PubMed

Wells, Gregory D; Tang, Qiong-Yao; Heler, Robert; Tompkins-MacDonald, Gabrielle J; Pritchard, Erica N; Leys, Sally P; Logothetis, Diomedes E; Boland, Linda M

2012-07-15

A cDNA encoding a potassium channel of the two-pore domain family (K(2P), KCNK) of leak channels was cloned from the marine sponge Amphimedon queenslandica. Phylogenetic analysis indicated that AquK(2P) cannot be placed into any of the established functional groups of mammalian K(2P) channels. We used the Xenopus oocyte expression system, a two-electrode voltage clamp and inside-out patch clamp electrophysiology to determine the physiological properties of AquK(2P). In whole cells, non-inactivating, voltage-independent, outwardly rectifying K(+) currents were generated by external application of micromolar concentrations of arachidonic acid (AA; EC(50) ∼30 μmol l(-1)), when applied in an alkaline solution (≥pH 8.0). Prior activation of channels facilitated the pH-regulated, AA-dependent activation of AquK(2P) but external pH changes alone did not activate the channels. Unlike certain mammalian fatty-acid-activated K(2P) channels, the sponge K(2P) channel was not activated by temperature and was insensitive to osmotically induced membrane distortion. In inside-out patch recordings, alkalinization of the internal pH (pK(a) 8.18) activated the AquK(2P) channels independently of AA and also facilitated activation by internally applied AA. The gating of the sponge K(2P) channel suggests that voltage-independent outward rectification and sensitivity to pH and AA are ancient and fundamental properties of animal K(2P) channels. In addition, the membrane potential of some poriferan cells may be dynamically regulated by pH and AA.

Expression and permeation properties of the K(+) channel Kir7.1 in the retinal pigment epithelium.

PubMed

Shimura, M; Yuan, Y; Chang, J T; Zhang, S; Campochiaro, P A; Zack, D J; Hughes, B A

2001-03-01

Bovine Kir7.1 clones were obtained from a retinal pigment epithelium (RPE)-subtracted cDNA library. Human RPE cDNA library screening resulted in clones encoding full-length human Kir7.1. Northern blot analysis indicated that bovine Kir7.1 is highly expressed in the RPE. Human Kir7.1 channels were expressed in Xenopus oocytes and studied using the two-electrode voltage-clamp technique. The macroscopic Kir7.1 conductance exhibited mild inward rectification and an inverse dependence on extracellular K+ concentration ([K+]o). The selectivity sequence based on permeability ratios was K+ (1.0) approximately Rb+ (0.89) > Cs+ (0.013) > Na+ (0.003) approximately Li+ (0.001) and the sequence based on conductance ratios was Rb+ (9.5) > K+ (1.0) > Na+ (0.458) > Cs+ (0.331) > Li+ (0.139). Non-stationary noise analysis of Rb+ currents in cell-attached patches yielded a unitary conductance for Kir7.1 of approximately 2 pS. In whole-cell recordings from freshly isolated bovine RPE cells, the predominant current was a mild inwardly rectifying K+ current that exhibited an inverse dependence of conductance on [K+]o. The selectivity sequence based on permeability ratios was K+ (1.0) approximately Rb+ (0.89) > Cs+ (0.021) > Na+ (0.003) approximately Li+ (0.002) and the sequence based on conductance ratios was Rb+ (8.9) > K+ (1.0) > Na+ (0.59) > Cs+ (0.23) > Li+ (0.08). In cell-attached recordings with Rb+ in the pipette, inwardly rectifying currents were observed in nine of 12 patches of RPE apical membrane but in only one of 13 basolateral membrane patches. Non-stationary noise analysis of Rb+ currents in cell-attached apical membrane patches yielded a unitary conductance for RPE Kir of approximately 2 pS. On the basis of this molecular and electrophysiological evidence, we conclude that Kir7.1 channel subunits comprise the K+ conductance of the RPE apical membrane.

In Vivo Evaluation of Wearable Head Impact Sensors.

PubMed

Wu, Lyndia C; Nangia, Vaibhav; Bui, Kevin; Hammoor, Bradley; Kurt, Mehmet; Hernandez, Fidel; Kuo, Calvin; Camarillo, David B

2016-04-01

Inertial sensors are commonly used to measure human head motion. Some sensors have been tested with dummy or cadaver experiments with mixed results, and methods to evaluate sensors in vivo are lacking. Here we present an in vivo method using high speed video to test teeth-mounted (mouthguard), soft tissue-mounted (skin patch), and headgear-mounted (skull cap) sensors during 6-13 g sagittal soccer head impacts. Sensor coupling to the skull was quantified by displacement from an ear-canal reference. Mouthguard displacements were within video measurement error (<1 mm), while the skin patch and skull cap displaced up to 4 and 13 mm from the ear-canal reference, respectively. We used the mouthguard, which had the least displacement from skull, as the reference to assess 6-degree-of-freedom skin patch and skull cap measurements. Linear and rotational acceleration magnitudes were over-predicted by both the skin patch (with 120% NRMS error for a(mag), 290% for α(mag)) and the skull cap (320% NRMS error for a(mag), 500% for α(mag)). Such over-predictions were largely due to out-of-plane motion. To model sensor error, we found that in-plane skin patch linear acceleration in the anterior-posterior direction could be modeled by an underdamped viscoelastic system. In summary, the mouthguard showed tighter skull coupling than the other sensor mounting approaches. Furthermore, the in vivo methods presented are valuable for investigating skull acceleration sensor technologies.

In vivo results of hydrogel composite pericardial substitutes.

PubMed

Blue, M A; Guilbeau, E J; Brandon, T A; Walker, A S; Bjotvedt, G; Fisk, R L

1991-01-01

In this study, two improved pericardial patches were developed and evaluated for their efficacy as pericardial substitutes. The patches are composites consisting of a hydrogel (PHEMA) that coats an underlying mesh (either ETFE or PET). Studies were conducted using subcutaneous implants in rats and pericardial patch implants in greyhound dogs. Adhesions between the substitute and pericardium and the epicardium were minimal. The ETFE composite patch caused an unacceptable epicardial reaction. The PET patch results were encouraging; the epicardium was largely unaffected by the patch.

A pliable electroporation patch (ep-Patch) for efficient delivery of nucleic acid molecules into animal tissues with irregular surface shapes.

PubMed

Wei, Zewen; Huang, Yuanyu; Zhao, Deyao; Hu, Zhiyuan; Li, Zhihong; Liang, Zicai

2015-01-05

Delivery of nucleic acids into animal tissues by electroporation is an appealing approach for various types of gene therapy, but efficiency of existing methodsis not satisfactory. Here we present the validation of novel electroporation patch (ep-Patch) for efficient delivery of DNA and siRNA into mouse tissues. Using micromachining technology, closely spaced gold electrodes were made on the pliable parylene substrate to form a patch-like electroporation metrics. It enabled large coverage of the target tissues and close surface contact between the tissues and electrodes, thus providing a uniform electric field to deliver nucleic acids into tissues, even beneath intact skin. Using this ep-Patch for efficiently delivery of both DNA and siRNA, non-invasive electroporation of healthy mouse muscle tissue was successfully achieved. Delivery of these nucleic acids was performed to intact tumors with satisfactory results. Silencing of tumor genes using the ep-Patch was also demonstrated on mice. This pliable electroporation patch method constitutes a novel way of in vivo delivery of siRNA and DNA to certain tissues or organs to circumvent the disadvantages of existing methodologies for in vivo delivery of nucleic acid molecules.

Superstatistics analysis of the ion current distribution function: Met3PbCl influence study.

PubMed

Miśkiewicz, Janusz; Trela, Zenon; Przestalski, Stanisław; Karcz, Waldemar

2010-09-01

A novel analysis of ion current time series is proposed. It is shown that higher (second, third and fourth) statistical moments of the ion current probability distribution function (PDF) can yield new information about ion channel properties. The method is illustrated on a two-state model where the PDF of the compound states are given by normal distributions. The proposed method was applied to the analysis of the SV cation channels of vacuolar membrane of Beta vulgaris and the influence of trimethyllead chloride (Met(3)PbCl) on the ion current probability distribution. Ion currents were measured by patch-clamp technique. It was shown that Met(3)PbCl influences the variance of the open-state ion current but does not alter the PDF of the closed-state ion current. Incorporation of higher statistical moments into the standard investigation of ion channel properties is proposed.

Selective block of late Na+ current by local anaesthetics in rat large sensory neurones

PubMed Central

Baker, Mark D

2000-01-01

The actions of lignocaine and benzocaine on transient and late Na+ current generated by large diameter (⩾50 μm) adult rat dorsal root ganglion neurones, were studied using patch-clamp techniques.Both drugs blocked whole-cell late Na+ current in a concentration-dependent manner. At 200 ms following the onset of a clamp step from −110 to −40 mV, the apparent K for block of late Na+ current by lignocaine was 57.8±15 μM (mean±s.e.mean, n=4). The value for benzocaine was 24.9±3.3 μM, (mean±s.e.mean, n=3).The effect of lignocaine on transient current, in randomly selected neurones, appeared variable (n=8, half-block from ∼50 to 400 μM). Half-block by benzocaine was not attained, but both whole-cell (n=11) and patch data suggested a high apparent K,>250 μM. Transient current always remained after late current was blocked.The voltage-dependence of residual late current steady-state inactivation was not shifted by 20 μM benzocaine (n=3), whereas 200 μM benzocaine shifted the voltage-dependence of transient current steady-state inactivation by −18.7±5.9 mV (mean±s.e.mean, n=4).In current-clamp, benzocaine (250 μM) could block subthreshold, voltage-dependent inward current, increasing the threshold for eliciting action potentials, without preventing their generation (n=2).Block of late Na+ current by systemic local anaesthetic may play a part in preventing ectopic impulse generation in sensory neurones. PMID:10780966

Inventing a co-axial atomic resolution patch clamp to study a single resonating protein complex and ultra-low power communication deep inside a living neuron cell.

PubMed

Ghosh, Subrata; Sahu, Satyajit; Agrawal, Lokesh; Shiga, Takashi; Bandyopadhyay, Anirban

2016-12-01

To read the signals of single molecules in vitro on a surface, or inside a living cell or organ, we introduce a coaxial atom tip (coat) and a coaxial atomic patch clamp (COAPAP). The metal-insulator-metal cavity of these probes extends to the atomic scale (0.1[Formula: see text]nm), it eliminates the cellular or environmental noise with a S/N ratio 10 5 . Five ac signals are simultaneously applied during a measurement by COAT and COAPAP to shield a true signal under environmental noise in five unique ways. The electromagnetic drive in the triaxial atomic tips is specifically designed to sense anharmonic vibrational and transmission signals for any system between 0.1[Formula: see text]nm and 50[Formula: see text]nm where the smallest nanopatch clamp cannot reach. COAT and COAPAP reliably pick up the atomic scale vibrations under the extreme noise of a living cell. Each protein's distinct electromagnetic, mechanical, electrical and ionic vibrational signature studied in vitro in a protected environment is found to match with the ones studied inside a live neuron. Thus, we could confirm that by using our probe blindly we could hold on to a single molecule or its complex in the invisible domain of a living cell. Our decade long investigations on perfecting the tools to measure bio-resonance of all forms and simultaneously in all frequency domains are summarized. It shows that the ratio of emission to absorption resonance frequencies of a biomaterial is around [Formula: see text], only a few in the entire em spectrum are active that regulates all other resonances, like mechanical, ionic, etc.

Novel regulatory mechanism in human urinary bladder: central role of transient receptor potential melastatin 4 channels in detrusor smooth muscle function

PubMed Central

Hristov, Kiril L.; Smith, Amy C.; Parajuli, Shankar P.; Malysz, John; Rovner, Eric S.

2016-01-01

Transient receptor potential melastatin 4 (TRPM4) channels are Ca2+-activated nonselective cation channels that have been recently identified as regulators of detrusor smooth muscle (DSM) function in rodents. However, their expression and function in human DSM remain unexplored. We provide insights into the functional role of TRPM4 channels in human DSM under physiological conditions. We used a multidisciplinary experimental approach, including RT-PCR, Western blotting, immunohistochemistry and immunocytochemistry, patch-clamp electrophysiology, and functional studies of DSM contractility. DSM samples were obtained from patients without preoperative overactive bladder symptoms. RT-PCR detected mRNA transcripts for TRPM4 channels in human DSM whole tissue and freshly isolated single cells. Western blotting and immunohistochemistry with confocal microscopy revealed TRPM4 protein expression in human DSM. Immunocytochemistry further detected TRPM4 protein expression in DSM single cells. Patch-clamp experiments showed that 9-phenanthrol, a selective TRPM4 channel inhibitor, significantly decreased the transient inward cation currents and voltage step-induced whole cell currents in freshly isolated human DSM cells. In current-clamp mode, 9-phenanthrol hyperpolarized the human DSM cell membrane potential. Furthermore, 9-phenanthrol attenuated the spontaneous phasic, carbachol-induced and nerve-evoked contractions in human DSM isolated strips. Significant species-related differences in TRPM4 channel activity between human, rat, and guinea pig DSM were revealed, suggesting a more prominent physiological role for the TRPM4 channel in the regulation of DSM function in humans than in rodents. In conclusion, TRPM4 channels regulate human DSM excitability and contractility and are critical determinants of human urinary bladder function. Thus, TRPM4 channels could represent promising novel targets for the pharmacological or genetic control of overactive bladder. PMID:26791488

Direct block of native and cloned (Kir2.1) inward rectifier K+ channels by chloroethylclonidine

PubMed Central

Barrett-Jolley, R; Dart, C; Standen, N B

1999-01-01

We have investigated the inhibition of inwardly rectifying potassium channels by the α-adrenergic agonist/antagonist chloroethylclonidine (CEC). We used two preparations; two-electrode voltage-clamp of rat isolated flexor digitorum brevis muscle and whole-cell patch-clamp of cell lines transfected with Kir2.1 (IRK1).In skeletal muscle and at a membrane potential of −50 mV, chloroethylclonidine (CEC), an agonist at α2-adrenergic receptors and an antagonist at α1x-receptors, was found to inhibit the inward rectifier current with a Ki of 30 μM.The inhibition of skeletal muscle inward rectifier current by CEC was not mimicked by clonidine, adrenaline or noradrenaline and was not sensitive to high concentrations of α1-(prazosin) or α2-(rauwolscine) antagonists.The degree of current inhibition by CEC was found to vary with the membrane potential (approximately 70% block at −50 mV c.f. ∼10% block at −190 mV). The kinetics of this voltage dependence were further investigated using recombinant inward rectifier K+ channels (Kir2.1) expressed in the MEL cell line. Using a two pulse protocol, we calculated the time constant for block to be ∼8 s at 0 mV, and the rate of unblock was described by the relationship τ=exp((Vm+149)/22) s.This block was effective when CEC was applied to either the inside or the outside of patch clamped cells, but ineffective when a polyamine binding site (aspartate 172) was mutated to asparagine.The data suggest that the clonidine-like imidazoline compound, CEC, inhibits inward rectifier K+ channels independently of α-receptors by directly blocking the channel pore, possibly at an intracellular polyamine binding site. PMID:10516659

Combinatorial materials research applied to the development of new surface coatings VII: An automated system for adhesion testing

NASA Astrophysics Data System (ADS)

Chisholm, Bret J.; Webster, Dean C.; Bennett, James C.; Berry, Missy; Christianson, David; Kim, Jongsoo; Mayo, Bret; Gubbins, Nathan

2007-07-01

An automated, high-throughput adhesion workflow that enables pseudobarnacle adhesion and coating/substrate adhesion to be measured on coating patches arranged in an array format on 4×8in.2 panels was developed. The adhesion workflow consists of the following process steps: (1) application of an adhesive to the coating array; (2) insertion of panels into a clamping device; (3) insertion of aluminum studs into the clamping device and onto coating surfaces, aligned with the adhesive; (4) curing of the adhesive; and (5) automated removal of the aluminum studs. Validation experiments comparing data generated using the automated, high-throughput workflow to data obtained using conventional, manual methods showed that the automated system allows for accurate ranking of relative coating adhesion performance.

Effect of Amphotericin B antibiotic on the properties of model lipid membrane

NASA Astrophysics Data System (ADS)

Kiryakova, S.; Dencheva-Zarkova, M.; Genova, J.

2014-12-01

Model membranes formed from natural and synthetic lipids are an interesting object for scientific investigations due to their similarity to biological cell membrane and their simple structure with controlled composition and properties. Amphotericin B is an important polyene antifungal antibiotic, used for treatment of systemic fungal infections. It is known from the literature that the studied antibiotic has a substantial effect on the transmembrane ionic channel structures. When applied to the lipid membranes it has the tendency to create pores and in this way to affect the structure and the properties of the membrane lipid bilayer. In this work the thermally induced shape fluctuations of giant quasi-spherical liposomes have been used to study the influence of polyene antibiotic amphotericin B on the elastic properties of model lipid membranes. It have been shown experimentally that the presence of 3 mol % of AmB in the lipid membrane reduces the bending elasticity of the lipid membrane for both studied cases: pure SOPC membrane and mixed SOPC-Cholesterol membrane. Interaction of the amphotericin B with bilayer lipid membranes containing channels have been studied in this work. Model membranes were self-assembled using the patch-clamp and tip-dip patch clamp technique. We have found that amphotericin B is an ionophore and reduces the resistance of the lipid bilayer.

Synergistic Effect of Light and Fusicoccin on Stomatal Opening 1

PubMed Central

Assmann, Sarah M.; Schwartz, Amnon

1992-01-01

Upon incubation of epidermal peels of Commelina communis in 1 millimolar KCl, a synergistic effect of light and low fusicoccin (FC) concentrations on stomatal opening is observed. In 1 millimolar KCl, stomata remain closed even in the light. However, addition of 0.1 micromolar FC results in opening up to 12 micrometers. The same FC concentration stimulates less than 5 micrometers of opening in darkness. The synergistic effect (a) decreases with increasing FC or KCl concentrations; (b) is dark-reversible; (c) like stomatal opening in high KCl concentrations (120 millimolar) is partially inhibited by the K+ channel blocker, tetraethyl-ammonium+ (20 millimolar). In whole-cell patch-clamp experiments with guard cell protoplasts of Vicia faba, FC (1 or 10 micromolar) stimulates an increase in outward current that is essentially voltage independent between - 100 and +60 millivolts, and occurs even when the membrane potential is held at a voltage (−60 millivolts) at which K+ channels are inactivated. These results are indicative of FC activation of a H+ pump. FC effects on the magnitude of inward and outward K+ currents are not observed. Epidermal peel and patch clamp data are both consistent with the hypothesis that the plasma membrane H+ ATPase of guard cells is a primary locus for the FC effect on stomatal apertures. PMID:16668799

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ullah, Ghanim; Demuro, Angelo; Parker, Ian

Amyloid beta (Aβ) oligomers associated with Alzheimer’s disease (AD) form Ca 2+-permeable plasma membrane pores, leading to a disruption of the otherwise well-controlled intracellular calcium (Ca 2+) homeostasis. The resultant up-regulation of intracellular Ca 2+ concentration has detrimental implications for memory formation and cell survival. The gating kinetics and Ca 2+ permeability of Aβ pores are not well understood. We have used computational modeling in conjunction with the ability of optical patch-clamping for massively parallel imaging of Ca 2+ flux through thousands of pores in the cell membrane of Xenopus oocytes to elucidate the kinetic properties of Aβ pores. Themore » fluorescence time-series data from individual pores were idealized and used to develop data-driven Markov chain models for the kinetics of the Aβ pore at different stages of its evolution. Our study provides the first demonstration of developing Markov chain models for ion channel gating that are driven by optical-patch clamp data with the advantage of experiments being performed under close to physiological conditions. As a result, we demonstrate the up-regulation of gating of various Ca 2+ release channels due to Aβ pores and show that the extent and spatial range of such up-regulation increases as Aβ pores with low open probability and Ca 2+ permeability transition into those with high open probability and Ca 2+ permeability.« less

Analysis of single ion channel data incorporating time-interval omission and sampling

PubMed Central

The, Yu-Kai; Timmer, Jens

2005-01-01

Hidden Markov models are widely used to describe single channel currents from patch-clamp experiments. The inevitable anti-aliasing filter limits the time resolution of the measurements and therefore the standard hidden Markov model is not adequate anymore. The notion of time-interval omission has been introduced where brief events are not detected. The developed, exact solutions to this problem do not take into account that the measured intervals are limited by the sampling time. In this case the dead-time that specifies the minimal detectable interval length is not defined unambiguously. We show that a wrong choice of the dead-time leads to considerably biased estimates and present the appropriate equations to describe sampled data. PMID:16849220

Compact Microscope Imaging System With Intelligent Controls Improved

NASA Technical Reports Server (NTRS)

McDowell, Mark

2004-01-01

The Compact Microscope Imaging System (CMIS) with intelligent controls is a diagnostic microscope analysis tool with intelligent controls for use in space, industrial, medical, and security applications. This compact miniature microscope, which can perform tasks usually reserved for conventional microscopes, has unique advantages in the fields of microscopy, biomedical research, inline process inspection, and space science. Its unique approach integrates a machine vision technique with an instrumentation and control technique that provides intelligence via the use of adaptive neural networks. The CMIS system was developed at the NASA Glenn Research Center specifically for interface detection used for colloid hard spheres experiments; biological cell detection for patch clamping, cell movement, and tracking; and detection of anode and cathode defects for laboratory samples using microscope technology.

Analysis of whole-cell currents by patch clamp of guinea-pig myenteric neurones in intact ganglia

PubMed Central

Rugiero, François; Gola, Maurice; Kunze, Wolf A A; Reynaud, Jean-Claude; Furness, John B; Clerc, Nadine

2002-01-01

Whole-cell patch-clamp recordings taken from guinea-pig duodenal myenteric neurones within intact ganglia were used to determine the properties of S and AH neurones. Major currents that determine the states of AH neurones were identified and quantified. S neurones had resting potentials of −47 ± 6 mV and input resistances (Rin) of 713 ± 49 MΩ at voltages ranging from −90 to −40 mV. At more negative levels, activation of a time-independent, caesium-sensitive, inward-rectifier current (IKir) decreased Rin to 103 ± 10 MΩ. AH neurones had resting potentials of −57 ± 4 mV and Rin was 502 ± 27 MΩ. Rin fell to 194 ± 16 MΩ upon hyperpolarization. This decrease was attributable mainly to the activation of a cationic h current, Ih, and to IKir. Resting potential and Rin exhibited a low sensitivity to changes in [K+]o in both AH and S neurones. This indicates that both cells have a low background K+ permeability. The cationic current, Ih, contributed about 20 % to the resting conductance of AH neurones. It had a half-activation voltage of −72 ± 2 mV, and a voltage sensitivity of 8.2 ± 0.7 mV per e-fold change. Ih has relatively fast, voltage-dependent kinetics, with on and off time constants in the range of 50–350 ms. AH neurones had a previously undescribed, low threshold, slowly inactivating, sodium-dependent current that was poorly sensitive to TTX. In AH neurones, the post-action-potential slow hyperpolarizing current, IAHP, displayed large variation from cell to cell. IAHP appeared to be highly Ca2+ sensitive, since its activation with either membrane depolarization or caffeine (1 mm) was not prevented by perfusing the cell with 10 mm BAPTA. We determined the identity of the Ca2+ channels linked to IAHP. Action potentials of AH neurones that were elongated by TEA (10 mm) were similarly shortened and IAHP was suppressed with each of the three Ω-conotoxins GVIA, MVIIA and MVIIC (0.3–0.5 μm), but not with Ω-agatoxin IVA (0.2 μm). There was no additivity between the effects of the three conotoxins, which indicates the presence of N- but not of P/Q-type Ca2+ channels. A residual Ca2+ current, resistant to all toxins, but blocked by 0.5 mm Cd2+, could not generate IAHP. This patch-clamp study, performed on intact ganglia, demonstrates that the AH neurones of the guinea-pig duodenum are under the control of four major currents, IAHP, Ih, an N-type Ca2+ current and a slowly inactivating Na+ current. PMID:11790812

Actin cytoskeleton and exocytosis in rat melanotrophs.

PubMed

Chowdhury, Helana H; Popoff, Michel R; Zorec, Robert

2000-01-01

We monitored secretory activity of single rat melanotrophs by the patch-clamp membrane capacitance measurements (C m ). Secretory activity was stimulated by cytosol dialysis with a patch-pipette solution containing 1μM [Ca 2+ ] i . Actin cytoskeleton was disaggregated by pretreating cells with Clostridium spiroforme toxin, which specifically ADP-ribosylates cellular actin. The extent of cytoskeleton disaggregation was monitored by phalloidin immunostaining. The maximal rate of secretion increases two folds in toxin-treated cells in comparison to controls, whereas the extent of calcium-induced secretory response was similar to that obtained in the non-treated cells. The results show that the subcortical actin network attenuates the rate of secretory activity, which we interpret to reflect a barrier function of cytoskeleton for exocytosis.

Actin cytoskeleton and exocytosis in rat melanotrophs.

PubMed

Chowdhury, H H; Popoff, M R; Zorec, R

2000-01-01

We monitored secretory activity of single rat melanotrophs by the patch-clamp membrane capacitance measurements (Cm). Secretory activity was stimulated by cytosol dialysis with a patch-pipette solution containing 1 microM [Ca2+]i. Actin cytoskeleton was disaggregated by pretreating cells with Clostridium spiroforme toxin, which specifically ADP-ribosylates cellular actin. The extent of cytoskeleton disaggregation was monitored by phalloidin immunostaining. The maximal rate of secretion increases two folds in toxin-treated cells in comparison to controls, whereas the extent of calcium-induced secretory response was similar to that obtained in the non-treated cells. The results show that the subcortical actin network attenuates the rate of secretory activity, which we interpret to reflect a barrier function of cytoskeleton for exocytosis.

Involvement of mitochondrial Na+–Ca2+ exchange in intestinal pacemaking activity

PubMed Central

Kim, Byung Joo; Jun, Jae Yeoul; So, Insuk; Kim, Ki Whan

2006-01-01

AIM: Interstitial cells of Cajal (ICCs) are the pacemaker cells that generate slow waves in the gastrointestinal (GI) tract. We have aimed to investigate the involvement of mitochondrial Na+-Ca2+ exchange in intestinal pacemaking activity in cultured interstitial cells of Cajal. METHODS: Enzymatic digestions were used to dissociate ICCs from the small intestine of a mouse. The whole-cell patch-clamp configuration was used to record membrane currents (voltage clamp) and potentials (current clamp) from cultured ICCs. RESULTS: Clonazepam and CGP37157 inhibited the pacemaking activity of ICCs in a dose-dependent manner. Clonazepam from 20 to 60 µmol/L and CGP37157 from 10 to 30 µmol/L effectively inhibited Ca2+ efflux from mitochondria in pacemaking activity of ICCs. The IC50s of clonazepam and CGP37157 were 37.1 and 18.2 µmol/L, respectively. The addition of 20 µmol/L NiCl2 to the internal solution caused a “wax and wane” phenomenon of pacemaking activity of ICCs. CONCLUSION: These results suggest that mitochondrial Na+-Ca2+ exchange has an important role in intestinal pacemaking activity. PMID:16521198

Insulin activates single amiloride-blockable Na channels in a distal nephron cell line (A6).

PubMed

Marunaka, Y; Hagiwara, N; Tohda, H

1992-09-01

Using the patch-clamp technique, we studied the effect of insulin on an amiloride-blockable Na channel in the apical membrane of a distal nephron cell line (A6) cultured on permeable collagen films for 10-14 days. NPo (N, number of channels per patch membrane; Po, average value of open probability of individual channels in the patch) under baseline conditions was 0.88 +/- 0.12 (SE)(n = 17). After making cell-attached patches on the apical membrane which contained Na channels, insulin (1 mU/ml) was applied to the serosal bath. While maintaining the cell-attached patch, NPo significantly increased to 1.48 +/- 0.19 (n = 17; P less than 0.001) after 5-10 min of insulin application. The open probability of Na channels was 0.39 +/- 0.01 (n = 38) under baseline condition, and increased to 0.66 +/- 0.03 (n = 38, P less than 0.001) after addition of insulin. The baseline single-channel conductance was 4pS, and neither the single-channel conductance nor the current-voltage relationship was significantly changed by insulin. These results indicate that insulin increases Na absorption in the distal nephron by increasing the open probability of the amiloride-blockable Na channel.

Cysteine-rich secretory protein 4 is an inhibitor of transient receptor potential M8 with a role in establishing sperm function

PubMed Central

Gibbs, Gerard M.; Orta, Gerardo; Reddy, Thulasimala; Koppers, Adam J.; Martínez-López, Pablo; Luis de la Vega-Beltràn, José; Lo, Jennifer C. Y.; Veldhuis, Nicholas; Jamsai, Duangporn; McIntyre, Peter; Darszon, Alberto; O'Bryan, Moira K.

2011-01-01

The cysteine-rich secretory proteins (CRISPs) are a group of four proteins in the mouse that are expressed abundantly in the male reproductive tract, and to a lesser extent in other tissues. Analysis of reptile CRISPs and mouse CRISP2 has shown that CRISPs can regulate cellular homeostasis via ion channels. With the exception of the ability of CRISP2 to regulate ryanodine receptors, the in vivo targets of mammalian CRISPs function are unknown. In this study, we have characterized the ion channel regulatory activity of epididymal CRISP4 using electrophysiology, cell assays, and mouse models. Through patch-clamping of testicular sperm, the CRISP4 CRISP domain was shown to inhibit the transient receptor potential (TRP) ion channel TRPM8. These data were confirmed using a stably transfected CHO cell line. TRPM8 is a major cold receptor in the body, but is found in other tissues, including the testis and on the tail and head of mouse and human sperm. Functional assays using sperm from wild-type mice showed that TRPM8 activation significantly reduced the number of sperm undergoing the progesterone-induced acrosome reaction following capacitation, and that this response was reversed by the coaddition of CRISP4. In accordance, sperm from Crisp4 null mice had a compromised ability to undergo to the progesterone-induced acrosome reaction. Collectively, these data identify CRISP4 as an endogenous regulator of TRPM8 with a role in normal sperm function. PMID:21482758

Buccal patches of atenolol formulated using fenugreek (Trigonella foenum-graecum L.) seed mucilage.

PubMed

Adhikari, Surya Narayan Ratha; Panda, Satyabrata

2017-01-01

The use of mucoadhesive natural polymers in designing mucoadhesive patch systems has received much attention. The study involved the development and evaluation of buccal patches of atenolol using fenugreek (Trigonella foenum-graecum L.) seed mucilage with hydroxylpropyl methyl cellulose (HPMC K4M) and a backing membrane (ethyl cellulose 5% w/v). These atenolol-releasing buccal patches were prepared using a solvent casting technique. The buccal patches prepared were evaluated for average weight, thickness, drug content, folding endurance and moisture content. Ex vivo mucoadhesive strength, force of adhesion and bonding strength were determined using porcine buccal mucosa. The mucosal permeation of atenolol through the porcine buccal mucosa was carried out using a Franz diffusion cell in phosphate buffer saline, pH 6.8. These buccal patches were also characterized by SEM and FTIR spectroscopy. The average weight, thickness, drug content, folding endurance and moisture content of these atenolol-releasing buccal patches were found satisfactory for all the patches. Amongst all, the F-4 buccal patch showed maximum mucoadhesive strength (31.12 ±1.86 g), force of adhesion (30.53 × 10-2 N) and bond strength (1748.89 N/m2). Ex vivo atenolol permeation from the buccal patches showed drug permeation across the excised porcine buccal mucosa over 12 h. The F-4 buccal patch showed maximum permeation flux (29.12 μg/cm2/h). The developed atenolol-releasing buccal patches can be beneficial over the conventional drug delivery systems to decrease the dosing frequency and enhance patient compliance.

Testosterone decreases urinary bladder smooth muscle excitability via novel signaling mechanism involving direct activation of the BK channels

PubMed Central

Hristov, Kiril L.; Parajuli, Shankar P.; Provence, Aaron

2016-01-01

In addition to improving sexual function, testosterone has been reported to have beneficial effects in ameliorating lower urinary tract symptoms by increasing bladder capacity and compliance, while decreasing bladder pressure. However, the cellular mechanisms by which testosterone regulates detrusor smooth muscle (DSM) excitability have not been elucidated. Here, we used amphotericin-B perforated whole cell patch-clamp and single channel recordings on inside-out excised membrane patches to investigate the regulatory role of testosterone in guinea pig DSM excitability. Testosterone (100 nM) significantly increased the depolarization-induced whole cell outward currents in DSM cells. The selective pharmacological inhibition of the large-conductance voltage- and Ca2+-activated K+ (BK) channels with paxilline (1 μM) completely abolished this stimulatory effect of testosterone, suggesting a mechanism involving BK channels. At a holding potential of −20 mV, DSM cells exhibited transient BK currents (TBKCs). Testosterone (100 nM) significantly increased TBKC activity in DSM cells. In current-clamp mode, testosterone (100 nM) significantly hyperpolarized the DSM cell resting membrane potential and increased spontaneous transient hyperpolarizations. Testosterone (100 nM) rapidly increased the single BK channel open probability in inside-out excised membrane patches from DSM cells, clearly suggesting a direct BK channel activation via a nongenomic mechanism. Live-cell Ca2+ imaging showed that testosterone (100 nM) caused a decrease in global intracellular Ca2+ concentration, consistent with testosterone-induced membrane hyperpolarization. In conclusion, the data provide compelling mechanistic evidence that under physiological conditions, testosterone at nanomolar concentrations directly activates BK channels in DSM cells, independent from genomic testosterone receptors, and thus regulates DSM excitability. PMID:27605581

Dynamics of T-Junction Solution Switching Aimed at Patch Clamp Experiments

PubMed Central

Auzmendi, Jerónimo A.; Smoler, Mariano; Moffatt, Luciano

2015-01-01

Solutions exchange systems are responsible for the timing of drug application on patch clamp experiments. There are two basic strategies for generating a solution exchange. When slow exchanges are bearable, it is easier to perform the exchange inside the tubing system upstream of the exit port. On the other hand, fast, reproducible, exchanges are usually performed downstream of the exit port. As both strategies are combinable, increasing the performance of upstream exchanges is desirable. We designed a simple method for manufacturing T-junctions (300 μm I.D.) and we measured the time profile of exchange of two saline solutions using a patch pipette with an open tip. Three factors were found to determine the timing of the solution switching: pressure, travelled distance and off-center distance. A linear relationship between the time delay and the travelled distance was found for each tested pressure, showing its dependence to the fluid velocity, which increased with pressure. The exchange time was found to increase quadratically with the delay, although a sizeable variability remains unexplained by this relationship. The delay and exchange times increased as the recording pipette moved away from the center of the stream. Those increases became dramatic as the pipette was moved close to the stream borders. Mass transport along the travelled distance between the slow fluid at the border and the fast fluid at the center seems to contribute to the time course of the solution exchange. This effect would be present in all tubing based devices. Present results might be of fundamental importance for the adequate design of serial compound exchangers which would be instrumental in the discovery of drugs that modulate the action of the physiological agonists of ion channels with the purpose of fine tuning their physiology. PMID:26177538

Insect Optic Glomeruli-Exploration of a Universal Circuit for Sensorimotor Processing

DTIC Science & Technology

2011-01-25

09 to 2-28-10. We have successfully achieved the first recordings from any laboratory of the small palisade output neurons from the lobula of...glomeruli. Using infrared illumination and optics, the cell bodies of such clones are directly observed. A patch clamp recording electrode, filled...neuron and electrolyte of the electrode has been established, the cell is recorded during the presentation of a sequence of visual stimuli: stripes

Inhibitory effects of sevoflurane on pacemaking activity of sinoatrial node cells in guinea-pig heart

PubMed Central

Kojima, Akiko; Kitagawa, Hirotoshi; Omatsu-Kanbe, Mariko; Matsuura, Hiroshi; Nosaka, Shuichi

2012-01-01

BACKGROUND AND PURPOSE The volatile anaesthetic sevoflurane affects heart rate in clinical settings. The present study investigated the effect of sevoflurane on sinoatrial (SA) node automaticity and its underlying ionic mechanisms. EXPERIMENTAL APPROACH Spontaneous action potentials and four ionic currents fundamental for pacemaking, namely, the hyperpolarization-activated cation current (If), T-type and L-type Ca2+ currents (ICa,T and ICa,L, respectively), and slowly activating delayed rectifier K+ current (IKs), were recorded in isolated guinea-pig SA node cells using perforated and conventional whole-cell patch-clamp techniques. Heart rate in guinea-pigs was recorded ex vivo in Langendorff mode and in vivo during sevoflurane inhalation. KEY RESULTS In isolated SA node cells, sevoflurane (0.12–0.71 mM) reduced the firing rate of spontaneous action potentials and its electrical basis, diastolic depolarization rate, in a qualitatively similar concentration-dependent manner. Sevoflurane (0.44 mM) reduced spontaneous firing rate by approximately 25% and decreased If, ICa,T, ICa,L and IKs by 14.4, 31.3, 30.3 and 37.1%, respectively, without significantly affecting voltage dependence of current activation. The negative chronotropic effect of sevoflurane was partly reproduced by a computer simulation of SA node cell electrophysiology. Sevoflurane reduced heart rate in Langendorff-perfused hearts, but not in vivo during sevoflurane inhalation in guinea-pigs. CONCLUSIONS AND IMPLICATIONS Sevoflurane at clinically relevant concentrations slowed diastolic depolarization and thereby reduced pacemaking activity in SA node cells, at least partly due to its inhibitory effect on If, ICa,T and ICa,L. These findings provide an important electrophysiological basis of alterations in heart rate during sevoflurane anaesthesia in clinical settings. PMID:22356456

Direct negative chronotropic action of desflurane on sinoatrial node pacemaker activity in the guinea pig heart.

PubMed

Kojima, Akiko; Ito, Yuki; Kitagawa, Hirotoshi; Matsuura, Hiroshi; Nosaka, Shuichi

2014-06-01

Desflurane inhalation is associated with sympathetic activation and concomitant increase in heart rate in humans and experimental animals. There is, however, little information concerning the direct effects of desflurane on electrical activity of sinoatrial node pacemaker cells that determines the intrinsic heart rate. Whole-cell patch-clamp experiments were conducted on guinea pig sinoatrial node pacemaker cells to record spontaneous action potentials and ionic currents contributing to sinoatrial node automaticity, namely, hyperpolarization-activated cation current (If), T-type and L-type Ca currents (ICa,T and ICa,L, respectively), Na/Ca exchange current (INCX), and rapidly and slowly activating delayed rectifier K currents (IKr and IKs, respectively). Electrocardiograms were recorded from ex vivo Langendorff-perfused hearts and in vivo hearts. Desflurane at 6 and 12% decreased spontaneous firing rate of sinoatrial node action potentials by 15.9% (n = 11) and 27.6% (n = 10), respectively, which was associated with 20.4% and 42.5% reductions in diastolic depolarization rate, respectively. Desflurane inhibited If, ICa,T, ICa,L, INCX, and IKs but had little effect on IKr. The negative chronotropic action of desflurane was reasonably well reproduced in sinoatrial node computer model. Desflurane reduced the heart rate in Langendorff-perfused hearts. High concentration (12%) of desflurane inhalation was associated with transient tachycardia, which was totally abolished by pretreatment with the β-adrenergic blocker propranolol. Desflurane has a direct negative chronotropic action on sinoatrial node pacemaking activity, which is mediated by its inhibitory action on multiple ionic currents. This direct inhibitory action of desflurane on sinoatrial node automaticity seems to be counteracted by sympathetic activation associated with desflurane inhalation in vivo.

KCa3.1 Modulates Neuroblast Migration Along the Rostral Migratory Stream (RMS) In Vivo

PubMed Central

Turner, Kathryn L.; Sontheimer, Harald

2014-01-01

From the subventricular zone (SVZ), neuronal precursor cells (NPCs), called neuroblasts, migrate through the rostral migratory stream (RMS) to become interneurons in the olfactory bulb (OB). Ion channels regulate neuronal migration during development, yet their role in migration through the adult RMS is unknown. To address this question, we utilized Nestin-CreERT2/R26R-YFP mice to fluorescently label neuroblasts in the adult. Patch-clamp recordings from neuroblasts reveal K+ currents that are sensitive to intracellular Ca2+ levels and blocked by clotrimazole and TRAM-34, inhibitors of intermediate conductance Ca2+-activated K+ (KCa3.1) channels. Immunolabeling and electrophysiology show KCa3.1 expression restricted to neuroblasts in the SVZ and RMS, but absent in OB neurons. Time-lapse confocal microscopy in situ showed inhibiting KCa3.1 prolonged the stationary phase of neuroblasts' saltatory migration, reducing migration speed by over 50%. Both migration and KCa3.1 currents could also be inhibited by blocking Ca2+ influx via transient receptor potential (TRP) channels, which, together with positive immunostaining for transient receptor potential canonical 1 (TRPC1), suggest that TRP channels are an important Ca2+ source modulating KCa3.1 activity. Finally, injecting TRAM-34 into Nestin-CreERT2/R26R-YFP mice significantly reduced the number of neuroblasts that reached the OB, suggesting an important role for KCa3.1 in vivo. These studies describe a previously unrecognized protein in migration of adult NPCs. PMID:23585521

A new design for a green calcium indicator with a smaller size and a reduced number of calcium-binding sites

PubMed Central

Barykina, Natalia V.; Subach, Oksana M.; Doronin, Danila A.; Sotskov, Vladimir P.; Roshchina, Marina A.; Kunitsyna, Tatiana A.; Malyshev, Aleksey Y.; Smirnov, Ivan V.; Azieva, Asya M.; Sokolov, Ilya S.; Piatkevich, Kiryl D.; Burtsev, Mikhail S.; Varizhuk, Anna M.; Pozmogova, Galina E.; Anokhin, Konstantin V.; Subach, Fedor V.; Enikolopov, Grigori N.

2016-01-01

Genetically encoded calcium indicators (GECIs) are mainly represented by two- or one-fluorophore-based sensors. One type of two-fluorophore-based sensor, carrying Opsanus troponin C (TnC) as the Ca2+-binding moiety, has two binding sites for calcium ions, providing a linear response to calcium ions. One-fluorophore-based sensors have four Ca2+-binding sites but are better suited for in vivo experiments. Herein, we describe a novel design for a one-fluorophore-based GECI with two Ca2+-binding sites. The engineered sensor, called NTnC, uses TnC as the Ca2+-binding moiety, inserted in the mNeonGreen fluorescent protein. Monomeric NTnC has higher brightness and pH-stability in vitro compared with the standard GECI GCaMP6s. In addition, NTnC shows an inverted fluorescence response to Ca2+. Using NTnC, we have visualized Ca2+ dynamics during spontaneous activity of neuronal cultures as confirmed by control NTnC and its mutant, in which the affinity to Ca2+ is eliminated. Using whole-cell patch clamp, we have demonstrated that NTnC dynamics in neurons are similar to those of GCaMP6s and allow robust detection of single action potentials. Finally, we have used NTnC to visualize Ca2+ neuronal activity in vivo in the V1 cortical area in awake and freely moving mice using two-photon microscopy or an nVista miniaturized microscope. PMID:27677952

Early Disruption of Extracellular Pleiotrophin Distribution Alters Cerebellar Neuronal Circuit Development and Function.

PubMed

Hamza, M M; Rey, S A; Hilber, P; Arabo, A; Collin, T; Vaudry, D; Burel, D

2016-10-01

The cerebellum is a structure of the central nervous system involved in balance, motor coordination, and voluntary movements. The elementary circuit implicated in the control of locomotion involves Purkinje cells, which receive excitatory inputs from parallel and climbing fibers, and are regulated by cerebellar interneurons. In mice as in human, the cerebellar cortex completes its development mainly after birth with the migration, differentiation, and synaptogenesis of granule cells. These cellular events are under the control of numerous extracellular matrix molecules including pleiotrophin (PTN). This cytokine has been shown to regulate the morphogenesis of Purkinje cells ex vivo and in vivo via its receptor PTPζ. Since Purkinje cells are the unique output of the cerebellar cortex, we explored the consequences of their PTN-induced atrophy on the function of the cerebellar neuronal circuit in mice. Behavioral experiments revealed that, despite a normal overall development, PTN-treated mice present a delay in the maturation of their flexion reflex. Moreover, patch clamp recording of Purkinje cells revealed a significant increase in the frequency of spontaneous excitatory postsynaptic currents in PTN-treated mice, associated with a decrease of climbing fiber innervations and an abnormal perisomatic localization of the parallel fiber contacts. At adulthood, PTN-treated mice exhibit coordination impairment on the rotarod test associated with an alteration of the synchronization gait. Altogether these histological, electrophysiological, and behavior data reveal that an early ECM disruption of PTN composition induces short- and long-term defaults in the establishment of proper functional cerebellar circuit.

Electrophysiological safety of DW-286a, a novel fluoroquinolone antibiotic agent.

PubMed

Kim, Eun-Joo; Kim, Ki-Suk; Shin, Won-Ho

2005-01-01

Inhibition of the potassium current I(Kr) and QT prolongation has been known to be associated with drug-induced torsades de pointes arrhythmias (TdP) and sudden cardiac death. We investigated the cardiac electrophysiological effects of DW-286a, a new class of fluoroquinolone antibiotics reported to prolong the QT interval. To investigate the electrophysiological safety of DW-286a, we used conventional microelectrode recording techniques in isolated guinea pig papillary muscles, whole-cell patch clamp techniques in human ether-à-go-go related gene (hERG)-transient transfected Chinese hamster ovary cells, and in vivo electrocardiogram (ECG) measurements in Sprague-Dawley (SD) rats by the use of a telemetry system. DW-286a at 300 microM significantly (P<0.01) prolonged action potentials at 50% repolarization (APD50) and 90% repolarization (APD90). For IHERG, the IC50 value was 89.00+/-37.85 microM with a Hill coefficient (nH) of -0.97+/-0.49. However, when DW-286a was orally administered to conscious SD rats at a high dose (1000 mg/kg), no significant effect on ECG in vivo was detected. From a previous study, we know that concentration at 19.8 microM is the antimicrobial end-point of DW-286a. Therefore, our data suggest that in the electrophysiological aspect, it can be thought that the effective concentrations of DW-286a are between 19.8 and 100 microM (concentration in serum).

Conductive polymer nanotube patch for fast and controlled ex vivo transdermal drug delivery.

PubMed

Nguyen, Thao M; Lee, Sebin; Lee, Sang Bok

2014-10-01

To uptake and release hydrophilic model drugs and insulin in a novel conductive polymer (CP) nanotube transdermal patch. The externally controlled transdermal delivery of model drugs and insulin were tested ex vivo and results were compared with CP films. The unique intrinsic properties of CPs provide electrostatic interaction between the model drugs and polymer backbone. When a pulsed potential was applied, the drug delivery release profile mimics that of injection delivery. With a constant potential applied, the release rate constants of the patch system were up to three-times faster than the control (0 V) and released approximately 80% more drug molecules over 24 h. The CP nanotube transdermal patch represents a new and promising drug method, specifically for hydrophilic molecules, which have been a large obstacle for conventional transdermal drug delivery systems.

Bidirectional control of spike timing by GABA(A) receptor-mediated inhibition during theta oscillation in CA1 pyramidal neurons.

PubMed

Kwag, Jeehyun; Paulsen, Ole

2009-08-26

Precisely controlled spike times relative to theta-frequency network oscillations play an important role in hippocampal memory processing. Here we study how inhibitory synaptic input during theta oscillation contributes to the control of spike timing. Using whole-cell patch-clamp recordings from CA1 pyramidal cells in vitro with dynamic clamp to simulate theta-frequency oscillation (5 Hz), we show that gamma-aminobutyric acid-A (GABA(A)) receptor-mediated inhibitory postsynaptic potentials (IPSPs) can not only delay but also advance the postsynaptic spike depending on the timing of the inhibition relative to the oscillation. Spike time advancement with IPSP was abolished by the h-channel blocker ZD7288 (10 microM), suggesting that IPSPs can interact with intrinsic membrane conductances to yield bidirectional control of spike timing.

Propylparaben reduces the excitability of hippocampal neurons by blocking sodium channels.

PubMed

Lara-Valderrábano, Leonardo; Rocha, Luisa; Galván, Emilio J

2016-12-01

Propylparaben (PPB) is an antimicrobial preservative widely used in food, cosmetics, and pharmaceutics. Virtual screening methodologies predicted anticonvulsant activity of PPB that was confirmed in vivo. Thus, we explored the effects of PPB on the excitability of hippocampal neurons by using standard patch clamp techniques. Bath perfusion of PPB reduced the fast-inactivating sodium current (I Na ) amplitude, causing a hyperpolarizing shift in the inactivation curve of the I Na, and markedly delayed the sodium channel recovery from the inactivation state. Also, PPB effectively suppressed the riluzole-sensitive, persistent sodium current (I NaP ). PPB perfusion also modified the action potential kinetics, and higher concentrations of PPB suppressed the spike activity. Nevertheless, the modulatory effects of PPB did not occur when PPB was internally applied by whole-cell dialysis. These results indicate that PPB reduces the excitability of CA1 pyramidal neurons by modulating voltage-dependent sodium channels. The mechanistic basis of this effect is a marked delay in the recovery from inactivation state of the voltage-sensitive sodium channels. Our results indicate that similar to local anesthetics and anticonvulsant drugs that act on sodium channels, PPB acts in a use-dependent manner. Copyright © 2016 Elsevier B.V. All rights reserved.

Engineering Lipid Bilayer Membranes for Protein Studies

PubMed Central

Khan, Muhammad Shuja; Dosoky, Noura Sayed; Williams, John Dalton

2013-01-01

Lipid membranes regulate the flow of nutrients and communication signaling between cells and protect the sub-cellular structures. Recent attempts to fabricate artificial systems using nanostructures that mimic the physiological properties of natural lipid bilayer membranes (LBM) fused with transmembrane proteins have helped demonstrate the importance of temperature, pH, ionic strength, adsorption behavior, conformational reorientation and surface density in cellular membranes which all affect the incorporation of proteins on solid surfaces. Much of this work is performed on artificial templates made of polymer sponges or porous materials based on alumina, mica, and porous silicon (PSi) surfaces. For example, porous silicon materials have high biocompatibility, biodegradability, and photoluminescence, which allow them to be used both as a support structure for lipid bilayers or a template to measure the electrochemical functionality of living cells grown over the surface as in vivo. The variety of these media, coupled with the complex physiological conditions present in living systems, warrant a summary and prospectus detailing which artificial systems provide the most promise for different biological conditions. This study summarizes the use of electrochemical impedance spectroscopy (EIS) data on artificial biological membranes that are closely matched with previously published biological systems using both black lipid membrane and patch clamp techniques. PMID:24185908

Chloride channel blockers promote relaxation of TEA-induced contraction in airway smooth muscle

PubMed Central

Yim, Peter D.; Gallos, George; Perez-zoghbi, Jose F.; Trice, Jacquelyn; Zhang, Yi; Siviski, Matthew; Sonett, Joshua; Emala, Charles W.

2014-01-01

Enhanced airway smooth muscle (ASM) contraction is an important component in the pathophysiology of asthma. We have shown that ligand gated chloride channels modulate ASM contractile tone during the maintenance phase of an induced contraction, however the role of chloride flux in depolarization-induced contraction remains incompletely understood. To better understand the role of chloride flux under these conditions, muscle force (human ASM, guinea pig ASM), peripheral small airway luminal area (rat ASM) and airway smooth muscle plasma membrane electrical potentials (human cultured ASM) were measured. We found ex vivo guinea pig airway rings, human ASM strips and small peripheral airways in rat lungs slices relaxed in response to niflumic acid following depolarization-induced contraction induced by K+ channel blockade with tetraethylammonium chloride (TEA). In isolated human airway smooth muscle cells TEA induce depolarization as measured by a fluorescent indicator or whole cell patch clamp and this depolarization was reversed by niflumic acid. These findings demonstrate that ASM depolarization induced contraction is dependent on chloride channel activity. Targeting of chloride channels may be a novel approach to relax hypercontractile airway smooth muscle in bronchoconstrictive disorders. PMID:24662476

Salicylate enables cochlear arachidonic-acid-sensitive NMDA receptor responses.

PubMed

Ruel, Jérôme; Chabbert, Christian; Nouvian, Régis; Bendris, Rim; Eybalin, Michel; Leger, Claude Louis; Bourien, Jérôme; Mersel, Marcel; Puel, Jean-Luc

2008-07-16

Currently, many millions of people treated for various ailments receive high doses of salicylate. Consequently, understanding the mechanisms by which salicylate induces tinnitus is an important issue for the research community. Behavioral testing in rats have shown that tinnitus induced by salicylate or mefenamate (both cyclooxygenase blockers) are mediated by cochlear NMDA receptors. Here we report that the synapses between the sensory inner hair cells and the dendrites of the cochlear spiral ganglion neurons express NMDA receptors. Patch-clamp recordings and two-photon calcium imaging demonstrated that salicylate and arachidonate (a substrate of cyclooxygenase) enabled the calcium flux and the neural excitatory effects of NMDA on cochlear spiral ganglion neurons. Salicylate also increased the arachidonate content of the whole cochlea in vivo. Single-unit recordings of auditory nerve fibers in adult guinea pig confirmed the neural excitatory effect of salicylate and the blockade of this effect by NMDA antagonist. These results suggest that salicylate inhibits cochlear cyclooxygenase, which increased levels of arachidonate. The increased levels of arachidonate then act on NMDA receptors to enable NMDA responses to glutamate that inner hair cells spontaneously release. This new pharmacological profile of salicylate provides a molecular mechanism for the generation of tinnitus at the periphery of the auditory system.

Accurate measurement of junctional conductance between electrically coupled cells with dual whole-cell voltage-clamp under conditions of high series resistance.

PubMed

Hartveit, Espen; Veruki, Margaret Lin

2010-03-15

Accurate measurement of the junctional conductance (G(j)) between electrically coupled cells can provide important information about the functional properties of coupling. With the development of tight-seal, whole-cell recording, it became possible to use dual, single-electrode voltage-clamp recording from pairs of small cells to measure G(j). Experiments that require reduced perturbation of the intracellular environment can be performed with high-resistance pipettes or the perforated-patch technique, but an accompanying increase in series resistance (R(s)) compromises voltage-clamp control and reduces the accuracy of G(j) measurements. Here, we present a detailed analysis of methodologies available for accurate determination of steady-state G(j) and related parameters under conditions of high R(s), using continuous or discontinuous single-electrode voltage-clamp (CSEVC or DSEVC) amplifiers to quantify the parameters of different equivalent electrical circuit model cells. Both types of amplifiers can provide accurate measurements of G(j), with errors less than 5% for a wide range of R(s) and G(j) values. However, CSEVC amplifiers need to be combined with R(s)-compensation or mathematical correction for the effects of nonzero R(s) and finite membrane resistance (R(m)). R(s)-compensation is difficult for higher values of R(s) and leads to instability that can damage the recorded cells. Mathematical correction for R(s) and R(m) yields highly accurate results, but depends on accurate estimates of R(s) throughout an experiment. DSEVC amplifiers display very accurate measurements over a larger range of R(s) values than CSEVC amplifiers and have the advantage that knowledge of R(s) is unnecessary, suggesting that they are preferable for long-duration experiments and/or recordings with high R(s). Copyright (c) 2009 Elsevier B.V. All rights reserved.

Cytochalasin E alters the cytoskeleton and decreases ENaC activity in Xenopus 2F3 cells.

PubMed

Reifenberger, Matthew S; Yu, Ling; Bao, Hui-Fang; Duke, Billie Jeanne; Liu, Bing-Chen; Ma, He-Ping; Alli, Ahmed A; Eaton, Douglas C; Alli, Abdel A

2014-07-01

Numerous reports have linked cytoskeleton-associated proteins with the regulation of epithelial Na(+) channel (ENaC) activity. The purpose of the present study was to determine the effect of actin cytoskeleton disruption by cytochalasin E on ENaC activity in Xenopus 2F3 cells. Here, we show that cytochalasin E treatment for 60 min can disrupt the integrity of the actin cytoskeleton in cultured Xenopus 2F3 cells. We show using single channel patch-clamp experiments and measurements of short-circuit current that ENaC activity, but not its density, is altered by cytochalasin E-induced disruption of the cytoskeleton. In nontreated cells, 8 of 33 patches (24%) had no measurable ENaC activity, whereas in cytochalasin E-treated cells, 17 of 32 patches (53%) had no activity. Analysis of those patches that did contain ENaC activity showed channel open probability significantly decreased from 0.081 ± 0.01 in nontreated cells to 0.043 ± 0.01 in cells treated with cytochalasin E. Transepithelial current from mpkCCD cells treated with cytochalasin E, cytochalasin D, or latrunculin B for 60 min was decreased compared with vehicle-treated cells. The subcellular expression of fodrin changed significantly, and several protein elements of the cytoskeleton decreased at least twofold after 60 min of cytochalasin E treatment. Cytochalasin E treatment disrupted the association between ENaC and myristoylated alanine-rich C-kinase substrate. The results presented here suggest disruption of the actin cytoskeleton by different compounds can attenuate ENaC activity through a mechanism involving changes in the subcellular expression of fodrin, several elements of the cytoskeleton, and destabilization of the ENaC-myristoylated alanine-rich C-kinase substrate complex. Copyright © 2014 the American Physiological Society.

Cytochalasin E alters the cytoskeleton and decreases ENaC activity in Xenopus 2F3 cells

PubMed Central

Reifenberger, Matthew S.; Yu, Ling; Bao, Hui-Fang; Duke, Billie Jeanne; Liu, Bing-Chen; Ma, He-Ping; Eaton, Douglas C.; Alli, Abdel A.

2014-01-01

Numerous reports have linked cytoskeleton-associated proteins with the regulation of epithelial Na+ channel (ENaC) activity. The purpose of the present study was to determine the effect of actin cytoskeleton disruption by cytochalasin E on ENaC activity in Xenopus 2F3 cells. Here, we show that cytochalasin E treatment for 60 min can disrupt the integrity of the actin cytoskeleton in cultured Xenopus 2F3 cells. We show using single channel patch-clamp experiments and measurements of short-circuit current that ENaC activity, but not its density, is altered by cytochalasin E-induced disruption of the cytoskeleton. In nontreated cells, 8 of 33 patches (24%) had no measurable ENaC activity, whereas in cytochalasin E-treated cells, 17 of 32 patches (53%) had no activity. Analysis of those patches that did contain ENaC activity showed channel open probability significantly decreased from 0.081 ± 0.01 in nontreated cells to 0.043 ± 0.01 in cells treated with cytochalasin E. Transepithelial current from mpkCCD cells treated with cytochalasin E, cytochalasin D, or latrunculin B for 60 min was decreased compared with vehicle-treated cells. The subcellular expression of fodrin changed significantly, and several protein elements of the cytoskeleton decreased at least twofold after 60 min of cytochalasin E treatment. Cytochalasin E treatment disrupted the association between ENaC and myristoylated alanine-rich C-kinase substrate. The results presented here suggest disruption of the actin cytoskeleton by different compounds can attenuate ENaC activity through a mechanism involving changes in the subcellular expression of fodrin, several elements of the cytoskeleton, and destabilization of the ENaC-myristoylated alanine-rich C-kinase substrate complex. PMID:24829507

Identification Of Cells With A Compact Microscope Imaging System With Intelligent Controls

NASA Technical Reports Server (NTRS)

McDowell, Mark (Inventor)

2006-01-01

A Microscope Imaging System (CMIS) with intelligent controls is disclosed that provides techniques for scanning, identifying, detecting and tracking mic?oscopic changes in selected characteristics or features of various surfaces including, but not limited to, cells, spheres, and manufactured products subject to difficult-to-see imperfections. The practice of the present invention provides applications that include colloidal hard spheres experiments, biological cell detection for patch clamping, cell movement and tracking, as well as defect identification in products, such as semiconductor devices, where surface damage can be significant, but difficult to detect. The CMIS system is a machine vision system, which combines intelligent image processing with remote control capabilities and provides the ability to autofocus on a microscope sample, automatically scan an image, and perform machine vision analysis on multiple samples simultaneously.

Tracking of Cells with a Compact Microscope Imaging System with Intelligent Controls

NASA Technical Reports Server (NTRS)

McDowell, Mark (Inventor)

2007-01-01

A Microscope Imaging System (CMIS) with intelligent controls is disclosed that provides techniques for scanning, identifying, detecting and tracking microscopic changes in selected characteristics or features of various surfaces including, but not limited to, cells, spheres, and manufactured products subject to difficult-to-see imperfections. The practice of the present invention provides applications that include colloidal hard spheres experiments, biological cell detection for patch clamping, cell movement and tracking, as well as defect identification in products, such as semiconductor devices, where surface damage can be significant, but difficult to detect. The CMIS system is a machine vision system, which combines intelligent image processing with remote control capabilities and provides the ability to autofocus on a microscope sample, automatically scan an image, and perform machine vision analysis on multiple samples simultaneously

Tracking of cells with a compact microscope imaging system with intelligent controls

NASA Technical Reports Server (NTRS)

McDowell, Mark (Inventor)

2007-01-01

A Microscope Imaging System (CMIS) with intelligent controls is disclosed that provides techniques for scanning, identifying, detecting and tracking microscopic changes in selected characteristics or features of various surfaces including, but not limited to, cells, spheres, and manufactured products subject to difficult-to-see imperfections. The practice of the present invention provides applications that include colloidal hard spheres experiments, biological cell detection for patch clamping, cell movement and tracking, as well as defect identification in products, such as semiconductor devices, where surface damage can be significant, but difficult to detect. The CMIS system is a machine vision system, which combines intelligent image processing with remote control capabilities and provides the ability to auto-focus on a microscope sample, automatically scan an image, and perform machine vision analysis on multiple samples simultaneously.

Operation of a Cartesian Robotic System in a Compact Microscope with Intelligent Controls

NASA Technical Reports Server (NTRS)

McDowell, Mark (Inventor)

2006-01-01

A Microscope Imaging System (CMIS) with intelligent controls is disclosed that provides techniques for scanning, identifying, detecting and tracking microscopic changes in selected characteristics or features of various surfaces including, but not limited to, cells, spheres, and manufactured products subject to difficult-to-see imperfections. The practice of the present invention provides applications that include colloidal hard spheres experiments, biological cell detection for patch clamping, cell movement and tracking, as well as defect identification in products, such as semiconductor devices, where surface damage can be significant, but difficult to detect. The CMIS system is a machine vision system, which combines intelligent image processing with remote control capabilities and provides the ability to autofocus on a microscope sample, automatically scan an image, and perform machine vision analysis on multiple samples simultaneously.

[Single channel analysis of aconitine blockade of calcium channels in rat myocardiocytes].

PubMed

Chen, L; Ma, C; Cai, B C; Lu, Y M; Wu, H

1995-01-01

Ventricular myocardiocytes from neonatal Wistar rats were isolated and cultured. Aconitine, Ca2+ channel blocker verapamil or Ca2+ channel activator BAY K8644 were added to the bath solution separately. Using the cell-attached configuration of the patch clamp technique, the single channel activities of L type Ca2+ channel were recorded before and after addition of all three drugs. The results showed the blocking effect of aconitine (50 micrograms.ml-1) on L type Ca2+ channels. Its mechanism may be relevant to the decrease in both open state probability and the mean open time of Ca2+ channel. The difference was statistically significant compared with control group (P < 0.01). The amplitude of Ba2+ currents, which flow through open L type Ca2+ channel was unchanged.

Observation and analysis of the Coulter effect through carbon nanotube and graphene nanopores.

PubMed

Agrawal, Kumar Varoon; Drahushuk, Lee W; Strano, Michael S

2016-02-13

Carbon nanotubes (CNTs) and graphene are the rolled and flat analogues of graphitic carbon, respectively, with hexagonal crystalline lattices, and show exceptional molecular transport properties. The empirical study of a single isolated nanopore requires, as evidence, the observation of stochastic, telegraphic noise from a blocking molecule commensurate in size with the pore. This standard is used ubiquitously in patch clamp studies of single, isolated biological ion channels and a wide range of inorganic, synthetic nanopores. In this work, we show that observation and study of stochastic fluctuations for carbon nanopores, both CNTs and graphene-based, enable precision characterization of pore properties that is otherwise unattainable. In the case of voltage clamp measurements of long (0.5-1 mm) CNTs between 0.9 and 2.2 nm in diameter, Coulter blocking of cationic species reveals the complex structuring of the fluid phase for confined water in this diameter range. In the case of graphene, we have pioneered the study and the analysis of stochastic fluctuations in gas transport from a pressurized, graphene-covered micro-well compartment that reveal switching between different values of the membrane permeance attributed to chemical rearrangements of individual graphene pores. This analysis remains the only way to study such single isolated graphene nanopores under these realistic transport conditions of pore rearrangements, in keeping with the thesis of this work. In summary, observation and analysis of Coulter blocking or stochastic fluctuations of permeating flux is an invaluable tool to understand graphene and graphitic nanopores including CNTs. © 2015 The Author(s).

Initial Characterization of a Gel Patch Dosimeter for In Vivo Dosimetry

PubMed Central

Matrosic, C; Culberson, W; Rosen, B; Madsen, E; Frank, G; Bednarz, B

2016-01-01

In vivo dosimetry is a greatly underutilized tool for patient safety in clinical external beam radiotherapy treatments, despite being recommended by several national and international organizations (AAPM, ICRU, IAEA, NACP). The reasons for this underutilization mostly relate to the feasibility and cost of in vivo dosimetry methods. Due to the increase in the number of beam angles and dose per fraction in modern treatments, there is a compelling need for a novel dosimeter that is robust and affordable while able to operate properly in these complex conditions. This work presents a gel patch dosimeter as a novel method of in vivo dosimetry. DEFGEL, a 6%T normoxic polyacrylamide gel, was injected into 1-cm thick acrylic molds to create 1-cm thick small cylindrical patch dosimeters. To evaluate the change in optical density due to radiation induced polymerization, dosimeters were scanned before and after irradiation using an in-house developed laser densitometer. The dose-responses of three separate batches of gel were evaluated and compared to check for linearity and repeatability. The response development time was evaluated to ensure that the patch dosimeter could be high throughput. Additionally, the potential of this system to be used as an in vivo dosimeter was tested with a clinically relevant end-to-end in vivo phantom test. All irradiations were performed with a Varian Clinac 21EX at the University of Wisconsin Medical Radiation Research Center (UWMRRC). The dose response of all three batches of gel was found to be linear within the range of 2–20 Gy. At doses below 0.5 Gy the statistical uncertainties were prohibitively large to make quantitative assessments of the results. The three batches demonstrated good repeatability in the range of 2 Gy to up to 10 Gy, with only slight variations in response at higher doses. For low doses the dosimeter fully developed within an hour while at higher doses they fully developed within four hours. During the in vivo phantom test the predicted patch absorbed dose was 4.23 Gy while the readout dose was evaluated to be 4.37 Gy, which corresponds to a 3.2% discrepancy. The dosimeter and densitometer pairing shows promise as an in vivo dosimetry system, especially for hypofractionated or MRI-guided radiotherapy treatments where higher doses are prescribed. PMID:27088207

Initial characterization of a gel patch dosimeter for in vivo dosimetry

NASA Astrophysics Data System (ADS)

Matrosic, C.; Culberson, W.; Rosen, B.; Madsen, E.; Frank, G.; Bednarz, B.

2016-05-01

In vivo dosimetry is a greatly underutilized tool for patient safety in clinical external beam radiotherapy treatments, despite being recommended by several national and international organizations (AAPM, ICRU, IAEA, NACP). The reasons for this underutilization mostly relate to the feasibility and cost of in vivo dosimetry methods. Due to the increase in the number of beam angles and dose per fraction in modern treatments, there is a compelling need for a novel dosimeter that is robust and affordable while able to operate properly in these complex conditions. This work presents a gel patch dosimeter as a novel method of in vivo dosimetry. DEFGEL, a 6% T normoxic polyacrylamide gel, was injected into 1 cm thick acrylic molds to create 1 cm thick small cylindrical patch dosimeters. To evaluate the change in optical density due to radiation induced polymerization, dosimeters were scanned before and after irradiation using an in-house developed laser densitometer. The dose-responses of three separate batches of gel were evaluated and compared to check for linearity and repeatability. The response development time was evaluated to ensure that the patch dosimeter could be high throughput. Additionally, the potential of this system to be used as an in vivo dosimeter was tested with a clinically relevant end-to-end in vivo phantom test. All irradiations were performed with a Varian Clinac 21EX at the University of Wisconsin Medical Radiation Research Center (UWMRRC). The dose-response of all three batches of gel was found to be linear within the range of 2-20 Gy. At doses below 0.5 Gy the statistical uncertainties were prohibitively large to make quantitative assessments of the results. The three batches demonstrated good repeatability in the range of 2 Gy to up to 10 Gy, with only slight variations in response at higher doses. For low doses the dosimeter fully developed within an hour while at higher doses they fully developed within four hours. During the in vivo phantom test the predicted patch absorbed dose was 4.23 Gy while the readout dose was evaluated to be 4.37 Gy, which corresponds to a 3.2% discrepancy. The dosimeter and densitometer pairing shows promise as an in vivo dosimetry system, especially for hypofractionated or MRI-guided radiotherapy treatments where higher doses are prescribed.

The Mechanosensitive Ca2+ Channel as a Central Regulator of Prostate Tumor Cell Migration and Invasiveness

DTIC Science & Technology

2010-01-01

Use time-lapse videomicroscopy and patch-clamp techniques to characterize the motility of eGFP-transfected PC-3 cells in which MScCa/TRPC1 has been...except for GsmTx-4 (Peptides International, Louisville, KY) and fluorescent agents (Invitrogen/Molecular Probes, Carlsbad, CA). Videomicroscopy ...and Ca2+-imaging. Cell migration was monitored at 37oC by time-lapse videomicroscopy using Nomarski optics with an Epifluorescent microscope (Nikon

Direct evidence for high affinity blockade of NaV1.6 channel subtype by huwentoxin-IV spider peptide, using multiscale functional approaches.

PubMed

Gonçalves, Tânia C; Boukaiba, Rachid; Molgó, Jordi; Amar, Muriel; Partiseti, Michel; Servent, Denis; Benoit, Evelyne

2018-05-01

The Chinese bird spider huwentoxin-IV (HwTx-IV) is well-known to be a highly potent blocker of Na V 1.7 subtype of voltage-gated sodium (Na V ) channels, a genetically validated analgesic target, and thus promising as a potential lead molecule for the development of novel pain therapeutics. In the present study, the interaction between HwTx-IV and Na V 1.6 channel subtype was investigated using multiscale (from in vivo to individual cell) functional approaches. HwTx-IV was approximatively 2 times more efficient than tetrodotoxin (TTX) to inhibit the compound muscle action potential recorded from the mouse skeletal neuromuscular system in vivo, and 30 times more effective to inhibit nerve-evoked than directly-elicited muscle contractile force of isolated mouse hemidiaphragms. These results strongly suggest that the inhibition of nerve-evoked skeletal muscle functioning, produced by HwTx-IV, resulted from a toxin-induced preferential blockade of Na V 1.6, compared to Na V 1.4, channel subtype. This was confirmed by whole-cell automated patch-clamp experiments performed on human embryonic kidney (HEK)-293 cells overexpressing hNa V 1.1-1.8 channel subtypes. HwTx-IV was also approximatively 850 times more efficient to inhibit TTX-sensitive than TTX-resistant sodium currents recorded from mouse dorsal root ganglia neurons. Finally, based on our data, we predict that blockade of the Na V 1.6 channel subtype was involved in the in vivo toxicity of HwTx-IV, although this toxicity was more than 2 times lower than that of TTX. In conclusion, our results provide detailed information regarding the effects of HwTx-IV and allow a better understanding of the side-effect mechanisms involved in vivo and of channel subtype interactions resulting from the toxin activity. Copyright © 2018 Elsevier Ltd. All rights reserved.

Diminished A-type potassium current and altered firing properties in presympathetic PVN neurones in renovascular hypertensive rats

PubMed Central

Sonner, Patrick M; Filosa, Jessica A; Stern, Javier E

2008-01-01

Accumulating evidence supports a contribution of the hypothalamic paraventricular nucleus (PVN) to sympathoexcitation and elevated blood pressure in renovascular hypertension. However, the underlying mechanisms resulting in altered neuronal function in hypertensive rats remain largely unknown. Here, we aimed to address whether the transient outward potassium current (IA) in identified rostral ventrolateral medulla (RVLM)-projecting PVN neurones is altered in hypertensive rats, and whether such changes affected single and repetitive action potential properties and associated changes in intracellular Ca2+ levels. Patch-clamp recordings obtained from PVN-RVLM neurons showed a reduction in IA current magnitude and single channel conductance, and an enhanced steady-state current inactivation in hypertensive rats. Morphometric reconstructions of intracellularly labelled PVN-RVLM neurons showed a diminished dendritic surface area in hypertensive rats. Consistent with a diminished IA availability, action potentials in PVN-RVLM neurons in hypertensive rats were broader, decayed more slowly, and were less sensitive to the K+ channel blocker 4-aminopyridine. Simultaneous patch clamp recordings and confocal Ca2+ imaging demonstrated enhanced action potential-evoked intracellular Ca2+ transients in hypertensive rats. Finally, spike broadening during repetitive firing discharge was enhanced in PVN-RVLM neurons from hypertensive rats. Altogether, our results indicate that diminished IA availability constitutes a contributing mechanism underlying aberrant central neuronal function in renovascular hypertension. PMID:18238809

Novel roles of ascorbate in plants: induction of cytosolic Ca2+ signals and efflux from cells via anion channels.

PubMed

Makavitskaya, M; Svistunenko, D; Navaselsky, I; Hryvusevich, P; Mackievic, V; Rabadanova, C; Tyutereva, E; Samokhina, V; Straltsova, D; Sokolik, A; Voitsekhovskaja, O; Demidchik, V

2018-02-17

Ascorbate is not often considered as a signalling molecule in plants. This study demonstrates that, in Arabidopsis roots, exogenous L-ascorbic acid triggers a transient increase of the cytosolic free calcium activity ([Ca2+]cyt.) that is central to plant signalling. Exogenous copper and iron stimulates the ascorbate-induced [Ca2+]cyt. elevation while cation channel blockers, free radical scavengers, low extracellular [Ca2+], transition metal chelators and removal of the cell wall inhibit this reaction. These data show that apoplastic redox-active transition metals are involved in the ascorbate-induced [Ca2+]cyt. elevation. Exogenous ascorbate also induces a moderate increase in programmed cell death symptoms in intact roots, but it does not activate Ca2+ influx currents in patch-clamped root protoplasts. Intriguingly, the replacement of gluconate with ascorbate in the patch-clamp pipette reveales a large ascorbate efflux current, which shows sensitivity to the anion channel blocker, anthracene-9-carboxylic acid (A9C), indicative of the ascorbate release via anion channels. EPR spectroscopy measurements demonstrates that salinity (NaCl) triggers the accumulation of root apoplastic ascorbyl radicals in A9C-dependent manner, confirming that L-ascorbate leaks through anion channels under depolarisation. This mechanism may underlie ascorbate release, signalling phenomena, apoplastic redox reactions, iron acquisition and control the ionic and electrical equilibrium (together K+ efflux via GORK channels).

Negative Inotropic Effects of High Mobility Box Group 1 Protein in Isolated Contracting Cardiac Myocytes

PubMed Central

Tzeng, Huei-Ping; Fan, Jinping; Vallejo, Jesus G.; Dong, Jian Wen; Chen, Xiongwen; Houser, Steven R.; Mann, Douglas L.

2013-01-01

HMGB1 released from necrotic cells or macrophages functions as a late inflammatory mediator, and has been shown to induce cardiovascular collapse during sepsis. Thus far, however, the effect(s) of HMGB1 in the heart are not known. We determined the effects of HMGB1 on isolated feline cardiac myocytes by measuring sarcomere shortening in contracting cardiac myocytes, intracellular Ca2+ transients using fluo-3, and L-type calcium currents using whole cell perforate configuration of the patch clamp technique. Treatment of isolated myocytes with HMGB1 (100 ng/ml) resulted in a 70% decrease in sarcomere shortening and a 50% decrease in the height of the peak Ca++ transient within 5 min (p <0.01). The immediate negative inotropic effects HMGB1 on cell contractility and calcium homeostasis were partially reversible upon washout of HMGB1. A significant inhibition of the inward L-type calcium currents also was documented by the patch clamp technique. HMGB1 induced the PKCε translocation and a PKC inhibitor significantly attenuated the negative inotropic effects of HMGB1. These studies show for the first time that HMGB1 impairs sarcomere shortening by decreasing calcium availability in cardiac myocytes through modulating membrane calcium influx, and suggest that HMGB1 maybe act as a novel myocardial depressant factor during cardiac injury. PMID:18223193

A new pH-sensitive rectifying potassium channel in mitochondria from the embryonic rat hippocampus.

PubMed

Kajma, Anna; Szewczyk, Adam

2012-10-01

Patch-clamp single-channel studies on mitochondria isolated from embryonic rat hippocampus revealed the presence of two different potassium ion channels: a large-conductance (288±4pS) calcium-activated potassium channel and second potassium channel with outwardly rectifying activity under symmetric conditions (150/150mM KCl). At positive voltages, this channel displayed a conductance of 67.84pS and a strong voltage dependence at holding potentials from -80mV to +80mV. The open probability was higher at positive than at negative voltages. Patch-clamp studies at the mitoplast-attached mode showed that the channel was not sensitive to activators and inhibitors of mitochondrial potassium channels but was regulated by pH. Moreover, we demonstrated that the channel activity was not affected by the application of lidocaine, an inhibitor of two-pore domain potassium channels, or by tertiapin, an inhibitor of inwardly rectifying potassium channels. In summary, based on the single-channel recordings, we characterised for the first time mitochondrial pH-sensitive ion channel that is selective for cations, permeable to potassium ions, displays voltage sensitivity and does not correspond to any previously described potassium ion channels in the inner mitochondrial membrane. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012). Copyright © 2012 Elsevier B.V. All rights reserved.

Analyzing and modeling the kinetics of amyloid beta pores associated with Alzheimer’s disease pathology

DOE PAGES

Ullah, Ghanim; Demuro, Angelo; Parker, Ian; ...

2015-09-08

Amyloid beta (Aβ) oligomers associated with Alzheimer’s disease (AD) form Ca 2+-permeable plasma membrane pores, leading to a disruption of the otherwise well-controlled intracellular calcium (Ca 2+) homeostasis. The resultant up-regulation of intracellular Ca 2+ concentration has detrimental implications for memory formation and cell survival. The gating kinetics and Ca 2+ permeability of Aβ pores are not well understood. We have used computational modeling in conjunction with the ability of optical patch-clamping for massively parallel imaging of Ca 2+ flux through thousands of pores in the cell membrane of Xenopus oocytes to elucidate the kinetic properties of Aβ pores. Themore » fluorescence time-series data from individual pores were idealized and used to develop data-driven Markov chain models for the kinetics of the Aβ pore at different stages of its evolution. Our study provides the first demonstration of developing Markov chain models for ion channel gating that are driven by optical-patch clamp data with the advantage of experiments being performed under close to physiological conditions. As a result, we demonstrate the up-regulation of gating of various Ca 2+ release channels due to Aβ pores and show that the extent and spatial range of such up-regulation increases as Aβ pores with low open probability and Ca 2+ permeability transition into those with high open probability and Ca 2+ permeability.« less

Cationic nanoparticles induce nanoscale disruption in living cell plasma membranes.

PubMed

Chen, Jiumei; Hessler, Jessica A; Putchakayala, Krishna; Panama, Brian K; Khan, Damian P; Hong, Seungpyo; Mullen, Douglas G; Dimaggio, Stassi C; Som, Abhigyan; Tew, Gregory N; Lopatin, Anatoli N; Baker, James R; Holl, Mark M Banaszak; Orr, Bradford G

2009-08-13

It has long been recognized that cationic nanoparticles induce cell membrane permeability. Recently, it has been found that cationic nanoparticles induce the formation and/or growth of nanoscale holes in supported lipid bilayers. In this paper, we show that noncytotoxic concentrations of cationic nanoparticles induce 30-2000 pA currents in 293A (human embryonic kidney) and KB (human epidermoid carcinoma) cells, consistent with a nanoscale defect such as a single hole or group of holes in the cell membrane ranging from 1 to 350 nm(2) in total area. Other forms of nanoscale defects, including the nanoparticle porating agents adsorbing onto or intercalating into the lipid bilayer, are also consistent; although the size of the defect must increase to account for any reduction in ion conduction, as compared to a water channel. An individual defect forming event takes 1-100 ms, while membrane resealing may occur over tens of seconds. Patch-clamp data provide direct evidence for the formation of nanoscale defects in living cell membranes. The cationic polymer data are compared and contrasted with patch-clamp data obtained for an amphiphilic phenylene ethynylene antimicrobial oligomer (AMO-3), a small molecule that is proposed to make well-defined 3.4 nm holes in lipid bilayers. Here, we observe data that are consistent with AMO-3 making approximately 3 nm holes in living cell membranes.

Increase in Cardiac Ischemia-Reperfusion Injuries in Opa1+/- Mouse Model

PubMed Central

Fauconnier, Jérémy; Cellier, Laura; Tamareille, Sophie; Gharib, Abdallah; Chevrollier, Arnaud; Loufrani, Laurent; Grenier, Céline; Kamel, Rima; Sarzi, Emmanuelle; Lacampagne, Alain; Ovize, Michel; Henrion, Daniel; Reynier, Pascal; Lenaers, Guy; Mirebeau-Prunier, Delphine

2016-01-01

Background Recent data suggests the involvement of mitochondrial dynamics in cardiac ischemia/reperfusion (I/R) injuries. Whilst excessive mitochondrial fission has been described as detrimental, the role of fusion proteins in this context remains uncertain. Objectives To investigate whether Opa1 (protein involved in mitochondrial inner-membrane fusion) deficiency affects I/R injuries. Methods and Results We examined mice exhibiting Opa1delTTAG mutations (Opa1+/-), showing 70% Opa1 protein expression in the myocardium as compared to their wild-type (WT) littermates. Cardiac left-ventricular systolic function assessed by means of echocardiography was observed to be similar in 3-month-old WT and Opa1+/- mice. After subjection to I/R, infarct size was significantly greater in Opa1+/- than in WTs both in vivo (43.2±4.1% vs. 28.4±3.5%, respectively; p<0.01) and ex vivo (71.1±3.2% vs. 59.6±8.5%, respectively; p<0.05). No difference was observed in the expression of other main fission/fusion protein, oxidative phosphorylation, apoptotic markers, or mitochondrial permeability transition pore (mPTP) function. Analysis of calcium transients in isolated ventricular cardiomyocytes demonstrated a lower sarcoplasmic reticulum Ca2+ uptake, whereas cytosolic Ca2+ removal from the Na+/Ca2+ exchanger (NCX) was increased, whilst SERCA2a, phospholamban, and NCX protein expression levels were unaffected in Opa1+/- compared to WT mice. Simultaneous whole-cell patch-clamp recordings of mitochondrial Ca2+ movements and ventricular action potential (AP) showed impairment of dynamic mitochondrial Ca2+ uptake and a marked increase in the AP late repolarization phase in conjunction with greater occurrence of arrhythmia in Opa1+/- mice. Conclusion Opa1 deficiency was associated with increased sensitivity to I/R, imbalance in dynamic mitochondrial Ca2+ uptake, and subsequent increase in NCX activity. PMID:27723783

Distribution and characterisation of Glucagon-like peptide-1 receptor expressing cells in the mouse brain

PubMed Central

Cork, Simon C.; Richards, James E.; Holt, Marie K.; Gribble, Fiona M.; Reimann, Frank; Trapp, Stefan

2015-01-01

Objective Although Glucagon-like peptide 1 is a key regulator of energy metabolism and food intake, the precise location of GLP-1 receptors and the physiological relevance of certain populations is debatable. This study investigated the novel GLP-1R-Cre mouse as a functional tool to address this question. Methods Mice expressing Cre-recombinase under the Glp1r promoter were crossed with either a ROSA26 eYFP or tdRFP reporter strain to identify GLP-1R expressing cells. Patch-clamp recordings were performed on tdRFP-positive neurons in acute coronal brain slices from adult mice and selective targeting of GLP-1R cells in vivo was achieved using viral gene delivery. Results Large numbers of eYFP or tdRFP immunoreactive cells were found in the circumventricular organs, amygdala, hypothalamic nuclei and the ventrolateral medulla. Smaller numbers were observed in the nucleus of the solitary tract and the thalamic paraventricular nucleus. However, tdRFP positive neurons were also found in areas without preproglucagon-neuronal projections like hippocampus and cortex. GLP-1R cells were not immunoreactive for GFAP or parvalbumin although some were catecholaminergic. GLP-1R expression was confirmed in whole-cell recordings from BNST, hippocampus and PVN, where 100 nM GLP-1 elicited a reversible inward current or depolarisation. Additionally, a unilateral stereotaxic injection of a cre-dependent AAV into the PVN demonstrated that tdRFP-positive cells express cre-recombinase facilitating virally-mediated eYFP expression. Conclusions This study is a comprehensive description and phenotypic analysis of GLP-1R expression in the mouse CNS. We demonstrate the power of combining the GLP-1R-CRE mouse with a virus to generate a selective molecular handle enabling future in vivo investigation as to their physiological importance. PMID:26500843

Acyclic Cucurbit[n]uril-Type Molecular Container Enables Systemic Delivery of Effective Doses of Albendazole for Treatment of SK-OV-3 Xenograft Tumors.

PubMed

Hettiarachchi, Gaya; Samanta, Soumen K; Falcinelli, Shane; Zhang, Ben; Moncelet, Damien; Isaacs, Lyle; Briken, Volker

2016-03-07

Approximately, 40-70% of active pharmaceutical ingredients (API) are severely limited by their extremely poor aqueous solubility, and consequently, there is a high demand for excipients that can be used to formulate clinically relevant doses of these drug candidates. Here, proof-of-concept studies demonstrate the potential of our recently discovered acyclic cucurbit[n]uril-type molecular container Motor1 (M1) as a solubilizing agent for insoluble drugs. M1 did not induce significant rates of mutations in various Salmonella typhimurium test strains during the Ames test, suggesting low genotoxicity. M1 also has low risk of causing cardiac toxicity in humans since it did not inhibit the human Ether-à-go-go-Related Gene channel as tested on transfected CHO cell lines via patch clamp analysis. Albendazole (ABZ) is a widely used antihelminthic agent but that has also shown promising efficacy against cancerous cells in vitro. However, due to its low aqueous solubility (2.7 μM) and poor pharmacokinetics, ABZ is clinically limited as an anticancer agent. Here we investigated the potential of M1 as a solubilizing excipient for ABZ formulation. A pharmacokinetic study indicated that ABZ escapes the peritoneal cavity resulting in 78% absolute bioavailability, while its active intermediate metabolite, albendazole sulfoxide, achieved 43% absolute bioavailability. The daily dosing of 681 mg/kg M1 complexed with 3.2 mg/kg of ABZ for 14 days did not result in significant weight loss or pathology in Swiss Webster mice. In vivo efficacy studies using this M1·ABZ inclusion complex showed significant decreases in tumor growth rates and increases in survival of mice bearing SK-OV-3 xenograft tumors. In conclusion, we provide substantial new evidence demonstrating that M1 is a safe and efficient excipient that enables in vivo parenteral delivery of poorly water-soluble APIs.

A Genetic Selection for dinB Mutants Reveals an Interaction between DNA Polymerase IV and the Replicative Polymerase That Is Required for Translesion Synthesis.

PubMed

Scotland, Michelle K; Heltzel, Justin M H; Kath, James E; Choi, Jung-Suk; Berdis, Anthony J; Loparo, Joseph J; Sutton, Mark D

2015-09-01

Translesion DNA synthesis (TLS) by specialized DNA polymerases (Pols) is a conserved mechanism for tolerating replication blocking DNA lesions. The actions of TLS Pols are managed in part by ring-shaped sliding clamp proteins. In addition to catalyzing TLS, altered expression of TLS Pols impedes cellular growth. The goal of this study was to define the relationship between the physiological function of Escherichia coli Pol IV in TLS and its ability to impede growth when overproduced. To this end, 13 novel Pol IV mutants were identified that failed to impede growth. Subsequent analysis of these mutants suggest that overproduced levels of Pol IV inhibit E. coli growth by gaining inappropriate access to the replication fork via a Pol III-Pol IV switch that is mechanistically similar to that used under physiological conditions to coordinate Pol IV-catalyzed TLS with Pol III-catalyzed replication. Detailed analysis of one mutant, Pol IV-T120P, and two previously described Pol IV mutants impaired for interaction with either the rim (Pol IVR) or the cleft (Pol IVC) of the β sliding clamp revealed novel insights into the mechanism of the Pol III-Pol IV switch. Specifically, Pol IV-T120P retained complete catalytic activity in vitro but, like Pol IVR and Pol IVC, failed to support Pol IV TLS function in vivo. Notably, the T120P mutation abrogated a biochemical interaction of Pol IV with Pol III that was required for Pol III-Pol IV switching. Taken together, these results support a model in which Pol III-Pol IV switching involves interaction of Pol IV with Pol III, as well as the β clamp rim and cleft. Moreover, they provide strong support for the view that Pol III-Pol IV switching represents a vitally important mechanism for regulating TLS in vivo by managing access of Pol IV to the DNA.

Acute effects of gentamicin on the ionic currents of semicircular canal hair cells in the frog.

PubMed

Martini, Marta; Canella, Rita; Prigioni, Ivo; Russo, Giancarlo; Tavazzani, Elisa; Fesce, Riccardo; Rossi, Maria Lisa

2011-12-01

The effects of acute gentamicin application on hair cells isolated from the frog semicircular canals have been tested by using the patch-clamp technique in the whole-cell configuration. Extracellular gentamicin (1 mM) mostly affected the Ca(2+) macrocurrent, I(Ca), and the Ca-dependent K(+) current, I(KCa). The drug, applied to the hair cell basolateral membrane through a fast perfusion system, produced a rapid and relevant decrease (∼34%) of I(Ca) amplitude, without apparently affecting its activation-deactivation kinetics. The I(KCa) component of the delayed I(KD) was similarly affected: peak and steady-state mean amplitudes were significantly reduced, by about 47 and 54%, respectively, whereas the time constant of the mono-exponential current rising phase did not change. The Ca(2+) independent fraction of I(KD), I(KV), and the fast IA current were unaffected. Transduction channels (permeable to and blocked by gentamicin) are not available in the isolated hair cell, so the effect of intracellular gentamicin was tested by applying the drug through the patch pipette (1 mM in the pipette): again, it significantly reduced both I(Ca) and I(KD) amplitude, without affecting currents kinetics. IA properties were also unaffected. The drug did not affect the onset and removal of I(KD) inactivation, although the changes were scaled to the reduced I(KD) amplitude. From these observations, it is expected that hair cells exposed to gentamicin 'in vivo' become unresponsive to physiological stimulation (block of the transduction channels) and transmitter release at the cytoneural junction be drastically depressed due to reduced Ca(2+) inflow. In particular, functional impairment ensues much earlier than biochemical events that lead to hair cell apoptosis. Copyright © 2011 Elsevier B.V. All rights reserved.

Glycogen synthase kinase 3 beta alters anxiety-, depression-, and addiction-related behaviors and neuronal activity in the nucleus accumbens shell

PubMed Central

Crofton, Elizabeth J.; Nenov, Miroslav N.; Zhang, Yafang; Scala, Federico; Page, Sean A.; McCue, David L.; Li, Dingge; Hommel, Jonathan D.; Laezza, Fernanda; Green, Thomas A.

2017-01-01

Psychiatric disorders such as anxiety, depression and addiction are often comorbid brain pathologies thought to share common mechanistic biology. As part of the cortico-limbic circuit, the nucleus accumbens shell (NAcSh) plays a fundamental role in integrating information in the circuit, such that modulation of NAcSh circuitry alters anxiety, depression, and addiction-related behaviors. Intracellular kinase cascades in the NAcSh have proven important mediators of behavior. To investigate glycogen-synthase kinase 3 (GSK3) beta signaling in the NAcSh in vivo we knocked down GSK3beta expression with a novel adeno-associated viral vector (AAV2) and assessed changes in anxiety- and depression-like behavior and cocaine self-administration in GSK3beta knockdown rats. GSK3beta knockdown reduced anxiety-like behavior while increasing depression-like behavior and cocaine self-administration. Correlative electrophysiological recordings in acute brain slices were used to assess the effect of AAV-shGSK3beta on spontaneous firing and intrinsic excitability of tonically active interneurons (TANs), cells required for input and output signal integration in the NAcSh and for processing reward-related behaviors. Loose-patch recordings showed that TANs transduced by AAV-shGSK3beta exhibited reduction in tonic firing and increased spike half width. When assessed by whole-cell patch clamp recordings these changes were mirrored by reduction in action potential firing and accompanied by decreased hyperpolarization-induced depolarizing sag potentials, increased action potential current threshold, and decreased maximum rise time. These results suggest that silencing of GSK3beta in the NAcSh increases depression- and addiction-related behavior, possibly by decreasing intrinsic excitability of TANs. However, this study does not rule out contributions from other neuronal sub-types. PMID:28126496

Electrical tuning and transduction in short hair cells of the chicken auditory papilla.

PubMed

Tan, Xiaodong; Beurg, Maryline; Hackney, Carole; Mahendrasingam, Shanthini; Fettiplace, Robert

2013-04-01

The avian auditory papilla contains two classes of sensory receptor, tall hair cells (THCs) and short hair cells (SHCs), the latter analogous to mammalian outer hair cells with large efferent but sparse afferent innervation. Little is known about the tuning, transduction, or electrical properties of SHCs. To address this problem, we made patch-clamp recordings from hair cells in an isolated chicken basilar papilla preparation at 33°C. We found that SHCs are electrically tuned by a Ca(2+)-activated K(+) current, their resonant frequency varying along the papilla in tandem with that of the THCs, which also exhibit electrical tuning. The tonotopic map for THCs was similar to maps previously described from auditory nerve fiber measurements. SHCs also possess an A-type K(+) current, but electrical tuning was observed only at resting potentials positive to -45 mV, where the A current is inactivated. We predict that the resting potential in vivo is approximately -40 mV, depolarized by a standing inward current through mechanotransducer (MT) channels having a resting open probability of ∼0.26. The resting open probability stems from a low endolymphatic Ca(2+) concentration (0.24 mM) and a high intracellular mobile Ca(2+) buffer concentration, estimated from perforated-patch recordings as equivalent to 0.5 mM BAPTA. The high buffer concentration was confirmed by quantifying parvalbumin-3 and calbindin D-28K with calibrated postembedding immunogold labeling, demonstrating >1 mM calcium-binding sites. Both proteins displayed an apex-to-base gradient matching that in the MT current amplitude, which increased exponentially along the papilla. Stereociliary bundles also labeled heavily with antibodies against the Ca(2+) pump isoform PMCA2a.

Can optical recordings of membrane potential be used to screen for drug-induced action potential prolongation in single cardiac myocytes?

PubMed

Hardy, M E L; Lawrence, C L; Standen, N B; Rodrigo, G C

2006-01-01

Potential-sensitive dyes have primarily been used to optically record action potentials (APs) in whole heart tissue. Using these dyes to record drug-induced changes in AP morphology of isolated cardiac myocytes could provide an opportunity to develop medium throughout assays for the pharmaceutical industry. Ideally, this requires that the dye has a consistent and rapid response to membrane potential, is insensitive to movement, and does not itself affect AP morphology. We recorded the AP from isolated adult guinea-pig ventricular myocytes optically using di-8-ANEPPS in a single-excitation dual-emission ratiometric system, either separately in electrically field stimulated myocytes, or simultaneously with an electrical AP recorded with a patch electrode in the whole-cell bridge mode. The ratio of di-8-ANEPPS fluorescence signal was calibrated against membrane potential using a switch-clamp to voltage clamp the myocyte. Our data show that the ratio of the optical signals emitted at 560/620 nm is linearly related to voltage over the voltage range of an AP, producing a change in ratio of 7.5% per 100 mV, is unaffected by cell movement and is identical to the AP recorded simultaneously with a patch electrode. However, the APD90 recorded optically in myocytes loaded with di-8-ANEPPS was significantly longer than in unloaded myocytes recorded with a patch electrode (355.6+/-13.5 vs. 296.2+/-16.2 ms; p<0.01). Despite this effect, the apparent IC50 for cisapride, which prolongs the AP by blocking IKr, was not significantly different whether determined optically or with a patch electrode (91+/-46 vs. 81+/-20 nM). These data show that the optical AP recorded ratiometrically using di-8-ANEPPS from a single ventricular myocyte accurately follows the action potential morphology. This technique can be used to estimate the AP prolonging effects of a compound, although di-8-ANEPPS itself prolongs APD90. Optical dyes require less technical skills and are less invasive than conventional electrophysiological techniques and, when coupled to ventricular myocytes, decreases animal usage and facilitates higher throughput assays.

Impact of Renal Hilar Control on Outcomes of Robotic Partial Nephrectomy: Systematic Review and Cumulative Meta-analysis.

PubMed

Cacciamani, Giovanni E; Medina, Luis G; Gill, Tania S; Mendelsohn, Alec; Husain, Fatima; Bhardwaj, Lokesh; Artibani, Walter; Sotelo, Renè; Gill, Inderbir S

2018-02-05

During robotic partial nephrectomy (RPN), various techniques of hilar control have been described, including on-clamp, early unclamping, selective/super-selective clamping, and completely-unclamped RPN. To evaluate the impact of various hilar control techniques on perioperative, functional, and oncological outcomes of RPN for tumors. We conducted a systematic literature review and meta-analysis of all comparative studies on various hilar control techniques during RPN using PubMed, Scopus, and Web of Science according to the Preferred Reporting Items for Systematic Review and Meta-analysis statement, and Methods and Guide for Effectiveness and Comparative Effectiveness Review of the Agency for Healthcare Research and Quality. Cumulative meta-analysis of comparative studies was conducted using Review Manager 5.3. Of 987 RPN publications in the literature, 19 qualified for this analysis. Comparison of off-clamp versus on-clamp RPN (n=9), selective clamping versus on-clamp RPN (n=3), super selective clamping versus on-clamp RPN (n=5), and early unclamped versus on-clamp (n=3) were reported. Patients undergoing RPN using off-clamp, selective/super selective, or early unclamp techniques had higher estimated blood loss compared with on-clamp RPN (weight mean difference [WMD]: 47.83, p=0.000, WMD: 41.06, p=0.02, and WMD: 37.50, p=0.47); however, this did not seem clinically relevant, since transfusion rates were similar (odds ratio [OR]: 0.98, p=0.95, OR: 0.72, p=0.7, and OR: 1.36, p=0.33, respectively). All groups appeared similar with regards to hospital stay, transfusions, overall and major complications, and positive cancer margin rates. Short- and long-term renal functional outcomes appeared superior in the off-clamp and super selective clamp groups compared with the on-clamp RPN cohort. Off-clamp, selective/super selective clamp, and early unclamp hilar control techniques are safe and feasible approaches for RPN surgery, with similar perioperative and oncological outcomes compared with on-clamp RPN. Minimizing global renal ischemia may provide superior renal function preservation. However, higher quality data are necessary for definitive conclusions in this regard. The objective of partial nephrectomy is to treat the cancer while maximizing renal function preservation. Clamping the main vessels is done primarily to reduce the blood loss during partial nephrectomy; however, vascular clamping can compromise kidney function. In order to avoid clamping, various techniques have been described. Our analysis showed that techniques that avoid main renal artery clamping during RPN are associated with better renal function preservation, yet deliver non-inferior perioperative and oncological outcomes as compared with robotic partial nephrectomy procedures that clamp the main vessels. Copyright © 2018 European Association of Urology. Published by Elsevier B.V. All rights reserved.

SU-D-213-07: Initial Characterization of a Gel Patch Dosimeter for in Vivo Dosimetry

DOE Office of Scientific and Technical Information (OSTI.GOV)

Matrosic, C; Culberson, W; Rosen, B

Purpose: In vivo dosimetry, despite being the most direct method for monitoring the dose delivered during radiation therapy and being recommended by several national and international organizations (AAPM, ICRU, NACP), is underutilized in the clinic due to issues associated with dose sensitivity, feasibility, and cost. Given the increasing complexity of radiation therapy modern treatments, there is a compelling need for a robust, affordable in vivo dosimetry option. In this work we present the initial characterization of a novel gel patch in vivo dosimeter. Methods: DEFGEL (6%T) was used to make 1-cm thick small cylindrical patch dosimeters. The optical density ofmore » each dosimeter was read before and after irradiation by an in-house laser densitometer. The dosimeters were irradiated using a Varian Clinac EX linac. Three separate batches of gel patches were used to create dose response curves and evaluate repeatability. The development time of the dosimeter was also evaluated. Results: The dose response of the dosimeter was found to be linear from a range of approximately 1-Gy to 20-Gy, which is a larger window of linearity compared to other in vivo dosimeters. At doses below 1-Gy, the cumulative uncertainties were on the order of the measured data. When compared, the three batches demonstrated repeatability from 1-Gy to approximately 13-Gy, with some variation at higher doses. For doses of >8-Gy, the dosimeter reached full optical density after 4-hours, whereas low doses developed within an hour. Conclusion: Initial results indicate that the gel patch dosimeter is a reliable and simple way to measure a large range of doses, including high doses such as those delivered during hypofractionated treatments (e.g. SBRT or MR-guided radiotherapy). The simple fabrication method for the dosimeter and the use of a laser densitometer would allow for the dosimeter to used and read in-house, cheaply and easily.« less

Properties of voltage-activated Na+ and K+ currents in mouse hippocampal glial cells in situ and after acute isolation from tissue slices.

PubMed

Steinhäuser, C; Kressin, K; Kuprijanova, E; Weber, M; Seifert, G

1994-10-01

In the present study, we were interested in a quantitative analysis of voltage-activated channels in a subpopulation of hippocampal glial cells, termed "complex" cells. The patch-clamp technique in the whole-cell mode was applied to identified cells in situ and to glial cells acutely isolated from tissue slices. The outward current was composed of two components: a sustained and a transient current. The transient K+ channel had electrophysiological and pharmacological properties resembling those of the channel through which the A-currents pass. In addition, this glial A-type current possessed a significant Ca2+ dependence. The current parameters determined in situ or in isolated cells corresponded well. Due to space clamp problems in situ, properties of voltage-dependent Na+ currents were only analysed in suspended glial cells. The tetrodotoxin (TTX) sensitivity and the stationary and kinetic characteristics of this current were similar to corresponding properties of hippocampal neurons. These quantitative data demonstrate that at an early postnatal stage of central nervous system maturation, glial cells in situ express a complex pattern of voltage-gated ion channels. The results are compared to findings in other preparations and the possible consequences of transmitter-mediated channel modulation in glial cells are discussed.

Bio-mimicking of Proline-Rich Motif Applied to Carbon Nanotube Reveals Unexpected Subtleties Underlying Nanoparticle Functionalization

NASA Astrophysics Data System (ADS)

Zhang, Yuanzhao; Jimenez-Cruz, Camilo A.; Wang, Jian; Zhou, Bo; Yang, Zaixing; Zhou, Ruhong

2014-11-01

Here, we report computational studies of the SH3 protein domain interacting with various single-walled carbon nanotubes (SWCNT) either bare or functionalized by mimicking the proline-rich motif (PRM) ligand (PPPVPPRR) and compare it to the SH3-PRM complex binding. With prolines or a single arginine attached, the SWCNT gained slightly on specificity when compared with the bare control, whereas with multi-arginine systems the specificity dropped dramatically to our surprise. Although the electrostatic interaction provided by arginines is crucial in the recognition between PRM and SH3 domain, our results suggest that attaching multiple arginines to the SWCNT has a detrimental effect on the binding affinity. Detailed analysis of the MD trajectories found two main factors that modulate the specificity of the binding: the existence of competing acidic patches at the surface of SH3 that leads to ``trapping and clamping'' by the arginines, and the rigidity of the SWCNT introducing entropic penalties in the proper binding. Further investigation revealed that the same ``clamping'' phenomenon exits in the PRM-SH3 system, which has not been reported in previous literature. The competing effects between nanoparticle and its functionalization components revealed by our model system should be of value to current and future nanomedicine designs.

In vivo evaluation of wearable head impact sensors

PubMed Central

Wu, Lyndia C.; Nangia, Vaibhav; Bui, Kevin; Hammoor, Bradley; Kurt, Mehmet; Hernandez, Fidel; Kuo, Calvin; Camarillo, David B.

2015-01-01

Inertial sensors are commonly used to measure human head motion.(R1–3) Some sensors have been tested with dummy or cadaver experiments with mixed results, and methods to evaluate sensors in vivo are lacking. Here we present an in vivo(R3–10) method using high speed video to test teeth-mounted (mouthguard), soft tissue-mounted (skin patch), and headgear-mounted (skull cap) sensors during 6–13g(R1–20) sagittal soccer head impacts. Sensor coupling to the skull (R1–3) was quantified by displacement from an ear-canal reference. Mouthguard displacements were within video measurement error (<1mm), while the skin patch and skull cap displaced up to 4mm and 13mm from the ear-canal reference, respectively. We used the mouthguard, which had the least displacement from skull (R1–5), as the reference to assess 6-degree-of-freedom skin patch and skull cap measurements. Linear and rotational acceleration magnitudes were over-predicted by both the skin patch (with 120% NRMS error for amag, 290% for αmag(R1–6)) and the skull cap (320% NRMS error for amag, 500% for αmag(R1–6)). Such over-predictions were largely due to out-of-plane motion. To model sensor error, we found that in-plane skin patch acceleration peaks in the anterior-posterior direction could be modeled by an underdamped viscoelastic system. In summary, the mouthguard showed tighter skull coupling than the other sensor mounting approaches(R1–7). Furthermore, the in vivo methods presented are valuable for investigating skull acceleration sensor technologies. PMID:26289941

Mispair-specific Recruitment of the Mlh1-Pms1 Complex Identifies Repair Substrates of the Saccharomyces cerevisiae Msh2-Msh3 Complex*

PubMed Central

Srivatsan, Anjana; Bowen, Nikki; Kolodner, Richard D.

2014-01-01

DNA mismatch repair is initiated by either the Msh2-Msh6 or the Msh2-Msh3 mispair recognition heterodimer. Here we optimized the expression and purification of Saccharomyces cerevisiae Msh2-Msh3 and performed a comparative study of Msh2-Msh3 and Msh2-Msh6 for mispair binding, sliding clamp formation, and Mlh1-Pms1 recruitment. Msh2-Msh3 formed sliding clamps and recruited Mlh1-Pms1 on +1, +2, +3, and +4 insertion/deletions and CC, AA, and possibly GG mispairs, whereas Msh2-Msh6 formed mispair-dependent sliding clamps and recruited Mlh1-Pms1 on 7 of the 8 possible base:base mispairs, the +1 insertion/deletion mispair, and to a low level on the +2 but not the +3 or +4 insertion/deletion mispairs and not on the CC mispair. The mispair specificity of sliding clamp formation and Mlh1-Pms1 recruitment but not mispair binding alone correlated best with genetic data on the mispair specificity of Msh2-Msh3- and Msh2-Msh6-dependent mismatch repair in vivo. Analysis of an Msh2-Msh6/Msh3 chimeric protein and mutant Msh2-Msh3 complexes showed that the nucleotide binding domain and communicating regions but not the mispair binding domain of Msh2-Msh3 are responsible for the extremely rapid dissociation of Msh2-Msh3 sliding clamps from DNA relative to that seen for Msh2-Msh6, and that amino acid residues predicted to stabilize Msh2-Msh3 interactions with bent, strand-separated mispair-containing DNA are more critical for the recognition of small +1 insertion/deletions than larger +4 insertion/deletions. PMID:24550389

Mispair-specific recruitment of the Mlh1-Pms1 complex identifies repair substrates of the Saccharomyces cerevisiae Msh2-Msh3 complex.

PubMed

Srivatsan, Anjana; Bowen, Nikki; Kolodner, Richard D

2014-03-28

DNA mismatch repair is initiated by either the Msh2-Msh6 or the Msh2-Msh3 mispair recognition heterodimer. Here we optimized the expression and purification of Saccharomyces cerevisiae Msh2-Msh3 and performed a comparative study of Msh2-Msh3 and Msh2-Msh6 for mispair binding, sliding clamp formation, and Mlh1-Pms1 recruitment. Msh2-Msh3 formed sliding clamps and recruited Mlh1-Pms1 on +1, +2, +3, and +4 insertion/deletions and CC, AA, and possibly GG mispairs, whereas Msh2-Msh6 formed mispair-dependent sliding clamps and recruited Mlh1-Pms1 on 7 of the 8 possible base:base mispairs, the +1 insertion/deletion mispair, and to a low level on the +2 but not the +3 or +4 insertion/deletion mispairs and not on the CC mispair. The mispair specificity of sliding clamp formation and Mlh1-Pms1 recruitment but not mispair binding alone correlated best with genetic data on the mispair specificity of Msh2-Msh3- and Msh2-Msh6-dependent mismatch repair in vivo. Analysis of an Msh2-Msh6/Msh3 chimeric protein and mutant Msh2-Msh3 complexes showed that the nucleotide binding domain and communicating regions but not the mispair binding domain of Msh2-Msh3 are responsible for the extremely rapid dissociation of Msh2-Msh3 sliding clamps from DNA relative to that seen for Msh2-Msh6, and that amino acid residues predicted to stabilize Msh2-Msh3 interactions with bent, strand-separated mispair-containing DNA are more critical for the recognition of small +1 insertion/deletions than larger +4 insertion/deletions.

Magnetophoresis for enhancing transdermal drug delivery: Mechanistic studies and patch design

PubMed Central

Murthy, S. Narasimha; Sammeta, Srinivasa M.; Bower, C.

2017-01-01

Magnetophoresis is a method of enhancement of drug permeation across the biological barriers by application of magnetic field. The present study investigated the mechanistic aspects of magnetophoretic transdermal drug delivery and also assessed the feasibility of designing a magnetophoretic transdermal patch system for the delivery of lidocaine. In vitro drug permeation studies were carried out across the porcine epidermis at different magnetic field strengths. The magnetophoretic drug permeation “flux enhancement factor” was found to increase with the applied magnetic field strength. The mechanistic studies revealed that the magnetic field applied in this study did not modulate permeability of the stratum corneum barrier. The predominant mechanism responsible for magnetically mediated drug permeation enhancement was found to be “magnetokinesis”. The octanol/water partition coefficient of drugs was also found to increase when exposed to the magnetic field. A reservoir type transdermal patch system with a magnetic backing was designed for in vivo studies. The dermal bioavailability (AUC0–6 h) from the magnetophoretic patch system in vivo, in rats was significantly higher than the similarly designed nonmagnetic control patch. PMID:20728484

Electrical coupling of single cardiac rat myocytes to field-effect and bipolar transistors.

PubMed

Kind, Thomas; Issing, Matthias; Arnold, Rüdiger; Müller, Bernt

2002-12-01

A novel bipolar transistor for extracellular recording the electrical activity of biological cells is presented, and the electrical behavior compared with the field-effect transistor (FET). Electrical coupling is examined between single cells separated from the heart of adults rats (cardiac myocytes) and both types of transistors. To initiate a local extracellular voltage, the cells are periodically stimulated by a patch pipette in voltage clamp and current clamp mode. The local extracellular voltage is measured by the planar integrated electronic sensors: the bipolar and the FET. The small signal transistor currents correspond to the local extracellular voltage. The two types of sensor transistors used here were developed and manufactured in the laboratory of our institute. The manufacturing process and the interfaces between myocytes and transistors are described. The recordings are interpreted by way of simulation based on the point-contact model and the single cardiac myocyte model.

The stress protein heat shock cognate 70 (Hsc70) inhibits the Transient Receptor Potential Vanilloid type 1 (TRPV1) channel.

PubMed

Iftinca, Mircea; Flynn, Robyn; Basso, Lilian; Melo, Helvira; Aboushousha, Reem; Taylor, Lauren; Altier, Christophe

2016-01-01

Specialized cellular defense mechanisms prevent damage from chemical, biological, and physical hazards. The heat shock proteins have been recognized as key chaperones that maintain cell survival against a variety of exogenous and endogenous stress signals including noxious temperature. However, the role of heat shock proteins in nociception remains poorly understood. We carried out an expression analysis of the constitutively expressed 70 kDa heat-shock cognate protein, a member of the stress-induced HSP70 family in lumbar dorsal root ganglia from a mouse model of Complete Freund's Adjuvant-induced chronic inflammatory pain. We used immunolabeling of dorsal root ganglion neurons, behavioral analysis and patch clamp electrophysiology in both dorsal root ganglion neurons and HEK cells transfected with Hsc70 and Transient Receptor Potential Channels to examine their functional interaction in heat shock stress condition. We report an increase in protein levels of Hsc70 in mouse dorsal root ganglia, 3 days post Complete Freund's Adjuvant injection in the hind paw. Immunostaining of Hsc70 was observed in most of the dorsal root ganglion neurons, including the small size nociceptors immunoreactive to the TRPV1 channel. Standard whole-cell patch-clamp technique was used to record Transient Receptor Potential Vanilloid type 1 current after exposure to heat shock. We found that capsaicin-evoked currents are inhibited by heat shock in dorsal root ganglion neurons and transfected HEK cells expressing Hsc70 and TRPV1. Blocking Hsc70 with matrine or spergualin compounds prevented heat shock-induced inhibition of the channel. We also found that, in contrast to TRPV1, both the cold sensor channels TRPA1 and TRPM8 were unresponsive to heat shock stress. Finally, we show that inhibition of TRPV1 depends on the ATPase activity of Hsc70 and involves the rho-associated protein kinase. Our work identified Hsc70 and its ATPase activity as a central cofactor of TRPV1 channel function and points to the role of this stress protein in pain associated with neurodegenerative and/or metabolic disorders, including aging. © The Author(s) 2016.

Formulation and evaluation of buccal patches for delivery of atenolol.

PubMed

Adhikari, Surya N Ratha; Nayak, Bhabani S; Nayak, Amit K; Mohanty, Biswaranjan

2010-09-01

Buccal patches for the delivery of atenolol using sodium alginate with various hydrophilic polymers like carbopol 934 P, sodium carboxymethyl cellulose, and hydroxypropyl methylcellulose in various proportions and combinations were fabricated by solvent casting technique. Various physicomechanical parameters like weight variation, thickness, folding endurance, drug content, moisture content, moisture absorption, and various ex vivo mucoadhesion parameters like mucoadhesive strength, force of adhesion, and bond strength were evaluated. An in vitro drug release study was designed, and it was carried out using commercial semipermeable membrane. All these fabricated patches were sustained for 24 h and obeyed first-order release kinetics. Ex vivo drug permeation study was also performed using porcine buccal mucosa, and various drug permeation parameters like flux and lag time were determined.

Electrophysiological responses of dissociated type I cells of the rabbit carotid body to cyanide.

PubMed Central

Biscoe, T J; Duchen, M R

1989-01-01

1. The carotid body is the major peripheral sensor of arterial PO2 in the mammal and is excited by cyanide (CN-). Type I cells, the presumed sites for transduction, were freshly dissociated from the carotid body of the adult rabbit and studied with the whole-cell patch clamp technique. 2. Type I cells were hyperpolarized by CN-, the action potential was shortened, and there was an increased after-hyperpolarization. 3. Under voltage clamp control, CN- increased a voltage-dependent outward current, which showed pronounced outward rectification. Tail currents increased by CN- reversed close to the predicted EK, the reversal potential of the CN--induced current depended on extracellular [K+], and the current was blocked by intracellular TEA+ and Cs+. 4. The i-V relation of the CN--induced conductance strongly mirrored that of voltage-gated Ca2+ entry, and the response was abolished by removal of extracellular Ca2+. We conclude that the increased gK is Ca2+ -dependent (gK(Ca]. 5. The Ca2+ current was attenuated by CN-, and showed an increased rate of inactivation. Thus, the increased gK(Ca) must result from an alteration in Ca2+ homeostasis independent of the Ca2+ current, and not an increased Ca2+ entry through voltage-activated channels. 6. Carbachol also hyperpolarized cells and increased a K+ conductance. 7. At depolarized holding potentials a steady-state outward current was increased by CN-. The current reversed close to EK, and was associated with increased current fluctuations. Noise analysis showed that a channel conductance of 3 pS carries the current. 8. The response to CN- was not impaired by the inclusion of 5 mM-MgATP in the patch pipette. 9. If signals to the CNS are initiated by the calcium-dependent release of transmitters from type I cells, transduction would appear to be the direct consequence of the energy dependence of Ca2+ homeostasis. PMID:2557439

Gut-derived factors promote neurogenesis of CNS-neural stem cells and nudge their differentiation to an enteric-like neuronal phenotype.

PubMed

Kulkarni, Subhash; Zou, Bende; Hanson, Jesse; Micci, Maria-Adelaide; Tiwari, Gunjan; Becker, Laren; Kaiser, Martin; Xie, Xinmin Simon; Pasricha, Pankaj Jay

2011-10-01

Recent studies have explored the potential of central nervous system-derived neural stem cells (CNS-NSC) to repopulate the enteric nervous system. However, the exact phenotypic fate of gut-transplanted CNS-NSC has not been characterized. The aim of this study was to investigate the effect of the gut microenvironment on phenotypic fate of CNS-NSC in vitro. With the use of Transwell culture, differentiation of mouse embryonic CNS-NSC was studied when cocultured without direct contact with mouse intestinal longitudinal muscle-myenteric plexus preparations (LM-MP) compared with control noncocultured cells, in a differentiating medium. Differentiated cells were analyzed by immunocytochemistry and quantitative RT-PCR to assess the expression of specific markers and by whole cell patch-clamp studies for functional characterization of their phenotype. We found that LM-MP cocultured cells had a significant increase in the numbers of cells that were immune reactive against the panneuronal marker β-tubulin, neurotransmitters neuronal nitric oxide synthase (nNOS), choline acetyltransferase (ChAT), and neuropeptide vasoactive intestinal peptide (VIP) and showed an increase in expression of these genes, compared with control cells. Whole cell patch-clamp analysis showed that coculture with LM-MP decreases cell excitability and reduces voltage-gated Na(+) currents but significantly enhances A-current and late afterhyperpolarization (AHP) and increases the expression of the four AHP-generating Ca(2+)-dependent K(+) channel genes (KCNN), compared with control cells. In a separate experiment, differentiation of LM-MP cocultured CNS-NSC produced a significant increase in the numbers of cells that were immune reactive against the neurotransmitters nNOS, ChAT, and the neuropeptide VIP compared with CNS-NSC differentiated similarly in the presence of neonatal brain tissue. Our results show that the gut microenvironment induces CNS-NSC to produce neurons that share some of the characteristics of classical enteric neurons, further supporting the therapeutic use of these cells for gastrointestinal disorders.

Using the genome aggregation database, computational pathogenicity prediction tools, and patch clamp heterologous expression studies to demote previously published long QT syndrome type 1 mutations from pathogenic to benign.

PubMed

Clemens, Daniel J; Lentino, Anne R; Kapplinger, Jamie D; Ye, Dan; Zhou, Wei; Tester, David J; Ackerman, Michael J

2018-04-01

Mutations in the KCNQ1-encoded Kv7.1 potassium channel cause long QT syndrome (LQTS) type 1 (LQT1). It has been suggested that ∼10%-20% of rare LQTS case-derived variants in the literature may have been published erroneously as LQT1-causative mutations and may be "false positives." The purpose of this study was to determine which previously published KCNQ1 case variants are likely false positives. A list of all published, case-derived KCNQ1 missense variants (MVs) was compiled. The occurrence of each MV within the Genome Aggregation Database (gnomAD) was assessed. Eight in silico tools were used to predict each variant's pathogenicity. Case-derived variants that were either (1) too frequently found in gnomAD or (2) absent in gnomAD but predicted to be pathogenic by ≤2 tools were considered potential false positives. Three of these variants were characterized functionally using whole-cell patch clamp technique. Overall, there were 244 KCNQ1 case-derived MVs. Of these, 29 (12%) were seen in ≥10 individuals in gnomAD and are demotable. However, 157 of 244 MVs (64%) were absent in gnomAD. Of these, 7 (4%) were predicted to be pathogenic by ≤2 tools, 3 of which we characterized functionally. There was no significant difference in current density between heterozygous KCNQ1-F127L, -P477L, or -L619M variant-containing channels compared to KCNQ1-WT. This study offers preliminary evidence for the demotion of 32 (13%) previously published LQT1 MVs. Of these, 29 were demoted because of their frequent sighting in gnomAD. Additionally, in silico analysis and in vitro functional studies have facilitated the demotion of 3 ultra-rare MVs (F127L, P477L, L619M). Copyright © 2017 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.

Involvement of Potassium and Cation Channels in Hippocampal Abnormalities of Embryonic Ts65Dn and Tc1 Trisomic Mice

PubMed Central

Stern, Shani; Segal, Menahem; Moses, Elisha

2015-01-01

Down syndrome (DS) mouse models exhibit cognitive deficits, and are used for studying the neuronal basis of DS pathology. To understand the differences in the physiology of DS model neurons, we used dissociated neuronal cultures from the hippocampi of Ts65Dn and Tc1 DS mice. Imaging of [Ca2+]i and whole cell patch clamp recordings were used to analyze network activity and single neuron properties, respectively. We found a decrease of ~ 30% in both fast (A-type) and slow (delayed rectifier) outward potassium currents. Depolarization of Ts65Dn and Tc1 cells produced fewer spikes than diploid cells. Their network bursts were smaller and slower than diploids, displaying a 40% reduction in Δf / f0 of the calcium signals, and a 30% reduction in propagation velocity. Additionally, Ts65Dn and Tc1 neurons exhibited changes in the action potential shape compared to diploid neurons, with an increase in the amplitude of the action potential, a lower threshold for spiking, and a sharp decrease of about 65% in the after-hyperpolarization amplitude. Numerical simulations reproduced the DS measured phenotype by variations in the conductance of the delayed rectifier and A-type, but necessitated also changes in inward rectifying and M-type potassium channels and in the hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. We therefore conducted whole cell patch clamp measurements of M-type potassium currents, which showed a ~ 90% decrease in Ts65Dn neurons, while HCN measurements displayed an increase of ~ 65% in Ts65Dn cells. Quantitative real-time PCR analysis indicates overexpression of 40% of KCNJ15, an inward rectifying potassium channel, contributing to the increased inhibition. We thus find that changes in several types of potassium channels dominate the observed DS model phenotype. PMID:26501103

A novel crystallization method for visualizing the membrane localization of potassium channels.

PubMed Central

Lopatin, A N; Makhina, E N; Nichols, C G

1998-01-01

The high permeability of K+ channels to monovalent thallium (Tl+) ions and the low solubility of thallium bromide salt were used to develop a simple yet very sensitive approach to the study of membrane localization of potassium channels. K+ channels (Kir1.1, Kir2.1, Kir2.3, Kv2.1), were expressed in Xenopus oocytes and loaded with Br ions by microinjection. Oocytes were then exposed to extracellular thallium. Under conditions favoring influx of Tl+ ions (negative membrane potential under voltage clamp, or high concentration of extracellular Tl+), crystals of TlBr, visible under low-power microscopy, formed under the membrane in places of high density of K+ channels. Crystals were not formed in uninjected oocytes, but were formed in oocytes expressing as little as 5 microS K+ conductance. The number of observed crystals was much lower than the estimated number of functional channels. Based on the pattern of crystal formation, K+ channels appear to be expressed mostly around the point of cRNA injection when injected either into the animal or vegetal hemisphere. In addition to this pseudopolarized distribution of K+ channels due to localized microinjection of cRNA, a naturally polarized (animal/vegetal side) distribution of K+ channels was also frequently observed when K+ channel cRNA was injected at the equator. A second novel "agarose-hemiclamp" technique was developed to permit direct measurements of K+ currents from different hemispheres of oocytes under two-microelectrode voltage clamp. This technique, together with direct patch-clamping of patches of membrane in regions of high crystal density, confirmed that the localization of TlBr crystals corresponded to the localization of functional K+ channels and suggested a clustered organization of functional channels. With appropriate permeant ion/counterion pairs, this approach may be applicable to the visualization of the membrane distribution of any functional ion channel. PMID:9591643

A novel crystallization method for visualizing the membrane localization of potassium channels.

PubMed

Lopatin, A N; Makhina, E N; Nichols, C G

1998-05-01

The high permeability of K+ channels to monovalent thallium (Tl+) ions and the low solubility of thallium bromide salt were used to develop a simple yet very sensitive approach to the study of membrane localization of potassium channels. K+ channels (Kir1.1, Kir2.1, Kir2.3, Kv2.1), were expressed in Xenopus oocytes and loaded with Br ions by microinjection. Oocytes were then exposed to extracellular thallium. Under conditions favoring influx of Tl+ ions (negative membrane potential under voltage clamp, or high concentration of extracellular Tl+), crystals of TlBr, visible under low-power microscopy, formed under the membrane in places of high density of K+ channels. Crystals were not formed in uninjected oocytes, but were formed in oocytes expressing as little as 5 microS K+ conductance. The number of observed crystals was much lower than the estimated number of functional channels. Based on the pattern of crystal formation, K+ channels appear to be expressed mostly around the point of cRNA injection when injected either into the animal or vegetal hemisphere. In addition to this pseudopolarized distribution of K+ channels due to localized microinjection of cRNA, a naturally polarized (animal/vegetal side) distribution of K+ channels was also frequently observed when K+ channel cRNA was injected at the equator. A second novel "agarose-hemiclamp" technique was developed to permit direct measurements of K+ currents from different hemispheres of oocytes under two-microelectrode voltage clamp. This technique, together with direct patch-clamping of patches of membrane in regions of high crystal density, confirmed that the localization of TlBr crystals corresponded to the localization of functional K+ channels and suggested a clustered organization of functional channels. With appropriate permeant ion/counterion pairs, this approach may be applicable to the visualization of the membrane distribution of any functional ion channel.

Design, Development, and Optimization of Dexibuprofen Microemulsion Based Transdermal Reservoir Patches for Controlled Drug Delivery

PubMed Central

Ali, Fatima Ramzan; Yousuf, Rabia Ismail; Ali, Syed Abid; Imtiaz, Muhammad Suleman; Bashir, Lubna; Naz, Shazia

2017-01-01

The aim of the study was to develop a reservoir-type transdermal patch for a controlled delivery of dexibuprofen and to evaluate its in vivo anti-inflammatory activity in Albino Wistar rats. In order to develop these patches, six formulations of dexibuprofen microemulsion comprising ethyl oleate, Tween 80: PG (2 : 1), and water were prepared by simplex lattice design and characterized. The reservoir compartment was filled with these microemulsions and in vitro release and skin permeation were assessed. The optimized patch was obtained on the basis of the responses: Q24 and flux. The impact of drug loading, surface area, membrane thickness, adhesive, and agitation speed on drug release and permeation was also studied. The skin sensitivity reaction and in vivo anti-inflammatory activity of optimized patch were evaluated. Stability study at three different temperatures for three months was carried out. The result suggests that a membrane based patch with zero-order release rate, Q24 of 79.13 ± 3.08%, and maximum flux of 331.17 µg/cm2h can be obtained exhibiting suitable anti-inflammatory activity with no visible skin sensitivity reaction. The outcomes of stability study recommend storage of patches at 4°C having shelf-life of 6.14 months. The study demonstrates that the reservoir-type transdermal patch of dexibuprofen microemulsion has a potential of delivering drug across skin in controlled manner with required anti-inflammatory activity. PMID:29090219

Modulation of inward rectifier potassium channel by toosendanin, a presynaptic blocker.

PubMed

Wang, Z F; Shi, Y L

2001-07-01

The effect of toosendanin, a presynaptic blocker, on the inward rectifier potassium channel (K(Kir)) of hippocampal CA1 pyramidal neurons of rats was studied by the single-channel patch-clamp technique. The results showed that toosendanin had an inhibitory effect on K(Kir) in an excised inside-out patch of the neuron under a symmetrical 150 mM K(+) condition. By decreasing the slower open time constant and increasing the slower close time constant, toosendanin (1x10(-6)-1x10(-4) g/ml) significantly reduced the open probability of the channel in a concentration-dependent manner. Meanwhile, a dose-dependent reduction in unitary conductance of the channel was also detected after toosendanin application. These data offer an explanation for toosendanin-induced facilitation of neurotransmitter release and antibotulismic effect of the drug.

Design and in vivo evaluation of a patch system based on thiolated polymers.

PubMed

Hoyer, Herbert; Greindl, Melanie; Bernkop-Schnürch, Andreas

2009-02-01

A new oral patch delivery system has been designed to increase the overall oral bioavailability of drugs within the gastrointestinal tract. The patch system consists of four layered films: a mucoadhesive matrix layer, a water insoluble backing layer, a middle layer and an enteric surface layer. The separation layer between the two matrix layers contained lactose, starch and confectioners' sugar. The matrix layer, exhibiting a diameter of 2.5 mm and a weight of 5 mg, comprised Polycarbophil-cysteine conjugate (49%), fluoresceine isothiocyanate-dextran (26%), glutathione (5%), and mannitol (20%). A standard tablet formulation consisting of the same matrix served as control. Entire fluoresceine isothiocyanate-dextran (FD(4)) was released from the delivery system within 2 h. For in vivo studies patch systems were administered orally to male Sprague-Dawley rats. Maximum FD(4) concentration in blood of the patch system was 46.1 +/- 8.9 ng/mL and was reached 3 h after administration. In contrast c(max) of control tablets displayed 50.5 +/- 14.9 ng/mL after 2 h and the absorption of FD(4) after administration in oral solution was negligible. The absolute bioavailability of orally administered patch systems and control tablets was 0.54% and 0.32% respectively. Results of this study indicate that a prolonged and higher oral bioavailability of FD(4) is obtained with patches than with tablets.

Pre-clinical evaluation of novel mucoadhesive bilayer patches for local delivery of clobetasol-17-propionate to the oral mucosa.

PubMed

Colley, H E; Said, Z; Santocildes-Romero, M E; Baker, S R; D'Apice, K; Hansen, J; Madsen, L Siim; Thornhill, M H; Hatton, P V; Murdoch, C

2018-06-14

Oral lichen planus (OLP) and recurrent aphthous stomatitis (RAS) are chronic inflammatory conditions often characterised by erosive and/or painful oral lesions that have a considerable impact on quality of life. Current treatment often necessitates the use of steroids in the form of mouthwashes, creams or ointments, but these are often ineffective due to inadequate drug contact times with the lesion. Here we evaluate the performance of novel mucoadhesive patches for targeted drug delivery. Electrospun polymeric mucoadhesive patches were produced and characterised for their physical properties and cytotoxicity before evaluation of residence time and acceptability in a human feasibility study. Clobetasol-17-propionate incorporated into the patches was released in a sustained manner in both tissue-engineered oral mucosa and ex vivo porcine mucosa. Clobetasol-17 propionate-loaded patches were further evaluated for residence time and drug release in an in vivo animal model and demonstrated prolonged adhesion and drug release at therapeutic-relevant doses and time points. These data show that electrospun patches are adherent to mucosal tissue without causing tissue damage, and can be successfully loaded with and release clinically active drugs. These patches hold great promise for the treatment of oral conditions such as OLP and RAS, and potentially many other oral lesions. Copyright © 2018. Published by Elsevier Ltd.

A novel NaV1.5 voltage sensor mutation associated with severe atrial and ventricular arrhythmias.

PubMed

Wang, Hong-Gang; Zhu, Wandi; Kanter, Ronald J; Silva, Jonathan R; Honeywell, Christina; Gow, Robert M; Pitt, Geoffrey S

2016-03-01

Inherited autosomal dominant mutations in cardiac sodium channels (NaV1.5) cause various arrhythmias, such as long QT syndrome and Brugada syndrome. Although dozens of mutations throughout the protein have been reported, there are few reported mutations within a voltage sensor S4 transmembrane segment and few that are homozygous. Here we report analysis of a novel lidocaine-sensitive recessive mutation, p.R1309H, in the NaV1.5 DIII/S4 voltage sensor in a patient with a complex arrhythmia syndrome. We expressed the wild type or mutant NaV1.5 heterologously for analysis with the patch-clamp and voltage clamp fluorometry (VCF) techniques. p.R1309H depolarized the voltage-dependence of activation, hyperpolarized the voltage-dependence of inactivation, and slowed recovery from inactivation, thereby reducing the channel availability at physiologic membrane potentials. Additionally, p.R1309H increased the "late" Na(+) current. The location of the mutation in DIIIS4 prompted testing for a gating pore current. We observed an inward current at hyperpolarizing voltages that likely exacerbates the loss-of-function defects at resting membrane potentials. Lidocaine reduced the gating pore current. The p.R1309H homozygous NaV1.5 mutation conferred both gain-of-function and loss-of-function effects on NaV1.5 channel activity. Reduction of a mutation-induced gating pore current by lidocaine suggested a therapeutic mechanism. Copyright © 2016 Elsevier Ltd. All rights reserved.

Optimization and charaterization of repaglinide biodegradable polymeric nanoparticle loaded transdermal patchs: in vitro and in vivo studies.

PubMed

Vijayan, V; Reddy, K Ravindra; Sakthivel, S; Swetha, C

2013-11-01

Biodegradable polymeric nanoparticles loaded Repaglinide were prepared by solvent extraction method. In this method chitosan, PLA and PCL were employed to prepare Repaglinide polymeric nanoparticles. Some of the formulation parameters were optimized to obtain high quality nanoparticles. The particles were spherical shape with sizes of 108.6 ± 3.4 nm to 220.6 ± 1.2 nm and the poly dispersity indexes were in the range of 0.06 to 0.44. The zeta potential was in the range between - 16.48 ± 2.02 and 30.52 ± 3.20 mV. The percentage entrapment efficiency (EE%) was 81.4 ± 1.8% to 92.7 ± 1.4%. The drug release behavior was studied by externally sink method and the release pattern of drug was found to follow zero order, Higuchi and Peppas equations. The optimized PLA-Repaglinide nanoparticles were loaded in Methocel transdermal patches. These transdermal patches were evaluated by physiochemical parameters, in vitro, ex vivo and in vivo studies. Based on in vivo hypoglycemic results, bioavailability parameters like AUC, AUMC, Cmax, Tmax, MRT, t1/2 and relative bioavailability were found to be 2218.88 μIU/mL/h, 381630.3 μIU/mL/h, 41.88 μIU/mL, 36 h, 83.24h, and 52.79 h respectively. The transdermal patch containing Repaglinide nanoparticles showed 76 fold effective than conventional oral administrations. Copyright © 2013 Elsevier B.V. All rights reserved.

Effect of serum nicotine level on posterior spinal fusion in an in vivo rabbit model.

PubMed

Daffner, Scott D; Waugh, Stacey; Norman, Timothy L; Mukherjee, Nilay; France, John C

2015-06-01

Cigarette smoking has a deleterious effect on spinal fusion. Although some studies have implied that nicotine is primarily responsible for poor fusion outcomes, other studies suggest that nicotine may actually stimulate bone growth. Hence, there may be a dose-dependent effect of nicotine on posterior spinal fusion outcomes. The purpose of this study was to determine if such a relationship could be shown in an in vivo rabbit model. This is a prospective in vivo animal study. Twenty-four adult male New Zealand white rabbits were randomly divided into four groups. All groups received a single-level posterolateral, intertransverse process fusion at L5-L6 with autologous iliac crest bone. One group served as controls and only underwent the spine fusion surgery. Three groups received 5.25-, 10.5-, and 21-mg nicotine patches, respectively, for 5 weeks. Serum nicotine levels were recorded for each group. All animals were euthanized 5 weeks postoperatively, and spinal fusions were evaluated radiographically, by manual palpation, and biomechanically. Statistical analysis evaluated the dose response effect of outcomes variables and nicotine dosage. This study was supported by a portion of a $100,000 grant from the Orthopaedic Research and Education Foundation. Author financial disclosures were completed in accordance with the journal's guidelines; there were no conflicts of interests disclosed that would have led to bias in this work. The average serum levels of nicotine from the different patches were 7.8±1.9 ng/mL for the 5.25-mg patch group; 99.7±17.7 ng/mL for the 10.5-mg patch group; and 149.1±24.6 ng/mL for the 21-mg patch group. The doses positively correlated with serum concentrations of nicotine (correlation coefficient=0.8410, p<.001). The 5.25-mg group provided the best fusion rate, trabeculation, and stiffness. On the basis of the palpation tests, the fusion rates were control (50%), 5.25 mg (80%), 10.5 mg (50%), and 21 mg (42.8%). Radiographic assessment of trabeculation and bone incorporation and biomechanical analysis of bending stiffness ratio were also greatest in the 5.25-mg group. Radiographic evaluation showed a significant (p=.0446) quadratic effect of nicotine dose on spinal fusion. The effects of nicotine on spinal fusion are complex, may be dose dependent, and may not always be detrimental. The uniformly negative effects of smoking reported in patients undergoing spinal fusion may possibly be attributed to the other components of cigarette smoke. Copyright © 2015 Elsevier Inc. All rights reserved.

Design of a Drug-in-Adhesive Transdermal Patch for Risperidone: Effect of Drug-Additive Interactions on the Crystallization Inhibition and In Vitro/In Vivo Correlation Study.

PubMed

Weng, Wei; Quan, Peng; Liu, Chao; Zhao, Hanqing; Fang, Liang

2016-10-01

The purpose of this work was to develop and design an appropriate drug-in-adhesive patch for transdermal delivery of risperidone (RISP). Various formulation factors were investigated by in vitro permeation study using excised rabbit skin. Increasing the drug concentration in the pressure sensitive adhesive (PSA) was used to enhance the drug permeation. To overcome the high crystallization tendency of the patch, several crystallization inhibitors such as PVP, PEG, and surfactants and fatty acids were evaluated by microscopy study. The mechanism of crystallization inhibition was investigated by differential scanning calorimetry, nuclear magnetic resonance spectrometer, and FT-IR studies. RISP and its active metabolite were determined after topical application of the optimized transdermal patch, and the in vivo pharmacokinetic parameters were compared with the intravenous administration group. The microscopy study indicated that fatty acid greatly inhibited the crystallization of RISP in PSA. The inhibition was attributed to the drug-additive interaction between amino group of RISP and the carboxyl group of fatty acid which was further confirmed by (1)H-NMR and FT-IR studies. The optimal permeation profile was obtained with the patches containing 5% RISP and 5% oleic acid in Duro-Tak(®) 87-2287. The in vivo pharmacokinetic study exhibited a sustained absorption and metabolism profile and well correlated with the in vitro permeation data. Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

Experimental and computational laser tissue welding using a protein patch.

PubMed

Small, W; Heredia, N J; Maitland, D J; Eder, D C; Celliers, P M; Da Silva, L B; London, R A; Matthews, D L

1998-01-01

An in vitro study of laser tissue welding mediated with a dye-enhanced protein patch was conducted. Fresh sections of porcine aorta were used for the experiments. Arteriotomies were treated using an indocyanine green dye-enhanced collagen patch activated by an 805-nm continuous-wave fiber-delivered diode laser. Temperature histories of the surface of the weld site were obtained using a hollow glass optical fiber-based two-color infrared thermometer. The experimental effort was complemented by simulations with the LATIS (LAser-TISsue) computer code, which uses coupled Monte Carlo, thermal transport, and mass transport models. Comparison of simulated and experimental thermal data indicated that evaporative cooling clamped the surface temperature of the weld site below 100 °C. For fluences of approximately 200 J/cm2, peak surface temperatures averaged 74°C and acute burst strengths consistently exceeded 0.14×106 dyn/cm (hoop tension). The combination of experimental and simulation results showed that the inclusion of water transport and evaporative losses in the computer code has a significant impact on the thermal distributions and hydration levels throughout the tissue volume. The solid-matrix protein patch provided a means of controllable energy delivery and yielded consistently strong welds. © 1998 Society of Photo-Optical Instrumentation Engineers.

Electrophysiological and morphological properties of pre-autonomic neurones in the rat hypothalamic paraventricular nucleus.

PubMed

Stern, J E

2001-11-15

1. The cellular properties of pre-autonomic neurones in the hypothalamic paraventricular nucleus (PVN) were characterized by combining in vivo retrograde tracing techniques, in vitro patch-clamp recordings and three-dimensional reconstruction of recorded neurones in adult hypothalamic slices. 2. The results showed that PVN pre-autonomic neurones constitute a heterogeneous neuronal population. Based on morphological criteria, neurones were classified into three subgroups. Type A neurones (52 %) were located in the ventral parvocellular (PaV) subnucleus, and showed an oblique orientation with respect to the third ventricle (3V). Type B neurones (25 %) were located in the posterior parvocellular (PaPo) subnucleus, and were oriented perpendicularly with respect to the 3V. Type C neurones (23 %) were located in both the PaPo (82 %) and the PaV (18 %) subnuclei, and displayed a concentric dendritic configuration. 3. A morphometric analysis revealed significant differences in the dendritic configuration among neuronal types. Type B neurones had the most complex dendritic arborization, with longer and more branching dendritic trees. 4. Several electrophysiological properties, including cell input resistance and action potential waveforms, differed between cell types, suggesting that the expression and/or properties of a variety of ion channels differ between neuronal types. 5. Common features of PVN pre-autonomic neurones included the expression of a low-threshold spike and strong inward rectification. These properties distinguished them from neighbouring magnocellular vasopressin neurones. 6. In summary, these results indicate that PVN pre-autonomic neurones constitute a heterogeneous neuronal population, and provide a cellular basis for the study of their involvement in the pathophysiology of hypertension and congestive heart failure disorders.

Electrophysiological and morphological properties of pre-autonomic neurones in the rat hypothalamic paraventricular nucleus

PubMed Central

Stern, Javier E

2001-01-01

The cellular properties of pre-autonomic neurones in the hypothalamic paraventricular nucleus (PVN) were characterized by combining in vivo retrograde tracing techniques, in vitro patch-clamp recordings and three-dimensional reconstruction of recorded neurones in adult hypothalamic slices. The results showed that PVN pre-autonomic neurones constitute a heterogeneous neuronal population. Based on morphological criteria, neurones were classified into three subgroups. Type A neurones (52 %) were located in the ventral parvocellular (PaV) subnucleus, and showed an oblique orientation with respect to the third ventricle (3V). Type B neurones (25 %) were located in the posterior parvocellular (PaPo) subnucleus, and were oriented perpendicularly with respect to the 3V. Type C neurones (23 %) were located in both the PaPo (82 %) and the PaV (18 %) subnuclei, and displayed a concentric dendritic configuration. A morphometric analysis revealed significant differences in the dendritic configuration among neuronal types. Type B neurones had the most complex dendritic arborization, with longer and more branching dendritic trees. Several electrophysiological properties, including cell input resistance and action potential waveforms, differed between cell types, suggesting that the expression and/or properties of a variety of ion channels differ between neuronal types. Common features of PVN pre-autonomic neurones included the expression of a low-threshold spike and strong inward rectification. These properties distinguished them from neighbouring magnocellular vasopressin neurones. In summary, these results indicate that PVN pre-autonomic neurones constitute a heterogeneous neuronal population, and provide a cellular basis for the study of their involvement in the pathophysiology of hypertension and congestive heart failure disorders. PMID:11711570

Fish scale-derived collagen patch promotes growth of blood and lymphatic vessels in vivo.

PubMed

Wang, Jun Kit; Yeo, Kim Pin; Chun, Yong Yao; Tan, Timothy Thatt Yang; Tan, Nguan Soon; Angeli, Véronique; Choong, Cleo

2017-11-01

In this study, Type I collagen was extracted from fish scales asa potential alternative source of collagen for tissue engineering applications. Since unmodified collagen typically has poor mechanical and degradation stability both in vitro and in vivo, additional methylation modification and 1,4-butanediol diglycidyl ether (BDE) crosslinking steps were used to improve the physicochemical properties of fish scale-derived collagen. Subsequently, in vivo studies using a murine model demonstrated the biocompatibility of the different fish scale-derived collagen patches. In general, favorable integration of the collagen patches to the surrounding tissues, with good infiltration of cells, blood vessels (BVs) and lymphatic vessels (LVs) were observed under growth factor-free conditions. Interestingly, significantly higher (p<0.05) number of LVs was found to be more abundant around collagen patches with methylation modification and BDE crosslinking. Overall, we have demonstrated the potential application of fish scale-derived collagen as a promising scaffolding material for various biomedical applications. Currently the most common sources of collagen are of bovine and porcine origins, although the industrial use of collagen obtained from non-mammalian species is growing in importance, particularly since they have a lower risk of disease transmission and are not subjected to any cultural or religious constraints. However, unmodified collagen typically has poor mechanical and degradation stability both in vitro and in vivo. Hence, in this study, Type I collagen was successfully extracted from fish scales and chemically modified and crosslinked. In vitro studies showed overall improvement in the physicochemical properties of the material, whilst in vivo implantation studies showed improvements in the growth of blood and lymphatic host vessels in the vicinity of the implants. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhou, Min; Yu, Yun; Hu, Keke

Investigating the collisions of individual metal nanoparticles (NPs) with electrodes can provide new insights into their electrocatalytic behavior, mass transport, and interactions with surfaces. Here we report a new experimental setup for studying NP collisions based on the use of carbon nanopipettes to enable monitoring multiple collision events involving the same NP captured inside the pipet cavity. A patch clamp amplifier capable of measuring pA-range currents on the microsecond time scale with a very low noise and stable background was used to record the collision transients. The analysis of current transients produced by oxidation of hydrogen peroxide at one IrOxmore » NP provided information about the origins of deactivation of catalytic NPs and the effects of various experimental conditions on the collision dynamics. Lastly, high-resolution TEM of carbon pipettes was used to attain better understanding of the NP capture and collisions.« less

Effects of tacrolimus on action potential configuration and transmembrane ion currents in canine ventricular cells.

PubMed

Szabó, László; Szentandrássy, Norbert; Kistamás, Kornél; Hegyi, Bence; Ruzsnavszky, Ferenc; Váczi, Krisztina; Horváth, Balázs; Magyar, János; Bányász, Tamás; Pál, Balázs; Nánási, Péter P

2013-03-01

Tacrolimus is a commonly used immunosuppressive agent which causes cardiovascular complications, e.g., hypertension and hypertrophic cardiomyopathy. In spite of it, there is little information on the cellular cardiac effects of the immunosuppressive agent tacrolimus in larger mammals. In the present study, therefore, the concentration-dependent effects of tacrolimus on action potential morphology and the underlying ion currents were studied in canine ventricular cardiomyocytes. Standard microelectrode, conventional whole cell patch clamp, and action potential voltage clamp techniques were applied in myocytes enzymatically dispersed from canine ventricular myocardium. Tacrolimus (3-30 μM) caused a concentration-dependent reduction of maximum velocity of depolarization and repolarization, action potential amplitude, phase-1 repolarization, action potential duration, and plateau potential, while no significant change in the resting membrane potential was observed. Conventional voltage clamp experiments revealed that tacrolimus concentrations ≥3 μM blocked a variety of ion currents, including I(Ca), I(to), I(K1), I(Kr), and I(Ks). Similar results were obtained under action potential voltage clamp conditions. These effects of tacrolimus developed rapidly and were fully reversible upon washout. The blockade of inward currents with the concomitant shortening of action potential duration in canine myocytes is the opposite of those observed previously with tacrolimus in small rodents. It is concluded that although tacrolimus blocks several ion channels at higher concentrations, there is no risk of direct interaction with cardiac ion channels when applying tacrolimus in therapeutic concentrations.

Metabolic Therapy for Temporal Lobe Epilepsy in a Dish: Investigating Mechanisms of Ketogenic Diet using Electrophysiological Recordings in Hippocampal Slices

PubMed Central

Kawamura, Masahito Jr.; Ruskin, David N.; Masino, Susan A.

2016-01-01

The hippocampus is prone to epileptic seizures and is a key brain region and experimental platform for investigating mechanisms associated with the abnormal neuronal excitability that characterizes a seizure. Accordingly, the hippocampal slice is a common in vitro model to study treatments that may prevent or reduce seizure activity. The ketogenic diet is a metabolic therapy used to treat epilepsy in adults and children for nearly 100 years; it can reduce or eliminate even severe or refractory seizures. New insights into its underlying mechanisms have been revealed by diverse types of electrophysiological recordings in hippocampal slices. Here we review these reports and their relevant mechanistic findings. We acknowledge that a major difficulty in using hippocampal slices is the inability to reproduce precisely the in vivo condition of ketogenic diet feeding in any in vitro preparation, and progress has been made in this in vivo/in vitro transition. Thus far at least three different approaches are reported to reproduce relevant diet effects in the hippocampal slices: (1) direct application of ketone bodies; (2) mimicking the ketogenic diet condition during a whole-cell patch-clamp technique; and (3) reduced glucose incubation of hippocampal slices from ketogenic diet–fed animals. Significant results have been found with each of these methods and provide options for further study into short- and long-term mechanisms including Adenosine triphosphate (ATP)-sensitive potassium (KATP) channels, vesicular glutamate transporter (VGLUT), pannexin channels and adenosine receptors underlying ketogenic diet and other forms of metabolic therapy. PMID:27847463

M2 receptors exert analgesic action on DRG sensory neurons by negatively modulating VR1 activity.

PubMed

De Angelis, Federica; Marinelli, Sara; Fioretti, Bernard; Catacuzzeno, Luigi; Franciolini, Fabio; Pavone, Flaminia; Tata, Ada Maria

2014-06-01

The peripheral application of the M2 cholinergic agonist arecaidine on sensory nerve endings shows anti-nociceptive properties. In this work, we analyze in vitro, the mechanisms downstream M2 receptor activation causing the analgesic effects, and in vivo the effects produced by M2 agonist arecaidine administration on nociceptive responses in a murine model of nerve growth factor (NGF)-induced pain. Cultured DRG neurons treated with arecaidine showed a decreased level of VR1 and SP transcripts. Conversely, we found an increased expression of VR1 and SP transcripts in DRG from M2/M4(-/-) mice compared to WT and M1(-/-) mice, confirming the inhibitory effect in particular of M2 receptors on SP and VR1 expression. Patch-clamp experiments in the whole-cell configuration showed that arecaidine treatment caused a reduction of the fraction of capsaicin-responsive cells, without altering the mean capsaicin-activated current in responsive cells. We also demonstrated that arecaidine prevents PKCϵ translocation to the plasma membrane after inflammatory agent stimulation, mainly in medium-small sensory neurons. Finally, in mice, we have observed that intraperitoneal injection of arecaidine reduces VR1 expression blocking hyperalgesia and allodynia caused by NGF intraplantar administration. In conclusion, our data demonstrate that in vivo M2 receptor activation induces desensitization to mechanical and heat stimuli by a down-regulation of VR1 expression and by the inhibition of PKCϵ activity hindering its translocation to the plasma membrane, as suggested by in vitro experiments. © 2013 Wiley Periodicals, Inc.

Carbon monoxide induces cardiac arrhythmia via induction of the late Na+ current.

PubMed

Dallas, Mark L; Yang, Zhaokang; Boyle, John P; Boycott, Hannah E; Scragg, Jason L; Milligan, Carol J; Elies, Jacobo; Duke, Adrian; Thireau, Jérôme; Reboul, Cyril; Richard, Sylvain; Bernus, Olivier; Steele, Derek S; Peers, Chris

2012-10-01

Clinical reports describe life-threatening cardiac arrhythmias after environmental exposure to carbon monoxide (CO) or accidental CO poisoning. Numerous case studies describe disruption of repolarization and prolongation of the QT interval, yet the mechanisms underlying CO-induced arrhythmias are unknown. To understand the cellular basis of CO-induced arrhythmias and to identify an effective therapeutic approach. Patch-clamp electrophysiology and confocal Ca(2+) and nitric oxide (NO) imaging in isolated ventricular myocytes was performed together with protein S-nitrosylation to investigate the effects of CO at the cellular and molecular levels, whereas telemetry was used to investigate effects of CO on electrocardiogram recordings in vivo. CO increased the sustained (late) component of the inward Na(+) current, resulting in prolongation of the action potential and the associated intracellular Ca(2+) transient. In more than 50% of myocytes these changes progressed to early after-depolarization-like arrhythmias. CO elevated NO levels in myocytes and caused S-nitrosylation of the Na(+) channel, Na(v)1.5. All proarrhythmic effects of CO were abolished by the NO synthase inhibitor l-NAME, and reversed by ranolazine, an inhibitor of the late Na(+) current. Ranolazine also corrected QT variability and arrhythmias induced by CO in vivo, as monitored by telemetry. Our data indicate that the proarrhythmic effects of CO arise from activation of NO synthase, leading to NO-mediated nitrosylation of Na(V)1.5 and to induction of the late Na(+) current. We also show that the antianginal drug ranolazine can abolish CO-induced early after-depolarizations, highlighting a novel approach to the treatment of CO-induced arrhythmias.

Reversal of Cocaine-Associated Synaptic Plasticity in Medial Prefrontal Cortex Parallels Elimination of Memory Retrieval.

PubMed

Otis, James M; Mueller, Devin

2017-09-01

Addiction is characterized by abnormalities in prefrontal cortex that are thought to allow drug-associated cues to drive compulsive drug seeking and taking. Identification and reversal of these pathologic neuroadaptations are therefore critical for treatment of addiction. Previous studies using rodents reveal that drugs of abuse cause dendritic spine plasticity in prelimbic medial prefrontal cortex (PL-mPFC) pyramidal neurons, a phenomenon that correlates with the strength of drug-associated memories in vivo. Thus, we hypothesized that cocaine-evoked plasticity in PL-mPFC may underlie cocaine-associated memory retrieval, and therefore disruption of this plasticity would prevent retrieval. Indeed, using patch clamp electrophysiology we find that cocaine place conditioning increases excitatory presynaptic and postsynaptic transmission in rat PL-mPFC pyramidal neurons. This was accounted for by increases in excitatory presynaptic release, paired-pulse facilitation, and increased AMPA receptor transmission. Noradrenergic signaling is known to maintain glutamatergic plasticity upon reactivation of modified circuits, and we therefore next determined whether inhibition of noradrenergic signaling during memory reactivation would reverse the cocaine-evoked plasticity and/or disrupt the cocaine-associated memory. We find that administration of the β-adrenergic receptor antagonist propranolol before memory retrieval, but not after (during memory reconsolidation), reverses the cocaine-evoked presynaptic and postsynaptic modifications in PL-mPFC and causes long-lasting memory impairments. Taken together, these data reveal that cocaine-evoked synaptic plasticity in PL-mPFC is reversible in vivo, and suggest a novel strategy that would allow normalization of prefrontal circuitry in addiction.

In vitro and ex vivo screening of candidate therapeutics to restore neurotransmission in nerve terminals intoxicated by botulinum neurotoxin serotype A1.

PubMed

Beske, Phillip H; Bradford, Aaron B; Hoffman, Katie M; Mason, Sydney J; McNutt, Patrick M

2018-06-01

Botulinum neurotoxins (BoNTs) are exceedingly potent neurological poisons that block cholinergic release in the peripheral nervous system and cause death by asphyxiation. While post-exposure prophylaxis can effectively eliminate toxin in the bloodstream, there are no clinically effective treatments to prevent or reverse disease once BoNT has entered the neuron. To address the need for post-symptomatic countermeasures, we designed and developed an in vitro assay based on whole-cell, patch-clamp electrophysiological monitoring of miniature excitatory post-synaptic currents in synaptically active murine embryonic stem cell-derived neurons. This synaptic function-based assay was used to assess the efficacy of rationally selected drugs to restore neurotransmission in neurons comprehensively intoxicated by BoNT/A. Based on clinical reports suggesting that elevated Ca 2+ signaling promotes symptomatic relief from botulism, we identified seven candidate drugs that modulate presynaptic Ca 2+ signaling and assessed their ability to reverse BoNT/A-induced synaptic blockade. The most effective drugs from the screen were found to phasically agonize voltage-gated calcium channel (VGCC) activity. Lead candidates were then applied to ex vivo studies in BoNT/A-paralyzing mouse phrenic nerve-hemidiaphragm (PND) preparations. Treatment of PNDs with VGCC agonists after paralytic onset transiently potentiated nerve-elicited muscle contraction and delayed progression to neuromuscular failure. Collectively, this study suggests that Ca 2+ -modulating drugs represent a novel symptomatic treatment for neuromuscular paralysis following BoNT/A poisoning. Published by Elsevier Ltd.

Arctigenin, a potential anti-arrhythmic agent, inhibits aconitine-induced arrhythmia by regulating multi-ion channels.

PubMed

Zhao, Zhenying; Yin, Yongqiang; Wu, Hong; Jiang, Min; Lou, Jianshi; Bai, Gang; Luo, Guo'an

2013-01-01

Arctigenin possesses biological activities, but its underlying mechanisms at the cellular and ion channel levels are not completely understood. Therefore, the present study was designed to identify the anti-arrhythmia effect of arctigenin in vivo, as well as its cellular targets and mechanisms. A rat arrhythmia model was established via continuous aconitine infusion, and the onset times of ventricular premature contraction, ventricular tachycardia and death were recorded. The Action Potential Duration (APD), sodium current (I(Na)), L-type calcium current (I(Ca, L)) and transient outward potassium current (I(to)) were measured and analysed using a patch-clamp recording technique in normal rat cardiomyocytes and myocytes of arrhythmia aconitine-induced by. Arctigenin significantly delayed the arrhythmia onset in the aconitine-induced rat model. The 50% and 90% repolarisations (APD50 and APD90) were shortened by 100 µM arctigenin; the arctigenin dose also inhibited the prolongation of APD50 and APD90 caused by 1 µM aconitine. Arctigenin inhibited I(Na) and I(Ca,L) and attenuated the aconitine-increased I(Na) and I(Ca,L) by accelerating the activation process and delaying the inactivation process. Arctigenin enhanced Ito by facilitating the activation process and delaying the inactivation process, and recoverd the decreased Ito induced by aconitine. Arctigenin has displayed anti-arrhythmia effects, both in vivo and in vitro. In the context of electrophysiology, I(Na), I(Ca, L), and I(to) may be multiple targets of arctigenin, leading to its antiarrhythmic effect. © 2013 S. Karger AG, Basel.

Artemin growth factor increases nicotinic cholinergic receptor subunit expression and activity in nociceptive sensory neurons.

PubMed

Albers, Kathryn M; Zhang, Xiu Lin; Diges, Charlotte M; Schwartz, Erica S; Yang, Charles I; Davis, Brian M; Gold, Michael S

2014-05-22

Artemin (Artn), a member of the glial cell line-derived growth factor (GDNF) family, supports the development and function of a subpopulation of peptidergic, TRPV1-positive sensory neurons. Artn (enovin, neublastin) is elevated in inflamed tissue and its injection in skin causes transient thermal hyperalgesia. A genome wide expression analysis of trigeminal ganglia of mice that overexpress Artn in the skin (ART-OE mice) showed elevation in nicotinic acetylcholine receptor (nAChR) subunits, suggesting these ion channels contribute to Artn-induced sensitivity. Here we have used gene expression, immunolabeling, patch clamp electrophysiology and behavioral testing assays to investigate the link between Artn, nicotinic subunit expression and thermal hypersensitivity. Reverse transcriptase-PCR validation showed increased levels of mRNAs encoding the nAChR subunits α3 (13.3-fold), β3 (4-fold) and β4 (7.7-fold) in trigeminal ganglia and α3 (4-fold) and β4 (2.8-fold) in dorsal root ganglia (DRG) of ART-OE mice. Sensory ganglia of ART-OE mice had increased immunoreactivity for nAChRα3 and exhibited increased overlap in labeling with GFRα3-positive neurons. Patch clamp analysis of back-labeled cutaneous afferents showed that while the majority of nicotine-evoked currents in DRG neurons had biophysical and pharmacological properties of α7-subunit containing nAChRs, the Artn-induced increase in α3 and β4 subunits resulted in functional channels. Behavioral analysis of ART-OE and wildtype mice showed that Artn-induced thermal hyperalgesia can be blocked by mecamylamine or hexamethonium. Complete Freund's adjuvant (CFA) inflammation of paw skin, which causes an increase in Artn in the skin, also increased the level of nAChR mRNAs in DRG. Finally, the increase in nAChRs transcription was not dependent on the Artn-induced increase in TRPV1 or TRPA1 in ART-OE mice since nAChRs were elevated in ganglia of TRPV1/TRPA1 double knockout mice. These findings suggest that Artn regulates the expression and composition of nAChRs in GFRα3 nociceptors and that these changes contribute to the thermal hypersensitivity that develops in response to Artn injection and perhaps to inflammation.

Beta-Estradiol Regulates Voltage-Gated Calcium Channels and Estrogen Receptors in Telocytes from Human Myometrium.

PubMed

Banciu, Adela; Banciu, Daniel Dumitru; Mustaciosu, Cosmin Catalin; Radu, Mihai; Cretoiu, Dragos; Xiao, Junjie; Cretoiu, Sanda Maria; Suciu, Nicolae; Radu, Beatrice Mihaela

2018-05-09

Voltage-gated calcium channels and estrogen receptors are essential players in uterine physiology, and their association with different calcium signaling pathways contributes to healthy and pathological conditions of the uterine myometrium. Among the properties of the various cell subtypes present in human uterine myometrium, there is increasing evidence that calcium oscillations in telocytes (TCs) contribute to contractile activity and pregnancy. Our study aimed to evaluate the effects of beta-estradiol on voltage-gated calcium channels and estrogen receptors in TCs from human uterine myometrium and to understand their role in pregnancy. For this purpose, we employed patch-clamp recordings, ratiometric Fura-2-based calcium imaging analysis, and qRT-PCR techniques for the analysis of cultured human myometrial TCs derived from pregnant and non-pregnant uterine samples. In human myometrial TCs from both non-pregnant and pregnant uterus, we evidenced by qRT-PCR the presence of genes encoding for voltage-gated calcium channels (Cav3.1, Ca3.2, Cav3.3, Cav2.1), estrogen receptors (ESR1, ESR2, GPR30), and nuclear receptor coactivator 3 (NCOA3). Pregnancy significantly upregulated Cav3.1 and downregulated Cav3.2, Cav3.3, ESR1, ESR2, and NCOA3, compared to the non-pregnant condition. Beta-estradiol treatment (24 h, 10, 100, 1000 nM) downregulated Cav3.2, Cav3.3, Cav1.2, ESR1, ESR2, GRP30, and NCOA3 in TCs from human pregnant uterine myometrium. We also confirmed the functional expression of voltage-gated calcium channels by patch-clamp recordings and calcium imaging analysis of TCs from pregnant human myometrium by perfusing with BAY K8644, which induced calcium influx through these channels. Additionally, we demonstrated that beta-estradiol (1000 nM) antagonized the effect of BAY K8644 (2.5 or 5 µM) in the same preparations. In conclusion, we evidenced the presence of voltage-gated calcium channels and estrogen receptors in TCs from non-pregnant and pregnant human uterine myometrium and their gene expression regulation by beta-estradiol in pregnant conditions. Further exploration of the calcium signaling in TCs and its modulation by estrogen hormones will contribute to the understanding of labor and pregnancy mechanisms and to the development of effective strategies to reduce the risk of premature birth.

On the cellular site of two-pore channel TPC1 action in the Poaceae.

PubMed

Dadacz-Narloch, Beata; Kimura, Sachie; Kurusu, Takamitsu; Farmer, Edward E; Becker, Dirk; Kuchitsu, Kazuyuki; Hedrich, Rainer

2013-11-01

The slow vacuolar (SV) channel has been characterized in different dicots by patch-clamp recordings. This channel represents the major cation conductance of the largest organelle in most plant cells. Studies with the tpc1-2 mutant of the model dicot plant Arabidopsis thaliana identified the SV channel as the product of the TPC1 gene. By contrast, research on rice and wheat TPC1 suggested that the monocot gene encodes a plasma membrane calcium-permeable channel. To explore the site of action of grass TPC1 channels, we expressed OsTPC1 from rice (Oryza sativa) and TaTPC1 from wheat (Triticum aestivum) in the background of the Arabidopsis tpc1-2 mutant. Cross-species tpc1 complementation and patch-clamping of vacuoles using Arabidopsis and rice tpc1 null mutants documented that both monocot TPC1 genes were capable of rescuing the SV channel deficit. Vacuoles from wild-type rice but not the tpc1 loss-of-function mutant harbor SV channels exhibiting the hallmark properties of dicot TPC1/SV channels. When expressed in human embryonic kidney (HEK293) cells OsTPC1 was targeted to Lysotracker-Red-positive organelles. The finding that the rice TPC1, just like those from the model plant Arabidopsis and even animal cells, is localized and active in lyso-vacuolar membranes associates this cation channel species with endomembrane function. © 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.

Characterization of Two-Pore Channel 2 by Nuclear Membrane Electrophysiology

PubMed Central

Lee, Claire Shuk-Kwan; Tong, Benjamin Chun-Kit; Cheng, Cecily Wing-Hei; Hung, Harry Chun-Hin; Cheung, King-Ho

2016-01-01

Lysosomal calcium (Ca2+) release mediated by NAADP triggers signalling cascades that regulate many cellular processes. The identification of two-pore channel 2 (TPC2) as the NAADP receptor advances our understanding of lysosomal Ca2+ signalling, yet the lysosome is not amenable to traditional patch-clamp electrophysiology. Previous attempts to record TPC2 single-channel activity put TPC2 outside its native environment, which not reflect TPC2’s true physiological properties. To test the feasibility of using nuclear membrane electrophysiology for TPC2 channel characterization, we constructed a stable human TPC2-expressing DT40TKO cell line that lacks endogenous InsP3R and RyR (DT40TKO-hTPC2). Immunostaining revealed hTPC2 expression on the ER and nuclear envelope. Intracellular dialysis of NAADP into Fura-2-loaded DT40TKO-hTPC2 cells elicited cytosolic Ca2+ transients, suggesting that hTPC2 was functionally active. Using nuclear membrane electrophysiology, we detected a ~220 pS single-channel current activated by NAADP with K+ as the permeant ion. The detected single-channel recordings displayed a linear current-voltage relationship, were sensitive to Ned-19 inhibition, were biphasically regulated by NAADP concentration, and regulated by PKA phosphorylation. In summary, we developed a cell model for the characterization of the TPC2 channel and the nuclear membrane patch-clamp technique provided an alternative approach to rigorously investigate the electrophysiological properties of TPC2 with minimal manipulation. PMID:26838264

Signaling of Pigment-Dispersing Factor (PDF) in the Madeira Cockroach Rhyparobia maderae

PubMed Central

Funk, Nico W.; Giese, Maria; Baz, El-Sayed; Stengl, Monika

2014-01-01

The insect neuropeptide pigment-dispersing factor (PDF) is a functional ortholog of vasoactive intestinal polypeptide, the coupling factor of the mammalian circadian pacemaker. Despite of PDF's importance for synchronized circadian locomotor activity rhythms its signaling is not well understood. We studied PDF signaling in primary cell cultures of the accessory medulla, the circadian pacemaker of the Madeira cockroach. In Ca2+ imaging studies four types of PDF-responses were distinguished. In regularly bursting type 1 pacemakers PDF application resulted in dose-dependent long-lasting increases in Ca2+ baseline concentration and frequency of oscillating Ca2+ transients. Adenylyl cyclase antagonists prevented PDF-responses in type 1 cells, indicating that PDF signaled via elevation of intracellular cAMP levels. In contrast, in type 2 pacemakers PDF transiently raised intracellular Ca2+ levels even after blocking adenylyl cyclase activity. In patch clamp experiments the previously characterized types 1–4 could not be identified. Instead, PDF-responses were categorized according to ion channels affected. Application of PDF inhibited outward potassium or inward sodium currents, sometimes in the same neuron. In a comparison of Ca2+ imaging and patch clamp experiments we hypothesized that in type 1 cells PDF-dependent rises in cAMP concentrations block primarily outward K+ currents. Possibly, this PDF-dependent depolarization underlies PDF-dependent phase advances of pacemakers. Finally, we propose that PDF-dependent concomitant modulation of K+ and Na+ channels in coupled pacemakers causes ultradian membrane potential oscillations as prerequisite to efficient synchronization via resonance. PMID:25269074

Ryanodine receptors decant internal Ca2+ store in human and bovine airway smooth muscle.

PubMed

Tazzeo, T; Zhang, Y; Keshavjee, S; Janssen, L J

2008-08-01

Several putative roles for ryanodine receptors (RyR) were investigated in human and bovine airway smooth muscle. Changes in intracellular Ca2+ concentration ([Ca2+]i) and membrane current were investigated in single cells by confocal fluorimetry and patch-clamp electrophysiology, respectively, whereas mechanical activity was monitored in intact strips with force transducers. RyR released Ca2+ from the sarcoplasmic reticulum in a ryanodine- and chloroethyl phenol (CEP)-sensitive fashion. Neither ryanodine nor CEP inhibited responses to KCl, cholinergic agonists or serotonin, indicating no direct role for RyR in contraction; in fact, there was some augmentation of these responses. In tissues pre-contracted with carbachol, the concentration-response relationships for isoproterenol and salmeterol were unaffected by ryanodine; relaxations due to a nitric oxide donor were also largely unaffected. Finally, it was examined whether RyR were involved in regulating [Ca2+]i within the subplasmalemmal space using patch-clamp electrophysiology as well as Ca2+ fluorimetry: isoproterenol increased [Ca2+]i- and Ca2+-dependent K+ current activity in a ryanodine-sensitive fashion. In conclusion, ryanodine receptors in airway smooth muscle are not important in directly mediating contraction or relaxation. The current authors speculate instead that these allow the sarcoplasmic reticulum to release Ca2+ towards the plasmalemma (to unload an overly full Ca2+ store and/or increase the Ca2+-buffering capacity of the sarcoplasmic reticulum) without affecting bronchomotor tone.

Contribution of Rho-kinase to membrane excitability of murine colonic smooth muscle.

PubMed

Bayguinov, O; Dwyer, L; Kim, H; Marklew, A; Sanders, K M; Koh, S D

2011-06-01

The Rho-kinase pathway regulates agonist-induced contractions in several smooth muscles, including the intestine, urinary bladder and uterus, via dynamic changes in the Ca(2+) sensitivity of the contractile apparatus. However, there is evidence that Rho-kinase also modulates other cellular effectors such as ion channels. We examined the regulation of colonic smooth muscle excitability by Rho-kinase using conventional microelectrode recording, isometric force measurements and patch-clamp techniques. The Rho-kinase inhibitors, Y-27632 and H-1152, decreased nerve-evoked on- and off-contractions elicited at a range of frequencies and durations. The Rho-kinase inhibitors decreased the spontaneous contractions and the responses to carbachol and substance P independently of neuronal inputs, suggesting Y-27632 acts directly on smooth muscle. The Rho-kinase inhibitors significantly reduced the depolarization in response to carbachol, an effect that cannot be due to regulation of Ca(2+) sensitization. Patch-clamp experiments showed that Rho-kinase inhibitors reduce GTPγS-activated non-selective cation currents. The Rho-kinase inhibitors decreased contractions evoked by nerve stimulation, carbachol and substance P. These effects were not solely due to inhibition of the Ca(2+) sensitization pathway, as the Rho-kinase inhibitors also inhibited the non-selective cation conductances activated by excitatory transmitters. Thus, Rho-kinase may regulate smooth muscle excitability mechanisms by regulating non-selective cation channels as well as changing the Ca(2+) sensitivity of the contractile apparatus. © 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.

Investigation of miscellaneous hERG inhibition in large diverse compound collection using automated patch-clamp assay

PubMed Central

Yu, Hai-bo; Zou, Bei-yan; Wang, Xiao-liang; Li, Min

2016-01-01

Aim: hERG potassium channels display miscellaneous interactions with diverse chemical scaffolds. In this study we assessed the hERG inhibition in a large compound library of diverse chemical entities and provided data for better understanding of the mechanisms underlying promiscuity of hERG inhibition. Methods: Approximately 300 000 compounds contained in Molecular Library Small Molecular Repository (MLSMR) library were tested. Compound profiling was conducted on hERG-CHO cells using the automated patch-clamp platform–IonWorks Quattro™. Results: The compound library was tested at 1 and 10 μmol/L. IC50 values were predicted using a modified 4-parameter logistic model. Inhibitor hits were binned into three groups based on their potency: high (IC50<1 μmol/L), intermediate (1 μmol/L< IC50<10 μmol/L), and low (IC50>10 μmol/L) with hit rates of 1.64%, 9.17% and 16.63%, respectively. Six physiochemical properties of each compound were acquired and calculated using ACD software to evaluate the correlation between hERG inhibition and the properties: hERG inhibition was positively correlative to the physiochemical properties ALogP, molecular weight and RTB, and negatively correlative to TPSA. Conclusion: Based on a large diverse compound collection, this study provides experimental evidence to understand the promiscuity of hERG inhibition. This study further demonstrates that hERG liability compounds tend to be more hydrophobic, high-molecular, flexible and polarizable. PMID:26725739

Measurement of the membrane potential in small cells using patch clamp methods

PubMed Central

Wilson, James R; Clark, Robert B; Banderali, Umberto

2011-01-01

The resting membrane potential, Em, of mammalian cells is a fundamental physiological parameter. Even small changes in Em can modulate excitability, contractility and rates of cell migration. At present accurate, reproducible measurements of Em and determination of its ionic basis remain significant challenges when patch clamp methods are applied to small cells. In this study, a mathematical model has been developed which incorporates many of the main biophysical principles which govern recordings of the resting potential of “small cells”. Such a prototypical cell (approx. capacitance, 6 pF; input resistance 5 GΩ) is representative of neonatal cardiac myocytes, and other cells in the cardiovascular system (endothelium, fibroblasts) and small cells in other tissues, e.g., bone (osteoclasts) articular joints (chondrocytes) and the pancreas (β cells). Two common experimental conditions have been examined: (1) when the background K+ conductance is linear; and (2) when this K+ conductance is highly nonlinear and shows pronounced inward rectification. In the case of a linear K+ conductance, the presence of a “leakage” current through the seal resistance between the cell membrane and the patch pipette always depolarizes Em. Our calculations confirm that accurate characterization of Em is possible when the seal resistance is at least five times larger than the input resistance of the targeted cell. Measurement of Em under conditions in which the main background current includes a markedly nonlinear K+ conductance (due to inward rectification) yields complex and somewhat counter-intuitive findings. In fact, there are at least two possible stable values of resting membrane potential for a cell when the nonlinear, inwardly rectifying K+ conductance interacts with the seal current. This type of bistable behavior has been reported in a variety of small mammalian cells, including those from the heart, endothelium, smooth muscle and bone. Our theoretical treatment of these two common experimental situations provides useful mechanistic insights, and suggests practical methods by which these significant limitations, and their impact, can be minimized. PMID:21829090

Complex role of STIM1 in the activation of store-independent Orai1/3 channels

PubMed Central

Zhang, Wei; González-Cobos, José C.; Jardin, Isaac; Romanin, Christoph; Matrougui, Khalid

2014-01-01

Orai proteins contribute to Ca2+ entry into cells through both store-dependent, Ca2+ release–activated Ca2+ (CRAC) channels (Orai1) and store-independent, arachidonic acid (AA)-regulated Ca2+ (ARC) and leukotriene C4 (LTC4)-regulated Ca2+ (LRC) channels (Orai1/3 heteromultimers). Although activated by fundamentally different mechanisms, CRAC channels, like ARC and LRC channels, require stromal interacting molecule 1 (STIM1). The role of endoplasmic reticulum–resident STIM1 (ER-STIM1) in CRAC channel activation is widely accepted. Although ER-STIM1 is necessary and sufficient for LRC channel activation in vascular smooth muscle cells (VSMCs), the minor pool of STIM1 located at the plasma membrane (PM-STIM1) is necessary for ARC channel activation in HEK293 cells. To determine whether ARC and LRC conductances are mediated by the same or different populations of STIM1, Orai1, and Orai3 proteins, we used whole-cell and perforated patch-clamp recording to compare AA- and LTC4-activated currents in VSMCs and HEK293 cells. We found that both cell types show indistinguishable nonadditive LTC4- and AA-activated currents that require both Orai1 and Orai3, suggesting that both conductances are mediated by the same channel. Experiments using a nonmetabolizable form of AA or an inhibitor of 5-lipooxygenase suggested that ARC and LRC currents in both cell types could be activated by either LTC4 or AA, with LTC4 being more potent. Although PM-STIM1 was required for current activation by LTC4 and AA under whole-cell patch-clamp recordings in both cell types, ER-STIM1 was sufficient with perforated patch recordings. These results demonstrate that ARC and LRC currents are mediated by the same cellular populations of STIM1, Orai1, and Orai3, and suggest a complex role for both ER-STIM1 and PM-STIM1 in regulating these store-independent Orai1/3 channels. PMID:24567509

Effect of an N-terminus deletion on voltage-dependent gating of the ClC-2 chloride channel

PubMed Central

Varela, Diego; Niemeyer, María Isabel; Cid, L Pablo; Sepúlveda, Francisco V

2002-01-01

ClC-2, a chloride channel widely expressed in mammalian tissues, is activated by hyperpolarisation and extracellular acidification. Deletion of amino acids 16–61 in rat ClC-2 abolishes voltage and pH dependence in two-electrode voltage-clamp experiments in amphibian oocytes. These results have been interpreted in terms of a ball-and-chain type of mechanism in which the N-terminus would behave as a ball that is removed from an inactivating site upon hyperpolarisation. We now report whole-cell patch-clamp measurements in mammalian cells showing hyperpolarization-activation of rClC-2Δ16–61 differing only in presenting faster opening and closing kinetics than rClC-2. The lack of time and voltage dependence observed previously was reproduced, however, in nystatin-perforated patch experiments. The behaviour of wild-type rClC-2 did not differ between conventional and nystatin-perforated patches. Similar results were obtained with ClC-2 from guinea-pig. One possible explanation of the results is that some diffusible component is able to lock the channel in an open state but does so only to the mutated channel. Alternative explanations involving the osmotic state of the cell and cytoskeleton structure are also considered. Low extracellular pH activates the wild-type channel but not rClC-2Δ16–61 when expressed in oocytes, a result that had been interpreted to suggest that protons affect the ball-and-chain mechanism. In our experiments no difference was seen in the effect of extracellular pH upon rClC-2 and rClC-2Δ16–61 in either recording configuration, suggesting that protons act independently from possible effects of the N-terminus on gating. Our observations of voltage-dependent gating of the N-terminal deleted ClC-2 are an argument against a ball-and-chain mechanism for this channel. PMID:12381811

Local Membrane Deformations Activate Ca2+-Dependent K+ and Anionic Currents in Intact Human Red Blood Cells

PubMed Central

Dyrda, Agnieszka; Cytlak, Urszula; Ciuraszkiewicz, Anna; Lipinska, Agnieszka; Cueff, Anne; Bouyer, Guillaume; Egée, Stéphane; Bennekou, Poul; Lew, Virgilio L.; Thomas, Serge L. Y.

2010-01-01

Background The mechanical, rheological and shape properties of red blood cells are determined by their cortical cytoskeleton, evolutionarily optimized to provide the dynamic deformability required for flow through capillaries much narrower than the cell's diameter. The shear stress induced by such flow, as well as the local membrane deformations generated in certain pathological conditions, such as sickle cell anemia, have been shown to increase membrane permeability, based largely on experimentation with red cell suspensions. We attempted here the first measurements of membrane currents activated by a local and controlled membrane deformation in single red blood cells under on-cell patch clamp to define the nature of the stretch-activated currents. Methodology/Principal Findings The cell-attached configuration of the patch-clamp technique was used to allow recordings of single channel activity in intact red blood cells. Gigaohm seal formation was obtained with and without membrane deformation. Deformation was induced by the application of a negative pressure pulse of 10 mmHg for less than 5 s. Currents were only detected when the membrane was seen domed under negative pressure within the patch-pipette. K+ and Cl− currents were strictly dependent on the presence of Ca2+. The Ca2+-dependent currents were transient, with typical decay half-times of about 5–10 min, suggesting the spontaneous inactivation of a stretch-activated Ca2+ permeability (PCa). These results indicate that local membrane deformations can transiently activate a Ca2+ permeability pathway leading to increased [Ca2+]i, secondary activation of Ca2+-sensitive K+ channels (Gardos channel, IK1, KCa3.1), and hyperpolarization-induced anion currents. Conclusions/Significance The stretch-activated transient PCa observed here under local membrane deformation is a likely contributor to the Ca2+-mediated effects observed during the normal aging process of red blood cells, and to the increased Ca2+ content of red cells in certain hereditary anemias such as thalassemia and sickle cell anemia. PMID:20195477

Local membrane deformations activate Ca2+-dependent K+ and anionic currents in intact human red blood cells.

PubMed

Dyrda, Agnieszka; Cytlak, Urszula; Ciuraszkiewicz, Anna; Lipinska, Agnieszka; Cueff, Anne; Bouyer, Guillaume; Egée, Stéphane; Bennekou, Poul; Lew, Virgilio L; Thomas, Serge L Y

2010-02-26

The mechanical, rheological and shape properties of red blood cells are determined by their cortical cytoskeleton, evolutionarily optimized to provide the dynamic deformability required for flow through capillaries much narrower than the cell's diameter. The shear stress induced by such flow, as well as the local membrane deformations generated in certain pathological conditions, such as sickle cell anemia, have been shown to increase membrane permeability, based largely on experimentation with red cell suspensions. We attempted here the first measurements of membrane currents activated by a local and controlled membrane deformation in single red blood cells under on-cell patch clamp to define the nature of the stretch-activated currents. The cell-attached configuration of the patch-clamp technique was used to allow recordings of single channel activity in intact red blood cells. Gigaohm seal formation was obtained with and without membrane deformation. Deformation was induced by the application of a negative pressure pulse of 10 mmHg for less than 5 s. Currents were only detected when the membrane was seen domed under negative pressure within the patch-pipette. K(+) and Cl(-) currents were strictly dependent on the presence of Ca(2+). The Ca(2+)-dependent currents were transient, with typical decay half-times of about 5-10 min, suggesting the spontaneous inactivation of a stretch-activated Ca(2+) permeability (PCa). These results indicate that local membrane deformations can transiently activate a Ca(2+) permeability pathway leading to increased [Ca(2+)](i), secondary activation of Ca(2+)-sensitive K(+) channels (Gardos channel, IK1, KCa3.1), and hyperpolarization-induced anion currents. The stretch-activated transient PCa observed here under local membrane deformation is a likely contributor to the Ca(2+)-mediated effects observed during the normal aging process of red blood cells, and to the increased Ca(2+) content of red cells in certain hereditary anemias such as thalassemia and sickle cell anemia.

Enhanced Stability of Inactivated Influenza Vaccine Encapsulated in Dissolving Microneedle Patches

PubMed Central

Chu, Leonard Y.; Ye, Ling; Dong, Ke; Compans, Richard W.; Yang, Chinglai; Prausnitz, Mark R.

2015-01-01

Purpose This study tested the hypothesis that encapsulation of influenza vaccine in microneedle patches increases vaccine stability during storage at elevated temperature. Methods Whole inactivated influenza virus vaccine (A/Puerto Rico/8/34) was formulated into dissolving microneedle patches and vaccine stability was evaluated by in vitro and in vivo assays of antigenicity and immunogenicity after storage for up to 3 months at 4, 25, 37 and 45°C. Results While liquid vaccine completely lost potency as determined by hemagglutination (HA) activity within 1–2 weeks outside of refrigeration, vaccine in microneedle patches lost 40–50% HA activity during or shortly after fabrication, but then had no significant additional loss of activity over 3 months of storage, independent of temperature. This level of stability required reduced humidity by packaging with desiccant, but was not affected by presence of oxygen. This finding was consistent with additional stability assays, including antigenicity of the vaccine measured by ELISA, virus particle morphological structure captured by transmission electron microscopy and protective immune responses by immunization of mice in vivo. Conclusions These data show that inactivated influenza vaccine encapsulated in dissolving microneedle patches has enhanced stability during extended storage at elevated temperatures. PMID:26620313

Induction of pancreatic duct cells of neonatal rats into insulin-producing cells with fetal bovine serum: A natural protocol and its use for patch clamp experiments

PubMed Central

Leng, San-Hua; Lu, Fu-Er

2005-01-01

AIM: To induce the pancreatic duct cells into endocrine cells with a new natural protocol for electrophysiological study. METHODS: The pancreatic duct cells of neonatal rats were isolated, cultured and induced into endocrine cells with 15% fetal bovine serum for a period of 20 d. During this period, insulin secretion, MTT value, and morphological change of neonatal and adult pancreatic islet cells were comparatively investigated. Pancreatic β-cells were identified by morphological and electrophysiological characteristics, while ATP sensitive potassium channels (KATP), voltage-dependent potassium channels (KV), and voltage-dependent calcium channels (KCA) in β-cells were identified by patch clamp technique. RESULTS: After incubation with fetal bovine serum, the neonatal duct cells budded out, changed from duct-like cells into islet clusters. In the first 4 d, MTT value and insulin secretion increased slowly (MTT value from 0.024±0.003 to 0.028±0.003, insulin secretion from 2.6±0.6 to 3.1±0.8 mIU/L). Then MTT value and insulin secretion increased quickly from d 5 to d 10 (MTT value from 0.028±0.003 to 0.052±0.008, insulin secretion from 3.1±0.8 to 18.3±2.6 mIU/L), then reached high plateau (MTT value >0.052±0.008, insulin secretion >18.3±2.6 mIU/L). In contrast, for the isolated adult pancreatic islet cells, both insulin release and MTT value were stable in the first 4 d (MTT value from 0.029±0.01 to 0.031±0.011, insulin secretion from 13.9±3.1 to 14.3±3.3 mIU/L), but afterwards they reduced gradually (MTT value <0.031±0.011, insulin secretion <8.2±1.5 mIU/L), and the pancreatic islet cells became dispersed, broken or atrophied correspondingly. The differentiated neonatal cells were identified as pancreatic islet cells by dithizone staining method, and pancreatic β-cells were further identified by both morphological features and electrophysiological characteristics, i.e. the existence of recording currents from KATP, KV, and KCA. CONCLUSION: Islet cells differentiated from neonatal pancreatic duct cells with the new natural protocol are more advantageous in performing patch clamp study over the isolated adult pancreatic islet cells. PMID:16437601

The GABA(A) receptor RDL acts in peptidergic PDF neurons to promote sleep in Drosophila.

PubMed

Chung, Brian Y; Kilman, Valerie L; Keath, J Russel; Pitman, Jena L; Allada, Ravi

2009-03-10

Sleep is regulated by a circadian clock that times sleep and wake to specific times of day and a homeostat that drives sleep as a function of prior wakefulness. To analyze the role of the circadian clock, we have used the fruit fly Drosophila. Flies display the core behavioral features of sleep, including relative immobility, elevated arousal thresholds, and homeostatic regulation. We assessed sleep-wake modulation by a core set of circadian pacemaker neurons that express the neuropeptide PDF. We find that disruption of PDF function increases sleep during the late night in light:dark and the first subjective day of constant darkness. Flies deploy genetic and neurotransmitter pathways to regulate sleep that are similar to those of their mammalian counterparts, including GABA. We find that RNA interference-mediated knockdown of the GABA(A) receptor gene, Resistant to dieldrin (Rdl), in PDF neurons reduces sleep, consistent with a role for GABA in inhibiting PDF neuron function. Patch-clamp electrophysiology reveals GABA-activated picrotoxin-sensitive chloride currents on PDF+ neurons. In addition, RDL is detectable most strongly on the large subset of PDF+ pacemaker neurons. These results suggest that GABAergic inhibition of arousal-promoting PDF neurons is an important mode of sleep-wake regulation in vivo.

Glutamate is an inhibitory neurotransmitter in the Drosophila olfactory system.

PubMed

Liu, Wendy W; Wilson, Rachel I

2013-06-18

Glutamatergic neurons are abundant in the Drosophila central nervous system, but their physiological effects are largely unknown. In this study, we investigated the effects of glutamate in the Drosophila antennal lobe, the first relay in the olfactory system and a model circuit for understanding olfactory processing. In the antennal lobe, one-third of local neurons are glutamatergic. Using in vivo whole-cell patch clamp recordings, we found that many glutamatergic local neurons are broadly tuned to odors. Iontophoresed glutamate hyperpolarizes all major cell types in the antennal lobe, and this effect is blocked by picrotoxin or by transgenic RNAi-mediated knockdown of the GluClα gene, which encodes a glutamate-gated chloride channel. Moreover, antennal lobe neurons are inhibited by selective activation of glutamatergic local neurons using a nonnative genetically encoded cation channel. Finally, transgenic knockdown of GluClα in principal neurons disinhibits the odor responses of these neurons. Thus, glutamate acts as an inhibitory neurotransmitter in the antennal lobe, broadly similar to the role of GABA in this circuit. However, because glutamate release is concentrated between glomeruli, whereas GABA release is concentrated within glomeruli, these neurotransmitters may act on different spatial and temporal scales. Thus, the existence of two parallel inhibitory transmitter systems may increase the range and flexibility of synaptic inhibition.

APOEε4 increases trauma induced early apoptosis via reducing delayed rectifier K(+) currents in neuronal/glial co-cultures model.

PubMed

Chen, Ligang; Sun, Xiaochuan; Jiang, Yong; Kuai, Li

2015-06-10

Traumatic brain injury (TBI) is a commonly encountered emergency and severe neurosurgical injury. Previous studies have shown that the presence of the apolipoprotein E (APOE) ε4 allele has adverse outcomes across the spectrum of TBI severity. Our objective was to evaluate the effects of APOE alleles on trauma induced early apoptosis via modification of delayed rectifier K(+) current (Ik(DR)) in neuronal/glial co-cultures model. An ex vivo neuronal/glial co-cultures model carrying individual APOE alleles (ε2, ε3, ε4) of mechanical injury was developed. Flow cytometry and patch clamp recording were performed to analyze the correlations among APOE genotypes, early apoptosis and Ik(DR). We found that APOEε4 increased early apoptosis at 24h (p<0.05) compared to the ones transfected with APOEε3 and APOEε2. Noticeably, APOEε4 significantly reduced the amplitude of the Ik(DR) at 24h compared to the APOEε3 and APOEε2 (p<0.05) which exacerbate Ca(2+) influx. This indicates a possible effect of APOEε4 on early apoptosis via inhibiting Ik(DR) following injury which may adversely affect the outcome of TBI. Copyright © 2015 Elsevier Inc. All rights reserved.

Application of bifurcation analysis for determining the mechanism of coding of nociceptive signals

NASA Astrophysics Data System (ADS)

Dik, O. E.; Shelykh, T. N.; Plakhova, V. B.; Nozdrachev, A. D.; Podzorova, S. A.; Krylov, B. V.

2015-10-01

The patch clamp method is used for studying the characteristics of slow sodium channels responsible for coding of nociceptive signals. Quantitative estimates of rate constants of transitions of "normal" and pharmacologically modified activation gating mechanisms of these channels are obtained. A mathematical model of the type of Hogdkin-Huxley nociceptive neuron membrane is constructed. Cometic acid, which is a drug substance of a new nonopioid analgesic, is used as a pharmacological agent. The application of bifurcation analysis makes it possible to outline the boundaries of the region in which a periodic impulse activity is generated. This boundary separates the set of values of the model parameter for which periodic pulsation is observed from the values for which such pulsations are absent or damped. The results show that the finest effect of modulation of physical characteristic of a part of a protein molecule and its effective charge suppresses the excitability of the nociceptive neuron membrane and, hence, leads to rapid reduction of pain.

Glycosides from edible sea cucumbers stimulate macrophages via purinergic receptors

PubMed Central

Aminin, Dmitry; Pislyagin, Evgeny; Astashev, Maxim; Es’kov, Andrey; Kozhemyako, Valery; Avilov, Sergei; Zelepuga, Elena; Yurchenko, Ekaterina; Kaluzhskiy, Leonid; Kozlovskaya, Emma; Ivanov, Alexis; Stonik, Valentin

2016-01-01

Since ancient times, edible sea cucumbers have been considered a jewel of the seabed and used in Asian folk medicine for stimulation of resistance against different diseases. However, the power of this sea food has not been established on a molecular level. A particular group of triterpene glycosides was found to be characteristic metabolites of the animals, responsible for this biological action. Using one of them, cucumarioside A2-2 (CA2-2) from the edible Cucumaria japonica species as an example as well as inhibitory analysis, patch-clamp on single macrophages, small interfering RNA technique, immunoblotting, SPR analysis, computer modeling and other methods, we demonstrate low doses of CA2-2 specifically to interact with P2X receptors (predominantly P2X4) on membranes of mature macrophages, enhancing the reversible ATP-dependent Ca2+ intake and recovering Ca2+ transport at inactivation of these receptors. As result, interaction of glycosides of this type with P2X receptors leads to activation of cellular immunity. PMID:28004778

Development of controlled release silicone adhesive-based mupirocin patch demonstrates antibacterial activity on live rat skin against Staphylococcus aureus

PubMed Central

David, Sheba R; Malek, Nurafiqah; Mahadi, Abdul Hanif; Chakravarthi, Srikumar; Rajabalaya, Rajan

2018-01-01

Background Peritonitis is the most serious complication of peritoneal dialysis. Staphylococcus aureus infections could lead to peritonitis which causes reversal of peritoneal dialysis treatment back to hemodialysis. The aim of this study was to develop a controlled release silicone adhesive-based mupirocin patch for prophylactic effect and analyze its antibacterial effectiveness against S. aureus. Methods The matrix patches were prepared by using different polymers, with and without silicone adhesive, dibutyl sebacate and mupirocin. The patches were characterized for mechanical properties, drug content, moisture content, water absorption capacity and Fourier transform infrared spectrum. In vitro release studies were performed by using Franz diffusion cell. In vitro disk diffusion assay was performed on the Mueller–Hinton Agar plate to measure the zone of inhibition of the patches. The in vivo study was performed on four groups of rats with bacterial counts at three different time intervals, along with skin irritancy and histopathologic studies. Results The patches showed appropriate average thickness (0.63–1.12 mm), tensile strength (5.08–10.08 MPa) and modulus of elasticity (21.53–42.19 MPa). The drug content ranged from 94.5% to 97.4%, while the moisture content and water absorption capacities at two relative humidities (75% and 93%) were in the range of 1.082–3.139 and 1.287–4.148 wt%, respectively. Fourier transform infrared spectra showed that there were no significant interactions between the polymer and the drug. The highest percentage of drug release at 8 hours was 47.94%. The highest zone of inhibition obtained was 28.3 mm against S. aureus. The in vivo studies showed that the bacterial colonies were fewer at 1 cm (7×101 CFU/mL) than at 2 cm (1.3×102 CFU/mL) over a 24-hour period. The patches were nonirritant to the skin, and histopathologic results also showed no toxic or damaging effects to the skin. Conclusion The in vitro and in vivo studies indicated that controlled release patches reduced the migration of S. aureus on the live rat skin effectively, however, a longer duration of study is required to determine the effectiveness of the patch on a suitable peritonitis-induced animal model. PMID:29563773

Development of controlled release silicone adhesive-based mupirocin patch demonstrates antibacterial activity on live rat skin against Staphylococcus aureus.

PubMed

David, Sheba R; Malek, Nurafiqah; Mahadi, Abdul Hanif; Chakravarthi, Srikumar; Rajabalaya, Rajan

2018-01-01

Peritonitis is the most serious complication of peritoneal dialysis. Staphylococcus aureus infections could lead to peritonitis which causes reversal of peritoneal dialysis treatment back to hemodialysis. The aim of this study was to develop a controlled release silicone adhesive-based mupirocin patch for prophylactic effect and analyze its antibacterial effectiveness against S. aureus . The matrix patches were prepared by using different polymers, with and without silicone adhesive, dibutyl sebacate and mupirocin. The patches were characterized for mechanical properties, drug content, moisture content, water absorption capacity and Fourier transform infrared spectrum. In vitro release studies were performed by using Franz diffusion cell. In vitro disk diffusion assay was performed on the Mueller-Hinton Agar plate to measure the zone of inhibition of the patches. The in vivo study was performed on four groups of rats with bacterial counts at three different time intervals, along with skin irritancy and histopathologic studies. The patches showed appropriate average thickness (0.63-1.12 mm), tensile strength (5.08-10.08 MPa) and modulus of elasticity (21.53-42.19 MPa). The drug content ranged from 94.5% to 97.4%, while the moisture content and water absorption capacities at two relative humidities (75% and 93%) were in the range of 1.082-3.139 and 1.287-4.148 wt%, respectively. Fourier transform infrared spectra showed that there were no significant interactions between the polymer and the drug. The highest percentage of drug release at 8 hours was 47.94%. The highest zone of inhibition obtained was 28.3 mm against S. aureus . The in vivo studies showed that the bacterial colonies were fewer at 1 cm (7×10 1 CFU/mL) than at 2 cm (1.3×10 2 CFU/mL) over a 24-hour period. The patches were nonirritant to the skin, and histopathologic results also showed no toxic or damaging effects to the skin. The in vitro and in vivo studies indicated that controlled release patches reduced the migration of S. aureus on the live rat skin effectively, however, a longer duration of study is required to determine the effectiveness of the patch on a suitable peritonitis-induced animal model.

Sortilin 1 knockout alters basal adipose glucose metabolism but not diet-induced obesity in mice.

PubMed

Li, Jibiao; Matye, David J; Wang, Yifeng; Li, Tiangang

2017-04-01

Sortilin 1 (Sort1) is a trafficking receptor that has been implicated in the regulation of plasma cholesterol in humans and mice. Here, we use metabolomics and hyperinsulinemic-euglycemic clamp approaches to obtain further understanding of the in vivo effects of Sort1 deletion on diet-induced obesity as well as on adipose lipid and glucose metabolism. Results show that Sort1 knockout (KO) does not affect Western diet-induced obesity nor adipose fatty acid and ceramide concentrations. Under the basal fasting state, chow-fed Sort1 KO mice have decreased adipose glycolytic metabolites, but Sort1 deletion does not affect insulin-stimulated tissue glucose uptake during the insulin clamp. These results suggest that Sort1 loss-of-function in vivo does not affect obesity development, but differentially modulates adipose glucose metabolism under fasting and insulin-stimulated states. © 2017 Federation of European Biochemical Societies.

Transbuccal delivery of chlorpheniramine maleate from mucoadhesive buccal patches.

PubMed

Sekhar, K Chandra; Naidu, K V S; Vishnu, Y Vamshi; Gannu, Ramesh; Kishan, V; Rao, Y Madhusudan

2008-01-01

This article describes buccal permeation of chlorpheniramine maleate (CPM) and its transbuccal delivery using mucoadhesive buccal patches. Permeation of CPM was calculated in vitro using porcine buccal membrane and in vivo in healthy humans. Buccal formulations were developed with hydroxyethylcellulose (HEC) and evaluated for in vitro release, moisture absorption, mechanical properties, and bioadhesion, and optimized formulation was subjected for bioavailability studies in healthy human volunteers. In vitro flux of CPM was calculated to be 0.14 +/- 0.03 mg.h(-1).cm(-2) and buccal absorption also was demonstrated in vivo in human volunteers. In vitro drug release and moisture absorbed were governed by HEC content and formulations exhibited good tensile and mucoadhesive properties. Bioavailability from optimized buccal patch was 1.46 times higher than the oral dosage form and the results showed statistically significant difference.

In vivo evaluation of hybrid patches composed of PLA based copolymers and collagen/chondroitin sulfate for ligament tissue regeneration.

PubMed

Pinese, Coline; Gagnieu, Christian; Nottelet, Benjamin; Rondot-Couzin, Capucine; Hunger, Sylvie; Coudane, Jean; Garric, Xavier

2017-10-01

Biomaterials for soft tissues regeneration should exhibit sufficient mechanical strength, demonstrating a mechanical behavior similar to natural tissues and should also promote tissues ingrowth. This study was aimed at developing new hybrid patches for ligament tissue regeneration by synergistic incorporation of a knitted structure of degradable polymer fibers to provide mechanical strength and of a biomimetic matrix to help injured tissues regeneration. PLA- Pluronic ® (PLA-P) and PLA-Tetronic ® (PLA-T) new copolymers were shaped as knitted patches and were associated with collagen I (Coll) and collagen I/chondroitine-sulfate (Coll CS) 3-dimensional matrices. In vitro study using ligamentocytes showed the beneficial effects of CS on ligamentocytes proliferation. Hybrid patches were then subcutaneously implanted in rats for 4 and 12 weeks. Despite degradation, patches retained strength to answer the mechanical physiological needs. Tissue integration capacity was assessed with histological studies. We showed that copolymers, associated with collagen and chondroitin sulfate sponge, exhibited very good tissue integration and allowed neotissue synthesis after 12 weeks in vivo. To conclude, PLA-P/CollCS and PLA-T/CollCS hybrid patches in terms of structure and composition give good hopes for tendon and ligament regeneration. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 1778-1788, 2017. © 2016 Wiley Periodicals, Inc.

Screening of E. coli β-clamp Inhibitors Revealed that Few Inhibit Helicobacter pylori More Effectively: Structural and Functional Characterization.

PubMed

Pandey, Preeti; Verma, Vijay; Dhar, Suman Kumar; Gourinath, Samudrala

2018-01-11

The characteristic of interaction with various enzymes and processivity-promoting nature during DNA replication makes β-clamp an important drug target. Helicobacter pylori ( H. pylori ) have several unique features in DNA replication machinery that makes it different from other microorganisms. To find out whether difference in DNA replication proteins behavior accounts for any difference in drug response when compared to E. coli , in the present study, we have tested E. coli β-clamp inhibitor molecules against H. pylori β-clamp. Various approaches were used to test the binding of inhibitors to H. pylori β-clamp including docking, surface competition assay, complex structure determination, as well as antimicrobial assay. Out of five shortlisted inhibitor molecules on the basis of docking score, three molecules, 5-chloroisatin, carprofen, and 3,4-difluorobenzamide were co-crystallized with H. pylori β-clamp and the structures show that they bind at the protein-protein interaction site as expected. In vivo studies showed only two molecules, 5-chloroisatin, and 3,4-difluorobenzamide inhibited the growth of the pylori with MIC values in micro molar range, which is better than the inhibitory effect of the same drugs on E. coli . Therefore, the evaluation of such drugs against H. pylori may explore the possibility to use to generate species-specific pharmacophore for development of new drugs against H. pylori .

Hyperpolarization-activated current (I(h)) in vestibular calyx terminals: characterization and role in shaping postsynaptic events.

PubMed

Meredith, Frances L; Benke, Tim A; Rennie, Katherine J

2012-12-01

Calyx afferent terminals engulf the basolateral region of type I vestibular hair cells, and synaptic transmission across the vestibular type I hair cell/calyx is not well understood. Calyces express several ionic conductances, which may shape postsynaptic potentials. These include previously described tetrodotoxin-sensitive inward Na(+) currents, voltage-dependent outward K(+) currents and a K(Ca) current. Here, we characterize an inwardly rectifying conductance in gerbil semicircular canal calyx terminals (postnatal days 3-45), sensitive to voltage and to cyclic nucleotides. Using whole-cell patch clamp, we recorded from isolated calyx terminals still attached to their type I hair cells. A slowly activating, noninactivating current (I(h)) was seen with hyperpolarizing voltage steps negative to the resting potential. External Cs(+) (1-5 mM) and ZD7288 (100 μM) blocked the inward current by 97 and 83 %, respectively, confirming that I(h) was carried by hyperpolarization-activated, cyclic nucleotide gated channels. Mean half-activation voltage of I(h) was -123 mV, which shifted to -114 mV in the presence of cAMP. Activation of I(h) was well described with a third order exponential fit to the current (mean time constant of activation, τ, was 190 ms at -139 mV). Activation speeded up significantly (τ=136 and 127 ms, respectively) when intracellular cAMP and cGMP were present, suggesting that in vivo I(h) could be subject to efferent modulation via cyclic nucleotide-dependent mechanisms. In current clamp, hyperpolarizing current steps produced a time-dependent depolarizing sag followed by either a rebound afterdepolarization or an action potential. Spontaneous excitatory postsynaptic potentials (EPSPs) became larger and wider when I(h) was blocked with ZD7288. In a three-dimensional mathematical model of the calyx terminal based on Hodgkin-Huxley type ionic conductances, removal of I(h) similarly increased the EPSP, whereas cAMP slightly decreased simulated EPSP size and width.

Cellular resolution circuit mapping with temporal-focused excitation of soma-targeted channelrhodopsin

PubMed Central

Baker, Christopher A; Elyada, Yishai M; Parra, Andres; Bolton, M McLean

2016-01-01

We describe refinements in optogenetic methods for circuit mapping that enable measurements of functional synaptic connectivity with single-neuron resolution. By expanding a two-photon beam in the imaging plane using the temporal focusing method and restricting channelrhodopsin to the soma and proximal dendrites, we are able to reliably evoke action potentials in individual neurons, verify spike generation with GCaMP6s, and determine the presence or absence of synaptic connections with patch-clamp electrophysiological recording. DOI: http://dx.doi.org/10.7554/eLife.14193.001 PMID:27525487

Expression of cystic fibrosis transmembrane conductance regulator corrects defective chloride channel regulation in cystic fibrosis airway epithelial cells

NASA Astrophysics Data System (ADS)

Rich, Devra P.; Anderson, Matthew P.; Gregory, Richard J.; Cheng, Seng H.; Paul, Sucharita; Jefferson, Douglas M.; McCann, John D.; Klinger, Katherine W.; Smith, Alan E.; Welsh, Michael J.

1990-09-01

The cystic fibrosis transmembrane conductance regulator (CFTR) was expressed in cultured cystic fibrosis airway epithelial cells and Cl- channel activation assessed in single cells using a fluorescence microscopic assay and the patch-clamp technique. Expression of CFTR, but not of a mutant form of CFTR (ΔF508), corrected the Cl- channel defect. Correction of the phenotypic defect demonstrates a causal relationship between mutations in the CFTR gene and defective Cl- transport which is the hallmark of the disease.

Whole-cell patch clamp recording of voltage-sensitive Ca²+ channel currents: heterologous expression systems and dissociated brain neurons.

PubMed

Hainsworth, Atticus H; Randall, Andrew D; Stefani, Alessandro

2005-01-01

Voltage-sensitive Ca(2+) channels (VSCC) play a central role in an extensive array of physiological processes. Their importance in cellular function arises from their ability both to sense membrane voltage and to conduct Ca(2+) ions, two facets that couple membrane excitability to a key intracellular second messenger. Through this relationship, activation of VSCCs is tightly coupled to the gamut of cellular functions dependent on intracellular Ca(2+), including muscle contraction, energy metabolism, gene expression, and exocytotic/endocytotic cycling.

Vibration suppression with approximate finite dimensional compensators for distributed systems: Computational methods and experimental results

NASA Technical Reports Server (NTRS)

Banks, H. T.; Smith, Ralph C.; Wang, Yun

1994-01-01

Based on a distributed parameter model for vibrations, an approximate finite dimensional dynamic compensator is designed to suppress vibrations (multiple modes with a broad band of frequencies) of a circular plate with Kelvin-Voigt damping and clamped boundary conditions. The control is realized via piezoceramic patches bonded to the plate and is calculated from information available from several pointwise observed state variables. Examples from computational studies as well as use in laboratory experiments are presented to demonstrate the effectiveness of this design.

Histological and molecular-biological analyses of poly(3-hydroxybutyrate) (PHB) patches for enhancement of bone regeneration.

PubMed

Gredes, Tomasz; Gedrange, Tomasz; Hinüber, Claudia; Gelinsky, Michael; Kunert-Keil, Christiane

2015-05-01

Tissue engineered cell-seeded constructs with poly(3)hydroxybutyrate (PHB) induced ectopic bone formation after implantation into the back muscle of rats. The objective of our in vivo study was to evaluate the osteogenic potential of pure PHB patches in surgically created cranial defects. For this, PHB patches were analyzed after implantation in surgically created defects on the cranium of adult male rats. After healing periods of 4, 8 and 12 weeks, the bone tissue specimens containing PHB patches were processed and analyzed histologically as well as molecular-biologically. After 4 weeks, the PHB patches were completely embedded in connective tissue. Eight weeks after PHB insertion, bone regeneration proceeding from bearing bone was found in 50% of all treated animals, whereas all PHB treated cavities showed both bone formation and embedding of the patches in bone 12 weeks after surgery. Furthermore, all slices showed pronounced development of blood vessels. Histomorphometric analysis presented a regenerated bone mean value between 46.4 ± 16.1% and 54.2 ± 19.3% after 4-12 weeks of healing. Caveolin-1 staining in capillary-like structures showed a 1.16-1.38 fold increased expression in PHB treated defects compared to controls. Real-time RT-PCR analyses showed significantly lower expressions of Alpl, Col1a1 and VEGFA in cranium defects after treatment with PHB patches compared to untreated bony defects of the same cranium. Within the limits of the presented animal investigation, it could conclude that the tested PHB patches featured a good biocompatibility and an osteoconductive character. Copyright © 2014 Elsevier GmbH. All rights reserved.

Collisions of Ir Oxide Nanoparticles with Carbon Nanopipettes: Experiments with One Nanoparticle.

PubMed

Zhou, Min; Yu, Yun; Hu, Keke; Xin, Huolin L; Mirkin, Michael V

2017-03-07

Investigating the collisions of individual metal nanoparticles (NPs) with electrodes can provide new insights into their electrocatalytic behavior, mass transport, and interactions with surfaces. Here we report a new experimental setup for studying NP collisions based on the use of carbon nanopipettes to enable monitoring multiple collision events involving the same NP captured inside the pipet cavity. A patch clamp amplifier capable of measuring pA-range currents on the microsecond time scale with a very low noise and stable background was used to record the collision transients. The analysis of current transients produced by oxidation of hydrogen peroxide at one IrO x NP provided information about the origins of deactivation of catalytic NPs and the effects of various experimental conditions on the collision dynamics. High-resolution TEM of carbon pipettes was used to attain better understanding of the NP capture and collisions.

Collisions of Ir oxide nanoparticles with carbon nanopipettes: Experiments with one nanoparticle

DOE PAGES

Zhou, Min; Yu, Yun; Hu, Keke; ...

2017-02-03

Investigating the collisions of individual metal nanoparticles (NPs) with electrodes can provide new insights into their electrocatalytic behavior, mass transport, and interactions with surfaces. Here we report a new experimental setup for studying NP collisions based on the use of carbon nanopipettes to enable monitoring multiple collision events involving the same NP captured inside the pipet cavity. A patch clamp amplifier capable of measuring pA-range currents on the microsecond time scale with a very low noise and stable background was used to record the collision transients. The analysis of current transients produced by oxidation of hydrogen peroxide at one IrOxmore » NP provided information about the origins of deactivation of catalytic NPs and the effects of various experimental conditions on the collision dynamics. Lastly, high-resolution TEM of carbon pipettes was used to attain better understanding of the NP capture and collisions.« less

Synchronization of action potentials during low-magnesium-induced bursting

PubMed Central

Johnson, Sarah E.; Hudson, John L.

2015-01-01

The relationship between mono- and polysynaptic strength and action potential synchronization was explored using a reduced external Mg2+ model. Single and dual whole cell patch-clamp recordings were performed in hippocampal cultures in three concentrations of external Mg2+. In decreased Mg2+ medium, the individual cells transitioned to spontaneous bursting behavior. In lowered Mg2+ media the larger excitatory synaptic events were observed more frequently and fewer transmission failures occurred, suggesting strengthened synaptic transmission. The event synchronization was calculated for the neural action potentials of the cell pairs, and it increased in media where Mg2+ concentration was lowered. Analysis of surrogate data where bursting was present, but no direct or indirect connections existed between the neurons, showed minimal action potential synchronization. This suggests the synchronization of action potentials is a product of the strengthening synaptic connections within neuronal networks. PMID:25609103

Synchronization of action potentials during low-magnesium-induced bursting.

PubMed

Johnson, Sarah E; Hudson, John L; Kapur, Jaideep

2015-04-01

The relationship between mono- and polysynaptic strength and action potential synchronization was explored using a reduced external Mg(2+) model. Single and dual whole cell patch-clamp recordings were performed in hippocampal cultures in three concentrations of external Mg(2+). In decreased Mg(2+) medium, the individual cells transitioned to spontaneous bursting behavior. In lowered Mg(2+) media the larger excitatory synaptic events were observed more frequently and fewer transmission failures occurred, suggesting strengthened synaptic transmission. The event synchronization was calculated for the neural action potentials of the cell pairs, and it increased in media where Mg(2+) concentration was lowered. Analysis of surrogate data where bursting was present, but no direct or indirect connections existed between the neurons, showed minimal action potential synchronization. This suggests the synchronization of action potentials is a product of the strengthening synaptic connections within neuronal networks. Copyright © 2015 the American Physiological Society.

Diadenosine tetraphosphate-gating of recombinant pancreatic ATP-sensitive K(+) channels.

PubMed

Jovanovic, S; Jovanovic, A

2001-02-01

Diadenosine tetraphosphate (Ap4A) has been recently discovered in the pancreatic beta cells where targets ATP-sensitive K(+) (K(ATP)) channels, depolarizes the cell membrane and induces insulin secretion. However, whether Ap4A inhibit pancreatic K(ATP) channels by targeting protein channel complex itself was unknown. Therefore, we coexpressed pancreatic K(ATP) channel subunits, Kir6.2 and SUR1, in COS-7 cells and examined the effect of Ap4A on the single channel behavior using the inside-out configuration of the patch-clamp technique. Ap4A inhibited channel opening in a concentration-dependent manner. Analysis of single channels demonstrated that Ap4A did not change intraburst kinetic behavior of K(ATP) channels, but rather decreased burst duration and increased between-burst duration. It is concluded that Ap4A antagonizes K(ATP) channel opening by targeting channel subunits themselves and by keeping channels longer in closed interburst states.

On the simple random-walk models of ion-channel gate dynamics reflecting long-term memory.

PubMed

Wawrzkiewicz, Agata; Pawelek, Krzysztof; Borys, Przemyslaw; Dworakowska, Beata; Grzywna, Zbigniew J

2012-06-01

Several approaches to ion-channel gating modelling have been proposed. Although many models describe the dwell-time distributions correctly, they are incapable of predicting and explaining the long-term correlations between the lengths of adjacent openings and closings of a channel. In this paper we propose two simple random-walk models of the gating dynamics of voltage and Ca(2+)-activated potassium channels which qualitatively reproduce the dwell-time distributions, and describe the experimentally observed long-term memory quite well. Biological interpretation of both models is presented. In particular, the origin of the correlations is associated with fluctuations of channel mass density. The long-term memory effect, as measured by Hurst R/S analysis of experimental single-channel patch-clamp recordings, is close to the behaviour predicted by our models. The flexibility of the models enables their use as templates for other types of ion channel.

A bursting potassium channel in isolated cholinergic synaptosomes of Torpedo electric organ.

PubMed Central

Edry-Schiller, J; Ginsburg, S; Rahamimoff, R

1991-01-01

1. Pinched-off cholinergic nerve terminals (synaptosomes) prepared from the electric organ of Torpedo ocelata were fused into large structures (greater than 20 microns) using dimethyl sulphoxide and polyethylene glycol 1500, as previously described for synaptic vesicles from the same organ. 2. The giant fused synaptosomes were easily amenable to the patch clamp technique and 293 seals with a resistance greater than 4 G omega were obtained in the 'cell-attached' configuration. In a large fraction of the experiments, an 'inside-out' patch configuration was achieved. 3. Several types of unitary ionic currents were observed. This study describes the most frequently observed single-channel activity which was found in 247 out of the 293 membrane patches (84.3%). 4. The single-channel current-voltage relation was linear between -60 and 20 mV and showed a slope conductance of 23.8 +/- 1.3 pS when the pipette contained 350-390 mM-Na+ and the bath facing the inside of the synaptosomal membrane contained 390 mM-K+. 5. From extrapolated reversal potential measurements, it was concluded that this channel has a large selectivity for K+ over Na+ (70.4 +/- 11.5, mean +/- S.E.M.). Chloride ions are not transported significantly through this potassium channel. 6. This potassium channel has a low probability of opening. The probability of being in the open state increases upon depolarization and reaches about 1% when the inside of the patch is 20 mV positive compared to the pipette side. 7. The mean channel open time increases with depolarization; thus the product current x time (= charge) also increases upon depolarization, showing properties of an outward rectifier. 8. The potassium channel in the giant synaptosome membrane has a bursting behaviour. Open-time distribution, closed-time distribution and a Poisson analysis indicate that the minimal kinetic scheme requires one open state and three closed states. PMID:1654418

Effect of novel mucoadhesive buccal patches of carvedilol on isoprenaline-induced tachycardia

PubMed Central

Verma, Navneet; Chattopadhyay, Pronobesh

2014-01-01

The main aim of the study was designed to develop bioadhesive buccal patches of carvedilol (CR) and evaluate for isoprenaline-induced tachycardia. Buccal patches of carvedilol were prepared by using chitosan (CH), sodium salt of carboxy methyl cellulose (NaCMC), and polyvinyl alcohol (PVA) as mucoadhesive polymers. The solvent evaporation method was used for the preparation of buccal patches. The patches were evaluated for their physical characteristics like patch thickness, weight variation, content uniformity, folding endurance, surface pH, residence time, in vitro drug release, and in vivo pharmacodynamic study. The swelling index of the patches was found to be proportional to the polymer concentration, whereas surface pH of all the formulated bioadhesive patches was found to lie between neutral ranges. In-vitro release study shows that 94.75% drug was release in 8 hours from the patch, which containing 2% w/v chitosan. The folding endurance result shows good elasticity in all the patches. Application of buccal patches on buccal mucosa of rabbit shows a significant result in % inhibition of isoprenaline-induced tachycardia. Prepared buccal patches of chitosan, NaCMC, and PVA containing Carvedilol meet the ideal requirement for the delivery of cardiovascular drugs and inhibit the isoprenaline tachycardia. PMID:24959419

Effect of novel mucoadhesive buccal patches of carvedilol on isoprenaline-induced tachycardia.

PubMed

Verma, Navneet; Chattopadhyay, Pronobesh

2014-04-01

The main aim of the study was designed to develop bioadhesive buccal patches of carvedilol (CR) and evaluate for isoprenaline-induced tachycardia. Buccal patches of carvedilol were prepared by using chitosan (CH), sodium salt of carboxy methyl cellulose (NaCMC), and polyvinyl alcohol (PVA) as mucoadhesive polymers. The solvent evaporation method was used for the preparation of buccal patches. The patches were evaluated for their physical characteristics like patch thickness, weight variation, content uniformity, folding endurance, surface pH, residence time, in vitro drug release, and in vivo pharmacodynamic study. The swelling index of the patches was found to be proportional to the polymer concentration, whereas surface pH of all the formulated bioadhesive patches was found to lie between neutral ranges. In-vitro release study shows that 94.75% drug was release in 8 hours from the patch, which containing 2% w/v chitosan. The folding endurance result shows good elasticity in all the patches. Application of buccal patches on buccal mucosa of rabbit shows a significant result in % inhibition of isoprenaline-induced tachycardia. Prepared buccal patches of chitosan, NaCMC, and PVA containing Carvedilol meet the ideal requirement for the delivery of cardiovascular drugs and inhibit the isoprenaline tachycardia.

Local Optogenetic Induction of Fast (20-40 Hz) Pyramidal-Interneuron Network Oscillations in the In Vitro and In Vivo CA1 Hippocampus: Modulation by CRF and Enforcement of Perirhinal Theta Activity.

PubMed

Dine, Julien; Genewsky, Andreas; Hladky, Florian; Wotjak, Carsten T; Deussing, Jan M; Zieglgänsberger, Walter; Chen, Alon; Eder, Matthias

2016-01-01

The neurophysiological processes that can cause theta-to-gamma frequency range (4-80 Hz) network oscillations in the rhinal cortical-hippocampal system and the potential connectivity-based interactions of such forebrain rhythms are a topic of intensive investigation. Here, using selective Channelrhodopsin-2 (ChR2) expression in mouse forebrain glutamatergic cells, we were able to locally, temporally precisely, and reliably induce fast (20-40 Hz) field potential oscillations in hippocampal area CA1 in vitro (at 25°C) and in vivo (i.e., slightly anesthetized NEX-Cre-ChR2 mice). As revealed by pharmacological analyses and patch-clamp recordings from pyramidal cells and GABAergic interneurons in vitro, these light-triggered oscillations can exclusively arise from sustained suprathreshold depolarization (~200 ms or longer) and feedback inhibition of CA1 pyramidal neurons, as being mandatory for prototypic pyramidal-interneuron network (P-I) oscillations. Consistently, the oscillations comprised rhythmically occurring population spikes (generated by pyramidal cells) and their frequency increased with increasing spectral power. We further demonstrate that the optogenetically driven CA1 oscillations, which remain stable over repeated evocations, are impaired by the stress hormone corticotropin-releasing factor (CRF, 125 nM) in vitro and, even more remarkably, found that they are accompanied by concurrent states of enforced theta activity in the memory-associated perirhinal cortex (PrC) in vivo. The latter phenomenon most likely derives from neurotransmission via a known, but poorly studied excitatory CA1→PrC pathway. Collectively, our data provide evidence for the existence of a prototypic (CRF-sensitive) P-I gamma rhythm generator in area CA1 and suggest that CA1 P-I oscillations can rapidly up-regulate theta activity strength in hippocampus-innervated rhinal networks, at least in the PrC.

BKCa currents are enriched in a subpopulation of adult rat cutaneous nociceptive dorsal root ganglion neurons

PubMed Central

Zhang, Xiu-Lin; Mok, Lee-Peng; Katz, Elizabeth J; Gold, Michael S.

2010-01-01

The biophysical properties and distribution of voltage-dependent, Ca2+-modulated K+ (BKCa) currents among subpopulations of acutely dissociated DiI labeled cutaneous sensory neurons from the adult rat were characterized with whole cell patch clamp techniques. BKCa currents were isolated from total K+ current with iberiotoxin, charybdotoxin, or paxilline. There was considerable variability in biophysical properties of BKCa currents. There was also variability in the distribution of BKCa current among subpopulations of cutaneous DRG neurons. While present in each of the subpopulations defined by cell body size, IB4 binding or capsaicin sensitivity, BKCa current was present in vast majority (>90%) of small diameter IB4+ neurons but was present in only a minority of neurons in subpopulations defined by other criteria (i.e., small diameter IB4−). Current clamp analysis indicated that in IB4+ neurons, BKCa currents contribute to the repolarization of the action potential and adaptation in response to sustained membrane depolarization, while playing little role in the determination of action potential threshold. RT-PCR analysis of mRNA collected from whole DRG revealed the presence of multiple splice variants of the BKCa channel α-subunit, rslo and all 4 of the accessory β subunits, suggesting that heterogeneity in the biophysical and pharmacological properties of BKCa current in cutaneous neurons, reflects, at least in part, the differential distribution of splice variants and/or β subunits. Because even a small decrease in BKCa current appears to have a dramatic influence on excitability, modulation of this current may contribute to sensitization of nociceptive afferents observed following tissue injury. PMID:20105244

Synthesis of conjugated chitosan and its effect on drug permeation from transdermal patches.

PubMed

Satheeshababu, B K; Shivakumar, K L

2013-03-01

The aim of this study was to synthesis the conjugated chitosan by covalent attachment of thiol moieties to the cationic polymer, mediated by a carbodiimide to improve permeation properties of chitosan. Thioglycolic acid was covalently attached to chitosan by the formation of amide bonds between the primary amino groups of the polymer and the carboxylic acid groups of thioglycolic acid. Hence, these polymers are called as thiomers or thiolated polymers. Conjugation of chitosan was confirmed by Fourier transform-infrared and differential scanning calorimetric analysis. Matrix type transdermal patches of carvedilol were prepared using the different proportions of chitosan and chitosan-thioglycolic acid conjugates (2:0, 1.7:0.3, 1.4:0.6, 1:1, 0.6:1.4 and 0.3:1.7) by solvent casting technique. Prepared matrix type patches were evaluated for their physicochemical characterization followed by in vitro evaluation. Selected formulations were subjected for their ex vivo studies on Wistar albino rat skin and human cadaver skin using the modified Franz diffusion cell. As the proportion of conjugated chitosan increased, the transdermal patches showed increased drug permeation. The mechanism of drug release was found to be nonFickian profiles. The present study concludes that the transdermal patches of carvedilol using conjugated chitosan with different proportions of chitosan were successfully developed to provide improved drug permeation. The transdermal patches can be a good approach to improve drug bioavailability by bypassing the extensive hepatic first-pass metabolism of the drug.

Allergic reaction to vanadium causes a diffuse eczematous eruption and titanium alloy orthopedic implant failure.

PubMed

Engelhart, Sally; Segal, Robert J

2017-04-01

Allergy as a cause of adverse outcomes in patients with implanted orthopedic hardware is controversial. Allergy to titanium-based implants has not been well researched, as titanium is traditionally thought to be inert. We highlight the case of a patient who developed systemic dermatitis and implant failure after surgical placement of a titanium alloy (Ti6Al4V) plate in the left foot. The hardware was removed and the eruption cleared in the following weeks. The plate and screws were submitted for metal analysis. The elemental composition of both the plate and screws included 3 major elements-titanium, aluminum, and vanadium-as well as trace elements. Metal analysis revealed that the plate and screws had different microstructures, and electrochemical studies demonstrated that galvanic corrosion could have occurred between the plate and screws due to their different microstructures, contributing to the release of vanadium in vivo. The patient was patch tested with several metals including components of the implant and had a positive patch test reaction only to vanadium trichloride. These findings support a diagnosis of vanadium allergy and suggests that clinicians should consider including vanadium when patch testing patients with a suspected allergic reaction to vanadium-containing implants.

Regulation of Drosophila transient receptor potential-like (TrpL) channels by phospholipase C-dependent mechanisms.

PubMed

Estacion, M; Sinkins, W G; Schilling, W P

2001-01-01

Patch clamp and fura-2 fluorescence were employed to characterize receptor-mediated activation of recombinant Drosophila TrpL channels expressed in Sf9 insect cells. TrpL was activated by receptor stimulation and by exogenous application of diacylglycerol (DAG) or poly-unsaturated fatty acids (PUFAs). Activation of TrpL was blocked more than 70% by U73122, suggesting that the effect of these agents was dependent upon phospholipase C (PLC). In fura-2 assays, extracellular application of bacterial phosphatidylinositol (PI)-PLC or phosphatidylcholine (PC)-PLC caused a transient increase in TrpL channel activity, the magnitude of which was significantly less than that observed following receptor stimulation. TrpL channels were also activated in excised inside-out patches by cytoplasmic application of mammalian PLC-b2, bacterial PI-PLC and PC-PLC, but not by phospholipase D (PLD). The phospholipases had little or no effect when examined in either whole-cell or cell-attached configurations.TrpL activity was inhibited by addition of phosphatidylinositol-4,5-bisphosphate (PIP2) to excised inside-out membrane patches exhibiting spontaneous channel activity or to patches pre-activated by treatment with PLC. The effect was reversible, specific for PIP2, and was not observed with phosphatidylethanolamine (PE), PI, PC or phosphatidylserine (PS). However, antibodies against PIP2 consistently failed to activate TrpL in inside-out patches. It is concluded that both the hydrolysis of PIP2 and the generation of DAG are required to rapidly activate TrpL following receptor stimulation, or that some other PLC-dependent mechanism plays a crucial role in the activation process.

Binding of indocyanine green in polycaprolactone fibers using blend electrospinning for in vivo laser-assisted vascular anastomosis.

PubMed

Schönfeld, Annemarie; Kabra, Zacharia Mbäıdjol; Constantinescu, Mihai; Bosshardt, Dieter; Stoffel, Michael H; Peters, Kirsten; Frenz, Martin

2017-12-01

The clinical application of laser-assisted vascular anastomosis is afflicted by unreliable and low bonding strengths as well as tedious handling during microvascular surgery. The challenge to be met arises from the flow-off of the chromophore during soldering that changes the absorption and stains the surrounding tissue, leading to an uncontrollable thermal damage zone. In this study, we investigated the feasibility to produce an indocyanine green (ICG)-loaded patch by electrospinning and tested its applicability to both in vitro and in vivo microvascular laser soldering. A blend of polycaprolactone and ICG was electrospun to produce a pliable patch. Prior to soldering, the patch was soaked in 40% wt. bovine serum albumin solution. The solder patch was wrapped in vitro around blood vessel stumps of rabbit aortas. An intraluminal balloon catheter enabled an easy alignment and held the setup in place. The soldering energy was delivered via a diffusor fiber from the vessel lumen using a diode laser at 810 nm. During the procedure, the surface temperature was observed with an infrared camera. Afterward, samples were embedded in methylmethacrylate and epon to study thermal damage. The quality of the fusion was assessed by measuring the tensile strength. After in vitro tests with rabbit aortas, eight large white pigs were subjected to an acute in vivo experiment, and the artery of the latissimus dorsi flap was anastomosed to the distal femoral artery. The ICG-loaded patch, produced by electrospinning, has a thickness of 279 ± 62 μm, a fiber diameter of 1.20 ± 0.19 μm, and an attenuation coefficient of 1,119 ± 183 cm -1 at a wavelength of 790 nm. The patch was pliable and easy to handle during surgery. No leakage of the chromophore was observed. Thermal damage was restricted to the Tunica adventitia and Tunica media and the area of the vessel wall that was covered with the patch. Six pigs were successfully treated, without any bleeding and with a continuous blood flow. The in vivo flap model yielded a similar tensile strength compared to in vitro laser-assisted vascular anastomoses (138 ± 52 vs. 117 ± 30 mN/mm 2 ). Our study demonstrated the applicability of the ICG-loaded patch for laser-assisted vascular anastomosis. By using electrospinning, ICG could be bound to polymer fibers, avoiding its flow-off and the staining of the surrounding tissue. This patch demonstrated several advantages over liquid solder as it was easier to apply, ensured a high and reliable bonding strength while maintaining a constant concentration of ICG concentration during the surgery. Lasers Surg. Med. 49:928-939, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Kvβ1.1 (AKR6A8) senses pyridine nucleotide changes in the mouse heart and modulates cardiac electrical activity.

PubMed

Tur, Jared; Chapalamadugu, Kalyan C; Katnik, Christopher; Cuevas, Javier; Bhatnagar, Aruni; Tipparaju, Srinivas M

2017-03-01

The present study investigates the physiological role of Kvβ1 subunit for sensing pyridine nucleotide (NADH/NAD+) changes in the heart. We used Kvβ1.1 knockout (KO) or wild-type (WT) mice and established that Kvβ1.1 preferentially binds with Kv4.2 and senses the pyridine nucleotide changes in the heart. The cellular action potential duration (APD) obtained from WT cardiomyocytes showed longer APDs with lactate perfusion, which increases intracellular NADH levels, while the APDs remained unaltered in the Kvβ1.1 KO. Ex vivo monophasic action potentials showed a similar response, in which the APDs were prolonged in WT mouse hearts with lactate perfusion; however, the Kvβ1.1 KO mouse hearts did not show APD changes upon lactate perfusion. COS-7 cells coexpressing Kv4.2 and Kvβ1.1 were used for whole cell patch-clamp recordings to evaluate changes caused by NADH (lactate). These data reveal that Kvβ1.1 is required in the mediated inactivation of Kv4.2 currents, when NADH (lactate) levels are increased. In vivo, isoproterenol infusion led to increased NADH in the heart along with QTc prolongation in wild-type mice; regardless of the approach, our data show that Kvβ1.1 recognizes NADH changes and modulates Kv4.2 currents affecting AP and QTc durations. Overall, this study uses multiple levels of investigation, including the heterologous overexpression system, cardiomyocyte, ex vivo, and ECG, and clearly depicts that Kvβ1.1 is an obligatory sensor of NADH/NAD changes in vivo, with a physiological role in the heart. NEW & NOTEWORTHY Cardiac electrical activity is mediated by ion channels, and Kv4.2 plays a significant role, along with its binding partner, the Kvβ1.1 subunit. In the present study, we identify Kvβ1.1 as a sensor of pyridine nucleotide changes and as a modulator of Kv4.2 gating, action potential duration, and ECG in the mouse heart. Copyright © 2017 the American Physiological Society.

Shortened Spadin Analogs Display Better TREK-1 Inhibition, In Vivo Stability and Antidepressant Activity

PubMed Central

Djillani, Alaeddine; Pietri, Mariel; Moreno, Sébastien; Heurteaux, Catherine; Mazella, Jean; Borsotto, Marc

2017-01-01

Depression is a devastating mental disorder that affects 20% of the population worldwide. Despite their proven efficacy, antidepressants present a delayed onset of action and serious adverse effects. Seven years ago, we described spadin (PE 12-28) as a promising endogenous peptide with antidepressant activity. Spadin specifically blocks the TREK-1 channel. Previously, we showed in vivo that, spadin activity disappeared beyond 7 h after administration. In order to improve in vivo spadin stability and bioavailability, we screened spadin analogs and derivatives. From the study of spadin blood degradation products, we designed a 7 amino-acid peptide, PE 22-28. In vitro studies on hTREK-1/HEK cells by using patch-clamp technique, showed that PE 22-28 displayed a better specificity and affinity for TREK-1 channel compared to spadin, IC50 of 0.12 nM vs. 40–60 nM for spadin. In the same conditions, we also pointed out that different modifications of its N or C-terminal ends maintained or abolished TREK-1 channel activity without affecting PE 22-28 affinity. In vivo, the antidepressant properties of PE 22-28 and its derivatives were demonstrated in behavioral models of depression, such as the forced swimming test. Mice treated with spadin-analogs showed a significant reduction of the immobility time. Moreover, in the novelty suppressed feeding test after a 4-day sub-chronic treatment PE 22-28 reduced significantly the latency to eat the food pellet. PE 22-28 and its analogs were able to induce neurogenesis after only a 4-day treatment with a prominent effect of the G/A-PE 22-28. On mouse cortical neurons, PE 22-28 and its derivatives enhanced synaptogenesis measured by the increase of PSD-95 expression level. Finally, the action duration of PE 22-28 and its analogs was largely improved in comparison with that of spadin, up to 23 h instead of 7 h. Taken together, our results demonstrated that PE 22-28 and its derivatives represent other promising molecules that could be an alternative to spadin in the treatment of depression. PMID:28955242

An In Vivo Study of Low-Dose Intra-Articular Tranexamic Acid Application with Prolonged Clamping Drain Method in Total Knee Replacement: Clinical Efficacy and Safety.

PubMed

Sa-ngasoongsong, Paphon; Chanplakorn, Pongsthorn; Wongsak, Siwadol; Uthadorn, Krisorn; Panpikoon, Tanapong; Jittorntam, Paisan; Aryurachai, Katcharin; Angchaisukisiri, Pantap; Kawinwonggowit, Viroj

2015-01-01

Recently, combined intra-articular tranexamic acid (IA-TXA) injection with clamping drain method showed efficacy for blood loss and transfusion reduction in total knee replacement (TKR). However, until now, none of previous studies revealed the effect of this technique on pharmacokinetics, coagulation, and fibrinolysis. An experimental study was conducted, during 2011-2012, in 30 patients undergoing unilateral TKR. Patients received IA-TXA application and then were allocated into six groups regarding clamping drain duration (2-, 4-, 6-, 8-, 10-, and 12-hours). Blood and drainage fluid were collected to measure tranexamic acid (TXA) level and related coagulation and fibrinolytic markers. Postoperative complication was followed for one year. There was no significant difference of serum TXA level at 2 hour and 24 hour among groups (p < 0.05). Serum TXA level at time of clamp release was significantly different among groups with the highest level at 2 hour (p < 0.0001). There was no significant difference of TXA level in drainage fluid, postoperative blood loss, blood transfusion, and postoperative complications (p < 0.05). Low-dose IA-TXA application in TKR with prolonged clamping drain method is a safe and effective blood conservative technique with only minimal systemic absorption and without significant increase in systemic absorption over time.

Clamping characteristics study on different types of clamping unit

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jiao, Zhiwei; Liu, Haichao; Xie, Pengcheng

2015-05-22

Plastic products are becoming more and more widely used in aerospace, IT, digital electronics and many other fields. With the development of technology, the requirement of product precision is getting higher and higher. However, type and working performance of clamping unit play a decisive role in product precision. Clamping characteristics of different types of clamping unit are discussed in this article, which use finite element numerical analysis method through the software ABAQUS to study the clamping uniformity, and detect the clamping force repeatability precision. The result shows that compared with toggled three-platen clamping unit, clamping characteristics of internal circulation two-platenmore » clamping unit are better, for instance, its mold cavity deformation and force that bars and mold parting surface suffered are more uniform, and its clamping uniformity and repeatability precision is also better.« less

Signaling of pigment-dispersing factor (PDF) in the Madeira cockroach Rhyparobia maderae.

PubMed

Wei, Hongying; Yasar, Hanzey; Funk, Nico W; Giese, Maria; Baz, El-Sayed; Stengl, Monika

2014-01-01

The insect neuropeptide pigment-dispersing factor (PDF) is a functional ortholog of vasoactive intestinal polypeptide, the coupling factor of the mammalian circadian pacemaker. Despite of PDF's importance for synchronized circadian locomotor activity rhythms its signaling is not well understood. We studied PDF signaling in primary cell cultures of the accessory medulla, the circadian pacemaker of the Madeira cockroach. In Ca²⁺ imaging studies four types of PDF-responses were distinguished. In regularly bursting type 1 pacemakers PDF application resulted in dose-dependent long-lasting increases in Ca²⁺ baseline concentration and frequency of oscillating Ca²⁺ transients. Adenylyl cyclase antagonists prevented PDF-responses in type 1 cells, indicating that PDF signaled via elevation of intracellular cAMP levels. In contrast, in type 2 pacemakers PDF transiently raised intracellular Ca²⁺ levels even after blocking adenylyl cyclase activity. In patch clamp experiments the previously characterized types 1-4 could not be identified. Instead, PDF-responses were categorized according to ion channels affected. Application of PDF inhibited outward potassium or inward sodium currents, sometimes in the same neuron. In a comparison of Ca²⁺ imaging and patch clamp experiments we hypothesized that in type 1 cells PDF-dependent rises in cAMP concentrations block primarily outward K⁺ currents. Possibly, this PDF-dependent depolarization underlies PDF-dependent phase advances of pacemakers. Finally, we propose that PDF-dependent concomitant modulation of K⁺ and Na⁺ channels in coupled pacemakers causes ultradian membrane potential oscillations as prerequisite to efficient synchronization via resonance.

Distribution and Function of HCN Channels in the Apical Dendritic Tuft of Neocortical Pyramidal Neurons

PubMed Central

Harnett, Mark T.; Magee, Jeffrey C.

2015-01-01

The apical tuft is the most remote area of the dendritic tree of neocortical pyramidal neurons. Despite its distal location, the apical dendritic tuft of layer 5 pyramidal neurons receives substantial excitatory synaptic drive and actively processes corticocortical input during behavior. The properties of the voltage-activated ion channels that regulate synaptic integration in tuft dendrites have, however, not been thoroughly investigated. Here, we use electrophysiological and optical approaches to examine the subcellular distribution and function of hyperpolarization-activated cyclic nucleotide-gated nonselective cation (HCN) channels in rat layer 5B pyramidal neurons. Outside-out patch recordings demonstrated that the amplitude and properties of ensemble HCN channel activity were uniform in patches excised from distal apical dendritic trunk and tuft sites. Simultaneous apical dendritic tuft and trunk whole-cell current-clamp recordings revealed that the pharmacological blockade of HCN channels decreased voltage compartmentalization and enhanced the generation and spread of apical dendritic tuft and trunk regenerative activity. Furthermore, multisite two-photon glutamate uncaging demonstrated that HCN channels control the amplitude and duration of synaptically evoked regenerative activity in the distal apical dendritic tuft. In contrast, at proximal apical dendritic trunk and somatic recording sites, the blockade of HCN channels decreased excitability. Dynamic-clamp experiments revealed that these compartment-specific actions of HCN channels were heavily influenced by the local and distributed impact of the high density of HCN channels in the distal apical dendritic arbor. The properties and subcellular distribution pattern of HCN channels are therefore tuned to regulate the interaction between integration compartments in layer 5B pyramidal neurons. PMID:25609619

The antipsychotic drug loxapine is an opener of the sodium-activated potassium channel slack (Slo2.2).

PubMed

Biton, B; Sethuramanujam, S; Picchione, Kelly E; Bhattacharjee, A; Khessibi, N; Chesney, F; Lanneau, C; Curet, O; Avenet, P

2012-03-01

Sodium-activated potassium (K(Na)) channels have been suggested to set the resting potential, to modulate slow after-hyperpolarizations, and to control bursting behavior or spike frequency adaptation (Trends Neurosci 28:422-428, 2005). One of the genes that encodes K(Na) channels is called Slack (Kcnt1, Slo2.2). Studies found that Slack channels were highly expressed in nociceptive dorsal root ganglion neurons and modulated their firing frequency (J Neurosci 30:14165-14172, 2010). Therefore, Slack channel openers are of significant interest as putative analgesic drugs. We screened the library of pharmacologically active compounds with recombinant human Slack channels expressed in Chinese hamster ovary cells, by using rubidium efflux measurements with atomic absorption spectrometry. Riluzole at 500 μM was used as a reference agonist. The antipsychotic drug loxapine and the anthelmintic drug niclosamide were both found to activate Slack channels, which was confirmed by using manual patch-clamp analyses (EC(50) = 4.4 μM and EC(50) = 2.9 μM, respectively). Psychotropic drugs structurally related to loxapine were also evaluated in patch-clamp experiments, but none was found to be as active as loxapine. Loxapine properties were confirmed at the single-channel level with recombinant rat Slack channels. In dorsal root ganglion neurons, loxapine was found to behave as an opener of native K(Na) channels and to increase the rheobase of action potential. This study identifies new K(Na) channel pharmacological tools, which will be useful for further Slack channel investigations.

Aldosterone down-regulates the slowly activated delayed rectifier potassium current in adult guinea pig cardiomyocytes.

PubMed

Lv, Yankun; Bai, Song; Zhang, Hua; Zhang, Hongxue; Meng, Jing; Li, Li; Xu, Yanfang

2015-12-01

There is emerging evidence that the mineralocorticoid hormone aldosterone is associated with arrhythmias in cardiovascular disease. However, the effect of aldosterone on the slowly activated delayed rectifier potassium current (IK s ) remains poorly understood. The present study was designed to investigate the modulation of IK s by aldosterone. Adult guinea pigs were treated with aldosterone for 28 days via osmotic pumps. Standard glass microelectrode recordings and whole-cell patch-clamp techniques were used to record action potentials in papillary muscles and IK s in ventricular cardiomyocytes. The aldosterone-treated animals exhibited a prolongation of the QT interval and action potential duration with a higher incidence of early afterdepolarizations. Patch-clamp recordings showed a significant down-regulation of IK s density in the ventricular myocytes of these treated animals. These aldosterone-induced electrophysiological changes were fully prevented by a combined treatment with spironolactone, a mineralocorticoid receptor (MR) antagonist. In addition, in in vitro cultured ventricular cardiomyocytes, treatment with aldosterone (sustained exposure for 24 h) decreased the IK s density in a concentration-dependent manner. Furthermore, a significant corresponding reduction in the mRNA/protein expression of IKs channel pore and auxiliary subunits, KCNQ1 and KCNE1 was detected in ventricular tissue from the aldosterone-treated animals. Aldosterone down-regulates IK s by inhibiting the expression of KCNQ1 and KCNE1, thus delaying the ventricular repolarization. These results provide new insights into the mechanism underlying K(+) channel remodelling in heart disease and may explain the highly beneficial effects of MR antagonists in HF. © 2015 The British Pharmacological Society.

Allergen challenge sensitizes TRPA1 in vagal sensory neurons and afferent C-fiber subtypes in guinea pig esophagus.

PubMed

Liu, Zhenyu; Hu, Youtian; Yu, Xiaoyun; Xi, Jiefeng; Fan, Xiaoming; Tse, Chung-Ming; Myers, Allen C; Pasricha, Pankaj J; Li, Xingde; Yu, Shaoyong

2015-03-15

Transient receptor potential A1 (TRPA1) is a newly defined cationic ion channel, which selectively expresses in primary sensory afferent nerve, and is essential in mediating inflammatory nociception. Our previous study demonstrated that TRPA1 plays an important role in tissue mast cell activation-induced increase in the excitability of esophageal vagal nodose C fibers. The present study aims to determine whether prolonged antigen exposure in vivo sensitizes TRPA1 in a guinea pig model of eosinophilic esophagitis (EoE). Antigen challenge-induced responses in esophageal mucosa were first assessed by histological stains and Ussing chamber studies. TRPA1 function in vagal sensory neurons was then studied by calcium imaging and by whole cell patch-clamp recordings in 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)-labeled esophageal vagal nodose and jugular neurons. Extracellular single-unit recordings were performed in vagal nodose and jugular C-fiber neuron subtypes using ex vivo esophageal-vagal preparations with intact nerve endings in the esophagus. Antigen challenge significantly increased infiltrations of eosinophils and mast cells in the esophagus. TRPA1 agonist allyl isothiocyanate (AITC)-induced calcium influx in nodose and jugular neurons was significantly increased, and current densities in esophageal DiI-labeled nodose and jugular neurons were also significantly increased in antigen-challenged animals. Prolonged antigen challenge decreased esophageal epithelial barrier resistance, which allowed intraesophageal-infused AITC-activating nodose and jugular C fibers at their nerve endings. Collectively, these results demonstrated that prolonged antigen challenge sensitized TRPA1 in esophageal sensory neurons and afferent C fibers. This novel finding will help us to better understand the molecular mechanism underlying esophageal sensory and motor dysfunctions in EoE. Copyright © 2015 the American Physiological Society.

Allergen challenge sensitizes TRPA1 in vagal sensory neurons and afferent C-fiber subtypes in guinea pig esophagus

PubMed Central

Liu, Zhenyu; Hu, Youtian; Yu, Xiaoyun; Xi, Jiefeng; Fan, Xiaoming; Tse, Chung-Ming; Myers, Allen C.; Pasricha, Pankaj J.; Li, Xingde

2015-01-01

Transient receptor potential A1 (TRPA1) is a newly defined cationic ion channel, which selectively expresses in primary sensory afferent nerve, and is essential in mediating inflammatory nociception. Our previous study demonstrated that TRPA1 plays an important role in tissue mast cell activation-induced increase in the excitability of esophageal vagal nodose C fibers. The present study aims to determine whether prolonged antigen exposure in vivo sensitizes TRPA1 in a guinea pig model of eosinophilic esophagitis (EoE). Antigen challenge-induced responses in esophageal mucosa were first assessed by histological stains and Ussing chamber studies. TRPA1 function in vagal sensory neurons was then studied by calcium imaging and by whole cell patch-clamp recordings in 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI)-labeled esophageal vagal nodose and jugular neurons. Extracellular single-unit recordings were performed in vagal nodose and jugular C-fiber neuron subtypes using ex vivo esophageal-vagal preparations with intact nerve endings in the esophagus. Antigen challenge significantly increased infiltrations of eosinophils and mast cells in the esophagus. TRPA1 agonist allyl isothiocyanate (AITC)-induced calcium influx in nodose and jugular neurons was significantly increased, and current densities in esophageal DiI-labeled nodose and jugular neurons were also significantly increased in antigen-challenged animals. Prolonged antigen challenge decreased esophageal epithelial barrier resistance, which allowed intraesophageal-infused AITC-activating nodose and jugular C fibers at their nerve endings. Collectively, these results demonstrated that prolonged antigen challenge sensitized TRPA1 in esophageal sensory neurons and afferent C fibers. This novel finding will help us to better understand the molecular mechanism underlying esophageal sensory and motor dysfunctions in EoE. PMID:25591867

Comparison of NMDA and AMPA Channel Expression and Function between Embryonic and Adult Neurons Utilizing Microelectrode Array Systems.

PubMed

Edwards, Darin; Sommerhage, Frank; Berry, Bonnie; Nummer, Hanna; Raquet, Martina; Clymer, Brad; Stancescu, Maria; Hickman, James J

2017-12-11

Microelectrode arrays (MEAs) are innovative tools used to perform electrophysiological experiments for the study of electrical activity and connectivity in populations of neurons from dissociated cultures. Reliance upon neurons derived from embryonic tissue is a common limitation of neuronal/MEA hybrid systems and perhaps of neuroscience research in general, and the use of adult neurons could model fully functional in vivo parameters more closely. Spontaneous network activity was concurrently recorded from both embryonic and adult rat neurons cultured on MEAs for up to 10 weeks in vitro to characterize the synaptic connections between cell types. The cultures were exposed to synaptic transmission antagonists against NMDA and AMPA channels, which revealed significantly different receptor profiles of adult and embryonic networks in vitro. In addition, both embryonic and adult neurons were evaluated for NMDA and AMPA channel subunit expression over five weeks in vitro. The results established that neurons derived from embryonic tissue did not express mature synaptic channels for several weeks in vitro under defined conditions. Consequently, the embryonic response to synaptic antagonists was significantly different than that of neurons derived from adult tissue sources. These results are especially significant because most studies reported with embryonic hippocampal neurons do not begin at two to four weeks in culture. In addition, the utilization of MEAs in lieu of patch-clamp electrophysiology avoided a large-scale, labor-intensive study. These results establish the utility of this unique hybrid system derived from adult hippocampal tissue in combination with MEAs and offer a more appropriate representation of in vivo function for drug discovery. It has application for neuronal development and regeneration as well as for investigations into neurodegenerative disease, traumatic brain injury, and stroke.

GluN2B N-methyl-D-aspartate receptor and excitatory amino acid transporter 3 are upregulated in primary sensory neurons after 7 days of morphine administration in rats: implication for opiate-induced hyperalgesia.

PubMed

Gong, Kerui; Bhargava, Aditi; Jasmin, Luc

2016-01-01

The contribution of the peripheral nervous system to opiate-induced hyperalgesia (OIH) is not well understood. In this study, we determined the changes in excitability of primary sensory neurons after sustained morphine administration for 7 days. Changes in the expression of glutamate receptors and glutamate transporters after morphine administration were ascertained in dorsal root ganglions. Patch clamp recordings from intact dorsal root ganglions (ex vivo preparation) of morphine-treated rats showed increased excitability of small diameter (≤30 μm) neurons with respect to rheobase and membrane threshold, whereas the excitability of large diameter (>30 μm) neurons remained unchanged. Small diameter neurons also displayed increased responses to glutamate, which were mediated mainly by GluN2B containing N-methyl-D-aspartate (NMDA) receptors, and to a lesser degree by the neuronal excitatory amino acid transporter 3/excitatory amino acid carrier 1. Coadministration in vivo of the GluN2B selective antagonist Ro 25-6981 with morphine for 7 days prevented the appearance of OIH and increased morphine-induced analgesia. Administration of morphine for 7 days led to an increased expression of GluN2B and excitatory amino acid transporter 3/excitatory amino acid carrier 1, but not of the α-amino-3-hydroxy-5-methyl-4-isoxazole propionate, kainate, or group I metabotropic glutamate receptors, or of the vesicular glutamate transporter 2. These results suggest that peripheral glutamatergic neurotransmission contributes to OIH and that GluN2B subunit of NMDA receptors in the periphery may be a target for therapy.

Enhanced inhibitory synaptic transmission in the spinal dorsal horn mediates antinociceptive effects of TC-2559

PubMed Central

2011-01-01

Background TC-2559 is a selective α4β2 subtype of nicotinic acetylcholine receptor (nAChR) partial agonist and α4β2 nAChR activation has been related to antinociception. The aim of this study is to investigate the analgesic effect of TC-2559 and its underlying spinal mechanisms. Results 1) In vivo bioavailability study: TC-2559 (3 mg/kg) had high absorption rate in rats with maximal total brain concentration reached over 4.6 μM within first 15 min after administration and eliminated rapidly with brain half life of about 20 min after injection. 2) In vivo behavioral experiments: TC-2559 exerts dose dependent antinociceptive effects in both formalin test in mice and chronic constriction injury (CCI) model in rats by activation of α4β2 nAChRs; 3) Whole-cell patch-clamp studies in the superficial dorsal horn neurons of the spinal cord slices: perfusion of TC-2559 (2 μM) significantly increased the frequency, but not amplitude of spontaneous inhibitory postsynaptic currents (sIPSCs). The enhancement of sIPSCs was blocked by pre-application of DHβE (2 μM), a selective α4β2 nicotinic receptor antagonist. Neither the frequency nor the amplitude of spontaneous excitatory postsynaptic currents (sEPSCs) of spinal dorsal horn neurons were affected by TC-2559. Conclusions Enhancement of inhibitory synaptic transmission in the spinal dorsal horn via activation of α4β2 nAChRs may be one of the mechanisms of the antinociceptive effects of TC-2559 on pathological pain models. It provides further evidence to support the notion that selective α4β2 subtype nAChR agonist may be developed as new analgesic drug for the treatment of neuropathic pain. PMID:21816108

A Novel Orally Available Asthma Drug Candidate That Reduces Smooth Muscle Constriction and Inflammation by Targeting GABAA Receptors in the Lung.

PubMed

Forkuo, Gloria S; Nieman, Amanda N; Kodali, Revathi; Zahn, Nicolas M; Li, Guanguan; Rashid Roni, M S; Stephen, Michael Rajesh; Harris, Ted W; Jahan, Rajwana; Guthrie, Margaret L; Yu, Olivia B; Fisher, Janet L; Yocum, Gene T; Emala, Charles W; Steeber, Douglas A; Stafford, Douglas C; Cook, James M; Arnold, Leggy A

2018-05-07

We describe lead compound MIDD0301 for the oral treatment of asthma based on previously developed positive allosteric α 5 β 3 γ 2 selective GABA A receptor (GABA A R) ligands. MIDD0301 relaxed airway smooth muscle at single micromolar concentrations as demonstrated with ex vivo guinea pig tracheal rings. MIDD0301 also attenuated airway hyperresponsiveness (AHR) in an ovalbumin murine model of asthma by oral administration. Reduced numbers of eosinophils and macrophages were observed in mouse bronchoalveolar lavage fluid without changing mucous metaplasia. Importantly, lung cytokine expression of IL-17A, IL-4, and TNF-α were reduced for MIDD0301-treated mice without changing antiinflammatory cytokine IL-10 levels. Automated patch clamp confirmed amplification of GABA induced current mediated by α 1-3,5 β 3 γ 2 GABA A Rs in the presence of MIDD0301. Pharmacodynamically, transmembrane currents of ex vivo CD4 + T cells from asthmatic mice were potentiated by MIDD0301 in the presence of GABA. The number of CD4 + T cells observed in the lung of MIDD0301-treated mice were reduced by an oral treatment of 20 mg/kg b.i.d. for 5 days. A half-life of almost 14 h was demonstrated by pharmacokinetic studies (PK) with no adverse CNS effects when treated mice were subjected to sensorimotor studies using the rotarod. PK studies also confirmed very low brain distribution. In conclusion, MIDD0301 represents a safe and improved oral asthma drug candidate that relaxes airway smooth muscle and attenuates inflammation in the lung leading to a reduction of AHR at a dosage lower than earlier reported GABA A R ligands.

Medullary 5-HT neurons: Switch from tonic respiratory drive to chemoreception during postnatal development

PubMed Central

Cerpa, Veronica J.; Wu, Yuanming; Bravo, Eduardo; Teran, Frida A.; Flynn, Rachel S.; Richerson, George B.

2016-01-01

Serotonin (5-HT) neurons contribute to respiratory chemoreception in adult mice, but it is unclear whether they play a similar role in neonatal mice. We studied breathing during development in Lmx1bf/f/p mice, which lack 5-HT neurons. From postnatal days 1–7 (P1–P7), ventilation of Lmx1bf/f/p mice breathing room air was 50% of WT mice (p < 0.001). By P12, baseline ventilation increased to a level equal to WT mice. In contrast, the hypercapnic ventilatory response (HCVR) of neonatal Lmx1bf/f/p and WT mice were equal to each other, but were both much less than adult WT mice. By P21 the HCVR of WT mice increased to near adult levels, but the HCVR of Lmx1bf/f/p mice had not changed, and was 42% less than WT mice. Primary cell cultures were prepared from the ventromedial medulla of neonatal mice, and patch-clamp recordings were made from neurons identified as serotonergic by expression of a reporter gene. In parallel with developmental changes of the HCVR in vivo, 5-HT neurons had little chemosensitivity to acidosis until 12 days in vitro (DIV), after which their response increased to reach a plateau around 25 DIV. Neonatal Lmx1bf/f/p mice displayed high mortality and decreased growth rate, and this worsened in hypoxia. Mortality was decreased in hyperoxia. These results indicate that maturation of 5-HT neurons contributes to development of respiratory CO2/pH chemoreception during the first few weeks of life in mice in vivo. A defect in the 5-HT system in early postnatal life decreases survival due in part to hypoxia. PMID:27619736

Caveolin-3 Overexpression Attenuates Cardiac Hypertrophy via Inhibition of T-type Ca2+ Current Modulated by Protein Kinase Cα in Cardiomyocytes*

PubMed Central

Markandeya, Yogananda S.; Phelan, Laura J.; Woon, Marites T.; Keefe, Alexis M.; Reynolds, Courtney R.; August, Benjamin K.; Hacker, Timothy A.; Roth, David M.; Patel, Hemal H.; Balijepalli, Ravi C.

2015-01-01

Pathological cardiac hypertrophy is characterized by subcellular remodeling of the ventricular myocyte with a reduction in the scaffolding protein caveolin-3 (Cav-3), altered Ca2+ cycling, increased protein kinase C expression, and hyperactivation of calcineurin/nuclear factor of activated T cell (NFAT) signaling. However, the precise role of Cav-3 in the regulation of local Ca2+ signaling in pathological cardiac hypertrophy is unclear. We used cardiac-specific Cav-3-overexpressing mice and in vivo and in vitro cardiac hypertrophy models to determine the essential requirement for Cav-3 expression in protection against pharmacologically and pressure overload-induced cardiac hypertrophy. Transverse aortic constriction and angiotensin-II (Ang-II) infusion in wild type (WT) mice resulted in cardiac hypertrophy characterized by significant reduction in fractional shortening, ejection fraction, and a reduced expression of Cav-3. In addition, association of PKCα and angiotensin-II receptor, type 1, with Cav-3 was disrupted in the hypertrophic ventricular myocytes. Whole cell patch clamp analysis demonstrated increased expression of T-type Ca2+ current (ICa, T) in hypertrophic ventricular myocytes. In contrast, the Cav-3-overexpressing mice demonstrated protection from transverse aortic constriction or Ang-II-induced pathological hypertrophy with inhibition of ICa, T and intact Cav-3-associated macromolecular signaling complexes. siRNA-mediated knockdown of Cav-3 in the neonatal cardiomyocytes resulted in enhanced Ang-II stimulation of ICa, T mediated by PKCα, which caused nuclear translocation of NFAT. Overexpression of Cav-3 in neonatal myocytes prevented a PKCα-mediated increase in ICa, T and nuclear translocation of NFAT. In conclusion, we show that stable Cav-3 expression is essential for protecting the signaling mechanisms in pharmacologically and pressure overload-induced cardiac hypertrophy. PMID:26170457

Gene Editing Vectors for Studying Nicotinic Acetylcholine Receptors in Cholinergic Transmission.

PubMed

Peng, Can; Yan, Yijin; Kim, Veronica J; Engle, Staci E; Berry, Jennifer N; McIntosh, J Michael; Neve, Rachael L; Drenan, Ryan M

2018-05-19

Nicotinic acetylcholine receptors (nAChRs), prototype members of the cys-loop ligand gated ion channel family, are key mediators of cholinergic transmission in the central nervous system. Despite their importance, technical gaps exist in our ability to dissect the function of individual subunits in the brain. To overcome these barriers, we designed CRISPR/Cas9 small guide RNA sequences (sgRNAs) for production of loss-of-function alleles in mouse nAChR genes. These sgRNAs were validated in vitro via deep sequencing. We subsequently targeted candidate nAChR genes in vivo by creating herpes simplex virus (HSV) vectors delivering sgRNAs and Cas9 expression to mouse brain. Production of loss-of-function insertions or deletions (indels) by these "all-in-one" HSV vectors was confirmed using brain slice patch clamp electrophysiology coupled with pharmacological analysis. Next, we developed a scheme for cell type-specific gene editing in mouse brain. Knockin mice expressing Cas9 in a Cre-dependent manner were validated using viral microinjections and genetic crosses to common Cre-driver mouse lines. We subsequently confirmed functional Cas9 activity by targeting the ubiquitous neuronal protein, NeuN, using adeno associated virus (AAV) delivery of sgRNAs. Finally, the mouse β2 nAChR gene was successfully targeted in dopamine transporter (DAT) positive neurons via CRISPR/Cas9. The sgRNA sequences and viral vectors, including our scheme for Cre-dependent gene editing, should be generally useful to the scientific research community. These tools could lead to new discoveries related to the function of nAChRs in neurotransmission and behavioral processes. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Hda Monomerization by ADP Binding Promotes Replicase Clamp-mediated DnaA-ATP Hydrolysis*S⃞

PubMed Central

Su'etsugu, Masayuki; Nakamura, Kenta; Keyamura, Kenji; Kudo, Yuka; Katayama, Tsutomu

2008-01-01

ATP-DnaA is the initiator of chromosomal replication in Escherichia coli, and the activity of DnaA is regulated by the regulatory inactivation of the DnaA (RIDA) system. In this system, the Hda protein promotes DnaA-ATP hydrolysis to produce inactive ADP-DnaA in a mechanism that is mediated by the DNA-loaded form of the replicase sliding clamp. In this study, we first revealed that hda translation uses an unusual initiation codon, CUG, located downstream of the annotated initiation codon. The CUG initiation codon could be used for restricting the Hda level, as this initiation codon has a low translation efficiency, and the cellular Hda level is only ∼100 molecules per cell. Hda translated using the correct reading frame was purified and found to have a high RIDA activity in vitro. Moreover, we found that Hda has a high affinity for ADP but not for other nucleotides, including ATP. ADP-Hda was active in the RIDA system in vitro and stable in a monomeric state, whereas apo-Hda formed inactive homomultimers. Both ADP-Hda and apo-Hda could form complexes with the DNA-loaded clamp; however, only ADP-Hda-DNA-clamp complexes were highly functional in the following interaction with DnaA. Formation of ADP-Hda was also observed in vivo, and mutant analysis suggested that ADP binding is crucial for cellular Hda activity. Thus, we propose that ADP is a crucial Hda ligand that promotes the activated conformation of the protein. ADP-dependent monomerization might enable the arginine finger of the Hda AAA+ domain to be accessible to ATP bound to the DnaA AAA+ domain. PMID:18977760

Hda monomerization by ADP binding promotes replicase clamp-mediated DnaA-ATP hydrolysis.

PubMed

Su'etsugu, Masayuki; Nakamura, Kenta; Keyamura, Kenji; Kudo, Yuka; Katayama, Tsutomu

2008-12-26

ATP-DnaA is the initiator of chromosomal replication in Escherichia coli, and the activity of DnaA is regulated by the regulatory inactivation of the DnaA (RIDA) system. In this system, the Hda protein promotes DnaA-ATP hydrolysis to produce inactive ADP-DnaA in a mechanism that is mediated by the DNA-loaded form of the replicase sliding clamp. In this study, we first revealed that hda translation uses an unusual initiation codon, CUG, located downstream of the annotated initiation codon. The CUG initiation codon could be used for restricting the Hda level, as this initiation codon has a low translation efficiency, and the cellular Hda level is only approximately 100 molecules per cell. Hda translated using the correct reading frame was purified and found to have a high RIDA activity in vitro. Moreover, we found that Hda has a high affinity for ADP but not for other nucleotides, including ATP. ADP-Hda was active in the RIDA system in vitro and stable in a monomeric state, whereas apo-Hda formed inactive homomultimers. Both ADP-Hda and apo-Hda could form complexes with the DNA-loaded clamp; however, only ADP-Hda-DNA-clamp complexes were highly functional in the following interaction with DnaA. Formation of ADP-Hda was also observed in vivo, and mutant analysis suggested that ADP binding is crucial for cellular Hda activity. Thus, we propose that ADP is a crucial Hda ligand that promotes the activated conformation of the protein. ADP-dependent monomerization might enable the arginine finger of the Hda AAA+ domain to be accessible to ATP bound to the DnaA AAA+ domain.

Redox Regulation of Epithelial Sodium Channels Examined in Alveolar Type 1 and 2 Cells Patch-clamped in Lung Slice Tissue*

PubMed Central

Helms, My N.; Jain, Lucky; Self, Julie L.; Eaton, Douglas C.

2008-01-01

The alveolar surface of the lung is lined by alveolar type 1 (AT1) and type 2 (AT2) cells. Using single channel patch clamp analysis in lung slice preparations, we are able to uniquely study AT1 and AT2 cells separately from intact lung. We report for the first time the Na+ transport properties of type 2 cells accessed in live lung tissue (as we have done in type 1 cells). Type 2 cells in lung tissue slices express both highly selective cation and nonselective cation channels with average conductances of 8.8 ± 3.2 and 22.5 ± 6.3 picosiemens, respectively. Anion channels with 10-picosiemen conductance are also present in the apical membrane of type 2 cells. Our lung slice studies importantly verify the use of cultured cell model systems commonly used in lung epithelial sodium channel (ENaC) studies. Furthermore, we identify novel functional differences between the cells that make up the alveolar epithelium. One important difference is that exposure to the nitric oxide (NO) donor, PAPA-NONOate (1.5 μm), significantly decreases average ENaC NPo in type 2 cells (from 1.38 ± 0.26 to 0.82 ± 0.16; p < 0.05 and n = 18) but failed to alter ENaC activity in alveolar type 1 cells. Elevating endogenous superoxide (\\documentclass[10pt]{article} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{pmc} \\usepackage[Euler]{upgreek} \\pagestyle{empty} \\oddsidemargin -1.0in \\begin{document} \\begin{equation*}{\\mathrm{O}}_{2}^{\\overline{.}}\\end{equation*}\\end{document}) levels with Ethiolat, a superoxide dismutase inhibitor, prevented NO inhibition of ENaC activity in type 2 cells, supporting the novel hypothesis that \\documentclass[10pt]{article} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{pmc} \\usepackage[Euler]{upgreek} \\pagestyle{empty} \\oddsidemargin -1.0in \\begin{document} \\begin{equation*}{\\mathrm{O}}_{2}^{\\overline{.}}\\end{equation*}\\end{document} and NO signaling plays an important role in maintaining lung fluid balance. PMID:18541535

Voltage-clamp analysis of the potentiation of the slow Ca2+-activated K+ current in hippocampal pyramidal neurons.

PubMed

Borde, M; Bonansco, C; Fernández de Sevilla, D; Le Ray, D; Buño, W

2000-01-01

Exploring the principles that govern activity-dependent changes in excitability is an essential step to understand the function of the nervous system, because they act as a general postsynaptic control mechanism that modulates the flow of synaptic signals. We show an activity-dependent potentiation of the slow Ca2+-activated K+ current (sl(AHP)) which induces sustained decreases in the excitability in CA1 pyramidal neurons. We analyzed the sl(AHP) using the slice technique and voltage-clamp recordings with sharp or patch-electrodes. Using sharp electrodes-repeated activation with depolarizing pulses evoked a prolonged (8-min) potentiation of the amplitude (171%) and duration (208%) of the sl(AHP). Using patch electrodes, early after entering the whole-cell configuration (<20 min), responses were as those reported above. However, although the sl(AHP) remained unchanged, its potentiation was markedly reduced in later recordings, suggesting that the underlying mechanisms were rapidly eliminated by intracellular dialysis. Inhibition of L-type Ca2+ current by nifedipine (20 microM) markedly reduced the sl(AHP) (79%) and its potentiation (55%). Ryanodine (20 microM) that blocks the release of intracellular Ca2+ also reduced sl(AHP) (29%) and its potentiation (25%). The potentiation of the sl(AHP) induced a marked and prolonged (>50%; approximately equals 8 min) decrease in excitability. The results suggest that sl(AHP) is potentiated as a result of an increased intracellular Ca2+ concentration ([Ca2+]i) following activation of voltage-gated L-type Ca2+ channels, aided by the subsequent release of Ca2+ from intracellular stores. Another possibility is that repeated activation increases the Ca2+-binding capacity of the channels mediating the sl(AHP). This potentiation of the sl(AHP) could be relevant in hippocampal physiology, because the changes in excitability it causes may regulate the induction threshold of the long-term potentiation of synaptic efficacy. Moreover, the potentiation would act as a protective mechanism by reducing excitability and preventing the accumulation of intracellular Ca2+ to toxic levels when intense synaptic activation occurs.

Expression and function of K(V)2-containing channels in human urinary bladder smooth muscle.

PubMed

Hristov, Kiril L; Chen, Muyan; Afeli, Serge A Y; Cheng, Qiuping; Rovner, Eric S; Petkov, Georgi V

2012-06-01

The functional role of the voltage-gated K(+) (K(V)) channels in human detrusor smooth muscle (DSM) is largely unexplored. Here, we provide molecular, electrophysiological, and functional evidence for the expression of K(V)2.1, K(V)2.2, and the electrically silent K(V)9.3 subunits in human DSM. Stromatoxin-1 (ScTx1), a selective inhibitor of K(V)2.1, K(V)2.2, and K(V)4.2 homotetrameric channels and of K(V)2.1/9.3 heterotetrameric channels, was used to examine the role of these channels in human DSM function. Human DSM tissues were obtained during open bladder surgeries from patients without a history of overactive bladder. Freshly isolated human DSM cells were studied using RT-PCR, immunocytochemistry, live-cell Ca(2+) imaging, and the perforated whole cell patch-clamp technique. Isometric DSM tension recordings of human DSM isolated strips were conducted using tissue baths. RT-PCR experiments showed mRNA expression of K(V)2.1, K(V)2.2, and K(V)9.3 (but not K(V)4.2) channel subunits in human isolated DSM cells. K(V)2.1 and K(V)2.2 protein expression was confirmed by Western blot analysis and immunocytochemistry. Perforated whole cell patch-clamp experiments revealed that ScTx1 (100 nM) inhibited the amplitude of the voltage step-induced K(V) current in freshly isolated human DSM cells. ScTx1 (100 nM) significantly increased the intracellular Ca(2+) level in DSM cells. In human DSM isolated strips, ScTx1 (100 nM) increased the spontaneous phasic contraction amplitude and muscle force, and enhanced the amplitude of the electrical field stimulation-induced contractions within the range of 3.5-30 Hz stimulation frequencies. These findings reveal that ScTx1-sensitive K(V)2-containing channels are key regulators of human DSM excitability and contractility and may represent new targets for pharmacological or genetic intervention for bladder dysfunction.

The luminal K+ channel of the thick ascending limb of Henle's loop.

PubMed

Bleich, M; Schlatter, E; Greger, R

1990-01-01

In vitro perfused rat thick ascending limbs of Henle's loop (TAL) were used (n = 260) to analyse the conductance properties of the luminal membrane applying the patch-clamp technique. Medullary (mTAL) and cortical (cTAL) tubule segments were dissected and perfused in vitro. The free end of the tubule was held and immobilized at one edge by a holding pipette kept under continuous suction. A micropositioner was used to insert a patch pipette into the lumen, and a gigaohm seal with the luminal membrane was achieved in 455 instances out of considerably more trials. In approximately 20% of all gigaohm seals recordings of single ionic channels were obtained. We have identified only one single type of K+ channel in these cell-attached and cell-excised recordings. In the cell-attached configuration with KCl or NaCl in the pipette, the channel had a conductance of 60 +/- 6 pS (n = 24) and 31 +/- 7 pS (n = 4) respectively. In cell-free patches with KCl either in the patch pipette or in the bath and with a Ringer-type solution (NaCl) on the opposite side the conductance was 72 +/- 4 pS (n = 37) at a clamp voltage of 0 mV. The permeability was 0.33 +/- 0.02 . 10(-12) cm3/s. The selectivity sequence of this channel was: K+ = Rb+ = NH4+ = Cs+ greater than Li+ much greater than Na+ = 0; the conductance sequence was K+ much greater than Li+ much greater than Rb+ = Cs+ = NH4+ = Na+ = 0. In excised patches Rb+, Cs+ and NH4+ when present in the bath at 145 mmol/l all inhibited K+ currents out of the pipette. The channel kinetics were described by one open (9.5 +/- 1.5 ms, n = 18) and by two closed (1.4 +/- 0.1 and 14 +/- 2 ms) time constants. The open probability of this channel was increased by depolarization. The channel open probability was reduced voltage dependently by Ba2+ (half maximal inhibition at 0 mV: 0.07 mmol/l) from the cytosolic side. Verapamil, diltiazem, quinine and quinidine inhibited at approximately 1 mumol/l -0.1 mmol/l from either side. Similarly, the amino cations lidocaine, tetraethylammonium and choline inhibited at 10-100 mmol/l. The channel was downregulated in its open probability by cytosolic Ca2+ activities greater than 10(-7) mol/l and by adenosine triphosphate greater than or equal to 10(-4) mol/l. The open probability was downregulated by decreasing cytosolic pH (2-fold by a decrease in pH by less than or equal to 0.2 units).(ABSTRACT TRUNCATED AT 400 WORDS)

Preparation of mucoadhesive oral patches containing tetracycline hydrochloride and carvacrol for treatment of local mouth bacterial infections and candidiasis.

PubMed

Obaidat, Rana M; Bader, Ammar; Al-Rajab, Wafa; Abu Sheikha, Ghassan; Obaidat, Aiman A

2011-01-01

The specific aim of this work was to prepare mucoadhesive patches containing tetracycline hydrochloride and carvacrol in an attempt to develop a novel oral drug delivery system for the treatment of mouth infections. The bilayered patches were prepared using ethyl cellulose as a backing layer and carbopol 934 as a matrix mucoadhesive layer. Patches were prepared with different loading amounts of tetracycline hydrochloride and carvacrol. The antimicrobial activity was assessed for the prepared patches using the disc-diffusion method against the yeast Candida albicans and five bacterial strains, including Pseudomonas aeruginosa, Escherichia coli, Bacillus cereus, Staphylococcus aureus, and Bacillus bronchispti. In this work, we highlighted the possibility of occurrence of a synergistic action between carvacrol and tetracycline. The best formulation was selected based on microbiological tests, drug release, ex-vivo mucoadhesive performance, and swelling index. Physical characteristics of the selected formulations were determined. These included pH, patch thickness, weight uniformity, content uniformity, folding endurance, and patch stability.

Investigating ion channel conformational changes using voltage clamp fluorometry.

PubMed

Talwar, Sahil; Lynch, Joseph W

2015-11-01

Ion channels are membrane proteins whose functions are governed by conformational changes. The widespread distribution of ion channels, coupled with their involvement in most physiological and pathological processes and their importance as therapeutic targets, renders the elucidation of these conformational mechanisms highly compelling from a drug discovery perspective. Thanks to recent advances in structural biology techniques, we now have high-resolution static molecular structures for members of the major ion channel families. However, major questions remain to be resolved about the conformational states that ion channels adopt during activation, drug modulation and desensitization. Patch-clamp electrophysiology has long been used to define ion channel conformational states based on functional criteria. It achieves this by monitoring conformational changes at the channel gate and cannot detect conformational changes occurring in regions distant from the gate. Voltage clamp fluorometry involves labelling cysteines introduced into domains of interest with environmentally sensitive fluorophores and inferring structural rearrangements from voltage or ligand-induced fluorescence changes. Ion channel currents are monitored simultaneously to verify the conformational status. By defining real time conformational changes in domains distant from the gate, this technique provides unexpected new insights into ion channel structure and function. This review aims to summarise the methodology and highlight recent innovative applications of this powerful technique. This article is part of the Special Issue entitled 'Fluorescent Tools in Neuropharmacology'. Copyright © 2015 Elsevier Ltd. All rights reserved.

Experimental Modal Analysis of Rectangular and Circular Beams

ERIC Educational Resources Information Center

Emory, Benjamin H.; Zhu, Wei Dong

2006-01-01

Analytical and experimental methods are used to determine the natural frequencies and mode shapes of Aluminum 6061-T651 beams with rectangular and circular cross-sections. A unique test stand is developed to provide the rectangular beam with different boundary conditions including clamped-free, clamped-clamped, clamped-pinned, and pinned-pinned.…

Dynamic analysis of clamp band joint system subjected to axial vibration

NASA Astrophysics Data System (ADS)

Qin, Z. Y.; Yan, S. Z.; Chu, F. L.

2010-10-01

Clamp band joints are commonly used for connecting circular components together in industry. Some of the systems jointed by clamp band are subjected to dynamic load. However, very little research on the dynamic characteristics for this kind of joint can be found in the literature. In this paper, a dynamic model for clamp band joint system is developed. Contact and frictional slip between the components are accommodated in this model. Nonlinear finite element analysis is conducted to identify the model parameters. Then static experiments are carried out on a scaled model of the clamp band joint to validate the joint model. Finally, the model is adopted to study the dynamic characteristics of the clamp band joint system subjected to axial harmonic excitation and the effects of the wedge angle of the clamp band joint and the preload on the response. The model proposed in this paper can represent the nonlinearity of the clamp band joint and be used conveniently to investigate the effects of the structural and loading parameters on the dynamic characteristics of this type of joint system.

A New Individually Addressable Micro-LED Array for Photogenetic Neural Stimulation.

PubMed

McGovern, B; Berlinguer Palmini, R; Grossman, N; Drakakis, E; Poher, V; Neil, M A A; Degenaar, P

2010-12-01

Here, we demonstrate the use of a micro light emitting diode (LED) array as a powerful tool for complex spatiotemporal control of photosensitized neurons. The array can generate arbitrary, 2-D, excitation patterns with millisecond and micrometer resolution. In particular, we describe an active matrix control address system to allow simultaneous control of 256 individual micro LEDs. We present the system optically integrated into a microscope environment and patch clamp electrophysiology. The results show that the emitters have sufficient radiance at the required wavelength to stimulate neurons expressing channelrhodopsin-2 (ChR2).

Measurement of Single Channel Currents from Cardiac Gap Junctions

NASA Astrophysics Data System (ADS)

Veenstra, Richard D.; Dehaan, Robert L.

1986-08-01

Cardiac gap junctions consist of arrays of integral membrane proteins joined across the intercellular cleft at points of cell-to-cell contact. These junctional proteins are thought to form pores through which ions can diffuse from cytosol to cytosol. By monitoring whole-cell currents in pairs of embryonic heart cells with two independent patch-clamp circuits, the properties of single gap junction channels have been investigated. These channels had a conductance of about 165 picosiemens and underwent spontaneous openings and closings that were independent of voltage. Channel activity and macroscopic junctional conductance were both decreased by the uncoupling agent 1-octanol.

Nervous System of Periplaneta americana Cockroach as a Model in Toxinological Studies: A Short Historical and Actual View

PubMed Central

Stankiewicz, Maria; Dąbrowski, Marcin; de Lima, Maria Elena

2012-01-01

Nervous system of Periplaneta americana cockroach is used in a wide range of pharmacological studies, including electrophysiological techniques. This paper presents its role as a preparation in the development of toxinological studies in the following electrophysiological methods: double-oil-gap technique on isolated giant axon, patch-clamp on DUM (dorsal unpaired median) neurons, microelectrode technique in situ conditions on axon in connective and DUM neurons in ganglion, and single-fiber oil-gap technique on last abdominal ganglion synapse. At the end the application of cockroach synaptosomal preparation is mentioned. PMID:22666245

Effects of pioglitazone on cardiac ion currents and action potential morphology in canine ventricular myocytes.

PubMed

Kistamás, Kornél; Szentandrássy, Norbert; Hegyi, Bence; Ruzsnavszky, Ferenc; Váczi, Krisztina; Bárándi, László; Horváth, Balázs; Szebeni, Andrea; Magyar, János; Bányász, Tamás; Kecskeméti, Valéria; Nánási, Péter P

2013-06-15

Despite its widespread therapeutical use there is little information on the cellular cardiac effects of the antidiabetic drug pioglitazone in larger mammals. In the present study, therefore, the concentration-dependent effects of pioglitazone on ion currents and action potential configuration were studied in isolated canine ventricular myocytes using standard microelectrode, conventional whole cell patch clamp, and action potential voltage clamp techniques. Pioglitazone decreased the maximum velocity of depolarization and the amplitude of phase-1 repolarization at concentrations ≥3 μM. Action potentials were shortened by pioglitazone at concentrations ≥10 μM, which effect was accompanied with significant reduction of beat-to-beat variability of action potential duration. Several transmembrane ion currents, including the transient outward K(+) current (Ito), the L-type Ca(2+) current (ICa), the rapid and slow components of the delayed rectifier K(+) current (IKr and IKs, respectively), and the inward rectifier K(+) current (IK1) were inhibited by pioglitazone under conventional voltage clamp conditions. Ito was blocked significantly at concentrations ≥3 μM, ICa, IKr, IKs at concentrations ≥10 μM, while IK1 at concentrations ≥30 μM. Suppression of Ito, ICa, IKr, and IK1 has been confirmed also under action potential voltage clamp conditions. ATP-sensitive K(+) current, when activated by lemakalim, was effectively blocked by pioglitazone. Accordingly, action potentials were prolonged by 10 μM pioglitazone when the drug was applied in the presence of lemakalim. All these effects developed rapidly and were readily reversible upon washout. In conclusion, pioglitazone seems to be a harmless agent at usual therapeutic concentrations. Copyright © 2013 Elsevier B.V. All rights reserved.

Diadenosine tetraphosphate-gating of cardiac K(ATP) channels requires intact actin cytoskeleton.

PubMed

Jovanović, S; Jovanović, A

2001-09-01

Diadenosine polyphosphates (ApnA) have been recently discovered in the heart, and their levels found to be regulated by ischemia. These signaling molecules are believed to regulate cellular processes that alarm a cell to metabolic stress. In particular, changes in cardiac diadenosine polyphosphates (ApnA) levels may contribute to the regulation of ATP-sensitive K+ (K(ATP)) channel activity, an ion channel that couples the cellular metabolic state with membrane excitability. A feature of myocardial ischemia is the disruption of the actin cytoskeleton which critically regulates the behavior of K(ATP) channels. Whether the integrity of actin microfilaments regulates the interaction of ApnA with K(ATP) channels is not known. The inside-out configuration of the patch-clamp technique was applied to cardiomyocytes isolated from guinea-pig heart. Following patch excision, the prototype dinucleotide, diadenosine tetraphosphate (Ap4A), inhibited K(ATP) channel opening. Treatment of the internal side of membrane patches with either cytochalasin B or DNase I, disrupters of the actin cytoskeleton, prevented Ap4A-induced inhibition of K(ATP) channel opening. Application of purified actin to DNase-treated membrane patches restored the ability of Ap4A to close K(ATP) channels. This study shows that inhibition of cardiac K(ATP) channel by Ap4A, a putative alarmone, requires intact subsarcolemmal actin network. Such interaction between K(ATP) channels, the cardiomyocyte cytoskeleton and intracellular Ap4A could affect different channel-dependent functions.

Transition metal ion FRET to measure short-range distances at the intracellular surface of the plasma membrane

PubMed Central

Senning, Eric N.; Aman, Teresa K.

2016-01-01

Biological membranes are complex assemblies of lipids and proteins that serve as platforms for cell signaling. We have developed a novel method for measuring the structure and dynamics of the membrane based on fluorescence resonance energy transfer (FRET). The method marries four technologies: (1) unroofing cells to isolate and access the cytoplasmic leaflet of the plasma membrane; (2) patch-clamp fluorometry (PCF) to measure currents and fluorescence simultaneously from a membrane patch; (3) a synthetic lipid with a metal-chelating head group to decorate the membrane with metal-binding sites; and (4) transition metal ion FRET (tmFRET) to measure short distances between a fluorescent probe and a transition metal ion on the membrane. We applied this method to measure the density and affinity of native and introduced metal-binding sites in the membrane. These experiments pave the way for measuring structural rearrangements of membrane proteins relative to the membrane. PMID:26755772

Aniracetam reduces glutamate receptor desensitization and slows the decay of fast excitatory synaptic currents in the hippocampus.

PubMed Central

Isaacson, J S; Nicoll, R A

1991-01-01

Aniracetam is a nootropic drug that has been shown to selectively enhance quisqualate receptor-mediated responses in Xenopus oocytes injected with brain mRNA and in hippocampal pyramidal cells [Ito, I., Tanabe, S., Kohda, A. & Sugiyama, H. (1990) J. Physiol. (London) 424, 533-544]. We have used patch clamp recording techniques in hippocampal slices to elucidate the mechanism for this selective action. We find that aniracetam enhances glutamate-evoked currents in whole-cell recordings and, in outside-out patches, strongly reduces glutamate receptor desensitization. In addition, aniracetam selectively prolongs the time course and increases the peak amplitude of fast synaptic currents. These findings indicate that aniracetam slows the kinetics of fast synaptic transmission and are consistent with the proposal [Trussell, L. O. & Fischbach, G. D. (1989) Neuron 3, 209-218; Tang, C.-M., Dichter, M. & Morad, M. (1989) Science 243, 1474-1477] that receptor desensitization governs the strength of fast excitatory synaptic transmission in the brain. PMID:1660156

Aniracetam reduces glutamate receptor desensitization and slows the decay of fast excitatory synaptic currents in the hippocampus.

PubMed

Isaacson, J S; Nicoll, R A

1991-12-01

Aniracetam is a nootropic drug that has been shown to selectively enhance quisqualate receptor-mediated responses in Xenopus oocytes injected with brain mRNA and in hippocampal pyramidal cells [Ito, I., Tanabe, S., Kohda, A. & Sugiyama, H. (1990) J. Physiol. (London) 424, 533-544]. We have used patch clamp recording techniques in hippocampal slices to elucidate the mechanism for this selective action. We find that aniracetam enhances glutamate-evoked currents in whole-cell recordings and, in outside-out patches, strongly reduces glutamate receptor desensitization. In addition, aniracetam selectively prolongs the time course and increases the peak amplitude of fast synaptic currents. These findings indicate that aniracetam slows the kinetics of fast synaptic transmission and are consistent with the proposal [Trussell, L. O. & Fischbach, G. D. (1989) Neuron 3, 209-218; Tang, C.-M., Dichter, M. & Morad, M. (1989) Science 243, 1474-1477] that receptor desensitization governs the strength of fast excitatory synaptic transmission in the brain.

Delamination detection in smart composite beams using Lamb waves

NASA Astrophysics Data System (ADS)

Ip, Kim-Ho; Mai, Yiu-Wing

2004-06-01

This paper presents a feasibility study on using Lamb waves to detect and locate through-width delamination in fiber-reinforced plastic beams. An active diagnostic system is proposed for clamped-free specimens. It consists of a piezoelectric patch and an accelerometer both mounted near the support. Such a system can locate damage in an absolute sense, that is, a priori knowledge on the response from pristine specimens is not required. The fundamental anti-symmetric Lamb wave mode is chosen as the diagnostic wave. It is generated by applying a voltage in the form of sinusoidal bursts to the piezoelectric patch. The proposed system was applied to locate delaminations in some fabricated Kevlar/epoxy beam specimens. With an appropriate actuating frequency, distortions of waveforms due to boundary reflections can be reduced. Based on their arrival times and the known propagating speed of Lamb waves, the delaminations can be located. The errors associated with the predicted damage positions range from 4.5% to 8.5%.

Rapid temperature jump by infrared diode laser irradiation for patch-clamp studies.

PubMed

Yao, Jing; Liu, Beiying; Qin, Feng

2009-05-06

Several thermal TRP ion channels have recently been identified. These channels are directly gated by temperature, but the mechanisms have remained elusive. Studies of their temperature gating have been impeded by lack of methods for rapid alteration of temperature in live cells. As a result, only measurements of steady-state properties have been possible. To solve the problem, we have developed an optical approach that uses recently available infrared diode lasers as heat sources. By restricting laser irradiation around a single cell, our approach can produce constant temperature jumps over 50 degrees C in submilliseconds. Experiments with several heat-gated ion channels (TRPV1-3) show its applicability for rapid temperature perturbation in both single cells and membrane patches. Compared with other laser heating approaches such as those by Raman-shifting of the Nd:YAG fundamentals, our approach has the advantage of being cost effective and applicable to live cells while providing an adequate resolution for time-resolved detection of channel activation.

Experimental Comparison of two Active Vibration Control Approaches: Velocity Feedback and Negative Capacitance Shunt Damping

NASA Technical Reports Server (NTRS)

Beck, Benjamin; Schiller, Noah

2013-01-01

This paper outlines a direct, experimental comparison between two established active vibration control techniques. Active vibration control methods, many of which rely upon piezoelectric patches as actuators and/or sensors, have been widely studied, showing many advantages over passive techniques. However, few direct comparisons between different active vibration control methods have been made to determine the performance benefit of one method over another. For the comparison here, the first control method, velocity feedback, is implemented using four accelerometers that act as sensors along with an analog control circuit which drives a piezoelectric actuator. The second method, negative capacitance shunt damping, consists of a basic analog circuit which utilizes a single piezoelectric patch as both a sensor and actuator. Both of these control methods are implemented individually using the same piezoelectric actuator attached to a clamped Plexiglas window. To assess the performance of each control method, the spatially averaged velocity of the window is compared to an uncontrolled response.

hERG K+ channel-associated cardiac effects of the antidepressant drug desipramine.

PubMed

Staudacher, Ingo; Wang, Lu; Wan, Xiaoping; Obers, Sabrina; Wenzel, Wolfgang; Tristram, Frank; Koschny, Ronald; Staudacher, Kathrin; Kisselbach, Jana; Koelsch, Patrick; Schweizer, Patrick A; Katus, Hugo A; Ficker, Eckhard; Thomas, Dierk

2011-02-01

Cardiac side effects of antidepressant drugs are well recognized. Adverse effects precipitated by the tricyclic drug desipramine include prolonged QT intervals, torsade de pointes tachycardia, heart failure, and sudden cardiac death. QT prolongation has been primarily attributed to acute blockade of hERG/I(Kr) currents. This study was designed to provide a more complete picture of cellular effects associated with desipramine. hERG channels were expressed in Xenopus laevis oocytes and human embryonic kidney (HEK 293) cells, and potassium currents were recorded using patch clamp and two-electrode voltage clamp electrophysiology. Ventricular action potentials were recorded from guinea pig cardiomyocytes. Protein trafficking and cell viability were evaluated in HEK 293 cells and in HL-1 mouse cardiomyocytes by immunocytochemistry, Western blot analysis, or colorimetric MTT assay, respectively. We found that desipramine reduced hERG currents by binding to a receptor site inside the channel pore. hERG protein surface expression was reduced after short-term treatment, revealing a previously unrecognized mechanism. When long-term effects were studied, forward trafficking was impaired and hERG currents were decreased. Action potential duration was prolonged upon acute and chronic desipramine exposure. Finally, desipramine triggered apoptosis in cells expressing hERG channels. Desipramine exerts at least four different cellular effects: (1) direct hERG channel block, (2) acute reduction of hERG surface expression, (3) chronic disruption of hERG trafficking, and (4) induction of apoptosis. These data highlight the complexity of hERG-associated drug effects.

An ideal clamping analysis for a cross-ply laminate

NASA Technical Reports Server (NTRS)

Valisetty, R. R.; Murthy, P. L. N.; Rehfield, L. W.

1988-01-01

Different elementary clamping models are discussed for a three layer crossply laminate to study the sensitivity of clamping to the definition of cross-sectional rotation. All of these models leave a considerable residual warping at the edges. Using a complimentary energy principle and principle of superposition, an analysis is conducted to reduce this residual warping. This led to the identification of exact interior solution corresponding to the ideal clamping. This study also suggests a presence of stress singularities at the corners and between different layers near the fixed edge.

Design, development, physicochemical, and in vitro and in vivo evaluation of transdermal patches containing diclofenac diethylammonium salt.

PubMed

Arora, Priyanka; Mukherjee, Biswajit

2002-09-01

In this study, matrix-type transdermal patches containing diclofenac diethylamine were prepared using different ratios of polyvinylpyrrolidone (PVP) and ethylcellulose (EC) by solvent evaporation technique. The drug matrix film of PVP and EC was casted on a polyvinylalcohol backing membrane. All the prepared formulations were subjected to physical studies (moisture content, moisture uptake, and flatness), in vitro release studies and in vitro skin permeation studies. In vitro permeation studies were performed across cadaver skin using a modified diffusion cell. Variations in drug release profiles among the formulations studied were observed. Based on a physicochemical and in vitro skin permeation study, formulation PA4 (PVP/EC, 1:2) and PA5 (PVP/EC, 1:5) were chosen for further in vivo experiments. The antiinflammatory effect and a sustaining action of diclofenac diethylamine from the two transdermal patches selected were studied by inducing paw edema in rats with 1% w/v carrageenan solution. When the patches were applied half an hour before the subplantar injection of carrageenan in the hind paw of male Wistar rats, it was observed that formulation PA4 produced 100% inhibition of paw edema in rats 12 h after carrageenan insult, whereas in the case of formulation PA5, 4% mean paw edema was obtained half an hour after the carrageenan injection and the value became 19.23% 12 h after the carrageenan insult. The efficacy of transdermal patches was also compared with the marketed Voveran gel and it was found that PA4 transdermal patches produced a better result as compared with the Voveran gel. Hence, it can be reasonably concluded that diclofenac diethylamine can be formulated into the transdermal matrix type patches to sustain its release characteristics and the polymeric composition (PVP/EC, 1:2) was found to be the best choice for manufacturing transdermal patches of diclofenac diethylamine among the formulations studied. Copyright 2002 Wiley-Liss, Inc.

Single- and multiple-dose pharmacokinetics of a novel tetramethylpyrazine reservoir-type transdermal patch versus tetramethylpyrazine phosphate oral tablets in healthy normal volunteers, and in vitro/in vivo correlation.

PubMed

Shen, Teng; Xu, Huinan; Weng, Weiyu; Zhang, Jianfang

2013-01-01

A novel reservoir-type transdermal system of 2,3,5,6-tetramethylpyrazine (TMP) was developed containing eucalyptus oil as a penetration enhancer. The single and multiple-dose pharmacokinetic profiles of TMP administrated by TMP transdermal patch were characterized in healthy volunteers using an in vivo, randomized, open-label, two-way crossover design. 2,3,5,6-Tetramethylpyrazine phosphate (TMPP) oral tablets were chosen as reference. Following single/multiple oral administration of 200/100 mg TMPP tablets, a TMP C(max) of 1284/613.5 ng/mL was observed within 0.75 h. Single/multiple applications of the TMP patch yielded mean C(max) of 309/325 ng/mL at a median T(max) of 5/4 h, with steady state achieved at second application. The mean C(min) of the patch was 131±30.38 ng/mL, contrasting to nearly zero for the tablet. Multiple applications of patch produced an accumulative effect over single application. At steady state 250 mg/20 cm(2) TMP patch given daily provided comparable exposure to 100 mg TMPP tablets three times daily (3753.91 versus 3563.67 ng·h/mL). TMP tablets and patch yielded similar steady-state plasma concentrations: C(av) (148.48±51.27, 156.41±40.31 ng/mL). The results demonstrated that TMP patch can achieve a therapeutic effect that is comparable to oral administration, exhibited prolonged and sustained plasma levels, fewer drug fluctuations, lower adverse effects, more convenience, and improved patient compliance. In-vitro permeation through human skin demonstrated zero-order kinetics with the flux of 364 µg/cm(2)/h. The predicted C(av) (163.9 ng/mL) was in agreement with the observed C(av) (156.4 ng/mL).

Reduction in spontaneous firing of mouse excitatory layer 4 cortical neurons following visual classical conditioning

NASA Astrophysics Data System (ADS)

Bekisz, Marek; Shendye, Ninad; Raciborska, Ida; Wróbel, Andrzej; Waleszczyk, Wioletta J.

2017-08-01

The process of learning induces plastic changes in neuronal network of the brain. Our earlier studies on mice showed that classical conditioning in which monocular visual stimulation was paired with an electric shock to the tail enhanced GABA immunoreactivity within layer 4 of the monocular part of the primary visual cortex (V1), contralaterally to the stimulated eye. In the present experiment we investigated whether the same classical conditioning paradigm induces changes of neuronal excitability in this cortical area. Two experimental groups were used: mice that underwent 7-day visual classical conditioning and controls. Patch-clamp whole-cell recordings were performed from ex vivo slices of mouse V1. The slices were perfused with the modified artificial cerebrospinal fluid, the composition of which better mimics the brain interstitial fluid in situ and induces spontaneous activity. The neuronal excitability was characterized by measuring the frequency of spontaneous action potentials. We found that layer 4 star pyramidal cells located in the monocular representation of the "trained" eye in V1 had lower frequency of spontaneous activity in comparison with neurons from the same cortical region of control animals. Weaker spontaneous firing indicates decreased general excitability of star pyramidal neurons within layer 4 of the monocular representation of the "trained" eye in V1. Such effect could result from enhanced inhibitory processes accompanying learning in this cortical area.

Inhibition of sympathetic sprouting in CCD rats by lacosamide.

PubMed

Wang, Yuying; Huo, Fuquan

2018-05-14

Early hyperexcitability activity of injured nerve/neuron is critical for developing sympathetic nerve sprouting within dorsal root ganglia (DRG). Since lacosamide (LCM), an anticonvulsant, inhibits Na + channel. The present study tried to test the potential effect of LCM on inhibiting sympathetic sprouting in vivo. LCM (50 mg/kg) was daily injected intraperitoneally into rats subjected to chronic compression DRG (CCD), an animal model of neuropathic pain that exhibits sympathetic nerve sprouting, for the 1st 7 days after injury. Mechanical sensitivity was tested from day 3 to day 18 after injury, and then DRGs were removed off. Immunohistochemical staining for tyrosine hydroxylase (TH) was examined to observe sympathetic sprouting, and patch-clamp recording was performed to test the excitability and Na + current of DRG neurons. Early systemic LCM treatment significantly reduced TH immunoreactivity density in injured DRG, lowered the excitability level of injured DRG neurons, and increased paw withdrawal threshold (PWT). These effects on reducing sympathetic sprouting, inhibiting excitability and suppressing pain behavior were observed 10 days after the end of early LCM injection. In vitro 100 μM LCM instantly reduced the excitability of CCD neurons via inhibiting Na + current and reducing the amplitude of AP. All the findings suggest, for the first time, that early administration of LCM inhibited sympathetic sprouting and then alleviated neuropathic pain. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Diabetes-induced changes in the alternative splicing of the slo gene in corporal tissue.

PubMed

Davies, Kelvin P; Zhao, Weixin; Tar, Moses; Figueroa, Johanna C; Desai, Pratik; Verselis, Vytas K; Kronengold, Jack; Wang, Hong-Zhan; Melman, Arnold; Christ, George J

2007-10-01

Erectile dysfunction is a common diabetic complication. Preclinical studies have documented that the Slo gene (encoding the BK or Maxi-K channel alpha-subunit) plays a critical role in erectile function. Therefore, we determined whether diabetes induces changes in the splicing of the Slo gene relevant to erectile function. Reverse transcriptase-polymerase chain reaction was used to compare Slo splice variant expression in corporal tissue excised from control and streptozotocin (STZ)-induced diabetic Fischer F-344 rats. Splice variants were sequenced, characterized by patch clamping, and fused to green fluorescent protein to determine cellular localization. The impact of altered Slo expression on erectile function was further evaluated in vivo. A novel Slo splice variant (SVcyt, with a cytoplasmic location) was predominantly expressed in corporal tissue from control rats. STZ-diabetes caused upregulation of a channel-forming transcript SV0. Preliminary results suggest that SV0 was also more prevalent in the corporal tissue of human diabetic compared with nondiabetic patients. The change in isoform expression in STZ-treated rats was partially reversed by insulin treatment. Intracorporal injection of a plasmid expressing the SV0 transcript, but not SVcyt, restored erectile function in STZ-diabetic rats. Alternative splicing of the Slo transcript may represent an important compensatory mechanism to increase the ease with which relaxation of corporal tissue may be triggered as a result of a diabetes-related decline in erectile capacity.

A Simplified, Langendorff-Free Method for Concomitant Isolation of Viable Cardiac Myocytes and Nonmyocytes From the Adult Mouse Heart

PubMed Central

Ackers-Johnson, Matthew; Li, Peter Yiqing; Holmes, Andrew P.; O’Brien, Sian-Marie; Pavlovic, Davor; Foo, Roger S.

2018-01-01

Rationale Cardiovascular disease represents a global pandemic. The advent of and recent advances in mouse genomics, epigenomics, and transgenics offer ever-greater potential for powerful avenues of research. However, progress is often constrained by unique complexities associated with the isolation of viable myocytes from the adult mouse heart. Current protocols rely on retrograde aortic perfusion using specialized Langendorff apparatus, which poses considerable logistical and technical barriers to researchers and demands extensive training investment. Objective To identify and optimize a convenient, alternative approach, allowing the robust isolation and culture of adult mouse cardiac myocytes using only common surgical and laboratory equipment. Methods and Results Cardiac myocytes were isolated with yields comparable to those in published Langendorff-based methods, using direct needle perfusion of the LV ex vivo and without requirement for heparin injection. Isolated myocytes can be cultured antibiotic free, with retained organized contractile and mitochondrial morphology, transcriptional signatures, calcium handling, responses to hypoxia, neurohormonal stimulation, and electric pacing, and are amenable to patch clamp and adenoviral gene transfer techniques. Furthermore, the methodology permits concurrent isolation, separation, and coculture of myocyte and nonmyocyte cardiac populations. Conclusions We present a novel, simplified method, demonstrating concomitant isolation of viable cardiac myocytes and nonmyocytes from the same adult mouse heart. We anticipate that this new approach will expand and accelerate innovative research in the field of cardiac biology. PMID:27502479

An In Vivo Study of Low-Dose Intra-Articular Tranexamic Acid Application with Prolonged Clamping Drain Method in Total Knee Replacement: Clinical Efficacy and Safety

PubMed Central

Sa-ngasoongsong, Paphon; Chanplakorn, Pongsthorn; Wongsak, Siwadol; Uthadorn, Krisorn; Panpikoon, Tanapong; Jittorntam, Paisan; Aryurachai, Katcharin; Angchaisukisiri, Pantap; Kawinwonggowit, Viroj

2015-01-01

Background. Recently, combined intra-articular tranexamic acid (IA-TXA) injection with clamping drain method showed efficacy for blood loss and transfusion reduction in total knee replacement (TKR). However, until now, none of previous studies revealed the effect of this technique on pharmacokinetics, coagulation, and fibrinolysis. Materials and Methods. An experimental study was conducted, during 2011-2012, in 30 patients undergoing unilateral TKR. Patients received IA-TXA application and then were allocated into six groups regarding clamping drain duration (2-, 4-, 6-, 8-, 10-, and 12-hours). Blood and drainage fluid were collected to measure tranexamic acid (TXA) level and related coagulation and fibrinolytic markers. Postoperative complication was followed for one year. Results. There was no significant difference of serum TXA level at 2 hour and 24 hour among groups (p < 0.05). Serum TXA level at time of clamp release was significantly different among groups with the highest level at 2 hour (p < 0.0001). There was no significant difference of TXA level in drainage fluid, postoperative blood loss, blood transfusion, and postoperative complications (p < 0.05).  Conclusions. Low-dose IA-TXA application in TKR with prolonged clamping drain method is a safe and effective blood conservative technique with only minimal systemic absorption and without significant increase in systemic absorption over time. PMID:26583092

The Effect of Single Pyramidal Neuron Firing Within Layer 2/3 and Layer 4 in Mouse V1.

PubMed

Meyer, Jochen F; Golshani, Peyman; Smirnakis, Stelios M

2018-01-01

The influence of cortical cell spiking activity on nearby cells has been studied extensively in vitro . Less is known, however, about the impact of single cell firing on local cortical networks in vivo . In a pioneering study, Kwan and Dan (Kwan and Dan, 2012) reported that in mouse layer 2/3 (L2/3), under anesthesia , stimulating a single pyramidal cell recruits ~2.1% of neighboring units. Here we employ two-photon calcium imaging in layer 2/3 of mouse V1, in conjunction with single-cell patch clamp stimulation in layer 2/3 or layer 4, to probe, in both the awake and lightly anesthetized states , how (i) activating single L2/3 pyramidal neurons recruits neighboring units within L2/3 and from layer 4 (L4) to L2/3, and whether (ii) activating single pyramidal neurons changes population activity in local circuit. To do this, it was essential to develop an algorithm capable of quantifying how sensitive the calcium signal is at detecting effectively recruited units ("followers"). This algorithm allowed us to estimate the chance of detecting a follower as a function of the probability that an epoch of stimulation elicits one extra action potential (AP) in the follower cell. Using this approach, we found only a small fraction (<0.75%) of L2/3 cells to be significantly activated within a radius of ~200 μm from a stimulated neighboring L2/3 pyramidal cell. This fraction did not change significantly in the awake vs. the lightly anesthetized state, nor when stimulating L2/3 vs. underlying L4 pyramidal neurons. These numbers are in general agreement with, though lower than, the percentage of neighboring cells (2.1% pyramidal cells and interneurons combined) reported by Kwan and Dan to be activated upon stimulating single L2/3 pyramidal neurons under anesthesia (Kwan and Dan, 2012). Interestingly, despite the small number of individual units found to be reliably driven, we did observe a modest but significant elevation in aggregate population responses compared to sham stimulation. This underscores the distributed impact that single cell stimulation has on neighboring microcircuit responses, revealing only a small minority of relatively strongly connected partners. Patch-clamp stimulation in conjunction with 2-photon imaging shows that activating single layer-2/3 or layer-4 pyramidal neurons produces few (<1% of local units) reliable single-cell followers in L2/3 of mouse area V1, either under light anesthesia or in quiet wakefulness: instead, single cell stimulation was found to elevate aggregate population activity in a weak but highly distributed fashion.

Periodic shunted arrays for the control of noise radiation in an enclosure

NASA Astrophysics Data System (ADS)

Casadei, Filippo; Dozio, Lorenzo; Ruzzene, Massimo; Cunefare, Kenneth A.

2010-08-01

This work presents numerical and experimental investigations of the application of a periodic array of resistive-inductive (RL) shunted piezoelectric patches for the attenuation of broadband noise radiated by a flexible plate in an enclosed cavity. A 4×4 lay-out of piezoelectric patches is bonded to the surface of a rectangular plate fully clamped to the top face of a rectangular cavity. Each piezo-patch is shunted through a single RL circuit, and all shunting circuits are tuned at the same frequency. The response of the resulting periodic structure is characterized by frequency bandgaps where vibrations and associated noise are strongly attenuated. The location and extent of induced bandgaps are predicted by the application of Bloch theorem on a unit cell of the periodic assembly, and they are controlled by proper selection of the shunting circuit impedance. A coupled piezo-structural-acoustic finite element model is developed to evaluate the noise reduction performance. Strong attenuation of multiple panel-controlled modes is observed over broad frequency bands. The proposed concept is tested on an aluminum plate mounted in a wooden box and driven by a shaker. Experimental results are presented in terms of pressure responses recorded using a grid of microphones placed inside the acoustic box.

Structure-function analysis of the extracellular domain of the pneumococcal cell division site positioning protein MapZ

NASA Astrophysics Data System (ADS)

Manuse, Sylvie; Jean, Nicolas L.; Guinot, Mégane; Lavergne, Jean-Pierre; Laguri, Cédric; Bougault, Catherine M.; Vannieuwenhze, Michael S.; Grangeasse, Christophe; Simorre, Jean-Pierre

2016-06-01

Accurate placement of the bacterial division site is a prerequisite for the generation of two viable and identical daughter cells. In Streptococcus pneumoniae, the positive regulatory mechanism involving the membrane protein MapZ positions precisely the conserved cell division protein FtsZ at the cell centre. Here we characterize the structure of the extracellular domain of MapZ and show that it displays a bi-modular structure composed of two subdomains separated by a flexible serine-rich linker. We further demonstrate in vivo that the N-terminal subdomain serves as a pedestal for the C-terminal subdomain, which determines the ability of MapZ to mark the division site. The C-terminal subdomain displays a patch of conserved amino acids and we show that this patch defines a structural motif crucial for MapZ function. Altogether, this structure-function analysis of MapZ provides the first molecular characterization of a positive regulatory process of bacterial cell division.

Oracle Applications Patch Administration Tool (PAT) Beta Version

DOE Office of Scientific and Technical Information (OSTI.GOV)

2002-01-04

PAT is a Patch Administration Tool that provides analysis, tracking, and management of Oracle Application patches. This includes capabilities as outlined below: Patch Analysis & Management Tool Outline of capabilities: Administration Patch Data Maintenance -- track Oracle Application patches applied to what database instance & machine Patch Analysis capture text files (readme.txt and driver files) form comparison detail report comparison detail PL/SQL package comparison detail SQL scripts detail JSP module comparison detail Parse and load the current applptch.txt (10.7) or load patch data from Oracle Application database patch tables (11i) Display Analysis -- Compare patch to be applied with currentmore » Oracle Application installed Appl_top code versions Patch Detail Module comparison detail Analyze and display one Oracle Application module patch. Patch Management -- automatic queue and execution of patches Administration Parameter maintenance -- setting for directory structure of Oracle Application appl_top Validation data maintenance -- machine names and instances to patch Operation Patch Data Maintenance Schedule a patch (queue for later execution) Run a patch (queue for immediate execution) Review the patch logs Patch Management Reports« less

Product Development and Cost Analysis of Fabricating the Prototype of Roller Clamp in Intravenous (I.V) Tubing Medical Devices using Fused Deposition Modeling (FDM) Technology

NASA Astrophysics Data System (ADS)

Way, Yusoff

2018-01-01

The main aim of this research is to develop a new prototype and to conduct cost analysis of the existing roller clamp which is one of parts attached to Intravenous (I.V) Tubing used in Intravenous therapy medical device. Before proceed with the process to manufacture the final product using Fused Deposition Modeling (FDM) Technology, the data collected from survey were analyzed using Product Design Specifications approach. Selected concept has been proven to have better quality, functions and criteria compared to the existing roller clamp and the cost analysis of fabricating the roller clamp prototype was calculated.

Methylene blue inhibits function of the 5-HT transporter

PubMed Central

Oz, Murat; Isaev, Dmytro; Lorke, Dietrich E; Hasan, Muhammed; Petroianu, Georg; Shippenberg, Toni S

2012-01-01

BACKGROUND AND PURPOSE Methylene blue (MB) is commonly employed as a treatment for methaemoglobinaemia, malaria and vasoplegic shock. An increasing number of studies indicate that MB can cause 5-HT toxicity when administered with a 5-HT reuptake inhibitor. MB is a potent inhibitor of monoamine oxidases, but other targets that may contribute to MB toxicity have not been identified. Given the role of the 5-HT transporter (SERT) in the regulation of extracellular 5-HT concentrations, the present study aimed to characterize the effect of MB on SERT. EXPERIMENTAL APPROACH Live cell imaging, in conjunction with the fluorescent SERT substrate 4-(4-(dimethylamino)-styryl)-N-methylpyridinium (ASP+), [3H]5-HT uptake and whole-cell patch-clamp techniques were employed to examine the effects of MB on SERT function. KEY RESULTS In EM4 cells expressing GFP-tagged human SERT (hSERT), MB concentration-dependently inhibited ASP+ accumulation (IC50: 1.4 ± 0.3 µM). A similar effect was observed in N2A cells. Uptake of [3H]5-HT was decreased by MB pretreatment. Furthermore, patch-clamp studies in hSERT expressing cells indicated that MB significantly inhibited 5-HT-evoked ion currents. Pretreatment with 8-Br-cGMP did not alter the inhibitory effect of MB on hSERT activity, and intracellular Ca2+ levels remained unchanged during MB application. Further experiments revealed that ASP+ binding to cell surface hSERT was reduced after MB treatment. In whole-cell radioligand experiments, exposure to MB (10 µM; 10 min) did not alter surface binding of the SERT ligand [125I]RTI-55. CONCLUSIONS AND IMPLICATIONS MB modulated SERT function and suggested that SERT may be an additional target upon which MB acts to produce 5-HT toxicity. PMID:21542830

Regulation of the epithelial Na+ channel by membrane tension.

PubMed

Awayda, M S; Subramanyam, M

1998-08-01

The sensitivity of alphabetagamma rat epithelial Na+ channel (rENaC) to osmotically or mechanically induced changes of membrane tension was investigated in the Xenopus oocyte expression system, using both dual electrode voltage clamp and cell-attached patch clamp methodologies. ENaC whole-cell currents were insensitive to mechanical cell swelling caused by direct injection of 90 or 180 nl of 100-mM KCl. Similarly, ENaC whole-cell currents were insensitive to osmotic cell swelling caused by a 33% decrease of bathing solution osmolarity. The lack of an effect of cell swelling on ENaC was independent of the status of the actin cytoskeleton, as ENaC remained insensitive to osmotic and mechanical cell swelling in oocytes pretreated with cytochalasin B for 2-5 h. This apparent insensitivity of ENaC to increased cell volume and changes of membrane tension was also observed at the single channel level in membrane patches subjected to negative or positive pressures of 5 or 10 in. of water. However, and contrary to the lack of an effect of cell swelling, ENaC currents were inhibited by cell shrinking. A 45-min incubation in a 260-mosmol solution (a 25% increase of solution osmolarity) caused a decrease of ENaC currents (at -100 mV) from -3.42 +/- 0.34 to -2.02 +/- 0.23 microA (n = 6). This decrease of current with cell shrinking was completely blocked by pretreatment of oocytes with cytochalasin B, indicating that these changes of current are not likely related to a direct effect of cell shrinking. We conclude that alpha beta gamma rENaC is not directly mechanosensitive when expressed in a system that can produce a channel with identical properties to those found in native epithelia.

Human spermatozoa possess a calcium-dependent chloride channel that may participate in the acrosomal reaction

PubMed Central

Orta, Gerardo; Ferreira, Gonzalo; José, Omar; Treviño, Claudia L; Beltrán, Carmen; Darszon, Alberto

2012-01-01

Motility, maturation and the acrosome reaction (AR) are fundamental functions of mammalian spermatozoa. While travelling through the female reproductive tract, spermatozoa must mature through a process named capacitation, so that they can reach the egg and undergo the AR, an exocytotic event necessary to fertilize the egg. Though Cl− is important for sperm capacitation and for the AR, not much is known about the molecular identity of the Cl− transporters involved in these processes. We implemented a modified perforated patch-clamp strategy to obtain whole cell recordings sealing on the head of mature human spermatozoa. Our whole cell recordings revealed the presence of a Ca2+-dependent Cl− current. The biophysical characteristics of this current and its sensitivity to niflumic acid (NFA) and 4,4′-diisothiocyano-2,2′-stilbene disulphonic acid (DIDIS) are consistent with those displayed by the Ca2+-dependent Cl− channel from the anoctamin family (TMEM16). Whole cell patch clamp recordings in the cytoplasmic droplet of human spermatozoa corroborated the presence of these currents, which were sensitive to NFA and to a small molecule TMEM16A inhibitor (TMEM16Ainh, an aminophenylthiazole). Importantly, the human sperm AR induced by a recombinant human glycoprotein from the zona pellucida, rhZP3, displayed a similar sensitivity to NFA, DIDS and TMEM16Ainh as the sperm Ca2+-dependent Cl− currents. Our findings indicate the presence of Ca2+-dependent Cl− currents in human spermatozoa, that TMEM16A may contribute to these currents and also that sperm Ca2+-dependent Cl− currents may participate in the rhZP3-induced AR. PMID:22473777

The blockade of excitation/contraction coupling by nifedipine in patch-clamped rat skeletal muscle cells in culture.

PubMed

Cognard, C; Rivet, M; Raymond, G

1990-04-01

The effects of the dihydropyridine derivative, nifedipine, well known as a blocker of calcium channels, were tested on cultured rat myoballs. Membrane currents and contractions were simultaneously recorded by means of the patch-clamp technique and a photoelectric transducing method. High concentrations of nifedipine (5 microM) inhibited the contractile responses and inward calcium current (ICa) elicited by long depolarizations. In the absence of ICa (1.5 mM cadmium in the bath), nifedipine inhibited both the ICa-independent contractile component and the outward current, supposed to depend on the intracellular calcium released during contraction. At low concentrations (0.5 microM) the blocking effects of nifedipine could be strongly enhanced by shifting the membrane potential towards less negative values (-60 mV) for 50 s prior to the test pulse. A blocking effect of nifedipine, at a usually ineffective concentration (0.1 microM), could also be observed when long-lasting (3 min) prepulses to 0 mV were applied from a reference membrane potential of -60 mV. This effect could be relieved by long-lasting cell hyperpolarizations (-90 mV). The blocking effects of nifedipine unrelated to ICa could be interpreted as an action on a molecule (voltage sensor) in the T-tubule membrane involved in the excitation/contraction coupling process and as a preferential binding of the dihydropyridine derivative on the inactivated form of this molecule, favored by the weak negative potentials or long-lasting depolarizations. The results provide data in favor of the existence of strong similarities between the calcium channels and voltage sensors since their operation was inhibited in a voltage-dependent manner by nifedipine.

Distribution and function of HCN channels in the apical dendritic tuft of neocortical pyramidal neurons.

PubMed

Harnett, Mark T; Magee, Jeffrey C; Williams, Stephen R

2015-01-21

The apical tuft is the most remote area of the dendritic tree of neocortical pyramidal neurons. Despite its distal location, the apical dendritic tuft of layer 5 pyramidal neurons receives substantial excitatory synaptic drive and actively processes corticocortical input during behavior. The properties of the voltage-activated ion channels that regulate synaptic integration in tuft dendrites have, however, not been thoroughly investigated. Here, we use electrophysiological and optical approaches to examine the subcellular distribution and function of hyperpolarization-activated cyclic nucleotide-gated nonselective cation (HCN) channels in rat layer 5B pyramidal neurons. Outside-out patch recordings demonstrated that the amplitude and properties of ensemble HCN channel activity were uniform in patches excised from distal apical dendritic trunk and tuft sites. Simultaneous apical dendritic tuft and trunk whole-cell current-clamp recordings revealed that the pharmacological blockade of HCN channels decreased voltage compartmentalization and enhanced the generation and spread of apical dendritic tuft and trunk regenerative activity. Furthermore, multisite two-photon glutamate uncaging demonstrated that HCN channels control the amplitude and duration of synaptically evoked regenerative activity in the distal apical dendritic tuft. In contrast, at proximal apical dendritic trunk and somatic recording sites, the blockade of HCN channels decreased excitability. Dynamic-clamp experiments revealed that these compartment-specific actions of HCN channels were heavily influenced by the local and distributed impact of the high density of HCN channels in the distal apical dendritic arbor. The properties and subcellular distribution pattern of HCN channels are therefore tuned to regulate the interaction between integration compartments in layer 5B pyramidal neurons. Copyright © 2015 the authors 0270-6474/15/351024-14$15.00/0.

Ion channels in plants.

PubMed

Hedrich, Rainer

2012-10-01

Since the first recordings of single potassium channel activities in the plasma membrane of guard cells more than 25 years ago, patch-clamp studies discovered a variety of ion channels in all cell types and plant species under inspection. Their properties differed in a cell type- and cell membrane-dependent manner. Guard cells, for which the existence of plant potassium channels was initially documented, advanced to a versatile model system for studying plant ion channel structure, function, and physiology. Interestingly, one of the first identified potassium-channel genes encoding the Shaker-type channel KAT1 was shown to be highly expressed in guard cells. KAT1-type channels from Arabidopsis thaliana and its homologs from other species were found to encode the K(+)-selective inward rectifiers that had already been recorded in early patch-clamp studies with guard cells. Within the genome era, additional Arabidopsis Shaker-type channels appeared. All nine members of the Arabidopsis Shaker family are localized at the plasma membrane, where they either operate as inward rectifiers, outward rectifiers, weak voltage-dependent channels, or electrically silent, but modulatory subunits. The vacuole membrane, in contrast, harbors a set of two-pore K(+) channels. Just very recently, two plant anion channel families of the SLAC/SLAH and ALMT/QUAC type were identified. SLAC1/SLAH3 and QUAC1 are expressed in guard cells and mediate Slow- and Rapid-type anion currents, respectively, that are involved in volume and turgor regulation. Anion channels in guard cells and other plant cells are key targets within often complex signaling networks. Here, the present knowledge is reviewed for the plant ion channel biology. Special emphasis is drawn to the molecular mechanisms of channel regulation, in the context of model systems and in the light of evolution.